[Abstract Title]. - Society for Neuroscience

When citing an abstract from the 2008 Annual Meeting please use the format below. 

[Authors]. [Abstract Title]. Program No. XXX.XX. 2008 Neuroscience Meeting Planner. 

Washington, DC: Society for Neuroscience, 2008. Online. 

2008 Copyright by the Society for Neuroscience all rights reserved. Permission to republish any 

abstract or part of any abstract in any form must be obtained in writing by SfN office prior to 

publication

Poster 

229. Neuronal and Glial Proliferation III 

Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm 

Program#/Poster#: 229.1/A1 

Topic: A.02.a. Proliferation 

Support: Korea Science Academy R&E Program (2007) 

Title: Resveratrol suppresses the proliferation of neural progenitor cells and impairs adult 

hippocampal neurogenesis 

Authors: J. BAEK 1 , H. BANG 1 , C. LEE 1 , N. CHOI 1 , M. PARK 2 , H. PARK 2 , J. RYU 1 , *J. 

LEE 2 ; 

1 Korea Sci. Acad., Busan, Republic of Korea; 2 Pharm., Pusan Natl. Univ., Busan, Republic of 

Korea 

Abstract: Due to lack of regenerative capacity of mature neuron, impairment and damage of the 

adult human central nervous system (CNS) are critical and incurable. However, the nervous 

system contains populations of proliferative cells called “neural progenitor cells (NPC)” that are 

capable of dividing, migrating and differentiating into neurons and glia. Thus, there is a growing 

hope in the possible therapeutic potential for ischemic, traumatic, and degenerative brain 

diseases. In the current study, we investigated the therapeutic potential of resveratrol in 

regulating neurogenesis of NPC. Resveratrol was not effective to stimulate the proliferation of 

mouse multi-potent NPC and high concentrations were even cytotoxic. We found that resveratrol 

decreased the levels of phosphorylated extracellular signal regulated kinases (ERKs) which are 

to be involved in the regulation of neuronal plasticity cell proliferation. Interestingly, 

administration of resveratrol to adult mice resulted in a significant decrease in the number of 

newly-generated cells in the dentate gyrus of hippocampus, indicating that resveratrol impairs 

adult hippocampal neurogenesis. The natural polyphenol resveratrol has been shown to extend 

lifespan, and offer prospect against age-related diseases such as cancer, diabetes, and 

neurodegenerative diseases. However, our findings suggest that the beneficial resveratrol could 

have negative effects on the proliferation of NPC, thus be harmful in developmental and adult 

hippocampal neurogenesis. 

Disclosures: J. Baek, None; H. Bang, None; C. Lee, None; N. Choi, None; M. Park, None; H. 

Park, None; J. Ryu, None; J. Lee , None. 

Poster





Support: NIH Grant R15-NS054761 

Title: Acute chemotherapy treatment yields long-lasting effects on cytogenesis in the mouse 

dentate gyrus 

Authors: *E. T. WEBER, K. A. VANDERGRIFT, C. M. MONDIE; 

Dept Biol, Rider Univ., Lawrenceville, NJ 

Abstract: The dentate gyrus of mammalian hippocampus is one of the few sites that produce 

neuronal progenitor cells in the adult brain. Proliferation in the dentate gyrus is acutely and dosedependently 

inhibited 50-75% by the antimitotic chemotherapy agent, thioTEPA, in young adult 

(8 weeks old) C57BL/6J mice, as assessed by BrdU immunohistochemistry. In this study, longterm 

effects of acute thioTEPA treatment on proliferative capacity in the dentate gyrus and 

subsequent performance in depression-related behavioral tests were examined. Of the BrdUlabeled 

cells that survive acute chemotherapy, none appear to persist for more than 3 weeks. 

Measurements performed at intervals following chemotherapy treatment show an approximate 

50% reduction in proliferative capacity in the dentate gyrus that persists for at least 12 weeks 

after thioTEPA treatment. Assessment of depression-related behavior by tail suspension test 

revealed no differences in performance between mice treated with thioTEPA or with vehicle 

when tested up to 12 weeks following treatment, though recent preliminary data suggests a 

significant increase when tested 30 weeks following chemotherapy treatment. No significant 

differences in performance in the Porsolt forced swim test were measured up to and including 30 

weeks post-treatment. 

These results suggest that acute treatment with thioTEPA yields an immediate and long-lasting 

effect on the capacity of the pool of progenitor cells in the dentate gyrus to proliferate. 

Furthermore, this may yield behavioral effects that are not evident for many months beyond 

treatment, suggesting a cumulative neurological deficit. 

Disclosures: E.T. Weber , None; K.A. Vandergrift, None; C.M. Mondie, None. 

Poster 


Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm



Title: Effects of 5-HT1A and 5-HT1B receptors disruption on adult hippocampal neurogenesis 

in mouse 

Authors: *Q. RAINER 1 , J.-P. GUILLOUX 1 , M. TOTH 2 , R. HEN 3 , A. M. GARDIER 1 , D. J. 

DAVID 1 ; 

1 Univ. Paris Sud XI, Chatenay-Malabry, France; 2 Dept. of Pharmacol., Weill Cornell Med. 

College, Cornell Univ., New York, NY; 3 Departments of Neuroscience, Pharmacol. & 

Psychiatry, Div. of Integrative Neuroscience, Columbia Univ., New York, NY 

Abstract: Recent clinical and preclinical observations have shown that adult hippocampal 

neurogenesis is decreased by stress and increased by chronic antidepressants suggesting that this 

process may be involved in both the pathogenesis and treatment of mood disorders [1]. Indeed, 

using focal X-ray irradiation, it has been demonstrated that adult hippocampal neurogenesis is 

required for behavioural activity of selective serotonin reuptake inhibitors (SSRIs) in the Novelty 

Suppressed Feeding. 

Among the various serotonin (5-HT) receptor subtypes, the 5-HT1A and 5-HT1B receptors were 

prominently implicated in the modulation of mood and anxiety-related behaviors [1, 2]. The 

implication of 5-HT1A receptors in SSRIs-induced increases in adult hippocampal neurogenesis 

seems to be controversial and depends on genetic background of mice [1, 3]. Regarding the 5- 

HT1B receptors, a pharmacological study reported that this receptor sub-type regulates cell 

proliferation in the subgranular layer of the dentate gyrus in the hippocampus[4]. 

The present study aimed to explore the consequences of dual 5HT1A and 5HT1B receptors‟ 

deletion, using 5-HT1A/5-HT1B receptors double knock-out mice (KO 5HT1A/1B), on basal 

adult hippocampus and also on SSRI-induced increases in proliferation, survival and maturation 

of newborn neurons. 

To assess the effects of 5HT1A and 5HT1B receptors‟ deletion on the number of BrdU-positive 

cell proliferation, KO 5HT1A/1B mice were administered with Bromodesoxyuridine (BrdU; 150 

mg/kg, i.p.) 2h before sacrifice, whereas BrdU was administered 2x/day during 3 days , 8 days 

before sacrifice to study survival of newborn progenitor neuronal cells. The same protocol was 

applied to study whether the effects of fluoxetine (18 mg/kg/day) -induced increase in progenitor 

cells require 5HT1A and 5HT1B receptors. The results obtained in 5HT1A/1B KO mice will be 

also compared to 5HT1A or 5HT1B single KO mice and their respective control littermates. 

Taken together our study should clarify the role of 5HT1A and 5HT1B receptors on adult 

neurogenesis in the hippocampus. 

[1] Santarelli et al., . Science 2003, 301, 805-809. 

[2] Gardier et al., J Neurochem 2003, 86(1):13-24. 

[3] Holick et al., Neuropsychopharmacology 2008, 33, 406-417. 

[4] Banasr et al., Neuropsychopharmacology 2004, 29, 450-460. 

Disclosures: Q. Rainer, None; J. Guilloux, None; M. Toth, None; R. Hen, None; A.M. 

Gardier, None; D.J. David, None.

Poster 





Support: NIH Grant NS41228 

NIH Grant NS048256 

Title: Striatal neurons provide a relay for olfaction and SVZ neurogenesis 

Authors: *K. A. DAVE, S. YOUNG, A. BORDEY; 

Dept Neurosurg, Yale Sch. Med., New Haven, CT 

Abstract: Olfactory activity, seizures, and neurodegenerative disorders influence the production 

of newly-born olfactory bulb (OB) interneurons within the subventricular zone (SVZ). However, 

how olfaction influences neurogenesis is unknown. We hypothesize that the striatum is a relay 

network for olfactory input and can influence SVZ neurogenesis. The SVZ is sandwiched 

between the lateral ventricle and the striatum. Here we test whether striatal neurons signal to 

SVZ neural progenitor cells. 

We used morphological characterization, electrophysiology, and calcium (Ca 2+ ) imaging to show 

that GABAergic striatal neurons contribute to signaling in the SVZ. Morphological data indicate 

that 75% of neurons within the striatum bordering the SVZ send processes into the SVZ near 

both neuroblasts and astrocytes; 80% of those are medium spiny neurons (n=130, postnatal day 

15-23). Patch clamp recordings indicate that medium spiny neurons fire action potentials (APs) 

in the range of 6-10 Hz with depolarization. APs are sensitive to both GABAA receptor blocker 

bicuculline and tetrodotoxin (TTX). Depolarizations of striatal neurons with patch clamp 

recordings elicit Ca 2+ responses in SVZ cells surrounding visualized neuronal processes. Elicited 

SVZ Ca 2+ responses are blocked by TTX. SVZ Ca 2+ activity increases 20-40% after action 

potential induction of a nearby striatal neuron (n=5 slices, n=81 cells analyzed). Striatal neurons 

and their processes are visualized with Alexa 568 in the patch pipette, and SVZ cells are loaded 

with Fluo4-AM Ca 2+ dye. 

Collecitively, our results demonstrate that the striatum is in the ideal position to relay inputs from 

the olfactory bulb to the SVZ. Future experiments will test whether striatal spiny neurons 

projecting to the SVZ receive functional connections from the OB. 

Disclosures: K.A. Dave , None; S. Young, None; A. Bordey, None.

Poster 





Title: The histon deacetylase inhibitor, sodium butyrate, stimulates cell proliferation in the 

ischemic brain: roles of BDNF-TrkB signaling 

Authors: *H. KIM, P. R. LEEDS, D.-M. CHUANG; 

Mol. Neurobiol Section, Natl. Inst. Mental Health/NIH, Bethesda, MD 

Abstract: Neurogenesis occurs in the rostral subventricular zone (SVZ) and hippocampal 

dentate gyrus (DG) in the adult brain. It is known that cerebral ischemia enhances neurogenesis 

in the ischemic brain of rodents. The present study investigated whether the beneficial effects of 

sodium butyrate (SB), an HDAC inhibitor, are associated with an enhancement of cell 

proliferation in the ischemic brain. Rats were subjected to permernant middle cerebral artery 

occlusion (pMCAO) followed by daily injections with SB (300 mg/kg, i.p.). Bromo-2' 

deoxyuridine (BrdU) was also given by injections (50 mg/kg, i.p.) after ischemia to label 

newborn cells. We confirmed that SB treatment markedly stimulated BrdU labeling in the SVZ, 

hippocampal DG and frontal cortex of the ischemic brain. SB treatment further increased the 

number of cells expressing doublecortin, nestin, GFAP, phospho-CREB and brain derived 

neurotrophic factor (BDNF) in various regions at 7 and 14 days after pMCAO. SB treatment also 

markedly increased the levels of acetylated histone H3 in cells expressing NeuN in the ischemic 

brain. Intraventricular injection of K252a, a TrkB receptor antagonist, markedly reduced SBinduced 

cell proliferation in the SVZ and DG, and blocked the drug-induced behavioral benefits, 

suggesting the involvement of BDNF-TrkB signaling in the enhanced proliferation. HDAC 

inhibitor-induced cell proliferation may contribute to the drug‟s long-term beneficial effects after 

ischemic injury. Given that there is no effective treatment for stroke, HDAC inhibitors such as 

SB should be evaluated for their potential use for clinical trials in stroke patients. 

Disclosures: H. Kim, None; P.R. Leeds, None; D. Chuang, None. 

Poster 

229. Neuronal and Glial Proliferation III




Support: NIH Grant RO1HD008299 

Title: The effect of the type 1 insulin-like growth factor receptor signaling on neurogenesis in 

the postnatal development of the dentate gyrus 

Authors: *W. LIU, P. YE, A. J. D'ERCOLE; 

Dept Pediatric, Univ. North Carolina-Chapel Hill, Chapel Hill, NC 

Abstract: It has been shown that Insulin-like growth factor-I (IGF-I) is essential to normal brain 

growth and development, and that most of its actions are mediated by the type 1 IGF receptor 

(IGF1R). To directly assess the in vivo effects of IGF-I-IGF1R signaling on neurogenesis, we 

have generated mutant mice with blunted IGF1R expression specifically in neuronal precursors. 

In these mutant mice, termed nes-igf1r -/Wt mice, the function of one igf1r allele is ablated by 

Cre-mediated deletion of exon 3 of the igf1r gene directed by nestin genomic regulatory 

elements. Previously we found that in the dentate gyrus of nes-igf1r -/Wt mice the volume of 

granule cell layer and the number of granule neurons were significantly reduced at postnatal day 

(P) 5 and P20. To further define the actions of IGF-I-IGF1R signaling during postnatal 

development of dentate gyrus, proliferation, survival, and developmental progression of neuron 

precursors were determined. As judged by immunostaining for Ki67, a cell cycle marker, 

proliferating cells in subgranule layer were reduced by 65% in nes-igf1r -/Wt mice at P20, as 

compared to their littermate controls. Consistently, following a single injection of BrdU 3 hr 

prior to tissue collection, the number of dividing cells in S-phase was reduced by 60% in P20 

nes-igf1r -/Wt mice. In contrast, the density of apoptotic cells, assessed by immunostaining for 

active caspase 3, was increased by 78% in the granule cell layer of P20 nes-igf1r -/Wt mice. The 

progression of neuron precursors was then determined by evaluating the number of cells that 

express transcription factors characteristic of early progenitors (Pax6), intermediate progenitor 

cells (Tbr2), and granule neurons (Tbr1). Compared to controls, P5 nes-igf1r -/Wt mice exhibited a 

significant decrease in the number of Pax6 positive cells (by 40%), Tbr2 positive cells (by 55%) 

and Tbr1 positive cells (51%). Similarly, the number of Pax6, Tbr2 and Tbr1 positive cells was 

reduced by 74%, 63% and 74%, respectively, in P20 nes-igf1r -/Wt mice. These results strongly 

indicate that IGF-I-IGF1R signaling plays a critical role in dentate gyrus neurogenesis by 

stimulating proliferation and by augmenting survival of neuron lineage cells. 

Disclosures: W. Liu , None; P. Ye, None; A.J. D'Ercole, None. 

Poster





Support: NJCSCR 07-2933-SCR-E-O 

NIH Training Grant MH019957-09 

Title: Effects of opioid system mutation on the regulation of cell proliferation and cell survival 

in the dentate gyrus and spinal cord 

Authors: *T. P. COMINSKI, M.-S. HSU, M. ANSONOFF, J. PINTAR; 

UMDNJ, Piscataway, NJ 

Abstract: Our previous data indicate that BrdU labeling in the dentate gyrus (DG) is increased 

by 60% (p< .05) in mice lacking the mu opioid receptor (MOR-1) when compared to wild type 

mice on both the C57BL/6 and the 129S6 genetic backgrounds, with over 80% of these 

proliferating cells expressing neuronal markers after 4 weeks in C57BL/6 mice. Since 

endogenous opioid receptors also include DOR-1 (delta receptor) and KOR-1 (kappa receptor), 

we wanted to examine the combined effects of all three opioid receptors on cell proliferation in 

the DG to compare to MOR-1 alone. To accomplish this, we quantified the amount of cell 

proliferation in the granule cell layer (GCL) of the DG in triple opioid receptor knockout (TKO) 

mice and wild type mice on both C57BL/6 and 129S6 genetic backgrounds. In order to label the 

entire proliferative population in the dentate gyrus, mice were injected with 50mg/kg BrdU once 

every 4 hours for a period of 12 hours and sacrificed 12 hours following the last injection; the 

brains were then processed for BrdU immunohistochemistry. Data indicates that BrdU labeling is 

increased by 50% (p< .05) in the GCL of the DG in TKO mice on the 129S6 background when 

compared to wild type. These results, in addition to the MOR-1 data, suggest that opioid system 

regulation of neurogenesis in the DG is primarily dependent on MOR-1. 

Since disruption of the opioid system leads to an increase in cell proliferation in the DG, we also 

wanted to examine the effects of opioid system disruption on cell proliferation and cell survival 

on the proliferative population that exists in the spinal cord, which unlike the DG gives rise to 

mostly glial cells. After quantification of the total number of BrdU positive cells in the thoracic 

region of the spinal cord using the procedure described above, our data indicate that 129S6 TKO 

mice do not exhibit increased levels of cell proliferation at baseline when compared to wild type. 

However, if we compare the number of BrdU positive cells present 12 hours following the BrdU 

injection paradigm to the number of cells present 4 weeks later, only 30% of the BrdU labeled 

cells in TKO mice survive after 4 weeks compared to 50% in wild type (p< .05). Finally, 

following a contusion spinal cord injury (SCI), TKO mice exhibit increased levels of cell 

proliferation for as many as 30 days following SCI as well as a lower amount of locomotor 

recovery, as measured by the BMS scale, when compared to wild type mice. The data suggest 

that glial cell proliferation, and perhaps formation of the glial scar, following SCI may be

exaggerated when opioid receptor function is disrupted and thus may lead to the reduction in 

functional recovery following SCI that we have observed in our TKO mice. 

Disclosures: T.P. Cominski , None; M. Hsu, None; M. Ansonoff, None; J. Pintar, None. 

Poster 





Support: NIAAA09838 

Title: Voluntary exercise and hippocampal neurogenesis: Effect of daily running duration and 

housing environment 

Authors: *J. L. HELFER, C. J. ROCHA, A. Y. KLINTSOVA; 

Psychology, Univ. Delaware, Newark, DE 

Abstract: Cell proliferation and neurogenesis in the rodent hippocampus is affected by 

numerous factors, including voluntary exercise. Wheel-running increases cell proliferation and 

adult neurogenesis as well as the survival of the newly generated cells and neurons in the 

hippocampal dentate gyrus, while improving performance on learning and memory tasks (Adlard 

et al. 2004, Vaynman et al. 2004). It has been demonstrated that twenty-four hour voluntary 

wheel running (24WR) in social housing conditions increases neurogenesis and cell proliferation 

in the hippocampal dentate gyrus (DG) of adolescent and adult rat (Stranahan et al., 2006; Helfer 

et al., in preparation). However, it is debated if mere exercise (wheel running), when done in 

isolation, is sufficient to increase cell proliferation and neurogenesis (Stranahan et al., 2006). The 

present study examined the effects of three voluntary exercise designs on cell proliferation and 

adult neurogenesis. The first design entailed two conditions: voluntary wheel running (24 

hours/day access, single housing) or inactive control (IC, single housing) from PD30-42. 

Animals were injected with BrdU (50mg/kg) every day during PD32-42 and perfused on PD42. 

The second design also had two conditions: voluntary wheel running (12 hour/day, three per 

cage) and IC (3 per cage) during PD30-50. The third wheel running paradigm included a 

voluntary wheel running condition (12 hour/day, single housed) and IC (single housed) during 

PD30-50. In the last two studies animals were injected with BrdU (50mg/kg) every other day 

during PD30-50 and perfused on PD50. Immunocytochemistry was used to localize cells 

containing BrdU and NeuN. BrdU positive cell counts were attained from the DG and confocal 

images were used to assess the colocalization of BrdU and NeuN. Within these three designs,

only the 24hrs WR access (single-housed rats) significantly increased cytogenesis in DG after 12 

days of exercise. These results suggest that voluntary exercise (24hr/day) in both social and 

isolated housing conditions promotes cell proliferation and neurogenesis. Additionally, these 

results suggest that repetitive removal from the running wheels (in the 12hrs exposure paradigm) 

thwarts the exercise effect on cell proliferation and neurogenesis. Supported by NIAAA09838. 

Disclosures: J.L. Helfer , None; C.J. Rocha, None; A.Y. Klintsova, None. 

Poster 





Support: DGAPA IN204206 

CONACYT V40286M 

Title: Pacing behavior increase the number of cells in the accessory olfactory bulb in the adult 

female rat 

Authors: R. CORONA, *R. G. PAREDES; 

Cognitivas, Inst. De Neurobiologia UNAM, Queretaro 76001 QRO, Mexico 

Abstract: Neurogenesis in the olfactory bulb of rodents persists in adult life. This production of 

new cells comes from the subventricular zone of the lateral ventricles, where cells originate and 

migrate undifferentiated along the rostral migratory stream until they reach the accessory (AOB) 

and main olfactory bulb (MOB) where they integrate as neurons or glia. The AOB is a structure 

located in the dorsal and medial portion of the olfactory bulb. This structure plays and important 

role in the modulation of behavioral responses elicited by pheromones, it processes 

chemosensory sexually relevant cues by the vomeronasal organ. During mating a lot of olfactory 

stimuli are provided that could influence the production of new cells. When the females pace 

their sexual interaction, physiological and behavioral changes occur that favor gestation in 

comparison to non-paced mating. The present experiment was aimed to determine if paced 

sexual behavior induces neurogenesis in the OB. Ovariectomized hormonally primed females 

were divided in different groups. One group was allowed to paced while another mated in a nonpaced 

condition. All subjects were injected intraperitoneally with the mitotic marker BrdU one 

hour before, at the end and one hour after mating. The rats were sacrificed 15 days after the test. 

Brain sections were obtained and processed immunohistochemically to reveal BrdU-labeling.

Our results shows an increase in BrdU+ cells in the granular layer of the AOB in the pacing 

group compared with the control groups. No significant differences in the number of BrdU+ cells 

were found in the main olfactory bulb. These results suggest that in sexually naïve female rats 

the integration of new cells in the granular layer of the AOB is necessary for processing novel 

sexual stimuli. Further studies are needed to determine the type of cells that increases in the 

AOB and if they are activated in the next sexual interaction. 

Disclosures: R. Corona, None; R.G. Paredes , None. 

Poster 





Support: Technology Development Program for Agriculture and Forestry, Ministry of 

Agriculture and Forestry Grant 105075-3 

Title: High fat diet impairs adult hippocampal neurogenesis without affecting spatial learning 

and memory 

Authors: *H. PARK, M. PARK, K. KONG, H. KIM, M.-S. KIM, J. LEE; 

Pusan Natl. Univ., Busan, Republic of Korea 

Abstract: Obesity has been progressively increasing worldwide and cause many diseases such as 

diabetes, hypertension, cardiovascular diseases, dementia and cancer. One of the factors in 

obesity is increased consumption of a saturated fat and sugar. High fat diet (HFD) has been 

shown to be related to neurodegenerative diseases, long-term memory loss and cognitive 

impairment. In addition, free fatty acid has known to induce apoptosis in differentiated PC12 

cells. In the present study, we investigated the impact of HFD on the adult hippocampal 

neurogenesis. Male C57/BL6 mice were divided into 2 groups, and maintained on either normal 

diet (ND) or HFD. 7-weeks HFD significantly decreased in numbers of newly generated cells in 

the dentate gyrus of hippocampus. Interestingly, short-term HFD (for 2 weeks) decreased the 

numbers of surviving BrdU-labeled cells without altering proliferation. However, HFDmaintained 

mice behaved normally, and it was not likely that HFD caused the cognitive changes 

and spatial learning and memory deficit. Furthermore, we evaluated the effects of fatty acid on 

neural progenitor cells (NPC). We found that free fatty acid, palmitate decreased the 

proliferation of C17.2 NPCs, and was even cytotoxic. These findings suggest that HFD intake 

could impair adult hippocampal neurogenesis and have negative influence on the developmental

NPC. 

Acknowledgments 

This study was supported by grant number 105075-3 from the Technology Development 

Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea. 

Disclosures: H. Park , None; M. Park, None; K. Kong, None; H. Kim, None; M. Kim, 

None; J. Lee, None. 

Poster 





Support: Région Poitou-Charentes 

Fondation de France 

CNRS 

Fondation de l'Avenir 

Title: Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors 

from the subventricular zone in adult mice 

Authors: *M. DECRESSAC, L. PRESTOZ, J. VERAN, A. CANTEREAU, M. JABER, A. 

GAILLARD; 

IPBC pole biologie Sante, CNRS UMR 6187, Poitiers, France 

Abstract: Introduction: Neuropeptide Y (NPY) is widely expressed in the central nervous 

system and has been shown to stimulate neurogenesis in the hippocampus, olfactory epithelium 

and retina. Recently, NPY has been shown to play a role in neurogenesis in the subventricular 

zone (SVZ). Therefore, we sought to analyse the effect of intracerebroventricular (ICV) injection 

of NPY on SVZ neural stem cells. 

Methods: Proliferation was assessed 48 hours after NPY injection by BrdU labelling. 

Distribution and differentiation of newly BrdU positive cells was also examined 3 weeks postinjection. 

Results: We demonstrate that ICV injection of NPY stimulates proliferation of neural stem cells 

in the SVZ of adult mice. Newly generated cells migrate through the rostral migratory stream to

the olfactory bulb and also directly to the striatum where they differentiate mainly into neurons 

but also into glial cells. Using specific NPY receptor agonists, antagonist and KO mice, we 

report that NPY-induced neuroproliferation is mediated by Y1 receptor subtype. Furthermore, 

we found that Y1 receptor is highly expressed in the SVZ both at the mRNA and protein levels 

and mainly by neuroblasts. 

Conclusion: Stimulating endogenous SVZ neural stem cells by NPY may be of a potential 

interest in cell replacement based therapies of neurodegenerative diseases affecting the striatum 

such as Huntington‟s disease. 

Disclosures: M. Decressac, None; L. Prestoz, None; J. Veran, None; A. Cantereau, None; M. 

Jaber, None; A. Gaillard, None. 

Poster 





Title: Differential post-lesional response of distinct populations of hippocampal precursors in 

the young and aged brain 

Authors: J. WALTER, S. KEINER, J. OBERLAND, A. KUNZE, O. WITTE, *C. 

REDECKER; 

Dept Neurol, Friedrich Schiller Univ., Jena D-07747, Germany 

Abstract: During hippocampal neurogenesis slowly dividing precursor cells with astrocytic, 

radial glia-like properties could be distinguished from highly dividing neuronal precursors in the 

subgranular zone (SGZ). Several studies indicate that these distinct subtypes of precursors are 

differentially stimulated under pathophysiological conditions. Even the relatively quiescent 

radial glia-like precursors (type 1 cells) increase their proliferative activity within hours after 

cortical infarcts. It is the aim of the present study, to analyze whether this proliferative response 

is also present in the aged brain. To this purpose, we used the photothrombosis model to induce 

focal infarcts in the forelimb cortex of 3 and 16 months old transgenic mice expressing greenfluorescent 

protein (GFP) under control of the stem cell marker nestin. Sham-operated mice 

served as controls. To label proliferating cells all mice received three single injections of 

bromodeoxyuridine (BrdU, 50 mg/kg i.p. every 2 h) at day 4 after the infarct. Two hours after 

the last injection the animals were transcardially perfused and processed for 

immunocytochemistry using antibodies against BrdU, Nestin-GFP, glial fibrillary acidic protein 

(GFAP) and doublecortin (DCX). Stereological analysis of BrdU-positive cells in the SGZ

evealed only 14 % of proliferating cells in aged compared with young controls. After cortical 

infarcts the total number of BrdU-positive cells remained stable in young animals whereas old 

mice showed a significant increase in proliferating cells (+40 %). Phenotype analysis of the 

proliferating cells using confocal laser scanning microscopy further showed that cortical infarcts 

stimulate radial glia-like as well as neuronal subpopulations in the young but only neuronal 

precursors in the aged brain. Our data demonstrate that hippocampal precursor cells in the aged 

brain maintain their ability to respond to cortical infarcts even though their number is strongly 

reduced. 

Disclosures: J. Walter, None; S. Keiner, None; C. Redecker , None; J. Oberland, None; A. 

Kunze, None; O. Witte, None. 

Poster 





Support: DFG (SFB/TR3, SE 774/3) 

EC (FP7-202167) 

Title: Gap junction coupling of radial glia-like precursor cells and its impact on neurogenesis in 

the adult dentate gyrus 

Authors: *A. KUNZE 1 , M. R. CONGRESO 2 , C. HARTMANN 2 , A. WALLRAFF-BECK 2 , K. 

HUETTMANN 2 , P. BEDNER 2 , G. SEIFERT 2 , R. REQUARDT 3 , C. REDECKER 1 , M. THEIS 2 , 

K. WILLECKE 3 , C. STEINHAEUSER 2 ; 

1 Dept. of Neurol., Univ. Jena, Jena, Germany; 2 Inst. of Cell. Neurosciences, Univ. Bonn, Bonn, 

Germany; 3 Inst. of Genetics, Univ. Bonn, Bonn, Germany 

Abstract: In the subgranular zone (SGZ) of adult dentate gyrus (DG), one of the neurogenic 

regions in the mature brain, cells with a radial glia (RG)-like morphology represent putative stem 

cells that can give rise to new neurons and glial cells. In the course of neurogenesis, RG-like 

cells divide to generate precursors expressing immature neuronal features. Although the different 

cell populations as well as the architecture of the SGZ have been recently characterized, still 

little is known about possible communication pathways of the distinct precursors. In contrast to 

progenitors that are committed to a neuronal fate, RG-like cells apparently do not receive 

synaptic input. Therefore, alternative environmental cues must be involved in the control of

proliferation and differentiation of RG-like cells in the dentate gyrus. In the present study, we 

addressed the question whether RG-like cells in the DG are functionally coupled and if so, which 

connexins might form gap junctions in these precursor cells. In a second step we investigated 

proliferation and neurogenesis in the adult dentate gyrus following deletion of connexin 

expression in RG-like cells. Combining patch-clamp techniques with different molecular genetic 

approaches, we demonstrate that a subpopulation of RG-like cells in the DG is coupled through 

gap junctions and predominantly expresses connexin 43 (Cx43) and connexin 30 (Cx30). We 

further provide evidence that the deletion of connexin expression in RG-like cells leads to a 

significant decrease of proliferation and neurogenesis in the adult DG. 

Supported by DFG (SFB/TR3, SE 774/3) and EC (FP7-202167) 

Disclosures: A. Kunze , None; M.R. Congreso, None; C. Hartmann, None; A. Wallraff- 

Beck, None; K. Huettmann, None; P. Bedner, None; G. Seifert, None; R. Requardt, None; C. 

Redecker, None; M. Theis, None; K. Willecke, None; C. Steinhaeuser, None. 

Poster 





Support: NIH RO1 MH67157 

NSF IBN 0344448 

Title: Adult neurogenesis in the crustacean brain: Influences of serotonin and lithium on the 

neurogenic niche, migratory streams, and proliferation zones 

Authors: *J. L. BENTON, C. R. KIRKHART, B. S. BELTZ; 

Neurosci. Program, Wellesley Col., Wellesley, MA 

Abstract: New neurons are incorporated into the adult brains of a variety of organisms, from 

humans and higher vertebrates, to non-vertebrates such as crustaceans. In virtually all of these 

systems serotonergic pathways appear to provide important regulatory influences over the 

machinery producing the new neurons. Serotonergic pathways have also been implicated in the 

mechanisms of action of lithium, which is known to increase brain levels of tryptophan, a 

precursor of serotonin. In the present studies we have examined the influences of serotonin and 

lithium on adult neurogenesis in the crustacean brain, where the neurogenic cellular lineage has 

been identified. Adult neurogenesis in decapod species begins with precursors that reside in a

neurogenic niche; daughters of these precursors migrate on glial fibers to proliferation zones 

where they divide at least one more time before differentiating into neurons. We have examined 

serotonergic influences on this pathway by incubating (1) whole animals, (2) animals whose 

eyestalks and associated sinus glands had been removed, and (3) organ-cultured brains in 

serotonin or lithium. We find that serotonin is effective in regulating neurogenesis at levels as 

low as 10 -10 M, suggesting that circulating serotonin may have hormonal influences on the stemlike 

precursor cells residing in the neurogenic niche. In addition, contrasting effects of serotonin 

at high concentrations (10 -4 M), dependent upon whether eyestalk tissue is present or absent, 

suggest that substances capable of suppressing neurogenesis reside in the lateral protocerebrum, 

and most likely in the sinus gland. We are also using NSD-1015 to examine the dependency of 

lithium effects on serotonin levels. The various effects of serotonin and lithium on the numbers 

of newborn cells labeled with BrdU suggest multiple mechanisms and pathways by which these 

substances may regulate neurogenesis in the crustacean brain. 

Disclosures: J.L. Benton, None; C.R. Kirkhart, None; B.S. Beltz, None. 

Poster 





Support: LNE Grant HD07228-26 

MH061994 

Title: Sex, steroids and cell proliferation in an adult songbird 

Authors: *A. MIRZATONI 1 , A. KATZ 2 , S. DONG 3 , S. ZHEN 3 , B. A. SCHLINGER 2 ; 

1 MCIP, 2 Physiological Sci. Department, Lab. of Neuroendocrinology, Brain Res. Inst., 

3 Physiological Sci. Dept., UCLA, Los Angeles, CA 

Abstract: Neurogenesis in the adult brain continues in a specialized cell layer surrounding the 

lateral ventricles, called the ventricular zone (VZ). We examined the acute impact of steroids on 

VZ proliferation in the adult zebra finch. Adult male and female brain slices containing the VZ 

were freshly prepared and proliferation revealed after 2 hrs exposure to 5-bromo-2‟deoxyuridine-5‟-monophosphate 

(BrdU). Slices from one hemisphere served as controls; 

contralateral slices received BrdU plus steroids or steroidogenic enzyme inhibitors. Proliferation 

along the male VZ was found to be approximately 50% greater than that of females. In addition,

acute administration of corticosterone significantly reduced proliferation in males but not in 

females. There were no significant effects on proliferation in males or females by acute exposure 

to sex steroids or to inhibitors of sex-steroid synthesis. Co-incubation of female slices with 

ovarian tissue significantly increased the number of BrdU-labeled cells suggesting a role for a 

non-steroidal ovarian factor on adult VZ proliferation. Sex differences in VZ proliferation may 

be one mechanism maintaining the sexually dimorphic song system of these birds. Stress may 

impact adult male neural plasticity by suppressing mitosis along the VZ. Supported by 

MH061994 and LNE Training Grant HD07228-26. 

Disclosures: A. Mirzatoni , LNE training grant, Schlinger, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received); A. 

Katz, None; S. Dong, None; S. Zhen, None; B.A. Schlinger, None. 

Poster 





Support: NIH grant NS 32401 

Title: Postnatal hippocampal neurogenesis is deficient in PAC1 receptor mutant mice 

Authors: *L. TASCAU 1 , A. FALLUEL-MOREL 1 , X. ZHOU 1 , P. BRABET 2 , E. DICICCO- 

BLOOM 1,3,4 ; 

1 Neurosci. and Cell Biol., UMDNJ, Piscataway, NJ; 2 INSERM U583, Montpellier, France; 

3 Dept. of Pediatrics, UMDNJ-RWJMS, New Brunswick, NJ; 4 Cancer Inst. of New Jersey, New 

Brunswick, NJ 

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) and its main receptor 

PAC1 are expressed as early as the development of the neural tube. The PACAP signaling 

system is known to regulate multiple processes including proliferation, survival and 

differentiation: in embryonic cortex, PACAP plays an antimitogenic role. Postnatally, mice with 

PACAP-PAC1 system disruption exhibit poor social interactions, hyperactivity, and deficits in 

learning, memory and mossy fiber LTP function, suggesting hippocampal abnormalities. In turn, 

we now define developmental functions in the postnatal hippocampus in PAC1 deficient mice, 

characterizing effects on cell proliferation and neuron production and survival. 

At postnatal day 7 (P7) hippocampal formation DNA content (marker of total cells) was 

decreased in PAC1 deficient mice (90.6±4.21% of control, p

dentate gyrus (DG) granule cell neurogenesis, we examined BrdU incorporation, a marker for S 

phase. PAC1 deficient mice exhibited a 31.6±4.1% reduction in BrdU labeling in the DG hilus, 

suggesting that cells failed to enter S phase and /or underwent cell death. The largest difference 

was observed in the caudal compartment (58.2±6.5% from control, p

projection neurons. The large size and the accessibility of the DGNs in crayfish and lobsters have 

made them ideal subjects for studies of their anatomy, physiology and development. Of 

particular interest, however, is their proximity to the only two populations of interneurons in the 

crustacean midbrain where cell proliferation occurs throughout the life of the animal. These local 

interneurons in Cluster 9 and projection neurons in Cluster 10, arborize in the same neuropils as 

the DGN, and their proliferation zones are adjacent to the AL. In addition a recently discovered 

vascular neurogenic niche in the brains of crayfish and lobsters lies on the surface of the AL. The 

migratory streams that link the niche to the proliferation zones of Cluster 9 and 10 also lie across 

the surface of the AL. Electrical stimulation of the surface of the OL or AL (cortex) results in a 

hyperpolarization of the DGN. Stimulation of the projection neurons (Cluster 10), the OGT or 

the AL medulla, depolarizes the DGN. In these studies, unilateral stimulation of a DGN in semiisolated 

brains was carried out over a period of 6 hours while perfusing with 0.5mg/ml 

bromodeoxyuridine (BrdU) in saline. We find that the number of labeled cells on the stimulated 

side is increased compared with the unstimulated side if the DGN is depolarized to its spiking 

threshold, but not if it is hyperpolarized. Counts of Cluster 10 cell proliferation, and HPLC 

measurements of the serotonin content of the ALs in unstimulated animals, reveal a high degree 

of symmetry between left and right side measurements. We conclude that stimulation of the 

DGN releases serotonin into the blood capillary system of the Al and cell clusters 9 and 10, 

where it acts to increase the rate of cell proliferation. This neuron therefore constitutes a neural 

pathway that regulates adult neurogenesis. 

Disclosures: D.C. Sandeman, None; A.M. Rose, None; J.L. Benton, None; B.S. Beltz, None. 

Poster 





Title: Induction of long-term depression suppresses hippocampal adult neurogenesis enhanced 

by long-term potentiation in the rat 

Authors: *S. CHUN 1,2 , M. JUNG 2 , W. SUN 1 ; 

1 Dept. of Anat., Korea Univ., Seoul, Republic of Korea; 2 Neurosci. Lab., Ajou Univ. Sch. of 

Med., Suwon, Republic of Korea 

Abstract: Neurogenesis persists in certain adult brain regions including hippocampal dentate 

gyrus (DG). Recently we have shown that LTP induction in the afferent pathway enhances adult 

neurogenesis in the DG. The effect of LTD induction on hippocampal adult neurogenesis is

currently unknown, however. To investigate this matter, we induced LTD in the perforant path 

input-to the DG by low frequency stimulation (LFS; 1Hz, 900 pulses) in one hemisphere in 

freely moving rats, and quantified the number of newly generated progenitor cells in the DG. 

The LTD-induced hemisphere exhibited a similar extent of progenitor cell proliferation 

compared to the control hemisphere. On the other hand, when LTD was induced a day before 

LTP induction, enhanced neurogenesis induced by LTP induction was markedly blunted, 

indicating that LTD selectively suppressed LTP-induced neural progenitor cell proliferation. 

Exploration of molecular mechanisms underlying selective influence of LTD on LTP-induced 

adult neurogenesis (as opposed to basal neurogenesis) is currently under way. 

Disclosures: S. Chun , None; M. Jung, None; W. Sun, None. 

Poster 





Title: Effects of intrathecal application of brain-derived neurotrophic factor on the perilesional 

response after focal cortical infarcts 

Authors: *S. KEINER, O. WITTE, C. REDECKER; 

Dept. of Neurol., Friedrich-Schiller Univ., Jena, Germany 

Abstract: Brain-derived neurotrophic factor (BDNF) is known as a member of the neurotrophin 

family of growth factors, which plays an important role in the complex cellular and functional 

changes after ischemic infarcts. Up to now only little is known about the effects of exogenous 

BDNF on cellular proliferation and differentiation in the peri-infarct zone. Here we analyzed the 

effects of BDNF on the perilesional cellular response after focal infarcts induced in the 

sensorimotor fore- and hindlimb cortex using the photothrombosis model in rats. BDNF or 

vehicle was continuously infused into the ipsilateral ventricle directly after infarct induction for 2 

weeks by osmotic minipumps. The proliferating cells were labelled with bromodeoxyuridine 

(BrdU) within the period of drug administration starting one day post surgery. At day 14 and 42 

after infarct induction, BrdU-positive cells were immunocytochemically stained and quantified 

in the peri-infarct zone using semiautomatic stereology. To further analyze the phenotypes, 

triple-immunofluorescence with antibodies against the immature neuronal marker (DCX), mature 

makers (NeuN, Hu), astrocytic markers (GFAP, S100β) and microglia/macrophages marker 

(CD68) was performed. Sensorimotor function was assessed using the limb-use asymmetry test, 

the ladder rung walking test as well as the tapered beam walking test at day 1, 3, 14, and 42 post

surgery. Our study demonstrates that intrathecal BDNF application significantly increased the 

number of DCX-positive neuroblasts in the perilesional area 2 weeks after infarct induction and 

provoked the generation of mature neurons 42 days post surgery. Furthermore, exogenous BDNF 

reduced the number of newly generated microglia/macrophages two weeks after the lesion, 

whereas S100β and GFAP expressing astrocytes were not significantly influenced. Intrathecal 

BDNF administration modified the cellular response in the perilesional area. However, 

functional recovery was not significantly improved. 

Disclosures: S. Keiner, None; O. Witte, None; C. Redecker, None. 

Poster 





Support: DVA Grant to MWM 

NIAAA Grant AA015413 to SMM 

NIAAA Grant AA07568 to MWM 

NIAAA Grant AA06916 to MWM 

Title: Postnatal neurogenesis in thalamus: effects of prenatal exposure to ethanol and the role of 

neurotrophins 

Authors: *S. M. MOONEY, M. W. MILLER; 

Dept Neurosci & Physiol, SUNY Upstate Med. Univ., Syracuse, NY 

Abstract: Neurotrophins such as nerve growth factor (NGF) and brain derived neurotrophic 

factor (BDNF) play a role in postnatal neurogenesis, notably in the dentate gyrus. Postnatal 

neurogenesis also occurs in the ventrobasal thalamus (VB). As prenatal exposure to ethanol 

alters the postnatal neurogenesis in the dentate gyrus, we tested the hypothesis that ethanol 

affects the postnatal cell proliferation in the VB, and whether this effect might be mediated by 

neurotrophins. Timed pregnant Long-Evans rats were given a liquid diet containing ethanol (Et; 

6.7% v/v) or were pair-fed an isocaloric, isonutritive diet (Ct) from gestational day (G) 11 

through G21. Dams were allowed to litter, and pups were surrogate-fostered by untreated dams 

within 24 hr of birth. Pup‟s brains were collected at time between postnatal day (P) 1 and P30.

Two pups per litter were euthanized for anatomical studies (by transcardial aldehyde perfusion) 

or biochemical analysis (decapitated and quick-frozen). The numbers of VB neurons rose more 

quickly and peaked sooner in Et-treated pups, but by P30 there was no treatment-induced 

difference in the numbers. Cell cycle kinetics for the proliferative population was determined on 

P6 using a cumulative bromodeoxyuridine (BrdU; 25 mg/kg) labeling method. Ethanol did not 

affect the proportion of cycling cells in the VB, but it did alter the cell cycle kinetics. The total 

length of the cell cycle (Tc) was shorter in Et-exposed rats (11.4 ± 0.9 hr) than in controls (17.3 ± 

1.1 hr). The length of S-phase (Ts) was also shorter in Et-treated pups (4.2 ± 0.3 hr) than in 

controls (7.0 ± 0.9 hr). Enzyme linked immunosorbant assays and immunoblots were used to 

determine the expression of NGF and BDNF and their receptors, respectively. Ethanol increased 

NGF expression at all ages examined, but it had no effect on the expression of either BDNF or 

the neurotrophin receptors. The results show that prenatal exposure to ethanol increases the 

proliferation of VB neural progenitors. It appears that this effect is mediated by NGF. 

Disclosures: S.M. Mooney , None; M.W. Miller, None. 

Poster 





Support: NSF Grant 541602 

Title: Circadian variations of cell proliferation in the dentate gyrus of the adult hamster 

Authors: *J. M. HEITZER, J. SWANN; 

Lehigh Univ., Bethlehem, PA 

Abstract: Variations in DNA synthesis have been found between day and night in many 

mammalian tissues, such as liver, skin, and intestinal epithelium, suggesting that circadian 

rhythms affect the timing of cell division in these tissues. However, there is little information 

about a diurnal rhythm of cell proliferation in the central nervous system of mammals. In the 

adult rodent, the dentate gyrus is one of the main sites of cell proliferation. The goal of this study 

was to determine whether there are circadian rhythms of cell proliferation in the dentate gyrus of 

adult male hamsters using the marker BrdU. BrdU is a thymidine analog incorportated into the 

cells‟ DNA during cell division. Adult male hamsters were maintained on a 14:10 light-dark 

cycle, with ad lib access to food and water and randomly assigned to four different groups. Each 

group was injected with BrdU (300 mg/kg, i.p.) at one of four time points, the time points

coinciding with the time of light onset, the middle of the light phase, time of dark onset and the 

middle of the dark phase. The animals were killed 24 hours after the single injection. Brains were 

removed and sectioned at 40 micron thickness and free floating sections were labeled for BrdU 

using immunocytochemistry. Preliminary results show a trend toward an increase in cell division 

during the time of light onset suggesting a circadian variation in hamster hippocampal cell 

proliferation. 

Disclosures: J.M. Heitzer , None; J. Swann, None. 

Poster 





Support: NMSS RG 379A6/1 

Title: Role of resident activated microglia during neurogenesis 

Authors: *A. M. NIKOLAKOPOULOU 1 , A. ZAREMBA 2 , R. H. MILLER 2 , B. D. TRAPP 1 ; 

1 Dept Neurosci, Lerner Res. Inst, Cleveland Clin., Cleveland, OH; 2 Neurosciences, Case 

Western Reserve Univ., Cleveland, OH 

Abstract: Neurodegeneration is the major cause of irreversible neurological disability in 

individuals with multiple sclerosis (MS). So far, no therapeutical approaches have been 

successful in reversing the effects of neurodegeneration either by inducing neurogenesis, which 

would replace the neurons destroyed, or by attenuating the factors that cause neuronal death. 

Preliminary studies from our lab indicate that neurons are destroyed during inflammatory 

demyelination and then replaced in some, but not all, MS lesions. Regions of MS lesions with 

increased neuronal densities contain a morphologically distinct population of activated 

microglia. Activated microglia, the resident immune cells of the central nervous system (CNS), 

play an important neuroprotective role in the CNS. 

The purpose of this study is to activate microglia by administration of lipopolysaccharide (LPS) 

and examine the effects on cell differentiation. Adult rats were injected intraperitoneally (i.p.) 

with saline (controls) or LPS (1 mg/kg of body weight) for four consecutive days, animals were 

sacrificed, the brains were removed and trypsinized. Microglia was isolated by percoll gradient 

and was stained by CD45+ and CD11b+ antibodies. Microglia (CD45 low and intermediate) and 

monocytes (CD45 high) were isolated by flow cytomety (FACS). These cells were then cocultured 

with dissociated optic nerve cells from P2-P6 pups. The optic nerve cells are a good in

vitro assay for factors that modulate progenitor cell production, since the optic nerve is not a 

neurogenic area per se. 

Image analysis of fluorescence intensity with Image Pro software showed that the optic nerve 

cells that were co-cultured with microglia isolated from LPS activated brains show an increase in 

the percentage of TUJ-1 positive cells (P=0.01). Monocytes have no effect on cell proliferation 

and differentiation when co-cultured with optic nerves from neonatal rats. 

To investigate the possible source of optic nerve generated neurons, A2B5 positive cells were 

panned and the number of TUJ-1 positive cells with the presence of microglia derived from LPS 

and control brains was counted. Interestingly, there is no difference in the number of TUJ-1 + 

cells derived from A2B5+cells that were co-cultured with activated or inactivated microglia. 

Disclosures: A.M. Nikolakopoulou, None; B.D. Trapp, None; A. Zaremba, None; R.H. 

Miller, None. 

Poster 





Support: NIH Grant R01-MH69686-05 

Title: The long-term effects of selective brain lesions on adult neurogenesis in the rhesus 

monkey brain 

Authors: *D. KIM, L. B. NGWENYA, L. A. WELKE, R. J. KILLIANY, M. B. MOSS, D. L. 

ROSENE; 

Anat. and Neurobio., BUSM, Boston, MA 

Abstract: The effects of brain damage on adult cell proliferation have been investigated in the 

hippocampal formation in rodents as well as other mammalian models, but there has been limited 

research conducted in the non-human primate. In the present study, the long-term effects on cell 

proliferation of selective brain lesions, produced as part of a separate behavioral study, were 

evaluated in the dentate gyrus (DG) of the hippocampus. A total of twelve young male monkeys 

(3.9-7.9 years of age) were studied. Six animals received aspiration lesions of the hippocampus 

in one hemisphere and the other six animals were unoperated controls. Each animal received 

intraperitoneal injections of the synthesis phase marker 5-bromo-2‟-deoxyuridine (BrdU), 21-22 

days prior to perfusion-fixation of the brain which occurred 7 to 18 months after the lesion. After 

immunohistochemical processing, BrdU positive cells in the DG were quantified in the intact

hippocampus using an exhaustive stereological sampling scheme. Results showed a significant 

increase of cell proliferation in monkeys who had received hippocampal lesions relative to the 

control monkeys. Lesioned monkeys showed a two-fold increase in the number of BrdU+ cells 

(lesioned: 4346.7±726.6 cells, control: 2073.3±254.8 cells, p=0.01). This increase in cell 

proliferation was sustained at 13 months after the lesion (lesion at 13 months: 3800 cells, 

p

subgranular zone (SGZ). In Experiment 1, adult male C57BL/6J mice received two sham or 25 

mg morphine pellets s.c. (0 and 48 hrs), followed by a single injection of bromodeoxyuridine 

(BrdU; i.p. 150 mg/kg) 2 hrs prior to sacrifice at 24, 72 or 96 hrs. Morphine decreased 

proliferation in the SGZ as the number of cells immunoreactive (IR) for the exogenous S phase 

marker BrdU and the endogenous cell cycle marker Ki67 (expressed in all phases of the cell 

cycle) were both reduced. However the percentage of BrdU-IR cells that were Ki67-IR was 

decreased at 24 but not 96 hrs. This suggests that exposure to morphine has a greater effect on S 

phase cycling cells. Cell death, as determined by activated-caspase 3 counts, was increased at 24 

hrs but not 96 hrs. In Experiment 2, we utilized nestin-GFP reporter mice to determine the 

impact of morphine on maturation through discrete stages of neurogenesis. Mice were given 

BrdU 1 day before receiving morphine pellet or sham surgery at 0 and 48 hrs, and sacrificed at 

96 hrs. The number of Ki67-IR cells was decreased, as in Experiment 1 however the number of 

BrdU-IR cells was unchanged. This suggests that as BrdU-IR cells mature, they are less sensitive 

to the effects of morphine. To clarify which stages of adult neurogenesis were susceptible to 

morphine, sections were stained for BrdU, DCX and GFP and cells were categorized into 

specific stages of progenitor cell maturation. The percent of BrdU-IR cells that were type 1 

(stem-like, triangular cell body, process extending into molecular layer, GFP+/DCX-) or type 2a 

(transiently amplifying, ovoid cell body, GFP+/DCX-) were unchanged. The percent of BrdU-IR 

cells that were type 2b increased (ovoid cell body, GFP+/DCX+), whereas immature cells 

decreased (round cell body, dendrite extending into molecular layer, GFP-/DCX+). These data 

suggest that chronic morphine decreases neurogenesis by inhibiting progenitor cell progression 

to a mature neuronal stage. Studies are ongoing to explore the cellular mechanism underlying the 

differential sensitivity of stages of adult neurogenesis to the negative effects of morphine. 

Disclosures: A.A. Arguello , None; G.C. Harburg, None; J. Schonborn, None; C.D. 

Mandyam, None; M. Yamaguchi, None; A.J. Eisch, None. 

Poster 





Title: Amygdala lesions attenuate stress-induced suppression of adult hippocampal cell 

proliferation 

Authors: *E. D. KIRBY 1 , D. KAUFER 1 , K. A. GOOSENS 2 ; 

1 Helen Wills Neurosci Inst., Univ. of California Berkeley, Berkeley, CA; 2 McGovern Inst. for 

Brain Res., MIT, Cambridge, MA

Abstract: Psychological stress and the associated elevations in circulating glucocorticoids (GCs) 

alter numerous aspects of hippocampal physiology and function. However, many of these 

hippocampal stress effects appear to be dependent on the basolateral complex of the amygdala 

(BLA); lesions of the BLA prevent stress/GC-induced decrements in both hippocampusdependent 

memory and hippocampal long-term potentiation (Roozendaal et al., 2008, Prog. 

Brain Res.). Moreover, the BLA appears to affect the hippocampal response to GCs through 

direct, ipsilateral projections, as BLA lesions prevent memory consolidation enhancements due 

to intrahippocampal GC receptor agonists only if the lesion is ipsilateral to the hippocampal 

infusion site (Roozendaal et al., 1999, PNAS). Both stress and elevated GC levels are also 

known to suppress the birth of new neurons in the adult hippocampus, but it is not known 

whether this effect is dependent on the BLA. To determine whether the regulation of adult 

hippocampal neurogenesis by psychological stress depends on the BLA, adult male rats received 

unilateral lesions of the BLA and, after one week of recovery, were exposed to daily 

immobilization stress for seven days. At the end of immobilization on the 7 th day, all rats 

received one injection of 5‟-bromodeoxyuridine (BrdU, 200mg/kg) to mark dividing cells and 

were sacrificed 24 hours later. BrdU+ cells were counted throughout the dentate gyrus of the 

hippocampus both ipsilateral and contralateral to the BLA lesion. There were significantly more 

BrdU+ cells in the dentate gyrus ipsilateral to the BLA lesion compared to the contralateral 

dentate. These results suggest that lesions of the BLA had a protective effect on adult 

hippocampal precursor cells, attenuating chronic stress-induced suppression of new cell 

proliferation. This dependence of the neurogenic stress response on BLA input implies a novel 

mechanism for modulation of adult hippocampal neurogenesis wherein GC stimulation may 

require local permissive signaling derived from the BLA to alter mitotic activity of neural 

precursor cells in the hippocampus. Future work will focus on determining the nature of this 

signal, as well as on the eventual differentiated fate of the BrdU+ cells. 

Disclosures: E.D. Kirby, None; D. Kaufer, None; K.A. Goosens, None. 

Poster 





Support: AA06916 

Dept. of Veterans Affairs 

AA07568

Title: Fetal programming in brain development: postnatal neurogenesis in the dentate gyrus of 

an adolescent monkey is reduced by prenatal exposure to ethanol 

Authors: *M. W. MILLER, A. V. FEDORCHAK; 

Dept Neurosci Physiol, SUNY Upstate Med. Univ., Syracuse, NY 

Abstract: Brain development is fetally programmed, that is, traumas or situations experienced 

during fetal development can have long-term, often transiently quiescent, consequences on later 

development. As such, fetal alcohol spectrum disorder (FASD) can lead to deficits in learning 

and memory in adolescents and adults. The present study tested the hypothesis that prenatal 

exposure to ethanol alters the generation of neurons in the adolescent dentate gyrus. To study 

this, monkeys were exposed to ethanol during gestation once per week for 6 (Et6) or 24 (Et24) 

weeks (n=5 or n=4, respectively). Six control macaques were treated with saline (Ct) once per 

week. Offspring were euthanized during adolescence and their temporal lobes were sectioned 

and immunostained for the expression of proteins associated with cell proliferation (Ki-67 and 

PCNA), cell cycle exit (p21), and neuronal differentiation (cyoskeletal proteins doublecortin 

(Dcx) and TuJ1). The frequency of immunoexpressing neurons in the proliferative (subgranular 

zone; SGZ and post-proliferative (granular cell layer; GCL) regions were quantified. No 

significant differences among the treatment groups were detected for any of the markers. The 

only exception was that the Et6-treated monkeys exhibited less Ki-67 expression in the 

SGZ+GCL than did the dentate gyri of Et24-treated monkeys, but no less than controls. That 

said, when the data were re-analyzed based on whether the offspring were exposed on gestational 

day (G) 19 or G20 (the time of gastrulation) or at another time during the fourth postnatal week 

(on G21 or G24), significant differences emerged. Expression of Ki-67 and PCNA was 

significantly lower in the SGZ of monkeys exposed on G21/G24. Furthermore, the expression of 

Dcx and TuJ1 were reduced in both regions of the dentate gyrus in monkeys exposed on 

G21/G24. Thus, prenatal exposure to ethanol can lead to a reduction in cell proliferation (either 

by a lengthening of the cell cycle or a reduction in the number of cycling cells) that leads to a 

reduction in neurons that are differentiating, but this only occurs if the exposure is during a 

critical time. This identifies a pattern of fetal programming that may underlie learning and 

memory deficits in adolescents with FASD. 

Disclosures: M.W. Miller , Upstate Medical University, A. Employment (full or part-time); 

AA06916 and AA 07568, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received); Dept. of Veterans Affairs, C. Other 

Research Support (receipt of drugs, supplies, equipment or other in-kind support); A.V. 

Fedorchak, None. 

Poster 

230. Cell Migration: Molecules Mediating Migration 



Topic: A.02.c. Cell migration 

Title: The Role of BDNF signaling in the migration of neural progenitors through the RMS 

Authors: *J. A. BAGLEY, L. BELLUSCIO; 

DNPU, NIH/NINDS, Bethesda, MD 

Abstract: Subventricular zone (SVZ) neural precursor cells generate neuroblasts that travel 

tangentially along the rostral migratory stream (RMS) toward the olfactory bulb (OB) where they 

differentiate into a variety of interneuron cell types. While the mechanisms responsible for this 

migration are largely unknown, a variety of cellular and molecular factors associated with 

cellular patterning and axonal guidance have been implicated, and include: slits/robos, 

semaphorins/neuregulin, netrins and neurotophins. One neurotrophin, Brain-derived 

Neurotrophic Factor (BDNF) has been shown to function as a chemoattractant for RMS 

neuroblasts in explant culture assays. Interestingly, several studies have recently noted that some 

neuroblasts within the RMS appear to migrate away from their olfactory bulb target. These 

findings suggest that RMS migration is bidirectional and may be more dynamically complex 

than simple chemoattraction. As a result, the RMS region provides an ideal model system to 

study the dynamics of neural migration while also exploring the role of potential guidance 

molecules in the process. Therefore, to understand how neuroblasts are guided to the OB, and 

how this process is regulated, we sought to develop a method to record and analyze the dynamic 

effects of BDNF signaling on RMS migration. Using a transgenic mouse line (GAD65-GFP) in 

which RMS neuroblasts are fluorescently labeled, we developed an acute slice preparation 

combined with time-lapse confocal imaging to individually track the migration of neurons at a 

population level. We also established an approach to analyze the complex dynamics of this 

population based upon two distinct categories: directionality (measured as the proportion of cells 

migrating toward or away from the OB) and motility (determined by displacement, total distance 

traveled, and velocity). Our results indicate that altering BDNF levels can affect both neuroblast 

motility and directionality. Futhermore, we determined that while the low affinity neurotrophins 

receptor, p75NTR is undetectable in the RMS, the high affinity TrkB receptor is clearly 

expressed and mediates the migratory affects observed with BDNF. As both BDNF and TrkB are 

themselves modulated by activity, it will be interesting to determine if expression levels could 

provide activity dependent regulation of RMS migration. 

Disclosures: J.A. Bagley , None; L. Belluscio, None. 

Poster 






Yale Brown-Coxe fellowship 

NIH Grant DC007681 

Title: Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in a whole 

mount preparation of the subventricular zone 

Authors: *J.-C. J. PLATEL, T. HEINTZ, S. YOUNG, A. BORDEY; 

Neurosurgery, Cell. & Molec, Yale Univ. Sch. Med., New Haven, CT 

Abstract: In the postnatal subventricular zone (SVZ), neuroblasts migrate in chains along the 

lateral ventricle towards the olfactory bulb. AMPA/kainate receptors as well as metabotropic 

glutamate receptors subtype 5 (mGluR5) are expressed in SVZ cells. However, the cells 

expressing these receptors and the function of these receptors remain unexplored. We thus 

discovered that SVZ neuroblasts express mGluR5 and Ca2+-permeable kainate receptors using 

immunostaining, patch clamp recordings and Ca2+ imaging in mouse brain slices. Doublecortinimmunopositive 

cells, identified as neuroblasts, express mGluR5 as well as GLUK5-7containing 

kainate receptors. Patch clamp data suggest that neuroblasts express functional 

kainate receptors, including GLUK5-containing kainate receptors. Activation of mGluR5 and 

GLUK5-containing kainate receptors induced Ca2+ increases in 50% and 60% of SVZ 

neuroblasts, respectively, while most SVZ neuroblasts displayed GABAA receptor-mediated 

Ca2+ responses. To examine the function of these receptors on the speed of neuroblast 

migration, we developed a whole mount preparation of the entire lateral ventricle. In postnatal 

day 20-25 transgenic mice carrying green fluorescent protein under the doublecortin promoter, 

the GABAA receptor antagonist bicuculline increased the speed of neuroblast migration by 27%, 

as previously reported using acute slices. While the mGluR5 antagonist MPEP did not affect the 

speed of neuroblast migration, the homomeric GLUK5 antagonist NS3763 increased the 

migration speed by 42%. These data show that tonic activation of GLUK5-containing kainate 

receptors, but not of mGluR5, reduces the speed of neuroblast migration along the lateral 

ventricle suggesting different downstream Ca2+ dependent intracellular mechanisms. 

Disclosures: J.J. Platel, None; T. Heintz, None; S. Young, None; A. Bordey, None. 

Poster 

230. Cell Migration: Molecules Mediating Migration




Support: Global COE MEXT Japan 

Title: Analysis of coactosin, an actin binding protein, in neural crest cell migration 

Authors: X. HOU, T. KATAHIRA, *H. NAKAMURA; 

Dept Molec Neurobiol, Tohoku Univ., Sendai 980-8575, Japan 

Abstract: Coactosin is a 17kDa actin binding protein that belongs to the ADF/cofilin homology 

family of actin-binding protein. Coactosin inhibits barbed-end capping of actin filament and to 

act as actin polymerization. Whole-mount in situ hybridization shows that Coactosin is 

expressed in cephalic and trunk neural crest cells. The neural crest cells travel long distance to 

form a wide range of derivatives. They have high motility, and respond to environments cues 

that guide them along specific routes to their final destination. This process requires dynamic and 

highly coordinated regulation of the actin cytoskeleton. So we are analyzing the roles of 

Coactosin in related to neural crest cells. Immunocytochemistry with anti-Coactosin antibody 

shows that Coactosin is localized in cytoplasm and associated with actin stress fibers in neural 

crest cells. We designed shRNA expression construct to interfere with coactosin, and performed 

in ovo electroporation. To our expectation, knockdown with Coactosin-siRNA resulted in 

impairment of neural crest cell migration in vivo and in vitro, and shrinkages of neural crest cells 

due to disruption of actin stress fibers. Our data suggest that in vertebrate development, 

regulation of Coactosin is critical for neural crest cell migration and cell shape maintenance. 

Disclosures: X. Hou, None; T. Katahira, None; H. Nakamura , None. 

Poster 





Support: BMBF 01GS0498 

Schilling Foundation

BMBF 01GS0117 

SFB484 

Title: Large-scale in vivo analysis of genes involved in neurogenesis, migration and 

differentiation of subventricular zone neuroblasts 

Authors: *K. KHODOSEVICH 1 , P. H. SEEBURG 2 , H. MONYER 1 ; 

1 Heidelberg Univ. Clin., Heidelberg, Germany; 2 Max-Plank Inst. Med. Res., Heidelberg, 

Germany 

Abstract: Neurogenesis in adult brain continues throughout life. New neurons that are added to 

different circuits are thought to underlie brain plasticity. Two brain areas, namely the dental 

gyrus (DG) of the hippocampus and the subventricular zone (SVZ), are associated with postnatal 

neurogenesis. While the DG supplies new neurons to the hippocampus, neuroblasts from SVZ 

migrate through the rostral migratory stream (RMS) to the olfactory bulb where they become 

granule cell and periglomerular neurons. Different signaling networks are involved in the 

orchestration and the coordination of the neurogenesis and subsequent migration and 

differentiation of newborn neuroblasts. To identify and analyze these networks, we isolated 

neuroblasts (identified by EGFP expression in transgenic mice) from two locations of the RMS 

(posterior and anterior) and from immature periglomerular neurons. We compared expression 

profiles of these cell populations and found differentially expressed genes. We identified 

signaling networks underlying each of the three major processes in the SVZ-RMS axis: 

neurogenesis, migration and differentiation. Using various recombinant virus types 

(oncoretrovirus, lentivirus and adeno-associated virus) expressing shRNAs we performed in vivo 

functional analysis of specific networks to confirm their involvement in neurogenesis, migration 

or differetiation. 

Disclosures: K. Khodosevich, None; P.H. Seeburg, None; H. Monyer, None. 

Poster 





Support: NIH grant 

march of dimes

Title: FEZ1 and NDEL1 interact with DISC1 in regulating integration of newly generated 

neurons in the adult brain 

Authors: E. KANG 1 , X. DUAN 1 , S. GANESAN 2 , J. CHANG 2 , B. LU 2 , H. SONG 1 , *G.-L. 

MING 1 ; 

1 Dept Neurol & Neurosci, Johns Hopkins Univ., Baltimore, MD; 2 Neurosci., Natl. Inst. of Hlth., 

Bethesda, MD 

Abstract: Disturbed adult neurogenesis is increasingly appreciated as a potential factor for 

pathogenesis of psychiatric disease. Disrupted-in-Schizophrenia 1 (DISC1) is one of the 

susceptibility genes for schizophrenia and other major mental illness. Our previous study 

demonstrated that newborn dentate granule cells with DISC1 knockdown exhibit aberrant 

morphogenesis, mispositioning and accelerated dendritic development in the adult brain. How 

DISC1 regulates different aspects of neural development in the adult brain is largely unknown. 

Based on previously identified DISC1 interacting proteins and their expression patterns, we 

investigated functions of two DISC1-interacting proteins: fasciculation and elongation proteinzeta1 

(FEZ1) and NDEL1 in newborn neurons of the adult mouse hippocampus. Interestingly, 

retrovirus-mediated knockdown of FEZ1 or NDEL1 in newborn neurons of the adult dentate 

gyrus differentially phenocopies the defects by DISC1 knockdown. Furthermore, synergistic 

effects were observed when any two of the three molecules were knock down simultaneously. 

Taken together, our study identified the functional interaction of DISC1 with FEZ1 and Ndel1 in 

regulating adult hippocampal neurogenesis. 

Disclosures: E. Kang, None; G. Ming , None; X. duan, None; S. Ganesan, None; J. chang, 

None; B. lu, None; H. Song, None. 

Poster 





Support: Hereditary Disease Foundation 

Title: SDK2 downregulation disrupts migration in the adult rostral migratory stream 

Authors: *S. S. MAGAVI 1 , W. KELSCH 1 , L. KAUFMAN 2 , H. SHIN 3 , J. R. SIMS 3 , P. 

KLOTMAN 2 , C. LOIS 1 ;

1 Picower Inst. Learning, MIT, Cambridge, MA; 2 Div. of Nephrology, Mt. Sinai Sch. of Med., 

New York, NY; 3 Neurol. and Radiology, MGH, Harvard Med. Sch., Boston, MA 

Abstract: Sidekicks are transmembrane proteins that have previously been established to play a 

role in synapse formation. Here, we present evidence that sidekick-2 plays a role in supporting 

neuronal migration. During adulthood, sidekick-2 (Sdk2) is expressed in the subventricular zone 

(SVZ) and rostral migratory stream (RMS), regions which contain tangentially migrating 

neurons. Sdk2 is expressed by GFAP positive astrocytes in the RMS, and is found closely 

apposed to doublecortin positive migrating neurons. During development, Sdk2 is expressed in 

proliferative regions, but especially in the medial and lateral ganglionic eminences, regions that 

produce tangentially migrating neurons. In subcortical regions of the developing brain, vimentin 

positive cells with radial glial morphologies express Sdk2. Embryonic cortex, which contains 

predominantly radially migrating cells, does not contain Sdk2-positive fibers with radial glial 

morphologies. These data suggest that Sdk2 may support the tangential migration of neurons. 

Interestingly, Sdk2, which is expressed extensively during development, is up regulated 

following injuries that are known to induce ectopic neurogenesis. Needle stick injuries extending 

through cortex to the SVZ induced Sdk2 expression in reactive astrocytes. By two weeks after 

lesion, doublecortin (Dcx)-positive cells were found near Sdk2-positive cells along the lesion. 

Ischemic lesions induced by medial cerebral artery occlusion induce Sdk2 up regulation, which 

increases significantly between 3 and 7 days after lesion. Sdk2 expression is particularly 

increased along vasculature, which has been suggested to form a neurogenic niche after ischemic 

injury. 

To examine the role of Sdk2 in migration, we downregulated SDK2 in the RMS of the adult 

brain using lentivirally mediated RNAi. Migrating neurons were unable to migrate past the 

injection site. By two weeks after injection, Dcx-positive cells were found posterior to the 

injection site but there were almost no Dcx-positive cells in the olfactory bulb. Mice injected 

with a mismatch control RNAi had normal patterns of Dcx expression. Injecting SDK2 into the 

posterior SVZ, which labeled neurons but almost no glia in the rostral migratory stream, had no 

effect on migration. These results suggest that SDK2 expression by the glia lining the RMS is 

important for adult-born neuronal migration. 

Disclosures: S.S. Magavi, None; W. Kelsch, None; L. Kaufman, None; H. Shin, None; J.R. 

Sims, None; P. Klotman, None; C. Lois, None. 

Poster 




Topic: A.02.c. Cell migration

Support: Manitoba Institute of Child Health 

Manitoba Health Research Council 

Title: Extracellular regulated kinase regulation of oligodendrocyte progenitor migration 

Authors: *P. F. VORA 1,4 , J. MUSTAPHA 4 , Z. ZHOU 2,4 , E. E. FROST 1,2,3,4 ; 

1 Human Anat. and Cell Sci., 2 Pathology, 3 Biochem. and Med. Genet., Univ. of Manitoba, 

Winnipeg, MB, Canada; 4 Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada 

Abstract: Oligodendrocytes (OL) are the myelinating cells of the CNS. Migration of immature 

OL is a prerequisite for normal myelination, as the OL progenitors (OP) originate in discrete 

areas of the developing CNS and migrate significant distances to their final site of myelination. 

OL originate in the embryonic telencephalon and migrate across the subpallial layer to the subventricular 

zone (SVZ). The OPs then migrate away from the SVZ to populate white matter 

tracts of the CNS in response to numerous local cues including soluble signaling proteins. One 

such factor, Platelet-derived growth factor (PDGF), is essential for normal development of the 

myelin system. PDGF modulates several different OP behaviours, including proliferation, 

migration, and survival. OP response to PDGF differs according to the signaling pathway 

activated by receptor phosphorylation, i.e. migration is regulated via activation of the ERK 

signaling pathway, whereas proliferation is regulated via the PI3K signaling pathway. Short 

periods of PDGF exposure (10ng/ml), 10 minutes, are sufficient to initiate and maintain OP 

migration for up to 72 hours. Further, PDGF-A induced OP migration is concentration 

dependent, as it is initiated by transient exposure to 10ng/ml PDGF, but not 1ng/ml PDGF. 

However, ERK1/2 phosphorylation occurs after transient exposure to both 10 and 1ng/ml PDGF, 

suggesting that ERK1/2 activation alone is not sufficient to activate OP migration.Our 

hypothesis is that ERK activation requires a threshold level of receptor activation that is not 

achieved by exposing OPC to low concentrations of PDGF for short periods of time. This 

supports previous studies that show that duration of receptor occupancy determines the 

downstream effects of ligands. This work was funded by the Manitoba Children‟s Hospital 

Foundation and the Manitoba Health Research Council. 

Disclosures: P.F. Vora, None; J. Mustapha, None; Z. Zhou, None; E.E. Frost, None. 

Poster 





Support: NIH Grant N5040449 

Univ. of MO Research Board 

Title: Two Wnt/planar cell polarity proteins, Vangl2 and Celsr1, are essential for facial 

branchiomotor neuron migration in the mouse brain stem 

Authors: D. M. GLASCO 1 , B. FRITZSCH 2 , G. C. VILHAIS-NETO 3 , O. POURQUIE 3 , J. N. 

MURDOCH 4 , *A. CHANDRASEKHAR 1 ; 

1 Dept Biolog Sci., Univ. Missouri, Columbia, MO; 2 Dept Biomed. Sci., Creighton Univ., 

Omaha, NE; 3 Stowers Inst. for Med. Res., Kansas City, MO; 4 MRC Mammalian Genet. Unit, 

Harwell, Oxon, United Kingdom 

Abstract: A fundamental process in brain development involves the migration of facial 

branchiomotor neurons (FBMNs) in the embryonic brain stem across several rhombomeres to 

their final locations. There they are integrated into neural circuits and innervate target muscles 

involved in facial expression, blinking, sound attenuation and holding food in the mouth. Using 

NeuroVue lipophilic dyes (Molecular Targeting Technolgies, Inc.), we report here that two 

molecules of the non-canonical Wnt/planar cell polarity (PCP) pathway, Vangl2 and Celsr1, are 

essential for the caudal migration of FBMNs in mice. Vangl2 is a four-pass transmembrane 

protein containing a PDZ binding domain, and Celsr1 is a calcium-binding cell adhesion 

molecule. In Vangl2 mutant embryos, FBMNs fail to undergo their characteristic caudal 

migration and migrate laterally like otic efferent neurons. In Celsr1 mutants, many FBMNs fail 

to migrate caudally but migrate rostrally instead. These rostrally-migrating FBMNs maintain 

normal expression of some motor neuron markers but not others, suggesting that cell identity 

may be affected. In both Vangl2 and Celsr1 mutants, the Ret guidance receptor is aberrantly 

expressed in non-migratory FBMNs, suggesting that these FBMNs may not be able to properly 

control their gene expression or respond to environmental cues. We are currently investigating 

the expression of other cell surface receptors in these mutants and whether these molecules of the 

Wnt/PCP pathway have broader roles in other types of neuronal migration. 

Disclosures: D.M. Glasco, None; B. Fritzsch, None; A. Chandrasekhar , None; G.C. Vilhais- 

Neto, None; O. Pourquie, None; J.N. Murdoch, None. 

Poster 





Support: NIH Grant NS-46616 (R01) 

Title: Altered interneuronal development in Arx conditional knockout mice 

Authors: *E. MARSH 1 , C. T. FULP 2 , R. RISBUD 1 , E. LIN-HENDEL 2 , J. A. GOLDEN 2 ; 

1 Div. Child Neurol, Childrens Hosp Philadelphia, Philadelphia, PA; 2 Dept of Pathology, 

Children's Hosp Philadelphia, Philadelphia, PA 

Abstract: Introduction: Mutations in the Aristaless-related homeobox protein, ARX, have been 

linked to a number of epilepsy and mental retardation (MR) phenotypes in humans. ARX 

functions in early cortical development, particularly in non-radial migration. However, how 

mutations in ARX disrupt development and result in epilepsy and MR is unknown. Our lab has 

generated a conditional mouse line, ArxloxP-exon2/3-loxP (Arxfl), and crossed it to the 

Dlx5/6cre-IRES-GFP (DlxG/C) mouse line. We found that the offspring develop early seizures 

but exhibit no major morphological abnormalities. In contrast, early developmental studies of 

these mice found abnormalities in the major non-radial migratory streams, cortical size, and 

olfactory bulb morphology. As a discrepancy exists between the prenatal and postnatal brain 

morphologies and the CKO is only in interneuronal populations, we studied interneuron 

development from embryonic day (E) 14.5 to adulthood to determine alterations in interneuron 

ontogeny resulting from conditional loss of Arx. 

Methods: Brains from Arx-/y/DlxC/G, Arx-/X/DlxC/G, DlxC/G, and Arxfl mice were collected 

at defined timepoints from E14.5 through adulthood (greater than P 60). The brains were 

prepared for histological and immunohistochemical analyses with interneuron subtype and 

cortical laminar specific markers. Counts of each interneuron subtype as well as measurements 

of layer and regional areas were recorded. Groups were compared using ANOVA and students ttests. 

Results: All genotypes were generated, with mendalian ratio of CKO males in prenatal litters but 

a low frequency of CKO males surviving postnatally. At early time points, in the CKO, 

embryonic brains showed reduced cortical size, altered hemispheric shape, and diminutive 

olfactory bulbs. From P7 through adulthood the cortex and olfactory bulbs were morphologically 

normal. Histological analysis revealed only a single stream of migrating interneurons in the 

marginal zone in Arx-/y/DlxC/G mice compared to the three typical migratory pathways. In 

contrast to the abnormalities during migration, mature Arx/Dlx KO males had normal appearing 

cortical layers but a decrease in the numbers of calbindin and calretinin interneurons and no 

change in Parvalbumin interneurons. 

Conclusion: Conditional loss of Arx in the interneurons results in a change in the migratory 

pattern of non-radially derived cells and gross morphological deficits at early time points. In 

contrast, postnatal ages show normal gross morphology, but interneuronal subtype deficits. 

These data suggest compensatory mechanisms are present in interneuronal development in Arx 

deficient mice. 

Disclosures: E. Marsh , None; C.T. Fulp, None; R. Risbud, None; E. Lin-Hendel, None; J.A. 

Golden, None.

Poster 






Title: Aberrant migration of sympathetic preganglionic neurons into the dorsal root ganglia of 

reeler and dab1KO mutants 

Authors: J. M. HAN 1 , M. HIROSE 1 , A. I. BASBAUM 2 , *P. E. PHELPS 1 ; 

1 Dept Physiological Sci., UCLA, Los Angeles, CA; 2 Depts Anat. and Physiol., UCSF, San 

Francisco, CA 

Abstract: Reelin is an extracellular matrix protein that is a critical contributor to cell positioning 

during development. Deletion of Reelin in reeler mice disrupts the Reelin-Disabled-1 (Dab1) 

signaling pathway, leading to the accumulation of high levels of Dab1 in neurons that migrate 

incorrectly in reeler mutants. The first neurons found to be mispositioned in reeler spinal cord 

were sympathetic preganglionic neurons (SPNs); they failed to stop in the intermediolateral horn 

and instead continued to migrate medially. Here we describe an aberrant migration of Dab1labeled 

spinal cord neurons into the PNS of embryonic reeler mice. These ectopic neurons 

migrated along the reeler ventral root and into the ventral aspect of the dorsal root ganglia 

(DRG) and the mixed spinal nerve. As we found these neurons only at thoracic and upper lumbar 

segmental levels we suspected that they were SPNs. To test this hypothesis we used antibodies to 

choline acetyltransferase (ChAT) and neuronal nitric oxide (nNOS), co-expressed by SPNs, and 

found ectopically located neuronal clusters in reeler DRG. The mispositioned neurons increased 

in number within the DRG between E12.5 and E14.5, decreased significantly by E17.5 and were 

absent in adult reeler DRG. ChAT and nNOS double labeling experiments found a small number 

of ChAT-only possible somatic motor neurons but most ectopic neurons expressed both ChAT 

and nNOS, consistent with a SPN identity. To unequivocally identify these neurons as displaced 

SPNs, we injected DiI into the sympathetic chain ganglia of reeler organotypic slices and found 

retrogradely-labeled neuronal clusters in the mutant, but not the control, DRG. To determine if 

the peripheral migratory errors were due to a loss of the Reelin-Dab1 signaling pathway, we 

asked if dab1ko mice had similar ectopic neurons. At E14.5, both ChAT and nNOS-positive 

neurons were in the ventral dab1ko mutant DRG, but not in dab1ko adult DRG. We conclude 

that most of the aberrant neurons that erroneously migrate out of the spinal cord into the 

periphery are SPNs. As these neurons die perinatally, the Reelin-Dab1 signaling pathway most 

likely contributes to both their positioning and survival. These results indicate that Reelin 

functions as a stop signal that prevents SPNs from migrating out of the CNS and maintains them 

in the intermediolateral horn.

Disclosures: J.M. Han, None; M. Hirose, None; A.I. Basbaum, None; P.E. Phelps , None. 

Poster 



Program#/Poster#: 230.11/B1 


Support: Swiss National Grant 3100A0-116496/1 

Title: Effect of serotonin on GABAergic interneuron migration in the embryonic cortex 

Authors: *A. G. DAYER 1 , O. RICCIO 1 , C. WALZER 1 , J. KISS 2 ; 

1 Dept of Adult Psychiatry, 2 Fundamental Neurosci., CMU, Geneva, Switzerland 

Abstract: Serotonin (5-HT) is a strong neurodevelopmental signal that can modulate a wide 

variety of cellular processes in the embryonic and early postnatal brain. In rodents serotonergic 

fibers reach the developing cortex during the late embryonic period, a period during which 

neuronal migration is taking place. Recent studies have implicated 5-HT in the regulation of 

embryonic axonal pathfinding and interneuron migration. In this study we make the hypothesis 

that an excess of 5-HT could affect embryonic cortical interneuron migration. Using time-lapse 

videometry to monitor the migration of interneurons in embryonic mouse cortical slices, we 

show that the application of 5-HT decreases interneuron migration in a reversible and dosedependent 

manner. Current experiments are designed to identify the serotoninergic receptor(s) 

implicated in this effect. These results shed new light on the neurodevelopmental alterations 

caused by an excess of 5-HT during the embryonic period and contribute to a better 

understanding of the cellular processes that could be modulated by genetically controlled 

differences in human 5-HT homeostasis. 

Disclosures: A.G. Dayer, None; O. Riccio, None; C. Walzer, None; J. Kiss, None. 

Poster 





Support: Startup funds from the University of Michigan (CEK) 

Title: SMN regulates motor neuron cell body positioning in the neural tube 

Authors: *C. E. KRULL 1 , F. SU 1 , M. SAHIN 2 ; 

1 Biologic and Materials Sci., Univ. of Michigan, Ann Arbor, MI; 2 Childrens' Hosp. and Harvard, 

Boston, MA 

Abstract: During development, motor neurons extend their axons from the neural tube towards 

their target muscles in the hindlimb. We are interested in how motor neurons accomplish this 

feat, in such a stereotyped manner. Our studies have focused on the gene SMN, which is 

responsible for Spinal Muscular Atrophy (SMA), a human neurodegenerative disease. At 

present, it is unclear why motor neurons are affected in this disease. To examine the function of 

SMN in motor neurons, we have used specific SMN shRNAs/EGFP to reduce SMN levels. At 

stage 21 and early stage 23, motor axons exhibited growth defects and perhaps deficits in 

fasciculation when SMN levels were reduced. Interestingly, at later stages, we found EGFP+ 

cells from the neural tube in the spinal nerve, when SMN levels were decreased using shRNAs. 

This departure begins at mid-late stage 23, after motor axons have arrived at the limb base and 

are sorting, and is maintained at least until stage 30. To determine which cells have left the 

neural tube, we labeled SMN shRNA-treated cells with various markers; Islet1-positive cells that 

co-express EGFP are found in the spinal nerve, suggesting that these cells are motor neurons. We 

used markers to assess whether boundary cap cells. a neural crest derivative, were present in 

SMN shRNA-treated embryos. Boundary cap cells were present but perhaps in reduced numbers. 

Experiments are in progress to examine exactly which neural tube cells "migrate" abberantly in 

the spinal nerve, their eventual fate, and to determine the rationale for their "migration". 

Disclosures: C.E. Krull, None; F. Su, None; M. Sahin, None. 

Poster 





Support: NIH Grant 2RO1NS036692

NIH Grant 5RO1NS031234 

Title: The role of the sodium-potassium-chloride cotransporter (NKCC1) in cell migration 

Authors: *B. R. HAAS, H. W. SONTHEIMER; 

Neurobiol, Univ. Alabama-Birmingham, Birmingham, AL 

Abstract: During brain development, neuronal and glial progenitor cells migrate over long 

distances through narrow and tortuous extracellular spaces, posing significant demands on the 

cells‟ ability to dynamically alter their volume. This phenotype is recapitulated in primary brain 

tumors, i.e. malignancies arising from such progenitors. As a result, they are some of the most 

deadly cancers. These tumors‟ ability to invade normal brain tissue and migrate far from the 

original tumor site makes surgical resection challenging and contributes to their poop prognosis. 

Glioma invasion requires rapid changes in cell volume to allow cells to traverse narrow 

extracellular brain spaces. Volume changes occur through osmolyte flux, accompanied by the 

obligatory movement of water. For glioma cells, chloride is the major osmolyte involved. For it 

to function as such, high concentrations must be accumulated intracellularly. This involves the 

activity of the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1). Inhibiton of 

NKCC1 with bumetanide, a loop diuretic, results in a decrease of [Cl-]i which also correlates 

with a decrease in cell size. In this study, we hypothesized the high [Cl-]i present in glioma cells 

provides the energetic driving force for cell volume changes observed in migrating cells. By 

using immnunocytochemistry, we demonstrated the presence of NKCC1 in glioma cells and 

showed evidence of higher expression at the leading edge of migrating cells. Bumetanide 

treatment reduced the ability of glioma cells to migrate across a Transwell barrier by 20-50%, the 

barrier mimicking the spatial constraints of the brain. It did not affect 2D-migration, as assessed 

by scrape motility assay which lacks spatial constraints. These findings encouraged us to 

examine the roll of NKCC1 in tumor invasion in vivo and the potential to prevent tumor 

migration pharmacologically. To this end, we treated SCID mice with xenograft tumors with 

intraperitoneal injections of bumetanide. These pilot studies suggest a reduced number of 

satellite tumors, and NKCC1 may be a valuable pharmacological target to contain glioma 

invasion. Furthermore, NKCC1 may play a similar role in the migration of other brain cells, for 

example, stem cells. 

Disclosures: B.R. Haas , None; H.W. Sontheimer, None. 

Poster 



Program#/Poster#: 230.14/B4


Title: In situ visualization of GnRH-1 neuronal migration: perturbation by GABA 

Authors: *F. CASONI, S. WRAY; 

NINDS-CDNS, NIH, Bethesda, MD 

Abstract: Reproduction in vertebrates requires migration of GnRH-1 neurons from nasal regions 

into the brain during prenatal development. Although much is known about these neurons, our 

understanding of mechanisms controlling their movement is still limited. In the present work, 

GnRH-1 neuronal migration was analyzed in situ in embryonic mouse nasal explants from 3-6 

div using time-lapse microscopy. Live views show that GnRH-1 neurons exhibited saltatory 

movement during migration and had a neuronal speed of ~13um/hr. However, as a population, 

the fastest movement was consistently recorded at 4 div [mean rate = 18um/hr]. Midline cues are 

known to influence migratory cells. Removal of the midline cartilage in nasal explants 

significantly reduced GnRH-1 cell movement. Chemokines such as SDF-1, known to be present 

in this region, will be re-introduced and changes in GnRH-1 cell movement evaluated. Among 

molecules involved in migration, GABA has been shown to modulate GnRH-1 neuronal 

development and be present in nasal explants. Thus, the effect of GABA on GnRH-1 neuronal 

movement was examined. Treating explants acutely with picrotoxin, a GABAA receptor 

antagonist, significantly increased GnRH-1 neuronal migration [even at 4 days =23um/hr]. 

Muscimol, a GABAA receptor agonist, attenuated GnRH-1 neuronal migration at all days 

examined (10um/hr) and GnRH-1 neuronal speed in nasal explants from GAD67 KO mice was 

significantly faster as compared to controls. This study reports new aspects of GnRH-1 neuronal 

migration such as speed, GABA influence and saltatory movement. These data indicate that 

GnRH-1 cells, which undergo neurophilic migration, share characteristic with other migratory 

neurons like cerebellar and cortical neurons, which undergo gliophilic migration. Moreover this 

study describes a model in which large numbers of primary neurons can be resolved, apposed to 

their native pathway, to evaluate specific molecules important for cell migration as well as 

changes in cellular components during movement. 

Disclosures: F. Casoni , None; S. Wray, None. 

Poster 





Support: NIH grants NS23022 and NS33091 (ATC) 

(PMP) 

Postdoctoral Fellowship from the National Multiple Sclerosis Society, FG1723A1/1 

Title: Golli myelin basic proteins regulate oligodendroglial progenitor cell migration through 

modulation of Ca ++ influx 

Authors: *P. M. PAEZ, D. J. FULTON, V. SPREUER, V. HANDLEY, C. W. 

CAMPAGNONI, A. T. CAMPAGNONI; 

Semel Inst. for Neurosci, UCLA, Los Angeles, CA 

Abstract: Recent findings have clearly established that golli proteins, products of the myelin 

basic protein gene, plays a critical role in regulating Ca ++ influx in oligodendrocytes (OLs) and 

in T cells (Feng et al., 2006; Jacobs et al., 2005). Previous work from this laboratory indicates 

that membrane-associated golli proteins can regulate voltage-gated Ca ++ channels (VOCCs) in 

OLs during process extension and retraction (Paez et al, 2007). Migration of OL progenitor cells 

(OPCs) from proliferative zones to their final location is an essential step in nervous system 

development, yet the mechanisms controlling this migration are largely unknown. In this work 

we studied the physiological consequences of golli expression on OPC migration in vivo and in 

vitro. Using time-lapse imaging of both isolated OPCs and acute brain slice preparations from 

golli KO and overexpressing mice (JOE) we measured the dynamics of OPC migration. We 

found that golli stimulates migration, and this enhanced motility was associated with increases in 

the amplitude of Ca ++ transients in the soma of OPCs that depended on VOCC mediated Ca ++ 

influx. Our results showed a positive correlation between [Ca ++ ]int of OPCs and the rate of 

migration of these cells. We found a significant increase in the migration speed of golli 

overexpressing OPCs vs control cells after high K + stimulation, a manipulation that may increase 

[Ca ++ ]int by depolarizing the membrane potential and activating VOCCs. Additionally, gollimediated 

modulation of OPC velocity disappeared when VOCC antagonists Cd ++ and Verapamil 

were added to the culture medium. Furthermore, under our experimental conditions Ca ++ 

imaging revealed spontaneous Ca ++ oscillations in the soma and processes of OPCs during their 

migration. It also indicated that the rate of soma translocation correlated positively with both the 

amplitude and frequency of Ca ++ transients under a variety of pharmacological treatments that 

perturb these transients. Interestingly, the Ca ++ transient amplitude in migrating JOE cells was 

higher than that observed in control cells suggesting that the presence of golli may act to increase 

the size of spontaneous Ca ++ transients in migrating OPCs. These Ca ++ oscillations required the 

presence of external Ca ++ and were abolished in cells incubated with Cd ++ and Verapamil, 

indicating that Ca ++ influx through VOCCs are essential for this phenomenon. In summary, these 

data are the first demonstration that VOCCs may regulate OPC migration in vitro and in vivo and 

provide further support for the role of golli in the modulation of voltage-gated Ca ++ signaling in 

OPCs. 

Disclosures: P.M. Paez , None; D.J. Fulton, None; V. Spreuer, None; V. Handley, 

None; C.W. Campagnoni, None; A.T. Campagnoni, None.

Poster 





Support: NIH Grant AG25525 

NIH Grant T32 HD049309 

Title: Local activation of a Src family kinase mediates reverse signaling via a specific GPIlinked 

Ephrin within migrating neurons during embryonic development 

Authors: *P. F. COPENHAVER 1 , T. L. SWANSON 2 , T. M. COATE 2 ; 

1 Dept Cell & Dev Biol, 2 Cell & Developmental Biol., Oregon Hlth. & Sci. Univ., Portland, OR 

Abstract: Bidirectional signaling by Eph receptors and their Ephrin ligands regulates many 

aspects of neuronal development, but the mechanisms by which GPI-linked (type A) ephrins 

mediate reverse signaling are poorly understood. Using a model systems approach, we have 

shown that reverse signaling via a specific GPI-linked Ephrin regulates the guidance of 

migrating neurons within the developing enteric nervous system (ENS) of the hornworm 

Manduca sexta. During the formation of the ENS, a population of ~300 neurons (EP cells) must 

migrate selectively along pre-formed muscle bands on the gut surface, while avoiding adjacent 

midline regions. Notably, the migrating EP cells express the sole Ephrin ligand in Manduca 

(MsEphrin, a GPI-linked ligand), whereas the midline muscle cells express the cognate Eph 

receptor (MsEph). Manipulations in cultured embryos verified that reverse signaling via 

MsEphrin prevents the EP cells from crossing the enteric midline, while forward signaling via 

MsEph receptors is not required. Recently, we discovered that MsEphrin reverse signaling is 

mediated by a Src family kinase (MsSrc). Hyperactivating MsEphrin reverse signaling (with 

MsEph-Fc) caused a dramatic increase in MsSrc activity in the EP cells and inhibited their 

motility, as demonstrated by quantitative confocal analyses and biochemical assays of the ENS 

from single embryos. In contrast, inhibiting endogenous MsSrc activity blocked reverse 

signaling and induced the same midline crossover phenotypes caused by blocking endogenous 

MsEphrin-MsEph interactions. These results indicate that endogenous MsSrc is necessary for the 

transmission of MsEphrin reverse signals in the migrating neurons. We have cloned both Src 

family kinases that are expressed in Manduca (MsSrc42 and MsSrc64), and we are currently 

testing whether one or both of these Src orthologues mediates MsEphrin reverse signaling. Using 

similar methods, we found that MsEphrin-dependent Src stimulation leads to the local activation 

of Rho GTPase in the EP cells, which in turn is required for their retraction/repulsion away from

the midline. Intriguingly, MsSrc may also integrate responses to other pathway cues encountered 

by migrating neurons in ENS, including attractive responses to the adhesion receptor Fasciclin II. 

We are currently using in vivo and in vitro methods to further characterize the signaling 

pathways by which input from multiple guidance cues converge to regulate neuronal guidance in 

the developing ENS. 

Disclosures: P.F. Copenhaver , None; T.M. Coate, None; T.L. Swanson, None. 

Poster 





Title: Migration of stellate/basket cells in developing cerebellar cortex 

Authors: *H. KOMURO 1 , D. B. CAMERON 1 , Y. JIANG 1 , K. KASAI 2 , Y. SAEKI 2 ; 

1 Dept Neurosci, NC30, Lerner Res. Inst. Cleveland Clin. Fndtn, Cleveland, OH; 2 Neurolog. 

Surgery, The Ohio state Univ., Columbus, OH 

Abstract: The precursors of cerebellar stellate/basket cells reside in the deep WM, but little is 

known about how these neurons attain their final positions within the ML. In this study, with the 

use of a HSV amplicon plasmid pHGY, which expresses EGFP under the control of the HSV 

IE4/5 promoter, we examined the migration of stellate/basket cells in the early postnatal mouse 

cerebellum. We found that stellate/basket cells sequentially go through four distinct phases of 

migration. The first phase (Phase I) is a period of when basket/stellate cells migrate radially from 

the bottom of the IGL through the PCL towards the top of the ML. Upon reaching the top of the 

ML, basket/stellate cells undergo the first change of the direction of migration from radial to 

tangential. The second phase (Phase II) is a period of when basket/stellate cells migrate 

tangentially in the rostro-caudal direction (perpendicular to the folia) at the top of the ML. 

Thereafter, basket/stellate cells undergo the second change of the direction of migration from 

tangential to radial. The third phase (Phase III) is a period of when basket/stellate cells migrate 

radially within the ML from the top to the bottom and vice versa with multiple branches of the 

leading processes. Subsequently, basket/stellate cells undergo the third change of the direction of 

the migration from radial to tangential in the rostro-caudal direction at the middle and the top of 

the ML. The fourth phase (Phase IV) is a period of when basket/stellate cells tangentially migrate 

in the rostro-caudal direction and complete their migration at the middle and the top of the ML. 

Stellate/basket cells exhibited the distinctive rate of migration during each phase of migration. 

The average rates of migration were ~14.9 κm/h in Phase I, ~15.4 κm/h in Phase II, ~7.2 κm/h in

Phase III, and ~4.6 κm/h in Phase IV. The average transit times were ~18.1 hours in Phase I, 

~16.8 hours in Phase II, ~19.3 hours in Phase III, and ~10.2 hours in Phase IV. The present 

results revealed that after entering the final destination, stellate/basket cells exhibit the multiple 

phases of migration before completing the migration. Furthermore, the present study provides the 

foundation for future studies examining the mechanisms underlying basket/stellate cell migration 

in the developing cerebellum. 

Disclosures: H. Komuro , None; D.B. Cameron, None; Y. Jiang, None; K. Kasai, None; Y. 

Saeki, None. 

Poster 





Support: CMRF of the University of Pittsburgh Medical Center (JWY) 

JSPS (SS, KN), MEXT of Japan, Ichiro Kanehara Foundation, Tokyo Biochemical 

Research Foundation, Sumitomo Foundation, Uehara Memorial Foundation, Novartis 

Foundation and Brain Science Foundation (KN). 

Title: Netrin effects on migration of sympathetic preganglionic neurons in the spinal cord 

Authors: *J. W. YIP 1 , S. SASAKI 2 , K. NAKAJIMA 2 ; 

1 Dept Neurobiol, Univ. Pittsburgh Sch. Med., Pittsburgh, PA; 2 Dept Anat., Keio Univ. Sch. of 

Med., Tokyo, Japan 

Abstract: 

Postmitotic sympathetic preganglionic neurons (SPN) of mammals migrate first from the neuroepithelium to the ventrolateral 

spinal cord. There they undergo a secondary dorsal migration along commissural fibers to form the intermediolateral column (IML). The cues 

that guide SPN migration to the IML are unknown. Netrin, a molecule secreted by the floor plate of the spinal cord and a chemoattractant for 

commissural fibers, has been known to act as a chemorepellent for neuronal migration during the development of various brain and brainstem 

nuclei. Here we investigate whether Netrin affects SPN migration by examining the locations of SPN in Netrin-1 -/- and DCC-KO mice at 

progressive stages of development. Result showed that SPN still migrated to form the IML in Netrin-1 -/- and DCC-KO mice. In addtion, 

immunostaining showed no immunoreactivity for Netrin repulsive receptors UNC5H2, UNC5H3, UNC5H4 and Neogenin on SPN, but some 

immunoreactivity for the Netrin attractive receptor DCC on SPN axons. Therefore, Netrin is not likely to be required for the ventral to dorsal 

migration of SPN. However, in Netrin-1 -/- and DCC-KO mice, the IML was more diffusely organized and there were more ectopic SPN in the 

ventral spinal cord than in their control littermates. Since Netrin also affects commissural fiber guidance, this result suggests that when 

commissural fibers are absent or are abnormal, SPN positioning is less precise and many more errors are made. Moreover, many migrating SPN

in Netrin-1 -/- and DCC-KO were multipolar instead of bipolar, suggesting that SPN use a different mode of migration when commissural fibers 

are absent. Thus Netrin may affect SPN migration through its effect on commissural fiber guidance. 

Disclosures: J.W. Yip , None; S. Sasaki, None; K. Nakajima, None. 

Poster 





Support: ANR-06-NEUR-010 

EU FP7-2007-ICT-216593 (SECO) 

Title: Role of p27kip1 in modulating radial migration in the neocortex 

Authors: *E. GAUTIER 1,2 , N. DOERFLINGER 1,2 , V. CORTAY 1,2 , C. HUISSOUD 3 , P. 

GIROUD 1,2 , M. BERLAND 4 , H. KENNEDY 1,2 , C. DEHAY 1,2 ; 

1 Inserm, U846, Stem Cell and Brain Res. Inst., BRON, France; 2 Univ. de Lyon, Lyon 1, UMR-S 

846, Lyon, France; 3 Hospices civils de Lyon, Hôpital de la Croix-Rousse, Lyon, France; 

4 Hospices civils de Lyon, Hôpital Lyon Sud, Pierre-Benite, France 

Abstract: Projection neuron precursors proliferate in the ventricular and subventricular zones, 

before migrating radially towards their final destination in the cortical plate. The cytoarchitecture 

of the neocortex depends crucially on the coordination of the rates of proliferation and migration. 

p27kip1is a putative candidate for coordinating proliferation and migration of cortical neurons 

(Nguyen et al., 2006). p27kip1 is a modular protein: via its N-term domain, it binds to the 

cyclin/Cdk complexes in the nucleus and inhibits the G1/S phase transition, whereas via its Cterm 

domain, it inhibits RhoA in the cytoplasm and so promotes cellular migration. Using gain 

of function (GOF) and downmodulation (DM) experiments based on electroporation of plasmids 

and siRNAs respectively in E15 mouse cortical precursors, we have explored the role of p27kip1 

on radial migration in the cortex. Immunohistochemistry and time-lapse videorecordings (TLV) 

on organotypic slices show that overexpression of p27kip1 leads to a decrease in the rate of 

proliferation and importantly to an increase in the speed of migration, whereas a 

downmodulation of p27kip1 by RNA interference gives the opposite results. Variations in the 

speed of migration are correlated with modulations of the expression level of cytoplasmic 

p27kip1 in migrating neurons. 

In the E77 -E80 monkey, the rate of proliferation in visual area A17 is higher than in area 18.

This area-specific difference in rates of proliferation is accompanied by higher levels of p27kip1 

expression in area 18 compared to area 17 precursors. Preliminary TLV recordings indicate that 

radial migration is faster in area 17 compared to area 18. Migrating neurons in area 17 show 

higher cytoplasmic levels of p27kip1 than area 18 neurons. These results point to the role of 

p27kip1 in the coordinated and area-specific regulation of rates of neuronal production and 

migration. 

Disclosures: E. Gautier, None; N. Doerflinger, None; V. Cortay, None; P. Giroud, None; H. 

Kennedy, None; C. Dehay, None; M. Berland, None; C. Huissoud, None. 

Poster 





Support: Canadian Institutes of Health Research (CIHR) Grant, MOP14460 

Nina Ireland, NIMH RO1 MH49428-01 and K05MH065670 

Title: Characterization of a distinct subpopulation of striatal projection neurons expressing the 

Dlx genes in the basal ganglia through the activity of the I56ii enhancer 

Authors: *M. YU 1 , N. GHANEM 1 , L. POITRAS 1 , J. L. R. RUBENSTEIN 2 , M. EKKER 1 ; 

1 CAREG, Dept. of Biol., Univ. of Ottawa, Ottawa, ON, Canada; 2 Nina Ireland Lab. of 

Developmental Neurobiology, Dept. of Psychiatry, Univ. of California at San Francisco, San 

Francisco, CA 

Abstract: Regulation of region-specific neuronal differentiation and migration in the developing 

forebrain is a complex mechanism requiring the involvement of various transcription factors 

such as the Dlx genes. At least four cis-acting regulatory elements (CREs) are responsible for the 

Dlx transcriptional regulation in the subcortical telencephalon and the rostral diencephalon. 

These include I12b and URE2 in the Dlx1/2 bigene cluster, and, I56i and I56ii in the Dlx5/6 

bigene cluster. We previously reported that URE2, I12b, and I56i, mark different progenitor cell 

populations in the ganglionic eminences as well as different subtypes of adult cortical 

interneurons (Ghanem et al, J Neurosci, 27, 5012-22, 2007). Here, we carried out a detailed 

spatial and temporal analysis of the I56ii CRE activity in the developing telencephalon between 

E10.5 and E15.5. Using the lacZ reporter gene expression in transgenic mice, we compared its 

activity with the other three Dlx CREs. We show that I56ii marks distinct group(s) of neurons

located in the superficial mantle of the lateral and medial ganglionic eminences (LGE and MGE) 

between E11.5 and E13.5. The I56ii-positive cells are Dlx- and GABA-immunoreactive. 

However, unlike the other CREs, I56ii does not label interneuron progenitors in the basal 

ganglia, nor tangentially migrating cells to the cortex at E13.5. Instead, I56ii-positive cells mark 

a subpopulation(s) of post-mitotic projection neurons that tangentially migrate from the LGE to 

the deep mantle of the MGE and reside between the subventricular zone and the globus pallidus 

during midgestation. The majority of these neurons express the striatal markers Meis2 and Islet1. 

Moreover, both Meis2 and Islet1 activate transcription of a reporter gene containing the I56ii 

sequence in co-transfection assays, indicating that these transcriptional factors may be potential 

upstream modulators of the Dlx genes in vivo. In summary, our data suggest a complex and 

dynamic regulation of Dlx gene expression during early stages of embryonic development 

through several regulatory elements with overlapping and distinct function(s). 

Disclosures: M. Yu, None; N. Ghanem, None; L. Poitras, None; J.L.R. Rubenstein, None; M. 

Ekker, None. 

Poster 





Title: On the chronological course of zinc distribution in the developing mouse cerebellum 

Authors: *M. BORGMANN 1 , S. FRISCHMUTH 2 , J. BAEURLE 2 ; 

2 Physiol., 1 Charite - Berlin, Berlin, Germany 

Abstract: Early development and normal formation of the cerebellum critically depends on zinc 

finger transcription factors. In the mature brain, Zn is considered to act as a co-transmitter and 

neuromodulator in certain brain regions. Moreover, elevated concentrations of Zn in 

neurodegenerative diseases have also suggested a role for Zn in neuronal cell death. Thus, Zn 

seems to be involved in a number of essential functions during different phases of brain 

development and maturation. 

However, only little is known about the spatio-temporal distribution or function of Zn during 

normal postnatal cerebellar development. 

Here we show the first chronological course of Zn distribution in the mouse cerebellum using 

immersion autometallography (AMG), a well established technique to visualize Zn in biological 

tissue. 

C57Bl/6J mice were sacrificed at postnatal day (P) 7, 14, 19, 22, 24, 29, 83 and 120. Brains were

immediately dissected and frozen on liquid nitrogen. 10µm cryo-sections were stained for Zn 

using the immersion-AMG technique. 

The pattern of silver-staining at various postnatal ages revealed clear alterations in Zn 

distribution and concentration during postnatal cerebellar development. Overall staining intensity 

was lowest at P7, continuously increased until the end of the fourth postnatal week, to remain 

almost constant after P29. At P7 Zn was mainly localized in premigratory granule cells (GC) of 

the external granular layer (EGL), whereas Purkinje cells (PC) were only sporadically labelled. 

From P14 to P19 highest staining intensities were observed in the inner granular layer (IGL). The 

number of stained PCs increased significantly during this phase, with highest Zn concentrations 

in PC nuclei. In the molecular layer (ML) some large cells were AMG-positive. At P22 the 

complete PC layer was stained for Zn, whereas the density of labelled cells in ML and IGL 

remained almost identical as in the foregoing time interval. From P24 onwards the number of 

positive PCs decreased continuously. In the adult brain (P83 - P120) only few positive PCs were 

present and staining in the IGL was much less intense than during development. Large positive 

cells in the ML were present even at this age in about the same density and distribution as 

observed at around P14. 

In conclusion, this differential spatio-temporal pattern of Zinc distribution in the developing 

mouse cerebellum suggests an important role for Zn in GCs and PCs during the phase of GC 

migration and PC-parallel fiber synaptogenesis. 

Disclosures: M. Borgmann , None; J. Baeurle, None; S. Frischmuth, None. 

Poster 




Topic: A.02.b. Cell lineage and cell fate specification 

Support: NIH Grant MH071679-01A1 

Title: FoxG1 regulates neuronal migration in the cerebral cortex 

Authors: *G. MIYOSHI 1 , V. SOUSA 1 , C. HANASHIMA 1,2 , G. FISHELL 1 ; 

1 Smilow Res. Ctr., New York Univ. SoM, New York, NY; 2 CDB Riken, Kobe, Japan 

Abstract: The Forkhead box transcription factor FoxG1 is known to have multiple roles during 

forebrain development. We have generated a floxed allele such that FoxG1 expression is lost 

following Cre mediated recomibination. When FoxG1 is removed globally from the pallium 

using the Emx1-Cre driver, the size of the cortex and olfactory bulb was severely reduced. In

addition, the pyramidal tract marked by reporter EGFP was largely diminished. Consistent with 

this, the expression of Ctip2, which is a gene required for the specification of the deep layer 

cortico-spinal tract neurons was absent in Emx1Cre/+ FoxG1c/c animals at E18.5. Surprisingly, 

these mutants live at least until the age of 3 weeks with a very thin layer of cortex with almost 

80% reduction. To better understand the role of FoxG1 in cortical plate formation, we removed 

this gene from the pyramidal neuron progenitors in a mosaic manner at a time coincident with 

them becoming postmitotic. The FoxG1 null cells failed to penetrate into the cortical plate and 

remained underneath the subplate, despite having initiated the expression of Ctip2. We also 

observed migration defects in ventral telencephalic neurons upon removal of FoxG1. This raises 

the possibility that FoxG1 is universally required for the telencephalic progenitors to migrate out 

from their respective progenitor domains. 

Disclosures: G. Miyoshi , None; V. Sousa, None; C. Hanashima, None; G. Fishell, None. 

Poster 





Support: BBSRC 

Title: Role of vascular endothelial growth factor and blood vessels in the development of GnRH 

neurons 

Authors: *A. CARIBONI 1,2 , C. RUHRBERG 1 , S. RAKIC 1 , K. DAVIDSON 1 , E. DOZIO 2 , R. 

MAGGI 2 , J. G. PARNAVELAS 1 ; 

1 Univ. Coll London, London, United Kingdom; 2 Univ. of Milan, Milan, Italy 

Abstract: Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered 

in the hypothalamus, play an essential role in reproductive function. During development, these 

neurons originate in the olfactory placode and migrate along olfactory/vomeronasal axons in the 

nasal compartment to gain access into the forebrain and reach their final positions in the 

hypothalamus. In humans, failure of GnRH neurons to migrate normally results in delayed or 

absent pubertal maturation and infertility. The movement of these cells through changing 

molecular environments suggests that numerous factors are involved in their migration. We have 

recently described that classical neuronal guidance molecules, such neuropilins (NRPs), are 

expressed by GnRH neurons, and established the importance of NRP2 in their migration in vivo. 

Using immortalised GnRH cells (GN11), we also found that two NRP ligands modulate their

migratory response: the class 3 semaphorins (SEMA3A/3F) and vascular endothelial growth 

factor A (VEGF). VEGF is a major regulator of vasculogenesis and vascular permeability, 

interacting with receptor tyrosine kinases Flt-1 and Flk-1 on endothelial cells. Recent evidence 

indicates that VEGF has additional non-vascular functions. In particular, the identification of 

NRPs as co-receptors for VEGF, as well as the detection of their receptors on neurons, suggests 

that VEGF could act directly on neurons to produce effects such survival and migration. In this 

study, the possible interactions between VEGF, blood vessels and the GnRH-system have been 

tested. We first visualized the presence of a network of blood vessels along peripherin-positive 

olfactory axons and migrating GnRH neurons. In embryonic mice, blood vessels, stained with 

lectin IsoB4, were seen around the olfactory placode, the vomeronasal organ, as well as in the 

nasal mesenchyme, during the appearance and migration of GnRH neurons. Then, by using RT- 

PCR and enzymatic staining of VEGF-LacZ reporter mice, we found that VEGF is abundantly 

expressed in the developing olfactory structures. Moreover, FACS-isolated embryonic GFP- 

GnRH neurons expressed specific transcripts for VEGF and its receptor Flt-1. We found that 

GN11 cells-used as a model of migrating GnRH neurons- also express these molecules and 

respond to VEGF. Functionally, we observed that the latter promotes their survival and 

stimulates the chemomigration of GN11 cells by activating PI3K and MAPK pathways. Taken 

together, these results indicate that GnRH neuron migration and development are modulated by 

VEGF signalling, suggesting the existence of a „cross-talk‟ between the vascular and GnRHneuronal 

systems. 

Disclosures: A. Cariboni , None; C. Ruhrberg, None; S. Rakic, None; K. Davidson, None; E. 

Dozio, None; R. Maggi, None; J.G. Parnavelas, None. 

Poster 

231. Dendrite Growth and Branching: Signaling 



Topic: A.04.i. Dendritic growth and branching 

Support: Canadian Institutes of Health Reserach FRN:72027622 to FJV and SR 

Natural Sciences and Engineering Research Council Doctoral Grant to LAT 

Natural Sciences and Engineering Research Council Grant to DAL 

Title: Teneurin C-terminal associated peptide (TCAP)-1 reverses the behavioral effects of 

chronic restraint stress and induces morphological changes in CA3 hippocampal neurons in rats

Authors: *L. A. TAN 1 , A. AL CHAWAF 1 , P. C. BOUTROS 2 , F. J. VACCARINO 3 , D. A. 

LOVEJOY 1 , S. ROTZINGER 3 ; 

1 Cell and Systems Biol., 2 Pharmacol., 3 Psychology, Univ. of Toronto, Toronto, ON, Canada 

Abstract: Teneurin C-terminal associated peptide (TCAP)-1 is a newly elucidated and wellconserved 

endogenous neuropeptide that has been shown to have both in vitro and in vivo 

effects. In vitro, exogenous TCAP-1 upregulates cytoskeletal elements and influences neurite 

outgrowth in immortalized neurons, while in vivo, repeated TCAP-1 administration has been 

shown to modulate behavioral responses to CRF when injected into the rat brain. The 

hippocampus is a neuroplastic region that is implicated in memory, learning, and emotional 

responses to stress. Chronic restraint stress (CRS) has been shown to decrease hippocampal 

dendritic branching, a process that can be attenuated by some antidepressant treatments. This 

study investigated the effect of repeated intracerebroventricular TCAP-1 administration on CRSinduced 

anxiety-like behaviors in the elevated plus maze as well as the effect on CRS-induced 

hippocampal dendritic atrophy. TCAP-1 (300 pmol) and CRS (2 h) was administered daily for 

10 days to male Wistar rats before being tested on the elevated plus maze on the 11 th day. Brains 

collected from these rats were then stained using the Golgi method to visualize CA3 neurons in 

the hippocampus. Results show that TCAP-1 administration reversed the effects of CRS in the 

elevated plus maze, resulting in near-basal behavioral profiles. TCAP produced morphological 

changes in both the apical and basilar dendrites approximately 250 um and 100 um from the 

soma, respectively. These results indicate that TCAP-1 may be a novel neuropeptide system 

involved in modulating stress-related behaviors and neuroplasticity. 

Disclosures: L.A. Tan , None; A. Al Chawaf, None; P.C. Boutros, None; F.J. Vaccarino, 

None; D.A. Lovejoy, None; S. Rotzinger, None. 

Poster 





Support: Financial Support: This work was supported by grant R01 AG 13620 from the NIH (to 

E.Z.). 

Title: Overexpression of delta-catenin promotes changes in dendritic morphology of 

hippocampal neurons: GRIP/ABP and PSD95 inhibit morphological changes

Authors: J. M. FERREIRA, *E. B. ZIFF; 

Dept Biochem, New York Univ. Med. Ctr., New York, NY 

Abstract: Delta-catenin (also called NPRAP) was first identified through its interaction with 

Presenilin-1, and has been implicated in the regulation of dendrogenesis and cognitive function. 

In neurons, delta-catenin is present in dendrites and is enriched in the postsynaptic density, 

where it is associated with classical cadherins. Coimmunoprecipitation studies from our lab have 

shown the association of delta-catenin with the scaffolds GRIP and PSD95 through a PDZ 

binding sequence (Silverman et al., 2007). Thus, delta-catenin links adherens junctions to the 

postsynaptic protein complex through a direct interaction with synaptic scaffolding proteins 

through its PDZ ligand. This interaction forms a macromolecular protein complex that includes 

the NMDA and AMPA receptors. Here we show that expression of GRIP/ABP and PSD95 

inhibits the ability of delta-catenin to form filopodia- and lamellipodia-like structures. Using 

hippocampal neuron cultures (18-21 DIV), we show by immunofluorescence that viral vector 

expression of delta-catenin induces a dramatic change in the morphology of dendrites, including 

the appearance of actin-containing filopodia-and lamellipodia-like structures, while a deltacatenin 

mutant lacking ARM domains is not able to promote these structures. Notably, structure 

formation is increased by deletion of the delta-catenin PDZ binding motif. Also, the coexpression 

of ABP or PSD95 with wild type delta-catenin in hippocampal neurons inhibits the 

ability of delta-catenin to promote actin polymerization, as reflected by the blockage of the 

filopodia and lamellipodia. Not only ABP PDZ domain Set I, which contains the delta-catenin 

binding site and is composed of PDZ domains 1,2,3, but Set II, including PDZ domains 4,5,6, 

inhibit morphological changes caused by delta-catenin over-expression. In addition, we have 

shown that expression of delta-catenin dominant negative mutants compromises GluR2 surface 

expression. We conclude that ABP/GRIP and PSD95 repress the effect of delta-catenin in 

branching and elongation of dendrites by a mechanism dependent upon the PDZ binding motif in 

delta-catenin. 

Financial Support: This work was supported by grant R01 AG 13620 from the NIH (to E.Z.). 

Disclosures: J.M. Ferreira, None; E.B. Ziff , Biochemistry Department, New York University, 

School of Medicine, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received). 

Poster 




Topic: A.04.i. Dendritic growth and branching

Support: NSERC 

Title: Elucidating a role for CAPON in synaptogenesis 

Authors: L. RICHIER 1 , M. O'BRIEN 1 , N. ZINCK 1 , S. KRUEGER 2 , T. PAWSON 4 , *J. P. 

FAWCETT 3 ; 

1 Pharmacol., 2 Physiol. and Biophysics, 3 Pharmacol. and Surgery, Dalhousie, Halifax, NS, 

Canada; 4 Samuel Lunenfeld Res. Inst., Toronto, ON, Canada 

Abstract: The synapse is a highly differentiated organelle designed to mediate the transmission 

of information between axons and dendrites on target cells. Both the axon and dendrite contain 

different complements of proteins that are necessary to give rise to their specialized function. 

Recent genetic and biochemical studies have revealed a number of conserved polarity proteins 

including Par3 and scribble in synaptic function. To gain insight into the function of scribble in 

the synapse we conducted a mass spectrometry screen using scribble as bait to identify novel 

interacting proteins. Our screen identified a number of proteins implicated in the synapse 

including carboxy-terminal PSD-95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON), G-protein 

coupled receptor interacting protein1 and 2 (Git1,2), p21 kinase activated (PAK) interacting 

exchange factor (β-Pix) and p21 activated kinase (PAK). CAPON contains an amino terminal 

phosphotyrosine binding (PTB) domain followed by a carboxyl terminal PDZ binding motif. The 

PTB domain has been shown to bind synapsin1 and DexRas while the carboxy-terminal PDZ 

binding motif has been shown to bind directly to the PDZ domain of nNOS. In our screen we 

also identified a novel CAPON splice variant (CAPONLong) that destroys the carboxyl terminal 

PDZ binding motif. CAPONLong fails to associate with nNOS suggesting a novel function for 

this isoform in the CNS. The association between scribble and CAPON is dependent on the PTB 

domain of CAPON and the fourth PDZ domain of scribble. Furthermore, scribble bridges the 

association between CAPON and a Git1/β-Pix complex. Overexpression of CAPON in 

dissociated hippocampal neurons induces filopodial outgrowth on dendrites. This outgrowth is 

dependent on the PTB domain of CAPON. Together these data suggest that CAPON plays an 

important role in synaptic development. 

Disclosures: L. Richier, None; J.P. Fawcett , None; M. O'Brien, None; N. Zinck, None; S. 

Krueger, None; T. Pawson, None. 

Poster 





Support: Grant-in-Aid for Scientific research (B) 

Title: Vezatin is a possible downstream effector of ADP-rybosilation factor 6 that regulates 

dendritic formation 

Authors: *M. SANDA 1,2 , K. FUKUNAGA 2 , H. SAKAGAMI 1 ; 

1 Anat., Kitasato Univ., sagamihara, Japan; 2 Pharmacol., Tohoku Univ. Grad. Sch. of Pharmaceut. 

Sci., Sendai, Japan 

Abstract: ADP-ribosylation factor 6 (ARF6) belongs to the ARF family of Ras-related small 

GTPases and mediates various neuronal functions through the regulation of actin cytoskeleton 

and membrane trafficking. Although ARF6 has recently been shown to regulate the dendritic 

formation, the detailed mechanisms by which ARF6 regulates the dendritic formation are poorly 

understood. To address this issue, it is crucial to identify interacting proteins with ARF6 as 

candidates for downstream targets. Therefore, we performed a yeast two hybrid screening of a 

mouse brain cDNA library with a constitutively active ARF6 mutant as bait. Among positive 

clones, three independent clones encoded vezatin. Vezatin has recently been identified as a 

transmembrane protein that interacts with myosin VIIA and α-catenin at adherens junctions, 

thereby constructing a functional and structural linkage between actin cytoskeleton and 

cadherin/catenin complex. In vitro binding assay showed that vezatin was able to bind 

specifically to GTP-bound ARF6 among the ARF family members. In the mature mouse brain, 

vezatin mRNA was widely expressed in the mature mouse brain with a high level in the olfactory 

bulb, cerebral cortex, hippocampus, striatum and cerebellar granular cell layer. In the embryonic 

brain, vezatin mRNA was already detected at a high level throughout the brain and spinal cord. 

The expression was observed in both mantle and ventricular zones with a higher level in the 

former. Further, northern blot analysis showed that vezatin mRNA was already detected in the 

mouse brain at E13 and peaked at E15. After birth, the expression of vezatin mRNA was 

gradually decreased by 50% compared to that at embryonic stages. In cultured hippocampal 

neurons, vezatin was present in cell bodies and dendrites, but not in axons. Confocal microscopic 

observation at high magnification of dendrites, the immunoreactivity for vezatin appeared as 

punctate inside the dendritic shafts, which were colocalized with EEA1, a marker for endosomes. 

Furthermore, the overexpression of full length of vezatin promoted dendritic length and 

complexity of cultured hippocampal neurons. Our present findings suggest that vezatin may 

function to regulate dendritic formation as a downstream effector for ARF6. 

Disclosures: M. Sanda, None; K. Fukunaga, None; H. Sakagami, None. 

Poster 





Support: NIH Grant DC000210 



Title: Role of TrkB in dendritic development of mitral cells in mouse olfactory bulb 

Authors: *F. IMAMURA, C. GREER; 

Dept. Neurosurg., Yale Univ,Sch Med., New Haven, CT 

Abstract: Mature mitral and tufted cells in the mammalian olfactory bulb (OB) have a single 

primary dendrite that arborizes within a single glomerulus. Here, we focused on factors 

regulating the development of mitral/tufted cell dendritic arbors within a glomerulus, and test the 

hypothesis that the neurotrophins are important determinants of this mechanism. First, we 

visualized the development of glomerular tufts of mitral cells in mice using intracellular lucifer 

yellow injections. We found that dendritic tufts in a glomerulus significantly increase their total 

length and number of branching points during early postnatal days, especially from postnatal day 

(P) 3 to 10. Second, using immunohistochemical analyses, we found that the full-length TrkB 

(TrkB.FL) was expressed by thick dendritic trunks of mitral/tufted cells, while the truncated 

isoform of TrkB (TrkB.T1) is localized at the tip of mitral/tufted cell dendrites including 

dendritic tips in the glomerular tuft. Interestingly, the TrkB.T1 expression in glomeruli 

colocalized with growth cone marker CDA 1, and was high during early postnatal days but 

disappeared by P10. Therefore, this suggested that the TrkB.T1 was expressed in dendritic 

growth cones when dendrites arborize in glomeruli. Third, to examine the role of TrkB in 

dendritic development, we cultured mitral/tufted cells and treated them with neurotrophins: 

BDNF, NT-3, and NT-4. In cultured mitral/tufted cells at 1 day in vitro (DIV), localization of the 

TrkB.T1 at the tip of neurites was observed, while the TrkB.FL was seen in whole neurites. 

When treated with TrkB ligands, BDNF or NT-4, from 0 to 4 DIV, mitral/tufted cells 

significantly increased the number of primary neurites and branching points, as well as their total 

neurite length compared with untreated controls. NT-3 treatment did not have a significant 

effect. Our findings strongly suggest that the TrkB.T1 expression during dendritic development 

plays a significant role in the elaboration of mitral/tufted cell glomerular dendritic arbors, 

consistent with our working hypothesis that neurotrophins are determinants of olfactory bulb 

circuitry. 

Disclosures: F. Imamura, None; C. Greer, None. 

Poster





Support: Scientific Research from the Ministry of Education, Science, Sports, and Culture of 

Japan 

the Takeda Science Foundation 

Title: Regulation of dendritic development by the Rac activator Dock4 in hippocampal neurons 

Authors: *H. KATOH, S. UEDA, S. FUJIMOTO, M. NEGISHI; 

Lab. Mol Neurobiol Grad Sch. Biostudies, Kyoto Univ., Kyoto, Japan 

Abstract: Dendrite development is required for establishing proper neuronal connectivity. It is 

already well known that members of the Rho family small GTPases are key regulators of the 

actin cytoskeleton in diverse cellular functions including neuronal morphogenesis However, the 

molecular mechanisms that control the activities of Rho GTPases in developing dendrites are not 

well understood. Dock4 is a member of an evolutionarily conserved superfamily of Dock180related 

proteins and functions as a guanine nucleotide exchange factor (GEF) for the small 

GTPase Rac. We found that Dock4 is highly expressed in the developing rat brain, 

predominantly in the hippocampal neurons. In dissociated cultured hippocampal neurons, the 

level of Dock4 protein is up-regulated between 3 and 8 days in culture, when dendrites begin to 

grow. Knockdown of endogenous Dock4 results in reduced dendritic growth and branching. 

Conversely, overexpression of Dock4 with its binding partner ELMO2 enhances the numbers of 

dendrites and dendritic branches. These morphological effects elicited by Dock4 and ELMO2 

require the Rac activation and the C-terminal region of Dock4. These findings demonstrate a 

new function of the Rac activator Dock4 in dendritic morphogenesis in hippocampal neurons. 

Disclosures: H. Katoh , None; S. Ueda, None; S. Fujimoto, None; M. Negishi, None. 

Poster 





Title: The role of phosphatase PP1 and PP2A isoforms in dendritic and axonal development 

Authors: D. MONROE, *C. C. FINK; 

Dept Biolog Sci., Univ. Wisconsin-Milwaukee, Milwaukee, WI 

Abstract: Protein phosphatase types 1 and 2A are serine/threonine phosphatases that are present 

in the hippocampus, a structure of the brain that has been implicated in learning and memory 

phenomena. These phosphatases have been shown to play a role in such neuronal processes as 

signal transduction, LTP induction of protein synthesis and the regulation of neuronal 

morphology. Four isoforms of the catalytic subunit of phosphatase 1 have been identified in 

mammals: α, β, γ1 and γ2. Two isoforms of the catalytic subunit of phosphatase 2A have also 

been identified in mammals: α and β. This study represents an attempt to determine what effect 

the different catalytic subunits of PP1 and PP2A have upon neuronal, and particularly, dendritic 

and axonal development. Hippocampal tissue cells taken from 1-2 day old Sprague Dawley rats 

were transfected with Dicer generated dsiRNA. Knockdown of protein expression was initiated 

four days after initial cell plating at a time when dendritic and axonal processes were still 

forming. This allowed us to evaluate the role played by the catalytic subunits relative to dendritic 

and axonal morphology. Our results indicate that PP2A catalytic isoforms may play a role in the 

regulation of dendritic and axonal length. However, the role played by PP2A catalytic isoforms 

in the regulation of the number of dendritic and axonal branches is uncertain. Our results indicate 

that PP1 regulation of the number of dendritic and axonal branches, and of the length of 

dendrites and axons, is uncertain. 

Disclosures: D. Monroe, None; C.C. Fink , None. 

Poster 





Support: Swiss National science Fonds (SNF) 

Title: Neuronal Nogo-A modulates growth and branching of neurites in the developing nervous 

system

Authors: *B. TEWS 1 , M. PETRINOVIC 2 , C. S. DUNCAN 2 , D. BOURIKAS 3 , O. 

WEINMANN 2 , A. GHOSH 2 , E. T. STOECKLI 3 , M. E. SCHWAB 2 ; 

1 Brain Res. Inst., ETH / Univ. Zürich, CH-8057 Zürich, Switzerland; 2 Brain Res. Inst., ETH / 

University Zürich, Switzerland; 3 Inst. of Zoology, University of Zurich, Switzerland 

Abstract: A striking feature of mammalian neurons is the complexity of their axonal and 

dendritic trees, by which they transmit, receive, process and integrate inputs and outputs. Shape 

and extent of a neuron‟s neuritic arbors strongly influence its function. Regulation of branching 

and tree size in differentiating neurons is achieved by a multi-step process involving numerous 

signaling molecules, many of which are known positive regulators. More recently, crucial roles 

of repulsive/ inhibitory interactions are coming to light. We concentrated on the neurite 

outgrowth-inhibitor Nogo-A which is enriched in CNS myelin where it restricts the capacity of 

the mature mammalian CNS to regenerate after injury. During development, Nogo-A expression 

occurs mainly in neurons where its function is currently unknown. 

We show that neutralization of neuronal Nogo-A leads to increased neurite outgrowth, increased 

fasciculation, and decreased branching in cultured dorsal root ganglion (DRG) and hippocampal 

neurons. Interestingly, very similar effects were obtained by treatments of the cultures with 

antibodies against NgR or ROCK inhibitor, which points to signaling via the NogoA receptorcomplex 

Rho/ROCK pathway. Nogo-A is unevenly distributed on the surface of dissociated 

neurons with specific enrichment on growth cones and at branch points. In vivo in the chicken 

embryo, injection of antibodies against Nogo-A leads to highly bundled peripheral nerves with 

reduced branching. Thus, Nogo-A acts as a negative regulator of neurite-neurite adhesion and as 

a positive regulator of branching in the developing nervous system. 

Disclosures: B. Tews, None; M. Petrinovic, None; C.S. Duncan, None; D. Bourikas, None; O. 

Weinmann, None; A. Ghosh, None; E.T. Stoeckli, None; M.E. Schwab, None. 

Poster 





Support: Carnegie Mellon University SURG program 

Howard Hughes Medical Institute 

Alfred P. Sloan Research Fellow

Searle Scholar Award 

Dana Program in Brain and Immuno Imaging 

Title: Protein kinase C activation by bryostatin increases spine density in hippocampal area CA1 

and neocortex in an activity independent manner 

Authors: *I. A. OLDENBURG 1 , J. C. CROWLEY 1,2,3 ; 

1 Biol. Sci., 2 Ctr. for the Neural Basis of Cognition, 3 Lane Ctr. for Computat. Biol., Carnegie 

Mellon Univ., Pittsburgh, PA 

Abstract: Memory encoding involves complex molecular processes and can result in physical 

changes in neurons; examples of this are changes to dendritic spine shape or number. In addition, 

changes in the activity level of neurons are correlated with changes in spine growth. It has been 

shown that activation of protein kinase C, PKC, can enhance the longevity and encoding rate of 

new memories. There is evidence to suggest that PKC can duplicate many of the anatomical 

correlates of learning in vitro. The compound bryostatin is a known activator of classical and 

atypical isoforms of PKC, primarily α, δ and ε. We track the change in the number of spines of 

individual GFP labeled neurons after two-hour incubations with bryostatin in pair wise 

comparisons across experimental groups of organotypic brain slices from juvenile mice. We find 

that bryostatin treatment increases spine density by 40% in hippocampal area CA1 and by 30% 

in neocortex. Blockade of GABA signaling augments the bryostatin effect by an additional 40% 

increase in spine density. Surprisingly, this process is independent of activity blockade with 

tetrodotoxin, TTX, (i.e. the bryostatin effect is unchanged). We have also found that bryostatin 

treatment in vivo has a similar effect after a seven day treatment paradigm modeled on the work 

of (Hongpaisan and Alkon, 2007). These preliminary data suggest a more reliable effect of 

bryostatin in CA1 than in neocortex. Thus, we see that PKC activation by bryostatin increases 

the density of spines in hippocampus in vivo and in vitro, by a mechanism that is complimentary 

to, but not dependent on neural activity levels. 

Disclosures: I.A. Oldenburg, None; J.C. Crowley, None. 

Poster 





Title: Interaction of very-KIND RasGEF with MAP2 regulates dendrite growth

Authors: *T. FURUICHI, J. HUANG, A. FURUYA, Y. SHINODA; 

Lab. for Mol. Neurogenesis, RIKEN Brain Sci. Inst., Wako, Japan 

Abstract: The kinase non-catalytic C-lobe domain (KIND) is a putative protein-protein 

interaction module identified in three proteins; the actin-nucleation factor Spir, non-receptor-type 

protein tyrosine phosphatase 13 (PTPN13), and the brain-specific Ras guanine nucleotide 

exchange factor (RasGEF) very-KIND (v-KIND). However, the function of the KIND domain 

has been unclear. v-KIND is unique in that it contains two tandemly-repeated KIND domains, 

KIND1 and KIND2 (see Figure). We previously showed that v-KIND is colocalized with 

microtubule-associated protein 2 (MAP2) in the dendrites of hippocampal neurons and cerebellar 

granule cells, and is involved in dendrite growth and branching by regulating the activation of 

Ras small GTPase, which probably leads to the threonine (Thr) phosphorylation of MAP2. 

Overexpression of v-KIND suppresses dendritic extension and branching of hippocampal 

neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression by 

RNAi promotes dendrite growth. In this study, to understand the role of v-KIND-Ras-MAP2 

signaling in microtubule-mediated dendrite growth, we analyzed the structural determinants and 

functional properties of the v-KIND-MAP2 interaction. Among four KIND domains that we 

tested, only the v-KIND KIND2 domain possesses MAP2 binding activity. Our study reveals the 

modules involved in the interaction between v-KIND KIND2 domain and MAP2, which confers 

the ability to localize v-KIND to dendrites where MAP2 is associated with microtubules. These 

results indicate that the interaction of v-KIND with MAP2 via the KIND2 domain is essential for 

the initial step of v-KIND-Ras-MAP2 signaling. (Figure: A model of the v-KIND-Ras-MAP2 

signaling in dendrite growth) 

Disclosures: T. Furuichi , None; J. Huang, None; A. Furuya, None; Y. Shinoda, None.

Poster 





Title: Vertebrate dpy-19 homologue, mdpy-19-1, regulates neuronal morphology 

Authors: *D. DELAUNAY 1 , L. A. KING 2 , A. FENSTERMAKER 1 , Y. ZOU 1 ; 

1 UCSD, San Diego, CA; 2 Univ. of Chicago, chicago, IL 

Abstract: In Caenorhabditis elegans, the dpy-19 gene is required for the appropriate polarization 

of Q neuroblasts, hallmarked by a cellular process pointing towards the direction of their 

subsequent migration along the anterior-posterior axis (Honigberg and Kenyon, 2000). We 

cloned three mouse homologs of C. elegans dpy-19 gene (mdpy-19-1, -2 and -3) and focused our 

studies on mdpy-19-1, the most abundant one in the embryonic cortex. Mdpy-19-1 encodes a 680 

amino acid protein with multiple transmembrane domains and is expressed in the cortical plate 

during the neurogenic period. By immunostaining in dissociated neurons and sections of 

embryonic brain, we show that mDPY-19-1 is enriched in a perinuclear region in cortical 

neurons that partially overlaps with the endoplasmic reticulum and the microtubule cytoskeleton. 

When overexpressed into COS7 cells, mdpy-19-1 changed the cell morphology. COS7 cells 

acquire thin and elongated processes. Conversely, preliminary data indicate that mdpy-19-1 

downregulation by RNAi in vitro reduces the length and arborization of the neuronal processes 

in cultured cortical neurons. Together, these data suggest that the vertebrate mDPY-19-1 protein 

may play a conserved role in cell polarization for proper differentiation and/or maintenance of 

cortical neurites. 

Disclosures: D. Delaunay, None; L.A. King, None; A. Fenstermaker, None; Y. Zou, None. 

Poster 





Support: CIHR Grant MOP 62738 

Title: The role of a novel calcium binding protein in neurite outgrowth and regeneration in 

central neurons of lymnaea 

Authors: *N. NEJATBAKHSH 1 , K. HUI 1 , I. CARDONA 1 , C. GUO 1 , R. VAN KESTEREN 2 , 

Z.-P. FENG 1 ; 

1 Physiol., Univ. Toronto, Toronto, ON, Canada; 2 Dept. of Mol. & Cell. Neurobio., Vrije Univ., 

Amsterdam, Netherlands 

Abstract: The transduction of intracellular signalling by calcium binding proteins implicates 

these proteins in a wide range of intracellular functions, including neurodevelopment and 

neurogeneration. Intracellular localization of these proteins has yielded crucial information 

regarding their function. Localization of protein transcripts of a novel calcium binding protein 

(LCaBP) in neurites of isolated lymnaea central neurons suggest that this particular calcium 

binding protein may have a direct function in local protein synthesis, leading to neurite 

elongation and eventual synaptogenesis. We hypothesized that down-regulation of the LCaBP 

transcript will lead to a reduction in outgrowth of intact and transected neurites. To test this 

hypothesis, we used a double strand RNA interference approach to effectively knock down the 

expression of the LCaBP gene in Lymnaea stagnalis PeA neurons, and compared the elongation 

and branching properties of transected and intact neurites under the control or LCaBP 

knockdown conditions. We found that the neurite outgrowth behaviour of the transected and 

intacted neurites was markedly reduced after the treatment of LCaBP dsRNA/siRNA, but not 

affected by the control RNA treatment. In transacted neurites, LCaBP treated cells experienced a 

net decrease in their total length, while control treated cells showed a net gain in length over a 

period of 48 hours. These results suggest that local translation of LCaBP to functional protein is 

required for de-novo synthesis of neurite proteins necessary for neurite elongation, and implicate 

a direct role for LCaBP in local protein synthesis. 

Disclosures: N. Nejatbakhsh , None; K. Hui, None; I. Cardona, None; C. Guo, None; R. van 

Kesteren, None; Z. Feng, None. 

Poster 





Support: NIH P20RR017703

NIH EY012190 

OUHSC 

NIH EY10608 

NIH grant EY07751 

Title: Reelin regulates the cell-specific dendritic and synaptic architecture of AII amacrine cells 

in the mouse retina 

Authors: *D. M. SHERRY; 

Cell Biol, Univ. Oklahoma Hlth. Sci. Ctr., Oklahoma City, OK 

Abstract: Dendritic organization is a key characteristic of neuronal type and is critical to 

establishing appropriate synaptic connections and neural function, however, the signals that 

specify cell type-specific dendritic organization and growth remain largely unknown. The 

extracellular signaling protein Reelin can regulate dendritic growth and synapse formation, but 

the role of Reelin in specifying cell type-specific dendritic architecture is unclear. In the retina, 

Reelin is positioned to specifically regulate the bistratified dendritic and synaptic architecture of 

the AII (A2) amacrine cell, which is the only retinal cell type with the machinery for Reelin 

signaling. The role of Reelin in regulating the cell-specific dendritic and synaptic organization of 

the AII cell was examined in the retinas of reeler mutant mice, which lack functional Reelin, and 

heterozygote and wildtype controls by intracellular dye injection. Additional immunolabeling 

studies examined the synaptic organization of A2 cells and their bipolar cell targets in the inner 

plexiform layer (IPL). Dye-injected A2 amacrine cells in wildtype and reeler heterozygote 

retinas showed normal bistratified dendrites, with large lobular dendrites in the distal IPL and 

thin arboreal dendrites in the proximal IPL. A2 cells in the reeler retina, however, showed 

aberrant dendritic architecture with mis-stratification of dendrites at inappropriate depths in the 

IPL and asymmetry of the two dendritic strata. Electrical synapses between A2 cells and ONcone 

bipolar cells and between A2 cells were reduced in the reeler retina, as was Connexin 36 

labeling where these electrical synapses are made and metabolic coupling between A2 cells and 

ON-cone bipolar cells. ON-cone bipolar cells in the reeler retina also showed errors in synaptic 

stratification similar to previously reported errors for rod bipolar cells. Laminar organization of 

other amacrine cell types in the IPL, however, was not disturbed. Thus, Reelin signaling 

contributes to the development of appropriate cell-specific bistratified dendritic architecture and 

synaptic organization of A2 amacrine cells, but is not a global “master” signal for patterning the 

functional organization of the IPL. Reelin signaling may be especially important in regulating 

electrical synapse formation. 

Disclosures: D.M. Sherry , None. 

Poster






Title: The change of dendrite morphology of spinal cord neurons with GluR1 knockdown 

Authors: *L. ZHANG 1 , M. WERNER 2 , J. MOJSILOVIC-PETROVIC 1 , G. XIONG 1 , M. 

HOLLMANN 2 , R. KALB 1 ; 

1 Dept Neurol, Children Hosp. Philadelphia, Univ. Pennsylvania, Philadelphia, PA; 2 Dept. of 

Biochem. I - Receptor Biochem., Ruhr Univ. Bochum, Bochum, Germany 

Abstract: The glutamate receptor subunit GluR1 is likely to play an essential role in motor 

neuron development. GluR1 is expressed abundantly by neonatal motor neurons (as well as 

surrounding interneurons) coincident with active dendrite growth. Although GluR1 expression is 

down regulated in mature motor neurons, reintroduction of GluR1 causes the remodeling of the 

architecture of dendrites of mature motor neurons. We used RNAi to investigate whether the 

actions of GluR1 are solely cell autonomous. We generated RNAi to GluR1 that specifically 

knockdown GluR1 in neurons. By linking this small RNA to the vector peptide Penetratin 1, we 

were able to knock down GluR1 in virtually all neurons in vitro and this led to a marked 

reduction in dendrite size and complexity. Next we engineered this small RNA into pSUPER so 

that only individual transfected neurons will have GluR1 knockdown. In this paradigm, 

knockdown of GluR1 also reduced dendrite size and complexity. These observations suggest that 

GluR1 promotes dendrite growth in a cell autonomous manner. Finally we generated a version of 

GluR1 that resists the knockdown of our RNAi. In cultures transfected with this construct and 

treated with penetratin-linked RNAi, the expression of GluR1 was reduced in all neurons except 

the one we analyzed that express wild type levels of GluR1. Surprisingly, in this paradigm, 

despite expressing GluR1, neurons had reduced dendrite size and complexity. Thus GluR1 does 

not operate in a strictly cell autonomous manner to promote dendrite elaboration. These results 

were also seen in vivo when we analyzed the dendritic tree of motor neurons from GluR1 (aka 

GluR-A) knock out animals as well as animals with a conditional knockout of GluR1 specifically 

in motor neurons These experiments provide insight into how GluR1 plays an important role in 

regulating the normal development of the motor system. 

Disclosures: L. Zhang, None; M. Werner, None; J. Mojsilovic-Petrovic, None; G. Xiong, 

None; R. Kalb, None; M. Hollmann, None. 

Poster





Support: 21st Century Frontier Research Program Grant M103KV010023-07K2201-02510 

Title: Role of inositol 1,4,5-trisphosphate 3 kinase-A in neuronal development 

Authors: S. HONG, I. KIM, B.-I. CHOI, S. HAN, J. MO, I. RHYU, W. SUN, *H. KIM; 

Dept Anat., Korea Univ. Col. Med., Seoul, Republic of Korea 

Abstract: The inositol 1,4,5-trisphosphate 3 kinases (IP3Ks) are known as regulators for the 

maintenance of intracellular calcium homeostasis. Among three IP3K isoforms (IP3K-A, B and 

C), IP3K-A appears to play a significant role in neuronal development, because it exhibits 

developmentally regulated neuron-specific expression. However, the precise molecular function 

of IP3K-A in neuronal development is currently unknown. 

In this study, we investigated the developmental expression pattern of IP3K-A in the rat brain 

with in situ hybridization histochemistry, and the roles of IP3K-A in neuronal development with 

gene perturbation experiments. 

The expression of IP3K-A was observed from neonate and gradually increased during the 

postnatal brain development. Spatially, strong expression was found in the hippocampal 

formation, cerebral cortex, caudate putamen and cerebellum. Over-expression of IP3K-A in 

primary cultured hippocampal and cerebral cortical neurons increased the number and length of 

dendritic protrusions compared to the control neurons. Conversely, suppression of IP3K-A with 

siRNA in primary cultured hippocampal neurons retarded the development of dendritic 

protrusions. Similar deficits in the dendritic spine development were also found in the 

hippocampus of IP3K-A knockout mice. 

These results suggest that IP3K-A plays a significant role in the development of dendritic spines. 

Disclosures: S. Hong, None; I. Kim, None; B. Choi, None; S. Han, None; J. Mo, None; I. 

Rhyu, None; W. Sun, None; H. Kim , None. 

Poster 





Support: NSF SOMAS Grant DUE-0426266 

Lycoming College Professional Development Grant 

Title: Purkinje neuron survival and dendrite structure: MAP and PI-3 kinase pathway 

contributions 

Authors: *M. E. MORRISON, A. C. CAMPBELL; 

Biol., Lycoming Col., Williamsport, PA 

Abstract: Purkinje neurons are the major information processing neurons in the cerebellum; they 

are responsible for coordination of locomotion, balance, and smooth eye movements. Granule 

neurons, the presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell 

differentiation. Purified cultures of Purkinje cells survive poorly, never developing mature 

dendrites, while Purkinje cells cocultured with granule neurons survive better and develop 

dendrites studded with synapse-bearing spines. The molecular conversation between the granule 

and Purkinje neurons is the main focus of our lab. 

BDNF/TrkB signaling regulates Purkinje cell spine number and morphology. We present 

experiments designed to elucidate the signal transduction steps that link BDNF/TrkB signaling to 

changes in neuronal morphology and function, using the MAP kinase pathway inhibitor 

PD98059 and the PI3-kinase pathway inhibitor LY294002. Preliminary data indicate that 

inhibiting either pathway decreases the total cell area (soma + dendrites), without altering the 

cell soma size. Inhibiting MAP kinase signaling decreases the length of the longest dendrite, 

while inhibiting PI3-kinase signaling does not. These data, taken together, reveal that MAP 

kinase pathway inhibition produces shorter and/or less branched dendrites, while PI3-kinase 

inhibition affects total dendrite outgrowth. We will further assess the relative contributions of 

these pathways to the survival and development of Purkinje cell dendrites in cultures of mixed 

cerebellar cells and cocultures of purified granule and Purkinje neurons. 

Disclosures: M.E. Morrison , None; A.C. Campbell, None. 

Poster 





Title: CaMKIIδ promotes axonal and dendritic development in hippocampal neurons 

Authors: *V. A. GLODOWSKI, J. MCDERMOTT, C. FINK; 

Biol. Sci., Univ. Wisconsin-Milwaukee, Milwaukee, WI 

Abstract: CaMKII is a protein serine/threonine kinase that is the most abundant protein in the 

hippocampus comprising up to 2% of hippocampal protein. There are four isoforms of CaMKII 

which are found in mammals: α, β, γ and δ. The catalytic and regulatory domains are highly 

homologous between the four isoforms. However, the association domain contains variable 

inserts which affect the ability of CaMKII isoforms to interact with different substrates. Previous 

studies have shown that CaMKII isoforms α and β play a role in the development of filopodia 

and in spine formation. Furthermore, CaMKIIδ has been shown to regulate neuronal morphology 

due to its ability to bind actin. This project explores whether CaMKIIδ, which binds to actin, also 

plays a role in neuronal development, specifically in axonal and dendritic development. Using 

cell cultures of hippocampal tissue taken from 1-2 day postnatal Sprague-Dawley rats and a 

Dicer RNAi system, we reduced protein levels of the CaMKIIδ isoform. The length of the 

primary axonal process, axonal branches and dendritic processes were significantly decreased in 

the δ isoform. These results suggest that the CaMKIIδ isoform may play a role in neuronal 

development. 

Disclosures: V.A. Glodowski, None; J. McDermott, None; C. Fink, None. 

Poster 





Support: Grant-in-Aid for Scientific Research (B) #19300119 form the Ministry of Education, 

Science, Sports and Technology of Japan 

Science 

Grant-in-Aid for JSPS Fellows #19-3007 from Japan Society for the Promotion of 

Title: Developmental gene expression of the CaMK cascade in hippocampus and its roles in the 

dendritic development of hippocampal neurons 

Authors: *A. KAMATA 1,2 , K. FUKUNAGA 2 , H. KONDO 3 , H. SAKAGAMI 1 ; 

1 Dept. of Anat., Kitasato Univ. Grad. Sch. of Med., Sagamihara, Japan; 2 Dept. of Pharmacol.,

Tohoku Univ. Grad. Sch. of Pharmaceut. Sci., Sendai, Japan; 3 Dept. of Rehabil., Tohoku Bunka 

Gakuen Univ. Sch. of Med. Sci. & Welfare, Sedai, Japan 

Abstract: The Ca 2+ /calmodulin-dependent protein kinase (CaMK) cascade, consisting of 

CaMKI, CaMKIV and CaMK kinase (CaMKK), is an evolutionally conserved Ca 2+ -triggered 

signal transduction pathway that plays important roles for a variety of neuronal functions 

including gene expression and synaptic plasticity. We have previously demonstrated distinct 

gene expression of CaMKI, CaMKIV and CaMKK in the adult brain. In this study, to examine 

the roles of this cascade in the hippocampal development, we first examined the gene expression 

of each isoform of CaMKI, CaMKIV and CaMKK in the developing mouse hippocampus by in 

situ hybridization histochemistry. In the adult hippocampus, CaMKIβ2 and δ are expressed at a 

higher level than CaMKIα and γ. During the hippocampal development, the expression of 

CaMKIβ2 mRNA in the dentate granule cell layer was progressively increased, whereas its 

expression in the CA1-3 pyramidal cell layers was gradually decreased during the postnatal 

development. CaMKIδ mRNA showed prominent expression in the CA1-3 pyramidal cell layers 

already at embryonic day 18 (E18) with a progressive increase in its expression during the 

postnatal development. CaMKIα and γ mRNA showed moderate to weak expression throughout 

the hippocampal development. The expression of CaMKIV mRNA was already prominent in the 

CA1-3 pyramidal cell layers at E18 and gradually increased with a peak around postnatal day 15. 

The expression of CaMKKα mRNA was almost negligible during embryonic development and 

gradually increased in the dentate granule cell layer after birth. CaMKKβ mRNA was already 

expressed abundantly in the hippocampus at E15 and progressively increased in the CA1-3 

pyramidal cell layers during the postnatal development. Finally, to examine the roles in the 

dendritic development, we overexpresssed the kinase-dead mutants of CaMKI, CaMKIV and 

CaMKK in primary hippocampal neurons. The overexpression of a kinase-dead mutant of 

CaMKI reduced the average dendritic length without any significant changes in the number of 

dendrites. In contrast, the overexpression of a kinase-dead mutant of CaMKIV and CaMKK 

resulted in decreased number of dendrites without any effects on the average dendritic length. 

STO-609, an inhibitor of CaMKK, treatment reduced the number of dendrites without any 

effects on the average dendritic length. All these findings suggest that CaMKI and CaMKIV are 

involved in distinct steps of dendritic formation of hippocampal neurons, namely dendritic 

extension and initiation, respectively. 

Disclosures: A. Kamata, None; K. Fukunaga, None; H. Kondo, None; H. Sakagami, None. 

Poster 





Support: NIH Grant EY016031 

Title: DSCAMs in the development of the mouse retina 

Authors: *R. W. BURGESS 1 , P. G. FUERST 2 , F. BRUCE 3 , A. KOIZUMI 4 , L. ERSKINE 3 , R. 

H. MASLAND 5 ; 

1 Res., The Jackson Lab., Bar Harbor, ME; 2 Jackson Lab., Bar Harbor, ME; 3 Inst. of Med. Sci., 

Univ. of Aberdeen, Aberdeen, United Kingdom; 4 Natl. Insistute for Physiological Sci., Okazaki, 

Japan; 5 Massachusetts Gen. Hosp., Boston, MA 

Abstract: Retinal neurons establish their spatial domains by arborizing their processes, which 

requires the self-avoidance of neurites from an individual cell, and by spacing their cell bodies, 

which requires positioning the soma and establishing a zone within which other cells of the same 

type are excluded. The mosaic patterns of distinct cell types form independently and can overlap. 

Therefore, there must be unique identifiers for each cell type that mediate these self-recognition 

and avoidance events. These molecular signals are now being identified in the vertebrate retina. 

We have shown that DSCAM in the mouse retina is required for some types of retinal amacrine 

cells to arborize their processes and to maintain the spacing of their cell bodies. In the mutant 

retina, cells that would normally express Dscam have hyperfasciculated processes, preventing 

them from creating an orderly arbor. The cell bodies of these neurons are randomly distributed or 

pulled into clumps rather than being spaced in regular mosaics. Retinal ganglion cells in the 

Dscam -/- mice also show a dendrite arborization phenotype, and cell bodies are abnormally 

spaced even at very early ages. Most axonal projections are initially normal, but extensive axonal 

remodeling occurs after eye opening. The ganglion cell expression of Dscam and the mutant 

phenotype in these cells is more complicated than in amacrine cells, and suggest that Dscam may 

function at multiple stages of ganglion cell development. Other retinal cell populations express 

related genes that may also function as recognition molecules. Dscam-like1 is expressed in a 

non-overlapping subset of amacrine cells and ON-bipolar neurons. In mice lacking Dscam-like1, 

there is a disorganization in these cell populations that is preliminarily similar to the defects 

observed in Dscam -/- retinas. Our results indicate that mouse DSCAMs mediate isoneuronal selfavoidance 

for arborization, and heteroneuronal self-avoidance within specific cell types to 

prevent fasciculation and to preserve mosaic spacing. These functions are highly analogous to 

those of Drosophila Dscam and Dscam2, are necessary for establishing orderly retinal circuitry, 

and suggest mammalian DSCAMs may serve similar roles in other regions of the central nervous 

system. 

Disclosures: R.W. Burgess, None; P.G. Fuerst, None; F. Bruce, None; A. Koizumi, None; L. 

Erskine, None; R.H. Masland, None. 

Poster





Title: Lutein enhances neurite outgrowth of rat primary hippocampal neurons 

Authors: *B. M. BERG 1 , W. STEWART 2 , B. MCILVAIN 2 , J. MOORE 2 , J. CHEN 2 , R. 

MARTONE 2 , J. DUNLOP 2 ; 

1 Nutr., Wyeth Pharmaceuticals, Collegeville, PA; 2 Discovery Neurosci., Wyeth Pharmaceuticals, 

Princeton, NJ 

Abstract: Providing optimal nutrition during the first months of life is an important factor in the 

development of mammalian organ systems, especially the brain. Lutein is an oxygenated, 

lipophillic carotenoid, or xanthophyll, present in breast milk throughout the world and recently 

included in commercial infant formula. Lutein is highly concentrated in the macula of the human 

retina, and has been quantified in human frontal cortex. However, the potential role of lutein in 

neurodevelopmental mechanisms has largely been unexplored. Lutein and the pharmacological 

positive control, β-estradiol, were compared for their ability to influence a fundamental process 

for normal neurodevelopment: the outgrowth of neurites from the neuronal cell body. 

Hippocampal neurons harvested from embryonic day 18 rats were exposed to 10 concentrations 

of each treatment and grown in culture for 2 and 7 days. The neurons were then stained and 

analyzed using the neuronal profiling bio-application on the Cellomics Arrayscan. 

Multiparametric image analysis of four independent assays revealed that the average neurite 

length of 6.3 uM lutein treated neurons was 1.3-fold higher (± 0.15 SEM) than vehicle treated 

neurons 2 days after treatment, a magnitude similiar to that induced by 3 uM β-estradiol (1.2 

fold, ± 0.29). Importantly, after 7 days of treatment the average neurite length of 2.1 and 6.3 uM 

lutein treated neurons was 2-fold higher (± 0.43 SEM) than vehicle treated neurons compared to 

the positive control β-estradiol (0.4 and 1.1 uM) which produced a 1.2 fold increase (± 0.1 

SEM). In a separate single experiment, 16.7 uM lutein caused a 3-fold increase in neurite length 

of N2A neuroblastoma cells after 4 days of treatment. These data suggest lutein could potentially 

play an important role in growth and organization of neural networks early in life. 

Disclosures: B.M. Berg, Wyeth Nutrition, A. Employment (full or part-time); W. Stewart, 

Wyeth Discovery Neuroscience, A. Employment (full or part-time); B. McIlvain, Wyeth 

Discovery Neuroscience, A. Employment (full or part-time); J. Moore, Wyeth Discovery 

Neuroscience, A. Employment (full or part-time); J. Chen, Wyeth Discovery Neuroscience, A. 

Employment (full or part-time); R. Martone, Wyeth Discovery Neuroscience, A. Employment 

(full or part-time); J. Dunlop, Wyeth Discovery Neuroscience, A. Employment (full or parttime).

Poster 





Support: Scientific Reseach from the Ministry of Education, Science, Sports, and Culture of 

Japan 

Title: The Cdc42 activator Zizimin1/Dock9 regulates dendritic development in hippocampal 

neurons 

Authors: *K. KURAMOTO, M. NEGISHI, H. KATOH; 

Grad Sch. Biostudies, Kyoto Univ., Kyoto, Japan 

Abstract: Rho family small GTPases are key regulators for morphological changes of various 

cells by regulating cytoskeleton. They serve as molecular switches between GDP-bound 

inactivated state and GTP-bound activated state. Activation of Rho family small GTPases 

requires GDP-GTP exchange catalyzed by guanine nucleotide exchange factors (GEFs). Cdc42, 

one of the most characterized members of Rho family small GTPases, is involved in actin 

cytoskeletal reorganization in axonal and dendritic outgrowth. Recent studies have identified 

Zizimin1, a member of Dock180-related protein family, as a Cdc42-spicific GEF. However, the 

physiological function of Zizimin1 is totally unknown. Here we studied expression pattern and 

neuronal function of Zizimin1 in developing rodent brains. In situ hybridization and western 

blotting analysis showed that Zizimin1 is strongly expressed in developing brain including 

hippocampus and cerebral cortex in late developmental stages. We investigated the function of 

Zizimin1 using primary cultured rat hippocampal neurons. Expression of Zizimin1 in cultured 

hippocampal neurons increased in the dendrite developing stage, and knockdown of Zizimin1 by 

shRNA reduced the dendrite length. In addition, expression of Zizimin1-LNP, a mutant lacking 

GEF activity, also suppressed dendritic development. On the other hand, Zizimin1 mutants that 

deleted CZH1 domain, conserved domain in Dock180-related protein family, or PH domain, 

both of which are reported to bind phosphoinositides, could not localize in membrane fraction. 

Zizimin1-LNP mutants that deleted one of these domains had no effects on the dendritic 

development. These results suggest that Zizimin1 plays an important role in dendritic 

development through the regulation of Cdc42 activity and N-terminal mediated membrane 

localization. 

Disclosures: K. Kuramoto, None; M. Negishi, None; H. Katoh, None.

Poster 





Support: NJ Governor's Council on Autism Pilot Grant 

NIH Training Grant T32MH/AG-19957 

March of Dimes Foundation Grant 1-FY04-107 (to B.L.F.) 

NSF Grant IBN-0234206 

Title: A biochemical analysis of the interaction between cypin and microtubules 

Authors: *E. SWEET 1 , M. L. PREVITERA 2 , B. L. FIRESTEIN 2 ; 

1 Cell Biol. and Neurosci., 2 Mol. Biosci., Rutgers Univ., Piscataway, NJ 

Abstract: Cypin (cytosolic PSD-95 interactor) is a guanine deaminase that contains a CRMP 

homology domain and is expressed in neurons. We previously reported that overexpression of 

cypin protein increases dendrite branching in primary cultures of hippocampal neurons. In 

addition, we have shown that cypin binds to tubulin heterodimers via cypin's CRMP homology 

domain. We now show that cypin binds to tubulin heterodimers in at least a 2:1 ratio. Cypin also 

binds to polymerized microtubules. Cypin's binding to tubulin heterodimers and polymerized 

microtubules suggests that cypin increases microtubule polymerization by aiding in the addition 

of tubulin to the + end of polymerizing microtubules. The addition of tubulin increases the Km 

and Vmax of polymerizing microtubules, thus encouraging nascent outgrowths to grow into full 

dendrites. 

Disclosures: E. Sweet, None; M.L. Previtera, None; B.L. Firestein, None. 

Poster 





Support: NIH grants (JC, MML and DRC) 

Research to Prevent Blindness (DRC and MML) 

Knights Templar Research Grant (XL) 

Research to Prevent Blindness (DRC and MML) 

That Man May See (DRC) 

Title: Neurotrophin-3 regulates early neonatal development of retinal ganglion cell dendrites 

Authors: *X. LIU 1,2,3 , M. L. ROBINSON 6 , A. M. SCHREIBER 2 , V. WU 2 , M. M. LAVAIL 2,3,4 , 

J. CANG 1 , D. R. COPENHAGEN 2,3,5 ; 

1 Neurobio. and Physiol., Northwestern Univ., Evanston, IL; 2 Ophthalmology, 3 Program in 

Neurosci., 4 Anat., 5 Physiol., Univ. of California San Francisco, San Francisco, CA; 6 Zoology, 

Miami Univ., Oxford, OH 

Abstract: Purpose: To investigate the role of Neurotrophin-3 (NT-3) in the maturation of neural 

retina in mouse. Methods: Transgenic mice expressing NT-3 driven by the αA-crystallin 

promoter in the lens were crossed with Thy-1-YFP transgenic mice. Confocal Z-stack images of 

YFP-expressing retinal ganglion cells (RGCs) were collected to examine their dendritic 

structure. Results: During postnatal development, the initially diffuse dendritic arbors of RGCs 

are refined into mono-laminated structures in the inner plexiform layer (IPL). The progressive 

refinement of RGCs starts before eye-opening and continues for two weeks after eye-opening. 

Overexpression of NT-3 in the eye accelerates RGC laminar refinement before eye-opening. 

Furthermore, the dendritic arbors of ON-OFF RGCs expand mainly before eye-opening, and NT- 

3 overexpression increases dendritic branching but inhibits dendritic elongation in ON-OFF 

RGCs. In contrast, dendrites of ON RGCs expand both before and after eye-opening, and NT-3 

overexpression does not affect dendritic maturation in these cells. In comparison, brain-derived 

neurotrophic factor (BDNF) overexpression facilitates the formation of new branches in ON but 

not ON-OFF RGCs. Taken together, our results suggest that NT-3 and BDNF play overlapping 

roles in laminar refinement and distinct roles in dendritic arborization during RGC postnatal 

development. 

Disclosures: X. Liu , None; M.L. Robinson, None; A.M. Schreiber, None; V. Wu, 

None; M.M. LaVail, None; J. Cang, None; D.R. Copenhagen, None. 

Poster 

231. Dendrite Growth and Branching: Signaling




Support: Fundacio La Caixa 2005x1045 

MEC(Spain) BFU2007-63033 

Title: NGF, Amyloid beta and dendrogenesis 

Authors: *A. RODRIGUEZ-TEBAR 1 , P. J. CHACON 1 , P. M. ROLDAN 1 , A. AREVALO 2 ; 

1 Dept Cell Signalling, CABIMER/CSIC, Seville, Spain; 2 Inst. Cajal, CSIC, Madrid, Spain 

Abstract: In cultured hippocampal neurons, we have studied the transduction pathway used by 

NGF to promote dendrite growth and GABAergic synaptic input upon binding to p75 NTR . We 

have also investigated how Amyloid beta, one of the main agents in Alzheimer‟s disease and a 

peptide known to oppose the effects of NGF, blocks the signalling induced by NGF. The cellular 

effects of NGF exposure depend on the expression of Enhancer-of-split 1. Such expression relies 

on the prior activation of NF-kappa-B, as witnessed through the nuclear translocation of p65/NFkappa-B. 

This activation of NF-kappaB requires NGF-induced tyrosine phosphorylation of Ikappa-B, 

which results in its degradation in the cytosol. In our search for tyrosine kinases 

capable of phosphorylating I-kappa-B, we proposed c-ABL kinase to be a likely candidate since 

NGF augments its activity. The tyrosine phosphorylation of I-kappa-B induced by NGF was also 

dependent on protein tyrosine phosphatase 1B, although its relationship with c-ABL remains to 

be clarified. However, pharmacological inhibition of c-ABL impaired NGF-induced NF-kappaB 

activation and NGF-induced expression of Enhancer-of split 1. In addition, pharmacological 

inhibition of c-ABL reduced cell viability and was associated with the production of shorter 

dendrites and a decrease in GABAergic input, effects similar to those observed when cultured 

neurons are exposed to Amyloid beta. Taken together, our results stress the importance of the 

correct activation of NF-kappa-B for neuron survival, dendrite growth and the establishment of 

GABAergic connectivity. Hyperactivation of this factor leads to inflammation and oncogenic 

transformation, while deficient activation may lead to neurodegeneration. Hence we are 

presently trying to quantitatively manipulate NF-kappa-B activation to see whether this can 

overcome the effects of Amyloid beta, increasing neuron survival and restoring dendrite 

patterning, as well as GABAergic connectivity. 

Disclosures: A. Rodriguez-Tebar, None; P.J. Chacon, None; P.M. Roldan, None; A. 

Arevalo, None. 

Poster





Support: Supported by DFG GRK-736. 

Title: Survival Promoting Peptide/ Y-P30 enhances expression of Syndecan-3 and axon growth 

but does not influence the dendrite morphology 

Authors: *S. DASH-WAGH 1 , P. LANDGRAF 2 , M. KREUTZ 2 , H.-C. PAPE 3 , P. WAHLE 1 ; 

1 Developmental Neurobio., Ruhr Univ., Bochum, Germany; 2 Leibniz-Institut für Neurobiologie, 

39118 Madgeburg, Germany; 3 Inst. für Physiologie I, Westfälische Wilhelms-Universität,, 48149 

Münster, Germany 

Abstract: Survival Promoting Peptide/ Y-P30 has been shown to promote the survival of 

thalamic neurons, and neurite outgrowth from thalamocortical and cerebellar microcultures 

(Landgraf et al., FASEB J. 2005). It is a small peptide of 30 amino acids. It is produced by 

peripheral blood mononuclear cells of pregnant rats and humans. It is imported to the brain of 

perinatal rat and humans where it enriches in cortical neurons. The present study focused to find 

the molecular mechanism and characterize the effects of Y-P30 on dendritic differentiation and 

axon growth. 

The microarray analysis of the cortical organotypic cultures (OTC) treated with exogenous Y- 

P30 revealed altered expression of some genes which were later also confirmed with the RT- 

PCR. One of these genes is Syndecan-3 which is known to promote neurite growth of 

hippocampal and cortical neurons. Syndecan-3 has recently been discovered as binding partner 

for Y-P30 and interaction of Y-P30 with syndecan-3 also enhances neurite out growth (Abstract; 

Landgraf et al., 5th FENS; 2006). Y-P30 also activates ERK signaling via MEK1/2. 

For investigating the effect of Y-P30 on dendritic differentiation we analysed pyramidal neurons 

and interneurons from 10 day in vitro cortical OTCs transfected with eGFP. We observed that 

the exogenous addition of Y-P30 has no affect on dendrites of pyramidal neurons while 

surprisingly the neutralization of endogenous cortical Y-30 with anti-Y-P30 antibodies promotes 

dendritic elongation and branching. The morphology of interneurons is unaltered in either of the 

condition. 

Next we analyze the axon growth, for this we established retinal explant cultures in roller tubes. 

Y-P30 treatment significantly increases the length of retinal ganglion cell axons. In order to 

unravel the molecular mechanism, we analyzed if Y-P30 alters the expression of Syndecan-3. 

Indeed, Y-P30 significantly enhanced the mRNA expression of Syndecan-3 in the retinal 

explants. 

Currently we are analyzing the protein levels of Syndecan-3 in OTCs and involvement of Src 

signaling in axon growth. 

Supported by DFG GRK-736.

Disclosures: S. Dash-Wagh , None; P. Landgraf, None; M. Kreutz, None; H. Pape, None; P. 

Wahle, None. 

Poster 





Support: NIH Grant NS39475 to AJK 

NIH Grant MH77306 to AJK 

Title: Rho inhibition through a β1 integrin-dependent pathway mediates dendritic spine 

morphogenesis and synapse stability in late postnatal hippocampal development 

Authors: *M. WARREN 1 , W. BRADLEY 1 , M. SFAKIANOS 1 , A. EISMAN 1 , S. GOURLEY 1 , 

J. TAYLOR 1 , C. GREER 1 , J. SETTLEMAN 2 , L. REICHARDT 3 , A. WILLIAMSON 1 , A. J. 

KOLESKE 1 ; 

1 Yale Univ., New Haven, CT; 2 MGH Cancer Ctr., Charlestown, MA; 3 Dept. of Physiology, 

UCSF, San Francisco, CA 

Abstract: Rho activation in developing neurons slows dendrite branch dynamics, yielding 

smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes 

dendritic spine loss and dendritic regression. Here we show that β1 integrins act through the Arg 

tyrosine kinase and p190RhoGAP to mediate the inhibition of Rho to promote dendritic spine 

morphogenesis and synapse and dendrite stability. In arg-/- mice, p190RhoGAP activity is 

reduced, leading to increased Rho activity. While hippocampal CA1 pyramidal dendrites develop 

normally in these mice and reach their mature size by postnatal day 21 (P21), their spines do not 

undergo normal morphological maturation and exhibit severe deficits in synaptic efficacy. These 

defects lead to synapse loss and dendritic regression by P42. Coincident with this synapse and 

dendrite loss, arg-/- mice exhibit progressive deficits in hippocampus-dependent behavioral 

tasks. While mice heterozygous for arg and either integrin β1 or p190rhogap display the 

dendritic regression phenotype, reducing gene dosage of the Rho effector ROCKII can suppress 

the dendritic regression observed in arg-/- mice. Together, these data indicate that integrin β1 

signaling through Arg and p190RhoGAP promotes dendritic spine maturation, synaptic function, 

and synapse stability by attenuating synaptic Rho activity.

Disclosures: M. Warren , None; W. Bradley, None; M. Sfakianos, None; A. Eisman, 

None; S. Gourley, None; J. Taylor, None; C. Greer, None; J. Settleman, None; L. Reichardt, 

None; A. Williamson, None; A.J. Koleske, None. 

Poster 

232. Developmental Cell Death: Biological Effects 



Topic: A.06.a. Developmental cell death: Biological effects 

Support: NIH CA77803-03 

Title: Mus Musculus Atp8a1 and Atp8a2 and plasma membrane phosphatidylserine 

translocation in neuronal cells 

Authors: K. LEVANO 1 , T. SOBOCKI 2 , F. JAYMAN 2 , *S. PURKAYASTHA 2 , P. 

BANERJEE 2 ; 

1 Biochem., The Grad. Center, the City Univ. New York, New york, NY; 2 CUNY- COLL 

STATEN, Staten Island, NY 

Abstract: Flippases, also known as aminophospholipid translocases (APLTs), sequester 

phosphatidylserine (PS) to the inner leaflet of the plasma membrane. Biochemical studies have 

shown that APLT is an ATP-dependent, P-type Mg 2+ -ATPase activity. In our quest to elucidate 

the protein or proteins responsible for the plasma membrane APLT activity in neuronal cells, we 

have been studying the roles of two P-type ATPases, Atp8a1 and ATPase IB, in the translocation 

of the fluorescent PS analogue, NBD-PS. Transient overexpression of Atp8a1 in the N18 

neuroblastoma cells causes no change in Km but a 1.7-fold increase in Vmax for the plasma 

membrane APLT activity, but similar overexpression of Atp8a2 does not elicit an increase in 

Vmax. In our earlier studies, similar high levels of overexpression of Atp8a1 in the hippocampal 

neuron-derived HN2 cells yielded elevated, plasma membrane APLT activity. However, stable 

overexpression of Atp8a1 in differentiated HN2 cells does not cause an increase in the plasma 

membrane APLT activity. PS internalization, which is an important property of healthy cells, is 

lost in apoptotic cells causing PS externalization and phagocyte recognition of these cells by PS 

receptor-containing mast cells. Using siRNA and antisense mediated suppression of both Atp8a1 

and Atp8a2 followed by rigorous kinetic analysis, we will test the hypothesis that Atp8a1 and/or 

ATPase 1B contribute toward or modify plasma membrane APLT activity in neuronal cells. 

Disclosures: K. Levano, None; S. Purkayastha , None; P. Banerjee, None; F. Jayman, 

None; T. Sobocki, None.

Poster 





Title: Undernutrition during the gestation and suckling periods does not cause loss of pyramidal 

neurons in the ca2-ca3 region of the rat hippocampus 

Authors: *K. S. BEDI 1 , G. PARTADIREDJA 2 ; 

1 Fac. of Hlth. Sci. and Med., Bond Univ., QLD, Australia; 2 Dept. of Physiology,, Fac. of 

Medicine, Gadjah Mada University,, Gadjah Mada University,, Indonesia 

Abstract: Undernutrition during early life is known to affect the morphology of the hippocampal 

formation including causing deficits in the numbers of dentate gyrus granule cells and neurons 

within the hilus region. Other studies have indicated that the pyramidal cells in the CA2-CA3 

region are relatively spared and their total numbers are unaffected by undernutrition during the 

gestation period. We hypothesised that this may not be the case in animals subjected to a 

lengthier period of undernutrition extending from conception until the end of the suckling period 

at 21 post-natal days-of-age. Wistar rats were undernourished during this period by restricting 

the amount of food given to their pregnant or lactating mothers to about half that eaten by control 

mothers. Rats were then weaned on to an ad libitum diet. Groups of well-fed control and 

undernourished were killed for examination at 21 and 62 days of age. The left hippocampal 

formation from each animal was dissected out and prepared for stereological analysis using 

routine histological procedures. The Cavalieri principle and disector methods were used, 

respectively, to estimate the layer volume and numerical densities of cells in the CA2-CA3 

subfield. These estimates were later used to calculate the total numbers of pyramidal cells in this 

subregion. There were between 180-212 thousand pyramidal cells in the CA2-CA3 region in 

control animals at both 21 and 62 days of age. Twenty-one-day-old undernourished rats had 

about 152 thousand such cells and this increased to about 206 thousand by 62 days-of-age. 

Analysis of variance tests on this data revealed a significant main effect of age but no group or 

interaction effects. Our experiments therefore confirm that these hippocampal pyramidal neurons 

are relatively spared the adverse effects of undernutrition during early life, even when this is 

extended to include both the gestation and suckling periods. 

Disclosures: K.S. Bedi , None; G. Partadiredja, None.

Poster 




Abstract: Withdrawn 

Poster 





Support: NIH/NICHD 

FDA/NCTR E-7189 

ORISE 

NTP/NIEHS 

Title: Genotoxicity of ketamine and potential protection by midazolam in neonatal monkey 

cerebral cortical culture 

Authors: *X. ZOU 1 , N. SADOVOVA 2 , T. PATTERSON 1 , B. DIVINE 2 , M. PAULE 2 , W. 

SLIKKER, WILLIAM 2 , C. WANG 2 ; 

1 Div. Neurotoxicol, 2 Toxicologic Pathology Associates, Natl. Cntr Toxicological Resch, 

Jefferson, AR 

Abstract: Previous data suggest that exposure of rodents to various anesthetics, including those 

that block NMDA glutamate receptors and those that activate GABAA receptors, triggers 

widespread neurodegeneration in the developing brain. Information on genetic damage or 

protection in infant monkeys after exposure to a combination of NMDA antagonists and GABAA 

agonists (i.e., anesthetic agents) is scarce. In this study, the alkaline single cell gel 

electrophoresis (Comet assay) was used to study the potential genotoxic and/or protective 

properties of ketamine (an NMDA antagonist) and midazolam (a GABAA agonist) in cells from

postnatal day 3 (PND-3) monkey cerebral cortical cultures. The Comet assay is commonly used 

to detect DNA strand breaks induced directly by genotoxic agents, as well as, DNA degradation 

due to cell death. In the present study, ketamine-induced DNA strand breaks and alkali-labile 

sites were measured as total Comet length and % tail DNA. The cells in the monkey cortical 

cultures were exposed to either ketamine or midazolam alone, or a combination of the two drugs 

at concentrations of 10 µM for 24 hours. After washout of ketamine and midazolam, cultures 

were kept in serum and glutamate-containing medium, then, processed for Comet assay. A 

remarkable increase in DNA damage (mean tail length and the % tail DNA) was apparent in the 

cultures exposed to ketamine alone, but not in those treated with midazolam alone. However, coadministration 

of midazolam effectively prevented the DNA damage and enhanced neurotoxicity 

induced by ketamine. These data suggest that the increase in DNA migration induced by 

ketamine was a result of cell death-associated DNA degradation. GABA agonists, such as 

midazolam, may reduce the risks associated with the use of NMDA antagonists for anesthesia. 

Disclosures: X. Zou, None; N. Sadovova, None; T. Patterson, None; B. Divine, None; M. 

Paule, None; W. Slikker, William, None; C. Wang, None. 

Poster 





Support: FAPERJ Jovem Cientista do Estado do Rio de Janeiro 

PRONEX 

CNPQ-Edital Universal 

Title: Large numbers of neurons are lost in congenital hypothyroidism in the rat 

Authors: *P. M. RODRIGUES 1 , A. KINUPE 2 , A. VARGAS 2 , S. HERCULANO-HOUZEL 2 ; 

1 Federal univ of Rio de Janeiro, Rio de Janeiro, Brazil; 2 Univ. Federal do Rio de Janeiro, Rio de 

Janeiro, Brazil 

Abstract: Hypothyroidism is a condition in which the body lacks sufficient thyroid hormone, 

causing reduced brain and body mass, delayed migration and cellular differentiation, and altered 

synaptogenesis. Little is know, however, about how hypothyroidism affects the cellular 

composition of the brain. To determine whether the reduced brain mass is due to diminished

numbers of neuronal and non-neuronal cells, we estimated the cellular composition of the brain 

in animals with congenital hypothyroidism. 

Sixty Wistar rats (29 controls and 31 hypothyroid animals) of 1, 2, 3, 4 and 5 months of age were 

euthanized and perfused with 4% paraformaldehyde. Their brains were dissected into five 

regions of interest (cerebral cortex, hippocampus, cerebellum, olfactory bulb and remaining 

areas) which were weighted and analyzed separately with the Isotropic Fractionator. 

Hypothyroidism was induced by ingestion of metimazole 0,002% from the 12th day of gestation 

until the experimental age. 

We find no significant differences in body mass, brain mass, and numbers of cells between 

controls and hypothyroid animals of one month of age. Beginning at 2 months, brain and body 

mass increase more slowly in hypothyroid animals compared to controls, such that, at 5 months, 

body mass is 47% smaller and brain mass 7% smaller in hypothyroids than in controls. In 

contrast to the slight reduction in brain mass, at 5 months we find in all brain structures a striking 

decrease in numbers of neurons compared to controls (cerebral cortex, -58%; hippocampus, - 

67%; cerebellum, 61%; olfactory bulb, -50%; remaining areas, -70%). This reduction is 

progressive and starts at 2 months in the olfactory bulb, at 3 months in the cerebral cortex, 

cerebellum and remaining areas, and at 4 months in the hippocampus. 

Our data indicate that the reduced brain size in hypothyroidism is not due to delayed 

development or generation of abnormal numbers of neurons in the brain, since cellular 

composition is normal at 1 month, but rather to a pronounced loss of neurons that begins at the 

age of 2-3 months. This striking loss of neurons cannot be predicted from the mild tissue loss, 

and suggests that a compensatory increase in neuronal cell size takes place in hypothyroidism. 

Disclosures: P.M. Rodrigues , None; A. Kinupe, None; A. Vargas, None; S. Herculano- 

Houzel, None. 

Poster 





Support: NH&MRC 

Title: Effect of betemethasone treatment on apoptotic cell death and neurosteriodogenic 

pathways in a guinea pig model of growth restriction 

Authors: *R. K. SULLIVAN 1,2 , H. K. PALLISER 1,2 , A. A. MCKENDRY 1,2 , D. M. YATES 1,2 , 

D. W. WALKER 3 , J. J. HIRST 1,2 ;

1 M&BRC and Sch. of Biomed Sc, Univ. Newcastle, Callaghan, Newcastle, Australia; 2 Hunter 

Med. Res. Inst., Newcastle, Australia; 3 Physiol., Monash Univ., Melbourne, Australia 

Abstract: Neurosteroids have potent neuroprotective actions in the fetal and adult brain. We 

have previously shown that the key neurosteroidogenic enzyme, 5Î± reductase (5Î±R), is 

upregulated in response to acute hypoxia in the fetus and the resultant increase in 5Î±R-reduced 

steroids decreases hypoxia-induced apoptotic cell death in the hippocampus. Repeated treatment 

with betamethasone (a synthetic corticosteroid), may suppress levels of 5Î±R, resulting in brain 

injury. 

The aim of this study was to determine the effect of repeated betamethasone treatment on the 

expression of key enzymes in the neurosteroid synthethesis pathway and on the level of apoptotic 

cell death in normal and growth restricted fetal brains. Placental insufficiency in guinea pigs was 

produced by the ablation of uterine artery branches at mid gestation (term 68d) in order to induce 

fetal growth restriction. Sham and growth restriction fetuses were treated with vehicle or 

betamethasone (1mg/kg/day) for 4 days prior to sacrifice (65d). Western blot and real time PCR 

were used to quantify the effects on key neurosteroidogenic enzymes (5Î±R and P450scc) in fetal 

brains, adrenal glands and placenta. Activated caspase-3 immunolabelling in the fetal brains 

were used to assessed levels of apoptotic cell death. Betamethasone treatment reduced 5Î±R 

expression in the placenta by 51% (P=0.029), but not in the brain. Apoptosis significantly 

increased in the cortex, CA1 and dentate regions of the hippocampus with betamethasone 

treatment. There was no additive effect when betamethasone was combined with growth 

restriction on either 5Î±R expression or apoptosis, however there were reductions in the 

expression of glutamate transporter GLAST in glial cells and reductions in microtubule 

associated protein 2 (MAP-2) in neurons in regions of the cortex and hippocampus of growth 

restricted animals. The decrease in 5Î±R in the placenta with betamethasone treatment may 

reduce fetal brain concentrations of neurosteroids. This reduction in fetal brain neurostreroids 

and the concomitant reductions in GLAST expression may impair neuroprotective mechanisms, 

increasing the sensitivity of the fetuses to excitotoxic brain injury. 

Disclosures: R.K. Sullivan, None; H.K. Palliser, None; A.A. McKendry, None; D.W. 

Walker, None; J.J. Hirst, None; D.M. Yates, None. 

Poster 





Support: NIH Grant HD055365

NIH Grant DA05072 


NIH Grant ES012443 

Title: Neonatal dexamethasone exposure produces permanent neuromotor deficits in mice 

Authors: S. MALONEY, S. B. CONYERS, K. K. NOGUCHI, J. W. OLNEY, N. B. FARBER, 

*D. F. WOZNIAK; 

Dept Psychiatry, Washington Univ. Sch. Med., St. Louis, MO 

Abstract: Corticosteroids (CORTs) such as dexamethasone (DEX) or betamethasone are 

administered for up to a month to prematurely born infants as a treatment for chronic lung 

disease. Results of clinical trials have shown that the use CORTs in these infants induces longterm 

deficits in neuromotor function and cognition. We have previously shown at these meetings 

that a single exposure to clinically relevant doses of DEX or other CORTs in the mouse during a 

period corresponding to the human perinatal period produces a dramatic increase in apoptotic 

cell death of neural progenitor cells in the developing cerebellum and permanent decreases in the 

number of cerebellar internal granule layer neurons. To examine possible behavioral correlates 

associated with CORT-induced apoptosis, we injected neonatal mouse pups with 3 mg/kg DEX 

on postnatal days (P) 7, 9, and 11 (to approximate more chronic clinical dosing regimens), and 

evaluated them on several behavioral measures. The results revealed that DEX-treated mice were 

impaired on several neuromotor-based tests. For example, DEX-treated mice displayed 

significantly less rearing (p=0.044) compared to vehicle controls during a 1-h locomotor 

activity/exploration test conducted on P21. This finding suggests dysfunction in the hindlimbs or 

balance disturbances in the DEX-treated mice since the groups did not differ on any other 

activity variable. DEX-treated mice also spent significantly less time on an inverted screen 

compared to control mice (P26), and were significantly impaired on a task involving running on 

a complex activity wheel (P86). The complex wheel contains unevenly spaced rungs or crossbars 

and thus higher-order motor co-ordination skills are required for wheel running. Group 

performance differences were not likely due to alterations in motivation or poor health because 

DEX-treated and control mice traveled similar distances on a standard activity wheel and during 

a 1-week acquisition period on the complex wheel when the mice were run for 1 h/day, 5 days 

/week. However, DEX-treated mice traveled significantly shorter distances on the complex 

wheel during the first post-acquisition week (p=0.045), and during each of two weeks when the 

mice were re-tested 6 weeks later (p=0.019 and 0.004, respectively). The results show that 

neonatal DEX exposure can produce longterm neuromotor deficits, suggesting that CORTinduced 

apoptosis of cerebellar neural progenitor cells might underlie similar deficits in humans. 

Further research using our mouse model for studying relevant mechanisms and developing 

protective strategies may allow for these important clinical agents to be used more safely. 

Disclosures: S. Maloney, None; S.B. Conyers, None; K.K. Noguchi, None; J.W. Olney, 

None; N.B. Farber, None; D.F. Wozniak , None.

Poster 





Support: RO1 MH071376 

Title: Serotonin 1a receptor-mediated signaling in neuroprotection and cell cycle regulation 

Authors: B. RANASINGHE 1 , *P. BANERJEE 2 ; 

1 Ph.D. Program in Biochem., The Grad. Center, the City Univ. New York, New York, NY; 2 Dept 

Chem & Neurosci Prog, City Univ. New York Staten Isla, Staten Island, NY 

Abstract: The early development of the serotonergic system in the brain has caused much 

speculation into the action of serotonin as a mitogenic and protective signal. Among the 15 

known 5-HT receptor subtypes the 5-HT1A receptor expression occurs as early as embryonic day 

14.5 in development and is expressed densely in the thalamus and hippocampus. Studies have 

shown that 5-HT1A receptor activation induces neurogenesis in both the dentate gyrus and 

subventricular zone even in the adult brain. There has been speculation that this increase is a 

result of faster progression through the cell cycle and/or increase in the population of 

proliferating cells. We have shown previously that BrdU-labeled cells occur in clusters in 5- 

HT1A receptor-activated hippocampal slices, supporting the former hypothesis. In our present 

study we have addressed this question in the hippocampal neuron-derived HN2-5 cell line where 

the 5-HT1A receptor is stably expressed. We hope to delineate the signal transduction pathway by 

which the 5-HT1A receptor regulates cell cycle progression. Our preliminary data suggests that 

the 5-HT1A receptor activation induces cell proliferation and cell cycle progression in HN2-5 

cells under adequate serum concentrations while inducing neuronal protection, terminal 

differentiation and cell cycle exit under starving conditions. This observation is in line with our 

previously reported studies that demonstrates the involvement of the 5-HT1A receptor in 

neuroprotection of post-mitotic HN2-5 cells. Using protein arrays specific for cell cycle proteins 

in synchronized cultures we hope to initially identify the downstream targets of 5-HT1A receptor 

activation, under both low and adequate serum concentrations. Consequently we hope to follow 

the expression profile of the targets at the genetic and protein level using RT-PCR/microarray 

and western blot analysis respectively. Further, using inhibitors, dominant negative mutants, and 

siRNA on targets we hope to reveal the significance of these proteins in 5-HT1A-induced cell 

cycle regulation and eventually extend this mechanistic study to cortical hippocampal slices. 

Disclosures: B. Ranasinghe, None; P. Banerjee , None.

Poster 





Support: FDA/NCTR E-7189, NTP/NIEHS and NICHD 

Title: Effects of ketamine on oxidative stress in monkey frontal cortical cultures during 

development 

Authors: *C. WANG 1 , N. SADOVOVA 1 , T. A. PATTERSON 1 , X. ZOU 1 , X. ZHANG 1 , M. M. 

PAULE 1 , J. P. HANIG 3 , W. SLIKKER 2 ; 

1 Divi Neurotoxicol, 2 Natl. Ctr. Toxicol Rese, Jefferson, AR; 3 CDER, Silver Spring,, MD 

Abstract: Previous studies have demonstrated that administration of ketamine, a pediatric 

anesthetic and an N-methyl-D-aspartate (NMDA) antagonist, results in a dose-related increase in 

neurotoxicity that is associated with a compensatory up-regulation of NMDA receptors. This 

study was designed to determine whether ketamine-induced neuronal cell death correlates with 

oxidative DNA damage and altered DNA repair. Frontal cortical cultures obtained from 5-6 day 

old rhesus monkeys were incubated for 24 hrs with 10 µM ketamine or normal culture medium 

(control). After washout of ketamine, cultures were kept in serum and glutamate-containing 

medium for 2, 6 and 24 hrs. In this study, 8-oxoguanine, a modification of guanine caused by 

reaction of endogenous and exogenous reactive oxygen species (ROS) with nucleic acids, was 

used as a biomarker for oxidative DNA damage. Antibodies to human 8-oxoguanine DNA 

glycosylase (hOGG1) an enzyme that catalyzes the removal of 8-oxoguanine through cleavage of 

the DNA-phosphodiester bond, was used to explore the activity of this regulatory DNA repair 

system. Immunostaining data indicate that 8-oxoguanine in both neurons and glia was localized 

primarily in nuclei and mitochondria. Ketamine administration remarkably up-regulated 8oxoguanine 

expression. hOGG1 expression was decreased in a manner that was dependent upon 

time after exposure. Ketamine (10 µM) also caused a marked reduction in immunostaining for 

PSA-NCAM (a surface marker for neurons), a substantial increase in lactate dehydrogenase 

(LDH) release, a significant increase in TUNEL-positive cells, and a reduction in mitochondrial 

metabolism of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Western 

analysis showed that co-administration of NR1 antisense oligonucleotide prevented the synthesis 

of NMDA NR1 subunit protein and effectively blocked the neurotoxic effects induced by 

ketamine. These data suggest that ketamine-induced neuronal loss in the developing monkey is 

probably both apoptotic and necrotic in nature. Ketamine-induced neurotoxic effects are 

associated with an up-regulation of NMDA receptor function. In the developing monkey, ROS

are associated with ketamine-induced oxidative DNA damage as indicated by increased 8oxoguanine 

expression and decreased expression of the DNA repair enzyme hOGG1. 

Disclosures: C. Wang, FDA/NCTR E-7189, NTP/NIEHS and NICHD, B. Research Grant 

(principal investigator, collaborator or consultant and pending grants as well as grants already 

received); N. Sadovova, None; T.A. Patterson, None; X. Zou, None; X. Zhang, None; M.M. 

Paule, None; J.P. Hanig, None; W. Slikker, None. 

Poster 





Support: Merit Award HD37100 


Title: Neonatal phencyclidine exposure induces strain-dependent effects on agonistic behaviors 

in ICR mice 

Authors: *C. M. YUEDE, S. E. MALONEY, C. E. CREELEY, N. B. FARBER, J. W. OLNEY, 

D. F. WOZNIAK; 

Dept Psychiatry, Washington Univ., St. Louis, MO 

Abstract: It is well established that NMDA receptor antagonists induce widespread apoptotic 

neurodegeneration in the developing rodent brain, and that various neuronal populations exhibit 

differential sensitivities to this damage depending on the age of exposure during the early 

neonatal period of synaptogenesis. Thus, exposing rodents to these agents at different times 

during early development may induce a wide range of functional disturbances later in life. We 

previously reported at these meetings (Yuede et al., 2006) that exposing male C57Bl/6J (B6) 

mice to phencyclidine (PCP) on postnatal days 2 and 7 (P2+P7) produced chronic, heightened 

aggression as measured by the resident intruder test, which was likely due to a permanent 

alteration of brain circuitry resulting from PCP-induced neuroapoptosis. Here we report that 

exposing male ICR mice to the same PCP treatment also produced changes in certain agonistic 

behaviors but not on the indices of aggression that were significantly increased in B6 mice. The 

present study included additional male control (deprived) mice that were injected with saline but 

kept from the dam for the same amount of time that the PCP-treated mice were away from their 

mothers (8 h). On P30 the mice were evaluated on a reactivity to handling test and PCP-treated

mice were found to be significantly more reactive compared to normal (suckle) controls 

(p=0.005), while the deprived group was not different from the normal controls. PCP-treated 

mice tended to be more reactive than the deprived group (p=0.09). However, unlike the results 

from the B6-PCP study, no group differences were observed on an aggression index derived 

from the results of the resident intruder test, which was conducted beginning on P70. On the 

other hand, results from the tube test (P85) suggested that the neonatal PCP treatment had an 

effect on dominance. Surprisingly, the deprived group was found to be the least dominant on this 

test in that it had significantly fewer mice with large numbers of "wins" compared to the PCPtreated 

and normal control groups. This finding had relevance for the PCP-treated mice since 

they were also deprived of maternal care for the same period of time as the deprived group, yet 

they still showed control-like levels of dominance, suggesting that PCP treatment led to an 

increase in dominance-related behaviors. No notable effects were observed on the social choice 

(P50) or elevated plus maze tests (P108). In conclusion, the differential strain effects of neonatal 

PCP exposure on aggression and other agonistic behaviors underscores the importance of genetic 

components in determining long-term behavioral consequences of NMDA-antagonist-induced 

neuroapoptosis. 

Disclosures: C.M. Yuede, None; S.E. Maloney, None; C.E. Creeley, None; N.B. Farber, 

None; J.W. Olney, None; D.F. Wozniak, None. 

Poster 





Support: NIAAA Grant R01-AA06069 

NIMH Grant K award for Lars Jarskog 

Title: Acute and long-term prefrontal cortical deficits following post-natal exposure to MK801: 

Linking neurotoxicity and neurodevelopmental disorders 

Authors: *L. G. COLEMAN 1 , L. F. JARSKOG 2 , F. T. CREWS 3 ; 

1 Univ. of NC- Chapel Hill, Chapel Hill, NC; 2 Columbia Univ., New York, NY; 3 Univ. of North 

Carolina at Chapel Hill, Chapel Hill, NC 

Abstract: Introduction: Treatment of rodents with ethanol at post-natal day seven (P7), a 

developmental age that corresponds to the third trimester in humans, can be mimicked by the

NMDA antagonist MK801. This study investigates both acute and long-term cellular and 

behavioral effects of post-natal MK801 in mice. This work shows that acute NMDA antagonism 

during the post-natal critical period of neurodevelopment has lasting consequences on prefrontal 

cortical structure and function. Methods: C57BJ/6 mice at post-natal day seven were treated with 

MK801 (1mg/kg) or saline at times 0, 8, and 16h. Mice were then either: 1.) sacrificed at 24h and 

fixed in 4% paraformaldehyde for 48 hours, followed by vibratome sectioning at 50κM to assess 

the acute effect of MK801 on apoptosis using caspase 3 immunohistochemistry (IHC) or 2.) left 

without further intervention until age P80 for behavioral studies, followed by sacrifice and 

perfusion with 4% paraformaldehyde and vibratome sectioning at 100κM for parvalbumin IHC. 

Behavioral studies were performed in the Neurodevelopmental Research Behavioral 

Measurement Core facility at UNC-CH. 

Results: MK801 caused an increase in activated caspase-3 in the mouse mediodorsal prefrontal 

cortex from 10 to 70 positively stained neurons (p

DGAPA-UNAM (IN223107) 

Title: Effects of potassium channel blockers on apoptotic death of cultured cerebellar granule 

cells 

Authors: *B. HERNANDEZ-ENRIQUEZ 1 , R. ARELLANO OSTOA 2 , J. MORÁN 1 ; 

1 Neurosci., Inst. of Cell Physiol., Mexico City, Mexico; 2 Dept. of Cell. and Mol. Neurobio., 

Neurobio. Inst., Querétaro, Mexico 

Abstract: Cerebellar granule cells (CGC) from 8-day-old undergo apoptosis when they are 

transferred from high potassium (25mM KCl [K+]e, K25) to low potassium (5mM KCl [K+]e, 

K5) or with staurosporine (0.5-1κM, STS). Recent evidences suggest a major role for potassium 

(K+) efflux in apoptotic cell death. In this study we found that CGC treated with K5 or Sts show 

the depletion of intracellular potassium ions (K+) by using the cell permeable form of K+ 

binding benzofuran isophtalate (PBFI-AM). We also observed that NGC treated with K5 or Sts 

showed an early cell shrinkage, which is characteristic of the apoptotic process. In addition, two 

different potassium channel blockers, cesium (Cs+) and tetraethylammonium (TEA+), prevented 

the apoptotic volume decrease induced by K5. Both, Cs+ and TEA+ also markedly decreased the 

caspase activity, nuclear condensation and death induced by K5. Surprisingly, we failed to 

observe anti-apoptotic effect of blockers on NGC when cell death was induced by Sts. These 

data suggest that K+ efflux plays an important role in the activation of the apoptotic death and 

apoptotic volume decrease induced by K5 in CGC. 

Disclosures: B. Hernandez-Enriquez , None; J. Morán, None; R. Arellano Ostoa, None. 

Poster 





Support: CONACyT (47158-Q) 

DGAPA-UNAM (IN223107) 

Title: Apoptosis induced by k5, but not staurosporine is mediated by jnk in cerebellar granule 

neurons

Authors: *Y. RAMIRO 1 , J. MORÁN 2 ; 

1 INSTITUTO DE FISIOLOGIA CELULA, MEXICO, D.F., Mexico; 2 INSTITUTO DE 

FISIOLOGIA CELULAR, Mexico, d.f., Mexico 

Abstract: Apoptotic cell death is an intricate process involving a wide range of signaling 

pathways, including the Jun N-terminal Kinases (JNK), a member of mitogen-activated protein 

kinases (MAPKs). It has been reported that JNK pathway is activated under stress in many type 

of cells. When cerebellar granule neurons (CGN) are cultured in a medium with high potassium 

(K25) during 6-8 days in vitro and then transferred to a medium with low potassium (K5) or 

staurosporine (Sts), most of the cells die after 24-48 hrs by apoptosis. We reported that apoptosis 

induced by K5 and Sts induces an early increase of ROS levels, followed by the activation of the 

apoptotic machinery. In the present study we found that the inhibition of JNK activation with 

SP600125 prevented caspase 3 activation, nuclear condensation and cell death induced by K5. In 

contrast, JNK inhibition did not affect cell death induced by Sts. On the other hand, antioxidants 

increased ASK1 phosphorylation and decreased JNK1/2 and c-Jun activation induced by K5. 

These results suggest that apoptosis induced by K5 is JNK-dependent and mediated by ROS, but 

apoptosis induced by Sts is not dependent of JNK, suggesting that other signaling pathway is 

responsible of cell death induced by Sts. 

Disclosures: Y. Ramiro, None; J. Morán, None. 

Poster 





Support: NIH P20 RR-16460 

Title: Rat cortical subplate comparisons across development and aging 

Authors: *T. J. TEAGUE-ROSS 1 , S. M. LANTZ 1 , C. M. DAVIS 1 , A. L. JAMES 1 , G. N. 

BRATTON 2 , B. CLANCY 1 ; 

1 Biol., 2 Mathematics, Univ. Central Arkansas, Conway, AR 

Abstract: Subplate neurons participate extensively in the development of the mammalian cortex 

and although many undergo apoptosis, a subset remains in adult brains. In conventional models 

of development, the persisting subplate cells are considered simply a remnant population. 

However, these neurons remain active across aging in advantageous positions in and above the

white matter, projecting in a widespread fashion across various functional cortical regions. The 

subplate neural population was reconstructed from serial sections in rat cortex on postnatal days 

(P) 0, P7, P30, 6 months, and 1 year using three-dimensional software (Neurolucida, 

MicroBrightField), generating the first anatomical models of this population from early ages 

through late adulthood. These cells, which stain darkly with Nissl, aggregate below a fasciculus 

of intercortical axons, which itself runs subjacent to cortical layer VI. At all ages, the subplate is 

apparent beginning at the level of the forceps minor of the corpus callosum and ending in visual 

cortex, although the thickness and distribution of this neural population is non-uniform. 

Extensions of the persisting subplate region protrude in a medial/lateral plane, particularly in 

secondary somatosensory cortex. Cell counts, densities, volumes, and areas were collected 

(Stereo Investigator, MicroBrightField) and compared across ages. Preliminary statistical 

analysis (Analysis of Means; Minitab) indicates that despite the profound decrease in subplate 

numbers that occurs across early ages (P0, P7, P30), numbers stabilize and remain constant in the 

mature mammalian brain (P30, 6 months, 1 year; alpha = 0.05). The persisting subplate neurons 

appear to be remarkably resilient across aging; such stability suggests a role in mature cortical 

function. 

Disclosures: T.J. Teague-Ross, None; S.M. Lantz, None; C.M. Davis, None; A.L. James, 

None; G.N. Bratton, None; B. Clancy, None. 

Poster 





Support: RO1ES015700 

RO1AA010114 

Title: Ethanol (ETOH) induced oxidative stress inhibits programmed cell death 4 (PDCD4) 

degradation in primary cortical neurons (PCN) 

Authors: *L. MAHIMAINATHAN 1 , M. L. RATHINAM 2 , S. MUMMIDI 3 , G. I. 

HENDERSON 2 ; 

2 Medicine/GI, 3 Med., 1 UTHSCSA, San Antonio, TX 

Abstract: Chronic consumption of alcohol leads to Fetal Alcoholic Syndrome that is 

characterized by neuronal degeneration. Previous studies in our lab have implicated oxidative

stress as a result of glutathione (GSH) depletion as a mediating factor in neuronal death. 

However, the mechanisms that lead to neuronal death following oxidative stress are poorly 

understood. To date, there have been no reports regarding the role of PDCD4, a translational 

initiation inhibitor on neuronal survival. In this study, we determined the role of oxidative insult 

by ethanol exposure on PDCD4 expression in PCN. ETOH treatment (4.0 mg/ml for 24 hrs) led 

to increased PDCD4 expression as assessed by Western Blotting. To determine the involvement 

of GSH in this increased expression, we used pharmacological GSH depletion using the GSH 

neosynthesis inhibitor buthionine sulfoximine (BSO), and demonstrated that GSH depletion 

primes PDCD4 expression. On the contrary, cysteinyl-glycine (CG), a GSH precursor 

supplement decreased the basal expression of PDCD4 and further CG pretreatment blocked 

ETOH-induced PDCD4 expression suggesting involvement of oxidative trigger in driving the 

PDCD4 expression. Degradation of PDCD4 is a widely reported phenomenon, therefore we 

hypothesized that the effect of ETOH in increasing PDCD4 expression might be due to inhibition 

of PDCD4 degradation. Therefore, we examined the effect of ETOH on PDCD4 protein turnover 

using cycloheximide (CHX). CHX significantly blocked the basal and ETOH-induced PDCD4 

expression. Similar studies were also performed using actinomycin D (ActD), a RNA 

polymerase inhibitor to inhibit transcription in the presence and absence of ETOH. ActD blocked 

ETOH-induced PDCD4 expression suggesting possible transcriptional regulation as well. Taken 

together, our data suggest that the primary mechanism responsible for PDCD4 up regulation 

following oxidative stress induced by ethanol may be through blocking of protein degradation, 

whereas transcriptional mechanisms may have a secondary effect. Further studies are in progress 

to investigate the signaling mechanisms involved in this regulation and the role of PDCD4 in 

neuronal survival. 

Disclosures: L. Mahimainathan , None; M.L. Rathinam, None; S. Mummidi, None; G.I. 

Henderson, None. 

Poster 





Support: Mr Gabriel Perez 

CONACYT, 

Doctoral Program of Biological Sciences UAM

DGAPA IN-214607-3 

Title: Rotenone-induced nigrostriatal dopaminergic neurons degeneration during early 

development stages in rats 

Authors: *M. GOMEZ-CHAVARIN 1 , R. MORALES 2 , M. C. RAMIREZ 3 , G. ROLDAN 1 , J. 

FERNANDEZ 1 ; 

1 Dept. de Fisiología, UNAM, Mexico D. F., Mexico; 2 Dept. de Microbiología y Parasitología 

Facultad de Medicina, UNAM, D.F., Mexico; 3 Dept. de Sistemas Biológicos, UAM-Xochimilco, 

D.F., Mexico 

Abstract: INTRODUCTION Epidemiological studies have linked environmental factors with 

increased risk of idiopathic Parkinson‟s disease (PD). Because PD is associated with a late-in-life 

onset, it has traditionally been considered as a disease of aging, despite a long history of 

speculation that environmental factors early in life can predispose an individual to develop 

parkinsonian symptoms. Recent reports have begun to identify specific environmental factors 

occurring in uterus or during perinatal development that may prompt nigrostriatal system 

damage. Rotenone (ROT) is a commonly used pesticide that has been associated with 

parkinsonism since it produces degeneration of dopaminergic neurons (DAN) at the nigrostriatal 

pathway. Chronic exposure to ROT in adult rats induces nigrostriatal DAN degeneration 

accompanied by aggregation of α-synuclein (α-syn) and motor deficits. OBJETIVE To analyze 

the effect of a low dose ROT treatment during gestation and/or nursing on biochemical markers 

associated with PD in adulthood. METHODS Pregnant Wistar rats were s.c administrated. with 

250 κg/day of ROT. One day after birth, offspring were divided in 3 groups: 1) Gestation 

Group which received ROT only during gestation phase and was nursed by an intact fostermother 

which has never been exposed to ROT, 2) Nursing Group which received ROT only 

during lactation thought a ROT-treated rat, and 3) Gestation/Nursing Group which received 

ROT during both stages. Offspring were sacrificed at the 15, 30 or 60 postnatal day and their 

brains were processed for tyrosine hydroxilase (TH) immunostain. In a second experiment, other 

three groups treated as described above were evaluated for motor activity and α-syn content in 

the striatum and substantia nigra, two months after birth. RESULTS All ROT-treated groups 

showed motor deficits and a progressive decrease in TH+ neurons in the substantia nigra, plus an 

increase in α-syn aggregation into striatum and substantia nigra which was highest in 

Gestation/Nursing Group. CONCLUSIONS The results show a deleterious effect of low doses 

ROT treatment during early stages of life. These effects were progressive, irreversible and more 

severe when the exposure to ROT occurred during gestation than during nursing, suggesting that 

the nigrostriatal system is more vulnerable at this phase. Therefore, exposure to xenobiotics 

during early stages of life should be considered in the etiology of neurodegenerative disorders in 

adulthood. 

Disclosures: M. Gomez-Chavarin, None; R. Morales, None; M.C. Ramirez, None; G. 

Roldan, None; J. Fernandez, None.

Poster 





Support: CONACyT Grant 47158-Q 

DGAPA-UNAM Grant IN222303 

Title: Role of inhibitory apoptosis proteins in the survival of cerebellar granule neurons in 

culture 

Authors: *S. S. BLANCAS 1 , J. MORÁN 2 ; 

1 Neurociencias, Inst. DE Fisiologia Celular, MEXICO, D.F., Mexico; 2 Neurociencias, Inst. de 

Fisiologia Celular, Mexico, d.f., Mexico 

Abstract: During cerebellar development occurs a massive death of cerebellar granule cells 

(CGC). The CGC in culture die in low potassium condition ( KCl 5-10 mM; K5 or K10, 

respectively), but the chronic depolarization with potassium (KCl 25 mM; K25) or the 

stimulation with the agonist of glutamate receptors, NMDA, promote the survival and 

differentiation of the CGC. It has been suggested that these effects promoted by K25 or NMDA 

in CGC mimic the activation of the glutamate receptors during cerebellar development. The cell 

death of CGC induced by these conditions shows features of apoptotic death. Apoptosis is 

characterized by many morphological and biochemical alterations accomplished by caspases. 

This process can be negatively regulated by the inhibitory apoptosis proteins (IAP) that may act 

directly on caspases. It is no clear, however, the role of the IAP on the survival process of the 

CGC induced by NMDA or depolarizing conditions. In this study we explored the role of IAP in 

the survival of CGC under survival conditions. We evaluated the endogenous levels of some IAP 

(survivin, cIAP1, cIAP2 and XIAP) by Western blot assays in primary culture of CGC. The 

treatment with K25 did not have any effect on the protein levels of cIAP1, cIAP2 and survivin, 

but increased the levels of XIAP at 7 days in vitro (DIV) as compared to K5. These results 

suggest a differential effect of the trophic conditions in the regulation of the IAP levels. 

This work was supported by CONACYT 47158-Q project and DGAPA-UNAM IN222303 

project. 

Disclosures: S.S. Blancas , None; J. Morán, None. 

Poster





Support: CONACYT 47158-Q 

DGAPA-UNAM IN223107 

Title: Role of apoptosis and autophagy in rat cerebellar granule neuron death 

Authors: *P. MAYCOTTE, J. MORÁN; 

Neurociencias, UNAM, Inst. Fisiologia Celular, Mexico, Mexico 

Abstract: Cerebellar granule neurons (CGN) in culture are a widespread model for the study of 

neuronal cell death. They survive when grown in depolarizing conditions (KCl 25 mM) and die 

when are treated with the protein kinase inhibitor staurosporine (STS) or when are deprived of 

serum and potassium (KCl 5 mM) after 7 days in vitro. These conditions produce morphologic 

and biochemical apoptotic characteristics such as chromatin condensation and caspase activation. 

On the other hand, CGN also show characteristics of type II programmed cell death (PCD) or 

autophagy when deprived of serum and potassium, including autophagosome formation and 

cathepsin activation. 

In this work we evaluated the contribution of both type I (apoptotic) and type II (autophagic) 

PCD in rat CGN death induced by serum and potassium deprivation (-S-K) and STS. Apoptotic 

death was blocked with QVD-OPH, a general caspase inhibitor, while autophagic death was 

inhibited by 3 methyl adenine (3MA), a phosphatidylinositol 3 kinase (PI3K) blocker. When 

CGN were deprived of serum and potassium, QVD-OPH decreased cell death at 24h but not at 

48h, while 3MA protected from cell death even at 48h. Moreover, both 3MA and QVD-OPH 

decreased the apoptotic characteristics nuclear condensation and DNA internucleosomal 

fragmentation. However, although 3MA decreased nuclear condensation at 48h, a synergistic 

effect is observed with inhibitors of both types of death. Moreover, although 3MA preserved 

viability of -S-K treated cells even at 48h, internucleosomal DNA fragmentation was inhibited at 

12h but not at 24h, suggesting that apoptosis and autophagy are different pathways activated 

when CGN are deprived of serum and potassium and 3MA treated cells finally showed apoptotic 

characteristics, suggesting only a delay in apoptotic cell death. 

On the other hand, when CGN were treated with STS, both 3MA and QVD-OPH protected from 

cell death at 24h while only QVD-OPH protected from cell death at 48h. Moreover, only QVD- 

OPH decreased nuclear condensation both at 24 and 48h in STS treated cells and 3MA inhibited 

internucleosomal DNA fragmentation at 12h but not at 24h, delaying only the appearance of 

apoptotic features. 

Our results suggest the activation of both apoptotic and autophagic pathways in CGN treated 

with -S-K or STS, being autophagy the main pathway in -S-K and apoptosis in STS treated cells.

Disclosures: P. Maycotte , None; J. Morán, None. 

Poster 





Support: IAG #224-07-007 

Title: Neonatal PCP, but not ketamine, treatment increases running wheel activity in adult 

Sprague-Dawley rats 

Authors: *S. Y. BOCTOR 1,2 , C. WANG 2,1 , S. A. FERGUSON 2,1 ; 

1 Interdisciplinary Biomed. Sci., UAMS, Little Rock, AR; 2 Neurotoxicology, Natl. Ctr. for 

Toxicological Research/FDA, Jefferson, AR 

Abstract: Neonatal treatment with ketamine (KET), phencyclidine (PCP) or other N-methyl-Daspartate 

(NMDA) receptor antagonists can trigger apoptotic neurodegeneration in rodents and, 

in the case of PCP treatment, cause long-term behavioral alterations. Here, the potential of 

neonatal KET or PCP to alter adult residential running wheel activity was assessed. Male and 

female Sprague-Dawley rats were subcutaneously injected with: 10 mg/kg PCP (1x/day) on 

PNDs 7, 9 and 11; 20 mg/kg KET (6 injections every 2 hrs on PND 7); or a similar regimen of 

KET and 250 mg/kg LC on PND 7, with a single injection of 250 mg/kg LC on PNDs 8-11 or 

saline. On PND 72, each rat (n=16/sex/treatment) was placed in a housing cage containing a 

running wheel and wheel revolutions were recorded for each 12-hour day and night period on 

PNDs 72-77. Daytime running wheel activity was higher in male and female rats neonatally 

treated with PCP (p

cortices), only neonatal PCP treatment caused lasting behavioral alterations. (Supported by IAG 

#224-07-007 between NCTR/FDA and NIEHS/NTP). 

Disclosures: S.Y. Boctor, None; C. Wang, None; S.A. Ferguson, None. 

Poster 





Support: NIH Grant R01 548528 

California Institute for Regenerative Medicine 

Title: Cell-intrinsic mechanisms regulate cell death of cortical interneurons 

Authors: *D. SOUTHWELL, A. ALVAREZ-BUYLLA; 

Univ. California, San Francisco, CA 

Abstract: Cortical interneurons are produced in the ganglionic eminences of the embryonic 

ventral forebrain, from which they undergo a dramatic tangential migration into the developing 

cortex. While recent studies have uncovered some of the genetic mechanisms that produce 

interneuron diversity, less is known about the processes that regulate the maturation and 

integration of these cells in the cortex. Developmental cell death is known to eliminate a large 

fraction of nascent neural cell types in many regions of the nervous system. In the rodent 

olfactory bulb, nearly forty to fifty percent of nascent interneurons die once they have migrated 

and reached morphological maturity in the granule cell layer. 

To examine the basic mechanisms guiding cortical interneuron cell death, we have performed 

heterochronic transplantations of interneuron precursors. After introducing embryonic cells into 

the neonatal cortex, we observe that around forty percent of grafted interneuron precursors are 

eliminated. Furthermore, over a range of transplant sizes, the same fraction of transplanted cells 

dies. Transplanted interneurons can increase the total cortical interneuron population by at least 

fifty percent, without affecting the size of the endogenous interneuron cohort. These results 

suggest that interneuron cell death does not necessarily follow from a simple intercellular 

competition for limited signals. Rather, the cortex can support large numbers of supernumerary 

interneurons, implying that programmed cell death does not fine-tune population size to match 

an environmentally defined capacity. This further points to the precise control of progenitor 

activity as a key determinant of the population size. Should a large fraction of developing

interneurons be fated to die, it also raises the intriguing question of how this fleeting subpopulation 

contributes to cortical development and function. 

Disclosures: D. Southwell , None; A. Alvarez-Buylla, None. 

Poster 





Title: The mechanism of neogenin-induced apoptosis 

Authors: *Y. FUJITA 1 , J. TANIGUCHI 2 , M. UCHIKAWA 3 , M. ENDO 1 , K. HATA 1 , T. 

YAMASHITA 1,2 ; 

1 Dept Mol Neurosci, Grad Sch. Med, Osaka Univ., Osaka, Japan; 2 Dept Neurobiol, Grad Sch. 

Med, Chiba Univ., Chiba, Japan; 3 Dept Dev Biol, Grad Sch. of Frontier Biosci, Osaka, Japan 

Abstract: The repulsive guidance molecule (RGM) is a glycosylphosphatidylinositol (GPI)anchored 

protein that shares no sequence homology with any other known proteins. And it was 

originally identified in the chick retinotectal system. RGM repels retinal axons in the optic 

tectum and induces the collapse of temporal but not nasal growth cones. Thus, it was thought that 

it might be involved in topographic map formation. Recently, neogenin was identified as an 

RGM receptor. In the ligand-free state, neogenin induces apoptosis. Neogenin overexpression or 

RGMa down-regulation by small interference RNA (siRNA) in the developing neural tube of 

chick embryos resulted induces apoptosis. However, the signal transduction mechanism that 

induces cell death is completely unknown and remains to be analyzed. 

In this study, we identified the serine/threonine kinase death associated protein kinase (DAPkinase) 

to be an interactor of neogenin. DAP-kinase is a crucial intracellular protein that 

mediates cell death via its serine threonine kinase activity. Here we show that DAP-kinase is 

required for the proapoptotic activity of neogenin in vitro and in vivo. In HEK 293T cells, 

neogenin enhances the activity of DAP-kinase in the absence of RGM but not in the presence of 

RGM. To examine whether DAP-kinase is involved in neogenin-mediated cell death in vivo, we 

analyzed cell death in neural tubes of chick embryos. We electroporated the control or neogenin 

expression vectors with or without dominant negative form of DAP-kinase (DAPK-d/n) with the 

GFP-expression vector in HH stage 10 chick embryos. In the neogenin electroporated side, there 

was an increase in the number of TUNEL-positive cell wheareas, in the neogenin and DAPkinase 

co-electroporated side, resulted in the disappearance of the TUNEL-positive cells. 

Furthermore, we examined RGM functions as a cell survival factor to counteract neogenin

proapoptotic activity. When RGM was reduced by RGM siRNA, the number of TUNEL-positive 

cells was increased in the chick neural tube. Then we examined whether DAP-kinase is 

associated with the apoptosis-inducing effect of the RGM silencing. the number of the TUNELpositive 

cells in the RGM siRNA and DAPK-d/n co-electroprated side was decreased. These 

results suggest that the activity of DAP-kanase is required for neogenin induced cell death in the 

developing chick neural tube. 

Disclosures: Y. Fujita, None; J. Taniguchi, None; M. Uchikawa, None; M. Endo, None; K. 

Hata, None; T. Yamashita, None. 

Poster 




Topic: A.06.c. Developmental cell death: Other trophic factors 

Support: Korea Research Foundation Grant KRF-2005-041-C00278 

Asan Institute for Life Sciences 2007-396 

Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea 

government R01-2006-000-10771-0 

Title: Intracellular zinc chelation by TPEN blocks the apoptotic cell death in the developing 

brain 

Authors: *J.-Y. LEE, E. CHO, H. JEONG, J.-Y. KOH; 

Asan Inst. Life Sci., Univ. Ulsan Col. Med., Seoul, Republic of Korea 

Abstract: In the previous study, we showed that all neural cells undergoing apoptosis in the 

developing rat brain contained labile zinc in their cytosols (Eur J Neurosci., 23; 435-442, 2006). 

However, it still remains unclear whether the intracellular zinc accumulation is the cause or 

effect of the apoptotic cell death. To determine the role of intracellular labile zinc in the 

developmental apoptosis, postnatal rats were daily treated with a zinc chelator TPEN (5-20 

mg/kg, s.c.) for 7 days. Beforehand, we examined that this repeated subcutaneous administration 

of TPEN effectually chelated the intracellular zinc without modulating the expression levels of 

zinc regulating proteins (Znt1, Znt3 or synaptophysin) in the adult brain of mice. In the brain of 

newborn rat with treatment of TPEN, the number of apoptotic neural cells significantly 

decreased compared with that in non-treated normal brain. Moreover, the activation of caspase-3

was also blocked by the TPEN-treatment. Therefore, we propose that intracellular zinc 

accumulation causes the event of programmed cell death in the developing brain. By contrast, 

although TPEN chelates other metal ion (e.g. iron or copper) as well as zinc, their involvements 

in the developmental apoptosis were ruled out because the intracellular accumulation of labile 

iron or copper ion was not found by Perl‟s stain or Phen Green SK (PGSK) fluorescent dye. 

Disclosures: J. Lee, None; E. Cho, None; H. Jeong, None; J. Koh, None. 

Poster 




Topic: A.06.c. Developmental cell death: Other trophic factors 

Support: Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza Univ Rome 

Ateneo 2006-2007 

Title: De novo transcription of the Thg-1pit gene and translocation of the protein product to the 

nuclear matrix landmark the onset of apoptosis in cerebellum granule neurons of the mouse 

Authors: S. CANTERINI 1 , A. BOSCO 1 , V. DE MATTEIS 1 , F. MANGIA 1,2 , *M. 

FIORENZA 1,2 ; 

1 Sapienza Univ. Rome, Rome, Italy; 2 Neurobio. Res. Ctr. "D. Bovet", Rome, Italy 

Abstract: Thg-1pit belongs to the transforming growth factor (TGF)-beta-stimulated clone 22 

domain (TSC22D) gene family, coding for proteins widely expressed in developing and adult 

mouse tissues (Kester et al., 2000; Treisman et al., 1995; Dohrmann et al., 2000). 

Proteins encoded by these genes act as transcriptional regulators and influence a number of 

biological processes, including cell proliferation, apoptosis and stress response. Based on our 

previous findings on Thg-1pit expression in developing and adult cerebellum granule neurons 

(CGN) (Canterini et al., 2005), we have exploited primary CGN in vitro cultures to gain insight 

on the function of this gene. CGN cultures have widely been used to investigate the anti/proapoptotic 

effect exerted on these cells by growth factors/cytokines as Insulin-like Growth Factor 

1 (IGF1) and Transforming Growth Factor-β (TGF-β) (rev. by Vaudry et al., 2003), as well as 

the dependence of CGN survival/apoptosis on membrane electrical activity (D‟Mello et al., 

1993). In fact, lowering the level of K + in the culture medium ([K + ]ext) from 25 mM to 5 mM 

triggers CGN apoptosis, while TGF-β apparently has opposite roles, by promoting apoptosis 

when administered with 5 mM [K + ]ext, and cell survival in the presence of 25 mM [K + ]ext (de

Luca et al., 1996). However, besides the demonstration that the anti-apoptotic effect of 25 mM 

[K + ]ext is mediated by a sustained increase in intracellular free [Ca 2+ ] (Gallo et al., 1987; Pearson 

et al., 1992; Franklin and Johnson, 1992) and that the pro-apoptotic effect of TGF-β1 involves a 

Ca 2+ -dependent pathway(s) (Kanzaki et al., 1990), the molecular events occurring when CGN 

are committed to apoptosis by TGF-β1 and/or 5 mM [K + ]ext are still obscure. 

We report that CGN engagement in the apoptotic pathway is accompanied by de novo Thg-1pit 

transcription and THG-1pit protein translocation to nucleus, the 5 mM [K + ]ext and TGF-β1 

treatments having idiosyncratic although synergic effects. We also show that THG-1pit is posttranslationally 

modified by phosphorylation and/or O-glycosylation and that the phosphorylated 

form is a normal chromatin component, while the O-glycosylated form is targeted to the nuclear 

matrix in concomitance with apoptosis induction. 

Disclosures: S. Canterini, None; A. Bosco, None; V. De Matteis, None; F. Mangia, None; M. 

Fiorenza , None. 

Poster 



Program#/Poster#: 232.24/C1 


Title: Ketamine activates cell cycle events and apoptosis in the neonatal rat brain 

Authors: *S. G. SORIANO, J. LI, Q. LIU, D. BAJIC, J. C. IBLA; 

Dept Anesthesia, Childrens Hosp., Boston, MA 

Abstract: Prolonged exposure to ketamine results in accelerated neurodegeneration and longterm 

neurocognitive deficits postnatal 7 day (P7) rat pups. The cell cycle is the biological process 

that mediates eukaryotic cell proliferation. Pathological entry into the cell cycle leads to 

tumorgenesis in most cell types. Mature neurons are postmitotic and typically do not proliferate. 

Experimental models of neurodegeneration have implicated pathological reentry of neurons into 

the cell cycle, leading to cell death. The role of anesthetic-induced reentry into the cell cycle has 

not been investigated. We hypothesize that ketamine induces cycle cell reentry that leads to 

neuronal cell death. We characterized the effect of ketamine on the cell cycle signaling pathway 

in the developing brain in vivo. 

Material and Methods: With the approval of the Investigational Review Board and adherence to 

the Guide for the Care and Use of Laboratory Animals, Sprague-Dawley P7 rat pups were 

utilized for the experiments. Each rat pup received 5 intraperitoneal doses of either saline or 

ketamine (5, 10 and 20 mg/kg/dose) at 90-minute intervals over 6 hours. Standard

immunobloting techniques was utilized to measure caspase-3 and cell cycle protein (cyclin D1, 

cdk4, E2F1, and BIM) expression. 

Results: Ketamine mediated a dose-dependent increase in cell cycle protein and caspase-3 

expression after 6 hours. 

Summary: Ketamine activates cell cycle proteins in a dose-dependent manner. Cell cycle reentry 

may be another mechanism for anesthetic-induced neuroapoptosis in the developing brain. 

Disclosures: S.G. Soriano , None; J. Li, None; Q. Liu, None; D. Bajic, None; J.C. Ibla, None. 

Poster 




Topic: D.09.c. Neural coding 

Support: C-18590551, Japan 

Title: Maintenance of morphology of lateral line tube in teleost scales 

Authors: S. N. DO 1 , L. LI 1 , H. KATSUYAMA 2 , S. ABE 3 , A. ISHIZAKI 1 , H. TANII 1 , *K. 

SAIJOH 1 ;

1 Dept Hyg., Kanazawa Univ. Sch. Med., Kanazawa 920-8640, Japan; 2 Kawasaki Med. Univ., 

Kurashiki, Japan; 3 Jikei Med. Col., Tokyo, Japan 

Abstract: Lateral line nerve of teleosts locating in the lateral line system is a sensor of current 

flow and supposed to be correspondent to deep sensory nerve of mammalian. In order to sense 

very minute flow, the wall of lateral line tube should be maintained very thin, whereas the tube 

itself becomes bigger according to scale growth. Thus, its morphology seems to be strictly 

regulated. When subtraction cloning was subjected to RNAs between scale - gill (SG) in order to 

explore mRNA species enriched in the scale, the clones highly homologous to SPARC, 

baculoviral IAP repeat containing 2 (birc2), and BCl2-related ovarian killer a (boka) were 

isolated. In situ hybridization confirmed birc2 and boca hybridization in flat cells along the ridge. 

Hybridization for birc2 was also observed in cells between the ridges. SPARC hybridization was 

apparent in flat and small cells between the ridges. birc2 and boka were isolated as mRNAs 

enriched in the normal scale but they also abundantly expressed in the cells along the lateral line 

tube. Although all the cells in the scale were designated as scleroblasts, there were several types 

of cells having different gene expression. Co-expression of birc2 and boka in the cells along the 

ridge also suggested that anti-apoptotic mechanism worked to maintain the ridges. birc2 positive 

cells along the tube were associated with RANK-positive cells suggesting that these cells were 

induced apoptosis by osteoclast-phenotype cells so that apoptosis seems play an important role to 

maintain the tube shape. 

Disclosures: S.N. Do, None; H. Katsuyama, None; S. Abe, None; K. Saijoh , None; L. Li, 

None; A. Ishizaki, None; H. Tanii, None. 

Poster 

233. Nicotinic Aacetylcholine Receptors: Regulation and Function II 



Topic: B.01.d. Monoamines: Intracellular signaling cascades 

Support: Philip Morris USA (MJE) 

SBU MD with Recognition Program (BB and JS) 

NIH Grant NS37685 (AST) 

Title: Nicotine stimulates signal transduction pathways in pheochromocytoma cells

Authors: *M. J. EVINGER 1 , M. HU 1 , B. BARR 1 , J. STRAUSS 1 , J. F. POWERS 2 , A. S. 

TISCHLER 2 ; 

1 Dept Pediatrics, Stony Brook Univ., Stony Brook, NY; 2 Pathology, Tufts-New England Med. 

Ctr., Boston, MA 

Abstract: Nicotine evokes rapid changes in neuronal cells resulting in stimulation of 

intracellular signaling cascades and release of the neurotransmitters dopamine, norepinephrine 

and epinephrine from catecholamine-synthesizing cells. In adrenergic mouse pheochromocytoma 

cells (MPC), nicotine elevates mRNAs for catecholamine synthetic enzymes and for specific 

neuronal nicotinic acetylcholine receptors (nAChRs). In other neuronal cells, nicotinic agonists 

activate the PI3K/Akt (phosphoinositide 3-kinase/ Akt kinase) pathway involved in cell survival 

and apoptosis and MAPK/ERK (mitogen associated phosphokinase/ extracellular signal related 

kinase) pathway regulating catecholamine release. We hypothesize that nicotine selectively 

activates PI3K/Akt and MAPK/ERK signal transduction pathways in MPC cells through 

nAChRs. 

MPC 10/9/96CRC1, an adrenergic continuous cell line, was incubated with nicotine or inhibitors 

for intervals to 12 hr. Cell extracts were fractionated by SDS-PAGE for western 

immunodetection using primary antisera to phospho- or total protein forms of pathway 

intermediates. Chemiluminescent visualization and densitometric scanning were employed for 

signal quantitation. 

Nicotine stimulates the PI3K/ Akt pathway in MPC 10/9 cells through rapid phosphorylation of 

pathway intermediates. Specifically, increases in phosphorylated upstream intermediates PI3K 

p85", Akt-Thr 308 , and Protein Kinase D1 are detectable within 15 min and are maximal by 60 

min nicotine treatment. A 50% increase in mRNA (3.5 h) indicates that PI3K gene expression 

also accelerates. Rapid phosphorylation of downstream target GSK-3 (but no change in Bad) 

suggests that nicotine facilitates MPC 10/9 cell survival. Moreover, incubation with wortmannin 

blocks nicotine-evoked phosphorylation of PI3K and downstream intermediates. 

As observed in other pheochromocytoma lines, nicotine activates the MAPK/ERK pathway. In 

MPC 10/9 cells, nicotine enhances production of phospho-forms of c-Raf, MEK1, and ERK. 

Phosphorylation of c-Raf proceeds most rapidly with activation of MEK and ERK following a 

delayed course. Although ERK 1 and 2 stimulation with nicotine (2-fold at 15 min) is less robust 

than that evoked by GDNF in the related MPC862 line, these data indicate that the MAPK/ERK 

pathway constitutes an integral aspect of cholinergic stimulation of catecholaminergic cells. 

Therefore, in addition to exerting pronounced effects on the transcriptional profile of 

pheochromocytoma cells, nicotine also stimulates intracellular signaling through MPC pathways 

associated with neuronal cell survival, catecholamine release and synaptic plasticity. 

Disclosures: M.J. Evinger, None; M. Hu, None; B. Barr, None; J. Strauss, None; J.F. 

Powers, None; A.S. Tischler, None. 

Poster 

233. Nicotinic Aacetylcholine Receptors: Regulation and Function II



Topic: B.02.c. Nicotinic acetylcholine receptors: regulation and function 

Support: ADCC pilot grant 

Title: Pathophysiological levels of amyloid-beta 1-42 induces selective up-regulation of α7nAChRs 

and cytotoxicity in differentiated SH-SY5Y cells 

Authors: *Q. LIU 1 , S. EMADI 2 , M. SIERKS 2 , J. WU 1 ; 

1 Neurol, Barrow Neurolog. Inst., Phoenix, AZ; 2 Chem. Engin., Arizona state university, phoenix, 

AZ 

Abstract: Dysfunction of basal forebrain cholinergic neurons (BFCNs) correlates with cognitive 

deficits in Alzheimer disease (AD). Amyloid-beta (Aβ) 1-42, a major toxic peptide that 

accumulates in the brain of AD patients, has been shown to directly interact with neuronal 

nicotinic acetylcholine receptors (nAChRs) expressed on BFCNs. It remains unclear how Aβ 

interacts with nAChRs expressed on BFCNs and modulates BFCN function. Our previous 

studies indicated that pathophysiological levels of Aβ acutely blocked nAChRs on BFCNs, thus 

affecting basal forebrain cholinergic transmission. In the present study, we investigated the 

effects of 10 days exposure to Aβ on nAChRs expressed on differentiated cholinergic SH-SY5Y 

cells. Molecular biology, electrophysiological, immunochemistry staining techniques, and LDH 

(Lactic Dehydrogenase ) assay, were employed to perform this study. Our results revealed that 

10 days exposure to Aβ selectively up-regulated α7-nAChR expression, which was accompanied 

by a significant reduction in cell viability. After 10 days exposure to Aβ, function of α7-nAChRs 

was comparable to non-treated α7-nAChRs, which was assessed by patch-clamp recordings in an 

Aβ-containing environment. A significant increase in α7-nAChR function following Aβ 

treatment was observed when cells were washed with Aβ-free medium. This up-regulation of α7containing 

nAChRs could be a compensatory response to maintain cholinergic activity during 

AD progression. Long-term interactions of α7-nAChRs with Aβ may serve as a pathogenic 

mechanism of cholinergic neuronal dysfunction in AD. 

Disclosures: Q. Liu , None; M. Sierks, None; J. Wu, None; S. Emadi, None. 

Poster 



Program#/Poster#: 233.3/C5

Topic: B.02.a. Nicotinic acetylcholine receptors in brain: Physiology and function 

Support: PMERP Postdoctoral Fellowship 

NIH Grant - NS012601-32 

TRDRP Grant - 16RT-0167 

NIH Grant - NS035469-13 

Title: Unique presynaptic effects of postsynaptic PSD-95 family members at nicotinic synapses 

Authors: *R. NEFF, W. G. CONROY, D. K. BERG; 

Biol Sci., Univ. Calif, San Diego, La Jolla, CA 

Abstract: All four members of the PSD-95 family of PDZ-containing proteins (PSD-93, PSD- 

95, SAP97, and SAP102) are expressed in chick ciliary ganglion (CG) neurons and collectively 

contribute to postsynaptic scaffolds at nicotinic synapses on the cells. Previous studies have 

shown that disruption of PDZ interactions by the entire family compromises synaptic input to the 

neurons in culture. The results indicated that one or more of the PDZ-proteins are likely to 

interact with transynaptic components to influence presynaptic events. We report here distinctive 

roles for individual members that reveal novel features of transynaptic control of transmitter 

release. 

We used RNAi to knockdown individual PSD-95 family members in CG neurons in culture. 

None of the four RNAi constructs expressed alone in the neurons changed the evoked excitatory 

postsynaptic currents (EPSCs) in the neurons assessed by patch-clamp recording. Co-expression 

of RNAi constructs for PSD-95 and SAP102 in the neurons, however, significantly reduced the 

amplitude of evoked EPSCs and the frequency (but not amplitude) of spontaneous miniature 

EPSCs (mEPSCs). The results suggest a reduction in functional release sites when both PSD-95 

and SAP102 levels are depressed. The number of synaptic contacts and the levels of postsynaptic 

nicotinic receptors, judged by immunostaining, were unchanged. Other pairwise combinations of 

RNAi constructs had no effect, indicating that only PSD-95 and SAP102 were likely to exert 

mutually redundant regulation of presynaptic events. 

RNAi for SAP97 yielded a different story. It had no effect on either evoked EPSCs or on mEPSC 

frequency or amplitude but it did reduce evoked release in the presence of strontium (replacing 

calcium). This suggests that SAP97 may influence asynchronous release. SAP97 RNAi had no 

effect on the number or size of synaptic contacts judged by immunostaining. 

The results indicate that individual members of the PSD-95 family expressed in a postsynaptic 

neuron can exert distinct transynaptic actions on transmitter release at nicotinic synapses. At 

least two kinds of mechanisms are involved, likely involving two distinct transynaptic linkers - 

one employed by PSD-95/SAP102 and the other by SAP-97. This regulation of transmitter 

release by postsynaptic PDZ-proteins occurs without changes in the number of synaptic contacts 

on the neuron. Thus, changes in the relative expression levels of PDZ-proteins in the 

postsynaptic neuron can dictate the properties of transmitter release onto the cell. 

Disclosures: R. Neff , None; W.G. Conroy, None; D.K. Berg, None.

Poster 





Title: A single amino acid substitution in the β9 strand of rat α7 nicotinic receptor which slows 

desensitization 

Authors: T. MCCORMACK, E. A. GAY, *J. L. YAKEL; 

Lab. of Neurobio., NIEHS F2-08, Res Triangle Pk, NC 

Abstract: The α7 subtype of nicotinic acetylcholine receptors (nAChRs) is widely recognized as 

a potential therapeutic target for the treatment of a variety of pathologic conditions, including 

schizophrenia, and Alzheimer's disease. One unique feature of α7 nAChRs that complicates 

functional assays, which would help identify selective drugs for these receptors, is the rapid 

desensitization of the agonist-evoked responses. A region of the ligand-binding pocket that is 

thought to move when agonist binds to the α7 nAChR is the F-loop (also known as the β8-β9 

linker). Whether and/or how this region interacts with other regions of the ligand-binding pocket 

and affects channel function is unknown. When the proline residue at position 180 (P180) in the 

β9 strand was mutated to a threonine residue (P180T), we observed a much slower rate of 

receptor desensitization; the half-time of decay increased from 111 ± 7 msec for wildtype to 

1260 ± 86 msec for the P180T mutation. In addition the EC50 value decreased to 56 κM for the 

mutant versus 120 κM for wildtype. When P180 was mutated to glycine, the channels‟ 

desensitization rate (242 ± 44 msec) was much closer to that of wildtype. Mutation of P180 to 

cysteine produced a decrease in peak current response to ACh and covalent modification of the 

mutant C180 with various MTS reagents (e.g. MTSEA) was also tested in order to investigate 

the effect of sulfhydryl reagents on channel function; however these reagents had no significant 

effect on channel function. Considering that α7 homology models consistently predict that the 

position 180 sidechain projects outward into the aqueous environment and thus should be 

accessible to covalent modification, we would predict that these MTS molecules are covalently 

modifying the C180 residue but with no noticeable effect on channel function. Together these 

data would suggest that the size, shape, and/or post-translational modification of the threonine 

residue at position 180, and not just the absence of the endogenous proline residue at this 

position, confers slow desensitization. Furthermore the P180T α7 mutation, unlike the α7-L248T 

gain-of-function mutant, exhibits properties that are remarkably similar to the wild-type α7 

receptor (except for desensitization onset), implicating it as a useful tool for identifying 

therapeutic agents targeting the α7 nAChR.

Disclosures: T. McCormack, None; E.A. Gay, None; J.L. Yakel , This research was supported 

by the Intramural Research Program of the NIH, National Institute of Environmental Health 

Sciences, A. Employment (full or part-time). 

Poster 





Title: (+/-)TC-5619 produce a profound desensitization of alpha-7 nicotinic receptor activated 

currents in vitro 

Authors: *F. W. JOW 1 , M. BOWLBY 1 , T. LOCK 1 , R. PERI 1 , D. KOWAL 1 , A. NENCINI 2 , S. 

HAYDAR 1 , C. GHIRON 2 , G. TERTSAPPEN 2 , J. DUNLOP 1 ; 

1 Neurosci. Discovery Res., Wyeth Res., Monmouth Junction, NJ; 2 Siena Biotech, Siena, Italy 

Abstract: Alpha-7 (α7) nicotinic acetylcholine receptor (nAChR) agonists are promising 

therapeutic candidates for the treatment of cognitive dysfunction associated with a variety of 

disorders including schizophrenia and Alzheimer‟s disease, and a number of selective agonists 

have now been disclosed. TC-5619 has recently been identified as a potent and full agonist at the 

α7 nAChR. In this study we have examined the effect of (+/-)TC-5619 on α7 nAChR agonistevoked 

currents in GH4C1 cells stably expressing the receptor. α7 nAChR agonist activity of 

(+/-)TC-5619 was confirmed in a FLIPR based assay measuring agonist activation of calcium 

flux. In electrophysiological experiments, sequential application of increasing concentrations of 

acetylcholine to GH4C1/α7 cells produced a concomitant concentration-dependent increase in 

the magnitude of evoked currents (EC50, 30 µM). Interestingly, sequential application of (+/- 

)TC-5619 to GH4C1/ α7 cells resulted in small currents in the presence of low drug 

concentration and no subsequent response when higher concentrations of compound were 

applied. Cells treated with low concentrations of (+/-)TC-5619 were also found to be 

unresponsive to subsequent addition of acetylcholine suggestive of a profound receptor 

desensitization to these low drug concentrations. Potent α7 nAChR agonist activity of (+/-)TC- 

5619 could be demonstrated by treating individual cells with a single concentration of drug and 

constructing a concentration-response curve from data derived from multiple cells (EC50, 0.11 

µM; Emax, 0.76). Similarly, a number of closely related (+/-)TC-5619 were found to be potent 

α7 receptor agonists but only when evaluation of each concentration of drug was restricted to a 

single cell. In contrast, a series of unrelated α7 nAChR agonists, representing diverse 

chemotypes, e.g., SEN12333, were found to produce a similar concentration-dependent increase 

in evoked currents, when applied sequentially to the same cell, as was observed with

acetylcholine. These results suggests unique properties of (+/-)TC-5619 with respect to α7 

nAChR desensitization when compared with other selective α7 agonists. 

Disclosures: F.W. Jow , None; M. Bowlby, None; T. Lock, None; R. Peri, None; D. Kowal, 

None; A. Nencini, None; S. Haydar, None; C. Ghiron, None; G. Tertsappen, None; J. 

Dunlop, None. 

Poster 





Support: Barrow Neurological Foundation 

Philip Morris USA Inc. and Philip Morris International through their External 

Research Program 

NIH U19 DA019377 

NIH R01 DA017980 

NIH R01 DA015389 

Title: Comparisons across techniques used to characterize the functional pharmacology of 

ligand-gated ion channels: Toward a universal scale of function 

Authors: B. EATON 1 , Q. LIU 2 , C. ZHENG 2 , B. DASH 1 , J. WU 2 , *R. J. LUKAS 1 ; 

1 Div. Neurobiol, 2 Div. Neurol., Barrow Neurol Inst., Phoenix, AZ 

Abstract: With the diversity of techniques available for characterizing the functional 

pharmacology of ligand-gated ion channels, there is a need to establish an objective means to 

compare data obtained from different labs using these different techniques and expression 

systems. As high-throughput techniques become more affordable and prevalent and novel 

techniques are created, this need will only increase. Potentially, even data obtained using the 

same technique in cells transfected with identical DNA constructs might differ subtly if the host 

cell line differs. Here we compare functional data for nicotinic acetylcholine receptor (nAChR) 

subtypes expressed in oocytes or mammalian cells and assessed using whole-cell current 

recording, isotopic ion flux assays, or fluorescence-based assays using voltage-sensitive dyes.

The goal of our analysis is to derive an objective measure that enables the comparison of data 

irrespective of technique applied. Results obtained in studies of α4β2-nAChR and other subtypes 

in response to agonists, interesting partial agonists such as varenicline, and in the presence of 

classic or novel competitive or non-competitive antagonists (e.g., analogs of bupropion or its 

metabolites) are presented. It is suggested that one can derive an objective scale of function that 

makes functional pharmacological results obtained using different techniques easier to interpret 

and compare. 

Support in part by NIH U19 DA019377, R01 DA015389 and R01 DA017980, Philip Morris 

USA Inc. and Philip Morris International through their External Research Program, and the 

Barrow Neurological Foundation. 

Disclosures: B. Eaton, None; Q. Liu, None; R.J. Lukas , None; C. Zheng, None; B. Dash, 

None; J. Wu, None. 

Poster 





Support: NIH grant DA020811 

Title: Discovery and in vitro evaluation of novel, potent and highly selective antagonist ligands 

for the α3β4 nicotinic acetylcholine receptors 

Authors: N. T. ZAVERI 1 , W. E. POLGAR 2 , F. JIANG 2 , C. OLSEN 2 , W. ZHOU 2 , X. S. XIE 2 , 

*L. R. TOLL 2 ; 

1 Medicinal Chem., Mol. Med. Res. Inst., Sunnyvale, CA; 2 Biosci., SRI Intl., Menlo Park, CA 

Abstract: Subtype-selective ligands for the neuronal nicotinic acetylcholine receptors (nAChRs) 

α4β2 and α7, have shown clinical benefit for smoking cessation and as analgesics and are 

currently being investigated as Alzheimer‟s disease therapeutics. The α3β4 nAChR, on the other 

hand, is relatively less understood as a therapeutic target. Several low affinity, non-selective noncompetitive 

ligands such as methyllycaconitine and its analogs, 18-methoxycoronaridine, 

bupropion, and mecamylamine have been reported to bind to and inhibit α3β4 nAChRs. We 

recently reported the discovery and structure-activity relationship (SAR) studies of a series of 

highly selective noncompetitive antagonists for the α3β4 nAChRs. Further SAR on this novel 

class of α3β4 nAChR ligands has led to the discovery of a new, related class of 

[3.3.1]azabicyclononanes that have high binding affinity for the α3β4 nAChR and displace

[ 3 H]epibatidine with nanomolar affinity on membranes from HEK cells transfected with α3β4 

receptors. This class of compounds possess greater than 100-fold selectivity versus α4β2 

receptors. Our lead compound, SR17080 has Ki of 2.6 nM at α3β4 receptors and 476 nM at 

α4β2, although the inhibition is non-competitive as it decreases the [ 3 H]epibatidine Bmax. In 

functional assays of calcium mobilization in HEK cells using FLIPR, these novel ligands were 

antagonists with respect to the agonist epibatidine. SR17080 blocks epibatidine induced Ca 2+ - 

flux with an IC50 of 150 nM, but induces an inhibition of the epibatidine maximal response 

indicating non-competitive antagonism. In whole cell patch-clamp electrophysiological 

experiments in HEK cells, 10 nM SR17080 inhibited the fast component of the epibatidineinduced 

inward current. Overall, the binding and functional experiments suggest that SR17080 

and its congeners represent a novel class of potent and selective noncompetitive antagonists at 

the α3β4 receptors. These ligands will be useful to study the role of the α3β4 subtype of nAChRs 

in various pathophysiologies and for therapeutic applications. 

Disclosures: N.T. Zaveri, None; W.E. Polgar, None; F. Jiang, None; L.R. Toll, None; C. 

Olsen, None; W. Zhou, None; X.S. Xie, None. 

Poster 





Support: NIH Grant 5P20RR016466 

Title: Mechanisms of des-formylflustrabromide potentiation of neuronal nicotinic acetylcholine 

receptors 

Authors: M. M. WELTZIN 1 , A. PANDYA 1 , B. E. EDMONDS 2 , *M. K. SCHULTE 1 ; 

1 Inst. Arctic Biol, Univ. Alaska Fairbanks, Fairbanks, AK; 2 Univ. Alaska Juneau, Juneau, AK 

Abstract: Neuronal nicotinic acetylcholine receptors (nAChR) are similar in structure and 

function to other members of the Cys-loop superfamily of ligand-gated ion channels. (GABAA, 

glycine and type 3 serotonin receptors). nAChRs, specifically α4β2 subtypes, are involved in 

modulating nicotinic tone in the CNS. Alterations of α4β2 nAChR expression are thought to play 

a role in a wide range of CNS disorders. Ligands which selectively modulate nicotinic receptor 

subtypes represent a promising new avenue for treatment of Autism, Alzheimer‟s and 

Parkinson‟s disease. Des-formylflustrabromide (dFBr) is a newly discovered, positive allosteric 

modulator selective for α4β2 nAChRs. We are investigating the mechanisms of dFBr

potentiation. We have previously shown that dFBr potentates acetylcholine response by over 

250%. In the current study we report that co-application of dFBr with partial agonist (ex: 

nicotine) produces Imax values equivalent to the potentiated acetylcholine response thus 

apparently converting nicotine to a full agonist. Experiments were conducted using human α4β2 

neuronal nAChR injected in Xenopus leavis oocytes and evaluated using two-electrode voltage 

clamp electrophysiology. Identification of dFBr mechanism will facilitate construction of ligands 

similar to dFBr, leading to the development of new therapeutic options for disorders involving 

alterations in CNS nicotinic tone. 

Disclosures: M.M. Weltzin, None; A. Pandya, None; B.E. Edmonds, None; M.K. Schulte , 

None. 

Poster 





Support: NCRR grant RR016466 

Virginia Center on Aging 

Title: Beta2 selective allosteric modulators of neuronal nicotinic receptors 

Authors: *A. A. PANDYA 1 , M. WELTZIN 1 , J. KIM 2 , N. GERMAN 2 , M. SCHULTE 1 , R. 

GLENNON 2 ; 

1 Dept Chem. & Biochem, Univ. Alaska, Fairbanks, Fairbanks, AK; 2 Dept. of Medicinal 

Chemistry, Sch. of Pharm., Virginia Commonwealth Univ., Richmond, VA 

Abstract: Allosteric modulators targeting neuronal nicotinic acetylcholine receptors (nAChRs) 

are emerging as lead molecules with potential applications in the treatment of a wide range of 

neurological disorders. Compounds such as galantamine are used clinically in the treatment of 

Alzheimer‟s disease and may be useful in the treatment of other disorders where nicotinic tone in 

the CNS is altered. Galantamine and Physostigmine are acetyl-cholinesterase inhibitors but also 

act as non-selective allosteric modulators of nAChRs. 

The metabolite des-formylflustrabromine (dFBr) obtained from the marine Bryozoan Flustra 

Foliacea was recently identified as a selective modulator of the alpha4/beta2 nAChR subtype. 

We previously reported the synthesis of dFBr and evaluated its action on heteromeric 

alpha4/beta2 and homomeric alpha7 nAChRs. dFBr selectively potentiates acetylcholine induced

esponses on the human alpha4/beta2 subtype of nAChRs but not homomeric alpha7 nAChRs. In 

this study, we evaluated a series of synthetic analogues of dFBr. Two compounds (JSK-062) and 

(NG-089) displayed potentiating effects similar to dFBr. Potentiation was observed on 

alph4/beta2 but not alpha7 nAChRs. All the compounds inhibited acetylcholine responses at 

higher concentrations. 

To compare the effects of dFBr to other acetylcholine potentiating ligands, we evaluated the 

effects of co-application of galantamine and physostigmine with dFBr. Co-application of 

Galantamine or Physostigmine with acetylcholine produced response amplitudes similar to 

acetylcholine alone. This is consistent with previous studies that show little observable 

potentiation by these compounds using acetylcholine receptors expressed in Xenopus oocytes. 

When co-applied with dFBr, however, both Galantamine and Physostigmine produced greater 

potentiation than with dFBr alone. This concerted effect suggests independent and interacting 

mechanisms of action for these compounds. The selectivity of dFBr for heteromeric alpha4/beta2 

nnAChR compared to the non-selective nature of Galantamine and Physostigmine for both 

alpha7 and alpha4/beta2 nnAChR also supports the hypothesis that the dFBr class of compounds 

exerts their actions through a distinct mechanism involving the beta2 subunit of the nAChR. The 

unique selectivity of these compounds makes them potentially valuable lead molecules in the 

treatment of disorders involving decreased nicotinic tone resulting from decreased alpha4beta2 

receptor expression. 

Disclosures: A.A. Pandya, None; M. Weltzin, None; M. Schulte, None; J. Kim, None; N. 

German, None; R. Glennon, None. 

Poster 





Support: Abbott 

Title: In vitro pharmacological profile of A-867744: a novel type II α7 nAChR PAM exhibiting 

a unique pharmacological profile 

Authors: *J. MALYSZ 1 , J. H. GRONLIEN 2 , D. J. ANDERSON 1 , M. HAAKERUD 2 , K. 

THORIN-HAGENE 2 , H. WEEN 2 , C. A. BRIGGS 1 , R. FAGHIH 1 , W. H. BUNNELLE 1 , M. 

GOPALAKRISHNAN 1 ; 

1 Neurosci. Research, GPR&D, Abbott, Abbott Park, IL; 2 Abbott, Oslo, Norway

Abstract: α7 nAChRs are considered as attractive targets for treating cognitive deficits in 

schizophrenia and AD. Positive allosteric modulators (PAMs) may offer an advantage over 

agonists by potentiating the function of α7 nAChRs. In this study, we describe a novel type II α7 

PAM, A-867744 that exhibits a unique pharmacological profile based on radioligand binding. In 

X. oocytes expressing hα7 nAChRs, A-867744 potentiated ACh evoked currents with an EC50 of 

~ 1.2 κM and ~ 6.8-fold increase in maximal current amplitude. In the presence of 5 κM A- 

867744, ACh evoked concentration-responses in oocytes expressing hα7 nAChRs were 

potentiated by increases in potency, maximum efficacy, and Hill slope. In contrast, at hα4β2 and 

hα3β4 subtypes expressed in HEK-293 cell lines, A-867744 did not potentiate nicotine-evoked 

Ca 2+ responses. Rather, moderate inhibition of α4β2 and α3β4 was noted (IC50 values of ~ 2 and 

20 κM, respectively). At h5-HT3A receptors expressed in oocytes, no effect of A-867744 (≤ 30 

κM) was observed. At native α7 nAChRs, examined by whole cell patch clamp in rat 

hippocampus CA1 or dentate gyrus brain slices, A-867744 (10 κM) enhanced α7 currents evoked 

by ACh or choline, increased recovery from inhibition/desensitization, and potentiated choline 

evoked spontaneous GABAergic synaptic activity. A-867744, similar to other α7 PAMs 

(NS1738, TQS, and PNU-120596) did not displace the binding of [ 3 H]MLA to α7* or [ 3 H] 

cytisine to α4β2* at ≤ 10 κM in rat cortex homogenates. However, unlike NS1738, TQS, and 

PNU-120596, A-867744 displaced the binding of the agonist radioligand, [ 3 H]A-585539, in rat 

cortex with a Ki of ~ 0.8 κM. In an α7-5-HT3A chimera composed of the extracellular N-terminus 

domain of the α7 nAChR, A-867744 neither potentiated α7 agonist evoked responses nor 

displaced [ 3 H]A-585539 binding, suggesting a site of action outside of the α7 N-terminal 

domain. In summary, this study identifies a novel and selective α7 nAChR PAM that exhibits 

unique pharmacological interaction with the receptor and will be further useful in addressing 

mechanisms involved in allosteric modulation of the α7 nAChR. 

Disclosures: J. Malysz, Abbott, A. Employment (full or part-time); J.H. Gronlien, Abbott, A. 

Employment (full or part-time); D.J. Anderson, Abbott, A. Employment (full or part-time); M. 

Haakerud, Abbott, A. Employment (full or part-time); K. Thorin-Hagene, Abbott, A. 

Employment (full or part-time); H. Ween, Abbott, A. Employment (full or part-time); C.A. 

Briggs, Abbott, A. Employment (full or part-time); R. Faghih, Abbott, A. Employment (full or 

part-time); W.H. Bunnelle, Abbott, A. Employment (full or part-time); M. Gopalakrishnan, 

Abbott, A. Employment (full or part-time). 

Poster 




Topic: B.02.a. Nicotinic acetylcholine receptors in brain: Physiology and function

Support: GM57481-05A2 

Title: Identification of multiple pharmacophores for the selective activation of nicotinic alpha7type 

acetylcholine receptors 

Authors: *R. L. PAPKE 1 , F. LEONIK 2 , N. A. HORENSTEIN 2 ; 

1 Dept Pharmacol & Therapeut, Univ. Florida, Gainesville, FL; 2 Chem., Univ. of Florida, 

Gainesville, FL 

Abstract: The activation of heteromeric and homomeric nAChR was studied in Xenopus 

oocytes in order to identify key structures of putative agonist molecules associated with the 

selective activation of homomeric alpha7 receptors. We observed that selectivity between alpha7 

and alpha4beta2 was more readily obtained than selectivity between alpha7 and alpha3beta4. 

Based on structural comparisons of previously characterized selective and nonselective agonists, 

we hypothesized that there existed at least three chemical motifs which, when present in 

molecules containing an appropriate cationic center, could be associated with the selective 

activation of alpha7 receptors. We identify three distinct structural motifs, based on prototypical 

drugs: the choline motif, the tropane motif, and the benzylidene motif. The choline motif 

involves the location of an oxygen-containing polar group such as a hydroxyl or carbonyl 

separated by two carbons from the charged nitrogen. The tropane motif provides alpha7selectivity 

based on the presence of multiple hydrophobic groups positioned away from the 

cationic center at specific orientations. We show that this motif can convert the nonselective 

agonists quinuclidine and ethyltrimethyl-ammonium to the alpha7-selective analogs methylquinuclidine 

and diethyldimethyl-ammonium, respectively. We have previously shown that the 

benzylidene group of GTS-21 converts anabaseine into an alpha7-selective agonist. The 

benzylidene motif was also applied to quinuclidine to generate another distinct family of alpha7selective 

agonists. Our results provide insight for the further development of nicotinic 

therapeutics and will be useful to direct future experiments with protein structure-based 

modeling and site-directed mutagenesis. 

Supported by grant GM57481-05A2 

Disclosures: R.L. Papke , Targacept, F. Consultant/Advisory Board; Servier, F. 

Consultant/Advisory Board; F. Leonik, None; N.A. Horenstein, None. 

Poster 




Topic: B.02.a. Nicotinic acetylcholine receptors in brain: Physiology and function

Support: Supported By: Cephalon, Inc. 

Title: Interaction of methamphetamine (MET) methylphenidate (MPH) and modafinil (MOD) 

with cloned human α7 and α4β2 nicotinic acetylcholine receptors (nAChRs) 

Authors: S. BERTRAND 1 , D. BERTRAND 1 , D. BOZYCZKO-COYNE 2 , *M. J. MARINO 2 , 

M. WILLIAMS 2 ; 

1 HiQScreen Sàrl, Geneva, Switzerland; 2 Discovery Res., Cephalon, West Chester, PA 

Abstract: The mechanism of action of MOD, a novel wake-promoting agent, is unknown, with 

weak interactions at the dopamine (DAT; IC50 = 2-6 µM) and norepinephrine (NET; IC50 = 36 

µM) transporters being the only molecular targets identified in a panel of more than 150 drug 

sites. While MOD has some similarity in its actions to classical psychostimulant DAT inhibitors 

(e.g. MET, MPH), it differs from these agents in regard to a lack of abuse liability and the ability 

to elicit wake without inducing rebound hypersomnolence. DAT inhibitors like MET and 

bupropion (BUP) inhibit α7 nAChRs. The effects of MOD on the function of human α7 and 

α4β2 nAChRs expressed in Xenopus oocytes were compared with those of BUP, GBR-12909, 

MET and MPH. All compounds dose-dependently inhibited α7 nAChR function with a rank 

order: BUP (IC50 = 3.5 ± 0.9 µM) > GBR-12909 ≈ MET ≈ MPH≈ MOD. At α4β2 nAChRs the 

rank order of potency was BUP (IC50 = 390 ± 5 nM) >> GBR-12909 ≈ MET ≈ MPH >>MOD. 

BUP was approximately 10-fold selective for α4β2 versus α7 nAChRs while MOD was 

approximately 10-fold selective for α7 versus α4β2 nAChRs with the remaining compounds 

being active in the low (2-20) micromolar range. The selectivity of MOD for α7 nAChRs 

distinguishes this novel wake-promoting agent from the other DAT inhibitors studied. The brain 

concentration of MOD associated with wake promotion is estimated to be 36 µM (Moachon et 

al., 1996, Drugs Today 32:327-337), indicating that the selective inhibition of α7 nAChRs, 

together with the weak DAT activity may have functional significance and distinguish MOD 

from classical DAT blocking psychostimulants in its wake promoting actions. 

Supported By: Cephalon, Inc. 

Conflict of Interest: DBC, MJM and MW are employed by Cephalon, Inc. 

Disclosures: S. Bertrand, HiQScreen Sàrl, A. Employment (full or part-time); D. Bertrand, 

HiQScreen Sàrl, A. Employment (full or part-time); D. Bozyczko-Coyne, Cephalon, Inc., A. 

Employment (full or part-time); M.J. Marino, Cephalon, Inc., A. Employment (full or parttime); 

M. Williams, Cephalon. Inc., A. Employment (full or part-time). 

Poster 





Support: Philip Morris USA and Philip Morris International 

Title: Molecular and pharmacological characterization of recombinant nicotinic receptor 

α6β4β3 @ (β3 @ =val273ser) and α6β4β3 @ α5 

Authors: *B. DASH 1 , Y. CHANG 2 , R. J. LUKAS 2 ; 

1 Barrow Neurolo Inst., PHOENIX, AZ; 2 Barrow Neurolog. Inst., PHOENIX, AZ 

Abstract: Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits have attracted 

attention because of the particularly high abundance of α6 subunits in pleasure-reward centers of 

the brain, where they may play roles in nicotine dependence. Also concentrated in those brain 

regions are nAChR β3 subunits, which thus are prime candidates as assembly partners for α6 

subunits. Radioligand binding and knock-out mouse studies support the idea that nAChR α6 and 

β3 subunits co-assemble. However, it has been difficult to demonstrate function of 

heterologously expressed nAChR containing α6 and β3 subunits. Recently Dash et al. 

(Neuroscience 2007, San Diego, Poster 39.19) reported that the presence of nAChR β3 subunits 

has a dominant-negative effect on the function of α6β4*-nAChR subtypes. In continuation of our 

studies we further characterized the α6β4*-nAChR by heterologously expressing human mutant 

(V273S) β3 subunits with α6β4 and α6β4α5-nAChR subunits in Xenopus oocytes. The level of 

functional responsiveness to nicotinic agonists and antagonists were assessed using twoelectrode 

voltage-clamp recording. The order of potency displayed by nAChR α6β4β3 @ is 

sazetidine-A>varenicline>DMPP (1,1-dimethyl-4phenylpiperazinium)>cytisine>nicotine>acetylcholine. 

Similarly the order of potency for 

α6β4β3 @ α5 nAChR is sazetidine-A>varenicline>nicotine>cytisine. Both d-tubocurarine and 

mecamylamine inhibited α6β4β3 @ -nAChR (IC50 1.05 κM vs 20.7 κM). In order to translate the 

results from oocyte studies, we transfected SHEP-1 cells with α6, β4 and β3 @ subunits. At least 

one of the three selected clones expressing all the respective mRNAs appeared to display specific 

3 H-EBDN binding. We are in the process of furthering this research work with a focus on stable 

expression of functional α6*-nAChR. The ramifications of expression of dominantly negative, 

wild-type b3 subunits in α6*-nAChR remain to be determined. However, functional 

pharmacological profiles displayed in oocyte expression work are useful toward development of 

nicotinic analogs selective for α6β4*-nAChRs, that might illuminate mechanisms involved in 

reward, and of potential therapeutic use in treatment of nicotine dependence. 

Supported by Philip Morris USA and Philip Morris International through their external research 

program. 

Disclosures: B. Dash , None; Y. Chang, None; R.J. Lukas, None. 

Poster 

233. Nicotinic Aacetylcholine Receptors: Regulation and Function II




Support: NIH (NIDA) DA014369 

NIH (NIDA) DA017637 

Title: Interation of Chrna4 T529A polymorphism with S530 modulates α4β2 nAChR expression 

Authors: *J. A. WILKING 1,2 , K. G. HESTERBERG 1 , J. A. STITZEL 1,2 ; 

1 Inst. Behavioral Genet., Univ. Colorado at Boulder, Boulder, CO; 2 Dept. of Integrative Physiol., 

Univ. of Colorado, Boulder, CO 

Abstract: The mouse α4 nicotinic acetylcholine receptor (nAChR) subunit has several potential 

phosphorylation sites within the large cytoplasmic loop between transmembrane domains (TMD) 

III and IV. One of the potential phosphorylation sites, a serine in the large at amino acid position 

530 (S530), is immediately carboxyl terminal to a previously described polymorphism in the 

mouse α4 subunit. The polymorphism leads to a threonine:alanine variation at amino acid 529 

(T529A). Phosphorylation of S530 has been predicted to be influenced by the polymorphism at 

amino acid position 529. In order to examine the potential interaction between the α4 T529A 

polymorphism and S530, site directed mutagenesis was used to mutate S530 to either an alanine 

(A530) or an aspartic acid (D530) in both theT529 and A529 variants of α4. Afterwards, 

HEK293T cells were transiently transfected with one of the α4 variants and β2 and the affect of 

the mutations on receptor expression was assessed. [ 125 I]Epibatidine (200pM) binding indicated 

significant differences in total binding between α4A529 S530 and α4 T529 S530 co-transected 

with β2 (431.65 and 1826.41 fmol/mg respectively). Additionally, mutating α4 A529 S530 to 

either α4 A529 A530 or α4 A529 D530 increased binding to a level comparable to the T529 

variant. In contrast, mutating S530 to either A530 or D530 did not alter binding in the T529 

variant of α4. These data suggest that the effect of the α4 alanine/threonine variation on the 

expression α4β2 nAChRs is dependent upon the serine at amino acid position 530. These studies 

will increase our understanding of how the T529A polymorphism affects α4β2 nAChR 

expression. 

Disclosures: J.A. Wilking , None; K.G. Hesterberg, None; J.A. Stitzel, None. 

Poster 





Support: NIH grant MH53631 

Title: Molecular basis for differential sensitivity of human and rat α9α10 nAChRs to αconotoxins 

RgIA and Vc1.1 

Authors: *L. AZAM 1 , J. M. MCINTOSH 2 ; 

1 Dept Biol, 2 Dept. of Psychiatry and Biol., Univ. Utah, Salt Lake City, UT 

Abstract: The α9 nicotinic acetylcholine receptor (nAChR) subunit has gained considerable 

attention in recent years. It was originally functionally characterized in rat cochlear hair cells, but 

subunit transcripts and /or protein have also been reported in neuronal, as well as non-neuronal, 

tissue such as dorsal root ganglia, lymphocytes, alveolar macrophages, sperm, keratinocytes and 

the pituitary gland. In many tissues, α9 is co-expressed with α10; heterologously expressed 

α9α10 nAChRs display much larger macroscopic currents than homomeric α9 nAChRs. Except 

for its function at synapses of cochlear hair cells, the physiological role of the α9α10 nAChR 

subtype is largely unknown. Recently, however, it was demonstrated that α-conotoxin RgIA, a 

selective and potent blocker of both homomeric α9 and heteromeric α9α10 nAChRs, is analgesic 

in a rat model of nerve injury pain. α-conotoxin Vc1.1, another selective blocker of α9α10 

nAChRs, is also analgesic in rat models of neuropathic pain. Vc1.1 (ACV1) entered human 

phase II clinical trials with Metabolic Pharmaceuticals; subsequently, however, both Vc1.1 and 

RgIA were found to be ~100-fold less potent at human vs. rat α9α10 nAChRs. In this study, we 

have investigated the molecular basis of this species difference. Expressed rat α9 with human 

α10 subunit displayed the same affinity for RgIA compared to rat α9α10 nAChRs, implicating 

the human α9 subunit as the cause of decreased conotoxin sensitivity. Confirming this, 

heterologous expression of human α9 with rat α10 subunit displayed the same sensitivity to the 

toxin as human α9α10 nAChRs. The amino acid sequence of the N-terminal binding region of rat 

and human α9 subunits exhibits high homology, differing by only 8 amino acids. We are now 

systematically exchanging individual residues that differ in the human vs. rat α9 subunit to 

determine the residue(s) that potentially underlie the species difference in affinities for RgIA and 

Vc1.1. 

Supported by NIH grant MH53631. 

Disclosures: L. Azam, Financial support from Metabolic Pharmaceutical, C. Other Research 

Support (receipt of drugs, supplies, equipment or other in-kind support); J.M. McIntosh, 

Financial contribution from Metabolic Pharmaceutical, C. Other Research Support (receipt of 

drugs, supplies, equipment or other in-kind support). 

Poster





Support: NIH Grant DA13199 

NIH Grant MH53631 


NIH Grant GM48677 

Title: Fluorescent analogs of α-conotoxin ArIB: novel ligands for the study of α7 nicotinic 

acetylcholine receptors 

Authors: *A. J. HONE 1,2 , Y. XIAO 4 , S. CHRISTENSEN 3 , D. YOSHIKAMI 3,1 , J. M. 

MCINTOSH 2,3,1 ; 

1 Neurosci., 2 Psychiatry, 3 Biol., Univ. Utah, Salt Lake City, UT; 4 Pharmacol., Georgetown Univ., 

Washington DC, DC 

Abstract: The α7 nicotinic acetylcholine receptor (nAChR) is widely distributed throughout the 

central nervous system, and it has also been found on non-neuronal cells including lymphocytes 

and macrophages. These receptors are thought to be homomeric pentamers with five ligand 

binding sites; however, there is some evidence that they may also combine with additional 

subunits to form heteromeric receptors. Although much is known about the localization and 

function of α7 receptors within the central nervous system, much less is known about the role of 

these receptors in the cellular processes of non-neuronal cell types. For example, historically α7 

receptors have been thought of as ligand-gated ion channels that are highly permeable to Ca ++ , 

but recently it has been suggested that in lymphocytes, α7 subunits form receptors that may 

modulate cellular function by a means not involving Ca ++ entry. The detection of α7 receptors on 

lymphocytes and the elucidation of their function have been complicated by the lack of subtypeselective 

ligands that can discriminate between α7 receptors and those that contain α1 or α9 

subunits. To address this problem, we have labeled several analogs of α-conotoxin ArIB 

(Whiteaker, et al., Biochemistry, 2007) with fluorescent probes to create novel ligands that 

selectively bind α7 receptors. The pharmacology of these fluorescent ligands was assessed by 

two electrode voltage clamp electrophysiology of Xenopus laevis oocytes expressing specific 

subtypes of nAChRs. The fluorescent properties were assessed by fluorescence microscopy of 

HEK293 cells stably transfected with the rat α7 subunit gene. The conotoxin analogs differed in 

their biochemical properties and possessed unique pharmacological profiles. We hope to exploit 

them to study α7 receptors, particularly in tissues where multiple nAChR subtypes are expressed.

Disclosures: A.J. Hone , None; Y. Xiao, None; S. Christensen, None; D. Yoshikami, 

None; J.M. McIntosh, None. 

Poster 





Support: The Royal Society 

Title: Effects of nitrated cytisine on α4β2 and α7 receptors 

Authors: *A. L. CARBONE 1 , P. SÁEZ-BRIONES 3 , M. REBOLLEDO-FUENTES 4 , G. 

ZAPATA-TORRES 4 , G.-H. MANUEL 4 , M. MORONI 2 , B. K. CASSELS 4 , I. BERMUDEZ 2 ; 

1 Sch. of Life Sci., 2 Oxford Brookes Univ., Oxford, United Kingdom; 3 Univ. de Santiago de 

Chile, Santiago, Chile; 4 Univ. de Chile, Santiago, Chile 

Abstract: Cytisine, an alkaloid of the quinolizidine family, is a potent agonist at α4β2 and α7 

nicotinic acetylcholine receptors (nAChR). Halogen substituents on position C(3) or C(5) bring 

about remarkable changes in the radioligand binding affinity and functional potency and efficacy 

of cytisine. In the present study, we have nitrated cytisine at position C(3) or C(5) and 

determined the pharmacological consequences of the nitration on α4β2 and α7 using a 

combination of binding, functional and molecular modeling approaches. Radioligand binding 

assays showed that 3-NO2cytisine induces an increase in affinity at α4β2 and α7 receptors, 

whilst 5-NO2-cytisine causes a significant increase in binding affinity at both receptor types. 

Functional studies on receptors expressed heterologously in Xenopus laevis oocytes revealed that 

3-NO2-cytisine behaves as a low efficacy partial agonist of both high and low sensitivity α4β2 

receptors and as a full agonist of low potency at a7 receptors. In contrast, neither α4β2 nor α7 

nAChR are activated by 5-NO2-cytisine. Docking experiments revealed that 3-NO2-cytisine 

may adopt different orientations inside the binding site at α7 but not at α4β2 receptors, compared 

to cytisine. These studies also revealed that position C(5) of cytisine points towards charged side 

chains, which may underlie the loss of agonist activity observed for 5-NO2-cytisine. Our 

findings are in agreement with the existence of different structural requirements at each receptor 

subtype associated to affinity, functional potency and efficacy. 

Disclosures: A.L. Carbone , None; P. Sáez-Briones, None; M. Rebolledo-Fuentes, None; G. 

Zapata-Torres, None; G. Manuel, None; M. Moroni, None; B.K. Cassels, None; I. 

Bermudez, None.

Poster 






Title: Negative transcriptional regulation of the neuronal nicotinic acetylcholine receptor β4 

subunit gene promoter 

Authors: M. D. SCOFIELD, Z. MOU, *P. D. GARDNER; 

Brudnick Neuropsych Res. Inst., Univ. Massachusetts Med. Sch., Worcester, MA 

Abstract: Heterogenous nuclear ribonucleoprotein K (hnRNP K) was originally identified as a 

component of the hnRNP complex, a major group of chromatin-associated RNA-binding 

proteins. Further investigation of the hnRNP K protein revealed not only its high affinity for 

pyramidine-rich single-stranded DNA sequences, but also its ability to act as a potent DNAbinding 

transactivator. Interestingly, the hnRNP K protein has since been shown to function as 

either an activator or repressor of transcription from promoters containing CT-rich regions. We 

previously showed that hnRNP K interacts with a CT-rich region of the nicotinic acetylcholine 

receptor (nAChR) β4 subunit gene promoter in vitro. Importantly, the hnRNP K protein was also 

shown to negatively impact Sp factor binding to the β4 promoter. This finding was consistent 

with additional results demonstrating hnRNP K‟s ability to inhibit Sp-mediated transactivation 

from the β4 promoter. Here we extend these studies and show, using chromatin 

immunoprecipitation, that hnRNP K interacts with the CT element present in the β4 subunit gene 

promoter in vivo. Experiments are currently being performed in order to gain a better 

understanding of the regulatory role that hnRNP K plays at the β4 promoter. Our lab has also 

shown that an additional single-strand DNA-binding protein, Purα, binds to a purine-rich region 

of the β4 promoter present on the negative strand of the CT element. This finding is of particular 

significance due to the fact that hnRNP K and Purα have been reported to act together to 

negatively regulate transcription from a number of cellular promoters. Currently, experiments are 

being performed to elucidate the hnRNP K/Purα-mediated transcriptional mechanisms 

underlying expression of the β4 subunit gene. 

Disclosures: M.D. Scofield, None; Z. Mou, None; P.D. Gardner , None.

Poster 





Support: Targacept 

Title: Differences in the mechanism of functional up-regulation of high and low sensitivity α4β2 

nicotinic receptors induced by low temperature: electrophysiological evidence 

Authors: *N. B. FEDOROV; 

Preclinical, Targacept, Winston-Salem, NC 

Abstract: Widely expressed in the brain, the α4β2 neuronal nicotinic receptor (NNR) is 

proposed to play a major role in the mechanisms of learning, memory and attention. Using the 

patch-clamp technique and pharmacological methods, we examined the effects of incubating SH- 

EP1 cells expressing the major human brain α4β2 receptor at low temperature (29 degree 

Celsius). The acetylcholine dose-response curves were biphasic and revealed both highsensitivity 

(HS) and low-sensitivity (LS) components with apparent EC50 values of 4 and 50 

uM. Ratios of the number of receptors expressed as the HS and LS isoforms were 25% and 75%, 

respectively. Exposure of cell cultures to low temperature increased the fraction of HS receptors 

by 60% and receptor function was augmented 5 to 6-fold, based on current amplitude. These 

changes were not associated with apparent changes in current kinetics. LS up-regulated 

acetylcholine-evoked currents were increased by approximately 1.5-fold and were associated 

with significant changes in response kinetics, showing features of functional up-regulation that 

reflected less profound desensitization (k=5.0 and k=3.4). Using a selective agonist for the HS 

receptor isoform we confirmed these observations. These data indicate clear differences in the 

mechanisms of up-regulation for HS and LS forms of human α4β2 NNRs following incubation at 

low temperature. 

Disclosures: N.B. Fedorov , Targacept, A. Employment (full or part-time). 

Poster 





Support: the Peter F. McManus Foundation to WNG 

PO1 DA019695 

NIH grants NS043782 

DA019695 

DA13602 

Title: Mechanims underlying nicotine-induced upregulation of nicotinic acetylcholine receptors 

(nAChRs) 

Authors: *A. P. GOVIND 1 , H. S. WALSH 2 , Y. VALLEJO 3 , V. MCSWEEN 1 , W. GREEN 1 ; 

1 Neurobio., Univ. of Chicago, Chicago, IL; 2 UCtech, Univ. of Chicago, Chicago, IL; 3 Natl. Inst. 

of Neurolog. Disorders and Stroke, Bethesda, MD 

Abstract: Chronic nicotine exposure upregulates the number of high-affinity agonist binding 

sites found on α4β2, as well as α3- and α6-containing nicotinic acetylcholine receptors 

(nAChRs). We have investigated whether nicotine-induced upregulation results from changes in 

nAChR numbers or other mechanisms. Both nicotine treatment and a temperature shift from 37 

to 30°C caused similar increases in the number of high-affinity binding sites on α6β2 and α3β2 

nAChRs. While the temperature-induced increase in high-affinity binding sites corresponded 

with an increase in nAChR numbers, no change in nAChRs, either cell-surface or intracellular 

pools, was observed after nicotine treatment. α4β2 receptors differed from the other nAChRs in 

that the nicotine upregulation had two different effects on α4β2 nAChRs. Like the other 

nAChRs, nicotine-induced increases of α4β2 high-affinity binding sites were largely independent 

of a change in receptor number. These upregulated receptors appeared to undergo a 

conformational change caused by nicotine treatment as evidenced by: 1) a change in α4 subunit 

migration on SDS-PAGE and 2) a change in the binding of subunit-specific antibodies and other 

ligands. However, nicotine did cause a significant increase in receptor numbers that 

corresponded to a fraction of the increase in high-affinity binding sites. The increased α4β2 

receptor numbers coincided with a slowing of the degradation rate of intracellular β subunits. 

Additional evidence for two different mechanisms underlying α4β2 receptor nicotine-induced 

upregulation was the reversal of upregulation. Removal of nicotine after nicotine-induced 

upregulation caused upregulated high-affinity binding sites to decay at two very different rates. 

The fast rate (tau~1.5 hrs) corresponded to the fraction of high-affinity binding sites independent 

of a change in nAChR numbers and may be the reversal of a nicotine-induced conformational 

change. The slow decay rate (tau~18 hrs) corresponded with the increased number of nAChRs 

and was consistent with nAChR degradation rates.

Disclosures: A.P. Govind, None; H.S. Walsh, None; Y. Vallejo, None; V. McSween, 

None; W. Green, None. 

Poster 





Title: Pharmacological characterization of functional upregulation of human α4β2 neuronal 

nicotinic acetylcholine receptors by reduced temperature 

Authors: *J. ZHANG, T. HAUSER, V. GRINEVICH, M. BENCHERIF; 

Targacept, Winston Salem, NC 

Abstract: The existence of two functional subpopulations of α4β2 neuronal nicotinic 

acetylcholine receptors (NNRs) has been demonstrated both in vivo and in vitro. While most of 

the studies have focused on upregulation of α4β2 NNRs by nicotine treatment, very few reports 

have addressed the effect of temperature. It has been demonstrated previously that lower 

temperature increases cell-surface expression and total radioligand binding of α4β2 NNRs 

transfected in human embryonic kidney cells. In the present study, using the SH-EP1 epithelial 

cell line stably transfected with human (h) α4β2 NNRs, we observed a time-dependent increase 

in the maximal response and decrease in the EC50 for nicotine- and acetylcholine (ACh)-induced 

elevation of intracellular calcium (Ca 2+ ) levels by maintaining cells at 29 o C, with a plateau 

reached around 24 hours. This 24 hour treatment condition was used in all later experiments. 

When normalized to the response of 10 κM nicotine at 37 o C, the maximal Ca 2+ influx responses 

were 110 % and 186 % for nicotine, and 129 % and 198 % for ACh in cells maintained at 37 o C 

and 29 o C respectively. A rank order of potency for agonist of epibatidine > nicotine > ACh > 1, 

1-dimethyl-4-phenylpiperazinium > cytisine, and for antagonist of dihydro-β-erythroidine > 

methyllycaconitine > mecamylamine on inhibition of 1 κM nicotine-induced Ca 2+ influx, were 

revealed in cells cultured at lower temperature. We also observed that the functional upregulation 

was still partially preserved 24 hours after switching the culture temperature back to 37 o C. In 

addition, the ligand binding properties of hα4β2 NNRs expressed under both temperature 

conditions were assessed with radioligand saturation binding using cell membrane preparations. 

We observed that the receptors expressed on membranes of cells cultured at 29°C exhibited 

different properties compared to receptors expressed by cells grown at 37°C, by means of 

binding capacity and affinity. Together, our results demonstrate that in response to lower 

temperature conditions, the Ca 2+ influx responses for hα4β2 receptors are increased, suggesting a 

functional upregulation of hα4β2 NNRs.

Disclosures: J. Zhang, None; T. Hauser, None; V. Grinevich, None; M. Bencherif, None. 

Poster 





Support: an anonymous donation 

Title: Do genes play a role in nicotine-induced α4β2 receptor upregulation? 

Authors: *V. HOSUR, R. H. LORING; 

Pharmaceut. Sci., Northeastern Univ., Boston, MA 

Abstract: Nicotine is the major reinforcing component of tobacco smoke and chronic tobacco 

smoking upregulates α4β2 nicotinic acetylcholine receptors (nAChRs). Post-mortem and clinical 

studies show elevated levels of α4β2 nAChRs in several cortical and sub-cortical regions of 

smokers' brain. The α4β2 nAChRs constitute the major proportion (90%) of high-affinity 

nicotinic receptor subtypes and have been implicated in neurodegenerative diseases, cognition, 

and attention-related disorders. We investigated the cellular and molecular mechanisms 

underlying nAChR upregulation based on previous results obtained from gene-chip analysis 

(Society for Neuroscience abstract # 723.22, 2005). Human neuroblastoma cells (SH-EP1) stably 

expressing human α4β2 nicotinic receptors were treated with 10κM nicotine for 1h, 8h and 24h. 

Quantitative RT-PCR validated the gene-chip predicted changes in mRNA expression of 

cytokines (IL-1β and IL-6), chemokines (CXCL2) and transcription factors (CREM). Exposure 

to nicotine significantly lowered the expression of IL-1β, IL-6 and CXCL2, and increased the 

expression of CREM, whereas untransfected wild type SH-EP1 cells did not show these nicotinemediated 

gene alterations. Co-application of dihydro-β-erythroidine (dHβE) or mecamylamine 

(Mec) potentiated nicotine-induced upregulation; however, both dHβE and Mec blocked 

nicotine-induced down-regulation of IL-1β and IL-6. In contrast, antagonist-mediated 

potentiation of α4β2 receptor upregulation correlates (0.98, p

chronic effects of nicotine in vivo. We thank Ron Lukas for hα4β2 SH-EP1 cells and Michael 

Marks for mouse brain samples. 

Disclosures: V. hosur, None; R.H. Loring, None. 

Poster 

234. A-Type Potassium Channels 



Topic: B.04.c. Potassium channels: Physiology 

Title: Activity-dependent A-type K+-channel trafficking in CA1 pyramidal dendrites in 

recordings from adult mouse hippocampal neurons 

Authors: *E.-T. HAHM, D. HOFFMAN; 

NICHD, NIH, Bethesda, MD 

Abstract: A-type K+ currents (IA) are rapidly activating and inactivating and have been shown 

to play an important role in regulating neuronal excitability. The A-type K+ channel subunit 

Kv4.2 underlies the A-current in the dendrites of cultured hippocampal CA1 neurons and live 

imaging showed that Kv4.2 internalization is induced rapidly upon glutamate receptor 

stimulation (Kim et al., 2007). We report here the activity-dependent decrease of IA in the 

dendrites of CA1 pyramidal neurons in acute hippocampal slices of adult mice. IA was measured 

in cell-attached patch-clamp recordings from the soma and at various locations on the primary 

apical dendrite (up to ~300 µm from soma). In somatic recordings, brief bath application of 

AMPA (5 min, 50 µM) caused a reduction in peak A-current amplitude, similar to our previous 

results reported in cultured hippocampal neurons (Kim et al., 2007). No change in the sustained, 

or slowly inactivating K+ currents was found upon AMPA treatment. The transient current peak 

amplitude in somatic recordings was 16.9 ± 2.0 pA pre-AMPA treatment and decreased to 8.3 ± 

1.9 pA (n = 7, p = 0.002) afterward. The sustained current peak amplitude was unchanged upon 

AMPA stimulation (pre: 10.4 ± 1.6 pA; AMPA: 8.4 ± 2.2 pA; n = 7; p = 0.84). In apical 

dendrites, AMPA caused a selective decrease in peak A-current amplitude from 11.9 ± 2.2 pA to 

6.9 ± 2.4 pA (n = 3; p = 0.046) with no change in sustained current amplitude (pre: 14.1 ± 4.0 

pA; AMPA: 14.9 ± 8.6 pA; n = 3; p = 0.50). 

Disclosures: E. Hahm , None; D. Hoffman, None.

Poster 




Topic: B.04.f. Channel trafficking 

Title: Kv4.2 C-terminal interaction with KChIP4a: effects of phosphorylation on binding and 

surface expression 

Authors: *L. LIN, A. M. WIKENHEISER, D. A. HOFFMAN; 

LCSN, NICHD-NIH, Bethesda, MD 

Abstract: Voltage-gated potassium (Kv) channels play important roles in regulating the 

excitability of myocytes and neurons. Kv4.2 is a key constituent of the A-type channel in CA1 

pyramidal neurons of the hippocampus and is critically involved in the regulation of dendritic 

excitability and plasticity. K + channel-interacting proteins, KChIPs (KChIP1-4), have been 

shown to associate with the N-terminal of Kv4.2 and modulate its biophysical properties (An et 

al., 2000). KChIP4a, which has a unique KIS (K-channel inactivation suppressor) domain 

(Holmqvist et al., 2002), abolishes fast inactivation of Kv4 currents in various cell types. In the 

present study, we investigated the role of the cytoplasmic C-terminal of Kv4.2 in the interaction 

with KChIP4a and the effects of Kv4.2 phosphorylation sites on surface expression regulation by 

KChIP4a. We made a series of Kv4.2 C-terminal truncation mutations (560Γ, 490Γ, 417Γ and 

406Γ), and CO-IP results show that only 560Γ and 490Γ can interact with KChIP4a. 

Immunostaining showed these two proteins co-localize in COS7 cells. These results suggested 

C-terminal Kv4.2 plays a critical role in the interaction. We then made site-directed mutations, 

changing the phosphosite serine (S) or Threonine (T) to Alanine (A) to remove the 

phosphorylation sites on PKA (mPKA), PKC (mPKC), ERK (mERK) and CaMKII (mCaMKII). 

We found that the interaction of Kv4.2 and KChIP4a requires the C-terminal of Kv4.2, but not 

phosphorylation of Kv4.2 on the C-terminal. Biotinylation experiments showed that cotransfection 

of Kv4.2 and KChIP4a in COS7 cells results in a 1.5-fold increase in Kv4.2 surface 

expression, compared to Kv4.2 alone. Enhanced surface expression by KChIP4a was abolished 

by KChIP4a siRNA, and reduced by mPKA, mERK or mCaMKII Kv4.2 mutations. These data 

suggest that PKA, ERK and CaMKII phosphorylation sites of Kv4.2 play an important role in 

the trafficking of Kv4.2 through its interaction with KChIP4a. 

Disclosures: L. Lin , None; A.M. Wikenheiser, None; D.A. Hoffman, None. 

Poster





Support: Academy of Finland 

Finnish Cultural Foundation 

Title: TIRF study of pHluorin-tagged Kv4.2 trafficking in cultured neurons 

Authors: *E. PRYAZHNIKOV 1 , S. COLEMAN 2 , K. KEINÄNEN 2 , A. JEROMIN 3 , L. 

KHIROUG 1 ; 

1 Neurosci. Ctr., 2 Div. of Biochem., Univ. of Helsinki, Helsinki, Finland; 3 Allen Inst. for Brain 

Sci., Seattle, WA 

Abstract: The A-type current plays a significant role in neuronal excitability by regulating the 

propagation of action potentials. Kv4.2 is a key subunit of the A-type channel. Therefore it is 

important to study the processes of Kv4.2 trafficking, internalization and insertion in plasma 

membrane. 

In our work we studied trafficking of pHluorin tagged Kv4.2 subunit in cultured hippocampal 

neurons using Total Internal Reflection Fluorescence (TIRF) microscopy. TIRF is an advanced 

optical microscopy method based on unique properties of evanescent field that is created on the 

surface of cell-bearing glass due to total internal reflection of the laser beam. Evanescent field 

allows selective visualization of those fluorescent molecules which are located in the immediate 

vicinity (~100 nm) of the plasma membrane of cultured cells. PHluorin has the distinctive 

feature that it is fluorescent at pH 7.4 and nonfluorescent at pH



Support: Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and 

Technology of Japan 

Keio Gijuku Academic Development Funds 

Kanehara Foundation Research Grant 

Title: Regulation of Kv4.2 by neuronal activity 

Authors: *M. NURIYA, T. ITO, M. YASUI; 

Pharmacol, Keio Univ., Shinjuku, Japan 

Abstract: Voltage gated ion channels are crucial to neuronal functions both in physiological and 

pathological conditions. Kv4.2 constitutes a major component of A-type potassium current and is 

localized specifically at dendrites and soma, where it plays critical roles in propagations of 

voltage information by regulating the excitability of dendrites. While functional modifications of 

Kv4.2 by its phosphorylations through several signal transduction pathways have been described, 

physiological stimuli that modify these phosphorylation states remain mostly unknown. Here, we 

report that neuronal activity induces rapid and specific dephosphorylation of Kv4.2 in primary 

cultured cortical neurons. Activation of N-methyl-D-aspartic acid (NMDA) receptor but not αamino-3-hydroxy-5-methylisoxazole-4-propionic 

acid (AMPA) receptor is necessary and 

sufficient for this dephosphorylation. As bulk calcium influx by ionomycin does not mimic this 

NMDA receptor activation effect, some specific signal transductions other than a mere rise of 

cytoplasmic calcium concentration appear to be responsible for this rapid dephosphorylation of 

Kv4.2. Finally, this reaction occurs both at the plasma membrane as well as inside the cell and 

does not change surface expression level of Kv4.2 by itself. These data indicate that Kv4.2 

phosphorylation is dynamically regulated in neurons and that neuronal activity and following 

activation of NMDA receptor is a strong regulator of Kv4.2. This activity dependent 

modification of Kv4.2 may underlie activity-dependent changes of dendritic excitability under 

physiological conditions. 

Disclosures: M. Nuriya, None; T. Ito, None; M. Yasui, None. 

Poster 





Support: NFR 170502 

Title: Local application of 4-AP shows that hippocampal CA3 cell axons have a latent burst 

behaviour that can increase transmitter release 

Authors: D. PALANI, *M. RAASTAD; 

Dept Neurophysiol, Inst. Basic Med. Sci., 0317 Oslo, Norway 

Abstract: At hippocampal CA3->CA1 synapses, 4-AP increases neurotransmitter release. 

Because our previous recordings from individual Schaffer collaterals showed that these axons 

can fire bursts of action potentials in response to single electrical stimuli when exposed to 1 - 2 

mM 4-AP, we wanted to investigate if such bursts contributed to the enhanced transmitter 

release. The alternative hypothesis was that broadening of the action potential accounted for all 

the increase in transmitter release. 

We recorded field excitatory synaptic potentials (fEPSPs) at Schaffer collateral paths in stratum 

radiatum after surgically removing CA3 somata. We blocked NMDA and GABA currents with 

APV (100 µM) and picrotoxin (20 µM). To be able to follow both the compound action potential 

and the fEPSP we reduced the AMPA currents by adding 1 µM CNQX. 

We recorded fEPSPs by activating axons with two stimulation electrodes positioned on opposite 

sides of the recording electrode along the Schaffer collateral paths in stratum radiatum. One of 

the electrodes was filled with 2 mM 4-AP, the other just with extracellular solution, and served 

as a control for direct influence of 4-AP that may have diffused from the test electrode to the 

recording site. 

We observed that the fEPSP in response to the 4-AP filled electrode increased by 124.6 % ± 54 

(n = 6) while the control response remained stable. The compound action potential, on the other 

hand, remained stable in amplitude with test and control stimulation. 

Our interpretation of the finding that the increase in fEPSC only occurred when the stimulation 

was applied by the 4-AP filled electrode is that this electrode induced a burst of action potentials 

due to block of potassium currents at the stimulation site. 

Thus, there may be two distinct excitability enhancing effects of 4-AP, one due to spike 

broadening, and one due to disclosure of a latent burst behaviour of the axons. The first effect is 

sensitive to low concentrations of 4-AP, and the second sensitive to high concentrations of 4-AP, 

and the effects may therefore be mediated by different types of potassium channels. 

Disclosures: D. Palani, None; M. Raastad , None. 

Poster 

234. A-Type Potassium Channels




Support: NIH National Institute of Neurological Disorders and Stroke 

Title: Molecular dissection of functional IA channels in cortical neurons 

Authors: *A. J. NORRIS 1 , J. M. NERBONNE 2 ; 

1 Developmental Biol., Washington Univ., St. Louis, MO; 2 Developmental Biol., Washington 

Univ., Saint Louis, MO 

Abstract: Somatodendritic IA channels, encoded by voltage-gated K+ channel (Kv) α-subunits, 

are important for the normal physiological functioning of cortical neurons. Previous studies have 

shown that the Kv4.2 α-subunit encodes a rapidly inactivating and rapidly recovering A-type Kv 

current in cortical neurons. Mounting evidence indicates that native Kv4 encoded channels 

function as macromolecular complexes composed of pore-forming Kv α-subunits, accessory 

subunits and additional regulatory molecules. Although the various Kv4 channel accessory 

subunits are hypothesized to control the surface expression and kinetic properties of native 

channels, delineation of the precise physiological roles of these subunits is confounded by the 

co-expression of many types of Kv channels in neurons. Specifically, studies in neurons lacking 

Kv4.2 (Kv4.2-/- ) demonstrated a second, rapidly inactivating, but slowly recovering, current. 

Subsequent experiments using a mutant Kv4.2 construct that functions as a dominant negative 

yielded similar results and the further addition of 1mM 4-AP blocked the remaining rapidly 

inactivating current suggesting that a Kv1 subfamily α-subunit encodes this current. Further 

experiments on neurons from mice lacking Kv1.4 demonstrated a marked reduction in the slowly 

recovering A-type current. Taken together, these results indicate that Kv1.4 and Kv4.2 encode 

kinetically distinct components of IA in cortical neurons. To elucidate the physiological roles of 

individual Kv α and accessory subunits in neurons, we have developed interfering RNA 

constructs and combined these with targeted genomic deletions and in vitro dominant-negative 

strategies. Plasmids yielding both efficient microRNA (miRNA) mediated knockdown of Kv4.2 

α and accessory subunits as well as robust fluorophore expression for visual identification of 

transfected neurons were generated. To examine the effects of acute, specific disruption of Kv4.2 

expression and to examine the turnover rate of Kv4 channel complexes in cortical neurons, 

miRNA targeting Kv4.2 was electroporated into isolated cortical neurons and whole cell 

electrophysiological recordings were obtained. Approximately 24 hours post transfection, the 

Kv4.2 encoded, rapidly inactivating, rapidly recovering current was markedly reduced revealing 

the second rapidly inactivating but slowly recovering (Kv1.4 encoded) current similar to findings 

in Kv4.2 -/- neurons. These results demonstrated that Kv4.2 channel complexes turnover quickly 

and that miRNA can be employed in combination with knockout mice to determine the specific 

roles of Kv4 channel accessory subunits in the generation of native IA. 

Disclosures: A.J. Norris, None; J.M. Nerbonne, None.

Poster 





Support: NIHR01066388 

Heartland affiliate of the American Heart Association 

WashU/Pfizer Biomedical Research Agreement 

Title: The sodium channel accessory subunit Navb1 associates with brain Kv4.2 and modulates 

the functional expression of Kv4.2-encoded channels 

Authors: C. MARIONNEAU 1 , A. NORRIS 1 , R. R. TOWNSEND 2 , A. J. LINK 3 , *J. M. 

NERBONNE 1 ; 

1 Dept Molec Biol & Pharmacol, 2 Cell Biol. and Physiol., Washington Univ. Sch. Med., St. 

Louis, MO; 3 Dept. of Microbiology and Immunol., Vanderbilt Univ. Med. Ctr., Nashville, TN 

Abstract: Somatodendritic A-type (IA) voltage-gated K+ (Kv) channels are key regulators of 

neuronal excitability, functioning to modulate the back-propagation of action potentials and 

synaptic plasticity. Although progress has been made in identifying the primary components of 

Kv4-encoded IA channels, increasing evidence suggests that additional regulators are involved in 

modulating the functional expression and the properties of these channels. To characterize the 

components of native Kv4.2-encoded IA channel complexes, a polyclonal anti-Kv4.2 antibody 

was used to immunoprecipitate Kv4.2 from mouse brain and co-purifying proteins were 

identified using a mass spectrometry (MS)-based proteomic approach. In addition to the Kv4 

pore-forming α subunits (Kv4.2, Kv4.3, Kv4.1) and the previously described accessory subunits 

KChIP1-4 and DPP6/DPP10, the sodium channel accessory subunit Navβ1 was identified. Eight 

tandem MS spectra identifying the Navβ1 peptide YENEVLQLEEDERFEGR were acquired 

from Kv4.2 immunoprecipitates from wild-type brain lysates, but were absent in control samples 

obtained from Kv4.2 targeted deletion (Kv4.2-/-) brains. Although Navβ1 has previously been 

reported to regulate the functional expression of voltage-gated Na+ channels in heterologous 

expression systems, these MS-based results suggest the interesting possibility that Navβ1 might 

also be a functional component of Kv4.2-encoded IA channels. The ability of Navβ1 to modulate 

the functional expression of Kv4.2 channels in HEK-293 cells was investigated, and compared 

directly with the effects of KChIP2. Co-immunoprecipitation experiments confirmed that Navβ1 

binds to Kv4.2, but not to KChIP2 or Kv2.1. Whole-cell voltage-clamp recordings revealed that,

similar to KChIP2, Navβ1 increases Kv4.2-encoded current densities. When both KChIP2 and 

Navβ1 were co-expressed (with Kv4.2), Kv current densities were increased dramatically 

compared with the effects of expression of either Navβ1 or KChIP2 alone. To explore the 

underlying mechanisms, total and cell surface expression of Kv4.2 were analyzed by Western 

blot and cell surface biotinylation assays, respectively. These experiments revealed that Navβ1 

increases total Kv4.2 protein expression by 3-fold whereas KChIP2 augments both total (by 4fold) 

and cell surface (by 3-fold) Kv4.2 protein expression. When the two accessory Navβ1 and 

KChIP2 subunits were co-expressed, the effects were cumulative and resulted in dramatic 

increases in total and cell surface expression of Kv4.2. Taken together, these results suggest that 

Navβ1 functions as a modulator of neuronal Kv4-encoded A-type K+ channels. 

Disclosures: C. Marionneau, None; J.M. Nerbonne , None; A. Norris, None; R.R. Townsend, 

None; A.J. Link, None. 

Poster 





Support: Wellcome Trust 

Hungarian Research Fund 49100 

Title: Synaptic input-specific localization of A-type potassium channels in the thalamus 

Authors: K. GIBER, M. KOLLO, H. BOKOR, Z. NUSSER, *L. ACSADY; 

Cell. and Network Neurobio., Inst. Exp. Med. Hung Acad Sci., Budapest H-1450, Hungary 

Abstract: A-type potassium-channels are responsible for a transient K+ current, which plays 

important role in modulating the integrative properties of dendrites. Among the voltagedependent 

K+ channels (Kv) the Kv4.2 and Kv4.3 subunits are responsible for the majority of Acurrents 

in the brain. The localization of these proteins is unknown in thalamic relay cells. Thus, 

in the present study the distribution of Kv4.2 and Kv4.3 subunits were studied in different 

thalamic nuclei and along various somato-dendritic domains of relay cells using 

immunocytochemistry at the light and electron microscopic level. 

At the light microscopic level Kv4.2 and Kv4.3 subunits gave a weak homogeneous neuropil 

immunostaining in most thalamic nuclei. In addition, both subunits intensely labeled short 

segments of relay cell proximal dendrites selectively in the primary sensory nuclei. Double

immunostaining with a marker for sensory afferents and either Kv subunits revealed that almost 

all Kv-positive dendritic segments (95%) are contacted by large subcortical terminals and the 

vast majority of these terminals (94%) innervate strongly Kv-positive parts of the dendrites. In 

sharp contrast, in higher order thalamic nuclei only the minority of subcortical afferents (20%) 

and none of the giant layer V cortical terminals contacted strongly Kv4.2 and Kv4.3 

immunopositive dendritic segments. The input-specific K channel localization in the primary 

sensory nuclei was confirmed with a preembedding immunogold labeling at the electron 

microscopic level. The density of both Kv4.2 and Kv4.3 subunit signals was highest in proximal 

dendritic membranes innervated by giant terminals, the labeling being the strongest in spine-like 

dendritic appendages of relay cells. 

Our data demonstrate input specific accumulation of A-type potassium channels in relay cells of 

primary sensory nuclei. The strong association of large subcortical afferents with Kv4.2 and 

Kv4.3 subunits on the postsynaptic side suggests an effective curtailing of subcortical sensory 

EPSPs by A-type K current. The data also indicate that in higher order thalamic nuclei giant 

excitatory terminals-evoked EPSPs are not modulated by A-current, suggesting differential 

summations of “driver” EPSPs in first- and higher-order under thalamic nuclei. 

Disclosures: K. Giber, None; L. Acsady , None; H. Bokor, None; M. Kollo, None; Z. Nusser, 

None. 

Poster 





Support: Hertie Foundation 

BMBF 

Title: A-type potassium channels contribute to the functional diversity of the dopaminergic 

midbrain system 

Authors: *S. KRABBE 1 , S. LAMMEL 1 , B. LISS 2 , J. ROEPER 1 ; 

1 Inst. for Neurophysiol., Goethe Univ. Frankfurt, Frankfurt, Germany; 2 Inst. for Gen. Physiol., 

Univ. Ulm, Ulm, Germany 

Abstract: The dopaminergic (DA) midbrain system is important for a variety of brain functions 

like motor control, working memory and reward, and is involved in related disorders, including

Parkinson disease, schizophrenia and drug addiction. We recently showed that this midbrain 

system consists of two highly distinct types of DA neurons with different anatomical, molecular 

and functional properties (Lammel et al. 2008, Neuron). To further investigate the 

electrophysiological characteristics of these distinct “classical” substantia nigra (SN) and 

“unconventional” ventral tegmental area (VTA) DA neurons, we combined selective retrograde 

tracing of two respective projection areas - the dorsal striatum (DS) and the nucleus accumbens 

(NAc) core - in 3-month old C57bl6 mice with in-vitro patch-clamp recordings. Using nucleated 

outside-out patches of retrogradly identified DA neurons, we studied the biophysical properties 

of subthreshold A-type potassium currents. The A-type currents recorded from DA VTA neurons 

projecting to NAc core inactivated with significantly slower kinetics (mean time constant of 

inactivation; taui=69.3±8.4 ms at -20 mV, n=11) compared to those in nigrostriatal DA neurons 

(taui=29.4±1.7 ms, n=17; p

Authors: *Z. M. KHALIQ, B. P. BEAN; 

Dept Neurobiol, Harvard Med. Sch., Boston, MA 

Abstract: A-type potassium current (IA) activates and inactivates at subthreshold voltages and is 

associated with slow tonic firing. Experiments in a variety of neurons have shown considerable 

overlap in the steady-state activation and inactivation curves for IA. This overlap implies the 

presence of steady-state or “window” IA in the region of overlap, at least for Hodgkin-Huxleylike 

models with independent activation and inactivation processes. We tested for the presence of 

steady-state IA in dopaminergic neurons of the VTA recorded in brain slices prepared from 2-3 

week old mice (34 ºC). VTA neurons are a favorable preparation because there is little 

subthreshold current other than IA and persistent sodium current. The voltage-dependence of 

activation and steady-state inactivation of IA could be fit reasonably well by Boltzmann curves. 

Activation and inactivation curves overlapped in the voltage range between -70 and -60 mV. 

Despite this overlap, however, there was no measurable steady-state voltage-dependent outward 

current in this voltage range when assayed by very slow ramps (5 mV/s) in the presence of 

tetrodotoxin. Clear voltage-dependent outward current corresponding to IA was however evident 

with ramps delivered at faster rates (>10 mV/s), activating between -70 and -65 mV and reaching 

a plateau near -45 mV. Thus, the overlap of activation and inactivation curves does not 

necessarily imply the existence of steady-state window current. A direct illustration of this was 

seen when a 2-sec prepulse to -68 mV was followed by a step to -48 mV to assay inactivation. 

During the step to -68 mV, IA activated and then inactivated with a time constant of about 200 

ms. Decay of the current at -68 mV was complete (within a resolution of ~3-5 pA). Despite this, 

however, steady-state inactivation at -68 mV was incomplete as assayed by the subsequent test 

pulse to -48 (the measurement used for defining inactivation curves), which elicited a sizeable 

transient current. Thus, at -68 mV there is some activation and incomplete inactivation (as 

assayed by a subsequent -48 mV test pulse), yet no measurable steady-state current. The gating 

behavior of IA was identical in nucleated patches where there is no concern about voltage 

control. From the resolution of the measurements, we estimate that any steady-state conductance 

at subthreshold voltages amounts to less than 0.05% of the peak transient conductance. This is 

very different than for gating of voltage-dependent sodium channels. This behavior of IA is ideal 

for enabling low frequency pacemaking because IA is not present during very slow spontaneous 

depolarizations but activates with faster depolarization rates to exert a slowing influence. 

Disclosures: Z.M. Khaliq , None; B.P. Bean, None. 

Poster 





Support: Wayne State University 

Title: The potassium channel kv4.2 is expressed in subtypes of retinal ganglion cells 

Authors: *L. CHEN, J. QU, K. L. MYHR; 

Dept Biol Sci., Wayne State Univ., Detroit, MI 

Abstract: Transient potassium currents mediated by Kv4.2, which are found in hippocampal 

neurons, regulate excitability by controlling action potential generation, excitatory postsynaptic 

potentials and back-propagating action potentials. The regulation of Kv4.2 channels by 

catecholamines can change the ability of hippocampal neurons to undergo long term potentiation 

(LTP). Kv4.2 is expressed in retinal ganglion cells (RGCs). Therefore, the regulation of Kv4.2 

may play a role in neural plasticity in the retina, such as the catecholamine-mediated regulation 

of light and circadian adaptations. Kv4.2 is expressed in only some RGCs and in two bands in 

the inner plexiform layer (IPL), which suggests expression in subtypes of RGCs, but the identity 

of the subtypes that express Kv4.2 is unknown. It is important to identify the subtypes of RGCs 

that express Kv4.2 to determine the role of Kv4.2 in adaptations of retinal function. We 

characterized the morphology and the fast, transient potassium currents of Kv4.2-IR positive and 

negative RGCs. The morphologies of the labeled RGCs were studied using three different filling 

methods: YFP transgenic mice, DiO injection of retinas first immunolabeled for Kv4.2 and patch 

filling with Alexa dye. Among Kv4.2-IR positive cells, three main morphological categories 

were identified, although several Kv4.2-IR positive RGCs did not fit into these three groups. Of 

the RGCs in which the transient potassium current, IA, was assayed, all of the RGCs that 

expressed Kv4.2 also had a fast, transient potassium current that had the expected characteristics 

of Kv4.2 currents. None of the RGCs that lacked IA expressed Kv4.2. Some RGCs that did not 

have Kv4.2 also had fast, transient potassium currents, which are likely to be due to other 

potassium channels. These findings support the hypothesis that Kv4.2-IR positive RGCs form 

several functional subtypes in the retina, in which Kv4.2-mediated currents contribute to the 

plasticity of retinal processing. 

Disclosures: L. Chen, None; K.L. Myhr, None; J. Qu, None. 

Poster 




Topic: B.04.c. Potassium channels: Physiology

Title: Messenger RNA and protein expression analysis of voltage-gated potassium channels, 

Kv1.4 and Kv4.2, in rat facial nucleus after axotomy 

Authors: *M. ARDEHALI, P.-P. VIDAL, I. VASSIAS, C. DE WAELE; 

Dept LNRS, CNRS, Paris, France 

Abstract: Facial nerve axotomy represents a model of the plasticity of cranial nerves. Several 

studies have tried to describe subcellular changes that contribute to plastic changes in facial 

motoneurons. In these experiments, after axotomy, a survey is made of the facial nucleus for a 

particular synaptic transmitter, synaptic transmitter receptor or subcellular signalling molecule. 

Morphological analyses in our laboratory by in situ hybridization and immunohistochemistry 

have already demonstrated modifications in the expression of subunits of GABA and 

glutamatergic receptors in axotomized facial motoneurons. 

It has been hypothesized that modifications in voltage-gated potassium channels contribute to 

plastic changes in facial nuclei neurons. The present study was designed to define whether the 

expression of Kv1.4 and Kv4.2 receptor mRNA and protein is modified in facial nucleus 

following unilateral axotomy. 

The study was carried out on 24 adult male pigmented Long Evans rats. The facial nerve trunk 

was unilaterally sectioned and the animals were sacrificed at 3, 8, 30, and 60 days after the 

surgery. Kv1.4 and Kv4.2 receptor mRNA and protein expressions were analyzed by in situ 

hybridization and immunohistofluorescence methods. 

Our data revealed no difference in Kv1.4 and Kv4.2 receptor mRNA and protein labeling 

between anotomized and intact facial nuclei at any time following the lesion. This was the same 

in the control group. 

In conclusion these findings demonstrate that Kv1.4 and Kv4.2 receptors do not play a role in the 

plasticity of facial nerve nuclei. Our study might give clues for the understanding of molecular 

mechanism of the plasticity of facial nerve motoneurons. 

Disclosures: M. Ardehali , None; P. Vidal, None; I. Vassias, None; C. de Waele, None. 

Poster 






NIH Grant NS30989

Title: Electrophysiological and behavioral characterization of DPPX (DPP6) knock out mice 

Authors: *E. W. ZAGHA 1 , J. K. MAFFIE 2 , H.-Y. JEONG 2 , M. NADAL 2 , B. CLARK 2 , E. 

KWON 2 , E. M. GOLDBERG 2 , B. RUDY 2 ; 

1 Dept Physiol & Neurosci, 2 Smilow Neurosci. Program, Dept. Physiol. and Neurosci., NYU Sch. 

of Med., New York, NY 

Abstract: The subthreshold-activating somato-dendritic A-type K+ currents in neurons (ISA) is 

essential for the proper functioning of the brain and the heart. The rapid, transient activation of 

this current in the subthreshold range of membrane potentials causes delayed excitation, helps 

determine the duration of the interspike interval thus regulating the frequency of repetitive firing 

and is proposed to have important roles in signal processing in dendrites. Evidence now supports 

the view that ISA channels in neurons are ternary complexes of a pore forming subunit of the 

Kv4 family as well as two types of associated proteins KChiPs and the Dipeptidyl peptidase like 

proteins DPPX and DPP10. These accessory proteins have been shown to associate with Kv4 

subunits and to restore the in vivo current properties to channels expressed in heterogonous cells. 

Furthermore most neurons that prominently express DPPX proteins also prominently express 

Kv4.2 proteins, and both are enriched in dendritic processes. Areas of DPPX expression include 

the hippocampus, cerebellar cortex, striatum and several brainstem nuclei. Moderate expression 

for both Kv4s and DPPX are observed in thalamus and neocortex. To further explore the role of 

this protein we generated a knockout animal using the Cre-Lox P system to remove exon II, 

thereby removing the conserved transmembrane domain and disrupting the reading frame. Use of 

the Cre- Lox P system will allow for cell type and temporally restricted removal of the protein. 

Deletion of exon two in the germline yields mice that lack immunohistologicaly detectable 

DPPX protein, are viable and lack gross anatomical defects. We explored the ISA in cerebellar 

granule cells in acute slices, given the suitability of these neurons for good quality whole cell 

voltage clamp recording. Consistent with expectations from experiments in heterologous cells, 

we observed depolarizing shifts in the activation and steady state inactivation curves in the 

knockouts, and we observed a significant change in the slope of the activation curve. Also 

consistent with experiments in heterologous cells, we observed a large decrease in the rate of 

recovery from inactivation. We explored LTP in CA1 hippocampal pyramidal neurons and 

observed increased LTP induction thresholds in knockout recordings. Behavioral analysis 

suggests changes in measures of social interaction. Theses studies are of particular interest 

considering a recently found association between DPPX and autism. 

Disclosures: E.W. Zagha , None; J.K. Maffie, None; H. Jeong, None; M. Nadal, None; B. 

Clark, None; E. Kwon, None; E.M. Goldberg, None; B. Rudy, None. 

Poster 





Support: NS045217 

Title: Characterization of a DPPX (DPP6) splice variant producing A-type currents with faster 

inactivation 

Authors: J. K. MAFFIE 1 , *B. RUDY 2 ; 

1 Smilow NeuroscienceProgram, Dept. Physiol. and Neurosci., NYU Sch. of Med., New York, 

NY; 2 Physiol & Neurosci, NYU Sch. Med., New York, NY 

Abstract: Transient A-type K(+) currents (I(SA)s) have fundamental roles in neuronal function. 

These subthreshold-activating, somato-dendritic currents produce delayed excitation, influence 

spike repolarization, modulate the frequency of repetitive firing, and are proposed to have 

important roles in signal processing in dendrites. We previously reported that DPPX proteins are 

key components of Kv4 channels, the channels that produce these A-type currents. (Nadal, M.S., 

Ozaita, A., Amarillo, Y., Vega-Saenz, E., Ma, Y., Mo, W., Goldberg, E.M., Misumi, Y., Ikehara, 

Y., Neubert, T.A., Rudy, B., 2003. The CD26-related dipeptidyl aminopeptidase-like protein 

DPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461). The DPPX 

gene encodes alternatively spliced transcripts that generate single-spanning transmembrane 

proteins with a short, splice specific intracellular domain and a large extracellular domain. The 

modulatory effects on Kv4.2-mediated currents and the rat brain distribution of three splice 

variants of the DPPX subfamily of protein have previously been described. (Nadal MS, Amarillo 

Y, Vega-Saenz de Miera E, Rudy B., 2006. Differential characterization of three alternative 

spliced isoforms of DPPX. Brain Res. 13;1094). The kinetics and voltage dependence of the 

currents produced by channels containing these isoforms were shown to be very similar in the 

presence or absence of KChIPs, the other putitive accessory protein of Kv4 channels. We cloned 

cDNA of an additional DPPX isoform, DPPX-E, and studied the ability of DPPX-E protein to 

modulate the properties of Kv4.2 channels in heterologous expression systems. We show that the 

kinetics of inactivation is dramatically faster than those produced by Kv4 channels expressed 

with other splice forms of DPPX. This parallels a similar acceleration seen in the DPP10-A 

splice variant, a protein with a similar role in Kv4 complexes. The DPP10-A splice variant has 

significant sequence similarity to DPPX-E supporting the finding that they have similar effects 

on the Kv4 current. The location of this isoform in mouse brain was explored using in situ 

hybridization. The diversity of DPPX splice variants opens up intriguing possibilities for the 

modulation of Kv4 channels and thereby for diversity in the electrophysiologic processes 

modulated by this current. 

Disclosures: J.K. Maffie, None; B. Rudy , None. 

Poster





Support: Thomas and Kate Jeffress Memorial Trust 

HHMI Grant to the University of Richmond 

Title: Determining the structural basis for fatty acid sensitivity of Kv4 potassium channels 

Authors: *L. M. BOLAND, G. TIMONEY, M. DRZEWIECKI, L. M. SPICER; 

Dept Biol., Univ. Richmond, Richmond, VA 

Abstract: Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA; C20:4) and 

docosahexaenoic acid (DHA; C22:6) modulate fast-inactivating Kv4 potassium channels which 

are localized to post-synaptic dendritic regions of CNS neurons. This modulation regulates 

neuronal firing frequency and the induction of hippocampal long-term potentiation. The goal of 

this project is to identify structural regions of Kv4 or the Kv4/KChIP channel complex which 

confer sensitivity to modulation by AA and DHA. Using a site-directed mutagenesis approach 

which is guided by homology modeling, we are probing functional regions of the channel which 

are involved in ionic conduction and inactivation gating. K + currents are recorded by twoelectrode 

voltage clamp of Xenopus oocytes injected with subunit-specific mRNAs. A conserved 

residue in the pore region of Kv4.2 was studied by mutagenesis and found to confer sensitivity to 

inhibition of the peak Kv4.2/KChIP1b potassium current in voltage clamp recordings. Kinetic 

modulation of gating was also reduced but not completely prevented by the pore mutation. Our 

results suggest that the PUFAs may require certain amino acids in the pore region in order to 

confer channel block whereas modulation of inactivation gating may be regulated by different 

regions of the channel. We are currently testing other regions of interest including regions that 

regulate gating in order to better understand the mechanisms of PUFA modulation of Kv4 

channel function and contribute to our understanding of the impact of fatty acids as signaling 

molecules. This research was supported by undergraduate fellowships from an HHMI grant to 

the University of Richmond (MD & GT) and a grant from the Thomas and Kate Jeffress 

Memorial Trust (LMB). 

Disclosures: L.M. Boland , None; G. Timoney, None; M. Drzewiecki, None; L.M. Spicer, 

None. 

Poster





Support: NIH Grant PO1 NS37444 

Title: Tuning of native A-current properties by expression of auxiliary subunit alternative 

transcripts 

Authors: *H. H. JERNG, P. J. PFAFFINGER; 

Neurosci., Baylor Col. Med., Houston, TX 

Abstract: Somatodendritic subthreshold A-type K + currents (ISA) regulate neuronal membrane 

excitability. Recently it has been established that the K + channels underlying ISA minimally 

consist of Kv4 pore-forming α-subunits in partnership with KChIP (Kv channel-interacting 

protein) and DPLP (dipeptidyl peptidase-like protein; DPP6 and DPP10) auxiliary subunits. 

Careful analysis of native ISA functional properties reveals significant functional variability 

between different neuronal populations. Here we propose that neuron specific expression of 

alternative KChIP and DPLP transcripts likely underlies much of the variability in native ISA 

functional properties. 

Through the analysis of ESTs, 5‟ RACE, and in situ hybridization, we and others have identified 

the expression of a large number of transcript variants for KChIP and DPLP. Consistent with this 

hypothesis, we find that that all variants tested thus far show unique expression patterns in the 

CNS. For KChIPs, the most dramatic functional effects are produced by the alternative 

expression of N-terminal transmembrane variant proteins by the KChIP2, KChIP3 and KChIP4 

genes. For DPLPs, the most dramatic functional effects are produced by the alternative 

expression of variants with a cytoplasmic N-type inactivation ball peptide. We examined the 

functional roles in the brain for these different auxiliary subunit combinations by recording from 

neuronal populations with different transcript specific expression patterns. For example, the 

DPP10a variant, containing an inactivation ball peptide, is expressed at high levels in cortical 

pyramidal neurons. Recording of ISA from cortical layers 2/3 pyramidal neurons shows that this 

current inactivates rapidly (η ~ 10 ms at +50 mV) with little or no voltage dependence, properties 

very similar to heterologous expression of A currents containing DPP10a. Similarly, DPP6a, 

another inactivation ball peptide variant, is expressed at high levels in cerebellar granule 

neurons, which also display rapid, relatively voltage independent inactivation. In contrast, ISA 

from cerebellar Purkinje cells shows inactivation kinetics that strongly slow with depolarization 

(η ~ 25 ms at +50 mV). Purkinje cells highly express the DPP10c and DPP10d isoforms, which 

lack N-type ball peptides. As expected, heterologous expression studies show that such subunit 

combinations produce A type currents with properties similar to those of ISA from Purkinje cells. 

We are currently mapping the functional correlations between the expression of different 

auxiliary subunit variants and the native ISA current properties as well as examining the 

functional importance of such kinetic tuning.

Disclosures: H.H. Jerng , None; P.J. Pfaffinger, None. 

Poster 





Support: NIHDK49430 

Title: Substance P and an NK2-selective agonist inhibit heteropodatoxin-sensitive low threshold 

inactivating K + -currents in DRG neurons 

Authors: W. C. DE GROAT 1 , *A. SCULPTOREANU 2 ; 

1 Pharmacol. and Chem. Biol., 2 Sch. of Med., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: We have shown previously that in capsaicin (CAPS)-responsive phasic neurons 

dissociated from L4-S3 DRG of adult male rats, SP (0.5 κM) lowered the threshold for initiating 

action potentials (AP) and increased the number of APs elicited by long depolarizing current 

pulses (600 ms). The effects of SP on firing in both types of neurons were similar to the effect of 

a Kv4 channel blocker heteropodatoxin II (0.005-0.5 κM) or low concentrations of 4aminopyridine 

(50 κM). Here we show that low threshold transient K + currents after block of 

remaining inactivating and non-inactivating currents with a combination of high TEA (60 mM) 

and (-) verapamil (5κM) and nifedipine (5κM) and substitution of Cl - with methyl sulfonate are 

inhibited by SP or a selective NK2 agonist, [βAla 8 ]-neurokinin A (4-10) (NKA, 0.5 κM). About 

80% of the current remaining after the Cl - substitution and K + channel blockers is an inactivating 

and α-dendrotoxin insensitive (50-500 nM) and heteropodatoxin sensitive (HPTX, 50-500 nM) 

K + current. The remaining currents after full voltage-dependent inactivation or HPTX blockade 

was a non-inactivating current of undetermined pharmacology. An NK3 selective agonist 

([MePhe 7 ]-neurokinin B, 0.5 κM), an NK1 selective agonist ([Sar 9 , Met 11 ]-substance P, 0.5 κM) 

or direct activation of PKC with phorbol 12,13-dibutyrate (0.5 κM) inhibited K + -currents 

activated over a wide range of voltages (0 to +90 mV) but as previously reported did not change 

firing. The effect of SP and NKA on K + channels was fully reversed by an NK2 receptor 

antagonist (MEN10376, 0.5 κM) but only partially reversed by a PKC inhibitor 

(bisindolylmaleimide, 0.5 κM). Both SP and NKA inhibited low voltage activated K + -currents 

(activated at voltages between -50 and -10 mV) with an apparent positive shift in the voltagedependence 

of activation. Our data suggest that the excitability of CAPS-sensitive phasic firing 

afferent neurons is increased by activation of NK2 receptors and that this is due in part to an 

inhibition of HPTX-sensitive low voltage activated inactivating K + -currents.

Disclosures: W.C. de Groat, None; A. Sculptoreanu , None. 

Poster 




Topic: B.04.f. Channel trafficking 

Support: NIH/NIGMS R01 GM83335 

Title: Dendritic localization of Shal (Kv4) K+ channels is mediated by the novel interactor SIDL 

Authors: J. C. CHAUFTY, F. DIAO, G. WARO, *S. L. TSUNODA; 

Boston Univ., Boston, MA 

Abstract: Shal K + (Kv4) channels are localized exclusively to somato-dendritic regions of 

neurons, where they function as key determinants of dendritic excitability. Kv4 channels have 

been shown to influence the integration of post-synaptic potentials, the shape of mEPSCs, the 

inhibition backpropagating action potentials into dendrites, as well as the induction of long-term 

potentiation. Although the function of Shal channels is clearly dependent on their somatodendritic 

localization, little is known about the mechanisms and proteins involved in achieving 

this polarized distribution. Dendritic localization of Shal channels, however, has been shown to 

be dependent on an evolutionarily conserved di-leucine motif (LL-motif) found in all Kv4 

channels. Here, we identify the novel Drosophila protein, SIDL, as the first target protein of this 

conserved LL-motif. We show that SIDL is a nervous system specific protein, which interacts 

specifically with the LL-motif of Drosophila and mouse Shal channels and co-localizes with 

GFP-Shal channels. To test the role of SIDL in Shal channel localization, we use the projection 

neurons (PNs) of the adult Drosophila brain as a model for polarized targeting. We first show 

that GFP-Shal is restricted to the cell bodies and dendrites of PNs, confirming that the somatodendritic 

localization of Shal channels is conserved in Drosophila. Next, we show that deletion 

of the LL-motif on Shal channels leads to a loss of polarized targeting in vivo. To examine the 

role of SIDL in the dendritic localization of Shal channels, we obtained a transgenic strain 

expressing an hpRNA predicted to knock-down expression of SIDL. We show that SIDL is 

indeed knocked-down, and as a consequence, these flies display a severe loss of GFP-Shal 

channels in PN dendrites. We further explore the role of SIDL in Shal channel localization by 

generating a transgenic line expressing the C-terminus of SIDL (SIDLC), which contains the 

Shal binding site. We predict that over-expression of SIDLC will compete with endogenous 

SIDL protein for binding Shal channels. With the disruption of the SIDL-Shal interaction, we 

again observe an absence of GFP-Shal signal in dendrites of PNs. Together, our data

demonstrates that SIDL is a key regulator required for dendritic localization of Shal K + channels. 

The identification of SIDL provides the first step for future investigation into molecular 

mechanisms regulating dendritic targeting of K + channels. 

Disclosures: J.C. Chaufty, None; F. Diao, None; S.L. Tsunoda , None; G. Waro, None. 

Poster 





Support: NIH Grant RO1 NS31234 

Title: 17β-estradiol modulates astrocytic kir4.1 following in vivo and in vitro spinal cord inury 

Authors: *M. L. OLSEN, C. L. FLOYD, H. SONTHEIMER; 

Dept Neurobiol, Univ. Alabama, Birmingham, Birmingham, AL 

Abstract: We have previously demonstrated that astrocytes in the spinal cord express the 

inwardly rectifying potassium channel (Kir) Kir4.1. This channel confers a high resting K + 

conductance and is responsible for the negative resting membrane potential required to drive 

glutamate uptake in these cells. These channels are believed to be responsible for K + buffering in 

brain. In spinal cord Kir4.1 expression and activity is developmentally upregulated and correlates 

with increasingly tight extracellular K + regulation in the spinal cord. A number of studies, mainly 

done in vitro, suggest a loss of Kir channel activity following injury when astrocytes become 

gliotic. If this occurred in vivo, it would lead to deficient K + and glutamate homeostasis. We 

examined this question by inflicting a crush spinal cord injury in adult rats. 7 days post injury 

Kir4.1 expression was decreased by nearly 80% by Western blot, and this loss was confirmed by 

immunohistochemistry and patch-clamp recording. Similar results were observed when we 

examined the glutamate transporter Glt-1. Recent studies suggest that 17β-estradiol confers 

neuroprotection in this injury model. When these animals were given physiological time-release 

17β-estradiol following injury, Kir4.1 and Glt-1 expression increased. Estrogen appears to 

directly modulate Kir4.1 and Glt-1 expression as treatment of cultured „injured‟ astrocytes with 

estrogen was sufficient to increase Kir4.1 channel expression and glutamate uptake. These 

findings suggest that some neuroprotective benefits seen with 17β-estradiol treatment following 

spinal cord injury may be due to increased Kir4.1 channel expression in astrocytes which would 

lead to improved K + regulation and enhanced glutamate homeostasis. These findings suggest a

potential therapeutic benefits of 17β-estradiol not only in treating CNS injury but other 

conditions in which a loss of K + and glutamate homeostasis. 

Disclosures: M.L. Olsen , None; C.L. Floyd, None; H. Sontheimer, None. 

Poster 






Title: Differential Kir4.1 channel expression in complex and passive astrocytes revealed by 

mouse transgenesis 

Authors: X. TANG, K. TANIGUCHI, *P. KOFUJI; 

Dept Neurosci, Univ. Minnesota, Minneapolis, MN 

Abstract: Results from several laboratories have indicated that the inwardly rectifying 

potassium channel Kir4.1 underlies the principal potassium conductance in astrocytes and retinal 

Müller cells and have presented evidences of its critical role in extracellular potassium buffering 

in the retina and brain. We have explored a genetic approach to provide high resolution cellular 

mapping of Kir4.1 expression by employing bacterial artificial chromosome (BAC) transgenic 

mice that express Enhanced Green Fluorescent Protein (EGFP) under the transcriptional control 

of the Kir4.1 gene. Analysis performed in brain sections of Kir4.1-EGFP transgenic mice 

showed that transgenic expression of EGFP was well correlated with Kir4.1 and was prominent 

in GFAP-expressing astrocytes. In addition, weak expression of EGFP was detected in NG2expressing 

glial cells. Whole cell voltage clamp recordings in hippocampus indicate that these 

two glial cell types defined by the EGFP levels of expression display the properties of the so 

called passive and complex astrocytes. The strongly fluorescent EGFP glial cells have linear 

current-voltage relationships and are strongly coupled via gap junctions. The weakly fluorescent 

EGFP glial cells display non-linear current voltage-relationships and are not electrically coupled 

extensively with other cells. Pharmacological blockade of Kir4.1 channels with desipramine or 

cesium indicate that Kir4.1 channels constitute the dominant resting potassium conductance in 

complex astrocytes but not in passive astrocytes. These results suggest differential roles of 

Kir4.1 channels in complex and passive astrocytes. 

Disclosures: X. Tang, None; K. Taniguchi, None; P. Kofuji , None.

Poster 





Support: RFBR 08-04-00423-a 

NIH-RCMI G12RR03035 

NIH-AABRE P20RR16470 

NIH-MBRS-GM5-340508/09 

NIH-NINDS-CNS S11NS08201 

Title: Biphasic block of glial Kir4.1 channels by interaction of sodium and spermine 

Authors: *Y. V. KUCHERYAVYKH 1 , S. M. ANTONOV 5 , Y. M. SHUBA 6 , L. Y. 

KUCHERYAVYKH 2 , M. INYUSHIN 3 , H. KURATA 7 , C. G. NICHOLS 7 , M. J. EATON 2 , S. N. 

SKATCHKOV 4 ; 

1 Biochem., Univ. Central del Caribe, Bayamon, Puerto Rico; 2 Biochem., 3 Physiol., 4 Biochem. 

and Physiol., Univ. Central del Caribe, Bayamon, PR; 5 Sechenov Inst. of Evol. Physiol. and 

Biochem, RAS, St. Petersburg, Russian Federation; 6 Intl. Ctr. of Mol. Physiology, NASU, Kiev, 

Ukraine; 7 Cell Biol., Washington Univ. Sch. of Med., St. Louis, MO 

Abstract: If glial Kir4.1 channels are dysfunctional, the CNS suffers different diseases 

(Rozengurt et al., 2003; Buono et al., 2004; Ferraro et al., 2004; Wangemann et al., 2004; Lenzen 

et al., 2005). Down-regulation of Kir4.1 by siRNA or by conditional knockout in glia results in 

decreased glutamate uptake and suggests a link between Kir4.1 and other glial functions. 

Recently, we have shown that Kir4.1 channels display an unusual biphasic rectification 

determined by the blocker interaction with low and high affinity binding sites within the 

channels. Further, we hypothesize that low affinity block is in part due to Na + ions transported to 

the cytoplasm by the glutamate transporter or ion channels. Here we investigated the mechanism 

of this biphasic rectification using inside-out patches from tsA201 cells expressing Kir4.1 

channels. We found that under symmetrical K + conditions (150 mM) application of Na + (3 - 

30mM) to the intracellular side of patches induced low affinity block of outward currents 

achieved at high voltages. Spermine (SP) at concentrations of 10 - 100 κM caused high affinity 

block starting at low voltages. When applied together Na + and SP produced additive and

complex rectification of Kir4.1 channels. Because of differences in kinetics, both blocks could be 

distinguished and are observed as two curvatures in the I/V relationship of Kir4.1 conductance. 

Under non-symmetrical conditions which are close to physiological (3 mM K + out -147 mM 

Na + out/ 3 mM Na + in-147 mM K + in), both types of block were found, however, the degree of SP 

block was weaker than in symmetrical conditions and the contribution of Na + block to 

rectification was more pronounced by SP. 

These data suggest that redistribution of K + and Na + gradients around the glial membrane during 

neuronal excitation can affect Kir4.1 rectification. Therefore, glial cells surrounding GABA or 

glutamate synapses may function differently in respect to Kir4.1 properties. Linear I/V curves 

are observed for Kir4.1 only in absence of both Na + and SP 4+ . Moreover, Na + accumulation 

stabilizing Kir4.1 rectification is inherent to support glial function. 

Disclosures: Y.V. Kucheryavykh, None; S.M. Antonov, None; Y.M. Shuba, None; L.Y. 

Kucheryavykh, None; M. Inyushin, None; H. Kurata, None; C.G. Nichols, None; M.J. 

Eaton, None; S.N. Skatchkov, None. 

Poster 





Support: RFBR 08-04-00423-a 

BMB+F (IZKF, 01KS 9504 project C5) 

NIH-NINDS and NCRR SNRP-U54 NS039408 

DFG Ve-187/1-2 

NIH-RCMI G12RR03035 

Title: Intracellular sodium block of Kir2.1 channels 

Authors: *S. M. ANTONOV 1 , Y. V. KUCHERYAVYKH 2 , Y. M. SHUBA 4 , R. W. VEH 5 , A. 

REICHENBACH 6 , H. KURATA 7 , C. G. NICHOLS 7 , M. J. EATON 2 , S. N. SKATCHKOV 3 ; 

1 Sechenov Inst. of Evolutionary Physiol. and Biochem. RAS, St-Petersburg, Russian Federation; 

2 Biochem., 3 Biochem. and Physiol., Univ. Central del Caribe, Bayamon, PR; 4 Intl. Ctr. of Mol. 

Physiology, NASU, Kiev, Ukraine; 5 Inst. Integrated Anat., Charite, Berlin, Germany; 6 Inst. of

Brain Res., Univ. of Leipzig, Leipzig, Germany; 7 Cell Biol., Washington Univ. Sch. of Med., St. 

Louis, MO 

Abstract: The Kir2.x family of potassium channels plays a vital role in many organs and brain. 

Neurons widely express Kir2.x including Kir2.1 (Pruss et al., 2006; Howe et al., 2008). 

Malfunctions of these channels may be severe: the T75M mutation results in Andersen-Tawil 

syndrome (Tani et al., 2007) and is associated with a distinct neurocognitive phenotype in 

humans (Yoon et al., 2006). It is well known that spermine (SP) can block Kir2.x (Lopatin et al., 

1994), but it has recently been shown that there are two blocking sites for SP in Kir2.1 and 

Kir2.2 (Ishihara & Yan, 2007). There is an assumption that other ions such as spermidine (SD), 

Mg 2+ , etc. interact with SP (Ishihara & Ehara; 2004; Yan & Ishihara, 2005). In fact, in brain and 

retina the polyamines, SP and SD, are accumulated in glia, not in neurons. Neurons employ Na + - 

and K + -channels, including Kir2.1 (Constani & Galvan, 1983) to generate spikes even with a 

lack of SP. Thus, we asked (i) what is the behavior of Kir2.1 channels under physiological 

conditions in the absence of SP and (ii) may Na + located in the cytoplasm be an effective 

modulator of these channels? We used inside-out patches of Kir2.1 expressed in tsA201 cells to 

demonstrate that intracellular Na + in the µM range effectively blocks outward currents through 

Kir2.1 channels. In the absence of SP 4+ , SD 3+ , Mg 2+ and Na + in symmetrical K + , the I/V curves 

of the Kir2.1 conductance remained linear. Addition of either Na + or SP to the intracellular side 

of patches induced a strong inward rectification. In our experiments, Kds for SP and Na + at Vm = 

0 mV differed by four orders of magnitude. Kd(0) values for Na + and SP were about 100 κM (efold 

change of 27 mV) and 10 nM (e-fold change of 9 mV), respectively. Considering the very 

high efficiency of Na + as a Kir2.1 channel blocker, we suggest that it may cause Kir2.1 channel 

rectification. As cytoplasmic [Na + ] rises up to 100 times higher than the Kd for Na + under 

physiological conditions, Na + block may be highly relevant for Kir2.1 rectification, and SP is not 

necessary for rectification in neurons. We conclude that neuronal Kir2.1 channels can exhibit 

rectification without SP, but in the presence of µM intracellular sodium. 

Disclosures: S.M. Antonov, None; Y.V. Kucheryavykh, None; Y.M. Shuba, None; R.W. 

Veh, None; A. Reichenbach, None; H. Kurata, None; C.G. Nichols, None; M.J. Eaton, 

None; S.N. Skatchkov, None. 

Poster 

235. Ion Channels in Disease I 



Topic: B.04.d. Ion channels and disease 

Title: Neuronal activation in familial hemiplegic migraine mice following CSD induction

Authors: *C. F. BARRETT 1 , L. A. M. BROOS 2 , R. R. FRANTS 2 , M. D. FERRARI 2 , A. M. J. 

M. VAN DEN MAAGDENBERG 2 ; 

1 Neurol and Human Genet., 2 Leiden Univ. Med. Ctr., Leiden, Netherlands 

Abstract: Migraine is one of the most common neurological disorders, affecting up to 12% of 

the general population on a chronic basis. In one third of migraine patients, the headache is 

preceded by an aura. Spreading depression is a self-propagating wave of transient depolarization 

of neuronal and glial cells. Spreading depression in the cerebral cortex (CSD) is believed to 

underlie the aura phase of the attack and may also trigger the headache phase itself, possibly by 

activating neurons within the trigeminovascular nucleus. However, whether CSD is either 

necessary and/or sufficient to induce a migraine attack is not known. In an attempt to understand 

what makes the migraine brain more susceptible to the effects of spreading depression, we 

examined which brain regions are activated by CSD events, using c-Fos expression as a marker 

of neuronal activation. We compared activation patterns between wild-type (i.e., non-migraine) 

mice and two lines of mice carrying missense mutations in the Cacna1a gene linked to familial 

hemiplegic migraine (FHM), a monogenic subtype of migraine with aura. The R192Q mutation 

models “pure” FHM in patients, while the S218L mutation, although quite close to position 192 

in primary structure, causes a much more severe clinical outcome, including not only FHM but 

also ataxia, seizures, and coma and cerebral edema following mild head trauma. Consistent with 

these overlapping yet different clinical phenotypes, we found that CSD activates both 

overlapping and distinct brain regions in the FHM mutant mice. Specifically, in the S219L brain, 

CSD events and seizures both drive strong activation of neurons in the cerebral cortex and 

hippocampus. However, CSD events but not seizures activate Purkinje neurons in the 

cerebellum. Moreover, in wild-type and R192Q mice, CSD events activate neurons in the 

cerebral cortex but not the hippocampus or cerebellum. These findings provide clues to the 

pathophysiology of these two seemingly distinct episodic events, and help explain the disparate 

clinical findings in R192Q and S218L patients. 

Disclosures: C.F. Barrett, None; L.A.M. Broos, None; R.R. Frants, None; M.D. Ferrari, 

None; A.M.J.M. van den Maagdenberg, None. 

Poster 





Support: European Integrated Project Epicure

Title: Self-limited hyperexcitability: functional effect of a familial hemiplegic migraine mutation 

of NaV1.1 (SCN1A) Na + channel 

Authors: *M. A. MANTEGAZZA 1,2 , S. CESTÈLE 3 , P. SCALMANI 1 , R. RUSCONI 1 , B. 

TERRAGNI 1 , G. BECHI 1 , S. FRANCESCHETTI 1 ; 

1 Neurophysiopathology, Inst. Neurologico Besta, Milano, Italy; 2 Equipe Avenir Inserm, IFR 95- 

Saints-Pères, Univ. Paris 5-Descartes, Paris, France; 3 Inserm U836 (Equipe 3),, Inst. of 

Neurosciences, Grenoble, France 

Abstract: Migraine is a common brain disorder with a strong genetic component. 35% of 

affected patients display aura, a phase preceding the headache characterized by focal 

neurological symptoms. 

Familial Hemiplegic Migraine (FHM) is a rare and severe autosomal dominant subtype of 

migraine with aura associated with hemiparesis during the attacks. FHM type 3 has been recently 

associated with mutations of the voltage-gated Nav1.1 Na + channel α subunit, but functional 

studies have been done using the cardiac Nav1.5 isoform and the observed effect were similar to 

those of some epileptogenic mutations. We studied the FHM mutation Q1489K in transfected 

tsA-201 cells and cultured neurons using the human clone of Nav1.1. 

Our results obtained with voltage-clamp recordings from tsA-201 cells show that the mutation 

modifies the gating properties of the channel consistently with both hyperexcitability and 

hypoexcitability. Simulations of high frequency discharges and long depolarizing pulses 

mimicking pro-migraine conditions revealed an initial gain of function effect consistent with 

increased excitability that during long lasting events became a loss of function consistent with 

decreased excitability. Voltage-clamp recordings from transfected neurons showed similar 

results. 

To disclose the overall effect of Q1478K on neuronal firing with more physiological 

experiments, we did current-clamp recordings of the firing of transfected neurons. Only 

transfected neurons were able to generate sustained firing during injections of 2.5s long 

depolarizing current pulses, whose threshold was more negative in neurons expressing wild-type 

channels. The input-output relationship (number of action potentials vs. injected current) showed 

an initial monotonic rising phase followed by a falling phase that was due to a depolarizing block 

of the firing during the injection of large depolarizing current, but there was a large scattering 

and no significant differences between wild type and Q1489K channels. 

Thus, we compared for each neuron the firing trace obtained in response to the largest current 

pulse capable to consistently evoke a train of overshooting action potentials and that did not 

induce a depolarizing block. The neurons transfected with Q1478K showed significantly higher 

frequency at the beginning of these discharges in comparison with the wild type, but in the final 

part of the discharge the curves tended to converge and the frequency was not significantly 

different. 

In conclusion, in our experimental system Q1478K induces a self-limited hyperexcitability that 

can account for the different pathophysiological mechanism between FHM and epilepsy. 

Disclosures: M.A. Mantegazza , None; S. Cestèle, None; P. Scalmani, None; R. Rusconi, 

None; B. Terragni, None; G. Bechi, None; S. Franceschetti, None.

Poster 





Support: Medical Research Council 

Human Frontier Science Program 

Title: Modulation of action potentials and neurotransmitter release by Kv1.1 mutations 

associated with Episodic Ataxia type 1 

Authors: J. H. HEEROMA 1 , *C. HENNEBERGER 2 , D. M. KULLMANN 1 ; 

1 Inst. of Neurol., Univ. Col. London, London, United Kingdom; 2 Inst. Neurol, London, United 

Kingdom 

Abstract: Point mutations of KCNA1, encoding Kv1.1, are associated with Episodic Ataxia type 

1 (EA1), which is characterised by paroxysmal cerebellar incoordination, neuromyotonia and 

sometimes with focal epilepsy. Because Kv1.1 is abundantly expressed in the axons of many 

neurons we have asked how two different mutations affect action potential initiation, shape and 

frequency, and neurotransmitter secretion. 

We used lentivectors to express wild type (WT) or either of two KCNA2 mutants in cultured 

autaptic rat hippocampal neurons: T226R, associated with EA1 complicated by epilepsy, or 

R417stop, associated with severe drug-resistant EA1. Both have previously been shown to exert 

a dominant negative effect in non-neuronal expression systems. WT over-expression 

significantly increased the rheobase, after hyper polarisation (AHP) amplitude, spike frequency 

and paired pulse ratio, and decreased spike width, first spike delay, AHP decay rate and 

probability of release. R417stop had opposite effects on all of these parameters, consistent with 

sequestration and/or degradation of endogenous Kv1.1. T226R did not affect spike 

characteristics (measured at the soma) but increased neurotransmitter release probability by 

approximately 50%. We propose that the distinct effects of R417stop and T226R may contribute 

to the differences in associated phenotypes. 

Disclosures: J.H. Heeroma, None; C. Henneberger, None; D.M. Kullmann, None.

Poster 





Support: University of Arizona Foundation and the Office of the Vice President for Research, 

Graduate Studies and Economic Development 

Title: Over-expression of the potassium channel Kir2.3 by an adenoviral vector using the 

GAD67 promoter changes the excitability of hippocampal neurons 

Authors: *T. A. FALK 1 , J. Y. XIE 1 , A. J. YOOL 2 , S. J. SHERMAN 1 ; 

1 Dept Neurol., 2 Dept Physiol, Univ. Arizona Col. Med., Tucson, AZ 

Abstract: Selective control of the electrical activity of GABA-ergic neurons in anatomically 

discrete brain regions would provide an important technique for the study of neural circuitry and 

could form the basis of novel therapeutic approaches for a variety of neurological diseases. 

Viral-vector mediated gene transfer utilizing a cell-type specific promoter to drive expression of 

ion channels could achieve the goal of selective neuronal control. In order to provide proof in 

principle of this concept, we have constructed an adenoviral gene transfer vector that utilizes a 

relatively small portion of the glutamic acid decarboxylase (67 kD isoform, GAD67) promoter 

region to over-express an inward rectifier type of K+ channel (Kir2.3). We have cloned a 900bp 

fragment of the GAD67 promoter region into an adenoviral shuttle vector containing green 

fluorescent protein (eGFP) as a living stain and also have subcloned the cDNA coding for the 

Kir2.3 channel into the construct. Kir2.3 and eGFP are separated by an internal ribosomal entry 

site (IRES) sequence allowing transcription of both genes under the control of the same 

promoter. The Kir2.3 channel was tagged with a Strep-tag which allows distinguishing the 

expression from that of endogenous Kir2.3. (1) We have found that this small portion of the 

GAD67 promoter region is able to confer cell type selectivity to an adenoviral vector in vitro 

using primary cultures of rat hippocampal neurons. (2) The current characteristics and kinetics of 

Kir2.3 have not changed after the addition of the Strep-tag as verified by patch clamp 

electrophysiology after heterologous expression in HEK293 cells. (3) Western analysis with anti- 

Strep-tag antibody verified expression of the tagged Kir2.3 channel construct in hippocampal 

neurons in vitro. (4) In addition, we were able to use this GAD67 promoter region to overexpress 

the inward rectifier K+ channel Kir2.3 at levels sufficient to counteract neurotoxininduced 

neuronal hyperactivity. We utilized cFOS expression as a marker for excitability, 

comparing eGFP-positive with control cells. Weak expression of eGFP was amplified using an 

anti-eGFP antibody. We applied anemone toxin II (ATX-II) to induce over-excitability in 

cultured hippocampal neurons. The ATX-II induced increase in expression of cFOS was reduced 

to baseline values in neurons over-expressing Kir2.3. To our knowledge, this is the first report to

use a neuronal cell-type specific promoter to modify neuronal activity in hippocampal neurons 

using ion channel over-expression. 

Disclosures: T.A. Falk , None; J.Y. Xie, None; A.J. Yool, None; S.J. Sherman, None. 

Poster 





Title: Neuroprotective effects of a novel sodium channel blocker (NW-3381) in rat cortical 

neurons 

Authors: P. SALVATI, L. CURATOLO, A. RESTIVO, C. SABIDO-DAVID, S. 

FRANCISCONI, *C. CACCIA; 

Preclinical Res., Newron Pharmaceuticals Spa, Bresso, Italy 

Abstract: There is increasing evidence that voltage-gated Na + channel blockade provides useful 

therapy not only for epileptic seizures, but also for prevention of neuronal cell death and 

treatment of neuropathic pain. 

In neuronal cells, the prolonged opening of Na + channels eventually results in a Ca 2+ -dependent 

neuronal cell degeneration. Cellular Na + loading induces a strong and persistent depolarization 

leading to a voltage-gated Ca 2+ channel opening with the consequent intracellular Ca 2+ rise and 

release of large amount of glutamate from the cytosol into extracellular space. Extracellular 

glutamate activates AMPA and NMDA-type receptors, causing further damaging effects. The 

neuronal cell death is prevented by tetrodotoxin (TTX) supporting that Na + channel blockade is a 

key step to inhibit neuronal depolarization, Na + influx and the following Ca 2+ influx and 

glutamate release, all detrimental events for neurons. 

The aim of the present study was to study the in vitro neuroprotective effects of a novel 

compound (NW-3381) endowed with potent Na + current blocking properties and 

pharmacological efficacy in animal models of epilepsy and psychiatric disorders. 

In ND7/23 cell line (a hybrid rat/mouse neuroblastoma cell line highly expressing TTX-sensitive 

Na + currents), NW-3381 inhibited the veratridine-induced Na + influx with an IC50 = 2 µM. The 

effects of NW-3381 on veratridine-induced Ca 2+ influx were evaluated in rat cortical neurons, as 

in ND7/23 cells, although expressing Ca 2+ channels, a very small increase in Ca 2+ influx after 

veratridine was found (about 6%). In rat cortical neurons, NW-3381 inhibited the veratridineinduced 

Ca 2+ influx with an IC50 = 6 µM without affecting the NMDA-induced Ca 2+ influx up to 

100 µM.

Incubation of rat cortical neurons for 24 h at 37°C in a serumless medium containing veratridine 

(30 κM) resulted in a significant increase in cell death. NW-3381, added 1 h before veratridine, 

was able to reduce the neuronal damage with an IC50 = 6 κM. 

Our results show that NW-3381 by functionally blocking the consequences of Na + channel 

activation (both the Na + influx and the subsequent Ca 2+ influx due to the prolonged 

depolarization) might have a potential for neuroprotection. 

Disclosures: P. Salvati, None; L. Curatolo, None; A. Restivo, None; C. Sabido-david, 

None; S. Francisconi, None; C. Caccia, None. 

Poster 





Support: 6TH FRAMEWORK EUROPEAN PROGRAM 

Title: Antiepileptic properties and antipsychotic potential of NW-3381, a novel potent sodium 

channel blocker 

Authors: E. IZZO 1 , M. CALABRESI 1 , *C. SABIDO-DAVID 1 , S. PARINI 1 , A. VEZZANI 2 , M. 

CARLI 2 , S. BALOSSO 2 , K. WEDZONY 3 , P. SALVATI 1 ; 

1 Newron Pharmaceut SPA, Bresso, Italy; 2 MARIO NEGRI INSTITUTE, MILANO, Italy; 

3 Polish Acad. of Sci., KRAKOW, Poland 

Abstract: Sodium channels have been implicated in a number of diseases including epilepsy, 

pain and psychiatric disorders. Several drugs in the market targeting sodium channels have 

shown to be effective against these diseases. NW-3381 was selected for its high potency (submicromolar 

range) for the target (sodium channels) and good preliminary profile in terms of 

selectivity and developability. 

To assess its antiepileptic activity, NW-3381 was tested in a range of seizure models in mice: the 

maximal electroshock test (MES), the kainic acid (KA)-induced status epilepticus (SE), and the 

ip pentylenetetrazol (PTZ)-induced generalized seizures, in comparison to the antiepileptic drug 

lamotrigine. In the MES test the compound resulted to be active both after intraperitoneal (ip) 

and oral (po) treatment with ED50 of 4.6mg/kg and 8.9mg/kg respectively. In the KA model the 

dose of 20mg/kg po significantly inhibited seizure generalization and the incidence of SE while 

lamotrigine at 30mg/kg ip was ineffective. In the PTZ model NW-3381 20mg/kg ip protected 

from generalized seizure whereas lamotrigine at 15 and 30mg/kg ip worsened seizure severity.

The therapeutic index, as determined by the rotarod test was within the range of the known anti 

epileptics. The onset of action was extremely fast showing its ability to quickly reach the brain. 

To explore possible use in psychiatric disorders, NW-3381 was evaluated in the 

amphetamine/chlordiazepoxide-induced hyperactivity test, a model of mania in which 

lamotrigine and other antiepileptics used in clinical practice for the treatment of bipolar disorders 

have been shown to be active. In this test NW-3381 was efficacious at relatively low doses of 5 

and 10mg/kg ip (bid) without affecting basal locomotion. Finally, the effects of NW-3381 and 

lamotrigine were tested in a pharmacological mouse model of cognitive dysfunctions in 

schizophrenia. Both lamotrigine (27 mg/kg ip) and NW-3381 (10mg/kg ip) were active in 

preventing the PCP-induced impairment in attentional performance in the 5-choice serial 

reaction task. 

Taking in account that NW-3381 has low potential for drug-drug interactions as well as low cell 

toxicity and fast onset of action, these in vivo data indicate NW-3381 as possible candidate for 

epilepsy and psychiatric disorders therapy. 

Disclosures: E. Izzo, None; M. Calabresi, None; C. Sabido-David , None; S. Parini, None; A. 

Vezzani, None; M. Carli, None; S. Balosso, None; K. Wedzony, None; P. Salvati, None. 

Poster 





Title: Electrophysiological characterization of NW-3381, a potent sodium channel blocker 

active in animal models for epilepsy and psychiatric disorders 

Authors: L. FARAVELLI, E. COLOMBO, C. SABIDO-DAVID, *P. SALVATI; 

Newron Pharmaceuticals SpA, Bresso MI, Italy 

Abstract: Sodium channels are involved in the neuronal hyper-excitability that can occur in 

pathological conditions such as epilepsy, pain and psychiatric disorders. 

Compound NW-3381 was selected from Newron‟ ion channels program, for its high affinity for 

sodium channels, good preliminary profile in terms of selectivity and developability and activity 

in animal models of epilepsy mania and schizophrenia. 

Patch clamp experiments in isolated rat cortical neurons showed that NW-3381 has a selective, 

sub-micromolar and voltage dependent inhibition of Na + currents with an apparent affinity for 

inactivated channels Ki of 0.2µM. Furthermore, in the same neurons, the sustained repetitive 

firing is significantly reduced by NW-3381 in a concentration range of 3-10µM, ten times more

potent than observed with the reference anti-epileptic drug (AED) lamotrigine. 

For safety purposes, the activity of NW-3381 against specific cardiac Na + , Ca 2+ and K + channels 

subtypes was tested in recombinant cell systems. NW-3381 showed a good safety profile, 

producing a weak inhibition of Nav1.5, L-type Ca 2+ currents and hERG K + currents. 

In conclusion, the favourable in vitro profile of NW-3381 both in terms of selective modulation 

of Na + channel activity as well as the capacity to modulate neuronal firing, in particular under 

conditions of hyper-excitability, can explain the efficacy of this compound in animal models for 

epilepsy and psychiatric disorders. 

Disclosures: L. Faravelli, None; E. Colombo, None; C. Sabido-david, None; P. Salvati , 

None. 

Poster 





Support: 1K08MH77220 

Title: SK3 potassium channels protect against excitotoxicity in dopaminergic neurons in vitro 

Authors: *B. A. BENITEZ 1 , A. ANASTASÍA 4 , D. MAMAH 1 , H. BELALCAZAR 1 , D. H. 

MASCÓ 4 , G. A. DE ERAUSQUIN 1,2,3 ; 

1 Psychiatry, 2 Neurol., 3 Hope Ctr. for Neurolog. Disorder, Washington Univ., St Louis, MO; 4 Ctr. 

de Biología Celular y Mol., Univ. Nacional de Córdoba, Córdoba, Argentina 

Abstract: Dopaminergic neurons (DNs) degenerate selectively in Parkinson's disease, and there 

are suggestions of a partial dopaminergic mesolimbic deficit in schizophrenia. Although a 

substantial amount is known about DNs, the molecular mechanisms that regulate the selective 

susceptibility to excitotoxicity in DNs are poorly understood. We have found that stimulation of 

DNs in culture by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) results in 

opening of L-type voltage-gated calcium channels, release of calcium from intracellular stores, 

loss of calcium homeostasis and generation of reactive oxygen species. These events trigger 

translocation and transcriptional activation of the transcription factor nuclear factor kappa B, and 

activation of the oncogene p53, and neuronal death. A gap in the knowledge of DNs' 

susceptibility is its relationship to the intrinsic physiological properties of DNs. The DNs are 

characterized in vitro by their long-duration action potentials and spontaneous pacemaker 

activity (marked afterhyperpolarization -IAHP- followed by an inward current). The IAHP has

several components. The fast component (fAHP) helps to repolarize the action potential and 

regulates spike interval, whereas subsequent slow components (sAHP) underlie spike-frequency 

adaptation. The channels underlying the sAHP are small conductance Calcium-activated K(+) 

channels (SK channels). Here, we test the role of SK3 channel in an in vitro model of selective 

susceptibility of DNs. Using whole-cell patch-clamp recordings we found that the exposure to 

100 κM AMPA or 20 nM Apamine for 2 h resulted in a 50 % reduction in SK current, alteration 

of the I/V relationship of the current, and reduction of the area under the curve at different 

holding potentials for AMPA. To assess the impact of reduction of the SK current on DNs 

survival, we exposed DNs for 16-24 h to increasing concentrations of AMPA alone, or in the 

presence of modulators of the SK current. The SK current antagonist apamine caused 

concetration-dependent selective toxicity to DNs, which at 100nM was comparable to that 

caused by 100 κM AMPA; however, its toxicity was not increased by AMPA at the 

concentrations and times tested, suggesting that apamin and AMPA toxicities are not additive. 

On the other hand, cotreatment with the selective SK potassium channel agonist 1-Ethyl-2benzimidazolinone 

(EBIO-1), at a concentration that increased SK current amplitude by 50% 

resulted in significant protection of DNs. Thus, it appears reasonably to postulate that the 

selective toxicity to DNs in vitro of AMPA requires the suppression of SK3 channels prior to 

expression of the toxic mechanism. 

Disclosures: B.A. Benitez, None; A. Anastasía, None; D. Mamah, None; H. Belalcazar, 

None; D.H. Mascó, None; G.A. de Erausquin, None. 

Poster 





Support: PHS F32NS055584 

PHS R01NS40470 

PHS R01NS33592 

Title: Abnormal release of store-operated calcium within the cerebellum of the Cav2.1 mouse 

mutant tottering is ameliorated with Kv7 channel agonists 

Authors: *R. S. RAIKE 1 , H. A. JINNAH 1 , E. J. HESS 1,2 ; 

1 Dept Neurol, 2 Neurosci., Johns Hopkins, Baltimore, MD

Abstract: Episodic channelopathies are characterized by intermittent dysfunction arising from 

ion channel mutations. Attacks in patients are commonly triggered by environmental stress, 

caffeine or ethanol. The episodic dystonia exhibited by tottering mice is also triggered by the 

same precipitants, making it a good model for studying channelopathies. Tottering mice express 

hypo-conductive Cav2.1 (P/Q-type) voltage-gated calcium channels, predicting dysfunction in 

cerebellar Purkinje cells (PCs), where these channels are abundantly expressed. Others have 

demonstrated that tottering PCs indeed exhibit disorganized patterned firing, but the 

neuroanatomical origin of the attacks remains unclear. Therefore we micro-perfused caffeine into 

tottering cerebellum or striatum and found that attacks were dose-dependently induced only 

when drug was applied to the cerebellum. Although the pathways through which the attacks are 

not understood, expression of Cav1.2 (L-Type) channels and ryanodine receptors (RyRs) in 

tottering PCs is altered, suggesting that abnormal store-operated calcium (SOC) release may play 

a role. Therefore, to determine if caffeine, a RyR agonist, triggers tottering attacks through a 

pathway involving SOC, we challenged tottering mice systemically with a panel of other RyR 

agonists and found that they too induced attacks dose-dependently. We hypothesized that 

abnormal SOC dynamics in tottering PCs may induce attacks by interfering with the function of 

other calcium-dependent ion channels. Kv7 (KCNQ) potassium channels, which play an 

important role in regulating the excitability of pacemaking neurons such as PCs, are inhibited by 

SOC release. Treatment with the KCNQ agonist, flupirtine, dose-dependently blocked tottering 

attacks induced by multiple precipitants. Overall, these results indicate that precipitants of 

attacks in channelopathies may work through a common pathway involving SOC and that Kv7 

channels should be considered important pharmacological targets for treatment. 

Disclosures: R.S. Raike , None; H.A. Jinnah, None; E.J. Hess, None. 

Poster 





Support: NIH Grants R01NS42926 and R01NS49470 (Z.G.X.) 

Title: Zinc-induced toxicity mediated by TRPM7 channels 

Authors: *K. INOUE, Z.-G. XIONG; 

Robert S. Dow Neurobio. Lab., Legacy Res., Portland, OR

Abstract: TRPM7 channels are Ca 2+ -permeable non-selective cation channels whose activation 

has been shown to be involved in ischemic neuronal cell death. In addition to Ca 2+ , previous 

studies have suggested that these channels might be zinc permeable. Here we show further 

evidence that activation of TRPM7 channels induces zinc accumulation, and that zinc entry 

through these channels plays an important role in cell injury. First, fluorescent imaging with 

FluoZin3 demonstrated higher intracellular zinc concentration in HEK293 cells over-expressing 

TRPM7. Second, over-expression of TRPM7 channels increased susceptibility of these cells to 

zinc-induced injury. Third, in cultured mouse cortical neurons, removal of Ca 2+ /Mg 2+ from 

extracellular solution, a condition known to facilitate the opening of TRPM7 channels, also 

augmented zinc-induced cell death and the degree of cell injury was correlated with the 

increment of intracellular zinc. Fourth, this zinc toxicity in neurons was attenuated by 

nonspecific TRPM7 blockers Gd 3+ and 2-APB. Taken together, these results suggest that 

activation of TRPM7 channels is associated with zinc-dependent toxicity. 

Disclosures: K. Inoue, None; Z. Xiong, None. 

Poster 





Title: TRPA1 is a potential target for the treatment of asthma 

Authors: M. M. MORAN 1 , D. DEL CAMINO 2 , J. STRICKER 3 , J. WITEK 4 , J. A. CHONG 5 , 

*N. J. HAYWARD 6 ; 

1 Director of Biol., 2 Project Leader, 3 Dept. of Pharmacol., 4 Director of Cell Biol., 5 Director of 

Target Discovery, 6 VP: Pharmacol. & Toxicology, Hydra Biosci., Cambridge, MA 

Abstract: TRPA1 is a member of the Transient Receptor Potential superfamily of calciumpermeable, 

non-selective cation channels. It is selectively expressed in neurons of the dorsal root, 

trigeminal and nodose ganglia. Recent work has shown that TRPA1 responds to a number of 

environmental irritants known to cause respiratory distress, including formaldehyde, acrolein and 

oxidants. Here we probed the role of TRPA1 in an ovalbumin-induced model of asthma using a 

selective TRPA1 antagonist we identified. We show that blocking TRPA1 not only decreases 

airway hyperresponsiveness, but also decreases infiltration of the lung by a range of immune 

cells. This work implicates TRPA1 as a possible target for the treatment of asthma and other 

pulmonary diseases. In addition, these data suggest that neurogenic inflammation plays a critical 

role in the development of pathology in the OVA-model.

Disclosures: M.M. Moran, Hydra Biosciences, A. Employment (full or part-time); N.J. 

Hayward , Hydra Biosciences, A. Employment (full or part-time); J. Stricker, Hydra 

Biosciences, A. Employment (full or part-time); J. Witek, Hydra Biosciences, A. Employment 

(full or part-time); D. del Camino, Hydra Biosciences, A. Employment (full or part-time); J.A. 

Chong, Hydra Biosciences, A. Employment (full or part-time). 

Poster 





Support: NIH 5RO1NS031234 

NIH 2RO1NS036692 

Title: TRPC1 in glioma cells: Store operated calcium entry and implications for glioma 

physiology 

Authors: *V. C. BOMBEN, H. W. SONTHEIMER; 

Neurobio., Univ. Alabama-Birmingham, Birmingham, AL 

Abstract: Gliomas, glial-derived primary brain tumors, are amongst the fastest growing human 

tumors and often kill patients within months of initial diagnosis. In terms of pathology, these 

cells proliferate and migrate extensively throughout the brain. In many cell types including 

cancer cells, pharmacological inhibition of ion channels has been shown to halt cell proliferation 

suggesting that ion channels play an important role in cell cycle progression. One ion channel 

family that has been suggested to impact cell proliferation is the transient receptor potential 

(TRP) family. The TRP canonical (TRPC) subfamily is a 7-member, non-selective cation 

channel family that is activated downstream of the phospholipase C cascade and has a high 

selectivity for calcium. We previously demonstrated that several TRPC channel genes are 

expressed in glioma cells and that pharmacological inhibition of TRPC channels using 

pharmacological inhibitors with some selectivity for TRPC impairs proliferation in D54 glioma 

cells. In this study we used the cell permeant calcium indicator dye FURA-2 to demonstrate that 

pharmacological inhibitors of TRPC channels (2-APB, SKF96365, and MRS1845) impair store 

operated calcium entry. This is also the case with an antibody generated specifically against an 

extracellular domain of the TRPC1 channel, implicating TRPC1 as a component of store 

operated calcium entry in glioma cells and suggesting that Ca 2+ signaling through this pathway is 

important in cell proliferation. To more unequivocally determine the role of TRPC1, we utilized

a shRNA knockdown approach to selectively reduced TRPC1 expression levels by transient 

transfection of multiple TRPC1 shRNA plasmids that target different sequences of the gene into 

D54MG cells. This led to a significant reduction in TRPC1 protein expression as determined by 

Western blot and significantly reduced TRPC1 mediated SKF96365 sensitive currents as 

determined by patch-clamp recordings. Furthermore, FURA-2 imaging studies show that store 

operated calcium entry was impaired in cells that have been transfected with shRNA plasmids. 

Finally, transfection with shRNA also slowed glioma proliferation and a proportion of 

transfected cells became large and multinucleated as we had previously observed with 

pharmacological inhibition of TRPC1 channels. These studies suggest that TRPC1 constitutes a 

molecularly defined component of the store-operated Ca 2+ entry pathway required to allow 

glioma cells to undergo normal cell divisions. (supported by NIH 5RO1 NS031234 & 2RO1 

NS036692). 

Disclosures: V.C. Bomben, None; H.W. Sontheimer, None. 

Poster 





Support: NIH Grant EY014852 and GM60448 

Title: Regulation of bestrophin Cl channels by calcium and phosphorylation: role of the Cterminus 

Authors: *Q. XIAO 1 , K. YU 1 , A. PRUSSIA 2 , Y. CUI 1 , H. C. HARTZELL 1 ; 

1 Cell Biol., 2 Dept. of Chem., emory university, Atlanta, GA 

Abstract: Bestrophins, a new family of chloride channels, are activated by Ca. However, the 

structures and mechanisms responsible for Ca sensitivity remain unknown. hBest1 contains a 

region of acidic amino acids that has been proposed as a potential Ca binding site. Of the 35 

mutants we made in this region, all but two were non-functional. Therefore, we searched for 

other Ca binding domains and identified a pair of motifs in the C-terminus that resemble EFhands. 

Using a homology model built on the crystal structure of calmodulin, the first EF hand of 

hBest1 was predicted to bind Ca with higher affinity than the third EF hand of calmodulin. We 

made point mutations in the first EF hand of hBest1 to test whether this region was involved in 

activation of the channel by Ca. The D312G mutant in the putative Ca binding loop (312-323) 

reduced Ca affinity by 20-fold, and also abolished Ca-dependent rundown. In contrast, mutations

in the second EF hand (369-380) had no effects on channel activation and rundown, suggesting 

EF2 domain may not bind Ca. To explore the function of the EF2 domain, we introduced stop 

codons to produce truncated protein. When the entire second EF hand was deleted by stops 

codons at amino acid 350, 360, or 370, the channels were nonfunctional. Channels truncated at 

390 and 400 were similar to wild type, but the mutant truncated at 380, which deleted the F-helix 

of the EF-hand, showed no rundown. Channel rundown was attenuated by the PKC activator 

PMA, and the protein phosphatase inhibitors, okadaic acid and calyculin. We identified a PKC 

phosphorylation site (S358) located between the two EF hands. The pseudo-phosphorylated 

mutation S358E abolished the rundown. Furthermore, coimmunoprecipitation of hBest1 with 

PP2A was enhanced by PMA and the phosphorylation-mimicking S358E mutation, and was 

inhibited by BIM and S358A mutation, suggesting channel rundown was finely controlled by 

PKC and PP2A at S358. These findings provide insight into how bestrophins are physiologically 

regulated by Ca, PKC and PP2A. 

Disclosures: Q. Xiao , None; K. Yu, None; A. Prussia, None; Y. Cui, None; H.C. Hartzell, 

None. 

Poster 





Title: Effect of malonate, a metabolic pathway inhibitor, on action potential peak shape and the 

relationship to cellular pathways 

Authors: *N. U. AKANDA 1 , P. MOLNAR 2 , J. J. HICKMAN 2 ; 

1 Nanosci Technol. Ctr., Univ. Central Florida, Orlando, FL; 2 NanoScience Technol. Ctr., Univ. 

of Central Florida, Orlando, FL 

Abstract: Malonate is a competitive inhibitor of succinate dehydrogenase, which is a key 

enzyme in the citric acid cycle and electron transport chain. This enzyme plays a central role in 

neuronal energy metabolism as about 80% of total ATP is produced by the mitochondrial citric 

acid cycle and electron transport chain. It has been generally accepted that intracellular ATP 

plays a protective role for cells by maintaining the optimal function of the plasma membrane ion 

pumps, membrane stability, as well as optimal concentration of important intracellular molecules 

and ions (e.g., ROS, Ca 2+ ). If intracellular ATP concentration is reduced for any reason cellular 

functions deteriorate. It has been consistently reported that defects in energy metabolism play a 

key role in progression of neurodegenerative diseases (e.g., amyotrophic lateral sclerosis,

Alzheimer‟s disease, Parkinson‟s disease, and Huntington‟s disease). Monitoring changes in 

action potentials peak shape can be a simple tool to measure the activity of excitable cells as the 

individual ion channels that contribute to the action potential are differentially connected to 

various intercellular pathways. In this study, we used an action potential shape analysis for the 

assessment of the involvement of the voltage-gated ion channels in the neuronal plasma 

membrane during exposure to a toxin. We present the effect of malonate on action potentials in a 

NG108-15 hybrid cell line (mouse neuroblastoma and rat glioma cells) to investigate the 

relationship between differences in ion channel conductance and how this relates to metabolic 

pathway changes. Perfusion of malonate changes the shape of action potentials in a characteristic 

fashion. However, when the cell culturing medium was supplemented with 5 mM Ketones (βhydroxybutyrate 

and acetoacetate) for 24 hours before experiments, no change in the shape of 

action potential was observed. To analyze the drug effects, we have used a computer model of 

the action potential generation of this cell type to discover the extent each ion channel is affected 

during the administration of different toxins. Our work is a first step towards establishing a new 

assay system, based on a complex pathway model of NG108-15 cells, to detect and identify 

toxins by action potential shape analysis. 

Disclosures: N.U. Akanda, None; P. Molnar, None; J.J. Hickman, None. 

Poster 





Support: Danish Medical Research Council 

Ludvig and Sarah Elsass Foundation 

Lundbeck Foundation 

Title: Abnormal peripheral nerve excitability in mice deficient of the myelin protein P0 

Authors: M. MOLDOVAN 1 , S. ALVAREZ 1 , R. MARTINI 2 , *C. KRARUP 3,1 ; 

1 Neurosci. and Pharmacol., Univ. of Copenhagen, Panum Inst., Copenhagen, Denmark; 

2 Neurology, Developmental Neurobio., Univ. of Wuerzburg, Wuerzburg, Germany; 3 Dept Clin. 

Neurophysiol NF 3063, Rigshospitalet, Copenhagen, Denmark

Abstract: Mice expressing half of the normal dose of protein zero (P0+/- mice) have almost 

normal myelin during the first months of life and later develop a slowly progressing 

demyelinating neuropathy resembling human Charcot-Marie-Tooth type 1B neuropathy. In 

contrast, mice completely deficient of P0 (P0-/- mice) display a compromised myelin 

compaction and axonal loss from birth. 

Recent immunohistochemical studies suggested that myelin dysfunction in P0 mutant mice is 

associated with abnormal function and disposition of ion channels at the nodes of Ranvier. The 

aim of this study was to investigate the peripheral nerve excitability of P0-deficient mice by 

“threshold-tracking”, which is an electrophysiological method that can offer clues about the 

nodal and internodal membrane function of peripheral nerves in vivo. 

Excitability and conduction studies were carried out under anesthesia in 2-10 months P0deficient 

mutant mice. Tibial nerves were stimulated at the ankle and the evoked motor 

responses were recorded from the plantar muscles using subcutaneous needle electrodes. 

P0-/- mice revealed marked abnormalities in both conduction and excitability studies. Motor 

responses were at least 200% delayed and had amplitudes below 10%. Accommodation appeared 

remarkably increased during depolarizing threshold electrotonus. Accommodation during 

hyperpolarizing threshold electrotonus was decreased. The recovery cycle appeared shifted 

upwards with increased refractoriness at the expense of the superexcitable period that normally 

follows a single impulse. 

During the investigated time-frame, conduction studies in P0+/- mice remained 

undistinguishable from controls. Nevertheless, both the nodal (refractoriness) and internodal 

(accommodation) excitability measures were abnormal. Deviations were largely similar to those 

observed in P0-/-, albeit of a lower magnitude. The only notable exception was the 

accommodation to depolarization that appeared unaffected, in contrast with the findings in P0-/- 

mice. 

Our data suggest that in mouse model of a demyelinating polyneuropathy, both the nodal and 

internodal membrane function of peripheral axons is abnormal, depending on the P0 expression 

levels. 

Disclosures: M. Moldovan, None; S. Alvarez, None; C. Krarup , None; R. Martini, None. 

Poster 

236. Neurotransmitter Release: Docking and Fusion 


Program#/Poster#: 236.1/D1 

Topic: B.06.c. Calcium dependence 

Support: GK-1328

Title: Electrical fusion pore measurements in mouse chromaffin cells: A high-resolution analysis 

to study the molecular mechanisms of exocytosis 

Authors: *P. KAMALI MOGHADAM, D. BRUNS; 

Physiol. 59, Saarland Univ., Homburg, Germany 

Abstract: The molecular mechanisms that constitute and regulate exocytotic fusion pores are 

little understood. Here, we study the properties of exocytotic fusion pores and the role of 

vesicular SNARE proteins in fusion of single chromaffin granules using time-resolved 

admittance measurements in the cell-attached configuration. We investigate the dynamics of the 

exocytotic fusion pore (the first connection between vesicular lumen and extracellular solution) 

in wild type and v-SNARE deficient cells. We find that the average membrane capacitance of a 

single granule in mouse chromaffin cells is 0.54 ± 0.46 fF. This calculates to a morphological 

granule radius of 63 ± 18 nm as also determined with electron microscopy. Furthermore, the size 

distribution of capacitance steps (reflecting fusion of single vesicles) compares well with the 

radius distribution of the chromaffin granules (CVCv^(1/2) = 0.37; CVRad^(1/2) = 0.38). These results 

suggest that capacitance steps detected during admittance measurements in the cell-attached 

configuration reflect exocytosis of single mouse chromaffin granules. In close correlation, 

secretory signals are abolished in the absence of the vesicle-associated SNARE proteins, 

cellubrevin and synaptobrevin II (dko cells). Moreover, acute viral driven expression of 

synaptobrevin II in dko cells fully restores exocytosis with fusion events showing properties 

(fusion pore expansion time, dimensions of initial fusion pore) similar to those of wild type cells. 

In ongoing experiments, we investigate how structural components of synaptobrevin II govern 

the fusion pore dynamics. 

Disclosures: P. Kamali Moghadam , None; D. Bruns, None. 

Poster 




Topic: B.06.a. Docking and fusion 

Support: CURE/USAMRMC Grant W81XWH 07254001 

New York State Spinal Cord Injury Grant C020925 

Title: Two distinct modes of exocytotic fusion pore formation in large astrocytic vesicles

Authors: *J. KANG, H. PENG, N. KANG, P. K. STANTON, J. H.-C. LIN; 

Dept Cell Biol & Anat, New York Med. Col., Valhalla, NY 

Abstract: Formation of the fusion pore is a central question for regulated exocytosis by which 

secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring 

exocytosis of glutamate-containing large vesicles in astrocytes, we found that an exocytotic 

fusion pore was generated from fusion at a ring of the docked membrane and movement of a 

fusion-produced membrane fragment. Two modes of fragment movement, 1) a “shift” fragment 

that shifted to the side to expand the fusion pore and 2) a “fall-in” fragment that fell into the 

collapsing vesicle to expand the fusion pore, were observed. Whole-vesicle patch-clamp 

recording showed that partial collapse with transient pore conductance was more frequently 

observed in the fall-in mode than in the shift mode. Electron microscopy of large astrocytic 

vesicles showed shift and fall-in membrane fragments. Two modes of exocytotic fusion pore 

formation demonstrate a novel mechanism for fusion pore expansion and full/partial collapse of

large secretory vesicles.

Disclosures: J. Kang, None; H. Peng, None; N. Kang, None; P.K. Stanton, None; J.H. Lin, 

None. 

Poster 





Support: NS44057

Title: Vesicle membrane bending energy drives fusion pore dilation in Ca 2+ -triggered exocytosis 

Authors: *M. B. JACKSON, E. HUI, E. R. CHAPMAN, Z. ZHANG; 

Dept Physiol, Univ. of Wisconsin Med. Sch., Madison, WI 

Abstract: Amperometry recording of catecholamine release from chromaffin cells and PC12 

cells reveals two sequential stages during the exocytosis of single vesicles. The first stage, the 

pre-spike foot, reports the slow leak of vesicle content through an open, metastable fusion pore. 

The second stage, the spike, reports the rapid expulsion of the remaining vesicle content as the 

vesicle membrane fuses with the plasma membrane. We noticed that the lifetime of the prespike 

foot of an individual event was on average longer when the subsequent spike was larger. This 

correlation was highly significant and could indicate that membrane bending energy drives 

fusion pore dilation. Smaller vesicles have greater tension and if this accelerates fusion pore 

dilation then fusion pore lifetimes will brief. Elasticity theory states that the membrane curvature 

of a sphere produces a force that is inversely proportional to the radius, R; R is proportional to 

the cube root of the total amperometric charge, Q. Taking tau as the mean pre-spike foot lifetime 

of events with similar Q, we found that a plot of 1/tau versus 1/Q^1/3 was well fitted by a line. 

This suggests that membrane bending tension acts as a force to drive fusion pore dilation. 

Changing vesicle size by adding reserpine or L-DOPA changed Q without changing the slope of 

this line. By contrast, lysophosphatidylcholine, arachidonic acid, and oleic acid, which induce 

membrane curvature, all changed the slope of the 1/tau vs 1/Q^1/3 plot. Thus, reagents known to 

alter membrane bending energetics yielded results consistent with the hypothesis that membrane 

bending drives fusion pore dilation. Tryptophan mutants close to the vesicle lumen in the 

membrane anchor of synaptotagmin also change the slope of the 1/tau vs 1/ Q^1/3 plot in a 

manner consistent with the hypothesis that membrane bending drives fusion pore dilation. A 

lipidic fusion pore should be stabilized by the addition of lysophosphatidylcholine to the outer 

membrane leaflet of the plasma membrane. We saw the opposite effect, so we conclude that in 

Ca 2+ -triggered exocytosis the fusion pore becomes lipidic after the onset of dilation. This 

indicates that initially the fusion pore consists of protein rather than lipid. 

Disclosures: M.B. Jackson , None; Z. Zhang, None; E. Hui, None; E.R. Chapman, None. 

Poster 





Support: NIH grant NS44057

Title: Phosphatidylserine regulates fusion pores in PC12 cells 

Authors: *Z. ZHANG 1 , E. HUI 2 , E. R. CHAPMAN 1 , M. B. JACKSON 1 ; 

1 Dept Physiol, UW-Madison, Madison, WI; 2 Biophysic program, Univ. of Wisconsin, Madison, 

Madison, WI 

Abstract: Synaptotagmin (syt) I senses Ca 2+ and triggers exocytosis by initiating a sequence of 

molecular interactions. Syt I binds to both SNARE proteins and lipid bilayers containing 

phosphatidylserine (PS) in a Ca 2+ -dependent manner, but the precise functions of these 

interactions in exocytosis have been difficult to establish. The present study investigated the role 

of PS binding by using carbon fiber amperometry to record Ca 2+ -triggered norepinephrine 

release from PC12 cells. The PS content of the inner leaflet of the plasma membrane was altered 

either by lipid addition to the growth medium or by overexpression of PS synthase 1, PS 

synthase 2, or their function-enhancing mutants. The PS content of PC12 cells was then 

determined by HPLC. All manipulations that elevated PS levels in PC12 cells enhanced 

exocytosis by increasing the frequency of vesicle fusion events triggered by Ca 2+ . Elevated PS 

levels also increased the lifetime of fusion pores that opened during release events. At the highest 

PS level attained, the average fusion pore duration was more than double that of controls. 

Addition of phosphatidylcholine had no effect on exocytosis or fusion pores. Analysis of fusion 

pore openings that close (kiss-and-run), together with fusion pore openings that dilate (fullfusion), 

indicated that PS had a weak, insignificant effect on the rate of fusion pore closure but 

strongly reduced the rate of fusion pore dilation. Elevating PS levels failed to alter vesicle size or 

voltage-activated calcium current. Staurosporine produced very small reductions in the effects of 

PS on fusion pore lifetime, indicating that the major component of PS action does not depend on 

protein kinase C. Stopped-flow kinetics measurements of Ca 2+ -stimulated binding of syt to PS 

containing liposomes showed that the binding became stronger as PS content was increased. 

Thus, the effects of PS revealed here may indicate roles for syt-lipid interactions in exocytosis 

and fusion pore stability. 

Disclosures: Z. Zhang, None; E. Hui, None; E.R. Chapman, None; M.B. Jackson, None. 

Poster 





Support: Slovenian Research Agency Grant P3 310

Title: Transient narrow fusion pore openings in resting neuroendocrine cells 

Authors: *N. VARDJAN 1,2 , J. JORGACEVSKI 1 , M. STENOVEC 2,1 , M. KREFT 1,2 , R. 

ZOREC 1,2 ; 

1 Inst. of Pathophysiology,LN-MCP, Ljubljana, Slovenia; 2 Celica Biomed. Ctr., Ljubljana, 

Slovenia 

Abstract: Spontaneous and stimulated exocytosis were thought to exhibit similar properties at 

elementary level, differing only in the probability of occurrence. However, recent studies 

indicate that spontaneous exocytosis differs from the stimulated one in many respects. For 

instance, spontaneous hormone discharge from a single lactotroph vesicle of the anterior 

pituitary is some 10-20 times slower than stimulated discharge because of the kinetic constraints 

of regular fusion pore openings. 

To see whether the slow release at rest reflects also a relatively narrow fusion pore, we 

performed additional electrophysiological and optical studies on resting and stimulated 

lactotrophs. Transient, kiss-and-run exocytosis, consisting of reversible fusion between the 

vesicle membrane and the plasma membrane, is considered to lead to full fusion upon 

stimulation of vesicles containing classical transmitters. Whether this is also the case in the 

fusion of peptidergic vesicles is unknown. 

We analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously 

forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent 

fluorescent fusion marker. Confocal imaging showed that in ~50% of the spontaneous exocytotic 

events fusion pore openings were associated with a change in synaptopHluorin fluorescence, 

indicating efflux of protons, but the pore was impermeable to FM 4-64 (molecular diameter = ~1 

nm) and HEPES (molecular diameter = ~0.5 nm). These findings, confirmed with capacitance 

measurements, indicate a fusion pore diameter 70% 

of exocytotic events exhibited a larger, FM 4-64-permeable pore (>1 nm). Capacitance 

measurements showed that the majority of exocytotic events in spontaneous and stimulated 

conditions were transient. However, stimulation increased the frequency of transient events and 

their fusion pore dwell-time, but decreased the fraction of events with lowest measurable fusion 

pore. 

Thus kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic 

vesicles. Furthermore, stimuli prolong the effective fusion pore opening, which expands from its 

resting subnanometer diameter to dimensions permitting hormone secretion. 

Disclosures: N. Vardjan , None; J. Jorgacevski, None; M. Stenovec, None; M. Kreft, 

None; R. Zorec, None. 

Poster 

236. Neurotransmitter Release: Docking and Fusion




Support: NIH Grant MH064070 

Title: Relationship between vesicle pools, release probability, and the prevalence of rapid vesicle 

retrieval (kiss and run) 

Authors: Q. ZHANG, *R. W. TSIEN; 

Dept Molec & Cell Physiol, Stanford Univ. Sch. Med., Stanford, CA 

Abstract: There is growing evidence for the existence of non-classical fusion (modes generally 

termed “kiss-and-run”, K&R) in various secretory systems, but the cellular basis and biological 

impact of K&R are far from clear. We have developed a quantum dot (Qdot)-based approach to 

monitor the fate of individual synaptic vesicles as they undergo K&R or classical full collapse 

fusion (FCF). After loading a single vesicle with an indivisible probe, we could identify a 

vesicle‟s pool of origin with a hypertonic sucrose challenge, then track the fate of the once-used 

vesicle during subsequent electrical stimulation. This allowed us to clarify the basis of a 

progressive change in the prevalence of K&R during electrical stimulation, falling from 65% to 

5% at steady state. We prefaced the electrical stimulation with a hypertonic challenge to provoke 

a restricted turnover of the readily releasable pool (RRP). The hypertonic challenge produced a 

mixture of Qdot signals corresponding to K&R and FCF events that were similar in amplitude, 

time course and prevalence to those evoked at the start of electrical stimulation on its own. More 

than half of the hypertonicity-induced events were K&R. However, in the wake of the hypertonic 

challenge and RRP release, subsequent electrical stimulation evoked only a low, flat level of 

K&R usage (~5%). Thus, RRP vesicles show a much higher prevalence of K&R than reserve 

pool vesicles, and the gradual change in prevalence reflects usage of the RRP. Put differently, 

RRP residents acquired an enhanced propensity to undergo K&R, enabling their subsequent 

reuse. However, the readiness for K&R can be dissipated. Interestingly, we found that a loading 

procedure that targeted RRP vesicles led to preferential loading of vesicles releasable by 

subsequent hypertonic challenges, supporting the idea of a preferential recycling of RRP vesicles 

back to their pool of origin. 

Disclosures: Q. Zhang, None; R.W. Tsien , None. 

Poster 





Support: DFG Grant GRK521- 

Title: The role of the SNARE complex in neuronal exocytosis 

Authors: J. P. WEBER, *J. B. SORENSEN; 

Dept of Membrane Biophys, Max-Planck Inst. for Biophys Chem, Gottingen, Germany 

Abstract: The neuronal SNARE complex is at the heart of neuronal exocytosis. It is a coiled coil 

consisting of four alpha-helical SNARE-motifs, which are provided by three proteins: Syntaxin- 

1, Synaptobrevin-2 and SNAP-25. The complex is held together by central layers made up of the 

side chains of every third or fourth amino acid in the α-helical SNARE domains. The formation 

energy of the complex is assumed to overcome the energy barrier for fusion, but exactly how and 

when this happens is unclear. According to the zipper hypothesis, assembly of the SNARE 

motifs towards the C-terminal membrane anchors of synaptobrevin and syntaxin provides the 

energy for fusing the membranes. 

We investigate the mechanism of SNARE-driven fusion, and the role of SNARE complex 

assembly in shaping synaptic transmission, especially short-term plasticity, recovery from 

presynaptic depression and spontaneous release. To this end, we introduced mutations in the 

different layers of the two SNARE motifs in SNAP-25, from the N- to the C-terminal end. We 

use cultured autaptic hippocampal neurons isolated from Snap-25 null mice infected (“rescued”) 

with recombinant lentivira expressing different SNAP-25 mutations/constructs. The cultured and 

rescued neurons were characterized electrophysiologically. 

We could phenotypically distinguish N-terminal mutations from mutations in the middle and in 

the C-terminal end of the complex. The C-terminal and middle phenotypes include differences in 

short-term plasticity and spontaneous release, whereas the N-terminal mutations markedly 

delayed recovery from synaptic depression induced by a long train of action potentials. 

Interestingly, the pool of vesicles released by hypertonic sucrose application (sucrose pool) was 

unchanged with N-terminal mutations, and recovery of the sucrose pool was also unaffected, 

showing that assembly of the N-terminal part of the SNARE-complex is not rate-limiting for 

release during hypertonic shock. C-terminal mutations suppress spontaneous release (mini 

events) almost completely, whereas mutations in the middle of the complex stimulate 

spontaneous release. Mutations in the C-terminal end of the complex lead to a shift from shortterm 

synaptic depression towards facilitation, indicative of a decreased vesicular fusion 

probability. 

These findings demonstrate that C-terminal assembly of the SNARE-complex is involved in 

setting the probability for spontaneous as well as evoked release, and the SNARE-complex 

affects both short-term synaptic plasticity and recovery from presynaptic depression. 

Disclosures: J.P. Weber, None; J.B. Sorensen , None.

Poster 





Support: NIH-NS036942 

Title: Vesicle release probability and immediately releasable pool size at hippocampal synapses 

determined with vGlut-pHluorin 

Authors: *P. ARIEL 1,2 , T. A. RYAN 1 ; 

1 Biochem., Weill Med. Col, Cornell, New York, NY; 2 David Rockefeller Grad. Program, The 

Rockefeller Univ., New York, NY 

Abstract: Neurotransmitter release from nerve terminals requires exocytosis of synaptic 

vesicles. Two parameters determine how many vesicles fuse in response to an action potential: 

the number of vesicles in a privileged pool awaiting only calcium entry to trigger their fusion 

(the immediately releasable pool, IRP) and the probability that each of them will fuse (Pv). Here 

we have made use of vGlut-pHluorin expressed in dissociated neurons in culture to determine the 

size of the IRP as well as Pv. High sensitivity optical recordings at 10 msec resolution allowed us 

to probe these biophysical properties at the single synapse level. We used two different methods 

to determine the size of the IRP. The first relied on rapid depletion of the pool using high 

frequency action potential firing. These measurements show that when stimulated at 100 Hz, the 

rate of synchronous exocytosis decreases to zero following exocytosis of 8.4+/-0.7% of the total 

recycling pool of vesicles, which we refer to as IRPHF. As an alternate approach we devised 

conditions to saturate the calcium sensitive release machinery present on synaptic vesicles and 

thus release the entire IRP with a single action potential. We refer to this pool as IRPCaSat .To this 

end, we applied individual action potentials in conditions of high extracellular calcium ion 

concentration ([Ca2+]e) and measured exocytosis. Independently, we measured the relative 

increase in intracellular ([Ca2+]i) in presynaptic terminals under the same conditions using 

calcium indicators. We showed that calcium influx saturates under these conditions of high 

[Ca2+]e and used action potential broadening with K+ channel blocker 4-AP to further increase 

[Ca2+]i. The combination of high [Ca2+]e and 4-AP allowed us to assay single action potential 

exocytosis as a function of relative changes in intracellular [Ca2+]i over a wide range. These 

data show that exocytosis is well described by a generalized Hill model giving an apparent 

cooperativity n~5 and an IRPCaSat size of 5.5+/-0.3% of the total recycling pool of vesicle, ~65% 

of the value of IRPHF. Cross-depletion studies show that IRPCaSat is a subset of IRPHF and thus a 

hierarchy of at least 2 pools appears to exist at the active zone of these nerve terminals. We 

determined the probability that a vesicle within IRPCaSat fuses in response to a single action 

potentials to be 0.06 but that this parameter varied over a wide range across synapses. The

molecular determinants of this variability remain an open question that can now be addressed 

using the methods we have developed. 

Disclosures: P. Ariel , None; T.A. Ryan, None. 

Poster 





Support: Deutsche Forschungsgemeinschaft (DFG) (SFB 530 and GRK1326) to D.B. 

HOMFOR to D.B. 

Title: Synaptobrevin II comprises a mechanism that stabilizes vesicles in the primed state 

Authors: *M. BORISOVSKA 1 , D. BRUNS 2 ; 

1 Oregon Hlth. Sci. Univ., Portland, OR; 2 Inst. for Physiol., Univ. of Saarland, Homburg, 

Germany 

Abstract: SNARE proteins play an essential role in multiple steps of regulated exocytosis. It is 

believed that by binding one another, SNAREs mediate priming of vesicles and initiation of 

membrane fusion. However little is known how structures outside the SNARE motif modulate 

exocytosis. Here we investigate the impact of the membrane proximal region of Synaptobrevin II 

on chromaffin granule exocytosis. For our experiments, we took advantage of double v-SNARE 

deficient mice devoid of both synaptobrevin II and cellubrevin. The possibility to acutely express 

the protein of interest on a genetic v-SNARE null background provides nearly ideal conditions 

for a structure-function analysis. Whole-cell membrane capacitance measurements in 

combination with flash-photolysis and calcium imaging allowed us to monitor exocytosis at a 

millisecond time scale. 

Amino acid insertions (1, 3 and 12 amino acids) between the SNARE motif and the tryptophanrich 

region result in strong linker-length dependent reduction of vesicle priming as well as in 

delayed stimulus-secretion coupling. These results are consistent with our previous observations 

(Kesavan et al., 2007), but show stronger effects with respect to the linker length-dependent 

attenuation suggesting a multifunctional role of juxtamembrane region of synaptobrevin II in 

vesicle exocytosis. Moreover, we find that the increase in distance and flexibility between the 

SNARE motif and the transmembrane anchor does not depend on the character of the inserted 

amino acids. In addition, increase in distance or flexibility between the tryptophan residues and

the transmembrane anchor of synaptobrevin II at position 94K causes vesicle exocytosis at subthreshold 

Ca2+ concentrations without changing the Ca2+-affinity for secretion. This indicates 

destabilization of vesicles in the primed state. Taken together these observations suggest a 

critical role of juxtamembrane region of synaptobrevin II in transferring and in keeping the 

vesicles in the release-ready state. 

Disclosures: M. Borisovska , None; D. Bruns, None. 

Poster 





Support: SFB 530 

Title: The intramolecular distance between SNARE motif and transmembrane domain of 

synaptobrevin II is crucial to synchronize neurotransmitter release 

Authors: *R. E. GUZMAN 1 , Y. SCHWARZ 2 , D. BRUNS 2 ; 

1 Univ. des Saarland, Homburg, Germany; 2 Physiol., Univ. of Saarland, Homburg, Germany 

Abstract: Synaptic neurotransmitter release is mediated by exocytotic fusion of synaptic 

vesicles. At least one SNARE protein is anchored in the vesicle membrane (v-SNARE), whereas 

another is anchored in the target membrane (t-SNARE). SNARE complex formation is believed 

to be essential for driving membrane fusion. SNARE assembly is initiated at the N-terminal ends 

of the SNARE motif and proceeds toward the C-terminal membrane anchor, clamping the 

membranes together and thus overcoming the energy barrier for fusion. We studied to what 

extent force transduction between the SNARE domain and the transmembrane domain (TMD) is 

required for neurotransmitter release. For this, we expressed Synaptobrevin II (Syb II) mutant 

proteins carrying an extended juxtamembrane region in autaptic and continental cultures of 

hippocampal neurons that are genetically deficient for Syb II. Overexpression of the mutants did 

not change the number of synapses or protein levels compared to expression of the 

wildtypprotein. We show that extending the juxtamembrane region by insertion of flexible 

linkers reduces the amplitude and charge of the synchronous release. It diminishes the RRP size, 

release probability and delays the time to peak of the evoked response in a linker lengthdependent 

fashion. Furthermore, it slows the time course of the glutamatergic mEPSC. Our 

results indicate that tight coupling between the complex-forming SNARE motif and TMD of Syb 

II drives exocytosis of small synaptic vesicles at the millisecond time scale.

Disclosures: R.E. Guzman , None; Y. Schwarz, None; D. Bruns, None. 

Poster 





Support: Ministry of Education, Science, Technology, Sports and Culture of Japan 

Sasakawa Scientific Research Grants 

Takeda Science Foundation 

Title: Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin 

Authors: *K.-I. NAGATA 1 , H. ITO 1 , K. ATSUZAWA 2 , R. MORISHITA 1 , N. USUDA 2 , K. 

SUDO 1 , I. IWAMOTO 1 , K. MIZUTANI 3 , R. KATOH-SEMBA 1 , Y. NOZAWA 3 , T. ASANO 1 ; 

1 Inst. Dev Res., Kasugai, Japan; 2 Fujita Hlth. Univ. Sch. of Med., Toyoake, Japan; 3 Gifu Intl. 

Inst. of Biotech., Gifu, Japan 

Abstract: Septins, a conserved family of GTP/GDP-binding proteins, are present in organisms 

as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, 

Sept8, Sept9 and Sept11, in various rat tissues by Western blot analyses and found all septins to 

be expressed in brain. We also examined the developmental changes of the expression of these 

septins in the rat brain and found that the level of Sept8 increased during post-natal development. 

Morphological analyses revealed that Sept8 is enriched at presynapses. Using yeast two-hybrid 

screening, we identified vesicle-associated membrane protein 2 (VAMP2), a SNARE protein, as 

an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 

to synaptic vesicles and dissociate prior to forming SNARE complex. We found Sept8 

suppresses the interaction between VAMP2 and synaptophysin through the interaction with 

VAMP2. On the other hand, Sept8 formed complex with syntaxin 1A but not with SNAP25 

while SNAP25 but not syntaxin 1A disrupted the interaction of VAMP2 with Sept8. These 

results suggest that Sept8 may participate in the process of SNARE complex formation and 

subsequent neurotransmitter release. 

Disclosures: K. Nagata, None; H. Ito, None; K. Atsuzawa, None; R. Morishita, None; N. 

Usuda, None; K. Sudo, None; I. Iwamoto, None; K. Mizutani, None; R. Katoh-Semba, 

None; Y. Nozawa, None; T. Asano, None.

Poster 





Support: 1211/07 to U.A 

EUSynapse project, contract No. 019055 to AJG 

Title: DOC2B acts as a calcium switch and enhances vesicle fusion 

Authors: *R. FRIEDRICH 1 , A. J. GROFFEN 2 , E. CONNELL 3 , J. R. T. VAN WEERING 2 , O. 

GUTMAN 1 , Y. I. HENIS 1 , B. DAVLETOV 3 , U. ASHERY 1 ; 

1 Tel Aviv Univ., Tel Aviv, Israel; 2 Dept. of Functional Genomics, Ctr. for Neurogenomics and 

Cognition Research, Vrije Univ. (VU) and VU Med. Ctr., Amsterdam, Netherlands; 3 Neurobio. 

Division, Med. Res. Council-Laboratory of Mol. Biol., Cambridge, Cambridge, United Kingdom 

Abstract: Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a 

synaptic protein that translocates to the plasma membrane (PM) upon small elevations in 

intracellular calcium concentration. The aim of this study is to investigate the role of DOC2B in 

calcium-triggered exocytosis and its interactions with other proteins of the exocytotic machinery. 

Using biochemical and biophysical measurements, we demonstrate that the C2A domain of 

DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of 

electrophysiological, morphological and total internal reflection fluorescent measurements, we 

found that DOC2B acts as a priming factor and increases the number of fusion-competent 

vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a 

mutated DOC2B that is constantly at the PM, showed that DOC2B enhances catecholamine 

secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert 

its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that 

DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion 

kinetics of single vesicles and interacts with the PM SNAREs. We conclude that DOC2B is a 

calcium-dependent priming factor and its activity at the PM enables efficient expansion of the 

fusion pore, leading to increased catecholamine release. The interaction of DOC2B with Munc18 

and/or Munc13 might be important for DOC2B's ability of to increase the exocytotic burst. 

Disclosures: R. Friedrich , None; A.J. Groffen, None; E. Connell, None; J.R.T. van Weering, 

None; O. Gutman, None; Y.I. Henis, None; B. Davletov, None; U. Ashery, None.

Poster 





Support: Ministry of Education, Culture, Sports, Science, and Technology, Japan 

Title: Synaphin/complexin directly associates with calcium sensor synaptotagmin 1 

Authors: *H. TOKUMARU 1 , C. SHIMIZU-OKABE 1 , K. ITOH 1 , T. SHINOHARA 1 , T. ABE 2 ; 

1 Pharmaceut. Sci., Tokushima Bunri Univ., Sanuki-shi, Japan; 2 Dept. Cell. Neurobiol., Brain 

Res. Inst. Niigata Univ., Niigata, Japan 

Abstract: Although the binding of synaphin (also called complexin) to the SNARE complex is 

critical for neurotransmitter release, the role of synaphin remains unclear. Here, we show that 

synaphin directly binds with synaptotagmin 1, a major Ca 2+ sensor for fast neurotransmitter 

release. The mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the 

C-terminal region is essential for the binding. The binding was sensitive to ionic strength, 

suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven 

consecutive glutamic acid residues (residues 108-114) at the C-terminal region of synaphin to 

alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. 

Furthermore, the synaphin C-terminal peptide blocked the binding of synaptotagmin 1 to 

synaphin, while the same C-terminal peptide with mutation of all the consecutive glutamic acid 

residues to alanine had no effect on the binding. Immunoprecipitation experiments with the brain 

membrane extract showed the presence of a complex of synaphin/synaptotagmin 1/SNAREs. We 

propose that synaphin recruits synaptotagmin 1 to fusion machinery and synergistically functions 

with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis. 

Disclosures: H. Tokumaru, None; C. Shimizu-Okabe, None; K. Itoh, None; T. Shinohara, 

None; T. Abe, None. 

Poster 





Support: MH 069791 

DOD/DMEA-H94003-06-2-0608 

Title: Investigation of parallel and anti-parallel configurations of syntaxin 1a and synaptobrevin2 

interaction 

Authors: W. LIU 1 , *V. MONTANA 1 , U. MOHIDEEN 2 , V. PARPURA 1 ; 

1 Dept Neurobiol, Univ. Alabama, Birmingham, AL; 2 Dept of Physics and Astronomy, Univ. of 

California, Riverside, CA 

Abstract: Syntaxin 1A and Synaptobrevin 2 (also known as vesicle-associated membrane 

protein 2) along with SNAP25 belong to the SNARE (soluble N-ethylmaleimide-sensitive fusion 

protein attachment protein receptors) family of proteins. They are involved in the exocytosis of 

synaptic vesicles. It has been shown that both parallel (N-termini of two proteins are aligned at 

same end) and anti-parallel (C-terminal of one protein and N-terminal of the other protein are 

aligned at same end) configurations are present, while details of the interactions between these 

proteins in these configurations have not yet been defined. We used an Atomic Force 

Microscope (AFM) in force spectroscopy mode to investigate the mechanical interactions 

between syntaxin 1A and synaptobrevin 2 at single molecule level. Various terminal 

configurations of proteins (N-N, N-C, C-N and C-C) were studied by attaching the recombinant 

proteins via their histidine 6 tags to nickel-coated AFM tips and glass coverslips. The syntaxinsynaptobrevin 

intermolecular interaction forces, extensions, spontaneous lifetimes and 

interaction energies were obtained. The measured interaction extensions difference (up to 6 nm) 

is related to alignment of different terminal configurations. Their activation energy (ΓH) 

difference is as large as 5 kBT, and implies that parallel configurations might be energetically 

favorable to tether/dock vesicles at the plasma membrane. These findings provide additional 

insight on the characteristics of the Sx1A-Sb2 binary complex and they aid better understanding 

of the possible role for various configurations of the complex in exocoytosis. 

Disclosures: W. Liu, None; V. Montana , None; U. Mohideen, None; V. Parpura, None. 

Poster 





Support: NSF 

Title: In vivo functions and interactions of NSF and SNAP in neurotransmitter release 

Authors: W. YU 1 , F. KAWASAKI 2 , *R. W. ORDWAY 2 ; 

1 Intercollege Grad. Program in Genet., 2 Biol & Ctr. Mol Cell Neurosci, Penn State Univ., 

University Park, PA 

Abstract: Previous work has defined the in vivo role of the Drosophila N-ethylmaleimide- 

Sensitive Factor 1 (dNSF1) protein in neurotransmitter release through analysis of a temperaturesensitive 

(TS) paralytic dNSF1 mutant, comatose. One extension of these studies involves 

analysis of dNSF1 tagged with enhanced green fluorescent protein (EGFP). Transgenic 

expression of dNSF1-EGFP has allowed live fluorescence imaging of wild-type and TS mutant 

dNSF1 at native neuromuscular synapses. Wild-type dNSF1-EGFP was found to rescue 

comatose TS paralytic and lethal mutants and is enriched within presynaptic boutons as observed 

for native dNSF1, indicating dNSF1-EGFP retains the functional properties of dNSF1. Because 

NSF interacts with the Soluble NSF Attachment Proteins (SNAPs) to disassemble SNARE 

protein complexes, which are thought to participate in synaptic vesicle fusion, complementary 

studies involve imaging a fluorescent Drosophila SNAP protein (tdTomato-dSNAP). Like 

dNSF1-EGFP, tdTomato-SNAP is enriched within presynaptic boutons of native synapses. 

Ongoing live imaging studies are employing fluorescence resonance energy transfer (FRET) and 

fluorescence recovery after photobleaching (FRAP) to define the distribution, mobility and 

interactions of NSF and SNAP at native synapses. These properties may be examined at resting 

synapses, during synaptic activity, and in the comatose (NSF) TS paralytic mutant to further 

define the in vivo roles of NSF and SNAP in synaptic vesicle trafficking. 

Disclosures: W. Yu, None; R.W. Ordway , None; F. Kawasaki, None. 

Poster 





Support: Max-Planck Society

Title: Neurosecretion mediated by hetero-oligomeric SNARE supercomplexes in chromaffin 

cells 

Authors: *R. MOHRMANN, J. B. SORENSEN; 

Dept of Membrane Biophys, Max-Planck Inst. f Biophys Chem, Göttingen, Germany 

Abstract: Vesicle fusion requires the orchestrated interaction of multiple key components of the 

release machinery, including the formation of SNARE-complexes. It is envisioned that several 

SNARE complexes accumulate at the prospective fusion site to form a ring-like supercomplex, 

in the middle of which the fusion pore forms. Here, we present a strategy to gain insight into 

supercomplex structure and function by progressive inhibition of fusion using a dominant 

negative SNAP25 variant. This SNAP25 version bears a double point mutation in layer +5, does 

not allow rescue when expressed in Snap-25 null cell, and abolish fusion when overexpressed in 

wildtype cells. The dependency of inhibition on the concentration of the dominant-negative 

variant can deliver information on the properties of supercomplexes. In order to measure the 

inhibition profile, wildtype SNAP25 and dominant-negative variant were co-expressed in Snap- 

25 null chromaffin cells using dicistronic Semliki-viruses harboring an internal ribosomal entry 

site (IRES) of either polio- or encephalomyocarditis-virus. To obtain different expression ratios, 

viruses with different succession of the reading frames of both SNAP25 versions and different 

IRES-elements were generated. Both SNAP25 versions were N-terminally tagged with GFP or 

mCherry and the expression ratio determined by fluorescence quantification. N-terminal tagging 

did not affect the function of the protein. Secretion was assayed by patch-clamp capacitance 

measurements and amperometry and elicited by flash photolysis of caged-calcium. Increasing 

concentrations of the inhibitory variant decreased the amount of exocytosis. The inhibition 

profile displayed a super-linear dependence upon the concentration of the inhibitory variant, 

indicating the existence of supercomplexes, i.e. fusion complexes containing more than one copy 

of SNAP-25. Inhibition affected the fast release component more dramatically than overall 

release, suggesting that ready-releasable vesicles depend on supercomplexes with a larger 

number of SNAREs - or with a more defined configuration of these complexes - than the 

sustained release component. This analysis shed new light on the dependence of exocytosis on 

the SNARE-complex, and delivers an explanation of the wide-spread observation that the fast 

release component is more dramatically affected than sustained released by genetic manipulation 

of exocytotic proteins. 

Disclosures: R. Mohrmann, None; J.B. Sorensen, None. 

Poster 



Program#/Poster#: 236.17/D17


Support: Heart and Stroke Foundation of Canada. 

National Science and Engineering Council of Canada 

Title: Simulation of vesicular mobility in secretory cells 

Authors: *M. I. GLAVINOVIC 1 , M. TAJPARAST 2 ; 

1 Dept Physiol, 2 Chem. Engin., McGill Univ., Montreal, QC, Canada 

Abstract: Vesicular size is known to be variable and vesicular location changes during secretion. 

Vesicular mobility has also been estimated and may change during stimulation. Several forces 

are expected to be important in determining the mobility of vesicles in secretory cells: a) 

cytoskeleton, b) viscosity of intracellular fluid and c) vesicular size. However, the electrostatic 

interactions caused by the surface charges at both vesicular and plasma membranes may also 

make a contribution. The vesicular mobility may thus also be influenced by the charge density on 

the vesicular and plasma membranes. To assess the importance of various forces in determining 

vesicular mobility and to gain better understanding of the dynamics of vesicular movement 

during secretion we consider the vesicle in the secretory cell as a nano-size electromechanical 

device operating in a liquid electrolyte. We simulate the vesicle as a charged sphere near a 

charged dielectric sheet (representing plasma membrane of a secretory cell) using a coupled 

system of Poisson-Nernst-Planck and Navier-Stokes equations. The external force acting on the 

vesicle is assumed to change in a step-like manner. Several parameters were varied from one 

simulation run to another - vesicular radius, vesicular distance from the plasma membrane, 

charge density and sign on both membranes, fluid viscosity, concentration of charged particles in 

the solution and their diffusion constants. Vesicular „impulse response‟ was determined from the 

movement of the vesicle following a step-like change of the external force. Vesicular „impulse 

response‟ and mobility are found to be dependent not only on the vesicular size and fluid 

viscosity but also on the distance between the vesicle and the plasma membrane, the charge on 

both the vesicle and plasma membrane as well as on the concentration of free charged particles 

(ions, transmitter, hormones,�). Moreover, an estimate is made of the external force needed to 

move the vesicle at speeds as measured experimentally. Finally, for each set of parameters 

characterizing the secretory cell and the vesicle, a distance is determined at which the external 

force pushing the vesicle toward the plasma membrane is balanced by the repulsive electrostatic 

forces. 

Disclosures: M.I. Glavinovic , None; M. Tajparast, None. 

Poster 





Title: Alpha-synuclein inhibits arachidonic acid-induced exocytosis and SNARE complex 

formation 

Authors: *F. DARIOS 1 , I. LOPEZ 2 , L. M. GUTIÉRREZ 2 , B. DAVLETOV 1 ; 

1 Lab. Mol Biol, Med. Res. Council, Cambridge CB2 0QH, United Kingdom; 2 Univ. Miguel 

Hernández, Inst. de Neurociencias, Alicante, Spain 

Abstract: Alpha-synuclein is a small cytosolic protein involved in several neurodegenerative 

diseases including Parkinson‟s disease, but its physiological function is unknown. Recently, 

alpha-synuclein has been proposed to regulate the function of SNAREs (soluble Nethylmaleimide-sensitive 

factor attachment protein receptors), a set of proteins involved in 

vesicle fusion. However, the mechanism of regulation remains unknown. Alpha-synuclein fails 

to interact with SNARE proteins suggesting an indirect mode of action. 

We have recently shown that polyunsaturated fatty acids act on one of the SNARE proteins, 

syntaxin, enhancing the formation of SNARE complex which is instrumental for 

neurotransmitter release. Since alpha-synuclein is also sensitive to polyunsaturated fatty acids, 

we analysed alpha-synuclein effects on fatty acid-mediated SNARE interactions. Here we show 

that arachidonic acid, an omega-6 polyunsaturated fatty acid involved in the regulation of 

neurotransmitter release, stimulates SNARE complex formation and exocytosis, whereas alphasynuclein 

blocks this action both in vitro and in vivo. This effect is due to the ability of alphasynuclein 

to sequester arachidonic acid, thereby blocking the activation of syntaxin. Our data 

shed new light on the action of alpha-synuclein in the modulation of neurotransmission. 

Disclosures: F. Darios, None; B. Davletov, None; L.M. Gutiérrez, None; I. Lopez, None. 

Poster 





Support: NSF Grant 0544031

Title: Regulation of levels of UNC-13 at synapses in Caenorhabditis elegans 

Authors: *B. M. KAAS, R. WENNELL, T. LIMOUZE, R. LAWSON, R. KOHN; 

Ursinus Col., Collegeville, PA 

Abstract: Synaptic neurotransmission is a highly regulated process. The nematode 

Caenorhabditis elegans has a well-characterized nervous system that has been intensively 

investigated in the study of neurotransmission. This organism‟s nervous system has functional 

similarity to those of higher organisms. The UNC-13 protein, which operates at the pre-synaptic 

membrane of C. elegans, participates in the fusion of synaptic vesicles to allow neurotransmitter 

release into the synapse. Defects in unc-13 result in decreased release of neurotransmitters and 

paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate 

in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin 

proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is 

tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. 

elegans knockout consortium isolated a strain with a large deletion in the coding region of the 

gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation 

of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in 

changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, 

developmental rate, and body bends per minute. Aldicarb assays were used to determine whether 

a deletion in the gene coding for the F-box protein alters the response to inhibitors of 

acetylcholinesterase. We found that the deletion resulted in changes in developmental rate and in 

aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding 

for the F-box protein and in unc-13. 

Disclosures: B.M. Kaas , None; R. Wennell, None; T. Limouze, None; R. Lawson, None; R. 

Kohn, None. 

Poster 





Title: Carboxypeptidase E cytoplasmic tail mediates docking/exocytosis of synaptic vesicles 

Authors: *J. J. PARK 1 , H. LOU 1 , N. X. CAWLEY 1 , A. SARCON 1 , T. ADAMS 2 , Y. LOH 1 ; 

1 NICHD, NIH, Bethesda, MD; 2 QIAGEN, Germantown, MD

Abstract: Activity-dependent secretion of neurotransmitters and neuropeptides is essential for 

brain function. Previously, we reported that CPE-KO mice showed a of lack glutamate-mediated 

b-wave in their retinograms. In this study, we show absence of high K+stimulated secretion of 

glutamate from cultured primary embryonic hypothalamic neurons (E16) and adult mouse 

synaptosomes isolated from CPE-KO mice, raising the possibility that CPE may be present on 

glutamate vesicles and play a role in mediating release of these vesicles. Indeed, subcellular 

fractionation studies revealed that a synaptophysin-enriched synaptic vesicle fraction from 

mouse brain contained CPE and the vesicular glutamate transporter, Vglut2, but not a dense-core 

granule-specific protein marker, chromogranin A (CgA). Moreover, intact synaptic vesicles were 

found to bind the extreme C-terminal antibodies but not IgGs, indicating the existence of a 

population of synaptic vesicles which contains transmembrane CPE with a cytoplasmic tail, as 

was previously found in peptidergic dense core vesicles. Electron microscopic analysis of 100 

hypothalamic synaptic densities revealed that 40% of synapses had no docked synaptic vesicles 

at the presynaptic density in CPE-KO mice in contrast to WT mice, implicating that in some 

neurons, CPE is involved in synaptic vesicle docking, possibly mediated by its cytoplasmic tail. 

In in vitro GST pulldown assays, GST-CPEC10 consisting of amino acid residues in the 

cytoplasmic tail, bound Rab27A, a small GTPase protein necessary for vesicle docking to the 

plasma membrane, from both brain and PC12 cell cytosol. A role of the CPE cytoplasmic tail 

(CPEC10) in exocytosis of RFP-synaptophysin labeled, CgA negative synaptic vesicles was 

demonstrated by TIRF microscopic studies. Expression of GFP-tagged CPEC10 in PC12 cells 

abolished high K+ stimulated exocytosis of RFP-synaptophysin labeled vesicles. From all the 

observations in this study, we propose that the vesicular CPE cytoplasmic tail plays a critical 

new role in the recruitment of Rab27A necessary for docking/exocytosis of glutaminergic 

vesicles in the hypothalamus. 

Disclosures: J.J. Park, None; H. Lou, None; N.X. Cawley, None; A. Sarcon, None; T. 

Adams, None; Y. Loh, None. 

Poster 






NIH Grant 1F30NS061494-01

Title: Identification and characterization of complexin 3 and 4 paralogs in the zebrafish visual 

system 

Authors: *G. J. ZANAZZI, G. G. MATTHEWS; 

Dept Neurobiol & Behavio, SUNY Stony Brook, Stony Brook, NY 

Abstract: In the vertebrate retina, the presynaptic terminals of photoreceptors and bipolar cells 

release neurotransmitter tonically and rapidly at active zones that contain synaptic ribbons, 

which are electron-dense organelles that tether vesicles. The molecular mechanisms that underlie 

efficient exocytosis at these synapses are not well-understood. However, a recent study identified 

specific expression of complexins 3 and 4 at ribbon-containing synapses in the retina. At 

conventional synapses, complexins appear to stabilize the SNARE complex in a fusion-ready 

state before calcium enters the terminal and binds to synaptotagmin. Therefore, the identification 

of ribbon-specific isoforms of these synaptic regulators is of special interest. By searching the 

ENSEMBL and GenBank databases, we identified and subsequently cloned five zebrafish 

orthologs that show 50-75% amino acid identity with mammalian complexins 3 and 4. 

Phylogenetic analysis reveals two complexin 3 paralogs and three complexin 4 paralogs. 

Utilizing polyclonal antibodies specific for complexin 3 and complexin 4, we demonstrate that 

these proteins are expressed in the outer plexiform layer (OPL) and inner plexiform layer (IPL) 

of the adult zebrafish retina. Complexin 3 co-localizes with zpr 1 in terminals of double cone 

photoreceptors in the OPL, and co-localizes with protein kinase C in ON bipolar cell terminals in 

the IPL. Complexin 4 appears to be expressed more diffusely in terminals in both the OPL and 

IPL. To characterize complexin 3 expression during development, we have performed 

immunocytochemistry with the complexin 3 polyclonal antibody and whole mount in situ 

hybridization with paralog-specific probes. Complexin 3 immunoreactivity appears in double 

cone terminals in the OPL at 2.5 days post-fertilization, and can also be observed in the lens, 

pineal photoreceptors, and optic nerve. By 3 days post-fertilization, complexin 3 also appears in 

the IPL, similar to adult staining. Whole mount in situ hybridization reveals that both paralogs 

are expressed in the retina during larval development. We are currently investigating possible 

roles for complexin 3 during zebrafish development and whether complexin 3 may regulate 

synaptic exocytosis in cones and bipolar cells. Taken together, these results suggest that 

complexins 3 and 4 may have varied effects in the developing and adult zebrafish visual system. 

Disclosures: G.J. Zanazzi , None; G.G. Matthews, None. 

Poster 





Support: NSF 0456578 

Title: Identification of a new locus functioning in synaptic transmission through a screen for 

genetic modifiers of the Drosophila calcium channel mutant, cac TS2 

Authors: A. LUTAS, S. ACHARJEE, *F. KAWASAKI; 

Biol & Ctr. Mol Cell Neurosci, Penn State Univ., University Park, PA 

Abstract: Neurotransmitter release is triggered by calcium influx through presynaptic voltagegated 

calcium channels. In Drosophila, the molecular basis of presynaptic calcium channel 

function was defined through analysis of cac TS2 , a temperature-sensitive (TS) paralytic mutant of 

the calcium channel α1 subunit gene, cacophony. The cac TS2 mutant has also served as a starting 

point for further genetic analysis to broaden our understanding of the functions and interactions 

of cac-encoded calcium channels. We have conducted a screen for second chromosome genetic 

modifiers of cac TS2 and have begun to characterize a new mutation recovered as an enhancer. 

Double mutants of cac TS2 and the new mutation, referred to as e(cac) 2902 , exhibited enhanced TS 

paralysis relative to cac TS2 . Electrophysiological analysis revealed that e(cac) 2902 also enhanced 

the TS synaptic phenotype of cac TS2 . Furthermore, the isolated e(cac) 2902 mutation in a cac + 

genetic background produced a TS reduction in the EPSC amplitude, indicating that the 

e(cac) 2902 gene product serves an important function in synaptic transmission. Meiotic mapping 

placed the e(cac) 2902 mutation within cytological region 53A and 57F on the distal right arm of 

the second chromosome and deficiency mapping further refined this position to a 9kb genomic 

region. This region contains four genes which have not been implicated in synaptic transmission 

and thus molecular and phenotypic characterization of e(cac) 2902 is expected to further define the 

in vivo roles of specific gene products at native synapses. 

Disclosures: A. Lutas, None; F. Kawasaki , None; S. Acharjee, None. 

Poster 





Support: Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science 

19592308

Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science 

18300128 

Title: Bimodal effect of peripherally-administered ghrelin on the mesolimbic dopamine system 

is dependent on the food consumption 

Authors: Y. KAWAHARA 1 , *H. KAWAHARA 2 , F. KANEKO 1 , M. YAMADA 1 , Y. NISHI 1 , 

E. TANAKA 1 , A. NISHI 1 ; 

1 Kurume Univ. Sch. Med., Kurume, Japan; 2 Dept Dent. Anesthesiol, Kyushu Dent. Col., 

Fukuoka 803-8580, Japan 

Abstract: Ghrelin is a powerful orexigenic peptide released from the stomach that targets CNS 

receptors to induce orexigenic behavior. The present study aimed to identify the neuronal 

interactions that mediate the effects of peripherally-administered ghrelin on mesolimbic 

dopaminergic neurons by intracerebral microdialysis. Peripheral administration of ghrelin 

inhibited dopamine levels in the nucleus accumbens (NAc) when food was deprived following 

ghrelin administration. This inhibitory effect was mediated through an increase in glutamatergic 

inputs to the ventral tegmental area (VTA) that activate GABA interneurons and subsequent 

inhibition of dopamine neurons via GABAA receptors. In contrast, when animals consumed food 

following ghrelin administration, dopamine levels increased robustly. This stimulatory effect was 

mediated through the increase in glutamatergic inputs that directly activate dopamine neurons via 

NMDA receptors. Importantly, both the inhibitory and stimulatory effects of ghrelin required 

activation of ghrelin receptors, growth hormone secretagogue 1 receptors (GHSRs), in the VTA, 

presumably localized on GABA interneurons and dopamine neurons, respectively. When ghrelin 

was injected locally into the VTA, dopamine release in the NAc increased regardless of food 

consumption, supporting the local action of ghrelin on dopamine neurons. In addition, the role of 

GHSRs in the VTA in the orexigenic effect of peripherally-administered ghrelin was 

demonstrated. We conclude that peripherally-administered ghrelin, acting at GHSRs in the VTA, 

induces bimodal effects on mesolimbic dopamine neurotransmission depending on the execution 

of food consumption. A model of neuronal pathways based on the current data is discussed. 

These findings propose a novel mechanism for ghrelin to regulate appetite and food 

consumption. 

Disclosures: Y. Kawahara, None; F. Kaneko, None; H. Kawahara , None; M. Yamada, 

None; E. Tanaka, None; Y. Nishi, None; A. Nishi, None. 

Poster 




Topic: B.06.b. Vesicle recycling and biogenesis 


Title: Multiphoton monoamine imaging reveals two modes of release by Raphe Nucleus 

serotonin neurons 

Authors: *L. A. COLGAN 1 , I. PUTZIER 2 , E. S. LEVITAN 2 ; 

1 Neurobiol, 2 Pharmacol., Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Currently, neurotransmission imaging techniques rely on markers of cycling 

membranes or vesicular proteins instead of direct detection of neurotransmitter. Here, a novel 

approach, Multiphoton Monoamine Imaging (MMI), is established to investigate monoamine 

dynamics in cultured cells and neurons in brain slices. Specifically, a fluorescent serotonin 

analog was visualized by multiphoton microscopy, allowing monoamine storage, release, and 

repackaging to be monitored. The analog was taken-up by PC12 cells expressing the serotonin 

transporter (SERT) and uptake was blocked by fluoxetine, a SERT inhibitor. Intracellularly, the 

analog was concentrated into growth cones where it colocalized with secretory vesicles. This 

localization was disrupted by reserpine, an inhibitor of the vesicular monoamine transporter 

(VMAT), suggesting packaging of the analog into secretory vesicles by VMAT. Furthermore, 

stimulation with high potassium led to reserpine sensitive release, with kinetics and magnitude 

comparable to peptidergic release. MMI was also feasible with Raphe neurons in rat brain slice. 

Incubation of slices with analog gave a fluoxetine sensitive signal in the somatodendritic region 

of neurons, which decreased upon stimulation with the glutamate receptor agonist AMPA. 

Further examination revealed two phases of release. Initial release required calcium and was 

blocked by reserpine, indicating exocytosis of vesicles. This release also decreased the cytosolic 

pool of analog, suggesting that recycling vesicles were rapidly refilled. This was followed by a 

slower, calcium and reserpine insensitive phase of release. Interestingly, this slow release was 

not blocked by fluoxetine, suggesting a mechanism that is independent of calcium, VMAT, and 

SERT. 

Disclosures: L.A. Colgan , None; I. Putzier, None; E.S. Levitan, None. 

Poster 




Topic: B.06.a. Docking and fusion

Support: KB is a recipient of the fellowship from the Algerian Government 

AGT, BHL and SK are supported by British Heart Foundation 

Title: Quantal characteristics of 5HT release from fluorescently identified central neurons of the 

rat 

Authors: K. BENZEKHROUFA 1 , B. LIU 2 , A. G. TESCHEMACHER 1 , *S. KASPAROV 2 ; 

1 Physiol. and Pharmacol., Univ. of Bristol, Bristol, United Kingdom; 2 Physiol. and Pharmacol., 

Univ. Bristol, Bristol, United Kingdom 

Abstract: Serotonin (5HT) is one of the principal neuromodulators in the brain. Characteristics 

of 5HT release from central 5HTergic neurones have not been studied. We have developed novel 

lenti- and adenoviral vectors (AVV) which selectively express transgenes in raphe 5HT neurons. 

Organotypic slice cultures of the rat pons/midbrain containing the raphe nuclei were prepared 

and transduced with two AVV which incorporate 3.6 kb-long fragments of the tryptophan 

hydroxylase 2 (TPH2) promoter and together form a transcriptional amplification system to 

express EGFP. Protocols for slice culture preparation and viral transduction were published 

previously(1). Slices were used between 7 and 10 days in vitro when EGFP expression was at its 

maximum. Immunohistochemistry for TPH2 performed on fixed slice cultures one week posttransfection 

confirmed the specificity of expression at >97% (~700 cells). Vesicular release of 

5HT can be detected with micro-amperometry, which employs positively charged carbon fiber 

microelectrodes placed adjacent to the release site . Oxidation spikes consistent with exocytotic 

release of 5HT were detected at fluorescently labelled varicosities and cell bodies of midline 

raphe 5HT neurons. The frequency of events was low (average 0.01 Hz and 0.03 Hz for 

varicosities and somata, respectively). At varicosities, events had a median quantal size of ~18 

fC, with a median amplitude of ~5pA and duration at half-height of 3.1 msec (106 events from 5 

release sites). At somatic release sites, the majority of events (97%) had features comparable to 

those detected at varicosities with a similar height (~5 pA), but a slightly higher median quantal 

content (~22fC) and longer duration (3.5 msec; 114 events in 5 recordings). At somata, a few 

larger events were observed with a quantal content higher than 200fC (average ~500fC). Footlike 

events, the putative signs of „partial‟ fusion, were present in less than 3% of events. We 

conclude that 5HT is released predominantly in quanta of ~28000 molecules from varicosities 

and ~34000 molecules from cell bodies. In addition, in somata, the rare large release events 

discharge on average ~800000 molecules. These categories of release events are comparable to 

those previously recorded from central catecholaminergic neurons, as were their time courses . 

However, the quantal sizes of 5HT release events are smaller and their frequencies are markedly 

lower than in catecholaminergic neurons (2). Like norepinephrine, 5HT is released by exocytosis 

from neuronal cell bodies as well as axonal varicosities. 

Citations 

1. Chiti, Z. & Teschemacher, A. G. FASEBJ 21, 2540(2007). 

2. Teschemacher, A. G., et al. ADDR 57, 79(2005). 

Disclosures: K. Benzekhroufa, None; B. Liu, None; A.G. Teschemacher, None; S. Kasparov 

, None.

Poster 

237. Synaptic Integration II 



Topic: B.07.c. Synaptic integration 

Support: NIH MH71401 

Title: Synaptic activity during hippocampal gamma oscillations in vivo 

Authors: *B. ATALLAH, M. SCANZIANI; 

Biol., UCSD, La Jolla, CA 

Abstract: Gamma oscillations, the synchronous spiking of neurons at 30-80 Hz, occur in many 

cortical areas and are thought to play a role in sensory processing. Several studies suggest that 

both inhibitory and excitatory neurons in cortical networks participate in these rhythms. 

However, the pattern of synaptic activity underlying gamma oscillations in vivo remains poorly 

understood. Here we determine the properties of inhibitory and excitatory synaptic currents 

recorded in hippocampal neurons during gamma oscillation in vivo. 

In vivo recordings were performed from 5-7 week old rats anesthetized with urethane. Ketaminexylazine 

was supplemented as needed. Whole cell voltage clamp recordings were made from the 

pyramidal cell layer of area CA3 in the dorsal hippocampus using the "blind-patch" method and 

a cesium based intracellular solution. The recording location was verified with post-hoc 

histology. The local field potential (LFP) was simultaneously recorded with a tungsten electrode 

placed in the stratum radiatum at ~0.5 mm from whole cell recording site. Excitatory synaptic 

currents were recorded by holding the cells at the reversal potential for inhibition (~-90 mV), 

while inhibitory synaptic currents were recorded at the reversal potential for excitation (~20 

mV). 

Gamma oscillations were routinely recorded in the LFP of stratum radiatum of area CA3. The 

amplitude of these oscillations fluctuated from cycle to cycle (time scale ~ 30 ms) ranging from 

0.05 mV to 1 mV. 

Simultaneous whole-cell recordings revealed that excitatory synaptic currents (-90mV) were 

phase locked with each gamma oscillation cycle. The mean excitatory synaptic conductance 

during an oscillation cycle was 0.4 nS (n=5 cells). 

Like excitatory currents, inhibitory current (recorded at 20 mV) were time-locked to each gamma 

oscillations cycle. The mean inhibitory synaptic conductance was 5 times larger than that of the 

excitatory conductance (1.9 nS; n = 6). 

The magnitude of excitatory and inhibitory synaptic conductances fluctuated from cycle to cycle 

over an order of magnitude and correlated with the amplitude of the LFP.

We conclude that the both excitatory and inhibitory synaptic currents are phase locked to gamma 

oscillations and their amplitudes fluctuate rapidly on a cycle to cycle basis. 

Disclosures: B. Atallah , None; M. Scanziani, None. 

Poster 





Support: NIDCD 1F31DC009366 

Title: Odor-evoked excitatory and inhibitory synaptic currents are coherent at beta band 

frequency in rat piriform cortex 

Authors: *C. POO, J. S. ISAACSON; 

Neurosci Grad Program, UCSD, La Jolla, CA 

Abstract: Rhythmic oscillatory activity in the olfactory system is thought to play an important 

role in the encoding of odor identity (Laurent, 2002). Odor-evoked oscillations at beta band 

frequency (15-30Hz) have been reported in the local field potential (LFP) of the olfactory bulb 

(OB) and piriform cortex (PC) of rats in odor discrimination tasks (Martin et al., 2004, Ravel et 

al., 2003, Vanderwolf and Zibrowski, 2001). Prominent odor-induced beta oscillations have also 

been observed in both the OB and PC in urethane anesthetized rats (Neville and Haberly, 2003). 

Lesioning studies have suggested a role for long-range centrifugal fibers from the PC to the OB 

(Neville and Haberly, 2003), however, the cellular mechanism that underlie odor-evoked beta 

frequency oscillations remains unknown. Here we investigate how inhibitory and excitatory 

synaptic currents vary with odor-evoked beta oscillations in vivo. 

In vivo recordings were performed from freely breathing urethane-anesthetized rats (p16-21). 

Whole-cell patch clamp recordings were made from the pyramidal cell layer (L2/3) in the 

anterior PC. Action potentials were recorded in cell-attached mode and subsequent whole-cell 

recording (Cs+ gluconate-based internal containing 5 mM Cl-) was used to examine excitatory (- 

80 mV) and inhibitory (0 mV) synaptic responses in the same cells. A tungsten electrode was 

used to simultaneous monitor beta oscillations in the LFP in Layer 1a at a distance of less than 

1mm from the whole-cell recording site. Odors were delivered via a computer controlled flowdilution 

olfactometer (5% saturated vapor, 2s duration). 

Odor application evoked prominent beta frequency oscillations in the LFP. We find that in 

simultaneously recorded cells, odor-evoked spiking activity is phase-locked to the LFP. Odor-

evoked excitatory and inhibitory currents occurred rhythmically with a period of approximately 

70ms. During each oscillation cycle of the LFP, IPSCs occurred after EPSCs with an average 

latency of 10ms. This time window may account for the tightly phase-locked spiking activity of 

the cell. Furthermore, the amplitude of LFP beta oscillations was reduced greater than 80% in the 

presence of the GABABR agonist baclofen, which spares excitatory synaptic transmission of the 

lateral olfactory tract (LOT) while suppressing both associational and local inhibitory inputs 

(Franks and Isaacson, 2006). In summary, we find that both odor-evoked excitatory and 

inhibitory synaptic currents in vivo are coherent with LFP beta oscillations. Our results suggest 

the participation of local inhibitory interneurons as well associational fibers in the generation of 

these odor-evoked beta oscillations. 

Disclosures: C. Poo , None; J.S. Isaacson, None. 

Poster 





Support: Wellcome Trust Grant 

Title: Hippocalcin signaling in spines of hippocampal neurons 

Authors: V. CHERKAS 1 , A. DOVGAN 1 , D. FITZGERALD 2 , A. TEPIKIN 2 , R. D. 

BURGOYNE 2 , *P. V. BELAN 1 ; 

1 Dept Gen Physiol Nervous Syst, Bogomoletz Inst. Physiol, Kiev, Ukraine; 2 Physiol., Univ. of 

Liverpool, Liverpool, United Kingdom 

Abstract: Hippocalcin (HPCA) is a Ca 2+ -binding protein, and its Ca 2+ -dependent activation in 

hippocampal neurons is one of the necessary steps involved in many signal transduction 

mechanisms. We have earlier shown that HPCA can translocate to certain sites in a neuronal 

dendritic tree of transiently transfected hippocampal cultured neurons as a result of ionotropic 

glutamate receptor activation. In this work we have examined if synaptic glutamate receptor 

activation can result in HPCA signaling in spines of hippocampal neurons. 

Spontaneous and evoked synaptic activity induced excitatory postsynaptic potentials (EPSPs) in 

the hippocampal neurons leading to AP bursts and HPCA-YFP translocation to many sites in a 

neuronal dendritic tree including dendritic spines. At the same time miniature EPSPs and short 

episodes of presynaptic activity did not result in HPCA-YFP translocation. Back propagating 

APs induced by current stimulations increased [Ca 2+ ]i in spines but also did not result in

translocation. In neurons clamped at -40 mV, episodes of presynaptic activity resulted in EPSCs 

associated with HPCA translocation mainly to dendritic spines while no translocation was 

observed at -70 mV . At the same time HPCA-YFP translocation to spines at -70 mV was present 

in Mg 2+ -free extracellular solution. NMDA receptor blocker, APV, partially blocked EPSCs and 

completely blocked the translocation. Thus, Ca 2+ influx via synaptic NMDARs in which Mg 2+ 

block is relieved by APs is a main reason for HPCA-YFP translocation. Different types of 

voltage activated Ca 2+ channels also contributed to the observed translocation. FRET 

(fluorescence resonance energy transfer) between HPCA tagged by Yellow and Cyan 

Fluorescent Proteins was increased in spines where the translocation was observed. It indicates 

that the translocation was due to HPCA-FPs insertion in patches of spine membrane resulting in 

decrease of protein concentration in the cytosol of spines and diffusion of new HPCA-FP 

molecules from the dendritic trunk. As shown above this processes required association of 

presynaptic glutamate release and AP generation in the postsynaptic neuron. Based on 

estimations of HPCA-YFP concentration in the plasma membrane we have shown that 

membranous HPCA concentration in spines is increased several times during synaptic NMDARdependent 

translocation. Thus, we have shown that hippocalcin may decode [Ca 2+ ]i changes in 

dendrites of hippocampal neurons, induced by simultaneous pre- and postsynaptic activity, into 

its translocation to spines via diffusion from the neighboring parts of dendritic tree and robust 

insertion in spine membranes. 

Disclosures: V. Cherkas, None; P.V. Belan , None; A. Dovgan, None; D. Fitzgerald, None; A. 

Tepikin, None; R.D. Burgoyne, None. 

Poster 





Support: MH71401 

MH7005 

Title: Excitation necessary to reach threshold varies with input strength through a mechanism 

involving feed forward inhibition 

Authors: *A. MARIN-BURGIN 1 , F. R. POUILLE 2 , M. SCANZIANI 2 ; 

1 Biol. -0634, UCSD, La Jolla, CA; 2 Biol., Univ. of California San Diego, La Jolla, CA

Abstract: We determined the peak excitatory conductance necessary to reach threshold for 

action potential (AP) generation in CA1 pyramidal cells (PCs) recorded in acute hippocampal 

slices obtained from 3-4 weeks old rats. PCs were initially recorded in the loose patch 

configuration and Schaffer collaterals stimulated at an intensity just sufficient to reach threshold 

for AP generation. We subsequently gained whole cell access to the neurons and determined the 

peak conductance of the underlying "threshold" excitatory postsynaptic current (EPSG) by 

holding the PC at the reversal potential for inhibition. We found that the threshold EPSG varied 

over a wide range, between 3 and 30 nS. The threshold EPSG correlated with the number of 

stimulated Schaffer collaterals. Both the variability in the threshold EPSG and the correlation 

with the number of activated Schaffer collaterals were abolished in the presence of the GABA A 

receptor antagonists gabazine. We determined the peak conductance of feed forward inhibitory 

postsynaptic currents (IPSGs) elicited in response to stimulation of Schaffer collaterals at 

threshold for AP generation and found that it strongly correlated with the evoked threshold 

EPSG. 

These data suggest that the threshold excitation in PCs is not fixed but varies as a function of 

input strength. This variability depends on the presence of feed-forward inhibition. By 

instantaneously adjusting pyramidal cell excitability to the strength of incoming activity, feedforward 

inhibitory circuits allow the pyramidal cell population to be sensitive to weak stimuli yet 

not to saturate with stronger ones. This results in an expansion of the dynamic range of the CA1 

region to incoming activity. 

Disclosures: A. Marin-Burgin , None; F.R. Pouille, None; M. Scanziani, None. 

Poster 





Support: Ribble Undergraduate Scholarship, University of Kentucky, Department of Biology 

Title: Comparison of the autonomic response of multiple sensory modalities in crayfish 

Authors: *M. M. ROBINSON, T. SPENCE, T. MCLAURINE, S. M. BIERBOWER, R. L. 

COOPER; 

Univ. Kentucky, Lexington, KY 

Abstract: Most organisms show diversity in the type and amount of peripheral sensors which 

allow for detection of different sensory stimuli within and across multiple sensory modalities.

Variation in sensory pathways allows organisms to monitor their environment, integrate sensory 

information from multiple sensory sources, and in turn, respond accordingly due to refined 

integration of information. Most invertebrates possess chemosensory neurons which allow for 

identification of the many chemicals in the environment and thus are able to behave differentially 

between chemical compounds based upon the sensory pathway stimulated (i.e., attractive and/or 

repellant). Current literature shows this is particularly true for decapod crustaceans in detecting 

chemical signals, especially in the cephalic and thoracic appendages. Crayfish are decapod 

crustaceans that rely on visual, as well as chemical cues in the environment. While behavior is 

often used as a bio-index for awareness to a changing environment, previous studies have shown 

that many organisms may show no outward behavioral change while internally readying (i.e., 

autonomic response) themselves for an impending predator attack. Thus, behavior alone often 

excludes “flight or flight” internal readiness changes and may conclude a lack of environmental 

awareness. To understand this sympathetic-like response, we examine the autonomic response 

(HR, heart rate and VR, ventilation rate) in crayfish, Procambarus clarkii, during chemical 

introduction, as well as establish chemical and/or modality sensitivities by targeting multiple 

sensory modalities. Preliminary findings suggest crayfish which show no behavioral response do 

in fact show an internal response through changes in HR and VR. In addition, crayfish show an 

increase in HR with attractant chemical introduction (i.e., cysteine, blood water) suggesting a 

natural response to a possible food source, while showing a more pronounced response to 

possible repellent compounds. Future research will include the examination antennule flicking as 

a possible quantitative measure of chemosensory reception for the multiple sensory modalities. 

Disclosures: M.M. Robinson , None; S.M. Bierbower, None; R.L. Cooper, None; T. Spence, 

None; T. McLaurine, None. 

Poster 





Support: KAKENHI(15082207) 

Title: Intracellular Ca 2+ elevation accompanying NMDA receptor-mediated plateau potential in 

hippocampal CA1 pyramidal neurons 

Authors: S. KODAMA 1 , S. WATANABE 1 , T. SUZUKI 2 , T. IZUMI 1 , *H. BABA 3 , M. 

INOUE 1 , H. MIYAKAWA 1 ; 

1 Cell. Neurophysiol., Tokyo Univ. of Pharm. and Life Sci., Hachiouji, Tokyo, Japan; 2 Dept. of

Neurosurg., Univ. of New Mexico Sch. of Med., Albuquerque, NM; 3 Dept Mol Neurobio, Tokyo 

Univ. Pharm., Hachioji, Tokyo, Japan 

Abstract: Extra-synaptic NMDA receptor (NMDAR) -mediated plateau potential can be induced 

either by synaptic stimulation in the presence of glutamate transporter antagonist TBOA or by 

iontophoresis of glutamate in hippocampal CA1 pyramidal neurons. To investigate if the 

NMDAR-mediated plateau potentials is accompanied by a rise in [Ca 2+ ]i, we performed wholecell 

recordings and Ca 2+ imagings simultaneously from single CA1 pyramidal neurons in 

hippocampal slices. Neurons were loaded with Ca 2+ indicator fluo-4 (100κM). NMDARmediated 

plateau potentials were induced by repetitively stimulating Schaffer collaterals or by 

applying glutamate iontophoretically in the presence of 10κM CNQX and 50κM DL-APV. 

Substantial increase in [Ca 2+ ]i accompanying the plateau potential was detected both at the 

dendrite and the soma. When TBOA was present, a slow rise in [Ca 2+ ]i was detected near the 

soma after the end of the plateau potentials. After adding Cd 2+ and antagonists for metabotropic 

glutamate receptors, most of the Ca 2+ rise was suppressed, leaving small but substantial Ca 2+ rise 

in the dendrites. This Ca 2+ elevation was abolished by applying 30κM 5,7-dichlorokynurenic 

acid, an antagonist for the glycine binding site of the NMDAR. Our results show that the 

NMDAR-mediated plateau potential is accompanied by substantial Ca 2+ elevation mainly due to 

Ca 2+ entry from voltage-gated Ca 2+ channels and partly due to Ca 2+ entry from NMDAR 

channels. 

Fig. Left, Fluorescence image of neuron filled with a Ca 2+ indicator fluo-4 (Scale bar, 10κm). 

Right, Spatial distribution of iontophoretically-induced Ca 2+ elevation in the presence of 10κM 

CNQX, 50κM DL-APV, 100κM Picrotoxin, 10κM MPEP, 50κM LY367385 and 1κM 

Tetrodotoxin. Ca 2+ signals was recorded from areas of the square written on Left image, (1-7), 

and whole-cell membrane potential was recorded from soma, (bottom).

Disclosures: S. Kodama, The Ministry of Education, Culture, Sports, Science and Technology 

of Japan, B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received); S. Watanabe, The Ministry of Education, Culture, Sports, 

Science and Technology of Japan, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); T. Suzuki, The Ministry of 

Education, Culture, Sports, Science and Technology of Japan, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received); T. 

Izumi, The Ministry of Education, Culture, Sports, Science and Technology of Japan, B. 

Research Grant (principal investigator, collaborator or consultant and pending grants as well as 

grants already received); H. Baba , The Ministry of Education, Culture, Sports, Science and 

Technology of Japan, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); M. Inoue, The Ministry of Education, 

Culture, Sports, Science and Technology of Japan, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); H. Miyakawa, 

The Ministry of Education, Culture, Sports, Science and Technology of Japan, B. Research Grant 


received). 

Poster 





Support: Dept of Biology, Univ of KY, G. Ribble fellowship to MD 

Title: Roles of the sodium calcium exchanger (NCX), the plasma membrane Ca2+-ATPase 

(PMCA) and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) in synaptic 

transmission at the crayfish and Drosophila neuromuscular junctions 

Authors: *M. S. DESAI, R. L. COOPER; 

Dept Biol, Univ. Kentucky, Lexington, KY 

Abstract: It has been previously shown that in crayfish as well as in Drosophila, short-term 

facilitation (STF) can be induced by repetitively stimulating the neuromuscular junction (NMJ). 

The basis of this STF has been shown to be the buildup of residual Ca2+ in the nerve terminal. 

This build up can be modified by modulating the activity of the different Ca2+ -ion channels like 

the NCX, the PMCA and the SERCA and thereby the amount of STF can also be changed. Here 

we have made an attempt to study the roles of these three ion channels in the regulation of

[Ca2+]i which impacts STF at the NMJ in both these model synaptic preparations. At the 

crayfish NMJ, STF is induced by a train of 30 pulses at 40 Hz and EPSPs measured on the 

opener muscle. At the Drosophila NMJ, STF is induced by applying a train of 10 pulses at 20 Hz 

and measuring EPSPs on muscle 12 or 13. The activity of the NCX is compromised by reducing 

the external Na+ in the saline by 2/3rd. The PMCA has been shown to be inactive at pH 8.8 so in 

order to block it, the pH of the external saline is increased to 8.8. For studying the role of 

SERCA in Drosophila, a temperature sensitive SERCA mutant (Kum170) is used, which renders 

the animals inactive when they are heat-shocked. For the crayfish the drug Thapsigargin (Tg) 

will be used to block the SERCA. A comparison of the roles of these Ca-channels in synaptic 

transmission will be made between the two model animals. 

Disclosures: M.S. Desai , None; R.L. Cooper, None. 

Poster 





Support: G. Ribble Dept of Biology, Univ of KY 

Title: The effect of CO2 on the neural circuitry of an identified behavior 

Authors: *S. M. BIERBOWER, R. L. COOPER; 

Dept Biol, Univ. Kentucky, Lexington, KY 

Abstract: Carbon dioxide (CO2) is universally found and impacts all organisms throughout their 

lifetime. Thus, most organisms constantly monitor their environment and alter behavior based 

upon sensory information according to various stimuli. Current literature, primarily in insects, 

shows that low levels of CO2 acts as an attractant, while higher concentrations induce paralytic 

effects in vertebrates and invertebrates alike. Interestingly, invertebrates and vertebrates are very 

different systemically, yet the effect of CO2 is not. Crayfish serve as an excellent model since 

these animals possess the complex ability to integrate sensory information, relay the information 

into motor output to target tissues and allow the „sympathetic-like‟ autonomic response to be 

easily studied. It is through this motor output (i.e., locomotor activity, heart and ventilatory 

measures) that we can assess the internal state of the organism. The effects previously identified 

in Drosophila melanogaster larvae were also shown in the crayfish with acute CO2 exposure. 

While attraction to low levels of CO2 has been documented in insects, this is a foundation study 

showing attraction/repellent behavioral responses in crustaceans. The identified effect (i.e.,

immobilization, cardiac arrest) is characterized by a cessation heart (HR) and ventilatory (VR) 

rates after approximately 10 minutes, a steady decrease in locomotor activity, as well as 

unresponsiveness to stimuli prior to HR and VR cessation. To identify the effect of CO2 on 

chemical synapses within a complete circuit, the „sensory-ganglia-motor nerve-muscle‟ of the 

abdominal superficial flexor muscle was used. Furthermore, to identify mechanisms of action, 

we examined synaptic transmission (EPSPs) at the skeletal NMJ by introducing an excitatory 

neurotransmitter (glutamate). Results indicate that the anesthetic effect is characterized by a 

decrease in synaptic transmission resulting from CO2 induced glutamate receptor block; thus this 

should be considered a paralytic effect. Due to glutamatergic insensitivity, the site of action was 

identified as post-synaptic at NMJs. 

Disclosures: S.M. Bierbower , None; R.L. Cooper, None. 

Poster 





Support: NSF-IBN-0131459 (RLC), Univ. KY (JYL), Korean Army & Korea Military 

Academy (JYL, WYC & NTL). 

Title: Pre and postsynaptic actions of kainate- Negative feedback at glutamate-ergic nerve 

terminals 

Authors: *J. LEE 1 , D. M. BHATT 3 , D. M. BHATT 4 , W.-Y. CHUNG 5 , N.-T. LEE 5 , R. L. 

COOPER 2 ; 

2 Biol., 1 Univ. of Ky, Lexington, KY; 3 Col. of Med., 4 Col. of Pharm., Univ. of KY, Lexington, 

KY; 5 Chem., Korea Military Acad., Seoul, Republic of Korea 

Abstract: Drosophila larval neuromuscular junctions (NMJs) serve as a model for various gene 

mutations of synaptically relevant molecules to characterize functional consequences in synaptic 

physiology. In addition, the molecular sequence of the glutamate receptors on the muscle fibers 

have been described; however, the pharmacological profile of glutamate receptor subtypes at the 

NMJ have not been fully elucidated. In this study, we present evidence for self regulation (i.e. 

negative auto-feedback) at the NMJ in Drosophila. Based on the pharmacological profile these 

presynaptic autoreceptors are likely a kainate subtype of glutamate receptors. Kainate does not 

depolarize the muscle, but evoked EPSP dampen when kainate is present and mini frequency is 

reduced. Quantal responses show a decreased amplitude and area under the voltage curve

indicative of reduced postsynaptic receptor sensitivity to glutamate transmission. ATPA, a 

kainate receptor agonist, mimics kainate action both pre- and post-synaptically. Domoic acid, a 

quisqualate receptor antagonist, blocks the postsynaptic receptors without depolarizing the 

muscle, which supports earlier findings of the muscle containing quisqualate subtype receptors. 

No effects are seen on the frequency of spontaneous events in low concentration of domoic acid 

which causes a 50% reduction in quantal amplitude. The results suggest a kainate pre-synaptic 

auto-inhibitory feedback on evoked release. The feedback could be partly responsible for the 

rapid depression with evoked high quantal release and facilitation with evoked low quantal 

release during repetitive stimulation. The mechanism of action within the presynaptic terminal 

remains to be elucidated. The mild direct postsynaptic action of kainate is likely due to partial 

antagonist action on the quisqualate receptors. 

Disclosures: J. Lee , None; D.M. Bhatt, None; D.M. Bhatt, None; W. Chung, None; N. Lee, 

None; R.L. Cooper, None. 

Poster 





Support: 1R21 NS056124-01A1 

RO1 MH065924 

RO1 AA010983 

Title: Concentration-dependent dopamine modulation of recurrent synaptic activity and 

enhancement of signal-to-noise ratio in the prefrontal cortex 

Authors: *S. KROENER 1 , P. E. M. PHILLIPS 2 , L. J. CHANDLER 1 , J. K. SEAMANS 3 ; 

1 Neurosciences, Med. Univ. South Carolina, Charleston, SC; 2 Dept. of Psychiatry & Behavioral 

Sci., Univ. of Washington, Seattle, WA; 3 Dept of Psychiatry and Brain Res. Ctr., Univ. of British 

Columbia, Vancouver, BC, Canada 

Abstract: The membrane potential of cortical neurons periodically shifts from a hyperpolarized 

down-state into a depolarized up-state which shapes synaptic integration and action potential 

generation. In the prefrontal cortex (PFC) the recurrent network activity that sustains the up-state 

may be modulated by dopamine (DA). Here we used organotypic co-cultures consisting of slices

of the PFC, hippocampus and VTA to study DA‟s effects on intrinsic excitability and synaptic 

activity in an active network. Patch-clamp recordings were obtained from deep layer PFC 

neurons and up-states were evoked by stimulation of the VTA or hippocampus. The VTA 

provided a tonic level of DA to the culture and we used fast-scan cyclic voltammetry at carbonfiber 

microelectrodes to measure extracellular DA concentrations in the PFC that resulted from 

phasic VTA stimulation used to evoke up-states. 

Acute blockade of DA receptors by receptor specific antagonists did not affect induction of 

VTA-evoked up-states, indicating that DA is not required for this type of network activity in the 

PFC. However, when DA concentrations were further increased by the addition of exogenous 

DA (>1uM), the duration of up-states was greatly reduced, an effect that was prevented by preapplication 

of DA receptor antagonists. Similarly, increasing endogenous DA via bath 

application of cocaine (1 or 10 uM) also significantly shortened VTA-evoked up-states. In 

contrast, in co-cultures from which the VTA was omitted thereby removing any basal DA tone, 

the addition of 10nM - 1uM of exogenous DA increased up-state duration and spike frequency. 

Surprisingly, regardless of its effect on network activity in all preparations bath application of 

DA consistently increased the number of spikes evoked by somatic current injection from the 

down-state, consistent with findings in acute slices. This suggests that the effects on up-states 

were independent of, or even opposite to DA-mediated changes in intrinsic membrane 

excitability. In a second set of experiments we investigated the modulation of local synaptic 

inputs at a concentration of DA (100 nM) that had little effect on the properties of the up-state 

itself. In the down-state, DA increased the summation of EPSPs during trains of 20 Hz synaptic 

stimulation, leading to enhanced depolarization by later EPSPs. During the up-state, DA 

increased the probability that synaptic stimulation would evoke spikes. Taken together these 

results provide support for an inverted u-curve of DA‟s effects on network activity in the PFC. In 

addition, DA can alter signal integration to enhance the effectiveness of a signal over the 

ongoing background network activity. 

Disclosures: S. Kroener, None; P.E.M. Phillips, None; L.J. Chandler, None; J.K. Seamans, 

None. 

Poster 





Title: The first central synapse: a key for reliable transmission of sensory information

Authors: N. ROTEM 1 , E. SESTIERI 3 , Y. YAROM 2 , *J. HOUNSGAARD 4 ; 

1 Dept. of Neurobiology/ The Otto Loewi Ctr., 2 Neurobio., Hebrew Univ., Jerusalem & Eilat, 

Israel; 3 Otto Loewi Minerva Ctr., Inter-University Inst., Eilat, Israel; 4 MFI, Panum Inst., 2200N 

Copenhagen, Denmark 

Abstract: Hair cell driven sensory systems track rapid changes in the environment. However, 

early processing of vestibular and auditory information is complicated by a diversity of CNS 

projections with specialized primary afferent terminals and target neurons. In elasmobranches the 

near-body electric field is monitored by hair cells in Ampullae Lorenzini. This information is 

transmitted exclusively to ascending efferent neurons (AENs) in the ipsilateral dorsal 

octavolateral brain stem nucleus. Thus, the precise goal-directed behaviors elicited by particular 

changes in the near-body electric field crucially depend on the fidelity of mapping primary 

afferent activity onto the AENs. Here we present the first study of transmission at this first 

synapse in early processing of electrosensory information in the CNS. 

Intracellular recordings were obtained from AENs in the isolated brainstem from Iago omanensis 

sharks. Stimulating the anterior lateral line nerve selectively activated primary afferents (AFFs). 

Only monosynaptic excitatory responses were evoked by the stimulus. The range in stimulus 

intensity from just threshold to supramaximal intensity was narrow. In this range the synaptic 

response typically increased in amplitude from 0.5 to 4.5 mV. We noted that this increase was 

non-continuous. Indeed, amplitude histogram revealed clustering around 2-5 amplitude means. 

In most AENs the synaptic response evoked by an AFF stimulus readily elicited an action 

potential. Furthermore, the voltage threshold for action potentials was distinctly lower for AFF 

evoked response than for depolarization by intracellularly injected current and the synaptic 

response evoked by a parallel fiber stimulus. This suggests that each AEN is innervated by only 

few afferents that form a highly efficient synaptic transmission. 

The response to AFF stimulation was depressed by paired stimulation. Paired-pulse depression 

reached a maximum with a delay of 30 ms and was observed at all stimulus intensities. The 

depression was not associated with a detectable postsynaptic conductance increase and inhibitory 

synaptic potential could not be discerned. Paired-pulse depression was blocked by bicculline and 

therefore most likely mediated by GABAA receptors. These observations suggest that pairedpulse 

depression is caused by presynaptic inhibition. 

In conclusion, electrosensory information in the shark is mapped from primary afferents to AENs 

with minimal convergence. Each AEN is dominated by 2-5 afferents that form robustly reliable 

excitatory synaptic connections. Temporal integration of electrosensory information is regulated 

by feed-forward presynaptic inhibition. 

Disclosures: N. Rotem, None; E. Sestieri, None; Y. yarom, None; J. Hounsgaard , None. 

Poster 





Title: Effects of acute PKC activation during behavioral training on dendritic spines and 

presynaptic axonal boutons in the CA1 hippocampus of aged rats 

Authors: *J. HONGPAISAN 1,2 , D. L. ALKON 1,3 ; 

1 BRNI, Morgantown, WV; 2 Lab. of Neurobiology, NINDS, NIH, Bethesda, MD; 3 Dept. of 

Neurol., West Virginia Univ. Med. Sch., Morgantown, WV 

Abstract: Activation of endogenous PKC occurs with a variety of associative learning and 

memory paradigms. We have recently shown (PNAS, 2007, 104:19571-19576) that bryostatin, a 

PKC-activating drug, enhances long-term memory in young rats by enhancing protein synthesis, 

the memory-dependent generation of mushroom spines, and a memory-dependent increase in 

presynaptic vesicles. Here, we further investigated the effects of acute PKC application during 

water maze training in aged (>24 months), brown Norway rats. 

Aged (vs. 4-5 months old) rats were behaviorally trained with spatial maze task for 8 days, 

followed by the probe test on day 9 to assess long-term memory retention. In these aged rats, I.V. 

bryostatin (2.5κg/kg, on day 1, 3, 5, and 7) enhanced short-term but not long-term memory. At 

24h after the probe test (i.e. 48h after training), morphological studies of dendritic spines were 

performed as quantified by double blind protocols. DiI staining visualized by confocal 

microscopy showed that bryostatin application during water maze training increased (p





Support: NIH 

Title: Mapping the functional connectivity between cerebellar granule cells and Purkinje cells 

Authors: *M. J. DIZON, K. KHODAKHAH; 

Neurosci., Albert Einstein Coll Med., Bronx, NY 

Abstract: One of the main functions of the cerebellum is to integrate sensory and cortical 

information to coordinate voluntary movements. The main site of this integration is the 

cerebellar cortex, with excitatory granule cells composing the input layer, and inhibitory Purkinje 

cells processing over 150,000 synaptic inputs to formulate the output. Classic in vivo 

experiments established that increases and decreases from the high-frequency spontaneous firing 

rate of these cells correlate with skeletal muscle contraction and extension, respectively. The 

highly stereotypical, repeating cytoarchitecture of the cerebellar cortex implies possible 

anatomical correlates to this phenomenon. Granule cells can either directly excite Purkinje cells, 

or indirectly inhibit them via a disynaptic pathway through GABAergic basket- and stellate-type 

interneurons. Such dichotomous signaling pathways likely underlie the bidirectional coding 

range of Purkinje cells. This study asks whether a single patch of granule cells is capable of both 

exciting and inhibiting different subsets of Purkinje cells. As a first pass, we assessed the 

efficacy with which different granule cell patches drive changes in Purkinje cell firing rates by 

employing physiological and noninvasive approaches, namely single-unit extracellular recording 

and serial glutamate uncaging using laser-scanning photostimulation. We found that in both 

sagittal and coronal rat cerebellar slices, pharmacologically blocking feed-forward inhibition 

consistently strengthens Purkinje cell receptive fields, and eliminates laterally elicited pauses in 

Purkinje cell firing, thereby also widening the region of granule cells that net excites a given 

Purkinje cell. In addition, the data suggest functional distinctions between basket and stellate 

cells. Taken together, the results point to a central role of these interneurons in shaping the 

spatial patterns of activation of Purkinje cells. These investigations are a crucial step toward 

understanding the functional neuronal organization of the cerebellar cortex and how it subserves 

fine tuning of movement. 

Disclosures: M.J. Dizon, None; K. Khodakhah, None. 

Poster





Support: CIHR 

Heart and Stroke Foundation of British Colubia and the Yukon 

Canadian Stroke Network 

Title: Pannexin-1 hemichannels open during low Mg2+-induced epileptiform bursting in the 

hippocampus 

Authors: *R. L. RUNGTA 1 , R. J. THOMPSON 1,2 , B. A. MACVICAR 1 ; 

1 the Brain Res. Ctr., Univ. British Columbia, Vancouver, BC, Canada; 2 Dept. of Cell Biol. & 

Anat., Univ. of Calgary, Calgary, AB, Canada 

Abstract: Enhancing NMDA receptor activation by removing external Mg 2+ triggers 

epileptiform Interictal spiking in hippocampal brain slices. In recent experiments we have found 

that NMDA receptor activation can open pannexin-1 (Px1) hemichannels in hippocampal 

neurons either acutely isolated or in brain slices. We hypothesized that by potentiating NMDAR 

activity in hippocampal slices by perfusion of low Mg 2+ /5 mM K + bathing solution, Px1 

hemichannels could open and contribute to the properties of interictal spiking. To show that Px1 

channels open in low Mg 2+ /5 mM K + , we measured neuronal uptake of sulforhodamine 101 

(SR101), which is normally excluded from neurons but can load when Px1 channels are opened. 

We observed neuronal loading of SR101 in the CA1 region under low Mg 2+ /5 mM K + 

conditions. Neuronal dye loading was blocked by pre-treatment with 50 κM APV (a NMDAR 

antagonist) or by a short peptide sequence targeted against an extracellular domain of Px1 

( 10 panx; 100 µM) which blocks Px1 hemichannels. To determine whether Pannexin-1 opening 

could then affect seizure like activity, we monitored extracellular field potentials in both the CA1 

and CA3 region. We confirmed that the initiation of low Mg 2+ -induced bursting was NMDARdependant 

by blocking the induction of spontaneous bursting with 50 κM APV. In slices 

displaying persistent CA1 bursting, perfusion of 10 panx reduced the interburst frequency by 28 ± 

3 %, with recovery to 108 ± 4% (n=4 slices) of control upon washout of the peptide. Mean 

amplitude of individual spikes was also reduced by 26 ± 10 %, with recovery to 87 ± 5 % of 

control. This effect of 10 panx was not due to inhibition of the fast component of synaptic 

currents, as 10 panx did not alter evoked field potentials in the presence of 2mM Mg 2+ . These data 

support the hypothesis that pannexin-1 hemichannels can be opened by synaptic activation of 

NMDARs, and may represent a novel target for intervention in epilepsy. 

This work was supported by grants to BAM from the Canadian Institutes of Health Research and 

the Heart and Stroke Foundation of British Columbia.

Disclosures: R.L. Rungta, None; R.J. Thompson, None; B.A. MacVicar, None. 

Poster 





Support: NIH NIDA DA017884 

Stanford Anesthesia 

Title: Multiple actions produced by propofol on subthalamic neurons recorded from humans 

Authors: M. B. MACIVER 1 , J. G. BROCK-UTNE 2 , *R. A. JAFFE 2 ; 

1 Dept Anesthesia, 2 Dept Anesthesia S-260, Stanford Univ. Med. Ctr., Stanford, CA 

Abstract: Subthalamic nucleus (STN) neuron discharge activity is correlated with motor 

abnormalities in Parkinson‟s disease (PD). In rat brain slices, STN neurons spontaneously 

discharge in the absence of synaptic input. In studies of human PD patients receiving STN 

electrode implants we showed that propofol sedation had little effect on overall discharge 

activity, however, both high frequency and low frequency tonic firing of STN neurons was 

slowed. To further characterize the target sites of propofol, we studied it's action on spontaneous 

spike firing in rat STN brain slices. Following IRB approval, 400 micron thick brain slices were 

prepared from male Sprague-Dawley rats (P 20 to 25). Slices were maintained in submerged 

chambers at 22 °C and continuously perfused with artificial cerebrospinal fluid (ACSF). Action 

potential activity was monitored with extracellular glass microelectrodes filled with ASCF or 

using whole cell patch clamp recordings with standard procedures. Sedative concentrations of 

propofol depressed spontaneous discharge of STN neurons by 20 to 30 %. Co-application of 

bicuculline (20 µM) or picrotoxin (100 µM) did not change the degree of depression produced 

by propofol, consistent with a direct propofol action on STN neurons not involving GABAergic 

inhibition. Whole cell recordings of STN neurons showed that propofol prolonged GABAmediated 

synaptic currents, similar to effects seen in other types of neurons. Neither of the 

inward rectifier K channel blockers Ba2+ (100 µM) nor Cs+ (1 mM) had any actions on 

discharge or on propofol‟s effect. The Ca channel antagonist nifedipine (10-100 µM) was also 

without effect, but when external Ca2+ was reduced from 2 mM to 1 mM spike rates increased 

~50%, although the percent depression produced by propofol remained unchanged. The 

persistent Na current (INaP) antagonist riluzole (10 µM) depressed STN neuron spike frequency 

by ~40% without affecting the spike amplitude, rise-time or repolarization. The SKCa antagonist

apamin (20 nM) increased spike rate variance but did not appreciably change mean spike rate in 

control conditions; however, apamin reversed the spike rate depression produced by propofol. 

Thus, propofol activation of SKCa channels appeared to contribute to the increased spike rate 

variance and depression of low frequency activity that we observed in human PD patients and in 

rat brain slice recordings. The spike rate depression produced by riluzole and lack of effect of 

varying calcium concentrations suggests that propofol may also target INaP, but not Ca channels, 

to depress excitability. These effects would add to the enhanced GABA-mediated inhibition that 

has been previously described. 

Disclosures: M.B. MacIver, None; J.G. Brock-Utne, None; R.A. Jaffe, None. 

Poster 





Support: NIH R01 MH065634 

Title: Probing the synaptic response function of deep cerebellar nuclei neurons with artificial 

small conductance Ca2+-mediated potassium (SK) conductance applied via dynamic clamping 

Authors: *S. S. FENG 1,2 , T. SANGREY 2 , D. JAEGER 2 ; 

1 Biomed. Engin., Georgia Inst. Tech., Atlanta, GA; 2 Biol., Emory Univ., Atlanta, GA 

Abstract: Small conductance Ca 2+ -mediated potassium currents (SK) play an important role in 

shaping the slow spike-afterhyperpolarization (sAHP) in multiple types of neurons, which 

furthermore can affect intrinsic firing properties such as pacemaking and mode-switching, 

synaptic plasticity, and synaptic integration through regulation of local dendritic Ca 2+ transients 

(Bond, Maylie, et al., 2005, Curr. Opin. Biol. 15: 305-311). Neurons in the deep cerebellar nuclei 

(DCN) show a pronounced SK dependent sAHP, but the significance of this current in 

processing synaptic input from Purkinje cells and mossy fibers has not been established. We 

used whole-cell patch clamp recordings from DCN neurons in acute rat brain slices to 

characterize the influence of SK current on synaptic processing. In this study dynamic clamping 

was used to apply simulated synaptic conductance patterns as previously described (Gauck and 

Jaeger, 2000, J. Neurosci., 20: 3006-3016). New custom-made dynamic clamp software written 

in RT-Labview (National Instruments, Inc) also allowed us to add or subtract simulated SK 

conductance to recorded neurons. Since SK conductance is gated by intracellular Ca 2+ , we also 

implemented the simulation of an intracellular Ca 2+ pool with inflow through a depolarization

gated HVA calcium current in our dynamic clamp circuit. As previously established, we found 

that in the presence of the SK channel blocker apamin (100nM), DCN neurons exhibited high 

frequency bursting behavior brought on by elimination of the sAHP. When simulated synaptic 

conductance patterns were applied before and after the addition of apamin, we found that 

synaptic responses remained precisely locked to specific input events in both conditions. 

However, responses to small depolarizing synaptic events were reduced after apamin application, 

whereas responses to larger depolarizing events were increased. In addition, the responses to 

larger depolarizing event consisted of short high frequency bursts rather than a single spike. The 

application of artificial SK conductance could restore the regular spontaneous firing pattern as 

well as a synaptic response function closely resembling that seen before apamin application. The 

short bursts to large synaptic events seen during apamin application were also restored to single 

spike responses. These data suggest that the SK currents play a role in defining the sensitivity 

and gain function of synaptic responses in DCN neurons. Modulation of the SK channels or the 

calcium dynamics triggering SK activation could thus allow for tuning of DCN responsiveness to 

synaptic input. 

Disclosures: S.S. Feng , None; T. Sangrey, None; D. Jaeger, None. 

Poster 






FRSQ 

Title: Impact of altered chloride extrusion capacity on cell excitability 

Authors: N. DOYON 1 , H. KROGER 2 , S. A. PRESCOTT 3 , *Y. DE KONINCK 4 ; 

1 Cell. Neurobio., 2 Laval Univ., Quebec, QC, Canada; 3 Neurobio., Univ. of Pittsburgh, 

Pittsburgh, PA; 4 Cell. Neurobiol, Laval Univ. / CRULRG, Quebec, Canada 

Abstract: Hyperpolarizing inhibition mediated by GABAA receptors is highly dependent on Cl 

homeostasis since its efficacy depends on the neuron‟s ability to maintain a strong 

transmembrane Cl gradient. A decrease in Cl extrusion capacity (e.g., resulting from diminished 

KCC2 activity or expression) can cause a change in the Cl gradient with consequences on the 

excitability of the cell (Prescott et al., Mol. Pain, 2007). In addition, a collapse in Cl gradient can

also dynamically arise from Cl flux through the GABAA channel (e.g., during high frequency 

bursts of GABAA receptor-mediated synaptic activity). Finally, shunting by GABAA receptor 

activation remains unaffected by changes in Cl gradients. These three phenomena interact nonlinearly 

making it difficult to predict their net impact on the input-output properties of the cell. 

While the impact of changes in Cl gradients and/or shunting inhibition can be relatively easily 

studied using conventional compartmental models (e.g., the NEURON environment), taking into 

account the dynamics of ion gradients is more complex. To address this, we developed an 

electro-diffusion multicompartment model in which Cl concentration and membrane potential 

are updated in each spatial elements of a finite element model of cellular compartments at each 

time step. We first built a subcellular model of a dendrite section with 200nm spatial resolution 

to identify relevant parameters and study the relationship between GABAA currents and Cl 

dynamics. We found that when the functional level of KCC2 is partly depleted, increasing 

intraburst frequency at synapses and/or prolonging the kinetics of GABAA synaptic events could 

easily cause a dynamic collapse of GABAA currents. The rate and extent of collapse depended 

heavily on the diameter of the dendritic compartments. Activity at a given synapse could affect 

EGABA of synapses located as far as 60um apart. These results confirmed the need for a whole 

cell electro-diffusion model to study the impact of changes in Cl extrusion capacity on the inputoutput 

properties of neurons. Using such model, we studied the impact of both random synaptic 

activity and bursting activity. In both scenarios, depletion of functional KCC2 led to an increase 

in excitability of the cells. This increase was however significantly more dramatic when both the 

inhibitory and excitatory input frequency was high or when synaptic activity occurred in bursts. 

In some conditions of low extrusion capacity and high synaptic activity, increasing the frequency 

of GABAA events failed to restore inhibition. Cl dynamics is thus an important factor to take into 

account to determine the impact of altered KCC2 expression or activity. 

Disclosures: N. Doyon, None; H. Kroger, None; S.A. Prescott, None; Y. De Koninck , None. 

Poster 





Support: The Grass Foundation 

The Neuroscience Institute of the MBL 

Title: Neurophysiological mechanisms underlying thermal acclimation in a neuron of the 

vertebrate central nervous system

Authors: *T. M. SZABO 1 , T. BROOKINGS 1 , D. S. FABER 2 , T. PREUSS 2 ; 

1 Biol Dept, Brandeis Univ., Waltham, MA; 2 Domick P Purpura Dept of Neurosci., Albert 

Einstein Col. of Med., New York, NY 

Abstract: An animal's ability to adapt and behave normally in the natural environment is crucial 

to its survival, and one important factor it must adjust to is temperature. To study the effect of 

temperature acclimation on intrinsic membrane properties and the integrated functioning of a 

neuron in the vertebrate central nervous system we used the Mauthner (M-) cell as a model. The 

M-cell can be studied both physiologically in the intact brain as well as in a behaving animal, 

since it triggers a startle response when it fires. Animals acclimated for at least one month to 5°, 

15° or 25°C had escape kinetics consistent with the general effects of temperature: slower in 

cold, faster in warm. Probability of escape increased and directionality decreased at 25°C, results 

that were also seen when acclimated animals were tested at 15°C. To examine the 

neurophysiology underlying these behavioral changes, we recorded from the M-cell 

intracellularly in animals acclimated under the same conditions. Some cellular properties were 

directly impacted by temperature including input resistance and action potential kinetics, 

although not to the extent seen in acute temperature exposure (Preuss and Faber, 2003). No 

differences were seen in length constant or threshold between the three populations. At VIIIth 

nerve-M-cell mixed synapses there was a reduction in the slope of the input-output relationship 

over many stimulation strengths at both 5° and 25° compared to 15°C. Kinetic differences in the 

coupling potential, which reflects presynaptic (VIIIth nerve) action potential kinetics, were 

consistent with general temperature effects, while tau of the chemical PSP was lower at both 5° 

and 25°C. An examination of feedback inhibition demonstrated a significant increase in coldacclimated 

animals and decrease in warm-acclimated animals, results that are consistent with an 

increased probability of escape in warm-acclimated animals. Together, these studies demonstrate 

that while various aspects of the M-cell circuit are directly impacted by temperature effects in a 

predictable manner even after a period of acclimation, the opposing mechanistic actions of 

various systems, for example inhibition vs. input resistance, act to help the cell and animal 

maintain stable levels of activity. In addition, it seems likely that any disturbance of the 

excitatory/inhibitory balance produces computational problems for directional escape. 

Disclosures: T.M. Szabo, None; T. Brookings, None; D.S. Faber, None; T. Preuss, None. 

Poster 




Topic: B.07.c. Synaptic integration


HHMI 

Title: Light stimuli alters the balance of two components of synaptic inhibition received by 

primate ON parasol ganglion cells 

Authors: J. P. CAFARO, *F. M. RIEKE; 

HHMI/Dept Physiol & Biophysics, Univ. Washington, Seattle, WA 

Abstract: The retina provides an excellent opportunity to examine the relationship between 

synaptic input and action potential generation because of the ability to record synaptic inputs 

elicited by physiological stimuli. We are exploring how excitatory and inhibitory synaptic input 

shape the spike output of primate ON parasol retinal ganglion cells. Consistent with work on 

other retinal ganglion cells, an increase in light intensity produced a rapid increase in excitatory 

synaptic input followed by a slower increase in inhibitory input. The timing of the inhibitory 

input was consistent with feedforward inhibition. Surprisingly, a decrease in light intensity 

produced a slow decrease in excitatory input and a rapid increase in inhibitory input. Thus ON 

parasol cells apparently receive inhibitory input through both ON and OFF pathways. Consistent 

with this idea, application of the mGluR6 agonist, L-APB, abolished excitatory synaptic input 

but had a modest impact on the inhibitory synaptic input elicited by decreases in light intensity. 

The kinetics and timing of the ON and OFF inhibitory components suggest that synaptic 

inhibition may play two distinct roles in shaping spike output - i.e. the ON component of 

synaptic inhibition follows excitation closely and may thus abbreviate the duration of the 

resulting depolarization, while the OFF component operates in a push-pull fashion with 

excitation. The relative contribution of the ON and OFF components of synaptic inhibition were 

not fixed, but instead depended on both mean light level and contrast. The ON component of 

synaptic inhibition was relatively stronger at rod light levels than cone light levels. Similarly, 

increasing contrast at either rod or cone light levels caused a decrease in the contribution of the 

ON component of synaptic inhibition relative to the OFF component. We are currently 

investigating how this shift in the timing and kinetics of synaptic inhibition alters the cell‟s spike 

output. 

Disclosures: J.P. Cafaro, None; F.M. Rieke , None. 

Poster 






Stanley Medical Research Institute 

Title: Synaptic proteome of entorhinal cortex in rhesus monkeys 

Authors: N. S. TANNU, *S. E. HEMBY; 

Physiol & Pharmacol, Wake Forest Univ. Sch. Med., Winston-Salem, NC 

Abstract: The entorhinal cortex (EC) in the parahippocampal region is critical in acquisition, 

consolidation and recovery of long-term memory traces. EC essentially acts as a „gatekeeper‟ for 

the declarative memory system with compromises in functional integrity evident in 

schizophrenia, Alzheimer‟s disease as well as temporal lobe epilepsy. While modulation of 

synaptic architecture forms the basis of synaptic plasticity, dysregulation of synaptic structure 

and function is a principal cellular substrate of the aforementioned disorders. In view of the 

involvement of synaptic plasticity and signaling in physiologic as well as pathological states of 

EC it is imperative to understand the normative abundance of proteins in the presynaptic fraction 

(PSF) and the post-synaptic density (PSD). Therefore, the present study documents the relative 

expression of proteins between PSF and PSD of EC by coupling the isobaric tag peptide labeling 

(iTRAQ) to multidimensional liquid chromatography (MDLC) and matrix-assisted laser 

desorption ionization-time of flight-time of flight tandem mass spectrometry (MALDI-TOF- 

TOF, MS/MS). The results show that the PSF and PSD exhibit signature expression patterns of 

proteins comprised of transporters, ion channels, neurotransmitter receptors, receptor-associated 

scaffold proteins, cytoskeletal proteins, kinases and other enzymes as well as proteins involved 

in neurotransmitter secretion and signaling. Various transporters (e.g. SNAP-25, Synapsin 1, 

Sodium-dependent glutamate/aspartate transporter 1 and 2, Synaptic vesicle glycoprotein 2 A 

and B, Syntaxin-1A, and Syntaxin-binding protein 1); ion channels (e.g. Phospholemman 

precursor, Syntaxin-1B2, and Glycoprotein M6 A and B isoform 1); and enzymes (e.g. Enolase 2 

and Tyrosine 3/tryptophan 5 -monooxygenase activation protein, zeta polypeptide) were found to 

be highly expressed in the PSF of EC. At the same time the cytoskeletal proteins (e.g. Ankyrin-3, 

tubulin α 6 and β [2A and 4] isoform); growth cone proteins (e.g. Brain acid soluble protein 1 

and neural phosphoprotein B-50); and the transmembrane signaling modulators were highly 

expressed in the PSD compared with the PSP. The current study documents the first proteomic 

signature of the entorhinal cortex. This comprehensive knowledge of the protein composition of 

the synapse offers promise to understand the synaptic mechanisms in physiology and 

pathological states involving the EC. 

Disclosures: N.S. Tannu, None; S.E. Hemby , None. 

Poster

238. LTD: Hippocampus and Cortex 



Topic: B.08.f. Long-term depression ( LTD ) 

Support: NIH grant P01 HD29587 

NIH grant R01 EY09024 

Title: Preferential blockade of extrasynaptic NMDARs by NO-Memantine prevents NMDAinduced 

long-term depression in hippocampus 

Authors: *Z. NIE, D. ZHANG, S. A. LIPTON; 

Neurosci, Aging, & Stem Cell, Burnham Inst., La Jolla, CA 

Abstract: NMDARs play an important role in physiological processes such as synaptic 

plasticity, learning, and memory. However, excessive or prolonged activation of NMDARs 

contributes to various pathological conditions, which can be blocked by NMDAR antagonists. 

Here we used rat hippocampal slices to test a new NMDAR antagonist NO-Memantine, a more 

effective neuroprotectant than Memantine. We previously showed that Memantine, which has 

been approved by the FDA for Alzheimer‟s disease, is an uncompetitive inhibitor that at 1-10 

µM blocks excessive extrasynaptic receptor activity preferentially over physiological synaptic 

activity. It is well known that bath application of NMDA stimulates AMPA receptor 

internalization and induces long-term depression (LTD) in CNS neurons. Interestingly, we found 

that 5 µM NMDA-induced LTD occurred after prior exposure to 5 µM MK-801 to persistently 

block synaptic NMDARs. Additionally, while 10 µM NO-Memantine plus 5 µM MK-801 totally 

blocked NMDA-induced LTD, 10 µM NO-Memantine blocked 50 to 60%, consistent with the 

notion that both synaptic and extrasynaptic NMDARs mediate NMDA-induced LTD. On the 

other hand, 10 µM NO-Memantine did not block LTP and had no effect on field excitatory 

postsynaptic potentials (fEPSPs). Additionally, 10 µM NO-Memantine had no effect on non- 

NMDAR-mediated excitatory postsynaptic currents (EPSCs) and only slightly decreased (10 to 

15%) NMDAR-mediated EPSCs in single CA1 pyramidal neurons. These data suggest that NO- 

Memantine blocks NMDA-induced LTD by inhibiting predominantly extrasynaptic NMDARs 

without blockade of LTP and excitatory synaptic transmission in the hippocampus. Therefore, 

NO-Memantine may be an effective drug to ameliorate excitotoxicity without blocking synaptic 

transmission, thereby avoiding severe side effects. 

Disclosures: Z. Nie, None; D. Zhang, None; S.A. Lipton, None. 

Poster





Support: NICHD Intramural research award to CMcB 

Title: Depolarization induced LTD (DiLTD) at immature mossy fiber-CA3 pyramid synapses 

produced by postsynaptic burst firing-like protocols 

Authors: *T. HO, K. A. PELKEY, C. J. MCBAIN; 

LCSN, NICHD, Bethesda, MD 

Abstract: Elucidating cellular mechanisms of synapse maturation is crucial to understanding 

CNS development. Mossy fiber-CA3 pyramid (MF-PYR) synapses develop postnatally 

providing a tractable model of synapse maturation in a defined circuit. Recently we showed that 

between 2-3 postnatal weeks AMPARs supporting MF-PYR transmission undergo a subunit 

switch: immature synapses contain mixed populations of GluR2-lacking and GluR2-containing 

AMPARs and then mature to a state dominated by GluR2-containing AMPARs. A similar 

subunit switch occurs during DiLTD, a postsynaptic form of plasticity observed only at 

immature MF-PYR synapses. DiLTD is triggered by sustained PYR depolarization for 3-5‟ to 

promote Ca2+ influx through L-type voltage-gated Ca2+ channels (L-VGCCs). Interestingly, the 

developmental window of DiLTD competence coincides with the emergence of action potential 

(AP) burst firing (BF) in PYRs, suggesting an attractive physiological mechanism to induce 

DiLTD. Here we investigated whether phasic AP-dependent L-VGCC activation can trigger 

DiLTD using BF-like protocols in acute hippocampal slices. To elicit PYR BF we repetitively 

injected a long depolarizing current pulse (125ms, 0.5nA) at 1Hz for 5‟ in current-clamp mode. 

This protocol reliably produced AP bursts (4-5 APs per pulse) and persistently depressed MF- 

PYR synapses (15‟ after induction EPSCs were 57.6+7.0% of control EPSCs obtained before the 

induction protocol, n=8). However, AP blockade by intracellular QX-314 did not prevent this 

LTD (56.3+8.3% of control 15‟ post-induction, n=4) suggesting that this protocol directly 

activates L-VGCCs independent of APs. Indeed, in the absence of APs the current step typically 

depolarized PYRs sufficiently to activate L-VGCCs and LTD was prevented by blockers of L- 

VGCCs (95.8+18.4% of control 15‟ post-induction, n=3). To avoid direct L-VGCC activation, 

we adopted a second protocol to drive brief trains of PYR APs at the frequency observed during 

our bursts without inducing a large plateau potential. We applied trains of brief current pulses (5 

pulses of 0.8nA, 5ms duration, 25ms apart) delivered at 1 Hz for 5‟. This produced robust LTD 

(41.8+4.3% of control 15‟ after induction, n=5) that was prevented by intracellular QX-314 

(93.4+14.5% of control 15‟ after induction, n=5), and preliminary data confirms a requirement 

for L-VGCCs. Like DiLTD AP train-induced LTD does not affect presynaptic function (PPR and 

CV) indicating postsynaptic expression. We conclude that DiLTD can be induced by phasic L- 

VGCC activation driven by trains of PYR APs suggesting a previously unsuspected role for PYR 

BF in MF-PYR synapse plasticity and maturation.

Disclosures: T. Ho, None; K.A. Pelkey, None; C.J. McBain, None. 

Poster 





Support: CIHR 

Title: The effects of ethanol on long-term depression in both the CA1 and dentate gyrus 

Authors: *J. D. SHIN 1 , R. PETERSEN 1 , B. R. CHRISTIE 2 ; 

1 Biol., 2 Div. of Med. Sci., Univ. Victoria, Victoria, BC, Canada 

Abstract: Alcohol is a widely consumed drug that has a multitude of effects on the brain and 

body. Numerous studies have shown that direct application of ethanol can block long-term 

potentiation, a biological model of learning and memory, less is known of its effects on longterm 

depression (LTD). Some studies have shown ethanol to enhance LTD (Hendricson et al., 

2002), while others have found ethanol blocks LTD (Izumi et al., 2005) in the CA1 region. Field 

excitatory post synaptic potentials (fEPSPs) were recorded from rat (p14-p28) hippocampal 

slices (400 micron). Hippocampal slices were recorded in normal aCSF at 30 degrees Celsius. 

After obtaining a stable baseline, a low frequency stimulus protocol (900 pulses at 1Hz) was 

applied in either the presence or absence of ethanol containing ACSF (50 or 100mM). In the 

CA1 region, 100mM ethanol had a greater inhibition on LTD than 50mM ethanol. In contrast, 

LTD was more severely attenuated by 50mM ethanol than 100mM in dentate gyrus (DG) 

recordings. In conclusion, our results indicate that ethanol reduces LTD in both the CA1 and DG 

subfields of the hippocampus. In the CA1 an increase in ethanol concentration led to an increase 

in LTD inhibition. However, in the DG, with a decrease in inhibition was observed with 

increasing ethanol concentration, suggesting that the effects of ethanol are at multiple receptor 

subtypes. 

Disclosures: J.D. Shin , None; R. Petersen, None; B.R. Christie, None. 

Poster






Title: Group I mGluRs regulate bidirectional plasticity at the mossy fiber synapse on CA3 

interneurons 

Authors: *E. J. GALVAN, G. BARRIONUEVO; 

Dept Neurosci, Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Activation of the group I mGluRs causes the release of calcium from the endoplasmic 

reticulum via IP3 and ryanodine receptors. Although the CA3 region of the hippocampus have a 

high density of ryanodine receptors, the role of calcium stores in synaptic plasticity at the mossy 

fiber (MF) synapse on CA3 interneurons has not been investigated. We have previously shown 

that high frequency stimulation (HFS) delivered to the MF input to s. lacunosum moleculare (L- 

M) interneurons in area CA3 induces a Hebbian form of NMDAR independent LTP that requires 

postsynaptic calcium influx produced by the activation of L-type channels and mGluR1α. Here, 

we further explore the role of the group I mGluRs, and the involvement of the internal calcium 

stores in synaptic plasticity at the MF to L-M interneuron synapse. In naïve hippocampal slices, 

HFS induces MF LTP (150±7%). However, the same HFS applied in slices previously treated 

with the antagonist of the mGluR1α, LY367385 (100 µM), induces MF LTD (74±2%). Using 

minimal stimulation, we determined that the locus of LY367385-mediated MF LTD is 

presynaptic because it was associated with a decrease in failure rate and increase in the CV -2 . In 

contrast, when slices were pretreated with the antagonist of the mGluR5, MPEP (25 µM) HFS 

induced MF LTP (169±1%). In another experiments, we found that the group I mGluR agonist 

DHPG did not produce significant changes in MF EPSP amplitude (94.4 ± 4.4% of control at 10 

min post DHPG; n=4). However, in slices pretreated with DHPG (50 µM) for 10 min prior to 

LY367385, HFS successfully induced LTP (180.3±1%). We also tested the possible involvement 

of the calcium stores in the induction of MF LTP with ryanodine in the patch pipette. In 

ryanodine loaded cells, HFS induces LTD, instead of LTP (72.6±1%). Together, these data 

indicate that mGluRs1α play a critical role in determining the direction of change in long-term 

synaptic plasticity at the MF to L-M interneurons. When mGluRs1α are available, the additional 

release of calcium from the internal stores following HFS leads to MF LTP. In the absence of 

mGluR1α activation, calcium entry through L-type channels leads to LTD. Support: NS24288 

from NINDS. 

Disclosures: E.J. Galvan , None; G. Barrionuevo, None.

Poster 





Support: NIH grant NS044421 

Title: Effects of chemically and stimulus-evoked long-term synaptic depression (LTD) and 

potentiation (LTP) on kiss-and-run release, vesicle recycling and pool exchange at Schaffer 

collateral-CA1 synapses in hippocampus from SynaptopHluorin-expressing mice 

Authors: *X.-L. ZHANG, Z. Y. ZHOU, C. UPRETI, P. K. STANTON; 

New York Med. Coll, Valhalla, NY 

Abstract: Long-term potentiation (LTP) and depression (LTD) are believed to be important 

mechanisms of information storage, learning, and memory. We have shown previously that a 

component of LTD is due to long-term reduction in vesicular release probability, while LTP is 

associated with long-term increases in vesicular release. In this study, we combined 

electrophysiology and two-photon imaging to measure the mode of vesicular release and 

exchange rates between vesicle pools before and after chemically or low-frequency stimulusinduced 

LTD in acute hippocampal slices from synaptopHluorin-expressing mice. We compared 

the balance of exocytosis and endocytosis during low-frequency (2Hz) and high-frequency 

(20Hz) stimulation by measuring fluorescence increases produced by synaptic stimulation in 

slices from mice expressing the pH-sensitive eGFP-tagged SNARE protein synaptopHluorin 

before and after treatment with the vacuolar ATPase inhibitor Bafilomycin (Baf, 1µM) to isolate 

rates of exocytosis by alkaline trapping of release vesicles. With low-frequency 2Hz stimulation, 

the ratio of rates of endocytosis to exocytosis was 0.86 (n=17 boutons in 4 slices), while higherfrequency 

20Hz stimulation produced an endocytosis to exocytosis ratio of 0.43 (n=26 boutons 

in 6 slices). These data suggest there is a larger “kiss-and-run” component to synaptic 

transmission during low versus higher-frequency transmission, because more kiss-and-run 

release allows endocytosis rates to keep up with exocytosis. Data will also be presented 

comparing the ratio of endocytosis and exocytosis before and after chemically or low-frequency 

stimulation induced LTD or LTP, to test the hypothesis that changes in the proportion of release 

mediated by kiss-and-run vs. full fusion modes of release could be a mechanism underlying 

presynaptic long-term plasticity. 

Disclosures: X. Zhang , None; Z.Y. Zhou, None; C. Upreti, None; P.K. Stanton, None.

Poster 





Support: NIH R01-EY014882 

NACS summer internship 

Title: Phosphorylation of GluR1-S845 regulates synaptic AMPA receptor subunit composition 

Authors: *K. HE 1,2 , L. SONG 1 , L. C. CUMMINGS 1 , J. GOLDMAN 2 , H.-K. LEE 1,2 ; 

1 Dept Biol, Univ. Maryland, College Park, MD; 2 Univ. of Maryland, 2. Neuroscience and 

Cognitive Sci. (NACS) Program, College Park, MD 

Abstract: AMPA receptor (AMPAR) is the major glutamatergic receptor, which mediates most 

of the fast excitatory synaptic transmission in the brain. AMPAR channel property and function 

are regulated by its subunit composition and phosphorylation. Recent evidence suggests that 

certain types of neural activity can recruit Ca2+ permeable AMPA receptors, such as GluR1 

homomers, to synapses. Here we report that the GluR1-S845 site phosphorylation can alter the 

subunit composition of synaptic AMPARs by stabilizing homomeric GluR1. Using mice 

specifically lacking the GluR1-S845 site (GluR1-S845A), which was generated in Dr. Richard 

Huganir‟s laboratory at Johns Hopkins University, we demonstrate that this site is necessary for 

maintaining homomeric GluR1 at synapses. Specifically, in the hippocampus of GluR1-S845A 

mutant mice, GluR1 homomers were absent from synapses. We have evidence suggesting that 

this is due to lysosomal degradation of GluR1-S845A homomers. This regulation is mimicked by 

acute desphosphorylation of the GluR1-S845 site in wild type mouse by NMDA application. Our 

findings suggest that GluR1-S845 is necessary for maintaining Ca2+ permeable AMPA receptors 

at synapses, and support the role of GluR1-S845 phosphorylation in the regulation of synaptic 

AMPAR subunit composition. 

Disclosures: K. He , None; L. Song, None; L.C. Cummings, None; H. Lee, None; J. 

Goldman, None. 

Poster 

238. LTD: Hippocampus and Cortex




Support: FORUN, Univ Rostock Med Faculty 

Title: MGluR-dependent LTD at schaffer collateral-CA1 synapses is enhanced by HCN channel 

inhibition 

Authors: *T. KIRSCHSTEIN, T. TOKAY, M. ROHDE, R. E. KOHLING; 

Dept Physiol, Univ. Rostock, Rostock, Germany 

Abstract: HCN channels are widely expressed in hippocampal pyramidal cells, and play a 

fundamental role in regulating the synaptic function. Here, we studied the involvement of HCN 

channels in basal synaptic transmission, short-term plasticity and the induction of metabotropic 

glutamate receptor-dependent long-term depression (mGluR-LTD) by bath application of (RS)- 

3,5-dihydroxyphenylglycine (DHPG) at Schaffer collateral-CA1 synapses using field excitatory 

postsynaptic potential recording techniques. Brief application of DHPG (100 µM, 10 min) 

caused a strong depression of CA1 fEPSPs resulting in stable LTD accompanied by an increase 

in the paired-pulse ratio (PPR) suggesting a presynaptic mechanism. When the slices were 

bathed with the HCN channel blocker ZD7288 (10 µM), a substantial increase in the fEPSP 

amplitude and a significant decrease in the PPR were observed. However, pre-treatment of 

ZD7288 significantly enhanced the DHPG-induced LTD, and the concomitant increase of the 

PPR was significantly higher than the PPR increase following DHPG without ZD7288 pretreatment. 

In addition, we observed an intermediate and strong enhancement of DHPG-induced 

LTD in heterozygous (HCN1+/-) and homozygous (HCN1-/-) mutants, respectively. In contrast, 

NMDA receptor-dependent LTD induced by low-frequency stimulation was not significantly 

altered in the HCN1-deficient mice. These results suggest a constraining effect of HCN1 

channels on Schaffer collateral-CA1 mGluR-dependent but not NMDA receptor-dependent LTD. 

Disclosures: T. Kirschstein , None; T. Tokay, None; M. Rohde, None; R.E. Kohling, None. 

Poster 




Topic: B.08.f. Long-term depression ( LTD )

Title: Role of dimerization for PICK1 function 

Authors: *I. AMMENDRUP-JOHNSEN 1 , K. L. MADSEN 1 , D. S. HAN 2 , M. B. JENSEN 1 , V. 

K. BHATIA 1 , H. A. WEINSTEIN 2 , U. GETHER 1 ; 

1 Univ. of Copenhagen, Copenhagen, Denmark; 2 Dept. of Physiol. and Biophysics, Weill Cornell 

Med. Col., Cornell Univ., New York, NY 

Abstract: PICK1 (Protein Interacting with C-Kinase 1) is a scaffolding protein that is widely 

expressed in the CNS and interacts via its N-terminal PDZ domain with several receptors, 

transporters and kinases including e.g. the AMPA receptor GluR2/3 subunits, the metabotropic 

glutamate receptor mGluR7, the dopamine transporter (DAT) and protein kinase Cα. PICK1 is 

believed to fulfil its biological role either by regulating trafficking of its binding partners, or by 

recruiting PKCα to facilitate their phosphorylation. PICK1 contains in its C-terminal half a BAR 

domain that can dimerize to yield a membrane curvature sensing unit with a key role in 

mediating these functions. Recently, we showed that in the absence of PDZ ligand the activity of 

the BAR domain is inhibited by the PDZ domain, and that unmasking of BAR domain activity is 

not just a consequence of ligand binding to the PDZ domain but requires as well recruitment of 

PICK1 to a specific membrane compartment. Here we hypothesize that activation of the BAR 

domain involves transitions between monomeric and dimeric/multimeric states of PICK1. To test 

this hypothesis we have employed FRET (Fluorescence Resonance Energy Transfer) based 

techniques. For studies in living cells, we have tagged full length PICK1 as well as two different 

truncation mutants with eCFP- and eYFP. Application of the three-filter FRET technique showed 

no FRET signal for cytoplasmic full-length PICK1; however, a significant FRET signal was seen 

for PICK1 co-expressed with a transmembrane ligand that localized the protein to recycling 

endosomes. A clear FRET signal was also observed for PICK1 with truncated PDZ domain 

(PICK1 Γ101) that constitutively localized to endosomes. Importantly, the FRET signal was 

reduced in a mutation of PICK1 Γ101 predicted based on homology modelling to disrupt BAR 

domain dimerization. Additional cellular and biochemical experiments should further clarify the 

significance of these observations and their functional implications. 

Disclosures: I. Ammendrup-Johnsen , None; K.L. Madsen, None; M.B. Jensen, None; V.K. 

Bhatia, None; H.A. Weinstein, None; U. Gether, None; D.S. Han, None. 

Poster 




Topic: B.08.f. Long-term depression ( LTD )

Support: Ray Thomas Edwards Foundation 

Whitehall Foundation 

Autism Speaks 

Alfred P. Sloan Foundation 

Silvio Varon Chair in Neurodegeneration 

Title: MHC class I modulates glutamate receptor function and trafficking in hippocampal 

neurons 

Authors: *L. FOURGEAUD, M. B. SPENCER, T. T. CHENG, L. M. BOULANGER; 

Neurobio., UCSD, La Jolla, CA 

Abstract: Proteins of the major histocompatibility complex, class I (MHCI) are known for their 

central role in adaptive immunity. However, recent studies have shown that MHCI proteins are 

also dynamically expressed by healthy neurons, and are unexpectedly required for normal 

synaptic plasticity. Specifically, in adult mice genetically deficient for cell surface MHCI (β2m -/- 

TAP -/- ), the bidirectional regulation of synaptic strength is shifted in favor of potentiation: 

hippocampal long-term potentiation (LTP) is enhanced, while long-term depression (LTD) is 

absent. However, how MHC class I regulates plasticity remains unclear. Activity-dependent 

modification of the trafficking and/or channel properties of glutamate receptors is thought to lead 

to LTP and LTD in the adult hippocampus. Therefore, we tested the hypothesis that MHCI 

regulates the expression, function or trafficking of hippocampal AMPA- or NMDA-type 

glutamate receptors. Basal total, surface and synaptic levels of GluR1, GluR2 and NR1 are 

indistinguishable from wild type (WT) in MHCI deficient β2m -/- TAP -/- hippocampal neurons, as 

measured immunocytochemically and biochemically. In WT hippocampal slices, brief bath 

application of NMDA induces a form of LTD that is mechanistically similar to that induced by 

low-frequency stimulation (LFS-LTD). Like LFS-LTD, NMDA-LTD is converted to LTP in 

MHCI-deficient slices. In parallel, NMDA induces changes in GluR1 trafficking in MHCIdeficient 

neurons in vitro that are reminiscent those seen during LTP. Whole cell and field 

recordings from hippocampal slices show that the AMPA/NMDA ratio of excitatory 

postsynaptic currents is reduced, and NMDAR-mediated field excitatory postsynaptic potentials 

are enhanced, in MHCI-deficient CA1 pyramidal neurons. These results demonstrate that 

endogenous MHC class I limits neuronal NMDAR function and regulates the coupling between 

NMDAR activation and appropriate regulation of GluR1 trafficking. In this way, changing levels 

of neuronal MHCI during brain development may permit graded fine-tuning of the rules that 

determine the sign and magnitude of synaptic plasticity. 

Disclosures: L. Fourgeaud, None; M.B. Spencer, None; T.T. Cheng, None; L.M. Boulanger, 

None.

Poster 





Support: NIH Grant NIA5R01AG027443-02 

Title: Soluble amyloid ß-protein facilitates NMDAR-dependent long-term synaptic depression 

(LTD) by a novel mechanism 

Authors: *S. LI 1 , S. HONG 2 , N. SHEPARDSON 2 , L. FEIG 3 , G. SHANKAR 2 , D. SELKOE 2 ; 

1 Neurol., Brigham and Women's Hosp., Boston, MA; 2 Brigham and Women's Hosp., Boston, 

MA; 3 Tufts Univ. Sch. of Med., Boston, MA 

Abstract: Alzheimer‟s disease (AD) is characterized by insidious and progressive memory loss 

accompanied by high levels of soluble amyloid-β proteins (Aβ) and the accumulation of amyloid 

plaques and neurofibrillary tangles. Perhaps the strongest histopathological correlate of the 

degree of dementia in AD subjects is the decrease in levels of cortical synapses, and APP 

transgenic mice have decreased dendritic spine densities. Long-term potentiation (LTP) is 

associated with spine formation or increase in spine volume, whereas the induction of long-term 

depression (LTD) results in spine shrinkage and elimination. We and others have reported that 

hippocampal LTP is inhibited by soluble oligomers of Aβ. Far less is known about the effect of 

Aβ on LTD induction. Cell-secreted human Aβ from a hAPP-transfected CHO cell line (7PA2 

cell medium) was used to study the effects of soluble Aβ on synaptic plasticity. Field excitatory 

postsynaptic potentials (fEPSP) were recorded in stratum radiatum of CA1 in mouse 

hippocampal slices, with the stimulating electrode in the Schaffer collaterals. LTD was induced 

by 300 or 900 pulses at 1 Hz. We found that the LTD induced by 900 pulses in slices exposed to 

cell-secreted Aß was resistant to the usual dose of NMDAR antagonist (50 κM AP5) that 

prevents LTD in the absence of soluble Aß, but this “Aβ-mediated LTD” was prevented when 

AP5 was combined with MK801. The Aβ-mediated LTD required extracellular Ca 2+ influx and 

active PP2B and GSK-3 signaling pathways, in contrast to conventional NMDAR-mediated LTD 

that requires low-level NMDAR activation and involves both extracellular and intracellular Ca 2+ 

sources and an active p38 MAPK signaling pathway. Using Ras-GRF1 knockout mice, in which 

NMDA-mediated LTD (900 pusles protocol) cannot be induced in CA1, we confirmed this 

distinct and novel Aβ-mediated LTD mechanism. Aβ-mediated LTD was mimicked by the 

glutamate uptake inhibitor, TBOA, suggesting that soluble Aβ facilitates an LTD state in part 

through reduced glutamate clearance. 

Disclosures: S. Li , None; S. Hong, None; N. Shepardson, None; L. Feig, None; G. Shankar, 

None; D. Selkoe, None.

Poster 





Support: NIH Grant HD056235 

The Coleman Institute 

Anna & John J. Sie Foundation 

Title: Effects of memantine on NMDA induced long-term depression in the Ts65Dn mouse 

Authors: *J. J. SCOTT-MCKEAN 1 , K. E. SMITH 2 , M. L. DELL'ACQUA 2,1 , A. C. S. 

COSTA 3,1 ; 

1 Neurosci. Training Program, Univ. of Colorado Denver, Aurora, CO; 2 Dept. of Pharmacol., 

3 Div. of Clin. Pharmacol. and Toxicology, Dept. of Med., Univ. of Colorado Denver, Anschutz 

Med. Campus, Aurora, CO 

Abstract: The Ts65Dn mouse is the most complete animal model for Down syndrome that is 

widely available. Recently, our research group has shown that the Ts65Dn mouse displays 

pharmacological responses consistent with a dysfunction in molecular pathways coupled to the 

gating of NMDA receptors; these include the finding that the uncompetitive NMDA receptor 

antagonist memantine rescues learning and memory deficits in these mice. In the present study, 

we used electrophysiological and biochemical techniques to probe potential mechanisms 

underlying these observations. NMDA mediated long-term depression (LTD) was induced in the 

CA1 region of acute hippocampal slices from Ts65Dn and euploid control mice. This form of 

chemically-induced LTD (Chem-LTD) was elicited by bath application of 20 µM NMDA for 3 

min, and the resulting depression of field postsynaptic potentials was assessed 60 min after 

application. We found that Ts65Dn mice showed a greater synaptic depression when compared 

to euploid littermate controls. To determine whether memantine could rescue this phenotype in 

the Ts65Dn mice, half of the hippocampal slices were treated with1 µM memantine before 

Chem-LTD treatment. Interestingly, we found that memantine-treated hippocampal slices from 

Ts65Dn mice presented control levels of Chem-LTD. Currently, phosphorylation of the AMPA 

receptor GluR1 subunit is being assessed on hippocampal slices from Ts65Dn and control mice, 

which were flash frozen under control and Chem-LTD conditions, with or without drug. Our 

goal is to determine whether Chem-LTD-induced dephosphorylation of this subunit is 

dysregulated in Ts65Dn mice and whether memantine treatment rescues this molecular

phenotype. 

This study confirms our previous finding that molecular pathways coupled to the gating of 

NMDA receptors are altered in the Ts65Dn mouse and brings us a step closer to understanding 

the role of NMDA receptors in the pathogenesis of the cognitive deficits associated with Down 

syndrome. 

Disclosures: J.J. Scott-McKean, None; K.E. Smith, None; M.L. Dell'Acqua, None; A.C.S. 

Costa, Forest Pharmaceuticals, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received). 

Poster 





Support: partly sponsored by Neurosearch A/S 

Title: Identification of a small molecule inhibitor of the PICK1 PDZ domain that attenuates LTD 

in CA1 neurons 

Authors: *T. S. THORSEN 1 , K. MADSEN 1 , N. REBOLA 2 , A. BACH 3 , T. BEUMING 4 , I. 

MOREIRA 4 , N. STUHR-HANSEN 3 , D. PETERS 5 , T. DYHRING 5 , H. WEINSTEIN 4 , K. 

STRØMGAARD 3 , C. MULLE 2 , L. RØNN 5 , U. GETHER 1 ; 

1 Univ. Copenhagen, Copenhagen N, Denmark; 2 Inst. des Neurosciences de Bordeaux, Univ. 

Bordeaux, Bordeaux, France; 3 Dept. of Medicinal Chem., Univ. of Copenhagen, Copenhagen Ø, 

Denmark; 4 Dept. of Physiol. and Biophysics,, Weill Med. Col. of Cornell Univ., New York, NY; 

5 Neurosearch A/S, Ballerup, Denmark 

Abstract: PICK1 (Protein Interacting with C Kinase 1) contains an N-terminal PDZ domain 

known to mediate interaction with the C-termini of the GluR2 subunit of the AMPA receptor, 

with the dopamine transporter (DAT) as well as with several G protein coupled receptors, ion 

channels and kinases including protein kinase Cα. PICK1 is necessary for AMPA receptorrelated 

neuroplasticity associated with sensitization processes underlying e.g. neuropathic pain 

and cocaine addiction. Specifically, PICK1 is believed to be responsible for intracellular 

accumulation of the AMPA receptor, a process necessary for expression of Long Term 

Depression (LTD). Inhibitors of the PICK1 PDZ domain may accordingly prove useful for 

treatment of diseases involving abnormal synaptic plasticity. To identify such inhibitors, we 

screened a library of 44,000 random compounds using a previously described fluorescence

polarization assay. The screen yielded several compounds with affinities in the low micromolar 

range. One compound (231) with desirable chemical properties was selected for further detailed 

characterization. Its interaction with the PICK1 PDZ domain was confirmed using a GST pulldown 

assay showing dose-dependent and reversible inhibition of the interaction between the 

DAT C-terminus and PICK1. According to both pull-down experiments and a fluorescence 

polarization assay, compound 231 displayed no affinity towards the three PDZ domains of 

PDS95. Molecular modeling and mutagenesis support that compound 231 bind in the binding 

groove of the PICK1 PDZ domain. Using a FRET setup, the compound was shown to inhibit the 

PICK1 - GluR2 interaction in living cells thereby demonstrating its membrane permeability. 

Moreover, compound 231 was shown to block LTD in CA1 pyramidal cells with similar efficacy 

as C-terminal peptides corresponding to the GluR2 C-terminus, Analogues of compound 231 are 

currently being synthesized to optimize affinity. 

Disclosures: T.S. Thorsen, None; K. Madsen, None; N. Rebola, None; A. Bach, None; T. 

Beuming, None; N. Stuhr-Hansen, None; D. Peters, Neurosearch A/S, A. Employment (full or 

part-time); T. Dyhring, Neurosearch A/S, A. Employment (full or part-time); H. Weinstein, 

None; K. Strømgaard, None; C. Mulle, None; L. Rønn, Neurosearch A/S, A. Employment (full 

or part-time); U. Gether, None; I. Moreira, None. 

Poster 





Support: Medical Research Council (UK) 

The Wellcome Trust 

BBSRC 

Title: Tyrosine dephosphorylation of GluR2 regulates AMPAR internalisation in mGluR-LTD 

but not in NMDAR-LTD 

Authors: *E. MOLNAR, C. M. GLADDING, Z. I. BASHIR, G. L. COLLINGRIDGE; 

MRC Ctr. Synaptic Plasticity, Univ. Bristol, Bristol, United Kingdom 

Abstract: Long-term depression (LTD) can be induced at hippocampal CA1 synapses, by 

activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors

(mGluRs) using their selective agonists. Recent studies revealed that mGluR-LTD is dependent 

on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently 

dephosphorylate tyrosine residues in AMPA receptors (AMPARs) [Moult et al., (2006) J. 

Neurosci. 26, 2544-2554]. In the present study we have investigated native AMPARs in 

hippocampal brain slices to reveal: (1) the involvement of individual AMPAR subunits in 

mGluR-LTD induction, (2) differential changes in AMPAR subunit phosphorylation in mGluR- 

LTD and NMDAR-LTD and (3) the role of tyrosine dephosphorylation in AMPAR endocytosis 

in mGluR-LTD. Here we show that while both GluR2 and GluR3 AMPAR subunits are tyrosinephosphorylated 

at basal (nonpotentiated) activity, only GluR2 is dephosphorylated in mGluR- 

LTD. The PTP-dependent tyrosine dephosphorylation of GluR2 is specific for mGluR-LTD and 

does not occur in NMDAR-LTD. Conversely, while NMDAR-LTD is associated with the 

dephosphorylation of GluR1-serine-845, mGluR-LTD does not alter the phosphorylation of this 

site. The increased AMPAR endocytosis in mGluR-LTD is PTP-dependent and involves tyrosine 

dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit 

selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in 

mGluR-dependent but not in NMDAR-mediated LTD in the hippocampus. 

Disclosures: C.M. Gladding, None; E. Molnar , None; Z.I. Bashir, None; G.L. Collingridge, 

None. 

Poster 





Support: Scottish Enterprise (Proof of Concept) 

Carlton Pharmaceutics 

The Cunningham Trust 

Title: Proteases induce the expression of long-term depression (LTD) 

Authors: *A. P. MALLON 1 , D. G. MACGREGOR 2 , T. W. STONE 3 , H. G. NIMMO 3 ; 

1 Brown Univ., MPPB, Providence, RI; 2 Strathclyde Univ., Glasgow, United Kingdom; 3 Glasgow 

Univ., Glasgow, United Kingdom

Abstract: Here we describe the discovery of a novel protein, cadeprin, which elicits a reliable 

and particularly profound long-term depression (LTD) in the CA1 area of the adult rat 

hippocampus. This protein was subsequently purified and identified as a new member of the 

chymotrypsin-like S8A serine protease family, several members of which were also tested and 

found to induce LTD by virtue of their proteolytic activity. 

The LTD induced was not blocked by NMDA antagonists and could not be occluded by the 

induction of NMDA receptor-dependent LTP. However, it was prevented and reversed by Group 

I mGluR antagonists indicating that this is a mGluR dependent form of LTD. Interestingly, 

during the initial perfusion of the protease no changes in baseline synaptic strength were 

observed that would be expected with the primary activation of the metabotropic glutamate 

receptor (mGluR). Intriguingly, this LTD protocol, whilst it is dependent on mGluRs for its 

expression it is not dependent on activating them during its induction. We speculate that this 

protease protocol circumvents the initial steps in agonist-induced mGluR LTD and act as a 

downstream mediator. Perhaps making specific structural modifications of dendritic spines and 

their associated postsynaptic densities (PSDs) leading to decreased synaptic strength. The 

emergence of this late stage protocol for inducing LTD without the contaminating influence of 

early stage factors may prove a very useful tool in investigating LTD. 

Disclosures: A.P. Mallon, None; D.G. MacGregor, None; T.W. Stone, None; H.G. Nimmo, 

None. 

Poster 






Title: Dynamic imaging of cyclic nucleotide concentration changes during long-term activitydependent 

synaptic plasticity using fluorescent sensors 

Authors: *Z. ZHOU 1 , X. L. ZHANG 1 , C. UPRETI 1 , V. O. NIKOLAEV 2 , P. K. STANTON 1 ; 

1 Dept Cell Biol/Anatomy, New York Med. Coll, Valhalla, NY; 2 Inst. of Pharmacol. and 

Toxicology, Würzburg, Germany 

Abstract: The cyclic nucleotide second messengers cAMP and cGMP play important roles in 

both presynaptic and postsynaptic long-term activity-dependent synaptic plasticity at a variety of 

synapses. Previously, our lab has shown that pairing elevation of [cGMP] with suppression of the

production of cAMP, or inhibition of PKA activity itself, is sufficient to elicit a presynaptic LTD 

of synaptic strength associated with a reduced probability of release from the rapidly-recycling 

pool of vesicles. Intracellular [cGMP] can undergo relatively rapid changes in response to 

natriuretic peptides and nitric oxide. While many techniques have been used to monitor 

activation of G protein-coupled receptors that act via cyclic nucleotides, they have been only 

poorly able to resolve cyclic nucleotide concentrations in space and time. We will describe data 

concerning the real-time measurement of [cAMP] and [cGMP] during induction and expression 

of chemical and stimulus-evoked long-term potentiation (LTP) and depression (LTD) of synaptic 

strength at Schaffer collateral-CA1 synapses in brain slices. Cyclic nucleotide concentration is 

imaged optically using two-photon excitation in hippocampal slices transfected with vectors 

expressing a fusion protein of cAMP or cGMP-selective binding domains with fluorescent 

biosensors that respond to binding-induced conformational changes that alter fluorescence via 

Förster resonance energy transfer. 

Disclosures: Z. Zhou , None; X.L. Zhang, None; C. Upreti, None; V.O. Nikolaev, None; P.K. 

Stanton, None. 

Poster 





Support: CIHR 

Title: Characterization of synaptic plasticity in the lateral perforant path input to the dentate 

gyrus in young rats 

Authors: *R. PETERSEN, B. CHRISTIE; 

Univ. Victoria, Victoria, BC, Canada 

Abstract: The lateral perforant path (LPP) input to the outer portion of the dentate molecular 

layer has traditionally been thought of as a segregated input, separate and distinct from the 

medial perforant path (MPP) input on the basis of electrophysiological, structural and 

pharmacological differences. In particular, early work has shown that the release probability in 

these two paths is markedly different, with the MPP having a higher probability of release than 

the LPP. In these experiments we sought to quantify differences in paired-pulse responses 

between the MPP and LPP in transverse slices taken from young rats (14-28 days old). In 

addition, we examined how varying the frequency of conditioning stimuli affected the induction

of synaptic plasticity in the lateral pathway. Our results show that in young animals, both lateral 

and medial afferents show paired-pulse facilitation and therefore appear to have a lower release 

probability than was initially suspected. In addition, we found that 1 Hz stimuli effectively 

induced large LTD in this pathway, while stimuli at 3-5Hz were less efficacious. LTP could be 

reliably induced with high-frequency stimulation (100 Hz). These findings help to further define 

the basic electrophysiological characteristics of this hippocampal afferent, and help to lay the 

groundwork for future studies on how drugs of addiction might alter its function. 

Disclosures: R. Petersen , None; B. Christie, None. 

Poster 





Support: NIH Grant AA014691 

NIH Grant AA09675 

Title: Long-term synaptic depression that is associated with GluR1-S845 dephosphorylation but 

not AMPA receptor internalization 

Authors: *K. D. DAVIES 1 , S. M. GOEBEL-GOODY 2 , S. J. COULTRAP 1 , M. D. 

BROWNING 1 ; 

1 Pharmacol., 2 Neurosci., Univ. of Colorado HSC, Aurora, CO 

Abstract: Long lasting changes in the strength of synaptic transmission in the hippocampus are 

thought to underlie certain forms of learning and memory. Accordingly, the molecular 

mechanisms that account for these changes are heavily studied. Post-synaptically, changes in 

synaptic strength can occur by altering the amount of neurotransmitter receptors at the synapse or 

by altering the functional properties of synaptic receptors. In this study, we examined the 

biochemical changes produced following chemically induced long-term depression in acute 

hippocampal CA1 minislices. Using three independent methods, we found that this treatment did 

not lead to an internalization of AMPA receptors. Furthermore, when the plasma membrane was 

separated into synaptic membrane-enriched and extra-synaptic membrane-enriched fractions, we 

actually observed a significant increase in the concentration of AMPA receptors at the synapse. 

However, phosphorylation of S845 on the AMPA receptor subunit GluR1 was significantly 

decreased throughout the neuron, including in the synaptic membrane-enriched fraction.

Phosphorylation of this residue has been demonstrated to control peak open probability of 

AMPA receptors. From these data, we conclude that the decrease in synaptic strength is likely 

the result of a change in the functional properties of AMPA receptors at the synapse and not a 

decrease in the amount of synaptic receptors. 

Disclosures: K.D. Davies , None; S.M. Goebel-Goody, None; S.J. Coultrap, None; M.D. 

Browning, PhosphoSolutions, E. Ownership Interest (stock, stock options, patent or other 

intellectual property). 

Poster 





Support: FRAXA Research Foundation 

NIH grant NS034007 

Alzheimer's Association 


Title: Regulation of protein synthesis-dependent mGluR-LTD by the eIF2α kinase PERK 

Authors: *M. A. TRINH 1 , H. KAPHZAN 1 , D. R. CAVENER 2 , E. KLANN 1 ; 

1 Ctr. for Neural Sci., New York Univ., New York, NY; 2 Dept. of Biol., Pennsylvania State 

Univ., University Park, PA 

Abstract: Alteration of gene expression and synthesis of de novo proteins are crucial for the 

expression of long-lasting forms of synaptic plasticity and long-term memory formation in 

hippocampal area CA1. In the hippocampus, generation of new proteins has been shown to be 

regulated during plasticity via factors involved in the initiation of mRNA translation. A key 

regulatory step in the initiation of translation is the formation of the preinitiation ternary 

complex, eIF2-GTP-Met-tRNAi Met , which is required to bind to the 40S ribosomal subunit prior 

to recognition of mRNA. Upon each round of translation initiation, the guanine-nucleotide 

exchange factor, eIF2B, is required to catalyze the exchange of eIF2-GDP for GTP-bound eIF2 

in order to regenerate the ternary complex. It has been shown that phosphorylation of the α 

subunit of eIF2 inhibits eIF2B activity and subsequently decreases general translation. We have

initiated studies to determine whether alterations in eIF2α phosphorylation are associated with 

metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). Our 

preliminary results indicate that eIF2α phosphorylation is enhanced upon activation of group I 

mGluRs with the agonist DHPG, and attenuated by antagonists of mGluR1 and mGluR5. The 

PKR-like endoplasmic reticulum eIF2α kinase (PERK) is one of four kinases shown to 

phosphorylate eIF2α thereby inhibiting general translation. Using biochemical and 

immunocytochemical approaches, we have found that PERK is expressed in hippocampal 

pyramidal neurons, and are currently determining whether it co-localizes with eIF2α. In order to 

investigate whether PERK regulates eIF2α phosphorylation during mGluR-LTD, we have begun 

studies with a forebrain-specific conditional PERK knockout mouse generated via the CaMKIIαdriven 

cre-lox expression system. In addition, studies are underway to examine hippocampusdependent 

memory in the PERK knockout mice. The results of our studies will provide insight 

into the regulation of eIF2α phosphorylation by PERK during translation-dependent synaptic 

plasticity and memory in the hippocampus. Supported by NIH grants NS034007 and NS047384, 

the Alzheimer's Association, and the FRAXA Research Foundation (E.K.). 

Disclosures: M.A. Trinh, None; H. Kaphzan, None; D.R. Cavener, None; E. Klann, None. 

Poster 





Support: MRC 

Title: Developmental regulation of the role of NR2B in hippocampal long term depression 

Authors: T. E. BARTLETT 1 , *S. M. FITZJOHN 1 , N. J. BANNISTER 2 , Z. I. BASHIR 2 , G. L. 

COLLINGRIDGE 2 ; 

1 Dept. of Anat., 2 Univ. of Bristol, Bristol, United Kingdom 

Abstract: N-methyl D-aspartate receptor (NMDAR)-dependent long term depression (LTD) 

mediates refinement of neuronal pathways early in development, yet remains important in the 

mature brain for learning and memory. Although variation in expression levels of NMDAR NR2 

subunits through development has been characterised, it is important to know whether there is a 

change in the NR2 subunit dependency of LTD induction with developmental stage. In this work 

the sensitivity of LTD induction to the best available NR2-selective antagonists was examined 

using in-vitro electrophysiology in CA1 of rat hippocampal slices at postnatal days 7, 14 and 42-

56 (P7, P14 and P42-56). We found that LTD induction is dependent on NR2B at P7, but is not 

sensitive to the NR2A or NR2B antagonists alone at P42-56. LTD at P14 but not at P42-56 is 

sensitive to a combination of the NR2A and NR2B-selective antagonists. These results show that 

the NMDAR subunit dependency of LTD induction changes through development, but do not 

reveal the identity of the necessary subtype at ages beyond two weeks. In an extension to this 

study we have tested some of the factors that may account for the obvious disparity in the 

literature concerning the sensitivity of in-vitro LTD induction to the NR2B-selective antagonist 

Ro 25-6981. 

Disclosures: T.E. Bartlett, None; Z.I. Bashir, None; S.M. Fitzjohn , None; G.L. Collingridge, 

None; N.J. Bannister, None. 

Poster 





Title: PICK1 decreases reinsertion rates of recycling interaction partners 

Authors: *K. L. MADSEN, T. R. CLEMMENSEN, U. GETHER; 

Neurosci. and Pharmacol., Panum Institute, Building 18.6, Copenhagen, Denmark 

Abstract: PICK1 (Protein Interacting with C Kinase 1) is a scaffolding protein that in its aminoterminal 

half possesses a single PDZ domain known to mediate interaction with the C-termini of 

several receptors, transporters and kinases expressed in the CNS, including e.g. the GluR2/3 

subunits of AMPA-type ionotropic glutamate receptors (AMPA receptors), the metabotrobic 

glutamate receptor mGluR7, the dopamine transporter (DAT), the norepinephrine transporter 

(NET) the Glt1b glutamate transporter and protein kinase Cα. In its C-terminal half, PICK1 

possesses a BAR (Bin/amphyphysin/Rvs) domain that is believed mediate curvature dependent 

recognition/tubulation of lipid membranes. For some interactions partners, evidence suggests that 

PICK1 is responsible for intracellular accumulation of the interaction partner, and this function 

of PICK1 in relation to the AMPA receptors is necessary for expression of LTD and potentially 

LTP in cerebellar granule cells and hippocampal neurons. For other interaction partners, such as 

the DAT, PICK1 seems to stabilize surface expression of the membrane protein. 

It is conceivable that these function are dependent on the BAR domain; however, it is not known 

how PICK1 differentially affects trafficking of its different interactions partners. Recently, we 

showed evidence that the BAR domain in PICK1 is autoinhibited and that unmasking of the lipid 

binding capacity depends on and coincides with recruitment of PICK1 to a membrane

compartment. Here, by application of a series of ELISA and confocal microscopy based assays 

in heterologous cells and neurons we have investigated how PICK1 affect trafficking rates 

during internalization and recycling of different chimeric and natural interaction partners. 

We find no evidence of PICK1 affecting internalization rates. Instead our data suggest that 

PICK1 affects reinsertion rates for proteins already directed to recycling. We show that this 

function of PICK1 is dependent on both a functional PDZ and BAR domain. Moreover using 

chimeric constructs of the β2 adrenergic receptor engineered to bind the PICK1 PDZ domain, we 

show that the ability of PICK1 to reduce recycling of interaction partners is calcium independent. 

Disclosures: K.L. Madsen , None; T.R. Clemmensen, None; U. Gether, None. 

Poster 





Support: Swedish Research Council, project number 01580 and 12600) 

Wilhelm and Martina Lundgren's Foundation 

Title: Neither NMDA nor metabotropic glutamate receptors are required for long-term 

depression of developing hippocampal CA3 - CA1 synapses 

Authors: *J. STRANDBERG, B. GUSTAFSSON; 

Dept Physiol, Sahlgrenska Academy, Goteborg Univ., Goteborg, Sweden 

Abstract: It has previously been described that brief (about 100 impulses) 0.2 - 0.05 Hz 

stimulation of naïve (previously non-stimulated) developing hippocampal CA3 - CA1 synapses 

leads to a substantial depression, explained by AMPA silencing. This depression does not require 

NMDA or mGluR activation for its induction, and it is reversed within half an hour following 

stimulus interruption. Using field recordings we have examined synaptic depression of naïve 

CA3-CA1 synapses in hippocampal slices from P8 - P13 rats using LFS (900 impulses at 1 Hz) 

as well as 900 impulses at 0.2 Hz. It was found that 900 impulses produced depression (about 60 

%) that was largely independent of whether 0.2 Hz (in some cases 0.05 Hz) or 1 Hz stimulation 

was used. Following 30 min of stimulus interruption the remaining depression (LTD) was about 

40 % and not significantly different. In the combined presence of D-AP5 (50 µM) and 

LY341495 (100 µM) to block NMDARs/mGluRs the 900 impulse stimulation produced 

depression of about 45 % independent of whether 0.2 or 1 Hz stimulation was used. Following

30 min of stimulus interruption the remaining depression (LTD) was 20 - 25 % and not 

significantly different. When using AP5 and LY 341495 separately significantly more depression 

was observed when NMDAR activation was allowed than when mGluR activation was allowed. 

When mGluR activation was allowed 1 Hz stimulation produced significantly more depression 

than did 0.2 Hz stimulation. Nevertheless, this depression was no more than that obtained when 

both NMDARs and mGluRs were blocked. In conclusion, in the developing rat prolonged 

stimulation at sub-LFS, as well as LFS, frequencies produce LTD that does not need 

NMDA/mGluR activation for its induction. It is proposed that LTD in the developing CA3 - 

CA1 synapse mainly reflects AMPA silencing facilitated by but not requiring NMDAR 

activation and counteracted (specifically at sub-LFS frequencies) by mGluR activation. 

Disclosures: J. Strandberg, None; B. Gustafsson, None. 

Poster 





Support: NIH-NINDS Grant RO1NS049661 

Title: Redistribution of CaMKII to inhibitory synapses following NMDA receptor activation 

Authors: *K. C. MARSDEN 1 , K. U. BAYER 2 , R. C. CARROLL 1 ; 

1 Dominick P. Purpura Dept Neurosci, Albert Einstein Coll Med., Bronx, NY; 2 Univ. of Colorado 

Denver Hlth. Sci. Program, Denver, CO 

Abstract: Maintaining a balance between excitatory and inhibitory synaptic inputs is critical for 

normal brain function, as disruptions in excitability can lead to diseases such as epilepsy, anxiety 

and even autism. This balance can be regulated through the strengthening or weakening of 

synapses, which in turn can happen through the insertion or removal of receptors from the 

postsynaptic membrane. We have previously demonstrated that activation of NMDA receptors, a 

model for long-term depression of excitatory synapses, simultaneously decreases surface AMPA 

receptor (AMPAR) expression and increases that of GABAA receptors in hippocampal neurons. 

This process potentiates inhibitory synaptic transmission and requires CaMKII for its expression. 

CaMKII is well known for its role in excitatory LTP and AMPAR trafficking, and it has been 

shown to translocate to excitatory synapses following LTP-like stimuli. Although it has 

previously been implicated in GABAAR trafficking and the potentiation of inhibitory synapses, 

CaMKII expression at inhibitory synaptic locations has not been examined. Here we provide data

from live imaging experiments of hippocampal neurons expressing mGFP-CaMKII as well as 

evidence from antibody labeling of the endogenous protein which demonstrate that CaMKII 

undergoes a redistribution following NMDAR activation. Preliminary data suggest that 

excitatory LTP-inducing NMDAR activation drives CaMKII preferentially to excitatory 

synapses, while inhibitory LTP-inducing NMDAR stimulation results in accumulation of 

CaMKII at inhibitory synapses. We are currently analyzing several mutant mGFP-CaMKII 

constructs to determine whether this redistribution requires CaMKII activation, 

autophosphorylation, or calmodulin binding. Biochemical analysis of protein derived from 

hippocampal slices will also allow us to determine whether CaMKII interacts with components 

of the inhibitory synapse following NMDAR activation. Our data suggest that redistribution of 

CaMKII may underlie the potentiation of inhibitory synapses, and future experiments will 

examine the mechanism and functional consequences of this process. 

Disclosures: K.C. Marsden , None; K.U. Bayer, None; R.C. Carroll, None. 

Poster 





Support: NIH GM 60655 

Alfred P. Sloan Foundation 

Title: Low-frequency potentiation of perforant path-dentate gyrus synapses in vivo 

Authors: *D. M. VILLARREAL, F. L. JONES, K. E. ANGUIANO, B. E. DERRICK; 

Dept Biol Sci., Univ. Texas San Antonio, San Antonio, TX 

Abstract: Low-frequency potentiation (LFP) is a sustained form of synaptic plasticity seen in a 

number of synapses, including afferents to the amygdala (Huang and Kandel, Learn. & Mem., 

2007). In an attempt to determine optimal stimulation parameters for the induction of LTD at 

perforant path-dentate gyrus synapses in behaving animals, we used paradigms previously found 

effective in inducing LTD at other hippocampal synapses. However, stimulation of either medial 

or lateral perforant path projections to the dentate (900 pulses, 1 Hz) produced a robust LTP. 

This was observed in both the medial and lateral perforant pathways, although the lateral 

perforant pathway appears particularly amenable to LFP. In this pathway, LFP was seen with as 

few as 25 pulses at 0.3 Hz. Although LTP induced in both pathways by LFS was robust (100%

above baseline) and showed no decrement after 4 hrs (suggesting protein synthesis-dependent 

“late” LTP), it nonetheless returned to baseline within 24 hrs. These data indicate LTP with 

distinct characteristics can be induced with different stimulation frequencies. Moreover, different 

stimulation parameters induce LTP of different durations, suggesting different patterns of 

afferent activity engage distinct molecular cascades of LTP maintenance. This indicates the 

diversity and versatility of LTP at a single synapse, and that distinct forms of LTP can be 

induced with distinct patterns of afferent activity, even within a single synaptic population. 

Disclosures: D.M. Villarreal , None; K.E. Anguiano, None; B.E. Derrick, None; F.L. Jones, 

None. 

Poster 





Support: Hearing Research, Inc. 

NIDCD 

Sandler Translational Research postdoctoral fellowship 

Jane Coffin Childs postdoctoral fellowship 

Title: Conservation of net synaptic input in cortical receptive field plasticity 

Authors: *R. C. FROEMKE, M. M. MERZENICH, C. E. SCHREINER; 

Dept Otolaryngology, UCSF, San Francisco, CA 

Abstract: Cortical synapses are modified by sensory experience and electrical activity. Synaptic 

modification in vivo alters receptive fields of sensory cortical neurons, with complex dynamics 

over an extended period of time. Here we used in vivo whole-cell voltage-clamp recordings to 

examine the dynamics of receptive field plasticity in primary auditory cortex of adult rats, 

focusing on coordination of synaptic modifications across multiple inputs and stimulus features. 

Neuromodulation is required for receptive field plasticity. We used electrical stimulation of the 

cholinergic nucleus basalis to enable synaptic receptive field modification of auditory cortical 

neurons (Froemke et al., Nature 2007). Basalis stimulation was paired with presentation of a 

sensory stimulus (pure tones of specific intensity and frequency). Before pairing, excitation and

inhibition were balanced. Afterwards, this balance was broken due to increased excitation and 

suppressed inhibition. These changes were specific to the intensity and frequency of the paired 

tone. In parallel, we observed changes to unpaired inputs, including reduced excitation at the 

original best input. Consequentially, receptive fields shifted in the direction of the paired input, 

while the total input was kept roughly constant. 

Suppression at the original best stimuli might be due to competitive Hebbian mechanisms. 

However, suppression at this unpaired input was also induced when low-intensity tones, 

subthreshold for spiking, were paired with basalis stimulation. Therefore, we used 

pharmacological agents and manipulation of activity levels to determine the network elements 

responsible for synaptic receptive field plasticity. 

A cascading sequence of activity- and calcium-dependent mechanisms was engaged to 

persistently shift cortical receptive fields without distending their overall structure. These 

processes actively normalize the net input received by cortical neurons within minutes, 

depending on the statistics of sensory input. This re-balancing of net excitation prevents 

saturation of synaptic strength and corresponding input-output relations, during and after periods 

of heightened attention or perceptual learning. 

Disclosures: R.C. Froemke , None; M.M. Merzenich, None; C.E. Schreiner, None. 

Poster 

239. Structural Plasticity at Synapses I 



Topic: B.08.i. Structural plasticity 

Support: BFU2006-07313/BFI 

BFU2004-00931 

MEC-FPU Grant AP2006-01953 

MEC-FPI Grant BES 2007-15757 

MEC-FPU Grant AP2005-4672 

Title: Dopamine acting through D2 receptors modulates the expression of PSA-NCAM, a 

molecule related to neuronal structural plasticity, in the medial prefrontal cortex of adult rats

Authors: *E. CASTILLO-GOMEZ 1 , M. GOMEZ-CLIMENT 2 , E. VAREA 2 , R. GUIRADO 2 , 

J. BLASCO-IBAÑEZ 2 , C. CRESPO 2 , J. NACHER 2 ; 

1 UNIVERSITAT DE VALÈNCIA, BURJASSOT (VALENCIA), Spain; 2 UNIVERSITAT DE 

VALÈNCIA, Burjassot (Valencia), Spain 

Abstract: A “neuroplastic” hypothesis proposes that changes in neuronal structural plasticity 

may underlie the aetiology of depression and the action of antidepressants. The medial prefrontal 

cortex (mPFC) is affected by this disorder and shows an intense expression of the polysialylated 

form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-associated molecule, which 

is expressed mainly in interneurons. The monoamines serotonin, dopamine and noradrenaline are 

the principal targets of antidepressant action. Pharmacological manipulation of serotonin levels 

regulates synaptophysin and PSA-NCAM expression in the adult mPFC. However, the 

involvement of structural plasticity on the antidepressant effects of dopamine has not been well 

explored yet. Using immunohistochemistry, we have studied the relationship between 

dopaminergic fibers and PSA-NCAM expressing neurons in the mPFC and the expression of D2 

receptors. In order to evaluate the effects of dopamine in neuronal structural plasticity and on 

inhibitory neurotransmission, we have analyzed the expression of synaptophysin, PSA-NCAM 

and GAD67 in the mPFC after cortical dopamine depletion with 6-OHDA and after chronic 

treatments with the D2 receptor antagonist haloperidol or the D2 receptor agonist PPHT. Many 

dopaminergic fibers were observed in close apposition to PSA-NCAM expressing neurons and 

76% of these cells co-expressed D2 receptor. Both haloperidol treatment and 6-OHDA injection 

reduced significantly PSA-NCAM, synaptophysin and GAD67 expression in the mPFC. 

Conversely, PPHT treatment increased the expression of these molecules. Our results give 

support to the “neuroplastic” hypothesis of depression, suggesting that dopamine acting on D2 

receptors may modulate neuronal structural plasticity and inhibitory neurotransmission through 

changes in PSA-NCAM expression. 

Disclosures: E. Castillo-Gomez , None; M. Gomez-Climent, None; E. Varea, None; R. 

Guirado, None; J. Blasco-Ibañez, None; C. Crespo, None; J. Nacher, None. 

Poster 





Support: ASI 

European Community

Italian MIUR 

San Paolo Foundation 

Ministry of Health 

Title: Activity-dependent involvement of GABAergic terminals in the heterologous competition 

for the mature Purkinje cell innervation 

Authors: *R. CESA 1 , L. MORANDO 2 , P. STRATA 1,3,4 ; 

1 Dept Neurosci., Univ. Turin, Turin, Italy; 2 Dept Anat., Univ Turin, Italy; 3 Natl. Inst. of 

Neurosci., Turin, Italy; 4 IRCCS Santa Lucia Fndn., Rome, Italy 

Abstract: Competition among different axons to reach the somatodendritic region of the target 

neuron is an important step during development in order to achieve the final architectural design 

typical of the mature brain. Such a competition is activity-dependent and can occur between 

homologous fibers or between two different neuronal populations (heterologous competition) 

innervating contiguous target territories. Such a heterologous competition has been described in 

the cerebellar cortex between the two main excitatory inputs which innervate the two dendritic 

domains of the Purkinje cell (PC); the parallel fibers (PFs) and the climbing fiber (CF). 

A prolonged block of electrical activity by tetrodotoxin (TTX) or of the ionotropic glutamate 

receptors leads to the appearance in the proximal dendritic region of the PC of a high number of 

new spines mainly innervated by PFs, an input normally restricted to the branchlets. In contrast, 

CFs undergo a morphological modification consisting of a reduced size of their varicosities and a 

loss of synaptic contacts with the PCs. This finding has led to the hypothesis that in the absence 

of activity the PC is intrinsically endowed with cues to be innervated by the PFs. The CFs, in 

order to achieve and maintain their dendritic territory, have to be active, thus displacing the cue 

of the excitatory competitor afferents. 

In addition to the two excitatory inputs, the PC receives also a strong inhibitory GABAergic 

input from both basket and stellate neurons which is distributed along the entire extent of the PC 

somatodendritic region, directly onto the dendritic shaft. Following the prolonged 

intraparenchymal administration of TTX, a new type of synapse appears made by GABAergic 

terminals onto the newly-formed PC spines in the proximal dendrites. The surprising observation 

of the presence of GABAergic terminals on spines which normally receive an excitatory input 

has prompted us to study the inhibitory input distribution both on the dendritic shaft and on the 

spines. Here we show that the density of labelled GABAergic terminals per area of molecular 

layer and per length of PC dendritic membrane is increased after the block of electrical activity 

and that such an expansion takes place only in the PC proximal domain. Thus, the competition 

for the innervation of the PC is not limited to the two excitatory inputs and activity of the CF has 

a fundamental role in the maintenance of the proper synaptic excitatory and inhibitory 

architectural wiring. 

Disclosures: R. Cesa, None; L. Morando, None; P. Strata, None.

Poster 





Support: NIH grant MH 071316 

AHA 

NARSAD 

NAAR 

Title: Autism-associated protein Epac2 promotes synapse destabilization and depression 

Authors: *K. WOOLFREY 1 , H. PHOTOWALA 1 , M. YAMASHITA 2 , M. V. BARBOLINA 3 , 

D. P. SRIVASTAVA 1 , M. E. CAHILL 1 , Z. XIE 1 , K. A. JONES 1 , L. A. QUILLIAM 4 , M. 

PRAKRIYA 2 , P. PENZES 1 ; 

1 Physiol., 2 Mol. Pharmacol. and Biol. Chem., 3 Chem. and Biol. Engin., Northwestern Univ., 

Chicago, IL; 4 Biochem. and Mol. Biol., Indiana Univ. Sch. of Med., Indianapolis, IN 

Abstract: Tight regulation of synapse assembly and structure is essential for cortical 

development and plasticity, and dysregulation of these processes can result in a variety of 

cognitive disorders, including the autism spectrum disorders (ASD). However, much remains 

unknown about the molecular mechanisms underlying synapse development and maturation and 

how they go awry in disease states. Genetic association studies have identified a number of genes 

associated with the autistic phenotype, many of which encode synaptic proteins. We have 

focused on one of these proteins, Epac2, which is a PKA-independent cAMP target and Rapguanine-nucleotide 

exchange factor (GEF). We report that Epac2 activation induces spine 

shrinkage, increases spine motility, reduces synaptic GluR2/3-containing AMPA receptors, and 

depresses excitatory transmission, effects blocked by Epac2 knockdown. Importantly, Epac2 also 

complexes with Neuroligins and Shank3, synaptic proteins previously implicated in the autistic 

phenotype. Collectively, these data identify a novel mechanism that promotes destabilization and 

depression of spiny synapses, malfunctioning of which may contribute to cortical dysfunction in 

ASD. 

Disclosures: K. Woolfrey , None; H. Photowala, None; M. Yamashita, None; M.V. 

Barbolina, None; D.P. Srivastava, None; M.E. Cahill, None; Z. Xie, None; L.A. Quilliam, 

None; K.A. Jones, None; P. Penzes, None; M. Prakriya, None.

Poster 



Program#/Poster#: 239.4/E1 


Support: KAKENHI 17021011 

KAKENHI 20019016 


Title: Acute and coordinated spine reorganization in behaviorally activated neurons 

Authors: *T. KITANISHI, Y. IKEGAYA, N. MATSUKI, M. K. YAMADA; 

Lab. Chem Pharmacol, Grad Sch. Pharm Sci, Univ. Tokyo, Tokyo, Japan 

Abstract: Morphological plasticity of dendritic spines underlies the fine-tuning of network 

connectivity. The relationship between spine plasticity and experience-dependent neuronal 

activities, however, is largely unknown. We simultaneously imaged spine morphology and Arc 

(Arg3.1, an immediate-early gene that serves as a cellular marker of past neuronal activity) 

expression using Thy1-mGFP transgenic mice after exploration in novel environment for either 

15 or 60 min. After the exploring sessions, Arc was detected in 25% of hippocampal CA1 

neurons. We found that, after 60 min of exploration, Arc-positive cells possessed fewer small 

spines and more large spines compared with Arc-negative cells, although these parameters 

showed no difference after 15 min of exploration. Further, the number of extra-large spines 

showed inverse correlation with that of small spines in the 60-min mice. These results provide 

the first evidence of acute (but not immediate) and coordinated morphological changes in spines 

selective in behaviorally activated Arc-expressing neurons. 

Disclosures: T. Kitanishi, None; Y. Ikegaya, None; N. Matsuki, None; M.K. Yamada, None. 

Poster 

239. Structural Plasticity at Synapses I




Title: “Fezzin” family member LAPSER1 takes part in synapto-nuclear translocation in 

hippocampal neurons 

Authors: *M. SCHMEISSER, A. M. GRABRUCKER, T. M. BOECKERS; 

Inst. of Anat., Ulm Univ., Ulm, Germany 

Abstract: The postsynaptic density (PSD), an electron dense structure underneath the 

postsynaptic membrane of excitatory synapses in the central nervous system, forms a protein 

platform composed of various scaffolding molecules, cytoskeletal elements and members of 

neuronal signaling cascades. Recently, we have identified a novel postsynaptic scaffolding 

molecule called LAPSER1 which belongs to the “Fezzin” familiy of PSD proteins and interacts 

with the PDZ domain of ProSAP/Shank. By in-vitro and in-vivo analysis, we identified a PSD 

protein complex that includes LAPSER1 and two of its interaction partners: spine-morphology 

regulator SPAR1 and Wnt-pathway member beta-Catenin. Interestingly, LAPSER1 and beta- 

Catenin translocate into the nucleus selectively upon NMDA application while SPAR1 stays at 

the synapse. 

Taken together, our findings show that “Fezzin” family member LAPSER1 has diverse functions 

within the neuron. It creates a scaffold within the PSD by homo- and heteromeric interaction 

with other Fezzin family members and might serve as an activity-dependent transport molecule 

to the nucleus. 

Disclosures: M. Schmeisser , None; A.M. Grabrucker, None; T.M. Boeckers, None. 

Poster 





Support: German Isreali Foundation (827/04) 

Deutsche Forschungsgemeinschaft (DE 551/8-1)

Israel Science Foundation (768/06) 

Title: Synaptopodin regulates plasticity in dendritic spines of cultured hippocampal neurons 

Authors: A. VLACHOS 1,2 , E. KORKOTIAN 1 , E. SCHONFELD 1 , T. DELLER 2 , *M. SEGAL 1 ; 

1 Dept Neurobiol, The Weizmann Inst., Rehovot 76100, Israel; 2 Goethe-Univ., Frankfurt 60590, 

Germany 

Abstract: Synaptopodin (SP), an actin-binding protein, is tightly associated with the spine 

apparatus of mature cortical neurons. SP has been suggested to play a role in synaptic plasticity, 

but it has not yet been linked mechanistically to synaptic functions. We studied endogenous and 

transfected SP in dendritic spines of cultured hippocampal neurons and found that spines 

containing SP (SP(+)) generated larger current responses to flash photolysis of caged glutamate 

than adjacent SP-negative ones in the same neurons. An NMDA-receptor mediated chemical 

LTP caused the delivery of GluR1-GFP into spine heads of control but not of neurons that were 

transfected with a specific shRNA to SP. Calcium stores are linked to SP, since the 

pharmacological blockade of ryanodine-sensitive stores eliminated the SP(+) enhancement of 

glutamate responses. Finally, caffeine-induced release of calcium from stores produced an SPdependent 

delivery of GluR1 into spines, which was not seen in shRNA-SP transfected neurons. 

Thus, SP plays a crucial role in the calcium store-associated ability of cultured neurons to 

undergo long-term plasticity. 

Disclosures: A. Vlachos, None; E. Korkotian, None; E. schonfeld, None; T. Deller, None; M. 

Segal , None. 

Poster 





Title: Estrous cycle-induced plasticity in the caudal brainstem in the female golden hamster 

Authors: *P. O. GERRITS 1 , H. DE WEERD 1 , R. KORTEKAAS 1 , A. ALGRA 1 , L. J. 

MOUTON 2 , H. J. J. L. VAN DER WANT 3 , J. G. VEENING 4,5 ; 

1 Dept Anat, Univ. Med. Ctr. Groningen, 9700 AD Groningen, Netherlands; 2 Dept. of Human 

Movement Sci., 3 Dept. of Cell Biology, Mol. Imaging and Electron Microscopy, Univ. Med. 

Ctr., Groningen, Netherlands; 4 Anat., Univ. Med. Ctr. St Radboud, Nijmegen, Netherlands; 

5 Dept of Psychopharmacology, UIPS, Utrecht, Netherlands

Abstract: During the short four day estrous cycle of the female hamster various behavioral 

(lordosis, vocalization and aggression) and autonomic adaptations occur. Presumably, these 

changes are under ovarial hormonal control. Recently, we described a distinct estrogen receptorα 

immunoreactive (ER-α-IR) cell group, now called nucleus para-retroambiguus (NPRA), in the 

caudal ventrolateral medulla. Neurons of this group project to the ipsilateral intermediolateral 

cell column in the thoracic and upper lumbar cord. Clearly, the NPRA is part of an estrogensensitive 

neuronal network and the same applies to the region containing the commissural part of 

the solitary tract nucleus (NTScom) and the A2 group, here called NTScom/A2. 

Estrogen is known to modulate neuronal ultrastructure in various brain areas and spinal cord, but 

not in the caudal brainstem. Because we assumed that the NPRA plays a role in estrous cycle 

related adaptations, we hypothesized the occurrence of plasticity in this nucleus. 

In the present study we examined morphological changes of axo-dendritic relationships in NPRA 

and NTScom/A2 in estrous, diestrous and ovariectomized (OVX) hamsters, using immunoelectron 

microscopy and the 1D5 anti-ER-α antibody. 

Ultrastructural analysis revealed that the ratio ”axon terminals surface/dendrite surface” was 

significantly increased in both the NPRA and NTScom/A2 during the estrous phase compared to 

the OVX and diestrous conditions. Enlargement of the axon terminal fields contacting more 

dendrites was the main cause for the “axonal terminal-dendritic-ratio” shift. 

Estrous cycle-induced plasticity is present in the NPRA, and in the NTScom/A2 group. Our 

findings support our hypothesis that estrogen-sensitive neuronal networks in the caudal 

brainstem display structural plasticity, probably to modulate steroid hormone dependent 

behaviors or autonomic adaptations. 

Disclosures: P.O. Gerrits , None; H. de Weerd, None; R. Kortekaas, None; A. Algra, 

None; L.J. Mouton, None; H.J.J.L. Van der Want, None; J.G. Veening, None. 

Poster 





Title: A role for ProSAP/Shank protein SAM domains in dynamic processes at postsynaptic 

densities of excitatory synapses 

Authors: *A. M. GRABRUCKER 1 , B. VAIDA 1 , A. DOLNIK 1 , C. PROEPPER 1 , M. R. 

KREUTZ 2 , E. GUNDELFINGER 2 , T. M. BOECKERS 1 ; 

1 Inst. of Anat., Univ. Ulm, Ulm, Germany; 2 Leibniz Inst. for Neurobio., Magdeburg, Germany

Abstract: Chemical synapses serve as specialized contact sites between neurons and enable 

communication within the central nervous system. 

The three members of mammalian ProSAP/Shank proteins serve as major scaffolding proteins 

within postsynaptic densities (PSDs) of excitatory synapses. Via the C-terminal SAM domain, 

ProSAP1/Shank2 and ProSAP2/Shank3 are able to build large platforms within the PSD. 

Recently it was shown that Zn2+ is incorporated into these platforms. We investigated 

ProSAP/Shank platforms and the localization of interacting proteins including Homer1, PSD95, 

and NMDA receptors in hippocampal culture neurons under different experimental conditions. 

ProSAP/Shanks are recruited to the PSD in a distinct time depending manner during 

synaptogenesis. The localization of Zn2+ and ProSAP/Shanks was investigated in HeLa cells 

and hippocampal neurons. Staining of hippocampal neurons with Zinquin and Newport Green 

showed Zn2+ enrichments colocalizing with ProSAP1/Shank2 and ProSAP2/Shank3. 

Fluorescence immunocytochemistry with antibodies directed against synaptic proteins was used 

to visualize synaptic contacts in wild type conditions and under treatment with ZnCl2 or zincchelators. 

Electron microscopy was used to investigate the ultra-structure of these synapses. 

Local, dynamic changes of PSD proteins within spines and synapses give new insights into 

additional mechanisms and pathways involved in “postsynaptic plasticity” that may be a crucial 

part of learning and memory formation. Our results indicate a role of Zn2+ ions at PSDs and 

reveal ProSAP/Shank family members as important components for the structural integrity of 

PSDs at excitatory synapses with an individual functional importance. 

Disclosures: A.M. Grabrucker , None; B. Vaida, None; A. Dolnik, None; C. Proepper, 

None; M.R. Kreutz, None; E. Gundelfinger, None; T.M. Boeckers, None. 

Poster 





Support: NARSAD 

AHA 

NIH-NIMH (MH071316) 

NAAR 

NIH-NINDS (NS44322)

Title: Rapid enhancement of two-step wiring plasticity by Estrogen and NMDA receptor activity 

Authors: *D. P. SRIVASTAVA 1 , K. WOOLFREY 1 , C. Y. SHUM 1 , K. A. JONES 1 , L. L. 

LASH 2 , G. T. SWANSON 2 , P. PENZES 1 ; 

1 Dept Physiol, 2 Dept Pharm, Northwestern Univ., Chicago, IL 

Abstract: Cortical information storage is achieved through both changes in neuronal 

connectivity and in synaptic strength. However, the cellular and molecular underpinnings of 

these types of plasticity remain unclear. In vivo studies have demonstrated that acute estrogen 

(E2) treatment enhances cortically based memory and recent evidence suggests that E2 rescues 

cortical memory deficits induced by ischemia. Given that E2 can be rapidly synthesized in the 

adult mammalian cortex, we examined the effects of acute E2 (< 1 hr) on spine morphogenesis, 

AMPAR trafficking and GTPase signaling in cortical neurons. 

We have found that acute E2 treatment results in a rapid, transient increase in spine density, 

accompanied by temporary formation of silent synapses through reduced surface-GluR1. These 

rapid effects of E2 are dependent on the small GTPase Rap and its downstream signaling targets 

AF-6 and ERK1/2, as revealed by selective inhibition of these components. Further, we have 

demonstrated that E2-mediated elevation in spine density was sustained for up to 24 hours by 

subsequent NMDAR activation. Additionally, E2-induced silent synapses were potentiated by 

NMDAR activation via synaptic AMPAR insertion. Together, the data describe the molecular 

mechanisms underlying a form of two-step wiring plasticity. Step one of the model is composed 

of an E2-induced temporary increase in dynamic dendritic spines that sample the surrounding 

neuropil for presynaptic contacts, forming silent synapses. In step two, a subsequent NMDARdependent 

input stabilizes these transient spines and potentiates them by delivering AMPARs to 

the synapse. Collectively, these data identify E2 and NMDAR signaling as mediators of a novel 

form of two-step wiring plasticity and suggest new molecular targets for aiding in recovery from 

brain injury via stimulation of cortical rewiring. 

Disclosures: D.P. Srivastava , None; K. Woolfrey, None; C.Y. Shum, None; K.A. Jones, 

None; L.L. Lash, None; G.T. Swanson, None; P. Penzes, None. 

Poster 





Support: NIH grants EY018119 and EY18082-01A2 (Kirschstein-NRSA)

Howard Hughes Medical Institute 

Title: RNAi suppression of BDNF expression inhibits cortical plasticity 

Authors: *J. T. PENA 1,3 , H. YAMAHACHI 2 , S. MARIK 2 , T. TUSCHL 2,4 , C. D. GILBERT 2 ; 

1 Lab. of Neurobio., 2 Rockefeller Univ., New York City, NY; 3 Weill-Cornell Med. Col., New 

York, NY; 4 Howard Hughes Med. Inst., New York, NY 

Abstract: During the course of adult cortical plasticity, a number of signal transduction 

mechanisms are brought into play. To study regulatory genes implicated in this process, we 

inhibited gene expression by harnessing the machinery of RNA interference (RNAi) via small 

hairpin RNAs (shRNA) delivered by viral vectors. Using this technology we sought to influence 

plasticity of the mouse vibrissal barrel cortex. In this model system, chronic whisker plucking 

reliably leads to the expansion of the cortical representation of the adjacent non-deprived 

whiskers. The mechanism underlying this process involves changes in synaptic efficacy and 

sprouting of axon collaterals. Initial studies describing the molecular events leading to 

synaptogenesis and remapping have implicated a number of signal transduction pathways, 

including neurotrophins such as brain derived neurotrophic factor (BDNF). Though previous 

experiments have shown upregulation of neurotrophins and their receptors in reorganized cortex, 

it is necessary to remove these factors to definitively prove their involvement in adult cortical 

plasticity. We therefore repressed neurotrophic gene expression in the mouse somatosensory 

cortex to determine if these trophic factors are essential for cortical remodeling after sensory 

deprivation. We blocked gene expression in vivo using non-replicative adeno-associated virus 

bearing genes encoding shRNA constructs. The shRNA nucleotide sequences were designed to 

target and destroy selected neurotrophin messenger RNAs by triggering the RNAi pathway, 

resulting in a reduction of BDNF protein levels by up to 80% in vivo. This knockdown of BDNF 

expression effectively repressed functional cortical reorganization induced by chronic whisker 

plucking. Manipulating gene expression via RNAi is therefore a useful approach towards 

establishing the molecular link between sensory experience and use-dependent changes in 

cortical circuits. 

Disclosures: J.T. Pena, None; H. Yamahachi, None; S. Marik, None; T. Tuschl, alnylam, F. 

Consultant/Advisory Board; C.D. Gilbert, None. 

Poster 




Topic: B.08.i. Structural plasticity

Support: NIH Grant MH 071316 

Title: Neuregulin-1 (NRG-1) enhances the structure and function of dendritic spines via kalirin-7 

activation 

Authors: *M. E. CAHILL 1 , Z. XIE 2 , P. PENZES 2 ; 

2 Physiol., 1 Northwestern Univ., Chicago, IL 

Abstract: Neuregulin-1 (NRG-1) is a trophic factor that exists in a soluble form and binds to the 

extracellular domain of ErbB tyrosine kinase receptors. ErbB4 is thought to be the predominant 

ErbB receptor isoform for NRG-1, and both ErbB4 and NRG-1 show genetic linkages to 

schizophrenia. Recently, NRG-1/ErbB4 signaling was shown to promote the maturation of 

dendritic spines, however, the link between ErbB4 receptor activation and synaptic structural and 

functional plasticity remain unknown. We show that the NRG-1/ErbB4 signaling complex is an 

upstream regulator of kalirin-7 function. Kalirin-7 is a RacGEF that is highly enriched in the 

postsynaptic densities of forebrain excitatory synapses where it is crucial for both the basal 

maintenance and activity-dependent plasticity of dendritic spine structure and function. We show 

that kalirin-7 and ErbB4 form a complex in cortical neurons and that NRG-1 causes the 

phosphorylation and subsequent activation of kalirin-7. Consequently, we show that NRG-1 

enhances the activation of the kalirin-7 effector molecule Rac1 whose activation is critical for 

inducing actin polymerization in spines. Finally, using kalirin-7 knockdown in cortical neurons, 

we show that the activation of kalirin-7 via NRG-1 is necessary for NRG-1 mediated 

enhancements in both spine structure and function. 

Disclosures: M.E. Cahill , None; Z. Xie, None; P. Penzes, None. 

Poster 





Title: Possible relation between the peak concentration of calcium ion in a spine and 

morphological plasticity 

Authors: *K. ICHIKAWA, K. NOZAWA; 

Col. of Biosci. Chem., Kanazawa Inst. of Technolo, Hakusan, Japan

Abstract: Recently structural plasticity of a spine, which is a change in the spine morphology by 

the stimulation of a synapse, has been reported from many laboratories. Some reports indicated 

the role of actin molecules in the structural plasticity, and the change in F-actin structure will 

play a pivotal role in the morphological change of a spine. Structure of F-actin is controlled by 

complex mechanisms including small GTPases, kinases, phosphatases and many kinds of actin 

binding proteins. In addition to these, Ca 2+ is also implicated to be involved in the structural 

plasticity. It is not known, however, how the increase in intracellular Ca 2+ concentration ([Ca 2+ ]i) 

is linked to the activation/inactivation of these proteins. In addition, it is also not known how 

[Ca 2+ ]i is changed by the change in spine morphology. 

Here we simulated the change in [Ca 2+ ]i in a 3D spine morphology to see the relation between 

spine morphology and [Ca 2+ ]i. The model is simple consisting of Ca 2+ entry from the spine head, 

Ca 2+ buffers, extrusion from the plasma membrane and diffusion within a model spine. The 

simulation results showed that the peak [Ca 2+ ]i was increased as the length of filopodium was 

increased as was expected. However, this increase was saturated and further increase was not 

seen at filopodium length longer than 1κm. When the diameter of spine head was increased, the 

peak [Ca 2+ ]i was decreased as was expected. However, the decrease stopped at spine head 

diameter larger than 0.4κm. From these simulation results, we proposed a model for the 

morphological change of a spine by the change in the peak [Ca 2+ ]i. This change will lead to the 

structural change in F-actin. 

Disclosures: K. Ichikawa , None; K. Nozawa, None. 

Poster 





Support: CIHR 

MUHC 

Title: Regulation of dendritic spine morphology through slingshot phosphatase signalling 

Authors: *L. ZHOU, E. JONES, M. HABER, K. MURAI; 

Ctr. for Res. in Neurosci., McGill Univ., Montreal, QC, Canada 

Abstract: Dendritic spines are small protrusions from the shaft of neuronal dendrites that are the 

primary sites of excitatory glutamatergic synapses in the brain. Although spines very in shape,

they generally have an enlarged head portion that is connected to the dendritic shaft through a 

narrow neck. This architecture enables spines to confine calcium ions and signalling events at 

synapses. The structural plasticity of spines is believed to help regulate synaptic efficacy and is 

dependent on actin filament remodelling. However, the signalling mechanisms that modulate 

actin filament organization in spines remain to be fully described. Here, we present data 

implicating the Slingshot 1L (SSH1L) phosphatase in controlling dendritic spine morphology. 

SSH1L is known to dephosphorylate cofilin, an actin depolymerising/severing protein that 

regulates actin filament dynamics and spine plasticity upon activity stimulation. We find that 

SSH1L expression peaks during the first postnatal week of the mouse hippocampus, and 

gradually declines toward adult stages. Interestingly, disruption of normal SSH1L signalling by 

expression a phosphatase-inactive SSH1L in CA1 pyramidal neurons in organotypic 

hippocampal slices cultures significantly alters dendritic spine morphology. The average length 

of the head portion of spines is significantly increased while the width is decreased, resulting in a 

larger percentage of elongated spines. Immunostaining for over-expressed wild type and 

phosphatase-inactive SSH1L proteins in CA1 cells shows that the wild type form is concentrated 

in the spine head region while the phosphatase-inactive form is enriched in the spine neck, with 

weak expression at the tip of spine head. Our results suggest that SSH1L helps to control actin 

cytoskeletal rearrangements that are important for regulating dendritic spine morphology. 

Disclosures: L. Zhou, None; E. Jones, None; M. Haber, None; K. Murai, None. 

Poster 





Support: CIHR 

Canadian Stroke Network 

MSFHR 

Title: Imaging the emergence of new structural and functional circuits in the somatosensory 

cortex of adult mice recovering from stroke in vivo 

Authors: *C. E. BROWN 1 , T. H. MURPHY 2 ; 

1 Dept Psychiatry, Univ. British Columbia, Vancouver, BC, Canada; 2 Psychiatry, Univ. of British 

Columbia, Vancouver, BC, Canada

Abstract: Previous work from our lab has shown that a new cortical forelimb (FL) 

representation emerges in peri-infarct motor (M1) and hindlimb (HL) cortex 2-3 months after 

unilateral focal ischemic stroke. Although these results clearly show extensive functional remapping 

in a stroke-recovered mouse, we still do not know how quickly these functional 

changes in cortical responsiveness occur after stroke; nor do we know how well functional remapping 

correlates temporally, with behavioural recovery of forelimb use. To this end, we 

utilized voltage sensitive dye imaging in vivo to examine forelimb-evoked cortical responses in 

adult mice at 1 week, 2-3 or 12 months after FL cortex stroke. Our preliminary analysis indicated 

that 1 week after stroke when behavioural deficits in forelimb use are most prominent, forelimbevoked 

responses were virtually absent in the contralateral M1/HL regions, with faint, but 

detectable responses in the surviving piece of FL cortex. Of note, neuronal responses in the periinfarct 

M1/HL cortex could still be evoked by hindlimb stimulation, suggesting that peri-infarct 

cortex was indeed functionally responsive. However after 2-3 or 12 months recovery, forelimbevoked 

responses first appeared in the surviving remnant of FL cortex which propagated to, and 

led to robust, sustained responses within peri-infarct M1/HL cortical areas (3-4 fold increase in 

response duration). Collectively, these results indicate that much of the functional re-mapping of 

the FL cortex occurs within the first 2-3 months after stroke, and is characterized by an initial 

loss of cortical responsiveness to the impaired forelimb which gradually recovers, and reemerges 

in peri-infarct M1/HL cortex. Currently, we are employing in vivo two-photon imaging 

to investigate what structural modifications (eg changes in intracortical or thalamocortical 

connectivity) might form the basis for the progressive recovery/re-mapping of the FL 

representation. 

Disclosures: C.E. Brown , None; T.H. Murphy, None. 

Poster 





Support: NIDA 5P01DA021633-02 

Office of Naval Research N00014-02-0879 

NIDA R37DA04294 

Title: Basal differences in spine density in a selectively bred population of rats with a 

predisposition for addictive behavior

Authors: J. M. WANG 1 , S. B. FLAGEL 1 , J. JEDYNAK 2 , S. M. CLINTON 1 , T. E. 

ROBINSON 3 , *S. J. WATSON 4 , H. AKIL 1 ; 

1 Molec Behav Neurosci Inst., 2 Neurosci. Program, 3 Psychology, Univ. Michigan, Ann Arbor, 

MI; 4 Univ. Michigan Sch. Med., Ann Arbor, MI 

Abstract: Some individuals are able to casually use drugs without ever becoming addicted, 

whereas others become hooked upon first exposure to the drug. The predisposing factors that 

render some individuals susceptible to addiction and protect others from developing the disorder 

remains to be determined. We have utilized a population of rats selectively bred on the basis of a 

novelty-seeking trait and known to differ on a number of other behaviors relevant to addiction to 

examine basal differences in neuronal morphology. High-responder (HR) bred rats are those that 

exhibit increased locomotor response in a novel environment (the trait they were selected for), 

greater propensity for psychomotor sensitization to cocaine, and increased acquisition of drugtaking 

behavior relative to their low-responder (LR) counterparts. HR-bred rats are also more 

impulsive and more aggressive compared to LR-bred rats. These two lines of rats also exhibit 

differences in stress responsiveness (e.g. corticosterone levels) and in basal gene expression in 

the hippocampus (e.g. glucocorticoid receptor mRNA) and striatum (dopamine D2 receptor 

mRNA). Thus, we used these selectively bred rats to examine differences in basal morphology in 

the hippocampus (HC) and nucleus accumbens (NAc), brain regions that have been implicated in 

their behavioral traits. Brains were obtained in the “basal state” from adult HR and LR rats from 

the 15 th generation of our selective breeding colony. 125 µm thick coronal sections were 

collected from the prefrontal cortex, NAc and HC using a vibratome. Sections were processed 

for ballistic labeling of neurons with the lipophilic dye, DiI. The dye was delivered using a “gene 

gun” which allowed for rapid visualization of dendrites and dendritic spines. Images of medium 

spiny neurons (MSNs) in the core of the NAc and of pyramidal neurons in the CA1 subregion of 

the HC were captured using a confocal microscope and spine density was analyzed using 

Neurolucida ® software. To date, we have found no significant HR/LR differences in basal spine 

density of MSNs in the NAc core or on basilar branches of pyramidal neurons in the CA1 

subregion of the HC. However, LR rats exhibit greater spine density on apical dendrites in the 

CA1 subregion of the HC relative to HR rats. Ongoing studies are examining basal differences in 

other brain regions of relevance to substance abuse. These findings are, to our knowledge, the 

first report of basal differences in spine morphology associated with differences in substance 

abuse vulnerability and represent an important prelude to examining the impact of drugs (e.g. 

cocaine) on this morphology. 

Disclosures: J.M. Wang, None; S.B. Flagel, None; J. Jedynak, None; S.M. Clinton, 

None; T.E. Robinson, None; S.J. Watson , None; H. Akil, None. 

Poster 





Support: NIH/NIMH 

NARSAD 

Title: Role of the serotonin 2a receptor in cortical dendritic spine morphogenesis 

Authors: *K. A. JONES 1 , D. P. SRIVASTAVA 1 , B. L. ROTH 2 , P. PENZES 1 ; 

1 Physiol., Northwestern Univ., Chicago, IL; 2 Pharmacol., Univ. of North Carolina at Chapel Hill, 

Chapel Hill, NC 

Abstract: Structural plasticity of excitatory synapses on cortical pyramidal neurons is a crucial 

determinant of cognition and behavior. Many neuropsychiatric disorders are associated with 

aberrant dendritic spine morphogenesis, but the molecular mechanisms that link synaptic 

structural plasticity and neuropsychiatric disorders remain unclear. The 5-HT2a receptor (2AR) 

is prominently expressed in the mammalian cortex and has been genetically and 

pharmacologically implicated in several psychiatric disorders, including schizophrenia and 

depression - diseases in which dendritic spine morphology in the cortex is altered. However, the 

precise role of the 2AR in the structural changes that accompany these disorders are not well 

understood. Here we investigate the role of the 2AR in dendritic spines in cultured cortical 

neurons. We demonstrate that the 2a receptor forms a complex with the PDZ protein Mupp1 and 

the RacGEF kalirin-7 in cortical dendritic spines. We also examine the relationship between 2AR 

signaling and activation of downstream effectors of actin reorganization that lead to dendritic 

spine morphogenesis. These data may provide an important link between serotonin signaling and 

dendritic spine morphogenesis in the cortex, a process that may contribute to the pathogenesis of 

neuropsychiatric disorders. 

Disclosures: K.A. Jones , None; D.P. Srivastava, None; B.L. Roth, None; P. Penzes, None. 

Poster 





Support: NEI grant EY018119

Max-Planck Society 

The Rockefeller University Women & Science Fellowship 

Title: Axonal sprouting of horizontal connections in adult mouse somatosensory cortex follows 

whisker trimming 

Authors: *S. A. MARIK 1 , H. YAMAHACHI 1 , W. DENK 2 , C. D. GILBERT 1 ; 

1 Lab. of Neurobio., The Rockefeller Univ., New York, NY; 2 Dept. of Biomed. Optics, Max- 

Planck Inst. for Med. Res., Heidelberg, Germany 

Abstract: Neural circuits in the adult brain can be rewired to recover function that is lost after 

stroke, sensory deprivation or neurodegenerative diseases. Evidence for this rewiring can be seen 

at the functional level through changes in receptive fields and at the anatomical level by 

synaptogenesis following sensory loss. Layer II/III cortical neurons are key players in cortical 

reorganization since their long range horizontal axons allow for communication between 

topographically distant cortical regions, and they are responsible for an increase in axonal 

density in response to sensory deprivation. To determine the circuit mechanisms underlying map 

reorganization in the mouse somatosensory cortex, we applied a technique that allowed us to 

explore the dynamics of axons and boutons in vivo. Specifically, we employed the use of viral 

vectors that were genetically engineered with the gene encoding YFP, which allowed us to 

fluorescently label neurons located in specific barrels. The structural dynamics of labeled 

neuronal processes were observed with two-photon imaging. We used the adult mouse 

somatosensory cortex as a model to examine the structural changes of axons undergoing cortical 

reorganization. Chronic whisker plucking reliably induces an expansion of the cortical 

representation of adjacent whiskers. The receptive fields of the barrel cortex were mapped 

electrophysiologically, and inputs from horizontal connections in the superficial cortical layers of 

the deprived whisker barrel were labeled by injecting the virus into the adjacent, non-deprived 

barrels. After full expression of the virus, the same labeled axons were imaged before whisker 

plucking and at varying times following the initiation of chronic whisker plucking. Over a period 

of weeks, there was a progressive sprouting of new axon collaterals into the deprived whisker 

barrels. At the same time, other parts of the original axonal arbors projecting to this region were 

pruned. Overall there was a net increase in axonal density from the non-deprived whisker barrel 

into the deprived barrels. The functional remapping of the whisker barrel system is likely due to 

this change in the plexus of horizontal connections. 

Disclosures: S.A. Marik, None; H. Yamahachi, None; W. Denk, None; C.D. Gilbert, None. 

Poster 





Support: Hjärnfonden 

M & A Wallenbergs Minnesfond 

Karolinska Institutet 

M & M Wallenbergs Stiftelse 

Swedish Research Counsil 

Title: A study of synaptic band pattern and density in normal and re-innervated hind limb 

muscles of mice lacking MHC class I 

Authors: *S. J. THAMS, S. CULLHEIM; 

Neurosci, Karolinska Inst., Stockholm, Sweden 

Abstract: The major histocompatibility complex class I (MHC-I) plays an important role in 

synaptic plasticity, particularly in the pruning of redundant synapses during development. One 

suggested function for post-synaptic MHC-I is the determination of stabilization vs. repulsion of 

pre-synaptic terminals, through interaction with a pre-synaptic receptor. 

MHC-I is expressed by subpopulations of neurons during different stages of development or 

activity and the expression pattern has been linked to brain regions with high synaptic plasticity. 

Using in situ hybridization we here demonstrate that adult spinal motoneurons strongly upregulate 

two classical MHC-I α-chain mRNAs in response to axonal injury. During this phase 

synapses are eliminated from the motoneuron surface by an unknown mechanism. In mice 

lacking functional MHC-I, more synapses are eliminated and the selectivity of the synaptic 

stripping is abolished, as previously shown. In addition, motoneurons from MHC-I mutants 

regenerate their axons poorly in comparison with wild type mice (Wt). 

In this study, we show that MHC-I protein is transported down the axon in both normal and 

uninjured motoneurons, indicating importance for axonal elongation and regeneration. In 

accordance with the previous finding of a central synaptic elimination defect in MHC-I mutants, 

we explored whether this is also the case at peripheral synapses in the muscle. We therefore 

studied the synaptic band pattern in normal hindlimb muscles from Wt mice and MHC-I mutants 

using confocal microscopy for α-bungarotoxin and synaptophysin. To study formation of new 

synapses we then analyzed re-innervated hind limb muscles 30 and 45 days after sciatic nerve 

crush in different MHC-I mutant mouse strains. At the first time-point the muscles can be 

expected to be hyper-innervated and at the later time point a substantial part of the normal 

synaptic pruning could be expected to have occurred. At 45 days postoperatively, mice lacking 

MHC-I display a broadening of the reformed synaptic bands innervating the gastrocnemius 

muscles, indicating a higher density of synapses and a disrupted organization of the innervation 

pattern. Reformation and subsequent pruning of neuromuscular synapses is a critical step in the

motor recovery of re-innervated muscles and we here provide an indication that MHC-I is 

important for this process. 

Disclosures: S.J. Thams , None; S. Cullheim, None. 

Poster 





Title: Oxidation-state dependent interconnections between dendritic spines provide early 

evidence in support of semblance hypothesis of memory 

Authors: *K. I. VADAKKAN 1,2 ; 

1 Neurosci., Neurosearch Ctr., Toronto, ON, Canada; 2 Buffer Labs., Calicut, India 

Abstract: Synaptic plasticity changes are critical at the stage of encoding of various types of 

memories. These changes leading to retrieval-efficient mechanisms are still lacking. Here, 

derivation of semblance hypothesis and evidence for functional interconnections between 

dendritic spines that support the hypothesis are explained. During one learning event, a specific 

set of postsynaptic membranes along different orders of neurons from the learned item are 

activated. It can be assumed that artificial activation of this specific set of postsynaptic 

membranes will induce memory for the learned event. Deriving from this, memory depends on 

the subset of postsynaptic membranes that are activated during retrieval out of the set of 

postsynapses that were activated during learning. For this, co-activation of fibers from the 

learned item and the cue stimulus during learning need to induce specific changes that will later 

allow the cue stimulus alone evoke activation of the set of postsynaptic membranes that represent 

the learned item. This entailed the generation of semblance hypothesis. Associative memory, in 

the presence of an internal or external cue stimulus, results from the ability to induce specific 

postsynaptic activation at the synapses of neurons from the learned item without the requirement 

of action potentials (APs) reaching their presynaptic sides. In the presence of a cue stimulus, the 

patterns of synapses named as shared extracellular matrices and shared postsynapses can evoke 

cellular illusion of an AP-induced synaptic transmission from presynaptic terminals belonging to 

the learned item. To examine the presence of oxidation-state-dependent functional 

interconnections between dendritic spines, a change in the pre-Golgi stain oxidation state was 

induced by allowing chemicals to reach towards presynaptic and postsynaptic terminals from 

outside the synapse through the capillaries. Cardiac perfusion of a reducing agent followed by 

Golgi staining showed a contrasting view of the presynaptic terminals on the dendritic spines.

Oxidation-state-dependent interconnections between spines were visible at the dendritic 

terminals in the cortex (8 ± 5.6; n = 113 clusters). In the CA3 region of the hippocampus, these 

clusters were very large (482 ± 106; n = 106 clusters). These interconnections due to oxidationstate 

dependent progression of chemical reaction between the dendritic spines open possibilities 

to examine similar oxygenation-state-dependent transient functional connections between the 

dendritic spines of adjacent neurons at sites of increased signal intensity seen in fMRI both 

during learning and memory retrieval. 

Disclosures: K.I. Vadakkan, None. 

Poster 





Support: The DANA Foundation 

Title: Ephrin-A2 is required for stabilization of dendritic spines in the developing mouse 

somatosensory cortex 

Authors: X. YU, T. XU, V. T. CHEW, *Y. ZUO; 

Mol. Cell and Devel Biol, UC Santa Cruz, Santa Cruz, CA 

Abstract: Tyrosine kinase EphAs and their ligand ephrin-As play important roles in axon 

guidance and topographic map formation during brain development. However, their roles in 

synapse formation and activity-dependent plasticity remain unclear. Using transcranial twophoton 

microscopy, we repeatedly imaged postsynaptic dendritic spines in barrel cortex of 

transgenic mice expressing Yellow Fluorescent Protein. Here we show that adolescent ephrin-A2 

knockout (KO) mice have significantly higher (>40%) spine elimination over short periods of 

time (2, 4 and 8 days) than littermate controls (P

Poster 





Support: NIH 

AFA 

Swedish Brain Foundation 

Swedish Research Council 

Loo and Hans Ostermans foundation for geriatric research 

Title: Consequences of Nogo receptor 1 over-expression in forebrain 

Authors: *T. K. KARLSSON 1 , A. KARLEN 2 , A. MATTSSON 2 , K. LUNDSTROMMER 2 , K. 

PERNOLD 2 , S. BRENE 3 , A. JOSEPHSON 2 , L. OLSON 2 ; 

1 Neurosci., Stockholm, Sweden; 2 Neurosci., 3 Dept. of Neurobiology, Caring Sci. and Society, 

Karolinska Institutet, Stockholm, Sweden 

Abstract: Interaction of myelin-associated inhibitors (Nogo, Mag and OMgp) with the Nogo 

receptor 1 (NgR1) activates the RhoA pathway, thereby causing growth cone collapse. We have 

previously demonstrated specific down-regulation of NgR1 mRNA in gray matter in response to 

increased neuronal activity. We hypothesize that this transient NgR down-regulation is critical 

for plasticity and formation of long-term memories. We have generated transgenic mouse lines 

with inducible over-expression of NgR in forebrain neurons. Observations from behavioral tests 

(passive avoidance, water maze) suggest that inability to down-regulate NgR in forebrain gray 

matter impairs the formation of very long-term memories. Here we investigate the consequences 

of NgR1 over-expression in forebrain in regards to synaptogenesis. Increased RhoA signaling 

has been shown to reduce the dendritic tree. Because RhoA activation is a downstream event of 

NgR1 activation, we hypothesize that dendritic tree complexity and spine density may be 

reduced in NgR1 over-expressing mice. To test this hypothesis we are currently investigating 

dendritic morphology using Golgi staining. Ongoing work also addresses possible changes of the 

expression of synaptic proteins in NgR1 over-expressing mice.

Disclosures: T.K. Karlsson , None; A. Karlen, None; A. Mattsson, None; K. Lundstrommer, 

None; K. Pernold, None; S. Brene, None; A. Josephson, None; L. Olson, None. 

Poster 





Support: NIH 

Loo and Hans Ostermans foundation for geriatric research 

Swedish Brain Foundation 

AFA 


Title: Nogo receptor 1 regulates formation of lasting memories 

Authors: *A. KARLEN 1 , T. E. KARLSSON 1 , A. MATTSSON 1 , K. LUNDSTOMER 1 , K. 

PERNOLD 1 , T. PHAM 2 , C. M. BACKMAN 3 , S. O. OGREN 1 , A. F. HOFFMAN 3 , M. A. 

SHERLING 3 , C. R. LUPICA 3 , B. J. HOFFER 3 , C. SPENGER 1 , A. JOSEPHSON 1 , S. BRENE 2 , 

L. OLSON 1 ; 

1 Dept Neurosci., Karolinska Inst., Stockholm, Sweden; 2 Dept. of Neurobiology, Caring Sci. and 

Society, Karolinska Institutet, Stockholm, Sweden; 3 NIDA/NIH, Baltimore, MD 

Abstract: Formation of lasting memories requires structural changes at the synaptic level. We 

had found that neuronal activity down-regulates Nogo receptor-1 (NgR1), and postulated this to 

be a key component of the plasticity required for formation of lasting memories. To test this, we 

developed mice with inducible overexpression of NgR1 in forebrain neurons. While these mice 

have normal long term potentiation and ability to remember 24 hours, their ability to remember 4 

weeks is severely impaired (open-field, passive avoidance and swim maze tests). Blocking 

transgene expression normalizes formation of lasting memories. Transcription of Nogo, Lingo-1, 

Troy, endogenous NgR1 and BDNF were not changed, suggesting the impaired ability to form 

lasting memories was directly coupled to the inability to down-regulate NgR1. Ongoing work 

addresses the possible effects of NgR1 on cell proliferation and/or survival in the dentate gyrus, 

as well as possible additional behavior endophenotypes. Our findings provide experimental

evidence that the Nogo signaling system plays a role in controlling synaptic plasticity in the 

brain and the complex process of transforming transiently stored new information into very long 

term memories. Understanding the molecular underpinnings of the synaptic structural 

rearrangements that carry lasting memories may help the development of better treatments for 

memory dysfunction. 

Disclosures: A. Karlen, None; T.E. Karlsson, None; A. Mattsson, None; K. Lundstomer, 

None; K. Pernold, None; T. Pham, None; C.M. Backman, None; S.O. Ogren, None; A.F. 

Hoffman, None; M.A. Sherling, None; C.R. Lupica, None; B.J. Hoffer, None; C. Spenger, 

None; A. Josephson, None; S. Brene, None; L. Olson, None. 

Poster 






Title: Analysis of protein interaction “managers” and their system-wide connections: 

universality and plasticity of synapse molecular organizations 

Authors: *A. KURAKIN; 

Pathology, BIDMC/Harvard Med. Sch., Boston, MA 

Abstract: We have developed a suit of biochemical and computational techniques that allows 

for the identification of interactors of PDZ, WW, SH3 and other signaling/scaffolding domains 

on the scale of a whole proteome. Identification of interacting partners of a given target 

domain/protein followed by clustering of identified interactors in the functional space of the 

proteome provides a comprehensive overview of the functional connections of the target 

domain/protein. 

Analysis of proteome-wide functional associations of the PSD95 scaffold indicates that, in 

addition to its well-recognized role in managing spatio-temporal dynamics of the post-synaptic 

specialization, PSD95 may play an underappreciated role in establishing and maintaining cell 

polarity. Two novel protein families found to interact with PSD95, membrane-associated 

ubiquitin ligases and viral proteins, will be discussed in the context of the PSD95 biology. 

Analysis of interactions of the CIN85/SETA/Ruk protein on the proteome-wide scale suggests 

that different intercellular specialized junctions, including neuronal and immunological synapses 

and adherens junctions, may represent functionally analogous molecular organizations built of

oth cell type-specific and cell type-invariant functional modules that are arranged and perform 

in an overall similar manner. 

Disclosures: A. Kurakin , None. 

Poster 





Support: Cincinnati Children's Hospital Trustee Award 

Title: Granule cell dysmorphogenesis in epilepsy: possible formation of recurrent basal dendrites 

by translocation of existing apical dendrites 

Authors: *B. L. MURPHY 1,2 , C. WALTER 1 , S. C. DANZER 1,2,3 ; 

1 Dept. of Anesthesia, Cincinnati Children's Hosp., Cincinnati, OH; 2 Program in Neurosci., 

3 Departments of Anesthesia and Pediatrics, Univ. of Cincinnati, Cincinnati, OH 

Abstract: The epileptic brain is characterized by a number of pathological changes of the 

hippocampal dentate gyrus, including the appearance of granule cells with dysmorphic dendritic 

structures. Despite intense interest in these phenomena as potential contributing factors to 

epileptogenesis, the cellular mechanisms by which these changes come about remain mysterious. 

Here, we examined the development of these phenomena by serial confocal imaging of living 

granule cells in organotypic hippocampal explant cultures made from Thy1-YFP expressing 

mice. In these mice, yellow fluorescent protein (YFP) is expressed in a subset of granule cells, 

revealing their complete morphology. Select neurons in these explants were imaged over a 

period of ten days to determine whether pathological changes reminiscent of those seen in the 

epileptic brain developed. Previous studies have demonstrated that explant preparation can lead 

to other changes characteristic of temporal lobe epilepsy, such as granule cell dispersion and 

mossy fiber reorganization, likely due to deafferantation and trauma. Surprisingly, the present 

study revealed that mature granule cells with initially normal morphologies could develop 

recurrent basal dendrites by movement of existing apical dendrites to the basal pole of the cell. 

This appeared to result as a consequence of the soma translocating into an adjacent apical 

dendrite. As the soma moved up this dendrite, neighboring dendrites were shifted to the basal 

pole of the cell. Movement of the soma also led to the absorption of dendritic branch points, 

increasing the number of primary dendrites possessed by the cell. The somatic movement 

producing these dendritic changes likely reflects granule cell dispersion, which also occurred in

these cultures. Together, these findings suggest that granule cell dispersion, in addition to 

disrupting the normally compact granule cell body layer, also disrupts granule cell dendritic 

structure. Moreover, these findings demonstrate in principal, that recurrent basal dendrites can 

form by movement of pre-existing apical dendrites. Whether similar cellular changes underlie 

granule cell pathology in patients with epilepsy remains to be determined. 

Disclosures: B.L. Murphy, None; C. Walter, None; S.C. Danzer, None. 

Poster 





Support: Cincinnati Children's Hosp. 

Title: Structure and complexity of CA3 pyramidal cell thorny excrescences 

Authors: *S. L. BRONSON 1,2 , B. L. MURPHY 1,2 , C. WALTER 1 , S. C. DANZER 1,2,3 ; 

1 Dept. of Anesthesia, Cincinnati Children's Hosp., Cincinnati, OH; 2 Program in Neurosci., 

3 Departments of Anesthesia and Pediatrics, Univ. of Cincinnati, Cincinnati, OH 

Abstract: Hippocampal dentate granule cell - CA3 pyramidal cell synapses constitute one part 

of the classic hippocampal “trisynaptic circuit”. This synaptic pathway is structurally and 

functionally unique. The presynaptic terminals making up this pathway are known as “giant 

mossy fiber boutons”, so named because of their unusually large size, which is five to ten times 

greater than typical presynaptic terminals in the CNS. These giant boutons contact CA3 

pyramidal cell postsynaptic spines termed “thorny excrescences”. Most dendritic spines of the 

CNS exhibit a simple structure of only one, or occasionally a few, spine heads per spine neck. In 

contrast, thorny excrescences exhibit multitudes of spine heads attached to the trunk dendrite by 

a single neck. The increased size and complexity of both presynaptic and postsynaptic structures 

at this synapse reflects its potency. Firing of a single granule cell is sufficient to fire its target 

pyramidal cell. Until recently, however, the complexity of CA3 pyramidal cell thorny 

excrescences has made it difficult to characterize their structure and plasticity. Here, we 

demonstrate that these structures can be quantified by combining high resolution confocal 

microscopy with the Thy1-GFP expressing mouse line. In this mouse line, a subset of CA3 

pyramidal cells is labeled with green fluorescent protein (GFP). Using this approach, it has 

become clear that thorny excrescences are even more complicated than previously appreciated. 

Specifically, thorny excrescences made up of more than thirty individual spine heads have been

identified. Indeed, some of these structures extend for more than 6 microns from the parent 

dendrite. In addition, analysis of more than 75 neurons so far reveals tremendous diversity 

among dendritic segments and among thorny excrescences on these segments. Dendritic 

segments, often from the same animals, may possess few or no thorny excrescences, occasional 

more complicated excrescences, or elaborate compilations of excrescences totaling hundreds of 

individual spine heads. This diversity suggests that, like other postsynaptic structures, thorny 

excrescences may exhibit significant plasticity during both normal and pathological processes, 

such as learning or epileptogenesis. The tools described here will facilitate studies of this 

intriguing possibility. 

Disclosures: S.L. Bronson, None; B.L. Murphy, None; C. Walter, None; S.C. Danzer, None. 

Poster 

240. Intrinsic Membrane Properties: Modulation of Neuronal Firing Properties by Inputs 

and Activity 



Topic: B.10.b. Modulation of neuronal firing properties 


Title: Accurate scaling of neural responses may depend on localization of modulatory inputs to 

proximal basal dendrites 

Authors: *M. JADI, B. W. MEL; 

Univ. So. California, Los Angeles, CA 

Abstract: Neurophysiological studies often report that sensory-evoked responses of cortical 

neurons can be scaled up or down by modulatory inputs - for example, multiplicative boosting of 

classical sensory responses by contextual or attentional inputs. However, the synaptic, neuronal, 

and circuit-level mechanisms that underlie this type of response scaling remain poorly 

understood. NMDA channels seem ideally suited to contribute: (1) they provide a large fraction 

of the excitatory drive to the synapse-dense thin branches of pyramidal neurons; (2) their 

voltage-dependence gives rise to a sigmoidal input-output relation with a strong accelerating 

phase; and (3) an accelerating nonlinearity - to the extent that it resembles an exponential 

function - provides a natural substrate for scaling: any response can be multiplied (or divided) by 

a factor F by incrementing (or decrementing) the input by log(F). Using a compartmental model 

of a neocortical pyramidal cell, we previously showed that scaling based on NMDA currents 

could be both accurate and precise over a substantial range of scaling factors (Jadi & Mel,

Society for Neuroscience Abstracts, 2007). In that work, we assumed the classical and 

modulatory inputs were co-distributed in the dendrites. In the current work, we discarded this 

assumption based on recent evidence from modeling and experimental studies of layer 5 

pyramidal cells showing that nonlinear "summation" of two inputs to the basal dendrites depends 

strongly on their absolute and relative locations (Behabadi et al., Society for Neuroscience 

Abstracts, 2007). We found that "scaling competence" was significantly improved when the 

modulatory input was delivered to the proximal portion of the dendritic branch. In that case, an 

accurate and repeatable multiplicative scaling of the classical response could be achieved over 

multiple octaves of input and output dynamic range. This finding supports the proposal of 

Behabadi et al. (2007) that attentional, contextual and other scaling inputs may target more 

proximal regions of neocortical basal dendrites. This work was supported by NIMH grant 

MH065918-01. 

Disclosures: M. Jadi , None; B.W. Mel, None. 

Poster 


and Activity 





Title: Electrophysiological characterization of sympathetic preganglionic neurons in HB9-GFP 

transgenic mice 

Authors: *A. L. ZIMMERMAN 1 , S. HOCHMAN 1,2 ; 

1 Biomed. Engin., Georgia Tech/Emory Univ., Atlanta, GA; 2 Physiol., Emory Univ., Atlanta, GA 

Abstract: Spinal cord sympathetic preganglionic neurons (SPNs) are the final common output of 

the sympathetic nervous system. Intracellular recordings have characterized several 

electrophysiological properties of SPNs in rat and cat, and here we extend these studies to 

mouse. We undertook whole-cell recordings of fluorescently-identified GFP + -HB9 SPNs in the 

intermediolateral column in young mice using horizontal and transverse slices. 

Preliminary results using a combination of current and voltage step protocols suggest that the 

active and passive membrane properties are comparable to those reported in neonatal rat. Action 

potentials exhibited the characteristic inflection during repolarization and prolonged 

afterhyperpolarization typical of SPNs (Pickering et al, Neuroscience Letters, 1991. 130: 237-

242). No significant differences in membrane electrical properties were observed between SPNs 

recorded in horizontal vs. transverse slices. When held at -80 mV, in response to intracellular 

current injection, most SPNs fired repetitively over a wide range of injected currents. However at 

potentials near resting membrane potential (-60 mV; mean Emr -59 mV), there was strong 

accommodation and several SPNs fired only single spikes. 

We then assessed the modulatory actions of serotonin (5HT), norepinephrine (NE) and dopamine 

(DA) on intrinsic membrane properties and glutamate receptor agonist-evoked currents. In the 

majority of SPNs recorded from, 5HT and NE depolarized the cell, accompanied by an increase 

in membrane input resistance and increased frequency response to current injection. DA alone 

and in combination, however, elicited a variety of responses, including hyperpolarizing and 

biphasic changes in membrane potential, increased and decreased input resistance, and altered 

current-frequency relations. Additionally, individual or combined monoamine application 

sometimes unmasked rebound excitation to hyperpolarizing current steps, supporting the 

presence of an anomalous rectifier (Miyazaki et al, Brain Research, 1996. 743: 1-10). In voltage 

clamp, NMDA and kainate evoked inward currents with characteristics I-V relations. 5HT, NE, 

and DA modulated these conductances. 5HT tended to facilitate while DA and NE tended to 

depress kainate and NMDA-evoked currents, although a diversity of responses were seen. 

Future studies are aimed at further detailing the neuromodulatory properties of specific 

monoamine receptor subtypes on SPN ligand- and voltage-gated ion channels. 

Disclosures: A.L. Zimmerman, None; S. Hochman, None. 

Poster 


and Activity 




Support: NIAAA (R01AA014366) (D.R.) 

NIH/NIDA Intramural Research Program (T.S.) 

funds provided by the State of California for medical research on alcohol and 

substance abuse through the University of California, San Francisco (D.R.) 

Title: Rapid actions of glial cell line-derived neurotrophic factor on firing of ventral tegmental 

dopaminergic neurons and dopamine release in the nucleus accumbens

Authors: *J. WANG 1 , S. CARNICELLA 1 , A. ZAPATA 2 , T. SHIPPENBERG 2 , D. RON 1,3 ; 

1 Dept. of Neurol., Gallo Res. Ctr., Emeryville, CA; 2 Integrative Neurosci. Section, NIDA, 

Baltimore, MD; 3 Dept. of Neurol., Univ. of California San Francisco, Emeryville, CA 

Abstract: The ventral tegmental area (VTA) dopaminergic neurons play an essential role in 

alcohol reward and addiction. Recently, we found that intra-VTA infusion of the Glial cell linederived 

neurotrophic factor (GDNF) produced a very rapid attenuation of rat operant selfadministration 

of alcohol (1). We hypothesized that the acute actions of GDNF in the VTA are 

due to changes in neuronal activity. To address this hypothesis we used slice cell-attached 

recording to examine whether GDNF alters the spontaneous firing of VTA neurons. We found 

that application of GDNF, but not its heat-inactivated form, induced a rapid increase in the firing 

frequency of Ih and tyrosine hydroxylase positive but not Ih negative neurons suggesting that 

GDNF selectively increases dopaminergic neuron firing in the VTA. To explore a possible 

mechanism underlying GDNF augmentation of firing, we used whole-cell current-clamp 

recordings and showed that GDNF application caused a depolarization of membrane potential of 

dopaminergic neurons. Enhancement of firing of VTA dopaminergic neurons may result in an 

increase in dopamine release in the nucleus accumbens (NAc). We therefore used in vivo 

microdialysis and observed an elevation in the level of dopamine in the NAc after infusion of 

GDNF into the VTA. Together, our results suggest that GDNF acutely and selectively increases 

the spontaneous firing of VTA dopaminergic neurons, which leads to an increase in dopamine 

release in the NAc. This excitatory effect of GDNF on VTA neuronal activity may underlie the 

mechanism by which GDNF in the VTA reduces alcohol consumption. 1. Carnicella et al. 2008 

PNAS, In Press. 

Disclosures: J. Wang, None; S. Carnicella, None; A. Zapata, None; T. Shippenberg, 

None; D. Ron, None. 

Poster 


and Activity 



Topic: B.10.a. Neural oscillators 

Support: INSERM 

ANR

Title: Alterations of Ih dynamics underlie a deficit of theta oscillations in a rat model of 

temporal lobe epilepsy 

Authors: *B. A. MARCELIN 1 , L. CHAUVIÈRE 1 , A. BECKER 2 , M. MIGLIORE 3 , M. 

ESCLAPEZ 1 , C. BERNARD 1 ; 

1 U751, INSERM, Marseilles, France; 2 Dept. of Neuropathology & Natl. Brain Tumor Reference 

Ctr., Univ. of Bonn Med. Ctr., Bonn, Germany; 3 Inst. of Biophysics, Palermo, Italy 

Abstract: Theta rhythm (4-12 Hz) production is directly built in the architecture of neuronal 

networks as some neurons, in particular CA1 pyramidal cells, respond preferentially to inputs in 

the theta frequency range, a property called resonance. Theta resonance is prominent in the distal 

dendrites of CA1 pyramidal cells where it depends upon hyperpolarizing-activated cyclic 

nucleotide-gated ion channels (HCN; Ih). Since experimental temporal lobe epilepsy (TLE) is 

associated with dendritic HCN channelopathy, we hypothesized that alteration in Ih function 

may modify dendritic resonance. We performed whole-cell dendritic and somatic patch clamp 

recordings during epileptogenesis in the pilocarpine model of TLE in rats. We injected wavelet 

current at different frequencies to measure the resonance parameters in relationship to Ih 

properties. We observed a decrease of all the resonance parameters in the distal dendrites of CA1 

pyramidal cells during epileptogenesis, which strongly correlated with a decrease of Ih 

availability. This decrease of Ih availability was due to: 1) an alteration of HCN channels 

kinetics (slower activation time constant and shift of the activation curve toward more 

hyperpolarized potential), 2) a significant reduction in HCN1/HCN2 subunit expression, leading 

to reduced current amplitude. Dendritic modeling showed that Ih reduction is sufficient to 

decrease theta resonance. Model predictions were verified experimentally. We suggest that 

decreased dendritic theta resonance during epileptogenesis is due to the decrease of Ih 

availability. 

In parallel we observed a decrease in theta power and theta frequency in vivo during 

epileptogenesis. Because theta activity is central to many cognitive and memory processes, 

altered theta oscillations may contribute to cognitive deficits found in TLE. Distal dendrites 

receive layer 3 enthorinal inputs, one of the major theta generators in the hippocampus. So 

alteration of Ih in the dendrites could contribute to decreased theta rhythm, and thus to the 

deficits in theta-dependent cognitive and memory processes associated to TLE. 

Disclosures: B.A. Marcelin, None; L. Chauvière, None; A. Becker, None; M. Migliore, 

None; M. Esclapez, None; C. Bernard, None. 

Poster 


and Activity 




Support: Epilepsy Foundation 

Postdoctoral Research Abroad Program, National Science Council of Taiwan (NSC- 

095-SAF-I-564-611-TMS) 

Title: Mild hyperthermia induces intrinsic firing of CA1 pyramidal neurons 

Authors: *H.-J. YAU, M. MARTINA; 

Dept. of Physiol., Northwestern Univ., Chicago, IL 

Abstract: Febrile seizures are the most frequent type of seizures, affecting 3-5 % of children 

between 6 months and 3 years of age; yet, the detailed cellular mechanisms of febrile seizures 

remain unclear. To increase our knowledge of such mechanisms, we studied the effect of 

hyperthermia on the electrophysiologic properties of hippocampal pyramidal neurons. 

Extracellular (loose-seal) and whole cell recordings obtained from CA1 pyramidal neurons 

showed that mild hyperthermia (39-40°C) depolarizes ~95% of the pyramidal cells and induces 

spontaneous firing in ~70 % of the cases, even in the absence of fast synaptic transmission, 

suggesting an effect on intrinsic neuronal properties. Hyperthermia-induced depolarization was 

still present after pharmacological blockade of voltage-gated channels and it was paralleled by a 

decrease in membrane resistance, suggesting that the depolarizing response is mediated by the 

activation of a temperature-dependent background conductance. These data suggest the possible 

involvement of TRPV channels. Indeed, the TRPV blocker ruthenium red increased the input 

resistance in ~40 % of pyramidal cells recorded at >37°C, while it was ineffective at 30-32°C, 

confirming that TRPV channels are expressed in pyramidal neurons and open at febrile 

temperature. Although hyperthermia depolarized ~95 % of the pyramidal cells, in ~40 % of the 

cells a biphasic response was detected, in which a transient hyperpolarization preceded the 

depolarization. This result suggests that other types of temperature-sensitive channels (possibly 

potassium selective) are also present in these cells and mediate the hyperpolarization. Thus, we 

tested the effect of flufenamic acid, an agonist of temperature-dependent KCNK channels, on the 

hyperthermic response of CA1 pyramidal neurons. Flufenamic acid potentiated a temperaturedependent 

potassium conductance and completely abolished hyperthermia-induced intrinsic 

firing. 

These results show that temperature sensitive ion channels mediate the response of pyramidal 

neurons to hyperthermia and may constitute new targets for pharmacological treatment of febrile 

seizures. 

Disclosures: H. Yau, None; M. Martina, None. 

Poster


and Activity 




Support: CIHR MOP-77566 

Title: TRPC channels mediate persistent activity in layer V principal neurons of the medial 

entorhinal cortex 

Authors: *Z. ZHANG, A. REBOREDA, A. ALONSO, P. A. BARKER, P. SEGUELA; 

Neurol. & Neurosurg., Montreal Neurolog. Inst., Montreal, QC, Canada 

Abstract: Principal neurons in layer V of the rat medial entorhinal cortex (MEC) generate selfsustained 

plateau potentials and persistent spiking following the application of a brief 

suprathreshold excitatory stimulus in the presence of the muscarinic receptor agonist carbachol 

(CCh). Plateau potentials and persistent firing activity, non-synaptic mechanisms of information 

storage contributing to working memory, have been shown to be mediated by a Ca2+ dependent, 

non-selective cation current (Ican). However the identity of the channel(s) that carry Ican in layer 

V MEC neurons is unknown. In this study, we investigated the molecular identity of the channels 

mediating CCh-evoked persistent activity using whole-cell patch clamp recordings in rat layer V 

MEC neurons in acute brain slices. Our data indicate that compounds that block phospholipase C 

(PLC) activation and TRP channels inhibited plateau potential and persistent activity in layer V 

neurons. U73122, a PLC blocker, and the TRPC channel blockers flufenamic acid, 2-APB and 

SKF 96365 inhibited plateau potential and persistent activity, while the TRPV channel blocker 

ruthenium red did not have any inhibitory effect. Interestingly, the diacylglycerol analog OAG 

which activates TRPC3/6/7 channels failed to induce the plateau potential and persistent firing. 

Voltage clamp analysis revealed a current-voltage relationship typical of TRPC4/5-containing 

channels with a double-rectifying current that reverses at 0 mV. A specific intracellular peptide 

designed to disrupt the interaction between the PDZ binding motif of TRPC4/5 channels and 

NHERF/PLCβ was found to be inhibitory. Finally, intracellular application of antibodies directed 

against the TRPC5 C-terminal domain also blocked CCh-evoked plateau potential and persistent 

activity. Taken together, our data show that receptor-operated TRPC channels are required for 

the expression of plateau potentials and persistent activity in layer V principal neurons of rat 

MEC. 

Disclosures: Z. Zhang , None; A. Reboreda, None; A. Alonso, None; P.A. Barker, None; P. 

Seguela, None.

Poster 


and Activity 




Title: The electrophysiological characterization of Substance P sensitive muscle afferent 

neurons 

Authors: *C.-C. J. LIN; 

Academia Sinica, Taipei, Taiwan 

Abstract: Peripheral substance P (SP) is well known for its role in inflammatory pain and its 

involvement in the phenomenon of neurogenic inflammation and central sensitization. 

Nontheless, its role in acid induced muscle pain still remained as a less-studied aspect of its 

biology. This study utilized whole-cell patch recording to characterize the properties of the 

muscle afferent DRG neurons. Lumbar dorsal root ganglion (DRG) neurons retrogradely labeled 

with 4 % fluoro-gold from gastrocnemius muscle were used. The current clamp experiment 

showed that the treatment of SP could attenuate or reverse the depolarizing membrane potential 

induced by the initial treatment of acid in ~76% of muscle afferent neurons. On the other hand, 

the voltage clamp study demonstrated that SP had different effects on acid-evoked current. 

Muscle innervated DRG neurons were more responsive to acidic treatment than non-muscle 

ones. Moreover, a subpopulation of neurons was found only within muscle afferent DRG 

neurons to exhibit outward current upon SP treatment. The data so far indicated that SP has the 

ability to modulate acid-sensing properties of muscle afferent neurons. 

Disclosures: C.J. Lin , None. 

Poster 


and Activity 



Topic: B.10.b. Modulation of neuronal firing properties

Support: NIH grant AA14973 

Title: Ethanol increases spontaneous action potential firing frequency of cerebellar Golgi 

neurons via membrane depolarization 

Authors: *P. BOTTA, C. F. VALENZUELA; 

Neurosci, Univ. New Mexico HSC, Albuquerque, NM 

Abstract: Cerebellar Golgi neurons (GNs) are spontaneously firing GABAergic interneurons 

situated in the cerebellar granule layer. These neurons play a pivotal role in filtering mossy fiberdependent 

excitation of granule cells. We have previously shown that acute ethanol (EtOH) 

exposure enhances GN spontaneous action potential firing (Carta et al. J Neurosci. 24:3746-51, 

2004) and that this effect can be observed in presence of antagonists of ionotropic glutamate, 

mGluR2, GABA-A, GABA-B and glycine receptors (Program No. 909.10. Neuroscience 

Meeting Planner. San Diego, CA: SFN 2007). These findings indicate that EtOH enhances GN 

firing by affecting intrinsic conductances rather than changing synaptic inputs. The goal of this 

study was to further characterize the action of EtOH at GNs. Parasagittal cerebellar vermis slices 

(200 micrometer-thick) were prepared from 23-25 day-old male Sprague-Dawley rats. GN firing 

was recorded using the perforated-patch current-clamp configuration (Iinject = 0 pA) at 32 o C. The 

artificial cerebrospinal fluid contained antagonists of the above-mentioned neurotransmitter 

receptors. EtOH (40 mM) reversibly increased the spontaneous action potential firing frequency 

(ctrl = 3.9 ± 0.9 Hz; EtOH = 4.6 ± 0.9 Hz; n = 9; p


Support: CIHR 

Title: Modulation of sensory proccesing by serotonin in ELL pyramidal cell 

Authors: *T. DEEMYAD, M. J. CHACRON; 

McGill Univ., Montreal, QC, Canada 

Abstract: The serotonergic system is involved in many neural disorders. There is evidence that 

serotonin induces burst firing in many area in CNS and capable of altering the neural processing 

of sensory information. However, the cellular mechanisms by which this is achieved are not well 

understood. 

Weakly electric fish, due to relatively simple and well characterized anatomy that shows 

important similarities with that of higher vertebrates are a suitable model for investigating this 

problem. The electrosensory lateral line lobe (ELL) is the first and only terminal of the 

peripheral sensory afferents. The ELL is comprised of three parallel somatotopic representations 

of sensory space: lateral (LS), centrolateral (CLS) and centromedial (CMS) segments that all 

receive the same afferent input. There are anatomical differences between these maps in terms of 

descending input, ion channel distributions, and neuromodulatory inputs. Previous studies have 

shown that pyramidal cells, the sole output neurons of the ELL, have a well-defined burst 

mechanism that signals particular stimulus features in vivo. The effect of serotonin on pyramidal 

cell activity and their processing of sensory input are unknown. Therefore, we studied the effect 

of serotonin application on electrical activity of pyramidal cells using sharp-electrode 

intracellular recordings. Action potential amplitude and duration were not affected by serotonin. 

Serotonin increased burst firing three fold in the lateral segment (LS) and had no significant 

effect in the centromedial segment. This augmentation in burst activity in LS pyramidal cells was 

achieved by a reduction of the spike afterhyperpolarization (AHP). Moreover, in presence of 

serotonin adaptation becomes slower in pyramidal cells. As the calcium activated potassium 

channels (SK) are underlies medium component of AHP besides speed of adaptation this finding 

suggests that effect of serotonin resulted from down regulation of SK channels. SK channels 

show graded patterns of expresion across the ELL maps that mirror those of serotonergic input. 

The nature of contribution of SK channels in effect of serotonin on electrical activity of ELL 

pyramidal cells in presence of apamin and EBIO, as well as their consequences on sensory 

processing, will be discussed. 

Disclosures: T. Deemyad , None; M.J. Chacron, None. 

Poster 


and Activity




Support: NINDS Grant NS37760 

Title: Responses of striatal cholinergic interneurons to globus pallidus stimulation in rat brain 

slices 

Authors: *A. BAUMGARTNER, C. J. WILSON; 

Dept Biol., Univ. Texas San Antonio, San Antonio, TX 

Abstract: Striatal cholinergic interneurons spontaneously generate action potentials that 

maintain the background cholinergic tone in the striatum. In behaving animals, a pause in firing 

followed by a burst of action potentials occurs when the animal is presented with a salient event 

such as a cue indicating reward. In slices, cholinergic interneurons show spontaneous firing 

patterns similar to those seen in vivo. We previously reported that stimulation in the globus 

pallidus in sagittal rat brain slices evokes a pause followed by a burst, which may have a 

common mechanism with the in vivo pause and burst, and that fast synaptic transmission was not 

required for the pause-burst response. We hypothesized that G-protein coupled receptors may 

underlie some or all of the response. We included 2 mM GDP-β-S in the pipette solution and 

found both the pause and burst were abolished in current clamp recordings. The pause and burst 

may be due to modulation of the ionic currents that underlie cholinergic interneurons‟ 

spontaneous activity: KIR, Ih, persistent Na + and Ca 2+ -dependent K+ currents. In cholinergic 

interneurons voltage clamped at their resting membrane potential of -60 mV, stimulation that 

triggered a pause and burst response in current clamp evoked a slow outward current followed by 

an even slower inward current. When we blocked KIR and Ih with 3 mM cesium and 30 µM 

ZD7288, the pause and burst remained. Blockade of Na + current with 5 mM QX-314 also had no 

effect. In voltage clamp under these conditions, the early outward current reversed at -87 mV and 

the late inward current reversed near -10 mV. Intracellular application of 5 mM BAPTA 

significantly reduced the conductance of the early response in voltage clamp, and reduced the 

hyperpolarizing response seen in current, with no effect on the late response. Because the late 

response was still present when the outward current was blocked by BAPTA, the burst is not 

dependent on the pause. We hypothesize that synaptic activation of one or more G-protein 

pathways mediates both the stimulus-induced pause and burst, the former of which requires an 

increase in cytoplasmic calcium. 

Disclosures: A. Baumgartner , None; C.J. Wilson, None. 

Poster


and Activity 



Topic: B.10.c. Activity-dependent plasticity of intrinsic membrane properties 

Support: Clarke Foundation 

Title: Molecular mechanisms of DCS induced changes in hippocampal neuron excitability 

Authors: *E. DONZIS, L. WILLMOT, L. T. THOMPSON; 

Behavioral & Brain Sci., Univ. of Texas at Dallas, Richardson, TX 

Abstract: Aging is associated with specific deficits in hippocampal-dependent learning tasks 

including trace eyeblink conditioning, spatial maze learning, and rule learning of odor 

discriminations tasks. Age-related impairments have been linked to decreases in the intrinsic 

excitability of hippocampal pyramidal neurons as reflected by increase amplitudes of postburst 

afterhyperpolarizations (AHPs) and increases in spike-frequency adaptation (accommodation). 

Slow learning young subjects also show larger AHPs and greater accommodation when 

compared to naive and especially to faster learning subjects. In rats, dose-response studies have 

shown that administration of 6 mg/kg of a partial NR1 agonist, D-cycloserine (DCS), improves 

acquisition on spatial memory tasks and increases intrinsic neuronal excitability. The exact 

mechanisms of DCS modulation of neuronal excitability are not fully understood, although 

calcium-signalling pathways are implicated. A possible molecular mechanism could be through 

activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Many components of this 

pathway have been shown to be involved in learning and memory, and activation of NMDA 

receptors has been linked to activation of the PI3K pathway. 

In these studies, we gave either a single injection of 6 mg/kg of DCS or a series of daily 

injections over one week to FBNF-1/HSD rats and sacrificed them at varying intervals afterward. 

Changes in phosphorylation of Akt in the hippocampus were examined by western blot and 

changes in hippocampal intrinsic excitability were examined by sharp electrode recording from 

ventral brain slices. Tissue from rats given DCS injections showed higher levels of phospho-Akt 

compared to saline injected rats after a single dose when sacrificed 1 hr post-injection. DCS 

injected rats also showed increases in excitability in the CA1 at the same time interval which 

correlated with increases in phosphorylation of Akt. Together, these findings suggest that DCS 

induces a temporally constrained increase in hippocampal pyramidal cell excitability through 

activation of the PI3K/Akt pathway. 

Disclosures: E. Donzis , None; L. Willmot, None; L.T. Thompson, None.

Poster 


and Activity 




Support: NINDS 24288 

Title: Modification of temporal summation and Ih following mossy fiber LTP induction in CA3 

interneurons 

Authors: *W. D. ANDERSON 1,2 , E. J. GALVAN 1 , G. BARRIONUEVO 1,2 ; 

1 Univ. Pittsburgh, Pittsburgh, PA; 2 Ctr. for the Neural Basis of Cognition, Pittsburgh, PA 

Abstract: Inhibitory interneurons in hippocampal area CA3 function as a dynamic filtering 

system for information transmitted to CA3 pyramidal neurons along mossy fibers (MF) and the 

perforant path (PP). We have previously demonstrated that a subset of interneurons in the s. 

lacunosum moleculare (SLM) layer exhibit MF activity dependent LTP and a subsequent 

increase in the interval over which sequentially activated MF and PP EPSPs summate 

supralinearly. Further, we have also shown that supralinearity is present at longer inter-stimulus 

intervals when h-channels are blocked by ZD7288. In this report we describe preliminary 

evidence which indicates that Ih is downregulated following MF LTP induction in a subset of 

SLM interneurons. 

First, input resistance (Rin) was increased to a similar extent following ZD7288 application and 

during LTP. Next, when cells were held at -60 mV in voltage clamp and stepped to -120 mV 

before and after LTP induction, the slowly activating inward current following the capacitive 

transient was reduced, suggesting a decrease in a hyperpolarization activated inward current 

putatively mediated by h-conductance. In a different set of cells, the application of forskolin - 

which raises intracellular cyclic AMP (cAMP) production by activating adenylate cyclase - 

resulted in both LTP and a reduction of the slowly activating inward current at -120 mV. 

Forskolin application was also shown to increase Rin. These data suggest (a) that Ih is reduced 

when LTP is induced at MF synapses and that this reduction contributes to increased supralinear 

summation, and (b) that both LTP and Ih downregulation can be induced by a cAMP dependent 

signaling pathway. Future experiments are being conducted to (1) determine whether LTP 

induction is necessary for the decrease in Ih and (2) establish an explanation for the apparent 

decrease in Ih (e.g., decrease in the channel number, single channel conductance, voltage 

dependence, and/or channel kinetics). Supported by NINDS 24288. 

Disclosures: W.D. Anderson , None; E.J. Galvan, None; G. Barrionuevo, None.

Poster 


and Activity 




Support: NIH AG 20506 

NIH R37 AG08796 

US-Israel Binational Science Foundation #2005370 

Title: Learning-related increases in synaptically-evoked intrinsic excitability 

Authors: *B. M. MCKAY, J. F. DISTERHOFT; 

Dept of Physiol., Northwestern Univ., Chicago, IL 

Abstract: The learning-related increase in the afterhyperpolarization (AHP), elicited by a train 

of action potentials delivered to the soma, is a well documented phenomenon. It has been 

established in the CA1 and CA3 of rabbits, CA1 of rats, and piriform cortex of rats to occur after 

learning tasks as divergent as trace eyeblink conditioning, water maze, and odor discrimination 

learning. However, in the behaving animal, the AHP is elicited by activation of synapses located 

in the dendrites. It has not yet been demonstrated that learning-related decreases in the AHP can 

be elicited using synaptic stimulation. To this end, we trained rats at trace eyeblink conditioning, 

a hippocampus-dependent task. In this task, animals receive repeated presentations of a tone 

(conditioned stimulus, CS) followed by an air puff to the cornea (unconditioned stimulus, US), 

separated by a 250msec stimulus-free interval. Pseudoconditioned control animals receive equal 

number of stimuli, never explicitly paired in time. After 5 training sessions, hippocampal slices 

were prepared. CA1 was isolated by cutting off CA3, and a stimulating electrode was placed in 

the stratum radiatum. Five action potentials at 50Hz were elicited by stimulation of the Schaffer 

collaterals (synaptic) or via the somatic recording pipette (somatic). Consistent with what has 

previously been published, I observed smaller somatically-evoked AHPs in cells from trained 

animals compared to naïve and pseudoconditioned controls. In unmodified aCSF, there was no 

difference in the hyperpolarization following 5 synaptically-evoked action potentials, which 

includes both the AHP and an inhibitory postsynaptic potential (IPSP). However, when measures 

were made in the presence of the GABA-A blocker gabazine and the GABA-B blocker 

SCH50911, the synaptically-evoked AHP was smaller in cells from trained animals compared to 

controls. These data demonstrate that a learning-related increase in intrinsic excitability can be

evoked by synaptic stimulation, and suggest an increase in feed-forward inhibition in CA1. 

Together, an increase in excitation and inhibition may act to increase the signal-to-noise ratio of 

information flowing through the hippocampus as the CS-US association is stored. 

Disclosures: B.M. McKay , None; J.F. Disterhoft, None. 

Poster 


and Activity 




Support: NAAR (DPS) 

NIH AG08796 & US-Israel Binational Sci Fnd #2005370 (JFD) 

NIH MH071316 & NARSAD (PP) 

NIH AG20506 (BMM) 

Title: Learning trace eyeblink conditioning altered protein levels of AHP contributors in 

CA1/subiculum 

Authors: *M. M. OH, D. P. SRIVASTAVA, B. M. MCKAY, K. SCHOEDEL, P. PENZES, J. 

F. DISTERHOFT; 

Dept Physiol., Northwestern Univ. Med. Sch., Chicago, IL 

Abstract: The postburst afterhyperpolarization (AHP) is a Ca 2+ -dependent K + current with 2 

distinct phases. Apamin sensitive SK2 channels mediate the medium AHP, and play a role in 

regulating NMDA receptor mediated synaptic plasticity in dendritic spines of CA1 neurons 

(Ngo-Ahn et al, 2005). The channel underlying the slow AHP is not yet identified, however, a 

neuronal calcium sensor, hippocalcin, was shown to be its intracellular mediator (Tzingounis et 

al, 2007). Blocking of L-type Ca 2+ channels causes a ~30% reduction in the slow AHP in CA1 

neurons (Power et al, 2002). Importantly, the AHP is reduced in hippocampal pyramidal neurons 

ex vivo after an animal has learned a hippocampus-dependent task (Disterhoft & Oh, 2006). 

Thus, we examined potential learning-related alterations in protein levels of these 3 AHP 

contributors in the CA1/subiculum (CA1/S) region from 3-4 mo F344xBN rats after trace 

eyeblink conditioning (EBC).

Rats were randomly assigned to one of 3 groups: trained, pseudoconditioned (pseudo), and naïve. 

The trained group received 30 paired tone CS and corneal airpuff US separated by a 250ms trace 

interval. Pseudo rats were given 30 CS and 30 US in an unpaired manner. Both groups had 5 

training sessions (2sessions/day). Hippocampal slices were cut in ice-cold aCSF 1 d after the last 

training session using a vibratome. The CA1/S was isolated from the hippocampal slice, and 

SK2, hippocalcin and CaV1.2 L-type Ca 2+ channel protein levels were determined by western 

blotting. Here we report an increase in SK2 and hippocalcin, but a decrease in CaV1.2 levels in 

CA1/S region from rats that learned as compared to naïve and pseudo rats. These data strongly 

suggest that the reduction in the amount of CaV1.2 is one source of the learning-related AHP 

reduction observed in CA1 neurons after trace EBC. Although hippocalcin is proposed to be a 

mediator of the slow AHP current, it does not contribute to the learning-related AHP reduction. 

Rather, the increase in hippocalcin levels suggests that a compensatory mechanism may occur 

after learning in response to a decreased cytosolic Ca 2+ level. The enhanced SK2 levels suggest 

that a homeostatic regulation may occur after learning. The increased SK2 level in the spines 

would filter out extraneous synaptic noise by establishing a higher threshold for the postsynaptic 

response to occur, but a strong signal that passes the SK2 filter would be readily passed on to the 

next element in the neural circuit because of the significantly reduced somatic slow AHP. 

Our present findings of molecular changes that occur with trace EBC directly link the reductions 

in CaV1.2 to the learning-related AHP reduction in CA1 neurons. 

Disclosures: M.M. Oh, None; D.P. Srivastava, None; B.M. McKay, None; K. Schoedel, 

None; P. Penzes, None; J.F. Disterhoft, None. 

Poster 


and Activity 




Support: NIH RR-015497 

NIH NS-35180 

NIH MH-067564 

NIH MH-074866

Title: Activity-dependent bidirectional plasticity of intrinsic burst firing in subicular pyramidal 

neurons 

Authors: *S. J. MOORE, N. SPRUSTON; 

Dept Neurobiol & Physiol, Northwestern Univ., Evanston, IL 

Abstract: The subiculum, which is the primary efferent pathway of the hippocampus, 

participates in memory for spatial tasks, relapse to drugs of abuse, and initiation of temporal lobe 

seizures. An important electrophysiological property of subicular pyramidal neurons is lowthreshold 

burst firing. We have previously demonstrated that burst firing can be regulated in an 

activity-dependent manner via synaptic stimulation of afferents from entorhinal cortex and CA1. 

Here, we extend these findings to show that, unlike synaptic plasticity in subicular pyramidal 

cells, plasticity of burst firing does not require postsynaptic depolarization, because an 

enhancement of burst firing was still induced when synaptic stimulation was given during 

somatic voltage clamp (to prevent action potential generation) or in the presence of ionotropic 

glutamate receptor antagonists. Instead, the enhancement of burst firing depended on synaptic 

stimulation to recruit synergistic activation of group I, subtype 1 metabotropic glutamate 

receptors (mGluRs) and the M1 subtype of muscarinic acetylcholine receptor (mAChR). When 

enhancement of burst firing was blocked, activation of group I, subtype 5 mGluRs resulted in 

suppression of burst firing. These results indicate that output of the subiculum can be strongly 

and bidirectionally regulated in a state-dependent manner through coordinated activation of 

glutamatergic inputs within the hippocampus and cholinergic afferents from the septal nucleus. 

Disclosures: S.J. Moore , None; N. Spruston, None. 

Poster 


and Activity 




Support: NINDS Grant NS47085 

NSF EF-0634588 

Title: Inactivation of persistent sodium current causes slow spike frequency adaptation in 

subthalalmic neurons

Authors: *D. BARRAZA 1 , C. J. WILSON 2 ; 

2 Biol., 1 UTSA, San Antonio, TX 

Abstract: Neurons of the subthalamic nucleus (STN) fire rhythmically in slices, even when 

disconnected from synaptic input. When driven with short (1 s) depolarizing steps, they exhibit 

reverse spike frequency adaptation and can fire at very high rates (up to 500 Hz). However, using 

the perforated patch method to study longer current pulses, a slowly developing spike frequency 

adaptation is apparent. Cells were driven by 2.5 minute current steps of various amplitudes. After 

the initial rate increase during the first 50-200 ms after the onset of current injection, firing rate 

gradually decreased over the next several seconds and stabilized at 18-63% of the maximal firing 

rate. The adaptation could be described using two exponentials, a fast one with a time constant of 

about 1.4 s and a longer one with a time constant averaging 23.0 s. Following current offset the 

spontaneous firing was abolished for several seconds, followed by a gradual recovery of firing 

rate with a time constant averaging 35.4 s. During adaptation, the variability of instantaneous 

firing rate was greatly increased. Variability was measured as a deviation from the mean firing 

rate during adaptation, and increased in parallel with adaptation and decreased during recovery. 

Thus the influence of adaptation on variability was independent of rate, but depended upon 

adaptation. Driving STN neurons with high frequency depolarizing voltage pulses in whole cell 

voltage clamp recordings resulted in the accumulation of a slow apparent outward current that 

outlasted the stimulus, and decayed with a time constant of about 1 second. We considered this 

current a candidate for the mechanism responsible for spike frequency adaptation. The slow 

outward current was reduced to zero below -70 mV, but never reversed, and increased in 

amplitude rapidly at voltages above -55 mV. Using a voltage ramp from -70 to -30 mV, we 

looked for changes in conductance that could be responsible for the outward current. We noted 

that there was a reduction in the negative conductance region of the steady state I/V curve during 

the period associated with the outward current, but no change in conductance at voltages outside 

that range. The negative conductance region is caused by activation of a persistent sodium 

conductance, as indicated by its sensitivity to TTX. Thus, the apparent outward current that 

follows high frequency driven firing is actually a reduction in the voltage-dependent persistent 

Na+ current that is known to be responsible for spontaneous firing. The slowly developing and 

long-lasting inactivation of persistent Na+ currents can account for all the properties of the slow 

spike frequency adaptation of STN neurons. 

Disclosures: D. Barraza , None; C.J. Wilson, None. 

Poster 


and Activity 


Program#/Poster#: 240.18/E40


Support: NINDS Grant 5F31NS055516 

Title: Cell-type specific maintenance of neocortical firing type by network activity 

Authors: *M. N. MILLER, S. B. NELSON; 

Biol., Brandeis Univ., Waltham, MA 

Abstract: The adult neocortical microcircuit is composed of multiple discrete cell-types that 

differ morphologically, physiologically, and transcriptionally, and these cell-type-specific 

properties permit distinct functional roles within the network. Maturation and maintenance of 

cell-type specificity is most likely regulated by a complex interaction between genetic and 

environmental factors, but the relative contributions of these have not been directly examined. 

We combined genetic and anatomical labeling of cortical cell-types with chronic local muscimol 

delivery in adult mice in vivo to ask whether cortical network activity is necessary for the 

maintenance of cell-type specificity. 48 hours of local network inactivation increased the 

excitability of fast-spiking interneurons (FS) and reduced the excitability of intratelencephalic 

corticostriatal pyramidal cells (IT), but had no effect on their respective characteristic firing 

types. In contrast, pyramidal-tract projecting pyramidal cells (PT), which under control 

conditions exhibit spike-frequency acceleration in response to current injection, either lost 

acceleration to become non-adapting or, in the majority of cases, exhibited spike-frequency 

adaptation accompanied by a dramatic increase in input resistance. The combination of spikefrequency 

adaptation and high input resistance is reminiscent of both IT pyramidal cells and 

immature PT pyramidal cells, suggesting that PT physiological properties quickly regress to an 

immature and less-differentiated state if not actively maintained by network activity. 

Furthermore, the effect of chronic activity blockade on PT but not IT or FS populations indicates 

that activity-dependent maintenance of neuronal phenotype is cell-type specific. 

Disclosures: M.N. Miller , None; S.B. Nelson, None. 

Poster 


and Activity 


Program#/Poster#: 240.19/F1 


Support: Neuroinformatics Doctoral Training Centre Grant (EPSRC)

Title: Conductance changes underlying homeostatic regulation of intrinsic excitability in 

hippocampal neurons 

Authors: *T. S. O'LEARY 1 , M. C. W. VAN ROSSUM 2 , D. J. A. WYLLIE 3 ; 

1 Neurosci. and Informatics, Univ. Edinburgh, Edinburgh, United Kingdom; 2 Informatics, 3 Ctr. 

for Neurosci. Res., Univ. of Edinburgh, Edinburgh, United Kingdom 

Abstract: For the nervous system to function properly, activity levels must be regulated. 

Excitable cells operate in a regime that is responsive to normal levels of input but also need to 

adjust their excitability to counter sustained changes in mean activity. Such changes in activity 

levels occur during development, and may also result from Hebbian learning. 

Neurophysiological correlates of Hebbian learning have been found to strengthen connectivity 

between excitatory cells in response to correlated firing patterns, and enhance intrinsic 

excitability at the same time [1]. In isolation, this constitutes a positive feedback loop which may 

destabilise network activity [3]. It is therefore likely that homeostatic mechanisms exist to coregulate 

activity on a cellular level, and several candidate mechanisms have been characterised to 

date - most of which address the effect of diminished activity [2,3]. Here we describe a 

previously uncharacterised in-vitro model for homeostatic control of intrinsic excitability. We 

find that cultured hippocampal neurons respond to chronic depolarisation over a period of days 

by attenuating their response to injected current. Cells grown in depolarising medium containing 

15 mM KCl exhibited a tenfold increase in the amount of steady current required to induce 

spiking (9.7±3.5 pA for n=25 control cells; 94.5±16.0 pA for n=18 treated cells). This effect was 

found to depend on the level of depolarisation and the length of treatment, and is accompanied 

by a decrease in voltage-gated sodium conductance in the cells. Consistent with these 

observations is a prominent hyperpolarising shift in resting membrane potential relative to 

control (-50±1 mV vs. -58±1 mV) and a drop in input resistance (790±140 MOhm vs. 330±60 

MOhm). Using these data to parameterise a conductance-based computer model offered insight 

as to whether they could account for the observed differences in excitability. Substituting sodium 

conductance and input resistance caused a shift in excitability which was comparable to that 

observed in experiments. When introduced separately, the changes in sodium conductance and 

input resistance failed to recapitulate the in vitro result. 

References 

[1] Chavez-Noriega LE, Halliwell JV, Bliss TV (1990) A decrease in firing threshold observed 

after induction of the EPSP-spike (E-S) component of long-term potentiation in rat hippocampal 

slices. Exp Brain Res 79:633-641. 

[2] Desai NS, Rutherford LC, Turrigiano GG (1999) Plasticity in the intrinsic excitability of 

cortical pyramidal neurons. Nat Neurosci 2:515-520 

[3] Turrigiano GG, Nelson SB (2004) Homeostatic plasticity in the developing nervous system. 

Nat Rev Neurosci 5:97-107 

Disclosures: T.S. O'Leary , None; M.C.W. van Rossum, None; D.J.A. Wyllie, None. 

Poster


and Activity 




Support: International Human Frontier Science Program Organization 

NIH Grants MH48432, MH44754, NS37444 

Title: Intracellular calcium store depletion in rat hippocampal neurons induces long-term 

increases in the h current 

Authors: *R. NARAYANAN, D. JOHNSTON; 

Ctr. Learning & Memory, Univ. Texas at Austin, Austin, TX 

Abstract: The role of intracellular calcium stores in numerous forms of activity-dependent 

synaptic plasticity is well established. However, long-term plasticity in voltage-gated ion channel 

properties mediated by intracellular calcium stores has not been well explored. In this study, 

employing whole-cell patch-clamp recordings from 4-7 week old male Sprague Dawley rat 

hippocampal CA1 pyramidal neurons, we report that store depletion induced through ten minute 

treatment with either 20 µM cyclopiazonic acid (CPA) or 10 µM thapsigargin (TG) led to a longterm 

increase in the hyperpolarization-activated nonspecific-cation (h) current. This was inferred 

from the following significant changes at 40 min after baseline: a 35-40% reduction in input 

resistance, lower action potential firing frequency for given step current injections, a 40-50% 

increase in resonance frequency, a 300-500% increase in the total inductive phase, a 15-20% 

increase in resonance strength and a 25% reduction in temporal summation of alpha current 

injections (CPA: n=7; TG: n=5). We then tested this inference pharmacologically using 20 µM 

ZD7288, a specific h-channel blocker, in the recording pipette (n=5). We found that CPAinduced 

reductions in input resistance, action potential firing frequency and temporal summation 

were all abolished in the presence of ZD7288, while all impedance parameters displayed lowpass 

characteristics through the entire experimental period. We also found that all CPA-induced 

changes were blocked by 20 mM BAPTA in the recording pipette (n=6) emphasizing the 

necessity of postsynaptic calcium in the increase of the h current. Finally, all CPA-induced 

changes were significantly suppressed in the presence of NMDA receptor antagonists 50 µM 

D,L-APV and 10 µM (+)MK801 in the bath (n=7), highlighting the possible role for calcium 

entry through NMDA receptors in this form of plasticity. We are performing additional 

experiments to explore the mechanisms underlying this form of plasticity, and to understand the 

somato-dendritic locus of such plasticity and its relevance to activity-dependent forms of 

plasticity of the h current. These results suggest an active role for intracellular calcium stores in 

regulating intrinsic excitability and postsynaptic signal integration in CA1 pyramidal neurons. 

Disclosures: R. Narayanan, None; D. Johnston, None.

Poster 


and Activity 




Title: Activity-dependent depression of the spike after-depolarization generates long-lasting 

intrinsic plasticity in hippocampal CA3 pyramidal neurones 

Authors: *J. T. BROWN, A. D. RANDALL; 

MRC Ctr. Synaptic Plasticity, Univ. Bristol, Bristol, United Kingdom 

Abstract: Intrinsic neuronal plasticity is the process by which the core excitability of neurones is 

modified in response to a conditioning stimulus. Long-lasting forms of intrinsic plasticity have 

the potential to substantially alter information processing in the CNS thereby producing a broad 

range of functional consequences. CA3 pyramidal neurones in rat hippocampal slices exhibited a 

diverse range of stimulus-driven action potential firing patterns, however all cells exhibit an 

initial high frequency (~80-200 Hz) burst of spikes. In experiments performed in the presence of 

pharmacological blockers of fast synaptic transmission we have identified conditioning stimuli 

that generate long-term intrinsic plasticity in these cells. Both tonic and phasic periods of 

depolarization to -14 mV in voltage clamp produced robust and long-lasting changes in a number 

of facets of CA3 neurone excitability. The spike afterdepolarization (ADP) following a 2 nA, 2 

ms current injection was substantially depressed by conditioning. This change was accompanied 

by a substantial prolongation of the first inter-spike interval generated by longer depolarizing 

current injections. These long-lasting changes to excitability were termed depolarisation-induced 

plasticity (DIP). Having established that depolarisation applied in voltage clamp was capable of 

inducing DIP, we sought to establish whether more physiological stimulation paradigms could 

induce DIP. Short, high frequency burst of 2-4 action potentials, repeated at 5 or 10 Hz for 1 

minute effectively induced DIP. This form of DIP was eliminated by the inclusion of BAPTA in 

the recording pipette. The Kv7/KCNQ/M-channel blocker, XE991, completely eliminated 

conditioning-induced depression of the ADP. Furthermore, the Kv7 channel opener, retigabine, 

which under control conditions suppresses the ADP, was rendered inactive following the 

induction of DIP. These data indicate that modulation of Kv7 channels plays a central role in this 

novel form of long-lasting intrinsic plasticity. We have also established that activation of 

mGluR1 by either an exogenous agonist or via glutamatergic synaptic activity induces longlasting 

depression of the ADP and associated changes to firing patterns. Our findings represent 

the first demonstration of long-term plastic modulation of hippocampal ADPs and may represent 

a novel and important form of intrinsic plasticity.

Disclosures: J.T. Brown, None; A.D. Randall, None. 

Poster 


and Activity 




Support: RO1 EY 014439 

Title: Monocular deprivation reduces the intrinsic excitability of layer 4 pyramidal neurons in 

binocular cortex 

Authors: *M. E. LAMBO 1 , A. MAFFEI 2 , G. G. TURRIGIANO 1 ; 

1 Biol., Brandeis Univ., Waltham, MA; 2 Dept. of Neurobio. and Behavior, SUNY Stony Brook, 

Stony Brook, NY 

Abstract: Visual deprivation induces profound changes in visual response properties, but the 

activity-dependent plasticity mechanisms underlying them are poorly understood. Monocular 

deprivation (MD) rapidly decreases responsiveness of the deprived eye to visual drive. Here we 

examine the changes in intrinsic and synaptic response properties in the binocular region of 

primary visual cortex following 2 days of MD. We sutured one eye at p18 for 2 days and used 

whole-cell and perforated patch clamp recordings of layer 4 pyramidal neurons. Recordings were 

obtained from hemispheres contralateral and ipsilateral to the deprived eye and in sham operated 

rats, with the majority of comparisons being made between contralateral and sham conditions. 

Recordings of spontaneous firing rates under conditions that preserved synaptic drive indicated 

that MD reduced spontaneous firing by a factor of three (sham: 0.15±0.04 Hz, n=34; contra: 

0.05±0.02 Hz; n=23; p

whether this reduction in excitability requires an imbalance in drive from the two eyes, we 

generated FI curves from rats that underwent 2 days of binocular lid suture (BD). BD did not 

induce a reduction in excitability (sham n=14; BD n=16; p>0.22 for all current injections). 

Overall, the data indicate that MD suppresses spontaneous activity, at least in part through a 

reduction in intrinsic excitability. Further, the reduction in intrinsic excitability can be partially 

explained by a decrease in input resistance (but likely also involves voltage gated channels), and 

is a process which requires competitive input from both eyes. One locus of activity dependent 

plasticity in visual cortex is thus the intrinsic excitability of cortical pyramidal neurons. 

Disclosures: M.E. Lambo , None; A. Maffei, None; G.G. Turrigiano, None. 

Poster 


and Activity 




Support: NIH EY014439 

DOD W81XWH-04-1-0158 

Title: Metaplasticity of intrinsic excitability of cortical layer 5 pyramidal neurons induced by 

visual deprivation 

Authors: *K. NATARAJ, G. TURRIGIANO; 

Dept Biol, Brandeis Univ., Waltham, MA 

Abstract: Changes in visual experience dramatically alter the organization of visual cortical 

circuits, but the cellular plasticity mechanisms that drive these changes are poorly understood. 

Here we examined the role of long term potentiation of intrinsic excitability (LTP-IE) of layer-5 

pyramidal neurons in the experience-dependent changes induced by monocular deprivation 

(MD). MD was achieved with eyelid suture for 48 hours, starting at P18, and whole-cell patch 

clamp recording were obtained from the monocular region of primary visual cortex (Vm1) from 

the deprived and control hemispheres. Neurons from the deprived hemisphere had lower 

spontaneous firing rates than control neurons, and exhibited a right-ward shift in their firing rate 

vs. current (FI) curves, indicating a reduction in intrinsic excitability. LTP-IE was induced in 

control and deprived neurons by making the neurons fire 15 spikes at 40 Hz every 4 seconds for 

10 minutes, in the presence of synaptic blockers (50µM APV, 20 µM DNQX, and 20 µM

Picrotoxin). The induction protocol caused a significant increase in intrinsic excitability in cells 

from both hemispheres. However, MD induced a far greater increase in the LTP-IE in the 

deprived hemisphere compared to the control hemisphere (control hemisphere: 142.72±2.14% of 

control, n = 7; deprived hemisphere: 200.21±24.13% of control, n = 10: p < 0.001). Further, 

LTP-IE caused a leftward shift of the FI curve, and reduced the current threshold in both 

hemispheres, but did not alter passive neuronal properties, suggesting the involvement of voltage 

dependent conductances. These data suggest that MD reduces the intrinsic excitability of L5 

pyramidal neurons by reducing LTP-IE. Further, the increased magnitude of LTP-IE in deprived 

cortex suggests that LTP-IE could play a role in recovery from an insult or visual deprivation 

Disclosures: K. Nataraj , None; G. Turrigiano, None. 

Poster 


and Activity 




Support: the EJLB–Canadian Institutes of Health Research (CIHR) Michael Smith Chair in 

Neurosciences and Mental Health 

Title: Plastic changes of electrophysiological properties of cingulate neurons after nerve injury 

Authors: *X. CAO, H. XU, L. WU, M. ZHUO; 

Physiol., Univ. of Toronto, Toronto, ON, Canada 

Abstract: The anterior cingulate cortex (ACC), a large region around the rostrum of the corpus 

callosum, undergoes dramatic changes under chronic pain conditions (Zhuo, 2005; Zhuo, 2006). 

Here, we have tested the hypothesis that electrophysiological changes occurred in ACC cells 

after neuropathic pain. Whole-cell patch-clamp recordings from ACC neurons in mice brain slice 

preparation were used to record active and passive membrane properties. Three physiological 

groups of pyramidal cells were defined according to their action potential firing behavior: (i) 

Regular spiking (RS) cells that fired single spikes, with each spike followed by a prominent 

sAHP and an obvious deflection of fAHP and ADP during the hyperpolarizing phase; (ii) 

Intermediate (IM) cells that fired single spikes and the action potential was followed by a fast 

AHP, ADP, and sAHP; (iii) Intrinsic bursting (IB) cells that fired an initial spike doublet, 

followed by single spikes. The most frequent firing patterns observed in neuropathic and wild 

type animals were the IM cells (neuropathic: 53%, wild type: 49%), IB cells (neuropathic: 31%,

wild type: 29%) and RS cells (neuropathic: 16%, wild type: 22%). Membrane excitability of RS 

and IB neurons from neuropathic or wild type animals was indistinguishable. For IM cells, the 

action potential in neuropathic pain neurons has wider half width (neuropathic: 1.57 ± 0.02 ms, 

sham-treated: 1.46 ± 0.05 ms), slower decay slope (neuropathic: -37.9 ± 3.1 mV/ms, wild type: - 

45.7 ± 1.9 mV/ms) and longer decay time (neuropathic: 1.96±0.12 ms, wild type: 1.63±0.06 ms). 

The present results indicate that the peripheral nerve injury, which gives rise to neuropathic pain, 

produces profound changes in the action potential waveform of cingulate pyramidal neurons in a 

cell type-specific fashion. 

Disclosures: X. Cao , EJLB–Canadian Institutes of Health Research (CIHR) Michael Smith 

Chair in Neurosciences, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); H. Xu, None; L. Wu, None; M. zhuo, None. 

Poster 

241. Glial Neuronal Interactions 



Topic: B.11.b. Cell biology and signalling 

Support: Startup Fund from Texas A&M University 

Title: Modulation of Notch signaling enhances CNS myelination in aggregate cultures 

Authors: *H. KOITO, J. LI; 

Dept of Vet Integrative Biosci, Texas A&M Univ., College Station, TX 

Abstract: Myelination is essential for the development and function of the nervous system in 

vertebrates. Dysfunction of myelin or myelin-forming oligodendrocytes (OLs) in the CNS 

contributes to many neurological deficits associated with, for example, multiple sclerosis, 

cerebral palsy, schizophrenia, and Alzheimer‟s diseases. However, the molecular mechanisms 

that control CNS myelination remain poorly understood. To address the question how CNS 

myelination is initiated and regulated, we developed an in vitro CNS myelination system in 

which cell aggregates prepared from rat E16 forebrains were co-cultured with oligodendrocyte 

precursors. The CNS cell aggregates were composed of progenitors of neurons, astrocytes and 

OLs, and were cultured in a serum-free medium for up to 4 weeks. Radial growth of axons was 

evident after 1 or 2 days in culture (DIV) and was extensive around 2 weeks. Glial progenitors 

migrated out of the aggregates and gradually differentiated into OLs and astrocytes over the 

course of culturing. Exogenous OL precursors were added to the aggregate cultures at DIV 10 to 

increase the number of OLs. Many OL processes aligned with axons and started to ensheath

axons at DIV 18-22. Using this system, we examined the effect of several neurotrophic factors 

that have been reported previously to promote myelination. Both ciliary neurotrophic factor and 

leukemia inhibitor factor markedly promoted OL differentiation and survival but only 

moderately increased myelin formation. In contrast, γ-secretase inhibitor DAPT did not promote 

OL survival and differentiation, but significantly increased the population of ensheathing OLs 

and enhanced myelination in a concentration-dependent manner. Notch signaling has been 

implicated in regulating OL development and is subjected to γ-secretase regulation. The Notch 

ligand contactin/F3 appears to promote OL differentiation whereas Jagged1 inhibits OL 

differentiation. To examine whether DAPT promotes myelination by blocking the Notch 

signaling, the effect of soluble contactin1 and Jagged1 was studied. While contactin1 

significantly increased the number of internodes, Jagged1 had no effect. Combination of 

contactin1 and DAPT resulted in further increases in myelin formation. These data suggest that 

modulation of Notch signaling pathways may provide a new strategy for promoting CNS 

myelination and myelin repair. 

Disclosures: H. Koito, None; J. Li, None. 

Poster 






Title: Regulation of connexins in proliferating Schwann cells 

Authors: *M. M. FREIDIN, S. ASCHE, C. K. ABRAMS; 

Dept Neurol, SUNY Downstate Med. Ctr., Brooklyn, NY 

Abstract: Our lab has recently reported that Glial Growth factor II (GGF2), a member of the 

neuregulin family of growth factors, increases Schwann Cell (SC) expression of Connexin32 

(Cx32) and promotes SC survival, proliferation, and differentiation. Increases in GGF2 and its 

receptors (ErbB2 & ErbB3) are associated with SC proliferation during Wallerian degeneration. 

In addition, we found proliferating and non-proliferating populations of wild type (WT) SCs 

express Cx32, while SCs from Cx32-null (32KO) mice show reduced proliferative response to 

Neg1 as compared to WT. GGF2 also increases junctional conductances in WT SCs. Some of 

these cell-cell channels are characteristic of Cx32; however, the patterns of coupling are 

sufficiently complex and varied to suggest the involvement of additional connexins. Other

connexins have been identified in cultured SCs and in the SCs in peripheral nerve, but their role 

in promyelinating or proliferating SC is not well understood. To determine whether loss of Cx32 

influences the expression of other connexins and/or the expression of SC-specific and 

promyelinating genes, we are using real time PCR with Sybr green detection to investigate the 

expression profiles of the 20 identified mouse connexins genes in proliferating WT and 32KO 

SCs in vitro. Specifically, GGF2 upregulates expression of Cx32, Cx45, and Cx29 in cultured 

SCs from WT mice as compared to untreated controls. As predicted, treatment of WT SCs with 

GGF2 increases the expression of several promyelinating genes, including P0, as well. Current 

studies are comparing connexin and promyelinating gene expression in cultured WT and 32KO 

SCs grown in the presence or absence of GGF2. Finally, to determine whether loss of Cx32 

alters the expression profile of these connexins and promyelinating SC-specific genes during 

peripheral nerve regeneration, real time PCR studies will determine the relative changes in 

connexin and SC-specific gene expression in regenerating WT and Cx32-null sciatic nerves 

following crush injury. Examining the regulation of SC connexins and the genes associated with 

myelinating and non-myelinating SC phenotype will help define the cellular and transcriptional 

processes that control Cx32 expression before and after myelin formation and clarify the 

mechanisms involved in the maintenance, repair, and re-myelination of peripheral nerves. 

Disclosures: M.M. Freidin, None; S. Asche, None; C.K. Abrams, None. 

Poster 





Support: BMRC grant 04/1/21/19/305 

NMRC grant 0946/2005 

Title: Distribution and possible funcitons of juxtanodin as a specific marker protein of 

sustentacular cells of rat olfactory epithelium 

Authors: *W. LI, J. TANG, J. H. TANG, F. LIANG; 

Anat., Natl. Univ. of Singapore, Singapore, Singapore 

Abstract: Juxtanodin (JN) is found in oligodendroglia and jaxtanodal terminal loops of myelin 

sheath in the central nervous system (CNS). In tissues or cell types other than the CNS, JN 

expression and function remain much unknown. With Western blotting and immunoperoxidase

histochemistry, the present study first surveyed peripheral nerve, skeletal muscle as well as 

various epithelial tissues of the rat for possible JN expression. Double immunofluorescence 

confocal microscopy and immunoelectron microscopy were then employed to map the cellular 

and subcellular localization of JN. Out of the peripheral tissues examined, only olfactory 

epithelium showed prominent JN expression in the glia-like sustentacular cells and in the 

epithelial cells of the ducts of Bowman‟s glands. Tips of the apical processes of the sustentacular 

cells, in particular, were enriched with JN protein under the roots of sustentacular microvilli and 

at the junctions of sustentacular cells with apical processes of olfactory receptor neurons. Except 

for the root part, sustentacular microvilli themselves were mostly devoid of JN 

immunoreactivity. JN distribution in olfactory epithelium differed from that of microtubuleassociated 

protein 2 (a marker of neurons) or nestin, but overlapped with a zone of intense Factin 

staining near olfactory epithelium mucous surface. Immunoreactivities for 2‟,-3‟-cyclic 

nucleotide-3‟-phosphodiesterase and myelin basic protein were not detected in rat olfactory 

epithelium. These results indicate that sustentacular cells are oligodendroglialike in olfactory 

epithelium. The high levels of JN in the apical band of olfactory sustentacular cells conforms to 

JN enrichment along the edges of CNS myelin sheaths such as in the juxtanodal and paranodal 

terminal loops. Similar to functions of JN in oligodendroglial differentiation, CNS myelination 

or myelin-axon interaction, the roles of JN in olfactory epithelium may involve maturation of 

sustentacular cells, outgrowth or motility of sustentacular microvilli, as well as molecular 

interaction of sustentacular cells with olfactory receptor neurons for olfactory signal transduction 

at the mucous surface of olfactory epithelium. 

Disclosures: W. Li, None; J. Tang, None; J.H. Tang, None; F. Liang, None. 

Poster 





Title: Mechanisms of myelin basic protein recruitment to the plasma membrane 

Authors: S. NAWAZ 1 , *A. M. KIPPERT 2 , T. LANG 3 , K.-A. NAVE 1 , M. SIMONS 2,3 ; 

1 MPI for experimental Med., Göttingen, Germany; 2 Univ. of Göttingen, Göttingen, Germany; 

3 MPI for biophysical Chem., Göttingen, Germany 

Abstract: Oligodendrocytes are the myelinating glia of the central nervous system. One of the 

major CNS myelin proteins is myelin basic protein (MBP). It is involved in the compaction of 

the myelin sheath. Although it is essential for the formation of the myelin sheath, it is not known

how MBP performs this task and how it interacts with the myelin membrane. Here we asked the 

question whether phosphoinositides play a role in this process. To explore the possibility, 

whether MBP interacts with PIP2 and PIP3, we overexpressed inositol 5-phosphatase domain of 

synaptojanin 1 which eliminates PIP2 from the membrane. Both membrane sheets generated 

from these cells and quantification of plasma membrane localisation studies showed a reduced 

level of MBP. Additionally, overexpression of an Arf6 constitutive active form, which leads to 

the formation of PIP2 enriched endosomal membranes, showed accumulation of MBP in these 

membranes. When cells were treated with ionomycin, which activates Phospholipase C through 

influx of Ca 2+ and which in turn hydrolyses PIP2, MBP dissociated from the plasma membrane. 

Additionally ATP depletion showed a similar effect. We hypothesize that the interaction of MBP 

with PIP2 and possibly PIP3 might lead to its plasma membrane recruitment, which is essential 

for the assembly of myelin lipids and proteins. These protein and lipid interactions may play a 

fundamental role in the formation of myelin in the central nervous system. 

Disclosures: S. Nawaz, None; A.M. Kippert , None; T. Lang, None; K. Nave, None; M. 

Simons, None. 

Poster 





Support: NIH DE017805 

Title: Differential expression of MAP kinases and MAP kinase phosphatases in ipsilateral and 

contralateral trigeminal ganglion in response to capsaicin injection in the TMJ capsule 

Authors: *P. L. DURHAM, S. THALAKOTI; 

Biol Dept, 225 Temple Hall, Missouri State Univ., Springfield, MO 

Abstract: Inflammation of the temporomandibular joint (TMJ) joint, which is implicated in the 

pathology of TMJ disorders, can be caused by the release of neuropeptides from trigeminal 

neurons that provide sensory innervation of the joint tissues. We have previously shown that 

stimulation of cultured trigeminal neurons leads to activation of MAP kinases and induction of 

MAP kinase phosphatases (MKPs) that control neuropeptide gene expression. Although MAP 

kinases and phosphatases are known to play important roles in regulating cellular events in 

response to inflammation in other joints, their in vivo regulation in response to capsaicin-induced 

TMJ inflammation has not been investigated. Thus, the goal of this study was to determine the

temporal and spatial expression of MAP kinases and MKPs in ipsilateral and contralateral 

trigeminal ganglion cells in response to unilateral capsaicin injection into the TMJ capsule. 

Capsaicin stimulation of V3 trigeminal nerves at 2 hrs was found to increase the nuclear 

expression of the MAP kinase protein p38 in both neurons and satellite glial cells, but not 

Schwann cells. In contrast, increased levels of ERK were only observed in the nucleus of 

neuronal cells, but not glial cells, 2 hrs after capsaicin injection. The levels of p38 and ERK 

staining were greater in the ipsilateral ganglion when compared to contralateral ganglion at the 2 

hrs. By 24 hrs post injection, p38 and ERK levels had returned to basal levels. Somewhat 

surprisingly, no increase in JNK staining was observed in either neurons or glia in response to 

capsaicin stimulation. MKP-1 expression was readily detected in satellite glial cells but not 

neuronal cells under basal conditions. In response to capsaicin, MKP-1 levels were increased 

similarly in both neurons and satellite glial cells in both ipsilateral and contralateral ganglion 2 

hrs after injection and then remained elevated even after 24 hrs. However, elevated levels of 

MKP-2 and MKP-3 were only observed in neurons in both ganglia at 2 and 24 hrs post injection. 

In summary, we have shown that stimulation of trigeminal neurons that provide sensory 

innervation of the TMJ leads to increased levels of active p38 and ERK MAP kinases as well as 

a sustained induction of MKPs in all regions of the ipsilateral and contralateral ganglia. We 

propose that the induction of MKPs in response to capsaicin injection into the TMJ capsule plays 

an important role in regulating neuronal gene expression in response to acute inflammatory 

stimuli by decreasing expression of active MAP kinases to basal levels in both neurons and 

satellite glial cells. 

Disclosures: P.L. Durham, NIH DE017805, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); S. Thalakoti, 

None. 

Poster 





Support: New York State Spinal Cord Injury Research Board 

NIH Grant CA128158-12 

Title: GFAP-expressing astrocytes respond to sonic hedgehog signaling in the adult CNS

Authors: *A. GARCIA 1 , L. ENG 3 , A. L. JOYNER 2 ; 

1 Rockefeller Res. Labs, 2 Developmental Biol., Mem Sloan Kettering Cancer Ctr., New York, 

NY; 3 New York Univ., New York, NY 

Abstract: The secreted signaling factor sonic hedgehog (Shh) is critical for proper development 

of the vertebrate CNS. Multiple roles for Shh signaling in the developing CNS have been 

identified, including ventral patterning, cell specification, regulation of proliferation and cell 

death, as well as axon pathfinding. Expression of Shh signaling pathway components persist into 

adulthood, but their role in the mature CNS has not been well characterized. Induction of the 

zinc-finger transcription factor Gli1 has been shown to be a reliable marker of cells responding 

to Shh signaling in the CNS. We used Gli1-lacZ knock-in mice, as well as a genetic inducible 

labeling strategy to examine the distribution and cell types responding to Shh signaling in the 

adult mammalian CNS. Our results indicate that Shh-responding cells are widely distributed 

throughout multiple nuclei of the adult forebrain. In the adult spinal cord, Shh-responding cells 

were observed primarily within dorsal gray matter, with scattered cells labeled in ventral gray 

matter. We performed double labeling, immunohistochemistry for β-galactosidase (β-gal) and 

cell type specific markers and performed single cell analysis. The majority of β-gal positive cells 

co-labeled with glial fibrillary acidic protein (GFAP), and exhibited an astrocytic morphology. A 

small percentage of β-gal labeled cells co-labeled with the oligodendrocyte marker, CAII. BrdU 

labeling experiments indicate that the majority of Shh-responding cells in the adult CNS are not 

proliferating, suggesting that in contrast to development, Shh signals primarily to a terminally 

differentiated cell population in the adult. Interestingly, not all GFAP-positive astrocytes respond 

to Shh signaling, as hippocampal CA1 astrocytes did not express β-gal. The functional 

significance of Shh signaling in astrocytes of the mature CNS is unknown, but these data raise 

the intriguing possibility that Shh might play a novel role in astrocyte function. 

Disclosures: A. Garcia, None; L. Eng, None; A.L. Joyner, None. 

Poster 



Program#/Poster#: 241.7/G1 


Support: Swiss National Science Foundation Grant 3100A0-105773 

Title: Noradrenaline activates the CREB coactivator TORC2 through the cAMP-signaling 

pathway in astrocytes

Authors: M. TOSIC 1 , I. ALLAMAN 2 , P. J. MAGISTRETTI 1,2 , *J.-R. CARDINAUX 1 ; 

1 Dept of Psychiatry-CHUV, Ctr. for Psych. Neurosci., Prilly-Lausanne, Switzerland; 2 Lab. of 

Neuroenergetics and Cell. Dynamics, Brain Mind Institute, Ecole Polytechnique Fédérale de 

Lausanne (EPFL), Lausanne, Switzerland 

Abstract: In the brain, many cAMP- and calcium-responsive genes are regulated by the 

transcription factor CREB, its coactivator CREB-Binding Protein, and a family of coactivators 

called Transducers of regulated CREB activity (TORCs). In neurons, synergistic effect of cAMP 

and calcium on CREB-mediated transcription depends mainly on TORC1 nuclear translocation 

and its binding to CREB (Kovács et al., 2007. PNAS 104, 4700-4705). Here we investigated 

whether one of these coactivators, TORC2, could serve as a cAMP and calcium-sensitive 

coincidence detector in mouse primary astrocytes. In these cells forskolin alone induced TORC2 

nuclear translocation and CRE-dependent transcription as efficiently as simultaneous exposure to 

forskolin and ionomycin, which respectively stimulate cAMP and calcium pathways. Similarly, 

massive TORC2 translocation from cytoplasm to nucleus and activation of a CRE-reporter gene 

were observed when astrocytes were exposed to 10κM noradrenaline (NA). Western blot 

analysis using a phospho-Ser133-specific antibody showed that NA does not activate CREB by 

increasing its phosphorylation. Activation of TORC2 by NA was drastically decreased by the 

beta-adrenergic receptor antagonist propranolol, whereas the alpha-adrenergic receptor 

antagonist phentolamine had a weak effect. Combination of both antagonists completely 

abolished TORC2 nuclear translocation and CREB-mediated transcription. Moreover, we 

showed that the beta-adrenergic receptor agonist isoproterenol activates TORC2 to the same 

extent as NA. Altogether these results strongly suggest that NA activates CREB-regulated gene 

expression in astrocytes through TORC2 mobilization using mostly or exclusively the cAMP 

pathway. 

Disclosures: M. Tosic, None; I. Allaman, None; P.J. Magistretti, None; J. Cardinaux , None. 

Poster 




Topic: B.11.c. In vivo approaches 

Support: NIH 

Title: Transgenic mice with photoswitchable GFP in Schwann cells

Authors: *W. J. THOMPSON 1 , Y. LEE 2 , Y. LI 1 , S. MAIKA 1 , M. MIKESH 1 , P. KRIEG 3 ; 

1 Sec Neurobiol, Univ. Texas, Austin, TX; 2 Univ. of Texas, Austin, TX; 3 Univ. of Arizona, 

Tucson, AZ 

Abstract: Green fluorescent proteins (GFPs) isolated from coral have been found whose 

fluorescence properties can be irreversibly altered by illumination. Two such proteins, Kaede and 

Dendra2, fluoresce green with blue light illumination. However, once illuminated with longer 

wavelengths, the proteins undergo a permanent conversion, lose their green fluorescence and 

emit red light when illuminated with green light. These properties allow these “photoswitchable” 

GFPs to be used as “optical highlighters” so that cells or even proteins tagged with these FPs can 

be marked and followed over time. Here we report our attempts to use Kaede and Dendra2 as 

cellular markers in mouse transgenics. We prepared mice that express Kaede or Dendra2 under 

the control of S100 promoter, a promoter that has been previously used to drive GFP expression 

in Schwann cells (SCs). Our experience with Kaede was disappointing. Only 1 of 3 established 

lines had any detectable green Kaede fluorescence (found in Langerhans cells of the skin but 

absent from SCs). On the other hand, Dendra2 proved to be a robust fluorescent marker in 

transgenic mice. A Dendra2 expressing pup can be easily identified under a fluorescence 

dissecting microscope due to green fluorescence in the Langerhans cells of the skin and a green 

lens in the eye. SCs in Dendra2 transgenic mice fluoresce green and can be photoswitched to red 

fluorescence using the UV illumination provided by a DAPI filter cube. We were able to 

optically highlight individual terminal SCs at a mouse neuromuscular junction by illuminating a 

soma; the processes quickly filled with the red fluorescent product. Although we are unable to 

achieve full green-to-red conversion, photoswitched Dendra2 persists at sufficient levels to 

clearly mark the photoconverted cells for at least 10 days in a living mouse. A second 

photoswitching can extend the observation period. Thus photoswitching can be used in 

transgenic mice expressing Dendra2 to identify individual cells and their processes and to 

optically highlight these cells so they can be re-identified during a subsequent imaging session. 

Supported by the NIH. 

Disclosures: W.J. Thompson , None; Y. Lee, None; Y. Li, None; S. Maika, None; M. Mikesh, 

None; P. Krieg, None. 

Poster 





Support: OMHF

OGS 

CIHR 

Title: Brain-specific cross-reactivity of connexin32 antibodies: problems and solutions 

Authors: *S. L. FOWLER 1 , A. C. MCLEAN 2 , S. A. L. BENNETT 2 ; 

1 Dept of Biochem, Micro, Immuno, Univ. of Ottawa, Ottawa, ON, Canada; 2 Univ. Ottawa, 

Ottawa, ON, Canada 

Abstract: Connexins are members of a highly homologous family of transmembrane-spanning 

proteins that oligomerize to form gap junction channels. Connexin32 (Cx32) is the most highly 

expressed connexin in the liver and is also present in oligodendrocytes and their progenitor cells. 

Previous work from our laboratory has implicated Cx32 as an important mediator of neural 

progenitor cell specification. However, further study has been complicated by problems with 

antigenic cross-reactivity in central nervous system (CNS) tissue when analyses are verified 

using the gold-standard control of the Cx32 null-mutant mouse. Here, we demonstrate that the 

vast majority of commercially available antibodies detect a protein in both wild-type (WT) and 

Cx32 null-mutant mice that migrates with a mobility of 27 kD. We show that this cross-reactive 

epitope is specific to CNS. Liver lysates derived from Cx32 null-mutant mice do not exhibit nonspecific 

labeling. We further demonstrate that WT Cx32 in liver lysates migrates with a 

molecular weight that is 4 kDa higher than the anti-Cx32-reactive protein in brain and spinal 

cord. We find that this artifactual labeling is only observed under denaturing conditions. Crossreactivity 

with a reactive protein of approximately the same mobility as WT Cx32 is not detected 

in brain lysates of Cx32 null-mutant mice when tertiary structure is maintained and protein 

expression is analyzed by immunoprecipitation, immunofluourescence, or ELISA. Finally, by 

sucrose gradient fractionation and mass spectrometry analysis of whole brain and corpus 

callosum, we have identified the subcellular compartments that contain the cross-reactive protein 

and characterized the cross-reactive epitopes. These carefully controlled antibody 

characterizations, comparing reactivity in WT and Cx32-null mutant CNS within the context of 

multiple protein analysis techniques, permit the proper detection and quantification of Cx32 from 

brain and spinal cord, and provide essential information for accurate Cx32 investigation in the 

brain. 

Disclosures: S.L. Fowler, None; A.C. McLean, None; S.A.L. Bennett, None. 

Poster 



Program#/Poster#: 241.10/G4


Support: CIHR 

OMHF 

Title: Citalopram mediated neural progenitor cell proliferation and survival 

Authors: *A. S. PETTIT, S. A. L. BENNETT; 

Dept Biochem, Micro, Immuno, Univ. of Ottawa, Ottawa, ON, Canada 

Abstract: Depression is associated with a decreased hippocampal volume that correlates with 

the length of the depressive episode. Converging evidence suggests that this pathology results 

from both progressive loss of neurons and glia and impaired regeneration. Successful 

amelioration of the depressive phenotype by chronic antidepressant treatment correlates with an 

increase in neural progenitor cell (NPC) proliferation and maturation in experimental models of 

disease matching the kinetics of clinical antidepressant efficacy. These correlations have led 

researchers to hypothesize that neurogenesis is required for antidepressant drug action. 

Citalopram, a selective serotonin reuptake inhibitor, is one of the most widely prescribed 

antidepressants on the market to date. Few studies have examined the impact of citalopram on 

cell proliferation and maturation in the subgranular zone (SGZ) of the dentate gyrus of the 

hippocampus, one of the primary sites of neurogenesis in the adult mammalian brain. Using 

halogenated thymidine analogue labeling, we have studied the effect of subchronic (7 or 14 day) 

and chronic (28 day) citalopram treatment on NPC proliferation and survival in the SGZ of 

wildtype mice identifying the progenitor subtypes responsive to antidepressant treatment. 

Disclosures: A.S. Pettit , None; S.A.L. Bennett, None. 

Poster 





Support: NINDS, NIH Intramural Funds 

Title: Site specific phosphorylation of human septin 5 (sept5) at serine 327 by cyclin dependent 

kinase 5 regulates secretion

Authors: *N. D. AMIN 1 , Y. ZHENG 1 , S. KESAVAPANY 2 , J. KANUNGO 1 , T. 

GUSZCZYNSKI 3 , R. K. SIHAG 4 , P. RUDRABHATLA 1 , W. ALBERS 1 , P. GRANT 1 , H. C. 

PANT 1 ; 

1 LNC, NINDS, NIH, Bethesda, MD; 2 Dept. of Biochem. , Yong Loo Lin Sch. of Med., Natl. 

Univ. of Singapore, Singapore, Singapore; 3 Lab. of Cell and Developmental Signaling, Natl. 

Cancer Inst., Frederick, MD; 4 Ctr. for biologics evaluation & resch, FDA, Bethesda, MD 

Abstract: Cyclin dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system 

where it is involved in neuronal migration, synaptic transmission and survival. The role of Cdk5 

in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins 

such as, synapsin, Munc18 and dynamin 1. Its multifunctional role at the synapse is complex and 

probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid 

screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known 

also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in 

GST- pulldown and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates 

SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A 

serine (S) to alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild 

type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild type 

and knock out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding 

to syntaxin-1. Moreover, mutant non-phosphorylated SEPT5 potentiated regulated exocytosis 

more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 

phosphorylation of human septin, SEPT5 at S327 plays a role in regulating exocytotic secretion. 

Disclosures: N.D. Amin, None; Y. Zheng, None; S. Kesavapany, None; J. Kanungo, None; T. 

Guszczynski, None; R.K. Sihag, None; P. Rudrabhatla, None; W. Albers, None; P. Grant, 

None; H.C. Pant, None. 

Poster 





Support: NIH 1R21NS047543-02 

Funds from Penn State University 

Title: Group I metabotropic glutamate receptors regulate the release of neuropeptide Y from 

dense core granules in cortical astrocytes

Authors: *P. RAMAMOORTHY, M. D. WHIM; 

Dept Biol, Pennsylvania State Univ., State College, PA 

Abstract: Neuropeptide Y is a widely distributed neuropeptide that is thought to modulate a 

variety of physiological processes including blood pressure, feeding and the stress response. We 

have previously found that cortical astrocytes can synthesize neuropeptide Y (NPY) and that the 

newly synthesized peptide enters the regulated secretory pathway. However the pathway(s) that 

control the secretion of neuropeptide Y are not clear. To address this question, cortical astrocytes 

were isolated and maintained in vitro. At 8-12 DIV astrocytes were stimulated with glutamate for 

1 min, then immediately fixed and stained for NPY-immunoreactivity (NPY-ir) without 

permeablization. After stimulation many cells displayed surface NPY-ir puncta. Control 

experiments indicated that the NPY-ir puncta only appeared after stimulation and were not due to 

intracellular staining. Thus the NPY-ir puncta arose from the fusion of NPY-containing granules 

with the cell membrane and subsequent display of the peptidergic core. Varying the time 

between stimulation and fixation indicated that the NPY-ir puncta disappeared with a time 

constant of ~ 1.6 mins. To clarify whether the disappearance of the puncta reflected NPY 

secretion (rather than internalization) we monitored secretion from astrocytes expressing NPYred 

fluorescent protein (NPY-RFP). In these cells following glutamate stimulation, the intensity 

of individual NPY-RFP puncta declined in a step-like manner indicating a regulated secretion of 

the granular contents. The glutamatergic-dependent appearance of the NPY-ir puncta was not 

prevented by the presence of CPP and NBQX, antagonists of ionotropic glutamate receptors. 

However t-ACPD (100 µM), an agonist of group I/II metabotropic glutamate receptors was as 

effective as glutamate in causing secretion. (S)-3,5-DHPG, an agonist of group I mGluR‟s also 

triggered the surface appearance of NPY-ir puncta, while (2R,4R)-APDC, an agonist of group II 

mGluR‟s was ineffective. Group I mGluR‟s are coupled to calcium signaling and experiments 

using fura-2 indicated that glutamate caused a transient rise in intracellular calcium. Collectively 

these results suggest that activation of group I mGlu receptors leads to the elevation of 

intracellular calcium which subsequently triggers the fusion of peptidergic granules and release 

of NPY from cortical astrocytes. Since NPY is a widespread and potent regulator of synaptic 

transmission, these results suggest that astrocytes could play a role in the peptidergic modulation 

of synaptic signaling in the CNS. 

Disclosures: P. Ramamoorthy , None; M.D. Whim, None. 

Poster 




Topic: B.11.b. Cell biology and signalling

Support: MA 037 

Title: Process formation in astroglia: scanning electron microscopy study 

Authors: *E. M. ABD-EL-BASSET; 

Dept Anat, Kuwait Univ. Fac Med., Safat 13100, Kuwait 

Abstract: This study describes and compares the appearance of astroglia and their processes 

during the differentiation of astroglia in colony cultures before and after treatment of astroglia 

with dibutyryl cyclic AMP (dBcAMP) and Cytochalasin D (CD) using scanning electron 

microscopy. The small cells (SC) in the center of astroglia colonies had many microvilli near the 

intercellular junctions. More peripheral medium sized cells (MC) and the large cells (LC) had 

many thin processes that were in contact with those of adjacent cells. The surface of the cell 

body in region adjacent to the processes had folds and blebs. The LC at the periphery of the 

colony had similar folds and blebs proximally from the lamellipodia which extended from their 

free surface. The mature small stellate astrocytes (SSA) had fewer, thicker and longer processes 

some of which branched and/or ended in resembling structures growth cones. When the astroglia 

were treated with dBcAMP the cells also acquired many thin processes which were similar to 

those seen in untreated MC and LC. In addition the cell body had complete or incomplete 

circular folds in the region from which the processes radiated. When both the untreated and the 

dBcAMP-treated astroglia were exposed to CD the thin processes disappeared and the cells 

acquired long thick processes instead. The formation of the thin processes in astroglia during 

their differentiation in colony cultures and after treatment with dBcAMP may be due to 

polymerization of actin filament bundles at the cell periphery followed by retraction of the 

cytoplasm away from these bundles. However, the formation of the much longer and thicker 

processes in SSA and CD treated cells after disassembly of actin filaments may instead be due to 

the retraction of cytoplasm away from the bundles of the more stable intermediate filaments (IF) 

which then persist in the longer processes. 

Disclosures: E.M. Abd-El-Basset , None. 

Poster 





Support: OMHF

CIHR 

Title: Connexin32 expression is under hormonal control in the adult female mouse spinal cord 

Authors: *A. C. MCLEAN 1 , S. A. L. BENNETT 2 ; 

2 Dept. of Biochemistry, Microbiology and Immunol., 1 Univ. of Ottawa, Ottawa, ON, Canada 

Abstract: Connexin32 (Cx32) is expressed by mature oligodendrocytes and a subset of early 

oligodendrocyte progenitor cells in the adult murine central nervous system. Connexin 

expression across various tissues has been proven to be under hormonal regulation by estrogen 

and/or progesterone. Previously, we have demonstrated that Cx32 expression is under estrogenspecific 

regulation within the hippocampus of the brain. Here we establish that Cx32 is under 

hormonal control within the spinal cord in adult female C57BL/6 mice. To determine expression 

levels over the estrous cycle, the T11-T13 region of spinal cord were dissected from females 

during estrus, metestrus/diestrus, and proestrus. mRNA was quantified using qPCR. Perfused 

tissue was sectioned and Cx32 was visualized by immunofluorescence. Cell types were 

delineated by colabelling with antigenic lineage markers for mature oligodendrocytes and early 

oligodendrocyte progenitors. Protein quantification was assessed by immunoprecipitation and 

western blot analysis. Cx32 was visualized by immunofluorescence and colabeled with antigenic 

lineage markers. To ensure specificity, congenic Cx32 null-mutant female mice were used as 

negative controls. MULAN promoter analysis was performed to detect estrogen or progesterone 

responsive elements in the Cx32 promoter. We have determined Cx32 mRNA and protein 

expression levels are suppressed during proestrus when estrogen and progesterone levels are 

highest and maximal during estrus when ovarian hormone levels are lowest. We show that Cx32 

expression at both the mRNA and protein level fluctuates in the T11-T13 region of the spinal 

cord over the course of estrous. These results demonstrate that Cx32 expression is under the 

control of ovarian steroid hormones in the central nervous system of adult female mice. 

Disclosures: A.C. McLean , None; S.A.L. Bennett, None. 

Poster 





Support: NMSS RG3669 

NS 25304

Title: Plp gene mutation results in dysfunction of calcium activated potassium channels in the 

MD rat 

Authors: *C. A. MAYER 1 , C. G. WILSON 1 , W. B. MACKLIN 2 , M. J. MILLER 1 ; 

1 Case Western Reserve Universit, Cleveland, OH; 2 Neurosciences, Cleveland Clin., Cleveland, 

OH 

Abstract: In the myelin deficient (MD) rat, a point mutation in the proteolipid protein (Plp) gene 

results in dysmyelination, seizures and lethal hypoxic ventilatory depression by postnatal day 21. 

The PLP protein is primarily expressed in oligodendrocytes. However, we have shown 

expression in second order neurons in an area of the brainstem involved in control of breathing, 

the nTS. We hypothesized that expression of the MD PLP protein in nTS neurons results in 

abnormal neuronal signaling at the age of early death (P21). To test this hypothesis, we used 

whole-cell patch-clamp recording to examine the electrophysiological properties of individual 

nTS neurons in 300µm brainstem slices from 14 (P14) and 21 (P21) day old MD rats and wild 

type (wt) littermates. Under current-clamp, cells were depolarized with injected current and 

action potentials were recorded. We quantified, from initiation of depolarization, latency of the 

action potential (AP) peak, afterhyperpolarization (AHP), and return to baseline. At P14, an age 

where MD rats do not show lethal hypoxic ventilatory depression, there were no significant 

differences between nTS cells from MD and wt pups in latency of the AP peak (p





Support: Mission Connect 


National Multiple Sclerosis Society 

Dr. Miriam and Sheldon G. Adelson Medical Research Foundation 

Title: Molecular mechanisms of CNS node of Ranvier formation 

Authors: *Y. OGAWA 1 , K. SUSUKI 1 , Y. ESHED 2 , E. PELES 2 , M. N. RASBAND 1 ; 

1 Baylor Coll Med., Houston, TX; 2 Mol. cell biology, Weizmann Inst. of Sci., Rehovot, Israel 

Abstract: In myelinated nerve fibers, action potential conduction depends on polarized axonal 

membrane domains including axon initial segments and nodes of Ranvier. High densities of 

voltage-gated Na+ (Nav) channels at the axon initial segment initiate action potentials, whereas 

nodal Nav channels mediate the currents necessary for rapid and efficient action potential 

conduction along the axons. However, despite their importance, the molecular mechanisms 

underlying formation of these domains are not fully understood. Several mechanisms have been 

proposed to mediate the accumulation of nodal molecules including: 1) restriction of channel 

mobility by paranodal junctions, 2) clustering and recruitment of nodal proteins by axonal 

cytoskeletal scaffolds such as ankyrinG, and 3) Nav channel clustering induced by a soluble, 

glial derived factor. In the peripheral nervous system, the glial derived extracellular matrix 

(ECM) molecule called gliomedin interacts with axonal cell adhesion molecule neurofascin (NF) 

186, and initiates clustering of the nodal molecules. In contrast, in the central nervous system 

(CNS), gliomedin is not present in the nodal region. Nevertheless, we show here that a 

specialized ECM is assembled at CNS nodes of Ranvier, and that a component of this ECM, a 

chondroitin sulfate proteoglycan called brevican, binds directly to NF 186, suggestive of a role 

similar to gliomedin. To test whether interactions between NF186 and the ECM could account 

for recruitment of NF186 to CNS nodes, we introduced NF186 truncated constructs into layer 

II/III pyramidal neurons, and observed nodes in corpus callosum. We found that the NF186 

ectodomain alone (i.e. no cytoskeletal scaffold binding) was sufficient to target this construct to 

CNS nodes. However, while deletion of the NF186 ectodomain (i.e. no ECM binding) resulted in 

failure of clearance from internodal axon, the truncated NF186 was still clustered at nodes. 

Furthermore, a careful developmental analysis indicated that paranodal junctions formed before 

the clustering of NF186 and the accumulation of nodal brevican. These results suggest that a 

single mechanism cannot account for the assembly of CNS nodes of Ranvier. We propose that

paranodal, ECM, and axonal cytoskeletal interactions all contribute to CNS node of Ranvier 

formation, and can compensate for one another. 

Disclosures: Y. Ogawa, None; K. Susuki, None; Y. Eshed, None; E. Peles, None; M.N. 

Rasband, None. 

Poster 





Support: AutoDesk 

IBM 

CIHR 

CFI 

OMHF 

Title: 3D modelling of connexin expression in postnatal neural progenitor cells 

Authors: *V. PESHDARY 1 , S. IMBEAULT 1 , K. WURTS 2 , L. G. GAUVIN 1 , S. A. L. 

BENNETT 1 , S. FAI 2 ; 

1 Dept of Biochem, Micro, Immuno, Univ. of Ottawa, Ottawa, ON, Canada; 2 Architecture, 

Carleton Univ., Ottawa, ON, Canada 

Abstract: New neurons and glia are generated in adult brain by rare populations of activated 

neural stem and progenitor cells (NPCs). As a result of asymmetric division, these single cells 

can both self-renew and produce intermediate progeny that terminally specify to functional 

central nervous system cells. Determining how the fates of two initially identical daughter cells 

diverge within the same microenvironment is key to facilitating therapeutic cell replacement. 

Evidence that postnatal NPCs express a significant number of connexin proteins has raised new 

questions about the roll of cell-cell interaction in determining NPC fate. Our understanding of 

these interactions is hampered by the fact that most studies rely on 2D representations to describe 

the 3D microenvironment. Here, we employed an interdisciplinary approach to assess connexin 

expression in NPCs cultured as 3D neurospheres. Using multiple digital simulation technologies

used by engineers, medical researchers, and architects, we show that specific connexins exhibit 

not only medial-lateral specificity (i.e. localize to cells in core or periphery of the neurosphere) 

but also a unique anterior-posterior polarity with respect to proximity to the air-media interface. 

Localization of specific connexins within this spatial gradient to different NPC lineages suggests 

a structural determination of NPC fate that can be altered by connexin deletion. 

Disclosures: V. Peshdary, None; S. Imbeault, None; K. Wurts, None; L.G. Gauvin, 

None; S.A.L. Bennett, None; S. Fai, None. 

Poster 




Topic: B.11.a. Synapses 

Support: Swiss National Science Foundation Grant # 3100A0-119827/1 

Title: Measurement of local metabolic responses in neocortical astrocytes in intact circuits by 

single-cell electroporation-mediated delivery of fluorescent probes 

Authors: *C. M. LAMY 1 , J.-Y. CHATTON 1,2 ; 

1 Dept. of Physiol., 2 Dept. Cell. Biol. Morphol., Univ. of Lausanne, Lausanne, Switzerland 

Abstract: Astrocytes play an active role in matching brain energy demands to neuronal activity. 

They express Na + -coupled excitatory amino-acid cotransporters (EAATs) that remove glutamate 

spilling over from the synaptic cleft. The resulting increase in astrocytic Na + leads to an 

enhancement of Na/K ATPase activity, ATP consumption, glucose uptake and glycolysis. 

Moreover, astrocytes propagate intercellular ATP-mediated Ca 2+ waves. Glutamate released by 

astrocytes during the Ca 2+ wave is recaptured by EAATs which results in the regenerative 

propagation of cytosolic Na + increase and a correlated augmentation of glucose uptake. Although 

Ca 2+ waves have been shown in slice models, evidence of subsequent metabolic responses in 

intact circuits are scarce due to limitations of dynamic fluorescent imaging techniques on brain 

slices. Bulk-loading of esterified forms of ion-sensitive dyes is hindered by poor signal-to-noise 

ratio. On the other hand delivering such dyes through a patch pipette is marred by interferences 

with intracellular medium composition and buffering of cellular responses by large pipette 

volume. We evaluated the use of single-cell electroporation (SCE) to deliver ion-sensitive dyes 

to astrocytes to study their metabolic responses in intact circuits. Acute slices were obtained 

from somatosensory cortex of Sprague-Dawley rats (P15-P23) and incubated in sulforhodamine 

101 to specifically stain astrocytes. Sodium-binding benzofuran isophthalate (SBFI) or the Ca 2+ -

sensitive dye Fura-2 were loaded in a patch pipette and electroporated into astrocytes selected in 

layer 2/3. High intracellular concentrations of both dyes were obtained and remained stable for 

more than one hour without any detectable photodamage in low-light level imaging conditions. 

Local application of ATP induced Ca 2+ spikes in Fura-2 loaded astrocytes while local application 

of glutamate produced an increase in cytosolic Na + in SBFI-loaded astrocytes, indicating intact 

cellular responses after SCE. To check whether endogenous release of glutamate would produce 

similar effects, we placed a concentric stimulation electrode in layer 4 of the same cortical 

column. Short trains of 10-20V pulses induced Na + increases in layer 2/3 astrocytes that were 

blocked by tetrodotoxin. EAATs inhibitors threo-β-benzyloxyaspartate and dihydrokainic acid 

impaired evoked astrocytic Na + responses evidencing a predominant role of glutamate uptake 

mechanisms. In conclusion, SCE is a powerful tool for optical imaging of physiological 

responses in the slice model. The responses recorded in intact circuits are reminiscent of 

metabolic waves described in primary cultures of astrocytes. 

Disclosures: C.M. Lamy , None; J. Chatton, None. 

Poster 



Program#/Poster#: 241.19/H1 


Support: NIH R01 NS054736-01 

Title: Geometric and physiological properties of functional connectivity patterns arising in 

spontaneously forming neuron networks 

Authors: *K. W. CHIAO, G. A. SILVA; 

UC San Diego, La Jolla, CA 

Abstract: Spontaneously forming neuronal networks provide a useful model system for probing 

mechanisms giving rise to and regulating intrinsic spontaneous activity. In addition, the 

application of electrophysiological and pharmacological manipulations, combined with optical 

imaging with calcium and voltage sensitive dyes, allow for the dissection of network dynamics 

into its component connectivities. Well-studied synapses provide examples of the range of 

diversity in synaptic connections, which include differences in both the onset (bursting, 

continuous, or delayed) and sustained (irregular, accommodating, non-accommodating, or 

stuttering) pattern of activity generated in the post-synaptic cell. The propensity of dissociated

neurons to form the connections that produce such recurring patterns of electrical activity is 

examined in the context of spontaneously forming networks in dissociated neuronal cultures. 

Disclosures: K.W. Chiao , None; G.A. Silva, NIH R01 NS054736-01, B. Research Grant 


received). 

Poster 






NIH GM3775 

CIHR 

NIH EY014127 

Title: Connexin 36 is implicated in control of post-natal murine neural progenitor cell survival 

and specification 

Authors: *C. D. SORBARA 1 , D. L. PAUL 3 , S. A. L. BENNETT 2 ; 

2 Biochem, Micro, Immuno, 1 Univ. Ottawa, Ottawa, ON, Canada; 3 Neurobio., Harvard Med. 

Sch., Boston, MA 

Abstract: The adult mammalian brain contains neural stem and neural progenitor cells (NPCs) 

that have the ability to self-renew and differentiate into functional neurons, astrocytes or 

oligodendrocytes. These cells can be identified in vivo by their expression of nestin and glial 

fibrillary acidic protein (GFAP). The progeny of these multipotential cells can generate 

doublecortin-positive (DCX+) neuroblasts committed to a neuronal lineage. Here, we examined 

the influence of connexin36 (Cx36) on NPC proliferation and survival in the hippocampus in 

vitro and in vivo in both wild type (WT) and Cx36-/- mice. Using bromodeoxyuridine (BrdU) to 

label actively proliferating cells in uninjured mice and following kainic acid-induced seizures, 

we show that Cx36 deletion does not alter cell proliferation in the subgranular zone (SGZ) of the 

dentate gyrus, a well-established neurogenic niche. However, using cultured neurospheres from 

P0-P3 mice pup hippocampi, we show that constitutive deletion of Cx36 decreases both NPC

survival and specification to a TuJ1+ neuronal lineage. We show that the impact of Cx36 

expression is further modulated by the addition of paracrine neurogenic factors. Together, these 

data implicate Cx36 in directing postnatal NPC fate in the postnatal hippocampus. 

Disclosures: C.D. Sorbara , None; S.A.L. Bennett, None; D.L. Paul, None. 

Poster 





Support: OGSST 

OMHF 

NSERC 

CIHR 

Title: Localization of connexin 29 to oligodendrocyte progenitor cells and role in progenitor 

proliferation 

Authors: *S. IMBEAULT, V. PESHDARY, H. TOEG, S. A. L. BENNETT; 

Dept of Biochem, Micro, Immuno, Univ. of Ottawa, Ottawa, ON, Canada 

Abstract: Connexins (Cxs) are a highly homologous protein superfamily which are the protein 

components of gap junctions. In recent years, evidence for hemichannel and channelindependent 

signalling has come to light in addition to mediating gap junctional intercellular 

communication. Connexin 29 is one of the most divergent connexins and is known to be 

expressed in myelinating fibers of the central and peripheral nervous systems. Here we report the 

expression of Cx29 by platelet-derived growth factor receptor alpha (PDGFαR) expressing cells 

in both the subgranular zone (SGZ) and the subventricular zone (SVZ) of the adult mouse brain 

and in primary neurosphere cultures obtained from these two areas in postnatal animals as 

simplified models of CNS development. Using null-mutant mice expressing a β-galactosidase (βgal) 

reporter gene under the control of the Cx29 promoter, co-expression of β-gal was observed 

in PDGFαR+ early oligodendrocyte progenitors and RIP+ oligodendrocytes but not in cells 

expressing the markers nestin (Type 1 and 2a neural progenitors), NG2 (polydendrocytes, early 

oligodendrocyte progenitors), A2B5 (intermediate oligodendrocyte progenitors), GFAP

(astrocytes/Type 1 neural progenitors) or NeuN (mature neurons) in vitro and in vivo. 

Incorporation of 5-bromo-2‟-deoxyuridine (BrdU) was used to assess cell proliferation in Cx29 

+/+ and -/- mice and postnatal neural progenitor neurosphere cultures. Both genotypes had 

comparable numbers of BrdU+ cells in the SVZ and SGZ in vitro and in vivo. In addition to 

BrdU labelling, we used the mitotic marker phospho-histone H3 to assess the mitotic index of 

our postnatal neurosphere cultures and again found no significant difference between 

neurospheres derived from +/+ and -/- animals. In sum, Cx29 was found to localize to a small 

subset of PDGFαR expressing early oligodendrocyte progenitor cells in both neurogenic niches 

of the mouse brain but was not required for cell proliferation in these areas. 

Disclosures: S. Imbeault, None; V. Peshdary, None; H. Toeg, None; S.A.L. Bennett, None. 

Poster 




Topic: C.10.a. Brain: Cellular and molecular mechanisms 

Support: National Basic Research Program (G 1999054005) 

National Natural Science Foundation (39930090, 39870248) 

Title: The expression changes of Neuropilin-1 and its ligand Sema3A and VEGF in the 

hippocampus following entorhinal deafferentation 

Authors: *W. WANG 1,2 , C. ZHOU 2 ; 

1 Picower Inst., MIT, Cambridge, MA; 2 Shanghai Inst. for Biol. Sciences, Chinese Acad. of Sci., 

Shanghai Inst. of Physiol., Shanghai, China 

Abstract: Neuropilin-1 (NRP-1) is a cell surface receptor shared by class3 semaphorins and 

vascular endothelial growth factor (VEGF), and plays multiple roles through different signal 

pathways by interacting with these two ligand families. Moreover, it has been shown that VEGF 

could antagonize the Sema3A induced apoptosis and growth cone collapse by competing with 

Sema3A for binding to NRP-1. Here we reported the expression changes of NRP-1 and its ligand 

VEGF and Sema3A in the mouse hippocampus after transections of the entorhinal afferents. 

Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis showed the 

significant expressed up-regulation of NRP-1 and VEGF mRNAs in the deafferented 

hippocampus with its maximal elevation at 7-15 days postlesion while the Sema3A mRNA level 

remained unchanged at any time point we examined. In situ hybridization and

immunohistochemisty revealed that the up-regulated expression of NRP-1 and VEGF occurred 

in the entorhinally denervated dentate out molecular layer and hippocampal stratum lacunosummolecular 

layer, and reactive astrocytes co-expressed both of them. Meanwhile NRP-1 was also 

expressed by the neuritis in the denervated area and the granule cells and its processes, which 

most likely belong to sprouting axons and/or regrowing dendrites. Double labeling of NRP-1 and 

GAP-43 also demonstrated the clustered expression of NRP-1 by regenerated axonal terminals 

surrounding the VEGF positive reactive astrocytes. Our data indicates that spatiotemporal shift 

of a balance from Sema3A/NRP-1 to VEGF/NRP-1 facilitates the layer-specific neurite 

outgrowth and circuits reorganization in the deafferented hippocampus and the crosstalk between 

neurons and reactive astrocytes may be involved in the plasticity events of the adult brain after 

injury. 

Disclosures: W. Wang, None; C. Zhou, None. 

Poster 






Title: Role of astrocyte FMRP on synapse development in Fragile X Syndrome 

Authors: *S. STEAD, M. NATHWANI, U. SHETH, L. C. DOERING; 

Pathology & Mol. Med., McMaster Univ., Hamilton, ON, Canada 

Abstract: Fragile X Syndrome (FXS) is the most common cause of inherited mental impairment 

that affects approximately 1 in 2000 males and 1 in 4000 females. FXS is caused by a 

trinucleotide repeat expansion in the FMR1 gene, resulting in gene silencing and lack of the 

protein product termed Fragile X Mental Retardation Protein (FMRP). Abnormal synapse 

morphology and function may contribute to the clinical features of FXS. 

We recently reported the expression of FMRP in cells of the astrocyte lineage (Pacey and 

Doering, 2007). Glial cells are known to play integral roles in the development and maintenance 

of synapses in the CNS. We therefore examined if neuronal process formation and synapse 

development are affected by FMRP in astrocytes. To address this question we utilized a coculture 

system of astrocytes and neurons as described by Kaech & Banker (2006), with 

modifications. Hippocampal neurons (E16-E17) and cortical astrocytes (P0-P1) were 

independently isolated from FMR1 knock out or wild type mice to establish the combinations of

cells summarized in Table 1. After 7 and 14 days in culture, the neurons were counted and 

studied by immunocytochemistry with antibodies to MAP2, PSD-95 and synaptophysin in the 

different co-culture conditions. 

The results of these experiments will offer insight into the developmental mechanisms 

underlying the normal formation of neural synapses, and those mechanisms that are defective in 

FXS. 

Table 1: Co-Culture Conditions 

Pacey, LKK and Doering LC, 2007, Glia 55:1601-1609 

Kaech, S and Banker, G, 2006, Nature protocols 1:2406-2415 

Disclosures: S. Stead, None; M. Nathwani, None; U. Sheth, None; L.C. Doering, None. 

Poster 





Support: Start up funds from Texas A&M University 

Title: Microfluidic co-culture platform for studying cns axon-glia interaction 

Authors: *J. PARK 1 , H. KOITO 2 , J. LI 2 , A. HAN 1 ; 

1 Electrical and Computer Eng., 2 Vet. Integrative Biosci., Texas A&M Univ., College Station, TX 

Abstract: A circular design microfluidic compartmentalized co-culture platform that can be used 

for axon-glia interaction research has been fabricated by polydimethylsiloxane (PDMS) 

softlithography. The PDMS co-culture platform is composed of one open compartment for 

neuronal soma (soma compartment) and one closed co-centric ring compartment (axon/glia 

compartment) for axons and oligodendrocytes (OLs). Two compartments are connected through 

arrays of shallow microfluidic channels that function as axon-guiding structures as well as a 

physical barrier between the compartments. This allows only axons to cross the microchannels 

and form axonal network layer inside the axon/glia compartment, thus OLs loaded into the 

axon/glia compartment interact only with axons but not with dendrites or neuronal somata. 

Primary cortical neurons were cultured in the soma compartment for two weeks prior to the 

loading of OLs into the axon/glia compartment to allow axons to form extensive networks inside 

the axon/glia compartment. Cells were then co-cultured for two additional weeks. The 

microfluidic device showed successful fluidic isolation between the two compartments. The 

separation of neuronal cell bodies and dendrites from axons growing through the arrays of

microchannels into the axon/glia compartment was verified by immunocytochemical labeling of 

cells. The proposed circular design showed excellent cell loading capability near the axonguiding 

microchannels that increased the number of axons crossing the channels as well as 

excellent cell viability. More than 79 % of the axon/glia compartment was covered with axons 

after four weeks of culture. OLs cultured on top of the axonal networks interacted extensively 

with axons and differentiated rapidly into mature OLs. This novel microfluidic co-culture device 

will provide a significant technical improvement in studying CNS axon-glia interaction in vitro 

and offer a potential high-throughput screening platform for drug candidates that promote myelin 

repair. 

Disclosures: J. Park, None; H. Koito, None; J. Li, None; A. Han, None. 

Poster 





Support: DFG SFB 645, B2 

DFG Wi 270/29-1 

DFG SFB/TR3 N01, C1 

DFG SPP1172 SE 774/3 

EU FP7-202167 

Title: Quality control of astrocyte-directed Cre transgenic mice: the benefits of a direct link 

between loss of gene expression and reporter activation 

Authors: *M. THEIS 1 , R. REQUARDT 2 , L. KACZMARCZYK 3 , P. DUBLIN 3 , A. 

WALLRAFF-BECK 3 , T. MIKESKA 4 , J. DEGEN 2 , A. WAHA 5 , K. WILLECKE 2 , C. 

STEINHÄUSER 3 ; 

1 Inst. of Cell. Neurosci, Univ. of Bonn, Bonn, Germany; 2 Inst. of Genet., Bonn, Germany; 3 Inst. 

of Cell. Neurosciences, Bonn, Germany; 4 Peter MacCallum Cancer Ctr., Melbourne, Australia; 

5 Dept. of Neuropathology, Bonn, Germany

Abstract: Cre recombinase activity for cell-type restricted deletion of floxed target genes (i.e. 

flanked by Cre recognition loxP-sites) is often measured by separate matings with 

recombination-activated reporter gene mice. Using a floxed Gja1 (Cx43) allele, we demonstrate 

the benefits of a direct link between reporter gene expression and target gene deletion to 

overcome critical limitations of the Cre/loxP system. The widely used human Glial Fibrillary 

Acidic Protein (hGFAP)-Cre transgene exhibits variable recombination activity and requires 

post-experimental validation. Such quality control is essential to correlate the extent of Cremediated 

Gja1 ablation with phenotypical alterations and to maintain the activity status of 

hGFAP-Cre in transgenic mouse colonies. We present alternative strategies to control for fidelity 

of Cre recombination in case identical or different reporter genes are present in multiple 

transgenic mice, including a multiple reporter gene approach. 

Disclosures: M. Theis , None; R. Requardt, None; L. Kaczmarczyk, None; P. Dublin, 

None; A. Wallraff-Beck, None; T. Mikeska, None; J. Degen, None; A. Waha, None; K. 

Willecke, None; C. Steinhäuser, None. 

Poster 

242. Glial Neuronal Interactions: Glutamate and Calcium 




Support: DFG SPP 1172 SE 774/3 

EC FP7-202167 

DFG SFB/TR3 

Title: Quantitative analysis of Kir4.1 channel expression in developing hippocampal astrocytes 

Authors: *G. SEIFERT 1 , C. HARTMANN 1 , D. K. BINDER 2 , K. HÜTTMANN 1 , A. 

WYCZYNSKI 1 , C. STEINHÄUSER 1 ; 

1 Inst. of Cell. Neurosci, Univ. Bonn, D-53105 Bonn, Germany; 2 Dept Neurolog. Neurosurg., 

Univ. of California, Irvine, CA 

Abstract: Astrocytes in the CNS possess inwardly rectifying K + (Kir) channels which are 

involved in extracellular K + buffering. Properties of Kir currents were investigated in mouse 

hippocampus using the patch clamp-technique combined with immunohistochemistry, 

immunoblot and single cell RT-PCR.

Hippocampal astrocytes were electrophysiologically identified by the large resting K + currents 

which dominated the current pattern of these cells beyond postnatal (p) day 3. These currents 

were sensitive to Ba 2+ at submillimolar concentration but were not pH-sensitive up to a pH of 

6.5. By using single cell RT-PCR, the subunit Kir4.1 was encountered in all cells while Kir5.1, 

another glial Kir subunit that co-assembles with Kir4.1, was co-expressed scarcely. In some 

cells, co-expression of Kir2 transcripts was detected. In Kir4.1-deficient mice, astrocytes were 

almost devoid of Ba 2+ -sensitive currents and lacked negative resting potentials, suggesting that 

Kir4.1 is mainly responsible for astroglial Kir currents in the hippocampus. 

A developmental up-regulation of Kir4.1 expression was found both on the mRNA and protein 

levels. Semi-quantitative real-time RT-PCR revealed a strong rise in the amount of Kir4.1 

mRNA between the p3 and p10, both in tissue from the stratum radiatum of the CA1 region as 

well as in single astrocytes. Quantification of grey levels of Kir4.1 tetramers in respect to the 

loading control of b-actin by Western blot analysis showed a 13-fold increase of Kir4.1 protein 

between p3 and p10. Electrophysiological analysis confirmed expression of Ba 2+ sensitive Kir 

currents in p3 astrocytes, but the amplitudes per cell surface area amounted to only 25% 

compared to astrocytes at p10. This increase in astroglial Kir4.1 channel density parallels a 

significant drop of the extracellular volume fraction of the developing hippocampus, suggesting 

an important role of Kir4.1 in extracellular K + buffering. 

Disclosures: G. Seifert , None; C. Hartmann, None; D.K. Binder, None; K. Hüttmann, 

None; A. Wyczynski, None; C. Steinhäuser, None. 

Poster 





Support: NHMRC 

Title: Role of ATP in memory formation 

Authors: *D. N. BOWSER 1 , M. E. GIBBS 2 ; 

1 Howard Florey Inst., Parkville, Australia; 2 Anatmoy and Cell Biol., Monash Univ., Melbourne, 

Australia 

Abstract: The nucleotide ATP is recognized as a neurotransmitter and amongst its various roles, 

is involved in neural-glial interactions in the brain. It functions both as an extracellular signalling 

molecule and as a gliotransmitter and it has been suggested to have a pivotal role in learning and

memory. ATP is released from both neurones and astrocytes and activates ionotropic (P2X) or 

metabotropic (P2Y) purine receptors found on neurones, astrocytes or capillaries in the central 

nervous system. ATP is hydryolsed to ADP and adenosine causing presynaptic inhibition. All 

three nucleotides activate different purinergic receptors with distinct pharmacology. We have 

discovered which receptors are involved in memory consolidation formation at 0-2.5 min and at 

30-35 min after training. 

In a discriminative avoidance task in young chicks where a single, brief (10 sec) learning 

experience with a bitter tasting bead is used to dissect out the roles for activation of the different 

purinergic receptors. The discrete timing of this task allows accurate „real-time‟ measurements 

for injection and testing of memory processes. A weak version of this task where memory lasts 

for only 30 min, presents the opportunity to enhance memory consolidation and facilitate the 

formation of long-term memory. 

In the chick hippocampus, glutamate release occurs for up to 2.5 min after training and then 

again around 30 min post-training, a time during which the previously labile memory 

consolidates into permanent memory. We have shown that activation or inhibition of NMDA 

receptors in the chick hippocampus can enhance or inhibit storage at these two times. AMPA 

receptors antagonists also inhibit memory at these same times. 

We have found that central, hippocampal injections of ATP (1nmol/hemisphere) promotes the 

formation of memory for weakly-reinforced learning. To understand which of the purinergic 

receptor subtypes are involved in memory processing and how, we have injected selective 

purinergic agonists and antagonists where available and examined their effects on memory. 

ATP-γ-S, ADP-β-S, and UTP all enhanced weak learning when administered at similar time 

periods. 

Disclosures: D.N. Bowser, None; M.E. Gibbs, None. 

Poster 





Support: MEC (BFU2007-64764) 

European Union (Health-F2-2007-202167) 

Title: Astrocyte-induced long-term potentiation of transmitter release requires nitric oxide 

signalling

Authors: *G. PEREA, A. ARAQUE; 

Dept Neural Plasticity, Cajal Inst., Madrid, Spain 

Abstract: Accumulating evidence indicates the existence of bidirectional communication 

between astrocytes and neurons. However, the effects of astrocytes on action potential-evoked 

synaptic transmission at single synapse level are largely unknown. 

We investigated the neuromodulatory role of astrocytes on synaptic physiology at single 

hippocampal synapses. Using electrophysiological and Ca 2+ imaging techniques on rat 

hippocampal slices, we performed paired recordings from CA1 pyramidal neurons and single 

astrocytes. Astrocytes were loaded with the Ca 2+ indicator Fluo 4 (50 κM) and the Ca 2+ -cage NP- 

EGTA (5mM) and were selectively stimulated by UV-flash photolysis (2Hz, 5s). Single synapses 

of Schaffer collaterals were stimulated at 0.5 Hz. 

We found that: 

The selective elevation of Ca 2+ in single astrocytes transiently increased the synaptic efficacy due 

to the potentiation of the probability of transmitter release, without affecting the amplitude of 

synaptic currents. 

This form of short-term plasticity was due to SNARE protein- and Ca 2+ -dependent release of 

glutamate from astrocytes, that activates presynaptic type I metabotropic glutamate receptors 

(mGluRs). 

The concurrent activity of astrocyte Ca 2+ elevation and postsynaptic neuron caused the persistent 

potentiation of synaptic transmission. Therefore, the temporal coincidence of neuronal and 

astrocytic signals induced the long-term potentiation (LTP) of hippocampal synaptic 

transmission. 

LTP was independent of NMDA receptor activation and postsynaptic intracellular Ca 2+ . 

However, LTP was prevented by blockage of presynaptic type I of mGluRs and synthesis of 

nitric oxide. 

We conclude that astrocytes potentiate synaptic transmission playing an active role in the 

transfer and storage of synaptic information by the nervous system. 

Disclosures: G. Perea , None; A. Araque, None. 

Poster 




Topic: B.11.a. Synapses

Title: Decreased glutamate clearance by glutamate transporters contributes to abnormal 

activation of NMDA receptors in the spinal substantia gelatinosa (SG) neurons of neuropathic 

rats 

Authors: *H.-R. WENG 1 , H. NIE 2 ; 

1 Dept of Anesthesiolo, Univ. of TX, Houston, TX; 2 Anesthesiol. and Pain Med., Univ. of Texas 

MD Anderson Cancer Ctr., Houston, TX 

Abstract: Activation of NMDA receptors, which is a key step in the pathogenesis of pain, 

depends on the spatiotemporal profile of glutamate in the synaptic cleft and extrasynaptic space. 

The homeostasis of glutamate in the CNS is ensured by a family of glutamate transporters in the 

plasma membrane of both glial cells and neurons. In this study, we asked if impaired glutamate 

transporters contributed to the abnormal activation of NMDA receptors in spinal substantia 

gelatinosa (SG) neurons of neuropathic rats. The expression of glutamate transporters was 

examined by immunohistology. Activation of NMDA receptors was analyzed by recording 

NMDA excitatory postsynaptic currents (EPSCs) of the spinal SG neurons evoked by graded 

electrical stimulation of the dorsal root entry zone in L4-5 spinal slices taken from adult normal 

and neuropathic rats on days 7-14 post-partial sciatic nerve ligation. Expression of glutamate 

transporters in the spinal dorsal horn was downregulated in neuropathic rats. The peak latency 

and duration of NMDA EPSCs in the spinal SG neurons were significantly greater in neuropathic 

rats than in normal rats, although the NMDA amplitudes were not significantly different. This 

abnormal activation of NMDA receptors could be simulated by pharmacological blockade of 

glutamate transporters in normal rats. Limiting extracellular glutamate spillover in neuropathic 

rats by adding 5% dextran to the perfusing solution resulted in shortening of the NMDA EPSC 

duration, decay time, and peak latency, indicating that glutamate spillover contributed to the 

abnormal activation of NMDA receptors in neuropathic rats. Dextran also increased the NMDA 

amplitude in neuropathic rats, indicating that more synaptic NMDA receptors were activated by 

the accumulated glutamate molecules inside the synaptic cleft owing to less glutamate spillover. 

In contrast, perfusion of 5% dextran had only minimal effects on the NMDA EPSCs in normal 

rats, indicating that glutamate spillover is minimal under normal conditions. These results 

indicate that impaired glutamate transporters lead to abnormal activation of NMDA receptors in 

neuropathic rats and suggest that repairing the damaged glutamate transporters may be a new 

avenue for the management of pathological pain. 

Supported by funds (to H.R.W.) provided by UT M. D. Anderson Cancer Center. 

Disclosures: H. Weng , None; H. Nie, None. 

Poster 





Support: NIMH 

NINDS 

Title: Astrocytes promote synaptic plasticity by activating mGluR5 

Authors: *S.-Y. LEE 1 , M. M. HALASSA 1 , O. PASCUAL 2 , P. G. HAYDON 1 ; 

1 Univ. Pennsylvania, Philadelphia, PA; 2 Biologie Cellulaire de la Synapse, INSERM, Paris, 

France 

Abstract: Though electrically non-excitable, astrocytes respond to external signals by an 

increase in intracellular Ca 2+ . Astrocytic Ca 2+ signaling can result in a number of physiological 

consequences, including the release of chemical transmitters from these cells, to impact nearby 

neuronal activity. The role of astrocytic Ca 2+ signaling in synaptic transmission and plasticity, 

however, remains poorly understood. Here, we show that elevating Ca 2+ selectively in astrocytes 

using photolysis of the Ca 2+ cage o-nitrophenyl-EGTA in area CA1 of the hippocampus triggers 

two forms of plasticity; long-term facilitation (LTF) and long-term depression (LTD). Whether a 

neuron exhibited LTF or LTD was dependent on its membrane potential at the time of astrocytic 

activation; elevation of astrocytic Ca 2+ coincident with neuronal depolarization resulted in LTF 

while coincidence with neuronal hyperpolarization resulted in LTD. Both forms of plasticity 

were dependent on metabotropic glutamate receptor 5 (mGluR5) while LTF was also dependent 

on NMDA receptor activity. To test whether astrocytic Ca 2+ signaling was necessary for known 

forms of plasticity in the hippocampus, we generated a transgenic animal in which IP3 5phosphatase 

was selectively and conditionally expressed in astrocytes to attenuate IP3-dependent 

Ca 2+ signaling. Transgenic inhibition of astrocytic Ca 2+ signaling resulted in attenuated Schaffer 

collateral-CA1 long-term potentiation (LTP). This transgenic phenotype was mimicked by 

dialysis of the Ca 2+ chelator BAPTA into the astrocytic syncytium to attenuate activitydependent 

astrocytic Ca 2+ signaling. These results demonstrate that astrocytic Ca 2+ signaling, 

which is recruited by neuronal activity, is required for synaptic plasticity. 

Disclosures: S. Lee , None; M.M. Halassa, None; O. Pascual, None; P.G. Haydon, None. 

Poster 



Program#/Poster#: 242.6/I1


Support: INSERM 

Title: Impact of neurotransmitters uptake by astrocytes on corticostriatal information processing 

Authors: *L. VENANCE 1 , V. GOUBARD 2 , J.-M. DENIAU 3 ; 

1 Dynamique et Physiopathol des Reseaux Neuronaux, INSERM U-667, Col. de France, Paris, 

France; 2 INSERM U667, College de France, France; 3 INSERM U667, College de france, France 

Abstract: Basal ganglia, an ensemble of sub-cortical interconnected nuclei, are involved in 

learning of contextual cognitivo-motor sequences related to environmental stimuli. Striatum, the 

main input nucleus of basal ganglia, receives glutamatergic inputs from the whole cerebral 

cortex. The striatal output neurons (medium-sized spiny neurons, MSNs), the main neuronal 

striatal population, act as detectors and integrators of distributed patterns of cortical activity. 

Modulation of release and uptake of neurotransmitters constitutes a key step in the information 

processing. Astrocytes play a predominant role in the uptake of neurotransmitters. However, the 

impact of astrocytes on corticostriatal transmission and synaptic plasticity remains to be 

determined. For this purpose, we have used rat brain slices in which connections between 

cortical pyramidal cells and MSNs were preserved. We performed electrical stimulation in layer 

V of the somatosensory cortex while recording MSNs and astrocytes by double patch-clamp in 

the dorsal striatum. Cortical stimulations evoked reliable glutamatergic excitatory postsynaptic 

currents (EPSCs) in MSNs and efficient inward currents in astrocytes. Such astrocytic evokedcurrents 

displayed latency of 4.74±0.32 ms, amplitude of 55.4±7.0 pA and rise time of 11.9±0.8 

ms (n=29). Astrocytic evoked-currents originated mainly from the activities of glutamate and 

GABA transporters. Indeed, dihydrokainic acid (300 mM; an inhibitor of glutamate transporter- 

1) and nipecotic acid (500 mM; a non-specific GABA transporter inhibitor) inhibited cortically 

astrocytic evoked-currents by 30.3±7.5 % and 29.2±5.5 % (n=12 and n=10) respectively, and 

47.8±6.0 % (n=5) when applied together. Inhibition of glutamate and GABA transporters led to 

significant decreases of the cortically MSN-evoked responses: 20.2±7.5 % (n=8) and 41.7±12.7 

% (n=5), respectively. We have determined the characteristics of short-term plasticity in MSNs 

and astrocytes. Namely, we have observed some facilitation and depression of the MSN EPSCs 

as well as astrocytic transporter currents, depending on the paired-pulses intervals. Finally, we 

have observed that magnitudes of facilitation and depression of cortically-evoked EPSCs were 

mainly increased after glutamate and GABA uptakes inhibitions. 

In conclusion, our experiments indicate that neurotransmitters uptake by astrocytes is particularly 

efficient in the corticostriatal axis and have a strong impact on transmission and synaptic 

plasticity. Such astrocytic properties could play a determinant role in the MSN coincidence 

detection of cortical activities. 

Disclosures: L. Venance , None; V. Goubard, None; J. Deniau, None. 

Poster



Program#/Poster#: 242.7/I2 



Human Frontier Science Program 

Medical Research Council 

Title: LTP induction changes the morphology of astrocytes in the CA1 region of the 

hippocampus in vitro 

Authors: *C. HENNEBERGER 1 , N. MEDVEDEV 2 , M. G. STUART 2 , D. A. RUSAKOV 1 ; 

1 Inst. of Neurol., Univ. Col. London, London, United Kingdom; 2 Dept. of Biol. Sci., The Open 

Univ., Milton Keynes, United Kingdom 

Abstract: Astrocytes are reported to sense and modulate neuronal activity. One important aspect 

of astrocyte function is the fast clearance of neurotransmitter in the vicinity of synapses. This 

relies on a high density of neurotransmitter transporters expressed on the membranes of fine 

astrocytic processes that surround synaptic structures. Whether the spatial relationship between 

astrocytes and synapses is static or whether it responds to neuronal plasticity remains poorly 

understood. 

We combined recordings of field potentials, whole-cell currents and two-photon excitation 

fluorescence imaging of astrocytes in the CA1 stratum radiatum in hippocampal slices obtained 

from 3-4 week old Sprague Dawley rats. Alexa 594 and Oregon Green BAPA-1 were included in 

the intracellular solution to monitor fine astrocyte morphology and Ca 2+ dynamics. 

Morphological changes were quantified in real time as changes in the tissue volume fraction 

occupied by the astrocyte, dendritic image entropy, fluorescence recovery after photobleaching 

and also using an image segmentation algorithm. 

We found that astrocyte morphology was modified by electrical stimulation protocols that 

induced long-term potentiation (LTP) of transmission at Schaffer collateral synapses. The 

observed changes, which were sensitive to NMDA receptor blockade, can be best explained as 

partial retraction of fine astrocyte processes. Electron microscopy investigation of biocytin-filled 

and reconstructed astrocytes that underwent potentiation protocols is currently under way. 

Disclosures: C. Henneberger , None; D.A. Rusakov, None; N. Medvedev, None; M.G. 

Stuart, None.

Poster 





Title: Impaired glutamate uptake increases activation of synaptic and extrasynaptic NMDA 

receptors in the spinal substantia gelatinosa (SG) neurons 

Authors: *H. NIE, H.-R. WENG; 

MD Anderson Cancer Ctr., Houston, TX 

Abstract: Activation of N-methyl-D-aspartate (NMDA) receptors in spinal dorsal horn neurons 

is a key process related to sensory transmission, neural plasticity, and pathogenesis of pain. In 

this study, we investigated how activation of NMDA receptors in spinal SG neurons is regulated 

by glutamate re-uptake through glutamate transporters located in the astrocytic and neuronal 

plasma membrane. Using visualized whole cell patch recording techniques, we analyzed NMDA 

excitatory postsynaptic currents (EPSCs) evoked by graded inputs from primary afferents 

recorded in spinal slices of young adult rats before and after pharmacological blockade of both 

glial and neuronal glutamate transporters by DL-threo-β-benzyloxyaspartate (DL-TBOA). 

Blockade of glutamate transporters led to increases in the number and duration of NMDA 

receptors activated by monosynaptic or polysynaptic inputs and exogenous glutamate. Blockade 

of glutamate transporters also resulted in increased activation of NR2B subunits by primary 

afferent inputs. The degree of enhancement in activation of NMDA receptors induced by DL- 

TBOA was greater in neurons having weaker synaptic input at baseline than in those having 

stronger synaptic input. We observed that impaired glutamate uptake caused glutamate spillover 

outside the active synaptic cleft and activation of NMDA receptors outside the active synapses, 

including extrasynaptic NMDA receptors and NMDA receptors in neighboring synapses. 

Blockade of glutamate transporters also caused glutamate accumulation and dwelling, leading to 

increased activation of unsaturated NMDA receptors and prolonged the duration of NMDA 

receptors activated. Taken together, our results indicate that the number, duration, and type of 

NMDA receptors activated in spinal SG neurons are regulated by glutamate transporters. 

Reversing impaired glutamate uptake may be a new avenue to relieve the pathological pain 

related to abnormal activation of NMDA receptors. 

Supported by funds from The University of Texas M. D. Anderson Cancer Center to H.R.W. 

Disclosures: H. Nie , None; H. Weng, None. 

Poster





Support: The Council of Scientific and Industrial Research (CSIR) Grant 

Title: Acute effect of 17β-estradiol on signaling between astrocytes and neurons in mixed 

hippocampal cultures 

Authors: *S. P. RAO 1 , S. K. SIKDAR 2 ; 

1 Psychiatry/MRRC, Semel Inst, UCLA, Los Angeles, CA; 2 Mol. biophysics unit, Indian Inst. of 

Sci., Bangalore, India 

Abstract: The ovarian steroid hormone 17β-estradiol, in addition to its classical genomic effects 

in the brain through intracellular estrogen receptors, can bring about actions that are rapid in 

onset via a plasma membrane-associated form of its receptor. In the hippocampus, estrogen 

receptors have been observed at extranuclear sites on both neurons and astrocytes suggesting a 

complex interplay between the two cell types in mediating the effects of the hormone. Neuronal 

activity can trigger intracellular calcium elevations in astrocytes, in turn leading to release of 

gliotransmitters that can modulate neuronal activity and synaptic transmission. 

We investigated the functional consequences of acute (5 min) estradiol treatment on astrocyteastrocyte 

and astrocyte-neuron signaling in rat hippocampal cultures using calcium imaging and 

electrophysiological techniques. Mechanical stimulation of an astrocyte in fluo-3 AM loaded 

cultures evoked a calcium rise in the stimulated astrocyte followed by calcium elevations in the 

surrounding astrocytes and neurons. Following acute treatment with estradiol, the amplitude of 

the calcium responses in the surrounding astrocytes and neurons was attenuated. Astrocyteevoked 

electrophysiological responses in neurons were affected by estradiol too. The incidence 

and amplitude of the astrocyte-evoked slow inward current in neurons was reduced in presence 

of estradiol. Also, acute estradiol treatment decreased the incidence of astrocyte-evoked increase 

in spontaneous postsynaptic current frequency in neurons. Astrocyte calcium responses to the 

metabotropic glutamate receptor agonist t-ACPD were reduced in presence of estradiol 

suggesting functional changes in astrocyte metabotropic glutamate receptors following acute 

treatment with estradiol. Our results indicate that glutamate-mediated astrocyte-astrocyte and 

astrocyte-neuron communication is sensitive to rapid estradiol-mediated hormonal control. 

Disclosures: S.P. Rao, None; S.K. Sikdar, None. 

Poster





Support: Israel Science Foundation, grant number 269/06 

Title: Astrocytic-derived nitric oxide modulates synaptic function 

Authors: *Y. BUSKILA, Y. AMITAI; 

Dept. of Physiol., Ben-Gurion Univ., Beer-Sheva, Israel 

Abstract: Nitric oxide (NO) is produced in the brain by neurons, astroglia and endothelial cells, 

and is known to participate in diverse signaling pathways. While neurons are capable of rapid 

release of small amounts of NO serving as neurotransmitter, most studies of NO production by 

astrocytes have assumed slow activation of an inducible form of NOS (NOS2) under a variety of 

stress stimulations such as ischemia or inflammation, occurring over a time-scale of hours and 

days. In the neocortex, the neuronal NOS isoform (NOS1) has been documented only in a small 

percentage of GABAergic neurons. Hence, one of the most intriguing questions regarding NO 

biology in the brain is the cellular source of NO acting as a retrograde synaptic messenger. 

We used NO imaging with the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and 

electrophysiological methods in acute neocortical slices from CD1 mice to test the hypothesis 

that astrocytic-derived NO participates in modulating synaptic strength. 

NO imaging experiments demonstrated that robust astrocytic NO production can occur on a time 

scale of seconds and minutes, and is not dependent on de-novo protein synthesis. SR-101, a 

selective marker for astrocytes, was highly co-localized with the diffuse staining pattern of DAF 

-2DA, confirming that most NO producing cells are astrocytes. Bath application of the selective 

NOS2 inhibitor 1400W (3 κM) blocked the astrocytic DAF-2DA fluorescence, but not the 

neuronal fluorescence. We next recorded from pyramidal neurons in layer 2/3 of the neocortex, 

and examined the effect of inhibiting NOS2 on excitatory synaptic transmission. 1400W reduced 

the frequency of spontaneous unitary EPSC's without affecting their amplitude. Moreover, when 

inducing LTP by pairing the pre- and the postsynaptic spikes, 1400W reduced the potentiation of 

the EPSC's amplitude from 144±18% to 122±18%. Taken together, our results suggest that 

astrocytic-derived NO is involved in modulating synaptic function. 

Disclosures: Y. buskila, None; Y. Amitai, None. 

Poster 

242. Glial Neuronal Interactions: Glutamate and Calcium




Support: NINDS 

NIMH 

Title: Astrocyte-specific transgene expression using adeno-associated virus 

Authors: *J. DONG, A. MUNGENAST, H. TAKANO, E. STAMBROOK, H. ZHOU, P. 

HAYDON; 

Univ. Pennsylvania Sch. Med., Philadelphia, PA 

Abstract: Recombinant adeno-associated virus (rAAV) is a highly effective vehicle for gene 

delivery into CNS. Towards the goal of manipulating gene expression in astrocyte specifically, 

we examined the transduction pattern and cellular tropism of AAV2 vector sequences 

pseudotyped with capsid sequences from serotypes 1, 2, 5, 7, 8, and 9. Quantification of the 

EGFP expression 7 days after in vivo injection showed that recombinant AAV2/1, AAV2/2, 

AAV2/7, AAV2/8 and AAV2/9 displayed predominantly neuronal tropism in both cortex and 

hippocampus. Comparatively, AAV2/5 was demonstrated to show significantly more astrocyte 

transduction. To further investigate whether the astrocyte specific GFAP promoter can enhance 

astrocyte specificity of the AAV2/5 pseudotype, we generated rAAV vectors with transgenes 

driven by the human GFAP promoter (AAV-Gfa2-eGFP), a downsized GFAP promoter (AAV- 

Gfa104-eGFP and AAV-Gfa104-Venus-DAGK). AAV2/5 containing the GFAP promoter 

exhibited efficient transduction both in vitro and in vivo and the transduction was restricted to 

astrocytes. This specificity was verified by colabeling using anti-GFAP and anti- glutamine 

synthetase antibodies, well-known markers for astrocytes, and a lack of co-labeling with other 

cell-specific antibodies including anti-NeuN, anti-NG2, anti-Oligodendrocyte and anti-Iba1. We 

also demonstrated the possibility of repeated imaging on the same astrocyte transduced by 

AAV2/5 in living mice using 2-photon microscopy, which allows us to study astrocyte dynamics 

and function in vivo over time. Our study suggests that specifically targeting astrocytes via the 

combination of the AAV2/5 pseudotype and the GFAP promoter will become a powerful tool to 

investigate the role of astrocytes in vivo. 

Disclosures: J. Dong , None; A. Mungenast, None; H. Takano, None; E. Stambrook, 

None; H. Zhou, None; P. Haydon, None. 

Poster 

242. Glial Neuronal Interactions: Glutamate and Calcium




Title: Eph receptors mediate gliotransmitter release from astrocytes 

Authors: *Z.-Y. ZHUANG, J. HUANG, C. NELERSA, J. SAGEN, D. J. LIEBL; 

The Miami Project to Cure Para, Univ. of Miami, Miami, FL 

Abstract: Astrocytes regulate synaptic transmission by releasing gliotransmitters such as 

glutamate, ATP and D-serine. Eph receptors, the largest family of receptor tyrosine kinases, and 

their membrane bound ligand, the ephrins, are very known for their functions in regulating 

axonal growth/guidance, synaptic formation/plasticity in the developing nervous system. Our 

previous data have shown that ephrinB3 KO null mutant mice exhibit reductions in CA3-CA1 

long-term potentiation that is associated with defective leaning and memory (Rodenas-Ruano et 

al., 2006). Here, we explored mechanisms regulating EphB3/EphA4-mediated release of 

gliotransmitters from astrocytes. First, we used reverse-transcription PCR and 

immunohistochemistry to study the mRNA and protein expression of ephrins (B1, B2, and B3) 

and Eph receptors B1, B2, B3, and A4) in purified cultured cortex astrocytes. We found 

ephrinB1, ephrinB3, EphB1, EphB2, EphB3 and EphA4 were strongly expressed in cultured 

astrocytes while ephrinB2 was weakly expressed in astrocytes. Second, we employed HPLC and 

chemiluminescence to measure the release of glutamate, D-serine and ATP from astrocytes, and 

found that ephrinB3 stimulation could increase glutamate, D-serine and ATP release. Our data 

demonstrate that Eph receptors can regulate gliotransmitter release from cultured astrocytes, 

which may have important implication on synaptic function and plasticity. 

Disclosures: Z. Zhuang , None; J. Huang, None; C. Nelersa, None; J. Sagen, None; D.J. 

Liebl, None. 

Poster 





Support: Ministerio de Educación y Ciencia (BFU2007-64764)

CAM–CSIC (200620M083) 

European Union (Health-F2-2007-202167) 

Title: Endocannabinoids mediate neuron-astrocyte communication 

Authors: *M. NAVARRETE, A. ARAQUE; 

CAJAL INSTITUTE, MADRID, Spain 

Abstract: Type 1 cannabinoid receptors (CB1Rs) play key roles in brain physiology but their 

expression and function in astrocytes remain unknown. Using electrophysiological and Ca 2+ 

imaging techniques in mice brain slices, we investigated the expression of CB1Rs by astrocytes 

in situ and their involvement in the neuron-astrocyte communication. 

We found: 

1) Local application of CB1R agonists increased the astrocyte Ca 2+ levels, which were 

insensitive to glutamatergic, GABAergic, cholinergic, and purinergic receptors but were 

abolished by the CB1 receptor antagonist AM251. These Ca 2+ elevations were absent in slices 

from CB1R -/- transgenic mice. 

2) Endocannabinoid-induced astrocyte Ca 2+ elevations were unaffected by pertussis toxin but 

were abolished by PLC inhibitors and thapsigargin. 

3) Endocannabinoids released by depolarization of pyramidal neurons elevated the astrocytic 

Ca 2+ . These effects were abolished by AM251. 

4) Astrocytic Ca 2+ elevations and endocannabinoid-mediated depolarization supression of 

inhibiton (DSI) depended on the duration of the neuronal depolarization, showing a high degree 

of correlation. 

5) Endocannabinoid-mediated astrocyte Ca 2+ elevations stimulate the release of glutamate from 

astrocytes, evoking NMDA receptor-dependent slow inward currents (SICs) in adjacent neurons. 

We conclude: 

1. Hippocampal astrocytes in situ express functional CB1Rs. 

2. CB1Rs in astrocytes are activated by endocannabinoids released from pyramidal neurons, 

increasing the astrocyte Ca 2+ levels and stimulating the release of glutamate, which activates 

NMDA receptors in pyramidal neurons. 

3. Present results show a new form of neuron-astrocyte communication based on 

endocannabinoid signaling, and reveal an endocannabinoid-glutamate signaling pathway, where 

astrocytes serve as a bridge for non-synaptic interneuronal communication. 

Disclosures: M. Navarrete , None; A. Araque, None. 

Poster 





Support: NIH Grant RO1-NS33938 

Title: Investigating the role of astrocyte Gs-linked GPCR signaling on mouse behavior 

Authors: K. B. CASPER, *K. D. MCCARTHY; 

Dept Pharmacol, Univ. North Carolina, Chapel Hill, NC 

Abstract: Evidence has linked alterations in the cAMP signaling pathway to changes in animal 

behavior and possible links to many psychiatric disorders. To assess what role the cAMP 

pathway in astrocytes may play in animal behavior, we have utilized an inducible cell specific 

knockout created in our laboratory to remove part of the gene encoding the stimulatory G protein 

alpha subunit (Gs alpha; Gs). The Cre/loxP system has been used extensively to generate 

conditional knockouts of genes. To create an astrocyte specific knockout of Gs, mice carrying 

floxed Gs gene (GNAS) were crossed with mice carrying an inducible, tamoxifen responsive cre 

recombinase driven by the human glial fibrillary acidic protein (GFAP) promoter. Mice were 

given tamoxifen in adulthood to avoid removal of Gs from neurons due to recombination 

occurring in GFAP+ precursor cells during development. Several weeks after giving tamoxifen, 

mice were subjected to a battery of behavioral tests to assess if loss of Gs and consequent cAMP 

signaling in astrocytes affects animal behavior. Gs conditional knockout (Gs cKO) mice 

exhibited loss of habituation and increased activity in open field testing. In addition, Gs cKO 

mice showed reduced prepulse inhibition in the acoustic startle response test. Finally, testing in 

the water maze revealed that loss of Gs from astrocytes may cause a learning deficit and less 

cognitive flexibility. We are further characterizing astrocyte specific Gs knockouts via 

electrophysiology and biochemical analyses. 

Disclosures: K.B. Casper, None; K.D. McCarthy , None. 

Poster 





Support: T32DE007309

NS059028 

DE018573 

NS060735 

Title: Alterations in astrocytic glutamate transporter GLT-1 activity in the RVM contribute to 

descending pain facilitation 

Authors: *W. GUO, M. GU, S. LAGRAIZE, S. ZOU, R. DUBNER, K. REN; 

Dept Biomed Sci., Dentl Sch, & Prog. in Neurosci, Baltimore, MD 

Abstract: The glutamate transporter GLT-1 (EAAT2), mainly expressed in astrocytes, plays a 

major role in maintaining glutamate homeostasis in the brain. We have shown that astrocytes are 

activated in the rostral ventromedial medulla (RVM), a pivotal structure in descending pain 

modulation, after peripheral tissue injury. In vitro studies show that activated astrocytes exhibit a 

"de-differentiated" phenotype with reduced GLT-1 expression and increased cytokine levels. 

Here we have tested the hypothesis that changes in GLT-1 activity contribute to RVM 

hyperexcitability and descending pain facilitation. Immunohistochemistry showed the 

localization of GLT-1 in the RVM. Double immunofluorescence staining showed selective 

colocalization of GLT-1 with glial fibrillary acidic protein (GFAP), a marker of astrocytes, in the 

RVM. We next examined the effect of complete Freund‟s adjuvant (CFA)-induced hind paw 

inflammation on GLT-1 expression in the RVM. The RVM tissues were punched out and total 

proteins isolated for Western immunoblot. While there appeared no change in GLT-1 expression 

at earlier time points, a decrease in the GLT-1 protein levels was seen at 5d after CFA, which 

remained low as late as 14d after injection of CFA. We finally tested whether changes in GLT-1 

activity in the RVM affected pain sensitivity. Dihydrokainate (DHK 0.1 mM, 500 nl), a GLT-1 

selective antagonist, was microinjected into the RVM of the naïve rats. Ten minutes after 

microinjection of DHK, paw withdrawal latencies to a noxious thermal stimulus were 

significantly decreased on both hind paws (p





Uehara Memorial Foundation 

Title: Astrocyte-to-pericyte communication mediated by ATP 

Authors: *S. KOIZUMI 1 , K. FUJISHITA 1 , K. SUEISHI 2 , F. TAKATA 2 , Y. KATAOKA 2 ; 

1 Dept. Pharmacol., Univ. Yamanashi, Facul. Med., Yamanashi, Japan; 2 Fukuoka Univ., Fukuoka, 

Japan 

Abstract: Astrocytes release gliotransmitters to communicate neighbouring cells as a form of 

Ca2+ wave. Although recent accumulating evidence show that astrocytic Ca2+ responses 

dynamically affect local circulations in the CNS, a direct regulation of vascular function by 

astrocytic ATP has received only limited attention. In the present study, we tested hypothesis that 

astrocytes transmit gliotransmitter ATP to pericytes, cells that are located abluminal site of 

capillary walls with patchy structure, and regulate microcirculation of capillaries by controlling 

tonus of pericytes. Pharmacological analysis by Ca2+ imaging showed that cultured pericytes 

express several functional P2 receptors, among which the UTP preferring P2Y2 receptors had a 

major role for the responses. Astrocytes released ATP in response to mechanical stimulation. In 

the astrocytes-pericytes co-cultures, mechanical stimulation of single astrocytes resulted in 

elevations in [Ca2+]i in the cells, which was followed by propagation of Ca2+ waves into 

adjacent astrocytes and preicytes in a P2Y1 and P2Y2 receptor-dependent fashion, respectively. 

Interestingly activation of P2Y2 receptors resulted in contraction of pericytes in three-dimension 

cultures and in situ. Prenchymal capillaries are almost completely surrounded by astrocytes that 

receive projection of vaso-active neurons. These findings suggest that astrocytes regulate tonus 

of brain pericytes via ATP/P2 receptors, which may contribute to direct and fine control of 

microcirculation within capillaries. 

Disclosures: S. Koizumi, None; K. Fujishita, None; K. Sueishi, None; F. Takata, None; Y. 

Kataoka, None. 

Poster 





Support: NIH/NINDS NS051509 

Title: Synaptic connectivity is a universal property of NG2+ cells that is lost upon differentiation 

Authors: *L. M. DE BIASE 1 , S. KANG 1 , A. NISHIYAMA 2 , D. E. BERGLES 1 ; 

1 Neurosci., Johns Hopkins Sch. of Med., Baltimore, MD; 2 Physiol. and Neuorbiology, Univ. of 

Connecticut, Storrs, CT 

Abstract: Glial cells expressing the NG2 proteoglycan (NG2+ cells) serve as progenitors for 

oligodendrocytes during development yet remain abundant in gray and white matter of the 

mature CNS, suggesting that they perform additional roles. Unlike other glia, NG2+ cells form 

synapses with neurons, perhaps to allow rapid, activity-dependent regulation of this ubiquitous 

group of progenitors. While synaptogenesis and circuit maturation among neurons has been well 

described, little is known about the developmental onset of neuron-NG2+ cell synaptic 

communication, the variability of synaptic connectivity among NG2+ cells, or the changes that 

occur in this form of signaling as NG2+ cells differentiate into oligodendrocytes. We used NG2- 

DsRed BAC transgenic mice to study how NG2+ cell properties change during development in 

the corpus callosum (CC), hippocampus (HC) and cerebellum (CB). To obtain an unbiased 

assessment of neuron-NG2+ cell synaptic connectivity, we made whole-cell recordings from 

NG2+ cells in acute brain slices and focally applied hypertonic solution to force fusion of primed 

synaptic vesicles. Hypertonic challenge reliably induced bursts of AMPA receptor-mediated 

mEPSCs that were blocked by NBQX. The number of mEPSCs evoked increased as animals 

matured (P5-8: 12 ± 11; P12-15: 88 ± 51; P20-26: 55 ± 43; P40-45: 89 ± 48), paralleling the 

development of neuron-neuron synapses. Although nearly all NG2+ cells exhibited voltage-gated 

Na+ channel (NaV) currents (141/144 cells; NaV current density: 22 ± 1 pA/pF), the extent of 

synaptic connectivity was not correlated with NaV current amplitude (P = 0.74) and NG2+ cells 

did not fire action potentials in response to current injection in any brain region or developmental 

time point sampled. To study how the membrane properties of NG2+ cells change as they 

differentiate, we generated NG2-DsRed;NG2-CreER;Z/EG triple transgenic mice. In these mice, 

tamoxifen injection induced GFP expression in NG2+ cells, yielding DsRed+GFP+ cells. As 

NG2+ cells differentiated, their progeny continued to express GFP while DsRed expression was 

lost as NG2 promoter activity ceased. A comparison of the physiological properties of 

DsRed+GFP+, weakly DsRed+GFP+, and DsRed-GFP+ cells, revealed that NG2+ cells rapidly 

lose synaptic contacts and cease expressing NaV as they began to differentiate. Our data suggest 

that NG2+ cell synaptic inputs are established and refined concurrently with neuronal 

synaptogenesis and that both synapses and NaV expression are maintained only prior to 

differentiation into oligodendrocytes. 

Disclosures: L.M. De Biase, None; A. Nishiyama, None; S. Kang, None; D.E. Bergles, None.

Poster 



Program#/Poster#: 242.18/J1 


Support: Aston University 

The Royal Society 

Wellcome Trust Grant 78068 

Title: Long term enhancement of glial signalling by sustained synaptic activity in the 

ventrobasal thalamus 

Authors: *T. M. PIRTTIMAKI, R. PARRI; 

Aston Univ., Birmingham, United Kingdom 

Abstract: In the ventrobasal (VB) thalamus, spontaneous astrocytic activity leads to neuronal 

NMDA-receptor mediated slow inward currents (SICs). These SICs can also be evoked by 

activation of astrocytic mGluRs, acting via intracellular Ca2+ release and presumed vesicular 

glutamate release. Since the VB thalamus receives glutamatergic inputs from the periphery 

(primary sensory PS) and cortex (corticothalamic CT) we examined the role of synaptically 

released glutamate in activating glial signalling. Fluorescence calcium imaging from astrocytes 

and patch clamp recordings from thalamocortical (TC) neurones were performed. Brief 

stimulation of PS or CT afferents evoked astrocytic [Ca2+]i increases, demonstrating functional 

neuron-astrocyte synaptic connectivity. Spontaneous SICs were compared to those following PS 

or CT afferent stimulation of varying frequency (1-500Hz) and duration (2msec-10sec). SIC 

incidence was not significantly increased after stimulus (control 0.16±0.03 SICs/min; stimulation 

0.12±0.02 SICs/min; n=71; p=0.2). However, sustained delivery of intermittent stimulation of PS 

and CT afferents led to a Long Term Enhancement (LTE) of SIC frequency lasting over 60 

minutes from 0.08±0.01 SICs/min, (n=70) to 0.31±0.06 SICs/min, (n=53; p=0.0001). LTE of 

SIC frequency persisted in the presence of TTX (1µM; n=7; p=0.16) indicating independence 

from possibly increased neuronal activity. SICs following LTE were inhibited by D-AP5 (50µM; 

n=6), and reduced in amplitude by ifenprodil (10µM) from 76±2.8pA to 56.2±1.7pA (n=178 and 

184 SICs respectively; p=4.8e-8) indicating that thalamic SICs are mediated by NR2B subunit 

containing NMDA-receptors. Synaptically induced postsynaptic currents were not significantly 

affected by ifenprodil, indicating likely non-synaptic expression of LTE. Induction of LTE was 

inhibited (0.06±0.02 SICs/min; n=8) by mGluR antagonists CPCCOEt (200µM) and MTEP 

(50µM) present during stimulation while the mGluR agonist DHPG (100µM) mimicked the 

stimulation effect at producing LTE (0.25±0.1 SICs/min; n=13; p=0.04). The study therefore 

shows that glial-neuronal glutamatergic signalling is not greatly affected by short synaptic

stimuli, but is potentiated by sustained synaptic activity. This mechanism may therefore be a 

form of glial “memory” of neuronal activity. 

Disclosures: T.M. Pirttimaki , None; R. Parri, None. 

Poster 





Support: BBSRC 

Title: NG2-glia have the mechanisms for communication with neurons and astrocytes at a 

tetrapartite synapse 

Authors: *R. J. WIGLEY, A. M. BUTT; 

Pharm. & Biomed. Sci., Cell. Neurophysiol., Portsmouth, United Kingdom 

Abstract: NG2-glia are identified by the expression of the chondroitin sulphate proteoglycan 

NG2 on their cell surface. NG2-glia form contacts with neurons at synapses and respond to 

neuronally produced glutamate (Bergles et al. 2000). We have studied these NG2-glial cells, 

together with astrocytes, to investigate their involvement at the tripartite synapse that may exist 

between pre- and postsynaptic neurons in the presence of a synaptic astrocyte. Experiments 

were carried out using a transgenic mouse line whose astrocytes express green fluorescent 

protein under the glial fibrillary acidic protein (GFAP) promoter. Mice aged post-natal day (P) 

15 and above were humanely killed according to the UK Home Office Animals (Scientific 

Procedures) Act 1986. Brains were removed and fixed in 4% paraformaldehyde before being 

sliced to sagital sections of 100µm on a vibrotome and stained for the presence of NG2-glia 

(NG2), synapses (synaptophysin), glutamate transport vesicles (vGLUT1) and ionotrophic 

AMPA glutamate receptors (GluR4). Images were captured on a Zeiss Meta 510 confocal 

microscope and analysed by deconvolution and colocalization studies using Volocity 

(Improvision, UK). The three-dimensional images show that NG2-glia are in direct proximity to 

neuronal synapses and form points of contact with astrocytes that express both vGLUT1 and 

GluR4. The results suggest that NG2-glia are a component of a four way communication system 

at a tetrapartite synapse and may have an important functional role in glutamate transmission. 

Thus NG2-glia are integrated into the neural-glial functional unit of the CNS. 

Disclosures: R.J. Wigley, None; A.M. Butt, None.

Poster 





Support: Swedish Research Council. Proj. 01580 and 12600. 

Title: Astrocyte-mediated negative feedback at hippocampal glutamatergic synapses 

Authors: *M. S. ANDERSSON, E. HANSE; 

Neurosci & Physiol, Gothenburg Univ., Gothenburg, Sweden 

Abstract: Presynaptic terminals express receptors whose activation modulates the release 

probability. 

These receptors can be activated by gliotransmitters and we have recently described an astrocytemediated 

transient heterosynaptic depression (tHeSD) in the CA1 area of rat hippocampal slices. 

A 3-impulse 50 Hz conditioning burst produces a 25% reduction in release probability of 

neighbouring synapses within 500 ms in slices from 3 week old rats (Andersson et al, 2007). 

Here we have addressed the question to what extent the synapses providing the conditioning 

stimuli are affected by this transient depression. To examine this, a paired-pulse stimulus was 

applied 500 ms after the conditioning burst (3 impulses, 50 Hz) to the same (homosynaptic) 

synaptic input. This protocol resulted in a depression of 50 %, measured using either field 

EPSPs, AMPA EPSCs or NMDA EPSCs, when comparing the 1 st response in the conditioning 

3-impulse burst, with the 1 st response in the paired-pulse stimuli. 

This transient homosynaptic depression (tHoSD) is mediated by astrocytes since it was blocked 

by high concentrations of calcium chelator (BAPTA) delivered via the patch pipette to the 

astrocytic network. In addition, the astrocyte-specific metabolic inhibitor fluoroacetate (FAC) 

blocked the tHoSD. Similar to the tHeSD, the tHoSD exhibited a clear developmental profile 

with only a 7% depression in slices from rats aging 6 to 11 postnatal days. Activation of several 

receptors seems to contribute to the tHoSD. It was reduced by antagonists to GABAB-, kainate- 

and adenosine A1- receptors. 

These results show that a short burst gives rise to a previously unrecognized form of short term 

plasticity - a synaptic negative feedback mediated by astrocytes. 

Disclosures: M.S. Andersson , None; E. Hanse, None.

Poster 





Support: Swiss National Science Foundation grant # 3100A0-119827/1 

Title: Sodium-dependent glutamate uptake induces mitochondrial matrix acidification in 

astrocytes 

Authors: G. AZARIAS 1 , D. POBURKO 2 , N. DEMAUREX 2 , *J.-Y. CHATTON 1 ; 

1 Dept Physiol & DBCM, Univ. Lausanne, Lausanne, Switzerland; 2 Dept Cell Physiol. and 

Metabolism, Univ. Geneva, Geneva, Switzerland 

Abstract: An increasing body of evidence collected in vitro and in vivo suggests that 

synaptically-released glutamate taken-up by astrocytes enhances the formation of lactate from 

aerobic glycolysis. Astrocytic sodium homeostasis has been identified as a major factor linking 

neuronal activity with astrocyte metabolism, but the mechanisms regulating the lactate shuttle 

remain to be elucidated. Since astrocytic processes ensheathing synapses contain mitochondria, 

we hypothesized that mitochondrial physiology is altered during glutamate capture. To tackle 

this issue, we made use of Mito SypHer, a new genetically encoded pH sensor targeted to the 

mitochondrial matrix. Cortical astrocytes in primary culture transfected with Mito SypHer 

exhibited a typical mitochondrial labeling colocalized with CoroNa Red, a mitochondrial specific 

dye. In situ calibration revealed that the ratiometric Mito SypHer signal increased monotonically 

with mitochondrial pH. Short-term (1.5 min) application of glutamate evoked substantial 

mitochondrial acidification in a concentration-dependent manner (e.g. delta pH = -0.30±0.02 for 

200uM glutamate). Na + -coupled glutamate transporters seemed to be mainly responsible for the 

mitochondrial acidification, as similar effects were obtained using D-aspartate and the glutamate 

effect was inhibited by the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate. To 

investigate the mechanisms responsible for mitochondrial acidification, we first challenged the 

role of the mitochondrial Na + /H + exchanger. Inhibition of this exchanger by ethyl-isopropyl 

amiloride decreased the rate but not the amplitude of glutamate-evoked mitochondrial 

acidification. We found that the cytosolic acidification associated with plasma-membrane 

glutamate transport activity was responsible for the mitochondrial acidification. Compensating 

cytosolic acidification using tri-ethylammonium during a pulse of glutamate almost abolished 

glutamate-evoked mitochondrial acidification, without markedly altering cytosolic and 

mitochondrial Na + responses. This study suggests that plasma membrane glutamate transport, in 

addition to producing a substantial energy load, causes mitochondrial pH to drop, likely 

weakening the driving force for mitochondrial ATP production. 

Disclosures: G. Azarias, None; J. Chatton , None; D. Poburko, None; N. Demaurex, None.

Poster 





Title: Molecular diffusion model of glutamate homeostasis in the nucleus accumbens to study 

geometric configurations of perisynaptic glia and tortuosity 

Authors: *S. PENDYAM 1 , A. MOHAN 1 , P. KALIVAS 2 , S. S. NAIR 1 ; 

1 Univ. Missouri-Columbia, Columbia, MO; 2 Med. Univ. of South Carolina, Charleston, SC 

Abstract: A typical chemical synapse has substantial morphological specialization to control 

movement of substances that reflect special functional requirements. It is established that 

synaptic geometry, molecules per vesicular release, neurotransmitter diffusion into the 

perisynaptic space after a release, its binding to transporter and uptake via the glial sheaths, nonsynaptic 

production (e.g., via cystine glutamate exchanger system located on the glial sheaths), 

and inhibition of synaptic vesicular release due to negative feedback activation of receptors such 

as the group II metabotropic autoreceptors contribute significantly to synaptic efficacy, plasticity 

and homeostasis in the extracellular space. 

Cellular adaptations in the reward circuitry of cocaine withdrawn rats are evident in the 

prefrontal cortex - nucleus accumbal (PFC-NAc) glutamatergic projection. Two neuroplasticity 

mechanisms observed are the functional down regulation of metabotropic glutamate receptors 

and reduction in the basal extracellular glutamate level as a result of the diminished efficacy of 

the glial cystine-glutamate exchange transporter. However, neither in vivo nor in vitro techniques 

presently provide physiological information of the spatial and temporal glutamate profiles 

needed to understand such mechanisms in the perisynaptic environment. Further, no 

computational model exists that incorporates known physiological parameters to study such 

mechanisms in cocaine pathologies. 

A mathematical framework is proposed to study the mechanisms contributing to homeostasis in 

the perisynaptic region surrounding a PFC-NAc synapse for control and cocaine cases. 

Candidate glial geometries are proposed to characterize its role in regulating both synaptic and 

non-synaptic glutamate uptake. The model can be used to predict homeostatic glutamate 

concentrations at different locations for basal, reward seeking and cocaine pathologies. The 

framework provides insight into the mechanisms of homeostasis, and enables identification of

key parameters involved using a sensitivity analysis. 

Disclosures: S. Pendyam , None; A. Mohan, None; S.S. Nair, None; P. Kalivas, None. 

Poster 





Support: HFSP 

DFG 

ANR 

Title: Connexin30 controls synaptic strength by regulating astroglial glutamate transport 

Authors: *U. PANNASCH 1 , P. EZAN 1 , E. BROUILLET 2 , K. WILLECKE 3 , C. GIAUME 1 , N. 

ROUACH 1 ; 

1 INSERM U840 Col. de France, Paris, France; 2 URA CEA-CNRS 2210 Inst. d'Imagerie 

Biomédicale, Orsay, France; 3 Inst. of Genetics, Univ. of Bonn, Bonn, Germany

Abstract: Astrocytes play active roles in brain physiology by dynamic interactions with neurons. 

Numerous astroglial properties are thought to regulate neurotransmission, but the detailed 

mechanisms remain unclear. Here we show that connexin30 (Cx30), one of the two main gap 

junction proteins expressed in mature astrocytes, modulates hippocampal synaptic transmission 

in CA1 pyramidal neurons. Field excitatory postsynaptic potentials are reduced in Cx30-/- mice, 

compared to wild type animals. Reduced glutamatergic transmission is neither due to altered 

intrinsic properties and excitability of CA1 pyramidal neurons nor postsynaptic receptor density. 

It is rather due to a decrease in glutamate synaptic concentration, as the frequency but not the 

amplitude of mEPSCs is reduced in Cx30-/- mice and the ratio of AMPA to NMDA synaptic 

currents is unchanged. This effect is specific to glutamatergic transmission of CA1 pyramidal 

cells, as miniature IPSCs of pyramidal cells, as well as mEPSCs of interneurons, are not affected 

in Cx30-/- mice. Cx30 plays also a role in short and long-term synaptic plasticity, as paired-pulse 

facilitation is increased, whereas post-tetanic and long-term potentiation are reduced in Cx30-/- 

mice. Cx30 regulation of neurotransmission is due to reduced synaptic glutamate levels, as 

demonstrated by experiments with gamma-DGG, a low affinity AMPA receptor antagonist. 

Therefore glutamate clearance was assessed by measuring synaptically-evoked glutamate 

transporter currents in astrocytes, which are increased by ~100% in Cx30-/- mice. In addition, 

reduction of astroglial glutamate transporter currents to wild-type levels by dihydrokainate, a 

specific inhibitor of GLT-1, restores normal excitatory transmission in Cx30-/- mice. Altogether 

these results demonstrate that astrocytic Cx30 controls synaptic strength of excitatory terminals 

by regulating glutamate clearance. 

Disclosures: U. Pannasch, None; P. Ezan, None; E. Brouillet, None; K. Willecke, None; C. 

Giaume, None; N. Rouach, None. 

Poster 

243. Abeta Assembly and Deposition 



Topic: C.01.b. Abeta assembly and deposition 

Support: NIH AG00538 

Larry C. Hillblom Foundation 

Title: Fibrillar oligomers are a distinct neurotoxic subset of soluble amyloid beta 

Authors: *J. W. WU, L. BREYDO, Y. G. KUZNETSOV, C. G. GLABE; 

MB&B, Univ. of California, Irvine, Irvine, CA

Abstract: Alzheimer‟s disease (AD) is characterized by extracellular Aβ peptide plaques and 

intracellular hyperphosphorylated tau tangles. Extensive evidence from human AD patients and 

transgenic mice support a central pathological role for soluble Aβ oligomers in AD. Aβ 

oligomers are known to target neurons at synapses and may be responsible for synaptic failure 

leading to neuritic cell death. Here we report at least two types of structurally distinct oligomers 

exist as defined by their mutually exclusive reactivity with conformation dependent antibodies: 

prefibrillar oligomers (PFOs) and fibrillar oligomers (FOs). Fibrillar oligomers react with a fibril 

specific antibody, but are soluble (100,000 x G) and smaller than amyloid fibrils Here we 

characterize the properties and toxicity of Aβ FOs. Aß FOs do not resemble amyloid fibrils but 

rather appear as small (1 - 2 nm height) spherical or elongated particles. There are no obvious 

morphological differences between FOs and PFOs although FOs may be slightly smaller than 

PFOs. FOs are Thioflavin-T negative, unlike mature fibrils. Aβ PFOs selectively bind to neurons 

at synapses, while FOs bind to neurons extensively at both synaptic and non-synaptic sites along 

the entire dendritic processes. In vitro, Aβ FOs induce an apoptotic pathway involving 

mitochondrial impairment in hippocampal and cortical neurons. Fibrils induce moderate cell 

toxicity whereas monomers have no effect. Based on their similar tinctorial and immunological 

properties, Aβ diffuse plaques may serve as reservoirs of FOs. 

Disclosures: J.W. Wu , None; L. Breydo, None; Y.G. Kuznetsov, None; C.G. Glabe, CG is a 

consultant for Kinexis, Inc, F. Consultant/Advisory Board. 

Poster 





Support: Grant of the Ministry of Education, Culture, Sports, Science, and Technology of Japan 

Title: Staging for amyloid angiopathy in human aging 

Authors: H. HATSUTA 1 , Y. SAITO 1 , R. SENGOKU 1 , T. ADACHI 1 , T. ARAI 2 , M. 

SAWABE 2 , H. MORI 3 , *S. MURAYAMA 1 ; 

1 Dept Neuropath, Tokyo Metropolitan Inst. of Gerontol, Tokyo 173-0015, Japan; 2 Dept 

Pathology, Tokyo Metropolitan Geriatric Hosp., Tokyo 173-0015, Japan; 3 Dept Neurosci, Osaka 

City Univ., Osaka, Japan 

Abstract: PURPOSE: The present staging schemes for amyloid angiopathy (AA) in the aging 

human brain are based on semi-quantitive evaluation of the severity of vascular changes. Thus,

the vulnerability of the staging method to inter-rater bias has yet to be resolved. We tried to 

establish an extension staging paradigm for AA in order to minimize this inter-rater bias. 

TISSUE SOURCE: The brains from 217 consecutive general autopsy cases at a general geriatric 

hospital since April 2005, as well as ten archival autopsy cases of AA with lobar hemorrhage 

since January 1995, which were registered to the Brain Bank for Aging Research (BBAR) were 

employed for this study. METHOD: The brains were fixed in formalin and embedded in paraffin. 

Six-κm-thick serial sections were obtained from blocks of the frontal pole (F), hippocampus (H), 

superior temporal gyrus (T1), interparietal sulcus (P), posterior cingulate gyrus (Ci), precuneus 

(Pc), occipital pole (O), insular cortex (I), putamen (Pu), thalamus (Th), cerebellar hemisphere 

(CH), midbrain (MB) and cervical cord (CC). The sections were evaluated with hematoxylin and 

eosin, Kluever- Barrera and Congo red stainings, and immunohistochemically stained with antiamyloid 

beta 11-28 and 1-40 antibodies. RESULT: Of the 217 cases overall, 174 cases 

demonstrated AA and 7 among these showed small cerebral cortical hemorrhages caused by AA. 

The incidence of AA in each anatomical location showed the following order of frequency: O, 

137 cases; P, 128 cases; T1, 119 cases; F, 116 cases; Pc, 115 cases; Ci, 115 cases; CH, 81 cases; 

I, 64 cases; H, 29 cases; CC, 29 case; MB, 19 cases; Pu, 13 cases and Th, 8 cases. The ten cases, 

showing lobar hemorrhage, exhibited AA in the F, T1, P, O, I, CH and H. The hierarchy of AA 

appearance was as follows: P/O> F/T1> CH> H> Pu. ApoE genotypes of the 17 cases showing 

lobar or small hemorrhage were as follows: e2/e3, 4 cases; e3/e3, 10 cases; and e4/e4, two cases. 

DISCUSSION and CONCLUSION: Our studies propose the following extension stage: Stage 0, 

no AA; Stage A, AA only in P/O; Stage B, extending to F/T1; Stage C, spreading to CH; Stage 

D, further involving H; and Stage E, reaching Pu. Those cases of lobar or small hemorrhages are 

categorized into Stage D or E. Immuno- stainings with anti- Abeta antibody of occipical pole and 

hippocampus, first, subsequently followed by that of cerebellum and putamen are sufficient for 

this staging system. This extension stage fo AA shows a good correlation with the present 

staging schemes for AA and is suitable to prepare morpholigical basis for amyloid imaging, 

which causes attention to the deposition of amyloid beta to putamen. Our study also confirms an 

association of apoE e2/e3 and e4/ e4 genotypes with AA. 

Disclosures: H. Hatsuta, None; S. Murayama, None; Y. Saito, None; R. Sengoku, None; T. 

Adachi, None; T. Arai, None; M. Sawabe, None; H. Mori, None. 

Poster 





Support: SCOR 2P50HL60296

The Children‟s Foundation for Sleep and Neurobiology Research 

SCOR 2P50HL65270 

Title: Human apolipoprotein E4 transgenic mice subjected to chronic intermittent hypoxia 

develop accelerated beta amyloid deposition in hippocampus and cortex 

Authors: N. KAUSHAL, *D. GOZAL, V. RAMESH; 

Dept Pediatrics, Kosiar Childrens Hosp Res. Inst., Louisville, KY 

Abstract: The allele E4 of apolipoprotein E (ApoE4) is a well established genetic risk factor for 

Alzheimer's disease, and has been associated with increased amyloid levels in brain. Although 

epidemiological associations have emerged between ApoE4, AD, and sleep apnea, no studies 

have specifically examined deposition patterns of beta amyloid in transgenic mice expressing 

human ApoE4 in CNS exposed to a murine model of sleep apnea. ApoE4 transgenic mice (n=2) 

and C57BL/6J WT mice (n=2) were chronically subjected to either intermittent hypoxia (IH; 

cycling of 5.7% or 21% oxygen every 3 min) starting from 7.00 am to 7.00 pm (light period) 

followed by room air (RA) until 7.00 am next day from age 7 months to 9.5 months. 

Hippocampal and cortex sections were double stained with Neu-N and beta amyloid in both WT 

and ApoE4 mice. Increased expression of beta amyloid was apparent in both hippocampus and 

cortex in ApoE4 mice when compared to the control mice. In hippocampus, the increase in beta 

amyloid appeared to be more prominent in the CA3 region. These preliminary results support the 

hypothesis that chronic IH during sleep, as occurs in sleep apnea, not only imposes global 

temporal effects on multiple physiological functions, but also enhances pathological changes in 

neuronal structures, the latter further leading to accentuated sleep disturbances, and to 

accelerated short-term memory and neurocognitive deficits. 

Disclosures: N. Kaushal, None; D. Gozal , None; V. Ramesh, None. 

Poster 





Support: Alzheimer's Association 

Title: Evidence for intraneuronal formation and accumulation of amyloid-beta oligomers in APP 

transgenic mice

Authors: M. ELIAVA 1 , *V. GRINEVICH 4 , P. N. LACOR 2 , R. VASSAR 3 , W. L. KLEIN 2 , P. 

OSTEN 1 ; 

1 Physiol., 2 Neurobio. and Physiol., 3 Cell and Mol. Biol., Northwestern Univ., Chicago, IL; 4 Mol. 

Neurobiol., Max Planck Inst. Med. Res., Heidelberg, Germany 

Abstract: Soluble amyloid-β (Aβ) oligomers have been proposed to critically contribute to early 

pathology of Alzheimer‟s disease (AD), especially to deficits in synaptic plasticity that may 

underlie memory impairments. However, whereas the evidence for pathological function of 

exogenously applied Aβ oligomers is well documented, little is known about how Aβ oligomers 

are formed in the brain. For example, it is not known whether Aβ oligomerization occurs 

intraneuronally following β- and γ-secretase-mediated proteolysis of amyloid precursor protein 

(APP) or whether Aβ oligomers are formed from secreted Aβ monomers extracellularly. Here we 

examine this question in two AD mouse models, 5XFAD mice with rapid Aβ accumulation due 

to expression of five FAD mutations (APP Swedish, Florida and London and presenilin-1 

M146L and L286V) and Tg2576 mice with much slower Aβ accumulation from expression of a 

single APP Swedish FAD mutation. We take advantage of a mosaic expression pattern common 

to most transgenic mice and ask whether neurons with high expression of the APP transgene also 

show high accumulation of intraneuronal monomeric and oligomeric Aβ, as would be expected 

from direct intraneuronal processing. These experiments are done by double-labeling 

immunohistochemistry, with 1) APP N- or C-terminal-specific antibodies applied to detect the 

level of APP transgene expression, and 2) Aβ40-, Aβ42- and Aβ oligomer-specific antibodies 

used to detect the level of intraneuronal accumulation of the respective Aβ forms. Our 

preliminary data suggest a close correlation between APP expression and intraneuronal 

accumulation of oligomeric Aβ forms. Furthermore, cortical neurons previously shown to be 

highly vulnerable to cell death in the 5XFAD model show the highest expression of APP and 

accumulation of Aβ oligomers. This suggests that Aβ oligomers formed intraneuronally may 

directly contribute to AD neurotoxicity. 

Disclosures: M. Eliava, None; V. Grinevich , None; P.N. Lacor, None; R. Vassar, 

None; W.L. Klein, None; P. Osten, None. 

Poster 





Support: NIH Grant AG18879


NIH Grant AG 027465:01A2 

NIH Grant AG 5AG016570 

Alzheimer's Association NIRG-03-6103 

Title: Elevated levels of soluble Aβ oligomers and Aβ42 in cortical synapses of late stage 

Alzheimer‟s disease 

Authors: *S. SOKOLOW 1 , A. J. HEADLEY 1 , K. E. BLAKELY 1 , C. A. MILLER 3 , H. V. 

VINTERS 4 , G. M. COLE 5 , K. H. GYLYS 2 ; 

1 Sch. of Nursing, 2 Brain Res. Inst. and Sch. of Nursing, UCLA, Los Angeles, CA; 3 Dept. of 

Pathology and Neurol., Keck Sch. of Med. of USC, Los Angeles, CA; 4 Dept. of Pathology and 

Lab. Med., UCLA Sch. of Med., Los Angeles, CA; 5 Sch. of Med. and Sepulveda VAMC 

GRECC, UCLA and Sepulveda VA Med. Ctr., Los Angeles, CA 

Abstract: Although fibrillar amyloid beta (Aβ) is the major component of senile plaques in 

Alzheimer‟s disease (AD), new evidence suggests that accumulation of soluble Aβ oligomers is 

the proximal cause of synapse dysfunction in AD. Thus accurate identification and quantification 

of Aβ species in AD synapses are crucial steps to elucidate Aβ toxicity in AD pathogenesis. In 

order to study synaptic changes in AD, our laboratory has developed methods for flow cytometry 

analysis of synaptosomes prepared from cryopreserved human tissue. Our previous results in AD 

synaptosomes showed a high correlation between flow quantification of Aβ and different Aβ 

assembly states identified by Western blot. The present study quantifies three Aβ species (Aβ40, 

Aβ42 and Aβ oligomers) in late AD cases (Braak & Braak score V and VI), with specific beadbased 

immunoassays using the xMAP ® technology (Luminex ® , Austin, Texas). We addressed 

Aβ solubility in AD synapses by performing serial protein extractions of human cryoprotected 

synaptosomes (crude P2) isolated from post-mortem parietal cortex (A39): i) P2 proteins were 

extracted by sonication in a detergent-free buffer followed by centrifugation at 26,000 g and ii) 

the remaining pellet was extracted twice by sonication in 1% N-lauroylsarcosyl (NLS) sucrose 

followed by centrifugation at 300,000 g. Levels of Aβ oligomers (aggregated Aβ), Aβ42 and 

Aβ40 were measured in each fraction using specific assays developed for the Luminex ® xMAP ® 

platform (Invitrogen, Camarillo). Our results showed significantly elevated synaptic levels of 

soluble Aβ oligomers in the detergent-free extracts of AD synaptosomes compared to normal. 

The levels of Aβ oligomers were undetectable in extracts of control patients (n = 2) and 57.57 ± 

25.17 pg/κg in AD (mean ± SD, n = 7, p

Disclosures: S. Sokolow, None; A.J. Headley, None; K.E. Blakely, None; C.A. Miller, 

None; H.V. Vinters, None; G.M. Cole, None; K.H. Gylys, None. 

Poster 





Support: NIH 

Title: N-terminally truncated pyroglutamate beta-amyloid, pyrogluAbeta(3-42), is colocalized 

with Abeta40 and Abeta42 in regulated secretory vesicles: implications for biosynthesis of Abeta 

peptides related to Alzheimer‟s disease 

Authors: *V. Y. HOOK 1 , T. TONEFF 2 , G. HOOK 3 ; 

1 Skaggs Sch. Pharm. & Pharmaceut Sci., Univ. Calif, San Diego, La Jolla, CA; 2 Skaggs Sch. of 

Pharm., Univ. of Calif., San Diego, La Jolla, CA; 3 American Life Sci. Pharmaceuticals, Inc., San 

Diego, CA 

Abstract: Investigations of amyloid plaques in Alzheimer‟s disease brains have identified the Nterminal 

truncated pyroglutamate form of beta-amyloid, pGluAbeta, as a significant portion of 

Abeta in AD brains (Saido et al., 1995), in addition to Abeta40 and Abeta42 peptide forms. The 

modified Abeta peptide starts at glutamate, the third amino-terminal residue of Abeta, which 

results from removal of two N-terminal residues of Abeta40/42; this N-terminal glutamate is 

converted to pyroglutamate. Interestingly, pGluAbeta facilitates seeding of Abeta peptides into 

oligomers and aggregates (Schilling et al., 2006). Thus, pGluAbeta represents an important 

modified Abeta peptide related to AD. Elucidation of the subcellular location of pGluAbeta will 

facilitate understanding of how it participates in AD. 

The majority of Abeta40 and Abeta42 peptides are produced intracellularly in the regulated 

secretory pathway. These Abeta peptides are produced from the amyloid precursor protein (APP) 

by proteolytic processing by proteases known as beta-secretase and gamma-secretase in the 

secretory pathway. Regulated secretory vesicles (RSV) isolated from chromaffin cells of the 

peripheral nervous system contain Abeta40, Abeta42, as well as full-length APP that undergoes 

processing within the RSV to generate Abeta peptides. These isolated RSV have provided an 

excellent model for peptide biosynthetic mechanisms that are utilized in brain for production of 

peptide neurotransmitters. 

To address the question of the subcellular localization and production of the pGluAbeta peptide, 

its colocalization with Abeta40 and Abeta42 was investigated with a specific ELISA to

pGluAbeta(3-42). The isolated regulated secretory vesicles contained substantial levels of 

pGluAbeta, combined with Abeta40 and Abeta42. Each of these Abeta peptide forms were 

present at similar levels. Furthermore, these Abeta peptides are colocalized in these RSV with 

multiple peptide neurotransmitters and catecholamines, suggesting the parallel secretory 

mechanisms for release of pGluAbeta with Abeta40 and Abeta42, as well as with 

neurotransmitter molecules, from large dense core vesicles. These results demonstrate that 

pGluAbeta peptide is generated under normal conditions with Abeta40 and Abeta42 in the 

regulated secretory pathway, which may provide a mechanism for their deposition in amyloid 

plaques of Alzheimer‟s disease. 

Disclosures: V.Y. Hook , American Life Science Pharmaceuticals, Inc., B. Research Grant 


received); American Life Science Pharmaceuticals, Inc., E. Ownership Interest (stock, stock 

options, patent or other intellectual property); T. Toneff, None; G. Hook, American Life Science 

Pharmaceuticals, Inc. (ALSP), A. Employment (full or part-time); ALSP, B. Research Grant 


received); ALSP, E. Ownership Interest (stock, stock options, patent or other intellectual 

property). 

Poster 



Program#/Poster#: 243.7/K1 


Support: Alzheimer‟s Association 

The Mitchell Foundation 

Title: Characterization of conformation and localization of different Aβ species of Alzheimer‟s 

mouse models 

Authors: *C. A. LASAGNA-REEVES, C. SOTO, R. KAYED; 

Dept Neurol, Univ. Texas Med. Br., Galveston, TX 

Abstract: Alzheimer‟s disease (AD) is a devastating neurodegenerative disorder and is one of 

several disorders collectively known as the amyloid diseases. Other amyloid diseases include 

Parkinson‟s disease (PD), Huntington disease (HD), type II diabetes, and the prion diseases. All 

of these disorders are characterized by the deposition of proteinaceous aggregates, extracellular

and/or intracellular. 

Recently it has been proposed that a hierarchical assembly mechanism in which amyloidogenic 

proteins begin with a random coil structure, are then misfolded and next aggregate into spherical 

oligomers. The spherical oligomers can then further aggregate into chain-like protofibrils and/or 

annular protofibrils, and finally form the fibrils present in amyloid. Many research groups are 

using a variety of techniques to detect and probe these aggregates, especially the soluble forms of 

Aβ recently described as the most toxic species implicated with synapse loss and synaptic 

function impairment in the early stages of the disease. 

In this study, we used a combination of sequence specific Aβ antibodies and novel conformation 

antibodies against different amyloid species, specifically anti-oligomers, anti-fibrils and antiannular 

protofibrils to investigate the location and conformation of each amyloid species in 

transgenic mouse models. Immunohistochemistry, Western Blot and immunoprecipitation were 

used to analyze and characterize the different amyloid species in these animals at different ages. 

We found the presence of both intracellular and extracellular Aβ oligomers, while the 

extracellular oligomers were similar to the ones detected in AD brains and located where 

amyloid plaques are. Intracellular and membrane associated oligomers were also detected, and 

are being analyzed. These results are important to understand the dynamic changes of Aβ 

deposits in both location and conformation and their role in the progression of the disease. 

Disclosures: C.A. Lasagna-Reeves , None; C. Soto, None; R. Kayed, None. 

Poster 





Support: NCCR Zurich Neural plasticity and repair 

SNF Grant 310000-117806 

Title: Reduced Reelin-dependent signaling affects amyloid-β plaque formation in-vivo 

Authors: *I. KNUESEL, S. KOCHERHANS, F. CRESTANI, C. SIDLER, J.-M. FRITSCHY; 

Inst. of Pharmacol. and Toxicology, Univ. of Zurich, Zurich, Switzerland 

Abstract: Besides the control of cortical lamination during neurodevelopment, several recent 

findings suggest novel roles for the glycoprotein Reelin and its receptors in the pathogenesis of 

Alzheimer‟s disease (AD). The highly conserved protein is a pivotal synaptic regulator which

modulates neuronal function and synaptic plasticity. It enhances the induction of long-term 

potentiation via its modulatory effects on NMDA receptor function and its ability to stimulate the 

translation of dendritically expressed transcripts. Reelin binds to the very-low-density lipoprotein 

receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). This induces their clustering and 

tyrosine phophorylation of the adapter protein Dab1 that interacts with cytoplasmic NPxY motifs 

of VLDLR, ApoER2, and amyloid precursor protein (APP). Reelin also affects the formation of 

amyloid-β plaques and neurofibrillary tangles, the two neuropathological hallmarks of AD: 

Reelin-induced receptor clustering is necessary for recruitment of fyn and src tyrosine kinases to 

Dab1 and the recruitment and activation of kinases that are key mediators of Tau 

phosphorylation. In addition, Reelin increases the intracellular interaction of Dab1 with both 

ApoER2 and APP and promotes their cell surface localization and activation of nonamyloidogenic 

APP processing in-vitro. Here, we tested the hypothesis that reduction in Reelin 

expression and signaling are causally involved in neurodegenerative processes of late-onset AD 

by favoring amyloidogenic processing of APP and amyloid-β plaque formation in-vivo. We 

crossed Reelin heterozygous knockout mice with transgenic ArcAβ mice. We assessed the 

cognitive status of the F1 wild-type, single- and double-transgenic mutant mice at 3, 6, and 9 

months of age using an object-in-context recognition task. The amyloid-β plaque burden was 

investigated in the hippocampus and cerebral cortex using immunohistochemistry and unbiased 

stereological quantification. This was complemented by biochemical examinations of soluble 

and insoluble fractions of brain homogenates using western blotting and ELISA focusing on 

Aβ40/42 peptide productions. We found a precocious formation of amyloid-β plaques in Reelindeficient 

ArcAβ double-transgenic mice compared to single-mutant AD mice, which was 

associated with selective hippocampal-dependent memory impairment. Our results confirm a 

critical role of Reelin-dependent signaling in APP processing and suggest that aging-related 

decline in Reelin expression represent an important driving force of senile plaque formation and 

cognitive decline associated with late-onset AD. 

Disclosures: I. Knuesel , None; S. Kocherhans, None; F. Crestani, None; C. Sidler, None; J. 

Fritschy, None. 

Poster 





Support: Alzheimer's Association Research Grant 

Title: Endogenous testosterone prevents amyloid deposition in transgenic animal brain

Authors: *R. LI, C. MCALLISTER, J. DOLE, J. LONG; 

LJ Roberts Ctr. Alzheimers, Sun Hlth. Res. Inst., Sun City, AZ 

Abstract: While both menopause and andropause refer to the decrease in sex steroids upon 

reproductive senescence in women and men, respectively, the rate at which these changes take 

place are drastically different, with menopause occurring very abruptly while the process of 

andropause is a gradual phenomenon. In fact, this gender-based difference in the rate and 

intensity of hormonal changes due to reproductive aging serves as the basis for hypotheses 

regarding the role of sex steroids in Alzheimer‟s disease (AD). Several recent studies have 

reported that low bioavailable testosterone is associated with AD in aged men. Testosterone 

administration in elderly men might help to improve their cognitive function. Because 

testosterone in the brain is converted into estradiol by a single enzyme, aromatase, to further 

identify and understand the underlying mechanism of testosterone in AD, we generated a new 

animal model using a gene-based approach, by crossing aromatase gene knockout mice with 

APP23 transgenic mice to produce sex hormone disrupted APP23 mice (APP/Ar+/-). Compared 

to APP23 transgenic control mice, the male APP23/Ar+/- mice exhibited a large increase in brain 

testosterone and a significant reduction in estrogen. Instead of an early-onset and increased Aβ 

deposition as is found in female APP23/Ar+/- mice, we found a significant delay in brain plaque 

formation in the male APP23/Ar+/- mice. These male mice also exhibited increased Aβ 

clearance/degradation and changes in Aβ metabolism compared to APP23 control mice. We 

examined cognitive behavior and our data suggest that increasing endogenous testosterone level 

not only prevents cognitive deficits in AD model mice, but also improves cognitive function 

compared to wildtype. Our results indicate that endogenous testosterone in the male brain may 

play a significant neuroprotective role in AD neuropathology. 

Disclosures: R. Li, None; C. McAllister, None; J. Long, None; J. Dole, None. 

Poster 







Title: Elucidating the roles of small molecules in modulating the dynamic assembly of Aß in 

solution by electrospray ionization high resolution mass spectrometry

Authors: N. NEMIEBOKA 1 , S. THOMAS 2 , Y. WAN 3 , Z. LIAO 2 , J. MCLAURIN 4 , *A. 

YANG 3 ; 

1 Program in Oncology, 2 Dept. of Radiation Oncology, 3 Greenebaum Cancer Ctr., Univ. 

Maryland, Baltimore, Baltimore, MD; 4 Ctr. for Resaech in Neurodegenerative Dis., Univ. of 

Toronto, Toronto, ON, Canada 

Abstract: In this study, we have developed a novel electrospray ionization mediated high 

throughput mass spectrometry-based platform to address the role of various small molecules in 

modulating the dynamic assembly of amyloid peptide (Aß). By coupling a capillary ultra high 

pressure HPLC system (UPLC) to either a triple quadrupole or a high resolution and high mass 

accuracy orbitrap mass spectrometer, we are able to detect the assembly of Aß-ligand complexes 

in solution at the low femtomole to attomole range, which provides a drastic improvement in the 

sensitivity of our detection in comparison to other traditional solution-based binding and 

biophysical approaches. In contrast to most MALDI-TOF mass spectrometry based methods, 

examination of the charge distribution of both Aß and Aß-ligand complexes using ESI and gas 

phase fractionation mass spectrometry preserves the structure of Aß-ligand complexes in 

solution under their physiological condition. 

To further determine the specificity of Aß-ligand complexes, we have also implemented topdown 

and collision induced dissociation mass spectrometry approaches. Our preliminary study 

demonstrates that by introducing several different types of gas phase fragmentation inside the 

collision cell of the mass spectrometer we are able to measure the binding stoichiometry of the 

Aß1-42-scyllo-inositol complex. Furthermore, we have demonstrated that scyllo-inositol 

preferentially binds to Aß1-42 rather than Aß1-40 under our conditions. Finally, by using a series 

of newly developed computational tools (both commercial and in-house) coupled with the 

sequencing capability of tandem mass spectrometry, we are able to specifically demonstrate the 

role of metal ions, such as copper, in modulating both the oxidation and structure of histidine 

residues within Aß molecules. 

Disclosures: N. Nemieboka, None; S. Thomas, None; Y. Wan, None; Z. Liao, None; J. 

McLaurin, None; A. Yang , None. 

Poster 



Program#/Poster#: 243.11/L1 


Title: Products of amyloid precursor protein metabolism are associated with mitochondria in 

synaptic fields

Authors: *K. KIPIANI, T. A. MILNER, G. K. GOURAS, F. M. BEAL, M. T. LIN; 

Neurol. & Neurosci., Weill Med. Col. Cornell Univ., New York, NY 

Abstract: Intracellular accumulation of amyloid-β peptide (Aβ) is increasingly recognized as a 

contributor to the Alzheimer‟s disease (AD) pathogenesis. Mitochondrial impairment is also 

implicated in the AD pathogenesis. To investigate whether mitochondria are a site of 

intracellular Aβ accumulation, we performed subcellular fractionation and immunoelectron 

microscopy studies on brains of Tg19959 mice, which express a doubly mutant human amyloid 

precursor protein (APP) and are characterized by accelerated production and deposition of Aβ42. 

We found that Aβ is present in mitochondrially enriched fractions, and is resistant to the 

treatment with nonionic detergent and to the proteolytic digestion, suggesting its localization to a 

membrane protected compartment of mitochondrial origin. Synthetic Aβ gets also incorporated 

within the same membrane-protected compartment. By immunoelectron microscopy, we found 

that some but not all mitochondria in Tg19959 mouse brains were immunoreactive for Aβ or 

APP C-terminal fragments (APP CTFs). We found this in both very young mice and after plaque 

deposition. In the hippocampus, mitochondria with Aβ immunoreactivity were most common in 

distal neuronal processes in layers containing synaptic fields. Mitochondria immunoreactive for 

APP CTFs occurred in both neuronal and astroglial processes. The astroglial processes 

containing APP CTF-immunoreactive mitochondria were typically heavily labeled by APP CTFs 

and were morphologically altered. Because mitochondria are important in synaptic function, 

mitochondrial Aβ and APP CTFs within neuronal and glial processes could contribute to 

synaptic dysfunction in AD. 

Disclosures: K. Kipiani , None; T.A. Milner, None; G.K. Gouras, None; F.M. Beal, 

None; M.T. Lin, None. 

Poster






Title: Assembly dynamics of the Alzheimer amyloid β-protein 

Authors: R. ROYCHAUDHURI 1 , A. LOMAKIN 2 , G. BENEDEK 2 , *M. M. CONDRON 1 , D. 

TEPLOW 1 ; 

1 Dept Neurol, David Geffen Sch. Med. at UCLA, Los Angeles, CA; 2 Dept Physics, MIT, 

Cambridge, MA 

Abstract: Alzheimer‟s disease (AD) is a fatal neurodegenerative disorder linked to aggregation 

of the amyloid β-protein (Aβ). Aggregation of a 42-residue form of Aβ, Aβ42, is thought to be 

particularly important pathologically. Unfortunately, the propensity of Aβ42 to aggregate makes 

its study difficult. To circumvent this problem, we have used a novel chemical approach 

involving synthesis of an Aβ42 isomer, Ser26-O-acyl-isoAβ42 (IAβ42). IAβ42 has superior 

handling properties compared to native Aβ and can be converted quasisynchronously into the 

native Aβ42 isomer by a simple pH change from acidic to neutral or basic. Importantly, the ester 

within this Aβ isomer occurs within a peptide segment that has been determined to form a turn 

structure that nucleates Aβ monomer folding. Use of this ester thus also allows study of the 

importance of the turn region in Aβ assembly. The O-N acyl shift that converts the IAβ peptide 

into native Aβ occurs rapidly (

Poster 





Support: Alzheimer Association IIRG-06-27077 

NIH/NIA Grant AG027973 



Title: Regulatory role of Abca1 and brain lipoproteins in Aβ oligomerization and deposition - 

comparative pathology in ABCA1ko and ApoA-Iko/ApoEko mice 

Authors: R. KOLDAMOVA 1 , N. FITZ 1 , A. A. CRONICAN 1 , P. LEFTEROV 1 , *I. M. 

LEFTEROV 2 ; 

1 Envrn. & Occup. Hlth., Univ. Pittsburgh, Grad. Sch. of Publ. Hlth., Pittsburgh, PA; 2 Enviro & 

Occup. Hlth., Univ. Pittsburgh, Grad. Sch. Pub, Pittsburgh, PA 

Abstract: The ATP-binding cassette transporter (ABCA1) and apolipoproteins regulate 

cholesterol and HDL formation. Recently, we and other groups demonstrated that APP 

transgenic mice with engineered disruption of Abca1 (ABCA1 ko ) have an increased level of 

amyloid plaques. In addition, Abca1ko mice have decreased levels of soluble apoA-I and apoE in 

brain whereas the level of insoluble apolipoproteins is not changed. In APP mice with global 

deletion of Abca1 the decrease of apoE and apoA-I is accompanied by an increase of insoluble 

Amyloid beta (A[[Unsupported Character - Symbol Font ]]). Our most recent 

experiments demonstrate that APP23 mice with one functional Abca1 allele (APP/Abca1 +/- ) have 

cognitive deficits when compared to APP23 with intact Abca1 (APP/Abca1 wt ). We hypothesize 

that Abca1-regulated generation of lipid-rich apoE, not the decreased levels of apoE and apoA-I, 

is a critical mediator of A[[Unsupported Character - Symbol Font ]] aggregation. To 

test this hypothesis we generated APP/PS1[[Unsupported Character - Symbol Font 

]]E9 transgenic mice with global deletion of ApoE and ApoA-I genes (APP/ApoA- 

I ko /ApoE ko ). In this study we have compared the amyloid deposition and cognitive decline in 

APP/ApoA-I ko /ApoE ko to those in APP/Abca1 -/- and APP/Abca1 wt . The initial results of our 

experiments show that the APP/Abca1 -/- mice have the highest level of amyloid deposition and 

soluble A[[Unsupported Character - Symbol Font ]] oligomers. In contrast APP/ApoA- 

I ko /ApoE ko double knockout mice have significantly less Thio-S positive plaques than 

APP/Abca1 wt and APP/Abca1 -/- mice. Surprisingly, the level of soluble A[[Unsupported 

Character - Symbol Font ]] oligomers in APP/ApoA-I ko /ApoE ko double knockout mice

is comparable to those in APP/Abca1 wt although lower than in APP/Abca1 -/- animals. 

The results from our study demonstrate that Abca1 and brain lipoproteins have a significant, yet 

not fully understood, impact on A[[Unsupported Character - Symbol Font ]] 

aggregation and, even more, the formation of A[[Unsupported Character - Symbol Font 

]] oligomer structures. Considering the regulatory role of ABCA1 on ApoE and ApoA- 

I lipidation - a prerequisite for their normal function related to brain cholesterol and phospholipid 

metabolism and transport, further studies will undoubtedly improve our understanding of central 

pathogenic mechanisms in Alzheimer Disease and will substantiate new therapeutic approaches. 

Disclosures: R. Koldamova, None; N. Fitz, None; A.A. Cronican, None; P. Lefterov, 

None; I.M. Lefterov , None. 

Poster 





Support: NIH 1F32 AG030256-01 (LMJ) 

R01 AG19121 (MJL) 

Alzheimer's Association NIRG-06-26957 (CY) 

Title: Fluorophore-labeled amyloid-beta 42 oligomers for cellular trafficking and toxicity studies 

in neuronal cells 

Authors: *L. M. JUNGBAUER, C. YU, M. LADU; 

Anat. and Cell Biol., Univ. of Illinois-Chicago, Chicago, IL 

Abstract: Revision of the amyloid cascade hypothesis over the past decade of Alzheimer‟s 

disease (AD) research has stimulated intense focus on soluble oligomeric assemblies of the 42 

amino acid form of the amyloid-beta peptide (Aβ42) as the proximate cause of neuronal injury, 

synaptic loss and the eventual dementia associated with AD. While neurotoxicity, 

neuroinflammation, and deficits in behavior and memory have all been attributed to Aβ 

oligomers in various in vitro and in vivo experimental paradigms, the specific roles of Aβ42 

oligomer toxicity in AD pathology remain poorly understood. Cellular trafficking of exogenous 

Aβ42 by neurons in vitro and in vivo is critical to our understanding of mechanisms underlying 

its neurotoxicity; however, studies have been hindered by the lack of reliable and well-

characterized forms of labeled Aβ42 oligomers. As researchers become increasingly 

conscientious of utilizing structurally uniform, well-characterized Aβ preparations, the same 

criteria must be imposed on derivitized Aβ assemblies such as fluorophore-labeled- and biotinlabeled-Aβ, 

prior to their widespread use as experimental tools. Therefore, the objective of this 

study is to obtain and characterize uniform preparations of fluorescently-labeled Aβ42 oligomers 

that are comparable to the unlabeled species in both structure and function. Previous studies have 

demonstrated the advantages of using tapping mode atomic force microscopy (AFM) to 

characterize the structurally discreet species formed by synthetic Aβ42 under specific solution 

conditions (e.g., oligomers, protofibrils and fibrils). Here, we extend these methods to 

characterize the effects of fluorophore conjugation on Aβ assembly state and to ensure 

fluorophore-labeled oligomers remain as uniform assemblies, free from fibrils, protofibrils or 

other conformations of the peptide. Finally, we compare the function of fluorophore-labeled- and 

unlabeled oligomeric Aβ42 by monitoring neurotoxicity and cellular uptake in neuronal cells. 

AFM-characterized fluorophore-Aβ42 oligomers are an exciting new reagent for use in a variety 

of studies designed to elucidate critical cellular and molecular mechanisms underlying the 

toxicity of this Aβ assembly form in AD. 

Disclosures: L.M. Jungbauer, None; C. Yu, None; M. LaDu, None. 

Poster 





Support: grants-in-aid for Scientific Research A (No.18208011) from Japanese Government 

Title: Verification of the intermolecular parallel β-sheet in E22K-Aβ42 (Italian) aggregates by 

solid-state NMR using rotational resonance: Implications for the supramolecular arrangement of 

the toxic conformer of Aβ42 

Authors: *K. IRIE 1 , Y. MASUDA 1 , A. NAKANISHI 2 , R. OHASHI 2 , K. TAKEGOSHI 2 , T. 

SHIMIZU 3 , T. SHIRASAWA 4 ; 

1 Grad. Sch. of Agr., 2 Grad. Sch. of Sci., Kyoto Univ., Kyoto, Japan; 3 Res. Team for Mol. 

Biomarkers, Tokyo Metropolitan Inst. of Gerontology, Tokyo, Japan; 4 Dept. of Ageing Control 

Med., Juntendo Univ. Grad. Sch. of Med., Tokyo, Japan 

Abstract: Aggregation of the 42-residue amyloid β peptide (Aβ42) plays a critical role in the 

pathogenesis of Alzheimer‟s disease (AD). Formation of the intermolecular β-sheet is a key

event in the aggregation of Aβ42. Recently, our proline scanning and solid-state NMR identified 

two conformers in Aβ42 aggregates: a physiological one with a turn at positions 25 and 26, and a 

toxic one with a turn at positions 22 and 23. Moreover, we suggested that E22K-Aβ42 (Italian), 

one of the mutants related to cerebral amyloid angiopathy, has a higher aggregative ability and 

neurotoxicity than wild-type Aβ42 because an ionic interaction between the side chains of Lys- 

22 and Asp-23 could promote the formation of a turn at positions 22 and 23 to increase the ratio 

of the toxic conformer (1). The establishment of a precise structural model of the E22K-Aβ42 

aggregates is thus essential for understanding the etiology of AD. However, it remains unclear 

whether the intermolecular β-sheet in the E22K-Aβ42 aggregates is parallel or antiparallel, and 

how each conformer assembles in relation to each other. To clarify these, we prepared the E22K- 

Aβ42 aggregates from an equal mixture of E22K-Aβ42 labeled at Cα and that labeled at C=O 

with 13C, whose intermolecular 13C-13C distance was estimated by solid-state NMR using 

rotational resonance (R2) (2). The intermolecular proximities of β-strands at positions 21 and 30 

were less than 6 Å (3), supporting the presence of the intermolecular parallel β-sheet in E22K- 

Aβ42 aggregates as well as in wild-type Aβ42 aggregates. The results also suggest that each 

conformer would not accumulate alternately but form a relatively large assembly consisting of an 

intermolecular parallel β-sheet in the aggregates. Since the aggregation of the toxic conformer is 

much faster than that of the physiological one, the toxic conformers would form a nucleus, 

resulting in the elongation of the physiological conformers. 

1) Bioorg. Med. Chem. 2005, 13, 6803 

2) Chem. Phys. Lett. 1988, 146, 71 

3) Biosci. Biotechnol. Biochem. 2008, 72, in press 

Disclosures: K. Irie, None; Y. Masuda, None; A. Nakanishi, None; R. Ohashi, None; K. 

Takegoshi, None; T. Shimizu, None; T. Shirasawa, None. 

Poster 

244. Abeta Toxicity I 



Topic: C.01.c. Abeta toxicity 

Support: Italian Ministry of Research and University FIRB (# RGNEO3PX83; PZ and SLS) 

PRIN 2006 (SLS) 

Title: Aluminum modulates effects of Beta-Amyloid 1-42 on neuronal calcium homeostasis and 

mitochondria functioning and is altered in the cortex of a triple transgenic mouse model of 

Alzheimer‟s disease

Authors: *S. SENSI 1,2 , D. DRAGO 4 , N. MASCETRA 3 , D. CIAVARDELLI 3 , P. ZATTA 5 ; 

1 Dept Neurol, Univ. California Irvine, Irvine, CA; 2 CeSI Ctr. for Excellence On Aging, Univ of 

Chieti-Pescara, Italy; 3 CeSI Ctr. for Excellence On Aging, Chieti, Italy; 4 Dept. of Biology, Univ. 

of Padua,, CNR-Institute for Biomed. Technologies- “Metalloproteins” Unit, Viale G. Colombo 

3-35121 Padua, Italy, Italy; 5 Dept. of Biology, Univ. of Padua,, CNR-Institute for Biomed. 

Technologies- “Metalloproteins” Unit, Padua, Italy 

Abstract: Recent findings suggest that beta-amyloid (A-beta) is potently neurotoxic when 

present in its oligomeric configuration rather than as monomers or fibrils. Previous work from 

our laboratories has shown that A-beta 

aggregation is strongly influenced by the conjugation of the peptide with metal ions (aluminum, 

copper, zinc, and iron) that are found in high concentrations in the core of senile plaques. 

Disruption of Ca2+ signaling and mitochondrial dysfunction are potent triggers of neuronal death 

and have been implicated in Alzheimer‟s disease (AD) related neuronal loss. In this study we 

explored in neuronal and isolated mitochondria preparations whether A-beta-metal complexes, 

by influencing A-beta conformation, can have detrimental effects on Ca2+ homeostasis and 

mitochondrial functioning. Results from this study indicate that, when conjugated with 

Aluminum (Al), A-beta strongly promotes perturbation of intraneuronal Ca2+ ([Ca2+]i) 

homeostasis and induces inhibition of mitochondrial respiration. Finally, we analyzed the content 

of these metals in several brain regions of a triple transgenic animal model of AD and found that 

Al is the only one to be increased in the cortex of the mice. 

Disclosures: S. Sensi , None; D. Drago, None; N. Mascetra, None; D. Ciavardelli, None; P. 

Zatta, None. 

Poster 





Support: SNRP NIH grant to AR and GH Fan 

Title: Clarification of the neuroprotective pathway functioning through the different chemokine 

receptors in Alzheimer's disease 

Authors: S. ZAJA-MILATOVIC 1 , D. RAMAN 1 , D. MILATOVIC 1 , G.-H. FAN 2 , *A. Y. 

DEUTCH 3 , A. RICHMOND 4 ; 

1 Dept. of Cancer Biol., Vanderbilt Univ., Nashville, TN; 2 Mehhary Med. Col., Nashville, TN;

3 Dept Psychiat & Pharmacol, Vanderbilt Univ. Sch. Med., Nashville, TN; 4 Dept. of Cancer Biol., 

Vanderbilt Univ. and VA Med. Ctr., Nashville, TN 

Abstract: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by senile 

plaque pathology, neurofibrillary tangles, and neuronal death, which result in impairment of 

memory and cognitive function. AD is characterized by the progressive loss of the neurons in 

specific brain regions. Chemokines are expressed not only in neuroinflammatory conditions, but 

also constitutively by different cell types, including neurons in the normal brain, suggesting that 

they may act as modulators of neuronal functions. It has been shown that MIP2, a mouse 

chemokine ligand for the chemokine receptor, CXCR2, prevents Aβ induced apoptosis in 

hippocampal neurons in vitro (Watson, K. et al. 2005). Based on these initial findings, 

chemokines in the CNS appear to be neuroprotective, in contrast to the neurotoxic effects of 

inflammatory cytokines. Our results indicate that pre-incubation of mixed primary neuronal 

cultures with SB225002, an antagonist specific for CXCR2, blocked the MIP-2 rescue of 

neurons from Aβ-induced apoptosis, indicating that the neuroprotective pathway functions 

through CXCR2. Also, N-methyl D-aspartate (NMDA), a ligand for glutamate receptors, 

attenuated CXCR2-mediated neuroprotection through persistent CXCR2 phosphorylation and 

blockade of receptor. Based upon our morphological study, pretreatment with the NMDA 

receptor antagonist memantine resulted in significant protection in vitro and in vivo against Aβ 

effects on spine density and total dendritic length. Quantitative morphometric analysis of 

pyramidal neurons from Aβ treated CXCR2 (-/-) neurons also showed protection by memantine 

as assessed in vitro by drebrin staining, and in vivo by Golgi impregnated, stained neurons. 

Pretreatment with SDF-1 (0.25µg/µl), ligand for the chemokine receptor CXCR4, showed only 

partial protection against Aβ induced neurotoxicity in vivo and in vitro in contrast to strong 

protection by MIP-2. Our results demonstrate that CXCR2 and CXCR4 offer neuronal protection 

from the toxic effects of Aβ in vivo and in vitro. 

Disclosures: S. Zaja-Milatovic, None; D. Raman, None; A.Y. Deutch , None; D. Milatovic, 

None; G. Fan, None; A. Richmond, None. 

Poster 





Title: Development of new High Content fluorescence based Assay of soluble amyloid beta 

polymer mediated cellular effects

Authors: S. LEONOV 1 , H. NORMAN 1 , P. WOLLBERG 1 , L. DAHLLUND 1 , *J. J. KRUPP 2 , 

K. SANDBERG 1 ; 

1 Mol. Pharmacol., Södertälje, Sweden; 2 Mol. Pharmacol, AstraZeneca R&D Soedertaelje, 

Södertälje, Sweden 

Abstract: Soluble polymers (SPs) of the Aβ peptide, also known as ADDLs can be the primary 

pathological species in AD responsible for binding to target receptor leading to aberrant 

activation of trophic signaling and to an incomplete array of downstream events that follows by 

tau hyperphosphorylation and neuronal dysfunction. To investigate the down-stream cellular 

events one need to have a functional assay with resolution of individual cell and sub cellular 

levels. The high content screening assays (HCSA) allows simultaneous multi-color analysis of 

defined parameters on the same population of cells expressing target receptor and marker 

fluorophore. We developed such an HCSA based on ImageXpress platform and MetaExpress 

software (Molecular Dynamics). SPs, made of the biotinylated human Aβ42 peptide, were used 

as a model ligand to study the link between SP binding, internalization and down-stream events. 

The binding and the internalization of SPs was traced by Neutravidin labeled respectively with 

Alexa 488 dye and either pHrodo (Molecular Probes) or CypHer 5E (GE Healthcare), the 

lysosome pH-sensitive dyes. Generation of reactive oxygen species (ROS) was detected by DCF 

fluorescence. Target cells were mouse 3T3 cells over expressing human four repeat-tau protein 

(3T3-4R), SHSY-5Y (human neuroblastoma) and ECV-340 (human endothelial) cell lines over 

expressing target receptor as well as AsRed2 fluorescent protein using an internal ribosome entry 

site. Both high (10-12-mers, HMWSP) and low (3-4-mers, LMWSP) molecular weight SPs 

demonstrated target receptor selective binding that was blocked by pretreatment with SP-specific 

decoy bio-molecules. Dose-dependent increase of receptor-mediated internalization of HMWSPs 

into cellular acidic compartment (presumably, lysosomes) of 3T3-4R cells was demonstrated. 

This process was followed by time-dependent increase of ROS formation in SHSY-5Y and by 

cytosolic accumulation of tau in β-sheet pleated form in 3T3-4R cells. Reverse sequence (42-1) 

Aβ42 peptide did not induce a similar effect. Receptor-specific binding, internalization and ROS 

generation was blocked by pretreatment with both SP- and receptor-specific decoy molecules, 

and by pretreatment with small molecule antagonists. Unspecific decoys did not interfere with 

neither binding/internalization of SPs nor with ROS formation. Our data support the view that 

receptor-mediated endocytosis of SPs (ADDLs) mediates both ROS generation in SHSY-5Y 

cells and tau deposition in 3T3 cells. HCSA based on IMAGExpress platform is a valuable tool 

to enable primary pharmacology of small molecules and biological decoy molecules for 

treatment of AD. 

Disclosures: S. Leonov, AstraZeneca R&D Södertälje, A. Employment (full or part-time); J.J. 

Krupp , AstraZeneca R&D Södertälje, A. Employment (full or part-time); P. Wollberg, 

AstraZeneca R&D Södertälje, A. Employment (full or part-time); L. Dahllund, AstraZeneca 

R&D, Södertälje, A. Employment (full or part-time); H. Norman, AstraZeneca R&D, 

Södertälje, A. Employment (full or part-time); K. Sandberg, AstraZeneca R&D, Södertälje, A. 

Employment (full or part-time).

Poster 





Support: Italian Ministry of Health, Project 2006-8. 

Title: Anabolic-androgenic steroid effects on cultured cortical neurons 

Authors: *F. CARACI 1 , V. PISTARÀ 2 , T. MUSUMECI 3 , A. CORSARO 2 , M. SORTINO 4 , F. 

NICOLETTI 5 , A. COPANI 6 ; 

1 Dept of Pharmaceut. Sci., 2 Dept. of Chem. Sci., 3 Dept. of Pharmaceut. Sci., 4 Dept. of Exptl. and 

Clin. Pharmacol., Univ. of Catania, Catania, Italy; 5 I.N.M. Neuromed, Pozzilli, Italy; 6 Dept. of 

Pharmaceut. Sci., University of Catania, Italy 

Abstract: Anabolic-androgenic steroids (AASs) are synthetic derivatives of testosterone, which 

are used to increase strength and muscle mass and to improve athletic performance. Abuse of 

AASs causes serious adverse effects in the central nervous system (CNS), such as mood 

disturbances and psychotic symptoms. Both neuroprotective and neurotoxic effects have been 

described for testosterone in different experimental models (1, 2), whereas little evidence exists 

about the neurotoxic potential of AASs (3). 

We compared the effects of testosterone with that of the AAS, nandrolone, in mixed cortical 

cultures containing both neurons and glia. Steroids were applied at different concentrations in the 

10 nM -10 µM range, for 24 hours and toxicity was assessed by Trypan blue staining of 

degenerated neurons. Testosterone neurotoxicity developed starting at a concentration of 1 µM 

and was prevented by the androgen receptor antagonist, flutamide. As opposed to testosterone, 

nandrolone was toxic at nanomolar concentrations (100nM). In addition, the cell-impermeable 

BSA-conjugated nandrolone exhibited more potently toxic effects than nandrolone, suggesting 

that activation of membrane androgen receptors might have a critical role in the neurotoxic 

properties of AASs. 

Interestingly, both testosterone and nandrolone were devoid of toxicity (up to a concentration of 

10 µM) when applied for 24 hours to pure neuronal cultures. These cultures are known to 

undergo neuronal apoptosis in response to ß-amyloid protein (Aβ) (4). Thus, we tested the effects 

of testosterone and nandrolone on apoptotic neuronal death induced by Aβ. Steroids were applied 

to the cultures 24 hours before Aß exposure. Both testosterone and nandrolone amplified Aβ 

toxicity at high concentrations (10κM). 

These data suggest that glia cells facilitate the neurotoxic effects of nanomolar concentrations of 

AASs, whereas micromolar concentrations of AASs might increase neuronal vulnerability to 

chronic insults independently of the contributing effect of glia. 

REFERENCES 

1) Estrada, M. et al. Elevated testosterone induces apoptosis in neuronal cells. J. Biol. Chem.

2006; 281, 24501-25492. 

2) Pike CJ et al. Androgen cell signaling pathways involved in neuroprotective actions. 

Hormones and behaviour 2008; in press. 

3) Orlando R et al. Nanomolar concentrations of anabolic-androgenic steroids amplify 

excitotoxic neuronal death in mixed mouse cortical cultures. Brain Res. 2007 Aug 24;1165:21-9. 

4) Copani A et al. Mitotic signaling by beta-amyloid causes neuronal death. FASEB J. 

1999;13:2225-34. 

Disclosures: F. Caraci, None; V. Pistarà, None; T. Musumeci, None; A. Corsaro, None; M. 

Sortino, None; F. Nicoletti, None; A. Copani, None. 

Poster 





Title: Autophagic pathway protects neuron from Aβ-induced cytotoxicity 

Authors: *S.-Y. HUNG 1 , W.-P. HUANG 2 , H.-C. LIOU 1 , W.-M. FU 1 ; 

1 Inst. Pharmacol, Nat'l Taiwan Univ., Taipei, Taiwan; 2 Dept Life Science, Nat'l Taiwan Univ., 

Taipei, Taiwan 

Abstract: Autophagy is a degradation pathway for the turnover of the dysfunction organelles or 

aggregated proteins in cells, particularly under stress or injury. Extracellular beta amyloid 

peptides (eAβ) have been reported to be a primary cause of Alzheimer's disease (AD) and large 

numbers of autophagic vacuoles accumulates in AD patient‟s brain. However, the effect 

regarding how autophagic process is involved in Aβ-induced neurotoxicity and how eAβ is 

transported into neuron and metabolized is still unknown. In order to study the role of autophagic 

process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH- 

SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35 or serum starvation induced strong 

autophagy response in SH-SY5Y/pEGFP-LC3 and caused much more cell apoptosis in SH- 

SY5Y than in SH-SY5Y/pEGFP-LC3. Co-treatment with autophagic process inhibitors 

wortmannin or bafilomycin A1 enhanced Aβ-induced neuronal apoptosis. Confocal doublestaining 

image shows that exogenous application of Aβ1-42 (1 κΜ) in medium caused the colocolization 

of Αβ1-42 with the autophagosome marker LC3 in the cytoplasm. Knockdown of 

Atg7 using siRNA inhibited the autophagosome formation at earlier step increased Hoechst (+) 

cells following Aβ25-35 treatment. Moreover, we found that treatment of Aβ with a selective 

α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in both SH-

SY5Y and SH-SY5Y/pEGFP-LC3 cells. On the other hand, nicotine, the α7nAChR agonist, 

enhanced the autophagic process and inhibited cell death following Aβ25-35 application. Nicotine 

but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. 

Confocal double-staining image shows that nicotine treatment enhanced the co-localization of 

α7nAChR with autophagosome. These results suggest that α7nAChR may act as a carrier 

binding with eAβ and internalized into cytoplasm and further detoxify Aβ-induced neurotoxicity 

via autophagic degradation pathway. Taken together, these results suggest that autophagy 

process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic 

regulation or Aβ-α7nAChR transport system will impair the clearance of Aβ and enhance 

neuronal death. 

Disclosures: S. Hung, None; W. Huang, None; H. Liou, None; W. Fu, None. 

Poster 





Support: Brain Korea 21 

National Core Research Center 

Title: Inhibition of soluble amyloid precursor protein alpha by amyloid beta in SH-SY5Y cells 

Authors: M.-H. CHAE 1 , J.-Y. OH 1 , *H.-J. KIM 2 ; 

1 Div. of Life & Pharmaceu. Sci., 2 Coll Pharmacy, Divi Life, Ewha Womans Univ., Seoul, 

Republic of Korea 

Abstract: Beta amlyoid peptide (βA4) is hypothesized to be toxic in Alzheimer's disease (AD) 

by forming its soluble oligomeric and protofibrillar assemblies, however, the role of Aβoligomers 

or mature Aβ-fibrils remains controversial and the underlying mechanisms of toxicity 

and the neuronal cellular signaling cascades activated by βA4 are not fully understood. Cleavage 

of amyloid precursor protin (APP) by α-secretase within the βA4 sequence results in the 

secretion of a soluble N-terminal APP fragment (sAPPα). We have studied the mechanisms of 

muscarinic recptor-mediated increase in sAPPα in human neuroblastoma (SH-SY5Y) cells. In 

the present study, effects of Aβ-oligomers and Aβ-fibrils on both basal and muscarinic receptormediated 

were investigated in SH-SY5Y cells. The cell viability and the level of phospho-AKT 

were not changed by treatment of aggregated βA4 prepared with different incubation conditions.

Both basal and muscarinic agonist-induced releases of sAPPα were significantly blocked by 

treatment of aggregated Aβ (possibly fibillar Aβ) prepared either by incubating at 4°C or by 

incubating at 37°C. By contrast, the release of sAPPα was differently regulated by oligomeric 

Aβ. To elucidate the intracellular biochemical bases of effects of aggregated Aβ forms, the 

expression level and activities of α-secretase candidates (ADAM 10 or TACE), or alteration of 

intracellular Ca 2+ levels were examined. Results from this study indicate that even though cell 

viability is not tightly correlated, the release of sAPPα is differently regulated by diverse forms 

of βA4, and their cellular mechanism will be presented and discussed. 

Disclosures: M. Chae, None; J. Oh, None; H. Kim , None. 

Poster 





Support: the Global COE Program (A-8) 

Title: Different effects of monocarboxylates on long term potentiation (LTP) and neuronal cell 

survival in the presence of β-amyloid 

Authors: *X. N. WANG 1 , T. TAKATA 1,2 , T. SAKURAI 1 , K. YOKONO 1 ; 

1 Kobe Univ., Kobe, Japan; 2 Saiseikai Nakatsu Hosp., Osaka, Japan 

Abstract: [Background and purpose] 

Glucose is a principal metabolic fuel in the central nervous system, but when glucose is 

unavailable, brain can utilize alternative metabolic substrates such as monocarboxylates (MCTs) 

to sustain brain function. Pyruvate is known to work protectively against ischemia and β-amyloid 

toxicity in primary cell culture system. But, the role of pyruvate as an alterative to glucose in the 

maintenance of synaptic activity and cell survival is still controversial. Therefore, even the 

current numerous findings do not fully elucidate the role of MCTs on brain function and 

survival. We studied the effects of two MCTs, pyruvate and lactate, on the synaptic activity and 

the cell survival, that were inhibited and accelerated, respectively, in the presence of β-amyloid. 

Possible mechanisms on the action of pyruvate against β-amyloid toxicity were also studied. 

[Method] 

Rat hippocampal slice cultures were used in our studies. After 10 days in vitro, long term 

potetiation (LTP) was induced by 100Hz-1s tetanus stimulation at the Schaffers Collaterals and 

the field excitatory postsynaptic potentials (fEPSPs) were recorded at the stratum radiatum of the

CA1. For evaluation of cell survival, the propidium iodide method was applied after 14 days in 

vitro. 

[Results and Conclusion] 

MCTs alone did not maintain the neuronal cell survival. Pyruvate, in the presence of glucose, 

showed the protective effects against β-amyloid cell toxicity as well as vitamin E analogue and 

nicotinamide. Lactate did not show any protective effects. Pyruvate, but not lactate, also reversed 

the β-amyloid induced inhibition of LTP. Pyruvate treatment led to the reduction of the ROS 

levels and the increase of the NAD levels in the slices. The involvement of CaMK-II or Protein 

Phosphatase-1 with these pyruvate protective actions against β-amyloid in the short term (LTP) 

and the long term consequences (cell survival) has been under exploring. 

Disclosures: X.N. Wang , None; T. Takata, None; T. Sakurai, None; K. Yokono, None. 

Poster 



Program#/Poster#: 244.8/M1 


Support: MU Research Board Grant URB-04-038 

NIH Grant 1P01 AG18357 

Alzheimer's Assoiciation Grant NIRG-06-25448 

NIH Grant 1R21NS052385 

Title: Amyloid-β peptide induces temporal change of astrocytic cell membranes through cPLA2 

Authors: *G. Y. SUN 1 , J. HICKS 2 , Y. LAI 2 , W. SHENG 3 , D. ZHU 2 , J. LEE 2 ; 

1 Dept Biochem, 2 Dept Biol. Engin., 3 Dept of Pathology and Anatom. Sci., Univ. Missouri, 

Columbia, MO 

Abstract: Amyloid-beta peptide (Aβ) has been widely implicated as the neurotoxic species in 

Alzheimer‟s disease (AD), and has been shown to deposit on cell membranes resulting in 

disruption of cell functions. However, mechanisms underlying Aβ‟s effects on cell membranes 

have not been fully characterized. In this study, oligomeric amyloid-β1-42 (Aβ42) caused a 

temporal biphasic change in astrocytic DITNC cell membranes, where membranes became more 

molecularly disordered after ~ 30min-1hr, but more molecularly ordered after ~ 3hr of Aβ42

treatment. However, Aβ42 caused artificial membranes made of rat brain lipid extract to become 

more molecularly disordered only, without the biphasic change induced in astrocytic DITNC 

cells. The trend for more molecularly ordered DITNC cell membranes was abrogated by the 

NADPH oxidase inhibitor apocynin, and an inhibitor of cytosolic phospholipase A2 (cPLA2), but 

not by an inhibitor of calcium independent PLA2 (iPLA2). Apocynin also suppressed superoxide 

anion production and the phosphorlyation of cPLA2 induced by Aβ42. Additionally, arachidonic 

acid (AA), a hydrolytic product of cPLA2, also caused DITNC cell membranes to become more 

molecularly ordered. These results suggest that in astrocytic DITNC cells, Aβ42 directly interacts 

with cell membranes with the immediate effect of making them more molecularly disordered, 

while also indirectly through the signaling pathway involving cPLA2 and NADPH oxidase, 

results in more molecularly ordered membranes. 

Disclosures: G.Y. Sun , None; J. Hicks, None; W. Sheng, None; J. Lee, None; Y. Lai, 

None; D. Zhu, None. 

Poster 





Support: Alzheimer's Association NIRG-06-27267 

Title: Correlation of amyloid-β aggregation state with tumor necrosis factor-α production in a 

human monocytic cell line 

Authors: *D. AJIT 1 , M. L. UDAN 2 , M. R. NICHOLS 2 ; 

2 Chem. and biochemistry, 1 Univ. Missouri, St. Louis, Saint Louis, MO 

Abstract: Pathological and biochemical studies have determined that fibrillar forms of 

aggregated amyloid-beta peptide (Aβ) comprise the characteristic neuritic plaques in 

Alzheimer‟s disease (AD). These studies have also revealed significant inflammatory markers 

such as activated microglia and cytokines surrounding the plaques. Although the plaques are a 

hallmark of AD, they are only part of an array of Aβ aggregate morphologies observed in vivo. 

Interestingly, not all of these Aβ deposits provoke an inflammatory response. Since structural 

polymorphism is a prominent feature of Aβ aggregation both in vitro and in vivo, we sought to 

clarify what Aβ morphology or aggregation species induces the strongest proinflammatory 

response using human THP-1 monocytes as a model system. These cells are sensitive to Aβ and 

readily secrete the cytokine tumor necrosis factor α (TNFα) in response. Freshly-reconstituted

Aβ(1-42) in water (100 κM) did not effectively stimulate the cells at a final Aβ concentration of 

15 κM. However, continued incubation of the peptide for 48-72 h at 4°C greatly increased TNFα 

production which, surprisingly declined upon further incubation of the peptide. Imaging of the 

Aβ(1-42) aggregation solutions by atomic force microscopy (AFM) and transmission electron 

microscopy (TEM) showed dense globular species immediately upon reconstitution and long 

flexible single fibers after continued incubation at 4°C. The appearance of fiber structures 

coincided with TNFα production although more concentrated or higher temperature Aβ(1-42) 

solutions which showed increased fibrillar content at earlier time points (24 h) were unable to 

stimulate the cells. Aβ(1-40) aggregation solutions under similar conditions were unable to 

stimulate TNFα production at any aggregation stage . The proinflammatory Aβ(1-42) species 

was found to be soluble as centrifugation for 10 mins at 18,000xg did not diminish its ability to 

induce TNF-α from THP-1 monocytes. Aβ(1-42) fiber morphology could be regulated by 

altering solution conditions. Incubation of the peptide in phosphate-buffered saline (pH 7.1) at 

4°C showed a meshwork of loosely-defined fibrillar structures by TEM which failed to evoke a 

proinflammatory response compared with incubation in water (pH 3.6). The findings suggest that 

an intermediate Aβ(1-42) aggregation species induces the peak proinflammatory response in a 

human monocytic cell line. 

Disclosures: D. Ajit , None; M.L. Udan, None; M.R. Nichols, None. 

Poster 





Title: Identification and initial characterization of a new binding-partner of the neuroprotective 

peptide Humanin 

Authors: *A. CARICASOLE 1 , L. ROSSINI 1 , L. TRABALZINI 2 , G. C. TERSTAPPEN 1 ; 

1 Siena Biotech, Siena, Italy; 2 Mol. Biol., Univ. of Siena, Siena, Italy 

Abstract: Humanin (HN) is a 24-residue peptide able to inhibit neurotoxicity induced by various 

insults which can be related to Alzheimer Disease (AD) as well as cell death induced by other 

stimuli, but little is known about its biological function in vivo. To date four proteins have been 

demonstrated to interact with HN, but none of them has been conclusively demonstrated to be 

essential for HN‟s neuroprotective activity. Thus, the identification of new binding partners 

and/or regulators of HN is fundamental to understand its physiological function and might also 

lead to the identification of novel therapeutic strategies for neurodegenerative disease. We have

isolated five new potential binding partners of HN by using a yeast two-hybrid (Y2H) approach. 

The observed Y2H interaction with HN was confirmed in mammalian cells for one of the 

binding partners in co-immunoprecipitation studies, a secreted protein of uncharacterized 

function. We present expression and functional data supporting a role for this novel Humanin 

binding partner in the modulation of Humanin‟s biological activity. 

Disclosures: A. Caricasole , Siena Biotech SpA, A. Employment (full or part-time); G.C. 

Terstappen, None; L. Rossini, None; L. Trabalzini, None. 

Poster 





Support: CIHR Grant RQ 

FRSQ-INSERM Grant RQ & SK 

Title: Age-dependent loss of hippocampal GABAergic interneurons in a transgenic model of 

Alzheimer‟s disease 

Authors: *S. M. KRANTIC 1 , N. MECHAWAR 2 , M. DAVOLI 2 , O. GUBKINA 1 , J. 

BROUILLETTE 2 , J.-G. CHABOT 2 , R. QUIRION 2 ; 

1 INSERM U29, Inst. Neurobiol Medit(INMED), Marseille, France; 2 Mc Gill Univ., Douglas 

Hosp. Res. Ctr., Montreal, QC, Canada 

Abstract: The neurotoxicity of β-amyloid (Aβ) observed in Alzheimer‟s disease (AD) is 

possibly related to glutamate toxicity resulting from the release of GABAergic interneuronmediated 

tonic inhibition of glutamate neurotransmission. We first assessed whether in vitro Aβ 

treatment of primary rat hippocampal cultures affects the survival of GABAergic GADimmunoreactive 

(GAD-IR) interneurons. These experiments pointed to a selective vulnerability 

of this neuronal population, with only 30-50% of these cells surviving after a 72 hours treatment 

with Aβ1-42 (25 µM) (SfN 2007 annual meeting, abstract 484.24). We then tested if this 

vulnerability also occurs in a more integrated model of AD. We thus quantified GAD-IR neurons 

in the entire hippocampal formation in 4 and 6 months old double transgenic CRND8 mice 

bearing Swedish KM670/671NL and Indiana V717F mutations in the gene encoding Aβprecursor 

protein 695. Our preliminary data indicate that at 4 months of age, there is no 

significant difference in the number of GAD-IR neurons between transgenic and wild-type mice

in either dentate gyrus or CA fields. In contrast, at 6 months of age, there was a strong trend 

towards a decrease (-35%) of the GAD-IR neurons in the CA fields of transgenic mice compared 

to their wild-type counterparts. No such trend was observed in the dentate gyrus. Given that a 

three-fold increase in hippocampal Aβ burden (reflecting extracellular Aβ accumulation) was 

observed in 6 months old compared to 4 monts old transgenic animals, these results suggest an in 

vivo relationship between Aβ levels and hippocampal interneuron vulnerability. 

Disclosures: S.M. Krantic , None; N. Mechawar, None; M. Davoli, None; O. Gubkina, 

None; J. Brouillette, None; J. Chabot, None; R. Quirion, None. 

Poster 





Support: American Health Assistance Foundation 

University of Arizona 

Title: Aβ1-42 and the endocytosis of AMPA-type glutamate receptors in cultured hippocampal 

neurons 

Authors: *P. A. ST JOHN; 

Dept Cell Biol & Anat., Univ. Arizona Col. Med., Tucson, AZ 

Abstract: Amyloid-beta (Aβ) peptides are the primary constituents of amyloid plaques in the 

brain in Alzheimer's disease (AD). A great deal of evidence supports the conclusion that Aβ 

peptides cause AD, but how they do so is not clear. Aβ peptides spontaneously aggregate, or 

self-assemble, to generate distinct macromolecular forms. In considering the range of molecular, 

cellular, and cognitive abnormalities observed in AD, in AD-model animals, and in neurons 

exposed to Aβ, it appears that different deficits may be caused by different assembly forms of 

Aβ, rather than all stemming from a single proximate cause. For example, recent evidence from 

our lab and others indicates that fibrillar forms of Aβ1-42 (Aβ42) selectively activate apoptotic 

pathways in CNS neurons, that is, they may be considered "neurotoxic," while several groups 

have provided evidence that less aggregated, soluble forms of Aβ are more "synaptoxic," that is, 

they more potently interfere with synaptic function, including synaptic plasticity, and even 

synaptic survival. This dichotomy of functional effects of different Aβ assembly forms is 

consistent with the suggestion that fibrillar and soluble forms of Aβ42 bind to different

subcellular locations and therefore different molecules on hippocampal neurons (St. John, 2007). 

A large and growing body of evidence indicates that the rapid trafficking of glutamate receptors 

onto and off of the neuronal surface plays a critical role in important forms of synaptic plasticity. 

It has been suggested that Aβ peptides interfere with synaptic plasticity - and possibly cause 

memory deficits in AD - by altering the normal dynamics of glutamate receptor trafficking in 

CNS neurons. We have examined the effects of different assembly forms of Aβ42 on the 

endocytosis of AMPA-type glutamate receptor subunits in cultured hippocampal neurons, using 

microscopic imaging to track and localize the internalized receptors (St. John and Gordon, 2001). 

Our results to date indicate that Aβ42 alters the constitutive endocytosis of GluR1 and GluR2 

subunits, even though it does not appear to bind to those receptors. Experiments in progress are 

examining the effects of Aβ42 on agonist-induced trafficking of glutamate receptors and 

comparing the effects of different Aβ42 assembly forms on glutamate receptor dynamics in these 

neurons. 

Disclosures: P.A. St John , None. 

Poster 





Support: BRNI Institute support 

NIH National Cancer Institute (CA31845) 

Title: A cellular model for Alzheimer‟s disease therapeutic efficacy: PKC activator bryostatin 

and a synthetic analog reverse Aβ toxicity on cultured fibroblasts 

Authors: *T. K. KHAN 1 , T. J. NELSON 1 , V. A. VERMA 2 , P. A. WENDER 2,3 , D. L. ALKON 1 ; 

1 Memory division, B Rockefeller Neurosci Institu, Morgantown, WV; 2 Chem., 3 Chem. and 

Systems Biol., Stanford Univ., Stanford, CA 

Abstract: PKC signaling deficits may themselves contribute to the origins of Alzheimer‟s 

disease (AD). Bryostatin, a potent PKC activator, has the potential to ameliorate both the 

neurodegeneration and the recent memory loss of AD. Bryostatin and a synthetic analog 

(picolog) were found to cause stimulation of non-amyloidogenic pathways by increasing αsecretase 

activity and thus lowering the amount of toxic Aβ1-42 produced. In the range of subnanomolar 

to nanomolar concentrations, both bryostatin and picolog, increased the secretion of

the α-secretase product (s-APP-α) of amyloid precursor protein (APP) in AD skin fibroblasts 

(N=4, for bryostatin: 289±67 (SEM); for picolog: 160±29 (SEM) with respect to 100 for control, 

respectively). An autopsy-validated peripheral AD-Biomarker was also developed by our 

laboratories (BRNI) based on bradykinin induced differential phosphorylation of Erk1 and Erk2. 

Drug efficacy was tested using the AD-Biomarker as follows. Values of the AD-Biomarker (AD- 

Index) were measured for control fibroblasts (Coriell Cell Repository) and were found to have 

the negative (normal) values of the AD-Biomarker. After soluble amyloidogenic Aβ1-42 treatment 

(1 κM, 16 hours), the fibroblasts were then found to have the AD-specific, positive AD- 

Biomarker values (N=6, AD-Index changes from -0.057±0.030 (SEM) to +0.130±0.035 (SEM)). 

Since the PKC activator, bryostatin, has shown both neuroprotective and cognitive enhancing 

efficacy, we then tested the possibility that bryostatin might prevent the Aβ1-42-induced 

abnormalities of the AD-Biomarker. Addition of 0.2 nM bryostatin protected against the Aβ1-42induced 

change of the AD-Biomarker into the positive values that would have indicated the 

presence of AD (see above). After bryostatin treatment, the AD-Index values returned back to 

the normal lower values (AD-Index= -0.034±0.049 (SEM)) for control fibroblasts. Similar 

results were obtained after picolog treatment. Both bryostatin (0.2 nM) and picolog (5 nM) 

treatment lowered the AD-Index values for AD fibroblasts (for bryostatin: N=6, AD-Index 

changes from 0.098±0.026 (SEM) to 0.025±0.03 (SEM); for picolog: N=4, AD-Index changes 

from 0.065±0.022 (SEM) to -0.017±0.013 (SEM), respectively), indicating that these compounds 

are efficacious at restoring a healthy phenotype of AD fibroblasts. The Aβ1-42-induction, and 

bryostatin/picolog reversal of the AD-Biomarker phenotype suggest the AD-Biomarker‟s 

potential in fibroblasts to monitor both disease progression and treatment response. Due to the 

scarcity of bryostatin, synthetic analogs such as picolog represent novel potential for the 


Disclosures: T.K. Khan, Full time, A. Employment (full or part-time); T.J. Nelson, 

None; V.A. Verma, None; P.A. Wender, None; D.L. Alkon, None. 

Poster 





Title: Receptor-mediated binding and internalization of soluble amyloid beta polymers mediates 

intracellular deposition of tau protein in 3T3 cells overexpressing 4-repeat human tau 

Authors: S. LEONOV, S. SVENSSON, *K. SANDBERG; 

Mol. Pharmacol., AstraZeneca R & D Sodertalje, Södertälje, Sweden

Abstract: Soluble polymers (SPs) of the Aβ peptide, also known as ADDLs might be the 

primary pathological species in AD, provoking deposition of hyperphosphorylated Tau, i.e. 

paired helical filaments (PHF-Tau). Suggested mechanism includes binding of SPs to cellular 

membrane receptor(s) leading to aberrant activation of trophic signaling and to an incomplete 

array of downstream events that follows by tau hyperphosphorylation and neuronal dysfunction. 

To test the mechanism and to demonstrate the feasibility of pharmacological interference 

between SPs and formation of PHF-Tau, we investigated the impact of SPs, made of the 

biotinylated human Aβ42 peptide, on the deposition of tau/PHF-Tau in 3T3 cells over expressing 

human 4R-Tau protein. The binding and subsequent internalization of SPs was traced by 

Neutravidin labeled with either Alexa 488 dye or lysosome pH-sensitive dye pHrodo 

(Molecular Probes), respectively. The deposition of PHF-Tau was detected by Thioflavin S 

(ThS) fluorescence. High Content Screening analysis was performed using ImageXpress 

platform and MetaExpress software (Molecular Dynamics). Both high (10-12-mers, HMWSP) 

and low (3-4-mers, LMWSP) molecular weight SPs demonstrated binding to cells and this 

binding was blocked by pretreatment with SP-specific decoy proteins. Dose-dependent increase 

of serine-protease sensitive receptor-mediated internalization of HMWSPs into cellular acidic 

compartment (presumably, lysosomes) was demonstrated. This process was followed by 

significant deposition of PHF-Tau surrounding or in close proximity to SP-containing cellular 

acidic compartments. Reverse sequence (42-1) Aβ42 peptide did not induce similar effect. 

Importantly, HMWSPs did not induce cellular DNA condensation, while SP content of 

lysosomes did not change up to 72 h of exposition. Both internalization and Tau deposition was 

blocked by pretreatment with both SP- and receptor-specific decoy proteins, and by pretreatment 

with small molecule protein kinase inhibitors. Unspecific decoys did not interfere with neither 

binding/internalization of SPs nor with tau deposition. Our results support a linkage between 

internalization of HMWSPs (ADDLs) and intracellular deposition of PHF-Tau form. The 

observed effect seems to be dependent on SP attachment to sites on cellular membrane and 

involves accumulation of lysosomal protease-resistant form of SP in acidic cytosolic 

compartment. Use of either SP-, receptor-blocking decoys or small molecules that interfere with 

SP-dependent down-stream signaling may represent an avenue for effective therapy of AD. 

Disclosures: S. Leonov, None; S. Svensson, None; K. Sandberg , AstraZeneca R&D, 

Södertälje, A. Employment (full or part-time); Molecular Pharmacology, A. Employment (full or 

part-time). 

Poster 




Topic: C.01.c. Abeta toxicity

Support: National Health and Medical Research Council-Australia 

Title: Copper bound to β-Amyloid catalyzes the formation of toxic soluble dityrosine crosslinked 

dimers 

Authors: *B. R. ROBERTS 1 , A. P. GUNN 1 , C. L. MASTERS 1,2 , A. I. BUSH 1,2,3 , R. A. 

CHERNY 1,2 ; 

1 Oxidation Biol. Lab., Mental Hlth. Res. Inst., Parkville, Australia; 2 Dept. of Pathology, Univ. of 

Melbourne, Parkville, Australia; 3 Genet. and Aging Res. Unit, Massachusetts Gen. Hosp., 

Charlestown, MA 

Abstract: The β-amyloid (Aβ) peptide that results from the cleavage of the amyloid precursor 

protein (APP) is generally considered to be the principle toxic species in Alzheimer‟s disease 

(AD). However, the mechanism of how Aβ is toxic remains a matter of debate. Our lab and 

others have shown that the binding of copper to Aβ, catalyzes the production of hydrogen 

peroxide and the formation of dityrosine cross-linked dimers. The amount of dityrosine in the 

AD brain is increased compared to age matched control brains, which is consistent with an 

enhanced production of reactive oxygen species. The objectives of this study are to characterize 

the rate of dityrosine formation and determine the amino acids that are oxidized by the reaction 

of Aβ-Cu with ascorbate. To accomplish these goals we used fluorescence spectroscopy to 

monitor dityrosine formation and mass spectrometry techniques to determine the amino acids 

that undergo oxidation. We found that Aβ-Cu, in the presence of ascorbate, catalyzed the 

formation of dityrosine with a half-life of ~20 minutes. Western blot analysis with an antibody 

specific for dityrosine (1C3) also confirmed the dityrosine formation as indicated by dityrosine 

positive Aβ dimers. Mass spectrometric analysis showed that the oxidation of several residues of 

the Aβ peptides occurred by 15 min. The addition of the copper binding agent clioquinol, 

inhibited the formation of dityrosine. This study demonstrates that Aβ/Cu complex can rapidly 

form dityrosine cross-linked dimers. Furthermore, we show that the oxidation of the Aβ peptide 

increases the solubility of both the Aβ peptide and dityrosine cross-linked dimers. Therefore the 

rapid formation of oxidized dityrosine cross-linked Aβ dimers catalyzed by the copper bound Aβ 

may represent a mechanism for the creation and stabilization of soluble toxic Aβ dimers. In 

conclusion our study shows that the formation of dityrosine cross-linked dimers occurs rapidly 

and that the oxidation of the Aβ peptide enhances the solubility of the dityrosine cross-linked 

dimers. 

Disclosures: B.R. Roberts, None; A.P. Gunn, None; C.L. Masters, None; A.I. Bush, 

None; R.A. Cherny, None. 

Poster 





Support: NIEHS RO1s ES10042 

NIEHS RO1s ES08089 

Title: The effect of Aβ(1-42) administration on Nrf2-ARE activation and differential sensitivity 

of Nrf2 knockout mice in vivo 

Authors: *G. JOSHI, M. VARGAS, D. JOHNSON, J. JOHNSON; 

Pharm., UW-Madison, Madison, WI 

Abstract: Abstract 

Alzheimer‟s disease (AD) is an age associated progressive neurodegenerative disorder leading to 

cognitive dysfunction and neuronal loss. It is pathologically characterized by deposition of 

amyloid-beta (Aß) peptide aggregated in the form of senile plaques and hyperphosphorylated tau 

as neurofibrillary tangles (NFT). Oxidative stress is known to be central to the AD pathology and 

Aβ(1-42)-mediated oxidative stress has been documented. Genetic mutations in amyloid beta 

precursor protein (APP), presenilin-1 and presenilin-2 are known to be involved in familial AD. 

Recently researchers have shown that a transgenic mouse model expressing mutant human APP 

and PS-1 had increased Aβ(1-42) production and plaque formation as well as increased 

susceptibility to exogenous oxidative insult. 

The antioxidant response element (ARE) is a regulatory enhancer found in 5‟ flanking region of 

many phase II detoxification enzymes. Up-regulation of ARE-dependent genes has shown to be 

neuroprotective. Various transcription factors and kinases are involved in ARE activation, 

however NF-E2 related factor 2 (Nrf2)-mediated ARE activation is central to the induction of 

phase II detoxification enzyme genes. We looked at the chronic effect of Aβ(1-42) injected intrahippocampally 

in wild type and the ARE-human placental alkaline phosphatase (hPAP) reporter 

mouse model. Initial studies indicate a loss of CA1 neuron and increased glial fribrillary acidic 

protein (GFAP) 14 days post-injection. There was also an increase in 3-nitrotyrosine levels, a 

marker of nitrosative stress, surrounding the damaged CA1 region, implicating peroxinitritemediated 

oxidative stress was occurring. We observed increased ARE-hPAP activity around the 

site of injection and damage indicating that the Nrf2-ARE pathway is being activated. We also 

injected Aβ(1-42) into the hippocampus of Nrf2 knockout (Nrf2-/-) mice and found that 

hippocampal neurons in Nrf2-/- mice were more vulnerable to Aβ(1-42)-induced toxicity. 

Finally, the effect of Aβ(1-42) injection in mice overexpressing Nrf2 selectively in astrocytes 

(GFAP-Nrf2 mice) is under examination. These data imply that the Nrf2-ARE pathway could 

play a role in the progression of AD. 

Disclosures: G. Joshi , None; M. Vargas, None; D. Johnson, None; J. Johnson, None.

Poster 





Support: FONDECYT 1080221 

Title: The inhibition of c-Abl/p73 signal pathway ameliorate neurologic damage in a 

Alzheimer‟s disease transgenic mouse model 

Authors: *G. I. CANCINO 1 , A. R. ALVAREZ 2 ; 

1 Facultad Ciencias Biologicas, P. Univ. Catolica Chile, Santiago, Chile; 2 Dep Biologia Celular y 

Mol., P. Univ. Catolica de Chile, Santiago, Chile 

Abstract: Alzheimer‟s disease (AD) is characterized by the presence of neurofibrillary tangles 

and senile plaques formed by amyloid β-peptide (Aβ). Aβ induces neuronal dystrophy, neuronal 

dysfunction and apoptosis. Nevertheless, the mechanisms by which the neurons degenerate are 

unknown. In previous in vitro and in vivo studies, we demonstrated that Aβ as fibrils activate the 

c-Abl/p73 pathway. Here, we analyzed the role of the c-Abl/p73 pathway in an AD double 

transgenic animal model, the APPswe/PSEN1ΓΔ9 mouse. Immunohistochemistry analysis 

showed that p73 and tau were phosphorylated and caspase-3 was activated in double transgenic 

mouse. Moreover, STI571, a c-Abl kinase inhibitor, significantly prevented its phenomenon. 

This inhibitor also reduced the spatial learning impairments, evaluated by Morris Water Maze 

task. The transgenic mice treated with intraperitoneal injection of STI571 presents significantly 

lower escape latency values and better navigation pattern than the transgenic mice treated with a 

saline solution indicating that STI571 treatment is able to reduce the cognitive impairments on 

spatial memory performance presents in the transgenic mice. Therefore inhibition of the c- 

Abl/p73 pathway may be an alternative for AD therapeutic design. 

This work was supported by FONDECYT 1080221 

Disclosures: G.I. Cancino , None; A.R. Alvarez, None. 

Poster 





Support: Alzheimer‟s Association NIRG-06-27267 

Title: Nonfibrillar, oligomeric, amyloid-β1-42 aggregates induce THP-1 monocyte adherence 

through formyl peptide receptor-like 1 

Authors: *N. R. CROUSE, D. AJIT, M. R. NICHOLS; 

Univ. Missouri, St Louis, St. Louis, MO 

Abstract: Amyloid-β (Aβ) is a naturally occurring peptide fragment known to aggregate and 

form deposits called senile plaques in the brains of Alzheimer‟s disease (AD) patients. The 

monomeric Aβ peptide is produced through proteolytic cleavage of a larger precursor protein and 

is found in both 40- and 42-residue lengths. In vitro studies have shown that Aβ monomers will 

undergo non-covalent self-assembly to form soluble intermediate species which progress to 

insoluble fibrils. Distinct Aβ species have also been observed in vivo prompting discussion on 

which species are responsible for diverse biological activities. Inflammatory markers such as 

activated microglia have been observed surrounding Aβ plaques in the AD brain. Some of these 

microglia are believed to originate from peripheral monocytes which infiltrate and differentiate 

in response to Aβ. We have modeled the differentiation process using the THP-1 human 

monocytic cell line, which can be transformed into an adherent morphology in response to many 

stimuli including Aβ. Our studies demonstrated that 15 κM Aβ1-42 was an effective stimulus for 

transformation of non-adherent THP-1 monocytes into an adherent phenotype. Aβ1-42 induced 

42±4% adherence after a 6 hr incubation, similar to another monocyte differentiating agent 

phorbol myristate acetate (PMA) (10 ng/ml, 43±4% adherence). Aβ1-42-induced monocyte 

adherence was inversely dependent on aggregation in that freshly reconstituted Aβ1-42 solutions 

were the most effective yet continued Aβ incubation for >48 hours at 4°C reduced the ability to 

induce adherence. Monocyte adherence disappeared completely at later stages of Aβ1-42 

aggregation. Interestingly, solutions of Aβ1-40 and a slower aggregating Aβ1-42 L34P mutant had 

little effect on monocyte adherence under the same conditions, implicating a specific Aβ1-42 

aggregation species in the process. Studies in which separate 50κM and 100κM Aβ1-42 solutions 

were prepared, yet final cell treatment concentrations were kept equal, showed that the 50κM 

solution induced much less monocyte adherence compared to the 100κM solution (16 and 58% 

respectively). This result suggests that nucleation of an oligomeric species is important for 

activity as opposed to monomer. Blocking the NF-kB pathway with pyrrolidine dithiocarbamate 

significantly attenuated PMA-induced adherence but showed little effect on Aβ1-42-induced 

adherence. However, the use of the small peptide WRW4, an agonist of the FPRL 1 receptor, 

inhibited 97% of Aβ1-42-induced adherence. Our studies presented here demonstrate that early, 

non-monomeric, Aβ1-42 aggregation species are able to potently induce THP-1 monocyte 

adherence via FPRL 1. 

Disclosures: N.R. Crouse, None; D. Ajit, None; M.R. Nichols, None.

Poster 





Support: Cure Alzheimer's Fund 

Extendicare Foundation 

NIH 

Title: Dimeric cross-linked abeta species (CAPS) disrupt and oxidize membranes 

Authors: *S. J. SOSCIA 1,2 , S. M. TUCKER 1 , R. E. TANZI 1 , R. D. MOIR 1 ; 

1 Genet. and Aging Res., Massachusetts Gen. Hosp., Charlestown, MA; 2 Anat. and Neurobio., 

Boston Univ. Sch. of Med., Boston, MA 

Abstract: Accumulation of the beta-amyloid protein (Abeta) as beta-amyloid deposits is a 

pathological hallmark of Alzheimer‟s disease (AD). Until recently the prevailing view held that 

insoluble fibrillar deposits of Abeta in brain were the cause of the massive neuronal death 

associated with the disease. However, evidence is mounting that soluble, oligomeric, crosslinked 

beta-amyloid protein species (CAPS), particularly dimeric forms, are highly neurotoxic 

and may, in fact, be the principal pathological agent in AD. Recent studies have shown that 

Abeta permeabilizes lipid bilayers and can disrupt other cellular membrane functions. In this 

study we report on the interactions of CAPS with lipid bilayers and conditions that modulate 

Abeta-mediated membrane disruption. We have developed novel methods to generate, 

fractionate, and purify dimeric and trimeric CAPS from Abeta isoforms containing 40 (Abeta40) 

or 42 (Abeta42) residues. Experiments compared monomeric Abeta and purified dimeric CAPS 

for membrane permiabilization and peroxidation. Initial experiments identified a novel CAPS 

heterodimer containing both Abeta40 and Abeta42 subunits. The Abeta heterodimer has 

potentiated redox activity. Our data suggest membrane permeabilization by CAPS is strongly 

modulated by the composition of the lipid bilayer and the peptide‟s access to metals. Membrane 

disruption was highest for CAPS/copper complexes against mitochondrial-like membranes. In 

addition, CAPS also appear to induce membrane peroxidation with a rank order of CAPS40 < 

CAPS42 < CAPS40/42. Our preliminary data suggest the association of dimeric CAPS with 

membranes may play an important role in AD pathology. 

Disclosures: S.J. Soscia , None; S.M. Tucker, None; R.E. Tanzi, None; R.D. Moir, None.

Poster 



Program#/Poster#: 244.20/N1 


Title: Aβ oligomers: larger than a monomer, smaller than a fibril--an analysis and comparison 

of various Aβ oligomer preparations 

Authors: *S. WRIGHT, P. SEUBERT, I. GRISWOLD-PRENNER; 

Dept Biol., Elan Pharmaceuticals, South San Francisco, CA 

Abstract: Aβ oligomers have garnered a great deal of attention as the putative species that 

causes the loss of cognitive function in Alzheimer‟s disease (AD)--presumably prior to the 

formation of the Aβ fibrils that compose the pathological hallmark of AD, the senile plaque. 

Physical characterization of an oligomer has primarily been by size, e.g., 

dimer/trimer/dodecamer on gel or morphology (not fibrillar). In this study we undertook a 

careful examination of various oligomer preparation methods reported in the literature and 

characterized each for ultrastructure by transmission electron microscopy (TEM), for secondary 

structure by circular dichroism (CD), for fibril formation by Thioflavin T binding, for molecular 

weight by gel electrophoresis and for overt cell toxicity using human primary neurons. 

Comparing wild-type Aβ40, wild-type Aβ42, Arctic Aβ40 and Arctic Aβ42 we see a variety of 

morphologies by TEM at time 0, which in many cases over time convert to fibrillar structures. 

There is a clear correlation with ability to further aggregate and the development of overt cell 

toxicity in human neurons, consistent with our previous findings of polymerization dependent 

toxicity (JNeurosci 25:1071, 2005). Interestingly, CD reveals instances of very clear beta sheet 

character that do not show fibrils by TEM, indicating a separation of these two characteristics, 

and vice versa, fibrillar Aβ by TEM can show CD spectra indicating a mix of secondary structure 

characteristics. Western blotting of the material can show a fibril preparation running as a 

monomer band, or as high molecular weight material, indicating some difference in the stability 

of the Aβ species under SDS/reducing conditions. This comprehensive analysis of the various 

oligomer preparations should assist in the development of oligomer “fingerprints”, with the hope 

of being able to correlate a particular set of properties with biological activity. 

Disclosures: S. Wright , Elan Pharmaceuticals, A. Employment (full or part-time); P. Seubert, 

Elan Pharmaceuticals, A. Employment (full or part-time); I. Griswold-Prenner, Elan 

Pharmaceuticals, A. Employment (full or part-time).

Poster 






Title: Foxo/Atrogin induction in skeletal muscle by intracellular β-amyloid 

Authors: *H.-K. LEE 1 , E. ROCNIK 1 , K. WALSH 2 , Q. FU 1 , K. M. ROSEN 1 , H. W. 

QUERFURTH 1 ; 

1 Neurol., Caritas St. Elizabeth's Med. Ctr., Boston, MA; 2 Cardiovasc. Resarch, Boston Univ. 

Sch. of Med., Boston, MA 

Abstract: Skeletal muscle atrophy is an outcome of: fasting, disuse, drug use (steroids), 

spaceflight, altitude, systemic cancer or inflammatory disorders, neurogenic as well as local 

dystrophic or myositic processes. In atrophic muscles, the ubiquitin ligase atrogin-1 (MAFbx) is 

induced. Accumulations of intracellular β-amyloid (Aβi), tau, and inflammatory markers are 

found pathologically in Inclusion Body Myositis (IBM) in which selective muscle groups 

undergo atrophy. Toxicity of Aβi has been shown in cultures of skeletal muscle and neuronal 

cells as well as in transgenic mice. We found that human IBM samples show increased 

expression of atrogin-1 mRNA. In differentiated C2C12 myotubes, intracellular Aβ expression 

caused increased expression of Atrogin mRNA and a reduction in myotube diameter, which 

could be partially reversed with myristoylated Akt and dnFoxo3A. Foxo3A was localized in the 

nucleus of select IBM myofibers as compared to age-matched controls. Similar results were seen 

in C2C12 myotubes expressing intracellular Aβ. In conclusion, Aβi is related to Akt 

deactivation, downstream activation of Foxo/Atrogin-1 and skeletal muscle atrophy. 

Disclosures: H. Lee , None; E. Rocnik, None; K. Walsh, None; Q. Fu, None; K.M. Rosen, 

None; H.W. Querfurth, None. 

Poster 

244. Abeta Toxicity I




Support: NIA P50AD16570 

NIA 050AG05142 

NIH 5AF016570 

NIH CA16042 

NIH NS43946 

Title: Synaptic amyloid beta is elevated in early and mid-stage Alzheimer disease cortex but 

decreased in late-stage AD cases 

Authors: *K. GYLYS 1 , S. SOKOLOW 1 , K. HENKIINS 1 , C. A. MILLER 3 , H. V. VINTERS 2 , 

G. M. COLE 4 ; 

1 Schl Nursing & Brain Res. Inst., 2 Pathology and Lab. Med., UCLA, Los Angeles, CA; 3 Depts of 

Pathology, Neurology, and Program in Neurosci., Keck USC Sch. of Med., Los Angeles, CA; 

4 Med. and Neurol., UCLA Sch. of Med. and Sepulveda VAMC GRECC, Sepulveda, CA 

Abstract: Early cognitive loss correlates with soluble amyloid beta (Aβ) in Alzheimer‟s disease 

(AD), and intraneuronal Aβ oligomers are hypothesized to contribute to synaptic dysfunction. 

We have developed flow cytometry protocols for quantification of synaptosomal 

immunolabeling to characterize and quantify Aβ in synaptic terminals from postmortem AD 

cases. Our previous work has demonstrated that synaptic Aβ correlates with oligomers, 

colocalizes with p-tau in synaptosomes and is associated with pathological changes (Fein et al., 

2008). Using the 10G4 antibody, we observe striking increases in Aβ immunofluorescence in 

AD synaptosomes (mean RFU=122±17, n=19 ) compared to aged normal controls (mean 

RFU=32 ±6, n=5). P-tau immunofluorescence using the pS422 antibody is also increased in AD 

(mean RFU=54±5, n=19) compared to aged normal controls (mean RFU=31.88±6, n=5). 

Synaptic Aβ and p-tau levels in cases with Parkinson‟s disease only (n=2) closely resemble 

control cases. Interestingly, two cases with neuropathologically documented tauopathy show 

strikingly elevations in synaptic p-tau with the AT100 antibody (mean RFU=129), while p-tau 

measured with the pS422 antibody (mean RFU=35.30) resembles the level in controls. A 

preliminary analysis of synaptic Aβ level by neuropathologic AD stage shows that synaptic Aβ 

level varies significantly with Braak stage (one way ANOVA, p=0.033); stepwise increases are 

observed for Braak stages III (mean RFU=115 ±23, n=3) , IV (mean RFU=126 ±44, n=5), and V 

(mean RFU=182 ±40, n=5). In late stage cases (Braak stage VI), synaptic Aβ levels decrease 

significantly from Stage V levels (mean RFU=87 ±10, n=7, p=.008), but remain higher than in 

aged normal controls. Considered together with elevated synaptic Aβ in early to mid-stage AD,

diminished Aβ immunofluorescence in late-stage AD cortical synaptosomes suggests a role for 

synaptic Aβ in early synaptic dysfunction and a potential site of deposit or release. 

Disclosures: K. Gylys, None; S. Sokolow, None; K. Henkiins, None; C.A. Miller, None; H.V. 

Vinters, None; G.M. Cole, None. 

Poster 





Poster 





Support: grants-in-aid for Scientific Research (A) (No.18208011 for K.I.) 

the Promotion of Science for young scientists (No.18.3327 for Y.M.) 

the Promotion of Science for young scientists (No.16.1215 and 19.403 for K.M.) 

Title: Isomerization and/or racemization at Asp23 of Aβ42 do not enhance its aggregation, 

neurotoxicity, and radical productivity in vitro 

Authors: *K. MURAKAMI 1,2 , M. UNO 2 , Y. MASUDA 2 , T. SHIMIZU 1 , T. SHIRASAWA 3 , K. 

IRIE 2 ; 

1 Res. Team Mol. Biomarkers, Tokyo Metro Inst. Gerontology, Tokyo, Japan; 2 Dept. of Agr., 

Kyoto Univ., Kyoto, Japan; 3 Sch. of Med., Juntendo Univ., Tokyo, Japan

Abstract: Aggregation of 42-mer amyloid β (Aβ42) peptide is a key event in the pathology of 

Alzheimer‟s disease (AD). Aβ42 aggregates to induce neurotoxicity mainly through the 

formation of radicals. Recent research suggested that the isomerization and/or racemization of 

Asp residues at position 1, 7, or 23 of Aβ42 might be associated with its pathology. Our previous 

study using systematic proline replacement and electron spin resonance (ESR) spectrometry 

suggested that turn formation at positions 22 and 23 of Aβ42 could be closely related to the 

radical production by the interaction between the side chains of Met35 and Tyr10. 1,2) To 

investigate the contribution of the posttranslational modifications (isomerization and/or 

racemization) at Asp23 to its pathology, three isomerized and/or racemized Aβ42 mutants (LisoAsp23-, 

D-Asp23-, and D-isoAsp23-Aβ42) were examined for its aggregative ability both by 

the sedimentation assay using HPLC and the thioflavin-T fluorescence, neurotoxicity in PC12 

cells by the MTT assay, and radical productivity by ESR using phenyl-tert-butylnitrone (PBN) as 

a trapping reagent. All Aβ42 mutants except for D-isoAsp23-Aβ42 showed moderate 

aggregative ability similar to wild-type Aβ42. However, the neurotoxicity of D-Asp23-Aβ42 was 

weaker than that of wild-type Aβ42, while that of L-isoAsp23-Aβ42 was as potent as that of 

wild-type Aβ42. In contrast, D-isoAsp23-Aβ42 showed weak aggregative ability without 

neurotoxicity. Their ability to produce radicals correlated well with their corresponding 

neurotoxicity. These results suggest that the isomerization and/or racemization of Asp23 might 

not be related to the pathogenesis of AD, but could be a consequence of chemical reaction during 

the long-term deposition of amyloid fibrils. If these modifications at Asp23 of Aβ42 were 

involved in the pathogenesis of AD, a possibility other than the neurotoxicity of Aβ42 peptides 

themselves, such as resistance to the enzymatic degradation, should be considered. 3) 

1) Murakami et al., J. Am. Chem. Soc. 2005, 127, 15168-15174 

2) Murakami et al., ChemBioChem 2007, 8, 2308-2314 

3) Murakami et al., Biochem. Biophys. Res. Commun. 2008, 366, 745-751 

Disclosures: K. Murakami , None; T. Shimizu, None; M. Uno, None; Y. Masuda, None; K. 

Irie, None; T. Shirasawa, None. 

Poster 





Support: A Grant-in-aid for Scientific Research (A) (No.18208011 for K.I.) from the Ministry 

of Education, Science, Culture, Sports, and Technology of the Japanese Government

A Grant-in-aid for Promotion of Science for Young Scientists (No. 18.3327 for Y.M.) 

from the Ministry of Education, Science, Culture, Sports, and Technology of the Japanese 

Government 

Title: Verification of the C-terminal intramolecular antiparallel β-sheet in Aβ42 aggregates using 

solid-state NMR: Implications for potent neurotoxicity through the formation of radicals 

Authors: *Y. MASUDA 1 , S. UEMURA 1 , A. NAKANISHI 2 , R. OHASHI 2 , K. TAKEGOSHI 2 , 

T. SHIMIZU 3 , T. SHIRASAWA 4 , K. IRIE 1 ; 

1 Grad Schl Agr., 2 Grad Schl Sci., Kyoto Univ., Kyoto, Japan; 3 Res. Team for Mol. Biomarkers, 

Tokyo Metropolitan Inst. of Gerontology, Tokyo, Japan; 4 Grad. Sch. of Med., Juntendo Univ., 

Tokyo, Japan 

Abstract: Alzheimer‟s disease (AD) is characterized by the abnormal deposition of 40- and 42residue 

amyloid β peptides (Aβ40 and Aβ42), the latter of which is more aggregative and 

neurotoxic. Recent investigations revealed that radicalization of Met-35 in the presence of 

transition metals is closely related to the neurotoxicity of the Aβ42. However, the resultant Soxidized 

radical cation at Met-35 is too short-lived to cause sufficient neurotoxicity in neuronal 

cells. We proposed previously a mechanism for its stabilization by the formation of an S-O bond 

with the C-terminal carboxylate anion through the intramolecular antiparallel β-sheet. 1) To verify 

this model, we analyzed the C-terminal structure of Aβ42 aggregates labeled with 13 C at Met-35 

and Ala-42 using solid-state NMR. Two sets of 13 C chemical shifts were observed for Met-35, 

indicating that Met-35 exists as two conformations or molecular species. The distance between 

the side chain of Met-35 and carboxyl group of Ala-42 in Aβ42 aggregates was estimated by 

rotational resonance (R2). 2) Significant intramolecular dipole-dipole interactions were observed 

between Cβ and/or Cγ of Met-35 and C=O of Ala-42, indicating that at least some part of the 

intramolecular distance between Cβ and/or Cγ of Met-35 and C=O of Ala-42 is shorter than 6 Å; 

this strongly supports the presence of the intramolecular antiparallel β-sheet structure in the Cterminus 

of the Aβ42 aggregates. 3) Although our result does not directly prove the existence of 

the S-O bond, it is certain that the S-oxidized radical cation at Met-35 is able to interact with the 

C-terminal carboxylate anion to form the S-O bond. This would cancel the electrical charges and 

form a hydrophobic core at the C-terminus that could accelerate the aggregation of Aβ42. The 

mechanism verified in this study clearly explains the stronger aggregative ability, neurotoxicity, 

and radical productivity of Aβ42 compared to Aβ40, whose C-terminal region does not form βsheet. 

1) Murakami, K. et al., J. Am. Chem. Soc. 2005, 127, 15168; 2) Raleigh, D. P. et al., Chem. 

Phys. Lett. 1988, 146, 71; 3) Masuda, Y. et al., Bioorg. Med. Chem. Lett. 2008, 18, in press. 

Disclosures: Y. Masuda, None; S. Uemura, None; A. Nakanishi, None; R. Ohashi, None; K. 

Takegoshi, None; T. Shimizu, None; T. Shirasawa, None; K. Irie, None. 

Poster

245. Tau and Alzheimer's disease 



Topic: C.01.h. Tau 

Support: NIH Grant R03AG030060 

Title: Fyn kinase regulation of Dab1 and tau phosphorylation 

Authors: *S. MINAMI 1 , H.-S. HOE 1 , M. P. BURNS 1 , Y. MATSUOKA 2 , G. W. REBECK 1 ; 

1 Dept Neurosci, 2 Dept Neurol., Georgetown Univ., Washington, DC 

Abstract: Fyn, a Src family tyrosine kinase, has recently been implicated in the pathogenesis of 

Alzheimer‟s disease. Fyn is known to be upregulated in the brains of AD patients and colocalizes 

with hyperphosphorylated tau. We have recently shown that Fyn also affects APP 

phosphorylation, trafficking, and processing in part through interaction with Dab1, a cytoplasmic 

adaptor protein itself known to affect APP processing and decrease Aβ. Fyn is highly enriched in 

lipid raft domains, so to determine whether the interaction between Fyn, Dab1, and APP 

occurred in lipid rafts, we isolated lipid rafts from brains of wild-type and Fyn knock-out mice. 

We found that Dab1 was distributed both in and out of lipid rafts in brains of both types of mice. 

In wild-type mouse brains, tyrosine-phosphorylated Dab1 was found predominantly in lipid rafts. 

However, tyrosine-phosphorylated Dab1 was observed equally in and out of lipid rafts isolated 

from Fyn knock-out mice, implicating an important role for Fyn in the phosphorylation of Dab1 

in lipid rafts. To examine the downstream effects of Fyn activation of Dab1, we treated primary 

neurons with PP2, an inhibitor of Src family kinases, and found increased levels of Dab1 and 

decreased phospho-Dab1, consistent with previously published studies, and decreased Gsk3β and 

Cdk5, two major tau kinases. In addition, we probed Fyn knock-out brain homogenates for 

phospho-tau and tau kinases and found decreased tau phosphorylation and decreased Cdk5 

compared to wild-type mice. Together, these results suggest a role for Fyn in the pathological 

processes related to APP processing and tau phosphorylation, and we will further examine how 

these processes are regulated through cellular lipid rafts. 

Disclosures: S. Minami , None; H. Hoe, None; M.P. Burns, None; Y. Matsuoka, None; G.W. 

Rebeck, None. 

Poster 





Support: NIH Grant 5AG016570 

Title: Fyn kinase is increased in synaptic terminals in Alzheimer‟s Disease 

Authors: K. M. HENKINS 1 , S. SOKOLOW 2 , *B. TETER 5 , C. A. MILLER 6 , H. V. VINTERS 7 , 

G. M. COLE 8,3 , K. H. GYLYS 4 ; 

2 Sch. of Nursing, 3 Sch. of Med. and Neurol., 4 Brain Res. Inst. and Sch. of Nursing, 1 UCLA, Los 

Angeles, CA; 5 Geriatrics, UCLA-SFVP MC151, Sepulveda, CA; 6 Dept. of Pathology and 

Neurol., Keck Sch. of Medicine, USC, Los Angeles, CA; 7 Pathology and Lab. Medicine, 

Neurol., UCLA Sch. of Med., Los Angeles, CA; 8 VAMC GRECC, Sepulveda VA Med. Ctr., 

Sepulveda, CA 

Abstract: Overexpression of Fyn, a tyrosine kinase in the Src family, and Aβ in double 

transgenic animals (FYN/hAPP J-9) creates a phenotype with cognitive impairment that is very 

similar to transgenic animals expressing much higher levels of Aβ (Palop et al. 2005) and 

removing Fyn all together blocks some types of Aβ induced neurotoxicity (Lambert et al. 1998, 

Chin et al. 2004). Fyn is relocated in AD brains from synapses to neuronal bodies where it was 

found to co-localize with neurofibrillary tangles (Ho et al. 2005). Using flow cytometry analysis 

of synaptosomes prepared from post-mortem AD cortex, we have previously demonstrated that 

synaptic Aβ elevations are accompanied by pathologic changes and are co-localized with p-tau. 

In the present experiments, synaptosomes from our cohort of AD cases and normal controls were 

immunolabeled with an antibody directed against Fyn kinase (BD Transduction Laboratories, 

Franklin Lakes, NJ), and immunolabeling was quantified by flow cytometry. AD cases (16.44% 

positive ± 7.98, n =14) demonstrated elevated Fyn labeling compared to aged normal control 

cases (7.12% positive ± 0.54, n = 4, p

Poster 





Support: Cure Alzheimer‟s Fund 

NIH NS049237 

Burroughs Wellcome Career Award in the Biomedical Sciences 

NIH AG13956 

Title: Towards understanding the link between traumatic brain injury and alzheimer‟s disease: 

amyloid beta and tau pathologies in an experimental traumatic brain injury mouse model 

Authors: *H. T. TRAN 1 , S. ODDO 3 , F. M. LAFERLA 3 , D. M. HOLTZMAN 2 , D. L. BRODY 2 ; 

1 Biol. and Biomed. Sci., 2 Neurol., Washington Univ. St Lo, St Louis, MO; 3 Neurobio. & 

Behavior, Univ. of California, Irvine, Irvine, CA 

Abstract: Traumatic brain injury (TBI) is one of the epidemiological factors predisposing a 

person to developing Alzheimer‟s disease (AD) later in life. Neurofibrillary tangles (NFTs) and 

neuritic plaques are two pathological hallmarks of AD; both have been observed in human 

patients with TBI. To date, the mechanisms underlying the association between TBI and AD 

remain unknown. This is in part due to the lack of an appropriate small animal model which 

recapitulates the pathologies observed in human patients with TBI. Here, we set out to develop 

such a model. We performed controlled cortical impact (CCI), an established experimental TBI 

paradigm, on 3xTg-AD mice which express human forms of amyloid precursor protein (APP) 

and tau, the main components of plaques and NFTs, respectively. Animals at 6 months of age 

were utilized since they have not yet developed prominent amyloid beta (Aβ) and tau 

pathologies. This is analogous to young human TBI patients who do not typically have plaques 

and NFTs prior to injury. Using immunohistochemistry, western blotting, and ELISA, we 

demonstrated that animals which have undergone TBI exhibited exacerbated Aβ and tau 

pathologies, as compared to sham injured animals. We observed intracellular immunoreactivity 

for Aβ in the ipsilateral fimbria of injured mice only. Aβ immunoreactive axonal varicosities 

were also immunoreactive for APP and PS1, a component of the γ-secretase machinery. In 

addition, we confirmed there was an increase in total Aβ levels in hippocampal lysates of injured 

mice via ELISA and western blots. Interestingly, there was a 50% increase in total tau 

immunoreactivity in hippocampal CA1 regions contralateral to impact site. PHF1 (phospho-tau 

at residues S396 and S404) immunoreactivity was also observed in the in fimbria, corpus 

collosum, and pericontusional areas of injured animals. Previous studies have demonstrated that

Aβ immunotherapy in uninjured 3xTg-AD mice reduced extracellular Aβ plaques and 

intracellular Aβ accumulation, which in turn led to clearance of early tau pathology. Thus, we 

hypothesize that tau pathology may be downstream of Aβ pathology in 3xTg-AD animals with 

TBI. Axonal transport of Aβ from the ipsilateral fimbria across the hippocampal commissure 

could exacerbate tau pathology in the contralateral hippocampal CA1. This hypothesis will be 

tested in future experiments. In summary, this experimental TBI mouse model has similar 

pathologies as observed in human TBI patients; thus, it could be potentially useful for 

investigation of the mechanisms connecting TBI to subsequent development of AD. 

Disclosures: H.T. Tran, None; S. Oddo, None; F.M. LaFerla, None; D.M. Holtzman, 

None; D.L. Brody, None. 

Poster 





Support: a grant-in-aid for Scientific Research (20591024) from the Ministry of Education, 

Culture, Sports, Science and Technology, Japan 

Title: FKBP12 is associated with neurofibrillary tangles in Alzheimer brain tissues 

Authors: H. SUGATA 1 , K. MATSUO 2 , T. NAKAGAWA 1 , M. TAKAHASHI 2 , H. MUKAI 2 , 

Y. ONO 2 , K. MAEDA 3 , *T. KAWAMATA 1 ; 

1 Dept Hlth. Sci., Kobe Univ. Grad Sch. Hlth. Sci., Kobe, Japan; 2 Biosignal Res. Center, Kobe 

Univ., Kobe, Japan; 3 Kobe Univ. Grad Sch. Med., Kobe, Japan 

Abstract: FKBP12 is a peptidyl-prolyl cis-trans isomerase, known to be important in protein 

assembly, folding and transportation, and also known to modulate the activity of calcineurin, 

protein phosphatase enriched in the brain, which dephosphorylates tau protein. We investigated a 

possible role for a FKBP12 in postmortem brain tissues from the elderly controls and the patients 

with Alzheimer's disease (AD) using immunoblotting, and light and confocal laser microscopies. 

FKBP12 was enriched and localized to neurons and neurites in control brains. Intense 

immunoreactivity was found in the large neurons such as pyramidal cells. In normal neurons, 

many FKBP12 positive granules were located in or around the nucleus, and in the proximal 

portion of dendrites and axons. In contrast, expression of FKBP12 in AD brains was lower than 

control brains. Furthermore, numerous intracellular neurofibrillary tangles (NFTs) were detected 

in the CA1 subfields of AD hippocampi. Neuritic pathology such as neuropil threads and

dystrophic neurites (DNs) within senile plaques (SPs) and some reactive astrocytes were also 

immunolabeled for FKBP12 in AD. Double immunofluorescence staining showed dual labeling 

of neurons for FKBP12 and tau detected with HT-7 monoclonal antibody. Similar results were 

obtained in glia for the combination of FKBP12 and glial fibrillary acidic protein (GFAP). In the 

peripheral neurites stained for highly-phosphorylated neurofilament protein, additional labeling 

for FKBP12 was dense in axons, but weak in dendrites. Most of the HT7-positive neurons were 

also labeled for FKBP12 in AD brains, whereas small number of neurons were only positive for 

FKBP12. In an analogous fashion, DNs were mostly double-labeled within SPs, but some DNs 

were only stained for FKBP12. Our results suggest that FKBP12 may be involved in the folding 

of tau protein and in the axonal transport in neurons. Given that neuronal FKBP12 acts as an 

isomerase in neurons, reduction in FKBP12 may lead to abnormal phosphorylation and 

misfolding of tau protein and to impairment in protein transport, causing abnormal assembly of 

tau protein and formation of NFTs. Thus, it may be speculated that FKBP12 is involved in the 

tau pathology that may be responsible for neuronal death in AD brains. 

Disclosures: H. Sugata, None; T. Kawamata , Toshio Kawamata; supported by a grant-in-aid 

for Scientific Research (20591024) from the Ministry of Education, Culture, Sports, Science and 

Technology, Japan, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); T. Nakagawa, None; K. Matsuo, None; M. 

Takahashi, None; H. Mukai, None; Y. Ono, None; K. Maeda, None. 

Poster 





Support: Alzheimer‟s Association NIRG-07-59659 (QL. M.) 

NIA AG021975 (S.A.F.) 

NIH AT003008 (G.M.C.) 

NIA AG13471 (G.M.C.) 

NIA AG16570 (G.M.C.) 

Title: Omega-3 fatty acid docosahexaenoic acid protects from aβ oligomer-induced neuronal tau 

pathology through pten signaling

Authors: *Q.-L. MA, O. J. UBEDA, W. BEECH, F. YANG, P. CHEN, B. HUDSPETH, S. A. 

FRAUTSCHY, G. M. COLE; 

GRECC(VA) & Med. (UCLA), UCLA, VA Med. Ctr., North Hills, CA 

Abstract: Several epidemiological studies have reported that low dietary fish or omega-3 fatty 

acid docosahexaenoic acid (DHA, C22:6, n=3) intake increases risk of Alzheimer‟s disease 

(AD). Different transgenic AD mouse models have confirmed that DHA supplementation 

reduces Aβ and may improve cognitive deficits. Further, DHA ameliorated tau pathology in the 

3xTg-AD mice and suppressed tau phosphorylation and pJNK but not other tau kinases. Here we 

show evidence for the additional involvement of PTEN, an upstream lipid phosphatase that limits 

another tau kinase, Akt, reported elevated in AD tangle vulnerable neurons. In cultured 

hippocampal neurons, we observed that Aβ42 oligomers significantly increased (>200%) soluble 

phospho-tau (ptau) at Ser214, Thr212 (AT100), Ser202 (AT8), Thr205 (AT8), Ser396 (PHF1), 

Ser404 (PHF1), Ser422, and total tau (tau5). Aβ42 oligomers also increased high molecular 

weight soluble tau aggregates, but not insoluble tau aggregates. Adding a JNK inhibitor, 

SP600125, not only suppressed known JNK tau phospho-epitopes at Ser202, Thr205, Ser396, 

Ser404 and Ser422, but also suppressed Akt/GSK phosphorylated tau epitope (Thr212) without 

altering pSer214. Interestingly, pre-treating the neurons with DHA (2.5µM) for 48 h blocked all 

of the Aβ oligomer-induced ptau, including Ser214. Moreover, soluble Aβ significantly 

suppressed PTEN protein (reported reduced in AD brain) while DHA pretreatment increased 

PTEN. To our knowledge, this is the first evidence that Aβ oligomers may drive the 

dysregulation of Akt and PTEN that has been reported in AD brain. In APP transgenic mice and 

3xTg-AD mice, dietary DHA also increased PTEN levels but reduced active tau kinases, GSK3β 

and JNK. These results suggest that DHA can mitigate the seemingly paradoxical simultaneous 

upregulation of both Akt and the downstream GSK3β that Akt normally suppresses. Thus, DHA 

may have the potential to protect against tau pathology in AD brain through both inhibition of 

tau kinase activation and upregulation of PTEN activity. Since tau knockout suppresses Aβmediated 

synaptic and cognitive deficits in AD APP Tg model mice, the correction of Aβinduced 

alterations in tau kinases, PTEN and ptau may be relevant to both synaptic dysfunction 

and tau pathology. 

Disclosures: Q. Ma, None; O.J. Ubeda, None; W. Beech, None; F. Yang, None; P. Chen, 

None; B. Hudspeth, None; S.A. Frautschy, None; G.M. Cole, None. 

Poster 




Topic: C.01.h. Tau

Support: Society for Progressive Supranuclear Palsy (Research Grant No. 448) 

Title: Defects in insulin signaling promote Ser214 tau hyperphosphorylation and microtubule 

dysfunction in a potential mechanism in Alzheimer's disease 

Authors: *H. KSIEZAK-REDING 1,2 , C. DIAZRUIZ 1 , R. LIPPELL 1 , S. THOMAS 1 , W. QIN 1 , 

G. M. PASINETTI 1,2 ; 

1 Dept. of Psychiatry, Mount Sinai Sch. of Med., New York, NY; 2 James J. Peters VA Med. Ctr., 

Bronx, NY 

Abstract: Dysfunction of neuronal insulin signaling mediated by protein kinase Akt has been 

implicated in the pathogenesis of Alzheimer's disease (AD). Such dysfunction is considered to 

play a role in hyperphosphorylation of tau, disruption of the microtubule (MT) network and 

accumulation of fibrillary tau inclusions in the brain. Our previous studies have shown that 

fibrillary tau is hyperphosphorylated at a disease-specific epitope AT100 (pThr212/pSer214) 

which contains consensus motif for Akt-dependent phosphorylation of tau in cell-free systems 

and in Akt-transfected cell cultures. Our recent studies focused on the relationship between 

insulin signaling and phosphorylation of tau using cortico-hippocampal neuronal cultures derived 

from mouse brain. Cultures were grown in neurobasal medium supplemented with insulin and 

growth factors for 5-8 days and then in plain DMEM medium for 2h prior to insulin treatment. 

We found that low concentrations of insulin (1µg/ml) upregulated pSer214 tau content in the 

mechanism dependent on insulin and Akt activity (Mechanism 1). Inhibition of insulin signaling 

by LY 294002 upstream of Akt significantly upregulated by 14-fold pSer214 tau content and 

revealed an alterntive pathway of Ser214 phosphorylation independent of insulin and Akt 

activity (Mechanism 2). Mechanism 2 resembles pathological mechanisms leading to AD. We 

next evaluated the effects of LY 294002 on stability of the detyrosinated microtubules (Glu MT). 

Glu MT are a characteristic component of the endosomal recycling compartment (ERC) involved 

in presenilin/gamma-secretase dependent protein trafficking of amyloid precursor protein. We 

found that LY 294002 induced derangement of the stable Glu MT network. Our studies suggest 

that inhibition of insulin signaling may promote upregulation of pSer214 tau and Glu MT 

instability, which may then lead to compromised ERC-dependent protein trafficking. Our studies 

link defects in insulin signaling to some of the alterations characteristic of AD and propose a 

unifying molecular mechanism of insulin resistance leading to neurodegenerative changes in AD. 

Our studies reveal potential pharmacological targets for AD treatment. Supported by the Society 

for Progressive Supranuclear Palsy (Grant No. 448 to HKR). 

Disclosures: H. Ksiezak-Reding , None; C. DiazRuiz, None; R. Lippell, None; S. Thomas, 

None; W. Qin, None; G.M. Pasinetti, None. 

Poster 

245. Tau and Alzheimer's disease


Program#/Poster#: 245.7/O1 


Support: American Federation for Aging Research 

NIH Grant DK26741 

Ellison Medical Foundation 

Clayton Medical Research Foundation 

Title: CRF receptor mediation of repeated stress effects on tau phosphorylation and solubility 

Authors: *R. A. RISSMAN, N. J. JUSTICE, P. E. SAWCHENKO; 

Lab. Neuronal Structure & Func, The Salk Inst., La Jolla, CA 

Abstract: Exposure and/or sensitivity to stress may confer increased risk of Alzheimer's Disease 

(AD). Although the specific links between stress and AD pathogenesis remain unsettled, our 

recent data provide evidence for a differential involvement of corticotropin-releasing factor 

receptors (CRFR1 and 2) in the regulation of hippocampal tau phosphorylation (tau-P) induced 

by acute exposure to an emotional stressor, restraint. Relative to wild type (wt) controls, tau-P 

responses were lacking in CRFR1-deficient mice, and exaggerated in CRFR2 mutants. Because 

acute stress-induced tau-P is a transient phenomenon, we used anatomical and biochemical 

analyses to assess the relationship between CRFRs and tau-P after exposure to repeated restraint 

stress (14 daily exposures). Dual immunofluorescence labeling in a transgenic CRFR1 reporter 

mouse line exposed to repeated stress revealed substantial overlap between hippocampal CRFR1 

expression and cells positive for phosphorylated tau (PHF-1). We next used Western analyses of 

soluble (RAB) and detergent-soluble (RIPA) fractions of hippocampal extracts to explore the 

pathogenic potential of stress-induced tau-P by comparing the effects and CRFR-dependence of 

acute versus repeated restraint stress exposure on the persistence of tau-P and its solubility. 

Whereas hippocampal tau-P was comparably increased at 20 min after a single (acute) or the last 

of 14 (repeated) restraint episode(s), chronic elevations (i.e., at 24 hr after stress) were detected 

only in the repeated condition. Moreover, a portion of tau-P in extracts from repeatedly stressed 

mice was present in an insoluble form (RIPA fraction), while acute restraint-induced tau-P was 

detectable in only the soluble (RAB) fraction. Relative to wt controls, each of these stress effects 

on tau-P and solubility was comparable, if not enhanced, in CRFR2-deficient mice, but was not 

detectable in CRFR1 or double knockout animals. Analysis of RIPA fractions from wt and 

CRFR2 animals by negative staining at the EM level and identified globular aggregates that 

displayed positive immunogold labeling for early conformational changes in tau (MC-1 and Alz- 

50), as well as tau-P (PHF-1). These results further support a push-pull involvement of CRFRs in 

regulating restraint stress-induced tau-P. Given that repeated stress exposure results in chronic 

increases in hippocampal tau-P and its sequestration in an insoluble (and potentially pre-

pathogenic) form, our data may define a novel link between stress exposure/sensitivity and an 

AD-related pathogenic mechanism. 

Disclosures: R.A. Rissman , None; N.J. Justice, None; P.E. Sawchenko, None. 

Poster 





Support: PSC-CUNY 

NYSTAR 

Title: Effect of Alzheimer like pseudophosphorylated tau on microtubules dynamics, tau 

aggregation and neurodegeneration 

Authors: *A. ALONSO, S. BROWNE, W. L'AMOREAUX; 

Biol. and Ctr. for Develop., Col. of Staten Island, Staten Island, NY 

Abstract: Understanding mechanistically the foundation of synaptic withering and loss that 

precedes cell death in Alzheimer‟s disease (AD) and other neurodegenerative diseases is pivotal. 

Accumulation of hyperphosphorylated tau and the disruption of microtubules are correlated with 

synaptic loss and pathology of AD. One hallmark lesion of neurodegenerative disease in AD is 

the initial appearance of neurofibrillary tangles inside neurons. These tangles are composed 

mainly of hyperphosphorylated tau, a microtubule-associated protein (MAP), polymerized into 

paired helical and straight filaments (PHFs/SFs). Impaired cognitive function and pathology of 

AD is correlated with this lesion (PHFs/SFs). In in vitro assays, hyperphosphorylated tau is 

disruptive to microtubule assembly, whereas tau, stimulates tubulin assembly and subsequent 

stabilization of microtubules. Microtubules are the “tracks” for axonal transport: disruption of 

the microtubules leads to compromised axoplasmic flow, dysfunction at the synaptic terminals, 

and eventually neuronal death. Microtubules are disrupted in AD. 

Here our objective is to study the influence of tau phosphorylation at Thr 212, Thr 231, and Ser 

262 for tau-tau binding, self-assembly and the effect of its expression on the cells. 

Pseudophosphorylated tau was generated by site directed mutagenesis. Pseudophosphorylated 

tau was transiently expressed in PC12 cells and in CHO cells. The effect of expression of 

mutated taus on self assembly and binding to microtubules was studied by 

immunocytochemistry. Binding of normal tau to pseudophosphorylated tau was studied by dot

overlay assays. Caspase activation and apoptosis were determined by immunocytochemistry 

after transient transfection. For dynamic studies, we performed time-lapse fluorescent 

microscopy with fluorescent-tagged tau constructs that we generated and co-transfected in CHO 

cells with fluorescent-tagged tubulin. We found that pseudophosphorylated tau aggregates in 

cells when Thr 212 is mutated to Glu, suggesting that phosphorylation at this site facilitates tau 

self-assembly. The expression of tau pseudophosphorylated at Thr212, Thr231, and Ser262 

triggers caspase 3 activation in as much as 85% of the transfected cells and apoptosis to a lesser 

degree. These findings suggest that tau phosphorylation at Thr 212 facilitates tau selfaggregation, 

and that the combination of phosphorylation at Thr212, 231 and Ser262 in the same 

tau molecule can trigger toxic reaction. Pseudophosphorylated tau, as Alzheimer phosphorylated 

tau, sequesters normal tau. 

Disclosures: A. Alonso , None; S. Browne, None; W. L'Amoreaux, None. 

Poster 





Support: NIH R03MH075832-01A1 to LHV 

Title: Studies of spontaneous, long-range coherence in normal adult mice and young htau 

Alzheimer‟s mice 

Authors: J. ROBSON 1 , N. ESIOBU 1 , R. HERMER-VAZQUEZ 2 , A. BROWN 2 , *L. L. 

HERMER-VAZQUEZ 3 ; 

1 Neurobio. and Cognitive Sci. Program, Univ. of Florida, Gainesville, FL; 2 Neurobio. and 

Cognitive Sci. Program, 3 Dept Behav Neurosci, Box 112250, Univ. Florida, Gainesville, FL 

Abstract: Synchronous brain activity plays a major role in attention, learning, and memory 

consolidation in humans. Several fMRI studies have detected spontaneous, correlated brain 

activity, in addition to task-related correlated activity, on a relatively slow timescale. However, a 

much higher-resolution and information-laden measure, with critical properties in both the time 

and spectral domains, has recently been discovered (R.H.-V.). Spontaneous frequency bursts 

(SFBs), as seen in spectrograms of rat local field potentials, are a biomarker of transient, longrange 

coherence among brain areas. They are most often seen in states of moderately low brain 

complexity, such as light anesthesia (isoflurane or ketamine/xylazine), and preliminarily, slowwave 

sleep. Previous studies in rats characterized SFBs across the extended olfactory circuit,

here, the anterior piriform, posterior piriform and entorhinal cortices, revealing four major 

properties beyond their spontaneous occurrence: broadband frequency (often up to 80-100 Hz); a 

brief (~250 ms) duration; near synchrony across even widely separated brain regions (often 

appearing within 5-6 ms of one another); and either bursty (two or more occurring on the same 

LFP channel less than 250 ms apart) or singular occurrences. 

We will present the findings of SFB activity across the extended olfactory circuit in two studies 

using a different species: mice. The first study demonstrated the occurrence of SFBs in normal 

adult mice as well as in juveniles, with properties extremely similar to those seen in rats. The 

second study, in progress at the time of abstract submission, is comparing young htau 

Alzheimer‟s model mice and normal controls. We hypothesize that the SFB metric will suggest 

early reductions in long-range coherence, prior to prevalent tau pathology in the neocortex and 

hippocampus, and well before neurofibrillary tangles are present. 

Disclosures: J. Robson, None; N. Esiobu, None; R. Hermer-Vazquez, None; A. Brown, 

None; L.L. Hermer-Vazquez, None. 

Poster 





Support: The Alzheimer's Association 

Title: Does retromer deficiency observed in Alzheimer‟s disease affect Tau phosphorylation? 

Authors: A. S. BENDER, E. PLANEL, K. DUFF, *S. A. SMALL; 

Taub Instt Res. & Alzh Dis & Aging Brain, Columbia Univ, Col. Physicians & Sur, New York, 

NY 

Abstract: Gene-expression and genetic studies have implicated the retromer sorting pathway in 

late-onset Alzheimer‟s disease. Previous studies in animal models have shown that retromer 

deficiency observed in Alzheimer‟s disease can cause hippocampal dysfunction, Abeta 

accumulation, and neurodegeneration. Nevertheless, whether retromer deficiency causes tau 

hyperphosphorylation, a key histological feature of the disease, remains unknown. 

We have begun addressing this question by using monoclonal antibody PHF1 to examine brain 

extracts of genetically-modified retromer-deficient mice, heterozygote knockouts for VPS26. In 

our first analysis we found no abnormalities in tau phosphorylation in whole brain extracts of 

retromer-deficient mice. Guided by recent studies suggesting that the retromer is differentially

expressed in the hippocampal formation, we completed a second study examining both 

hippocampal and cerebellar extracts in older retromer-deficient mice. Results suggest an 

interesting interaction, such that retromer-deficient mice have increases in tau phosphorylation in 

the hippocampus versus the cerebellum, compared to the between-region difference observed in 

wildtype mice. To confirm, extend and clarify these observations we are currently A) replicating 

this analysis in a larger number of subjects; B) relying on brain slice histology to identify which, 

if any, hippocampal subregion has the greatest effect; and, C) testing for tau 

hyperphosphorylation in retromer-deficient mice expressing human amyloid precursor protein. 

Disclosures: A.S. Bender, None; E. Planel, None; S.A. Small , None; K. Duff, None. 

Poster 





Title: The novel calpain-inhibitor A-705253 prevents stress-induced tau hyperphosphorylation 

in vitro and in vivo 

Authors: *R. BITNER 1 , A. L. NIKKEL 1 , S. MARKOSYAN 1 , M. W. DECKER 1 , A. HAHN 2 , J. 

SCHOEMAKER 2 , A. MOELLER 2 ; 

1 Abbott Labs., Abbott Park, IL; 2 Abbott Labs., Ludwigshafen, Germany 

Abstract: Calpains are a family of calcium-dependent cysteine proteases expressed by numerous 

cell types that participate in signal transduction, cytoskeletal architecture and apoptosis. In 

neuronal cells, calcium-mediated overactivation of calpain has been implicated in several 

neurodegenerative disorders, including Alzheimer‟s disease (AD). Hyperphosphorylation of the 

microtubule-associated protein tau and the subsequent aggregation of tau filaments resulting in 

the formation of neurofibrillary tangles are recognized etiological pathways in AD pathology. 

Cyclin-dependent kinase 5 (cdk5) is a major kinase responsible for tau hyperphosphorylation in 

AD that in part may involve calpain-mediated conversion of a cdk-5 regulatory activating protein 

(p35 to p25). In the present study, we examined the effects of calpain inhibition both in vitro and 

in vivo on tau phosphorylation. In hippocampal slices, lowering medium temperature to 33°C 

increased tau phosphorylation at the AT8 site as measured by Western blot analysis. Incubation 

with the novel small molecule calpain inhibitor A-705253 blocked the 33°C-induced tau 

phosphorylation with an IC50 of 100 nM. In vivo, pentobarbital-induced hypothermia or acute 

systemic LPS treatment increased tau phosphorylation in Mossy Fibers of the CA3 hippocampus 

of non-transgenic mice, as measured immunohistochemically at the AT8 site. A-705253 (3-10

mg/kg s.c.) pretreatment reduced the stress-induced tau hyperphosphorylation near control levels 

in both models. Results of these studies suggest that calpain inhibition has potential utility in 

reducing tau hyperphosphorylation and may represent a novel disease modifying approach in the 


Disclosures: R. Bitner, None; A.L. Nikkel, None; S. Markosyan, None; M.W. Decker, 

None; A. Hahn, None; J. Schoemaker, None; A. Moeller, None. 

Poster 





Support: UNMC Faculty Retention Fund 

Vada Oldfield Alzheimer‟s Research Awards 

John Douglas French Alzheimer‟s Foundation 

Title: Over-expression of tau-tubulin kinase 1 gene in P301L tau mice results in enhanced prefibrillar 

tau formation, spatial learning impairment and motor dysfunction 

Authors: *J. XU, S. SATO, S. OKUYAMA, M. JACOBSEN, E. STRUNK, R. SWAN, T. 

IKEZU, T. IKEZU; 

Univ. Nebraska Med. Ctr., Omaha, NE 

Abstract: Neuron-specific tau-tubulin kinase-1 (TTBK1) directly phosphorylates tau, a 

cytoskeletal microtubule-associated protein, at specific Serine/Threonine residues found in 

paired helical filaments of Alzheimer‟s disease (AD). To better understand the effects of TTBK1 

on tau pathology in vivo, we developed bigenic TTBK1/P301L Tau mice by crossing P301L tau 

transgenic mice with TTBK1 transgenic mice harboring human full-length TTBK1 genomic 

DNA. TTBK1/P301L Tau mice demonstrated age-dependent spatial learning impairment as 

determined by radial arm water maze, exacerbated locomotive dysfunction, and motor neuron 

loss in ventral horn as compared with age-matched P301L Tau littermates. TTBK1/P301L Tau 

mice developed enhanced tau phosphorylation at AT8, pS422 and 12E8 recognition sites, which 

was associated with increased levels of p25, an activator of cyclin-dependent kinase 5, and 

Tyr216-phosphorylation of glycogen synthase kinase-3β. This series of phosphorylation events 

correlated with increased sarkosyl-soluble tau aggregates, a pre-fibrillar form, in TTBK1/P301L

Tau mice as compared with P301L Tau littermates. These data indicate that TTBK1 can induce 

memory impairment through activation of specific protein kinases and increased formation of 

pre-fibrillar tau. 

Disclosures: J. Xu , None; S. Sato, None; S. Okuyama, None; M. Jacobsen, None; E. Strunk, 

None; R. Swan, None; T. Ikezu, None; T. Ikezu, None. 

Poster 





Support: UNMC Faculty Retention Fund (TI) 

Vada Oldfield Alzheimer‟s Research Awards (SS and TI) 

John Douglas French Alzheimer‟s Foundation (SS) 

Title: Tau-tubulin kinase 1 is upregulated in Alzheimer‟s disease brain and induces spatial 

learning impairment in transgenic mice in vivo 

Authors: *S. OKUYAMA, S. SATO, J. XU, L. B. MARTINEZ, S. M. WALSH, M. T. 

JACOBSEN, R. J. SWAN, T. IKEZU; 

Pharmacology/Exp Neurosci, Univ. Nebraska Med. Ctr., Omaha, NE 

Abstract: Tau-tubulin kinase-1 (TTBK1) is a neuron specific protein kinase exclusively 

expressed in brain that directly phosphorylates tau-a cytoskeletal microtubule associated proteinat 

specific Serine/Threonine residues found in paired helical filaments, suggesting its role in 

Alzheimer‟s disease (AD) and tauopathy pathogenesis. Indeed, we found that TTBK1 levels 

were upregulated in brains of human AD patients as compared to age-matched non-AD controls. 

To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring 

human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 was highly 

expressed in subiculum and cortical pyramidal layers, and induced phosphorylated neurofilament 

aggregation in V1-V2 cortical regions, entorhinal cortex, and subiculum. TTBK1-Tg mice also 

showed enhanced microgliosis and astrogliosis, and developed significant age-dependent 

memory impairment as determined by radial arm water maze test. This was associated with 

enhanced tau and neurofilament phosphorylation, increased levels of p25/p35, two activators of 

cyclin-dependent protein kinase 5 (CDK5), and increased p25/p35-associated CDK5 activity in

vivo. These data suggest that TTBK1 may be involved in AD pathogenesis, including learning 

impairment, astro/microgliosis, CDK5 activation, and phosphorylation of cytoskeletal proteins. 

Disclosures: S. Okuyama, full, A. Employment (full or part-time); S. Sato, None; J. Xu, 

None; L.B. Martinez, None; S.M. Walsh, None; M.T. Jacobsen, None; R.J. Swan, None; T. 

Ikezu, None. 

Poster 





Support: NIH intramural grant 

Title: Dab1 deficiency leads to tau hyperphosphorylation in postnatal mice 

Authors: M. ZAKA, *T. MATSUKI, B. W. HOWELL; 

Neuronal Migration Disorders Unit, NINDS/NIH, Bethesda, MD 

Abstract: Hyperphosphorylation of the microtubule binding protein tau is a pathological feature 

observed in Alzheimer‟s and related neurological diseases. Tau is primarily expressed in 

neuronal axons and unphosphorylated tau stabilizes microtubules. Under disease conditions, tau 

becomes phosphorylated to a high degree, resulting in the dissociation from microtubules, 

microtubule depolymerization, and ultimately the formation of neurofibrillar tangles. We have 

investigated the role of Disabled-1 (Dab1), a cytoplasmic adaptor protein, in the 

hyperphosphorylation of tau. Dab1 is an important downstream target of the Reelin signaling 

pathway that is tyrosine phosphorylated in response to Reelin binding to the apolipoprotein-Ereceptor-2 

and the very-low-density-lipoprotein-receptor. Phosphorylation of Dab1 is critical for 

positioning of migrating neurons during brain development. Dab1-mutant mice exhibit 

developmental defects in neuronal positioning and laminar organization in several regions of the 

brain, including the cerebral cortex, hippocampus, cerebellum, the olfactory bulb and the spinal 

cord. Previously it has been reported that Dab1-null mice display hyperphosphorylation of tau in 

the hippocampus and fatality occurring at weaning. However, it is not clear whether the 

inactivation of Dab1 and the interruption of Reelin signaling, or the profound neurological 

condition of the Dab1 mutant mice results in enhanced phosphorylation of tau in postnatal 

animals. To address this, we have inactivated the Dab1 gene in mice at postnatal day eleven after 

most neuronal positioning is complete. These animals have normal life spans and improved brain 

anatomy compared to Dab1-null animals. We found that inactivation of a conditional allele of

Dab1 by Cre-mediated recombination in postnatal mice led to tau hyperphosphorylation in the 

hippocampus. Furthermore, Dab1-inactivation increased levels of phosphorylated Erk1/2 and 

MEK1/2. These results suggest that loss of Dab1 leads to the activation of the MAP kinase 

pathway and increases tau phosphorylation in adult mice. Therefore, elucidating the roles of the 

Reelin/Dab1 signaling pathway and the suppression of tau hyperphosphorylation has implication 

for understanding tauopathies including Alzheimer‟s disease. 

Disclosures: M. Zaka, None; T. Matsuki, None; B.W. Howell, None. 

Poster 





Support: NIH Grants NS020591; NS029494; AG10124 

Alzheimer's Association 

Title: Specific tau phosphorylation is induced by DNA double strand breaks: a possible link to 

neurodegenerative diseases 

Authors: J. CHEN 1 , H. LI 1 , M. MARTINEZ-LAGE 2 , *J. Q. TROJANOWSKI 2 , K. HERRUP 1 ; 

1 Cell Biol. and Neurosci., Rutgers Univ., Piscataway, NJ; 2 Dept Pathol & Lab. Med., Univ. 

Pennsylvania Sch. Med., Philadelphia, PA 

Abstract: Phosphorylation of tau at specific residues, for example phospho-serine 214 (pS214), 

occurs normally in proliferating cells during metaphase. At the same time, hyperphosphorylated 

tau is the main constituent of the paired helical filaments (PHF) that are one of the pathological 

hallmarks of Alzheimer‟s disease (AD). Indeed, monoclonal antibodies against specific phosphotau 

epitopes, including pS214, are commonly used to stain PHF. This is curious, as neurons are 

supposedly postmitotic. S214 is a specific substrate of both Akt (a kinase and survival factor that 

is up-regulated in AD) and Pten (a phosphatase and a tumor suppressor that is down-regulated in 

AD). To explore a possible role for pS214 in neurodegeneration, brain sections were 

immunostained using a pS214-specific antibody. As expected, we detected strong pS214 

neuronal staining in affected regions of the AD brain. In these neurons, pS214 was 

predominantly cytoplasmic. Similar cytoplasmic pS214 staining was detected in some cases of 

corticobasal syndrome and frontotemporal disease brains as well as in amyotrophic lateral 

sclerosis CNS samples. pS214 staining was present in control specimens as well, but here its

location was predominantly nuclear. Thus, the shift of pS214 from the nucleus to the cytoplasm 

is tightly correlated with disease. In culture, we observed strong nuclear pS214 staining in mouse 

and human neuroblastoma cells but only during mitosis. On the other hand, clear nuclear pS214 

staining was found in many cultured mouse cortical neurons, even with no detectable 

chromosome condensation. The pS214-stained neurons had well defined nuclei and were 

activated caspase 3-negative. We speculated that DNA damage might serve as a trigger for the 

cytoplasmic shift of pS214, so N2a neuroblastoma cells were exposed to γ-rays to induce DNA 

breaks. Nuclear foci of H2AX staining quickly formed in treated cells while cytoplasmic pS214 

staining increased, eventually coalescing into distinct aggregates that grew in size over time. 

Taken together, our data suggest that tau, and it pS214 isoform may play an unexpected role in 

the DNA damage response system of a neuron. 

Disclosures: J. Chen, None; M. Martinez-Lage, None; J.Q. Trojanowski , None; K. Herrup, 

None; H. Li, None. 

Poster 





Title: In vitro neuroprotection by α 7 nAChR ligands: analysis of effects on Aβ 1-42-induced 

neurite outgrowth and tau phosphorylation 

Authors: *M. HU, M. E. SCHURDAK, P. S. PUTTFARCKEN, R. EL KOUHEN, M. 

GOPALAKRISHNAN, J. F. WARING, J. LI; 

Abbott Labs., Abbott Park, IL 

Abstract: Alzheimer‟s disease is an age-related neurodegenerative disorder characterized by 

progressive loss of memory accompanied with cholinergic neurodegeneration. There is 

substantial evidence indicating that Aβ1-42 is one of the major factors in AD pathogenesis, but the 

underlying mechanism(s) remains unclear. In this study, we first established an in vitro model of 

Aβ1-42-induced neurotoxicity in rat neonatal primary cortical cells, using high content screen 

(HCS) microscopy for measurement of neurite outgrowth, to address the neurotoxicity effect of 

Aβ1-42. Neurite outgrowth was found to be significantly reduced by Aβ1-42 (300 nM - 30 µM), but 

not by the scrambled control peptide control, in a time- and concentration-dependent manner. 

The total number of neurons was not affected. The Aβ1-42-induced reduction of neurite outgrowth 

was partially attenuated by α7 nicotinic acetylcholine receptor (nAChR) selective agonist PNU- 

282987 and also by the antagonist methyllycaconitine (MLA). This suggested that the observed

neuroprotective effects could arise from interference of Aβ1-42 interactions with α7 nAChRs. 

Emerging evidence also indicates that Aβ1-42 can potentiate hyperphosphorylation of tau in cell 

lines and in transgenic mice. To further examine the basis of neuroprotective effects, we 

evaluated effects in an in vitro model of Aβ1-42-induced tau hyperphosphorylation in 

differentiated PC12 cells using in-cell Western blot technique. Exposure of the cells to Aβ1-42 

increased phosphorylation of tau at serine-202 and phosphorylation of GSK-3β at tyrosine-216 

(GSK-3β-pY216), which were both partially inhibited by the GSK-3ß inhibitor, CHIR98023. 

Again, both α7 nAChR selective agonists including A-582941 and PNU-282987, and 

antagonists, MLA and α-bungarotoxin partially attenuated the increased phosphorylation tau and 

phosphorylation GSK-3β-pY216. This study provide evidence for a role for α7 nAChR 

mechanisms underlying Aβ1-42 toxicity, tau phosphorylation and neuroprotection, which, if 

translated in vivo, could provide additional basis for their utility of α7 nAChR ligands in 

treatment of Alzheimer‟s disease. 

Disclosures: M. Hu, None; M.E. Schurdak, None; P.S. Puttfarcken, None; R. El Kouhen, 

None; M. Gopalakrishnan, None; J.F. Waring, None; J. Li, None. 

Poster 





Support: Grant-in-Aid for Matching Fund Subsidy for Private Universities from The Promotion 

and Mutual Aid Corporation for Private Schools of Japan 

Grant-in-Aid for Scientific Research 11680746 from the Japanese Ministry of 

Education, Science, and Culture 

Title: Retinoic acid induced activation of GSK3β promoter in neuronal cells 

Authors: *O. MURAYAMA, A. SUMIKURA, S. NAKANISHI, Y. SASAKI, M. MATSUDA; 

Lab. MolBiol, Sch. Enviro Hlth. Sci., Azabu Univ., Kanagawa, Japan 

Abstract: Glycogen synthase kinase 3β (GSK3β) is known as a key protein that involved in 

developing Alzheimer‟s disease (AD) via tau phosphorylation. Regulation of GSK3β activity 

may be an important target of AD treatment or therapeutics. Previously age dependent increasing 

of GSK3β in human brain (Takashima, A. et al. 1998). This suggests that a factor contributing to 

GSK3β expression is involved in generating and/or progressing of AD. By using reporter gene

constructs, we studied mechanism for regulating GSK3β gene expression in neuron under 

differentiating condition. SH-SY5Y, a human neuroblastoma cell, stably expressing luciferase 

gene directed by GSK3β promoter was treated with retinoic acid (RA). Luciferase activity in SH- 

SY5Y cells was increased by RA treatment. This RA-induced activation of the promoter was 

confirmed by RT-PCR for endogenous GSK3β mRNA of SH-SY5Y cells. On the contrary, the 

RA-induced activation was not observed in HEK293, a human embryonic kidney cell. Because 

the effect of RA was enhanced by depolarization induced by high K+, the neuron specific RAinduced 

activation of GSK3β expression might be depended on activity of neuronal cells. 

Recently it has been reported that RA enhanced the protein level of GSK3β resulting in AD 

linked phosphorylation of tau in SH-SY5Y cells (Jamsa, A. et al., 2004). This phosphorylation 

was inhibited by lithium chloride a GSK3β inhibitor. In our study, lithium reduced the RAinduced 

activation of GSK3β promoter. Thus, inhibition of tau phosphorylation by lithium may 

be based on modifying not only the kinase activity but also transcription of GSK3β. Our results 

may promote to develop a new therapeutic agent for treating AD or related disorders. 

Disclosures: O. Murayama, None; A. Sumikura, None; S. Nakanishi, None; Y. Sasaki, 

None; M. Matsuda, None. 

Poster 






N. Bud Grossman Ctr for Memory Research and Care Fund 

NIH NS033249 

Title: The effect of tau phosphorylation on dendritic spine morphology in rodent hippocampal 

and cortical neurons 

Authors: *B. R. HOOVER 1 , K. H. ASHE 2 , D. LIAO 3 ; 

1 N Bud Grossman Ctr. Dept Neurol, 2 N Bud Grossman Ctr, Dept Neurol, GRECC, VAMC, 

3 Dept Neurosci., Univ. Minn, Minneapolis, MN 

Abstract: Tau is a major microtubule-binding protein localized primarily to neuronal axons. In 

the axons of healthy neurons, tau acts to stabilize microtubules and promote their assembly in the

process of neurite outgrowth. Tau function is regulated by a variety of post-translational 

modifications including phosphorylation by tau kinases. Recent studies have suggested that tau 

dysfunction occurs following increases in its phosphorylation state. Tau hyperphosphorylation 

leads to its dissociation from microtubules, aggregation into paired helical filaments, and 

formation of intraneuronal neurofibrillary tangles (NFTs), one of the major histopathological 

hallmarks of Alzheimer‟s disease. Once formed, this conformationally abnormal, insoluble, 

hyperphosphorylated form of tau relocalizes from the axons to the somatodendritic region, 

leading to changes in microtubule and actin stability, impaired axonal transport, and ultimately 

neurodegeneration in brain regions such as the hippocampus and cortex. Accordingly, NFTs 

correlate strongly with cognitive deficits and neuronal loss in humans. A recent study showed 

that the human P301L tau mutant, which results in frontotemporal dementia associated with 

Parkinsonism (FTDP-17), is more readily phosphorylated than wild-type human tau. To test the 

hypothesis that mis-targeting of human P301L tau to dendritic spines plays a role in human 

tauopathies, we transfected dissociated rat hippocampal neurons with either P301L tau or wildtype 

tau. We find that the P301L human tau mutant is localized to the dendritic spines of rat 

dissociated hippocampal neurons more often than wild-type human tau. To investigate the role of 

phosphorylation in P301L tau mis-localization to hippocampal dendritic spines, 14 mutations 

that target serine-threonine phosphorylation sites have been introduced into the P301L human tau 

to produce either a hyperphosphorylated or unphosphorylatable tau isoform. The localization of 

these mutant isoforms in comparison with the P301L tau mutant will be examined in rat 

hippocampal neurons. To complement our findings in rat hippocampal neurons, mixed corticalhippocampal 

neuronal cultures prepared from rTg4510 mice overexpressing the P301L human 

tau mutant will also be utilized. Specifically, dendritic spine morphology and the expression 

patterns of different synaptic proteins will be evaluated. Our results will shed light on the role of 

tau in neuron dysfunction in tauopathies such as FTDP-17 and Alzheimer‟s disease. 

Disclosures: B.R. Hoover, None; K.H. Ashe, None; D. Liao, None. 

Poster 



Program#/Poster#: 245.19/P1 


Title: Paired helical filament core associated to neuronal membrane as a nucleation center for 

tau aggregation 

Authors: *K. LIRA-DE LEON 1 , M. A. DE ANDA-HERNANDEZ 1 , P. FIGUEROA- 

CORONA 1 , M. CARDENAS-AGUAYO 2 , V. CAMPOS-PEÑA 3 , M. A. MERAZ-RIOS 1 ;

1 Mol. Biomedicine, CINVESTAV-IPN, Distrito Federal, Mexico; 2 Kline Inst., New York, NY; 

3 Insituto Nacional de Neurología y Neurocirugía Manuel Velazco Suárez, Distrito Federal, 

Mexico 

Abstract: Alzheimer Disease (AD) is characterized by hallmarks lesions: neuritic plaques and 

neurofibrillary tangles (NFT). Progressive dementia is correlated with the appearance of NFT in 

different regions of hippocampus and enthorinal cortex. These NFT are mainly composed by 

filamentous aggregates of Tau protein as paired helical filaments (PHF). Previous studies 

showed a proteolytic resistant fragment named as PHF core, containing a main portion of the Tau 

microtubule binding domain, suggesting that this fragment is important for Tau abnormal 

polymerization in AD. In the present study, we modified neural precursor cells primary cultures 

(NPCs) by a retroviral system, in order to of evaluated the factor involve in the aggregates 

generation of Tau. PHF core was used in two ways: 1) as a soluble fraction and 2) with a 

membrane localization (IFNγR1-NMF), to elucidate the possible role of the membrane substrate 

as a nucleation center for Tau polymerization. We observed that soluble PHF-core was able to 

form a moderate quantity of β-sheet structures, but interestingly the presence of these structures 

was higher in cells with IFNγR1-NMF expression. These data suggest that the interaction 

between PHF core and plasma membrane could regulate the formation of abnormal fibrillary 

deposits. 

Disclosures: K. Lira-De Leon , None; M.A. De Anda-Hernandez, None; P. Figueroa- 

Corona, None; M. Cardenas-Aguayo, None; V. Campos-Peña, None; M.A. Meraz-Rios, 

None. 

Poster 





Title: Expression of soluble and chimerical truncated tau (151-391) in neuronal cultures 

Authors: *M. A. DE ANDA-HERNANDEZ 1 , K. I. LIRA-DE LEON 1 , P. FIGUEROA- 

CORONA 1 , V. CAMPOS-PEÑA 2 , M. A. MERAZ-RIOS 1 ; 

1 Mol. Biomedicine, CINVESTAV-IPN, Mexico, Mexico; 2 Inst. Nacional de Neurologia y 

neurocirugia manuel velazco suarez, mexico, Mexico 

Abstract: Alzheimer disease (AD) is a neurodegenerative progressive encephalic disorder, 

characterized by behavioral changes associated to memory lost and disorientation. Is the most

common dementia among older people and is the 4th cause of death in developed countries. 

Many studies are focused on Tau protein which is the principal component of neurofibrillary 

tangles (NFT). Tau localization is changed during the pathology development. However it had 

not been reported the analysis of Tau membrane localization and its importance in the 

pathological Tau precipitation. The purpose of this job was to develop a cellular model to 

analyze the possible role of the miss-localization of truncated Tau in AD neurodegeneration. We 

used 2 gene constructs: 1) soluble truncated Tau (151-391 aa) and 2) a chimerical form with a 

membrane anchored signal (IFNγR1-151-391). Through retroviral system and transfection 

analysis, we expressed the constructions in neuronal culture. We noticed the presence of β-sheet 

structures when we induce differentiation from neural stem cells to neuronal culture in the 

transduced cell that expressed the Tau anchored to membrane. These data suggests the 

participation of neuronal membrane as nucleation center to abnormal aggregation of Tau and 

sustain the importance of truncation as a post-translation modification to the development of AD. 

Disclosures: M.A. De Anda-Hernandez, None; K.I. Lira-De Leon, None; P. Figueroa- 

Corona, None; M.A. Meraz-Rios, None; V. Campos-Peña, None. 

Poster 




Topic: C.01.j. Cognitive function 

Support: NIH grant AG 05146 

Intramural Research Program of the NIA, NIH. 

Burroughs Wellcome Fund for Translational Research 

Title: No quantitative differences in 1-42β-amyloid and hyperphosphorylated-tau tangles loads 

in the brains of subjects with asymptomatic AD and mild cognitive impairment (MCI). The 

BLSA study 

Authors: *D. IACONO 1 , R. O' BRIEN 2 , S. M. RESNIK 3 , A. ZONDERMAN 3 , O. 

PLETNIKOVA 1 , G. RUDOW 1 , B. CRAIN 1 , J. C. TRONCOSO 1 ; 

1 Dept Pathol, Div. Neuropathol, 2 Dept of Neurol., Johns Hopkins Univ., Baltimore, MD; 

3 Personality and Cognition Lab., Natl. Inst. of Aging, Baltimore, MD

Abstract: Objectives. Morphometric studies from the Baltimore Longitudinal Study of Aging 

(BLSA) showed marked neuronal hypertrophy in asymptomatic AD (ASYMAD) subjects 

compared to age-matched controls (Controls), MCI and Alzheimer‟ disease (AD) subjects. 

ASYMAD is a state characterized by comparable AD lesions (β-amyloid neuritic plaques and 

neurofibrillary tangles) as those found in autopsy brains of MCI subjects and some definite AD 

cases, as assessed by the CERAD criteria and Braak staging. The aim of this study was to 

quantify the amounts of different forms of 1-42β-amyloid (Aβ): diffuse β-amyloid (Aβ-diffuse), 

Aβ-diffuse plaques (Aβ-DP), Aβ-neuritic plaques (Aβ-NP), Aβ-cored plaques (Aβ-CP), and 

hyperphosphorylated-tau protein in the form of neurofibrillary tangles (NFT). We examined the 

same BLSA postmortem brains and anatomical regions as those in morphometric studies: CA1hippocampus 

(CA1), anterior and posterior cingulate gyrus (ACG and PCG), and the primary 

visual cortex (PVC). We obtained a large set of quantitative data to analyze and compare the 

characteristic lesions of AD in different brain areas and clinical-pathological states. 

Material and Methods. We examined 60 brains (45 males and 15 females) from BLSA: 

Controls (n=15), ASYMAD (n=15), MCI (n=15) and AD (n=15) patients. AD lesions were 

stereologically estimated using the area fraction fractionator (AFF) probe on randomly selected 

sections (10 κm thick) immunostained for 1-42 β-amyloid and hyperphosphorylated-tau 

associated with tangles. One-way ANOVA analyses were performed across all groups. 

Results. The analysis did not show differences for 1-42β-amyloid or hyperphosphorylated-tau 

amounts in ASYMAD compared to MCI or AD group for any region, except for Aβ-NP. 

Neuritic plaques were significantly more abundant in AD cases compared with all other groups 

in all regions (p


Topic: C.01.j. Cognitive function 

Support: Abercrombie Foundation and the Kleberg Foundation 

NIA 1P30AG028383 

NIH AG05146 

NIA K04AG00553 

NIA R01AG09862 

Title: Hypertrophy of CA1-hippocampal neurons in asymptomatic AD: findings from the nun 

study 

Authors: *J. C. TRONCOSO 1 , D. SNOWDON 3 , W. R. MARKESBERY 3 , O. PLETNIKOVA 1 , 

G. RUDOW 1 , E. PATEL 3 , D. TUDOR 3 , P. ZANDI 2 , D. IACONO 1 ; 

1 Neuropathol Lab., 2 Mental Hlth., Johns Hopkins Univ., Baltimore, MD; 3 Neurol. and Sanders- 

Brown Ctr. on Aging, Univ. of Kentucky, Lexington, KY 

Abstract: Objectives. The pathology of Alzheimer‟s disease (AD) evolves decades before the 

disease becomes clinically manifest. Consequently, it is common to find substantial Aβ-plaques 

(NP) and neurofibrillary tangles (NFT) in the autopsy brains of older subjects with normal 

cognition, a state we term asymptomatic AD (ASYMAD). Since the CA1 region of the 

hippocampus is highly vulnerable to AD, here we examine whether CA1 neurons undergo 

morphometric changes of their cell bodies, nuclei, and nucleoli in ASYMAD subjects. 

Material and Methods. We examined the autopsy brains of subjects with AD pathology and no 

cognitive deficits (ASYMAD) (n=10) and compared them to age-matched controls (n=13), mild 

cognitive impairment (MCI) patients with autopsy-confirmed AD pathology (n=5) and AD 

patients (n=10) from the Nun Study. All subjects underwent neuropsychological evaluations 

within a year before death. We used the nucleator, a stereological probe, to measure the volumes 

of neuronal cell bodies, nuclei, and nucleoli in CA1. 

Results. We found a significant hypertrophy of the neuronal cell bodies, nuclei, and nucleoli in 

CA1 of ASYMAD subjects compared to both controls (p

This work was supported by the Johns Hopkins University Alzheimer‟s Disease Research Center 

(NIH grant AG05146), the Nun Study (NIA grants: R01AG09862, K04AG00553 and 

1P30AG028383), the Abercrombie Foundation and the Kleberg Foundation. 

Disclosures: J.C. Troncoso , None; O. Pletnikova, None; G. Rudow, None; D. Iacono, 

None; D. Snowdon, None; W.R. Markesbery, None; E. Patel, None; D. Tudor, None; P. 

Zandi, None. 

Poster 

246. Alzheimer's Imaging and Biomarkers II 



Topic: C.01.o. Imaging and biomarkers 

Support: U24-RR021382 

NCRR P41- RR14075 

NIA R01-AG29411 

K23-AG22509 

R21-AG029840 

Title: Executive dysfunction in patients with posterior cortical atrophy 

Authors: *A. M. NEGREIRA, D. SAPOLSKY, A. BAKKOUR, B. C. DICKERSON; 

Massachusetts Gen. Hosp., Charlestown, MA 

Abstract: Objective: Posterior cortical atrophy (PCA) is a neurodegenerative disorder marked 

by focal atrophy of the parietal lobes and a constellation of cognitive impairments such as 

progressive visuospatial dysfunction, alexia, optic ataxia, ocular apraxia, simultanagnosia and 

visual agnosia. Deficits in executive functioning, however, are not reported as typical 

characteristics of PCA, although they are sometimes anecdotally noted in neuropsychological 

reports. 

The current study sought to characterize impairment of executive functioning, particularly 

complex attention as measured by the WAIS Backward Digit Span (WBDS), in patients with 

PCA. Additionally, we compared PCA performance on this measure with that of patients with 

Alzheimer‟s Disease (AD).

Methods: Data from the WBDS was collected on six patients (4F:2M; Age = 62.0, SD 10.7) 

patients with PCA. Participants were selected if they illustrated prominent impairment in 

visuospatial processing and neuroimaging findings revealed focal parietal lobe atrophy. Cortical 

thickness measures were derived from T1-weighted anatomic MRI data and analyzed to identify 

areas where thickness was reduced in patients with PCA as compared to a group of older 

controls; thickness from these regions was used as independent structural variables in a 

correlation with degree of impairment on WBDS. 

Results: Patients with PCA demonstrated impaired performance on the WBDS (Mean Z-score 

(Z) = -1.10). Patients with PCA exhibited significant focal thinning of the parietal cortex as 

compared to a group of older controls (Z =-2.23, SD= 2.0) whereas thickness of the frontal 

cortex was relatively spared (Z = -0.12, SD = .60). Patients with PCA also performed worse on 

WBDS as compared to patients with AD (t(32)=-2.16, p=.02). AD patients demonstrated milder 

parietal thinning than patients with PCA (Z = -1.23, SD = 1.44) with minimal frontal thinning (Z 

= -0.35, SD =1.33). 

Conclusions: Although these preliminary findings should be interpreted with caution due to 

small sample size, executive dysfunction may play a more prominent role in PCA than 

previously reported. Further investigation of the role of the parietal cortex in executive function 

is warranted. 

Disclosures: A.M. Negreira , None; D. Sapolsky, None; A. Bakkour, None; B.C. Dickerson, 

None. 

Poster 





Support: Innovative Research Incentive Schemes of the Netherlands Organization for Scientific 

Research 

Calouste Gulbenkian Foundation 

Title: Impaired temporal correlations in temporo-parietal oscillations in early-stage Alzheimer's 

disease 

Authors: *K. LINKENKAER-HANSEN 1,2 , T. MONTEZ 3 , S.-S. POIL 1,2 , B. F. JONES 4 , I. 

MANSHANDEN 4 , J. P. A. VERBUNT 4,6 , B. W. VAN DIJK 4,6 , A. B. BRUSSAARD 1,2 , A. VAN 

OOYEN 1,2 , C. J. STAM 4,7 , P. SCHELTENS 5,8 ;

1 Dept Exp Neurophysiol., VU Univ. Amsterdam, Amsterdam, Netherlands; 2 Ctr. for 

Neurogenomics and Cognitive Res. (CNCR), Amsterdam, Netherlands; 3 Inst. of Biophysics and 

Biomed. Engin., Lisbon, Portugal; 4 Dept Clin. Neurophysiol., 5 Dept Neurol., VU Univ. Med. 

Ctr., Amsterdam, Netherlands; 6 Dept. of Physics and Med. Technol., Amsterdam, Netherlands; 

7 MEG Ctr., Amsterdam, Netherlands; 8 Alzheimer Ctr., Amsterdam, Netherlands 

Abstract: Sustained oscillatory activity on time scales of seconds is commonly observed for 

different frequency bands in the retention interval of a working-memory task [1,2]. Interestingly, 

during resting-state conditions, which are known to be associated with prominent retrieval and 

manipulation of information in working memory, ongoing oscillations also exhibit amplitude 

modulations on multiple time scales, as indicated by long-range temporal correlations (LRTC) 

up to tens of seconds [3,4]. We reasoned that correlations in oscillations over time might be 

important for memory and could therefore be abnormal in Alzheimer's disease (AD). To test this 

hypothesis, we measured magnetoencephalography (MEG) during eyes-closed rest in 19 patients 

diagnosed with early-stage AD and 16 age-matched control subjects and characterized temporal 

correlations in ongoing oscillations using detrended fluctuation analysis and a novel "avalanche 

analysis" that quantifies the life- and waiting-time probability distributions of oscillation bursts 

[5]. We found that Alzheimer‟s patients had markedly weaker long-range temporal correlations 

in the alpha band (6-13 Hz) over temporo-parietal regions on time scales of 1-25 seconds. On 

shorter time scales (< 1 second), abnormal dynamics of alpha oscillations in AD patients were 

expressed as a strongly reduced probability for the occurrence of oscillation bursts with long lifetimes 

or for long waiting-times between bursts in the temporo-parietal regions. These regions 

have been associated with mnemonic functions in healthy subjects and show metabolic and 

structural deficits in AD, suggesting that the tendency for ongoing alpha oscillations to carry a 

memory of their own amplitude dynamics is important for cognition. 

References 

[1] Raghavachari et al. (2001) J Neurosci 21:3175-3183. 

[2] Jensen et al. (2002) Cereb Cortex 12:877-882. 

[3] Linkenkaer-Hansen et al. (2001) J Neurosci 21:1370-1377. 

[4] Linkenkaer-Hansen et al. (2007) J Neurosci 27:13882-13889. 

[5] Poil, van Ooyen, Linkenkaer-Hansen (2008) Human Brain Mapping (10.1002/hbm.20590). 

Disclosures: K. Linkenkaer-Hansen , None; T. Montez, None; S. Poil, None; B.F. Jones, 

None; I. Manshanden, None; J.P.A. Verbunt, None; B.W. van Dijk, None; A.B. Brussaard, 

None; A. van Ooyen, None; C.J. Stam, None; P. Scheltens, None. 

Poster 



Program#/Poster#: 246.3/P7


Support: Investigator Initiated Grant from Pfizer 

Title: Pre-clinical evaluation of [18F]-fluoroethoxy-benzovesamicol: Metabolism and PET 

imaging brain distribution in rat 

Authors: E. LANDRY ST-PIERRE 1 , M.-A. BEDARD 2 , P. ROSA 1 , A. ALIAGA 1 , G. 

MASSARWEH 1 , *J.-P. SOUCY 1 ; 

1 Brain Imaging Ctr., Montreal Neurolog. Inst., Montreal, QC, Canada; 2 Psychology - 

Neuropsychology Div., UQAM, Montreal, QC, Canada 

Abstract: Background: [18F]-Fluoroethoxy-benzovesamicol ([18F]-FEOBV) is a positron 

emitting ligand of the ACh vesicular transporter which reversibly binds to its target. It holds 

promise as a potential ACh system pre-synaptic marker. [18F]-FEOBV could be useful for early 

detection of neurodegenerative diseases where acetylcholine (ACh) neurotransmission is altered 

such as Alzheimer‟s or Progressive Supranuclear Paralysis. 

Study #1: Seven male Sprague-Dawley rats were anaesthetized with isofluorane 2% and placed 

in a CTI Concorde R4 microPET scanner. Physiological parameters (respiration rate, EKG and 

temperature) were recorded throughout the imaging sessions. Animals received an i.v. dose of 

4.3 to 16.7 MBq (117 to 450 uCi) of high specific activity [18F]-FEOBV. Emission scans were 

obtained for 60 min. Images were co-registered to a rat brain anatomical (MRI) template. Study 

#2: Five male Sprague-Dawley rats received an average i.v. dose of 37 MBq (1mCi) of high 

specific activity [18F]-FEOBV. They were sacrificed at 5, 7, 10, 24 and 70 minutes. Blood was 

drained and centrifuged and plasma was analyzed for metabolites using reverse-phase HPLC. 

Results#1: Physiological parameters remained constant during the experiment. Rats manifested 

no overt acute nor subacute (days) signs of toxicity following scanning. Distribution of the tracer 

was found to be as expected from the literature on Ach systems anatomy. There was a fast 

washout of radioactivity from the cerebellum. On parametric images, the highest binding 

potentials were detected in the caudate, amygdala, hippocampus and basal forebrain. Results#2: 

Blood analysis showed one hydrophilic metabolites (still to be identified). The parent compound 

had a mean retention time of 120 seconds with the HPLC set-up we used. Importantly, no 

lipophilic metabolite was found. Although no physiologic monitoring was done, rats showed no 

overt sign of physiological distress until sacrifice (up to 70 minutes), except for one animal who 

received significantly more mass of the compound. 

Conclusions: [18F]-FEOBV is a promising ligand for imaging the innervations density of the 

cholinergic system, a potentially important parameter in neurodegenerative diseases assessment. 

In this (and other) experiment, FEOBV induced no overt toxicity, an encouraging result in terms 

of its potential utilisation in humans. Its metabolism is fairly rapid and its hydrophilic metabolite 

allows rapid elimination. We are now proceeding to more advanced toxicology testing and are 

working on optimisation of the tracer imaging and quantification protocol. 

Disclosures: E. Landry St-Pierre, None; M. Bedard, Pfizer Canada, A. Employment (full or 

part-time); P. Rosa, None; A. Aliaga, None; G. Massarweh, None; J. Soucy, Researcher 

Initiated Grant from Pfizer, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received).

Poster 





Support: NIH 5K23AG022476-05 

Title: Plasma amyloid-beta peptides and amygdala atrophy: probable biomarkers of prodromal 

depression of alzheimer‟s disease 

Authors: *W. Q. QIU; 

Tufts-New England, Boston, MA 

Abstract: Background Our previous study has shown that amyloid-associated depression 

defined by high plasma Amyloid-β peptide (Aβ 40) and low Aβ42 is associated with impaired 

episodic memory, the early sign of Alzheimer‟s disease (AD). Therefore, we postulated that 

plasma Aβ is associated with brain structures regulating mood, and are related to AD. 

Methods Homebound elders (N=362) in a cross-sectional, population-based study were used. 

Plasma Aβ1-40 and Aβ1-42 were measured. The volumes of total brain, amygdala, hippocampal 

and white matter hyperintensities (WMHI) were measured. 

Results Subjects with concomitant MCI and depression had lower level of plasma Aβ42, smaller 

volumes of amygdala and hippocampus than those with MCI alone. Smaller amygdala was 

associated with lower plasma Aβ42 level, the determining factor of Aβ40/Aβ42 ratio, after 

adjusting for AD, depression and other potential confounders. Among those who were dementiafree, 

decreasing quartiles of plasma Aβ42 and increasing quartiles of plasma Aβ40/Aβ42 ratio as 

well were associated with a smaller volume of amygdala. Subjects with amyloid-associated 

depression defined by having a high plasma Aβ1-40/1-42 ratio had the smallest left amygdalar 

volume compared to those with non-amyloid-associated depression and those without 

depression. 

Interpretation Since the atrophy of amygdala, the brain structure mediating emotional memory, 

predicts the risk of AD, the combination of a high plasma Aβ1-40/1-42 ratio and amygdalar 

atrophy might represent an early sign of underlying AD pathology, and serve as biomarkers of 

prodromal depression. 

Disclosures: W.Q. Qiu , None.

Poster 





Support: This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of 

Health & Welfare, Republic of Korea.( A050079) 

Title: The changes of cerebellar cortical gray matter of the alzheimer disease according to 

clinical dementia rating: Voxel Based Morphometric Study 

Authors: *Y.-C. YOUN 1 , P.-S. KIM 1 , S. CHAE 2 , S. KIM 3 ; 

1 Neurol., 2 Pediatrics, Chung-ang Univ. Hosp, Seoul, Republic of Korea; 3 Neurol., Seoul Natl. 

Univ. Bundang Hasp., Bundang, Republic of Korea 

Abstract: Introduction 

The symptoms of Alzheimer's disease (AD) have a correlation with cerebral cortical atrophy. 

This study is designed to compare the change of gray matter feature of AD patient according to 

CDR with voxel-based morphometry (VBM). 

Methods 

The 77 patients aged of 60 years and above with impairment of memory and cognition 

underwent assessments of CDR, history of the disease, full neuropsychological tests including 

memory, language, visuospatial, frontal executive functions, and 3D volumetric brain MRI. We 

selected a group of patients who are fulfilling the AD criteria suggested by DSM-IV and 

NINCDS-ADRDA. As a result, only 22 patients were selected finally. For control, 22 normal 

individuals without any specific health problem or medication history that can affect cognitive 

function were recruited, and like the patients, 3D volumetric brain MRI were obtained. The 

patients were divided into patient group 1 of CDR 0.5, patient group 2 of CDR 1, and patient 

group 3 of CDR 2, and the control subjects were assigned for each patient group as control group 

1, 2, and 3 by matching the sample sizes and ages via sampling with replacement. For 

quantitative analysis, the 3D volumetric brain MRI images from each individual of patient 

groups and control groups were reconstructed with the presented standard VBM protocol: 

realignment, spatial normalization, segmentation, and smoothing. 

Results 

In test 1 that patient group 1 vs, control group 1, the tendency of cortical atrophy to occur mainly 

in medial temporal lobe including hippocampal region could be found. In test 2 that patient 

group 2 vs, control group 2 and test 3 that patient group 3 vs, control group 3, atrophy emerged 

in temporal lobe, gradually involved parietal lobe, and then extended into frontal lobe; finally, 

atrophy of overall cortex progressed. In test 4 that patient group 1 vs, patient group 2, it was 

revealed that cortical atrophy of cerebellum was more prominent in patient group 2. And in test 5 

that patient group 2 vs, patient group 3, the progression of atrophy in cerebellum was also

observed as a result consistent with that of test 4. 

Discussion 

The results of test 1, test 2 and test 3 are also considered to be similar to the patterns of cortical 

atrophy in AD that were observed in previous studies. In this study, cortical atrophy was 

apparently present in cerebellum. It is not yet clarified whether the cerebellar change is related to 

the clinical progression or prognosis of AD. If there is a relation between them, however, it is 

thought that the studies of cerebellum could contribute to the treatment and management of AD. 

Therefore, a variety of studies for cerebellums, as well as cerebrums in AD is required from now 

on. 

Disclosures: Y. Youn , None; P. Kim, None; S. Chae, None; S. Kim, None. 

Poster 





Title: Subcellular localization of the neuronal retromer: Insight into its role in hippocampal 

function and Alzheimer‟s disease 

Authors: *A. BHALLA 1 , G. DI PAOLO 2 , S. A. SMALL 3 ; 

1 Taub Inst., 2 Taub Inst. and Dept. of Pathology, 3 Taub Inst. and Dept. of Neurol., Columbia 

Univ., New York, NY 

Abstract: First described in yeast, the retromer sorting pathway consists of a multimeric 

retromer complex and a retromer-binding receptor, VPS10. Mammalian homologues of the 

retromer complex have been identified, and mammals express a family of VPS10-containing 

molecules, all highly expressed in the brain. To date two members of this family, sorLA and 

sortilin, have been shown to act as retromer-binding receptors in the brain. 

A range of gene-expression and genetic studies in humans, and biochemical and behavioral 

studies in animal models, have implicated the retromer sorting pathway in late-onset Alzheimer‟s 

disease. Furthermore, the retromer complex is differentially expressed in the hippocampal 

formation. Nevertheless, the precise role the retromer sorting pathway plays within neurons 

remains unknown. 

Here, we provide initial insight into this question by performing a series of co-labeling 

immunocytochemistry studies to determine the subcellular localization of the retromer complex 

in cultured hippocampal neurons. Although axonal staining could not be excluded, neuronal 

VPS35, the core element of the retromer complex, was found to have a predominant

somatodendritic distribution. A series of synaptic markers were then used to further clarify this 

observation. An absence of co-localization was noted between VPS35 and synaptophysin, 

suggesting the retromer is excluded from the pre-synaptic terminals. In contrast, VPS35 was 

found juxtaposed, but not overlapping the post-synaptic marker PSD95. Indeed, the spatial 

distribution of VPS35 suggested that it is localized near dendritic spines, in the general locale of 

dendritic endosomes and Golgi outposts. These observations immediately generate a number of 

predictions about the role the retromer plays in hippocampal neurons, and how retromer 

dysfunction contributes to late-onset Alzheimer‟s disease. 

Disclosures: A. Bhalla , None; G. Di Paolo, None; S.A. Small, None. 

Poster 





Support: Howard Hughes Medical Institute 

the Alzheimer‟s Association 

NIA Grants RO1-AG029411, K23-AG22509, P50-AG05134, P50-AG05681, and P01- 

AG03991 

the MIND Institute 

NCRR Grants P41-RR14075 and U24-RR021382 

Title: The differential effects of aging and AD on the cerebral cortex: MRI-based quantification 

of regional cortical thickness 

Authors: *A. BAKKOUR 1 , J. C. MORRIS 3 , R. L. BUCKNER 4 , B. C. DICKERSON 2 ; 

1 Psychiatry, 2 Neurol., Massachusetts Gen. Hosp., Charlestown, MA; 3 Neurol., Washington 

Univ., St. Louis, MO; 4 Psychology, Harvard Univ., Cambridge, MA 

Abstract: Objective: Although both normal aging and Alzheimer‟s disease (AD) are associated 

with regional atrophy of the cerebral cortex, data are sparse on the comparative spatial patterns 

and magnitude of effects of these processes. We sought to investigate the differential effects of 

age and of AD on cerebral cortical thickness across the entire cortical mantle.

Methods: Subjects in the present study included a sample of young controls (YC, N=142, age 18- 

30), older controls (OC, N=87, age 65-94) and AD patients (N=29, age 66-96). Previously 

conducted exploratory analyses identified a set of regions of interest (ROIs) of cortical thinning 

in both aging and AD. Mean thickness values within these ROIs were used to calculate percent 

thinning due to aging or AD. 

Results: Specific regions identified from the “cortical signature of aging” were predominantly 

affected by age and minimally more by AD. For example, the calcarine cortex exhibited 22% 

thinning in OC vs. YC with an additional 3.8% when comparing AD to OC. Other regions 

identified from the “cortical signature of AD” were predominantly affected by AD with minimal 

effects of age. For example, the inferior temporal cortex exhibited only 1.2% thinning in OC vs. 

YC with an additional 11.8% thinning in AD vs. OC. Finally, other regions were affected by 

both aging and AD. For example, the supramarginal gyral cortex showed 8.4% thinning in aging 

and an additional 8.4% thinning in AD. 

Conclusions: The processes of aging and AD have both differential and partially overlapping 

effects on specific regions of the cerebral cortex. Further work is warranted to determine the 

behavioral and clinical relevance of these findings. 

Disclosures: A. Bakkour , None; J.C. Morris, None; R.L. Buckner, None; B.C. Dickerson, 

None. 

Poster 





Support: NIA Grants R01-AG29411, R21-AG029840, and K23-AG22509 

MIND Institute 

NCRR Grants P41- RR14075 and U24-RR021382 

Title: Temporal pole anatomy and semantic deficits in primary progressive aphasia 

Authors: D. SAPOLSKY, A. BAKKOUR, A. NEGREIRA, *B. C. DICKERSON; 

Dept Neurol, Massachusetts Gener Hosp., Charlestown, MA 

Abstract: Objective: Although the functional role of the temporal pole in semantic processing 

has been illuminated through studies of groups of patients with focal lesions and

neurodegenerative syndromes, little detailed morphometric data exist on the magnitude of 

temporal polar cortical atrophy as it relates to semantic processing, in part due to challenges in 

the development of measurement protocols. Further, it is not clear whether measurement of 

temporal polar anatomy at the individual subject level can predict the presence or relative 

severity of semantic deficits within the progressive aphasia clinical phenotype. 

Methods: Subjects in the present study included a group of patients with primary progressive 

aphasia (PPA, N=13). Cortical thickness of the temporal pole was obtained from MRI data. 

These thickness measures were transformed to Z scores based on a group of older cognitively 

intact controls. These measures were related to semantic processing ability, determined through 

use of a battery of language and semantic memory testing. 

Results: Across all subjects, patients with semantic deficits (N=5) had strikingly reduced 

temporal pole thickness (left: Z=-4.1 SD=1.6; right: Z=-2.8 SD=1.3) while patients without 

semantic deficits had temporal pole thickness within the range of normal (left: Z=0.2 SD=2.0; 

right: Z=-0.1 SD=1.1). At the individual subject level, 100% of patients with semantic deficits 

had temporopolar thickness greater than 1.5 SD below normals. Further, the patient with the 

mildest semantic impairment had the smallest magnitude of thinning of the left temporopolar 

cortex (Z=-1.8). 

Conclusions: Quantitative measurement of temporopolar anatomy in vivo is possible at the 

individual subject level in patients with neurodegenerative disease. This method has the potential 

to provide insight into the functional role of the temporal pole in semantic processing, and 

possibly assist in clinical classification. 

Disclosures: D. Sapolsky, None; B.C. Dickerson , None; A. Bakkour, None; A. Negreira, 

None. 

Poster 



Program#/Poster#: 246.9/Q1 


Poster 





Support: NIA AG024904 

State of Arizona and ADHS 

NIH Foundation 

Title: Twelve month MRI gray matter declines in Alzheimer's dementia evaluated by voxelbased 

morphometry with multivariate network analyses: Findings from the Alzheimer's Disease 

Neuroimaging Initiative 

Authors: *K. D. HANSON 1,4,2 , K. L. BERGFIELD 3,4,2 , K. CHEN 5,4 , E. M. REIMAN 5,4 , M. A. 

BERNSTEIN 6 , J. KORNAK 7 , D. J. HARVEY 10 , N. W. SCHUFF 8,11 , P. M. THOMPSON 12 , M. 

W. WEINER 8,9,11 , C. R. JACK, Jr 6 , G. E. ALEXANDER 1,4,2,3 ; 

1 Dept. of Psychology, 2 Evelyn F. McKnight Brain Inst., 3 Neurosci. Grad. Program, Univ. 

Arizona, Tucson, AZ; 4 Arizona Alzheimer's Consortium, Phoenix, AZ; 5 Banner Alzheimer's 

Inst., Banner Good Samaritan Med. Ctr., Phoenix, AZ; 6 Dept. of Radiology, Mayo Clin. Col. of 

Med., Rochester, MN; 7 Dept. of Radiology, Epidemiology and Biostatistics, 8 Dept. of 

Radiology, 9 Dept. of Med. and Psychiatry, Univ. of California San Francisco, San Francisco, 

CA; 10 Dept. of Publ. Hlth. Sci., Univ. of California Davis Sch. of Med., Davis, CA; 11 Dept. of 

Veterans Affairs Med. Ctr., San Francisco, CA; 12 Lab. of Neuro Imaging, Dept. of Neurol., 

UCLA Sch. of Med., Los Angeles, CA 

Abstract: Previous studies in Alzheimer‟s disease (AD) patients have demonstrated brain 

volume reductions on magnetic resonance imaging (MRI), with medial temporal structures 

preferentially affected compared to healthy aging. We used voxel-based morphometry (VBM) 

with multivariate network analysis to investigate twelve-month declines in the regionally 

distributed patterns of gray matter atrophy in AD patients and healthy controls from the 

Alzheimer‟s Disease Neuroimaging Initiative (ADNI; www.loni.ucla.edu/ADNI). Analyses 

included 56 AD (mean age = 76.0±7.7; M/F = 32/24) patients and 100 healthy controls (mean 

age = 76.0±5.4; M/F = 59/41), who did not differ in age or gender. Using volumetric T1 

MPRAGE MRIs obtained at baseline and after a twelve-month follow up visit, longitudinal 

VBM processing was performed with statistical parametric mapping (SPM5) to produce 

smoothed gray matter maps. To identify a regional pattern of gray matter reductions that 

distinguished the groups and to test for a group x time interaction in pattern expression over the 

twelve-month interval, multivariate Scaled Subprofile Model (SSM) analysis was performed. 

SSM analysis of the two groups combined identified a linear combination of component patterns 

that best distinguished the AD patients from controls (R 2 = 0.48, p ≤ 0.0000001) with each 

significantly contributing to the model. This combined pattern was characterized mainly by 

bilateral medial and lateral temporal, perisylvian, and parietal reductions with relative 

preservation in visual cortical regions. Using the SSM network subject scores derived from each 

subject‟s baseline and twelve month follow up scan, a group x time interaction (p ≤ 0.01) was 

observed with the AD patients showing greater pattern expression from baseline to 12 months

compared to the controls. After controlling for baseline Mini Mental State Exam (MMSE) 

scores, greater longitudinal increases in pattern expression in the AD patients were associated 

with greater declines in MMSE performance over the twelve month period (p ≤ 0.003) and this 

association was not observed in the controls. Together, the findings indicate a regionally 

distributed MRI gray matter pattern associated with AD that shows a progression of gray matter 

reductions over 12 months which is not observed in healthy elderly and which is associated with 

the rate of cognitive decline in the AD patients. SSM network analysis with MRI longitudinal 

VBM may aid in tracking the progression of AD and potentially assist in evaluating treatments 

and prevention therapies. 

Disclosures: K.D. Hanson, None; K.L. Bergfield, None; K. Chen, None; E.M. Reiman, 

None; M.A. Bernstein, None; J. Kornak, None; D.J. Harvey, None; N.W. Schuff, None; P.M. 

Thompson, None; M.W. Weiner, None; C.R. Jack, None; G.E. Alexander, None. 

Poster 





Support: Brain Research Center of the 21st Century Frontier Research Program funded by the 

Ministry of Science and Technology of Republic of Korea: M103KV010021-03K2201-02130) 

Title: Neural correlate of cognitive reserve: The role of precuneus in protecting the dementias 

Authors: *J. CHEY 1 , M. J. KIM 2 , K.-M. SHIN 1 , M. SHIN 1 ; 

1 Dept Psychol, Seoul Natl. Univ., Seoul, Republic of Korea; 2 Dept Psychological and Brain Sci., 

Dartmouth Col., Hanover, NH 

Abstract: The concept of cognitive reserve has been proposed to explain the discrepancy 

between the neuropathology and the clinical symptoms of people with the Alzheimer disease. 

Individuals with superior IQ develop dementia symptoms later on in the illness (Snowdon et al., 

1996), and low education has been repeatedly observed as a risk for Alzheimer‟s disease and 

dementia in general (Del Ser et al., 1999). It has been proposed that individuals with greater 

cognitive reserve can withstand the effects of neurodegenerative processes, and would delay the 

clinical manifestation of dementia, while individuals with smaller cognitive reserve would 

manifest the symptoms very early in the disease process (Stern, 2002.) 

The neural correlate of this cognitive reserve has been speculated to be the whole brain size or 

the general synaptic density of the brain (Katzman et al., 1988; Mori et al., 1997), but no specific

ain regions have been proposed. In this study, we examined the risk of having a small 

cognitive reserve in a non-demented community sample of 243 elderly people with various 

education levels. Small cognitive reserve or low cognitive performance (LCP) was defined as 

scores less than one standard deviation below the mean compared to the age- and education- 

specified norms on cognitive measures, such as the Dementia Rating Scale. Utilizing the voxelbased 

morphometry, we measured the regional brain volumes of these LCP individuals and 

compared to those of the normal cognitive performance (NCP) individuals who were matched in 

terms of age and years of education. Non-demented elderly individuals with low cognitive 

performance had significantly smaller volume of the precuneus. Further, after a seven-year 

follow-up, the LCP individuals had over four times the risk of developing dementia caused by 

the Alzheimer‟s disease and vascular diseases, compared to the elderly people with normal 

cognitive performance. Therefore, we propose that the precuneus is one of the key sites in the 

brain that buffers the impact of neuropathology caused by Alzheimer‟s disease and vascular 

diseases. This was supported by the fact that the baseline volume of precuneus was significantly 

correlated not only with the Total score of the Dementia Rating Scale (.73) but also with an 

independent test of general cognition, the Clock Drawing performance (.56). 

Precuneus is a multimodal association cortex which has rich connections with cortical and 

subcortical regions (Goldman-Rakic, 1988; Petrides & Pandya, 1984). Further, the precuneus has 

been demonstrated to be involved in a variety of cognitive functions (Cavanna & Trimble, 2006), 

which is consistent with its role as a cognitive reserve. 

Disclosures: J. Chey, None; M.J. Kim, None; K. Shin, None; M. Shin, None. 

Poster 





Title: Evaluation of cerebral metabolic profile and volumetry in Tg2576 mouse model of 

Alzheimer‟s disease by 1H-MR spectroscopy and T2-MRI 

Authors: *J. M. YRJANHEIKKI 1 , K. LEHTIMAKI 1 , T. HEIKKINEN 1 , T.-K. MIETTINEN 1 , 

A. NURMI 1 , O. GROHN 2 , J. PUOLIVALI 1 ; 

1 Neurosci., Cerebricon Ltd, Kuopio, Finland; 2 A.I.Virtanen Inst. Univ. of Kuopio, Kuopio, 

Finland 

Abstract: Alzheimer‟s disease (AD) is the most common cause of dementia in the elderly and 

the need for translational biomarkers of the disease progression is of crucial importance. Non-

invasive in vivo magnetic resonance imaging and spectroscopy (MRI, MRS) have been recently 

employed for CNS diseases as translational tools. In human AD, metabolic changes including 

increase in myo-inositol and decrease in N-acetyl-aspartate (NAA) have been reported. Similarly 

MRI has been applied to demonstrate brain atrophy. Here we characterize the Tg2576 mouse 

model of AD, carrying a mutated form of the human amyloid precursor protein and exhibiting 

increased Aβ levels and plaque deposition in the brain. Under isoflurane anesthesia we applied 

T2-weighted MRI and 1H-MRS using a 4.7T magnet at 3, 8 and 13 months of age in order to 

study hippocampal metabolic profile and cerebral volumetric changes over time in female 

Tg2576 mice. 

Tg2576 mice showed increased amyloid plaque load at the age of 13 months as assessed by 

amyloid beta 1-42 immunohistochemistry and ELISA. The plaque load was more robust in 

ventral cortex than in hippocampus. 

MRS data analysis did not show any statistically significant differences between transgenic (TG) 

and wild type (WT) mice in the hippocampal concentrations of NAA, myo-inositol, glutamate, 

glutamine, taurine, choline and GABA when studied at 3, 8 and 13 months of age. Volumetric 

analysis of total brain, cortex and hippocampus revealed that total brain and cortex were readily 

smaller in TG as compared to WT mice already at the age of 3 months. Total brain, cortical and 

hippocampal volumes were 7.2, 8.1, and 1.1% smaller in TG than WT mice at 3 months of age. 

Interestingly, the difference between TG and WT brain structure volumes increased over age. 

The total brain, cortical and hippocampal volumes were 11.2, 14.8 and 12.9 % smaller in TG 

than WT mice at 13 months of age.As a conclusion, no significant metabolic differences were 

observed in the hippocampus of Tg2576 mice when compared to WT mice, indicating that in the 

Tg2576 model the metabolic changes in hippocampus may occur at later age, whereas possibly 

earlier in the ventral cortex where the plaque load is more robust already at 13 months of age. 

TG mice have smaller brains than WT mice already at 3 months of age, but this difference is 

slightly enhanced over time, possibly reflecting increased amyloid beta load related changes. 

Disclosures: J.M. Yrjanheikki , None; T. Heikkinen, None; T. Miettinen, None; A. Nurmi, 

None; O. Grohn, None; J. Puolivali, None; K. Lehtimaki, None. 

Poster 



Program#/Poster#: 246.13/R1 


Support: NIH P01 AG09466

Title: Frontal lobe activation changes during the digit symbol substitution test in Mild Cognitive 

Impairment 

Authors: *M. A. TRIVEDI, C. M. MURPHY, R. C. SHAH, J. D. E. GABRIELI, S. 

WHITFIELD-GABRIELI, G. T. STEBBINS; 

Neurolog Sci., Rush Univ. Med. Cntr, Chicago, IL 

Abstract: The digit symbol substitution test (DSST) is a complex attention test. Previous studies 

suggest that the DSST is also sensitive to the cognitive changes that occur during the 

pathogenesis of Alzheimer‟s disease (AD). In the present study, we used a modified version of 

the DSST to evaluate fMRI activation changes during DSST performance in individuals with 

amnestic mild cognitive impairment (AMCI), a risk factor for AD. The two groups included in 

this study were an ONC group (n = 15) and an AMCI group (n = 6). fMRI data processing was 

conducted using the standard SPM2 routines. To examine fMRI activation changes associated 

with DSST performance we created a contrast of “hits > misses”, that would allow us to 

determine brain regions in which the AMCI group displayed increased or decreased brain 

activation relative to the ONC group. Whole brain analysis failed to reveal any significant group 

differences after FDR correction for multiple comparisons. However, follow-up region of 

interest (ROI) analyses restricted to frontal cortex revealed that the AMCI group displayed 

significantly greater activation than the ONC group bilaterally in the inferior frontal cortex and 

in the left superior frontal cortex (p = 0.001). In contrast, there were no brain regions in which 

the ONC group displayed significantly greater activation than the AMCI group. These 

preliminary results might suggest that individuals with AMCI display greater activation in frontal 

lobe regions important for complex attentional processes, possibly due to pathophysiological 

changes associated with AD. A larger group of subjects will help to confirm these findings. 

Disclosures: M.A. Trivedi , P01 AG09466, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); C.M. Murphy, 

None; R.C. Shah, None; J.D.E. Gabrieli, None; S. Whitfield-Gabrieli, None; G.T. Stebbins, 

None. 

Poster 




Abstract: Withdrawn

Poster 





Support: NINDS intramural grant 

Title: Brain iron in high field magnetic resonance imaging: susceptibility contrast as function 

field strength 

Authors: *B. YAO 1 , T.-Q. LI 2 , P. VAN GELDEREN 2 , K. SHMUELI 2 , J. A. DE ZWART 2 , J. 

H. DUYN 2 ; 

1 AMRI, LFMI, NINDS, 2 Amri, lfmi, ninds, NIH, Bethesda, MD 

Abstract: Magnetic susceptibility provides an important contrast mechanism for MRI. 

Increasingly, susceptibility-based contrast is being exploited to investigate brain tissue 

microstructure and to detect abnormal levels of brain iron as these have been implicated in a 

variety of neuro-degenerative diseases. However, it remains unclear to what extent magnetic 

susceptibility-related contrast at high field relates to actual brain iron concentrations. In this 

study, we performed susceptibility weighted imaging as a function of field strength on healthy 

brains in vivo and post-mortem brain tissues at 1.5 T, 3 T and 7 T. Nine normal volunteers 

(female/male = 4/5) with mean age = 31 ± 5 (mean ± standard deviation) participated in the 

study and each completed three scan sessions (1.5, 3 and 7 T) under a human subjects protocol 

approved by the Institutional Review Board. Axial T2 * -weighted gradient echo (GRE) images 

were acquired at several echo times (TEs) at each field strength with an in-plane resolution of 

0.94 × 0.94 mm 2 . R2 * and phase images were calculated from the GRE images. For comparison, 

iron histology was performed on the samples from two brain tissue specimens. The calculated 

susceptibility-related parameters R2 * and signal frequency shift in four iron-rich regions 

(putamen, globus pallidus, caudate, and thalamus) showed an almost linear dependence (r ≥ 0.90 

for R2 * ; r ≥ 0.83 for phase, p < 0.01) on field strength, suggesting that potential ferritin saturation 

effects are not relevant to susceptibility-weighted contrast for field strengths up to 7 T. The R2 * 

dependence on the putative (literature-based) iron concentration was 0.048 Hz/Tesla/ppm. The 

histological data from brain samples confirmed the linear dependence of R2 * on field strength 

and showed a slope against iron concentration of 0.0099 Hz/Tesla/ppm dry-weight, which is 

equivalent to 0.05 Hz/Tesla/ppm wet-weight and closely matched the calculated value in vivo. 

These results confirm the validity of using susceptibility-weighted contrast as an indicator of iron 

content in iron-rich brain regions. The absence of saturation effects opens the way to exploit the 

benefits of MRI at high field strengths for the detection of iron distributions with high sensitivity 

and resolution.

Disclosures: B. Yao , None; T. Li, None; P. van Gelderen, None; K. Shmueli, None; J.A. de 

Zwart, None; J.H. Duyn, None. 

Poster 




Topic: C.01.n. Genetics and functional genomics 

Support: RO1 AG023173 

P50 AG005142 

Title: Mild cognitive impairment (MCI): accelerated aging or prodromal AD? 

Authors: *N. C. BERCHTOLD 1 , J. ROGERS 3 , P. COLEMAN 3 , C. W. COTMAN 2 ; 

1 Inst. Brain Aging & Dementia, 2 Inst. for Brain Aging & Dementia, Univ. California Irvine, 

Irvine, CA; 3 Sun Hlth. Res. Inst., Sun City, AZ 

Abstract: Mild cognitive impairment (MCI) is a clinical diagnosis for individuals who are not 

cognitively normal for age and yet do not have overt dementia. It has been alternatively argued 

that MCI represents accelerated aging, or that it is prodromal Alzheimer‟s disease (AD). To test 

these ideas, we use microarray analysis of several brain regions to identify the gene signature 

associated with MCI, and compare the expression patterns to those observed in normal aging and 

in AD. MCI diagnosis was based on the Petersen criteria, with a clinical dementia rating (CDR) 

≤ 0.5. The regions assessed were the hippocampus (HC), entorhinal cortex (EC), superior frontal 

gyrus (SFG) and post-central sensory gyrus (PCG). In MCI, the HC is the primary region to 

show functional changes, as evidenced by HC-dependent cognitive tests and fMRI. Tissue was 

obtained from 11 MCI (age 83-90 yrs, mean age 87.5), 26 AD (age 83-90 yrs, mean age 87.5) 

and 16 normal controls (age 82-91, mean age 86), all groups sex-balanced, from Alzheimer‟s 

disease research center (ADRC) brain banks (UC Irvine, Sun Health Research Institute, Johns 

Hopkins, USC, U Rochester, Mayo Clinic). RNA was individually hybridized to Affymetrix Hg- 

U133A chips, and only probe sets flagged significant (p

Disclosures: N.C. Berchtold , None; C.W. Cotman, None; P. Coleman, None; J. Rogers, 

None. 

Poster 





Support: commercial 

Title: Development of novel biomarkers for Alzheimer's disease based on tau oligomers 

Authors: *E. J. DAVIDOWITZ 1 , S. VASAN 3 , J. G. MOE 2 ; 

1 OLIGOMERIX, Inc., 2 OLIGOMERIX, Inc., New York, NY; 3 Consultant, New York, NY 

Abstract: There is a critical unmet need for novel biomarkers for differential diagnosis of 

Alzheimer‟s disease (AD) at early onset and to stage disease progression. Presently, definitive 

diagnosis of AD requires tissue biopsy or autopsy of the brain, which can occur only after the 

patient dies. There are no validated biomarkers identified in patient samples such as 

cerebrospinal fluid (CSF), blood, and urine that can be used to definitely monitor the progression 

or regression of AD. Most approaches focus on quantitative changes of individual proteins such 

as tau and Aβ in patient samples as biomarkers. These studies have led to a consensus that an 

increase in total and p-tau and a concomitant decrease in Aβ1-42 in CSF may be indicative of 

neurodegeneration. However, these changes may not be specific indicators of AD and may also 

occur in some other forms of dementia. The development of novel biomarkers based on the 

paradigm of protein-protein interactions, rather than on specific protein levels, may create 

opportunity for earlier and more specific diagnosis and disease monitoring for better patient 

management. Using the approach of measuring pathological interactions between proteins may 

provide urgently needed novel AD biomarkers. These biomarkers can also serve as drug targets 

for the identification of new therapeutic entities to treat AD and to monitor different therapeutic 

effects when used to treat AD. Here, data is presented demonstrating that tau oligomers in CSF 

have potential as a biomarker for AD. Novel results from an ELISA formatted to detect these 

complexes indicate that they are present in CSF and that they are elevated in AD samples 

compared to non-AD samples. The choice of capture antibody for tau was critical for 

identification of tau oligomers that correlated with disease. Although the sample size is small in 

these pilot experiments, these preliminary results support the notion of developing tau oligomers 

as biomarkers for AD.

Disclosures: E.J. Davidowitz, OLIGOMERIX, Inc., A. Employment (full or part-time); 

OLIGOMERIX, Inc., E. Ownership Interest (stock, stock options, patent or other intellectual 

property); S. Vasan, OLIGOMERIX, Inc., F. Consultant/Advisory Board; J.G. Moe, 

OLIGOMERIX, Inc., A. Employment (full or part-time); NIH, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received); 

OLIGOMERIX, Inc., E. Ownership Interest (stock, stock options, patent or other intellectual 

property). 

Poster 





Support: NIH Grant P01 AG09466 

Title: Ventral processing stream dissociation of fMRI activation during explicit and implicit 

memory in AD, MCI and healthy controls 

Authors: *G. T. STEBBINS 1 , M. TRIVEDI 1 , C. MURPHY 1 , R. SHAH 2 , S. W. GABRIELI 3 , J. 

D. E. GABRIELI 3 ; 

1 Neuroloical Sci., 2 Alzheimer's Dis. Ctr., Rush Univ. Med. Ctr., Chicago, IL; 3 Brain and 

Cognitive Sci., MIT, Cambridge, MA 

Abstract: Memory function is dependent to a large extent on the functioning of the ventral 

processing stream. Higher order visual processing occurs in the fusiform gyrus and information 

is then transferred to medial temporal lobes structures critical for memory formation. 

Pathological changes have been documented in this processing stream in patients with 

Alzheimer‟s disease (AD), and some evidence suggest these changes may be present in 

individuals with amnestic mild cognitive impairment (MCI) who have an increased risk of 

developing AD. In the present study, we examined differences in fMRI activation during 

encoding and repetition priming in participants with no cognitive impairment (NCI), MCI, and 

mild AD. Whole brain fMRI analyses demonstrated increased activation during encoding in all 

groups in multiple regions, including occipital lobe, parietal lobe, temporal lobe, frontal lobe, the 

ventral process stream (fusiform gyrus), and hippocampus/parahippocampal regions. 

Examination of between groups encoding differences revealed a significant reduction in 

encoding related activation in MCI and AD relative to NCI in many regions, including ventral 

processing stream and hippocampus/parahippocampal locations. Regions of interest (ROI) 

examination of a priori defined regions, including the hippocampal/parahippocampal region,

fusiform gyrus, and Brodmann area 17 (BA 17) of the primary visual cortex, revealed significant 

reductions of encoding related fMRI signal in the AD and MCI groups in the 

hippocampal/parahippocampal and fusiform ROI, but not in the ROI encompassing BA 17 of the 

primary visual cortex. Examination of these same ROIs for repetition priming revealed a 

different pattern with no significant group differences in any of the examined regions. These 

results indicate that during the earliest stages of memory impairment, such as occurs in MCI, 

there are alterations in encoding related fMRI activation in medial temporal lobe structures that 

extends into the fusiform gyrus of the ventral processing stream, but spares primary visual 

regions. This disruption of the ventral processing stream is encoding specific and does not 

evidence during repetition priming. The alteration of ventral processing in AD and MCI during 

encoding may reduce connectivity between primary visual areas involved in stimulus perception 

and affect higher order visual processing prior to medial temporal lobe processing. 

Disclosures: G.T. Stebbins , None; M. Trivedi, None; C. Murphy, None; R. Shah, 

None; S.W. Gabrieli, None; J.D.E. Gabrieli, None. 

Poster 





Support: NIH Grant RO1 MH057899 

State of Arizona Grant 211002 

NIH Grant 9R01AG031581-10 

Title: Hypertension is associated with hypometabolism in brain regions affected by Alzheimer‟s 

disease and normal aging: preliminary results 

Authors: *J. B. S. LANGBAUM 1 , K. CHEN 1 , W. LEE 1 , C. RECSHKE 1 , S. REEDER 1 , D. 

BANDY 1 , G. E. ALEXANDER 2 , R. J. CASELLI 3 , E. M. REIMAN 1 ; 

1 Brain Imaging, Banner Alzheimer's Inst., Phoenix, AZ; 2 Psychology, Univ. of Arizona, Tucson, 

AZ; 3 Neurol., Mayo Clin. Arizona, Scottsdale, AZ 

Abstract: Background: Longitudinal studies have suggested that hypertension in mid-life is a 

risk factor for subsequent cognitive decline and probable Alzheimer‟s disease (AD). We have 

proposed using fluorodeoxyglucose (FDG) positron emission tomography (PET) measurements

of the regional cerebral metabolic rate for glucose (CMRgl) as a presymptomatic quantitative 

endophenotype to evaluate putative genetic and non-genetic risk factors for AD. 

Objective: To test the hypothesis that hypertension is associated with lower CMRgl in brain 

regions affected by AD or normal aging. Secondly, examine whether higher higher systolic 

blood pressure (SBP) and diastolic blood pressure (DBP) are inversely correlated with CMRgl in 

these brain regions. 

Methods: A brain mapping algorithm (SPM5) was used to characterize respective relationships 

between higher SBP, higher DBP, and hypertension and lower CMRgl in 26 cognitively normal 

persons (mean Mini-Mental State Examination [MMSE] score 29.4 ± 1.1) 60.6 ± 6.7 years of 

age, including 6 apolipoprotein E (APOE) ε4 homozgotes (HM), 11 heterozygotes (HT), and 9 

noncarriers (NC). 

Results: Participants with and without hypertension did not differ significantly in their age, 

gender, educational level or MMSE scores. Participants with hypertension had lower CMRgl 

than those who did not in parietal regions previously associated with AD and in frontal regions 

previously associated with AD and normal aging (p < 0.05 corrected for multiple comparisons). 

Similarly, higher SBP and DBP were each associated with lower CMRgl in temporal regions 

previously associated with AD and in frontal regions previously associated with normal aging (p 

< 0.005 uncorrected for multiple comparisons). 

Conclusions: These preliminary brain-imaging findings provide additional evidence that 

hypertension along with higher SBP and DBP are associated with an increased risk of AD. This 

study provides a foundation for using FDG PET to rapidly evaluate the efficacy of antihypertensive 

treatments in the primary prevention of AD. 

Disclosures: J.B.S. Langbaum, None; K. Chen, None; W. Lee, None; C. Recshke, None; S. 

Reeder, None; D. Bandy, None; G.E. Alexander, None; R.J. Caselli, None; E.M. Reiman, 

None. 

Poster 

247. Parkinson's Disease Interventions: Animal and Clinical Models 



Topic: C.02.d. Therapies 

Support: (M103KV010021-06K2201-02110) from BRC 

(R15-2006-020) from NCRC (ROK) 

Title: Novel functional role of oxi gene family during oxidative stress induced dopamine 

neuronal cell death

Authors: *S. KIM, K. CHOI, J. HA, J. SON; 

Ewha Women Univ., Seoul, Republic of Korea 

Abstract: Oxidative stress exerts a significant pathological role for apoptosis and/or autophagic 

cell death of nigral dopamine (DA) neurons in experimental models of Parkinson‟s disease (PD) 

and PD patients. However, it is largely unknown what molecules are responsible for the selective 

susceptibility of DA neurons to oxidative stress and how these molecules affect the DA neuronal 

cell death. Using a DA cell line model, SN4741, the previous microarray analysis help identify a 

group of novel genes, called Oxi gene family including Oxi-α and Oxi-β. Oxi-α protein is quite 

specifically expressed in nigral DA neurons and phylogenetically conserved. Oxidative stress 

significantly down-regulated the expression of Oxi-α while the level of Oxi-β was dramatically 

upregulated during the oxidative stress-induced DA cell death. Of interest Oxi-β was specifically 

increased by oxidative stress and PD-related toxins. Oxi-α and Oxi-β share a very significant 

homology (63%) in amino acid sequence. We are further investigating how Oxi-α and Oxi-β 

regulate oxidative stress-induced apoptotic and autophagic cell death in DA neurons and 

exploring their usage as biomarkers to monitor DA neuronal degeneration. Our study may 

provide significant insights into the novel neuroprotective strategy against oxidative stress in PD. 

Disclosures: S. Kim, None; K. choi, None; J. Ha, None; J. Son, None. 

Poster 





Support: P01NS036302 

Title: Over-expression of GDNF in the nigrostriatal tract induces greater weight loss than 

hypothalamic over-expression in aged obese rats 

Authors: F. P. MANFREDSON 1 , *K. R. NASH 2 , N. TUMER 1 , P. J. SCARPACE 1 , B. 

ERDOS 1 , C. S. BROXSON 1 , L. F. SULLIVAN 1 , T. LANDA 1 , Y. ZHANG 1 , R. J. MANDEL 1 ; 

1 Neurosci., 2 Univ. Florida, Gainesville, FL 

Abstract: Intraventricular administration of glial cell line-derived neurotrophic factor (GDNF) 

in primate and human trials for treatment of Parkinson disease have revealed the potential for 

GDNF to induce weight loss. We have previously shown that bilateral hypothalamic overexpression 

of GDNF via recombinant adeno-associated virus (rAAV) results in significant

failure to gain weight in young rats and weight loss in aged rats. However, in the previous study, 

we could not determine an underlying biological mechanism for the weight loss effect in 

hypothalamus. We hypothesized therefore that since the nigrostriatal tract passes through the 

lateral hypothalamus, increased activity mediated by nigrostriatal DA may have been responsible 

for the observed effect on body weight. In this study we compared bilateral injections of rAAV- 

GDNF in hypothalamus versus substantia nigra in aged Brown Norway X Fischer 344 rats. 

Nigrostriatal GDNF over-expression resulted in significantly greater weight loss than rats treated 

in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to food 

intake or activity levels of the rats. Moreover, hypothalamic or striatal catecholamine levels did 

not account for the observed effects on body weight. In contrast, significant DA increases in 

nucleus accumbens was observed when GDNF was over-expressed either in hypothalamus or 

SN. However, GDNF-induced increases in accumbens DA levels were larger in the 

hypothalamic-treated group than in the SN treated group. Therefore, while increased accumbens 

DA may partially account for the observed weight loss, increased DA cannot completely account 

for the robust weight loss observed when GDNF is over-expressed in the nigrostriatal tract. 

Disclosures: F.P. Manfredson, None; R.J. Mandel, None; K.R. Nash , None; N. Tumer, 

None; P.J. Scarpace, None; B. Erdos, None; C.S. Broxson, None; L.F. Sullivan, None; T. 

Landa, None; Y. Zhang, None. 

Poster 






Title: The antiepileptic drug zonisamide protects in MPTP model of Parkinson's disease 

Authors: *P. K. SONSALLA, L.-Y. WONG; 

Dept of Neurol, UMDNJ RW Johnson Med. Sch., Piscataway, NJ 

Abstract: Parkinson‟s disease (PD) is a debilitating progressive neurodegenerative disorder in 

which loss of motor function results from the degeneration of nigrostriatal dopamine (DA) 

neurons. There is at present no cure for PD. Zonisamide is an FDA-approved antiepileptic drug 

that has neuroprotective actions against hippocampal neuronal damage in seizure models. 

Moreover, in a randomized clinical study, zonisamide improved motor function in PD patients 

possibly due to its ability to increase DA release. Preliminary reports by others suggest that

zonisamide 1) elevates the brain content of glutathione, an important brain antioxidant, 2) 

provides neuroprotection against 6-hydroxydopamine, and 3) modifies DA neurotransmission. 

Based on these observations, we hypothesized that zonisamide would protect against MPTPinduced 

damage to DA neurons. Mice were treated once daily for 3 days with MPTP (30, 30 and 

15 mg/kg, sc). Mice receiving zonisamide (20 mg/kg, ip) were pretreated 15 min before each 

MPTP administration. Mice were killed 7 days after the last treatment. MPTP treatment 

produced a profound lesioning of striatal DA nerve terminals as evidenced by 84 ± 6% and 92 ± 

4% reductions in tyrosine hydroxylase (TH) and DA, respectively. In mice treated with 

zonisamide and MPTP, the reductions in TH and DA were only 21 ± 11% and 50 ± 13%, 

respectively, and were statistically different from MPTP-only treatment. DA and TH content 

were not altered in mice treated with zonisamide alone. The IC50 for zonisamide inhibition of 

MAO B was ~300 uM. Moreover, in ex vivo studies, zonisamide treatment of mice did not 

interfere with the irreversible binding of systemically administered deprenyl to MAO B. 

Zonisamide does not interfere with DA uptake into synaptosomes (Okada et al. Epilepsy Res, 

22:193-205, 1995). Thus, zonisamide does not appear to alter pharmacokinetic or 

pharmacodynamic properties of MPTP/MPP+ although experiments will be conducted to 

measure the actual striatal content of MPP+ in both groups of mice. The mechanism(s) by which 

zonisamide protects as well as whether it protects in chronic PD models remains to be 

established. However, the finding that a drug which is already used clinically might retard DA 

neurodegeneration in PD is very encouraging. If additional pre-clinical studies in other PD 

models confirm the neuroprotective effect of zonisamide, they would pave the way for clinical 

trials examining the neuroprotective properties of zonisamide in PD patients. Supported by NIH 

NS052733. 

Disclosures: P.K. Sonsalla, None; L. Wong, None. 

Poster 





Support: Fondation de France (032153-2005013853) 

Title: Consequences of chronic subthalamic nucleus high-frequency stimulation on striatal 

activity in intact and parkinsonian rats: in vitro and in vivo recordings 

Authors: P. GUBELLINI 1 , S. LARDEUX 2 , *P. P. SALIN 3 , D. PALERESSOMPOULLE 2 , C. 

BAUNEZ 2 ;

1 IBDML, CNRS UMR6216, Marseille, France; 2 Lab. de Neurobiologie de la Cognition, CNRS 

UMR6155, Marseille, France; 3 IBDML, CNRS, Marseille, France 

Abstract: High-frequency stimulation (HFS) of the subthalamic nucleus (STN) is, to date, one 

of the most efficient treatments for the symptoms of Parkinson‟s disease (PD). However, despite 

its efficacy, the consequences of STN HFS on the circuitry and the neuronal activity within the 

basal ganglia (BG) are still a matter of debate. In parkinsonian rats, it has been demonstrated in 

vitro that the output neurons of the striatum show an increased spontaneous synaptic activity 

mediated by glutamate, and that such hyperactivity is reversed by chronic (5 days) STN HFS, 

which also ameliorates akinesia. However, since slice recordings do not allow the preservation of 

the BG circuitry, an in vivo approach is necessary to fully evaluate the consequences of this 

treatment on striatal neurons. For this reason, we have performed a parallel electrophysiological 

study from freely moving rats undergoing chronic STN HFS, both control and parkinsonian (6- 

OHDA lesion). Interestingly, also in vivo recordings showed a marked decrease of striatal 

neurons activity, in terms of spike frequency, triggered by STN HFS. Moreover, while in non 

stimulated rats we recorded a mixed tonic/burst spike mode, most of the residual spikes in 

stimulated animals occurred within bursts. Overall, these data show that STN HFS inhibits both 

glutamate synaptic transmission and spike activity of striatal neurons, confirming that the 

striatum is deeply involved in the pathophysiological mechanisms of PD and that its function is 

strongly affected by STN HFS. 

Disclosures: P. Gubellini, None; P.P. Salin , None; C. Baunez, None; S. Lardeux, None; D. 

Paleressompoulle, None. 

Poster 





Support: FRST NZ postdoctoral fellowship 

Title: Ghrelin promotes nigrostriatal dopamine function and protects dopamine cells in a mouse 

model of Parkinson‟s disease 

Authors: *Z. B. ANDREWS 1 , D. ERION 2 , R. BEILER 3 , J. ELSWORTH 4 , R. ROTH 4 , T. L. 

HORVATH 1 ; 

1 OB/GYN, 2 Intrnl. Med., 3 Comparative Med., 4 Psychiatry, Yale Univ., New Haven, CT

Abstract: Ghrelin targets the hypothalamus to regulate food intake and adiposity. Endogenous 

ghrelin receptors (growth hormone secretagogue receptor, GHSR) are also present in 

extrahypothalamic sites where they promote circuit activity associated with learning and 

memory, and reward seeking behavior. Here, we show that the substantia nigra pars compacta 

(SNpc), a brain region where dopamine (DA) cell degeneration leads to Parkinson‟s disease 

(PD), expresses GHSR. Ghrelin binds to SNpc cells, electrically activates SNpc DA neurons, 

increases midbrain mitochondrial respiratory capacity, tyrosine hydroxylase mRNA and 

increases DA concentration in the dorsal striatum. Exogenous ghrelin administration for 14 days 

to mice decreased SNpc DA cell loss and diminished striatal dopamine levels after 1-methyl-4phenyl-1,2,5,6 

tetrahydropyridine (MPTP) treatment. Genetic ablation of ghrelin increased SNpc 

DA cell loss and lowered striatal dopamine levels after MPTP treatment. Ghrelin-induced 

neuroprotection was mediated by enhanced uncoupling protein 2 (UCP2)-dependent 

mitochondrial function and biogenesis. Exogenous ghrelin administration for 14 days to UCP2 

knockout mice did not rescue SNpc TH cell loss in MPTP treated mice, as observed in UCP2 

wild type. Taken together, our data reveals that ghrelin plays an important role in the 

maintenance and protection of normal nigrostriatal dopamine function thereby offering a novel 

therapeutic strategy to combat neurodegeneration associated with PD. 

Disclosures: Z.B. Andrews, None; T.L. Horvath, None; R. Roth, None; J. Elsworth, None; D. 

Erion, None; R. Beiler, None. 

Poster 



Program#/Poster#: 247.6/S1 


Support: Swedish Brain Foundation 


Swedish Brain Power 

Swedish Parkinson Foundation 

Title: A2A receptor antagonist treatment counteract symptoms in the MitoPark mouse model of 

progressive Parkinson‟s disease

Authors: *D. J. MARCELLINO 1 , K. FUXE 1 , J. HOCKEMEYER 2 , C. MÜLLER 2 , L. 

OLSON 1 , D. GALTER 1 ; 

1 Dept Neurosci, Karolinska Inst., Stockholm, Sweden; 2 Pharmaceut. Inst. of Bonn, Univ. of 

Bonn, Bonn, Germany 

Abstract: Dopamine replacement therapy is effective in the treatment of early motor symptoms 

of Parkinson‟s disease (PD); however, its association with the development of disabling motor 

complications limit the usefulness in later stages and suggests that non-dopaminergic therapies 

capable of positively modulating the effects of L-DOPA represent a valuable alternative. Based 

on the known antagonistic A2A/D2 receptor interactions, development of specific A2A 

antagonists were initiated to effectively target A2A/D2 heteromers in the dorsal striato-pallidal 

GABA pathway as a potential PD treatment. A2A antagonists act by enhancing D2 signaling 

which leads to a reduction in the activity of the striato-pallidal pathway and allow the motor 

drive to be restored. Our hypothesis is that treatment with an A2A antagonist as a monotherapy 

will only have modest effects in late-stage PD, while exhibiting beneficial anti-parkinsonian 

effects in early stages. To test this hypothesis, acute studies were performed using MSX-3, a prodrug 

of the highly specific A2A antagonist MSX-2, in the recently characterized MitoPark 

mouse model of progressive Parkinson‟s disease. MSX-2 was found to effectively bind to murine 

A2A receptors in striatal membrane preparations from both wild-type and MitoPark animals, and 

no differences were found between A2A receptor agonist binding characteristics. In both young 

and old wild-type mice, MSX-3 treatment was found to significantly increase locomotor activity 

in a dose-dependent manner. Young MitoPark mice were found to respond similarly to MSX-3 

treatment as wild-type animals, whereas aged MitoPark mice failed to respond to higher dosages 

of MSX-3. Additionally, treatment of the aged MitoPark mice with a low threshold dose of L- 

DOPA in combination with a low dose of MSX-3 induced a robust increase in locomotor 

activity, to which the animals did not become sensitized. To determine whether stereotypic 

behavior could develop after prolonged treatment, aged MitoPark mice were repeatedly injected 

with either the low dose combination of L-DOPA and MSX-3 or with a high dose of L-DOPA 

alone. Animals treated with L-DOPA as mono-therapy developed stereotypic behaviors after a 

few days while those treated with the combination therapy did not. Our results indicate that the 

A2A antagonist could be tested in PD patients as mono-therapy in early stages of the disease in 

order to delay the use of L-DOPA and in combination with low doses of L-DOPA close to 

threshold doses at later stages of the disease. 

Disclosures: D.J. Marcellino , None; K. Fuxe, None; J. Hockemeyer, None; C. Müller, None; 

L. Olson, L.O. is co-owner of a company owning commercial rights to the MitoPark mice., E. 

Ownership Interest (stock, stock options, patent or other intellectual property); D. Galter, None. 

Poster 





Support: Supported by: University of Cincinnati Millenium Fund (TJC). 

Title: Chronic antidepressant treatment is neuroprotective in a rat model of Parkinson's disease 

Authors: *K. L. PAUMIER, C. E. SORTWELL, B. F. DALEY, B. T. TERPSTRA, L. M. 

MADHAVAN, N. D. LEVINE, A. M. HEMMERLE, J. W. DICKERSON, K. B. SEROOGY, T. 

J. COLLIER; 

Neurol., Univ. Cincinnati, Cincinnati, OH 

Abstract: There is considerable evidence that Parkinson‟s disease (PD) results from a 

combination of genetic determinants, environmental factors and a failure of 

neuroprotective/compensatory mechanisms. While current pharmacological therapies aim to 

restore dopamine (DA) tone in the nigrostriatal system, they do not address the progression of 

the disease. Therefore, a neuroprotective therapy that prevents patients from reaching the 

advanced stages of the disease may provide a more thorough approach to treatment for PD. It has 

recently been demonstrated that administration of antidepressants such as tricyclics and selective 

serotonin reuptake inhibitors modulate the signaling pathways involved in cell survival and 

plasticity. In vitro and in vivo data provide direct evidence that antidepressants increase trophic 

factors such as glial cell-line derived neurotrophic factor (GDNF) (Hisaoka et al., 2001; Mercier 

et al., 2004) and brain derived neurotrophic factor (BDNF) (Chen et al., 2001; Rogoz and 

Legutko, 2005) which are known to attenuate the lesion-induced loss of DAergic neurons in 

animal models of PD (Klein et al., 1999; Kirik et al., 2000). This suggests that antidepressant 

treatment may prevent cell degeneration in PD via upregulation of trophic factors relevant to 

nigrostriatal DA neuron viability. The current study investigated whether chronic antidepressant 

treatment increases endogenous trophic factor levels in the nigrostriatal system, thereby creating 

a neurotrophic-enriched environment that will ultimately protect DA neurons from toxic insult in 

a rodent model of PD. Adult male Wistar rats received either daily or once per week injections of 

low dose amitriptyline hydrochloride (AMI; 5mg/kg.) or saline for a period of six weeks, 

beginning two weeks prior to unilateral 6-OHDA infusion into the striatum. Four weeks postlesion 

animals were sacrificed and processed for immunohistochemistry. THir neurons were 

counted and analyzed using unbiased stereology. Estimated total population counts revealed a 

significant sparing (p=0.01) of THir neurons in the SN of rats that received chronic AMI 

treatment compared to saline control animals. There was some sparing in animals that received 

weekly AMI administration, but it did not reach significance. This study provides evidence that 

tricyclic antidepressants provide neuroprotection to nigral DA neurons when administered daily. 

Although preliminary, these results highlight the ability of non-dopaminergic drugs, such as 

antidepressants, to induce endogenous changes within the basal ganglia that may protect dying or 

injured DA neurons. 

Disclosures: K.L. Paumier, None; C.E. Sortwell, None; B.F. Daley, None; B.T. Terpstra, 

None; L.M. Madhavan, None; N.D. Levine, None; A.M. Hemmerle, None; J.W. Dickerson, 

None; K.B. Seroogy, None; T.J. Collier, None.

Poster 





Support: KRF-2005-500-J00702 

Kyung Hee University 

Title: Preventive effects of CAA on 6-hydroxydopamine-induced dopaminergic dysfunction in 

mice 

Authors: *L. JIN 1,2,3 , H. KU 4 , Y. CHOI 6,5 , M. JEONG 6,5 , Y. KIM 7 , Y. SON 8 , J. LEE 9 , S. 

LIM 10,11,12 ; 

2 Dept. of Meridian and Acupuncture, Col. of Basic Oriental Medicinal Sci., 3 Dept. of Applied 

Korean Med., 4 Dept. of Applied Korean Medicine, Grad. School, Kyung Hee Univ., 5 WHO 

Collaborating Ctr. for Traditional Medicine, East-West Med. Res. Inst., 1 Kyung Hee Univ., 

Seoul, Republic of Korea; 6 Dept. of Meridian and Acupuncture, Col. of Basic Oriental Medicinal 

Science, Kyung Hee Univ., Seoul, Republic of Korea; 7 Dept. of Pharmacology, Col. of 

Medicine, Seoul Natl. Univ., Seoul, Republic of Korea; 8 Dept. of Genet. Engineering, Col. of 

Life Science, Kyung Hee University,, Youngin, Republic of Korea; 9 Dept. of Herbology, Col. of 

Korean Medicine, Dongguk University, Seokjang-dong, Gyeongju, Gyeongju, Republic of 

Korea; 10 WHO Collaborating Ctr. for Traditional Medicine, East-West Med. Res. Institute, and 

Depar, kyung Univ., Seoul, Republic of Korea; 11 Dept. of Meridian and Acupuncture, Col. of 

Basic Oriental Medicinal Sci., Kung Hee Univ., Seoul, Republic of Korea; 12 Dept. of Applied 

Korean Med., kyung Hee Univ., Seoul, Republic of Korea 

Abstract: This study was conducted to evaluate the effects of a combined extract of CAA on 

dopaminergic dysfunction caused by 6-hydroxydopamine (6-OHDA) in mice. CAA is a 

combined extract of Cinnamomi Ramulus, Anemarrhenae Rhizoma, and Alpiniae Officinari 

Rhizoma. Levels of dopamine and its metabolites (3,4-dihydroxyphenylacetic acid (DOPAC) and 

homovanilic acid (HVA)) in the striatum were markedly reduced respectively, compared to the 

control group seven days after 6-OHDA injection. However, CAA administration increased 

significantly striatal dopamine and its metabolites in a dose-dependent manner. Tyrosine 

hydroxylase (TH) and dopamine transporter (DAT) expression was reduced by 6-OHDA 

injection, but increased significantly by CAA administration. Such results suggest that CAA 

could prevent the dopaminergic dysfunction caused by 6-OHDA in mice and may be useful in 

preventing and treating Parkinson‟s disease (PD). Because our study has not dealt with CAA‟s

neuroprotective mechanism, further research is needed to study mechanism of CAA‟s 

neuroprotective action on dopaminergic cells. 

Disclosures: L. Jin, None; H. Ku, None; Y. Choi, None; M. Jeong, None; Y. Kim, None; Y. 

Son, None; J. Lee, None; S. Lim, None. 

Poster 





Support: Antonio Mendoza-Camargo of the CIR-UADY (Assistance) 

CONACYT grant 47763 to JLGA 

UADY grant PRIORI-06-011 to JLBG 

Title: Intrastriatal implants of nanostructured silicon dioxide reservoirs impregnated with 

dopamine restore postural adjustment in hemiparkinsonian rats 

Authors: *F. ALVAREZ-CERVERA 1 , A. S. CASTILLA-TIRADO 1 , J. L. BATA-GARCIA 1 , 

T. LOPEZ-GOERNE 2 , F. J. HEREDIA-LOPEZ 1 , P. QUINTANA-OWEN 3 , D. M. ESQUIVEL- 

GOMEZ 4 , G. ARANKOWSKY-SANDOVAL 1 , J. L. GONGORA-ALFARO 1 ; 

1 Neurociencias, Univ. Autonoma de Yucatan, Merida, Mexico; 2 Unidad Iztapalapa, Univ. 

Autonoma Metropolitana, Mexico, DF, Mexico; 3 Unidad Merida, CINVESTAV, Merida, 

Mexico; 4 Univ. de Guanajuato, Guanajuato, Mexico 

Abstract: Parkinson‟s disease is a chronic disorder characterized by akinesia and postural 

instability. Although the dopamine (DA) precursor levodopa is the most effective pharmacologic 

treatment, during chronic administration its efficacy is hampered by the development of 

dyskinesias and episodic motor freezing. These motor fluctuations have been ascribed to the 

oscillatory concentrations of DA occurring in all brain nuclei during intermittent oral levodopa 

administration. Theoretically, this drawback could be overcome by placing a constant DArelease 

device in the neostriatum. In the present study the therapeutic effect of an intrastratial 

implant consisting of a nanostructured silicon dioxide cylinder impregnated with dopamine 

(SiO2-DA) was evaluated in hemiparkinsonian rats. When rats were manually displaced over a 

smooth surface, they showed a significant reduction in the number of adjusting steps performed 

with the forepaw on the contralateral side to the SNc lesion. This motor impairment was fully

everted following striatal embedding of the SiO2-DA reservoir. Notably, the effect was 

maintained up to 113 days after implantation without the development of dyskinesias. Rats that 

did not receive an implant or that were implanted with an empty dioxide silicon reservoir did not 

show any recovery during the study, suggesting that the therapeutic effect on the postural 

adjustment observed in the hemiparkinsonian rats was due to the continuous dopamine release 

from the SiO2-DA reservoir. 

Disclosures: F. Alvarez-Cervera, None; A.S. Castilla-Tirado, None; J.L. Bata-Garcia, 

None; J.L. Gongora-Alfaro, None; G. Arankowsky-Sandoval, None; F.J. Heredia-Lopez, 

None; T. Lopez-Goerne, None; P. Quintana-Owen, None; D.M. Esquivel-Gomez, None. 

Poster 





Support: The Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea 

government(MEST) (R11-2005-014) 

The Second Stage of Brain Korea 21 project in 2008 

Title: The 2-DE analysis of the neuroprotective effect of electroacupuncture in the MPTPinjected 

mouse striatum 

Authors: *S.-T. KIM 1,2 , H.-J. YOO 2 , S.-N. KIM 1 , C. YIN 2 , W. MOON 2 , H.-J. LEE 2 , H.-J. 

PARK 1 ; 

1 Col. of Oriental Med., Kyung Hee Univ., Seoul, Republic of Korea; 2 Acupuncture & Meridian 

Sci. Res. Center, KyungHee Univ., Seoul, Republic of Korea 

Abstract: Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in 

substantia nigra (SN), and dopaminergic neuronal fibers in striatum (ST) were also destructed. 

But the pathogenic mechanism remains still unknown. 

Using a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, we 

investigated whether electroacupuncture normalizes the proteomic changes by MPTP in the mice 

ST. 

C57BL/6 mice were injected i.p. with 30 mg/kg of MPTP at 24 h interval for 5 days, and 100 Hz 

EA treatment was performed at GB34 and GB39 once a day for 12 days consecutively from the 

first injection. Then we performed tyrosine hydroxylase (TH)-immunohistochemistry in the ST

and two-dimensional electrophoretic (2-DE) separations of proteins extracted from ST tissues 

from saline, MPTP and MPTP+EA treated mice. Protein profiles of control and treated tissues 

were compare by the PDQuest 2D-gel analysis software, and the separated proteins were 

analyzed by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry. 

2-DE separation showed that MPTP upregulated three proteins such as cytosolic malate 

dehydrogenase, Munc18-1 and hydroxyacyl glutathione hydrolase, and downregulated a novel 

protein similar to cytochrome c oxidase subunit Vb in ST. With EA treatment, this MPTPinduced 

proteomic changes were reduced. The proteins were related to mitochondrial 

metabolism or synaptic vesicle secretion. 

100 Hz EA at GB34 reduced the destruction of dopaminergic neuronal fibers, and normalized the 

proteomic changes by MPTP in the mouse striatum. These EA effects may be due to normalizing 

mitochondrial metabolism and synaptic vesicle secretion. 

Disclosures: S. Kim, This work was supported by the Korea Science and Engineering 

Foundation (KOSEF) grant funded by the Korea government(MEST) (R11-2005-014), B. 


grants already received); H. Yoo, None; S. Kim, None; C. Yin, None; H. Lee, None; H. Park, 

None; W. Moon, None. 

Poster 





Support: Chicago Institute of Neurosurgery and Neuroresearch (CINN) 2007-2008 

Title: Therapeutic use of neural induced human embryonic stem cells (hESC) in Parkinson's 

disease 

Authors: L. P. KELLY 1 , M. B. NEWMAN 1 , A. P. SMITH 2 , *R. A. BAKAY 3 ; 

1 Neurosurg. & Pharmacol., 2 Neurosurg., 3 Neurol Surgery, Rush Univ. Med. Ctr., Chicago, IL 

Abstract: Parkinson‟s disease (PD) is recognized as an idiopathic neurodegenerative disorder in 

which the striatum becomes deficient in dopamine (DA) secondary to the degeneration of DAproducing 

neurons in the substantia nigra pars compacta (SNpc). Current therapeutic options 

only alleviate symptoms without repairing or halting disease progression. Cell transplantation, 

especially human stem cells, used to treat various CNS diseases is of interest because this area of 

research has the greatest potential to provide a cure. The objective of the current study is to

determine the viability and potential for human embryonic stem cells (HESC), when induced 

towards a DA phenotype in culture or not, to repair, replace, or regenerate the loss of DA in the 

striatum in rats pre-treated with a DA neurotoxin. We are therefore aiming to inhibit the 

progression of PD and not to treat the symptoms. The HESC cells were cultured in serum-free 

defined expansion media and in serum free defined neural induction media 7 to 21 days prior to 

transplantation. After culturing, the HESCs were assessed for the number of tyrosine 

hydroxylase (THIR) immunoreactive cells to establish the cell pre-direction towards a DA 

phenotype. Male Spragey-Dawley rats were either lesioned with 6-OHDA (DA neurotoxin) at 

two sites in the medial forebrain bundle (MFB), or were sham lesioned. The lesioned and sham 

rats were tested for rotational behavior 1-2 days before surgery (baseline) and again 1-2 days 

before cell transplantation for inclusion in the study. Animals not meeting the rotational criteria 

after 6-OHDA treatment were excluded from the study. In this model of PD, the rats will have 

lost at least 60% of DA neurons in the SNpc (~80% of DA levels in the striatum) and will show 

full PD-like motor deficits by 3 weeks post surgery. Unilateral transplantations were performed 

into the striatum with either (1) untreated HESCs, (2) neuronal induced HESCs or (3) no cells, 

with 7 rats per group. After transplantation, behavioral testing was performed every other week 

until brains were taken and processed for dependent measures. These included the number of 

human grafted cells, cell counts for DA neurons in substantia nigra, other human neural markers, 

and observation of human cells in the peripheral blood, bone marrow, and spleen in addition to 

the target area of the brain. The results thus far have indicated modest behavioral improvements, 

with 3 different motor behavioral tests, and in vitro immunocytochemistry has shown these stem 

cells can be induced to DA phenotype (35 to 40% of cells at 4 DIV) along with all neural lineage 

markers. Completed results will be presented. 

Disclosures: L.P. Kelly, None; M.B. Newman, None; A.P. Smith, None; R.A. Bakay , None. 

Poster 





Support: Chicago Institute of Neurosurgery and Neuroresearch (CINN) 2007-2008 

Title: Therapeutic use of neural induced human umbilical cord blood (hUCB) stem cells in 

Parkinson‟s disease 

Authors: *M. B. NEWMAN 1 , L. P. KELLY 1 , A. P. SMITH 2 , R. A. E. BAKAY 1 ; 

1 Depts.NeuroSurg & Pharmacol, 2 NeuroSurgery, Rush Univ. Med. Ctr., Chicago, IL

Abstract: The present study was designed to determine the potential for hUCB stem cells, the 

CD133 subset of the hematopoietic mononuclear fraction, to repair, replace, or induce 

regeneration of the damaged tissue, loss of dopamine (DA) in the striatum, or the loss of 

dopaminergic neuron in adult rats lesioned with the DA neurotoxin, 6-hydroxydopamine, which 

model‟s Parkinson‟s disease (PD). Presently, one published study investigated the transplantation 

of hUCB cells into an animal model of PD, and showed the lifespan of PD mice were extended 

compared to PD mice receiving congenic bone marrow cells or no treatment. The mononuclear 

fraction of hUCB cells are well-established and a reliable source cells that are known to be rich 

in hematopoietic stem cells, and have been used successfully for over 15 year to treat children 

with malignant and non-malignant diseases. 

In this study, the CD133 stem cells were first cultured in proliferation media [Stemline 

Hematopoietic media (Sigma)] treated with the human recombinant proteins stem cell factor, 

thrombooietin, and interleukine-3 for 5 to 10 days in vitro (DIV). The media was either changed 

to neural induction media consisting of the Stemline media with never growth factor and all-trans 

retinoic acid for 7 to 21 DIV or the Stemline media without the neural induction factors. The 

CD133 stem cells formed neurospheres with cells differentiating by 5 DIV as they migrated 

away from the aggregate. Therefore, CD133 neural induced and non-induce cells were 

transplanted at day 4 in culture, which is before they fully differentiation and are still consider 

neural stem cells. A total of 1 X 10 6 cells, were transplanted into the striatum of rats 4 weeks 

after receiving 6-OHDA in middle forebrain bundle at two lesion sites. Behavioral testing was 

preformed pre-lesion, post-lesion, and post-cell transplant. Rats were sacrificed 2 months after 

cell transplants. 

Cell culture results showed CD133 cells, cultured in neural induction media, were immunopositive 

for all 3 neural lineages, with an average of 45 to 50% of cells labeling positive for DA 

and Nurr1 early during the neural induction process (4 to 7 DIV), with 40 to 45% of cells 

labeling positive for DA after 7 to 21 DIV, and a decrease in the number of Nurr1 positive cells 

after 7 DIV. Several other neuronal markers were also examined in vitro and in vivo. The 

behavioral tests have shown behavioral improvements in the 6-OHDA lesioned/CD133 

transplanted rats. All of the behavioral tests and the other dependent measures which include DA 

cell counts in the striatum and substantia nigra, human specific marker cell counts in several 

areas of the brain, the peripheral blood, bone marrow, and spleen will be presented. 

Disclosures: M.B. Newman, None; L.P. Kelly, None; A.P. Smith, None; R.A.E. Bakay, None. 

Poster 




Topic: C.02.d. Therapies

Title: SKA-PD-01 attenuates behavioral impairments and neurotoxicities in MPTP- and 6- 

OHDA-induced animal and cellular models of Parkinson‟s disease 

Authors: J. LEE, J. CHUNG, C. RYU, M. JI, H. MIN, Y. YOON, M. LIM, *C. PARK; 

Drug Dev Ctr., SK Corp., Daejeon, Republic of Korea 

Abstract: Parkinson‟s disease (PD) is a progressive neurodegenerative disorder characterized by 

motor abnormalities including resting tremor, rigidity, postural instability and bradykinesia. The 

neuropathological feature is the selective loss of the nigrostiatal dopaminergic neurons within the 

substantia nigra pars compacta (SNpc) and nerve terminals extended to the striatum. Recent 

study suggests that inhibition of monoamine oxidase B (MAO-B) partially contributes to the 

improvement of behavioral disturbances and prevention of neuronal loss by decreasing dopamine 

metabolism and oxidative stress. 

We investigated the anti-PD and neuroprotective efficacies of a new compound, SKA-PD-01, in 

the MPTP and 6-hydroxydopamine (6-OHDA)-induced in vitro and in vivo models of PD. SKA- 

PD-01 efficiently inhibited MAO-B activities with high selectivity (IC50 of MAO-A and MAO-B 

is ~100 uM and 0.011 uM, respectively). Administration of SKA-PD-01 (20 mg/kg, ip) at 30 min 

before and after MPTP treatment significantly ameliorated behavioral impairments, neuronal loss 

in the SNpc, and dopamine depletion in the striatum of mice. In 6-OHDA-induced PD animal 

models, dopaminergic activity by L-dopa was considerably increased by SKA-PD-1 treatment 

(20 mg/kg, ip). MPP + or 6-OHDA-induced neuronal loss was attenuated by SKA-PD-1 treatment 

in SH-SY5Y and C57BL6 mice. This compound also significantly reversed reserpine-induced 

akinesia in reserpine akinesia model (140.1%* at 20mg/kg, ip), and reduced haloperidol-induced 

catalepsy (ED50 was 3.55 mg/kg, ip and 4.54mg/kg, po). 

In rat pharmacokinetic study, bioavailability was 21.1% and half-life was 0.6 hour. A single 

neurotoxic dose was 210 mg/kg,ip in rotor rod test. Taken all together, our findings indicate that 

SKA-PD-01 as a new selective MAO-B inhibitor and neuroprotectant might have therapeutic 

potential for other neurodegenerative diseases as well as PD. 

Disclosures: J. Lee, None; J. Chung, None; C. Ryu, None; M. Ji, None; H. Min, None; Y. 

Yoon, None; M. Lim, None; C. Park , None. 

Poster 





Support: SFI Grant 05/RF/ENM047

Title: Effect of tissue capacitance and dispersion in an inhomogeneous model of deep brain 

stimulation 

Authors: *P. F. GRANT, M. M. LOWERY; 

Univ. Col. Dublin, Dublin 4, Ireland 

Abstract: Deep Brain Stimulation (DBS) has been shown to be highly effective for suppressing 

the symptoms of Parkinson's Disease. Despite its widespread use, the mechanisms of action of 

DBS remain unknown, and the region of tissue activated is unclear. In this study, a threedimensional, 

inhomogeneous, finite element model of the head was constructed to examine the 

time-dependent distribution of the electric field in the vicinity of the subthalamic nucleus during 

DBS. Typical DBS stimuli, approximating square waves, carry energy through a wide range of 

frequency components. Many biological tissues have frequency-dependent conductivities and 

permittivities, caused by alpha dispersions in water molecules. For example, the relative 

permittivity of white matter at 100 Hz is over twenty times that reported in the literature at 1 

kHz. It is likely that the broad spectral nature of the stimulus pulse, coupled with the frequency 

dependent material properties will have a significant effect on the magnitude of the electric field 

in the region of neural tissue close to the electrode. Most bioelectric models of DBS to date have 

assumed that tissues are purely resistive. To properly quantify the capacitive effects in the tissue, 

it is necessary to incorporate the effect of dispersion. In this study a new model has been 

developed to examine the effect of both tissue capacitance and dispersion over a wide range of 

frequencies. Using this model, the electric potentials in the region of neural tissue close to the 

stimulating electrode were calculated for varying capacitive and dispersive material properties. 

The electric potential observed in the region of neural tissue close to the electrode during 

current-controlled stimulation was found to be sensitive to variations in the frequency at which 

material properties were estimated. The peak magnitude of the electric potential at a point 5 mm 

posterior to the stimulating electrode where material properties were estimated at 1 kHz, was 

over 2.5 times that observed when material properties were estimated at 100 Hz. Furthermore, 

results indicate a further effect on both the magnitude and shape of the electric potential 

observed when dispersive material properties are incorporated. It is, therefore, concluded that 

inclusion of the frequency-dependent material properties is necessary for accurate computation 

of the temporal electric field in the vicinity of DBS electrodes. 

Disclosures: P.F. Grant, None; M.M. Lowery, None. 

Poster 



Program#/Poster#: 247.15/S10


Support: Michael J, Fox Foundation for Parkinson‟s disease 

NCRR Grant P51 RR000167 

Kinetics Foundation 

Title: Neuroanatomical effects of chronic oral administration of pioglitazone to MPTP-treated 

monkeys 

Authors: C. SWANSON 1,2 , J. MOIRANO 3 , V. JOERS 1 , K. BRUNNER 1 , H. SIMMONS 1 , J. 

RASCHKE 1 , V. BONDARENKO 1 , M. VARGUS 4 , D. JOHNSON 4 , J. KEMNITZ 1,2 , J. 

JOHNSON 2,4 , *M. E. EMBORG 1,2,3 ; 

1 Wisconsin Natl. Primate Res. Ctr., Univ. Wisconsin, Madison, WI; 2 Neurosci. Training 

Program, 3 Med. Physics, 4 Sch. of Pharm., Univ. of Wisconsin, Madison, WI 

Abstract: Pioglitazone is a thiazoledinedione, currently used as an anti-diabetic drug due to its 

ability to stimulate insulin-mediated glucose transport and metabolism. It targets the peroxisome 

proliferators-activated receptor gamma and has anti-inflammatory properties. Neurosphere 

cultures derived from ARE-hPAP reporter mouse exposed to pioglitazone show a dosedependent 

increase in ARE activity, suggesting activation of the Nrf2 anti-oxidative pathway. In 

MPTP mice models of PD, oral administration of pioglitazone prevents dopaminergic nigral cell 

loss. Based on this information we examined the neuroanatomical effects of chronic oral 

administration of pioglitazone to parkinsonian monkeys. Sixteen male rhesus monkeys (Macaca 

mulatta), age 5-7 years old, received one single intracarotid injection of MPTP, Twenty- four 

hours later therapy treatment was initiated after they were assessed using a clinical rating scale, 

matched and assigned to one of three groups: placebo (n=5), 15 mg (n=6), 30 mg (n=5). Three 

months after daily oral treatment the animals were necropsied. Our results revealed a modest but 

significant protection of the nigrostriatal pathway measured as a significantly higher tyrosine 

hydroxylase (TH) putaminal optical density (p= 0.0003) and significantly higher stereological 

cell counts of TH (p= 0.02) and VMAT2 (p= 0.039, positive nigral neurons in the pioglitazonetreated 

monkeys compared to control. We also found modulation of microglia activation 

observed as significantly less CD68 immunostaining in animals treated with pioglitazone 30 mg 

compared to placebo (p= 0.047). There was also an increase in GFAP positive cells in the 

substantia nigra ipsilateral to MPTP administration compared to the contralateral side in all 

monkeys, yet comparison between groups did not reach statistical significance. Morphological 

and behavioral datasets showed significant correlations. For example, better clinical performance 

was associated with more TH positive nigral cells (p = 0.003) and less CD68 immunostaining 

(p= 0.002). Our results strongly suggest that oral administration of pioglitazone has protective 

effects against the functional and anatomical consequences of MPTP administration and could be 

a potential therapy in neurodegenerative disorders such as Parkinson‟s disease. 

Disclosures: C. Swanson, None; M.E. Emborg, None; J. Moirano, None; J. Raschke, 

None; V. Bondarenko, None; V. Joers, None; K. Brunner, None; H. Simmons, None; M. 

Vargus, None; D. Johnson, None; J. Kemnitz, None; J. Johnson, None.

Poster 





Title: Effect of T-817MA on 1-Methyl-4-phenylpyridinium (MPP+) and Amyloid β (Aβ) 

induced dying-back degeneration in primary culture of rat cortical neurons 

Authors: *K. HIRATA 1,2 , M. SUGIMORI 1 , R. LLINAS 1 ; 

1 Dept Physiol Neurosci, New York Univ. Med. Cntr, New York, NY; 2 Res. Labs., Toyama 

Chem. Co., Ltd., Toyama, Japan 

Abstract: Fast axonal transport (FAT) defects are present in a variety of neurodegenerative 

diseases, probably associated with a lost of synaptic function characteristic of a “dying-back” 

neuropathy. We previously demonstrated that 1-Methyl-4-phenylpyridinium (MPP + ) and 

amyloid-β peptide 1-42 (Aβ) impaired mitochondria FAT in primary culture of rat cortex neurons. 

Here we demonstrate that bath application of either MPP + or Aβ induce dying back degeneration 

in rat cortical primary culture. We further studied whether such “dying back” event could be 

prevented by T-817MA (1-{3-[2-(1-Benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a 

pharmacological agent developed specifically for neurodegenerative disorders such as 

Alzheimer‟s disease. Primary cortical cell cultures were prepared from E17 Sprague-Dauley rat. 

At 7-10 days of culture, cells were exposed to 30 κM MPP + or 10 κM Aβ, both of which can 

cause mitochondria FAT impairment. Dying back phenomena was determined optically with a 

Olympus IX-71 microscope and MagnaFire digital CCD camera, using three criteria: (1) 

Morphological observation under bright field microscopy. (2) Fluorescence imaging (FITC) with 

conjugated Annexin V, which binds specifically to phosphatidylserine translocated to the outer 

plasma membrane during apoptosis. (3) Imaging active synaptic terminals based on FM1-43 

endocytosis . MPP + treatment at 30κM for 24 hr induced mitochondria FAT impairment with no 

morphological changes. On the other hand, longer treatment (more than 3 days) of MPP + induced 

membrane beading and shrinking in distant neurites. Annexin V-FITC binding was identified on 

neurite beads and blebs in MPP + treated cultures with little binding on neuronal cell somata . In 

these terminals synaptic activity, judged by the endocytosis of FM1-43, was reduced. These 

results indicate that MPP + can induce dying back phenomena at the same concentration that 

induced mitochondria FAT impairment. Aβ, which has been known to induce dying back 

degeneration, also induced similar dying back changes as seen in MPP + treatment in our culture. 

Simultaneous application of 1 κM of T-817MA with MPP + or Aβ prevented the dying back 

phenomena induced by the neurotoxins. This result indicates that T-817MA can rescue dying 

back degeneration in the neurons and might have benefit for neurodegenerative disease

Disclosures: K. Hirata, Toyama Chemical Co., Ltd., A. Employment (full or part-time); M. 

Sugimori, None; R. Llinas, None. 

Poster 





Title: Effects of T-817MA on the nigrostriatal dopaminergic systems and spatial motor learning 

in MPTP-treaded mice 

Authors: *T. KIMURA 1,2,3 , A. H. TRAN 2,3 , H. NISHIJO 2,3 , T. ONO 2,3 ; 

1 2nd Res. Dept., Toyama Chem. Co, Ltd, Toyama, Japan; 2 Syst. Emotional Sci., Univ. of 

Toyama, Toyama, Japan; 3 Crest, JST, Tokyo, Japan 

Abstract: T-817MA is a newly synthesized agent with neuroprotective action against toxicity 

and neurite outgrowth promoting action in in vitro study for treatment of neurodegenerative 

disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), etc. Furthermore, systemic 

administration of T-817MA displayed neurogenetic action in in vivo AD model using amyloid-ß 

peptide-infused rats, and ameliorated cognitive dysfunctions in this model. The neurotoxin 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine 

(MPTP) has been used to produce an animal model 

of PD in mice by decreasing brain dopaminergic neurons. 

In the present study, we investigated a role of dopaminergic neurons in locomotor activity and 

spatial motor learning using MPTP-treated mice, and effects of T-817MA (30 mg/kg, p.o.) on 

motor and cognitive disturbances in MPTP-treated mice. Three days after MPTP treatment (10 

mg/kg, s.c. x 4 with 2-h intervals), the mice were tested with intracranial self-stimulation (ICSS) 

behavior, spontaneous locomotor activity in an open field, and spatial motor learning task in 

which the mice were required to alternatively visit two reward places in an open field to obtain 

ICSS as rewards. 

The results indicated that MPTP (1) did not apparently change ICSS behavior, (2) tended to 

decrease spontaneous locomotor activity, (3) raised locomotor activity during the spatial motor 

learning task, (4) disturbed learning of the spatial motor learning task, and (5) reduced THimmunoreactivity 

in the substantia nigra, but not in the ventral tegmental area. On the other 

hand, T-817MA, against MPTP toxicity, (1) reduced hyper-locomotor activity during the spatial 

motor learning task, (2) significantly improved learning disturbance in the spatial motor learning 

task, and (3) prevented a decrease in TH-immunoreactivity in the substantia nigra. 

These results indicated that MPTP incompletely impaired the nigrostriatal dopaminergic pathway 

rather than the mesolimbic dopaminergic pathway, and consequently suggest that it induced

hyperactivity during the task and failure of spatial motor learning. The results further suggest 

that T-817MA is useful for treatment of neurodegenerative disorders of dopaminergic neurons 

exhibiting psychiatric symptoms and/or cognitive dysfunctions. 

Disclosures: T. Kimura, None; A.H. Tran, None; H. Nishijo, None; T. Ono, None. 

Poster 



Program#/Poster#: 247.18/T1 


Support: NIH Grant K08NS060948 

American Parkinson Disease Association Cotzias Memorial Fellowship 

Title: Differential neuroprotective effects of 14-3-3 proteins in PD models 

Authors: *T. A. YACOUBIAN 1 , S. R. SLONE 1 , S. HAMAMICHI 2 , A. J. HARRINGTON 2 , K. 

A. CALDWELL 2 , G. A. CALDWELL 2 , D. G. STANDAERT 1 ; 

1 Dept Neurol, Univ. Alabama Birmingham, Birmingham, AL; 2 Dept Biol. Sci., Univ. Alabama, 

Tuscaloosa, AL 

Abstract: Alpha-synuclein (α-syn) plays a central role in Parkinson‟s disease, yet its mechanism 

of toxicity to dopaminergic neurons is poorly understood. We have previously shown that three 

14-3-3 isoforms - 14-3-3ε, γ, and ζ - are significantly downregulated in the cortex of transgenic 

mice overexpression human α-syn under the PDGF promoter. These same three isoforms have 

been shown to reduce α-syn aggregation in cell-based model of α-syn aggregation. Because the 

14-3-3s negatively regulate apoptotic signaling pathways, we hypothesized that overexpression 

of 14-3-3 proteins could be neuroprotective in PD. To address this question, we used the 

dopaminergic cell line SK-N-BE(2)-M17 to create seven stable cell lines that overexpress each 

of the 14-3-3 isoforms, and tested each line for their resistance to the neurotoxins rotenone and 

MPP+. Cells were treated with rotenone at concentrations ranging from 0.04 to 25 κM for 48 

hours, and cell death was assessed by LDH release. For the MPP+ experiments, cells were 

treated with 2 to 10 mM MPP+ for 24 hours. We found that those cell lines overexpressing either 

14-3-3ε, γ, or ζ were resistant to toxicity in response to both toxins. Cell death in response to 

1κM rotenone in the 14-3-3ζ was reduced to 45% of that in control stable lines transfected with 

the empty vector. Similarly, rotenone-induced cell death was reduced to 65% and to 55% of 

control levels in the 14-3-3ε and 14-3-3γ lines, respectively. MPP+-induced death (5mM) was

educed to 60%, 76%, and 56% of control levels in the 14-3-3ζ, 14-3-3ε, and 14-3-3γ lines, 

respectively. Cells overexpressing 14-3-3β or 14-3-3δ were only mildly resistant to similar doses 

of rotenone and did not show a significant reduction of MPP+-induced cell death as compared to 

control. Cell lines overexpressing 14-3-3ε or 14-3-3ζ showed no significant reduction of cell 

death in response to rotenone or MPP+ but instead showed a trend of increased toxicity. 

We are currently evaluating whether 14-3-3ε, γ, or ζ can reduce the dopaminergic cell loss seen 

in transgenic C. elegans that overexpress α-syn. Preliminary results of transgenic worms 

overexpressing 14-3-3ζ show a reduction in dopamine cell loss. Our findings suggest that certain 

14-3-3 proteins may serve as potential targets for neuroprotective therapy in PD. 

Disclosures: T.A. Yacoubian , None; S.R. Slone, None; S. Hamamichi, None; A.J. 

Harrington, None; K.A. Caldwell, None; G.A. Caldwell, None; D.G. Standaert, None. 

Poster 





Support: PDF 

NS26836 

DAMD17-03-1-0492 

NS38370 

Title: Restoration of the nigrostriatal dopaminergic pathway in mouse models of Parkinson‟s 

disease: effects of a constitutively active form of AKT 

Authors: *X. CHEN 1 , T. KAREVA 1 , M. RZHETSKAYA 1 , C. WANG 3 , M. DURING 3 , N. 

KHOLODILOV 1 , R. E. BURKE 1,2 ; 

1 Dept Neurol, 2 Pathology and Cell Biol., Columbia Univ., New York, NY; 3 Human Cancer 

Genet. Program, The Ohio State Univ., Columbus, OH 

Abstract: Restoration of the nigrostriatal dopaminergic projection and reinstatement of 

dopamine function presents one of the greatest challenges for the development of new 

therapeutic strategies for Parkinson‟s disease (PD). We have previously reported that 

myristoylated Akt (Myr-Akt), a constitutively active form of Akt, has striking neuroprotective

effects on dopaminergic neurons in murine models of PD, not only when it is administered 

before neurotoxin, but also when it is administered 3 weeks after, a paradigm that better 

simulates the clinical context. To further characterize the restorative effects of Myr-Akt, we 

injected mice with 10µg 6-OHDA unilaterally into the striatum. Pronounced (approximately 50- 

60%) and persistent losses of tyrosine hydroxylase (TH) positive nigral neurons were observed at 

all time points over a 12-week period post 6-OHDA lesion. The losses of dopaminergic cell 

bodies in the substantia nigra (SN) were accompanied by comparable and persistent decreases in 

axonal projections of the pathway, as determined by TH immunostaining of the medial forebrain 

bundle (MFB) and the striatum on horizontal sections. Constitutive activation of Akt was 

achieved by AAV-mediated gene delivery of Myr-Akt into the SN 3 weeks after 6-OHDA 

lesion. Three weeks after the AAV injection (6 weeks post lesion), Myr-Akt began to produce 

remarkable neurorestorative effects on the entire nigrostriatal pathway. TH positive neurons 

demonstrated increased size and staining intensity. The restorative effects of Myr-Akt extended 

to the axonal projections of nigrostriatal dopaminergic neurons. More TH reactive fibers were 

present in the MFB, and they were more intensely labeled with TH, as compared to YFP injected 

controls. In the striatum, TH immunostaining revealed partial to full restoration of striatal 

dopamine innervation. The marked morphologic recovery of the nigrostriatal pathway induced 

by Myr-Akt in this partial striatal lesion was long-lasting and stable over a nine week time 

course. In future studies we will examine the generality of the restorative effects of Myr-Akt in 

alternative models, and its cellular and molecular basis. 

Disclosures: X. Chen, None; T. Kareva, None; M. Rzhetskaya, None; C. Wang, None; M. 

During, None; N. Kholodilov, None; R.E. Burke, None. 

Poster 





Support: CONACYT 

NIH 

PAPIIT (UNAM) 

Fundacion Aleman

Title: Enzymatic digestion of chondroitin sulphate proteoglycans and transplantation of ES cellderived 

dopamine neurons in the substantia nigra of hemi-parkinsonian rats 

Authors: *N. DIAZ, F. DIAZ, I. VELASCO; 

Univ. Natl. Autonoma Mexico, Inst. Fisiol Ceular, Mexico City, Mexico 

Abstract: Cell therapy in animal models of Parkinson‟s disease has been successful when 

dopaminergic cells are grafted in the striatal area. Intranigral transplantation of these cells has 

only been effective when combined with intrastriatal grafting, and the therapeutic benefit has 

been associated with striatal dopamine release. One of the major difficulties for nigral grafting 

and its association with behavioral recovery is that the adult brain is a restrictive environment for 

axonal growth due to expression of components of the extracellular matrix especially chondroitin 

sulphate proteoglycans (CSPG). In this work, we evaluated if intranigral transplantation of 

dopamine neurons differentiated from embryonic stem (ES) cells, combined with enzymatic 

digestion of CSPG with chondroitinase ABC (ChABC) might contribute to anatomical and 

functional regeneration of the nigro-striatal pathway in a rat model of the Parkinson‟s disease. 

We differentiated ES cells to dopamine neurons in vitro and grafted them in 3 experimental 

groups: 1) striatum; 2) angle injection to reach the substantia nigra; 3) intranigral transplantation 

together with ChABC injection in the striatal direction. In all animals, we performed behavioral 

tests to establish functional recovery of the experimental procedures. We found that intrastriatal 

transplantation caused recovery in pharmacological and non-pharmacological behavioral test. 

Intranigral transplantation without ChABC did not cause behavioral recovery. We observed an 

increase in the reactivity of CSPG and glial fibrillary acidic protein along the injection in the 

groups transplanted in the substantia nigra. Addition of ChABC showed partial degradation of 

proteoglycans as evidenced by the use of 2B6 antibody, which recognizes a partially degraded 

form of CSPG. The group with transplant in the substantia nigra and enzymatic degradation 

presented significant improvement in the stepping test, in contrast with the group transplanted in 

the substantia nigra without ChABC. However, the rotational test did not show recovery in the 

nigral graft plus ChABC. These results indicate that the addition of ChABC might be used to 

promote partial functional recovery in the diseased adult brain. 

Disclosures: N. Diaz , None; F. Diaz, None; I. Velasco, None. 

Poster 




Topic: C.02.d. Therapies

Support: 2005-2006 American Foundation for Pharmaceutical Education Predoctoral 

Fellowship 

NCI-NSF IGERT Nanomedicine S&T award 

Michael J. Fox Foundation for Parkinson's Research 

Northeastern University Research Scholarship Development Fund 

Bouve College Bridging Grant 

Title: Intranasal GDNF is neuroprotective in a rat model of Parkinson‟s disease 

Authors: *M. M. MIGLIORE, R. ORTIZ, R. B. CAMPBELL, M. M. AMIJI, B. L. 

WASZCZAK; 

Pharmaceut. Sci., Northeastern Univ., Boston, MA 

Abstract: Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert 

neurotrophic and neuroprotective effects on substantia nigra (SN) dopamine neurons and has 

great potential in the treatment of Parkinson‟s disease (PD). However, its inability to cross the 

blood-brain barrier has required invasive intracerebral infusions rendering GDNF administration 

too risky for widespread clinical use. The purpose of the present study was to assess the 

therapeutic efficacy of intranasally administered GDNF in a rat 6-hydroxydopamine (6-OHDA) 

model of PD. The intranasal route of administration was selected because it bypasses the bloodbrain 

barrier, avoids systemic absorption, and limits potential peripheral side effects. Rats were 

intranasally administered GDNF in either phosphate-buffered saline (PBS) or in cationic 

liposomes. A single dose of 50 µg GDNF was administered 1 hr prior to a 6-OHDA lesion, or 

three 50 µg doses were given over 3 days (1 day prior, 1 hr prior, and 1 day after the lesion). 

Rats were sacrificed 3-4 weeks later, and lesions were quantified by tyrosine hydroxylase 

immunohistochemistry. Administration of a single intranasal dose of GDNF decreased lesion 

severity in the SN from 75 ± 7% in controls to 54 ± 9% (n.s.) and 41 ± 8% (p

Poster 





Title: Evidence that practice of multi-directional stepping with auditory stimulation improves 

movement performance in patients with Parkinson‟s disease 

Authors: *Z. KADIVAR 1 , M. JOHNSON 1 , J. JONES 1 , T. BOYANCE 1 , M. DUET 1 , J. M. 

HONDZINSKI 1 , J. FOTO 2 ; 

1 Kinesiology, LSU, Baton Rouge, LA; 2 Natl. Hansen's Dis. Programs, Baton Rouge, LA 

Abstract: Parkinson‟s disease (PD) is a debilitating neurodegenerative disorder causing many 

physical limitations most of which are not alleviated by medication. External cues, including 

rhythmic auditory stimulation (RAS), have become important modalities for improving various 

aspects of movement in this population. Although effectiveness of RAS is confirmed for muscle 

activation pattern, step length, step duration and step duration variability for straight path 

walking, it is not clear whether RAS can enhance the ability to walk in more complex 

environments. The response to complex environmental demands can trigger signs and symptoms 

of PD, explaining why these patients have difficulties with gait initiation and continuation and 

greater fall rates with direction changes and obstacle avoidance. We reasoned that a program 

incorporating multidirectional stepping accompanied with the use of RAS would help target a 

more complex aspect of movement and a movement used during common daily activities for 

those with PD. The purpose of this study was to investigate whether such practice would transfer 

to a different context of functional and perceived gait indicated by dynamic gait index (DGI) and 

Fall and Gait questionnaire (FGQ), respectively. The DGI was used because it requires 

participants to perform more complex aspects of gait (i.e. walking around cones, clearing 

obstacles). In addition, the Unified Parkinson‟s disease rating scale (UPDRS_motor section) was 

used to provide insights to motor performance associated specifically to PD. Five individuals 

with PD (stages II-III) trained 3 days a week for 6 weeks. Participants were evaluated on the first 

day, last day and 1 week and 4 weeks after practice termination. Results from the repeated 

measures ANOVA indicated that participants significantly improved their DGI, UPDRS-motor 

and FGQ scores upon practice termination and retained the improvements for 1 week (p

support the idea that RAS may encourage use of alternative pathways that bypass the basal 

ganglia in PD patients. Further studies to add to the evidence are currently underway. 

Disclosures: Z. Kadivar, None; M. Johnson, None; J. Jones, None; T. Boyance, None; M. 

Duet, None; J.M. Hondzinski, None; J. Foto, None. 

Poster 





Title: Onset of disease at young age or L-DOPA treatment at young age: Which is a more 

important risk factor for L-DOPA-induced dyskinesia in Parkinson disease? 

Authors: T. KIMURA 1 , M. TOMIYAMA 1 , A. ARAI 1 , *K. KANNARI 2 , M. SHOUJI 1 ; 

1 Neurol., Hirosaki Univ. Grad Sch. Med., Hirosaki, Japan; 2 Intrnl. Med., Tokiwa-Kai Hosp., 

Minami-Tsugaru-Gun, Japan 

Abstract: Patients with young-onset Parkinson disease (PD) often develop L-DOPA-induced 

dyskinesia. However, it remains unknown whether the high incidence of L-DOPA-induced 

dyskinesia in young-onset PD results from onset of the disease at young age or L-DOPA 

treatment at young age. To determine which factor is more important in the development of L- 

DOPA-induced dyskinesia, we examined dyskinestic movements in the following three groups 

of 6-hydroxydopamine-lesioned hemiparkinsonian rats: young-onset (6-OHDA-lesioning at 10 

wks of age) and young-L-DOPA-treated (from 15 to 17 wks of age) (YOYD) group, young-onset 

and old-L-DOPA-treated (from 93 to 95 wks of age) (YOOD) group, and old-onset (6-OHDAlesioning 

at 88 wks of age) and old-L-DOPA-treated (OOOD) group. Rats in each group 

received intraperitoneal injections of L-DOPA methyl ester, 10 mg/kg in combination with 

benserazide, 5 mg/kg twice daily (morning and evening) for 2 weeks. We also evaluated the 

expression of dynorphin mRNA in the striatum and the volume of the medial globus pallidus on 

the lesioned side. The YOYD group and YOOD group displayed severer dyskinesia than the 

OOOD group, but no difference between the YOYD group and YOOD group. The expression of 

dynorphin mRNA was significantly increased in the YOYD group compared to the OOOD 

group. The YOYD and YOOD groups showed increased volume of the medial globus pallidus 

on the lesioned side compared to the OOOD group. These results suggest that both onset of PD 

at young age and L-DOPA treatment at young age contribute to the development of L-DOPAinduced 

dyskinesia, but onset of PD at young age appeared to be a more important risk for L- 

DOPA-induced dyskinesia.

Disclosures: T. Kimura, None; K. Kannari , None; M. Tomiyama, None; A. Arai, None; M. 

Shouji, None. 

Poster 





Support: Solvay Pharmaceuticals Research Laboratories 

Title: Differential expression of abnormal involuntary movements with short and long acting 

dopamine receptor agonists in levodopa treated and untreated 6-hydroxydopamine-lesioned rats 

Authors: *M. PAPATHANOU 1 , A. MCCREARY 2 , S. ROSE 1 , P. JENNER 1 ; 

1 NDRG, King's Col. London, London, United Kingdom; 2 Solvay Pharmaceuticals Res. Labs., 

Weesp, Netherlands 

Abstract: Long term L-3,4-dihydroxyphenylalanine (L-Dopa) treatment in Parkinson‟s disease 

results in the expression of abnormal involuntary movements (AIMs) or dyskinesia. The severity 

of dyskinesia may be related to the duration of dopaminergic stimulation. In L-Dopa primed and 

drug naïve (unprimed) non-human primates continuous dopaminergic stimulation with long 

acting dopamine receptor agonists (DRAs) produces less severe dyskinesia than intermittent 

stimulation with short acting DRAs or L-Dopa (Maratos et al., 2003). Whether this differential 

effect is also seen in 6-hydroxydopamine (6-OHDA)-lesioned rats is not known and is the 

purpose of this study. 

Male Wistar rats were lesioned with 6-OHDA in the medial forebrain bundle. Rats in Group 1 

(n=24) were primed to induce AIMs with L-Dopa (6.25mg/kg + benserazide 15mg/kg i.p. s.i.d.) 

(LD/B) for 3 weeks followed by LD/B and the short acting DRAs apomorphine (0.0625mg/kg 

s.c.) and ropinirole (0.4mg/kg s.c.) (n=12) or LD/B and the long acting DRAs pramipexole 

(0.0375mg/kg s.c.) and pergolide (0.03125mg/kg s.c.) (n=12) in a cross over design. Group 2 

(n=36) was divided into 6 groups (n=6) and treated with saline, LD/B, apomorphine, ropinirole, 

pramipexole or pergolide for 3 weeks. In both groups AIMs was assessed (scale from 0-4) 

(Carlsson et al., 2005) for up to 6 hours after drug administration. The above doses of the DRAs 

showed similar total number of rotations at peak activity as LD/B, obtained previously with 

rotometry. 

In L-Dopa primed animals (group 1) LD/B induced maximum AIMs scores at peak activity, with 

an overall duration of activity of 210 mins. Similarly apomorphine and ropinirole expressed 

maximum peak AIMs scores with a duration of activity 135 and 150 mins respectively. In

contrast, pramipexole and pergolide had a longer duration of activity (≥360 mins) but evoked 

only mild-moderate peak AIMs. 

In unprimed animals (group 2) daily administration of LD/B and apomorphine induced 

maximum peak AIMs scores, ropinirole produced moderately high AIMs (score=3), while 

chronic treatment with pergolide expressed mild peak AIMs (score=1-2). Pramipexole induced 

only mild orolingual AIMs (score=1). 

These data show that in animals primed to express AIMs, LD/B and short acting DRAs express 

severe AIMs but long acting DRAs produce only mild to moderate AIMS. In non primed rats 

chronic treatment results in the expression of AIMs such that short acting DRAs evoke moderate 

to severe AIMs, whereas long acting DRAs show mild or no AIMs. These data confirm the 

hypothesis that continuous dopaminergic stimulation with DRAs induces the expression of mild 

AIMs compared to the severe AIMs seen with pulsatile dopaminergic stimulation. 

Disclosures: M. Papathanou, None; A. McCreary, None; S. Rose, None; P. Jenner, None. 

Poster 





Support: Solvay Pharmaceuticals Research Laboratories 

Title: Investigation into the chronic effect of pardoprunox (SLV308) treatment upon L-DOPAinduced 

dyskinesia in MPTP-treated common marmosets 

Authors: *K. STOCKWELL 1,2 , M. J. JACKSON 1,2 , K. TAYARANI-BINAZIR 1,2 , S. ROSE 1,2 , 

A. C. MCCREARY 3 , P. G. JENNER 1,2 ; 

1 NDRG, King's Col. London, London, United Kingdom; 2 Proximagen Ltd, London, United 

Kingdom; 3 Solvay Pharmaceuticals Res. Labs., Weesp, Netherlands 

Abstract: Introduction: Chronic treatment with levodopa (LD) in Parkinson‟s disease (PD) 

induces motor complications, notably dyskinesia. Dyskinesia may also be induced to a lesser 

extent by full dopamine agonists. When administered with LD, however, severe dyskinesia are 

expressed. Whether this is also true for the partial dopamine (DA) agonist pardoprunox is not 

known and forms the aim of this study in the MPTP-treated marmoset model of PD. 

Methods: MPTP-treated marmosets were primed with LD (12.5 mg/kg) and were maintained on 

LD, 3-10 mg/kg, po (+ carbidopa (CD), 12.5 mg/kg, po) such that they had comparable levels of 

locomotor activity, motor disability and dyskinesia. Group 1 was treated with LD/CD alone (3-

10 mg/kg, po) whilst Groups 2 and 3 received LD/CD plus pardoprunox (0.0125 and 0.025 

mg/kg, po) respectively for 14 days. Subsequently, in the 3 weeks following drug withdrawal, 

animals were challenged weekly with LD/CD (12.5 mg/kg, po). Locomotor activity, motor 

disability and dyskinesia were assessed by standard procedures. 

Results: Pardoprunox (0.0125 and 0.025mg/kg) plus LD/CD maintained locomotor activity and 

improved the reversal of motor disability compared to LD/CD alone. Pardoprunox 

(0.0125mg/kg) plus LD/CD produced less dyskinesia than previous treatment with LD/CD alone. 

In the first two LD/CD challenges, previous treatment with pardoprunox in Groups 2 and 3 

reduced locomotor activity and tended to reduce dyskinesia compared to LD/CD alone. The 

reversal of motor disability was similar in all groups. This effect was not observed on the third 

LD/CD challenge. 

Conclusion: Concomitant pardoprunox and LD/CD treatment maintained or improved the LDinduced 

reversal of motor deficits whilst the low dose combination significantly reduced LDinduced 

dyskinesia. Subsequent LD/CD challenges suggest that chronic treatment with 

pardoprunox produced desensitization to LD. These data suggest that pardoprunox may improve 

the therapeutic benefit produced by a sub-maximal dose of LD whilst reducing dyskinesia. 

Disclosures: K. Stockwell, None; A.C. McCreary, None; M.J. Jackson, None; K. Tayarani- 

Binazir, None; S. Rose, None; P.G. Jenner, None. 

Poster 





Title: Antidyskinetic effect of BN82451 on a new model of L-DOPA-induced dyskinesia in 

hemiparkinsonian rats 

Authors: *P. E. CHABRIER, B. SPINNEWYN, C. CHARNET, S. BERNETIERE, P. 

ROUBERT, L. VIGNAUX, J. CAMARA, M. AUGUET; 

Neurol., IPSEN, Les Ulis, France 

Abstract: Chronic L-DOPA pharmacotherapy in parkinson's disease (PD) is often accompanied 

by motor complications known as dyskinesia. Although the underlying mechanism of these 

abnormal involuntary movements (AIMs) remains to be clarified, recent findings suggest that 

excitotoxicity, oxidative stress and mitochondrial dysfunction may play a role. Since BN82451, 

acts on neuronal excitotoxicity oxidative stress and possesses mitochondrial protective properties 

its symptomatic effects have been tested on L-DOPA-induced dykinesia in rats.

Unilateral intrastriatal injection of 6-hydroxydopamine (2x6µg) in rats was chosen as a model of 

parkinson‟s disease. Four weeks later, administration of escalating doses of L-DOPA during 4 

weeks led to AIMs in the majority of rats. Thus, we have examined the effect of acute or 

subchronic treatment with BN82451 (from 1 to 30 mg/kg; p.o.) on L-DOPA-induced locomotive, 

axial, orolingual and forelimb dyskinesia. For acute treatment, BN82451 was administered just 

after the first AIM measurement (30 minutes after L-DOPA injection) and for subchronic 

determination, treatment was pursued the 4 consecutive days, twice a day according to its 

pharmacokinetics and AIMs were measured at Day 1 and Day 5. Plasma L-DOPA, striatal 

dopamine were not affected by BN82451 administration at 10 mg/kg; p.o. 

BN82451 markedly and dose dependently reduced the severity of dyskinesia. The results also 

show that a single administration of BN82451 alleviates dyskinesia induced by L-DOPA in 

hemiparkinsonian rats and demonstrate that subchronic treatments necessitate lower doses to 

achieve the same antidyskinetic efficacy. 

This protocol, using gradual development of dyskinesia induced by increasing doses of L-DOPA 

result in a sound model of L-DOPA induced dyskinesia with a high rate of dyskinetic rats. In 

these experimental conditions, BN82451 reduces AIMs and might represent a treatment option 

for managing dyskinesia in PD. 

Disclosures: P.E. Chabrier , IPSEN, A. Employment (full or part-time); B. Spinnewyn, 

IPSEN, A. Employment (full or part-time); C. Charnet, IPSEN, A. Employment (full or parttime); 

S. Bernetiere, IPSEN, A. Employment (full or part-time); P. Roubert, IPSEN, A. 

Employment (full or part-time); L. Vignaux, IPSEN, A. Employment (full or part-time); J. 

Camara, IPSEN, A. Employment (full or part-time); M. Auguet, IPSEN, A. Employment (full 

or part-time). 

Poster 





Title: New treatment (BN82451) and assessment for L-Dopa-induced dyskinesias in 

parkinsonian macaques 

Authors: *R. ARON BADIN 1,2 , E. BROUILLET 1,2 , B. SPINNEWYN 3 , M. AUGUET 3 , P.-E. 

CHABRIER 3 , P. HANTRAYE 1 ; 

1 Molec Imag Res. Center(MIRCen), CEA,Inst Biomed Imaging (I2BM), Fontenay aux Roses, 

France; 2 CNRS, URA 2210, Orsay, France; 3 IPSEN, Inst. Henri Beaufour, Les Ulis, France

Abstract: The aim of this study was to study the effect of a subchronic treatment with a novel 

multitargeting molecule, BN82451, on levodopa induced dyskinesias in a primate MPTP model 

of Parkinson‟s disease. 

Five adult male macaques (Macaca fascicularis) received daily injections of 1-methyl-4-phenyl- 

1,2,3,6-tetrahydropyridine (MPTP; 0.2 mg/kg i.m.) until they presented stable parkinsonian 

symptoms, followed by chronic administration of increasing daily oral doses of L-Dopa 

(100:25mg levodopa:benserazide) in order to induce abnormal involuntary movements (AIMs). 

After stabilisation of the parkinsonian syndrome and of the AIMs, subchronic 5 day treatment 

with a daily subcutaneous injection of BN82451 (5 mg/kg; base form), Amantadine (2.5 mg/kg) 

or their solvent polyethylenglycol 400 (PEG 400) was administered after appropriate washout 

times. L-Dopa was co-administered and animals were filmed for 6 hours on the first and last day 

of treatment. Ethovision® software was used to track spontaneous locomotor activity (total 

distance moved, TDM) and The Observer® software to quantify the incidence of dyskinesias in 

7 different parts of the body (face, neck, trunk, both left and right legs and arms). 

On day 1 of BN82451 injection the AIMs score was reduced in 3 monkeys and remained 

unchanged in the other 2 as compared to their solvent control. At day 5, all animals benefited 

from the treatment with an overall AIMS decrease of 34%. This effect was not associated with a 

reduction in TDM. Moreover, the treatment affected all types of abnormal movements in the 

different parts of the body studied. 

In day 1 or day 5 of Amantadine treatment, the AIMs score was reduced in 3 monkeys, increased 

in 1 monkey and remained unchanged for the last one. However, at day 5, this reduction in the 

incidence of dyskinesias was associated to a concomitant decrease in TDM, observed in all 3 

animals presenting a reduction of their AIM score. 

In conclusion, these results show that a subchronic administration of BN82451 is more 

efficacious than the standard benchmark molecule Amantadine since it significantly decreases 

the total AIMs score in all parkinsonian animals without impairing their spontaneous locomotor 

activity. 

Disclosures: R. Aron Badin, None; B. Spinnewyn, None; M. Auguet, None; P. Chabrier, 

None; E. Brouillet, None; P. Hantraye, None. 

Poster 





Support: Michael J Fox Foundation

Title: L-DOPA-induced dyskinesia in a rat model of Parkinson‟s disease; correlation with 

indexes of dopaminergic and serotonergic transmission 

Authors: *D. M. RYLANDER, E. M. STROME, F. MELA, A. M. CENCI; 

BMC, Lund Univ., Lund, Sweden 

Abstract: Nigrostriatal dopamine (DA) denervation plays an essential role in L-DOPA induced 

dyskinesia in Parkinson‟s disease. However degeneration of serotonin (5-HT) and noradrenaline 

(NA) systems may also play a role. 

In this experiment rats with severe unilateral 6-hydroxydopamine (6-OHDA) of the medial 

forebrain bundle (MFB) were selected for a 3-week course of L-DOPA-treatment with ratings of 

abnormal involuntary movements (AIMs). The 5-HT, NA and DA innervation densities in the 

striatum and the cortex were assessed autoradiographically using tritiated ligands for specific 

neurotransmitter transporters (DAT, SERT and NAT). In addition, the integrity of DA cell 

bodies was examined in the ventral tegmental area (VTA) and the substantia nigra pars compacta 

(SNpc) by in situ hybridization analysis of tyrosine hydroxylase (TH). 

The results showed that all rats had a severe (> 80%) loss of striatal DAT binding and TH 

mRNA levels in SNpc and VTA ipsilaterally to the lesion. The extent of DA denervation did not 

correlate with the L-DOPA-induced AIM scores. In addition, the 6-OHDA lesions caused an 

ipsilateral depletion of SERT and NAT binding in the striatum and the sensorimotor cortex 

(~60% and ~75 % for SERT and NAT, respectively). The density of SERT binding in the 

striatum was positively correlated with the L-DOPA-induced AIM scores (r = 0.537, P = 0.002), 

being significantly larger in L-DOPA-treated rats that developed AIMs compared to nondyskinetic 

cases. No correlation was found between AIMs and striatal or cortical NAT binding 

densities. 

In conclusion, in a Parkinson model with nearly complete DA denervation, the severity of L- 

DOPA induced dyskinesia does not correlate with DAT radioligand-binding, but is positively 

and linearly related to SERT binding levels in the striatum. This suggests that, for the same 

extent of nigrostriatal lesion, rats endowed with a higher capacity for L-DOPA conversion in 

serotonergic neurons have a higher risk to develop AIMs. 

Disclosures: D.M. Rylander, None; E.M. Strome, None; F. Mela, None; A.M. Cenci, None. 

Poster 

248. Dystonia 



Topic: C.03.j. Dystonia

Support: NINDS NS037409 (XOB) 

DC002281 (DPC) 

Title: Metabolic biotinylation of torsinA allows visualization of intracellular fate 

Authors: J. E. FARLEY 1 , B. P. NILAND 1 , F. C. NERY 1 , J. W. HEWETT 1 , *D. P. COREY 2 , 

X. O. BREAKEFIELD 1 , B. A. TANNOUS 1 ; 

1 Dept. of Neurol., Massachusetts Gen. Hosp., Boston, MA; 2 Dept Neurobiol, Harvard Med. Sch., 

Boston, MA 

Abstract: TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic 

reticulum (ER) and nuclear envelope (NE). Its relative position in these contiguous 

compartments is thought to be determined by interaction with transmembrane proteins specific 

each of these regions. A specific deletion of a glutamic acid residue in the carboxy terminal 

region of torsinA (torsinAΓE) leads to the most common cause of early onset torsion dystonia 

(DYT1). Here we describe a method to visualize the intracellular location of torsinA protein 

within cells by incorporating within it a 15 amino acid biotin acceptor peptide (BAP) sequence 

which is biotinylated upon co-expression of the bacterial biotin ligase directed to the ER. 

Endogenous, metabolic biotinylation is a specific and sensitive technique which allows protein 

labelling within or on the surface of cells and is compatible with immunocytochemistry and 

electron microscopy for high resolution imaging. Here we monitored the effects of insertion of 

the BAP sequence in five different regions within torsinA by co-transfection of BAP-fusion 

expression constructs and biotin ligase in Gli36 cells. Some of these insertions acted as 

mutations, biasing the relative cellular distribution of torsinA between the NE and ER as 

visualized using streptavidin-fluorophore conjugates. Fusing the BAP sequence to the Cterminus 

of torsinA favored NE localization, whereas an N-terminal insertion of BAP 

(immediately following the signal sequence, amino acid 21) predisposed this protein to 

concentrate preferentially in the ER. Inserting the BAP sequence at other positions in the protein, 

such as position 71 which lies just after the hydrophobic region of the protein shows the typical 

relatively even ER and NE distribution. These studies support the usefulness of BAP sequences 

in determining the cellular localization of torsinA, as well as the use of BAP insertion sites as a 

means of regional domain analysis of this and other proteins. 

Disclosures: J.E. Farley, None; B.P. Niland, None; F.C. Nery, None; J.W. Hewett, 

None; D.P. Corey , None; X.O. Breakefield, None; B.A. Tannous, None. 

Poster 

248. Dystonia 


Program#/Poster#: 248.2/U1 

Topic: C.03.j. Dystonia 





Title: Characterization of anxiety- and depression-like behaviors in dyt1 Γgag knock-in mice 

Authors: *Y. LI 1 , F. YOKOI 1 , M. DANG 2 ; 

1 Dept Neurol, Univ. Alabama at Birmingham, Birmingham, AL; 2 Univ. of Illinois at Urbana- 

Champaign, Urbana, IL 

Abstract: DYT1 early-onset torsion dystonia is caused by mutations in DYT1 (TOR1A) with 

approximately 30% penetrance. A previous epidemiologic study for psychiatric status of the 

patients suggested an association of early-onset recurrent major depression with DYT1 ΓGAG 

mutation regardless of the manifestation of dystonic symptoms. However, more recent study 

suggested no significant association between the DYT1 ΓGAG mutation and depression. It was 

also reported that some of DYT1 dystonia patients had an additional mutation in SGCE that is 

known to cause diverse psychiatric symptoms including anxiety and depression. Therefore, it 

remains unclear whether DYT1 ΓGAG mutation alone truly causes depression. In this study, we 

analyzed anxiety and depression-like behaviors in Dyt1 ΓGAG knock-in mice that model DYT1 

dystonia. Although knock-in mice did not show significant anxiety-like behaviors in the openfield 

test, they exhibited alterations of anxiety-like behavior in elevated-plus maze and light-dark 

box tests. The depression-like behaviors of knock-in mice were investigated using forced 

swimming and tail suspension tests. The knock-in mice did not exhibit significant alterations in 

total immobility time in both tests. However, male knock-in mice exhibited significant shorter 

latency to the first episode of immobility in tail suspension test, suggesting a depression-like 

behavior. The results suggest that DYT1 ΓGAG mutation may contribute to the high incidence of 

depression in patients reported in DYT1 carriers. 

Disclosures: Y. Li , None; F. Yokoi, None; M. Dang, None. 

Poster 

248. Dystonia 




Support: Bachmann-Strauss Dystonia Foundation 

Title: Molecular cloning and characterization of zebrafish Torsin 1 

Authors: *J. J. SAGER 1,2 , G. E. TORRES 1,3 , E. A. BURTON 4,5,2,6,7 ; 

1 Neurobio., 2 Pittsburgh Inst. of Neurodegenerative Dis., 3 Pharmacol., 4 Neurol., 5 Mol. Genet. and 

Biochem., Univ. Pittsburgh, Pittsburgh, PA; 6 Div. of Movement Disorders, Univ. of Pittsburgh 

Med. Ctr., Pittsburgh, PA; 7 Pittsburgh VA Healthcare Syst., Pittsburgh, PA 

Abstract: Early Onset Torsion Dystonia (EOTD) is a movement disorder caused by a mutation 

in the dyt1 gene encoding TorsinA, an ATPase of the AAA+ superfamily. The disorder is 

inherited as an autosomal dominant Mendelian trait with 30-40% penetrance. Patients with 

EOTD have an in frame trinucletide deletion, resulting in the loss of a single glutamic acid near 

the carboxyl terminal of Torsin A. It is believed that the mutant protein exerts a dominant 

negative function, perhaps by effecting relocalization of wild-type protein to the nuclear 

envelope from its usual position in the endoplasmic reticulum. The cellular functions of TorsinA 

are unknown; the neuronal mechanisms that are perturbed by expression of mutant Torsin A are 

a major unresolved issue in understanding the pathophysiology of dystonia. We aim to use the 

genetically manipulable zebrafish as a model vertebrate central nervous system in which to 

elucidate the functions and pathophysiology of TorsinA in vivo. As a first step towards this goal, 

we have identified and cloned the zebrafish tor1 gene encoding the ortholog of human torsin 1A. 

The tor1 gene shows alternative 5‟ exons that splice into a common transcript at exon 2; both 

splice variants are expressed in zebrafish brain. Sequence analysis shows that the zebrafish and 

human proteins share approximately 60% identity and almost 80% similarity, with complete 

conservation of the catalytic domains, suggesting conserved biochemical functions and implying 

that the zebrafish system will be informative for understanding the molecular physiology of 

human torsin. Current studies are focused on delineating the expression pattern of zebrafish 

Torsin1 and the effects of under or over expression in larval zebrafish. 

Disclosures: J.J. Sager , None; G.E. Torres, None; E.A. Burton, None. 

Poster 

248. Dystonia 


Program#/Poster#: 248.4/U3


Support: Grant from the Forschungskommission of the Freie Universität Berlin 

Title: Pharmacological manipulations of the dopaminergic system in a transgenic mouse model 

of early-onset dystonia 

Authors: N. LANGE 1 , M. HAMANN 1 , P. SHASHIDHARAN 2 , *A. RICHTER 1 ; 

1 Dept Pharmacol& Toxicol, Depart. Vet. Med. Fu Berlin, Berlin, Germany; 2 Dept. of Neurol., 

Mount Sinai Sch. of Med., New York, NY 

Abstract: A trinucleotide deletion (ΓGAG) in the DYT1 gene is implicated in early-onset 

torsion dystonia. This movement disorder is characterized by involuntary, sustained muscle 

contractions and has an autosomal dominant inheritance pattern with ~30-40% penetrance. The 

mutation leads to the loss of a glutamic acid residue in the carboxy region of torsinA. The 

pathophysiological role of the mutation in causing dystonia is poorly understood. Well 

characterized animal models can help to understand the underlying mechanisms and thereby to 

develop new therapeutic strategies. Shashidharan et al. described a transgenic mouse model 

(DYT1 mice) with the human gene defect and overexpression of mutant torsinA, leading to 

hyperactivity, dystonic-like movements, abnormal shaking of the head and circling behavior. In 

the present study, different behavioral investigations combined with the administration of 100 

mg/kg L-DOPA + 10 mg/kg carbidopa (CD) in DYT1 and control mice should clarify the 

pathophysiological role of the dopaminergic system. It was described that the severity of 

symptoms increased with age in the DYT1 mice (Shashidharan et al., 2005). Thus, all 

experiments were done in 3 and 6 month old DYT1 and age-matched control mice. DYT1 mice 

in both age periods showed hyperactivity in comparison to controls. The administration of L- 

DOPA/CD reduced the locomotor activity in both, DYT1 and age-matched control mice in 

comparison to vehicle treatment. Naïve and vehicle treated transgenic mice at the age of 3 month 

expressed higher neuromuscular strength in the grip strength test. The application of L- 

DOPA/CD reduced the neuromuscular strength in 3 month old DYT1 mice, while no effect 

could be observed in controls. In 6 month old mice, no significant differences between the 

different animal or treatment groups could be observed. In comparison to vehicle control, the 

administration of L-DOPA/CD reduced the latency to fall from the rotarod in both, transgenic 

and control mice without age-dependency. The severity of dystonic-like movements, which was 

rated by a score-system, increased after the administration of L-DOPA/CD in DYT1 mice in 

both tested age periods. However, the present data revealed that also wild-type mice exhibit 

similar movements. The present results may indicate that the dopaminergic system is affected in 

DYT1 mice, although the observed differences to control animals were only moderate. Ongoing 

pharmacological studies with dopamine receptor agonists and antagonists as well as a dopamine 

reuptake inhibitor have to further clarify the relevance of distinct parts of the dopaminergic 

system. 

Supported by a grant from the FK of the Freie Universität Berlin. 

Disclosures: N. Lange, None; M. Hamann, None; A. Richter , None; P. Shashidharan, None.

Poster 

248. Dystonia 




Support: NIH grant R01NS47151 

Title: Quantification of gait in dystonic Gunn rats 

Authors: K. D. CHANIARY 1 , *A. C. RICE 2 , M. S. BARON 2 , P. A. WETZEL 1 , S. M. 

SHAPIRO 2 ; 

1 Dept Biomed. Engin., 2 Dept Neurol., MCV - VCU, Richmond, VA 

Abstract: The objective of this study was to develop a simple, non-invasive method to quantify 

motor deficits in dystonic jaundiced Gunn rats congenitally deficient in UDP-glucuronosyl 

transferase. These rats, when exposed to sulfadimethoxine, develop bilirubin encephalopathy 

(kernicterus) with hearing loss and dystonia, closely resembling the human syndrome. We 

recently characterized electromyograms (EMGs) in this model supporting our clinical impression 

of dystonia. On day 16, Gunn rats were treated with 100 mg/kg of sulfadimethoxine and on day 

31, gait analysis measurements were obtained. Systematic measurement of hindlimb spread, step 

length ratio, stance/swing ratio and walking speed were carried out during free, spontaneous 

locomotion in jaundiced dystonic, non-jaundiced non-dystonic and jaundiced non-dystonic 

littermates. Data analysis demonstrated a significant increase in hindlimb spread 

(67.72+0.88mm) in sulfa treated jaundiced animals as compared to controls (50.85+0.57mm) 

including a significant decrease in step length ratio in sulfa treated jaundiced animals 

(0.975+0.010) as compared to controls (1.002+0.011). This study helps us to develop a technique 

to objectively characterize dystonia in rats. Systematic studies of motor control in this model 

may lead to better understanding and treatment of movement disorders associated with 

kernicterus in humans. 

Disclosures: K.D. Chaniary, None; A.C. Rice , collaborator, B. Research Grant (principal 


M.S. Baron, collaborator, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received); P.A. Wetzel, None; S.M. Shapiro, PI, B. 


grants already received).

Poster 

248. Dystonia 




Title: Deep brain stimulation of the entopeduncular nucleus induces striatal c-fos expression in 

the dystonic hamster 

Authors: *W. MEISSNER 1 , R. REESE 1 , G. CHARRON 1 , A. NADJAR 1 , I. AUBERT 1 , M.-L. 

THIOLAT 1 , M. HAMANN 2 , A. RICHTER 2 , E. BEZARD 1 ; 

1 Lab. Neurophysiol, CNRS UMR 5227, Bordeaux, France; 2 Inst. of Pharmacol. and Toxicology, 

Dept. of Vet. Med., Freie Univ., Berlin, Germany 

Abstract: Pallidal deep brain stimulation (DBS) is an established therapy to alleviate symptoms 

of focal and generalized dystonia. Its mechanisms of action are, however, only incompletely 

understood. The dtsz hamster is a well characterized model of non-kinesiogenic paroxysmal 

dystonia where animals display age-dependent long-lasting attacks of generalised dystonia in 

response to mild stress. Bilateral DBS of the entopeduncular nucleus (EP, corresponds to the 

human internal pallidum) significantly reduces the severity of dystonic symptoms in these 

animals. We here assess the effect of entopeduncular nucleus deep brain stimulation on the 

expression of c-fos and cytochrome oxidase subunit I (COI) in the cortex-basal ganglia network 

in dystonic hamsters. Unilateral subchronic EP-DBS was performed in anesthetized dtszhamsters 

and controls. Expression of c-fos and COI was assessed in the cortex-basal ganglia 

network by using immunohistochemistry. C-fos expression was generally lower in dtsz hamsters 

than in controls, while COI expression was higher in most assessed structures. EP-DBS 

significantly increased striatal c-fos expression in dtsz hamsters and controls, while COI 

expression was not modified. Previous results suggest an age dependent deficiency in 

parvalbumin positive striatal GABAergic interneurons as a feature of dystonic hamsters. It is 

tempting to speculate that EP-DBS may particularly increase c-fos expression in these striatal 

interneurons which may positively affect abnormal information processing in the basal ganglia 

and finally underlie clinical improvement 

Disclosures: W. Meissner , None; G. Charron, None; R. Reese, None; A. Nadjar, None; I. 

Aubert, None; M. Thiolat, None; E. Bezard, None; M. Hamann, None; A. Richter, None.

Poster 

248. Dystonia 




Support: NIH NS40470 

Bachmann-Strauss Dystonia and Parkinson Foundation 

NIH F32 052040 

Title: Female gonadal hormones and paroxysmal dystonia in rocker mice 

Authors: *H. A. JINNAH, T. L. SHIRLEY, M. C. TERZI, E. J. HESS; 

Dept Neurol, Johns Hopkins Univ., Baltimore, MD 

Abstract: Ion channel dysfunction is known to cause several forms of episodic neurological 

disease in humans. Different mutations in CACNA1A, the pore-forming subunit of CaV2.1 

calcium channels, underlie several distinct disorders that include familial hemiplegic migraine, 

episodic ataxia type 2, spinocerebellar ataxia 6, epilepsy, and dystonia. Such phenotypic 

diversity also occurs in mice carrying different mutations in Cacna1a (tottering, rolling mouse 

Nagoya, leaner, rocker, and two knockouts). Intermittent dystonic attacks resembling 

paroxysmal dyskinesia have only been described for tottering and rocker mice. The most 

distinctive feature of attacks in rocker mice is their sexual dimorphism, with attacks occurring 

almost exclusively in adult females. This sex difference could be due to the effects of gonadal 

sex hormones on the central nervous system. Orchidectomy was performed in young male rocker 

mice to determine whether gonadal androgens are suppressive, but this failed to induce attacks in 

adult males. Conversely, ovariectomy in young female rocker eliminated attacks in adult females 

suggesting that female gonadal hormones are required for dystonia attacks. Combining 

ovariectomy with chronic estrogen (in the form of 17Β-estradiol) or progesterone replacement 

using silastic tubing implants will be conducted to determine which gonadal hormone is required 

for dystonia attacks. Cross-replacement in orchidectomized males will help determine whether 

hormone alone is sufficient. Sex bias occurs in many prevalent human neurological diseases such 

as migraine, epilepsy and dystonia. Rocker mice provide a model of sexually dimorphic 

neurological dysfunction that could offer new insight into the mechanisms of sex hormone 

modulation of disease. 

Disclosures: H.A. Jinnah , None; T.L. Shirley, None; M.C. Terzi, None; E.J. Hess, None.

Poster 

248. Dystonia 




Support: Ministero Salute (Prog. Finalizzato and Art. 56) 

Bachmann-Strauss Dystonia & Parkinson‟s Foundation 

Istituto Superiore Sanità (Malattie Rare) 

Dystonia Medical Research Foundation 

Title: Dysregulation of striatal D2 receptors leads to altered GABA transmission in a mouse 

model of DYT1 dystonia 

Authors: G. SCIAMANNA 1 , A. TASSONE 1 , *P. BONSI 1 , A. TSCHERTER 1 , N. SHARMA 2 , 

D. G. STANDAERT 3 , G. MARTELLA 1 , A. PISANI 1 ; 

1 Neurophysiol., Fondazione Santa Lucia, Rome, Italy; 2 Massachusetts Gen. Hosp., , Harvard 

Med. Sch., Boston, MD; 3 Univ. of Alabama at Birmingham, Birmingham, AL 

Abstract: DYT1 dystonia is caused by a deletion in a glutamic acid residue in the C-terminus of 

the protein torsinA,but the mechanisms which results in symptoms of the disease are unknown. 

Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. We 

recorded GABA- and glutamate-mediated synaptic currents from striatal neurons obtained from 

a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing 

human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, 

of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents 

(mIPSCs), whereas glutamatergic activity was normal. No alterations were found in mice 

overexpressing normal human torsinA (hWT). To identify the possible sources of the increased 

GABAergic tone, we recorded GABAergic Fast-Spiking (FS) interneurons that exert a feedforward 

inhibition on MSNs. Both sEPSC and sIPSC recorded from hMT FS interneurons were 

comparable to hWT and controls. In physiological conditions, dopamine (DA) D2 receptor act 

presynaptically to reduce striatal GABA release. Notably, application of the D2-like receptor 

agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to 

hWT and controls. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in 

MSNs and FS interneurons from hMT mice. Our findings demonstrate a disinhibition of 

GABAergic synaptic activity, that can be partially attributed to a D2 DA receptor dysregulation. 

A rise in GABA transmission would result in a profound alteration of striatal output, that might 

be relevant to the pathogenesis of dystonia.

Disclosures: G. Sciamanna, None; A. Tassone, None; P. Bonsi , None; A. Tscherter, None; N. 

Sharma, None; D.G. Standaert, None; A. Pisani, None; G. Martella, None. 

Poster 

248. Dystonia 




Support: NIH/NINDS (K02 NS058450) 

Title: Differences in torsinA degradation caused by the disease-linked mutation reveal potential 

therapeutic approaches for DYT1 dystonia 

Authors: *K. L. GORDON 1 , P. GONZALEZ-ALEGRE 2 ; 

1 Program in Neurosci, 2 Grad. Programs in Neurosci, Genetics, and Mol & Cell Bio, and Dept of 

Neurol., Univ. Iowa, Iowa City, IA 

Abstract: DYT1 dystonia is a disabling neurological disease caused by a glutamic acid deletion 

in torsinA (torsinA(ΓE)). The mutation causes torsinA(ΓE) to redistribute from the endoplasmic 

reticulum (ER) to the nuclear envelope (NE) and to form abnormal intermolecular disulfide-link 

dependent oligomers. Although how neurons handle disease-linked proteins has been addressed 

in many disorders, how cells dispose of torsinA remains unexplored. As an ER protein, torsinA 

is a potential substrate for degradation through macroautophagy or the ubiquitin proteasome 

system. Here, we aimed to identify the preferential catabolic pathway for torsinA and to 

determine if the change in localization and oligomerization of torsinA(ΓE) modulates this 

process. 

Initial experiments in overexpression systems showed that both forms of torsinA can be cleared 

by the proteasome and macroautophagy, but suggested that high levels of overexpression likely 

saturate their normal degradation route and recruit alternative pathways. We optimized a cellular 

system with expression at near physiological levels and determined that wild type torsinA is 

preferentially degraded by macroautophagy. This was confirmed for endogenous torsinA in SH- 

SY5Y cells. In contrast, torsinA(ΓE) is equally cleared by the proteasome and macroautophagy. 

In subsequent studies, we determined that torsinA(ΓE) oligomerization is not a result of NE 

localization but is linked to abnormal torsinA function, suggesting this molecular event might be 

important in DYT1 pathogenesis. Interestingly, torsinA(ΓE) oligomers were cleared by 

macroautophagy, and not the proteasomal system as is the monomeric form. 

These studies suggest the turnover of torsinA is normally mediated by macroautophagy, but the 

disease-linked mutation targets torsinA to the proteasome. However, the presence of abnormal

disulfide-bonds in torsinA(ΓE) oligomers would prevent retrotranslocation to the ER for 

proteasomal degradation, requiring macroautophagy for their clearance. As a result, 

pharmacological induction of autophagy should be explored as a potential therapeutic approach 

for DYT1 dystonia. 

Disclosures: K.L. Gordon, None; P. Gonzalez-Alegre, None. 

Poster 

248. Dystonia 




Support: NIH Grant P01-NS037409 

Title: TorsinA influences neurogenesis and neuronal migration in the embryonic brain 

Authors: *P. G. BHIDE 1 , D. M. MCCARTHY 2 , J. R. SIMS 3 , J. ZENG 3 , Y. LI 4 , X. 

BREAKEFIELD 3 ; 

1 Developmental Neurobiol, Massachusetts Gen. Hosp, Charlestown, MA; 2 Developmental 

Neurobio., 3 Neurol., Massachusetts Gen. Hosp., Charlestown, MA; 4 Neurol., Univ. of Alabama, 

Birmingham, AL 

Abstract: Early onset torsion dystonia (DYT dystonia) is caused by a three base-pair GAG 

deletion in the DYT1 gene on chromosome 9. TorsinA, the product of DYT1, is implicated as an 

AAA+ protein in intracellular signaling functions, organelle biogenesis and membrane 

trafficking. The majority of torsinA is localized within the nuclear envelope and the contiguous 

endoplasmic reticulum and it is known to act as an intermediary for linking the nuclear envelope 

to cytoskeletal proteins such as actin. Therefore, torsinA could play a role in nuclear movement, 

a critical event in cell division and migration. To address this possibility, we analyzed 

neurogenesis and neuronal migration in the embryonic striatum in DYT1 knockout mice, which 

lack torsinA. DYT1 +/- mice were crossed and the timed-pregnant dams carrying 15-day old 

embryos were administered a single injection of the S-phase marker bromodeoxyuridine (BrdU; 

50mg/kg; i.p.). Following 2.0 hr survival, embryonic brains were fixed in ethanol and processed 

for BrdU immunohistochemistry. BrdU labeling index was significantly higher (~40%) in the 

lateral ganglionic eminence (precursor of the striatum) in the DYT1 -/- embryos compared to 

DYT1 +/+ littermates. The average number of phosphohistone-3 labeled nuclei (presumptive Mphase 

nuclei) was also significantly higher (~50%) in the DYT1 -/- embryos. In other experiments 

we analyzed neuronal migration in explants of the embryonic striatum from E15 DYT1 -/- and

DYT1 +/+ littermates cultured for 24 hr in 3-dimensional Matrigel matrix. The size and origin of 

the explants were standardized by using a biopsy needle to punch out striatal tissue from 250µm 

coronal Vibratome sections of the forebrain. We found ~46% decrease in the average number of 

cells migrating from the DYT1 -/- explant compared to the number migrating from the wild-type 

explant. These data suggest that torsinA plays a role in neurogenesis and neuronal migration in 

the embryonic brain. 

Disclosures: P.G. Bhide , None; D.M. McCarthy, None; J.R. Sims, None; J. Zeng, None; Y. 

Li, None; X. Breakefield, None. 

Poster 

248. Dystonia 




Support: NIH P01 for dystonia 

Title: TorsinA participates in linkage between nuclear envelope and cytoskeleton 

Authors: F. C. NERY 1 , J. ZENG 1 , B. NILAND 1 , J. HEWETT 1 , J. FARLEY 1 , D. IRIMIA 2 , Y. 

LI 3 , G. WICHE 4 , A. SONNENBERG 5 , *X. O. BREAKEFIELD 1 ; 

1 Molec Neurogenetics Unit, Massachusetts Gen Hosp, Charlestown, MA; 2 BioMEMS Resource 

Ctr., Massachusetts Gen. Hosp., Charlestown, MA; 3 Neurol., Univ. of Alabama, Birmingham, 

AL; 4 Dept. of Mol. Cell Biol. & Max F. Perutz Labs., Univ. of Vienna, Austria, Viena, Austria; 

5 Dept. of Cell Biol., Netherlands Cancer Inst., Amsterdam, Netherlands 

Abstract: A specific mutation (ΓE) in torsinA underlies most cases of the dominantly inherited 

movement disorder, early onset torsion dystonia (DYT1). TorsinA is an AAA+ protein located 

within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER) in most cells. 

We investigated an association between torsinA and nesprin-3, which spans the outer nuclear 

membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin- 

3α, plectin and vimentin co-immune precipitated with torsinA, and the carboxy terminal region 

of torsinA was found to bind to the KASH domain of nesprin-3α in the lumen of the NE in an 

ATP-dependent manner. TorsinAΓE binds more tightly to nesprin-3 than does torsinA. TorsinA 

also binds to the KASH domains of nesprin-1 and -2 (SYNE-1 and -2) which link to actin. 

TorsinA is involved in maintaining the nuclear localization of nesprin-3α with mouse embryonic 

fibroblasts (MEFs), with cells lacking torsinA showing a re-distribution of nesprin-3 from the 

NE to the ER, and a resulting decrease in the perinuclear collapse of vimentin in response to

protein kinase C (PKC) activation. When a nesprin-3 fluorescent fusion protein is overexpressed 

in normal fibroblasts it maintains a primary NE localization, but in DYT1 fibroblasts it 

accumulates in aggregates throughout the cells. TorsinA null MEFs have normal NE structure, 

but nuclear polarization and cell migration are delayed in a wound healing assay as compared to 

wild-type cells. These studies support a role for torsinA in influencing dynamic interactions 

between the KASH domains of nesprins in the ONM and their known partners, such as SUN 

proteins, in the INM of the NE which participate in cytoskeletal-mediated positioning of the 

nucleus during cell migration. 

Disclosures: F.C. Nery, None; D. Irimia, None; Y. Li, None; G. Wiche, None; A. 

Sonnenberg, None; X.O. Breakefield , None; J. Zeng, None; B. Niland, None; J. Hewett, 

None; J. Farley, None. 

Poster 

248. Dystonia 




Support: R01-NS048458 


R03-NS050185 

Title: Cytochrome oxidase metabolic mapping reveals dysfunctional networks in DYT1 dystonia 

Authors: *Y. ZHAO 1 , N. SHARMA 2 , M. LEDOUX 1 ; 

1 Neuro, Univ. of Tennessee, Memphis, TN; 2 Neurol, Massachusetts Gen Hosp, Charlestown, 

MA 

Abstract: DYT1 dystonia is characterized by torsional movements which typically begin in 

distal portions of a limb and oftentimes spread to involve twisting movements and fixed postures 

in other regions of the body. DYT1 dystonia is an autosomal dominant disorder of reduced 

penetrance caused by a GAG deletion in Exon 5 of the TOR1A gene which encodes torsinA. 

Magnetic resonance imaging and postmortem neuropathological studies suggest that DYT1 

dystonia is a functional disorder of the nervous system associated with microstructural 

abnormalities of neurons and/or glia. Positron emission tomography (PET) has localized 

increased metabolic activity to the putamen/globus pallidus, cerebellum, and supplementary

motor areas in manifesting and non-manifesting carriers of the DYT1 mutation. To supplement 

human PET studies with data of higher spatial resolution, cytochrome oxidase (CO) 

histochemistry was used to map metabolic activity in a mouse model of DYT1 dystonia. CO 

activity within neuroanatomical regions of interest (ROIs) was measured in transgenic mice 

expressing either human wild-type (hWT) or mutant (hMT1) torsinA and their non-transgenic 

(WT) littermates and converted to Z-scores. None of the hMT1 mice exhibited overt 

manifestations of dystonia. Compared to WT littermates, hMT1 mice showed increased CO 

activity in all layers of cerebellar cortex (molecular, Purkinje cell, granule cell), the medial 

cerebellar nucleus, and the ventral posterior thalamic nucleus; and decreased CO activity in the 

rostral caudate-putamen, substantia nigra pars reticulata, somatosensory 1 hindlimb region, 

somatosensory 1 forelimb region, and ventrolateral geniculate nucleus (P < 0.05, for all). These 

findings suggest that a net shift of energy demand from basal ganglia output to the cerebellum is 

a feature of the DYT1 carrier state. 

Disclosures: Y. Zhao, None; N. Sharma, None; M. Ledoux, None. 

Poster 

248. Dystonia 




Support: Bachmann-Strauss Dystonia & Parkinson Foundation 

NS047692 


NS54246 

Title: Characterization of cerebral cortex-specific and Purkinje cell-specific Sgce conditional 

knockout mice 

Authors: *F. YOKOI 1 , M. T. DANG 2 , Y. LI 1 ; 

1 Neurol., Univ. Alabama Birmingham, Birmingham, AL; 2 Univ. of Illinois at Urbana- 

Champaign, Urbana, IL 

Abstract: DYT11 myoclonus-dystonia is a movement disorder characterized by myoclonic jerks 

and rapid muscle contraction combined with dystonic postures and repetitive movements that

often exhibits diverse psychiatric symptoms. In the previous studies, we reported that Sgce 

knockout mice lacking exon 4 exhibit myoclonus, motor deficits and alterations in emotional 

responses and striatal monoamine metabolism. We also reported that striatum-specific 

conditional knockout mice exhibited motor deficits in rotarod and beam-walking tests. To 

analyze the contribution of other brain regions to the pathogenesis of this disease, we produced 

Purkinje cell-specific Sgce conditional knockout and cerebral cortex-specific conditional 

knockout mice and performed rotarod and beam-walking tests. Although cerebral cortex-specific 

KO mice did not exhibit any motor defects in these tests, Purkinje cell-specific KO mice exhibit 

significant motor learning defects in beam-walking test. The result suggested that ε-sarcoglycan 

have an essential function in Purkinje cells for the motor learning. 

Disclosures: F. Yokoi , None; M.T. Dang, None; Y. Li, None. 

Poster 

248. Dystonia 




Title: Physiological conditions induce dissociation of the 900 kDa neurotoxin type A complex 

from Clostridium Botulinum 

Authors: K. H. EISELE, H. V. TAYLOR, *K. B. FINK; 

Biotech., Merz Pharmaceuticals, Frankfurt, Germany 

Abstract: The objective of this study was to assess the stability of the 900 kDa botulinum 

neurotoxin type A complex from clostridium botulinum and its dissociation kinetics under 

physiological conditions. 

Botulinum neurotoxin type A finally cleaves SNAP25 thus inhibiting exocytotic neurotransmitter 

release. It is clinically used for treatment of various neurological disorders such as cervical 

dystonia. The medicinal botulinum neurotoxin type A product Xeomin(1) consists of the 150 

kDa neurotoxin molecule free of complexing proteins. In contrast, first-generation botulinum 

neurotoxin type A products contain the 900 kDa neurotoxin complex consisting of the 150 kDa 

neurotoxin molecule and several non-toxic proteins known as complexing proteins as produced 

by the Clostridium Botulinum bacterium. It has been suggested that these complexing proteins 

serve to prolong neurotoxin persistence and inhibit neurotoxin diffusion into adjacent tissues. 

The 900 kDa botulinum neurotoxin type A complex was exposed to pH values from 6.0 to 7.6 

and then separated by anion exchange HPLC to differentiate between the resulting neurotoxin 

entities. Separation conditions were qualified by Western blotting using antibodies directed

against the botulinum neurotoxin type A heavy or light chain or against the full complex. 

Separation fractions were functionally analyzed using the mouse hemidiaphragma assay. 

The 150 kDa neurotoxin type A molecule is released from the 900 kDa complex in less than one 

minute when exposed to physiological pH values (pH 7.2-7.6). 

It is concluded that the 150 kDa botulinum neurotoxin type A molecule is released from the 900 

kDa complex in less than one minute when exposed to physiological pH values. This time frame 

is extremely short in comparison to the onset of the therapeutic effect which is measured in days. 

Therefore, the complexing proteins cannot stabilize the neurotoxin or inhibit its diffusion as 

claimed. Accordingly, the necessity of these complexing proteins in medicinal formulations must 

be questioned. Finally, these data aid in understanding the clinical equipotency of Xeomin(1), the 

150 kDa botulinum neurotoxin type A molecule free of complexing proteins, and botulinum 

neurotoxin type A preparations containing complexing proteins. 

(1) not approved for marketing in the USA 

Disclosures: K.H. Eisele, Merz Pharmaceuticals, A. Employment (full or part-time); K.B. Fink 

, Merz Pharmaceuticals, A. Employment (full or part-time); H.V. Taylor, Merz Pharmaceuticals, 

A. Employment (full or part-time). 

Poster 

248. Dystonia 




Title: The effects of a comprehensive sensorimotor retraining program on individuals with 

Focal Hand Dystonia vs. normal control participants 

Authors: *A. L. MCKENZIE 1 , N. BYL 2 ; 

1 Physical Therapy, Chapman Univ., Orange, CA; 2 Physical Therapy and Rehabil. Sci., Univ. of 

California at San Francisco, San Francisco, CA 

Abstract: Background and Purpose: Focal hand dystonia (FHD), also known as occupational 

hand cramps, writer‟s cramp, or musician‟s cramp, is a condition characterized by involuntary, 

sustained, co-contraction of the flexors and extensors of the affected digits, resulting in 

decreased motor control of the hand during specific tasks. Previously, we reported the effects of 

a comprehensive sensorimotor retraining on patients with FHD using both clinical outcomes and 

magnetoencephalography, but the effects of such training on normal, healthy controls were not 

known. The purpose of this study was to compare clinical parameters for musicians and writers 

with FHD to those for normal, healthy control subjects to delineate differences in responses to

training between the groups. 

Participants: 10 individuals with a diagnosis of FHD and 10 normal, healthy control subjects 

who heavily used their hands for sustained fine motor movements (e.g., keyboarding, 

handwriting) were recruited for participation in this study. 

Methods: Each participant completed a battery of sensory, motor, and physical parameter tests, 

as well as a questionnaire on functional tasks. Supervised sensorimotor retraining sessions were 

conducted for each subject twice/week for one month, augmented by a home program. As part of 

a larger longitudinal study, subjects with FHD continued to undergo retraining with weekly 

supervision for an additional two months. For the current study, baseline and one month followup 

data were compared to determine group differences. Each dependent variable was considered 

an independent family. 

Results: Both subjects with FHD and normal healthy control subjects exhibited changes in 

clinical parameters following sensorimotor retraining. For control subjects, the greatest changes 

were seen in tests for kinesthesia, motor accuracy and motor control. Subjects with FHD 

exhibited changes in sensory, motor, and physical parameters; subjects continued to improve at 3 

months, with sustained changes observed at 6 months. 

Conclusion: The findings of this study suggest that while normal, healthy control subjects do 

demonstrate some changes in response to sensorimotor retraining, such changes are different and 

less pronounced than those seen in subjects with FHD. 

Disclosures: A.L. McKenzie , None; N. Byl, None. 

Poster 

248. Dystonia 




Support: Canadian Institutes of Health Research (CIHR) 

CIHR fellowship to AJN 


Title: Short and long interhemispheric inhibition during unimanual contraction of hand muscles 

in writer‟s cramp dystonia and controls

Authors: *A. J. NELSON 1,2 , C. GUNRAJ 2 , T. HOQUE 2 , Z. NI 2 , R. CHEN 2 ; 

1 Dept Kinesiol, Univ. Waterloo, Waterloo, ON, Canada; 2 Div. of Neurol. and Krembil Neurosci. 

Ctr., Toronto Western Res. Institute, Univ. of Toronto, Toronto, ON, Canada 

Abstract: Interhemispheric interactions between homologous muscle representations may be 

probed using a paired-pulse TMS technique whereby a conditioning pulse is applied to the 

muscle representation in one hemisphere followed by a test pulse to the contralateral hemisphere. 

The amplitude of the motor evoked potential (MEP) in the muscle contralateral to the test pulse 

is suppressed when the inter-stimulus interval (ISI) is between 6 and 50 ms suggesting that 

interhemispheric inhibition (IHI) dominates the interaction during this time frame. The present 

study investigates IHI from the dominant to non-dominant hemisphere and vice versa during rest, 

and isometric contraction of right or left first dorsal interosseous muscle. Isometric contraction at 

20% MVC in FDI occurred by a pinch between the thumb and index finger and also by holding a 

pen to paper to simulate the context of writing. IHI was measured at ISIs of 6, 8, 10, 12, 30, 40, 

and 50 ms. Results indicate that 1) during rest, the strength of IHI is similar in both directions, 

from the dominant to non-dominant hemisphere and vice versa, 2) contraction of FDI 

contralateral to the CS or the TS reduced IHI at almost all ISIs tested, and 3) the magnitude of 

reduced IHI is similar between isometric contraction and pen holding in controls, yielding no 

contextual modulation on IHI. Preliminary results suggest that isometric contraction during pen 

holding reduces IHI more than isometric contraction alone in Writer‟s cramp patients. 

Disclosures: A.J. Nelson , None; C. Gunraj, None; T. Hoque, None; Z. Ni, None; R. Chen, 

None. 

Poster 

248. Dystonia 




Support: BSIK 03016 

Title: Dystonia explained by aberrant proprioceptive reflexes - a modeling study 

Authors: *W. MUGGE, A. C. SCHOUTEN, F. C. T. VAN DER HELM; 

BioMechanical Engin., Delft Univ. of Technol., Delft, Netherlands 

Abstract: Dystonia is a motor disorder which may develop in neural disorders like Complex 

Regional Pain Syndrome (CRPS) and entails sustained muscle contractions that result in

abnormal postures. 

It is often hypothesized that a lack of inhibition results in hyperreflexia (exaggerated reflexes), 

effectively causing dystonia. Humans control their movements using proprioceptive feedback 

from muscle afferents: muscle force by Golgi tendon organs and muscle stretch by muscle 

spindles. To assess whether proprioceptive reflexes can explain the features of dystonia a 

neuromusculoskeletal model was developed. 

The model describes the human wrist joint with an antagonistic muscle pair and proprioceptive 

reflexes originating from Ia, II and Ib afferents: muscle stretch, stretch velocity and force. 

Various scenarios were tested and the results were rated to criteria based on characteristic 

features of dystonia: abnormal posture, sustained muscle contraction, increased stiffness, activity 

dependent rigidity and loss of supraspinal control. 

The reflex mechanisms were investigated in three scenarios: 1) hyperreflexia, overexaggerated 

reflex sensitivity in both antagonists; 2) biased imbalance, a constant bias to the reflex output of 

one of the antagonists; and 3) strength imbalance, increased reflex sensitivity in only one of the 

antagonists. 

With hyperreflexia, the model shows that despite distinct motor dysfunction, none of the reflex 

pathways can fully account for dystonia, since none of them result in an abnormal posture. The 

imbalance in the other two scenarios was introduced to provoke an abnormal posture. Although 

biased imbalance does result in an abnormal posture, again it can not fully account for dystonia, 

since other criteria are no longer satisfied. Nevertheless, strength imbalance closely resembles 

the features of dystonia: effectively satisfying all criteria. 

In conclusion dystonia may be explained by an imbalance in excitatory muscle force feedback. 

The other reflex pathways did not satisfy the criteria for dystonia, but based on the oscillatory 

characteristics may explain features in clonus, tremor, etc. 

This study shows that dystonia may be explained by proprioceptive reflexes, however it does not 

rule out other candidates like hyperactive pain withdrawal reflexes. More research is needed to 

elucidate the cause of dystonia. 

Disclosures: W. Mugge, None; A.C. Schouten, None; F.C.T. van der Helm, None. 

Poster 

248. Dystonia 




Support: NIH Grant

Title: Rapid onset dystonia-parkinsonism requires interaction between dysfunctional cerebellar 

and basal ganglia motor control loops 

Authors: *D. P. CALDERON, K. KHODAKHAH; 

Neurosci., Albert Einstein Col. Med., Bronx, NY 

Abstract: Dystonia is characterized by co-contraction of opposing agonist and antagonist 

muscles and can be acquired or genetic in origin. One of the most intriguing types of dystonia is 

Rapid-onset Dystonia-Parkinsonism (RDP) or DYT12, which is caused by mutations in the α3 

isoform of the sodium/potassium ATPase pump (sodium pump). Subjects carrying this mutated 

gene show a sudden onset of symptoms often triggered by an extremely stressful 

experience.These symptoms typically include dystonia, parkinsonism, dysarthria, dysphagia, 

slurred speech, postural instability, and wide stance. 

Even though the genetic basis of this disorder is known, it is currently unclear how dysfunction 

of the sodium pump results in RDP. Furthermore, the anatomical brain structures that 

malfunction in this disease have not been identified. Many of the symptoms such as dystonia and 

parkinsonism suggest the involvement of the basal ganglia in this disorder. However, other 

prominent symptoms such as a wide stance or slurred speech are most frequently caused by 

cerebellar dysfunction. We took advantage of a potent and exquisitely selective blocker of 

sodium pumps to generate a pharmacological animal model of RDP in mice. We found that 

concurrent local chronic perfusion of the basal ganglia and the cerebellum with ouabain 

concentrations that partially blocked sodium pumps accurately reproduced the features of RDP. 

Prior to a stressful event the mice showed some reduced locomotion and mild ataxia/gait 

disturbance. However, only extreme stress triggered severe dystonia, as determined by 

electromyogram recordings. Dystonia persisted for days after the stressful episode. Remarkably, 

we found that an interaction between dysfunctional basal ganglia and cerebellar motor loops is 

essential to reproduce RDP. While perfusion of ouabain in the basal ganglia or cerebellum alone 

produced clear, but distinct, motor phenotypes, stress did not worsen these phenotypes or trigger 

dystonia. This animal model highlights the concomitant involvement of cerebellar and basal 

ganglia motor control loops in RDP and may be valuable in understanding its etiology. 

Disclosures: D.P. Calderon, None; K. Khodakhah, None. 

Poster 

248. Dystonia 



Topic: C.03.j. Dystonia

Support: Deutsche Forschungsgemeinschaft Grant Ri 845/1-3 

Title: Density of enkephalin expressing striatal projection neurons and entopeduncular 

dynorphin immunoreactivity are unaltered in a model of paroxysmal dystonia 

Authors: *M. HAMANN, A. KREIL, A. RICHTER; 

Dept. of Vet. Med., Freie Univ. Berlin, Berlin, Germany 

Abstract: The pathophysiology of hereditary dystonia is still unknown, but it is regarded as a 

basal ganglia disorder. Recent studies in the dt sz hamster, an animal model of paroxysmal 

dystonia, demonstrated a reduced density of striatal GABAergic interneurons at an age of 

maximum severity of dystonia (30-42 days of life) in comparison to non-dystonic controls. The 

reduced density coincides with a decreased neuronal activity and an altered neuronal pattern in 

the entopeduncular nucleus (EPN), a basal ganglia output structure, which receives inhibitory 

input from the striatum via the so-called direct pathway. In the present study, the density of 

striatal met-enkephalin (ENK) expressing GABAergic neurons, which project to the globus 

pallidus (indirect pathway) and the immunoreactivity of dynorphin A (DYN), which is expressed 

in striatal neurons of the indirect pathway, were determined in dt sz and control hamsters by using 

an image analysis system in a blinded fashion to clarify a possible role of an altered ratio 

between striatal interneurons and projection neurons. Neither the density of striatal ENK + 

neurons nor the immunoreactivity of DYN, determined in the EPN, significantly differed 

between mutant and control hamsters. Consequently, alterations seem to be restricted to 

GABAergic interneurons in the dt sz mutant. Thus, the present results support the hypothesis that 

an altered ratio between striatal interneurons and projection neurons leads to imbalances in the 

basal ganglia network and results in a reduced neuronal activity and altered pattern in the EPN. 

Supported by the Deutsche Forschungsgemeinschaft (Ri 845/1-3). 

Disclosures: M. Hamann, None; A. Kreil, None; A. Richter, None. 

Poster 

248. Dystonia 





Title: Evidence for brain microstructural abnormalities in spasmodic dysphonia

Authors: *M. L. MAKHLOUF 1,2,3 , N. SHARMA 4,8,3 , T. J. MULTHAUPT-BUELL 4 , J. 

KUSTER 4 , R. E. HILLMAN 1,5,6,3 , A. J. BLOOD 2,4,3,7 ; 

1 Dept. of Hlth. Sci. and Technol., Harvard-MIT, Cambridge, MA; 2 Athinoula A. Martinos Ctr. 

for Biomed. Imaging, Charlestown, MA; 3 Harvard Med. Sch., Boston, MA; 4 Dept. of Neurol., 

5 Ctr. for Laryngeal Surgery and Voice Rehabil., 6 Dept. of Surgery, 7 Dept. of Psychiatry, 

Massachusetts Gen. Hosp., Boston, MA; 8 Brigham and Women's Hosp., Boston, MA 

Abstract: Spasmodic dysphonia (SD) is a disabling neurological disorder characterized by 

involuntary contractions of one or more muscles of the larynx during speech. SD is classified as 

a focal laryngeal form of the movement disorder, dystonia. Currently there is no specific test for 

SD and the disorder is often misdiagnosed as muscle tension dysphonia. We have previously 

shown an abnormal microstructural asymmetry medial to the pallidum in hand and cervical 

dystonia patients (Blood et al., 2006). To test whether this abnormality may be a biomarker for 

focal dystonia more generally, we investigated brain microstructure in SD. Diffusion tensor 

imaging (DTI) was used to screen for white matter abnormalities in regions between the 

pallidum and thalamus in two SD patients. Scanning was performed when botulinum toxin 

treatment was least effective (i.e. just before the patient‟s next set of injections). A region of 

interest (ROI) analysis was performed in which 20 a priori ROIs were drawn on the fractional 

anisotropy (FA) maps for each hemisphere and for each patient. Both SD patients exhibited an 

abnormal hemispheric asymmetry in a focal region where the ansa lenticularis fibers are known 

to project between the pallidum and thalamus. These findings indicate that a microstructural 

abnormality previously observed in focal hand and cervical dystonia patients may also exist in 

SD patients, suggesting the abnormality may be a potential biomarker for multiple forms of focal 

dystonia. 

Reference: Blood AJ, Tuch DS, Makris N, Makhlouf ML, Sudarsky LR, Sharma N. White matter 

abnormalities in dystonia normalize after botulinum toxin treatment. NeuroReport 2006; 

17(12):1251-5. 

Disclosures: M.L. Makhlouf, None; N. Sharma, None; T.J. Multhaupt-Buell, None; J. 

Kuster, None; R.E. Hillman, None; A.J. Blood, None. 

Poster 

249. Dyslexia, SLI, and Other Disorders of Cognition and Behavior 



Topic: C.05.e. Other 

Support: K. Leroy Irvis Fellowship

Title: Effects of early exposure to the antibiotic ceftriaxone on cognitive and social behavior 

later in life 

Authors: *M. MATTHEWS, B. MOGHADDAM; 

Neurosci., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: Ceftriaxone, a beta (β)-lactam class of antibiotics, has recently been shown to increase 

the glutamate transporter, GLT1, function in the rat brain and increase glutamate uptake. 

Glutamate plays a critical role in brain development. The common use of ceftriaxone in infants 

and young children for the treatment of bacterial infections can have negative effects on a 

developing brain because of ceftriaxone's potential ability to increase the brain's expression of 

glutamate transporters and its biochemical and functional activity early in development. This 

study explores the effects of pediatric ceftriaxone administration on cognitive and social 

behavior later in life by mimicking the administration of a common antibiotic to infants and 

small children for the treatment of various bacterial infections. To achieve this, 30 rat pups were 

either injected with ceftriaxone or vehicle during postnatal day 10, an age equivalent to a new 

born human, and were tested on tasks that measure social behavior, working memory and 

cognition performances at adolescence and again at adulthood. During adolescence, ceftriaxonetreated 

animals showed reduced social interaction compared to vehicle-treated animals. This 

difference was exacerbated in adulthood. Ceftriaxone-treated adolescent animals showed reduced 

open field locomotion compared to vehicle treated animals treated animals. No changes were 

observed in working memory. These findings indicated that early exposure to ceftriaxone during 

development may lead to long-term changes in social and anxiety related behaviors. 

Disclosures: M. Matthews, None; B. Moghaddam, None. 

Poster 




Topic: C.05.d. ADHD, SLI, dyslexia and other specific disorders of neurobehavior 

Support: HD 20806 

Title: Postnatal phenotypes following embryonic transfection of the candidate dyslexia 

susceptibility gene Kiaa0319 in rats 

Authors: *V. J. PESCHANSKY 1 , T. J. BURBRIDGE 1 , A. J. VOLZ 1 , C. G. FIONDELLA 2 , J. 

J. LOTURCO 2 , A. M. GALABURDA 1 , G. D. ROSEN 1 ;

1 Neurol., Beth Israel Deaconess Med. Ctr., Boston, MA; 2 Physiol. and Neurobio., Univ. of 

Connecticut, Storrs, CT 

Abstract: We have previously reported that disruption of candidate dyslexia susceptibility gene 

(CDSG) homolog function in the rat results in neuronal migration disorders. Most recently, we 

have shown that knockdown of Dyx1c1 and Dcdc2 function by in utero electroporation of short 

hairpin RNAs (shRNAs) targeted against these genes causes abnormal migration, cortical 

malformations and changes in neuronal morphology (Cereb Cortex, 17:2562,2007; 

Neuroscience, 152:723, 2008). Here we examine the postnatal phenotype resulting from 

embryonic disruption of another CDSG, KIAA0319, on neuronal migration and dendritic 

morphology of neocortical neurons. 

Embryos from pregnant rats (E16) were divided into four groups: Group 1 consisted of rats that 

were co-transfected with plasmids encoding short hairpin RNA (shRNA) targeted against 

Kiaa0319 along with eGFP. Group 2 was co-transfected with bicistronic plasmids encoding 

eGFP and the KIAA0319 protein, and with mRFP plasmids while Group 3 was co-transfected 

with Kiaa0319 shRNA, KIAA0319 overexpression-eGFP and mRFP. Group 4 was transfected 

with a scrambled version of the Kiaa0319 shRNA. Mothers were also injected with BrdU at E18. 

Animals were sacrificed at P21 and their brains were removed, cut in the coronal plane, and a 1in-10 

series of sections stained with Thionin. Adjacent series of sections were processed for 

immunohistochemical detection of eGFP, mRFP, BrdU, and the laminar markers Cux1, Foxp2 

and/or Ctgf. The position of transfected neurons was charted using Neurolucida, and a migration 

distance analysis performed. The dendritic trees from at least 45 neurons in each condition were 

traced and quantified using Neurolucida. 

Qualitative analysis revealed severe neuronal migration disorders in those rats embryonically 

transfected with shRNA targeted against Kiaa0319, including large white matter heterotopias. 

These heterotopias contained shRNA-transfected neurons as well as a number of non-transfected 

neurons, suggesting that there are non-cell autonomous effects of shRNA transfection. Many of 

the neurons in the heterotopias were Cux-1 and BrdU-positive, indicating misplaced layer 2-3 

neurons. Taken together, these results support the contention that there are both cell- and noncell-autonomous 

effects of embryonic CDSG shRNA electroporation. 

Disclosures: V.J. Peschansky, None; T.J. Burbridge, None; A.J. Volz, None; C.G. Fiondella, 

None; J.J. LoTurco, None; A.M. Galaburda, None; G.D. Rosen, None. 

Poster 




Topic: C.05.d. ADHD, SLI, dyslexia and other specific disorders of neurobehavior


Title: Assessment of episodic memory performance following early interference with a dyslexiarisk 

gene (Dyx1c1) in male Sprague-Dawley rats 

Authors: *C. E. CLEARY 1 , J. R. HINMAN 1 , K. DIPINTO 1 , D. MALLOY 1 , S. W. 

THRELKELD 1 , Y. WANG 1 , G. D. ROSEN 2 , J. J. CHROBAK 1 , R. H. FITCH 1 ; 

1 Univ. Connecticut, Storrs, CT; 2 BIDMC/Harvard Med. Sch., Cambridge, MA 

Abstract: Subtle alterations in the development of the neocortex are associated with cognitive 

deficits in humans and other mammals. A number of genes contribute to the development of the 

neocortex, and research into the behavioral phenotype associated with specific gene 

manipulations is advancing rapidly. Findings include evidence that anomalies in the human gene 

DYX1C1 may be associated with an increased incidence of dyslexia, and concurrent research has 

shown that the rat homolog for this gene modulates critical parameters of early cortical 

development. Moreover, recent studies have shown auditory processing and spatial learning 

deficits in rats following in utero transfection of an RNA interference vector (RNAi) of the rat 

homolog Dyx1c1 gene. In order to further pursue possible neurobehavioral relationships between 

genetic anomalies and subsequent behavioral disruptions that may be relevant to human clinical 

conditions such as dyslexia, the current study examined the effects of in utero RNAi of Dyx1c1 

on episodic memory performance in Sprague-Dawley rats. This task was chosen based on 

replicable evidence of short-term memory deficits in dyslexic populations, as well as more recent 

evidence of an association between memory deficits and DYX1C1 anomalies in humans. 

Episodic memory performance was assessed using a relatively novel match to place radial water 

maze task that allows evaluation of memory for a single brief (~4-10 second) swim to a new goal 

location each day. A ten-minute retention interval was used, followed by a test trial. All testing 

was performed with researchers blind to the subjects‟ treatment condition (Dyx1c1 RNAi or 

sham), which was revealed through the identification of red fluorescent protein in sham subjects 

and no fluorescence in Dyx1c1 RNAi subjects during post mortem brain tissue analysis. 

Dyx1c1 RNAi treated rats exhibited a subtle, but significant and persistent impairment (seen 

over 12 weeks of testing) compared to shams, with Dyx1c1 RNAi treated rats making more 

errors as a group, and a larger population of Dyx1c1 RNAi rats making one or more errors, 

compared to shams. Post-mortem histological analysis revealed the formation of hippocampal 

dysplasias, collections of unmigrated neurons in the white matter, and molecular layer ectopias 

in the Dyx1c1 RNAi treated rats. These findings demonstrate that disruption of the Dyx1c1 gene 

leads to disordered neuronal migration and results in subtle learning and/or memory deficits. 

Disclosures: C.E. Cleary , None; J.R. Hinman, None; K. DiPinto, None; D. Malloy, 

None; S.W. Threlkeld, None; Y. Wang, None; G. D. Rosen, None; J.J. Chrobak, None; R.H. 

Fitch, None. 

Poster





Support: Tourette Syndrome Association 

Mittelstaedt family 

NINDS K23 NS41920 

Title: Tourette syndrome blood gene expression profiles distinguish comorbid ADHD and OCD 

Authors: *L. LIT 1 , F. R. SHARP 2 , D. L. GILBERT 3 ; 

2 Neurol. and M.I.N.D. Inst., 1 Univ. California Davis, Sacramento, CA; 3 Pediatric Neurol., 

Cincinnati Children‟s Hosp. Med. Ctr., Cincinnati, OH 

Abstract: Tourette syndrome (TS) is characterized by verbal and motor tics that occur for more 

than one year and are not attributed to another condition. TS is often comorbid with attention 

deficit hyperactive disorder (TS+ADHD), obsessive-compulsive disorder (TS+OCD) or both 

(TS+ADHD/OCD). Underlying dysfunctional cortico-striato-thalamo-cortical (CSTC) circuitry, 

affecting functionally distinct yet anatomically overlapping regions, is suspected to contribute to 

this comorbidity. All three disorders reflect failure to inhibit unwanted actions: motor/verbal tics 

(TS), repetitive behaviors (OCD), or inappropriate behaviors (ADHD). Elucidation of molecular 

mechanisms is needed to understand etiology and pathophysiology and develop specific 

treatments. Here we show that TS-only, TS+ADHD, TS+OCD, and TS+ADHD/OCD have 

distinct blood RNA expression profiles based upon the expression of 400-500 genes. These 

findings support a distinct pathophysiology and pharmacology of ADHD and OCD within TS. 

Moreover, the data is the first to suggest two distinct biological types of ADHD and two types of 

OCD. One type of ADHD and one type of OCD occur when they are co-morbid with TS alone 

(TS+ADHD1 or TS+OCD1); and the second types of ADHD and OCD occur when both are 

comorbid with TS (TS+ADHD2/OCD2). 

Disclosures: L. Lit , None; D.L. Gilbert, None; F.R. Sharp, None. 

Poster 






Title: Embryonic methylazoxymethanol (MAM) exposure leads to rapid auditory discrimination 

and learning impairments in rats 

Authors: *S. W. THRELKELD 1 , C. A. HILL 1 , C. CLEARY 1 , G. D. ROSEN 2 , R. H. FITCH 1 ; 

1 Univ. Connecticut, Storrs, CT; 2 BIDMC/Harvard Univ., Boston, MA 

Abstract: Developmental malformations of neocortex (microgyria, ectopia) are associated with 

reading impairments in humans, and with rapid auditory processing deficits in rodent models. It 

has further been suggested that language-based learning impairments in humans (e.g., dyslexia) 

could result from underlying abnormalities in auditory temporal processing. In rodent models, 

deficits in rapid auditory processing can be induced via disruption of cortical plate formation 

through genetic, epigenetic or injury-based manipulations. Interestingly, recent human clinical 

evidence suggests that periventricular nodular heterotopias (PNH) are also associated with poor 

reading skills (in the presence of normal intelligence; Chang et al., 2005). We sought to extend 

our rodent model by assessing the behavioral effects of PNH, induced via embryonic day (E) 15 

methylazoxymethanol (MAM) treatment, on auditory processing and maze learning in rats. Male 

sham and MAM groups were tested with simple and complex auditory stimuli in juvenile (P22- 

P45) and adult (P60+) periods using a modified startle response paradigm. At the conclusion of 

acoustic testing, a subset of subjects was evaluated for spatial and non-spatial learning on two 

water maze tasks. Juvenile results showed no difference in the detection of silent gaps in white 

noise, indicating simple temporal processing remained intact in MAM subjects as compared to 

shams. However, on an oddball two-tone discrimination task, MAM subjects were significantly 

impaired relative to shams at detecting the stimulus reversal across interstimulus intervals (ISI) 

of 275 and 225 ms (p < .01). In adulthood, both groups were again tested for oddball two-tone 

discrimination and also an oddball FM sweep detection task. Importantly, deficits in oddball 

two-tone detection observed in juvenile MAM subjects were no longer seen in adulthood. 

However, on the novel FM sweep discrimination task, MAM subjects were significantly 

impaired relative to shams at detecting sweep reversals of 225, 175, 125 and 75 ms (p < .001). In 

addition, maze results showed a significant effect of MAM on spatial (p = .05) but not nonspatial 

water maze learning (MAM worse than shams). Overall results indicate significant 

auditory temporal processing impairments in juvenile and adult rats with E15 MAM exposure. 

These data show that E15 MAM exposure produces robust but selective deficits in the 

discrimination of complex acoustic stimuli and spatial learning, which may parallel deficits 

observed in human learning impaired populations. 

Disclosures: S.W. Threlkeld , None; C.A. Hill, None; C. Cleary, None; G.D. Rosen, 

None; R.H. Fitch, None.

Poster 





Support: Eemil Aaltonen Foundation 

Centre of Excellence program of Academy of Finland (44858 and 213486) 

Title: Training with speech sounds affects electric brain responses of dyslexic children 

Authors: *P. H. LEPPANEN 1 , S. TUOVINEN 1 , J. HAMALAINEN 2 , A. TANSKANEN 1 , A. 

OKSANEN 1 , K. LOHVANSUU 1 ; 

1 Dept Psychol, Univ. Jyvaskyla, Jyvaskyla, Finland; 2 Ctr. for Neurosci. in Educ., Univ. of 

Cambridge, Cambridge, United Kingdom 

Abstract: Dyslexia, impairment in learning to read, is faced by 5-10 percent of population. In a 

sub-group of dyslexic children, an underlying problem is suggested to be related to speech 

perception and phonemic representations, which in turn affect grapheme-phoneme conversion 

ability. Here we studied, using auditory brain event-related potentials (ERPs), how combined 

training of low level perceptual skills (bottom-up processing) followed by training of higher 

level skills of linking speech sounds to letters (top-down processing) would affect speech 

processing in 9-10 year-old dyslexic children. The focus in speech perception training was in 

temporal differences. Combined training group (N=10) received speech perception training 

which was followed by grapheme-phoneme training. G-P-only training group (N=9) received 

grapheme-phoneme training only. Matched control group (N=10) attended only to regular school 

based educational program and did not receive any training. Training effects were examined 

from the ERPs before and after training periods. Naturally produced pseudoword stimuli with 

varying stop consonant durations (ata and atta, with silent gaps of 95 ms and 255 ms, 

respectively) were presented in a passive oddball paradigm with an interstimulus-interval of 610 

ms. The analyses of current source density maps of the N250 response at about 250 ms after the 

initial vowel or second syllable onset revealed training effects. N250 for the initial /a/-vowel 

(followed with both short and long stop consonants) showed larger increase in the area involving 

negative fronto-central activation in the post-training measurement in Combined training group 

than in the other two groups. The same pattern was observed also for the second syllable /ta/ 

following the short gap. This topographical pattern is in line with studies showing that N250 

shifts towards fronto-central distribution with maturation in typical readers. The ERP related to 

the differentiation between short and long /t/-duration (in ata vs. atta) showed larger area changes 

in both training groups in comparison to Matched control group. The results thus showed 

training effects on basic auditory processing, manifested in changes of brain responses to the 

stimulus onset, and on rapid rate auditory processing (of the second syllable onset following a

short silent gap), as well as in pre-attentive discrimination of temporal differences. Overall, these 

findings suggest that in dyslexic children training of bottom-up processes of speech perception 

results in larger changes in brain activation for speech cues than training of mere higher level, 

top-down, processes. 

Disclosures: P.H. Leppanen, None; S. Tuovinen, None; J. Hamalainen, None; A. Tanskanen, 

None; A. Oksanen, None; K. Lohvansuu, None. 

Poster 





Support: Joseph Young, Sr. Fund from State of Michigan 




Title: Neural substrates of number processing are impaired in young adults prenatally exposed to 

alcohol 

Authors: *M. J. AVISON 1 , E. E. GARCIA 1 , N. C. DODGE 3 , M. J. BURDEN 3 , R. L. 

COWAN 2 , R. KLORMAN 4 , S. W. JACOBSON 3 , J. L. JACOBSON 3 ; 

1 Dept. of Radiology and Radiological Sci., 2 Dept. of Psychiatry, Vanderbilt Univ. of Med., 

Nashville, TN; 3 Wayne State Univ. Sch. of Med., Detroit, MI; 4 Univ. of Rochester, Rochester, 

NY 

Abstract: Prenatal alcohol exposure (PNAE) is associated with specific deficits in number 

processing. Functional imaging studies have identified networks of left frontal (dorsolateral 

prefrontal cortex - dlPFC, precentral sulcus - PCS) and bilateral parietal (intraparietal sulcus - 

IPS, angular gyrus - AG) brain areas that are engaged in number processing. The goal of this 

study was to identify the neural correlates of impaired number processing in young adults with 

PNAE. Participants were 10 exposed and 8 non-exposed right-handed, African American young 

adults (18-20 yr) from the Detroit Prenatal Alcohol Exposure Cohort. Maternal alcohol and drug 

use were ascertained prospectively during pregnancy. The fMRI task consisted of an Exact

Addition paradigm with a letter matching control condition. Both the exposed and control groups 

performed well on the task (M % correct = 89.8 and 88.1, respectively, p > 0.50). Both groups 

activated the fronto-parietal networks previously identified; however the exposed participants 

showed significantly (p

Abstract: We have previously reported that disruption of candidate dyslexia susceptibility gene 

(CDSG) homolog function in the rat results in neuronal migration disorders. Most recently, we 

have shown that knockdown of Dyx1c1, Dcdc2 and Kiaa0319 function by in utero 

electroporation (IUE) of short hairpin RNAs (shRNAs) targeted against these genes causes 

abnormal migration, cortical malformations and changes in neuronal morphology (Cereb Cortex, 

17:2562, 2007; Neuroscience, 152:723, 2008). Previous work has also shown that spontaneous 

and induced cortical malformations including ectopias and microgyrias show altered connectivity 

(Cereb Cortex, 10: 1005, 2000; J Comp Neurol, 418:423, 2000). In order to more effectively 

determine connectivity in neurons in which CDSG function has been knocked down, our lab has 

developed a novel method of delivery of the wheat germ agglutinin (WGA) transgene using IUE. 

Rat embryos were assigned one of four groups at E16: Group 1 consisted of rats that were cotransfected 

with plasmids encoding short hairpin RNA (shRNA) targeted against the CDSGs, 

eGFP, and a plasmid encoding the WGA transgene (courtesy of Y. Yoshihara). Group 2 was cotransfected 

with bicistronic plasmids encoding eGFP and the CDSG protein, along with the 

plasmids containing the WGA transgene and mRFP, while Group 3 was co-transfected with 

plasmids containing CDSG shRNA, CDSG protein and the WGA transgene. Group 4 was cotransfected 

with a scrambled version of the CDSG shRNA, the WGA transgene, and eGFP. 

Animals were sacrificed at postnatal day 21 and their brains removed and processed for 

immunohistochemical detection of WGA and fluorescent proteins (to identify transfected cells). 

WGA-positive neurons were charted throughout the brain using Neurolucida. Preliminary 

analysis reveals that WGA is expressed in transfected neurons, and is transported both intra- and 

interhemispherically. 

Figure Legend: Confocal microscopic images of postnatal rat showing two neocortical neurons 

(arrows) that were co-transfected with eGFP (A) and the WGA transgene (B, 

immunohistochemically stained)

Disclosures: G.D. Rosen, None; A.J. Volz, None; V.J. Peschansky, None; T.J. Burbridge, 

None; M. Paramasivan, None; J.J. LoTurco, None; A.M. Galaburda, None. 

Poster 





Support: Wellcome Trust 079314 

Title: Prenatal exposure to nicotine induces impulsivity and inattentiveness in adult rats 

Authors: T. SCHNEIDER 1 , L. BIZARRO 3 , P. ASHERSON 2 , *I. P. STOLERMAN 4 ; 

1 Section of Behavioural Pharmacol., 2 SGDP, Inst. of Psychiatry, King's Col. London, London, 

United Kingdom; 3 Dept. de Psicologia do Desenvolvimento e da Personalidade, Univ. Federal

Do Rio Grande do Sul, Porto Alegre, Brazil; 4 Section of Behav Pharmacol, Inst. Psych, Kings 

Col. London P049, London, United Kingdom 

Abstract: Inattention, hyperactivity, and poor impulse control (impulsivity) are the main 

characteristics of Attention-Deficit Hyperactivity Disorder (ADHD). About 60% of children 

diagnosed with ADHD retain the condition as adults. The aetiology of the disorder is not known. 

Recently, maternal cigarette smoking during pregnancy has been proposed as a risk factor for 

ADHD. Attention and impulsivity deficits in humans can be assessed by the continuous 

performance test (CPT) requiring responding to a specific visual stimulus. Children with ADHD 

show overall lower scores on this test due to increased impulsivity and inattention. The 

preclinical analogue of the CPT is the 5-choice serial reaction time test (5-CSRTT) that is 

performed in an operant chamber with one curved wall containing a horizontal array of five 

apertures. Nose-pokes into a briefly illuminated aperture are reinforced with food. This visual 

discrimination paradigm involves sustained, selective and divided attention, and impulsivity to 

varying extents, depending upon the scheduled task demands. Here we present the first 

experimental evidence of a link between prenatal exposure to nicotine and increased 

inattentiveness and impulsivity in adult rats. Pregnant Lister hooded rats consumed nicotine 

solution (0.06 mg/ml) throughout pregnancy as the only source of drinking water. The mean 

nicotine daily consumption was 4.2±0.5 mg/kg body weight. Weight loss in females exposed to 

nicotine was not more than 15%, the consequences of which need further investigation. Male 

offspring of these females were tested in adulthood using the 5-CSRTT. There was no difference 

between control- and nicotine- exposed animals during acquisition; however, at the final stage 

when a 1 s stimulus duration was used, the performance of rats prenatally exposed to nicotine 

was compromised during the first twelve days of training. These rats displayed decreased 

accuracy (inattentiveness; 77.1±3.5 vs. 84.9±2, p


Support: NICHD (P50 HD40095) 

Title: The anatomical profile of dyslexic females: a voxel-based morphometry study 

Authors: *T. M. GERNER 1 , A. E. WALL 1 , E. M. NAPOLIELLO 1 , L. D. FLOWERS 1,2 , G. F. 

EDEN 1 ; 

1 Ctr. for the Study of Learning, Georgetown Univ., Washington, DC; 2 Wake Forest Univ., 

Winston-Salem, NC 

Abstract: Developmental dyslexia is a neurologically-based specific learning disorder affecting 

7-12% of the population. It is characterized by reading deficits unrelated to intellectual ability or 

access to instruction and is more prevalent in males (Rutter et al., 2004). Structural imaging 

studies have revealed regions of reduced grey matter volume in dyslexia (Eckert 2004). These 

investigations have been limited to groups of purely male participants or groups consisting of 

both males and females. The former have revealed left lateralized differences and the latter 

(mixed groups) have shown bilateral temporal lobe differences in dyslexia. However, anatomical 

differences have not yet been examined in purely female dyslexic samples, although it is known 

that the functional anatomy of language in women is different from that found in men (Shaywitz 

1995), suggesting that the brain-basis for a language learning disability in women might have a 

unique neuroanatomical manifestation. 

Using Voxel-Based Morphometry (Ashburner & Friston, 2000) we compared 8 adult dyslexic 

females (age = 24.8 years, SD = 1.4) and 8 adult typically-reading females (age = 22.1 years, SD 

= 2.6). We found decreased grey matter density in right superior parietal lobe and right inferior 

temporal gyrus in addition to the previously described differences in left inferior frontal gyrus 

and left superior parietal lobe. To determine whether these right hemisphere differences are also 

present in children, we compared 9 female children with dyslexia (age = 9.5 years, SD = 1.3) and 

8 typically-reading females (age = 10.1 years, SD = 3.0) employing the same analysis as that 

used in the adults. Large areas of reduced grey matter density were found in the dyslexic group 

in the inferior frontal gyrus, bilaterally. Also, we observed differences in left inferior and middle 

temporal gyrus and cerebellum, as well as right hemisphere cingulate gyrus, insula and inferior 

temporal gyrus. 

Together these results demonstrate that reduced grey matter density is observed in females with 

dyslexia in the left inferior frontal and right inferior temporal gyri as children and adults. 

However, an adult-specific morphometric difference in bilateral parietal grey matter density 

suggests an age-dependant anatomical difference in females with dyslexia. This age-dependant 

change in parietal cortices could be induced developmentally or as a consequence of 

compensatory mechanisms associated with difficulties in reading acquisition. We conclude that 

the brain basis for dyslexia varies as a function of gender and age. 

Disclosures: T.M. Gerner, None; A.E. Wall, None; E.M. Napoliello, None; L.D. Flowers, 

None; G.F. Eden, None.

Poster 





Support: NIH Grant R03HD050372 

NIH Grant R01HD42527 

Title: Changed motor cortical excitability in children with Developmental Coordination Disorder 

Authors: *F. A. KAGERER 1,2,3 , J. WHITALL 2 ; 

1 Dept Kinesiol, Grad Prog Neurosci, Univ. Maryland, College Park, MD; 2 Dept. of Physical 

Therapy & Rehabil. Sci., Univ. of Maryland, Baltimore, MD; 3 NACS Program, Univ. of 

Maryland, College Park, MD 

Abstract: Recent studies in adults and children have demonstrated that cortical inhibition is 

altered in attention-deficit/hyperactivity disorder (ADHD). Little is known whether and how 

other developmental dysfunctions such as Developmental Coordination Disorder (DCD) show 

altered characteristics of cortical excitability and inhibition, even though certain motor 

behavioral patterns - increased movement times, or increased occurrence of mirror movements - 

would support this notion. Using transcranial magnetic stimulation (TMS), the present study 

investigated whether cortical excitability parameters in children with DCD are different from 

those in typically developing children. 

Three typically developing children (between 8.5 and 11.6 years old) and two children with DCD 

(9.9 and 10.9 years old) were assessed with single- and paired-pulse TMS, using a Magstim 200 

magnetic stimulator (Magstim, UK), and a figure 8-shaped coil (45 mm inner diameter). 

Electromyogram (EMG) signals were recorded from the abductor pollicis brevis (APB) muscle 

in both hands with a sampling rate of 2kHz, amplified, and filtered at 30 Hz and 1 kHz. During 

stimulation, the APB was activated with 25% of the maximum voluntary contraction. Using the 

single pulse paradigm, active motor thresholds for both hands were determined. Using the shortinterval 

cortical inhibition (SICI) paradigm, intracortical inhibition and facilitation were 

determined, using interstimulus intervals of 2ms and 16ms, and conditioning and test stimuli at 

20% below and 20% above the active motor threshold, respectively. 

The preliminary findings indicate that children with DCD show substantially higher active motor 

thresholds than the typically developing children in both hemispheres. Assessment of SICI in the 

typically developing children showed that a 2ms interstimulus interval between conditioning and 

test stimulus produced a suppression of the motor evoked potential (MEP) amplitude, compared 

to MEP amplitudes resulting from single pulse stimulation at the same stimulation intensity. In 

the children with DCD, suppression was found only for the dominant hand, whereas stimulation 

of the non-dominant hand (or hemisphere) did not show this clear pattern.

Disclosures: F.A. Kagerer, None; J. Whitall, None. 

Poster 




Topic: C.05.c. Non- syndromic forms of general cognitive dysfunction. 


NSF9728742 

KAKENHI 18591156, 20591224 

the Second Research Project of Kochi University 

Inamori Foundation 

Title: Mice lacking a mental retardation gene, motopsin/neurotrypsin/prss12, show enhanced 

social behavior and impaired spatial memory 

Authors: *S. MITSUI 1 , Y. OSAKO 1 , F. YOKOI 2 , M. T. DANG 3 , K. YURI 1 , Y. LI 2 , N. 

YAMAGUCHI 4 ; 

1 Dept Neurobiol & Anat, Kochi Med. Sch., Nankoku, Japan; 2 Univ. of Alabama, Birmingham, 

AL; 3 Beckman Inst., Univ. of Illinois, Urbana-Champaign, IL; 4 Mam Flora Hosp., Nagaokakyo, 

Japan 

Abstract: Motopsin is a mosaic serine protease secreted from neuronal cells in various brain 

regions including the hippocampus. The loss of motopsin function causes nonsyndromic mental 

retardation in humans and impairs long-term memory formation in Drosophila. Here we describe 

behavioral phenotype of motopsin-deficient mice. A social recognition test showed that motopsin 

knockout mice had the ability to recognize two unfamiliar mice, however, we noticed that they 

investigated an unfamiliar mouse for a longer time than wild-type mice did. In a resident-intruder 

test, the knockout mice showed significantly prolonged social interactions with an intruder 

mouse compared with wild-type mice. In particular, the mutant mice showed longer sniffing 

during the first minute after an intruder mouse was presented. In a social novelty test, motopsin 

knockout mice spent a longer time investigating a familiar mouse than wild-type mice did, 

although the mutant mice discriminated the familiar and the unfamiliar mice. In addition to the 

abnormal social behaviors, motopsin-deficiency moderately impaired spatial memory. Consistent

with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not 

on basal dendrites, of hippocampal pyramidal neurons of motopsin knockout mice. Social 

interaction and spatial learning induced the phosphorylation of cAMP responsive element 

binding protein (CREB) in hippocampal neurons of wild-type mice, whereas phosphorylated 

CREB was barely detected in mutant mouse brains. Our results indicate that motopsin 

preferentially affects social interest and spatial memory by modulating the functions of 

hippocampal neurons. 

Disclosures: S. Mitsui, None; Y. Osako, None; F. Yokoi, None; M.T. Dang, None; K. Yuri, 

None; Y. Li, None; N. Yamaguchi, None. 

Poster 




Topic: C.05.c. Non- syndromic forms of general cognitive dysfunction. 

Support: EU IP LSHG-CT-2003-035367 

Title: Characterisation of MEGAP/SrGAP3 mental retardation mouse model 

Authors: *R. WALTEREIT 1 , U. LEIMER 1 , V. ENDRIS 3 , O. VON BOHLEN UND 

HALBACH 4 , J. PANKE 1 , V. BAIER 1 , S. TILL 5 , J. HADERER 1 , S. KUTSCHERJAWY 1 , S. 

NESCHOLTA 1 , M. HRABÉ DE ANGELIS 6 , A. SARTORIUS 2 , G. RAPPOLD 3 , D. 

BARTSCH 1 ; 

1 Mol. Biol., 2 Translational Imaging, Central Inst. Mental Hlth., Mannheim, Germany; 3 Human 

Genet., 4 Neuroanatomy, Univ. of Heidelberg, Heidelberg, Germany; 5 Ctr. for Neurosci. Res., 

Univ. of Edinburgh, Edinburgh, United Kingdom; 6 Inst. of Exptl. Genet., Helmholtz Zentrum 

München, München, Germany 

Abstract: Mental retardation (MR) affects up to two percent of the population and is 

accompanied by impaired learning and memory. MR is frequently associated with mutations in 

genes involved in Rho signal transduction cascades. Rho signal transduction pathways regulate 

axonal and dendritic growth during embryonic development and morphological adaptations of 

dendritic spines underlying synaptic plasticity in adult neurons. Loss of function mutation of the 

MEGAP/SrGAP3 (Mental disorder associated GAP protein) gene was diagnosed in a patient 

with MR and ataxia. The MEGAP protein is part of the Rho signal transduction cascades. To 

obtain a mouse model to study MEGAP depletion, we have generated a knockout mouse. 

Homozygous knockout mice are viable, but about 30% mice develop a hydrocephalus and die

within 45 days after birth. During the intrauterine period, about half of the homozygous mice do 

not come to term. To further characterise MEGAP ablation, we assessed neuroanatomy and 

neuronal migration, emotional and cognitive behaviour, and we studied Rho signal transduction 

cascades in brain and primary neuronal cell cultures of homozygous and heterozygous knockout 

mice and their wild-type littermates. MEGAP knockout mice show reduced activity during daytime 

period and increased anxiety in the light/dark-box. Normal behaviour was observed in 

resident-intruder test, contextual and auditory cue fear conditioning and susceptibility to 

chemically-induced seizure. They also present increased phosphorylation of p44/42-MAPK in 

the hippocampus. Further results from behavioural, histological and molecular analysis will be 

presented, along with discussion of the findings for the role of MEGAP in neuronal migration 

and mental retardation. 

Disclosures: R. Waltereit, None; U. Leimer, None; V. Endris, None; O. von Bohlen und 

Halbach, None; J. Panke, None; V. Baier, None; S. Till, None; J. Haderer, None; S. 

Kutscherjawy, None; S. Nescholta, None; A. Sartorius, None; G. Rappold, None; D. Bartsch, 

research grant (principal investigator), B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); M. Hrabé de Angelis, None. 

Poster 





Support: NIH R01NS054994 

Title: Decreased number of parvalbumin and cholinergic interneurons in the striatum of 

individuals with Tourette Syndrome 

Authors: *Y. KATAOKA 1 , H. GRANTZ 1 , C. SAPER 3 , J. F. LECKMAN 1 , F. M. 

VACCARINO 1,2 ; 

1 Child Study Ctr., 2 Neurobio., Yale Univ. Sch. Med., New Haven, CT; 3 Neurol. & Neurosci., 

Harvard Med. Sch., Boston, MA 

Abstract: A 5% decrease in caudate (Cd) nucleus volume is the most consistent structural 

finding in the brain of patients with Tourette Syndrome (TS), but the cellular abnormalities that 

underlie this decrease are not known. The density of different types of interneurons and medium 

spiny neurons (MSNs) was assessed in the postmortem brains of TS subjects as compared with 

matched normal controls (NC) by unbiased stereological analyses. TS patients demonstrated a

50-60% decrease of both parvalbumin (PV)+ and choline acetyltransferase (ChAT)+ cholinergic 

interneurons in the head of the Cd. ChAT+ interneurons were also significantly decreased in the 

body of the Cd, whereas in the Putamen (Pt), only PV+ interneurons were significantly 

decreased. There was no significant effect of TS diagnosis on DARPP-32+ MSN density in these 

regions. The decrease in the number of PV+ and ChAT+ interneurons in the striatum of TS 

subjects suggests an impaired cortical and thalamic control and modulation of striatal neuron 

firing in TS. The deficiency of these crucial cell populations may lead to the first animal model 

of TS and to novel approaches in TS treatment. 

Disclosures: Y. Kataoka, None; H. Grantz, None; C. Saper, None; J.F. Leckman, 

None; F.M. Vaccarino, None. 

Poster 





Support: WFUSM Small Animal MR Imaging Facility 

NIH Grant HD050201 

WFUSM Parker Neurosciences Research 

Title: MR spectroscopic analysis of neonatal rat striatum: An early biomarker for adult 

hyperemotionality following perinatal asphyxia 

Authors: *A. E. RONCA 1 , G. A. KLEVEN 1 , C. TULBERT 1 , G. G. WINN 1 , T. F. ATWOOD 2 , 

J.-M. ZHU 2 ; 

1 Dept Obstetrics/Gynecol, 2 Dept Radiation Oncology & Biomed. Engin., Wake Forest Univ. 

Sch. Med., Winston Salem, NC 

Abstract: In vivo proton (1H) MR spectroscopy (MRS) can be used to identify neurological 

impairments in human and animal subjects. We hypothesized that neurochemical profiles 

acquired from one-week-old perinatally asphyxiated (PA) rats could be used to predict adult 

hyperemotionality. Gestational day 22 pregnant rat dams were administered spinal anesthesia 

and the uterus externalized into a heated (37.5 C) saline bath. Controlled asphyxia was produced 

by occluding the blood supply feeding one uterine horn (12 min). The other uterine horn 

remained undisturbed (non-occluded control). Fetuses were immediately delivered by cesarean

section, then fostered to newly parturient dams. On Postnatal day 7, high resolution in vivo 

proton MR spectra were acquired from the striatum using a 7T small animal MRI scanner using 

single-voxel (3 x 3 x 3 cu mm) MRS imaging. The point-resolved spectroscopy (PRESS) 

sequence was applied, and positioning of MRS voxels based on T2-weighted images. 

Neurometabolites derived from spectral analysis were corrected for creatine + phosphocreatine 

(Cr + pCr). Significant changes in N-acetyl aspartate (NAA) and glutamate and glutamine (Glx) 

were observed in PA rats as compared to non-asphyxiated controls. At 8-weeks of age, subjects 

were singly housed (24hrs), placed in an open field and exposed to a 52min test comprised of a 

concatenation of novel stimuli. Videographic analysis revealed magnified emotional responses to 

novelty in PA rats as measured by position within the open field, responses to novel stimuli, and 

social interactions. In vivo MRS is a sensitive imaging tool for detecting neurochemical changes 

in early life that can be used to predict adult hyperemotionality. 

Disclosures: A.E. Ronca , None; G.A. Kleven, None; C. Tulbert, None; G.G. Winn, 

None; T.F. Atwood, None; J. Zhu, None. 

Poster 



Program#/Poster#: 249.16/V1 


Support: NIH AA009838 

Title: Rats exposed to binge-like ethanol on PD4-9 show altered basal dendritic complexity in 

medial prefrontal cortex neurons 

Authors: *G. F. HAMILTON, L. T. WHITCHER, A. Y. KLINTSOVA; 

Univ. Delaware, Newark, DE 

Abstract: Prenatal exposure to alcohol in humans can result in a wide range of deficits 

collectively referred to as Fetal Alcohol Spectrum Disorders. Of these deficits, cognitive 

impairments are among the most debilitating and long-lasting. Moreover, cognitive impairments 

in executive functioning suggest damage to the prefrontal cortex (PFC). Recent findings 

demonstrate that long-term potentiation (LTP) in medial PFC (mPFC) could not be induced in 

the animal model of binge-like drinking during the third trimester equivalent (postnatal days 4-9, 

PD4-9). Induction and maintenance of LTP is known to lead to more complex interactions 

among neurons via increased dendritic tree complexity, branch length and spine density (Yuste 

& Bonhoeffer, 2001; Nimchinsky et al., 2002). It is known that several external stimuli, such as

morphine, nicotine, caffeine, chronic stress, and maternal stress have been found to alter the 

dendritic structure of cells within the PFC. Three groups of rat pups were used in this study: 

intubated with alcohol (5.25g/kg/day; AE - alcohol exposed), sham intubated (SI), or remained 

with the mother (SC - suckle control) on PD4-9. On PD26-30 rats were anesthetized and 

perfused with phosphate buffer and brains were processed for Golgi-Cox staining. Basal dendrite 

complexity (Sholl analysis) and spine density were evaluated for layer III neurons in the mPFC 

using NeuroLucida software (MicroBrightField, Inc.). 

Results indicate that AE rats have an altered basal dendritic tree complexity due to both a 

significant decrease in the number of intersections in proximity to the neuronal soma and a 

significant decrease in total length of basal dendrites. These effects were not seen in the apical 

dendrites. Moreover, spine density patterns show an increase in Order 4 basal branches, whereas 

a significant decrease was found in Order 5 apical branches in the previous work. Together, this 

indicates that the innervations of the soma and basal dendrites by thalamic projections may play 

a role (Shibata, 1993; Cerqueira et al., 2007). Thus, our data demonstrates that postnatal 

exposure to alcohol produces permanent changes in the prefrontal cortex neuronal organization 

that may affect the performance on behavioral tasks. Supported by NIH AA009838. 

Disclosures: G.F. Hamilton , None; L.T. Whitcher, None; A.Y. Klintsova, None. 

Poster 





Support: Conte Center grant P50 MH077970 

John C & Edw. Coleman Memorial Fund 

Title: Cortical biasing by zona incerta 

Authors: *N. NAGARAJAN 1 , M. M. MERZENICH 2 ; 

1 UCSF, san francisco, CA; 2 Keck Ctr. for Integrative Neuroscience, OHNS, UCSF, San 

francisco, CA 

Abstract: The mechanisms by which cortical and sub-cortical structures prioritize relevant, 

novel stimuli in healthy individuals, and by which focused attention is disrupted by irrelevant, 

distracting stimuli in ADHD individuals are largely unknown. A large body of evidence has led 

to the hypothesis that specific sub-cortical structures are novelty or “alerting stimulus” detectors

that amplify and increase the selectivity of cortical responses to surprising stimulus events. 

However, the possible role for this sub-cortical biasing in the genesis of ADHD remains 

unexplored. To further understand this possible link, we have examined the role of the Zona- 

Incerta, a deep brain nucleus, in cortical plasticity and cortical map reorganization. Using a 

combination of electrical and sound stimulation paradigms we have determined that selective 

activation of ZI using electrical stimulation paired to variable sound frequencies results in the 

systematic re-tuning of cortical responses of the primary auditory cortex (A1) to lower 

frequencies (< 8 kHz). To explore the detailed function of ZI in inducing this cortical bias, we 

either passively exposed young (P0 up to P40) or trained adult rats (from P35 to 7 months of age) 

to recognize 5-8 kHz tonal stimuli. Our results demonstrate that passive exposure or behavioral 

training leads to an over-representation of cortical responses within this 5-8 kHz band. ZI 

stimulation in these animals results in an immediate and moderately persistent re-tuning of 

selective frequency responses to greatly exaggerate the < 5 kHz range. 

Disclosures: N. Nagarajan , None; M.M. Merzenich, None. 

Poster 





Support: COFIN-MIUR 2005 

Title: Excitatory amino acids in the forebrain of the Naples High-Excitability rats: behavioral 

effects of subchronic D-Aspartate and its Diethyl prodrug treatment 

Authors: A. DI PIZZO 1 , L. A. RUOCCO 2 , U. A. GIRONI CARNEVALE 2 , D. MELISI 3 , A. 

CURCIO 3 , S. DE LUCIA 3 , M. RIMOLI 3 , C. ARRA 4 , E. TOPO 1 , A. D'ANIELLO 1 , *A. G. 

SADILE 5 ; 

1 Biochem., Zoological Station A.Dohrn, Naples, Italy; 2 Exptl. Med., Second Univ. of Naples, 

Naples, Italy; 3 Pharmaceut. and Toxicol. Chem., Univ.Federico II, Naples, Italy; 4 Animal 

facility, Natl. Cancer Inst., Naples, Italy; 5 Exptl. Med., Second Univ. Napoles, Napoli, Italy 

Abstract: Naples High Excitability (NHE) rats model the mesocortical variant of ADHD. 

Differential display experiments have shown a higher expression of the NR1 subunit of NMDA 

receptors in the prefrontal cortex of NHE rats compared to Random Bred (NRB) controls. Aim 

of this study was to investigate whether this was associated to an altered level of putative 

excitatory amino acids, i.e. D and L glutammate and aspartate isomers. Thus, prepuberal NHE

and NRB rats were given a daily i.p. injection of D aspartate (D-Asp 0.5 M), or D-Asp dietilester 

hydrochloride (DEE 0.5 M) or vehicle for 17 days. One hour after the 15th injection rats 

were exposed to a spatial novelty, a Làt-maze, for 10 min. Horizontal (HA) and vertical (VA - 

freq. and duration) activity were monitored. On the 17th day 1h after last injection rats were 

exposed to a radial maze (Olton maze) for 10 min, sacrificed and brains dissected out as 

prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypotalamus (HYP). Amino 

acids were detected by HPLC in duplicates. Results indicate that D-Asp treated NHE rats 

showed a significant increment of HA. Conversely in NRB rats DEE treatment decreased it. No 

treatment effect was found for VA in NHE and NRB for frequency and duration of rearing. In 

addition, in the Olton maze no treatment effect was found on HA in both rat lines. In contrast, 

VA was decreased in NHE by D-Asp treatment whereas the same effect was obtained by DEE in 

the NRB. Furthermore D-Asp treatment impaired attention in NRB decreasing number of arm 

visited before first repetition. Moreover forebrain excitatory amino acids were higher in the NHE 

vs. NRB under basal conditions. Subchronic D-Asp or DEE treatment reduced D-Asp levels in 

the NHE and increased it in the NRB rats. In particular this effect pertained to all areas but 

hypotalamus in the NRB and to all areas but hippocampus in the NHE. Finally D-Asp level in 

the brain region was negatively correlated with behavioral components in both rats lines. In 

conclusion, these findings demonstrate differential effects of subchronic D-Asp and its diethyl 

prodrug. This in turn could be explained by the different amino acid basal level in NHE and 

NRB rats. 

Disclosures: A. Di Pizzo, None; L.A. Ruocco, None; U.A. Gironi Carnevale, None; D. Melisi, 

None; A. Curcio, None; M. Rimoli, None; C. Arra, None; E. Topo, None; A. D'Aniello, 

None; A.G. Sadile, None; S. De Lucia, None. 

Poster 





Support: NIH/NCRR P41 RR013642 

NIAAA U01 AA017122 

NIDA R21 DA15878 

NIAAA U24 AA014808

NIDA RO1 DA017831 

Title: Increased frontal activation during language processing in children with Fetal Alcohol 

Spectrum Disorders 

Authors: *C. NUNEZ 1 , M. DAPRETTO 1,2 , L. H. LU 4 , S. Y. BOOKHEIMER 2,3 , M. 

O'CONNOR 3 , E. R. SOWELL 1,2 ; 

1 Lab. of Neuro Imaging, UCLA Dept. of Neurol., Los Angeles, CA; 2 Interdepartmental Ph.D. 

Program for Neurosci., 3 Dept. of Psychiatry and Biobehavioral Sci., UCLA, Los Angeles, CA; 

4 Dept. of Psychology, Roosevelt Univ., Chicago, IL 

Abstract: Previous studies have documented language deficits in children with Fetal Alcohol 

Spectrum Disorders (FASD), but little is known about their functional activation during language 

processing. Recent reports indicate increased activation in frontal regions in FASD children on 

tasks with an attentional/working memory component compared to unexposed children. The 

purpose of this study was to examine brain activation differences during a sentence 

comprehension and judgment task in FASD children. Given previous findings, it was predicted 

that FASD children would demonstrate more activation in frontal regions than unexposed 

children. Twenty-nine children (ages 7-15, mean=11.0, SD=2.6; 12 females) participated in a 

study of neurocognitive development in FASD. Nine children with significant prenatal alcohol 

exposure comprised the FASD group, four of whom had facial dysmorphology sufficient for a 

full or partial Fetal Alcohol Syndrome diagnosis. Twenty children were in the unexposed group 

(CON). There were no group differences in age or gender. IQ was significantly lower in the 

FASD group. With fMRI at 3 Tesla (EPI gradient echo, slice thickness=3mm/1 skip, 36 total 

slices, TR=2.5s), participants performed a sentence comprehension and judgment task presented 

in a blocked design. They listened to pairs of sentences and decided if their meaning was the 

same. fMRI data were analyzed with FSL 3.3, and group effects for task vs. rest contrast were 

evaluated. Z-statistic images were thresholded with clusters determined by Z > 1.7 and a 

corrected cluster significance of p = 0.05. No group differences in reaction time or accuracy were 

observed. The FASD group had more activation than the CON group in bilateral, anterior and 

dorsal frontal regions. Since IQ was significantly lower in the FASD group, IQ as a covariate of 

non-interest was evaluated, and increased activation in bilateral frontal regions for the FASD 

group remained significant. Children with prenatal alcohol exposure had different patterns of 

cortical activation on a language processing task when compared to unexposed children. FASD 

children had increased activation in frontal regions, with effects independent of IQ differences. 

While both groups performed similarly during the task, FASD children appeared to rely more on 

frontal cortices to accomplish similar performance, and this may reflect differences in 

organization of language processing systems. Alternatively, activation in these areas may reflect 

differences in executive functioning in the FASD group, specifically with decision-making, as 

these regions are associated to executive functions in typically developing individuals. 

Disclosures: C. Nunez, None; M. Dapretto, None; L.H. Lu, None; S.Y. Bookheimer, 

None; M. O'Connor, None; E.R. Sowell, None.

Poster 





Title: Voluntary physical exercise produces sex-dependent changes in attentional orienting and 

social behavior in spontaneously hypertensive rats 

Authors: *M. SHARMA, M. E. HOPKINS, D. J. BUCCI; 

Psychological and Brain Sci., Dartmouth Col., Hanover, NH 

Abstract: Voluntary physical exercise improves cognition and increases neurogenesis and the 

levels of neurotrophic factors in hippocampus. We have recently examined the effects of exercise 

on cognitive function in spontaneously hypertensive rats (SHR), a well-studied model of 

Attention-Deficit/Hyperactivity Disorder (ADHD). SHRs exhibit locomotor hyperactivity and 

have deficits in attentional function, working memory, and response inhibition that are 

reminiscent of impairments typically observed in ADHD. Our recent studies also indicate that 

SHRs exhibit hyper-social behavior. The present study examined the effects of voluntary wheel 

running on attentional orienting and social interaction in male and female SHRs. Exercise and 

control rats were housed in groups of 4 and were 8 weeks old at the start of the experiment. Rats 

in the exercise groups were provided 24 hour access to a running wheel attached to the side of 

the home cage for 2 weeks prior to behavioral training; control rats did not have access to a 

wheel. In the attentional orienting task, rats were placed in individual chambers and first received 

12 non-reinforced presentations of a visual stimulus (a panel light) over the course of a single 

session. In all groups, presentation of the light evoked unconditioned orienting defined as rearing 

up on the hind legs towards the light. The unconditioned orienting response habituated over the 

course of the 12 trials as the rats learned that it had no behavioral relevance. Overall, female 

SHR rats exhibited more rearing behavior than males. Physical exercise reduced the amount of 

rearing observed in females but did not affect rearing behavior in males. In the second phase of 

the experiment the light was paired with the delivery of food reward. There were no effects of 

exercise on conditioned responding and male and female rats exhibited similar levels of 

conditioning. In the social interaction task, the same SHR rats were placed individually in an 

open field arena for 10 min with a Wistar-Kyoto rat (control strain) of the same sex. Exercise 

decreased the amount of social interaction exhibited by both male and female SHRs. Subsequent 

analyses indicated that there were no sex differences or effects of exercise on locomotor activity, 

indicating that the results of the orienting and social interactions studies cannot be attributed to 

exercise-induced changes in activity or fatigue. Together, these data indicate that exercise has 

beneficial effects on cognitive and social function in SHRs. Moreover, the sex differences in 

attentional orienting behavior are in line with recent studies indicating that females diagnosed 

with ADHD are more cognitively impaired than males.

Disclosures: M. Sharma , None; M.E. Hopkins, None; D.J. Bucci, None. 

Poster 





Support: COFIN MIUR 2005 

Title: Subchronic galactosylated dopamine desensitizes behavioral response to novelty in the 

Naples High-Excitability rats 

Authors: L. A. RUOCCO 1 , U. A. GIRONI CARNEVALE 1 , R. CONTE 1 , C. TRENO 1 , M. 

MUROLO 1 , C. ARRA 2 , D. MELISI 3 , A. CURCIO 4 , S. DE LUCIA 5 , M. RIMOLI 4 , *A. G. 

SADILE 6 ; 

1 Dept Exptl Med., Second Univ. of Naples, Naples, Italy; 2 Animal Facility, Natl. Cancer Inst. 

“G. Pascale” Fndn., Naples, Italy; 3 Pharmaceut. and Toxicol. Chem., Univ. “Federico II”, 

Naples, Italy; 4 Pharmaceut. and Toxicol. Chem., Univ. “Federico II”, Naples, Italy; 5 Pharmaceut. 

and Toxicol. Chem., Univ. of Naples “Federico II”, Naples, Italy; 6 Dept Exptl Med., Second 

Univ. Naples (SUN), Naples, Italy 

Abstract: Alterations of specific dopamine (DA) pathways have been connected to pathological 

conditions, such as Parkinson‟s disease and Attention Deficit Hyperactivity Disorder (ADHD). 

Galactosylated form of dopamine (GAL-DA) crosses the blood brain barrier and modulates 

activity of mesocorticolimbic system. This, in turn, leads to reduced hyperactivity and increase 

scanning time in the Naples High Excitability (NHE) rats, an animal model of the mesocortical 

variant of ADHD (Neurocience, vol. 152: 234-44, 2008). Since these effects followed an acute 

administration of 100 mg/kg of GAL-DA, aim of this study was to verify the plasticity of this 

response. Thus, adult male NHE rats, received GAL-DA (10, 100 mg/kg or vehicle) daily for 15 

days (exp 1) or 5 days (exp 2). In both cases, 3h after the last injection, rats were tested for 

activity and non-selective attention in the Làt-maze. The day after, rats were injected again and 3 

hours later tested in the Olton maze. In both experiments no effect was observed for indices of 

activity (horizontal and vertical) or scanning duration, in both spatial novelty setups i.e. Làtmaze 

and Olton maze. Moreover, in the radial maze no treatment effect was observed for number 

of arm visited before first repetition occurred (FE) and number of arm visited before completion 

of eight arm visit (NVTC). Besides, in the exp 1, 10 mg/kg of GAL-DA increased scanning 

duration in the second part of the Làt-maze test. Therefore, subchronic GAL-DA treatment

appears to desensitises response to novelty of NHE rats. This in turn may reveal a differential 

sensitivity of mesencephalic and forebrain sites to repetitive DA challenge. 

Disclosures: L.A. Ruocco, None; U.A. Gironi Carnevale, None; R. Conte, None; C. Treno, 

None; M. Murolo, None; C. Arra, None; D. Melisi, None; A. Curcio, None; S. De Lucia, 

None; M. Rimoli, None; A.G. Sadile, None. 

Poster 





Support: Finnish Cultural Foundation 

The Academy of Finland Grant 1211486 

Title: 6-year-old children at risk for dyslexia and the discrimination of 5 changes in speech 

sounds as determined with the multi-feature paradigm 

Authors: *R. K. LOVIO 1 , S. PAKARINEN 1 , M. HUOTILAINEN 1,2 , P. ALKU 3 , R. 

NÄÄTÄNEN 1 , T. KUJALA 1 ; 

1 Cognitive Brain Res. Unit, Dept.Psychology, Univ.Helsinki, Helsinki, Finland; 2 Finnish Ctr. of 

Excellence in Interdisciplinary Music Res., Univ.Jyväskylä, Jyväskylä, Finland; 3 Dept. of Signal 

Processing and Acoustics, Helsinki Univ. of Technol., Espoo, Finland 

Abstract: Objective: The aim of this study was to determine auditory processing skills of 

children having an elevated risk for dyslexia. Their cortical discrimination of 5 changes in 

Finnish syllables critical in speech perception and language development (consonant, vowel, 

vowel length, pitch and intensity changes) was compared with that of children without dyslexia 

risk. 

Methods: From a group of 59 pre-school-aged children, 19 children meeting the subject criteria 

and succeeding EEG measurements were selected for the final analyses. The parents were 

carefully interviewed about their family history of dyslexia. The children were tested in prereading 

skills. Children who had at least one close relative with dyslexia and problems in prereading 

skills were chosen to the “at risk” group (10 children). Children without family history of 

developmental reading or language disorders and with normally developing reading skills were 

chosen to the control group (9 children). Children‟s mismatch negativity (MMN) to five changes 

in speech sounds was recorded. The stimuli were presented in a multi-feature paradigm in which

five deviants are presented in an alternating fashion with the standards. Also data from a control 

oddball condition was recorded. 

Results: Children at risk for dyslexia had smaller MMN amplitudes than their controls for vowel, 

vowel length and intensity changes, both in the right and left side of the head, and smaller 

MMN-amplitudes for the consonant change in the right side of the head in the multi-feature 

paradigm. This suggests that the children at risk for dyslexia had difficulties in pre-attentive 

discrimination of various speech sound changes. 

Conclusions: The children at risk for dyslexia seem to have problems in discriminating 

consonant, vowel, vowel length and intensity changes in speech sounds. The present results are 

encouraging by suggesting that the multi-feature MMN paradigm with changes in speech sounds 

could be used for identifying sound-discrimination impairments already before the school age. 

Disclosures: R.K. Lovio, PhD. student Grant from the Finnish Cultural Foundation, B. 


grants already received); The Academy of Finland (grant number 1211486), C. Other Research 

Support (receipt of drugs, supplies, equipment or other in-kind support); P. Alku, Helsinki 

University of Technology, Department of Signal Processing and Acoustics, A. Employment (full 

or part-time); S. Pakarinen, Cognitive Brain Research Unit, Department of Psychology, 

University of Helsinki, A. Employment (full or part-time); The Academy of Finland, B. 


grants already received); M. Huotilainen, Finnish Centre of Excellence in Interdisciplinary 

Music Research, University of Jyväskylä, A. Employment (full or part-time); The Academy of 

Finland, B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received); T. Kujala, Cognitive Brain Research Unit, Department of 

Psychology, University of Helsinki, A. Employment (full or part-time); The Academy of 

Finland, B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received); R. Näätänen, The Academy of Finland, B. Research Grant 


received). 

Poster 




Topic: F.01.d. Language 

Support: NICHD Grant HD29891

Title: The neurobiological profile of adult disabled readers supports the phonological deficit 

hypothesis of dyslexia 

Authors: A. ZUMBERGE 1 , J. L. BRUNO 2 , J. G. GOLDMAN 2 , *C.-P. KO 1 , Z.-L. LU 2 , F. R. 

MANIS 2 ; 

1 Dept Biol. Sci., 2 Dept Psychology, USC, Los Angeles, CA 

Abstract: The cognitive profile of reading disability is marked by phonological deficits. The 

neurobiological profile that has emerged recently includes decreased activity in posterior brain 

regions such as the superior temporal gyrus (STG; which is associated with phonological access), 

and particularly the occipitotemporal region (OT; which is associated with orthographic 

processing). Researchers have interpreted increased activity in more anterior regions (inferior 

frontal gyrus; IFG) as compensation for deficits in orthographic and phonological processing. 

The present study aimed to replicate previous fMRI findings of reduced OT activation and 

elevated IFG activation in lower skilled adult readers. Regions of interest were localized on an 

individual basis using a separate block-design rhyming task, and BOLD responses within these 

regions during an event-related phonological lexical decision task were compared across skill 

groups and correlated with cognitive measures of reading and phonological skill. Surprisingly, 

no significant differences in activation pattern were found in OT, suggesting that dysfunction in 

OT is not always present in individuals with reading disability. In STG and IFG, the reading 

disabled group displayed deficits in phonological sensitivity; activation by phonologically 

familiar pseudohomophones was equivalent to that by phonologically unfamiliar pseudowords. 

Correlations between activation level and language skills were positive in STG and negative in 

IFG. These observations in regions commonly associated with phonological processing may 

account for the inefficient decoding and slow access to phonological representations observed in 

numerous behavioral studies. They suggest that, at least in adults functioning at the college level, 

persistent reading difficulties are related to deficits at the phonological, rather than orthographic, 

level. This lends neural support to the behaviorally-based phonological hypothesis for dyslexia. 

Disclosures: A. Zumberge, None; C. Ko , None; J.L. Bruno, None; J.G. Goldman, None; Z. 

Lu, None; F.R. Manis, None. 

Poster 





Support: NIDCD Intramural Research Program

Title: Functional neuroimaging and network-level abnormalities underlying foreign accent 

syndrome 

Authors: *J. A. RILEY 1 , J. A. BUTMAN 2 , W. IDSARDI 1 , A. R. BRAUN 3 ; 

1 Univ. Maryland CP, College Park, MD; 2 Diagnos. Radiology Dept., Clin. Ctr. of the Natl. Inst. 

of Hlth., Bethesda, MD; 3 Language Section, Natl. Inst. on Deafness and Other Communication 

Disorders, Bethesda, MD 

Abstract: We present a case study of a patient presenting with Foreign Accent Syndrome (FAS) 

with an unusual pattern of lesions. A highly focal unilateral lesion in motor cortex was revealed 

by structural magnetic resonance imaging (MRI), with another small, similar lesion in parietal 

cortex. A functional magnetic resonance imaging (fMRI) study was performed while the patient 

carried out hand, jaw, and tongue maneuvers. The results indicated that the lesion in primary 

motor cortex was located within the area representing the tongue. Combined with the parietal 

lobe damage, this lesion pattern is atypical of previous cases of FAS, as there is no indication of 

subcortical damage. 

A traditional phonetic analysis of the patient‟s productive deficits revealed classic productive 

symptoms of FAS. The results of this analysis are assessed within the context of the FAS 

literature. 

An additional series of fMRI studies will be conducted comparing activity in the patient with 

normal age-matched controls in order to investigate network-level abnormalities. These studies, 

along with the traditional phonetic analysis and lesion localization provided by the combined 

structural and 

functional MRI studies is likely to shed light on the neural mechanisms implicated in FAS. 

Figure 1. Extent of cortical 

laminar infarct (white arrows, A-C) in the expected location of the face representation of the 

motor strip. Left lateral (A) and supero-lateral (B) views demonstrate the extent of cortical 

laminar infarct as hyperintense signal (white arrow) confined to the precentral gyrus involving 

the expected location of the face representation on the motor strip. Focal cortical infarct is also 

noted in the parietal lobe (white arrowhead). BOLD fMRI activations (C) from fingertapping 

task alternating between the right (blue) and left (orange) hands confirm that the hand

epresentation is superior to the region of laminar cortical infarct (white arrow) involving the 

motor strip. Focal cortical infarct in the parietal lobe (white arrowhead) is also seen. 

Disclosures: J.A. Riley , None; J.A. Butman, None; W. Idsardi, None; A.R. Braun, None. 

Poster 





Support: MRC 

Title: Analysis of grey matter volume in persistent developmental stuttering: a voxel-based 

morphometry study 

Authors: *P. M. GOUGH 1,2 , S. DAVIS 3 , D. WARD 4 , P. HOWELL 3 , K. E. WATKINS 1,2 ; 

1 Exp Psychology, 2 Fmrib, Univ. Oxford, Oxford, United Kingdom; 3 Psychology, UCL, London, 

United Kingdom; 4 Psychology, Univ. Reading, Reading, United Kingdom 

Abstract: Functional imaging studies of people who stutter (PWS) are reaching a consensus 

regarding the brain areas that are abnormally active during speech. Similarly, structural analyses 

of white matter integrity show a remarkably similar pattern of deficits in the tracts connecting 

motor and sensory cortices in PWS. In contrast, structural analyses of grey matter show a more 

varied pattern of results across studies. We carried out a Voxel-Based Morphometry analysis of 

grey matter volume in a large group of PWS. We compared 29 PWS (age range 14-42 years, 

16M, 13F) with 22 age-matched and fluent-speaking controls (age range 14-42 years, 11M, 11F). 

T1-weighted 1-mm isotropic structural images of the whole brain were acquired at 1.5T. A 

study-specific symmetrical template was constructed. In each subject, an image of grey matter 

was created using an automated tissue classifier (FAST, FSL; http://www.fmrib.ox.ac.uk/fsl). 

These grey matter segmented images were nonlinearly transformed (FNIRT, FSL) to the 

template image and the Jacobian determinant was used to modulate the grey matter signal at each 

voxel to reflect its original size. These images were smoothed to 10-mm FWHM. A t-test 

between the two groups was performed voxel-wise across the whole brain. Images were 

thresholded at p

the right inferior frontal sulcus, and in the left superior frontal sulcus and intraparietal sulcus. In 

some regions (e.g. the inferior frontal gyrus), an area of increased grey matter volume was found 

next to an area of decreased volume. This suggests that a shape difference may exist in these 

locations that persists even after nonlinear registration. Such differences are consistent with 

studies revealing abnormal sulcal anatomy in PWS (e.g. Foundas et al., 2001). 

Disclosures: P.M. Gough, None; S. Davis, None; D. Ward, None; P. Howell, None; K.E. 

Watkins, None. 

Poster 





Support: Medical Research Council UK 

Title: Functional connectivity analyses in developmental stuttering during speech with normal 

and altered auditory feedback 

Authors: *K. E. WATKINS 1 , T. E. J. BEHRENS 1 , S. DAVIS 2 , P. HOWELL 2 ; 

1 Exptl. Psychology, Univ. Oxford, Oxford, United Kingdom; 2 Psychology, Univ. Col. London, 

London, United Kingdom 

Abstract: Imaging studies of people who stutter (PWS) show abnormalities in motor and 

sensory areas of the brain. Reduced auditory cortex activity is thought to be due to abnormal 

efference copy from motor to sensory cortex. On the other hand, overactivity in cortical and 

subcortical motor structures is consistent with the notion of abnormal basal ganglia function in 

PWS. Using functional MRI, we scanned PWS and controls while they received normal, delayed 

or frequency-shifted auditory feedback during speech. Altering feedback can improve fluency in 

PWS. In contrast, normally fluent speakers become disfluent under delayed feedback. Altered 

feedback increased activity in auditory areas bilaterally. Under delayed, but not frequencyshifted 

feedback, controls showed increased activity in the right inferior frontal gyrus (IFG), 

cerebellum and supplementary motor area (SMA). Using functional connectivity analyses, we 

examined the correlation in activity between (i) left auditory and motor cortex and (ii) right IFG 

and the midbrain. The correlation between activity in motor cortex (face representation) and 

auditory cortex increased from baseline in controls but decreased in PWS during speech with 

normal feedback. The correlation during altered feedback was increased relative to baseline in 

both groups. This finding is consistent with the theory proposing abnormal interactions between

sensory and motor cortex during speech production in stuttering, possibly due to abnormal 

efference copy. For the right hemisphere, correlations between the midbrain and IFG during 

speech increased from baseline in PWS and decreased in controls. We believe that the increased 

activity in right IFG, seen here in controls under delayed feedback and reported previously in 

PWS, relates to the inhibition of the motor act of speech. Previous studies have revealed a 

network of regions comprising the right IFG, SMA and the subthalamic nucleus involved in 

response inhibition of motor acts including speech. Thus the basal ganglia, and possibly 

dopamine, may play an overactive role in this “braking” mechanism during speech disfluencies. 

In conclusion, functional connectivity analyses has revealed abnormal coupling of activity in two 

separate networks consistent with two different explanations of stuttering. 

Disclosures: K.E. Watkins , None; T.E.J. Behrens, None; S. Davis, None; P. Howell, None. 

Poster 





Support: Charles A. Dana Foundation 

NIDCD Grant 04418 

Title: White matter pathways underlying reading skills in profoundly deaf adults 

Authors: E. HIRSHORN 1 , M. MANI 2 , A. GUIDON 2 , *D. BAVELIER 1 ; 

1 Brain & Cog Sci., Univ. Rochester, Rochester, NY; 2 Rochester Ctr. for Brain Imaging, Univ. of 

Rochester, Rochester, NY 

Abstract: Deaf readers on average only reach a 4th grade reading level. While greater residual 

hearing predicts better reading skills in this population, some profoundly deaf readers, with little 

to no access to the sounds of English, still reach high levels of reading proficiency. The current 

study aims to characterize structural white matter tracts involved in reading processes in such 

individuals. Deaf individuals with a profound, congenital dB loss, native proficiency in 

American Sign Language and a college level of education (N=20) were compared to age, gender 

and education matched hearing individuals (N=20) in order to investigate the underlying neural 

substrates of reading in the deaf and possible alternate neural routes to achieving literacy. 

Diffusion tensor imaging (DTI), which provides useful information about white matter 

anisotropy, was used in addition to obtaining standardized reading scores using the PIAT. Group

differences using fractional anisotropy (FA) revealed two kinds of regions. First, we observed 

regions in which FA values did not co-vary with reading scores, suggesting structural differences 

unrelated to reading, per se. For example, lower FA was noted in deaf as compared to hearing 

individuals in the auditory cortices bilaterally, as expected from the lack of auditory inputs to 

these regions from birth. Second, there were regions in which FA values did co-vary with 

reading skill, indicating a functional-structural link between reading ability and white matter 

structure. Among these regions was the left posterior corona radiata. Importantly, past research 

shows that the integrity of this region's microstructure is associated with reading skills in hearing 

individuals. For example, hearing individuals with reading disability show reduced FA in the left 

posterior corona radiata as compared to control subjects, along with greater FA in this region as 

reading proficiency increases. In the current study, deaf individuals also showed reduced FA in 

this region as compared to hearing controls. Unlike what has been reported with hearing reading 

disabled participants, however, reduced FA was associated with better reading skills in the deaf 

group in the left posterior corona radiata. Thus, this white matter pathway may be efficient for 

sound-based approaches to reading. Yet, in the absence of auditory input, recruiting this structure 

may become detrimental to reading proficiency. Pathways that mediate reading proficiency in 

the absence of auditory inputs will be further discussed. 

Disclosures: E. Hirshorn, None; M. Mani, None; A. Guidon, None; D. Bavelier , None. 

Poster 

250. Epilepsy: Networks 



Topic: C.06.f. Circuits and systems 

Support: NS 052302 

Epilepsy Foundation 

NIH AG21981 

Title: Seizures and long-term changes in network excitability in the dentate gyrus of mice after 

experimental brain injury 

Authors: *R. F. HUNT 1 , S. W. SCHEFF 2 , B. N. SMITH 1 ; 

1 Physiol., 2 Anat. & Neurobio., Univ. of Kentucky, Lexington, KY

Abstract: Temporal lobe epilepsy is a common consequence of traumatic brain injury, but 

fundamental aspects of epileptogenesis after trauma remain poorly understood. Identifying 

experimental models that exhibit similar pathology to injury-induced epilepsy in humans is 

necessary to elucidate underlying mechanisms by which the injured brain becomes epileptic. 

Using the controlled cortical impact (CCI) injury model, we examined whether mice developed 

spontaneous seizures after moderate (0.5mm injury depth) or severe (1.0mm injury depth) brain 

injury and how subsequent posttraumatic mossy fiber sprouting was associated with excitability 

in the dentate gyrus 42-71d post-injury. Days to weeks after injury, spontaneous seizures were 

observed in 36% of mice with severe and 20% of mice after moderate injury. Timm staining 

revealed mossy fiber sprouting in the inner molecular layer of the dentate gyrus in 55% of mice 

after severe injury and 20% of mice after moderate injury. Sprouting was typically localized in 

septal areas of the dentate gyrus ipsilateral to the injury. Extracellular field-potential recordings 

were made in isolated hippocampal brain slices to examine excitability after injury. Stimulation 

of perforant path revealed a significant reduction (P < 0.01) in paired-pulse responses in dentate 

granule cells at 20ms (47%) and 40ms (59%) interpulse intervals in slices from injured animals 

with mossy fiber sprouting compared to controls; no difference was detected at 80ms or 160ms 

intervals. Responses in slices from injured animals that did not display mossy fiber sprouting 

were not different from controls at any interval. In the presence of Mg 2+ - free ACSF containing 

100µM picrotoxin, slices with mossy fiber sprouting were characterized by spontaneous and 

hilar-evoked epileptiform activity in the dentate gyrus. These data suggest the development of 

spontaneous posttraumatic seizures as well as structural and functional network changes 

associated with temporal lobe epilepsy in the mouse dentate gyrus by 71d after CCI injury. 

Disclosures: R.F. Hunt , University of Kentucky, A. Employment (full or part-time); Epilepsy 

Foundation, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); S.W. Scheff, University of Kentucky, A. Employment 

(full or part-time); NIH AG21981, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); B.N. Smith, University of 

Kentucky, A. Employment (full or part-time); NS 052302, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received). 

Poster 





Support: Dr. Ralph and Marian Falk Medical Research Trust

Title: Modulating inflammatory pathway and epileptogenesis in mouse neocortex 

Authors: H. KOCH 1 , S. MARLER 1 , *M. TURNER 2 , J.-M. RAMIREZ 1 ; 

1 Organismal Biol. & Anat., 2 Neurosurg., Univ. Chicago, Chicago, IL 

Abstract: Traumatic brain injury is a common cause of epileptogenesis. The underlying 

mechanisms are poorly understood. Thus, it is also unknown how epileptogenesis can be 

prevented. Here we investigated the possible involvement of the COX-2 dependent inflammatory 

pathway in the progression from injury to seizure activity. The short term effects of PGE-2 were 

studied by directly applying low concentration of PGE-2 to both cultured and acute neocortical 

slices. The long-term consequences of manipulating the COX-2 pathway were explored in 

organotypic cultures that were incubated for 48 hours in PGE-2. In acute slices (n=6) PGE-2 

decreased the amplitude of evoked excitatory postsynaptic potentials (EPSP) by 32.59 ± 17.01 % 

compared to controls The specific PGE-2 receptor (EP-3) agonist sulprostone mimicked the 

effect of PGE-2 and reduced in acute slices (n=5) the amplitude of EPSPs by 29.52 ± 10.07 % . 

PGE-2 reversibly suppressed spontaneous network activity in acute and spontaneously occurring 

upstates (amplitude: 13.95 ± 4.46 mV) in slices cultured in normal media. Chronic treatment 

with PGE-2 for 48 hours dramatically altered the network behavior of organotypic slice cultures. 

In PGE-2 treated slices, upstates triggered paroxysmal depolarization shifts (PDSs) that were 

characterized by significantly larger depolarization amplitudes (36.65 ± 9.11 mV;n=11). The 

triggering of high amplitude PDSs suggests a possible mechanism for epileptogenesis. 

Specifically we propose that prolonged PGE-2 exposure resulting in prolonged activity 

deprivation evokes a homeostatic response that increases synaptic transmission and induces 

high-amplitude paroxysmal depolarization shifts at the network level. 

Disclosures: H. Koch, None; S. Marler, None; M. Turner , None; J. Ramirez, None. 

Poster 




Topic: C.06.h. In vivo mechanisms 

Support: MEST-CT-2005-019217 

Academy of Finland 

Title: Pilocarpine-induced status epilepticus triggers angiogenesis in adult rat hippocampus

Authors: *X. E. NDODO-EKANE, J. NISSINEN, A. PITKÄNEN; 

A.I.V Inst., KUOPIO, Finland 

Abstract: Angiogenesis is one of the processes associated with the development of epilepsy. 

However, the spatiotemporal evolution of this process and how it influences the subsequent 

progression of epilepsy is not yet understood. To address some of these questions we used 

pilocarpine-induced status epilepticus (SE) model of temporal lobe epilepsy and determined 

using unbiased stereological methods, vessel length changes and number of proliferating 

endothelial cells in the CA1 and CA3 subfields of the hippocampus and molecular layer of the 

dentate gyrus at 2 d, 4 d, and 14 d post-SE. There was a decrease in total vessel length in the 

hippocampus at 2 d post-SE (P

1 Mario Negri Inst. for Pharmacol. Res., Milano, Italy; 2 Inst. de la Santé et de la Recherche 

Medicale U661, Montpellier, France; 3 Dept Neurophysiol., Inst. Neurologico, 20133 Milan, Italy 

Abstract: Rationale: Brain inflammation, angiogenesis and increased blood-brain barrier (BBB) 

permeability occur in adult rodent and human epileptogenic brain tissue. No information is 

available on whether inflammation is responsible for neovascularization and BBB damage; 

moreover, the contribution of these factors to epileptogenesis is largely unexplored. 

Aim: This study addresses the role of these events in epileptogenesis using a developmental 

approach since the propensity to develop spontaneous seizures, therefore the induction of 

epileptogenesis, is age-dependent and increases with brain maturation. 

Method:Immunohistochemical analysis of inflammation, angiogenesis and BBB permeability 

was done in postnatal day (PN)9 and PN21 rats, at 4 h, 1 week and 4 months after pilocarpineinduced 

status epilepticus. Brain inflammation was evaluated by reactive gliosis and 

interleukin(IL)-1β immunohistochemistry. Microvessels were identified using an anti-laminin 

antibody or by intraluminal signal of intracardial FITC-albumin, and their density was 

quantified. BBB integrity was evaluated by extravascular IgG immunostaining or by detection of 

parenchymal extravasation of FITC-albumin. 

Results: Status epilepticus in PN9 rats did not induce either inflammation or angiogenesis or 

changes in BBB permeability and these rats did not develop spontaneous seizures in adulthood. 

Differently, status epilepticus in PN21 rats induced chronic inflammation, angiogenesis and BBB 

leakage in the hippocampus in about 70% of rats. Inflammation invariably preceded the onset of 

angiogenesis and BBB damage. In the remaining 30% of rats, only transient inflammation was 

observed. Epilepsy developed in about 70% of PN21 rats exposed to SE and these epileptic rats 

showed the three phenomena in their hippocampi. About 30% of PN21 rats exposed to SE did 

not develop epilepsy; these rats did not show angiogenesis and BBB damage in any of the 

forebrain areas analyzed. 

Conclusion: Our data show that angiogenesis and BBB permeability are concomitantly altered 

by status epilepticus in an age-dependent manner; moreover, these phenomena are spatiotemporally 

correlated with the extent of inflammation in various forebrain areas. This evidence 

suggests the occurrence and persistence of these phenomena in the hippocampus only if 

epileptogenesis is induced, thus highlighting the possibility that they are implicated in the 

mechanisms predisposing to the occurrence of spontaneous seizures. 

Disclosures: T. Ravizza, None; M. De Curtis , None; B. Gagliardi, None; J. Marcon, 

None; F. Noe', None; A. Vezzani, None; M. Morin, None; M. Lerner-Natoli, None. 

Poster 



Program#/Poster#: 250.5/V17


Title: Selective suppression of neuronal NF-θB signaling: effect on intensity of kainic acid 

induced seizures and hippocampal neurodegeneration 

Authors: *P. RATHORE 1 , S. J. LISTWAK 2 , M. HERKENHAM 2 ; 

1 Section of Functnl Neuroanatom, NIMH , NIH, Bethesda, MD; 2 Section of Functional 

Neuroanatomy, Natl. Inst. of Mental Health, NIH, Bethesda, MD 

Abstract: Nuclear Factor-kappaB (NF-kB) is a transcription factor activated in response to 

inflammation and injury. In brain, NF-kB has a role in cell survival and also in synaptic 

plasticity, learning, and memory consolidation. It is reported that kainic acid (KA)-induced 

excitotoxic seizures activate NF-kB in brain, but the consequences of such activation are not 

known in part because the role played by NF-kB in neurons versus glia may be different. The 

present study uses tetracycline-inducible NSE-tTA + tet-IKK2-DN double transgenic mice to 

investigate the role of NF-kB activation in neurons following KA injection (30 mg/kg). In these 

mice, the tetracycline-regulated IkB kinase2-dominant negative (tet-IKK2-DN) construct is 

under control of neuron-specific enolase (NSE-tTA) promoter. This allows suppression of NFkB 

signaling in neurons upon removal of doxycycline (Dox Off) from the diet. KA-treated mice 

were scored for seizure intensity using the Racine scale. Changes in body and brain weight 

following seizures were measured. We found that Dox-Off NSE-IKK2-DN mice and C57BL/6 

wildtype (WT) mice exhibited very severe KA-induced seizures in contrast to relatively milder 

seizures seen in Dox-On NSE-IKK2-DN mice. Alterations in body and brain weight correlated 

with the intensity of seizures. Fluorojade B staining at 3 days revealed that WT and Dox-Off 

NSE-IKK2-DN mice showed neurodegeneration in the CA3 region of hippocampus in contrast 

to no degeneration detected in Dox-On NSE-IKK2-DN mice. qRT-PCR analysis showed 

manifold increase in expression of cytokines like MCP-1, GRO-alpha, TIMP-1, and IL-6 in the 

cortex from WT and Dox-Off NSE-IKK2-DN mice. In Dox-Off NSE-IKK2-DN mice, the 

increased expression of genes was blunted relative to WT. DNA binding assays (EMSAs) and 

Western blot analysis in nuclear extracts from cortex showed involvement of p50 and p65 NF-kB 

subunits. Neuroanatomical localization of the up-regulated genes following seizures is being 

detected using in situ hybridization technique. The study so far suggests a pivotal role for 

neuronal NF-kB in transcriptional regulation of cytokines and chemokines in brain, which may 

be dictating the fate of neurons during seizures. We will explore further why in comparing Dox- 

On and Dox-Off animals, neuronal NF-kB expression appears to be neuroprotective, whereas in 

comparing Dox-Off with WT, it appears to be deleterious. 

Disclosures: P. Rathore, None; S.J. Listwak, None; M. Herkenham, None. 

Poster 

250. Epilepsy: Networks




Support: NIH Grant R37 NS-32403 

Title: Epilepsy-associated dysregulation of recurrent feedback inhibition in hippocampal area 

ca1 

Authors: C. YUE, C.-W. ANG, *F. WEISSINGER, D. COULTER; 

Children's Hosp. Philadelphia, Philadelphia, PA 

Abstract: Temporal lobe epilepsy (TLE) is the most prevalent seizure disorder in adults. It is an 

acquired disorder, defined by seizures involving limbic structures, including the hippocampus. 

Most studies examining mechanisms of TLE have focused on changes in the synaptic and 

intrinsic properties of hippocampal neurons. However, it is not clear how reorganization in the 

local circuitry affects integration of cortical inputs into the hippocampus. In the normal rodent, 

direct cortical input into the hippocampus, the temporoammonic (TA) pathway, is highly 

segregated to the distal apical tuft of CA1 pyramidal neurons by feedforward inhibition mediated 

by stratum lacunosum moleculare intereneuons, and TA interaction with Schaffer collateral 

EPSPs is regulated by feedback inhibition, mediated by a population of stratum oriens 

interneurons. Previous studies showed that, in pilocarpine-treated rats exhibiting spontaneous 

seizures, the disruption of feedforward inhibitory control of the TA pathway results in a loss of 

spatial segregation of TA EPSPs to CA1 stratum lacunosum moleculare, with propagation of the 

TA EPSPs to stratum radiatum and stratum pyramidale. To examine how alterations in feedback 

inhibitory regulation may further dysregulate TA inputs after pilocarpine-induced SE, we used 

voltage sensitive dye imaging combined with dendritic whole cell recording. To compare the 

spatiotemporal patterns of feedback inhibition mediated by oriens/alveus interneurons in control 

and chronic epileptic animals, we stimulated CA1 pyramidal cell axons antidromically by 

activating the alveus, using varying stimulation frequencies. We found that, in slices from 

epileptic rats, there was a significant lack of oriens/alveus mediated inhibition in response to 

alvear stimulation, both in proximal and distal dendritic areas. This deficit in oriens/alveus 

mediated recurrent inhibition results in an inability of CA1 circuitry to restrict and/or gate TA 

inputs impinging onto CA1 distal dendrites, allowing propagation of TA EPSPs to the CA1 

soma. These changes in the local circuitry contribute further to persistent hyperexcitability in the 

hippocampal area CA1 that may underlie the generation of paroxysmal activity in the 

hippocampus. 

Disclosures: C. Yue, None; F. Weissinger, None; C. Ang, None; D. Coulter, None. 

Poster





Support: F30-DA023758 to PTN 

VCU AD Williams Award to LSS 

U01-NS058213-01 to RJD 

R01-NS051505-01 to RJD 

R01-NS052529-01 to RJD 

Title: Long-term regional differences in cb-1 receptor-mediated g-protein activity following 

pilocarpine-induced status epilepticus 

Authors: K. W. FALENSKI 1 , P. T. NGUYEN 2 , *L. S. DESHPANDE 1 , R. E. BLAIR 1 , L. J. 

SIM-SELLEY 2 , R. J. DELORENZO 1,2,3 ; 

1 Dept Neurol, 2 Dept Pharmacol. & Toxicology, 3 Biochem. & Mol. Biophysics, Virginia 

Commonwealth Univ., Richmond, VA 

Abstract: The endocannabinoid system has been shown to play a role in modulating seizure 

activity in several in vivo and in vitro models. Studies from our laboratory have demonstrated 

that CB1 receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy, 

and that a unique plasticity of the CB1 receptor occurs in the hippocampus of these epileptic rats 

using agonist-stimulated [ 35 S]GTPγS autoradiography (Wallace et al., JPET 2003; 307[1]:129- 

37; Falenski et al., Neuroscience 2007; 146[3]:1232-44). However, to date, other regions have 

not been thoroughly evaluated for changes in CB1 receptor-mediated G-protein activity 

following epileptogenesis. Therefore, this study was initiated to evaluate the long-term effect of 

pilocarpine-induced status epilepticus (SE) on CB1 receptor function in epileptic rats using a 

whole-brain analysis approach. Coronal sections from control and chronically epileptic animals 

(1 year post-SE) were cut in a cryostat at equal intervals throughout the entire neuroaxis and 

processed for agonist-stimulated [ 35 S]GTPγS autoradiography with maximally effective 

concentrations of the cannabinoid agonist WIN55,212-2 (WIN, 10 µM). In addition to areas of 

the hippocampus, widespread increases in total WIN-stimulated [ 35 S]GTPγS binding occurred in 

the cortex and thalamus of epileptic animals. More specifically, increases in the cingulate, 

frontal, lateral orbital, infralimbic, insular, and temporal cortices were observed, most notably in 

deep layers (layers V and VI). WIN-stimulated [ 35 S]GTPγS binding levels were also increased in 

the thalamus, particularly in the ventrolateral, ventromedial, ventroposterolateral, 

ventroposteromedial, reticular, laterodorsal, anteromedial, and lateral and medial geniculate 

thalamic nuclei. Increases in WIN-stimulated [ 35 S]GTPγS binding were also observed in other

egions including caudate-putamen. Interestingly, there were no large areas of the brain that 

exhibited decreases in WIN-stimulated [ 35 S]GTPγS binding, but several regions illustrated no 

significant changes, including the substantia nigra, globus pallidus, and cerebellum. Overall, 

WIN-stimulated [ 35 S]GTPγS autoradiography revealed a unique global profile of receptormediated 

G-protein activity in epileptic animals, and thus illuminated specific regions of the 

epileptic brain that may serve as targets for cannabinimimetic compounds or manipulation of the 

endocannabinoid system. 

Disclosures: K.W. Falenski, None; L.S. Deshpande , None; P.T. Nguyen, None; R.E. Blair, 

None; L.J. Sim-Selley, None; R.J. DeLorenzo, None. 

Poster 





Support: P50-DA005274 

U01-NS058213-01 

R01-NS051505-01 

R01-NS052529-01 

Title: Pilocarpine-induced status epilepticus in rats causes an acute decrease in CB1 receptor 

protein levels and mRNA expression 

Authors: *D. S. CARTER, K. W. FALENSKI, R. E. BLAIR, R. J. DELORENZO; 

Neurol., Virginia Commonwealth Univ., Richmond, VA 

Abstract: Acquired epilepsy is characterized by the occurrence of spontaneous recurrent 

seizures. This form of epilepsy is caused by an injury to the brain such as stroke or status 

epilepticus (SE). Following the initial injury, there is a 1-4 month epileptogenic period during 

which time many plasticity changes occur prior to the onset of spontaneous seizures. It has been 

well established that cannabinoids exhibit anticonvulsant properties through their interaction 

with the CB1 receptor (Wallace et al, 2001, Eur J Pharmacol; Marsciano et al, 2003, Science). 

There is growing evidence that the endocannabinoid system regulates excitatory and inhibitory 

synaptic transmission during epilepsy. Our lab has recently shown that a long term and

permanent redistribution of hippocampal CB1 receptors occur in the epileptic rat (Wallace, et al, 

2003, JPET; Falenski et al, 2007, Neuroscience). This study was conducted to determine 

potential changes in the CB1 receptor in the hippocampus acutely following a single, one hour 

episode of SE. Using the pilocarpine model to induce SE, rats were sacrificed at 1h, 24h, and 1 

week following SE onset and the hippocampi were harvested for both protein and RNA. Using 

western blot analysis of isolated hippocampal membranes, CB1 receptor levels were decreased at 

1h, 24h, and 1 week post SE. An initial decrease in the surface levels of the CB1 receptor was 

observed following one hour of SE. This decrease became more pronounced at both 24h and 1 

week post SE. RT-PCR was performed to determine if changes in the CB1 mRNA expression 

occurred at these same time points. One hour following SE, mRNA expression of the CB1 

receptor was not significantly decreased. However, by 24h post SE and at 1 week post SE, CB1 

mRNA expression was significantly decreased. These results indicate that the endocannabinoid 

system rapidly responds to a single, one hour episode of SE resulting in a decrease of the surface 

levels of the CB1 receptor protein and CB1 mRNA. These finding are consistent with the 

growing evidence implicating the involvement of the endocannabinoid system in the 

pathophysiology of epilepsy. 

Supported by U01-NS058213-01, R01-NS052529-01, R01-NS051505-01, and P50-DA005274 

Disclosures: D.S. Carter, None; K.W. Falenski, None; R.E. Blair, None; R.J. DeLorenzo, 

principle investigator, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 







Title: Altered excitability of the thalamic reticular neurons following cortical thrombotic lesions 

Authors: J. PAZ, D. PRINCE, *J. R. HUGUENARD; 

Dept Neurol & Neurol Sci., Stanford Univ. Sch. Med., Stanford, CA 

Abstract: Focal cortical injuries result in death of cortical neurons, corticothalamic axons and 

ultimately in death or damage of thalamocortical relay (TCR) neurons. TCR cells are a normal

target of the inhibitory output of thalamic neurons of reticular nucleus (nRT), which generally 

survive such insults. nRT cells are the recipients of major excitatory cortical and thalamic 

outputs and can thus read out cortical activity and influence thalamocortical excitability in 

powerful ways. The survival and reorganization of nRT following cortical injury is expected to 

play a role in recovery of thalamocortical circuits following injury. However, the physiological 

properties and connectivity of the survivors remain unknown. In order to study the possible 

alterations in nRT neurons, we used the rat photothrombosis model of cortical stroke using 

intravenous Rose Bengal injections along with focal light exposure to the skull (Watson et al., 

1985). Using in vitro patch clamp recordings from thalamic slices obtained at various times 

following the photothrombotic injury we show that localized strokes in the somatosensory cortex 

induce changes in intrinsic excitability of nRT cells at the end of the first week after the injury. 

These nRT neurons are denervated of their inputs from cortex and also gradually disconnected 

from retrogradely degenerating TCR cells following cortical injury. Specifically in nRT neurons 

from rats with cortical lesions (1) membrane input resistance is decreased, and (2) low threshold 

calcium burst responses are strongly decreased or absent. Moreover, evoked excitatory responses 

in nRT cells are strongly reduced. These changes could alter thalamic oscillatory activity and 

lead to abnormal operation of thalamocortical circuits. Indeed, such an alteration in the 

excitability in nRT cells could lead to loss of nRT-mediated inhibition in relay nuclei, increasing 

surviving TCR cell output and enhancing thalamocortical excitation which may facilitate 

recovery of thalamic and cortical sensory circuits. However, it is also possible that the circuit 

reorganization in nRT that occurs following cortical thrombosis may promote the abnormal 

thalamocortical epileptic activity that has been observed in such models. 

Disclosures: J. Paz, None; D. Prince, None; J.R. Huguenard , None. 

Poster 




Topic: B.09.d. Oscillations and synchrony: Other 

Support: NCRR 

NINDS 

NIMH 

Title: The role of Kv7 mediated potassium currents and recurrent excitation in stellate cells of 

the entorhinal cortex in a dynamic clamp based model of temporal lobe epilepsy

Authors: *T. J. KISPERSKY 1 , J. A. WHITE 2 , H. G. ROTSTEIN 3 ; 

1 Program Neurosci, Boston Univ., Boston, MA; 2 Dept. of Bioengineering, Univ. of Utah, Salt 

Lake City, UT; 3 Dept. of Mathematical Sci., New Jersey Inst. of Technol., Newark, NJ 

Abstract: Recent experimental work has suggested that the epileptic state in the temporal lobe of 

the rat brain is associated with a decrease in recurrent inhibition but no change in excitation. In 

modeling work we have explored the generation of epileptic-like behaviors in networks without 

inhibition and find that once recurrent excitation reaches a threshold level, sustained, high 

frequency firing is induced. These networks synchronize slightly out of phase so they can be 

approximated by single stellate cells connected via an autapse. To study this phenomenon in 

vitro we used slices of rat entorhinal cortex and dynamic clamp to couple single stellate cells to 

themselves to mimic such recurrent excitation. We find that autaptic coupling, at a threshold 

level of conductance, does indeed cause a sudden transition to an epileptic-like state. Unlike the 

computational model, which lacks slow potassium currents known not to contribute to 

subthreshold oscillations, the in vitro model does not sustain firing but instead enters into a 

bursting regime in which periods of high frequency firing are interspersed by periods of 

quiescence. To understand the biophysical mechanisms underlying this behavior we investigated 

the role of the Kv7 mediated potassium current (m-current) since its introduction into the model 

caused bursting behavior similar to that seen in vitro. We find that pharmacologically blocking 

the m-current with linopirdine in recurrently connected stellate cells causes an increase in burst 

duration but not a change in the interspike interval (ISI) during bursts. In control conditions, the 

slow buildup of adaptation mechanisms including the m-current lead to the termination of a burst 

after a few spikes. As supported by our theoretical studies, we hypothesize that m-current block 

attenuates these adaptation mechanisms and thus permits longer periods of burst firing. We 

conclude that recurrent excitation is sufficient to induce epileptic-like behavior in single stellate 

cells and that this behavior is modulated by the presence of the m-current. 

Disclosures: T.J. Kispersky , None; H.G. Rotstein, None; J.A. White, None. 

Poster 





Support: NIMH 

NINDS

NIMH 

Title: The transition to hyperexcitability in stellate cells (SCs) from layer II of the medial 

entorhinal cortex during temporal lobe epilepsy: A modeling study 

Authors: *H. G. ROTSTEIN 1 , T. KISPERSKY 2 , J. A. WHITE 3 ; 

1 Dept Math Sci., New Jersey Instit. Technol., Newark, NJ; 2 Program in Neurosci., Boston Univ., 

Boston, MA; 3 Dept. of Bioengineering, Univ. of Utah, Salt Lake City, UT 

Abstract: Previous studies have shown that stellate cells (SCs) are hyperexcitable in animal 

models of temporal lobe epilepsy. A recent 'in vitro' study using pilocarpine-treated rats (Kumar 

et al. 2007) found evidence for the existence of recurrent excitatory connections among SCs, 

reduced recurrent inhibition among SCs in epileptic animals and no change in recurrent 

excitation. In this work we investigate the biophysical mechanism that governs the transition 

from normal to hyperexcitable spiking activity in SCs. We use biophysical (conductance based) 

modeling, simulations, dynamical systems techniques and dynamic clamp experiments. The SC 

model includes a persistent sodium, an h- and an M- currents. We show that a minimal network 

model including SCs and interneurons is able to qualitatively reproduce the experimental 

findings. This model displays an abrupt transition between the two frequency regimes as the 

result of small changes in the amount of inhibition. This abrupt transition also occurs in the 

absence of inhibition, as a result of small changes in the amount of recurrent excitation. To 

further investigate the biophysical mechanism that governs these phenomena we considered a 

single SC connected to itself via an autapse. This approximation mimics the network activity and 

is justified since SCs synchronize in phase and slightly out of phase in the theta and 

hyperexcitable regimes respectively. We show that the abrupt changes in firing frequency can be 

induced by increasing the amount of autaptic conductance, but not by increasing the level of the 

applied (tonic) current in an isolated SC; i.e., they are the result of phasic but not tonic 

excitation. 'In vitro' experimental resuts using a single, isolated SC and dynamic clamp to 

generate an autapse confirmed our theoretical predictions. Our results show that a single, isolated 

SCs have intrinsic dynamic properties that endow them with the potential ability to evolve on 

both fast and slow time scales, and that the combination of excitatory and inhibitory network 

properties determines the SC's frequency regime. 

Disclosures: H.G. Rotstein, None; T. Kispersky, None; J.A. White, None. 

Poster 



Program#/Poster#: 250.12/V24


Support: Burroughs Wellcome Fund 1001749 

NIH Grant EB005736 



Title: Two-photon imaging of cell-type specific firing patterns and neuronal network 

correlations during epileptiform activity in hippocampal slice 

Authors: *K. P. LILLIS 1 , M. A. KRAMER 2 , J. MERTZ 1 , J. A. WHITE 3 ; 

1 Biomed Engin., 2 Dept. of Mathematics, Boston Univ., Boston, MA; 3 Bioengineering, Univ. of 

Utah, Salt Lake City, UT 

Abstract: A recently developed laser-scanning strategy allows the simultaneous measurement 

from many neurons distributed across a large area with high spatial and temporal resolution. 

Here we use this technique, Targeted Path Scanning (TPS), in conjunction with two-photon 

excitation of bath-applied, calcium-sensitive dyes, Calcium Green-1 AM or Indo-1 AM, to image 

epileptiform activity in the hippocampal formation. With TPS, user-selected neurons separated 

by a distance of >2mm could be sampled at rates exceeding 100Hz without sacrificing single cell 

resolution. In this study, TPS was employed to record up to four minutes of 4-AP-induced 

epileptiform activity. Dozens of cells in the dentate gyrus (DG) and CA3 were sampled at a rate 

of 30-50 Hz. The resulting data provided an independent calcium signal for each cell. Cellular 

calcium dynamics were evaluated both visually and using statistical techniques to pick out 

stereotypical motifs. Preliminary analysis reveals at least two classes of cells in the DG: those 

which are active at the beginning of a seizure-like event (SLE) only and those which sustain 

firing through the SLE. To better understand the role these cells play in neuronal network 

dynamics, multidimensional correlation analyses were also performed. Correlations were 

computed between calcium signals recorded from each pair of cells during SLE and used to 

establish graph representations of the data. In these graphs, each cell was represented as a node, 

and edges were drawn between sufficiently coupled pairs of nodes (i.e., strongly correlated pairs 

of cells). To analyze the complicated topological properties of the graphs determined during 

SLE, techniques from network analysis were implemented. Two measures of network 

connectivity were calculated for each node: in-degree, the sum of correlations for which other 

cells lead the node (as determined by peak correlation); and out-degree, the sum of correlations 

for which the node leads other cells. Preliminary results suggest that DG cells exhibiting 

transient firing during SLE tend to have a higher in-degree than out-degree. Conversely, DG 

cells exhibiting prolonged activity during SLE tend to have higher out-degree than in-degree. 

Together these results support a hypothesis that, in the latter phase of SLE, a class of cells in the 

DG, which sustain high levels of activity throughout the SLE, drive a second class of cells, 

which transiently fire at the beginning of the SLE. In coming experiments, we plan to use both 

GAD/67 GFP mice and patch-clamp recordings with intracellular dye application to better 

categorize these dynamically distinct cell types.

Disclosures: K.P. Lillis, None; M.A. Kramer, None; J. Mertz, None; J.A. White, None. 

Poster 





Support: NSF CAREER Grant PHY-0547647 

Title: Spatiotemporal patterns of phase synchronization in voltage-sensitive dye imaging of 

4AP-induced epileptic seizures in the rat neocortex 

Authors: *D. TAKESHITA, V. TSYTSAREV, S. BAHAR; 

Dept Physics & Astronomy, Univ. Missouri-St. Louis, St. Louis, MO 

Abstract: We use voltage-sensitive dye imaging to investigate how spatiotemporal patterns of 

synchronization change during drug-induced in vivo neocortical seizures in rats. Focal seizures 

were caused by the potassium channel blocker 4-aminopyridine (4AP). 

The neocortex was stained with the voltage-sensitive dye RH-1691. The intensity changes in dye 

fluorescence were imaged using a 16-bit CCD camera and are consistent with the signal from 

local field potential recording. After the administration of 4AP, seizure events were triggered by 

electrical stimulation. Imaging data before electrical stimulation was collected as a reference. 

The power spectrum was calculated, using a sliding window, for voltage sensitive dye recordings 

both before and during the seizure event. The signal from each pixel after electrophysiological 

seizure onset was classified as part of the seizure event if the power spectrum was higher a 

threshold value determined from the pre-seizure state. 

The epileptic area increases quickly at the beginning of a seizure event and stays almost constant 

throughout the duration of the seizure. A sudden decrease of the area is observed at the end of 

each seizure event. We applied phase synchronization analysis to the voltage-sensitive dye 

signals from multiple pairs of pixels throughout the seizure area. We observe a dramatic increase 

in synchronization during the seizure events. We also observe that closer pairs of pixels show 

stronger synchronization than pairs separated by a larger distance. Even widely separated pairs of 

pixels within the seizure area, however, show a significant increase in synchronization during the 

seizure event. 

Disclosures: D. Takeshita, None; V. Tsytsarev, None; S. Bahar, None.

Poster 





Support: Australian Research Council 

Title: Modeling absence seizure dynamics: implications for network and cellular mechanisms 

Authors: *J. A. ROBERTS 1,3 , P. A. ROBINSON 1,2,3 ; 

1 Sch. Physics, 2 Fac. Med., Univ. Sydney, Sydney, Australia; 3 Brain Dynamics Center, Westmead 

Millenium Inst., Westmead Hosp. and Univ. Sydney, Westmead, Australia 

Abstract: The relative contributions of network and cellular mechanisms to absence seizures are 

not yet fully understood. Oscillations in corticothalamic loops have been observed, with debate 

over the roles of local mechanisms in the cortex and thalamus. We use a physiologically-based 

mean-field model of the interconnected cortex and thalamus to determine the relative 

contributions of axonal and intrinsic cellular delays to the waveforms of absence seizures. Our 

model has previously been shown to successfully reproduce both the healthy human 

electroencephalogram and 3 Hz oscillations corresponding to absence seizures under plausible 

parameter changes in the corticothalamic loop. The predicted period of the absence seizure 

depends linearly on model parameters describing thalamocortical, corticothalamic, intracortical, 

synaptic, and dendritic delays, and these dependences are linked to the seizure mechanism by 

showing how time intervals between peaks in the waveforms depend on the parameters. We 

show that a corticothalamic loop mechanism for absence seizures is consistent with intrathalamic 

cellular properties being the leading determinant of the frequency of spike-wave discharges in rat 

genetic models, while properties of the corticothalamic loop play a greater role in humans. 

Furthermore, the lower frequency of human absence seizures compared to spike-wave discharges 

in the rat can be attributed to longer propagation delays in the corticothalamic loop, without 

needing different cellular properties. 

Disclosures: J.A. Roberts, None; P.A. Robinson, None. 

Poster





Support: NIBIB grant EB003583 (MZ) 

University of Michigan Center for Computational Medicine and Biology (All authors) 

NSF grant DBI-0340687 (VB) 

NIMH grant 076280 (VB) 

Title: Interaction of cellular and network mechanisms in spatio-temporal pattern formation in 

neuronal networks and its role in seizure generation 

Authors: A. BOGAARD 1 , V. BOOTH 2 , *M. R. ZOCHOWSKI 1 ; 

1 Physics, 2 Dept. of Mathematics, Anasthesiology, Univ. of Michigan, Ann Arbor, MI 

Abstract: We investigate combined effects of network and cellular mechanisms that can 

contribute to spontaneous seizure generation in neuronal networks. We constructed a large 

network of simplified multicompartmental model cells based on CA1 hippocampal pyramidal 

neurons. The model neurons consist of a dendritic cable with a soma compartment containing 

Hodgkin-Huxley Na+ and K+-delayed rectifier currents as well as the K+ A-type current and the 

hyperpolarization-activated, non-specific cation current Ih that are known to occur in these cells. 

The cells were coupled locally with dendritic excitatory synaptic connections. To investigate 

influences of pathological changes in cellular properties, we generated four classes of neurons 

having significantly different cellular dynamics ranging from Type I to Type II membrane 

excitability. At the same time, we simulated effects of axonal sprouting that is known to occur 

during epileptogenesis by varying network connectivity levels as well as wiring patterns using 

the Small World Network paradigm. In homogeneous networks, in which all neurons are of the 

same excitability type, we find that network activity is strongly affected by cellular properties for 

different topologies. Networks of Type II-like cells displayed synchronous bursting, a pattern 

associated with seizure activity, for most values of connectivity and rewiring while networks of 

Type I-like cells display different high frequency activity patterns before the constraints of high 

connectivity and random rewiring force synchronous bursting behavior. We link the phase 

response curves of each neuron class to the phase diagram of the observed spatio-temporal 

patterning in these homogeneous networks to show how the modifications of membrane currents 

can significantly alter the network response. In heterogeneous networks, in which cells of 

different excitability type are mixed, we find that small changes in cell type composition can 

have a significant effect on spatio-temporal patterning displayed by the network. By quantifying 

these changes in network activity with measures of frequency, phase coherence and bursting, our

esults pinpoint the combined interaction of intrinsic properties of network elements and overall 

network structure in the generation of network activity. 

Disclosures: A. Bogaard, None; V. Booth, None; M.R. Zochowski , None. 

Poster 

251. Demyelinating Disorders: Animal Models and Human Studies I 



Topic: C.08.c. Human studies 

Support: UCB Pharma 

Title: The effect of levetiracetam on cerebellar tremor in patients with multiple sclerosis 

Authors: *K. M. FISHER 1 , P. P. NICHOLS 2 , M. R. BAKER 1,2 , S. N. BAKER 1 ; 

1 Inst. of Neurosci., Newcastle Univ., Newcastle-upon-Tyne, United Kingdom; 2 Dept. of Clin. 

Neurol., Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom 

Abstract: Cerebellar tremor is estimated to be present in up to 70% of chronic multiple sclerosis 

(MS) patients. In general, therapeutic options, both pharmacological and surgical, have met with 

only limited success. Cerebellar tremor therefore presents a major therapeutic problem in MS. 

Although a recent open-label study in 14 MS patients suggested that levetiracetam might be 

effective in controlling cerebellar tremor (Striano et al., 2006), the tremor was only quantified 

using clinical rating scales. Tremor rating scales are subject to intra- and inter-rater variability, 

and are also insensitive to small but significant changes in tremor power. We have therefore 

undertaken a double blind placebo controlled crossover trial to quantify the effects of 

levetiracetam on cerebellar tremor, using accelerometry in addition to standard clinical rating 

scales. 

Treatment was titrated up from 250mg BD until the maximum tolerated dose was reached. We 

recorded electromyogram (EMG), electroencephalogram (EEG) and tremor whilst patients 

performed a series of manual tasks. Tremor was quantified using accelerometers attached to the 

index finger and elbow. An independent clinician reviewed blinded video footage and rated 

tremor using a standard rating scale. Patients were also asked to provide an assessment of their 

tremor using a visual analogue scale. 

Frequency domain analysis of the accelerometry data has been performed to assess the effect of 

levetiracetam on relative tremor power. Preliminary results show a significant reduction in 

cerebellar tremor in 4/6 patients following a 6 week course of levetiracetam. 

This study provides objective evidence for the efficacy of levetiracetam in the treatment of MS

tremor. 

References 

Striano et al., Levetiracetam for cerebellar tremor in multiple sclerosis: an open-label pilot 

tolerability and efficacy study. J Neurol. 2006; 253(6):762-6. 

Disclosures: K.M. Fisher , UCB Pharma, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); P.P. Nichols, 

UCB Pharma, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); M.R. Baker, UCB Pharma, B. Research Grant 


received); S.N. Baker, UCB Pharma, B. Research Grant (principal investigator, collaborator or 


Poster 





Support: The Göteborg Medical Society 

Stiftelsen för Gamla Tjänarinnor 

The Swedish Association of Persons with Neurological Disabilities 

The Swedish Research Council 

The Anna-Lisa and Bror Björnsson Foundation 

Title: Reduced cerebrospinal fluid BACE1 activity in multiple sclerosis 

Authors: *N. MATTSSON 1 , M. AXELSSON 3 , S. HAGHIGHI 3 , C. MALMESTRÖM 3 , G. 

WU 4 , R. ANCKARSÄTER 5 , S. SANKARANARAYANAN 4 , U. ANDREASSON 2 , S. 

FREDRIKSSON 6 , A. GUNDERSEN 7 , L. JOHNSEN 7 , T. FLABY 7 , A. TARKOWSKI 8 , E. 

TRYSBERG 9 , A. WALLIN 2 , H. ANCKARSÄTER 10 , J. LYCKE 3 , O. ANDERSEN 3 , A. J. 

SIMON 4 , K. BLENNOW 2 , H. ZETTERBERG 2 ; 

1 Inst. of Neurosci and Phys, 2 The Sahlgrenska Acad., Mölndal, Sweden; 3 Dept. of Neurology, the 

Sahlgrenska Acad. at Univ. of Gothenburg, Inst. of Neurosci. and Physiol., Gothenburg, Sweden; 

4 Alzheimer‟s Research, Merck Res. Labs., West Point, PA; 5 Kungälv Hosp., Dept. of

Anaesthesiology and Intensive Care, Kungälv, Sweden; 6 Dept. of Clin. Neuroscience, Karolinska 

Institutet, Stockholm, Sweden; 7 Univ. of Oslo, Dept. of Neurol. at Akershus Univ. Hosp., Oslo, 

Norway; 8 The Sahlgrenska Acad. at Univ. of Gothenburg, Dept. of Rheumatology and 

Inflammation Res., Göteborg, Sweden; 9 Dept. of Rheumatology, Karolinska Institutet, 

Stockholm, Sweden; 10 Inst. for Clin. Sciences, Malmö Univ. Hospital, Lund Univ., Malmö, 

Sweden 

Abstract: Cell and animal experiments have shown that beta-site APP-cleaving enzyme 1 

(BACE1) is required for neuregulin 1 (NRG1)-mediated activation of myelination by 

oligodendrocytes. In this study we assess the association of cerebrospinal fluid (CSF) BACE1 

activity with multiple sclerosis (MS). BACE1 activity and levels of secreted amyloid precursor 

protein (APP) and amyloid-beta (A-beta) isoforms were analyzed in CSF from 100 MS patients 

and 114 neurologically healthy controls. Patients with systemic lupus erythematosus (SLE), 26 

with and 41 without cerebral engagement, were also included to enable comparisons with regards 

to another autoimmune disease. A subset of MS patients and controls underwent a second lumbar 

puncture after 10 years. MS patients had lower CSF BACE1 activity than controls (p=0.003) and 

patients with cerebral and non-cerebral SLE (p

Title: Myelin analysis reveals hypomyelination in RLS brains 

Authors: *P. PONNURU 1 , C. EARLEY 3 , R. P. ALLEN 3 , J. R. CONNOR 2 ; 

1 Neurosurg., 2 Penn State coll Med., Hershey, PA; 3 Neurol., Johns Hopkins Inst., Baltimore, MD 

Abstract: Restless Legs syndrome (RLS) is a neurological disorder characterized by a strong 

urge to move the legs. RLS patients experience chronic sleep deprivation, due to worsening of 

symptoms both when at rest and during the night. Studies have provided profound insights into 

the key role of iron regulation in the pathophysiology of restless legs syndrome and that RLS is 

associated with low brain iron levels. Iron is essential for the process of myelination. Disruption 

in iron availability in the brain can affect oligodendroglial cell maturation and myelinogenesis 

causing hypomyelination. Therefore we hypothesized that hypomyelination could exist in RLS 

brains because of insufficient iron in the brain. To test this hypothesis, we isolated crude myelin 

from RLS (n=4) and control (n=5) cortex brain tissues obtained at autopsy and analysed for 

myelin proteins, Myelin basic protein (MBP), 3 5 -Cyclic nucleotide phosphohydrolase 

(CNPase) and Proteolipid protein (PLP) by western blotting. Results showed a significant 

decrease in the expression of all the three myelin proteins in RLS compared with the controls. 

Our results suggest that hypomyelination exists in RLS brain. 

Disclosures: P. Ponnuru, None; C. Earley, None; R.P. Allen, None; J.R. Connor, None. 

Poster 





Support: Italian Multiple Sclerosis Foundation Project 2004/R/3 

Italian Multiple Sclerosis Foundation Project 2007/R/16 

Title: Role of corpus callosum in bimanual coordination 

Authors: *L. BONZANO, A. TACCHINO, L. ROCCATAGLIATA, G. ABBRUZZESE, G. L. 

MANCARDI, M. BOVE; 

Univ. Genoa, Genoa, Italy 

Abstract: Injury to corpus callosum (CC) impairs bimanual motor performance as demonstrated 

by callosotomy studies. CC pathology occurs in Multiple Sclerosis (MS) patients, associated

with altered performance of tasks requiring interhemispheric integration of sensory and motor 

information. Here we investigated the CC functional role in bimanual coordination combining 

motor behavioral with Magnetic Resonance Diffusion Tensor Imaging (DTI) data in MS 

patients. 

We asked 14 MS patients to perform repetitive finger opposition movements of thumb to index, 

medium, ring and little fingers with the two hands simultaneously, paced with a metronome set 

at 1, 1.5 and 2Hz. We defined the Inter Hand Interval (IHI) as the absolute time difference 

between the corresponding touch onsets occurring in the two hands: the larger is the IHI, the 

more severe is the impairment in bimanual coordination. Sub-regions were selected in the CC: 

CC1 included the genu and the rostrum, CC5 covered the splenium; CC2, CC3, CC4 were 

identified in the remaining portion of the CC, subdividing the “anterior to posterior” distance 

into 3 parts. Fractional Anisotropy (FA) values were obtained by DTI data to quantify damage in 

each CC area. 

In a first series of experiments, an eyes closed paradigm was chosen to exclude confounding 

effects due to the integration of acoustic and visual information. Interestingly, a correlation 

between mean IHI and FA values was found only in CC1 and CC2 for all the rates. 

To investigate the role of posterior CC in bimanual coordination, a sub-group of 5 MS patients 

underwent a new experimental paradigm where a visual cue substituted the acoustic cue as 

metronome. In addition, a visual input was given to the patient in correspondence of the finger 

touch for the two hands separately, providing real-time feedback about the bimanual 

performance. After a phase of motor task execution at a specific rate set by the visual cue, the 

patient continued the task trying to preserve the same rhythm as before, always with the signals 

representing the two hands but without the visual metronome. Results showed a trend to a 

correlation between mean IHI and FA values in the posterior CC. 

We showed that anterior and body callosal connections are essential to perform simultaneous 

bimanual finger opposition movements, while the posterior CC seems to be involved only when 

a visual paradigm is adopted. These findings suggest that bimanual coordination relies on 

communication through CC in different sub-regions according to the specific motor task 

modality. 

Disclosures: L. Bonzano, None; A. Tacchino, None; L. Roccatagliata, None; G. Abbruzzese, 

None; G.L. Mancardi, None; M. Bove, None. 

Poster 




Topic: C.08.b. Animal models


NMSS Grant RG3900-A-2 

Title: Oligodendrocyte lineage cells from BDNF +/- mice exhibit blunted responses to cuprizone 

Authors: *M. R. WOOD 1,2 , J. ZHANG 1,2 , J. ALPERT 1 , C. F. DREYFUS 1 ; 

1 Dept Neurosci/Cell Biol, UMDNJ/RWJMS, Piscataway, NJ; 2 Grad. Sch. of Biomed. Sci., 

UMDNJ, Piscataway, NJ 

Abstract: Previous work in culture has shown that oligodendrocyte lineage cells (OLCs) 

respond to BDNF by increasing DNA synthesis and differentiation. In vivo analysis utilizing 

BDNF knockout animals (BDNF tm1Jae on a 129/BalbC/C57Bl6 background, Jackson 

Laboratories) confirmed that OLCs in the basal forebrain (BF) and the corpus callosum express 

the trkB receptor and are capable of responding to BDNF. Quantitative analysis indicated that 

BDNF +/- mice exhibit reduced numbers of OLG progenitor cells and MBP protein levels, 

suggesting that BDNF plays a role in OLC development in these regions. The present work 

investigates the role BDNF may play in the repair of a demyelinating lesion by utilizing the 

cuprizone model of demyelination. Eight week old BDNF +/+ and +/- mice were fed control feed 

or 0.2% cuprizone milled into feed (Harlan Teklad). The corpus callosum was evaluated by 

Western blot and immunocytochemical analyses to define effects on BDNF levels, NG2+ 

progenitors, and myelin traits. Mature BDNF protein levels in wildtype mice were reduced after 

3 to 8 weeks of cuprizone treatment, suggesting that the demyelinating lesion elicits a reduction 

in BDNF synthesis. To define the roles of BDNF in response to cuprizone we evaluated BDNF 

+/- mice. These mice exhibited a blunted increase in the number of NG2+ cells beginning at 4 

weeks of cuprizone and continuing until 5 weeks. BDNF +/- mice also exhibited decreased levels 

of myelin basic protein (MBP). These data indicate that BDNF may play a role in response to a 

demyelinating lesion by regulating numbers of progenitors and the ability of differentiating cells 

to express MBP. 

Disclosures: M.R. Wood , None; J. Zhang, None; J. Alpert, None; C.F. Dreyfus, None. 

Poster 




Topic: C.08.b. Animal models 

Support: SFB 665

Title: Oligodendrocytes form coupled networks in mouse corpus callosum largely promoted by 

Connexin47 

Authors: *M. MAGLIONE 1 , O. TRESS 2 , B. HAAS 3 , K. WILLECKE 2 , H. KETTENMANN 1 ; 

1 Cell. Neurosci., MDC für Molekulare Medizin, Berlin, Germany; 2 Inst. für Genetik, Univ. 

Bonn, Bonn, Germany; 3 Col. de France, Paris, France 

Abstract: Cx32 and Cx47 are the major gap junction proteins expressed by oligodendrocytes 

and Schwann cells. In addition to their role in cell communication, the expression of these 

proteins is essential for the formation and maintenance of myelin as it has become evident in 

mutations. Based on immunocytochemical and recombinant studies it was suggested that 

oligodendrocytes in white matter are not coupled among each other, but form gap junctions with 

astrocytes. We have now studied functional coupling among oligodendrocytes and astrocytes in 

the corpus callosum. Using the patch-clamp technique we tested for coupling by injection of 

biocytin, a gap-junction-permeable tracer, into oligodendrocytes in murine acute slices of corpus 

callosum (postnatal day 10-15). Oligodendrocytes were identified by their typical membrane 

current pattern and morphology. In 16 out of 17 cells the tracer spread on average to 24 

neighboring cells. To identify the coupled cells in the network we combined biocytin labeling 

with CNPase and GFAP immunostaining, i.e. markers for oligodendrocytes and astrocytes, 

respectively. Most biocytin labeled cells were CNPase positive, but very few were GFAP 

positive. In Cx47 knock-out mice, biocytin spread on average to four other cells from 10 out of 

15 injected cells. These data suggest that Cx47-deficiency significantly decreased the size of the 

network of coupled cells, but did not abolish it. This small network consisted only of 

oligodendrocytes, since the Cx47 knock-out mice express eGFP under control of the Cx47 

promoter. These results indicate that oligodendrocytes in the corpus callosum are coupled among 

each other, but not to astrocytes and that coupling depends to a large extent on expression of 

Cx47. 

Disclosures: M. Maglione, None; O. Tress, None; B. Haas, None; K. Willecke, None; H. 

Kettenmann, None. 

Poster 





Support: NIH Grant NS27336

Title: Absence of oligodendroglial glucosylceramide synthesis does not cause myelin 

abnormalities or change the dysmyelinating phenotype of CGT-deficient mice 

Authors: L. SAADAT 1 , G. DAWSON 2 , J. DUPREE 3 , R. PROIA 4 , M. TRAKA 1 , *B. J. 

POPKO 1 ; 

1 Dept Neurol, 2 Dept Pediatric, Univ. Chicago, Chicago, IL; 3 Dept. of Anat. and Neurobio., 

Virginia Commonwealth Univ., Richmond, VA; 4 Genet. of Develop. and Dis. Branch, Natl. Inst. 

of Diabetes and Digestive and Kidney, NIH, Bethesda, MD 

Abstract: To examine the function of gangliosides in oligodendrocytes we generated 

Ugcg flox/flox ;Cnp/Cre mice, which lack expression of the enzyme UDP-glucose ceramide 

glucosyltransferase (Ugcg) specifically in these cells. These mice, which are incapable of 

synthesizing glucosylceramide (GlcCer) in their myelinating cells, did not show an abnormal 

behavioral or pathological phenotype. Total brain lipid extracts from postnatal day (PND) 18 Wt 

and Ugcg flox/flox ;Cnp/Cre mice were analyzed by HPTLC. All GlcCer-based gangliosides 

including GM1, GD1a, GD1b, and GT1b showed normal expression levels in the mutant 

indicating that oligodendrocytes are not a major source of GlcCer-based ganglioside biosynthesis 

in the mouse brain. Preliminary electron microscopy analysis has not revealed any myelin 

defects in the CNS of Ugcg flox/flox ;Cnp/Cre mice, indicating that ganglioside elimination in 

oligodendrocytes is not detrimental to myelination. 

Furthermore, in order to investigate the potential compensatory or toxic effect of the 

accumulated hydroxy-fatty-acid-(HFA)-containing GlcCer previously detected in the 

galactolipid-deficient (Cgt -/- ) mice (Coetzee, et al 1996 Cell, Vol. 86, 209-219), we generated 

Ugcg flox/flox ;Cnp/Cre;Cgt -/- mutants, which are incapable of synthesizing HFA-GlcCer in 

oligodendrocytes. The behavioral phenotype of the double mutants was similar to Cgt -/- mice and 

morphometric analysis of myelinated fibers in the ventral white matter of the cervical spinal cord 

segment showed that both genotypes suffered from the same degree of dysmyelination. Thus, 

accumulation of GlcCer in Cgt -/- mice does not appear to functionally compensate for the loss of 

the myelin galactolipids or contribute to the pathological defects of the Cgt -/- mice. 

Disclosures: L. Saadat, None; J. Dupree, None; R. Proia, None; M. Traka, None; B.J. Popko 

, None; G. Dawson, None. 

Poster 



Program#/Poster#: 251.8/W1 

Topic: C.08.b. Animal models

Support: B. Deverman is supported by a fellowship from CIRM 

National Institute of Neurological Disorders and Stroke 

Title: Exogenous LIF stimulates oligodendrocyte progenitor cell proliferation and remyelination 

in vivo 

Authors: *B. E. DEVERMAN 1 , S. BAUER 2 , P. H. PATTERSON 1 ; 

1 California Inst. Tech., Pasadena, CA; 2 Physiologie Neurovégétative, UMR 6153 CNRS, 1147 

INRA, Univ. P. Cézanne-Aix-Marseille-3, Marseille, France 

Abstract: The development of therapies that enhance the repair capabilities of the adult brain by 

stimulating the proliferation of endogenous neural stem and progenitor cells and/or directing 

their subsequent differentiation into functional neurons and glia is a relatively neglected area of 

current stem cell research. We have found that injection of an adenovirus expressing leukemia 

inhibitory factor (LIF) into the adult brain promotes neural stem cell (NSC) self-renewal and 

stimulates the proliferation oligodendrocyte progenitor cells (OPCs). Based on these findings, in 

particular the effect of LIF on OPCs, we hypothesized that if LIF could enhance the OPC 

response in the context of chronic demyelination it may, in turn, promote the generation of new 

oligodendrocytes and aid remyelination. To test this, we have been feeding mice a diet 

containing cuprizone for 12 weeks, a course of treatment that induces demyelination in the 

corpus callosum (CC), hippocampus, and cortex, and injecting the mice with either a LIF- or 

lacZ-expressing adenovirus (Ad-LIF or Ad-lacZ) in the lateral ventricle. Three weeks after 

adenovirus injection and removal of cuprizone from the diet, mice that received Ad-LIF exhibit 

an increase in the number of proliferating OPCs in the demyelinated hippocampus. Many of 

these OPCs survive and differentiate so that by 6 weeks after removal of cuprizone, the number 

of mature oligodendrocytes in LIF-treated mice is restored to near normal numbers in the CA3 

region of the hippocampus, where LIF-induced Stat3 activation is the greatest. Remarkably, 

remyelination in the CA3 region is more extensive in LIF-treated mice and is accompanied by 

the reformation of nodal, paranodal, and juxtaparanodal domains on a subset of axons. Our 

findings that LIF can promote oligodendrocyte generation and remyelination in vivo taken 

together with its known ability to protect oligodendrocytes from death in the EAE model of MS, 

as well as following spinal cord injury, suggest that LIF has multiple activities that could be of 

therapeutic benefit for demyelinating diseases. 

Disclosures: B.E. Deverman , None; P.H. Patterson, None; S. Bauer, None. 

Poster 





Support: J. Sagol doctoral fellowship 

Title: Experimental autoimmune encephalomyelitis (EAE) in mice causes sex- and brain regiondependent 

increases in peripheral benzodiazepine receptors (PBR) 

Authors: *Y. DERI 1,2 , A. KATZAV 2 , J. CHAPMAN 1,3 , A. BIEGON 2,4 ; 

1 Sackler Fac. of Med., Tel Aviv Univ., Tel Aviv, Israel; 2 The Joseph Sagol Neurosci. Ctr., 3 Dept. 

of Neurol., Sheba Med. Ctr., Ramat Gan, Israel; 4 Brookhaven Natl. Labs., Long Island, NY 

Abstract: Multiple sclerosis is a chronic inflammatory disease of the central nervous system. 

The regional intensity of the neuroinflammatory response can be examined by [3H]PK11195 

autoradiography, which had been found to be an excellent quantitative marker for 

neuroinflammation and microlesions. Using a common animal model of MS, MOG-induced 

EAE, we explored region-selective neuroinflammation and its relationship to disease course in 

male and female mice. Ten male and 18 female C57bl mice, 11 weeks old, were injected with 

MOG. The animals' symptoms were scored daily on a 7 point scale ranging from 0 (no 

symptoms) to 6 (death). Control mice (10 females and 10 males) were handled daily during the 

same time. At the end of a 5 weeks follow up period, animals were decapitated and brains were 

quickly removed and frozen for cryo-sectioning in multiple consecutive series. Coronal brain 

sections at the level of striatum and hippocampus were incubated with the selective non 

competitive PBR antagonist [ 3 H]PK11195. Non specific binding was assessed on consecutive 

sections in the presence of excess unlabeled PK11195. Washed and dried sections were digitally 

scanned and PBR density measured in anterior cingulate cortex, striatum, retrosplenial granular 

cortex, perirhinal cortex and hippocampal regions (CA1, CA3 and dentate gyrus) using ImageJ 

software. Two EAE males (20%) and 6 EAE females (33%) died during the follow up period. 

EAE mice had significantly higher PBR levels in all brain regions measured compared to 

controls, which was also sex dependent [significant 3 way ANOVA by group (p

Poster 






NMSS RG3618 

Title: Correlation between transverse bands and lifespan in dysmyelinated mice 

Authors: *J. ROSENBLUTH 1,4 , A. MIERZWA 2 , J. AREVALO 5 , M. CHAO 3,6 ; 

1 Dept Physiol., 2 Physiol. and Neurosci., 3 Mol. Neurobio. Program, New York Univ. Sch. Med., 

New York, NY; 4 Rusk Inst., New York, NY; 5 INCYL-Facultad de Medicina, Salamanca, Spain; 

6 Skirball Inst. of Biomolecular Med., New York Univ. Sch. of Med., New York, NY 

Abstract: The spontaneous mouse mutant „shaking‟ (Mierzwa et al., SFN Abstract 277.17, 2005 

and Mierzwa et al., SFN Abstract 975.20, 2004) has minimal PNS pathology but severe CNS 

dysmyelination. Homozygous mice display a mild kinetic tremor and seizures in 10-20% of the 

animals. Their hindlimbs do not clasp when the mice are lifted by the tail, and they can hang by 

front or hindlimbs, indicating no signs of muscle weakness. They differ strikingly from other 

„myelin mutants‟ with comparable CNS dysmyelination in that their lifespan is near-normal, 

with animals living past two years of age. Other mutants, such as Caspr -/- and CGT -/-, with 

grossly similar levels of dysmyelination have more severe behavioral phenotypes and lifespans 

under six months. In this poster, we present data showing that „shaking‟ mice differ markedly 

from other dysmyelinating mutants with respect to the transverse bands (TB) and other features 

of their paranodes. Cervical dorsal spinal cord of control, CST -/- and shaking animals aged 

1.5mo to 12mo was sectioned and examined at 25K magnification by EM. The percentage of 

paranodal loops directly contacting the axolemma was similar in control and shaking animals but 

significantly reduced in the CST-/-. Correspondingly, the percentage of paranodes with TBs was 

comparable in control and „shaking‟ mice but significantly reduced in the CST-/- animals, and 

those paranodes lacking transverse bands were more likely to have everted loops. Previous 

studies have shown that CGT-/- and Caspr-/- mice, which are more severely affected than 

„shaking‟, lack transverse bands entirely. CST-/- mice show decreasing numbers of TBs with age 

corresponding to the worsening phenotype. In mutants lacking TBs, axolemmal domains are 

abnormal in that the nodal Na+ 

channel domain lengthens and may become non-circumferential, and the fast voltage-gated K+ 

channels, normally confined to the juxtaparanode, move into the paranode. Our findings suggest 

that the presence of transverse bands is important in maintaining paranodal structure, axoglial 

domains, and subsequently function. Those mutants that lack or have grossly reduced numbers of 

TBs have more severe deficits and shortened lifespans.

Disclosures: J. Rosenbluth, None; A. Mierzwa, None; J. Arevalo, None; M. Chao, None. 

Poster 





Support: FP6 program of the European Union (LSHM-CT-2005-018637; Neuropromise) 

HOMFOR (research program of the Medical Faculty of the University of the Saarland) 

Title: The role of the calcium channel subunit β3 in a mouse model of optic neuritis 

Authors: *S. K. WILLIAMS 1 , R. FAIRLESS 1 , V. FLOCKERZI 2 , M. FREICHEL 2 , A. 

CAVALIE 2 , R. DIEM 1 ; 

1 Abteilung Neurologie, 2 Experimentelle und Klinische Pharmakologie und Toxikologie, Univ. of 

the Saarlandes, Homburg/Saar, Germany 

Abstract: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central 

nervous system (CNS). For many years research has been predominately aimed at understanding 

the involvement of the immune system and it has only recently become evident that neuronal 

pathology plays a significant and possibly decisive role in the clinical progression of the disease. 

In many MS patients, optic neuritis is the first clinical manifestation of the disease, making the 

optic nerve an ideal system in which to investigate neuronal pathology in MS. In a mouse model 

of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune 

encephalomyelitis (EAE), animals develop optic neuritis characterized by inflammation, 

demyelination, and axonal degeneration of the optic nerve and a loss of retinal ganglion cells 

(RGCs), the cells whose axons form the optic nerve. 

Recent data suggest that in both MS and EAE, a dysregulation of ion channels and an 

accumulation of intracellular calcium contribute to neuronal and axonal pathology. Voltagegated 

calcium channels (VGCCs) are one of the major routes of calcium entry into neuronal cells 

and are comprised of a central pore-forming α1 subunit, together with accessory β, α2δ, and γ 

subunits. The administration of specific VGCC blockers was previously shown to both reduce 

the severity of EAE and to decrease inflammation and axonal loss, demonstrating a significant 

role for these channels in inflammatory demyelinating disease. The aim of our present study was 

to investigate the role of the VGCC subunit β3 during MOG-induced optic neuritis in mice. 

Intracellular β subunits are known to modulate the trafficking of the pore-forming α1 subunit and 

to affect the functional expression of VGCCs. Therefore, EAE was induced in both transgenic

mice lacking the β3 subunit of VGCCs and in respective wild type mice. The course of EAE was 

found to be more severe in transgenic mice lacking the β3 subunit, in comparison to wild type 

controls. Histopathological results will be compared between transgenic and wild type mice in 

order to determine the effect of altered functional VGCC expression on demyelination, 

inflammatory infiltration, axonal degeneration and neuronal loss. These results point to a role for 

not only α1, but also β VGCC subunits in inflammatory demyelinating disease. 

Disclosures: S.K. Williams, None; R. Fairless, None; V. Flockerzi, None; M. Freichel, 

None; A. Cavalie, None; R. Diem, None. 

Poster 





Support: ANR 2007 

Title: Brain magnetic resonance elastography as a biological marker of demyelination : 

implication in demyelinating neuropathologies of central nervous system 

Authors: *E. DIGUET 1 , B. LARRAT 1 , E. YANG 2 , M. FINK 1 , R. SINKUS 1 ; 

1 ESPCI, LOA-UMR7587, ESPCI, Paris, France; 2 The Univ. of Hong Kong, Hong Kong, China 

Abstract: The novel MR based imaging technology called magnetic resonance elastography 

(MRE) has been already reported to allow the assessment of mechanical properties of different 

human brain structures. We demonstrated that this technique could be extended to mouse brain 

thanks to MR-compatible piezo-transducers. These piezo-transducers generate mechanical waves 

at high frequency (1000Hz) beneath the head of the animal leading to measurable propagating 

waves within brain (~1µm amplitude and 2mm wavelength) at an isotropic image resolution of 

300µm. Preliminary results on a double transgenic APP/PS1 mouse model of Azheimer‟s disease 

showed that disease progression significantly correlates with changes of viscoelastic properties 

within corpus callosum when compared to age-matched controls. Using the well-established 

cuprizone-challenged mouse, we attempted to study the effect of demyelination and 

remyelination on mechanical properties of corpus callosum. C57Bl/6 mice were fed with 

cuprizone 0.2% for eight weeks (ingestion phase) and then placed on a normal diet for another 

eight weeks (recovery phase). MRI examinations were conducted at 0, 4, 8, 12 and 16 weeks 

using a 7T MRI scanner. MRI protocol combined T2-weighted images (RARE), diffusion tensor 

imaging (DTI) and elastography. Currently, this study is still ongoing. Correlation between

different MRI scans, clinical global motor score and histopathological data will be conducted. A 

methodology based on mechanical measurements of brain structures such as MRE might open 

new possibilities for the early detection and the management of central demyelinating disorders 

such as multiple sclerosis or Alzheimer‟s disease. 

Disclosures: E. Diguet , None; B. Larrat, None; M. Fink, None; R. Sinkus, None; E. Yang, 

None. 

Poster 






DBSCIP Grant F170TP 

NMSS Grant RG3515 

Title: Axonal involvement in early stage cuprizone demyelination of mouse corpus callosum 

Authors: *J. E. TOBIN 1 , M. XIE 2 , T. Q. LE 1 , D. MCDANIEL 1 , S.-K. SONG 2 , R. C. 

ARMSTRONG 1 ; 

1 Anatomy, Physiology,& Genet., USUHS, Bethesda, MD; 2 Washington Univ., St. Louis, MO 

Abstract: Animal models of demyelinating disease facilitate analysis of pathogenesis and repair 

mechanisms as well as development of novel imaging modalities. We used the model of 0.2% 

cuprizone fed to adult male C57Bl/6 mice to induce reproducible demyelination of the corpus 

callosum (CC). Previously, we demonstrated axonal injury in the CC early in the course of 

cuprizone treatment (2 - 6 wks, most extensive at 4 wks) reflected as decreased axial diffusion 

measured by diffusion tensor imaging (DTI). Axon damage during this early treatment phase was 

also indicated by β-amyloid precursor protein accumulation. In the current study, we more 

specifically examined axonal parameters in mice fed cuprizone for 4 wks. In vivo DTI of treated 

and non-treated mice indicated rostro-caudal differences in pathology from analysis of midline 

regions-of-interest taken as 0.5 mm coronal slices encompassing the entire CC. Axial diffusivity 

was not altered in the most rostral CC (bregma +1.0 mm) while a significant decrease was seen 

in all other regions, with the largest decrease in the most caudal slice (bregma -2.5 mm). 

Ultrastructural analysis showed a higher proportion of demyelinated axons in the caudal versus

ostral CC, while myelinated or demyelinated axons had respectively similar diameters within 

each region. Neurofilament dephosphorylation, an indictor of disrupted axonal cytoskeletal 

structure, was also more extensive in the caudal versus rostral CC. Our data in this cuprizone 

model demonstrate that the early progression of pathology is more advanced in the caudal CC. 

Furthermore, DTI axial diffusivity may be more sensitive to the disruption of neurofilament 

structural integrity and less indicative of axonal atrophy. Supported by NIH NS39293, NMSS 

RG3515 and DBSCIP F170TP. 

Disclosures: J.E. Tobin, None; M. Xie, None; S. Song, None; T.Q. Le, None; D. McDaniel, 

None; R.C. Armstrong, None. 

Poster 





Support: grant EU-NEST 1702 

SNSF Grant PP00A-106714 

Grant GM-28140 

Grant SSMBS1140 

Title: Phosphatidic acid mediates demyelination in Lpin1 mutant mice 

Authors: K. NADRA 1 , A.-S. DE PREUX CHARLES 1 , J.-J. MÉDARD 1 , W. T. HENDRIKS 2 , 

G.-S. HAN 3 , S. GRÈS 4 , G. M. CARMAN 3 , J.-S. SAULNIER-BLACHE 4 , M. H. G. 

VERHEIJEN 2 , *R. CHRAST 1 ; 

1 Dept Med. Genet, Univ. Lausanne, Lausanne, Switzerland; 2 Dept. of Mol. and Cell. Neurobio., 

Vrije Univ., Amsterdam, Netherlands; 3 Dept. of Food Sci., Rutgers Univ., New Brunswick, NJ; 

4 Dept. of Metabolism and Obesity, INSERM, U858/I2MR, Toulouse, France 

Abstract: Lipids play crucial roles in many aspects of glial cell biology, affecting processes 

ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role 

of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 

gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary 

for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral

neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and 

proliferation, and a reduction in nerve conduction velocity. The observed demyelination is 

mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid 

(PA). Our results therefore reveal a surprising role for PA in Schwann cell fate determination and 

provide evidence of a direct link between diseases affecting lipid metabolism and abnormal 

Schwann cell function. 

Disclosures: K. Nadra, None; R. Chrast , None; A. De Preux Charles, None; J. Médard, 

None; W.T. Hendriks, None; M.H.G. Verheijen, None; G. Han, None; G.M. Carman, 

None; S. Grès, None; J. Saulnier-Blache, None. 

Poster 





Title: Chronic installation of lysophosphatidylcholine (c24:0) induces demyelination 

Authors: *K. J. GAROFALO 1 , J.-Q. REN 1 , D. KULJIS 1 , R. RANSOHOFF 2 , F. EICHLER 1 ; 

1 Mass Gen Hosp, Charlestown, MA; 2 Neuroinflam. Res. Ctr., Learner Res. Inst., Cleveland, OH 

Abstract: We previously reported that intracerebral injections of very long chain fatty acids 

(VLCFA) incorporated into lysophosphatidylcholine (LPC) induce microglial apoptosis in rodent 

brain. Elevations in VLCFA occur in patients with X-linked adrenoleukodystrophy (ALD) and 

may play a role in the pathogenesis of cerebral demyelination. In the current study we assess the 

impact of chronic LPC (C24:0) exposure upon brain myelin and microglia in rodents. We 

compared a one time stereotactic injection of LPC (C24:0) versus PBS with repeated injections 

of LPC (C24:0) versus PBS (4 injections every 4 days). Mice injected only once were assessed 

after 4 days and 3 weeks, and mice with repeated injections were assessed after 3 weeks only. 

Repeated LPC (C24:0) injections induced microglial apoptosis and demyelination at three weeks. 

By contrast, four days after a one-time injection of LPC (C24:0), but not PBS, microglial 

activation and apoptosis was present without evidence of demyelination. Three weeks after a one 

time exposure brain tissue had healed completely. We conclude that chronic LPC (C24:0) 

exposure is necessary to induce cerebral demyelination. Following a one-time injection, brain 

myelin is not affected and in the absence of repeated exposure complete healing can occur. The 

data support the notion that lipid-reducing measures may alleviate the pathology in cerebral 

ALD.

Disclosures: K.J. Garofalo , None; J. Ren, None; D. Kuljis, None; R. Ransohoff, None; F. 

Eichler, None. 

Poster 





Support: ANPCYT, PICT 05-32975 

Title: Bone marrow mononuclear cells participation in the Wallerian degeneration process 

Authors: V. USACH, B. GOITIA, *P. C. SETTON-AVRUJ; 

Biol. Chem., Sch. of Pharm. and Biochem, Buenos Aires, Argentina 

Abstract: We have previously described the reorganization as clusters of myelin major proteins 

in the distal stump of the ligated sciatic nerve. Concerning myelin composition, we have 

observed a decrease in P0 and MBP levels and an increase in a 14 kDa band that was resolved as 

α - and β- globin. In the same model we have demonstrated the migration, either spontaneous or 

after intraortical injection, of fresh bone marrow mononuclear cells (BMMC) CD34 + to the distal 

stump of the nerve. According to these, the aim of the present work was to study the temporary 

window where the 14 kDa band is present in a reversible model of Wallerian degeneration (WD) 

promoted by the crush of the sciatic nerve, to evaluate the migration of BMMC and 

mesenchymal stem cells to the distal zone of the crushed sciatic nerve, and to characterise both 

groups as to identify the population that would migrate to the injured nerve. 

Adult Wistar rats were submitted to the crush of the right sciatic nerve and were sacrificed at 

different survival times. The nerve was dissected into proximal and distal areas; the left sciatic 

nerve, contralateral nerve, was taken as a control. Myelin isolation was done according to Iyer et 

al. procedure (J Neurosci. Res. 46: 531-539, 1996). BMMC were isolated from the bone marrow 

extruded from tibia and femur bones. A group was immediately processed and the other was 

seeded. After 24-48hs the non-adherent cells were separated and the adherent cells were grown 

to confluence. Both groups were analyzed by Western blot and immunocytochemistry. Fresh 

isolated or cultured BMMC were dyed with a fluorescent probe and injected intravenously 

immediately after crushing the sciatic nerve in order to evaluate the migration of these cells to 

the injured area. Immunohystochemical analyses of the crushed nerve were done to study the 

changes suffered by the nerve. 

Our results demonstrate the appearance of the 14 kDa band in isolated myelin of the distal zone 

and the migration of fresh isolated BMMC to the crushed area of the sciatic nerve during the first

week post injury. The hystological analysis of the nerve confirmed the degeneration process 

from the crush area to the distal zone. 

The characterisation of cultured BMMCs shows a heterogeneous population of small-rounded, 

spindle-shaped and large-flattened morphology; they showed fibroblast-like morphology at 

reaching confluence. 

After 24-48hs in culture the adherent and non-adherent cells are CD34 + , while adherent cells are 

CD45 - , CD11b - , vimentin + , Thy 1.1 + . 

Our results demonstrate the existence of a progenitor population in BMMC that spontaneously 

migrates to the injured nerve to participate in the degeneration-regeneration process. 

Disclosures: V. Usach, None; B. Goitia, None; P.C. Setton-Avruj , None. 

Poster 





Title: Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the 

expansion of regulatory T cell population 

Authors: *H. TAKEUCHI, J. WANG, Y. SONOBE, S. JIN, T. MIZUNO, A. SUZUMURA; 

Dept Neuroimmunol, Res. Inst. Env Med, Nagoya Univ., Nagoya, Japan 

Abstract: CD4+CD25+ regulatory T (Treg) cells are crucial mediators of autoimmune tolerance. 

The factors that regulate Treg cells, however, are largely unknown. Here, we show that 

deficiency in midkine (MK), a heparin-binding growth factor involved in oncogenesis, 

inflammation, and tissue repair, attenuated experimental autoimmune encephalomyelitis (EAE) 

due to an expansion of the Treg cell population in peripheral lymph nodes and decreased 

numbers of autoreactive T-helper type 1 and T-helper-17 cells. MK decreased the Treg cell 

population ex vivo in a dose-dependent manner by suppression of STAT5 phosphorylation that is 

essential for Foxp3 expression. Moreover, administration of anti-MK RNA aptamers 

significantly expanded the Treg cell population and alleviated EAE symptoms. These 

observations indicate that MK serves as a critical suppressor of Treg cell expansion, and 

inhibition of MK using RNA aptamers may provide an effective therapeutic strategy against

autoimmune diseases, including multiple sclerosis. 

Disclosures: H. Takeuchi , None; J. Wang, None; Y. Sonobe, None; S. Jin, None; T. Mizuno, 

None; A. Suzumura, None. 

Poster 





Support: NIH AA016566 

NMSS RG3618 

NIH NS37475 

Title: Evidence that CNS rather than PNS defects underlie the „shaking‟ mouse phenotype 

Authors: *A. J. MIERZWA 1 , J. ROSENBLUTH 1,2 , E. J. LANG 1 ; 

1 Neurosci & Physiol, New York Univ. Sch. Med., New York, NY; 2 Rusk Inst., New York Univ., 

New York, NY

Abstract: In previous abstracts, we have presented a spontaneously occurring „shaking‟ mouse 

mutant characterized by severe CNS dysmyelination, minimal PNS pathology, a mild kinetic 

tremor, seizures in 10-20% of animals, and near-normal longevity (Mierzwa et al., SFN Abstract 

277.17, 2005 and Mierzwa et al., SFN Abstract 975.20, 2004). We now present data on CNS and 

PNS conduction velocity measured in vivo in „shaking‟ and control animals. Mice were 

anesthetized with Nembutal, placed on a ventilator and paralyzed with Flaxedil. The sciatic 

nerve and two sites on the dorsal columns (C1 and low thoracic) were exposed. To obtain CNS 

conduction velocity the exposed sciatic nerve was stimulated with a bipolar electrode and 

recordings were made with bipolar electrodes placed on the dorsal columns. The difference in 

latency between rostral and caudal recording sites was measured and used to calculate CNS 

conduction velocity. Maximum CNS conduction velocity is decreased fourfold in the „shaking‟ 

mutant (shaking=5.2 ± 1.2 m/s, n=4 [mean ± SD] vs control=22.7 ± 5.1 m/s, n=9; p

Authors: *M. CORTÉS 1 , M. L. ITA 1 , D. M. MENDIOLA 1 , A. UGARTE 1 , J. R. EGUIBAR 2 ; 

1 Inst. Physiol., 2 Inst. Physiol. and Secrtearia Gen., B. Univ. Autonoma De Puebla, Puebla, 

Mexico 

Abstract: Taiep rats are a myelin mutant with a progressive motor syndrome characterized by: 

tremor, ataxia, immobility, epilepsy and paralysis. The main cause of the alteration is a 

hypomyelination followed by a progressive demyelination. At 7 months of age the rat´s showed 

immobility episodes (IEs) induced by gripping the animals from the tail, during IEs a REM-like 

EEG characteristics were obteined. The IEs increase with α1 agonists and with prazosin, an α1 

antagonist, and diminished with α2 antagonists. In the present experiment we analyze the effects 

of systemic injection of two D3 agonists. 

The rats were maintained under standard conditions with free access to water and balanced 

rodent pellets (Zeigler, USA). At 8 mo of age male taiep rats with an increased dose scheme 7- 

OH-DPAT (1 to 1,000 µg/kg) and PD-128907 (1 to 1,000 µg/kg) at 900 AM 

Our results showed that 7-OH-DPAT produce an increase in the frequency to 13.3 ± 0.9 of IES 

respect to control with 6.8 ± 1.1 with highest dose (P

Abstract: Multiple sclerosis (MS) is an inflammatory and demyelinating CNS disease that 

affects ~400,000 adults in the US; however, currently available treatments remain unsatisfactory. 

Clinical MRI is routinely utilized for diagnosis and prognosis of MS and as a primary outcome 

measure in clinical trials. Preclinically, experimental autoimmune encephalomyelitis (EAE) is 

characterized by many of the same pathophysiological processes observed in MS and is therefore 

commonly employed for development of new therapeutics for MS. Recently, ultrasmall 

superparamagnetic iron oxide (USPIO)-enhanced T2-weighted MRI was described as an 

invaluable tool that allows in vivo monitoring of infiltrating macrophages, the predominant 

effector cells in EAE, allowing visualization of new lesions during disease development. Here, 

we describe how we have developed imaging methods to visualize these inflammatory responses 

(macrophage infiltration and blood-brain barrier breakdown) using molecular MRI in EAE rats 

immunized with myelin oligodendrocyte glycoprotein (MOG). With a goal to characterize the 

CNS lesions and elucidate the implications on observed neurological deficits, behavioral 

symptoms were scored daily, and rats were imaged longitudinally during acute, remission, and 

relapse phases using a 7T MRI scanner. We demonstrated a distinct spatiotemporal pattern of 

CNS lesions that unveils the involvement of spino-olivocerebellar pathways. In particular, 

lesions of the inferior olives were observed primarily in the acute phase whereas lesions of 

cerebellum and spinal cord were observed during the relapse phase and appeared to correlate 

with severity. Histological and immunochemical studies in the same animal confirm the presence 

of USPIO uptake as well as activated macrophages in demyelinated lesion areas. Further, clinical 

scores recorded from these animals revealed observations of loss of tail tone, abnormal gait, and 

paralysis that agree with the functional role of spino-olivocerebellar pathways in coordination 

and movement. Collectively, our results provide a new perspective for further understanding the 

underlying pathology of this MS model. 

Disclosures: M.V. Pai, None; P.F. Bousquet, None; A. Schwartz, None; B.L. Mcrae, 

None; E.M. O'Connor, None; C.M. Nelson, None; V.P. Hradil, None; B.F. Cox, None; G.B. 

Fox, None; C. Chin, None. 

Poster 





Support: NINDS, 5R01NS056427 

Sanitätsrat Dr. Emil Alexander Huebner & Gemahlin Foundation, Germany

National Multiple Sclerosis Society, Grant RG3712A1 (V.G.) 

Title: Hyperoxia causes astrogliosis in the developing brain 

Authors: *T. SCHMITZ, L.-J. CHEW, V. GALLO; 

Ctr. for Neurosci. Res., Children's Natl. Med. Ctr., Washington, DC 

Abstract: Periventricular white matter damage is a major cause of impaired neurological 

development in premature infants. Contributing factors include perinatal infection, inflammation 

and hypoxia/ischemia. High oxygen (80%) concentrations have also been shown to cause 

oligodendrocyte progenitor cell death and hypomyelination. In many animal models of perinatal 

brain damage, the observation of astrocyte activation (astrogliosis) has often been associated 

with CNS inflammation and white matter deficiency. The role of astroglial changes in white 

matter loss following oxygen exposure however has not been explored and is the focus of this 

study. We investigated the effects of high oxygen concentrations on astrocytes both in vivo and 

in vitro. In a transgenic mouse line bearing astrocyte-targeted reporter expression (glial fibrillary 

acidic protein promoter, GFAP-EGFP), exposure of postnatal day 6 (P6) pups to 48h hyperoxia 

decreased myelin basic protein (MBP) expression in subcortical white matter regions when 

analyzed by immunocytochemistry and Western blotting after a 4-day recovery in room air at 

P12. These observations at P12 were associated with markedly elevated GFAP-EGFP and GFAP 

expression levels (P


Support: MDA4266 

R37DK018024 

K99NS057903 

Title: Investigating the function of the inactive myotubularin related phosphatase Mtmr13 in 

mice: a model of CMT4B2 and an investigation of Mtmr13 localization in Schwann cells 

Authors: *F. L. ROBINSON 1 , I. R. NIESMAN 2 , K. K. BEISWENGER 3 , J. E. DIXON 1 ; 

1 Pharmacol., 2 Anesthesiol., 3 Pathology, Univ. California San Diego, La Jolla, CA 

Abstract: Charcot-Marie-Tooth disease type 4B (CMT4B) is a demyelinating peripheral 

neuropathy characterized by slowed nerve conduction velocity (NCV), severe axonal loss, and 

myelin outfolding and infolding. CMT4B is caused by recessive mutations in either 

myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are 

a large family of phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 

3-phosphate (PtdIns3P) and PtdIns(3,5)P2, two PIs that regulate membrane traffic within the 

endosomal-lysosomal system. Intriguingly, nearly half of the metazoan myotubularins are 

catalytically inactive. MTMR2 and MTMR13 are active and inactive PI 3-phosphatases, 

respectively, and the two proteins have been shown to directly associate, although the functional 

significance of this association is not well understood. 

In hopes of establishing a mouse model of CMT4B2, we have disrupted the Mtmr13 gene. 

Biochemical and genetic analyses confirmed the disruption of Mtmr13. We used a combination 

of NCV testing, standard histology and electron microscopy to evaluate whether the loss of 

Mtmr13 leads to a CMT4B2-like condition in the mouse. Peripheral nerve pathology was 

examined in sciatic and saphenous nerves. We find that Mtmr13-deficient mice develop a 

peripheral neuropathy characterized by reduced NCV and myelin outfolding and infolding. At 

the earliest time point examined (14 days), myelin infoldings and outfoldings are present, 

suggesting that this pathology arises concomitantly with myelination. Notably, axonal loss and 

hypomyelination, two prominent features of CMT4B2, are largely unobserved in Mtmr13deficient 

mice at 14 months of age. Experiments are currently underway to examine peripheral 

nerve pathology in older mice (20 months or older), to determine if axonal loss and 

hypomyelination are more prominent in such animals. In summary, loss of Mtmr13 in mice leads 

to a dysmyelinating peripheral neuropathy with many of the key features of CMT4B2. Our 

mouse model will likely be an essential tool for future studies of how the loss of MTMR13 leads 

to CMT4B2. 

In a second aspect of our study, we used immunofluorescence to examine the localization of both 

Mtmr2 and Mtmr13 in Schwann cells of teased fibers from sciatic nerves. Preliminary 

immunofluorescence results indicate the presence of Mtmr2 and Mtmr13 in all cytoplasmcontaining 

domains of myelinating Schwann cells. Preliminary double-labeling experiments 

using antibodies specific for Mtmr2, Mtmr13 and markers of endosomal and lysosomal 

compartments have suggested unexpected localizations of Mtmr2 and Mtmr13 in Schwann cells.

Disclosures: F.L. Robinson, None; I.R. Niesman, None; K.K. Beiswenger, None; J.E. Dixon, 

None. 

Poster 





Support: Barrow Neurological Foundation 

Title: APOE 4 polymorphism results in early cognitive deficits in the EAE mouse 

Authors: *J. SHI, J.-L. TU, T. VOLLMER, C.-B. ZHAO; 

Dept Neurol, Barrow Neurol Inst., Phoenix, AZ 

Abstract: Background: Recent studies have identified an association between APOE 4 and 

cognitive deficits in multiple sclerosis, which is most striking in the youngest cohort of patients. 

Previous studies in transgenic APOE animal models have demonstrated cognitive deficits in aged 

mice, emphasizing its effect in diseases such as Alzheimer‟s. Understanding the unique role of 

APOE 4 in MS requires animal models which manifest cognitive deficits at an earlier age and 

stage. 

Objective: To determine the role of APOE 4 polymorphism on MS and produce a model 

whereby the unique early effect can be studied, we measured cognitive performance in the 

transgenic APOE 4 mouse after EAE induction before the onset of motor symptoms. 

Methods: Cognitive function of APOE knockout (KO) and human APOE 4 knockin (E4) mice 

were compared with wild type on day 7-9 (age 6-8 weeks ) after EAE induction prior to the onset 

of motor manifestations (day 13-15). Behavioral characteristics of the cognitive deficits were 

determined using the Morris water maze. Neurohistochemical studies of the hippocampus were 

accomplished to confirm the deficits. 

Results: After EAE induction, KO and E4 showed significant deficits in spatial learning and 

recall suggesting deficits in the cholinergic function. Evidence of neuronal damage in the 

hippocampal CA region was identified and correlated with regional increases in cholinesterase 

and decreased levels of acetylcholine in the E4 and KO groups relative to the wild type. 

Conclusions: Understanding the unique role of APOE on MS requires accurate animal models. 

We present evidence of the role of APOE in EAE prior to the onset of motor symptoms 

suggesting, that cognitive deficits may precede motor deficits in a predisposed population. 

Identifying early cognitive deficits early may broaden the diagnostic and therapeutic window and 

provide valuable information regarding the roles of inflammation and APOE in disease.

Disclosures: J. Shi , None; J. Tu, None; T. Vollmer, None; C. Zhao, None. 

Poster 






NMSS RG3515 

Title: Assessment of sensorimotor and social interaction paradigms to detect corpus callosum 

demyelination in mice 

Authors: N. E. HARWOOD 1 , R. PANNU 2 , T. J. WU 3 , *R. C. ARMSTRONG 2 ; 

1 Neurosci. Program, 2 Dept Anat. & Cell Biol/Neurosci, 3 Dept Ob Gyn, USUHS, Bethesda, MD 

Abstract: For studies of demyelinating disease processes, the corpus callosum (CC) offers 

advantages as the largest myelinated tract in the mammalian brain, and, as located adjacent to the 

subventricular zone, a potential source of neural stem cells. However, the major disadvantage of 

the CC in rodents is difficulty in identifying functional deficits associated with demyelination, 

especially for longitudinal analysis throughout the course of demyelination and remyelination. 

We tested two different behavioral measures, each attributed to CC involvement, for the ability 

to distinguish functional deficits at specific stages of CC demyelination in male C57Bl/6 mice 

fed 0.2% cuprizone, a copper chelator that induces extensive CC demyelination. No overt 

neurological deficits were apparent in treated or non-treated mice. After completion of 

behavioral analyses, mice were perfused for immunohistochemistry to verify CC demyelination. 

One behavioral assessment was running on a “complex” wheel with varied rung intervals which 

requires rapid bilateral limb sensorimotor coordination and integration between cerebral 

hemispheres. The daily maximum running velocity was not different between mice during acute 

CC demyelination (6 wks of cuprizone) as compared to before treatment or after recovery (6 wks 

after cuprizone removed from diet), or as compared to non-treated mice. A second functional 

assessment used a resident-intruder test of social interaction. Behavior was video taped for each 

“resident” mouse during a 15 min period of acclimation to a new cage followed by a 15 min 

period with a second mouse introduced into the cage as an “intruder”. Recordings were scored 

for a battery of specific activities that were subsequently classified as either interactive or 

independent behaviors. Preliminary analyses of initial cohorts showed that the ratio of 

independent to interactive behaviors was decreased for resident mice treated with cuprizone for 6

wks (acute CC demyelination) or 12 wks (chronic CC demyelination), as compared to nontreated 

age-matched control resident mice. These results indicate that the complex wheel task is 

not a good indicator of CC demyelination. However, social interaction tests, such as the residentintruder 

paradigm, might reveal CC functional abnormalities. 

Disclosures: N.E. Harwood, None; R.C. Armstrong , None; R. Pannu, None; T.J. Wu, None. 

Poster 





Support: NMSS RG 3128 

NIH/NINDS R01 39569 

Title: Impact of chronic restraint stress during early Theiler‟s virus infection on CNS disease 

severity in SJL mice 

Authors: *E. E. YOUNG 1 , A. N. SIEVE 1 , E. G. VICHAYA 1 , L. M. CARCOBA 1 , A. 

AMBRUS 2 , R. STORTS 2 , C. R. YOUNG 3 , C. J. R. WELSH 3 , M. W. MEAGHER 1 ; 

1 Behav Cell Neurosci, 2 Vet. Pathobiology, Col. of Vet. Med., 3 Vet. Integrative Biosci., Texas A 

and M Univ., College Station, TX 

Abstract: Theiler‟s murine encephalomyelitis virus (TMEV) infection, a model of multiple 

sclerosis, is characterized by an acute CNS inflammatory phase followed by a chronic 

demyelinating disease in genetically susceptible strains of mice. Susceptibility to the chronic 

phase of disease has been shown to depend on a combination of genetic and environmental 

factors, including strain, sex, and stress exposure. Previous research from our laboratory 

indicates that chronic restraint stress during the first 4 weeks of infection exacerbates behavioral 

signs of disease in both male and female SJL mice. The present study examined the impact of 

sex and restraint stress exposure on spinal cord inflammation, glial activation, demyelination, 

and axonal loss during chronic TMEV infection. Histological analyses indicated that male SJL 

mice showed increased CNS inflammation, demyelination and axonal loss in the chronic phase 

of infection. These findings of increased CNS disease severity are in agreement with previous 

research from our laboratory showing significant restraint-induced exacerbation of the behavioral 

syndrome and motor impairment associated with chronic TMEV infection (Sieve et al., 2004). 

The present study provides evidence that exposure to chronic stress during the acute phase of

TMEV infection subsequently augments the development of chronic demyelinating disease and 

that the impact of stress on disease course may be partially dependent on genetic factors such as 

sex. These findings may shed light on clinical findings that stress exacerbates MS disease course 

(Mohr et al., 2004) and that men develop more severe clinical symptoms and deteriorate more 

rapidly than women (Cottrell et al., 1999). 

Disclosures: E.E. Young , None; A.N. Sieve, None; E.G. Vichaya, None; L.M. Carcoba, 

None; A. Ambrus, None; R. Storts, None; C.R. Young, None; C.J.R. Welsh, None; M.W. 

Meagher, None. 

Poster 





Support: Department of Veterans Affairs Biomedical Laboratory R&D Grant 


National Multiple Sclerosis Society Grant RG3405-B-5 


Title: Estrogen affects IL-17 levels in female mice with experimental autoimmune 

encephalomyelitis 

Authors: *M. A. YATES 1,2 , S. SINHA 1,2 , L. J. KALER 1,2 , H. OFFNER 1,2,3 ; 

1 Neurol., Oregon Hlth. & Sci. Univer, Portland, OR; 2 Neuroimmunology Res., Veterans Affairs 

Med. Ctr., Portland, OR; 3 Dept. of Anesthesiol. and Perioperative Med., Oregon Hlth. & Sci. 

Univ., Portland, OR 

Abstract: Estradiol (E2) treatment has previously been demonstrated to protect against the 

development of experimental autoimmune encephalomyelitis (EAE) in female mice. However, 

the effects of E2 treatment on production of interleukin-17 (IL-17), which is important in the 

pathogenesis of EAE, have not been explored. To further investigate the mechanism behind 

estrogen‟s protective effects, female C57Bl/6 mice were implanted with 2.5mg 17β-estradiol 

pellets (60 day release) before immunization to induce EAE. Compared to controls, females 

implanted with E2 were protected against the development of EAE. Brains, spleens, and lymph

nodes were collected at different points in the disease course in order to compare cytokine 

production. Luminex assay at 38 days post-immunization showed that E2 treated animals had 

dramatically reduced secretion of IL-17 in cells isolated from brain compared to control animals. 

Production from splenocytes also showed a decrease, but IL-17 levels were unchanged in the 

lymph node. The effects of IL-23 and the role of the receptors ER-α and ER-β are also being 

explored. 

Disclosures: M.A. Yates, None; S. Sinha, None; L.J. Kaler, None; H. Offner, Effective 

Pharmaceuticals, E. Ownership Interest (stock, stock options, patent or other intellectual 

property). 

Poster 





Support: NIH NINDS Grant 5F31NS056502 

Title: Effect of ASPA deficiency on neural cells during early postnatal development 

Authors: *N. S. MATTAN 1 , R. MATALON 2 , J. DE VELLIS 1 ; 

1 Dept Neurobiol, UCLA MRRC, Los Angeles, CA; 2 UTMB - Dept. of Pediatrics, Galveston, TX 

Abstract: Canavan Disease (CD) is a neurodegenerative disease, caused by a deficiency in the 

enzyme aspartoacylase (ASPA), and characterized by mental retardation, macrocephaly, head lag 

and hypotonia. Loss of ASPA activity causes a consequent increase in its substrate Nacetylaspartate 

(NAA) and decreased production of aspartate and acetate. Recently, ASPA has 

been localized in oligodendrocytes (OL), however, it is still undefined how ASPA deficiency 

may affect OL development. To investigate the effects of ASPA deficiency on the development 

of OL cells, we studied postnatal OL maturation in ASPA CD and wild-type (WT) mice at 

different ages. ASPA levels in WT mice had an expression pattern that correlates well with OL 

lineage progression and its expression peaked at postnatal day 30 (P30) in different brain regions 

suggesting a role for ASPA in OL development and myelination. CD and WT mice showed 

similar number of Sox2 and Sox 9 positive cells as well as immature OL at early postnatal ages 

(P1 and P7). However, more progenitor cells expressing Sox2 were found up to P30 in CD brains 

opposed to WT mice. Defects in OL maturation and myelinogenesis started to be seen in CD 

brains as early as P10 and P15. Decreased MBP and PLP immunoreactivity was found in both 

the corpus callosum (CC) and the CB of CD mice. MBP protein levels were decreased by P15 in

cortex and CB tissue lysates of CD mice and its levels were further decreased with age in CD 

mice. In agreement, the number of GSTpi/CNPase positive cells was significantly reduced in 

P30 CD brains compared to age-matching WT. At later postnatal ages, the central nervous 

system of CD mice displayed a more generalized de/dysmyelination. Improper myelination in 

CD brains was also confirmed by electron microscopy. Massive damage could be visualized as 

early as P17 in CD mice compared to the concentric and nicely formed myelin in the WT mice. 

In parallel, increased NG2, GFAP and s100beta expressions were found in CD brains starting at 

P15, indicative of gliosis. These effects were accompanied by decreased expression of markers 

for immature and mature neurons in CD brains. Our findings strongly suggest that, early in 

postnatal development, ASPA deficiency primarily affects OL maturation leading to impaired 

myelination and neuronal dysfunction. 

Disclosures: N.S. Mattan, None; J. de Vellis, None; R. Matalon, None. 

Poster 





Support: NIH Grant NS-30900 

Myelin Repair Foundation 

Title: Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune 

response and promote endogenous repair in animal models of Multiple Sclerosis 

Authors: L. BAI, *R. H. MILLER; 

Dept Neurosci, Case Western Res. Univ. Sch. Med., Cleveland, OH 

Abstract: Cell based therapies are attractive approaches to promote myelin repair. Recent 

studies demonstrated a reduction in disease burden in mice with EAE treated with mouse 

mesenchymal stem cells (MSCs). Here we examined the efficacy of human bone marrow derived 

MSCs (BM-hMSCs) to promote functional recovery in MOG peptide 35-55 induced EAE 

models, an animal model for Multiple Sclerosis (MS). We traced their immigration into the 

injured CNS and assayed their ability to modulate the host immune response. Injected BMhMSCs 

accumulated in the CNS, reduced the extent of damage and increased oligodendrocyte 

lineage cells in lesion areas. Inflammatory T-cell producing cytokines and Th17 producing T 

cells and their associated cytokines were substantially reduced along with concomitant increases

in the anti-inflammatory cytokines. Together these data suggest the BM-hMSCs represent a 

viable option for therapeutic approaches. 

Disclosures: L. Bai, None; R.H. Miller , None. 

Poster 





Support: Myelin Repair Foundation 

Title: Progression of functional recovery in lysolecithin demyelinating models in adult rats 

Authors: *Y.-H. HSIEH 1 , R. MILLER 1 , C. WILSON 2 ; 

1 Dept Neurosci., 2 Dept Pediatrics, Case Western Res. Univ., Cleveland, OH 

Abstract: The failure of remyelination can lead to pathophysiologic impairment that mimics 

Multiple Sclerosis including axonal damage, astrocytosis, and prominent inflammatory infiltrate. 

However, there is little data on functional changes over time in central nervous system neural 

circuits and the precise contribution of demyelination/remyelination to functional conduction in 

white matter axons is not clearly defined. Models of demyelinating spinal cord lesions involving 

the injection of lysolecithin (LPC) have been developed to better understand the biology of 

remyelination and this model allows quantification of neural pathway recovery using 

electrophysiological methods. We hypothesize that functional recovery parallels 

histological/morphologic markers of full remyelination. To test our hypothesis, we use an in vivo 

adult rat model (Sprague Dawley, 325-350 g) to evaluate both functional deficits in neural 

transmission along these tracts as well as time course and degree of recovery following LPC 

lesions. We electrically stimulate (L1) and record (C3) in anatomically identified axonal tracts 

containing LPC lesions (T12) in the dorsal column of the spinal cord to examine the compound 

evoked potential and conduction velocity. Black-Gold staining was utilized to morphologically 

identify the extent of demyelination/remyelination. Our preliminary functional data show that 

despite remyelination at 14 days post injection (dpi), incomplete recovery of signal transmission 

persists (10% when comparing the area of the evoked compound potential from the lesion to the 

non-lesioned control side). By 21 dpi, a 40% recovery is observed. Full recovery is observed by 

28 dpi. These results suggest that histological markers of remyelination do not correlate with 

functional recovery of transmission.

Disclosures: Y. Hsieh , None; R. Miller, None; C. Wilson, None. 

Poster 





Support: FSS, Denmark 

Title: Demyelination in experimental cerebral malaria 

Authors: *C. HEMPEL 1,2,4 , L. WIESE 5,4,3 , J. KURTZHALS 4,2 , M. PENKOWA 3 ; 

1 Univ. Copehagen, Cophenhagen, Denmark; 2 Ctr. for Med. Parasitology, 3 Section of 

Neuroprotection, Univ. of Copenhagen, Copenhagen, Denmark; 4 Dept. of Clin. Microbiology, 

Copenhagen Univ. Hosp., Copenhagen, Denmark; 5 Dept. of Infectious Dis., Hvidovre Hosp., 

Hvidovre, Denmark 

Abstract: Cerebral malaria (CM) is a serious and often fatal complication of Plasmodium 

falciparum malaria. It is an acute encephalopathy with loss of consciousness, convulsions and 

fever. The underlying pathology includes dysregulated cytokine expression, increased 

permeability of the blood-brain barrier and sequestration of host cells. Neurological sequelae are 

common in survivors but no pathological processes responsible for the sequelae have been 

identified. Since the neurological sequelae are often reversible, we hypothesised that 

demyelination, followed by remyelination, could be involved. 

We used the murine CM model (P. berghei infection in C57BL/6 mice) to study the possibility 

that CM results in demyelination. Experimental murine and human CM share several clinical and 

pathological characteristics. Brains were perfusion fixed with Zamboni‟s fixative and embedded 

in paraffin. Myelin was visualised immunohistochemically with antibodies specific for myelin 

basic protein. Apoptosis was visualised with antibodies specific for caspase-3. 

Extensive demyelination was detected in terminally ill mice. Demyelination was particularly 

evident in the cerebellum and in the corpus callosum. Apoptosis was present in infected mice but 

the number of caspase-postitive cells was not statistical different from uninfected mice. The 

underlying mechanisms remain to be elucidated as well as the extent of remyelination and 

neuronal damage. 

Disclosures: C. Hempel, None; L. Wiese, None; J. Kurtzhals, None; M. Penkowa, None.

Poster 





Title: Notch pathway and the effect of apotransferrin on remyelination 

Authors: *A. M. ADAMO, V. L. MILLANOVICH; 

Biol. Chem., Sch. of Pharm. and Biochem, Buenos Aires, Argentina 

Abstract: In previous studies, we have demonstrated that a single intracranial injection (ICI) of 

apotransferrin (aTf) in 3-day-old rats produces an increase in the different myelin components. 

This promyelinating effect of aTf has been clearly shown in animals suffering demyelination 

after cuprizone (CPZ) feeding. ATf induces a marked increase in the myelin deposition in 

comparison with the spontaneous remyelination observed in control animals (C). In this study, 

we examined the possible participation of the Notch signalling pathway in the aTf mediated 

reversion of demyelination. Twenty one day old Wistar rats were fed a diet containing 0.6% CPZ 

during 2 weeks. After withdrawal of CPZ, animals were treated with either a single ICI of aTf or 

saline. At different times after ICI, rats were used to dissect the subventricular zone (SVZ) and 

corpus callosum (CC) for Western blot and the brain for immunohistochemistry. We studied the 

colocalization of Notch intracellular domain (NICD) with different oligodendroglial cells 

(OLGc) markers and the expression of down stream genes by RT-PCR to explore Notch pathway 

activation in the population of OLGc. Results show that the level of Notch receptor is lower in 

SVZ from brain of C in comparison with CPZ treated rats and that 6 h after the ICI of aTf the 

level of Notch increases in the SVZ from C while it decreases in CPZ treated animals. The ICI of 

aTf induces a significant decrease in the activation of Notch signalling pathway in SVZ NG2+ 

OLGc of CPZ demyelinated rats concomitantly with a decrease in the level of Jagged in GFAP+ 

cells in these animals. However, in CC the ICI of aTf induces an increase in the level of Deltex 

associated to NICD in CPZ treated animals, demonstrated by immunoprecipitation experiments 

using anti-Deltex specific antibody. Preliminary results obtained with gama-secretase inhibitor 

injected with aTf in CPZ and control rats show similar outcomes in the remyelination process to 

those observed with the ICI of aTf. These results seem to indicate the involvement of Notch 

pathway in the aTf effect on the remyelination process. 

Disclosures: A.M. Adamo, None; V.L. Millanovich , None.

Poster 

252. Demyelinating Disorders: Mechanisms and Therapeutics II 



Topic: C.08.a. Molecular and cellular mechanisms 

Support: American Academy of Neurology Foundation 

NIH IDDRC P30HD40677 

Title: Alterations in Olig1 and MBP after ER stress in oligodendrocyte progenitor cells 

Authors: *A. TAKANOHASHI, A. VANDERVER; 

Ctr. for Genet. Med., Children's Natl. Med. Cntr, Washington, DC, DC 

Abstract: Introduction: Recent studies have suggested that endoplasmic reticulum (ER) stress 

may be involved in variety of disorders of the white matter of the brain. We hypothesize that an 

altered stress response in oligodendrocytes leads to altered myelin related protein transcription 

and expression. Clarification of the impact of ER stress on oligodendrocytes and myelin proteins 

will lead to improved understanding of white matter disorders in which ER stress has been 

implicated, such as multiple sclerosis, ischemia and leukodystrophies. 

Methods: OliNeu, a mouse oligodendrocyte progenitor cell line, was cultured in basal medium 

and differentiated to mature oligodendrocytes with 2mM dibutyryl cyclic adenosine 3‟, 5‟monophosphate 

(dcAMP) or de-differentiated with 10ng/mL of PDGF and 10ng/mL of bFGF. 

Cells were subjected to ER stress inducing compounds, such as thapsigargin (TG) and 

tunicamycin (TUN), in dose and time series experiments. Cell activity/viability was assessed by 

MTT assay, and the effects of stress on myelin related proteins were analyzed by 

immunocytochemistry and western blot. 

Results: 

At early time points, 3 hr to 6 hr, TG (0.5 µM-2.5µM) stimulated MTT reduction in OliNeu 

cells, while longer treatment, such as 15 hr and 24 hr, decreased MTT to 80% and 60%, 

respectively. Various concentrations of TUN (0.1-5µg/mL) showed a 20% and 60% reduction of 

MTT compared to controls in OliNeu at 6 hr and 24 hr, respectively. Neither differentiation 

treatments dcAMP or PDGF/bFGF altered the expression of myelin basic protein (MBP) or 

Olig1, but TG decreased both MBP and Olig1 protein expression in a dose dependent fashion 

(p

interest, since MBP is thought to be synthesized in free polysomes and not in the ER. ER stressrelated 

regulation of myelin protein transcription factors and downstream decreased expression 

of MBP could help explain myelin loss in certain acquired and heritable white matter disease. 

Understanding the effects of ER stress on glial cells could lead to targeted therapeutic 

interventions in these disorders. 

Disclosures: A. Takanohashi , None; A. Vanderver, None. 

Poster 





Support: New Jersey Commission on Spinal Cord Research 

New Jersey Commission on Brain Injury Research 

Title: Schwann cell proliferation following PNS demyelination: Are they coupled to one 

another? 

Authors: *D. YANG, H. A. KIM; 

Biol. Sci., Rutgers Univ., Newark, NJ 

Abstract: Injury to peripheral nerves initiates a sequence of injury responses in the distal 

Schwann cells termed Wallerian degeneration: Schwann cell demyelination is followed by 

dedifferentiation and proliferation. The nature of the signal that initiates the Schwann cell 

response is unknown. Glial growth factor (GGF) is a potent Schwann cell mitogen which 

belongs to the neuregulin-1 (Nrg-1) growth factor family. Treatment of myelinating co-culture 

with GGF induces demyelination followed by Schwann cell proliferation, indicating the 

demyelinating function of the growth factor is coupled to its mitogenic function. To address the 

issue, we tested the functions of other Schwann cell mitogens, TGF-b, PDGF, FGF-2 and EGFdomain 

of Nrg-1, on initiating demyelination. 

These growth factors, when combined with elevated levels of cAMP, have been shown to 

stimulate Schwann cell proliferation. We also tested the effect of CRD-Nrg-1, a Nrg-1 isoform 

that has been shown to promote myelination. 

Under a mitogenic condition, FGF-2 induced demyelination in the co-culture system as seen with 

GGF. However, TGF-b and PDGF failed to do so, indicating that the mitogenic function of a 

growth factor is not necessarily coupled to demyelination. Interestingly, the EGF-domain of Nrg-

1 and CRD-Nrg, under a condition that did not stimulate Schwann cell proliferation, were still 

able to induce demyelination. Biochemical analysis of the receptor downstream signaling 

pathways indicated that the demyelinating function of the growth factors were associated with a 

prolonged activation of the p42/44 Erk pathways. Altogether, our results suggest that the 

Schwann cell proliferation that normally follow PNS demyelination might not necessarily occur 

as a consequence of the previous event or by the signal that initiates demyelination. It is possible 

that the two events are regulated by independent signals that are generated by injured axons, or 

by an insult to the Schwann cells. 

Disclosures: D. Yang , None; H.A. Kim, None. 

Poster 





Title: VEGF-A induces blood-brain barrier permeability via downregulation of endothelial 

Claudin-5 

Authors: A. TADESSE ARGAW 1 , *G. R. JOHN 1,2 , B. T. GURFEIN 1 , Y. ZHANG 1 , A. 

ZAMEER 1 , C. F. BROSNAN 2 ; 

1 Mount Sinai Sch. Med., New York, NY; 2 Pathology and Neurosci., Albert Einstein Col. of 

Med., Bronx, NY 

Abstract: Permeability of the blood-brain barrier (BBB) is an early and significant event in CNS 

inflammation, but the underlying mechanisms remain elusive. The barrier exists at the level of 

brain microvascular endothelial cells (BMVEC) which restrict paracellular diffusion via complex 

tight junctions, the properties of which depend on transmembrane proteins of the claudin (CLN) 

family, particularly CLN-5. Formation and regulation of the endothelial barrier depend on other 

lineages, notably astrocytes and pericytes, and these cells have also been implicated in BBB 

permeability, acting via the vascular endothelial growth factor VEGF-A. Here, we show for the 

first time that astrocyte-derived VEGF-A promotes BMVEC permeability via downregulation of 

CLN-5. We further demonstrate that this occurs as part of a larger coordinated change in 

expression of CLN family members involving both BMVEC and reactive astrocytes. In human 

BMVEC cultures, both CLN-5 and occludin were lost in response to VEGF-A. In human 

astrocyte cultures, IL-1β or TGFβ1 induced VEGF-A, and also triggered expression of CLN-1, 

CLN-4, and JAM-1. In animal models of CNS inflammation, BBB permeability was associated 

with a striking reduction of endothelial CLN-5, and induction of astrocytic CLN-1 and CLN-4.

Functional studies showed that HBMVEC cultures expressing CLN-5 under the control of a 

VEGF-insensitive promoter were protected from VEGF-induced permeability. Taken together, 

these data implicate VEGF-induced downregulation of CLN-5 as an important mechanism in 

BBB permeability. They further suggest that loss of the endothelial barrier is accompanied by 

formation of an astrocytic organization that may regulate macromolecular traffic in the CNS 

parenchyma. 

Disclosures: A. Tadesse Argaw, None; G.R. John , None; B.T. Gurfein, None; Y. Zhang, 

None; A. Zameer, None; C.F. Brosnan, None. 

Poster 





Support: NIH (NS043432) 

VA Medical and RR&D Services 

NMSS CA1009A10 

Title: CNPase expression in olfactory ensheathing cells 

Authors: *C. RADTKE 1,2 , K. L. LANKFORD 1 , M. SASAKI 1 , V. GALLO 3 , J. D. KOCSIS 1 ; 

1 Yale Univ. Sch. Med., West Haven, CT, CT; 2 Reconstructive Surgery, Hannover Med. Sch., 

Hannover, Germany; 3 Children's Natl. Med. Ctr., Children's Res. Inst., Washington, DC 

Abstract: The olfactory bulbs were studied in transgenic mice expressing the enhanced green 

fluorescent protein (eGFP) under the control of the 2‟,3‟-cyclic nucleotide 3‟-phosphodiesterase 

(CNPase) promoter. CNPase is present in myelinating cells in both the central and peripheral 

nervous systems (oligodendroctyes and Schwann cells). eGFP was observed in the nuclei and 

cytoplasm of glial cells in the outer nerve layer (ONL) of the olfactory bulb where olfactory 

ensheathing cells (OECs) reside, and in oligodendrocytes in the interior of the olfactory bulb. 

Immunohistochemical analysis for CNPase in the olfactory bulb demonstrated weak expression 

of CNPase in the ONL, but intense expression in oligodendrocytes. However, dissociated OECs 

derived from the olfactory bulb and maintained in culture for 4 days had both intense eGFP 

expression and CNPase immunoreactivity. These data indicate that CNPase expression of OECs 

in vivo is weak, but present as evidenced by accumulated intracellular eGFP in OECs of the

ONL of the olfactory bulb in CNPase transgenic mice. Moreover, in culture the OECs maintain 

strong eGFP expression, but also show intense immunoreactivity for CNPase. Thus, while OECs 

do not normally form myelin on the fine calibre olfactory nerve axons, their upregulation of 

CNPase in culture is commensurate with reports that transplantation of cultured OECs can form 

myelin when transplanted into demyelinated spinal cord. 

Disclosures: C. Radtke, None; K.L. Lankford, None; M. Sasaki, None; V. Gallo, None; J.D. 

Kocsis, None. 

Poster 





Support: Nancy Davis MS Center Without Walls 

Medical Research Service 

Department of Veterans Administration 

Medical Rehabilitation Research Service 

Title: Sodium channel Nav1.6 contributes to macrophage function 

Authors: *P. ZHAO 1,2 , J. BLACK 1,2 , S. WAXMAN 1,2 ; 

1 Dept Neurol, Yale Univ. Med. Sch., West Haven, CT; 2 Rehabil. Res. Ctr., Vetarans Affairs 

Connecticut Healthcare Syst., West Haven, CT 

Abstract: Voltage-gated sodium channels are expressed in neurons where they are responsible 

for the action potential genesis and conduction. Sodium channels are also expressed in nonexcitable 

cells, including glia, immune cells and keratinocytes, but the functional roles of sodium 

channels in these cells are not well-understood. As part of studies to determine the contribution 

of macrophages to the pathogenesis of inflammatory/demyelinating disorders, we have examined 

the expression of sodium channels in macrophages and the contribution of sodium channels to 

macrophage functions. Following sciatic nerve crush in adult Sprague-Dawley rats, we observed 

the recruitment of large numbers of activated macrophages into the degenerating nerve. In 

immunocytochemical studies, sodium channel Nav1.6, but not Nav1.5, was robustly expressed in 

these macrophages, and was co-localized with the plasma membrane markers Na/K-ATPase and

CD14 and the cytoskeleton proteins actin, vimentin and β3-tubulin. Cultured macrophages 

obtained by peritoneal lavage displayed a similar distribution of Nav1.6 immunolabeling as 

exhibited by macrophages in vivo. To examine whether blockade of sodium channels affects 

macrophage function, we assayed macrophage phagocytosis and migration in the presence or 

absence of the sodium channel blockers phenytoin and tetrodotoxin (TTX). LPS (100 ng/ml)induced 

phagocytosis was significantly reduced by sodium channel blockade (reduction with 

phenytoin: 26%; TTX: 32%). Similarly, ATP (50 κM)-induced macrophage migration was 

decreased by exposure to phenytoin (36%) and TTX (26%). These results demonstrate that 

peripheral macrophages in vivo express Nav1.6 and suggest that these sodium channels 

contribute to macrophage function. 

Disclosures: P. Zhao, None; J. Black, None; S. Waxman, None. 

Poster 





Support: DFG SFB 581 

DFG MA1053/5-1/2 

Title: The Monocyte Chemoattractant Protein-1 is a pathogenetically relevant modulator of 

inherited peripheral neuropathies in animal models for Charcot-Marie-Tooth Type-1 disorders 

Authors: *B. D. KOHL 1 , S. M. FISCHER 1 , J. GROH 1 , A. WEISHAUPT 2 , J. TROPPMAIR 3 , R. 

MARTINI 1 ; 

1 Dept Neurol, Dev Neurobiol, 2 Dept Neurol, Neuroimmunology/Multiple Sclerosis Res., 

Wuerzburg Univ., Wuerzburg, Germany; 3 Dept. of Gen. and Transplant Surgery, Daniel- 

Swarovski-Research Lab., Innsbruck Univ., Innsbruck, Austria 

Abstract: The monocyte chemoattractant protein-1 (MCP-1) is an important mediator of 

macrophage and T-lymphocyte attraction into inflamed tissue. We could previously show that in 

its absence infiltration of macrophages and T-lymphocytes into peripheral nerves is diminished 

in mice heterozygously deficient for P0 (P0+/-), a model for Charcot-Marie-Tooth 1B (CMT1B). 

Surprisingly, the complete absence of MCP-1 (MCP-1-/-) did not alleviate the demyelinating 

phenotype, while reducing the dose of MCP-1 by 50% (MCP-1+/-) leads to a substantial 

reduction of the demyelinating pathology. Additionally, we identified the mitogen activated

protein kinase cascade MEK1/2-ERK1/2 as an important mediator of MCP-1 expression in 

mutant Schwann cells. To elucidate if MCP-1 has a common role in mouse models for other 

CMT1-forms, we examined the function of MCP-1 in peripheral myelin protein 22 

overexpressing mice (PMP22tg, C61) and mice deficient in connexin32 (Cx32-/-), which are 

models for CMT1A and for the X-linked, dominant form of CMT1 (CMT1X), respectively. We 

crossbred PMP22tg and the Cx32-/- mice with MCP-1 mutants and used immunohistochemistry, 

electron microscopy, real-time PCR and western blots for analyses of single and double mutant 

animals. Similarly to the findings in P0+/-mice, femoral quadriceps nerves of 6 months old 

PMP22tg/MCP-1+/- and Cx32-/-/MCP-1+/- mice showed reduced numbers of macrophages and 

exhibited a significant amelioration of the pathological phenotype. Peripheral nerves of 

PMP22tg/MCP-1-/- mice showed also reduced numbers of macrophages but alleviation was not 

as striking as in PMP22tg/MCP-1+/- mice. Contrary, peripheral nerves of Cx32-/-/MCP-1-/- 

showed similar to P0+/-/MCP-1-/- mice an aggravation of disease, accompanied by only slightly 

reduced numbers of macrophages in comparison to Cx32-/-/MCP-1+/-. Thus, reduction but not 

complete absence of MCP-1 substantially alleviates the pathological phenotype in three distinct 

mouse models for CMT1. The search for regulatory pathways of MCP-1-upregulation in 

PMP22tg and Cx32-/- mice, identified ERK1/2 in the PMP22tg mice, whereas in Cx32-/- mice 

MCP-1 might be upregulated independently from ERK1/2. Our findings show that the 

expression of MCP-1 is crucial for the pathological mechanisms in three mouse models for 

CMT1, whereas the regulation of MCP-1 expression might require different intracellular 

pathways. 

Disclosures: B.D. Kohl , None; S.M. Fischer, None; J. Groh, None; A. Weishaupt, None; J. 

Troppmair, None; R. Martini, None. 

Poster 




Topic: C.08.d. Therapeutic strategies 

Support: National Multiple Sclerosis Society 

Title: Low dose naltrexone (LDN) and opioid growth factor (OGF) prevent or delay 

experimental autoimmune encephalomyelitis (EAE) 

Authors: *K. A. RAHN 1 , R. H. BONNEAU 2 , P. J. MCLAUGHLIN 1 , I. S. ZAGON 1 ; 

1 Neural & Behavioral Sci., 2 Microbiology & Immunol., Penn State Univ. Coll Med., Hershey, 

PA

Abstract: Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous 

system that affects 2.5 million individuals worldwide. While some treatments are available, more 

effective therapies that have few side-effects need to be explored. Opioid peptides affect a 

variety of physiological processes, including analgesia and growth, but the role of opioids in 

autoimmune diseases is unknown. The purpose of this research is to determine how modulation 

of endogenous opioid systems affects the progression of MS using the animal model 

experimental autoimmune encephalomyelitis (EAE). Mice immunized with MOG were given 

daily injections of saline (MOG-vehicle), the opioid receptor agonist opioid growth factor (OGF, 

also termed met-enkephalin, 10 mg/kg, MOG-OGF), or the opioid receptor antagonist naltrexone 

in a low (0.1 mg/kg, MOG-LDN) or high (10 mg/kg, MOG-HDN) dose. LDN provides a 4-6 

hour opioid receptor blockade and HDN a 24 hour opioid receptor blockade. Disease scores were 

recorded daily. In comparison to 100% of MOG-vehicle animals that developed EAE by day 30, 

45% of the MOG-LDN (p

Title: A novel free radical scavenger:platinum nanoparticle improves neurological signs and 

electrophisiological findings in a rat EAE model via quenching ROS and NO 

Authors: *H. YOSHIDA, M. SAGIC, J. SCHWERMAN, K. J. SMITH; 

Neuroinflammation, Neurol, Univ. Coll London, London, United Kingdom 

Abstract: Platinum nanoparticles were prepared by an alcohol reduction method and 

complementarily stabilized with polyacrilic acid(PAA-Pt). The average diameter of PAA-Pt was 

about 2nm. They were well dispersed in water and became colloidal solution. PAA-Pt efficiently 

quenched superoxide anion(O(2)(-)), Hydrogen peroxide(H(2)O(2))and Nitric oxicide(NO). This 

quenching activity against ROS and NO persisited like catalysis such as SOD or catalase. 

Therefore, PAA-Pt may be a useful scavenger which is effective on medical treatment of 

oxidative stress diseases. In fact, we have shown that platinum nanoparticles are effective on 

animal models of various ROS-related diseases like stroke, Alzheimer's disease, Huntinton 

disease and so on. And this time, we evaluated the effect of PAA-Pt on experimental 

autoimmune encephalomyelitis(EAE) using a rat. Intravenous injection of PAA-Pt improves 

neurological signs and electrophysiological findings in an EAE rat model. 

Disclosures: H. Yoshida, None; M. Sagic, None; J. Schwerman, None; K.J. Smith, None. 

Poster 





Support: National Multiple Sclerosis Society PP1247 

Title: Potential experimental therapy to improve Multiple Sclerosis (MS) symptoms in an animal 

model 

Authors: P. K. BOSE 1,3 , J. HOU 3 , *C. PHADKE 4 , R. SMITH 3 , R. PARMER 3 , Y. CHENG 3 , P. 

M. HOFFMAN 2 , W. STREIT 3 , F. J. THOMPSON 3 ; 

1 BRRC, 2 Res. Service, North Florida/South Georgia VAMC, Gainesville, FL; 3 Neurosci., Univ. 

of Florida, McKnight Brain Inst., Gainesville, FL; 4 Univ. Florida, Gainesville, FL 

Abstract: Symptoms and disabilities of MS appear across a broad spectrum, including spasticity, 

weakness, cognitive impairment, and balance disorder. Our recent Experimental Autoimmune 

Encephalomyelitis (EAE) rat model has revealed significant neurophysiological, locomotor,

alance and cognitive changes which have collectively demonstrated the feasibility of 

reproducing significant features of human spasticity and other disabilities in this model. Thus 

this animal model became amenable to applications of rigorous outcome measures and potential 

treatment approaches that can have direct translational potential. We used two rehabilitative 

approaches using a custom made bicycle locomotor training and Transcranial magnetic 

stimulation (TMS) to expand the evidence base for new rehabilitative interventions to document 

the extent to which these can safely reduce symptoms of MS-disabilities, and how these 

interventions influence the underlying neurobiology. By week-5 post-inoculation (pi, MBP in 

CFA), the EAE rats revealed significant velocity-dependent ankle extensor spasticity which 

contained both dynamic (higher velocities) and tonic component (at low velocities) and increased 

over time (to pi week 17). Moreover, TMS motor-evoked potentials (tcMMEPs) amplitudes 

increased in soleus (SOL) and tibialis anterior (TA) and forelimb flexor (FF) at pi week 3 (in 

acute stage of the disease) and drastically decreased or absent (~ 50%) especially in SOL & TA 

and remained increased in FF from pi week 5 through 17. Interestingly, there was a recovery in 

TA tcMMEPs amplitudes at pi week 17 which was not observed in SOL. In addition, these 

animals showed significant motor weakness in the forelimbs, a cognitive deficit for serial 

learning in MWM, and a significant reduction in balance tested on a rotorod. Our data to date 

indicate that EAE animals treated using cycle training (two 20 min. sessions for 3 weeks) or 

TMS (25 single magnetic pulses with graded stimulus intensities from 30% to 70% of Max) 

revealed significantly decreased spasticity, increased forelimb grip strength, improved scores for 

serial learning, and increased balance performance. We propose that a significant portion of 

these disabilities are companion disorders correlated with decreased noradrenergic (NE) function 

in neural regions that are critical to these functions. We hypothesize that the therapeutic 

treatments using locomotor training or TMS significantly improved MS symptoms through a 

upragulation of NE function in selected spinal, brainstem, and cortical regions. These data 

support the exploration of these treatment modalities for delaying and reducing the severity of 

MS symptoms. 

Disclosures: P.K. Bose, None; J. Hou, None; C. Phadke , None; R. Smith, None; R. Parmer, 

None; Y. Cheng, None; P.M. Hoffman, None; W. Streit, None; F.J. Thompson, None. 

Poster 





Title: Reinforced cinematic feedback for the treatment of balance disorders in multiple sclerosis

Authors: *L. A. PIRON 1 , A. TUROLLA 1 , M. TIOZZO 1 , M. DAM 2 , P. TONIN 1 ; 

1 Neurorehabilitation, I.R.C.C.S. San Camillo Hosp., Venice, Italy; 2 Neurosci., Univ. of Padova, 

Padova, Italy 

Abstract: We performed a study to evaluate and treat the balance deficits in patients affected by 

Multiple Sclerosis (MS) afferenting to our rehabilitation centre. We applied an innovative 

therapeutic procedure based on the use of the reinforced feedback of their body center of gravity 

(COG) motion during specific balance exercises. The reinforced feedback consisted of the 

knowledge of performance and the knowledge of results of the motion of the patient‟s COG 

visually displayed as an interactive trajectory on the computer screen. Several experimental 

studies have already shown that cinematic information about a movement could promote 

neurophysiological mechanisms of learning. Along this line we evaluated if this paradigm could 

be useful for exploiting new abilities in the balance deficits of MS patients. 

Two homogeneous samples of MS patients were recruited; the study group underwent a 

reinforced feedback training and control group underwent a balance-oriented traditional 

treatment. Each group was composed by twenty-four patients that were affected by a secondaryprogressive 

MS with various degrees of balance deficits. The study group received the treatment 

by the means of a computerized postural force platform. The subjects were requested to move 

their body COG virtually reaching several targets displayed in the monitor located in front of 

them, while they were safely standing on the SBM postural platform. They could have the online 

feedback of their body movement displayed on the screen and, at the end of each trial, they 

could observe the graphic report of the morphology of their COG trajectories. 

Before and after 15 daily treatment sessions, all the patients were examined with the Fugl-Meyer 

scale (balance sub-score - FM), the Rivermaid motor assessment scale (general activities, lower 

limb and trunk scores) and the Tinetti‟s test. Some cinematic parameters were performed in both 

groups before and after the treatment: the reaction time (RT), the maximum endpoint excursus 

(EPE), the directional control (DCL) and the movement velocity (MVL). 

Non parametric tests (Wilcoxon, U Mann - Whitney and Spearman‟s π ) was used to determine 

the statistical significance within and between groups of the clinical scales mean scores and 

cinematic parameters before and after therapy. 

Only the study group showed a statistically significant improvement of the mean scores of the 

three clinical scales after treatment. Only the FM and DCL showed a statistical significance 

between groups and also a significant correlation. 

These preliminary data indicated that an exposition to an artificially generated feedback could be 

used as a therapeutic approach for balance disorders in MS patients. 

Disclosures: L.A. Piron , IRCCS San Camillo, A. Employment (full or part-time); A. Turolla, 

IRCCS San Camillo, A. Employment (full or part-time); M. Tiozzo, IRCCS San Camillo, A. 

Employment (full or part-time); M. Dam, University of Padova, A. Employment (full or parttime); 

P. Tonin, IRCCS San Camillo, A. Employment (full or part-time). 

Poster



Program#/Poster#: 252.11/X1 


Support: NIH/NINDS NS41268 

NMSS CA1044A1 

Title: Tuftsin (TKPR) abrogates symptoms in experimental autoimmune encephalomyelitis 

(EAE): a switch to Th2 response 

Authors: *M. WU 1 , S. TSIRKA 2 ; 

1 Neurosci, 2 Pharmacol., Stony Brook Univ., Stony Brook, NY 

Abstract: Multiple sclerosis (MS) is a demyelinating autoimmune disease. One of the 

commonly used animal models for MS is experimental autoimmune encephalomyelitis (EAE), 

which mimics many pathological and histological hallmarks of MS in human. During the 

progression of MS/EAE, microglia, the immunocompetent cells of the brain, become activated 

and accumulate around demyelinated lesions. Tuftsin (TKPR) is a macrophage/microglial 

activator, as it promotes phagocytic activity for cells of monocytic origin that express tuftsin 

receptors. Previous studies in our lab found that infusion of tuftsin using micro-osmotic pumps 

severely abrogated EAE in wild-type mice, and resulted in upregulation of GATA-3, which is a 

Th2 cytokine transcription factor. Our current studies further investigate the role of tuftsin during 

EAE both in vivo and in culture. We culture primary spleen T cells and measure cytokine 

production after different stimulations. Tuftsin added directly to T cells does not have any effect 

on cytokine production; however, neuronal conditional medium and tuftsin-treated microglia 

modulate the T cell response by causing upregulation of Th2 cytokines. PCR arrays on T cell 

RNA extracts reveal that several potential genes may be involved. Western blot and 

immunostaining on EAE mice spinal cord extracts are used to confirm the results. Our findings 

suggest that early activation of microglia by tuftsin may modulate T cell phenotypes to favor 

upregulation of Th2 genes and promote anti-inflammatory response. Early sensitization of 

microglia could modify the immune response and potentially induce protective autoimmunity. 

Disclosures: M. Wu, None; S. Tsirka, None. 

Poster 

253. Parkinson's Disease Models: Neuroprotective Mechanisms I




Poster 

253. Parkinson's Disease Models: Neuroprotective Mechanisms I 



Topic: C.11.j. Neuroprotective mechanisms: Parkinson‟s disease models 

Support: F. M. Kirby Foundation 

Title: The neuroprotective potential of taurine and dextromethorphan in acute and subacute 

MPTP-treated mice 

Authors: *D. W. ANDERSON 1 , J. S. SCHNEIDER 2 ; 

1 Dept Pathol, Anat & Cell Biol, 2 Pathology, Anat. and Cell Biol., Thomas Jefferson Univ., 

Philadelphia, PA 

Abstract: No single factor has been identified as the primary cause of dopamine (DA) neuron 

death in the substantia nigra pars compacta (SNc) in idiopathic Parkinson‟s disease (PD). 

However, both mitochondrial dysfunction and oxidative stress may play important roles in the 

pathogenesis of PD, with postmortem studies suggesting complex 1 defects in PD patients and 

animal studies showing DA neuron degeneration from oxidative stress and oxidizing toxins. 

There is also no consensus as to the mode of DA neuron death in PD, with some postmortem 

studies of PD brains detecting signs of apoptosis in the SNc while others have found no evidence 

of apoptosis. Additionally, there are some shortcomings in the way animal models of PD have 

been utilized in the search for neuroprotective therapies. We have previously shown that the 

efficacy of putative neuroprotective agents can vary greatly depending upon the way in which 

the toxin MPTP is administered to mice (i.e., acute vs. sub-acute administration) and the type of 

cell death that is produced (necrotic vs. apoptotic). The complexity of cell death in PD and the 

way in which animal models have been utilized to date (mostly relying on acute MPTP 

administration) likely underlie the disappointing results in the clinic in the search for an effective 

neuroprotective agent. The current study assessed the efficacy of two putative neuroprotective 

agents with glutamatergic actions, taurine and dextromethorphan (DM), on DA cell survival in 

two MPTP models. Taurine is a sulfonated amino acid found in high concentration in many brain 

regions that, among a variety of other physiological functions, can presynaptically attenuate

glutamate release and postsynaptically attenuate the effects of high glutamate concentrations by 

maintaining calcium homeostasis. Dextromethorphan is a codeine analog whose primary 

metabolite acts as a non-competitive antagonist at the NMDA receptor that has previously been 

shown to attenuate glutamate-induced damage to DA cells. Adult male C57Bl/6 mice received 

either taurine (43 mg/kg s.c.) or DM (10 mg/kg i.p.) prior to either acute (4 injections in 1 day at 

2 hr intervals) or sub-acute (2 injections per day at 4 hr intervals for 5 days) MPTP 

administration and for 14 days after the last MPTP admosistration. In the acute MPTP model, 

only taurine had a significant neuroprotective effect whereas in the subacute model, only DM 

had a significant protective effect. These data underscore the need to assess putative 

neuroprotective therapies in more than one PD model and suggest the possible usefulness of 

glutamate modulatory therapies in PD. 

Disclosures: D.W. Anderson , None; J.S. Schneider, None. 

Poster 





Support: UAB Neuroscience Core Facilities (NS47466 and NS57098) 

American Parkinson's Disease Association 

UAB Faculty Development Fund 

Alzheimer Disease Research Center 

Michael J. Fox Foundation 

Title: Cathepsin D haploinsufficiency exacerbates the phenotype of mice exposed to 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine 

Authors: *D. M. CRABTREE, Z.-L. XIE, J. ZHANG; 

UAB, Birmingham, AL 

Abstract: Cathepsin D (CD) is a lysosomal aspartic protease vital to protein turnover and overall 

cellular homeostasis. The importance of CD is evidenced by the homozygous knockout (KO) 

mice, which show tremor, muscle wasting, neurodegeneration and early lethality by postnatal

day 26. Alpha-synuclein (α-syn), the primary protein implicated in the pathogenesis of 

Parkinson‟s Disease (PD), is cleaved in vitro by CD at Tyr125, lending further evidence to the 

observation that decreased protein metabolism is one possible mechanism that results in the 

formation of Lewy Bodies, proteinaceous inclusions composed of α-syn and other proteins that 

serve as histological hallmarks of PD. Using mice that were either wild type (WT) or 

heterozygous (HET) for cd, we tested the hypothesis that cd haploinsufficiency results in greater 

sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropryridine (MPTP). Previous 

studies have demonstrated that α-syn null mice are MPTP resistant, therefore, a protease such as 

CD, which is capable of metabolizing α-syn may be neuroprotective and thus a potential drug 

target for PD therapy. Acute (20mg/kg X 4 injections/day X 1 day) (n=3 WT, n=3 HET) and 

chronic (30mg/kg X 1 injection/day X 10 days) (n=4 WT, n=4 HET) intraperitoneal MPTP 

injection protocols were tested. Fine, ambulatory and total movement was measured by photocell 

beam breaks both pre- and post-MPTP injection. Total ambulatory hypolocomotor activity 

showed the greatest significance at day 3 post-MPTP injection with the WT being significantly 

higher than the HET after acute injection (p

Aging, LKS Fac. of Med., 4 State Key Lab. of Brain and Cognitive Sci., The Univ. of Hong 

Kong, Hong Kong, China 

Abstract: Oxyresveratrol (OXY) is a natural hydroxylated stilbene existing in mulberry. OXY is 

antioxidant, blood-brain-barrier permeable and readily water-soluble. Numerous reports have 

shown its neuroprotective effects against Alzheimer‟s disease (AD) and stroke. As little is known 

about its neuroprotective effects on Parkinson‟s disease (PD), we investigate whether OXY can 

prevent parkinsonian mimetic 6-hydroxydopamine (6-OHDA) neurotoxicity. In SH-SY5Y cells, 

both pretreatment and post-treatment with OXY significantly reduced lactate dehydrogenase 

release and caspase-3 activity. Moreover, OXY exhibited a wide effective window compared to 

resveratrol. 

Our experimental results have demonstrated that OXY could penetrate cell membrane by HPLC 

analysis of cell extract. In this notion, OXY may act as intracellular antioxidants to significantly 

repress the generation of reactive oxygen species (ROS) triggered by 6-OHDA. Therefore, OXY 

markedly attenuated 6-OHDA-induced phosphorylation of JNK and c-Jun. Apart from the 

conventional antioxidant activity, we proved that OXY up-regulated the expression of SirT1 

repressed by 6-OHDA, and prevented the acetylation of downstream p53. 

Taken together, both antioxidant activity and up-regulation of SirT1 by OXY may be responsible 

for preventing 6-OHDA-induced neurotoxicity, and dietary OXY can be a potential candidate for 

nutritional supplement preventing neurodegeneration from PD. 

Disclosures: J.F. Chao, None; M.S. Yu, None; Y.S. Ho, None; M. Wang, None; R.C.C. 

Chang, None. 

Poster 





Support: NIH 1R01NS049433-02 

Title: Tumor necrosis factor-induced p38 MAP kinase activation in dopaminergic 

neurodegeneration 

Authors: *M. G. TANSEY, L. T. ALTO, I. TREVINO; 

Dept Physiol, UT Southwestern Med. Sch. Dallas, Dallas, TX

Abstract: Tumor necrosis factor alpha (TNF) has been implicated in degeneration of nigral 

dopaminergic (DA) neurons in Parkinson‟s disease (PD). Previous results from our lab show that 

blocking TNF signaling can inhibit or delay progressive degeneration of DA neurons in the 

substantia nigra in 6 hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) models of PD, 

but mechanisms by which TNF might contribute to DA neuron death remain unclear. TNF can 

signal through three MAPK pathways, p38 MAPK, ERK and JNK. The p38 MAPK pathway has 

been implicated in pro-apoptotic signaling during oxidative stress-induced injury and systemic 

administration of p38 inhibitors blocks TNF-mediated inflammatory bone destruction in a rat 

arthritis model. Based on these findings, we hypothesize that activation of the p38 MAPK 

pathway is critical for TNF-mediated degeneration of DA neurons. Specifically, we hypothesize 

that p38 signaling downstream of TNF contributes to death of DA neurons in 2 ways: 1) TNF 

receptors expressed by DA neurons activate the p38 MAPK pathway resulting directly in 

apoptosis of DA neurons and 2) TNF signaling in microglia activate the p38 MAPK pathway 

causing increased production of TNF and generation of reactive oxygen species that kill DA 

neurons. To test this hypothesis we are applying selective p38 MAPK inhibitors to mixed and 

neuron-enriched postnatal mouse mesencephalon cultures treated with TNF to determine whether 

p38 MAPK is a potential therapeutic target that could block or delay TNF-mediated degeneration 

of DA neurons. If in vitro experiments are successful, we will use proprietary p38 MAPK 

inhibitor compounds, administered systemically in a mouse model of PD, to test whether p38 

MAPK inhibition could be useful as a therapeutic for delaying or blocking the progressive 

degeneration of DA neurons in PD. 

Disclosures: M.G. Tansey , None; L.T. Alto, None; I. Trevino, None. 

Poster 





Support: NIH Grant K08-NS059576-01 

APDA Research Grant 

Title: Mitochondrial dynamics: influence in a chronic model of Parkinson‟s disease and agerelated 

neuronal changes 

Authors: *S. B. BERMAN, B. A. ARNOLD; 

Neurol., Univ. of Pittsburgh PIND, Pittsburgh, PA

Abstract: Mitochondrial dynamics (fission/fusion and transport) are important in synapse 

formation, mitochondrial DNA maintenance, and cell death mechanisms, and are increasingly 

linked to neurodegenerative diseases. In Parkinson‟s disease (PD), where mitochondrial 

dysfunction is implicated, the dynamic properties of mitochondria may be important in the 

degeneration of susceptible axons. In fact, genetic studies have suggested a role for PD-related 

genes in mitochondrial dynamics (Poole et al and Yang et al, PNAS, 2008). We have previously 

developed methodology for directly quantifying mitochondrial dynamics in living neurons, and 

in order to examine mitochondrial dynamics in models of PD, we first developed a chronic 

rotenone model of PD in differentiated dopaminergic cells. NGF-differentiated PC6-3 cells were 

treated with vehicle or low concentrations of rotenone. Chronic treatment with up to 10 nM 

rotenone for 3 weeks did not result in an increase in cell death over control conditions, and 3 

weeks of 25 nM rotenone treatment resulted in minimal cell death. However, similar to patients 

with PD and animal models, our chronic low-dose rotenone treatment resulted in significant loss 

of neuronal processes, which may represent early toxicity. We then asked whether altering 

mitochondrial dynamics could influence these pathologic changes by altering the mitochondrial 

fission mediator Drp1. One could hypothesize either that inhibition or promotion of fission could 

be protective in PD. We found that overexpression of the dominant-negative form of Drp1, 

dnDrp1 K38A , which inhibits mitochondrial fission in neurons, protected against the loss of 

neuronal processes caused by 2 and 3 week treatment with 5 and 25 nM rotenone. This supports 

a role for mitochondrial dynamics in the early pathologic changes in PD. 

Because PD is a disease of aging neurons, yet little is known about age-related changes in 

mitochondrial dynamics, we also directly characterized mitochondrial dynamics as neurons age. 

Using methodology we previously developed (utilizing mitochondrially targeted DsRed2 and 

photoactivatable GFP and direct live imaging of fusion, fission, and transport), we quantified 

mitochondrial dynamics in rat cortical neurons as they aged in culture. The rate of mitochondrial 

fission in neuronal processes was higher at 7 days (young neurons) than at 14 or 28 days, with 

the lowest ratio of fission to fusion at 14 days (mature neurons), which may reflect the role of 

fission in mitochondrial distribution and synapse formation. Similar dynamic processes can be 

observed in a chronic PD model, and their characterization may help in elucidating the role of 

mitochondrial dynamics in PD. 

Disclosures: S.B. Berman, None; B.A. Arnold, None. 

Poster 




Topic: C.11.j. Neuroprotective mechanisms: Parkinson‟s disease models

Support: The National Research Council of Thailand 

Title: Curcumin reduced loss of dopaminergic neurons and glial reaction in the substantia nigra 

pars compacta of 6-OHDA lesioned mice 

Authors: *W. TRIPANICHKUL; 

Anat., Srinakharinwirot Univ., Bangkok, Thailand 

Abstract: Parkinson's disease (PD) is a neurodegenerative disease commonly characterized by 

the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Until 

recently, it is not exactly known what underlies the degeneration of dopaminergic nigral neurons, 

yet inflammatory reaction has been shown contributing to the demise of these neurons. Curcumin 

is one of the main curcuminoids isolated from Curcuma longa Linn (Turmeric) and posseses 

anti-inflammatory properties. Accumulated evidence indicate neuroprotective effects of 

curcumin in brain ischemia, kainic acid neurotoxicity and Alzheimer‟s disease. To determine 

whether curcumin-elicited neuroprotection in PD animal model, adult male ICR mice were 

treated with curcumin for a total of 10 days. Following 2 days of pretreatment with curcumin, the 

animals were injected intrastriatally with 6-hydroxydopamine (6-OHDA). The brains were 

collected on day 7 after 6-OHDA injection. Immunohistochemistry and quantitative study were 

used to assess the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the SNpc 

and that of activated astrocytes and activated microglia in the SNpc and the striatum. Curcumin 

decreased the loss of TH-IR nigral neurons and diminished the deficit of TH-IR striatal fibers 

triggered by 6-OHDA. The neuroprotective effects of curcumin were coincident with a downregulation 

of glial response within the nigra and the striatum. These findings suggest that the 

neuroprotective effects of curcumin in 6-OHDA-lesioned mice might mediate through an 

inhibition of reactive glia. 

Disclosures: W. Tripanichkul , None. 

Poster 





Support: VA Merit grant 

American Parkinson Disease Association

Title: Rotenone reduces Mg2+-dependent block of NMDA currents in substantia nigra dopamine 

neurons 

Authors: Y.-N. WU 1 , *S. W. JOHNSON 1,2 ; 

1 Dept Neurol, Oregon Hlth. & Sci. Univ., Portland, OR; 2 Portland Veterans Affairs Med. Ctr., 

Portland, OR 

Abstract: Rotenone is a pesticide that has been successfully used to produce a rodent model of 

Parkinson‟s disease. We reported previously that rotenone potently augmented NMDA-evoked 

currents in rat dopamine neurons via a tyrosine kinase-dependent mechanism. In this study, we 

investigated the effect of rotenone on the current-voltage relationship of NMDA-induced 

currents in substantia nigra zona compacta neurons recorded with whole-cell patch pipettes in 

slices of rat brain. Currents were measured during a continuous voltage ramp from -120 to - 

30mV (4s duration). In a physiologic concentration of Mg2+ (1.2mM), a 30min perfusion with 

rotenone (100nM) produced marked increases in NMDA currents especially when measured at 

relatively hyperpolarized currents. At -100mV, for example, NMDA (20µM) evoked 819±50pA 

of inward current in the presence of rotenone compared to 288±39pA in control conditions. In 

the presence of rotenone, NMDA currents lost the characteristic region of negative slope 

conductance that is normally produced by voltage-dependent block by Mg2+. In the presence of 

0.2mM Mg2+, NMDA produced 1338±125pA of inward current at -110mV, and this current was 

not significantly increased after 30min perfusion with rotenone (1638±134pA). Furthermore, an 

elevated extracellular concentration of Mg2+ (6mM) nearly completely blocked the ability of 

rotenone to potentiate NMDA-induced inward currents. Voltage-dependent augmentation of 

NMDA currents by rotenone was blocked by the tyrosine kinase inhibitor genistein (100µM). 

These results suggest that rotenone potentiates NMDA currents by a tyrosine kinase-dependent 

process that attenuates voltage-dependent Mg2+ block of NMDA-gated channels. In that 

augmentation of NMDA-gated channels may mediated some aspects of neurotoxicity, our data 

suggests that elevated levels of Mg2+ might be neuroprotective in animal models of Parkinson‟s 

disease. 

Disclosures: Y. Wu, None; S.W. Johnson , None. 

Poster 





Support: Davis Phinney Foundation

University of Cincinnati 

Title: Development of a clinically relevant platform for the evaluation of the neuroprotective 

effects of HF STN-DBS in parkinsonian rats 

Authors: *A. SPIELES-ENGEMANN, M. M. BEHBEHANI, B. T. TERPSTRA, T. J. 

COLLIER, K. STEECE-COLLIER, L. MADHAVAN, K. PAUMIER, S. WOHLGENANT, S. 

GOMBASH, C. E. SORTWELL; 

Univ. Cincinnati, Cincinnati, OH 

Abstract: High-frequency deep brain stimulation of the subthalamic nucleus (HF STN-DBS) is 

the most frequently practiced surgical therapy for treatment of Parkinson‟s disease (PD). 

However, its clinical use has proceeded without a complete understanding of its mechanism of 

action. Therefore, we set out to develop a rodent model that closely approximates its clinical 

application in PD. To target the STN accurately and consistently, we utilized extracellular 

microelectrode recordings to guide placement of the stimulator. Targeting was confirmed via dye 

injections in the electrode trajectory and with Kluver-Barrera staining. To determine the volume 

of tissue activated, we conducted dual stimulation-recording experiments with a stimulating 

electrode placed in the STN and a recording electrode placed at varying distances away. 

Recordings were made at each site before and after STN stimulation (130 Hz, 60 κs pulse width, 

80-100 κA). Our studies indicate that STN-DBS at routine clinical parameters causes current 

spread approximately 250 κm from the stimulator tip. Subsequently, stimulators were placed in 

the anterior portion of the STN to minimize current spread to the SN. To establish a clinicallyrelevant 

model of nigrostriatal dopamine denervation, a number of different lesion protocols 

were tested using differing volumes of 6-hydroxydopamine (1-3 κl) injected into varying 

locations in the striatum. Lesions were assessed via stereological counts of tyrosine hydroxylase 

immunoreactive neurons in the substantia nigra (SN). The functional effects of lesioning were 

assessed via the cylinder task. Our studies have identified a protocol that is similar to the clinical 

presentation of Parkinson‟s disease in that it causes a progressive lesion (2 wk = 50% SN lesion, 

4 wk = 70%, 6 wk = 75%) that generated significant deficits in forelimb akinesia and rotational 

asymmetry. Finally, we lesioned animals with a progressive lesion protocol, implanted STN 

stimulators utilizing extracellular microelectrode recordings, and stimulated animals for 2 weeks. 

Placement of stimulators was confirmed histologically and functional effects of stimulation were 

assessed via the cylinder task. Results of the long-term stimulation experiments revealed 

functional improvement in forelimb akinesia and accurate long-term targeting of the STN. In 

conclusion, these studies show that long-term stimulation of STN without substantial current 

spread into the SN improves akinesia and indicates that the procedures we have developed can 

be used for a reproducible, clinically-relevant long-term model of HF STN-DBS that has a wide 

variety of applications. 

Disclosures: A. Spieles-Engemann, None; M.M. Behbehani, None; B.T. Terpstra, None; T.J. 

Collier, None; K. Steece-Collier, None; L. Madhavan, None; K. Paumier, None; S. 

Wohlgenant, None; S. Gombash, None; C.E. Sortwell, None.

Poster 





Support: NRSA F32 DK075253 

NPF mega projects in Parkinson Research 

Title: PGC-1 alpha and Parkinson‟s disease: a putative transgenic model of neuroprotection 

during oxidative stress 

Authors: *J. M. SILVAGGI 1 , C. CHUNG 2 , A. SHEETS 1 , J. CLARK 3 , D. K. SIMON 3 , O. 

ISACSON 2 , B. M. SPIEGELMAN 1 ; 

1 Dana-Farber Cancer Inst., Boston, MA; 2 Harvard Med. Sch., Boston, MA; 3 Beth Israel 

Deaconess Med. Ctr., Boston, MA 

Abstract: Many studies suggest that the neurodegeneration observed in Parkinson‟s disease is 

associated with mitochondrial dysfunction and damage through excess oxidative stress. 

Unfortunately, current pharmacological approaches for the treatment of Parkinson‟s disease 

patients do not alter the progression of the disease, but only aid in symptoms of the 

neurodegeneration. New therapeutic approaches using gene therapy and delivery methods are 

currently being investigated in pre-clinical trials, providing future avenues for the delay of 

progression of the disease. An intriguing target of interest is the transcriptional coactivator PGC- 

1 alpha, which is a master regulator of several pathways related to mitochondria and energy 

homeostasis in a wide variety of tissues. PGC-1 alpha has also been shown to induce several 

genes involved in the detoxification response for reactive oxygen species (ROS), and is itself 

induced by increases in ROS. PGC-1 alpha null mice display defects in several regions of the 

brain including severe lesions in the striatum and increased sensitivity to chemicals which model 

the effects of Parkinson‟s disease and seizure. We are investigating the neuroprotective effects of 

PGC-1 alpha in the substantia nigra (SN), the neuronal region affected in Parkinson‟s disease. 

Neuroprotection will be assayed using oxidative stressors (MPTP) in a transgenic mouse model 

in which PGC-1 alpha is overexpressed in the SN. Ultimately, microarray analysis will be 

performed on laser captured SN cells to compare transgenic lines versus wild type mice, to 

determine the global effect of PGC-1 alpha expression in dopaminergic neurons in vivo. In 

parallel to in vivo studies, we are also investigating the transcription factor through which PGC-1 

alpha coactivates the ROS detoxifying enzyme manganese superoxide dismutase (SOD2). Using 

luciferase fusion constructs, we have found that PGC-1 alpha significantly coactivates the PPAR 

(peroxisome proliferator activated receptor) transcription factors on the mouse SOD2 promoter. 

Promoter analysis revealed putative peroxisome proliferator response elements (PPREs) in the 

SOD2 promoter which when mutated blunt this coactivation effect. Electrophoretic mobility shift

assays further confirm the ability of PPAR gamma to bind to predicted PPREs. These results 

implicate PGC-1 alpha and PPAR gamma in a coordinated response during cellular oxidative 

stress. Altogether this project will determine the feasibility of PGC-1 alpha overexpression as a 

protective strategy against ROS-induced neuronal cell death, and provide a better understanding 

of the mechanisms by which PGC-1 alpha acts in neuronal cells. 

Disclosures: J.M. Silvaggi, None; C. Chung, None; A. Sheets, None; J. Clark, None; D.K. 

Simon, None; O. Isacson, None; B.M. Spiegelman, None. 

Poster 





Title: Effect of the C-terminal chain of tetanus toxin on the motor behaviors in hemiparkinonian 

rats 

Authors: *L. M. MARTINEZ MENDIETA 1 , B. VENEGAS 2 , A. PATRICIO 2 , I. 

MARTÍNEZ 2 , J. AGUILERA 3 , I. LIMÓN 2 ; 

1 Dept Pharmacy, Neuropharmacol, Benemerita Univ. Autonoma de Puebla, Puebla, Mexico; 

2 Dept Pharmacy, Neuropharm., Benemérita Univ. Autónoma de Puebla, Puebla, Mexico; 

3 Bioquímica i de Biologia Mol., Univ. Autónoma de Barcelona, Barcelona, Spain 

Abstract: Currently treatments for PD alleviating symptoms of the disease so only restore the 

motor function temporally but not stop the dopaminergic neurodegeration. Thus, it is essential to 

develop new alternatives preventing ongoing cell death in this disorder. A successful therapeutic 

use the glial derived neurotrophic factor (GDNF), a potent trophic factor that improves motor 

behaviors, and protects tyrosine hydroxylase (TH) nigral neurons and enhance dopamine levels. 

Unfortunately, all the neurotrophins doesn´t pass the blood-brain barriers and need repeated 

administration for this reason is more difficult their use for PD. Recently, has been shown that 

the C-terminus fragment of the tetanus toxin (Hc-TeTx) is transported retrogradly and had shown 

neuroprotective effects, preventing neuronal death by apoptosis in cerebellar granule neurons in 

cultures. Moreover the first results have shown the efficacy of Hc-TeTx on ameliorating 

behavior deficits and improvement of dopaminergic system, were evaluated in turn behavior, 

fine motor skills, asymmetry of both forelimbs in MPP + -lesioned rats. The aim of this study was 

to evaluate the effects of Hc-TeTx in 6-OHDA or MPP + treated rats using motor behavioral tests 

and striatal survival. Male Wistar rats (250-300g) were unilateral injected with 2 µM of Hc-TeTx 

previous to 6-OHDA 2 µl (8 µg/µl) or MPP + 1µL (10µg/µL) in the nigrostriatal pathways or left

striatum respectively, and another group were injected only with 6-OHDA or MPP + in the same 

brain area. All animals were assessed by turn behaviour and after were evaluated the asymmetry 

with the cylinder test and the fine motor movements respectively. The viability cell into left and 

right striatum of rat was examined by MTT assay. In rotational behavior task Hc-TeTx/6-OHDA 

group displayed a decreased number of contralateral turns respect to control group. In the case of 

cylinder task the group Hc-TeTx/6-OHDA enhances the percentage of contralateral forelimb use 

with respect to 6-OHDA group. For the group Hc-TeTx/MPP + had been shown that decrease the 

ipsilateral turns respect to control group, whereas the fine motor movements: pronation, grasp 

and supination with the contralateral forelimb, increase when injected the Hc-TeTx/MPP + with 

respect to MPP + group. Finally was found that Hc-mediated survival cell in left striatum. The 

present study clearly shows that Hc-TeTx improves different motor behaviours probably by their 

enhance DA system so strongly favor the hypothesis of neuroprotective properties of Hc-TeTx 

fragment even in animals with dopaminergic denervation by 6-OHDA or MPP + 

Disclosures: L.M. Martinez Mendieta , None; B. Venegas, None; A. Patricio, None; I. 

Martínez, None; J. Aguilera, None; I. Limón, None. 

Poster 





Support: Parkinson Society Canada 

Title: Chronic treatment with omega-3 polyunsaturated fatty acids and underlying mechanisms 

of neuroprotection in an animal model of Parkinson‟s disease 

Authors: *M. BOUSQUET 1,3 , M. SAINT-PIERRE 2 , C. JULIEN 1,3 , N. SALEM 5 , F. 

CICCHETTI 2,4 , F. CALON 1,3 ; 

1 Endocrinologie moleculaire, 2 Ctr. de recherche Neurosciences, CHUL, Québec, QC, Canada; 

3 Faculté de pharmacie, 4 Faculté de médecine, Univ. Laval, Québec, QC, Canada; 5 NIH, 

Rockville, MD 

Abstract: Parkinson‟s disease (PD) is a neurodegenerative disorder characterized a massive and 

largely preferential targeting of the dopaminergic nigrostriatal system. Despite the fact that the 

etiology of PD is still unknown for most cases, environmental aspects, such as low omega-3 

polyunsaturated fatty acids (n-3 PUFAs) intake, have recently been suspected as risk factors, as 

also suggested in Alzheimer‟s disease. We have recently reported the beneficial effects of n-3

PUFA in PD mice although the underlying mechanisms of action remain to be elucidated. Here, 

we exposed mice to either a control or a high n-3 PUFA diet from 2 to 12 months of age and then 

treated them with MPTP (140 mg/kg in 5 days), neurotoxin that targets the dopaminergic 

pathway. The high n-3 PUFA consumption modified the brain fatty acids profile. It also 

completely prevented the MPTP-induced decrease of tyrosine hydroxylase (TH)-labeled nigral 

cells (p < 0.01 versus MPTP mice on control diet), Nurr1 mRNA levels (p < 0.01 versus MPTP 

mice on control diet) and dopamine transporter mRNA levels (p < 0.05 versus MPTP mice on 

control diet) in the substantia nigra. Although n-3 PUFA dietary treatment had no effect on 

striatal dopaminergic terminals, the high n-3 PUFA diet protected against the MPTP-induced 

decrease in dopamine (p < 0.05 versus MPTP mice on control diet) and its metabolite 

dihydroxyphenylacetic acid (DOPAC) (p < 0.05 versus MPTP mice on control diet) in the 

striatum. Thus far, we have investigated three main mechanistic pathways involving: 1) 

inflammation, 2) heat shock proteins and 3) neurotrophic factors. PK11195 binds to peripheral 

benzodiazepine receptors on glial cells, which are upregulated during inflammatory response. As 

assessed with autoradiography, the specific binding of [3H]-PK11195 remained unchanged in 

mice fed the high n-3 PUFA diet. In situ hybridization revealed no differences in heat shock 

protein (hsp70) mRNA levels between animals on either diet. However, we have detected a 

higher expression of brain-derived neurotrophic factor (BDNF) mRNA in the cortex of mice fed 

the high n-3 PUFA diet (p < 0.05 versus mice on control diet). In light of these preliminary 

observations, we are now focusing mainly on mechanisms related to neurotrophic factors and 

expression of their receptors that could underlie the neuroprotective action of n-3 PUFA 

observed here. Regulation of oxidative damage is also being investigated. Since the prevalent 

low consumption of n-3 PUFA might be an important modifiable risk factor of PD, our data call 

for further investigation on the neuroprotective effect of n-3 PUFA in neurodegenerative 

diseases. 

Disclosures: M. Bousquet, None; M. Saint-Pierre, None; C. Julien, None; N. Salem, None; F. 

Cicchetti, None; F. Calon, None. 

Poster 





Support: SEOULRNBD program(10524) 

Title: Polygala radix protects dopaminergic cell from toxicity induced by MPTP in mice model 

of Parkinson's disease

Authors: J. SHIM 1 , H. KIM 1 , M. JU 1 , *Y. KIM 2 , J. LEE 3 , M. OH 1 ; 

1 Col. of Pharmacy, Kyung Hee Univ., Seoul, Republic of Korea; 2 Neurosci., Kyunghee Univ., 

Seoul, Republic of Korea; 3 Col. of Medicine, Konkuk Univ., Seoul, Republic of Korea 

Abstract: Parkinson‟s disease (PD) is a chronic progressive neurodegenerative disorder 

characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), 

which causes a consequent reduction of dopamine levels in the striatum. Polygala radix, the root 

of Polygala tenuifolia Willd., has been used to treat amnesia, neurasthenia, palpitation, and 

insomnia in Oriental traditional medicine. In this study, we assessed the neuroprotective effects 

of water extracts of polygala radix (PRW) on 6-hydroxydopamine-induced apoptosis in PC12 

cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of PD. In 

3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, co-treatment with PRW (1- 

100 κg/ml) significantly attenuated 6-hydroxydopamine (300 κM)-induced cell death in a dose 

dependent manner. In subacute MPTP mice model (30 mg/kg/day, 5 days, i.p.), PRW (500 

mg/kg/day, 3 days, p.o.) showed improvement of PD-like symptoms (bradykinesia) in a 

behavioral test. Histological assays showed that PRW had protective effects on dopaminergic 

neurons against MPTP toxicity in SNc and striatum. These results demonstrate that PRW has 

protective effect against neurotoxicity in PD models. 

Disclosures: J. Shim, None; H. Kim, None; M. Ju, None; Y. Kim , None; J. Lee, None; M. 

Oh, None. 

Poster 





Support: Canadian Institute of Health Research 

Parkinson's Disease Society 

Natural Sciences and Engineering Research Council of Canada 

Title: Interferon-gamma deficiency influences the behavioural and neurochemical alterations 

provoked by paraquat in a murine model of Parkinson's disease 

Authors: *E. N. MANGANO 1 , D. LITTELJOHN* 2 , S. HAYLEY 2 ; 

1 Psychology, 2 Carleton Univ., Ottawa, ON, Canada

Abstract: While the profound motor disturbances engendered by degeneration of the 

nigrostriatal pathway remain the hallmark clinical features of Parkinson‟s disease (PD), 

accumulating evidence suggests that PD is often characterized, and even preceded, by co-morbid 

neuropsychiatric symptoms, most notably anxiety and depression. Separate lines of evidence 

have suggested that inflammatory processes associated with the actions of several cytokine 

immunotransmitters, including interferon-gamma (IFN-g), may be fundamental to the 

development and progression of both PD and major depression. Accordingly, we assessed the 

impact of the ecologically relevant dopaminergic toxin, paraquat, upon behavioural indices of 

motor functioning and anxiety-like states, in addition to central monoamine activity, 

dopaminergic neuron survival, and brain phosphoprotein levels, among wild-type and IFN-gdeficient 

mice. To this end, paraquat induced profound motor impairment and anxiety-like 

behaviour among wild-type mice and loss of IFN-g abrogated these effects. Additionally, IFN-g 

deficiency in mice attenuated the paraquat-induced loss of nigral dopaminergic cells and changes 

in dopaminergic and noradrenergic activity within the striatum, dorsal hippocampus and/or 

medial prefrontal cortex. Moreover, paraquat administration in mice provoked alterations in the 

level of several phosphoproteins that have important signaling functions linked to neuronal 

survival and plasticity. The magnitude of these changes varied both across brain regions and, in 

many cases, as a function of IFN-g deficiency. Taken together, these results suggest that IFN-g 

may be critical in mediating both the motor impairment and the putative anxiogenic effects of an 

environmental toxin implicated in PD; and that brain region-specific modulation of several 

intracellular signaling pathways by IFN-g may be a fundamental determinant of such effects. 

Disclosures: E.N. Mangano, None; D. Litteljohn*, None; S. Hayley, None. 

Poster 





Support: The Parkinson Council (National Parkinson's Foundation) 

Title: VTA neurons mediate neuroprotection in dopamine neurons of the substantia nigra 

Authors: *S. PHANI, L. IACOVITTI; 

Farber Inst. Neurosci, Thomas Jefferson Univ., Philadelphia, PA 

Abstract: Parkinson‟s disease and its characteristic symptoms are thought to arise from the 

progressive degeneration of specific midbrain dopamine (DA) neurons. In humans, DA neurons

of the Substantia Nigra (SN) and their projections to the striatum show selective vulnerability, 

while neighboring DA neurons of the Ventral Tegmental Area (VTA) are relatively spared from 

degeneration. Notably, this selective neurodegeneration is mimicked in humans, non-human 

primates and C57BL/6J mice by the neurotoxin MPTP. In this study, we aimed to test the 

hypothesis that genes/proteins in the VTA confer neuroprotection against MPP+ mediated 

toxicity. We have developed an in vitro cell culture system in which the SN and VTA are grown 

separately by utilizing embryonic day 13 midbrains from transgenic C57BL/6J mice carrying the 

human TH promoter driving GFP (hTH-GFP) expression. To determine the neuroprotective 

potential of VTA derived factors, SN cells grown in VTA-CM were treated with MPP+. 

Following treatment, cultures were fixed with 4% paraformaldehyde and GFP postive cells were 

counted and quantified. Importantly, we have found that the cells of the SN are susceptible to 

MPP+ in the presence of VTA-CM. Our preliminary results show that a concentration of 7.5µM 

MPP+ caused approximately 50% cell death in SN cultures plated in 50% VTA-CM. Notably, 

there is an inverse relationship between the concentration of VTA-CM and the amount of 

protection against MPP+. We found that MPP+ caused approximately 62%-40% SN cell death in 

the presence of 25%-75% VTA-CM respectively. Immunocytochemical staining of VTA 

cultures at the time conditioned media was drawn (3 days in vitro) demonstrated positive staining 

for the neuronal marker β-III tubulin, and negative staining for the astrocytic markers GFAP and 

s100β. This suggests that the factors conferring protection against MPP+ toxicity have a 

neuronal origin. Importantly, the origin of these factors is specific to the VTA, as media 

conditioned by cortical neurons do not appear to offer protection against MPP+ toxicity. In order 

to isolate specific VTA-derived factors as potential neuroprotectants, we are currently using 

microarray analysis to create gene expression profiles of the SN and VTA during the progression 

of DA neuron death in an MPTP mouse model of PD. 

Disclosures: S. Phani , None; L. Iacovitti, The Parkinson Council, B. Research Grant (principal 


Poster 





Support: NSERC Grant 

OMHF Grant

Title: The effects of environmental enrichment with exercise on the antipsychotic-induced 

reduction of tyrosine hydroxylase-immunopositive nigrostriatal neurons 

Authors: *K. B. REYNOLDS 1 , L. E. S. MACGILLIVRAY 1 , P. I. ROSEBUSH 2 , M. F. 

MAZUREK 2 ; 

1 Hlth. Sci., 2 Psychiatry and Behavioural Neurosciences, McMaster Univ., Hamilton, ON, Canada 

Abstract: Background and Objectives: Patients treated with antipsychotic drugs (APDs) are 

prone to developing parkinsonism. We have previously reported a long-term downregulation of 

nigrostriatal dopaminergic neurons following treatment with APDs similar to the changes 

observed in animal models of idiopathic Parkinson‟s Disease (PD). Environmental enrichment 

(EE) prior to or following toxin exposure, has been shown to attenuate both the behavioural 

consequences and the neurochemical changes seen in animal models of PD. We postulated that 

EE with exercise may attenuate the toxic effects associated with APD treatment. 

Methods: At 3 weeks of age male Sprague-Dawley rats (n=41) were placed into either a 

standard or an enriched environment. In the standard environment, animals were housed 

individually with no enrichment objects. In the enriched environment, animals were housed ten 

animals per cage with two running wheels as well as other enrichment items that were changed 

and rearranged regularly. After approximately 9 weeks, the rats were assigned to one of four 

different treatment groups. One group of rats in enriched housing (n=10) and one group in 

standard housing (n=11) received oral haloperidol treatment (HAL; 2 mg/kg/day) for 7 days 

while the other two groups (n=10; n=10) received saline. Sections of the substantia nigra (SN) 

were processed for tyrosine hydroxylase (TH) immunoreactivity, and cells were counted 

manually. Results were analyzed by pre-planned t-tests. Significance was set at p


Topic: C.15.d. Schizophrenia: Mutant animal models 

Support: NIMH IRP 

Title: NMDA receptor ablation restricted to forebrain GABAergic interneurons results in 

psychiatric-like behavior 

Authors: *J. E. BELFORTE 1 , V. ZSIROS 1 , Y. LI 3 , G. KUNOS 2 , K. NAKAZAWA 1 ; 

1 NIMH, 2 NIAAA, NIH, Bethesda, MD; 3 Dept. of Neurol., Univ. of Alabama at Birmingham, 

Birmingham, AL 

Abstract: Local GABAergic interneurons sculpt activity in cortical circuits to control 

excitability and timing of firing in principal neurons. Recently, converging clinical evidence 

suggests that altered GABAergic function may underlie the pathophysiology of psychiatric 

disorders such as bipolar disorder and schizophrenia. It has been proposed that GABAergic 

dysfunction in these disorders may result from hypofunction of NMDA-type glutamate receptors 

(NRs) in cortical interneurons. To explore this hypothesis we knocked out the NMDA receptor 

subunit 1 (NR1) in forebrain GABAergic cells by crossing floxed-NR1 mice with a Cre 

transgenic line, Cre#4127, in which Cre recombinase is expressed in ~50% of cortical and 

hippocampal GABAergic cells, preferentially parvalbumin positive ones, without affecting 

excitatory cells (SfN abstract # 99.5, 2007). Non-radioactive double in situ hybridization 

immunocytochemistry using mRNA probes against GAD67 and NR1 uncovered an absence of 

NR1 labeling in approximately 50 % of the GAD67 positive cells across cortices in sections 

from 4 weeks-old mutants, suggesting that the knockout occurred before adolescence. In 

addition, whole-cell patch-clamp recordings from labeled neurons in mutant mice crossed with 

the R26R floxed-EYFP reporter line, showed no NMDA-mediated EPSCs in most of the targeted 

cells. Tests for behavioral phenotyping of mutant mice (8-18 weeks old) revealed increased 

levels of anxiety-like behaviors when measured in elevated plus maze and open field tests, 

without displaying any abnormal motor function in these tests or in accelerated rotarod 

performance. Significant hyperlocomotion was observed only during the first several minutes of 

exploration in new open fields, which could reflect psychomotor agitation. Mutants also 

displayed decreased prepulse inhibition with a normal auditory startle response. Moreover, nest 

construction, breeding and mating performances, and social interaction were impaired in 

mutants. Social isolation-induced stress precipitated the mutant phenotypes observed in the 

elevated plus maze and nesting behavior. Assessment of anhedonia by a two-bottle saccharine 

preference test resulted in decreased preference for saccharine in 16 week-old mutants but not in 

younger animals. A similar age-dependent enhancement of behavioral phenotypes was observed 

for the elevated plus maze, mating performance and nesting behavior. These results suggest that 

NR ablation in cortical and hippocampal GABAergic cells evokes psychiatric-like phenotypes. 

Disclosures: J.E. Belforte, None; V. Zsiros, None; Y. Li, None; G. Kunos, None; K. 

Nakazawa, None.

Poster 

254. Schizophrenia: Mutant Animal Models 




Support: T32 DA016224 (IGS) 

K02 DA000356 (SR) 

P50 MH066171 (SR) 

Title: Differential expression of AMPA and NMDA receptors in the hippocampus of 

Glutaminase-deficient mice 

Authors: *I. GAISLER-SALOMON 1,2 , Y. WANG 1,2 , S. M. MCKINNEY 1,2 , A. J. RAMSEY 3 , 

E. L. SIBILLE 4 , S. RAYPORT 1,2 ; 

1 Columbia Univ., NYC, NY; 2 Mol. Therapeut., New York State Psychiatric Inst., New York 

City, NY; 3 Cell Biol., Duke Univ. Med. Ctr., Durham, NC; 4 Psychiatry, Univ. of Pittsburgh, 

Pittsburgh, PA 

Abstract: Glutamate (GLU) signaling in the hippocampus (HIPP) is fundamental to learning and 

memory; its disruption has been linked to schizophrenia (SZ) and several other disorders. We 

showed previously that glutaminase-deficient mice (GLS1 hets), with a presynaptic defect in 

GLU recycling, have reduced glutamate levels in several brain regions, but a focal reduction in 

HIPP activity, measured using in-vivo imaging and electrophysiological techniques, and a 

selective deficit in HIPP-dependent contextual learning (Gaisler-Salomon et al., SFN 2007). 

Extending this work, we asked: (1) Are other genes involved in glutamine-GLU recycling 

pathway, or related metabolic pathways, affected in the HIPP of GLS1 hets? (2) Is NMDA and 

AMPA receptor expression altered in GLS1 hets? (3) How are learning and memory-related 

genes affected? Using Affymetrix gene chips, we found that (1) other than GLS1, which was 

downregulated by about 50%, enzymes involved in glutamine-GLU recycling or related 

metabolic pathways were unaffected. (2) The GluR2 receptor subunit of the AMPA receptor was 

downregulated by 40%, while NMDA receptor expression was unaffected. RT-PCR performed 

on HIPP and frontal cortex samples confirmed the HIPP-specific reduction in GluR2 expression 

(3) Although no single molecule implicated in learning-related cascades was drastically affected, 

Ingenuity Pathway analysis showed dysregulation of the long-term plasticity pathway. This was 

confirmed using RT-PCR, which also showed that the effect was specific to the HIPP. In order to 

examine the relevance of changes in gene expression observed in GLS1 hets to SZ-related

pathology, we compared the expression levels of GLS1, GluR2, the NMDA receptor subunit 

NR1 and CamKII (a gene involved in long-term plasticity pathways) in the HIPP of GLS1 hets 

and NR1 hypomorphic mice, which have 10% of normal NR1 expression levels and a SZ-like 

behavioral phenotype (Mohn et al., Cell 1999). GLS1 hets displayed a reduction in GLS1, GluR2 

and CamKII, but not NR1. In contrast, NR1 hypomorphs displayed a reduction in NR1 

expression only. These distinctly different gene expression profiles indicate that a reduction in 

GLS1, and in presynaptic GLU, does not mimic the gene expression effects of reduced NMDA 

receptor expression, consistent with our previous findings showing that a reduction in GLS1 

leads to an array of functional, behavioral and neurochemical phenotypes that are the inverse of 

what is typically observed in SZ. In line with current thinking that SZ is related to excessive 

GLU signaling via AMPA receptors, possibly the beneficial effects of reducing GLS1 expression 

are mediated through the associated reduction in GluR2. 

Disclosures: I. Gaisler-Salomon , None; Y. Wang, None; S.M. McKinney, None; A.J. 

Ramsey, None; E.L. Sibille, None; S. Rayport, None. 

Poster 





Support: MH051290 . 

P50 MH060450 

Title: Relationship between NMDA receptor function and GABAergic neuropathology in serine 

racemase and glycine transporter I mutant mice 

Authors: M. A. BENNEYWORTH, K. SUZUKI, *A. C. BASU, J. T. COYLE; 

Psychiatry - McLean Hosp, Harvard Med. Sch., Belmont, MA 

Abstract: Reduction in presynaptic markers for parvalbumin(PV)-positive GABAergic 

interneurons in the cortex is the most highly replicated postmortem neurochemical finding in 

schizophrenia. Meanwhile, mounting evidence from genetic and pharmacologic studies 

implicates hypofunction of NMDA receptor-mediated glutamatergic signaling as a critical 

component of the pathophysiology of schizophrenia. A potential relationship between these 

findings exists in the context of the circuitry of the cortex, where PV-positive GABAergic 

interneurons provide feedback inhibition to the cortical pyramidal cells. We asked whether a

constitutive genetic perturbation of NMDA receptor function through the glycine site modulators 

D-serine and glycine can result in changes in the GABA system. We and colleagues have 

established two mouse genetic models of altered NMDAR modulation: Serine racemase exon 1 

knockout (SR-/-) mice have a > 80% reduction in cortical D-serine, resulting in decreased 

occupancy of the glycine modulatory site (GMS) and decreased NMDAR-mediated 

glutamatergic activity. Mice heterozygous for a null mutation of glycine transporter 1 (GlyT1+/- 

mice) exhibit increased occupancy of the NMDA receptor glycine modulatory site. In the present 

study, we have investigated the expression of GABAergic markers in SR-/- and Glyt1+/- mice 

using a combination of quantitative PCR, western blotting, and immunohistochemical 

techniques. Here, we report significant reduction in the expression of PSD-95, PV and glutamate 

decarboxylase (GAD67) in the hippocampus of SR-/- mice. In contrast, GlyT1+/- mice exhibit 

an increased expression of PSD-95 and PV in the hippocampus. Preliminary 

immunohistochemical investigations demonstrate a reduction in the number of GAD67-postive 

neurons the prefrontal cortex (pre- and infralimbic cortices) and all areas of the hippocampus in 

SR -/- mice. Thus, we demonstrate that genetic manipulation of the availability of GMS ligands 

can influence the expression of GABAergic markers in either direction. These results are 

consistent with the hypothesis that the NMDARs on the PV-positive GABAergic interneurons 

are the subset of NMDA receptors critical to the pathophysiology of schizophrenia. 

Disclosures: M.A. Benneyworth, None; A.C. Basu , None; J.T. Coyle, Patent relating to the 

use of d-serine in the treatment of neuropsychiatric disorders. Patent is held by McLean Hospital 

and JTC., E. Ownership Interest (stock, stock options, patent or other intellectual property); K. 

Suzuki, None. 

Poster 





Title: Loss of glutamate transporter GLAST (EAAT1) causes 'schizophrenia-like' phenotype in 

mice 

Authors: *R.-M. KARLSSON 1 , M. HEILIG 1 , A. HOLMES 2 ; 

1 NIH, Bethesda, MD; 2 NIH, Rockville, MD 

Abstract: Glutamatergic dysfunction is increasingly implicated in the pathophysiology of 

schizophrenia. Current models postulate that dysfunction of glutamate and its receptors underlies 

many of the symptoms in schizophrenia. However, the mechanisms involved are not well

understood. Glutamate receptor signaling is tightly regulated by five different glutamate 

transporters which function to clear glutamate from the extracelluar space. A recent human study 

showed mutation to GLAST (SLC1A3) to be linked to schizophrenia. Elucidating the role for 

glutamate transporter GLAST in the disease has been limited by the absence of pharmacological 

tools that selectively target the transporter. We have recently shown that GLAST knockout mice 

display novelty-induced locomotor hyperactivity, which was rescued by treatment with the 

metabotropic glutamate (mGlu2/3) receptor agonist, LY379268. Mutants also showed an 

enhanced locomotor stimulant response to the NMDA receptor antagonist, MK-801 

(dizocilpine); which mimics the hypersensitivity to psychotomimetics in schizophrenia. We have 

expanded our analysis of the GLAST knockout mice for behaviors thought to model other 

symptoms of schizophrenia. GLAST knockouts were compared to wild-type (WT) littermate 

controls for social behaviors, sensorimotor gating, and cognitive functions. GLAST mutants 

displayed abnormal social interaction, modest sensorimotor gating deficits, and impaired visual 

discrimination learning. These data add to profile of „schizophrenia-related‟ abnormalities in 

GLAST knockout mice, and further suggest that GLAST dysfunction may contribute to 

glutamatergic abnormalities underlying schizophrenia. 

Disclosures: R. Karlsson , None; M. Heilig, None; A. Holmes, None. 

Poster 





Support: 5T32NS048004-04 

RL1-MH083269 

Title: Assessment of a behavioral and structural pre-frontal phenotype in dysbindin mutant mice 

Authors: *K. H. KARLSGODT 1 , E. S. LUTKENHOFF 2 , T. D. CANNON 3,4 , J. D. 

JENTSCH 3,4 ; 

1 Semel Inst., 2 Interdisciplinary Neurosci. Program, 3 Dept. of Psychology, 4 Dept. of Psychiatry 

and Biobehavioral Sci., UCLA, Los Angeles, CA 

Abstract: Dysbindin (DTNBP1) has been implicated in numerous studies as a candidate gene 

for schizophrenia. This gene is particularly relevant to known schizophrenia phenotypes, as it has 

effects on synaptic signaling, and in particular, on glutamate release. Variations in dysbindin

have been shown to influence cognitive processes, including working memory, which is known 

to rely on a complex system of glutamatergic and dopaminergic interactions. As patients with 

schizophrenia have demonstrated deficits in working memory, as well as decreased thickness of 

the pre-frontal cortex, we are investigating these measures in dysbindin null mutant mice 

(“sandy” mice) as a translational model of schizophrenia. Our laboratory has previously 

investigated working memory function in sandy mice on a DBA background using a spatial 

delayed non-match to position task and found decreased accuracy in both null mutant and 

heterozygous mice compared to wild-type littermate controls. In the current investigation, we 

probed frontal lobe function by assessing performance on the same task on sandy mice 

backcrossed onto a C57Bl/6 background. Preliminary analyses during the training phase indicate 

that at delays up to 3 seconds, wild-types significantly out-perform mutants, with a trend towards 

higher performance than heterozygotes. As the delay period increased up to 9 seconds, the 

performance of wild-types dropped to the level of the mutants and heterozygotes, indicating the 

presence of delay dependent changes in task difficulty. In the second part of the current study, 

frontal lobe structure was assessed using Micro-MRI on a 7T Bruker small-bore scanner at 

UCLA. In-vivo MSME MRI images were obtained in anesthetized animals using i.p. manganese 

(62.5mg/kg) as a contrast agent to aid in differentiation of grey and white matter. Preliminary 

analyses using mutant and wild-type DBA sandy mice indicate a trend (p=.061, 1-tailed) towards 

a decrease in thickness of the medial prefrontal cortex. These analyses will be extended to the 

C57Bl/6 sandy mice, in order to allow the direct comparison of imaging results and behavioral 

performance. 

Disclosures: K.H. Karlsgodt , None; E.S. Lutkenhoff, None; J.D. Jentsch, None; T.D. 

Cannon, None. 

Poster 





Support: NIH Grant RL1-MH083269 (AL, DJJ) 

College of Charleston MAYS Grant (NN) 

Title: Effects of dysbindin-1 null mutation on markers of glutamate signaling in prefrontal 

cortex and hippocampus

Authors: *N. N. NEW 1,2 , J. D. JENTSCH 3 , A. LAVIN 2 , D. B. CARR 2 ; 

1 Col. Charleston, Charleston, SC; 2 Neurosci., Med. Univ. of South Carolina, Charleston, SC; 

3 Psychology, UCLA, Los Angeles, CA 

Abstract: Dtnbp-1 is a schizophrenia susceptibility gene that encodes dystrobrevin-binding 

protein-1 (dysbindin-1). In schizophrenic patients, expression of dysbindin is reduced in the 

prefrontal cortex (PFC) and hippocampus, two areas of particular significance for schizophrenia 

pathology. While the precise cellular functions of this protein in the brain have not been fully 

elucidated, studies by our lab and others have suggested important roles for dysbinindin in 

glutamatergic transmission, both pre- and post-synaptically. These roles of dysbindin are of 

particular significance for schizophrenia research, given the evidence for glutamatergic (and in 

particular NMDA receptor) hypofunction in schizophrenia. In order to better understand the role 

of dysbindin in glutamatergic signaling, we have been studying excitatory synaptic transmission 

in the PFC and hippocampus of dysbindin null mice. To expand upon these findings, we are also 

examining markers of glutamate signaling in these areas using immunohistochemical methods. 

We are utilizing markers of terminal density, synapse density and spine density, as well as 

antisera directed against specific glutamate receptor subtypes to compare levels and laminar 

distribution of these proteins in PFC and hippocampal tissue from dysbindin null mice and their 

wild-type littermate controls. Preliminary pair-wise comparisons of the medial PFC indicates a 

decrease in the expression of NR1, spinophillin, PSD95 and synapsin immunoreactivity in the 

dysbindin mutant animals as compared to their wild-type littermates. Although very preliminary, 

our observations suggest that genetic deletion of dysbindin may impair prefrontal glutamatergic 

transmission via pre- and post-synaptic effects. 

Disclosures: N.N. New , None; J.D. Jentsch, None; A. Lavin, None; D.B. Carr, None. 

Poster 





Poster 

254. Schizophrenia: Mutant Animal Models




Support: NIH Grant RL1-MH083269 

NIH Grant NIGMS K12GM081265 

Title: Effects of null mutation of the gene encoding dysbindin-1 on cortical fast-spiking 

interneurons 

Authors: *H. TRANTHAM-DAVIDSON 1 , J. D. JENTSCH 2 , A. LAVIN 3 ; 

1 Dept Neurosci, Med. Univ. SC, Charleston, SC; 2 PSYCHOLOGY, UNIVERSITY OF 

CALIFORNIA, LOS ANGELES, Los angeles, CA; 3 Neurosciences, MEDICAL UNIVERSITY 

OF SOUTH CAROLINA, CHARLESTON, SC 

Abstract: Dtnbp-1, a schizophrenia susceptibility gene that encodes dystrobrevin-binding 

protein-1 (dysbindin), has been associated with the hallmark negative and cognitive symptoms of 

schizophrenia. Recent studies have indicated that this protein is expressed both presynaptically 

and postsynaptically in various brain regions and is selectively decreased in the prefrontal cortex 

(PFC) of schizophrenic patients. Previously, we examined cortical pyramidal neurons and 

showed that mutation of the dysbindin gene impairs glutamatergic transmission. Given that 

postmortem studies of brain tissue from schizophrenic patients suggest that functions of the fastspiking 

subtype of cortical interneuron may be affected in patients with this disorder, we decided 

to examine whether decreased dysbindin expression impacts inhibitory synaptic transmission in 

the PFC. In order to study the changes produced by the genetic deletion of dysbindin we used 

electrophysiological techniques, namely whole-cell patch clamp recordings made from cortical 

fast-spiking (FS) interneurons of dysbindin null mutant, heterozygous and wild type mice. 

Our initial studies have revealed a decrease in eEPSC amplitude (WT=198.7 +/- 13.2 pA; 

mutants=89.5 +/- 8.4 pA) and an increase in the AMPA:NMDA current ratio in the fast-spiking 

interneurons of mutant mice (2.5 ± 0.3, n=4) vs. WT (1.8 ± 0.3, n=5). Western blots will be 

performed to assess overall cortical levels of glutamate receptors for both genotypes. 

Additionally, sEPSC frequency appears to be reduced in null mutant vs. WT slices, suggesting 

that dysbindin deletion reduces cortical glutamate release. Paired pulse ratio and mEPSCs will 

also be assessed in order to further test this preliminary result. Finally, basic electrophysiological 

properties do not appear to be affected by dysbindin deletion. These data suggest that a reduction 

of dysbindin expression may have both presynaptic and postsynaptic effects on cortical FS 

interneurons. 

Disclosures: H. Trantham-Davidson , None; J.D. Jentsch, None; A. Lavin, None.

Poster 





Support: RL1-MH083269 (AL) 

Title: Genetic dysbindin deletion reduces synaptic plasticity in the CA1 region of the mouse 

hippocampus 

Authors: *W. B. GLEN 1 , D. JENTSCH 2 , A. LAVIN 1 ; 

1 Neurosci., Med. Univ. of SC, Charleston, SC; 2 Psychology, UCLA, Los Angeles, CA 

Abstract: Dtnbp1, which encodes the protein Dysbindin, is one of several recently identified 

susceptibility genes for schizophrenia. Recent studies show behavioral alterations, as well as 

electrophysiological changes, in the prefrontal cortex of Dysbindin null mutant mice, effects that 

may model simple aspects of schizophrenia. Here we assess glutamatergic transmission and 

synaptic plasticity in the hippocampus, a major site for the symptomology in schizophrenia. 

We examined synaptic plasticity in acute 300uM horizontal slices taken from the hippocampus 

of wild type, heterozygous and Dysbindin null mutant mice. Field recordings were taken from 

the CA1 hippocampal region while stimulating the Schaffer Collateral projections arising from 

the CA3. Following a 10 minute baseline, LTP or LTD was induced by either tetanus or low 

frequency stimulation. Measurements were taken from the slope of the field evoked excitatory 

potential. Our preliminary results suggest that mutant mice show a decrease in induced early 

LTP and LTD. In particular, it has proven very difficult to induce potentiation at all in mutant 

mice. LTD has been reliably induced in mutants but appears to be reduced. No difference has 

been observed in baseline response. Paired with behavioral deficits in cognitive tasks such as 

spatial learning and memory, these data suggest that Dysbindin plays a crucial role in 

glutamatergic processes in the hippocampus. 

Disclosures: W.B. Glen , None; D. Jentsch, None; A. Lavin, None. 

Poster 






P50 MH077972 

Title: Impacts of disruption of DISC1 at different post-natal time points on the 

pathophysiology of schizophrenia in inducible transgenic mice 

Authors: *W. LI 1 , T. CANNON 2 , A. SILVA 3 ; 

1 Neurobiology, Psychiatry, 2 Psychology and Psychiatry and Biobehavioral Sci., 3 Neurobiology, 

Psychiatry and Biobehavioral Sciences, Psychology and Brain Res. Inst., UCLA, Los Angeles, 

CA 

Abstract: DISC1 was initially discovered through analysis of a large Scottish kindred multiply 

affected with schizophrenia, recurrent major depression, and other mental disorders. A region of 

chromosome 1 was found to have a balanced translocation with 

chromosome11(1;11)(q42.1;q14.3). Using an inducible and reversible transgenic system, we 

have previously reported that induction of a C-terminal portion of DISC1 at postnatal day 7, but 

not in adult mice, results in a cluster of schizophrenia-related phenotypes, including depressivelike 

traits, abnormal spatial working memory and reduced sociability. Our findings demonstrate 

that alterations in DISC1 function during brain development contribute to schizophrenia 

pathogenesis. Here we utilize the Ligand Binding Domain of the Estrogen Receptor inducible 

system to investigate the impact of disruption of DISC1 at various post-natal time points on the 

pathophysiology of schizophrenia-related phenotypes. This work was supported by a NARSAD 

award to WL and by NIH P50 MH077972 to AJS. 

Disclosures: W. Li, NARSAD, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); T. Cannon, NARSAD, NIH, 

B. Research Grant (principal investigator, collaborator or consultant and pending grants as well 

as grants already received); A. Silva, NIH, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received). 

Poster 



Program#/Poster#: 254.11/X28


Support: the National Basic Research Project (973 program) of the Ministry of Science and 

Technology of China 2006CB500800 

Title: Potential role of Ddysbindin in synaptic transmission and olfactory memory 

Authors: *L. SHAO 1 , Y. SHUAI 1 , Z. LI 1 , Y. ZHAO 1 , C. HUANG 1 , L. WANG 1 , X. XIAO 2 , Y. 

ZHONG 1,3 ; 

1 Biol. Science&Technology, Tsinghua Univ., Beijing, China; 2 Beijing Univ. of Posts and 

Telecommunications, Beijing, China; 3 Cold Spring Harbor Lab., Cold Spring Harbor, NY 

Abstract: Schizophrenia is a debilitating mental disorder characterized by psychotic symptoms 

and cognitive dysfunction. Recent advance in identifying genetic candidate risk factor starts to 

shed light on the pathogenesis of this complex yet highly heritable disease. The DNTBP1 gene, 

encoding dystrobrevin-binding protein-1 (Dysbindin), is among the best-supported susceptible 

genes for schizophrenia. Moreover, this locus has been linked to general cognitive ability 

variation in both schizophrenia patients and normal population. Recent studies have implicated 

Dysbindin in regulation of glutamatergic and dopaminergic transmission. However, the exact 

neurobiological function of dysbindin is still obscure. Here, we intended to use Drosophila as a 

model system to investigate this problem. Drosophila homolog of Dysbindin (Ddysbindin) shares 

~30% homology with the human Dysbindin. A piggybac insertion mutant with the Ddysbindin 

expression level reduced by half in RT-PCR was used throughout this study. Severe defect of the 

excitatory junctional currents in Drosophila larval neuromuscular junction was observed in this 

Ddysbindin mutant, consistent with the study in vertebrate, which validated the model. We then 

tested the same mutant in the pavlovian olfactory avoidance memory and found the performance 

of the mutant was diminished in all memory tasks tested. Taken together, the preliminary data 

suggesting the Ddysbindin is required for optimal performance of the fruit fly nervous system, 

further studies are undertaken to pursue the underlying mechanisms and possible implication for 

human disease. 

Disclosures: L. Shao, None; Y. Shuai, None; Z. Li, None; Y. Zhao, None; C. Huang, None; L. 

Wang, None; X. Xiao, None; Y. Zhong, None. 

Poster 



Program#/Poster#: 254.12/Y1 

Topic: C.15.d. Schizophrenia: Mutant animal models

Support: CIHR Grant MOP-81123 

FRSQ 

Title: Subregional hippocampal decrease in vesicular glutamate transporter 1 (vglut1) expression 

density in mice with natural mutation in dysbindin-1 gene 

Authors: *L. MARSAN 1,3 , M. DANIK 3 , L. K. SRIVASTAVA 2,3 , S. WILLIAMS 2,3 ; 

1 Neurol. and Neurosurg., 2 Psychiatry, McGill Univ., Montreal, QC, Canada; 3 Douglas Mental 

Hlth. Univ. Inst., Montreal, QC, Canada 

Abstract: Genetic studies in a number of populations have revealed polymorphisms in 

dystrobrevin binding protein-1 (DTNBP-1), a gene encoding dysbindin-1, as a potential risk 

factor for schizophrenia. Significant levels of dysbindin-1 expression has been detected in the 

mouse brain and the protein is highly expressed both pre- and post-synaptically. Postmortem 

analysis of brains of patients diagnosed with schizophrenia showed a reduction in dysbindin-1 

protein expression in certain brain structures, notably the hippocampus. Dysbindin-1 has been 

linked to glutamatergic transmission however the mechanisms by which its down-regulation is 

involved in the aberrant cortical/limbic glutamatergic synapses in schizophrenia remain 

undefined. Using a mouse model with a natural deletion in the dysbindin-1 gene, we studied 

hippocampal subregional differences in protein expression of vesicular glutamate transporter 1 

(vGLUT1), a protein involved in packaging glutamate into vesicles, the vesicle marker 

synaptophysin (SYP) and the presynaptic membrane marker syntaxin-1 (SYN). Double-labeling 

fluorescent immunohistochemical analysis for vGLUT1/SYP and single-labeling fluorescent 

immunohistochemical analysis for SYN were performed on 20κm coronal brain slices of P21-25 

wild-type DBA/2J and DBA/2J mice with natural homozygous (SDY/SDY) mutation of 

DTNBP-1 (n=5 and n=2 per genotype, respectively). Subregional intensity line profile analysis 

of CA1, CA3 (str. alveus-oriens, str. pyramidale, str. radiatum and str. lacunosum-moleculare) 

and Dentate Gyrus (DG; granular cell layer, molecular layer, hilus and polymorphic layer) was 

conducted using Image J over a regional representative area of 4500κm 2 . We found a significant 

decrease in SYP and vGLUT1 across subregions of the CA1, CA3 and DG in SDY/SDY mice 

compared to DBA/2J. Interestingly, no significant subregional change was observed in SYN 

expression in SDY/SDY mice compared to DBA/2J. The present study provides further evidence 

supporting the role of dysbindin-1 in glutamatergic synapses and shows the possible involvement 

of dysbindin-1 in the destabilization of glutamate vesicular trafficking. The reductions of 

dysbindin-1 expression reported in schizophrenia perhaps contribute to glutamate hypofunction 

by decreasing the number of glutamatergic synaptic vesicles or vesicular glutamate content while 

likely preserving presynaptic terminal density. In vitro electrophysiological recordings from 

hippocampal slices are in progress to investigate whether the decrease in vGLUT1 expression 

has a significant effect on glutamatergic synaptic transmission in the hippocampal network. 

Disclosures: L. Marsan, None; M. Danik, None; L.K. Srivastava, None; S. Williams, None.

Poster 






DK07328 

Title: The role of intracellular processing of Type III Neuregulin 1 in cortical development 

Authors: *Y. CHEN 1 , M. HANCOCK 1 , D. A. TALMAGE 2 , L. W. ROLE 1 ; 

1 Neurobio. and Behavior, 2 Dept. of Pharmacol., SUNY at Stony Brook, New York, NY 

Abstract: Neuregulin 1 is a schizophrenia susceptibility gene. Among Neuregulin 1 proteins, 

Type III Neuregulin 1 (Nrg1) isoforms differ in their ability to signal in a contact-dependent 

manner. Ligand binding and depolarization have been shown to induce an intramembranous, γsecretase 

dependent cleavage event resulting in release of the intracellular domain of Nrg1 

(Nrg1-ICD) and Nrg1-ICD translocation to the nucleus. Using site-directed mutagenesis, we 

have identified amino acid residues in Nrg1 required for γ-secretase-mediated cleavage and 

nuclear translocation in CHO cells and cortical neurons. Interestingly, the sequences required for 

γ-secretase processing include Valine 321, a residue altered by a polymorphism associated with 

increased risk for developing schizophrenia. Furthermore, using mutant forms of Nrg1 defective 

in proteolytic cleavage and nuclear localization we are studying whether aberrant intracellular 

processing can contribute to cortical dysfunction phenotypes similar to those seen in 

schizophrenia. 

Disclosures: Y. Chen, None; M. Hancock, None; D.A. Talmage, None; L.W. Role, None. 

Poster 




Topic: C.15.d. Schizophrenia: Mutant animal models

Support: Burroughs Wellcome Fund Career Award in the Biomedical Sciences (Brodkin, PI) 

KO8MH068586 (Brodkin, PI) 

P50 MH064045 (Gur, PI) 

R01MH080718 (Brodkin, PI) 

NARSAD Distinguished Investigator Award (Gur, PI) 

Title: Neuregulin-1 (NRG1) mutant mice as a model of reduced sociability, a schizophreniarelated 

phenotype 

Authors: *A. KREIBICH, H. C. DOW, Y. LIANG, R. GUR, S. J. SIEGEL, E. S. BRODKIN; 

Psychiatry, Univ. of Pennsylvania, Philadelphia, PA 

Abstract: Deficits in social interactions are profoundly disabling symptoms of schizophrenia 

(scz); however, the neurobiological basis of these deficits is not well understood. We sought to 

test the overall hypothesis that the neuregulin1 gene (NRG1) may play a role in reduced 

sociability (tendency to seek social interaction) in scz. Several lines of evidence, including 

human genetic and postmortem studies of patients with scz, strongly implicate Neuregulin-1 

(NRG1) as a scz susceptibility gene. In addition, a scz risk allele of NRG1 has been associated 

with poor social functioning in humans. To investigate the possible role of NRG1 in sociability 

deficits in scz, we tested the hypothesis that a novel NRG1 mutation--a heterozygous mutation of 

the EGF-like domain of NRG1 (neo replacement of exons 7, 8, and 9)-- would be associated with 

reduced sociability of mice. Using a social choice assay in a 3-chambered apparatus, we 

measured sociability of adult, male and female heterozygous NRG1 mutant test mice and adult 

male and female wildtype controls toward gonadectomized A/J stimulus mice. Our data 

demonstrate a significant reduction in sociability in adult NRG1 mutant mice, relative to 

wildtype controls, as reflected by reduced social sniffing scores in both male and female mutants 

(main effect of genotype: F1,17 = 22.7, P < 0.001; n= 8 and 11 for wild type and mutant mice, 

respectively). These data differ from previous studies of social behaviors in mice heterozygous 

for a mutation in the transmembrane (TM) domain of NRG1. The latter mutant mouse showed no 

difference from the wildtype mouse in sociability toward an unfamiliar stimulus mouse, but 

decreased social interest toward a subsequently presented second stimulus mouse (decreased 

social novelty preference). While the TM domain mutants also showed increased aggressive 

behaviors, our data in the EGF-like domain mutants showed no significant effect of genotype on 

locomotor activity or aggressive behavior toward gonadectomized stimulus mice. Our data 

indicate that NRG1 may be involved in sociability deficits in scz. Future experiments will 

delineate the developmental progression of reduced sociability through the post-pubescent 

period. Additional studies will be aimed at elucidating the neural circuitry and molecular 

mechanisms underlying these sociability deficits. 

Disclosures: A. Kreibich , None; H.C. Dow, None; Y. Liang, None; R. Gur, None; S.J. Siegel, 

None; E.S. Brodkin, None.

Poster 




Topic: C.15.l. Other 

Support: MRC G0600214 

Title: Identification of novel DISC1-interacting proteins by a proteomics approach 

Authors: *F. OGAWA; 

Med. Genet. Section, Mol Med. Ctr, Univ. of Edinburgh, Edinburgh, United Kingdom 

Abstract: Accumulating evidence indicates that Disrupted-In-Schizophrenia 1 (DISC1) protein, 

a convincing risk factor for schizophrenia, plays an important role in brain development and 

function. A large number of studies employing the yeast two-hybrid system to comprehensively 

understand protein-protein interaction networks in which DISC1 is involved, have demonstrated 

that DISC1 potentially possesses diverse biological functions. Although numerous proteins may 

bind to DISC1, it remains to be determined which of these proteins DISC1 complexes with in 

vivo. 

Here, we identify novel DISC1-interacting proteins that have not been isolated in previous yeast 

two-hybrid screens. In this study, we utilized a proteomics approach, combining affinity 

purification of DISC1 complexes from HEK293T cells with mass spectrometric analysis using 

MALDI-TOF. The proteins identified were confirmed by immunoblotting. A potentially cleaved 

form of DISC1 was identified, suggesting that DISC1 may undergo posttranslational processing. 

We also observed coprecipitation of NUDEL, a known DISC1 interactor. Interestingly, each of 

the proteins identified shows divergent intracellular localization. These results will provide new 

insights into DISC1-mediated signaling pathways and posttranslational modifications of DISC1 

that may influence protein-protein interactions. 

Disclosures: F. Ogawa , None. 

Poster 

254. Schizophrenia: Mutant Animal Models




Title: Gene-environment interactions modulate phenotypic manifestations in transgenic mutant 

DISC1 mice 

Authors: *M. V. PLETNIKOV, O. NIKOLSKAIA, Y. AYHAN, J. NOMURA, M. VOGEL, C. 

ROSS; 

Dept Psychiat & Behav Sci., John Hopkins Univ. Sch. Med., Baltimore, MD 


Interactions between susceptibility genes and adverse prenatal and postnatal environmental 

factors play a major role in the etiology of major mental diseases. Recent findings in psychiatric 

genetics of schizophrenia, i.e., identification of the Disrupted-In-Schizophrenia 1 (DISC1) gene, 

have provided a novel approach to studying gene-environment interactions by utilizing 

genetically-engineered mice . Here, we use our mouse model of inducible expression of mutant 

human Disrupted-In-Schizophrenia-1 (hDISC1), a predicted truncated protein product of the 

chromosomal translocation previously shown to segregate with psychiatric illness. 

Methods 

We sough to evaluate interactions between mutant hDISC1 and the prenatal immune challenge 

with a synthetic analog of double-stranded RNA, Poly IC, to mimic intrauterine viral infections 

during pregnancy. 

Results 

The prenatal immune activation produced up-regulation of pro-inflammatory cytokines, IL1-β, 

TNF-α and IL-6, in serum of pregnant mice and brains of control and mutant fetuses. Compared 

to saline-treated mutant DISC1 mice, poly IC-challenged DISC1 mice exhibited elevated 

anxiety, decreased exploration of novel social and inanimate objects and elevated floating time 

(depression-like behavior) in Porsolt test. The neurobehavioral effects of prenatal immune stress 

in DISC1 mice are being investigated. 

Conclusions 

The preliminary findings indicate that expression of mutant human DISC1 and prenatal 

challenge can produce mood disorders-like phenotypic features unseen in DISC1 transgenic mice 

without environmental stress. Our mouse model provides an intriguing experimental system for 

future mechanistic studies of gene-environment interactions relevant to the pathogenesis of 

major mental illnesses. 

Disclosures: M.V. Pletnikov , None; O. Nikolskaia, None; Y. Ayhan, None; J. Nomura, 

None; M. Vogel, None; C. Ross, None. 

Poster





Support: Stanley Medical Research Institute 

Title: Comparison of distinct DISC1 animal models: roles for dopaminergic pathway and 

thalamus 

Authors: *H. JAARO-PELED 1 , S. TANKOU 1 , S. SESHADRI 1 , R. MURAI 4 , M. NIWA 4 , C. 

A. FOSS 2 , T. HIKIDA 1 , M. GALLAGHER 3 , T. NABESHIMA 4 , M. G. POMPER 2 , A. SAWA 1 ; 

1 Psychiatry, 2 Radiology, 3 Psychological and Brain Sci., Johns Hopkins Univ., Baltimore, MD; 

4 Dept. of Chem. Pharmacol., Meijo Univ., Nagoya, Japan 

Abstract: DISC1 is a major susceptibility factor for several neuropsychiatric diseases. The 

DISC1 protein is expressed in the cerebral cortex and hippocampus, but also in the thalamus and 

hypothalamus. Thus, it would be desirable to compare more than one DISC1 animal model to 

explore its multiple roles in vivo. Here we compare two distinct DISC1 transgenic mouse 

models. The first expresses a dominant-negative mutant DISC1 under the control of the 

αCaMKII promoter (Hikida et al 2007, PNAS). The other expresses the same mutant under the 

control of the prion protein (PrP) promoter. The αCaMKII promoter turns on the transgene 

expression postnatally mainly in the forebrain pyramidal neurons, whereas the PrP promoter 

activates the mutant DISC1 from E13 in several types of cells all over the brain. Both types of 

transgenic mice display a hyperdopaminergic phenotype: hyperactivity especially following an 

amphetamine challenge; increased binding of the dopamine D2 receptor antagonist raclopride to 

the striatum in PET scan; and a prepulse inhibition deficit. These results support the notion that a 

postnatal DISC1 disturbance in the cortex and hippocampus is associated with the classic 

dopamine hypothesis of schizophrenia. The PrP promoter directed expression of dominatnegative 

DISC1 is more similar to the endogenous DISC1 expression both temporally and 

spatially, which might explain the stronger phenotype. Notably, in contrast to the αCaMKII 

promoter-driven mutant DISC1, the endogenous DISC1 and the PrP promoter-driven mutant 

DISC1 are strongly and broadly expressed in the thalamus and hypothalamus. We are currently 

investigating potential behavioral consequences of disturbance of endogenous DISC1 in the 

thalamus. 

Disclosures: H. Jaaro-Peled , None; S. Tankou, None; S. Seshadri, None; R. Murai, 

None; M. Niwa, None; C.A. Foss, None; T. Hikida, None; M. Gallagher, None; T. 

Nabeshima, None; M.G. Pomper, None; A. Sawa, None.

Poster 





Title: Prenatal expression of mutant human DISC1 determines schizophrenia-like alterations in 

mice: support for neurodevelopmental hypothesis 

Authors: Y. AYHAN 1 , O. NIKOLSKAIA 1 , S. BROWN 1 , M. VOGEL 2 , *C. A. ROSS 1 , M. 

PLETNIKOV 1 ; 

1 Div. Neurobiol, Johns Hopkins Med. Sch., Baltimore, MD; 2 MPRC, Univ. of Maryland, 

Baltimore, MD 

Abstract: Background: The Disrupted in Schizophrenia-1 (DISC-1) is a strong candidate gene in 

the generation of major psychiatric disorders, first identified in a pedigree with a balanced 

choromosomal translocation disrupting DISC1. The hypothetical product of the gene, mutant 

human DISC1 can act as a dominant negative. Disruption of the normal gene function causes 

abnormal neural migration and maturation during neurodevelopment. Although several DISC1 

mouse models have been developed, the timing of the effects of mutant human DISC1 

(mhDISC1) remains incompletely understood. 

Methods: To evaluate the timing effects of mhDISC1, we used our Tet-off transgenic mouse 

model of inducible expression of mhDISC1 restricted to forebrain areas. The expression of the 

protein was regulated by feeding mice with doxycycline-containing food to generate three groups 

of mice with expression of mhDISC1 prenatally and postnatally; prenatally only; or without 

expression. 

Results: Prenatal but not postnatal expression of mhDISC1 was associated with decreased 

spontaneous alternation in Y maze, decreased social interaction and increased aggressiveness, 

enhanced responses to a NMDA antagonist, MK-801, or amphetamine. In addition, prenatal but 

not postnatal expression of the protein produced decreased cortical volume and increased 

dendritic spine density in granule cells of the dentate gyrus. The effects of prenatal expression on 

synaptic markers and DISC1 interactors are being evaluated and will be presented. 

Conclusions: The results indicate that the prenatal expression of mhDISC1may determine its 

schizophrenia-like phenotypic effects. Our findings provide a direct experimental support for the 

neurodevelopmental hypothesis of schizophrenia and other psychiatric conditions. 

Disclosures: Y. Ayhan, None; O. Nikolskaia, None; S. Brown, None; M. Vogel, None; C.A. 

Ross , None; M. Pletnikov, None.

Poster 





Support: NIMH MH0701667 (to E.C.) 

MH070855 (to (A.G.) 

Title: Studies on age-related cerebellar Purkinje cell loss and changes in open field motor 

activity in wild type (WT) and heterozygous reeler mice (HRM) 

Authors: *E. MALOKU, I. R. COVELO, A. GUIDOTTI, E. COSTA, I. HANBAUER; 

Psychiatric Institute/Psychiatry, Univ. Illinois Chicago, CHICAGO, IL 

Abstract: The trophism of cerebellar GABAergic Purkinje cells (PC) appears to depend on 

reelin secreted by cerebellar granule cells that send parallel fibers to impinge on the PC. 

Extracellular reelin appears to regulate axonal buttons and dendritic spine plasticity. To 

understand the role of reelin on PC survival, we measured PC numbers in various cerebellar 

regions of male wild type (WT) and HRM mice. 

Cerebellar serial sections (10 κm) in the sagittal plane from adult (4-11 months) and old (22 -26 

months) WT and HRM mice were stained with 1% cresyl violet. PC were counted in various 

regions of the cerebellum (10X magnification, Olympus microscope). 

The number of PC in old WT mice was lower than in adult WT mice. Particularly in the 

flocculus, the PC number decreased by 32% (4.2±0.3 versus 6.1±0.4; p

sec/15 min) than in adult HRM mice (353±25.1; p

mutations on the genes of molecules involved in CN signaling pathways. Ryanodine receptors 

(RyRs) are family of intracellular calcium channels and mediate calcium-induced calcium 

release (CICR) from the endoplasmic reticulum. Here we show the behavioral profile of 

ryanodine receptor type3 (RyR3) knockout mice. 

Disclosures: N. Matsuo, None; K. Tanda, None; N. Yamasaki, None; K. Toyama, None; S. 

Ohsako, None; K. Takao, None; H. Takeshima, None; T. Miyakawa, None. 

Poster 





Support: Ilanna Starr Scholar Fund 

Adler Foundation 

NINDS RO1 NS41438 and P01 NS047308 

Alzheimer‟s Association 

NIA P50 AG05146 

Title: BACE2 does not contribute to the schizophrenia-like endophenotypes observed in BACE1 

null mice 

Authors: *A. V. SAVONENKO 1 , J. W. YU 2 , T. MELNIKOVA 2 , H. D. KIM 2 , D. L. PRICE 2 , P. 

C. WONG 2 ; 

1 Pathol, Johns Hopkins Univ., Baltimore, MD; 2 Pathology, Johns Hopkins Univ. Med. Sch., 

Baltimore, MD 

Abstract: β-site APP cleaving enzyme (BACE1) is required for the penultimate cleavage of the 

amyloid-β precursor protein (APP) leading to the generation of amyloid-β peptides that is central 

to the pathogenesis of Alzheimer‟s disease. In addition to APP, the identification of several other 

putative BACE1 substrates strongly supports the idea that this enzyme plays multiple 

physiological roles. As a developmentally regulated aspartyl protease, BACE1 participates in the 

proteolytic processing of neuregulin 1 (NRG1) and influences the myelination of both central 

and peripheral axons. Furthermore, NRG1 has been genetically linked to schizophrenia and

NRG1+/- mice exhibit schizophrenia-like traits. BACE1-/- mice have been shown to have 

hypomyelination that seems to be a result of impaired processing of NRG1. In addition, we 

demonstrated that BACE1-/- mice exhibit multiple schizophrenia-like phenotypes including 

deficits in prepulse inhibition, novelty-induced hyperactivity, hypersensitivity to a glutamatergic 

psychostimulant (MK-801), working memory impairments and alterations in social interactions. 

Thus, BACE1-/- mice exhibit a variety of abnormalities reminiscent of those identified in 

schizophrenia that can be linked to altered BACE1-dependent NRG1 signaling. BACE2, a 

homolog of BACE1, shares some substrates with BACE1 such as APP. However, only BACE1 

participates in proteolyses of NRG1. Taking advantage of availability of BACE2-/- mice, we 

asked whether ablation of BACE2 leads to schizophrenia-like phenotypes observed in BACE1-/- 

mice. We tested a number of behaviors including novelty-induced exploration, prepulse 

inhibition, spatial working memory and anxiety levels. Unlike BACE1 null mice, mice lacking 

BACE2 performed similarly to wild type littermates in all of these tasks. Importantly, ablation of 

BACE2 did not modify behavioral deficits occurring in BACE1-/- mice, outcomes that are 

consistent with the view that BACE2 does not contribute to the BACE1-dependent pathways that 

mediate schizophrenia-like behaviors occurring in BACE1 null mice. 

Disclosures: J.W. Yu, None; A.V. Savonenko , None; T. Melnikova, None; H.D. Kim, 

None; D.L. Price, None; P.C. Wong, None. 

Poster 




Topic: C.15.e. Schizophrenia: Experimental pharmacotherapeutics 

Support: the Mitsubishi Pharma Research 

Title: Identification of susceptibility genes for antipsychotic -induced tardive dyskinesia 

Authors: *A. SYU 1 , H. ISHIGURO 2 , Y. HORIUCHI 2,3 , M. KOGA 2,3 , T. INADA 4 , M. 

TAKAHASHI 5 , M. ITOKAWA 6 , A. KAKITA 7 , H. NAWA 7 , H. TAKAHASHI 7 , T. 

ARINAMI 2,3 ; 

1 Tsukuba Univ., 2 Tsukuba Univ., Tsukuba, Japan; 3 CREST, Wako, Japan; 4 Seiwa Hosp., 

Shinjyuku, Japan; 5 Niigata Univ., Niigata, Japan; 6 TOKYO INSTITUTE OF PSYCHIATRY, 

Kodaira Tokyo, Japan; 7 Brain Reserch Institute,Niigata Univ., Niigata, Japan 

Abstract: Antipsychotic -induced tardive dyskinesia (TD) is an involuntary movement disorder 

that develops in patients who have undergone long-term treatment with antipsychotic

medications. A genome-wide association screening in 50 Japanese schizophrenia patients with 

TD and 50 patients without TD and subsequent replicate analysis of 36 patients with TD and 136 

patients without TD yielded an association between the HSPG2 gene and TD. The risk allele for 

TD was significantly correlated with lower expression of HSPG2 in human postmortem 

prefrontal cortex of non- schizophrenic subjects, though this association was not observed in 

those of schizophrenic subjects. Hspg2 expression levels was not significantly different between 

mice treated daily with vehicle and those with haloperidol 0.5mg/kg i.p. for 4 weeks. However, 

Hspg2 gene expression level in brains from haloperidol treated mice for 50 weeks was two-fold 

compared with those treated with vehicle for 50 weeks. Vacuous chewing movements (VCMs) in 

rats are widely accepted as an animal model of TD. The Hspg2 hetero knock-out female mice 

with haloperidol-reserpine showed more frequent VCMs than wild type female littermates. The 

findings in human and mice suggest that genetically determined lower expression of HSPG2 may 

increase the risk of the development of TD during antipsychotic medication. 

Disclosures: A. Syu, None; H. Ishiguro, None; Y. Horiuchi, None; M. Koga, None; T. Inada, 

None; M. Takahashi, None; M. Itokawa, None; A. Kakita, None; H. Nawa, None; H. 

Takahashi, None; T. Arinami, None. 

Poster 




Topic: C.15.b. Schizophrenia: Functional defecits 

Support: NICHD IRP 

NIMH IRP 

Title: Alteration in dopamine D2 receptor trafficking and D2 regulation of cortical neurons in 

Sandy mice 

Authors: Y. JI 1 , *F. YANG 1 , W. GAO 2 , B. LU 1,3 ; 

1 LCSN, Natl. Inst. Child Hlth. & Human Dev, Bethesda, MD; 2 Drexel Univ. Col. of Med., 

Philadelphia, PA; 3 Genes, Cognition and Psychosis Program (GCAP), NIMH, Bethesda, MD 

Abstract: Dysbindin, a risk factor for schizophrenia, is a component of the biogenesis of 

lysosome-related organelles complex 1 (BLOC1). Little is known about how dysbindin controls 

neuronal circuitry underlying behaviors. Recent studies showed that transfection of dysbindin 

siRNA resulted in an increase in cell surface dopamine D2 receptor (D2) in cell lines and

primary neurons. It is unclear how dysbindin regulates D2 trafficking. Here we used the sandy 

mouse (sdy/sdy, backcrossed to C57/BL6 background), a mutant line with natural deletion of 

dysbindin gene, to explore the cellular function of dysbindin and its underlying mechanisms. 

Combined imaging and biochemical experiments revealed a robust and selective increase in cell 

surface D2, but not D1, in cultured neurons from sdy/sdy cortex. Spontaneous and dopamineinduced 

endocytosis of D2 was not affected in sdy/sdy neurons. In contrast, these neurons 

exhibited a rapid re-insertion of D2 on the surface of neuronal soma and dendrites, following a 

dopamine-induced endocytosis of D2. Consistent with an elevated D2 signaling, the frequency 

and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from layer 5 

pyramidal neurons were decreased in prefrontal cortex (PFC) slices. Taken together, these results 

suggest that dysbindin controls PFC function by facilitating D2-mediated regulation of 

GABAergic transmission. 

Disclosures: Y. Ji, None; F. Yang , None; W. Gao, None; B. Lu, None. 

Poster 







Title: Behavioral analysis of Pdlim5 knockout mice with methamphetamine induced behavioral 

sensitization 

Authors: *Y. HORIUCHI 1,2 , H. ISHIGURO 1,2 , M. ISHIKAWA 1 , M. KOGA 1,2 , A. SYU 1 , T. 

ARINAMI 1,2 ; 

1 Med. Genet., Tsukuba Univ., Tsukuba, Japan; 2 CREST, Wako, Japan 

Abstract: The PDLIM5 (PDZ and LIM domain 5) gene contains LIM and PDZ domains and 

regulates intracellular calcium levels by linking calcium channel and protein kinase C. The 

expression of PDLIM5 is up-regulated in the postmortem brains of patients with schizophrenia. 

Previous studies showed that some SNPs in PDLIM5 were associated with schizophrenia and 

these SNPs influenced gene expression. Clinical studies have shown that common symptoms of 

repeated and long-term use of methamphetamine (METH) leads to dependence on the drug and 

psychotic symptoms such as delusion and elicits enhancement of locomotor activity known as

ehavioral sensitization. Behavioral sensitization may be a useful animal model of 

schizophrenia. The mechanism of METH induced behavioral sensitization is still unknown, but 

several evidences suggested dopaminergic system is underlying its fundamental roles. In the 

previous study, we obtained preliminary data indicating that PDLIM5 may play a role in the 

pathogenesis of schizophrenia through it‟s involvement in the dopaminergic signaling cascade. 

To investigate the involvement of Pdlim5 in an animal model of schizophrenia, we generated 

Pdlim5 knockout mice using Pdlim5 genetrap embryonic stem (ES) cells. An ES cell line 

containing genetrap vector was inserted in intron 8 of Pdlim5. Mice lacking Pdlim5 die during 

early developmental stage. Pdlim5 hetero knockout and wild type littermate male mice were 

treated with chronic METH (3mg/kg, once a day, intraperitoneal (i.p.)) for 10 days and 

developed behavioral sensitization to METH challenge injection (1mg/kg, i.p.) on withdrawal 

day 14. However, the Pdlim5 hetero knockout mice have attenuated the maintenance of 

behavioral sensitization of METH compared with wild type mice (p

myelin associated glycoprotein (MAG) and quaking (QKI), but little is known about the resulting 

myelin deficits. Here, we examined the cingulum bundle and cingulate cortex in two putative 

mouse models of dysmyelination, the MAG knockout, and the Quaking spontaneous mutant. 

These mice have specific behavioral deficits, particularly in fine motor tasks. In these models, 

we investigated the effect of alterations in myelin formation and oligodendrocyte deficits on 

prefrontal neuronal integrity, white matter coherence and integrity, and neuronal organization. In 

the MAG model, myelinated fiber density was assessed in the cingulum bundle and was found to 

be unchanged compared to wildtype control mice. Diffusion anisotropy imaging of the same 

mice also revealed unchanged fractional anisotropy in the cingulum. Advanced sterelogical tools 

were used to investigate the density and distribution of oligodendrocytes in the cingulum, as 

well, in both models. 

Our results suggest that the effects of dysmyelination in the cingulum in these mutant mouse 

models, if any, may be very subtle. In this context, we are conducting 3-dimensional analysis of 

the dendritic and spine integrity of pyramidal cells in both models at different ages based on 

preliminary observations in the MAG knockout showing an attrition of the basal dendritic tree at 

3 months of age. Further ultrastructural studies may be necessary to pinpoint the precise 

neuropathological alterations at the synaptic level. 

Disclosures: M. Hoistad , None; D. Segal, None; D. Carpenter, None; C.Y. Tang, None; P.R. 

Hof, None. 

Poster 





Support: NH&MRC Grant 

Trust Australia Scholarship 

Title: Behavioral endophenotypes and dysregulation of RGS4 gene expression in the PLC-β1 

knockout mice 

Authors: *C. E. MCOMISH 1,2 , E. BURROWS 2 , M. HOWARD 2 , A. J. HANNAN 2 ; 

1 Developmental Neurosci., RFMH, NY, NY; 2 Howard Florey Inst., Melbourne, Australia 

Abstract: The complexity of the genetics underlying schizophrenia is highlighted by the 

multitude of molecular pathways which have been reported to be disrupted in the disorder

including muscarinic, serotonergic and glutamatergic signaling systems. It is of interest, 

therefore, that phospholipase C-β1 (PLC-β1) acts as a point of convergence for these pathways 

during cortical development and plasticity. These signaling pathways, furthermore, are 

susceptible to modulation by RGS4, one of the more promising candidate genes for 

schizophrenia. PLC-β1 knockout mice were behaviorally assessed on tests including fear 

conditioning, elevated plus maze, and the Y maze. In situ hybridization was used to assess RGS4 

expression. We found that PLC-β1 knockout mice display abnormal anxiety profiles on some, 

but not all measures assessed, including decreased anxiety on the elevated plus maze. We also 

show memory impairment and a complete absence of acquisition of hippocampal-dependent fear 

conditioning. Furthermore, at a molecular level, we demonstrate dramatic changes in expression 

of RGS4 mRNA in selective regions of the PLC-β1 knockout mouse brain, particularly the CA1 

region of the hippocampus. These results validate the utility of the PLC-β1 knockout mouse as a 

model of schizophrenia, including molecular and cellular evidence for disrupted cortical 

maturation and associated behavioral endophenotypes. 

Disclosures: C.E. McOmish , None; E. Burrows, None; M. Howard, None; A.J. Hannan, 

None. 

Poster 

255. Mood Disorders: Animal Models and Treatment Effects II 



Topic: C.15.h. Affective disorders: Animal models 

Support: CIHR 

Title: Corticolimbic cannabinoid CB1 receptor activity regulates stress coping behavior in the 

forced swim test 

Authors: *R. J. MCLAUGHLIN 1 , M. N. HILL 2 , A. C. MORRISH 2 , B. B. GORZALKA 2 ; 

1 Dept Psych, 2 Psychology, Univ. of British Columbia, Vancouver, BC, Canada 

Abstract: In recent years, the endocannabinoid system has emerged as an important new target 

in the development of novel antidepressant drugs. However, a discrepancy exists in the literature 

such that antidepressant effects have been demonstrated following systemic administration of 

both CB1 receptor agonists and antagonists. These differential effects are likely mediated by 

CB1 receptor activation/antagonism within distinct neural circuits. Administration of a CB1 

receptor agonist into the dorsal hippocampus or ventromedial prefrontal cortex (vmPFC) has 

been found to elicit antidepressant-like effects in the forced swim test; however, no study to date

has determined the neuroanatomical site mediating the antidepressant effect of CB1 receptor 

antagonism. Given the importance of corticolimbic structures, such as the prefrontal cortex and 

amygdala, in the regulation of coping response to stress, the current study sought to determine if 

CB1 receptor signaling in subregions of the frontal cortex and the amygdala differentially 

regulate stress-coping behavior. To this extent we assessed the effects of discrete infusions of a 

CB1 receptor agonist (HU-210) or antagonist (AM251) directly into the vmPFC or the anterior 

cingulate cortex (ACC) within the frontal cortex, as well as within the basolateral, central and 

medial nuclei of the amygdala, on behavioral responses in the Porsolt forced swim test. 

Administration of the CB1 receptor agonist HU210 into the vmPFC decreased immobility and 

increased swimming in the forced swim test, indicating that this treatment evoked an 

antidepressant response, while the CB1 receptor antagonist AM251 had no effect. Alternately, 

infusions of AM251 into the ACC resulted in a dose-dependent antidepressant response as 

revealed by a reduction in immobility, while infusion of HU-210 had no significant effects on 

behavioral responses in the forced swim test. Within the amygdala, we have determined that 

administration of AM251 into the basolateral amygdala has no effect on stress coping behavior 

in the forced swim test. Ongoing studies are examining the effects of AM251 and HU-210 into 

the medial and central nuclei of the amygdala in the forced swim test. Collectively, these data 

argue that CB1 receptor signaling in distinct fronotocortical subregions has differential effects on 

emotional behavior, and that the ACC may be an integral neuroanatomical site mediating the 

reported antidepressant effects seen following systemic administration of a CB1 receptor 

antagonist. 

Disclosures: R.J. McLaughlin , None; M.N. Hill, None; A.C. Morrish, None; B.B. Gorzalka, 

None. 

Poster 





Support: FAPESP 

CNPQ 

FAEPA 

Title: Activation of CB1 cannabinoid receptors in Medial Prefrontal Cortex modulate the 

expression of contextual fear conditioning in rats

Authors: *L. B. RESSTEL, F. M. A. CORRÊA, F. S. GUIMARÃES; 

Pharmacol, Schl Med, FMRP-USP, Ribeirao Preto- SP, Brazil 

Abstract: Introduction: The glutamatergic system present in the ventral portion of medial 

prefrontal cortex (vMPFC) of the rat is involved in modulation of behavior and cardiovascular 

response associated with contextual fear conditioning. It has been described the expression of 

CB1 cannabinoid receptors in the vMPFC. Moreover, the CB1 receptors are able to modulate 

glutamate release. Methods: To better understand the role of the vMPFC-endocannabinoid 

system on the expression of contextual fear responses, male Wistar rats had direct 

administration,200 nl, into the vMPFC of the CB1 cannabinoid receptor agonist anandamide 

(AEA, 5 pmol) or the anandamide transport inhibitor AM404 (50 pmol) would attenuated the 

behavioral (freezing) and cardiovascular (MAP - mean arterial pressure or HR - heart rate) 

responses of rats submitted to a contextual fear conditioning paradigm. The CB1 antagonist 

AM251 (100 pmol) was administread before both drugs in half of animals. Footshock stimulus 

was used as conditioner. Bilateral microinjections were made immediately before conditioned 

response to context was tested. Results: After AEA, conditioned animals had reduced freezing 

duration when compared with vehicle-injected animals (21 ± 8 % vs 77 ± 7 %; t= 5.1, p

Title: Anxiolytic-like effect of TRPV1 receptor antagonism in the medial prefrontal cortex and 

central gray 

Authors: *A. B. TERZIAN 1 , D. C. AGUIAR 1 , F. A. MOREIRA 2 , F. S. GUIMARÃES 1 ; 

1 Phamacology, FMRP - USP, Ribeirao Preto, Brazil; 2 Univ. of Mainz, Mainz, Germany 

Abstract: Introduction: The so-called capsaicin vanilloid receptor, VR1, is a member of the TRP 

family of ion channels (TRPV1) and is widely found on small- and medium-sized primary 

afferent neurons and in diverse areas of the central nervous system, including the dorsolateral 

periaqueductal gray (dlPAG) and medial prefrontal cortex (mPFC). Both structures have been 

closely associated with defensive responses but the role of local TRPV1 receptors in these 

responses is largely unknown. In a previous study we found that intra-dlPAG administration of 

capsazepine (CPZ, 10-60 nmol), a TRPV1 receptor antagonist, produced anxiolytic-like effects 

at the higher (60 nmol) dose in the elevated plus maze. The aim of the present study was to 

confirm these anxiolytic-like effects in another model of anxiety, the Vogel conflict test (VCT). 

In the same test we also investigated the effects of CPF microinjection into the mPFC. Methods: 

Water-deprived male Wistar rats (n=3-6) with cannula aimed at the dlPAG (unilateral) or the 

mPFC (bilateral) received local microinjections of CPZ (60 nmol/0.2microL) or vehicle (DMSO) 

and were submitted to the VCT. Results: CPZ significantly increased the number of punished 

licks indicating an anxiolytic-like effect (Vehicle, dlPAG: 62.6+/-8.6, CPZ, dlPAG: 178.0+/-13.0 

F2,11= 6.49, p

Title: Mechanisms of the antidepressant-like effects of Cannabidiol in the forced swimming test 

Authors: *F. S. GUIMARAES 1 , C. BIOJONE 1 , T. V. ZANELATI 1 , S. R. L. JOCA 2 ; 

1 Sch. Med. Ribeirao, Ribeirao Preto, Brazil; 2 Dept. of Physics and Chem., FCFRP, Ribeirao 

Preto, Brazil 

Abstract: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa with 

anxiolytic and antipsychotic properties. It also showed antidepressant-like effects in the forced 

swimming test (FST). The mechanisms of these effects are still unknown, but may involve 

activation of 5HT1A receptors. Facilitation of serotonin-mediated neurotransmission can 

increase BDNF expression in the hippocampus and is a proposed mechanism for antidepressant 

effects. The objectives of the present investigation were to confirm the involvement of 5HT1A 

receptors in the antidepressant effects of CBD and to verify if these effects are associated with an 

acute increase in hippocampal BDNF expression. Male Swiss mice received i.p. injections of 

saline or WAY100635 (0.1 mg/Kg), a 5HT1A receptor antagonist, followed 30 min later, by i.p. 

injections of vehicle or CBD (30-100 mg/kg). They were submitted to the FST 30 min after the 

last injection. In this test the animals were left in the cylinder filled with water for 6 min and the 

total duration of immobility was measured during the last 4-min period. Classical antidepressant 

such as imipramine typically decrease immobility time. Independent groups received CBD (30 

mg/kg) or vehicle and were sacrificed immediately after the FST under deep urethane anesthesia. 

Their hippocampi were isolated and BDNF expression was measured by ELISA. The results 

confirmed that CBD (30 mg/kg) is able to decrease immobility time in the FST, an 

antidepressant-like effect. This effect was prevented by WAY100635. It was not associated, 

however, with changes in BDNF expression. The results suggest that CBD exerts antidepressantlike 

effects in the FST by activating 5HT1A receptors. These effects, however, do not seem to 

involve acute changes in hippocampal BDNF expression. 

Disclosures: F.S. Guimaraes , None; C. Biojone, None; T.V. Zanelati, None; S.R.L. Joca, 

None. 

Poster 





Support: Fapesp 

CNPq

Title: Cannabidiol prevents stress-induced behavioral and cardiovascular consequences: possible 

involvement of 5HT1A-receptors 

Authors: *S. S. JOCA 1 , L. B. M. RESSTEL 2 , R. TAVARES 2 , S. LISBOA 2 , F. M. A. 

CORREA 2 , F. S. GUIMARÃES 2 ; 

1 Physics-Chemistry/Pharmacology, FCFRP-USP, Ribeirao Preto-SP, Brazil; 2 Pharmacol., 

FMRP-USP, Ribeirao Preto-SP, Brazil 

Abstract: Introduction: Cannabidiol (CBD) is a non-psychotomimetic compound from 

Cannabis sativa which induces anxiolytic- and antipsychotic-like effects in rodents. CBD effects 

have been proposed to be mediated by facilitation of the endocannabinoid system or by the 

activation of 5-HT1A receptors. Since either facilitation of endocannabinoid neurotransmission 

or 5-HT1A receptor activation may promote adaptation to inescapable stress, the aim of the 

present work was to test the hypothesis that CBD would attenuate behavioral and autonomic 

consequences of restraint stress (RS). 

Methods: Male Wistar rats received i.p. injections of vehicle or CBD (1, 10 or 20 mg/kg) and, 

30 min later, were submitted to 60 min of restraint where their cardiovascular responses were 

recorded. One day later the animals tested in the elevated plus-maze (EPM), an animal model of 

anxiety. The protocol of the second experiment was similar to the first one except that the 

animals received i.p. injections of WAY100635 (0.1 mg/ Kg), a 5-HT1A receptor antagonist, 

before CBD treatment. 

Results: None of the treatments changed baseline MAP and HR. RS produced pressor and 

tachycardic responses that were attenuated by CBD (10 and 20 mg/kg; MAP: F 3,16=19.9, 

p

Support: CAPES 

FAPESP 

CNPq 

Title: Facilitation of endocannabinoid signaling in the dorsolateral periaqueductal gray 

modulates anxiety via activation of CB1 receptors 

Authors: *S. F. LISBOA, L. B. M. RESSTEL, D. C. AGUIAR, F. S. GUIMARÃES; 

Pharmacol., Univ. of São Paulo - FMRP, Ribeirão Preto, Brazil 

Abstract: Introduction: Activation of cannabinoid CB1 receptors by the endocannabinoid 

anandamide (AEA) microinjected into the dorsolateral periaqueductal gray induces anxiolyticlike 

effects in the elevated pus-maze, Vogel conflict test and contextual fear conditioning. Aim: 

The aim of this work was to verify if facilitation of local AEA neurotransmission in the 

dorsolateral periaqueductal gray, by inhibition of Fatty Acid Amide Hydrolase (FAAH) enzyme 

responsible by AEA degradation, would also evoke anxiolytic-like effects via CB1 receptor 

activation in the Vogel conflict test. Methods: Male Wistar rats (n=5-9) with cannulae aimed at 

the dorsolateral periaqueductal gray received a first microinjection of vehicle or AM251 

(cannabinoid CB1 receptor antagonist; 100 pmol/ 0.2 ul) followed, 5 min later, by a second 

microinjection of vehicle or URB597 (an inhibitor of FAAH, 0.1 or 0.01 nmol/ 0.2 ul). The 

animals were water deprived for 24 h and pre-exposed to the apparatus where they were allowed 

to drink for 3 min. After another 24 h of water deprivation they received the microinjections and, 

10 min later, were placed into the experimental box. In this box they received an electrical shock 

(0.5 mA, 2 s) in the spout of a drinking bottle at every twenty licks. Results: URB597 (0.01 

nmol, n=9) increased the total number of licks (F4,38=4.41, P0.05). Conclusion: These results support the proposal that facilitation 

of endocannabinoid signaling in the dorsolateral periaqueductal gray via CB1 receptors can 

modulate defensive responses, suggesting that this structure is a possible neuroanatomical site 

for the anxiolytic-like effects mediated by cannabinoid agonists. 

Table of Contents 

Disclosures: S.F. Lisboa, None; L.B.M. Resstel, None; D.C. Aguiar, None; F.S. Guimarães, 

None. 

Poster 



Program#/Poster#: 255.7/Y22


Support: Fapesp 

CNPQ 

Title: Stress influence on anxiety modulatory role of the endocannabinoid system in the ventral 

hippocampus 

Authors: *A. C. CAMPOS, J. I. LEMOS, F. R. FERREIRA, F. S. GUIMARAES; 

Dept. of Pharmacol., Sch. Med. Ribeirao-USP, Ribeirão Preto, Brazil 

Abstract: Introduction: Several studies have suggested that the endocannabinoid system 

modulates anxiety and stress adaptation. There is, however, a paucity of data regarding the 

involvement of endocannabinoids such as anandamide (AEA) on brain sites related to these 

effects. The ventral hippocampus (VHC) has been related to anxiety behaviors and has a high 

expression of cannabinoid-1 (CB1) receptors. Moreover, endocannabinoid signaling in the 

hippocampus is proposed to regulate stress adaptation. Thus, the aim of this study was to 

investigate the effects of AM404, an AEA uptake inhibitor, and AM251, a CB1 receptor 

antagonist, microinjected into the VHC of stressed and non-stressed rats. 

Methods: Male Wistar rats (240-270g) with cannulae aimed at the VHC were submitted to 

restraint stress for 2h. Non-stressed animals remained in their home cages. Twenty four hours 

later they received bilateral injections of vehicle (V, 0.5 microL), AM-404 (5 or 50 pmol, N=8- 

11) or AM251 (1-100 pmol; N= 5-11) and 10 minutes later were submitted to the elevated plus 

maze (EPM). 

Results: Restraint stress induced a significant decrease in the percent of entries and time spent in 

the open arms. AM-404 (50 pmol) reduced the percentage of entries and time spent in the open 

arms in non-stressed animals. On the other hand, AM404 (5 pmol) increased the open arm 

exploration in previously stressed animals. AM251 (1 pmol) tended to increase the time spent in 

the open arms of the non stressed animals but did not change EPM exploration of stressed 

animals. 

Discussion: The results suggest that the endocannabinoid system in the VHC modulates anxietylike 

behavior depending on previous stressful experience of the animal. 

REFERENCES: Hill et al., Neuropsychopharmacology, 2005; Rubino et al. 

Neuropharmacology, 2008. Financial support: FAPESP, CNPQ 

Disclosures: A.C. Campos, None; J.I. Lemos, None; F.R. Ferreira, None; F.S. Guimaraes, 

None. 

Poster 

255. Mood Disorders: Animal Models and Treatment Effects II




Title: Antidepressant efficacy screening of novel targets in the chick anxiety-depression model 

Authors: *J. E. WARNICK 1 , C. PULASKI 2 , S. R. SLAUSON 3 , Y. B. KIM 3 , J. M. 

RIMOLDI 3,4 , K. J. SUFKA 2,4,5 ; 

1 Dept Behav Sci., Arkansas Tech. Univ., Russellville, AR; 2 Dept. of Psychology, 3 Dept. of 

Medicinal Chem., 4 Res. Inst. of Pharmaceut. Sci., 5 Dept. of Pharmacol., Univ. of Mississippi, 

Oxford, MS 

Abstract: Research from this laboratory has developed a novel, clinically-relevant, non-rodentbased 

model of a neuropsychiatric syndrome that appears useful as a high-utility, dual-drug 

screen for anxiolytic/antidepressant compounds. This chick anxiety-depression model possesses 

face, construct and predictive validity. The current study investigated the efficacy of a number of 

novel antidepressant targets including: memantine (NMDA antagonist; 2.5-20.0 mg/kg), 

ketamine (NMDA antagonist; 1-10 mg/kg), DOV216,303 (monoamine triple reuptake inhibitor; 

5-20 mg/kg), CGP36742 (GABAB antagonist; 2.5-15.0 mg/kg) and antalarmin (CRF1 antagonist; 

1-30 mg/kg). Five to seven day-old chicks received either vehicle or drug probe IM 15 min prior 

to a two hr social separation period. Dependent measures were distress vocalizations: 0-5 min 

DVoc rate indexed anxiety phase and 30-120 DVoc rate indexed depression phase. Ketamine, 

DOV216,303 and CPG36742 showed antidepressant activity. However, CPG36742 displayed 

anxiogenic activity in the anxiety phase. Memantine and antalarmin failed to show 

antidepressant effects. This pattern of effects parallels human clinical data and highlight two 

important qualities of the screening model. First, the model is a better predictor of Phase II and 

III clinical failures (e.g., memantine). Second, the model has the potential to reveal 

contraindications of compounds (i.e., CPG36742, a GABAB antagonist) in some individuals that 

display anxiety symptoms concomitant with their depressive episode. These findings argue 

strongly that the chick anxiety-depression model may be a useful addition to the current rodentbased 

anxiolytic and antidepressant preclinical screening assays. 

Disclosures: J.E. Warnick , None; C. Pulaski, None; S.R. Slauson, None; Y.B. Kim, 

None; J.M. Rimoldi, None; K.J. Sufka, None. 

Poster 





Title: Acute stress responsiveness of the neurotrophin BDNF in rat hippocampus is modulated 

by chronic antidepressant treatment 

Authors: *M. A. RIVA 1 , F. CALABRESE 1 , A. CATTANEO 2 , M. MANCINI 3 , M. 

GENNARELLI 2 , G. RACAGNI 1 , R. MOLTENI 1 ; 

1 Dept Pharmacol Sci., Univ. Milan, Milan, Italy; 2 IRCCS Fatebenefratelli San Giovanni di Dio, 

Brescia, Italy; 3 Eli Lilly Italia S.p.A., Sesto Fiorentino (FI), Italy 

Abstract: Compelling evidence suggests that mood disorders are characterized by reduced 

neuronal plasticity that might ultimately be normalized by pharmacological intervention. 

However, the potential of psychotropic drugs to improve neuronal plasticity can also be due to 

the modulation of specific proteins and systems under a challenging condition. On this basis, our 

study aimed to establish whether chronic antidepressant treatment could alter the modulation of 

the neurotrophin brain-derived neurotrophic factor (BDNF) under a stressful condition. 

In order to do so, adult male Sprague-Dawley rats were treated for 21 days with vehicle or with 

the SNRI duloxetine (DLX, 10 mg/kg). Twenty-four hours after the last injection, half of the 

animals were exposed to an acute swim stress (5 min) and were killed 15 min later. We found 

that, although minor changes on hippocampal BDNF mRNA levels were observed under resting 

conditions, chronic DLX treatment was able to affect rapid transcriptional changes produced by 

the acute swim stress. Indeed while the mRNA levels of BDNF exon IV were up-regulated by 

stress in vehicle as well as in DLX-treated rats, a significant increase of exon VI was only found 

in rats that were pretreated with the antidepressant. These differential effects are in part due to 

selective changes in stress-induced activation of signalling pathways involved in the control of 

BDNF transcription. Moreover, we found that chronic duloxetine treatment can also modulate 

the intracellular trafficking of BDNF protein following the acute stress. In fact the levels of 

mBDNF in the synaptosomal compartment were significantly increased following stress in DLXtreated 

rats, but not in control animals. Conversely the levels of mBDNF in the whole 

homogenate were not affected by stress exposure. 

In summary, our results consolidate the idea that an up-regulation of the neurotrophin BDNF 

may represent a common event of antidepressant treatment. Moreover, we provide evidence for a 

novel degree of modulation of synaptic plasticity, which refers to the possibility that 

antidepressant drugs might alter rapid transcriptional changes of neurotrophin exons and can also 

enhance the synaptic pool of the neurotrophin under challenging conditions. 

Disclosures: M.A. Riva , None; F. Calabrese, None; A. Cattaneo, None; M. Mancini, Eli 

Lilly Italia S.p.A., Sesto Fiorentino, Italy, A. Employment (full or part-time); M. Gennarelli, 

None; G. Racagni, None; R. Molteni, None. 

Poster





Support: DFG Research Center Molecular Physiology of the Brain (CMPB), University of 

Goettingen. 

GlaxoSmithKline, Verona, Italy. 

Title: Effects of chronic social stress and fluoxetine treatment on the vascular niche and 

neurogenesis in the hippocampus 

Authors: *B. CZEH, E. FUCHS; 

Clin. Neurobio. Lab., German Primate Ctr., Goetttingen, Germany 

Abstract: Findings in experimental animals showing that stress inhibits adult neurogenesis, 

whereas antidepressant treatment has the opposite effect, have led to the formulation of the 

“neurogenic theory” of depression. Earlier studies also demonstrated that adult neurogenesis 

occurs within an angiogenic niche, but little is known about the effect of chronic stress and 

antidepressant treatment on angiogenesis or vasculature in the hippocampal dentate gyrus. 

We examined in rats the effect of chronic social defeat stress and treatment with the 

antidepressant fluoxetine (FLX) on vascular supply and adult neurogenesis in the subgranular 

zone. Our aim was to investigate whether changes in neurogenesis and vascular niche are 

running in parallel as a result of the different treatments. 

Adult male Wistar rats were subjected to 5 weeks of daily social defeat by an aggressive 

conspecific and received concomitant, daily, oral FLX (10 mg/kg) during the last four weeks. 

Rat-endothelial-cell-antigen-1 (RECA-1) labeling of capillaries and systematic density analysis 

of microvessels was used to evaluate the impact of the treatments on capillary number in the 

subgranular zone. By using bromodeoxyuridine (BrdU) labeling and stereological cell counting 

we assessed in the same animals the effects of stress and FLX treatment on adult neurogenesis. 

Chronic social defeat stress resulted in a significant decrease (-55%) in the number of BrdUlabeled 

cells. Treatment of stressed animals with FLX resulted in a significant increase in the 

number of BrdU-labeled cells compared to the stressed animals without FLX. Thus, the number 

of newborn cells was in a same range in non-stressed and stressed + FLX treated animals. 

Phenotypic analysis of the newly generated cells revealed that the majority of the newborn 

BrdU-positive cells in the dentate gyrus expressed the neuronal marker NeuN, whereas a 

minority of the BrdU-labeled cells was double labeled with GFAP, an astroglia marker. 

Furthermore, we observed a dense network of RECA-1-positive capillaries in the subgranular 

zone. Stress resulted in a significant decrease (-28%) of microvessel density. FLX treatment had 

no effect on vascular supply since in the Stress + FLX group we observed a very similar decrease 

by 27%. 

We report here that chronic stress significantly decreased the vascular supply of the subgranular

zone, which is regarded as a neurogenic niche for adult hippocampal neurogenesis; importantly 

FLX treatment did not counteract this effect. At the same time FLX normalized the stressinduced 

reduction in adult neurogenesis, suggesting that the effect of FLX on adult neurogenesis 

is independent from the vascular niche. 

Disclosures: B. Czeh , None; E. Fuchs, None. 

Poster 




Topic: C.15.f. Affective disorders: Pathology 


Title: Fluoxetine shares aspects of TCA and MAOI effects on factors relevant to hypothalamicpituitary-adrenal 

axis activity: could this explain broad efficacy of SSRIs? 

Authors: *W. A. HEYDENDAEL, L. JACOBSON; 

Ctr. for Neuropharmacol & Neurosci, Albany Med. Coll, Albany, NY 

Abstract: Abnormally high or low levels of glucocorticoids caused by dysregulation of the 

hypothalamic-pituitary-adrenal (HPA) axis can affect mood and may add to depression 

symptoms. Tricyclic antidepressants (TCA) have been used to treat forms of depression with 

elevated HPA activity, while patients suffering from atypical depression, which is often 

associated with decreased HPA activity, show a preferential responsiveness to monoamine 

oxidase inhibitors (MAOI). However, selective serotonin reuptake inhibitors (SSRIs) appear to 

be effective in both melancholic and atypical depression. Our lab has previously shown 

differential effects of the TCA imipramine and the MAOI phenelzine on HPA activity and HPArelevant 

factors. Because SSRIs can be effective for many forms of depression, we hypothesized 

that the SSRI fluoxetine would have effects that would be shared with either TCA or MAOI. To 

test this hypothesis, we measured gene expression for HPA-relevant factors such as 

glucocorticoid receptors (GR), mineralocorticoid receptors (MR), locus coeruleus tyrosine 

hydroxylase (TH) dorsal raphé tryptophan hydroxylase (TPH2), and amygdala corticotropinreleasing 

hormone (CRH) in male C57BL/6 mice treated chronically with fluoxetine (10 mg/kg/d 

ip for 5 weeks). Mice were sham-adrenalectomized or adrenalectomized and given fixed 

glucocorticoid levels to control for any fluoxetine-induced changes in glucocorticoid secretion 

that might alter GR and MR expression via autoregulation. Fluoxetine had no effect on MR 

expression in the brain regions examined. However, fluoxetine did decrease GR expression in

the prefrontal cortex, amygdala, locus coeruleus and dorsal raphé. Fluoxetine also decreased 

amygdala CRH expression and increased locus coeruleus TH and dorsal raphé TPH2 expression. 

Since glucocorticoids increase amygdala CRH and inhibit expression of locus coeruleus TH and 

dorsal raphé TPH2, these fluoxetine effects are consistent with fluoxetine-induced decreases in 

GR. We previously found that imipramine increased prefrontal cortex GR and decreased dorsal 

raphé GR while increasing dorsal raphé TPH2 and decreasing locus coeruleus TH. Phenelzine, in 

contrast, decreased prefrontal cortex and locus coeruleus GR while increasing locus coeruleus 

TH. Thus, in support of our hypothesis, fluoxetine had effects that were similar to aspects of both 

TCA and MAOI action. These effects may be relevant to fluoxetine‟s efficacy in treating both 

melancholic and atypical depression. Also, since elevated amygdala CRH has been linked to 

increased anxiety, fluoxetine-induced decreases in amygdala CRH may provide insight into the 

anxiolytic effects of SSRIs. 

Disclosures: W.A. Heydendael , None; L. Jacobson, None. 

Poster 





Support: CIHR grant MOP42438 

Croatian Ministry of Science, Education and Sport grant 219-1081970-2032 

Title: Chronic treatment with citalopram changes density of the serotonin transporter in some 

brain regions in FSL and SD rats 

Authors: *I. SKELIN 1 , T. KOVACEVIC 2,3 , M. DIKSIC 2,3 ; 

2 Neurol. and Neurosurg., 1 McGill Univ., Montreal, QC, Canada; 3 Fac. of Med., J.J. Strossmayer 

Univ., Osijek, Croatia 

Abstract: The Flinders Sensitive Line (FSL) rat is an animal model of depression [Neurosci 

Biobehav Rev 29(2005)739-59], selectively bred from the Sprague-Dawley (SD) strain for 

increased cholinergic sensitivity. FSL rats exhibit some behavioral, sleep and immunological 

abnormalities similar to those found in depressed humans. Citalopram is a selective serotonin 

reuptake inhibitor (SSRI), with the highest selectivity for the serotonin transporter (SERT) 

among the clinically used SSRIs. Chronic therapy with citalopram reduces the immobility in FSL 

rats in the forced swim test, considered to be a predictor of an antidepressant response in

humans. Citalopram binds to the SERT and blocks the reuptake of the serotonin into the synaptic 

terminals. The aim of this study was to assess the effect of citalopram treatment on the density of 

the SERT in FSL (FSL-CTP group) and SD (SD-CTP) rats [14-day therapy; 10 mg/kg/day of 

citalopram delivered by the osmotic minipump]. The controls received saline (FSL-SAL and SD- 

SAL groups). Following the treatment, the rats were decapitated and the brains were frozen in 

isopentane and stored at -80º C. The brains were cut in a microtom on slides 20 µm thick. The 

binding experiment was performed as described in Psychopharmacology (Berl.) 178 (2005)276- 

85 using 20 pM [ 125 I]-(-)-2β-Carbomethoxy-3β-(4-iodophenyl)tropane ( 125 I-RTI-55) with the 

presence of 200 nM 1-(2-Diphenylmethoxyethyl)-4-(3-phenylpropyl)piperazine dihydrochloride 

(GBR12935) for 120 min. Non-specific binding was measured in the presence of 1 mM of 

paroxetine along with 200 nM GBR12935. The slides were contacted with Fuji 14 C imaging 

plates along with the 125 I standards for 24 hours and scanned in a Fuji BAAS 5000 scanner. The 

images were quantified with the MCID image analyzer calibrated with standards. The regional 

values were assessed in 24 brain regions and transformed to receptor concentrations (fmol/mg). 

The statistical analysis was performed using STATISTICA software. One-way ANOVA 

revealed: 1) significant differences in 5 regions between the FSL-CTP and FSL-SAL groups, 

F(2,9)=39.8; p

This abstract has been authored by Brookhaven Science Associates, LLC under 

Contract No. DE-AC02-98CHI-886 with the U.S. Department of Energy. 

Title: Behavioral concomitants of congenital learned helplessness and its reversal by a 

monoamine oxidase B inhibitor 

Authors: *D. SCHULZ, M. M. MIRRIONE, F. A. HENN; 

Med. Dept., Brookhaven Natl. Lab., Upton, NY 

Abstract: Wild type Sprague Dawley rats were selectively bred for over 50 generations to yield 

two strains which differ in their susceptibility to develop learned helplessness. For further 

characterization of the animals, 3-4 months old male congenital learned helpless (cLH, n = 10) 

and non-helpless (cNLH, n = 12) rats as well as wild types (WT, n = 8) were subjected to three 

days of open field testing. On the first two days, the animals were exposed to an empty arena for 

10 min each. On the third day, an object exploration paradigm was carried out. Two days later, 

helplessness training consisted of 120 inescapable foot-shocks at 0.4 mA with a variable duration 

of 5-15 s. The next day, the animals were tested for helplessness by exposure to 15 escapable 

foot-shocks which lasted 60 s unless turned off earlier by one lever press. As expected, cLH rats 

turned off the foot-shock on fewer trials and required more time to complete the 15 runs 

compared to cNLH and WT rats. cNLH rats, in turn, pressed the lever more often than WT 

controls and completed the 15 trials sooner. On the first day of open field testing, cLH were 

more active than WT rats as measured by the total distance moved. Moreover, over the first two 

trials, cLH rats decreased activity levels more than cNLH and WT rats, indicative of enhanced 

habituation learning. In cNLH and WT rats, a smaller decrease in activity levels over trials or a 

greater resistance to habituate was correlated with more lever presses in the helplessness 

paradigm. In cLH rats, indices of „anxiety‟, including less time spent in the center of the open 

field, were correlated with fewer lever presses, in line with the known relationship between 

depression and anxiety in humans and other animal models of depression. In a different group of 

cLH rats, 14 days of treatment with a high dose of deprenyl hydrochloride (10mg/kg; i.p.), which 

may cause inhibition of monoamine oxidase (MAO)-A as well as MAO-B activity, attenuated 

the escape deficit in the helplessness test; compared to saline-treated controls (n = 5), deprenyltreated 

cLH rats (n = 5) turned off the foot-shock on more trials and required shorter times to 

complete the 15 runs. Moreover, administration of deprenyl resulted in significant weight loss in 

cLH rats, which were found to be overweight compared to cNLH controls (n = 12 and 8, 

respectively) at the age of weaning and onwards. In conclusion, the cLH strain shows 

consistency with a „depressive‟ phenotype which may be related to abnormalities in MAO-A 

and/or MAO-B functioning. 

Disclosures: D. Schulz , None; M.M. Mirrione, None; F.A. Henn, None. 

Poster 

255. Mood Disorders: Animal Models and Treatment Effects II




Support: NIH AWARD K02 DA22346 to SLD 

Brookhaven National Laboratory Directed Research and Development Program funded 

by the U.S. Department of Energy (LDRD-07-096 to FAH) 

NIH GRANT R01 DA15041 to SLD 

This abstract has been authored by Brookhaven Science Associates, LLC under 

Contract No. DE-AC02-98CHI-886 with the U.S. Department of Energy 

Title: Imaging neuronal activity in the learned helplessness model of depression using 18 FDG 

microPET 

Authors: *M. M. MIRRIONE 1 , D. SCHULZ 1 , S. L. DEWEY 1,2 , F. A. HENN 1,3 ; 

1 Med. Dept, Brookhaven Natl. Lab., Upton, NY; 2 Psychiatry Dept., New York University, Sch. 

of Med., New York, NY; 3 Psychiatry Dept., Mount Sinai Sch. of Med., New York, NY 

Abstract: Neuroimaging studies in humans using 2-deoxy-2[ 18 F]fluoro-D-glucose ( 18 FDG) have 

revealed unique patterns of neuronal activity in depressed patients compared to healthy controls. 

These studies show complex metabolic changes in the prefrontal cortex, thalamus, limbic 

system, and basal ganglia, which can be associated with expression of symptoms over many 

years. Here, we use small animal positron emission tomography (microPET) to measure acute 

changes in neuronal activity in vivo associated with an animal model of depression. We have 

chosen the „learned helplessness‟ paradigm, which has been validated pharmacologically, but not 

yet explored in depth for dysfunctional neurocircuitry. Our hypothesis was that there would be 

regional differences in 18 FDG uptake between learned helpless (LH) and non-learned helpless 

(NLH) wild type Sprague Dawley rats. Parallel experiments are also being conducted in 

congenitally helpless (cLH) and non-helpless (cNLH) rats which have been selectively bred for 

over 50 generations and differ in their susceptibility to develop learned helplessness. In our 

study, subjects underwent training and testing sessions (120 inescapable or 15 escapable footshocks 

respectively, at 0.4 mA with a variable duration of 5-15 s), followed 1-2 hours by an 

intraperitoneal injection of 18 FDG (~2 mCi/kg) in their home cage with a 45 minute uptake 

period (where radiotracer distributes through the brain/body becoming trapped in metabolically 

active cells). Subjects were then anesthetized (100 mg/kg Ketamine/Xylazine), and given a 10 

min static microPET scan (n = 10 LH, n = 10 NLH). Data were then processed, and quantified 

with a voxel based analysis using Statistical Parametric Mapping software (SPM2). The resulting 

metabolic profiles of LH and NLH subjects were directly compared. Significantly higher 18 FDG 

uptake was observed in several brain regions of the LH group including the medial 

thalamus/habenula, retrosplenial cortex and cerebellum. These data demonstrate the potential for 

characterizing metabolic patterns of helplessness to reveal areas associated with initiating and

propagating depressive-like behavior. Recently, evidence has emerged supporting a role for 

glutamate/GABA dysfunction underlying depressive symptoms. We hypothesize the differences 

found in the medial thalamus/habenula reflect dysregulation of glutamate/GABA in addition to 

monoamines, and that modulation of this or other target areas can reverse depressive-like 

behavior. 

Disclosures: M.M. Mirrione , None; D. Schulz, None; S.L. Dewey, None; F.A. Henn, None. 

Poster 





Support: Croatian Ministry of Science, Education and Sport (219-1081970-2032) 

The National Foundation for Science, Higher Education and Technological 

Development of the Republic of Croatia 

CIHR (MOP-42438) 

Title: Chronic fluoxetine treatment has significant effect on the density of serotonin transporters 

in FSL and FRL rats, with a greater effect in the FSL rats 

Authors: T. KOVACEVIC 1,3 , I. SKELIN 2 , M. DIKSIC 2,3 ; 

2 Montreal Neurolog. Inst., 1 McGill Univ., Montreal, QC, Canada; 3 Fac. of Med., Univ. J. J. 

Strossmayer, Osijek, Croatia 

Abstract: FSL (Flinders Sensitive Line) and FRL (Flinders Resistant Line) rats were bred from 

Sprague Dawley (SDR) rats to produce rats with increased (FSL) and decreased (FRL) 

sensitivity to a cholinergic drug, diisopropylfluorophosphate, an inhibitor of cholinesterase. FSL 

rats show reduced appetite and psychomotor function, sleep and immune abnormalities, while 

FRL rats are, in many behavioral aspects, similar to the SDR rats and are therefore usually used 

as control animals for studies of FSL rats. The objective of the present investigation was to 

assess the changes in serotonin transporter (SERT) density in a rat depression model (FSL rats) 

relative to changes in the FRL rats following chronic fluoxetine treatment. The rats received 

fluoxetine or saline, every day for 14 days (10 mg/kg, i. p.). On the 15 th day, the rats were 

decapitated, the brains were extracted, frozen in isopantane, and stored at -85°C. The rat brains 

were cut into 20 µm thick coronal sections in a cryostat. The slides were immersed in the pre-

incubation buffer (10 mM Na-Phosphate buffer, pH 7.4) and specific binding was assessed in 

mailers containing 20 pM 125 I-RTI-55 ([ 125 I]-(-)-2β-Carbomethoxy-3β-(4-iodophenyl)tropane] 

specific activity of 2200 Ci/mmol), and 200 nM GBR12935 (1-(2-Diphenylmethoxyethyl)-4-(3phenylpropyl)piperazine 

dihydrochloride) for 120 min. Non-specific binding was measured in 

the presence of 1 mM of paroxetine and 200 nM GBR12935. The brain slices were washed twice 

for 20 minutes in a cold buffer, and then quickly immersed in ice cold water, and left over night 

to air dry. The sections, along with I-125 standards, were exposed to the Fuji fluorescence plates 

for several hours and calibrated as nCi/mg-tissue. The plates were scanned and images were 

quantified by a calibration curve obtained from standards in a MCID imaging system. The 

binding was quantified in twenty five brain regions. Regional readings were transformed to 

bindings (fmol/mg) to assess any possible influence of fluoxetine treatment on the SERT 

densities in the FSL and FRL groups. 

Four groups ANOVA showed highly significant brain region*group interaction; F(72,480)=12.5; 

p

suffer from panic disorder. The CCK-4 challenge model has been used clinically to study panic 

disorder and has been proposed as a translational model to evaluate the antipanic potential of 

anxiolytic compounds such as novel metabotropic glutamate (2/3) receptor agonists and GABAergic 

antiepileptic drugs. Prevention of CCK-4-induced anxiogenic-like effects has been seen 

following pretreatment with CCKB antagonists, tricyclic antidepressants, selective serotonin 

reuptake inhibitors and benzodiazepines. Increases in plasma arginine-vasopressin (AVP) and 

oxytocin (OT) concentrations have also been seen following CCK-4 administration suggesting 

that AVP and OT may play a role in the HPA axis activity associated with CCK-4-induced 

anxiety responses. The aim of this study was to characterize CCK-4-induced anxiogenic-like 

effects in the rat elevated plus maze (EPM), a well-validated pre-clinical model for screening 

anxiolytic drugs, and to evaluate the antipanic/anxiolytic potential of a novel selective V1b 

receptor antagonist, A-913727.0, in this preclinical model. A-913727.0 exhibits nanomolar 

affinity to the rat V1b receptor (Ki= 8.9 nM) and subnanomolar affinity to the human V1b 

receptor (Ki= 0.31nM) expressed in CHO cells. A-913727.0 is greater than 100-fold selective 

versus the human vasopressin V1a, V2 and oxytocin receptors. Male Wistar rats were housed in 

a reversed-light cycle for at least two weeks before experimental testing, which was performed in 

dimmed light conditions. CCK-4 was administered 30 min prior to testing and in those 

experiments with combined treatment, A-913727.0 (10 mg/kg) or diazepam were injected 45 min 

prior to testing. Consistent with both clinical and preclinical data, administration of CCK-4 (100 

κg/kg, s.c.) induced significant anxiogenic-like effects in the model. These effects were 

attenuated by pretreatment with the benzodiazepine, diazepam (2 mg/kg, i.p.). A-913727.0 alone 

did not induce anxiolytic-like effects in the EPM model; however, it was able to prevent the 

effects of CCK-4. The present results support the utility of CCK-4 induced anxiogenic-like 

effects as a translational model for compounds advancing to clinical phase of the drug 

development process as well as the anxiolytic/antipanic potential of selective V1b receptor 

antagonists. 

Disclosures: N.A. Bratcher, Abbott Laboratories, A. Employment (full or part-time); L.E. 

Rueter, Abbott Laboratories, A. Employment (full or part-time); W. Wernet, Abbott 

Laboratories, A. Employment (full or part-time); A.M. Basso, Abbott Laboratories, A. 

Employment (full or part-time). 

Poster 





Support: VA Merit Review funds

Title: Rapid modulation of TRH and TRH-like peptide levels in rat brain and peripheral tissues 

by prazosin 

Authors: *A. E. PEKARY 1,3 , A. SATTIN 2 , J. BLOOD 1 ; 

1 Res., 2 Psychiatry, VA Greater Los Angeles Hlthcare Syst, Los Angeles, CA; 3 Med., UCLA, Los 

Angeles, CA 

Abstract: Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder 

(PTSD). Prazosin, a brain-active α1-adreoreceptor blocker originally used for the treatment of 

hypertension has recently been reported to alleviate trauma nightmares, sleep disturbance and 

improve global clinical status in war veterans with PTSD. In rats it reduces the acoustic startle 

response following an inescapable stressor. Compelling evidence of a role for thyrotropinreleasing 

hormone (TRH, pGlu-His-Pro-NH2) in the pathophysiology and treatment of 

neuropsychiatric disorders such as major depression, and PTSD has recently been reported. 

Mutation of the TRH type I receptor in mice results in behavioral responses consistent with panic 

disorder and major depression (Zeng et al., 2007). To determine whether TRH or TRH-like 

peptides (pGlu-X-Pro-NH2, where “X” can be any amino acid residue) mediate the therapeutic 

effects of prazosin, young adult, male Lewis and Sprague-Dawley (SD) rats were injected ip with 

4 mg/kg prazosin and peptide levels in brain and peripheral tissues were measured 0, 2, 4 and 6 h 

later. Changes in peptide levels at 2 h are most like due to altered release and clearance since 

peptide synthesis and posttranslational processing take more time. Among the 11 brain regions: 

pyriform cortex (PYR), entorhinal cortex (ENT), cerebellum (CBL), nucleus accumbens (NA), 

frontal cortex (FCX), amygdala (AY), posterior cingulate (PCNG), striatum (STR), hippocampus 

(HC), medulla oblongata (MED) and anterior cingulate (ACNG) and 8 peptides measured there 

were 10 significant changes in SD and 6 in Lewis rats at 2 h when compared to the 

corresponding control peptide levels. For the 5 peripheral tissues (adrenals, testes, epididymides, 

pancreas and prostate), there were 13 significant changes in SD and 13 in Lewis rats. Responses 

for a given peptide and brain region were correlated between SD and Lewis rats. The number of 

significant within-brain correlations (given in parentheses) were: PYR (7), ENT (6), FCX (5), 

CBL (5), NA (3), AY (3), PCNG (2), STR (2), HC (1), MED (1), ACNG (0). The corresponding 

list for peripheral tissues was: adrenal (5), prostate (5), pancreas (3), testes (3) and epididymides 

(2). The extent of prazosin effects on TRH and TRH-like peptide levels in a given brain region or 

peripheral tissue correlates with the local levels of norepinephrine and α1-adrenoreceptors, 

suggesting that these peptides may play a role in the therapeutic effects of this brain-active drug. 

Disclosures: A.E. Pekary, VA Greater Los Angeles Healthcare System, A. Employment (full 

or part-time); VA Merit (AEP & AS), B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); A. Sattin, VA Merit, B. 


grants already received); J. Blood, None. 

Poster





Support: Sepracor, Inc. 

Title: Eszopiclone facilitation of the antidepressant efficacy of fluoxetine using a social defeat 

stress model in the mouse 

Authors: D. M. NOEL 1 , B. A. HUGHES 1 , B. SHEPPARD 1 , K. N. THOMPSON 2 , G. A. 

ORDWAY 3 , *R. W. BROWN 1 ; 

1 Psychol, East TN State Univ., Johnson City, TN; 2 Anat Cell Biol, 3 Pharmacol, East TN State 

Univ. Quillen Coll Med., Johnson City, TN 

Abstract: Eszopiclone facilitation of the antidepressant efficacy of fluoxetine using a social 

defeat stress model in the mouse. Daniel M. Noel 1 , Benjamin A. Hughes 1 , A. Brianna Sheppard 1 , 

Gregory A. Ordway 2 , Russell W. Brown 1 . 1 Department of Psychology, East Tennessee State 

University; 2 Department of Pharmacology, Quillen College of Medicine, East Tennessee State 

University, Johnson City, TN 37614 . This study was designed to analyze whether the sleep aid 

eszopiclone may facilitate the efficacy of fluoxetine on social defeat stress (SDS) in the mouse. 

Subjects were adult male „intruder‟ C57/B6 mice that that were exposed to a retired „resident‟ 

male breeder ICR mouse in this animal‟s home cage for a 5 min period for 10 consecutive days. 

During this interaction, the resident would establish physical dominance of the intruder. After 

confrontation, the intruder was housed in a cage that allowed sensory contact with the resident 

mouse but that would not allow physical interaction during the 10-day period. Once the 10 days 

of interaction were complete, all animals were assigned to one of four drug treatment groups, and 

treatment was given for up to 18 days: saline treatment, fluoxetine (10 mg/kg) only, eszopiclone 

only (3 mg/kg), or fluoxetine + eszopiclone. A social interaction test was given on days 1, 5, 10, 

and 15 of drug treatment. Sucrose preference was also tested during the interaction test. Results 

showed that eszopiclone facilitated the action of fluoxetine on the interaction test at days 1 and 5, 

and these two groups were equivalent on days 10 and 15. SDS produced a significant decrease in 

sucrose preference that was not affected by drug treatment. This study demonstrates that 

eszopiclone facilitates the action of fluoxetine in the SDS paradigm, paralleling clinical studies 

that showed facilatory effects of the drug combination in depressed patients. Future work will 

examine neurochemical mechanisms underlying the effects of this drug combination. 

Disclosures: D.M. Noel, None; R.W. Brown , None; B.A. Hughes, None; B. Sheppard, 

None; K.N. Thompson, None; G.A. Ordway, None.

Poster 





Support: NIDA-NIH 1R21 DA024099-01 

Title: The antibiotic minocycline increases phosphorylation and membrane insertion of the 

neuronal GluR1 glutamate/AMPA receptor subunit 

Authors: *M. IMBESI, T. UZ, N. DIMITRIJEVIC, R. MANEV, R. SHARMA, H. MANEV; 

Psychiatry, UIC, Chicago, IL 

Abstract: Increased phosphorylation of the glutamate receptor subunit GluR1 at Ser845 and 

Ser831 is associated with the increased membrane insertion of this AMPA-sensitive receptor. 

GluR1 phosphorylation plays a role in the antidepressant action of various antidepressants 

(phosphorylation is increased) and in the addictive components of cocaine. Recently, we 

demonstrated that 5-lipoxygenase (5-LOX) inhibitors increase GluR1 phosphorylation, that they 

may have antidepressant activity, and also may influence the behavioral effects of cocaine. 

Minocycline is a neuroprotective tetracycline antibiotic that also inhibits 5-LOX (Brain Res 

2006, 1085:57-67) and exerts antidepressant-like activity (Prog Neuropsychopharmacol Biol 

Psychiatry 2008, 32:380-6). We hypothesized that minocycline may share similarities with 5- 

LOX inhibitors with respect to GluR1 and behavior. In this study, we investigated the effects of 

minocycline on GluR1 phosphorylation and membrane insertion in primary cultures of mouse 

striatal neurons and in the prefrontal cortex of adult male mice treated with intraperitoneal 

minocycline injections. GluR1 phosphorylation was measured using GluR1 phospho-Ser845 and 

phospho-Ser831 antibodies (Brain Res 2007, 1147:148-53) and GluR1 membrane insertion was 

measured using the BS3 assay (J Neurosci 2007, 27:10621-35). In-vitro, low micromolar 

concentrations of minocycline increased GluR1 phosphorylation at Ser845 and Ser831 and 

increased the membrane content of GluR1. Also, in-vivo minocycline treatment increased GluR1 

phosphorylation. In a forced swim test, minocycline increased climbing behavior and decreased 

immobility; i.e., produced antidepressant-like behavior (Prog Neuropsychopharmacol Biol 

Psychiatry 2008). Interestingly, a single case report described the antidepressant effects of 

minocycline in humans (Am J Psychiatry, 1996, 153:582), suggesting that our findings might 

have clinical implications. Further studies should investigate whether the effects of minocycline 

on GluR1 are involved in the behavioral activity of this antibiotic (e.g., antidepressant and 

modification of cocaine's behavioral effects), and whether the effects of minocycline involve 5- 

LOX inhibition. 

Disclosures: M. Imbesi , None; T. Uz, None; N. Dimitrijevic, None; R. Manev, None; R. 

Sharma, None; H. Manev, None.

Poster 



Program#/Poster#: 255.20/Z1 


Title: Olfactory bulbectomy induces deficits of hippocampus-dependent learning and memory; 

Environmental enrichment reverses OBX-induced hyperactive behaviour 

Authors: *R. S. OOSTING, D. MEULENDIJKS, T. DOUMA, E. HENDRIKSEN, M. 

BREUER, K. WESTPHAL, B. OLIVIER; 

Dept Psychopharmacol, Univ. Utrecht, Utrecht, Netherlands 

Abstract: Depression, especially in elderly patients, is associated with poor cognitive 

functioning and cognitive decline (Dotson et al, Am J. Geriatr. Psychiatry 2008, 16: 318-30). 

Olfactory bulbectomized (OBX) rodents are widely used as a screening model for 

antidepressants. OBX leads to hyperactivity in the open field test, which is reversed by chronic 

but not by acute administration of antidepressants. To further validate the OBX rat as a 

depression model, we studied the cognitive deficits that result from OBX. OBX affected in 

particularly hippocampus-dependent learning and memory, as measured in different paradigms: 

T-maze spontaneous alternation, the novel place recognition task and the step-through passive 

avoidance behaviour. Hippocampus-independent learning, as determined in the novel object 

recognition task, was not affected by OBX. Kim and Fanselow (Science 256, 675 (1992), 

showed that the hippocampus becomes less important over time (> 2 weeks) for recall of 

contextual fear. OBX rats had almost entirely lost memories acquired during a passive avoidance 

acquisition trial given either 48 hours or 28 days before surgery. These findings together with the 

Kim and Fanselow‟s data suggest that the functional damage in the OBX rat extends beyond the 

hippocampus. We also studied the effects of environmental enrichment/voluntary exercise 

(EE/VE) on OBX rats. In rodents and probably also in humans, EE/VE has many positive effects 

on behaviour, brain function and structure. We started with the EE/EV procedure 6 weeks after 

OBX and continued for 5 weeks. EE/VE normalized OBX-induced hyperactivity in open field, 

while having no effect on the cognitive measures. 

The lack of effect of EE/VE on cognitive parameters, while normalizing hyperactivity, suggests 

different neuronal mechanisms and pathways underlying OBX-induced behavioral changes. Our 

results indicate that the OBX model has a much broader use in depression research. In addition 

to serving as a screening model for (serotonergic) antidepressants, the OBX model may be used 

for mechanistic studies in the area of neurocognitive decline.

Disclosures: R.S. Oosting , None; D. Meulendijks, None; T. Douma, None; E. Hendriksen, 

None; M. Breuer, None; K. Westphal, None; B. Olivier, None. 

Poster 





Support: University of Vermont 

Title: The 5-HT1A receptor antagonist WAY100635 dose-dependently decreases social 

interaction when infused into the bed nucleus of the stria terminalis (BNST) 

Authors: *K. M. RHODES, B. A. GRIMMIG, M. KOCHO-SCHELLENBERG, R. A. 

SUGARMAN, M. R. WILLIAMS, S. E. HAMMACK; 

Psychology, Univ. of Vermont, Burlington, VT 

Abstract: Substantial evidence suggests that serotonin (5-HT) activation within the brain 

modulates anxiety-like behavior. The bed nucleus of the stria terminalis (BNST) and 

serotonergic caudal dorsal raphe nucleus (DRN) have both been argued to mediate anxiety-like 

behavioral responding; hence, the activation of 5-HT neurons in the caudal DRN may modulate 

anxiety-like behavior via the release of 5-HT within the BNST. BNST neurons exhibit a complex 

response pattern to exogenous 5-HT, exhibiting both inhibitory and excitatory responses, which 

are mediated by different 5-HT receptor subtypes. Prior studies suggest that the 5-HT1,7 receptor 

agonist 5-carboxyamidotryptamine (5-CT) is anxiolytic, which is consistent with a reduction in 

BNST activity mediated by 5-HT1A postsynaptic receptor activation. However the anxiolytic 

effects of 5-CT could also be mediated by 5-HT7 receptor activation, due to high affinity of 5- 

CT to this receptor subtype. To determine the effects of 5-HT1A receptor activation on anxietylike 

behavior, we infused the 5-HT1A antagonist WAY-100635 (0, 0.04, 0.4 and 4 µg / 0.5 µl) 

into the BNST of rats immediately before social interaction testing. In order to observe a 

behavioral effect of the 5-HT1A antagonist against a background of elevated 5-HT, we 

administered two 5-sec 1-mA footshocks immediately after infusion, because this treatment has 

been shown to induce serotonin release in projection regions of the DRN. Rats were then tested 

for social interaction in a different room and testing apparatus. Two animals receiving the same 

drug treatment were placed into a testing box and social interaction was recorded for 10-min. 

Pairs were scored for the total amount of time interacting (i.e. sniffing, boxing, following, 

grooming, etc.) and locomotor activity (total number of line crosses). Social interaction was 

defined as the amount of time interacting per line cross. WAY-100635 dose dependently

decreased social interaction, indicative of increased anxiety. The anxiogenic effect of the WAY- 

100635 is consistent with earlier reports suggesting that BNST 5-HT1A activation is anxiolytic. 

These data further support that the activation of brain 5-HT systems may modulate anxiety-like 

behavior by altering activity within the BNST. 

Disclosures: K.M. Rhodes , None; B.A. Grimmig, None; M. Kocho-Schellenberg, None; R.A. 

Sugarman, None; M.R. Williams, None; S.E. Hammack, None. 

Poster 





Title: Different efficacy of mood stabilizer and antipsychotic drugs to prevent the hyperactivity 

induced by amphetamine/chlordiazepoxide mixture or amphetamine alone in mice 

Authors: A. IERACI, S. MELI, P. SALVATI, *E. IZZO; 

Discovery, NEWRON PHARMACEUTICALS SPA, Bresso MI, Italy 

Abstract: Bipolar disorder is a common, severe and chronic mood disorder characterized by 

alternate episodes of mania or hypomania and depression. Identifying novel drugs acting as 

mood stabilizers is complicated by the absence of suitable animal models. The hyperactivity 

induced by a mixture of amphetamine (Amph) and chlordiazepoxide (CDP) in rodents placed in 

an unfamiliar environment, has been proposed by some authors as a predictive model of mania 

since mood-stabilizer drugs prevent the hyperactivity induced by the mixture but not the 

hyperactivity induced by Amph alone. Other authors instead, reported that mood stabilizers can 

reduce the hyperactivity induced by Amph alone similarly to most of clinically effective 

antipsychotic drugs with no data so far being available on the effect of antipsychotics on 

hyperactivity induced by Amph/CDP mixture. 

With the aim of comparing the Amph/CDP versus the Amph-induced hyperactivity model, we 

evaluated the effects of mood stabilizers and antipsychotics with different mechanism of action 

on the two tests in mice. In particular, the mood stabilizers lithium chloride (1, 2 and 4meq/kg 

ip), lamotrigine (5, 10 and 20mg/kg po) and carbamazepine (10 and 20mg/kg po) were used, 

while the antipsychotics, haloperidol (0.05, 0.1 and 0.2mg/kg ip) and clozapine (0.25, 0.5 and 

1mg/kg ip) were tested. Locomotor activity was recorded using the automated acti-track system 

(Panlab). 

The results showed that all three mood stabilizers reduced more efficiently the hyperactivity 

induced by Amph/CDP mixture than induced by Amph alone. Conversely, the typical

antipsychotic haloperidol was stronger at preventing the hyperactivity induced by Amph alone 

compared to the one induced by the mixture. Interestingly, the atypical antipsychotic clozapine, 

at all tested doses had no significant effect on hyperactivity induced by Amph alone and only the 

highest dose of 1mg/kg prevented the hyperactivity induced by the mixture. For each compound 

all tested doses had no effect on basal locomotor activity. 

These results suggest that the hyperactivity induced by Amph/CDP mixture could be a useful and 

sensitive tool to discriminate the effect of a potential mood stabilizer versus a typical but not an 

atypical antipsychotic drug. 

Disclosures: A. Ieraci, None; S. Meli, None; P. Salvati, None; E. Izzo , None. 

Poster 

256. Anxiety Disorders: Human Biomarkers 



Topic: C.15.k. Anxiety disorders 

Support: Finnish L‟Oréal-UNESCO Women in Science fellowship 

Yrjö Jahnsson Foundation 

Academy of Finland NEURO program 

Biocentrum Helsinki Foundation 

Sigrid Juselius Foundation 

Title: ALAD, DYNLL2, EPB41L4A, and PSAP are associated to anxiety disorders in the 

Finnish population 

Authors: *I. HOVATTA 1,2,4 , J. DONNER 1 , S. PIRKOLA 4 , L. KANANEN 1 , K. SILANDER 5 , 

J. D. TERWILLIGER 3 , L. PELTONEN 2 , J. LÖNNQVIST 4 ; 

1 Mol. Neurol. Program, 2 Dept. of Med. Genet., 3 Fac. of Med., Univ. Helsinki, Helsinki, Finland; 

4 Dept. of Mental Hlth. and Alcohol Res., 5 Dept. of Mol. Med., Natl. Publ. Hlth. Inst., Helsinki, 

Finland 

Abstract: Background: Human anxiety disorders are complex diseases with largely unknown 

etiology. We have taken a cross-species approach to identify genes that regulate anxiety-like 

behavior using inbred mouse strains that differ in their innate anxiety levels as a model. We

previously identified 17 genes with expression levels that correlate with anxiety behavior across 

six inbred mouse strains in at least one of the seven studied brain regions. In the present study, 

we tested their 13 known human homologues as candidate genes for human anxiety disorders 

with a genetic association study. 

Methods: We used an anxiety disorder study sample derived from a Finnish population-based 

Health 2000 Survey. It consists of 321 patients and 653 carefully matched controls, all 

interviewed using CIDI to obtain DSM-IV diagnoses of panic disorder, generalized anxiety 

disorder, agoraphobia, social phobia, or phobia NOS. We genotyped altogether 208 SNPs (all 

non-synonymous SNPs, SNPs that alter potential microRNA binding sites, and gap-filling SNPs 

selected based on HapMap information) from the investigated anxiety candidate genes. 

Results: Specific alleles and haplotypes of six of the examined genes revealed some evidence for 

association (p ≤ 0.01). The most significant evidence for association to different anxiety disorder 

subtypes were: p = 0.0009 to ALAD (delta-aminolevulinate dehydratase) in social phobia, p = 

0.009 to DYNLL2 (dynein light chain 2) in generalized anxiety disorder, and p = 0.004 to PSAP 

(prosaposin) in panic disorder. In addition, we examined trends in pointwise association across 

individual genes and noticed that EPB41L4A was overrepresented among the top findings, with 

a clear trend towards more significant p-values (rank test p=0.0001). 

Conclusions: Our findings suggest that variants in these genes may predispose to specific human 

anxiety disorders, although replication in other cohorts is needed. These results illustrate the 

potential utility of cross-species approaches in identification of candidate genes for psychiatric 

disorders. 

Disclosures: I. Hovatta, None; J. Donner, None; S. Pirkola, None; L. Kananen, None; K. 

Silander, None; J.D. Terwilliger, None; L. Peltonen, None; J. Lönnqvist, None. 

Poster 






Title: Ventral Hippocampus and medial prefrontal cortex synchronize in theta range during 

anxiety 

Authors: *A. ADHIKARI 1 , R. HEN 2 , J. A. GORDON 2 ; 

2 Psychiatry, 1 Columbia Univ., New York, NY

Abstract: The dorsal hippocampus (dHpc) has been implicated in spatial learning, while lesion 

and anatomical studies support a role for the ventral hippocampus (vHpc) in innate, conflictbased 

tests of anxiety. We hypothesize that the vHpc influences anxiety by acting on 

downstream structures such as the medial prefrontal cortex (mPFC), an area known to modulate 

fear and anxiety-related behaviors. In agreement with this hypothesis we previously reported that 

during exposure to an anxiogenic open field, theta power (4-12 Hz) in the vHpc and mPFC 

(Adhikari et al., 2007, SfN abstract 269.5) was increased relative to a control environment. We 

now report that this effect on mpfc and vHpc theta can be observed in an additional anxiety 

paradigm, and that the increases in mPFC theta power correlate with behavioral measures of 

anxiety in both tasks. We recorded local field potentials (LFPs) from the vHpc, dHpc and mPFC, 

an area required for normal behavior in conflict-based anxiety paradigms that receives direct 

vHpc but not dHpc projections. Multisite recordings were made from 129/SvEvTac mice in a 

familiar environment and two anxiogenic tasks: a brightly lit open field and the elevated plus 

maze. In all environments the mPFC LFP is highly coherent with vHpc but not dHpc at both 

gamma (30-80 Hz) and theta ranges, presumably reflecting the direct connectivity between vHpc 

and mPFC. In the open field and in the plus maze increases in both mPFC and vHpc theta power 

were found. The increase in mPFC theta power correlated significantly with behavioral measures 

of anxiety such as time spent in the center of the open field and time in the open arms of the plus 

maze. Interestingly, power correlations at the theta range between the mPFC and vHpc but not 

dHpc increased in both anxiety paradigms. We also found a trend towards increased phaselocking 

of mPFC single unit firing to local theta in the open field, demonstrating that increases in 

mPFC theta power in anxiogenic environments affect single unit firing. These findings support 

the hypothesis that the hippocampus drives the mPFC specifically during states of increased 

anxiety. Further studies recording single units in the mPFC are underway, to confirm and extend 

the field potential findings reported here. 

Disclosures: A. Adhikari, None; R. Hen, None; J.A. Gordon, None. 

Poster 





Support: NIMH Grant T32-MH017140 

UCLA Depression Research Program

Title: Modulatory effects of comorbid anxiety on neuronal circuitry in depression using current 

cource coherence 

Authors: *H. FARAHBOD 1,2 , I. A. COOK 2 , D. SCHAIRER 2 , A. KORB 2 , A. F. LEUCHTER 2 ; 

1 NPI, 2 UCLA Lab. of Brain, Behavior, and Pharmacology, Semel Inst., UCLA, Los Angeles, CA 

Abstract: Major Depressive Disorder (MDD) with comorbid anxiety symptoms has a poorer 

treatment outcome than MDD without anxiety. This difference in outcome may reflect 

neurophysiologic differences between these two groups. We hypothesized that MDD subjects 

with higher levels of anxiety would have stronger neural connectivity between brain regions that 

connect the insula and amygdala. We used quantitative electroencephalographic (QEEG), current 

source coherence (CSC) to assess real-time connectivity between deeper brain regions. CSC 

expands the conventional surface EEG coherence measure by examining signals from deeper 

structures determined via LORETA transformation, and yields estimates of connectivity in terms 

of shared activity. Regions of interest (ROIs) included the dorsolateral prefrontal cortex 

(DLPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), medial-temporal cortex 

(MT), insula, and the amygdala (Amyg). 8 adults with MDD and different levels of anxiety, and 

8 healthy controls participated in this study. Severity of each condition was determined using the 

Hamilton Depression Rating Scale and the Hamilton Anxiety Scale. QEEG was recorded during 

performance of cognitive tasks while viewing images of faces with fearful expressions. Thetaband 

CSC was calculated for regions linking the amygdala, DLPFC, ACC, OFC, MT, and insula. 

We found significant connectivity differences in MDD patients relative to controls in ROIs, e.g., 

left Insula-Amyg circuit (t(13)=2.32, p

Systems, Eli Lilly & Company, Novartis Pharmaceuticals, and MEDACorp., F. 

Consultant/Advisory Board. 

Poster 





Support: HealthEmotions Research Institute 

MH046729 

Meriter Hospital 

MH069315 

Title: Individual differences in anxious temperament are predicted by 5-HTT binding in the bed 

nucleus of the stria terminalis 

Authors: *S. E. SHELTON 1 , A. S. FOX 2,3 , J. A. OLER 1 , T. R. OAKES 3 , B. T. CHRISTIAN 1,3 , 

R. J. DAVIDSON 1,2,3 , N. H. KALIN 1,2,3 ; 

1 Psychiatry, 2 Psychology, 3 Waisman Lab. for Brain Imaging and Behavior, Univ. of Wisconsin - 

Madison, Madison, WI 

Abstract: Pharmacotherapies targeting the pre-synaptic serotonin re-uptake transporter (5-HTT) 

are increasingly being utilized to treat affective disorders, including depression and anxiety, and 

differential availability of 5-HT may be related to the vulnerability for developing affective and 

anxiety disorders. Our previous research in rhesus monkeys demonstrated that anxious 

temperament, defined using a composite measure of threat-induced freezing behavior, 

separation-induced vocalizations and stress-induced cortisol levels, is positively correlated with 

glucose metabolism within several emotion- and stress-related regions (bed nucleus of the stria 

terminalis [BNST], amygdala and hippocampus). The relationship between 5-HTT binding 

potential (BP) and anxious temperament was examined in regions that demonstrated significant 

correlations between glucose metabolism and anxious temperament. We predicted that in these 

regions 5-HTT BP would account for the relationship between glucose metabolism and anxious 

temperament. To investigate the localized effects of 5-HTT brain systems on anxious 

temperament, dynamic high-resolution microPET images were acquired to measure 5-HTT BP 

using the tracer C11-DASB in adolescent rhesus monkeys. 5-HTT BP was positively correlated

with anxious temperament in the BNST and amygdala/hippocampal region. Using hierarchical 

linear regression techniques, we found that BNST 5-HTT BP significantly contributed to anxious 

temperament. The contributions of 5-HTT BP to temperament were both unique to 5-HTT BP 

and also overlapped with glucose metabolism. The BNST, a major component of the “extended 

amygdala”, has been shown to play an important role in anxiety-like behavior in rodents (Walker 

& Davis, 1997). The present results suggest the BNST is one site where 5-HT availability may 

mediate the neural, behavioral, and endocrine responses associated with anxious temperament in 

rhesus monkeys. 

Disclosures: S.E. Shelton, None; A.S. Fox, None; J.A. Oler, None; T.R. Oakes, None; B.T. 

Christian, None; R.J. Davidson, None; N.H. Kalin, None. 

Poster 





Support: Department of Veterans Affairs Medical Research Funds 

Title: Vagal tone and information processing in recently returned combat veterans 

Authors: *J. GINSBERG 1,2 , M. BERRY 1 , D. A. POWELL 1,3,4 ; 

1 Shirley L. Buchanan Neurosci. Lab., Dorn VA Med. Ctr., Columbia, SC; 2 Pharmacology, 

Physiology, and Neurosci., 3 Neuropsychiatry and Behavioral Sci., Univ. of South Carolina Sch. 

of Med., Columbia, SC; 4 Psychology, Univ. of South Carolina, Columbia, SC 

Abstract: Reduced heart rate variability (HRV) has been reported in anxiety disorders in 

humans, including combat-related PTSD. Reduction of HRV indicates loss of vagal tone 

(defined as parasympathetic cardiac output from the vagus nerve that results in cardiac 

deceleration) and increased sympathetic autonomic activity. 

Growing evidence also suggests that deficits in attention and immediate memory (ATTN/IM) are 

associated with PTSD in combat veterans. However, the unique contribution that reduced HRV 

makes to ATTN/IM deficits in PTSD+ combat veterans has not been well-studied. We tested this 

association by assessing the effect that improving heart rate variability coherence through 

biofeedback training has on objective performance measures of ATTN/IM in recently-returned 

PTSD+ combat veterans. 

Five PTSD+ combat veterans aged 25 - 39 and 4 non-veteran (VA professional employees) 

control subjects were enrolled in a pilot study. The controls received HRV coherence

iofeedback training, but did not have ATTN/IM variables measured. Baseline HRV was 

measured during a 10 minute period before the first coherence training session, with no visual 

feedback. Subjects then received weekly instruction on coherence biofeedback training over a 

four week period. Pre- and Post-Training ATTN/IM were assessed with the Conners 

Performance Test (CPT) of sustained attention, Digit Span (DS), and Rey Auditory-Verbal 

Learning Test (RAVLT). Artifacts were eliminated from the heart rate variability data before 

analysis. 

There was clear evidence from inspection of individual power spectrum density that the veterans 

were in coherence after training. Changes in the distribution of HRV power occurred across all 

frequency bands, with greatest change being increase in the low frequency (LF) band from 253.1 

to 833.6 ms 2 /Hz. The Post-training peak frequency of all subjects (veterans and controls) also 

shifted into the LF range (0.09-.014 Hz). 

Statistically significant improvements in ATTN/IM were indicated by: decrease in Commissions 

(False Alarms) on CPT; increase in DS Backwards and Scaled Scores; and increase in AVLT 

Total and Trial 1 scores. LF power significantly negatively correlated with False Alarms. 

These preliminary data provide evidence that improvements in ATTN/IM do co-occur with 

increases in HRV that result from HRV coherence training in recently returned, PTSD+ combat 

veterans. The 0.1 Hz peak power negative correlation with False Alarms on CPT was robust, but 

may be spurious given the extremely small sample. These results re-confirm the hypothesis that 

vagal tone indexes the ability to attend and appropriately respond to information in the 

environment. 

Disclosures: J. Ginsberg, None; M. Berry, None; D.A. powell, None. 

Poster 





Support: National Alliance for Research on Schizophrenia and Depression (NARSAD) 

Department of Veterans Affairs Medical Center, Minneapolis, Minnesota 

RO1 AG120852 

Minnesota Medical Foundation 

Title: PET study of script-driven fear imagery: Combat veterans with active vs. remitted PTSD

Authors: *S. E. FAHNHORST 1 , M. A. KUSKOWSKI 2 , J. T. LEE 3 , B. ENGDAHL 4 , R. 

EBERLY 4 , J. V. PARDO 3 ; 

1 Dept Cognitive Neuroimaging Ct, 2 GRECC, 3 Dept Cognitive Neuroimaging Ctr., 4 Psychology 

Services, Minneapolis VA Med. Ctr., Minneapolis, MN 

Abstract: Background: Limbic and medial prefrontal regions are implicated as sources of 

neural dysfunction in patients with PTSD. This study examines brain structures associated with 

active PTSD (aPTSD) and remitted PTSD (rPTSD). 

Methods: Male combat veterans, unmedicated and free of comorbidity, underwent [ 15 0]-H2O 

PET to examine changes in regional cerebral blood flow (rCBF) during script-driven imagery. 

Three autobiographic scripts (fear/neutral/sad) were presented to induce corresponding 

emotional states immediately before scanning. PET data were analyzed using SPM, regions of 

interest, as well as brain-behavior/seed-voxel partial least squares. 

Results: Consistent with previous findings, the aPTSD group in the fear minus neutral 

conditions significantly activated the dorsal anterior cingulate (dACC) and anterior insula while 

deactivating the rostral anterior cingulate (rACC). By comparison, the rPTSD group had no 

rACC or insular changes. This group activated the dorsomedial prefrontal gyrus and deactivated 

the gyrus rectus as well as the right amygdala. These changes were specific to fear as they did 

not arise in the sad minus neutral contrast. 

Conclusion: Remission from PTSD disengages a circuit involving affective and interroceptive 

systems. Key abnormalities in aPTSD are reversible. We report for the first time a deactivation 

of the right amygdala in rPTSD during script-driven fear-imagery. aPTSD subjects did not 

activate the amygdala. The differential patterns of between-group activation provide candidate 

structures participating in remission from PTSD, a component of resilience from trauma. 

Disclosures: S.E. Fahnhorst, None; M.A. Kuskowski, None; J.T. Lee, None; B. Engdahl, 

None; R. Eberly, None; J.V. Pardo, None. 

Poster 





Support: This research was supported by the U.S. Army RDECOM Acquisition Center, Natick 

Contracting Division, and U.S. Defense Advanced Research Projects Agency under Contract No. 

W911QY-07-C-0002 awarded to Michael J. Telch, Ph.D

Title: Frontolimbic white matter pathway differences associated with a serotonin transporter 

polymorphism (5-HTTLPR): Prospective analyses of soldiers deploying to Iraq 

Authors: *M. T. SATHISHKUMAR 1 , D. L. STOTE 2 , J. PACHECO 2 , R. E. FERRELL 3 , A. 

HARIRI 4 , D. M. SCHNYER 2 , M. J. TELCH 2 ; 

1 Imaging Res. Ctr., Univ. Texas Austin, Austin, TX; 2 Dept. of Psychology, Univ. of Texas, 

Austin, Austin, TX; 3 Dept. of Human Genet., 4 Dept. of Psychiatry, Univ. of Pittsburg, Pittsburg, 

PA 

Abstract: Previous studies show that the low-expressing(S) polymorphism of the serotonin 

transporter gene (5-HTTLPR) correlates with vulnerability to anxiety disorders and stressinduced 

depression. This polymorphism has been linked to heightened fear and anxiety, 

vulnerability to posttraumatic stress disorder, greater amygdala activation in response to fearful 

faces, diminished volume in anterior cingulate (ACC), and functional uncoupling between the 

ACC and amygdala (Lesch et al., 1996; Hariri et al 2002, 2005; Lee et al., 2005; Pezawas et al., 

2005). 

The goal of the current study is to examine the relationship between 5-HTTLPR status and 

frontolimbic fiber tracts (left and right uncinate fasciculus, UF). Prior to deploying to Iraq, 160 

United States Army soldiers underwent MRI and were genotyped for the 5-HTTLPR. A random 

subset of data (n = 54) was used in the current analysis. Diffusion tensor imaging data were 

collected in a GE 3T magnet using single shot echo planar imaging, and a twice-refocused spin 

echo pulse sequence, optimized to minimize eddy current (TR = 12000 ms, TE = 71.1 ms, FOV 

= 24, slice thickness = 3mm, 0 gap, B = 1000, 128 x128 matrix, 1 T2 + 25 DWI, 47 slices). 

Diffusion tensor and fractional anisotropy (FA) were calculated on a voxel by voxel basis using 

standard reconstruction methods in FSL. A probabilistically defined track of the left and right UF 

was subdivided into temporal and frontal sections. Data were sorted by genotype as either 

containing a low-expressing allele (S/S and S/L; n=40) or not (L/L; n=14). Mean FA values were 

computed for the UF from each section and hemisphere, and were submitted to statistical 

analysis. 

There was a significant effect of group, reflecting overall lower FA values for the individuals 

with at least one short allele relative to those with none. This effect interacted with location and 

hemisphere reflecting a reliable between-group difference in the left frontal UF pathway. FA 

values were also extracted from 2 left hemisphere control regions, the posterior forceps and the 

longitudinal fasciculus. These data are consistent with preliminary results in college-aged 

females (Pacheco, et al., 2008) indicating a correspondence between the presence of a short 

allele of the 5-HTTLPR promoter region and the white matter integrity of frontolimbic circuits, 

which have been shown to play a critical role in the regulation of fear, anxiety, and mood. 

Disclosures: M.T. Sathishkumar , None; J. Pacheco, None; R.E. Ferrell, None; A. Hariri, 

None; D.M. Schnyer, None; D.L. Stote, None; M.J. Telch, None. 

Poster





Support: Pfizer unrestricted educational grant 

Wyeth-Ayerst unrestricted educational grant 

Title: Salivary cortisol in males does not differ between PTSD and control subjects and fails to 

correlate with emotional arousal ratings for pleasant pictures 

Authors: *E. J. NEAFSEY 1 , A. SPAHIC-MIHAJLOVIC 2 , J. CRAYTON 3 ; 

1 Dept Cell Biol Neurobiol/Anat, Loyola Univ. Stritch Sch. Med., Maywood, IL; 2 Psychiatry, 

Univ. of Illinois Chicago, Chicago, IL; 3 Psychiatry, Hines Veterans Admin. Hosp., Hines, IL 

Abstract: Our previous work has demonstrated that the symptom of emotional numbing in 

PTSD appears to be selective, only affecting emotional arousal responses to positive IAPS 

pictures (Lang's Looking at Pictures test) in both male and female Bosnian refugee subjects (J 

Anx Disorders 19:383-402, 2005). In the male subjects in this study we also measured baseline 

(8AM-day1) salivary cortisol levels and the values (8AM-day2) after dexamethasone (Dex, 0.5 

mg) adminstration at 11PM of day1. No difference was seen in baseline (8AM-day1) salivary 

cortisol levels (nmol/l (SEM)) of Bosnian control (7.95 (2.14), n=12) and Bosnian PTSD (8.80 

(2.16), n=10) males, nor was any difference seen between groups in their post-Dex (8AM-day2) 

salivary cortisol levels (Control=6.77 (2.16), PTSD=4.55 (1.41)). Both groups did show a 

significant reduction from baseline (day1) levels; a two way ANOVA with diagnosis 

(Control/PTSD) and day (pre-Dex, post-Dex) factors only found a signficant effect of day on the 

log-transformed salivary cortisol values (F(1,20)=6.97, p=.016). Next a regression analysis was 

performed to see if day1 baseline or day2 post-Dex salivary cortisol values were related to 

each subject's mean arousal rating for pleasant pictures. None of the regression analyses were 

significant, indicating no relationship between cortisol levels and the arousal ratings for pleasant 

pictures. Although PTSD subjects have been proposed to have a hyperresponsive HPA axis by 

some studies, the present results do not support this concept. 

Disclosures: E.J. Neafsey , Unrestricted educational grants from Pfizer and Wyeth-Ayerst, C. 

Other Research Support (receipt of drugs, supplies, equipment or other in-kind support); A. 

Spahic-Mihajlovic, None; J. Crayton, None. 

Poster





Support: U.S. Army RDECOM Acquisition Center, Natick Contracting Division 

U.S. Defense Advanced Research Projects Agency #W911QY-07-C-0002 

Title: Prospective assessment of the neurological correlates of PTSD: Pre-deployment 

neuroimaging data from soldiers going to a combat zone in Iraq 

Authors: *D. L. STOTE 1 , A. R. HARIRI 3 , R. E. FERRELL 4 , Y. LI 2 , C. LANTRIP 2 , M. T. 

SATHISHKUMAR 2 , M. J. TELCH 1 ; 

1 Dept Psychol, 2 Imaging Res. Ctr., Univ. Texas, Austin, Austin, TX; 3 Dept Psychiatry, 4 Dept of 

Human Genet., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: Post-traumatic stress disorder (PTSD) is a common source of mental illness in the 

general population and is a major problem facing soldiers returning from combat duty. Data 

suggest that one in six soldiers returning from the current war in Iraq meets diagnostic criteria for 

PTSD. Prior research has examined the emotional brain mechanisms of PTSD after the 

emergence of the pathology, however such studies do not reveal why someone succumbs to 

PTSD. This study is unique in that we are collecting prospective data from soldiers prior to 

deploying to a combat zone (Iraq) from Fort Hood, TX. Indices include genetic screening, MRI 

scans, and psychological assessments prior to deployment. While in Iraq, soldiers will respond to 

an online questionnaire assessing details of their experiences and perceptions of the combat zone. 

Upon return from their tour of duty (approximately 15 months), the soldiers will undergo another 

series of MRI scans and interviews to determine the presence of PTSD or other emotional illness 

indicators. Using an interdisciplinary, multi-level approach we are currently collecting data for 

the realms of brain imaging, genetics, hormones, fear, memory, clinical pathology, family & life 

history, and the specifics of chronic stress or a traumatic event reported from in-theatre. The data 

presented here represent initial brain imaging analyses from pre-deployment (baseline) 

assessments and include volumetric and fMRI analyses of hippocampus and amygdala as a 

function of allele type (5-HTTLPR), and analyses of the relationship of between those variables 

and standard clinical indices of proneness for anxiety and mood disorders. 

Disclosures: D.L. Stote , None; A.R. Hariri, None; Y. Li, None; C. Lantrip, None; M.T. 

Sathishkumar, None; M.J. Telch, None; R.E. Ferrell, None. 

Poster





Support: Merit Review from the Department of Veterans Affairs 

Title: Low cerebrospinal fluid Neuropeptide Y (NPY) concentrations in posttraumatic stress 

disorder (PTSD) 

Authors: *R. SAH 1 , N. N. EKHATOR 2 , J. R. STRAWN 3 , F. R. SALLEE 3 , P. S. HORN 2 , T. D. 

GERACIOTI 2 ; 

1 Dept Psychiat, Univ. Cincinnati, Cincinnati, OH; 2 VA, Cincinnati, OH; 3 Psychiatry, Univ. of 

Cincinnati, Cincinnati, OH 

Abstract: BACKGROUND: Neuropeptide Y (NPY), a peptide transmitter that regulates stress 

and anxiety, has been proposed as a stress resilience factor in humans. Post-traumatic stress 

disorder (PTSD) is a stress-related anxiety disorder. We hypothesized that central nervous 

system NPY is dysregulated in PTSD. However, data on central NPY in PTSD are lacking. 

METHODS: We determined NPY concentrations in cerebrospinal fluid collected by lumbar 

puncture from ten male subjects with chronic combat-related PTSD and from thirteen normal 

healthy volunteers. NPY-like immunoreactivity was measured by EIA and compared between 

groups. RESULTS: As compared with the normal subjects, PTSD patients had significantly low 

concentrations of CSF neuropeptide Y [Mean NPY values 360.0 ± 17.7 controls; and 233.6 ± 

28.7 PTSD (p = .0008)]. NPY concentrations adjusted for age and BMI still revealed significant 

reduction in NPY in the PTSD group (p=0.003). CONCLUSIONS: Low central NPY 

concentrations may be a pathophysiological feature of PTSD and may be associated with anxiety 

and deficits in stress resilience. This novel observation, if replicated, suggests that the NPY 

system could be a target of experimental therapies for PTSD. 

Disclosures: R. Sah , None; N.N. Ekhator, None; J.R. Strawn, None; F.R. Sallee, None; P.S. 

Horn, None; T.D. Geracioti, None. 

Poster 



Program#/Poster#: 256.11/Z14


Title: Neuropeptide Y (NPY) and posttraumatic stress disorder (PTSD): studies in a rodent 

model of chronic variable stress 

Authors: *J. L. MCGUIRE 1 , J. P. HERMAN 2 , F. R. SALLEE 3 , R. SAH 3 ; 

1 Psychiatry, Univ. Cincinnati, Cincinnati, OH; 2 Neurosci., 3 Psychiatry, Univ. of Cincinnati, 

Cincinnati, OH 

Abstract: The development of PTSD has been associated with abnormalities in the major stress 

response systems of the body, the hypothalamic-pituitary-adrenal (HPA) axis, the CNS 

noradrenergic system, and the sympathetic nervous system. Pathological anxiety in PTSD is 

indicative of impairment in anxiety regulating mechanisms in the CNS. A major transmitter that 

is linked to the regulation of stress and anxiety is neuropeptide Y (NPY), which is increasingly 

suspected to be a potential “stress resilience factor”. Currently, pathophysiological relevance of 

NPY in PTSD is not known. The objective of this study was to a) develop a chronic variable 

stress (CVS)-recovery model that elicits “PTSD-like” behavior and b) investigate whether NPY 

expression is dysregulated in the CVS-recovery model. Adult male Long Evans rats were 

subjected to variable stressors for 7 days followed by 7 d recovery period. Animals were tested 

for PTSD-pertinent behavior (contextual fear conditioning, extinction and re-activation), and 

NPY mRNA and protein expression. Rats exposed to CVS showed significant potentiation of 

freezing response following fear-reactivation (p=.0247, t=2.540, df=13), as well as delayed 

impairment of fear extinction (p=.008, t=3.177, df=12). NPY expression was attenuated in the 

hippocampus after 16 hours (35.29% below controls, p=.0251, t-2.632, df=10) and amygdala 

after 7 days (38.4% below controls, p=.045, t=2.258, df=10). These are regions relevant to 

contextual fear-conditioning. Our studies indicate that persistent NPY dysregulation in limbic 

regions of CVS animals may contribute to enhanced fear responses upon re-exposure as well as 

impaired extinction. NPY may be relevant to the pathophysiology of PTSD. 

Disclosures: J.L. McGuire , None; J.P. Herman, None; F.R. Sallee, None; R. Sah, None. 

Poster 





Support: NIMH Division of Intramural Research Program

Title: Regulation of human serotonin transporter by microRNAs 

Authors: P. R. MOYA, J. R. WENDLAND, *D. L. MURPHY; 

Lab. Clin. Sci., NIMH, NIH, Bethesda, MD 

Abstract: Alterations in serotonin transporter (SERT) expression have been shown to have a 

role in psychiatric diseases including anxiety and depressive disorders, as well as autism and 

other developmental spectrum disorders. Most prior research has focused on polymorphisms in 

the promoter and coding regions of this gene, while investigations of 3‟-untranslated regulatory 

region (UTR) in its mRNA is scant. This information gap is critical since the recent discovery of 

the role of microRNA (miRNAs, a family of Pol II- transcribed small non-coding RNAs)in the 

regulation of gene expression is being increasingly recognized. Using bioinformatic tools, we 

found a cluster of predicted miRNA binding sites in the 3‟-UTR of the human SERT mRNA. 

Cloning of the 696 bp SERT 3‟UTR into a reporter luciferase vector markedly reduced its 

expression compared to empty control vector in RN46 and JAR cell lines. Transfecting synthetic 

miRNA precursors comprising four of the predicted miRNAs further decreased gene expression 

at different levels. No effect was found when negative pre-miRNAs controls were transfected. 

Via qPCR we detected medium abundance of 3 of the predicted miRNAs in those serotonergic 

cell lines. Transfection of the corresponding anti-miRNA rescued the expression of reporter 

vector to levels comparable to control. Similar results were obtained when cloning a small, 75 bp 

region spanning the predicted miRNA binding site; the same region cloned in opposite direction 

abolished the repression, thus validating the role of this region as a miRNA binding site. When 

evaluating the effect on endogenous SERT expression using plasmid-based overexpression, we 

found a microRNAs able to effectively downregulate SERT expression in cultures of 

serotonergic cell lines. We are currently developing lentiviral-mediated miRNA overexpression 

technique in raphe nuclei of mice to extend these investigations in vivo, to provide evidence of 

relevance to the many human disorders found to be associated with altered SERT function and 

expression. 

Disclosures: P.R. Moya, None; D.L. Murphy , None; J.R. Wendland, None. 

Poster 





Title: Diffusion tensor imaging of amygdala-hippocampus connectivity in human subjects with 

high and low trait anxiety

Authors: *K. C. KLEMENHAGEN 1 , T. K. YANAGIHARA 1 , R. HEN 2 , J. HIRSCH 3 ; 

1 Dept. of Neurosci., 2 Depts. of Neuroscience, Psychiatry, and Pharmacol., 3 Depts. of 

Neuroscience, Radiology, and Psychology; Program for Imaging & Cognitive Sci., Columbia 


Abstract: The role of the amygdala and the hippocampus in normal and pathological fear and 

anxiety has been a focus of recent research in humans using behavioral paradigms such as 

contextual and trace fear conditioning. While the amygdala has long been associated with the 

processing of anxiogenic stimuli, the role of the hippocampus in anxiety is poorly understood. 

Extensive anatomical connections between the two structures suggest that the hippocampus may 

contribute toward fear and anxiety behavior. In rodent studies, lesions of the ventral 

hippocampus (homologous to the anterior hippocampus in humans) affect emotional behavior 

related to anxiety and fear in tests such as the elevated plus maze and light/dark test. In contrast, 

lesions of the rodent dorsal hippocampus (human posterior homologue) primarily affect spatial 

learning in tests such as the Morris water maze. To investigate how anatomical connectivity 

between the amygdala and hippocampus may influence emotional behavior in human volunteers, 

we have applied a structural imaging technique, diffusion tensor imaging (DTI), to examine 

anatomical connections in healthy volunteers with differing scores on the trait portion of the 

Spielberger State Trait Anxiety Inventory (STAI-T). The trait anxiety score is a stable 

characteristic of an individual that describes their tendency to respond to threatening situations 

with anxiety (Spielberger, 1970). Diffusion weighted images were obtained on a 1.5T GE Signa 

scanner and anatomical connectivity was assessed using FMRIB's Diffusion Toolbox (FDT v2.0, 

FMRIB, Oxford, UK). Our results indicate that subjects with relatively high trait anxiety (STAI- 

T score >50) have a unique pattern of connections between the amygdala and hippocampus 

favoring stronger connectivity between the amygdala and anterior hippocampus, and weaker 

connectivity between the amygdala and posterior hippocampus. In subjects with low trait anxiety 

(STAI-T score

Topic: C.15.l. Other 

Title: Differences of white matter tracts in fronto-striato-thalamic circuit between patients with 

obsessive-compulsive disorder and normal adults: A diffusion spectrum imaging study 

Authors: Y.-C. LO 1 , C.-H. CHIU 2 , W.-Y. CHIANG 3 , F.-C. YEH 3 , F.-S. JAW 1 , *W.-Y. 

TSENG 4,1,3 ; 

1 Inst. of biomedical engineering, Natl. Taiwan Univ., Taipei, Taiwan; 2 Dept. of Psychiatry, 

Taipei City Hospital, Songde Campus, Taipei, Taiwan; 3 Ctr. for Optoelectronic Biomedicine, 

Natl. Taiwan Univ. Col. of Med., Taipei, Taiwan; 4 Natl. Taiwan Univ. Med., Taipei, Taiwan 


Obsessive-compulsive disorder (OCD) is a psychiatric anxiety disorder characterized by an 

individual's unwanted thoughts and/ or repetitive behaviors. According to past neuroanatomical 

research of OCD, regional abnormalities in the gray matter nodes of the implicated frontostriato-thalamic 

circuit have been reported, including orbitofrontal cortex, anterior cingulate 

cortex, caudate nucleus, basal ganglia, thalamus, and parietal cortex. There are three main tracts 

in the circuit, namely, cingulum bundles (CB), stria terminalis (ST), and anterior thalamic 

radiations (ATR); they form the primary connections among the frontal lobe, the limbic system, 

and the thalamus, integrating functions of emotion control, memory, anxiety, and decision 

making. To date, the integrity of these three white matter tracts in patients with OCD remains 

unclear. 

Methods 

20 right-handed adult patients with OCD (10 males and 10 females) and 20 age- and sexmatched 

healthy adults were examined using diffusion spectrum imaging (DSI). Images were 

acquired on a 3T MRI system with an eight-channel head coil. The DSI experiment was 

performed by applying 203 diffusion gradient vectors. DSI analysis was computed based on the 

Fourier relationship between diffusion echo signals and the probability density function (PDF). 

Tractography was based on a simple algorithm that was adapted for DSI data and using fiber 

tracking to define the CB, ST, and ATR. Furthermore, mean path analysis, a method that 

projected the anisotropy of PDF (generalized fractional anisotropy [GFA]) onto a single mean 

path of the specific white matter tract bundle, was used to analyze subtle changes in 

microstructure coherence along individual tract bundles. The asymmetric differences of CB, ST, 

and ATR were assessed statistically by calculating GFA values. 

Results 

In CB, reduced left-greater-than-right asymmetry of GFA was noted in the male OCD group. In 

ST, no specific asymmetry was found for both OCD and healthy group. In ATR, increased leftgreater-than-right 

asymmetry of GFA was found in the male OCD group. Compared with OCD 

patients, healthy controls consistently showed higher GFA values in all individual tracts. 

Conclusion 

Our results suggest that generalized decrease of GFA might reflect the neurobiological defects of 

OCD, and male OCD patients tend to reveal abnormal patterns of asymmetry in CB and ATR. 

Disclosures: Y. Lo, None; C. Chiu, None; W. Chiang, None; F. Yeh, None; F. Jaw, None; W. 

Tseng , None.

Poster 

257. Alcohol Intake and Preference 



Topic: C.16.a. Alcohol 

Support: MIUR 2006057754 

Banca d'Italia 2007 

Fondazione Banco di Sardegna 2006.0454 

Title: Reduction of ethanol-derived acetaldehyde induced motivational properties by L-cysteine 

Authors: *A. T. PEANA 1 , A. R. ASSARETTI 1 , G. MUGGIRONI 2 , M. MEREU 2 , D. SIRCA 2 , 

A. GOLOSIO 2 , P. ENRICO 3 , M. DIANA 2 ; 

1 Dept. di Scienze del Farmaco, 2 Drug Sci., 3 Dept. Biomed. Sci., Univ. of Sassari, Sassari, Italy 

Abstract: Acetaldehyde (ACD), the first metabolite of ethanol (EtOH), is produced peripherally 

by alcohol dehydrogenase (ADH) and centrally by catalase. We have recently suggested that 

ACD contributes to the positive motivational properties of EtOH ingested, as assessed by the 

place conditioning paradigm; indeed, we found that by reducing ACD production and/or by 

using ACD-sequestrating agents, EtOH is deprived from its motivational properties. Thiol 

products, such as the amino acid cysteine, are known to be effective ACD-sequestering agents. 

Indeed, cysteine is able to covalently bind ACD forming a stable, non toxic 2-methylthiazolidine-4-carboxylic 

acid compound. Thus, we treated rats intraperitoneally with saline or 

L-cysteine (10, 20 or 30 mg/kg), before intragastric administration of saline, EtOH (1 g/kg) or 

ACD (20 mg/kg). The specificity of L-cysteine effect was addressed using morphine-induced 

conditioned place preference (cpp) (2.5 mg/kg, i.p.). L-cysteine dose-dependently prevented both 

EtOH and ACD-induced cpp but did not interfere with morphine-induced cpp, suggesting that Lcysteine 

specifically modulates the motivational properties of EtOH-derived ACD. The present 

results further underscore the role of EtOH-derived ACD in EtOH-induced motivational 

properties. L-cysteine, by binding EtOH-derived ACD, would deprive it of its rewarding 

properties and reduce its abuse liability. EtOH self-administration experiments are currently in 

progress to further evaluate the role of L-cysteine as a potential treatment for alcohol abuse. 

Disclosures: A.T. Peana , None; A.R. Assaretti, None; G. Muggironi, None; M. Mereu, 

None; D. Sirca, None; A. Golosio, None; P. Enrico, None; M. Diana, None.

Poster 






EB2092 

Title: Influence of low dose oral ethanol on patterns of activity in cortico-mesolimbic system 

related to responding for water 

Authors: *D. J. WOODWARD, A. V. AZAROV; 

Neurosci., Neurosci Resch Inst. North Carolina, Winston-Salem, NC 

Abstract: The goal of this study was to describe activity patterns in cortico-mesolimbic neurons 

during operant water intake to satiation when influenced by low doses of volitional oral ETOH. 

Nucleus accumbens (NAC) and medial prefrontal cortex (mPFC) were implanted with arrays of 

microwires. Neuron activity was recorded at 1 msec intervals during events defined by nose 

pokes, tones and licks at a spout. Sprague Dawley rats water (W) deprived 24 hours responded 

with an FR1 nose poke for 0.1 cc water during 15-hour sessions overlapping the dark phase of 

normal lighting cycle. Food was provided ad lib in the light phase between the sessions and W - 

for 24-48 hours after the session. Upon completion of the initial 64 FR1 W responses of the 

session, ethanol was made available as a 15% v/v solution in tap water in a non-operant mode by 

simple licking from an alternative spout, in the doses of 0.3, 0.4, 0.5 and 0.6 g/kg. Rapid (≤10 

min/dose) ETOH intake was observed, which led in 10 to 30 min to interruption of W 

responding. This interruption lasted from about 30 min at 0.3 g/kg dose and up to 5 hours at 0.6 

g/kg. The estimated time to metabolize 0.6 g/kg ETOH in rat is about 1 hour. NAC and mPFC 

neurons sustained unique activity patterns linked to nosepoke and CS tone events across the 

initial 64 self-initiated FR1 water trials. These signals appeared in altered form within 30 min 

upon the completion of ETOH intake. Later in the session with water responding resumed, 

relative fading of those distinctly modified by ETOH neural response patterns was observed, but 

not a restoration of the initial coding. This change of pattern has possibly resulted either from 

developing satiation to water, fading of the pharmacological effects of the consumed ethanol, 

and/or session global timing. This sequence of pattern transition shows that altered neural coding 

of operant behavior by corticomesolimbic system (as in mPFC and NAC) is lasting over times 

longer than required to metabolize a measured low dose of volitional oral ETOH. (AA13199 and 

EB2092 to DJW)

Disclosures: D.J. Woodward , None; A.V. Azarov, None. 

Poster 





Support: NIAAA, R01AA014366 

State of California for medical research on alcohol and substance of abuse 

Title: GDNF: a potent regulator of alcohol-drinking and relapse 

Authors: *S. CARNICELLA, V. KHARAZIA, P. H. JANAK, D. RON; 

EGCRC-UCSF, Emeryville, CA 

Abstract: We previously demonstrated that the mechanism of action of the natural alkaloid, 

Ibogaine, to reduce alcohol (ethanol) consumption, is the upregulation of the glial derived 

neurotrophic factor (GDNF) expression in the ventral tegmental area (VTA) (1). Here we set out 

to characterize GDNF‟s actions on ethanol drinking behaviors. We found that GDNF infusion 

into the VTA dose-dependently reduced ethanol operant self-administration in rats. The 

reduction of ethanol self-administration was very rapid and long-lasting. Moreover, we found 

that intra-VTA infusion did not alter sucrose self-administration, and infusion into the 

neighboring substantia nigra did not affect operant responses for ethanol, suggesting that the 

action of GDNF is specific to ethanol and to the VTA. In addition, we showed that the GDNFmediated 

decrease in ethanol self-administration was also observed in rats with a history of high 

voluntary ethanol intake. Importantly, we demonstrated that GDNF infused into the VTA 

blocked rats‟ responses in a model of relapse. In line with these results, we found that reduction 

of endogenous levels of GDNF or of the GDNF receptor GFRα1 in heterozygote knockout mice 

resulted in an increase in ethanol, but not saccharin, consumption compared to wild-type 

littermates after a period of abstinence. Finally, we showed that intra-VTA infusion of GDNF 

rapidly activated the mitogen-activated protein kinase (MAPK) signaling pathway in the VTA, 

and that selective inhibition of the MAPK pathway in the VTA blocked the reduction of ethanol 

self-administration induced by GDNF. Taken together, these results suggest that GDNF regulates 

ethanol-drinking behaviors via the MAP kinase pathway and is a potent agent to prevent relapse. 

(1) He D-Y et al. (2005). J Neurosci, 25:619-628. 

Disclosures: S. Carnicella , None; V. Kharazia, None; P.H. Janak, None; D. Ron, None.

Poster 






Title: RNAi-mediated inhibitions of BDNF expression in the nucleus accumbens do not alter 

alcohol-drinking behaviors in the Wistar rat 

Authors: *Q.-S. YAN, S.-E. YAN; 

Dept Cancer Biol & Pharmacol, Univ. Illinois Col. Med., Peoria, IL 

Abstract: Previous studies showed that brain-derived neurotrophic factor (BDNF) protein levels 

in the nucleus accumbens (NACC) of alcohol-preferring (P) rats were significantly lower than 

those of the alcohol-nonpreferring (NP) rats, suggesting that this innate difference may 

contribute to the disparate alcohol intakes between the P and NP rats. This study was to assess 

further the role of BDNF in the NACC in the regulation of the alcohol-drinking behavior. To this 

end, a lentivirus, which was capable of delivering and expressing short hairpin RNA (shRNA) 

that targeted BDNF mRNA, was generated. This virus has been proved to be effective in 

silencing the BDNF gene both in vitro and in vivo. To determine whether RNA interference 

(RNAi)-mediated inhibitions of BDNF expression in the NACC alter alcohol-drinking behaviors, 

this virus (active shRNA), scrambled shRNA, or saline was bilaterally infused into the NACC of 

the Wistar rat, the progenitor line from which the P and NP rats were originally derived. The 

effects of the infusion on daily alcohol consumptions were assessed by a two-bottle free-choice 

method. BDNF expression at the protein level in the NACC after the treatments was measured 

via the enzyme-linked immunosorbent assay (ELISA). The result showed that no significant 

differences in total liquid or alcohol consumptions were observed after the infusion among the 

groups. However, administration of active shRNA did significantly decrease BDNF proteins in 

the NACC. This reduction was not observed in either the saline or scrambled shRNA group. The 

data suggest that BDNF signaling in the NACC may not be involved in the regulation of the 

alcohol-drinking behavior since reductions in BDNF proteins in this region did not cause 

significant changes in alcohol consumptions. Together with the results obtained from 

heterozygous BDNF (+/-) mice, the data may suggest that reductions of BDNF activity in other 

brain areas, but not the NACC, may be associated with the augmentation of alcohol intake 

observed in those mice. The data also suggest that the observed innate difference in NACC 

BDNF between the P and NP rats may not contribute to the disparate alcohol-drinking behavior 

in these lines of animals.

Disclosures: Q. Yan, None; S. Yan, None. 

Poster 





Support: William Evans Visiting Fellowship Fund of the University of Otago 

University of Delaware Funds 

Title: Neonatal one-day binge-like ethanol exposure results in the loss of neurons in the 

CA1/CA3 subfields of rat hippocampus 

Authors: C. E. SCHNELL 1 , R. M. A. NAPPER 2 , *A. Y. KLINTSOVA 1 ; 

1 Psychology, Univ. Delaware, Newark, DE; 2 Univ. of Otago, Dunedin, New Zealand 

Abstract: A single binge exposure to ethanol via intragastric intubation on postnatal day four 

(PD4) results in significant apoptotic cell death in the hippocampus of the rat 10 hours after 

ethanol delivery. This study reports the long-term effects of a binge-like ethanol exposure on 

PD4 on the number of neurons in CA1 and CA3 subfields of the hippocampus. On PD4, Long 

Evans male and female rat pups were divided between three treatment conditions: alcoholexposed 

(AE), pups received intubations of ethanol in milk substitute (4.5g/kg of ethanol in two 

feeds two hours apart); sham-intubated (SI), pups were intubated as for AE but no fluid was 

delivered; and suckle control (SC), pups remained with the dam. On PD50, animals were 

anesthetized and transcardially perfused with fixative. Using unbiased stereology (estimation of 

total cell number using optical fractionator on a systematic random set of every 10 th horizontal 

section through the entire hippocampus) the total neuron number in CA1 and CA3 hippocampal 

regions was estimated. There was a significant effect of postnatal treatment on the total number 

of CA1 neurons (F=8.48; p=0.003). Post-hoc comparison revealed that AE animals had lower 

number of neurons in the CA1 subfield of hippocampus than SC (p=0.002) and SI rats (p=0.05). 

Importantly, data also indicated a negative correlation between the peak blood alcohol 

concentration, measured on PD4 after the second AE intubation, and the total number of CA1 

neurons on PD50 (r = - .45). Preliminary data showed a trend toward a deficit in CA3 neurons as 

a result of postnatal ethanol exposure (F=2.95, p=0.088). This data demonstrates that even a 

single binge ethanol exposure during the brain growth spurt can result in permanent brain 

damage.

Disclosures: C.E. Schnell, None; R.M.A. Napper, None; A.Y. Klintsova, None. 

Poster 






Center for Development and Behavioral Neuroscience at Binghamton University 

Title: Binge-like alcohol intake is increased during early adolescence in C57BL/6J and DBA2/J 

mice 

Authors: *E. M. MOORE, J. N. MARIANI, L. C. BLACKMAN, D. N. LINSENBARDT, S. L. 

BOEHM, II; 

Psychology, Binghamton Univ., Binghamton, NY 

Abstract: Alcohol use is a common occurrence during the adolescent years, a time at which a 

number of crucial neurobiological, hormonal and behavioral changes occur. Binge-alcohol intake 

is a particular problem during adolescence, comprising approximately 56% of drinking among 

youth aged 12-17 years old in 2003. In order to more fully understand the complex interaction 

between alcohol use and these age-typical neurobiological changes, animal models must be 

utilized. Rodents experience a developmental period similar to that of adolescence. Although rat 

models have shown striking adolescent-specific differences in sensitivity to ethanol, little work 

has been done in mice despite the fact that the C57BL/6J (B6) and DBA2/J (D2) mice have been 

shown to markedly differ in ethanol preference drinking and exhibit widely different sensitivities 

to ethanol. The current study examined adolescent-specific voluntary binge-like alcohol drinking 

patterns in B6 and D2 mice using a limited access alcohol exposure paradigm called Drinking in 

the Dark (DID). We hypothesized that adolescent (P25-45) mice would exhibit greater binge-like 

alcohol intake compared to adults (P60-80), and that B6 mice would exhibit greater binge-like 

alcohol intake compared to D2 mice. Moreover, we predicted that relative difference in bingelike 

alcohol intake between adolescents and adults would be greater in D2 mice. Results showed 

that adolescents of both mouse strains consumed more alcohol than adults in this voluntary 

binge-like alcohol intake model. However, this effect was most pronounced during early 

adolescence (P25-32) with the relative difference between early adolescent and adult D2 mice 

being greater than that of early adolescent and adult B6 mice. Future work will determine 

whether binge-like alcohol exposure during adolescence in these strains increases alcohol intake

during adulthood. This work was supported in part by AA015434 and the Center for 

Development and Behavioral Neuroscience at Binghamton University. 

Disclosures: E.M. Moore , None; J.N. Mariani, None; L.C. Blackman, None; D.N. 

Linsenbardt, None; S.L. Boehm, None. 

Poster 





Support: USF Dept of Psychology 

Title: Adult female rats consume more ethanol than male rats following repeated binge ethanol 

exposure during adolescence 

Authors: K. K. ALIPOUR, A. M. MALDONADO, *C. L. KIRSTEIN; 

Psychol Dept PCD 4118G, Univ. South Florida Tampa, Tampa, FL 

Abstract: Recently, studies have found a significant increase in the rate of binge drinking among 

females, as there has been a dramatic change in the trend of alcohol abuse in men and women. 

These findings are important, as binge alcohol consumption is a rising concern in the United 

States, especially during adolescence. It is during this developmental period that binge alcohol 

consumption is usually initiated. Binge drinking models have been established in animals. In 

these models, animals are repeatedly administered high doses of ethanol (EtOH) over a period of 

three or four days. While such research has examined the effects of a single four-day and 

repeated three-day binge, little work has investigated the behavioral consequences of repeated 

four-day binges. The present experiment examined the effects of repeated binge EtOH 

administration during adolescence on voluntary EtOH intake in adulthood in male and female 

rats. Results indicate that repeated binge EtOH administration during adolescence, regardless of 

dose, enhanced voluntary EtOH intake in adulthood. Rats showed increased EtOH intake across 

days in adulthood. Females pretreated with EtOH consumed significantly more EtOH in 

adulthood relative to males. Together, these data indicate repeated binge EtOH exposure during 

adolescence has long-term consequences on voluntary EtOH consumption in adulthood, and 

females may be especially at risk to the long-term behavioral effects of early binge EtOH 

exposure. Given alcohol use is initiated during adolescence, and the trend of alcoholism among 

women has recently grown, the present experiment demonstrates the importance of elucidating 

the impact of early binge-pattern EtOH exposure on the subsequent predisposition to drink later

in life. Future studies should not only investigate the behavioral and neural mechanisms that 

mediate these sex differences, but also examine the biological mechanisms related to the longterm 

consequences of early EtOH administration on subsequently elevated patterns of EtOH 

intake in adulthood. 

Disclosures: K.K. Alipour, None; C.L. Kirstein , None; A.M. Maldonado, None. 

Poster 






AFOSR FA9550-06-1-0083 

Title: Innate and post-ethanol differences in protein expression levels in liver from selectively 

bred alcohol-preferring (P), -nonpreferring (NP) and Harlan outbred Wistar rats 

Authors: *R. L. BELL 1 , H. RINGHAM 2 , D. JI 3 , Z. RODD 1 , T.-K. LI 4 , F. A. WITZMANN 2 ; 

1 Inst. Psych Res., 2 Biochem. & Mol. Biol., Indiana Univ. Sch. Med., Indianapolis, IN; 3 Salk 

Inst., San Diego, CA; 4 Natl. Inst. on Alcohol Abuse and Alcoholism, Bethesda, MD 

Abstract: P rats develop ~15% ethanol (E) metabolic tolerance after free-choice E drinking 

suggesting changes in E metabolic, or associated, processes. The present study examined 

whether protein expression levels in the liver after acute (AE), or subchronic (CE), E would 

differ across P, NP and Harlan Wistar (HW) rat lines. P, NP and HW rats were intragastrically 

(i.g.) administered water (W) or E (18%, 4 g/kg) once a day for 5 consecutive days. The AE 

group received 4 days of W followed by E on the 5th day and the CE group received 5 days of E. 

A control group received W i.g. for 5 days. The animals were euthanized 2 hr after the 5th 

treatment, perfused with ice cold saline, the ventral-rostral liver was dissected, flash frozen, and 

maintained at -80 F until 2-DE analyses. Gels were stained with colloidal Coomassie Brilliant 

Blue, scanned, and the resulting images analyzed with Progenesis SameSpots software. 

Statistical significance (alpha was set at p=0.01) was determined using 2-way (dose by line) 

ANOVAs. Expression of 115 protein spots was significantly different across dose and/or line. 

Tryptic digests of 82 protein spots were analyzed by tandem mass spec., and 64 were identified 

with 99.9% confidence or better. Because 16 proteins differed amongst the 3 rat lines under the 

control condition, all AE and CE protein expression levels were computed as difference from

W/control values. After AE, 11 proteins had differential expression across the rat lines; whereas, 

after CE, 23 proteins had differential expression across the rat lines. Actb (actin), Aldh6a1 

(methylmalonate semialdehyde dehydrogenase), Atp5b (ATP synthase subunit beta), Gmpr2 

(guanosine monophosphate reductase 2), Rad23b and Sord (sorbitol dehydrogenase) were 

differentially expressed under both AE and CE conditions. P rats displayed both decreased 

levels, and the lowest levels of the 3 lines for all of these proteins except Aldh6a1. For Aldh6a1, 

P rats displayed increased levels and the highest levels of the 3 lines. GO analysis of 

differentially expressed proteins revealed innate rat line differences, notably in mitochondrial 

proteins of the HW compared with the P and NP lines, with the P and NP lines differing little in 

this regard. These data suggest line-specific responses to E exposure that may be related to 

unique innate phenotypes. 

Disclosures: R.L. Bell , None; H. Ringham, None; Z. Rodd, None; F.A. Witzmann, None; D. 

Ji, None; T. Li, None. 

Poster 





Support: NIH Grant R01 AA015741 

NIH Grant R24 AA015512 

Title: Enhanced orosensory-mediated responding to alcohol and sucrose in selectively bred 

alcohol-preferring (P) rats compared to alcohol-nonpreferring (NP) and Wistar rat lines 

Authors: *B. C. SILBAUGH, M. KETCHUM, S. M. BRASSER; 

Dept Psychol, San Diego State Univ., San Diego, CA 

Abstract: Alcohol is a salient, complex orosensory stimulus that directly activates the sweet 

taste system (Hellekant et al., 1997; Kiefer and Mahadevan, 1993; Lemon et al., 2004) as well as 

oral trigeminal circuits (Carstens et al., 1998) responsible for the detection and processing of 

irritant stimuli. Research in rodents and humans has supported a genetically based positive 

association between preference for alcohol and sweet substances, with enhanced consumption of 

sweet-tasting solutions being one of the strongest phenotypic predictors of alcohol intake 

common across alcohol-preferring rodent lines/strains (Overstreet et al., 1993; Sinclair et al., 

1992; Stewart et al., 1994; Woods et al., 2003). Although the mechanisms underlying inherited

co-variation in alcohol and sweet intake remain to be determined, recent findings indicate that 

sweet taste substrates are involved in alcohol intake and preference in C57BL/6J mice (Brasser et 

al., 2006; Blednov et al., 2008), suggesting that the processing of alcohol‟s sweet taste 

component may be influencing its ingestion. The present study compared self-initiated 

orosensory responding to alcohol and prototypic sweet, bitter, and oral trigeminal stimuli among 

P, NP, and non-selected Wistar rat lines to assess the relationship between genetically-influenced 

alcohol preference and orosensory sensitivity to these stimuli. Rats were tested for immediate 

short-term lick responses to alcohol (3, 5, 10, 15, 25, 40%), sucrose (0.01, 0.03, 0.06, 0.1, 0.3, 1 

M), quinine (0.01, 0.03, 0.1, 0.3, 1, 3 mM) and capsaicin (0.003, 0.01, 0.03, 0.1, 0.3, 1 mM) in a 

brief-access paradigm designed to index orosensory-mediated behavior while minimizing the 

influence of post-ingestive factors. P rats displayed elevated short-term lick responses to both 

alcohol and sucrose relative to NP and Wistar lines across a wide range of concentrations of each 

stimulus. There were no line differences in orosensory avoidance of capsaicin, and Wistar rats 

showed a moderately greater aversion to quinine compared to P and NP lines, which did not 

differ in quinine sensitivity. These data indicate that genetic selection for alcohol preference in P 

rats is associated with enhanced sensory responses to alcohol and sweet stimuli, consistent with 

prior evidence that differences in sweet taste processing may contribute to differences in alcohol 

intake. 

Disclosures: B.C. Silbaugh, None; M. Ketchum, None; S.M. Brasser, None. 

Poster 





Support: NIH-NIAAA R56AA012386 GDF 

Title: Do GABAARs containing alpha 2/3/5 subunits contribute to synaptic dysfunction after 

early postnatal binge-ethanol exposure in medial septum / diagonal band (MS/DB) neurons? 

Authors: *G. D. FRYE, H. WANG; 

Neurosci & Exptl. Therap, Texas A&M Univ. Syst Hlth. Sci. C, College Station, TX 

Abstract: „Binge-like‟ ethanol exposure on PD 4-9 exaggerates allopregnanolone and blunts 

zolpidem potentiation of GABAA receptor (GABAARs) mediated miniature postsynaptic 

current (mPSC) decay kinetics in MS/DB neurons in brain slices prepared on PD 11-16 (Wang 

and Frye SfN Abst, 34: 329.2, 2006; Wang et al., EB Abst, 1439:1127.12 2008). This treatment

also up-regulates alpha 4 subunit protein expression in septal tissues and alters GABA mPSC 

decay kinetics reactivity to alpha 4 selective positive or negative allosteric modulators suggesting 

that alpha 4 subunit containing GABAARs are recruited in to postsynaptic receptor clusters by 

binge ethanol exposure during early neonatal brain development. In the present study, we 

investigated the possible contribution of alpha 2/3/5 containing GABAARs to mPSC kinetics 

before and after binge ethanol exposure on PD 4-9. L655,708 (10 microM), a negative allosteric 

modulator of alpha 5 subunit containing GABAARs, did not alter mPSC mean amplitude on PD 

4-9 or for either PD 11-16 group, but 100 microM L655,708 reduced mPSC peak current of the 

PD 11-16 ethanol treated group by 22%. GABA mPSC frequency was reduced by 35% for PD 4- 

9 cells by 10 microM L655,708, but increased by 23% in PD 11-16 Control and 61% in Ethanol 

neurons. At 100 microM, L655,708 further increased frequency in both Control (65%) and 

Ethanol (85%) groups. L655,708 (10 microM) accelerated both phases of GABA mPSC decay 

kinetics in PD 4-9 neurons (fast tau 1: -37% and slow tau 2: -29%), however, PD 11-16 Control 

and Ethanol groups showed only a small acceleration of GABA mPSC decay times with 100 

microM L655,708. Finally, SB--205384 (0.3-100 microM), a selective positive allosteric 

modulator of alpha 2/3 containing GABAARs caused concentration dependent increases in 

mPSC amplitude for PD 11-16 Control and Ethanol neurons but a biphasic increase followed by 

inhibition of peak currents in PD 4-9 cells. Frequency of mPSCs was also increased but then 

decreased at higher concentrations for all groups. SB-205384 activated strong concentrationdependent 

increases in GABA mPSC decay time constants for PD 4-9 neurons which decreased 

with age in PD11-16 Control cells (100 microM = tau 1: 173% vs 139%; tau 2: 209% vs 168%). 

SB-205384 100 microM differentially accelerated fast tau 1: -20%) but prolonged slow tau 2: 

275%) decay in Ethanol cells. These results suggest GABAARs containing alpha 2/3/5 subunits 

may play a decreasing role in postsynaptic function as synapses mature in the early postnatal 

period. Furthermore binge-like ethanol exposure on PD 4-9 may distort developing GABAergic 

circuits by exaggerating the postsynaptic contribution of alpha 2/3 containing GABAARs. 

Disclosures: G.D. Frye , None; H. Wang, None. 

Poster 





Support: NIDA, NIAAA (Intramural Research Program, LNI) 

the U.S. Department of Energy under contract DEAC02-98CH10886

Title: Ethanol binge drinking increased nucleus accumbens dopamine levels in late adolescent 

rats: A microdialysis study 

Authors: *F.-C. YANG 1 , O. RICE 2 , G.-J. WANG 1 , N. D. VOLKOW 1,3 , P. K. THANOS 1,3,4 ; 

1 Med. Dept., Brookhaven Natl. Lab., Upton, NY; 2 Psychology, Furman Univ., Greenville, SC; 

3 Lab. of Neuroimaging, NIAAA, NIH, Bethesda, MD; 4 Dept. of Psychology, Stony Brook Univ., 

Stony Brook, NY 

Abstract: The mesolimbic dopaminergic (DA) system has been related to the reinforcing 

mechanisms to alcohol abuse. Studies using microdialysis have shown that acute systematic 

administration (ip) of ethanol induced a dose- and time-dependence surge in a wide range (15- 

215%) of DA in the NAc (Gonzales et al. 2004). Oral administration of ethanol, either with 

operant self administration or non-operant methods, increased DA in the NAc (17- 139%) in rats 

too (Weiss et al. 1993, Nurmi et al. 1998, De Montis et al. 2004). It's suggested that the 

mechanism was due to enhanced DA release but not inhibition of DA uptake (Yim et al. 2000). 

Chronic ethanol treatment during adolescence increased the extracellular DA level in the NAc of 

rats comparing with the controls (Badanich et al. 2007). Adolescent rats (PND 45) also showed a 

highest basal DA level and a lowest DOPAC/DA ratio than that of the preadolescent (PND 25, 

35) and young adult (PND 60). These results suggested that, in rats, adolescence is a critical 

period for the development of the mesolimbic DA system and thus, when exposed to ethanol, its 

vulnerability to ethanol was increased. The present study examined the effects of acute binge 

drinking during late adolescence, various doses of ethanol (control, 0.2g/kg, 0.7g/kg, 1.7g/kg) on 

DA levels within the NAc using in vivo microdialysis sampling and HPLC analysis. Recently we 

showed in a similar binge drinking paradigm using microPET in vivo imaging that there's a 

significant decrease in DA D2 availability following 0.7g/kg or 1.7g/kg ethanol (Thanos et al. 

2008). The present study will seek to measure the corresponding DA levels in these conditions. 

Male, 42-day rats were separated into 4 groups: water; near beer (EtOH 0.2g/kg), EtOH-high 

(1.7g/kg) and EtOH-low (0.7g/kg) dose. Rats were trained to drink daily limited (10 min) access 

of water/near beer in the test cages. Rats then received dialysis cannula into the NAc. During in 

vivo microdialysis, samples were collected at 20-min intervals. After baseline was taken, rats had 

access to one of the following: 20 ml of water, near beer, or EtOH/near beer solution for 10 min. 

Five additional samples were collected following treatment. DA was analyzed by HPLC. Rats 

received the same treatment the next day after microdialysis and decapitated 5 min or 20 min 

after 10-min of drinking. BAC of trunk blood sera were measured. Preliminary data indicated 

that starting from the second day of training, rats significantly consumed more near beer than 

water; this was also true when high or low dose of EtOH was added into the solution during 

microdialysis. HPLC results showed that DA levels in the NAc were mildly increased after low 

or high dose of EtOH oral administration. 

Disclosures: F. Yang, None; G. Wang, None; N.D. Volkow, None; P.K. Thanos, None; O. 

Rice, None. 

Poster





Support: NIAAA (AA015434) 

Integrative Neuroscience Initiative on Alcoholism - West 

Title: Synthetic cannabinoid antagonist SR141716A decreases binge-like alcohol intake in 

C57BL/6J mice 

Authors: *D. N. LINSENBARDT, C. WALSH, E. MOORE, S. BOEHM, II; 

Dept Psychol, Univ. Binghamton, Binghamton, NY 

Abstract: Research in rodents has suggested a role for the CB1 receptor system in modulating 

voluntary ethanol intake and preference. Whereas, cannabinoid agonists increase ethanol 

consummatory behaviors such as ethanol-maintained responding (Gallate et al., 1999), and 2 

bottle choice preference (Colombo et al., 2002), the cannabinoid antagonist SR141716A 

effectively blocks these effects. Interestingly, SR141716A has also been shown to decrease 

ethanol consummatory behaviors in various strains of mice (Arnone et al., 1997; Hungund et al., 

2002) and rats (Colombo et al., 1998; Freedland et al., 2001). Along these lines, previous 

research from our lab has shown that the cannabinoid agonist WIN 55-212,2 is capable of 

increasing binge-like intake using a modified version of the „Drinking In the Dark‟ (DID) model. 

The goal of the present work was to further examine the extent to which the endocannabinoid 

system is involved in binge-like alcohol intake through antagonism of the CB1 receptor system 

using SR141716A. C57BL/6J mice were allowed six days of once a day access to DID. On day 

7, mice were injected with one of three concentrations of SR141716A or vehicle and binge-like 

alcohol intake and corresponding blood alcohol concentrations were recorded. SR141716A dosedependently 

decreased alcohol consumption compared to vehicle injected controls. These 

findings provide further support for the growing literature implicating the endogenous 

cannabinoid system in ethanol preference and intake and suggest that this system may mediate 

binge-like consumption. 

Disclosures: D.N. Linsenbardt , None; C. Walsh, None; E. Moore, None; S. Boehm, None. 

Poster 

257. Alcohol Intake and Preference




Title: Acute effects of acamprosate and MPEP on ethanol drinking-in-the-dark in C57BL/6J 

mice 

Authors: *T. GUPTA 1 , A. REVIS 1 , S. MILLER 1 , Y. SYED 1 , M. MARTINEZ 1 , K. COHN 1 , M. 

DEMEYER 1 , K. PATEL 1 , J. S. RHODES 2 ; 

2 Psychology, 1 Univ. Illinois, Urban, Urbana, IL 

Abstract: Recently, a simple procedure in mice, Drinking-in-the-Dark (DID), was hypothesized 

to have value for medication development for human alcoholism. In DID, mice are offered 

intermittent, limited access to ethanol over a series of days during the dark phase that results in 

rapid drinking to intoxication in predisposed genotypes. We measured effects of acamprosate or 

MPEP, metabotropic glutamate 5 receptor (mGluR5) antagonist, on intake of 20% ethanol, plain 

tap water or 10% sugar water using the DID procedure in male C57BL/6J mice. Acamprosate 

(100, 200, 300, or 400 mg/kg) dose dependently decreased ethanol drinking with 300 mg/kg 

reducing ethanol intake by approximately 20% without affecting intake of plain water or 10% 

sugar water. MPEP (1, 3, 5, 10, 20, or 40 mg/kg) was more potent than acamprosate with 20 

mg/kg reducing ethanol intake by approximately 20% and for longer duration without affecting 

intake of plain water or 10% sugar water. These results support the hypothesis that mGluR5 

signaling is required for excessive ethanol intake in DID and suggest DID may have value for 

screening novel compounds that reduce overactive glutamate signaling for potential 

pharmaceutical treatment of excessive ethanol drinking behavior. 

Disclosures: T. Gupta , None; A. Revis, None; S. Miller, None; Y. Syed, None; M. Martinez, 

None; K. Cohn, None; M. Demeyer, None; K. Patel, None; J.S. Rhodes, None. 

Poster 





Support: Academy of Finland Grant 118325

Title: Extracellular levels of GABA and glutamate in the ventral pallidum of the alcohol 

preferring AA and alcohol avoiding ANA rats after administration of ethanol 

Authors: *K. KIIANMAA, N. RAIVIO, H. NURMI, H. KEMPPAINEN; 

Dept Mental Hlth. & Alcohol Res., Natl. Publ. Hlth. Inst., 00251 Helsinki, Finland 

Abstract: Ventral pallidum serves as a major terminus for accumbal efferent projections, which 

are GABAergic. These projections have been viewed as the final common link in the brain 

reward circuitry. The purpose of the present study was to clarify the role of the ventral pallidum 

in the regulation of ethanol consumption by studying the extracellular levels of GABA and 

glutamate in the ventral pallidum after acute administration of ethanol. Studies were performed 

with in vivo microdialysis in drug naive alcohol preferring AA (Alko Alcohol) and alcohol 

avoiding ANA (Alko Non-Alcohol) rats. Microdialysis probes were implanted in the ventral 

pallidum and artificial cerebrospinal fluid was pumped in awake and freely moving animals at a 

rate of 1,5 κl/min. Dialysate was collected at 20-minute intervals. Following a baseline period 

(120 minutes, 6 samples), the rats were injected with physiological saline or ethanol (1 or 2 g/kg, 

IP). The collection of the samples was continued for 240 min. The concentrations of GABA and 

glutamate were determined with a HPLC system using fluorescent detection. Ethanol 

significantly decreased the extracellular levels of GABA. The ANA rats were more sensitive to 

this effect of ethanol than AA rats. Ethanol didn‟t have any effect on the concentrations of 

glutamate in either rat line. The suppressive effect of ethanol on the extracellular levels of 

GABA in ventral pallidum suggest a role for pallidal GABAergic transmission in the control of 

ethanol consumption. 

Disclosures: K. Kiianmaa , None; N. Raivio, None; H. Nurmi, None; H. Kemppainen, None. 

Poster 





Support: SC-INBRE 

NIAAA/NIH Intramural Research Program 

Title: The effects of chronic ethanol intake on brain glucose utilization in mice lacking the 

cannabinoid 1 receptor

Authors: *O. RICE 1 , M. KRANTZ 2 , D. SCHONHAR 2 , Y. PIYIS 3 , M. MICHAELIDES 3 , N. 

VOLKOW 4 , P. THANOS 3 ; 

1 Psychology and Neurosci., 2 Furman Univ., Greenville, SC; 3 Brookhaven Natl. Lab., Upton, NY; 

4 Natl. Inst. on Drug Abuse, Bethesda, MD 

Abstract: Cannabinoids undoubtedly play a role in the reinforcing properties of abused drugs, 

including alcohol. We previously reported that cannabinoid knockout (CB1 KO) mice consumed 

and preferred significantly less ethanol (EtOH), in a two bottle choice drinking paradigm, than 

did wild-type (WT) or heterozygous (HT) mice. In addition, we demonstrated that CB1 KO mice 

did not develop a conditioned place preference (CPP) to EtOH, while the wild-type animals did 

develop a CPP. The aim of this study was to further characterize the brain‟s CB1 receptor‟s 

involvement in mediating, or modulating, chronic reinforcing effects of EtOH, by evaluating any 

metabolic differences in brain regions associated with reward or the CB1 receptor, using [ 3 H ]2deoxy-2-fluoro-D-glucose 

(2DG) and β-imager autoradiography (ARG). CB1 WT and KO mice 

(N=6/genotype) were randomly assigned to either a high (1.5 g/kg) or low (0.5 g/kg) dose 

ethanol consumption group. In order to simulate binge drinking all mice were trained to binge 

drink water using a limited access drinking paradigm (1 hr/day). After the animals were trained 

to binge drink for 1 hr/day, water was substituted for non alcoholic beer, which was eventually 

spiked with either a high or low concentration of ethanol. The animals then drank either 

concentration daily for 4 consecutive weeks, which constituted the chronic phase. The 2DG 

procedure was initiated on the last day after 4 weeks of daily EtOH binge drinks. On 2DG days 

mice were injected with 2DG via the lateral tail vein, immediately returned to their home cages 

and allowed to binge drink for one hour. The animals were then sacrificed, and their brains were 

harvested and sectioned for ARG. Blood was collected in order to measure blood glucose and 

ethanol concentrations. 

Disclosures: O. Rice , None; M. Krantz, None; D. Schonhar, None; Y. Piyis, None; M. 

Michaelides, None; N. Volkow, None; P. Thanos, None. 

Poster 





Support: NIAAA Grant AA12600 

Title: A novel compound, JR-220, reduces voluntary alcohol consumption in a limited-access 

"drinking in the dark" paradigm

Authors: B. LEWIS 1 , J. M. FAROOK 1 , K. A. WELLMANN 1 , P. A. CROOKS 2 , J. M. 

LITTLETON 2 , *S. BARRON 1 ; 

1 Dept Psychol, 2 Pharm., Univ. Kentucky, Lexington, KY 

Abstract: Recovering alcoholics often relapse into drinking behavior. We have been generating 

novel compounds to reduce the likelihood of relapse. The neurotransmitter glutamate, and more 

specifically the NMDA receptor (NMDAR) have been linked to this behavior. Agents that block 

NMDAR activity have been rejected as viable pharmacotherapies due to their inherent toxicity, 

however, drugs that can modulate NMDAR activity may have significant clinical value. 

Polyamines potentiate NMDAR activity by binding to modulatory sites on the receptor. Agents 

that block these polyamine binding sites have been shown to reduce excitotoxicity during ethanol 

withdrawal in vitro, using organotypic hippocampal slices. JR-220, a newly synthesized agent 

within a series of iminoguanidino compounds, inhibits the effects of polyamines on NMDA 

receptor [3H]MK801 binding. Previous studies with JR-220 have shown that this agent can 

reduce seizure susceptibility during ethanol (ETOH) withdrawal, reduce relapse behavior in an 

alcohol-deprivation effect paradigm, and reduce increases in ETOH consumption associated with 

chronic stress. The current study examined the effects of JR-220 using a “drinking in the dark” 

paradigm. This procedure, developed by Rhodes and colleagues, involves daily access to a 20% 

v/v ETOH solution during a 4 hr window early in the dark phase of the light/dark cycle. 

C57BL/6J mice were given daily access to 20% v/v ETOH for four weeks (5 days on, 2 days off) 

prior to the beginning of the experiment. Male mice (N=20) were administered JR-220 (20 

mg/kg, i.p.) or saline in a counterbalanced fashion. Injections were followed immediately by 4 h 

ETOH access. ETOH consumption was allowed to return to baseline between injection days. JR- 

220 dramatically reduced ETOH consumption relative to the saline controls. This finding 

demonstrates that JR-220, a putative polyamine binding site antagonist and NMDA receptor 

modulator, may reduce voluntary drinking and provides further support for JR-220 as a potential 

therapeutic agent for alcohol-related disorders. (Supported, in part, by AA12600 to JML). 

Disclosures: B. Lewis, None; K.A. Wellmann, None; S. Barron , None; J.M. Farook, 

None; J.M. Littleton, None; P.A. Crooks, None. 

Poster 





Support: Supported by use of facilities at the VA Puget Sound Health Care Sysem, Seattle, 

Washington.

P rats provided by the Indiana Alcohol Res Ctr, supported by NIH grant R24 

AA015512 

NIH Grants AA10567 and AA13881 

Title: Daily administration of melatonin reduces ethanol drinking in female ethanol-preferring 

(P) rats 

Authors: D. D. RASMUSSEN 1 , *T. H. WOLDEN-HANSON 2 , K. M. MCKELVEY 1 ; 

1 116-MIRECC, VA Puget Sound Hlth. Care Syst and Dept Psychiatry, Univ. Washington, 

Seattle, WA; 2 S-182-GRECC, VA Puget Sound Hlth. Care Syst., Seattle, WA 

Abstract: Chronic ethanol and withdrawal disrupts secretion of the hormone melatonin. 

Melatonin has been demonstrated to regulate circadian rhythm physiology, autonomic function, 

anxiety, forebrain endogenous opioid activity, and stress responses - i.e., functions which all are 

altered during and after chronic ethanol abuse and which all have been suggested to have roles in 

modulating ethanol intake and relapse. Accordingly, we tested the hypothesis that melatonin 

treatment will suppress ethanol drinking in rats selectively-bred for increased ethanol preference 

and intake (P line). Adult male and female P rats were provided food and three-bottle choice 

between 0, 15 and 30% (v/v) ethanol 24 hours/day, 7 days/week. After 7 weeks, Melatonin (1 

κg/ml) or vehicle Control (0.01% ethanol) was added to all three fluids, which were consumed 

primarily (84%) at night. Two weeks after initiation of Melatonin vs Control treatment, the 15 

and 30% ethanol solutions were removed for 2 weeks and then reinstated for 2 weeks, repeated 

for a total of 4 consecutive ethanol deprivation/reinstatement cycles. Pre-treatment baseline total 

(15% + 30%) ethanol intake was 6.2+/-0.3 and 5.2+/-0.3 g/kg BW/24 hours for the females and 

males, respectively. For females, ethanol intake decreased (p


Program#/Poster#: 257.18/AA1 



AA014874 

Title: Naltrexone does not attenuate ethanol stimulated dopamine release in the nucleus 

accumbens shell in ethanol naive rats 

Authors: *M. JOB, R. A. GONZALES; 

Pharmacol., Univ. Texas-Austin, Austin, TX 

Abstract: Naltrexone is approved for the management of alcoholism, and though it‟s mechanism 

of action has not been fully elucidated, it is thought to be due to the attenuation of ethanol 

evoked mesolimbic dopamine by blockade of the µ opioid receptors. The effect of naltrexone 

(ntx) on ethanol (etOH) stimulated dopamine (DA) release in the nucleus accumbens shell was 

studied in adult, male Long Evans rats using in vivo microdialysis in freely moving animals, in 

conjunction with HPLC for DA detection and GC for etOH detection. Rats weighing 280-407 g 

were implanted with microdialysis probes 15-20 hours before the experiment. On the next day, 

baseline samples were taken and afterwards the animals were intravenously (i.v.) pretreated with 

ntx (0, 0.3 or 1 mg/kg bolus) 20 min before i.v. infusion with etOH (10% w/v, 1 g/kg in a 

volume of 10 mL/kg delivered at a rate of 4 mL/min) or an equivalent volume of saline. 

Sampling continued for an hour. The administration of both doses of ntx caused a transient 

increase (first sample after infusion) in DA compared to baseline. The infusion of 1 g/kg etOH 

increased extracellular DA concentrations to approximately 130% of baseline in the saline 

pretreated group (n = 8). The two ntx pretreatment dose groups (n = 8 each) were not different 

from the control in both the increase in DA (% of basal) over time and area under the curve 

(AUC) (p > 0.05). The infusion of saline did not show an increase in DA for any of the 

pretreatment doses (n = 4 - 6). Ntx did not affect the peak concentration and clearance of ethanol 

in the brain. A positive control experiment was done to ensure that the ntx doses used were 

effective in blocking the µ opioid receptors. Rats were pretreated i.v. with ntx (0, 0.01, 0.03, 0.1, 

0.3 mg/kg), followed 20 min later by i.v. morphine (1 mg/kg). Morphine increased DA in the 

control group (n = 7) to about 150% of baseline within 5 min, followed by an escalating, 

prolonged increase for the next 25 min. Ntx dose dependently inhibited the effect of morphine on 

DA release over time and AUC (within 30 min after morphine infusion) (n = 5 - 7). Interestingly, 

ntx (0.3 mg/kg) (n = 7) was able to completely abolish the morphine evoked DA release, though 

in the previous experiment it didn‟t attenuate etOH stimulated DA release. The results suggest 

that the µ opioid receptors are not involved in the mechanism of etOH stimulated DA release in 

the nucleus accumbens shell in ethanol-naïve male rats. This may suggest that the mechanism of 

action of naltrexone in the management of alcoholism does not involve mesolimbic dopamine, 

although further research is required using alcohol dependent models.

Disclosures: M. Job, None; R.A. Gonzales, None. 

Poster 





Support: Academy of Finland grant 

Finnish Foundation for Alcohol Studies grant 

Title: Histamine H3 receptor confers alcohol intake in rodents 

Authors: *P. PANULA 1 , P. HYYTIÄ 2 , K. KARLSTEDT 1 , J. KASLIN 1 , K. KIIANMAA 2 , M. 

LINTUNEN 1,3 ; 

1 Neurosci. Ctr., Univ. of Helsinki, Helsinki, Finland; 2 Dept. of Mental Hlth. and Alcohol Res., 

Natl. Publ. Hlth. Inst., Helsinki, Finland; 3 Dept. of Biol., Abo Akademi Univ., Turku, Finland 

Abstract: Mechanisms of alcohol addiction and intake in animal models have been difficult to 

assess, due to obvious involvement of several different neuronal systems and transmitters. 

Previous studies have shown that brain histamine levels in alcohol-preferring AA rats are high 

compared to alcohol non-preferring ANA rats, and alcohol self-administration of AA rats is 

bidirectionally regulated by histamine H3 receptor ligands. Here we show using HPLC, that the 

production of histamine in the brains of alcohol-preferring AA rats is enhanced, and the 

expression of H3 receptor isoforms studied with quantitative in situ hybridization differs from 

that of an alcohol non-preferring ANA strain. Lowering the brain histamine in AA rats did not 

affect operant alcohol intake, suggesting that H3 receptor is more important than histamine 

through effects on other receptors for this behaviour. Mice lacking H3 receptor showed low 

alcohol preference ratios in a two-bottle choice test compared to control mice. No difference in 

alcohol preference ratio was observed between control animals and those lacking endogenous 

histamine (histidine decarboxylase KO animals). The results suggest that histamine H3 receptor 

is important in regulation of alcohol intake. 

Disclosures: P. Panula, Johnson & Johnson Pharmaceutical Research & Development, L.L.C. 

(San Diego, CA, U.S.A.) donated the H3R KO mice, C. Other Research Support (receipt of 

drugs, supplies, equipment or other in-kind support); P. Hyytiä, None; K. Karlstedt, None; J. 

Kaslin, None; K. Kiianmaa, None; M. Lintunen, None.

Poster 





Support: Samuel Johnson Foundation for Genomics of Drug Addiction Program at Mayo Clinic 

NIH, AA015164, to D.-S. C. 

Title: The neurotensin analog NT69L potentiates alcohol sensitivity and decreases alcohol 

consumption in mice 

Authors: *M. LEE 1 , J. Y. SONG 1 , C. CHOO 1 , H. JOHNG 1 , S. UNAL 2 , E. RICHELSON 3 , D.- 

S. CHOI 1 ; 

1 MPET, Mol Neurosci, 2 Psychiatry and Psychology, Mayo Clin. Col. Med., Rochester, MN; 

3 Psychiatry and Psychology, Mayo Clin. Col. Med., Jacksonville, FL 

Abstract: Neurotensin is known to regulate locomotor, hypothermia, nociception, and alcohol 

responses in the brain. In this study, we found that neurotensin analog NT69L, which can cross 

the blood-brain barrier, contributes to alcohol sensitivity and consumption. First, alcohol-induced 

ataxia response was significantly increased when mice were treated at a dose of 1 mg/kg of 

NT69L on the rotarod test. In the open field activity test, we found that NT69L-treated mice 

travel significantly less compared to the saline-treated mice in a dose-dependent pattern. These 

results demonstrate that NT69L synergistically increased sensitivity to ethanol-induced 

locomotor activity. At a hypnotic dose of ethanol (3.6 g/kg ethanol), NT69L promoted ethanolmediated 

hypothermic effect, but not in loss of righting reflex (LORR). To examine if the 

increased acute response to ethanol with treatment of NT69L are due to alcohol metabolism, we 

measured blood alcohol concentrations 1-3 hours after intraperitoneal (i.p.) injection of ethanol. 

NT69L-treated mice showed reduction in ethanol metabolism compared to those of saline-treated 

mice. Since the high level of alcohol sensitivity is known to inversely correlate with ethanol 

consumption, we hypothesized that NT69L might reduce alcohol consumption. Therefore, we 

evaluated oral ethanol self-administration using a two-bottle choice paradigm. We found that 

NT69L-treated mice significantly consumed less ethanol than the saline-treated mice. In 

conclusion, our results suggest that activation of neurotensin signaling can be a possible 

therapeutic target for alcoholism. 

Disclosures: M. Lee, None; J.Y. Song, None; C. Choo, None; H. Johng, None; S. Unal, 

None; E. Richelson, None; D. Choi, None.

Poster 

258. Molecular and Neurochemical Basis of Nicotine Addiction 



Topic: C.16.l. Addiction: Neurobiology 

Support: Philip Morris USA Inc. 

Title: Inhibition of anandamide hydrolysis blocks nicotine, but not morphine and cocaine, effects 

on mesolimbic dopamine neurons 

Authors: *A. MUNTONI 1 , G. PILLOLLA 2 , A. LUCHICCHI 2 , M. MELIS 2 , M. PISTIS 2 ; 

1 Inst. of Neurosci., CNR, Cagliari, Italy; 2 B.B. Brodie Dept of Neuroscience, Univ. of Cagliari, 

Cagliari, Italy 

Abstract: Increasing evidence suggests that the endogenous cannabinoid (endocannabinoid) 

system is involved in the general neurobiological substrates underlying drug addiction and in the 

mechanisms of action of different drugs, including nicotine, opiates and psychostimulants. For 

instance, it has been shown that pharmacological blockade or genetic ablation of cannabinoid 

receptors type-1 (CB1) decreases nicotine and opiates self-administration, while acute 

administration of cocaine increases the levels of the endocannabinoid anandamide in the 

striatum. All these addictive drugs are well-known to interact with the mesolimbic dopamine 

(DA) system, which arises from the ventral tegmental area (VTA) and is critical for the central 

processing of reward and motivation. Here we investigated whether the enhancement of 

endocannabinoid levels produced by the irreversible fatty acid amide hydrolase (FAAH) 

inhibitor URB597 would affect modulation of VTA DA neurons by nicotine, morphine and 

cocaine. To this aim, single unit extracellular recordings from VTA DA neurons were performed 

in chloral hydrate anaesthetized rats. Animals were pre-treated with URB597, or vehicle, and 

then the effects of nicotine, morphine or cocaine were studied on firing rate and discharge pattern 

of VTA DA cells. As expected, nicotine (0.2 mg/kg i.v.) stimulated the firing rate and the 

percent of burst firing of VTA DA neurons. On the other hand, in URB597 (0.1 mg/kg i.v., 1-2 

hours before recordings) pre-treated animals, we observed a complete blockade of the excitation 

induced by nicotine. Morphine also (4.0 mg/kg i.v.) increased the firing rate and the percent of 

burst firing of VTA DA neurons, but its effects were not antagonized by URB597 pre-treatment. 

Similarly, the inhibitory actions of cocaine (1.0 mg/kg, i.v.) on neuronal activity of VTA DA 

cells were not prevented by URB597. These findings suggest that URB597 selectively 

antagonizes nicotine-induced effects, being ineffective on morphine- and cocaine-evoked 

excitation or inhibition, respectively, of DA neuronal activity. Thus, our data point to a specific 

vulnerability of nicotinic acetylcholine receptors towards the negative modulation induced by

endocannabinoids. The molecular mechanisms underlying these selective actions are currently 

under investigation. 

Disclosures: A. Muntoni, None; G. Pillolla, None; A. Luchicchi, None; M. Melis, None; M. 

Pistis, None. 

Poster 




Topic: C.16.p. Neuroplasticity and addiction 

Support: FLADOH 05NIR-594 

Title: Differential effects of cannabinoid (CB) 1 receptor antagonist or bupropion in the mossy 

fibre reorganization following behavioral sensitization to nicotine in a rat model of noveltyseeking 

Authors: *A. BHATTI, C. ISGOR; 

Florida Atlantic Univ., BOCA RATON, FL 

Abstract: We have previously shown that a novelty-seeking phenotype in the outbred rat has 

predictive value in the expression of behavioral sensitization to nicotine. Locomotor reactivity to 

a novel environment is used to rank high (HR, locomotor scores ranking in the highest 1/3 rd of 

the population) versus low (LR, locomotor scores ranking at the lowest 1/3 rd of the population) 

responsiveness. HR rats are shown to express locomotor sensitization to a low dose nicotine 

challenge following a high dose of nicotine training and an associated increase in the mossy fibre 

terminal field (MF) size. Moreover, we previously showed that a cannabinoid (CB) 1 receptor 

antagonist administered during nicotine abstinence reverses the locomotor sensitization to 

nicotine challenge in the HR rats. In the present work we investigate effects of CB1 receptor 

antagonist and an FDA approved nicotine cessation agent, bupropion, on the mossy fibre 

plasticity following nicotine sensitization in the LRHR phenotype. LRHR rats underwent 

intermittent nicotine training with 4 injections at a medium dose (0.35 mg/kg s.c.) administered 

at 3-d intervals during the peripubertal-juvenile period (postnatal days 28-40). Following 

nicotine training, CB1 receptor antagonist AM251 (5 mg/kg; i.p.), bupropion (40 mg/kg; s.c.) or 

saline (1 ml/kg; s.c.) were administered on 1 st , 3rd and 5 th day of the 1 wk of abstinence. 

Expression of behavioral sensitization to nicotine is assessed in response to a low dose (0.1 

mg/kg s.c.) nicotine challenge. Five days after challenge, rats were sacrificed; hippocampi were 

sectioned and stained with Timm‟s silver sulfide method. MF size was estimated using

contemporary stereology. HR but not LR rats showed locomotor sensitization to the low dose 

nicotine challenge following nicotine training and abstinence. Opposite trends were observed in 

total MF size following nicotine training and challenge showing a decrease in LRs and an 

increase in HRs. Moreover, independent of pretraining condition, AM251 resulted in an increase 

in suprapyramidal (SP), intra-infrapyramidal (IIP) and total MFs in LRs. Conversely following 

bupropion treatment, a phenotype effect is observed in nicotine pretrained LRs and HRs; 

showing an increase in SP-MF size in LRs and a decrease in IIP-MF size in HRs. These results 

indicate that mossy fibres from LRs and HRs respond differently to nicotine and the therapeutic 

agents in their structural reorganization. Neurotransmitter mechanisms potentially underlying 

these phenotype differences induced by nicotine, CB1 receptor antagonist and bupropion are 

discussed. This work is supported by the Florida Department of Health 05NIR-594 granted to Dr. 

Isgor. 

Disclosures: A. Bhatti , None; C. Isgor, None. 

Poster 




Topic: C.16.k. Nicotine 

Support: NIH NIDA DA-005274 and DA-009789 

Title: Role of endocannabinoids in nicotine dependence 

Authors: P. PATEL 1 , L. MERRITT 1 , A. LICHTMAN 1 , B. R. MARTIN 1 , *M. DAMAJ 2 ; 

1 Pharmacol., 2 Dept Pharmacol & Toxicol, Virginia Commonwealth Univ., Richmond, VA 

Abstract: A growing body of evidence suggests that the endogenous cannabinoid system 

modulates the addictive properties of nicotine, the main component of tobacco that produces 

rewarding effects. In our study, complementary transgenic and pharmacological approaches were 

used to test the hypothesis that the endocannabinoid system modulates nicotine reward and 

dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in 

CB1 knockout (KO) mice and mice treated with the CB1 antagonist rimonabant. However, 

disruption of CB1 receptor signaling blocked nicotine reward, as assessed in the conditioned 

place preference (CPP) paradigm. Conversely, genetic deletion or pharmacological inhibition of 

fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the 

endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the 

expression of spontaneous nicotine withdrawal (14-days, 24 mg/kg/day nicotine) was unaffected

in CB1 KO mice, acute administration of rimonabant (3 mg/kg) ameliorated somatic withdrawal 

signs in wild type mice. Increasing endogenous levels of anandamide through genetic or 

pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine 

withdrawal in a milder withdrawal model (7-days, 24 mg/kg/day nicotine). Moreover, FAAHcompromised 

mice displayed increased conditioned place aversion in a mecamylamineprecipitated 

model of nicotine withdrawal. These findings indicate that endocannabinoids play a 

role in the rewarding properties of nicotine as well as nicotine dependence liability. Specifically, 

increasing endogenous cannabinoid levels magnifies, while disrupting CB1 receptor signaling 

attenuates nicotine reward and withdrawal. Collectively, these results support the hypothesis that 

cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence. 

Disclosures: P. Patel, None; A. Lichtman, None; B.R. Martin, None; M. Damaj , NIDA DA- 

005274 and DA-009789, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received); L. Merritt, None. 

Poster 





Support: NIH Grant DA-024932 

NIH Grant DA-023915 

Title: Orexin signaling in the insula regulates nicotine reinforcement 

Authors: *J. A. HOLLANDER, P. J. KENNY; 

Dept Mol. Therapeut., The Scripps Res. Inst., Jupiter, FL 

Abstract: Smokers with damage to the insula have less difficulty quitting the tobacco habit. 

Nevertheless, little is known about how this neural substrate modulates tobacco consumption. 

Orexin transmission is emerging as a critical regulator of drug seeking, but its role in the 

reinforcing effects of nicotine, considered the key addictive component of tobacco, is unclear. 

We found that the selective orexin-1 receptor antagonist SB-334867 (0.5-6 mg/kg IP) dosedependently 

attenuated the lowering effects of nicotine (0.25 mg/kg SC) on intracranial selfstimulation 

(ICSS) thresholds in rats, considered a measure of the stimulatory effects of nicotine 

on brain reward systems that that may motivate nicotine consumption. In addition, we found that 

SB-334867 (0.5-4 mg/kg IP) dose-dependently decreased responding for intravenous nicotine

infusions (0.03 mg/kg/infusion) but not for food rewards (45 mg) under fixed and progressive 

ratio schedules of reinforcement. These data suggest that orexin transmission plays a central role 

in regulating the stimulatory effects of nicotine on brain reward systems, and thereby controls the 

motivation to consume the drug. Finally, we found that infusion of SB-334867 (.04-5 microg) 

directly into the insula decreased intravenous nicotine self-administration (0.03 mg/kg/infusion) 

under a fixed ratio schedule. As such, these findings suggest that insular orexin transmission may 

represent a critical substrate necessary for maintaining tobacco dependence. 

Disclosures: J.A. Hollander , None; P.J. Kenny, None. 

Poster 






Title: Cyclic GMP-dependent protein kinase type II contributes to nicotine cue reactivity in mice 

Authors: *D. SCOTT 1 , M. LEE 2 , F. HOFMANN 3 , N. HIROI 1,2 ; 

1 Dominick P. Purpura Dept Neurosc, 2 Psychiatry and Behavioral Sci., Albert Einstein Coll. 

Med., Bronx, NY; 3 Inst. für Pharmakologie und Toxicologie, Technische Univ. München, 

Munich, Germany 

Abstract: Cues associated with smoking and withdrawal contribute to persistent smoking in 

smokers and relapses in ex-smokers. However, because the molecular mechanisms underlying 

cue reactivity are still poorly understood, effective treatment to extinguish the impact of cues is 

not available. We have previously observed that a single nicotine injection up-regulated cGMPdependent 

protein kinase (cGK) activity in the amygdala and caudate-putamen of adolescent 

mice, and in the nucleus accumbens and ventral tegmental area of adult mice. We have now 

examined the role of cGK type II (cGKII) in approach behavior toward cues associated with 

nicotine and in avoidance triggered by cues associated with precipitated withdrawal in the place 

conditioning paradigm. Congenic cGKII wild-type (WT), heterozygous (HT), and knockout 

(KO) mice were repeatedly tested for expression of conditioned place preference (CPP) 

following a single pairing of nicotine (0.2 mg free base/kg, s.c.). WT mice showed potentiation 

of CPP during the first 4 nicotine-free test days and extinction thereafter, but KO mice exhibited 

complete extinction by the second test; HT mice showed an intermediate trend. We have now 

begun examining the role of cGKII in avoidance triggered by cues associated with withdrawal.

In nicotine-dependent C57BL/6J mice, a single pairing of the nicotinic acetylcholine receptor 

antagonist mecamylamine (2.5 mg free base/kg, s.c.) was sufficient to induce conditioned place 

aversion (CPA). Work is in progress to ascertain the role of cGKII in withdrawal-induced cue 

avoidance in congenic cGKII WT, HT, and KO mice. These results suggest that cGMPdependent 

protein kinase type II contributes to persistent nicotine cue reactivity. 

Disclosures: D. Scott , None; N. Hiroi, None; M. Lee, None; F. Hofmann, None. 

Poster 




Topic: C.16.p. Neuroplasticity and addiction 

Support: FLADOH 05NIR-594 

Title: Effects of HDAC2 silencing on the neurotrophic factor family and inflammatory cytokine 

gene expression in a rat model of novelty-seeking phenotype: implications for behavioral 

sensitization to nicotine 

Authors: *O. OZTAN, C. AYDIN, A. BHATTI, D. TOBIANSKY, C. ISGOR; 

Florida Atlantic Univ., Boca Raton, FL 

Abstract: In the presence of the mild stress of a novel environment, experimentally naïve rats 

exhibit varying degrees of exploratory activity. Some rats display high rates of locomotor 

reactivity to novelty (high responders; HR), and others display low rates (low responders; LR). 

We have shown that this model can differentiate behavioral sensitization to nicotine between 

LRs and HRs. HR rats show enhanced expression of locomotor sensitization to nicotine and 

increased hippocampal mossy fiber plasticity. Chromatin remodeling may be responsible for 

nicotine-induced structural changes. DNA that is tightly coiled in nucleosomes is inaccessible to 

DNA binding proteins. Histone acetylation is associated with DNA relaxation and gene 

expression, whereas histone deacetylation catalyzed by histone deacetylases (HDACs) leads to 

tighter DNA coiling and gene silencing. HDAC2 is also shown to be an important regulator of 

gene expression for proinflammatory cytokines such as tumor necrosis factor alpha (TNFa). In 

preliminary studies we showed that HDAC2 is downregulated in response to nicotine in the HR 

hippocampus. In this study, we silenced HDAC2 by siRNA technology. We microinfused two 

doses (20nM, 40nM) of HDAC2 siRNA or vehicle in vivo in the hippocampal hilus along with a 

transfection agent to assess downstream changes in gene transcription particularly in 

neurotrophic factor family. We used reverse transcriptase-polymerase chain reaction (RT-PCR)

to assess changes in gene transcript levels. We have seen successful HDAC2 inhibition between 

1-8hr following 20nM dose, and between 24-48hr following 40nM dose. Consequently, we 

found decreased BDNF transcript levels between 3-24hr that was normalized at 48hr following 

silencing with the 20nM dose of siRNA. Associated increase in TrkB receptor transcript levels 

were observed between 8-48hr. Interestingly we also found increased TNF-a transcript levels 

between 6-12hr following 20nM silencing. These findings suggest that HDAC2 can regulate 

gene transcription for neurotrophic factors and receptors such as BDNF and TrkB in the 

hippocampal hilus, which may be associated with mossy fibre sprouting in response to repeated 

nicotine in the HR phenotype. Other members of the neurotrophic factor family such as nerve 

and fibroblast growth factors are investigated in phenotype-specific link to chromatin remodeling 

in nicotine sensitization. Silencing of HDAC2 appear to be associated with increased 

inflammatory response, and implications of phenotype- and/or nicotine-specific regulation of 

inflammation are discussed. This work is supported by the Florida Department of Health 05NIR- 

594 to Dr. Isgor. 

Disclosures: O. Oztan , None; C. Aydin, None; A. Bhatti, None; D. Tobiansky, None; C. 

Isgor, None. 

Poster 





Support: NIH Grant DA 09407 

NIH Grant DA 00378 

Title: Nicotine-induced changes in globus pallidus met-enkephalin systems are mediated by 

nicotinic and dopamine d-2 receptor mechanisms 

Authors: *M. E. ALBURGES, P. S. FRANKEL, A. J. HOONAKKER, G. R. HANSON; 

Dept Pharmacol & Toxicol, Univ. Utah, Salt Lake City, UT 

Abstract: We previously reported that the dopamine (DA)-related neuropeptides, neurotensin 

and substance P, associated with limbic and extrapyramidal pathways, are affected by nicotine 

administration through activation of nicotinic and D-2 DA receptors. Because metenkephalin 

(Menk) systems are also closely related to both limbic and extrapyramidal DA projections, we 

determined if nicotine treatment alters this neuropeptide. We focused primarily on the expression

of Menk levels in the globus pallidus due to the feedback importance of the D-2 receptordominated 

Menk pathway in the extrapyramidal system. Male Sprague-Dawley rats received 

multiple administrations of (+/-) nicotine totaling 4.0 mg/kg/day (0.8 mg/kg, i.p.: 5 X 2h 

intervals) in the presence or absence of selective DA receptor antagonists (D-1, SCH23390; D-2, 

eticlopride) or a non-selective nicotinic receptor antagonist (mecamylamine; 3.0 mg/kg, s.c.) and 

were sacrificed 18 h after treatment. The nicotine injections significantly reduced pallidal Menk 

levels, an effect that was blocked by pretreatment with either the D-2 or nicotinic, but not the D- 

1, receptor antagonists. For comparison, in these same experiments the Menk levels in other 

regions were also determined. Nicotine treatment significantly lowered Menk in the nucleus 

accumbens shell and the substantia nigra, but not in the nucleus accumbens core or the ventral 

tegmental area. The Menk responses in the accumbens shell and the nigral brain regions were 

blocked by both D-1 and D-2 as well as the nicotinic receptor antagonists. These findings 

suggest that Menk systems associated with extrapyramidal and limbic brain regions are 

influenced by nicotinic activation through both D-1 and D-2 receptors. This suggests the 

existence of a functional interaction between nicotinic, dopaminergic and enkephalinergic 

systems in these brain regions and that opioid systems may contribute to some of the effects of 

tobacco use. 

Disclosures: M.E. Alburges , None; P.S. Frankel, None; A.J. Hoonakker, None; G.R. 

Hanson, None. 

Poster 





Support: NIH Grant U01 MH69062 

Propondis Foundation Fellowship to Styliani Vlachou 

Title: Both GABAB receptor activation and blockade exacerbated anhedonic aspects of nicotine 

withdrawal in rats 

Authors: *S. VLACHOU 1 , N. PATERSON 1 , S. GUERY 2 , W. FROESTL 2 , K. KAUPMANN 2 , 

A. MARKOU 1 ; 

1 Psychiatry, UCSD, Sch. of Med., La Jolla, CA; 2 Neurosci. Research, Novartis Inst. for 

BioMedical Research, Novartis Pharma AG, Basel, Switzerland

Abstract: Nicotine dependence is maintained by the aversive, depression-like effects of nicotine 

withdrawal and the rewarding effects of acute nicotine. GABAB receptor antagonists exhibit 

some anti-depressant-like effects in preclinical studies, while GABAB receptor agonists 

attenuate the rewarding effects of acute nicotine. Recent studies with GABAB receptor positive 

modulators show that these compounds represent potentially improved therapeutic compounds 

due to fewer side-effects than GABAB agonists. Thus, both GABAB receptor agonists and 

antagonists, as well as GABAB receptor positive modulators, may have efficacy as smoking 

cessation aids. The present study assessed the effects of the GABAB receptor agonist (3-amino- 

2[S]-hydroxypropyl)-methylphosphinic acid (CGP44532), the GABAB receptor antagonist [3- 

{1-(S)-[{3-cyclohexylmethyl)hydroxy phosphinyl}-2-(S) hydroxy propyl]amino}ethyl]benzoic 

acid (CGP56433A) and the GABAB receptor positive modulator N-[(1R,2R,4S)bicyclo[2.2.1]hept-2-yl]-2-methyl-5-[4-(trifluoromethyl)phenyl]-4-pyrimidinamine 

(BHF177) on 

the anhedonic effects of nicotine withdrawal in rats. Wistar rats were prepared with electrodes 

implanted in the posterior lateral hypothalamus. After the establishment of stable ICSS 

thresholds, rats were prepared with subcutaneous osmotic minipumps containing either nicotine 

(3.16 mg/kg/day, base) or saline. After 7 or 14 days of nicotine/saline infusion, the pumps were 

removed to precipitate nicotine/saline withdrawal. ICSS thresholds were assessed 6 hours after 

pump removal and at 24 hour intervals thereafter. Thirty hours after pump removal, either the 

GABAB receptor agonist CGP44532 (0, 0.25 or 0.5 mg/kg, salt) or the GABAB receptor 

antagonist CGP56433A (0, 5 or 10 mg/kg, salt) or the GABAB receptor positive modulator 

BHF177 (0, 7.5 or 15 mg/kg, salt) were administered subcutaneously, 30 minutes prior to ICSS 

testing. Both GABAB receptor activation (CGP44532 or BHF177) and blockade (CGP56433A) 

elevated ICSS thresholds associated with nicotine withdrawal in all groups; saline-exposed 

controls exhibited elevated ICSS thresholds also after the administration of these compounds 

although the magnitude of the effect was less than that seen in nicotine withdrawing subjects. 

This pattern of results showing that both the GABAB receptor activation and blockade 

exacerbated nicotine withdrawal is surprising and perhaps reflects differential efficacy of these 

compounds at presynaptic hetero- and autoreceptors versus postsynaptic GABAB receptors that 

may have contributed to these findings. 

Disclosures: S. Vlachou , None; N. Paterson, None; S. Guery, None; W. Froestl, None; K. 

Kaupmann, None; A. Markou, None. 

Poster 




Topic: C.16.k. Nicotine

Title: Behavioral evidence for baclofen as anti-relapsing medication in nicotine detoxification 

Authors: *L. FATTORE 1,2 , M. S. SPANO 2 , G. COSSU 2 , M. SCHERMA 2 , W. FRATTA 2 , P. 

FADDA 2 ; 

1 Dept Neuroscience, Univ. Cagliari, CNR Inst. of Neurosci., Monserrato (CA), Italy; 2 Dept. 

Neurosci., Dept. Neuroscience, Univ. of Cagliari, Cagliari, Italy 

Abstract: The gamma-aminobutyric acid (GABA)-B receptor agonist baclofen is known to 

reduce the intake of most drugs of abuse, to decrease the alcohol deprivation effect in 

genetically alcohol-preferring rat lines, and to prevent reinstatement of cocaine- and 

heroin-seeking behaviour in abstinent rats. This study was undertaken to investigate 

whether baclofen may be effective in reducing drug-induced relapse to nicotine. To this 

aim, we used the extinction/reinstatement model of nicotine-induced conditioned place 

preference (CPP) in mice who developed firm nicotine (0.3 mg/kg s.c.) CPP, and the 

extinction/reinstatement model of nicotine-seeking in abstinent rats previously trained to 

self-administer nicotine (0.03 mg/kg/inf) under a continuous (FR1) schedule of 

reinforcement and lever-pressing as operandum. Baclofen was tested on reinstatement of 

nicotine CPP and seeking behaviour at doses (0.612, 1.25 and 2.5 mg/kg) known to have an 

effect on animals’ behavioural performances without altering motor activity (Fattore et al. 

2001, 2002; Spano et al. 2007). Results showed that baclofen (0.612, 1.25 mg/kg) completely 

prevented reinstatement of extinguished nicotine CPP in mice, while higher doses of 

baclofen were able to significantly (p


Support: Academy of Finland 

Sigrid Jusélius Foundation 

Title: Effects of acute and chronic nicotine treatment on ventral tegmental area GABA - 

interaction with morphine 

Authors: *T. VIHAVAINEN, T. R. A. RELANDER, R. K. TUOMINEN, L. AHTEE, P. 

PIEPPONEN; 

Fac. of Pharm., Univ. Helsinki, Helsinki, Finland 

Abstract: We have shown earlier that chronic oral nicotine treatment enhances morphineinduced 

behaviors and dopamine turnover/metabolism and release in mouse brain. Using in vivo 

brain microdialysis of conscious mice, we have now studied the role of substantia nigra/ventral 

tegmental area (SN/VTA) GABAergic system in the effects of nicotine and in nicotine-morphine 

-interaction. We investigated whether the GABA output provoked by an inhibitor of GABA 

uptake, nipecotic acid, in SN/VTA is affected by acute nicotine, or by seven-week oral nicotine 

treatment. In addition, we studied whether a single administration of morphine differently affects 

GABA output in control mice and mice chronically treated with nicotine. The guide cannula for 

the microdialysis probe was aimed above SN/VTA (coordinates: A/P -3.1; L/M +0.8; D/V -3.8 

mm relative to bregma) of NMRI male mice under isoflurane anesthesia one week before the 

microdialysis experiments. The concentrations of GABA were measured with HPLC and a 

fluorescence detector with pre-column derivatization from the dialysates collected in 15 min 

intervals. After stable baselines were achieved, nipecotic acid (100 µM in the dialysis fluid) was 

perfused through the probes for 90 min. Drugs were administered 15 min after starting the 

nipecotic acid perfusion. Acute nicotine (0.3 mg/kg or 0.5 mg/kg i.p.) dose-dependently 

decreased extracellular GABA in SN/VTA. Also, at 24 h after cessation of chronic nicotine 

treatment the extracellular GABA concentration was decreased in the SN/VTA as compared with 

controls. The effects of nicotine on GABA might be due to desensitization of alpha4beta2nicotinic 

acetylcholine receptors by nicotine, which leads to weakening of acetylcholine control 

on GABA release. In control mice, a single dose of morphine (15 mg/kg s.c.) decreased 

extracellular GABA as expected, but in mice withdrawn from chronic nicotine treatment 

morphine surprisingly increased GABA levels. Thus, chronic nicotine modifies the GABAergic 

transmission in the VTA/SN which may reflect the changes observed in mesolimbic 

dopaminergic neurotransmission. Chronic nicotine also unmasks a stimulatory component of 

opioidergic control on GABA release. In conclusion, our results suggest that changes in VTA/SN 

GABAergic transmission by chronic nicotine treatment are involved in the cross-sensitization to 

the effects of morphine, as reflected by augmented locomotor activity and striatal dopaminergic 

transmission. 

Disclosures: T. Vihavainen, None; T.R.A. Relander, None; R.K. Tuominen, None; L. Ahtee, 

None; P. Piepponen, None.

Poster 





Support: NIH Grant DA-021801 

Title: Topographically patterned influence of acute nicotine on serotonin and GABA in the 

ascending raphe nuclei 

Authors: *R. E. SPERLING 1,2 , K. G. COMMONS 1,2 ; 

1 Anesthesia, Children's Hosp Boston, Boston, MA; 2 Harvard Med. Sch., Boston, MA 

Abstract: Studies have shown that chronic nicotine reduces anxiety while nicotine 

withdrawal is anxiogenic. The serotonergic system has been implicated in 

mediating these effects, specifically neurons from the dorsal raphe nucleus 

(DR) and median raphe (MR), which project to areas in the forebrain 

responsible for controlling anxiety state. Previous studies have reported 

that acute nicotine produces an oscillatory increase of serotonin (5-HT) and 

GABAergic neuronal activity within the DR. Moreover, 5-HT-1A receptors may 

play a role directing 5-HT cell activity during nicotine exposure. The aim 

of the present study was to establish the topography of 5-HT and GABA 

neurons in the DR and MR that are activated in response to acute nicotine 

administration as measured by Fos immunoreactivity. In addition, we 

examined the role of 5-HT-1A receptors in controlling Fos activation 

specifically in 5-HT cells. Adult male Sprague-Dawley rats received a 

single dose of nicotine (0.2-0.8 mg/kg) or saline. Tissue sections through 

the MR and DR were processed for dual immunolabeling of Fos and either 5-HT 

or GABA. Acute nicotine injections resulted in a moderate increase in Fos 

expression in 5-HT neurons that was most pronounced in the rostral-dorsal 

DR. Immunolabeling of Fos with GABA identified many dually labeled cells, 

predominantly in the rostral DR, as well as laterally. To determine if Fos 

expression in 5-HT cells was dampened by 5-HT-1A-dependent processes, we 

assayed the effect of the 5-HT-1A receptor antagonist (WAY-100635, 0.1 

mg/kg) in nicotine (0.4 mg/kg) and saline treated rats. Nicotine in 

combination with WAY-100635 produced a notable increase in Fos activation in 

5-HT cells in the dorsal DR. This effect peaked rostrally and, while 

remaining dorsal, extended through the caudal DR. The results of the

present study demonstrate that exposure to nicotine produces a topographic 

activation of Fos in 5-HT and GABA neurons, most notably at the rostral and 

dorsal pole of the DR. Moreover, nicotine activates 5-HT-1A dependent 

processes, which appear to temper activation of 5-HT cells in the same 

location and elsewhere within the DR. The results are consistent with the 

idea that both 5-HT and GABAergic neurotransmission are engaged in the DR 

during nicotine exposure and furthermore suggest topographic organization of 

these effects. These results give new insight into how nicotine modulates 

activity of the 5-HT nuclei that innervate forebrain areas responsible for 

managing anxiety states. Adaptations in the ascending raphe nuclei with 

chronic exposure to nicotine may contribute to the development of nicotine 

addiction. 

Disclosures: R.E. Sperling , None; K.G. Commons, None. 

Poster 





Support: NIH Grant U19DA17548 

NIH Grant T32DA007304 

Title: N,N‟-Dodecane-1,12-diyl-bis-3-picolinium dibromide inhibits nicotine-evoked 

norepinephrine release through an allosteric mechanism of action 

Authors: *A. M. SMITH, G. DHAWAN, Z. ZHANG, P. A. CROOKS, L. P. DWOSKIN; 

Pharmaceu Sci., UK Col. Pharm., Lexington, KY 

Abstract: Norepinephrine (NE) is believed to significantly contribute to the etiology of 

depression, an affective disorder that is often comorbid with nicotine dependence. As a result, 

nicotinic acetylcholine receptors (nAChRs) responsible for mediating nicotine-evoked NE 

release from presynaptic terminals in the hippocampus have become a potential target for drug 

design aimed at treating both nicotine addiction and depression. Previous research from our 

laboratory found that N,N’-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) potently 

inhibits (IC50 = 2 nM) nicotine-evoked striatal [ 3 H]dopamine (DA) release in vitro via an 

orthosteric mechanism. Furthermore, bPiDDB also inhibits nicotine-evoked [ 3 H]NE release from

the hippocampus in vitro (IC50 = 430 nM), revealing a 200-fold lower potency at nAChRs 

mediating this response as compared with its potency at nAChR subtypes mediating nicotineevoked 

[ 3 H]DA release. The current study sought to determine whether bPiDDB inhibits 

nicotine-evoked [ 3 H]NE release through an orthosteric or allosteric mechanism. Also, for the 

sake of comparison, the mechanism through which mecamylamine inhibits nicotine-evoked 

[ 3 H]NE release was determined. Hippocampal slices were superfused for 60 min with Krebs‟ 

buffer containing pargyline (a monoamine oxidase inhibitor), and then for 28 min in the absence 

or presence of either mecamylamine (100 nM - 10 µM) or bPiDDB (10 nM - 1 µM). Then, 

nicotine (1 - 300 µM) was added to the buffer and superfusion continued for an additional 28 

min. As expected, mecamylamine-induced inhibition was not overcome by increasing nicotine 

concentrations, a result consistent with an allosteric mechanism. bPiDDB also produced 

inhibition that could not be surmounted by increasing concentrations of nicotine, and Schild 

analysis revealed a linear regression with a slope that was significantly different from 1.0, 

suggesting allosteric inhibition. Thus, bPiDDB inhibits nicotine-evoked [ 3 H]NE release in an 

allosteric manner, whereas bPiDDB inhibition of nicotine-evoked [ 3 H]DA release is consistent 

with an orthosteric mechanism of action. These results support the previous suggestion that 

different subtypes of nAChRs mediate these responses, and that bPiDDB inhibits both nAChR 

subtypes via different mechanisms. Supported by: NIH Grant U19DA17548 & T32DA007304. 

Disclosures: A.M. Smith, None; G. Dhawan, None; Z. Zhang, None; P.A. Crooks, None; L.P. 

Dwoskin, None. 

Poster 





Support: MIUR 2005054943_004 

Glaxo research agreement G3166 

Title: Chronic nicotine treatment differentially affects the function of presynaptic NMDA 

receptors modulating dopamine release from rat prefrontal cortex and nucleus accumbens 

Authors: *M. MARCHI 1,2 , M. GRILLI 1 , A. PITTALUGA 1,2 , E. MERLO-PICH 3 ; 

1 Dept Exp Med., Pharmacol & Toxicol Sect, Genova 16148, Italy; 2 Ctr. of Excellence for 

Biomed. Research, Univ. of Genoa, Genova, Italy; 3 Medicines Res. Ctr., GlaxoSmithKline 

S.p.A, Verona, Italy

Abstract: It is widely accepted that several effects of nicotine are mediated by increased 

dopamine (DA) release in the terminal fields of the mesencephalic dopaminergic systems. 

However, there is also evidence that nicotine may influence several other neurotransmitters that 

can indirectly affect the DA systems. In particular presynaptic interaction between glutamatergic 

and cholinergic systems has been reported to play an important role in the mechanism of action 

of nicotine, possibly via modulation of DA. 

The objective of this study was to investigate the effects of chronic administration of (-)nicotine 

on the function of the N-methyl-D-aspartate (NMDA) presynaptic receptors modulating [3H]DA 

release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc) using rat superfused nerve 

endings. In the PFC synaptosomes, prelabelled with [3H]DA, NMDA in a concentrationdependent 

manner evoked [3H]DA release in rats chronically treated with vehicle (14 days; via 

osmotic mini-pumps) with an apparent affinity of 13.1 ± 2 κM. The NMDA evoked overflow of 

the [3H]DA in PFC nerve endings of rats treated with (-)nicotine (14 days; 0.125 mg/Kg/h) was 

significantly lower (-43%) than in vehicle treated rats. The apparent affinity was 9 ± 1.4 κM. 

Exposure of NAc synaptosomes of rats treated with vehicle to different concentration of NMDA 

produced an increase of [3H]DA overflow with an EC50 of. 14.5 ± 5.5 κM. This effect was 

significantly potentiated in synaptosomes from animals chronically treated with (-)nicotine 

suggesting an up-regulation of the NMDA presynaptic heteroreceptor. The EC50 was 10.5 ± 0.5 

κM. The K+-evoked exocytotic release of [3H]DA was not modified by the long term (-)nicotine 

administration. Both the up-and downregulation of the NMDA receptors disappeared after 14 

days withdrawal. The results show 

that chronic (-)nicotine differentially affects the function of presynaptic NMDA receptors which 

regulate [3H]DA release in the PFC and NAc of the rat. 

This work was supported by research contract with GlaxoSmithKline SpA and by Italian MIUR. 

Disclosures: M. Marchi , GlaxoSmithKline SpA, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); E. Merlo-Pich, 

None; M. Grilli, None; A. Pittaluga, None. 

Poster 






TRDRP 15FT-0073

NS47162 

Title: Chronic nicotine treatment alters dopamine release in ventral but not dorsal striatum in 

non-human primates 

Authors: M. QUIK 1 , N. PARAMESWARAN 1 , J. M. MCINTOSH 2 , *X. A. PEREZ 1 ; 

1 Basic Res., Parkinson's Inst., Sunnyvale, CA; 2 Dept. of Biol. and Psychiatry, Univ. of Utah, Salt 

Lake City, UT 

Abstract: Tobacco addiction and neurological disorders such as Parkinson‟s disease are linked 

to alterations in nicotinic acetylcholine receptor (nAChR) expression that lead to changes in 

dopamine neurotransmission. Recent rodent studies show that acute nicotine, through activation 

of nAChRs, influences the amount of dopamine released with varying stimulus frequencies, 

however the effects of chronic nicotine treatment are unknown. We used voltammetry to assess 

the role of nAChR subtypes on non-burst and burst-stimulated endogenous dopamine release 

from striatal slices derived from non-human primates. NAChRs mediated ~55% of dopamine 

release in control striatum, of which the greater majority was mediated by α3/α6β2* nAChRs. In 

ventral and dorsal striatum, α3/α6β2* nAChRs evoked 80% and 100%, respectively, of non-burst 

stimulated dopamine release, with α4β2* nAChRs mediating the remainder. By contrast, 

blockade of α3/α6β2* and α4β2* nAChRs led to a smaller decrease in burst stimulated dopamine 

release in ventral striatum and had no effect in dorsal striatum. These results indicate that the 

different nAChR subtypes have a differential effect on dopamine release that depends on both 

striatal subregion and neuronal firing frequency. Next, we investigated the effects of chronic 

nicotine administered to monkeys via the drinking water for 8 months. Although chronic nicotine 

treatment did not affect total nAChR-mediated dopamine release from monkey striatal slices, it 

did modify α3/α6β2* and α4β2* nAChR-mediated release. In ventral striatum of nicotine-treated 

monkeys, nAChR blockade no longer decreased dopamine release with either non-burst or burst 

stimulation. By contrast, nicotine treatment did not modify release with either stimulation pattern 

in dorsal striatum. Altogether, these data supports a predominant role for α3/α6β2* nAChRs in 

the regulation of endogenous striatal dopamine release in control monkeys. They also show that 

chronic nicotine treatment modifies nAChR-mediated release, which may have implications for 

the development of therapies for addiction and neurological disorders with nAChR dysfunction. 

Disclosures: M. Quik, None; N. Parameswaran, None; J.M. McIntosh, None; X.A. Perez , 

None. 

Poster 



Program#/Poster#: 258.15/AA18


Title: Activation of the rat VTA-N. Accumbens dopaminergic system by nicotine occurs via 

interactions in the N. Accumbens rather than in the VTA 

Authors: M. VAN DER HART 1 , J. KLEIJN 3 , *H. ROLLEMA 4 , T. CREMERS 2 ; 

1 Brains On-Line, San Francisco, CA; 2 Brains On-Line, Groningen, Netherlands; 3 Univ. of 

Groningen, Groningen, Netherlands; 4 Neurosci., Pfizer Global Res. & Dev, Groton, CT 

Abstract: Background 

The interaction of nicotinic compounds with the Ventral Tegmental Area-Nucleus Accumbens 

(VTA-NAC) system plays a crucial role in nicotine addiction . The „addictive‟ effect is thought 

to occur in the shell of the NAC, while the interaction with nicotinic compounds takes place in 

the cell body region, the VTA. Removal of VTA β2 nicotinic acetylcholine receptors (nAChRs) 

in mice abolishes addictive behavior as well as dopaminergic responses in the NAC. Consecutive 

reconstitution of nAChRs in VTA restores baseline behavior and corresponding dopaminergic 

pharmacology (Maskos et al 2005) 

Mice vs rats 

Whereas interactions with nAChRs in mice predominantly occur in the VTA, the current study 

evaluates whether that is also true in rats. We used microdialysis in awake Sprague Dawley rats 

to measure the effects of nicotine (0.32 and 1 mg/kg s.c.) on DA, GABA and Glutamate release 

in VTA, NAC core and NAC shell. 

The effects of intravenous nicotine administration (0.02-0.3 mg/kg i.v.) on VTA cell firing was 

measured using single cell in vivo electrophysiology in anaesthetized Sprague Dawley rats. 

Results 

While DA levels in NAC increased approximately 2-fold following s.c. administration of 

nicotine, we did not observe a significant difference between the effects in the core and the shell 

region. In addition, only small DA elevations were observed in the VTA and only relatively high 

nicotine doses that produced pronounced DA increases in the NAC (≥0.1 mg/kg i.v.), elicited 

cell firing in rat VTA. This is in contrast to the robust increases in VTA firing that have been 

observed in mice at very low doses (Maskos et al 2005). 

Conclusions 

In rats, nicotine increases DA levels more potently in the NAC than in the VTA. It is 

hypothesized that in contrast to mice, nicotinic interactions with the DA-ergic system in rats 

occur in the NAC rather than in the VTA, . The consequences of this observation for human 

pharmacology is discussed. 

Maskos U, Molles BE, Pons S, Besson M, Guiard BP, Guilloux JP, Evrard A, Cazala P, Cormier 

A, Mameli-Engvall M, Dufour N, Cloëz-Tayarani I, Bemelmans AP, Mallet J, Gardier AM, 

David V, Faure P, Granon S, Changeux JP. Nicotine reinforcement and cognition restored by 

targeted expression of nicotinic receptors.Nature. 2005 Jul 7;436(7047):31-2. 

Disclosures: M. Van der Hart, Brains On-Line, A. Employment (full or part-time); T. 

Cremers, Brains On-Line, A. Employment (full or part-time); J. Kleijn, None; H. Rollema , 

pfizer, A. Employment (full or part-time).

Poster 





Support: European consortium BACS FP6-IST-027140 

Marie Curie Team of Excellence Fellowship BIND MEXT-CT- 2005-024831 

Title: Nicotine control of dopamine signaling in the Ventral Tegmental Area 

Authors: *M. GRAUPNER, B. GUTKIN; 

ENS, Collège de France, Group for Neural Theory, DEC, Paris, France 

Abstract: Midbrain dopaminergic (DA) neurons signal motivational properties of natural 

reinforcers and addictive drugs. Nicotine, like other drugs of abuse, boosts DA output from the 

ventral tegmental area (VTA). This increase results from direct stimulation of nicotinic 

acetylcholine receptors (nAChRs) expressed in the VTA. However, how the DA signal is 

constructed in the VTA and how nicotine subverts this signal remains controversial. In 

particular, in vivo and in vitro experimental paradigms reach contradictory conclusions about the 

key target of nicotine action: direct DA cell stimulation or indirect effects mediated through the 

GABAergic interneurons. We address these issues through computational modeling of the VTA 

circuitry and nAChR function. 

The VTA contains DAergic and GABAergic neurons receiving cholinergic (ACh) and 

glutamatergic (Glu) afferent inputs from subcortical and cortical structures. The DA response to 

endogenous acetylcholine and exogenous nicotine is mediated by various nAChR subtypes 

expressed on three different cell types: (i) on the DA neurons, (ii) on the GABAergic neurons, 

and (iii) on presynaptic Glu terminals. We implement this structure in a neuronal network which 

accounts for the local VTA connectivity as well as the location of the nAChRs. Based on known 

activation and desensitization properties of the nAChR subtypes, we investigate the VTA DA 

neuron responses to nicotine. 

We show that the apparent data mismatch between in vitro and in vivo recordings can be 

reconciled by differences in the afferent input activity. We find that the GABA cells are principle 

in causing nicotine dependent DA signals. We pin-point the specific contributions of various 

nAChRs to the DA signal. Moreover, we make predictions about how Glu and ACh input to the 

VTA is translated into DA neuron activity and how such processing is altered in the presence of 

nicotine. We furthermore investigate the specific contributions of various input pathways to the 

firing modes of the VTA DA neurons thought to be important for reward signaling: the regular

spiking and the bursts. These results can help understand how the VTA mediates the rewarding 

properties of nicotine and therefore may have clinical implications for nicotine replacement 

therapy. 

Disclosures: M. Graupner, None; B. Gutkin, None. 

Poster 





Support: IMHR Pilot Grant 

Philip Morris External Research Grant 

ABRC Research Grant 

Title: Cellular mechanisms of systemic nicotine-treatment-induced increase in the 

AMPA/NMDA receptor ratio in rat VTA dopaminergic neurons 

Authors: *J. WU 1 , M. GAO 2 , R. J. LUKAS 3 ; 

1 Dept Neurol, Barrow Neurolog Inst., Phoenix, AZ; 2 Neurol., 3 Neurobio., Barrow Neurolog. 

Inst., Phoenix, AZ 

Abstract: Tobacco use is estimated to be the largest single cause of premature death in Western 

society. Nicotine dependence likely involves actions at nicotinic acetylcholine receptors 

(nAChRs), which are widely distributed throughout the mammalian central nervous system and 

are expressed as diverse subtypes on cell bodies, dendrites and/or nerve terminals. Alterations in 

dopaminergic (DAergic) neuronal function in the ventral tegmental area (VTA) are thought to 

contribute to nicotine addiction. However, mechanisms of nicotine action and roles of specific 

nAChR subtypes in direct or indirect modulation of DAergic neuron function remain poorly 

understood. It has been reported that systemic exposure to several addictive drugs, including 

nicotine, increases the ratio of AMPA/NMDA receptor-mediated currents, which may serve as a 

common mechanism underlying drug addiction. In the present study, we investigated the 

possible mechanisms of systemic nicotine-treatment-induced increase in the AMPA/NMDA 

receptor ratio using patch-clamp recordings from rat midbrain slices. The results fall into four 

categories: (1) single (0.5 mg/kg, i.p.) or repetitive (0.5 mg/kg/d for 7 d, i.p.) injections of 

nicotine doubled the AMPA/NMDA receptor ratio compared to saline exposure; (2) block of

either α7- (by methyllycaconitine, MLA) or α4β2-nAChRs (by dihydro-β-erythroidine, DHβE) 5 

min before nicotine exposure failed to prevent nicotine-induced increase in the AMPA/NMDA 

receptor ratio, but pretreatment with MLA and DHβE together abolished nicotine-induced 

increase in the AMPA/NMDA receptor ratio; (3) in both wild-type and nAChR β2 subunit 

knockout mice, a single injection (0.5 mg/kg, i.p.) of nicotine doubled the AMPA/NMDA 

receptor ratio; and (4) a single injection of nicotine reduced both the ratio of paired-pulseinduced 

glutamatergic responses and maximal evoked inhibitory postsynaptic current. These 

results suggest that either α7- or α4β2-nAChRs participate in mediation of systemic nicotinetreatment-induced 

increase in the AMPA/NMDA receptor ratio via activation of α7-containing 

nAChRs on glutamatergic terminals and by desensitization/down-regulation of α4β2-nAChRs on 

GABAergic neurons. 

Disclosures: J. Wu , None; M. Gao, None; R.J. Lukas, None. 

Poster 





Support: Supported by an unrestricted grant from Philip Morris USA 

Title: Sazetidine-A selectively desensitizes alpha4beta2 nicotinic receptors and significantly 

reduces nicotine and cocaine self-administration in rats 

Authors: E. D. LEVIN 1 , D. HAMPTON 1 , S. SLADE 1 , M. CAULEY 1 , A. PETRO 1 , C. 

PERRAUT 1 , E. KHOLDEBARIN 1 , A. H. REZVANI 1 , Y. XIAO 2 , M. L. BROWN 3 , M. A. 

PAIGE 3 , B. E. MCDOWELL 3 , *K. J. KELLAR 2 ; 

1 Duke Univ. Med. Ctr., Durham, NC; 2 Dept Pharmacol, 3 Dept of Oncology, Georgetown Univ. 

Sch. Med., Washington, DC 

Abstract: Nicotine's effects on nicotinic cholinergic receptors (nAChRs) are crucial to the 

mechanisms underlying tobacco addiction, but one reason for less than optimal smoking 

cessation treatments is a poor understanding of these specific pharmacologic effects. In addition 

to its stimulatory actions on nAChRs, nicotine also markedly desensitizes these receptors. It is 

currently not known how much stimulation vs. desensitization each contribute to nicotine‟s 

varied behavioral effects. Sazetidine-A, a partial agonist at alpha4beta2 nAChRs, potently 

desensitizes these receptors. This study was conducted to determine if sazetidine-A would reduce 

nicotine self-administration (SA) in rats. After initial food pellet training and 10 sessions of

nicotine SA training (0.03 mg/kg/infusion, IV), Sprague-Dawley rats were administered 

sazetidine-A (1 or 3 mg/kg, SC) or the saline vehicle. Sazetidine-A at the 3 mg/kg dose 

significantly decreased nicotine SA relative to performance after saline injections. These results 

suggest that nAChR desensitization plays a critical role in blocking nicotine SA. In addition to 

reducing nicotine SA, sazetidine-A at a dose of 0.3 mg/kg significantly reduced cocaine SA. 

Sazetidine-A in this dose range had only modest effects on locomotor activity and no overall 

decrease in activity in the figure-8 maze. No impairment in average choice accuracy was seen in 

the repeated acquisition radial-arm maze test. This line of research is directed at developing a 

better understanding of the roles of nAChR desensitization in behavioral reinforcement, which, 

in turn, can lead to development of new nAChR desensitization-based treatments for tobacco and 

other addictions. 

Disclosures: E.D. Levin, None; D. Hampton, None; S. Slade, None; M. Cauley, None; A. 

Petro, None; C. Perraut, None; E. Kholdebarin, None; A.H. Rezvani, None; Y. Xiao, 

Georgetown University holds a patent on Sazetidine-A, E. Ownership Interest (stock, stock 

options, patent or other intellectual property); M.L. Brown, None; M.A. Paige, None; B.E. 

McDowell, None; K.J. Kellar , Georgetown University Holds a patent on Sazetidine-A, E. 

Ownership Interest (stock, stock options, patent or other intellectual property). 

Poster 





Support: CNR Italy 

Collège de France 

Institut Pasteur 

ARC 

ANR 

Title: Crucial role of α4 and α6 nicotinic acetylcholine receptor subunits from ventral tegmental 

area in systemic nicotine self-administration

Authors: S. PONS 1 , L. FATTORE 2 , G. COSSU 2 , S. TOLU 1 , E. PORCU 2 , M. MCINTOSH 3 , J.- 

P. CHANGEUX 1 , *U. MASKOS 1 , W. FRATTA 4 ; 

1 Neurosci. CNRS URA 2182, Inst. Pasteur, Paris, France; 2 CNR, Cagliari, Italy; 3 Univ. Utah, 

Salt Lake City, UT; 4 Dept Neurosci, Cagliari, Italy 

Abstract: The identification of the molecular mechanisms involved in nicotine addiction and its 

cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were 

developed to match this aim. While the β2 subunit of the neuronal nicotinic acetylcholine 

receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement 

properties of nicotine, little is known about the contribution of the different α subunit partners of 

β2 (i.e. α4 and α6), the homo-pentameric α7, and the brain areas other than the ventral tegmental 

area (VTA) involved in nicotine reinforcement. In this study, nicotine (0.025 to 0.15 mg/kg/inf) 

self-administration was investigated with drug-naïve mice deleted (KO) for the β2, α4, α6 and α7 

subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR 

subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT 

mice, β2-VEC mice with the β2 subunit re-expressed exclusively in the VTA, α4-VEC mice with 

selective α4 re-expression in the VTA, and α7-KO mice promptly self-administer nicotine intravenously, 

while β2-KO, β2-VEC in the substantia nigra, α4-KO and α6-KO mice do not respond 

to nicotine. We thus define the necessary and sufficient role of α4β2*- and α6β2* nAChRs 

present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drugnaïve 

mice. 

Disclosures: S. Pons, None; L. Fattore, None; G. Cossu, None; S. Tolu, None; E. Porcu, 

None; M. McIntosh, None; J. Changeux, None; U. Maskos , None; W. Fratta, None. 

Poster 





Support: Supported by an unrestricted grant from Philip Morris USA 

Title: Nicotinic alpha7 and beta2 null mice: Single and double knockout effects on behavioral 

function 

Authors: A. PETRO, C. MIRANDA, N. POLLARD, H. SEXTON, N. C. CHRISTOPHER, *E. 

KHOLDEBARIN, A. H. REZVANI, E. D. LEVIN; 

Dept Psychiatry, Duke Univ. Med. Cntr, Durham, NC

Abstract: Nicotinic acetylcholine receptors are known to be important for the neural substrate 

for spatial learning and memory and addiction. High affinity nicotinic receptors containing beta2 

subunits have been implicated both with cognitive function as well as nicotine reinforcement. 

The neurobehavioral roles of alpha7 nicotinic receptors are less well characterized. We have 

assessed alpha7 and beta2 knockout mice and wildtype controls (C57 BL/6) for spatial learning 

on the 8-arm radial maze and nicotine consumption with two-bottle continuous access procedure. 

In the radial-arm maze task male beta2 KO mice showed a sex-selective accuracy impairment. 

Alpha7 KO mice showed a significant radial-arm maze choice accuracy impairment with no 

differential effect between sexes. With nicotine consumption, the beta2 KO mice showed a 

significant reduction relative to controls for the first three weeks. The alpha7 KO mice did not 

differ in nicotine preference from controls initially, but interestingly did have an emergent 

decrease in nicotine consumption that emerged after the first few weeks and persisted for the rest 

of the 5-month experiment. In a separate study in rats we found that prenatal nicotine exposure 

caused more persistent nicotine self-administration in the offspring. The alpha7 and beta2 

knockout mice are being used to determine which nicotinic receptor may be the target for this 

developmental effect. Recently, we have succeeded in producing mice with combined alpha7 and 

beta2 receptor knockouts. They appear to be healthy and produce offspring. Neurobehavioral 

effects are being tested. Better understanding of the differential and interactive roles of alpha7 

and beta2-containing nicotinic receptors is key for developing better treatments for tobacco 

addiction and other syndromes. 

Disclosures: A. Petro, None; E. Kholdebarin , None; C. Miranda, None; N. Pollard, 

None; H. Sexton, None; N.C. Christopher, None; A.H. Rezvani, None; E.D. Levin, None. 

Poster 




Topic: C.17.d. Other 

Support: NSF DUE-0426266 

Title: A cembranoid from tobacco alleviates nicotine- but not carbamylcholine-induced 

behavioral effects in planarian worms 

Authors: *O. R. PAGAN 1 , A. ROWLANDS 1 , A. FATTORE 1 , T. COUDRON 1 , K. URBAN 1 , 

A. BIDJA 1 , V. A. ETEROVIC 2 ; 

1 Dept Biol, West Chester Univ., West Chester, PA; 2 Biochem., Univ. Central del Caribe, 

Bayamon, PR

Abstract: Using an adaptation of published behavioral protocols, we have determined that acute 

exposure to the cholinergic compounds nicotine and carbamylcholine (an acetylcholine analog) 

decreased planarian motility in a concentration-dependent manner, with IC50 values of 75 +/- 9 

µM and 151 +/- 14 µM for nicotine and carbamylcholine respectively. The tobacco cembranoid 

(1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R), also decreased planarian motility 

with an IC50 of 20 +/- 5 uM. At concentrations at or below 1 µM, 4R alone did not decrease 

planarian motility nor had any apparent behavioral or toxic effects in this organism. Experiments 

in the presence of 1 µM 4R did not change the IC50 for carbamylcholine-induced decrease in 

planarian motility (p = 0.350, F-test). In contrast, 1 µM 4R significantly increased the IC50 for 

the nicotine-induced decrease in planarian motility (p < 0.0001, F-test). When planarians were 

exposed for 24 hours to either nicotine or carbamylcholine at concentrations near their respective 

IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholineexposed 

worms displayed distress behaviors resembling withdrawals symptoms. In experiments 

where planarians were pre-exposed to 100 µM nicotine for 24 hours in the presence of 1 µM 4R, 

the withdrawal-like effects were significantly reduced. These results parallel published data 

describing the relative higher potency of 4R to inhibit nicotine- vs. acetylcholine-induced 

currents through α4β2 neuronal nicotinic acetylcholine receptors. 

Disclosures: O.R. Pagan , None; A. Rowlands, None; A. Fattore, None; T. Coudron, 

None; K. Urban, None; A. Bidja, None; V.A. Eterovic, None. 

Poster 

259. Cochlear Mechanoreceptors 



Topic: D.02.a. Mechanoreceptors and cochlea 

Support: USyd R&D grant 2007 

Title: Efferent innervation is present to both low- and high-frequency regions of the auditory 

basilar papilla of lizards 

Authors: E. WIBOWO, J. BROCKHAUSEN, *C. KOEPPL; 

Sch. of Med. Sci., Univ. Sydney, Sydney, Australia 

Abstract: The inner ear of vertebrates contains hair cells for sensory transduction. Hair cells are 

generally innervated by afferent neurons to transmit the sensory information to the brain as well 

as efferent neurons to receive feedback from the brainstem. The function of this efferent 

feedback system is poorly understood and may have changed during evolution as different

vertebrate groups acquired sensitivity to airborne sound and extended their hearing ranges to 

higher frequencies. Lizards show a unique subdivision of their basilar papilla into a lowfrequency 

and a high-frequency (>1 kHz) region, with many characteristic differences. 

Interestingly, the high-frequency region was reported to have lost its efferent innervation (review 

by Miller, 1992, in: The Evolutionary Biology of Hearing), suggesting it was insignificant or 

even functionally detrimental at higher frequencies. 

We re-examined the innervation to the auditory organs of 6 different species of skink lizards and 

one pygopod (close relatives of geckos), using immunocytochemistry. As efferent neurons are 

generally cholinergic, choline acetyltransferase (ChAT) was used as an efferent marker. Western 

blotting confirmed antibody specificity in lizard tissue. Wholemounts and cryosections of the 

cochlear duct were analyzed with a Zeiss LSM 510 Meta Confocal Microscope. ChATimmunoreactivity 

was observed along the whole length of the basilar papilla, including the 

regions which had previously been reported to be devoid of efferent terminals. ChAT labeling 

was present around the basal regions of the hair cells and occasionally further up their lateral 

wall, consistent with the typical distribution of synapses. To confirm the synaptic nature of the 

label, cryosections of the cochlear duct were double-labeled with anti-ChAT and anti-SV2 (a 

synaptic vesicle marker). ChAT-labeled presumed synapses were typically double-labeled for 

SV2. In addition, there were SV2-only labeled terminals, i.e. presumed afferents. 

Contrary to earlier descriptions, our findings suggest that efferent innervation is a general feature 

of the hair cells in the basilar papilla of lizards, irrespective of tonotopic location. This reenforces 

the notion that.efferent feedback control of hair cells is a fundamental and important 

property of all vertebrate hearing organs. 

Disclosures: E. Wibowo, None; J. Brockhausen, None; C. Koeppl, None. 

Poster 





Support: NIDCD R01 DC04928 

NASA GSRA56000135 

NIDCD R01 DC06685 

Title: Efferent control of hair cell mechanical power output

Authors: *R. D. RABBITT 1,2 , K. D. BRENEMAN 3 , S. M. HIGHSTEIN 4 , R. BOYLE 5 ; 

1 Dept Bioengineering, Univ. Of Utah, Salt Lake City, UT; 2 Marine Biol. Lab., Woods Hole, MA; 

3 Univ. of Utah, Salt Lake City, UT; 4 Washington Univ., Saint Louis, MO; 5 NASA, Ames, CA 

Abstract: The mammalian inner ear is endowed with an extensive efferent innervation that is 

used by the brain to control sensitivity of the cochlear amplifier and vestibular end organs. A 

significant fraction of this innervation contacts basal poles of mechano-sensitive hair cells. 

Activation of these axo-somatic endings alters the gain and tuning of afferent discharge 

responses to sound or vestibular stimuli. We provide evidence from the vestibular semicircular 

canals that the canonical event following activation of efferent neurons is a large increase in the 

electrical admittance of target hair cell somata. For a given hair bundle displacement, this is 

accompanied by increase in the mechano-electrical-transduction current and by a decrease in 

receptor potential modulation. To explore how this admittance change might alter hair cell 

amplification we formulated biophysically based models of prestin-dependent somatic motility 

(outer hair cells) and flexoelectric hair-bundle motility (all hair cells). Model results clearly 

indicate that the increase in somatic admittance would sharply attenuate the power-output and 

efficiency of both the somatic motor and the hair-bundle motor. This is particularly relevant at 

auditory frequencies where hair cell motors are key to amplification. Results may explain a 

fundamental principle used by the efferent system to provide exquisite control over amplification 

and tuning in a broad range of inner-ear sensory organs. 

Disclosures: R.D. Rabbitt , University of Utah, A. Employment (full or part-time); R01 

DC04928, R01 DC06685, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received); NIH, B. Research Grant (principal 


K.D. Breneman, GSRA56000135, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); NASA Ames, B. Research 

Grant (principal investigator, collaborator or consultant and pending grants as well as grants 

already received); S.M. Highstein, None; R. Boyle, None. 

Poster 





Title: Characterizing the physiological and morphological inputs from the anterior lateral line 

nerve onto the goldfish Mauthner cell

Authors: M. MIRJANY, *D. S. FABER; 

Dominick P.Purpura Dept Neurosci, Albert Einstein Coll Med., Bronx, NY 

Abstract: The goldfish Mauthner (M-) cells, a bilateral pair of reticulospinal neurons initiate the 

auditory evoked escape behavior. These neurons and their connections have been well 

characterized, and their large size makes them accessible to in-vivo electrophysiology. The 

behavior is appropriately directional; in the open field a fish reliably turns away 

from the sound source. So far the mechanisms that underlie this directionality remain unknown. 

Our previous studies have suggested that the lateral line (LL) mechanosensory organ, a velocity 

and acceleration detector, transmits 

directional information to the M-cell (Mirjany et al., SFN 2005). We hypothesize that this 

information is transmitted to the M-cell via the anterior branch of the LL (aLL), which innervates 

the large population of canal neuromasts on the head of the fish. The inputs from the aLL to the 

M-cell have not been characterized to date. We postulate that the input to the M-cell from the 

aLL should be monosynaptic, so that it is fast enough to summate with mixed electrotonic inputs 

from the inner ear. 

Electrophysiological data obtained by recording intracellular responses to aLL nerve stimulation 

indicate this is the case. The latency of the evoked EPSP is 1.15± 0.11ms, which is within the 

2ms processing window of the M-cell. Superimposed on the evoked EPSP is a shunting 

inhibition with a delay of 1ms, which is characteristic of other sensory inputs to the M-Cell. 

Repetitive stimulation at 10Hz reveals a component of the aLL evoked EPSP is electrotonic, a 

property favoring speed of transmission. In addition, preliminary morphological and 

electrophysiological mapping data indicate this input is localized to the lateral dendrite proximal 

to the site of input from the inner ear, suggesting this system provides a model for studying 

dendritic interaction in the analysis of responses evoked by natural stimuli. 

Disclosures: M. Mirjany, None; D.S. Faber , None. 

Poster 





Support: The Hartwell Foundation 

ALSAC 

NIH Grant 1F31DC009393



Title: Potential roles of Pkd1 in mechanoelectrotransduction of mouse cochlear hair cells 

Authors: *K. A. STEIGELMAN 1,2 , X. WU 1 , J. GAO 1 , F. QIAN 3 , K. B. PIONTEK 3 , G. 

GERMINO 3 , J. ZUO 1 ; 

1 Developmental Neurobio., St. Jude Children's Res. Hosp., Memphis, TN; 2 Anat. and Neurobio., 

Univ. of Tennesee Hlth. Sci. Ctr., Memphis, TN; 3 Johns Hopkins Med. Sch., Baltimore, MD 

Abstract: The components of the mechanoelectrotransduction (MET) channel in the cochlear 

hair cells are still unknown. We hypothesize that as a member of the TRP family, polycystic 

kidney disease (Pkd1) is part of the MET channel in the hair cells because Pkd1 plays a role in 

fluid-flow sensation and Ca2+ ion uptake in kidney cilia. It also forms cation channels with 

conduction similar to that of MET channels. Recent studies on Pkd1L3, a close relative of Pkd1, 

demonstrated its involvement in olfactory sensory epithelia 1 . We created and analyzed two 

mutant mouse models with two independent alleles of Pkd1, a knockin model (Pkd1T3041V) that 

disrupts the normal cleavage of the protein and a hair cell specific conditional knockout model 

(Pkd1cko) using a recently characterized Atoh1-CreER line 2,3 . 

In wildtype cochleae, we have localized the mRNA of Pkd1 to both the inner and outer hair cells 

through RT-PCR from laser-captured individual hair cells and the protein to the hair 

bundle/cuticular plate of IHCs and OHCs through immunohistochemistry. In both Pkd1T3041V 

and Pkd1cko models, auditory brainstem response (ABR) tests revealed a 25-30 dB hearing loss 

compared to wild type littermates. SEM imaging displayed abnormal hair cell morphology in 

both models. However, FM1-43 dye uptake experiments show similar results to the wildtype. 

These results demonstrate that the hearing loss is due specifically to a phenotype in the hair cells 

and not other cell types of the cochlea. We plan to further analyze both models to provide in vivo 

evidence that Pkd1 is a component of the MET channel in the hair cells and to determine the 

roles of Pkd1 in the inner ear. 

Disclosures: K.A. Steigelman, None; X. Wu, None; J. Gao, None; F. Qian, None; K.B. 

Piontek, None; G. Germino, None; J. Zuo, None. 

Poster 



Program#/Poster#: 259.5/BB1 

Topic: D.02.a. Mechanoreceptors and cochlea

Support: NIH R01 DC00092 

NIH 5T32GM007469 

Title: Calcium-activated potassium (BK) channels are encoded by two Slo1 genes in teleost 

fishes 

Authors: *K. N. ROHMANN, D. L. DEITCHER, A. H. BASS; 

Neurobio. & Behavior, Cornell Univ., Ithaca, NY 

Abstract: Calcium-activated potassium (BK) channels are encoded by a single Slo1 gene in 

tetrapods. A high level of functional diversity in these channels is achieved in large part via 

extensive alternative splicing. Despite their important neurophysiological roles, ranging from 

electrical tuning of auditory hair cells to shaping the firing properties of central neurons, these 

channels have not been investigated at the molecular level in ray-finned fishes 

(actinopterygians), most of which are teleosts that represent approximately half of all extant 

vertebrate species. Slo1 genes were identified here in both teleost and non-teleost fishes with 

PCR using degenerate primers designed to highly conserved regions of the single tetrapod Slo1 

gene. All of the non-teleosts examined pre-date the proposed whole-genome duplication event 

that resulted in seven Hox clusters (compared to four in tetrapods) with the origin of teleosts 

about 300-350 million years ago. Like tetrapods, a single Slo1 gene is expressed in the brain of 

chondrichthyan fish (little skate, Raja erinacea) that pre-date actinopterygians, and several 

groups of non-teleost actinopterygians (bichir, Polypterus senegalus; longnose gar, Lepisostus 

osseus; bowfin, Amia calva). In contrast, two Slo1 genes, both of which are expressed in the 

brain, are present in several distantly related teleosts (zebrafish, Danio rerio; threespine 

stickleback, Gasterosteus aculeatus; plainfin midshipman, Porichthys notatus; cichlid, 

Neolamprologus pulcher). Searches of genome databases show two Slo1 genes in yet other 

teleosts (medaka, Oryzias latipes; puffer fishes, Takifugu rubripes and Tetraodon nigroviridis). 

The presence of two Slo1 co-orthologs across teleosts, together with the lack of evidence for 

duplicate genes in non-teleosts, suggest that the Slo1 gene was duplicated as part of a teleost 

whole-genome duplication event. The retention of Slo1 co-orthologs in such a diverse and large 

group of vertebrates suggests strong positive selection for this trait. While duplicate Slo1 genes 

may reflect the subfunctionalization and/or neofunctionalization of Slo1 co-orthologs, the high 

degree of alternative splicing known for the single tetrapod Slo1 gene may yet be retained in 

either or both Slo1 co-orthologs. 

Disclosures: K.N. Rohmann , None; D.L. Deitcher, None; A.H. Bass, None. 

Poster 





Support: EuroHear FP5 512063 

Title: Cochlear hair cell currents and tuning in adult rats 

Authors: J. HERING 1,2 , *C. PETIT 1,2,3 ; 

1 INSERM UMRS 587, Inst. Psteur, Paris Cedex 15, France; 2 Univ. Pierre et Marie Curie, Paris 

VI, France; 3 Collège de France, Paris, France 

Abstract: The sensory cells (hair cells) of the mammalian cochlea are involved for sound 

amplification (outer hair cells) and transduction into electrical signals (inner hair cells). The 

motion of the sensory epithelium (organ of Corti) is driven by the movement of the underlying 

basilar membrane. In non-mammalian species, auditory hair cells display electrical tuning, which 

enables each hair cell to respond preferentially to a “best frequency” of the basilar membrane 

movement (tonotopy). In mammals, electrical tuning of the hair cell response has neither been 

shown nor totally excluded. To address this issue, we have used the hemicochlea technique that 

preserves the integrity of the organ of Corti. Our setup combines electrophysiology, 

interferometry, and IR observation of the preparation. The recordings were made at 35-37°C in 

Wistar rats aged from P25 to 6 months. The hair cells were stimulated by the means of a tungsten 

probe (driven by a piezoelectrical actuator) applied on the basilar membrane, and delivering sine 

stimulations comparable to pure tones. Displacement and velocity of the basilar membrane were 

measured with a commercial interferometer (Polytec, France). The receptor potentials and 

currents of hair cells were recorded with the patch-clamp technique in whole-cell configuration. 

Because the stimulation and receptor potential acquisition were under computer control, we were 

able to determine the best frequency of each patched hair cell, which allowed us to record 

mechanoelectrical currents and receptor potentials at this particular frequency. In addition, the 

different hair cell conductances could be characterized by their pharmacological and biophysical 

properties. This unique system, combining different techniques, allowed us to study for the first 

time hair cell properties along the entire tonotopic axis of a mature cochlea. 

Disclosures: J. Hering, None; C. Petit , None. 

Poster 



Program#/Poster#: 259.7/BB3


Support: NIH NIDCD DC 00141 

NIH NIDCD DC 006273 

Title: Measurement of sound-evoked vibrations in the organ of Corti of living guinea pigs using 

optical coherence tomography 

Authors: J. ZHENG 1 , F. CHEN 1 , N. CHOUDHURY 2 , S. L. JACQUES 2 , *A. L. NUTTALL 1 ; 

1 Oregon Hearing Res. Ctr., 2 Biomed. Engineering, Oregon Grad. Inst., Oregon Hlth. Sci. Univ., 

Portland, OR 

Abstract: Measurement of sound-evoked vibrations in the organ of Corti of living guinea pigs 

using optical coherence tomography 

In mammals, the cochlear sensitivity depends on the function of an active mechanism termed 

“cochlear amplification” (CA) in the organ of Corti, in which the outer hair cells (OHCs) 

generate mechanical forces to enhance the basilar membrane (BM) vibration.CA causes the 

sharp tuning and exquisite sensitivity of hearing. In such a unique mechanically- physiologically 

coupled system with active mechanical force generation, knowledge of the differential motion of 

the key components within the organ of Corti is of crucial importance in understanding cochlear 

amplification. Using a newly developed optical coherence tomography (OCT) system and 

homodyne interferometry, we observed sound-induced differential motions of two importance 

surfaces, the BM and reticular laminar (RL), in the sensitive cochlea of living guinea pigs. We 

found that at the same radial location, the vibrations of BM and RL exhibit similar sharp tuning 

and sound-level dependent magnitude compression, which are features of mechanical responses 

in the sensitive cochlea. Differential motion between the BM and RL was observed, as shown by 

differences in magnitude and phase. We observed that the RL has both a higher level of vibration 

and a relative phase lead compared the BM. These data represent the first in vivo measurements 

of the micromechanical motions of the organ of Corti. The data support the CA mechanism 

involves a phase shift for the production of outer hair cell force such that vibration generated 

viscous energy losses are compensated. Supported by NIH NIDCD DC 00141 and DC 006273. 

Disclosures: J. Zheng, None; F. Chen, None; N. Choudhury, None; S.L. Jacques, None; A.L. 

Nuttall , None. 

Poster 





Support: BMBF Grant to TM and AN 

DFG Fellowship to NS 

Jack Kent Cooke Foundation 

NIDCD Grant P30 DC 00529 to MCL 

NIDCD Grant RO1 DC 00188 to MCL 

Title: Synaptic ribbons in cochlear hair cells are required for reliable coding of stimulus onset 

Authors: *B. N. BURAN 1,2 , N. STRENZKE 3,2 , A. NEEF 4 , T. MOSER 3,4 , M. C. 

LIBERMAN 1,5,2 ; 

1 Div. of Hlth. Sci. and Technol., Harvard-MIT, Cambridge, MA; 2 Eaton-Peabody Lab., 

Massachusetts Eye and Ear Infirmary, Boston, MA; 3 Inner Ear Lab, Dept. of Otolaryngology, 

4 Bernstein Ctr. for Computat. Neurosci., Univ. of Goettingen, Goettingen, Germany; 5 Dept. of 

Otology and Laryngology, Harvard Med. Sch., Boston, MA 

Abstract: Synaptic ribbons are electron-dense structures surrounded by vesicles and tethered to 

the presynaptic membrane of photoreceptors, retinal bipolar cells and hair cells. Such synapses 

are characterized by sustained exocytosis that is graded with stimulus intensity and can achieve 

very high rates of release. 

To gain insight into the function of this specialized presynaptic machinery, we characterized the 

response properties of single auditory nerve (AN) fibers in a mouse with targeted deletion of 

bassoon, the gene for a presynaptic scaffolding protein, in which synaptic ribbons are no longer 

tethered to the active zone. Prior in vitro studies showed a strong reduction of hair cell 

exocytosis in these mutants. While informative, whole-cell responses reflect the summed activity 

of many synapses, which are heterogeneous with respect to spontaneous and sound-evoked rates. 

Since each mammalian AN fiber receives input from a single inner hair cell active zone, to 

which, typically, a single ribbon is tethered, single-fiber recordings in bassoon mutants and 

control mice offer a sensitive functional metric of the contribution of individual synaptic ribbons. 

Response properties of mutant AN fibers were similar, in many respects, to wild-type. Spike 

intervals retained their normal irregularity, thresholds were unaffected, dynamic range was 

unchanged, spike synchronization to stimulus phase was unimpaired, post-onset adaptation and 

recovery from adaptation were normal, and the ability to sustain discharge throughout a longduration 

stimulus was unaffected. However, striking abnormalities were seen in absolute 

discharge rates. Mean spontaneous rate was decreased by a factor of 4 and sound-evoked rates 

were decreased by a factor of 2. A corresponding increase was observed in the variance of the 

first spike latency in response to moderate level tone bursts. This increased jitter likely arises 

from a reduction in synaptic drive through a mechanism such as decreased probability of 

simultaneous vesicle release, and may degrade the ability of the auditory system to resolve 

stimulus features such as interaural time differences that mediate sound localization and other 

key aspects of binaural hearing.

Disclosures: B.N. Buran, None; N. Strenzke, None; A. Neef, None; T. Moser, None; M.C. 

Liberman, None. 

Poster 





Support: NIH Grant DC05989 

Title: Identification and amplification of gap junction genes and proteins from celloidinembedded 

human cochlear sections 

Authors: X.-H. WANG, *H.-B. ZHAO; 

Dept Otolaryngol, Univ. Kentucky Med. Sch., Lexington, KY 

Abstract: Gap junctional coupling plays a crucial role in hearing. Connexin mutations are 

associated with a high incidence of nonsyndromic hearing loss. However, its pathological 

mechanisms remain largely undetermined. In this study, the expressions of gap junction genes 

and proteins in the celloidin-embedded human temporal bone sections with formalin fixation 

were investigated. After de-celloidin, the genomic DNA was successfully extracted from the 

long-term storage human cochlear slice (10 years) using RecoverAll TM Total Nucleic Acid 

Isolation kit (Ambion). The formalin-fixed, celloidin-embedded human cochlear sections (10-20 

years) were also processed by the antigen-retrieval treatment for immunofluorescent staining. 

Connexin 26 (Cx26), which is a predominant isoform of gap junctions in the cochlea, was 

identifiable from the PCR product and immunofluorescent staining. Clearly labeling for Cx26 

could be detected in the supporting cells in the organ of Corti and at the stria vascularis (SV) and 

spiral ligament (SPL) in the cochlear lateral wall. We also examined the expression of Pannexin 

(Panx), which is a new found gap junction gene family in vertebrate, in the human cochlea. As 

our finding in the rat and mouse cochlea, the Panx1 expression was also detected in the human 

cochlea. Our preliminary data show successful extraction of DNA and immunofluorescent 

staining in the long-term storage human cochlear sections. These studies provide a 

methodological fundament for further identification of gap junction gene expression and 

pathogenic mechanisms of deafness mutants in the human cochlea. 

Supported by NIH R01 DC05989 

Disclosures: X. Wang, None; H. Zhao, None.

Poster 





Support: NIH Grant CA21765 

NIH Grant 1F31DC009393 

ALSAC, Hartwell individual biomedical research award 



Title: Effect of p16 Ink4a deletion on cochlear hair cells after damage with ototoxic drugs 

Authors: *B. C. COX 1 , S. PAPAL 1 , K. A. STEIGELMAN 1,2 , J. ZUO 1 ; 

1 Dev Neurobiol, St Jude Children's Res. Ho, Memphis, TN; 2 Anat. and Neurobio., Univ. of 

Tennessee Hlth. Sci. Ctr., Memphis, TN 

Abstract: Exposure to harmful noise or ototoxic drugs can induce hearing loss which is 

primarily caused by damage to sensory hair cells of the inner ear. Humans and other mammals 

cannot replace damaged hair cells; however, chicken, fish and amphibians can, by proliferation 

and transdifferentiation of neighboring supporting cells. Based on non-mammalian models, we 

hypothesize that mammalian hair cells release a signal when damaged that triggers supporting 

cells to re-enter the cell cycle and later to transdifferentiate into hair cells. Unblocking the 

inhibition of cell cycle re-entry in postmitotic, quiescent supporting cells appears to be the first 

and most important step in this process. Recently, many researchers have studied hair cell 

regeneration by manipulating the retinoblastoma protein as well as cyclin-dependent kinase 

inhibitors. These studies have obtained mixed results depending on the timing and the cell-type 

specificity of the gene deletion. The cyclin-dependant kinase inhibitor, p16 Ink4a, is not expressed 

until animals are aged or have experienced an insult; thus, deletion of this protein is attractive as 

it may allow supporting cells to respond to the signal naturally released by damaged hair cells. 

We have recently begun studies with p16 Ink4a -null mice and found that these mice have normal 

hearing and normal structure and morphology of the inner ear and hair cells. We are currently 

conducting studies with ototoxic drugs (gentamicin and cisplatin) in p16 Ink4a -null mice and will

examine potential cell cycle re-entry in supporting cells as well as possible regeneration of hair 

cells. 

Disclosures: B.C. Cox , None; S. Papal, None; K.A. Steigelman, None; J. Zuo, None. 

Poster 





Support: NSF grant BES-0522862 

The Clayton Foundation 

NIH grant DC006671 



Title: Altered tectorial membrane anatomy produces cochlear dysfunction in alpha tectorin 

C1509G transgenic knockin mice 

Authors: *J. OGHALAI 1,2 , A. XIA 1 , S. GAO 2 , T. YUAN 1 , M. PFISTER 3 , F. PEREIRA 1,2 ; 

1 Baylor Col. Med., Houston, TX; 2 Rice Univ., Houston, TX; 3 Univ. of Tübingen, Tübingen, 

Germany 

Abstract: The tectorial membrane (TM) is an extracellular matrix that attaches to the stereocilia 

of outer hair cells (OHCs) and guides mechanoelectrical transduction. Alpha tectorin is a 

glycoprotein that is thought to be involved in cross-linking collagen and possibly other 

glycoproteins within the TM. We have created a transgenic knockin of a cysteine to glycine 

mutation at codon 1509 in alpha tectorin, which was identified as a mutation in patients with 

autosomal dominant sensorineural hearing loss. We have now characterized the morphological, 

histological, and physiological consequences of this mutation. Light microscopy demonstrated 

that the TM was shortened in heterozygotes and bulbous in homozygotes. Immunostaining 

demonstrated that wild-type, heterozygote, and homozygote mice all expressed alpha tectorin, 

beta tectorin, and otogelin within the TM. Transmission electron microscopy of the TM revealed 

that the marginal band, Kimura‟s membrane, and the covernet bundles had reduced staining

density in heterozygotes. These changes were even more pronounced in homozygotes. Auditory 

brainstem response measurements identified a 40 dB threshold elevation in heterozygotes and a 

60 dB threshold elevation in homozygotes relative to wild-type littermates. Distortion product 

otoacoustic emission measurements revealed that heterozygotes had a 10-20 dB threshold 

elevation and homozygotes had a 40 dB threshold elevation. The cochlear microphonic 

amplitude was moderately reduced in heterozygotes and severely reduced in homozygotes below 

stimulus levels of 90 dB SPL. The cochlear microphonic was in phase with the stimulus in wildtype 

mice but led the stimulus by 90 degrees in homozygote mice. In heterozygotes, the cochlear 

microphonic was in phase with the stimulus at lower intensity levels, but progressively shifted to 

lead the stimulus as the intensity was increased. Electrically-evoked otoacoustic emissions were 

larger in heterozygotes and homozygotes compared to wild-type mice. These findings indicate 

that the alpha tectorin C1509G mutation alters the structure of the TM so that its interactions 

with the OHC stereocilia vary according to mutation copy number. These data are consistent 

with a model whereby all OHCs are stimulated by the TM in wild-type mice, only the first row 

of OHCs are stimulated by the TM in heterozygote mice, and no OHCs are stimulated by the TM 

in homozygote mice. Our results confirm that this C1509G mutation in alpha tectorin is the 

causative mutation in our patients with autosomal dominant sensorineural hearing loss. 

Disclosures: J. Oghalai , NIH, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); The Clayton Foundation, B. 


grants already received); A. Xia, None; S. Gao, None; T. Yuan, None; M. Pfister, None; F. 

Pereira, NSF, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); NIH, B. Research Grant (principal investigator, 


Poster 





Support: NIH NIA Grant P01 AG09524 

NIH NIDCD Grant P30 DC05409 

Title: Major upregulation of apoptosis genes in the aging mouse cochlea

Authors: *R. D. FRISINA 1,2 , S. F. TADROS 1,3 , X. ZHU 1,2 , M. D'SOUZA 1,2 ; 

1 Otolaryngology Dept, Univ. Rochester Med. Sch., Rochester, NY; 2 Int Ctr. Hearing Speech 

Res., Rochester Inst. Tech., Rochester, NY; 3 Physiol. Dept, Univ. New S. Wales, Sydney, 

Australia 

Abstract: Apoptosis - endogenous programmed cell death involves alteration of the cell cycle. 

Various apoptotic pathways play different roles according to the tissue type involved in 

pathological or physiological states. To understand possible roles apoptosis may play in agerelated 

hearing loss (presbycusis), gene expression patterns of different apoptotic pathways were 

compared, using GeneChip arrays and real-time (qPCR) microarrays, as a function of age and 

hearing loss for the CBA mouse cochlea. Mice were divided into 4 groups based upon age and 

hearing measurements: young adult control with good hearing (N=8, age=3.5 +/- 0.4 mon), 

middle-aged with good hearing (N=17, age=12.3 +/- 1.3 mon), old with mild presbycusis (N=9, 

age=27.7 +/- 3.4 mon) and old with severe presbycusis (N=6, age=30.6 +/- 1.9 mon). GeneChip 

expression patterns of 318 apoptosis-related probes and differences in signal-log-ratio and fold 

changes between subject groups were analyzed. Thirty eight probes (35 genes) showed 

significant differences in expression. The significant gene families include Caspases, B-cell 

leukemia/lymphoma2 (Bcl2) family, P53, Calpains, Mitogen activated protein kinase (MAPK) 

family, Jun oncogene (Jun), Nuclear factor of kappa light chain gene enhancer in B-cells 

inhibitor (Nf-kB) -related, and tumor necrosis factor (TNF) -related genes. These genes that 

showed significant changes were classified according to their expression (up- or downregulation) 

and according to the apoptosis pathway (extrinsic, death receptor and survival factor 

withdrawal pathways; intrinsic pathways; and downstream pathways. The genechip results of 31 

of these genes were validated using the new TaqMan® Low Density Array (TLDA). Eight genes 

showed highly correlated results with the GeneChip data. These genes are: activating 

transcription factor3 (Atf3), B-cell leukemia/lymphoma2 (Bcl2), Bcl2-like1 (Bcl2l1, also known 

as Bcl-xL), caspase4 apoptosis-related cysteine protease 4 (Casp4, also known as Casp11), 

Calpain2 (Capn2), dual specificity phosphatase9 (Dusp9, also known as Mkp4), tumor necrosis 

factor receptor superfamily member12a (Tnfrsf12a, also known as TweakR / Fn14 receptor), and 

Tumor necrosis factor superfamily member13b (Tnfsf13b, also known as BAFF, THANK, 

TALL1, zTNF4). All of these genes except Capn2 showed upregulation with age and hearing 

loss. It is hoped that increased knowledge of cell death pathways in the aging auditory system 

may someday lead to interventions to slow or prevent presbycusis. 

[Work supported by NIH Grants: NIA P01 AG09524 and NIDCD P30 DC05409] 

Disclosures: R.D. Frisina , NIH Grants, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received); S.F. Tadros, None; X. Zhu, 

NIH Grants, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); M. D'Souza, NIH Grant, B. Research Grant (principal 


Poster





Support: NIH R01 DC006283 

NIH P30 DC04665 

NIH T32 DC00022 

Title: Valproic acid inhibits sensory epithelia development of the embryonic mouse cochlea 

through histone deacetylase inhibition 

Authors: *E. L. SLATTERY, J. M. JONES, M. E. WARCHOL; 

Otolaryngology, Washington Univ, Sch. Med., St. Louis, MO 

Abstract: The developing mouse cochlea undergoes rapid embryonic differentiation that is 

accompanied by extensive changes in gene expression (Sajan, S et al., 2007). The aim of the 

present study was to examine the role of epigenetic influences on gene expression during 

cochlear development. One class of epigenetic gene regulation is mediated by histone 

acetylation, which leads to relaxation of DNA coiling and permits increased interaction with 

transcriptional protein complexes. Histone acetylation is a dynamic process, and treatment with 

histone deacetylase (HDAC) inhibitors leads to maintained histone acetylation, along with 

corresponding changes in gene expression. We found that treatment with the HDAC inhibitor 

valproic acid (VPA), a commonly used antiepileptic drug, caused decreased differentiation of the 

developing sensory epithelium in the mouse cochlea. Embryonic cochleae were dissected from 

mice at E13.5, placed in organ culture, and treated for 5 days with 1 mM VPA. This treatment 

resulted in a marked disruption of cochlear development. Along the length of the organ of Corti, 

we observed scattered sensory regions, which appeared as small patches and were located near 

the region that would normally be populated by inner hair cells. The patches contained reduced 

numbers of inner hair cells (which lacked stereocilia bundles), no outer hair cells, and decreased 

numbers of p75-labeled pillar cells. Expression of Sox2, E-cadherin and Jagged 1 was confined 

to cells immediately adjacent to the sensory patches. Cochleae exposed to sodium butyrate and 

trichostatin A, biochemically-distinct HDAC inhibitors, possessed similar phenotypes, 

suggesting that the effects were due to inhibition of histone deacetylases. Notably, we observed 

no increase in pyknotic nuclei following HDAC inhibitor treatment, indicating that the structural 

changes were not caused by enhanced cell death in the developing organ of Corti. The results 

suggest that histone acetylation status plays an important role in cochlear development. 

Disclosures: E.L. Slattery, None; J.M. Jones, None; M.E. Warchol, None.

Poster 





Support: Fondi di Ateneo 2006/07- UCSC-Italy 

Title: Antioxidant property of Ferulic acid in Noise induced hearing loss (NIHL) 

Authors: M. RALLI 1 , A. FETONI 1 , G. PALUDETTI 1 , *D. TROIANI 2 ; 

1 Otolaryngology, UCSC, Rome, Italy; 2 Inst. Human Physiol, UCSC, Rome 00168, Italy 

Abstract: Ferulic Acid (FA) is known for its antioxidant properties and its free-radical 

scavenging activity towards peroxynitrite, hydroxyl radicals and oxidized low-density proteins. 

FA has been studied as a neuroprotector in neurodegenerative disorders and cerebral ischemia 

injury. In this study, we tested the hypothesis that FA might exert, by virtue of its antioxidant 

properties, beneficial protective effects against noise-induced hearing loss (NIHL). Loud noise 

induces hearing impairment by increasing the production of reactive oxygen species (ROS) and 

toxic free radicals which, in turn through a series of reactions, induce lipid peroxidation and cell 

damage in the organ of Corti. We evaluated the protective effect of FA against NIHL in guinea 

pigs (n=22). Noise-induced hearing loss was induced by exposing animals to a continuous pure 

tone of 6kHz, 120dB for 1 h; FA was injected intraperitoneally 1 h before acoustic trauma and 

once daily for 3 days. Auditory function was investigated by recording auditory brainstem 

responses (ABR) at 2-20 kHz; morphological studies were performed with scanning electron 

microscopy (SEM); immunohistochemistry was performed with terminal deoxynucleotidyl 

transferase-mediated dUTP nick end labeling assay (TUNEL) for identification of missing and 

apoptotic cells, free radical activity was determined via 4-hydroxynonenal (4HNE) and 

nitrotyrosine (3NT) as markers of reactive oxygen species and reactive nitrogen species action. 

Guinea pigs in the FA group showed significantly smaller auditory threshold shifts than 

unprotected control animals, indicative of a lesser extent of both apoptotic activation and hair 

cell loss in the organ of Corti. Our preliminary data confirm the antioxidant properties of FA and 

show its protective function in NIHL. This might be because FA inhibits the process of lipid 

peroxidation as documented by 4HNE immunohistochemistry. Our results suggest a therapeutic 

potential of FA in NIHL through its antioxidant property. 

Disclosures: M. Ralli, None; A. Fetoni, None; G. Paludetti, None; D. Troiani , None.

Poster 





Poster 





Support: University of Illinios SURGE Fellowship 

National Science Foundation Graduate Research Fellowship 

NIH Sensory Neuroscience Training Grant 

Title: A dynamical point process model for auditory nerve spiking encoding complex sounds 

Authors: *A. TREVINO 1 , R. E. WICKESBERG 2 , J. B. ALLEN 3 , T. P. COLEMAN 3 ; 

2 Psychology, 3 EE, 1 Univ. Illinois CU, Urbana, IL 

Abstract: Statistical point process analysis has recently proved quite successful for modeling 

neural spiking activity in many brain regions. We apply a dynamic point process approach to 

model the neural encoding of complex sounds in the auditory nerve. Specifically, voiced phones 

at different sound levels are presented to the cochlea of chinchillas, and subsequent auditory 

nerve spiking is recorded. We develop a parametric dynamic statistical point process model to 

perform maximum-likelihood estimation and apply statistical goodness-of-fit procedures to 

verify our model. For this auditory model, we focus on spontaneous firing rate, envelope phaselocking, 

cochlear nonlinearities, and onset/offset characteristics. This rich dataset, combined with

compact parametric point process methodology, allows for the testing of several different 

modeling hypotheses without the need for new experiments. 

Disclosures: A. Trevino, None; R.E. Wickesberg, None; J.B. Allen, None; T.P. Coleman, 

None. 

Poster 

260. Objects and Faces in Humans II 



Topic: D.04.j. Processing of objects and faces 

Support: NIH Grant RR00166 

NIH Grant EY05864 



Title: A developmental approach to studying the neural mechanisms of “crowding”: an animal 

model for lateral spatial interactions 

Authors: *M. A. HAGAN, D.-P. LI, G. VON TRAPP, L. KIORPES; 

Ctr. for Neural Sci., New York Univ., New York, NY 

Abstract: Crowding is the deleterious effect of neighboring contours on visual discrimination. 

Although it has been widely studied, the neural mechanisms remain unsolved. Because crowding 

suppresses certain visual within a scene, understanding the neural basis of crowding may inform 

our understanding of object recognition. There have been very few studies of the neural 

mechanisms involved in crowding, but psychophysics has provided support for several different 

theories. One theory suggests that long-range horizontal connections in V1 account for the 

spatial effects of crowding. Other theories suggest that the locus of crowding lies beyond V1 and 

results either from magnification errors in downstream feedback connections or effects of topdown 

attentional processes. We used a developmental approach to investigate the neural basis of 

crowding. 

The purpose of this study was to measure crowding at various stages of development in macaque 

monkeys. It is believed that the local connections within primary visual cortex are fully 

developed in macaques in the early post-natal weeks, while feedback connections that may

govern more global types of processing are not fully developed until six months of age or older 

(e.g., Batardieré et al., (2002) Cerebral Cortex. 12, 453-465). Therefore, if feedback connections 

were involved in crowding, one would not expect to see crowding effects in infant macaques. 

We tested seven pig-tailed macaque monkeys in three different age ranges: five weeks (infant), 

six months (juvenile) and two or more years (adults). To test whether “crowding” is observed 

during development, subjects were trained to discriminate a band-limited C-pattern comprised of 

Gabor patches in the presence of Gabor patch flankers placed at various distances from the C 

along the horizontal axis. This type of stimulus has been used in many human psychophysical 

studies of crowding. We found that macaques at all test ages showed a crowding effect. The 

“critical distance” or range of crowding was 2.45 standard deviations for the infant, 2.04 for the 

juvenile, and 2.58-2.64 for the adults. Our macaque adults perform similarly to humans: critical 

distances for two adult humans tested with the same stimuli were 2.26 and 2.44. These data 

suggest that crowding is mediated by connections that are already fully developed near birth. Our 

results further indicate that macaque monkeys are a useful model for studying lateral spatial 

interactions related to object processing, and provide important insight into the neural 

mechanisms of crowding. 

Disclosures: M.A. Hagan, None; D. Li, None; G. von Trapp, None; L. Kiorpes, None. 

Poster 





Support: NIMH 

Autism Speaks 

Title: Improved functional MRI of the macaque ventral visual pathway at 3 T using multi-echo 

EPI and dynamic, field map corrected image reconstruction 

Authors: *M. A. PINSK 1,2 , M. ARCARO 1,2 , C. S. KONEN 1,2 , X. LI 1,2 , S. KASTNER 3,1,2 , S. J. 

INATI 4 ; 

1 Dept Psychol, 2 Ctr. for the Study of Brain, Mind & Behavior, 3 Princeton Neurosci. Inst., 

Princeton Univ., Princeton, NJ; 4 Ctr. for Brain Imaging, New York Univ., New York, NY 

Abstract: The macaque ventral visual pathway along the temporal lobes has been shown to be 

an important region in visual object perception, recognition, and memory (Desimone and

Ungerleider, 1989; Ungerleider and Mishkin, 1982). Neurons responding to complex pictures 

such as faces have been found throughout the superior temporal sulcus (STS) and inferotemporal 

(IT) cortex of the temporal lobes. Most recently, functional magnetic resonance imaging (fMRI) 

studies in awake macaques have revealed several areas, mainly in the STS, selectively 

responsive to images of faces and body parts (Tsao et al. 2003; Pinsk et al. 2005; Hadj-Bouziane 

et al. 2008). However, due to strong susceptibility gradients in the region of the ear canals, fMRI 

of the IT cortex is typically compromised by signal loss and distortion (Goense et al. 2008). 

Here, we attempted to improve fMRI of this region with an optimized multi-echo gradient echo 

sequence in combination with a novel image reconstruction algorithm. The multi-echo echo 

planar imaging (EPI) sequence collects data at several echo times, under reversed gradient 

readouts. This allows for simultaneous estimation of the magnetic field, and the reconstruction of 

distortion free images with partial recovery of susceptibility induced signal loss. We compared 

fMRI activation of the temporal lobe using this optimized method to a typical single-shot 

gradient echo sequence. 

Video clips (~2 s in length) of faces and scenes were used as stimuli to robustly activate the 

ventral visual pathway. The stimuli subtended ~24 deg of the visual field and were presented in 

17.5 s blocks interleaved with equally long blank periods while the animal maintained fixation 

within a 4 deg window. The animal was scanned in a horizontal human 3 T MR scanner with a 

12 cm surface coil (Pinsk et al. 2005). The ventral visual pathway was strongly activated by the 

video clip periods compared to the blank periods. Results indicate that our optimized methods 

led to more robust and widespread activations of IT cortex, including areas typically affected by 

the ear canals. These activations were also more precisely aligned with anatomical scans due to 

image distortion correction. We believe that this combination of multi-echo gradient echo EPI 

and non-linear image reconstruction has the power to greatly improve investigations of the 

macaque ventral visual pathway at 3 T. 

Disclosures: M.A. Pinsk, None; M. Arcaro, None; C.S. Konen, None; X. Li, None; S. 

Kastner, None; S.J. Inati, None. 

Poster 






NIH S10 RR19186

Title: Adaptation of local field potentials in the fusiform face area (FFA) and fusiform body area 

(FBA) 

Authors: *R. T. ASH 1 , D. K. MURPHEY 1 , D. YOSHOR 2 , M. S. BEAUCHAMP 3 ; 

1 Neurosci., 2 Neurosurg., Baylor Col. Med., Houston, TX; 3 UT-Houston, Houston, TX 

Abstract: fMRI adaptation exploits the principle that neurons adapt to repeated presentations of 

effective stimuli to study the response properties of human visual areas. This allows studies of 

the selectivity of neurons in high-level visual areas, such as the fusiform face area, which have 

no clear homolog in non-human primates. Recording studies reveal important properties of 

adaptation in single neurons, but adaptation properties may vary between monkey and human 

visual areas. We sought to use intracranial recording from subdural electrodes implanted in 

human subjects to bridge the gap between primate single neuron and human fMRI studies of 

adaptation. 

fMRI using retinotopic mapping and functional localizers was performed before electrode 

implantation to identify visual areas in three subjects. Following electrode implantation, local 

field potentials (LFPs) were recorded as subjects viewed visual stimuli from different categories. 

In two subjects, electrodes located over the fMRI-defined fusiform face area (FFA) showed 

greater LFPs to face stimuli than to other stimuli, with a broad selectivity to many different 

faces. In one subject, an electrode located over the fusiform body area showed greater LFPs to 

bodies than to other stimuli. Early electrodes (V1/V2/V3) in each subject responded to many 

objects without category selectivity. 

To study adaptation, we presented foveal stimuli for 125 ms every 500 ms. The root-mean square 

power of the LFP was measured in a window of 50 to 500 ms following stimulus onset. 

Reference LFPs were recorded to preferred category stimuli (faces for FFA, bodies for FBA) 

whose preceding stimulus was not from the preferred category. Within-category LFPs were 

recorded to preferred category stimuli whose preceding stimulus was a different exemplar from 

the same category. Direct-repeat LFPs were recorded from preferred-category stimuli whose 

preceding stimulus was the exact same stimulus. 

Averaged across subjects, in FFA and FBA strong adaptation was observed to direct-repeat 

stimuli (85 uV vs. 110 uV for reference LFPs). Weaker adaptation was observed to withincategory 

stimuli (104 uV vs. 110 uV). In early visual areas, no adaptation was observed. 

Late but not early areas adapted to repeated presentation of complex face and body stimuli, 

suggesting that adaptation effects arise in late areas which have the greatest selectivity for the 

adapting stimulus. Because within-category adaptation is weaker than direct-repeat adaptation, 

individual neurons in FFA/FBA may be relatively sharply tuned, suggesting a model of an 

intermixed population of individual neurons with sharply tuned, but differing, response 

selectivities. 

Disclosures: R.T. Ash , None; D.K. Murphey, None; D. Yoshor, None; M.S. Beauchamp, 

None. 

Poster





Title: Human brain areas involved in perception of surface glossiness 

Authors: A. WADA 1,2 , Y. SAKANO 1,2 , *H. ANDO 1,2 ; 

1 Universal Media Res. Ctr., Natl. Inst. Inf. & Comm. Technol., Kyoto, Japan; 2 ATR Cognitive 

Inf. Sci. Labs., Kyoto, Japan 

Abstract: Glossiness is one of the object surface properties which can be used as a cue for 

identifying materials of objects in our daily life. Although psychophysical studies of glossiness 

perception are getting active recently, little is known yet about the neural substrates of the 

glossiness perception. Here we conducted functional magnetic resonance imaging (fMRI) 

experiments to clarify the neural correlates of the perception of surface glossiness. 

The stimulus was a computer-generated display which simulated a 3D object with a certain level 

of surface glossiness. The glossy surfaces usually contain bright specular highlights on them. In 

our experiments, in order to dissociate the neural activations evoked by glossiness from those 

evoked by luminance, we set four conditions of the stimulus with two levels of lighting as well 

as two levels of glossiness. The luminance of the stimulus was controlled so that the luminance 

of the specular highlight was higher in the [bright-lighting low-gloss] condition than in the [darklighting 

high-gloss] condition. If some brain areas are activated in the contrasts of the latter 

condition minus the former condition, as well as the high-gloss conditions minus the low-gloss 

conditions with the same lighting levels, the areas might be involved in perception of surface 

glossiness. In contrast, if other brain areas are activated in the contrasts of the bright-lighting 

minus dark-lighting conditions regardless of the gloss levels, the areas might be involved in 

perception of luminance or lighting level. In each condition, there were two slightly different 

levels of glossiness of the stimuli. We adopted a block-design paradigm, where in each trial these 

two stimuli were presented sequentially. The subjects pressed the buttons according to which 

stimulus appeared to be glossier. We scanned the whole brain area using a 3T scanner. 

The surface-based t-test analysis showed that the BA17 area was significantly activated in all the 

contrasts of bright-lighting minus dark-lighting conditions, while the ventral occipital region was 

significantly activated in all the contrasts of high-gloss minus low-gloss conditions. In addition, 

the posterior part of the intra-parietal sulcus and the transverse occipital region were activated in 

some contrasts of these conditions. These results suggest that the ventral occipital region, and 

possibly, the posterior part of the intra-parietal sulcus and the transverse occipital region are 

involved in the perception of the surface glossiness, while BA17 is involved in the perception of 

luminance or lighting level. 

Disclosures: A. Wada, None; H. Ando, None; Y. Sakano, None.

Poster 





Title: Enhanced activation in face regions during false categorisation of Mooney images as 

faces 

Authors: *E. GENC 1,2 , C. TILLMANN 1,2,3 , W. SINGER 1,2,4 , P. UHLHAAS 1,2,3 , A. 

KOHLER 1,2 ; 

1 Dept. of Neurophysiol., Max Planck Inst., Frankfurt am Main, Germany; 2 Brain Imaging Ctr., 

Frankfurt am Main, Germany; 3 Lab. for Neurophysiol. und Neuroimaging, 4 Frankfurt Inst. for 

Advanced Studies, J.W. Goethe Univ., Frankfurt am Main, Germany 

Abstract: There is a large body of evidence that several areas in the ventral temporal cortex are 

involved in face processing. Research has focused on a region in the fusiform gyrus that 

responds stronger to faces compared to other objects. To date, there are few studies addressing 

the whole network that subserves face perception. Here, we used fMRI and ambiguous face 

stimuli to study the neural correlates of categorizing stimuli as faces. 

Functional MR images with standard BOLD contrast were obtained from 22 healthy subjects. 

Using a rapid event-related design, subjects were presented with a random sequence of upright 

and inverted-scrambled Mooney stimuli. Mooney stimuli are degraded pictures of human faces 

in which contrast is maximized to black-and-white. Stimulus duration was 200 ms and subjects 

indicated whether they detected a face or not via button press after each stimulus. Subjects 

performed four functional runs within one scanning session. High-resolution anatomical images 

for coregistration and display of functional data were obtained from each subject in the same 

session. 

Four stimulus-response constellations were considered for analysis: i.e. upright stimuli 

recognized as faces („hits‟, 83% of the trials), upright stimuli classified as non-faces („misses‟, 

14%), inverted stimuli recognized as non-faces („correct rejections‟, 82%), and inverted stimuli 

classified as faces („false alarms‟, 14%). BOLD signal time courses for the different 

constellations were estimated with a deconvolution analysis, based on a general linear model. 

Following previous studies using Mooney stimuli (Andrews & Schluppeck, 2004), the difference 

in BOLD activity was computed for events when a face was perceived (hits and false alarms) 

compared to when no face was perceived (misses and correct rejections). This contrast revealed 

an increase in BOLD activity in the fusiform face area (FFA) (bilateral), the superior temporal 

sulcus (STS) (right hemisphere), and the occipital face area (OFA) (right hemisphere). Amygdala 

and orbito-frontal areas also showed significant increases in activity when a face was perceived.

BOLD signal time courses for the four constellations showed the strongest increase in activity 

for false alarms. To analyze the activity associated with perceived faces in more detail, we 

subsequently contrasted BOLD responses for hits with those for false alarms. We found that 

activity was significantly stronger for false alarms compared to hits in FFA, STS, OFA, parietal 

and frontal areas. Apparently, false classification of images as faces correlates with enhanced 

activation of cortical regions involved in face processing. 

Disclosures: E. Genc, None; C. Tillmann, None; W. Singer, None; P. Uhlhaas, None; A. 

Kohler, None. 

Poster 





Support: NIH Grant K23 MH076054 

NARSAD Young Investigator Award 

NIH Grant ROI EY017081 

Title: Monitoring personal space: regions with a „looming‟ bias in the human brain 

Authors: *B. S. CASSIDY 1 , X. YUE 2 , J. C. YOUNG 1 , S. HUANG 3 , S. K. LYNN 4 , R. B. H. 

TOOTELL 2 , D. J. HOLT 1 ; 

1 Psychiatry, Massachusetts Gen Hosp, Charlestown, MA; 2 Radiology, Massachusetts Gen. 

Hosp., Charlestown, MA; 3 Behavioral Neurosci., Boston Univ. Sch. of Med., Boston, MA; 

4 McLean Hosp., Belmont, MA 

Abstract: Studies in non-human primates have identified a network of polymodal regions 

(including the ventral intraparietal area (VIP), area 7b, premotor cortex and putamen) that 

responds preferentially to objects and movements near the body. This near-space network is 

proposed to protect the body surface from harm, and to facilitate contact with rewarding stimuli 

(e.g. food or a mate). Some neurons within this network respond selectively to 'looming' stimuli 

(e.g. expanding versus contracting), but the frequency and distribution of such responses are 

unknown. 

Using fMRI, we tested for regions in the human brain that exhibit a looming bias. Stimuli 

included faces, 1) to permit assessment of face-selective areas (e.g. FFA) for this bias and 2)

ecause in humans, a looming bias to an enlarging face (allowing for rapid classification of an 

approaching person as friend or foe) might be particularly strong. Thus, during functional MRI 

scanning, subjects were presented with approaching and receding computer-generated human 

faces (FaceGen, 6 identities, 3 males, each advancing or receding at ~ 1m/second) in two blockdesign 

experiments. In one experiment, subjects passively viewed the stimuli, while in another, 

subjects performed a task that ensured that attention was allocated similarly in the approach and 

recede conditions. FMRI responses during the approach versus recede condition were compared 

using a spherical coordinate system. 

Looming-selective areas included regions identified as near-space preferring in non-human 

primates: posterior parietal cortex (apparently the putative human homologue of VIP), premotor 

cortex and putamen. In addition, looming biases were found in lower-tier regions (V1, MT, 

dorsolateral occipital cortex) and in FFA, precuneus and posterior cingulate cortex. Results of 

the attention task experiment suggested that there was a relative enhancement of activity by 

attention in these looming biased areas. 

Overall, these findings suggest that within portions of the human visual system, there is a 

perceptual bias for symmetrically expanding stimuli, allowing objects on a collision course with 

the body to be automatically prioritized for further processing. 

Disclosures: B.S. Cassidy, None; X. Yue, None; J.C. Young, None; S. Huang, None; S.K. 

Lynn, None; R.B.H. Tootell, None; D.J. Holt, None. 

Poster 





Support: NIH Intramural Program Funds 

Title: Position information throughout the ventral stream 

Authors: *D. J. KRAVITZ, N. KRIEGESKORTE, C. I. BAKER; 

NIMH, NIH, Bethesda, MD 

Abstract: Despite behavioral evidence suggesting that object recognition is position invariant 

(e.g. Biederman and Cooper, 1991), recent studies in humans and non-human primates have 

reported strong effects of position in anterior regions of the ventral visual pathway thought to be 

critical for object recognition. Here we examine the effects of position at both a behavioral and 

physiological level in human participants. Specifically, we tested the hypothesis that object

ecognition might depend on representations that are tied to limited ranges of retinotopic 

positions. Thus, the same object presented in different positions might evoke only partially 

overlapping or even completely distinct representations, leading to distinct physiological 

responses in anterior object-selective cortex and limited behavioral position invariance. To test 

our behavioral hypothesis, participants were asked to indicate whether a briefly presented (66ms) 

stimulus was a whole or scrambled line drawing of an object. Many trials later (e.g. 256) 

participants saw the same stimulus again in either the same or a different position. The amount of 

priming for the repeated stimuli scaled with the distance between the initial and repeated 

positions. Priming was also qualitatively reduced when the positions were in different 

hemifields. To test our physiological hypothesis, we presented 24 of the objects used in our 

behavioral studies in each of the four quadrants (5° from fixation) in an event-related fMRI 

paradigm. The objects were divided evenly between six categories (faces, mammals, body parts, 

objects, tools, vehicles), allowing us to test for the relative effects of category and position 

changes. Participants performed a color-matching task on the stimuli and fixation cross. In a set 

of independently defined ventral stream ROIs (LO, PFs, FFA), we then established the similarity 

of responses to the stimulus conditions using multivariate pattern analysis. We correlated the 

response pattern of each of the 96 stimulus conditions (24 objects * 4 quadrants) with every other 

condition within each ROI. We found a very clear effect of hemifield, with far lower correlations 

between presentations of objects occurring in different hemifields. Response patterns could also 

be grouped within a hemifield into the upper and lower quadrants. In many cases, the effect of 

position was stronger than that of category. The results indicate the presence of positiondependent 

object representations in anterior regions of the ventral stream, and establish that these 

representations have predictable behavioral consequences. 

Disclosures: D.J. Kravitz, None; N. Kriegeskorte, None; C.I. Baker, None. 

Poster 





Support: NIH-NEI 

Title: A semantic encoding model that provides an unbiased description of object category 

selectivity in visual cortex 

Authors: *R. J. PRENGER 1 , T. NASELARIS 2 , K. KAY 3 , M. D. OLIVER 4 , A. T. VU 5 , J. L. 

GALLANT 2 ;

1 Dept Pysics, UC Berkeley, Berkeley, CA; 2 Helen Wills Neurosci. Inst., 3 Psychology, 4 Vision 

Sci. Group, 5 Bioengineering, Univ. of California, Berkeley, Berkeley, CA 

Abstract: While the posterior portion of human visual cortex consists of several distinct areas 

that represent structural aspects of the visual world, the anterior portion appears to represent 

semantic information about the categories of objects in a given image. Most previous studies 

have examined object-category selectivity in anterior visual cortex by measuring BOLD 

responses to images drawn from a small, pre-selected set of object categories. This classical 

approach makes it difficult to arrive at conclusions about object-category selectivity that 

generalize beyond the pre-selected categories, as it does not measure responses to other potential 

categories. To address this issue, we have developed a quantitative semantic encoding model that 

characterizes responses of voxels in object-category selective areas to arbitrary natural images. 

Importantly, the model allows for different degrees of object-category selectivity, and does not 

incorporate strong assumptions about the specific object categories that activate a given voxel. 

Thus, our model is far less prone to biases introduced by pre-selection of a small number of 

object categories. According to the semantic encoding model, each voxel performs an abstract 

mapping between a complex natural image and a set of object categories that are specific to the 

voxel. We use an Expectation-Maximization algorithm to learn the object categories represented 

by each voxel directly from BOLD activity elicited by ~2000 distinct natural images. We fit the 

semantic encoding model to ~25,000 voxels from visual cortex, encompassing areas V1 through 

V4, lateral occipital cortex, and posterior portions of ventral occipital-temporal cortex. We report 

three main findings. First, this model produces accurate predictions of voxel responses to natural 

images in higher-level visual areas near lateral occipital cortex. Second, the semantic encoding 

model provides far better predictions of responses in these areas than does a structural encoding 

model developed previously in our laboratory (see Kay et al., Nature, 2008, v.452, 352-355). 

Finally, inspection of the fitted semantic encoding models for voxels in the most posterior parts 

of object-category selective cortex reveal that they do not merely represent a single object 

category, but rather represent a probabilistic distribution of multiple object categories. 

Disclosures: R.J. Prenger , None; T. Naselaris, None; K. Kay, None; M.D. Oliver, 

None; A.T. Vu, None; J.L. Gallant, None. 

Poster 





Support: NIH / NEI

Title: A Bayesian decoder that reconstructs perceptual experiences from human BOLD activity 

Authors: *T. NASELARIS 1 , R. J. PRENGER 2 , K. KAY 3 , M. OLIVER 4 , J. L. GALLANT 1 ; 

1 Helen Wills Neurosci. Inst., 2 Physics, 3 Psychology, 4 Vision Sci. Group, Univ. CA, Berkeley, 

Berkeley, CA 

Abstract: We show that it is possible to reconstruct arbitrary natural images shown to a human 

observer, based only on measurements of BOLD activity evoked by those images. 

Reconstruction is based on a Bayesian framework that optimally combines various sources of 

information: measurements of BOLD activity from multiple visual areas of the human brain; 

encoding models that describe the relationship between structural or semantic scene information 

and evoked activity; and the prior probabilities of structural and semantic features in natural 

images. We employ two distinct encoding models: a structural model that describes evoked 

activity in early visual areas in terms of structural image features such as orientation, spatial 

frequency, and spatial location (see Kay et al., Nature, 2008, v.452, 352-355); and a semantic 

model that describes evoked activity in later visual areas in terms of semantic attributes of 

natural images. The structural and semantic encoding models leverage the distinct and 

specialized information represented at different levels of visual processing. When combined with 

the natural image prior these models produce reconstructions that capture both the structural and 

semantic composition of observed natural images. The modular nature of the Bayesian 

framework allows us to isolate the distinct contributions made by different brain areas to the 

representation of the visual scene, and to infer the likely role of prior experience in natural 

perception. Our decoding approach provides the first reconstructions of arbitrary natural images 

from human brain activity, and so provides a qualitative improvement over previous classifierbased 

studies. 

Disclosures: T. Naselaris , None; R.J. Prenger, None; K. Kay, None; M. Oliver, None; J.L. 

Gallant, None. 

Poster 






Title: How reliable are "context" effects in the parahippocampal place area?

Authors: *R. A. EPSTEIN, E. J. WARD; 

Dept Psychol, Univ. PA, Philadelphia, PA 

Abstract: The parahippocampal place area (PPA) is a region in human posterior 

parahippocampal cortex that responds preferentially to visual scenes (e.g. images of landscapes 

or cityscapes). Epstein and Kanwisher (1998) proposed that this scene-preferential response 

indicates the encoding of spatial layout information obtained from visual scenes. In contrast, Bar 

and colleagues propose that the PPA encodes "context", which they define as information about 

which objects "typically cooccur in the environment around us" (Bar et al., 2008). Evidence for 

the context hypothesis comes from reports that the PPA responds more strongly to "highcontext" 

than to "low-context" objects (Bar & Aminoff, 2003) and more strongly to famous faces 

(for which contextual associations are available) than to nonfamous faces (Bar et al., 2008). Here 

we examined the reliability of these two effects by scanning subjects with fMRI while they 

viewed high- and low-context objects, scrambled versions of the same stimuli, and famous and 

nonfamous faces. In contrast to earlier studies that used slow presentation rates (0.33-0.5 hz) 

which might have permitted subjects to form mental images of scenes, we presented stimuli at a 

faster presentation rate (1.25 Hz) in order to discourage scene imagery. Although we observed a 

small advantage in PPA response for high- vs. low-context objects, the same advantage was 

observed for uninterpretable scrambled versions of the same stimuli. Furthermore, contrary to 

previous reports, we found no evidence that famous faces activated the PPA more strongly than 

nonfamous faces. Based on these results, we hypothesize that previously reported "context" 

effects in the PPA might be explainable in part by scene imagery or by low-level physical 

differences in the stimulus sets rather than by activation of "contextual" representations. 

Disclosures: R.A. Epstein , None; E.J. Ward, None. 

Poster 





Support: NIMH Training Grant 

NIH EY07023 

Jim and Marilyn Simons Fund 

NEI R21EY015521-01

Title: Building vision: Splitting the difference between stimulus-driven and prior information 

Authors: *S. GORLIN 1 , J. SHARMA 1,2,3 , M. MENG 1 , H. SUGIHARA 1,2 , M. SUR 1,2 , P. 

SINHA 1 ; 

1 Brain and Cognitive Sci., 2 Picower Inst. for Learning and Memory, MIT, Cambridge, MA; 

3 Martinos Ctr. for Biol. Imaging, Massachusetts Gen. Hosp., Charlestown, MA 

Abstract: Prior information and experience with visual stimuli enhance our ability to recognize 

images, but where and how does this facilitation occur in the brain? Using degraded stimuli we 

can tease apart the effects of bottom-up visual processes, and top-down, experience-dependent 

processes, as prior knowledge of the fully coherent images makes them easier to recognize. 

Using machine learning algorithms like Support Vector Machines (SVM's), we can then quantify 

the amount of information a given brain region contains about the stimulus as the subject learns 

the coherent image. Here we show how distinct brain regions from prefrontal cortex to V1 

contain more information about degraded stimuli with prior knowledge, and that regional 

information in the brain persists in line with behavior. Interestingly, this effect depends critically 

on the complexity of the stimuli, so that prior information seems to be encoded over complex, 

real-world features, but not simple stimuli such as oriented gratings. 

Disclosures: S. Gorlin, None; J. Sharma, None; M. Meng, None; H. Sugihara, None; M. Sur, 

None; P. Sinha, None. 

Poster 





Support: NIMH/DIRP 


Title: Stimulus timing induced plasticity of face perception 

Authors: *D. B. MCMAHON, S. A. JENNINGS, D. A. LEOPOLD; 

Lab. Neuropsychol, NIH, Bethesda, MD 

Abstract: Object recognition in humans is extremely plastic, as evidenced by our ability to 

recognize and remember new faces after a few seconds of exposure. This form of visual learning

is presumably mediated by experience dependent changes in synaptic efficacy among neurons in 

ventral visual cortical areas that are involved in face recognition. In this psychophysical study we 

investigated the potential mechanisms that could underlie the plasticity of face perception in 

human observers. Specifically, we tested whether face perception can be modified through a 

conditioning protocol in which the relative timing of visual stimulation was manipulated. 

Previous whole cell recording studies have shown that the strength of synaptic connections in 

early sensory areas is modifiable in a manner that is highly sensitive to the relative timing of pre- 

and post-synaptic events. Synaptic strength increases when an EPSP is followed by a 

postsynaptic spike within a few tens of milliseconds, and decreases when an EPSP is preceded 

by a postsynaptic spike. This phenomenon, known as spike timing dependent plasticity, has been 

linked to changes in the stimulus selectivity of V1 neurons measured physiologically, and to 

shifts in perceived orientation of vertical lines in human subjects measured psychophysically. In 

the current study we asked whether face perception is similarly sensitive to stimulus timing. To 

test this hypothesis, we measured the psychophysical functions of human observers using a series 

of face stimuli generated by progressively morphing between two distinct images, face A and 

face B. In each trial the subject was presented with a parametrically morphed face and reported 

whether the face more closely resembled face A or face B. After determining the morph level 

that corresponded to the subjects' perceptual midpoint between A and B, the subjects were 

exposed to a block of conditioning stimulation that consisted of 100 pairings of two brief (10 ms) 

presentations of faces A and B. When face A was followed by face B with a stimulus onset 

asynchrony of 20 ms, the midpoint of the subjects' psychometric function shifted towards face B. 

When the order of stimulus presentation was reversed (B followed by A), the psychometric 

function was shifted in the opposite direction towards face A. The magnitude of perceptual shift 

decreased substantially or was abolished altogether when the stimulus onset asynchrony was 

increased to 60 ms. We conclude that face perception in humans is sensitive to stimulus timing 

dependent plasticity. This effect is likely due to changes in connection strength among neurons 

in ventral visual areas involved in face processing. 

Disclosures: D.B. McMahon , None; S.A. Jennings, None; D.A. Leopold, None. 

Poster 





Support: Natural Sciences and Engineering Research Council 

Michael Smith Foundation for Health Research

Title: Beamforming event-related fields to highly experienced single-letter and word stimuli 

Authors: *J. M. GASPAR, A. T. HERDMAN; 

Simon Fraser Univ., Burnaby, BC, Canada 

Abstract: Event-related fields (ERFs) were recorded using whole-head 

magnetoencephalography (MEG) to investigate the early spatiotemporal 

patterns of neural activity related to letter and simple word 

perception. Twelve right-handed adult participants were presented 

letters, non-letters, or symbols, either alone or in triplicate (letters 

spelling a word when presented in triplicate). We used a beamformer 

approach called event-related synthetic aperture magnetometry (ER-SAM) 

to localize the ERFs. Behavioural reaction times were significantly 

shorter to letter stimuli versus non-letter or symbol stimuli (p

Title: Discriminating your left foot from my right foot: body representations in human 

extrastriate visual cortex 

Authors: *A. CHAN, D. J. KRAVITZ, C. I. BAKER; 

NIMH, Bethesda, MD 

Abstract: Recent fMRI studies have identified two body selective regions in human ventral 

visual cortex: the extrastriate body area (EBA) and the fusiform body area (FBA). However, the 

nature of the representations in these regions remains unclear. Do these regions contain specific 

representations of individual body parts or is their response more categorical? These regions 

show a similar response magnitude to isolated body parts and headless bodies suggesting little 

sensitivity to specific body parts. However, very few studies have investigated whether this 

information is available in the spatial pattern of response across EBA and FBA. Here, we 

conducted a multivariate analysis on the response of visual body selective regions to investigate 

the effect of visual field, identity (laterality), and viewpoint on the representation of a single 

body part, the foot. Specifically, we conducted a visual field (left, right, 3.2° from fixation) x 

identity (left foot, right foot) x viewpoint (allocentric, egocentric) blocked-design fMRI 

experiment with 10 participants. Grayscale photographs of 20 individuals‟ right feet served as 

base images. To create left feet the images were flipped horizontally, while different viewpoints 

were created with vertical flips. The stimuli were projected onto a screen placed above the 

participants‟ knees, which obscured the view of their own feet. On each trial within a block, a 

single naked foot was presented and participants maintained fixation while performing a oneback 

task. Bilateral EBA and FBA were identified within each participant from an independent 

localizer scan, contrasting body parts with common objects. Within both ROIs we compared the 

pattern of activation across voxels for all eight conditions using correlations. The correlations 

between foot presentations in the same hemifield (contralateral or ipsilateral) were much higher 

than those across hemifields, although this effect was weaker in FBA than EBA. Furthermore, 

even within a given hemifield, the patterns of response differed between the egocentric and 

allocentric views of the left and right feet. In general, the patterns of response within a given 

hemifield were more distinct in the FBA than the EBA, suggesting a higher-level representation 

within the more anterior body selective region. Taken together, these results suggest that the 

representations of body parts in extrastriate cortex, at least for feet, are specific to the visual 

field, identity and viewpoint of the body part. 

Disclosures: A. Chan, None; D.J. Kravitz, None; C.I. Baker, None. 

Poster 





Title: The effect of priming on feature binding theory: stimulus feature distortion? 

Authors: *E. M. PARZYCH; 

Franklin & Marshall Col., Stone Harbor, NJ 

Abstract: TreismanAs Feature Binding Theory (FBT) states that visual features sharing the 

same spatial location are bound together through attention, resulting in the perception of a whole 

object. In situations where attention or focus is limited, binding errors are made that result in 

illusory conjunctions (1998). This study further explored the FBT and the formation of illusory 

conjunctions through temporal manipulation. A colored line acted as the prime, followed by a 

square of the opposite color serving as the target stimulus. Participants were asked to describe 

the color and shape they perceived. Results of multiple Related-Sample T-tests indicated 

significant shape and color distortion, with some indications of complete illusory conjunctions, 

where the target was reported to be the color of the prime. These results reproduce the Shape- 

Contrast Effect previously described by Suzuki and Cavanagh (1998) and also support 

TreismanAs FBT, although illusory conjunctions were not expected; color mixing was the 

primary predicted result. 

Disclosures: E.M. Parzych , None. 

Poster 






Title: The sum of its parts? Decoding the representation of multiple simultaneous stimuli in 

human object-selective cortex 

Authors: *S. P. MACEVOY, R. A. EPSTEIN; 

Dept. of Psychology, Univ. of Pennsylvania, Philadelphia, PA 

Abstract: During normal visual behavior, the brain must simultaneously encode information 

about multiple objects within the environment. Although previous fMRI studies have shown that 

information about the category of single objects is contained in distributed patterns of activity in

human object selective cortex, little is known about how multiple, simultaneous objects are 

encoded. Here we examined whether the neural representations of multiple simultaneouslyviewed 

objects reflect the patterns evoked by each component object, or whether they reflect 

entirely novel patterns of activity. We applied multivariate analyses to fMRI data collected while 

subjects viewed objects belonging to four categories (brushes, chairs, shoes, and cars), presented 

either singly or in pairs. Subjects performed a one-back task that required them to attend to all 

items on the screen (i.e. either a single item, or both items in a pair.) We found that information 

about the identities of object pairs in activity patterns of the lateral occipital complex (LOC) was 

finely grained, permitting pattern classification techniques to reliably discriminate among all 

pairs. To understand the rules governing the representation of object pairs, we performed a 

voxel-by-voxel analysis of the relationship between responses evoked by each pair and responses 

evoked by its component objects. Within individual voxels, variations in response strength across 

object pairs were well-predicted by the mean of responses to the corresponding component 

objects. We assessed the generality of this rule by measuring the ability of the classifier to 

identify object pairs after training with synthetic “pair” responses derived from the voxelwise 

means of the responses to component single items. Pair classification accuracy after training with 

synthetic pair input was comparable to accuracy after training with actual pair input. These 

results suggest that the representation of multiple objects in LOC is governed by response 

normalization mechanisms similar to those reported in single neuron studies of macaque 

extrastriate and inferotemporal cortices. 

Disclosures: S.P. MacEvoy, None; R.A. Epstein, None. 

Poster 





Title: TMS evidence for category-selectivity in lateral occipital cortex: Triple dissociation 

between faces, objects, and bodies 

Authors: *D. J. PITCHER 1 , L. CHARLES 2 , J. DEVLIN 1 , V. WALSH 1 , B. DUCHAINE 1 ; 

1 Univ. Col. London, Instit of Cog Neurosciene, London, United Kingdom; 2 École Normale 

Supérieure, Paris, France 

Abstract: Does object recognition depend on representations distributed across high-level visual 

cortex or on representations in specialized modules? Functional magnetic resonance imaging 

(fMRI) studies have identified category-selective areas, but these areas also carry information

about non-preferred objects. We tested the specificity of the occipital face area (OFA), the 

extrastriate body area (EBA), and lateral occipital area (LO) in the right hemisphere by 

delivering TMS during discrimination tasks involving faces, bodies, and novel objects. Target 

areas were individually localized using fMRI in fifteen subjects who were then tested in three 

separate TMS experiments. Each experiment compared performance on same-different matching 

tasks from two object classes while TMS was delivered over the cortical areas selective for those 

classes (e.g. face and object tasks with TMS over OFA and LO). Participants also performed 

each task without TMS to act as a behavioral baseline. All three experiments showed a double 

dissociation with task performance impaired only on the site selective for that object class. 

Namely, TMS over OFA impaired discrimination of faces but not objects or bodies; TMS over 

LO impaired discrimination of objects but not faces or bodies; TMS over EBA impaired 

discrimination of bodies but not faces or objects. These results indicate that recognition of items 

from each category relies on representations in the respective category-selective areas and not on 

a more distributed representation. 

Disclosures: D.J. Pitcher , None; L. Charles, None; J. Devlin, None; V. Walsh, None; B. 

Duchaine, None. 

Poster 





Support: CIHR 

Title: Cue-integration and competition between shading and structure-from-motion 

Authors: *R. FARIVAR-MOHSENI, A. CHAUDHURI; 

Dept. of Psychology, McGill Univ., Montreal, QC, Canada 

Abstract: Natural vision requires integration of several co-localized depth cues, such as shading, 

stereopsis, and structure-from-motion. Typically, experiments investigating cue combinations 

have utilized an additive or subtractive approach, by adding or removing additional depth cues 

from the structure of an object. Here we investigated the possibility that different cues may 

integrate across space in order to drive a coherent percept. If cue-chimeria is resolved prior to 

processes of object recognition, then illusory effects seen in object recognition, such as the 

composite face effect, should be unaffected by cue-chimeric stimuli. If, on the other hand, 

different cues give rise to separate object descriptors, then the composite face effect should

disappear for cue-chimeric stimuli. We designed such stimuli, combining structure-from-motion 

and shaded faces to form cue-chimeric stimuli. We found that although the composite face effect 

decreases for cue-chimeric stimuli, it remains significant, suggesting that multiple cues are 

integrated prior to recognition mechanisms. In a second experiment, we assessed whether 

conflicting global cues give rise to bistable percepts or whether the two cues compete. We found 

that the perception of a structure-from-motion face was completely obliterated by the presence of 

a phase-locked shaded face. Reducing the overlap between the two facial surfaces allowed both 

to be perceived, but without overlap, the shaded face dominated the percept, even at low contrast 

and luminance levels. This pattern of result suggests that multiple cues may integrate only if they 

relate to a single resolvable surface_as soon as additional surfaces need to be resolved from one 

another, surface competition may lead the strongest surface structure to win. 

Disclosures: R. Farivar-Mohseni , None; A. Chaudhuri, None. 

Poster 






NSF Grant BCS-0617688 

Klingenstein Fellowship in Neuroscience 

NIH Grant 1R21EY017741 

Title: Fiber tracts connecting mid-fusiform and lateral occipital face-selective regions 

Authors: *J. YOON, G. GOLARAI, M. BEN SHACHAR, R. F. DOUGHERTY, S. HONG, A. 

LIBERMAN, K. GRILL-SPECTOR; 

Psychology, Stanford Univ., Stanford, CA 

Abstract: Introduction: Functional neuroimaging studies of face processing reveal a network of 

regions involved in face processing including a region in the fusiform gyrus (FFA), a region in 

lateral occipital cortex (OFA) and a region in the posterior STS. Models of face processing 

suggest a projection from OFA to the FFA (e.g., Haxby et al, 2000). Lesions to the FFA or the 

OFA are sufficient to induce prosopagnosia even when the rest of the network is structurally

intact. This suggests that the FFA and OFA are necessary for successful face identification 

(Barton et al, 2002; Rossion et al, 2003; Steeves et al, 2006). However, little is known about the 

anatomical connections between the FFA and OFA. 

Method: We acquired functional magnetic resonance imaging (fMRI) and diffusion tensor 

imaging (DTI) data in healthy adults and adolescents on a 3T GE scanner. In fMRI, subjects 

viewed images of faces, objects, scenes and textures in short blocks. FFA was defined as a faceselective 

region (faces > scenes + objects; p

Authors: *S. IWAKI 1 , J. W. BELLIVEAU 2 ; 

1 Inst. Human Sci. & Biol Engineer, AIST, Ikeda, Osaka, Japan; 2 Mass Gen. Hosp., Boston, MA 

Abstract: Two-dimensional motions of the retinal images were known to be an important cue to 

perceive 3-D structure of objects. Recent neuroimaging studies implicate both the dorsal and 

ventral visual pathways, as well as motion perception area MT, in the perception of 3D structure 

from 2D motion (3D-SFM). However, the neural dynamics underlying the reconstruction of a 3D 

object from 2D optic flow is not known. Here we combined neuromagnetic 

magnetoencephalography (MEG) and hemodynamic functional MRI (fMRI) measurements to 

investigate the spatiotemporal brain dynamics during 3D-SFM. 

We manipulated parametrically the coherence of randomly moving groups of dots to create 

different levels of 3D perception and to study the associated changes in brain activity. 

Neuromagnetic signals were measured with a 306-channel MEG system. The fMRI scanning 

was conducted using a 3-T scanner with the EPI imaging sequence. The results of the fMRI 

analysis were used to impose plausible constraints on the MEG inverse calculation using the 

„weighted‟ minimum-norm approach to improve spatial resolution of the spatio-temporal activity 

estimates. 

The infero-temporal (IT), parieto-occipital (PO), and intraparietal (IP) regions showed at 

different latencies increased neural activity during highly coherent motion conditions in which 

subjects perceived a robust 3D object. Causality analysis between these regions indicated a 

significant causal influence from IP to IT and from IT to PO only in conditions where subjects 

perceived a robust 3D object. Current results suggest that the perception of a 3D object from 2D 

movement includes integration of global motion and 3D mental image processing as well as 

object recognition that are accomplished by interactions between the dorsal and ventral visual 

pathways. 

Disclosures: S. Iwaki , None; J.W. Belliveau, None. 

Poster 

261. Object and Faces: Neuronal Representation I 





Title: Object representations in monkey posterior parietal cortex

Authors: *C. S. KONEN 1,2 , M. A. PINSK 1,2 , M. ARCARO 1,2 , X. LI 1,2 , S. J. INATI 3 , S. 

KASTNER 1,2,4 ; 

1 CSBMB, 2 Dept. Psychology, Princeton Univ., Princeton, NJ; 3 Ctr. for Brain Imaging, New 

York Univ., New York City, NY; 4 Princeton Neurosci. Inst., Princeton Uni., Princeton, NJ 

Abstract: The visual system is broadly divided into two separate pathways (Ungerleider & 

Mishkin 1982), a dorsal stream to posterior parietal cortex (PPC) that is associated with visuallyguided 

action, and a ventral stream to temporal cortex that is involved with object recognition. 

Despite this classical view, we have recently reported object-selectivity, and size and viewpoint 

invariance in areas along the dorsal pathway (Konen & Kastner 2008). Here, we investigated 

object-selective responses in monkey PPC using fMRI adaptation and compared them with those 

in human PPC. 

Two monkeys viewed geometric 2D-objects that alternated with scrambled pictures while 

maintaining fixation (Siemens Allegra 3T). In the adapted condition, one object was presented 

15 times. In the non-adapted condition, 15 different objects were presented once. In the sizeinvariance 

experiment, the adapted condition consisted of 15 presentations of one object in 

different sizes. Regions of interest were defined on the basis of anatomical locations (Saleem & 

Logothetis 2007). 

The comparison between adapted and non-adapted conditions revealed object-selective 

responses in monkey V4, TE, and TEO in the ventral pathway and LIP and VIP in the dorsal 

pathway. Similarly, ventral V4 and LOC as well as dorsal IPS1 and IPS2 in humans exhibited 

object-selectivity. In contrast to humans, intermediate areas of the dorsal pathway did not 

respond object-selective in monkeys. Further, invariant responses evoked by 2D-objects 

presented in different sizes were found in monkey LIP and VIP as well as in human IPS1 and 

IPS2 (in addition to ventral stream areas). Taken together, the organization of the ventral 

pathway in humans and non-human primates appears to be similar, while the organization of 

dorsal pathway may be fundamentally different in the two species. 

Disclosures: C.S. Konen, None; M.A. Pinsk, None; M. Arcaro, None; X. Li, None; S.J. Inati, 

None; S. Kastner, None. 

Poster 





Support: NIH Grant EY011797

Title: Structural basis of IT responses to natural objects 

Authors: *K. BOWMAN 1 , E. T. CARLSON 2 , C. E. CONNOR 1 ; 

1 Dept. of Neurosci., 2 Dept. of Biomed. Engin., Johns Hopkins Univ., Baltimore, MD 

Abstract: The fundamental question concerning higher-level ventral pathway cortex is how 

neurons encode objects. This question is usually addressed by studying selective neural 

responses to natural objects, but the neural coding scheme that explains this selectivity remains 

unknown. We sought to understand responses to natural objects in terms of neural tuning for 

geometric object structure. We studied responses of neurons in CIT/AIT (central and anterior 

inferotemporal cortex) of awake macaque monkeys performing a fixation task. The same 

neurons were tested both with natural object photographs and with abstract shapes that evolved 

in response to neural feedback. The natural object set comprised 60 stimuli in 8 categories: faces, 

hands, bodies, fruits, animals, manmade objects, plants, and predators. Abstract shapes were 

constructed with piecewise Bezier spline functions that defined external boundary shape and 

internal contrast. For each neuron, an initial generation of 40 abstract stimuli was created by 

randomizing parameters controlling spline function shape. Subsequent stimulus generations 

included partially morphed descendants of higher response stimuli from previous generations. 

Descendants were probabilistically morphed at the local and global levels. Over the course of 6-8 

generations, this method produced dense sampling in the tuning range of the cell. Convergence 

of independent lineages confirmed that this evolutionary method discovered a global maximum 

in most cases. Dense sampling with multiple lineages allowed us to fit linear/nonlinear geometric 

models describing neural sensitivity to shape. Application of these models to the natural stimulus 

responses showed that in many cases those responses can be understood in terms of metric 

tuning for geometric object structure. 

Disclosures: K. Bowman, None; E.T. Carlson, None; C.E. Connor, None. 

Poster 



Program#/Poster#: 261.4/CC1 


Support: Max Planck Society 

RA1025-1/2 

DFG SFB550

Title: Simultaneous LFP recordings in primate V4 and PF cortex during visual perception of 

natural images 

Authors: *S. LIEBE, N. K. LOGOTHETIS, G. RAINER; 

Dept Physiol Cognitive Proc, MPI Biol. Cybernetics, Tuebingen, Germany 

Abstract: Both the extrastriate area V4 and the dorsolateral prefrontal cortex (PFC) play an 

important role in the processing of visual information. Their role can be best understood by 

examining single unit responses as well as local field potentials (LFPs) that are indicators of 

integrative dendro-somatic events. Here, we examine tuning properties of LFPs simultaneously 

in both areas during a visual memory task. We first focus on the visual evoked potential (VEP). 

We employed a set of natural images as stimuli that were shown in chromatic and achromatic 

conditions at different degradation levels. 

A majority of recorded sites (110/151 in V4, 91/140 in PF in two monkeys) showed significant 

deflections in stimulus-locked averaged LFP waveforms or VEPs in both areas. Across sites, the 

VEP in V4 tended to exhibit two peaks with approximate latencies of 80 and 200 ms. In PFC the 

VEP was characterized by a unimodal peak at a latency of about 200ms, which corresponded in 

terms of its onset dynamics very closely to the late peak in V4 (200 msec). This suggests that the 

arrival of sensory information is reflected in an early peak in V4, and that both areas process 

information jointly during a period characterized by a late VEP peak. 

VEPs in both regions exhibited tuning for stimulus identity and color. In V4 the amplitude of 

both peaks carried information about stimulus identity (p

Support: Sofja Kovalevskaja Award of the Alexander von Humboldt Foundation and the BMBF 

the German Ministry of Science (Grant 01GO0506, Bremen Center for Advanced 

Imaging) 

the German Science Foundation (DFG FR1437/3-1) 

Title: A direct link between face-selective regions in temporal and prefrontal cortex 

Authors: *S. MOELLER 1,2 , W. A. FREIWALD 1,2 , D. Y. TSAO 1,2 ; 

1 Inst. Brain Res., Univ. Bremen, Bremen, Germany; 2 Ctr. for Advanced Imaging, Univ. of 

Bremen, Bremen, Germany 

Abstract: Macaques possess several distinct patches of face-selective cortex. One set of six face 

patches is arranged along the anterior-posterior axis of the temporal lobe. This set includes one 

posterior patch, two middle patches, and three anterior patches, including one patch anterior and 

lateral to the AMTS („AM‟). Another set of face-selective patches can be found in the frontal 

lobe. This set includes a bilateral pair of patches in the lateral orbital sulcus (PO), a rightlateralized 

patch in the inferior convexity (PL), and, in some animals, a patch within the anterior 

bank of the lower ramus of the arcuate sulcus. 

We have previously shown that the inferotemporal patches are strongly and specifically 

interconnected (Moeller, Freiwald, Tsao (2008), Science, in press). Here, to identify connections 

of the temporal face patches with prefrontal cortex, we microstimulated the most anterior 

temporal face patch, AM, in one rhesus macaque while performing fMRI (3 T, Siemens Allegra, 

with Sinerem (Guerbet) contrast agent) to image activated areas across the anterior temporal and 

the frontal lobes. 

We first identified temporal and prefrontal face patches in this macaque by contrasting activation 

to faces versus non-face objects. We recorded from the anterior face patch AM to 

physiologically confirm its face specificity. We then electrically stimulated AM inside the 

scanner. During stimulation the monkey performed a simple fixation task. Stimulation of AM 

produced strong, spatially-specific activation in prefrontal cortex, restricted to the location of PL 

and PO within the same hemisphere. 

Our results indicate that at least one of the macaque temporal lobe face patches is directly 

connected to face patches in prefrontal cortex. Thus the temporal and prefrontal face patches 

likely form a unified system for face processing. 

Disclosures: S. Moeller , None; W.A. Freiwald, None; D.Y. Tsao, None. 

Poster 





Support: NINDS K08 NS048871 

Title: Face-category selective neuronal activity in human‟s temporal cortex 

Authors: *A. E. IPATA 1,2 , J. BESLE 1,3 , A. L. GEE 1,2 , C. A. SCHEVON 1,4 , R. EMERSON 1,4,5 , 

J. CAPPELL 1,4,5 , R. R. GOODMAN 1,6 , G. M. MCKHANN 1,6 , A. WAZIRI 1,6 , M. E. 

GOLDBERG 1,4 ; 

2 Neurosci., 3 Psychiatry, 4 Neurol., 5 Pediatrics, 6 Neurolog. Surgery, 1 Columbia Univ., New York, 

NY 

Abstract: Primates can efficiently determine identities, emotions and intentions by looking at 

faces. In human as in monkey temporal cortex, there are classes of neurons that respond better to 

faces than other objects. However, it is unclear whether this selectivity is purely sensorial or also 

relates to the cognitive meaning of the visual stimulus. To answer this question, we recorded one 

epileptic patient implanted both with a 4x4mm 2-dimensional intracortical array of 96 

microelectrodes (Cyberkinetics, Foxboro, MA) in posterior temporal cortex and with subdural 

macro-electrode grids over the major part of the temporal cortex. The patient performed a 

modified match to sample task in which she had to maintain sustained attention to the category 

of an object and not to a specific object identity. Each trial consisted of a visual cue in one of 5 

possible categories (fruits, cars, furniture, buildings and faces) sustained for 1000 ms, followed 

by a sequence of 1 to 7 non-face objects (“delay period”). The subject was required to report 

when an object of the same category of the cue appeared. Action potentials were detected and 

classified offline. We analyzed the activity of 97 cells that were classified as visually responsive 

(out of 165 identified neurons). 81/97 neurons responded better to the appearance of faces than 

other objects (non-parametric Wilcoxon p





Support: DIRAC 

HFSP 

GSKE 

EF 

GOA 

Title: Position invariance of adaptation of macaque inferior temporal spiking activity and local 

field potentials 

Authors: *W. DE BAENE, R. VOGELS; 

Lab. voor Neuro- en Psychofysiologie, K.U. Leuven, Leuven, Belgium 

Abstract: In many cortical areas, repeated presentation of a specific stimulus is commonly 

associated with a reduced neuronal response, i.e. adaptation. Recently, two single-cell studies in 

IT cortex suggested that adaptation is stimulus selective: Sawamura et al (2006) found much 

smaller adaptation when two highly dissimilar, but equally effective images were presented 

compared to the repetition of the same image. De Baene and Vogels (2007) used parametric 

shape sets and found that repetition of a suboptimal stimulus reduced the neuronal response 

strength more than when this stimulus followed the preferred stimulus. In these studies, both the 

first and second stimulus (the adapter and test stimulus, respectively) were presented foveally. In 

the present study, we presented both stimuli at different positions, to examine the effect of 

position on adaptation both for single-cell spiking activity and local field potentials (LFPs). 

Out of 4 parametric sets of 6 shapes (each created by morphing between 2 complex 3D shapes), 

we selected two stimuli from within one shape set for which the neuron was judged to be 

selective for the subsequent test. In this subsequent task, the two monkeys needed to passively 

fixate the fixation point while sequences of 2 stimuli (stimulus durations 300ms, ISI 300ms) 

were presented. The shapes were presented at an eccentricity of 4° above or below the fixation 

point. In this fully crossed design, both selected stimuli could serve as an adapter and as a test 

stimulus, and they could be presented at both positions, resulting in 16 different conditions. We 

restricted our analyses to conditions in which the most effective stimulus served both as the 

adapter and test stimulus. 

Our results (n = 40 neurons) show that, although the response reduction was less when adapter 

and test stimulus were presented at different positions compared to when both stimuli were 

presented at an identical position, adaptation did not disappear completely in the former case.

Additionally, the degree of adaptation was dependent on the relation between the positions of the 

adapter and the test stimulus: we found more response reduction when a stimulus at the worst 

position (i.e. where the response to the adapter was lowest) was repeated compared to when the 

test stimulus at the worst position followed the adapter at the best position (i.e. where the 

response to the adapter was highest). These results were found both for the spiking activity as for 

the power in the gamma band (60-100Hz). These results suggest that adaptation depends on the 

relation between the positions of the adapter and test stimulus in a similar way as it does on the 

relation between shape features of the first and second stimulus. 

Disclosures: W. De Baene , None; R. Vogels, None. 

Poster 





Support: Human Frontiers Science Program 

NIH-NCRR 

McKnight Foundation 

Whitehall Foundation 

Title: Stimulus duration and stimulus content interact in neural responses to images in 

inferotemporal cortex during the classification of visual stimuli by macaque monkeys 

Authors: Y. LIU, *B. JAGADEESH; 

Physiol & Biophysics Dept., Univ. Washington, Seattle, WA 

Abstract: Responses of inferotemporal neurons, which underlie object vision, change 

dynamically during the static presentation of a photographic image. The changing dynamics of 

this processing suggest that visual stimulus perception depends on both the content and duration 

of a visual image, and that short duration stimuli might produce qualitatively different perceptual 

experiences of the stimulus compared to long duration stimuli. To test this hypothesis we 

recorded behavior and neural responses in rhesus macaques while monkeys performed a 

classification task in which both the content of the image and the duration of the image were 

manipulated. Image durations were chosen from a range of durations near threshold performance

for classifying photographic images (Allred and Jagadeesh, 2007). Image content was varied by 

morphing between the two photographic images that served as the classification choices in a 2alternative-forced-choice 

delayed match to sample task (2AFC-DMS task) (Liu and Jagadeesh, 

2008). In the 2AFC-DMS task, a sample stimulus appeared, was masked, and followed after a 

delay by two choice images; the monkey‟s task was to select the image that matched the sample 

from the choices by making a saccade to it. The sample images were identical to one of the two 

choice images, or were one of nine morphed variants between the two choice images (and could 

be displayed for different stimulus durations). In this task, classification depended on the 

duration of the stimulus as well as the content of the image (the morph level). The proportion of 

choices of an image changed linearly as a function of morph level for short duration stimuli. As 

the image was shown for longer durations, the classification became more categorical, with 

images of several morph levels resulting in the same choice. Simultaneously collected neural 

responses showed a similar interaction between stimulus content (morph level) and stimulus 

duration. In addition, stimulus duration integration times were short, suggesting that in the 

presence of a masking stimulus, integration of long duration responses did not substantially 

improve neural selectivity for a stimulus that produced a small neural response. These data 

contribute to our understanding of the neural dynamics of object vision that follows the rapid, 

instantaneous impression of a visual stimulus. 

Disclosures: Y. Liu, None; B. Jagadeesh , None. 

Poster 






Title: Investigating category-selective regions in monkey inferior temporal cortex using fMRI 

and single unit recordings 

Authors: N. MALECEK 1 , *A. H. BELL 1 , F. HADJ-BOUZIANE 1 , R. B. H. TOOTELL 1,2,3 , L. 

G. UNGERLEIDER 1 ; 

1 Lab. Brain & Cognition, NIMH/NIH, Bethesda, MD; 2 Athinoula A. Martinos Ctr. for Biomed. 

Imaging, Massachusetts Gen. Hosp., Charlestown, MA; 3 Dept. of Radiology, Harvard Med. Sch., 

Boston, MA

Abstract: Electrophysiological recordings in monkey inferior temporal (IT) cortex have 

frequently revealed neurons selective for complex visual stimuli. More recently, functional 

magnetic resonance imaging (fMRI) has identified regions within IT cortex selective for 

categories of visual stimuli (e.g., faces, body-parts). In comparing the distribution of fMRIidentified 

category selective regions with single unit recordings, the present study sought to 

understand the neuronal properties of category-selective cortex, as well as the relationship 

between single unit activity and the BOLD signal obtained with fMRI. 

Regions selective for faces, body-parts, places, and objects were identified in monkeys in a 

previous study using fMRI. Subsequently, recording chambers were implanted over areas TE and 

TEO in the left hemisphere of two monkeys. This placement allowed access to two fMRIidentified 

face-selective regions as well as adjacent regions selective to other categories. 

Monkeys were trained using a rapid serial visual presentation task, which required them to 

maintain fixation while 100 images comprising five semantic categories (faces, body-parts, fruit, 

objects, places) were presented in random order. 

At present, we have recorded 391 neurons from 19 sites throughout IT cortex. Approximately 

75% of neurons encountered were responsive to visual stimuli. Interestingly, the majority of 

these neurons were strongly category-selective (~70%) but showed relatively weak stimulus 

selectivity. Face-selective neurons were the most prevalent type encountered (~34%), followed 

by body-part selective neurons (~29%). The general bias suggests an encoding scheme 

encompassing semantic classification. In recordings so far, the distribution of category-selective 

neurons in IT cortex did not strictly correspond to the boundaries of category-selective regions 

identified by fMRI, suggesting that single unit activity may not, by itself, account for the BOLD 

response that defines these regions. 

Disclosures: N. Malecek, None; A.H. Bell , None; R.B.H. Tootell, None; L.G. Ungerleider, 

None; F. Hadj-Bouziane, None. 

Poster 





Title: Different elements of time-varying neural activations in response to a face stimulus 

Authors: *E. TANAKA 1 , K. INUI 1,2 , T. KIDA 1,3 , R. KAKIGI 1,2 ; 

1 NIPS, Okazaki, Japan; 2 Grad. Univ. for Advanced Studies, Hayama, Japan; 3 NIH, Bethesda, 

MD

Abstract: Face stimuli involve many visual processing. Segregation of different components of 

neural activity evoked by a face stimulus would promote the understanding of mechanisms 

underlying face recognition. To segregate luminance-, face-, and non-specific components, we 

recorded cortical responses to face appearance (Onset), disappearance (Offset), and change 

(Change) using magnetoencephalography. Activity in and around the primary visual cortex 

showed luminance-dependent behavior. Any of the three events evoked activity in the middle 

occipital gyrus (MOG) at 150 ms and temporo-parietal junction (TPJ) at 250 ms after onset. 

Onset and Change activated the fusiform gyrus (FG), while Offset did not. These results show 

the responses of MOG and TPJ to be associated with non-specific processes such as responses to 

abrupt changes. Activity in FG seemed to be related to object recognition though its faceselectivity 

was unclear. Therefore, this study demonstrated at least four different elements of 

time-varying neural activations in response to a face stimulus. 

Disclosures: E. Tanaka, None; K. Inui, None; T. Kida, None; R. Kakigi, None. 

Poster 





Support: MEXT (17022025) 

CREST 

Title: Spatial frequency tuning in face responsive neurons of the macaque temporal cortex 

Authors: *M. INAGAKI, I. FUJITA; 

Grad Sch. Frontier Biosci., Osaka Univ., Toyonaka, Japan 

Abstract: Most face responsive neurons in higher visual areas of the monkey temporal cortex 

are tuned to the spatial frequencies (image-based spatial frequency, characterized by cycles per 

image) of a face at a certain image size. A small face image produces a spectrum of relatively 

high spatial frequencies in the retina (retina-based spatial frequency, characterized by cycles per 

degree of visual angle), whereas a larger face image produces a spectrum of relatively low retinabased 

spatial frequencies. If face responsive neurons are purely tuned to image-based spatial 

frequencies and not to retina-based ones, changes in retinal size of face image, which accompany 

changes in the spectrum of retina-based spatial frequencies, should not influence the neurons‟ 

tunings to image-based spatial frequencies. Here we tested whether face responsive neurons in

the temporal visual cortex of monkeys are tuned to image-based spatial frequencies or retinabased 

spatial frequencies by changing the retinal size of the face images. We used a face image 

set that combined the center image-based spatial frequencies (2, 2.8, 4, 5.7, 8, 11.3, 16 

cycles/image) of 7 band-pass filters and 5 image sizes (3.8, 5.4, 7.7, 11, 15.3 degrees). We 

recorded 60 face responsive neurons in the inferior temporal gyrus and the upper and lower 

banks of the superior temporal sulcus (A18 - A24) from an awake, fixating monkey (Macaca 

fuscata). Among them, 43 neurons were selective for center image-based spatial frequencies in at 

least one image size. We examined the effects of image size on image-based spatial frequency 

tuning by assessing the shift in preferred image-based spatial frequencies. Some neurons showed 

a preferred image-based spatial frequency across different image sizes. Others showed a shift in 

the preferred image-based spatial frequencies depending on image size. Overall, despite a wide 

variety of shifts in the tuning curves, image-based spatial frequency tuning as a whole depended 

only weakly on image size. Furthermore, the neurons were tuned to image-based spatial 

frequency rather than retina-based spatial frequency. The results suggest that the neural 

representation of the face in the temporal visual cortex is made by integrating various ranges of 

retina-based spatial frequencies and is minimally dependent on the retinal size of the face image. 

Disclosures: M. Inagaki , None; I. Fujita, None. 

Poster 





Support: DARPA 

Lions Foundation 

NDSEG Fellowship 

Title: Neural representations of cluttered scenes in macaque ventral visual cortex 

Authors: *E. M. MEYERS 1 , H. EMBARK 2 , T. SERRE 1 , G. KREIMAN 3 , W. A. FREIWALD 2 , 

T. POGGIO 1 ; 

1 Brain & Cognitive Sci., MIT, Cambridge, MA; 2 Ctr. for Cognitive Science, Brain Res. Inst., 

Univ. of Bremen, Bremen, Germany; 3 Ophthalmology and Program in Neurosci., Children's 

Hosp. Boston, Harvard Med. Sch., Boston, MA

Abstract: Humans and other primates can rapidly categorize objects even when they are 

embedded in complex visual scenes (Thorpe et al. 1996, Delorme et al. 2000). Studies by Serre 

et al. (2007) have shown that the ability of humans to detect animals in brief presentations of 

natural images decreases as the amount of clutter increases, and that a feedforward 

computational model of the ventral visual system originally developed to account for 

physiological properties of neurons, shows a similar pattern of degraded classification 

performance with increasing clutter. Motivated by these studies, we recorded single and multi 

unit spiking activity from macaque superior temporal sulcus (STS) and anterior inferior temporal 

cortex (AIT), as a monkey passively viewed images of natural scenes. The stimuli consisted of 

600 images of animals in natural scenes, and 600 images of nature scenes without animals, 

captured at four different distances and were the same images used by Serre et al. to allow for a 

direct comparison between human psychophysics, computational models, and neural data. We 

also recorded fMRI signals from 3 macaques to a subset of these stimuli. To analyze the data, we 

applied population „readout‟ methods (Hung, Kreiman et al. 2005) to decode from the neural 

activity whether an image contained an animal. Results from analyzing both the fMRI signals 

and the firing rates of neurons showed a similar pattern of degraded decoding performance with 

increasing clutter as was seen in the human psychophysics and computational model results. 

Disclosures: E.M. Meyers , None; H. Embark, None; T. Serre, None; G. Kreiman, 

None; W.A. Freiwald, None; T. Poggio, None. 

Poster 





Support: Max Planck Society 

DFG RA 1025/1-2 

Title: Timing of local field potential (LFP) responses in primate inferior temporal (IT) cortex 

distinguishes between monkey faces, human faces and objects 

Authors: *G. R. SIGALA ALANIS, J. VEIT, N. LOGOTHETIS, G. RAINER; 

Phys Cognitive Processes, MPI Biol Cybernetics, Tuebingen, Germany 

Abstract: It is well established that the inferior temporal (IT) cortex of the macaque monkey 

contains cells that respond selectively to faces. How information about faces is represented and

organized at the network level remains largely unknown. Here we simultaneously recorded local 

field potentials (LFPs) and spiking activity in the IT cortex of two monkeys fixating at realistic 

human, monkey faces and objects, to investigate the neural representation of these stimulus 

classes. Our previous results indicate that spike information recorded from single neurons clearly 

differentiates between these three classes of stimuli. Here we investigate whether LFPs also 

contain information about these three types of stimuli. From the visual evoked potentials (VEP), 

we reliably and automatically extracted (in 44/65 sites in monkey M1 and in 20/68 sites in 

monkey 20) different features that convey time or amplitude information about stimulus class. 

Specifically, we focused on the timing and amplitude of the so called “N70” (negative deflection 

after about 70 ms of stimulus presentation), “P100” (positive deflection at about 100 ms) and 

“N170” (positive deflection at about 170 ms) components of the VEP. We grouped the VEPs 

into three classes according to the stimulus: humans, monkeys and objects VEPs. We found in 

both monkeys that the onset time of the face VEPs was significantly faster compared to the 

object VEPs (ttests, P

monkeys were exposed first to human faces for a week. Soon after, their preference changed 

drastically. They preferred upright human faces but lost preference for monkey faces. 

Furthermore, they lost preference for human faces presented in reversed contrast. Using fMRI, 

their brain activity for face stimuli was measured while they viewed photographs of human 

faces. Control monkeys were also scanned while viewing photographs of monkey faces. We 

found face selective regions along the STS in both monkeys. The location of the regions was 

comparable to each other. These results indicate that face deprivation, therefore, seems to delay 

the normal maturation and keeps the face-processing system in an immature state. 

Disclosures: Y. Manaka, None; Y. Sugita , None. 

Poster 





Support: HHMI Grant 52003749 

Title: Spatiotemporal analysis of scanpaths in monkeys viewing images of conspecific facial 

expressions 

Authors: *R. R. GIBBONI 1 , C. M. LAINE 2 , P. E. ZIMMERMAN 1 , K. M. GOTHARD 2 ; 

2 Dept. of Physiol., 1 Univ. Arizona, Tucson, AZ 

Abstract: Visual exploration of images progress through a series of saccades and fixations 

(scanpaths), which are the final output of a vast network of structures that guide the appropriate 

temporal and spatial allocation of visual attention. Dysfunction in this network leads to abnormal 

visual scanning, particularly when viewing socially relevant stimuli such as faces. 

Neurobehavioral studies aimed at understanding how various brain regions contribute to face 

viewing require a system of quantifying scanpaths. Typical scanpath measures include total 

scanpath length, number and duration of fixations, and total amount of time spent looking at 

specific face regions (e.g. eyes, mouth), but fail to consider the temporal pattern of region 

visitation. We evaluated the probability of the subject exploring a particular face region over the 

3 s course of an image presentation as well as the temporal order of region visitation. We 

analyzed scanpaths obtained from three adult male rhesus macaques (Macaca mulatta) as they 

viewed conspecific faces displaying appeasing, aggressive, and neutral facial expressions with 

different gaze directions. Preliminary results confirm previous findings of a general trend 

towards eyes being the first and most frequently visited target, however, deviations from this

trend occurred on an individual basis for certain facial expression-gaze combinations. For two 

monkeys, the order of region visitation was widely variable over many trials, whereas the third 

monkey produced highly stereotypical patterns of face scanning, particularly within the first 

second of image viewing. For example, the probability of this monkey exploring the eye region 

of a direct threat 300ms after image onset was 0.75 compared to 0.20 after 500ms (pooled over 8 

images, about 45 presentations each). This decrease in eye looking predictably corresponded to 

an increase in mouth looking. For all three monkeys, the probability of looking at a particular 

face region became relatively fixed after the first second, however these probabilities for pooled 

data were not necessarily predictive of the individual scanpaths. The amount of variability 

between monkeys suggests that large differences between individuals should be considered in the 

design of experiments where scanpaths are the dependent variable. 

Disclosures: R.R. Gibboni , None; C.M. Laine, None; P.E. Zimmerman, None; K.M. 

Gothard, None. 

Poster 





Support: Canadian Foundation for Innovation, Leaders Opportunity Fund 

Max Planck Society 

NSERC Discovery Grant 

Alfred P. Sloan Fellowship 

Title: The influence of saccadic eye movements on neural activity in the temporal lobe 

Authors: S. OVAYSIKIA 1 , A. M. BARTLETT 1 , N. K. LOGOTHETIS 3,4 , *K. L. HOFFMAN 2 ; 

1 Psychology Dept., 2 Psychol Dept, York Univ., Toronto, ON, Canada; 3 Max Planck Inst. for 

Biol. Cybernetics, Tuebingen, Germany; 4 Univ. of Manchester, Manchester, United Kingdom 

Abstract: The responses of cells in the temporal lobe to faces and objects has been the topic of 

extensive research; however, the eye movement constraints during these experiments varies 

considerably. Some require central eye fixation during image presentation, whereas others allow 

unconstrained viewing of stimuli. Here, we explore the influence of saccadic eye movements on

neural responses in the temporal lobe. Awake macaques were presented with a set of objects or 

faces while we recorded simultaneously from neurons in the upper-bank of the superior temporal 

sulcus (STS) and in the auditory cortex. Responses during three categories of saccades were 

evaluated: 1) those occurring prior to fixation onset (spontaneous saccades), 2) those made at the 

time of central cue fixation (visually-guided saccades) and 3) saccades made during scanning of 

an image (image-guided saccades). Saccade modulation was observed for all categories of 

saccades, though not always in the same cells, and irrespective of a cell‟s image selectivity. 

Single unit responses typically occurred during negative components of the local field potential 

(LFP). Whereas broad peri-saccadic modulation was seen in both STS and auditory cortex, only 

cells in STS showed precise, short-latency responses at the termination of a saccade. The results 

of our study demonstrate that even those cells that respond to face and object stimuli can show 

modulation to eye movements. In addition, saccade modulation is not limited to classicallydefined 

image-responsive regions of the brain: broad peri-saccadic modulation was also 

observed in auditory cortex. The observation that actions can influence sensory responses will be 

considered within the framework of embodied cognition. 

Disclosures: S. Ovaysikia, None; A.M. Bartlett, None; N.K. Logothetis, None; K.L. Hoffman 

, None. 

Poster 

262. Visually-Guided Reaching II 



Topic: D.05.d. Visually guided reaching 

Support: NSF Graduate Fellowship to LMMM 

National Eye Institute (R01 EY015679) 

Whitehall Foundation (2004-08-81-APL) 

NIMH Conte Center (P50-MH-077970) 

Title: Neural representation of reaches to visual and proprioceptive targets in Area 5 and MIP of 

the rhesus macaque 

Authors: *L. M. MCGUIRE, P. N. SABES; 

Dept. of Physiol., UCSF, San Francisco, CA

Abstract: The sensory inputs used to plan and execute movements arrive through distinct 

sensory pathways in very different forms. Nevertheless, primates make remarkably similar 

movements irrespective of the sensory signals that are available, apparently integrating the 

available information across modalities. In order to address how visual and proprioceptive 

information are represented and used by the motor system we recorded from the superior parietal 

cortex, putatively Areas 5 and MIP, of a rhesus macaque monkey trained to reach to visual, 

combined visual and proprioceptive, and proprioceptive targets. Reaches were preformed to a 

target array with two fixation points, a design aimed at distinguishing between retinotopic and 

body-centered coding of target position or movement vector. Consistent with previous findings 

(Buneo et al., Nature 2002), we observed that representations tend to be more body-centered in 

character on the surface of the cortex (Area 5) and more retinotopic in character in the sulcus 

(MIP). Further, the coordinate frame representation of a given cell did not depend on task, 

suggesting that the reference frames in which information is represented in a given neuron does 

not depend on the sensory information available. However, many cells in MIP show greater 

modulation in firing rate during reaches to visual targets than to proprioceptive targets, while 

cells in Area 5 have similar responses across all trial types. This suggests that the strength of the 

representation in a given area, and thus its influence on movement planning and execution, 

depends on the sensory information available. 

Disclosures: L.M. McGuire, None; P.N. Sabes, None. 

Poster 





Support: EU FP6 Grant IST-027574 

PENED Grant 01ED111 

Title: Parietal cortical areas of the monkey brain engaged in visual and somatosensory guidance 

of reaching-to-grasp 

Authors: *V. RAOS 1,2 , M. N. EVANGELIOU 1,2 , H. E. SAVAKI 1,2 ; 

1 IACM, FORTH, Heraklion, Greece; 2 Basic Sci., Univ. of Crete Med. Sch., Heraklion, Greece 

Abstract: The 14 C-deoxyglucose method was employed to study whether different areas of the 

primate parietal cortex are involved in different aspects of grasping behavior. To this aim, we

mapped the functional activity in the cortex of the lateral and medial parietal convexity, the 

intraparietal and the parietoccipital sulci of monkeys which reached and grasped a 3D-object 

either in the light or in the dark. The extent and intensity of activations in the hemispheres 

contralateral to the moving forelimb were compared to those of the corresponding ipsilateral 

hemispheres as well as to those of control monkeys either in the light or in the dark. The 

forelimb representation in the primary somatosensory cortex of the postcentral convexity, areas 

PF and PFG of the inferior parietal convexity, areas PEip and 7VIP of the intraparietal sulcus 

and area V6A in the anterior bank of the parietoccipital sulcus were activated in all monkeys 

regardless of whether the movements were performed under visual or nonvisual (somatosensory 

and memory-related) guidance of the forelimb. In contrast, area PEc of the superior parietal lobe, 

area PG of the inferior parietal lobe, areas AIP, MIP, 5IPp and 5VIP of the intraparietal sulcus, 

area V6 in the anterior bank of the parietoccipital sulcus and areas PGm and 29/30 in the medial 

wall of the hemisphere were activated only for visually-guided reaching-to-grasp and remained 

unaffected when no visual feedback was available for reaching-to-grasp in complete darkness. 

This study provides evidence for the involvement of different cortical regions of the parietal lobe 

in the process of visual, somatosensory and memory-related signals which guide reaching-tograsp 

movements. 

Disclosures: V. Raos, None; H.E. Savaki, None; M.N. Evangeliou, None. 

Poster 





Support: NSF GRFP 

NIH NS 30256 

NSF BCS 0726685 

Title: The olivocerebellar response to motor errors 

Authors: *J. E. SCHLERF 1 , R. B. IVRY 1 , J. DIEDRICHSEN 2 ; 

1 Helen Wills Neurosci. Inst., Univ. California - Berkeley, Berkeley, CA; 2 Psychology, Bangor 

Univ., Bangor (Gwynedd), United Kingdom

Abstract: In the influential Marr-Albus-Ito model, climbing fiber input from the inferior olive 

provides an error signal that shapes cerebellar processing to produce the correct output (see 

Albus 1971). This model has provided a mechanistic account of cerebellar function across a wide 

range of error-based learning tasks. A neurophysiological investigation of the inferior olive in 

cats, however, argues that olivary discharge is linked to unexpected somatosensory stimulation, 

independent of movement error (Horn et al. 2004). 

We suggest a third hypothesis, building on the premise that there may be two distinct types of 

on-line errors. Filled errors are directly conveyed by an unexpected sensory event; for example, 

stepping onto a sidewalk that is higher than expected may cause a person to trip on the curb. In 

contrast, empty errors occur when an expected sensory event is absent; when a boxer throws a 

punch, the lack of contact with his opponent indicates an error. We propose that the 

olivocerebellar system may be more sensitive to filled errors, perhaps because the time of the 

error is precisely defined. 

To test this prediction, human participants used an MR-compatible robotic manipulandum to 

move a cursor during an fMRI scanning session. In one condition, the goal was to pass through a 

virtual window. Contact with the surrounding walls was felt as a bump, a situation in which 

unexpected sensory information conveys an error. In a second condition, the goal was to hit a 

bump the same size as the window. Errors for this condition would be indicated by the absence 

of an expected sensory signal. Both conditions required identical reaching movements for correct 

performance. To ensure comparable error rates, we randomly rotated the cursor's trajectory. 

When participants made errors, activation was observed in the inferior olive, as well as motor 

regions of the cerebellum (ipsilateral lobule V and bilateral VIII). In a direct contrast of the two 

error types, filled errors were associated with greater activation in vermal lobule III and 

contralateral VIII, as well as the caudal aspect of the olive. Empty errors evoke activation in the 

rostral olive, as well as ipsilateral lobule V and bilateral Crus I. The olivocerebellar system, then, 

seems to react to both empty and filled errors, although with different subregions. 

Disclosures: J.E. Schlerf, None; J. Diedrichsen, None; R.B. Ivry, None. 

Poster 





Support: CIHR Grant 74634 

Title: Oscillatory activity in different monkey premotor areas during a dissociated reaching task

Authors: P. SAYEGH 1 , B. NEAGU 1 , K. HOFFMAN 2 , X. YAN 2 , J. D. CRAWFORD 2 , *L. E. 

SERGIO 1 ; 

1 Sch. Kinesiol & Hlth. Sci., 2 Psychology, York Univ., Toronto ON, ON, Canada 

Abstract: We are interested in the contribution of different brain areas to reaching tasks having 

an increasing dissociation between the visual stimulus and the motor output. A visual-to-motor 

transformation can be considered standard when the visual stimulus guiding a movement is the 

target of the action itself. A transformation can be considered nonstandard when a visual 

stimulus provides information about the direction of a required movement but is not the target of 

the motor output. We perform nonstandard tasks (e.g. using a computer mouse) effortlessly, yet 

this ability is not innate and can be compromised under neuropathological conditions(1). 

Previous human imaging work has demonstrated that activity in a network of brain regions that 

includes premotor and superior parietal cortex can vary as a visually-guided reaching task 

becomes progressively nonstandard(2). Here we examine the contribution of the rostral and 

caudal portions of dorsal premotor cortex (PMdr, PMdc) under these same conditions, by 

analysing local field potentials (LFP) recorded in awake behaving primates. Monkeys (macaca 

mulatta) were trained to move their eyes and displace a cursor reflecting finger position from a 

central to a cued peripheral target under standard and nonstandard visuomotor conditions. In the 

standard condition, the animal moves its finger along a customized touch screen placed at waist 

height in a horizontal plane so that the cursor is under its finger. In the nonstandard condition, 

the cursor and targets are displayed on a monitor positioned in a frontal plane 40 cm in front of 

the animal. The full trajectory of the hand and eye are recorded to ensure that the motor task is 

similar between conditions. We observed both changes in the power spectrum and changes in the 

temporal structure of the LFP between task conditions during the cue period. These data suggest 

that communication between local cell assemblies in PMdr/PMdc comprise a necessary part of 

the visuomotor transformation required for non-standard reach planning. References: 1. Tippett 

et.al. (2007) Eur. Neurology. May; 58(1):1-11, 2. Gorbet et al (2004) Neuroimage 23:1100-1111. 

Disclosures: P. Sayegh, None; B. Neagu, None; K. Hoffman, None; X. Yan, None; J.D. 

Crawford, None; L.E. Sergio , None. 

Poster 





Support: MIUR

Fondazione del Monte di Bologna e Ravenna 

FP6-IST-027574-MATHESIS 

Title: Cortical connections of macaque parietal area PEc 

Authors: S. BAKOLA, M. GAMBERINI, L. PASSARELLI, P. FATTORI, *C. GALLETTI; 

Dept. Human & Gen. Physiol., Univ. Bologna, Bologna, Italy 

Abstract: The present report examines the connectivity patterns of area PEc, a cortical area 

located in the caudal part of the posterior parietal lobe. Retrograde fluorescent tracers were 

injected or placed directly as crystals on the exposed cortical convexity of six cynomolgus 

monkeys (Macaca fascicularis) within the cytoarchitectonic limits of PEc. We observed 

ipsilateral cortical projections from visuomotor and somatosensory areas as well as from motor 

areas with somatotopic organization. The heaviest projections were from the neighboring areas 

of the parietal lobe. Labeled cells were in area PE, immediately rostral to the injection sites, in 

areas MIP and PEip in the medial intraparietal bank, and in area V6A in the anterior bank of the 

parieto-occipital sulcus. Dense input arose also from area PEci at the caudal tip of the cingulate 

sulcus, whereas primary and secondary somatosensory areas provided weaker labeling. In 

addition, modest numbers of labeled cells were noted in areas PFG, on the parietal convexity, 

and in the parietal opercular area PGop. In the frontal lobe, labeling originated mostly in the 

dorsal part of area F2, around the superior precentral dimple, and to a lesser extent in medial area 

F3 and primary motor cortex. Further projections arose from the mesial cortical surface of the 

hemisphere, in and around the cingulate sulcus, mainly from its posterior part. There was no 

significant labeling in early visual, superior temporal and prefrontal areas or in the known 

oculomotor centers. An anteroposterior gradient was evident so that more posterior injection 

sites were targeted by visuomotor areas and more anterior ones by somatosensory-related areas. 

Our findings are in agreement with the described physiological properties of PEc neurons 

(visual, somatosensory, and bimodal neurons; Breveglieri et al, 2006; 2008), but emphasize a 

skeletomotor function for this area. We suggest that PEc relays proprioceptive, rather than visual, 

information about the position of body parts that can be used by motor cortices with direct output 

to the spinal cord. The anatomical link from PEc to premotor areas that host a representation of 

the upper and lower limbs, but not the hands or face, presumably implies a role in locomotion 

and coordinated body movement in the environment. Visual information in PEc is likely used to 

integrate somatosensory information for the control of body movements. 

Disclosures: S. Bakola, None; C. Galletti, None; M. Gamberini, None; L. Passarelli, None; P. 

Fattori, None. 

Poster 

262. Visually-Guided Reaching II




Support: Marie Currie Fellowship 

Research School, Ruhr-University 

German Science Foundation (DFG) SFB-509 Neurovision 

Title: Primate superior colliculus neurons active during hand-target contact 

Authors: *A. MIKULIC, K.-P. HOFFMANN; 

Allgemeine Zool und Neurobio, Ruhr-Univ, BOCHUM, Germany 

Abstract: The primate superior colliculus (SC) is recognized as a crucial brain structure for 

gaze-orienting behavior. However, other aspects of its functional repertoire have not been 

extensively studied. Here we report about neural activity within the primate SC related to handtarget 

contact in a task involving goal-directed arm movements during visual fixation. A rhesus 

macaque monkey was trained to visually fixate a dot of light while reaching for and pressing 

illuminated targets on the working panel. To initiate a trial the subject was required to put a hand 

on the start position in a horizontal plane and to fixate a dot of light that appeared shortly after on 

the vertical working panel. One button on the panel was then lighted red followed by a change to 

green, which was a „go‟ signal for the subject to press the button. After pressing and maintaining 

the contact, the subject finished the trial by returning his hand to the start position. During the 

whole trial visual fixation was required. We found previously reported neural activity within 

intermediate and deep layers of the SC that was enhanced during the contact phase of the trial. 

Importantly, these units seem to be active during a contact with an object that is a target of a 

movement, but not during task-irrelevant somatosensory stimulation of a hand. In addition, we 

report neural activity that was attenuated during the hand-target contact. To examine the origin 

of contact-gated SC neural responses and their modulation by external events we introduced 

trials with force perturbation triggered by the subject‟s contact with the target. Upon subject‟s 

contact with the button, on some trials a real-time force perturbation system induced abrupt and 

brief panel motion. During these trials the subject had to maintain normal performance in the 

task i.e. visual fixation and contact with the panel. Importantly, some neural responses were 

significantly higher during perturbed trials and, moreover, some others were present exclusively 

following perturbation. Together, our findings suggest that specific neurons within the primate 

SC might be involved in signaling sensory consequences of animal‟s interaction with the target 

during goal directed movements. This may help elucidate a new role of the primate SC in 

sensory-motor processing. 

Disclosures: A. Mikulic, None; K. Hoffmann, None.

Poster 





Support: James S. McDonnell - Higher Brain Function Grant 

Title: Directional encoding in motor cortex with ipsilateral ECoG activity 

Authors: *N. ANDERSON 1 , M. SHARMA 1 , K. WISNESKI 1 , T. BLAKELY 2 , G. SCHALK 3 , 

D. MORAN 1 , E. C. LEUTHARDT 4 ; 

1 Biomed. Engin., Washington Univ., Saint Louis, MO; 2 Univ. of Washington, Seattle, WA; 

3 Wadsworth Ctr., New York State Dept. of Hlth., Albany, NY; 4 Neurolog. Surgery, Washington 

Univ. in St. Louis, St. Louis, MO 

Abstract: Hemispheric stroke renders one side of the brain non-functional. Injury to primary 

motor cortex leads to impaired operation of the contralateral side of body. This most notably 

affects the hand in terms of chronic dysfunction. Although treatments of stroke (medicinal[7, 8], 

stenting[9], therapy based[10], stem cell based[6] and others) are under investigation for the 

protection and salvage of affected areas of the brain there is a need to develop methodologies to 

restore function that has been lost and can not be recovered with rehabilitation. Previously, our 

group has demonstrated the utility of electrocorticography (ECoG) as a platform for Brain- 

Computer Interfaces (BCIs) utilizing signals from motor cortex associated with contralateral 

hand movements[1, 2]. Recent findings by our group and Buch et al. have enabled external 

device control with ipsilateral brain activity using ECoG [4] and magnetoencephalography 

(MEG) [5]. Control thus far, however, has been simple one dimensional tasks. In this study we 

explored to what degree more complex motor tasks can be discriminated from ipsilaterally 

derived ECoG signals. 

In four epilepsy patients requiring an invasive unilateral hemispheric electrode array, the cortical 

physiology associated with a center-out task performed with the ipsilateral hand (same side as 

grid) and the contralateral hand (opposite side of grid) were compared. ECoG activity was 

recorded while the patients performed basic horizontal and vertical joystick movements with 

either the left or the right hand. Spectral components of the ECoG signal pertaining to different 

directions were compared for both ipsilateral and contralateral conditions. 

The results show that ECoG can be used to differentiate the two-dimensional joystick 

movements of the ipsilateral hand. This information for ipsilateral movements was 

predominantly captured in lower frequencies around 40 Hz, whereas the information for 

contralateral movements was captured by components in higher frequencies.

These findings demonstrate that information about specific ipsilateral motor intentions is indeed 

encoded within ipsilateral cortical physiology. This information about ipsilateral hand 

movements may have substantial implications for an ipsilateral based BCI (iBCI) for 

hemispheric dysfunction because the neuroprosthetic may allow for restoration of more complex 

motor function in the affected limb by utilizing signals taken from the unaffected hemisphere. 

Disclosures: N. Anderson , None; M. Sharma, None; K. Wisneski, None; T. Blakely, 

None; G. Schalk, None; D. Moran, None; E.C. Leuthardt, None. 

Poster 





Title: Brain areas related to execution of visually guided reaching movements: a positron 

emission tomography study with macaque monkey 

Authors: M. KOMATSU 1 , Y. TSUTSUMI 1 , H. ONOE 2 , H. TSUKADA 3 , *E. MIYASHITA 1 ; 

1 Compu Intell & Sys Sci, Intrdsc Grad Sch. Sci/Eng, Tokyo Inst. Tech., Yokohama, Japan; 

2 Functional Probe Res. Laboratory, Mol. Imaging Res. Program, RIKEN, Kobe, Japan; 3 Central 

Res. Lab., Hamamatsu Photonics, Hamamatsu, Japan 

Abstract: To visualize the brain areas related to execution of visually guided reaching 

movements, we measured the regional cerebral blood flow (rCBF) of a macaque monkey using a 

positron emission tomography (PET). A monkey (Macaca fuscata) was over-trained to perform 

center-out reaching movements on the horizontal plane manipulating a handle of a 2-dimensional 

Space Interface Device for Artificial Reality. The monkey performed 3 different reaching tasks 

with similar kinematics under 0, 1.5, and 3 N external loads applied to the handle and also 

performed a control task watching playback movies without actual movements in which the 

visual and reward conditions were the same as in the reaching tasks. The PET scan was initiated 

~20 s after the start of the task and when the radioactivity of [ 15 O]H2O in brain was greater than 

30 kcps. The scanned data of the first 60 s of 15 scans for each task condition were used for the 

statistical analysis (SPM99, the significance level was thresholded at p < 0.01). Subtractions of 

the control task from the reaching tasks revealed the following results. Significant increases in 

rCBF were observed in the ipsilateral dorsal premotor, primary motor (M1), primary 

somatosensory (S1), parietal cortices (the anterior and lateral intraparietal areas, area 5, and 

parietal opercullar part of the area 7), and the thalamus. The ipsilateral cerebellar cortices (the 

simple lobule, paramedian lobule, and cerebellar lobule 5), and the lateral cerebellar nucleus also

showed significant increases in rCBF. In contrast, significant decreases in rCBF were observed 

in the contralateral caudate nucleus and the ventral striatum. Among these areas, a part of M1, 

S1, and the parietal area 5 showed significant modulations of rCBF associated with application 

of the external loads. These results suggest that the parieto-frontal and somato-motor connections 

together with the cerebellar and basal ganglia loops play an essential role for execution of the 

visually guided reaching movements, and that the somato-motor connection together with the 

parieto-motor connection calculate the arm dynamics. 

Disclosures: M. Komatsu, None; Y. Tsutsumi, None; H. Onoe, None; H. Tsukada, None; E. 

Miyashita , None. 

Poster 





Support: International Graduate School of Neuroscience 

Title: Contribution of the superior colliculus to error correction in the skeletomotor system 

Authors: *S. HEBA, K.-P. HOFFMANN; 

Gen. Zoology & Neurobio., Ruhr-University Bochum, Bochum, Germany 

Abstract: Goal-directed reach movements were thought to be of a ballistic nature, i.e. their 

neural commands are fully planned in advance. In order to achieve precision, the final phase of 

the movement might be adapted through feedback mechanisms. This doctrine has been revised in 

favour to an “on-line”-monitoring and -correction of skeletomotor commands. 

Cortical areas like the posterior parietal cortex, frontal as well as prefrontal areas and the 

cerebellum compose the neural correlate. In addition, previous studies also point towards a

subcortical contribution to on-line corrections, and experiments performed in our laboratory 

revealed neuronal responses during correction of arm movements in the superior colliculus (SC) 

of the macaque monkey. Thus, the SC known to be primarily involved in oculomotor functions, 

acts also in response to skeletomotoric operations. 

Still, we could determine neither the origin, nor the relevance of these neuronal reach-signals 

beyond any doubt. 

In order to examine these topics, we combined electrophysiological multi-electrode single- cell 

recordings with electromyograms of trunk muscles and induced a reversible inactivation of the 

SC with applications of the GABAA-agonist muscimol. 

Within earlier experiments, physical push buttons mounted on an opaque fronto-parallel board 

were offered as targets within a centre-out reach paradigm, thus arm movements homed in on 

permutations of overtrained predefined positions. Now, the animal performs a modified centreout 

task towards a touch-sensitive TFT-monitor, giving the opportunity to present a variable set 

of visual reach targets without any orienting landmarks. 

So far the inactivation of the SC by injections of muscimol altered the duration of reach 

movements significantly (monkey N). Whether these effects (1) give a hint to an autonomous 

contribution of the SC in reach movements, (2) are caused by an impairment of a top-down or 

basal ganglia route, or (3) are just an effect of fatigue, remains uncertain. 

Disclosures: S. Heba , None; K. Hoffmann, None. 

Poster 





Support: NIH EY09223 

Title: Neuronal plasticity in sensorimotor transformations in macaque inferior parietal cortex 

due to shifting prisms 

Authors: *A. KARNIK, B. HEIDER, R. M. SIEGEL; 

CMBN, Rutgers Univ., Newark, NJ 

Abstract: Inferior parietal neurons in areas 7a and the dorsal prelunate (DP) are spatially tuned 

for angle of gaze, retinal location of the stimulus and reaching location (MacKay 1992, Heider et 

al., 2006; Heider et al., submitted). The current study explores the plasticity of the sensorimotor 

transformation during reaching by distorting the visual field with prisms. The simplest prediction

was that eye position would dominate the response via the gain field. 

Neural activity was recorded in areas 7a and DP contralateral to the reaching arm. The monkey 

was required to fixate one of 9 positions on a touch screen while a visual stimulus (expansion 

optic flow, radius 6º) appeared behind the fixation point. The change from structured to 

unstructured motion cued the monkey to make a ballistic hand movement towards the target and 

hold his hand in place. A binocular Fresnel prism introduced a 10º shift in the same direction or 

the opposite direction as the spatial gain field of the unit; the monkey had to reach to the physical 

location of the target which did not match the perceived location. The trained monkey correctly 

reached to the target within a few trials demonstrating swift adaptation to the distortion. Changes 

in firing rate were assessed across 3 events of the task: (1) fixation onset, (2) visual stimulus 

onset (“visual signal”), and (3) lifting of hand from rest (“reach signal”). Linear regressions with 

categorical variables denoting the presence or absence of the prism distortion quantified changes 

in spatial tuning. 

Spatial tuning of single unit activity was compared during the “pre-prism” block, the “prism” 

block, and the “post-prism” block (33/60); the remainder were not tested “post prism”. The 

spatial tuning typically returned to pre-prism levels once the prism was removed. Alteration of 

the spatial tuning with insertion of the prisms to at least one event was found in 26/33 units. If 

the cells merely responded to the shift in eye position in the prism block, the firing rate would be 

expected to vary in proportion to the slope of the gain field. This did not occur indicating more 

complex interactions between the prism and neuronal activity. 

In many cases, the prism distortion altered the slope of the reach field. For some cells, the change 

in the shape of the reach field acted in a manner to correct for the prisms (10/24). In others, there 

was no clear relation between the prism and the change in the reach field. 

Similar variations in spatial tuning were observed for the fixation onset and the visual signal. 

These dramatic changes in spatial tuning across all measured neural events suggest that there is a 

range of remapping of the sensorimotor transformations that guide reaching. 

Disclosures: A. Karnik, None; B. Heider, None; R.M. Siegel, None. 

Poster 





Support: Natural Sciences and Engineering Research Council of Canada 

National Institutes of Health

Canadian Institutes of Health Research 

Title: fMRI activation during observation of others' reach errors 

Authors: *N. MALFAIT 1,2 , K. F. VALYEAR 2 , J. C. CULHAM 2 , L. E. BROWN 3 , J.-L. 

ANTON 1 , P. L. GRIBBLE 2 ; 

1 Univ. de Provence, CNRS, Marseille, France; 2 Univ. of Western Ontario, London, ON, Canada; 

3 Trent Univ., Peterborough, ON, Canada 

Abstract: When exposed to novel dynamical conditions (e.g. externally imposed forces), 

neurologically intact subjects easily adjust motor commands on the basis of their own reaching 

errors. Subjects can also benefit from visual observation of others‟ kinematic errors. Here, using 

functional magnetic resonance imaging (fMRI), we scanned subjects watching movies depicting 

another person learning to reach in a novel dynamic environment created by a robotic device. 

Passive observation of reaching movements (whether or not they were perturbed by the robot) 

was associated with increased activation in fronto-parietal regions that are normally recruited in 

active reaching. We found significant clusters in the parieto-occipital cortex, the intraparietal 

sulcus, as well as in the dorsal premotor cortex. Stronger activation was seen contralateral to the 

arm shown in the movies. Moreover, it appeared that part of the network that has been shown to 

be engaged in processing self-generated reach-error is also involved in observing reach errors 

committed by others. Specifically, activity in the left intraparietal sulcus and the left dorsal 

premotor cortex (contralateral to the arm shown reaching in the movies), as well as in the right 

cerebellar cortex (ipsilateral to the reaching arm) was modulated by the amplitude of observed 

kinematic errors. 

Disclosures: N. Malfait, None; J. Anton, None; K.F. Valyear, None; J.C. Culham, None; P.L. 

Gribble, None; L.E. Brown, None. 

Poster 





Support: MSTP, BioX, NDSEG, NSF, NINDS/CRCNS 5-R01-NS054283-02 

Gatsby Charitable Foundation 

University of Pittsburgh

Center for the Neural Basis of Cognition 

Burroughs Wellcome Fund 

Title: Absence of fast-timescale correlations in macaque dorsal premotor cortex 

Authors: *C. A. LEHOCKY 1 , G. SANTHANAM 2 , A. AFSHAR 2,3 , B. M. YU 2,6,4 , S. I. RYU 2 , 

J. P. CUNNINGHAM 2 , V. GILJA 5 , K. V. SHENOY 2,4 , A. P. BATISTA 1,7 ; 

1 Bioengineering, Univ. Pittsburgh, Pittsburgh, PA; 2 Electrical Engin., 3 Sch. of Med., 

4 Neurosciences, 5 Computer Sci., Stanford Univ., Stanford, CA; 6 Gatsby Computat. Neurosci. 

Unit, Univ. Col. London, London, United Kingdom; 7 Ctr. for the Neural Basis of Cognition, 

Pittsburgh, PA 

Abstract: Spike time correlations between pairs of neurons were found to be virtually 

nonexistent in the dorsal aspect of premotor cortex (PMd) in two monkeys performing a delayed 

center-out reaching task. Neurons were recorded on a 96-electrode array (400 κm minimum 

electrode spacing) in two macaque monkeys. Data for eight sessions from Monkey 1 conducted 

over 34 days, and one session from Monkey 2 were analyzed. Analyses were performed 

separately for each of the eight reach directions. For each direction, well-isolated neurons that 

fired at greater than 10 spikes/second during a 500 ms epoch of the delay period were analyzed. 

Each analysis included between 25 and 114 neurons for Monkey 1, and 130-135 neurons for 

Monkey 2. We looked for correlations in 111,579 unique combinations of neuron pair and reach 

direction for Monkey 1, and 69,706 combinations for Monkey 2. Monkey 1 performed between 

18 and 164 successful trials (mean 95 trials) across all combinations, and monkey 2 performed 

160-197 (mean 178) trials across combinations. Covariograms (cross-correlation histograms 

minus shuffle corrector) were generated for every combination of cell pair and reach direction. 

Correlations were deemed significant when at least one bin of the covariogram (binned at 9ms) 

exceeded the 95% confidence limit around the distribution expected for no correlation. 

Significant correlations were observed in only 889 (0.80%) of all tested combinations (856 

unique pairs of neurons) for Monkey 1; in Monkey 2, 931 (1.34%) combinations were correlated. 

We calibrated the sensitivity of the technique using synthetic data with known correlations: we 

found our recorded data sets were sufficient to detect correlations that consisted of 3% or more 

coincident spikes between two neurons. From this surprising lack of spike time correlation in 

PMd we conclude that fast-timescale interactions between cortical neurons in nearby columns 

are weak (





Title: Postural instability modulates the gain of involuntary manual response elicited by visual 

motion during reaching movement 

Authors: *K. KADOTA 1 , H. GOMI 1,2 ; 

1 Shimojo Implicit Brain Function Project, ERATO-JST, Kanagawa, Japan; 2 NTT 

Communication Sci. Labs., Kanagawa, Japan 

Abstract: We have reported about the manual following response (MFR), which is a reflexive 

manual response during arm reaching evoked by a sudden visual background motion. From 

functional view point, this response may contribute to adjust the arm trajectory affected by 

postural motion and disturbances because the response direction is a parallel with visual motion. 

If this is true, it would be expected that a gain of the MFR will be modulated according to the 

state of posture and its stability. To clarify this hypothesis, we compared the MFR amplitude 

when participants performed reaching under different postural stabilities. 

First, to clarify the postural effect on the MFR gain, we compared the MFR amplitude with 

participants in a standing or a sitting positions. The participants sat or stood in front of a large 

screen (62 x 46 cm) with their eyes 50cm away from the screen surface, where a visual stimulus 

was projected. The stimulus consisted of a sinusoidal grating pattern having a horizontal gray bar 

and a small white target-marker placed at the center of gray bar. The participants were asked to 

reach for the target. In two third of the trials, the grating pattern started to move horizontally 

either leftward or rightward at constant velocities, which was initiated 60 ms after the handmovement 

start. In the results, the MFR amplitude was greater when reaching was performed in 

the standing than in the sitting position. In addition, this tendency varied according to the 

velocity of the visual motion. In the next experiment, we examined the effect of postural stability 

on the MFR gain. Like the first experiment, participants required performing reaching for the 

visual target projected on the screen, but they were standing on a stable or an unstable (motordriven) 

platform. In the unstable platform condition, the participant required to maintain their 

posture during reaching because of an induced platform instability at every five trial-interval. In 

contrast, there was no postural disturbance in the stable platform condition. In the unstable 

condition, the participants generated greater MFR than that in the stable condition. Thus, the 

postural instability could enhance the MFR amplitude. 

These results would support the idea that the MFR gain is modulated according to the postural 

stability states. Therefore, it is reasonable to assume that the MFR could contribute an online 

correction of hand trajectory deviation caused by postural disturbance during reaching. 

Disclosures: K. Kadota , None; H. Gomi, None.

Poster 





Title: Short-latency manual responses of monkey are impaired by lesions in the MST 

Authors: *A. TAKEMURA 1,2 , T. OFUJI 3 , N. ABEKAWA 4 , K. KAWANO 5 , H. GOMI 2,4 ; 

1 Neurosci Res. Inst., AIST, Tsukuba, Ibaraki, Japan; 2 SHIMOJO Implicit Brain Function Project, 

ERATO, JST, Kanagawa, Japan; 3 Grad Sch. of Comprehensive human sci, Univ. of Tsukuba, 

Tsukuba, Ibaraki, Japan; 4 NTT Communication Sci. Labs, Atsugi, Kanagawa, Japan; 5 Grad Sch. 

of Med, Kyoto Univ., Kyoto, Japan 

Abstract: When a sudden drifting motion of a surrounding visual stimulus is given during 

reaching movement of an upper limb, the limb trajectory is deviated towards the direction of 

motion. This rapid manual response is elicited with an ultra-short latency (~70 ms in monkeys), 

termed „Manual Following Response (MFR)‟ (Saijo et al. 2005, Gomi et al. 2006). This 

visuomotor response might be functional in quickly adjusting the reaching movement during 

self-movements accompanied by visual motion of the background. On the other hand, 

unexpected target shifts also elicit another adjustment of ongoing reaching movement with a 

short latency (~100 ms in monkeys). 

The medial superior temporal (MST) area of the monkey‟s cortex contains a preponderance of 

neurons which encode the direction of motion of visual stimuli, including many that respond 

selectively to full-field visual motion (optic flow). 

In the present study, to determine whether the MST plays a role in eliciting these manual 

responses by on-line control mechanisms during reaching, we examined the effects of injecting 

muscimol (a GABA agonist) into the left MST of two monkeys. The monkey faced a CRT in a 

dark room with its head fixed in the standard stereotaxic position. The CRT was 366 mm in front 

of the eyes and the visual stimulus was subtended 62° x 50° along the vertical and horizontal 

meridian. The visual stimulus was a pair of vertical sinusoidal grating patterns (anti-phase) of a 

fixed contrast (50%). When the monkey pressed the button, a red fixation target appeared in the 

center of the CRT. After a random delay, the target color became green, which instructed the 

monkey to release the button. The monkey had to release the button and touch the CRT to get a 

reward. The size of the reward was inversely proportional to the magnitude of the error to 

encourage accurate reaching in the training period. 

When we gave the monkey a total of two muscimol injections (10 mg/ml, 4.0 κl total) into the 

MST of the left hemisphere, the MFR was abolished one hour after the injections. As for the 

other manual response to the target shift, the initial response was decreased substantially

following injections in comparison to the pre-injection controls but the peak of response was 

spared the deficit. One day after, these manual responses were recovered. Overall the results 

strongly support the hypothesis that the MST is a primary site for producing these two 

visuomotor responses for adjustments for limb reaching movements at ultra-short and short 

latencies. 

Disclosures: A. Takemura, None; T. Ofuji, None; N. Abekawa, None; K. Kawano, None; H. 

Gomi, None. 

Poster 





Title: Spatial updating for involuntary manual response during goal directed movement 

Authors: *N. ABEKAWA 1 , H. GOMI 1,2 ; 

1 NTT Communication Sci. Labs., NTT, Kanagawa, Japan; 2 Shimojo Implicit Brain Function 

Project, ERATO-JST, Kanagawa, Japan 

Abstract: Visual motion during reaching movement induces an involuntary manual response in 

the direction of visual motion, called manual following response (MFR). This response is 

induced with short latency, but is modulated by a spatial relationship between the gaze point and 

reaching target as reported previously (Abekawa et al. 2006, 2007 SFN). The modulation manner 

appears to be represented in the retinal coordinate: high MFR gain for the target at retinal center 

and low MFR gain for the target at retinal periphery. Therefore the MFR seems to be 

functionally modulated by the distance between the gaze and target, but it remains unclear when 

the MFR gain is updated: whether the gain modulation is completed at the motor planning stage 

(before reaching movement) or the gain is modulated at the motor execution stage (during 

reaching movement). To examine this issue, we here conducted an experiment in which the gaze 

location changed during reaching movement. 

In the experiment, every trial was started with the presentation of vertical sine-wave gratings and 

of the reaching target (always shown in the lower part of the screen) and the fixation marker 

(shown in the upper or lower part of the screen). In the “No gaze-change” task, participants were 

asked to reach the target at the lower position while looking at either upper or lower fixation 

marker. In the “gaze-change” task, fixation point vertically jumped to another position at the 

time of hand movement start, and participants were instructed to make saccade to the new 

location as fast as possible during reaching movement. The fixation marker was changed from

upper to lower (U-L) position or from lower to upper (L-U) position. In both tasks, 350 ms after 

the reaching start, gratings moved rightward or leftward to induce the MFR. The task was 

randomly changed in every trial. The results showed that, in the “No gaze-change” task, the 

MFR amplitude for the lower fixation was greater than that for the upper fixation. In the “gazechange” 

task, the MFR for the U-L saccade condition was greater than that for the L-U saccade 

condition, indicated that the MFR gain changed according to the gaze-target configuration after 

saccade. This result suggests that the spatial updating for the gain setting of the involuntary 

visuomotor response was not completed in the motor planning process, but it was dynamically 

modulated in the motor execution process according to the new gaze-target configuration. 

Disclosures: N. Abekawa , None; H. Gomi, None. 

Poster 





Title: Target-reaching and switching movements during suppression of the visual cortex in 

humans 

Authors: *Y. OHKI 1 , S. SHIBUYA 1 , H. SEKIGUCHI 2,3 , H. KADOTA 2,4 , S. TAKEUCHI 2 , Y. 

NAKAJIMA 2 ; 

1 Physiol, Kyorin Univ. Sch. Med., Tokyo, Japan; 2 Rehab Sensory Funct, Res. Inst. NRCD, 

Saitama, Japan; 3 ASMeW, Waseda Univ., Saitama, Japan; 4 Shibaura Inst. Tech., Saitama, Japan 

Abstract: During target-reaching movements, humans can make mid-flight adjustments when 

the target jumps to new positions (switching movements). It was reported previously that such 

switching movements could be induced without conscious experiences of the new target, by 

suppressing the primary visual cortex (V1) in normal human subjects (Christiansen et al., 2008). 

In the present study, we further examined involvement of V1 in especially early part of the 

switching movement (latency ≥120ms). Four normal human subjects, who gave informed 

consents, participated in experiments. They sat ca. 40 cm in front of a CRT monitor, with their 

chin supported by a chin rest. A figure-of-eight coil for transcranial magnetic stimulation (TMS) 

was held over the primary visual cortex as described previously (Kammer et al., 2005), whose 

position was checked on MRI brain images from each subject (Navigation System, NexStim). 

When subjects gazed at a 1 cm square target in the center of the screen, sound GO signal was 

given to reach to the target. After 25 ms from the movement onset, the center target sometimes 

disappeared, and a new target could appear either transiently (duration ca. 8 ms) or constantly.

The new target appeared 10 cm left or right to, and 2 cm below the central target. TMS was 

sometimes delivered over V1, 70 or 100 ms after disappearance of the center target. When the 

new target appeared, subjects were instructed to reach it, and their arm positions were monitored 

by Optotrak system (NDI). After each trial, subjects were required to report if they saw a new 

target (1-4 scale) and where it was (forced choice between left and right). Brightness of the 

background was adjusted, so that subjects could detect the new target in trials without TMS. 

Subjects reached around the center of the monitor, when new target did not appear. In addition 

without TMS, they could reach toward the new target, irrespective of the duration of its 

appearance. When TMS was delivered, they sometimes missed detecting the new target. 

Especially when using TMS with 100 ms delay, they occasionally made switching toward the 

new target, even if they did not perceive it. However with TMS of 70 ms delay, the switching 

movement was deteriorated in both frequency and amplitude. In trials without conscious 

perception of the new target, subjects‟ judgments about the position of the new target were rather 

accurate, and were above the chance level. We conclude that early switching movements could 

be induced without conscious visual experiences, though V1 could partly be involved in the 

movement.. 

Disclosures: Y. Ohki, None; S. Shibuya, None; H. Sekiguchi, None; H. Kadota, None; S. 

Takeuchi, None; Y. Nakajima, None. 

Poster 






Title: Gaze-biased misreaching in optic ataxia does not generalize to other spatial planes 

Authors: *J. A. GRANEK 1 , L. PISELLA 2 , A. BLANGERO 2 , Y. ROSSETTI 2 , L. E. SERGIO 1 ; 

1 Kinesiol & Hlth. Sci., York Univ., Toronto, ON, Canada; 2 Inserm U864, Lyon, France 

Abstract: In our everyday movements, we typically interact directly with the object that we are 

viewing. Hence, the visual stimulus guiding the action is itself the target of the action. Such 

behaviour can be considered “standard”, or “direct”. However, the evolution of the capacity for 

tool-use in primates has resulted in situations where the correspondence between vision and 

action is not direct. Rather, the mapping between stimulus and response must be learned and 

calibrated. This situation has been referred to as “non-standard” sensorimotor mapping. In non-

standard visuomotor transformations, the visual information used to perform a motor task does 

not come from the object one‟s hand is interacting with. A common example is a reach for a 

peripherally-viewed computer mouse sitting on a flat tabletop while viewing an icon on a 

computer monitor, and then sliding the mouse forward on a horizontal plane in order to displace 

a cursor vertically to touch the icon. In this situation, the direction of gaze and the manipulated 

object (the cursor) are in different depths or spatial planes. Our previous work has begun to 

characterize the contributions of different brain pathways to the processing of different levels of 

visuomotor compatibility. In this study we test the hypothesis that neural pathways involving the 

superior parietal lobule are involved in basic visuomotor transformations and in decoupling the 

movements of the eyes and the hand, a skill required for some non-standard mapping tasks. We 

examined the performance of a bilateral optic ataxic patient in reaching tasks which involved 

different levels of dissociation (spatial plane changes, rotated visual feedback, arbitrary 

associations). We observed a gaze-direction dependent reaching bias when the visual feedback of 

hand location was rotated. Importantly, we found that these misreaches did not change when the 

hand was also moving in a different spatial plane. That is, this patient, who is missing most of 

both superior parietal lobules, is successful in transforming the reach target into a different plane 

in space. However, she is unable to decouple the eye and the hand fully in this new motion plane. 

These data suggest that the different levels of dissociation examined here are processed by 

independent neural pathways. 

Disclosures: J.A. Granek , None; L.E. Sergio, None; L. Pisella, None; A. Blangero, None; Y. 

Rossetti, None. 

Poster 





Support: Heart and Stroke Foundation Centre for Stroke Recovery 

Title: Post-stroke patients with substantial right parietal cortex injury demonstrate an ability to 

perform successful visuospatial transformations 

Authors: *W. J. TIPPETT, S. E. BLACK; 

Cogntive Neurol., Sunnybrook Hlth. Sci. Cen, Toronto, ON, Canada 

Abstract: Introduction: The ability to perform successful visuospatial/visuomotor 

transformations relies on effective processing primarily within the right posterior parietal cortex

(PPC). To identify the extent of visuomotor deficits using traditional and non-traditional 

visuospatial tasks, we examined 10 post-stroke patients with right parietal lesions, and 10 agematched 

controls. 

Participants: Neurologically healthy controls (Age: 60.3 ± 1.3, MMSE 29.3 ±.2) and stroke 

patients (Age: 57.4 ± 4.5, MMSE 28.7 ±.4). Average duration post-stroke episode was 8.9 years 

(range 3.3-19) and average percent right inferior parietal volume loss was 32% (range 0-89%). 

Methods: Neuropsychological tasks included: Benton Judgement of Line Orientation, Trails A & 

B, Rey-Osterrieth Complex Figure (Copy, Immediate recall, Delayed recall), Clock Drawing 

(Lezak MD. 1995) and a visuomotor task (visually guided pointing tasks on a touch sensitive 

computer screen measuring kinematic movements) (Tippett WJ. et al. 2006). 

Results: Post-stroke participants displayed no significant differences from the control sample on 

any of the neuropsychological measures. In addition, the stroke sample demonstrated no 

significant deficits in completing the visuomotor task. 

Conclusions: Past research suggests populations with lesions within the right PPC will have 

difficulty performing visuomotor transformations (Buneo CA. et al. 2002). This study‟s chronic 

stroke patient sample did not confirm this finding. Their competence despite loss of most of the 

relevant parietal cortex for several patients in this sample was unexpected. Patient results suggest 

that their ability in completing visually-guided transformations relies on compensatory 

mechanisms from the left hemisphere, a hypothesis that will be tested with the use of fMRI 

experiments. 

Disclosures: W.J. Tippett , None; S.E. Black, None. 

Poster 





Support: U.S. Army Medical Research and Materiel Command W81XWH-05-1- 

Title: Upper extremity amputees adapt to their prostheses during reaching movements, but 

exhibit abnormalities with their intact arm 

Authors: A. J. METZGER 1,2 , P. S. LUM 1,2 , C. N. SCHABOWSKY 1,2 , R. J. HOLLEY 3 , B. 

MONROE 4 , *A. W. DROMERICK 5 ; 

1 Dept. of Biomed. Engin., The Catholic Univ. of America, Washington, DC; 2 Ctr. for Applied 

Biomechanics and Rehabil. Res., 3 Inpatient Occup. Therapy, Natl. Rehabil. Hosp., Washington,

DC; 4 District Amputee Care, Washington, DC; 5 Rehab Med. & Neurol, Natl. Rehab Hosp, 

Washington, DC 

Abstract: This study investigated the role of vision in upper extremity reaching movements of 

unilateral below elbow prosthetic users. Subjects used a robotic manipulandum to reach to two 

targets, one contralateral and one ipsilateral, located in a horizontal plane. These trials were 

performed with both the prosthetic arm and the intact arm. Visual guidance was then eliminated 

from the environment and the subjects had to reach to the same targets, relying on their sense of 

proprioception. Endpoint error, trajectory error and variability were calculated and compared to 

that of control subjects. We predicted that performance of the prosthetic device would be less 

accurate than controls and that the intact arm would be comparable to normal performance. 

Contrary to our hypothesis, results showed no significant difference between the performance of 

the prosthetic arm and the controls in the vision and no-vision conditions. Analysis did however 

reveal significant abnormalities in performance of the intact limb. When compared to controls, 

the intact arm of the prosthetic users had significantly larger medial endpoint errors for the 

ipsilateral target without visual guidance (P = 0.001). This was consistent with data from the 

intact arm in the vision condition, where a significantly larger medial trajectory error (P = 0.003) 

was found. In the vision condition, this trajectory error was corrected with visual feedback, 

which allowed the subjects to correct their errors and complete the reach without endpoint error. 

The intact arms also demonstrated significantly higher variability in their reaching endpoints in 

the no-vision condition. (P = 0.010). These findings regarding the prosthetic arm reveal an 

adaptation to the altered inertial properties of the arm. The findings regarding the intact arm may 

reflect the cortical reorganization that occurs after amputation of a limb, which is consistent with 

recent theories regarding hemispheric lateralization of motor control. 

Disclosures: A.J. Metzger, None; P.S. Lum, None; C.N. Schabowsky, None; R.J. Holley, 

None; B. Monroe, None; A.W. Dromerick , None. 

Poster 




Topic: D.05.c. Eye-hand coordination 

Support: Dutch Neurofibromatosis Foundation 

Sophia Foundation for Medical Research 

Hersenstichting Nederland

Prinses Beatrix Fonds 

Title: Motor deficits in children with Neurofibromatosis type I 

Authors: *J. N. VAN DER GEEST 1 , L. C. KRAB 1,2 , A. DE GOEDE-BOLDER 2 , C. I. DE 

ZEEUW 1 , H. A. MOLL 2 , Y. ELGERSMA 1 ; 

1 Neurosci., 2 Gen. Pediatrics, Erasmus MC, Rotterdam, Netherlands 

Abstract: Neurofibromatosis type 1 (NF1) is characterized by various neurocutaneous 

symptoms and cognitive impairments. In addition, children with NF1 have frequently been 

reported to display problems in fine and gross motor functioning. However, their potential 

deficits in motor performance and motor learning capacities have so far not been investigated at 

a quantitative and systematic level. 

Here, we investigated motor performance and motor learning in 70 children with NF1 and 19 

healthy age-matched controls (8-16 years) using various quantitative tests. We used the Beery 

Developmental test for Visual-Motor Integration (Beery VMI) to assess fine motor performance 

controlled by mainly cerebral processing, and paradigms for saccadic eye movement adaptation 

and prism-induced hand movement adaptation to assess motor perfomance and motor learning 

capacities controlled by mainly cerebellar processing. 

NF1 children scored significantly lower on the Beery VMI, showing problems in both visuomotor 

integration as well as in fine motor coordination. While no significant impairments were 

observed in motor performance of either eye or hand movements, NF1 children did show deficits 

in motor learning during prism-induced hand movement adaptation. The change in hand 

movement angle before and after wearing prism glasses was more variable, and on average 

smaller in children with NF1 than in control subjects (see figure). In contrast, saccadic eye 

movement adaptation appeared not to be affected in NF1. No correlation was observed between 

scores on any of the three paradigms assessed. 

Taken together, our results suggest that the motor problems of children with NF1 in daily life 

may partly be related to deficits in motor learning. These behavioral deficits may be caused by 

aberrations within specific regions of the cerebellar and cerebral cortex, but not by a ubiquitous 

disorder of these brain regions as a whole.

Disclosures: J.N. Van Der Geest, None; L.C. Krab, None; A. De Goede-Bolder, None; C.I. 

De Zeeuw, None; H.A. Moll, None; Y. Elgersma, None. 

Poster 

263. Eye Movements: Eye and Head Control 


Program#/Poster#: 263.1/DD1 

Topic: D.06.c. Eye and head control 

Support: In partial fulfillment of the requirements for awarding the Ph.D. degree in the Brain & 

Mind Sciences to AAK (co-funded by the European Social Fund and National resources) 

French Ministry of Research grant ACI 2003 N°03 5 45 

Greek Secretariat of Research and Technology grant 03ED803 

Title: A new model of primate eye-head gaze shifts 

Authors: *A. K. MOSCHOVAKIS 1,2 , A. A. KARDAMAKIS 1,2 , A. GRANTYN 3 ; 

1 Comp Neurosci, Inst. Appl & Comput Math, Crete, Greece; 2 Basic Sci., Univ. of Crete, 

Heraklion, Greece; 3 Lab. de Physiol. de la Percept. & Action, C.N.R.S.-College de France, Paris, 

France 

Abstract: We present a neural network that simulates the portion of the primate brain 

responsible for eye-head gaze shifts and evaluate its performance. Although it assumes that it is 

the eyes and the head that are controlled independently rather than their sum (gaze), our model 

replicates the psychophysics of eye-head gaze shifts and is consistent with neurophysiology. 

Firstly, it replicates the accuracy and the time course of gaze shifts to targets at several different 

positions both head free and head fixed as well as eye plateaus and the results of head 

perturbation experiments. Secondly, it replicates the relative contributions of the eye and the 

head such that the latter increases and the former decreases together with the size of the gaze 

shifts while the absolute size of eye movements saturates at about 30 deg. Thirdly, it replicates 

the fact that the relative contributions of the eye and the head depend on the initial positions of 

the eyes as well as the position sensitivity of electricaly evoked head-fixed sacades. Moreover, it 

demonstrates that EBN units would be classified as eye-related cells and IBN units as gaze 

related cells on the basis of the linear regression line between NB and saccade size despite the 

fact that the former receive information about head movements and the latter do not. Finally, our 

model makes a number of predictions. For example, it predicts that the slope of the linear 

regression line between NB and saccade size should depend on the initial position of the eyes.

Disclosures: A.K. Moschovakis , None; A.A. Kardamakis, None; A. Grantyn, None. 

Poster 





Support: Dept. of Veterans Affairs Merit Award 

Title: Laboratory measures of eye-head coupling predict spontaneous behavior in natural 

environment 

Authors: Z. C. THUMSER 1 , *J. S. STAHL 2,1 ; 

1 Neurol., Cleveland Dept. of Veterans Affairs Med. Ctr., Cleveland, OH; 2 Dept Neurol, Case 

Western Reserve Univ., Cleveland, OH 

Abstract: The tendency to couple head movements to eye movements during gaze shifts varies 

from person to person, and these “head movement tendencies” can be quantified in the 

laboratory (Exp Brain Res 126(1999):41-54). It is unknown whether the laboratory measures 

predict eye-head coupling during spontaneous viewing in a natural environment. We addressed 

this question in 20 normal humans. Subjects were recorded in the laboratory making horizontal, 

centrifugal saccades to a sequence of illuminated targets along a ±90° semicircular array, and 

during an similar 30-minute period of spontaneous viewing while seated in an open park. Eye 

movements were recorded using a lightweight head-mounted video oculography system. Head 

movements were recorded indoors using an electromagnetic tracker and outdoors using a 

geomagnetic heading sensor. Head movement tendencies in the laboratory were quantified by the 

customary ocular motor range (COMR, the span of eye-in-head eccentricity in which the eyes 

are customarily (probability 0.9) restricted), customary head orientation range (CHOR, the span 

of head-on-neck range in which the head is customarily (probability 0.9) restricted), and eyeonly 

range (EOR, the range of target eccentricities (with respect to the head) in which the gaze 

shift is ordinarily (probability>0.5) made without a head movement). Head movement tendencies 

in the outdoor environment were quantified by COMR, CHOR, and the Percentage of time in 

which the Head was in Motion (HMoP). Indoor measures were based solely on the eye and head 

positions following centrifugal saccades, whereas the outdoor measures were based on the entire 

30-minute recording. The outdoor index HMoP correlated significantly with the indoor COMR 

(r=-0.63), EOR (r=-0.55), and CHOR (r=+0.67), in each case indicating that stronger inlaboratory 

head movement tendencies predicted that subjects spent more time with the head in 

motion during spontaneous viewing. Indoor and outdoor CHOR were also significantly

correlated (r=+0.59). In contrast, the correlation between indoor and outdoor COMR was not 

significant, although it was in the predicted direction (r=+0.21, p=0.36). One factor that may 

dissociate indoor and outdoor COMR is the fact that the range of gaze positions was controlled 

experimentally indoors, but determined spontaneously (and idiosyncratically) by each subject 

while outdoors. The results suggest that, despite there being many factors that determine whether 

or not the head is moved during each specific saccade, there are consistent head movement 

tendencies that influence subject performance across very different behavioral contexts. 

Disclosures: Z.C. Thumser, None; J.S. Stahl , None. 

Poster 






Title: Sources of input to the central mesencephalic reticular formation in macaque monkeys 

Authors: *S. WARREN 1 , L. ZHOU 1 , P. J. MAY 2 ; 

1 Dept Anat., 2 Dept Anatomy, Ophthalmology & Neurol., Univ. Mississippi Med. Ctr., Jackson, 

MS 

Abstract: The central mesencephalic reticular formation (cMRF) is a region of the primate 

midbrain where electrical stimulation elicits horizontal saccadic eye movements and recordings 

show activity related to the eye and head components of gaze. We previously demonstrated that 

the superior colliculus (SC) provides an intense input to the cMRF that terminates on cells 

projecting back to the SC. The cMRF also provides downstream projections to raphe interpositus 

(RIP), the paramedian pontine reticular formation (PPRF), medullary reticular formation (MdRF) 

and cervical spinal cord. In the present study, we sought to identify the sources of input to the 

cMRF, by injecting retrograde tracers into this structure in M. fascicularis monkeys. In the 

diencephalon, labeled neurons were observed in the zona incerta (ZI) and the rostral interstitial 

nucleus of the medial longitudinal fasciculus (riMLF), ipsilaterally. In the midbrain, we found 

that the tectoreticular projection showed injection site specific differences: the upper sublamina 

of the intermediate gray layer (SGI) was preferentially labeled from lateral cMRF injections, but 

injections that also included the medial cMRF labeled cells throughout SGI. Based on the known 

SGI projections, the medial and lateral cMRF appear to receive inputs from SGI sublaminae that 

are more head and eye related, respectively. The main source of midbrain input to the cMRF

outside the SC was the ipsilateral substantia nigra pars reticulata (SNr), although cells were also 

present in the contralateral cMRF. The medial pons was relatively free of label, with a few cells 

present ipsilaterally in RIP and the PPRF. However, labeled neurons were found in and around 

the contralateral nucleus prepositus hypoglossi (PH). Ventrolaterally, labeled neurons were 

found adjacent to the exiting facial nerve. Retrogradely labeled neurons were also present in the 

interposed and fastigial portions of the deep cerebellar nuclei. Just a few neurons were present in 

the MdRF, and in the cervical spinal cord, labeled neurons were scattered in the ventral horn. 

These findings have implications for our understanding of cMRF function. It has been suggested 

that the cMRF might provide a conduit to the SC for ascending information on saccade duration 

and direction. While some feedback was observed for most of the cMRF‟s downstream targets, 

extensive connections to support this proposed function were not found. Instead, the cMRF 

seems to receive its greatest input from structures that also supply the SC, like the SNr, ZI, and 

PH. If the cMRF reticulotectal projection is inhibitory, then the GABAergic cells in SNr and ZI 

appear to be in position to release this inhibition. 

Disclosures: S. Warren, None; L. Zhou, None; P.J. May, None. 

Poster 





Support: EY014263 

Title: The macaque central mesencephalic reticular formation sends inhibitory feedback to 

ipsilateral tectal neurons 

Authors: N. WANG 1 , S. WARREN 1 , *P. J. MAY 2 ; 

1 Dept Anat., 2 Dept Anat, Ophthalmol & Neurol, Univ. Mississippi Med. Ctr., Jackson, MS 

Abstract: The central mesencephalic reticular formation (cMRF) is believed to play a role in the 

control of gaze, as its cells exhibit saccade-related activity and its primary input is from 

collaterals of crossed descending tectal fibers. Previously, it has been demonstrated that cMRF 

neurons receiving collicular input provide a bilateral projection back to the superior colliculus 

(SC), which terminates predominantly in the intermediate gray layer (SGI). However, while it is 

presumed that tectoreticular inputs excite cMRF neurons, it is not known whether cMRF 

reticulotectal axons provide an excitatory or inhibitory feedback. Nor have the neuronal targets 

of this feedback been specified. The present study provides a detailed ultrastructural description

of cMRF reticulotectal axonal terminals and SC tectoreticular neurons. Biotinylated dextran 

amine (BDA) was injected into the cMRF of macaque monkeys (M. fascicularis) in order to 

anterogradely label reticulotectal terminals and retrogradely label tectoreticular neurons. To 

better specify the nature of this circuit, postembedding GABA immunochemistry was used. BDA 

labeled terminals in the ipsilateral SGI were examined with the electron microscope. These 

terminals, which could be identified by electron density precipitate, were moderately packed 

with small, clear pleomorphic vesicles. Their synaptic densities were predominately symmetric. 

Based on their diameters and cytoplasmic contents, the profiles lying postsynaptic to BDA 

labeled terminals were mostly distal dendrites, and occasionally proximal dendrites, with few 

somatic relationships. A total of 60 BDA labeled terminals were categorized following GABApostembedding. 

Fifty-seven of them (95%) were GABA-positive. All of the profiles postsynaptic 

to BDA labeled terminals were exclusively GABA-negative. In addition, 70 profiles from 

retrogradely labeled tectoreticular cells in ipsilateral SGI were characterized. These BDA labeled 

profiles, primarily dendrites, had homogeneous electron dense cytoplasm and were 

overwhelmingly GABA-negative, although a few were GABA-positive. Finally, BDA labeled 

terminals were observed to contact BDA labeled dendrites, indicating the feedback loop 

connecting the cMRF and SC can be monosynaptic. These results suggest that the cMRF 

projection to the ipsilateral SC is mainly inhibitory, and that neurons providing the crossed 

descending output are among its targets. Since SC outputs drive cMRF feedback neurons, this 

circuit may allow SC output neurons to either turn off their own activity during presaccadic 

bursts, or suppress the activity of other tectal efferents in order to regulate the saccade vector. 

Disclosures: N. Wang, None; P.J. May , None; S. Warren, None. 

Poster 





Support: Supported by: CIHR (Canada). 

JDC holds a Canada Research Chair. 

Title: Intrinsic reference frames for visuomotor receptive fields in head-free gaze shifts I: 

Methods and simulations

Authors: *G. P. KEITH 1 , J. F. X. DESOUZA 2 , X. YAN 4 , H. WANG 4 , J. D. CRAWFORD 3 ; 

1 Dept Psychol, 2 Psychology, 3 Psychology, Biol. and Kinesiology, York Univ., Toronto, ON, 

Canada; 4 Ctr. for Vision Res., Toronto, ON, Canada 

Abstract: We developed a method for determining the intrinsic reference frame of neuronal 

activity associated with head-unrestrained gaze shifts. The use of head-unrestrained animals 

allows for more natural behavior and distinguishing between head and space/body frames. 

However, it also necessitates the measurement of three-dimensional gaze and head orientations, 

which are torsionally noisy and enter the range of motion where non-linear effects become 

significant. To develop our method we used a data set (see accompanying abstract) where 

monkeys made head-unrestrained gaze shifts from a central home target to a variety of secondary 

targets throughout the general receptive field of units recorded in the superior colliculus. Rather 

than training the animal to dissociate eye, head, and body, we allowed the animal to dissociate 

these through the natural variability of its behavior. We developed our technique and explored its 

limitations using simulations of other possible conditions. Real and simulated unit activity were 

plotted as a function of both target location and gaze saccade metrics in eye, head, and 

body/space frames using mathematically correct 3-D transformations that accounted for 

variabilities in torsional as well as horizontal and vertical initial and final eye and head position. 

Simulations of known but noisy receptive fields in various frames showed that a neuron‟s 

intrinsic reference frame was identifiable by the greater coherence of that neuron's activity for 

trials plotted in this frame. The statistical determination of this coherence was made using the 

PRESS statistic of nonparametric regression (used since position values were continuous rather 

than discrete), which measured the predictability of the fit relative to the data. If the reference 

frame showing the best fit was significantly better than that made in the other reference frames, 

then it was identified as the candidate for that neuron's intrinsic reference frame. Further 

simulations showed that this method was robust for different receptive field shapes and sizes, 

and pointed toward various possible improvements in the experimental methodology. We further 

showed how these statistics can be combined across units and how this method can be used to 

analyze intermediate reference frames or units having gain-field-like activity modulations. The 

application of this data to our current data set is shown in the accompanying abstract, but the 

method can be applied to essentially any spatially-related unit activity. 

Disclosures: G.P. Keith, None; J.F.X. DeSouza, None; X. Yan, None; H. Wang, None; J.D. 

Crawford, None. 

Poster 



Program#/Poster#: 263.6/DD6


Support: CIHR (Canada) 

JDC holds a Canada Research Chair 

Title: Intrinsic reference frames for visuomotor receptive fields in head-free gaze shifts II: 

Superior colliculus units in the monkey 

Authors: *J. F. DESOUZA 1 , G. P. KEITH 1 , X. YAN 1 , H. WANG 1 , J. D. CRAWFORD 1,2 ; 

1 Psychology, Ctr. Vision Res., Toronto, ON, Canada; 2 Biol., York Univ., Toronto, ON, Canada 

Abstract: We trained two macaque monkeys to make gaze shifts to visual targets in a headunrestrained 

condition and where the targets were projected onto a tangent screen in front of the 

monkey. Three dimensional (3-D) eye and head orientations were recorded. A vertical recording 

chamber was placed at streotaxic centre to provide electrode access to the midbrain. Animals 

were trained to fixate a central target in the dark and then make gaze saccades toward briefly 

presented targets, with no memory delay, and with no constraints on their natural patterns of eye 

head coordination. Gaze-related activity of intermediate and deep layer superior colliculus (SC) 

neurons was identified using single-unit recording. The visuomotor receptive fields of 10 units 

were fully characterized using these methods, requiring animals to make gaze shifts toward at 

least 9 to a maximum of 16 targets with a repetition of approximately 10 trials per target. A 

standard analysis of gaze-related activity in space coordinates showed a variety of units with 

both „closed‟ and „open‟ receptive fields, with „hot spots‟ ranging from 14 to 44.7º amplitude. 

The activation for a given unit was highly variable for a given target. However, the initial 

combinations of eye and head position at centre fixation during viewing were highly variable, 

with 9.9º variation in horizontal-vertical head orientation and 11.3º variation in head torsion, and 

7.7º variation in eye-in-space torsion. A 3-D kinematic analysis was used to rotate both the 

saccade target positions and the gaze saccade trajectories themselves into space and head 

coordinates, and unit activity was re-plotted in all 6 combinations (gaze trajectory and target vs. 

three frames). The fit coherence of the units in these different plots was then compared using the 

non-parametric fit/PRESS statistic method described in the companion abstract (Keith et al. 

2008). Not every unit has been analyzed at this time, but preliminary results suggest that the 

receptive field activity of most units fit best to target position in eye coordinates, and at this time, 

at the population level these fits are statistically better than fits in head or space coordinates. 

These preliminary findings agree with our previous investigation of SC frames using electrical 

microstimulation (Klier et al. 2001). 

Disclosures: J.F. DeSouza, None; G.P. Keith, None; X. Yan, None; H. Wang, None; J.D. 

Crawford, None. 

Poster





Support: CIHR 

HFSP 

Canada Research Chair 

Title: Superior colliculus activity during cued planning of sequenced head-free gaze shifts 

Authors: M. AVILLAC 1,3 , *X. YAN 1 , J. ASENSCIO-MONTEON 1 , J. D. CRAWFORD 1,2 ; 

1 Ctr. Vision Res., 2 Departments of Psychology, Biology, and Kinesiology & Hlth. Sci., York 

Univ., Toronto, ON, Canada; 3 Neurobio. of Learning, Memory and Communication Laboratory, 

CNRS UMR 8620, Paris-Sud Univ., Orsay, France 

Abstract: Humans and non-human primates use different patterns of gaze sequences in eye-head 

coordination as a function of the behavioral context. For example, both people and monkeys 

show different eye-head kinematics when they plan a centrifugal-centripetal sequence of gaze 

shifts vs. a centrifugal-centrifugal sequence, either in repetitive patterns (Oommen et al., Exp. 

Brain. Res. 2004) or in response to a color cue (Ascensio-Monteon et al. Soc. Neurosci. Abst. 

2007). The latter study also showed that these context-dependent behaviors are retained in gaze 

saccades evoked during stimulation of the frontal eye fields, suggesting the presence of statedependent 

signals in the brain. The aim of this study is to test the role of the superior colliculus 

(SC) in processing these context-dependent signals. One rhesus monkey was trained to perform a 

cued head-free gaze saccade sequence from a central fixation point, either out-in (back to centre), 

or out-out (two equal sized gaze shifts) along any radial direction. The animal was cued to the 

type of paradigm (red fixation light for out-in and green for out-out), but the saccade target and 

required gaze displacement for the first saccade was constant. Eye and head movements were 

tracked using search coils and a chamber was implanted over both SCs. After training, the outout 

paradigm was always accompanied by a much larger head movement. Movement tuning 

fields were characterized for 101 SC neurons, which were then tested in both paradigms along 

their preferred and anti-preferred directions. 13 of these neurons were classified as primarily 

visual (stimulus onset related), 36 as primarily motor (saccade related), 22 as visual and motor 

(stimulus and saccade related), and 20 as primarily fixation-related. Our preliminary analysis 

suggests that some units with a strong motor response show a greater response in their preferred 

direction when the animal is cued that this will be followed by another saccade in the same 

direction. Activity during subsequent movements was often predictable, but some units showed 

unexpected activity in their off-direction before the second movement of the cued sequence. This 

might reflect „confusion‟ over the meaning of the cue and the expected direction of the second

movement. A more complete analysis will be provided. This data provides direct evidence for 

state-dependent signals at the level of the SC. 

Disclosures: M. Avillac, None; X. Yan , None; J. Asenscio-Monteon, None; J.D. Crawford, 

None. 

Poster 






Title: Changes in neuromuscular responses evoked by stimulation of the primate FEF as a 

function of initial gaze position 

Authors: *B. B. CHAPMAN 1 , J. K. ELSLEY 2 , B. D. CORNEIL 2 ; 

1 Neurosci., Univ. Western Ontario, London, ON, Canada; 2 Physiol. & Pharmacol., Univ. of 

Western Ontario, London, ON, Canada 

Abstract: It is well known that eye-head coordination within gaze shifts varies with initial eye 

and head position; however, the underlying neural mechanisms are not known. Previous research 

has used stimulation of oculomotor structures (e.g., SC, FEF, SEF) as one approach to examine 

this question. This approach is motivated by the similarity between volitional and evoked 

movements across different initial positions. However, given the biomechanics of the head, 

substantially different patterns of neck muscle recruitment can produce similar movement 

kinematics. The purpose of the present project is to examine the patterns of neck muscle 

recruitment following stimulation of the FEF, specifically examining how the evoked responses 

change as a function of initial gaze position. We also compared these evoked responses to those 

accompanying volitional gaze shifts. 

Stimulation (50µA, 300 Hz, 100 ms) was applied to the FEF as the monkey was fixating targets 

located between ±20° of centre. Electromyographic (EMG) responses were recorded from 

bilateral horizontal head turning muscles. Sessions were run with either the head-restrained (17 

sites) or -unrestrained (12 sites). 

Consistent with previous results, the amplitude of evoked gaze shifts decreased when initial gaze 

position was deviated contralateral to stimulation. We observed strong modulation of the evoked 

levels of EMG activity as a function of initial gaze position. Importantly, this modulation 

mirrored the baseline levels of EMG activity attained prior to stimulation, consequent to the

eccentric gaze position. The facilitation of contralateral (agonist) muscles increased for 

progressively more contralateral gaze positions (even though the evoked gaze shift was smaller). 

In contrast, the suppression of ipsilateral (antagonist) muscles increased for progressively more 

ipsilateral gaze positions. Our interpretation of these results is that the cephalomotor command 

following FEF stimulation summates with baseline levels of neck EMG activity associated with 

different initial gaze positions. Importantly, these evoked patterns of recruitment differ 

substantially from those associated with head movements during volitional gaze shifts. These 

results suggest that the neural mechanisms that vary eye-head coordination across initial gaze 

position are overridden by FEF stimulation (as with SC stimulation), and that observed changes 

in eye-head coordination following stimulation results from a straightforward combination of 

biomechanical factors with an invariant stimulation-evoked response. 

Disclosures: B.B. Chapman , None; J.K. Elsley, None; B.D. Corneil, None. 

Poster 





Support: CIHR 

Title: Neck muscle activation during gaze fixations in the monkey: 3-D postural fields and 

relationship to 3-D behavioral constraints 

Authors: *F. FARSHADMANESH 1,2 , G. P. KEITH 2,3 , X. YAN 2 , H. WANG 2 , B. D. 

CORNEIL 4 , J. D. CRAWFORD 1,2,3 ; 

1 Dept Biol., 2 Ctr. for Vision Res., 3 Psychology, York Univ., Toronto, ON, Canada; 4 Physiology, 

Pharmacol. and Psychology, Univ. of Western Ontario, London, ON, Canada 

Abstract: The head-neck motor system has more muscles than are theoretically required to 

specify a particular orientation and velocity. Although some neck muscles are reported to have a 

preferred direction (PD) of activation, because of inherent redundancy in the system, and 

interaction between muscles, relating the activation for each neck muscle to a given head 

movement is difficult. Moreover, the role of neck muscle mechanics in the implementation of 

three-dimensional (3-D) postural constraints (i.e., Donders‟ law of the head and the Fick 

strategy) are not known. Most previous studies on this topic have been performed in cats or 

humans, and generally used various stabilization and/or load-imposing tasks. The current study 

investigates the simultaneous activation of several neck muscles in relation to 3-D head posture

and movement in alert, head-unrestrained monkeys. We recorded 3-D head orientation along 

with EMG (electromyographic) activity in the following six bilateral neck muscles in two rhesus 

monkeys: sternocleidomastoid (SCM), splenius capitis (SP), complexus (COM), biventor 

cervicis (BC), rectus capitis posterior major (RCPmaj.) and occipital capitis inferior (OCI). 

Animals were allowed to make unconstrained eye-head gaze movements between targets that 

appeared randomly within a large (60 X 60 degrees) grid. Preliminary analysis in one animal has 

confirmed that the torsional head position range was smaller than horizontal and vertical ranges, 

forming a „Fick-like‟, noisy Donders‟ surface. Muscle posture fields were fit using a nonparametric 

method. All recorded muscles showed 1) PDs for upward head orientation, 2) highly 

non-linear horizontal recruitment patterns that were symmetric between bilateral pairs, and 3) 

PDs that were more linearly related to vertical head orientation. The PDs of the muscles were 

determined as vectors in 3-D. These PD vectors showed a narrow torsional distribution that 

appeared to align with the Donders‟ range of the head. Moreover, when data were recalculated 

relative to the Donders‟ surface, there was no systematic variation of EMG activity for any 

muscle with torsional head position. This suggests that the variation in head positions from a 

pure Donders‟ surface results not from the systematic activation of individual muscles, but rather 

random variation in the combination of these muscles. An analysis that includes positiondependent 

movement dynamics is planned. 

Disclosures: F. Farshadmanesh , None; G.P. Keith, None; X. Yan, None; H. Wang, 

None; B.D. Corneil, None; J.D. Crawford, None. 

Poster 






Title: Properties of ocular counter-rotation during gaze shifts 

Authors: *B. P. BECHARA 1 , N. J. GANDHI 2 ; 

1 Dept of Bioengineering, 2 Dept of Otolaryngology, Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Abducens motor neurons (ABD) receive an oculomotor command to produce an eye 

movement and a head velocity related signal to counter-rotate the eyes in the orbits. We asked: Is 

there a difference in the properties of the observed movements when the oculomotor inputs are 

matched during head-restrained (HR) and head-unrestrained (HU) gaze shifts? Moreover if a

difference exists in the eye-in-head component, is it related to the head velocity produced in HU 

trials? Also can this difference be explained by the firing rate of VN neurons (positionvestibular-pause 

and eye-head cells) known to project to the ABD? To answer these questions, 

the activities of excitatory burst neurons and their inhibitory counterpart, collectively referred to 

as burst neurons (BNs), were recorded during HR and HU gaze shifts performed by non human 

primates. The burst profile recorded on each HU trial was matched, in a root-mean-squared-error 

sense, with a burst measured during HR trials, thus satisfying the assumption that the oculomotor 

input to the ABD was the same. Matched trials were found to have similar gaze amplitudes, but 

the amplitude and peak velocity of the saccadic eye component was lower for HU movements. 

Thus, the difference in the eye velocity waveforms of HR and HU trials reflects the ocular 

counter-rotation induced by the head movement. The counter-rotation gain, defined as the ratio 

of the difference in the eye velocities and the head velocity, was high (>10) and variable at the 

onset of the movement, decreased smoothly during the gaze shift and stabilized at one after gaze 

shift offset. Finally a computer simulation was used to check if the difference in the eye 

velocities could be explained by VN inputs. The simulation modeled the difference in ABD 

firing rate (ΓABD) between HR and HU matched trials as the weighted sum of the difference in 

the firing rate of VN (ΓPVPc, ΓPVPi and ΓEHc) input. The oculomotor input was not used in 

this model because for matched trials the difference equals zero. Linear regression algorithm was 

used to find the optimal set of weights to fit this difference model. The result of this simulation 

showed that the weight of EHc cells input was the highest and accounted for most of the 

difference in the ABD and consequently the difference in the eye velocities. This leads to the 

implication that EHc cells play a major role in reducing the eye velocity during head 

unrestrained gaze shifts. 

Disclosures: B.P. Bechara, None; N.J. Gandhi, None. 

Poster 





Support: NSERC (Canada) 

Botterell Fund (Queen‟s University, Canada) 

Marie Curie (EU) 

IAP (Belgium), ARC (UCLouvain, Belgium)

FNRS (Belgium), ESA (EU) 

Title: Compensation for smooth eye and head movements by gaze saccades during headunrestrained 

tracking 

Authors: *P. M. DAYE 1 , G. BLOHM 2 , P. LEFÈVRE 2 ; 

1 CESAME-UCL, Univ. Catholique Louvain, Louvain-la-Neuve, Belgium; 2 CESAME, Univ. 

Catholique de Louvain, Louvain-la-Neuve, Belgium 

Abstract: Gaze saccades are generally composed of a combination of eye and head movements. 

In head fixed conditions, smooth eye movements that occur during the saccade latency period are 

taken into account by the saccade if the nervous system has enough time to integrate eye 

velocity. In the present study we investigate this compensatory mechanism when the head was 

unrestrained in order to possibly differentiate the compensation for eye movements from the 

compensation for head movements occurring during the saccade latency period. Subjects sat in 

front of a 1-m distant tangential screen. They were instructed to pursue with their eyes and head 

a sinusoidal target (Frequency [0.6 to 1.2 Hz]) moving along a straight line in 2D (Orientation [0 

to 360°] and amplitude [20 to 25°] randomly chosen). 1.2-1.8s after target motion onset, a second 

target was briefly flashed at a random position on the screen. The position of both eyes was 

recorded by a video-based recording device (200 Hz), head position was recorded by active 

infrared marker tracking cameras (200 Hz) and gaze orientation was reconstructed (Ronsse & al., 

2007). We analyzed how the orienting gaze shift towards the flash was programmed and how 

these saccades compensated for the smooth eye (SED) and head (SHD) displacements during the 

latency period. Multiple regression analysis (R=0.94, N=899, p



Support: MRC 

Title: Evidence for sampling and storage of visual motion information in the predictive control 

of head and eye during head-free pursuit 

Authors: *R. ACKERLEY, S. COLLINS, G. R. BARNES; 

Fac. of Life Sci., Univ. of Manchester, Manchester, United Kingdom 

Abstract: During head-free pursuit of a target, both the eyes and head move together using 

visual feedback and internal (extra-retinal) mechanisms. This necessitates that the vestibuloocular 

response is suppressed so that gaze (head + eye) and head move in the same direction. If a 

moving target is extinguished, the continuation of any movement must be controlled by internal 

mechanisms alone. Experiments were conducted to investigate the use of internal representations 

in reproducing target motion. After a period of fixation, the target moved with a randomized 

step-ramp horizontal motion (10-40 deg/sec, left or right) and subjects were instructed to follow 

the target with both eyes and head. In control trials the target was illuminated continuously. In a 

second set of trials (mid-ramp extinction), the target was visible in the first part of the ramp and 

then extinguished for 400-600ms before reappearing for the second part of the ramp. Some midramp 

extinction catch trials (10%) were embedded in the control trials. In a third set of trials 

step-ramp stimuli were presented in pairs of identical velocity. In the first of each pair (shortramp 

extinction), target motion was equivalent to the first part of the mid-ramp extinction but the 

second part (reappearance) was absent. Subjects tracked the target when visible and attempted to 

continue following it when extinguished, predicting its future trajectory. In the second of each 

pair (initial extinction), target motion was again equivalent to the mid-ramp extinction but the 

target was visible in the second part only. Subjects were encouraged to initiate eye and head 

movement so as to track the unseen target in anticipation of its appearance, using the extinction 

of fixation as a „go‟ cue. In all tasks, subjects were able to continue smooth eye and head 

movement even in the absence of visual input, a manifestation of the action of internal feedback. 

Moreover, they were able to grade their gaze and head velocity on the basis of expected target 

velocity. In both the mid-ramp extinction and catch trials, gaze and head velocity were sustained 

throughout target extinction. In these conditions motion information was derived from the initial 

part of the same trial and depended on expectation of target reappearance. For the initial 

extinction, eye and head movements were initiated ~200ms after extinction of fixation and prior 

to target appearance. In this case, grading of gaze and head velocity must have been derived from 

the prior short-ramp. In both cases, results indicate that sampled velocity information can be used 

to both generate and sustain eye and head responses when the target is not visible but when the 

future target trajectory can be predicted. 

Disclosures: R. Ackerley , None; S. Collins, None; G.R. Barnes, None.

Poster 





Support: CHIR to AW 

Title: Effects of earth-fixed vs head-fixed target on ocular counterroll: implications for 

mechanisms of VOR negation during eye-head pursuit 

Authors: *E. NIECHWIEJ 1,3 , M. CHANDRAKUMAR 3 , H. C. GOLTZ 2,3 , A. BLAKEMAN 2 , 

G. MIRABELLA 2,3 , A. M. F. WONG 2,3 ; 

1 Rehabililation Sci., 2 Ophthalmology and Vision Sci., Univ. of Toronto, Toronto, ON, Canada; 

3 Ophthalmology and Vision Sci., The Hosp. for Sick Children, Toronto, ON, Canada 

Abstract: Introduction. During combined eye-head pursuit of a moving target, the 

vestibuloocular reflex (VOR) must be negated for image stabilization. Two mechanisms have 

been proposed: VOR cancellation by a smooth-pursuit signal, and VOR suppression by a central 

process. Ocular counter-roll (OCR) generates compensatory torsional eye movements during 

static head tilt. Because there is no torsional pursuit, we postulate that if pursuit is the only 

mechanism responsible for VOR negation, OCR gain (ratio of change in eye position to change 

in head position) should not differ between viewing of an earth-fixed vs head-fixed target. 

Methods. Fifteen healthy subjects were recruited. The target consists of a central fixation cross 

against a high-contrast grid pattern at 33 cm. The target was mounted on a vertical wall (earthfixed), 

or coupled to the head (head-fixed). Eye movements were recorded using a video-based 

system. Change in torsional eye position was plotted as a function of head position step (0±25° 

in 5° steps), and a linear regression was performed. Mean OCR gain was calculated from the 

slope of the regression line. 

Results. Mean OCR gain with a head-fixed target was 43% lower than an earth-fixed target 

(p=0.003). 

Conclusion. The partial decrease in OCR gain during viewing of a head-fixed target indicates 

that both pursuit and VOR suppression are likely responsible for VOR negation during combined 

eye-head pursuit. 

Disclosures: E. Niechwiej , None; M. Chandrakumar, None; H.C. Goltz, None; A. 

Blakeman, None; G. Mirabella, None; A.M.F. Wong, None. 

Poster





Support: McCormick Fellowship 

NIH Program Project Grant 

Title: Eye movement performance during training predicts learned changes in timing 

Authors: *S.-L. SHIN, J. L. RAYMOND; 

Dept Neurobiol, Stanford Univ., Stanford, CA 

Abstract: Motor learning can induce changes in both the amplitude and timing of movements to 

optimize motor performance. The eye movements driven by the vestibulo-ocular reflex (VOR) 

can be altered by 30 min of visual-vestibular training. A decrease in VOR gain (amplitude of eye 

movement relative to head movement) can be induced by pairing vestibular stimulation with 

movement of a visual stimulus exactly with the head (called x0 training, since the ideal VOR 

gain to stabilize the image would be zero). In mice, x0 training induces not only a decrease in 

VOR gain, but also a phase lead (eye movement advances head movement). This phase change is 

apparently maladaptive, since it does not improve image stabilization. We evaluated whether a 

phase lead is a necessary byproduct of the changes supporting VOR gain decreases or whether it 

reflects an independent process in the circuit. Here we show that a subtle change in the visualvestibular 

training stimulus (x0.2 training) yielded a pure decrease in gain, with no significant 

change in phase. Thus, VOR gain changes are not necessarily accompanied by a change in VOR 

phase. We analyzed the eye movements and retinal image motion present during x0, x0.2, and 

closely-related training paradigms. Our results suggest that the phase of the tracking eye 

movements during visual-vestibular training influence the induction of changes in VOR phase. 

Disclosures: S. Shin , None; J.L. Raymond, None. 

Poster 





Support: Austrian Science Fund (FWF L425-N15) 

Upper Austrian University of Applied Sciences 

Title: Optimizing video-oculographic (VOG) eye movement recordings of rapid head impulses 

Authors: M. PLATZ 1 , J. SCHARINGER 2 , *T. P. HASLWANTER 1 ; 

1 Med. Technol., Upper Austrian Univ. o Appl Sci., Linz, Austria; 2 Dept. of Computat. 

Perception, Johannes Kepler Univ., Linz, Austria 

Abstract: Video-based eye movement recording, often called "Video-Oculography" (VOG), is 

replacing other techniques as the standard tool for measuring eye movements, for clinical 

applications as well as for research. Since new types of video cameras now regularly offer 

sampling rates above 100 Hz, the biggest remaining problem is the artifacts caused by camera 

movement with respect to the head: an uncompensated camera movement of only 1mm causes 

measurement errors of approximately 5deg. This is particularly important for VOG recordings 

during "Rapid Head Impulses", a standardized procedure for testing the functional status of the 

vestibular system: there camera slippage of a few millimeters is almost impossible to prevent. 

The most successful approach to compensate camera slippage with respect to the head has been 

utilization of the location of reflections of external illumination sources on the corneal surface. 

This technique works well when illumination and camera are at a distance that is much larger 

than the focal length of the camera. When camera and illumination are head-mounted, though, 

the suitability of this approach has yet to be shown. 

Using biomechanical simulations of the pupil center as well as of specular reflections of 

illumination points on the human cornea, we investigate the suitability and the limitations of this 

approach. In particular, we compare to which extent consideration of the actual corneal shape 

and slippage path can improve the elimination of camera slippage during VOG recordings, 

compared to more established approaches like linear or polynomial curves fit to the distance 

between pupil center and corneal reflections. 

For practical applications, our compensation scheme is applied to VOG recordings during rapid 

head impulses. For VOG we use the "EyeSeeCam" system, which can provide 3D eye 

movements at up to 500 Hz; the actual camera slippage is determined by measurements of 3D 

head- and camera movement with a "Lukotronic" system; and the results are compared with our 

compensation scheme, which tries to eliminate the camera slippage using only the information 

from the video images. 

Disclosures: M. Platz, None; J. Scharinger, None; T.P. Haslwanter, None. 

Poster 

263. Eye Movements: Eye and Head Control




Support: NASA Human Research Program 

Space Human Factors Engineering 

Title: Ocular counterroll revisited 

Authors: *D. B. LISTON 1,2 , L. STONE 1 ; 

1 Human Systems Integration Div., NASA Ames Res. Ctr., Moffett Field, CA; 2 San Jose State 

Univ. Fndn., San Jose, CA 

Abstract: Introduction: One vestibular ocular reflex (VOR) is the torsional counter-rotation of 

the eye that compensates for roll head tilt (i.e., rotation in coronal plane). However, unlike 

rotational or translational VOR, the gain of the Ocular Counter-roll Response (OCR) has 

generally been reported to be low, and is reduced further in both monkeys and astronauts after 

spaceflight (Dai et al., 1998; Moore et al., 2003). Thus, the OCR is thought to compensate only 

marginally for static head tilts (i.e., does little to align the vertical axis of the visual image on the 

retina). The OCR has been quantified for a wide range of head tilts, under a variety of 

circumstances, with various measurement methodologies. Reported OCR gain ranges from 5% to 

30% (Howard and Templeton, 1966), but most studies have reported gains only around 10%. 

Recent advances in digital photography (image resolution >10 Megapixels) and in computers 

(multi-Gigabyte RAM and multi-Gigahertz processors) allow for easier and more precise 

quantification of the OCR and invite a reinvestigation. 

Methods: We measured the rotation of the head and eyes of human observers using digital 

photographs. To measure head rotation, we either used a calibrated bite bar, or marked two 

fiducial points above and below one eye and measured the rotation of these points across photos 

of different head tilts. To measure eye rotation, we extracted the circular band that contains the 

iris from the eye image, and used cross-correlation to compute the rotation between the two 

images. 

Results: Our cross-correlation procedure yielded eye-rotation measurements with a resolution of 

0.25 deg. First, we measured the OCR using a bite bar oriented either horizontally or tilted 

rightward by 21.5 degrees. Observers (n=3) generated OCR with gain of 19%, 29%, and 31%. 

Second, we measured OCR for a series of self-generated static head tilts ranging up to 30 deg. 

Again, OCR gain (n=3) was reasonably high (22%, 24%, and 27%). 

Conclusion: Using digital photography, we measured the OCR and found its gain to be larger 

than typically reported, consistent with the highest reported values (Walton, 1948). The variation 

across previous gain measurements may be due to a number of factors, including: visual 

conditions, hysteresis, age, idiosyncratic inter-subject variability, and diurnal variation. Our 

simple methodology enables one to revisit these interesting issues. 

Disclosures: D.B. Liston, None; L. Stone, None.

Poster 




Topic: D.06.d. Saccades and Pursuit 

Support: Scientific Research (C) (Number 19500497) supported by Ministry of Education, 

Science, Sports, and Culture of Japan 

Title: Sports exercise effect on shortening of saccadic reaction time and decrease of spontaneous 

blink frequency during neck flexion 

Authors: *K. KUNITA 1 , K. FUJIWARA 3 , M. KATAYAMA 2 , H. WATANABE 1 ; 

1 Resch Ctr. Urban Hlth. and Sports, 2 Grad. Sch. of Human Life Sci., Osaka City Univ., Osaka, 

Japan; 3 Grad. Sch. of Med. Sci., Kanazawa Univ., Kanazawa, Japan 

Abstract: We investigated sports exercise effect on shortening of saccadic reaction time and 

decrease of spontaneous blink frequency during neck flexion. We selected two groups of subjects 

based on the questionnaire. They were the untrained group that have never belonged to any 

sports clubs until the university from the elementary school (30 persons; mean age 19.4 years) 

and the high-speed ball group that belong to the sports club of table tennis, tennis or baseball for 

4 years or more (30 persons; mean age 20.1 years). No subject reported any history of 

neurological or orthopedic impairments, and all provided informed consent to the experimental 

protocol. Subjects sat on a chair with their trunk stabilized by a restraining device. Saccadic 

reaction time and spontaneous blink frequency were measured in rest neck position and 20degree 

neck flexion. Visual stimuli for the saccadic task were produced by alternately lighting 

the left and right emitting diodes for random durations of 2-4 s. No significant difference of the 

reaction time between the two groups was found in the rest neck position (untrained group: mean 

reaction time 192.2 ms; high-speed ball group: mean reaction time 183.3 ms). For only highspeed 

ball group, the reaction time was significantly shorter in the neck flexion than in the rest 

neck position. No significant difference of the spontaneous blink frequency between the two 

groups was found in the rest neck position. The blink frequency per minute was 17.1 in the 

untrained group and 19.6 in the high-speed ball group. For only high-speed ball group, the blink 

frequency was significantly less in the neck flexion than in the rest neck position. These results 

indicated the sports exercise effect on shortening of saccadic reaction time and decrease of 

spontaneous blink frequency during the neck flexion. This suggested that for the high-speed ball 

group, the information processing of the saccadic neural pathway was facilitated and the 

emergence of the spontaneous blink causing the blank of visual information processing was

suppressed due to non-specific brain activation in association with the neck extensor muscle 

activity. 

Disclosures: K. Kunita , None; K. Fujiwara, None; M. Katayama, None; H. Watanabe, 

None. 

Poster 

264. Eye Movements: Smooth Pursuit 




Support: National Science Foundation Graduate Fellowship 

MH077970 

HHMI 

Title: Changes in single unit activity in the frontal eye fields (FEFSEM) during pursuit learning 

Authors: *J. X. LI, S. G. LISBERGER; 

UCSF, San Francisco, CA 

Abstract: Repeated exposure to a consistent change in target motion allows the smooth pursuit 

eye movement system to rapidly learn to emit an eye movement that predicts the time and 

direction of change in target motion (Medina, et.al. 2005). To understand how the neural signals 

for learning are represented and modified as they progress through the pursuit circuit, we 

recorded from the smooth eye movement region of the frontal eye field (FEFSEM). The 

FEFSEM is a motor cortex for pursuit that processes the visual motion input from area MT to 

drive downstream structures. Pursuit neurons in the FEFSEM do not respond in a stereotyped 

manner to pursuit. Rather, the coding of pursuit is distributed so that each neuron is most active 

during specific epochs of the movement (Schoppik, et.al. 2008). This raises the possibility that a 

neuron is selectively involved in learning at the time when it is most active. 

We recorded from 55 single units in the macaque FEFSEM to explore how these neurons alter 

their activity during pursuit learning. In these experiments, the target changed direction 250 ms 

after the onset of target motion, and the direction of target change was chosen to match the 

neuron's preferred direction. We found that the learning-related changes in responses varied 

widely across neurons. The heterogeneity could not be explained by disparities in the magnitude 

of the learned behavior, which remained fairly consistent from day to day. Instead, the amount of

neural learning was directly correlated with the neuron‟s normalized activity around 250ms 

during pursuit in its preferred direction. Higher normalized rates were associated with larger 

expressions of learning. 

To probe the hypothesis that pursuit learning generates the desired behavior by selectively 

modifying the activity of the most active neurons at the relevant time, we performed an 

additional experiment on 11 neurons where the pursuit system learned, in a subsequent block of 

trials, a change in target direction either 150, 350, or 450 ms after the onset of target motion. For 

8 of the 11 neurons, neural learning was largest when the target changed direction at the time of 

maximal firing. Two of the neurons that failed to show this effect learned poorly at all time 

points tested, suggesting that the learned movement may have been out of their dynamic range. 

Taken together, these findings indicate that one of the advantages of a temporally distributed 

coding for pursuit in the FEF may be to allow the system to efficiently and selectively modify 

movement kinematics. 

Disclosures: J.X. Li , None; S.G. Lisberger, None. 

Poster 




Topic: F.02.a. Attention 

Support: MEXT 

PRESTO-JST 

Title: Neuronal modulation during covert tracking of a moving object in primate frontal cortex 

Authors: A. MATSUSHIMA 1 , *M. TANAKA 1,2 ; 

1 Physiol, Hokkaido Univ. Sch. Med., Sapporo, Japan; 2 PRESTO, JST, Tokyo, Japan 

Abstract: Focus of attention can shift rapidly from one location to another, or move 

continuously with a moving object. Most of the previous studies examined attentional 

modulation of cortical neurons when subjects directed their attention to a fixed location. To 

explore the attentional modulation for moving objects, we devised a novel task in which 

monkeys track a moving target covertly in the presence of a moving distractor. After initial 

cueing, both visual stimuli (2°) were identical in color and shape, and moved at 20°/s in different 

directions within a 40° square. Monkeys were required to maintain fixation throughout the 

motion (3 s) and the delay (1 s) periods, and made a saccade to the target after the fixation point

offset. The properties of neuronal responses to the target or to the distractor during the motion 

period were assessed using the reverse correlation technique. 

Consistent with the previous studies, many neurons in the frontal eye field (FEF) responded 

more to the target than to the distractor located in the receptive field (Attentional type). In the 

lateral prefrontal cortex (LPFC), we also found two additional types of neurons; one responded 

more to the distractor than to the target (Distractor type), while the other modulated the activity 

depending on the locations of both the target and the distractor (Conditional type). These three 

different types of neuronal activity could be described by a simple summation model with a 

variable gain ratio between the responses to the target and the distractor. Almost all of these 

neurons displayed visual and/or delay period activity in the memory-guided saccade task. To test 

whether the Conditional type responses found in the LPFC were related to the active process 

distinguishing the target from the distractor, neuronal activity was examined in the additional 

covert tracking task in which a second moving distractor was presented with the target and the 

original distractor. The second distractor was different in color from the other two stimuli, so that 

monkeys could ignore it easily without any effort to track the moving stimuli. Although the 

locations of both the target and the original distractor determined neuronal activity critically, the 

location of the second distractor did not affect the neuronal firing considerably. 

Our data show that, in addition to the well-documented attentional enhancement, neurons in the 

LPFC also take account of the location of the distractor when its appearance is indistinguishable 

from the target. These signals might play roles in contrasting the target and the distractor 

internally, enabling monkeys to select the target reliably in the presence of equally salient visual 

distractors. 

Disclosures: A. Matsushima, None; M. Tanaka , None. 

Poster 






Title: Supplementary eye field (SEF) neurons interpret an oculomotor decision rule 

Authors: *S. J. HEINEN 1 , H. HWANG 1 , S.-N. YANG 1,2 ; 

1 Smith-Kettlewell Eye Res. Inst., San Francisco, CA; 2 Col. of Optometry, Pacific Univ., Forest 

Grove, OR

Abstract: Previously we demonstrated that SEF neurons appear to evaluate the trajectories of 

moving targets according to the rule of our novel go/nogo ocular baseball task (Kim et al., 2005). 

In ocular baseball, a central, visible zone (plate) is first displayed on the screen. Then, a small 

(0.5 deg) target begins to move from the periphery. The rule is to follow the target with a smooth 

pursuit eye movement if it intersects the plate (strike), and maintain fixation otherwise (ball). A 

population of SEF neurons discriminates between the rule states specified by strike and ball 

trajectories, despite different motion directions within each state. These neurons also modify 

their activity for the same trajectory for different rule states when plate size is changed, 

suggesting that, unlike MT neurons, SEF neurons continue to interpret the decision rule when the 

decision boundary is changed. Alternatively, these cells might merely classify the different plate 

sizes, as baseball-related activity begins when the plate appears and before the target moves. We 

present results here from a two-plate two-trajectory angle (2P2T) experiment designed to test 

between these alternatives, where neither plate size nor trajectory alone specified a given rule 

state. Single neurons were recorded from the SEF while monkeys participated in the 2P2T task. 

Plate sizes were 8 and 16 deg, and trajectory angles were 10 and 20 deg. A single plate size and 

trajectory was presented on each trial, and plate size and trajectory were randomized making any 

combination equally likely. Of 19 neurons tested, 12 correctly interpreted the rule state for all 

combinations. Furthermore, these neurons interpreted the change in the decision boundary with 

the proficiency of the monkey. The results suggest that SEF neurons interpret the decision rule of 

ocular baseball on a trial-by-trial basis and do not simply classify stimulus attributes. 

Disclosures: S.J. Heinen, None; H. Hwang, None; S. Yang, None. 

Poster 






Title: Contributions of visual area MT to variation in smooth pursuit eye movements 

Authors: *S. S. HOHL, S. G. LISBERGER; 

Physiol., UCSF, San Francisco, CA 

Abstract: Movements are inherently variable. Behavioral measurements have suggested that 

variation in smooth pursuit eye movements arises from variation in estimation of the visual 

motion parameters necessary for guiding eye movements. Extrastriate area MT provides sensory

input to the pursuit system and neurons in MT are tuned for both direction and speed of motion. 

Therefore MT is an ideal candidate area to be a source of sensory variation. If pursuit variation 

arises from MT, then there should be significant trial-by-trial correlations between the variation 

in individual MT responses and the associated pursuit eye velocity vector. We recorded from 

single neurons in macaque area MT during a standard step-ramp pursuit task. The pursuit target, 

a patch of coherently moving random dots, appeared in the receptive field of a neuron and 

moved in a given direction at a given speed. For each neuron, we tested different directions 

and/or speeds and each condition was balanced with equal numbers of target motions in the 

opposite direction to minimize anticipatory responses. We found significant trial-by-trial 

correlations between the firing rate of MT neurons and eye velocity. The correlations appeared 

consistently and were uniformly positive in many trial conditions across many neurons at the 

onset of the transient neural response. Whether firing rate and velocity were correlated depended 

on the stimulus rather than the neuron under study. The correlation values were greater for trial 

conditions that evoked higher firing rates. At times later in the neural response but still in the 

first 100 ms of pursuit, we found trial-by-trial correlations with statistical significance in excess 

of chance, but with equal numbers of positive and negative correlations. Our results show that 

neurons as early in the pursuit pathway as visual area MT contribute to variation in the initiation 

of smooth pursuit eye movements. The patterns of correlations can help us constrain how 

downstream areas decode the population response in MT to guide pursuit eye movements. 

Disclosures: S.S. Hohl, None; S.G. Lisberger, None. 

Poster 






RR00165 

Title: Neural activity in MSTd during short perturbations of ongoing smooth pursuit eye 

movements 

Authors: *U. R. BUTTNER 1 , S. ONO 2 , M. J. MUSTARI 2 , S. GLASAUER 1 , U. NUDING 1 ; 

1 Dept Neurol Klinik Grosshadern, Univ. Munchen, 81377 Munchen, Germany; 2 Yerkes Natl. 

Primate Res. Center, Emory Univ., Atlanta, GA

Abstract: Several structures in the cortex, brainstem and cerebellum are involved in the control 

of smooth pursuit eye movements (SPEM). Major areas in the cortex include the frontal eye field 

(FEF) and the medial superior temporal area (MST). The overall functions of these areas in 

smooth pursuit are still incompletely understood. For example, it has been shown that 

perturbations (high frequency sinusoidal oscillations) of target motion during ongoing pursuit 

lead to larger eye movement responses than would be observed during fixation (dynamic gain 

control, DGC). In order to obtain a better understanding of this nonlinear gain control 

mechanism we recorded neural activity in MSTd of trained monkeys (Macaca mulatta) during 

step-ramp stimuli (5-30 º/s constant velocity) with (one sinusoid 5 Hz; ± 10 º/s) and without 

perturbation. MSTd neurons responding during smooth pursuit in an otherwise dark environment 

(non-retinal component) were also modulated during large field visual motion. The preferred 

direction for SPEM and large field visual motion was always opposite in our population of 

neurons. During step-ramp SPEM, MSTd neurons increased their activity 100-120 ms after eye 

movement onset (200-250 ms after stimulus onset). Perturbation consistently led to eye 

movement changes which for most neurons were not accompanied by neuronal activity changes. 

However, 35 % of the neurons did show some modulation with perturbation. This perturbation 

related response followed (100 ms) onset of eye movement at a latency similar to that observed 

for SPEM initiation. Further analysis revealed that those MSTd neurons with higher acceleration 

sensitivity tended to show a perturbation response. In conclusion, the eye movement related 

response of MSTd neurons follows the onset of SPEM and perturbation, suggesting a role in 

maintenance but not initiation of SPEM. 

Disclosures: U.R. Buttner , None; S. Ono, None; M.J. Mustari, None; S. Glasauer, None; U. 

Nuding, None. 

Poster 





Support: HHMI 

MH0779970 

Title: Analysis of smooth pursuit circuit by recording cerebellar responses during electrical 

stimulation in the smooth eye movement region of the frontal eye fields (FEFsem)

Authors: *A. ROITMAN 1 , S. G. LISBERGER 1,2,3 ; 

1 Dept Physiol, Univ. Calif San Francisco, San Francisco, CA; 2 HHMI, San Francisco, CA; 3 Keck 

Ctr. for Integrative Neurosciences, San Francisco, CA 

Abstract: Both the cerebellum and the FEFsem are involved in controlling smooth pursuit eye 

movements. We have analyzed the activity of Purkinje cells (PCs) in the floccular complex of 

the cerebellum during the smooth eye movements evoked by micro-stimulation in the FEFsem. 

The monkey pursued step-ramp target motion to the left or the right. In half the trials (randomly 

interleaved), a 200-ms train of 50-uA pulses was delivered to the FEFsem 400 ms after the onset 

of target motion. The responses of PCs during stimulation of the FEFsem were quite different 

from those expected on the basis of the evoked smooth eye velocities. The observed Purkinje cell 

responses could not be explained by inputs related to either retinal image motion or efference 

copy of the eye movement. The previously reported enhancement of the stimulation effect on the 

eye velocity during pursuit compared to that during fixation was confirmed. However, the 

enhancement was markedly absent in the PC responses. We suggest that the floccular responses 

reflect the gain control signal that emanates from the FEFsem. 

The population response to electrical stimulation of the FEFsem provided a command signal that 

followed the time course of the evoked eye velocity, but was considerably smaller than predicted 

given the responses of the same PCs during smooth eye movement in the absence of the 

stimulation. We conclude that the signals from FEFsem may activate another structure, besides 

the floccular complex, that also drives smooth eye velocity. 

Disclosures: A. Roitman, None; S.G. Lisberger, None. 

Poster 




Topic: D.06.b. Cortex and thalamus 


Aginsky Estate 

NSF Graduate Research Fellowship 

Title: Target selection for smooth pursuit eye movements in the frontal pursuit area

Authors: *S. P. MAHAFFY 1,2 , R. J. KRAUZLIS 1 ; 

1 Systems Neurobio. Lab., Salk Inst., La Jolla, CA; 2 Neurosciences Grad. Program, UCSD, La 

Jolla, CA 

Abstract: Primates are adept at selecting moving targets for smooth pursuit eye movements, 

even in the presence of competing distracter motion. The superior colliculus has been implicated 

recently in the spatial selection of targets for smooth pursuit, but the neural mechanism for 

selecting the direction of pursuit motion is unknown. Based on its connectivity and previously 

documented role in pursuit gain control, the frontal pursuit area is a reasonable candidate for the 

site that helps determine the direction of pursuit. In particular, the frontal pursuit area contains 

directionally selective cells that respond during the acceleration and maintenance of smooth 

pursuit. To test his idea, we have recorded in the frontal pursuit area during a target selection 

task to determine whether neurons in this area discriminate pursuit targets in time to underlie 

pursuit selection. 

The task was a two-alternative forced choice match-to-sample selection of isoluminant colored 

dots moving in a step-ramp towards fixation. The monkey‟s task was to smoothly pursue the 

stimulus whose color matched the cue presented earlier in the trial. Dots always moved parallel 

and opposite to each other slightly offset from fixation, and their angle was adjusted to match the 

preferred direction of the neuron under study. Pursuit latencies were measured for each trial with 

a hinge model. Neural discrimination latencies, identifying when the cell‟s firing rate 

discriminated the direction of pursuit, were measured with an ROC analysis with a precision of 

one millisecond. 

We have recorded from 34 pursuit-related cells in the frontal pursuit area of one monkey. Pursuit 

in the preferred direction typically evoked a vigorous and sustained response during the target 

selection task, with some cells exhibiting a burst of activity during the initiation of pursuit. 

Pursuit in the non-preferred direction left the firing rate unchanged or suppressed, although some 

cells showed a transitory increase in activity before pursuit onset regardless of pursuit direction. 

We found that 15 out of the 34 (44%) cells discriminated target from distracter before pursuit 

onset. For these cells, the mean lead time for neural discrimination was 13 ms +/- 12ms (S.D.). 

These results show that a substantial minority of cells in the frontal pursuit area discriminate 

target from distracter prior to the onset of pursuit. This suggests that the frontal pursuit area may 

be involved in the process of determining the direction of pursuit eye movements when the target 

is selected in the presence of distracters. 

Disclosures: S.P. Mahaffy , None; R.J. Krauzlis, None. 

Poster 






RR 00165 

Title: Information processing in parallel cortical-brainstem pathways for smooth pursuit 

Authors: *M. J. MUSTARI 1,2 , S. ONO 1,2 , V. E. DAS 1,2 ; 

1 Neurol., Emory Univ., Atlanta, GA; 2 Div. of Sensory-Motor Systems, Yerkes Natl. Primate Res. 

Ctr., Atlanta, GA 

Abstract: Smooth pursuit eye movements are used to maintain the image of a moving object 

relatively stable on or near the fovea. The smooth pursuit system therefore must decode visual 

motion and create commands for eye movements. This visual-oculomotor transformation 

involves cortical, brainstem and cerebellar regions that carry complimentary signals for 

supporting volitional smooth pursuit. We have examined information processing related to 

smooth pursuit in different cortical areas including frontal eye field (FEF), middle temporal 

(MT) and medial superior temporal (MST). Signals in the cortical pursuit system influence the 

cerebellum through the dorsolateral pontine nucleus (DLPN), nucleus reticularis tegmenti pontis 

(NRTP) and pretectal nucleus of the optic tract (NOT). We used multiple linear-regression 

modeling to estimate the relative sensitivities of cortical (MT, MST, FEF), pontine (NRTP, 

DLPN) and pretectal nucleus of the optic (NOT) neurons to eye-motion parameters (position, 

velocity and acceleration) and retinal-error parameters (position, velocity and acceleration) 

during step-ramp smooth pursuit of macaques (Macaca mulatta). We found that a large 

proportion of pursuit-related MST and DLPN neurons primarily code eye-velocity information. 

Similarly, a large proportion of FEF and rostral NRTP neurons code eye-acceleration 

information. In contrast, foveal/parafoveal NOT neurons are most sensitive to retinal image 

velocity. Anatomical studies indicate that the FEF-NRTP pathway selectively targets the vermis, 

MST-DLPN pathway targets the ventral paraflocculus and MT-NOT pathways target the 

flocculus and ventral paraflocculus. Therefore, parallel channels of information originating in 

occipital, parietal and frontal cortical areas influence different regions of the cerebellum to 

initiate, maintain and adjust volitional smooth pursuit. 

Disclosures: M.J. Mustari , None; S. Ono, None; V.E. Das, None. 

Poster 






RR00165 

Title: The pretectal nucleus of the optic tract (NOT) is essential for smooth pursuit adaptation 

Authors: *S. ONO 1,2 , K. C. VITORELLO 2 , M. J. MUSTARI 1,2 ; 

1 Neurol., Emory Univ., Atlanta, GA; 2 Yerkes Natl. Primate Res. Ctr., Atlanta, GA 

Abstract: The smooth pursuit system has the capacity to adapt to changes associated with 

development, injury and new behavioral demands. The sources of signals that support smooth 

pursuit adaptation are incompletely understood but could involve cortical and brainstem areas 

involved in generation of pursuit and retinal image motion processing. Previous lesion and single 

unit recording studies show that the macaque cerebellum (e.g., ventral paraflocculus and vermis) 

plays an essential role in smooth pursuit initiation, maintenance and adaptation. Here, we focus 

on the pretectal nucleus of the optic tract (NOT), which provides retinal error information to the 

cerebellum (e.g., through the dorsal cap of the inferior olive) during smooth pursuit. The aim of 

this study is to characterize the potential roles of the NOT in smooth pursuit adaptation. 

We studied smooth pursuit adaptation in two juvenile rhesus monkeys (Macaca mulatta), using 

double-steps of target speed that step-up (10°/s to 30°/s) or step-down (25°/s to 5°/s). The target 

begins moving at one speed for first 100 ms and then changes to either a higher or lower speed. 

We used muscimol blockade (≤ 2%; 0.15µl) to reversibly inactivate (lesion) the NOT, 

unilaterally. The inactivation of NOT produced a partial deficit in smooth pursuit towards the 

side of injection. Muscimol injection of NOT also produced nystagmus with contralesional slow 

phases. We have found that inactivation of NOT significantly impaired smooth pursuit 

adaptation in the step-up paradigm for ipsilesional tracking (P



Title: Neural activities related to smooth pursuit and saccade eye movements in the primate 

cerebellar hemispheric lobules VI/VII 

Authors: *H. KITAZAWA 1 , M. OHKI 2 , T. KITAMURA 3 , J. YAMADA 3 , S. NAGAO 1,4 ; 

1 Lab. Motor Learning Control, RIKEN BSI, Wako, Japan; 2 Otolaryngol., Grad. Sch. of Med., 

Univ. of Tokyo, Tokyo, Japan; 3 Histo&Neuroanat, Tokyo Med. Univ., Tokyo, Japan; 4 SORST, 

Saitama, Japan 

Abstract: Primate cerebellar hemispheric lobules VI/VII (H-VI/VII), as well as the vermal 

lobules VI/VII, receive inputs issued from the frontal eye field. We previously reported that 

lesions of H-VI/VII appreciably depressed velocities of smooth pursuit eye movements and 

delayed the onsets of saccades. In the present study, we recorded single-unit activities of H- 

VI/VII Purkinje cells from two macaque monkeys to examine their responses to smooth pursuit 

and visually-guided saccade eye movements. We classified simple spike responses of H-VI/VII 

Purkinje cells into the following 4 groups. Group 1(51/138) increased simple spike discharges 

transiently at both the saccade and pursuit onsets. The increase in spike activities advanced the 

onsets of saccade eye movements by 10-100ms, and those of smooth pursuit by 10-50ms. 

Purkinje cells exhibited no directional preferences. Group 2 (46/138) and group 3 (17/138) 

increased simple spike discharges only at the onsets of saccade and smooth pursuit, respectively. 

One-third of the group 1 and 3 cells tested showed smooth pursuit velocity- or accelerationrelated 

responses. Group 4 (5/138) decreased simple spike discharges transiently at the onsets of 

saccade or smooth pursuit. Slow or quick eye movements, predominantly in the oblique 

directions, were induced by microstimulation in 30 % of the areas where these Purkinje cells 

were recorded. Taken together these results with those of previous lesion study, we suggest that 

the H-VI/VII plays an important role in the velocity tuning of the early (open-loop) component 

of smooth pursuit, and also in the initiation timing of saccade. 

Disclosures: H. Kitazawa, None; M. Ohki, None; T. Kitamura, None; J. Yamada, None; S. 

Nagao, None. 

Poster 




Topic: D.06.b. Cortex and thalamus

Support: DFG SFB 509 

Title: Development of the neural substrate of the optokinetic reflex in macaque monkeys 

Authors: *C. DISTLER, K.-P. HOFFMANN; 

Dept Zoology & Neurobiol, Ruhr Univ. Bochum, D-44780 Bochum, Germany 

Abstract: In infant macaques, monocular horizontal OKR (mhOKR) can be elicited both in 

temporo-nasal and, at lower gain, in naso-temporal direction already shortly after birth. 

Symmetry depends on stimulus velocity: mhOKR at low stimulus velocities becomes 

symmetrical at about 3 weeks of age, at higher stimulus velocities it reaches symmetry at about 

4-5 months of age (Distler, Vital-Durand, Korte, Korbmacher, Hoffmann, Vis Res. 39: 3909- 

3919, 1999). 

We recorded from retinal slip neurons in the nucleus of the optic tract and dorsal terminal 

nucleus (NOT-DTN) of 4 anaesthetized and paralyzed infant macaques (age: P9, P12, P14, P60). 

As in adults, retinal slip neurons in the NOT-DTN were already direction selective for 

ipsiversive stimulus movement shortly after birth (P9). The large majority (96%) of NOT-DTN 

neurons in P9, P12 and P14 were binocular (OD groups 2, 3, and 4). However, more neurons 

were dominated by the contralateral eye (OD group 2) in infants than in adults which could 

explain the mild asymmetry of mhOKR at that age. Already in the youngest animals, NOT-DTN 

neurons responded well to different stimulus velocities, their velocity tuning, however, was 

narrower than in adults, trunkated towards low stimulus velocities. 

Already at P12, electrical stimulation in V1 elicited orthodromic responses both in the superior 

colliculus and the NOT-DTN. However, the incidence of orthodromically activated neurons was 

much lower in infants (40-60% of the tested NOT-DTN neurons) than in adults (97%). 

Orthodromic latencies from V1 were significantly longer in P14 (x=12.2ms ± 8.9) than in adults 

(x=3.51ms±0.81). By contrast, electrical stimulation in motion sensitive area MT was 

significantly more efficient in activating NOT-DTN neurons (80% of the tested cells) and 

yielded shorter latencies than V1 (x= 7.8ms ± 3.02; adult x= 2.99ms ± 0.85). 

Thus, cortical input in addition to bilateral retinal input to retinal slip neurons in the NOT-DTN 

is present at latest at 2 weeks after birth. The lower incidence of orthodromically activated cells 

and the long latencies indicate that the cortical projection to the NOT-DTN is quite immature at 

that point. The projection from motion sensitive are MT is more efficient than that from V1. 

Disclosures: C. Distler , None; K. Hoffmann, None. 

Poster 





Title: Smooth pursuit adaptation (SPA) exhibits features useful to compensate changes in the 

properties of the smooth pursuit eye movement system due to usage 

Authors: *S. DASH 1 , P. THIER 2 ; 

1 Cognitive Neurol., Hertie Inst. for clinical brain res., Tuebingen, Germany; 2 Cognitive Neurol., 

Hertie Inst. for Clin. Brain Research., Tuebingen, Germany 

Abstract: During the first 100-150ms of smooth pursuit eye movements (SP) made in an attempt 

to compensate a ramp-like movement of a visual target, the eyes basically move open-loop due 

to the inherent delays of the visual system. Smooth-pursuit adaptation (SPA) refers to the fact 

that the pursuit gain in this early response phase can be adjusted based on experience. For 

instance, if the target moves initially at a constant velocity for around 100-200ms and then steps 

to a higher velocity, subjects learn to upregulate the pursuit gain evoked by the initial target 

velocity (gain increase SPA) in order to reduce the retinal error resulting from the velocity step. 

Correspondingly, a step to a lower target velocity leads to a decrease in gain (gain decrease 

SPA). Unadapted SP shows a characteristic initial acceleration profile with distinct peak 

acceleration and duration. Gain increase SPA is a consequence of expanding the duration of this 

profile while keeping peak acceleration constant. Conversely, gain decrease SPA exhibits 

reduced peak acceleration but unaltered duration (Takagi et al. J Neurophysiol. 83: 2047ff). 

In the present study we asked two monkeys to carry out stereotyped SP guided by ramp-like 

target movements (15 or 20°/s, fixation duration 500-800ms, ramp duration 600ms, and no intertrial 

interval). After 200-300 trials we observed a clear drop in initial peak acceleration which 

was compensated by an increase in the duration of the acceleration profile, thereby keeping 

initial pursuit gain constant. This drop in peak acceleration is similar to the one found in gaindecrease 

SPA and must reflect changes of the oculomotor periphery or other parts of SP system 

due to usage (“fatigue”). The compensatory increase in the duration of the acceleration profile 

during pursuit onset is reminiscent of the one leading to gain-increase SPA. This striking 

congruence in the kinematic changes during fatigue compensation and SPA suggests a common 

functional basis of the two: specifically, gain-decrease SPA seems to be at least in part a passive 

process building on the acceleration loss due to fatigue. On the other hand, both gain-increase 

SPA and fatigue compensation exploit the ability of the system to expand the duration of the 

initial acceleration response. The ability to adjust acceleration duration depends on the integrity 

of the oculomotor vermis (OV). This is suggested by the consequences of surgically lesioning 

the OV in one of the two monkeys we have studied. 

Disclosures: S. Dash , None; P. Thier, None. 

Poster 

264. Eye Movements: Smooth Pursuit




Support: Oppenheimer/Stein Endowment Award 

NIH EY-12816 

DARPA FA8650-06-C-7633 

NIH EY-18322 

Title: White noise analysis of smooth pursuit eye movements 

Authors: A. TAVASSOLI 1 , *D. L. RINGACH 2 ; 

1 Neurobio., 2 Dept Neurobiol, UCLA, Los Angeles, CA 

Abstract: Smooth pursuit eye movements allow the stabilization of small moving targets on the 

retina. The system can be thought of as a negative feedback controller, where the velocity of the 

target must be matched by that of the eye as closely and rapidly as possible (Rashbass, 1961). 

Here, we studied the (steady-state) maintenance phase of pursuit by measuring smooth pursuit 

eye movements when a target visual stimulus (a high-contrast Gabor) had a velocity determined 

by white noise superimposed on top of a mean baseline velocity. We estimated the first-order 

kernels of the system by cross-correlating fluctuations in eye velocity with fluctuations in target 

speed in saccade-free segments of pursuit. We performed the experiment for a range of baseline 

velocities and standard deviations of the noise. 

We found that (a) the first-order kernels are narrow in time, with half-width half-heights of ~50 

ms and a time-to-peak of ~100 ms, (b) there is a „gain control‟ mechanism at work, such that the 

overall gain of the system is modulated by the mean velocity and the standard deviation of the 

target velocity, (c) the standard deviation of eye velocity is proportional to mean target velocity 

and only weakly correlated with the standard deviation of the target velocity standard deviation, 

(d) two basic predictions of linearity between input and output fluctuations hold: the DC gain of 

the system is proportional to the integral of the impulse response, and the variance in the eye 

velocity is proportional to the energy of the impulse response multiplied by the variance of the 

stimulus velocity, (e) smooth pursuit gain decreased from ~1 at low noise levels to about ~0.9 at 

the highest noise levels tested, for which there was no measurable correlation between eye 

velocity fluctuations and stimulus velocity fluctuations, suggesting that smooth pursuit 

movements can be performed even in the absence of first-order visual motion measurements. 

Our results show that smooth pursuit maintenance is quasi-linear and amenable to white noise 

analysis.

Disclosures: A. Tavassoli, None; D.L. Ringach , None. 

Poster 





Support: conte center P50 MH077970 

HHMI 

Title: Single trial learning in smooth pursuit eye movements 

Authors: Y. YANG 1 , *S. G. LISBERGER 2 ; 

1 Dept. of physiology, HHMI, Keck Ctr. Integrative Neuroscience, UCSF, San Francisco, CA; 

2 Dept Physiol, Univ. California Sch. Med., San Francisco, CA 

Abstract: Motor skills improve with repeated attempts at the same task. To analyze the dynamic 

neural basis of learning, however, we have developed a new approach to study the effect of 

instructional stimuli in one behavioral trial on neural responses in the subsequent trial. 

Experiments consisted of a sequence of instructional trials where the target always started by 

moving in one direction and then, after 250 ms, added an instructional component of motion in 

the orthogonal direction. When the starting motion was rightward, the direction of the second, 

instructional motion varied randomly to be upward or downward. We then asked how the 

direction of the instructional motion on each of 10 previous trials affected the learned eye 

movement in a given trial. We found that the most recent trial had a large impact on the learned 

eye movement in any given trial, with rapidly decreasing weight given to each earlier trial. In 

addition, to the large and rapid learning induced by a single trial, we found a slower, learning 

when the same instructional motion was presented repeatedly. Because of the large amount of 

single trial learning in pursuit, the results might reflect anticipation rather than learning. To test 

this possibility, we presented cues that lasted 50, 150, or 250 ms and consisted of target motion 

to the right. As before, the instructional target motion started to move upward or downward 250 

ms after the start of the cue. The 250 ms duration cue provided the same target motion used to 

demonstrate single trial learning; the shorter cues provided the same information about the time 

of the instructional target motion, but engaged pursuit less effectively. If single trial learning 

were due to anticipation, then the amount of single trial learning should be independent of cue 

duration. If single trial learning represented pursuit motor learning, then we would expect more 

learning for longer duration cues. This is what we found. We conclude that a single instructional

trial causes impressive pursuit learning, that the learning is related to the pursuit motor action 

rather than to cognitive expectation, and that single trial learning comprises the rapid component 

of a full learning repertoire that also includes a more slowly acquired component. 

Disclosures: Y. Yang, None; S.G. Lisberger , None. 

Poster 





Support: Department of Veterans Affairs 

Evenor Armington Fund 

NIH R01-EY06717 

Title: Effects of horizontal divergence on vertical ocular following responses (OFR) 

Authors: A. C. JOSHI 1 , M. J. THURTELL 1 , M. F. WALKER 2 , A. SERRA 2 , *R. LEIGH 3 ; 

1 Biomed. Engin., 2 Neurol., Case Western Reserve Univ., Cleveland, OH; 3 Neurol. Service 127 

(W), Va Med. Ctr., Cleveland, OH 

Abstract: Human OFR is a pre-attentive, short-latency visual field-holding response, which is 

enhanced if a moving stimulus is applied in the wake of a saccade (Gellman, Carl & Miles, 

1990). Since most natural gaze shifts incorporate both saccades and vergence movements, we 

asked whether the OFR was also enhanced during vergence movements. Five subjects viewed 

vertically moving sine-wave gratings on a video monitor at 45 cm that had a temporal frequency 

of 16.7 Hz, contrast of 32%, and spatial frequency of 0.17, 0.27 or 0.44 cycles/degree. In 

fixation/OFR experiments, subjects fixed upon a white central dot on the video monitor, which 

disappeared at the beginning of each trial, as the sinusoidal grating started moving up or down. 

In vergence/OFR experiments, subjects initially fixed upon a red spot at ~15 cm; when the red 

spot went out, the white spot appeared on the monitor, which was the cue to diverge. After ~150 

ms, during the horizontal divergence movement, the white spot turned off and the sinusoidal 

grating elicited vertical OFR. Eye movements were monitored using the magnetic search coil 

technique, and we measured the change in eye position in the interval 70 - 150 ms following 

stimulus onset, correcting for drifts prior to onset of OFR. All subjects showed larger OFR when 

the moving stimulus was presented during divergence compared with following fixation,

especially if a correction was made for any vertical drift due to divergence (p =0.003, paired ttest). 

Since gaze shifts from near-to-far are common during locomotion, we postulate that the 

enhancement of OFR during divergence movements serves to stabilize the visual field when the 

eyes arrive at the new fixation point. 

Disclosures: A.C. Joshi, None; M.J. Thurtell, None; M.F. Walker, None; A. Serra, None; R. 

Leigh , None. 

Poster 






Evenor Armington Fund 

NIH R01-EY06717 

Title: Vertical saccadic and smooth pursuit eye movements violate Listing‟s law in normal 

humans 

Authors: M. J. THURTELL 1 , *M. F. WALKER 1 , A. C. JOSHI 2 , R. J. LEIGH 1 ; 

1 Neurol, 2 Biomed. Engin., Case Western Reserve Univ., Cleveland, OH 

Abstract: For Listing‟s law to be obeyed during eye movements, the „half-angle rule‟ must be 

satisfied, such that the eye velocity axis must tilt by exactly half the angle of eye eccentricity 

from primary position. In the present study, we aimed to determine how closely the half-angle 

rule is obeyed during vertical compared with horizontal saccadic and smooth pursuit eye 

movements. Using the magnetic search coil technique, three-dimensional eye and head rotation 

data were acquired from normal humans. With the head fixed, saccades were recorded in 

response to 40 deg jumps in laser target position. Horizontal saccades were recorded at five 

elevations (20 deg up, 10 deg up, 0 deg, 10 deg down, and 20 deg down), while vertical saccades 

were recorded at five azimuths (20 deg left, 10 deg left, 0 deg, 10 deg right, and 20 deg right). In 

the pursuit paradigm, subjects followed a laser target moving at 20 deg/s with a peak-to-peak 

amplitude of 40 deg. Horizontal pursuit was recorded at the same elevations as for horizontal 

saccades, while vertical pursuit was recorded at the same azimuths as for vertical saccades. Data 

were rotated into Listing‟s coordinates and the pursuit data were desaccaded. Eye velocity axis

orientation was determined from spatial plots of the eye-in-head velocity data. To determine how 

well the half-angle rule was obeyed, the tilt-angle coefficient was calculated as the angle of eye 

velocity axis tilt divided by the angle of eye position eccentricity. During horizontal saccadic and 

smooth pursuit eye movements, the half-angle rule was approximately obeyed, with tilt-angle 

coefficients being close to 0.5. In contrast, the half-angle rule was not satisfied during vertical 

saccadic and smooth pursuit eye movements, with tilt-angle coefficients being significantly less 

than 0.5. We suggest that this discrepancy could arise due to differences in the degree of eye 

position-dependent change in tendon pulling directions for the horizontal recti versus the vertical 

recti and oblique muscles. 

Disclosures: M.J. Thurtell, Department of Veterans Affairs, A. Employment (full or parttime); 

A.C. Joshi, None; M.F. Walker, Department of Veterans Affairs, A. Employment (full or 

part-time); R.J. Leigh, Department of Veterans Affairs, A. Employment (full or part-time); NIH 

R01-EY06717, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); Evenor Armington Fund, B. Research Grant (principal 


Poster 





Support: CIHR 

Canada Research Chair program 

Title: Task dependent spatial updating of saccade targets during smooth pursuit 

Authors: R. RADIK 1 , X. YAN 1 , *H. WANG 2 , J. F. X. DESOUZA 3 , J. CRAWFORD 1 ; 

1 Dept. Biology, Ctr. of Vision Res., 2 Dept Psychol, 3 Dept. Psychology, York Univ., Toronto, 

ON, Canada 

Abstract: It is known that monkeys are able to spatially update the location of saccade targets 

across an intermediate saccade or smooth pursuit (SP) movement (A. McKenzie and S. G. 

Lisberger, J. Neurophysiology, 1986). Less is known about the dynamics of the underlying 

neural signals during this updating, and how they might depend on instruction. In a recent 

modeling study, we found that neural network models developed different types of spatial 

remapping strategies depending on whether the metrics of an intervening SP movement were

predictable or unpredictable (Keith et al. Soc. Neurosci. Abst. 2007). Here, we reexamined the 

ability of primates to update saccade targets across SP movements, specifically testing the 

hypothesis that if animals are cued when and where SP will end, memory guided saccades will 

have lower latency and increased accuracy. To date, a complete behavioral data set has been 

obtained from 2-D search coil recordings in one rhesus monkey. The monkey was trained to 1) 

fixate a home fixation point 10º from the center in either direction along one of the 4 cardinal 

and oblique lines, and 2) then a memory target was flashed at a right angle (in either direction) 

from this line, also 10º from centre. 3) Following the memory flash, the fixation target began to 

move at 14º/s through the centre position and the animal was required to pursue the target until it 

extinguished. 4) At that point the animal had to saccade toward the memory target to obtain a 

reward. This created a total combination of 16 general SP-saccade direction pairs. In the un-cued 

condition, the pursuit target unpredictably traversed 1-30º before extinguishing, making the exact 

direction of the required saccade unpredictable. In the cued condition (a fixation cross instead of 

a dot) the SP target always traversed 20º, so in principle saccade direction was predictable. 

During initial training only, the SP end point was shown along with the cue to re-enforce this 

association. Blocks of cued and un-cued were used in training whereas all testing paradigms 

were randomly interleaved. The animal learned to perform all aspects of the spatial updating task 

with general accuracy (we required a 6º degree window for the final saccade target), but made 

idiosyncratic direction-dependent errors. Comparing between 20º SP runs (368 trials in total) we 

found a statistically lower saccade latency (p

Abstract: When generating saccades toward a moving target, the target displacement that occurs 

during the period preceding saccade end must be taken into account in order to accurately 

foveate the target. Previous studies have shown that these saccades are characterized by a 

prolonged deceleration phase. In some cases, a second peak eye velocity appears during the 

deceleration, presumably reflecting the late influence of a mechanism that compensates for the 

target motion. The goal of this work is to further determine the dynamics of this compensatory 

mechanism. 

Saccades were studied in 3 head restrained rhesus monkeys trained to track a small Gaussianblurred 

disk (diam. 0.5°). After a 200 ms gap, the target stepped from a central (straight ahead) 

position toward a peripheral one (8° or 16°) along the cardinal axes (horizontal or vertical). 

Then, the target started to move at a constant speed after a delay that ranged between 0 and 300 

ms. The target motion was always orthogonal to the target step (e.g. rightward or leftward 

motion after an upward or downward step). This arrangement was designed in order to easily 

decompose the saccade into two components: a component induced by the target step and 

another induced by the target motion. Different target speeds (7, 14 or 21°/s) were tested in order 

to estimate when the build-up of the motion-induced component was completed. Oblique 

saccades to stationary targets located at positions comparable to those reached by saccades 

toward moving targets were also tested. 

Our results show that the amplitude of the step-induced component did not vary with the presaccadic 

exposure duration, i.e. the period during which the target moved before the saccade is 

launched. In contrast, the motion-induced component, which was saccadic, increased with the 

pre-saccadic exposure duration. The temporal increase in amplitude differed between the three 

target speeds. For exposure durations longer than 70 ms, the motion-induced amplitude was 

clearly larger for faster target motions. The changes in amplitude started to differ from baseline 

approx. 50 ms after the onset of target motion. Interestingly, the motion-induced component was 

often delayed relative to the step-induced component, leading to a curvature of saccades. This 

asynchrony contrasted with the synchronized onsets of horizontal and vertical components 

during oblique saccades. 

Our study provides a direct estimate of the time taken for visual motion-related signals to update 

the programming and execution of saccades. The difference in saccade curvature between 

stationary and moving targets provides another illustration of the dynamic control of saccade 

trajectory by visual input. 

Disclosures: J. Fleuriet, None; S. Hugues , None; L. Goffart, None. 

Poster 

265. Nociceptors II 


Program#/Poster#: 265.1/EE2

Topic: B.02.t. TRP Channels 


Title: The development of peripheral cold neural circuits based on TRPM8 expression 

Authors: *Y. TAKASHIMA, D. D. MCKEMY; 

Univ.Southern Calif, Los Angeles, CA 

Abstract: Our peripheral somatosensory system is composed of many different types of sensory 

neurons that are responsible for detecting a broad range of environmental cues. Cold 

temperatures are primarily detected by neurons expressing the cold and menthol receptor 

TRPM8. We and others have previously shown these neurons to be a diverse and heterogeneous 

cell population that is likely responsible for a wide array of cold perceptions. How then is this 

heterogeneity established developmentally? To address this question, we have investigated the 

developmental time course of TRPM8 expression in a mouse transgenic line (Trpm8GFP) in 

which green fluorescent protein (GFP) expression is driven by the TRPM8 transcriptional 

promoter. We have analyzed Trpm8GFP expression throughout different embryonic and 

postnatal stages, and correlated their neurochemical phenotypes with that of other neuronal 

markers. Specifically, we have examined Trpm8GFP expression with markers for specific fiber 

types, trophic factor dependence, and somatosensory and nociceptive signaling. With this 

approach, we have established the developmental profile of the neurons that express TRPM8 as 

means to understand how distinct cold neural circuits are established. 

Disclosures: Y. Takashima, None; D.D. McKemy, None. 

Poster 



Program#/Poster#: 265.2/EE3 



Title: Cold neural circuitry identified by TRPM8-dependent c-Fos expression 

Authors: W. M. KNOWLTON, *D. D. MCKEMY; 

Bio. Sciences, Neurobio., Univ. of Southern California, Los Angeles, CA

Abstract: Cold temperatures are known to activate a small subset of sensory neurons, yet the 

perception of cold varies from innocuously cool to painfully cold. Remarkably, even with this 

diversity of cold responses, the majority are mediated by a single molecular sensor, the cold and 

menthol receptor TRPM8. We are interested in understanding how TRPM8 mediates the diverse 

sensations of cold, and have shown previously that TRPM8 neurons are anatomically 

heterogeneous in vivo. Our data suggests that TRPM8 neurons comprise multiple and 

functionally distinct neural circuits. To address this hypothesis, here we use expression of the 

immediate early gene c-Fos and examine neural activity evoked by both cooling compounds and 

cold temperatures in second order neurons in the spinal cord dorsal horn. Specifically, we found 

that the super-cooling agent icilin, a TRPM8 agonist, induces robust c-Fos expression in the 

superficial laminae of the dorsal horn when applied to the mouse hindpaw. These observations 

were extended to activity evoked by a range of cold stimuli and the dependence of these 

responses on functional TRPM8 expression. These data will begin to define and characterize the 

neural circuitry responsible for the wide array of sensations evoked by cold. 

Disclosures: W.M. Knowlton, None; D.D. McKemy, None. 

Poster 





Support: NIH/NINDS Grant NS054069 

Title: Mechanisms underlying TRPM8 adaptation 

Authors: *R. L. DANIELS 1 , Y. TAKASHIMA 1 , H. N. WATERS 2 , E. R. LIMAN 2 , D. D. 

MCKEMY 2 ; 

1 Prog Neurosci., 2 Biol. Sciences, Neurobio. Section, USC, Los Angeles, CA 

Abstract: Cold-sensing peripheral nerve fibers participate in innocuous temperature detection 

and underlie thermoregulatory behaviors in many organisms. During a prolonged cold stimulus, 

the firing rate of these fibers steadily decreases over time, an adaptive process thought to provide 

the peripheral nervous system with a means to discriminate subtle changes in temperature. At the 

molecular level, cold temperatures below 26°C are detected by TRPM8, a nonselective cation 

channel that is expressed in a subset of peripheral afferent fibers. Recombinant TRPM8 channels 

also adapt to prolonged cold stimuli with a time course similar to that observed with cold fibers, 

a finding which suggests that adaptation occurs by reduced TRPM8 activity. Previous studies

have shown that TRPM8 adaptation is dependent upon intracellular calcium, and that adaptation 

can be recovered in a temperature dependent manner. Moreover, TRPM8 activity is sensitive to 

the membrane phospholipid phosphoinositol-4,5-bisphosphate (PIP2), a substrate for the enzyme 

phospholipase C (PLC). These results suggest a model whereby TRPM8-mediated calcium 

influx leads to decreased levels of PIP2 as a result of increased activity of Ca2+-sensitive PLC 

isoforms. We have tested this model using recombinant TRPM8 channels and have found that 

direct activation of PLC reduces TRPM8 activity. Moreover, cellular manipulations that reduce 

PIP2 levels also diminish cold- and menthol-evoked currents in heterologous cells. Finally, we 

provide evidence that PLC and PIP2 regulate TRPM8 activity in cold-sensing neurons. These 

findings extend previous studies in heterologous systems to native cells and provide novel 

insight into the functional regulation of TRPM8. 

Disclosures: R.L. Daniels, None; Y. Takashima, None; H.N. Waters, None; E.R. Liman, 

None; D.D. McKemy, None. 

Poster 




Topic: D.08.a. Pain transduction molecules and channels 

Support: RO1 NS051551 

the American Cancer Society 

Title: In vivo function of TRPV2 in nociception 

Authors: U. PARK 1,2,3 , Y. GUAN 4 , S. RAJA 4 , *M. CATERINA 1,2,3 ; 

1 Biol. Chem., 2 Ctr. for Sensory Biol., 3 Neurosci., Johns Hopkins Sch. of Med., Baltimore, MD; 

4 Depts. of Anesth. and Crit. Care Med., Johns Hopkins Univ., Baltimore, MD 

Abstract: Electrophysiological recordings from sensory afferents in rat and monkey skin have 

revealed the existence of a class of myelinated A-fiber nociceptors that are activated by 

extremely high temperature over 52°C. One candidate transducer molecule for this nociceptor 

class is the heat-gated ion channel TRPV2. TRPV2 is expressed strongly in medium to large 

diameter neurons in sensory ganglia, and is activated at temperatures greater than 52°C in vitro. 

In order to examine whether TRPV2 is truly a transducer of noxious temperatures in vivo, we 

generated knockout mice lacking TRPV2 protein expression. TRPV2 knockout mice were born 

at approximately half the expected Mendelian ratio, but surviving animals were generally

healthy. These mice were subjected to a battery of mechanical and thermal pain behavioral 

assays under normal and injury conditions such as inflammation and spinal nerve ligation. Thus 

far, we have not detected any differences between wild type and TRPV2 knockout mice in these 

behavioral assays. We excluded the potential masking effect of TRPV1 or influence of TRPV1positive 

afferents by examining TRPV1/TRPV2 double knockout mice and RTX-treated mice. 

Our behavioral assay results demonstrate that TRPV2, unlike the initial expectation, does not 

play a crucial role in detection of noxious heat in the uninjured state or under skin-sensitizing 

conditions. It needs to be noted that high threshold heat-evoked responses of mice and rats are 

reportedly different qualitatively and quantitatively, and hypotheses of TRPV2 function in 

detection of noxious heat derived from rat studies may not be tested appropriately in mice. 

TRPV2 expressed in mouse sensory neurons in DRG may function in a capacity other than that 

of a primary transducer of painful heat. 

Disclosures: U. Park, None; Y. Guan, None; M. Caterina, None; S. Raja, None. 

Poster 





Support: KOSEF Grant R01-2008-000-10891-0 

Title: TRPV1 and TRPA1 channels induced currents from acutely dispersed nodose ganglion 

neurons but the channels distributed heterogeneously 

Authors: S.-I. BANG, B. SUN, *Y.-H. JIN; 

Sch. Med. Dept Physiol, Kyunghee Univ., Seoul, Republic of Korea 

Abstract: Temperature and chemical sensing transient receptor potential (TRP) channels 

distributed various sensory neurons and those axon terminals in the CNS. The pain transmission 

related function and distribution of TRP channels extensively studied from the dorsal root 

ganglion (DRG) neurons. The same TRP channels also exist in the visceral sensory afferent 

nerve but their distribution in the nerve and contribution on visceral signal transmission are not 

clear yet. Vagus nerve, which innervate visceral organs, have their cell body in the nodose 

ganglion (NG) and the nerve axon directly connected nucleus tractus solitaries neurons (NTS). 

Capsaicin sensitive TRPV1 channel exclusively present slow conducting non myelinated NG 

neurons and its axon terminals in the NTS. Recent immunohistochemical studies show low 

temperature and mustard oil sensitive TRPA1 channel coexist with TRPV1 channel sensitive

cells in some vagus nerves. TRPA1 channels response ingredients of multiple spices for example 

mustard, garlic and onion. These spices used as seasoning for a long time and recently their 

beneficial effects for the health by decreasing blood pressure, cholesterol level and body weight 

have been frequently reported. Together TRPA1 channel activity in the vagus nerve may relate 

with the spices effect on autonomic response in the body. However the cellular mechanism is not 

known yet. In this study we have test TRPA1 and TRPV1 agonists effects on nodose ganglion 

neurons using patch clamp method. NG neurons taken from 1 to 7 day-old rats and treated with 

protease before dispersed with Pasture pipette. 

TRPA1 channel agonist allyl isothiocyanate (100 κM, MO) induced inward current in acutely 

dispersed nodose ganglion neurons (66.4 ± 20.49 pA, n = 8) and the response have blocked by 

TRPA1 channel antagonist ruthenium red (100 κM). TRPA1 channel agonist allyl isothiocyanate 

(MO) and TRPV1 channel agonist capsaicin (CAP, 200 nm) induced inward currents from 

acutely isolated NG neurons by the reversible manner. TRPA1 receptor inhibitor ruthenium red 

(0.2 mM) blocked MO induced currents. 23% of the NG cells respond to of MO and capsaicin 

(CAP, 200 nM) but 43% of the NG cells respond neither of them. 34% of the NG neurons 

respond to only one of the MO or CAP.These TRPA receptors mediated neuronal activities 

change in vagus nerve and its axon terminals may have been related with autonomic reflex 

alteration after spice ingestion. 

Disclosures: S. Bang, None; Y. Jin , R01-2008-000-10891-0 (Jin, Y-H), B. Research Grant 


received); B. Sun, None. 

Poster 




Topic: D.08.b. Nociceptors 

Support: Supported in part by NIH R01 DA19585 and T32 DE14318. 

Title: ACEA modulation of TRPA1 

Authors: *N. B. RUPAREL 1 , A. M. PATWARDHAN 2 , A. N. AKOPIAN 2 , K. M. 

HARGREAVES 2 ; 

1 CELL STRUCTURAL BIOL, UTHSCSA, San Antonio, TX; 2 Endodontics, Univ. of Hlth. Sci. 

Ctr. at San Antonio, San Antonio, TX

Abstract: Peripheral anti-hyperalgesic effects of cannabinoids have long been studied. Recent 

reports demonstrate that sensory neuron-specific CB1-/- mice show loss of anti-hyperalgesia 

produced by WIN in vivo (Agarwal et al., 2007). Additionally, we now show that WIN, a CB1, 

CB2 and TRPA1 agonist, at peripherally selective doses, significantly attenuates capsaicin 

(CAP)-induced nocifensive behavior in WT mice (Akopian et al., 2008). However, this effect 

was fully abolished in CB1-/- and TRPA1-/- mice (Akopian et al., 2008). These studies suggest 

heterologous desensitization of TRPV1 by a TRPA1 cannabinoid agonist and hence implicates 

the role of TRPs in mediating part of the peripheral effects of cannabinoids in vivo. Moreover, 

arachidonoly-2 chloroethanolamine (ACEA) evoke similar effects via TRPV1 on desensitizing 

mustard oil (MO)-evoked responses. Using CHO cells, as well as sensory neurons, application of 

ACEA, a CB1 and TRPV1 cannabinoid agonist, significantly inhibited MO-evoked inward 

currents (Akopian et al., 2008). Moreover, ACEA, at peripherally restricted doses, attenuated 

MO-induced nocifensive behavior in mice. However, this effect was abolished in TRPV1-/- 

mice. Collectively these studies demonstrate that cannabinoids such mediate their peripheral 

antihyperlagesia effects, in part, via TRP channels. 

The present study sought to evaluate: 1) the potential sites required for functional ACEA 

activation of TRPV1 using five capsaicin-insensitive (S511Y, Y512A, Arg-114, Glu-761 and 

T550I) and two anandamide-insensitive (S511Y, Y512A) TRPV1 mutations; and 2) the effects 

of selected TRPV1 mutations on ACEA-induced inhibition of MO responses in vitro using 

calcium imaging. Data were analyzed using ANOVA. 

Results: Our results demonstrate that 1) in CHO cells transfected with TRPV1 mutations alone, 

all mutations lost >90% of ACEA activation of TRPV1; 2) in CHO cells transfected with WT 

TRPV1 and TRPA1, 100uM ACEA fully abolished MO-induced calcium influx. Interestingly, 

mutations such as S511Y, Arg-114 and Glu-761, when doubly transfected with TRPA1, led to 

restoration of ACEA function including the ability to inhibit MO-induced calcium influx. 

Conclusion: Collectively, we have shown that ACEA inhibits MO response in vitro and in vivo. 

Moreover, ACEA gating of TRPV1 is nearly abolished in TRPV1 mutants and that coexpression 

of TRPV1 mutations with TRPA1 restored ACEA function. These studies suggest a 

possible conformational change in TRPV1 upon physical interaction with TRPA1. Overall, these 

studies provide insight into the potential mechanisms by which certain cannabinoids mediate 

peripheral anti-nociceptive effects via TRP channels. 

Disclosures: N.B. Ruparel, None; A.M. Patwardhan, None; A.N. Akopian, None; K.M. 

Hargreaves, None. 

Poster 



Program#/Poster#: 265.7/EE8

Topic: D.08.b. Nociceptors 

Support: NIH Predoctoral Fellowship F31 

Title: TRPA1 mediates the noxious effects of sesquiterpene deterrents 

Authors: *J. J. ESCALERA, C. A. VON HEHN, B. F. BESSAC, M. SIVULA, S.-E. JORDT; 

Pharmacol., Yale Univ., New Haven, CT 

Abstract: Plants, fungi and animals generate a diverse array of deterrent natural products that 

induce avoidance behavior in biological adversaries. The largest known chemical family of 

deterrents are terpenes characterized by reactive α,β-unsaturated dialdehyde moieties, including 

the drimane sesquiterpenes and other terpene species. Deterrent sesquiterpenes are potent 

activators of mammalian peripheral chemosensory neurons, causing pain and neurogenic 

inflammation. Despite of their widespread synthesis and medicinal use as desensitizing 

analgesics their molecular targets remain unknown. Here we show that isovelleral, a noxious 

fungal sesquiterpene, excites sensory neurons through activation of TPRA1, an ion channel 

involved in inflammatory pain signaling. TRPA1 is also activated by polygodial, a drimane 

sesquiterpene synthesized by plants and animals. TRPA1-deficient mice show greatly reduced 

nocifensive behavior in response to isovelleral, indicating that TRPA1 is the major receptor for 

deterrent sesquiterpenes in vivo. Isovelleral and polygodial represent the first fungal and animal 

small molecule agonists of nociceptive TRP channels. 

Disclosures: J.J. Escalera, None; C.A. von Hehn, None; B.F. Bessac, None; M. Sivula, 

None; S. Jordt, None. 

Poster 





Support: Brain Research Center of the 21st Century Frontier Research Program (code 

M103KV010015-06K2201-01510) funded by the Ministry of Science and Technology, the 

Republic of Korea 

The Korea Health 21 R&D Project (code A060341) funded by Ministry of Health and 

Welfare, the Republic of Korea

Title: Transient receptor potential A1 mediates acetaldehyde-evoked pain sensation 

Authors: *S. HWANG, S. BANG, K. KIM, S. YOO; 

Grad Sch. Med., Korea Univ., Seoul, Republic of Korea 

Abstract: Six transient receptor potential (TRP) ion channels expressed in the sensory afferents 

are playing an important role as body thermosensors and also peripheral pain detectors. It‟s 

known that a number of natural compounds specifically activate those thermo sensitive TRP 

channel and a well-known example is cinnamaldehyde for TRPA1. Here we show that human 

and mouse TRPA1 is activated by acetaldehyde, an intermediate substance of ethanol 

metabolism, in HEK293T cell heterologous expression system and culture of mouse trigeminal 

neurons. Acetaldehyde failed to activate other temperature-sensitive TRP channels expressed in 

sensory neurons. A TRPA1 antagonist camphor and a general TRP blocker ruthenium red 

inhibited TRPA1 activation by acetaldehyde. Camphor and ruthenium red also suppressed acute 

nociceptive behaviors induced by intradermal administration of acetaldehyde into the mouse 

footpad. Intradermal coapplication of prostaglandin E2 with acetaldehyde greatly potentiated the 

acetaldehyde-induced nociceptive responses and the effect was reversed by treatment of the 

TRPA1 antagonist camphor. These results suggest that acetaldehyde causes nociception via 

activation of TRPA1. Our data also may help elucidation of acetaldehyde-related pathological 

symptoms such as hangover pain. 

Disclosures: S. Hwang , None; S. Bang, None; K. Kim, None; S. Yoo, None. 

Poster 





Support: KFO 100/1-2 

Title: Modulation of TRPV1 activity during Opioid withdrawal 

Authors: V. SPAHN, *M. SCHAEFER, C. ZOELLNER; 

Anesthesiol and Intensive Care Med., Charité, Campus B. Franklin, Berlin, Germany 

Abstract: Vanilloid receptor type 1 (TRPV1) is a ligand-gated ion channel expressed on sensory 

nerves that responds to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 

plays a critical role in the development of tissue injury, inflammation or nerve lesions. Opioids

such as morphine have been used widely for the treatment of many types of acute and chronic 

pain. Application of morphine leads to a dissociation of G-proteins and causes a reduced activity 

of adenylylcyclases (AC), resulting in a lower amount of cAMP. However, opioid withdrawal 

following chronic activation of the µ opioid receptor induces AC superactivation and 

subsequently an increase in cAMP and Protein Kinase A (PKA) activity. 

In the current project we investigated wether an increase in cAMP during opioid withdrawal 

increases the activity of TRPV1. In whole cell patch clamp and calcium imaging experiments 

opioids significantly increase capsaicin induced TRPV1 activity in a nalaxone and pertussis toxin 

sensitive manner. The role of different PKA phosphorylation sites at TRPV1 was investigated 

using site-directed mutagenesis. In summery, our results demonstrate that opioid withdrawal can 

increase the activity of TRPV1. These observations show a new mechanism underlying 

hyperalgesia during opioid withdrawal. 

Disclosures: V. Spahn, None; M. Schaefer, None; C. Zoellner, None. 

Poster 





Title: Involvement of protein kinase C phosphorylation sites of TRPV1 in acute sensitization by 

nerve growth factor 

Authors: *W. SONG, W. ZHU, G. OXFORD; 

Stark Neurosci. Res. Inst., Indiana Univ. Sch. of Med., Indianapolis, IN 

Abstract: The mammalian transient receptor potential vanilloid 1 (TRPV1) is a transmembrane 

protein critically involved in nociceptive responses of sensory neurons. Studies in adult rat dorsal 

root ganglion (DRG) neurons have demonstrated that many proinflammatory molecules, 

including nerve growth factor (NGF) can enhance pain sensation through the sensitization of 

TRPV1. A common signaling element linking several inflammatory mediators to TRPV1 

sensitization is protein kinase C (PKC). In vitro PKC can directly phosphorylate the receptorchannel 

at specific serine residues leading to enhanced TRPV1 sensitivity. However, it is still not 

clear whether phosphorylation of these sites can be induced by NGF and are actually involved in 

NGF mediated pain sensitization. 

We sought to determine which of the PKC phosphorylation sites in TRPV1 are involved in NGF 

mediated sensitization by combining biochemical and electrophysiological approaches. Using 

both a sensory neuron derived cell line F11 that endogenously expresses trkA, and HEK293 cells

stably expressing trkA receptors we explored modulation of TRPV1 by NGF. In whole-cell 

recordings of capsaicin evoked currents from F11 cells, 100ng/ml NGF acutely increased current 

amplitude. However, NGF mediated TRPV1 sensitization was not observed in HEK293-trkA 

cells. Accordingly, Western blot analysis revealed HEK293 cells to express significantly lower 

levels of PKC epsilon than F11 cells. We propose that PKC epsilon is an important molecule that 

is involved in NGF mediated TRPV1 sensitization. Following treatment of adult DRG neurons 

for 10-30 minutes with 100ng/ml NGF, serine phosphorylation of immunoprecipitated native 

TRPV1 was increased (Western blot with phosphoserine antibodies). We are investigating the 

PKC specific TRPV1 phosphorylation events using mass spectrometry of TRPV1 from native rat 

DRG neurons following NGF treatment. Furthermore, we are comparing wildtype and p-site 

mutant TRPV1 for sensitization in cells coexpressing trkA and TRPV1 constructs. 

Disclosures: W. Song , None; W. Zhu, None; G. Oxford, None. 

Poster 






Title: Up-regulation of transient receptor potential vanilloid-1 in primary afferent neurons is 

mediated by protein kinase C and involved in neurogenic inflammation 

Authors: *X. XU 1 , P. WANG 1 , X. ZOU 1 , D. LI 1 , L. FANG 2 , Q. LI 2 ; 

1 Neurosci & Cell Biol, 2 Surgery, UTMB, Galveston, TX 

Abstract: A recent study by our group indicated that the transient receptor potential vanilloid-1 

(TRPV1) is activated by intradermal injection of capsaicin (CAP) to initiate neurogenic 

inflammation by the release of neuropeptides in the periphery. In order to investigate further 

whether up-regulation of TRPV1 in dorsal root ganglion (DRG) neurons following CAP injection 

is subject to modulation by the phosphorylated protein kinase C (p-PKC) and whether this 

participates in neurogenic inflammation, immunohistochemistry, real-time PCR and Western 

blots were used to examine the molecular and cellular changes that occur in the expression of 

TRPV1, p-PKC and calcitonin gene-related peptide (CGRP) in DRG neurons following CAP 

injection in anesthetized rats. Results show that CAP injection evoked increases in the expression 

of TRPV1 both in mRNA and protein levels and in the numbers of single labeled TRPV1, p-PKC 

and CGRP neurons in ipsilateral L4-5 DRG. Co-expressions of TRPV1 with p-PKC and/or CGRP

(double and triple labeling) in DRG neurons were also significantly increased after CAP 

injection. These evoked expressions both at molecular and cellular levels were significantly 

inhibited after TRPV1 receptors were blocked by 5‟-iodoresiniferatoxin (5 µg) or PKC was 

inhibited by chelerythrine chloride (5 µg). Taken together, these data provide evidence that upregulation 

of TRPV1 receptors in DRG nociceptive neurons is closely associated with 

phosphorylation of PKC during neurogenic inflammation induced by CAP injection. Thus, the 

study strongly suggests a critical role of TRPV1 in initiating neurogenic inflammation and 

supports the idea that the functional activity of TRPV1 receptors is subject to modulation by the 

PKC cascade. (Supported by NIH grant NS40723). 

Disclosures: X. Xu, None; P. Wang, None; X. Zou, None; D. Li, None; L. Fang, None; Q. Li, 

None. 

Poster 






Title: Sensitization of primary afferent nociceptors during neurogenic inflammation is mediated 

by proinflammatory chemokines 

Authors: *D. LI 1 , X. XU 1 , P. WANG 1 , L. FANG 2 , Q. LIN 1 ; 

1 Dept. Neurosci & Cell Biol, 2 Div. of Neurosurgery, Dept. of Surgery, UTMB, Galveston, TX 

Abstract: Our recent studies have revealed that neuropeptides, such as calcitonin gene-related 

peptide (CGRP), are involved in the sensitization of primary afferent nociceptors in the model of 

capsaicin (CAP)-induced neurogenic inflammation. In this process, CGRP was suggested to be 

released from primary afferent terminals, which is driven by dorsal root reflexes (DRRs). 

However, it is not clear whether CGRP sensitizes nociceptors directly or via proinflammatory 

agents. Chemokines are known to participate in the immune-mediated inflammation through 

activation and recruitment of monocytes/macrophages. Macrophage-inflammatory protein-1α 

(MIP-1α/CCL3), a proinflammatory chemokine, is suggested to sensitize CAP receptors. In this 

study, afferent activity and response threshold to mechanical stimuli were recorded 

eletrophysiologically from single Aδ and C primary afferent nociceptive fibers in the tibial nerve 

in anesthetized rats. Under dorsal rhizotomized conditions that removed DRRs, intra-arterial 

injection of CGRP (10 κg) produced a remarkable increase in responses to mechanical stimuli

and decrease in response threshold of both Aδ and C fibers. Surprisingly, the enhanced response 

activity and reduced threshold elicited by CGRP were inhibited by peripheral pre-treatment with 

anti-MIP-1α/CCL3 neutralizing antibodies (10 κg). These observations suggest that CGRP 

sensitizes primary afferent nociceptors via the release of a proinflammatory chemokine, MIP-1α 

/CCL3. 

Disclosures: D. Li, None; X. xu, None; P. wang, None; L. Fang, None; Q. lin, None. 

Poster 






NS48499 

Title: Genetic visualization of the heat and capsaicin receptor, TRPV1 

Authors: D. J. CAVANAUGH 1 , D. JULIUS 2 , N. M. SHAH 3 , *A. I. BASBAUM 3 ; 

2 Physiol., 3 Dept Anat, 1 UCSF, San Francisco, CA 

Abstract: The heat and capsaicin receptor, TRPV1, is a critical contributor to the cellular and 

behavioral responses to noxious thermal stimulation. Although TRPV1 is concentrated in 

primary afferent nociceptors, there are reports of its widespread CNS and non-neuronal 

expression. Here, we generated a TRPV1 reporter knock-in mouse, in which there is coexpression 

of placental alkaline phosphatase (PLAP) and nuclear lacZ (nlacZ), allowing for 

sensitive delineation of TRPV1 processes and cell bodies, respectively. We demonstrate that in 

the adult mouse, neuronal TRPV1 is largely restricted to primary afferent neurons and to their 

central and peripheral (e.g. skin and bladder) axonal arbors, faithfully recapitulating the pattern 

of TRPV1 antibody staining. In addition to known projections of primary afferent neurons, we 

found PLAP expression in areas not previously shown to receive input from TRPV1 cells, 

including the external lateral parabrachial nucleus. This suggests that the parabrachial nucleus is 

monosynaptically activated by primary afferent nociceptors. Although we did not detect PLAP in 

cells intrinsic to the brain, we observed nlacZ in Cajal-Retzius neurons of the hippocampus 

(confirmed by co-expression of reelin). We also saw limited nlacZ in other brain areas, including 

the arcuate nucleus of the hypothalamus, interpeduncular nucleus and periaqueductal gray, but 

overall could not corroborate the extensive CNS expression reported by others. Finally, we found

significant TRPV1 expression in a subset of arteriolar smooth muscle cells. This result is of 

particular relevance to mechanisms through which heat and pH regulate vascular tone and to the 

temperature changes observed after systemic capsaicin administration. 

Disclosures: D.J. Cavanaugh, None; A.I. Basbaum , None; N.M. Shah, None; D. Julius, 

None. 

Poster 





Support: University of Kentucky Start-up Funds 

Tissue sharing with Dr. Lu-Yuan Lee 

Title: TRPV1 activation up-regulates TNFR1 expression in DRG neurons through reactive 

oxygen species 

Authors: *F. MA, L. ZHANG, K. N. WESTLUND; 

Physiol., Univ. of Kentucky, Lexington, KY 

Abstract: Tumor necrosis factor α, a proinflammatory cytokine, is involved in nociceptive 

responses causing hyperalgesia through TNF receptor type 1 (TNFR1) activation. Transient 

receptor potential vanilloid subtype 1 (TRPV1) also plays an important role in inflammatory 

pain. In this study, the mechanistic relationship between TRPV1 and TNFR1 in nociception was 

investigated. The TNFR1 expression in primary cultured dorsal root ganglion (DRG) neurons 

after TRPV1 activation and the involvement of reactive oxygen species (ROS), was examined 

with live cell imaging and immunocytochemistry. 

C57BL/6 strain mice, both TRPV1 knock out and wild type, were used in this study. The L4 and 

L5 DRGs were dissected bilaterally and cultured overnight. TRPV1 was activated with capsaicin 

or its potent analog, resinferatoxin. ROS production was measured with live cell imaging using 

ROS-sensitive fluorescent dye, 5-(and-6)-chloromethyl-2‟, 7‟-dichlorodihydrofluorescein 

diacetate, acetyl ester (CM-H2-DCFDA). Expression of TNFR1 was detected with 

immunofluorescence in aldehyde fixed primary DRG cells on coverslips. The TRPV1 antagonist, 

capsazepine or a nonspecific radical scavenger, PBN (N-tert-Butyl-α-phenylnitrone) was 

employed to further confirm the functional relationship between TRPV1 and TNFR1 in both 

studies.

The results demonstrate that 1) TRPV1 activation by capsaicin or resinferatoxin increases ROS 

production and up-regulates TNFR1 expression; 2) Capsazepine or PBN non-selectively block 

increases in ROS and the up-regulation TNFR1 expression in DRG neurons of wild type mice; 3) 

No changes in ROS production or TNFR1 expression are detected in DRG neurons from TRPV1 

knock out mice following capsaicin or resinferatoxin stimulation. These data suggest that 

TRPV1 activation up-regulates TNFR1 expression in DRG neurons with increases in ROS 

production. 

Disclosures: F. Ma, None; L. Zhang, None; K.N. Westlund, NIH, B. Research Grant (principal 


University of Kentucky Start-up funds, C. Other Research Support (receipt of drugs, supplies, 

equipment or other in-kind support); American Pain Society Fred Kerr Award, D. Speakers 

Bureau/Honoraria (speakers bureau, symposia, and expert witness); World Pain Congress 

Honoraria, D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness). 

Poster 





Support: NIH Grant SAM NS31826 

NIH Grant BMD DK06392 

Title: TRP channel function is modulated by growth factors in identified skin, muscle and 

visceral primary afferents 

Authors: *S. A. MALIN, D. C. MOLLIVER, J. A. CHRISTIANSON, B. M. DAVIS; 

Medicine/GI, Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Growth factor expression is modulated during injury and disease; these changes are 

critical to initiation and maintenance of chronic pain. We examined modulation of TRPA1 and 

TRPV1 channels by acute application of NGF, artemin, neurturin and GDNF in target-identified 

primary afferent neurons to probe the interaction of growth factors and TRP channels in 

nociception. We injected retrograde markers into saphenous nerve (Alexa488WGA; skin) 

femoral nerve (Alexa488WGA; muscle), or colon wall (Alexa488CTX; colon) to identify 

primary afferents projecting to skin, muscle and colon of adult C57Bl/6 mice. Responses in 

isolated target-identified neurons were recorded with fura-2 calcium imaging. Neurons were first

challenged with a depolarizing stimulus (50mM K + ), then exposed to 1κM capsaicin, to elicit 

TRPV1 responses, or 100κM mustard oil, to elicit TRPA1 responses. TRPA1 and TRPV1 

channels show profound desensitization; we have previously shown that acute application of 

growth factors can prevent this process and potentiate nociceptive signaling. To identify growth 

factors effective in different target-identified populations, cells were then perfused with growth 

factors (NGF, artemin, neurturin or GDNF) at varying concentrations for 7 minutes and TRPA1 

or TRPV1 responses elicited at 10 minute intervals. TRPV1 responses and TRPA1 responses 

were dramatically different in skin, muscle and colon primary afferents. TRPV1 responses were 

3-4 fold larger and twice as prevalent (67%) in colon and muscle, as compared to skin, afferents. 

Interestingly, TRPA1 responses were most prevalent in muscle afferents (52%), compared to 

colon (35%) and skin (16%) afferents. However, like TRPV1, TRPA1 responses were 4-5 fold 

larger in colon and muscle, compared to skin, afferents. Growth factor responsiveness was also 

distinct in target-identified populations: artemin was most effective in skin afferents (80%); 

NGF, artemin and neurturin were all effective in muscle afferents; neurturin is most effective in 

colonic afferents (70%). Growth factor modulation was distinct for TRPV1 and TRPA1 in 

identified afferents. TRP channel function and modulation is innervation target-dependent, 

suggesting that distinct populations of sensory neurons are affected in different chronic pain 

syndromes. 

Disclosures: S.A. Malin , None; D.C. Molliver, None; J.A. Christianson, None; B.M. Davis, 

None. 

Poster 




Topic: D.08.k. Inflammatory pain 

Support: NIDCR T-32 DE14318 

American Pain Society 

Title: The role of GPR30 sensitization of trigeminal neurons in inflammatory hyperalgesia 

Authors: *J. C. FEHRENBACHER, K. M. HARGREAVES; 

Endodontics, UTHSCSA, San Antonio, TX 

Abstract: Considerable evidence implicates estrogens as critical factors in sex-dependent 

differences in pain, especially in conditions where inflammation is present. Acute peripheral

treatment with estradiol increases thermal hyperalgesia in animal models of inflammation, but 

has no effect on acute thermal behavior. We previously demonstrated that inflammation also 

induces novel expression of mRNA in sensory neurons that encode GPR30, which is activated 

by estradiol (Fehrenbacher and Hargreaves, 2007), however the activity of this receptor and 

functional relevance for hyperalgesia in males and females is unclear. In this study, we evaluated 

the hypothesis that inflammation increases peripheral levels of estradiol and that the hormone 

activates the GPR30 receptor on sensory neurons to mediate estradiol-enhancement of 

inflammatory thermal hyperalgesia. 

We evaluated the effects of inflammation on estradiol levels; inflammation induced a 55% 

increase in peripheral skin levels in both females (OVX + E2 replacement) and males and a 40% 

increase in female trigeminal ganglion with no change in males. We next used real-time RT-PCR 

to determine that inflammation induced trigeminal ganglia increases in mRNA for GPR30 in 

males (1.6-fold increase) as well as females (2.7-fold increase) and demonstrated the functional 

ability of the GPR30 to mediate estradiol increases in the sensitivity of nerve terminals in 

females by examining the acute effects of a specific GPR30 agonist, G-1 (1 κM), on the 

capsaicin-stimulated (30 κM) release of neuropeptide from skin biopsies from non-inflamed and 

inflamed tissues. Although G-1 did not have a direct effect on iCGRP release or on the 

stimulated release from noninflamed skin biopsies, superfusion of the skin with G-1 for 15 

minutes prior to and throughout stimulation elicited an increase in the stimulated release of 

iCGRP from inflamed biopsies from females. Finally, we expanded the previous findings that 

estradiol augments inflammation-induced thermal hyperalgesia by demonstrating that similar 

increases in hyperalgesia are mimicked by subcutaneous injection of G-1 (5 κg) in CFA-injected 

females but not in saline-injected females as determined by ANOVA, supporting the notion that 

GPR30 receptor at least partly mediates the enhancement of thermal hyperalgesia induced by 

estradiol during inflammation. 

The pharmacology of the GPR30 distinguishes it from other estrogen receptors, thus the findings 

here may provide an opportunity for the development of therapeutics which can selectively 

reverse GPR30 activity to attenuate inflammatory pain without affecting the actions of estrogens 

at the classical receptors. 

Disclosures: J.C. Fehrenbacher, None; K.M. Hargreaves, None. 

Poster 





Support: NIH Grant RO1DE17696 to A.N.A.

We would like to thank Dr. A.F. Parlow (NHPP-Harbor-UCLA), Gaby Helesic and 

Griffin Perry for technical assistance. 

P. E. S. is a Canadian NSERC PGS-D award holder. 

Title: Prolactin sensitizes TRPV1 and produces hyperalgesia in male rats 

Authors: *P. E. SCOTLAND 1 , A. DIOGENES 2 , N. A. JESKE 3 , A. M. PATWARDHAN 2 , M. 

A. HENRY 2 , K. M. HARGREAVES 2,1 , A. N. AKOPIAN 2 ; 

1 Pharmacol., 2 Endodontics, 3 Oral and Maxillofacial Surgery, Univ. Texas Hlth. Sci. Ctr. San 

Antonio, San Antonio, TX 

Abstract: In female rats, prolactin (PRL) is a novel hyperalgesic neuromodulator that is 

expressed in trigeminal ganglia (TG) sensory neurons and activates PRL receptors leading to 

increased TRPV1 responsiveness. However, a role of PRL as a TRPV1 modulator in male rats 

has not previously been examined. Therefore, we evaluated the expression of PRL and prolactin 

receptors (PRL-R), and the modulation of TRPV1 responses in male rat TG neurons. 

Immunohistochemical analysis was used to characterize the expression of PRL and the PRL-R 

and the modulatory effect of PRL on TRPV1 responses was evaluated by Ca 2+ imaging and 

whole-cell patch clamp. The effect of PRL on the TRPV1 phosphorylation status was evaluated 

by the incorporation of P 32 in the channel and the effect of PRL on capsaicin nocifensive 

behavior and paw withdrawal latencies to thermal stimuli was also evaluated. Data were 

analyzed by ANOVA. The results demonstrate that PRL and the PRL-R are co-expressed with 

TRPV1 in trigeminal sensory neurons and peripheral fibers innervating the cornea and paw skin, 

suggesting that PRL may modulate TRPV1 responses in male rats. PRL increased 

phosphorylation of TRPV1 in TG cultures and also sensitized TRPV1 responses in cultured TG 

neurons, including increased capsaicin-evoked responses as measured by calcium accumulation 

and inward currents (p


Title: Involvement of c-src kinase, PLCγ1, and PKC in the acute sensitization of TRPV1 by 

artemin 

Authors: *W. ZHU 1 , G. OXFORD 2 ; 

1 Dept Pharmacol & Toxicol, 2 Stark Neurosciences Res. Inst., Indiana Univ. Schl Med., 

Indianapolis, IN 

Abstract: Acute sensitization of TRPV1 by inflammatory mediators, like nerve growth factor 

(NGF) or activin, contribute significantly to hypersensitivity of nociceptors, which underlie the 

induction and maintenance of persistent pain associated with inflammation or injury. Recent 

studies indicate that another neurotrophic factor artemin (ARTN), a member of the glial cell 

derived neurotrophic factor (GDNF) family of ligands, can acutely sensitize TRPV1 in cultured 

DRG neurons and can induce thermal hyperalgesia after injection into the animal paw. While 

ARTN and NGF are likely to exert their actions on the same subset of DRG neurons, whether 

they use the same signaling pathways to sensitize TRPV1 is unknown. We combined patch 

clamp electrophysiology and pharmacology to explore the signaling pathways underlying 

TRPV1 sensitization by ARTN in cultured adult rat DRG neurons. ARTN acutely sensitized 

capsaicin-induced current in ~70% of recorded neurons. Unlike NGF, ARTN acutely sensitizes 

TRPV1 when neurons were pre-treated with the MEK specific inhibitor U0126 (5 µM), or the 

PI3K specific inhibitor wortmannin (100 nM), despite their biochemically verified inhibition of 

p-ERK and p-Akt, respectively. In contrast, inhibition of c-src kinase by PP2 (10 µM), of PLCγ1 

by U73122 (10 µM), or of PKC (1 µM) by BIM, all abrogated acute sensitization of TRPV1 by 

ARTN. These data suggest that activation of c-src kinase, PLCγ1 and PKC are implicated to 

acute sensitization of TRPV1 by ARTN, whereas Erk1/2 and PI3K are not involved. We 

conclude that although NGF and ARTN both acutely sensitize TRPV1 through binding to their 

respective neurotrophic receptors (TrkA and GFRα3-RET), the post-receptor signaling cascades 

for each ligand are distinct. 

Disclosures: W. Zhu , None; G. Oxford, None. 

Poster 





Title: TRPM2 inhibits neurite outgrowth via LPA receptor

Authors: Y. JANG, H. CHO, H. KIM, Y. YANG, S. PARK, M. TAK, T. KIM, Y. CHO, *U. 

OH; 

Sensory Res. Ctr., Seoul Natl. Univ. Col. Pharm, Seoul 151 742, Republic of Korea 

Abstract: Neurogenesis and plasticity of neurons are correlated with neurite outgrowth. Recently 

the importance of transient receptor potential (TRP) channels for the neuronal differentiation and 

proliferation has been exhibited. But, it is still not well understood which types of TRP channels 

are involved in neurite outgrowth. So we screened types of TRP channels are required for the 

neuronal outgrowth, using real time quantitative-PCR technique in PC12 cells. As a result, we 

hypothesized that TRPM2 is related to neurite outgrowth. 

TRPM2 is highly expressed in the brain, but its role in the nervous system is largely unknown. 

We report that its expression with small interfering RNA increased neurite outgrowth in live cell 

imaging. Its overexpression decreased the length of neurite. Also the length of neurite is 

increased by its inhibitors such as econazole, clotrimazole and flufenamic acid. These results 

suggest that TRPM2 inhibits nuerite outgrowth as negative regulator. We found that expression 

of TRPM2 and LPA receptor is colocalized in the neuronal cell. Actually, LPA-induced Ca 2+ 

influx is higher in TRPM2 overexpressed in PC12 cell than those in control, using whole-cell 

recording and Ca 2+ imaging. Furthermore, LPA-induced neurite retraction is recovered by 

blocking TRPM2 channel. Therefore These findings suggest that TRPM2 inhibits neurite 

outgrowth via LPA receptor. 

Disclosures: Y. Jang, None; U. Oh , None; H. cho, None; H. kim, None; Y. Yang, None; S. 

park, None; M. Tak, None; Y. cho, None; T. kim, None. 

Poster 





Title: Use of laser Doppler imaging in rats to evaluate activation of Transient Receptor Potential 

channels in rats 

Authors: V. A. KENIGS, *M. R. BRANDT, C. M. FLORES; 

Analgesics Drug Discovery, Johnson & Johnson Pharmaceut R&D, Spring House, PA 

Abstract: Transient Receptor Potential (TRP) channels are a family of non-selective cation 

channels having a multitude of physiologic functions such as sensory transduction, taste, and 

thermosensation. In recent years, many naturally occurring chemicals have been identified that

have agonist properties at selective TRP channels. Some of these chemicals produce a 

neurogenic inflammatory response, which is produced by a locally-, as well as a centrallymediated 

release of vasoactive peptides and thought to be important in some types of 

inflammatory pain. For example, the TRPV1 agonist, capsaicin, produces an intense „burning‟ 

pain sensation followed by radiating areas of hyperalgesia and allodynia following intradermal or 

topical administration. This inflammatory response is visible as a “flare” surrounding the site of 

injection. Quantification of flare can be accomplished by using laser Doppler imaging, which 

measures superficial blood flow in the skin. 

In the present study, laser Doppler imaging was used to evaluate the flare response to TRP 

agonists. The TRPV1, TRPA1, and TRPM8 agonists capsaicin (0.1 - 3 mg), mustard oil (0.3%- 

10%), and menthol (50%), respectively, were topically administered in anesthetized rats. 

Capsaicin dose-dependently produced flare, with the maximal flare response occurring 45 min 

(191% increase from baseline) after application of 1 mg. When administered 120 min before 

capsaicin, the selective TRPV1 antagonist, JNJ-17203212 (30 mg/kg; p.o), blocked the flare 

response induced by topical administration of 0.3 mg capsaicin. Mustard oil, produced the 

greatest flare response at 30 min (120% increase from baseline) at a concentration of 0.3%. 

Higher concentrations of mustard oil produced less flare at the site of application, though flare 

outside the area of application was substantially greater. The flare response to menthol was not 

substantially different than that of vehicle. These results suggest that laser Doppler imaging can 

be useful for evaluating compounds for their ability to modify the flare response produced by 

TRP agonists. 

Disclosures: V.A. Kenigs, Johnson & Johnson PR&D, A. Employment (full or part-time); M.R. 

Brandt, Johnson & Johnson PRD, A. Employment (full or part-time); C.M. Flores, Johnson & 

Johnson PR&D, A. Employment (full or part-time). 

Poster 





Title: Development of a high-content automated electrophysiological assay combining TRPV1 

pH and voltage-dependent activations 

Authors: O. RADRESA, *S. ZICHA, M. VALIQUETTE, J. DUCHARME; 

AstraZeneca R&D Montreal, St-Laurent, QC, Canada

Abstract: TRPV1 is a multimodal ligand-gated ion channel that is activated by a variety of 

agents. Among physiological activators are anandamide, arachidonic acid metabolites (NADA), 

heat and protons. Capsaicin, camphor and resiniferatoxin (a capsaicin analog from Euphorbia 

resinifera Berg.) also act as VR1 activators. In addition to its ligand-gated properties, the channel 

displays voltage-dependent rectification upon depolarization to increasing positive potentials. 

Ironically, the detailed mechanisms by which the channel integrates these individual activation 

modes and eventual cross-sensitization are still poorly understood. 

In this context, we set out to develop an automated high-content electrophysiological assay that 

could lead us to rapidly profile the mode of action of selected compounds. We took advantage of 

the PatchXpress (Molecular Devices) ability to deliver higher throughput electrophysiology 

data to combine, in a single test, TRPV1 pH and voltage-dependent activations so as to gain 

insight into the possible modes of action of pharmacological agents. Furthermore, specific 

mechanisms such as compound state-dependency and effects on inward (Na/Ca currents) and 

outward conductances (K+) were investigated under relevant inflammatory conditions, over a 

wide range of physiological potentials. Data analyses were automated using the DataXpress2 

software (Molecular Devices). These studies allowed us to elucidate further the various signals 

integrated by the TRPV1 channel and show that the proposed assay configuration, by allowing 

us to measure several ranges at a time, is a useful and cost-effective way to profile the 

mechanism of action of selected drug candidates. 

Disclosures: O. Radresa, None; S. Zicha , None; M. Valiquette, None; J. Ducharme, None. 

Poster 

266. Pain: Psychophysics and Behavior 



Topic: D.08.h. Psychophysics and behavior 

Support: This work was supported by Advanced Neuromodulation Systems through an ANS 

sponsored clinical study. 

Title: Constant current versus constant voltage in spinal cord stimulation: Patient perceived 

differences 

Authors: R. CATLIN 1 , *S. N. WASHBURN 2 , K. BETHEL 3 , O. FLORETE 4 , E. SHADID 5 ; 

1 Chattanooga Ctr. for Pain Med., Hixon, TN; 2 Clin. Res., ANS, Plano, TX; 3 The Bethel Clin., 

Mesa, AZ; 4 Florida Inst. of Med. Res., Orlando, FL; 5 Southwest Neuromodulation Inst., 

Oklahoma City, OK

Abstract: Electrical stimulation can be supplied to the spinal cord using either a constant current 

or a constant voltage power source. A constant current source provides a consistent current to the 

tissue by adjusting the voltage in response to changes in tissue impedance. A constant voltage 

source adjusts the current in response to a change in the impedance thereby maintaining the 

voltage constant. Both systems produce paresthesia and both have been shown to treat chronic 

pain in clinical trials. However, it has been suggested that patients prefer constant current over 

constant voltage. This study compares patient preference for the stimulation sensation elicited by 

constant current and constant voltage systems. 

This study is an IRB approved, prospective, randomized, double blinded, multi-centered, 

crossover study during a 6-day stimulation trial period. Patients were randomized into 2 

treatment groups; Group A received constant voltage stimulation and Group B received constant 

current stimulation. Patients completed a baseline evaluation prior to trial implantation. Patients 

returned 1 day post-operatively for randomization and received their stimulation programs. Three 

days following initial programming, patients were evaluated and crossed over into the alternate 

treatment group. The same trial programs, including electrode configuration, pulse width and 

frequency, were used throughout the study. At 6 days post-operatively, patients returned for the 

final evaluation. Patient well being, pain, satisfaction/QOL, preference and stimulation sensation 

were evaluated. 

Thirteen patients have completed the study and six patients were dropped due to programming 

changes. Nine patients (69.2%) preferred the constant current stimulation after experiencing both 

treatments. During constant voltage stimulation, 7 patients (53.8%) were satisfied or very 

satisfied and 6 patients (46.2%) were unsatisfied or very unsatisfied. During constant current 

stimulation, 12 patients (92.3%) were satisfied or very satisfied and only 1 patient (7.7%) was 

unsatisfied. The mean patient reported percent pain relief was 59.6 for constant voltage and 63.5 

for constant current. The only descriptor for stimulation sensation used more often to describe 

constant voltage over constant current was “soothing.” Interestingly, this descriptor was also 

associated with patient satisfaction. 

Overall, patients preferred and experienced greater satisfaction, and pain relief with the constant 

current system. Animal studies are currently underway to investigate the mechanism underlying 

this effect. 

Disclosures: R. Catlin, ANS-A St. Jude Medical Company, F. Consultant/Advisory Board; 

ANS-A St. Jude Medical Company, C. Other Research Support (receipt of drugs, supplies, 

equipment or other in-kind support); ANS-A St. Jude Medical Company, D. Speakers 

Bureau/Honoraria (speakers bureau, symposia, and expert witness); S.N. Washburn , ANS-A 

St. Jude Medical Company, A. Employment (full or part-time); K. Bethel, ANS-A St. Jude 

Medical Company, C. Other Research Support (receipt of drugs, supplies, equipment or other inkind 

support); O. Florete, ANS-A St. Jude Medical Company, C. Other Research Support 

(receipt of drugs, supplies, equipment or other in-kind support); E. Shadid, ANS-A St. Jude 

Medical Company, C. Other Research Support (receipt of drugs, supplies, equipment or other inkind 

support). 

Poster





Support: Korea grant IITA-2008-C1090-0801-0002 

NIH grant F05-AT003770 

Title: Multi-organ autonomic response to verum and phantom acupuncture 

Authors: *K. PARK 1,2 , J. LEE 1 , J. KWON 1 , W. CHOI 3 , S. LEE 3 , V. NAPADOW 2,4 ; 

1 Dept biomed engineering, Kyunghee Univ., Yongin, Republic of Korea; 2 Dept. of Radiology, 

Martinos Ctr. for Biomed. Imaging, Charlestown, MA; 3 Dept. of Oriental Neuropsychiatry, 

Sangji Univ., Wonju, Republic of Korea; 4 Dept. of Radiology, Logan Col. of Chiropractic, 

Chesterfield, MO 

Abstract: BACKGROUND: Acupuncture is known to modulate autonomic tone. However, as 

most previous studies did not incorporate placebo controls, it is not known which aspect of 

acupuncture induces autonomic modulation. In order to separate the somatosensory from the 

visual aspect of acupuncture stimulation, we developed a novel form of placebo acupuncture - 

dubbed phantom acupuncture, which maintained the visual aspect of acupuncture, with no 

somatosensory stimulation, and variable credibility. 

METHODS: 20 healthy subjects (F, 21.8 ± 2.6 yrs) received verum (VA) and phantom (PA) 

acupuncture. VA was performed in session 1. Needle stimulation was video-recorded and 

simultaneously displayed to the subject. After 1 week, session 2 and 3, separated by at least 

40min, were performed with VA or PA, respectively. 

For VA, a 2min resting baseline was followed by needle insertion on right PC6. Needle 

stimulation comprised eight stimuli (3sec duration) at pseudo-randomized ISI (κ=19.5sec) for 

3min. For PA, the acupuncturist did not perform acupuncture, but the video recording from the 

previous VA session was replayed to the subject creating an illusion of needle 

insertion/stimulation. 

After session 3, subjects were separated into blinded (high credibility, n=11) and unblinded (low 

credibility, n=9) groups using a questionnaire and interview. Analyses used 4 groups: VA 

blinded, Vb; PA blinded, Pb; VA unblinded, Vu; and PA unblinded, Pu. Measurement included 

heart rate (HR), skin conductance (SC), pupil size (PS), respiration, and subjective acupuncture 

sensation using the MASS for each session. We computed a tonic response (3min stimulation - 

2min baseline) and a phasic event related response for HR, SC, and PS. 

RESULTS: For tonic HR response, PA, only when credible, can induce HR decrease(Pu Γ: - 

1.26±1.94bpm, p=0.09; PbΓ: -3.55±3.09bpm, p

p

increase in the grip force with the prolongation of the time which never be observed in ablebodied 

subjects. Taken together with the pathology of CIP, observed deficit of grip force control 

can attribute to the lack of sensory input mediated by Aδ and C-fibers. The present results 

strongly suggest that sensory information mediated by Aδ and C-fibers is important for grip 

force control. 

Disclosures: N. Kawashima, Japan Society for the Promotion of Science, B. Research Grant 


received); M.O. Abe, None; N. Haga, None; M. Iijima, None; K. Shirakawa, None; K. 

Nakazawa, None; T. Iwaya, None. 

Poster 





Support: AT001433 

T32 MH75884 

Title: Polymorphisms in the GTP cyclohydrolase gene (GCH1) are associated with ratings of 

capsaicin pain 

Authors: C. CAMPBELL 1 , R. EDWARDS 1 , C. CARMONA 2 , M. UHART 1 , G. WAND 1 , A. 

CARTERET 2 , Y. KIM 4 , J. FROST 3 , *J. N. CAMPBELL 2 ; 

1 Med., 2 Dept Neurosurg., 3 Radiology, Johns Hopkins Univ., Baltimore, MD; 4 Med., Seoul Natl. 

Univ., Seoul, Democratic People's Republic of Korea 

Abstract: Genetic variability is becoming a widely accepted factor with substantial impact on 

pain perception. Though it is clear that genomic variability plays an integral role in pain 

sensibility, controversy exists over which genes are involved. While recent evidence suggests 

evidence for a “protective” (i.e., less pain) haplotype in the GTP cyclohydrolase (GCH1) gene, 

other research has failed to confirm this association. Possibly, the effects of single nucleotide 

polymorphisms (SNPs) vary depending on the pain task. The current investigation analyzed the 

association of five GCH1 SNPs with ratings of pain induced by topical high concentration (10%) 

capsaicin applied to the skin of 39 normal human volunteers. Participants rated their pain over a 

90-minute period. Multiple GCH1 polymorphisms were associated with lower pain ratings. 

Participants expressing the uncommon alleles for SNPs previously identified by Tegeder et al

(2006) exhibited lower pain ratings. This relationship was particularly striking for rs4411417 (p 

= .005). For this SNP, those having the common variant reported 77% more pain than those 

exhibiting the uncommon allele. Significant effects were also evident for rs3783641, and 

rs752688 (p‟s < 0.05). When combined, these three SNPs accounted for a surprisingly high 35% 

of the inter-individual variance in pain ratings. We conclude that SNPs of the GCH1 gene 

powerfully affect ratings of pain induced by capsaicin. 

Disclosures: C. Campbell, None; J.N. Campbell , Jim Campbell has an employment 

arrangement with InterWest Partners, which in turn has an investment in Solace Pharmaceuticals, 

which in turn has a development program related to GCH1., A. Employment (full or parttime); 

R. Edwards, None; C. Carmona, None; M. Uhart, None; G. Wand, None; A. Carteret, 

None; J. Frost, None; Y. Kim, None. 

Poster 






Title: Persistence of chemo-neuropathy related pain and sensory dysfunction at long-term reexamination 

Authors: *P. M. DOUGHERTY, A. W. BURTON, B. HAMID, L. C. DRIVER, H.-R. WENG; 

Div. Anesthesiol & Critical Care Med., Univ. Texas, Anderson Cancer Inst., Houston, TX 

Abstract: Neuropathic pain is one of the chief dose-limiting toxicities associated with each of 

the major cancer chemotherapeutic drugs. Previous reports have shown pronounced sensory 

deficits, both within and in surrounding areas, of on-going pain in patients with chronic 

chemotherapy-related pain. Here we report pain ratings, analgesic use, and quantitative sensory 

findings obtained from patients with chronic chemo-neuropathy at a one-year or greater followup 

re-re-examination. Patients completed pain diagrams, selection of word descriptors, pain 

medication history inventories and then underwent a repeat quantitative sensory examination to 

assess touch (Aβ), sharpness (Aδ) and heat pain (C-fiber) sensibility. The results show that all 

indices, except daily maximum pain, remain unchanged over the follow-up interval for all 

patients that originally entered the study with chemotherapy-related pain of greater than 3 

months duration. Daily maximum pain ratings were modestly reduced. These results indicate that 

chemotherapy-related neuropathy is both very persistent and also refractory to therapy once the

transition to chronicity is crossed. Therapies for this condition would therefore most likely best 

be directed at protection as opposed to palliation. 

Disclosures: P.M. Dougherty , None; A.W. Burton, None; B. Hamid, None; L.C. Driver, 

None; H. Weng, None. 

Poster 





Support: VZ 0021620816 

CNS 1M0517 

Title: Is there a “birthday phenomenon” in nociceptive sensitivity in women? 

Authors: A. YAMAMOTOVÁ 1 , H. PAPEZOVA 2 , *R. ROKYTA 3 ; 

1 Dpt of Normal, Pathological and Clin. Physiol., Charles Univ. in Prague, Third Fac. of Med., 

Prague, Czech Republic; 2 Dept. of Psychiatry, Charles Univ. in Prague, First Fac. of Med., 

Prague, Czech Republic; 3 Dept Physiol & Clin. Physiol, 3rd Fac Med. Charles Univ. Prague, 

Prague 120 00, Czech Republic 

Abstract: Physiological functions and behavior are known to be influenced by genetic as well as 

environmental factors in adults. These factors have an effect on perinatal neurodevelopment and 

in adulthood they can affect personality traits, neurotransmitter turnover (Chotai, Adolfsson 

2002) or can contribute to predisposition to several diseases. Patients with eating disorders 

(anorexia and bulimia nervosa) are more likely to be born in summer months and they have 

decreased nociceptive sensitivity when compared with healthy women. In this retrospective 

study we analyzed the effect of the month of measurement, the month of birth, and their 

interaction on nociception in 68 healthy women (the mean age 23.3 ± 5.6 years) and 112 patients 

with eating disorders (23.6 ± 5.8 years). Thermal pain threshold was measured on the dorsal 

surface of three fingers using Analgesia Meter, Model 33IITC, Life Sciences. Data were 

analyzed by ANOVA and cosinor analysis. We found a tendency to higher pain threshold in 

winter experiments, although the skin temperature follows a biphasic curve with spring and 

autumn maximum. We found no relation between nociception and the month of birth. The 

greatest variations in nociception were observed when data were analyzed on the scale of elapsed 

time between month of measurement and month of birth (F(11,168)=3.03, p=0.001). In patients

and controls, the lowest pain threshold was observed when measured during the month of birth. 

As a birthday may be a potential psychosocial stressor triggering vascular events (Saposnik et al. 

2006) as well as mortality (Phillips et al. 1992), the further studies in various pain symptoms 

together with these factors remain to be explore. 

Supported by VZ 0021620816 and CNS 1M0517. 

Disclosures: A. Yamamotová, None; R. Rokyta , None; H. Papezova, None. 

Poster 



Program#/Poster#: 266.7/FF1 



Title: Evidence that nociceptive sensations at innocuous temperatures are inhibited by warmth 

and cold 

Authors: *B. G. GREEN 1 , G. MIHAILA 2 ; 

1 John B Pierce Lab. & Yale Schl Med., New Haven, CT; 2 The John B. Pierce Lab., New Haven, 

CT 

Abstract: Sensations such as burning, stinging and pricking can be stimulated by cooling or 

heating the skin to temperatures (e.g., 30° and 37°C) that are below the thresholds of classically 

defined cold- and heat-sensitive nociceptors. It has been hypothesized that these sensations, 

referred to as „Low-threshold Thermal Nociception‟ (LTN), result from stimulation of 

temperature-sensitive fibers that have thresholds in the range of warm and cold fibers but which 

synapse on nociceptive spinothalamic tract (STT) neurons. However, LTN is not reported by all 

subjects, which has led to the hypothesis that LTN can be inhibited by central neurons that 

encode warmth and cold, and that the extent of this inhibition varies across individuals. This 

hypothesis was tested in the present study by measuring the perceived intensity of nociceptive 

sensations evoked by innocuous thermal stimuli (warming to 39° or 41°C; cooling to 28° or 

25°C) that were either delivered alone or within a field of milder warming or cooling 

(background stimulation). Background stimulation (cooling to 31°C from a base of 35°C; 

warming to 35° from a base of 31°C) was mild enough to produce warmth or cold without also 

causing significant nociceptive sensations. All stimuli were generated using a 16-channel, 4x4 

matrix of Peltier thermodes that contacted the forearm. The thermal test stimuli were delivered 

via 4 thermodes interspersed among the other 12 thermodes that delivered the background

stimulus. The results showed that for both heating and cooling the intensity of nociceptive 

sensations was significantly reduced in the presence of the background stimulus. This inhibition 

occurred despite relatively small increases in the perceived intensity of warmth and cold. 

Although inhibition of cold pain by cold fiber stimulation has been inferred from studies in 

which pressure or ischemic block of myelinated fibers has caused innocuous cooling to be 

perceived as burning, the present results show that nociceptive sensations are normally inhibited 

by stimulation in both thermal pathways. Research is continuing to determine whether individual 

differences in thermal inhibition of nociceptive stimulation may be associated with individual 

differences in perception of LTN and/or in the thresholds for cold pain and heat pain. 

Disclosures: B.G. Green, None; G. Mihaila, None. 

Poster 





Support: NSERC/CIHR CHRP Grant 350980 

Canadian Paraplegic Association Ontario 

Title: Intensity of the thermal grill illusion of pain depends on body location 

Authors: *J. P. HUNTER 1 , R. DRANGA 2 , M. POPOVIC 3 , J. DOSTROVSKY 4 ; 

1 Dept Physical Therapy, Univ. Toronto, Toronto, ON, Canada; 2 Applied Sci. and Engin., 3 Inst. of 

Biomaterials and Biomed. Engin., 4 Dept. of Physiol., Univ. of Toronto, Toronto, ON, Canada 

Abstract: Purpose: The thermal grill (TG) illusion of pain occurs in response to the 

simultaneous application of interlaced warm (40°C) and cool (20°C) temperature bars. It is 

proposed that the response reflects the relative activity of COLD vs. HPC cells in response to the 

TG stimulus and is based on simple integration of activity in these two channels. Furthermore, it 

is proposed that the relative activation of these channels by the TG reflects receptive field size of 

cool vs. heat/pain fibers. We evaluated the effect of relative receptive size on the TGI response 

by comparing the TGI response from the palm and forearm skin. Methods: Thermal stimuli 

were produced by a 1x6 matrix of Peltier-controlled thermodes. Three bar configurations were 

tested: all bars 20°C (cool), all bars 40°C (warm), and alternating bars 20°C/40°C (TG) at two 

sites (palm vs. forearm). Stimulus duration was 120s. After completion of each run, subjects 

rated the pain intensity and the unpleasantness of the primary thermal sensation on an 11 point

numeric rating scale (0 - 10). Palm and forearm ratings were compared for each bar 

configuration (ANOVA). Results: The intensity of pain and the unpleasantness in response to 

the TG stimulus (Mean (SD)) was significantly higher at the palm - 2.9(2.3) and 3.83(2.4) than at 

the forearm - 1.81(1.8), p

in polymodal nociceptors. CA and MO induce heat hyperalgesia and mechanical allodynia in 

human skin, and sensitize responses of wide dynamic range (WDR)-type dorsal horn neurons to 

noxious skin heating in rats. We presently tested if these TRPA1 agonists induce behavioral 

evidence of thermal hyperalgesia and mechanical allodynia in rats. After habituation training, 

adult male rats underwent baseline testing of hindpaw withdrawal latency to noxious heat 

(Hargreaves test), or the threshold for paw withdrawal elicited by a mechanical probe from 

below (electronic von Frey). In separate sessions, one hindpaw received intraplantar 

microinjection of CA (5-20%= 398 mM-1.56 M, 10 µl) or vehicle. Thermal withdrawal latencies 

or mechanical thresholds for both hindpaws were then obtained 3, 15, 30, 45, 60 and 120 min 

post-injection. CA induced a significant, concentration-dependent reduction in thermal paw 

withdrawal latencies on the injected side. The peak latency change (to 61.7% of pre-injection 

baseline) occurred by 30 min with partial recovery at 120 min. Thermal withdrawal latencies of 

the uninjected (contralateral) paw did not change significantly. CA also induced a significant, 

concentration-dependent reduction in mechanical withdrawal threshold of the injected paw that 

peaked sooner (3 min) and was more profound (44.4% for 20% CA). Mechanical withdrawal 

thresholds for the uninjected paw were not significantly affected. A similar series is being 

conducted with MO. The present results are consistent with reports of allodynia and thermal 

hyperalgesia induced by TRPA1 agonists in humans. The thermal hyperalgesia is consistent with 

CA and MO enhancement of rat WDR responses to noxious heat. However, the mechanical 

allodynia observed presently is inconsistent with the lack of effect of CA or MO on WDR 

responses to graded mechanical stimuli, possibly due to different routes of administration of the 

TRPA1 agonists (intraplantar vs. topical). 

Disclosures: M.G. Tsagareli , None; N. Tsiklauri, None; G. Gurtskai, None; E. Abzianidze, 

None; E. Carstens, None; M. Iodi Carstens, None; K. Zanotto, None. 

Poster 





Support: NINDS P01 NS047399. 

Title: Capsaicin and histamine produce itch when delivered via inactivated-cowhage spicules 

Authors: *P. SIKAND 1 , S. G. SHIMADA 1 , B. G. GREEN 2,3 , R. H. LAMOTTE 1 ; 

1 2 3 

Dept Anesthesiol, Dept Surgery, Yale Univ. Sch. Med., New Haven, CT; Sensory Neurosci., 

The John B. Pierce Lab., New Haven, CT

Abstract: Capsaicin-desensitization reduces or blocks histamine- or cowhage-evoked itch 

suggesting that pruriceptive neurons respond to capsaicin (CAP). However, topical or 

intradermal injection of CAP evokes burning pain possibly mediated by nociceptive specific 

neurons that block the effects of activity in pruriceptive neurons. To test whether such 

pruriceptive neurons and nociceptive specific neurons might be selectively activated and their 

sensory effects dissociated by restricting chemical application to a very small locus, the tip of a 

single heat-inactivated cowhage spicule, previously soaked with one of different concentrations 

of CAP (1-200 mg/ml) or histamine (HIST, 0.01 - 100 mg/ml) was inserted superficially into the 

skin of the forearm of human volunteers. Magnitude ratings of itch, prick/sting and burn were 

obtained every 30s for up to 20 min using the generalized labeled magnitude scale and compared 

with ratings evoked by a native cowhage spicule. CAP and HIST spicules reliably evoked 

sensations of itch in a dose-dependent manner that were most often accompanied by prick/sting 

and less often burn. These sensations, and the similar qualities of sensation evoked by cowhage, 

were accompanied, in some cases, by the presence of one or more pruritic dysesthesias 

(alloknesis to stroking, hyperalgesia and/or hyperknesis to pricking). The peak magnitudes and 

durations of itch and prick/sting evoked by the highest concentration of CAP and HIST spicule 

were comparable to those elicited by cowhage. The incidence of one or more pruritic 

dysesthesias and their areas were independent of the peak magnitude of itch or prick/sting and 

not invariably associated with a flare. Our results indicate that CAP, when delivered via a 

spicule, elicits a predominant sensation of itch and is associated in some cases with pruritic 

dysesthesias similar to those produced by HIST and cowhage. Thus, CAP, HIST and cowhage, 

applied by spicule, most likely activate common neural mechanisms mediating pruritic 

sensations and the associated dysesthesias. 

Supported by NINDS P01 NS047399. 

Disclosures: P. Sikand , None; S.G. Shimada, None; R.H. LaMotte, None; B.G. Green, None. 

Poster 





Support: CIHR grant #MOP 81091 

MCRP-AZ Postdoctoral fellowship to P. Schweinhardt 

Title: The effect of dopamine depletion on pain perception in humans: Influence of personality 

traits

Authors: *P. SCHWEINHARDT 1 , M. CEKO 1 , M. LEYTON 2 , M. BUSHNELL 1 ; 

1 Ctr. for Res. on Pain, 2 Psychiatry, McGill Univ., Montreal, QC, Canada 

Abstract: Increased striatal dopamine (DA) transmission in animals decreases pain behaviors. In 

comparison, the functional significance of DA for pain perception in humans remains unknown. 

To investigate the association in humans, we hypothesized that depleting brain DA would 

increase pain sensitivity, and that individual differences in this response would be related to 

putative monoamine-associated personality traits. 

16 healthy young males took part in a double-blind crossover study. Acute DA depletion was 

achieved by administering an amino acid mixture deficient in the DA precursors, phenylalanine 

and tyrosine. In the control condition, subjects ingested a nutritionally balanced mixture. Pain 

testing (short heat stimuli) was performed both before (Pre) and 5 hrs after ingestion of each 

mixture (Post). Pain intensity was rated from 0 to 200 (0 - no sensation, 100 - pain threshold, 200 

- worst pain tolerable). Personality traits were assessed with the Temperament and Character 

Inventory (TCI), the BIS/BAS scale, and the Temporal Experience of Pleasure Scale (TEPS). To 

test for an effect of DA depletion on pain perception, we compared difference scores (Post-Pre) 

between the two conditions (Post-Pre) depleted vs. (Post-Pre) control. The changes in pain 

intensity in the depleted condition relative to the control condition were then correlated with the 

personality trait scores. 

The mean (SD) pain intensity ratings were: control condition Pre: 148 (17.2), Post: 142(32.5); 

depleted condition Pre: 150 (12.5), Post: 153 (24.4). The change in pain sensitivity did not differ 

significantly between the two conditions (p=0.29). However, individual differences in the effect 

of DA depletion correlated negatively with Anticipatory Pleasure scores (AP, TEPS) (r=-0.67, 

p=0.009) and positively with Harm Avoidance scores (HA, TCI) (r=0.54, p=0.044). A median 

split indicated that DA depletion significantly increased pain sensitivity in subjects with high 

(p=0.043) but not low HA scores (p=0.45), and at the trend level in subjects with low (p=0.069) 

but not high AP scores (p=0.46). 

In conclusion, DA depletion had no significant effect on pain perception on a group level. 

However, personality traits thought to be related to endogenous DA function influenced 

significantly changes in pain sensitivity following DA depletion. The observation that low AP 

subjects are more vulnerable to the pain enhancing effects of DA depletion supports the 

proposition that DA has analgesic properties in humans and that these susceptible subjects may 

have a low DA trait. 

Disclosures: P. Schweinhardt, None; M. Ceko, None; M. Leyton, None; M. Bushnell, None. 

Poster 



Program#/Poster#: 266.12/FF6


Support: NIH NINDS 1K23N2S4335 

NIH NIDCR U24 DE016509 

Title: Nociceptive stimulation affects sensitizing and inhibitory systems differently in a 

fibromyalgia patient and a matched healthy control 

Authors: *F. WONG 1 , A. RODRIGUES 2 , S. SCHMIDT 3 , A. P. MAUDERLI 4 ; 

1 Prosthodontics, Univ. Florida, Gainesville, FL; 2 Tuft Univ., Boston, MA; 3 Community heatlh 

and family medicine, 4 Prosthodontics, Univ. of Florida, Gainesville, FL 

Abstract: Clinicians and scientists know that different patients with fibromyalgia syndrome 

(FMS) may respond differently to the same treatments and experimental interventions. This 

suggests that the diagnosis of FMS combines a heterogeneous group of patients and that data 

from individual subjects may reveal phenomena that may not be obvious in group-based 

research. The goal of this study of a single 29 year old female FMS patient and a matched 

healthy control subject was to investigate whether exposure to a strong nociceptive conditioning 

stimulus affects pain sensitivity differently in an FMS patient and a healthy individual. 

Pain sensitivity of the thenar eminence of the hand was tested with a Peltier thermode of 1” sq. 

The intensity of evoked pain was rated on an electronic visual analog scale (eVAS) that was 

integrated with the stimulator. Two episodes (2 min each) of continuous thermal stimulation 

were administered to track changes in pain sensitivity. These tests were separated by a 30-60 min 

break during a strong nociceptive conditioning stimulus in some of the sessions. A 

methodological key feature was that pain intensity was kept near a predetermined value (20, 25 

or 35%) by constantly and automatically adjusting the thermode temperature as a function of the 

rating on the eVAS (“pain intensity clamping”). Temperature changes through each 2 min test 

were used to infer changes in pain sensitivity. The conditioning stimulus consisted of a series of 

thermal pulses (3sec duration, 30 sec pulse interval) during which the temperature first increased 

in 0.5 0 C steps from 43 to 49 0 C and then decreased back to 43 0 C. 

As expected, the average temperature to elicit predetermined pain intensity was much lower in 

the FMS patient than in the control subject. Without a conditioning stimulus the change in pain 

sensitivity within and between the 2 min pain intensity clamping tests were similar and minimal 

in both subjects. The conditioning stimulus had a markedly different effect on the two subjects: it 

led to a substantial increase in pain sensitivity in the FMS patient (lower temperature during the 

second test), and sensitization continued throughout at least part of the second test. Conversely, 

in the healthy subject the conditioning stimulus resulted in a significant decrease in pain 

sensitivity. It appears that a strong nociceptive stimulus can activate sensitizing and inhibitory 

systems. In the FMS patient the former was more responsive; in the healthy subject it was the 

latter. FMS- related pain symptoms may predominantly be a result of enhanced sensitization in 

some FMS patients, reduced inhibition in others leading to different therapeutic needs. 

Disclosures: F. Wong, None; A. Rodrigues, None; S. Schmidt, None; A.P. Mauderli, None.

Poster 

267. Pain: Pain Models II 



Topic: D.08.j. Pain models 

Support: The work was in part supported by the IRPF 

Title: The antinociceptive effects of L-kynurenine in the writhing test may be mediated by 

interaction kynurenic acid-GPR35 

Authors: *C. COSI, V. CARLÀ, G. MANNAIONI, D. MARATEA, F. MORONI; 

Dept Pharmacol, Univ. Florence, Florence, Italy 

Abstract: Kynurenine is the primary degradation product of tryptophan and the origin of the 

“kynurenine pathway”, a cascade of enzymatic steps that generate several biologically active 

compounds, including kynurenic acid (KYNA) which has both neuroprotective and 

antinociceptive properties. KYNA parenterally administrated crosses the blood brain barrier 

poorly but s.c. administration of 100 mg/kg kynurenine can increase kynurenic acid in the blood 

and in the brain (Chiarugi et al., J. Neurochem 67, 692, 1996) this effect being potentited by 

probenecid, an inhibitor of organic anion transport. GPR35, a formerly “orphan receptor” 

activated by KYNA, is highly abundant in DRG and possibly involved in nociception (Ohshiro 

et al., BBRC 365, 344, 2008). The intraperitoneal injection of 0.6% acetic acid in mice induces 

the contraction of the abdominal muscles together with stretching of the hind legs (“writhes”). 

This so-called writhing test is used for antinociceptive screening and enables detection of 

compounds activity on acute pain. 

We examined the effects of L-kynurenine (KYN) alone and in combination with probenecid on 

the writhing and monitored the levels of plasma KYNA from trunk blood after sacrifice at one 

hour following the drug treatments of mice. KYN (30, 100, 300 mg/kg s.c.) decreased the 

number of writhes by 16, 29 and 60% of controls, indicating an antinocicaptive/anti-inflamatory 

effect of the compound. KYN 100 mg/kg and 300 mg/kg dose-dependently increased plasma 

KYNA to 1068±190 pmol/ml and 2201±213 pmol/ml (controls: 49±10 pmol/ml), respectively, 

suggesting that the antinociceptive effect of KYN was due to the increased availability of 

KYNA. Co-administration of probenecid (200 mg/kg s.c) potentiated the antinociceptive effect 

of KYN (100 mg/kg) and increased plasma KYNA levels to 4610±712 pmol/ml. Probenecid by 

itself decreased the number of writhes and increased plasma KYNA to 268±87 pmol/ml. These 

results indicate that KYN has antinociceptive effects in the writhing test and suggest that KYNA 

mediated these effects.

Disclosures: C. Cosi, None; V. Carlà, None; G. Mannaioni, None; D. Maratea, None; F. 

Moroni, None. 

Poster 





Support: NIH Grant 5R01DE018531-02 

Title: Experimental trigeminal root compression in rodents 

Authors: *M. KLYUKINOV 1 , C. I. GOMES 1 , N. MANERING 1 , J. M. CUELLAR 2 , D. C. 

YEOMANS 2 ; 

1 Anesthesia, 2 Pain Research, Anesthesia, Stanford Univ., Stanford, CA 

Abstract: Trigeminal neuralgia (TN) is a chronic pain syndrome of the orofacial area that causes 

intense, stabbing pain in the face along the distribution of the trigeminal nerve. Symptoms 

associated with TN include weight loss, spontaneous tics/twitches, and increased sensitivity to 

light touch of the face. TN most commonly results from vascular compression of the trigeminal 

nerve root, which is believed to cause focal demyelination of trigeminal sensory fibers and to 

alter sodium channel distribution along the axon. Current treatments available for TN, including 

carbamazepine, provide incomplete management of the patient‟s pain. The development of better 

treatment options is limited by the lack of an adequate animal model for study. Our goal is to 

develop an animal model that closely parallels the human pathology seen in TN. 

In order to produce trigeminal nerve root compression (TRC), we injected a superabsorbant, 

biocompatible, volume-expanding polymer adjacent to the trigeminal nerve root in rats. TRC 

rats, but not sham rats, showed initial weight loss after surgery and slowed weight gain 

thereafter, suggesting decreased feeding as a consequence of paroxysmal pain. Spontaneous 

blinking was significantly increased ipsilaterally in TRC rats as compared to baseline and sham 

controls with some TRC rats showing ipsilateral eye twitching. Facial allodynia was measured 

by mechanical stimulation of the cheek with von Frey filaments of different weights, brushes of 

varying stiffness, and air puffs. A significant bilateral allodynia was observed in TRC rats, but 

not sham rats, at most time points, with a stronger response on the ipsilateral side. Facial 

allodynia resulting from TRC surgery, as measured with von Frey stimulation, was observed in 

the distribution of the maxillary branch, with ophthalmic and mandibular divisions being less 

affected. 

Trigeminal tissue from TRC rats that demonstrated paroxysmal behavior was removed and

stained for voltage-gated sodium channels (Nav) and Nodes of Ranvier and examined using 

differential interference contrast (DIC) light microscopy and laser scanning confocal microscope. 

Compressed entry zone tissue demonstrated clear dysmyelination and a notable increase in 

overall level and a clumping of Navs in compressed tissue without obvious infiltration of 

inflammatory cells into the damaged area. 

Behavioral and histological data obtained suggests that TRC surgery produces pathology in rats 

consistent with that seen in TN patients. Development of this novel animal model will allow us 

to study the pain mechanisms underlying TN and can provide us with an avenue for testing new 

therapies. 

Disclosures: M. Klyukinov, None; C.I. Gomes, None; N. Manering, None; J.M. Cuellar, 

None; D.C. Yeomans, None. 

Poster 





Support: Work supported by the Clinical development department of Pierre Fabre laboratories 

Title: Linguistic validation of five quality of life questionnaires for fibromyalgia patients in 

twelve languages 

Authors: A. NADJAR 1 , Y. MAINGUY 2 , M. GALISSIÉ 2 , M. LE GAL 3 , *L. BARDIN 4 , D. 

ALLAIN 2 ; 

1 MAPI Res. Inst., Lyon, France; 2 Clin. Develop., Pierre Fabre, Castres, France; 3 MAPI Values, 

Lyon, France; 4 Neurobiol II, Res. Ctr. Pierre Fabre, Castres, France 

Abstract: Objectives: Fibromyalgia's complex symptomatology means that several instruments 

and questionnaires for assessing Quality of Life are used to collect a wide variety of information. 

The concepts used by the questionnaires must be equivalent, regardless of the language used. 

Five questionnaires (Multidimensional Fatigue Inventory MFI, Multiple Ability Self-report 

Questionnaire MASQ, State-Trait Anxiety Inventory STAI, Fibromyalgia Impact Questionnaire 

FIQ, Beck Depression Inventory-II BDI-II) were translated into twelve European languages, 

using a standardized, validated and recognized process, in order to use them in international 

studies. 

Methods: The process of linguistic validation in a target language comprised the following steps: 

a forward translation by two translators who were native speakers of the target language, a

ackward translation by a translator who was a native speaker of the questionnaire source 

language, a review of the version obtained by an expert clinician and a comprehension test 

involving five healthy subjects from the country where the target language was spoken. The 

process was carried out in collaboration with the questionnaire authors. 

Results: Three questionnaires (MFI, MASQ and STAI) were linguistically validated in the 

twelve target languages. The FIQ and BDI were validated in the eight and four missing 

languages. Irrespective of the questionnaire and dimensions studied, the same issues were 

encountered in their validation. i) Issues were encountered in translation in a narrow sense. For 

example, the word "things" was translated as "something" and "rug" was translated as "carpet"; 

ii) Literal translation was possible but largely culturally irrelevant. For example, the expression 

"to walk several blocks", a completely abstract idea in Europe, was translated as "to walk more 

than one kilometer"; iii) Tenses needed to be reformulated or changed for idiomatic reasons. For 

example, for the MFI questionnaire, the present perfect does not exist in German, and so the 

present simple was used in the first version. However, as subjects had difficulty understanding 

the use of present simple with adverbs such as "lately" during the comprehension test, the 

imperfect was eventually used. 

Conclusions: Linguistic validation carried out using a rigorous and recognized method ensures 

that the concepts assessed are equivalent in twelve languages and allows their use in large scale 

studies measuring quality of life in fibromyalgia patients. 

Disclosures: A. Nadjar, Pierre Fabre, F. Consultant/Advisory Board; Y. Mainguy, Pierre Fabre 

laboratories, A. Employment (full or part-time); M. Galissié, Pierre Fabre laboratories, A. 

Employment (full or part-time); D. Allain, Pierre Fabre laboratories, A. Employment (full or 

part-time); M. Le Gal, Pierre Fabre, F. Consultant/Advisory Board; L. Bardin , Pierre Fabre 

laboratories, A. Employment (full or part-time). 

Poster 






Title: Place preference validates sustained lifting/grooming as selective indicator of rat pain after 

peripheral nerve injury 

Authors: *H.-E. WU, G. GEMES, T. KAWANO, V. ZOGA, Q. HOGAN; 

Dept Anesthesiol, Med. Col. Wisconsin, Milwaukee, WI

Abstract: We have previously demonstrated that simple paw-withdrawal from von Frey tactile 

stimulation fails to distinguish rats with peripheral nerve injury from control rats, whereas a 

complex non-learned lifting and grooming response after needle stimulation (hyperalgesia 

response, HR) is selectively expressed in neuropathic animals (Hogan et al., 2004). To validate 

this behavior as an indicator of pain, the aversiveness of the experience associated with HR was 

determined in sciatic nerve ligated (SNL) rats using the acquisition of conditioned place 

preference (step-down passive-avoidance test). Groups (n=8-16) of male SD rats received either 

SNL or skin incision alone. After 17d, the rat was placed on a platform (10.3 cm in diameter and 

2.5 cm in height) located in a 30 x 28 x 28 cm clear plastic enclosure with a ¼-in wire grid floor. 

Latency to step down from the platform was measured (cutoff 150s), and the rat was taken back 

to its cage immediately. This was repeated 9 times at 10min intervals. Rats were habituated with 

no stimuli the first day. The next day, rats received 0, 1 or 3 plantar, nonpenetrating touches by a 

22g needle stimulus upon stepping down at each trial, producing either an HR or a brief reflexive 

withdrawal. Without stimulation, all animals maintained baseline latency. Latency was 

prolonged by stimulation in SNL animals (3.56s/trial for 1 touch, R 2 = 0.8068; 4.01s/trial with 3 

touches, R 2 = 0.9204), whereas control animals had unchanged latency. Among SNL animals, 18 

animals that failed to demonstrate HR maintained their pre-stimulation baseline latency, whereas 

animals that exhibited HR increased their step-down latency both in 1 touch (7.05s/trail, R 2 = 

0.7152) and 3 touch (6.50s/trial, R 2 = 0.9072) groups. These findings indicate that the needleinduced 

HR is aversive and a valid indicator of rat pain, whereas mere withdrawal from 

mechanical stimulation, such as occurs with determination of withdrawal threshold using von 

Frey fibers, is not aversive, and therefore is a poor tool for indicating pain. 

Disclosures: H. Wu , None; G. Gemes, None; V. Zoga, None; Q. Hogan, None; T. Kawano, 

None. 

Poster 





Title: The effect of neuropathic pain on psychiatric and cognitive measurements in the rat 

Authors: *M. J. PIESLA, A. JOSHI, T. A. CUMMONS, J. E. HARRISON, L. LEVENTHAL, 

G. T. WHITESIDE; 

Discovery Neurosci., Wyeth Res., Princeton, NJ

Abstract: Chronic pain is detrimental to the quality of daily life in humans. In addition, 

depression, anxiety, cognitive impairment, an inability to handle stress and poor sleep patterns 

are often co-morbid with chronic pain conditions. Animals in chronic pain may also display 

symptoms of anxiety, depression or cognitive impairment and additional stress may exacerbate 

these symptoms. This study was undertaken to determine the effect of neuropathic pain and/or 

stress on measurements of anxiety, depression and cognition in the rat. Several groups of animals 

received partial sciatic nerve ligation (PSNL) and were exposed to various behavioral tests. 

Locomotor activity, open field, fear potentiated startle and novel object interaction were utilized 

as tests of activity and/or anxiety. Sucrose preference, with or without chronic mild stress, was 

utilized as a test of depression. Cognition tests examined were the Morris water maze and novel 

object recognition. In the locomotor activity test (light vs. dark), sham animals were less active 

in the light while PSNL animals were less active under both conditions. PSNL animals also 

displayed a significantly greater startle response in brightly illuminated test chambers. Thus, the 

neuropathic animals exhibited an anxious phenotype in the locomotor activity and fear 

potentiated startle tests. However, there was no difference between sham and PSNL animals in 

the open field (light vs. dark) or novel object interaction tests. Sucrose preference tests were 

employed to operationally define anhedonia (an inability to feel joy). Specifically, anhedonia in 

animals is defined as a reduction in sucrose intake relative to the control group and baseline 

intake values. Chronic mild stress decreased sucrose intake in all animals, however there was no 

significant difference between sham and PSNL rats. There was no significant difference between 

sham and PSNL rats in either the Morris water maze or novel object recognition test. Normal 

animals performed poorly in these assays, therefore, Sprague-Dawley rats may not be the 

optimal strain for use in cognition tests. In conclusion, while significant differences between 

sham and PSNL animals were observed in locomotor activity and fear potentiated startle, there 

was little difference between sham and PSNL rats in behavioral tests of depression and 

cognition. It is possible that PSNL develops insufficient ongoing or spontaneous pain to produce 

robust effects in these models. 

Disclosures: M.J. Piesla , Wyeth Research, A. Employment (full or part-time); A. Joshi, 

Wyeth Research, A. Employment (full or part-time); T.A. Cummons, Wyeth Research, A. 

Employment (full or part-time); J.E. Harrison, Wyeth Research, A. Employment (full or parttime); 

L. Leventhal, Wyeth Research, A. Employment (full or part-time); G.T. Whiteside, 

Wyeth Research, A. Employment (full or part-time). 

Poster 




Topic: D.08.j. Pain models

Support: Supported by CIHR 

Title: Bone cancer pain: A new way to fight pain ? 

Authors: *V. V. OTIS, L. GENDRON; 

Physiologie et Biophysique, Univ. de Sherbrooke, Sherbrooke, QC, Canada 

Abstract: In North America, cancer appears in one third of the population and leads to bone 

tumour invasion in 60% of cases. Bone metastasis are usually seen in advance stages of cancer 

especially those originating from breast or prostate cancer. Chronic pain due to osteosarcoma can 

achieve high levels of background and breakthrough pain, therefore increasing the difficulty to 

treat patients. As of today, morphine and its derivatives are among the most powerful and 

commonly used opioids to alleviate pain. Even with their strong analgesic potency, opioids 

acting at mu opioid receptors are often accompanied by various side-effects, possibly including 

increased osteolytic activity and bone loss. Over the past few years, compounds activating the 

delta opioid receptor (DOPR) were shown to have significant analgesic effects without any 

significant side-effects usually associated with narcotics. The aim of this study is to evaluate the 

potential roles of selective DOPR agonists for the treatment of bone cancer pain induced by 

injection of 30 000 syngenic MRMT-1 cells (mammary carcinoma) in the femur of Sprague- 

Dawley rats. As other mammary tumours, our results revealed the presence of DOPR mRNA in 

MRMT-1, although at low levels. Thus, DOPR agonists might potentially have an influence on 

tumour progression. Fourteen days after cancer induction, rats had develop significant allodynia 

in their ipsilateral hindpaw, an effect accompanied by an unilateral activation of glial cells 

(astrocyte and microglia) in L2-L4 lumbar spinal cord segments. Our results suggest that this 

bone cancer model might be a promising tool to evaluate the role of DOPR agonists for the 

treatment of bone cancer-induced pain. 

Disclosures: V.V. Otis, None; L. Gendron, None. 

Poster 





Support: NIH R21 NS 055896 

P32 NS07375

Title: Expression of c-Fos in a rodent model of central pain syndrome reveals the anatomical 

loci of altered pain sensation 

Authors: *G. WANG, S. M. THOMSON; 

Dept Physiol, Med. Sch. UMB, Baltimore, MD 

Abstract: Central pain syndrome (CPS) is defined as pain associated with a lesion of the central 

nervous system and is a common consequence of spinal cord injuries. CPS patients experienced 

severe, excruciating pain, either spontaneously or in response to non-noxious stimulation, and 

are highly resistant to opioid analgesia. The mechanisms underlying the genesis of CPS remain 

unclear. We have generated a rodent model of CPS by making unilateral electrolytic lesions 

centered on the spinothalamic tract in rats (T8-T9). We observe thermal hyperalgesia and 

mechanical allodynia bilaterally in both hind and forepaws beginning 7 days postlesion and 

persisting for >31 days postlesion. We also observe an increased probability of burst responses in 

the ventral posterior lateral nucleus (VPL), a postsynaptic target of the lesioned spinothalamic 

tract axons, in thalamic brain slices after lesion. Both bursting and altered pain sensation are 

eliminated by the T-type calcium channel blocker ethosuximide. 

In the present study, we used the immunocytochemical detection of the immediate early gene, c- 

Fos, to map brain regions displaying pathologically elevated activity levels. We compared c-Fos 

expression patterns in brain sections from rats with spinothalamic tract lesions to the pattern of c- 

Fos expression induced acutely as a consequence of hind paw inflammation produced by 

injection of Complete Freunds Adjuvant. c-Fos expression was negligible in sham control 

animals. In CPS animals, in contrast, high levels of nuclear c-Fos expression were observed in 

the anterior cingular cortex, somatosensory cortex, thalamic midline nuclei, posterior regions of 

the VPL, the tegmentum, and dorsal part of periaqueductal gray. The distribution pattern of c- 

Fos expression in CPS animals was similar as that induced by acute hind paw inflammation. In 

CPS rats, c-Fos expressing cells were not co-labeled with antibodies against either enkephalin or 

GABA. 

The pathological bursting activity of VPL neurons and the ability of ethosuximide to block this 

bursting and relieve pain behaviors indicates a primary role for the somatosensory thalamic 

nuclei in CPS. The widespread pattern of elevated c-Fos expression, however, suggests that the 

changes of pain behaviors induced by spinothalamic tract lesion are also accompanied by plastic 

changes in many pain related brain regions. 

Disclosures: G. Wang , None; S.M. Thomson, None. 

Poster 



Program#/Poster#: 267.8/FF14


Support: NIH Grant R44NS046951 

Title: Conduction velocity and thermal threshold of c vs a nociceptors activated by diode laser 

Authors: N. MANERING 1,2 , J. M. CUELLAR 1,2 , *M. I. NEMENOV 1,3,2 , M. KLYUKINOV 1,2 , 

D. C. YEOMANS 1,2 ; 

1 Anesthesia, 2 Pain Res. Ctr., Stanford Univ., Stanford, CA; 3 R&d, LASMED LLC, Mountain 

View, CA 

Abstract: Methods: Infrared diode laser stimulation provides relatively uniform heating of skin, 

allowing a margin of laser power amplitude and duration between stimulation of pricking pain 

and burning pain and providing a margin of safety over CO2 and Thulium lasers. Laser pulses 

were applied to trigeminal innervated (facial) skin of rats. Two types of stimuli were used: 1) 

brief (200 ms, 6.3mm2), relatively high power pulses, which produce clear pricking pain in 

humans or 2) long (15 s, 15.4mm2), relatively low power pulses, which produce burning pain in 

humans. Laser evoked skin temperatures were also measured using a high-speed thermal camera. 

In some animals, the trigeminal nerve was dissected to determine actual conduction distances. 

Results: C fiber nociceptors predominantly responded only to long pulse stimuli at an average 

skin temperature of approximately 46 (we have recording with 49 C so better: 47 +/- 2 )+/- 1 o C 

with an average CV of 0.5 m/s. Some A fibers responded only to the short pulses at an average 

skin temperature of approximately 48 +/- 3 o C at an average CV of 2.7 m/s. Some intermediate 

fibers responded to both long and short pulse stimuli, responding to long pulses at an average 5.8 

o C below their responses to short pulses at an average CV of 1.33 m/s. 

Conclusions: These results provide presumptive evidence that C fiber thermonociceptors are 

selectively activated by long, low heating rate pulses whereas some A fibers are activated solely 

by high rate brief stimuli; other fibers are activated by either. The relatively slow CV and the 

threshold temperature range observed suggests that the A fibers were likely AMH type II, and 

that all nociceptors recorded likely made use of TrpV1 heat transduction. The developed 

trigeminal protocol could be used for quantitative evaluation of the effects of novel analgesic or 

local anesthetic agents on these distinct nociceptor types. 

Acknowledgements: NIH Grant R44NS046951 

Reference: Jiang N et al., Proc. SPIE (2007) Vol. 6428, 642809, Tzabazis A. et al., Brain Res., 

1037 (2005):148-56, Nemenov MI et al., Patent Application (2003) # 20050027336. 

Disclosures: N. Manering, None; J.M. Cuellar, None; M.I. Nemenov , LASMED LLC, 

MOuntain View, CA, A. Employment (full or part-time); PI, B. Research Grant (principal 


LASMED LLC, E. Ownership Interest (stock, stock options, patent or other intellectual 

property); M. Klyukinov, None; D.C. Yeomans, None. 

Poster





Title: Uv-induced heat pain thresholds: a translational preclinical/clinical biomarker model for 

pain research 

Authors: *L. CORRADINI 1 , T. PITCHER 1 , N. BENSON 1 , H. MANSIKKA 2 , J. G. 

WHEELER 1 , B. TRAN 1 , F. KHEDACHE 1 , I. MACHIN 1 , K. TAN 1 , M. J. FIELD 1 ; 

1 Discovery Biol., Pfizer Ltd, Sandwich, United Kingdom; 2 Cambridge science park, 

Mundipharma Res. Ltd, Cambridge, United Kingdom 

Abstract: UltraViolet (UV) irradiation of the skin results in an inflammatory reaction 

characterised by erythema and hypersensitivity to noxious and non noxious stimuli (i.e. 

hyperalgesia and allodynia) in rats and humans (Bishop et al, 2007; Davies et al, 2005; Gustorff 

et al, 2004; Harrison et al, 2004; Hamilton et al, 1999). We have pharmacologically characterised 

thermal endpoints in both human and rat UV models using ibuprofen and oxycodone. 

The aim of this study was to explore the preclinical/clinical PK/PD translation of this model. 

To generate the model, the plantar surface of the rat hind paw exposed to UV light source (300 

mJ/cm2). Oxycodone (1-0.3 mg/kg, SC) and Ibuprofen (30-300 mg/kg, PO) were administered 2 

days post UV irradiation and thermal hypersensitivity assessed at various time pre- and postdose. 

All experiments were randomized by treatment and days, and conducted accordingly to a 

fully blind protocol. 

Clinically, we conducted a double-blind, placebo-controlled, randomised, 4-way crossover. All 

subjects were white male (mean age: 40.7 years). A minimum erythema dose (MED) was 

determined during training session and 3xMED applied during the study periods. Subjects were 

randomised to receive 20 mg oral oxycodone CR formulation, ibuprofen 800 mg IR, or placebo. 

Heat pain perception threshold (HPPT) and heat pain tolerance threshold (HPTT) were assessed 

in the area of erythema at 24 hours following UV irradiation (predose: baseline measurement) 

and 2, 4 and 6 hours post dose in the area of erythema and in the control site (on the opposite 

leg). 

Ibuprofen and Oxycodone significantly reversed thermal hyperalgesia in a dose dependent 

manner in rats and demonstrated a significant reversal of HPPT and HPTT in man (p≤0.05). 

Oxycodone, but not ibuprofen, had a frank analgesic effect on heat pain measures on nonirraditated 

control sites in both species. 

PK/PD analysis of ibuprofen and oxycodone multi dose experiments in the rat UV model showed 

that the data could be described by a direct Emax model, affording EC50 as a summary parameter 

for efficacy. These values were compared to the reported EC50 in man for ibuprofen (Kowalski et 

al, 2007), or in the case of oxycodone to the known exposure profile at a commonly given 

clinical efficacious dose (Bensinger et al, 1997). This analysis of data suggested that the potency 

of these two compounds appears to be approximately 10-20 fold lower in the rat than man. 

In conclusion, using UV-induced heat hypersensitivity model and PKPD methodology, a

database of comparative potency across a spectrum of mechanisms of action could be created, 

providing a basis for cross species scaling to man and hence efficacious dose prediction. 

Disclosures: L. Corradini, None; T. Pitcher, None; N. Benson, None; J.G. Wheeler, None; B. 

Tran, None; F. Khedache, None; I. Machin, None; K. Tan, None; M.J. Field, None; H. 

Mansikka, None. 

Poster 





Support: CIHR 

FRSQ. 

Title: Evaluation of chronic pain induced by injections of acidic saline into the masseter muscles 

of rats 

Authors: N. L. CARAM-SALAS 1,3 , *A. KOLTA 1,4 , J. P. LUND 2,4 ; 

1 Dept Physiol, 2 Physiologie, Univ. Montreal, Montreal, QC, Canada; 3 Dent. Fac., 4 Dent. faculty, 

Mc Gill Univ., Montreal, QC, Canada 

Abstract: Animal models are needed to help uncover the aetiology of chronic orofacial muscle 

pain conditions. Lactic acid produced by heavy exercise is a natural stimulus for muscle 

nociceptors, and Sluka et al (Muscle Nerve. 2001, 24(1):37-46) have shown that repeated 

injections of acidic saline (AS; pH 4) into leg muscles of rodents induce long-lasting signs of 

allodynia/hyperalgesia. Similar injections into jaw muscles have been yielding contradictory 

results. In rats, Ambalavanar et al. (Neuroscience. 2007, 149(3):650-9) reported no changes in 

nociceptive behaviour or in CGRP expression in muscle afferent neurons in the trigeminal 

ganglion, while Morris and Widmer (Soc. Neurosci. Abstr., 2007) did see changes in mastication 

and in the expression of several peptides in mice. We have previously shown changes in the 

excitability of jaw-closing muscle spindle afferents or rats following 2 injections of AS that 

appeared within 24hrs after the first injection and that lasted between 35 and 42 days. The aims 

of this study were: (1) To show that repeated AS injections into the masseter muscle induce longlasting 

nocifensive behaviour in rats, (2) To determine whether a single injection could be used 

and (3) To evaluate whether the effects are bilateral or unilateral. Nocifencive behaviour was 

evaluated at different time points before and after injections by determining the frequency of

head withdrawal to application of von Frey filaments. The data was normalized by dividing the 

number of head withdrawals at every time point by the average baseline number. Comparisons 

were made between groups receiving normal saline (NS) and AS. With 2 injections, increases in 

withdrawal frequency (ratios 1.13 to 1.46) were observed even with NS up to 28 days after the 

injections. However, ratios obtained with AS were significantly greater (3.1 to 5.1) for between 

38 and 45 days (P < 0.005). There was no difference between groups given one or two bilateral 

AS injections, whereas single unilateral injections did not produce any clear effect. Currently, we 

are using the model to test the bilateral effect produced by 2 unilateral injections of AS and the 

participation of glutamate in the development of chronic pain using different glutamate receptor 

antagonists. These results suggest that injections of acidic saline into the masseters of rats 

produce chronic jaw muscle pain and that this can be used as a model of chronic trigeminal 

myalgia. 

Disclosures: N.L. Caram-Salas, None; A. Kolta , None; J.P. Lund, None. 

Poster 





Title: Reversal of CFA-induced decrease in running wheel activity in mice 

Authors: *D. J. STONE, R. W. COLBURN, C. M. FLORES, M. R. BRANDT; 

Analgesics Drug Discovery, Johnson & Johnson Pharma R&D, Spring House, PA 

Abstract: Intraplantar (i.pl.) injection of Complete Freund‟s Adjuvant (CFA) into the hindpaw 

of rodents produces a long lasting thermal hypersensitivity that can be reversed by drugs of 

various chemical classes. However, measurement of thermal hypersensitivity involves an evoked 

response (e.g., paw withdrawal from a heat source), which can be confounded by other 

behavioral effects of drugs (e.g., sedation). The goal of these studies was to determine the utility 

of using a non-evoked, behavioral endpoint (i.e., running wheel activity) in assessing the efficacy 

of antinociceptive compounds. 

Male CD-1 mice were singly housed in rat-sized cages each containing an 8" diameter Wodent 

Wheel Jr. (2.6" wide, 2.1" entry holes, overall height 9.1") affixed with reed switches and 

neodymium magnets, which was interfaced via National Instruments FieldPoint modules to a 

custom LabVIEW application. The 12-hour light cycle was time-shifted so that lights turned off 

at 12pm, and all treatments were administered at 9am (i.e.- three hours before lights-out). 

Running wheel activity is expressed as the number of wheel revolutions per day.

The mice were acclimated to the running wheel cages for 3-4 days before the beginning of the 

study, and baseline levels of activity for each mouse was determined by allowing 6 days of 

unrestricted running. Pilot studies showed that running wheel activity begins 2-3 hours before the 

beginning of the dark cycle, peaks around the time of lights-out, and gradually declines steadily 

over the next 12 hours. Subjects were stratified into treatment groups based on activity levels and 

were then dosed i.pl. with either CFA, carrageenan or vehicle. 

CFA administration reduced running wheel activity to 40% of baseline levels for the first day 

after injection, with a gradual recovery over the next four days, whereas i.pl. saline did not have 

a significant effect on running. This reduction was dose-dependently reversed by oral 

administration the NSAID indomethacin (0.3, 3, and 30 mg/kg). The mixed opioid receptor 

ligand buprenorphine (1 mg/kg, p.o.), reversed CFA-induced reductions in wheel running to 90% 

of control. The antidopaminergic drug haloperidol (1 mg/kg, p.o.) was also fully efficacious in 

this model, in contrast to purported non-antinociceptive effects in rodents. These initial studies 

suggest that pain-suppressed wheel running might be an alternative behavioral endpoint to the 

typical evoked pain endpoints. 

Disclosures: D.J. Stone , Johnson & Johnson Pharmaceutical Research and Development, A. 

Employment (full or part-time); R.W. Colburn, Johnson & Johnson Pharma R&D, A. 

Employment (full or part-time); C.M. Flores, Johnson & Johnson Pharma R&D, A. 

Employment (full or part-time); M.R. Brandt, Johnson & Johnson Pharma R&D, A. 


Poster 





Support: WSU, Department of Anesthesiology Research Fund 

R01 DA-16736 (MPG). 

Joe Young Research Fund in Psychiatry 

Title: Metabolic profile of the rat brain using streptozotocin as a model for neuropathic pain: 

studies with proton magnetic resonance spectroscopy (MRS)

Authors: A. A. CHERRO 1 , *H. MARSH 1 , G. M. MCKELVEY 1 , S. O'LEARY-MOORE 2 , R. 

M. GUFFEY 1 , A. GLAESER 1 , F. GHODDOUSSI 2 , M. P. GALLOWAY 2 ; 

1 Dept Anesthesiol, 2 Psychiatry and Behavioral Neurosci., Wayne State Univ., Detroit, MI 

Abstract: Diabetes is a chronic metabolic disorder that affected 21 million Americans in 2005. 

A common complication of diabetes is peripheral and autonomic neuropathy. Streptozocin (STZ) 

is a pancreatic beta-cell toxin as well as an established agent to induce animal models of diabetes 

and neuropathic pain. The aim of this study was to evaluate the neurochemical changes in the 

periaqueductal grey area (PAG, a regulator of spinal cord functioning) and lumbar dorsal horn 

(LDH) in male rats after inducing diabetes with STZ. We hypothesized that STZ-induced 

diabetes would decrease pain thresholds and modify the neurochemical profile in the PAG 

(increased GLU) and the dorsal horn of the lower lumbar spinal chord (altered glycine GLY). 

Sprague-Dawley rats (n=16) were grouped as control or STZ (50 mg/kg IP) treated. Body 

weight, water and food consumption, urine output, locomotor activity (LMA), blood glucose and 

hindpaw withdrawal thresholds (Von Frey) were determined on days 1, 3, and 7 after STZ 

exposure. On day 7, animals were sacrificed and brain punches (1.5 x 2mm) from the PAG and 

LDH were collected and immediately frozen. HR-MAS 1H-MRS of the intact tissue (~3 mg) 

was performed with standard CPMG techniques using a Bruker Avance 500 11.7T magnet and a 

custom LCModel used for absolute quantification of each neurochemical. Five of the 10 STZ 

animals demonstrated profound diabetic symptoms. By day 7, STZ-treated animals exhibited a 

15% decrease in hindpaw withdraw threshold and a 22% reduction in LMA compared to 

controls. In the PAG and LDH, MR-visible lactate and creatine were increased (p




Title: Epidural cells express S100B and NeuN in patients with low back pain 

Authors: *S. S. PARHIZGAR 1 , J. E. HEAVNER 1 , H. A. BOSSCHER 1 , C. SAMATHANAM 2 , 

P. J. SYAPIN 3 , J. J. GIORDANO 4 , M. V. BOSWELL 1 ; 

1 Anesthesiol., 2 Pathology, 3 Pharmacol. and Neurosci., Texas Tech. Univ, HSC, Lubbock, TX; 

4 Med., Georgetown Univ. Med. Ctr., Washington, DC 

Abstract: Treatments for back pain are often ineffective or effective for only a short time. 

Studies in rats have shown that astroglial and neuronal cells are involved in pain pathologies. 

The hypothesis of this study is that astroglial and neuronal cells are involved in low back pain. 

This study utilized epidural cells collected from patients undergoing routine diagnostic 

epiduroscopy for back pain in order to understand the pathology of back pain and/or 

radiculopathy. Human epidural cells were incubated in Dulbecco's Modified Eagle's Medium in 

the presence of brain derived neurotrophic factor, nerve growth factor, or ciliary neurotrophic 

factors in the presence of fetal bovine serum in vitro. Epidural cells from 5 patients that grew to 

confluence in fetal bovine serum alone were immunostained for S100B, an astroglial cell marker, 

and NeuN, a marker for neuronal nuclei, on glass slides. Epidural cells on thin preparations were 

stained for S100B, CD34 (endothelial), smooth muscle actin, and CD68 (macrophage) cell 

markers. Growth factors did not promote growth of epidural cells. However, fetal bovine serum 

alone facilitated the growth of epidural cells. Epidural cells on thin preparations showed staining 

for S100B and CD68 but not for CD38 and smooth muscle actin. Epidural cells showed staining 

for S100B and NeuN on glass slides. Thus, epidural cells may have astroglial and neuronal cell 

origins, which has not been shown before. These cells may be involved in pain pathology of low 

back pain. 

Disclosures: S.S. Parhizgar, None; J.E. Heavner, None; H.A. Bosscher, None; C. 

Samathanam, None; P.J. Syapin, None; J.J. Giordano, None; M.V. Boswell, None. 

Poster 




Topic: D.08.j. Pain models

Support: DFG KFO 107 

Kompetenzzentrum Schmerz BW 

Title: Nerve growth factor causes sustained nociceptor sensitization in human skin 

Authors: R. RUKWIED 1 , A. MEYER 2 , M. SCHLEY 1 , O. KLUSCHINA 1 , *M. SCHMELZ 1 ; 

1 Dept Anesthesiol, Univ. Heidelberg, Mannheim, Germany; 2 CRS-Mannheim, Mannheim, 

Germany 

Abstract: We explored the sensitizing effect of nerve growth factor NGF on C-nociceptor 

activation in human skin as measured psychophysically to thermal and mechanical stimuli and 

indirectly by electrically induced axon reflex area. 

1 - 3 - 7 - 21 - 49 days after intradermal injection of 1µg NGF to 16 subjects the heat and cold 

pain thresholds were assessed using a contact thermode (9x9mm). Mechanical impact stimuli 

were delivered at 4 - 7 - 10m/s using a compressed air accelerated plastic bullet impact 

stimulator and pain sensation recorded on a visual analogue scale. Axon reflex erythema was 

induced by electrical stimuli (1.25 - 2.5 - 5mA, 1Hz, 2min) and recorded by Laser Doppler 

Imaging. Areas of mechanical and heat sensitization were also assessed. 

NGF injections did not cause any acute pain or flare responses. Moreover no spontaneous pain or 

local skin response was induced during the entire observation period. In comparison to NaCl 

injection a significant and sub-acute sensitization to heat (p




Support: NINDS grants NS 14627 

NS 47113 

NS 48499 

Title: A behavioral model of episodic cranial pain in mice conditions a place aversion 

Authors: *A. H. AHN 1 , E. MARTIN 2 , A. I. BASBAUM 3 ; 

1 Neurol, 2 Anat., 3 Anatomy, Physiol. and WM Keck Ctr. for Integrative Neurosci., Univ. 

California San Francisco, San Francisco, CA 

Abstract: We have adapted a widely-used behavioral model of inflammation and tissue-injury 

pain, the formalin test of the hindpaw, to model behaviors associated with episodic cranial pain. 

In wild-type CD1 male mice, a midline intradermal injection of 10 microliters of dilute formalin, 

overlying the coronal suture (Bregma), produces stereotypical, localized grooming behaviors that 

develop in intensity over 10-20 min and resolve by approximately 45 min after injection. The 

time the animals spend in this directed grooming behavior is largely non-existent in animals 

injected with saline, but is prominent and dose-dependent, in a range of formalin concentrations 

from 0.5 to 3.0 %. Unlike the traditional formalin test of the hindpaw, or even formalin injection 

of the lip or vibrissal pad, the cranial formalin test does not show a clear pause in the nociceptive 

behaviors after the first 5 min after injection. With 2.0% formalin used for subsequent tests, the 

behaviors are not reduced by the pre-treatment of 5.0 mg/kg morphine, but are reduced by 10.0 

mg/kg morphine. Two hours after the injection of formalin, the stimulus elicits Fos expression, a 

cellular marker of neuronal activation, in the dorsal horn of the upper cervical spinal cord and 

trigeminal nucleus caudalis, as well as in the external parabrachial nuclei. Another marker of 

cellular activation, phosphorylated extracellular signal-related kinase (pERK) is induced in the 

central nucleus of the amygdala, suggesting that this stimulus is aversive. 

To functionally test whether this stimulus is a behaviorally aversive signal, we used a 

Conditioned Place Aversion (CPA) paradigm. We conditioned 12-14 mice with daily sessions of 

2% formalin injection in one room, paired with saline on alternate days. We used a balanced 

CPA design in which half of the animals were conditioned in each room of a two-chamber 

system, with half of the animals receiving formalin on the first day, and half with saline. Cranial 

formalin produced a dose-dependent aversion for the formalin-paired room. Furthermore, low 

doses of morphine, at 5.0 mg/kg, were sufficient to completely block CPA, suggesting that the 

aversive association can be blocked independently of the expression of nociceptive behavior. 

Disclosures: A.H. Ahn , None; A.I. Basbaum, None; E. Martin, None.

Poster 





Support: McMaster University Grant 

Title: Cantral Post-stroke pain: tactile and cold hypersensitivity in an animal model 

Authors: J. L. HENRY 1 , *K. YASHPAL 2 , S. BOUSEH 1 , *K. YASHPAL 1 , V. PITELKA 1 ; 

1 Behavioural neuroscience and psychiatry, McMaster Univ., Hamilton, ON, Canada; 2 Dept 

Physio & Pharmacol, Univ. of Western Ont, London, ON, Canada 

Abstract: Central post-stroke pain (CPSP) was defined as a thalamic syndrome 100 years ago. 

CPSP is a central neuropathic pain that is refractory to any present drug or invasive medical 

treatment and the approximate 8% of stroke victims are faced with a lifetime of pain (Cenntral 

Post-Stroke Pain. IASP Press. 281 pp, 2007; Central poststroke pain: an abstruse outcome. Pain 

Res & Management 13: 41-49, 2008). Progress has been slowed due to the lack of any inroads 

into understanding underlying mechanisms. To address this knowledge gap, we have developed 

an animal model of central post-stroke pain. A localized haemorrhagic stroke was induced by 

stereotaxic microinjection of collagenase unilaterally to interrupt the local vascular barrier in the 

region of the thalamus on one side in the anaesthetized male Sprague Dawley rat. Following 

recovery, animals were tested in various nociceptive and pain paradigms. Tactile sensitivity 

assessed with von Frey filaments and cold sensitivity assessed in the cold plate test indicated a 

site-dependent decrease beginning in both parameters 5 days after induction that persisted for up 

to 23 days. Plasma extravasation was assessed by Evans blue injection i.v. and indicated blood 

leakage at the site of the lesion from 12 hours to 7 days following injection of collagenase. 

Injection sites outside the sensory thalamus did not yield any sensory or motor deficits. These 

results suggest that this model may provide a vehicle for understanding mechanisms underlying 

the central neuropathic pain that is due to damage following haemorrhagic stroke. 

Supported by McMaster University. 

Disclosures: J.L. Henry, None; K. Yashpal , None; S. Bouseh, None; V. Pitelka, None. 

Poster





Support: Center for Pain Research 

Univ. Florida Office of Research 

Title: Effects of duloxetine (Cymbalta) on escape and lick/guard behaviors in normal rats 

Authors: *M. K. GREEN 1 , D. P. DEVINE 2 , C. J. VIERCK, Jr 3 , R. P. YEZIERSKI 1 ; 

1 Orthodontics, 2 Psychology, 3 Neurosci., Univ. Florida, Gainesville, FL 

Abstract: Antidepressants reduce behavioral responses in reflexive tests of nociception; 

however, little work has been done examining the effects of antidepressants in operant tests 

involving the cognitive, emotional, and motivational aspects of pain perception. Therefore, we 

examined the effects of a selective serotonin/norepinephrine reuptake inhibitor, duloxetine, on 

escape behaviors in an operant-based thermal escape task, and compared these effects to those in 

a reflex-based lick/guard test. Female Long Evans rats (n=15) were trained to escape hot 

(44.5 o C) and cold (10 o C) plates by entering a brightly lit compartment with a thermally neutral 

platform. Baseline measures were established for latency to escape and cumulative duration of 

escape. Rats were then injected with a single dose of duloxetine (3 or 10 mg/kg) or vehicle 

control and tested on 10 o C and 44.5 o C escape tasks. On separate weeks, rats were tested on a hot 

(44.5 o C) plate without the option for escape, and the latency and duration of licking and guarding 

behaviors were recorded. Rats were then injected with duloxetine (3 or 10mg/kg) or a vehicle 

control and tested on the 44.5 o C plate. In the 10 o C escape task, all rats spent more time on the 

thermally neutral platform after injection (vehicle and duloxetine) compared to their baseline 

measures. This was attributed to an injection induced stress hyperalgesia. In the 44.5 o C escape 

task, rats receiving 10mg/kg duloxetine spent significantly more time on the escape platform 

compared to baseline. This duloxetine-induced heat hyperalgesia was accompanied by 

significantly increased guarding behavior during reflex testing. The results suggest a dosedependent 

increase in thermal sensitivity following administration of the antidepressant 

duloxetine. 

Disclosures: M.K. Green, None; D.P. Devine, None; C.J. Vierck, None; R.P. Yezierski, 

None. 

Poster





Support: DA11471 (DAS) 

Grant-in-Aid Program of the Graduate School of the University of Minnesota 

Title: Local inhibition of 2-AG degradation reduces mechanical hyperalgesia in a murine model 

of bone cancer pain 

Authors: *I. KHASABOVA 1 , C. HARDING-ROSE 3 , A. CHANDIRAMANI 2 , D. SIMONE 3 , 

V. SEYBOLD 4 ; 

1 Dept Oral Sci., 2 Col. of Biol. Sci., Univ. Minnesota, Minneapolis, MN; 3 Diagn/Biol Sci., 4 Dept 

Neurosci., Univ. of Minnesota, Minneapolis, MN 

Abstract: Cannabinoid receptors and their endogenous ligands, anandamide (AEA) and 2arachidonoyl-glycerol 

(2-AG), constitute an endocannabinoid system that is involved in 

modulation of nociception. Recently we have shown peripheral anti-hyperalgesic effects of AEA 

and URB597, an inhibitor of AEA degradation, in a murine bone cancer model. Whether 2-AG 

attenuates cancer-related mechanical hyperalgesia is unknown. Tumors were generated in mice 

by injection of mouse NCTC clone 2472 fibrosarcoma cells unilaterally into and around the 

calcaneous bone. Mechanical hyperalgesia ( 70% frequency of withdrawal to a force of 3.7 mN 

applied to the plantar surface of the ipsilateral hindpaw) developed within a few days after 

implantation of tumor cells. Intraplantar injection of 2-AG (18 µg, ipl) into the tumor-bearing 

hindpaw reduced mechanical hyperalgesia and had no systemic effect. The anti-hyperalgesic 

effect of 2-AG was blocked by co-injection of the CB2 receptor antagonist AM630 (4 µg, ipl) 

suggesting that the inhibitory effect of 2-AG was mediated by CB2 receptors. We investigated 

whether URB602, an inhibitor of monoacylglycerol lipase (MGL) that blocks 2-AG hydrolysis, 

decreased tumor-related hyperalgesia. Injection of URB602 (20-70 µg, ipl) into the tumorbearing 

hindpaw produced a long-lasting dose-dependent reduction in mechanical hyperalgesia. 

Dose-related effects were observed for both the magnitude and duration of anti-hyperalgesia. 

When the largest dose of URB602 was used, recovery to baseline withdrawal frequency was 

delayed until 26 h after injection of drug. Importantly, the anti-hyperalgesic effect of URB602 

was mediated by activation of CB2 receptors because co-administration of the CB2 receptor 

antagonist AM630 (4 µg, ipl) with URB602 blocked the inhibitory effect of URB602. Together, 

these data indicate that the local turnover of 2-AG can be manipulated peripherally to promote 

antinociception thereby supporting the therapeutic potential of endocannabinoid-based 

approaches for the treatment of cancer pain. 

Disclosures: I. Khasabova , None; C. Harding-Rose, None; A. Chandiramani, None; D. 

Simone, None; V. Seybold, None.

Poster 





Title: Structural and functional mitochondrial pathology in fibromyalgia 

Authors: *A. D. KONSTANTINIDOU 1 , I. FOUZAS 2 , D. G. TSAMIS 1 , A. N. FLARIS 3 , D. I. 

DARDAVESIS 1 , M. K. N. FLARIS 1 ; 

1 Pharmacol, Protypon Neuromuscular Ctr., Thessaloniki 54635, Greece; 2 Transplant Surgery, 

3 Sch. of Med., Aristotelian Univ.of Thessaloniki, Thessaloniki 54635, Greece 

Abstract: Fibromyalgia is a disease whose very existence is still debated and its pathology 

remains elusive. A total of 39 patients (white, 5 men, aged 36-51, mean 39, and 34 women, aged 

17-78, mean 49) were evaluated in our outpatient neuromuscular center, previously examined by 

other physicians (including rheumatologists), with the main symptom of continuous pain of more 

than 3 months with a history of widespread pain and tender points, carrying a diagnosis of 

fibromyalgia. The patients had previously tried several medications including NSAIs and 

antidepressants without clear help. All underwent a standard detailed neurologicalneuromuscular 

examination and an extensive laboratory investigation including CBC, ESR, 

ANA, CPK and lactate. They all had a modified lactate/NH3 ischemia test done (Munsat T.L., 

Neurology,1970). Resting lactate levels averaged 11.73mg/dL (nl 4.5-22mg/dL) and 15' lactate 

levels averaged 17.4mg/dL. EMGs were performed and were abnormal: 31/38 with evidence of 

active ongoing disease, 7/38 with chronic involvement as evidenced by polyphasic potentials. 

The final test was a muscle biopsy, snap frozen in the OR. All biopsies were abnormal. Biopsies 

had varying degrees of mitochondrial pathology (39/39), with Ragged Red Fibres in 28/39 and 

Cytochrome Oxidase Negative Fibres in 38/39. Metabolic dysfunction was frequently noted in 

35/39 biopsies, with lipid accumulation (30/39) and glycogen accumulation (25/39). 

Inflammatory changes (MHC I positive in 29/39) were noted in 30/39 patients, with clear focal 

inflammation in 13/39 biopsies. Following these investigations, the patients were told of the 

results and placed on our standardized treatment (Q10, carnitine, B12, folic acid). When the 

inflammation was severe, prednisone (2/39) or methotrexate (1/39) was added. When it was less 

severe, a combination of quercetin, glucosamine and chondroitin with kernel olive oil was added 

(10/39). All patients related improvement within 3 months (some as early as 1 month), although 

significant improvement was seen a year later, coupled with improved scores in follow-up 

dynamometry testing and reduced resting lactate levels. Follow up EMGs were done in 4 patients 

and all improved with 3 out of 4 rendered inactive. It appears that fibromyalgia, at least some 

forms of it, are related to mitochondrial/ metabolic /inflammatory muscle pathology and

functional metabolic impairment amenable to relatively safe treatment protocols, with substantial 

relief for the patient. 

Disclosures: A.D. Konstantinidou, None; A.N. Flaris, None; M.K.N. Flaris, None; D.G. 

Tsamis, None; D.I. Dardavesis, None; I. Fouzas, None. 

Poster 





Support: National Institutes of Health 1R01DA023593 

Title: Endothelin-1 exposure sex-dependently sensitizes rodents to endothelin-1 induced 

nociception 

Authors: *A. D. MCKELVY, T. E. HAYMOND, S. M. SWEITZER; 

Dept Pharmacol, Physiol & Neurosci, Univ. South Carolina, Sch. Med., Columbia, SC 

Abstract: A little over 10 years ago a series of clinical studies were published showing that 

untreated pain in the neonatal period (circumcision in male infants) enhanced subsequent pain 

responses in infancy and childhood. The mechanisms underlying this sensitization remain 

unknown. Endothelin-1, an endogenous protein released in response to injury and disease, can be 

used to model an acute surgical-like pain early in postnatal development. We hypothesize that 

exposure to endothelin-1 early in development will result in sex-dependent change in endothelin- 

1-induced nociception later in development. To model acute pain early in infancy, postnatal day 

7 male and female rats were administered intraplantar saline or endothelin-1 (3.3 nmol) followed 

by a challenge with endothelin-1 on postnatal day 11 (equivalent to a young child). Exposure to 

endothelin-1 on postnatal day 7 (infancy) sex-dependently shifted the dose-response curve for 

endothelin-1 induced nociception at postnatal day 11. Exposure to endothelin-1 in infancy 

sensitized males, as shown by a left-ward shift in the dose-response curve to endothelin-1 on 

postnatal day 11. In contrast, exposure to endothelin-1 in infancy de-sensitized females, as 

shown by a right-ward shift in the dose-response curve to endothelin-1 on postnatal day 11. In 

addition, the sensitization in male animals could be prevented by treatment with an androgen 

receptor antagonist at the time of first exposure to endothelin-1. Sex-dependent changes in 

endothelin-1 induced nociception on postnatal day 11 were accompanied by a down-regulation 

and an up-regulation in endothelin B expression in the skin of postnatal day 11 males and 

female, respectively. These results indicate that sensitization to nociception is sex-dependent.

These results highlight the importance of the endothelin system in modulating long term 

nociception following acute injury. Understanding the role of the endothelin B receptor in pain 

sensitization and de-sensitization is a critical first step in determining whether the endothelin 

system is a viable therapeutic target to minimize pain and prevent pain-induced sensitization in 

infants and children. 

Disclosures: A.D. McKelvy , None; T.E. Haymond, None; S.M. Sweitzer, None. 

Poster 





Title: Opioid, barbiturate, and capsaicin effects on rat respiration using unrestrained whole-body 

plethysmography 

Authors: *D. M. CAIN 1 , D. MENARD 2 ; 

1 Physiol., Algos Therapeut. Inc, St Paul, MN; 2 Algos Therapeut., Saint Paul, MN 

Abstract: The efficacy and qualification of analgesic compounds in animals relies heavily on 

conventional methods of behavioral pharmacology. Unfortunately, these methods are 

characterized by frequent variability of diverse origin. There is a need for increased application 

of quantitative physiological biometrics to supplement behavioral evaluations. 

With this goal in mind, we conducted plethysmography studies using male Sprague Dawley rats 

(283-440g) to observe the effects of an opioid (fentanyl), a barbiturate (pentobarbital), and 

capsaicin (evoked pain) on Penh values, tidal volume, and breathing frequency. Penh values 

reflect the elongated pauses due to bronchial congestion or respiratory distress. Mean values for 

each parameter were obtained at 5 or 10 minute intervals for at least two hours. Fentanyl (130 

κg/kg, s.c.) significantly increased Penh values and decreased both breathing frequency and tidal 

volume for approximately one hour compared to naïve (no treatment) rats (ANOVA, p < 0.0001, 

Dunnett‟s Multiple Comparison Test). Naloxone (1 mg/kg) administered i.p. prior to fentanyl 

blocked the opioid effect. 

Pentobarbital affected respiratory parameters dose-dependently at 100 and 50 mg/kg, i.p. but not 

at 25 mg/kg. Mean Penh values were significantly elevated while tidal volume and breathing 

frequency were decreased compared to values from naïve animals. The pentobarbital effect 

lasted 60-80 minutes for the 50 mg/kg dose and for over two hours with the 100 mg/kg dose. 

To determine whether respiratory endpoints can provide a broader evaluation of analgesic 

compounds, we obtained plethysmographic results from rats in which either chronic or acute

pain had been induced. Respiratory parameters in hyperalgesic rats (n = 8) having undergone 

spinal nerve ligation showed no change compared to naïve animals. However, injection of 

capsaicin (s.c.) into the plantar surface of the hindpaw increased respiratory frequency 

significantly compared to naive animals. After capsaicin injection (100 κg, s.c.) the significant 

increase lasted for approximately 60 minutes (ANOVA, p = 0.0427, Dunnett's Multiple 

Comparison Test). 

Our initial results encourage further studies to examine correlations between various pain states 

and respiratory response parameters. Achieving predictive quality by refining the application of 

such biometrics will substantially enhance the reliability of the process by which analgesic 

compounds are evaluated. 

Disclosures: D.M. Cain , None; D. Menard, None. 

Poster 





Title: Neuropathic pain and histochemical changes of the trigeminal subnuclei caudalis in facial 

cancer model of rats 

Authors: N. HARANO 1 , *O. NAKANISHI 3 , K. ONO 4 , K. INENAGA 2 ; 

1 Dent. anesthesiology, 2 Pyhsiology, Kyusyu Dent. Col., Kitakyusyu, Japan; 3 Kyushu Dent. Col., 

2-6-1 Manazuru, Fukuoka 803, Japan; 4 Physiol., Kyushu Dent. Col., Kitakyushu, Japan 

Abstract: It is well known that patients with orofacial cancer suffer from cancer-induced 

pain.To understand m- 

echanisms of pain by facial cancer ,we have recently created a rat facial cancer model by 

inoculation 

with Walker carcinosarcoma256B-cells into the right vibrissal pad.The aim of the present study 

was to 

investigate changes of pain-related behavior and ingestive behavior in the model,along with c- 

Fos ex- 

pression in the trigeminal subnuclei caudalis which is a center of facial pain sensation,by using 

both 

behavior and immunohistochemical techniques.Mechanical allodynia and thermal hyperalgesia 

deve- 

loped on the inoculated facial side on days 4 and 7 post-inoculation.After day 10,the sensitivities

of the 

rats to mechanical and thermal stimuli were reduced,and there were physical difficulties in 

ingestion. 

Furthermore,the periods of facial grooming were prolonged post-inoculation,as compared with 

those in the sham group.In the trigeminal subnuclei caudalis,the level of c-Fos immunoreactivity 

of the ips- 

ilateral side was increased significantly on days 4,7 and 10 post-inoculation,as compared with 

those of 

the contlateral side and in the sham group.However,substance and calcitonin gene-related 

peptide levels in the trigeminal subnuclei caudalis were not changed in the facial cancer model 

whereas they 

were increased in facial inflammation model of rat.These results suggest that neuropathic pains 

in the 

facial cancer model are probably caused by different pathway of inflammation-induced. 

Disclosures: N. Harano, None; O. Nakanishi, None; K. Ono, None; K. Inenaga, None. 

Poster 





Support: GACR 305/07/0242 

RG 0021620816 

GACR 305/06/1188 

Title: Age-related changes of nociceptive threshold in rats 

Authors: *S. VACULIN, M. FRANEK; 

Dpt of Physiol., UK,3th Fac. of Med., Praha, Czech Republic 

Abstract: The effect of age on pain thresholds has not been fully explained in both human and 

animal research. Several studies described pain threshold increase in older adults compared to 

young adults in men, however, reduction of noxious inhibitory system was revealed during 

aging. The aim of the present study was to determine nociceptive threshold in rats during their 

lifetime. Nociceptive thresholds were evaluated using paw withdrawal latency to thermal and

mechanical stimulation, and tail withdrawal latency to thermal stimulation. The thresholds were 

evaluated on postnatal day 7, 16, 23, and at the age of 1, 2, 3, 6, 9, 12, 15 and 18 months in 

forelimbs, hinds and tail. As far as thermal stimulation is concerned, we could observe 

oscillation of withdrawal latencies. Nociceptive thresholds were lowest at PD 7 and then 

increased to maximum at weaning period. Since the age of 1 month the thresholds remained 

unchanged but with two peaks of decrease - at the age of 2 and 9 months. The oscillations were 

expressed most in tail withdrawal latency and least in forelimb withdrawal latency. Comparing 

fore and hind limb withdrawal latency it was found that the latency was shorter in forelimbs 

since weaning. No increase of nociceptive threshold was seen in old rats. Concerning mechanical 

stimulation, withdrawal pressure increased until weaning period and remained unchanged 

afterwards with decrease in the oldest groups (at the age of 15 and 18 months). It is suggested 

that nociceptive threshold depends on age until the age of 1 month, while in adult animals the 

withdrawal latency to thermal stimulation depends rather on external factors like season, air 

temperature and/or humidity. In the oldest groups moderate decrease of withdrawal pressure 

suggests relative importance of inhibitory pain system deficit. 

Disclosures: S. Vaculin, None; M. Franek, None. 

Poster 





Title: A retrospective study assessing the quality of reporting materials and methods in the 

preclinical pain primary literature 

Authors: *J. M. TALL, J. BACKO; 

Biol. Sci., Youngstown State Univ., Youngstown, OH 

Abstract: Numerous studies have shown that external factors, such as housing conditions and 

diet, affect data collected from laboratory animals. To promote consistency among laboratories, 

it is essential that all components of the animal treatment, laboratory environment, and animal 

care facility be clearly reported when publishing the results from preclinical investigations. This 

inquiry was conducted to assess the quality of reporting materials and methods in the preclinical 

primary literature by conducting a retrospective study. Research databases used to search for the 

journal articles included ISI Web of Knowledge, Medline, and Biological Abstracts. Within 

these databases, the search was limited to peer-reviewed articles, written in English, and 

published in 2007. Inclusion criteria included the words "pain" AND "rats" AND "behavior" in

all fields. All search results were imported into EndNote, and duplicate records were omitted. A 

total of 250 manuscripts met the implemented search criteria. Data analysis suggested that some 

elements, such as adhering to the proper care and use guidelines set by the Institutional Animal 

Care and Use Committee, were commonly stated in the methods section of most studies. Other 

important aspects however, such as experimenter-blinding, rat diet, and the number of rats 

housed per cage, were not consistently reported. A variety of factors affect laboratory animals, 

and more precise reporting of the materials and methods utilized in preclinical investigations will 

improve the reproducibility of data among laboratories. 

Disclosures: J.M. Tall , None; J. Backo, None. 

Poster 






Title: Temporal analysis of nocifensive reflex or operant behaviors during prolonged thermal 

stimulation in the rat 

Authors: *R. H. KLINE IV 1 , J. MCDANIEL 2 , L. L. LEMONS 3 , K. CHATTERJEE 6 , S. 

DATTA 4 , M. T. HARVEY 7 , C. H. KENNEDY 5 , R. G. WILEY 8 ; 

1 Neurol, 2 Special Educ., 3 Ctr. for Cognitive and Intergrative Neurosci., 4 Anesthesiol., 5 Special 

Education, Pediatrics, Vanderbilt Univ., Nashville, TN; 6 Neurol., VA TVHS, Nashville, TN; 

7 Psychology, Florida Inst. of Technol., Melbourne, FL; 8 Neurology, Pharmacol., VA TVHS, 

Vanderbilt Univ., Nashville, TN 

Abstract: Contact thermal stimulation has been widely used in psychophysical and 

neurophysiological studies of nociception, most often measuring the length of time taken to 

produce innate reflex withdrawal responses that can be seen in decerebrate rats. Typically, 

thermal withdrawal testing protocols measure the first response latency and use temperatures of 

50° C, or more. These stimulus temperatures preferentially activate A-delta thermal nociceptors. 

These phasic, high intensity spinal and bulbospinal reflex tests differ in significant ways from the 

persistent pain that is so important clinically. Three approaches to address these issues are: 1 - 

use of lower intensity stimuli that preferentially activate C nociceptors, 2 - use of sustained 

exposure to the stimuli, and 3 - use of responses that require cerebral processing. In the present 

study, we sought to characterize the temporal characteristics of pain-related behaviors during

periods of prolonged thermal nociceptive stimulation in un-restrained rats with and without the 

option to escape a thermal plate (e.g. escape task and thermal place preference task). Hindpaw 

nocifensive reflex responses to 44-45° C were compared to operant escape or thermal preference 

after: 1- 38° C pre-warming (shift from lick/guard to operant escape responding), 2- 0.5 mg/kg, 

s.c. morphine (strong operant effect), 3- 0.8 mg/kg, s.c. naloxone (strong operant effect) , 4- 48 

hr of sleep deprivation (operant responses revealed sleep deprivation-induced hyperalgesia and 

age differences), 5- lumbar i.t. injection of NPY-sap + bilateral hindpaw CFA (operant superior 

in showing analgesic effect of toxin), and 6- bilateral chronic constriction injury of the sciatic 

nerve (both reflex and operants strongly altered by lesion). Differential effects between reflex 

and operant responses were found. Sequential temporal analysis of nocifensive behavior more 

clearly reveals the differences between reflex and operant responding and provides provides 

insight into endogenous modulation of nociception. The results illustrate the usefulness of 

thermal plate testing protocols that include operant responses and time-course analysis of 

responses to prolonged activation of C thermal nociceptors. Temporal analysis of responses to 

prolonged, low intensity thermal stimulation may provide valuable new and clinically relevant 

information. 

Disclosures: R.H. Kline IV , None; J. McDaniel, None; L.L. Lemons, None; K. Chatterjee, 

None; S. Datta, None; M.T. Harvey, None; C.H. Kennedy, None; R.G. Wiley, Advanced 

Targeting Systems, F. Consultant/Advisory Board. 

Poster 

268. Neuropathic Pain Mechanisms: Ion Channels 





Title: Sensitivity of Ica to CaMKII blockers after axotomy of rat DRG neurona 

Authors: *J. MCCALLUM 1 , H.-E. WU 2 , Q. HOGAN 2,3 ; 

1 Dept Anesthesiol, Med. Coll of WI, Milwaukee, WI; 2 Anesthesiol., Med. Col. of Wisconsin, 

Milwaukee, WI; 3 Anesthesiol., Clement J. Zablocki VA Med. Ctr., Milwaukee, WI 

Abstract: Neuropathic pain is a complex chronic pain state following nerve injury. In animal 

models of neuropathic pain, studies have shown that decreased Ca 2+ flux after nerve ligation 

contributes to excitability by prolonging action potential duration or increasing firing 

frequency.[1, 2] We have found no evidence for altered channel conductance or expression, so

we are examining channel modulation. Ca 2+ /calmodulin (CaM)-dependent protein kinase II 

(CaMKII) is abundant in the dorsal root ganglion (DRG), and is a modulator of Ca 2+ channels 

and downstream effectors of Ca 2+ . We therefore examined CaMKII regulation of Ca 2+ after 

neuronal injury using patch clamp recordings from acutely dissociated neurons to measure ICa in 

the presence of the CaMKII blocker KN-93 (2 µM) or the inactive analog KN-92 (2-5 µM), in 

axotomized L5 neurons after spinal nerve ligation (SNL), compared to neurons from nonoperated 

controls. Internal solutions included 11 mM EGTA to clamp [Ca 2+ ]c, and external 

solutions contained 2 mM Ba 2+ as charge carrier. 100 ms step pulses to maximal ICa were applied 

every 10 s from a HP of -100 mV. KN-93 reduced ICa in control neurons by 42.8±3.8%, while ICa 

in axotomized neurons was reduced by 13.8±11.8% (p < 0.02 vs control) by KN-93. KN-92 had 

minimal effects in either population. These results indicate that CaMKII facilitates Ca 2+ entry at 

the plasmalemmal level, and this facilitation is reduced after neuropathic injury. 

1. 

Sapunar, D., et al., Distinct membrane effects of spinal nerve ligation on injured and adjacent 

dorsal root ganglion neurons in rats. Anesthesiology, 2005. 103(2): p. 360-76. 

2. 

McCallum, J.B., et al., Painful Peripheral Nerve Injury Decreases Calcium Current in 

Axotomized Sensory Neurons. Anesthesiology, 2006. 105(1): p. 160-168. 

Disclosures: J. McCallum , None; H. Wu, None; Q. Hogan, None. 

Poster 




Topic: D.08.l. Neuropathic pain: Mechanisms 

Support: Grants-in-Aid for Scientific Research (H.F. and M.Y.) from Japanese MEXT 

Title: In vivo patch-clamp analysis of nociceptive synaptic transmission through calciumpermeable 

AMPA receptors in substantia gelatinosa neurons of the rat spinal dorsal horn 

following inflammation 

Authors: *H. FURUE, M. YOSHIMURA; 

Integrative Physiology, Grad Sch. Med. Sci., Kyushu Univ., Fukuoka, Japan 

Abstract: Recent studies have suggested that that plastic changes in nociceptive synaptic 

transmission in the spinal dorsal horn may contribute to underlying mechanisms for the 

development of hyperalgesia or allodynia following tissue injury or nerve damage. In this study,

we examined whether the functional change of AMPA-type glutamate receptors is involved in 

the plastic changes of the spinal nociceptive synaptic transmission in vivo following 

inflammation. In inflamed rats 24 h after intraplantar injection of complete Freund‟s adjuvant, 

whole-cell recordings were obtained from substantia gelatinosa (SG, lamina II of the spinal cord) 

neurons with resting membrane potentials of about -60 mV. In the presence of bicuculline, 

strychnine and D-APV, all neurons of inflamed rats tested exhibited spontaneous EPSCs at a 

holding potential of -70 mV. Large amplitudes of EPSCs were frequently observed in SG 

neurons that had receptive fields located on the inflamed skin on the hind paw or lower leg 

compared with the amplitudes of SG neurons that had receptive fields located on the uninflamed 

skin on the upper leg or thigh. The spontaneous events were completely inhibited by CNQX and 

GYKI52466. Pinch stimulation applied to the skin elicited a barrage of EPSCs with large 

amplitudes. Whereas the I-V relationship of the pinch-evoked current charges in the SG neurons 

that had receptive fields located on the uninflamed skin was linear, that in the SG neurons having 

receptive fields located on the inflamed skin was inwardly rectified. The average rectification 

index (RI) of pinch-evoked EPSCs in the SG neurons received from inflamed skin was lower 

than that from the uninflamed skin. Application of PhTx, a Ca 2+ -permeable AMPA receptore 

(CP-AMPAR) antagonist, markedly inhibited the pinch-evoked responses in SG neurons that had 

receptive fields located on the inflamed skin, though EPSCs with large amplitudes were still 

elicited at lower frequencies than those in the control in the absence of PhTX, thus suggesting 

that the excitatory synaptic inputs in single SG neurons were mostly mediated by CP-AMPAR. 

The amplitude of current charges of the pinch-evoked EPSCs was significantly decreased in the 

presence of PhTx. Even at the same spinal level in the same rats, however, SG neurons with 

receptive fields located on the uninflamed skin were insensitive to PhTX. These results suggest 

that CP-AMPARs in the superficial spinal dorsal horn received from inflamed skin have an 

important role in the induction of central sensitization following inflammation. 

Disclosures: H. Furue , None; M. Yoshimura, None. 

Poster 





Title: Behavioral and electrophysiological analyses of spinal nociceptive transmiss ion in a 

murine model of cancer-induced bone pain 

Authors: *Y. YANAGISAWA 1,2 , H. FURUE 2 , T. KAWAMATA 3 , D. UTA 2 , A. NAMIKI 3 , Y. 

IWAMOTO 1 , M. YOSHIMURA 2 ;

1 Orthopaedic surgery, Kyushu Univ., Fukuoka, Japan; 2 Dept. of Integrative Physiol., Grad. Sch. 

of Med. Sciences, Kyushu Univ., Fukuoka, Japan; 3 Dept. of Anesthesiol., Sapporo Med. 

University, Sch. of Med., Sapporo, Japan 

Abstract: Cancer-induced bone pain is a major clinical problem for which current treatments 

lack satisfactory efficiency. We investigated pain behavioral changes and plasticity of neural 

excitatory synaptic input to the superficial dorsal horn in a murine model of cancer-induced bone 

pain. 

A mouse model of bone cancer pain was produced by the implantation of osteosarcoma cells 

(NCTC2472, 10×10 5 in 20κL of medium) into the mouse femur. The first of all, bone histology 

and pain-related behaviors were assessed on day 7 and 14 after sarcoma injection. Another 

behavioral changes were tested by assessing the development of mechanical allodynia and 

thermal allodynia at hind paw only in the model mice that had apparent pain-related behaviors. 

Plantar mechanical and thermal allodynia was assessed by von Frey test and plantar test. When 

there were significant behavioral alterations in the model mice, compared to naïve, 

electrophysiological experiments were carried out on day 15-21 after surgery. Voltage patchclamp 

recordings from superficial dorsal horn neurons of the slices at lumbar spinal cord level 

(L3-5) were performed. 

The behavioral studies showed that mechanical, but not thermal, pain threshold of hind paw, 

over the lesion level, was significantly increased with hind paw lifting and flinching which were 

typical behavioral signs of spontaneous pain. Electrophysiological recordings demonstrated 

significant increase of both frequency and amplitude of spontaneous excitatory postsynaptic 

current (sEPSC), respectively, by 157% and 118%. 

These results suggest that central sensitization was occurred due to the increased excitatory 

synaptic input by cancer-related spontaneous pain stimulation, result in allodynia and 

hypersensitivility. The threshold and amplitude of root-evoked EPSCs should be tested for the 

next step. 

Disclosures: Y. Yanagisawa , None; H. Furue, None; T. Kawamata, None; D. Uta, None; A. 

Namiki, None; Y. Iwamoto, None; M. Yoshimura, None. 

Poster 



Program#/Poster#: 268.4/GG1 


Title: Anti-allodynic effect of the HCN antagonist, ZD7288, after nerve injury in HCN1-null 

mice

Authors: *W. A. ECKERT, M. MAHER, D. H. LEE, H. AO, T. BANKE, A. D. 

WICKENDEN, S. R. CHAPLAN; 

Pain & Related Disorders, Johnson & Johnson PR&D LLC, San Diego, CA 

Abstract: Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN1-3 and 

possibly 4) are each expressed on primary afferent neurons, with HCN1 and HCN2 being most 

abundant. Previously, we reported that blockade of HCN channels with ZD7288 (10 mg/kg, i.p.) 

reversed tactile allodynia and ectopic activity of dorsal root ganglion neurons following 

peripheral nerve injury in the rat (Chaplan et al., 2003 J Neurosci). However, the specific 

contributions of HCN channel subtypes to neuropathic pain remain unclear. In order to 

understand the contribution of HCN1, we evaluated the phenotype of HCN1 null mice. The 

mouse HCN1 gene was disrupted by targeted deletion of a region in the S1-S2 linker. Mice were 

generally healthy and viable, but exhibited a mild gait abnormality. Heart rate was reduced by 

approximately 12 - 20% compared to wild type controls. By rt-PCR, HCN1 was absent and there 

was no obvious upregulation of HCN2 - 4 expression in DRG from KO animals. To assess 

development of neuropathic pain, we evaluated tactile thresholds in wildtype and HCN1-null 

mice before and following injury using the mouse spared tibial nerve injury model of 

neuropathic pain (Shields et al., 2003 J Pain). Both HCN1-null and wildtype mice developed 

tactile allodynia to a similar degree from 2 days to 21 weeks following injury. ZD7288 

completely reversed tactile allodynia in the HCN1-null mice. From these data, we conclude that 

HCN1 plays a role in motor coordination and may contribute to sinoatrial pacemaker function in 

mice. In addition, activity of HCN2, 3 or 4 alone or in combination contributes to the tactile 

allodynia observed in HCN1 KO mice. 

Disclosures: W.A. Eckert, Johnson & Johnson, Pharma ceutical R. & D, L.L.C., A. 

Employment (full or part-time); M. Maher, Johnson & Johnson PR&D LLC, A. Employment 

(full or part-time); D.H. Lee, Johnson & Johnson PR&D LLC, A. Employment (full or parttime); 

H. Ao, Johnson & Johnson PR&D LLC, A. Employment (full or part-time); T. Banke, 

Johson & Johnson PR&D LLC, A. Employment (full or part-time); A.D. Wickenden, Johnson & 

Johnson PR&D LLC, A. Employment (full or part-time); S.R. Chaplan, Johnson & Johnson 

PR&D LLC, A. Employment (full or part-time). 

Poster 





Support: NIH Grant R01 NS040538-07

Title: Changes in TRPV1 function in A-fiber neurons after nerve injury 

Authors: *D. VILCEANU, C. L. STUCKY; 

Dept Cell Biol, Neurobiol & An, Med. Col. Wisconsin, Milwaukee, WI 

Abstract: Clinical manifestations of peripheral nerve injury include hypersensitivity to heat and 

mechanical stimuli, but the underlying molecular mechanisms are not yet clear. The TRPV1 

channel is an important player that contributes to both heat hypersensitivity and pain sensation as 

it is activated by noxious heat ( >42°C) and by many endogenous inflammatory chemicals 

including protons. TRPV1 is normally expressed mainly in small-diameter ( 26 um) which likely have A fiber 

axons. We assessed heat responsiveness by applying a fast ramp of heated buffer (24 to 50°C in 

3 sec). The criterion for a heat response was a rapid inward current > 100 pA that exhibited a 

clear heat threshold. 

Among medium-large neurons from naive controls, only 9% (4/45) of L3/L4 DRG neurons 

responded to heat, whereas in sham-operated mice, 15.4% (8/52) neurons responded to heat. 

After SNL injury, the percentage of heat responsive neurons from the injured L4 ganglia 

decreased to just 2% (1/48, p = 0.03 compared with sham neurons). Surprisingly however, the 

percentage of heat-responsive neurons from the adjacent L3 DRG was only 6% (3/49) and not 

increased compared to either the naive or sham controls. All heat-evoked responses appeared to 

be entirely due to TRPV1 activation because they had an average heat threshold of 44±0.2°C. 

These data indicate that spinal nerve ligation in mice does not alter TRPV1 function in A-fiber 

type neurons adjacent to the injury and thus, TRPV1 does not likely contribute to nerve injuryinduced 

evoked heat hyperalgesia. 

Disclosures: D. Vilceanu, None; C.L. Stucky, None. 

Poster 



Program#/Poster#: 268.6/GG3


Support: NIH R01 NS 40471-04 

NIH K08 DE14571-05 

Title: TRPV1 and TRPA1 contribute to thermal hyperalgesia and mechanical allodynia in 

platinum drug-induced neuropathy in mice 

Authors: *A. J. BIEBER, L. E. TA, P. A. LOW, A. J. WINDEBANK; 

Mol. Neurosci. Program, and Dept. of Neurol., Mayo Clin. Col. of Med., Rochester, MN 

Abstract: Cisplatin is primarily used for treatment of ovarian and testicular cancer. Oxaliplatin 

is the only effective treatment for metastatic colorectal cancer. Both are known to cause dose- 

related, cumulative toxic effects on the peripheral nervous system and 30-40% of cancer patients 

receiving these agents experience painful peripheral neuropathy. The mechanisms underlying 

painful platinum-induced neuropathy remain poorly understood. Previous studies demonstrated 

important roles for TRPV1, TRPM8, and TRPA1 in inflammation and injury-induced pain. In 

this study, using real-time, reverse transcriptase, polymerase chain reaction (RT-PCR), we 

analyzed the expression of TRPV1, TRPM8, and TRPA1 induced by cisplatin or oxaliplatin in 

vitro and in vivo. For in vitro studies, cultured E15 rat dorsal root ganglions (DRG) neurons were 

treated for up to 48 hours with cisplatin or oxaliplatin. For in vivo studies, trigeminal ganglia 

(TG) were isolated from mice treated with cis- or oxalplatin for three weeks. We showed that 

cis- and oxaliplatin-treated DRG neurons had significant increase in TRPV1, TRPA1, and 

TRPM8 mRNA expressions. TG neurons from cisplatin-treated mice had significant increase in 

TRPV1 and TRPA1 mRNA expressions. Furthermore, compared to the cisplatin-treated wildtype 

mice, cisplatin-treated TRPV1-null mice sustained mechanical allodynia but did not exhibit 

enhancement of thermally evoked responses. These results indicate that TRPV1 and TRPA1 

could contribute to the development of thermal hyperalgesia and mechanical allodynia following 

cisplatin-induced painful neuropathy. 

Disclosures: A.J. Bieber, None; L.E. Ta, None; P.A. Low, None; A.J. Windebank, None. 

Poster 




Topic: D.08.l. Neuropathic pain: Mechanisms


Erwin Schroedinger Fellowship, Austrian Science Fund 

Title: Hyperpolarization-activated current (Ih) contributes to excitability of primary sensory 

neurons in rats 

Authors: *Q. H. HOGAN, M. POROLI, M. RIGAUD; 

Dept Anesthesiol, Med. Coll of WI, Milwaukee, WI 

Abstract: In various excitable tissues, the hyperpolarization-activated, cyclic nucleotide-gated 

current (Ih) contributes to burst firing by depolarizing the membrane after a period of 

hyperpolarization. Alternatively, conductance through open channels Ih channels of the resting 

membrane may impede depolarization and excitability. Since primary sensory neurons of the 

dorsal root ganglion show both loss of Ih and elevated excitability after peripheral axonal injury 

(ref. 1), we examined the contribution of Ih to excitability of these neurons. A sharp electrode 

intracellular technique was used to record from neurons in nondissociated ganglia, in order to 

avoid potential artefacts due to tissue dissociation and cytosolic dialysis. Ih induced by 

hyperpolarizing voltage steps was completely blocked by ZD7288 (approximately 10µM), which 

concurrently eliminated the depolarizing sag of transmembrane potential during hyperpolarizing 

current injection. Ih was most prominent in rapidly conducting Aα/β neurons, in which ZD7288 

produced resting membrane hyperpolarization, slowed conduction velocity, prolonged action 

potential (AP) duration, and elevated input resistance. The rheobase current necessary to trigger 

an AP was elevated by ZD7288. Of 8 Aα/β neurons that fired repetitively during sustained 

depolarization, firing was inhibited by ZD7288 in 7, indicating an excitatory influence of Ih. 

Less Ih was evident in more slowly conducting Aδ neurons, resulting in diminished effects of 

ZD7288 on AP parameters. Of 6 repetitively firing Aδ neurons, ZD7288 inhibited firing in 5, 

and the peak frequency of AP transmission during tetanic bursts was diminished by ZD7288. 

Slowly conducting C-type neurons showed minimal Ih, and no effect of ZD7288 on excitability 

was seen. After spinal nerve ligation, axotomized neurons had less Ih compared to control 

neurons and showed minimal effects of ZD7288 application. We conclude that Ih supports 

sensory neuron excitability, and loss of Ih is not a factor contributing to increased neuronal 

excitability after peripheral axonal injury. 

Reference 1. Anesthesiology 103:360-76, 2005 

Disclosures: Q.H. Hogan , None; M. Poroli, None; M. Rigaud, None. 

Poster 





Title: Role of spinal NMDA NR2B receptors in neuropathic pain following peripheral nerve 

injury in rats 

Authors: Y. KIM 1 , H.-Y. CHO 1 , J. KIM 2 , H. HAN 1 , *Y. W. YOON 1 ; 

1 Dept Physiol, Korea Univ. Col. Med., Seoul, Republic of Korea; 2 Dept. of Physical Therapy, 

Korea Univ. Coll. Hlth. Sci., Seoul, Republic of Korea 

Abstract: Peripheral nerve injury may lead to chronic neuropathic pain, which has not been 

successfully managed. Neuropathic pain may result from abnormal peripheral nerve activity and 

persistent peripheral inputs to the spinal cord that leads to the alteration in synaptic activity and 

excitability of the dorsal horn cells. NMDA receptors are known to play an important role in pronociceptive 

sensitization. Blockade of NMDA receptors has shown to be effective to ameliorate 

neuropathic pain in the experimental and clinical setting. However, their use as an analgesic drug 

is restricted by unacceptable side effects. NR2B receptors are interesting because selective 

antagonists have been available and the localization of NR2B subunits is more restricted. The 

aim of this study was to explore the effects of NR2B antagonist on neuropathic pain and 

temporal profile of NR2B expression in the spinal cord following peripheral nerve injury. 

Neuropathic injury was made by tight ligation of L5 spinal nerve unilaterally in Sprague-Dawley 

rats under enflurane anesthesia. Paw withdrawal threshold to mechanical stimulation with a 

series of von Frey filaments was assessed. Ipsi- and contralateral L4 and L5 spinal cords were 

removed 3, 7, 10, 14 and 21 days after spinal nerve ligation to assess the level of NR2B receptor 

expression with western blot. To evaluate the analgesic efficacy of NR2B antagonists, ifenprodil 

and Ro25-6981 were injected intrathecally. 

Systemic (20 mg/kg) and intrathecal ifenprodil (100 nM) or intrathecal Ro25-6981 (200 nM) 

induced no significant attenuation of mechanical allodynia. From 3 days to 21 days after spinal 

nerve ligation, western blot showed a significant increase in density for NR2B receptor protein in 

the L5 spinal cord.These data suggest that NR2B receptors play little role in maintenance of 

mechanical allodynia after spinal nerve ligation and selective NR2B antagonists may be 

ineffective for controlling neuropathic pain in this condition. 

Disclosures: Y. Kim, None; Y.W. Yoon , None; H. Cho, None; H. Han, None; J. Kim, None. 

Poster 





Title: N-methyl-D-aspartate receptor-mediated synaptic transmission in neurons containing 

serotonin or GABA in the ventrolateral subdivision of the periaqueductal gray matter of the 

mouse 

Authors: N. FURUKAWA 1 , T. TAKASUSUKI 1 , *S. YAMAGUCHI 3 , M. MORITA 1 , T. 

TERASHIMA 1 , T. KITAJIMA 1 , M. MAEKAWA 2 , Y. HORI 2 ; 

1 Dept. of Anesthesiol., 2 Dept. of Physiol., Dokkyo Med. Univ., Mibu, Japan; 3 Dept Anesthesiol, 

Dokkyo Univ. Sch. Med., Mibu, Japan 

Abstract: The midbrain periaqueductal gray (PAG) performs a variety of functions, including 

the transmission of pain and analgesia, fear and anxiety, vocalization, and cardiovascular control. 

The PAG comprises the ascending pathway for pain transmission and receives afferents from 

nociceptive neurons in the spinal cord. It has been shown that N-methyl-D-aspartate (NMDA) 

receptors within the PAG are involved in the perception of tonic pain. The PAG is also involved 

in a descending pain inhibitory system. Electrical or chemical activation of the PAG inhibits 

nociceptive neurons in the dorsal horn of the spinal cord. This inhibition has been shown to be 

regulated by the GABAergic and serotonergic mechanisms. Serotonergic and GABAergic 

neurons and NMDA receptors have been identified in the PAG. However, the precise synaptic 

mechanisms underlying the nociceptive modulation in the PAG are not fully understood. In the 

present experiments, we made tight-seal whole-cell recordings from neurons in the ventrolateral 

subdivision of the PAG, and attempted to characterize NMDA receptor-mediated excitatory 

postsynaptic currents (EPSCs) in serotonergic and GABAergic neurons. Serotonergic and 

GABAergic neurons were identified by means of single-cell RT-PCR methods, which detected 

the expression of mRNA for tryptophan hydroxylase (TPH), serotonin synthesizing enzyme, and 

glutamate decarboxylase (GAD), GABA synthesizing enzyme. We also studied whether and how 

the properties of NMDA receptor-mediated synaptic transmission in the PAG are affected during 

the development of neuropathic pain induced by peripheral nerve injury. The ratio of the NMDA 

component to the non-NMDA component of EPSCs and the time decay course of the NMDA 

component of EPSCs in the non-serotonergic GABAergic neurons are significantly larger than 

those of non-serotonergic non-GABAergic neurons and those of serotonergic GABAergic 

neurons. We also found that the time decay course of the NMDA component of the nonserotonergic 

non-GABAergic neuron was significantly longer in nerve-ligated neuropathic mice 

than in sham-operated control mice. These results suggest that non-serotonergic GABAergic 

neurons might play a greater role in nociceptive transmission than other types of neurons in the 

PAG, and that the subunit composition of the NMDA receptor in the PAG changes during the 

development of neuropathic pain. 

Disclosures: N. Furukawa, None; T. Takasusuki, None; S. Yamaguchi , None; M. Morita, 

None; T. Terashima, None; T. Kitajima, None; M. Maekawa, None; Y. Hori, None. 

Poster




Topic: D.08.k. Inflammatory pain 

Support: Supported by Wellcome UK grant to SNL & GR 

Title: The distribution of the two-pore domain K + (K2P) channel TREK2 (KCNK10) in dorsal 

root ganglion (DRG) neurons after axotomy or skin inflammation 

Authors: *G. REID 1 , B. MARSH 2 , R. S. SHAH 2 , K. MORGAN 2 , R. PAREKH 2 , C. BERRY 2 , 

B. CARRUTHERS 2 , L. DJOUHRI 3,2 , S. N. LAWSON 2 ; 

1 Dept of Physiol., Univ. Col. Cork, Cork, Ireland; 2 Dept. of Physiol. and Pharmacol., Univ. of 

Bristol, Bristol, United Kingdom; 3 Dept. of Pharmacol. & Therapeut., Univ. of Liverpool, 

Liverpool, United Kingdom 

Abstract: Changes in membrane potential (Em) in DRG neurons may influence firing frequency 

in chronic pain states. Spontaneous firing occurs in some A-fibre neurons after axotomy, and in 

C and Aδ neurons during cutaneous inflammation (1). 

Altered expression of background (K2P) potassium channels could influence resting excitability 

and thus spontaneous firing. We have examined DRG expression of the K2P channel TREK2 

(KCNK10) in chronic pain models using both real time RT-PCR and ABC 

immunocytochemistry. We examined ipsilateral and contralateral L5 DRGs 1 week after L5 

spinal nerve axotomy (SNA; neuropathic pain model), and L5/L4 DRGs 1 and 4 days after 

intradermal injection of complete Freund‟s adjuvant in one hindpaw (CFA; chronic 

inflammatory pain model). For PCR, single DRGs were measured with GAPDH as reference 

gene. For immunocytochemistry, cytoplasmic staining of neuronal profiles containing nuclear 

sections was measured within small circles placed in the edge, mid, and juxta-nuclear regions of 

the cytoplasm. 

TREK2 mRNA expression did not differ between ipsi- and contralateral DRGs in any group, so 

ipsi- and contra-lateral data sets were combined. There was no detectable change in CFA day 1 

(n=4) or CFA day 4 (n=4) DRGs from normal (n=6). Axotomised DRGs showed higher 

expression than combined CFA DRGs (p500κm 2 ) was decreased throughout the cytoplasm in all treatments. In small 

(≤500κm 2 ) neurons staining was unchanged 1 day after CFA but significantly reduced 

throughout the cytoplasm 4 days after CFA. After axotomy, juxta-nuclear staining increased but 

edge staining decreased.

TREK2 is thus mainly expressed in small IB4 positive C nociceptors. Its expression alters in 

both large and small neurons in chronic pain models. Changes in protein but not mRNA 

expression may reflect post-transcriptional mechanisms. Increases in cytoplasmic protein near 

the nucleus in small neurons after axotomy may reflect increased mRNA production. 

1. Djouhri et al, J Neurosci 2006 26:1281-92. 

2. Fang et al, J Neurosci 2006 26:7281-92. 

Disclosures: G. Reid, None; B. Marsh, None; R.S. Shah, None; K. Morgan, None; R. Parekh, 

None; C. Berry, None; B. Carruthers, None; L. Djouhri, None; S.N. Lawson, None. 

Poster 





Support: Wellcome Trust UK grant to SNL 

Title: Intact L4 Aα/β nociceptive and non-nociceptive low threshold mechanoreceptive (LTM) 

DRG neurons show similar electrophysiological changes after L5 spinal nerve axotomy in rat in 

vivo 

Authors: *S. N. LAWSON 1 , L. DJOUHRI 2 ; 

1 Dept Phys&Pharm, Bristol Univ., Bristol, United Kingdom; 2 Dept. of Pharmacol. & Therapeut., 

Sch. of Biomed. sciences, Liverpool, United Kingdom 

Abstract: Partial nerve injury can lead to neuropathic pain. Spontaneous pain behaviour is 

related to spontaneous firing of the intact C- nociceptors (Djouhri et al., 2006) but other types of 

intact afferents intermingling with damaged afferents after partial nerve injury have not been 

studied. We examine whether they show electrophysiological changes that may contribute to 

neuropathic pain. 

We compared electrophysiological properties of L4/L5 DRG neurons in untreated rats with intact 

(L4) neurons in rats with SNA (L5 Spinal Nerve Axotomy) and modified SNA (mSNA), that is 

SNA plus inflammation-inducing chromic-gut loose-ligation of the L4 spinal nerve. Female rats 

(150-180g) were anaesthetised with pentobarbitone (60 mg kg -1 , i.p.), paralysed with 

pancuronium (0.5 mg/kg i.a.) and artificially ventilated.. Intracellular recordings of somatic 

action potentials (APs) evoked by dorsal root electrical stimulation were made from: a) normal 

L4/L5 DRG neurones in control rats and b) adjacent L4 neurons in mSNA and SNA rats. 

Neurones were classified as C, Aδ or Aα/β units from their dorsal root conduction velocities and

as non-nociceptive (LTM) or nociceptive units according to their responses to natural stimuli. 

1 week after nerve injury, intact L4 neurons showed electrophysiological changes. Although C 

nociceptors showed greater changes in mSNA than SNA rats (presumably due to greater 

inflammatory influences), changes in Aα/β units were generally greater in SNA rats, except 

action potential (AP) height/overshoot in nociceptors that was greater in mSNA rats. We 

therefore compared changes in Aα/β nociceptors and LTMs in SNA rats. Remarkably, these 

changes were similar in both groups. Furthermore, although numbers were too small for 

significance in all cases, the muscle spindle and different subgroups of cutaneous afferents (Field 

and G hair, slowly adapting and rapidly adapting LTMs) all showed similar changes. These 

included increased AP height and overshoot, and decreased AP duration and rise time, despite 

decreased maximum rates of AP rise and fall. 

These findings suggest that some global changes (perhaps in expression or properties of certain 

types of ion channel) occur in all rapidly conducting intact L4 afferents in in a partially injured 

nerve in SNA rats. Although there is no spontaneous firing in these A-fibres, our findings raise 

the question of a) whether these changes are associated with changed functional properties of 

these Aα/β neurons, and if so b) whether these changes occur in nociceptors and/or LTMs. 

Supported by The Wellcome Trust, UK. 

Disclosures: S.N. Lawson , Astrazeneca, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received); L. Djouhri, None. 

Poster 





Support: NINDS Grant KNS049420A 

Title: Morphological distribution of sulfonylurea receptor 1 (SUR1) in peripheral sensory 

neurons: the effect of nerve injury 

Authors: *V. ZOGA 1 , M.-Y. LIANG 2 , T. KAWANO 2 , G. GEMES 2 , H.-E. WU 2 , S. ABRAM 2 , 

Q. HOGAN 2 , C. SARANTOPOULOS 2 ; 

2 Anesthesiol., 1 MEDICAL COLL WISCONSIN, MIlwaukee, WI 

Abstract: KATP channels, containing the SUR1 subunit, have been identified in primary 

afferents 1 but details about their histological distribution remain unknown. Because KATP current 

is altered by nerve injury 2 , we examined the localization of the SUR1 subunit in primary

afferents, and its alteration by nerve injury that does, or does not result in hyperalgesia. 

Methods: Male rats subjected to spinal nerve ligation (SNL) or sham skin (SS) operation were 

tested for hyperalgesic behavior to distinguish animals with SNL-induced pain (SNLR) from 

non-responders (NR) 3 . Neurons from SS rats, were studied in comparison with SNLR or NR. 

Dorsal root ganglia, fixed in 4% paraformaldehyde (PFM) were cryoprotected in 30% sucrose, 

sectioned (12 µm), plated onto slides, blocked in 4% goat serum, and post-fixed in 4% PFM. 

Samples were incubated in polyclonal rabbit antibodies to SUR1 (1:50) (Santa Cruz), and labeled 

with goat anti-rabbit Texas Red secondary immunofluorescent antibody (1:500). Slides were 

examined at a Nikon E600 fluorescent microscope, images were digitized and stored for further 

analysis. 

Results: In most control (SS) neurons intense SUR1 staining was observed on plasmalemmal 

and nuclear membranes in a continuous pattern. Nuclear membranes stained more than 

plasmalemmal. Cytosol stained less intensely (in a pin-point pattern in some cells). Two patterns 

of axonal staining were present: 1) discrete, intense staining, of triangular or crescent shape, on 

distinct points along thicker axons; 2) diffuse, continuous, less intense staining on thinner axons. 

The same patterns of staining were observed on SNLR and NR neurons, without any obvious 

morphological differences from SS, although intensity of staining was weaker, and fluorescent 

points along the axons were fewer and linear in shape (versus crescent) in SNLR, but not in NR. 

Conclusion: The novel finding of SUR1 expression on peripheral sensory pathways indicates the 

presence of KATP channels on DRG neuronal somata, myelinated and non-myelinated axons. No 

qualitative or morphological changes were observed among intact or axotomized neurons, except 

a different pattern of staining on thick axons in SNLR. The presence of SUR1 after injury 

indicates a potential pharmacological target for control of excitability after nerve injury. 

References: 

1. McCallum JB, et al. 2007 SfN Meeting Abstract 509.9/PP12. 

2. Sarantopoulos C, et al. 2007 SfN Meeting Abstract 285.21/KK4 

3. Hogan Q, et al. Anesthesiology 2004; 101: 476-87 


Disclosures: V. Zoga , None; M. Liang, None; T. Kawano, None; G. Gemes, None; H. Wu, 

None; S. Abram, None; Q. Hogan, NIH NS42150 grant, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received); C. 

Sarantopoulos, NINDS Grant KNS049420A, B. Research Grant (principal investigator, 


Poster 






Title: Alterations of primary afferent neuronal subpopulations expressing SUR1 

immunofluorescence after spinal nerve ligation 

Authors: M.-Y. LIANG 1 , V. ZOGA 1 , *T. KAWANO 2 , G. GEMES 1 , J. MCCALLUM 1 , D. 

WEIHRAUCH 1 , Q. HOGAN 1 , C. SARANTOPOULOS 1 ; 

1 Anesthesiol., 2 Med. Col. Wisconsin, Milwaukee, WI 

Abstract: Primary afferents express Kir6.2/SUR1 channels1, conveying KATP current which 

diminishes in large neurons after painful-, but not after non-painful axotomy 2 . In order to 

distinguish if changes are associated with parallel changes in channel expression, we compared 

the distribution of SUR1 immunofluorescence among subpopulations of DRG neurons from rats 

with or without hyperalgesia after axotomy, in comparison to intact neurons. 

Methods: Male rats subjected to spinal nerve ligation (SNL) or sham skin (SS) operation were 

tested for mechanical hyperalgesia to distinguish animals with SNL-induced pain from nonresponders 

3 . Then we studied L5 DRG from rats with- (R-SNL), or without hyperalgesia (NR) 4 

after SNL in comparison with DRG from control rats with normal behavior after SS. Ganglia 

were fixed in 4% paraformaldehyde (PFM) followed by cryoprotection in 30% sucrose, then 

sectioned (12 µm), plated onto slides and post-fixed in 4% PFM. After blocking with 4% normal 

goat serum, samples were incubated in polyclonal rabbit antibodies to SUR1, and labeled with 

goat anti-rabbit Texas Red secondary immunofluorescent antibody. Ganglia were also co-labeled 

with mouse NF200 antibody, labeled with goat anti-mouse Alexa 488 green secondary antibody, 

to distinguish large myelinated from small non-myelinated neurons. 

Results: Axotomized primary afferents from R-SNL rats, but not from NR, exhibited less SUR1 

immunofluorescence of weaker intensity, along myelinated, NF200 positive axons. In either 

NF200 negative or NF200 positive neuronal subpopulations, rates of SUR1 positive DRG 

somata were higher after axotomy among either NF200 negative (p=0.002) or positive neurons 

(p=0.037), without differences between R-SNL and NR SNLs. 

Conclusion: Higher percentage of SUR1 positive neurons after nerve injury may indicate 

increased survival of cells expressing KATP channels, that may provide protection against DRG 

cell death that accompanies axotomy. Presence of KATP channel after nerve injury may be a 

suitable target for drug interventions. Decreased channel expression along axonal sites in R-SNL 

may explain increased neuronal excitability leading to hyperalgesia. 

References: 

1. McCallum JB, et al. 2007 SfN Meeting Abstract 509.9/PP12. 

2. Sarantopoulos C, et al. 2007 SfN Meeting Abstract 285.21/KK4 

3. Hogan Q, et al. Anesthesiology 2004; 101: 476-87 

4. Lawson SN, et al. J Physiol 1991; 435: 41-63 

Support was provided by the NIH K08 NS049420-A grant 

Disclosures: M. Liang, None; V. Zoga, None; T. Kawano , None; G. Gemes, None; J. 

McCallum, None; D. Weihrauch, None; Q. Hogan, NIH NS42150 grant, B. Research Grant 

(principal investigator, collaborator or consultant and pending grants as well as grants already

eceived); C. Sarantopoulos, NINDS Grant KNS049420A, B. Research Grant (principal 


Poster 






Title: Single channel parameters of KATP current in primary afferent neurons: the effect of 

painful nerve injury 

Authors: T. KAWANO, V. ZOGA, G. GEMES, B. MCCALLUM, W.-M. KWOK, Q. 

HOGAN, *C. SARANTOPOULOS; 

Dept Anesthesiol, Med. Coll of WI, Milwaukee, WI 

Abstract: Neuronal KATP currents mediate excitability and neuroprotection, functions pertinent 

to neuropathic pain. Because whole-cell KATP currents are present in DRG neurons and decrease 

after painful axotomy 1 , we investigated their single-channel properties and pharmacology in rats 

with painful axotomy compared with controls. 

Methods: Male rats subjected to sham skin (SS) operation or spinal nerve ligation 2 (SNL) were 

subsequently tested for hyperalgesia 3 . Then we compared single KATP channel parameters 

between control neurons from SS rats and axotomized neurons from rats with hyperalgesia after 

SNL. Neurons were dissociated 17-21 days post-surgery, stratified by size into large (L) or small 

(S), and studied within 8 hours, using the inside-out (IO) or cell-attached (CA) patch 

configurations. Pipette solution in both modes, as well as the bath in CA contained (in mM): 140 

KCl, 10 HEPES, 2.6 CaCl2, and 1.2 MgCl2. In IO, the bath contained 140 KCl, 10 HEPES, 5 

EGTA, and 2 MgCl2. Open events were detected by a conventional 50% threshold level 

criterion. Open probability (Po) was determined from the ratios of the area under the peaks in 

amplitude histograms fitted by a Gaussian distribution. Channel activity was calculated as NPo, 

where N is the number of observed channels in the patch, and determined from steady state data 

samples at 30s (IO) or 60s (CA). In IO configuration, NPo was normalized to baseline values 

(relative channel activity). Results were analyzed by ANOVA/t-tests, for the main effect of 

neuronal size (indicative of functional modality) and injury-status (SS or SNL). 

Results: 1) IO patches showed significant ATP-sensitive, inwardly-rectifying single channel 

activity of unitary conductance 72-78 pS at -60 mV, without any difference with regard to the 

main effect of size (p=0.21) or injury (p=0.99). Diazoxide enhanced, while ATP, glybenclamide

and tolbutamide blocked NPo, without differences in sensitivity between SS and SNL, or L and 

S neurons. 2) In CA recordings, L SNL neurons exhibited significantly decreased mean open 

times (p

potassium channel subunit Kv9.1 in DRG neurons from adult male Wistar rats. Additionally, we 

examined the regulation of the subunit following peripheral nerve injury. 

Using in situ hybridization, Kv9.1 mRNA was specifically detected in medium to large size 

DRG neurons, but was absent from small size nociceptor neurons as identified using the markers 

CGRP and IB4. Greater than 95% of large neurons (>40 κm diameter) and almost half of 

medium size neurons (30-40 κm diameter) expressed Kv9.1 mRNA. All Kv9.1 positive neurons 

also labelled for NF200 immunoreactivity, suggesting that they are myelinated neurons. Fourteen 

days after L5 spinal nerve transection, Kv9.1 transcript levels were dramatically reduced in the 

injured DRG. This down-regulation could be seen as early as 3 days post-injury and correlated 

well with the development of mechanical allodynia. 

Reduced Kv9.1 expression is predicted to increase excitability of DRG neurons. The specific 

detection of the channel in myelinated neurons is in agreement with the observation that 

following axotomy, spontaneous activity is almost exclusively recorded from injured A-fibres. 

We hypothesize that the attenuation of Kv9.1 expression as early as 3 days post-injury is an 

important factor in the generation of this ectopic activity. 

Disclosures: C. Tsantoulas, None; G.J. Michael , None; S.B. McMahon, None; J. Grist, 

None. 

Poster 





Support: NIH/NINDS R01NS45594 

Univ Cincinnati Millennium Fund 

NIH/NINDS R01NS55860 

Title: NF-kB-mediated enhancement of potassium currents by the chemokine CXCL1/growth 

related oncogene in small diameter sensory neurons 

Authors: *R.-H. YANG, J. A. STRONG, J.-M. ZHANG; 

Univ. Cincinnati, Cincinnati, OH 

Abstract: Inflammatory processes play important roles in both neuropathic and inflammatory 

pain states, but the effects of inflammation per se within the sensory ganglia are not well

understood. The cytokine GRO/KC (growth-related oncogene; CXCL1) shows strong, rapid 

upregulation in dorsal root ganglion (DRG) in both nerve injury and inflammatory pain models. 

We examined the direct effects of GRO/KC on small diameter DRG neurons, which are 

predominantly nociceptive. Whole cell voltage clamp technique was used to measure voltageactivated 

K currents in acutely cultured adult rat small diameter sensory neurons. Fluorescently 

labeled isolectin B4 (IB4) was used to classify cells as IB4-positive or IB4-negative. In IB4negative 

neurons, voltage-activated K current densities of both transient and sustained 

components were increased 1.6 fold after overnight incubation with GRO/KC (1.5 nM), without 

changes in voltage dependence or kinetics. The average values for the slow and fast decay time 

constants at 20 mV were unchanged by GRO/KC. The amplitudes of the fast inactivating 

component and the steady-state component were increased significantly with no shift in the 

voltage dependence of inactivation. These effects on K currents were completely blocked by coincubation 

with protein synthesis inhibitor cycloheximide (CHX) or NF-kB inhibitor pyrrolidine 

dithiocarbamate (PDTC). In contrast, the voltage-activated K current of IB4-positive neurons 

was unchanged by GRO/KC. Results suggest that GRO/KC has important effects in 

inflammatory process via its direct actions on sensory neurons, and activation of NF-kB is 

involved in its enhancement of K currents. 

Disclosures: R. Yang, None; J.A. Strong, None; J. Zhang, None. 

Poster 





Support: McMaster University 

Title: Increased excitability of dorsal root ganglion sensory neurones in an animal model of 

neuropathic pain 

Authors: Y. ZHU, Q. WU, *J. L. HENRY; 

Inst. for Pain Rese & Care, McMaster Univ., Hamilton, ON, Canada 

Abstract: Supportive evidence exists that pain following peripheral nerve injury is maintained 

by ectopic activity in primary sensory neurons, which is thought to arise from the dorsal root 

ganglion (DRG) somata, the axons and peripheral nerve terminals. Studies aimed at investigating 

the role of primary afferents in neuropathic pain (NeP) models have so far focused on small 

diameter neurones despite evidence of hyperexcitability of spinal neurones in response to both

noxious and innocuous cutaneous stimuli. We therefore used intracellular recordings from the 

somata of DRG neurons in vivo using the Mosconi and Kruger model to identify changes in all 

DRG neuron types. On the basis of preliminary studies it is not possible to draw conclusions 

regarding changes in neurones associated with C- and Aδ-fibres (n=40 neurones). However, all 

Aβ fibre types exhibited increased excitability, including low threshold mechanoreceptor 

neurones (LTM) and high threshold mechanoreceptor neurones (HTM). This consisted of an 

increase in the AP amplitude (n=66 and 127 neurones in control vs. NeP animals, respectively). 

In Aβ HTMs spontaneous discharge was seen in 6 of 29 neurones in NeP animals and in 0 of 15 

in controls. In muscle spindle (MS) afferents a single electrical stimulus to dorsal roots never 

elicited more than one action potential (AP) in controls (n=23 neurones), while in NeP animals 

up to 8 APs were observed (n= 50 neurones). Similarly, injection of a 3 nA depolarizing current 

for 20 ms elicited a maximum of 6 APs in controls (n=18) and 10 APs in NeP animals (n=36). 

These observations provide the first demonstration of altered MS and Aβ HTM DRG neurones 

associated with peripheral neuropathy and suggest that these neurones may contribute to 

symptoms of allodynia and spontaneous pain following peripheral nerve injury. 

Disclosures: Y. Zhu, None; Q. Wu, None; J.L. Henry , None. 

Poster 





Title: The increase of spontaneous nerve activity of primary afferent neurons in oxaliplatininduced 

neuropathy in mice 

Authors: P. GAUCHAN 1 , T. ANDOH 1 , A. KATO 2 , A. SASAKI 1 , *Y. KURAISHI 1 ; 

1 Dept Applied Pharmacol, 2 Dept. Hosp. Pharm, Univ. Toyama, Toyama, Japan 

Abstract: Chemotherapy-induced peripheral neuropathy is a common, dose-limiting side effect 

of cancer chemotherapeutic agents, including the platinum derivatives such as oxaliplatin. 

Neuropathic pain is characterized by spontaneous and evoked pain components, with a 

continuous or paroxysmal burning sensation including allodynia and/or hyperalgesia. 

Spontaneous activity of the injured primary afferents is one of the mechanisms that could lead to 

abnormal spontaneous pain. In the present study, we examined the electrophysiological 

properties of saphenous nerve (spontaneous discharge and mechanical sensitivity) in oxaliplatintreated 

mice. C57BL/6 mice (6 weeks old at the start of experiment) were injected with a single 

dose of oxaliplatin (3 mg/kg) intraperitoneally. Mechanical allodynia was tested by using von

Frey filament with strength of 0.68 mN. The behavior was observed by video recording and the 

duration of licking behavior was measured. The action potentials of the saphenous nerve were 

extracellularly recorded with a bipolar hook electrode. Oxaliplatin produced mechanical 

allodynia from day 3 after injection, reached the peak at day 10 and completely subsided by day 

21. Increased spontaneous licking behavior was observed at day 10 after the injection. 

Electrophysiological study showed that the spontaneous nerve activity was significantly higher 

in oxaliplatin-treated mice than in the control ones. Furthermore, mechanical sensitivity was 

significantly higher in oxaliplatin-treated mice in comparison to vehicle-treated ones. These 

results suggest that oxaliplatin causes neuropathic pain at least through the development of 

spontaneous discharge and increase in mechano-sensitivity of the afferent nerve. 

Disclosures: P. Gauchan, None; Y. Kuraishi , None; T. Andoh, None; A. Sasaki, None; A. 

Kato, None. 

Poster 

269. Pain: Visceral Pain II 



Topic: D.08.n. Visceral pain 

Support: Science Foundation Ireland 

Glaxo Smith Kline 

Industrial Development Agency, Ireland 

Title: Altered cortical cellular activation in an animal model of early-life stress: Effects of 

psychological and colorectal distension -induced stressors in adulthood 

Authors: *S. M. GIBNEY 1 , R. D. GOSSELIN 1 , T. G. DINAN 2 , J. F. CRYAN 3 ; 

1 NeuroGastroenterology, Alimentary Pharmabiotic Ctr., Cork, Ireland; 2 Psychiatry, 3 Pharmacol. 

and Therapeut., Univ. Col. Cork, Cork, Ireland 

Abstract: A clear aetiology and understanding of the mechanisms underlying irritable bowel 

syndrome (IBS) have not been fully elucidated. However, there is evidence that early life trauma 

and ongoing psychological stress in humans can affect the clinical course of this disorder. 

Alterations in stress responsivity and visceral pain are among the many hallmarks of irritable 

bowel syndrome (IBS). The pattern of altered physiological responses to psychological and 

visceral stressors reported in IBS patients is comparable to those seen in the maternally separated

(MS) model in rats, suggesting that it is a suitable animal model of IBS. Dysfunctional 

corticolimbic structures have been implicated in IBS, however the relative contribution of 

various divisions within the cortex to the altered stress responsivity of MS rats remain unknown. 

The immediate early gene, c-fos, is rapidly expressed in several brain regions in response to 

various stressors and is therefore a reliable indicator of activated cell populations in the central 

nervous system. The aim of this study was to analyse the cellular activation pattern of the 

prefrontal cortex in response to an acute psychological stressor (open field) and colonic rectal 

distension (CRD). Defecation rates and behavioural analysis in the open-field were quantified. In 

addition, CRD-induced alterations in the number of abdominal contractions were also 

determined. Several cortical-limbic structures were analysed for the presence of c-fos positive 

immunoreactivity including the prelimbic cortex, infralimbic cortex and the rostral and caudal 

anterior cingulate cortices. Our data demonstrate distinct activation patterns within these cortical 

regions following exposure to the open field or CRD, with significant (p

glutamate transporter EAAT2 is the main mediator of glutamate 

clearance to terminate glutamate-mediated responses. Transgenic mice 

over-expressing human EAAT2 (EAAT2 mice), which exhibited a 1.4-2.5 

fold enhanced glutamate uptake, showed 30% less writhing response to 

intraperitoneal acetic acid than nontransgenic littermates. Moreover, 

EAAT2 mice showed a 50-63% reduction in visceromotor response (VMR) to colo-rectal 

distension (CRD) when assessing the response to 15- 60 

mmHg pressures. Corroborating the involvement of enhanced glutamate 

uptake, wild-type mice treated for 1 week with ceftriaxone (200mg/kg 

daily), a stimulator of EAAT2 expression, showed a 52-70% reduction in 

VMR to graded increases in colo-rectal distension. The data suggest 

that enhanced glutamate uptake provides protective effects against 

mechanical distension-induced pain. Future studies will explore 1) the 

physiological site and mechanism by which enhanced glutamate transport 

activity mediates the antinociceptive effect and 2) enhanced glutamate 

transport effects on inflammogen-augmented visceral pain response. 

Disclosures: Y. Lin, None; J. Travers, None; R. Stephens, None; G. Lin, None. 

Poster 





Support: Science Foundation Ireland 

GlaxoSmithKline 

IDA Ireland 

Title: Altered spinal expression of the astrocytic glutamate transporter EAAT1 in the rat 

maternal separation model of visceral hypersensitivity 

Authors: *R. D. GOSSELIN, N. DWYER, P. FITZGERALD, T. G. DINAN, J. F. CRYAN; 

Univ. Col. Cork, Cork, Ireland 

Abstract: Alterations in gastrointestinal function and elevations in visceral pain in adulthood 

represent one of the most troublesome consequences of early-life stress. However, the molecular

connections between early-life stress and visceral hypersensitivity remain unresolved. A growing 

body of evidence suggests that an alteration in spinal sensory neurotransmission is critically 

involved in visceral pain. In this regard, glial cells, in particular astrocytes, are of particular 

interest as they play pivotal functions in glutamate and gamma-amino butyric acid (GABA) 

reuptake and are strongly implicated in various chronic pain states. Here, we investigate the 

influence of early life stress on astrocytic markers in the rat spinal cord. We study the expression 

of GFAP, the astrocytic glutamate transporters EAAT1 and EAAT2 as well as GABA 

transporters GAT1 and GAT3. Adult male Sprague-Dawley rats (250-300g) exposed to maternal 

separation (MS) between post-natal day 2 and 12 were used for the study. Immunofluorescence 

and western-blot were used to evaluate the spinal protein expression. Visceral hypersensitivity 

was quantified by counting abdominal contractions during colorectal distension (CRD, 10 

minutes, 40 mm/Hg). We show that rats submitted to MS present a visceral hypersensitivity to 

colorectal distnsion. Additionally, the basal expression of GFAP, GAT1, GAT3 and EAAT2 

appears to be unaltered by MS. However, a significant reduction in the expression of glutamate 

transporter EAAT1 is reported in MS rats. The reactivity of EAAT1 expression to a noxious 

visceral stimulus was studied following CRD. No change in EAAT1 expression was observed in 

non-separated rats; however, a significant increase in EAAT1 immunoreactivity was present in 

the spinal cord of MS animals two hours post-CRD. Taken together, our results represent a key 

insight into understanding the influence of early-life stress on visceral hypersensitivity. 

Moreover, it has direct implications for understanding pain states associated with a variety of 

stress-related psychiatric disorders and irritable bowel syndrome (IBS). 

This work was funded by Science Foundation Ireland, IDA Ireland and GlaxoSmithKline. 

Disclosures: R.D. Gosselin , GlaxoSmithKline, C. Other Research Support (receipt of drugs, 

supplies, equipment or other in-kind support); N. Dwyer, None; P. Fitzgerald, 

GlaxoSmithKline, C. Other Research Support (receipt of drugs, supplies, equipment or other inkind 

support); T.G. Dinan, GlaxoSmithKline, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); J.F. Cryan, 

GlaxoSmithKline, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 





Support: BBSRC

GlaxoSmithKline (CASE studentship) 

Title: Lumbosacral-bulbo-spinal loop relayed by RVM on-cells regulates visceral nociception 

and modulates inhibitory effects of pregabalin and ondansetron 

Authors: *S. SIKANDAR, A. H. DICKENSON; 

Univ. Col. London, London, United Kingdom 

Abstract: Descending controls from brainstem nuclei including the rostral ventromedial medulla 

(RVM) have been shown to play an important role in visceral pain, and compounds modulating 

serotonergic receptor activity and compounds targeting the α2δ subunit of voltage-gated calcium 

channels have demonstrated clinical efficacy in providing symptomatic relief in patients with 

visceral hyperalgesia. We investigated the role of RVM on-cells and a serotonergic lumbosacralbulbo-spinal 

loop in visceral hyperalgesia and examined the antihyperalgesic effects of 

ondansetron and pregabalin in modifying visceral pain responses to colorectal distension (CRD) 

in rats. 

An in vivo model of visceral pain was established involving CRD and a reliable EMG recording 

protocol for measuring activity in the external oblique muscle following CRD for quantifying 

evoked visceromotor responses (VMR) in Sprague-Dawley rats. Changes in VMR evoked by 

CRD in a range of 10-80 mmHg were recorded following administration of ondansetron (50 

κg/kg i.t.) and pregabalin (30 mg/kg s.c.) in naïve rats and rats pretreated with 0.25% 

intracolonic mustard oil (MO) to induce colonic hyperalgesia. Moreover, RVM on-cells were 

selectively ablated with injection of the neurotoxin saporin conjugated to the κ-receptor agonist 

dermorphin (DermSAP) using stereotaxic techniques. Twenty-eight days post-injection, the 

VMR were compared between naïve, SAP and Derm-SAP rats in control conditions and 

following intracolonic MO. 

CRD produced graded VMR responses that were facilitated by intracolonic MO. Both 

ondansetron and pregabalin were shown to effectively reduce evoked VMR to CRD in naïve rats 

and MO pretreated rats by antagonizing spinal 5-HT3 receptors and by binding to the α2δ subunit 

of voltage-gated calcium channels, respectively. Moreover, DermSAP pretreatment was shown 

to reduce overall evoked VMR, and the antihyperalgesic efficacies of ondansetron and 

pregabalin were also shown to be modified by the loss of on-cells in DermSAP rats. 

Furthermore, we verified immunohistochemically RVM on-cell ablation in DermSAP rats and 

quantified RVM 5-HT cell intensity between naïve, SAP and DermSAP rats. 

This study illustrates the role of 5-HT3-mediated descending facilitatory controls in visceral pain 

as well as providing evidence for the antihyperalgesic efficacy of the second generation α2δ 

ligand pregabalin in the CRD model. Moreover, evidence is provided for a facilitatory 

serotonergic lumbosacral-bulbo-spinal loop relayed by RVM on-cells that is evoked by CRD and 

modulates efficacies of pregabalin and ondansetron. 

Acknowledgments: 

Supported by the BBSRC and GlaxoSmithKline, UK. 

Disclosures: S. Sikandar, None; A.H. Dickenson, None.

Poster 





Support: CIHR Grant DPO73908 

FRSQ Postdoctoral Fellowship 12839 

Title: The localization of CB1 receptors in the mouse urinary bladder shows co-expression with 

P2X3 receptors in the urothelium 

Authors: *J.-S. WALCZAK 1 , T. J. PRICE 2 , F. CERVERO 1 ; 

1 Anesthesia Rsrch, McGill Univ., Montréal, QC, Canada; 2 Pharmacol., Univ. of Arizona, 

Tucson, AZ 

Abstract: Aim of the study: Cannabinoids have been shown to produce diverse physiological 

responses in the urinary bladder and indirect studies have shown the possible presence of 

cannabinoid receptors in the bladder. In addition, the exact expression and the localization of 

cannabinoid receptors in the urinary bladder is not known. In previous experiments we have 

shown that CB1 cannabinoid receptor mRNA was present in the mouse urinary bladder. The aim 

of this study was to evaluate the localization of CB1 receptor protein in the mouse urinary 

bladder and also to assess the co-expression with the purinergic P2X3 receptor which is a major 

player in transduction of sensory events by bladder afferents. 

Methods: Female C57BL/6 mice (20-25g) were used in this study. The animals were 

transcardially perfused with Phosphate Buffered Saline and fixed with 4% paraformaldehyde 

solutions. Immunofluorescence experiments were then performed on cryosections of the whole 

urinary bladder. Negative controls were sections incubated without the primary antibodies and 

the time of exposure was the same during acquisition of negative control images. 

Results: Cannabinoid receptor CB1 immunofluorescence was observed in the urothelium 

including the umbrella cells, as well as in nerve fibers. Co-localization studies showed that CB1 

and P2X3 receptors are co-expressed in urothelial cells. However, P2X3 immunofluorescence 

observed in the basal lamina did not colocalize with CB1-immunoreactivity. 

Conclusion: These results show evidence for the presence of cannabinoid CB1 receptor in the 

mouse urinary bladder in urothelial cells and in nerve fibers. In addition, the CB1 receptor is coexpressed 

with the purinergic P2X3 receptor in the urothelium which supports the hypothesis of 

an interaction between the cannabinoid and the purinergic system in the urinary bladder. 

Disclosures: J. Walczak, None; T.J. Price, None; F. Cervero, None.

Poster 





Support: NHMRC Grant 400053 

Title: Vagal afferent neurons innervating the adult mouse jejunum differ from spinal afferents in 

their neurochemical and structural features 

Authors: L. L. TAN 1 , J. C. BORNSTEIN 1 , *C. R. ANDERSON 2 ; 

1 Dept Physiol., 2 Dept Anat. & Cell Biol, Univ. Of Melbourne, Parkville 3010, Australia 

Abstract: Sensory information from the gastrointestinal tract is conveyed to the central nervous 

system by primary afferent fibres originating from the vagal sensory (nodose ganglion) and 

dorsal root ganglion (DRG) neurons. We have recently shown that the cell bodies of spinal 

afferents projecting to the jejunum of the adult mouse are medium-sized and are neurochemically 

defined by combinations of transient receptor potential vanilloid type 1 (TRPV1), nitric oxide 

synthase (NOS), calcitonin-gene related peptide (CGRP) and substance P (SP). It is unknown if 

vagal afferent neurons innervating the small intestine have similar characteristics. Multiple subserosal 

injections (each 0.1 κL) of cholera toxin B (CTB) were made into the jejunum of 

anesthetized C57Bl/6 mice (ketamine 100 mg/kg and xylazine 10 mg/kg; n=13). A thin layer of 

cyanoacrylate glue was applied over each injection site to prevent dye leakage. Multi-labelling 

immunofluorescence of CTB-labelled nodose cells with antibodies specific for TRPV1, CGRP, 

SP, NOS as well as binding to isolectin B4 (IB4) were performed 7 days post-operative. Soma 

sizes were assessed by measuring the cross-sectional areas of nucleated cell profiles using Image 

J software (NIH). CTB-labelled nodose ganglion neurons supplying the jejunum are significantly 

smaller than DRG neurons supplying the same region (median areas: 295 vs 540κm 2 

respectively; p

within the intestinal wall, terminals of these vagal afferents can be distinguished from those of 

enteric and spinal neurons. 

Disclosures: L.L. Tan, None; C.R. Anderson , None; J.C. Bornstein, None. 

Poster 





Title: The visceral antinociception of sigma1 receptor antagonist and its synergy with other 

analgesics in mice 

Authors: T.-W. KIM 1 , H.-W. KIM 2 , K.-W. KIM 1 , *Y. KWON 1 ; 

1 Phamacol, Med. Sch. Chonbuk Natl. Univ., Chonju, Republic of Korea; 2 Dept. of Physiol., Col. 

of Med. and Res. Inst. for Med. Sciences, Chungnam Natl. Univ., Daejeon, Republic of Korea 

Abstract: It has been demonostrated that sigma 1 receptor antagonist, BD1047, produces 

analgesic effect in formalin induced pain behavior. The aim of present study was to determine 

the antinociceptive effect of spinal sigma 1 receptor in the model of visceral acute tonic pain, the 

acetic-acid writhing test in mice. Furthermore, present study was undertaken to characterize the 

interactions between BD1047 and several type of analgesic candidates. Intrathecal pretreatment 

of BD 1047 (10, 30, 100 nmol) produced dose-dependent antinociceptive effect against acetic 

acid-induced writhing. Intraperitoneal injection of paroxetine, fluoxetine, nixosetine, morphine, 

ketamine and acetaminophen dose-dependently produced anti-nociceptive effect in acetic acid 

induced pain model. Combination of low analgesic doses of these drugs with low analgesic doses 

of BD 1047 (30 nmol) resulted in synergistic interactions. Therefore, the spinal blockage of 

sigma 1 receptor has potent analgesic effect in visceral pain. Furthermore, the combination of 

potential analgesics with BD 1047 is synergistic, and should allow either to increase their 

analgesic efficacy and/or to reduce their side effects. 

Disclosures: T. Kim, None; H. Kim, None; K. Kim, None; Y. Kwon , None. 

Poster





Support: NIH award NS 19912 

Title: P2X3 receptor contribution to colon hypersensitivity in the mouse 

Authors: *M. SHINODA, J. LA, K. BIELEFELDT, G. F. GEBHART; 

Ctr. for Pain Res., Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Irritable bowel syndrome is a functional gastrointestinal disorder of unknown etiology 

characterized by pain in the absence of colon inflammation. ATP is released during colon 

distension and increases responses of colon afferent fibers to stretch. In the present experiments, 

we examined the role of purinergic (P2X) receptors in colon hypersensitivity. Changes in 

visceral nociceptive behavior as the visceromotor response to colon distension and ATP release 

from colon by colon distension were measured after intracolonic treatment with zymosan (which 

reproduces major features of IBS - persistent hypersensitivity without colon inflammation) or 

saline. Intracolonic zymosan (3 mg in 100 κl daily for 3 days), but not saline, produced 

significant colon hypersensitivity, which was significantly reduced in both P2X3 +/- and P2X3 -/- 

mice relative to control (wildtype) mice. However, ATP release did not change after intracolonic 

zymosan or saline in either wild type or P2X3 -/- mice. In whole cell patch clamp studies, resting 

membrane potential, input resistance and rheobase were decreased in colon sensory neurons 

from mice treated with zymosan compared to saline. Similarly, the A-type K + current was found 

to be decreased, revealing increased excitability of colon sensory neurons associated with colon 

hypersensitivity. Immunohistochemically, the number of P2X3 receptor-positive colon sensory 

neurons was increased by intracolonic treatment with zymosan. These results suggest that P2X3 

receptors may be important peripheral mediators of colon mechanosensation and development of 

hypersensitivity in this model. 

Disclosures: M. Shinoda, None; J. La, None; K. Bielefeldt, None; G.F. Gebhart, None. 

Poster 





Title: Assessment of naturally occurring cannabinoids in response to visceral pain 

Authors: *L. BOOKER, A. LICHTMAN; 

Pharmacol., Virginia Commonwealth Univ., Richmond, VA 

Abstract: Considerable attention has been focused on examining the antinociceptive effects of 

Γ 9 -tetrahydrocannabinol (Γ 9 -THC), the major psychoactive constituent of Cannabis sativa. In 

addition to Γ 9 -THC, there are approximately 70 cannabinoids present in cannabis. The primary 

purpose of this study was to determine the analgesic effects of prevalent phytocannabinoids, 

which include Γ 9 -THC, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), and 

Γ 8 -tetrahydrocannabivarin (THCV), in the acetic acid stretching test, a visceral pain model. Γ 9 - 

THC (ED50 = 0.32 mg/kg) and CBN produced analgesic effects, though CBN was only effective 

at 50 mg/kg. The CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, 

SR144528, blocked the analgesic effects of both compounds. Conversely, CBD and CBN failed 

to decrease stretching. While THCV alone failed to affect acetic acid-induced stretching, it 

displayed antagonistic properties when co-administered with Γ 9 -THC. Because the CB2 receptor 

has been associated with inflammatory states in the GI tract, we examined whether 

administration of a CB2 receptor agonist, O-3223, would elicit analgesic effects in the acetic acid 

stretching test. Although O-3223 was without apparent activity, a selective CB1 receptor agonist 

arachiodonyl 2‟-chloroethylamide produced antinociception. All compounds having an analgesic 

effect did so at doses that did not reduce spontaneous locomotor activity, arguing against 

impaired motor function as a confounding variable. These data indicate that Γ 9 -THC and CBN 

reduce nociception in the acetic-acid stretching test through a CB1 receptor mediated mechanism. 

Moreover, the observation that THCV blocked the antinociceptive effects of Γ 9 -THC raises the 

possibility that phytocannabinoids present in marijuana interact to influence antinociception. 

Disclosures: L. Booker , None; A. Lichtman, None. 

Poster 





Support: NIH Grant HL75524

Title: Responses of T3 spinal neurons to cardiosomatic stimulation in proestrous female vs. male 

rats 

Authors: *J. M. LITTLE, J. P. FARBER, R. W. BLAIR, R. D. FOREMAN; 

Physiol., Univ. Oklahoma HSC, Oklahoma City, OK 

Abstract: Gender differences in cardiac pain have been reported from clinical studies: compared 

to men, women experience chest pain from angina that is longer in duration, more severe, and 

more frequent. Referred pain areas associated with cardiac pathophysiology are also different, 

suggesting differences in processing of cardiosomatic input. The mechanisms underlying gender 

differences in cardiac pain are unknown. One possible mechanism for the observed differences 

could be related to the circulating levels of estradiol. The present study was done to test the 

hypothesis that there were gender differences related to estradiol levels in the responses of T3 

spinal neurons to cardiosomatic information in an animal model that has been shown to exhibit 

cardiac pain. Estradiol levels peak between 50 and 150 pg/ml in female rats during proestrus. In 

male rats, estradiol levels range from 10 to 15 pg/ml. In this study, adult Sprague-Dawley male 

(n=10) and proestrous female rats (n=11) were anesthetized with sodium pentobarbital and 

paralyzed. Extracellular potentials of T3 spinal neurons were recorded in response to mechanical 

somatic stimulation and chemical cardiac stimulation. A volume of 0.2 ml saline (control) was 

injected into the pericardial sac and removed after 60 seconds. This was followed by an injection 

of 0.2 ml algesic chemicals (noxious stimuli) into the pericardial sac. Somatic fields were located 

by applying brush, pressure, and pinch to the upper body. Sizes of the somatic fields were 

evaluated before and after intrapericardial injection of chemicals to determine if the chemicals 

sensitized the neurons. Thirty eight percent and 57% of neurons responded to noxious chemical 

cardiac stimuli in female and male rats, respectively. Of those neurons receiving input from the 

heart, 62% in female and 56% in male rats also received input from somatic structures. In female 

rats, 76% of neurons responding to somatic stimuli had somatic receptive fields located on the 

side of the upper body, while 24% of neurons had somatic fields located on the chest. Most of 

the somatic fields in male rats were also located on the side of the upper body (90%), with only a 

few located on the chest (10%). The size of the receptive fields between female and male rats 

was not significantly different. Somatic field sizes in 69% of neurons in females and 76% of 

neurons in males did not change after intrapericardial injection of noxious chemicals. These 

results indicate there are no significant differences (p>0.05) in the responses of T3 spinal 

neurons to mechanical somatic stimulation and noxious chemical cardiac stimulation between 

male and proestrous female rats, despite differences in estradiol levels. 

Disclosures: J.M. Little, None; J.P. Farber, None; R.W. Blair, None; R.D. Foreman, None. 

Poster 






Title: Mice lacking P2X3 have decreased gastroesophageal mechanosensitivity 

Authors: *S. L. MCILWRATH 1 , B. M. DAVIS 2 , K. BIELEFELDT 2 , G. F. GEBHART 1 ; 

1 Dept Anesthesiol., 2 Dept Med., Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Bladder and colonic epithelium release ATP upon distension, which activates sensory 

neurons through ionotrophic P2X and metabotropic P2Y purinergic receptors. Prior studies have 

demonstrated that most gastric sensory neurons within the nodose ganglia respond to purinergic 

agonists. Based on these findings, we hypothesized that activation of P2X3 receptors contribute 

to gastric sensation. 

Stomach afferents from wildtype (WT) and P2X3 -/- (KO) mice were labeled using 2% Alexa 

Fluor 488 conjugated choleratoxin B. Mice were sacrificed 4 days after surgery and nodose 

ganglia dissociated for whole cell recordings. In other experiments, an ex vivo esophagus / 

stomach/ nodose ganglion preparation was used to characterize response properties of vagal 

afferents. Sharp electrode recordings were performed from single cells and their responses to an 

ascending series of stepwise fluid distensions (0, 10, 20, 30 and 40 cm H2O for 20 s each) as well 

as to perfusion with pH 4 solution for 3 min of the stomach recorded. Gastric emptying was 

determined as a ratio of ingested and retained food 3 h after food intake. 

While 85% of vagal stomach afferents from both genotypes responded to ATP, the current 

density differed significantly between the groups (WT = 50.4 ± 13.1 pA/pF; KO = 13.4 ± 5.1 

pA/pF; p

Poster 






Title: Identification and characterization of anterogradely traced extrinsic primary afferents of 

the mouse colon 

Authors: *P. R. BRUMOVSKY, J. LA, G. F. GEBHART; 

Ctr. for Pain Res., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: Nerve fibers innervating the mammalian colon are divided into extrinsic (sensory and 

autonomic axons) and intrinsic (enteric neurons producing intracolonic axons). Interestingly, 

most published anatomical data on both systems in the colon is based on whole mount 

preparations, where only the tangential distribution of nerve fibers is shown. The main objective 

of the present study is thus to reveal the distribution and phenotype of extrinsic primary afferent 

fibers through the different layers of the mouse colon. Distal colon and innervating pelvic nerves 

of C57Bl/6 mice were dissected out and placed in a chamber with circulating oxygenated Krebs 

solution at 34 °C. The colon was opened longitudinally and pinned flat to the bottom of the 

chamber. The pelvic nerves, passed through a gate into a second chamber with mineral oil, were 

split into 10-12 bundles and explored electrophysiologically to certify the presence of 

mechanosensitive fibers. This was done by mechanical stimulation of the colon using a 1g von 

Frey filament. Subsequently, the nerve bundles were exposed for 20-24 h to 5-10% biotinamide 

(BTA), used as an anterograde tracer. During this time, the Krebs solution was exchanged with 

oxygenated DMEM-12 containing fetal bovine serum, calcium chloride and nifedipine. 

Afterwards, the colon was transversely cryo-sectioned in the longitudinal axis and processed for 

immunohistochemistry. Traced fibers were detected by incubation with Streptavidin-Alexa Fluor 

(488), and their phenotype as primary afferents evaluated using calcitonin gene-related peptide 

(CGRP). We found that extrinsic nerves penetrating the colon extend both longitudinally and 

transversely. Moreover, some traced fibers were observed in the mucosal layer, supporting 

previous electrophysiological studies suggesting the presence of mucosal and muscular-mucosal 

afferents in the colonic wall. A proportion of the traced nerves expressed CGRP, although BTA- 

or CGRP-only fibers were also observed. The present data contribute to the anatomical 

description of nerve fibers of extrinsic origin in the colon, and will be useful for future 

correlations with the neurophysiological events occurring in the colonic wall. Supported by NS 

19112. 

Disclosures: P.R. Brumovsky, None; J. La, None; G.F. Gebhart, None.

Poster 





Support: POCTI/SAU-NSE/55983/2004 

Title: Human urothelial cells respond to inflammatory mediators trough a TRPV1-dependent 

mechanism 

Authors: *A. A. SILVA 1 , A. CHARRUA 1 , C. REGUENGA 1 , J. M. CORDEIRO 2 , M. 

DUARTE-ARAÚJO 2 , P. CORREIA-DE-SÁ 2 , C. PAULE 3 , I. NAGY 3 , F. CRUZ 4 ; 

1 Inst. of Hist and Embryol, Fac Medicine, IBMC, Univ. of Porto, Portugal, Porto, Portugal; 

2 Laboratório de Farmacologia/UMIB, ICBAS, Porto, Portugal; 3 Imperial Col. London, London, 

United Kingdom; 4 Inst. of Hist and Embryol, Hosp. de São João, IBMC, Univ. of Porto, 

Portugal, Porto, Portugal 

Abstract: Human urothelial cells express the TRPV1 receptor. Recent works have indicated that 

TRPV1 might be responsible for the control of pain and micturiction control during chronic 

cystitis. Here, we studied the functionality of the TRPV1 channel in cultured human urothelial 

cells. For that, we performed cobalt uptake experiments, an indirect method to measure the 

calcium influx into cells, in cells exposed to normal media, to a solution composed of 

inflammatory mediators and a solution composed of inflammatory mediators and capsazepine (a 

TRPV1 antagonist). Our data shows that inflammatory mediators induced a TRPV1-dependent 

increase of cobalt uptake, since this effect was completely blocked by capsazepine. Previous 

results from our group have shown that capsaicin induces cobalt uptake from cultured human 

urothelial cells, an effect completely reverted by capsazepine. In order to understand the 

consequence of human urothelial TRPV1 activation, we analysed by luminometry the capsaicininduced 

ATP release. Capsazepine significantly inhibited the release of ATP by the urothelial 

cells, thereby implicating TRPV1 in ATP secretion from human urothelium. This work shows 

further evidence that TRPV1 expressed in human urothelial cells have an important role in 

micturition control under pathological conditions such as cystitis. 

Disclosures: A.A. Silva, None; A. Charrua, None; C. Reguenga, None; J.M. Cordeiro, 

None; M. Duarte-Araújo, None; P. Correia-de-Sá, None; C. Paule, None; I. Nagy, None; F. 

Cruz, None.

Poster 







Title: The biochemical properties of cultured mouse splanchnic and pelvic nerve dorsal root 

ganglion neurons change over time 

Authors: *D. R. ROBINSON, J.-H. LA, G. F. GEBHART; 

Ctr. for Pain Res., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: Functional studies of sensory neurons can require the use of cultured dorsal root 

ganglia (DRG) cells. Previous studies have suggested that the phenotype of these cells change in 

a culture time dependent manner. Here we report changes in Calcitonin Gene-Related Peptide 

(CGRP)-like immunoreactivity and the binding of Isolectin B4 (IB4) in DRG somata with 

specific emphasis on those innervating the colon. 

DRG neurons innervating the descending colon were retrogradely labeled in adult male C57 

BL/6 mice using Fast Blue. Four weeks later, splanchnic (TL; T8 - L1) and pelvic (LS; L6 - S1) 

DRG neurons were dissected and cultured. Neurons were enzymatically treated, triturated, and 

plated onto coverslips that were fixed 2, 6, 9, 12, 24, 48, and 72 hours (TL) or 2, 12, 24, and 72 

hours (LS) later. Fixed coverslips were preblocked and sequentially incubated with primary 

antibody, Cy3-conjugated secondary antibody, and AlexaFluor 488-conjuagated IB4. Analysis 

was performed with NIH image analysis software, ImageJ. 

23% of all DRG neurons were CGRP-like immunoreactive (LI) 2 hours after plating. This 

increased to approximately 34% after 12 - 24 hours, then decreased to 25%. The number of 

CGRP-LI cells in the colonic DRG population was greater: 68% after 2 hours, 91% after 24 

hours. IB4 binding was relatively unchanged over time in the whole population of cells 

(approximately 26%) but colonic DRG neurons showed a decrease from 49% after 2 hours to 

approximately 34% after 12 hours. The relative number of CGRP-LI cells in TL and LS 

populations was similar but more LS neurons bound IB4. A similar trend was seen for those cells 

that were both CGRP-LI and bound IB4. After 9 hours, nearly all (90%) of IB4 binding colonic 

DRG neurons were also CGRP-LI, compared to only 14% of unlabeled cells. 

In conclusion, time- and marker-dependent changes were seen in cultured DRG neurons that 

varied among specific populations (e.g., colonic afferents). When considering the colonic DRG

population, fewer TL than LS neurons bound IB4, suggesting a different composition of neurons 

in the nerves innervating the colon, perhaps extending to functional differences that have been 

previously reported. This, together with the observation that nearly all colonic IB4 binding 

neurons were CGRP-LI, adds weight to the concept of a unique subtype of neuron that may be 

specific to the colon. 

Disclosures: D.R. Robinson, None; J. La, None; G.F. Gebhart, None. 

Poster 





Title: Antinociceptive effects of nociceptin/orphanin FQ receptor agonists in the mouse 

writhing test 

Authors: *A. RIZZI 1 , G. MARZOLA 1 , R. GUERRINI 2 , S. SALVADORI 2 , D. REGOLI 1 , G. 

CALO' 1 ; 

1 Exp Clin. Med, Pharmacol, Univ. Ferrara, Ferrara, Italy; 2 Pharmaceut. Scie and Biotech Ctr., 

Univ. of Ferrara, Ferrara, Italy 

Abstract: Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are widely expressed in the 

brain, spinal cord and peripheral nervous system and are involved in the modulation of pain 

transmission. In this study, the effects of N/OFQ and [(pF)Phe 4 Aib 7 Arg 14 Lys 15 ]N/OFQ-NH2 

(UFP-112), a potent and selective NOP agonist, were investigated in a mouse model of visceral 

pain: the abdominal acetic acid-induced writhing test. Mice were injected intraperitoneally (i.p.) 

with 0.1 ml 0.6% acetic acid aqueous solution to produce the writhing response characterized by 

contractions of the abdominal musculature followed by the extension of the hind limbs. The 

number of writhes was counted during a 30 min observation period. In control Swiss mice the 

injection of acetic acid into the peritoneal cavity of mice resulted in an average of 53 ± 4 writhes 

during the 30 min period. N/OFQ (10-1000 nmol/mouse) administered i.p. 5 min before the 

injection of acetic acid inhibited in a dose dependent manner the writhing response with maximal 

effects corresponding to 64% inhibition. This N/OFQ effect was mimicked by UFP-112 (1-100 

nmol/mouse) which was however 10 fold more potent than the natural peptide. In a separate 

series of experiments performed in NOP +/+ and NOP -/- mice, NOP -/- mice displayed a clear 

nociceptive phenotype since they were more sensitive to acid acetic injection compared to wild 

type animals (NOP +/+ : 36 ± 5; NOP -/- : 59 ± 6*). N/OFQ and UFP-112 tested at the higher 

effective doses (1000 and 100 nmol, respectively) produced robust inhibitory effects (50%

inhibition for both peptides) in NOP +/+ mice while being inactive in NOP -/- mice. In conclusion 

the present results demonstrated that selective activation of NOP receptors produces 

antinociceptive effects in the mouse writhing test and that endogenous N/OFQ - NOP receptor 

signaling inhibits this animal behavior. 

Disclosures: A. Rizzi , None; G. Marzola, None; R. Guerrini, None; S. Salvadori, None; D. 

Regoli, None; G. Calo', None. 

Poster 





Support: IASP Early Career Research Grant 

NS 19112 

Title: Expression of vesicular glutamate transporters in neurons innervating the mouse colon 

Authors: *G. F. GEBHART 1 , D. R. ROBINSON 2 , J. LA 2 , T. HÖKFELT 3 , M. WATANABE 4 , 

P. R. BRUMOVSKY 2 ; 

1 Ctr. for Pain Resch, W1444 BST, Pittsburgh, PA; 2 Ctr. for Pain Res., Univ. of Pittsburgh, 

Pittsburgh, PA; 3 Dept. of Neurosci., Karolinska Institutet, Stockholm, Sweden; 4 Dept. of Anat., 

Hokkaido Univ., Sapporo, Japan 

Abstract: Presence of vesicular glutamate transporters (VGLUTs) has been described in various 

neuronal systems. In primary afferent neurons innervating the hindpaw and hairy skin of rodents 

VGLUT1 and -2 are strongly expressed. In the present study, we analyzed these two transporters 

in neurons innervating the mouse colon. The colonic wall of C57 BL/6 mice was injected with 

the retrograde tracers Fast Blue or DiI. Two to three weeks later, animals were transcardially 

perfused with a mixture of paraformaldehyde and picric acid, and the distal colon and 

appropriate lumbosacral and thoracolumbar dorsal root ganglia (DRGs) were dissected and 

processed for immunohistochemistry. In other mice injected with the dye, DRGs were fixed after 

four days in culture, and processed for immunocytochemistry. Previously characterized 

antibodies against VGLUT1 and -2 were used. In tissue sections, many Fast Blue- or DiI-positive 

DRG neurons showed VGLUT2 -like immunoreactivity (-LI), while VGLUT1 -LI was virtually 

absent in retrogradely labeled colonic neurons. Occasional retrogradely labeled colonic neurons 

lacking VGLUT2-LI were also detected. VGLUT2-immunoreactive (IR) DRG neurons, both

etrogradely labeled and unlabeled, spanned all size ranges. In agreement with the observations 

in DRG tissue sections, VGLUT2 -LI was also detected in a large number of colonic neurons in 

culture. Abundant VGLUT2-IR fibers were detected in all the layers of the colon, including the 

myenteric plexus and the mucosal layer. VGLUT1-LI was not detectable in the colonic wall. In 

conclusion, we show (1) that DRG neurons innervating the colon express VGLUT2 but not 

VGLUT1, and (2) presence of abundant VGLUT2-IR fibers innervating the different layers of 

this organ. Supported by an IASP Early Career Research Grant and NS 19112. 

Disclosures: G.F. Gebhart, None; D.R. Robinson, None; J. La, None; T. Hökfelt, None; M. 

Watanabe, None; P.R. Brumovsky, None. 

Poster 





Support: NIH Grant NS 41384 

Title: GABA-A receptor mediated presynaptic inhibition contributes to the differential 

processing of visceral nociception in the rat thoracolumbar and lumbosacral spinal cord segments 

Authors: B. TANG 1 , Y. JI 1 , M. S. GOLD 2 , *R. J. TRAUB 1 ; 

1 Dept Biomed Sci., Univ. Maryland Dent. Sch., Baltimore, MD; 2 Dept. of Medicine, Div. of 

Gastroenterology, Hepatology and Nutr., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: Visceral structures are dually innervated. In the rat, colonic afferents project to the 

lumbosacral (LS) and thoracolumbar (TL) spinal cord segments via the pelvic nerve and 

hypogastric/lumbar colonic nerves, respectively. Previous studies in our lab have shown that TL 

and LS spinal segments differentially contribute to acute and inflammatory visceral nociceptive 

processing. LS visceroceptive dorsal horn neurons are significantly more excitable to colorectal 

distention (CRD) than TL neurons in normal rats. Colonic inflammation increases the activity of 

TL and LS visceroceptive dorsal horn neurons. The mechanism for this difference is unclear. We 

hypothesize that the lower response of TL visceroceptive dorsal horn neurons to CRD in normal 

rats results from greater pre-synaptic inhibition of the afferent signal in the TL spinal cord and 

that following colonic inflammation, a decrease in this pre-synaptic inhibition increases activity 

in TL neurons. To test this hypothesis, the effect of spinal administration of the GABA-A 

receptor antagonist gabazine (0.15µg / 5µl) on the visceromotor response (VMR) and activity of 

dorsal horn neurons to noxious CRD was examined in normal and chronic colonic inflamed rats

induced by intracolonic injection of deoxycholic acid. Intrathecal administration of gabazine to 

the TL segments significantly increased the VMR while having no effect when administered to 

the LS segments in normal rats. In the presence of chronic colonic inflammation, intrathecal 

administration of gabazine to the TL or LS segments significantly decreased the VMR. Spinal 

administration of gabazine significantly increased the response of TL dorsal horn neurons to 

noxious CRD in normal rats, but significantly decreased the response of TL dorsal horn neurons 

in chronic colonic inflamed rats. Spinal administration of gabazine had no effect on the response 

of LS dorsal horn neurons to noxious CRD in both normal and colonic inflamed rats. These 

results suggest that GABA-A receptors contribute to the differential processing of visceral 

nociception in TL and LS spinal cord segments. 

Disclosures: B. Tang, None; Y. Ji, None; M.S. Gold, None; R.J. Traub , None. 

Poster 





Support: POCTI/SAU-NEU/55983/2004, FCT, Portugal. 

Title: BDNF is important for bladder hyperactivity and pain in a rat model of chronic bladder 

inflammation 

Authors: *C. CRUZ 1 , R. PINTO 1,2 , S. ALLEN 3 , D. DAWBARN 3 , S. MCMAHON 4 , F. 

CRUZ 1,2 ; 

1 Inst. Histology and Embryology,, Fac Med, IBMC, Univ. of Porto, Portugal, Porto, Portugal; 

2 Dept. of Urology, Hosp. de S.Joao, Porto, Portugal; 3 Mol. Neurobio. Unit, Univ. of Bristol, 

CSSB, Dorothy Hodgkin Building, Bristol, United Kingdom; 4 Neurorestoration Group, London 

Pain Consortium, King's Col. London, Wolfson CARD, Wolfson Wing, London, United 

Kingdom 

Abstract: Brain derived nerve factor (BDNF) is a trophic factor belonging to the neurotrophin 

family that is upregulated in various inflammatory conditions, where it may contribute to altered 

pain states. In the case of bladder inflammation, little is known about the relevance of BDNF in 

bladder pain and hyperactivity, a matter we aimed to clarify in the present study. 

Female rats (n=4/group) were treated with cyclophosphamide (CYP; 200 mg/Kg). For a period 

of 3 days, animals received daily injections in the tail vein of sterile saline or TrkB-Ig2 (100 or 

200 ug). TrkB-Ig2 is a recombinant immunoglobulin-like domain that binds to BDNF, with

picomolar affinity, neutralising its activity. Then, bladder function was monitored for 2 hours in 

anaesthetized rats, followed by perfusion-fixation. Spinal cord segments L6 were collected, postfixed 

and cryoprotected in sucrose. Segments were cut into 40 κm sections and immunoreacted 

against activated ERK and c-Fos. 

In CYP-injected rats treated with intravenous saline, the number of bladder contractions per 

minute was significantly increased in comparison with naïve rats (1.17±0.16 versus 0.60±0.08, 

respectively; p

Authors: *B. C. YOBURN 1 , S. V. DIGHE 1 , E. A. WALKER 2 , S. SIROHI 1 ; 

1 Col. of Pharm., St. John's Univ., Jamaica, NY; 2 Sch. of Pharm., Temple Univ., Philadelphia, PA 

Abstract: In this study, we examined if the analgesic efficacy of fentanyl predicts the magnitude 

of tolerance and κ-opioid receptor regulation. In order to estimate efficacy, mice were injected 

i.p. with saline or clocinnamox (CCAM), an irreversible κ-opioid receptor antagonist, (0.32 - 

25.6 mg/kg) and 24 hr later fentanyl cumulative dose response studies were conducted (tail 

flick). CCAM dose dependently shifted the fentanyl dose-response function to the right and the 

apparent efficacy (τ) of fentanyl, based on the operational model of agonism, was estimated as 

58; indicating that fentanyl is a high analgesic efficacy agonist. Next, mice were infused with 

fentanyl (1, 2 or 4 mg/kg/day) for 7 days. Controls were implanted with placebo pellets. At the 

end of 7 days, morphine cumulative dose response studies or κ-opioid receptor saturation 

binding studies ([ 3 H] DAMGO) were conducted. Fentanyl infusions dose-dependently decreased 

morphine potency with the highest fentanyl dose reducing morphine potency by ≈ 6 fold. 

Chronic infusion with fentanyl (4mg/kg/day) significantly reduced κ opioid receptor density by 

28% without altering affinity, whereas lower infusion doses had no significant effect. These data 

were compared to previous results and confirmed that fentanyl produced less tolerance than 

lower analgesic efficacy agonists at equieffective doses. Taken together, the present results 

strengthen the proposal that opioid analgesic efficacy predicts κ-opioid receptor regulation and 

the magnitude of tolerance. 

Disclosures: B.C. Yoburn, None; S.V. Dighe, None; E.A. Walker, None; S. Sirohi, None. 

Poster 

270. Pain: Opioids and Other Analgesics II 


Program#/Poster#: 270.2/HH1 

Topic: D.08.r. Opioids and other analgesics 

Support: Conacyt Grant A S-M 200325 

Title: Delay in tolerance development with the combined administration of d-propoxyphene and 

metamizol (dipyrone) in rats 

Authors: *S. L. CRUZ, F. LÓPEZ-MUÑOZ, A. SILVA-MORENO; 

Dept. Pharmacobiology, CINVESTAV, IPN, Mexico City, Mexico 

Abstract: Dextropropoxyphene (DXP) is an opiate analgesic commonly prescribed for the relief 

of mild to moderate pain. Chronic administration of DXP is limited by tolerance development

and the occurrence of adverse side effects if high doses are used. Metamizol (MET), also known 

as dipyrone, is a nonsteroidal antiinflamatory drug (NSAID) extensively used in Latin America 

and Europe. There is evidence that the combined administration of low doses of opiates with 

NSAIDs reduces unwanted side effects and improves antinociception, but there are no studies for 

this particular analgesic combination. The objective of this work was to evaluate the effects of 

repeated administration of DXP and MET alone or in combination (DXP+MET), on the tail flick 

model in rats. Male Wistar rats (200 g ± 20 g) were i.v. injected twice a day (8 h apart) with 0.31 

mg/kg DXP, 400 mg/kg MET or the combination of these drugs at the same doses. This 

administration schedule was used until the antinociceptive effect disappeared. For each drug 

administration, a time course of the effects was determined. The results were expressed as area 

under the curve, percentage of maximal possible effect, and absolute peak effect. Both MET and 

DXP produced mild antinociception at the doses used. When these drugs were given repeatedly, 

tolerance developed at the 6 th administration for both compounds. The combination of 

DXP+MET resulted in potentiation, observed as an increased in tail flick latency higher than 

predicted by the arithmetic sum of the individual drug effects. Moreover, a recovery of the 

antinociceptive efficacy was observed when the combination DXP+MET was given to rats 

already tolerant to DXP or MET. These results suggest that repeated coadministration of DXP 

and MET produces good antinociception and delays tolerance development. 

Disclosures: S.L. Cruz , None; F. López-Muñoz, None; A. Silva-Moreno, None. 

Poster 





Support: NEWMOOD Project, 6th European Framework Program 

Title: Transcriptome and biochemical pathways analysis in brain of mice selected for high and 

low stress-induced analgesia 

Authors: P. LISOWSKI 1 , G. JUSZCZAK 2 , *A. H. SWIERGIEL 3,2 , J. GOSCIK 4 , M. 

WIECZOREK 5 ; 

1 Mol. Biol., 2 Animal Behavior, Inst. Genet. & Animal Breeding, Polish Acad. of Sci., 

Jastrzebiec, Poland; 3 Dept Pharmacol, Toxicol & Neur, Louisiana State Univ. Hlth. Sc, 

Shreveport, LA; 4 Software Engin., Bialystok Tech. Univ., Bialystok, Poland; 5 Biology, Lab. of 

Neurophysiol., Univ. of Lodz, Lodz, Poland

Abstract: Forced swim produces a decrease in pain sensitivity in laboratory rodents, known as 

swim stress-induced analgesia (SSIA). Pain perception and sensitivity to analgesics strongly 

depend on genotype. Mice selectively bred for magnitude of SSIA display differential 

emotionality in a variety of behavioral tests, different responses to SSRIs, and markedly 

divergent morphine analgesia (so called ,,correlated traits”). These differences appear to be 

determined by a number of unknown major genes. The present study examined gene expression 

in brain structures in lines of mice bred for high (HA) and low (LA) SSIA. All mice were 

experimentally naive (untreated). Expression profiling using microarrays allows for detailed 

characterization of the gene networks that regulate animal‟s analgesia and the correlated traits. 

To characterize individual differences in genetic correlates of SSIA and the correlated traits we 

used 24 K oligo microarrays and transcriptome analyses were performed in hypothalamus, 

hippocampus, raphe nucleus and frontal cortex. We found a different number of genes in each 

brain structures differently expressed in HA and LA lines. These genes are involved in regulation 

of biochemical pathways such as: gap junction, longterm potentiation, long-term depression, 

axon guidance, neuroactive ligand-receptor interaction. Different activity of these pathways may 

affect biological processes involved in SSIA, different emotionality in behavioral tasks of HA 

and LA mice and different responses to desipramine, venlafaxine and s-citalopram. The results 

suggest that selection for SSIA produces differential effects on transcriptome and activation of 

biological processes in brain. 

Disclosures: P. Lisowski, None; G. Juszczak, None; A.H. Swiergiel, None; J. Goscik, 

None; M. Wieczorek, None. 

Poster 





Support: NIDA Grant DA 19959 (to B.C.Y.) 

Title: Tolerance following acute, intermittent and chronic treatment with oxycodone and 

etorphine 

Authors: *P. A. MADIA, S. V. DIGHE, S. SIROHI, B. C. YOBURN; 

Col. of Pharm., St. John's Univ., Jamaica, NY 

Abstract: Opioid analgesic treatment protocol may play a role in determining the magnitude of 

tolerance. In this study, we evaluated the effect of treatment protocol on tolerance to morphine in

mice using the high efficacy opioid agonist etorphine and the lower efficacy agonist oxycodone. 

The treatment protocols consisted of acute (single s.c. injection), intermittent (1 injection/day for 

7 days) and 7 day continuous infusion (s.c. osmotic minipump) of oxycodone (29.5- 

118mg/kg/day; ≈50-175 times the analgesic ED50) or etorphine (50-400κg/kg/day; ≈50-400 

times analgesic ED50). The total amount of drug administered per day was the same for all 

protocols for each drug. Control groups were injected with saline or implanted with placebo 

pellets. Twenty four hours after the last injection or 16 hours after the end of the infusion (pumps 

removed), the analgesic potency of morphine (tail flick) was determined in cumulative doseresponse 

studies. Acute treatment with either drug produced minimal tolerance (≈1 - 2 fold 

decrease in potency). Similarly, 7 day intermittent injections produced minimal tolerance for 

both oxycodone (≈1 - 4 fold decrease in potency) and etorphine (≈2 fold decrease in potency). 

On the other hand, continuous infusions of both oxycodone and etorphine produced substantially 

more tolerance (as much 13-fold decrease in potency) than acute or intermittent administration of 

the same daily dose. Taken together, these data are consistent with the suggestion that continuous 

infusion of both high and low efficacy opioid agonists produces substantially more tolerance 

than acute or intermittent treatment with the same daily dose. 

Disclosures: P.A. Madia, None; S.V. Dighe, None; S. Sirohi, None; B.C. Yoburn, None. 

Poster 





Support: Canadian Institutes of Health Research 

Title: Mechanisms underlying increase in levels and effects of adrenomedullin in morphine 

tolerance 

Authors: *J.-G. CHABOT 1 , Y. HONG 2 , A. FOURNIER 3 , R. QUIRION 2 ; 

1 Douglas Mental Hlth. Univ. Inst., McGill Univ., Montreal, QC, Canada; 2 Dept. of Psychiatry, 

McGill Univ., Douglas Mental Hlth. Univ. Inst., Montreal, QC, Canada; 3 INRS- Inst. Armand 

Frappier, Laval, QC, Canada 

Abstract: We have demonstrated that up-regulation of adrenomedullin (AM), a member of the 

CGRP family, is involved in the induction of morphine tolerance (see Y. Hong et al, this 

meeting). The current study was designed to explore the mechanisms involved in this 

upregulation. Intrathecal administration of morphine (20 µg, daily) for 6 days induced an

increase in expression of CGRP in small and medium dorsal root ganglia (DRG) neurons and 

CGRP content in the spinal dorsal horn. These responses were significantly blocked when AM22- 

52 (10 nmol), an AM receptor antagonist, was co-administered daily with morphine. An in vitro 

study showed that cultured explants of trigeminal ganglia and DRG from adult rats treated with 

morphine for 6 days at concentrations of 1 and 3.3 µM (but not 10 µM) increased AM levels. 

These increases were attenuated in the presence of protein kinase C inhibitor Gö-6976 (10, 100 

and 1000 nM). The content of CGRP was increased following treatment with AM (0.1, 1 and 10 

nM) at 24 (but not 2) hours. However, treatment with CGRP (1, 10 and 100 nM) for 24 hours did 

not alter the content of AM. Taken together, these data suggest that chronic morphine increased 

AM synthesis at the spinal level through the activation of protein kinase C, and that AM-evoked 

loss of efficacy of morphine analgesia was mediated by increase in CGRP levels. 

Disclosures: J. Chabot , None; Y. Hong, None; A. Fournier, None; R. Quirion, None. 

Poster 





Title: Acute progesterone injection in males rapidly invokes female-typical morphine 

hyperalgesia 

Authors: *C. A. AROUT 1 , A. R. WAXMAN 2 , B. KOWALCZYK 3 , W. F. STERNBERG 5 , B. 

KEST 4 ; 

1 Queens Col, CUNY, Flushing, NY; 2 Neuropsychology, Queens Col., New York, NY; 

3 Neuropsychology, 4 Psychology/Neuroscience, City Univ. of New York, New York, NY; 

5 Psychology, Haverford Col., Haverford, PA 

Abstract: We have previously reported that hyperalgesia during continuous morphine is subject 

to qualitative sex differences in mice. Specifically, infusing a relatively large morphine dose (40 

mg/kg/24 h) caused hyperalgesia of equal magnitude and duration in male and female mice, but 

this hyperalgesia is reversed by NMDA receptor antagonists in males only. A lower morphine 

infusion dose (1.6 mg/kg/24 h) caused hyperalgesia in both sexes which dissipated in males 

within 7 days while continuing unabated in females. Both sex differences were completely 

abolished by ovariectomy and reinstated by chronic estrogen treatment in females, indicating that 

females possess male-like hyperalgesic systems but female sex hormones preclude their use. 

Here, we studied whether males possess female-typical morphine hyperalgesic systems by 

assessing their nociceptive responses on the tail-withdrawal test before and for 30-120 min after

an acute progesterone injection during morphine infusion. Whereas the NMDA receptor 

antagonist MK-801 reversed hyperalgesia during infusion of the large morphine dose, it had no 

effect on males previously injected with progesterone. During infusion of the lower morphine 

dose, progesterone injection rapidly re-instated persistent hyperalgesia in males whose 

hyperalgesia had already dissipated. These data show that intact males rapidly display a femaletypical 

hyperalgesic response to continuous morphine infusion after a single progesterone 

injection, indicating that they possess female-like mechanisms of hyperalgesia that are activated 

by female sex hormones. The ability of progesterone to rapidly feminize the pattern of male 

responses suggests that these effects are mediated by cell surface progesterone receptors, or are 

mediated by the neuroactive steroid metabolites of progesterone that are active at the GABA 

receptor complex. 

Disclosures: C.A. Arout , None; A.R. Waxman, None; B. Kowalczyk, None; W.F. Sternberg, 

None; B. Kest, None. 

Poster 





Support: ARC, Queen's Univ 

CFI 

CIHR 

CRC 

NIDA 

Title: Non-stereoselective effects of the ultra-low dose opioid receptor antagonist on attenuating 

opioid analgesic tolerance 

Authors: *T. M. MATTIOLI, M. SUTAK, B. MILNE, K. JHAMANDAS, C. M. CAHILL; 

Pharmacol. & Toxicology, Queen's Univ., Kingston, ON, Canada 

Abstract: Ultra-low doses of the opioid receptor antagonist, naltrexone, inhibit the development 

of spinal morphine antinociceptive tolerance. In adult rats, administration of three intrathecal

injections of morphine (15 ug) at 90 min intervals significantly decreased the antinociceptive 

effect and agonist potency in both the tail-flick (TF) and paw-pressure (PP) tests. Cumulative 

dose response curves for morphine performed the following day demonstrated a significant 

increase in morphine ED50 value (PP: 31 ug, TF: 29 ug) compared to saline (PP: 5.7 ug, TF: 4.6 

ug). In the PP test, co-injection of ultra-low dose (+) naloxone (0.005 ng) with morphine blocked 

the decrease in both the morphine-induced antinociception and agonist potency (ED50: 2.6 ug). 

Interestingly, this effect was not stereoselective as the (+) isomer of naloxone also blocked both 

responses. In the TF, co-administration of (+) naloxone (0.005 ng) also attenuated the decrease in 

morphine-induced antinocieption as well as the loss of agonist potency (ED50: 2.1 ug). 

However, this stereoisomer did not completely prevent the decline in morphine-induced 

antinociception, but significantly prevented the loss of morphine potency (ED50: 2.2 ug). The 

results demonstrate that ultra-low dose naloxone blocks morphine analgesic tolerance. This 

effect, unlike its classical antagonist action, lacks stereoselctivity and may result from a novel 

receptor mechanism. 

Disclosures: T.M. Mattioli, None; M. Sutak, None; B. Milne, None; K. Jhamandas, 

None; C.M. Cahill, None. 

Poster 





Support: University of Kentucky start-up funds 

ACS RSG-00-027-04-CDD and NIDA Drug Supply 

Title: Effects of transdermal cannabidiol on experimental arthritis 

Authors: *L. ZHANG 1 , F. MA 1 , S. M. ABSHIRE 1 , D. HAMMELL 2 , A. STINCHCOMB 2 , K. 

N. WESTLUND 1 ; 

1 Dept Physiol, 2 Pharmaceut. Sci., Univ. Kentucky, Lexington, KY 

Abstract: Cannabidiol (CBD), a non-psychoactive cannabinoid has been examined for its 

therapeutic potential for relief of neuropathic pain, cancer pain and inflammation by others. 

Transdermal delivery of CBD allows for a more controlled and sustained drug delivery rate. In 

the case of pain control, transdermal delivery would allow for treatment of specific target sites. 

In the present study, a transdermal formulation of CBD gel was evaluated using an animal

arthritis model for effectiveness in inflammation control and pain relief, as well as for 

psychoactive side-effects. Monoarthritis was induced with complete Freund's adjuvant (CFA) 

injected into one knee joint cavity of Sprague Dawley rats after baseline behavioral testing. 

Secondary thermal hyperalgesia and knee joint inflammation reach the peak three days after 

CFA injection. The joint was swollen and animals exhibited less weight bearing. Secondary 

thermal hyperalgesia was assessed with paw withdrawal latency (PWL) testing. Exploratory 

activities were quantified with an open field photobeam activity system (PAS) to evaluate the 

psychoactive effects. The 1% CBD gel was applied on the fourth day of post-CFA injection for 4 

consecutive days to a 3.5 cm 2 , 17.5 cm 2 , or 35.0 cm 2 area of skin on the rats‟ shaved back, 

required for the appropriate dosing of 0.62 mg, 3.1 mg, and 6.2 mg, respectively. Behavioral 

testing was done on each day four hours after drug application. After the fourth day of dosing 

and behavioral testing, animals were sacrificed and blood samples harvested. Blood plasma was 

analyzed for CBD absorption levels. With the 6.2 mg CBD dose, knee joint swelling and 

secondary thermal hyperalgesia were reduced; Subjective pain related posture scores (0-5) 

improved from 4 to 1 in arthritic animals treated with CBD. These studies indicate that 

transdermal CBD has therapeutic potential for effective treatment of arthritis symptomatology. 

Disclosures: L. Zhang, None; F. Ma, None; S.M. Abshire, None; D. Hammell, None; A. 

Stinchcomb, Altranz Corp, A. Employment (full or part-time); ACS RSG-00-027-04-CDD, B. 


grants already received); NIDA Drug Supply, C. Other Research Support (receipt of drugs, 

supplies, equipment or other in-kind support); provisional patent applied for, E. Ownership 

Interest (stock, stock options, patent or other intellectual property); K.N. Westlund, University 

of Kentucky start-up funds, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received); World Congress on Pain honorarium, D. 

Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); American Pain 

Society Fred Kerr Award, D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert 

witness). 

Poster 





Title: Psychostimulant and lesion in the neural motive circuit alter the morphine induced 

analgesia properties

Authors: A. CARRASCO, M. J. LEE, *J. C. WAYMIRE, A. C. SWANN, N. DAFNY; 

Dept Neurobiol & Anat, Univ. Texas- Houston Med. Sch., Houston, TX 

Abstract: The psychostimulant methylphenidate (MPD) is used to treat attention-deficit 

hyperactivity disorder. The target of psychostimulant action, such as MPD, is known to be at the 

motive circuit, which includes ventral tegmental area (VTA), claudate nucleus (CN), and 

prefrontal cortex (PFC). In a previous study, we found out that the pre-treatment with MPD 

modulates intact animals‟ reaction to morphine. This study was conducted to assess the effects of 

bilateral lesions in either VTA, CN, or PFC on the reaction to morphine in the male Sprague- 

Dawley rats pre-treated with MPD. Six groups of rats were used. Three groups of rats had 

chemical lesions in either VTA (N = 7), CN (N = 8), or PFC (N = 7), and other three groups had 

sham-operations in either VTA (N = 5), CN (N = 4), or PFC (N = 4). Chemical lesions were 

created with 6-hydroxydopamine. The MPD treatment protocol was as follows: one day of 

saline, six consecutive daily injections of 2.5 mg/kg MPD, three days of washout, and a rechallenge 

injection of 2.5 mg/kg MPD on the last day, which was followed by three incremental 

morphine injections (0.1 mg/kg, 0.5 mg/kg, and 1.0 mg/kg). The tail-flick latency assay and the 

core body temperature were measured before surgery, one day after surgery, and after the MPD 

and morphine treatment. The result indicated that VTA and PFC lesions strengthened the effect 

of morphine in tail-flick latency (i.e. increased the latency) compared to rats with sham 

operations while the CN lesions decreased the tail-flick latency. The body temperature showed 

only minor, insignificant fluctuations throughout the experimental groups. 

Disclosures: A. Carrasco, None; J.C. Waymire , None; M.J. Lee, None; A.C. Swann, 

None; N. Dafny, None. 

Poster 





Support: Epilepsy Therapy Project 

University of Utah Startup Fund 

Epilepsy Foundation of America 

Title: Systemically active, bioavailable galanin analogs for the treatment of pain

Authors: *E. M. ADKINS 1,3 , M. D. SMITH 1 , G. BULAJ 2 , H. S. WHITE 1 ; 

1 Pharmacol. and Toxicology, 2 Medicinal Chem., Univ. of Utah, Salt Lake City, UT; 

3 NeuroAdjuvants, Inc., Salt Lake City, UT 

Abstract: The neuropeptide galanin displays antinociceptive activity upon both direct injection 

into the brain as well as intrathecal delivery (Gu, et al., Behav. Brain Res., 179:331, 2007; 

Xiong, et al., Regul. Pept. 132:85, 2005). However, neither of these routes of delivery are 

feasible in humans. Furthermore, galanin displays low permeability through the blood-brainbarrier 

(BBB) as well as low metabolic stability (~5 min half-life in plasma). Here we describe a 

modified galanin analog, NAX 5055, that is centrally localized and exhibits antinociceptive 

activity in multiple rodent models of pain when administered systemically. NAX 5055 is a 

rationally designed analog of the truncated galanin neuropeptide that contains lipoamino acid and 

basic residues at the C-terminus. NAX 5055 was chemically synthesized and purified via HPLC. 

The antinociceptive effects of NAX 5055 were evaluated in the formalin test in both male CF-1 

mice and male Sprague-Dawley rats. For formalin tests, NAX 5055 or vehicle was administered 

i.p. to animals at the time-to-peak effect (1 hour) before hindlimb plantar injection of 20 µl of 

5% (mice) or 50 µl of 2.5% (rats) v/v formalin. Animals were observed for flinching behavior 

and the total area under the curve (AUC) and the percent of control AUC were calculated. NAX 

5055 displayed potent antinociceptive activity in both the mouse and rat formalin tests. At 1.5 

mg/kg (mice), and 0.5 mg/kg (rats), flinching decreased (p< 0.01) in the acute phase by 54% and 

64% and in the inflammatory phase by 74% and 64%, respectively. The ability of NAX 5055 (1 

mg/kg, i.p.) to attenuate mechanical allodynia in the rat partial sciatic nerve ligation model was 

evaluated using the von Frey test (Seltzer et al., Pain. 43: 205, 1990). NAX 5055 (2 mg/kg, i.p.) 

elevated the paw withdrawal threshold (507 + 123% of predrug, p






National Natural Science Foundation of China 

Title: Role of adrenomedullin in the development of morphine tolerance 

Authors: *Y. HONG 1,2 , P. CHEN 1 , W. MA 2 , J.-G. CHABOT 2 , R. QUIRION 2 ; 

1 Anat. and Physiol., Fujian Normal Univ., Fuzhou, China; 2 Dept. of Psychiatry, McGill Univ., 

Douglas Mental Hlth. Univ. Inst., Montreal, QC, Canada 

Abstract: The development of opioid tolerance is usually associated with enhancement of 

excitatory neurotransmission. Since adrenomedullin-like immunoreactivity (AM-IR), a 

pronociceptive peptide, was enhanced in the spinal cord and dorsal root ganglia (DRG) during 

chronic inflammation (Hong et al., 2007 Soc. Neurosci Abstr. 33, 25), we became interested to 

investigate if AM at the spinal level could also be up-regulated in morphine tolerance. Intrathecal 

administration of morphine (20 µg, daily) for 6 days induced a progressive decline of its 

antinociceptive effect and a shift to the right of the morphine dose-response curve in the tail flick 

test. Co-administration of the AM receptor antagonist, AM22-52 , in a dose-dependent manner (2- 

10 nmol, i.t.), prevented these responses. However, acute administration of AM22-52 did not alter 

the antinociceptive efficacy of morphine. Chronic treatment with morphine induced significant 

increase in the expression of AM-IR in the superficial layers of the dorsal horn as well as in 

small and medium subpopulations of DRG neurons. Furthermore, chronic administration of 

morphine evoked an increase in the expression of neuronal nitric oxide synthase (nNOS) in small 

and medium DRG neurons. Co-treatment with AM22-52 (10 nmol, i.t.) completely abolished 

morphine-evoked increase in AM-IR and nNOS in the spinal cord and/or DRG. The present 

study suggests that increase in AM quantity at the spinal level is involved in the development of 

morphine tolerance, and that the effect of AM is mediated at least partly by the activation of NO 

signal pathway. 

Disclosures: Y. Hong , None; P. Chen, None; W. Ma, None; J. Chabot, None; R. Quirion, 

None. 

Poster 

270. Pain: Opioids and Other Analgesics II




Title: In vivo characterization of novel, peripherally-acting opioid antagonists 

Authors: *M. S. TODTENKOPF, R. L. DEAN, K. A. RICHIE, K. REITANO, D. ARNELLE, 

D. R. DEAVER; 

Life Sci., Alkermes, Cambridge, MA 

Abstract: Opioid-induced constipation (OIC; an effect of chronic opioid use) is a severe side 

effect that hinders pain management. A potential strategy is to treat with a peripherally acting 

opioid antagonist without affecting analgesia. To that end, a series of non-selective, peripherallyacting 

opioid antagonists were synthesized and preliminary in vivo characteristics were 

examined. These compounds were initially identified as having similar properties as 

methylnaltrexone, but may have greater efficacy and/or bioavailability in rodent models of gut 

motility. Here we report data for four of these compounds (RDC-4253, RDC-0092, RDC-1036 

and RDC-2166) in comparison to methylnaltrexone (MNTX), via intraperitoneal (IP) and oral 

(PO) administration. Each compound was tested in mice in a test of gut motility. PGE2 (a 

prostaglandin) induces diarrhea in mice within 15 minutes of an IP injection (0.1 mg/kg). 

Pretreatment with morphine (1.0 mg/kg, IP) blocks this effect. We administered our compounds 

15 minutes prior to morphine to test their ability to block morphine‟s effects. In addition, we 

examined the duration of action of the minimum effective oral dose for each compound by 

increasing the time between test compound and morphine administration. When given IP, each 

test compound blocked morphine‟s effect comparable to MNTX. When given orally (30 mg/kg), 

RDC-4253 and RDC-0092 were comparable or more effective at blocking morphine‟s effects 

than MNTX, while RDC-1036 blocked morphine‟s effects at a considerably lower dose (10 

mg/kg). Furthermore, when given orally (10 mg/kg), RDC-1036 had a greater effect, quicker 

onset and longer duration of action (up to 8 hours) than MNTX. Finally, the ability of these 

novel antagonists to block morphine-induced antinociception was assessed (PO) in the hot plate 

and tail flick tests. For MNTX, 30 mg/kg (the same effective dose in the PGE2 model) reduced 

morphine-induced antinociception in the hot plate test whereas doses up to 30 mg/kg did not 

affect morphine-induced antinociception in the tail flick test. For RDC-4253, RDC-0092 and 

RDC-2166, 30 mg/kg did not reduce morphine-induced antinociception in the hot plate or tail 

flick test. For RDC-1036, 30 mg/kg slightly reduced morphine-induced antinociception in the hot 

plate test but 10 mg/kg (the minimal effective dose in the PGE2 model) did not. In addition, the 

effective doses in the PGE2 model did not affect morphine‟s analgesic effect in the tail flick test. 

In summary, these novel opiate antagonists are peripherally-acting, orally active, more potent 

and longer acting than MNTX, and may be potential therapeutics for OIC with minimal effects 

on morphine analgesia. 

Disclosures: M.S. Todtenkopf , Alkermes, Inc., A. Employment (full or part-time); R.L. Dean, 

Alkermes, Inc., A. Employment (full or part-time); K.A. Richie, Alkermes.com, A. Employment 

(full or part-time); K. Reitano, Alkermes, Inc., A. Employment (full or part-time); D. Arnelle,

Alkermes, Inc., A. Employment (full or part-time); D.R. Deaver, Alkermes, Inc., A. 


Poster 





Support: NIH Grant DA 19959, to BCY 

Title: Hydromorphone efficacy and treatment protocol impact on tolerance and κ-opioid receptor 

regulation 

Authors: *P. KUMAR, S. SIROHI, S. V. DIGHE, B. C. YOBURN; 

Pharmaceut. Sci., St. Johns Univ., Jamaica, NY 

Abstract: This study examined the role of treatment protocol on hydromorphone-induced 

tolerance and regulation of κ-opioid receptor density. We have previously determined the 

analgesic efficacy (η) of hydromorphone using the operational model of agonism and the 

irreversible κ-opioid receptor antagonist clocinnamox. The calculated η value for 

hydromorphone was 35, which suggested that hydromorphone is a low efficacy opioid agonist. 

To examine hydromorphone-induced tolerance, mice were continuously infused s.c. with 

hydromorphone (2.1-31.5mg/kg/day; ≈ 10-150 times the analgesic ED50) for 7 days. Other 

groups of mice were injected with hydromorphone (2.2-22 mg/kg/day; ≈ 10-100 times the 

analgesic ED50) once, or intermittently every 24 hr for 7 days. The total amount of 

hydromorphone administered per day for injection and infusion studies was the same for all but 

the highest dose group. Twenty-four hr after the last injection, or 16hr following the end of 

infusion, mice were tested using morphine cumulative dosing studies (tail flick). The magnitude 

of tolerance following infusion was similar to that of other low efficacy opioids and greater than 

that previously reported for high efficacy opioid agonists. There was substantially more tolerance 

following infusion treatments with hydromorphone (3.0-24.1-fold decrease in potency) 

compared to intermittent or acute hydromorphone treatment (1.2-2.9-fold decrease in potency). 

Neither infusion nor injection treatments with hydromorphone altered spinal cord κ-opioid 

receptor density. These results support suggestions that analgesic efficacy is correlated with 

tolerance magnitude and regulation of κ-opioid receptors when opioid agonists are continuously 

administered. In addition, treatment protocol (continuous, intermittent, acute) is important in 

determining the magnitude of tolerance.

Disclosures: P. Kumar, None; S. Sirohi, None; S.V. Dighe, None; B.C. Yoburn, None. 

Poster 





Support: NIH/NINDS R01NS043095 

Title: Opioid-induced tactile hypernociception is attenuated by the CXCR4 antagonist, 

AMD3100 

Authors: *N. M. WILSON 1 , H. JUNG 2 , M. S. RIPSCH 3 , R. J. MILLER 2 , F. A. WHITE 3,4 ; 

1 Pharmacol, Loyola Univ. Med. Ctr., Maywood, IL; 2 Molec Pharmacol and Biolog Chem, 

Northwestern Univ., Chicago, IL; 3 Cell Biology, Neurobiology, and Anat., 4 Anesthesiol., Loyola 

Univ. Chicago, Chicago, IL 

Abstract: Morphine and related compounds are the first line of therapy in the treatment of 

moderate to severe pain. Over time, individuals taking opioids can develop an increasing 

sensitivity to noxious stimuli, even evolving into a painful response to previously non-noxious 

stimuli (opioid-induced hyperalgesia; OIH). The mechanism underlying OIH is not well 

understood although complex intracellular neural mechanisms, including opioid receptor 

desensitization and down-regulation, are believed to be major mechanisms underlying OIH. 

However, OIH may also be associated with changes in gene expression. A growing body of 

evidence suggests that cellular exposure to mu agonists upregulate chemokines/receptors and 

recent work from our lab implicates chemokine upregulation in a variety of neuropathic pain 

behaviors. Here we characterized the degree to which chemokines/receptors are upregulated in 

primary afferent neurons of the dorsal root ganglion (DRG) following chronic morphine 

treatment and correlated these changes with hypernociceptive behavior in rodents. We 

demonstrate that the chemokine, stromal-derived factor-1 (SDF-1/CXCL12) is upregulated in 

sensory neurons of both the mouse and the rat. We also determined that there is pronounced 

CXCR4 expression in both satellite glial cells and some sensory neurons of the DRG. Moreover, 

intraperitoneal administration of the specific CXCR4 antagonist, AMD3100, completely 

reversed OIH in the rat. Collectively, the data suggest that opioid-induced SDF-1/CXCR4 

signaling is central to the development of OIH and that receptor antagonists represent a 

promising novel approach to the management of the side effects associated with long term 

opiates for chronic pain control.

Disclosures: N.M. Wilson , None; F.A. White, None; H. Jung, None; R.J. Miller, None; M.S. 

Ripsch, None. 

Poster 





Support: CONACyT 52659 

INP3280SC136 

Title: Antinociceptive effect of Rosmarinus officinalis L. and analysis of some mechanisms of 

action involved 

Authors: *A. L. MARTINEZ-MARTINEZ 1 , M. E. GONZALEZ-TRUJANO 2 , F. J. LOPEZ- 

MUNOZ 1 ; 

1 Pharmacobiology, Cinvestav, Ciudad de México, Mexico; 2 Inst. Nacional de Psiquiatria, Ciudad 

de México, Mexico 

Abstract: Rosmarinus officinalis Linn (RO) is a Labiatae from Mediterranean origin. It is well 

known as a common spice and household plant widely used around the world for medicinal 

purposes. In folk medicine, its aerial parts are used in oral administration to relief renal colic and 

dysmenorrhoea and as antispasmodic. It is prepared as maceration in ethanol and used as topical 

administration to relief rheumatic pain in Mexico. A tea made of the boiled leaves is used to 

improve digestion and to relief stomachache. The purposes of this investigation were: 1) to 

determine the antinociceptive effect of an ethanol extract of RO aerial parts, using an 

experimental model of arthritic pain: “pain-induced functional impairment model in the rat 

(PIFIR model)”. 2) to determine the system opioid and 5-HT1A receptors participation in the 

antinociceptive effect of RO extract. The antinociceptive efficacy of RO extract (30 to 3000 

mg/kg, p.o.) was evaluated using male Wistar rats (180-200 g). The antinociceptive effect of RO 

extract was compared with the antinociceptive effect of tramadol (TR: 3.2 to 31.6 mg/kg, i.p.) 

and acetylsalicylic acid (AA: 31.6 to 562.3 mg/kg, p.o.). Nociception was induced by injection of 

50 κl of 20% acid uric into knee joint of the right hind limb. An electrode was attached on the 

plantar surface of each hinds paw, next the rats was forced to walk on rotatory cylinder and the 

percentage of the functionality index (FI%) was recorded. A pretreatment with antagonist drugs 

like naloxone (1 or 3.2 mg/kg, i.p.) or WAY 100635 (0.12 mg/kg, s.c.) was applied to elucidate 

the system opioid and 5-HT1A receptors participation in the antinociceptive effect. Time courses

and dose-response curves were plotted to detect the antinociceptive efficacies. RO extract like 

either TR or AA, produced a significant (P < 0.001) dose-dependent antinociceptive response in 

rats (RO: ED50 = 222.8 mg/kg versus TR: ED50 = 11.1 mg/kg and AA: ED50 = 206.1 mg/kg). 

Nevertheless, nociception produced by RO extract in the PIFIR model was not modified in 

presence of naloxone but the antinociceptive effect was reverted in presence of WAY 100635 (P 

< 0.05). These results suggest that 5-HT1A receptors are involved in the antinociceptive effect of 

RO extract determined in the PIFIR model. These results strongly suggest that aerial parts of RO 

possess antinociceptive activity, and reinforce the use of this plant in folk medicine. In addition, 

the specific mechanism of action of RO extract is unknown, but this study suggests that 5-HT1A 

receptors are involved in its antinociceptive effect. 

Disclosures: A.L. Martinez-Martinez, None; M.E. Gonzalez-Trujano, None; F.J. Lopez- 

Munoz, None. 

Poster 





Support: Adolor Corporation 

Title: Multifaceted evaluation of opioid-induced microglial responsiveness 

Authors: P. E. STEVIS, M. S. FESCHENKO, H. GUAN, S. W. POSER, *R. T. WINDH; 

Cell and Mol. Signaling, Adolor Corp, Exton, PA 

Abstract: The involvement of glia in the initiation and maintenance of chronic pain states is 

becoming an established premise, fueling intense research efforts aimed at decoding the 

underlying mechanisms. Of particular interest is the possibility that drugs used clinically to treat 

pain may promote glial activation, potentially limiting their analgesic efficacy. For example, mu 

opioid receptor (MOR) agonists such as morphine are reported to activate microglia and 

astrocytes, whereas opioid receptor antagonists such as naloxone inhibit glial activation by LPS. 

However, the mechanisms of these actions have not been conclusively identified, and effective 

concentrations of MOR ligands in these assays are often several orders of magnitude above the 

affinity for MOR. To evaluate the direct effects of MOR ligands on microglial activity and 

explore potential mechanisms, the murine microglial cell line, BV-2, was treated with MOR 

agonists and antagonists. Kinase activation (extracellular signal-regulated kinase (ERK) and p38 

phosphorylation), transcriptional regulation (NF-θB reporter gene activity) and release of factors

(nitric oxide and inflammatory cytokines) were measured. Under conditions where established 

glial-activating stimuli such as LPS and INFγ generated robust responses, no effect of morphine 

or other MOR agonists on microglial activation markers were observed when MOR agonists 

were used alone or with submaximal concentrations of activating stimuli. Furthermore, although 

inhibitors of signaling pathways blocked responses induced by LPS and INFγ, no inhibition by (- 

)- or (+)-naloxone was observed. Comparison with responses in primary rat microglial cultures is 

ongoing. These data suggest that the mechanisms for glial activation by opioids may require the 

presence of other cell types or specific physiological conditions not modeled in these studies. 

Disclosures: P.E. Stevis, Adolor, A. Employment (full or part-time); Adolor, E. Ownership 

Interest (stock, stock options, patent or other intellectual property); R.T. Windh , Adolor 

Corporation, A. Employment (full or part-time); Adolor Corporation, E. Ownership Interest 

(stock, stock options, patent or other intellectual property); M.S. Feschenko, Adolor, A. 

Employment (full or part-time); Adolor, E. Ownership Interest (stock, stock options, patent or 

other intellectual property); H. Guan, Adolor, A. Employment (full or part-time); Adolor, E. 

Ownership Interest (stock, stock options, patent or other intellectual property); S.W. Poser, 

Adolor, A. Employment (full or part-time); Adolor, E. Ownership Interest (stock, stock options, 

patent or other intellectual property). 

Poster 





Support: DA-03816 

Title: Blockade of morphine antinociception by inhibitors of cytochrome P450 and arachidonate 

epoxygenase 

Authors: J. L. CONROY 1 , *J. NALWALK 1 , M. A. VANALSTINE 1 , B. I. KNAPP 2 , J. M. 

BIDLACK 2 , O. P. ZUIDERVELD 3 , R. LEURS 3 , Z. SHAN 4 , S. ZHANG 4 , M. P. WENTLAND 4 , 

L. B. HOUGH 1 ; 

1 Ctr. for Neuropharmacol & Neuroscie, Albany Med. Col., Albany, NY; 2 Dept. of Pharmacol. 

and Physiol., Univ. of Rochester Sch. of Med. and Dent., Rochester, NY; 3 Leiden/Amsterdam 

Ctr. for Drug Res., Vrije Univ., Amsterdam, Netherlands; 4 Dept. of Chem., Rensselaer 

Polytechnic Inst., Troy, NY

Abstract: It is well established that morphine acts at κ-opioid receptors in the brain and spinal 

cord to produce analgesia, but the biochemical cascade following receptor activation remains 

unclear. Recently, a new cimetidine derivative named CC12 was reported to inhibit morphine 

antinociception by an action independent of opioid receptors (Hough et al., 2007). Because CC12 

has been found to have potent cytochrome P450- (CYP-) inhibitory activity (Stadel et al., 2008), 

the effects of several CYP and epoxygenase inhibitors were presently studied on morphine 

antinociception in rats. Nociceptive thresholds were assessed with the hot-plate and tail-flick 

tests. Intracerebroventricular (i.c.v.) administration of morphine (20 κg) produced significant 

increases in both tail-flick and hot-plate latencies, effects which were nearly completely 

abolished by pretreatment with a very low dose (10 nmol i.c.v.) of the CYP inhibitor miconazole. 

However, miconazole had activity at κ, δ, and θ- opioid, as well as histamine H3 receptors (Ki = 

0.6 - 4.5 µM ). A new CC12 derivative, MW-06-25 (N-((4-iodobenzyl) thiomethyl)-imidazole), 

was a potent CYP inhibitor in vitro (CYP2C19, IC50 = 19.1 nM), and blocked morphine 

antinociception in vivo (10 nmol; i.c.v.). MW-06-25 (10 µM) also lacked activity at arachidonate 

cyclooxygenase, 12-lipoxygenase, opioid receptors and H3 receptors. Furthermore, pretreatment 

with the epoxygenase inhibitor MS-PPOH (a non-imidazole, fatty-acid derivative, 30 and 300 

nmol; i.c.v.) resulted in significant, dose-dependent inhibition of morphine antinociception. 

These findings suggest a role for the PLA2/ arachidonic acid/ epoxygenase pathway in κ-opioid 

mediated morphine antinociceptive signaling. Elucidation of morphine‟s downstream mediators 

will enhance understanding of the biochemistry of analgesic circuits. 

Disclosures: J.L. Conroy, None; J. Nalwalk , None; M.A. VanAlstine, None; B.I. Knapp, 

None; J.M. Bidlack, None; O.P. Zuiderveld, None; R. Leurs, None; Z. Shan, None; S. Zhang, 

None; M.P. Wentland, None; L.B. Hough, None. 

Poster 





Support: Department of Anesthesiology 

HL40881 

HL57120 

HL65272

MH45361 

Title: Increase in antinociception caused by microinjection of hypocretin-1 (hcrt-1) into the 

pontine reticular nucleus, oral part (PnO) of Sprague Dawley rat is blocked by the hcrt receptor-1 

(hcrt-r1) antagonist SB-334867 

Authors: *S. L. WATSON, A. S. BATTERSBY, C. J. WATSON, H. A. BAGHDOYAN, R. 

LYDIC; 

Dept Anesthesiol, Univ. Michigan, Ann Arbor, MI 

Abstract: Sleep disruption is a common side effect of pain, but the mechanisms by which pain 

inhibits sleep are not understood. Hcrt-1 is a hypothalamic peptide that enhances wakefulness 

(reviewed in The Orexin/Hypocretin System: Physiology and Pathophysiology, edited by Nishino 

and Sakurai, 2005), and microinjection of hcrt-1 into rat posterior hypothalamus (Pain 109:367, 

2004) or PnO (J Pain 9 Suppl. 2:7, 2008) decreases responses to nociceptive input. This study is 

testing the hypothesis that PnO microinjection of the hcrt-r1 antagonist SB-334867 blocks the 

increase in antinociception caused by PnO microinjection of hcrt-1. Adult male rats (n=11) were 

implanted with microinjection guide tubes. Paw withdrawal latency (PWL) was quantified using 

an IITC Model 336T Paw Stimulator Analgesia Meter with a focused beam of light providing the 

thermal, nociceptive stimulus. In separate experiments, the PnO was microinjected with 100 nL 

of saline (control), hcrt-1 (35.6 ng; 0.1 mM), SB-334867 (0.34 ng; 0.01 mM), and hcrt-1 plus 

SB-334867 (35.6 ng and 0.34 ng, respectively). Post-injection measurements of PWL (s) were 

taken at 10, 20, 30, 60, 90, and 120 min. The PWL measurements were expressed as percent 

maximum possible effect (%MPE) to account for individual animal differences and the thermal 

stimulus cutoff time. Upon completion of testing, all microinjection sites were histologically 

confirmed to be within the PnO. ANOVA for repeated measures indicated a significant drug 

effect on %MPE (F=4.38; df=3, 33; p=0.0114). Tukey/Kramer post hoc comparisons test 

revealed that hcrt-1 significantly (p




Support: NIH Grant GM64830 


Title: Role of M2, M3, and M4 muscarinic receptor subtypes in muscarine-induced spinal 

analgesia revealed using siRNA in rats 

Authors: Y.-Q. CAI 1 , *S.-R. CHEN 1 , H.-D. HAN 2 , G. LOPEZ-BERESTEIN 2 , H.-L. PAN 1 ; 

1 Dept Anesthesiol & Pain Med., 2 Dept of Exptl. Therapeut., Univ. Texas MD Anderson Cancer 

Ctr., Houston, TX 

Abstract: Muscarinic acetylcholine receptors (mAChRs) are involved in the control of 

nociception in the spinal cord. The M2, M3, and M4 subtypes are present in the spinal dorsal 

horn. However, the role of individual mAChR subtypes in the analgesic effect produced by 

mAChR agonists is uncertain. In this study, we determined the contribution of M2, M3, and M4 

subtypes to spinal muscarinic analgesia by using small interference RNA (siRNA) targeting 

specific mAChR subtypes in rats. The neuronal uptake and distribution of siRNA-chitosan 

tagged with a fluorescent dye in the spinal cord and dorsal root ganglion (DRG) were confirmed 

after intrathecal injection. The control and gene-specific siRNA/chitosan complexes were 

injected intrathecally for three consecutive days. Quantitative PCR analysis showed that 

treatment with siRNA targeting M2, M3, and M4 subtypes induced a large reduction in their 

mRNA level in both the DRG and spinal dorsal horn. Also, the receptor protein level in the 

spinal cord was significantly downregulated, as determined by the immunoprecipitation and 

receptor binding assay. Furthermore, intrathecal treatment with the M2 or M4 siRNA 

significantly attenuated the effect of muscarine on the thermal nociception. However, the 

antinociceptive effect produced by intrathecal injection of muscarine was not significantly 

changed by the M3 siRNA treatment. These findings provide new evidence that M2 and M4, but 

not M3, mAChR subtypes contribute to the muscarinic analgesic effect in the spinal cord. The 

chitosan nanoparticles can be used for effective delivery of siRNA into the neuronal tissues in 

vivo. 

Disclosures: Y. Cai, None; S. Chen , None; H. Han, None; G. Lopez-Berestein, None; H. 

Pan, None. 

Poster 

270. Pain: Opioids and Other Analgesics II




Support: NIDA Grant DA 19959 (to B.C.Y) 

Title: Effect of dosing protocol on morphine tolerance 

Authors: *S. V. DIGHE, S. SIROHI, P. MADIA, B. C. YOBURN; 

Col. of Pharm., St. John's Univ., Jamaica, NY 

Abstract: Dosing protocol has been proposed to be an important determinant of the magnitude 

of opioid tolerance. The present study examined the effect of acute, intermittent and continuous 

treatment with morphine on analgesic tolerance. Mice were implanted s.c. with one or two 25mg, 

or one 75mg morphine pellet for 7 days. Other mice were injected with morphine once (18.5- 

185mg/kg/day; ≈ 10-100 times ED50 for morphine analgesia) or once/day for 7 days. Controls 

were implanted with a placebo pellet or injected with saline. Preliminary analysis of drug release 

from morphine pellets indicated that a single 25mg pellet released 15.3±0.6mg morphine in 

7days (≈ 67mg/kg/day; ≈36 times the ED50 for morphine analgesia) and the 75mg pellet released 

23.9±0.9mg in 7days (≈ 123mg/kg/day; ≈66 times the ED50 for morphine analgesia). Pellets were 

removed and 16 hr following the end of implantation, or 24 hr following the last injection, 

morphine cumulative dose-response studies were conducted (tail flick). Continuous treatment 

with morphine using pellet implantation produced a dose-dependent shift in the morphine ED50 

by 3.4, 5.2 and 6.7 for one 25mg pellet, one 75mg pellet and two 25 mg pellets, respectively. 

Intermittent and acute treatment produced minimal tolerance compared to continuous treatment. 

The maximum shift in the ED50 was 1.6 for acute treatment and 2.7 for 7 day intermittent 

treatment, despite the larger total dose. The present results indicate that continuous treatment 

with morphine (i.e., pellets) results in greater development of analgesic tolerance than acute or 

intermittent treatment. These results are consistent with the suggestion that intermittent dosing 

has reduced risk of producing opioid tolerance. 

Disclosures: S.V. Dighe, None; S. Sirohi, None; P. Madia, None; B.C. Yoburn, None. 

Poster 

271. Mechanoreceptors 


Program#/Poster#: 271.1/HH20

Topic: D.09.b. Receptors 

Support: CIHR 

NSHRF 

Title: Mechanisms of GABAA receptor mediated excitation of spider mechanosensory neurons 

Authors: K. PFEIFFER, U. HOEGER, A. S. FRENCH, *P. H. TORKKELI; 

Dept Physiol & Biophys, Dalhousie Univ., Halifax, NS, Canada 

Abstract: Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter. In 

mechanosensory afferents entering the vertebrate spinal cord, and arthropod central ganglia, 

activation of ionotropic GABAA receptors causes inhibition via primary afferent depolarization 

and increased membrane conductance (shunting). Similar inhibition by depolarization and 

shunting occurs in mechanosensory neurons of VS-3 slit-sense organ of the spider (Cupiennius 

salei) when the GABAA receptor agonists GABA or muscimol are applied while the neurons are 

stimulated with mechanical or electrical step stimuli. 

Here we found that when the VS-3 neurons were stimulated with more natural stimuli of varying 

amplitude and frequency (pseudorandom noise) during muscimol application, the initial 

inhibition was followed by excitation lasting up to ten minutes. Frequency response analysis 

during this excitation, followed by power law fitting, indicated that the firing rate, sensitivity, 

adaptation (fractional exponent) and information capacity all increased significantly. 

Using voltage clamp controlled current clamp (VCcCC), which allows action potential recording 

while mean membrane potential is clamped to a desired value, we tested whether depolarization 

of VS-3 neurons alone would be sufficient to elicit the excitatory response. Depolarization by 

10-20 mV during mechanical noise stimulation caused significant increases in the firing rate and 

information capacity, while sensitivity and fractional exponent did not change. However, when 

neurons were clamped to the resting potential during muscimol application, firing rate, 

sensitivity and information capacity increased significantly, while fractional exponent was not 

affected. 

Muscimol increases intracellular Ca 2+ concentration in VS-3 neurons by opening low-voltageactivated 

Ca 2+ channels during depolarization (Panek et al. 2008, J Neurophysiol 99:1596-1606). 

To learn if the excitatory muscimol response depended on Ca 2+ influx, we blocked Ca 2+ channels 

with 100 µM Ni 2+ . In most experiments this led to a 50% reduction in the muscimol-induced 

increase in sensitivity, without significant changes in the fractional exponent or information 

capacity. 

Our results show that when spider VS-3 neurons were stimulated with randomly varying signals, 

the initial inhibitory response to GABAA receptor activation was followed by a long lasting 

excitation. This excitation may be partially mediated by Ca 2+ influx during depolarization. 

However, it also occurred when the neurons were clamped at their resting potential, suggesting 

that additional mechanisms are probably involved. 

Disclosures: K. Pfeiffer, None; U. Hoeger, None; A.S. French, None; P.H. Torkkeli , None.

Poster 




Topic: D.09.a. Cellular and molecular mechanisms of transduction 


Dalhousie Medical Research Foundation 

Nova Scotia Health Research Foundation 

Title: Regional calcium changes in spider mechanoreceptors during sensory transduction 

Authors: U. HOEGER, S. MEISNER, P. H. TORKKELI, *A. S. FRENCH; 

Dept Physiol & Biophysics, Dalhousie Univ., Halifax, NS, Canada 

Abstract: Intracellular Ca 2+ modulates mechanotransduction in auditory and vestibular hair 

cells, where it enters through transduction channels and regulates dynamic sensitivity via several 

mechanisms. There is also indirect evidence for Ca 2+ involvement in vertebrate cutaneous 

mechanoreception, based on labeling of Ca 2+ pumps and Ca 2+ binding proteins. One modulating 

mechanism found in both vertebrate and invertebrate mechanoreceptors is voltage dependent 

negative feedback via Ca 2+ activated K + channels. 

The compound slit sense organ VS-3 in the patella of the spider, Cupiennius salei, consists of 7-8 

cuticular slits, each innervated by a pair of bipolar mechanosensory neurons. VS-3 neurons are 

accessible to intracellular recording and mechanical stimulation in the periphery, where 

mechanotransduction occurs. VS-3 neurons have a low voltage activated Ca 2+ current that can 

produce action potentials when voltage-activated Na + and K + currents are blocked. During 

mechanotransduction, Ca 2+ enters VS-3 neurons via voltage activated Ca 2+ channels when they 

are opened by action potentials. 

VS-3 neurons have three distinct regions in the periphery, comprising the sensory dendrites, 

somata and axons. Some efferent modulation is differentially distributed to these three regions, 

with octopamine and GABAA receptors primarily located on the somata and axons while 

GABAB receptors are concentrated on dendrites and inhibitory glutamate receptors are present in 

all regions. 

Previous experiments showed that intracellular [Ca 2+ ] rises in all regions of VS-3 neurons during 

continuous action potential firing but did not identify the regional distribution of Ca 2+ entry. We 

have now used Ca 2+ sensitive fluorescence measurements to estimate the time course and 

amplitude of intracellular [Ca 2+ ] changes in the three peripheral regions of VS-3 neurons

following single, mechanically elicited action potentials. We also used an antibody against low 

voltage activated Ca 2+ channels to map their distribution across the three regions. Our results 

indicate that Ca 2+ channels are located in all three regions. Mechanical stimulation produces 

action potentials in the sensory dendrites that propagate rapidly through the neurons, causing a 

similar time course and amplitude of intracellular [Ca 2+ ] elevation in all three regions. 

Disclosures: U. Hoeger, None; A.S. French , None; S. Meisner, None; P.H. Torkkeli, None. 

Poster 





Support: JSPS Grant 19791571 

Title: Invovement of GDNF in the periodontal Ruffini endings during regeneration 

Authors: M. OISHI 1 , F. HARADA 1 , *S. WAKISAKA 2 , T. MAEDA 1 ; 

1 Niigata Univ. Grad. Sch. Med. & Dent. Sci., Niigata, Japan; 2 Dept Oral Anat & Dev Biol, 

Osaka Univ. Grad Sch. Dent., Osaka, Japan 

Abstract: The Ruffini ending, categorized in a low-threshold type II stretch mechanoreceptor in 

the periodontal ligament, has a high potential for neuroplasticity. Even in adulthood, this 

mechanoreceptor has been reported to express immunoreactions for TrkB and p75-NGFR, both 

of which can bind BDNF and NT-4/5 to mediate neurotrophic effects. Recently, our research 

group has revealed that the expression of glial cell line-derived neurotrophic factor (GDNF) in 

the periodontal Ruffini endings, suggesting that multiple nurotrophins are involved in their 

development, regeneration, survival and maintenance. However, little information is available 

for the involvement of GDNF in the periodontal Ruffini endings during regeneration. The 

present study was undertaken to examine the expression pattern of GDNF in the periodontal 

Ruffini endings after nerve injury to inferior alveolar nerve (IAN) by Immunohistochemistry. 

Furthermore, the semi-quantitative analysis was employed to clarify the changes in expression of 

GDNF mRNA in trigeminal ganglia during regeneration. Wistar rats (8-week-old) received a 

transaction of IAN. At postoperative day 1, 3, 7, 14 and 28, the animals were fixed with 4% 

paraformaldehyde. Frozen sections of trigeminal ganglia and decalcified lower jaws including 

incisors were prepared and processed for immunohistochemistry for GDNF. Changes in GDNF 

mRNA in the trigeminal ganglia were assessed by real time PCR. In non-surgery group, GDNFimmunoreaction 

was found in the terminal Schwann cells associated with the periodontal Ruffini

endings, never in their axons. In experimental group, transection to inferior alveolar nerve 

induces a disappearance of GDNF in the cellular elements of the periodontal ligament. At 

postoperative day 14 when the terminal branches became thick and expanded, a few terminal 

Schwann cells became positive for GDNF-immunoreaction. At postoperative day 28, 

immunoexpression pattern and number of the terminal Schwann cells with GDNFimmunoreaction 

returned to those in non-surgery group. An analysis with real time PCR showed 

a remarkable elevation of GDNF mRNA at 14 days post-operation. However, immunoexpression 

pattern of GDNF-immunoreaction did not differ in the trigeminal between non-surgery and 

experimental groups; it was localized in the satellite cells, not in the trigeminal neurons 

throughout the experimental period. These findings indicated that GDNF might be involved in 

the regeneration of the periodontal Ruffini endings at maturation stage. 

Disclosures: M. Oishi, None; S. Wakisaka , None; F. Harada, None; T. Maeda, None. 

Poster 





Support: JSPS Grant 20390464 

Title: Immunolocalization of Caveolin-1 and Ca 2+ -ATPase in the periodontal Ruffini endings 

Authors: A. SUZUKI, K. NOZAWA-INOUE, Y. KAWANO, H. AJIMA, *T. MAEDA; 

Dept Oral Anat, Niigata Univ. Grad Sch. Med. Dent., Niigata 951-8514, Japan 

Abstract: Caveolae are omega-shaped plasmalemmal invaginations which play a role in clathrin 

independent endocytosis, trancytosis, signal transduction, and tumor suppression which depend 

on their main constituent proteins caveolin family. Caveolin-1 (Cav-1) has been considered as a 

biochemical marker for caveolae because of its localization in most cells and the absence of 

caveolae formation in Cav-1 deficient mice. The Ruffini ending, a low-threshold type II stretch 

receptor, is a primary mechanoreceptor in the periodontal ligament. Ultrastructural investigations 

have reported that this mechanoreceptor develops caveolae on the cell membrane of both the 

axon terminals and Schwann sheaths, suggesting the existence of axon-Schwann cell interaction 

in the periodontal Ruffini endings. However, little information is available for the functional 

significance of these caveolae. The present study was undertaken to examine immunolocalization 

of Cav-1, -3 and Ca 2+ -ATPase (PMCA) in the periodontal Ruffini endings of the rat incisor for 

clarifying the role of caveola-like structures in mechanotransduction. Decalcified sections of the

upper jaws were processed for immunocytochemistry for CAV-1, -3 and PMCA at the levels of 

light and electron microscopy. Some immunostained sections were treated with histochemistry 

for non-specific cholinesterase (nChE) activity. The periodontal Ruffini endings were 

immunopositive for Cav-1, but not Cav-3. In immunoelectron microscopy, immunoreactive 

products for Cav-1 were confined to caveolae in the cell membrane of the cytoplasmic 

extensions and cell bodies of the terminal Schwann cells associated with the periodontal Ruffini 

endings. However, the axonal membranes of the terminals did not express any Cav-1 

immunoreaction. Immunocytochemistry for PMCA demonstrated intense immunoreaction in the 

periodontal Ruffini endings. Double staining with PMCA and PGP 9.5 disclosed the colocalization 

of immunoreactions in the axonal branches of the periodontal Ruffini endings, but 

not in the nerve bundles in the periodontal ligament. Double labeling with PMCA and S-100 

protein showed the lack of immunoreaction for PMCA in the terminal Schwann cells. Since Ca 2+ 

plays important role in mechanotransduction, these characteristic immunolocalization shows 

Cav-1/ Ca 2+ -ATPase might be involved in quick elimination of intracellular Ca 2+ in 

mechanotransduction. 

Disclosures: A. Suzuki, None; K. Nozawa-Inoue, None; Y. Kawano, None; H. Ajima, 

None; T. Maeda , None. 

Poster 






Title: Rapid adaptation in the Pacinian corpuscle is partially due to GABAergic inhibition 

emanating from the capsular lamellar cells 

Authors: *L. J. PAWSON 1 , L. T. PRESTIA 2,1 , G. K. MAHONEY 1 , B. GUCLU 3,1 , P. J. 

COX 2,1 , A. K. PACK 2,1 ; 

1 Biomed & Chem Eng, Syracuse Univ., Syracuse, NY; 2 Biol., Utica Col., Utica, NY; 3 Biomed 

Eng Inst., Bogazici Univ., Istanbul, Turkey 

Abstract: The Pacinian Corpuscle (PC) is an onion-shaped cutaneous tactile mechanoreceptor, 

which rapidly adapts to sustained pressure (indentation, displacement or force). The receptor 

consists of a nerve ending (neurite), which is encapsulated first by inner-core hemi-lamellae that 

develop from Schwann cells, and then by outer-core lamellar cells that originate from fibroblasts.

The neurite's cell body resides in either the dorsal root or trigeminal ganglia, and the first 

classical synapse for this receptor is thought to be in the spinal cord or medulla. For this reason, 

the mechanism of rapid adaptation (RA) has been attributed solely to the mechanical properties 

of the capsule of the PC. Past experiments in our laboratory have shown positive 

immunoreactivity for glutamate receptors, glutamate transporters, synaptobrevin (VAMP2) and 

SNAP-23 on the inner-core lamellae of the PC. Present results show positive labeling for 

VAMP2 and GABA receptor 1A in the vicinity of the filopodial tips of the neurite. RT-PCR 

results show gene expression for synaptobrevin in the lamellar cells. These results led us to 

hypothesize that chemical inhibition, emanating from the lamellar cells, plays a role in the RA 

response. Electrophysiological results show that isolated mesenteric PCs have an RA response to 

ramp-and-hold stimuli when bathed in Krebs saline. This means that action potentials appear 

during the dynamic (start and end of the on and off ramps) portion of the stimulus while little or 

no spikes appear during the static (hold) portion. However, when GABA antagonists (0.3mM 

gabazine or 4mM picrotoxin) are applied to the bathing solution many spikes appear in the static 

portion of the stimulus and these static spikes disappear completely with the application of 0.5M 

GABA. Hypothesizing that glutamate (either from the lamellae or from the neurite itself) is 

responsible for the static spikes, 20mM kynurenate (a glutamate antagonist) was applied to PCs 

that had some small static response. Once again, the static spikes disappeared while the dynamic 

ones remained. Together these results lead us to suggest a "mechanochemical" rather than purely 

mechanical model for rapid adaptation to sustained pressure in the PC. This glial-neuronal 

interaction is a completely novel finding for the Pacinian corpuscle and for somatosensation. 

Disclosures: L.J. Pawson, None; L.T. Prestia, None; G.K. Mahoney, None; B. Guclu, 

None; P.J. Cox, None; A.K. Pack, None. 

Poster 





Support: HHMI 

GMO55831 

GM067762 

Title: Mechanical responses of cutaneous afferents in ASIC triple KO mice

Authors: *L. XU 1 , M. PRICE 2 , R. BANIK 3 , A. SUBIETA 2 , M. WELSH 2 , T. J. BRENNAN 2 ; 

1 Dept Anesthesia, Univ. Iowa, Iowa city, IA; 2 Univ. of Iowa, Iowa city, IA; 3 New Jersey 

Neurosciences Inst., Edison, NJ 

Abstract: The members of the acid sensing ion channel (ASIC) family are thought to participate 

in mechanosensation. In this study, we utilized the triple knock-out (TKO) mice lacking ASIC1, 

ASIC2 and ASIC 3 genes. Withdrawal to responses to mechanical stimulation and afferent fiber 

responses to mechanical stimulation were measured and compared to wild-type (WT) mice. 

We generated mice with simultaneous disruptions of ASIC1a, -2, and -3 genes by crossing 

previously reported single ASIC null animals and produced them on a congenic C57Bl6 

background. . For behavioral studies, hindpaw withdrawal to frequency to application of 

Semmes-Weinstein monofilaments (0.9, 1.5, 3.4, 6.2, 14.0, and 27.3mN) was measured. We also 

recorded single afferent fiber responses to graded mechanical stimuli in vitro using the mouse 

saphenous skin nerve preparation. 

There was an increased mechanical responsiveness in the TKO vs the WT on days 1 and 3 of 

testing (P

Support: AFOSR MURI Biologically Inspired Flight for Micro-Air Vehicles 

Title: Separate contributions of dorsal and ventral wing-surface tactile receptors to bat flight 

behavior 

Authors: *J. M. ZOOK 1 , B. FALK 2 , S. J. STERBING-D'ANGELO 2 , C. F. MOSS 2 ; 

1 Dept Biol. Sci., Ohio Univ., Athens, OH; 2 Psychology, Univ. of Maryland, College Park, MD 

Abstract: The rich arrays of tactile receptors found on the Microchiropteran bat wing 

membranes are thought to provide feedback on boundary layer air flow necessary for estimations 

of on-going aerodynamic properties of the bat‟s flexible hand-wings (see Zook, 2006). During 

aerial prey capture, the pattern of tactile sensitivity of the wings may play an additional role in 

locating and manipulating prey caught in wing membranes. Cortical and primary afferent 

recordings suggest that certain populations of wing receptors preferentially respond to 

stimulation of one side of the wing membrane with no response to equal or greater stimulation of 

the opposite surface. This apparent mechanical isolation is surprising given that the typical bat 

wing membrane is paper-thin. Here we explore this wing-receptor response segregation and 

suggest some possible roles that it may play in both bat flight and aerial predation. 

Discrimination of dorsal and ventral wing surfaces could enhance tactile feedback to improve the 

bat‟s flight control and prey-capture. In flight, surface-specific responses could enhance the 

detection of boundary flow patterns along either surface possibly through a differential 

amplification of stimuli that are not common to both surfaces. In predation, differential 

amplification of surface specific responses might enhance the resolution of ventral surface 

receptors when handling prey during wing contact and capture. 

Preliminary behavioral experiments with free-flying bats suggest that flight performance can be 

degraded by the removal of wing-dome hairs. In these experiments, we used stereo high-speed 

cameras to track flying bats in 3-D and an array of microphones to track their vocal orientation. 

Baseline data collected from bats before wing manipulations show that they can maneuver to 

avoid obstacles and successfully intercept tethered prey. Experiments are aimed at exploring the 

specific influence of dorsal and ventral wing hair manipulations on flight control in obstacle 

avoidance and prey capture tasks. Future physiological and anatomical studies will be directed 

towards understanding the functional and structural basis of the surface-specific receptor 

response and to test the differential amplification hypothesis. 

JM Zook, 2006, Somatosensory Adaptations in Flying Mammals, In: Evolution of Nervous 

Systems Vol. 3, pp.215-226, J.H. Kaas, Ed., Academic Press: Oxford 

Disclosures: J.M. Zook, None; B. Falk, None; S.J. Sterbing-D'Angelo, None; C.F. Moss, 

None. 

Poster 

271. Mechanoreceptors




Support: NIH Grant DK065742 

EAM award from SIU SOM 

Title: Activation of TRPV4 by direct mechanical force 

Authors: *D. CAO, L. S. PREMKUMAR; 

Pharmacol., Southern Illinois Univ. Sch. Med., Springfield, IL 

Abstract: TRPV4 was first described as an osmosensor and recently it has been shown to be 

activated by heat (>27°C), low pH, anandamide and the phorbol ester derivative, 4aPDD. 

TRPV4 has been shown to be required for hypotonic stimulus-induced nociception and has been 

considered as a mechanotransducer in primary afferent nociceptive nerve fibers. However, it is 

still not clear whether TRPV4 is activated by direct mechanical force. In this study, we have 

investigated the mechanosensitivity of TRPV4 and determined its modulation by PKC. Patch 

clamp technique was used to record single channel current. Application of negative pressure via 

the patch pipette activated single TRPV4 channel activity in cell-attached patches from 

HEK293T cells heterologously expressing TRPV4, but not in vector transfected cells. The single 

channel current has an amplitude of 5.6 ± 0.1 pA corresponding to a conductance of 93 ± 2 pS at 

+ 60 mV. The single channel characteristics are similar to those activated by application of 

4aPDD, a specific TRPV4 agonist. Negative pressure- and 4aPDD- induced single channel 

activity was further potentiated by application of phorbol 12,13-dibutyrate (PDBu), a PKC 

activator. The specificity of PKC action was confirmed by using the PKC inhibitor, 

bisindolylmaleimide. In addition, TRPV4 channel activity could also be induced by sheer flow of 

fluid and by hypotonic solution. These results suggest that TRPV4 is a mechanosensitive channel 

and is sensitized by activation of PKC, which may play a crucial role in mechanosensitive 

responses (This study was supported by grants from NIH (DK065742) and EAM award from 

SIU SOM to L.S.P.). 

Disclosures: D. Cao, None; L.S. Premkumar, NIH DK065742 to L.S.P., B. Research Grant 


received); EAM award from SIU SOM to L.S.P., B. Research Grant (principal investigator, 


Poster 

271. Mechanoreceptors




Support: Natural Sciences and Engineering Research Council of Canada 

Canada Research Chairs Program 

Title: Site of stimulation effects on the prevalence of the tactile motion aftereffect 

Authors: *P. PLANETTA, P. SERVOS; 

Dept Psychol, Wilfrid Laurier Univ., Waterloo, ON, Canada 

Abstract: The motion aftereffect (MAE) refers to the apparent motion of a stationary stimulus 

following adaptation to a continuously moving stimulus. There is a growing consensus that the 

fast-adapting rather than the slowly-adapting mechanoreceptors mediate the tactile version of the 

MAE. In the present study we investigated the basis of the tactile MAE by measuring its 

duration, frequency, and vividness on areas of skin that differ in their distribution of fastadapting 

mechanoreceptors, namely the cheek (hair follicle receptors), volar forearm (hair 

follicle receptors and Pacinian corpuscles), and palmar hand (Meissner and Pacinian corpuscles). 

Thirty healthy right-handed volunteers (25 female, 5 male) participated in 3 sessions with 15 

trials in each (5 trials per skin area). On each trial a ridged cylindrical drum (sine-wave pattern) 

rotated on the skin at 60 rpm for 120 s. Although there was no difference in duration or vividness 

between the skin surfaces tested, the tactile MAE was reported approximately twice as often 

(62%) on the hand trials than the cheek (36%) and forearm (29%) trials, which did not differ 

significantly from one another. This suggests that the tactile MAE can be induced by adapting 

the hair follicle receptors (cheek and forearm) and Meissner corpuscles (hand), but that adapting 

the Pacinian corpuscles (forearm and hand) does not contribute significantly to the prevalence of 

the tactile MAE. 

Disclosures: P. Planetta , None; P. Servos, None. 

Poster 




Topic: D.09.b. Receptors

Support: TUBITAK-0301-COST 

TUBITAK (104S228) 

Title: Effects of serotonin on the frog slowly-adapting Type I mechanoreceptive fibers 

Authors: S. MUTLU, *B. GUCLU; 

Biomed. Engin. Inst., BOGAZICI UNIVERSITY, Istanbul, Turkey 

Abstract: It was previously hypothesized that serotonin may be involved with mechanoelectric 

transduction in the Merkel cell-neurite complex. We recorded single-unit electrophysiological 

responses from the dorsal cutaneous nerves of the common water frog‟s (Rana ridibunda) skin 

perfused in a tissue bath. Static mechanical stimulation was applied at suprathreshold levels by 

von Frey hairs calibrated at 0.16, 0.4, 1, 1.4, and 2 g-bending forces. After classification of 

slowly adapting Type-I fibers, the effect of serotonin was tested at three concentrations: 10 µM 

(n=8), 100 µM (n=8), 1000 µM (n=6). The spike rate increased as a linear function of the 

stimulus level at baseline and all tested concentrations. Additionally, serotonin had significant 

effects on the spike rate in general. Spike rate increased significantly at 10 µM (paired t-test; 

p

Support: NIH MBRS-SCORE grant 1s06GM067078-01A2 

Title: The effect of uncontrolled diabetes on rat movement behavior 

Authors: M. MARTIN, B. SOWELL, C. SPEARS, *V. K. HAFTEL; 

Dept of Biol., Morehouse Col., Atlanta, GA 

Abstract: Over 230 million people in the world are affected by diabetes (type I and type II). At 

least 177 million of these people suffer from secondary complications. Neuropathy is one such 

complication that can lead to numbness, tingling, pain and weakness of hands and feet, and 

movement disorders. Movement disorders in diabetic patients may be due, at least in part to 

deficits in function of proprioceptors mediating posture and gait. Electrophysiological records of 

proprioceptor function collected in this lab (Benedict et al., 2005, SFN abstr.) demonstrate 

significantly increased conduction velocity and altered firing patterns in proprioceptive spindle 

afferent axons following 3 wks of STZ-induced diabetes in rats. Subsequent studies at this early 

stage showed that proprioceptor function could not be explained by gross morphological 

changes, and that movement was not apparently affected (Adams et al., 2007, SFN abstr.). This 

study seeks to expand these data to determine whether longer periods (up to 8 wks) of 

uncontrolled diabetes would cause changes in rats‟ ability to walk across a beam or grid. To do 

so, as before, rats were injected with streptozotocin (55 mg/kg, IP) to create a model of diabetes. 

Rats were checked for diabetes after 48 hours by measuring blood and urine glucose. Blood and 

urine was tested weekly thereafter. The number of hind foot slips was counted when walking 

across a wide metal beam, a grid, or a challenging narrow metal beam each week for 8 wks 

following confirmation of hyperglycemia. Although the average number of slips increased from 

wk 3 to wk 8 for grid and narrow beam walking, it was not significant (wide beam: wk 3, 1.0 +/- 

0.57 slips vs. wk 8, 0.33 +/- 0.33 slips, p = 1.00; grid: wk 3, 0 slips vs. wk 8, 2.0 +/- 1.2 slips, p = 

1.00; narrow beam: wk 3, 0.67 +/- 0.33 slips vs. wk 8, 1.33 +/- 0.88 slips, p = 1.00; mean number 

slips +/- S.E.; repeated measures ANOVA with Tukey HSD posthoc test). Rats did seem to 

compensate their movements (crouching and crawling, hopping, limping) in order to cross the 

apparatus, but these compensations could not easily be quantified. Additional analysis of gait 

was done using the Sciatic Function Index (Bain et al., 1989) to determine whether movement 

disruptions could be detected. Rats demonstrated a loss of function with SFI values < 0 (left 

foot= -21.6, right foot = -18.0, where 0 is normal and -100 is total dysfunction). These data show 

that neuronal changes may take even longer to be reflected in movement behavior, and that 

behavioral changes may manifest in subtle ways. Further information on proprioceptor 

morphology and function will be gathered for the 6-8 week time period, as well as motoneuron 

function. 

Disclosures: M. Martin, None; B. Sowell, None; V.K. Haftel , None; C. Spears, None. 

Poster 

272. Kinematics and EMG: Diseases


Program#/Poster#: 272.2/II1 

Topic: D.12.b. Diseases 

Support: Cleveland Clinic grant (RPC6700) 

Department of Defense grant (DAMD17-01-1-0665) 

NIH grant (NS37400) 

Title: Motor task failure in cancer related fatigue is more attributed to central than muscular 

fatigue 

Authors: *V. SIEMIONOW 1,2 , K. KISIEL-SAJEWICZ 1,4 , A. WYANT 1 , V. K. 

RANGANATHAN 2 , J. HOU 2 , D. SEYIDOVA-KHOSHKNABI 3 , M. T. DAVIS 3 , T. D. 

WALSH 3 , G. H. YUE 1,2 ; 

1 Dept Biomed Eng, 2 Physical Med. and Rehabil., 3 Harry R. Horvitz Ctr. for Palliative Med. 

Taussig Cancer Ctr., The Cleveland Clin., Cleveland, OH; 4 Kinesiology, Fac. of Physiotherapy, 

Univ. Sch. of Physical Educ., Wroclaw, Poland 

Abstract: Our group observed impaired neuromuscular junction (NMJ) transmission function 

and worsened central fatigue with minimal muscle fatigue during voluntary motor activities in 

patients with cancer-related fatigue (CRF). Based on these findings, we hypothesized that muscle 

signals during prolonged motor activities in CRF would experience less changes compared with 

healthy controls. Twelve patients with CRF and 12 age- and gender-matched healthy controls 

performed a sustained isometric elbow flexion contraction of the right arm at 30% maximal level 

(S30) until self-perceived exhaustion. During the S30, surface EMG signals of the elbow flexor 

and extensor muscles and elbow flexion force were recorded. EMG amplitude and power at 

peak, median and mean frequencies were analyzed at the beginning (non-fatigue condition) and 

end (fatigue condition) of the S30. Similar to the reports in the literature, the median frequency 

decreased with muscle fatigue in both groups. However, the value of the median frequency in 

CRF under the non-fatigue condition was similar to the median frequency in controls under the 

fatigue condition of the S30. The power at both the peak and median frequencies was similar 

between the two groups at the beginning of the S30 but the patients experienced a significantly 

smaller increase in median power of the frequency spectrum (from 1.92 mV 2 to 8.23 mV 2 ) than 

the control subjects (from 2.21 mV 2 to 13.87 mV 2 ) at the end of the S30. Similarly, the mean 

power of the spectrum increased from 0.273 mV 2 to 1.029 mV 2 for CRF group and 0.362 mV 2 to 

3.76 mV 2 for controls. Furthermore, the amplitude (root mean square) of the EMG was similar 

between the two groups at the beginning of the S30 but it had a significantly smaller increase at 

the end of the S30 in CRF versus control group. Finally, at the end the S30, frequencies of the 

peak and median power, the power values, and EMG amplitude in CRF were not much different 

from the corresponding values of the controls at the beginning of the S30. These findings suggest 

that although the CRF patients and controls had a similar level of perceived fatigue (they felt 

exhausted and terminated the S30), the level of muscle fatigue at the end of the S30 was less in

CRF than controls. The feeling of exhaustion (severe fatigue) at the end of the S30 in CRF is 

more attributed to central rather than peripheral (muscle) mechanisms. 

Supported by a Cleveland Clinic grant (RPC6700), NIH grant (NS37400) and Department of 

Defense grant (DAMD17-01-1-0665). 

Disclosures: V. Siemionow, None; K. Kisiel-Sajewicz, None; A. Wyant, None; V.K. 

Ranganathan, None; J. Hou, None; D. Seyidova-Khoshknabi, None; M.T. Davis, None; T.D. 

Walsh, None; G.H. Yue, None. 

Poster 

272. Kinematics and EMG: Diseases 




Support: NIH P20 RR16458 

NIH S10RR022396 

Title: Adaptation to perturbed walking in people with medial knee osteoarthritis 

Authors: D. KUMAR, D. S. REISMAN, *K. S. RUDOLPH; 

Physical Therapy, Univ. of Delaware, Newark, DE 

Abstract: Background and Significance: People with knee osteoarthritis (OA) use higher muscle 

activation when exposed to an unexpected perturbation during walking. High muscle cocontraction, 

decreased knee excursions and increased adduction moments can increase knee 

loading, potentially leading to more rapid OA progression. The aim of this study is to investigate 

if people with knee OA fail to adapt their muscle activation patterns during perturbed walking in 

ways that would worsen joint loads. 

Methods: 6 subjects with knee OA and 5 controls walked for 10 trials (locked) and over a 

platform that translated laterally (lateral) at initial contact (IC). Kinematic (120 Hz) and kinetic 

(1080 Hz) were captured for 50 trials. Surface EMG was collected at 1080Hz from quadriceps, 

hamstrings and gastocnemeii . Data were analyzed over 3 intervals - Preparation, loading 

response (LR) and midstance (MSt). Differences between locked and lateral data over the first 5 

trials were compared. 

Results: Prep phase: Controls used similar knee flexion and EMG as in locked trials; OA 

subjects flexed more at IC and adapted by flexing less over 5 trials; OA subjects‟ EMG was 

higher in all muscles and 2 of 3 muscles‟ EMG fell progressively over the 5 trials.

Loading Response: Controls showed no change in knee flexion or EMG from locked trials 

whereas the OA subjects flexed their knees less across all trials and increased knee flexion from 

Trial 2 thru 5. OA subjects had greater EMG for 2 of the 3 muscles across all trials and showed 

no adaptation. 

Midstance: Both groups limited knee extension in trial 1. Controls adapted within 1 trial whereas 

the OA subjects showed a more gradual adaptation over all 5 trials. OA subjects had higher 

EMG all 3 muscles throughout the 5 trials and both groups appeared to adapt over 5 trials. 

Discussion: Differences appeared in preparation for IC where the OA subjects used a stiffening 

strategy involving higher muscle activity and greater knee flexion. During MSt OA subjects 

continued to use higher EMG and less motion but adapted similar to controls. Greater knee 

flexion with higher muscle activity can be a compensation for knee instability but can also 

increase joint loads. The minimal adaptation seen by both groups in prep and LR may be due to 

inadequate time for tuning the long loop reflexes or because adaptation does not occur under 

conditions where joint loads are high. In MSt the higher EMG and slower adaptation in OA 

indicates that joint loads may remain higher than normal which might influence the integrity of 

the cartilage in the joint. The mechanisms and effect of these neuromuscular responses warrants 

further investigation. 

Disclosures: D. Kumar, None; D.S. Reisman, None; K.S. Rudolph , None. 

Poster 





Support: CREST, JST 

Title: Mirror movements induced by inactivation of primary motor cortex is caused by increased 

activation in the contralateral primary motor cortex in monkeys 

Authors: *F. TSUBOI 1,3 , Y. NISHIMURA 4 , K. SAITO 3 , T. ISA 2,3,5 ; 

1 Sch. of Life Sci., 2 Sch. of Life science, Grad.Univ.Advanced Studies, Okazaki, Japan; 3 Dev. 

Physiol., Nat. Inst. Physiol Sci., Okazaki, Japan; 4 Physiol & Biophysics, Univ. of Washington, 

Seattle, WA; 5 CREST, JST, Kawaguchi, Japan 

Abstract: In humans, mirror movements (MMs) often occur after stroke or spinal cord injury. 

Functional brain imaging studies on patients of stroke and spinal injury have shown that activity 

of bilateral motor cortex (M1) is increased accompanying the MMs. However it is not clear

whether the increased activation was the cause of MMs or the result of MMs. To clarify the 

neural mechanism of MMs, we investigated kinematic and dynamic property of MMs using 

acute primate model of M1 lesion induced by reversible inactivation of the hand area in M1 by 

microinjection of muscimol, a GABAA receptor agonist (0.5-3κl). Two monkeys were trained to 

retrieve a morsel of food from the tube positioned in the front with a free hand, while the other 

hand was restricted. EMG activity was recorded from a total of 22 muscles of both hands. After 

inactivation of the hand area of the right M1, movements of the left hand was partially impaired 

when the monkey was required to reach for and grasp a small piece of food. MMs appeared on 

the right hand when the monkey grasped a food piece and/or released it to the mouth with the left 

hand. Mirror EMG activity was then observed in both flexor and extensor hand muscles. Peak 

value of cross-correlogram between homologous muscles on both hands at time-zero lag 

increased after inactivation the right M1, which indicated emergence of mirror activity in both 

hands. Then, muscimol was injected into the hand area of the left M1. After inactivation of the 

left M1, the peak of cross-correlogram disappeared. This reflected disappearance of the MMs 

and mirror EMG activity. These results suggests that following the inactivation of the right M1, 

the activity of the left M1 was increased and led to induction of the MM that appeared in the 

right hand. 

Disclosures: F. Tsuboi, None; Y. Nishimura, None; K. Saito, None; T. Isa, None. 

Poster 





Support: Natural sciences and engineering research council of Canada 

Fond de la recherche en santé du Québec 

Canada foundation for innovation 

Title: Amplitude of normal physiological tremor can be self-modulated 

Authors: *B. CARIGNAN, J.-F. DANEAULT, D. BEAUDOIN, C. DUVAL; 

UQAM, Montreal, QC, Canada 

Abstract: INTRODUCTION: Normal physiological tremor (NPT) is a very small unconscious 

movement, which has an amplitude of ~0.097mm. We recently showed that the amplitude of

oscillations within mechanical components (3-8Hz) was ~0.045mm and central components (8- 

12 & 16-30Hz) were ~0.034mm and ~0.009mm, respectively. 

OBJECTIVE: Determine whether it is possible to self-modulate NPT. 

METHODS: We recorded the postural index finger tremor of 30 subjects (mean 25.7 ± 3.8 

years) with a laser in four conditions: A) eyes closed, without any attempt to self-modulate NPT 

amplitude, B) no visual feedback, trying to reduce NPT amplitude, C) visual feedback, trying to 

reduce NPT amplitude. For B & C, subjects were asked to avoid using muscle contraction as a 

means to stabilise the finger. Finally, D) subjects were asked to reduce NPT amplitude using 

voluntary muscle contraction. We used EMG to monitor the activity of extensor digitorum 

communis and flexor digitorum superficialis. 

RESULTS: We observed that total amplitude of NPT did not change significantly between 

conditions A and B (p>0.05, 0.121 ± 0.053mm & 0.117 ± 0.044mm, respectively). With visual 

feedback (C), a significant decrease of NPT amplitude was observed (p

Support: NIH 1R15NS053442-01A1 

NIH 1R01NS060774-01A2 (pending) 

Title: Breathing controlled electrical stimulation and its long lasting effect on spasticity 

reduction 

Authors: *S. LI 1,2 ; 

1 Sch. Physical Therapy & Rehabil., Univ. Montana, Missoula, MT; 2 Gen. Surgery, Montefiore 

Med. Center/Albert Einstein Col. of Med., Bronx, NY 

Abstract: A new intervention, breathing-controlled electrical stimulation, has been developed 

for finger flexor spasticity management. According to the new intervention, a single electrical 

pulse is triggered and delivered to the finger extensors of the impaired hand when the inspiration 

airflow rate reaches and exceeds the pre-determined threshold. The intensity of electrical 

stimulation depends on patient‟s tolerance. Each of four patients with chronic neurological 

disorders (3 stroke, 1 TBI, a history of at least 18 months) received a 30-min intervention session 

of breathing-controlled electrical stimulation. On average, the Ashworth score was 2.9 before 

and 1.4 immediately after the intervention. Patients were followed up weekly with their regular 

activities and therapies for four weeks. Reduction in spasticity had lasted for four weeks. The 

Ashworth score remained the same (1.4) at the end of follow up. Spasticity reduction has 

significantly improved hand function in one patient (Figure). The results indicated the long 

lasting effect of breathing-controlled electrical stimulation on spasticity reduction in patients 

with chronic neurological disorders. The results also support the neurophysiological mechanisms 

mediating the proposed hypothesis of finger flexion-expiration, finger extension-inspiration 

coupling.

Disclosures: S. Li , NIH grant (1R15NS053442-01A1); Pending: 1R01NS060774-01A2, B. 


grants already received).

Poster 





Support: Creighton University School of Pharmacy and Health Professions Faculty 

Development Grant 

Title: Quantitative relationship between bradykinesia and rigidity in Parkinson‟s disease 

Authors: *R. XIA 1 , H. HAIDER 2 ; 

1 Dept of Physical Therapy, Creighton Univ., Omaha, NE; 2 Dept. of Orthopaedic Surgery and 

Rehabil., Univ. of Nebraska Med. Ctr., Omaha, NE 

Abstract: Bradykinesia and rigidity are primary motor symptoms in Parkinson‟s disease (PD). 

Both symptoms are responsive to therapeutic treatment. Thus, bradykinesia and rigidity are used 

as criteria for clinical diagnosis and as tools for evaluating the efficacy of treatment. The 

objective of this study was to examine the relationship between bradykinesia and rigidity using 

physiological and biomechanical quantification of the two symptoms. Five subjects with PD 

participated in the study under a temporary withdrawal from the treatment. Subject‟s more 

impaired hand was tested while each subject sat on an adjustable chair. To assess bradykinesia, 

subjects were instructed to perform voluntary wrist flexion and extension movements, from 30° 

extended position to 30° flexed position and back to original position, for 10 seconds. Each 

subject conducted movements either at the fastest pace or by following a metronome at 0.4 Hz. 

Subjects were provided with visual feedback regarding their wrist positions. To evaluate rigidity, 

a servomotor generated wrist flexion and extension movement within the same range of motion 

at 50°/sec and 280°/sec. Joint position, wrist torque and electromyograms (EMGs) of the flexor 

and extensor muscles of the wrist and fingers were recorded during both voluntary and 

servomotor-induced movements. Bradykinesia was quantified by the number of voluntary 

flexion/extension cycles completed within 10s duration. Performance of the voluntary movement 

was assessed by the movement error, measured as average degree of over- and under-shooting 

compared to peak amplitude (30°). Rigidity was measured by integrating torque resistance with 

time during movement phases. Pearson‟s correlation coefficients were calculated between 

variables characterizing bradykinesia and rigidity. Moderate correlations exist between 

movement error and torque integral (r = 0.682 for the slower motion; r = 0.564 for the faster 

one). Correlation between the number of movement cycle and torque integral was r = -0.424 

(applicable for the faster movement only). Pilot data suggest that there are correlations between

adykinesia and rigidity. More rigid the wrist joint is, more impaired the voluntary movement of 

the wrist appears. Preliminary findings will be tested on larger sample size. 

Disclosures: R. Xia, None; H. Haider, None. 

Poster 





Title: Improved motor function and cortical activation in Parkinson‟s disease patients following 

acute forced-exercise 

Authors: *J. L. ALBERTS 1 , A. L. RIDGEL 2 , J. L. VITEK 2 , M. J. LOWE 2 , M. D. PHILLIPS 2 ; 

1 Biomed Engin., 2 Cleveland Clin., Cleveland, OH 

Abstract: Previously, we have shown that a long-term lower extremity forced-exercise 

intervention resulted in significant global improvements in motor function of mild to moderate 

Parkinson‟s disease (PD) patients. The global nature of motor improvements suggest that forcedexercise 

may result in changes in central nervous system functioning. The aim of this project was 

to determine the motor and neural effects of an acute bout of forced-exercise in a group of PD 

patients. Motor function and functional MRI examinations were completed, in six mild to 

moderate PD patients, under two randomized conditions: no exercise and three hours after 

forced-exercise. The forced-exercise intervention consisted of one 40-minute session in which 

patients exercised on stationary tandem cycle with an able-bodied trainer. The trainer maintained 

a pedaling rate between 80-90 revolutions per minute (RPMs) which was approximately 35 

percent faster than the patient‟s voluntary pedaling rate. Clinical motor function, assessed with 

the Unified Parkinson‟s disease Rating Scale-III (UPDRS), was nearly 40 percent better 

following forced-exercise compared to the no-exercise condition. During functional MRI 

scanning patients performed a force-tracking task in which they matched a projected sinusoidal 

or constant line. Motor performance was 35 percent better following forced-exercise compared 

to no exercise. Functional MRI analysis of changes in cortical activation volume comparing after 

forced-exercise to no exercise. Findings were most prominent in the SMA which demonstrated 

increased activation after forced exercise in comparison to the no exercise state. This increase in 

activation was a general observation across tasks performed with each limb. Improvements in 

upper extremity function and increased levels of activation within cortical structures such as the 

SMA indicate that forced-exercise may facilitate central motor control processes in PD patients.

Disclosures: J.L. Alberts, None; A.L. Ridgel, None; J.L. Vitek, None; M.J. Lowe, 

None; M.D. Phillips, None. 

Poster 





Title: Visuomotor prism adaptation deficits in Parkinson's disease 

Authors: *R. K. CHONG 1 , K. LEE 1 , J. LEE 2 ; 

1 Dept Phys Therapy, Med. Coll Georgia, Augusta, GA; 2 Sports Sci., Sungkyunkwan Univ., 

Suwon, Republic of Korea 

Abstract: Although researchers seem to generally agree that the basal ganglia (BG) is involved 

in certain aspects of prism adaptation based on the lesion (monkey) and Parkinson‟s disease (PD) 

patient studies, some imaging studies did not show BG activation during kinematic adaptation. 

Even among those studies showing the adaptation deficits, the patterns of deficit vary. Some 

studies report greater spatial errors during the prism-on period accompanied by smaller aftereffects 

while others report normal after-effects. In a recent study, Seidler et al. (2006) suggested 

that the left caudate and GPi involvement in combination with the SMA may contribute to action 

selection processes of adaptation. Based on this idea, we investigated the possibility of „set‟ 

changing (changing internal constraints on action) function of the BG by analyzing the aftereffects 

during re-adaptation following the conventional prism finger-pointing paradigm. 

Adaptations were examined under Full and Terminal vision conditions (1 Hz per cycle). In the 

full vision condition, subjects could see their upper extremity throughout the pointing movement. 

In the terminal vision condition, vision of the upper extremity was blocked and the fingertip only 

became visible when subjects attained terminal pointing. Movement error patterns during the 

prism adaptation phase were not significantly different between age-matched controls and the PD 

groups in both Full and Terminal conditions. However, strikingly, PD patients did not show an 

after-effect in the Full condition, i.e., no significant re-adaptation. In the Terminal condition, on 

the other hand, PD patients showed normal re-adaptation and after-effect. Teulings et al. (2002) 

showed that PD patients did not adapt to changes in the gain of handwriting movements, but 

instead relied on visual feedback. The PD subjects in our study also appeared to have difficulties 

in realigning their sensory frames of reference, and depended heavily on the visual feedback of 

the reaching movement for corrections. Although the error-correcting mechanism may improve 

the performance during the adaptation, the after-effect would not be formed because of the lack 

of realignment processes. Normal adaptation pattern among the PD patients in the terminal

vision condition is a bit puzzling. We believe that the removal of visual feedback during the 

movement makes the error correction impossible and may facilitate a different type of adaptation 

such as trial-to-trial adjustments not requiring the BG circuitry. 

Disclosures: R.K. Chong , None; K. Lee, None; J. Lee, None. 

Poster 





Support: NIH Grant T32 HD041899 

NIH Grant K01 HD050369 

Title: Interlimb coupling during adaptation to a unilateral leg perturbation while walking: results 

in healthy adults and individuals with post-stroke hemiparesis 

Authors: *D. N. SAVIN JR 1 , S.-C. TSENG 1 , S. M. MORTON 1,2 ; 

1 Phys Ther & Rehab Sci., 2 Anat. and Neurobio., Univ. of Maryland Sch. of Med., Baltimore, 

MD 

Abstract: While strict interlimb coupling in the legs is required for proper human locomotion, it 

must also be adaptable in order to accommodate changes in the environment, the body or the 

behavioral goal. Presently, however, little is known about central nervous system mechanisms to 

control and adapt interlimb coupling during human gait and whether the adaptive mechanisms 

are impaired in individuals with post-stroke hemiparesis. To investigate this, we tested a group of 

healthy adults and a group of subjects with chronic stroke and hemiparesis while they walked on 

a treadmill in three successive conditions: Baseline (5 minutes), Adaptation (10 minutes) and 

Post-adaptation (10 minutes). In the Adaptation condition, a novel perturbation was introduced to 

one leg: a weight (mass, 0.9-1.4 kg) was added via a rope and pulley system that attached to the 

shank and resisted forward movement of the leg. We recorded 3D kinematics and 

electromyography from both legs during all conditions to quantify the adaptation and any 

possible aftereffects. We were particularly interested in measuring adaptations of step length and 

toe clearance because stroke commonly results in asymmetric step lengths and poor toe clearance 

during swing phase on the paretic side. All control subjects were able to successfully adapt and 

deadapt to the unilateral lower extremity perturbation within 10 minutes. Interestingly, we 

observed bilateral adaptations in response to this unilateral perturbation. For example during

Adaptation, step lengths initially decreased on the weighted leg and increased on the unweighted 

leg. Both returned to near-baseline values by the end of Adaptation. During Post-adaptation, 

negative aftereffects were also present bilaterally: step lengths initially increased on the 

(previously) weighted side and shortened contralaterally. We also observed aftereffects in 

minimum toe clearance during swing: toe clearance was increased bilaterally for the first several 

strides of Post-adaptation. Some subjects with stroke were able to produce adaptations and 

aftereffects in step lengths and toe clearance that were similar to controls. If the weight was 

applied to the leg with the shorter step length initially, aftereffects produced a temporary 

improvement in symmetry of step lengths and increased toe clearance on the paretic side. Our 

results provide evidence of neural coupling between the legs during locomotion. Adaptations 

occur bilaterally even when a unilateral response is sufficient. In addition, the ability to adapt 

these interlimb coordination patterns may only be minimally affected by stroke involving the 

corticospinal tract. 

Disclosures: D.N. Savin Jr, None; S. Tseng, None; S.M. Morton, None. 

Poster 





Title: Neural control of turning in people with stroke 

Authors: K. DUVAL, K. LUTTMANN, *T. LAM; 

Sch. Human Kinetics, Univ. British Columbia, Vancouver, BC, Canada 

Abstract: Introduction: Gait impairments due to stroke-related hemiparesis can result in severe 

mobility restrictions that limit participation in daily activities. Turning while walking make up a 

significant proportion of all steps taken in a day. However, despite the significance of turning 

during walking in everyday life, as well as the higher risk of falls and injuries while turning, 

there has been very little research on turning capacity in ambulatory individuals with stroke. The 

purpose of this study was to examine how a central nervous system injury, such as a stroke, 

affects the ability to adapt muscular output for curved walking. It was hypothesized that stroke 

participants will show difficulty turning associated with a reduced ability to adapt muscle 

activity in the paretic leg with different degrees of turning compared to able-bodied controls. 

Methodology: Participants with a history of stroke resulting in hemiparesis (>6 months postinjury) 

and age-matched controls were recruited into this study. Participants were asked to walk 

at their self-selected walking speed along four paths of different curvatures: 1) straight path, 2)

large circle (radius = 2 m), 3) medium circle (radius = 1 m) and 4) small circle (radius =0.5 m). 

Surface electromyography (EMG) was used to record activity of several lower limb muscles. 

Adaptations in muscle activity patterns during turning were described by examining changes in 

the amplitude and timing of EMG activity over the gait cycle. Results: All participants tended to 

walk slower when traveling along the small circle compared to the large circle or straight paths. 

Even after walking speed was taken into account, the EMG activity of all muscles in the control 

group tended to decrease from large to small circular paths. In contrast, stroke participants 

showed little adaptation in muscle activity patterns across the different path curvatures. 

Conclusion: Difficulties in turning in people with stroke could be associated with reduced 

capacity to adapt muscle activity patterns to different path curvatures. The results from this study 

could be used to guide the development of gait rehabilitation strategies aimed at maximizing 

functional mobility by including the ability to turn. 

Disclosures: K. Duval, None; T. Lam , None; K. Luttmann, None. 

Poster 





Support: CIHR MOP81366 

NSERC 

Title: Assessment of ipsi- and contralateral reaching following stroke 

Authors: *A. M. CODERRE 1 , S. P. DUKELOW 6 , K. D. MOORE 1,2 , M. DEMERS 6 , H. 

BRETZKE 1 , J. I. GLASGOW 3 , K. E. NORMAN 4 , S. D. BAGG 6 , S. H. SCOTT 1,2,5 ; 

1 Ctr. Neurosci Studies, 2 Anat. and Cell Biol., 3 Sch. of Computing, 4 Sch. of Rehabil. Therapy, 

5 Sch. of Med., Queen's Univ., Kingston, ON, Canada; 6 Physical Med. and Rehabil., St. Mary's of 

the Lake Hosp., Kingston, ON, Canada 

Abstract: Clinical assessments are important in all facets of patient care including diagnosis, 

prognosis and monitoring of rehabilitation programs. Unfortunately, current clinical tests for 

evaluating limb motor function are often subjective and provide little information about the 

underlying neurological impairment. Here we examine whether a visually-guided reaching task 

that requires coordinated participation of many different cortical and subcortical brain areas can 

be used to address this clinical need. The performance of 43 stroke patients was compared to that

of 65 age-matched control subjects using standard clinical assessment tests (Chedoke-McMaster, 

functional independent measure, modified Ashworth and Purdue Peg Board) and a centre-out 

reaching task using KINARM robot. Subjects were included in this study if they had no history 

of other neurological disorders and a Folstein Mini-Mental exam score >24. Subjects were 

instructed to move their hand quickly and accurately from a virtual start target to one of eight 

peripheral goal targets (distance of 10 cm). Ipsilesional and contralesional deficits were 

examined by having all subjects complete the task with both their left and right arms. We 

characterized subject performance by breaking the task into five categories: postural control, 

visuomotor response, total movement metrics, feed-forward control and feedback control. 

Performance during these categories was assessed using 15 movement-related features (e.g. 

postural hand speed, reaction time, total movement time and number of hand speed maxima). 

Using these results we identified 60% of right-affected and 80% of left-affected patients as being 

different from controls. Interestingly, Chedoke-McMaster arm scores only identified 40% of 

right-affected and 37% of left-affected patients as being different from control subjects. In 

addition, while right- and left-affected patients had similar Chedoke-McMaster scores, leftaffected 

patients performed worse with their paretic and non-paretic arms than right-affected 

patients. Finally, to assess the reliability of this robotic technology, we examined inter-rater 

reliability for 12 subjects that were setup in the robot by two different experimenters. We found 

strong inter-rater reliability (r>0.8) for 14/15 of the movement-related features. 

Disclosures: A.M. Coderre, None; S.P. Dukelow, None; K.D. Moore, None; M. Demers, 

None; H. Bretzke, None; J.I. Glasgow, None; K.E. Norman, None; S.D. Bagg, None; S.H. 

Scott, BKIN Technologies, Kingston, Ontario, E. Ownership Interest (stock, stock options, 

patent or other intellectual property). 

Poster 





Support: CIHR 

NSERC 

Title: Robotic assessment of arm position sense in stroke patients 

Authors: *T. M. HERTER 1,2 , S. P. DUKELOW 6,3 , K. MOORE 1,2 , M. DEMERS 6 , H. 

BRETZKE 1 , J. I. GLASGOW 1,4 , K. E. NORMAN 1,5 , S. D. BAGG 6,3 , S. H. SCOTT 1,2,3 ;

1 Ctr. Neurosci Studies, 2 Dept. of Anat. and Cell Biol., 3 Sch. of Med., 4 Sch. of Computing, 5 Sch. 

of Rehabil. Therapy, Queen's Univ., Kingston, ON, Canada; 6 Dept. of Physical Med. and 

Rehabil., St. Mary‟s of the Lake Hosp. and Queen's Univ., Kingston, ON, Canada 

Abstract: Clinical assessments are important for detecting sensory and motor deficits in stroke 

patients, but most tests for evaluating limb position sense are not objective, quantifiable, and/or 

reliable. To address this need, we have developed a novel test of arm position sense using robotic 

technology (KINARM). While the subjects‟ arms and hands are occluded from vision, one arm is 

passively moved by the robot to one of nine spatial locations in the horizontal plane. The 

subjects then actively move their other arm to the mirror location in space. Matching 

performance of forty stroke subjects was compared with fifty age-matched controls by 

quantifying the Cartesian hand positions at the end of each trial. The position of the passive hand 

was first mirrored across the x coordinate, thereby permitting us to compare the actual (active) 

and desired (passive) hand positions. Three quantitative measures of matching performance were 

subsequently calculated: 1) systematic shifts between active and passive hand positions, 2) trialto-trial 

variability of the active hand, and 3) contraction/expansion of the overall spatial area 

spanned by the active hand. Compared to control subjects, approximately two thirds of leftaffected 

and one third of right-affected patients displayed larger systematic shifts, greater 

variability, or increased contraction/expansion. Similar performance was observed whether their 

affected or unaffected arm was employed as the active arm. Furthermore, test-retest experiments, 

carried out by two different assessors, revealed that each measure displayed excellent inter-rater 

reliability despite low accordance with a common clinical evaluation (thumb localization test). 

These results suggest that robotic assessment is potentially valuable for assessing deficits in limb 

position sense in stroke patients. They also indicate that the right hemisphere may make a greater 

contribution to limb position sense. 

Disclosures: T.M. Herter , None; S.P. Dukelow, None; K. Moore, None; M. Demers, 

None; H. Bretzke, None; J.I. Glasgow, None; K.E. Norman, None; S.D. Bagg, None; S.H. 

Scott, SHS is associated with BKIN Technologies which commercializes the robotic technology 

used in this experiment., E. Ownership Interest (stock, stock options, patent or other intellectual 

property). 

Poster 





Support: NSERC

CSN 

Title: Motor patterns typical of early development occur after stroke in skilled reaching 

Authors: *A. FOROUD, I. Q. WHISHAW; 

Neurosci., Canadian Ctr. Behavioural Neurosci,, Lethbridge, AB, Canada 

Abstract: The purpose of this study was to investigate whether the early developmental patterns 

of skilled reaching identified in a previous study occur after stroke in adults. The temporal 

phases of the gestures performed during skilled reaching were compared between twelve stroke 

subjects, their matched controls, and the developmental patterns during early infancy. Results 

show that developmental patterns occur after stroke with respect to skilled reaching. Implications 

for the organization of reaching and recovery after stroke are discussed. 

Disclosures: A. Foroud , None; I.Q. Whishaw, None. 

Poster 





Support: NIDRR Grant H133G070089 

Title: Quantification of finger flexion forces as a function of limb loading in chronic hemiparetic 

stroke and the effects of finger extensor stimulation 

Authors: *L. C. MILLER 1 , M. WIESER 2 , T. KELLER 2 , J. P. A. DEWALD 1 ; 

1 Northwestern Univ., Chicago, IL; 2 ETH Zurich, Zurich, Switzerland 

Abstract: Abnormal torque coupling between shoulder abduction and elbow flexion in 

hemiparetic stroke has been well documented and elbow flexion has been shown to become more 

pronounced with increasing levels of shoulder abduction torque or limb support. However, no 

studies have investigated how this effect is manifested at the fingers and wrist. Therefore, the 

first part of this study investigated whether isometric finger flexion (FF) forces would increase as 

a function of active limb support. 

Using the Arm Coordination Training 3-D (ACT-3D) device, which can alter shoulder abduction 

torques by generating forces along its vertical axis, the paretic arm of 7 individuals with 

hemiparetic stroke and the non-paretic arm of 4 controls were progressively loaded through 11

possible active support levels. At the 5 most difficult active support levels for each participant, 

the isometric forces generated by the fingers/wrist were measured. Subsequently, the effect of 

electrical stimulation of the extensor digitorum communis (EDC) on abnormal FF activity during 

hand opening as a function of active limb support was investigated in the second part of this 

study. At each of the 5 active support levels used in part 1, participants lifted the paretic arm, 

maximally extended the elbow, and tried to open the hand. Each participant completed trials both 

with stimulation of the EDC (STIM) to aid hand opening and without stimulation (NO-STIM). 

Statistical analysis revealed that FF force was significantly higher for the paretic limb compared 

to non-paretic controls at support levels 2-5 (P < 0.001). Furthermore, a significant interaction 

between support level and limb (P < 0.0001) indicated that for the paretic limb, FF forces 

increased incrementally over the support levels, whereas forces for the non-paretic limb 

remained constant. The total change in FF forces from support level 1to 5 over all trials, was 

calculated and the Spearman correlation test indicated a significant linear relationship (rho = 

0.96, P = 0.003) between total change in FF force and impairment level, as measured by Fugl- 

Meyer Assessment Score. For the EDC stimulation protocol, the percent of isometric FF force 

that remained during hand opening as compared to rest was significantly lower for the STIM 

condition compared to the NO-STIM condition, for all support levels (P < 0.0001). 

In conclusion, results show that progressive limb loading of the paretic upper limb incrementally 

increases the FF that occurs in chronic hemiparetic stroke thus extending our previous work 

showing this phenomenon at the elbow. Finally, electrical stimulation of the EDC can overcome 

the FF bias at all support levels. 

Disclosures: L.C. Miller, None; M. Wieser, None; T. Keller, None; J.P.A. Dewald, None. 

Poster 

273. Basal Ganglia: Transmitters and Neuromodulation I 



Topic: D.15.a. Transmitters and neuromodulation 

Support: CIHR MOP-5781 

Title: Dopamine and serotonin innervation of the striatum in Parkinson's and Huntington's 

diseases 

Authors: *C. BEDARD 1 , M.-J. WALLMAN 1 , M. PARENT 2 , A. PARENT 1 ; 

1 Anat. & Physiol., Laval Univ., Quebec, QC, Canada; 2 Montreal Univ., Montreal, QC, Canada

Abstract: The primate basal ganglia receive a dense serotonin (5-HT) innervation, the 

anatomical and functional organization of which is poorly known compared to that of the 

dopamine (DA) innervation. Particularly lacking is information regarding the involvement of 5- 

HT in neurodegenerative diseases affecting basal ganglia. This study aims at characterizing the 

status of the dopamine and serotonin innervation of the striatum in Parkinson‟s (PD) and 

Huntington‟s (HD) diseases. An immunohistochemical approach was applied to post-mortem 

material gathered from patients that suffered from PD and HD and age-matched controls. The 5- 

HT and DA neuronal profiles were visualized with antibodies raised against the 5-HT transporter 

(SERT) and the DA synthesizing enzyme tyrosine hydroxylase (TH), respectively. The pattern 

and the density of each type of innervation were determined by a detailed microscopic scanning 

of coronal sections taken through anterior (pre-commissural), middle (commissural) and 

posterior (post-commissural) levels of the human striatum. Dopamine innervation: A marked loss 

of TH+ fibers and axon varicosities was noted throughout most of the dorsal striatum in PD 

patients. This reduction was more severe at middle level, especially for the caudate nucleus. The 

number of striatal TH+ profiles was also decreased in HD patients compared to controls, with a 

more profound loss in the caudate nucleus. Particularly notorious was the presence of a thin 

(400-500 µm) but intense TH immunoreactive zone located along the ventricular border of the 

atrophied caudate nucleus. A similar zone was detected in controls, but it appeared much less 

intensely stained than in HD patients. Serotonin innervation: The number of SERT+ fibers and 

axon varicosities was increased in PD patients compared to controls. The caudate nucleus in HD 

patients appeared to contain more SERT+ fibers than the same structure in controls while the 

nucleus accumbens contained a decreased number of theses fibers compared to controls. Our 

findings indicate that the dopamine and serotonin innervation of human striatum are differently 

affected in PD and HD, two neurodegenerative disorders characterized by opposite motor 

anomalies. In addition, the increase in SERT+ fibers in PD supports the hypothesis of a 

compensatory serotoninergic mechanism in PD. 

Disclosures: C. Bedard, None; M. Wallman, None; A. Parent, None; M. Parent, None. 

Poster 





Support: United States Public Health grant NS 047452 (ARW) 

NARSAD

Title: Dopaminergic modulation of nitric oxide synthase activity in the nucleus accumbens: 

Histochemical analysis of regional subdivisions 

Authors: *K. E. HOQUE, R. P. INDORKAR, S. SAMMUT, A. R. WEST; 

Rosalind Franklin Univ., North Chicago, IL 

Abstract: The integration of motivational processes within limbic and basal ganglia circuits is 

vital for normal cognition and goal-directed behavior. The nucleus accumbens (NAc) serves as 

an essential functional interface between these two systems. Nitric oxide (NO) is a gaseous 

neurotransmitter synthesized by aspiny interneurons containing neuronal NO synthase (nNOS). 

nNOS activity in the striatal complex can be detected with NADPH-diaphorase staining, a 

histochemical technique that utilizes the NADPH reducing activity of nNOS in fixed tissue. 

nNOS activation in the striatal complex is believed to be NMDA receptor dependent and 

regulated by activation of dopamine D1 and D2 like receptors. In this study, nNOS activity was 

determined using optical density measures of NADPH-diaphorase staining preformed in 

subdivisions (core, medial shell, intermediate shell and lateral shell) of the NAc. Awake 

behaving rats received i.p. injections of either saline, the D1 receptor agonist SKF 81297, the D1 

receptor antagonist SCH 23390 followed by SKF 81297, or the nNOS inhibitor N G -propyl-Larginine 

(NPA) followed by SKF 81297, approximately 25 minutes prior to harvesting the brain 

for staining and analysis. In saline treated animals a distinct medial-lateral histochemical 

gradient of NADPH-diaphorase staining was observed. This gradient was characterized by 

moderate NADPH-diaphorase staining in the core and medial shell and robust staining in the 

intermediate and lateral shell which was approximately three-fold higher than more medial 

subregions. SKF 81297 administration preferentially increased NADPH-diaphorase staining twofold 

in the medial shell. Pretreatment with both SCH 23390 and NPA attenuated the SKF 81297induced 

increase in NADPH-diaphorase staining in the medial shell and decreased staining in the 

intermediate shell as compared to saline controls. These observations demonstrate that nNOS 

activity measured in regionally distinct populations of NOS interneurons in the NAc is 

differentially modulated by D1 receptor activation. Similar studies assessing the impact of 

glutamatergic and D2 receptor activation on nNOS activity in NAc subregions are ongoing. 

Further characterization of the regulation of NOS activation in NAc subregions may facilitate the 

development of novel pharmacotherapies for treating disorders of cognition and motivation. 

Disclosures: K.E. Hoque, None; R.P. Indorkar, None; S. Sammut, None; A.R. West, None. 

Poster 



Program#/Poster#: 273.3/II18


Support: Fondation de France 

NIH-NINDS Grant NS041280 

CNRS 

NIH-NINDS Grant NS040705 

Title: Impact of chronic dopamine depletion on GABAergic transmission in the subthalamic 

nucleus 

Authors: *J. M. BAUFRETON 1 , A. RETAILLEAU 1 , E. KIRKHAM 2 , M. D. BEVAN 2 ; 

1 Inst. des Neurosciences, Univ. Bordeaux 2, Bordeaux, France; 2 Dept. of Physiol., Northwestern 

Univ., Chicago, IL 

Abstract: The activity patterns of subthalamic nucleus (STN) neurons, which are intimately 

related to normal movement and abnormal movement in idiopathic/experimental Parkinson‟s 

disease (PD), are regulated by GABAergic inhibition from the reciprocally connected globus 

pallidus (GP). Pathological hypoactivity of the GP may contribute the high rate of STN activity 

in PD, whereas abnormal patterns of GP input may contribute to emergent, correlated, rhythmic 

bursting STN activity in PD. Dopaminergic regulation of STN neuronal excitability is mediated, 

in part, by presynaptic D2/3 dopamine receptors, which reduce activity-dependent synaptic 

depression at GABAergic GP-STN synapses through a reduction in the initial probability of 

transmitter release. Chronic depletion of dopamine has also been reported to lead to an increase 

in whole-cell GABAA current in STN neurons. We therefore utilized patch clamp recording to 

test the hypothesis that chronic depletion of dopamine leads to a profound alteration in the 

magnitude and pattern of GP-STN synaptic transmission. 

Young rats (17- to 24-days old) were first dopamine-depleted through unilateral stereotaxic 

injection of 6-hydroxydopamine in the medial forebrain bundle or substantia nigra. The degree of 

dopamine depletion was assessed using a forelimb use asymmetry test 6-13 days after surgery 

and immunocytochemistry for tyrosine hydroxylase. Spontaneous and electrically evoked 

GABAA receptor-mediated synaptic currents were then recorded 7-14 days after surgery from 

slices ipsilateral and contralateral to lesions of midbrain dopamine neurons and age-matched 

control animals. 

Dopamine depletion increased the frequency but had no effect on the conductance or kinetics of 

miniature GABAergic transmission recorded in the presence of tetrodotoxin. Electrically evoked 

synaptic transmission also exhibited more profound synaptic depression following dopamine 

depletion. Together these and other recently published data suggest that dopamine depletion 

increases the initial probability of GABA release and/or number of GP-STN synapses. The 

effect(s) of dopamine neuron degeneration on GP-STN transmission may therefore increase the 

capability of the GP to pattern pathological STN activity in PD. 

Disclosures: J.M. Baufreton, None; A. Retailleau, None; E. Kirkham, None; M.D. Bevan, 

None.

Poster 





Title: Intranasal progesterone administration drives dopaminergic activity in the basolateral 

amygdala and neostriatum of rats 

Authors: M. A. DE SOUZA SILVA 1 , B. TOPIC 1 , J. P. HUSTON 1 , *C. MATTERN 2 ; 

1 Inst. of Physiological Psychology, Univ. of Düsseldorf, Düsseldorf, Germany; 2 Mattern 

Pharmaceuticals AG, Stans, Switzerland 

Abstract: The female steroid hormone progesterone has several physiological actions and 

regulates sexual and reproductive behaviors. It also influences memory and emotionality. 

Therapeutically, progesterone has been used as a contraceptive for the treatment of menopausal 

symptoms, and there is evidence that it is neuroprotective following central nervous system 

injury. Since progesterone undergoes a high first-pass metabolism, the intranasal route would 

seem be a more efficient alternative for delivery. In this study we examined the effects of 

intranasal administration of progesterone on dopaminergic activity in the basolateral amygdala 

and neostriatum. We also compared the effectiveness of intranasal vs systemic administered 

progesterone. Male Wistar rats were implanted with guide cannulae aimed at basolateral 

amygdala and neostriatum. Three to 5 days latter, they were anesthetized with urethane, and 

microdialysis probes were inserted into the basolateral amygdala and neostriatum. After a twohour 

stabilization period, four 30-min samples were collected. Thereafter, the treatment (vehicle, 

composed of a viscous castor oil mixture (M et P Pharma AG, Stans, Switzerland; 0.5, 1.0 or 2.0 

mg/kg of progesterone) was applied in a volume of 10µl (5 µl in each nostril). In other animals, a 

subcutaneous injection of progesterone (1.0, 2.0 or 4.0 mg/kg) or vehicle (sesame oil) was given, 

and samples were collected at 30 min interval for 4 h. Samples were immediately analyzed by 

HPLC-ED. After the intranasal administration of 2mg/kg of progesterone there was an 

immediate significant increase in dopamine in the basolateral amygdala. In the neostriatum, the 

2mg/kg dose caused a delayed significant increase of dopamine. Subcutaneous administration of 

4mg/kg of progesterone led to a delayed significant increase of dopamine, both, in the basolateral 

amygdala and neostriatum, but smaller in magnitude in comparison with the intranasal treatment. 

This is the first evidence of neurochemical action of progesterone on the basolateral amygdala. 

We conclude that the intranasal route is more efficient in inducing an increase of dopaminergic 

activity in the brain and that intranasally administered progesterone has differential effects on the 

mesolimbic and nigrostriatal dopaminergic systems.

Disclosures: M.A. de Souza Silva, None; B. Topic, None; C. Mattern , None; J.P. Huston, 

None. 

Poster 





Support: USPHS NS33538 

Title: NR2A and NR2B receptor subunits differentially contribute to NMDA currents in D1- and 

D2 receptor-expressing medium-sized striatal neurons 

Authors: *E. L. JOCOY, V. M. ANDRE, Y. E. FISHER, M. S. LEVINE; 

MRRC, UCLA, Los Angeles, CA 

Abstract: Subpopulations of medium-sized striatal neurons (MSNs) primarily express either D1 

(D1R) or D2 (D2R) dopamine receptors. Both subpopulations express N-methyl-D-aspartate 

receptors (NMDARs) with NR1, NR2A and/or NR2B subunits. This study determined if NR2 

subunit composition affects NMDAR-mediated currents differentially in D1R- and D2Rexpressing 

MSNs. Mice expressing the reporter gene enhanced green fluorescent protein (EGFP) 

under the control of promoters for either D1Rs or D2Rs were crossed with mice in which the 

NR2A subunit was removed (NR2AKO). MSNs from striatal slices were acutely isolated, 

voltage-clamped at -70 mV in 0 Mg 2+ and NMDA was applied to evaluate NMDAR-mediated 

currents. NMDA dose-responses (100, 500 and 1000 κM) were compared between MSNs from 

wildtype (WT) and NR2AKOs. Deletion of NR2A led to significant reductions in NMDARmediated 

currents and current densities in D1R-expressing MSNs compared to those from WT 

littermates. In contrast, currents and current densities did not differ between D2R-expressing WT 

and D2R-expressing NR2AKOs, suggesting the lack of a significant role for NR2A subunits in 

D2R-expressing MSNs. To more specifically examine the role of NR2B subunits, the NR2B 

antagonist ifenprodil (.1, 1 and 10 κM) was applied in the presence of NMDA. Peak NMDARmediated 

currents from D2R-expressing WTs were attenuated by ifenprodil significantly more 

than those from D1R-expressing WTs, suggesting that D2R-expressing MSNs have more 

functional NR2B-containing receptors than D1R-expressing MSNs. Although, ifenprodil 

sensitivity was higher in NR2AKOs than in WTs for both D1R- and D2R-expressing MSNs, no 

differences were observed between D1R-expressing NR2AKOs and D2R-expressing NR2AKOs. 

These data suggest that deletion of NR2A results in similar changes to NR2B-containing 

receptors in D1R- and D2R-expressing neurons and that the lack of a difference in NMDA dose-

esponses between D2R-expressing WT and D2R-expressing NR2AKO cells cannot be 

explained by a simple increase in functional NR2B-containing receptors. In conclusion, NR2A 

and NR2B subunits contribute differently to NMDA currents in D1R- and D2R-expressing 

MSNs. While NR2A contributes a larger component to NMDAR-mediated currents in D1Rexpressing 

MSNs, NR2B does the opposite and contributes a larger component to NMDA 

currents in D2R-expressing MSNs. A better understanding of the differences in NMDA and 

dopamine receptor interactions between D1R- and D2R-expressing MSNs could lead to 

improved therapeutic approaches for Huntington‟s and Parkinson‟s diseases in which these two 

subpopulations are differentially affected in the disorders. 

Disclosures: E.L. Jocoy, None; V.M. Andre, None; Y.E. Fisher, None; M.S. Levine, None. 

Poster 





Support: United States Public Health grant NS 047452 (ARW) 

Title: Impact of striatal NMDA and dopamine D1 receptor interactions on neuronal NOS 

activity: Studies combining in vivo amperometry and reverse microdialysis 

Authors: *D. PARK, A. WEST; 

Neurosci., Rosalind Franklin Univ., North Chicago, IL 

Abstract: Striatal nitric oxide (NO) signaling plays an important role in regulating the 

membrane excitability and synaptic plasticity of striatal neurons. Neuronal NO synthase- (nNOS) 

containing interneurons receive corticostriatal synaptic inputs and are potently activated by 

NMDA receptor stimulation. Our previous studies have shown that electrical and chemical 

stimulation of the substantia nigra and systemic dopamine (DA) D1 receptor agonist 

administration robustly increase striatal NO efflux via a nNOS-dependent mechanism. To study 

the impact of striatal D1 and NMDA receptor interactions on nNOS activity in the intact brain, 

we combined the techniques of amperometry and reverse microdialysis. Amperometric 

recordings of NO efflux were performed in the vicinity of a microdialysis probe implanted in the 

dorsal striatum of urethane anesthetized rats. Consistent with our previous work using systemic 

drug administration, intrastriatal infusion of both SCH-23390 and 7-nitroindazole significantly 

attenuated the increase in NO efflux evoked by systemic SKF 81297 administration. Moreover, 

intrastriatal infusion of the nonspecific ionotropic glutamate receptor antagonist kynurenic acid

or the specific NMDA receptor antagonist CPP significantly attenuated SKF 81297-induced NO 

efflux indicating that the activation of nNOS by D1 receptor stimulation requires concurrent 

activation of striatal NMDA receptors. No significant change in striatal NO efflux was observed 

following glycine co-administration. In separate studies, the ipsilateral frontal cortex was 

activated using electrical train stimulation (30 Hz) and NO efflux was measured during 

intrastriatal infusion of vehicle or SKF 81297. Intrastriatal infusion of SKF 81297 transiently 

increased cortically-evoked NO efflux (measured 10 minutes post-drug) over levels observed 

during local infusion of vehicle. These data demonstrate for the first time that striatal D1 receptor 

activation potentiates nNOS activity stimulated via corticostriatal transmission. Taken together, 

these studies indicate that reciprocal D1-NMDA receptor interactions play a critical role in 

regulating striatal nNOS activity. Given that disruption of nNOS activity has profound effects on 

motor behavior, the characterization of nNOS regulation by DA and glutamate interactions will 

further our understanding of sensorimotor integration within striatal networks involved in normal 

motor function and pathophysiological states. 

Disclosures: D. Park, Rosalind Franklin University, A. Employment (full or part-time); 

Anthony West, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); A. West, Rosalind Franklin University, A. 


Poster 





Title: Dopaminergic and serotonergic activity in the neostriatum and nucleus accumbens is 

increased by intranasal administration of testosterone in rats 

Authors: *J. P. HUSTON 1 , M. A. DE SOUZA SILVA 1 , B. TOPIC 1 , C. MATTERN 2 ; 

1 Inst. Physiological Psychology, Univ. Duesseldorf, Duesseldorf, Germany; 2 M & P Pharma AG, 

Stans, Switzerland 

Abstract: Testosterone decline associated to aging in males is an important factor affecting 

quality of life. Besides the known regulation of reproductive physiological functions and 

behavior, it has also been associated to the regulation of emotionality, reinforcement and 

memory processes. There is evidence that testosterone may be protective in neurodegenerative 

disorders, e.g. Alzheimer's disease, mild cognitive impairment and depression. For targeting the 

brain, the intranasal route of administration of testosterone could be advantageous. In this study

we investigated the effects of the intranasal administration of testosterone on the activity of 

dopaminergic and serotonergic neurons in the brain, and compared the effects to the systemic 

testosterone administration. Male Wistar rats were implanted with guide cannulae at the dorsal 

border of the neostriatum and nucleus accumbens. Three to 5 days later, they were anesthetized 

with urethane and microdialysis probes were inserted. After a stabilization period of 2 hours, 

four baseline samples were collected. Thereafter, the treatment (vehicle, composed of a viscous 

castor oil mixture (M et P Pharma AG, Stans, Switzerland; 0.5, 1.0 or 2.0 mg/kg of testosterone) 

was applied (5µl in each nostril). In other animals, a subcutaneous injection of testosterone (2.0, 

4.0 or 8.0 mg/kg) or vehicle (sesame oil) was given. Samples were collected at 30-min intervals 

for 5 h and analyzed by high performance liquid chromatography. Intranasal administration of 

testosterone significantly increased dopamine (DA) and serotonin (5-HT) levels in the 

neostriatum and nucleus accumbens. In the neostriatum, an immediate and gradual increase of 

DA occurred after the 2mg/kg dose, while a significant increase in 5-HT levels was seen after the 

1mg/kg dose. In the nucleus accumbens, DA and 5-HT significantly increased after, both, the 

1mg/kg and 2mg/kg doses. Subcutaneous administration of testosterone (8mg/kg) caused 

significant increases of DA and 5-HT in the neostriatum. No significant effect was seen in the 

nucleus accumbens after systemic administration. We conclude that the intranasal administration 

of testosterone has immediate effects on the dopaminergic and serotonergic neurotransmission 

and is an efficacious way for targeting the brain. 

Disclosures: J.P. Huston , None; M.A. de Souza Silva, None; B. Topic, None; C. Mattern, 

None. 

Poster 





Support: UPHS NIH NS-23805 

UPHS NIH DA-020654 

Title: The ventromedial tegmental nucleus: a potent contributor to the circuitry of the 

mesopontine tegmentum and likely modulator of multiple ascending neuromodulatory pathways, 

that is targeted by robust outputs from the lateral habenula 

Authors: *D. S. ZAHM 1 , T. C. JHOU 2 , S. GEISLER 3 , M. MARINELLI 4 ; 

1 Dept Pharmacol & Physiol Sci., St Louis Univ. Sch. Med., St. Louis, MO; 2 Dept. of

Psychological and Brain Sci., Johns Hopkins Univ., Baltimore, MD; 3 Behavioral Neurosci. Br., 

Natl. Inst. on Drug Abuse, Baltimore, MD; 4 Cell. & Mol. Pharmacol., Rosalind Franklin Univ. of 

Med. and Science/Chicago Med. Sch., North Chicago, IL 

Abstract: A conspicuous cluster of Fos-immunoreactive neurons dorsolateral to the caudal 

interpeduncular nucleus and in the mesencephalic tegmentum behind it was first noticed 

following the administration of the arousing drug, modafinil. Subsequent reports to be cited in 

the poster indicated that these Fos-ir neurons and likely others at the same site [1] project 

robustly to the VTA/substantia nigra compacta, in part by neurons that express GAD 67, [2] 

modulate conditioned and unconditioned freezing in response to fear-arousing stimuli, [3] 

express Fos in response to various events, including footshock and acute and subchronic 

administration of psychostimulants, and [4] express deltaFosB following chronic 

psychostimulants. Here, we designate this site as the ventromedial tegmental nucleus (VMTg) 

and further describe its neuroanatomy and connections. Non-isotopic hybridization 

histochemistry combined with retrograde labeling reveals distinctly robust GAD 67 expression in 

VMTg and confirms its GABAergic projection to the VTA. VMTg is robustly immunreactive for 

mu opioid receptor and somatostatin. Its cortical afferents, revealed with the aid of Fluoro-Gold, 

occupy rostral polar, prelimbic, infralimbic, dorsal peduncular, orbital and cingulate areas. 

Prodigious subcortical afferents represent all cortico-subcortical functional-anatomical systems, 

including dorsal and ventral striatopallidum, extended amygdala, and septal-preoptic system, as 

well as lateral and posterior hypothalamus, hypothalamic paraventricular n., thalamic 

parafascicular n., zona incerta, supramammillary n., periaqueductal gray, mammillary n., ventral 

lateral geniculate n., deep layers of the contralateral superior colliculus, deep mesencephalic n., 

VTA, nigral compacta and reticulata, red n., pedunculopontine and laterodorsal tegmental nn., 

dorsal raphe, cuneiform n., parabrachial n., deep cerebellar nn., and the pontine and medullary 

reticular formation. The densest retrograde labeling, however, occupies the lateral habenula 

(LHb) and dense, focused anterograde labeling is observed in the VMTg following PHA-L 

injections in LHb. While VMTg outputs to forebrain reach mainly the ventral pallidum-preopticlateral 

hypothalamic continuum, heavier outputs to the brainstem terminate prominently in the 

VTA/substantia nigra compacta, medial tegmentum, pedunculopontine and laterodorsal 

tegmental nuclei, dorsal raphe and locus ceruleus/subceruleus. VMTg may integrate forebrain 

and brainstem inputs in relation to a dominant LHb input. Its outputs to neuromodulatory 

projection systems likely coordinate with direct LHb projections to those structures. 

Disclosures: D.S. Zahm, USPHS NIH NS-23805, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); S. Geisler, 

None; M. Marinelli, USPHS NIH DA-020654, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); T.C. Jhou, 

None. 

Poster 

273. Basal Ganglia: Transmitters and Neuromodulation I




Support: Conacyt Grant 49371M 

Title: Effect of histamine H3 receptor ligands on depolarization-evoked [3H]-D-Aspartate 

release from rat globus pallidus slices 

Authors: A. OSORIO-ESPINOZA, J. RAMOS-JIMENEZ, M. SOTELO-GUZMÁN, *J.-A. 

ARIAS-MONTANO; 

Dept Neurosci, Cinvestav-IPN, Mexico City DF 07000, Mexico 

Abstract: The histamine H3 receptor (H3R) was initially characterized as an autoreceptor 

controlling the synthesis and release of histamine. Further work has shown that H3R activation 

decreases the release of other neurotransmitters, including glutamate, in the central and 

peripheral nervous system. The globus pallidus (GP) is a nucleus involved in the control of 

motor behavior and expresses a high density of H3Rs. The lack of mRNA in this nucleus 

suggests that such receptors are presynaptically located. We therefore set out to determine 

whether presynaptic H3Rs regulate glutamate release in rat GP. 

[ 3 H]-N-methyl-histamine binding to GP membranes revealed a receptor density (Bmax) of 162 ± 

29 fmol/mg protein and dissociation constant (Kd) of 0.58 ± 0.09 nM. Competition binding 

assays resulted in the following rank order of potency: immepip (pKi 9.49 ± 0.13) > clobenpropit 

(pKi 8.83 ± 0.07) > thioperamide (pKi 7.75 ± 0.16). 

For neurotransmitter release assays, GP slices were dissected out from coronal brain slices (350 

κm thick). The slices were then pre-incubated with [ 3 H]-D-aspartate (200 nM) for 30 min before 

being superfused with Krebs-Henseleit solution. [ 3 H]-D-aspartate release induced by high K + (20 

mM) was significantly reduced (-79.3 ± 11.6%, P = 0.02, paired t-test) by the H3R agonist 

immepip (100 nM; see figure). H3R function in GP is not yet established. However, our results 

suggest that H3R activation modulates glutamate release in rat GP.

Supported by Cinvestav and Conacyt (grant 49371M), Mexico. 

Disclosures: A. Osorio-Espinoza, None; J. Ramos-Jimenez, None; M. Sotelo-Guzmán, 

None; J. Arias-Montano , None. 

Poster 







Title: Profiles of Fos expression elicited in multiple brain structures by self- and investigatoradministered 

cocaine and vehicle after one and six sessions 

Authors: *M. MARINELLI 1 , M. L. BECKER 2 , A. J. FREIMAN 2 , S. GEISLER 3 , D. S. 

ZAHM 2 ; 

1 Cell & Molec Pharmacol, Rosalind Franklin Univ/Chicago Med., North Chicago, IL; 

2 Pharmacol. and Physiological Sci., St. Louis Univ. Sch. of Med., Saint Louis, MO; 3 Behavioral 

Neurosci. Br., Natl. Inst. on Drug Abuse, Baltimore, MD

Abstract: Neuroadaptations are widely viewed as a basis for the enduring propensity to relapse 

that persists during long periods following extinguished psychostimulant drug seeking or 

enforced abstention from it. Investigation of such neuroadaptations has long centered on the 

meso-accumbens-prefrontocortical axis, such that less is known about possible neuroadaptations 

in much of the rest of the brain. Inasmuch as psychostimulant-elicited expression of the 

immediate-early gene, c-fos, is commonly a starting point for neuroadaptation, we sought to have 

a fresh look at Fos, the c-fos protein product, in a broad variety of brain structures, including 

some that have been the object of previous attention and are known to neuroadapt. Standard 

immunohistochemistry and computer-assisted counting was used to evaluate the density of Fos 

neurons in several groups of rats: [1] vehicle (n=6) and [2] cocaine (n=6) self-administering for a 

1 hour session; [3] vehicle (n=8) and [4] cocaine (n=9) self-administering for 6 daily 1 h 

sessions; [5] investigator administered-cocaine for 6 daily sessions (n=8); [6] saline (n=6) and 

[7] cocaine (n=4) self-administering for 5 daily 1 h sessions, sacked on day 6. To date groups 1- 

5, have been evaluated in 15 structures. The most common profile observed, Fos expression 

enhanced in all cocaine groups relative to similar saline groups, was observed in the lateral 

septum, lateral part of the lateral habenula (LHb), and several basal ganglia structures, including 

ventral pallidum, caudate-putamen, globus pallidus, substantia nigra reticulata and subthalamic 

nucleus. In several accumbens subterritories, Fos was weakly enhanced relative to the vehicle 

control after 1 session, but not after 6 sessions. Indeed, cocaine-elicited Fos was suppressed in 

the caudomedial accumbens shell relative to the vehicle control after six sessions. In the anterior 

cingulate cortex and medial part of the LHb, cocaine produced enhanced Fos in the 1 day self- 

and 6 day investigator-administered, but not in the 6 day self-administered group. While 

counting remains to be completed in numerous structures, the existing data indicate that distinct 

profiles of cocaine-elicited Fos expression characterize different brain structures and may have 

differing implications vis a vis neuroadaptation. Furthermore, subjective impressions in the day 6 

cocaine-omitted group, still to be verified by counts, indicate that baseline Fos expression is 

suppressed in some structures between self-administration sessions in the repeated cocaine selfadministration 

group (group 7) relative to its control (group 6). Data for all groups will be 

completed in 32 structures by meeting time. 

Disclosures: M. Marinelli , USPHS NIH DA-020645, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already 

received); M.L. Becker, None; A.J. Freiman, None; S. Geisler, None; D.S. Zahm, USPHS 

NIH NS-23805, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 

274. Basal Ganglia: Transmitters and Neuromodulation II 


Program#/Poster#: 274.1/II26


Support: NIH Grant R01NS049474-04 

RR-000165 (Yerkes Center grant) 

Title: Effects of D2-like dopamine receptor activation on neuronal activity in substantia nigra 

pars reticulata and globus pallidus in monkeys 

Authors: *A. HADIPOUR-NIKTARASH 1 , H. LEE 1 , Z. U. KHAN 2 , Y. SMITH 1,3 , T. 

WICHMANN 1,3 ; 

1 Div. Sensory and Motor Systems, Yerkes Natl. Primate Ctr., Atlanta, GA; 2 Dept. of Cel Biol., 

Univ. of Malaga, Malaga, Spain; 3 Dept. of Neurol., Emory Univ., Atlanta, GA 

Abstract: The basal ganglia are a group of subcortical structures that interact with the cerebral 

cortex and thalamus. The striatum, which is the principal recipient of topographically organized 

inputs from cerebral cortex to the basal ganglia circuitry, receives dopaminergic projections from 

substantia nigra pars compacta (SNc). The dopaminergic input from the SNc to the striatum and 

the action of dopamine on striatal neurons have been the subject of extensive analysis. Along 

with nigrostriatal projections, the SNc also sends projections to extrastriatal basal ganglia nuclei 

such as both segments of the globus pallidus (GP) and the substantia nigra pars reticulata (SNr). 

The actions of dopamine on GP and SNr neurons are not well understood. The aim of the present 

study is to assess the effects of D2-like dopamine receptor activation on neuronal activity in SNr 

and both segments of GP in Rhesus monkeys. 

In one monkey, we used combined microelectrode recording-injection probes to microinject a 

D2-like dopamine receptor agonist, quinpirole (2.5 µg/0.5µl, injected in 200 s), into SNr or GP 

while recording extracellular single-neuron activity in close proximity to the injection sites. The 

microinjection of quinpirole reduced the spontaneous firing rates of the majority of recorded 

neurons in the SNr (5/6 cells) and in the internal segment of globus pallidus (GPi, 5/6 cells), 

while it increased spontaneous firing in most neurons in the external pallidal segment (GPe, 7/8 

cells). Each of these structures receives GABAergic and glutamatergic projections, which could 

be modulated by activation of presynaptic D2-receptors. The changes in firing rate could also be 

mediated through activation of postsynaptic D2-like receptors located on target structures. At the 

electron microscopic level, moderate to strong pre-synaptic D2 receptor immunoreactivity was 

found in unmyelinated axons throughout the GPe. Although pre-synaptic D2 labeling was also 

found in the GPi and SNr, the prevalence of labeled elements was lower than in GPe. In the SN, 

strong dendritic labeling was frequently encountered. D2-immunoreactive glial processes were 

found in all areas examined. 

These results show that D2-like receptors activation in the GP and SNr may have strong effects 

on the activity of these extrastriatal nuclei. These effects may help to understand some of the 

effects of dopamine replacement therapies in Parkinson‟s disease. 

Disclosures: A. Hadipour-Niktarash, None; H. Lee, None; Y. Smith, None; T. Wichmann, 

None; Z.U. Khan, None.

Poster 






NIH Grant F32DA020427 

NIH Grant F31DA023340 

NIH Grant F31MH076422 


Title: Cyclic AMP contributes to dopaminergic regulation of corticostriatal plasticity 

Authors: *J. P. BRITT 1 , M. A. KHEIRBEK 1 , J. A. BEELER 1 , Y. ISHIKAWA 3 , X. ZHUANG 1 , 

D. S. MCGEHEE 1,2 ; 

1 Committee on Neurobio., 2 Dept. of Anesthesia & Critical Care, Univ. Chicago, Chicago, IL; 

3 Dept. of Cell Biol. and Mol. Med., New Jersey Med. Sch., Newark, NJ 

Abstract: Dopamine receptor activation is integral to both reinforcement learning and 

corticostriatal synaptic plasticity. The mechanisms underlying dopamine-dependent 

corticostriatal plasticity are presently unknown. Dopamine receptors regulate cyclic AMP 

(cAMP) levels via adenylyl cyclase (AC). The dominant AC isoform expressed in the striatum is 

AC5, which is coupled to dopamine receptors and inhibited by calcium and protein kinase A, 

unlike AC1 that is stimulated by calcium and contributes to hippocampal plasticity. AC5 

deficient (AC5KO) mice have impaired dopamine receptor induced changes in cAMP levels and 

impaired motor learning. Here we test the hypothesis that dopamine-dependent plasticity of 

corticostriatal excitatory synapses is sensitive to changes in AC5 activity and cAMP levels. A 

well characterized form of corticostriatal synaptic plasticity is long term depression (LTD) of 

corticostriatal synaptic inputs to the dorsolateral striatum. On average, a high frequency tetanus 

paired with post-synaptic depolarization in striatal slices from WT mice produces LTD. In slices 

from AC5KO mice, this protocol does not produce any overall long-term changes in synaptic 

strength. LTD at this synapse is dependent upon endocannabinoid suppression of presynaptic 

function. In both AC5KO and WT slices we found robust suppression of presynaptic glutamate 

release by bath applied endocannabinoids. This indicates that in AC5KO mice, the LTD

induction protocol does not result in endocannabinoid release. The basic physiological 

characteristic properties of medium spiny neurons are comparable between the genotypes. We 

hypothesized that in WT mice the LTD induced by high frequency stimulation is dependent on a 

coincident D2 receptor-induced reductions in AC5 activity and cAMP levels. Including 1 mM 

cAMP in the internal solution in WT recordings, which prevents the postsynaptic cell from 

recognizing the drop in cAMP produced by D2 receptor activation, changed the plasticity 

induced by high frequency stimulation on average from LTD to LTP. Overall, these experiments 

suggest that dopamine regulates corticostriatal plasticity by modulating AC5 activity and cAMP 

levels. 

Disclosures: J.P. Britt , None; M.A. Kheirbek, None; J.A. Beeler, None; Y. Ishikawa, 

None; X. Zhuang, None; D.S. McGehee, None. 

Poster 





Support: CONACYT grants 60662 and 46-161-M 

Title: Chronic low-level arsenic (As) exposure causes alterations in locomotor activity of mice 

Authors: U. PACHECO-BARDULLAS 1 , *M. GIORDANO 1 , L. CARRIZALES 2 , J. LIMÓN- 

PACHECO 1 , S. MENDOZA 1 , V. M. RODRIGUEZ 1 ; 

1 Cognitive and Behavioral Neurobio., Inst. Neurobiologia, Querétaro, Qro. 7623, Mexico; 

2 Facultad de Medicina, Univ. Autónoma de San Luis Potosí, San Luis Potosí, SLP, Mexico 

Abstract: Arsenic is a toxic metalloid widely present in the environment. Arsenic exposure is a 

health problem associated with the development of skin cancer, black foot disease, and 

hypertension. Although there are few studies on the neurotoxic effects of arsenic exposure in 

humans, some studies have reported decrements in IQ, and sensory and motor alterations after 

chronic exposure. On the other hand, studies using rodents exposed to doses as high as 10 mg 

As/kg b.w. have found alterations in locomotor activity, brain neurochemistry, behavioral tasks, 

changes in sensory and motor nerves and oxidative stress. In the present study both male and 

female mice (C57BL/6J) were exposed to environmentally relevant doses of arsenic such as 

0.05, 0.5, 5.0 or 50 ppm of As in drinking water for four months, and locomotor activity was 

assessed every month. Male mice treated with 0.5 ppm As showed increments in horizontal

activity and stereotypic counts during the dark phase of the light-dark cycle after four months of 

As exposure. While female mice exposed to 0.05, 0.5 and 5.0 ppm As presented increments in 

horizontal activity and stereotypic counts after two, three, and four months of arsenic exposure. 

These data show that chronic exposure to low doses of As causes hyperactivity in both male and 

female mice, female mice being more susceptible to arsenic. Moreover, dopamine content in 

striatum of female mice decreased with the highest concentrations of arsenic after four months 

exposure (5.0, 50 ppm) (F(4,30)=2.73, p

peristimulus time histograms of dopaminergic neurons recorded in the SNpc showed the 

presence of an excitatory response which was similar in mean duration, mean latency and 

success rate of activation. As previously shown (Morera-Herreras et al., Neuroscience (2008) 

151: 817-823), systemic administration of Γ 9 -THC (1 mg/kg) increased the firing rate of 

dopaminergic neurons. In addition, Γ 9 -THC significantly inhibited the excitation induced by 

electrical stimulation applied in the STN (post-stimulus firing and success rates before and after 

Γ 9 -THC were significantly different (p

availability can affect mitochondrial respiration and therefore modulate DA release indirectly via 

H2O2. KATP-channel-dependent suppression of evoked DA release in striatal slices was the 

primary indicator of changes in modulatory H2O2 generation; H2O2-responsive chloromethyl 

dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence imaging was used to 

confirm changes in levels of reactive oxygen species. Striatal ATP content was also determined 

after incubating slices in varying glucose concentrations. 

Axonal DA release was evoked by either one-pulse or pulse-train stimulation (30 pulses, 10 Hz) 

and extracellular DA concentration ([DA]o) was monitored using carbon-fiber microelectrodes 

and fast-scan cyclic voltammetry in striatal slices in control (10 mM), low (1 mM), and high (30 

mM) glucose media; ionic strength was maintained by NaCl substitution. Inhibition of 

glutathione (GSH) peroxidase with mercaptosuccinate (MCS, 1 mM), which amplifies levels of 

endogenous H2O2, had no effect on one-pulse evoked [DA]o (n = 7), but suppressed pulse-train 

evoked [DA]o by 35% (n = 6) in 10 mM glucose, as previously. Decreasing glucose to 1 mM had 

no effect on either one-pulse or pulse-train evoked [DA]o (n = 6-7), despite a significant 14% 

decrease in tissue ATP content and increased H2O2 generation (n = 12). However, in 1 mM 

glucose, the effect of MCS on pulse-train evoked [DA]o was enhanced to a 55% decrease from 

control (n = 6). Elevating glucose to 30 mM did not alter ATP content (n = 29), but suppressed 

H2O2 generation. Consequently, H2O2-modulated pulse-train evoked [DA]o was significantly 

enhanced in 30 mM glucose (n = 6). Moreover, the usual suppression of pulse-train evoked 

[DA]o by MCS was markedly attenuated in 30 mM glucose (n = 7). By contrast, exogenous H2O2 

(1.5 mM) produced a 40% decrease in pulse-train evoked [DA]o in 30 mM glucose, indicating 

that normal H2O2/KATP-channel dependent inhibition of DA release was intact (n = 6). Overall, 

these data show that glucose modulates pulse-train stimulation evoked DA release by altering 

H2O2 generation, but not ATP content. 

Disclosures: L. Bao , None; C.R. Lee, None; M.E. Rice, None. 

Poster 



Program#/Poster#: 274.6/JJ1 


Support: NIH/NIDA/IRP 

Title: The apparent uniformity of projections to the accumbens and caudate-putamen from the 

ventral tegmental area constitutes a composite of merged patches: A different perspective on the 

mesostriatal projection

Authors: *S. GEISLER, R. A. WISE; 

Behavioral Neurosci. Br., NIH/NIDA, Baltimore, MD 

Abstract: The mesostriatal projection is broadly topographic, such that the medial ventral 

tegmental area (VTA) innervates predominantly the accumbens shell, whereas the lateral VTA 

projects to the accumbens (Acb) lateral shell and core and ventral and medial caudate-putamen 

(CPu), and the substantia nigra compacta (SNc) projects to central, dorsal and lateral parts of the 

CPu, thereby forming dorso-ventrally oriented territories that receive distinct but overlapping 

complements of mesostriatal innervation. Because the striatum projects in part to districts in the 

VTA-SNc more lateral than those from which it receives mesostriatal input, information is said 

to "spiral" lateralward within striato-nigro-striatal circuits. To better understand the nature of 

mesostriatal outputs, we placed small injections of the anterograde tracers PHA-L or BDA into 

different parts of the VTA and visualized the anterogradely labeled fibers 

immunocytochemically. Small and larger injections into rostral and caudal VTA produced 

multiple, patch-like distributions of anterogradely labeled fibers in the Acb shell, core and 

caudate-putamen. Even very small VTA injections resulted in multiple tufts of anterogradely 

labeled fibers widely separated from each other, usually in more than one striatal territory. Small 

injections into the rostral-lateral VTA resulted in dense patches of labeling in the Acb core, and 

at the same time, in patches in the medial and ventromedial Acb shell and central and ventral 

CPu. Injections into the caudal VTA produced several patches of anterogradely labeled fibers in 

the dorsomedial and medial shell, and also in the ventral shell, core, dorsomedial and ventral 

CPu. This mesostriatal pattern of labeling differs considerably from that of the VTA projections 

to, e.g., the septum and hypothalamus, where patches were not observed and single tracer 

injections produced contiguous labeling. While these new mesostriatal data are consistent with 

the broadly overlapping domains of retrogradely labeled neurons reported to characterize the 

VTA-nigral complex following injections of retrograde tracer in the striatum, the data indicate 

that neighboring VTA neurons do not necessarily innervate adjacent areas of the striatum. To the 

contrary, adjacent neurons may simultaneously modulate the activity of widely separated 

neuronal ensembles in the Acb shell and core and the CPu. How this kind of organization 

integrates with the "spirals" and whether it contributes to recently described functional 

microenvironments in the Acb remains to be learned. 

Disclosures: S. Geisler , None; R.A. Wise, None. 

Poster 




Topic: D.15.a. Transmitters and neuromodulation

Support: This research was supported by a grant (#R13-2002-005-04001-0) from MRC for Cell 

Death Disease Research Center Program funded by the Ministry of Science and Technology of 

Republic of Korea. 

Title: Involvement of cannabinoid receptors on ethanol induced depression of corticostriatal 

synaptic transmission 

Authors: *H. CHO 1,3 , K. KIM 1,3 , S. JEUN 1,3 , S. CHOI 1,3 , S. HAHN 2,3 , K.-W. SUNG 1,3 ; 

1 Dept. of Pharmacol., 2 Dept. of Physiol., The Catholic Univ. of Korea, Seoul, Republic of Korea; 

3 MRC for Death Dis. Center, The Catholic Univ. of Korea, Seoul, Republic of Korea 

Abstract: It has been implicated that the dorsal striatum plays a key role in habit formation, and 

compulsive alcohol or drug taking and seeking behavior. The dorsal striatum receives 

glutamatergic afferent inputs from the cortex and other brain areas, and these glutamatergic 

synaptic transmissions may play an important role in behavioral actions related to habit 

formation. The purpose of this study was to examine the role of endogenous cannabinoid 

systems on ethanol induced depression of synaptic transmission in the rat dorsal striatum. 

Acute treatment of ethanol (10 - 150 mM) for 10 minute decreased the corticostriatal excitatory 

postsynaptic currents (EPSCs) in a dose-dependent manner. In the presence of AM 251 (5 κM), a 

selective cannabinoid 1 (CB1) receptor antagonist, the inhibition of corticostriatal EPSCs by 

ethanol was significantly attenuated. However, AM 404 (2 κM), an anandamide transport 

inhibitor, had no significant effect on the inhibition of corticostriatal EPSCs by ethanol. These 

results suggest that ethanol may not relate with endocannabinoid release, but might act directly 

on CB1 receptors. To further clarify our hypothesis, brain slices were pre-treated with WIN 

55,212-2 (1 κM), a CB1 receptor agonist, and ethanol did not inhibit corticostriatal EPSCs under 

this condition. This data indicates that when CB1 receptors are previously activated by its 

agonist, the effect of ethanol on corticostriatal EPSCs is blunted. 

Taken together, our results suggest that CB1 receptors are involved in the inhibition of 

corticostriatal synaptic transmission by ethanol and might be a direct target for ethanol. 

Disclosures: H. Cho, None; K. Kim, None; S. Jeun, None; S. Choi, None; S. Hahn, None; K. 

Sung, None. 

Poster 





Support: CONACyT 50427Q 

Title: Dopamine modulates the input from globus pallidus to thalamic reticular nucleus via 

presynaptic D4 receptos 

Authors: D. GASCA 1 , A. HERNÁNDEZ 1,2 , A. SIERRA 1 , R. VALDIOSERA 1 , B. FLORÁN 1 , 

D. ERLIJ 1 , *J. ACEVES 1 ; 

1 Dept Physiol Biophy & Neurosci, Ctr. Investigacion Del IPN, Mexico DF 07000, Mexico; 2 Inst. 

de Fisiología Celular, UNAM, Mexico D.F., Mexico 

Abstract: Thalamic reticular nucleus (TRn) receives dopaminergic afferents from substantia 

nigra pars compacta (SNc) and GABAergic afferents from globus pallidus in the rat. In addition, 

D1 and D4 dopamine receptors are expressed in the TRn. Our laboratory previously has shown 

that D4 dopamine receptors inhibit the depolarization-induced [H 3 ]GABA release in TRn slices 

(Florán et al., 2004a), suggesting a presynaptic modulation of the GABAergic transmission. Here 

we have studied the source of the inhibitory synaptic currents modulated by D4 dopamine 

receptors in TRn slices from neonatal (14-21 days) rats. 

The inhibitory postsynaptic currents were evoked by local electrical stimulation (eIPSCs) in the 

presence of CNQX and AP-5. The miniature inhibitory posynaptic currents (mIPSCs) were 

recorded in the presence of TTX. At the end of the experiments the synaptic currents were 

blocked by bicuculline, confirming their GABAergic nature. 

The amplitude of the eIPSCs was reduced 40% by PD168,077 (selective D4 agonist) and this 

effect was blocked by L-745870 (selective antagonist). This effect was accompanied by a 23% 

increase in the paired pulse ratio, indicating a presynaptic effect. It was also observed that 

PD168,077 reduced by 40% the frequency of the mIPSCs without affecting their amplitude. This 

effect was blocked by L-745870, indicating that it was mediated by a specific activation of D4 

dopamine receptors. Dopamine also inhibited both eIPSCs and mIPSCs, and the inhibition was 

blocked by L-745,870. These results together indicate a dopamine modulation of GABAergic 

synaptic transmission by presynaptic D4 dopamine receptors in the TRn. Previous lesion of the 

globus pallidus eliminated completely the effect of the activation of D4 dopamine receptors on 

the GABAergic transmission, judged by its effect on evoked or spontaneous IPSCs. These results 

indicate that D4 dopamine receptors are located on the GABAergic pallidal terminals in the TRn, 

where they modulate the pallidal input. 

Disclosures: D. Gasca, None; J. Aceves , None; A. Hernández, None; A. Sierra, None; R. 

Valdiosera, None; B. Florán, None; D. Erlij, None. 

Poster 





Support: NIAAA DISCBR 

Title: Effects of D1 and D2 type dopamine receptor blockade on striatal activity and synchrony 

Authors: *J. M. BURKHARDT 1,2 , R. M. COSTA 1 ; 

1 Natl. Inst. of Alcoholi, Natl. Inst. of Hlth., Bethesda, MD; 2 Physiol. & Pharmacol., Wake Forest 

Univ., Winston-Salem, NC 

Abstract: In Parkinson's disease (PD) loss of the dopaminergic (DA) neurons in the substantia 

nigra pars compacta eventually leads to motor dysfunction and akinesia. We have previously 

shown that DA depletion leads to both changes in firing rate and in synchrony in the basal 

ganglia. Since D1 and D2 receptors are preferentially expressed in striatonigral and 

striatopallidal medium spiny neurons, respectively, we decided to investigate the role of D1 and 

D2 receptors in DA control of corticostriatal synchrony and voluntary movement. We 

chronically implanted mice with multielectrode arrays in the dorsolateral and dorsomedial 

striatum and investigated the effects of blocking both D1 and D2 receptor types, or D1 or D2 

alone, on movement, firing rate, and synchrony of striatal medium spiny neurons. Intraperitoneal 

administration of a D1 antagonist (SCH-23390, 0.4 mg/kg i.p.) or a D2 antagonist (raclopride, 

2.0 mg/kg i.p.) substantially reduced locomotion, and co-administration of D1 and D2 

antagonists rendered the mice completely akinetic. In vivo recordings were performed for 30 

minutes prior to and 60 minutes subsequent to dopamine receptor blockade or saline 

administration. We found that blockade of D1+D2 receptor types changed the firing rate of the 

majority of putative medium spiny neurons recorded, with a net decrease on average firing rate. 

Blockade of both D1+D2 receptors also caused an increase in crosscorrelated activity and in the 

number of neurons with activity entrained to the local field potential (LFP) oscillations. 

Blockade D2 receptors alone was sufficient to produce changes in striatal firing rate with a net 

decrease in rate after injection, and also a substantial increase in synchrony. However, D1 

blockade seemed to affect rate differently than D2. Although the majority of neurons changed 

firing rate after D1 blockade, the net population vector did not change. Also, the effects of D1 

blockade on synchrony seemed less pronounced than the effects of D2 blockade, and D1+D2 

blockade. Taken together, these results suggest that both D1 and D2 receptors contribute to the 

changes observed in basal ganglia circuits after dopamine depletion, but D2 receptors seem to 

have a more prominent role in decreasing average firing rate and increasing synchrony. Because 

D2 receptors are expressed in different cell types in the basal ganglia, and also in different 

compartments within the cell (pre and post synaptic), we need to investigate if these effects are 

caused by direct changes in medium spiny neurons, or by changes in the circuit, for example by 

modulation of fast-spiking striatal interneurons or large aspiny cholinergic interneurons. 

Disclosures: J.M. Burkhardt, None; R.M. Costa, None.

Poster 





Title: Nigral motor activity-induced dopamine release is locally regulated by muscarinergic 

receptors but not nicotinergic 

Authors: *D. R. ANDERSSON, F. BERGQUIST, H. NISSBRANDT; 

Dept Pharmacol, Univ. Gothenburg, Gothenburg, Sweden 

Abstract: In the last decade, a role of nigral somatodendritic dopamine (DA) release in 

regulating motor activity and muscle tone has been demonstrated. Recently, we showed that a 

reduction in the release of DA in the substantia nigra (SN) decreases motor performance as 

measured by rotarod in rats, without affecting striatal DA release, indicating a more direct role of 

somatodendritic DA than that of regulating nigral dopaminergic autoreceptors. In the same study, 

we also demonstrated an increase in nigral DA release during motor activity, but the regulatory 

mechanisms of this increase are as of yet unknown. 

In the current study, we utilized simultaneous microdialysis and rotarod testing to investigate a 

possible regulatory role on motor activity-induced DA release by cholinergic input into SN. This 

was done by perfusing SN with either the muscarinergic antagonist scopolamine or the 

nicotinergic antagonist mecamylamine (at doses that did not exhibit any dopaminergic effects in 

themselves) before and during motor activity. Perfusion of the SN with 100 µM of scopolamine 

significantly enhanced the increase in DA release during motor activity (153±11 per cent of 

baseline, compared to 118±8 per cent of baseline in non-treated controls), but DA concentrations 

during perfusion with 100 µM of mecamylamine (112±6 per cent of baseline) did not differ from 

non-treated controls. In another experiment however, treatment with 100 µM of scopolamine 

failed to alter motor performance even though DA concentrations increase during testing. This 

indicates that even though there is a dynamic nigral response to motor activity, it does not exert 

any intrinsic effect on motor performance. We therefore hypothesize that nigral DA release 

exerts a permissive action on motor performance. 

Disclosures: D.R. Andersson , None; F. Bergquist, None; H. Nissbrandt, None. 

Poster 

274. Basal Ganglia: Transmitters and Neuromodulation II




Title: Effect of 1-methylxanthine, a possible adenosine A2A receptor antagonist on striatopallidal 

activity 

Authors: *L. JIMENEZ BOTELLO 1 , J. CORREA BASURTO 2 , M. AVILA COSTA 1 , E. 

QUEREJETA VILLAGÓMEZ 2 ; 

1 Lab. de Neuromorfologia, FES-Iztacala UNAM, Tlalnepantla, Mexico; 2 Sección de Estudios de 

Posgrado e Investigación, Escuela Superior de Medicina IPN, México City, Mexico 


In recent years, A2A adenosine receptor antagonists, most of them caffeine derivates, have 

emerged as an alternative treatment to Parkison´s disease. 

1-methylxanthine is a caffeine biotransformation product. 

Objective 

To determinate 1-methylxanthine potential as A2A antagonist receptor in striatopallidal pathway. 

Methods 

Docking.- In order to prove if 1-methylxanthine, shows affinity for A2A adenosine receptors, a 

computational simulation of bind ligand-receptor (AUTODOCK3 software) was performed 

comparing it with, caffeine. 

Electrophysiology.- To determinate if 1-methylxanthine presents antagonic activity over 

striatopallidal A2A adenosine receptors, electrophysiological recordings of external globus 

pallidus of rats in vivo were performed. 

Results 

Docking.- Results showed that 1-methylxanthine occupies the same binding sites than caffeine 

and has similar affinity for this receptor. Figure 1 and 2. 

Electrophysiological recordings.- Intrapallidal application of adenosine (100µM) produced a 

reduction of 50% in spontaneous pallidal activity, while application of 1-methylxanthine in same 

dosis increased its activity above 150% (p

efficacy as a A2A antagonist and as a possible antiparkinsonian agent. 

Disclosures: L. Jimenez Botello, None; J. Correa Basurto, None; M. Avila Costa, None; E. 

Querejeta Villagómez, None. 

Poster 






Sackler Grant 

NARSAD Grant 

Title: Chronically elevated 5-HT signaling alters basal ganglia physiology and impairs motor 

coordination 

Authors: *E. MORELLI 1,3 , M. S. ANSORGE 2 , T. J. REBELLO 2 , J. A. GINGRICH 2 ; 

2 Psychiatry, 1 Columbia Univ., New York, NY; 3 Dept. Physiol., Universita' degli Studi G. 

d'Annunzio, Chieti, Italy

Abstract: Anatomical data suggest a functional interaction between the serotonin (5-HT) and 

dopamine (DA) systems. The striatum, for example, receives both DA and 5-HT input and 

DAergic neurons themselves are innervated by 5-HT neurons. To investigate 5-HT/DA system 

interaction at the level of behavior and physiology we turned to the well characterized basal 

ganglia system. Specifically, we analyzed the effect of genetic and pharmacologic 5-HTT 

manipulations on motor behavior and basal ganglia physiology. 

We found that 5-htt-/- mice display progressive motor coordination deficits as assessed by the 

rotarod, foot print, and beam walking test. These motor coordination deficits can be phenocopied 

by chronic but not acute 5-HTT blockade during adulthood in a dose dependent and reversible 

fashion. Importantly, developmental 5-HTT blockade did not elicit motor coordination deficits, 

effectively dissociating the previously reported emotional phenotype of 5-htt-/- mice from the 

motor coordination phenotype. Chronic fluoxetine (FLX) treatment did not worsen motor 

coordination in 5htt-/- mice, demonstrating that the effect of FLX is mediated via 5-HTT 

blockade. 

Disruption of cerebellar function impairs motor learning whereas disruption in basal ganglia 

function impairs motor learning as well as already acquired motor coordination abilities. Because 

chronic FLX treatment after acquisition of the rotarod task impairs performance, we 

hypothesized that chronic 5-HTT blockade impairs basal ganglia function. In support of this 

hypothesis, we detected reduced dopamine turnover specifically in the striatum of 5htt-/- mice. 

Furthermore, l-DOPA treatment reversed motor deficits in 5htt-/- mice, demonstrating that 

increasing dopamine signaling can rescue this behavioral phenotype. 

In order to investigate the physiological state of the basal ganglia system, we assess and will 

present electrophysiological properties of DAergic substantia nigra pars compacta (SNc) neurons 

and GABAergic substantia nigra pars riticulata (SNr) interneurons. Preliminary data indicate that 

SNc neurons are hypoactive after chronic fluoxetine treatment. These results suggest that 

chronically elevated 5-HT tone inhibits DA activity, which leads to a Parkinson-like state of the 

basal ganglia system, resulting in motor impairment. 

Our results shed light on the behavioral and physiological relevance of 5-HT/DA system 

interaction and might have implications in understanding the etiology and pathophysiology of 

motor disorders and extrapyramidal side effects associated with pharmacotherapy. 

Disclosures: E. Morelli , None; M.S. Ansorge, None; T.J. Rebello, None; J.A. Gingrich, 

None. 

Poster 





Support: BFI2003-02909 

Ramón y Cajal‟s program of the Spanish Science and Technology Ministry 

BFU2006-01189 

Title: Distribution of GABAergic interneurons and dopaminergic cells in the human striatum 

Authors: *J. BERNACER MARIA 1,2 , L. PRENSA 3 , J. GIMENEZ-AMAYA 3 ; 

1 PET Lab, - Psych, Mount Sinai Sch. Med., New York, NY; 2 Lab. de Neuromorfologia 

Funcional, Clinica Universitaria. Univ. de Navarra, Pamplona, Spain; 3 Dept. de Anatomia, 

Histologia y Neurociencia, Univ. Autonoma de Madrid, Madrid, Spain 

Abstract: The striatum (caudate nucleus, CN, and putamen, Put) integrates different types of 

cortical and thalamic inputs and projects to the output and intrinsic nuclei of the basal ganglia. 

Striatal interneurons contribute greatly to this integrative role. The GABAergic ones are the most 

abundant and they contain either calretinin (CR) or parvalbumin (PV). Conversely, dopaminergic 

cells (whose role as interneurons is still unclear) are scarce. This study aims at analyzing the 

morphology, distribution and compartmental organization of these two populations of neurons in 

the following regions of the CN and Put: precommissural head, postcommissural head, body, 

gyrus and tail of CN; precommissural and postcommissural Put. We used immunohistochemical 

methods to visualize CR, PV and tyrosine hydroxylase (TH) positive striatal neurons. CR-ir 

interneurons were divided in 3 groups according to the size of their soma: small (average volume 

of the soma = 662±13 κm 3 ), medium (1767±37 κm 3 ) and large (6837±138 κm 3 ). Somata of PV- 

and TH-ir cells were more homogeneous in size and averaged 1526±932 and 1896±1598 κm 3 , 

respectively. The distribution of these subsets of neurons was assessed by stereological methods 

applied to every striatal region abovementioned. CR-ir cells were by far the most abundant 

(1250±850 cells/mm 3 ), being the density of PV- (47±69) and TH-ir (10±27) cells much lower. 

CR-ir interneurons were more abundant in the tail of the CN, followed by the precommissural 

head, body, postcommissural head and gyrus of this nucleus, whereas their lowest density 

appeared in the precommissural and postcommissural Put. In contrast, PV-ir interneurons 

populated mainly the postcomissural Put, followed by the CN gyrus, precommissural Put, body, 

postcommissural and precommissural head of CN. PV-ir cells were not found in the CN tail in 

this stereological study. TH neurons appeared especially in the posterior regions of the CN 

(gyrus and body), followed by the postcommissural Put and postcommissural head of the CN, 

and the precommissural aspects of CN and Put. TH-ir cells were not detected in the CN tail. 

About their compartmental organization, PV- and TH-ir neurons were so infrequent that their 

integrative role between the matrix and striosomes is unexpected. CR-ir interneurons abounded 

in both the matrix and the striosomes and, within the striosomes, they were in their center, 

periphery and borders between these two compartments. This study demonstrates that the 

amount of CR-, PV- and TH-ir neurons varies greatly among the various striatal territories 

considered here. These findings suggest that each striatal territory might possess different 

integrative capabilities 

Disclosures: J. Bernacer Maria, None; L. Prensa, None; J. Gimenez-Amaya, None.

Poster 





Support: AG 21937 

Title: Rat strain affects sensitivity of striatal dopamine and glutamate synapses to 3-NP toxicity 

Authors: *J. P. WALSH 1 , G. AKOPIAN 2 , M. CAPPELLETTI 2 , M. JAKOWEC 3 , G. 

PETZINGER 3 , C. CRAWFORD 4 ; 

1 Dept Gerontol, Univ. So California, Los Angeles, CA; 2 Gerontology & Neurosci., 3 Neurol., 

USC, Los Angeles, CA; 4 Psychology, California State University, San Bernardino, San 

Bernardino, CA 

Abstract: The striatum is particularly sensitive to the irreversible inhibitor of succinate 

dehyrdrogenase 3-nitropropionic acid (3-NP). In the present study we examined early changes in 

behavior, dopamine and glutamate synaptic physiology created by a single systemic injection of 

3-NP in Fischer 344 rats. Hind limb dystonia was seen 2 hours after 3-NP injections and rats 

performed poorly on balance beam and rota-rod motor tests 24 hours later, with improvement by 

48 hours. Systemic 3-NP caused a similar temporal pattern for increased expression of a NMDA 

receptor-dependent form of long-term potentiation (LTP) at corticostriatal synapses. The 3-NP 

induced corticostriatal LTP was not due to increased NMDA receptor number or function, since 

3-NP did not change MK-801 binding and NMDA/AMPA receptor current ratios. The LTP seen 

24 hours after 3-NP was D1 receptor-dependent and reversed by exogenous addition of 

dopamine or a D2 receptor agonist to brain slices. High performance liquid chromatography and 

fast scan cyclic voltammetry revealed a decrease in dopamine content and release in rats injected 

24 hours earlier with 3-NP, and much like the enhanced LTP, dopamine changes were reversed 

by 48 hours. Tyrosine hydroxylase expression was not changed and there was no evidence of 

striatal cell loss at 24-48 hours after 3-NP exposure. Sprague-Dawley rats showed similar 

physiological responses to systemic 3-NP, albeit with reduced sensitivity. Thus, 3-NP causes 

significant changes in motor behavior marked by parallel changes in striatal dopamine release 

and corticostriatal synaptic plasticity. 

Disclosures: J.P. Walsh , This research was supported by a grant from the NIH (AG 21937), 


as grants already received); G. Akopian, None; M. Jakowec, None; G. Petzinger, None; C. 

Crawford, None; M. Cappelletti, None.

Poster 





Support: CONACyT Grant 50427Q 

Title: Dopamine modulates via D4 receptors pallido-nigral transmision to sustantia nigra 

Authors: *R. CRUZ-TRUJILLO 1 , A. SIERRA 2 , R. VALDIOSERA 2 , D. ERLIJ 3 , B. 

FLORAN 2 , J. ACEVES 2 ; 

1 CINVESTAV IPN, Mexico, D.F., Mexico; 2 CINVESTAV-IPN, Mexico DF, Mexico; 3 Sunny 

Dowstate Med. Center, State Univ. of NY, New York, NY 

Abstract: Inmunocytochemical studies have been shown the presence of D4 receptors in the rat 

sustantia nigra. Lack of mRNA in the nucleus suggested that D4 receptors are located on 

afferents. mRNA for D4 receptor is expressed in the globus pallidus, so it is possible that the 

receptors are located on pallidal afferents to sustantia nigra. Here we have examined this 

assumption using whole-cell recordings of visualized SNr neurons from neonatal (14-21days) 

rats. To test the source of the afferents some experiments were done in slices from rats lesioned 

unilaterally with ibotenic acid in the globus pallidus. Spontaneous miniature inhibitory synaptic 

currents (mIPSCs) were recorded in the presence of TTX, CNQX and D-AP5, to block 

propagated action potentials and participation of glutamatergic mEPSCs. Bicuculline abolished 

the mIPSCs, indicating their GABAergic nature. Experiments were performed in the presence of 

SCH 23390 to block D1 receptors from striato-nigral afferents. Addition of dopamine reduced by 

31.56% the frequency of the mIPSCs without affecting their amplitude, indicating a presynaptic 

locus of the involved receptors. The selective D4 agonist, PD 168,077 also reduced the 

frequency (30.56 %), and, as dopamine, without modifying the amplitude of the mIPSCs. The 

effect of dopamine and PD168 077 was blocked by L-745,870, a selective D4 antagonist. In 

slices from lesioned rats, the effect of PD 168,077 was lost. These results indicate that dopamine 

modulates the GABAergic transmission in the SNr via activation of D4 receptors located on the 

GABAergic terminals from globus pallidus origin. Through this action, dopamine may control 

ADHD, where D4 receptors appear to be involved, and which is controlled by elevating the 

levels of endogenous dopamine by blocking its capture either with amphetamine or 

methylphenidate. 

Disclosures: R. Cruz-Trujillo , None; A. Sierra, None; R. Valdiosera, None; D. Erlij, 

None; B. Floran, None; J. Aceves, None.

Poster 





Support: NIH Grant EB007830 

Title: Adenosine A1 receptor mediate both electrically-stimulated dopamine and adenosine 

concentration in rat caudate-putamen 

Authors: *S. CECHOVA, B. J. VENTON; 

Univ. Virginia, Charlottesvile, VA 

Abstract: Adenosine A1 and A2A receptors and dopamine D1 and D2 receptors are present in 

caudate-putamen, where their interactions can modulate the release of neurotransmitters in the 

extracellular space. To observe the effects of these receptors activation or inactivation on 

transient changes in dopamine and adenosine concentration we used our newly developed 

technique for simultaneous measurements of concentration of these two important 

neurochemicals. 

Electrical stimulation trains were applied to the substantia nigra every 3 minutes and the 

chemical changes were measured in the caudate-putamen of anesthetized rats. Fast-scan cyclic 

voltammetry at the carbon-fiber microelectrode detected adenosine and dopamine 

simultaneously with subsecond temporal resolution. 

Drugs were dissolved in 1 mL of saline and injected i.p. The transient changes in dopamine and 

adenosine concentration were measured every 3 minutes for period of 90 minutes after drug 

injection. Effects 45 minutes after administration are given for the following drugs: alphamethyl-DL-tyrosine 

methyl ester (AMPT, 250 mg/kg); SCH-23390 (0.1 mg/kg); MSX-3 (3 

mg/kg); N6-cyclopentyladenosine (CPA, 5 mg/kg); 8-cyclopenthyl-theophylline (CPT, 10 

mg/kg) and quinpirole (QIN, 1 mg/kg). 

First we tested the effects of dopamine drugs on adenosine concentrations. The dopamine 

synthesis inhibitor AMPT decreased the adenosine concentration only slightly, by 20%, while 

dopamine concentration was decreased by 50 %. SCH-23390, a D1R antagonist increased 

adenosine concentrations by 20%, but did not affect the dopamine release. Quinpirole, a D2R 

agonist decreased both dopamine and adenosine concentrations by 50%. 

Then we tested the effects of adenosine receptor agents on the stimulated dopamine and 

adenosine concentration. MSX-3, an A2A antagonist, decreased dopamine by 20% and 

adenosine concentrations by 40%. CPA, an A1 agonist, decreased the dopamine release by 50%, 

while the adenosine peak completely disappeared after drug injection. Interestingly, we also

observed that adenosine A1 receptor antagonist CPT decreased the concentration of both 

adenosine and dopamine by 40%. Adenosine A1 and dopamine D2 receptors are colocalized in 

dopaminergic nerve terminals and their interaction is known to modulate the concentration of 

dopamine. When we administered both D2 receptor agonist and A1 receptor antagonist together, 

the concentration of adenosine has been restored, even slightly increased, but dopamine 

concentration remained decreased. 

Our results suggest that adenosine A1 receptors play an important role in mediating the 

interaction between adenosine and dopamine in caudate-putamen. 

Disclosures: S. Cechova , None; B.J. Venton, None. 

Poster 





Poster 





Support: PDF individual research grant 2007 

Parkinsonfonden (Swedish Parkinson Foundation) 

Title: Serotonin autoreceptor agonists for the treatment of L-DOPA-induced dyskinesia 

Authors: *M. CARTA 1 , A. MUÑOZ 1 , Q. LI 2 , F. GARDONI 3 , E. MARCELLO 3 , T. 

CARLSSON 1 , C. QIN 2 , D. KIRIK 1 , M. DI LUCA 3 , A. BJÖRKLUND 1 , E. BEZARD 4 ;

1 Wallenberg Neurosci Ctr., Lund, Sweden; 2 China Acad. of Med. Sci., Beijing, China; 3 Univ. of 

Milano, Milano, Italy; 4 Univ. Victor Segalen Bordeaux, Bordeaux, France 

Abstract: The use of L-DOPA provides the best effect against the motor symptoms in 

Parkinson´s disease patients at early stages of the disease. However, the appearance of 

dyskinesia is a common problem of long-term L-DOPA treatment and represents a major 

limitation for the pharmacological management of the motor symptoms in advanced disease 

stages. We have recently demonstrated that dopamine released from serotonin neurons is 

responsible for L-DOPA-induced dyskinesia in 6-OHDA-lesioned rats, raising the possibility 

that blockade of serotonin neuron activity by combination of 5-HT1A and 5-HT1B agonists could 

reduce L-DOPA-induced dyskinesia. Here, we have investigated the efficacy of 5-HT1A and 5- 

HT1B agonists to counteract L-DOPA-induced dyskinesia in MPTP-treated macaques, the gold 

standard model of Parkinson´s disease. In addition, we have studied the chronic effect of this 

treatment to prevent development of L-DOPA-induced dyskinesia in 6-OHDA-lesioned rats. The 

results demonstrate the existence of a potent synergistic effect between 5-HT1A and 5-HT1B 

agonists in their ability to dampen L-DOPA-induced dyskinesia in the MPTP-treated macaques. 

Sub-threshold doses of the drugs, which individually produced no effect, were able to reduce the 

abnormal involuntary movements by up to 80% when administered in combination, without any 

worsening effect on the antiparkinsonian efficacy of L-DOPA. Furthermore, combination of low 

doses of the agonists was able to prevent development of dyskinesia, and reduce upregulation of 

FosB, after chronic treatment with L-DOPA in the rat 6-OHDA model. The demonstration of a 

synergistic effect between 5-HT1A and 5-HT1B receptor agonists in primates may have interesting 

clinical implication for the treatment of L-DOPA-induced dyskinesia in PD patients, where 

targeting of the 5-HT1A alone has not produced the expected efficacy. 

Disclosures: M. Carta , None; A. Muñoz, None; T. Carlsson, None; D. Kirik, None; A. 

Björklund, None; E. Bezard, None; F. Gardoni, None; E. Marcello, None; M. Di Luca, 

None; Q. Li, None; C. Qin, None. 

Poster 





Title: Differential DAergic control over Locomotor activity and GABA level in the Bagal 

ganglia

Authors: *E. JUNG 1 , D.-S. KIM 2 , K.-S. KIM 3 , J. KIM 4 , I. SHIM 5 ; 

1 Depart of Integrative Med., The Catho Univ. of korea, seoul, Republic of Korea; 2 Korea 

Advanced Inst. of Sci. and Technol., Daejeon, Republic of Korea; 3 Dept. of Integrative Med., 

4 Inst. of Med. Sci., The Catholic Univ. of Korea, Seoul, Republic of Korea; 5 The Catholic Univ. 

of Korea, Dept. of Integrative Med, Republic of Korea 

Abstract: Dopamine (DA) regulates motor function by influencing GABA level in the basal 

ganglia. GABAergic cells in the basal ganglia have DA receptors which divide into two main 

branches, D1 family (comprising the D1 and D5 subtype) and the D2 family (comprising the D2, 

D3, and D4 subtypes). In order to establish the functional role of the different DA subtypes on 

GABAergic system in basal ganglia, D1 selective agonist (SKF 38393) and D2 selective agonist 

(Quinpirole) were administered to mice. Motor activity was assessed for 60 min immediately 

after the injection of DA agonists SKF 38393 and Quinpirole (0,1,5,10 mg/kg). Mice were 

sacrificed after behavior test and the brains were dissected into region of the basal ganglia for 

analysis of GABA level with HPLC. Mice treated with D1 and D2 agonists showed significant 

increase in locomotor activity. In the analysis of GABA level measured by using HPLC-coupled 

with fluorescence detector, D1 agonist increased GABA level in the striatum and GPi whereas 

D2 agonist decreased its level in the same region. Our results suggest that two type of DA 

receptors differencially regulate motor function by regulating each GABA level of the basal 

ganglia. 

Disclosures: E. Jung , None; D. Kim, None; K. Kim, None; J. Kim, None; I. Shim, None. 

Poster 





Support: CONACyT Grant 50427Q 

Title: D4 receptors inhibit depolarization-induced [ 3 H]GABA release from pallido-nigral 

terminals 

Authors: J. ACOSTA 1 , A. SIERRA 1 , *R. ESCARTIN-PEREZ 2 , D. ERLIJ 3 , J. ACEVES 1 , B. 

FLORAN 1 ; 

1 CINVESTAV-IPN, Mexico DF, Mexico; 2 Lab. Eating Neurobiol, Univ. Natl. Autonoma 

Mexico, Tlalnepantla, Mexico; 3 Sunny Dowstate Med. Center, State Univ. of NY, New York, 

NY

Abstract: Immunocytochemical studies have been shown the presence of D4 receptors in 

substantia nigra pars reticulate (SNr). On the other hand, it is known that neurons of the globus 

pallidus (GP) express mRNA coding for D4. Taking this into consideration we have here 

explored the modulation of GABA release by D4 receptors assumed to be located on pallidal 

terminals in SNr. We studied the effect of dopamine and D4 selective agonists and antagonist on 

[ 3 H]GABA release from depolarized (20 mM K + ) SNr slices. To avoid endogenous dopamine 

effects, the slices were isolated from reserpinized rats. A set of experiments were performed on 

slices from rats with a previous kainic acid-induced lesion of either the globus pallidus or the 

caudate-putamen to test the location of the receptors on pallidal or striatal terminals. 

In reserpinized slices PD 168,077, a selective D4 agonist, and dopamine dose-dependently 

inhibited GABA release (log EC50= -9.27±0.24 and -5.96±0.42 respectively), and their effect 

was antagonized by L-745,870 (100 nM). To prevent activation of D1 receptors by dopamine, 

the effect of dopamine was tested in presence of SCH 23390. The effect of PD 168,077 was lost 

in slices from GP lesioned rats, but the effect remained on slices from the intact side of the same 

animal. By contrast, the inhibitory effect of PD 168,077in slices from striatal-lesioned rats 

persisted, although the facilitation of GABA release by the activation of D1 receptors with SKF 

38393 was lost in these slices, indicating the loss of most of the striatonigral terminals. These 

results suggest that the D4 receptors modulating GABA release in SNr are located exclusively on 

pallido-nigral terminals. 

The present results signal the role of D4 receptors in the output nucleus of the basal ganglia. By 

modulating the input from the globus pallidus to the output nuclei, they may play a relevant role 

in the control of movement and other functions processed by the basal ganglia. 

Disclosures: J. Acosta, None; A. Sierra, None; R. Escartin-Perez , None; D. Erlij, None; J. 

Aceves, None; B. Floran, None. 

Poster 

275. Posture: Kinematics and Muscle Activity 



Topic: D.16.d. Kinematics and Muscle Activity 

Support: NHMRC Project Grant 456017 

Title: Change in anticipatory postural adjustments in novel and familiar tasks 

Authors: *L. M. HALL, S. G. BRAUER, P. W. HODGES; 

Univ. Queensland, Brisbane, Australia

Abstract: Anticipatory postural adjustments (APA‟s) serve to prepare posture in advance of 

perturbations expected from voluntary movement. This study examined whether APA‟s are 

adjusted when postural support is increased using familiar (grip) and novel (bite) strategies. 12 

healthy subjects (4 male, 8 female) stood on dual force platforms and performed 20 randomised 

left and right rapid leg lift tasks in response to a visual cue under 4 conditions: unsupported (US), 

bilateral handgrip (HG), bite (BP) and a combined handgrip and bite (H+B) condition. APA 

onset and onset of unloading of the lift limb were identified from vertical ground reaction forces 

and used to calculate APA duration and amplitude. These were compared between conditions 

and repetitions. Lower limb acceleration was recorded by accelerometers placed on the patella. 

APA duration was affected by support condition and was shortest with maximum support (H+B), 

followed by HG, BP and US. APA amplitude was smallest in the H+B and HG conditions and 

significantly larger in BP and US trials. Lower limb acceleration was greater during US trials 

compared to HG and H+B, but was not different between HG, BP and H+B conditions. Thus, 

changes in APA duration and amplitude between the latter 3 conditions were related to external 

support and not due to variations in acceleration. 

To determine how long it takes the neuromuscular system to refine postural strategies after 

presentation of a new support, an average of the amplitude and duration of the first 2, middle 2 

and last 2 repetitions of each leg movement were compared within conditions. The APA had 

adapted within the first trial of each task and no further adaptation occurred in APA duration for 

any condition. However, amplitude in the most novel condition, BP, was decreased between the 

first and middle repetitions. Tasks in which familiar external supports were available required no 

further refinement of APA‟s. 

The results suggest that with increased external support and sensory cues, less preparation 

(decreased APA amplitude and duration) is required and the central nervous system (CNS) can 

adapt within the first trial to optimise balance. This supports the hypothesis that postural 

preparation and voluntary movement are controlled by parallel processes that allow re-weighting 

of postural commands to meet specific demands. In healthy subjects it is evident that the CNS is 

able to rapidly adapt to, and integrate multiple sensory and support strategies, from the first 

repetition to maintain balance during internal perturbations. This suggests that a healthy nervous 

system can predict the effect of changes in postural set and make immediate adjustments to 

postural preparation. 

Disclosures: L.M. Hall , National Health and Medical Research Council of Australia, B. 


grants already received); S.G. Brauer, National Health and Medical Research Council of 

Australia, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); P.W. Hodges, National Health and Medical Research 

Council of Australia, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 

275. Posture: Kinematics and Muscle Activity





Title: Lower limb muscle responses to leftward-rightward translations during pedaling 

movement 

Authors: *N. NOROUZI GHEIDARI 1,2 , M. BELANGER 3,2 ; 

1 McGill Univ., Montreal, QC, Canada; 2 Ctr. de Recherche Interdisciplinaire en Réadaptation 

(CRIR) du Montreal Metropolitain, Montreal, QC, Canada; 3 Kinanthropologie, Univ. du Quebec 

a Montreal, Montreal, QC, Canada 

Abstract: Leftward and rightward perturbations are common in everyday life, for instance, 

walking in a crowd or walking inside a mass transit vehicle while it makes a sudden turn. The 

goal of this study was to assess the compensatory strategies to perturbations perpendicular to the 

plane of progression. A modified stationary cycle ergometer has been used as a model of 

rhythmic movements. However, in this model the effects of balance are greatly diminished. 

Subjects were asked to pedal under 4 different conditions: dynamic active (DA) whereby they 

pedaled at 1 Hz frequency with the help of a metronome and a visual display; dynamic passive 

(DP) in which the recorded pedal motions from DA were replayed through a torque motor; static 

active (SA) in which the subject was asked to match the Soleus activity to that of the DA 

condition; static passive (SP) whereby the subject just oriented the lower limbs in different 

pedaling positions. Approximately 1 g leftward and rightward translations were randomly 

applied with an electrical cylinder during one of 4 phases of the pedaling cycle: propulsion (P), 

recovery (R), transition PR and transition RP. EMG of the Soleus, Medial Gastronemius, Tibialis 

Anterior, Vastus Lateralis, Biceps Femoris (Brevis) and Tensor Fascia Latae were recorded and 

analyzed. The EMG responses were divided into 2 epochs (E) based on the latency, E1 (80- 

250ms) and E2 (250-400ms). In both E1 and E2, left- and rightward translations evoked 

condition - and phase- dependent responses in the TFL, condition-, phase-, and directiondependent 

responses in the BF, phase-dependent responses in VL, condition-dependent 

responses in MG and SOL, and direction-dependent responses in TA. On the other hand, the E1 

responses were attenuated after several trials whereas the E2 EMG, which tended to be much 

smaller, remained relatively constant as the number of trials increased. In conclusion, despite 

situations in which the balance is relatively well controlled, leftward and rightward translations 

evoke complex and specific condition-, phase-, and direction-dependent muscle responses during 

cycle ergometry. 

Disclosures: N. Norouzi Gheidari , None; M. Belanger, None.

Poster 





Support: MIZUNO Sports Promotion Foundation 

Title: Less ankle stiffness causes less standing balance in the elderly 

Authors: *K. MASANI 1 , A. H. VETTE 2 , M. O. ABE 3 , K. NAKAZAWA 4 , M. R. POPOVIC 2 ; 

1 Lyndhurst Ctr., Toronto Rehab Inst., Toronto, ON, Canada; 2 Inst. of Biomaterials and Biomed. 

Engin., Univ. of Toronto, Toronto, ON, Canada; 3 Dept. of Kinesiology, The Pennsylvania State 

Univ., University Park, PA; 4 Dept. of Motor Dysfunction, Res. Inst. of Natl. Rehabil. Ctr. for 

Persons with Disabilities, Tokorozawa, Japan 

Abstract: It is well known that aging decreases the balance ability during standing, which can be 

assessed via the postural behavior during quiet standing. Many studies have reported a difference 

of postural sway between the young and elderly. However, the underlying variation in their 

control strategies remains unclear. We have recently developed a methodology by which a 

neural-mechanical control scheme can be identified using quiet standing data. In the present 

study, we aimed to investigate the postural control mechanism of the elderly by quantifying the 

neural-mechanical control scheme using this methodology. Twenty-three young (27.3+-4.7 yrs) 

and twenty-two elderly (66.5+-4.9 yrs) subjects were asked to stand still on a force platform to 

acquire data for 30 s. The fluctuation of the body angle, the electromyogram of the right soleus 

muscle, and the ankle torque were measured. A postural control model was build, which 

consisted of a neural controller (a proportional-derivative controller), a neural feedback delay of 

80 ms, the torque generation process (2nd order filter), a mechanical controller (stiffness and 

small viscosity), and an inverted pendulum. In the first procedure, the natural frequency of the 

torque generation process and the stiffness were optimized using the measured muscle activity 

and postural sway as the inputs and the ankle torque as the output of the model. In the second 

procedure, the gains of the neural controller were optimized using the identified variables in the 

first procedure and the measured postural sway as the input and the ankle torque as the output of 

the model. The results revealed that the stiffness normalized by the subjects‟ body dimensions 

was smaller in the elderly than the young. To compensate this lower stiffness, the contribution of 

the neural controller to the torque generation was higher in the elderly. Further, to overcome the 

neural feedback delay, the neural derivative gain was higher in the elderly. These different 

characteristics in the control scheme between the elderly and the young must cause the observed 

differences in postural sway and stability. In conclusion, the present results suggest that the 

lower stability in the elderly is primarily caused by the smaller stiffness, and that the resulting 

higher dependency on the neural controller adds an additional destabilizing factor to the overall 

control system as the neural controller needs to compensate for a long feedback time delay.

Disclosures: K. Masani, None; A.H. Vette, None; M.O. Abe, None; K. Nakazawa, 

None; M.R. Popovic, None. 

Poster 





Support: Canada Foundation for Innovations Grant 986 and 3103 

CIHR- Multidisciplinary Team in Locomotor Rehabilitation 

Title: A virtual reality-based paradigm to study anticipatory postural control during slope 

walking 

Authors: *A. OATES 1,2 , C. PEREZ 1,2 , A. DAREKAR 1,2 , J. FUNG 1,2 ; 

1 Fac. Med., Sch. of P & OT, McGill Univ., Montreal, QC, Canada; 2 Jewish Rehabil. Hosp., 

Laval, QC, Canada 

Abstract: Locomotion is a complex task requiring sensorimotor integration to travel safely 

within the environment and adapt appropriately to environmental challenges. Vision provides 

feedforward information about one‟s location in space and the environment. Proprioceptive input 

provides feedback on posture and movement with respect to oneself, the environment, and the 

motor plan. Voluntary movements are generated by incorporating feedforward, visual 

information with proprioceptive feedback to anticipate perturbations and to monitor stability, 

respectively, in order to maintain balance and complete the locomotor task. 

Anticipatory postural adjustments must be made with ongoing visual updates and are monitored 

by the proprioceptive system to ensure stability and to maintain forward progression. Haptic 

information, in the form of proprioceptive input through light contact with a stable surface, has 

been shown to improve postural stability in healthy and neurologically impaired populations. 

Haptic information is suggested to create an additional proprioceptive reference which is 

integrated to increase stability in both anticipated and unexpected locomotor perturbations. 

A virtual-reality based paradigm was created to simulate sensory, physical and environmental 

challenges encountered during walking with changing slopes, and to compare postural 

adjustments in healthy subjects with those who suffer a central nervous system lesion, such as 

stroke or spinal cord injury. Subjects walk on a self-paced treadmill mounted on a moveable 

platform towards a virtual environment (VE) that is rear projected onto a large screen placed in 

front of the treadmill. The treadmill speed, scene and surface motions, as well as the subject‟s

motion captured by a 6-camera Vicon MX system, are synchronized using the MOTEK 

CAREN3 system. The VE contains scenes with a transition from a level surface to either a 5º upslope 

or 5º down-slope which are matched by physical tilts of the walking surface. In half of the 

trials, participants lightly touch a sensor to provide haptic information. We hypothesize that 

locomotor stability and performance will improve with the addition of haptic information. These 

locomotor improvements will be examined through centre of mass stability, step parameters, and 

locomotor speed. The pattern and size of muscle activity will also be examined across groups 

and conditions. Preliminary data suggests that the transition between a level and a sloped surface 

is challenging as seen in a decreased stride length during the step onto the slope. The effect of 

haptic information and sensorimotor capabilities are currently being evaluated. 

Disclosures: A. Oates, None; C. Perez, None; A. Darekar, None; J. Fung, None. 

Poster 





Title: Motor redundancy facilitates multi-task performance 

Authors: *W.-L. HSU 1 , G. SCHÖNER 2 , J. P. SCHOLZ 3 ; 

1 Dept. of Human Physiol., Univ. Oregon, Eugene, OR; 2 Inst. Neuroinformation, Ruhr-Univ. 

Bochum, Bochum, Germany; 3 Physical Therapy, Univ. Delaware, Newark, DE 

Abstract: The control of posture is typically embedded in the simultaneous control of other 

motor tasks. Recent theorizing based on the Uncontrolled Manifold (UCM) hypothesis suggests 

that this ability is enhanced by a neural control scheme that takes advantage of motor 

redundancy. The purpose of this study was to address this hypothesis by investigating joint 

coordination during the simultaneous performance of multiple tasks while standing. The 

structure of joint configuration variance was examined with respect to control of the center of 

mass (COM) position using the UCM approach. Subjects executed a targeting task alone or in 

combination with an additional ball-balancing task while standing. UCM analysis revealed that 

most of the increased joint variance that occurred when performing targeting compared to 

standing quietly, or when adding the ball-balancing task to targeting, reflected flexible 

coordination of the joints to achieve a stable COM position. Only a small amount of the 

increased variance led to variation of the COM position. Although increasing targeting difficulty 

by changing increasing the index of difficulty affected targeting accuracy, it had little effect on 

COM control. The results support the hypothesis that the nervous system takes advantage of

available motor redundancy to achieve the successful performance of multiple tasks 

concurrently. Future work will need to investigate the limits of this control scheme. 

Disclosures: W. Hsu, None; J.P. Scholz, None; G. Schöner, None. 

Poster 





Title: Understanding bistable human postural coordination dynamics to guide bio-inspired 

control of humanoids 

Authors: V. BONNET 1 , *J. F. LAGARDE 3 , P. FRAISSE 2 , N. RAMDANI 2 , S. RAMDANI 3 , B. 

BARDY 3 ; 

1 obotics, 2 Robotics, Lirmm, Montpellier, France; 3 Motor Efficiency Deficiency la, Montpellier 1 

Univ., Montpellier, France 

Abstract: The present study examines mechanical contributions to human postural coordination 

dynamics and a bio-inspired control of humanoids robots. Previous studies have shown that 

upright posture is characterized by two stable patterns, inphase and antiphase, captured by the 

relative phasing between ankle and hip joint angles. Parametric experiments have also shown an 

abrupt switch, with hysteresis, between the two coordination patterns. Here we use a numerical 

optimization under constraints framework and validation tests on two different humanoid robots 

to investigate further these postural dynamics. Two experiments on humans are accomplished to 

check the predictions. Results from the optimization using the minimum torque change as cost 

function, and as constraints, the limits of base of support for the centre of pressure displacement 

and the head- target distance, show that the transition is dependent on both the minimum torque 

change and the base of support constraints. Two minima of the amplitude of centre of pressure 

displacement are found, one for the inphase pattern at lower frequency and one for antiphase 

pattern at higher frequency, showing the contribution of resonance phenomena at two 

eigenfrequencies. The tests accomplished on the two robots confirm these results. In the first 

experiment on human subjects (N = 7), subjects are asked to oscillate inphase back and forth at 

their preferred frequency (trial duration = 60 sec., N trials = 6), “as if they had to do it all day”. 

We measured the angular movement at ankle and hip joints and centre of pressure displacement. 

Data are recorded by an optical 3D tracking device and a force plateform. Subjects prove very 

consistent in adopting a frequency close to the predicted values (mean peak power spectral 

density = 0.357, SD = 0.027). In the second experiment, subjects (N = 11) are instructed to track

with the head a linearly actuated physical target placed in front of them, which moved 

periodically in the sagittal plane. The frequency of the visual stimulus was increased from 0.1 Hz 

to 0.65 Hz in steps of 0.05 Hz, every 15 cycles. Amplitude of the stimulus motion was 10 cm. 

Results show amplitude of motion larger for the hip than the ankle, in agreement with our 

simulations, but no clear cut minimum of the centre of pressure displacement at the predicted 

frequency. For the modelling part, since the visual tracking task is redundant, we propose a 

simple closed loop control in the operational space with a generalized inverse matrix. By adding 

a dynamical saturation on the ankle torque, the system is able to guarantee the centre of pressure 

constraint. Our model reproduces qualitatively most postural phenomena including hysteresis. 

Disclosures: V. Bonnet, None; J.F. Lagarde , None; P. Fraisse, None; N. Ramdani, None; S. 

Ramdani, None; B. Bardy, None. 

Poster 





Title: Optimal positioning and activation of a postural muscle to assess training-induced 

alterations in cortico-motor excitability 

Authors: *E. A. PITSCH 1 , A. WU 2 , C. POWERS 1 , B. FISHER 1 ; 

1 Univ. Southern CA, Los Angeles, CA; 2 Neurol., UCLA, Los Angeles, CA 

Abstract: Introduction: Lack of adequate recruitment of proximal lower extremity muscles is a 

contributor to movement dysfunction across the continuum of motor ability, from elite athletics 

to daily function after brain injury. As a result, there has been an increase in attention towards 

training strength and neuromuscular control of proximal muscles, such as the Gluteus Maximus 

(GM). However, the mechanism by which increased neuromuscular control is acquired is poorly 

understood, especially in relation to the acquisition of proximal control. Transcranial magnetic 

stimulation (TMS) is a non-invasive imaging method to detect changes in the brain in response 

to training (activity-dependent neuroplasticity). Previous studies have established that changes in 

cortico-motor excitability (CE) using TMS for distal muscles may reflect a central mechanism 

for training-induced improvement in motor skill. Thus, TMS has potential for identifying 

changes in proximal muscles associated with improved postural control. However, the 

methodology for assessing CE of GM has not been established. Objective: The purpose of this 

pilot study was to establish a reliable method for measuring CE for GM. Methods: A preactivation 

paradigm of GM was used to lower the threshold for eliciting a motor evoked potential

(MEP). Three active contraction positions- standing resisted hip extension, single leg bridging 

(SLB), and double leg bridging were tested for consistency in eliciting contraction of GM at 20% 

of maximum voluntary contraction. Five healthy individuals participated in the study. A double 

cone coil was used to identify the „hotspot‟ for GM in motor cortex, then a single stimulus 

intensity was selected for each subject that yielded a MEP of at least 50 κV (40-60% maximum 

stimulator output (MSO)) in the first test position. The same MSO was used in each test 

condition, in which 6-10 single TMS pulses were delivered every 5-10 seconds. The dependent 

measures were frequency count of MEPs per condition and MEP amplitude. Results: SLB 

yielded the most consistent and largest MEP. Conclusions: Future studies using SLB to assess 

CE of GM may reveal central mechanisms associated with training-induced improvements in 

postural control. 

Disclosures: E.A. Pitsch, None; A. Wu, None; C. Powers, None; B. Fisher, None. 

Poster 





Support: 1R01AR053608-01A2 

T32HD07418-12 

Title: Evolution of lower limb muscle design for standing posture 

Authors: *A. M. JARC, M. C. TRESCH; 

Northwestern Univ., Chicago, IL 

Abstract: The majority of studies that examine the control of movement focus on how the 

central nervous system (CNS) forms simplifying strategies to confront the complexities 

associated with movement. These studies treat the musculoskeletal system as simply a plant to be 

controlled. However, several researchers have taken a slightly different point of view, suggesting 

that the musculoskeletal system is part of the overall controller and performs „computations‟ in 

the periphery. In this research, we leverage this perspective to computationally evolve designs of 

the musculoskeletal system to better understand the role of the CNS in coordinating complex 

behaviors. We believe that one can learn aspects of how the CNS controls movement by 

thoroughly examining the musculoskeletal system and how task performance fluctuates with 

changes in its design. We developed a computational framework to optimally design the muscles

of the human lower limb for standing posture. A specific focus of our designs was the effect of 

bi-articular muscles. In support of previous literature, we demonstrate that bi-articular muscles 

improve the ability to coordinate and stabilize the lower limb during perturbations. 

The goal of this research is to design the human lower limb musculature to maximize the 

performance of a standing posture task. We used a genetic algorithm to evolve the muscle origin 

and insertion points on a fixed skeleton. Eight muscles modeled as passive springs were initially 

placed at random locations on the skeleton. From these initial conditions, the muscle locations 

were evolved to produce standing posture. We investigated designs that only allowed monoarticular 

muscles and other designs that allowed both mono- and bi-articular muscles. This 

enabled us to specifically address the influence of bi-articular muscles on the performance of a 

stabilization task where inter-joint coupling is thought to play a significant role. 

The evolved designs using only mono-articular muscles and both mono- and bi-articular muscles 

successfully stood upright. We evaluated the effectiveness of these successful designs by 

supplying horizontal perturbations to the uppermost segment‟s center of mass. The muscle 

designs that used bi-articular muscles were significantly more robust to perturbations than those 

only using mono-articular muscles. This suggests that bi-articular muscles are important for 

maximizing the stability of multijoint tasks such as standing posture. Furthermore, it 

demonstrates that the design of the musculoskeletal system should be considered when 

investigating the control of movement since it can significantly improve performance without 

expending neural control. 

Disclosures: A.M. Jarc, None; M.C. Tresch, None. 

Poster 





Support: Canada Foundation for Innovation 


Title: Effects of ankle fatigue and attentional demands on leg muscles activation in a single leg 

stance 

Authors: *E. BISSON 1,3 , D. W. MCEWEN 1 , Y. LAJOIE 1 , M. BILODEAU 2,1,3 ; 

1 Human Kinetics, 2 Rehabil. Sci., Univ. of Ottawa, Ottawa, ON, Canada; 3 Aging & Movement 

Res. Lab., Élisabeth Bruyère Res. Inst., Ottawa, ON, Canada

Abstract: Introduction: The purpose of this study was to examine the effects of ankle muscle 

fatigue on electromyographic (EMG) activity of the leg muscles during uni-pedal stance, and in a 

dual-task paradigm. Methods: Fourteen young adults (mean age = 22.50±3.23) were required to 

stand on the dominant leg for 30-s trials before, immediately and 30 min after a fatiguing 

protocol. Half of the trials were performed in a dual task condition where subjects in addition to 

standing (primary task) had to respond verbally to an auditory stimulus (secondary task). The 

fatigue protocol comprised repeated plantar- and dorsi-flexion maximal concentric contractions 

(30 o /s and 120 o /s, respectively) on a dynamometer, until the torque produced decreased below 

50% of the pre-fatigue maximal torque recorded in both directions. EMG activity was recorded 

from the tibialis anterior, gastrocnemius medialis, vastus lateralis, biceps femoris, rectus femoris 

and gluteus maximus. EMG amplitude was quantified with the root mean square (RMS) value 

calculated over the middle 20-s of a given 30-s trial. The effects of task (single versus dual) and 

fatigue were assessed with analysis of variance (ANOVA) for repeated measures models. 

Results: In general, all muscles showed a decrease in activity during uni-pedal stance with 

fatigue. However, significant reduction were found only for the gastrocnemius medialis, rectus 

femoris and gluteus maximus. No differences were found between the single and dual task 

conditions. Conclusions: The present results suggest that fatigue of the ankle induces changes in 

the neuromuscular mechanisms involved in the control of standing on one leg. This was found to 

be independent of differences in attentional demands between a single task and a dual task 

involving the addition of a verbal reaction time component. 

Disclosures: E. Bisson , None; D.W. McEwen, None; Y. Lajoie, None; M. Bilodeau, None. 

Poster 





Support: Canada Foundation for Innovation 


Title: Ankle fatigue has limited impact on postural sway and attentional demands in young 

healthy adults 

Authors: *D. W. MCEWEN 1 , E. BISSON 1,3 , Y. LAJOIE 1 , M. BILODEAU 2,3,1 ; 

1 Human Kinetics, 2 Rehabil. Sci., Univ. of Ottawa, Ottawa, ON, Canada; 3 Aging & Movement 

Res. Lab., Élisabeth Bruyère Res. Inst., Ottawa, ON, Canada

Abstract: Introduction. Studies on fatigue of lower limb muscles in relation to postural stability 

are limited, and thus its significance is not well known. Furthermore, studies using dual-task 

paradigms have shown that the control of upright posture can be influenced by the attentional 

demands of a given task. The purpose of this study was to assess the effects of ankle muscle 

fatigue on postural sway and reaction time during a unipedal stance task. Methods. Fourteen 

young adults (mean age = 22.50±3.23) were required to stand on the dominant leg for 30-s trials 

before, immediately and 30 min after a fatiguing protocol. Half of the trials were performed in a 

dual task condition where subjects in addition to standing (primary task) had to respond verbally 

to an auditory stimulus (secondary task). The fatigue protocol comprised repeated plantar- and 

dorsi-flexion maximal concentric contractions (30 o /s and 120 o /s, respectively) on a 

dynamometer, until the torque produced decreased below 50% of the pre-fatigue maximal torque 

recorded in both directions. Sway amplitude (RMS) in the antero-posterior and medio-lateral 

plane, the 95% eclipse area, and the average velocity of the center of pressure (COP) were 

collected using a force platform. Voice reaction time was recorded seated and during the dual 

task condition to assess attentional demands. Results. As expected, sway amplitudes were 

significantly greater in the medio-lateral plane compared to the antero-posterior plane. Sway 

measurements were not different between the single and the dual stance tasks, except for average 

velocity, which was significantly faster during the dual-task (unfatigued, fatigued and recovery). 

Fatigue did not affect any of the computed sway measurements. As expected, reaction time was 

increased from the seated to the standing trials, illustrating an increase in attentional demands 

when performing the one-leg stance. However, fatigue had no effect on reaction time. 

Conclusions. During a dual-task, subjects were able to perform the one-leg stance task with low 

sway amplitudes, but had more difficulty controlling their center of pressure movements 

(increased velocity). This suggest that more attention is needed in dual-task and/or the voice 

response itself might be affecting the sway. However, muscle fatigue of the ankle failed to affect 

healthy young adult‟s postural sway measurements or the attentional demands associated with 

the task. 

Disclosures: D.W. McEwen, None; E. Bisson, None; M. Bilodeau, None; Y. Lajoie, None. 

Poster 





Support: CRIR 

McGill University

Title: Equifinality in single-step and double-step unloading movements 

Authors: *P. S. ARCHAMBAULT 1,2 , A. KHAZRAIYAN-VAFADAR 1 ; 

1 Sch. of Phys & Occ Therapy, McGill Univ., Montreal, QC, Canada; 2 Ctr. for Interdisciplinary 

Res. in Rehabil. (CRIR), Montreal, QC, Canada 

Abstract: Equifinality, during arm reaching movements, relates to the capacity of the 

neuromuscular system to attain the same final position in the presence or absence of transient 

perturbations. A brief elastic perturbation, applied during a fast arm movement or just before its 

initiation, typically does not affect final arm position. On the other hand, several experiment 

have shown that velocity-dependent perturbations, such as coriolis force or negative damping, 

while transient in nature, have a significant effect on final arm position when compared to 

unperturbed movements. It remains unclear, however, if these changes are due to the nature of 

motor control in the nervous system, to a correction in the motor commands during movement or 

to peripheral factors (muscle or reflex properties). In this study, we analyzed the effects on final 

arm position of suddenly decreasing a static load maintained by subjects. Unloading movements 

are mediated by spinal reflexes, as long as subjects do not anticipate the decrease in load. We 

compared final arm position after decreasing the load in either one or two steps, to the same final 

value, thus allowing us to study the role of reflexes with respect to equifinality. Subjects 

maintained an initial load produced by a two-joint manipulandum moving in the horizontal 

plane. The load was suddenly decreased, either in one or two successive steps with different time 

intervals, resulting in a rapid reflex change in arm position. The initial holding time and the 

unloading conditions were randomized to prevent anticipation. Electromyographical recordings 

confirmed that unloading resulted in significant changes in muscle activity. Further, muscle 

activity during the stable phases of movement was constant, indicating that subjects did not 

modify their muscle commands. It was found that the final hand position was shorter for double- 

versus single-step unloading if the time between two successive changes in load was greater than 

100 ms. For shorter intervals, the final hand positions were the same. This difference was 

inversely proportional to the hand velocity at the time of the second change in load. A trial-bytrial 

analysis of all conditions also showed a significant correlation between peak velocity and 

displacement. Thus, both the change in load and the velocity at the time of the change may 

influence the magnitude of the unloading reflex. This may be indicative of a dependence of 

stretch reflexes on velocity and may explain the absence of equifinality observed in other studies. 

Disclosures: P.S. Archambault , None; A. Khazraiyan-Vafadar, None. 

Poster 



Program#/Poster#: 275.12/JJ27


Support: NIH grant R37-AG006457 

the Swedish Brain Foundation 

Title: Hand haptic touch modifies postural hip tone 

Authors: *E. FRANZEN 1,2 , V. S. GURFINKEL 1 , W. G. WRIGHT 3 , P. J. CORDO 1 , F. B. 

HORAK 1 ; 

1 Neurolog. Sci. Inst., Oregon Hlth. & Sci. Univ., Portland, OR; 2 Karolinska Instituet, Stockholm, 

Sweden; 3 PT Dept., Temple Univ., Philadelphia, PA 

Abstract: Axial muscle tone provides a stable reference frame for the execution of limb 

movements and stabilizes the torso during gait and stance. It has been proposed that tonic muscle 

activity for stance posture is supported, if not generated, by the resting discharge of sensory 

afferents from over the entire body. Earlier studies have shown that manual contact with a 

surface, perceived to be fixed in space, increases postural stability, even though light haptic 

touch results in a purely sensory interaction between the body and the floor (Lackner, et al,1999). 

This enhanced stability from hand haptic input may be a result from alterations in postural tone. 

To investigate why the body is stabilized by light touch we explored the influence of heavy and 

light haptic touch on postural tone in 14 healthy adults (20-55 and 85 yrs of age). We measured 

hip torsional torque and the position of the center of pressure under the feet of subjects standing 

on a slowly rotating force plate (Gurfinkel et al, 2006). This unique device twisted the hips by 

virtue of rotating the feet with the pelvis fixed to a solid external frame and was not giving 

information concerning the reference. Normal sway was not restricted by the pelvis fixation so 

the subjects remained in a state of active postural control during the three tested stability 

conditions: 1) quiet standing with the arms at the sides, 2) firmly gripping a rigid bar, or 3) 

lightly touching a rigid bar. The subjects stood quietly with eyes closed as a platform rotated for 

±10 deg at 1deg/s. As expected, the RMS of the center of pressure decreased during gripping and 

touching the bar (p

Poster 





Support: Natural Sciences and Engineering Research Council (NSERC) 

Title: Balance corrective responses associated with arm perturbations during treadmill 

locomotion 

Authors: *J. FORERO, J. MISIASZEK; 

Univ. Alberta, Edmonton, AB, Canada 

Abstract: It has been shown that the arms play an important role when balance corrective 

strategies are to be used; leg muscle responses are altered depending on whether hands are 

touching, grabbing or holding an object. Rapid balance reactions during walking are also 

required when perturbations to balance are delivered through the arms (Nashner and 

Forssberg1986). However, unlike the legs, the arms are not required to participate in locomotion 

or maintaining balance, but may be simultaneously engaged in other activities during walking. 

We hypothesized that balance reactions induced by perturbations applied through the arms 

depend upon the task conditions at the time of perturbation. To test this, seven subjects were 

asked to hold a set of handles while walking on a treadmill. Forward and backward perturbations 

were delivered to the handles at four different phases during the step cycle. Subjects were asked 

to A) react to the perturbation as quickly as possible and return to the starting arm posture, or B) 

be compliant and allow the handles to be displaced. Muscle activity was recorded from the 

Tibialis Anterior (TA), Soleus (SOL), Vastus Lateralis (VL), Biceps Femoris (BF), Anterior 

Deltoid (AD), Posterior Deltoid (PD), Biceps Brancii (BB) and Triceps Brancii (TB). 

Electrogoniometers recorded joint angles of the knee, ankle and elbow. Displacement of the 

handles and forces applied to the handles were also recorded. Responses to the perturbations 

were only evoked in either the leg or arm muscles when subjects were instructed to react to the 

perturbations. Therefore, displacement of the handles themselves was not a sufficient event to 

trigger a corrective response, in either the arm or leg muscles. Moreover, the evoked responses in 

the react condition were characterized by responses in the leg muscles preceding those of the 

arms, despite the locus of the perturbation. The implication is that the balance maintenance 

demands of the task supersede the arm task as the balance correction occurred prior to the arm 

correction. Furthermore, because the muscle activation pattern is present only during the react 

condition and occurs before the body gets perturbed from its rhythmic movement, evidence of a 

program being activated and deactivated depending on the task is presented. 

Disclosures: J. Forero, None; J. Misiaszek, None.

Poster 





Support: Canadian Institutes for Health Research (MOP- 64414) 

Michael Smith Foundation for Health Research Fellowship 

Natural Sciences and Engineering Research Council of Canada (RGPIN/239735) 

Title: The effect of initial movement dynamics on human responses to postural perturbations 

Authors: *C. D. MURNAGHAN, S. N. ROBINOVITCH; 

Sch. of Kinesiology, Simon Fraser Univ., Burnaby, BC, Canada 

Abstract: Introduction: Falls are a major cause of injury, and often occur while walking, 

reaching or bending [1]. Yet, we have little understanding of how the cognitive and 

biomechanical demands associated with such tasks influence our ability to recover balance. In 

the current study, we used a rocking paradigm to determine how the nature of the baseline task 

influences muscle activation patterns and kinematics following an external perturbation to 

balance. 

Methods: Fourteen women (mean age 23.5±3.5 (SD) yrs) participated in trials where a sudden 

backward translation of the support surface was applied as they either stood quietly, or rocked 

about the ankles or hips. In all trials, participants were instructed to “recover balance,” and the 

perturbation was applied at an instant when the trunk was vertical. Data analysis focused on 

comparing static versus dynamic trials in terms of step characteristics, joint angles (acquired with 

an 8 camera, 120 Hz motion analysis system), and the time intervals (latencies) between 

application of the perturbation and increased activity in various lower extremity muscles 

(acquired through surface electromyography at 960 Hz). 

Results: We found the nature of the activity at the time of the perturbation influenced both the 

pattern of muscle activation in the early-stage balance recovery response, and the subsequent 

joint kinematics and tendency to execute a step. When participants rocked forward (in ankle 

rocking) or held a static position at the time of the perturbation, we observed early gastrocnemius 

(at 94±2.7 (SE) ms) and biceps femoris activity (at 117±3.1 ms), representative of an ankledominant 

response [2]. However, when rocking backward, there was early abdominal (at 98±3.7 

ms) and rectus femoris activity (at 114±12.0 ms), and later gastrocnemius and biceps femoris 

activity (at 124±3.5 ms and 149±3.8 ms), representative of a hip-dominant response. Stepping

was 71% more common, and hip angular displacements were 13 degree greater, when rocking 

forward than backward. Similar trends were observed during hip rocking. 

Conclusion: These observations indicate the nature of the ongoing task at the time of imbalance 

influences the organization of the early-stage balance recovery response, and the subsequent 

tendency to execute a step. Of particular interest is the use of a hip-dominated response when 

accelerating backward and an ankle-dominant response when accelerating forward or when 

stationary, which highlights the ability of the CNS to adapt the recovery response to the 

biomechanical constraints and cognitive demands associated with ongoing activities. 

References: [1] Talbot et al. 2005; [2] Horak & Nashner 1986; [3] Horak et al 1997 

Disclosures: C.D. Murnaghan, None; S.N. Robinovitch, None. 

Poster 





Support: CFI 10653/3103 

HSFC 

CIHR 77548 

Title: Context-dependent characteristics of locomotor steering guided by optic flow 

Authors: *J. R. BERARD 1,3 , J. FUNG 2,3 , A. LAMONTAGNE 2,3 ; 

2 Physical and Occup. Therapy, 1 McGill Univ., Montreal, QC, Canada; 3 Jewish Rehabil. Hospital, 

research site of CRIR, Laval, QC, Canada 

Abstract: Optic flow is one of the visual cues used to guide locomotion whereby heading 

direction is indicated by the focus of expansion (FOE) during typical, straight ahead walking. 

Under some circumstances, however, the optic flow may be conflicting with other types of 

sensory information whereby the heading direction may no longer correspond to the FOE 

location. We investigated the context-dependent characteristics of visually guided locomotor 

steering by manipulating 1) the type of optic flow; 2) the location of the FOE; and 3) the 

instruction to use or disregard visual information presented. Subjects were evaluated while 

walking overground and wearing a head mounted display (HMD; NVisor). The scene presented 

in the HMD was of a large room with vertical and horizontal cues but no obstructions. Kinematic

data of the head and whole body were recorded using a 12-camera Vicon motion capture system. 

Movements of the head were tracked in real-time via three markers placed on the HMD. Head 

position data was computed and fed to the CAREN-2 (MOTEK) system, allowing for real-time 

movements of the head to be synchronized and displayed in the HMD. Subjects were instructed 

to either use optic flow information and walk straight with respect to the scene in the HMD 

(virtual environment: VE) or ignore visual information and walk straight with respect to the 

physical environment (PE). For both conditions, the flow could either be presented as rotational 

(FOE rotated along the anterioposterior axis) or translational (FOE shifted linearly with respect 

to the subject). For each type of flow presented, the FOE could be displaced by either 0°, 40° to 

the left, or 40° to the right. Results showed that participants kept their head aligned with the 

desired heading direction. When asked to walk straight with respect to the PE, the head was 

aligned with the physical neutral direction and the centre of mass (CoM) showed little 

mediolateral deviations in the PE. When asked to walk straight with respect to the VE, the head 

was aligned towards the virtual neutral and CoM showed small deviations in the VE. However, 

the strategy to maintain a straight heading differed between rotational and translational flows. 

With a rotational flow, subjects reoriented their body by rotating their head and trunk in the yaw 

direction. In contrast, with translational flows, reorientation was achieved using a side-stepping 

strategy. This study suggests that the head is consistently aligned with the heading direction, that 

locomotor reorienting strategies are specific to the type of optic flow presented and that healthy 

young individuals have the ability to reweight sensory information in a task-dependent manner. 

Disclosures: J.R. Berard , Canadiant Institutes of Health Research, B. Research Grant 


received); Heart and Stroke Foundation of Canada, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); J. Fung, 

Canadian Institutes of Health Research, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); Heart and Stroke Foundation, 


as grants already received); A. Lamontagne, Canadian Institutes of Health Research, B. 


grants already received); Heart and Stroke Foundation, B. Research Grant (principal investigator, 


Poster 




Topic: D.16.d. Kinematics and Muscle Activity

Support: NIH Grant R21HD055386 

Title: Posture assisted locomotion (pal) training enhances step initiation in Parkinson‟s disease 

Authors: *M. W. ROGERS 1 , I. FUDOKOS 1 , L. HANSON 2 , A. HAUPTSCHEIN 2 , T. 

RADECKI 2 , M. J. HILLIARD 2 , C. MACKINNON 2 , K. MARTINEZ 2 , T. SIMUNI 2 , Y. 

ZHANG 2 ; 

1 Physical Therapy & Human Movement Sci., 2 Northwestern Univ., Chicago, IL 

Abstract: Posture Assisted Locomotion (PAL) Training Enhances Step Initiation in Parkinson‟s 

disease. M. W. Rogers, J. Fudokos, A. L. Hanson, Hauptschein, T. Radecki, M.J. Hilliard, C. D. 

MacKinnon, K.Martinez, T. Simuni, Y. Zhang. Department of Physical Therapy and Human 

Movement Sciences, and Department of Neurology, Feinberg School of Medicine, Northwestern 

University, Chicago, IL 

Start hesitation and freezing of gait (FOG) are poorly understood manifestations of Parkinson‟s 

disease (PD). In gait initiation, anticipatory postural adjustments (APAs) for propulsion and 

weight transfer prior to stepping suggest a role for motor prediction in coordination. Thus, the 

release of the step cycle may normally be delayed until expected postural state conditions occur. 

In PD, absent or prolonged and reduced APA forces could delay gait initiation due to adaptive 

changes in neural control. With external postural assistance, impaired APAs could be enhanced 

to improve stepping. To examine this possibility, postural perturbations that augmented APA 

forces during step initiation were applied to 7 PD subjects (Stage 2.5- 3) ON medication. Ground 

reaction forces and motion kinematics were recorded. Unperturbed pre-training trials were 

compared with post-training trials following 6 weeks of biweekly training sessions involving 60 

rapid self-initiated stepping trials with a sudden pneumatically-driven vertical drop (1.5 cm in 

150 ms) of the single stance limb applied in the early phase APA. Immediately post-training the 

APA durations were reduced by 41% (antero-posterior) and 30 % (medio-lateral) and 

respectively remained shorter by 71 % and 57% after a 6 week retention period (p < 0.05). 

Additionally, first step speed was faster by 11% (p < .05), and step width narrowed 2-3 fold (p < 

.05). The time to complete the 7 m Stand-Walk-Sit Test was also reduced by 21%. These 

findings suggested that modifying preparatory state conditions through PAL training enhances 

locomotion possibly through adaptive changes in the forward model for step initiation. 

Disclosures: M.W. Rogers , None; I. Fudokos, None; L. Hanson, None; A. Hauptschein, 

None; T. Radecki, None; M.J. Hilliard, None; C. MacKinnon, None; K. Martinez, None; T. 

Simuni, None; Y. Zhang, None. 

Poster 





Support: NIH K01 HD050369 

ISB Predoctoral Award 

Title: Impaired postural adjustments to a predictable perturbation during step initiation in the 

elderly 

Authors: *S.-C. TSENG 1 , S. J. STANHOPE 3,4 , S. M. MORTON 1,2 ; 

1 Phys Ther & Rehab Sci., 2 Anat. & Neurobio., Univ. of Maryland Sch. of Med., Baltimore, MD; 

3 Health, Nutr. & Exercise Sci., Univ. of Delaware, Newark, DE; 4 Mechanical Engin., Univ. of 

Delaware, Newark, DE 

Abstract: Prior to and during gait, postural adjustments counteract the resultant forces induced 

by self-movement, offset unexpected perturbations, and correct movement errors, thereby 

minimizing postural instability and preventing falls. Here, we investigated acquisition of postural 

adjustments by healthy elderly subjects in response to a novel but predictable perturbation during 

a visually-guided step. We recorded 3D kinematics and ground reaction forces from 18 healthy 

elderly adults and an equal number of young controls. Subjects performed, as quickly and 

accurately as possible, a reactive, visually-guided stepping movement with the right foot toward 

an illuminated target located on the floor. During a block of baseline trials, the target appeared 

directly in front the stepping leg, at a distance of 40% of body height. During a block of 

perturbation trials, the target first appeared in the baseline position but was replaced, 

instantaneously during mid-step, by a second target that was shifted either toward the right or the 

left of the baseline location by a distance of 10% of body height. Subjects were randomly 

assigned to perform the perturbation block with either rightward- or leftward-shifting targets. To 

measure the acquisition of altered postural adjustments, we quantified changes in several 

variables over the duration of the perturbation block: the durations of three movement phases, 

the timing and magnitudes of foot path modifications, and the timing and magnitudes of force 

modifications. These variables were compared over three key time periods: initially, early, and 

late in the perturbation block. We found that the young group was able to quickly alter their 

stepping in response to the predictable perturbation, completely adjusting their performance by 

the end of the early perturbation period. In contrast, the elderly group required additional 

practice; they were unable to adjust lateral propulsive forces, shorten foot path modification 

times, and stabilize several other temporal variables by the end of the early perturbation period. 

By the late perturbation period, however, the elderly performed similarly to the young group on 

most measures. We also found that stepping accuracy showed direction-specific effects in both 

groups, probably because of an increase in the challenge to medial-lateral postural stability in the 

leftward step. Our results indicate that during step initiation, elderly subjects have a slowed but 

intact ability to adjust postural responses to a predictable perturbation. The findings suggest that 

elderly subjects may have impaired mechanisms for correcting postural adjustments using trialand-error 

practice.

Disclosures: S. Tseng , None; S.J. Stanhope, None; S.M. Morton, None. 

Poster 





Title: Effects of age and target height on dynamic balance control during reaching 

Authors: *M.-H. HUANG, M. A. MOUSIGIAN, S. H. BROWN; 

Div. Kinesiol, Univ. Michigan, Ann Arbor, MI 

Abstract: The control of balance during functional tasks such as reaching from standing requires 

the production of appropriate anticipatory (APAs) and compensatory postural responses. In older 

individuals, deterioration in the magnitude and timing of postural responses frequently occurs, 

leading to instability and increased risk of falls. While some studies have indicated that reaching 

to high targets produces increased instability (Row and Cavanagh, 2007), to what extent target 

location affects dynamic balance control during free standing in the elderly has not been 

systematically addressed. This study, therefore, examined the effects of age and reach target 

height on the control of center of pressure (COP). 

Twelve young (20 ± 1 yr) and 16 older (74 ± 5 yrs) participants reached to targets at various 

heights (above head, shoulder, low), with the dominant or non-dominant hand as fast as possible. 

Targets were at 110% of arm‟s length away and vertically aligned with the shoulder joint of the 

reaching arm. Kinematics of hand movements were measured with a 3-D motion analysis system 

(MotionStar). COP data were derived from ground reaction forces recorded by a force plate 

(AMTI AccuSway Plus). 

The magnitude of COP posterior displacement associated with APAs was significantly greater in 

older than young adults (p=0.03) at the lowest compared to the higher targets (p

to low targets. Although reach performance was comparable between both age groups, APA 

magnitude increased significantly in older adults, suggesting that, with aging, APA magnitude 

can be modified in response to task demands. 

Disclosures: M. Huang , None; M.A. Mousigian, None; S.H. Brown, None. 

Poster 





Support: National Health and Medical Research Council, Australia 

Physiotherapy Research Foundation, Australia 

Title: Reorganisation of the motor cortex in chronic low back pain 

Authors: *P. W. HODGES 1 , H. TSAO 1 , M. GALEA 2 ; 

1 CCRE SPINE, Univ. of Queensland, Brisbane, Australia; 2 The Univ. of Melbourne, Melbourne, 

Australia 

Abstract: Changes in motor control of the trunk muscles are common in chronic low back pain 

(LBP) (J Electromyogr Kinesiol, 2003 13: 361-370). These deficits can be improved with motor 

training (Exp Brain Res, 2007 181: 537-546). However, it is unclear how these changes in motor 

control are mediated in the nervous system. As deficits in anticipatory postural activation of 

transversus abdominis (TrA) are common in LBP, and these responses involve input from the 

motor cortex, the present study aimed to investigate the representation of TrA at the motor cortex 

in individuals with and without chronic LBP and whether this could be changed with motor 

training in individuals with chronic LBP. Eleven healthy volunteers and 20 individuals with 

chronic LBP were recruited. Chronic LBP individuals were randomly allocated into two training 

groups: specific motor control training that involved repeated voluntary contractions of TrA, or 

walking exercise, twice per day for two weeks. Recordings of electromyographic activity (EMG) 

of TrA were made bilaterally with intramuscular electrodes. Motor control of TrA was assessed 

using single rapid arm flexion and extension tasks. Transcranial magnetic stimulation (TMS) was 

delivered over pre-marked scalp sites to examine the cortical representation of TrA. EMG 

amplitude of the responses to TMS at each site was superimposed over the grid to produce a map 

of response amplitude relative to scalp site. All procedures were repeated two weeks after 

training for the LBP group. Onset of TrA EMG relative to deltoid, and centre of gravity (CoG) of

TMS map were compared between individuals with and without chronic LBP, and between pre- 

and post-training in individuals with chronic LBP. The representation of TrA was located 2 cm 

anterior and lateral to the vertex in healthy individuals. However, individuals with chronic LBP 

showed a significant posterior and lateral shift in the CoG. The change in location of TrA 

cortical representation was associated with timing of activation during rapid arm movement 

tasks. Following two weeks of specific training of TrA, motor cortical representation shifted 

towards that observed in healthy individuals. Changes in representation were not observed for 

the walking exercise group. These findings provide evidence of reorganisation of trunk muscle 

representation at the motor cortex in individuals with chronic LBP, and that cortical changes may 

contribute to deficits in motor control. Furthermore, training data suggest specific training of the 

trunk muscles can induce plasticity of the motor cortex, which may be associated with 

improvements in motor control of the trained muscle. 

Disclosures: P.W. Hodges, National Health and Medical Research Foundation, Australia, B. 


grants already received); Physiotherapy Research Foundation, Australia, B. Research Grant 


received); H. Tsao, National Health and Medical Research Foundation, Australia, B. Research 


already received); Physiotherapy Research Foundation, Australia, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already 

received); M. Galea, None. 

Poster 



Program#/Poster#: 275.20/KK1 


Support: NIH Grant R37-AG006457 

NIH Grant T32-NS045553 

Title: Sensory re-weighting in response to postural perturbations in patients with Parkinson‟s 

disease 

Authors: *K. J. FELLER 1 , R. J. PETERKA 1,2 , F. B. HORAK 1,3,2 ; 

1 NSI, OHSU, Beaverton, OR; 2 Biomed. Engin. Dept., 3 Dept. of Neurol., OHSU, Portland, OR

Abstract: Patients with Parkinson‟s Disease (PD) have an impaired ability to respond to 

transient postural perturbations and impaired kinesthesia (Horak et al. 1992; Dimitrova et al. 

2004). To determine whether patients with PD have impaired steady-state postural responses 

similar to patients with sensory deficits, we measured body sway during continuous, saggital 

plane, support surface rotations. Eight patients with moderately severe PD (age=64±7.2 yrs) and 

8 age-matched controls (age=64±7.5 yrs) performed tests during which the support surface was 

rotated using pseudorandom stimuli having peak-to-peak amplitudes of 1°, 2°, or 4°. PD patients 

were tested both on (HY= 2.0±0.80) and off (HY=2.8±1.1) medication, and all subjects were 

tested with eyes closed. To quantify the relationship between stimulus (support surface rotation) 

and response (COM rotation), we used spectral analysis to calculate transfer functions. We 

interpreted the results using a simplified feedback model of the postural control system (Peterka 

2002). By fitting the model to our experimental transfer functions, we estimated sensory 

weighting, postural (active and passive) stiffness and damping, time delay, and force-feedback 

parameters. Here, we focus on sensory re-weighting, which reflects active regulation of the 

sensory integration process. Consistent with previous results in younger controls, our older 

control subjects re-weighted the relative contribution of sensory information for different 

stimulus amplitudes (p

Authors: *S. LEE 1 , J. E. MISIASZEK 2,3 ; 

1 Rehabil. Med., 2 Occup. Therapy, 3 Neurosci., Univ. of Alberta, Edmonton, AB, Canada 

Abstract: Recent studies showed that the amplitude of balance corrective responses in the leg 

muscles modulated in a context-dependent manner during walking according to the demands of 

the tasks. For example, the amplitude of corrective responses in the leg muscles was increased 

when young adults were asked to walk with their arms crossed (Misiaszek & Krauss, 2005), 

compared to when the arms were freely swinging. In contrast, when holding supportive handles, 

leg muscle activity was diminished (Misiaszek et al. 2000). Others have shown that corrective 

responses in older adults are only modestly slower and of lower amplitude, compared to young 

adults (Schillings et al. 2005, Tang and Woollacott 1999). Tang and Woollacott (1999) showed 

that corrective responses in older adults modulated in a phase-dependent manner, much like 

young adults. The purpose of this study was to determine whether the context-dependent 

regulation of balance corrections witnessed in young adults was also retained in older adults. 

Specifically, we hypothesized that the pattern of modulation of corrective responses in older 

adults would be similar to that observed in young adults, but that the magnitude of these 

adaptations would be smaller in older adults. Twenty healthy young (YA) and older adults (OA) 

walked on a treadmill and periodically received balance disturbances with 1) arms swinging 

naturally, 2) arms folded across the chest, and 3) holding stable handles. Electromyographic 

responses to the perturbations were recorded in several muscles of the leg. The pattern of muscle 

activation was similar between age-groups and between tasks. For example, backward 

perturbations applied at heel strike were characterized by early latency responses TA and VL. In 

both age groups the amplitudes of these responses in TA and VL were significantly decreased 

while holding stable handles, compared to the other conditions. However, the magnitude of this 

decrease was more pronounced in the young adults. An interesting finding was that responses in 

the YA group typically involved an excitatory response in SOL (i.e. co-contraction with TA), 

whereas an inhibitory response was evoked in the OA group. These results suggest that although 

many features of the regulation of balance corrective responses are preserved in older adults they 

do not exhibit the same magnitude of modulation as young adults. The limited capacity to 

modulate balance reactions between tasks may contribute to an increased risk of falling in this 

population. 

Disclosures: S. Lee, None; J.E. Misiaszek, Alberta Center on Aging, B. Research Grant 


received); NSERC, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 





Support: The Wellcome Trust 

The Whitaker Foundation 

The European SENSOPAC Project (IST-2005-028056) 

Title: Generalization of dynamic learning for posture and movement 

Authors: *A. A. AHMED, D. M. WOLPERT; 

Engin., Univ. Cambridge, Cambridge, United Kingdom 

Abstract: Skillful movement depends upon our ability to predict the consequences that our 

actions have on our bodies and environment. It is widely accepted that this predictive control 

relies on a central representation (internal model) of the internal and external dynamics. 

However, it is not known whether learning novel dynamics of the arm in a seated position will 

generalize to the same movement performed while standing. In addition, will the postural system 

be able to generalize to standing and predict and compensate for the destabilizing consequences 

of these dynamics on posture? 

Initially, seated subjects made reaching movements to targets in the horizontal plane while 

grasping the handle of a robotic, force-generating, manipulandum. Their reaching movements 

were perturbed with a force proportional to their movement velocity, but perpendicular in 

direction. Following the seated trials, subjects stood on a force plate and performed another set 

of reaching movements in the presence of the force perturbations. Handle position, robotgenerated 

forces, and forceplate forces were recorded at 500 Hz. To assess learning of the 

dynamics in both the seated and standing conditions, we examined movement error on each trial. 

Quality of learning was assessed with random catch trials, where hand movement was restricted 

to a path along the target vector, using a force channel. Feedforward control of posture in the 

standing position was measured as the position and velocity of the center of pressure at the onset 

of hand movement. Movement of the center of pressure in the direction of the impending 

perturbing force indicates anticipatory control. 

All participants learned to make relatively accurate reaching movements to the target while 

seated, and significantly reduced their movement error. Upon switching to a standing posture, 

hand error remained reduced. Similarly, by the end of the sitting phase, participants generated 

significant anticipatory forces on catch trials and this performance was maintained upon 

changing to a standing posture. However, on standing subjects did not initially compensate for 

the postural perturbation in an anticipatory manner and generated similar postural responses as 

found when reaching in the absence of a force field. Over the course of several trials, 

anticipatory postural responses developed. These findings indicate that learned dynamics of the 

arm generalizes from sitting to standing but that the postural system does not generalize 

anticipatory mechanisms between body postures. 

Disclosures: A.A. Ahmed, None; D.M. Wolpert, None.

Poster 





Support: FHS111199 

IRND110830 

Title: Age-related changes in the first step generated for balance recovery 

Authors: *N. BUGNARIU MARHAO; 

Sch. of Rehabil. Scie, Univ. of Ottawa, Ottawa, ON, Canada 

Abstract: Regardless of the various factors that place a person at risk for falls, the execution of a 

change in support strategy by generating a fast and accurate stepping reaction is critical to 

prevent the fall. The control of stepping reactions is complex because it requires the initiation 

and execution of a rapid sequence of movements involving both lower extremities. In addition, 

step kinematics need to be adjusted to match the disturbance in terms of direction, amplitude and 

constraints imposed by surrounding environment. The demands of controlling these complex 

stepping reactions may create difficulties for older adults, due to age-related impairments in the 

neural and musculoskeletal systems. When the first step generated in response to loss of balance 

does not restore equilibrium, multiple later steps often emerge. This strategy may become a 

further risk for falls in itself due to increased chances of limb collisions. The purpose of these 

experiments were to determine: i) the characteristics of a first step that is successful for balance 

recovery in terms of kinematics and muscle activity patterns and ii) the age-related changes 

occurring in the first step generated for balance recovery. 

The kinematic and electromyographic (EMG) responses to unpredictable translations of the 

support surface in the anterior-posterior and medio-lateral directions were contrasted between 

healthy young (18-35 years old, n=5) and old (>65 years old, n=5) adults. The platform 

perturbations forced a stepping reaction by either starting to move unpredictably at peak velocity 

of 5m/s and translate for 20 cm, or it oscillate ± 10 cm at a 0.5 Hz frequency and stop abruptly 

after an unpredictable amount of time. All participants undergo a sensorimotor assessment of 

foot sensitivity, ankle proprioception and lower limb muscle strength. 

In comparison to young adults who recover their balance by taking one large, directionallyappropriate 

step, older adults took multiple (2-3) smaller steps and they failed to produce the 

anticipatory postural adjustments required before lifting and swinging the foot to make the step 

in response to external perturbations. The sequence of muscle activation pattern for the first step

was maintained, but aging resulted in 40-60 ms longer EMG latencies. Preliminary results 

suggest that impaired plantar sensation in old adults appears to be an important factor that could 

affect the execution of compensatory step reaction to prevent falls, due to the inability to detect 

center of pressure displacement under the feet. 

Disclosures: N. Bugnariu Marhao, None. 

Poster 





Title: Effect of unilateral stroke on trunk control in sitting position 

Authors: *S. PERLMUTTER 1 , M. MAKHSOUS 1 , Y.-C. WANG 2 , F. LIN 1 ; 

1 Phys Ther & Hum Move Sci., Northwestern Univ., Chicago, IL; 2 Rehabil. Inst. of Chicago, 

Chicago, IL 

Abstract: Background: Unlike the predominantly contralateral motor control of the extremities, 

the trunk is bilaterally innervated from each of the cerebral hemispheres[1]. Although there is 

much literature discussing the various aspects of trunk impairment post stroke, few studies have 

focused on quantifying specific dysfunctions. This study investigated post-stroke somatic trunk 

motion during static and dynamic tasks. 

Methods: Individuals with chronic hemisphere stroke (N=8) and healthy controls (N=5) were 

evaluated in the sitting position. Trunk control was assessed using forward flexion with both 

arms extended and lateral elbow-to-surface touch tasks. The elbow-to-surface task required 

touching of each elbow to a seat-level surface as close as possible. Moments exerted onto seat, 

location of subjects‟ Center of Pressure (COP), movement of trunk, and the location of elbow 

touch were recorded. For postural stability, three 60-second trials were used: eyes closed (EC), 

target staring (TS), and augmented feedback (FB). FB tasks required keeping a real-time COP 

cursor inside a displayed frame. COP sway area was obtained and further sub-divided into 4 

quadrants. Task Effort (TE) was quantified as integration of torque about flexion axis divided by 

absolute integration of all torques created. All trials were tested with and without footrest. 

Preliminary Results: Stroke subjects had significantly larger (P

than controls, with significance in no footrest trials (P

standard clinical measures for balance & gait (DGI, ABC, Berg). During balance testing, head 

position data were also recorded while patients and controls observed static and dynamic 

checkerboard images on stereoscopic video goggles to induce a vestibular-ocular reflex (VOR) 

and balance response. Functional magnetic resonance images (fMRI) of brain activity in the 

brainstem & cerebellum were recorded while patients and controls observed the same static and 

dynamic videos. Results from tests of balance, gait, head motion, and fMRI data before and after 

NINM training were scored and statistically analyzed. 

Results. Based on preliminary data we expect patient's post-NINM training scores will exhibit 

clinically significant improvement in functional balance and gait scores. We also expect patient's 

head-based stabilograms to exhibit a smaller RMS trajectory after NINM training, indicating a 

more normal VOR. Finally, we anticipate observing changes in the fMRI data showing localized 

activation of the brainstem and cerebellum in response to the NINM training, and in cortical 

activation patterns that correlate with the visual stimuli and VOR. 

Conclusion. Based on our prior research, we postulate that NINM induces resynchronization of 

sensory-motor coordination of both head and body control, leading to improved functional 

balance and gait. We believe that neuromodulation of the brainstem and cerebellum via cranial 

nerves that enervate the tongue (CN-V and CN-VII), exciting primarily the trigeminal nuclei 

complex, nucleus tractus solitarius is principally responsible for the observed this functional 

neurorehabilitation. 

Disclosures: Y.P. Danilov, None; M.E. Tyler, None; J.C. Wildenberg, None; M.E. 

Meyerand, None. 

Poster 





Support: NIH (K25HD047194) 

Nebraska Research Initiative 

NIDRR (H133G040118) 

Title: Largest Lyapunov exponent shows infant sitting postural sway is chaotic 

Authors: J. E. DEFFEYES 1 , R. T. HARBOURNE 2 , A. KYVELIDOU 1 , W. A. STUBERG 2 , 

*N. STERGIOU 1,3 ;

1 Dept Hlth. Phys Ed & Recreation, Univ. Nebraska-Omaha, Omaha, NE; 2 Munroe Meyer 

Institute, Univ. of Nebraska Med. Ctr., Omaha, NE; 3 Col. of Publ. Health, Univ. of Nebraska 

Med. Ctr., Omaha, NE 

Abstract: INTRODUCTION: Upright sitting is one of the first motor skills an infant learns. 

Understanding the sway mechanisms during infant sitting posture may be useful in 

understanding motor skill development, and early identification of neurodevelopmental 

disorders, such as cerebral palsy. Currently it is unclear whether this postural sway is similar to a 

single pendulum (periodic) or similar to a double pendulum (deterministic chaos) model. 

Furthermore, an often used measure to characterize chaos is the largest Lyapunov exponent 

(LyE). If a system is chaotic, LyE will be positive. However, calculating the LyE from 

experimental data is controversial because the presumably random dynamics of experimental 

noise can give a positive value for LyE, even if the underlying system has simple periodic 

dynamics. Thus, we tested the hypothesis that experimental noise will give rise to a positive LyE 

in sway data from a simple pendulum, a non-chaotic system. Additionally, we examined whether 

postural sway data from infant sitting is more similar to a periodic single pendulum, or more 

similar to a chaotic double pendulum. METHODS: Typically developing infants were recruited 

when they were just developing the ability to sit upright (age 5.06 months, sd=.76 months, 

n=30). Infants were screened for normal development by a physical therapist using a 

standardized development test prior to admission into the study. For sway data acquisition, 

infants sat on an AMTI force plate, interfaced to a Vicon data acquisition system. LyE was 

calculated using the commercially available Chaos Data Analyzer software. For the pendulum 

data, the data acquisition was the same, except that physical pendulums were placed on the force 

plate and set into motion prior to data acquisition. Thirty trials were collected from each 

pendulum (single and double). LyE values were calculated for single pendulum, double 

pendulum, and infant sitting from both medial-lateral and anterior-posterior direction. LyE 

values were compared using t-tests and compared with a value of zero, with significance set at 

p< .05/10= .005 to correct for multiple comparisons. RESULTS: The LyE from the single 

pendulum was positive and significantly different from zero. The LyE from infant sitting 

postural sway was found to be significantly higher than the LyE for the single pendulum, and 

significantly lower than the double pendulum. CONCLUSION: Experimental noise can lead to a 

low, but positive, LyE. The LyE from infant sitting postural sway is significantly higher than the 

value from the non-chaotic single pendulum, as was data from a chaotic double pendulum, 

consistent with postural sway being chaotic. 

Disclosures: J.E. Deffeyes, None; R.T. Harbourne, None; A. Kyvelidou, None; W.A. 

Stuberg, None; N. Stergiou , None. 

Poster 





Title: The effect of early stage Parkinson‟ disease on dynamic postural stability during turning 

activities 

Authors: J. SONG 1 , *B. E. FISHER 1 , S. SIGWARD 1 , G. PETZINGER 2 , G. J. SALEM 1 ; 

1 Biokinesiol & Physical Therapy, 2 Neurol., USC, Los Angeles, CA 

Abstract: PURPOSE: Persons with early stage Parkinson‟s disease (EPD) typically show 

minimal levels of impairment and disability, as quantified by scores of less than or equal to two 

on the Hoehn and Yahr scale. Previous studies have found no differences in gait parameters 

between healthy control (HC) participants and persons with EPD suggesting that more complex 

tasks may be needed to discriminate between these groups. Turning tasks impose a greater 

demand on postural stability as they require coordination of speed modulation with reorientation 

of the body. Given this, evaluation of postural stability during turning may be more sensitive to 

detect impairments due to EPD. Therefore, the purpose of this pilot study was to compare 

measures of dynamic postural stability between persons with EPD and HC participants during 

turning activities. 

SUBJECTS: Two persons diagnosed with EPD and two HC subjects participated in the study. 

MATERIALS AND METHODS: Subjects were instructed to walk 4 meters and perform a 90 0 

turn at a designated location. Three-dimensional kinematics (8 camera, VICON Motion System, 

60 Hz) and segment inertial parameters (Dempster, 1971) were used to calculate the whole body 

center of mass (COM). Center of pressure (COP) was determined from force plate measures 

during single and double limb stance (AMTI force plate 1.2m x 1.2m, 1560Hz). To account for 

the effects of COM velocity on postural stability during turning, an extrapolated COM (eCOM) 

was calculated using COM position and velocity as previously described by Hof et al., 2005. 

Dynamic postural stability was defined as the distance from COP to the eCOM in the mediallateral 

(ML) and anterior-posterior (AP) directions. RESULTS: When compared to HC 

participants, both subjects with EPD demonstrated a greater AP distance during turning activities 

indicating a greater threat to stability in the AP direction. In contrast, the subjects with EPD 

exhibited a shorter ML distance indicating a smaller threat to stability in the ML direction. 

CONCLUSION: Persons with EPD utilized different control strategies during turning tasks than 

HC participants. These preliminary data suggest that measures of dynamic postural stability 

during turning are sensitive enough to discriminate between persons with EPD and HC 

participants. However, it remains unclear if the control strategies employed by persons with EPD 

are associated with an inability to modulate COM velocity or position relative to COP. Dynamic 

postural stability during turning may be an early predictor of fall risk in persons with PD. 

Disclosures: J. Song, None; B.E. Fisher , None; S. Sigward, None; G. Petzinger, None; G.J. 

Salem, None.

Poster 




Topic: D.17.a. Kinematics and muscle activity 

Title: Human coordination variability and postural task performance 

Authors: *E. G. JAMES 1 , K. M. NEWELL 2 ; 

1 Pennsylvania State Univ., University Park, PA; 2 Kinesiology, The Pennsylvania State Univ., 

University Park, PA 

Abstract: Previous research from the dynamic systems perspective has found that the human 

motor system maintains standing posture in the face of internal or external perturbations through 

the use of inphase and antiphase ankle-hip coordination pattern attractors. During a tracking task 

involving postural sway, these ankle-hip coordination patterns have been found to emerge from 

task and organismic constraints. In previous research the variability of these coordination 

patterns has not been explicitly examined in relation to the level of task performance. Also, some 

subjects have not produced a modally distributed ankle-hip coordination pattern, with the data 

from these subjects being excluded from further analyses. 

This study examined the relation between ankle-hip coordination variability and task error while 

performing a postural tracking task at two movement frequencies. Subjects stood with arms 

folded across the chest and were instructed to move their head in an anterior-posterior direction 

to track the movement of a target projected onto a screen. Head movement was measured 

through the use of a string-potentiometer while hip and ankle movements were measured with 

electrogoniometers. The target oscillated at an amplitude of 10 cm and frequency conditions of 

0.15 and 0.75 Hz were performed. 

Significantly lower task error was produced in the 0.15 Hz tracking task condition than in the 

0.75 Hz condition. Also, in the 0.15 Hz condition a significantly negative correlation was found 

between ankle-hip relative phase and task error, indicating that better task performance was 

associated with greater coordination pattern variability. This demonstrates the motor system‟s 

use of multiple redundant strategies, rather than a fixed coordination pattern, to satisfy motor 

task demands. This indicates that a less stable attractor within the human motor system can 

produce greater functional task performance than a more stable attractor. 

Disclosures: E.G. James , None; K.M. Newell, None. 

Poster





Title: Always moving: A new solution to the posture-movement paradox of neuro-muscular 

control 

Authors: *D. A. ROSENBAUM; 

Psychology, Pennsylvania State Univ., University Park, PA 

Abstract: The neural control of motion reflects a seeming paradox. Mechanisms exist to keep 

the body in stationary postures, but those posture-maintaining mechanisms seem to be turned off 

when movements are made. How can motion sometimes be allowed and sometimes be 

disallowed? // Sherrington (1910) recognized this problem and identified the principle of 

reciprocal inhibition as a means of overcoming it. According to this principle, activation of 

muscle groups inhibits activation of stretch reflexes in opposing muscle groups. Von Holst and 

Mittelstaedt (1950) offered the reafference principle in much the same spirit. According to the 

reafference principle, feedback signals that are produced as a result of motor commands are 

discounted. Another solution to the posture-movement paradox is embodied in the equilibriumpoint 

principle (Asatryan & Feldman, 1965). According to this principle, muscle stretch 

thresholds are changed in anticipation of movements to new postures. // Support for these three 

principles need not imply that the posture-movement paradox actually exists. The three 

principles could reflect means of regulating movements either between postures or around 

postures. Indeed, it could be that there is no fundamental difference between these two kinds of 

movement. Holding still and moving could be essentially the same. This hypothesis is 

parsimonious, is consistent with Fourier‟s principle that any periodic signal can be decomposed 

into superposed sine waves, and with the law of thermodynamics that states that no finite number 

of steps can cool a body to absolute zero (i.e., complete cessation of movement is impossible, at 

least at the atomic level). // This new, always-moving, hypothesis is evaluated via simulations, 

studies of the relation between tremor and voluntary movements, and studies of the relation 

between position variability and speed in oscillatory movements. 

Disclosures: D.A. Rosenbaum , None. 

Poster 

276. Voluntary Movement: Stroke, Damage, or Disease 




Title: Simultaneous recordings of head and hand tremor in subjects with essential tremor: An 

investigation of coherence in tremor frequency 

Authors: *M. C. MACDONALD 1 , M. E. HEROUX 2 , K. E. NORMAN 3 ; 

1 Neurosci., Queen's Univ., Kingston, ON, Canada; 2 Rehabil. Therapy, Queen's Univ., Kingston, 

ON, ON, Canada; 3 Rehabil. Therapy, Queen's Univ., Kingston, ON, Canada 

Abstract: BACKGROUND: The pathophysiology of essential tremor (ET) is not clearly 

understood but is known to involve abnormal oscillatory activity in olivo-cerebellar pathways. 

While hand tremor has been the focus of much research, little is known about head tremor, which 

affects 41% - 46% of the ET population. The purpose of this study was to describe in greater 

detail head tremor in ET and to investigate the possible relationship between head and hand 

tremor. 

METHODS: Fourteen ET subjects were recruited (3 male, 11 female). Head and hand tremors 

were recorded simultaneously with surface electromyography (EMG) of the wrist extensors and 

various neck muscles, laser displacement sensors (hand tremor), a load cell (hand tremor) and an 

accelerometer (head tremor). While seated, subjects performed four tasks: 1) constant force (10% 

maximum) wrist extensions (with and without visual feedback); maintenance of the hands in a 

horizontal posture against gravity while 2) seated upright in a chair, 3) seated in a reclined chair 

(20° backward, head not supported); and 4) seated upright in a chair and producing steady 

submaximal hip adduction forces. 

RESULTS: Head tremor spectral peaks were found between 3.5 and 7 Hz in neck muscle EMG 

and the accelerometer signal. Wrist tremor (EMG and laser data) was slightly higher in 

frequency with a range of 4 -10 Hz. Five of the 14 subjects had the same peak tremor frequency 

in the head and both hands. Two other subjects showed the same peak tremor frequency in the 

head and one hand. Preliminary coherence analysis between wrist and neck muscle EMG data 

revealed a significant peak in coherence at the tremor frequency in 10 of 14 subjects in one or 

more of the experimental conditions. Coherence was most commonly observed between the 

Trapezius Descendens muscles and wrist extensors. 

DISCUSSION: Given the greater mass of the head it might be expected that head tremor might 

occur at a slightly lower frequency than the wrists, as was seen in our results. Our coherence 

analysis implies that hand-head coherence is observable in a majority of individuals with ET 

affecting both body parts. Interestingly, wrist tremor EMG showed coherence mostly commonly 

with EMG recorded from the Trapezius Descendens: a more peripheral muscle than either the 

Sternocleidomastoid or the Splenius Capitis. It is important to note that these two neck muscles 

often did have spectral EMG peaks, but they were not coherent with the wrist tremor. This 

finding may indicate that the oscillatory activity driving more peripheral muscles is different 

from that in the neck muscles thought to be most responsible for head tremor. 

Disclosures: M.C. MacDonald, None; M.E. Heroux, None; K.E. Norman, None.

Poster 





Support: NIH Grant HD050123 

VA Grant VA-B37091 

VA Grant VA-B2801R 

VA Grant VA-3790R 

Title: EMG coupling between synergist muscles is functionally relevant in patients post stroke 

Authors: K. KISIEL-SAJEWICZ 1,3 , Y. FANG 1 , V. SIEMIONOW 1 , J. J. DALY 4 , V. 

SAHGAL 2 , *G. H. YUE 1 ; 

1 Dept Biomed Engineer/ND20, 2 Physical Med. and Rehabil., Cleveland Clin. Fndtn, Cleveland, 

OH; 3 Dept. of Kinesiology, Univ. Sch. of Physical Educ. in Wroclaw, Wroclaw, Poland; 

4 Depatment of Res., Cleveland VA Med. Ctr., Cleveland, OH 

Abstract: AIM: A reaching movement toward a front target consists primarily of shoulder 

flexion and elbow extension. It has been shown that in patients post-stroke, coordination of the 

shoulder flexor and elbow extensor muscles during a reaching movement is impaired and that 

contributes to poorly performed reaching. Coherence is a measure of strength of functional 

coupling between two signals on frequency domain. We hypothesized that a potential 

mechanism behind poor coordination in reaching in stroke patients was weakened coupling 

(measured by coherence) between EMG signals of the shoulder flexor and elbow extensor. 

METHODS: Twenty one stroke patients (59.6±8.6 years) and eight healthy controls (60.6±6.3 

years) participated in the study. The subjects performed a reaching task of the affected (patients) 

and dominant (controls) arm. Surface EMG from the anterior deltoid (ATD) and triceps brachii 

(TB) muscles and force were measured during the task. Coherence between EMG signals of the 

two muscles as a function of movement time was calculated to determine if functional coupling 

between the two synergists in stroke patients was weakened. Correlation between the coherence 

and force variation (standard deviation [SD] of the mean force) was evaluated. 

RESULTS: Coherence between EMGs of the ATD and TB during the reaching movement was 

significantly lower in stroke compared to healthy subjects at a frequency range 0-13Hz (Table 1). 

In this frequency range a significant negative correlation (P

of the force (force smoothness) was observed in the stroke patients. 

CONCLUSIONS: The lower coherence between signals of the ATD and TB during reaching in 

stroke suggests weakening of functional coupling between the two synergists. This could be 

contributed by a loss of common drive at the frequency band as a result of stroke-induced 

interruption of the corticospinal network involved in controlling the movement. The significant 

correlation between the coherence and force variation suggests that the coherence is functionally 

relevant. Weakening of functional coupling between synergist muscles may be an important 

mechanism underlying poor motor performance in patients post stroke. 

Coherence between EMGs of anterior deltoid and triceps brachii at the frequency range 0-25Hz 

Group 0-4Hz 4-8Hz 8-13Hz 13-25Hz 

Control 0.287±0.065* 0.267±0.049* 0.190±0.023* 0.133±0.022 

Stroke 0.203±0.077 0.194±0.061 0.161±0.031 0.140±0.030 

* P

Abstract: Previous studies have suggested that limb trajectory and limb posture are controlled 

independently by the nervous system, and may be lateralized. We have proposed a hypothesis of 

lateralization that attributes control of limb impedance to right hemisphere processes and control 

of intersegmental coordination to left hemisphere processes. An alternative hypothesis contends 

that the right hemisphere is specialized for closed-loop processing, and the left hemisphere is 

specialized for open-loop processing. We now test these alternative hypotheses by employing a 

double-step task, in which the location of the target jumps to a new position at movement onset. 

Eight hemiparetic stroke (4 left, 4 right) patients and 8 control subjects were instructed to reach 

without visual feedback to one of 3 targets located in the ipsilateral hemispace. Control subjects 

used their right or left arms; patients used their ipsilesional arm. During jump trials, the target 

location unexpectedly changed ±40° at movement onset. For left stroke patients, corrective 

responses occurred earlier in the movement and were primarily driven by changes in shoulder 

joint torque. However, these responses were poorly executed due to inappropriate coordination 

of elbow muscle torques with interaction torques produced by the shoulder motion. The right 

stroke group showed delays in both baseline reaction time and corrective response initiation 

compared to other subjects. These responses were straight and coordinated, yet appeared to be 

pre-planned movements to a new target rather than online corrections. Although both stroke 

groups showed impaired final accuracy, we found that position deficits in the left stroke group 

were related to the magnitude of joint interaction torque, reflecting poor compensation of 

intersegmental dynamics. These data suggest that left and right stroke patients may employ 

different “corrective” strategies in response to rapid changes in target location that may reflect 

different cost functions that are optimized by the intact hemisphere. Rather than differentiating 

between these two hypotheses of lateralization, our current findings suggest a hybrid model: Left 

hemisphere specialization for planning dynamic aspects of movement, and right hemisphere 

specialization for feedback-mediated corrective processes. It remains unclear whether right 

hemisphere specialization is only related to impedance control for adjusting final position, or 

rather is dependent on feedback-mediated corrections, irrespective of spatial requirements; thus, 

we are currently conducting studies to differentiate between these alternatives. 

Disclosures: S. Schaefer , None; R. Sainburg, None; K. Haaland, None. 

Poster 





Support: NIH Grant R01NR010786

Title: Estimating the degree of neural activation in post-stroke muscles 

Authors: T. M. KESAR 1 , R. PERUMAL 2 , A. L. MEYERS 1 , *S. A. BINDER-MACLEOD 2 ; 

1 Interdisciplinary Program in Biomechanics and Movement Sci., 2 Dept Physical Therapy, Univ. 

Delaware, Newark, DE 

Abstract: Muscle weakness is a common consequence of stroke. It is challenging, if not 

impossible, to parse the weakness in post-stroke muscles caused by atrophy versus deficits in 

neural activation of muscle. Also, because post-stroke individuals cannot fully activate their 

muscles, it is difficult to estimate the true force generating ability of post-stroke muscles. 

In this study, we compared two methods of estimating the maximum force generating ability and 

the degree of volitional activation of the paretic plantarflexor muscles in 9 post-stroke 

individuals. In the 1 st method (interpolated twitch technique (ITT)), the force response to a 

supramaximal electrical pulse was compared with the subject‟s muscle at rest and during a 

maximum volitional contraction (MVC) to asses the degree of volitional activation (VAITT). The 

VAITT and MVC values were then used to estimate the maximum force generating ability 

(MaxVA). In the 2 nd method, a sub-maximal twitch to tetanus ratio (TTR) and the maximum 

twitch force were used to estimate the maximum force generating ability (MaxTTR). Also, the 

ratio between the MVC and the MaxTTR provided a second measure of VATTR. 

Our results showed that the peak MVC forces (102.1± 46.4 N) were markedly lower than the 

estimated maximum forces (MaxVA=229.8±77.0 N and MaxTTR=272.1±107.1 N). There was no 

significant difference (p=0.35) between the MaxVA and the MaxTTR. The VAITT was 44.3±15.5% 

and the VATTR was 44.3±33%, implying marked impairments in neural activation post-stroke. 

Also, the VAITT and the VATTR were not significantly different (p=1). This study demonstrates 

the ferasibility of 2 methods (ITT and the TTR) for assessing post-stroke muscle weakness. Our 

results suggest that there were no differences in the estimated maximum force generating ability 

and the degree of volitional activation calculated using the 2 techniques. Future work is needed 

to compare the validity and reliability of these 2 techniques in post-stroke individuals. 

Disclosures: T.M. Kesar, None; S.A. Binder-Macleod , Principal investigator, B. Research 


already received); R. Perumal, None; A.L. Meyers, None. 

Poster 




Topic: D.17.a. Kinematics and muscle activity

Support: NIH Grant R01-NS052509 

Title: Effects of sensory manipulations on targeted arm movements after stroke 

Authors: *M. O. CONRAD, R. A. SCHEIDT, B. D. SCHMIT; 

Marquette Univ., Milwaukee, WI 

Abstract: Stroke survivors commonly experience hemiparesis of the arm, resulting in decreased 

strength and sensation in the limb as well as increased spasticity, discoordination, reduced range 

of motion and instability of endpoint control. Because prior research suggests that sensory 

information from hand and wrist musculature influences control of proximal joints, we 

hypothesized that control of proximal musculature could be improved post-stroke by providing 

augmented feedback from distal sensory systems. Here we study the influence of enhanced Ia 

afferent input from distal forearm muscles on the kinematics of point-to-point reaching 

movements (eg. endpoint stability and range of motion at the shoulder and elbow). Ten 

hemiparetic, chronic stroke survivors were seated comfortably as they grasped the handle of a 

horizontal planar two joint robot with their impaired hand. Participants made center-out reaching 

movements in 8 directions in the horizontal plane. Movements were cued by the appearance of 

targets on a screen that obstructed the view of the participant‟s arm. Visual feedback of hand 

position was also projected as a cursor on the screen. After 80 baseline trials, custom designed 

tendon vibrators (Faulhaber Group, Clearwater, FL) applied 70Hz vibration to the forearm flexor 

tendons for 40 trials. Hand position, velocity and grip force data were compared prior to, during 

and post tendon vibration. At baseline, stroke survivors generally experienced more success at 

medial and lateral mobility than forward and backward movement, and often demonstrated 

instability of the endpoint position about the target. Vibratory stimulation of the forearm altered 

control of planar arm movements in stroke subjects. Endpoint stabilization was measured as the 

variance in the tangential velocity after the primary reach towards a target was complete. 

Vibratory stimulation enhanced stabilization in the upper arm as evidenced by a general decrease 

in variance in tangential velocity at the end of the reaching movements. Removal of the stimulus 

resulted in even greater increases in endpoint stabilization for those movements requiring 

shoulder flexion and extension. Additionally, the presence of a vibratory stimulus tended to 

decrease movement extent. Taken together, these results suggest that sensory stimulation of the 

wrist and finger musculature alters motor control of the upper arm, particularly enhancing a 

flexor response in the upper arm during planar reaches. After the removal of a vibratory 

stimulus, additional improvements in endpoint stability suggest that neural plasticity may be 

manipulated to promote improvements in movement kinematics post-stroke. 

Disclosures: M.O. Conrad, None; R.A. Scheidt, None; B.D. Schmit, None. 

Poster 

276. Voluntary Movement: Stroke, Damage, or Disease




Title: The effect of contraction intensity and visual feedback on force fluctuations in individuals 

with essential tremor 

Authors: *M. E. HEROUX 1 , N. J. BADKE 1 , G. PARI 2,3 , K. E. NORMAN 1 ; 

1 Rehabil. Therapy, 2 Sch. of Med. - Div. of Neurol., Queen's Univ., Kingston, ON, Canada; 

3 Neurol. - Movement Disorders Clin., Kingston Gen. Hospial, Kingston, ON, Canada 

Abstract: Introduction: It is unknown how the 4-10 Hz rhythmic muscle activity in essential 

tremor (ET) affects steady force production and whether tremor-associated force fluctuations are 

scaled in a signal-dependent manner as seen in healthy subjects. The purpose of this study was to 

assess the impact of contraction intensity on force fluctuation amplitude and structure produced 

during isometric wrist extension in ET. The effect of manipulating visual feedback was also 

investigated. Methods: Subjects with ET (n = 32, age = 63.6 ± 13.1) and healthy controls (CN, n 

= 23, age = 61.9 ± 14.4) were recruited. Subjects performed three maximal voluntary 

contractions (MVC) and were then asked to produce steady isometric wrist extension 

contractions at various intensities (5%, 10%, 20%, 30% of MVC). Visual feedback of the target 

force and the amount of force being produced was provided for 8 s, after which visual feedback 

of both was removed and subjects maintained the same intensity for another 8 s. Repeatedmeasures 

ANOVA were used to determine group (ET vs. CN), visual feedback condition 

(present vs. absent) and contraction intensity (5%, 10%, 20%, and 30%) effects on dependent 

measures (standard deviation and time-dependent structure of force; force spectral measures). 

Results: Force fluctuation amplitude was greater in the ET group; there was a group by intensity 

interaction caused by a greater between group difference at 5% and 10% MVC. Increasing 

contraction intensity led to more structured force output in both groups; this effect was greater in 

the CN group at higher intensities (group by intensity interaction). Removal of visual feedback 

led to reduced force fluctuation amplitude and a reduction in time dependent structure in both 

groups; the latter effect was greater in the CN group at 5% and 10% MVC (group by intensity 

interaction). The power in the 1-3Hz and 3-12Hz bands increased with increasing contraction 

intensity. Interestingly, the amplitude of the spectral peak associated with ET was not 

significantly different at 5%, 10% and 20% MVC. Discussion: Although force fluctuations in the 

ET group were larger, they tended to scale in response to increasing contraction intensity and the 

presence/absence of visual feedback similarly to CN. The most noticeable deviation between 

groups was at lower contraction intensities. This observation and the large tremor-associated 

spectral peak amplitude at low contraction intensities indicates that ET force fluctuations are 

proportionally greater at lower contraction intensities and raises many questions regarding the 

mechanism by which descending oscillatory inputs access the motorneuron pool. 

Disclosures: M.E. Heroux , None; K.E. Norman, None; N.J. Badke, None; G. Pari, None.

Poster 





Support: United Cerebral Palsy Research and Education Foundation 

Title: The role of reflexes in voluntary movement in childhood dystonia 

Authors: *S. N. KUKKE 1 , T. D. SANGER 2 ; 

1 Dept Bioengineering, 2 Stanford Univ., Stanford, CA 

Abstract: Reflex responses to muscle stretch are clearly a prominent feature of human motor 

control. For decades, the role of reflexes in voluntary movement has been explored (Stein and 

Capaday 1988, Gottlieb et al. 1970, Soechting et al. 1981, Houk 1979), however the answer is 

still unclear in unimpaired humans. More recently, the role of reflexes in various pathologies 

leading to spasticity has been studied (Kamper and Rymer 2000, Woolacott and Burne 2006, 

Jobin and Levin 2000) since spasticity is associated with an exaggerated stretch reflex. This 

study extends the investigation of reflexes during movement to dystonia in children with cerebral 

palsy (CP). Since dystonia is associated with abnormal postures, we investigate whether postural 

reflexes may be detectable and enhanced during movement in children with dystonia due to CP. 

Seven volunteers with dystonic CP (but without exaggerated reflexes on clinical exam) and 

seven controls were included. Participants were seated comfortably with their testing arm 

strapped into a custom-built robotic arm. The testing apparatus maintained the arm at shoulderlevel 

in the horizontal plane with the shoulder fixed and recorded surface electromyographic 

(EMG) activity of biceps and triceps. A servomotor on the robotic arm provided one of two 

levels of resistance to the participants‟ natural elbow extension. Occasionally the motor stretched 

the biceps to elicit short- and long-latency reflex responses. Multiple linear regression was used 

to analyze effects of subject group, level of resistance and age on short- and long-latency 

reflexes, at a significance level of 0.05. 

Results showed that children with dystonia have larger short- (p < 0.001) and long-latency (p = 

0.0344) responses to stretch during movement than unimpaired control subjects. Younger 

subjects had larger long-latency reflexes (p = 0.0437). There was no significant effect of 

resistance to extension movement in biceps stretch responses. 

These preliminary data suggest spinal reflexes during movement may contribute to the motor 

disorder in childhood dystonia. Future work will incorporate data from more subjects with 

dystonic CP as well as other types of CP to better characterize dystonic muscle activity during 

movement.

Disclosures: S.N. Kukke , None; T.D. Sanger, None. 

Poster 





Support: Department of Occupational Therapy Scholarship Advancement Award 

Title: Comparison of kinematic reaching patterns of stroke survivors completing Rhythmic 

Auditory-Motor Entrainment versus Constraint-Induced Therapy 

Authors: *C. L. MASSIE 1 , M. MALCOLM 1 , M. THAUT 2 ; 

1 Occup. Therapy, 2 Ctr. for Biomed. Res. in Music, Colorado State Univ., Fort Collins, CO 

Abstract: Background: In recent years, principles of motor control, motor learning, and 

sensorimotor facilitation have provided the foundation for conceptual shifts in upper-extremity 

stroke rehabilitation. Constraint-Induced Therapy (CIT) and Rhythmic Auditory Stimulation 

(RAS) are two prominent new approaches to stroke rehabilitation showing strong potential in 

clinical outcome studies. Both techniques, however, address motor recovery in very different 

ways - CIT by forced-use to reverse learned non-use of the hemiparetic arm, RAS by accessing 

sensory codes and feedforward/feedback mechanisms. Whereas CIT drives primarily quantitative 

motor activity by forced use of the affected side, RAS is based on facilitation of qualitative 

motor control in the hemiparetic limb. This project compares stroke-affected reaching kinematics 

of participants completing either CIT or RAS training. 

Methods: Ten stroke survivors completed 2 weeks of CIT incorporating massed practice of 

functionally-based reaching tasks 6 hours daily. Six stroke survivors completed 2 weeks of RAS 

training 3 hours daily. During RAS, participants reached in time with a metronome beat between 

an array of target locations on a template. During kinematic data collection (pre and posttest), 

participants were instructed to reach back and forth with their hemiparetic arm between two 

targets. Kinematic measures included: 1) segmental contribution of shoulder, elbow, and trunk; 

2) shoulder abduction ROM; 3) movement time. 

Results: Following CIT, participants used significantly more shoulder flexion (p=.034, d=1.22) 

to reach but the amount of elbow extension (p=.22) and compensatory trunk movement (p=.67) 

did not change. Participants also used significantly more shoulder abduction following CIT 

(p=.018, d=0.30). Following RAS, participants used significantly more shoulder flexion (p=.004, 

d=0.61) and significantly less trunk movement during reach (p=.032, d=0.9). Compensatory 

shoulder abduction significantly decreased (p

significantly change. Reaching times significantly decreased post CIT and RAS (p=.002, d=1.88; 

p

The paretic upper and lower extremities were assessed using standard assessments to determine 

the level of impairment and functional ability. Actical TM accelerometers were used to measure 

the physical activity in the upper and lower extremities. Actical is a tri-axial, water-proof 

accelerometer with excellent test re-test reliability. At each of the four time points, the 

participants wore one accelerometer on their hip and one accelerometer on each wrist for 3 

consecutive weekdays. Movement of the upper extremities (UE) was reported in total activity 

counts (AC) per day (mean ± standard deviation). Hip AC measured the lower extremity (LE) 

physical activity and provided the number of steps/day. 

Results: The preliminary data (N=7) demonstrate great variance in the degree of weakness, 

functional ability and amount of use of the paretic UE on admission. The paretic UE was used 

less (154054 ± 176916 AC) compared to the non-paretic UE (211575 ± 143208 AC). A decrease 

in the use of the paretic (191341±230143 AC) and non-paretic (236914 ± 189984 AC) UE from 

admission to discharge was seen in four participants who reached discharge (83320±66381, 

186067 ± 98156 AC respectively). On admission the participants had mild weakness of their 

lower extremity (LE) with three participants walking independently. Despite this fact, the levels 

of LE physical activity were very low (8797 ± 3564 AC, 185 ± 86 steps/day) but an increase on 

discharge was seen (24844 ± 1369 AC, 963 ± 795 steps/day). 

Conclusions: The accelerometers provided a real-life measure of physical activity of the upper 

and lower extremities after stroke and can serve as an outcome measure of stroke rehabilitation. 

The accelerometer readings revealed low levels of physical activity, which appeared lower than 

the participant‟s actual ability and is far too low for a meaningful exercise training effect. Since 

the focus in rehabilitation is to prepare patients for the routines of daily living, clinicians should 

encourage participants to be physically active during rehabilitation as well as after discharge. 

Disclosures: D. Rand, None; J.J. Eng, None. 

Poster 





Title: A preliminary study of physiological tremor for precise movements 

Authors: *H. ENDO 1 , M. WADA 2 ; 

1 Human Sci. & Biomed Eng, Natl. Inst. Adv Ind Sci. & Tech., Tsukuba, Japan; 2 Info Sci. & 

Tech., Hokkaido Univ., Hokkaido, Japan

Abstract: [Objective] The purpose of this study was to investigate the relationship between 

physiological tremor and finger dexterity. 

[Method] Position and force tremors were assessed with a test of pinching precision respectively. 

Position tremor was measured from a position of pinched object with an accelerometer and a 

laser displacement-transducer. Force tremor was measured to match a finger tip force to 1N with 

a load cell. A target line was displayed on a screen of an oscilloscope. Position and force 

fluctuations were evaluated with the standard deviation (SD) of a lower frequency component 

(

determine the maximum load they could lift. Next, subjects were asked to hold their hand in a 

horizontal position (forearm supported and secured) while supporting one of four test loads (0%, 

5%, 15% and 25% 1-RM; order of presentation randomized). For each load, two 16 s trials were 

collected with a 2-min break between each trial. Postural tremor was measured with a LASER 

displacement sensor and wrist extensor activity was recording using electromyography (EMG). 

Repeated-measures ANOVA was used to determine group and load effects on dependent 

measures (postural tremor amplitude and time-dependent structure; postural tremor and EMG 

spectral measures). Results: There was a load by group interaction for postural tremor amplitude 

caused by lower tremor amplitude in the ET group at 0% and 5% 1-RM compared to 15% and 

25% 1-RM; load did not influence tremor amplitude in the CN group. There was a load by group 

interaction for the measure of time-dependent structure. This interaction was caused by greater 

time-dependent structure in the CN group between the 0% and 5% 1-RM loads and the 15% and 

25% 1-RM; there was no load effect in the ET group. In ET subjects, total extensor EMG 

spectral area (1-40Hz band) increased with each additional load, reflecting increased muscle 

work. Interestingly, the percent of the total power accounted for by the tremor-related peak 

declined incrementally at 5%, 15% and 25% 1-RM. Discussion: The key finding was that ET 

postural tremor amplitude was greatest at lighter loads. Two factors likely contributed to this 

finding. First, the EMG spectral peak caused by tremor became proportionally smaller at 

increasing loads and thus less able to drive the wrist at the tremor frequency. Second, 

preliminary analysis showed that synchronization of the tremor frequency with the mechanical 

resonant frequency of the wrist joint, which was reduced with increasing load, was greatest at 

0% and 5% 1-RM loads for most individuals in the ET group. 

Disclosures: K.E. Norman , None; M.E. Heroux, None; N.J. Badke, None; G. Pari, None. 

Poster 





Support: ONR Grant N00014-05-1-0844 

NIH Grant R01 HD045639 

Stanford University Bio-X Bioengineering Graduate Student Fellowship 

NSF Grant BCS-0450218

Stanford University Department of Neurology 

Title: The role of noise on motor learning in children with dyskinetic cerebral palsy 

Authors: *V. W. CHU 1 , D. STERNAD 3 , T. D. SANGER 2 ; 

1 Dept Bioengineering, 2 Dept. of Neurol., Stanford Univ., Stanford, CA; 3 Dept. of Kinesiology, 

Pennsylvania State Univ., University Park, PA 

Abstract: Children with dyskinetic cerebral palsy (CP) display highly variable movements due 

to involuntary muscle contractions and spasms. The increased movement variability displayed 

when these children attempt to perform a task acts like excess, random noise. In control and 

machine learning theories, noise can be disruptive and negatively affects control and learning 

systems. Therefore, these extra movements in children with CP likely affect their ability to learn 

new movements. 

In order to study the effect of noise on learning, we used a virtual throwing task developed by 

Müller and Sternad (2004). In this task, participants swing a ball hanging from a center post in a 

simulated environment to hit a target. The task was experimentally constrained so that the ball's 

path was fully characterized by two parameters: position and velocity at release. Task error was 

quantified by the minimum distance between the ball's path and the target. The task is redundant 

with multiple position-velocity combinations that achieve the same outcome. Children with CP 

and age-matched controls practiced throwing at three different targets, with 100 trials for each 

target. 

We analyzed learning of this task in the framework of optimal control. The children were told 

their goal was to minimize the task error, or cost V in optimal control terms. Since performance 

in this task is completely determined by two release variables, cost V is a function of x, a vector 

of the release variables. We assumed that the children were able to optimize the expected value 

of the task error as they explored the space. We used the first few terms in the Taylor expansion 

on V(x) around the expected value of x, E[x]. Taking the expected value of the resulting 

expression, we obtain the equation: 

E[(V(x)] = V(E[x]) + ½ E[(x-E[x]) T (d 2 V/dx 2 )(x-E[x])] + ... 

where d 2 V/dx 2 is the Hessian (H) of the cost function computed at E[x]. This decomposition 

allows to separate the contribution to task performance based on adjusting their average x (the 

first term) or changing their variability in x (the second term). Results from this study showed 

that children in both groups were able to minimize the task error by moving E[x] to a region of 

low error or by controlling the variability in x, or both. However, children with CP showed 

changes that were slower over time and they had greater residual error. This showed different 

levels of effectiveness in their ability to control the variability between the patient and the 

control group. These results provide a better understanding of how learning progresses in the 

presence of excess noise. 

Disclosures: V.W. Chu , None; D. Sternad, None; T.D. Sanger, None. 

Poster




Topic: D.17.b. Cortical planning and execution 

Support: VA RR&D Associate Investigator Award N4725H 

NIA Pepper Center Grant 5P60AG14635 

Title: Identifying the relationship between post-stroke upper extremity motor ability and motor 

function with Rasch analysis: a pilot study 

Authors: *M. L. WOODBURY 1,2 , C. A. VELOZO 2,3 , L. G. RICHARDS 2 , S. A. 

STUDENSKI 4 , S. MIN-LAI 5 , P. W. DUNCAN 6 ; 

1 Univ. Florida, Gainesville, FL; 2 Brain Rehabil. Res. Ctr., NF/SG Veterans Hlth. Syst., 

Gainesville, FL; 3 Dept. of Occup. Therapy, Univ. of Florida, Gainesville, FL; 4 Dept. of Med., 

Univ. of Pittsburgh, Pittsburgh, PA; 5 Univ. of Kansas, Kansas City, KS; 6 Doctor of Physical 

Therapy Div., Duke Univ., Durham, NC 

Abstract: Objective: A critical issue for stroke rehabilitation research is to determine when 

improved motor ability translates to improved motor function. The Fugl-Meyer Upper Extremity 

Assessment (FMA-UE) is widely used to measure UE motor ability. However, the FMA-UE 

does not provide information about specific functional abilities. The purpose of this study was to 

link the impairment-level FMA-UE to the more functional Wolf Motor Function Test (WMFT) 

with Rasch analysis. The conceptual basis of linking existing assessments is that items measuring 

a single underlying motor behavior from different assessments can be measured on a single 

metric. Methods: Rasch analysis was applied to PRE and POST FMA-UE and WMFT item 

scores from 91 individuals enrolled in a rehabilitation clinical trial. Subjects were ages 69.4 ± 

10.3 yrs, and 76.1 ± 27.1 days post a minor (54.9%) or moderate (39.6%) stroke. FMA-UE and 

WMFT items combined into a single dataset were examined for dimensionality using 

confirmatory factor analysis. Rasch co-calibration of the combined dataset identified the 

relationship between the assessments and differential item functioning analysis determined the 

longitudinal stability of co-calibrated items. Results: 94% of the items measured a single latent 

trait. Figure 1 shows items arranged hierarchically according to difficulty; easier items at the 

bottom, more difficult items at the top of the scale. FMA-UE items spanned the metric with 

WMFT items overlapping at the more challenging, “functional” end of the scale. The shaded 

area corresponds to the “amount” of UE motor ability (measured by the FMA-UE) needed to 

have a 50% probability of achieving functional tasks (measured by the WMFT). The itemhierarchy 

was invariant at PRE and POST intervention, suggesting recovery proceeded from 

reacquisition of UE movements (FMA-UE items) to simple functional tasks (WMFT items). 

Conclusion: This pilot study demonstrates that linking assessments with Rasch analysis may be a

novel approach to exploring the relationship between motor ability and motor function. 

Disclosures: M.L. Woodbury , None; C.A. Velozo, None; L.G. Richards, None; S.A. 

Studenski, None; S. Min-Lai, None; P.W. Duncan, None. 

Poster 





Title: BART: A novel laboratory-based instrument to quantify preferred limb use in patients 

after stroke 

Authors: *S.-Y. CHEN 1 , C. E. HAN 2 , N. PARIKH 4 , J. LEE 1 , J. Y. LEE 2 , E. XU 3 , C. J. 

WINSTEIN 1 , N. SCHWEIGHOFER 1 ; 

1 Biokinesiology&PhysicalTherapy, 2 Computer Sci., 3 Neurosci, USC, Los Angeles, CA; 4 Biol. 

Systems Engin., Univ. Calif, Davis, CA 

Abstract: After stroke-hemiparesis, one of the most important patient-centered goals is 

voluntary use of the paretic arm in daily life. Limb specification is an important anticipatory premovement 

parameter that has received little attention from the neurorehabilitation research 

community. The Motor Activity Log (MAL) is the most commonly used instrument to measure 

arm use in daily life for patients post-stroke. However, it is a subjective, semi-structured

interview that relies on memory of limb use from the previous few days or week. Here, we 

propose a novel laboratory-based instrument to quantify limb specification for aiming in the 

workspace, the Bilateral Arm Reaching Task (BART). In BART, participants are instructed to 

reach to targets placed in a horizontal workspace, under a free-choice condition. Specifically, 

targets are distributed along 6 consecutive semi-circles with radii that range from 10 to 27 cm. At 

the onset of each trial, a target indicates the home position, on which the participant positions 

each hand. After the target shifts to a new position, the participant is to lift one hand from home 

and reach for the target as fast as possible. Two magnetic sensors (miniBIRD) are placed on each 

index finger tip to record hand-selection and reaching performance. We measure the use of the 

(paretic) arm by computing the “volume of reach” with a non-linear regression model which 

outputs the probability to use one arm at every {x,y} position, where the {0,0} coordinates 

correspond to the home position, the x axis is parallel to the coronal plane and y axis in the 

saggital plane. Using volume of reach as the outcome measure for the right arm, we tested 13 

non-disabled subjects (6 right handed) and found test-retest reliability was good (r = 0.858), and 

no learning effect was observed (p > 0.1). Further, a sensitivity analysis showed that 150 reach 

trials were sufficient to achieve a stable measure. We then tested the feasibility of BART in a 

pilot study (N = 4) with chronic, post-stroke volunteers, who participated in a minimum of 3 test 

sessions. We determined test-retest reliability, learning effect, and the validity of BART using 

the MAL as a reference measure. Our pilot study suggests that BART may provide an objective 

quantification of arm use in daily life after stroke. A larger study (N = 12) is needed to be 

conclusive. 

Disclosures: S. Chen, None; C.E. Han, None; N. Parikh, None; J. Lee, None; J.Y. Lee, 

None; E. Xu, None; C.J. Winstein, None; N. Schweighofer, None. 

Poster 






Title: 15-30Hz corticomuscular and intermuscular coherence is absent in patients with Primary 

Lateral Sclerosis 

Authors: *M. R. BAKER 1,2 , K. M. FISHER 1 , T. L. WILLIAMS 2,3 , S. N. BAKER 1 ; 

1 Inst. of Neurosci., Univ. Newcastle, Newcastle upon Tyne, United Kingdom; 2 Dept. of Neurol., 

3 Motor Neurone Dis. Care Ctr., Newcastle Gen. Hosp., Newcastle upon Tyne, United Kingdom

Abstract: The contribution of the corticospinal tract (CST) to 15-30 Hz human corticomuscular 

coherence (CMC) is somewhat controversial. Although some studies have identified phase lags 

between cortex and muscle consistent with conduction via the fastest CST axons (Mima et al., 

2000), others have not (Conway et al., 1995). Evidence that 15-30 Hz intermuscular coherence 

(IMC) is mediated by CST is equally circumstantial (Norton & Gorassini, 2006). 

Primary Lateral Sclerosis (PLS) is part of the Motor Neurone Disease (MND) spectrum. In PLS 

there is selective destruction of motor cortical layer V pyramidal neurones and degeneration of 

CST, without involvement of anterior horn cells. Patients usually present with a slowly 

progressive spastic tetraparesis. Hand function is often impaired but sufficient to perform simple 

repetitive manual tasks. In PLS therefore there is an opportunity to address the question: Is an 

intact CST necessary for 15-30 Hz CMC and IMC? 

In 8 PLS patients, who were asked to perform a simple repetitive precision grip task, we found 

no significant 15-30 Hz CMC or IMC. Although the presence of significant 15-30 Hz CMC was 

variable in control subjects, 15-30 Hz IMC was observed in all healthy age-matched controls 

(n=12) and patient controls with the progressive muscular atrophy variant of motor neurone 

disease (n=2). 

We conclude that CMC and IMC in the 15-30 Hz range are dependent upon intact CST. 

Furthermore, we propose that 15-30 Hz IMC could be used diagnostically to assess the integrity 

of CST in MND. 

References 

Mima et al., Electroencepalographic measurement of motor cortex control of muscle activity in 

humans Clin Neurophys 2000; 111(2): 326-37 

Conway et al., Synchronization between motor cortex and spinal motoneuron pool during the 

performance of a maintained motor task in man J Physiol 1995; 489: 917-24 

Norton & Gorassini, Changes in cortically related intermuscular coherence accompanying 

improvements in locomotor skills in incomplete spinal cord injury J Neurophys 2006; 95(4): 

2580-9 

Disclosures: M.R. Baker, None; K.M. Fisher, None; T.L. Williams, None; S.N. Baker, None. 

Poster 




Topic: D.17.d. Plasticity 

Support: Heart and Stroke Foundation of BC and Yukon 

Michael Smith Foundation for Health Research

Canadian Institute of Health Research 

Title: The effects of a graded repetitive arm supplementary program (GRASP) in individuals 

with stroke: a multi-site randomized controlled trial 

Authors: *J. E. HARRIS 1 , J. J. ENG 2 , W. C. MILLER 3 , A. S. DAWSON 4 ; 

2 Physical Therapy, 3 Occup. Sci. and Occup. Therapy, 4 Med., 1 Univ. British Columbia, 

Vancouver, BC, Canada 

Abstract: Purpose: To determine the effectiveness of a four week in-patient graded repetitive 

arm supplementary (GRASP) program on upper limb recovery in stroke. 

Methods: This was a multi-site single blind randomized controlled trial. All individuals who 

sustained a stroke and were admitted to one of 4 inpatient stroke rehabilitation sites were 

screened for study eligibility. 

Measures: Chedoke Arm and Hand Activity Inventory (primary outcome), Action Research Arm 

Test, Motor Activity Log, Grip strength, SF-12 (secondary). Baseline measurements were taken 

within 72 hours of study admission, post intervention at the end of the 4 week intervention and 

retention at 3 months. 

Protocol: Participants were randomly assigned to either the control or experimental group. The 

experimental group received the GRASP protocol, a home-work based exercise program 

designed to improve paretic upper limb strength, active range of motion, and functional 

performance. Three protocols (mild, moderate, and severe) were developed into exercise binders 

and kits. Participants were asked to complete the exercises 6 days per week, 60 minutes each day 

on their own or with family. The control group received an education binder with 4 modules (e.g. 

information on arm recovery, bone health). The study coordinator taught and monitored both 

protocols. All participants received usual care in addition to the study protocols. 

Statistical analysis: Intention-to-treat analyze was performed. Group differences were tested 

using analysis of covariance (ANCOVA). At each evaluation period the baseline score was used 

as the covariate. 

Results: Fifty four individuals were randomized to the GRASP protocol and fifty three to the 

control. At the end of the 4 week intervention, participants receiving the GRASP protocol 

showed greater improvement on the CAHAI compared to the control group (change score of 

14.1 versus 7.9, p

277. Voluntary Movement: Cortical Planning and Execution II 




Title: Corticospinal excitability is enhanced by combining action observation and motor imagery 

Authors: *M. SAKAMOTO 1 , T. MURAOKA 2 , N. MIZUGUCHI 3 , K. KANOSUE 1 ; 

1 Sport Sci., 2 ASMeW, 3 Grad. Sch. of Sport Sci., Waseda Univ., Tokorozawa, Japan 

Abstract: The size of motor-evoked potential (MEP) increased during action observation and 

motor imagery. The present study investigated whether the size of MEP further increased when 

humans simultaneously performed action observation and motor imagery. MEPs induced by 

transcranial magnetic stimulation were recorded from the biceps brachii muscle. The participants 

performed four tasks: 1) they just passively observed the elbow action presented on the screen 

(OBS), 2) they closed their eyes and imagined flexing and extending their right elbow (IMAG), 

3) they opened their eyes and imagined the same action while observing the elbow action 

presented on the screen (OBS+IMAG), 4) they observed the fixed point presented on the screen 

and held their muscles at rest (control). The size of MEP during IMAG and OBS+IMAG 

significantly increased compared to that during the control. In addition, the degree of facilitation 

of MEP size during OBS+IMAG was significantly greater than that during OBS and IMAG. 

Furthermore, the facilitation of MEP size during OBS+IMAG was significantly larger than the 

algebraic sum of the effect caused by OBS and IMAG. However, when the participants imagined 

the elbow flexion-extension while they observed the same action on the screen 180° out of phase, 

MEP size did not increased compared to that during only imagery of the elbow action. These 

findings suggest that the combination of action observation and motor imagery could enhance 

the corticospinal excitability, possibly through the modulation of the mirror systems. 

Disclosures: M. Sakamoto, None; T. Muraoka, None; N. Mizuguchi, None; K. Kanosue, 

None. 

Poster 




Topic: D.17.b. Cortical planning and execution

Support: NIH Grant 1T32 AR050959 

NIH Grant R01 HD40984 

NIH Grant R01 NS 35130 

Title: Motor command for activating and relaxing leg muscles originates from different parts of 

the brain 

Authors: *L. ZHANG 1 , V. SIEMIONOW 1 , K. KISIEL-SAJEWICZ 3,1 , J. ANDRISH 2 , G. H. 

YUE 1 ; 

1 Dept.of Biomed. Engin., 2 Dept. of Orthopaedic Surgery, Cleveland Clin., Cleveland, OH; 3 Dept. 

of Kinesiology, Fac. of Physiotherapy, Univ. Sch. of Physical Educ. in Wroclaw, Wroclaw, 

Poland 

Abstract: Electroencephalogram (EEG)-derived movement-related cortical potential (MRCP) 

preceding a voluntary motor activity represents cortical command to initiate the activity. This 

study investigated whether MRCP locations in the brain for activating and relaxing the knee 

extensor muscles are different. This information is currently not available. Twelve young healthy 

volunteers (24.2 ± 5.8 years, 9 men) participated in this study. Subjects performed intermittent 

isometric knee extensions (45 trials) using their right leg at 20% maximal voluntary contraction 

level. Each contraction lasted 10 s with a 10-s rest period between adjacent contractions. EEG 

data were recorded from the scalp using a 128-channel system during all contractions along with 

knee extension force and muscle EMG signals. MRCP was derived by force-triggered averaging 

of the EEG data from each channel and measured at times right before the contraction and 

relaxation. The EEG Data were processed and the sources (locations of the MRCP in the brain) 

were estimated using the BESA software. The sources were localized on x (left-right), y (frontback), 

and z (top-bottom) coordinates in the Talairach standard brain. MRCP before the 

EMG/force onset was quantified and considered as the command for activating the muscles and 

that before the muscles relaxing from the contraction was regarded as the command for ceasing 

the contraction. The averaged MRCP locations on the y axis were -18.2 (in frontal lobe) for the 

activation and -38.4 (in parietal lobe) for relaxation. The locations on the x axis (-10.5 for 

activation and -10.2 for relaxation) were in the left hemisphere and similar between the two 

conditions. The average locations on the z axis (55.4 for activation and 50.3 for relaxation) 

indicate that the MRCP source for relaxation was deeper (towards the neck) compared with 

activation. This study provides first-time information showing that the primary control area 

generating the command to initiate voluntary knee extension is in the frontal lobe near the motor 

cortices and that to relax the extension is in the parietal lobe close to the sensorimotor integration 

regions. Detailed analysis is underway to pinpoint exact command locations for the muscle 

activation and relaxation activities. These findings are important for guiding sports training, 

sports medicine practice, and neuromuscular function rehabilitation. 

Disclosures: L. Zhang, None; V. Siemionow, None; K. Kisiel-Sajewicz, None; J. Andrish, 

None; G.H. Yue, None.

Poster 






Medical Research Council (UK) 

Title: Estimating sensory delays to primate M1: a comparison of peri-stimulus time histograms 

and coherence phase-frequency regression 

Authors: *B. ZAAIMI, D. S. SOTEROPOULOS, S. N. BAKER; 

Inst. of Neurosci., Newcastle Univ., Newcastle upon Tyne, United Kingdom 

Abstract: During slow finger movements which generate ~10Hz tremulous discontinuities, 

motor cortical oscillations show coherence with EMG at this frequency. Directed coherence 

(Granger causality) analysis reveals the dominant interaction to be in the ascending (sensory) 

direction. Previous work demonstrated strong sensory input to M1, but focussed on measuring 

the onset latency of cortical responses following isolated peripheral stimuli. This is unlikely to be 

relevant to coherence calculations, which are instead sensitive to the average delay of the entire 

response profile. 

In order to estimate such delays, we recorded single unit discharge from the hand representation 

of primary motor cortex in a single macaque monkey sedated with ketamine and diazepam. 

Electrical stimuli were delivered to the median nerve in the upper arm at motor threshold. 

Stimuli were first given regularly at 2Hz, and responses assessed from peri-stimulus time 

histograms (PSTHs). The same stimuli were then delivered as a random Poisson train (mean rate 

10Hz, exponentially distributed inter-stimulus intervals), and coherence was calculated between 

the stimulus event sequence and the spike train. 

The cell illustrated in the figure was typical. A facilitation was seen in the PSTH (panel A), with 

onset latency of 10ms (arrowhead) and duration 6ms; this was followed by a suppression which 

lasted until ~200ms post-stimulus. Significant coherence was seen robustly for frequencies up to 

~50Hz (panel B), with coherence phase showing an approximately linear relationship with 

frequency over this range (panel C). The slope of the phase-frequency regression in this example 

(red lines, panel C) implied a delay of 18ms. 

We conclude that care must be taken in predicting the delays expected from coherence 

measurements on the basis of published onset latencies. This has important implications for

computational modelling of the sensorimotor oscillatory feedback loop. 

Disclosures: B. Zaaimi, None; D.S. Soteropoulos, None; S.N. Baker, None. 

Poster 






MRC 

Title: The role of the motor cortex in discontinuities during slow finger movements 

Authors: *S. N. BAKER, E. R. WILLIAMS, D. S. SOTEROPOULOS; 

Inst. Neurosci, Newcastle Univ., Newcastle upon Tyne, United Kingdom

Abstract: Slow finger movements in man are not smooth but characterised by 8-12 Hz 

discontinuities in finger acceleration. The primary motor cortex (M1) has been proposed to be a 

source of these oscillations. We trained two macaque monkeys to track a moving target by 

performing index finger flexion/extension movements and recorded local field potentials (LFPs) 

and extracellular spiking activity from M1. There was significant coherence between both LFPs 

and spikes with finger acceleration in the ~10 Hz range. Subsets of the M1 cells were 

antidromically identified as pyramidal tract neurons (PTNs). Some PTNs were further identified 

as corticomotoneuronal (CM) cells by spike triggered averaging of rectified EMG. There was 

significant coherence between PTNs and CM cells and finger acceleration between 6-13 Hz. 

LFP-acceleration coherence was similar for flexion and extension movements (0.094 at 9.8 Hz 

and 0.11 at 6.8 Hz respectively), but substantially smaller during periods of steady position 

holding (0.0067 at 9.35 Hz). The coherence phase showed a significant linear relationship with 

frequency over the 6-13 Hz range, as expected for a constant conduction delay; the slope 

indicated that LFP lagged acceleration by 17.1±7.4 ms. Directed coherence (Granger causality) 

analysis supported the conclusion that the dominant interaction was in the acceleration to LFP 

(i.e. sensory) direction. The phase relationships between both LFPs and cell spikes and finger 

acceleration shifted by approximately π radians in flexion compared to extension trials. We 

conclude that although M1 activity is synchronised with peripheral oscillations during slow 

finger movements, M1 is unlikely to be the sole origin of these movement discontinuities. 

Disclosures: S.N. Baker , None; E.R. Williams, None; D.S. Soteropoulos, None. 

Poster 





Support: Academic Frontier Project from the Ministry of Education, Culture, Sports, Science 

and Technology of Japan to Japan Women's College of Physical Education 

a Grant-in-Aid for Young Scientists (B, #19700532) to Kenichi Shibuya 

Title: Reduced contribution of the ipsilateral primary motor cortex to force modulation with 

short-term motor learning in humans 

Authors: *K. SHIBUYA 1 , M. IWADATE 2 , T. SADAMOTO 1 ; 

1 Res. Inst. of Physical Fitness, JWCPE, Tokyo, Japan; 2 Nihon Univ., Narashino, Japan

Abstract: The brain-machine interface (BMI) enables support devices such as artificial hands to 

restore lost human capabilities. The development of these devices would be a breakthrough for 

neuroscientists in the field of movement control. Enabling individuals who have lost their hands 

to drink a cup of coffee through the use of an artificial hand would offer immense gratification to 

them. BMI technology can achieve this goal. However, if the artificial hand is unable to decipher 

the information transmitted by the brain for force modulation, it would breach or release the held 

object. Force modulation of an artificial hand by the brain is a key factor in the development of 

artificial hands through the application of BMI technology. 

Studies to confirm the relationship between oxygenation in the contralateral primary motor 

cortex (M1) and power output in humans have yielded contradictory results. Recently, it was 

confirmed that the oxygenation in the ipsilateral M1 is considerably higher than that in the 

contralateral M1. This finding was attributed to precise force control during contractions. 

Further, the function of the ipsilateral M1 complements or inhibits that of the contralateral M1. 

In case of force modulation, the ipsilateral M1 may function complementarily to the contralateral 

M1. 

The validation of M1 oxygenation measured at each trial by brain mapping techniques especially 

fails to explain the force modulation. If the ipsilateral M1 modulates the muscle force in a 

complementary manner, the oxygenation in the ipsilateral M1 should decrease with the 

habituation of an exercise task. On the other hand, if the ipsilateral M1 does not control muscle 

force in a complementary manner or if the ipsilateral M1 modulates muscle force predominantly, 

then the oxygenation in the ipsilateral M1 should not decrease with the habituation of an exercise 

task. 

We aimed to investigate the effect of motor learning on the contribution of the changes in the 

ipsilateral M1 to force modulation. We monitored the oxygenation in the bilateral M1 during a 

repetitive handgrip task using near-infrared spectroscopy (NIRS). Changes in bilateral M1 

oxygenation were measured by NIRS during 5 repetitions of the handgrip task [exercise: 10 s, 

rest: 75 s; the tasks were performed at 40% and 60% of maximal voluntary contraction]. The 

purpose of the present study was to examine the effect of repeated motor task on ipsilateral M1 

oxygenation.Then we found significant decrease in ipsilateral M1 oxygenation at fifth trail 

compared with the first trial session. The results in the present study indicate that the 

contribution of the ipsilateral M1 to force modulation might be complementary to that of the 

contralateral M1. 

Disclosures: K. Shibuya, None; M. Iwadate, None; T. Sadamoto, None. 

Poster 



Program#/Poster#: 277.6/KK32


Support: Wellcome Trust, 

RIKEN BSI 

MRC 

Title: Macaque M1 pyramidal tract neurons active during precision grip are also activated during 

tool use 

Authors: *A. KRASKOV 1 , M. QUALLO 1,2 , S. SHEPHERD 1 , A. IRIKI 2,1 , R. N. LEMON 1,2 ; 

1 Sobell Dept., UCL Inst. of Neurol., London, United Kingdom; 2 Lab. for Symbolic Cognitive 

Development, RIKEN Brain Sci. Inst., Wako-shi, Saitama, Japan 

Abstract: The fast-conducting component of the corticospinal tract, which also gives rise to the 

cortico-motoneuronal (CM) system, is known to be active during skilled use of the hand and 

digits such as in precision grip. It has also been speculated that this system, which is most highly 

developed in dexterous primates, might also be important for the use of tools. To investigate this 

question we took advantage of the discovery that macaque monkeys can be trained to use a rake 

to retrieve out-of-reach food (Iriki et al., 1996). We trained a Rhesus macaque to perform both 

this task and a precision grip task. 

Although the rake task might appear to be characterized by use of the proximal arm as the 

monkey reaches out with the rake and then pulls in the reward with it, analysis of EMG activity 

recorded using implanted subcutaneous electrodes, revealed that the intrinsic hand muscles were 

also active as the monkey manipulated the exact position and orientation of the rake. EMG from 

intrinsic hand muscles showed features similar to that seen during precision grip, such a 

fractionation of activity across muscles acting upon different digits. 

Over an 8 week period we used a Thomas multiple electrode system to record from pyramidal 

tract neurons (PTNs) in the M1 hand area during performance of both tasks. PTNs were 

identified antidromically from stimulating electrodes implanted in the medullary PT. 

Intracortical stimulation evoked low-threshold hand/digit movements at 73 of 113 sites at which 

a PTN was recorded. 42/113 PTNs had short antidromic latencies (< 1.0 ms), and some were 

identified as CM cells by spike-triggered averaging. The discharge of many of these „fast‟ PTNs 

was as deeply modulated during the precision grip task as during the rake task, particularly 

showing bursts of activity during grasp and manipulation of the rake. Modulation of discharge 

during the rake task was quite different to that seen when the monkey was not using the rake, but 

simply reached for and retrieved the food reward. 

These preliminary results show that fast-conducting PTNs that are active during independent 

finger movements required for the precision grip task, are also active during manipulation of a 

rake tool and are consistent with the CM system being recruited during tool use by primates. 

Reference 

Iriki A et al (1996). Neuroreport, 7, 2325-2330. 

Disclosures: A. Kraskov, None; M. Quallo, None; S. Shepherd, None; A. Iriki, None; R.N. 

Lemon, None.

Poster 





Title: Relationship between finger movement rate, effort and regional cerebral blood flow 

Authors: A. A. FURUSAWA 1 , *H. NISHIJO 2,1 , H. ONISHI 1 ; 

1 Dept of Physical Therapy, Sch. of Hlth. Sci., Niigata Univ. of Hlth. and Welfare, Niigata City, 

Japan; 2 Syst. Emotional Sci, Grad Sch. Med., Univ. of Toyama, Toyama, Japan 

Abstract: Objectives: the purpose of this study was to investigate the relations between cerebral 

activation, and amplitude and frequency of finger movement by means of near infrared 

spectroscopy (NIRS). The subjects were required to perform an index finger tapping (IFT) task 

with five different conditions of finger extension. 

Methods: The subjects sat in a comfortable chair in a semi-darkened shielded room with their 

eyes closed and were instructed to be awake. The regional cerebral hemodynamics (oxy-, deoxy-, 

and the total-Hb) were recorded using 24-channel NIRS from 6 neurologically health subjects 

under five different EIFT tasks. These are 5 conditions: 100% of maximal amplitude (MA) with 

maximal frequency of tapping (T1), 100% MA with minimum frequency (T2), 50% MA with 

maximal frequency (T3), 50% MA with minimum frequency (T4) and a random free tapping 

(T5). In the separate experiment, the maximal amplitude and frequency of tapping were 

determined. The subject‟s wrist and fingers were immobilized by straps except the index finger. 

Each flexion movement of the index finger was triggered by a beep sound and the amplitude of 

the movement was kept constant so that the subjects were required to touch a ruler above the 

finger. Five cycles were performed and each of tapping cycles lasted 20 s, followed by a 40 s rest 

period. Electromyograms (EMGs) from the finger extensor muscle (extensor indicis muscle) 

were recorded. 

Results: the all subjects showed that activation in the motor-related areas was stronger in the 

condition of 100% MA with maximal frequency than the others conditions, consistent with the 

differences in EMG activities. 

Conclusions: the main finding of the study was that oxy-Hb levels decreased progressively 

(T1>T2>T3>T5>T4). These results indicated that brain activation in the motor-related areas 

were dependent on both the movement frequencies and amplitudes. In the MA tapping, EMG 

activity was correlated to oxy-Hb levels in the motor cortex. 

Disclosures: A.A. Furusawa, None; H. Nishijo, None; H. Onishi, None.

Poster 





Support: Patterson Trust 

Princeton University 

Title: Imaging the micro-representation of the motor repertoire with cellular resolution in the 

cortex of awake mice 

Authors: *D. A. DOMBECK 1,2 , D. W. TANK 1,2 ; 

1 Mol Bio, 2 Princeton Neurosci. Inst., Princeton Univ., Princeton, NJ 

Abstract: Intracortical micro-stimulation (ICMS) studies in mammalian motor cortex have 

demonstrated that a macroscopic ethological organization exists in which subregions emphasize 

different ethologically relevant categories of complex movements (Graziano, Ann. Rev. 

Neurosci, 2006). Because in vivo microscopy can provide an unbiased view of the population 

code within the cortical microcircuitry, we used our recently developed methods (Dombeck et. 

al, Neuron, 2007) to investigate the representation of complex mouse movements at the 

microscopic scale. We optically recorded the activity of layer 2/3 neurons in the forelimb motor 

cortex (identified by ICMS) in mobile, head-restrained mice during two distinct complex 

movements (running and grooming); the head-restrained mice remained mobile because their 

limbs were resting on a spherical treadmill. Neural activity was monitored at cellular resolution, 

during running and grooming, using two-photon microscopy measurements of calcium transients 

from populations bolus loaded with Calcium Green-1-AM. The timing of the two movements, 

recorded with a CCD camera, were converted into state vectors which were then cross-correlated 

with the fluorescence traces from the ~100-200 neurons in the ~200-300 micron fields of view. 

Different subpopulations of neurons were observed: those with activity patterns correlating 

primarily to running, primarily to grooming, or to both movements. When the neurons within an 

image were coded according to the magnitude of their movement correlations, the separate 

subpopulations of neurons were found to be spatially ordered: movement correlated neurons 

were more likely to be physically near neurons with the same movement correlation, with 

neurons correlated to both movements mixed throughout the field. For example, grooming 

correlated neurons were found to be physically closer to other grooming correlated neurons 

rather than to running correlated neurons. Though sharp physical boundaries between the 

subpopulations and purely homogeneous spatially ordered subpopulations were rarely observed,

the spatial segregation between the subpopulations was significant; T-test p-values of

simulate the conditions of active movement by applying frequency modulated stimulus trains to 

single M1 cortical sites. We used sinusoidally modulated stimulus trains with base rates of 50 - 

250 Hz and modulation rates of 4 - 28 Hz. This procedure yielded corresponding modulations of 

EMG activity whose latency could be measured relative to the applied stimulus train 

modulations. Our data show typical delays between the modulations in peak sinusoidal cortical 

stimulation and the corresponding EMG responses of 10-20 ms, with a mean of 14.5 ± 5.8 ms 

(n=316), averaged across the entire range of frequencies tested. This is consistent with 

expectations based on conduction time from cortex to muscles. There was a significant 

difference between the onset latencies when the stimulus was presented during the agonist phase 

of movement in the presence of background EMG activity (9.0 ± 2.7 ms; n=145) and the 

antagonist phase (11.8 ± 12.4 ms; n=149). This data supports the hypothesis that the disparity in 

time delay is due to the time needed to bring motoneurons to firing threshold from a 

hyperpolarized state. We conclude that the best estimate of the delay between modulations in 

cortical cell firing output and their impact on muscle activity during active movement is the 

physical conduction time from cortex to muscle. 

Disclosures: G.M. Van Acker , None; P.D. Cheney, None; D.M. Griffin, None. 

Poster 



Program#/Poster#: 277.10/LL2 


Support: NIH Grant T32 DA022616-01 

NIH Grant R01 NS31350 

NIH Grant R90 DK071500-04 

NIH Grant F32 NS047798 

Title: Relation between motor cortical firing and simultaneously recorded joint kinematics of 

hand shaping during reach-to-grasp in the monkey 

Authors: *J. F. KRAYENHAGEN PROSISE 1 , C. M. HENDRIX 2 , T. J. EBNER 2 ; 

1 Biomed. Engin., Univ. Minnesota, Minneapolis, MN; 2 Neurosci., Univ. of Minnesota, 

Minneapolis, MN

Abstract: Primary motor cortex (M1) is known to be important for planning and execution of 

grasping movements; however, the control strategy utilized by M1 in hand-shaping is not known. 

A better understanding of this strategy could aid in the development of control mechanisms for 

prosthetic limbs. To examine this, we are studying the firing of M1 neurons in relationship to the 

kinematics of hand shaping during a reach-to-grasp task that allowed the examination of the 

progression of hand shape with time. The discharge of task-related neurons in M1 hand area was 

recorded as two female rhesus monkeys reached to and grasped a set of 23 objects that were 

designed to systematically vary hand shape. Grasp force was explicitly controlled. The 

metacarpophalangeal (MCP) and interphalangeal (IP) joints of the long fingers, as well as the 

MCP joint of the thumb were tracked using a video-based motion analysis system. Joint angles 

differ significantly across objects (ANOVA, p

Title: Primary motor cortex reflects a statistical model of limb mechanics 

Authors: *T. P. LILLICRAP, S. H. SCOTT; 

Ctr. for Neurosci. Studies, Queen's Univ., Kingston, ON, Canada 

Abstract: Our lab has previously examined how neurons in primary motor cortex (M1) 

modulate their firing in response to constant torque loads applied to the shoulder and elbow 

joints. We found that the preferred torque directions (PTDs) of neurons in M1 are bimodally 

distributed - neurons tend to be most active for flexor torques at one joint in combination with 

extensor torques at the other. Why does this bias in the distribution of PTDs exist? We 

hypothesize that this bias reflects knowledge of the peripheral properties of the musculoskeletal 

system. To illustrate this idea we optimized a simple feedback control law composed of neuronlike 

units to move an arm with different musculoskeletal properties. When the the 

musculoskeletal system is made simple (e.g. biarticular muscles are removed and intersegmental 

dynamics are abolished), the distribution of PTDs for the neuron like elements in our optimized 

feedback control law is uniform. On the other hand, when the musculoskeletal system was made 

to include intersegmental dynamics and biarticular muscles, the distribution of PTDs was 

bimodal and qualitatively matched the observed empirical distribution. We believe that the 

example explored here illuminates a general principle: that the response properties of neurons in 

primary motor cortex are dictated by the interplay between simple optimization rules and the 

musculoskeletal system. 

Disclosures: T.P. Lillicrap, None; S.H. Scott, BKIN Technologies, Kingston Ontario, E. 

Ownership Interest (stock, stock options, patent or other intellectual property). 

Poster 





Title: Fatiguing exercises based on repetitive finger opposition movements can influence 

corticospinal excitability and motor behavior: a time course study 

Authors: *M. BOVE 1 , L. AVANZINO 2 , A. TACCHINO 1 , A. GIANNINI 1 , M. GHILARDI 3 , A. 

QUARTARONE 4 , G. ABBRUZZESE 2 ; 

1 Dept. of Exptl. Med., 2 Dept. of Neurosciences, Ophthalmology and Genet., Univ. of Genoa, 

Genoa, Italy; 3 Dept. of Physiol. and Pharmacol., CUNY Sch. of Med., New York, NY; 4 Dept. of 

Neuroscience, Psychiatric and Anaesthesiological Sci., Univ. of Messina, Messina, Italy

Abstract: Central fatigue is a subjective phenomenon which can be examined using transcranial 

magnetic stimulation (TMS). Motor Evoked Potentials (MEPs) are shown to be reduced up to 15 

min after a fatiguing exercise. Similarly, repetitive finger movements performed at maximal 

velocity for 1 min are capable to induce a long lasting depression of MEP. In literature there are 

no studies comparing data from corticospinal excitability and motor behavior during and after a 

fatiguing exercise. Aim of this study was therefore to compare changes in corticospinal 

excitability and motor behavior parameters in normal subjects during and after a fatiguing motor 

task based on repetitive finger opposition movements. 

Seven normal subjects (5 females and 2 males, mean age 27.5) participated in this study after 

giving their informed consent. Subjects wore a sensor-engineered glove on their right hand and 

performed a repetitive finger motor sequence (opposition of thumb to index, medium, ring and 

little fingers) at a metronome rate of 2Hz. 

Subjects were asked to perform for two times the finger motor sequence lasting 5 min (1st (1S) 

and 2nd SEQUENCE (2S)) with an interval of 1 min during which the MEP recording was 

performed. MEPs were recorded from the right opponens pollicis muscle. The stimulus intensity 

producing MEP amplitudes of 1 mV (SI1mV) at rest was established. Then, 10 responses at 

SI1mV have been collected before 1S, immediately after 1S and 2S and every 5 min for 20 min 

after 2S. 

Further, we considered as motor behavior parameters the time between the touch onset and the 

corresponding tone in its absolute value (ABSTE) and the percentage of advanced and delayed 

finger movements with respect to the tone occurrence. 

When subjects performed a fatiguing exercise MEP amplitude was significantly reduced 

immediately after the 1S and still remained around this value up to 10 minutes after 2S after that 

it recovered to its initial value. Regarding motor behavior data, the percentage of sequences with 

advanced onset significantly decreased from the 1st minutes to the 5th. Similarly, ABSTE value 

significantly increased during the performance of the task from the 1st minutes to the 5th. 

Interestingly, differently from corticospinal excitability, at the beginning of 2S motor behavior 

parameters completely recovered to their initial values (1st minute of 1S). 

We showed that fatiguing exercises based on repetitive finger opposition movements can 

influence corticospinal excitability and motor behavior. However, since the time course of these 

two phenomena is different we suggest that fatigue influences them through two different neural 

mechanisms. 

Disclosures: M. Bove, None; L. Avanzino, None; A. Tacchino, None; A. Giannini, None; M. 

Ghilardi, None; A. Quartarone, None; G. Abbruzzese, None. 

Poster 



Program#/Poster#: 277.13/LL5


Support: Ida Baruch fund 

Rosetrees Trust 

Binational Science foundation (BSF) 

Johnson&Johnson Fund for Innovative Science 

Israel Science foundation 

Title: Learning-induced modulation of motor cortical activity during adaptations to force fields 

with and without visual feedback 

Authors: *F. I. ARCE 1 , I. NOVICK 1 , Y. MANDELBLAT-CERF 1 , E. VAADIA 1,2 ; 

1 Physiol. Dept,Faculty of Med., Hebrew Univ., Jerusalem, Israel; 2 Interdisciplinary Ctr. for 

Neural Computation, Hebrew Univ., Jerusalem, Israel 

Abstract: We have recently shown that prior knowledge and state estimates based on vision and 

proprioception differentially influence the choice of a trajectory plan when humans adapt to 

dynamic force fields and visuomotor rotations (Arce et al., 2007). Here we use the same 

experimental design in monkeys to examine the neuronal activity in the motor cortex before, 

during, and after adaptations to viscous curl force fields with or without visual feedback (VFB). 

Each recording session was composed of three blocks of trials: (1) pre-learning standard, (2) 

force field with or without VFB, and (3) post-learning standard. For the behavioral analysis, we 

measured trajectory and endpoint deviations. We examined the activity of each cell during two 

different epochs of a trial: preparatory activity (PA) covers activity during a hold period after 

target onset (600 ms), and movement-related activity (MRA) during 600 ms after go-signal. Only 

cells that were stable during recording, with an average firing rate of >1 Hz, and directionally 

tuned (bootstrap, p

Reference list 

Arce F, Novick I, Shahar M, Ghez C, Vaadia E (2007) Adaptive strategies differ based on 

sensory estimates of initial and final reach positions. Abstract, Society for Neuroscience, 2007 

Disclosures: F.I. Arce , None; I. Novick, None; Y. Mandelblat-Cerf, None; E. Vaadia, None. 

Poster 





Poster 





Title: Corticospinal and EMG correlates of internal model formation during force field motor 

adaptation 

Authors: *T. P. HUNTER 1 , P. SACCO 2 , D. TURNER 2 ; 

2 Hlth. and Biosci., 1 Univ. of East London, London, United Kingdom 

Abstract: There is an increase in muscle co-contraction during arm reaching in the early stages 

of motor adaptation to a novel force-field. This co-contraction gradually declines as subjects 

form a new internal model to predicatively counteract the force-field. We used transcranial 

magnetic stimulation (TMS) to study whether changes in corticospinal excitability are associated 

with changes in voluntary EMG during motor adaptation. 10 right-handed subjects performed 

visually instructed upper limb ballistic, planar, centre out reaching movements with a robotic 

manipulandum to different directions (135° / 270°) in 2 separate sessions (7 days apart). Each 

session included 11 reaching blocks of 48 trials (move ratio = 50%). Blocks 1-3 were

unperturbed reaching movements (baseline) with TMS applied in Block 3. Blocks 4-8 were 

movements in a novel velocity-dependent robot-induced force field (25 Nsm -1 ; adaptation) with 

TMS in Block 8. Blocks 9-11 were unperturbed reaching movements (deadaptation) with TMS 

in Block 11. Single pulse TMS (120% of resting motor threshold) was applied to the 

contralateral motor cortical biceps representation at 0, 130, 160, 190 and 220ms after the visual 

go-signal and before initiation of voluntary EMG during the test Blocks (i.e. 3, 8, 11). Peak to 

peak amplitude of motor evoked potentials (MEPs; mV) and voluntary EMG were recorded from 

biceps and triceps. During the first 10 trials of force-field exposure at the 135° target there was 

an increase in biceps, but not triceps, muscle activity (p

Abstract: Introduction: Chronic, hemiparetic stroke survivors usually have difficulty in 

independently controlling adjacent joints in the paretic upper limb. The neural mechanisms 

underlying the loss of independent joint control are largely unknown. Our recent results have 

shown evidence for a significant association between abnormally increased overlap of cortical 

joint representations and the loss of independent joint control following stroke. In this study, we 

continued to investigate changes of cortical overlap from the preparation to execution phases of 

isometric single joint motor tasks in stroke subjects. 

Methods: Cortical activity during the generation of single joint isometric motor tasks was 

recorded using high-resolution (160 channel) EEG in 8 severely impaired chronic hemiparetic 

stroke and 8 able-bodied control subjects. The experimental setup also included the simultaneous 

measurements of 15-channel surface EMG and 6 degree-of-freedom (DOF) force/moment 

signals. Motor tasks performed by the subjects were isometric single DOF shoulder abduction 

(SABD) or elbow flexion (EF), all normalized to 25% of the subject‟s maximum voluntary 

torque generating ability. After EEG data processing and averaging, cortical current density 

reconstructions were created with the CURRY software program and analyzed in MATLAB. 

Results: Stroke subjects show significant abnormal coupling between SABD and EF during both 

motor tasks as measured by the torque-coupling ratio. Comparing to the control subjects, in 

stroke subjects the overlap of cortical shoulder/elbow joint representations significantly increases 

starting at the motor preparation phase (about 400 ms before the onset of motor task). 

Furthermore, the overlap increases over time from the motor preparation to the execution phases 

in severely impaired stroke survivors (Fugl-Meyer score < 20). However, in able-bodied control 

subjects the overlap decreases. 

Conclusions: Our results present evidence that changes in the overlap of cortical joint 

representations following stroke occur during the motor preparation phase and increases over 

time contrary to what is observed in able-bodied subjects. These increases in cortical overlap 

may explain the loss of independent joint control observed in severely impaired stroke survivors. 

Disclosures: J. Yao , None; J.P.A. Dewald, None. 

Poster 






Title: Changes in corticospinal excitability during probabilistic sequence learning

Authors: *S. BESTMANN 1 , M. MUNNEKE 2 , L. M. HARRISON 3 , R. MARS 4 , J. C. 

ROTHWELL 2 ; 

1 Sobell Dept, Inst. of Neurol, London, United Kingdom; 2 Sobell Dept., Inst. of Neurol., 

University College London, United Kingdom; 3 Wellcome Trust Ctr. for Neuroimaging, Univ. 

Col. London, London, United Kingdom; 4 Psychology, Univ. of Oxford, Oxford, United 

Kingdom 

Abstract: We often prepare and select actions based on regularities in our physical world. These 

regularities enable us to predict forthcoming events, and prepare actions in anticipation of these. 

Here we tested whether corticospinal excitability (CSE), prior to action, is influenced by changes 

in probabilistic context, i.e. the conditional uncertainty about sequential events (stimuli) in a 

sequential reaction time task. 

Four subjects performed 4-5 sessions (12 blocks with 60 trial per session; separated by several 

days), with informed consent, yielding a minimum of 2880 trials for each subject. One of 4 

arbitrary stimuli was presented o each trial. Responses were made with the left and right index 

and small finger, respectively. We generated 1 st order Markov sequences of different 

predictability. Therefore, the event on the current trial Ti was conditionally dependent on the 

previous trial Ti-1. The transition matrix specifying the probability of transition between 

consecutive events was changed for each block. Each event (and hence each action) was equiprobable 

over the course of the experiment. CSE was measured from the right FDI and ADM 

muscle, 200ms before presentation of a visual stimulus, using transcranial magnetic stimulation 

to left primary motor cortex. We could therefore measure how CSE was sculpted by changes in 

probabilistic context, before an event occurred 1 . 

Information theoretic measures of the conditional uncertainty were used to predict behavioural 

and CSE data during each block. Specifically, we computed the mutual information (MI) among 

events that provides a quantification of the conditional uncertainty on each trial 2 . The influence 

of MI on CSE (and RTs) was estimated using a hierarchical general linear model (GLM) whose 

parameters were optimized by empirical Bayes procedures 1 . We modelled subjects as ideal 

Bayesian observers, starting with flat prior at the beginning of each block 2 . 

Initial analyses show that CSE was larger when the conditional uncertainty about what action 

will be required next decreased (0.14mV/bit). These effects were effector-specific, i.e. dependent 

on the prior expectation that a certain response had to be made. Correspondingly, reaction times 

were faster when MI increased (55ms/bit) 2 . 

Our results suggest that CSE prior to action is shaped on a trial-by-trial basis by the probabilistic 

context in which events guiding our actions occur. This may help to specify actions in 

anticipation of an event. Moreover, it indicates how activity in the motor system is biased by 

probabilistic context, which itself is learned and represented in the brain. 

1.Bestmann et al (2008) Curr Biol 

2.Harrison et al (2006) Neural Netw 

Disclosures: S. Bestmann , None; M. Munneke, None; J.C. Rothwell, None; R. Mars, 

None; L.M. Harrison, None.

Poster 





Support: McDonnell Foundation 


Title: Complex movements evoked by microstimulation of motor cortex in the California ground 

squirrel (Spermophilus beecheyi) 

Authors: *D. F. COOKE 1 , J. PADBERG 1 , T. ZAHNER 2 , B. GRUNEWALD 1 , L. 

KRUBITZER 1,2 ; 

1 Ctr. Neurosci, 2 Dept. of Psychology, UC Davis, Davis, CA 

Abstract: Primates are famously dexterous. Does the basis for this dexterity, the primate motor 

system, have a fundamentally different organization from that of other mammals, or is motor 

organization in mammals different only by degree? Electrical microstimulation of primate motor 

cortex has revealed several aspects of organization, including regions coding complex 

movements resembling eating or defensive behaviors. Here we explore motor organization in the 

California ground squirrel, using the same technique. The California ground squirrel, a diurnal 

rodent with excellent vision, exhibits a number of well-characterized complex motor behaviors, 

such as a distinct set of movements seen only in confrontations with snakes, and skillful 

manipulation with the forepaws. Squirrels thus make an interesting comparison with monkeys 

because both are highly visual arboreal or semiarboreal mammals with varied manual behaviors. 

With the same parameters used in primate studies (500 ms train of biphasic 0.2-ms pulses at 200 

Hz, 50-150 κA), we stimulated motor cortex in anesthetized squirrels and recorded evoked 

movements on video. At the end of stimulation sessions, marking lesions were placed at several 

stimulation sites, animals were perfused, and the brain was flattened and sectioned tangentially. 

Tissue was stained for myelin and lesions were used to relate the location of stimulation sites to 

myeloarchitecture and therefore, to cortical field borders. 

Stimulation at different sites in squirrel motor cortex evoked a variety of movements. A coarse 

somatotopy was seen, with sites associated with hindlimb, forelimb, and face movements located 

progressively more laterally. As in other mammals, internal, fine-scale somatotopy was unclear. 

Forelimb movements usually converged on a particular end posture rather than moving in the 

same direction regardless of starting position. Many forelimb movements were complex and 

included movements of the wrist, elbow and shoulder or movements of multiple body parts, such 

as the face, forelimb and hindlimb. Some movements appeared to have a specific function: at one 

site, stimulation caused the contralateral (right) forelimb to move up and lateral, the left forelimb 

to move down and medial (as if to push the body up and left) and the mouth to open. These

elements are also part of a squirrel avoidance movement during a snake strike. At several sites, 

repetitive, bilateral digging or running movements (3-4 Hz for each limb) were evoked. In 

conclusion, motor cortex in squirrels and monkeys shares several organizational features, 

including the coding of complex behaviorally relevant movements at specific sites. 

Disclosures: D.F. Cooke , None; J. Padberg, None; B. Grunewald, None; T. Zahner, 

None; L. Krubitzer, None. 

Poster 






Title: Is the primary motor cortex engaged during hypnoticomotor tasks? Evidence from burst 

analysis of motor activity 

Authors: *V. GALEA 1 , H. SZECHTMAN 2 , E. Z. WOODY 3 ; 

1 Sch. Rehab Sci., 2 Psychiatry and Behavioural Neurosciences, McMaster Univ., Hamilton, ON, 

Canada; 3 Dept. of Psychology, Univ. of Waterloo, Waterloo, ON, Canada 

Abstract: A large percentage of classic hypnotic effects involve alterations in the perception of 

control of motor movements. The neural mechanisms underlying how hypnosis can modify 

mental processes in perception and consciousness is presently and area of intensive study. We 

chose to analyze two typical challenge tasks under hypnotic suggestion. One task involved the 

suggestion that the subject‟s arm was becoming very stiff and then challenging them to try to 

bend the arm whilst the other task involved the suggestion that the arm was immobile and then 

asked them to try to move it. In this study we used the method proposed by Rudroff et al (2007) 

to analyze the electromygraphic (EMG) profiles for the number of bursts of activity throughout 

the whole task and during the time of the challenge in particular. The driving hypothesis for this 

study was that burst activity in the prime movers of the shoulder or elbow would decrease (or not 

change) during the challenge portion if the primary motor cortex was not engaged in the action 

demanded by the challenge. We tested 12 subjects who had been pre-screened for 

hypnotizability. All of these subjects scored at least a 9 (range 1-12) on the HGSHS:A a 

standardized test of hypnotizability. Burst analysis was conducted on the surface evoked activity 

of the Upper Trapezius, Pectoralis Major, Anterior Deltoid, Posterior Deltoid, Biceps Brachii and 

Triceps Brachii. Our analysis revealed that muscles moving the shoulder girdle and shoulder

joint were more active during the arm rigidity task in 5 out of 12 subjects where burst activity 

was either non-existent or severely decreased around the elbow. It is likely that the burst activity 

from muscles controlling the shoulder girdle was primarily postural in nature, i.e. activity 

required to maintain the raised position of the arm, in these subjects. For the immobility task we 

expected to see higher burst activity in the muscles moving the shoulder girdle and arm if the 

subjects actually did move an immobilized arm. 7 out of 12 subjects exhibited a decrease or no 

burst activity in those muscles thereby exhibiting the same neural pattern as in the arm rigidity 

task. In conclusion, highly hypnotized participants exhibited varying motor control of the upper 

limb in response to the paradoxical suggestion of experiencing a very stiff arm and then 

challenged to try to bend it or being told that their arm was immobile and being asked to move it. 

Since some of our subjects did exhibit increased burst activity in the agonist muscles during the 

challenge portion of the task the question of primary motor cortical engagement remains 

unanswered at this point. 

Disclosures: V. Galea, None; H. Szechtman, None; E.Z. Woody, None. 

Poster 






NICHD center grant HD02528 

Title: Somatotopic organization of hindlimb motor cortex in rhesus macaques 

Authors: *H. M. HUDSON 1 , D. M. GRIFFIN 1 , A. BELHAJ-SAIF 2 , P. D. CHENEY 1 ; 

1 Mol. & Integrative Physiol, Univ. Kansas Med. Ctr., Kansas City, KS; 2 Dept. of Med., Univ. of 

Fribourg, Fribourg, Switzerland 

Abstract: Muscle-based maps of forelimb primary motor cortex (M1) in rhesus macaques show 

a core distal muscle (wrist, digit, and intrinsic hand) representation surrounded by a horseshoeshaped 

proximal muscle (shoulder and elbow) representation (Park et al., 2001). In addition, 

these two areas are separated by a large region representing combinations of distal and proximal 

muscles. In this study we investigated the possibility that a similar representation of distal and 

proximal muscles exists for hindlimb M1. The hindlimb representation of two rhesus monkeys 

was systematically mapped using stimulus triggered averaging of EMG activity at intensities of

15, 30, and 60κA. The stimulus frequency was 15 Hz. EMG activity was recorded chronically 

from 20 muscles of the hindlimb while the monkey grasped a post with its foot and performed 

alternating push-pull movements of the leg. This task engaged both distal and proximal muscles 

in reliable and stereotyped patterns of activation. Repetitive intracortical microstimulation was 

used to identify output to muscles from regions outside of the primary hindlimb M1 

representation (e.g., trunk and tail). Manual manipulation of the hindlimb was used to evoke 

sensory responses. Muscle-based maps of hindlimb M1 showed consistent features in two 

monkeys. The hindlimb representation extended 8-11 mm anterior from the central sulcus where 

it bordered an area producing no post-stimulus effects. Lateral from the midline, the hindlimb 

area extended 9-10 mm where it was bordered by the M1 trunk representation. Maps constructed 

using a 60κA stimulus showed a large region representing combinations of distal and proximal 

muscles with smaller, bordering representations of distal only (ankle, digit, and intrinsic foot) 

and proximal only (hip and knee) muscles. At lower stimulus intensities, the area representing 

combinations of distal and proximal muscles diminished in size while the distal only 

representation expanded and occupied a core region of the overall representation. Proximal only 

regions continued to form a peripheral border surrounding these areas. Our results demonstrate 

that the basic features of distal and proximal muscle representation in hindlimb M1 are similar to 

forelimb M1. 

Disclosures: H.M. Hudson , None; A. Belhaj-Saif, None; D.M. Griffin, None; P.D. Cheney, 

None. 

Poster 






Title: Interactions between inhibitory intracortical pathways and the modulation of 

interhemispheric inhibition in the human motor cortex during voluntary movement 

Authors: *M. E. GODDARD, W. R. STAINES; 

Kinesiology, Univ. of Waterloo, Waterloo, ON, Canada 

Abstract: Voluntary unilateral movements of the upper limb can increase the cortical 

excitability of the ipsilateral homologous motor representations. However, the mechanism 

underlying this modulation of excitability is not clear. The purpose of this study was to

investigate the role of short and long latency intracortical inhibition in the previoiusly observed 

interhemispheric modulation of motor cortical excitability. We delivered focal transcranial 

magnetic stimulation (TMS) to healthy, right-handed subjects targeting either the extensor carpi 

radialis (ECR) or first dorsal interosseous (FDI) motor representations. A paired-pulse protocol 

was applied to the left primary motor cortex using interstimulus intervals of 3 ms (short interval 

intracortical inhibition, SICI) and 100 ms (long interval intracortical inhibition, LICI). The 

conditioning stimulus was set at 80% and 120% of resting motor threshold (MT) for the SICI and 

LICI trials respectively. In both cases the test stimulus was suprathreshold at 120% MT. Motorevoked 

potentials (MEPs), recorded using surface electrodes, were measured while the 

homologous muscle groups contralateral to the right target hand were both active and at rest. In 

the active condition, EMG activity from the initiation of a dynamic contraction (10% of the 

maximal voluntary contraction) of either the ECR or FDI ipsilateral to the stimulated motor 

cortex was used to trigger the TMS. Motor thresholds were adjusted in the active conditions. For 

SICI and LICI conditions, the peak-to-peak MEP amplitude was averaged for each condition and 

the resulting data was normalized as a percentage change from the average MEP for single pulse 

stimulation. Results showed that discrete unilateral contractions of both ECR and FDI increased 

the excitability of the contralateral homologous muscle representation compared to when the 

muscle was at rest. Further, voluntary contraction of the contralateral ECR significantly reduced 

SICI whereas contraction of the FDI had a marginal effect on SICI in the homologous motor 

areas of the opposite hemisphere. Active conditions for both ECR and FDI had little effect on 

LICI. Our preliminary findings suggest that the interhemispheric modulation of motor cortical 

excitability between homologous muscle representations is primarily mediated through pathways 

acting on GABA(A) mediated inhibitory intracortical interneurons. 

Support Contributed By: NSERC 

Disclosures: M.E. Goddard , None; W.R. Staines, None. 

Poster 






NICHD center grant HD02528 

Title: Comparison of output effects on EMG activity from primary motor cortex obtained with 

different methods of microstimulation

Authors: *D. M. GRIFFIN 1 , H. M. HUDSON 1 , A. BELHAJ-SAIF 2 , P. D. CHENEY 1 ; 

1 Dept. Molec & Integ Physiol, Univ. of Kansas Med. Ctr., Kansas City, KS; 2 Dept. of Med., 

Univ. of Fribourg, Fribourg, Switzerland 

Abstract: Various forms of intracortical microstimulation (ICMS) have been used to quantify 

output effects from motor cortex to muscles of the limbs (Baker et al. 1998; Boudrias et al. 2006; 

Davidson and Buford 2006; Hummelsheim et al. 1986; Moritz et al. 2006; Perlmutter et al. 1998; 

Schieber 2002; Park et al. 2004) and to map the distribution of output to forelimb muscles 

(Godschalk et al. 1995; Hatanaka et al. 2001; Luppino et al. 1991; Mitz and Wise 1987; Park et 

al. 2001; Raos et al. 2003). However, few studies have documented how measures of motor 

output change with different ICMS stimulus parameters. The primary objective of this study was 

to compare output effects from primary motor cortex (M1) elicited by three forms of ICMS: 

stimulus triggered averaging of EMG activity (StTA), repetitive short duration ICMS (RS-ICMS, 

10 pulses @ 330 Hz) and repetitive long duration ICMS (RL-ICMS, 100 pulses @ 200 Hz). 

Averages of EMG activity from 24 forelimb muscles were collected from a male rhesus macaque 

during an isometric push-pull task and a forelimb reaching task. Twenty-two layer V sites were 

identified and microstimuli were applied at a low rate (15 Hz) to obtain output effects with StTA 

and at a high rate to obtain output effects with RS-ICMS and RL-ICMS. At 15 µA, effects in 

StTAs matched 76% of the effects elicited with RS-ICMS and 63% of effects elicited with RL- 

ICMS at the same sites. At higher stimulus intensities, the percent of muscles with effects 

matching the effects in StTAs showed modest improvement in most cases. The extent of 

matching effects across stimulation methods improved substantially when only the distal muscles 

were considered. This is probably attributable to the fact that distal muscles also had the 

strongest stimulus evoked effects. While significant disparities exist between effects obtained 

with StTA and short or long duration ICMS, overall the results might be viewed as surprisingly 

consistent given the potential for physiological spread and expansion of effects with repetitive 

stimulation methods. These results can also be related to output effects with spike triggered 

averaging. Cheney and Fetz (1985) found that the match between CM cell post-spike effects and 

post-stimulus effects, at low intensity (10 µA) in distal muscles, was 95%. Taking this result 

together with the results of the present study suggests a relatively high degree of similarity, at 

least for distal muscles, in output effects from high frequency repetitive stimulation methods 

relative to output effects from single CM cells at the site of stimulation. 

Disclosures: D.M. Griffin , None; H.M. Hudson, None; A. Belhaj-Saif, None; P.D. Cheney, 

None. 

Poster 



Program#/Poster#: 277.23/LL15


Support: NIH AG024106 (RS) 

NIH T32 AG000114 (JL) 

UM Pepper Center Human Subjects Core (NIH AG 08808) 

Title: Age-related changes in functional connectivity of the motor system 

Authors: *J. LANGAN 1,2 , S. J. PELTIER 3 , J. BO 1 , R. D. SEIDLER 1,2,4,5 ; 

1 Div. of Kinesiology, 2 Inst. of Gerontology, 3 Dept. of Biomed. Engin., 4 Dept. of Psychology, 

5 Neurosci. Program, Univ. of Michigan, Ann Arbor, MI 

Abstract: Older adults (OA) performing the same motor task as younger adults (YA) tend to 

recruit more brain regions and have less lateralized brain activation (Mattay, 2002). One 

explanation offered for the difference in brain activation patterns relates to task difficulty. OA 

may be more challenged by the task and require increased resources to successfully perform the 

task. Another possibility is that age associated changes in the corpus callosum (CC) may disrupt 

interhemispheric balance resulting in disinhibition between hemispheres (Talelli et al, 2008). To 

better understand the influence of interhemispheric balance on functional brain activation we 

asked right handed YA (n=18, 21.4 + 2.1 yrs, 9 males) and OA (n=18, 71.7 + 5.8 yrs, 7 males) to 

participate in two forms of magnetic resonance imaging (MRI) 1.) functional connectivity MRI 

(fcMRI) was used to establish interhemispheric balance, specifically between sensorimotor 

cortices at rest and 2.) functional MRI (fMRI) was used to examine the brain activation pattern 

during a motor task involving moving a cursor with a joystick into a target circle. We 

hypothesized that OA who were less lateralized in fMRI activation would show stronger 

interhemispheric connectivity. We also hypothesized that motor performance would be 

positively correlated with decreased lateralization, and therefore increased interhemispheric 

connectivity in OA. The OA did not perform as well as the YA on the joystick task, exhibiting 

longer reaction times (RT) and larger errors. Similar to previous studies the OA showed 

recruitment of additional brain regions and exhibited less lateralized activity in comparison to 

YA in the fMRI scans. In contrast to our hypotheses, we found that OA who recruited more of 

the right sensorimotor cortex had slower RT. Furthermore, we found that greater lateralization in 

the fMRI task was associated with greater interhemispheric connectivity in fcMRI for the OA. 

This indicates that stronger interhemispheric connectivity between sensorimotor cortices is 

associated with more lateralization of fMRI data. The results suggest that greater 

interhemispheric connectivity in OA allows for inhibition of the ipsilateral sensorimotor cortex, 

and better motor task performance. 

Supported by NIH AG024106 (RS), the UM Pepper Center Human Subjects Core (NIH AG 

08808), and NIH T32 AG000114 (JL). 

Disclosures: J. Langan, None; S.J. Peltier, None; J. Bo, None; R.D. Seidler, None.

Poster 





Title: Contralateral activity during a unilateral contraction mirrors the direction of movement 

rather than the active muscle assemblies 

Authors: *I. ZIJDEWIND, M. POST, P. MULDER, R. BAKELS; 

Med. Physiol., Univ. Med. Ctr., Groningen, Netherlands 

Abstract: Strong unilateral contractions are accompanied by unintended activation (associated 

contractions) of the contralateral hand. It is not known whether these associated contractions 

mirror the activity of the voluntarily activated target muscles or rather the direction of the 

voluntarily target movement. We assessed the distribution of contralateral activation for the left 

index finger during a sustained maximal abduction of the right index finger (target). Subjects 

(n=8) performed the task in 4 different experiments. The position of the left and right hand were 

systematically varied between experiments. In experiment 1 both hands were held vertically 

(lower arms midway between pronation and supination) in a symmetrical position and in 

experiment 2 both hands were held horizontally (palm of the hands downwards). In experiment 3 

and 4 hands were in an asymmetrical position; one hand palm downwards and the other hand 

vertical. 

In experiments 1 and 3 the direction of the contralateral associated force was upwards, i.e. in the 

symmetrical hand position the contralateral force increased mainly in abduction direction 

whereas in the asymmetrical hand position the contralateral force increased in the extension 

direction. In experiments 2 and 4 the main direction of the contralateral associated force was to 

the midline, i.e. in the symmetrical hand position the contralateral force increased mainly in 

abduction direction whereas in the asymmetrical hand position the contralateral force increased 

in the flexion direction. However, a small upwards (abduction) force was also seen in experiment 

4 (target hand: palm of the hand downwards; non-target hand: vertical). Thus, the contralateral 

contractions mirrored the direction of the target movement rather than simply the activity of the 

muscles activated on the target side. Hence, our results indicate that (contralateral) motor 

activation is mainly organized in an external, direction related reference frame rather than in a 

simple muscle related reference frame. 

Disclosures: I. Zijdewind, None; M. Post, None; P. Mulder, None; R. Bakels, None. 

Poster





Support: DFG grant KR 1392/7-5 

Title: Corticomuscular coherence extensively elevated after isometric dynamic contraction 

Authors: W. OMLOR 1 , M.-C. HEPP-REYMOND 2 , L. PATINO 1 , *R. G. KRISTEVA 1 ; 

1 Dept Neurol, Univ. Freiburg, Freiburg, Germany; 2 Inst. of Neuroinformatics, Univ. and ETH, 

Zürich, Switzerland 

Abstract: During isometric muscle contractions, human sensorimotor cortex shows oscillatory 

activity that is coherent with contralateral EMG at beta- and gamma-frequencies. The 

corticomuscular gamma-band coherence is supposed to reflect a processing mode related to the 

generation of dynamic force in an anticipated force profile. In contrast, corticospinal betaoscillations 

may function to favour a stationary motor state and correlates with performance and 

the displacement before the contraction, but the functional significance of both mechanisms still 

remains cryptic.We hypothesized that the corticospinal beta-range coherence also depends on the 

engagement of the sensorimotor system prior to a steady contraction task, thus providing a 

versatile tool for the adjustment to a stationary state. 

To test this hypothesis we instructed eight healthy subjects to compensate an isometric static 

force after rest as well as after compensating a periodical dynamic force with their right index 

finger. Visual feedback about the position of the index finger was provided to the subjects 

through a target cycle on a screen. We calculated EEG-EMG coherence and cortical motor 

spectral power as well as the force error during both conditions. 

The sinusoidal force was accompanied by corticomuscular gamma-band coherence which was 

absent or reduced during rest. Corticomuscular beta-band coherence during static force was 

extensively elevated if the subjects exerted sinusoidal force instead of rest afore. The increased 

beta-range coherence was neither associated with improved performance nor with increased 

displacement afore. 

We suggest corticospinal gamma-oscillations may function to generate more precise dynamic 

force. Corticospinal beta oscillations may in turn serve to overpower active motor routines 

related to the alteration of force and thereby facilitate the transition to a stationary motor state. 

Therefore, the corticomuscular beta-band coherence is highly dependent on the engagement of 

the sensorimotor system before a steady contraction task. 

Disclosures: W. Omlor, None; M. Hepp-Reymond, None; L. Patino, None; R.G. Kristeva , 

None.

Poster 





Support: Department of Defense, Army Research Office, W911NF0510538 

Title: Electrocortical dynamics during competitive psychomotor performance 

Authors: *J. C. RIETSCHEL 1 , M. E. COSTANZO 1 , R. N. GOODMAN 1 , A. J. HAUFLER 1 , 

L.-C. LO 1 , C. G. MCDONALD 2 , M. SAFFER 1 , B. D. HATFIELD 1 ; 

1 Neurosci. and Cognitive Sci., Univ. of Maryland, College Park, MD; 2 Dept. of Psychology, 

George Mason Univ., Fairfax, VA 

Abstract: Placing individuals in a competitive situation induces variability of their motor 

performance; while some „rise to the occasion‟ others may respond by „choking.‟ Despite this 

robust behavioral phenomenon, the neurobiological changes that underlie this performance 

variability are unclear. The psychomotor efficiency hypothesis posits that superior performance 

occurs through a reduction of task-irrelevant cerebral cortical processing (Hatfield and Hillman, 

2001). Specifically, this reduction has been inferred through increases in alpha power which is 

thought to reflect cortical „idling‟ in areas not essential for task execution. However, 

investigation of this hypothesis has been limited to novice-expert contrasts and motor learning 

studies. Thus, the purpose of this study was to examine the changes in cerebral cortical dynamics 

and kinematics during competition in an effort to explain performance variability during a 

precision aiming task. Nineteen participants from the U. Maryland Reserve Officers' Training 

Corps (ROTC) program completed a pistol shooting task under both performance alone (PA) and 

competitive (C) conditions. EEG, autonomic measures and subjective self report were recorded 

from subjects while they completed the PA condition independently. These same measures were 

assessed during the C condition where the two participants were monitored simultaneously. The 

EEG montage consisted of 30 electrodes and was sampled at 1000Hz. Further, spectral averages 

in the alpha band (8-13Hz) were calculated for one-second bins in the 4 seconds preceding 

trigger pull. Alpha power was subjected to a 2 (Order) X 2 (Condition) X 2 (Hemisphere) X 5 

(Region) X 4 Second) ANOVA. Changes in Spielberger State Anxiety Inventory (STAI), heart 

rate, cortisol and skin conductance provide evidence of a systematic difference in the 

psychological state between conditions, specifically, increased state anxiety during the C 

condition. Further, performance (i.e. aiming trajectories) was more variable during the 

competition along with a significant decrease in alpha power. These results provide support for 

generalizing the psychomotor efficiency hypothesis to a competitive situation. Beyond the 

spectral assessment of EEG alpha power, an addition principal component analysis was 

employed to further elucidate the temporal dynamics and spatial specificity of the event-related 

alpha power.

Disclosures: J.C. Rietschel, None; M.E. Costanzo, None; R.N. Goodman, None; A.J. 

Haufler, None; L. Lo, None; C.G. McDonald, None; M. Saffer, None; B.D. Hatfield, None. 

Poster 





Support: Grant-in-Aid for Young Scientists (B) 19700480 from the MEXT Japan 

Title: Lateralized power and phase dynamics in single-trial EEG related to the motor response 

Authors: *K. YAMANAKA, Y. YAMAMOTO; 

Dept Physical & Hlth. Educ, Univ. Tokyo, Tokyo, Japan 

Abstract: It is generally considered that lateralized readiness potential (LRP), which is the 

averaged potential negativity in the motor area contralateral to the response hand around the 

response onset time, is an index of the hand-specific motor preparation. However, the generation 

mechanism of such asymmetry in the averaged waveforms, that is, the underlying basis of the 

LRP waveforms, has not been reported. Therefore, we recorded the whole scalp surface 

electroencephalography (EEG) for 15 subjects during visual Go/NoGo tasks, and examined the 

EEG data over the bilateral motor areas around the response onset time in correctly responded 

Go trials by combining a detailed analysis of power and phase dynamics with a traditional LRP 

analysis. In the theta frequency bands, power increase and inter-trial phase-locking occurs 

broadly over the motor areas, including both hemispheres, around the response times. This result 

indicates an existence of polarity- and latency-fixed deflections whose cycle-length corresponds 

to theta band. However, around the response onset, the phase angles of the theta band responses 

at the ipsilateral motor area consistently precede those at the contralateral motor area, suggesting 

the difference of timing in the theta band additive deflections depending on the response hand. In 

the alpha frequency band, from the time period before the Go stimulus presentation, the power 

values in the contralateral motor area are smaller than those in the ipsilatelal motor area. And 

then, the alpha band power values bilaterally decrease around the response onset. However, these 

alpha band power decreases are hardly reflected in the averaged waveforms, because they are not 

accompanied by the inter-trial phase-locking. From these results, we conclude that, the LRP 

waveforms are mainly attributed to the different time courses of the theta band additive response 

in the motor areas ipsi- and contralateral to the response hand. Furthermore, we suggest that the 

lateralized power and phase dynamics around the response times, including alpha band power

dynamics which is not substantially reflected in the LRP waveforms, might in part reflect human 

motor preparation and execution function. 

Disclosures: K. Yamanaka, None; Y. Yamamoto, None. 

Poster 






GK-12 STEP NSF 

Georgia Space Consortium 

Title: Corticomuscular coherence between EEG-EMG is not associated with motor output 

variability during dual task 

Authors: *A. N. JOHNSON 1 , S. VOHRA 2 , M. SHINOHARA 2 ; 

1 Electrical & Computer Eng., 2 Applied Physiol., Georgia Inst. Technol., Atlanta, GA 

Abstract: An increase in motor output variability after immobilization has been reported to be 

accompanied with a reduction in beta band (12 - 30 Hz) coherence between EEG and EMG in a 

hand muscle. Recent studies have also suggested a modulation of corticomuscular coherence 

with dual task. The purpose of this study was to determine the potential association between beta 

band coherence and motor output variability in a hand muscle during single and dual tasks that 

involved motor and/or cognitive task. Six (3 men, 3 women) healthy right-handed adults (28 ± 

5.5 yrs) participated in the study. Subjects performed cognitive task, unilateral motor task, 

bilateral motor task, and concurrent motor-cognitive task in a randomized order. The cognitive 

task involved arithmetic and memory while the motor task involved isometric contraction of the 

first dorsal interosseus muscle in the right or both hands. In motor tasks, subjects received visual 

feedback of force and were instructed to match 5% and 10% of their maximal force by abducting 

the index finger of the right or both hands. Surface EMG was recorded over the first dorsal 

interosseus muscle in the right hand and EEG was recorded over the primary motor cortex in the 

left hemisphere. Statistical analysis resulted in no significant effect of contraction level in any 

measure of interest. There was a significant decrease in beta band coherence during dual tasks (P 

< 0.01). However there was no variation in the degree of this decrease between the bilateral

motor task and motor-cognitive task. On average, coherence at 25 - 26.5 Hz decreased from 

0.095 during unilateral motor task to 0.063 during bilateral motor task and 0.058 during motorcognitive 

task. The coefficient of variation of force (1.6% - 1.9%) and rectified EMG (13.2% - 

13.6%) were constant across tasks. Although dual task did not influence motor output variability, 

there was a tendency for decrease in cognitive performance with dual tasks. On average, 

cognitive performance (accuracy of arithmetic and memory) decreased from 86.5% during 

cognitive task to 74.0% during motor-cognitive task due to a decline in 5 out of 6 subjects. These 

results suggest beta band coherence decreases when attention is divided during dual tasks 

irrespective of the type of additional task, but there is no association between corticomuscular 

coherence and motor output variability in a hand muscle during these tasks. 

Disclosures: A.N. Johnson, None; M. Shinohara, None; S. Vohra, None. 

Poster 





Support: FNRS research Grant 1.5.047.06 

Title: Neural correlates of automatic motor control: an event-related fMRI study 

Authors: *G. GARRAUX 1 , P. PEIGNEUX 3 , K. D'OSTILIO 2 ; 

1 Cyclotron Res. Ctr., 2 Univ. of Liège, Liège, Belgium; 3 Fac. of Psychological and Educational 

Sci., Free Univ. of Brussels, Brussels, Belgium 

Abstract: The neural correlates of automatic motor control in humans is poorly known. To 

investigate this issue, we used event-related BOLD fMRI at 3T to record brain activity in 10 

healthy volunteers (5F/5M; age range: 20-26) as they performed a subliminal priming task (1). 

Arrows pointing to the left or to the right were used as prime and target stimuli. Primes were 

rendered invisible to the subjects using short presentation duration and visual backward masking. 

The target stimulus, which cued a response with the left or the right hand, was displayed at the 

same time as the random pattern mask. As expected, the group mean reaction time was shorter 

when the prime and target pointed to the same direction than when they pointed to opposite 

directions (mean ± sd: 413 ± 119,4 ms vs. 428 ± 119,3 ms; paired t-test, p=0.02). In a prime 

identification task, subjects‟ performance was at chance level for primes presented for 17 ms as 

in the main experiment. Brain activity was stronger in several areas including caudal medial 

frontal regions (i.e., medial premotor and anterior cingulate areas) and striatum (p

uncorrected) during compatible trials as compared with incompatible trials. No significant 

difference was observed in primary motor cortex. These preliminary results suggest that 

automatic control of movements is subserved by a cortico-subcortical network that partially 

overlap with that that has been implicated in the conscious control of voluntary movements. 

Reference 

(1) Eimer M, Schlaghecken F. Response facilitation and inhibition in subliminal priming. Biol 

Psychol 2003; 64(1-2):7-26. 

Disclosures: G. Garraux, None; P. Peigneux, None; K. D'Ostilio, None. 

Poster 

278. Sex Differences I 



Topic: E.01.e. Sexual differences 

Support: NSF Grant IOS-0721462 

Title: Sex differences during development of the MPN mag in Syrian hamsters using NeuN 

labeling 

Authors: *H. RICHENDRFER, J. SWANN; 

Dept Biol Sci., Lehigh Univ., Bethlehem, PA 

Abstract: Sexual behavior is critical for ensuring the survival of many species. To date, male 

sex behavior in all vertebrate species is regulated by the medial preoptic area (MPOA). In the 

Syrian hamster, male mating behavior is regulated by a subdivision of the MPOA, the 

magnocellular division of the medial preoptic nucleus (MPNmag). Lesions of the MPNmag 

eliminate male mating behavior. Adult males have significantly greater number and density of 

neurons in this nucleus compared to females. Our lab has shown that the MPN mag becomes 

sexually dimorphic in the total number of cells between postnatal days 5 and 10. Yet, it is not 

known whether these differences in cell number reflect differences in neuron number or another 

cell type. NeuN is a protein found exclusively in neurons. The goal of this experiment was to 

determine if there is a sex difference in the number of neurons in the MPN mag on postnatal days 

5 and 10 by examining the number of NeuN labeled cells in the MPN mag. 

Male and female hamsters were perfused 5 or 10 days after birth. Brains were removed, cut into 

40 um sections on a freezing microtome, mounted on slides and stored at -20C until labeled for 

NeuN using immunocytochemistry. The number of neurons was determined using the particle

counting program in ImageJ. Our data suggests that sex differences in neuron number are present 

as early as postnatal day 5. 

Disclosures: H. Richendrfer , None; J. Swann, None. 

Poster 





Support: NSF Grant IOS-0721462 

Title: The role of apoptosis in sexual differentiation of the MPN mag of the Syrian hamster 

Authors: J. WETZEL, H. RICHENDRFER, *J. M. SWANN; 

Dept Biol Sci., Lehigh Univ., Bethlehem, PA 

Abstract: There are areas of the brain that are sexually dimorphic. The ways that these 

differences develop are not completely understood. Apoptosis and differentiation of cell 

phenotype appear to be major contributors to the development of sex differences found in the 

central nervous system. Sex differences have been described in a nucleus in the preoptic area 

referred to as the magnocellular subdivision of the medial preoptic nucleus (MPN mag) of the 

Syrian hamster. The MPN mag plays a critical role in the regulation of male sex behavior. 

Bilateral ablation of the MPN mag eliminates copulation in male Syrian hamsters. There are sex 

differences in neuron density and number in the adult hamster MPN mag. The male MPN mag is 

larger and has more neurons by postnatal day 10. It is, therefore, hypothesized that there is a 

significantly greater amount of apoptosis in the female MPN mag thus leading to more neurons 

in the male MPN mag and sexual differentiation of the nucleus. The goal of this experiment is to 

determine the differences in the amount of apoptosis in the MPN mag of neonatal male and 

female Syrian hamsters, and how these differences relate to its sexual differentiation and 

development. It is expected that in studying both P-5 and P-10 male and female Syrian hamsters, 

P-5 male hamsters will present a lesser incidence of apoptosis in the MPN mag compared to P-5 

females. An antibody to Caspase-3, a precursor to apoptosis, was used to indicate apoptosis in 40 

um sections. Higher levels of cells expressing the marker in females will suggest a mechanism 

for the sexual differentiation of this nucleus. 

Disclosures: J. Wetzel, None; J.M. Swann , None; H. Richendrfer, None.

Poster 





Support: Italian Ministry of Research Grant 2006072719_004 

Title: Aromatase distribution during sheep brain development and evaluation on the sexually 

dimorphic nucleus 

Authors: S. GADAU, A. MURA, G. LEPORE, M. ZEDDA, B. ARRU, *V. FARINA; 

Univ. Sassari, Sassari, Italy 

Abstract: During foetal development, a series of crucial events may induce stable modifications 

in cell number and morphology of selected brain nuclei generating specific neuronal networks. 

An important step in the main process of foetal brain sexual differentiation is the aromatization 

of testosterone to estradiol by P450 aromatase, being its onset and time length depending on the 

length of gestation. In the foetal brain of sheep, a long-gestation species, P450 aromatase activity 

starts from embryonic day E30 to E100. From this time onward, aromatase activity is evident in 

a cell group of the caudal preoptic area corresponding to the sexually dimorphic nucleus (SDN) 

of adults in a lot of species. As to the sheep, its presence is still controversial. 

In this study, the possible distribution of P450 aromatase was assessed in the hypothalamic 

preoptic area of foetal sheep brain at different stages of development (E30 to E115). In addition, 

the existence of SDN has been investigated in adult sheep. Coronal sections of foetal 

hypothalami were processed by immunoperoxidase technique (ABC method) employing the 

following polyclonal antibodies: anti-P450 aromatase, anti-estrogen receptor (ERβ) and antiandrogen 

receptor (AR). In addition, coronal sections of hypothalami from adult sheep of both 

sexes were processed with the monoclonal antibody anti-calbindin, a widely considered marker 

of SDN. 

A high immunoreactivity was found for aromatase, ERβ and AR throughout the foetal period 

under examination in different brain regions being mainly distributed in the periventricular area 

where the bulk of cells were stained. Moreover, no calbindin-immunoreactive cells were seen in 

hypothalamic coronal sections of adult sheep of both sexes. SDN cannot be identified in adult 

sheep, neither on the basis of morphological observations, nor of calbindin immunoreactivity. 

However, that species is well-characterized by the wide extension of aromatase-immunopositive 

brain regions, even in comparison with species taxonomically close, such as the cow. In situ 

hybridation studies are being carried out at our labs in order to confirm aromatase 

immunoreactivity results.

Disclosures: S. Gadau, None; V. Farina , None; A. Mura, None; B. Arru, None; M. Zedda, 

None; G. Lepore, None. 

Poster 






Title: Peripubertal development of sex differences in androgen receptor associated membranes 

and fibers 

Authors: *L. M. TEJADA 1 , N. SHAH 2 , E. RISSMAN 1 ; 

1 Univ. Virginia, Charlottesville, VA; 2 Univ. of California San Francisco, San Francisco, CA 

Abstract: Puberty is considered by some to be a second critical period when gonadal hormones 

may organize differences in adult brain and behaviors. The medial preoptic area (mPOA), 

contains several well studied sexual dimorphisms. For example, a sexually dimorphic region has 

been identified with Nissl staining and with calbindin immunoreactivity, and within the 

anteroventral periventricular nucleus tyrosine hydroxylase immunoreactivity has revealed 

another sexual dimorphism. The number of cells containing androgen receptor (AR) protein or 

mRNA is also sexually dimorphic in adults. To assess the peripubertal contributions to AR 

sexual dimorphisms in this region, we used transgenic mice (C57BL/6J) bearing a modified AR 

gene. In these mice, cells that express AR also express two reporter molecules, LacZ for nuclear 

visualization and placental alkaline phosphatase (PLAP), which stains cell membranes and 

associated fibers. One advantage of this method is that the activity of the PLAP is not affected by 

steroid hormones thus sex differences are not a reflection of differences in circulating 

testosterone levels. We collected brains from male and female mice before, during, and after 

puberty (PN21, 35 and 50). PLAP-staining revealed sex differences in both the mPOA (p

Disclosures: L.M. Tejada , None; E. Rissman, None; N. Shah, None. 

Poster 





Support: NSF IOS-0642050 

NINDS 1 F31 NS058258 

Title: The effects of sex and breeding status on perineal muscles and motoneurons in 

Damaraland mole-rats 

Authors: *M. L. SENEY 1 , L. BENGSTON 1 , B. GOLDMAN 2 , N. G. FORGER 1 ; 

1 Ctr. Neuroendocrine Studies, UMASS Amherst, Amherst, MA; 2 Ecology and Evolutionary 

Biol., Univ. of Connecticut, Storrs, CT 

Abstract: The striated perineal muscles and their innervating motoneurons are sexually 

dimorphic in essentially every mammal studied to date. Males have larger bulbocavernosus 

(BC), ischiocavernosus (IC), and levator ani (LA) muscles and the motoneurons innervating 

these muscles (located in the spinal nucleus of the bulbocavernosus or Onuf‟s nucleus, 

depending on species) are greater in size and/or number in males. We recently reported an 

exception to this rule: this system is not sexually dimorphic in the naked mole-rat (Peroulakis et 

al., 2002; Seney et al., 2006). Naked mole-rats also did not have a bulb of the penis and therefore 

no true BC muscle, although they did have a large urethral muscle (UM) that may be 

homologous to the BC. Naked mole-rats live in colonies of 60-90 individuals and are considered 

to be eusocial. Each colony has only 1 breeding female (the queen) and 1-3 breeding males. All 

other individuals are subordinates, which do not breed, but help with foraging for food, 

maintaining the tunnel system, defending against predators, and caring for the young. In nature, 

less than 1% of naked mole-rats achieve breeding status. We hypothesize that the lack of sex 

differences seen in naked mole-rats is due to their unusual social structure. Here, we tested this 

hypothesis by examining the perineal muscles and motoneurons in another species considered to 

be eusocial, the Damaraland mole-rat (Cryptomys damarensis). These animals are found in arid 

regions of Africa. They live in colonies of 15-20 individuals comprised of 1 breeding female (the 

queen), 1-3 breeding males, and about a dozen non-reproductive subordinates. While breeders 

fulfill the duties of reproduction, subordinates forage for food, defend the colony, maintain the 

burrow system, and care for young. However, compared to naked mole-rats, Damaraland mole-

ats have much smaller mean colony size and significantly less reproductive skew (8% of 

Damaraland mole-rats mate while less than 1% of naked mole-rats mate). We are examining 

motoneuron number in Onuf‟s nucleus and size of the UM, LA, and IC muscles in subordinate 

and breeding Damaraland mole-rats. Similar to naked mole-rats, preliminary findings indicate a 

lack of sex difference in the number of motoneurons in Onuf‟s nucleus of subordinate 

Damaraland mole-rats (p > 0.5). The size of the LA also was not different in male and female 

subordinates (p > 0.3), and like naked mole-rats, there was no bulb of the penis. However, male 

subordinates had larger UMs than female subordinates (p < 0.04). We are currently examining 

breeding Damaraland mole-rats to test the hypothesis that sex differences in perineal muscles 

and motoneurons develop when animals become breeders. 

Disclosures: M.L. Seney , None; L. Bengston, None; N.G. Forger, None; B. Goldman, None. 

Poster 





Support: NIH PO1 HD044232 

Title: Estrogen receptor immunoreactivity in late-gestation fetal lambs: effects of sex and 

prenatal steroids 

Authors: *R. I. WOOD 1 , L. M. GORTON 1 , M. M. MAHONEY 2 , J. M. MAGORIAN 1 , T. M. 

LEE 2 ; 

1 Dept Cell & Neurobiol, Keck Sch. Med. USC, Los Angeles, CA; 2 Dept. of Psychology, Univ. of 

Michigan, Ann Arbor, MI 

Abstract: In sheep, prenatal exposure to androgens has profound organizational effects on 

reproductive function and behavior. Male sheep experience an earlier pubertal onset of tonic 

pulsatile secretion of luteinizing hormone (LH), but the preovulatory LH surge is abolished. 

Likewise, rams demonstrate aggression and masculine sexual behavior, while ewes show 

receptive behaviors during estrus. The critical period for sexual differentiation in sheep occurs 

during the middle of gestation: treatment of pregnant ewes with testosterone masculinizes sexual 

behavior and patterns of LH in female lambs. Reproductive neuroendocrine function and 

behavior are sensitive to gonadal steroid hormone action in the brain, principally via binding to 

estrogen receptors (ER). Considering the substantial sex differences in steroid-dependent 

physiology and behavior, we hypothesized that male and female sheep differ in the number and

distribution of ER-containing neurons. If so, we predicted that sex differences in ER should be 

sensitive to prenatal hormones. We evaluated immunocytochemistry for ER (1D5 antibody, 

1:1000) in male and female fetal lambs at the end of gestation. To determine if prenatal 

androgens modify ER in females, we compared ER in fetal females exposed to testosterone or 

dihydrotestosterone (DHT) during the prenatal critical period. ER staining was concentrated in 

the neuronal cell nucleus, with limited perinuclear cytoplasmic immunoreactivity. ER-positive 

neurons were abundant in BST, MPOA, VMH, ARC, Me and AHA. In female lambs, ARC had 

the largest number of stained cells (475.6±57.4 particles per 0.173 mm2), while staining intensity 

was greatest in MPOA (mean gray level: 31.3±5.3). Integrated gray level was similar in the two 

regions (ARC: 0.63±0.13, MPOA: 0.51±0.08). Integrated gray level in Me (0.31±0.10) and BST 

(0.21±0.06) was low, while ER immunoreactivity in AHA (0.36±0.10 integrated gray level) and 

VMH (0.37±0.07) was intermediate between these extremes. The distribution and intensity of 

ER-positive neurons was similar in male lambs. For all brain regions examined, the average 

integrated gray levels in females exceeded those in males. However, there were no statistically 

significant sex differences in the number of stained cells, mean gray level, or integrated gray 

level in any brain region (p>0.05). Likewise, there were no differences on ER immunoreactivity 

among fetal females exposed prenatally to testosterone, DHT or vehicle (p>0.05). These results 

suggest that the distribution of ER in the brains of male and female fetal lambs is unlikely to 

account for sex differences in reproductive neuroendocrine function and sexual behavior. 

Disclosures: R.I. Wood , None; L.M. Gorton, None; J.M. Magorian, None; M.M. Mahoney, 

None; T.M. Lee, None. 

Poster 





Support: National Institute of Mental Health (NIMH) Grants R37 MH39085 (MERIT Award) 

the Boyd and Elsie Welin Professorship 

R01 MH67968 

Title: Interaction of SRY (sex-determining region on the Y chromosome) and Sp1 activated 

monoamine oxiase A promoter activity in neuroblastoma cells

Authors: *B. WU 1 , K. CHEN 1 , Y. LI 3 , Y.-F. C. LAU 3 , J. C. SHIH 2 ; 

1 Pharmacol & Pharm. Sci., 2 Pharmacol & Pharm. Sci., Cell & Neurobio., Univ. Southern Calif, 

Los Angeles, CA; 3 Dept. of Med., Univ. of California, San Francisco, San Francisco, CA 

Abstract: The transcriptional regulation of monoamine oxidase (MAO) A has been extensively 

studied in this laboratory. Sp1 activates MAO A core promoter via several Sp1-binding sites. 

Two novel transcription factors R1 (RAM2/CDCA7L/JPO2) and EAPP compete with Sp1 and 

repress MAO A promoter by binding to Sp1-binding sites. This is the first study showing that 

SRY (sex-determining region on the Y chromosome) interacts with Sp1 and activates MAO A 

promoter activity in human male neuroblastoma BE(2)C cells. 

The SRY gene, encoding a putative transcription factor, plays a key role in testis determination 

and differentiation during embryogenesis. Although many pathways regulating sexual 

developmental process have been elucidated, direct downstream targets of SRY are still unclear. 

MAO A has been recently identified as a potential target by ChIP-chip assay. 

Transient co-transfection and luciferase assay showed that human SRY but not mouse Sry 

significantly increased human MAO A promoter activity in BE(2)C and human prostate 

carcinoma C42B cell lines by 3-7 folds and 8-15 folds, respectively. This activation was SRY 

concentration-dependent. siRNA-mediated endogenous SRY knockdown reduced MAO A 

promoter activity by 60% in BE(2)C cells. Moreover, serial deletion analysis of the MAO A 

promoter revealed that putative SRY-binding sites were located in the 0.2 kb core promoter 

region, which contained several Sp1-binding sites as shown previously. Co-transfection of 

various amounts of Sp1 enhanced SRY activation of MAO A promoter in a Sp1-concentration 

dependent manner, indicating that Sp1 interacted with SRY in MAO A promoter. The MAO A 

promoter activity reduced significantly by 50% after Sp1 was knocked down by siRNA, 

however, the fold of the increase of the SRY activation of MAO A promoter activity remained 

the same, suggesting that SRY may interact with other transcription factors in addition to Sp1. 

Taken together, this study has demonstrated that the interaction of SRY with Sp1 and other 

transcription factors activated MAO A promoter activity. Considering the important 

physiological role of SRY, this study provides new insights into the novel function of MAO A in 

SRY-associated developmental processes. 

Disclosures: B. Wu , None; K. Chen, None; Y. Li, None; Y.C. Lau, None; J.C. Shih, None. 

Poster 



Program#/Poster#: 278.8/MM1 

Topic: E.01.e. Sexual differences

Support: NIH grant RO1 NS055218 

Title: Androgen regulates sexually dimorphic H3 acetylation in the cortex and hippocampus of 

neonatal mice 

Authors: H.-W. TSAI, *E. F. RISSMAN; 

Dept Biochem and Molec Genet., Univ. Virginia Med. Sch., Charlottesville, VA 

Abstract: Sexual differentiation of the brain during early development is the critical process by 

which both reproductive and non-reproductive behaviors and their underlying neural structures 

become sexually dimorphic. The best documented mechanism that gives rise to sex differences 

in the brain is the male-only perinatal rise in testosterone (T). Androgens act on steroid receptors 

to cause differential gene expression, leading to the formation of masculine brain structures and 

behaviors. Post-translational modification of histone has recently emerged as one of the most 

significant mechanisms for regulating chromatin function and thus gene transcription. Here we 

test the hypothesis that histone modification is critically involved in sexual differentiation of the 

brain. Whole brains were collected from mouse embryos on gestational days 12 (E12), 16 (E16), 

and 18 (E18). In addition, on E18, postnatal (PN) days 0 (the day of birth) and 6, brains were 

dissected into specific regions and collected. Histones extracted from the brain tissues were 

immunoblotted with antibodies for acetylated (K9/14), or trimethylated H3 (K9). Relative levels 

of modified H3 were normalized with total H4. In the male cortex/hippocampus, an increase in 

H3 acetylation was observed on both E18 and PN0, relative to females. Next we asked if the sex 

differences in H3 acetylation in the cortex/hippocampus were caused by T. Timed pregnant 

female mice were treated with either vehicle or testosterone propionate (TP, 0.05 ml; 40 κg/ml 

sesame oil) daily starting from E16. On PN0, pups were collected and the cortex/hippocampi 

were processed as above for acetylated H3. In vehicle-treated controls, a sex difference on H3 

acetylation was observed in the cortex/hippocampus. TP increased H3 acetylation in the 

cortex/hippocampus of female neonates, which was similar to concentrations found in vehicle- or 

TP-treated males. Our data demonstrate that during early development, transient dimorphic 

changes in histone acetylation between the sexes occur in the cortex/hippocampus. Sex 

differences in histone H3 acetylation in the cortex/hippocampus appear to be mediated by sex 

steroids. Our results suggest important and novel roles of epigenetic mechanisms in brain sexual 

differentiation, which might be targeted for developing new treatments of sex-biased diseases, 

especially those that involve cortical function. This work is supported by NIH grant RO1 

NS055218. 

Disclosures: H. Tsai, None; E.F. Rissman , None. 

Poster 




Topic: E.01.d. Development 

Support: NIH Grant RO1 MH052716 

NIH Grant T32 NS07375 

Title: The cannabinoid agonist, WIN 55,212-2, decreases the number of new cells that express 

GFAP in the developing medial amygdala of female rats 

Authors: *D. L. KREBS-KRAFT, M. M. MCCARTHY; 

Dept of Physio, Program in Neurosci., Univ. of Maryland Baltimore, Sch. of Med., Baltimore, 

MD 

Abstract: The amygdala is an important brain region in the regulation of social, cognitive, and 

emotional behaviors. There are volumetric sex differences in the medial amygdala of 

peripubertal and adult rats, such that the regional volume of the medial amygdala is larger in 

males than females (Cooke, 2007; Mizukami et al, 1983). In most brain regions, volumetric sex 

differences are attributed to higher rates of cell death in one sex versus the other; however, cell 

proliferation may also be important. Cannabinoids are developmentally regulated retrograde 

messengers that have been implicated in brain development and can modulate cell proliferation 

and differentiation of neural cell types (Aguado et al, 2005, 2006; Galve-Roperh et al, 2006). 

There are sex differences in cannabinoid effects on behavior in adults, such that females are 

more susceptible to the effects of cannabinoid activation. We find females have more new cells, 

detected by 5-bromo-2‟-deoxyuridine-5‟-monophosphate (BrdU), in the developing amygdala 

than males. Treatment of neonates with a cannabinoid receptor agonist, WIN 55,212-2 (WIN), 

reduces the number of new cells in females to that of males. Astrocytes play a critical role in 

neurotransmission, axon guidance, and synapse formation and cannabinoids modulate glial 

differentiation (Aguado et al, 2006). The present study tested the hypothesis that cannabinoids 

modulate glial cell proliferation or survival in the developing medial amygdala of females. The 

number of newly generated glial cells was detected by double-label immunohistochemistry using 

a monoclonal antibody specific for BrdU, a marker of cell genesis, and glial fibrillary acidic 

protein (GFAP), a maker for mature astrocytes. Specifically, we determined whether treating 

males and females at postnatal day 0 and 1 with WIN (1 mg/kg) would decrease the number of 

BrdU, GFAP, and BrdU/GFAP immunoreactive cells in the rat medial amygdala at postnatal day 

4 or 14, in comparison to vehicle treated controls. Consistent with our previous findings, females 

had significantly more BrdU+ and BrdU+/GFAP+ cells in the medial amygdala than did males at 

PN4 and PN14. More importantly, treatment with WIN significantly decreased the number of 

BrdU+ and BrdU+/GFAP+ cells in females but not males. These findings suggest that 

cannabinoids modulate glial cell proliferation, survival, and/or differentiation in the developing 

medial amygdala. 

Disclosures: D.L. Krebs-Kraft , None; M.M. McCarthy, None.

Poster 





Support: NIH MH52716-010 to MMM 

Title: PGE2 mediates the perinatal organization of male sexual behavior: Is protein kinase A 

signaling required? 

Authors: *C. L. WRIGHT 1 , M. M. MCCARTHY 2,3 ; 

1 Program Neurosci, Univ. Maryland, Baltimore, MD; 2 Program in Neurosci., 3 Departments of 

Physiol. and Psychiatry, Univ. of Maryland, Sch. of Med., Baltimore, MD 

Abstract: Masculinization of the brain and behavior is mediated by gonadal steroids during a 

sensitive perinatal window. In the rat, estradiol aromatized from testosterone masculinizes the 

brain by upregulating the cyclooxygenase enzymes (COX-1 & -2) and their major product, 

prostaglandin E2 (PGE2), in the preoptic area. Consequently, PGE2 induces permanent changes 

in preoptic area neuroanatomy, including a two-fold increase in the density of dendritic spines 

and the level of spinophilin protein (Amateau and McCarthy, 2004, Nat. Neurosci.). Spinophilin 

is enriched in dendritic spines and is an excellent proxy marker for dendritic spine formation. We 

recently determined that the EP2 and EP4 receptors for PGE2 are necessary and sufficient for the 

organization of male sexual behavior. EP2 and EP4 are coupled to Gs-proteins which increase 

cAMP production by adenylyl cyclase and subsequently recruit protein kinase A (PKA) 

phosphorylation of substrates. We have now tested if PKA signaling is necessary for the 

induction of neonatal POA spinophilin protein and organization of male sexual behavior by 

utilizing the cell permeable form of an AKAP-PKA binding inhibitor, Ht31. PKA signaling 

depends upon its coupling to AKAP proteins which localize the enzyme near both adenylyl 

cyclase and enzymatic targets. Ht31 mimics the AKAP binding sequence for PKA, dissociating 

the two molecules. With PKA bound to Ht31, PKA is no longer near its substrate, thereby 

inhibiting signaling. Neonatal females were injected intracerebroventricularly with Ht31 then 

PGE2 on day of birth and half were assayed neonatally for the induction of spinophilin. The 

remaining animals were grown until adulthood, implanted with silastic testosterone capsules, and 

assessed for the expression of male sexual behavior. Treatment with Ht31 prevented the 

induction of neonatal POA spinophilin protein in response to PGE2 exposure, suggesting that 

PKA is necessary for changes in POA neuroanatomy including the formation of dendritic spines. 

The pending behavioral results will determine if PKA signaling is necessary for the organization 

of male sexual behavior in response to PGE2 exposure.

Disclosures: C.L. Wright , None; M.M. McCarthy, None. 

Poster 





Support: NIH MH 52716-010 MMM 

Title: Estradiol upregulates SPAK in the developing brain: a possible mechanism for estradiolmediated 

enhancement of depolarizing GABA 

Authors: B. M. NUGENT 1 , J. M. SCHWARZ 1 , *M. M. MCCARTHY 2,3,1 ; 

1 Program in Neurosci., 2 Dept Physiol, 3 Dept Psychiatry, Univ. Maryland Sch. Med., Baltimore, 

MD 

Abstract: During brain development the normally inhibitory transmitter, γ-Aminobutyric acid 

(GABA), exerts an excitatory effect. This is because immature neurons have higher levels of [Cl - 

]i relative to [Cl - ]e due to elevated levels of the Na-K-2Cl - co-transporter (NKCC1), which brings 

Cl - into the cell, and low expression levels of the K-Cl - co-transporter (KCC2), which removes 

Cl - from the cell (Plotkin et al., J Neurobiol 1997:33; Rivera et al., Nature 1999:397). 

Consequently, activation of GABAA receptors results in Cl - efflux and membrane depolarization, 

resulting in Ca 2+ influx through L-type voltage-gated Ca 2+ channels (Nunez et al., Eur J Neurosci 

2005:21). Estradiol is an important regulator of bran development. Our lab has shown that 

estradiol enhances GABA-mediated Ca 2+ influx in the neonatal brain and that expression of 

NKCC1 is increased in neonatal rats exposed to estradiol. In addition, we have shown that 

phosphorylation of the NKCC1 co-transporter is increased in animals treated with estradiol for 

the first two days after birth (Perrot-Sinal et al, J Neuroendo 2007:19). NKCC1 activity is 

determined by its phosphorylation state, and the Ste20-related proline alanine-rich kinase 

(SPAK) has been implicated in regulation of NKCC1 activity (Delpire and Gagnon, Acta Physiol 

2006:187). We hypothesized that estradiol might increase the activity of NKCC1 via SPAK 

upregulation. To assess changes in SPAK protein levels following estradiol treatment, we 

administered 17β-estradiol to female pups on their day of birth and collected hypothalamic and 

hippocampal tissue for western blot analysis 6 and 24 hours following hormone administration. 

SPAK levels were significantly elevated in neonatal female rat pups following 24 but not 6 hours 

of estradiol treatment. This data provides a possible mechanism by which estradiol increases 

NKCC1 activity, resulting in increased GABA-mediated neuronal excitation. Supported by: NIH 

MH 52716-010 to MMM.

Disclosures: B.M. Nugent, None; J.M. Schwarz, None; M.M. McCarthy , None. 

Poster 





Support: NIH Grant KO2 MH01497 

NIH Grant RO1 MH068482 

NIH Grant R01 MH47538 

Title: Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis in 

mice 

Authors: *E. K. MURRAY, A. HIEN, G. J. DE VRIES, N. G. FORGER; 

Neurosci & Behavior Program, Univ. Massachusetts, Amherst, Amherst, MA 

Abstract: The principal nucleus of the bed nucleus of the stria teminalis (BNSTp) is larger and 

contains more cells in male mice than in females. These sex differences arise from a higher rate 

of cell death during early postnatal life in females. Perinatal differences in testosterone appear to 

create this difference because neonatal testosterone treatment reduces cell death in females to the 

level seen in males. There is a delay of about six days between testosterone exposure and the 

peak of cell death, indicating that cells somehow „remember‟ whether or not they have been 

exposed to high levels of testosterone. This suggests that epigenetic mechanisms control cell 

fate. We examined whether histone acetylation, which is typically associated with activation of 

gene expression, plays a role in the sexual differentiation of the BNSTp. We manipulated the 

balance between histone acetylation and deacetylation by treating animals with the histone 

deacetylase inhibitor valproic acid (VPA) at the critical time for sexual differentiation. Males, 

females, and females treated neonatally with testosterone were treated with 50mg/kg VPA or 

saline on postnatal days 1 and 2. Animals were sacrificed on postnatal day 21 and volume and 

cell number of the BNSTp was determined. VPA treatment did not influence volume or cell 

number in control females but significantly reduced both of these parameters in males and 

testosterone-treated females, thereby eliminating the sex difference. No volume changes were 

noted in the suprachiasmatic nucleus, suggesting that the VPA effect was specific to the BNSTp. 

These findings suggest that a disruption in histone deacetylation blocks the masculinizing actions 

of testosterone in the BNSTp.

Disclosures: E.K. Murray , None; A. Hien, None; G.J. de Vries, None; N.G. Forger, None. 

Poster 





Support: NIH grant R01MH072956 

NIH grant T32MH75880 

Title: Sex specific influence of reduced Mecp2 expression within the developing amygdala on 

juvenile social behavior 

Authors: *A. P. AUGER 1 , J. R. KURIAN 1 , M. E. BYCHOWSKI 2 , R. M. FORBES- 

LORMAN 1 , C. J. AUGER 1 ; 

1 Dept Psych, 2 Neurosci. Training Program, Univ. Wisconsin, Madison, Madison, WI 

Abstract: Methyl-CpG-binding protein 2 (MeCP2) binds methylated DNA and recruits corepressor 

proteins to modify chromatin and alter gene transcription. Mutations of the MECP2 

gene can cause Rett syndrome (RTT), while subtle reductions of MeCP2 expression may be 

associated with male dominated social and neurodevelopmental disorders. We report that 

transiently decreased amygdala Mecp2 expression using siRNA during a sensitive period of 

brain sexual differentiation disrupts the organization of sex differences in juvenile social 

behavior. This disruption does not present as grossly atypical behavior and does not exhibit 

motor abnormalities present in models of RTT. Rather, typical sex differences in juvenile social 

behaviors are eliminated, and these behavioral modifications occurred only in males. This 

suggests Mecp2 may have an overlooked role in the organization of sexually dimorphic 

behaviors and that male juvenile behavior is particularly sensitive to Mecp2 disruption during 

this period of development. Further, modified amygdala Mecp2 expression is related to atypical 

male juvenile social behavior. 

Disclosures: A.P. Auger , None; J.R. Kurian, None; M.E. Bychowski, None; R.M. Forbes- 

Lorman, None; C.J. Auger, None.

Poster 





Support: NIH grant R01MH072956 

Title: Hormonal profiling of neophobic and neophilic rats at the onset of puberty 

Authors: *N. S. HASEN 1 , M. E. BYCHOWSKI 2 , R. M. FORBES-LORMAN 3 , A. P. AUGER 3 ; 

1 Dept Zoology, Univ. Wisconsin, Madison, WI; 2 Neurosci. Training Program, 3 Psychology, 

Univ. of Wisconsin-Madison, Madison, WI 

Abstract: Recent work (Cavagelli and McClintock, 2006) has shown strong correlations 

between how female rats respond to novelty as infants and their latency to mammary 

tumorigenesis as adults, with neophobic females developing mammary tumors earlier than their 

neophilic sisters. We were interested to see if this difference is reflected in levels of hormones 

during the early stages of puberty, when breast tissue is developing under the influence of 

estrogen and prolactin. We assessed neophobia and neophilia in three litters of rats at 19 days 

post partum. Each animal was placed in a plastic container and the container then placed in the 

center chamber of a larger, three-chambered apparatus. The two outer chambers each contained a 

novel object (binder clip or water bottle nozzle). She then had 10 minutes in which to explore the 

apparatus. Digital recordings of each animal‟s behavior were scored off line. Behaviors scored 

were: latency to leave the plastic container, bouts of investigation of each novel, entrances into 

each chamber, durations of investigation and durations of each visit to each chamber. At the end 

of her test, the rat was returned to her home cage. Animals were weaned at 21 days and remained 

group-housed with their sisters until 35 days post partum, when the animals were sacrificed. 

Brains, mammary glands and serum were collected from each rat. We will examine correlations 

between these behaviors and serum concentrations of estradiol, prolactin, progesterone and 

corticosterone. 

Disclosures: N.S. Hasen , None; M.E. Bychowski, None; R.M. Forbes-Lorman, None; A.P. 

Auger, None. 

Poster 

278. Sex Differences I




Support: R01MH072956 

Title: Sex differences in the expression of Nuclear Receptor Corepessor (NCoR) in the rat 

amygdala during development 

Authors: *H. M. JESSEN 1 , M. H. KOLODKIN 2 , A. P. AUGER 1 ; 

1 Dept Psychol, 2 Neurosci. Training Program, Univ. Wisconsin Madison, Madison, WI 

Abstract: Steroid hormones play an important role in the developing rat brain, causing many 

physiological and behavioral changes. Steroid hormone action is regulated at the level of the 

DNA through a balance of recruitment of complexes which cause acetylation of histones and 

increases in gene expression and through the recruitment of complexes, such as corepressors (i.e. 

NCoR), which cause deacetylation and decreases in gene expression. Methyl-CpG-binding 

protein 2 (MeCP2), which is thought to cause transcriptional repression through the recruitment 

of corepressors and consequent deacetylation of histones, has been shown to have sex differences 

in the developing rat brain. Females have been shown to express higher levels of MeCP2 mRNA 

and protein in the amygdala and ventral medial hypothalamus (VMH), but not the pre optic area 

(POA) on postnatal day 1 (PN1). This parallels our previous results, showing that females 

express higher levels of the corepressor NCoR mRNA in the VMH but not the POA on PN1 as 

compared to males. However, we have not investigated the levels of NCoR in the amygdala. We 

used real-time RT-PCR to examine potential sex differences in NCoR in the amygdala of the 

developing rat brain. On PN1 we have found that within the amygdala females express a greater 

relative amount of NCoR mRNA as compared to males, interestingly this difference is still 

present on post natal day 10 (PN10). The function of NCoR during this sensitive period may be 

to protect females from steroid hormones or steroid hormones may act by decreasing NCoR 

which would lead to a deaccetylation of histones resulting in an increase in gene expression. 

Taken together these data suggest a dynamic model of chromatin remodeling which may drive 

sex differences in the developing rat brain. 

Disclosures: H.M. Jessen , None; A.P. Auger, None; M.H. Kolodkin, None. 

Poster 





Support: NIH Grant R01MH072956 

Title: Sex differences in the expression of DNA methyltransferase 3a (DNMT3a) in the rat 

amygdala during development 

Authors: *M. H. KOLODKIN 1 , A. P. AUGER 2 ; 

1 Neurosci. Training Program, Univ. of Wisconsin-Madiso, Madison, WI; 2 Psychology, Univ. of 

Wisconsin-Madison, Madison, WI 

Abstract: Sexual differentiation of the neonatal rat brain is regulated by dynamic processes 

occurring at the level of DNA. Steroid hormone receptors act partly in the developing brain by 

recruiting co-activators, thereby increasing acetylation of histones and gene expression. The 

mechanism by which gene expression is concurrently downregulated has not yet been fully 

elucidated. Methylation is an epigenetic process that decreases gene expression without altering 

the original DNA sequence. Co-repressor complexes are recruited when DNA cytosine-5methyltransferases 

(DNMTs) tag DNA promoters by catalyzing the transfer of a methyl group to 

the cytosine in CpG dinucleotides. Sex differences in the expression of components of the 

methylation machinery may influence methylation patterns and result in divergent phenotypes. 

We investigated whether sex differences in the expression of DNMT1 and DNMT3a were 

apparent in the sexually dimorphic amygdala, preoptic area, and ventromedial hypothalamus at 

different time points during development. Using real-time polymerase and western 

immunoblotting, we have found that females express significantly more DNMT3a mRNA and 

protein in the amygdala at PND1, but not PND10. No sex difference was observed in the 

expression of DNMT1. Our results suggest that increased gene expression in males leading to 

sexual differentiation may be complemented by a relief of methylation-induced gene suppression 

by DNMT3a. 

Disclosures: M.H. Kolodkin, None; A.P. Auger, None. 

Poster 




Topic: E.01.e. Sexual differences

Support: NIH Grant T32 MH75880 

NIH Grant R01MH072956 

Title: Epigenetic contribution to sex differences in the brain 

Authors: *J. KURIAN, K. M. OLESEN, A. P. AUGER; 

Psychol, Univ. Wisconsin-Madison, Madison, WI 

Abstract: Early experience, such as maternal care, is known to modify gender typical rat 

behaviors including juvenile social and adult sexual behaviors (Olesen, in preparation; Cameron, 

2008). The mechanisms associated with these modifications are not understood, however, it is 

likely that an epigenetic mechanism such as DNA methylation of specific gene promoters is 

involved. For example, natural variations in maternal care are associated with differential ERα 

promoter DNA methylation and gene expression in female pups (Champagne, 2006). As 

maternal anogenital licking is also naturally sexually dimorphic, with males experiencing more 

than female pups, we chose to examine the methylation patterns of ERα in males and females. 

Bisulfite sequencing of the stat5b region of this promoter indicated ERα promoter methylation in 

the anterior hypothalamus is sexually dimorphic on post-natal day 8 (PN8), with males 

exhibiting greater methylation than females. This methylation pattern was correlated with ERα 

mRNA and protein expression such that increased methylation was associated with decreased 

expression. Interestingly, methylation of this promoter was increased and ERα expression was 

decreased in PN8 females that were either treated with estradiol (two 100µg peripheral injections 

on PN0-1) or simulated maternal grooming (SMG). These data suggest that sexually dimorphic 

gene expression in a region associated with gender typical rat behavior can be modified through 

epigenetic mechanisms. They further suggest that internal (i.e. hormonal) and external cues from 

the social environment converge within the developing brain to modify DNA and have lasting 

effects on social development. 

Disclosures: J. Kurian , None; A.P. Auger, None; K.M. Olesen, None. 

Poster 





Support: Natural Sciences and Engineering Research Council of Canada

Canada Foundation for Innovation 

Michael Smith Foundation for Health Research 

Title: DHEA and estradiol levels in plasma and brain of male and female zebra finches across 

development 

Authors: *A. H. SHAH 1 , E. H. CHIN 2 , K. L. SCHMIDT 1 , C.-M. HUANG 1 , K. K. SOMA 1 ; 

1 Univ. British Columbia, Vancouver, BC, Canada; 2 Trent Univ., Peterborough, ON, Canada 

Abstract: The zebra finch (Taeniopygia guttata) is a songbird species with highly sexually 

dimorphic song behavior and song control nuclei. In zebra finches, only males sing, and males 

have larger song nuclei than females. Traditionally, sexual differentiation has been thought to be 

driven by gonadal steroids, but recent studies suggest that steroids synthesized in the adrenals 

and/or brain may also be important. For example, dehydroepiandrosterone (DHEA), a precursor 

to androgens and estrogens, can be synthesized in the gonads, adrenals and/or brain, and may 

play a role in sexual differentiation. Exogenous 17β-estradiol (E2) during development partially 

masculinizes female zebra finches, and endogenous E2 might be synthesized from circulating 

DHEA or de novo from cholesterol in the brain. In this study, we examined DHEA and E2 levels 

in plasma, brain tissue, and peripheral tissues of zebra finches. Plasma was collected from either 

the brachial or jugular vein on hatch day (P0), P3, P6, P12, P30, and P90+ (adults). Brain and 

peripheral tissues were collected on P0, P3, P30, and P90+ (adults). Steroids were extracted 

using solid phase extraction, and DHEA and E2 were measured using sensitive and specific 

radioimmunoassays. Brain DHEA levels were low in both sexes at P0 and P3, but at P30, DHEA 

levels in the rostral telencephalon were higher in females than males. Higher brain DHEA in 

females suggests either higher DHEA synthesis in females or higher DHEA metabolism in 

males. Brain E2 levels did not differ between males and females at P30. We are currently 

measuring DHEA and E2 in remaining tissue and plasma samples. 

Disclosures: A.H. Shah , None; E.H. Chin, None; K.L. Schmidt, None; C. Huang, 

None; K.K. Soma, None. 

Poster 




Topic: E.01.a. HPG axis regulation 

Support: NIH Grant RO1 HD042634

Endocrine Society Bridge Award 

Title: Fibroblast growth factor 8 deficiency affects olfactory placode morphogenesis from which 

gonadotropin-releasing hormone neurons emerge 

Authors: *W. C. CHUNG, P.-S. TSAI; 

Dept Integrative Physiol, Univ. Colorado Boulder, Boulder, CO 

Abstract: Gonadotropin-releasing hormone (GnRH) neurons are scattered throughout the 

preoptic-hypothalamus and represent the most upstream neuroendocrine regulators of the 

hypothalamus-pituitary-gonadal axis. Previous studies showed that mouse GnRH neurons are 

first detected in the olfactory placode (OP) around embryonic day (E) 10.5-11.5. Recently, we 

showed that GnRH neurons failed to emerge from the olfactory placode (OP) of E11.5 mouse 

embryos hypomorphic for fibroblast growth factor (FGF) 8. To better understand the mechanism 

underlying this disruption, we examined the morphogenesis of the OP in E11.5 Fgf8 

hypomorphs. First, we examined whether FGF8 deficiency causes a general dysgenesis of the 

olfactory epithelium (OE) by verifying the presence of Olf-1 (a marker for olfactory receptor 

neurons (ORN)) in the OE of E11.5 Fgf8 hypomorphs. Second, because FGF8 is important for 

developmental cell survival, we examined whether the E11.5 OP in Fgf8 hypomorphs underwent 

aberrant levels of apoptosis. Our results showed that Olf-1 immunoreactive (IR) ORNs were 

present in the E11.5 OP of wildtype, heterozygous, and homozygous Fgf8 hypomorphs. 

Although homozygous Fgf8 hypomorphic embryos possessed a morphologically distinct OP 

with normal primary invagination, a secondary recess that presumably gives rise to the 

vomeronasal organ and GnRH neurons was reduced or absent. These results indicate that failure 

of GnRH neurons to emerge from the OP is not due to global dysgenesis of the OE. Instead, 

FGF8 deficiency seems to target the development of the GnRH system with some selectivity. 

Furthermore, we found that the Olf-1-IR region of the OP contained very few apoptotic cells, 

and there were no gross differences in apoptosis between E11.5 wildtype, heterozygous and 

homozygous Fgf8 hypomorphs. Therefore, the altered OP morphology in Fgf8 hypomorphs 

cannot be attributed to increased apoptosis at E11.5. Future studies will address if apoptosis prior 

to E11.5 contributes to OP abnormality in Fgf8 hypomorphs, and if Fgf8 deficiency leads to 

reduced proliferation of the progenitors fated to become GnRH neurons. 

Disclosures: W.C. Chung, None; P. Tsai, None. 

Poster 



Program#/Poster#: 278.20/MM13



Title: Relationship among GABA, nitric oxide, and brain derived neurotrophic factor: Potential 

effector molecules for the development of the paraventricular and ventromedial hypothalamic 

nuclei 

Authors: *K. MCCLELLAN, S. A. TOBET; 

Biomed. Sci., Colorado State Univ., Fort Collins, CO 

Abstract: Development of a hypothalamic nuclear group requires factors that influence 

proliferation, migration, and differentiation. It is known that transcriptional regulation of various 

genes is necessary to form functional paraventricular (PVN) and ventromedial (VMN) 

hypothalamic nuclei (e.g. Sim-1 and SF-1 respectively). We are investigating three molecules 

that may play roles in the migration and differentiation of the PVN and VMN by virtue of their 

effecting cell-cell communication. Roles for nitric oxide (NO), produced by neuronal nitric oxide 

synthase (nNOS), have been postulated for proliferation, migration, or differentiation of various 

cell phenotypes and similar roles have been postulated for brain derived neurotrophic factor 

(BDNF) and GABA. Work of others has shown that GABA can influence BDNF expression and 

that BDNF might enhance nNOS expression. Early in development (E15 - E17) in the mouse, 

nNOS and BDNF are found in specific regions within the hypothalamus. Two dense regions of 

nNOS and BDNF immunoreactivity (ir) are the PVN and VMN. These regions are also unique 

for being surrounded by a network of cells and fibers containing ir-GABA early in development 

(Tobet et al., 1999; McClellan et al., SfN 2007abstr). We used a GABABR1 subunit knockout 

mouse to look at the potential role of GABAB receptor signaling on cell positions within the 

VMN (McClellan et al., 2008) and now the PVN. We found a significant decrease in ir-BDNF in 

the PVN of GABABR1 subunit knockout mice in a region dependent manner (p


Topic: E.01.g. Behavioral neuroendocrinology: Other 

Title: Determining the critical postnatal exposure level of the brominated flame retardant DE-71 

required to induce learning and attention deficits 

Authors: J. KAPLAN, J. KRAUT, J. FITZPATRICK, *L. L. DRISCOLL; 

Dept Psychol, Colorado Col., Colorado Springs, CO 

Abstract: Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants added to 

polymers in the manufacturing of common household appliances and many textiles. Human 

PBDE exposure occurs primarily through inhalation and ingestion; in infants, high doses are 

ingested in breast milk. Due to PBDEs‟ bioaccumulative properties, concentrations of these 

compounds in humans have increased significantly over the last 30 years. The role of early 

postnatal exposure to PBDEs in provoking thyroid hormone disruption, coupled with the known 

effects of hypothyroidism on the developing cholinergic system, suggests that early PBDE 

exposure may have lasting consequences on cholinergic-dependent behaviors, such as learning 

and sustained attention. Previously in our laboratory, learning deficits were observed in rats 

exposed to DE-71, a commercial PBDE mixture, at a dose of 30 mg/kg/day during the second 

week of life. The purpose of the current study was to determine if this effect is seen at lower 

doses of DE-71. Rats received one of three daily oral treatment doses of DE-71 (0 mg/kg, 5 

mg/kg, or 15 mg/kg) from postnatal days 6-12 and were tested as adults on variations of a 5choice 

serial reaction time task. Learning was measured by acquisition rate of the initial visual 

discrimination task, in which the rats were trained to make a nosepoke in response to the random 

illumination of one of five ports. Following this, attentional ability was assessed by measuring 

the rats‟ response accuracy and error types when the visual cues were made brief and 

unpredictable. Finally, rats were administered a drug challenge with the muscarinic antagonist 

scopolamine on the sustained attention task. No significant effect of DE-71 treatment group was 

found on any of the dependent variables, suggesting that the investigated doses were below 

threshold to induce cognitive impairment or that the sample size was insufficient to reveal 

effects. Scopolamine effectively impaired attention but failed to differentially affect the 

treatment groups. Therefore, future research should investigate doses between 15 mg/kg and 30 

mg/kg in order to determine the critical levels required to generate learning impairments. 

Disclosures: J. Kaplan, None; L.L. Driscoll , None; J. Kraut, None; J. Fitzpatrick, None. 

Poster 

279. Steroids and Plasticity I 




Support: NIH Grant KO2 MH01497 

NIH Grant RO1 MH47538 

Title: Steroid-sensitive vasopressin innervation in male and female mice 

Authors: *B. D. ROOD, G. J. DE VRIES; 

Neurosci. & Behavior, Univ. Massachusetts, Amherst, MA 

Abstract: Vasopressin-immunoreactive fibers derive from a few distinct nuclei in the mouse 

brain. Large magnocellular neurons in the paraventricular nucleus of the hypothalamus and 

supraoptic nucleus project to the posterior pituitary, where they release vasopressin into the 

bloodstream. Smaller neurons in the paraventricular nucleus, but also in the suprachiasmatic 

nucleus, bed nucleus of the stria terminalis (BNST), and medial amygdala, project throughout the 

brain, where they release vasopressin as a neurotransmitter or neuromodulator. The distribution 

of fibers emanating from these nuclei is thought to be overlap minimally suggesting that 

vasopressin may have several mutually exclusive functions in the brain. The first step in 

understanding the relationship between structure and function in the vasopressin system is to 

elucidate the location and site of origin of the different projections. Because vasopressin 

expression is steroid- dependent in the BNST and medial amygdala of the mouse, we were able 

to use hormonal manipulation to determine which brain regions likely receive vasopressin 

innervation from these nuclei. Male and female mice were gonadectomized or sham operated, 

and vasopressin-immunoreactivity was examined in brains collected 15 weeks following surgery. 

Castration nearly eliminated vasopressin-immunoreactivity in fibers and terminals in numerous 

areas, many of which are part of, or associated with the limbic loop, suggesting they originate in 

the BNST or medial amygdala (e.g., lateral septum, ventral striatum and pallidum, bed nucleus 

of the stria terminalis, amygdala, lateral habenular nucleus, and mediodorsal nucleus thalamus). 

In contrast, some brain areas such as the paraventricular nucleus of the thalamus and 

dorsomedial hypothalamus retained both fiber and terminal staining suggesting different sources 

of innervation. Evaluation of vasopressin innervation of the limbic system may shed light on the 

role that vasopressin plays in social behavior. 

Disclosures: B.D. Rood, None; G.J. de Vries, None. 

Poster 





Poster 




Topic: E.01.c. Steroids and plasticity 


MH081503-01A1 

Title: The role of androgen receptors in determining the sex difference in neuron number in the 

posterodorsal medial amygdala (MePD) 

Authors: *A. DURAZZO, C. L. JORDAN, S. M. BREEDLOVE; 

Dept Neurosci, Michigan St Univ., East Lansing, MI 

Abstract: Steroid hormone receptors play a crucial role in the sexual differentiation of the rodent 

brain. In this study we examined the effect of the testicular feminization mutation (tfm), which 

results in a dysfunctional androgen receptor protein, on the morphology of the rat posterodorsal 

subregion of the medial amygdala (MePD), a sexually dimorphic brain region that is an 

important component of the rodent mating circuit. Adult (90 day old) male, female and tfm Long 

Evans rats were sacrificed and both volumes and neuronal numbers were quantified. In 

accordance with previous findings from our lab (Morris et al., 2008), we observed significantly 

greater volumes in males than females, and a main effect of laterality with the right hemisphere 

generally being larger than the left. Preliminary analysis indicates that the number of MePD 

neurons in tfm males did not differ from wild type males, indicating that androgen receptors are 

not required for the masculinization of MePD neuronal numbers and, by elimination, that 

estrogen receptors may be involved in the sexual differentiation of this parameter. 

Disclosures: A. Durazzo , None; C.L. Jordan, None; S.M. Breedlove, None. 

Poster





Support: NIH NS045T95 

Title: Muscle-specific loss of brain-derived neurotrophic factor impairs motor function 

Authors: *J. E. LEBOEUF 1,2 , E. N. OTTEM 4 , S. M. BREEDLOVE 3 , C. L. JORDAN 3 ; 

2 Neurosci., 3 Psychology, 1 Michigan State Univ., East Lansing, MI; 4 Biol., Northern Michigan 

Univ., Marquette, MI 

Abstract: We set out to examine the role of skeletal muscle fiber-produced brain-derived 

neurotrophic factor (BDNF) in the survival and plasticity of an androgen-sensitive 

neuromuscular system, the spinal nucleus of the bulbocavernosus (SNB). To do this, we 

genetically deleted BDNF from skeletal muscle fibers using the Cre/lox recombination system. 

We crossed mice having a floxed BDNF gene (Jackson stock number 004339) with transgenic 

mice possessing Cre recombinase driven by a human skeletal actin promoter resulting in 

expression only in skeletal muscle fibers. Data based on PCR indicate a selective recombination 

of the BDNF gene in skeletal muscle and not in other tissues. While the loss of muscle-derived 

BDNF did not appear to interfere with the survival of the SNB system in male mice, it did lead to 

a generalized disturbance in motor function. Adult BDNF skeletal muscle fiber knock-out 

(BNDF mfKO) mice show deficits in rearing behavior and impaired muscle strength based on 

hang and grip strength tests. BDNF smfKO mice also exhibited a clasping reflex consistent with 

neuromuscular dysfunction. Such deficits were seen in both male and female BDNF smfKO 

mice, indicating the effect of deleting muscle BDNF on motor function does not depend on sex. 

These results suggest that muscle-derived BDNF plays a critical role in motor function. 

Disclosures: J.E. LeBoeuf, None; E.N. Ottem, None; S.M. Breedlove, None; C.L. Jordan, 

None. 

Poster 



Program#/Poster#: 279.5/MM19



NIH Predoctoral F31 MH78273 

Title: The organizational role of androgens and the androgen receptor in anxiety-related 

behaviors and sensorimotor gating 

Authors: *D. G. ZULOAGA 1 , C. L. JORDAN 2 , S. M. BREEDLOVE 2 ; 

1 Dept Psychol and Prog Neurosci, 2 Michigan State Univ., East Lansing, MI 

Abstract: In rats, exposure to testosterone (T) early in life, which can act through both androgen 

and estrogen receptors (ARs; ERs), organizes adult behaviors. In the current study we compared 

behaviors in wild type (wt) male rats and AR deficient rats with the testicular feminization 

mutation (Tfm), that were gonadectomized (Neo-Gdx) or sham-operated (Neo-Sham) on the day 

of birth. In adulthood, rats were either gonadectomized or sham-operated and implanted with T 

capsules to equilibrate adult circulating T and compared in tests of anxiety (open field, novel 

object exposure, light dark box, elevated plus maze) and sensorimotor gating (prepulse inhibition 

(PPI) of the acoustic startle response (ASR)). Compared to Neo-Sham rats, Neo-Gdx rats showed 

decreased indices of anxiety in all tests with no differences between wt or Tfm males within 

treatment groups. PPI was increased, while ASR was decreased in Neo-Gdx rats, with the 

difference in ASR largely due to increased ASR in Neo-Sham Tfm males. Following behavior 

testing, blood corticosterone levels were measured at baseline or 20 minutes after exposure to an 

open field with a novel object. Corticosterone levels were elevated in Neo-Gdx rats at baseline 

with no differences in novel object exposed rats. Analysis of brain weight revealed an effect of 

neonatal castration largely due to heavier brains in Neo-Sham wt males compared to both Neo- 

Gdx groups. These findings indicate a role of T prior to adulthood in the organization of anxietyrelated 

behaviors and sensorimotor gating in rats, which appears to be primarily ARindependent. 

Disclosures: D.G. Zuloaga, None; C.L. Jordan, None; S.M. Breedlove, None. 

Poster 




Topic: E.01.c. Steroids and plasticity


Title: Acute and sub-chronic estrogen and progesterone differentially regulate histone 

modifications 

Authors: *E. M. WATERS 1 , K. J. MCCARTHY 1 , T. A. MILNE 2 , R. G. HUNTER 1 , C. D. 

ALLIS 2 , B. S. MCEWEN 1 ; 

1 Lab. of Neuroendocrinology, 2 Lab. of Chromatin Biol. and Epigenetics, Rockefeller Univ., New 

York, NY 

Abstract: Neuronal plasticity in the hippocampus is modulated by both acute and sub-chronic 

estrogen (E) and progesterone (P) treatment. Although neither estrogen receptors (ERs) nor 

progesterone receptors (PRs) are found in nuclei of hippocampal principal cells, extranuclear 

ERs and PRs are found in many sites including dendritic spines and axon terminals. Whether 

ERs and PRs in the hippocampus have the majority of their effect through local, rapid synaptic 

signaling and protein translation or whether these signaling pathways can also regulate gene 

transcription is unknown. Interestingly, E-induced PRs are found in the hippocampus suggesting 

that E-induced signaling pathways can have genomic effects. One of the first steps in gene 

transcription is chromatin remodeling involving posttranslational modifications of histones that 

regulate DNA availability. Posttranslational modifications of histone such as acetylations and 

methylations regulate transcriptional activation or repression. We show that acute E and P 

replacement (6 hours) in ovariectomized female rats reduced acetylation of histones 3 and 4 (H3, 

H4) and trimethylation of H3 at lysine 9 in the hippocampus. After treatment twice with E and 

examination 72 hours after the start of E treatment, E also reduced H4 acetylation but did not 

affect H3 acetylation and H3 lysine 9 trimethylation in comparison to oil treatment. In addition, 

acute treatment with P in tissue pretreated with E increased the expression of all three histone 

modifications above the E treatment levels. These results suggest that E and P co-opt signaling 

pathways originating in the synapses and other cytosolic sites to regulate chromatin remodeling. 

Disclosures: E.M. Waters, None; K.J. McCarthy, None; T.A. Milne, None; R.G. Hunter, 

None; C.D. Allis, None; B.S. McEwen, None. 

Poster 





Support: NIH Grant NS07080



Title: BDNF variant Val66Met regulates brain function in female mice in an estrous cycledependent 

manner 

Authors: *J. L. SPENCER 1 , E. M. WATERS 1 , T. A. MILNER 1,2 , F. S. LEE 2 , B. S. 

MCEWEN 1 ; 

1 Lab. Neuroendocrinol, Rockefeller Univ., New York, NY; 2 Weill Cornell Med. Col., New 

York, NY 

Abstract: Brain-derived neurotrophic factor (BDNF) is critical for neuronal survival, growth, 

differentiation, and synaptic plasticity. The Val66Met variant of the human BDNF gene, found in 

30% of Caucasians, is a point mutation in the coding sequence for the pro-region. Neurons 

expressing the Met variant show impaired trafficking of pro-BDNF and decreased activitydependent 

BDNF secretion. Human Met carriers have decreased hippocampal activation and 

cognitive performance. Similarly, male mice expressing the Met variant have smaller 

hippocampi and impaired freezing during hippocampal-dependent fear conditioning. Clinical 

studies suggest that the Val66Met variant influences susceptibility to psychiatric and neurologic 

disease, but in a gender-dependent manner. These gender differences could stem from an 

interaction of the Met variant with ovarian steroids. In animal studies, estrogen (E) modulates 

BDNF expression and activation of its receptor TrkB, which may be necessary for E 

enhancement of hippocampal synaptic plasticity. We hypothesized that the Val66Met variant 

alters the natural regulation of brain function by ovarian steroids in female mice homozygous for 

the Val or Met variant. Ten V/V (wild-type) and 10 M/M mice were tested on the object 

placement and object recognition tests at four different phases of the estrous cycle (proestrus, 

estrus, diestrus 1, and diestrus 2). Brains of mice in proestrus or diestrus were examined for 

synaptic protein expression and TrkB activation. M/M animals exhibited enhanced anxiety 

behavior, as well as impaired object placement and object recognition behavior. On the object 

placement task, genotype differences in performance were dependent on estrous cycle phase, 

suggesting differential modulation by ovarian steroids in V/V and M/M animals. In agreement 

with our previous studies, V/V mice had increased TrkB activation and PSD-95 expression 

during proestrus (high E) relative to diestrus (low E). In M/M animals, only TrkB activation, but 

not increased PSD-95 expression, was observed during proestrus. This suggests that intact 

activity-dependent secretion of BDNF is necessary for ovarian steroid modulation of synaptic 

protein expression, but not for signaling through the TrkB receptor. We conclude that BDNF 

variant Val66Met modulates hippocampal function in female mice in an estrous-cycle dependent 

manner. The dependence of the effects of variant BDNF on estrous cycle stage suggest that in 

humans, this variant may interact with ovarian steroids to influence susceptibility to psychiatric 

and neurological disorders, particularly those disorders known to be influenced by the menstrual 

cycle. 

Disclosures: J.L. Spencer , None; E.M. Waters, None; T.A. Milner, None; B.S. McEwen, 

None; F.S. Lee, None.

Poster 






NIH Grant 5P01 AG16765 

Title: PELP1 associates with PSD-95 and localizes to dendrites in hippocampal neurons 

Authors: *K. T. AKAMA 1 , K. L. MITTERLING 2 , T. A. MILNER 1,2 , B. S. MCEWEN 1 ; 

1 Neuroendocrinol, Rockefeller Univ., New York, NY; 2 Neurol. and Neurosci., Weill Cornell 

Med. Col., New York, NY 

Abstract: Dendritic spines are often the sites of excitatory synapse formation, and the steroid 

hormone estrogen stimulates an increase in spine formation and subsequent synaptogenesis in the 

hippocampus. Recent studies have demonstrated that the estrogen receptors (ER) can be 

immuno-localized to non-nuclear sites in hippocampal CA1 pyramidal cell dendrites, particularly 

spines. Such spine-targeting of ER‟s potentially allows for local signaling from hormonal stimuli 

to rapidly modify dendritic spine structure or downstream neuronal responses. Our objective is to 

identify specific protein binding partners that might selectively direct a rapid, estrogenstimulated 

non-genomic response, and therefore we are investigating the molecular interactions 

of ER‟s with spine proteins. A key protein that we are addressing here is the ER scaffolding 

protein, PELP1 (proline-, glutamic acid-, leucine-rich protein 1). ERα is known to associate with 

PELP1 (also known as MNAR, modulator of non-genomic activity of ER), and an ERα-PELP1 

interaction can result in estrogen-stimulated Src kinase signal transduction in breast cancer cells. 

Consistent with previous studies, PELP1 immuno-reactivity was detected in the nuclei of 

hippocampal pyramidal cells. Additionally, electron microscopic studies revealed PELP1 

immuno-reactivity in dendritic shafts and spines in CA1 stratum radiatum. Likewise, such 

PELP1 localization might be facilitated via the spine scaffolding protein PSD-95 (post-synaptic 

density protein-95). We further demonstrate here in vitro by co-immunoprecipitation assays that 

PELP1 protein associates with PSD-95. Thus, these findings suggest that certain non-genomic 

estrogen actions at the dendritic spine may occur via the cellular localization and the molecular 

function of PELP1. 

Disclosures: K.T. Akama, None; B.S. McEwen, None; T.A. Milner, None; K.L. Mitterling, 

None.

Poster 






NIH-MSTP Grant GM07739 

Title: Sex differences in the level and subcellular distribution of delta-opioid receptor 

immunoreactivity in hippocampal principal cells 

Authors: *T. J. WILLIAMS 1,2 , A. TORRES-REVERON 1 , T. A. MILNER 1,3 ; 

1 Neurol and Neurosci, Weill Cornell Med. Coll, New York, NY; 2 Weill 

Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY; 3 Lab. 

of Neuroendocrinology, The Rockefeller Univ., New York, NY 

Abstract: Our previous studies revealed that ovarian steroids influence endogenous opioid 

peptide levels and trafficking of mu-opioid receptors (MORs) in the rat hippocampal formation 

(HF). However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors 

(DORs) in the HF is unknown. Here, the brains of proestrus (as assessed by vaginal smear 

cytology) female, diestrus female, and male adult (2-3 month old) Sprague-Dawley rats were 

perfusion fixed with paraformaldehyde and acrolein. Vibratome cut coronal sections through the 

HF were processed for quantitative immuno-peroxidase light microscopy or immuno-gold 

electron microscopy using antisera directed against the DOR (Chemicon, rabbit polyclonal; 

Cheng et al., 1995, guinea pig polyclonal). Consistent with previous studies in males (Commons 

and Milner 1996, 1997), DOR-immunoreactivity (-ir) localized to select interneurons and 

principal cells in the female HF. Light microscopic densitometry analysis, performed by 

hippocampal region using NIH Image, revealed that in comparison to males, proestrus (high 

estrogen) females display reduced DOR-ir in the CA1 pyramidal cell layer while diestrus 

females show reduced DOR-ir in the dentate granule cell layer. No differences were observed 

between males and females within CA1 or dentate hilar interneurons. Electron microscopic 

analysis showed reduced total DOR labeling per dendrite and reduced localization of DORs to 

the dendritic plasmalemma within CA1 stratum radiatum in females (regardless of estrous phase) 

compared to males. Further analysis suggested that diestrus females had reduced total DOR per 

dendrite while proestrus females exhibited decreased plasmalemmal distribution of DORs. 

Postsynaptic DOR activation is thought to increase potassium conductance and hyperpolarize 

membranes. Thus, proestrus females, with fewer plasmalemmal DORs available for ligand

activation on principal cell dendrites, may more readily transmit excitatory signals than their 

diestrus or male counterparts. These findings suggest that ovarian steroids can alter hippocampal 

excitability through direct effects on DORs in principal cells. Analysis of DOR trafficking 

changes in interneurons is underway to determine if DORs, like MORs, are also positioned to 

facilitate indirect excitation of principal cells. 

Disclosures: T.J. Williams, None; A. Torres-Reveron, None; T.A. Milner, None. 

Poster 






The Gary R. Helman Foundation 

Title: Dynamic regulation of hippocampal histone H3 methylation by acute stress 

Authors: *R. G. HUNTER 1 , K. J. MCCARTHY 1 , T. A. MILNE 2 , R. D. ROMEO 3 , E. M. 

WATERS 1 , C. ALLIS 2 , B. S. MCEWEN 1 ; 

1 Lab. Neuroendocrinol, 2 Lab. of Chromatin Biol. and Epigenetics, Rockefeller Univ., New York, 

NY; 3 Psychology, Barnard Col., New York, NY 

Abstract: Modification of epigenetic histone marks has been observed in a number of models of 

human neurological and mental disorders such as ischemia, drug abuse and depression, all of 

which are influenced by stress and the actions of corticosteroid hormones. Here we examine the 

effect of both acute and chronic stress as well as acute and chronic corticosterone on the 

methylation status of histone H3 lysine 9 (H3K9). In our studies we used acute and chronic (21 

day) restraint stress as well as acute (10mg/kg s.c.) and chronic corticosterone (400ug/ml 

dissolved in drinking water with 2.5% ETOH) on the levels of H3K9 trimethylation, a marker of 

transcriptional repression in the hippocampal formation of the male rat. We found that the 

chronic manipulations produced small to non-significant reductions in H3K9 trimethylation. In 

contrast, acute restraint produced a robust (114 ±45%, p

stress on H3K9 methylation status, suggesting that the acute stress effects we observed are 

occurring independently of the adrenal steroids and may in fact result from the actions of another 

stress messenger such as corticotrophin releasing hormone or norepinephrine. The ability of 

acute stress to affect a global increase in H3K9 methylation status in a region and tissue specific 

manner is as remarkable as the fact that the typically stable H3K9 trimethyl mark can be 

modulated so rapidly. Further exploration of this phenomenon should provide insights of interest 

to both neuroscientists and chromatin biologists. 

Supported by MH41256 and The Gary R. Helman Foundation. 

Disclosures: R.G. Hunter , None; K.J. McCarthy, None; T.A. Milne, None; R.D. Romeo, 

None; E.M. Waters, None; C. Allis, None; B.S. McEwen, None. 

Poster 





Support: Bioinformatics JST Japan 

CREST JST Japan 

Title: Rapid modulation of spines and LTD/LTP by estradiol in rat hippocampus: neurosynaptocrinology 

Authors: *S. KAWATO, H. MUKAI, G. MURAKAMI, Y. HOJO, Y. HATANAKA, S. HIGO, 

T. KIMOTO; 

Dept Biophys & Life Sci, Grad Sch. Arts & Sci., Univ. of Tokyo, Tokyo, Japan 

Abstract: Estradiol-induced rapid modulation (within 90 min) was analyzed about spinogenesis 

and the long-term depression (LTD) in adult rat hippocampal slices. Spine analysis was 

performed by dye-injected single pyramidal neurons in the hippocampal slices. Estradiol at 1-10 

nM rapidly increased (within 120 min) the density of spines, in CA1 pyramidal neurons. On the 

other hand, in CA3 estradiol decreased the density of (spine-like) thorns which are postsynaptic 

pairs of mossy fiber terminals. MAP kinase was involved in these processes. Multielectrode 

analysis showed that LTD was significantly enhanced by 1- 10 nM estradiol in CA1 and CA3. 

ERalpha agonist but not ERbeta agonist, induced the same effect as that of estradiol in both 

spinogenesis and LTD. These rapid responses were driven via ERalpha that was found at 

synapses of glutamatergic neurons. Mass-spectrometric analysis demonstrated that even after

castration to deplete circulating testosterone, the male hippocampal estradiol level was not 

decreased, indicating that endogenously synthesized estradiol in male hippocampus plays an 

essential role for rapid synaptic plasticity. 

Disclosures: S. Kawato, Bioinformatics JST Japan, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); CREST JST 

Japan, B. Research Grant (principal investigator, collaborator or consultant and pending grants as 

well as grants already received); H. Mukai, None; G. Murakami, None; Y. Hojo, None; Y. 

Hatanaka, None; S. Higo, None; T. Kimoto, None. 

Poster 






Title: Increased mossy fiber BDNF expression and mossy fiber transmission in hippocampus 

after gonadectomy in adult male rats 

Authors: V. A. SKUCAS 1 , T. M. HINTZ 1 , S. BAROUK 1 , N. J. MACLUSKY 2 , *H. E. 

SCHARFMAN 1,3 ; 

1 CDR, Nathan Kline Inst., Orangeburg, NY; 2 Biomed. Sci., Univ. of Guelph, Guelph, ON, 

Canada; 3 New York Univ., New York, NY 

Abstract: Gonadal steroids have many effects in the CNS, and one that has attracted great 

interest is the regulation of neuronal plasticity. In hippocampus, it appears that estrogen exerts 

some of its effects on plasticity by increasing the expression of the neurotrophin BDNF (brainderived 

neurotrophic factor). Little, however, is known about the potential for testosterone or its 

metabolites to regulate BDNF in hippocampus. Of particular interest is the mossy fiber (MF) 

pathway, because BDNF expression is most prominent in this pathway, relative to other areas of 

hippocampus. Therefore, we asked whether BDNF expression would be altered, and whether MF 

transmission would be influenced, when testosterone levels were dramatically reduced by 

gonadectomy in adult male rats. Anesthetized rats were gonadectomized bilaterally or subject to 

sham surgery at 4 or 8 weeks of age, and examined 1 or 8 weeks later. To study BDNF 

expression, anesthetized rats were perfused using 2% paraformaldehyde, followed by 30% 

sucrose postfixation, and brains were sectioned using a cryostat. Free-floating sections from each 

experimental group were processed concurrently using an antibody to BDNF (Sigma,

monoclonal, 1:1000) with diaminobenzidine as a chromagen and nickel intensification. 

Gonadectomized rats demonstrated robust increases in MF BDNF compared to sham controls, 

quantified using Image J analysis of the MF terminal field. MF BDNF immunoreactivity 

increased after gonadectomy in both young and adult rats, and was evident at 1 or 8 weeks after 

gonadectomy (n=5 rats/group). In contrast to CA3, there was no detectable change in BDNF 

immunoreactivity in CA1. In other rats, hippocampal slices were prepared in the horizontal plane 

using standard methods, and MF transmission was evaluated by stimulation of the dentate gyrus 

subgranular zone, and extracellular recording in area CA3b. Comparisons were made to 

stimulation of the Schaffer collateral pathway, and recording in area CA1b. Maximal population 

spike amplitude was increased in CA3 in gonadectomized rats compared to sham controls, but 

not in CA1 of the same slices (n=>5 slices/group). These results suggest that male gonadal 

steroids, presumably testosterone and its metabolites, normally suppress MF synaptic 

transmission and reduce MF BDNF expression, but have less effect in area CA1. The subfieldspecific 

effects may help resolve discrepancies reported in the literature regarding hippocampal 

effects of testosterone. The results are consistent with previous reports that BDNF potentiates 

MF transmission, and gonadal hormones have a profound influence on the activity of area CA3 

pyramidal cells of rat hippocampus. 

Disclosures: V.A. Skucas, None; T.M. Hintz, None; S. Barouk, None; N.J. MacLusky, 

None; H.E. Scharfman, None. 

Poster 






Epilepsy Foundation 

Title: Hippocampal sharp wave (SPW) frequency fluctuates across the estrous cycle in the adult 

female rat 

Authors: *P. PEARCE 1,2 , D. FRIEDMAN 2 , H. SCHARFMAN 1,2 ; 

1 NYU Sch. Med., New York, NY; 2 Ctr. for Dementia Res., Nathan Kline Inst., Orangeburg, NY 

Abstract: Estrogen and progesterone exert powerful effects on hippocampal neurons, 

influencing structure, function as well as behavior. Recent studies suggest that CA3 neurons of

hippocampus are major targets of steroid hormones, yet few studies have examined the effects of 

steroid hormones on neuronal activity in vivo. Therefore, we tested whether a type of behaviorrelated 

synchronized CA3 activity, SPWs, would change across the estrous cycle. Twisted 

bipolar electrodes were implanted in area CA3 of adult female Sprague-Dawley rats using 

stereotaxic methods. Cycle stage was monitored by vaginal cytology each morning, and only 

those animals with 4-day estrous cycles were included. Starting 1 week after surgery, continuous 

recordings were made for 30 minutes during sleep, immobility and exploration in the home cage. 

After 4-5 estrous cycles, animals were euthanized to confirm electrode locations. SPWs were 

defined as 70-200 msec spikes in the baseline recordings that were greater than two standard 

deviations of the noise, and 2 minutes of continuous, artifact-free record were used to count 

SPWs for each behavioral state. SPW frequency was low during exploratory periods, and the 

frequency did not vary significantly with cycle stage (p>0.05; n=5). Mean SPW frequency 

during sleep and immobility varied significantly across the estrous cycle (one way ANOVA; 

p=0.017, n=5) increased at proestrus (123.8 and 130.7% respectively, normalized to diestrus 2), 

and remained elevated at estrus (136.1, 135.8%). The results suggest that the preovulatory 

estrogen surge initiates an increase in CA3 SPWs, which lasts for at least 48 hours. The increase 

in SPWs appears to follow the rise in brain-derived neurotrophic factor (BDNF) that potentiates 

mossy fiber transmission, suggesting an underlying mechanism. The changes in SPW during 

sleep and immobility may preferentially increase memory consolidation for events occurring 

during wakefulness, and ultimately facilitate reproductive success. 

Disclosures: P. Pearce, None; D. Friedman, None; H. Scharfman, None. 

Poster 





Support: NIH grant NIDA R01 DA019921 

NIH Grant 2 P20 RR016479 

NIH COBRE P20 RR15567 

Title: Estrogen regulation of proteins in the rat ventromedial nucleus of the hypothalamus

Authors: *M. TELEFONT 1 , B. MO 1 , E. CALLEGARI 2 , K. J. RENNER 1 ; 

1 Biol., Univ. South Dakota, Vermillion, SD; 2 Div. of Basic Biomed. Sci., Sanford Sch. of Med., 

Vermillion, SD 

Abstract: The expression of gonadal hormone-dependent female sexual behavior in rodents 

provides an excellent model for studies directed towards understanding how hormonal signals 

are translated in the central nervous system to produce behavioral responses. Estrogen-induced 

protein synthesis in the pars lateralis of the ventromedial nucleus of the hypothalamus (VMNpl) 

is critical to the activation of female sexual behavior. In this study, two-dimensional 

electrophoresis (2-DE) was used to separate proteins extracted from VMNpl tissue punches 

obtained from brains of ovariectomized (ovx) rats treated with either E2 or sesame oil vehicle 

(V). We focused on proteins that were undetectable on 2-DE maps obtained from groups without 

hormone replacement but were dramatically up-regulated by E2 replacement. These proteins 

represented a greater than 10-fold increase in stain intensity when compared to background as 

defined by PDQuest 8.0 (Bio-Rad Laboratories, Hercules, CA). A comparison of protein profiles 

obtained from the VMNpl of rats treated with E2 or vehicle (V) showed the presence of 30 spots 

that fit this criteria. Identification of the proteins was accomplished using RPLC-nanoESI- 

MS/MS analysis. Of the 29 proteins that were successfully identified, the majority have roles in 

the promotion of neuronal plasticity and signaling. Examples of E2-induced proteins include: 

alpha- and beta- soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs), 

cofilin2, rab GDI alpha, chromatin modifying protein 4B (CHMP4B), vesicle associated 

membrane protein 2, cytoplasmic dynein intermediate chain 2B, cytoskeleton-associated protein 

1, Ulip2/TOAD-64 (turn on after division protein 64 kD)/CRMP2 (collapsing response mediator 

protein-2), glial fibrillary acidic protein delta (GFAP), N-myc downstream regulated gene 2 

(NDRG2), and alpha isoform of regulatory subunit A of protein phosphatase 2. The E2-induced 

proteins identified in the current study are consistent with earlier reports that suggest a function 

of E2 in the VMNpl is to stimulate changes in synaptic spine density and/or alter signal 

transduction. 

Disclosures: M. Telefont, None; K.J. Renner, None; B. Mo, None; E. Callegari, None. 

Poster 





Support: NIH NSRA T32 DA007267

NINDS Grant NS48141 

Title: Using microdialysis coupled on-line to capillary electrophoresis to study the rapid effects 

of estradiol on GABA release in the striatum 

Authors: *K. N. SCHULTZ 1 , M. HU 2 , S. MUSATOV 3 , M. G. KAPLITT 4 , R. T. KENNEDY 1 , 

J. B. BECKER 2 ; 

1 Chem., Univ. Michigan, Ann Arbor, MI; 2 Mol. & Behavioral Neurosci. Inst., Univ. of 

Michigan, Ann Arbor, MI; 3 Neurobio. Behavior, Rockefeller Univ., New York, NY; 

4 Neurosurg., Weill-Cornel Med. Col., New York, NY 

Abstract: Sex differences exist across all stages of addiction from initiation to relapse with 

females being more susceptible to the addictive effects of drugs. Work in our lab has shown that 

estradiol has a profound effect on drug taking behavior and potentiates stimulated dopamine 

release in the striatum. A potential mechanism for this effect is that estradiol modulates GABA 

release which in turn modulates release of dopamine. To test the hypothesis that estradiol 

regulates GABA release, we have investigated the effect of manipulation of estrogen signaling 

on GABA release in the striatum measured using microdialysis coupled on-line to capillary 

electrophoresis. We have found that estradiol benzoate (EB) injected subcutaneously into 

ovariectomized (OVX) female rats decreases K+-evoked GABA release by 50% (n = 7). To 

determine if this effect is mediated by the alpha estrogen receptor (ERα), we overexpressed this 

receptor by microinjection of an adeno-associated viral vector containing DNA for human ERα 

into the striatum. To control for effects associated with the viral vector implant, half of the OVX 

animals received a vector containing DNA for alkaline phosphatase (ALP). We found that with 

ERα overexpressed, estradiol was even more potent in inhibiting K+-evoked GABA release with 

a two fold greater reduction seen in EB+ERα animals compared to EB+ALP animals (53% [n=8] 

vs. 23% [n=9] compared to vehicle+ALP animals). This result supports the hypothesis that 

estradiol inhibits GABA function through ERα. We next began to evaluate the mechanism of 

estradiol signaling. Our hypothesis is that estradiol exerts its effect through a mitogen activated 

protein kinase (MAPK) pathway. To test this hypothesis, PD 98059, an inhibitor of mitogenactivated 

protein kinase kinase (MEK), was microinjected into the striatum of OVX rats 

pretreated with EB. Compared to vehicle treated controls PD 98059 (0.1 mM, n=3) increased 

K+-evoked GABA release 30% suggesting that estradiol binding to ERα activates the MAPK 

pathway to inhibit GABA release from the medium spiny neurons. Our results suggest that 

estradiol inhibits GABA function through ERα and MAPK pathways. These results suggest that 

previously observed enhancing effects of estradiol on dopamine may be mediated by suppression 

of GABA release with subsequent disinhibition of DA. This estradiol dependent GABA release 

may contribute to sex differences in drug addiction and further investigation into the signaling 

mechanisms may identify new therapeutic targets. 

Disclosures: K.N. Schultz, None; M. Hu, None; S. Musatov, None; M.G. Kaplitt, None; J.B. 

Becker, None; R.T. Kennedy, None.

Poster 





Support: NINDS NS48141 

T32 HD007048 

Title: Investigation into the mechanisms mediating the rapid effect of 17ß-estradiol in the 

striatum 

Authors: *H. YANG 1 , E. M. PECKHAM 2 , J. B. BECKER 1 ; 

1 MBNI, 2 Psychology, Univ. Michigan, Ann Arbor, MI 

Abstract: 17ß-Estradiol (E2) acts rapidly and directly on the striatum to enhance amphetamine 

(AMPH) stimulated dopamine (DA) release. In these experiments, we investigated the 

mechanisms mediating the action of estradiol in the striatum. We have shown in previous studies 

that estradiol also rapidly inhibits L-type calcium channels in medium spiny neurons from 

striatum. Since medium spiny neurons are GABAergic, we hypothesized that estradiol inhibits 

the release of GABA, resulting in reduced GABA activity at GABA-B receptors and enhanced 

stimulated DA release through a release of inhibition. In one series of experiments we 

investigated whether the effect of estradiol on AMPH-induced DA in dialysate can be blocked by 

the GABA-B agonist baclofen. Female rats were ovariectomized (OVX) and 2 weeks later 

received bilateral guide cannula implants aimed at the striatum. Estradiol was administered s.c. 

(5 µg estradiol benzoate, EB) or intrastriatal via the probe (5 ng E2/ml). Intrastriatal E2 or 

systemic EB enhanced the AMPH-induced (2.5 mg/kg) increase in DA. This increase was 

blocked by systemic baclofen (5 mg/kg), but not intrastriatal baclofen (300 µM) delivered via the 

probe. Since we did not see an effect of intrastriatal baclofen, we went on to investigate other 

possible mechanisms mediating the effect of estradiol in the striatum. Experiments from the 

Mermelstein laboratory have indicated that the effect of estradiol in the striatum is mediated by 

the coupling of the estradiol receptor ERα to MGluR5 receptors. So, we investigated the effect of 

blocking mGluR5 with 2-methyl-6-(phenylethynyl) pyridine (MPEP, 10 mg/kg) on intrastriatal- 

E2 enhancement of AMPH-induced DA in dialysate. Preliminary results indicate that MPEP 

prevents the effect of intrastiatal E2 to enhance AMPH-induced DA in dialysate. 

Disclosures: H. Yang , None; E.M. Peckham, None; J.B. Becker, None.

Poster 





Support: NIH HD042635 

NIH DA013185 

Title: Surface biotinylation reveals an estradiol regulation of membrane ERα levels 

Authors: *R. DOMINGUEZ, G. BONDAR, P. MICEVYCH; 

Neurobio., David Geffen Sch. of Medicin, Los Angeles, CA 

Abstract: Estradiol regulates reproduction by binding to and activating both intracellular and 

membrane estrogen receptor-α (ERα) in the hypothalamus and limbic system. Activation of 

intracellular ERα results in the formation of dimers, which bind to specific sites on DNA and 

mediate the transcription of estradiol sensitive genes. There is a rapid component of estradiol 

action that also regulates reproduction through the interaction of a membrane-associated ERα 

with a metabotropic glutamate receptor-1a to activate intracellular signaling pathways. While the 

localization and activation of ERα in the plasma membrane has been demonstrated, the question 

of whether ERα is a transmembrane protein remains an open question. The present study tested 

the hypothesis that a portion of the ERα is exposed to the extracellular environment and that 

estradiol regulates the levels of ERα in the membrane. Surface biotinylation was used to identify 

membrane proteins. Treatment of neurons and astrocytes in vitro with 10 nM estradiol for 0-120 

min had a biphasic effect on levels of biotinylated ERα in the plasma membrane. The amount of 

biotinylated ERα increased for the first 30-60 min of estradiol treatment. Hypothalamic tissue 

analyzed 30 min after the third cycle of 5 µg estradiol benzoate every fourth day, also had an 

increase in membrane ERα immunoreactivity revealed by western blot. These results indicate 

that estradiol regulates the levels of ERα in the membrane suggesting a mechanism of regulation 

that involves translocation to and internalization from the plasma membrane. Supported by NIH 

HD042635 & DA013185. 

Disclosures: R. Dominguez , None; G. Bondar, None; P. Micevych, None. 

Poster 

279. Steroids and Plasticity I




Support: NIDA R01DA 10547-12 

Title: Adolescent anabolic steroids alter 5ht2a localization in hamster brain 

Authors: R. H. MELLONI, Jr., *L. A. RICCI; 

Dept of Psych, Northeastern Univ., Boston, MA 

Abstract: Anabolic-Androgenic steroid (AAS) exposure throughout adolescence produces 

aggressive behavior in male Syrian hamsters. This AAS-induced aggressive behavioral 

phenotype has been observed in animals demonstrating reduced serotonin (5HT) innervation to 

brain regions implicated in aggression control. The reduction of 5HT activity associated with 

AAS-induced aggression may be due to a pharmacodynamic increase in the expression of the 2A 

subtype (5HT2A), as antagonizing 5HT2A activity using atypical antipsychotics such as 

risperidone produces marked reductions in aggressive behavior. To test this hypothesis, we 

examined the effects of adolescent AAS exposure on 5HT2A receptor expression, using 

immunohistochemistry, in aggressive male hamsters. AAS treatment produced a two-fold 

increase in 5HT2A immunoreactivity in a specific subregion of the anterior hypothalamus (AH), 

the lateral anterior hypothalamus (LAH). Interestingly, no changes in 5HT2A immunoreactivity 

were observed in other specific nuclei, which have repeatedly shown changes in neuochemical 

systems correlated with adolescent AAS-induced aggression including the medial amygdala 

(CeA), and the ventrolateral hypothalamus (VLH). These data suggest that alterations of 5HT2A 

expression and/or function, in response to AAS-induced decreased 5HT innervation, in the LAH 

may play an important role in AAS-induced aggressive behavior. 

Disclosures: R.H. Melloni, None; L.A. Ricci , None. 

Poster 





Support: NIH grant RR000163

NIH grant HD18185 

NIH grant MH62677 

NIH grant T32HD07133 

NIH grant F32MH076555 

Title: Effects of ovarian steroids on kynurenine mono-oxygenase in the non-human primate 

dorsal raphe nucleus 

Authors: *J. A. HENDERSON, C. L. BETHEA; 

Dept Neurosci/Reproductive Biol, Oregon Hlth. Sci. Univ., Beaverton, OR 

Abstract: Estrogen (E) has been shown to have neuroprotective properties in both laboratory 

and clinical studies across a number of brain regions and against a diverse range of insults. Einduced 

neuroprotection encompasses several different processes and is thus likely to proceed 

through several distinct interrelated mechanisms. We found that E treatment of ovariectomized 

(OVX) monkeys reduced kynurenine mono-oxygenase (KMO) gene expression in the dorsal 

raphe nucleus (DRN). Therefore, we hypothesize that ovarian hormones may improve serotonin 

neuronal survival by promoting cellular resilience through the KMO pathway. KMO is an 

enzyme involved in tryptophan metabolism which regulates the balance of neuroprotective to 

neurotoxic kynurenine metabolites. We hypothesize that E down-regulates KMO thus shifting 

tryptophan metabolism towards the pathways producing neuroprotective kynurenic acid and 

away from the pathway producing several neurotoxic kynurenines. To test this hypothesis, adult 

rhesus macaques (Macaca mulatta) were OVX for 3-6 months then treated with Silastic implants 

of either placebo (OVX), E for 28 days (E), no hormone for 14 days then progesterone (P) for 14 

days (P), or estrogen for 28 days supplemented with P for the final 14 days of the 28 days (E+P). 

The brains were collected and the DRN was processed for Western blotting or 

immunocytochemistry using an antibody generated against a KMO peptide (amino acids 28-44, 

Pacific Immunology Corp.). Analysis of Western blots using a 1-way ANOVA (OVX, E, EP) 

found animals treated with E or EP had lower KMO protein levels compared to OVX animals 

(df=2, F= 7.75, p=0.008; SNK post hoc test OVX vs. E p=0.032, OVX vs. EP p=0.009). The 

antibody to KMO demonstrated robust staining of the large serotonergic neurons of the DRN. 

Double immunofluorescent histochemistry with both affinity purified rabbit-anti KMO and a 

sheep anti-tryptophan hydroxylase to detect serotonin neurons showed KMO was present in both 

serotonin neurons and in other DRN cells. Analysis of 7 sections at 250 µm intervals through the 

DRN using a 2 (E, no E) x 2 (P, no P) ANOVA revealed that animals with E treatment (E and EP 

groups) had lower KMO positive area (p=0.01) and KMO positive neurons (p=0.02) compared to 

animals without E treatment (OVX and P groups). Decreased levels of KMO may be 

neuroprotective through increased accumulation of the neuroprotective metabolite kynurenic 

acid and/or decreased levels of the neurotoxic metabolites. Supported by NIH grants MH62677 

(CLB), NRSA 1 F32 MH 076555 (JAH), T32 HD07133 (Director: Dr. Judy Cameron), U54- 

HD18185 and RR000163. 

Disclosures: J.A. Henderson , None; C.L. Bethea, None.

Poster 





Support: Generalitat Catalunya DURSI 

DOE grant MO-081 

NIH grants RO1MH064845 and KO2MH073090 

NSF grant BCS0224221 

Title: Structural changes in the human brain across the menstrual cycle 

Authors: *A. BIEGON 1 , A. CONWAY 2 , C. EPPERSON 3 , N. ALIA-KLEIN 1 , D. PARETO 4 , R. 

CONSTABLE 3 , T. CANLI 2 ; 

1 Med. Dept, Brookhaven Natl. Lab., Upton, NY; 2 Stony Brook Univ., Stony Brook, NY; 3 Yale 

Univ., New Haven, CT; 4 Inst. Alta Tecnologia, Barcelona, Spain 

Abstract: Postmenopausal women on hormone therapy show greater gray and white matter 

volumes than age-matched controls. This observation has been interpreted in terms of protective 

effects of ovarian hormones against aging-related volume decreases. Alternatively, the presence 

of ovarian hormones may directly promote structural changes, which should be observable in 

younger women. To test this hypothesis, 14 young healthy women with regular menstrual cycles 

were each scanned during the early follicular phase, when estrogen and progesterone plasmalevels 

are low, and the midluteal phase, when levels of both hormones are high. High-resolution 

(1mmx1mmx1mm) structural scans were obtained using a 3T MRI scanner. On each scan day, 

the subjects were evaluated for mood (POMS) and blood samples were taken for estrogen and 

progesterone measurement. Regional brain gray and white matter volume and density were 

measured and compared across menstrual cycle phases using voxel-based morphometry (VBM). 

There were no significant menstrual-cycle related changes in mood. Estrogen and progesterone 

levels were both significantly higher in the midluteal compared to the follicular stage, as 

expected (p

hippocampus (p

for 14 days to allow for direct social confrontation between the pair. In each pairing, a dominantsubordinate 

hierarchy was clearly established, assessed by measures of defense strategies utilized 

during conflicts. Subordinate mice were next individually transferred to a standard small mouse 

cage (IE) or to a large rat cage containing running wheels and a variety of enrichment objects 

(EE). After 2 weeks of IE or EE housing, submissive animals were reintroduced to the pair 

housing for two additional weeks. The test mice were then injected i.p. with 0.1mg/kg LPS. After 

60 min, mice were rapidly decapitated, trunk blood was collected, and brains were examined for 

proinflammatory cytokine (PIC) activity via qRT-PCR. In basal state, IE-exposed mice showed 

significantly elevated levels of TNFα and IL-1b in forebrain and hippocampus compared to EEexposed 

mice. IE mice also showed a significantly diminished PIC response to LPS compared to 

EE mice. 

Chronic stressful conditions similar to the current experiment are known to adversely affect 

immune function. In IE-housed subordinate mice, the immune system showed chronic elevations 

of basal activity and a blunted response to immune challenge. In contrast, the EE-housed 

subordinate mice exhibited normal basal immune function and normal response to LPS, 

suggesting that EE reduces the deleterious influence of social stress on immune function. 

Disclosures: M.L. Lehmann , None; R. Brachman, None; M. Herkenham, None. 

Poster 

280. Stress and the Brain: Stress and Neuroimmunology I 


Program#/Poster#: 280.2/NN2 

Topic: E.06.c. Stress and neuroimmunology 


Title: The interactive effects of an immune challenge and psychosocial stressor provoke 

contextually dependant behavioral and neurochemical alterations 

Authors: *J. E. GIBB, M.-C. AUDET, B. P. WANN, H. ANISMAN; 


Abstract: The concomitant application of a psychosocial stressor and an immune challenge 

interactively influences neurochemical processes that could, if sustained, lead to the development 

of behavioral disorders. When immune activation, either through the administration of the 

bacterial endotoxin lipopolysaccharide [LPS], synthetic double stranded RNA, Poly I:C, or 

exogenous cytokines, is elicited following exposure to a psychosocial stressor (e.g., social 

disruption), typical inflammatory-induced increases of sickness behavior, as well as circulating

corticosterone, brain monoamine utilization, and brain cytokine mRNA expression are 

exaggerated. As behavioral variations following an immune challenge are often dependent on 

environmental context, it was of interest to investigate whether synergistic or additive effects 

would also be present when the immune challenge preceded the social stressor. In fact, there was 

reason to believe that sickness behavior represents a luxury that animals cannot afford in the face 

of ongoing threat, and hence would be less pronounced under these conditions. Mice received 

acute LPS (10 κg) treatment immediately before or following a 1-hour social disruption stressor 

(e.g., regrouping mice after a prolonged period of isolation) and the effects on sickness behavior, 

and on neuroendocrine, cytokine and monoamine variations were assessed. As predicted, when 

LPS was administered following the social stressor, sickness behavior, corticosterone as well as 

monoamine utilization within the hippocampus and prefrontal cortex were markedly increased. 

However, when the stressor was applied after LPS administration, the sickness behavior 

ordinarily elicited by LPS was abolished. In addition, plasma corticosterone levels were 

dramatically increased by the social stressor, to the degree that a ceiling effect largely precluded 

detection of further elevations by LPS treatment. Identical findings were also obtained with 

norepinephrine utilization within the prefrontal cortex. Together, these findings suggest that 

synergistic effects between psychosocial stressors and LPS treatment are only apparent when the 

immune challenge is administered following the social stressor. Although the interaction 

between LPS and psychosocial stressors produce dramatic behavioral and neurochemical effects, 

these appear to be mediated by the context in which the challenge is administered, and is 

influenced by the timing and order of the treatments. 

Disclosures: J.E. Gibb , None; M. Audet, None; B.P. Wann, None; H. Anisman, None. 

Poster 





Support: NSF IOS-05-54514 (to RS) 

NICHD 05751-33 (to DP) 

NIMH 67782 (to RS) 

Title: Mast cells in the brain influence anxiety-like behaviors

Authors: *K. M. NAUTIYAL 1 , A. C. RIBEIRO 2 , D. PFAFF 2 , R. SILVER 1 ; 

1 Psyc, Columbia Univ., New York, NY; 2 Lab. of Neurobio. and Behavior, The Rockefeller 


Abstract: There is increasing evidence of a bidirectional relationship between affect and the 

immune system. Much of the work in behavioral neuroimmunology has focused on microglia as 

the primary immune cell in an otherwise „immune-privileged‟ brain, however other cells 

including mast cells, are also resident in the central nervous system. Although they are best 

known for their role in allergy, broader functions of mast cells have recently been discovered. 

Brain mast cells release cytokines and inflammatory agents as well as neuroactive mediators 

including serotonin and histamine, implicated in the regulation of behavior (Silver et al, 1996). 

Here we used two models to study the role of brain mast cells in the regulation of emotional 

reactivity. 

First, using the mast cell deficient Kit W-sh/W-sh “sash” mouse (which lacks all mast cells) we 

measured behavior in the open field arena, elevated plus maze and cued fear paradigm. Sash 

mice, compared to wild-type (WT) C57BL/6J and heterozygous littermate controls, showed 

more anxiety-like and fearful phenotype. Physiologically sash mice were also more responsive to 

stressors than controls, showing higher defecation rates (up to 4 times higher) and larger 

increases in corticosterone (73.6 ng/ml more). 

Second, using pharmacological blockade of mast cell degranulation with disodium cromoglycate 

(cromolyn) injected into the brain and periphery, we explored how different populations of mast 

cells impact anxiety-like behavior. Cromolyn injected centrally into the lateral ventricle of WT 

mice increased anxiety-like responses in the open field arena (2 times longer latency to enter the 

center) and elevated plus maze (6 times fewer open arm entries) compared to saline injected 

controls. Interestingly, there were no behavioral differences between animals injected with saline 

or cromolyn peripherally (i.p., cromolyn does not cross the blood brain barrier). The results 

indicate that a lack of brain, rather than peripheral, mast cells underlies the differences in 

emotional reactivity. 

These data from genetic and pharmacological models taken together implicate brain mast cells in 

the regulation of emotional reactivity and affect. 

Disclosures: K.M. Nautiyal, None; R. Silver, None; D. Pfaff, None; A.C. Ribeiro, None. 

Poster 




Topic: E.06.c. Stress and neuroimmunology

Support: NSF grant 0549987 to TD 

HDRF grant 6-008 to TD 

Title: Regulation of stress induced neuroinflammatory factors by norepinephrine and 

corticosterone 

Authors: *P. BLANDINO, C. J. BARNUM, B. S. LANKOW, S. ZOLA, T. DEAK; 

Dept Behav Neurosci, SUNY-Binghamton, Binghamton, NY 

Abstract: We recently reported that exposure to an acute session of footshock increased IL-1 

and CD14 mRNA in the hypothalamus, while CD200 receptor (CD200R) mRNA was downregulated 

in the same tissue. Previous data have also shown that the noradrenergic antagonist 

propranolol blocked the increase in hypothalamic IL-1 protein, and that adrenalectomy 

augmented hypothalamic IL-1 protein in response to stress. However, it remains unclear whether 

norepinephrine and corticosterone regulate changes in CD14 and CD200R mRNA in response to 

acute stressor exposure as well. Therefore, the goals of the following experiments were to 

determine whether changes in multiple neuroinflammatory factors provoked by footshock (a) all 

occur as a result of increased noradrenergic signaling; and (b) whether endogenous 

corticosterone constrains these neuroinflammatory responses. To do this, adult male Sprague- 

Dawley rats were exposed to 80 inescapable footshocks (0.8 mA, 5 sec each, 90 sec variable 

inter-trial interval) over approximately 2 hr or remained in the homecage as non-stressed 

controls. In Exp 1, rats were pre-treated with propranolol (20 mg/kg ip) or vehicle 15 min prior 

to footshock. As predicted, propranolol blocked the hypothalamic IL-1 mRNA response. 

Interestingly, propranolol also blocked the increase in CD14 mRNA, but had no effect on the 

footshock-dependent suppression of CD200R mRNA. In experiment 2, rats were injected with 

metyrapone (100 mg/kg sc) and aminoglutethimide (100 mg/kg sc) or equivolume vehicle, 2.5 

and 1 hr respectively, prior to footshock exposure to suppress corticosterone synthesis. As 

expected, blockade of corticosterone synthesis augmented the hypothalamic IL-1 mRNA 

response to footshock. Interestingly, metyrapone significantly increased hypothalamic CD14 

mRNA in combination with footshock exposure, but had no effect on the CD200R mRNA 

downregulation. Together, these data demonstrate the induction of the neuroinflammatory factors 

IL-1 and CD14 mRNA within the hypothalamus during acute stress, are driven by 

norepinephrine and constrained by corticosterone. 

Disclosures: P. Blandino , None; C.J. Barnum, None; B.S. Lankow, None; S. Zola, None; T. 

Deak, None. 

Poster 





Support: FONACIT G-1389 

Title: Effects of two stress models on serotonin transporter of rat lymphocytes 

Authors: M. MEDINA, M. URBINA, *E. H. JAFFE, L. LIMA; 

Lab. Neuroquimica CBB, IVIC, Caracas 1020 A, Venezuela 

Abstract: Modifications produced at lymphocyte serotonergic system could be associated to 

immune alterations observed in chronic stressed rats and depressive patients. The dysregulation 

of the hypothalamic-pituitary-adrenal axis and the increase of blood glucocorticoid levels 

reported in these patients might be involved in such changes. To investigate the effects of stress 

on some functional aspects of serotonin transporter (5-HTT) from rat lymphocytes we applied 

two stress models to adult male rats: 1) physical restraint stress 5 hours/day for 5 days or 2) 

intraperitoneal injection of 2,5 mg/kg/day of reserpine for 3 days with the aims to: a) measure the 

proliferative response of lymphocytes to the mitogen concanavalin A (Con A) in the presence or 

in the absence of fluoxetine and b) calculate affinity constants (Kd), number of binding sites 

(Bmax) and Hill coefficients (nH) from [ 3 H]-paroxetine binding using lymphocytes membranes. 

Morning serum corticosterone concentration was measured with an EIA kit of DSL. 

Corticosterone values in restrained: 544±52 ng/ml and controls: 232±74 ng/ml (p





Clayton Medical Research Foundation 

Title: The effects of dietary probiotic supplements on CNS and hormonal responses to acute 

physiological and emotional stressors 

Authors: *L. FEIGHERY, C. ARIAS, R. OGDEN, P. E. SAWCHENKO; 

Salk Inst., La Jolla, CA 

Abstract: The intestinal microflora comprises an enormous biomass of commensal and 

symbiotic bacteria that play important roles in gut structure and function, nutrient processing and 

in both innate and adaptive immune responses. Altering the composition of the gut microflora 

through the ingestion of certain beneficial, probiotic, bacteria has been reported to have efficacy 

in managing stress-related inflammatory diseases of the lower GI tract, including ulcerative 

colitis and irritable bowel syndrome. Here we address unanswered questions as to whether and 

how probiotic supplements may affect the CNS processing of stress-related information and 

adaptive responses to it, notably those of the hypothalamo-pituitary-adrenal (HPA) axis. 

C57BL/6 mice were fed a probiotic cocktail that is marketed for human consumption (VSL#3; 

mixture of viable lyophilized bifidobacteria, lactobacilli and S. thermophilus) in their drinking 

water (4x108 CFU/ml) with 10% skimmed milk added for 4 weeks. Controls were maintained on 

10% skimmed milk in water over this interval. On the day of testing, separate cohorts of VSL#3 

and control animals were subjected acutely to representative physiological/immune (10 κg/kg 

LPS, ip) or emotional (30 min restraint) stressors) or control procedures. Mice were anesthetized 

and perfused for histochemistry 3 hr after LPS challenge and 2 hr post restraint. Mice fed VSL#3 

displayed a reduction in LPS-induced neuronal activation responses (Fos induction) throughout 

the brain. Separate series of sections stained for cyclooxygenase-2 (COX-2), to provide an initial 

index of the extent to which the stimulus presented by LPS may have been altered by probiotic 

treatment, revealed that cerebrovascular COX-2 labeling was reduced but not eliminated. 

Maintenance on the probiotic supplement also significantly attenuated restraint stress-induced 

Fos expression in the PVH, among other brain regions. Paradoxically, radioimmunoassay of 

stress hormones in blood samples from separate groups of similarly prepared mice failed to 

reveal any reliable reduction in plasma ACTH or corticosterone responses to either stressor as a 

consequence of VSL#3 treatment. These findings indicate that probiotic supplements can alter 

the impact of acute immune or emotional stress on the CNS. The basis for the disparity between 

responses of the central and peripheral arms of the HPA axis under the supplemented condition 

is unclear. 

Disclosures: L. Feighery , None; C. Arias, None; R. Ogden, None; P.E. Sawchenko, None.

Poster 






Title: Prior experience with aggressive social interactions influences behavioral, hormonal, and 

cytokine responses to a subsequent bacterial endotoxin challenge 

Authors: *M.-C. AUDET, B. WANN, J. GIBB, H. ANISMAN; 


Abstract: Repeated activation of the inflammatory immune system may sensitize behavioral, 

neurochemical and inflammatory responses upon re-exposure to a similar challenge. Aggressive 

social interactions in mice have been shown to alter behavioral expression, hypothalamicpituitary-adrenal 

(HPA) activity, monoamines functioning, and expression of pro-inflammatory 

cytokines (signaling molecules of the immune system) within the brain. These behavioral and 

physiological effects varied between submissive and dominant mice. The aim of the current 

experiment was to examine whether repeated experience with aggressive social interactions in 

mice would sensitize behavioral, endocrine, and central cytokine expression in response to an 

immune challenge with the bacterial endotoxin lipopolysaccharide (LPS). Further, it was of 

interest to determine whether LPS-induced effects would be sensitive to 

submissiveness/dominance status. CD-1 male mice (N = 14/group) were introduced to the homecage 

of a pre-selected aggressive mouse for 15 min on each of 7 consecutive days, or were not 

exposed to any social stressor. Seventy-two hours after the last stressor session, mice were 

injected with LPS or saline. Home-cage activity and sickness behaviors were monitored for 90 

min, after which brains were removed and blood was collected. The sickness behaviors 

ordinarily induced by LPS were exacerbated in dominant mice. Likewise, LPS increased plasma 

corticosterone levels, but this outcome was smaller in submissive mice. The mRNA expression 

of pro-inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis 

factor-α (TNF-α) within the medial prefrontal cortex (mPFC) and the hippocampus was 

increased after LPS treatment in all mice. However, the elevation of IL-6 within the mPFC was 

less pronounced in submissive mice. It seems that repeated experience with social victories 

worsened sickness symptoms in response to LPS. It also appears that HPA activation and 

elevated prefrontal IL-6 expression typically reported after LPS treatment were attenuated in 

submissive mice. Given that psychosocial stressors and activation of the inflammatory immune 

system may contribute to the pathogenesis of stress-related disorders, including depression, it is

suggested that dominant and submissive characteristics might be differentially related to 

pathological conditions. In this regard, however, some of these outcomes may be more evident in 

dominant than in submissive mice. 

Disclosures: M. Audet , None; B. Wann, None; J. Gibb, None; H. Anisman, None. 

Poster 





Support: NIH Grant AGO20633 

Title: Chronic exposure to glucocorticoids worsens neuron death in a mouse model of 

excitotoxicity 

Authors: *S. F. SORRELLS, C. D. MUNHOZ, R. M. SAPOLSKY; 

Dept Biolog Sci., Stanford Univ., Stanford, CA 

Abstract: During neurological injury, long-term exposure to stress levels of glucocorticoids 

(GCs) has a detrimental effect on neuron survival. An extensive literature now demonstrates that 

exposure to stress levels of GCs, signaling through GR, can impair the ability of neurons in the 

hippocampus, cortex, and striatum to survive a variety of necrotic neurological insults [1]. GCs 

worsen neuron death in these situations in part because they inhibit glucose uptake, which 

exacerbates declining ATP concentrations, mitochondrial potential, and reuptake of excitotoxic 

glutamate from the synapse. GCs also compromise the ability of post-synaptic neurons to 

sequester and/or extrude free cytosolic calcium, and to contain and quench oxygen radicals [3]. 

Most of this work has been done in rats; however there is also some evidence that GCs adversely 

affect injured neurons in mice [4-7]. 

We sought to determine whether multiple GC injections exacerbate the neurotoxicity caused by 

kainic acid (KA) in mice. We treated C57Bl/6 male mice with vehicle or 10mg/kg corticosterone 

(the rodent GC) per day for three days prior to KA administration and measured the amount of 

excitotoxic neuron death in the hippocampus. Alternatively, animals were given s.c. pellets of 

varying corticosterone concentrations for the same duration. All treatments yielded circulating 

blood corticosterone concentrations in the physiological mid- to high-stress range. 

By 24 hours post-injury, there was a trend towards an increased number of fluorojade B positive 

neurons in the GC treated animals. This indicated that GC treatments might be increasing 

neurodegeneration. After 72 hours, GC treatment caused a measurable increase in lesions visible

y cresyl violet staining. Both vehicle and GC-treated animals had mild to substantial CA3 

neuron loss; however, GC-treatments dramatically exacerbated CA1 neuron death. These 

findings suggest that chronic stress levels of GCs can worsen excitotoxic injury in C57Bl/6 mice 

just as they can in rats. 

Disclosures: S.F. Sorrells, None; C.D. Munhoz, None; R.M. Sapolsky, None. 

Poster 





Support: ACS fellowship PF-08-086-01-TBE 

Brain Research Foundation Grant 

Title: Tumors induce depression and alter neuroimmune and neuroendocrine systems 

Authors: *L. M. PYTER, V. M. PINEROS, J. A. GALANG, M. K. MCCLINTOCK, B. J. 

PRENDERGAST; 

Psychology, Univ. of Chicago, Chicago, IL 

Abstract: The relationship between peripheral health and mental health is reciprocal. A striking 

example of this interaction is the high incidence of depression and anxiety disorders in cancer 

patients. A number of factors may contribute to this comorbidity, however, the extent to which 

tumor-derived chemical messengers (i.e., cytokines) contribute to the central states of depression 

and anxiety has never been investigated. We tested the hypothesis that tumors produce cytokines 

and dysregulate the HPA axis, which in turn, affect the brain and behavioral measures of 

affective state. Female Wistar rats received an injection of nitrosomethylurea (NMU, chemical 

carcinogen) to induce mammary tumors. Within 2 weeks of tumor palpation, depression and 

anxiety-like behaviors were examined in NMU-treated, tumor-bearing and saline-treated control 

rats. In a separate cohort of similarly treated rats, pro- and anti-inflammatory cytokines were 

measured in blood, tumors, and limbic brain regions. In a second study, the effects of tumors on 

the HPA axis were assessed by measuring corticosterone responses to a stressor and 

dexamethasone, and by quantifying glucocorticoid receptor expression in the brain. Preliminary 

findings suggest that tumors increase depressive-like behaviors and cytokine production and 

increase HPA negative feedback sensitivity. These results suggest that the mechanism by which

cancer increases the prevalence of mood disorders may be due to neuroimmune consequences of 

the tumors themselves. 

Disclosures: L.M. Pyter, None; V.M. Pineros, None; J.A. Galang, None; M.K. McClintock, 

None; B.J. Prendergast, None. 

Poster 





Support: NIH HL088083-02 

Title: Hypothalamic action of interleukin 18 

Authors: S. ALBONI 1,3 , D. CERVIA 1,4 , B. ROSS 1,2 , F. TASCEDDA 3 , M. SANCHEZ- 

ALAVEZ 1,2 , E. ZORRILLA 1,2 , *B. CONTI 1,2 ; 

1 MIND, 2 The Harold L. Dorris Neurolog. Res. Inst., Scripps Resch Inst., La Jolla, CA; 3 Univ. of 

Modena and Reggio Emilia, Modena, Italy; 4 Univ. of Pisa, Pisa, Italy 

Abstract: The pleiotropic inflammatory cytokine interleukin 18 (IL-18) is a central anorexigenic 

agent and a regulator of energy efficiency. In order to understand the sites and the mechanisms 

of action of interleukin 18 in the central nervous system, we characterized members of the IL-18 

family in the mouse brain. IL-18 and the two IL-18 receptor (IL-18R) heterodimer IL-18RI and 

IL-18 RAcP, as well as the soluble IL-18 inhibitor IL-18 Binding Protein (IL-18BP), were 

cloned in the CNS of normal C57B/6 mice. In situ hybridization showed extensive neuronal 

localization of IL-18R in the cortex, the hippocampus, and the hypothalamus. In addition to the 

canonical IL-18RI, a truncated variant of this receptor subunit was identified. In vitro studies on 

hypothalamic cell lines demonstrate that in neurons IL-18 action activates different signaling 

than in cells of the immune system. 

Disclosures: S. Alboni, None; D. Cervia, None; B. Ross, None; F. Tascedda, None; M. 

Sanchez-Alavez, None; E. Zorrilla, None; B. Conti , None. 

Poster





Support: NSF grant 0549987 

HDRF grant 6-008 

Title: Peri-adolescent immune challenge leads to profound changes in social investigation and 

blunted cytokine responses to stress that persist through adulthood 

Authors: *T. DEAK, P. BLANDINO JR, J. EBERLE, K. ARAKAWA, H. ARAKAWA; 

Dept Psychol, Behavi Neurosci Program, SUNY-Binghamton, Binghamton, NY 

Abstract: Adolescence is a critical period for gonadal system maturation and transition to adultlike 

social relationships. In addition, development of the stress response has been linked to 

abnormal social behavior later in life and/or vulnerability to later stress-related pathology. The 

developmental consequences of juvenile endotoxin exposure were assessed in male Sprague- 

Dawley rats. At 27 d of age, males were injected ip with 0, 100, or 1000 µg/kg of 

lipopolysaccharide (LPS) and social interaction was assessed at several times during 

development. As expected, LPS provoked a dose-dependent reduction in social investigation 4 

hour after injection. Subsequent social investigation tests on P30 and P50 demonstrated that male 

rats injected 1000 κg/kg of LPS as juveniles displayed increased social investigation compared to 

saline-injected controls. On P80, rats remained in their homecages as non-stressed controls or 

were exposed to footshock (80 shocks, 1.0 mA, 5 sec each, Variable ITI of 90 sec) and killed 

immediately afterwards. Footshock significantly increased expression of the pro-inflammatory 

cytokine IL-1β in the paraventricular nucleus of the hypothalamus (PVN), replicating our 

previous findings. However, no increase in IL-1β mRNA was observed in rats injected with 1000 

µg/kg as juveniles, suggesting that peri-adolescent immune challenge altered cytokine reactivity 

to stress as adults. Importantly, when rats were injected with 1000κg of LPS as adults (P73) 7 

days prior to the footshock session, footshock increased IL-1 mRNA in the PVN quite normally, 

though basal IL-1 expression remained higher than controls. Together, these findings show that 

peri-adolescent exposure to high doses of LPS produces persistent disruption of social interaction 

and altered stress reactivity into adulthood, suggesting peri-adolescence as a critical period for 

vulnerability to immune challenge which may predispose individuals to stress-related pathology. 

Disclosures: T. Deak , None; P. Blandino Jr, None; J. Eberle, None; K. Arakawa, None; H. 

Arakawa, None.

Poster 





Title: The neurovestibular system and immune function 

Authors: *C. A. FULLER 1 , T. M. HOBAN-HIGGINS 1 , E. S. INGHAM 1 , G. SONNENFELD 2 ; 

1 Dept Neurobiol, Physiol & Beha, Univ. California, Davis, Davis, CA; 2 Dept of Biol. Sci., 

Binghamton Univ, State Univ. of New York, Binghamton, NY 

Abstract: The vestibular system is critical for detecting and communicating information about 

the ambient force environment (i.e., gravity) to the central nervous system. In both humans and 

animals, exposure to spaceflight conditions has been shown to alter immune function, producing 

changes in cytokine production, lymphocyte proliferation and natural killer cell function. These 

immune responses may be caused by multiple factors present during spaceflight, including 

microgravity, radiation and stress. The present study tested the hypothesis that the vestibular 

linear acceleration sensory organs are involved in mediating immune responses, as this system 

modulates other homeostatic, autonomic and circadian control systems. To test this hypothesis, 

this study used the head tilt mouse model (abbr. het), which lacks macular otoconia and therefore 

cannot detect changes in linear/gravitational accelerations. Female and male het mice and agematched 

phenotypically wildtype littermates (n=6/group) were exposed to 2.5 weeks of 

hypergravity (2G) via chronic centrifugation. Control cohorts were maintained at ambient 

Earth‟s gravity (1G). All animals were housed on a 12 h light:12 h dark cycle (LD12:12) with ad 

libitum food and water. At the end of the hypergravity exposure, spleens were immediately 

harvested from all mice. Splenocytes were stimulated with the T-cell mitogen concanavalin A 

(ConA) and, as a measurement of immune competence, production levels of the cytokines IL-2, 

IL-10, IL-5, and GM-CSF were quantified using the Bio-Plex cytokine assay. At 1G, wildtype 

mice generally exhibited higher cytokine production levels than their het mice counterparts. 

Exposure to 2G significantly reduced production of IL-2 in both wildtype female and male mice 

compared to 1G controls (p = 0.012 and 0.030, respectively). 2G-exposed wildtype females also 

showed significant reductions in IL-10, IL-5 and GM-CSF production (p = 0.022, p < 0.001 and 

p = 0.010, respectively) compared to equivalent 1G wildtype controls. Conversely, production 

levels of all four cytokines were not significantly altered in either female or male het mice 

compared to parallel 1G het controls. These results demonstrate a probable role for the macular 

gravity receptors in modulating cell-mediated immune responses, especially in response to 

altered gravity conditions. As in prior studies, gender differences in immunoregulatory responses 

were also identified; female wildtype mice had consistently larger responses to hypergravity 

compared to males.

Disclosures: C.A. Fuller , None; T.M. Hoban-Higgins, None; E.S. Ingham, None; G. 

Sonnenfeld, None. 

Poster 




Topic: E.06.b. HPA axis: Brain systems and behavior 

Title: Dysphoric-like behavior correlates with lack of chronic stress-induced increases in 

glucocorticoids: relevance to atypical depression? 

Authors: N. BOWENS, *L. JACOBSON; 

Dept Pharmacol & Neurosci, Albany Med. Coll, Albany, NY 

Abstract: Evidence has shown a correlation between atypical depression and abnormally low 

hypothalamic-pituitary-adrenal (HPA) axis activity that may explain some symptoms of this 

disorder. To develop better treatments for atypical depression, an animal model is needed to 

identify causes and factors that can reverse this hypoactivity. Chronic social stress has been 

reported to cause HPA hypoactivity in rats. We hypothesized that the resident intruder model 

would produce similar HPA hypoactivity in mice. We placed male mice 5 minutes daily in the 

home cage of another male mouse, whereas the controls were only handled. Morning 

corticosterone levels were divergent enough for us to separate the intruders into two groups with 

corticosterone levels at the control mean (low morning cort: LMC), and two standard deviations 

above the control mean (high morning cort: HMC). Tail suspension immobility, a measure of 

depression-like behavior, was significantly lower in the LMC intruders. Although this measure 

suggested greater resilience, LMC mice showed other evidence of maladaptive behavior and 

physiology, including increased tail suspension-induced defecation, an autonomic response to 

stress, and reduced sucrose preference, a measure of depression-like anhedonia. After social 

defeat, c-fos, a marker of neuronal activity, was significantly lower in the hypothalamus of HMC 

versus control mice. This reduction suggests a habituation to chronic stress, which the LMC mice 

did not show. Adrenocortical 11-beta hydroxylase gene expression was significantly increased in 

HMC versus control mice. Overall the results show that compared to the HMC mice, the LMC 

mice show reduced adrenocortical activity relative to hypothalamic activity. This hypoactivity in 

the LMC mouse may prove useful in elucidating the role of HPA hypoactivity in atypical 

depression. 

Disclosures: N. Bowens, None; L. Jacobson , None.

Poster 




Topic: E.06.b. HPA axis: Brain systems and behavior 

Support: T32MH14654 

PHS Grant MH 58250 

NIMH 067651 

PHS Grant MH 40008 

Title: Individual differences in response to social defeat: Behavior, CRF expression and 

neuroendocrine function 

Authors: *S. K. WOOD, H. WALKER, V. IYER, R. J. VALENTINO, S. BHATNAGAR; 

Stress Neurobio., Children's Hosp Philadelphia, Philadelphia, PA 

Abstract: Chronic exposure to social stress is reported to precipitate psychological disorders in 

susceptible individuals. Individual differences in response to stress may determine vulnerability 

and resilience to the adverse physiologic and psychological consequences of chronic stress. In 

the present studies, we observed that during a 30 minute social defeat (resident-intruder) 

paradigm, Sprague Dawley intruder rats separated into two equal populations that exhibited 

distinct behavioral responses to an aggressive Long Evans resident over the course of 7 daily 

defeats. One population responded with relatively passive defensive behaviors (eg. freezing) and 

exhibited short latencies to assume a defeat posture (SL; ave 197±15 sec). The remaining 

population displayed more confrontational defensive behaviors (eg. boxing, resisting defeat) and 

long defeat latencies (LL; ave 438±12 sec). This dichotomous behavioral response emerged by 

the third consecutive defeat exposure with LL rats becoming more confrontational with repeated 

exposures. To identify a basis for these individual differences and their consequences, CRF 

mRNA, neuroendocrine endpoints and behavior were quantified 24 h after the 7 th defeat. Adrenal 

hypertrophy was negatively correlated with defeat latency. Moreover, when challenged with the 

novel stress of 30min restraint exposure, SL rats exhibited a blunted adrenocorticotropic 

hormone (ACTH) response, whereas LL rats showed the typical facilitated ACTH response. We 

also observed that CRF1 receptor protein levels were decreased in the pituitary of SL rats and 

CRF mRNA in the paraventricular nucleus of the hypothalamus (PVN) was significantly reduced 

in SL, but not LL rats compared to controls. The observed downregulated CRF1 in pituitary and

exaggerated adrenal hypertrophy in SL rats are consistent with an initial CRF hypersecretion and 

dysregulation of the hypothalamic-pituitary-adrenal axis, similar to what has been proposed to 

occur in melancholic depression. Supporting this, SL rats exhibited depressive-like behaviors in 

the Porsolt forced swim test, whereas LL rats were similar to controls. Thus, the alterations in 

behavior, neuroendocrine function, pituitary CRF1, and adrenal hypertrophy reported in SL rats 

are frequently observed in human depression and some other psychiatric disorders. Together, 

these results indicate that individual differences in the phenotypic response to a social stressor 

may predict vulnerability or resiliency to the neuroendocrine and behavioral repercussions of 

social stress. 

Disclosures: S.K. Wood, None; H. Walker, None; V. Iyer, None; R.J. Valentino, None; S. 

Bhatnagar, None. 

Poster 

281. Stress and the Brain: Early Life Experience I 



Topic: E.06.e. Early life experience 



Title: Re-wiring of CRH-expressing neurons in the PVN is involved in the neuroplasticity of the 

HPA axis after early-life handling 

Authors: *A. KOROSI 1 , M. SHANABROUGH 2 , Z.-W. LIU 2,4 , E. BOROK 2 , H. DUENAS 2 , L. 

PHAN 1 , X.-B. GAO 2 , T. HORVATH 2,3 , T. Z. BARAM 1 ; 

1 Pediatrics, Univ. California Irvine, Irvine, CA; 2 Obstetrics, Gynecology & Reproductive Sci., 

3 Neurobio., Yale Sch. of Med., New Haven, CT; 4 Neurobio., Yunyang Med. Col., Hubei, China 

Abstract: Rationale: The „tone‟ (reactivity) of the HPA system contributes to an individual‟s 

stress responses and vulnerability to depression and premature cognitive decline. In rat models, 

early-life experience, including bursts of maternal care, induces enduring neuroplasticity in this 

system, reducing corticotropin releasing factor (CRH) expression in the paraventricular nucleus 

of the hypothalamus (PVN), increasing hippocampal glucocorticoid receptor (GR) expression 

and downregulating hormonal stress responses. Increased maternal care provides sensory signals 

that reduce CRH mRNA expression in PVN of offspring already on postnatal day (P)9, as found 

using the handling paradigm (Fenoglio J Neurosci, 2006). However, the mechanisms by which

augmented maternal care is converted to reduced CRH gene expression in PVN neurons remain 

unclear. Here we tested the hypothesis that modified innervation of CRH-PVN neurons was 

responsible for reduced CRH expression. 

Methods: Pups were handled (15 min separation of pups from the dams) daily from P2-P8, and 

sacrificed on P9. Levels of vGAT (inhibitory terminal marker) and vGlut2 (excitatory terminals 

marker) were quantified using Western blots. Symmetric (inhibitory) and asymmetric 

(excitatory) synapses on CRH-immunoreactive neurons were quantified using electron 

microscopy (EM). Functional alterations of excitatory and inhibitory input onto CRH neurons 

were assessed by electrophysiology. 

Results: vGlut2 protein levels were significantly reduced (by 42%) in anterior hypothalamus of 

handled rats compared to undisturbed cohorts. Numbers of asymmetric synapses on PVN-CRH 

immunolabeled cells, assessed by EM, was reduced by 70% in handled vs undisturbed groups. 

The number of miniature excitatory postsynaptic currents (mEPSCs) onto parvocellular PVN 

cells from handled pups was reduced in preliminary studies. Changes in inhibitory synapses and 

markers (vGAT) were variable. 

Conclusion: The reduction of CRH expression in PVN of rats experiencing bursts of maternal 

care (handled) may derive from reduced excitatory signals to CRH neurons, which may attenuate 

intracellular signaling cascades governing CRH expression in these neurons. Because 

neuroplasticity of CRH and GR expression culminates in the „handled‟ phenotype conferring 

cognitive and emotional resilience, understanding its underlying mechanisms will provide 

valuable insights into this important plasticity. 

Disclosures: A. Korosi, None; M. Shanabrough, None; Z. Liu, None; E. Borok, None; H. 

Duenas, None; L. Phan, None; X. Gao, None; T. Horvath, None; T.Z. Baram, None. 

Poster 






NH73136 

Title: A novel mouse model for acute and long-lasting consequences of early life stress 

Authors: *C. J. RICE 1 , C. A. SANDMAN 2 , Y. CHEN 3 , T. Z. BARAM 1,3 ; 

1 Anat & Neurobiol, 2 Psychiatry & Human Behav, 3 Pediatrics, Univ. California Irvine, Irvine, CA

Abstract: Chronic early life stress (ES) exerts profound acute and long-lasting effects on the 

hypothalamic pituitary adrenal system, with relevance to cognitive function and affective 

disorders. Our ability to determine the molecular mechanisms underlying these effects should 

benefit greatly from appropriate mouse models, because these would enable utilization of 

powerful transgenic methods. Therefore, we have characterized a mouse model of chronic ES, 

that was provoked in mouse pups by abnormal, fragmented, interactions with the dam. Dam-pup 

interaction was disrupted by limiting the nesting and bedding material in the cages, a 

manipulation that affected this parameter in a dose-dependent manner. At the end of their weeklong 

rearing in the limited-nesting cages, mouse pups were stressed, as apparent from elevated 

basal plasma corticosterone levels. In addition, steady-state mRNA levels of corticotropin 

releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) of ESexperiencing 

pups were reduced, without significant change in mRNA levels of arginine 

vasopressin. 

Rearing mouse pups in this stress-provoking cage environment resulted in enduring effects: 

Basal plasma corticosterone levels were still increased and CRH mRNA levels in PVN remained 

reduced in adult ES mice compared with those of controls. In addition, hippocampus-dependent 

learning and memory functions were impaired in 4-8 month old ES mice. In summary, this 

novel, robust model of chronic early life stress in the mouse results in acute and enduring 

neuroendocrine and cognitive abnormalities. This model should facilitate the examination of the 

specific genes and molecules involved in the generation of this stress, as well as in its 

consequences. 

Disclosures: C.J. Rice, None; C.A. Sandman, None; Y. Chen, None; T.Z. Baram, None. 

Poster 






MH73136 

Title: Dysfunctional nurturing behavior in rat dams with limited access to nesting material: a 

clinically relevant model for early-life stress

Authors: *A. S. IVY 1 , K. L. BRUNSON 1 , K. FOK 1 , C. SANDMAN 2 , T. Z. BARAM 1,3 ; 

1 Dept Anat & Neurobiol, 2 Dept Psychiatry and Human Behavior, 3 Dept Pediatrics, Univ. 

California-Irvine, Irvine, CA 

Abstract: Background: Early-life emotional stress may be associated with affective and 

cognitive disorders later in life, yet satisfactory animal models for studying the underlying 

mechanisms are limited. Because maternal presence and behavior critically influence molecular 

and behavioral stress responses in offspring, we sought to create a model of dysfunctional, 

fragmented maternal nurturing behavior that would, in turn, provoke chronic early-life stress in 

the offspring. 

Methods: Dams‟ nursing and nurturing behaviors were altered by limiting their ability to create 

satisfactory nests during postpartum days 2-9. Maternal behavior was observed throughout the 

diurnal cycle, and the frequency and duration of nurturing behaviors were scored. In addition, 

potential stress and anxiety of the dams were assessed using behavioral, molecular and hormonal 

measures. 

Results: Both the quantity and the quality of dams‟ care of their pups were profoundly influenced 

by restriction of nesting materials in their cages: licking/grooming activities decreased and the 

frequency of leaving the pups increased, resulting in fragmented interactions between the dams 

and pups. The abnormal activity patterns of the dams were accompanied by increased anxietylike 

behavior in the open field, but not in the elevated plus maze tests. Additionally, dams‟ 

plasma corticosterone levels and adrenal weights were augmented, suggesting chronic stress of 

these dams. By the end of the limited-nesting, stress-inducing period, hypothalamic corticotropin 

releasing hormone (CRH) mRNA expression was reduced in the limited-nesting dams, while 

arginine-vasopressin (AVP) mRNA levels were not significantly affected. 

Conclusion: Limiting dams‟ ability to construct a nest for their pups leads to an abnormal 

repertoire of nurturing behaviors, possibly as a result of chronic stress and mild anxiety of the 

dams. Because the fragmented and aberrant maternal behavior provoked chronic stress in the 

pups, the limited-nesting paradigm provides a useful tool for studying the mechanisms and 

consequences of such early-life stress experience in the offspring. 

Disclosures: A.S. Ivy, None; K.L. Brunson, None; K. Fok, None; C. Sandman, None; T.Z. 

Baram, None. 

Poster 




Topic: E.06.e. Early life experience

Support: NIH Grant 2 P20 RR015567-07 

Title: Role of early life stress on anxiety-like behavior in adolescence 

Authors: *K. E. TSCHETTER 1 , L. B. CALLAHAN 3 , P. J. RONAN 2,3 ; 

1 Basic Biomed Sci., 2 Neurosci., Univ. South Dakota Sch. Med., Sioux Falls, SD; 3 Brain Res., 

Avera Res. Inst., Sioux Falls, SD 

Abstract: It is well known that stress is a primary factor driving both acquisition and 

maintenance of addictive behaviors. Early life stress is a risk factor for increased vulnerability to 

drug addiction during the particularly vulnerable period of adolescence. Addictive behaviors that 

develop during adolescence often persist into adulthood. Maternal separation (MS) is a widely 

used animal model of early life stress. Very few studies have evaluated the effects of MS on 

brain stress systems during adolescence. In this experiment we sought to evaluate the effects of 

MS on the expression of CRF receptors and anxiety-like behavior during adolescence. Rats 

experiencing maternal separation were removed from the dam once a day, for postnatal days 

(PND) 2-14, with the separation lasting either 15 minutes (MS15) or 180 minutes (MS180). We 

utilized two control groups; animal facility raised (AFR), which received twice weekly cage 

changes with no other separation, and non-handled (NH) which received no cage changes except 

on PND11 and no separation. Beginning on PND15 all groups received twice weekly cage 

changes. Pups were weaned on PND21-22. Rats were then allowed to mature to periadolescence 

(PND40) before testing for anxiety-like behavior on the elevated plus maze (EPM). Females 

exposed to MS spent less time on the open arms when compared to the controls. Males exposed 

to AFR and MS180 conditions showed a decrease in percent time spent on the open arms when 

compared to NH and MS15. There were no significant differences in the total distance moved 

(cm) between any groups. These results demonstrate that early life stress affects anxiety-like 

behavior during adolescence. We also used a microdissection technique coupled with 

quantitative immunoblotting to determine the effects of MS on components of the corticotropin 

releasing factor (CRF) system; one of the key stress-coordinating systems in brain. Females 

exposed to MS180 showed an upregulation of CRF1, CRF2, and CRF-BP in the nucleus 

accumbens (NAc) compared to controls. Females exposed to MS180 also showed an 

upregulation of CRF-BP in the bed nucleus of the stria terminalis (BNST). 

Disclosures: K.E. Tschetter , None; L.B. Callahan, None; P.J. Ronan, None. 

Poster 



Program#/Poster#: 281.5/NN19


Support: Wellcome Trust Research Career Development Fellowship RCDF 068558/Z/02/Z to 

CAL 

PMP 

Neuroendocrinology Charitable Trust Grant PMS/VW-01/02-808 to CAL 

BBSRC Grant BBS/B/06806 to SLL 

Silvio O. Conte Center for the Neuroscience of Mental Disease MH58922 Project 1 to 

NIMH Grants MH50113 to PMP 

Title: Adverse early life experience and adult social defeat interact to increase serotonin 

transporter mRNA expression in the dorsal raphe nucleus 

Authors: *C. A. LOWRY 1,2 , K. L. GARDNER 2 , S. L. LIGHTMAN 2 , P. M. PLOTSKY 3 ; 

1 Dept Integrative Phys, Univ. Colorado, Boulder, CO; 2 Henry Wellcome Labs. for Integrative 

Neurosci. and Endrocrinol., Univ. of Bristol, Bristol, United Kingdom; 3 Dept. of Psychiatry and 

Behavioral Sci., Emory Sch. of Med., Atlanta, GA 

Abstract: Genetic influences, together with adverse experience, either during early life or during 

adulthood, contribute to the vulnerability of individuals to anxiety disorders and depressive 

illness. One mechanism through which these factors may influence vulnerability to stress-related 

neuropsychiatric disorders is through effects on global brainstem monoaminergic systems, 

including brainstem serotonergic systems, which are thought to play an important role in the 

development and pathophysiology of anxiety and depression. In this study we tested the 

hypothesis that early life experience and subsequent stressful experience during adulthood 

interact to regulate serotonin transporter (5-HTT) mRNA expression in the dorsal raphe nucleus 

(DR). To evaluate the effects of early life experience, adverse experiences during adulthood, and 

potential interactions between these factors on 5-HTT mRNA expression in rats, we investigated 

the effects of maternal separation (180 min/day from days 2-14 of life; MS180; a model of 

vulnerability to a depression-like syndrome), neonatal handing (15 min/day from days 2-14 of 

life; MS15; a model of increased resilience to stress), or normal animal facility rearing control 

conditions (AFR) with or without subsequent exposure to adult social defeat on 5-HTT mRNA 

expression in the DR. There were no differences in 5-HTT mRNA expression between MS15 

and AFR rats. Likewise, there were no differences in 5-HTT mRNA expression among MS15, 

AFR, or MS180 rats exposed to a novel cage control condition. In contrast, MS180 rats exposed 

to social defeat as adults had increased 5-HTT mRNA expression compared to both MS15 and 

AFR controls. Social defeat increased 5-HTT mRNA expression, but only in MS180 rats and 

only in the “lateral wings” of the DR. Overall these data demonstrate that adverse early life 

experience and stressful experience during adulthood interact to determine 5-HTT mRNA 

expression. These data are consistent with the hypothesis that genetic influences, early life 

experience, and major stressful life events contribute to dysregulation of serotonergic systems in 

stress-related neuropsychiatric disorders.

Disclosures: C.A. Lowry , None; K.L. Gardner, None; S.L. Lightman, None; P.M. Plotsky, 

None. 

Press Conferences 

PC-1373. Epigenetics: How Does the Environment Influence the Brain's Genetic Program? 

Time: Monday, November 17, 2008, 2:00 pm - 3:00 pm 

Program#/Poster#: Poster Board Number: 



McKnight Brain Research Foundation 

NARSAD 

Title: Early-life adversity and its impact on DNA Methylation patterns in the amygdala 

Authors: *T. ROTH, J. SWEATT; 

Dept Neurobiol, Univ. Alabama Birmingham, Birmingham, AL 

Abstract: Neural mechanisms responsible for the enduring effects of childhood maltreatment on 

mental health remain undefined. On a molecular level, one such mechanism may be aberrant 

programming of DNA methylation, an epigenetic mechanism that represses gene expression. 

Indeed, aberrant DNA methylation continues to be highlighted for its role in the etiology and 

expression of several mental disorders. In this study, we addressed whether early-life adversity in 

the form of caregiver abuse and neglect disrupts DNA methylation in the adolescent and adult 

amygdala. To model abuse and neglect, rat neonates were exposed to a stressed caregiver 30 min 

daily during the first postnatal week. Littermate controls were exposed to either a non-stressed 

caregiver or remained in the home cage. Results indicate that the quality of early postnatal 

experiences profoundly influences DNA methylation patterns and gene expression in the 

developing and adult amygdala. Such alterations may provide a framework for enduring effects 

of early stressors on mental health. 

Disclosures: T. Roth , None; J. Sweatt, None. 

Poster







NARSAD 

Title: Early-life adversity and its impact on DNA Methylation patterns in the amygdala 

Authors: *T. ROTH, J. SWEATT; 

Dept Neurobiol, Univ. Alabama Birmingham, Birmingham, AL 

Abstract: Neural mechanisms responsible for the enduring effects of childhood maltreatment on 

mental health remain undefined. On a molecular level, one such mechanism may be aberrant 

programming of DNA methylation, an epigenetic mechanism that represses gene expression. 

Indeed, aberrant DNA methylation continues to be highlighted for its role in the etiology and 

expression of several mental disorders. In this study, we addressed whether early-life adversity in 

the form of caregiver abuse and neglect disrupts DNA methylation in the adolescent and adult 

amygdala. To model abuse and neglect, rat neonates were exposed to a stressed caregiver 30 min 

daily during the first postnatal week. Littermate controls were exposed to either a non-stressed 

caregiver or remained in the home cage. Results indicate that the quality of early postnatal 

experiences profoundly influences DNA methylation patterns and gene expression in the 

developing and adult amygdala. Such alterations may provide a framework for enduring effects 

of early stressors on mental health. 

Disclosures: T. Roth , None; J. Sweatt, None. 

Poster 





Support: MEXT Grant for Young Scientists 

JSPS Grant for Scientific Research 

Title: Role of REST/NRSF-mediated gene regulations in the risk of developing depression-like 

behavior 

Authors: *S. UCHIDA, K. HARA, A. KOBAYASHI, Y. WATANABE; 

Dept Neurosci, Yamaguchi Univ. Sch. Med., Ube, Yamaguchi, Japan 

Abstract: Early life stress in human enhances the risk of developing psychiatric disorders, 

including depression. In rodents, maternal separation is a potent early life stressor including 

long-lasting changes in neuroendocrine and molecular responsiveness to stress, associated with 

depression- and anxiety-like symptoms. However, little is known about the involvement of early 

life events in the susceptibility to subsequent repeated stress exposure during adulthood in 

rodents. The present study characterized the effects of maternal separation on the neuroendocrine 

and behavioral responses to repeated restraint stress in the adult rats. Rat pups were separated 

from the dams for 15 (HMS15) or 180 min (HMS180) per day from postnatal day 1 through 14, 

and they were repeatedly restrained for 14-days during adulthood. We found that non-restrained 

HMS180 rats showed the increased HPA axis response to stress and the increased anxiety 

behavior in the novelty-suppressed feeding test, compared to HMS15 and non-handling (NH) 

rats. However, depression-like behaviors in non-restrained HMS180 rats were comparable to that 

observed in HMS15 and NH rats. Importantly, repeatedly restrained HMS180 rats showed the 

increased depression-like behavior and anhedonic response, when compared to the nonrestrained 

HMS180, restrained HMS15 and restrained NH rats. Further, we found the aberrant 

expression of transcription factor REST/NRSF protein and a variety of its target genes (eg. CRH, 

alphaCaMKII, microRNAs) in the medial prefrontal cortex of HMS180 rats. Thus, the aberrant 

REST/NRSF-mediated gene regulations in the HMS180 rats might be involved in the 

vulnerability to stressful situations. 

Disclosures: S. Uchida, None; K. Hara, None; A. Kobayashi, None; Y. Watanabe, None. 

Poster 





Support: FILAS regione Lazio, Italy

Telethon Grant GGP05220, Italy 

Title: Maternal helper at the nest improves cognitive performance in the offspring of NMRI 

mice 

Authors: A. MOLES 1 , C. ZANETTINI 1 , C. SGOBIO 2 , M. AMMASSARI-TEULE 1,2 , *F. R. 

D'AMATO 1 ; 

1 Inst. of Neurosci., CNR, Roma, Italy; 2 Santa Lucia Fndn., Roma, Italy 

Abstract: The role of maternal care in shaping individual emotionality, responsiveness to stress, 

and cognition has been extensively demonstrated in rats. Specifically,the offspring of high 

licking/grooming mothers shows lower emotionality and a faster glucocorticoids feedback after 

stress exposure. To explore the role of maternal care on offspring emotionality and cognition in 

the mouse, we reared pups either with one female (the mother), or with two females: the mother 

plus an additional lactating female (L+L), and the mother plus a cycling female (L+C). In the 

L+L condition, the offspring of the first delivering female was removed so that female pairs took 

care of the second female offspring. All litters were cut to eight pups and only males were 

retained for physiological, behavioral and neuromorphological measurements. During 

development, the amount of maternal care pups received and their body weight gain were 

measured. At adulthood, motor activity (open field), emotionality (plus maze), corticosterone 

response, and cognitive performance (reactivity to spatial or to object change, active avoidance, 

contextual fear conditioning, long term object recognition) were assessed. Finally, spine density 

and dendrite branching were measured on neurons laying in the CA1 region of the hippocampus. 

Our results show that mice reared by two lactating females showed highest body weight from 

postnatal day 7 till adulthood. These mice also received more arched-back posture by the two 

lactating females and, as mice reared in the L+C condition, a greater amount of licking and 

grooming compared to control mice. Surprisingly, no effect of the rearing condition was found 

on emotional behavior (plus maze) or on corticosterone levels measured in baseline conditions or 

in response to stress. In the same fashion, motor activity, active avoidance, habituation of object 

exploration and short term reactivity to object change were unaffected by the rearing condition. 

However, mice reared by two females (L+L and L+NL) exhibited stronger contextual fear 

conditioning than mice reared by one female, with L+L mice showing higher scores than L+C 

mice. In addition, only L+L mice were capable to recognize an object 24 hours after they were 

exposed to. In agreement with the behavioral data, spine density values and dendrite branching in 

the basal compartment of CA1 neurons differed among groups with L+L mice showing the 

highest values. Altogether our findings indicate that coupling increased nutrition to increased 

maternal cares results in a consistent improvement of cognition and hippocampal neuronal 

connectivity in the mouse offspring but produces few effects on emotionality. 

Disclosures: A. Moles, None; C. Zanettini, None; M. Ammassari-Teule, None; F.R. 

D'Amato , None; C. Sgobio, None. 

Poster





Title: Early trauma induces heritable maladaptive behaviours via epigenetic mechanisms 

Authors: *T. B. FRANKLIN, H. RUSSIG, J. GRÄFF, N. LINDER, S. VIZI, I. M. MANSUY; 

Brain Res. Inst., Univ. of Zurich/ETH, Zurich, Switzerland 

Abstract: Chronic exposure to physical or mental stress early in development is a contributing 

factor to the onset of behavioural and emotional disorders in adulthood. Not only are these 

disorders persistent throughout life, but they can also be transmitted to the subsequent 

generation. To investigate potential mechanisms of occurrence and transmission of stress-related 

affective disorders, we modeled early trauma in the mouse using chronic and unpredictable 

maternal separation, and examined its impact on adult behaviour in consecutive generations. 

Mice subjected to maternal separation as pups behaved abnormally when exposed to novel or 

stressful conditions, and displayed depressive-like behaviours as adults. Remarkably, the 

offspring of these animals exhibited similar impairments despite being reared in normal 

conditions, suggesting that behavioural abnormalities induced by early trauma can be transmitted 

across generations. Although the mechanisms of this behavioural transmission are not fully 

understood, initial evidence indicates that they involve epigenetic alterations in the germline. 

This evidence uniquely points to the involvement of transmissible modifications of the 

epigenome in the epidemiology of stress-related affective disorders. 

Disclosures: T.B. Franklin, None; H. Russig, None; J. Gräff, None; N. Linder, None; S. Vizi, 

None; I.M. Mansuy, None. 

Poster 





Support: NIMH 5R01MH073175

Title: Early-life stress is associated with a neural bias towards emotionality 

Authors: *N. L. TOTTENHAM 1 , T. A. HARE 2 , A. MILLNER 1 , T. GILHOOLY 1 , B. CASEY 1 ; 

1 Sackler Inst, Cornell Med. Sch., New York, NY; 2 Caltech, Pasadena, CA 

Abstract: Early-life stress is associated with a neural bias towards emotionality 

Adverse rearing environments can produce long-lasting change in responsivity to arousing 

stimuli and the ability to regulate emotion. Children who had been institutionally reared during 

infancy (n=19) were scanned using fMRI while engaged in an emotional go-nogo task (Hare et 

al., 2005, 2008), which measures cognitive control in the context of highly arousing stimuli. 

There were no group differences in accuracy on the task (F(1,35) = 0.45) relative to an agematched 

sample (N=18). However, there were differences in the neural systems that the two 

groups recruited to perform the task. Unlike typical rearing, which resulted in a recruitment of a 

perceptuo-cognitive network, including fusiform gyrus and inferior frontal gyrus, early life 

adversity was associated with a greater tendency to engage an emotional network, including 

amygdala and ventral medial prefrontal cortex. Thus, although behavioral performance did not 

differ between groups, the neural circuit recruited by the previously institutionalized children 

tended towards an emotional one. These findings extend our earlier work showing an association 

between early life stress and atypically large amygdala volume and suggest that stressful 

experiences in infancy increase the bias towards recruitment of emotional systems. 

Disclosures: N.L. Tottenham , None; T.A. Hare, None; A. Millner, None; T. Gilhooly, 

None; B. Casey, None. 

Poster 





Support: PEDECIBA 

PDT 

SNB-ANII 

Title: Prolonged exposure to a stressful situation elicits precocial expression of fear responses in 

female and male rat pups

Authors: *M. J. ZULUAGA, D. AGRATI, A. FERREIRA; 

Seccion Fisiologia y Nutricion, Facultad de Ciencias, UdelaR, Montevideo, Uruguay 

Abstract: In rat pups, fear responses to a predator odor, like an adult male, do not emerge until 

postnatal day 14, when the stress hypo-responsive period ends. Corticosterone administration to 

male pups accelerates the expression of fear responses. As the emotional response of adults is 

sexually dimorphic, it could be speculated that stressful events early in life affect the emotional 

reactivity in a sex dependent manner. To test this hypothesis, lactating dams together with their 

litters were exposed for eight hours to an unfamiliar or a familiar male (that copulated and 

cohabitated with the mother until parturition) or an unfamiliar diestrous or proestrous female 

inside a cage, or an empty cage, during the first four postpartum days. To determine the 

influence of the mother‟s behavior on the litters‟ responses, the maternal behavior and aggression 

toward the stressor were assessed. After the stress exposure period, fear responses of 8 days-old 

male and female pups toward an anesthetized male were recorded. Male pups showed fear 

responses when confronted to an anesthetized male reflected as a significant reduction in the 

number of ultrasonic vocalizations and an increased in immobility, only if they were previously 

exposed to an unfamiliar male. Conversely, female pups showed fear responses toward an 

anesthetized male if they were previously exposed to unfamiliar or familiar males or to 

proestrous females. Present study shows that stressful experiences during the early postnatal 

period accelerate the appearance of fear responses toward a potential predator in 8 days-old pups 

and that this effect is sex dependent. The higher susceptibility of female pups to stressful 

situations is in line with previous studies showing that physiological and behavioral 

consequences of stress are greater in females than in males. Thus, it has been reported that during 

perinatal period, testosterone is critical for the maturation and programming of stress response. It 

could be speculated that this hormone might be protecting the male pups from the influences of 

stressful situations on the development of precocial fear responses. A direct effect of the 

mother‟s behavior during the stressful period might account for the pups‟ fear response, as 

mothers attacked more the unfamiliar males and the proestrous females and spent more time 

away from the nest. However, the frequency off-nest seems not to be an important factor on the 

acceleration of pups‟ fear responses, as the exposure to diestrous females did not hasten pups‟ 

fear expression. Future experiments will determine whether the adult‟s presence is affecting 

directly the pup or indirectly by altering the behavior of the mother. 

Disclosures: M.J. Zuluaga, None; D. Agrati, None; A. Ferreira, None. 

Poster 



Program#/Poster#: 281.12/NN26


Support: DK 064086 

UPenn-SOM 

Title: Epigenetic modulation of dopamine gene expression by prenatal and postnatal diet 

Authors: *Z. VUCETIC 1 , K. TOTOKI 2 , T. M. REYES 2 ; 

1 Univ. of Pennsylvania, Philadlephia, PA; 2 Univ. of Pennsylvania, Philadelphia, PA 

Abstract: A growing body of research, both in humans and in animal models, is linking prenatal 

and postnatal environmental factors including chemical agents, diet, and lifestyle with an 

increased risk of developing cancer, diabetes, obesity and behavioral disorders. The molecular 

mechanism underlying the onset of disease following environmental insult may involve 

epigenetic dysregulation of key genes involved in disease pathogenesis. Epigenetic changes (e.g., 

DNA methylation and chromatin modifications) can induce persistent alteration of gene 

expression as a response to environmental insults. We aimed to explore if either prenatal or 

postnatal nutritional manipulations have the potential to induce permanent molecular changes 

involving epigenetic regulation of gene expression and result in altered behavior. We studied 

nutritionally challenged mice; either during prenatal period (low protein (LP) diet to pregnant 

dams) or in adult life (high fat (HF) diet fed from weaning). Mice exposed to either gestational 

low protein diet or long-term postnatal high fat diet have significantly lowered preference for 

natural reward, sucrose, (n=5, p< 0.02) compared to controls. Additionally, both prenatal LP and 

postnatal HF diet led to increased expression of genes involved in the synthesis and transmission 

of dopamine signal in the hypothalamus (TH, DARPP-32, DAT). Both prenatal and postnatal 

dietary alterations resulted in 20% lower content of cytosine-methylated DNA in the 

hypothalamus compared to animals fed standard chow. Global hypomethylation of hypothalamic 

genomic DNA was accompanied with 2-fold up-regulation of nucleic acid demethylating 

enzyme, Fto in the case of LP animals and 35% reduction in mRNA levels of MeCP2 and Dnmt1 

in postnatal HF group. Additionally, methylated DNA immunoprecipitation (MeDIP) assay 

showed that prenatal and postnatal dietary alterations affected promoter DNA methylation of 

distinct gene targets. Animals on postnatal HF diet showed decreased enrichment of methylated 

DNA in the proximal promoter region of Th and DAT compared to controls and prenatal LP. In 

contrast, animals exposed to prenatal LP diet had reduced levels of Cdkn1c promoter 

methylation and increased expression of a subset of Cdkn1c-Igf2 locus imprinted genes (Cdkn1c, 

Tssc4, Igf2) important for dopamine neuron differentiation. Taken together data suggest that 

prenatal and postnatal dietary alterations have the ability to modulate expression of dopamine 

related genes through diverse epigenetic mechanisms. 

Disclosures: Z. Vucetic, None; K. Totoki, None; T.M. Reyes, None. 

Poster






UPenn-SOM 

Title: Alterations in dopamine-related behaviors in a mouse model of intrauterine growth 

retardation 

Authors: K. TOTOKI 1 , H. SCHOCH 2 , Z. VUCETIC 1 , *T. M. REYES 3 ; 

1 Pharmacol., 2 Cell and Mol. Biol., Univ. of Pennsylvania, Sch. of Med., Philadelphia, PA; 

3 Pharmacol., Univ. Pennsylvania, Philadelphia, PA 

Abstract: Intrauterine growth retardation (IUGR) affects approximately 10% of all US infants. 

These small-for-gestational age (SGA) babies face not only an increased risk for immediate 

morbidity and mortality, but also an increased risk for neurobehavioral disabilities (e.g., attention 

deficit hyperactivity disorder (ADHD), schizophrenia spectrum disorders, depression and 

addiction). The causes for IUGR are numerous and common. Risk factors include multiple order 

births, maternal malnutrition, smoking or drug use, and, common conditions that can lead to a 

decrease in nutrient flow to the fetus, including maternal hypertension, hyperemesis gravidum, 

and uterine or placental dysfunction. Using a well-characterized rodent IUGR model (low 

protein diet in pregnant mice), we have found alterations in preference for sucrose, hyperactivity, 

and a differential response to cocaine in our IUGR offspring; behaviors which are components of 

ADHD and addiction. Alterations in dopamine (DA) signaling affect these behavioral responses 

(reward processing and locomotor behavior) in both animal models of and human patients with 

ADHD and addiction disorders. Our IUGR offspring have altered expression of genes that 

control dopamine synthesis and activity, as well as altered levels of tyrosine hydroxylase (TH) 

producing neurons in the ventral tegmental area (VTA). Using Fos-mapping techniques in the 

nucleus accumbens, the IUGR animals also show a differential Fos response to cocaine injection. 

These data suggest that dopaminergic function is altered as a result of the low protein diet and 

causes the observed neurobehavioral changes. 

Disclosures: K. Totoki, None; Z. Vucetic, None; T.M. Reyes , None; H. Schoch, None. 

Poster 

281. Stress and the Brain: Early Life Experience I




Title: Early neonatal persistent inflammation affects adult pain reactivity and anxiety related 

traits: genetic background counts 

Authors: C. BENATTI 1 , S. ALBONI 1 , F. CAGGIA 1 , N. BRUNELLO 1 , F. TASCEDDA 1 , *J. 

M. BLOM 2 ; 

1 Dept Biomed. Sci., 2 Dept Ped, Univ. Modena & Reggio Emilia, Modena, Italy 

Abstract: Rodents represent a valuable resource to enhance our understanding concerning the 

long term consequences of neonatal pain. However, each rodent strain is characterized by its 

unique pattern of behavior and its genetic inheritance influences behavioral traits. In this study 

two different mouse strains were used, an outbred strain (CD1) and a F1 hybrid strain (B6C3F1). 

Their intra-strain differences were characterized by measuring anxious behavior and thermal 

sensitivity to a radiant heat source. B6C3F1 animals showed a more anxious phenotype with 

respect to CD1 mice. Objectives: 1) to study whether timing of postnatal exposure to persistent 

inflammation alters responsiveness to thermal pain in the adult animal; 2) to assess whether 

animals experiencing early postnatal chronic inflammation display alterations in anxiety related 

behavior; 3) to study the importance of genetic background. Newborn male mice CD1 or 

B6C3F1 received a single injection of CFA or saline on either postnatal day 1 or 14 (PND1 and 

PND14) into the left hind paw. A 5 minute session on an elevated plus maze (EPM) was 

performed at 11 weeks of age. At twelve weeks of age paw withdrawal latency (PWL) of each 

animal was tested both in basal condition and 24h after an unilateral injection of 100 κL of CFA 

in the left hind paw. In short: 1) CD1 outbred mice exposed to a robust long lasting local 

inflammatory insult early during development, grow into adults with an increase responsiveness 

to thermal pain Adult animals display low anxiety traits only after receiving a CFA injection on 

PND1 and not on PND 14; 2) B6C3F1 hybrid adult mice exposed to a robust long lasting local 

inflammatory insult on PND14 are characterized by hyperalgesia, while a treatment with CFA on 

PND1 fails to affect adult reactivity to thermal nociception. Moreover, adult animals display low 

anxiety traits only if they received a neonatal persistent inflammation on PND1 and not on 

PND14. 

Our findings indicate that neonatal persistent inflammation has a differential effect on adult 

behavioral responsiveness in outbred mice (CD1) and hybrid mice (B6C3F1). These results 

suggest a role for genetic background in modulating long term consequences of neonatal 

persistent inflammation. 

Disclosures: C. Benatti, None; S. Alboni, None; F. Caggia, None; N. Brunello, None; F. 

Tascedda, None; J.M. Blom , None.

Poster 






Title: Conditioned place aversion and 22-kHz distress vocalizations in socially subjugated male 

rats 

Authors: R. J. BROWN, A. R. LUMIA, R. L. CUNNINGHAM, K. A. FRAHM, *M. Y. 

MCGINNIS; 

Dept Pharmacol, UTHSCSA, San Antonio, TX 

Abstract: The early experience of social subjugation (SS) results in negative behavioral 

consequences in adulthood. In the present study, we wanted to determine if exposure to cues 

previously associated with SS elicits a behavioral response in subjugated rats. The importance of 

odor, the presence of a subjugator, and the home cage environment were examined. Specifically, 

we hypothesized that odors and environmental cues associated with SS would induce a 

conditioned place aversion and elicit 22-kHz ultrasonic distress vocalizations. Gonadally intact, 

Long-Evans male rats were divided into two groups: SS and controls. SS involved exposure to an 

aggressive, adult male for 10 min every other day over an 18-day period. On postnatal day 38 

(P38), SS rats underwent subjugation in the presence of lemon odor. Controls were placed in a 

novel cage with lemon odor. On alternate days both groups were exposed to a novel cage with 

almond odor. Beginning on P59, behavioral tests were performed in two different settings: a 

conditioned place preference (CPP) apparatus and the home cage (HC) of a subjugating male. 

CPP tests were used to determine if SS rats developed a conditioned place aversion to either the 

odor or to the presence of the subjugator. The first test was performed in a 3-chambered CPP 

apparatus with lemon odor in one chamber, almond odor in the other, and no odor in the middle 

chamber. The amount of time spent in each chamber was recorded. The second CPP test was 

performed with a subjugating male placed behind a partition in one side chamber while the other 

two chambers were empty. HC tests were used to examine the importance of the subjugation 

environment. These tests involved exposure to the HC alone, the HC with the SS-paired odor, 

and the HC with the subjugator placed behind a partition. 22-kHz vocalizations were recorded 

using a bat detector. In the CPP tests, SS rats spent less time with lemon and more time with 

almond compared to controls, though this was not significant. SS animals spent significantly less 

time in the chamber containing the subjugator than in the other two chambers combined, while 

controls did not. In the HC tests, SS rats only vocalized when the subjugator was present. 

Controls did not emit 22-kHz vocalizations in any HC test condition. The results from CPP and 

HC tests suggest that the physical presence of a subjugating male is the most salient cue. 

Although the presentation of an SS-paired odor may be sufficient to induce a conditioned place

aversion, further research is needed to confirm this effect. This study demonstrates that cues 

associated with the early experience of social subjugation elicit aversive and distressful 

behavioral responses that continue into adulthood. 

Disclosures: R.J. Brown, None; A.R. Lumia, None; R.L. Cunningham, None; K.A. Frahm, 

None; M.Y. McGinnis , None. 

Poster 





Support: NCRR Grant RR11122 (MAN) 

The Division of Intramural Research, NICHD, NIH (SJS) 

Title: Maternal environment and rh5-HTTLPR genotype differentially affect long-term infant 

rhesus macaque (Macaca mulatta) HPA activity across the first two years of life 

Authors: *A. M. DETTMER 1 , M. A. NOVAK 2 , S. J. SUOMI 3 , J. S. MEYER 2 ; 

1 Neurosci & Behavior, 2 Psychology, Univ. Massachusetts, Amherst, MA; 3 Lab. of Comparative 

Ethology, NIH, The Eunice Kennedy Shriver Natl. Inst. of Child Hlth. & Human Develop., 

Poolesville, MD 

Abstract: The serotonergic system is known to regulate the hypothalamic-pituitary-adrenal 

(HPA) axis, and strong heritability between mother/infant serotonergic and mother/infant HPA 

activity has been well-documented. However, little is known about maternal effects of serotonin 

functioning on infant HPA activity. We studied the effects of maternal environment and 

serotonin transporter length promoter polymorphism (rh5-HTTLPR) genotype on infant rhesus 

macaque (Macaca mulatta) long-term HPA activity across the first two years of life. Dams were 

genotyped for the long (l/l) and short (l/s) allele of the 5HTTLPR. Fifty-eight infant macaques 

were reared either with their mothers and peers (MPR; n=22; l/l=12, l/s=10), with peers only 

(PR; n=17; l/l=13, l/s=4), or with inanimate surrogates and given daily play sessions (SPR; n=19; 

l/l=12, l/s=7). Hair samples were obtained from these infants at six months of age and analyzed 

via enzyme immunoassay for hair cortisol concentrations, a newly established method for 

analyzing long-term HPA activity. A subset of these infants (n=24; MPR=9, l/l=7, l/s=2; PR=7, 

l/l=6, l/s=1; SPR=8, l/l=5, l/s=3) were studied every six months through 18 months of age. 

ANOVA was employed for all statistical analyses. Results show a significant effect (p=0.02) of

maternal rh5-HTTLPR on infant hair cortisol concentrations at month 6, with infants of l/l 

mothers exhibiting higher cortisol values than those of l/s mothers. No effect of maternal 

environment was found at month 6. The picture was reversed, however, at months 12 and 18: at 

each of these time points there was no effect of maternal rh5-HTTLPR on infant hair cortisol, but 

significant rearing effects did emerge for the subset of infants studied (month 12: p=0.02; month 

18: p=0.01). At month 12, hair cortisol concentrations were lowest in PR and highest in SPR 

infants; at month 18 hair cortisol concentrations were lowest in MPR and highest in SPR infants. 

These results suggest that maternal genetics may strongly influence infant HPA activity early in 

life, but that this effect is not long-lasting and that an infant‟s early life experience exerts 

stronger influences on its long-term HPA activity later in infancy and into adolescence. 

Disclosures: A.M. Dettmer, None; M.A. Novak, None; S.J. Suomi, None; J.S. Meyer, None. 

Poster 





Title: Harmful effects of close exposure of growing Wistar rats to telephone mast base station 

Authors: *P. U. NWOHA, I. O. ALATISE, A. I. ACHUDUME; 

Anat. & Cell Biol., Obafemi Awolowo Univ., Ile-Ife, Nigeria 

Abstract: The study assessed the harmful effects of close exposure of young growing Wistar rats 

to telephone mast base stations. Thirteen rats, 7 males and 6 females, weaned from their mothers 

at day 25 were utilized for this work. Seven (4 males and 3 females) rats form the experimental 

group, while 6 (2 males and 4 females) were the control. Animals in the experimental group were 

housed in a cage and kept in a hut located 29.6 meters from two telephone mast base stations. 

Control animals were similarly housed in a hut 5km away from a base station. Animals in both 

groups were fed mouse chow and had free access to tap water. The animals were monitored in 

there cages for behaviour and skin changes. Body weights were taken intermittently. On day 70, 

behavioural studies were done using Y-maze, light/dark box, and open field. The animals were 

also tested for reproductive performance by introducing freshly acquired fertile females into 

male cages for 3 days. All animals were subsequently sacrificed and biochemical studies 

conducted. Results showed heavy pigmentation of the skin of all male and one female rats in the 

experimental group. None of the control animals was pigmented. All females mated by control 

males became pregnant and delivered live litters, while none of those mated by experimental 

males was pregnant. On the Y maze, the experimental rats committed significantly higher errors

than the controls (p

measured by AUC-I, when controlling for prenatal cocaine exposure, group size, smoking, age, 

hours of sleep, and how stimulating the home environment was at age 4. Females were 

significantly less reactive to the stressor, as measured by AUC-I. The effects of parental 

nurturance and gender were independent of the peak anxiety level reported throughout the 

protocol as well as stressor appraisal. Parental nurturance and gender were not related to total 

cortisol exposure, as measured by AUC-G. Hours of sleep the night before testing was negatively 

related to AUC-G and prenatal cocaine exposure was positively related to AUC-G. Higher levels 

of parental nurturance within the range of normal variation at age 4 thus predict an increased 

cortisol response to social stress in adolescence, independent of the effects of a stimulating home 

environment, in a low-income, African-American sample. These results suggest that parental 

nurturance has a long-lasting effect on the HPA axis and is a promising candidate as a 

mechanism underlying the relationship between early adverse experience and cognitive and 

socioemotional outcomes. 

Disclosures: D.A. Hackman, None; M.J. Farah, None; L. Kobrin, None; L. Betancourt, 

None; H. Hurt, None. 

Poster 





Support: SFB 779 

Title: Prenatal stress affects dendritic and synaptic development in the hippocampus and 

amygdala in a sex-specific manner and can partly be reversed by intensifying postnatal maternal 

care 

Authors: *A. BRAUN 1 , M. S. MURMU 1 , M. WEINSTOCK-ROSIN 2 , J. BOCK 1 ; 

1 Zoolog/Developmental Neurobiol, Inst. Biol, Otto von Guericke U, Magdeburg, Germany; 

2 Dept. Pharmacol., Hebrew Univ. Med. Ctr., Jerusalem, Israel 

Abstract: It has become increasingly evident that deleterious and stressful life events 

experienced by a mother during pregnancy adversely affects the development of the offspring 

and constitutes a risk factor for developing psychopathology. The effects of prenatal stress (PS) 

on dendritic morphology was analyzed in neurons of the hippocampal CA1, CA3 regions and the 

dentate gyrus and the amygdala of rats. At the age of 21 days apical dendritic length of 

pyramidal neurons in the CA3 region was reduced in PS males (-27%) and females (-22%)

compared to unstressed controls. Similarly, shorter basal dendritic length was found in PS males 

(-19%) and females (-27%). The stress-induced change of apical dendritic length was 

“normalized” in PS animals, which were handled (intensifying maternal behaviour) during the 

first 9 postnatal days. The density of dendritic spines in CA3 was only reduced (-17%) on the 

basal dendrites of PS females. In the CA1 region, PS males showed shorter apical dendrites (- 

14%), whereas in PS females both apical (-26%) as well as basal (-25%) dendrites were shorter 

compared to unstressed controls. Postnatal handling normalized these changes only in PS males 

but not in the PS females. Sex differences were also observed for spine density in CA1, where 

PS males, unlike their sisters, displayed elevated spine density in the apical (+39%) and basal 

(+12%) dendrites. Sexually-dimorphic effects of PS was also found for spine density and 

dendritic length in dentate granule cells. In the inner blade of the dentate gyrus, PS females 

showed shorter dendrites (-19%) and reduced spine density (-25%), whereas their PS brothers 

displayed longer dendrites (+30%) and elevated spine-density (+28%). Only in PS males 

handling normalized stress-induced changes of spines. Stress-induced elongation (males) and 

shrinkage (females) of dendrites was amplified by postnatal handling. In the basolateral 

amygdala (BLA) shorter apical dendrites of large pyramidal neurons were observed only in PS 

males (-27%), handling reversed these changes back to normal levels. Spine density was not 

affected in PS animals. 

These findings demonstrate that prenatal stress exposure significantly interferes with dendritic 

and synaptic development in a highly sex-specific manner, and that these changes can partly be 

“normalized” in a gender-specific manner by increasing postnatal maternal care. Furthermore, 

our data indicate that the magnitude and direction in which pre- and postnatal emotional 

experience affects neuronal development is highly sex-specific. 

Disclosures: A. Braun , None; J. Bock, None; M.S. Murmu, None; M. Weinstock-Rosin, 

None. 

Poster 





Support: NIAAA Intramural Program 

NARSAD 

Title: A functional snp in the rhesus crh promoter increases stress reactivity and alcohol 

consumption as a function of early adversity

Authors: *C. BARR 1 , R. DVOSKIN 2 , M. GUPTE 3 , S. G. LINDELL 3 , M. L. SCHWANDT 3 , D. 

HIGLEY 5 , S. J. SUOMI 4 , D. GOLDMAN 2 , M. HEILIG 2 ; 

1 NIAAA, NIH, Poolesville, MD; 2 NIAAA, NIH, Bethesda, MD; 3 NIAAA-DICBER, LCTS-Sect 

Primate Models, NIH, Poolesville, MD; 4 NICHD, NIH, Bethesda, MD; 5 Brigham Young Univ., 

Provo, UT 

Abstract: The Corticotropin Releasing Hormone (CRH) system is critical to stress responding 

and survival, but chronic overactivity of the system can lead to stress-related pathology. We 

wanted to determine whether a SNP in a functionally important region of the rhesus macaque 

CRH gene promoter (-248 C>T) was functional and whether it influenced behavioral and 

endocrine responses to stress. Because increased CRH system activity drives alcohol drinking in 

rodents, we also wanted to examine whether this variant influenced voluntary alcohol 

consumption. We performed electrophoretic mobility shift and in vitro reporter assays to 

determine functional consequences of the CRH -248 T allele. Rhesus macaques (Macaca 

mulatta) were reared in natural conditions (mother reared, MR) or in a neonatal nursery (PR), a 

model for early adversity. At 6 months of age, they (N=155) were subjected to social separation 

stress. Behavior was scored and adrenocorticotropin (ACTH) and cortisol levels were determined 

at baseline and during stress. At approximately 4 years of age, macaques were given 

simultaneous access to an aspartame-sweetened 8.4% v/v ethanol solution and vehicle for 1 

h/day, 5 days/week. Animals were genotyped for CRH -248 C/T using a 5‟-Exonuclease assay. 

ANOVA was performed to determine whether the T allele influenced stress responding and 

voluntary alcohol consumption as a function of early adversity. CRH -248 C>T resulted in 

altered DNA protein interactions and disrupted glucocorticoid-mediated prevention of forskolinstimulated 

CRH promoter activity in vitro. Consistent with this mechanism, we found that 

endocrine and behavioral responses to stress were higher among carriers of the T allele, 

particularly among those exposed to early adversity in the form of peer rearing. As adults, peer 

reared macaques that were carriers of the T allele also consumed more alcohol. Like rhesus, 

humans exhibit variation in the proximal promoter of the CRH gene. It may be that human CRH 

promoter variation could increase risk for stress-related disorders, including alcohol dependence. 

Disclosures: C. Barr, None; R. Dvoskin, None; S.G. Lindell, None; M.L. Schwandt, 

None; D. Higley, None; M. Gupte, None; M. Heilig, None; D. Goldman, None; S.J. Suomi, 

None. 

Poster 



Program#/Poster#: 281.21/OO1 


Title: Maternal deprivation affects hippocampal neurogenesis in a sex-dependent manner 

Authors: *C. OOMEN, C. E. NEVES GIRARDI, E. C. VERBEEK, R. CAHYADI, H. 

KRUGERS, M. JOËLS, P. J. LUCASSEN; 

SILS-Center for neuroscience, Univ. Amsterdam, Amsterdam, Netherlands 

Abstract: Hippocampal structural and functional plasticity are highly susceptible to stress and 

stress hormones. A striking example of structural plasticity in the hippocampus is adult 

neurogenesis. Unlike other brain areas, the dentate gyrus of the hippocampus continues to 

generate new neurons into adulthood. Adult neurogenesis is influenced by various environmental 

factors and is inhibited by acute and chronic stress and stress hormones. This could potentially 

affect hippocampal functioning. 

The impact of stress is generally more pronounced when applied early in life. There is evidence 

that several properties of the hypothalamic-pituitary-adrenal axis can be life-long altered due to 

early life experiences. In rat pups, maternal behaviour ensures a “stress-hyporesponsive” period 

between postnatal day (PND) 3 and 14. This period coincides with postnatal development of the 

hippocampal dentate gyrus and with maximal levels of neurogenesis. We hypothesized that 

maternal deprivation at the beginning of this period will influence neurogenesis and alters the 

structural make up of the adult hippocampus and that this could potentially influence network 

properties later in life. 

To investigate this, we subjected rat pups to 24 hours of maternal deprivation at PND 3 and, at 

that time, labelled dividing cells by injecting BrdU (bromodeoxyuridine). Changes in maternal 

care were monitored during the experimental procedure. At PND4 we studied the acute effect of 

maternal deprivation on proliferation and at PND21 we analysed the different phases of 

neurogenesis by quantifying cell-survival, proliferation, neuronal differentiation and the number 

of astrocytes. 

We found no differences between groups in cell survival or proliferation rate at PND21. Analysis 

of the population of immature neurons (doublecortin-positive cells) revealed an increase in 

males, but a decrease in females due to maternal deprivation. However, the volume of granular 

cell layer was unchanged due to experimental procedure. Maternal care was increased after the 

deprivation day, but this increase was similar for male and female pups. We are presently 

studying whether the effect on immature neurons is transient or persisting into adulthood and 

what the functional consequences are for long-term potentiation in the dentate gyrus. 

We conclude that maternal deprivation induces sex-dependent differences in adult neurogenesis, 

without affecting newborn cell survival or volume of the granular cell layer. This points to sexdependent 

changes in maturation speed of young neurons, or a difference in cell-fate 

determination following exposure to early life stress. 

Disclosures: C. Oomen, None; C.E. Neves Girardi, None; E.C. Verbeek, None; R. Cahyadi, 

None; H. Krugers, None; M. Joëls, None; P.J. Lucassen, None. 

Poster




Topic: E.01.g. Behavioral neuroendocrinology: Other 

Support: IMSS-FIS R-2007-1602-6 

Title: Maternal separation decreases hippocampal neurogenesis during early postnatal life 

Authors: *N. LAJUD, L. TORNER; 

Ctr. Investigacion Biomed Michoacan, IMSS, Morelia, Mich., Mexico 

Abstract: Early life stress results in persistent abnormalities in behavioral and neuroendocrine 

functions (1) as well as reductions of neurogenesis in the dentate gyrus (DG) of adult rats (2). In 

rodents most of the granular cells of the DG are generated in the postnatal period. Maternal 

separation (MS) is a potent stressor that leads to the activation of the hypothalamo pituitary 

adrenal axis even during the hiporesponsive period (3) and could influence the neurogenesis of 

the DG since early stages. Here we evaluated the effect of MS on the survival of the DG cells 

during development. 

Maternally Separated (3hr daily from PN1-14) and control undisturbed litters of Sprague Dawley 

rats were injected with Bromodeoxyuridine (BrdU, 50mg/kg, ip) twice at PN5 to evaluate the 

survival of the DG cells. The litters were sacrificed at PN15 and brains were removed. Series of 

systematically selected brain sections (240 κM apart or every sixth section) within the extent of 

the hippocampus were immunostained to evaluate the number of BrdU-positive cells in the 

Granular and sub granular layer of the DG. 

The total number of BrdU positive cells estimated in the DG was similar in male and female 

control pups. However, a significant difference in the cell density was observed between male 

and female control pups, MS significantly reduced both the total number and density of BrdU- 

positive cells at PN15, and this effect was stronger in female than male pups. In addition a 

comparative analysis through the sagital plane of the hippocampus showed that the BrdU- 

labeled cells are homogeneously distributed and that MS affects cell number but not cell 

distribution along the hippocampus. 

A double immunostaining for the neuronal progenitor marker doublecortin (DCX) and BrdU, 

showed that the BrdU labeled cells evaluated here were mostly located in the zone of 

hippocampal progenitor activity, which was also labeled with Dcx. 

Additional litters were allowed to grow to confirm behavioral alterations. Maternally separated 

adult male, (but not female), rats showed an increased anxiety-related behavior on the Elevated 

plus Maze, and displayed a passive coping strategy in the Forced Swimming test than their 

control counterparts. In conclusion, the decreased survival of DG cells caused by MS is an early 

onset phenomenon and it is related to behavioral, sex dependent, abnormalities in the adult. 

1. Eur J Neurosci. 2007 May;25(10):3091-8. 

2. Brain Res. 2002 Sep 20; 950(1-2):52-63. 

3. Brain Res. 1986 Mar;396(1):64-76.

Disclosures: N. Lajud, None; L. Torner, None. 

Poster 





Title: Maternal enrichment alters postnatal GABA-A receptor activation in offspring 

hippocampus 

Authors: *R. MASHOODH 1 , J. NUÑEZ 3 , C. J. SINAL 2 , T. S. PERROT-SINAL 2 ; 

1 Dept. of Psychology, 2 Dalhousie Univ., Halifax, NS, Canada; 3 Michigan State Univ., East 

Lansing, MI 

Abstract: Events during pre- and post-natal developmental phases set trajectories for cognitive 

and emotional development throughout life. Sex differences in the incidence of stress-related 

disease or cognitive impairment are consistently reported, suggesting that differences in affective 

processing between the sexes may be, at least in part, rooted in development. Not surprisingly, 

the critical developmental period during which affective systems are shaped coincides with 

another critical developmental process, the sexual differentiation of the brain. Gammaaminobutyric 

acid (GABA)-A receptor activation, a major source of excitation and a divergence 

point for sexual differentiation in the developing brain, appears to be optimally positioned to 

integrate these two processes. Thus, we sought to characterize how manipulations of the 

maternal environment influence excitatory GABA action in the developing hippocampus. To this 

end, on gestational day (GD) 16, pregnant rat dams were introduced into an enriched 

environment (EE) where they lived for the remainder of the experiment. In the control condition 

(CC), pregnant dams were housed in a standard laboratory cage. Offspring (both male and 

female) born and raised in these conditions were sacrificed on postnatal day (PND) 0, 3, and 7. 

Hippocampi were dissected and processed for mRNA levels of the chloride co-transporters, 

NKCC1 and KCC2 and the GABA synthesizing enzyme, GAD (both 65 and 67 kDa isoforms). 

In a second experiment, we investigated the effect of the enriched maternal environment on 

GABA-A receptor-mediated calcium transients in primary hippocampal cultured neurons, 

derived from offspring born in the CC and EE environments on PND0 and PND3, using the 

calcium sensitive dye, Fura-2-AM. The results reveal a complex relationship between maternal 

enrichment and GABAergic activity in the developing hippocampus during the first postnatal 

week. Maternal enrichment initially promotes a greater degree of hyperpolarization just after 

birth, but gradually induces a slower rate of switch towards hyperpolarization, potentially 

culminating in a faster and more pronounced termination of excitatory GABA action as

compared to control-reared offspring. Furthermore, the early switch from depolarizing to 

hyperpolarizing GABAergic responses in control female offspring was prevented by maternal 

enrichment. 

Disclosures: R. Mashoodh, None; J. Nuñez, None; C.J. Sinal, None; T.S. Perrot-Sinal, None. 

Poster 





Support: NIH grant HD049336 

Title: Maternal care effects on SNB motoneurons and male copulatory behavior: The mediating 

role of sensory afferent activity 

Authors: *K. M. LENZ, D. R. SENGELAUB; 

Psychol & Brain Sci., Indiana Univ., Bloomington, IN 

Abstract: Early maternal care shapes the sexual behavior of male rats, with reductions in 

maternal licking of the perineum producing adult copulatory deficits. Maternal care also 

influences the development of the spinal nucleus of the bulbocavernosus (SNB), a sexually 

dimorphic motor nucleus in the lumbosacral spinal cord that controls the penile reflexes involved 

in copulation. Previous research has shown that reductions in maternal licking produce decreased 

adult SNB motoneuron number, size and dendritic length in offspring, especially in the rostral 

portion of the nucleus. We have begun to study the mechanisms through which maternal licking 

influences SNB development, with a focus on cutaneous sensory afferents from the perineal skin. 

Our previous work has shown that these sensory afferents are distributed in a caudally biased 

manner relative to the SNB dendritic field, suggesting that these sensory afferents may 

sufficiently support the caudal SNB dendritic arbor even when maternal licking is decreased, but 

render the rostral arbor more vulnerable to decreases in afferent activity. In the current studies, 

we used immediate early gene analysis to investigate whether licking-like tactile stimulation of 

the perineum increases local spinal cord activity in the area of the SNB dendritic field. 

We simulated maternal licking by stroking neonatal rats with a paintbrush, and subsequently 

measured the expression of the immediate early gene product, Fos, in the lumbosacral spinal 

cord using immunohistochemistry. Relative to unstimulated controls, pups that received lickinglike 

stimulation showed a dramatic increase in the number of Fos-positive neurons in the area of 

the SNB dendritic field, suggesting that maternal licking increases local neuronal activity during

SNB motoneuron development. In addition, we found a sex difference in the number of Fospositive 

cells following licking-like stimulation, with male pups showing greater Fos expression 

than female pups. We also mapped the rostrocaudal distribution of Fos-positive cells following 

licking-like tactile stimulation, to determine if regional differences in sensory afferent 

distribution produce a corresponding bias in local spinal cord activity. Preliminary data show a 

greater number of Fos-positive cells are located in the caudal portion of the SNB dendritic field, 

matching the caudal bias in sensory afferent distribution. As a whole, these data support the 

hypothesis that the maternal effects on the development of the SNB are mediated by the local 

spinal cord activity conferred by tactile stimulation. These findings add to other evidence of 

activity-dependence in dendritic development of the SNB. 

Disclosures: K.M. Lenz, None; D.R. Sengelaub, None. 

Poster 




Topic: A.09. Evolution of Developmental Mechanisms 

Title: Correlation between maternal anogenital licking and adult maze learning in mice 

Authors: *A. STAVNEZER, P. WEIGAND, C. BURDEN, E. N. WRIGHT; 

Dept Psych, Col. of Wooster, Wooster, OH 

Abstract: Male rats receive a greater amount of anogenital licking than female rats (Moore & 

Morelli, 1979). In addition, the overall amount of anogential licking, as provided by high- or 

low-licking dams, has been correlated with the density of neurons and length of dendritic spines 

in the hippocampus (Bredy et al., 2003; Lenz & Sengelaub, 2006). With the known relationship 

between hippocampal connectivity and spatial maze performance, we conducted an experiment 

with mice to determine if the sex difference in anogenital licking was directly correlated with the 

sex differences noted in Morris water maze. We used two strains of sex-reversed mice and 

control C57BL/6J mice to investigate this question since there is some evidence that sex 

differences in spatial learning are under both gonadal hormone and sex chromosome control. 

Both anogential and other maternal licking was recorded and scored at P4, 7 and 10. These 

scores were then correlated with open field activity, Morris water maze and complex pattern 

discrimination at 4-6 months of age for all mice. Results will be discussed in the context of 

interactions between sex, sex chromosome status and early maternal behavior on later behavioral 

capabilities and differences.

Disclosures: A. Stavnezer , None; P. Weigand, None; C. Burden, None; E.N. Wright, None. 

Poster 





Title: Brief maternal separation increases gastric pre-autonomic viral transport to the 

paraventricular nucleus of the hypothalamus in juvenile rats 

Authors: *L. BANIHASHEMI, L. RINAMAN; 

Dept. of Neurosci., Univ. of Pittsburgh, Pittsburgh, PA 

Abstract: A large literature has demonstrated that brief handling and maternal separation of rat 

pups for 15 minutes daily for one to two weeks postnatal (MS15) decreases later hormonal and 

behavioral responses to stress compared to responses in nonhandled (NH) controls. However, it 

is unclear whether MS15 alters neural circuits that mediate stress responses, such as descending 

central autonomic circuits that control visceral motor responses to stress. These circuits undergo 

substantial postnatal assembly and are susceptible to the effects of early life experience 

(Rinaman et al. 2000, J. Neurosci, 20:2731; Card et al. 2005, J. Neurosci. 25:9102). The present 

study examined the effects of early life experience (MS15 or NH) on central autonomic circuits 

in juvenile rats using the transynaptic viral tracer, pseudorabies virus (PRV). PRV (Bartha strain, 

2 µl; 10 7 pfu/ml) was microinjected into the ventral stomach wall of 28-day old male and female 

rats with a developmental history of MS15 or NH. Rats were perfused 72 hours later and tissue 

sections were processed to reveal PRV-positive neurons. SimplePCI imaging software was used 

to quantify the tissue area occupied by PRV-positive labeling within the area postrema (AP), bed 

nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and 

paraventricular nucleus of the hypothalamus (PVN). ANOVA revealed that MS15 rats displayed 

significantly more transneuronal PRV labeling within the PVN compared to NH controls (P < 

0.014; MS15 n=22, NH n=27). This difference was localized to the rostrocaudal level of the 

PVN that contains the dorsal and ventral pre-autonomic subnuclei and was only seen in male 

rats. No significant group differences in PRV labeling were observed in other analyzed forebrain 

regions. Interestingly, Card and colleagues reported that 10-day old MS15 rat pups (males and 

females) displayed fewer retrogradely labeled pre-autonomic neurons in the BNST, CeA, and 

PVN compared to NH control pups (Card et al. 2005). Considered together, these findings 

suggest that MS15 delays the early postnatal assembly of pre-autonomic circuits evident in 10day 

old pups, but that these circuits undergo further changes during the subsequent 3 weeks of 

development such that the hypothalamic PVN ultimately provides increased levels of gastric pre-

autonomic control in juvenile (i.e., 28-31 day old) male MS15 rats. Thus, these findings may 

reflect an altered ability of the PVN to modulate autonomic outflow in response to stress in rats 

with a developmental history of MS15. 

Disclosures: L. Banihashemi , None; L. Rinaman, None. 

Poster 





Title: A potential gastrointestinal link between postnatal maternal care and adult anxiety 

Authors: *L. M. RINAMAN, B. C. WEBER, H. N. MANFREDO; 

Dept Neurosci, Univ. Pittsburgh, Pittsburgh, PA 

Abstract: Previous studies have demonstrated that rat pups separated from their dam for 15 

min/day (MS15) during the first two weeks postnatal display reduced anxiety-like behavior as 

adults. MS15 increases the daily maternal care received by pups (e.g., licking/grooming, archedback 

nursing), thereby promoting increased somatic and visceral sensory stimulation. Other 

studies have shown that maternal reunion after separation increases plasma cholecystokinin 

(CCK) levels in rat pups. Increased signaling at CCK-1 receptors activates gastrointestinal vagal 

afferents, which could alter the development of central visceral circuits and thereby affect adult 

anxiety responses. We hypothesized that concurrent CCK-1R blockade would reverse the 

anxiolytic effects of MS15. To test this, rat pups were injected with devazepide (CCK-1R 

antagonist; 2.3 nmol/100 nl s.c.), or with vehicle (5% DMSO, 5% Tween 80 in 0.15M NaCl), or 

received no injection each day from P1-P10, in combination with MS15. Pups received no 

further treatments after P10, and were weaned on P21. As young adults (~P50-60), rats were 

tested for anxiety-like behavior in the EPMZ and in a novelty-suppressed feeding test. Compared 

to results in non-separated control rats, MS15 reduced anxiety-like behavior in both behavioral 

tests. The anxiolytic effects of MS15 were maintained in vehicle-injected MS15 rats, but were 

reversed in devazepide-injected MS15 rats. Thus, the ability of MS15 to reduce later anxiety-like 

behavior was eliminated by concurrent pharmacological blockade of CCK-1 receptors in rat 

pups. We speculate that increased maternal care promotes increased CCK release from the gut 

and increased vagal afferent signaling, and that this alters the developmental assembly of central 

visceral circuits that impact adult emotionality and stress responsiveness. 

Disclosures: L.M. Rinaman , None; B.C. Weber, None; H.N. Manfredo, None.

Poster 

282. Stress and the Brain: Cellular Actions of Stress 



Topic: E.06.d. Cellular actions of stress 

Support: NHMRC, Australia 

ARC, Network in Genes Environment and Development 

Title: Early cerebrovascular responses to brief global asphyxia in the late gestation fetal sheep 

Authors: *M. CASTILLO-MELENDEZ, A. A. BABURAMANI, D. W. WALKER; 

Monash Univ., Melbourne, Australia 

Abstract: Angiogenesis and neovascularisation, including up-regulation of vascular endothelial 

growth factor (VEGF), occur in adult and neonatal brain after cerebral hypoxia. Whether such 

responses occur in the fetal brain in utero after asphyxia caused by perturbations of placental 

blood flow is unknown. The objective of this study was to determine if brief global asphyxia 

produced by umbilical cord occlusion (UCO) results in increased VEGF expression and cell 

proliferation in the late gestation fetal brain. 

Singleton fetal sheep were subjected to 10 mins global asphyxia at 132 days gestation (term=147 

days) by inflation of an umbilical cuff implanted 7 days previously (n=5); control animals were 

subjected to a sham occlusion (n=5). The fetal brain was collected 24 h after UCO, and after 

transcardial perfusion with 4% paraformaldehyde . Immunohistochemistry was carried out on 10 

κm, sections using either mouse monoclonal anti-VEGF and a rabbit monoclonal anti-Ki67 

(proliferative marker) primary antibodies and visualized with metal-enhanced diaminobenzydine. 

Images were quantified with ImageJ using light microscopy. 

After UCO, VEGF expression was increased 1.5-2.5x in the corpus callosum (CC), claustrum, 

caudate nucleus (CNu), external (EC) capsule, cortex (layers I-VI) and subcortical white matter 

(SCWM), and 10-20x in the internal capsule (IC), stria terminalis (ST), putamen (Put), 

subventricular zone (SVZ) and periventricular white matter (PVWM), compared to the agematched 

sham-UCO fetal brain. VEGF expression occurred predominantly (70-100%) in blood 

vessels in all these brain regions of the UCO group, compared to none, or 60% (cortex) and 30% 

(SCWM) in sham-UCO fetuses. Cell proliferation (Ki67 immunoreactivity) associated with 

endothelial cells increased 2-5x in the CC, claustrum, CNu, and PVWM, 6-15x in the SCWM, 

EC and Put, and 30-60x in the SVZ and cortex. 

Brief global asphyxia results in robust up-regulation of VEGF expression throughout the late 

gestation fetal brain. Endothelial cell proliferation was markedly increased in brain regions

known to be susceptible to hypoxic brain injury. We suggest that VEGF-dependent angiogenic 

responses may alter vascular patterning and cerebrovascular reactivity in the neonatal brain, and 

that newly-formed blood vessels may be fragile and prone to injury. 

Disclosures: M. Castillo-Melendez , Monash University, A. Employment (full or part-time); 

National Health and Medical Research Council, Australia, B. Research Grant (principal 


A.A. Baburamani, Monash University, A. Employment (full or part-time); D.W. Walker, 

Monash University, A. Employment (full or part-time); National Health and Medical Research 

Council, Australia, B. Research Grant (principal investigator, collaborator or consultant and 


Poster 





Support: Wellcome Trust Research Fellowship RCDF 068558/Z/02/Z 

Title: Corticotropin-releasing factor in vitro and aversive acoustic stimulation in vivo increase 

tryptophan hydroxylase activity in the caudal dorsal raphe nucleus 

Authors: *A. K. EVANS 1,2 , J. HEERKENS 2 , C. A. LOWRY 1,2 ; 

1 Integrative Physiol., Univ. of Colorado, Boulder, CO; 2 Henry Wellcome Labs. for Integrative 

Neurosci. and Endocrinolgy, Univ. of Bristol, Bristol, United Kingdom 

Abstract: Corticotropin-releasing factor (CRF) is implicated in regulation of serotonergic 

systems modulating adaptive stress-related physiological and behavioral responses. 

Unpredictable, aversive acoustic stimulation increases activity of the rate-limiting enzyme for 

serotonin synthesis, tryptophan hydroxylase (TPH), in the median raphe nucleus and a 

mesolimbocortical serotonergic projection system. Although the mechanisms involved in these 

stress-induced increases in mesolimbocortical serotonergic neurotransmission have not been 

elucidated, the central amygdaloid nucleus has been implicated, and direct microinjections of 

CRF or related neuropeptides into the dorsal raphe nucleus increase mesolimbocortical 

serotonergic activity. In addition, CRF and its related family of neuropeptides, particularly at 

higher doses, can increase serotonergic neuronal firing rates in the caudal dorsal raphe nucleus. It 

is possible that actions of CRF in the caudal dorsal raphe nucleus may underlie the effects of 

aversive acoustic stimulation on TPH activity in both the median raphe nucleus and a

mesolimbocortical serotonergic system. In this study an in vivo aversive acoustic stimulation 

paradigm and an in vitro living brain slice preparation were designed to test the hypothesis that 

stress-related stimuli and CRF receptor activation have convergent actions on TPH activity in the 

caudal part of the dorsal raphe nucleus. In both studies, we measured 5-hydroxytryptophan (5- 

HTP) accumulation following inhibition of aromatic amino acid decarboxylase (with NSD-1015) 

as an index of TPH activity. To examine the effects of acoustic stimulation on TPH activity in 

the midbrain raphe complex, male Wistar rats were treated with NSD-1015 and then immediately 

exposed to a 30 minute sham, predictable or unpredictable acoustic stimulation paradigm; brains 

were collected, rapidly frozen and sectioned for microdissection and HPLC analyses of tissue 

concentrations of 5-HTP. To examine the effect of CRF-receptor activation on TPH activity in 

the midbrain raphe complex, living rat brain slices were exposed to a 10 minute CRF application 

(0-2000 nM) in the presence of NSD-1015; brain slices were frozen and microdissected for 

HPLC analysis of 5-HTP. We observed increased TPH activity in the caudal dorsal raphe 

nucleus in both the unpredictable acoustic stimulation paradigm and in the brain slice preparation 

following exposure to CRF (1000 nM). These findings are consistent with the hypothesis that 

stress-induced increases in TPH activity are mediated in part via effects of CRF on a 

mesolimbocortical serotonergic system originating in the caudal dorsal raphe nucleus. 

Disclosures: A.K. Evans, None; J. Heerkens, None; C.A. Lowry, None. 

Poster 





Support: MIUR (Prin 2004) 

Title: Previous stress potentiates the inhibitory effects of nociceptin/orphanin-fq on dorsal raphe 

nucleus neurons: potential role of corticotropin-releasing factor 

Authors: *C. NAZZARO 1 , M. BARBIERI 2 , A. SINISCALCHI 2 , R. J. VALENTINO 3 ; 

1 Dept Neurosci, Italian Inst. Technol., Genova, Italy; 2 Exptl. and Clin. Medicine, Pharmacol. 

Sec., Univ. of Ferrara, Ferrara, Italy; 3 Anesthesiology, Stress Neurobio. sec., Children's Hosp. of 

Philadelphia, Philadelphia, PA 

Abstract: The Nociceptin/Orphanin-FQ (N/OFQ)-NOP receptor has been implicated in mood 

disorders, such as anxiety and depression. One potential target of N/OFQ is the dorsal raphe 

nucleus (DR)-serotonin (5-HT) system, a system that is regulated by stress and involved in

stress-related psychiatric disorders, such as depression. Here we studied the effects of N/OFQ on 

activity of DR neurons in unstressed and previously stressed rats. 

Single-unit extracellular recordings from DR neurons were performed both in vivo and in vitro. 

In unstressed anesthetized rats, intraraphe (i.r.) injection of N/OFQ (0.03; 0.1 and 30 ng) reduced 

the discharge rate of putative 5HT neurons respectively by 80 ± 3 %; 44 ± 10 % and 8 ± 8 % the 

baseline rate. Previous exposure to swim stress (SS) increased the response to N/OFQ (0.1 ng) 

measured 24 h later to a 15 ± 8% inhibition. In contrast, SS did not modify the magnitude of 

inhibition induced by the 5HT1A agonist, 8-OHDPAT, ruling out non-specific effects of SS on 

K+ conductance. Corticotropin-releasing factor (CRF) a primary mediator of the stress response 

regulates activity of the DR system during stress. Convergent findings indicated that SS 

potentiation of N/OFQ was mediated by CRF. Thus, pretreatment with the CRF1 antagonist, 

NBI-30775 (20 mg/kg i. p.) 30 min before SS prevented the ability of SS to potentiate N/OFQ. 

The magnitude of inhibition produced by SS after NBI-30775 was 49 ± 16 % compared to 26 ± 

11 % after vehicle pretreatment. Additionally, like SS, CRF (30 ng, ir) administered before 

N/OFQ to unstressed rats potentiated the magnitude of inhibition (80 ± 3% vs 17 ± 10%; ctrl vs 

CRF). 

In vitro in DR slices from unstressed rats, bath perfusion of N/OFQ reduced, in a concentrationdependent 

manner (EC50 14.48 ± 1.65 nM), the firing rate induced by phenylephrine 10 µM. In 

slices from rats exposed to SS, the concentration-response curve to N/OFQ was significantly 

shifted to the left (EC50 2.1 ± 3.5 nM). The inhibitory effect of N/OFQ was prevented by the 

selective N/OFQ antagonist UFP-101 (pA2 = 6,86). In animals pretreated with the CRF1 

antagonist, antalarmin (20mg/kg i.p.) 30 min before FS, the responsiveness to N/OFQ was 

similar to that of the controls (EC50 16.45 ± 3.43 nM). 

In conclusion: Stress potentiates N/OFQ inhibitory effect on DR-5HT neurons and this is 

mediated through interactions with endogenous CRF . The interaction between the two 

peptidergic systems may be important in adapting to acute stressors. 

Disclosures: C. Nazzaro , None; M. Barbieri, None; A. Siniscalchi, None; R.J. Valentino, 

None. 

Poster 





Support: NARSAD Young Investigator Award 

NSF Grant IRFP0502379

NSF Grant IBN0220473 

Wellcome Trust Fellowship RCDF 068558/Z/02/Z 

Title: Expression of organic cation transporter 3, a corticosterone-sensitive monoamine 

transporter, in monoaminergic projection areas in the rat brain: potential role in stress-induced 

modulation of monoaminergic neurotransmission 

Authors: *P. J. GASSER 1 , M. ORCHINIK 3 , I. RAJU 2 , C. A. LOWRY 4 ; 

1 Biomed. Sci., 2 Biomed Sci., Marquette Univ., Milwaukee, WI; 3 Sch. of Life Sci., Arizona State 

Univ., Tempe, AZ; 4 Integrative Physiol., Univ. of Colorado, Boulder, CO 

Abstract: Organic cation transporters (OCTs) are high capacity, corticosterone-sensitive 

transporters, primarily studied in the periphery, whose substrates include norepinephrine (NE), 

dopamine (DA), serotonin (5-HT), and histamine. Our previous studies demonstrated OCT3 

expression and corticosterone-sensitive transport of OCT substrates in the rat dorsomedial 

hypothalamus, a region that accumulates NA, DA, and 5-HT in response to acute stress or 

corticosterone administration. We have hypothesized that inhibition of OCT3-mediated 

monoamine transport represents a general mechanism by which corticosterone acutely modulates 

monoaminergic neurotransmission, leading to altered physiological and behavioral responses. In 

order to begin to understand the importance of OCT3 in the regulation of monoaminergic 

neurotransmission, we used immunohistochemical methods to characterize the distribution of 

OCT3-like immunoreactive (OCT3-ir) cells throughout the rostrocaudal extent of the male rat 

brain. The specificity of the OCT3 antiserum was tested in western blots of membrane proteins 

from rat cerebellar homogenates. Western blot revealed a single major OCT3-ir band of 

approximately 53 kDa. Consistent with an important role for OCT3 in the transport of 

monoamine neurotransmitters, high densities of OCT3-ir cells were observed within many areas 

that are densely innervated by monoaminergic projection systems. OCT3-ir cells were detected 

in the retrosplenial granular cortex, granule cell layers of the main and accessory olfactory bulbs, 

superior and inferior colliculi, lateral habenular nuclei, islands of Calleja, medial mammillary 

nucleus, lateral septum, dorsal subiculum, and lateral hypothalamic nucleus. In most areas, 

OCT3-immunostaining was observed in small (6-8 µm diameter), glial- or granule neuron-like 

cells. These data provide anatomical evidence for a role for OCT3 in the postsynaptic clearance 

of monoamines within various brain regions. The data also are consistent with the hypothesis 

that corticosterone-induced blockade of OCT3-mediated monoamine clearance rapidly alters 

monoaminergic neurotransmission, modulating ongoing physiological and behavioral activity 

during stress responses. 

Disclosures: P.J. Gasser, None; M. Orchinik, None; I. Raju, None; C.A. Lowry, None. 

Poster 

282. Stress and the Brain: Cellular Actions of Stress




Title: Activation of locus coeruleus neurons projected from corticotropin-releasing factor 

neurons in the hypothalamic paraventricular nucleus is involved in arousal response 

accompanied by yawning behavior in rats 

Authors: *N. KUBOTA, T. OTSUKA, S. YANAGITA, C. MOTOKI, I. KITA; 

Tokyo Metropolitan Univ., Hachioji, Japan 

Abstract: Our previous studies have showed that an arousal response accompanied by yawning 

behavior can be evoked by electrical and chemical stimulation of the hypothalamic 

paraventricular nucleus (PVN) in rats. Furthermore, we have suggested that activation of 

corticotropin-releasing factor (CRF) neurons in the PVN is involved in a stereotyped yawning 

response, which was characterized by a single large inspiration, an initial fall blood pressure 

(BP), and an arousal shift in the electrocorticogram (ECoG), using c-Fos immunohistochemistry 

in rats. Recently, we found that intracerebroventricular (icv) administration of CRF antagonist 

attenuates arousal response accompanied by yawning behavior in rats. Although these findings 

suggest that CRF neurons in the PVN are primarily responsible for the arousal response during 

yawning, the CRF-containing pathway involved in the arousal response accompanied by 

yawning evoked by the PVN stimulation is still unknown. CRF is known to serve as an 

excitatory neurotransmitter in the locus coeruleus (LC), which is one of the neuronal aggregates 

within the brainstem reticular formation responsible for cortical activation and one of the major 

sources of noradrenalin (NA) in the central nervous system. Several neuroanatomical studies 

have suggested that the LC receives CRF afferents from PVN, and projects to various brain areas 

including regions of cortex. Therefore, it is possible that activation of the noradrenergic system 

by the PVN CRF neurons projecting to the LC is responsible for the arousal response during 

yawning induced by PVN stimulation. In the present study, we examined the involvement of LC 

neurons projected from PVN CRF neurons in the arousal response during yawning with doublestaining 

for c-Fos and CRF in the PVN, and for c-Fos and NA in the LC after CRF antagonist 

(icv) in anesthetized, spontaneously breathing rats. In addition, we also recorded the ECoG to 

evaluate the effect of CRF antagonist on arousal responses during yawning. Microinjection of Lglutamate 

into the PVN induced a frequent yawning accompanied by an arousal shift in the 

ECoG even after CRF antagonist (icv), but the arousal response was significantly suppressed 

compared to that after saline (icv). The effect of CRF antagonist (icv) on arousal response during 

yawning was associated with c-Fos expression in the LC, and with activation of the CRF neurons 

in the PVN. These results suggest that the arousal response accompanied by yawning behavior 

might be mediated by LC neurons projected from PVN CRF neurons. 

Disclosures: N. Kubota, None; T. Otsuka, None; S. Yanagita, None; C. Motoki, None; I. 

Kita, None.

Poster 





Support: MH075047 to Beck 

MH067651 to Bhatnagar 

ONR NN0014-03-1-0311 to Beck 

Title: Orexin alters firing pattern of neurons in the posterior paraventricular thalamus (pPVTh) 

after repeated stress 

Authors: *S. BHATNAGAR, D. PIEHL, V. IYER, S. BECK; 

Dept Anesthesiol., Univ. Pennsylvania, Children's Hosp Philadelphia, Philadelphia, PA 

Abstract: Our previous work suggests that activation of pPVTh neurons primarily inhibits 

neuroendocrine and behavioral responses in repeatedly stressed compared to control or acutely 

stressed rats. Interestingly, the PVTh receives dense orexin inputs from cell bodies in the lateral 

hypothalamus and pPVTh cells contain the two orexin receptor subtypes. Since orexin is a 

peptide important for arousal and attention, we hypothesized that orexin inputs to the pPVTh are 

important for regulating responsivity in repeatedly stressed rats. In a related abstract, we show 

that blockade of orexin receptors in the pPVTh attenuates ACTH responses to acute restraint in 

rats exposed to repeated swim but not in acutely restrained rats. Here, we examined the effects of 

orexin administration on pPVTh neurons at a cellular level and hypothesized that the response of 

pPVTh cells to orexin would be different in control and repeatedly stressed rats. We exposed rats 

to no stress or to 4 days of repeated swim for 15min per day. On day 5, we obtained 200 micron 

thick brain slices containing the pPVTh. Whole cell recordings were used to record from pPVTh 

neurons. Cell characteristics were measured using current clamp. Stress did not alter the resting 

membrane potential, resistance or time constant of the neurons. However, the firing pattern of 

the cells was different. The cells from the control rats exhibited primarily a bursting firing 

pattern, whereas the majority of the cells from the swim stressed rats did not exhibit any firing. 

Orexin (100 nM) was bath applied for 3 min. Preliminary results indicate that orexin induced a 

greater depolarization and increase in resistance in the cells from the swim stressed rats. The 

firing pattern of the cells recorded from the control rats primarily retained their bursting pattern, 

whereas the cells from the swim stressed rats switched to a firing pattern of single actions. In 

addition, we recorded excitatory postsynaptic potentials (EPSC) using voltage clamp techniques

and found that there was a greater amplitude and shorter decay time of the AMPA/kainate 

receptor-ionophore in cells recorded from control versus swim stressed rats. These results 

indicate that repeated swim stress influences the pPVTh by altering basic firing properties, 

afferent input and receptor mediated responses. Furthermore, the effect of orexin is not the same 

on the firing pattern of repeated swim compared to control rats. Together, these results indicate 

that repeated stress alters orexin actions in the pPVTh. These alterations may underlie the 

differential neuroendocrine and behavioral response observed in repeatedly vs. acutely stressed 

rats following manipulation of orexin receptors. 

Disclosures: S. Bhatnagar , None; D. Piehl, None; V. Iyer, None; S. Beck, None. 

Poster 





Title: Effects of low intensity exercise on neuronal activation of serotonergic neurons in the 

dorsal raphe nucleus and anxiety-like behavior in rats 

Authors: T. OTSUKA, N. KUBOTA, S. YANAGITA, *I. KITA; 

Dept Human Hlth. Sci., Tokyo Metropolitan Univ., Hachioji, Tokyo, Japan 

Abstract: Several studies have suggested that depression and anxiety respond to the same 

treatments, including selective serotonin reuptake inhibitors and physical exercise, and that 

serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) are implicated in 

antidepressant/anxiolytic properties. It is, thus, possible that physical exercise produces its 

antidepressant/anxiolytic properties by affecting neuronal activity of DRN 5-HT neurons. 

Chronic exercise using running wheel or treadmill is known to increase the activity of 5-HT 

neurons in the DRN and turnover of 5-HT in the cortex. On the other hand, it is reported that 

chronic wheel running could attenuate stress-induced c-Fos induction in 5-HT neurons in the 

DRN and increase mRNA level of 5-HT1A autoreceptor in the DRN, suggesting that activation 

of 5-HT neurons during acute physical exercise might mitigate excessive 5-HT activity to stress 

events. However, effects of acute physical exercise on neuronal activity of DRN 5-HT neurons, 

or relationship between the exercise intensity and the neuronal activity of 5-HT are unknown. In 

addition, since stressful life events are related to incidence and symptom of depression and 

anxiety, corticotropin-releasing factor (CRF) neurons in the hypothalamic paraventricular 

nucleus (PVN), which play an important role as one of the central activators of integrated stress 

response, may be also involved in antidepressant/anxiolytic properties by physical exercise. The

present study examined the activities of DRN 5-HT neurons and PVN CRF neurons to 30 min of 

treadmill running with different speeds (25m/min; 15m/min; control, just sitting on a treadmill) 

in male Wistar rats, using c-Fos/5-HT or CRF immunohistochemistry. We also performed an 

open field test after the treadmill running to evaluate the effect of physical exercise on anxietylike 

behavior. Low intensity exercise, but not high intensity induced a significant increase of c- 

Fos positive 5-HT neurons compared to control. The number of c-Fos positive CRF neurons was 

increased speed-dependently. Locomotor activities in the open field test were decreased by 

physical exercise, especially with low intensity. These results suggest that low intensity exercise 

can effectively induce antidepressant/anxiolytic properties of physical exercise. 

Disclosures: T. Otsuka, None; I. Kita , None; N. Kubota, None; S. Yanagita, None. 

Poster 





Support: SHRF 

NSERC 

Title: Downregulation of reelin in an animal model of depression is reversed by antidepressants 

and correlates with alterations in neural integration in the adult dentate gyrus 

Authors: *E. Y. FENTON 1 , N. M. FOURNIER 1 , A. L. LUSSIER 1 , H. J. CARUNCHO 2 , L. E. 

KALYNCHUK 1 ; 

1 Dept. of Psychology, Univ. of Saskatchewan, Saskatoon, SK, Canada; 2 Dept. of Cell Biol., 

Univ. of Santiago de Compostela, Galicia, Spain 

Abstract: Current hypotheses about the neurobiology of depression are focused on hippocampal 

neurogenesis. The idea is that decreased neurogenesis may play a causal role in depression. 

However, recent evidence has challenged this hypothesis. For example, post mortem analyses 

have revealed that human patients with depression do not have decreased neurogenesis. It may 

therefore be important to focus attention on the maturation and function of surviving cells, rather 

than the loss of cells per se. Reelin is a glycoprotein that may play a key role in cell survival and 

migration. Interestingly, reelin has been implicated in a number of neuropsychiatric disorders, 

including depression. Although reelin is important for neuronal migration during embryonic 

development, it also appears to play a prominent role in synaptic plasticity, memory processing,

and cognition in the adult brain. To evaluate the potential relationship between stress-induced 

changes in reelin expression and cell maturation, we administered the stress hormone 

corticosterone (CORT, 40 mg/kg, s.c.) or vehicle for 21 days to naïve male rats. Imipramine was 

administered at a high (15 mg/kg, i.p.) or intermediate (10 mg/kg, i.p.) dose to some of the 

CORT-treated rats to evaluate its protective effects. Immunohistochemistry revealed that animals 

treated with CORT had significantly fewer reelin positive cells in the hilus and subgranular zone 

of the hippocampus compared to all other groups. Analysis of the immature neuronal marker 

doublecortin also indicated that there were fewer immature neurons in the subgranular zone in 

groups receiving CORT alone or CORT + intermediate amounts of imipramine compared to rats 

receiving CORT + a high dose of imipramine. Further analysis revealed that granule cells born 

under conditions of stress had shorter apical dendrites and less complex dendritic arborizations, 

and that these effects were reversed by the high dose of imipramine. In addition, rats treated with 

CORT alone or CORT + intermediate amounts of imipramine showed abnormal numbers of 

granule cells with basal dendrites. Finally, CORT treatment decreased nestin expression while 

leaving GFAP untouched. Collectively, these results suggest that chronic stress may slow the 

maturation of newly born cells, and that reelin may play an important role in this maturational 

process. 

Disclosures: E.Y. Fenton, None; N.M. Fournier, None; A.L. Lussier, None; H.J. Caruncho, 

None; L.E. Kalynchuk, None. 

Poster 





Support: DA05014 

NS39415 

Title: Cyclic AMP-mediated stimulation of tyrosine hydroxylase mRNA translation is mediated 

by polypyrimidine-rich sequences within its 3‟UTR and poly(C) binding protein 2 (PCBP2) 

Authors: *L. XU, C. R. STERLING, A. W. TANK; 

Pharmacol. & Physiol., Univ. of Rochester, Rochester, NY 

Abstract: Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis 

of catecholamines. TH gene expression is tightly controlled by both transcriptional and post-

transcriptional mechanisms. In previous studies, we have shown that treatment with the cAMP 

analog, 8-CPT-cAMP or forskolin leads to induction of TH protein without concomitant 

induction of TH mRNA in rat midbrain slice explant cultures and mouse MN9D cells. This 

induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH 

mRNA translation. Furthermore, we have shown that cAMP activates mechanisms that regulate 

TH mRNA translation via cis-acting sequences within its 3‟ UTR. In this report, we continue to 

investigate this cAMP-mediated translational response. RNA-protein binding and UV crosslinking 

assays show that MN9D cell and rat midbrain cytoplasmic proteins bind to the TH 

mRNA 3‟UTR. RNase T1 mapping demonstrates binding of proteins to a 27 nt polypyrimidinerich 

sequence within the TH mRNA 3‟UTR. Mutations within this polypyrimidine-rich region 

inhibit binding of MN9D cell proteins. One of these MN9D cell proteins appears to be poly(C) 

binding protein 2 (PCBP2). Overexpression of PCBP2 induces TH protein without concomitant 

induction of TH mRNA in MN9D cells, which mimics the cAMP response. Our results support a 

model in which cAMP induces PCBP2, leading to increased interaction with its cognate 

polypyrimidine binding site within the TH mRNA 3‟UTR. This increased interaction enhances 

TH mRNA translation in dopaminergic neurons. This finding may help to explain the control of 

TH expression and consequently dopamine biosynthesis in midbrain neurons under different 

physiological and pathological conditions. This work was supported by NIH grants DA05014 

and NS39415. 

Disclosures: L. Xu, None; C.R. Sterling, None; A.W. Tank, None. 

Poster 





Support: ONR N00014-02-1-0325 

Title: Profiling alterations in gene expression in rat adrenal medulla with cold stress 

Authors: *E. L. SABBAN 1 , X. LIU 1 , R. KVETNANSKY 2 , L. SEROVA 1 , A. SOLLAS 1 , R. 

ZILINSKI 1 ; 

1 Dept Biochem & Mol Biol, New York Med. Coll, Valhalla, NY; 2 Inst. of Exptl. Endrocrinol., 

Bratislava, Slovakia 

Abstract: Prolonged cold stress elicits sensitization of various neuronal systems, including the 

adrenal medulla, to a novel or heterotypic stressor (reviewed in Sabban and Serova, 2007, Stress,

10:137-143). Here, microarray profiling was used to elucidate global changes in gene expression 

in rat adrenal medulla with different durations of cold stress. RNA was isolated from adrenal 

medulla of rats exposed for 1, 7 or 28 days to 4°C and used for expression profiling (Affymetrix 

RAE 230 2.0). The number of transcripts significantly changed >1.5 fold were similar during the 

various days of cold stress with 46, 69 and 58 up regulated and 28, 32, and 25 down regulated on 

days 1, 7, and 28, respectively. Less than half of the upregulated, and 20% of the downregulated 

transcripts were changed throughout the 28 days of cold. The greatest increase was 

seen in those transcripts categorized as related to gene transcription, secretion/neuropetides, 

signaling pathways, growth factors and metabolism. The transcripts down regulated also 

encompassed these same categories with several transcripts also related to chemokines. We 

confirmed, by real time RT-PCR, the induction of mRNA for CREM, phospholipase C epsilon, 

and two factors involved in synaptic vesicle function: vesicular monoamine transporter 2 

(VMAT2) and EH domain containing protein. Immunofluoresence was used to localize the 

VMAT2. The adrenal medulla expresses both VMAT1 and VMAT2. Under basal conditions, the 

more efficient VMAT2 was found to be exclusively present in norepinephrine synthesizing 

chromaffin cells (TH positive, PNMT negative). Even one day cold stress already increased the 

expression of VMAT2. The results enable us to propose that the adaptation of the adrenal 

medulla to cold stress is mediated by enhanced synaptic vesicle function and/or changes in 

specific signaling pathways. Some of the prolonged changes in gene expression following 28 

days of cold are proposed to mediate the sensitization of the adrenal medulla of cold adapted 

animals to a heterotypic stressor. 

Disclosures: E.L. Sabban, None; X. Liu, None; R. Kvetnansky, None; L. Serova, None; A. 

Sollas, None; R. Zilinski, None. 

Poster 





Support: Slovak grant APVV-0148-06 

VEGA 2/0133/08 

Title: Stress-induced changes in gene expression of β-adrenoceptors (especially β3), their 

proteins and binding sites in rat heart

Authors: *R. KVETNANSKY 1 , M. NOVAKOVA 2 , J. PETRAK 3 , O. KRIZANOVA 4 , J. 

MYSLIVECEK 2 , A. TILLINGER 3 ; 

1 Inst. Exp Endocrinol, Slovak Acad. Sci., Bratislava, Slovakia; 2 First Fac. of Medicine, Charles 

Univ., Prague, Czech Republic; 3 Inst. of Exptl. Endocrinology, SAS, Bratislava, Slovakia; 4 Inst. 

of Mol. Physiol. and Genetics, SAS, Bratislava, Slovakia 

Abstract: Catecholamines (norepinephrine-NE and epinephrine-EPI) are among the first 

circulating compounds released during stress response of the organism. They prepare heart to 

“fight or flight” situation via stimulation of β-adrenoceptors (β-ARs). There are only few papers 

dealing with changes of cardiac β-ARs in stress situations. The aim of the present work was to 

measure gene expression, protein levels and binding sites of β-ARs in the heart of rats exposed to 

acute and repeated immobilization (IMO) stress. Single and repeated IMO significantly increased 

EPI conc. in the heart ventricles, while NE was only slightly changed. We found a significant 

decrease of β2-AR mRNA, protein levels and binding sites in the left ventricle of animals 

exposed mainly to repeated IMO stress. These changes were not accompanied by similar changes 

in the right ventricle. No significant changes of β1-AR gene expression and proteins were 

observed in ventricles of rats exposed both, to a single or repeated IMO. The most interesting 

changes were found in β3-AR mRNA levels, proteins and binding sites, which were significantly 

increased in the left ventricle especially of repeatedly IMO rats. As by β2-AR, the changes of β3- 

AR were not observed in right ventricle. Taken together, we found decreased number of cardiostimulating 

β2-AR and increased number of cardio-inhibitive β3-AR in the left ventricle of 

repeatedly stressed rats. Moreover, repeated stress substantially reduced the basal and forskolinstimulated 

adenylyl cyclase activity in left ventricles. Our results suggest that maintaining of 

cardiac homeostasis under stress conditions is achieved by balance among the β-AR. We have 

demonstrated ventricle specific changes in β2- and β3-AR gene expression, which is probably 

related to different physiological function of ventricles. This is the first report showing on 

molecular genetic level the importance of cardiac β3-AR receptors coping with stress. 

Disclosures: R. Kvetnansky, None; M. Novakova, None; J. Petrak, None; O. Krizanova, 

None; J. Myslivecek, None; A. Tillinger, None. 

Poster 





Support: Lundbeck Inc.

MH 38273 

HD 05751 

Title: Corticotrophin-releasing hormone-induced excitability in a catecholaminergic neuronal 

cell line 

Authors: *A. W. LEE 1 , S. A. RABACCHI 2 , I. A. ANTONIJEVIC 2 , K. E. SMITH 2 , Q. 

ZHANG 1 , L.-M. KOW 1 , D. W. PFAFF 1 ; 

1 Neurobio. & Behavior, Rockefeller Univ., New York, NY; 2 Lundbeck Res. USA, Inc., Paramus, 

NJ 

Abstract: Hyperactivity of neurons secreting corticotrophin-releasing hormone (CRH) is a 

serious condition that is likely to underlie the pathophysiology of many disorders. CRH regulates 

the neuroendocrine stress response, resulting in stimulation of corticotrophin and subsequent 

glucocorticoid secretion. The locus coeruleus is activated by stress, resulting in increased 

noradrenaline secretion. CATH.a cells are a CNS catecholaminergic cell line immortalized from 

the locus coeruleus. Patch clamp recordings were carried out in current clamp mode to examine 

the effects of corticosterone (CORT), dexamethasone (DEX), CRH, or a combination DEX and 

CRH on CATH.a cells. Bath application of DEX (1 or 2 µM, 5-10 min) or CORT (1 µM, 5-10 

min) depolarized about one third of the cells. CRH alone at (1 or 10 µM, 3 min) reversibly 

depolarized CATH.a cells. When the combination of DEX (1 µM) and various doses of CRH 

(0.01 - 10 µM) was tested, there was a synergistic effect at the highest concentration of CRH; 

DEX and CRH (10 µM) worked synergistically to depolarize the membrane potential to levels 

more than 50% greater than DEX or CRH alone. Analysis of these cells by reverse 

transcriptase/polymerase chain reaction revealed the presence of mRNAs for glucocorticoid 

receptors and CRH receptors type 1 and 2. These data indicate that CATH.a cells are sensitive to 

CRH, and that under acute DEX treatment, are still more sensitive to CRH. Current studies 

address chronic exposure to glucocorticoids. Further studies are also being conducted on CAD 

cells, a variant of CATH.a in which morphological differentiation can be initiated. 

Disclosures: A.W. Lee, None; S.A. Rabacchi, None; I.A. Antonijevic, None; K.E. Smith, 

None; Q. Zhang, None; L. Kow, None; D.W. Pfaff, None. 

Poster 




Topic: E.06.d. Cellular actions of stress


Title: Chronic variable social stress: behavioral and biochemical studies in an animal model 

Authors: *N. J. WEINSTOCK, C. A. MARCINKIEWCZ, D. P. DEVINE; 

Psychology - Behav Neurosci, Univ. of Florida, Gainesville, FL 

Abstract: Major depression is a debilitating emotional disorder affecting millions of adults 

annually. Our current understanding of the etiology of major depression underscores the 

significance of emotional stress in conferring vulnerability to develop this devastating disorder. 

We have previously investigated the behavioral and biochemical consequences of emotional 

stressors using social defeat stress. We have shown that repeated exposure to social defeat 

produces an increase in behavioral despair in the Porsolt Test, as well as elevations in the basal 

concentrations of circulating adrenocorticotropic hormone (ACTH) and corticosterone (CORT). 

Most importantly, we found changes in gene expression of connexin36 within the limbic system. 

We have now developed a rat model of chronic variable stress (CVS) that utilizes unpredictable 

naturalistic stressors that produce significant emotional consequences in the rats. This regimen 

includes social defeat, blasts of loud white noise, cage tilt, strobe light, and novel environment 

stressors that are presented on a pseudo-random schedule with 2-3 stressors per day for a period 

of 14 days. On the final day, predator odor (cat urine) is used to evaluate responsiveness to an 

acute novel stressor. In contrast to our previous work with social defeat, this regimen introduces 

greater variability in the stressful stimuli, and allows us more control over the intensity of 

individual stressors. We have found that this regimen produces larger and more consistent 

physiological effects, including thymus involution and reduction in body weight gain. We are 

continuing to evaluate this regimen as a model of stress-induced psychopathology and as a tool 

to investigate emotional stress-induced changes in limbic gene expression and neurochemistry. 

Disclosures: N.J. Weinstock, None; C.A. Marcinkiewcz, None; D.P. Devine, None. 

Poster 





Title: Chronic oxotremorine treatment reverses stress-induced impairment of hippocampal longterm 

potentiation and spatial learning in rats

Authors: *B. N. SRIKUMAR, V. PRIYA, V. BHAGYA, J. VEENA, T. R. RAJU, B. S. 

SHANKARANARAYANA RAO; 

Dept Neurophysiol, Natl. Inst. Mental Hlth. Neu Sci., Bangalore, India 

Abstract: Severe, traumatic stress or repeated exposure to stress can result in long-term 

deleterious effects on the hippocampus, including dendritic atrophy and cell death, which, in 

turn, result in memory impairments and precipitate neuropsychiatric disorders like depression 

and anxiety. Further, chronic stress is known to affect hippocampal long-term potentiation 

(LTP), a synaptic model of memory. In an earlier study, we reported that the cholinergic 

muscarinic agonist, oxotremorine reverses chronic restraint stress-induced impairment in the 

performance of the radial arm maze task [Srikumar et al (2006) Neuroscience 143:679-688]. 

However, the cellular mechanisms recruited by oxotremorine to produce its effects are not 

known. Accordingly, in the present study, we assessed the effects of oxotremorine treatment on 

LTP in the Schaffer collateral-CA1 synapses and spatial learning and memory in the Morris 

water maze. Male Wistar rats were subjected to restraint stress for 21 days (6h/day) and 

oxotremorine treatment (0.1 and 0.2 mg/kg, i.p, once daily) for 10 days followed by behavioral 

evaluation in the water maze. In LTP experiments, rats underwent 10 daily injections of 

oxotremorine (0.2 mg/kg, i.p) after 21 days of restraint stress. Following 20 minutes of baseline 

recording of extracellular field potentials at the Schaffer collateral-CA1 synapses, LTP was 

induced by the high frequency stimulation (HFS) protocol. Stressed rats showed impairment in 

hippocampal CA1-LTP and the acquisition of the water maze task. Oxotremorine treatment 

significantly reversed the stress-induced impairment of the hippocampal CA1-LTP and learning 

deficits. These results together with our earlier findings demonstrate the cholinergic system as a 

good target in the development of drugs for the treatment of stress and stress-related disorders. 

Disclosures: B.N. Srikumar , None; V. Priya, None; V. Bhagya, None; J. Veena, None; T.R. 

Raju, None; B.S. Shankaranarayana Rao, None. 

Poster 





Support: NIH Grants NS28912, MH73136 

NARSAD young investigator award 

Title: Stress induces dendritic spine loss within hours in adult hippocampus: novel mechanisms

Authors: *Y. CHEN, C. DUBÉ, C. RICE, A. KOROSI, A. IVY, T. BARAM; 

Dept Ped/ Anat. Neurobio., Univ. Cal Irvine, Irvine, CA 

Abstract: Rationale: Chronic stress leads to dendritic remodeling in the hippocampus, and this is 

likely a result of loss of dendritic spines. However, whether acute stress provokes loss of spines 

in hippocampal pyramidal cells has not been fully investigated. In addition, the nature of the 

mechanisms underlying spine and dendritic reduction with stress is unclear. Corticotropinreleasing 

hormone (CRH) is released in hippocampus by stress, and activates the CRH receptor 

CRFR1 on pyramidal neurons (Chen et al., 2004, 2006). CRH exposure leads to a rapid spine 

loss in hippocampal organotypic cultures, which is blocked by a selective CRFR1 antagonist 

(Chen et al., 2008). Here, we tested whether exposure of adult rodents to acute stress induces loss 

of dendritic spines, as well as the functional consequences. 

Methods: Adult Thy1-YFP transgenic mice were exposed to a 5 hrs restraint and noise stress. At 

the end of this stress experience, spine densities on apical dendrites of CA3 pyramidal cells were 

compared among the following groups: (1) stressed, (2) stress-free controls, (3) pre-infused (icv) 

with the CRFR1 antagonist NBI 30775 (15 κg in 1 κl) 30 min prior to the 5 hr stress, and (4) 

stress-free and antagonist-infused. 

Results: Stress induced a rapid reduction of spine density in apical dendrites of CA3 neurons, 

primarily on the 3rd and 4th order dendritic branches, the main postsynaptic target of excitatory 

commissural/associational fibers. The selective CRFR1 antagonist, abolished the stress-induced 

decline of spine density. In further support of a role for CRH in rapid spine loss after stress, twophoton 

live imaging demonstrated an increased spine retraction within minutes after the infusion 

of the peptide to organotypic cultures of hippocampus. 

Conclusions: (1) Acute stress induces a rapid (within hours) spine loss in selective dendritic 

domains of adult hippocampus; (2) The activation of CRFR1, most likely by its endogenous 

ligand CRH, is involved in this process; and (3) CRH may promote spine loss via selective 

acceleration of spine retraction. 

Disclosures: Y. Chen, None; C. Dubé, None; C. Rice, None; A. Korosi, None; A. Ivy, 

None; T. Baram, None. 

Poster 





Title: Effects of repeated venlafaxine treatments on chronic mild stress-induced changes in 

corticosteroid receptor immunoreactivity in the rat hippocapmpus

Authors: *K. J. LEE 1 , H.-J. KIM 1 , H.-J. KIM 1 , S.-W. KANG 1 , S.-G. SHIN 1 , S.-H. CHOI 1 , K.- 

H. SHIN 1 , M.-S. LEE 2 ; 

1 Dept of pharmacology, Korea Univ, college of medicine, Seoul, Republic of Korea; 2 Psychiatry, 

Korea university, Seoul, Republic of Korea 

Abstract: Dysregulation of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) 

has been implicated in depression and antidepressant effect. Despite the fact that chronic stress 

or repeated antidepressant treatment changes GR or MR expression in the hippocampus, less is 

known about the effect of chronic mild stress (CMS), one of the animal models of depression, on 

the changes in GR and MR as well as the antidepressant pretreatment effect on these changes. To 

address this issue, we observed the effects of venlafaxine pretreatment on CMS-induced changes 

in immunoreactivities of GR and MR in the hippocampus. Venlafaxine, serotonin and 

norepinephrine reuptake inhibitor, is known for its rapid and effective antidepressant activity. 

CMS significantly increased GR immunoreactivity in granule cell layer of hippocampus (GCL) 

and venlafaxine pretreatment abolished the CMS-induced increase in GR-IR. Similar changes in 

GR-IR by venlafaxine pretreatment and CMS were observed in the CA1 of hippocampus. On the 

contrary, CMS slightly decreased MR immunoreactivity (MR-IR) in the CA1 and these changes 

were recovered by venlafaxine pretreatment. Interestingly, venlafaxine pretreatment alone 

significantly increased MR-IR in the CA1. In addition to changes in corticosteroid receptors, 

CMS significantly increased plasma corticosterone levels, but venlafaxine pretreatment prior to 

CMS did not significantly affect these changes. These results show that CMS significantly 

increased and decreased GR and MR in the hippocampus, respectively and that these changes 

were restored to normal by venlafaxine pretreatment. Thus, the present study suggests that 

venlafaxine may exert antidepressant effect through opposing the chronic stress-induced changes 

in GR and MR in the hippocampus. 

Disclosures: K.J. Lee, None; H. Kim, None; H. kim, None; S. Kang, None; S. Shin, None; S. 

Choi, None; K. Shin, None; M. Lee, None. 

Poster 





Support: MH 41256 

5P50MH58911

Title: Decreased hippocampal spine density as a morphological marker of chronically stressed 

adult male mice with intact neurotrophic support 

Authors: *A. M. MAGARINOS, B. S. MCEWEN; 

Lab. Neuroendocrinol, Rockefeller Univ., New York, NY 

Abstract: Chronic restraint stress (CRS) results in a specific apical dendritic retraction and 

simplification -reduced branching- of the pyramidal cells of the hippocampal CA3 subregion of 

both rats and mice. The dendritic remodeling is not accompanied by signs of cell damage and is 

no longer detected when the CRS is discontinued pointing to a reversible plasticity within the 

hippocampus. To further investigate the consequences of CRS we tested the hypothesis that the 

stress-induced CA3 dendritic retraction is associated with a decreased spine density, providing 

an additional morphological substrate for the changes in synaptic connectivity. Male adult 

C57BL/6 mice (n=6/experimental group) were either left undisturbed or subjected to 21 days of 

daily restraint stress and their hippocampi processed for the Golgi impregnation technique. For 

the estimation of spine density, spines were classified in stubby, thin and mushroom, based on 

their morphology. Dendritic segments in a single plane of focus were sampled from lateral thirdorder 

dendrites located in the middle third of the stratum radiatum of both CA1 and CA3 

hippocampal pyramidal neurons. In addition to dendritic retraction and simplification, the CA3 

dendritic tree of stressed mice showed a significant decrease in total spine density (Cont = 11.94 

+ 0.47 vs. Stress = 10.15 + 0.45 spines/10um, p< 0.05, Student‟s t-test). As a control, we 

estimated the spine density of the apical trees of CA1 pyramidal neurons which do not show 

dendritic retraction and simplification after the chronic stress paradigm. Interestingly, we found 

that stress also significantly decreased CA1 spine density (Cont = 14.74 + 0.56 vs. Rest = 11.65 

+ 0.60 spines/10um, p




SHRF 

Title: Repeated exposure to stress produces differential effects on reelin and synaptophysin 

expression in the amygdala and hippocampus 

Authors: *A. L. LUSSIER 1 , H. J. CARUNCHO 2 , L. E. KALYNCHUK 1 ; 

1 Psychology, Univ. Saskatchewan, Saskatoon, SK, Canada; 2 Cell Biol., Univ. of Santiago de 

Compostela, Galicia, Spain 

Abstract: Repeated stress is associated with extensive dendritic remodeling in the hippocampus 

and amygdala. However, it is also associated with neuronal atrophy within the hippocampus and 

neuronal growth in the amygdala. As chronic stress is a risk factor for depression, this has led to 

the suggestion that stress-induced changes in neuronal connectivity within these structures may 

be important for the development of depression. Recent studies have shown that changes in the 

glycoprotein reelin may be involved in the pathophysiology of several neuropsychiatric disorders 

including depression. Although reelin guides the migration of neurons during embryonic 

development, new evidence has shown that it may also be important for dendritic spine plasticity 

in the adult brain. Nineteen naive male rats were split into one of four groups; repeated restraint 

stress (6 hrs/day), repeated corticosterone injections (CORT, 40 mg/kg, s.c.), handled, or vehicle. 

The rats were treated for 21 consecutive days, and then sacrificed via transcardial perfusion. We 

focused our immunohistochemical analysis on the hippocampus and amygdala. For the 

hippocampus, we found that CORT treatment decreased the number of reelin positive cells 

within the stratum oriens of the CA1, stratum lacunosum, and granule cell layer of the dentate 

gyrus. Restraint stress had no effect. For the amygdala, we found that CORT treatment increased 

reelin cells in the lateral amygdala and decreased reelin cells in the medial and cortical 

amygdala. No changes were found in the basomedial, basolateral and central amygdala. Restraint 

stress decreased reelin positive cells in the basolateral and cortical amygdala only. Finally, 

densitometric analysis revealed that CORT treatment increased synaptophysin expression 

primarily in the subgranular zone and the hilus, but restraint had no consistent effects on 

synaptophysin. These results suggest that the differential effects of stress on hippocampal and 

amygdala circuitry may be partly due to alterations in reelin. This may be relevant to the 

pathophysiology of depression. 

Disclosures: A.L. Lussier , None; L.E. Kalynchuk, None; H.J. Caruncho, None. 

Poster





Support: The Center for the Study of Traumatic Stress 

Title: Cerebellar expression of GABA receptor subunit mRNA in male and female rats after 

stress 

Authors: *X. JIANG 1,2 , G. XING 2 , J. CARLTON 2 , L. ZHANG 2 , H. LI 2 , C. FULLERTON 2 , R. 

URSANO 2 ; 

1 Pharmacol, Univ. Virginia, Charlottesville, VA; 2 USUHS, Bethesda, MD 

Abstract: γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, 

acting through two types of pharmacologically and molecularly distinct GABAA and GABAB 

receptors. Most of GABAA subuits are highly expressed in the cerebellum that may play an 

important role in both motor-related functions (such as anterograde amnesia, ataxia, motor 

incoordination) as well as stress-related psychological functions such as attention, learning and 

memory. With real-time PCR, we determined the mRNA expression of GABAA receptor 

subunits (α1-6, β1-3, δ, γ1-3) in the cerebellum of both male and female young adult rat after 3day 

repeated restraint tail-shock stress and compared with that of the non-stressed control rats 

(with the house-keeping gene beta-actin served as the reference). GABAA receptor subunit 

mRNA did not change significantly in male rat cerebellum after 3d-repeated stress. GABAA δ 

mRNA, however, decreased significantly (P



Support: NIH Grant 5F32MH075506-03 

Title: Effects of chronic stress and estrogen on spine density in the prefrontal cortex 

Authors: *R. SHANSKY 1 , C. HAMO 1 , B. S. MCEWEN 2 , J. H. MORRISON 1 ; 

1 Dept Neurosci, Mount Sinai Sch. Med., New York, NY; 2 Lab. of Neuroendocrinology, 

Rockefeller Univ., New York, NY 

Abstract: Chronic restraint stress has been shown to induce morphological changes in neurons 

of the anterior cingulate, prelimbic and infralimbic regions of the medial prefrontal cortex 

(mPFC). To date, however, this work has only been done in male rats, and neither sex 

differences nor the potential effects of estrogen on this phenomenon have been investigated. 

Moreover, the specific targets of these neurons are unknown. Stress-related mental illnesses like 

Major Depressive Disorder and PTSD are twice as prevalent in women as in men and are 

characterized by abnormalities in mPFC and amygdala activity. These regions maintain 

reciprocal connectivity, forming a circuit that governs learning, memory and behavior based on 

emotionally salient information. The current study investigated whether chronic immobilization 

stress can cause spine changes in infralimbic neurons that project to the basal nucleus of the 

amygdala (BA), and what effects estrogen might have on these neurons. The retrograde tracer 

Fast Blue (FB) was injected into the BA of male and ovariectomized female rats with (OVX + E) 

and without (OVX) estrogen replacement. Animals were then exposed to 10 days of chronic 

immobilization stress, sacrificed and their brains removed. FB-labeled neurons in layers II/III of 

the infralimbic (IL) cortex were filled with Lucifer Yellow, and the filled cells were analyzed for 

spine density. Unstressed OVX rats showed a decrease in spine density compared to unstressed 

OVX + E rats, but both female groups showed a stress-related increase in spine density, such that 

there were no differences between the two stressed groups. In contrast to previous reports, there 

were no spine density differences between stressed and unstressed male rats. These results 

suggest that the effects of stress in the mPFC may be circuit-specific, and that the mPFCamygdala 

circuit may be particularly resilient against stress-induced atrophy in males. 

Additionally, the observed estrogen-stress interaction implies that hormones can tightly modulate 

stress-related changes in neuronal morphology. 

Disclosures: R. Shansky , None; C. Hamo, None; B.S. McEwen, None; J.H. Morrison, None. 

Poster 





Title: Rapid effect of corticosterone in the amygdala 

Authors: *H. KARST 1 , S. BERGER 2 , G. SCHÜTZ 2 , M. JOËLS 1 ; 

1 SILS-CNS, Univ. of Amsterdam, Amsterdam, Netherlands; 2 German Cancer Res. Inst., 

Heidelberg, Germany 

Abstract: In several brain structures e.g. the hypothalamus and the hippocampus, rapid effects of 

corticosterone have been described. These effects are most likely mediated via membrane 

located corticosteroid receptors. We reported a rapid increase by corticosterone of miniature 

EPSC frequency via mineralocorticoid receptors located on the membrane (mMRs) of 

hippocampal CA1 neurons (PNAS, 2005, 102, 19204).The receptors that are responsible for this 

effect appear to have the same genetic origin as the nuclear receptors, but have a 10 fold lower 

affinity for corticosterone. Therefore in contrast to the nuclear MRs, which are already activated 

with basal corticosterone levels, the mMRs might affect excitability of CA1 neurons only during 

periods of stress. The mMRs are located presynaptically and upon activation cause an increase in 

the release of glutamate via the MEK-ERK1/2 pathway. 

Another limbic structure that is in involved in stress related behavior is the amygdala. Especially 

memory consolidation of emotionally salient events depends on this structure. Recently, 

membrane located glucocorticoid receptors were demonstrated in the amygdala (Neuroscience, 

2005, 136, 289). In the current study we investigated a possible rapid modulation by 

corticosterone of the spontaneous mEPSCs in two substructures of the amygdala, the basolateral 

amygdala (BLA) and the central amygdala (CeA). In the CeA there was no rapid effect of 

corticosterone, neither on the frequency nor on the mean amplitude of the mEPSCs. However, in 

the BLA, application of 100 nM corticosterone induced an increase in the frequency within 10 

minutes. The mean amplitude of the mEPSCs was not affected by corticosterone. In contrast to 

the hippocampus the effect in the BLA was not reversible, indicating that rapid effects might 

develop into long term effects. In inducible neuron-specific GR knockout mice the rapid effect 

persisted. However in inducible neuron-specific MR knockout mice, the rapid effect was entirely 

absent. The data suggests that in the BLA too corticosterone affects the glutamate release 

probability via the MRs. However, in the BLA these effects seem to be far more persistent. This 

could mean that stressors involving activation of the amygdala have a more lasting impact. We 

are currently investigating the possible mechanism underlying the long lasting effect of 

corticosterone on the mEPSC frequency. 

Disclosures: H. Karst, None; S. Berger, None; G. Schütz, None; M. Joëls, None. 

Poster 

282. Stress and the Brain: Cellular Actions of Stress




Title: The comparison of behavior and structural reorganization in motor cortex of rats after 

immobilization 

Authors: *V. N. MATS, I. LEVSHINA, N. PASIKOVA, N. SHUYKIN; 

Inst. Higher, Moscow, Russian Federation 

Abstract: INTRODUCTION: 

While studying behavioral responses to the experimental stress in animals most researchers 

prefer using emotional stress rather than any other physical or painful stimuli. Numerous data 

about behavioral changes in stressed animals have been reported. However the influence of the 

emotional stress on morphological status of brain structure has not been documented sufficiently. 

We investigated behavioral changes in rats and accompanying morphological changes in their 

motor cortex after the emotional stress. 

Methods: 

Wistar rats (n=20) were stressed by the interrupted immobilization, wich carried out for 7-8 

hours a day during one week .“Open field” test was used to test behavior in experimental and 

control rats before and after immobilization. In the end of experiment rats were decapitated, their 

brains were fixed and frontal slices of forebrain were stained by Nissle method. The number of 

neurons, neuroglia, and cells square in motor cortex were estimated. 

Results: 

No differences were found in size,and density of glial cells and neurons between experimental 

and control animals. However, morphometric analyses showed significant increase of hypoxical 

neurons density in motor cortex of stressed rats. After 1 week of immobilization, the number of 

dark, stretched neurons with the corkscrew apical dendrite was estimated as 177% of those in 

control rats. Hypoxical neurons were concentrated asymmetrically with those prevailing in the 

left hemisphere in control rats and in the right hemisphere in stressed rats. Behavioral changes of 

immobilized rats were characterized by increased horizontal and vertical locomotor activity and 

duration of III and IV faze of grooming (comfortable grooming) in the “open field” test. 

CONCLUSIONS: 

Results showed the increased motor activity in animals despite the increase in concentration of 

hypoxical neurons after immobilization.. Experimental rats are considered as a model of 

redistribution of functional brain activity with preferential intensification of the left-brain 

hemisphere. 

Disclosures: V.N. Mats , None; I. Levshina, None; N. Pasikova, None; N. Shuykin, None. 

Poster






Title: BDNF deficiency and stress have overlapping effects on apical dendrites and excitatory 

transmission in medial prefrontal layer V pyramidal cells 

Authors: *R.-J. LIU, G. K. AGHAJANIAN; 

Dept Psychiatry, Yale Sch. Med., New Haven, CT 

Abstract: Recently, we reported that stress-induced apical dendritic atrophy is associated with 

diminished excitatory responses to apically-targeted excitatory inputs regulated by serotonin (5- 

HT) and hypocretin in medial prefrontal cortex (mPFC) 1 . However, it remains unclear what 

neurotrophic mechanisms may underlie these stress-induced changes. There is a growing body of 

evidence that stress leads to decreased expression of brain-derived neurotrophic factor (BDNF) 

in mPFC and other limbic structures, which could be related to changes in cortical dendrites 2 . 

Previously, in BDNF conditional knockout mice, we have found a marked decrease in 5-HT2Amediated 

excitatory postsynaptic currents (EPSCs) in layer V mPFC pyramidal cells 3 . Therefore, 

we hypothesized that deficits in BDNF could contribute to the stress-induced changes in 

morphology and function in layer V pyramidal cells. Using whole cell recording and two photon 

laser scanning in brain slice, we examined both electrophysiological and morphological changes 

in the same layer V pyramidal cells in wild type and heterologous BDNF knock out mice 

(BDNF +/- ) after exposure to repeated mild immobilization stress. As in our previous rat studies, 

we found that repeated stress in wild type mice results in a reduction in the frequency of 5-HT- 

and hypocretin-induced EPSCs as well as a reduction in apical but not basal dendritic branch 

length. In BDNF +/- mice, such deficits in dendritic branch length and EPSC responses were 

already present, even in the absence of externally applied stress. Surprisingly, in BDNF +/- mice 

there was no additional reduction in dendritic branch length and 5-HT-induced EPSCs after 

repeated stress. In contrast, there was a further decrement in the frequency of hypocretin-induced 

EPSCs after stress in the BDNF +/- mice. These results indicate that BDNF deficient mice have a 

constitutive deficit in both apical dendritic branch length and 5-HT and hypocretin-induced 

excitatory responses in layer V pyramidal cells. However, only hypocretin-modulated excitatory 

EPSCs showed a further decrease in BDNF +/- mice after repeated stress, implying the 

involvement of factors in addition to BDNF. 

1. Liu and Aghajanian, Proc Natl Acad Sci U S A. (2008)105:359-64. 

2. Gorski et al., J Neurosci. (2003) 23:6856-65. 

3. Rios et al., J Neurobiol. (2006) 66:408-20. 

Disclosures: R. Liu, None; G.K. Aghajanian, None.

Poster 

283. Stress-Regulated Pathways II 



Topic: E.06.f. Stress modulated pathways 

Support: Hazewinkel-Beringer Fellowship 

Title: Sex specific modifications of signaling cascades in the limbic system of rats after longterm 

stress and recovery: implications for depression 

Authors: *R. WICHMANN, Y. LIN, P. BAKKER, G. J. TER HORST, C. WESTENBROEK; 

Rijksuniversiteit Groningen, Groningen, Netherlands 

Abstract: Depression is currently one of the leading causes of disability worldwide and women 

show a higher prevalence for this affective disorder than men. 

The inability to experience pleasure (anhedonia), one of the core symptoms of depression, is 

likely caused by disturbances in the dopaminergic reward system. 

Stress is considered to be a profound factor mediating the onset of depression and chronic stress 

exposure can induce an anhedonic-like state in rodents. 

However, the biological basis of the gender difference in depression and the effects of chronic 

stress and recovery after long-term stress still remain poorly understood. 

Therefor the aim of this study was to assess sex differences in the reward system of rats in 

response to different durations of footshock stress (1 day; 42 days and 21 days followed by a 21 

day recovery period). 

Increased hypothalamic-pituitary-adrenocortical (HPA) axis activation was demonstrated by 

elevated plasma corticosterone levels. This elevation was seen in all stress groups which 

indicates that no habituation to the stress procedure occurred over time. 

Sex and brain region differences where found in the expression of cAMP response elementbinding 

protein (CREB) and the phosphorylated variant (pCREB). 

Within the nucleus accumbens shell, a major brain reward region, western blot analysis showed a 

significantly reduced ratio of pCREB to CREB levels in all stress groups in female but not male 

rats. The opposite was true for the nucleus accumbens core. Here the males showed a reduced 

ratio of pCREB to CREB compared to females. 

These effects were not normalized by a recovery period after the chronic stress exposure. 

On the basis of this results we examined different cellular signaling cascades known to influence 

CREB phosphorylation. Unexpectedly we found a stress induced increase in the ratio of 

phosphorylated to total calcium/calmodulin-dependent kinase IV (pCaMK IV/CaMK IV) as well 

as the ratio of cAMP-protein kinase A (pPKA/PKA) in the nucleus accumbens shell of female

ats. However these changes where not visible in male rats. 

The results suggest that male and female rats respond to stress and recovery in a different way 

and by using different strategies and probably second messenger pathways. This might be a 

factor which explains the gender differences in the predisposition for depressive disorder. 

Disclosures: R. Wichmann , None; Y. Lin, None; P. Bakker, None; G.J. Ter Horst, None; C. 

Westenbroek, None. 

Poster 





Support: KAKENHI 

Title: Modulation of the human emotional system by subacute fatigue load: an fMRI study 

Authors: *N. YAHATA 1 , T. SASAKI 3 , S. MATSUMOTO 3 , T. MATSUDA 4 , H. SUZUKI 1 , Y. 

OKUBO 2 , K. SAKAI 3 ; 

1 Dept Pharmacol, 2 Dept Neuropsychiatry, Nippon Med. Sch., Tokyo, Japan; 3 Inst. Sci. of Labor, 

Kawasaki, Japan; 4 Tamagawa Univ., Machida, Japan 

Abstract: Identifying neural correlates of fatigue and its effects on cognitive functions has 

significant bearing on understanding the etiology and prevention of stress-induced, 

neuropsychiatric disorders. In particular, to investigate how the balance within the human 

emotional circuit, including both limbic structures and prefrontal cortex, can be perturbed under 

chronic fatigue is crucial for understanding the mechanisms of the development of mood and 

anxiety disorders. Toward this end, we present here an fMRI study that aimed to quantitatively 

evaluate the temporal changes in activities in the human emotional network that were triggered 

by subacute fatigue load. Sixteen healthy males (mean age 27.9 years) participated in a 10-day 

fatigue-loading session, during which they underwent a monotonic typing task (11 hr), shortened 

sleep time (5 hr) and other medical tests every day. Before and after this session, they underwent 

an fMRI session wherein they viewed and rated the pleasure valence of pictures selected from 

the International Affective Picture System (IAPS, Lang et al. 2005). Another sixteen agematched 

controls (mean age 28.3 years) with no fatigue load were independently recruited and 

were scanned under the same protocol. This study was approved by the institutional ethics 

committees. All subjects gave informed consent prior to the study, confirming that they could 

decline their participation at any time during the experiment. Our major findings were that (1) in

the post-fatigue scan, the fatigue group exhibited decreased activities in several components of 

the limbic system including the anterior cingulate while viewing both unpleasant and pleasant 

pictures, whereas (2) the control group exhibited rather increased activities in these regions. 

Whilst previous studies on depression suggest that the limbic system plays a pivotal role in the 

pathophysiology of depression, our study further indicates that this structure is the primary site 

susceptible to even a subacute form of fatigue load. 

Disclosures: N. Yahata , None; T. Sasaki, None; S. Matsumoto, None; K. Sakai, None; H. 

Suzuki, None; T. Matsuda, None; Y. Okubo, None. 

Poster 





Support: NICHD U54 HD 18185 

NICHD T32 HD 07133 

Title: Evidence for a non-neuroendocrine role of corticotropin-releasing hormone (CRH) in 

female cynomolgus monkeys sensitive to stress-induced reproductive dysfunction 

Authors: *S. M. HEROD 1,2 , M. L. CENTENO 1,3 , C. L. BETHEA 1,3 , J. L. CAMERON 1,2,3,4 ; 

1 Oregon Natl. Primate Res. Ctr., OHSU, Beaverton, OR; 2 Dept of Behavioral Neurosci., 3 Dept of 

Neurosci., 4 Dept of Obstetrics & Gynecology, OHSU, Portland, OR 

Abstract: In response to mild stress, some individuals develop reproductive dysfunction while 

others maintain normal menstrual cyclicity. Female monkeys, when exposed to mild stress, can 

be categorized as Highly Stress-Resilient (“HSR”; maintain normal menstrual cycles in response 

to stress), Medium Stress-Resilient (“MSR”; slowly become anovulatory in response to 

prolonged stress), or Stress-Sensitive (“SS”; rapidly become anovulatory in response to stress). 

The most robust neurochemical difference that we have found between groups is that SS 

monkeys have lower physiological release of serotonin and lower expression of genes in the 

serotonergic pathway compared to HSR animals. We have also reported increased CRH gene 

expression in the caudal paraventricular nucleus of the hypothalamus in SS monkeys, suggesting 

that heightened adrenal axis activity might contribute to suppression of reproductive function in 

these animals. To examine whether sensitivity to stress-induced reproductive dysfunction is 

associated with elevated activity of the adrenal axis 16 adult female cynomolgus monkeys were

fitted with indwelling catheters that allow undisturbed blood collection. SS (n=5) monkeys did 

not differ from HSR (n=6) or MSR (n=5) monkeys in cortisol secretion over a 24-hour period 

(F13,2=2.135, p=0.158), in response to an acute psychological stressor (F13,2=0.353, p=0709), or 

in response to dexamethasone negative feedback (F13,2=0.463, p=0.639). In another group of 

animals categorized for degree of sensitivity of the reproductive axis to stress, we collected brain 

tissue in the early follicular phase of a control, nonstressed menstrual cycle. 

Immunocytochemical staining of CRH fibers was examined in SS (n=3) and HSR (n=4) 

monkeys. The immunostained area (µm) was significantly greater in both the dorsal raphe 

nucleus (HSR: 64,083±5,670; SS: 116,785±12,734; p=0.009) and median raphe nucleus (HSR: 

57,842±7,347; SS: 89,225±8,632; p=0.039) of SS animals. We conclude that increased 

sensitivity to stress-induced reproductive dysfunction is not associated with elevated adrenal axis 

activity, but that CRH neurons in the caudal PVN might be contributing in a non-neuroendocrine 

manner to suppress serotonergic function in the raphe nucleus of animals that show increased 

sensitivity to stress-induced reproductive dysfunction. 

Disclosures: S.M. Herod, None; M.L. Centeno, None; C.L. Bethea, None; J.L. Cameron, 

None. 

Poster 





Support: NWO Grant 864.05.008 

EURON MEST-CT-2005-020589 

Title: Exon-specific regulation of BDNF transcripts in the urocortin1 neurons of the nonpreganglionic 

Edinger-Westphal nucleus 

Authors: *B. GASZNER 1,2 , N. DERKS 2 , M. MAREIKE 2 , E. W. ROUBOS 2 , T. KOZICZ 2 ; 

1 Anat., Univ. of Pecs, Pecs, Hungary; 2 Donders Ctr. for Neurosci., Dept. of Cell. Animal 

Physiol., Nijmegen, Netherlands 

Abstract: Brain-derived neurotrophic factor (BDNF), a growth factor regulated through the 

expression of nine exons that each have their specific promoter, is expressed mainly in the limbic 

forebrain and hippocampus, where it is involved in neuronal growth and differentiation. BDNF 

was implicated in the pathogenesis of stress-related mood disorders such as depression, which

show a higher prevalence in females than in males. The highly complicated BDNF gene structure 

offers many opportunities to regulate the expression of the gene products. New insights into the 

site, gender, and exon-specific regulation of BDNF may add to our knowledge about 

pathogenesis of psychopathologies. In the non-preganglionic Edinger-Westphal nucleus (npEW), 

a nucleus also involved in stress and stress-related mood disorders, BDNF mRNA expression 

was found to be decreased in male suicide victims but increased in female ones. The CRF 

peptide family member urocortin 1 (Ucn1) is predominantly expressed in the npEW. Recently 

we have shown that Ucn1 neurons contain BDNF and beta estrogen receptors, suggesting 

gender-specific differences in the npEW. Based on these observations we hypothesized that 

BDNF mRNA and protein contents of the npEW change upon stress and differ between male and 

female rats; in addition the BDNF gene is differentially regulated by the different types of 

stressors in the npEW. To confirm our hypotheses, we examined acutely and chronically stressed 

male and female rats vs. controls. In situ hybridization and immunohistochemistry revealed the 

presence of BDNF mRNA and protein in the Ucn1 neurons while RT-PCR demonstrated an 

increase in BDNF mRNA expression after chronic stress in males and after acute and chronic 

stress in females. Quantitative RT-PCR showed that exon 3, 4, 7, 8, are not expressed in the 

npEW, in contrast, acute stress induced expression of exons 6 and 9 in both males and females, 

while expression of exons 1 and 2 were induced in males only. BDNF protein was increased by 

acute stress in females but unaffected in males. We therefore propose that BDNF plays a role in 

the response of npEW Ucn1 neurons to stress, in a stressor- and gender-specific fashion. Further 

research on the significance of these findings could help to understand how the well known 

gender differences in stress-related brain diseases occur. 

Disclosures: B. Gaszner , None; N. Derks, None; M. Mareike, None; E.W. Roubos, None; T. 

Kozicz, None. 

Poster 



Program#/Poster#: 283.5/PP1 


Support: BK21 program 

Title: Identification of glucocorticoid and mineralocorticoid receptors on spinally- and 

medullary- projecting neurons in the hypothalamic paraventricular nucleus 

Authors: S. SHIN, T. HAN, S. LEE, *P. RYU; 

Dept. Vet. Pharmacol., Col. Vet. Med, Seoul Natl. Univ., Seoul, Republic of Korea

Abstract: When living organisms are exposed to stressors, sympathetic systems are activated in 

the level of periphery and brain. The hypothalamic paraventricular nucleus (PVN) is well known 

integrative center for autonomic responses to stressors. To study whether corticosteroids 

(CORTs) can act directly to the preautonomic neurons in the PVN which modulate sympathetic 

outflow, we aimed to show the expression of two corticosteroid receptors mediating the action of 

CORTs, glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs), on the PVN 

preautonomic neurons. To identify the preautonomic neurons in the PVN, the retrograde tracer, 

fluospheres red was injected into the intermediolateral cell column (IML) of spinal cord and 

rostral ventrolateral medulla (RVLM). Single cell RT-PCR and immunohistochemistry were 

performed to show the expression of GRs and MRs in the retrogradely labeled preautonomic 

neurons. We found that GR and MR mRNAs were expressed in the PVN preautonomic neurons 

projecting to the IML and RVLM. We also confirmed the presence of GR and MR proteins on 

preautonomic neurons in the PVN by immunohistochemistry. Collectively, this study shows the 

presence of two types of corticosteroid receptors on the preautonomic neurons in the PVN. Our 

results suggest that CORTs can directly modulate the sympathetic outflow by acting on PVN 

preautonomic neurons at the level of hypothalamus. 

Disclosures: S. Shin, None; T. Han, None; S. Lee, None; P. Ryu , None. 

Poster 





Support: MH 52619 to AS 

Title: Anxiety-like responses in the rat panic-model are regulated by orexin in the BNST 

Authors: *W. A. TRUITT, P. L. JOHNSON, Y. LEE, A. D. DIETRICH, S. D. FITZ, A. I. 

MOLOSH, A. SHEKHAR; 

Psychiatry, Indiana Univ. Sch. Med., Indianapolis, IN 

Abstract: Intravenous (i.v.) infusions of 0.5M sodium lactate provokes panic attacks that are 

comprised of severe anxiety and cardiorespiratory responses in the majority of panic disorder 

patients compared to controls. Similarly, in our animal model of panic, inhibiting GABA 

synthesis (using the GABA synthesis inhibitor l-allylglycine: l-AG) in the dorsomedial 

hypothalamus and perifornical nucleus (DMH/PeF) produces panic-prone rats that display panic 

attack-like responses (i.e., anxiety-like behavior and cardiorespiratory responses) following an

i.v. sodium lactate. Recently, we reported that the anxiety-like responses observed in this model 

have been localized to the bed nucleus of the stria terminalis (BNST; Johnson and Truitt et al 

2008, Neuropsychopharmacology). Additionally, we demonstrated that all aspects of the lactateinduced 

panic-like responses are blocked with systemic orexin (ORX) 1 receptor antagonists or 

silencing of ORX gene. Here we investigate the role of ORX within the BNST in regulating 

anxiety-like behaviors in this panic model. As previously observed, challenging panic-prone rats 

(l-AG treated) with sodium lactate reduced social interaction (SI) time compared to pre-l-AG 

responses, indicating an increase in anxiety-like behavior, which was blocked when the orexin 1 

receptor antagonist (SB3344867), but not vehicle, was infused into the BNST prior to the lactate 

challenge. In another experiment, we also determined that injecting an orexin 1 receptor agonist 

(orexin A) into the BNST 30 min prior to SI testing reduced SI times compared to vehicle and 

baseline SI times. These experiments suggest orexin is capable of inducing anxiety and that a 

locus of this action is the BNST. Furthermore, the anxiety-like effects observed in the rat panicmodel 

may be a result of orexin 1 receptor activation in the BNST. Activation of the orexin 1 

receptor has been reported to specifically potentiate NMDA receptors but not AMPA receptors, 

and NMDA activation in the BNST has been linked to increased anxiety/fear responses in the 

BNST. Thus we further investigated the role of orexin in the BNST on NMDA receptors versus 

AMPA receptor activation within the BNST using c-Fos as an indication of cellular responses. 

Disclosures: W.A. Truitt , None; P.L. Johnson, None; Y. Lee, None; A.D. Dietrich, 

None; S.D. Fitz, None; A.I. Molosh, None; A. Shekhar, None. 

Poster 





Support: Office of Naval Research N00014-02-1-0879 (HA & SJW) 

L'Oréal USA Fellowship for Women in Science (SMC) 

NIDA 5P01DA021633-02 (HA and SJW) 

Title: High- and low-novelty seeking females exhibit different maternal styles: impact of crossfostering 

on offspring‟s behavioral phenotype

Authors: *T. A. BEDROSIAN, S. M. CLINTON, A. D. ABRAHAM, S. J. WATSON, H. 

AKIL; 

Molec Behav Neurosci Inst., Univ. Michigan, Ann Arbor, MI 

Abstract: Our laboratory has selectively bred rats which exhibit natural differences in locomotor 

response to novelty. High Responder (HR) rats show exaggerated novelty-induced locomotion 

compared to Low Responder (LR) animals which show little activity in a novel environment. HR 

rats also exhibit enhanced aggression, impulsivity, psychostimulant self-administration, and 

neuroendocrine stress reactivity, and decreased anxiety- and depression-like behavior compared 

to LRs. Few studies have explored early environmental factors which may contribute to the 

emergence of HR-LR traits. While our breeding studies point to the heritability of the HR/LR 

traits, we also discovered important early life environmental factors, namely distinct HR vs. LR 

maternal care styles, which may also contribute to the phenotypes. We found that LR dams are 

generally more attentive to their pups (spending more time licking/grooming and arched-back 

nursing) during the first 2 postpartum weeks compared to HR dams. HR mothers spent more 

time engaged in self-directed behaviors such as eating, drinking, and self-grooming. The current 

studies utilized a cross-fostering paradigm to determine how HR vs. LR maternal style impacts 

offspring. In the first study, HR- and LR-bred pups were raised by their biological mother, or 

fostered to an HR or LR foster mother. In a second study, we cross-fostered intermediate 

responder (IR) rats which were created by interbreeding the HR/LR lines. IR rats typically 

exhibit a behavioral phenotype which is intermediate compared to the extreme behavior of the 

bred HR/LR lines. Adult offspring were tested to assess locomotor response to novelty, anxiety- 

and depression-like behavior, and neuroendocrine stress reactivity. Cross-fostering had minimal 

impact on pure-bred HR/LR offspring, most likely due to the strong genetic factors which have 

been enriched over several generations of breeding. The IR offspring, on the other hand, were 

more readily influenced by HR vs. LR maternal style, probably because their baseline behavioral 

phenotype was more moderate. IR offspring raised by HR mothers showed slight, but 

significantly increased locomotor response to novelty, reduced anxiety-like behavior, and 

slightly exaggerated neuroendocrine sensitivity compared to IRs raised by LR mothers. Ongoing 

studies will evaluate the neural underpinnings of these behavioral effects. 

Disclosures: T.A. Bedrosian , None; S.M. Clinton, None; A.D. Abraham, None; S.J. 

Watson, None; H. Akil, None. 

Poster 




Topic: E.06.f. Stress modulated pathways

Support: MRC Project Grant (UK) 

Marie Curie Excellence Grant (European Commission) 

Title: The effect of restraint stress on gene expression in mouse amygdala - microarray study 

Authors: M. MUCHA, *R. PAWLAK; 

Univ. of Leicester, Leicester, United Kingdom 

Abstract: The amygdala is an almond shaped structure that mediates various kinds of emotions 

including emotional learning and stress-induced anxiety. It has been demonstrated that stress 

induces dendritic growth in the lateral amygdala that may contribute to behavioural changes 

usually associated with stress. 

To identify genes involved in stress-induced changes in the mouse amygdala we performed 

microarray analysis using mRNA extracted from this region after six hours of restraint stress. We 

have identified 1611 transcripts that were significantly (p

NIDA 5P01DA021633-02 (HA & SJW) 

Title: Selectively-bred low novelty-seeking rats exhibit exaggerated anxiety- and depression-like 

behavior: a novel animal model of depression? 

Authors: *A. D. ABRAHAM, S. M. CLINTON, I. A. KERMAN, T. A. BEDROSIAN, S. J. 

WATSON, H. AKIL; 

Molec Behav Neurosci Inst., Univ. Michigan, Ann Arbor, MI 

Abstract: We have selectively bred Sprague-Dawley (SD) rats according to innate differences in 

“novelty-seeking”, a trait in rodents that predicts several key facets of emotional reactivity, 

including anxiety-like behavior, neuroendocrine stress response, and propensity to selfadminister 

drugs of abuse. Our High Responder (HR)-bred rats vigorously explore new 

environments compared to Low Responders (LR), which exhibit very little exploration. Notably, 

LR rats exhibit exaggerated anxiety- and depressive- like behaviors compared to their HR 

counterparts, suggesting that LR-bred rats may represent a novel animal model of depression. 

The present experiment directly compared LR-bred rats with an established animal model of 

depression - the Wistar-Kyoto (WKY) rat, which is known to exhibit a variety of depressive-like 

behavioral and physiological abnormalities. Adult HR- and LR- bred males from the 17 th 

generation of our breeding colony as well as WKY and SD rats purchased from Charles River 

(N=12 per group) were subjected to a test battery to assess (1) depression-like behavior in the 

Forced Swim Test, (2) locomotor response to novelty, and (3) anxiety-like behavior in the Light- 

Dark Box and the Elevated Plus Maze. The data demonstrate that LR and WKY rats show 

similarly high levels of immobility on the Forced Swim Test, with both groups exhibiting greater 

immobility than HRs and purchased SD rats. WKY rats, like LR rats, exhibit very low levels of 

novelty-induced locomotor activity. HR rats show very high levels of activity in the novel 

environment, with purchased SD rats exhibiting an intermediate level of activity. LR and WKY 

rats behave similarly in the anxiety tests, both showing exaggerated anxiety-like behaviors 

compared to HR and purchased SD rats. Overall these findings support the notion that LR rats 

represent a novel animal model of depression with concurrent anxiety. What distinguishes the 

LR “depression model” from other models, such as the WKY rat, is that its depressive phenotype 

cannot be attributed to strain differences, but rather, is likely linked to underlying genetic 

differences which have been uncovered via selective breeding. 

Disclosures: A.D. Abraham , None; S.M. Clinton, None; I.A. Kerman, None; T.A. 

Bedrosian, None; S.J. Watson, None; H. Akil, None. 

Poster 





Support: Office of Naval Research N00014-02-1-0879 (HA & SJW) 

L'Oréal USA Fellowship for Women in Science (SMC) 

NIDA 5P01DA021633-02 (HA and SJW) 

Title: Low novelty-seeking rats exhibit exaggerated anxiety- and depression-like behavior 

compared to high novelty-seekers: impact of chronic paxil treatment 

Authors: *H. R. ORR, S. M. CLINTON, A. D. ABRAHAM, T. A. BEDROSIAN, S. J. 

WATSON, H. AKIL; 

Molec and Beh Neurosci Inst., Univ. Michigan, Ann Arbor, MI 

Abstract: During recent years we selectively bred Sprague-Dawley rats for high (high 

responder, HR) and low (low responder, LR) novelty-seeking behavior, a trait that predicts a 

variety of differences in emotional reactivity, including differences in neuroendocrine stress 

response, anxiety, aggression, and propensity to self-administer drugs of abuse. More recently, 

we discovered that our HR- and LR-bred lines also show differences in depression-like behavior, 

with LR rats showing increased immobility on the forced swim test (FST), and diminished 

sucrose preference compared to their HR counterparts. The first aim of the present study was to 

determine whether the LR “depressive-phenotype” could be attenuated via chronic treatment 

with the antidepressant paroxetine. HR and LR adult males (N=12 per group) received 

paroxetine (10 mg/kg/day) for 28 days via drinking water, and were then subjected to a test 

battery to assess locomotor response to novelty, anxiety- and depression-like behavior. Chronic 

paroxetine treatment reduced LRs‟ immobility in the FST, but did not affect HRs‟ performance. 

Paroxetine treatment did not affect locomotor response to novelty, and did not alter HR/LR 

anxiety-like behavior in the Elevated Plus Maze. The second aim of our study was to evaluate 

FST-induced neuronal activation patterns in HR and LR rats using expression of the immediate 

early gene c-Fos as a marker. We hypothesized that a) vehicle-treated HR-LR rats would express 

divergent patterns of c-Fos mRNA expression which would correspond to their marked 

differences in FST behavior, and b) that such activation differences would be attenuated by 

paroxetine treatment. Thus, vehicle- and paroxetine-treated HR/LR rats were euthanized 30 

minutes after the FST; brains were removed, frozen, and cryostat sectioned. Ongoing in situ 

hybridization studies are assessing patterns of c-fos mRNA expression in corticolimbic circuits 

relevant to depression-like behavior to examine stress-induced neuronal activation in a novel 

model of depression (LR rats), and its potential reversal with antidepressant treatment. 

Disclosures: H.R. Orr, None; S.M. Clinton, None; A.D. Abraham, None; T.A. Bedrosian, 

None; S.J. Watson, None; H. Akil, None.

Poster 





Support: NIH Grant MH 52619 

Title: Hypertonic sodium lactate or sodium chloride, but not D-mannitol, induce panic-like 

responses in an animal model of panic disorder involving reduced GABA inhibition in the 

dorsomedial hypothalamus 

Authors: *P. L. JOHNSON 1 , A. MOLOSH 1 , S. D. FITZ 1 , J. A. DIMICCO 2 , D. G. RAINNIE 3 , 

J. P. HERMAN 4 , A. SHEKHAR 1 ; 

1 Psychiatry, 2 Pharmacol. Toxicology, Indiana Univ. Med. Sch., Indianapolis, IN; 3 Psychiatry, 

Emory Univ., Atlanta, GA; 4 Psychiatry, Univ. of Cincinnati, Cincinnati, OH 

Abstract: Panic disorder in humans is characterized by recurrent panic attacks comprised of 

severe anxiety and marked increases in cardiorespiratory responses that can be provoked by 

ordinarily mild interoceptive stressors such as intravenous (i.v.) infusions of 0.5M sodium 

lactate. Similarly, chronic inhibition of GABAergic tone [using l-allylglycine, a glutamate 

decarboxylase (GAD) inhibitor] in the dorsomedial hypothalamus/perifornical region 

(DMH/PeF) of rats induces a vulnerability to panic-like behavior and cardiorespiratory responses 

following i.v. infusion of 0.5 M sodium lactate. We have previously shown that the region 

surrounding the anterior 3 rd ventricle (A3V) provides a critical afferent pathway to the DMH/PeF 

that is involved in the sodium lactate response in this panic model. Additionally, using wholecell 

patch clamp, we have shown that bath applications of sodium lactate, but not lactic acid or 

D-mannitol, induces firing in 33% of neurons in the “osmo/sodium-sensitive” region of the A3V 

that are retrogradely labeled from the DMH/PeF. Presently, we combined retrograde tracing 

(fluorogold) from the DMH/PeF with in situ hybridization for vesicular glutamate transporter 

and GAD65 to demonstrate that the majority of DMH/PeF-projecting neurons in the A3V are 

glutamatergic. In another experiment, we also determined that i.v. infusions of 0.5M sodium 

lactate or sodium chloride, but not D-mannitol, provoked “anxiety” (decreased social interaction) 

and “flight” (increased locomotion) behavior, as well as tachycardia and pressor (only sodium 

lactate) responses. These data are consistent with human studies that demonstrate that hypertonic 

sodium chloride (Peskind et al., 1998) or sodium bicarbonate (Gorman et al., 1989) provoke 

panic attacks in most panic disorder patients, suggesting that human panic disorder may involve 

analogous neural substrates. 

Disclosures: P.L. Johnson, MH 52619, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received); A. Molosh, None; S.D. 

Fitz, None; J.A. DiMicco, None; D.G. Rainnie, None; J.P. Herman, None; A. Shekhar, MH

52619, B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received). 

Poster 





Support: NIMH Grant 067651 

Title: Changes in intracellular mechanisms in the basolateral amygdala (BLA) involved in 

repeated stress 

Authors: *N. GRISSOM 1,2 , S. LUZ 2 , J. SINGARAVELU 2 , H. CHUNG 2 , S. BHATNAGAR 3,2 ; 

1 Dept Psychol, Univ. Michigan, Ann Arbor, MI; 2 Anesthesiol., Children's Hosp. of Philadelphia, 

Philadelphia, PA; 3 Anesthesiol., Univ. of Pennsylvania Med. Sch., Philadelphia, PA 

Abstract: Repeated exposure to a stressor leads to adaptive changes in responses to the familiar 

stressor. For example, while a first exposure to restraint elicits a high degree of hypothalamicpituitary-adrenal 

(HPA) activity, by the 5th restraint, the HPA response is significantly reduced, 

a phenomenon known as habituation. Habituation of HPA activity is associated with activity in a 

distributed network of limbic brain regions. One such region is the basolateral amygdala (BLA). 

We have previously shown that administration of the beta-receptor antagonist propranolol into 

the BLA immediately following each of 4 daily exposures to 30 minute restraint prevents normal 

habituation to the 5th restraint. Recently we have begun investigating intracellular changes, 

including epigenetic alterations, occurring in the BLA and other limbic brain regions which 

might underlie the effects of beta-adrenergic receptor blockade in the BLA on habituation to 

stress. Our preliminary results indicate that repeatedly restrained animals show decreased 

acetylation of the histone H3 in the BLA compared to acutely stressed rats. Histone acetylation 

changes are one form of epigenetic alteration; decreases in acetylation of H3 are associated with 

decreased gene expression. In contrast to the changes seen in the BLA, no change in acetylation 

of H3 was seen in the dorsal hippocampus of repeatedly stressed animals as compared to acutely 

stressed rats. We are currently investigating the effect of propranolol administration on repeatedstress-related 

changes in histone acetylation in the BLA, as well as other intracellular changes in 

the BLA and other regions. The current data indicate that a long-term changes occur in the BLA 

as a result of repeated stress, and may be important for normal adaptation to stress.

Disclosures: N. Grissom, None; J. Singaravelu, None; H. Chung, None; S. Bhatnagar, 

None; S. Luz, None. 

Poster 





Title: Somatostatin-2 receptors in the basolateral amygdala of rats modulate behavioral priming 

and anxiety-like responses 

Authors: *D. L. GASKINS, P. L. JOHNSON, P. E. KELLY, A. D. DIETRICH, W. A. 

TRUITT, A. SHEKHAR; 

Dept. of Psychiatry, Indiana Univ. Sch. Med., Indianapolis, IN 

Abstract: Previous studies from our laboratory have shown that Urocortin I (Ucn1), a CRF 

receptor agonist, given repeatedly into the basolateral complex of the amygdala (BLA) at subanxiogenic 

doses (priming) enhance synaptic transmission and results in long-term anxiety-like 

behaviors in social interaction (SI) as well as the elevated plus maze tests. The development of 

this behavior profile is similar to that which is observed in individuals who are vulnerable to 

anxiety disorders and are exposed to chronic stress. To determine the neurotransmitter system 

involved we used the RT-Profiler PCR array screen and found the somatostatin-2 receptor 

(sstr2) mRNA to be significantly down regulated in the BLA after Ucn1 priming. Emerging 

evidence suggests that the sstr2 has a role in depression and anxiety. Therefore, the present study 

was conducted to investigate the role of sstr2 activation in the BLA on anxiety using the sstr2 

agonist BIM-23027. Three experiments were conducted: BIM-23027 alone, pre-treatment with 

BIM-23027 prior to an acute anxiogenic dose of Ucn1 (an acute stress-like challenge), and pretreatment 

with BIM-23027 prior to Ucn1 priming. For all experiments male wistar rats were 

implanted bilaterally with chronic guide cannulae targeting the BLA and allowed to recover. For 

the first experiment, SI was measured 30 minutes after a bilateral injection of either vehicle or 1 

of 5 doses of BIM-23027(either100 fmole/100 nl/side or 1, 10, 30, or 90 pmol/100 nl/side). For 

the second experiment rats were infused with vehicle or BIM-23027 (30 or 90 pmoles/100 

nl/side) 30 min prior an acute anxiogenic dose of Ucn1 (100 fmoles/100 nl/side). SI was 

measured 30 min after the Ucn1 injection. Thirdly, rats were infused with vehicle or BIM-23027 

(90 pmoles/100 nl/side) 30 minutes prior to vehicle or Ucn1 (6 fmoles/100 nl/side) priming. SI 

was measured on injection days 1, 3, and 5 during priming, as well as 3 days after the last 

Ucn1/Veh injection. BIM-23027 alone did not significantly alter SI. However, 90 pmoles of 

BIM-23027 did block the reduction of SI induced by an acute anxiogenic dose of Ucn1. In the

priming experiments (daily bilateral injection of 6 fmoles of Ucn1 into the BLA for 5 

consecutive days), a significant decrease in SI was seen by day 3 and by day 5 the decrease in SI 

was persistent. BIM-23027 pre-treatment delays Ucn1 priming in that a significant decrease in SI 

was not observed until day 5 and thereafter. These findings suggest that the somatostatin-2 

receptor in the BLA has a role in regulating anxiety and we hypothesize that the somatostatin-2 

receptor agonists may be effective in treating anxiety disorders. 

Disclosures: D.L. Gaskins , None; P.L. Johnson, None; P.E. Kelly, None; A.D. Dietrich, 

None; W.A. Truitt, None; A. Shekhar, None. 

Poster 






Title: Effects of the organic cation transporter 3 (OCT3) inhibitor, normetanephrine, on 

yohimbine-induced anxiety and cocaine-seeking behavior in rats 

Authors: E. N. GRAF, J. R. MANTSCH, *D. A. BAKER, P. J. GASSER; 

Dept Biomed. Sci., Marquette Univ., Milwaukee, WI 

Abstract: Stressor-induced increases in noradrenergic neurotransmission have been implicated 

in anxiety and drug-seeking behavior. Yohimbine, an antagonist at the alpha-2 adrenergic 

receptor, has been used as a pharmacological stressor by virtue of its ability to disinhibit 

ascending noradrenergic activity, resulting in central increases in norepinephrine (NE) release. 

Recent studies have suggested that organic cation transporter 3 (OCT3), a high-capacity, lowaffinity 

monoamine transporter, contributes to clearance of extracellular NE in the brain when 

higher affinity NE transporters are saturated. The goal of the present study was to examine the 

effects of OCT3 inhibition on yohimbine-induced anxiety-like and drug-seeking behavior in rats. 

In the first set of studies, rats were tested for behavior on the elevated plus maze or in the 

light/dark box following administration of yohimbine (0.3, 1.0, and 3.0 mg/kg, ip) alone or in 

combination with the OCT3 inhibitor, normetanephrine (5.0 mg/kg, ip). Yohimbine dosedependently 

decreased activity on the open arms of the elevated plus maze and in the light 

compartment of the light/dark box. Normetanephrine, by itself or in combination with low doses 

of yohimbine that failed to alter anxiety-related behavior, also reduced open-arm and lightcompartment 

activity, suggesting that OCT3 blockade may be anxiogenic and may potentiate

yohimbine-induced anxiety. In the second study, yohimbine reinstated extinguished cocaineseeking 

behavior in rats with a history of self-administration (SA) under long-access (LgA) 

conditions (6 hrs daily) but not in rats with a history of short-access (ShA) SA (2 hrs daily). The 

effects of normetanephrine on yohimbine-induced reinstatement in ShA rats will be reported. 

The results of these studies should clarify the role of OCT3 in modulating noradrenergic 

neurotransmission underlying anxiety and the relapse of drug use during periods of stress. 

Disclosures: E.N. Graf, None; J.R. Mantsch, None; D.A. Baker , None; P.J. Gasser, None. 

Poster 





Support: Wellcome 069280 

Title: MiR-214 regulates Hsc70 expression and clathrin uncoating in response to heat stress in 

cortical neurons 

Authors: *I. BANTOUNAS 1 , Y.-B. LEE 1 , J. L. HOWARTH 1 , P. VERKADE 2 , J. B. UNEY 1 ; 

1 HW-LINE, 2 Wolfson Imaging Ctr., Univ. of Bristol, Bristol, United Kingdom 

Abstract: In this study we exposed rat cortical neurons to a sub-lethal heat shock to identify 

miRNAs that may be involved in regulating the cells‟ response to stress. The miRNA expression 

profile of heat-shocked neurons was compared to controls by microarray analysis. Changes in the 

expression of a number of miRNA were detected and miR-214, which was the most significantly 

down-regulated, was chosen for further investigation. Endogenous miR-214 expression levels 

were found to be significantly down-regulated 24h following heat shock but had recovered to 

normal levels 72h later. When neurons were transduced with a lentivirus overexpressing miR- 

214 there was a dramatic decrease in Hsc70 expression. Furthermore, when a more severe heat 

shock was applied, endogenous miR-214 expression increased 3-4 fold compared to baseline 

levels. This was accompanied by a decrease in Hsc70 protein levels by >80%. Further 

investigation showed that Hsc70 promoter activity was decreased in the presence of miR-214 via 

an effect on the availability of p53. Finally, overexpression of miR-214 resulted in an increase of 

clathrin-coated vesicles within the cells, suggesting that miR-214 may play an important role in 

the gross regulation (via Hsc70) of endocytic processes such as synaptic vesicle recycling.

Disclosures: I. Bantounas, None; Y. Lee, None; J.L. Howarth, None; P. Verkade, None; J.B. 

Uney, None. 

Poster 





Support: NARSAD Young Investigator Award (IAK) 

Pritzker Neuropsychiatric Disorders Research Consortium (SJW) 

NIDA 5P01DA021633-02 (HA & SJW) 

Office of Naval Research N00014-02-1-0879 (HA & SJW) 

Title: Are emotional-somatomotor circuits modifiable by exposure to increased environmental 

complexity? 

Authors: *S. M. CLINTON, I. A. KERMAN, P. R. BURGHARDT, A. D. ABRAHAM, T. A. 

BEDROSIAN, H. ORR, N. VACHHANI, N. MUZAMHINDO, C. SHABRANG, J. BEALS, S. 

J. WATSON, H. AKIL; 

Molec & Behav Neurosci Inst., Univ. Michigan, Ann Arbor, MI 

Abstract: Exposure to increasing environmental complexity (EC) has the potential to diminish 

depressive- and anxiety- like behaviors in rodents. The current study sought to determine 

whether such manipulation may also alter the organization of emotional-somatomotor circuits 

which likely mediate these behaviors. We recently found altered organization of such circuits in 

the Low Responder (LR) rat model of co-morbid depression and anxiety. When compared to 

their High Responder (HR) counterparts, LR rats exhibit increased depressive- and anxiety- like 

behaviors on a variety of assays and also contain fewer neurons in the paraventricular nucleus of 

the hypothalamus (PVN) with poly-synaptic projections to skeletal muscle (but not to the adrenal 

gland). To determine whether these HR/LR behavioral and associated anatomical differences 

may be reversible, we exposed LR rats to 4 weeks of increased EC. A group of LR rats (N=12) 

was housed from postnatal day (P)25 to P53 (28 days total) in a 3‟ x 3‟ x 3‟ wire mesh cage that 

contained different toys and obstacle courses. Animals were free to explore within this 

environment, and its complexity was increased by a daily addition of a novel object until the 

final day (P53) when the environment reached its maximum complexity. Control HR and LR rats

(N=12 per group) were pair-housed under standard laboratory conditions, and from P49-53 all 

animals were tested to assess locomotor response to novelty, anxiety- and depressive- like 

behaviors on the elevated plus maze (EPM) and the forced swim test (FST), respectively. 

Exposure to increasing EC dramatically reduced depressive-like behavior on the FST in LR 

animals, significantly reducing immobility and increasing climbing and swimming behaviors. 

Exposure to increasing EC modestly reduced LR rats‟ anxiety-like behavior in the EPM, leading 

to a reduced latency to enter the open arms, but did not impact their novelty-induced locomotor 

activity. Following behavioral testing, 7 rats from each experimental group were selected for 

trans-synaptic tract-tracing studies to delineate the organization of central circuits that regulate 

skeletal muscle and adrenal gland functions. Preliminary analysis indicates that exposure to 

increased EC “re-wired” LRs‟ emotional-somatomotor circuits by increasing the numbers of 

PVN neurons with poly-synaptic projections to skeletal muscle. These data suggest that 

emotional-somatomotor circuits are modifiable by environmental factors and mediate expression 

of depressive-like behaviors. 

Disclosures: S.M. Clinton , None; I.A. Kerman, None; P.R. Burghardt, None; A.D. 

Abraham, None; T.A. Bedrosian, None; H. Orr, None; N. Vachhani, None; N. Muzamhindo, 

None; C. Shabrang, None; J. Beals, None; S.J. Watson, None; H. Akil, None. 

Poster 





Support: HHMI grant for Undergraduate Education through the Department of Biological 

Sciences 

Clark Scholar's Program 

Title: Neuronal distribution of urocortin-1 and urocortin-3 in an anuran amphibian 

Authors: *N. B. BERGFELD, S. BULIN, N. BARAWID, J. A. CARR; 

Texas Tech. Univ., Lubbock, TX 

Abstract: The 41 amino acid neuropeptide corticotropin-releasing factor (CRF) plays a critical 

role in coordinating behavioral and physiological responses to stressors. The CRF gene family 

consists of 4 paralogous members, CRF, urocortin-1 (UCN-1), UCN-2, and UCN-3. Genes 

encoding UCN-1 and UCN-3 have been identified in at least one amphibian and two fish species,

ut the biological role and tissue distribution of the peptides encoded by these genes is not well 

studied. We examined the distribution of urocortin-1 (UCN-1) and UCN-3 producing neurons in 

a model anuran species, the Korean fire-bellied toad Bombina orientalis. We used polyclonal 

antisera raised in rabbits against an oligopeptide sequence from frog UCN-3 or an evolutionarily 

conserved region in the C-terminus of rat UCN-1. Intensely stained UCN-3-immunoereactive (ir) 

perikarya were found in the infundibular hypothalamus and preoptic area, while immunostained 

fibers were found throughout the septal nuclei, in the bed nucleus of the stria terminalis, 

amygdala, and throughout the hypothalamus. Interestingly, many UCN-3-ir fibers (probably 

dendrites) penetrated the ependyma to contact cerebrospinal fluid. Isolated UCN-1-ir neurons 

were observed in periventricular areas of the preoptic area and hypothalamus. Intensely-stained 

UCN-1-ir neurons were observed in the vicinity of the motor nucleus of the oculomotor nerve, a 

UCN-1 cell group that may be homologous to the well-described Edinger-Westphal (EW) UCN- 

1 cell group first reported in mammals. Our data suggest intense innervation of forebrain limbic, 

basal ganglia, and hypothalamus by UCN-3 neurons, providing strong support for a role of UCN- 

3 in regulating autonomic and endocrine homeostasis. The finding of UCN-1 in putative 

oculomotor regulatory areas of midbrain is consistent with the fact that the EW nucleus is the 

principal site of UCN-1 in the mammalian brain. 

Disclosures: N.B. Bergfeld , None; S. Bulin, None; N. Barawid, None; J.A. Carr, None. 

Poster 





Support: NIMH Grant MH 52619 

Eli Lilly & Co. 

Title: CBiPES, a potent group II metabotropic glutamate receptor allosteric potentiator, prevents 

sodium lactate-induced panic-like response in panic-prone rats 

Authors: *S. D. FITZ 1 , P. L. JOHNSON 1 , K. A. SVENSSON 2 , J. S. SCHKERYANTZ 2 , A. 

SHEKHAR 1 ; 

1 Psychiatry, Indiana Univ. Schl Med., Indianapolis, IN; 2 Eli Lilly & Co, Indianapolis, IN 

Abstract: Adult male rats with chronic inhibition of GABA synthesis (with l-allyglycine, a 

glutamic acid decarboxylase inhibitor) in the dorsomedial hypothalamus/perifornical area

(DMH/PeF) are highly anxious and exhibit panic-like cardio-respiratory responses to intravenous 

(i.v) sodium lactate infusions similar to patients with panic disorder (PD rats). We have 

previously shown that agents that block NMDA receptors (Johnson and Shekhar, 2006) or a 

metabotropic glutamate receptor type 2/3 receptor agonist (Shekhar and Keim, 2000) can block 

such a lactate response, suggesting a glutamate mechanism contributing to this panic-like state. 

Using this animal model of panic, we tested the efficacy of CBiPES (see Johnson MP et al., 

2005), a selective group II metabotropic glutamate type 2 receptor allosteric potentiator 

(30mg/kg i.p.), in preventing the lactate-induced panic-like behavioral and cardiovascular 

responses. Alprazolam (3mg/kg i.p.), a clinically effective anti-panic benzodiazepine, was used 

as a positive control. As predicted, PD rats given prior systemic vehicle injections displayed 

lactate-induced panic-like cardiovascular (i.e., tachycardia and hypertensive responses) 

responses and “anxiety” (i.e., decreased social interaction time) and “flight” (i.e., increased 

locomotion)-associated behavior. However, systemically injecting PD rats with either CBiPES or 

alprazolam prior to sodium lactate blocked all lactate-induced panic-like behavior and 

cardiovascular responses. These data suggest that selective group II metabotropic glutamate type 

2 receptor allosteric potentiators could be a novel target for developing anti-panic drugs that are 

as effective as alprazolam in acute treatment without the deleterious side effects (e.g, addiction, 

sedation and cognitive impairment) associated with benzodiazepines. 

Disclosures: S.D. Fitz , Eli Lilly & Co., C. Other Research Support (receipt of drugs, supplies, 

equipment or other in-kind support); P.L. Johnson, Eli Lilly & Co., C. Other Research Support 

(receipt of drugs, supplies, equipment or other in-kind support); K.A. Svensson, Eli Lilly & Co., 

A. Employment (full or part-time); J.S. Schkeryantz, Eli Lilly & Co, A. Employment (full or 

part-time); A. Shekhar, Eli Lilly & Co., C. Other Research Support (receipt of drugs, supplies, 

equipment or other in-kind support). 

Poster 





Support: NIDA 5P01DA021633-02 (HA and SJW) 


Office of Naval Research N00014-02-1-0879 (HA & SJW) 

NARSAD Young Investigator Award (IAK)

Title: Altered wiring of emotional-somatomotor, but not limbic-autonomic, circuits in rat models 

of depression 

Authors: *I. A. KERMAN, S. M. CLINTON, P. R. BURGHARDT, N. MUZAMHINDO, N. 

VACHHANI, C. SHABRANG, J. BEALS, H. AKIL, S. J. WATSON; 

Molec & Behav Neurosci Inst., Univ. Michigan, Ann Arbor, MI 

Abstract: In addition to cognitive and affective disturbances, major depression is characterized 

by physical symptoms, including dysregulation of motor control and autonomic dysfunction. We 

hypothesized that such functional alterations may be due to differential organization of brain 

circuits that regulate these functions. To test this hypothesis, we used a virally-mediated transsynaptic 

tract-tracing approach to delineate emotional-somatomotor, limbic-autonomic and 

somatomotor-sympathetic circuits in the rat using distinct pseudorabies virus (PRV) 

recombinants. Experiments were carried out in two models of depression: the Wistar-Kyoto 

(WKY) rat (compared to the Sprague-Dawley (SD) rat) and the Low Responder (LR) rat 

(compared to the High Responder (HR) rat). Behavioral studies confirmed the expression of 

depressive-like phenotypes in WKY and LR rats. Specifically, WKY rats exhibited significantly 

more immobility (91.3 ± 2.7% vs. 12.6 ± 5.5%; p < 0.01), and significantly less swimming (8.7 ± 

2.7% vs. 70.8 ± 6.6%; p < 0.01) and climbing (0% vs. 16.6 ± 4.7%; p < 0.01) compared to SD 

controls on the forced swim test (FST). Likewise, LR rats were significantly more immobile 

(68.5 ± 9.3% vs. 28.6 ± 7.5%; p < 0.01) and swam significantly less (24.0 ± 4.8% vs. 52.5 ± 

5.3%; p < 0.01) on the FST compared to their HR counterparts. Virally-mediated transsynaptic 

tract-tracing studies revealed large differences in the numbers of neurons with poly-synaptic 

projections to skeletal muscle, but not to the adrenal gland, in the paraventricular nucleus of the 

hypothalamus (PVN) in both depression models compared to their respective controls. 

Accordingly, SD rats contained approximately three times as many neurons with poly-synaptic 

projections to skeletal muscle compared to WKY rats, while similar differences were detected 

between HR and LR rats. There were no WKY/SD or LR/HR differences in the numbers of 

neurons with poly-synaptic projections to the adrenal gland, or in the numbers of neurons with 

collateralized poly-synaptic projections to both muscle and adrenal gland. Likewise, there were 

no WKY/SD or LR/HR differences in the numbers of labeled gastrocnemius motoneurons, 

suggesting that observed differences in the PVN were not due to disparate PRV entry into firstorder 

neurons. Overall, these data suggest differential wiring of emotional-somatomotor circuits 

in two different genetic rat models of depression, which may underlie their distinct behavioral 

phenotypes. 

Disclosures: I.A. Kerman , None; S.M. Clinton, None; P.R. Burghardt, None; N. 

Muzamhindo, None; N. Vachhani, None; C. Shabrang, None; J. Beals, None; H. Akil, 

None; S.J. Watson, None. 

Poster 

283. Stress-Regulated Pathways II




Support: MOST/KOSEF(R11-2005-014) 

Oriental Medicine R&D Project(03-PJ9-PG6-SO02-0001) 

Title: Cortico-entorhinal-hippocampal circuit involvement in the hypothalamo-pituitary-adrenal 

axis response during acute immobilization stress: a 18F-Fluorodeoxyglucose micro-pet imaging 

study 

Authors: *D. JANG 1 , Z.-H. CHO 1 , Y.-B. KIM 1 , K.-K. SUNG 2 , S. LEE 3 , M. KIM 4 ; 

1 Neurosci. Res. Inst., Gachon Univ., Incheon, Republic of Korea; 2 Dept. of Neurosci. & Oriental 

Med., 3 Wonkwang Univ., Iksan, Republic of Korea; 4 Dept. of psychology, Chonnam Natl. Univ., 

Gwangju, Republic of Korea 

Abstract: Although there are many studies on neural substrates related to stress, there is 

little neuroimaging data of the stress response in animals. We used the [F-18]FDG-PET 

neuroimaging technique to investigate changes in brain activity related to acute stress in 

rats. Immobilization stress given for an hour induced activation of the hypothalamus, 

entorhinal, insular, and pyriform cortices, and significant deactivation of the cortex, dorsal 

hippocampus, thalamus, and cerebellum, with increasing plasma corticosterone levels. In 

the 1 h stress plus 1 h rest condition and the prolonged stress condition, activation in the 

hypothalamus and entorhinal cortex, and deactivation in cortex and hippocampus, 

changed toward their respective baselines. Neuronal activity in the cortex, entorhinal 

cortex, and hippocampus strongly correlated with hypothalamic activity. Our findings 

suggest that the cortico-entorhinal-hippocampal circuit is involved in the regulation of the 

HPA axis during acute immobilization stress. 

Disclosures: D. Jang , None; Z. Cho, None; Y. Kim, None; K. Sung, None; S. Lee, None; M. 

Kim, None. 

Poster 



Program#/Poster#: 283.21/PP17


Support: Jan Kornelis de Cock Stichting 

Title: Fluoxetine treatment versus social housing in chronically stressed female rats 

Authors: *C. WESTENBROEK, R. WICHMANN, M. DE HAAN, J. HEUKELOM, P. 

BAKKER, G. J. TER HORST; 

Dept Neurosci., Univ. of Groningen, Groningen, Netherlands 

Abstract: Social support reduces the risk of developing a depressive episode and can prevent 

relapse. Social housing reduces the impact of chronic stress on female rats, simulating the effects 

of social support in humans. In the current study we have investigated if social housing affects 

recovery after chronic stress exposure. Effects of social housing were compared to the effects of 

chronic treatment with the antidepressant fluoxetine (FLX). 

Isolated female rats were exposed to 3 weeks of footshock stress after which the treatment was 

started. Treatment consisted of 3 weeks fluoxetine, social housing, or a combination of both. To 

investigate the effect of continuation of stress during treatment, for half of the groups stress 

continued (stress groups), for the other half the stress was stopped (recovery groups). An 

individually and a socially housed control group were not exposed to stress or fluoxetine. 

Behavior was measured in an elevated plus maze. Weight gain, plasma corticosterone levels, 

adrenal and thymus weights were used to measure the impact of stress. Changes in neuronal 

plasticity were examined using (p)CREB, Fibroblast Growth Factor 2 (FGF-2) and Brain 

Derived Neurotrophic Factor (BDNF) expression in the dentate gyrus (DG) and prefrontal cortex 

(PFC). 

Fluoxetine had no effect on behavior in the plus maze, in contrast to social housing which 

resulted in a preference for the open arms. Surprisingly fluoxetine augmented stress effects in 

individually housed females on adrenal and thymus weights, whereas social housing reduced 

basal corticosterone levels and increased thymus weights. 

After a recovery period, BDNF levels were increased only in isolated vehicle rats, which were 

prevented by fluoxetine. FLX also decreased FGF-2 levels in the DG of isolated recovery rats, 

while social housing in combination with FLX prevented this decrease. Neither Fluoxetine nor 

social housing resulted in the anticipated increase in pCREB, BDNF and FGF-2 levels in the DG 

and PFC compared to control levels when treatment is started after chronic stress exposure. 

Disclosures: C. Westenbroek, None; R. Wichmann, None; M. de Haan, None; J. Heukelom, 

None; P. Bakker, None; G.J. ter Horst, None. 

Poster 






5T32 E507148 

NIEHS P30 ES05022 

Title: Effects of intermittent pairing of chronic restraint stress with footshock on habituation of 

the c-fos response in the mouse brain 

Authors: *A. W. KUSNECOV 1 , D. URBACH-ROSS 2 ; 

1 Dept Psychol, 2 Toxicology, Rutgers Univ., Piscataway, NJ 

Abstract: It has been shown in rats that the c-Fos response habituates during chronic restraint 

stress. Habituation is considered a learning process, and in the case of restraint, predictable 

“safe” outcomes (eg., returning to home cage) following restraint may contribute to diminished 

c-Fos responses. The current study tested this question using male C57BL/6 mice about which 

little is known concerning habituation of c-Fos expression to chronic stress. Specifically, we 

asked if the c-fos response to repeated restraint would be attenuated if restraint sessions were 

intermittently followed by a session of unpredictable footshock. As a result, it was expected that 

restraint sessions would not always predict return to a safe, home cage environment, but rather 

may be followed by relocation of the mouse to the shock chamber. Mice were placed for 90 mins 

daily into aerated 50 ml conical tubes for one (acute restraint) or ten days (chronic restraint), 

while controls remained in the home cage. Of the chronically restrained mice, on Days 1, 4, 6, 7 

and 9 of restraint, one group was returned to the home cage, while another was transferred to 

separate chambers and received five 0.2 mA footshocks over a 15 min period before going to the 

home cage. On the assessment day (D11), mice were perfused for brain c-Fos 

immunohistochemistry after receiving restraint (Rst + Sh - test Rst; Rst+HC - test Rst) or home 

cage (Rst + Sh - test HC). In addition to separate HC groups that were tested for acute restraint 

effects, a group that received shock immediately before sacrifice after chronic restraint stress was 

included (Rst+HC - test Sh). The results showed that chronic restraint produced a habituated, but 

increased c-Fos response in the paraventricular nucleus, lateral septum, bed nucleus of the stria 

terminalis, and the central nucleus of the amygdala. Surprisingly, pairing restraint with shock did 

not block the attenuation in these areas. Interestingly, a heterotypic stressor (viz., shock) given 

after chronic restraint (Rst + HC) dramatically elevated c-Fos in hippocampal CA1 and CA3, 

whereas this was not evident after acute or chronic restraint. This suggests that these particular 

areas may be less susceptible to activation under mild stress conditions but become activated 

after a severe stressor such as shock. In conclusion, mice showed an elevated, but attenuated c- 

Fos response in stress-related circuitry in response to repeated restraint. This attenuation was not 

changed when the restraint was paired with shock. Therefore, the restraint itself did not appear to 

serve as a conditioned stimulus to anticipate shock. 

Disclosures: A.W. Kusnecov, None; D. Urbach-Ross, None.

Poster 






Title: Reduced somatostatin neurotransmission in the basolateral amygdala may be one 

mechanism by which “CRF-priming” induces persistent increases in anxiety-like behavior in the 

rat 

Authors: *A. MOLOSH 1 , W. TRUITT 1,2 , P. KELLEY 1 , A. DIETRICH 1 , S. FITZ 1 , G. 

OXFORD 3 , A. SHEKHAR 1,3 ; 

1 Dept Psychiatry, 2 Dept. Anat. and Cell Biol., 3 Stark Neurosciences Res. Inst., IU Sch. of Med., 

Indianapolis, IN 

Abstract: Our previous studies have shown that repeated activation of corticotrophin releasing 

factor (CRF) receptors ("CRF/Ucn1 priming") in the basolateral amygdala (BLA) results in a 

behavioral syndrome characterized by persistent anxiety and panic-like responses. While this 

phenomenon has been described in detail the underlying mechanisms remain elusive. To address 

this question we first investigated the effects of priming on mRNA levels of receptors and 

enzyme proteins from the BLA. Rats were primed, by administering small doses of the CRF 

receptor agonist urocortin 1 (Ucn1, 6 fmol/100nl) or vehicle directly into the BLA once a day for 

5 days. As has been observed previously, rats primed with Ucn1, but not vehicle, had significant 

reductions in social interaction (SI) time, a correlate for increased anxiety-like behaviors, which 

persisted for at least 3 days following the last injections. Five days after the last injection rats 

were sacrificed and the mRNA levels in the BLA of vehicle and Ucn1 primed rats were 

determined using RT-Profiler PCR-arrays for neuroscience (SuperArray). Of the 79 genes 

screened, the mRNAs for somatostatin 2 and 4 receptors (sst2, sst4) and muscarinic cholinergic 

receptor 4 were the only genes that were significantly different between treatment groups. Since 

somatostatin/sst2 has been linked to anxiety-like behavior, we further studied the effect of 

somatostatin (1 κM) on the properties of the BLA projection neurons utilizing the whole-cell 

path-clamp of the naïve and Ucn1-primed rats. Bath application of somatostatin (3min) induced a 

transient outward. Also, perfusion with somatostatin caused a significant decrease in amplitude 

and frequency of spontaneous EPSC, as well as on spontaneous IPSC of the BLA projection 

neurons of naïve rats. Further experiments using paired-pulse stimulation revealed that 

somatostatin also induced a significant decrease of the amplitude of evoked IPSC and had no 

effect on the amplitude of evoked EPSC. Taken together, our data suggests that somatostatin

hyperpolarizes BLA neurons corresponding to the induction of an outward ionic current most 

likely through Gβγ activation of Kir3.x family channels. Based on our data, we speculate that 

somatostatin responses are through sst2 receptors, would be inhibitory to BLA projection cells, 

and that reduction in sst2 receptor expression by Ucn1-priming would bias these neurons toward 

hyperexcitability. Currently, experiments on primed animals are in progress to test this 

hypothesis. 

Disclosures: A. Molosh, None; W. Truitt, None; P. Kelley, None; A. Dietrich, None; S. Fitz, 

None; G. Oxford, None; A. Shekhar, None. 

Poster 





Support: NIMH MH60668 

NIMH MH069558 

Title: Chronic stress selectively reduces hippocampal volume in rats 

Authors: *F. J. HELMSTETTER 1,2 , T. LEE 1 , T. JAROME 1 , S.-J. LI 2 , J. J. KIM 3 ; 

1 Dept Psychol, Univ. of Wisconsin Milwaukee, Milwaukee, WI; 2 Med. Col. of Wisconsin, 

Milwaukee, WI; 3 Univ. of Washington, Seattle, WA 

Abstract: It is well-documented that stress can exert physiological changes in the hippocampus, 

a structure implicated in the formation of long-term declarative memory in humans and spatial 

memory in rodents. As it increases in duration and/or intensity, uncontrollable stress will 

exacerbate neuronal endangerment in rodent hippocampus and correlate with hippocampal 

volume reduction in humans, particularly in posttraumatic stress disorder (PTSD) patients such 

as combat veterans. However, since human imaging studies cannot address the causal link 

between stress and hippocampal changes, the notion of stress-induced diminution of 

hippocampal volume remains controversial. 

To address these issues, the present study used a longitudinal, within-subjects design and 

compared hippocampal volumes in rats before and after exposure to chronic stress. Twenty 

Long-Evans rats (weight: 300-325 g) initially received a magnetic resonance imaging (MRI) 

scan to acquire T1-weighted brain images (spatial resolution: .068 x .068 x .75 mm) at 9.4T and 

then were divided into two groups: stress (n=10) and control (n=10). For 21 days, the stress

group received 6-hour daily restraint/immobilization while controls stayed in their home cage. 

During the restraint, controls were deprived of food and water for the same amount of time to 

match food access in the stress group. After stress, all the animals received the same strructural 

MRI scan again to measure volume changes in the brain. 

Comparisons of hippocampal volume revealed approximately 3% of hippocampal volume loss in 

the stress group. However, stress related volume changes were not found in other structures, such 

as the anterior cingulate cortex, retrosplenial granular cortex, or the adrenal gland. In addition to 

hippocampal volume loss, restraint procedures produced a drop in food intake and body weight 

compared to controls. In sum, chronic stress selectively reduces hippocampal volume within 

subjects without significantly modifying other brain structures or adrenal size. These novel 

findings directly support the idea that stress can alter the size of the hippocampus. 

Disclosures: F.J. Helmstetter , None; T. Lee, None; T. Jarome, None; S. Li, None; J.J. Kim, 

None. 

Press Conferences 

PC-1391. Stressing Out Our Brains 

Time: Tuesday, November 18, 2008, 2:00 pm - 3:00 pm 

Program#/Poster#: Poster Board Number: 


Support: NIMH MH60668 

NIMH MH069558 

Title: Chronic stress selectively reduces hippocampal volume in rats 

Authors: *F. J. HELMSTETTER 1,2 , T. LEE 1 , T. JAROME 1 , S.-J. LI 2 , J. J. KIM 3 ; 

1 Dept Psychol, Univ. of Wisconsin Milwaukee, Milwaukee, WI; 2 Med. Col. of Wisconsin, 

Milwaukee, WI; 3 Univ. of Washington, Seattle, WA 

Abstract: It is well-documented that stress can exert physiological changes in the hippocampus, 

a structure implicated in the formation of long-term declarative memory in humans and spatial 

memory in rodents. As it increases in duration and/or intensity, uncontrollable stress will 

exacerbate neuronal endangerment in rodent hippocampus and correlate with hippocampal 

volume reduction in humans, particularly in posttraumatic stress disorder (PTSD) patients such 

as combat veterans. However, since human imaging studies cannot address the causal link 

between stress and hippocampal changes, the notion of stress-induced diminution of 

hippocampal volume remains controversial.

To address these issues, the present study used a longitudinal, within-subjects design and 

compared hippocampal volumes in rats before and after exposure to chronic stress. Twenty 

Long-Evans rats (weight: 300-325 g) initially received a magnetic resonance imaging (MRI) 

scan to acquire T1-weighted brain images (spatial resolution: .068 x .068 x .75 mm) at 9.4T and 

then were divided into two groups: stress (n=10) and control (n=10). For 21 days, the stress 

group received 6-hour daily restraint/immobilization while controls stayed in their home cage. 

During the restraint, controls were deprived of food and water for the same amount of time to 

match food access in the stress group. After stress, all the animals received the same strructural 

MRI scan again to measure volume changes in the brain. 

Comparisons of hippocampal volume revealed approximately 3% of hippocampal volume loss in 

the stress group. However, stress related volume changes were not found in other structures, such 

as the anterior cingulate cortex, retrosplenial granular cortex, or the adrenal gland. In addition to 

hippocampal volume loss, restraint procedures produced a drop in food intake and body weight 

compared to controls. In sum, chronic stress selectively reduces hippocampal volume within 

subjects without significantly modifying other brain structures or adrenal size. These novel 

findings directly support the idea that stress can alter the size of the hippocampus. 

Disclosures: F.J. Helmstetter , None; T. Lee, None; T. Jarome, None; S. Li, None; J.J. Kim, 

None. 

Poster 





Support: 2002 NARSAD Independent Investigator award 

EU 6th program grant #512012 

Title: Altered GABAA receptor alpha subunit pattern of expression in a rat model of Posttraumatic 

Stress Disorder 

Authors: *G. RICHTER-LEVIN, S. JACOBSON-PICK; 

Lab. Behav Neurosci/Dept Psychol, Univ. Haifa, Haifa, Israel 

Abstract: Profound evidence indicates that GABAARs are important in the control of the 

physiological response to stress and anxiety. The GABAAR are best distinguished by their type 

of α subunit. The alpha 2, 3, 5 subunits are predominantly expressed in the brain during the

embryonic and early postnatal period, whilst alpha1 are most prominent during later 

developmental stages. We have shown before that Juvenile stress impairs coping behavior in 

adulthood. The present study examined the effects of Juvenile stress on GABAA subunit 

expression in both the amygdala and hippocampus. The open field, startle response test, and the 

elevated plus maze were used to assess anxiety level alterations 1 month following the exposure 

to Juvenile stress. Following the behavioral assessment, abnormal pattern of alpha1, 2 and 3 

subunits expression was found in the amygdala and hippocampus. To assess for the functional 

significance of such alterations the response of Juvenile stressed rats to benzodiazepines (BZs) 

(Diazepam, Brotizolam) was evaluated. Results suggest that these alterations in GABAA subunit 

expression are associated with altered sensitivity to BZs. The results suggest that altered 

GABAergic functionality contributes to Juvenile-stress induced impaired ability to cope with 

challenges in adulthood. 

Disclosures: G. Richter-Levin , None; S. Jacobson-Pick, None. 

Poster 






NIH Grant 5P50 MH58911 

Title: Short term and long term effects of different stressors on CART mRNA expression 

Authors: *M. M. MILLER, R. G. HUNTER, K. J. MCCARTHY, B. S. MCEWEN; 

Lab. Neuroendocrinol, The Rockefeller Univ., New York, NY 

Abstract: The neuropeptide CART (cocaine-amphetamine-regulated-transcript) is found in 

many areas of the brain that are involved in the stress response. CART has been shown to cause 

elevated ACTH levels and anxiogenic-like effects when administered into the ventricles. Recent 

studies have shown an increase in CART mRNA expression in central amygdala two hours after 

an acute restraint stress and an increase in expression in the dentate gyrus after three weeks of 

chronic restraint stress. In the current study, we examined two types of stressors, immobilization 

in a restraining bag and exposure to cat odor, and their effect on CART expression and anxiety. 

Male Sprague-Dawley rats were exposed to a single 2-hour immobilization stress or a single 10minute 

exposure to used cat litter and a ball of cat hair. Groups of rats were sacrificed either 2

hours or 10 days after the acute stress exposure. Additionally, a group was stressed chronically 

with the 2-hour immobilization stress for 10 days. Baseline anxiety measures were examined in 

the open field before stress exposure, and post-stress anxiety was measured in the elevated plus 

maze immediately prior to sacrifice in the acute stress, 10-day delay groups and the 10-day 

chronic immobilization stress group. In situ hybridization was used to examine CART mRNA 

expression in the dentate gyrus, central amygdala, medial amygdala, nucleus accumbens shell 

and core, and layer II/III of the prefrontal cortex. Similar to previous results, an increase in 

CART mRNA expression was found in the dentate gyrus of the 10-day chronic immobilization 

group compared to control and no changes were evident in the central nucleus of the amygdala. 

Further, there was an increase in CART expression in the medial amygdala and a decrease in 

expression in the prefrontal cortex. Surprisingly, the 10 minute cat odor exposure caused similar 

increases in CART expression in the medial amygdala and dentate gyrus, but only after the 10day 

delay. In contrast, the acute immobilization stress with 10-day delay did not appear to cause 

any change to CART expression in the amygdala or dentate gyrus. These results suggest that 

stressor type may influence how the brain responds to stress and that studying CART expression 

may provide a way to visualize these differences. 

Disclosures: M.M. Miller, None; R.G. Hunter, None; K.J. McCarthy, None; B.S. McEwen, 

None. 

Poster 





Support: PHS grant MH58250 

Title: Functional consequences of differential trafficking of corticotropin-releasing factor 

receptor subtypes in the dorsal raphe nucleus 

Authors: *M. WASELUS 1 , C. NAZZARO 2 , B. S. REYES 3 , E. J. VAN BOCKSTAELE 3 , R. J. 

VALENTINO 2 ; 

1 Molec & Behav Neurosci. Ins, Univ. of Michigan, Ann Arbor, MI; 2 The Children's Hosp. of 

Philadelphia, Philadelphia, PA; 3 Thomas Jefferson Univ., Philadelphia, PA 

Abstract: Corticotropin-releasing factor (CRF) exerts its effects in the rat dorsal raphe nucleus 

(DRN) via two receptors, CRF1 and CRF2. Previous studies from our laboratory showed that 

CRF receptors in the DRN differentially associate with the dendritic plasma membrane in

stressed vs. unstressed rats. In particular, in unstressed rats CRF1 is more abundant on the plasma 

membrane while CRF2 is largely restricted to the cytoplasm. Following a single exposure to 

swim stress (24 h later), CRF2 is recruited to the plasma membrane and although CRF1 tended to 

be internalized, this change was not significant. The present studies examined the consequences 

of stress-related receptor trafficking and whether CRF1 activation was necessary for this effect. 

CRF (30 ng, intra-raphe) decreased DRN neuronal activity of unstressed rats, as previously 

reported. In contrast, this same dose activated DRN neurons when administered 24 h after swim 

stress. The CRF-elicited excitation of DRN neurons was abrogated when stressed rats were 

pretreated with the selective CRF2 antagonist antisauvagine-30. Indeed, CRF administered in 

previously stressed rats that were pretreated with antisauvagine-30 inhibited DRN activity by a 

similar magnitude as was seen in unstressed rats. Finally, ultrastructural analysis revealed that 

administration of the selective CRF1 antagonist antalarmin prior to swim stress prevented CRF2 

recruitment to the plasma membrane 24 hours later. Taken with our previous studies, these data 

support a model whereby stress induces the recruitment of CRF2 from the cytoplasm to the 

plasma membrate in DRN neurons resulting in a qualitative shift of the CRF response from 

inhibition to excitation. Further, the unmasking of CRF-induced inhibition by antisauvagine-30 

in stressed rats is consistent with the continued presence of CRF1 at the plasma membrane 

sufficient to mediate an inhibitory response. Finally, the attenuation of CRF2 redistribution to the 

plasma membrane following blockade of CRF1 suggests the initial CRF1 activation is necessary 

for CRF2 trafficking. In summary, these findings suggest that actions at CRF1 or CRF2 

predominate in the DRN of unstressed and stressed rats, respectively, and that CRF2 availability 

may depend on the accessibility of CRF1 in the DRN when there is a history of prior stress. 

Disclosures: M. Waselus , None; C. Nazzaro, None; R.J. Valentino, None; B.S. Reyes, 

None; E.J. Van Bockstaele, None. 

Poster 

284. Regulation of Food Intake and Body Weight: Integration of Peripheral Signals: 

Systems 



Topic: E.07.b. Integration of peripheral signals: Systems 

Support: NIH AG 027697 

Biomedical Health Research Initiative, MSU 

Title: Chronic metformin treatment reverses the obese phenotype in diet-induced obese (DIO) 

rats placed on a high-fat (HF) diet

Authors: *A. C. SHIN 1 , S. M. J. MOHANKUMAR 2 , K. D. LINNING 3 , M. P. SIRIVELU 4 , G. 

D. FINK 2 , J. R. HAYWOOD 2 , K. J. CLAYCOMBE 5 , P. S. MOHANKUMAR 3 ; 

1 Neurobiol & Nutr., Pennington Biomed Res. Ctr., Baton Rouge, LA; 2 Pharmacol. & 

Toxicology, 3 Pathobiology & Diagnos. Investigation, 4 Comparative Med. & Integrative Biol., 

5 Food Sci. & Human Nutr., Michigan State Univ., East Lansing, MI 

Abstract: Neuroendocrine changes are involved in both causes and effects in the development of 

obesity. We have previously shown that selectively bred, diet-induced obese (DIO) rats under 

chronic high-fat (HF) diet manifest neuroendocrine dysregulation of the HPA axis at multiple 

levels that could be due to leptin insensitivity in the brainstem noradrenergic neurons. Since 

leptin has been shown to suppress central norepinephrine (NE), a potent activator of the HPA 

axis, the observed leptin resistance in the brainstem may contribute to the abnormal HPA 

function and promote obesity in these animals. The insulin-sensitizing agent, metformin (MET), 

has been recently suggested to increase leptin sensitivity in the rat hypothalamus. We 

hypothesized that it may improve brainstem leptin sensitivity in DIO rats and normalize the HPA 

function and reverse the obese phenotype. 9-week-old DIO male rats were given 7 weeks of 

chow or HF diet. At the end of week 3, the animals in each group were further divided into three 

groups and received either 0, 60 or 300mg/kg BW of MET orally in drinking water for the 

remaining 4 weeks. Weekly body weight and caloric intake were recorded. At the end of 

treatment, the animals were sacrificed. Their brains were collected, frozen in dry ice, and 

sectioned. The paraventricular nucleus (PVN) and median eminence (ME) of the hypothalamus 

were microdissected and analyzed for NE and CRH, respectively. Serum from trunk blood was 

analyzed for leptin and corticosterone levels. The abdominal fat pads were removed and 

weighed. Noradrenergic areas in the brainstem - A1, A2, A6 - were also analyzed for leptin 

signaling molecules by western blot. HF-fed DIO rats had higher body weight, adipose tissue 

weight, and greater feed efficiency than chow-fed rats (p




Support: IBRO 

FAPESP 

FAEPA 

CNPq 

Title: Activation of corticotrophin-releasing factor neurons in the paraventricular nucleus of 

hypothalamus in response to food intake is modulated by glucocorticoids 

Authors: *E. T. UCHOA, J. ANTUNES-RODRIGUES, L. L. K. ELIAS; 

Physiology-School of Med., Univ. of Sao Paulo (USP), Ribeirao Preto, Brazil 

Abstract: Adrenalectomy is associated with a decrease of food intake. Corticotropin-releasing 

factor (CRF) has been shown to play an important role on feeding behavior. In the present study, 

we evaluated the effect of adrenalectomy (ADX) and corticosterone replacement on the 

activation of CRF neurons of the paraventricular nucleus of hypothalamus (PVN) induced by 

feeding. Male Wistar rats (200-250g, n=9-10 per group) were subjected to ADX or Sham 

surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received 

corticosterone in drinking water (B: 25mg/L, ADX+B). Six days after surgery, animals were 

fasted for 16 hours; on next day, they were transcardiacally perfused, before or 2 hours after 

refeeding. The brain was processed for immunohistochemical double labeling of Fos and CRF of 

medial parvocellular (PaMP) and posterior parvocellular (PaPo) subdivisions of the PVN. In the 

PaMP subdivision, Fos expression and Fos-CRF double labeled neurons were increased (P

Poster 


Systems 




Support: NIH-DK065971 

NIH-DC000014 

Title: Brain Fos expression in the musk shrew induced by an emetic dose of a cancer 

chemotherapy agent 

Authors: *B. C. DE JONGHE, C. C. HORN; 

Monell Chem Senses Ctr., Philadelphia, PA 

Abstract: Cancer chemotherapy drugs, such as cisplatin, are extremely potent for producing 

nausea and vomiting. Acute effects of these treatments are partly controlled by using anti-emetic 

drugs, but the delayed effects (>24 h), especially nausea, are much more difficult to treat. It is 

unknown what brain pathways might be responsible for this delayed sickness. Previous data from 

our laboratory indicates that brain Fos expression is increased in the hindbrain and forebrain for 

at least 48 h after cisplatin treatment in the non-vomiting rat. Here we extend these observations 

using house musk shrews, a species with an emetic response. Since the cytoarchitecture of the 

musk shrew brain is relatively unknown, we used cresyl violet staining of the whole brain and 

retrograde tracer staining of the dorsal motor nucleus to identify the location of 

immunohistochemically labeled nuclei for Fos protein. We injected (i.p.) 30 mg/kg cisplatin or 

saline control and euthanized animals at 6, 24, or 48 h to determine brain Fos expression. 

Cisplatin induced Fos expression in the hindbrain for up to 48 h, however, the highest level of 

Fos staining occurred at 6 h post injection and decreased over 48 h. Fos positive cells were most 

abundant in the nucleus of the solitary tract (NTS), area postrema (AP), and the dorsal motor 

nucleus (DMN). Cisplatin also stimulated Fos expression in the amygdala at all three time points. 

These results indicate that cisplatin activates a neural system that includes the dorsal vagal 

complex (NTS, AP, and DMN) and amygdala of the shrew, which parallels the effects of 

cisplatin treatment in the rat. Our findings suggest neural systems that could be targeted to 

reduce delayed nausea and vomiting in cancer patients receiving chemotherapy. 

Disclosures: B.C. De Jonghe , NIH grant DK065971, DC000014, B. Research Grant (principal 


C.C. Horn, NIH grant DK065971, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received).

Poster 


Systems 




Support: HD46501 

RR00165 

Title: Progesterone reverses the adverse consequences of psychosocial stress and estradiol on 

body weight 

Authors: *V. MICHOPOULOS, J. B. HOFFMAN, E. KESSLER, M. E. WILSON; 

Yerkes Natl. Primate Rese Ctr., Emory Univ., Lawrenceville, GA 

Abstract: Chronic stress exposure and the upregulation of corticotropin releasing hormone 

(CRH) in animals reduce both body weight (BW) and intake of standard low fat, high fiber diets. 

Food intake and BW are also attenuated by estradiol (E2) exposure as BW decreases in response 

to E2 and fall during the cycle when E2 is elevated. While E2‟s action is thought to be due to a 

change in orexigenic signaling, progesterone promotes food intake and weight gain, as seen 

during the luteal phase and pregnancy. However, it remains unclear how progesterone interacts 

with E2 to influence BW. Additionally, the relationship between stress exposure and ovarian 

hormones on the regulation of BW is poorly understood. In order elucidate these relationships, 

the present study tested the hypothesis that E2 would exacerbate the adverse consequences of 

stress on BW and that these effects would be attenuated by co-administration with progesterone. 

Socially housed female rhesus monkeys provide an ethologically valid model to investigate the 

adverse consequences of psychosocial stressors as subordinate females are under continuous 

harassment by dominant animals, leading to a dysregulation of the hypothalamic-pituitaryadrenal 

axis. Ovariectomized females fed a low fat, high fiber chow ad libitum were studied 

under four conditions (control, E2, progesterone, and E2 plus progesterone) in a counterbalanced 

manner over 4 weeks. Social status had a significant effect on BW, as dominant females were 

consistently heavier than subordinates throughout the 16-week study (p=0.026). E2 significantly 

reduced BW (p=0.010), regardless of status (p=0.503). Progesterone significantly increased BW 

in all females even during co-administration with E2 (p

differences in ghrelin secretion and changes in food intake. These data suggest that E2 does not 

further reduce BW during chronic psychosocial stressor exposure and that the ability of 

progesterone to promote increases in weight is less in subordinate females. Further studies 

directly evaluating food intake and energy expenditure in this model will further show how 

subordinate females are at risk for negative energy balance. 

Disclosures: V. Michopoulos, None; J.B. Hoffman, None; E. Kessler, None; M.E. Wilson, 

None. 

Poster 


Systems 




Title: Efficacy of sleep-phase antagonism of T-type calcium channels in animal models of 

obesity 

Authors: *V. N. UEBELE 1 , C. E. NUSS 1 , R. L. KRAUS 1 , S. M. DORAN 1 , S. L. GARSON 1 , J. 

C. BARROW 2 , T. S. REGER 2 , Z.-Q. YANG 2 , J. E. BALLARD 3 , C. TANG 3 , J. M. METZGER 4 , 

S.-P. WANG 4 , K. S. KOBLAN 1 , J. J. RENGER 1 ; 

1 Neurosci., 2 Medicinal Chem., 3 Drug Metabolism, Merck, West Point, PA; 4 Pharmacol., Merck, 

Rahway, NJ 

Abstract: While the epidemic and consequences of metabolic syndrome and obesity are widely 

recognized, the correlation of obesity with sleep restriction is only recently gaining attention. 

Human and animal studies support a bidirectional regulation between sleep and weight control, 

with some studies suggesting diet affects sleep and others indicating that sleep disruption may 

increase the risk for pathologic weight gain. However, the pharmacological investigation of this 

link remains unexplored. T-type voltage-gated calcium channels play an important role in the 

thalamocortical loop which regulates sleep-wake EEG oscillations. Here, we examine the role of 

T-type calcium channels in the recently established link between sleep and metabolism. Mice 

lacking the CaV3.1 channel resisted diet-induced weight gain. Because no selective T-type 

calcium channel antagonists were available to explore this finding pharmacologically, a 

medicinal chemistry effort to optimize the potency, selectivity and pharmacokinetic properties of 

a T-type calcium channel antagonist was initiated and resulted in TTA-1. TTA-1 is a state 

dependent T-type calcium channel antagonist with potencies of 9.4 ± 4.4 (n=7) and 384 ± 266 

(n=8) nM in depolarized and hyperpolarized fluorometric imaging plate reader (FLIPR)

functional assays, respectively and 98 and 4200 nM in CaV3.3 voltage clamp assays at -80 and - 

100 mV holding potential, respectively. Selectivity for T-type calcium channels was assessed in 

more than 150 functional and binding assays with no activities identified with an IC50 less than 

10 uM. Dosing rodents with a short half-life, potent and selective T-type calcium channel 

antagonist prior to the sleep phase decreased wake and protected against weight gain in animals 

simultaneously starting treatment and high fat diet, without decreasing food intake. These 

changes occur in the absence of compound exposure during the active (feeding) phase, and 

despite treated animals ingesting more food when normalized to body weight. Administration of 

compound to already obese animals prior to the sleep phase reduced body weight and fat mass 

while increasing lean mass. Additionally, treated animals fed a normal diet did not show weight 

changes after 13 weeks of treatment. This sleep phase-restricted pharmacological treatment 

confirms the important role of sleep in metabolic regulation and suggests a novel therapeutic 

strategy for addressing the rapidly growing epidemics of obesity and metabolic syndrome. 

Disclosures: V.N. Uebele , Merck Research Labs, A. Employment (full or part-time); Merck 

Research Labs, E. Ownership Interest (stock, stock options, patent or other intellectual property); 

C.E. Nuss, Merck Research Labs, A. Employment (full or part-time); Merck Research Labs, E. 

Ownership Interest (stock, stock options, patent or other intellectual property); R.L. Kraus, 

Merck Research Labs, A. Employment (full or part-time); Merck Research Labs, E. Ownership 

Interest (stock, stock options, patent or other intellectual property); S.M. Doran, Merck 

Research Labs, A. Employment (full or part-time); Merck Research Labs, E. Ownership Interest 

(stock, stock options, patent or other intellectual property); S.L. Garson, Merck, A. Employment 

(full or part-time); Merck, E. Ownership Interest (stock, stock options, patent or other intellectual 

property); J.C. Barrow, Merck, A. Employment (full or part-time); Merck, E. Ownership 

Interest (stock, stock options, patent or other intellectual property); T.S. Reger, Merck, A. 

Employment (full or part-time); Merck, E. Ownership Interest (stock, stock options, patent or 

other intellectual property); Z. Yang, Merck, A. Employment (full or part-time); Merck, E. 

Ownership Interest (stock, stock options, patent or other intellectual property); J.E. Ballard, 

Merck, A. Employment (full or part-time); Merck, E. Ownership Interest (stock, stock options, 

patent or other intellectual property); C. Tang, Merck, A. Employment (full or part-time); 

Merck, E. Ownership Interest (stock, stock options, patent or other intellectual property); J.M. 

Metzger, Merck, A. Employment (full or part-time); Merck, E. Ownership Interest (stock, stock 

options, patent or other intellectual property); S. Wang, Merck, A. Employment (full or parttime); 

Merck, E. Ownership Interest (stock, stock options, patent or other intellectual property); 

K.S. Koblan, Merck, A. Employment (full or part-time); Merck, E. Ownership Interest (stock, 

stock options, patent or other intellectual property); J.J. Renger, Merck, E. Ownership Interest 

(stock, stock options, patent or other intellectual property); Merck, A. Employment (full or parttime). 

Poster 


Systems


Program#/Poster#: 284.6/QQ1 


Support: NIH Grant F010384 

NIH Grant F007995 

Title: High temporal resolution monitoring of leptin-modulated dopamine release by 

microdialysis coupled to MEKC 

Authors: *M. L. PERRY, M. SHOU, G. M. LEINNINGER, M. G. MYERS, Jr., R. T. 

KENNEDY; 

Univ. of Michigan, Ann Arbor, MI 

Abstract: Conventionally, microdialysis has been coupled to HPLC for off-line detection of in 

vivo-sampled analytes of interest. The dialysate must be analyzed in 10-30 minute fractions to 

meet the concentration detection limits for these assays. The low temporal resolution of analysis 

by HPLC prevents observation of rapid changes in extracellular neurotransmitter concentrations. 

Micellar electrokinetic chromatography (MEKC), a variant of capillary electrophoresis, requires 

smaller sample volumes and permits higher sampling frequency. A MEKC-based separation has 

been developed that resolves naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized dopamine in 

90. The high mass sensitivity of MEKC and the fast separation time facilitate on-line monitoring 

of rapid in vivo dopamine release. We applied this near real-time monitoring technique to the 

study of leptin-modulated dopamine release in the nucleus accumbens (NAc). Leptin-receptor 

expressing neurons in the lateral hypothalamus (LHA) project to the ventral tegmental area 

(VTA), and intra-LHA leptin infusion of leptin in ob/ob mice increased tyrosine hydroxylase 

expression in the VTA and tissue DA content in the NAc. This work aimed to determine whether 

leptin modulates DA release in the NAc of Sprague-Dawley rats by either causing an increase in 

basal extracellular dopamine concentration or by increasing amphetamine-stimulated dopamine 

release. Rats received either 1 mg/kg leptin or vehicle i.p. followed several hours later by 1 

mg/kg amphetamine s.c. Leptin had no effect on dopamine release under basal conditions but 

increased the amphetamine-stimulated release as compared to vehicle. These results suggest the 

importance of the LHA to VTA pathway in the regulation of striatal DA homeostasis in response 

in normal animals. 

Disclosures: M.L. Perry, None; M. Shou, None; G.M. Leinninger, None; M.G. Myers, 

None; R.T. Kennedy, None. 

Poster


Systems 




Support: Creight Foundation Grant, Drexel University 

Bucknell University 

Title: High fat intake alters expression of 5-HT and NMDA receptors in hippocampus of adult 

male rats 

Authors: *K. C. PAGE 1 , R. E. MALIK 1 , J. A. SCHAEFFER 1 , E. K. ANDAY 2 ; 

1 Bucknell Univ., Lewisburg, PA; 2 Pediatrics, Drexel Univ. Col. of Med., Philadelphia, PA 

Abstract: Recent studies have shown that leptin-deficient or leptin-insensitive rodents have 

disturbances in serotonergic function as well as impairments in hippocampal synaptic plasticity 

which is associated with leptin‟s capacity for enhancing NMDA receptor function. Our 

hypothesis was that rodents subjected to excess levels of saturated fat during early life and/or 

postweaning into adult life are more likely to exhibit disturbances in hippocampal serotonergic 

and glutamatergic systems. Rat dams were fed a control or high fat diet (45% kcal fat) prior to 

mating, pregnancy, and lactation. Male offspring from control dams and high fat-fed dams were 

weaned to control diet (CC or HFC) or weaned to the high fat diet (CHF or HFF) and tested for 

immobility using the forced swim test at 120 days. All animals were fasted overnight and 

sacrificed 24 hours after completion of behavioral testing. Radioimmunoassay was used to 

measure serum leptin. Real-time RT-PCR was used to analyze hippocampal expression of 5- 

HT1A, 1B and 2A as well as NMDA R1, R2A and R2B. Our results demonstrate that exposure 

to excess saturated fat during gestation and lactation did not significantly affect hippocampal 5- 

HT or NMDA receptors in the HFC compared to control animals (CC), however, a significant 

reduction in all of these receptors was measured in animals transitioned to the high-fat diet after 

weaning (CHF; P

Disclosures: K.C. Page , None; R.E. Malik, None; E.K. Anday, None; J.A. Schaeffer, None. 

Poster 


Systems 




Support: NIH R01 DK57768 

AHA Grant 0615602Z 

Title: Integration of metabolic & reproductive signals by leptin receptor-expressing neurons in 

the hypothalamic ventral premammillary nucleus 

Authors: *R. L. LESHAN 1 , G. LOUIS 2 , C. RHODES 3 , M. G. MYERS, Jr 2 ; 

1 Univ. Michigan, Ann Arbor, MI; 2 Intrnl. Med., Univ. of Michigan, Ann Arbor, MI; 3 Kovler 

Diabetes Ctr., Univ. of Chicago, Chicago, IL 

Abstract: The brain senses and integrates hormonal, sensory, and metabolic cues to 

appropriately regulate physiological processes. One such cue is leptin, which signals the status of 

long-term energy stores through the long form of its receptor (LepRb), and informs the 

appropriateness of expending resources on energy-intensive processes that are not immediately 

critical to life, such as reproduction. Using a mouse strain in which LepRb neurons express 

enhanced green fluorescent protein (LepRb GFP mice) we identified a population of LepRbexpressing 

cells in the ventral premammillary nucleus of the hypothalamus (PMv) similar in 

magnitude to the population in the arcuate nucleus of the hypothalamus (ARC). Leptin activated 

the majority of these PMv LepRb neurons by the criteria of increased c-fos immunoreactivity in 

vivo as well as directly activating them during electrophysiological recordings in acute slice 

preparations, in vitro. Since the PMv has been implicated in the integration of seasonal and 

odorant cues to regulate the reproductive axis, we examined the responsiveness of PMv LepRb 

neurons to sexual odorants by exposing male and female LepRb GFP mice to bedding soiled by the 

opposite sex, which stimulated FosIR in these PMv LepRb neurons in both male and female 

mice. Finally, we injected an adenoviral tract-tracer, in which cre recombinase induces 

expression of a farnesylated EGFP, to the PMv of lepr Cre mice revealing the neural processes of 

PMv LepRb neurons and demonstrating their projection to the preoptic and anteroventral 

periventricular areas, regions containing gonadotropin-releasing hormone and kisspeptin cells 

and known to regulate reproduction. Taken together, our results suggest that the PMv LepRb

neurons respond to metabolic and sexual odorant cues and may integrate these inputs to regulate 

the neuroendocrine reproductive system. 

Disclosures: R.L. Leshan, None; G. Louis, None; C. Rhodes, None; M.G. Myers, None. 

Poster 


Systems 





Minnesota Partnership for Biotechnology and Genomics 


Title: Evaluation of a quantitative magnetic resonance imaging system for whole body 

composition analysis in rodents 

Authors: *J. P. NIXON 1 , M. ZHANG 3 , C. M. NOVAK 3 , J. A. LEVINE 3 , C. KOTZ 2,4 ; 

1 MinnCResT, Sch. of Dent., 2 Food Sci. and Nutr., Univ. of Minnesota, Minneapolis, MN; 3 Mayo 

Clin., Rochester, MN; 4 VA Med. Ctr., Minneapolis, MN 

Abstract: In studying the neural mechanisms underlying propensity for weight gain, it is often 

desirable to quickly obtain accurate, repeated measurements of body composition. We evaluated 

the precision and accuracy of the EchoMRI quantitative magnetic resonance (QMR) body 

composition method in comparison to traditional whole-body chemical carcass analysis (CCA) 

methods, using an EchoMRI system designed to analyze both rats and mice to compare data 

obtained from QMR and CCA analyses. Three groups of rats were examined: Fisher outbred rats 

(n = 6), and obesity-prone (n = 6) and obesity-resistant (n = 5) Sprague-Dawley rats. For all 

animals, live fat and lean mass measurements were performed using QMR. Following QMR 

measurements, fat and lean mass measurements were obtained using CCA. For CCA, lean mass 

measurements were defined as water weight plus ash and protein weights. Body composition 

analysis using EchoMRI QMR provided fat and lean mass values comparable to those obtained 

by CCA. Deming regressions showed positive correlations between both fat and lean mass as 

determined by QMR and CCA measurements (Fat mass slope = 1.230 ± 0.0310; Lean mass slope 

= 0.860 ± 0.017). This regression analysis shows that QMR fat mass measurements are

approximately 1.2 times higher than fat mass measurements using CCA, while QMR lean mass 

measurements are approximately 0.86 times those obtained using CCA. The consistent linear 

correlations between measurement methods indicate that studies using either method can be 

directly compared. This study also demonstrates that QMR measurements have higher precision 

for both fat and lean mass than measurements obtained using CCA. The coefficient of variation 

(CoV) from repeated measures was used to estimate precision of methods. In both fat and lean 

mass measurements, QMR showed significantly less variation between measurements than did 

CCA analysis (Fat mass: QMR Live = 0.942 ± 0.122; CCA = 5.286 ± 1.457; p = 0.0095; Lean 

mass: QMR Live = 0.225 ± 0.030; CCA = 0.975 ± 0.182; p = 0.0010). No significant differences 

in fat or lean mass CoV by strain were observed. We conclude that QMR offers a fast, nonterminal, 

and accurate method of body composition analysis, yielding measurements comparable 

to that obtained by CCA without the need for time-consuming chemical analyses of the latter 

method. This study and previous investigations using mice demonstrate that the EchoMRI QMR 

system is a valid method for body composition analysis in obesity research. 

Disclosures: J.P. Nixon, None; M. Zhang, None; C.M. Novak, None; J.A. Levine, None; C. 

Kotz, None. 

Poster 


Systems 




Support: Unrestricted gift from Vivilac, Inc, Ashton, MD 

Title: Growth and body composition of Charles River Sprague-Dawley rats 

Authors: J. LEGER 1 , *R. R. SAKAI 2 , T. CASTONGUAY 1 ; 

1 Nutr. and Food Sci., Univ. of Maryland, College Park, MD; 2 Dept Psych, North, Univ. 

Cincinnati GRI, Cincinnati, OH 

Abstract: Laboratory rats have been used for studying the interaction between diet and body 

composition for more than 100 years. One of the assumptions that an experimenter makes in 

using rats is that if animals are fed the same diet and housed in the same environment that their 

growth and body composition will be the same. Differences in growth and body composition 

have been associated with strain, sex, cage size, activity level and a host of other environmental 

conditions (temperature, humidity, light cycle etc.). During the past year, we had the opportunity

to evaluate the validity of the assumption that the same strain of rat, purchased from the same 

vendor, housed in the same cages, fed the same diet, and maintained under the same conditions 

would provide growth and body composition data that was the same. Four shipments of male 

Sprague-Dawley (CD strain) rats weighing ~250g were purchased from Charles River Labs: the 

first in August, the second in December, the third in February, and the last in April. One group 

from each shipment (n=8) was assigned to an experimental control group and was given ad 

libitum access to food (Teklad 7012 rat diet) and water. The remaining rats were assigned to 

various diet groups. All rats were individually housed in standard stainless steel hanging cages, 

and were maintained in the same vivarium, on the same day/night cycle, and at the same time 

every day. Each rat was weighed and its 24 h food intake was determined daily. The control 

group from shipment one had a significantly lower growth rate and lower mean daily caloric 

intake than the controls from the other three shipments (p

Authors: *G. M. LEINNINGER 1 , Y.-H. JO 3 , R. L. LESHAN 1 , G. W. LOUIS 1 , J. G. 

BARRERA 4 , H. WILSON 4 , H. YANG 5 , M. L. PERRY 2 , J. C. JONES 1 , Y. GONG 1 , C. J. 

RHODES 6 , S. CHUA, Jr. 3 , R. T. KENNEDY 2 , R. J. SEELEY 4 , J. B. BECKER 5 , H. 

MUNZBERG 1 , M. G. MYERS, Jr. 1 ; 

1 MEND, Intrnl. Med., 2 Chem., Univ. Michigan, Ann Arbor, MI; 3 Dept. of Med., Albert Einstein 

Col. of Med., Bronx, NY; 4 Dept. of Psychiatry and Genome Res. Inst., Univ. of Cincinnati, 

Cincinnati, OH; 5 Mol. and Behavioral Neurosci. Inst., Univ. of Michigan, Ann Arbor, MI; 

6 Kovler Diabetes Ctr., Univ. of Chicago, Chicago, IL 

Abstract: The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area 

(VTA) and other components of the mesolimbic dopamine (DA) system to control the incentive 

to feed. While a variety of data suggest that homeostatic signals of energy balance modulate the 

mesolimbic dopamine system, the mechanisms underlying this regulation remain incompletely 

understood. Here we show that leptin, an anorexigenic hormone and signal of body energy 

status, directly regulates a large population of leptin receptor (LepRb)-expressing neurons in the 

LHA, and that these LHA LepRb neurons densely innervate the VTA. Furthermore, leptin action 

via LHA LepRb neurons decreases feeding and body weight and regulates gene expression and 

DA content in the mesolimbic DA system. LHA LepRb neurons, which are distinct from 

previously described orexigenic LHA neuronal populations, contain the neurotransmitter GABA. 

A subpopulation of the LHA LepRb neurons also express neurotensin (Nts) and genetic deletion 

of Nts receptor-1 reverses the anorectic effect of intra-LHA leptin treatment, suggesting the 

importance of Nts to the regulation of feeding by LHA LepRb neurons. Thus, leptin action via 

these novel LHA LepRb neurons blunts feeding and modulates the mesolimbic DA system. 

Disclosures: G.M. Leinninger, None; Y. Jo, None; R.L. Leshan, None; G.W. Louis, 

None; J.G. Barrera, None; H. Wilson, None; H. Yang, None; M.L. Perry, None; J.C. Jones, 

None; Y. Gong, None; C.J. Rhodes, None; S. Chua, None; R.J. Seeley, None; J.B. Becker, 

None; M.G. Myers, None; H. Munzberg, None; R.T. Kennedy, None. 

Poster 


Systems 





NIH Grant DK47348

NIH Grant RR021945 

Title: Innervation of skeletal muscle by leptin receptor containing neurons 

Authors: *T. BABIC 1 , M. N. PURPERA 2 , H. MUENZBERG 3 , M. G. MYERS, Jr. 3 , B. W. 

BANFIELD 4 , H.-R. BERTHOUD 1 , C. D. MORRISON 2 ; 

1 Neurobio. of Nutr., 2 Neurosignalling, Pennington Biomed Res. Ctr., Baton Rouge, LA; 3 Univ. 

of Michigan, Ann Arbor, MI; 4 Hlth. Sci. Ctr., Univ. of Colorado, Denver, CO 

Abstract: Leptin alters skeletal muscle metabolism in part by acting in the central nervous 

system, but the neural pathways that mediate these effects are not fully understood. To determine 

whether leptin receptors are expressed by sympathetic premotor neurons projecting to skeletal 

muscle, the gastrocnemius muscle of transgenic mice expressing green fluorescent protein (GFP) 

in leptin receptor (LepRb) neurons was injected with a modified pseudorabies virus (PRV) 

expressing red fluorescent protein (RFP). In the hypothalamus, large populations of PRVlabelled 

neurons were observed in the paraventricular nucleus (PVN) and the lateral 

hypothalamus, with lower numbers in the arcuate nucleus, retrochiasmatic area, ventromedial 

and dorsomedial hypothalamus. GFP-containing (LepRb) neurons were observed in these same 

brain areas, with the exception of the PVN. Significant colocalization was detected only within 

portions of the retrochiasmatic area, where 30 + 3% of PRV-labelled neurons contained GFP 

(LepRb), and 7.6 + 0.9 % of GFP-positive neurons contained PRV. In the brainstem, PRVlabelled 

neurons were observed in the raphe pallidus, gigantocellular reticular formation, 

ventrolateral medulla, and a few in the nucleus of the solitary tract (NTS), where a few GFPpositive 

(LepRb) neurons were also found. Within this small population, 30 + 3% of the PRVpositive 

neurons contained GFP and 14 + 4 % of GFP-positive neurons contained PRV. These 

data indicate that relatively few LepRb positive neurons project to gastrocnemius muscle, with 

most of these localized to the restrochiasmatic area. Given that POMC containing neurons in the 

retrochiasmatic area project directly to sympathetic preganglionic neurons in the spinal cord, it is 

likely that these PRV-positive LepRb neurons also contain POMC. Also, considering the 

evidence that leptin-sensitive neurons in the arcuate nucleus project heavily to the 

paraventricular nucleus and lateral hypothalamus, it is possible that longer survival times might 

reveal additional colocalization reflecting these higher order connections. 

Disclosures: T. Babic, None; M.G. Myers, None; H. Muenzberg, None; B.W. Banfield, 

None; C.D. Morrison, None; M.N. Purpera, None; H. Berthoud, None. 

Poster 


Systems 




Support: NIH grant DK 40498 

Juvenile Diabetes Research Foundation (JDRF) 

Title: Signals derived from adipose stores and from deficits in fat oxidation control feeding by 

independent mechanisms 

Authors: *M. F. WIATER, A. EMANUEL, B. HUDSON, S. RITTER; 

Dept VCAPP, Washington State Univ., Pullman, WA 

Abstract: Leptin is a circulating adipose-derived protein that acts centrally to decrease food 

intake and increase energy metabolism. Circulating leptin levels are positively correlated with 

the amount of body adiposity, such that elevated leptin levels signal excess adiposity and exert 

negative feedback control on energy intake. The lipoprivic control is a stimulatory control of 

feeding that can be experimentally activated by a number of drugs, including beta 

mercaptoacetate (MA), that acutely reduce fatty acid oxidation. The physiological context in 

which this control is operative is not known. Here we report results of two experiments in which 

we assessed the possible reciprocity of these signals of fat excess and fat deficit with respect to 

food intake. Both experiments utilized chronic leptin treatment in order to examine the effect of 

lipoprivation under the different metabolic environments associated with chronic treatment. 

Leptin (3 ug) or artificial cerebrospinal fluid (aCSF) was administered daily into the lateral 

ventricle. In experiment 1, feeding induced by MA (68 mg/kg, ip) was tested on days 0, 3 and 10 

of leptin treatment (with 0.9% saline baseline tests on days -1, 2 and 9). Baseline intakes did not 

differ across days between groups. Feeding was significantly elevated by MA in both aCSF and 

leptin groups and there were no significant between or within group differences across test days. 

Thus, MA stimulated feeding under basal conditions (day 0) and under two different leptininduced 

metabolic states. MA increased feeding on day 3 when rats were in a highly lipolytic 

state, inhibition of 24-hr food intake was maximal (55% of baseline) and body weight was 

rapidly falling. MA also increased intake on day 10 when body weight was stabilizing at its 

fatless nadir (80% of baseline), but food intake was increasing toward control (was 70% of 

baseline). These results suggest that the feeding response to MA is not modified directly by 

leptin or by fat stores per se, and support the strong linkage of the lipoprivic control to ongoing 

metabolic events. In experiment 2, we treated rats systemically with capsaicin to eliminate the 

lipoprivic control and examined the effect of this lesion on feeding and body weight changes 

during chronic central leptin administration for 20 days. Absence of the lipoprivic control did not 

alter leptin-induced changes in feeding, body weight or triglyceride levels at any time point. 

These data indicate that signals derived from adipose stores and from deficits in fat oxidation 

control feeding by independent mechanisms. 

Disclosures: M.F. Wiater , NIH grant DK 40498 and Juvenile Diabetes Research Foundation 

(JDRF), B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received); S. Ritter, NIH grant DK 40498 and Juvenile Diabetes

Research Foundation (JDRF), C. Other Research Support (receipt of drugs, supplies, equipment 

or other in-kind support); A. Emanuel, None; B. Hudson, None. 

Poster 


Systems 




Support: AHA053298N 

NARSAD 

P/F DK020572-30 

Title: Leptin receptor neurons in the dorsomedial hypothalamus as important regulator of 

preautonomic and neuroendocrine function in the paraventricular nucleus 

Authors: M. FAOUZI 1 , I. KERMAN 1 , G. LOUIS 1 , R. LESHAN 1 , G. M. LEINNINGER 1 , J. C. 

JONES 1 , S. J. WATSON 1 , C. J. RHODES 2 , *H. MUNZBERG 1 ; 

1 Univ. Michigan, Ann Arbor, MI; 2 Univ. of Chicago, Chicago, IL 

Abstract: Leptin acts in the central nervous system (CNS) to coordinate metabolism and energy 

balance. Deficiency of leptin or its receptor (LepRb) results in profound neuroendocrine failure, 

autonomic dysfunction, and hyperphagia. While leptin action via well-studied POMC- and 

AgRP/NPY-expressing LepRb neurons in the arcuate nucleus of the hypothalamus (ARC) 

contributes importantly to overall leptin action, it has become clear that these neurons can only 

account for a fraction of leptin action, and that leptin acts via a distributed network of LepRb 

neurons in the CNS. The dorsomedial hypothalamus (DMH) integrates information about energy 

balance, body temperature, and neuroendocrine status to modulate autonomic nervous system 

(ANS) function as well as stress and ingestion. Importantly, the DMH contains a major 

population of LepRb neurons. In order to study the role of DMH/LepRb neurons in leptin action, 

we stereotaxically injected cre-inducible adenoviral tract-tracers in LepRb Cre mice to investigate 

their projections and terminal fields. This analysis demonstrated dense projections to the 

parvocellular paraventricular nucleus (PVN) and median preoptic area (mPOA) as well as 

reciprocal projections to the ARC. The reciprocal interconnection of the DMH/LepRb and 

ARC/LepRb systems suggests that these cell groups function as a unit to coordinate leptin action. 

The mPOA also contains a dense population of LepRb neurons and is known to interconnect

with the DMH as an important pathway for thermoregulatory adaptations via the preautonomic 

and neuroendocrine PVN. Furthermore, using pseudorabies virus (PRV) injections into brown 

adipose tissue (BAT) we found that indeed DMH/LepRb neurons are involved in the regulation 

of the sympathetic BAT input. In addition, PRV was detected within LepRb neurons of the 

retrochiasmatic hypothalamus/ARC and mPOA, areas that contain neurons that sense the 

circulation (e.g. leptin levels) and project to the DMH, suggesting that they may serve as sensory 

inputs into the DMH to regulate sympathetic PVN outputs to the BAT. Therefore, DMH/LepRb 

neurons may mediate important aspects of leptin action on the ANS and neuroendocrine 

function. Grant support: AHA053298N and P/F DK020572-30 to HM; NARSAD Young 

Investigator Award to IAK. 

Disclosures: M. Faouzi, None; I. Kerman, None; G. Louis, None; R. Leshan, None; G.M. 

Leinninger, None; J.C. Jones, None; S.J. Watson, None; C.J. Rhodes, None; H. Munzberg , 

None. 

Poster 


Systems 





NIH Grant HD47643 

NIH Grant DK56373 

Title: TNF activates astrocytes and catecholaminergic neurons in the solitary nucleus 

Authors: R. C. ROGERS, *G. E. HERMANN; 

Lab. Autonomic Neurosci, Pennington Biomed. Res. Ctr., Baton Rouge, LA 

Abstract: Tumor necrosis factor [TNF] produces a profound cachexia and anorexia associated 

with gastrointestinal stasis. Our work has suggested that the principal site of action of TNF to 

cause this change in gastric function is via vagal afferents within the nucleus of the solitary tract 

[NST]. Excitation of these afferents presumably causes gastric stasis by activating downstream 

NST neurons that, in turn, suppress gastric motility via action on neurons in the dorsal motor 

nucleus of the vagus that project to the stomach. Results from our parallel studies on gastric

vago-vagal reflexes suggest that noradrenergic neurons in the NST are particularly important to 

the generation of reflex gastroinhibition. Convergence of these observations led us to 

hypothesize that TNF action within the NST may preferentially affect putative noradrenergic 

neurons. The current study demonstrates a dose-dependent TNF activation of cells [as indicated 

by cFOS production] in the NST; ~42% of these TNF-activated neurons in the NST were 

phenotypically identified as tyrosine-hydroxylase (TH)-positive. In contrast, less than 10% of the 

nitrergic neurons were activated after TNF exposure. To our surprise, another 45% of the cFOS 

activated cells in the NST were phenotypically identified to be astrocytes. These results suggest 

that TNF may work within the hindbrain to produce gastroinhibition by activating the same 

phenotypic NST neurons that have been associated with driving inhibitory gastric reflexes (i.e., 

TH-positive) as well as astrocytes to, ultimately, modulate the responsiveness of NST neurons. 

These observations also suggested that NST astrocytes should be activated by vagal afferent 

stimulation in much the same way that NST neurons are. Indeed, calcium imaging studies show 

that astrocyte activation by vagal afferent stimulation does occur. 

Taken together with previous observations, the present results suggest that intense or prolonged 

vagal afferent activity [induced by visceral pathway activity, the action of gut hormones or the 

action of cytokines like TNF] can alter local astrocyte immediate early gene expression that, in 

turn, can provoke long-term, perhaps permanent changes in the sensitivity of vagal-reflex 

circuitry. Such modulation of neuronal responsiveness as a consequence glial-neuronal 

communication may have precedence in migraine pathology (Thalakoti et al., 2007), enteric 

celiac disease (Esposito et al., 2007), and astrocyte-neuron vulnerability to prenatal stress effects 

on brain development (Barros et al., 2006). This relationship between glia and adjacent neurons 

may provide another target for therapeutic intervention in autonomic disease states. 

Disclosures: R.C. Rogers, None; G.E. Hermann , None. 

Poster 


Systems 




Title: Neural activity in rat forebrain regions induced by post-ingestive nutrients 

Authors: *T. TSURUGIZAWA, T. KONDOH, A. UEMATSU, K. TORII; 

Inst. Life Sci., Ajinomoto Co., Inc., Kawasaki, Japan

Abstract: Recent studies have shown the expression of nutrient-sensing systems transmitting 

nutritive information from the gastrointestinal tract to the brain. However brain activity induced 

by ingested nutrients has not been investigated. The first goal of the present study was, using 

functional magnetic resonance imaging, to determine dynamic neural activity following 

intragastric nutrients (glucose, L-glutamate and NaCl) administration. Rats were anesthetized 

with alpha-chloralose and after 30 min, MRI scanning was started. Thirty min after the start of 

scanning, 60 mM NaCl, monosodium L-glutamate (MSG), glucose, or 150 mM NaCl solution 

was infused into the stomach through a cannula for 10 min and then scanning was lasted for 50 

min. Our data clearly demonstrated that post-ingestive nutrient-specific neural activity was 

observed in several forebrain regions including the nucleus accumbens, hypothalamus, and 

limbic system with different timings. Second, we assessed a role of the vagal afferent nerve on 

transmitting nutrient signals from gastrointestinal tract to the brain using rats with 

subdiaphragmatic total vagotomy (TVX). TVX eliminated substantially the MSG and NaClinduced 

activation. On the contrary, TVX did not affect glucose-induced brain activity. These 

results indicate that postingestive nutrient-induced neural activity in the forebrain is due to the 

distinct pathway transmitting from the gut to the brain. 

Disclosures: T. Tsurugizawa, None; T. Kondoh, None; A. Uematsu, None; K. Torii, None. 

Poster 


Systems 





Title: Novel molecular tracing techniques to study the neural mechanisms of leptin action 

Authors: *G. W. LOUIS 1 , G. M. LEINNINGER 1 , C. J. RHODES 2 , M. G. MYERS 1 ; 

1 Univ. Michigan, Ann Arbor, MI; 2 Univ. of Chicago, Chicago, IL 

Abstract: Leptin, which is secreted by adipocytes as a signal of energy stores, acts via the long 

form of the leptin receptor (LepRb) in the hypothalamus and other areas of the brain to regulate 

energy balance and neuroendocrine function. Many regions involved in the regulation of energy 

balance contain large groups of LepRb-expressing neurons, including intensively studied “satiety 

centers" (such as the arcuate nucleus (ARC)), and “feeding centers” (such as the lateral 

hypothalamic area (LHA)). The LHA contains two well-described populations of orexigenic

neurons that are defined by their expression of melanin concentrating hormone (MCH) and 

orexin (OX); the LHA also contains a distinct population of LepRb-expressing neurons. In order 

to understand the LepRb-expressing neural afferents of LHA neurons, we generated and 

employed several novel adenoviral and transgenic systems in which cre recombinase promotes 

the expression of the anterograde tracer, wheat germ agglutinin (WGA), or the retrograde tracer, 

tetanus toxin C (TTC). Utilizing these systems in conjunction with LepRb cre mice initially 

demonstrated the direct innervation of both MCH and OX neurons by anatomically undefined 

groups of LepRb neurons, and revealed the synaptic coupling of OX, but not MCH neurons with 

LHA LepRb neurons. Using the trans-synaptic tracers in combination with ARC-specific Pomc cre 

and Agrp cre mice demonstrated that these ARC LepRb neurons directly innervate both OX and 

MCH neurons, but not LHA LepRb neurons. The use of these novel neuroanatomical tracing 

tools permits the analysis of the synaptic connectivity of distinct populations of intermingled 

neurons, and has revealed the mechanisms by which specific groups of LepRb neurons modulate 

distinct aspects of LHA physiology. 

Disclosures: G.W. Louis, None; G.M. Leinninger, None; C.J. Rhodes, None; M.G. Myers, 

None. 

Poster 


Systems 




Support: NIH grant DK065971 

Title: Fasting reduces vomiting in the musk shrew 

Authors: *C. C. HORN 1 , C. E. FITZGERALD 2 , M. I. FRIEDMAN 1 ; 

1 Monell Chem. Senses Ctr., Philadelphia, PA; 2 Univ. of Pennsylvania, Phildadelphia, PA 

Abstract: Nausea and vomiting are systems that protect the body from ingested toxins. These 

symptoms are ubiquitous in clinical care and treatment, including stimulation by various 

medicines (cancer chemotherapy drugs, anesthetics, opioids), pregnancy, and radiotherapy, but 

the neurobiology of these processes is not well understood. The neural system for emesis is 

generally considered to be a “hard-wired” reflex that is determined by the strength of the 

stimulus. Prior reports show that appetite stimulants (ghrelin and endocannabinoids) can reduce 

emesis in laboratory animals, suggesting that stimulation of feeding can modulate emesis. Here

we further tested the idea that stimulation of food intake reduces emesis by using food 

deprivation to stimulate appetite. Twenty-four h, but not 3 h, of fasting was sufficient to 

stimulate feeding in house musk shrews (Suncus murinus) and completely blocked emetic 

episodes produced by the chemotherapy agent cisplatin (20 mg/kg; a peripheral stimulus), and 

reduced the emetic effects of nicotine (2 mg/kg; a central stimulus). Emetic episodes need not 

produce an actual expulsion of gastric contents because animals will still retch in response to 

toxins. Contents of the stomach did not account for the reduction of emesis by 24 h food 

deprivation because only 3 h of food restriction emptied the stomach but was not sufficient to 

reduce the number of vomiting episodes. These data indicate that nutritional status has a strong 

effect on emesis and suggest that nutritional strategies could be employed as an adjunct to antiemetic 

drugs to help control emesis. 

Disclosures: C.C. Horn , None; C.E. Fitzgerald, None; M.I. Friedman, None. 

Poster 


Systems 




Support: NIDA 5P01DA021633-02 (HA and SJW) 


Office of Naval Research N00014-02-1-0879 

Title: Dietary self-selection in aged metabolic syndrome rats 

Authors: *P. R. BURGHARDT 1 , S. BRITTON 2 , L. KOCH 2 , S. J. WATSON 1 , H. AKIL 1 ; 

1 Molec & Behav Neurosci Inst., Univ. Michigan, Ann Arbor, MI; 2 Physical Med. & Rehabil., 

Univ. of Michigan, Ann Arbor, MI 

Abstract: Metabolic syndrome increases risk for developing a number of other diseases. 

Biological changes that occur during aging influence energy management however the 

interaction between age and energy management is not understood. Selective breeding of rats for 

the ability to run to exhaustion on a treadmill has produced lines with divergent running capacity 

termed high-capacity runners (HCR) and low capacity runners (LCR). In order to determine the 

role of age and reproductive status on dietary selection, 12- and 18-month old female HCR and

LCR rats were given ad libitum access to self-select from three purified macronutrient diets 

(Carbohydrate, Protein, or Fat). Eighteen month old LCR rats showed substantial weight gain 

over the course of the dietary self-selection intervention. In addition, LCR rats showed elevated 

fasting blood-glucose levels regardless of age. To better understand the interaction between 

aging and metabolism, we are also investigating additional hormonal markers of energy 

management and gene-expression responses in hypothalamic energy management regions. 

Disclosures: P.R. Burghardt , None; S. Britton, None; L. Koch, None; S.J. Watson, 

None; H. Akil, None. 

Poster 


Systems 




Support: Swedish Cancer Foundation 070630 

Landstinget i Östergötland 

Swedish Research Council 7879 

Title: Cancer-elicited anorexia is associated with induced Cox-2 expression in the brain vascular 

cells and can be reversed by Cox inhibition without affecting tumor size 

Authors: *J. P. RUUD 1 , W. WANG 3 , K. LUNDHOLM 3 , A. BLOMQVIST 2 ; 

1 Div. of Cell Biol., 2 Clin. and Exptl. Med., Linköping, Sweden; 3 Dept. of Surgery, The Inst. of 

Clin. Sci., Gothenburg, Sweden 

Abstract: Several lines of evidence indicate that pro-inflammatory cytokines are involved in the 

development of anorexia-cachexia, a syndrome associated with cancer and several other chronic 

diseases. Because peripheral cytokines have been shown to induce prostaglandin synthesis in the 

brain (Ek et al., Nature 410: 430-1, 2001), and since the deletion of prostaglandin synthesizing 

enzymes abolishes cytokine-induced anorexia (Elander et al., Am J Physiol 292: R258-67, 2007), 

has it been suggested that the anorexia-cachexia syndrome is prostaglandin dependent. Here we 

examined the mechanisms of reduced food intake in a mouse model of cancer anorexia. A 

sarcoma (MCG 101) was transplanted onto C57BL/6 mice resulting in reduced food intake, 

without affecting water intake, after about 7 days. The anorexia was associated with induction of

the cyclooxygenase-2 (Cox-2) gene (two-fold) in the hypothalamus, the hunger and satiety center 

of the brain, of tumor-bearing mice, as shown by quantitative real-time PCR. The Cox-2 protein 

was found to be induced in cells lining the cerebral blood vessels, suggesting a tumor-induced 

signalling via brain vascular cells to the brain. As a next step, tumor-bearing mice were given the 

Cox-inhibitor indomethacin in the drinking water (6.7 κg/ml) during ten days, which completely 

abolished the development of anorexia. This treatment also resulted in reduced tumor growth as 

shown previously (Cahlin et al., Cancer Res 60: 1742-9, 2000). To differentiate between the 

effect of reduced tumor burden and the Cox-inhibition on the food intake, indomethacin was 

given to tumor-bearing mice before lights out at day 7, during established anorexia. The results 

show that a single day of treatment restored food intake to normal levels in tumor bearing mice. 

These findings indicate that cancer-associated anorexia in this model occurs by inflammatory 

mechanisms that involve cyclooxygenases, and show that the beneficial effect of indomethacin 

on food intake is not a consequence of reduced tumor size. 

Disclosures: J.P. Ruud, None; W. Wang, None; K. Lundholm, None; A. Blomqvist, None. 

Poster 


Systems 




Title: Putative flip-flop regulation of running and eating in rats 

Authors: *M. BANNAI 1,2 , K. KASUH 2 , K. NAKAHARA 2 , N. MURAKAMI 2 , M. 

TAKAHASHI 1 ; 

1 Inst. of Life Sci., Ajinomoto Co., Inc., Kawasaki-shi, Japan; 2 Dept of Vet. Physiol, Univ. of 

Miyazaki, Miyazaki, Japan 

Abstract: Rats run up to 10 km/day if they are kept in the cage equipped with a running wheel. 

Running and eating never occur simultaneously suggesting a substantial flip-flop regulation. We 

have previously reported interaction between wheel running and food consumption where food 

consumption decreased either slightly or significantly by deprivation of a running wheel or 

giving “re-run” condition, respectively. In this study, daily running period was restricted in order 

to know the implication of “running deprivation”. Male Wistar rats were housed individually in a 

cage with running wheel and food consumption was recorded daily. The controls were kept in a 

cage without a running wheel. The experimental group was allowed to access freely to the wheel 

from day 1 to 19 (unbound-running-1), access to the wheel only 3 hours in the dark phase from

day 20 to 40 (restricted-running), and access freely again from day 41 to 59 (unbound-running- 

2). Wheeling activity gradually increased from day 1 to 7, and stabilized around at 1,500 (an 

arbitrary unit) after day 8. During the restricted-running, wheeling activity decreased to 

approximately 300, however the wheeling activity jumped up to 700 during the unboundrunning-2, 

though the level was still lower than that in the unbound-running-1 period, which may 

probably be due to the change of set point of the wheel running during the running restriction. 

During the restricted-running period, food intake was significantly higher than that during both 

unbound-running-1 and -2 periods, suggesting that occasional running restriction changes 

regulation of food intake positively. This escalation is rather contradictory from the view of 

energy homeostasis, but for rats, they would have much time to eat foods instead of the time for 

running. When they could run for 24 hrs again, food intake returned to the levels of unboundrunning-1. 

Thus, the complete running deprivation and restricted running differently affected the 

relation between running and food consumption. For the start or re-start of full running activity 

in rats, an introduction of anorexic stage must be indispensable. One possible explanation may be 

that rats try to find “running time” at expense of “eating time” until the putative flip-flop 

regulatory system is established. This regulation, if any, would have substantial influence on 

body weight control. 

Disclosures: M. Bannai, Ajinomoto Co., Inc., A. Employment (full or part-time); K. Kasuh, 

None; K. Nakahara, University of Miyazaki, A. Employment (full or part-time); N. Murakami, 

University of Miyazaki, A. Employment (full or part-time); M. Takahashi, Ajinomoto Co., Inc., 

A. Employment (full or part-time). 

Poster 

285. Sleep: Molecular, Cellular and Pharmacology I 



Topic: E.08.c. Sleep: Molecular, cellular, and pharmacology 



Title: Pontine-wave (P-wave) generator activation-dependent memory processing involves 

protein kinase A (PKA) activation-mediated brain-derived neurotrophic factor (BDNF) 

expression in the CA3 subfield of dorsal hippocampus 

Authors: *S. DATTA, J. L. SHEA; 

Dept Psychiatry, Boston Univ. Sch. Med., Boston, MA

Abstract: The pontine-wave (P-wave) is generated by the activation of a group of glutamateric 

cells in the pons and is one of the most prominent phasic events of REM sleep. Recently we have 

demonstrated that the P-wave generator activation-dependent two-way active avoidance 

(TWAA) memory processing involves expression of brain-derived neurotrophic factor (BDNF) 

in the CA3 subfield of the dorsal hippocampus (CA3-DH). The present study was designed to 

examine whether TWAA memory processing-related BDNF expression in the CA3-DH is 

mediated by the activation of protein kinase A (PKA). Adult male Sprague-Dawley rats (n=30) 

were chronically implanted with sleep-wake recording electrodes and bilateral guide tubes for 

microinjections into the CA3-DH. After postoperative recovery, rats were exposed to shuttle box 

context (thirty minutes) and baseline sleep-wave activities were recorded between 10 AM and 4 

PM for two consecutive days. One day after final baseline recording session, rats were placed in 

the shuttle box and subjected to a session of 30 TWAA learning trials. Immediately after training 

trials, rats were microinjected bilaterally with either KT-5720 (2.0 κmol in 200 nl/site; n=10 

rats), a specific inhibitor of cAMP-dependent PKA activation, or control saline (200 nl/site; n=10 

rats) into the CA3-DH. After microinjections, rats were recorded for sleep-wake activities for 

six-hours (between 10 AM and 4 PM). Twenty-four hours after the training session, rats were 

tested for TWAA memory. Another 10 rats received microinjection of KT-5720 in one side and 

saline in other side. These rats were killed 3h after the end of microinjection to collect brain 

tissue for measuring BDNF. The sleep-wake data revealed that compared to after control saline, 

KT-5720 microinjections into the CA3-DH did not produce any significant changes in the 

wakefulness, slow-wave sleep, or REM sleep. In the test trials of TWAA learning, however, KT- 

5720 microinjected rats showed a significant deficit in the retention of TWAA memory. BDNF 

results show that the amount of BDNF in the CA3-DH of KT-5720 treated side is 115 % less 

than the saline treated side. These findings suggest that the activation of cAMP-PKA in the CA3- 

DH is an important step for the consolidation and/or retention of TWAA memory but may not be 

involved in the regulation of sleep-wake behavior. These results also indicated that the P-wave 

generator activation-mediated expression of BDNF in the CA3-DH is mediated by the activation 

of cAMP-PKA system. 

Disclosures: S. Datta , None; J.L. Shea, None. 

Poster 





Support: Dept. of Veteran Affairs

HL60296 

MH63323 

Title: Sleep-related c-Fos expression in neurons projecting from preoptic hypothalamus to 

periaqueductal gray 

Authors: *J.-C. HSIEH 1,3 , A. USCHAKOV 1,3 , D. MCGINTY 2 , R. SZYMUSIAK 1,3 ; 

1 Med., 2 Psychology, UCLA, North Hills, CA; 3 Res. Service, V. A. Greater Los Angeles 

Heathcare Syst., North Hills, CA 

Abstract: The midbrain periaqueductal gray (PAG) is involved various physiological functions 

including autonomic regulation and sleep-wake control. The preoptic hypothalamus is an 

important source of afferents to the PAG. The preoptic area also contains populations of putative 

sleep-regulatory neurons that are activated during sleep. To investigate the extent to which 

preoptic-PAG projection neurons are sleep-active, we combined retrograde tracer injection into 

the PAG with c-Fos protein immunostaining in the preoptic area. 

Adult male Sprague-Dawley rats (n=6) were used in this study. Retrograde tracer, Fluoro-gold 

(FG), was pressure-injected into the PAG with a Hamilton microsyringe controlled by an 

infusion pump. Cortical EEGs and nuchal EMG electrodes were also implanted at the time of 

tracer injection. The animals were allowed to recover from surgery and entrained to a 12/12 

light/dark cycle. Nine days after tracer injection, the animals were subjected to 2-hours of sleep 

deprivation by gentle handling, starting at the onset of light period. This was followed by a 2hour 

undisturbed recovery period. Animals were then promptly anesthetized at the end of 

recovery period and perfused transcardially with saline followed by paraformaldehyde. The 

brains were sectioned and processed for c-Fos protein immunohistochemistry. Cells labeled with 

c-Fos and FG were mapped and quantified. 

In two of 6 animals, injection sites were centered in the ventrolateral PAG, while in the other 4 

animals, the injection was centered at the lateral PAG and extended over most of the dorsal to 

ventral aspects ipsilateral PAG. Similar and consistent retrograde labeling was found in the 

rostral hypothalamus in all animals, with highest amount of labeling in the ipsilateral medial 

preoptic nucleus (MPO) and the median preoptic nucleus (MnPO). Other regions containing 

retrogradely-labeled cells included lateroanterior hypothalamic nucleus, bed nucleus of stria 

terminalis, and lateral preoptic area. Among these areas, significant numbers of c-Fos positive, 

retrogradely-labeled neurons were found in MnPO and MPO. 

In conclusion, we report the existence of sleep-active neurons in the preoptic hypothalamus that 

project to the periaqueductal gray. These neurons may play a role in mediating sleep-related 

change in cardiovascular responses. 

Disclosures: J. Hsieh, None; A. Uschakov, None; D. McGinty, None; R. Szymusiak, None. 

Poster





Support: Fideicomiso-UNAM given to R. D-C 

UNAM/DGAPA/PAPIIT (IN208206-2) 

Title: Microinjections of cannabidiol into lateral hypothalamus enhance wakefuless in rats 

Authors: *E. MURILLO-RODRIGUEZ 1 , D. MILLÁN-ALDACO 2 , M. PALOMERO- 

RIVERO 2 , R. MECHOULAM 3 , R. DRUCKER-COLÍN 2 ; 

1 Fac Med., Lab. de Neurobiol, Campeche, Mexico; 2 Neurociencias, Inst. de Fisiología Celular, 

Universidad Nacional Autónoma de México. México DF, Mexico; 3 Dept. of Medicinal Chem. 

and Natural Products, Sch. of Pharm., Hebrew University. Jerusalem, Israel 

Abstract: Cannabidiol (CBD) is a constituent of Cannabis sativa that induces non-psychotropic 

effects and some of its biological actions in sleep have been described by our group. Here, we 

report that when administered (10, 20µg/1µL) during the lights-on period directly into wakeinducing 

brain areas, such as lateral hypothalamus (LH) or dorsal raphe nuclei (DRN), enhanced 

wakefulness (W) and decreased slow wave sleep (SWS), as well as rapid eye movement sleep 

(REMS), is noted. We found that the changes in sleep after CBD injection into LH or DRN were 

due to the result of modifications on the sleep parameters. For example, the number of bouts in 

W was statistically enhanced with the highest dose of CBD whereas both doses of this drug 

diminished SWS as well as REMS. On the other hand, all tested doses of CBD enhanced the 

mean duration of W whereas decreased it for SWS. We also found that CBD enhanced latency 

for SWS and REMS. Additionally, we found that microinjection of this cannabinoid into either 

LH or DRN increased alpha and theta but diminished delta power spectra. Finally, it was 

observed that the injection of CBD into either LH or DRN increased the c-Fos expression in 

these brain nuclei. Our findings suggest that CBD behaves as a wake-inducing compound that 

presumably activates neurons in LH and DRN. Despite the current lack of pharmacological 

evidence indicating that CBD modulates sleep, one might consider the use of this compound to 

treat excessive somnolence. This sleep disturbance affects mood and perturbs focusing on tasks. 

Disclosures: E. Murillo-Rodriguez, None; D. Millán-Aldaco, None; M. Palomero-Rivero, 

None; R. Mechoulam, None; R. Drucker-Colín, None. 

Poster





Title: Characterization of APD916, a novel histamine H3 antagonist with wake promoting 

properties 

Authors: *A. J. GROTTICK 1 , G. BARRERA 1 , J. EDWARDS 2 , E. HAUSER 1 , H. LAM 4 , J. 

LOREA 1 , N. MAIDMENT 4 , V. J. SANTORA 3 , G. SEMPLE 3 , B. M. SMITH 3 , J. M. SMITH 3 , 

M. SUAREZ 1 , B. THOMSEN 1 , K. WHELAN 1 , A. SIEGEL 6,5 , J. M. SIEGEL 6,5 , M.-F. WU 6,5 , 

W. CHEN 2 ; 

1 CNS pharmacology, 2 DMPK, 3 Medicinal chemistry, Arena Pharmaceuticals Inc., San Diego, 

CA; 4 Dept. Physiol., 5 Dept. Psychiatry, UCLA, Los Angeles, CA; 6 VA GLAHS, Sepulveda, CA 

Abstract: APD916 bound H3 receptors across three species with high affinity (rat H3; Ki 

=1.2nM, human H3; Ki=4.2nM dog H3; IC50=3.8nM) and displayed greater than 1000-fold 

selectivity against a standard panel of >70 reference sites, including the other known histamine 

receptors. When assessed in a functional assay, APD916 was a potent and highly efficacious 

inverse agonist of the recombinant human receptor (GTPγS binding, IC50=0.7nM). In vivo 

pharmacokinetic (PK) studies in the rat demonstrated rapid absorption (Tmax=0.3h), a relatively 

short half-life (T½=1-1.5h), good oral bioavailability (%F=27%) and high brain penetrance 

(brain/plasma ratio ~2). Functional in vivo efficacy in the rat was established in a 

pharmacological screen (blockade of R-α-methylhistamine (RAMH)-induced polydipsia) with a 

minimum effective oral dose of 0.3mg/kg. Efficacy in this screen was maintained after ten days 

of subchronic dosing. Using the same paradigm, we demonstrated that at twice the minimum 

effective dose (0.6mg/kg PO), APD916 was no longer active 6h after administration. EEG 

recordings in the rat found APD916 to increase wakefulness (minimum effective oral dose of 

0.3mg/kg) without concomitant changes in locomotor activity. These wake promoting properties 

of APD916 were more pronounced during the rodent subjective night, and were maintained 

throughout five days of dosing. Lastly, when administered to the narcoleptic Doberman, 

APD916 significantly reduced both number and total duration of palatable food-induced 

cataplectic attacks at one and four hours after administration, an effect which no longer remained 

at 24h post-dose. Taken together, these data suggest APD916 to be a potent and selective 

antagonist/inverse agonist of histamine H3 receptors across species, with a moderate duration of 

action. APD916 may therefore have utility in the treatment of disorders characterized by 

excessive daytime sleepiness, including narcolepsy. 

Disclosures: A.J. Grottick, Arena Pharmaceuticals, A. Employment (full or part-time); G. 

Barrera, None; J. Edwards, Arena Pharmaceuticals, A. Employment (full or part-time); E. 

Hauser, Arena Pharmaceuticals, A. Employment (full or part-time); J. Lorea, Arena 

Pharmaceuticals, A. Employment (full or part-time); V.J. Santora, Arena Pharmaceuticals, A. 

Employment (full or part-time); G. Semple, Arena Pharmaceuticals, A. Employment (full or

part-time); B.M. Smith, Arena Pharmaceuticals, A. Employment (full or part-time); J.M. Smith, 

Arena Pharmaceuticals, A. Employment (full or part-time); M. Suarez, Arena Pharmaceuticals, 

A. Employment (full or part-time); B. Thomsen, Arena Pharmaceuticals, A. Employment (full 

or part-time); K. Whelan, Arena Pharmaceuticals, A. Employment (full or part-time); W. Chen, 

Arena Pharmaceuticals, A. Employment (full or part-time); M. Wu, VA GLAHS, A. 

Employment (full or part-time); Arena Pharmaceuticals Inc., C. Other Research Support (receipt 

of drugs, supplies, equipment or other in-kind support); A. Siegel, VA GLAHS- 

Sepulveda/UCLA, A. Employment (full or part-time); Arena Pharmaceuticals Inc., C. Other 

Research Support (receipt of drugs, supplies, equipment or other in-kind support); H. Lam, 

UCLA, A. Employment (full or part-time); N. Maidment, UCLA, A. Employment (full or parttime); 

J.M. Siegel, VA GLAHS-Sepulveda/UCLA, A. Employment (full or part-time); NIH 

Grant R01MH064109-05A1, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); Arena Pharmaceuticals Inc., C. 

Other Research Support (receipt of drugs, supplies, equipment or other in-kind support). 

Poster 





Support: NIH grant HL60292 

Fondation pour la Recherche Medicale 


Title: Orexin acts in the basal forebrain to stabilize wakefulness 

Authors: C. ALEXANDRE 1 , T. MOCHIZUKI 1 , E. ARRIGONI 1 , E. CLARK 1 , J. N. 

MARCUS 3 , J. K. ELMQUIST 4 , B. B. LOWELL 2 , *T. E. SCAMMELL 1 ; 

1 Dept Neurol, 2 Dept Endrocrinol., Beth Israel Deaconess Med. Ctr., Boston, MA; 3 Merck Res. 

Labs, Boston, MA; 4 Dept Intrnl Med. Pharmacol, Univ. Southwestern Med. Ctr., Dallas, TX 

Abstract: The orexin/hypocretin-producing neurons play an essential role in promoting and 

maintaining full alertness and wakefulness, but little is known about the neuronal pathways 

through which orexin activates the cortex and maintains arousal and wakefulness. We propose 

that orexin promotes and stabilizes wakefulness by exciting basal forebrain (BF) neurons, 

especially those of the substantia inominata (SI) that project to and activate the cortex. We tested

whether focal rescue of orexin receptors (OX1 and OX2 receptors) in the SI improves the 

sleepiness of mice otherwise lacking orexin receptors. 

We produced mice in which the orexin receptor genes are preceded by transcriptional disruptor 

sequences that prevent expression of functional orexin receptors. These blocking sequences are 

flanked by loxP sites, and exposure to Cre recombinase deletes the disruptor sequences and 

normalizes receptor expression. In the absence of Cre recombinase, these transcription-disrupted 

(TD) mice lack normal OX1/2 mRNAs and proteins. Also, they exhibit severely fragmented 

sleep/wake behavior and fail to sustain long wake bouts during the dark period compared to WT 

littermates. 

To induce focal expression of orexin receptors in the BF, we bilaterally microinjected an adenoassociated 

viral vector (AAV) coding for Cre into the SI of male OX1/2 TD mice. Other OX1/2 

TD mice received injections of an AAV coding for green fluorescent protein (GFP) as negative 

(sleepy) controls, and WT mice served as positive (alert) controls. All mice were instrumented 

for EEG/EMG as well as for locomotor activity and body temperature recordings. 

Microinjections of AAV-Cre into the SI improved wake maintenance as reflected in longer wake 

bouts during the dark period, compared to the AAV-GFP controls. Like WT mice, OX1/2 TD 

mice injected with AAV-Cre had often wake bouts lasting > 42 min, while AAV-GFP-injected 

controls rarely produced such long wake bouts. 

These findings demonstrate that orexin signaling through the BF is sufficient to stabilize 

wakefulness. Most likely, in the absence of orexin signaling, BF neurons are less active, resulting 

in shorter wake bouts. Further microinjections into other brain regions should help test the 

anatomic specificity of this response. 

Disclosures: C. Alexandre, None; T. Mochizuki, None; E. Arrigoni, None; E. Clark, 

None; J.N. Marcus, None; J.K. Elmquist, None; B.B. Lowell, None; T.E. Scammell , None. 

Poster 





Support: direccion general de apoyo al personal academico of the National Autonomous 

University of Mexico by Victor Leyva-Grado 

HD36520 by JM Krueger 

NS31453 by JM Krueger

NS25378 by JM Krueger 

Title: Influenza virus inoculation in male mice increases interleukin-1 beta- and tumor necrosis 

factor alpha-immunoreactive cells in the piriform cortex, olfactory tubercle and central amygdala 

Authors: *L. CHURCHILL 1,2 , V. LEYVA-GRADO 1 , T. J. WILLIAMS 1 , P. TAISHI 1 , J. A. 

MAJDE 1 , J. M. KRUEGER 1 ; 

1 Dept VCAPP, 2 Washington State Univ., Pullman, WA 

Abstract: During influenza virus infections, the cytokines interleukin-1 beta (IL1β) and tumor 

necrosis factor alpha (TNFα) increase as part of the acute phase response. We have demonstrated 

that viral replication intermediates increase in the olfactory bulb (OB) as early as 4 h after nasal 

inoculation of C57Bl/6 male mice with live, mouse-adapted PR8/H1N1 influenza virus and 

continue to be present at 15 h. The mice respond to the viral infection with hypothermia 

beginning at 11-13 h after viral challenge. We have also demonstrated that cytokineimmunoreactive 

(IR) cells increase within the external plexiform layer (EPL) of the OB at 15 h 

after inoculation of live PR8 virus relative to boiled (heat-inactivated) PR8. In this study we 

hypothesize that the cytokine-IR cells in the EPL will project to the piriform cortex, olfactory 

tubercle and central amygdala to stimulate cytokine-IR cells. Preliminary data indicate that 

cutting of the olfactory nerve delays the onset of hypothermia suggesting that the entry of the 

influenza virus through the olfactory nerve is important in the acute phase response. Antibody 

specificity for TNFα and IL1β was verified by the loss of specific staining in IL1β- and TNFαknockout 

mice as well as by preabsorbtion with the appropriate mouse recombinant proteins. 

Western blot analyses demonstrated that the proform for IL1β and TNFα was labeled in the 

olfactory bulb and preabsorbed with the appropriate mouse recombinant proteins. Live PR8 

inoculation increased the number of IL1β- and TNFα-IR cells in the piriform cortex, olfactory 

tubercle and central amygdala (brain regions that receive innervation from the olfactory bulb) at 

15 h but not by 10 h which was prior to the physiological response to the virus. Double labeling 

with the neuronal nuclear protein marker, NeuN, indicated that the IL1β- and TNFα-IR were 

present in neurons in the piriform cortex, olfactory tubercle and central amygdala. These data 

suggest that the viral replication intermediates present in the olfactory bulb after the influenza 

virus inoculation may activate cytokine production in neurons that communicate with other 

centrally localized neurons to induce cytokines in these neurons as well. 

Disclosures: L. Churchill, None; V. Leyva-Grado, None; T.J. Williams, None; P. Taishi, 

None; J.A. Majde, None; J.M. Krueger, None. 

Poster 





Support: NIMH Grant P20 MH077967 (CC) 

NIH Director‟s Pioneer award (GT) 

Title: Cortical spike activity during NREM sleep up-states reflects sleep homeostasis 

Authors: *V. V. VYAZOVSKIY 1 , Y. M. LAZIMY 1 , S. K. ESSER 1 , J. WILLIAMS 2 , C. 

CIRELLI 1 , G. TONONI 1 ; 

1 Psychiatry, WISPiC, Madison, WI; 2 Biomed. Engin., Madison, WI 

Abstract: Changes in firing rates of cortical neurons during sleep and waking have been studied 

extensively. However, it is unknown whether cortical firing changes with sleep homeostasis. 

Adult male WKY rats (n=4) kept in a 12:12 light-dark cycle were chronically implanted with 16channel 

TDT microwire arrays in the barrel cortex (layers IV-V). Extracellular multiunit activity 

(MUA), local field potentials (LFPs), surface electroencephalographic (EEG) and muscle activity 

were recorded in freely-behaving animals for 2-3 weeks (> 6d after surgery; 2-3d of stable 

recording/rat) during baseline and after 4h of sleep deprivation (SD). On average 10.1±0.4 

channels per animal, per day showed robust MUA. Mean 24h MUA firing rates were high in 

waking (71 Hz), intermediate in REM (43 Hz) and low in NREM (37 Hz). Population MUA in 

NREM sleep showed regular periods of silence (down states) alternated with periods of jointly 

elevated activity (up states). Computing wave-triggered MUA averages revealed that the 

suppression of neuronal firing lasted for ~ 200 ms in association with the negative part of the 

surface EEG slow wave. Episodes of high sleep pressure (early light phase, beginning of post-SD 

recovery sleep), relative to episodes of low sleep pressure (late light phase, end of recovery 

sleep) were associated with longer interspike intervals (~100-400 ms). Importantly, MUA during 

the last ~150 ms before a down state was higher under high rel. to low sleep pressure: more 

channels showed spiking activity (light period: +14.7%; recovery: +20.2%), suggesting increased 

recruitment of neurons at the end of the up state. During high sleep pressure, compared to low 

sleep pressure, this recruitment surge was followed by significantly faster decline in the number 

of channels showing spiking activity (decruitment) while going into the down state (light period: 

+21 %; recovery: +36 %). SWA time course was positively correlated with: a) firing rates during 

the up state (light period: r=0.73, p

Poster 





Support: Grant-in-Aid for Scientific Research from the Japanese 21st Century COE Program, 

"Human Nutritional Science on Stress Control" 

Title: Bezafibrate, a PPARs agonist, enhances EEG delta oscillation during NREM sleep in mice 

Authors: *H. SEI 1 , S. CHIKAHISA 1 , K. TOMINAGA 2 , T. KAWAI 2 , K. OISHI 3 , N. ISHIDA 3 , 

K. ROKUTAN 2 ; 

1 Dept Integrative Physiol., 2 Dept Stress Sci., Univ. Tokushima Grad. Sch., Tokushima, Japan; 

3 Clock Cell Biol. Res. Group, Natl. Inst. of Advanced Industrial Sci. and Technol., Tsukuba, 

Japan 

Abstract: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription 

factors belonging to the nuclear receptor family. We examined whether chronic treatment with 

bezafibrate, a PPARs pan agonist, would alter sleep and body temperature. Mice were fed with a 

control diet for two weeks, and body temperature, electroencephalogram and electromyogram 

were recorded for 48 hours, under light-dark conditions. After the baseline recording, the mice 

were provided with bezafibrate-supplemented food for two weeks, after which the same 

recordings were carried out. Two-week feeding of bezafibrate decreased body temperature 

especially during the latter half of the dark period. Body temperature rhythm and sleep/wake 

rhythm were phase-advanced about two to three hours by bezafibrate treatment. Bezafibrate 

treatment also increased the EEG delta power in non-rapid eye movement (NREM) sleep 

compared to the control diet throughout 24 hours. Furthermore, bezafibrate-treated mice showed 

no rebound of EEG delta power in NREM sleep after six hours of sleep deprivation. DNA 

microarray and real time RT-PCR analysis in the hypothalamus showed that bezafibrate 

treatment increased Neuropeptide Y mRNA and decreased pro-opiomelanocortin-alpha mRNA. 

These findings demonstrate that PPARs participate in the control of body temperature and sleep 

regulation, which accompanied with gene expression in the hypothalamus. Activation of PPARs 

may enhance deep sleep and improve resistance against sleep loss. 

Disclosures: H. Sei, None; S. Chikahisa, None; K. Tominaga, None; T. Kawai, None; K. 

Oishi, None; N. Ishida, None; K. Rokutan, None.

Poster 





Support: NSF Pickwick Postdoctoral Research Fellowship 

Title: Role of gene transcription and translation during sleep-dependent cortical plasticity 

Authors: *J. SEIBT, S. J. ATON, T. COLEMAN, M. C. DUMOULIN, M. G. FRANK; 

Neurosci., Univ. Pennsylvania, Philadelphia, PA 


Sleep is hypothesized to consolidate memory, but the cellular mechanisms governing this process are unknown. One critical step 

in the consolidation of both memory and synaptic plasticity is gene transcription and translation. We examined the role of both processes in a 

classic form of in vivo plasticity that is triggered by monocular deprivation (MD) and consolidated by sleep (ocular dominance plasticitym, ODP). 

We used western blots and quantitative RT-PCR to assess changes in mRNA and protein expression of plasticity related genes in primary visual 

cortex in animals undergoing different manipulations of binocular vision and sleep. Immunochemistry was also used to determine activation of 

mTOR and eEF2 protein synthesis regulatory pathways. In separate experiments, we determined the necessity of protein synthesis for sleep- 

dependent ODP by locally inhibiting (during sleep) the mTOR pathway with rapamycin and global cortical protein synthesis with cycloheximide. 

Following intracortical infusion, ODP was assessed with intrinsic signal imaging and micro-electrode recording 

Preliminary western-blot analyses suggest a simultaneous activation of the mTOR and the eEF2 pathways in the remodeling V1 during sleep 

consistent with a coordinate process of enhanced translation initiation and reduced peptide chain elongation. RT-PCR performed on the same 

tissues showed a decrease of 50 to 80% of immediate-early genes (Arc, c-Fos) during sleep specifically after MD compared to control sleeping 

animals (no MD). Surprisingly, this decrease in Arc mRNA expression is accompanied by an increased expression of the corresponding protein in 

post-MD sleep. The requirement of de novo protein synthesis during sleep-dependent ODP was further confirmed by our results showing that 

both cycloheximide and rapamycin infusions during sleep impair the consolidation of ODP. 

Taken together, our results indicate that sleep promotes mechanisms implicated in „local‟ dendritic protein synthesis (activation of mTOR 

pathway, increase in Arc protein expression) and that this process is accompanied by a shut-down of general translation (eEF2 phosphorylation) 

and transcription (decrease mRNA expression of IEGs). 

Disclosures: J. Seibt , None; S.J. Aton, None; T. Coleman, None; M.C. Dumoulin, 

None; M.G. Frank, None.

Poster 





Support: F Hoffmann-La Roche 

Title: Photic induction of novel genes in the suprachiasmatic nuclei (SCN) and ventrolateral 

preoptic nucleus (VLPO) 

Authors: *S. H. GODINHO 1 , R. BUTLER 1 , M. EBLING 2 , S. GATTI 2 , J. G. WETTSTEIN 2 , 

M. HANKINS 1 , R. FOSTER 1 , S. PEIRSON 1 ; 

1 Nuffield Lab. of Ophthalmology, Univ. of Oxford, Oxford, United Kingdom; 2 F Hoffmann-La 

Roche, Basel, Switzerland 

Abstract: Circadian rhythms in physiology and behaviour are a product of intracellular 

transcriptional-translational feedback loops (1). The primary pacemaker in the mammalian 

circadian system are the suprachiasmatic nuclei (SCN) of the hypothalamus, which receive light 

input via the retinohypothalamic tract (RHT). The RHT also projects to the hypothalamic 

ventrolateral preoptic nuclei (VLPO), which in turn project to the nuclei of the ascending arousal 

system. Neurons of the VLPO are primarily active during sleep, and photic modulation of these 

nuclei may play an acute role in sleep regulation (2, 3). The mechanism by which light 

modulates the phase of the SCN clock is thought to involve Ca 2+ -Calmodulin, cAMP and MAPK 

signalling pathways (4). Based upon our previous microarray studies we predicted that the 

genetic network encoding this signalling pathway would undergo a light-induced transcriptional 

adjustment or “recalibration” of those genes whose protein products are part of the transduction 

pathway (5). Male C3H mice were housed under a 12:12 LD cycle for 2 weeks prior to exposure 

to a 30 minute light pulse of 400-500 lux at ZT16. Animals were sacrificed and brain sectioning 

was carried out in darkness with an infrared viewer. SCN and VLPO punches were dissected 

from 1 mm thick brain slices at 30, 60 and 120 mins after the onset of the light pulse. Total RNA 

was extracted using a lipid micro RNeasy column method (Qiagen). To ensure that punches of 

the defined areas were accurately dissected, qPCR of a number of SCN and VLPO specific 

mRNAs such as Six-6 and Gal was conducted, revealing selective enrichment. In addition, we 

performed qPCR on a range of target transcripts in the SCN in response to light. As well as 

reliably detecting immediate-early genes expression such as in the case of Fos, Nr4a1 and Egr3 

coupled with known clock genes such as Per1-2 in the SCN, we have identified another set of 

genes that are induced by light with no defined clock function. Moreover, in the VLPO, there is 

specific effect on gene transcription in response to light as early as 30 min after the onset of light 

exposure. This study outlines a powerful approach to identify novel genes involved in the 

specific light signalling retino-hypothalamic pathways regulating circadian rhythms and sleep.

References: 

1. S. M. Reppert, D. R. Weaver, Nature 418, 935 (Aug 29, 2002). 

2. C. B. Saper, T. C. Chou, T. E. Scammell, Trends Neurosci 24, 726 (Dec, 2001). 

3. C. B. Saper, J. Lu, T. C. Chou, J. Gooley, Trends Neurosci 28, 152 (Mar, 2005). 

4. J. H. Meijer, W. J. Schwartz, J Biol Rhythms 18, 235 (Jun, 2003). 

5. S. N. Peirson et al., Curr Biol 17, 1363 (Aug 21, 2007). 

Disclosures: S.H. Godinho, F Hoffmann-La Roche, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); R. Butler, F 

Hoffmann-La Roche, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); M. Ebling, F Hoffmann-La Roche, A. 

Employment (full or part-time); S. Gatti, F Hoffmann-La Roche, A. Employment (full or parttime); 

J.G. Wettstein, F Hoffmann-La Roche, A. Employment (full or part-time); M. Hankins, 

F Hoffmann-La Roche, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); R. Foster, F Hoffmann-La Roche, B. 


grants already received); S. Peirson, F Hoffmann-La Roche, B. Research Grant (principal 


Poster 





Support: Cephalon, Inc. 

Title: Biochemical, pharmacological, and behavioral characterization of the dopaminergic 

stimulant sydnocarb in rats 

Authors: *J. A. GRUNER, D. G. FLOOD, J. R. MATHIASEN, L. D. AIMONE, M. J. 

MARINO, M. GASIOR; 

CNS Biol., Cephalon, Inc, West Chester, PA 

Abstract: Sydnocarb is a psychomotor stimulant structurally similar to amphetamine that has 

been used in Russia since the 1970s for the treatment of a variety of neuropsychiatric 

comorbidities (e.g., asthenia, apathy, and adynamia). It has been reported to have lower toxicity 

and abuse potential than prototypical dopaminergic psychostimulants such as cocaine and 

amphetamine. Microdialysis studies have revealed sydnocarb-induced facilitation of

dopaminergic transmission (Gainetdinov et al, Eur J Pharmacol., 1997), but the nature of this 

action (dopamine release vs. dopamine transporter (DAT) inhibition) is not clear. The aim of the 

present study was to investigate sydnocarb‟s pharmacological actions on the dopaminergic 

system and to characterize its wake-promoting and other behavioral effects in rats and mice. In 

rat synaptosomes, sydnocarb (100 κM) did not directly release dopamine. However, it potently 

(Ki = 10.4 ± 1 nM) blocked DAT in human recombinant CHO-K1 cells; it also blocked the 

norepinephrine transporter at higher concentrations (Ki = 10.2 ± 1.5 µM). Sydnocarb [10 µM] 

showed no activity at 64 other targets. In a modified rat Irwin test, sydnocarb was well tolerated 

at 30 mg/kg ip in Sprague Dawley (SD) rats, but at 100 mg/kg ip, it produced amphetamine-like 

behavioral changes (sniffing, rearing, biting, marked stereotypy, etc.). In the EEG/EMG sleepwake 

model (7 am - 7 pm lights on, dosing at 5 h after lights on), sydnocarb (3, 10, and 30 mg/kg 

ip) increased wake in SD rats. At 30 mg/kg, sydnocarb enhanced wake for about 7 h and 

produced a maximum cumulative wake surplus of ~260 min at 7.5 h post dosing, which declined 

to 168 min at 22 h post dosing. In contrast to dextroamphetamine (Lin et al, J Sleep Res., 2000), 

there was no evidence of rebound hypersomnolence following enhanced wake activity. Since 

sydnocarb is used in the treatment of neuropsychiatric comorbidities, and prepulse inhibition 

(PPI) is impaired in schizophrenia and bipolar disorders, sydnocarb (1 - 30 mg/kg ip) was tested 

in an animal model of PPI. Sydnocarb (30 mg/kg ip) improved PPI of the startle response in 

DBA/2 mice. In summary, sydnocarb acts as a selective DAT inhibitor, is well tolerated up to 30 

mg/kg ip, produces robust increases in wake without amphetamine-like rebound 

hypersomnolence, and shows efficacy in a mouse model of the sensorimotor gating deficits in 

schizophrenia. 

Disclosures: J.A. Gruner, Cephalon, Inc., A. Employment (full or part-time); Cephalon, Inc., 

E. Ownership Interest (stock, stock options, patent or other intellectual property); D.G. Flood, 

Cephalon, Inc., A. Employment (full or part-time); Cephalon, Inc., E. Ownership Interest (stock, 

stock options, patent or other intellectual property); J.R. Mathiasen, Cephalon, Inc., A. 

Employment (full or part-time); Cephalon, Inc., E. Ownership Interest (stock, stock options, 

patent or other intellectual property); L.D. Aimone, Cephalon, Inc., A. Employment (full or parttime); 

Cephalon, Inc., E. Ownership Interest (stock, stock options, patent or other intellectual 

property); M.J. Marino, Cephalon, Inc., A. Employment (full or part-time); M. Gasior, 

Cephalon, Inc., A. Employment (full or part-time). 

Poster 




Topic: E.08.c. Sleep: Molecular, cellular, and pharmacology

Title: T-type (Ca[V]3) calcium channel function in rodent rest / activity cycles and setting 

circadian phase 

Authors: V. N. UEBELE 1 , *A. L. GOTTER 1 , D. R. REISS 1 , J. BRUNNER 1 , T. S. REGER 2 , K. 

E. RITTLE 2 , Y. SHU 2 , Z. YANG 2 , J. C. BARROW 2 , J. J. RENGER 1 ; 

1 Depression Circadian Disorders, 2 Medicinal Chem., Merck Res. Labs, West Point, PA 

Abstract: The role of T-type channels in sleep cycle stability has recently been appreciated. The 

α1g and α1i subtypes are expressed in thalamocortical and reticular thalamic nuclei where burst 

firing associated with non-REM sleep is thought to be dependent upon the kinetic activity of 

these channels. In fact, α1g knockouts display more brief awakenings and diminished delta wave 

activity during non-REM sleep. Calcium conductance, however, is also required for progression 

of the endogenous circadian clock and may contribute to setting circadian phase and the timing 

of the sleep cycles. Studies in which 24 hour rest / activity cycles were monitored illustrated 

significant differences between wildtype mice and those lacking both α1g and α1i subtypes of Ttype 

channels, particularly during the early active phase in Light:Dark cycles. Inhibition of Ttype 

conductance with a novel potent and selective T-type antagonist (TTA-1) administered 1 

hour prior to lights on acutely reduced locomotor activity in advance of the active phase and 

induced a trend in active phase activity similar to that seen in constitutive mutant animals. 

Importantly, these effects of TTA-1 were not observed in α1i/α1g double knockouts, 

demonstrating reagent specificity. Future studies evaluating mouse EEG will more closely 

examine these effects in both knockout and antagonist treated animals. Telemetry-based core 

temperature monitoring in rats over 24 hour Light:Dark cycles indicated a similar effect of Ttype 

antagonism when administered earlier in the active phase; an advance in the circadian 

temperature profile typical of the inactive phase and subsequent active phases were seen. The 

potential for T-type channels to set the phase of the endogenous circadian pacemaker was 

investigated by monitoring mouse running wheel activity in constant conditions following a 

single administration of a TTA. Significant phase advances were observed in response to acute 

inhibition of T-type conductance while circadian period remained unchanged. Remarkably, these 

resetting effects were also observed when antagonist was administered in the light phase prior to 

release into Dark:Dark, indicating that T-type inhibition may either directly affect the clock 

mechanism or modulate inputs from other brain areas. Together these results not only 

demonstrate the rest promoting effects of T-type channel antagonism in mice, but also the 

capacity of these novel therapeutics to reset the endogenous circadian pacemaker. 

Disclosures: V.N. Uebele, Merck Research Labs, A. Employment (full or part-time); A.L. 

Gotter , Merck Research Labs, A. Employment (full or part-time); D.R. Reiss, Merck Research 

Labs, A. Employment (full or part-time); J. Brunner, Merck Research Labs, A. Employment 

(full or part-time); T.S. Reger, Merck Research Labs, A. Employment (full or part-time); K.E. 

Rittle, Merck Research Labs, A. Employment (full or part-time); Y. Shu, Merck Research Labs, 

A. Employment (full or part-time); Z. Yang, Merck Research Labs, A. Employment (full or parttime); 

J.C. Barrow, Merck Research Labs, A. Employment (full or part-time); J.J. Renger, 

Merck Research Labs, A. Employment (full or part-time).

Poster 





Support: NIH Grant P01AG11412 

NIH Grant R01HL075020 

Title: Sleep-wake regulation is altered in leptin resistant (db/db) genetically obese and diabetic 

mice 

Authors: A. LAPOSKY 1 , M. BRADLEY 2 , D. WILLIAMS 2 , J. BASS 3 , *F. W. TUREK 1 ; 

1 Ctr. Sleep & Circadian, 2 Northwestern Univ., Evanston, IL; 3 Evanston Northwestern Healthcare 

and Res. Inst., Evanston, IL 

Abstract: Recent epidemiological and clinical studies indicate that the control of sleep-wake 

states may be an important factor in the regulation of energy metabolism. Leptin is a peripheral 

hormone that has critical signaling properties in the brain for the control of long-term energy 

homeostasis. In this study, we examined the hypothesis that leptin represents an important 

mechanistic link in the coordination of sleep-wake states and metabolism. 

The db/db mouse, a genetic model of leptin resistance, harbors a mutation in the leptin receptor 

gene (LRb). In adult (3-4 months) male db/db (N=13) and wild-type (wt, N=13) mice, EEG and 

EMG recordings were performed under a 48-hr baseline condition followed by 6-hrs of sleep 

deprivation and a 16-hr recovery opportunity. Spontaneous locomotor activity was recorded 

using infrared sensors. Animals were maintained on a 12:12 L:D cycle with free access to food 

and water. 

Db/db mice exhibited many alterations in sleep regulation, including increased 24-hr sleep time 

(+ 56 minutes, p < 0.05), due to a combined elevation in NREM (+ 66 minutes, p < .01) and a 

decrease in REM (-10 minutes, p < .05) sleep vs. wt mice. Db/db mice generated excessive sleep 

fragmentation, with more arousals from sleep (+ 27%, p < .001), an increased number of wake (+ 

48%, p < .001) and NREM (+ 34%, p < .001) bouts, and a reduced duration of wake (- 67%, p 

explanation for the increased sleep time in db/db mice is a compensation for increased sleep 

fragmentation. This pattern is indicative of sleep disorders that impair sleep consolidation and 

result in excessive daytime sleepiness. However, even the extra sleep time in db/db mice is likely 

to be non-restorative and these animals may be in a state of chronic sleep debt. In summary, the 

db/db mouse represents an important genetic model to examine the effects of disrupted leptin 

signaling, sleep fragmentation, and chronic sleep disruption on sleep regulation itself, as well as 

metabolic homeostasis. 

Disclosures: A. Laposky, None; F.W. Turek , None; D. Williams, None; M. Bradley, 

None; J. Bass, None. 

Poster 






HL65272 

MH45361 

HL57120 

HL40881 

Title: The hypocretin-1 (hcrt-1)-induced increase in wakefulness and decrease in sleep is 

blocked by bicuculline or the hypocretin receptor-1 (hcrt-r1) antagonist SB-334867 

Authors: *H. N. BREVIG 1,2 , C. J. WATSON 1 , R. LYDIC 1 , H. A. BAGHDOYAN 1,2 ; 

1 Dept. of Anesthesiol., 2 Dept. of Pharmacol., Univ. of Michigan, Ann Arbor, MI 

Abstract: Disruption of hypocretin signaling leads to narcolepsy-cataplexy in mouse, dog, and 

human (Curr Opin Neurobiol 12:339, 2002). Hcrt-1 is an arousal promoting peptide that 

increases wakefulness when microinjected into several brainstem sites, including the pontine 

reticular nucleus, oral part (PnO) (Sleep 31:453, 2008). The PnO is the rostral portion of the 

rodent pontine reticular formation and contributes to the generation of cortical arousal and REM 

sleep (Anesthesiology 103:1268, 2005). Dialysis delivery of hcrt-1 to the PnO increases GABA

levels, and GABA in the PnO is wakefulness promoting (J Neurophysiol 82:2015, 1999; Sleep 

31:453, 2008). No studies have evaluated the interaction between PnO GABA and enhancement 

of wakefulness by hcrt-1. The present study is testing the hypothesis that the hcrt-1-induced 

increase in wakefulness and decrease in sleep is blocked by coadministration of either the 

GABAA receptor antagonist bicuculline or SB-334867. Adult male rats (n=4) were anesthetized 

with isoflurane and implanted with electrodes for recording the cortical electroencephalogram 

and neck electromyogram. A guide cannula was aimed stereotaxically for the PnO. Rats were 

then conditioned to the recording chamber for a minimum of seven days. Two hour sleep 

recordings began after microinjecting (100 nl) Ringer‟s (vehicle control), hcrt-1 (35.6 ng, 100 

κM), bicuculline (0.1 ng; 2 κM) plus hcrt-1 (35.6 ng), or SB-334867 (3.4 ng, 100 κΜ) plus hcrt- 

1 (35.6 ng). Data were scored in 10 s bins as wakefulness, NREM sleep, or REM sleep using 

Icelus software (Physiol Behav 63:67, 1998). Microinjection sites were histologically confirmed 

to be in the PnO. In the first hour post injection, hcrt-1 increased wakefulness and decreased both 

NREM sleep and REM sleep. These hcrt-1 effects were blocked by bicuculline and by SB- 

334867. There was no block of the hcrt-1-induced decrease in REM sleep by bicuculline or SB- 

334867. The present data and the previous report that administering hcrt-1 to the PnO increases 

PnO GABA levels (Sleep 31:453, 2008) suggest that the hcrt-1- induced increase in wakefulness 

and decrease in NREM sleep are caused by a hcrt-r1 mediated increase in PnO GABAergic 

transmission. 

Disclosures: H.N. Brevig, None; C.J. Watson, None; R. Lydic, None; H.A. Baghdoyan, 

None. 

Poster 






NIH Grant HL40881 


NIH Grant HL57120 

NIH Grant HL65272

Title: In C57BL/6J (B6) mouse the GABAA receptor antagonist bicuculline blocks the increase 

in wakefulness and decrease in sleep caused by the GABAA receptor agonist muscimol 

Authors: *R. R. FLINT 1,2 , R. LYDIC 1 , H. A. BAGHDOYAN 1,2 ; 

1 Dept. of Anesthesiol., 2 Dept. of Pharmacol., Univ. of Michigan, Ann Arbor, MI 

Abstract: The pontine reticular formation contributes to the generation of wakefulness and rapid 

eye movement sleep (REM) (Anesthesiology 103:1268, 2005). GABAergic transmission and 

GABAA receptors in the pontine reticular nucleus, oral part (PnO) promote wakefulness in both 

cat and rat (J Neurophysiol 82:2015, 1999; Neurosci Lett 129:95, 1991; Sleep 31:453, 2008) but 

no studies have systematically examined the sleep effects of GABAergic transmission in mouse 

PnO. Muscimol microinjected into the PnO of B6 mouse causes a concentration dependent 

increase in wakefulness and decrease in sleep (Soc Neurosci Abstr Prog No 631.5, 2007). As a 

further effort to determine whether the sleep and wake responses to PnO muscimol in B6 mouse 

are mediated by GABAA receptors, the present study is testing the hypothesis that coadministration 

of bicuculline with muscimol blocks the increase in wakefulness and decrease in 

sleep caused by muscimol. Adult, male B6 mice (n = 3) were implanted with electrodes for 

recording the electroencephalogram and electromyogram, and with a microinjection guide tube 

stereotaxically aimed for the PnO. Each mouse received randomized microinjections (50 nL) of 

muscimol (5.71 ng; 1 mM), muscimol and bicuculline (2.5 ng; 0.1 mM), and saline (vehicle 

control) followed by a 4 h recording. States of wakefulness, non-REM sleep (NREM), and REM 

were digitized and analyzed manually in 10 s bins using Icelus Data Acquisition and Analysis 

software (Physiol Behav 63:67, 1998). The data were evaluated for statistical significance using 

ANOVA and Tukey/Kramer multiple comparisons test. All microinjection sites were 

histologically localized to the PnO. Muscimol caused a significant (p




Sepracor 


NIH grant HL40881 

Title: Microdialysis delivery of eszopiclone, zolpidem and diazepam to the pontine reticular 

formation (PRF) of rat increases PRF acetylcholine (ACh) release 

Authors: *V. S. HAMBRECHT, H. A. BAGHDOYAN, R. LYDIC; 

Dept Anesthesiol, Univ. Michigan, Ann Arbor, MI 

Abstract: Cholinergic and GABAergic neurotransmission in the PRF interact to regulate sleep 

and wakefulness (Anesthesiology 103:1268, 2005). Administering the GABAA antagonist 

bicuculline to the PRF increases PRF ACh release (J Neurophysiol 92:2198, 2004), decreases 

wakefulness, and triggers rapid eye movement sleep (ibid 92:2198, 2004; ibid, 82:2015, 1999). 

Increasing GABAergic transmission in the PRF increases wakefulness and decreases sleep 

(Sleep 31:453, 2008). The non-benzodiazepine sedative hypnotics eszopiclone and zolpidem, as 

well as the benzodiazepine diazepam enhance transmission at the GABAA receptor complex and 

increase sleep. The GABAA receptor subtypes through which these drugs may alter ACh release 

in the PRF have not been identified. This study is testing the hypothesis that microdialysis 

delivery of eszopiclone, zolpidem, and diazepam to the PRF causes concentration-dependent 

alterations in ACh release. A CMA/11 microdialysis probe was placed in the PRF of male 

Sprague-Dawley rats (n=22) and PRF ACh release was measured during dialysis with Ringer‟s 

(control) followed by dialysis with Ringer‟s containing either eszopiclone, zolpidem, or 

diazepam (10, 100 or 1000 κM). ACh release was quantified by high performance liquid 

chromatography with electrochemical detection. Repeated measures ANOVA and Tukey/Kramer 

post hoc test revealed that all three drugs increased ACh release (p

Baghdoyan, Sepracor, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); R. Lydic, Sepracor, B. Research Grant 


received). 

Poster 





Support: NIH Grant HL40881 

Department of Anesthesiology, University of Michigan 


Title: Microdialysis delivery of the adenosine A1 receptor antagonist 1,3-Dipropyl-8cyclopentylxanthine 

(DPCPX) to the pontine reticular formation (PRF) of C57BL/6J (B6) mouse 

increases PRF acetylcholine (ACh) release and decreases recovery time from isoflurane 

anesthesia 

Authors: *G. C. GETTYS, H. A. BAGHDOYAN, R. LYDIC; 

Dept. of Anesthesiol., Univ. Michigan, Ann Arbor, MI 

Abstract: The purinergic nucleoside adenosine modulates sleep and PRF ACh release (J 

Neurochem 96:1750, 2006). Laterodorsal and pedunculopontine tegmental (LDT/PPT) terminals 

release ACh in the PRF (J Neurosci 17:774, 1997) and adenosine inhibits LDT/PPT neurons 

(Science 263:689, 1994). Previous experiments with microdialysis delivery of adenosine A1 

receptor agonist N6-sulfylphenyl adenosine (SPA) to the PRF of B6 mice have shown a 

concentration dependent decrease in ACh and increase in the recovery time from isoflurane 

anesthesia (Sleep 31:A8, 2008). The present study is testing the hypothesis that microdialysis 

delivery of DPCPX to the PRF of B6 mouse increases PRF ACh release and decreases time to 

resumption of wakefulness after anesthesia. Adult male B6 mice (n=6) were anesthetized with 

isoflurane. A CMA/7 dialysis probe was placed into the PRF and perfused with Ringer‟s 

containing dimethyl sulfoxide (DMSO, 1%) (control) followed by Ringer‟s containing DMSO 

and DPCPX (0.01 or 0.1 mM). ACh release was quantified by HPLC/ECD. Mice were allowed 

to recover from anesthesia and time to resumption of righting was recorded. Dialysis probe 

placement in the PRF was confirmed histologically. Repeated measures one-way ANOVA and

Tukey/Kramer post hoc test revealed that DPCPX caused a concentration-dependent increase in 

ACh release (p

immobilisation stress in 5-HTT-/- mice and their WT littermates (CD1 background). Moreover, 

sleep-wake cycles were monitored by polysomnographic recordings in parallel. Finally, the 

effects on sleep of pharmacological blockade of hcrt 1 receptor (SB-334867, i.p.) immediately 

before the stress session were analysed. 

Compared to WT mice, 5-HTT-/- mutants exhibited an enhanced hypocretinergic 

neurotransmission but reduced activation of serotonergic neurons (as evidenced by the number of 

double labelled c-Fos and Tryptophan hydroxylase 2 neurons) after immobilisation stress. 

Polysomnographic recordings showed that stress was followed by a delayed increase in REM 

sleep in WT mice but not in 5-HTT-/- mutants. Although acute blockade of hcrt-R1 by SB- 

334867 did not significantly affect vigilance states in either mouse strain under control 

conditions, this treatment restored the stress-induced augmentation of REM sleep in 5-HTT-/- 

mice. 

Altogether, the present data show that 5-HT plays a key role in REM sleep homeostasis after a 

stress challenge, and that hypocretinergic neurotransmission might underlie the disrupted sleep 

response to stress observed in mice with genetically-driven loss of the 5-HTT response. Finally, 

this study emphasizes the role of hypocretin/serotonin interactions in mediating normal sleep 

response to stress. 

Disclosures: A. Rachalski, None; C. Alexandre, None; M.B. Emerit , None; J. Bernard, 

None; M. Hamon, None; J. Adrien, None; V. Fabre, None. 

Poster 





Support: USPHS grant NS20246 

USPHS grant RR20146 

Title: Cholinergic responses and intrinsic membrane properties of developing parafascicular 

neurons 

Authors: *M. YE, E. GARCIA-RILL; 

Neurobio., Univ. Arkansas Med. Sci., Little Rock, AR 

Abstract: Parafascicular neurons (Pf) receive cholinergic input from the pedunculopontine 

nucleus (PPN), which is active during waking and REM sleep. There is a developmental

decrease in REM sleep in humans between birth and puberty, and between 10-30 days in the rat. 

This study aimed at identifying responses of Pf cells to cholinergic input during this critical 

period. Whole-cell patch clamp recordings were performed in Pf neurons in 9-20 day rat 

parasagittal slices in artificial CSF, and their responses to the mixed cholinergic agonist 

carbachol (CAR) determined. Three types of cholinergic responses were identified: inhibitory 

(55.34%), excitatory (31.07%) and biphasic (fast inhibitory followed by a slow excitatory, 

6.8%). 6.8% of cells showed no response. The proportion of CAR-inhibited Pf neurons increased 

with development, conversely, the proportion of excited cells decreased during this period. 

Experiments using cholinergic antagonists revealed that M2 receptors were responsible for all 

inhibitory modulation, however, the excitatory modulation required the participation of M1, 

nicotinic and probably M3 or M5 receptors. Analyses of the current-voltage relationship under 

both voltage- and current-clamp modes showed significant differences in certain intrinsic 

membrane properties of Pf neurons. In general, CAR-excited Pf cells demonstrated 1) smaller 

time constants, 2) higher density of rebound currents (normalized to membrane capacitance), 3) 

higher density of normalized hyperpolarization-activated inward (Ih) current, 4) lower input 

resistance, 5) lower action potential threshold, and 6) shorter halfwidth duration of action 

potentials. Some Pf cells exhibited spikelets, which are rhythmic, subthreshold depolarizing 

potentials thought to reflect firing in the electrically coupled neurons, and all were excited by 

CAR. These studies suggest that PPN modulation of Pf neurons differentially affects separate 

populations, including electrically coupled cells, tending to show decreased activation during the 

developmental decrease in REM sleep. 

Disclosures: M. Ye , None; E. Garcia-Rill, None. 

Poster 






VA Merit Review 

Title: GABAA receptor subunits implicated in REM sleep control in pontine reticular formation 

of rat

Authors: C.-L. LIANG 1 , *G. A. MARKS 2,1 ; 

1 Veterans Affairs Med. Ctr., Dallas, TX; 2 Dept Psych, Univ. Texas Southwestern Med. Ctr., 

Dallas, TX 

Abstract: Microinjection of GABAA receptor antagonists into the rat nucleus pontis oralis (PnO) 

induces a long lasting increase in REM sleep similar to that produced by cholinergic agonists. 

We have shown that this REM sleep induction can be completely blocked by a muscarinic 

antagonist, indicating that the REM sleep-inducing effect of GABAA receptor blockade is 

dependent upon the cholinergic system. Coupled with the finding that GABAA receptor 

antagonists microdialyzed into PnO of cat results in increased levels of acetylcholine, it appears 

that GABA may act to inhibit acetylcholine release from presynaptic terminals. We hypothesize 

that GABAA receptors located on cholinergic terminals in the PnO, through disinhibition of 

acetylcholine release, are responsible for the REM sleep induction. Here we report our initial 

investigation to determine if GABAA receptors are present on cholinergic terminals in PnO and 

what receptor subtypes they may be. 

Rats were sacrificed by intracardial perfusion under deep pentobarbital anesthesia and their 

brains prepared for double or triple immunofluorescence, laser scanning, confocal microscopy. 

Cholinergic terminals were identified in PnO by labeling the vesicular acetylcholine transporter 

(VAChT). GABAA receptor subunits were identified by antibodies directed against one of 

several GABAA receptor subunits. In some tissue, a third antibody was directed against glutamic 

acid decarboxylase-67 to label presynaptic GABAergic terminals. 

We found that GABAA receptor subunits can be localized to cholinergic terminals in PnO and, 

thus far, identify one as gamma2. GABAA receptors containing gamma2 subunits have 

benzodiazepine binding sites and tend to cluster at synaptic locations. Identification of additional 

subunits is in progress and will inform as to the properties of the GABAA receptors in PnO 

mediating effects on REM sleep. 

Disclosures: C. Liang, None; G.A. Marks , None. 

Poster 





Support: IRSC Grant MOP 74472 

NSERC Grant # 155211

Title: REM sleep insomnia and decreased PPT cholinergic neurons following myocardial 

infarction in the rat 

Authors: *T. M. BAH 1,2 , F. LAPLANTE 2,4 , S. KALOUSTIAN 1,3 , R. SULLIVAN 4,2 , G. 

ROUSSEAU 1,3 , R. GODBOUT 1,2 ; 

1 Res., Sacre-Coeur Hosp., Montreal, QC, Canada; 2 Psychiatry, 3 Pharmacol., Univ. of Montreal, 

Montreal, QC, Canada; 4 Fernand-Seguin Res. Center, Louis-H Lafontaine Hosp., Montreal, QC, 

Canada 

Abstract: Introduction: We have already shown that myocardial infarction (MI) in the rat is 

followed within a few weeks by cell loss in the limbic system due to apoptosis, together with a 

“post MI behavioral syndrome” characterized by signs of anxiety and depression (Wann et al., J. 

Psychiatry Neurosci. 2007; 37: 11-16). Here we show that the post MI syndrome is accompanied 

by selective losses of Paradoxical (or REM) sleep (PS) and of cholinergic neurons in the 

pedunculopontine tegmental area (PPT). 

Methods: Ten adult Sprague-Dawley rats were implanted with chronic EEG and EMG 

electrodes; baseline sleep was recorded seven days after, for 24h. The following morning MI was 

induced by occluding the left coronary artery for 40 minutes in four rats while the six other rats 

were used as sham controls. Sleep was recorded again two weeks after MI. At the end of the 

protocol, the rats were perfused and quantification of choline acetyltransferase (ChAT) 

expressing neurons was carried out in the pedunculopontine tegmental area (PPT) and the 

laterodorsal tegmental area (LDT), using immunohistochemistry. Results in both groups of rats 

were compared using t-tests for independent samples. 

Results: Compared to sham rats, MI rats displayed less total sleep time and less time in PS, 

particularly at the light-dark transition. We also found that, compared to sham rats, MI rats 

showed a significant reduction (20%) of ChAT neurons in the PPT area, not in the LDT. 

Conclusion: The present results extend the apoptotic effects of MI in the limbic system to 

brainstem cholinergic area known to control PS. The selective loss of at the light-dark transition 

is not typical of anxiety and depression models and this needs to be further investigated. 

Disclosures: T.M. Bah , None; F. Laplante, None; S. Kaloustian, None; R. Sullivan, 

None; G. Rousseau, None; R. Godbout, None. 

Poster 




Topic: E.08.c. Sleep: Molecular, cellular, and pharmacology

Support: NIMH (P20 MH077967) 

NIH Director's Pioneer award to GT 

Title: Opposite and progressive changes in extracellular glutamate levels during wakefulness 

and sleep 

Authors: *M. B. DASH 1,2 , C. L. DOUGLAS 2 , C. CIRELLI 2 , G. TONONI 2 ; 

1 Neurosci. Training Program, 2 Psychiatry, Univ. of Wisconsin, Madison, WI 

Abstract: It is currently unclear whether the release of glutamate varies across the sleep-waking 

cycle. Previous microdialysis studies in rats have reported a decrease in glutamate concentration 

during non-rapid eye movement (NREM) sleep in a few brain regions (nucleus accumbens, 

orbitofrontal cortex) but not in others (prefrontal cortex). However, microdialysis has poor 

temporal resolution and in physiological conditions it may detect changes in glutamate levels 

that do not reflect changes in synaptic activity (van der Zeyden, 2007). Here we chronically 

recorded (~72 h) the extrasynaptic concentration of glutamate in freely behaving adult WKY rats 

by means of fixed-potential amperometry and a self-referencing microelectrode array 

(Burmeister, 2000), which may be sensitive to a variety of glutamate sources, including spillover 

from synapses, reversal of glutamate transporters, and glial exocytosis (Sem‟yanov, 2005). 

Simultaneous recordings of the electroencephalogram (EEG), electromyogram (EMG), and intracortical 

local field potentials (LFP) were used to score behavioral states in 4-sec epochs. Across 

all rats (n=12) and days a consistent pattern of state-dependent changes in extracellular glutamate 

concentration was observed in both prefrontal and motor cortex: in most cases glutamate 

progressively increased during spontaneous waking and REM sleep and progressively decreased 

during NREM sleep. In a subset (~10%) of all waking episodes, however, glutamate levels 

decreased: this occurred when the waking periods were interspersed within prolonged (>10min) 

NREM periods (~65% of cases) or when they immediately preceded a prolonged NREM period 

(~20% of cases), suggesting that under high sleep pressure glutamate accumulation in 

wakefulness may be substantively different than under low sleep pressure. To further test this 

hypothesis a subset of animals (N=5) were prevented from sleeping for 3 h starting at light onset 

by introducing novel objects in their cage. This treatment produced a progressive increase in 

sleep pressure as indicated by the increased number of sleep attempts (4, 6.8, and 8.2 during hour 

1, 2, and 3, respectively). Meanwhile, glutamate concentration increased during the first hour of 

sleep deprivation, reached a plateau during the second, and began to decline during the third. A 

large, progressive decrease in glutamate concentration was observed throughout recovery sleep. 

Thus, cortical glutamate concentration increases progressively during both spontaneous and 

forced wakefulness, until sleep pressure starts acting as a counterbalancing factor. In all cases, 

during ensuing sleep the level of glutamate returns to lower values. 

Disclosures: M.B. Dash, None; C.L. Douglas, None; C. Cirelli, None; G. Tononi, None. 

Poster





Support: CONACyT Grant 50633 

Title: Participation of muscarinic receptors in the regulation of sleep at hypothalamic preoptic 

area and brain stem pedunculopontine tegmentum 

Authors: *A. JIMENEZ-ANGUIANO, J. RAMÍREZ-ACOSTA, A. BARRETO- 

MALFAVON, J. VELAZQUEZ-MOCTEZUMA; 

Dept Biol de la Reproduccion, Univ. Autónoma Metropolitana, Mexico, Mexico 

Abstract: It has been reported that muscarinic-M2 agonists administered into the pontine 

reticular formation (PRF) modulates preferentially REM sleep in relation with selective M1 

agonist. However, the cholinergic inputs to this site during the physiological onset of REM sleep 

are given by the brain stem pedunculopontine tegmentum (PPT). On the other hand, 

hypothalamic preoptic area (POA) also regulates sleep, contains cholinergic neurons and 

receives inputs from the PPT. However, unknown if the stimulation or blockade of muscarinic 

M2 receptors (M2R) in the PPT and in the POA has the same effect in the architecture of sleep 

and the possible relation between PPT and POA in the regulation of sleep. The purpose of the 

present study was to analyze the possible participation of M2R subtype of POA and PPT in the 

regulation of sleep. Five cats (2.5-3.5 kg) were chronically implanted with the standard set of 

electrodes for sleep recording. In addition, stainless steel guide cannulas were implanted oriented 

towards the PPT and in the POA. After two weeks of recovery period, the animals were recorded 

for 8 hrs after the following injections: A: 0.1 µl of saline into the PPT. B: Methoctramine 

(MET), a M2 muscarinic antagonist (1.8 µM/0.1µl). C: Cis-Dioxolane (CISD) a M2 muscarinic 

agonist (1 µM/0.1µl). D: CISD into the PPT in combination with MET into the POA. In addition, 

the same injections were performed into the POA, but additionally tested one group with 

Trihexiphenydil (TRIH) (10 ng/0.1µl), a M1 muscarinic antagonist. Results showed that CISD 

into the PPT increases REM sleep and decreases SWS II. MET in PPT decreases REM sleep and 

SWS II with an increase of wakefulness. When CIS-DIOXO was administered into the PPT in 

combination with MET into the POA the increases of REM sleep was not observed, but the total 

time was similar to the controls. In POA not effect was observed with the infusion of CISD and 

MET, however, TRIH decreases REM sleep. The present results suggest a differential effect 

between muscarinic M1 and M2 receptors subtype into the POA and PPT on REM sleep 

regulation. 

Disclosures: A. Jimenez-Anguiano , CONACyT # 50633, B. Research Grant (principal 

investigator, collaborator or consultant and pending grants as well as grants already received); J. 

Ramírez-Acosta, None; A. Barreto-Malfavon, None; J. Velazquez-Moctezuma, None.

Poster 





Support: NIH Grant HL60292 


Title: Ecto-5‟-nucleotidase (CD73) knockout mice have less rebound NREM sleep after sleep 

deprivation 

Authors: *T. MOCHIZUKI 1 , E. L. CLARK 1 , L. F. THOMPSON 2 , T. E. SCAMMELL 1 ; 

1 Neurol., Beth Israel Deaconess Med. Ctr., Boston, MA; 2 Microbiol & Immunol, Oklahoma 

Med. Res. Fndn., Oklahoma City, OK 

Abstract: Adenosine is an endogenous somnogen that increases in the brain during forced 

wakefulness and then decreases during recovery sleep. Adenosine is formed by the breakdown of 

ATP, and ecto-5‟-nucleotidase (e5NT, also known as a lymphocyte surface protein CD73) 

catalyzes this step at the synaptic cleft. Researchers hypothesize that high levels of neuronal 

activity during wake increases extracellular ATP that is catalyzed into adenosine by e5NT, and 

the adenosine then promotes sleep. To test this hypothesis, we studied sleep/wake behavior of 

e5NT knockout (KO) mice under baseline conditions and with 2 and 4 hr of sleep deprivation. 

We recorded EEG/EMG, core body temperature, and locomotor activity of 4 month old, male 

e5NT KO mice and their wild-type (WT) littermates. e5NT KO mice had normal amounts of 

wake, NREM, and REM sleep on the baseline day. The number and duration of sleep/wake bouts 

were also normal in these mice, but they had less EEG delta power (1.5-2.5 Hz) than WT mice 

during spontaneous NREM sleep. After 2 or 4 hr of sleep deprivation at light onset, WT mice 

recovered their NREM and REM sleep deficits in the following recovery period. Their NREM 

sleep bouts were lengthened and EEG slow-wave activity was enhanced in the rest of light 

period, suggesting elevated NREM sleep pressure after the sleep deprivation. Surprisingly, e5NT 

KO mice recovered only their REM sleep deficit. They had little rebound in NREM sleep and 

their NREM bouts were not prolonged. As a result, their EEG slow-wave activity was enhanced 

for a longer period than in WT mice, suggesting impairment of the sleep recovery process. 

Accumulation of sleepiness appeared normal as estimated by the number of sleep entries during 

sleep deprivation and the latency toward the first episode of sleep after sleep deprivation. Body 

temperature and locomotor activity were also normal. 

These findings demonstrate that ecto-5‟-nucleotidase plays a necessary role in the homeostatic

control of NREM sleep. In e5NT KO mice, homeostatic sleep pressure accumulates normally 

and may not depend on rising adenosine levels. However, the impaired recovery sleep in these 

mice suggests that increases in adenosine, mediated by e5NT, are necessary for the normal 

recovery of NREM sleep. 

Disclosures: T. Mochizuki, None; E.L. Clark, None; L.F. Thompson, None; T.E. Scammell, 

None. 

Poster 

286. Imaging the Nervous System 



Topic: E.09.e. Functional imaging 

Support: ANR-06-NEURO-004-01 Astroglo 

Title: Intrinsic and autofluorescent optical signals and the neuro-astrocytic activities in the 

rodent olfactory glomeruli 

Authors: *H. GURDEN, B. L'HEUREUX, F. LEFEBVRE, L. PINOT, B. JANVIER, R. 

MASTRIPPOLITO, P. LANIECE, F. PAIN; 

IMNC, CNRS UMR8165, Orsay, France 

Abstract: Changes in the neuro-glial activity induce variations in optical properties of brain 

tissues. Imaging of Intrinsic Optical Signals (IOS) allow mapping of sensory-evoked activity but, 

because they are due to local blood-related changes, these signals are indirectly linked to neural 

activity. We use them in the rodent olfactory bulb (OB) to map odor-induced activation of 

olfactory glomeruli. We previously studied the cellular triggers of odor-induced IOS in rats and 

reported that glutamate release and uptake through astrocytic transporters are at their origin. In 

this context, we are developing our research following two complementary ways. 

i- Further studies of IOS triggers. According to our preliminary results, 2 DeoxyGlucose uptake 

is impaired in GLAST-Knock Out (KO) but not in GLT1-KO mice (see poster of Claire Martin 

in this meeting). We observed that GLAST-KO mice present intact glomerular IOS maps for a 

given odorant. To probe the dose-response curves between KO and wild-type, we are currently 

comparing odor-induced IOS intensity for several odor concentrations. 

ii- Recording autofluorescence (AF) signals from the intracellular compartment. This technique 

relies on AF properties of flavoproteins. To evaluate the feasibility of AF recording in the OB, 

we performed Monte Carlo simulations of photons travel through biological tissues. We 

implemented a multi-layer model for the OB but also for the somatosensory cortex that we used

as a reference since „barrel' AF has been reported. The simulations were performed in two steps : 

i) study of excitation photons absorption ii) study of the emission, propagation and detection of 

fluorescence photons. The first step provided with the coordinates of absorbed photons and 

allowed to build the fluorescence emission matrixes. Then, using these matrixes, we derived for 

each location in the 3-dimensional models the probability of a photon scattering to that location. 

Finally, we determined the origin and intensity of the detected fluorescence signals and the 

amount of detected photons at the surface of the somatosensory cortex and OB. We showed that 

most of the detected fluorescence come below the surface from 400 µm in the former and 200 

µm in the latter structure. Considering the same amount of fluorescence emission, the recorded 

intensity of the flavoprotein fluorescence is about 7 times higher compared to NADH in the 

somatosensory cortex and twice higher in the OB. Overall, AF intensity in the OB and 

somatosensory cortex are similar. We are currently comparing the results of our simulations with 

experimental data previously reported for the somatosensory cortex and with our preliminary AF 

imaging in the rat OB. 

Disclosures: H. Gurden, None; B. L'Heureux, None; F. Lefebvre, None; L. Pinot, None; B. 

Janvier, None; R. Mastrippolito, None; P. Laniece, None; F. Pain, None. 

Poster 





Support: University of Pennsylvania-AstraZeneca Master Research Collaboration 

Title: Identification of stress circuitry using ex vivo manganese enhanced magnetic resonance 

imaging (MEMRI) 

Authors: *D. A. BANGASSER 1 , Y. PEREZ 1 , B. BROCKEL 2 , R. J. VALENTINO 1 ; 

1 Stress Neurobio., Children's Hosp Philadel, Philadelphia, PA; 2 Dept. of Neurosci. Biol., 

AstraZeneca R&D, Wilmington, DE 

Abstract: Manganese (Mn 2+ ), a paramagnetic calcium analogue, is taken up though activitydependent 

voltage-gated calcium channels, transported down axons, and secreted at synapses. 

These properties of Mn 2+ led to the development of a Mn 2+ enhanced magnetic resonance 

imaging (MEMRI) technique, which has been used to identify functionally activated nuclei and 

fiber tracts in non-human primates, rats, and mice. Here we used MEMRI to identify pathways 

that are differentially regulated by swim stress in male rats. Rats were implanted with an

intracerebroventricular (i.c.v.) cannula guide. At least one week later, Mn 2+ (500mM, i.c.v.) was 

administered 1 h prior to a 15 min swim stress (n=10) in room temperature water (30 cm depth). 

Control rats (n=10) were handled 1 h after Mn 2+ administration. Rats were transcardially 

perfused with 4% paraformaldehyde and sacrificed 6 h, 24 h, 48 h, or 72 h after swim stress and 

brains were removed. The brains were scanned with a 9.4 T vertical bore magnet and areas of 

increased activity were analyzed using a program written in IDL virtual machine. The presence 

of Mn 2+ was detected in brain in a time-dependent manner. Thus, at 6 h Mn 2+ was present 

primarily in the ventricles and there was little difference between swim stress and control rats. 

By 24 h and 48 h, Mn 2+ was visualized in specific brain nuclei, and fiber tracts became visible at 

48 h. By 72 h there was little enhancement left. Swim stress increased Mn 2+ labeling at both 24 h 

and 48 h in specific brain regions. Enhancements were observed in the bed nucleus of the stria 

terminalis and the dorsal hippocampus, areas that mediate stress related behaviors and regulate 

the hypothalamic-pituitary adrenal axis. Additionally, enhanced labeling was seen in the lateral 

periaqueductal gray (lPAG), a brain region that has been proposed to mediate escape behaviors 

and active coping strategies. This labeling is consistent with the escape behavior (climbing and 

swimming) observed during initial exposure to swim stress. Through these experiments we have 

found that MEMRI is a rapid way of identifying structures activated by stress in 3 dimensions. 

These are the first studies, to our knowledge, to use MEMRI to examine brain circuitry activated 

by an acute stressor. This technique has much promise for revealing the mechanisms by which 

stress impacts on the brain and how this might be altered by pharmacological agents. 

Disclosures: D.A. Bangasser , None; Y. Perez, None; B. Brockel, Full time employee of 

AstraZeneca, A. Employment (full or part-time); Stock options for AstraZeneca, E. Ownership 

Interest (stock, stock options, patent or other intellectual property); R.J. Valentino, None. 

Poster 




Topic: E.09.b. Blood flow 



Title: In vivo two-photon measurement of single vessel diameter changes using astrocytic 

indicator SR101

Authors: I. C. TENG 1 , P. TIAN 1 , A. M. DALE 2 , *A. DEVOR 3 ; 

1 Neurosciences, 2 Neurosciences and Radiology, UCSD, La Jolla, CA; 3 Martinos Ctr. Biomed 

Imaging, MGH, Harvard Med. Sch., Charlestown, MA 

Abstract: Neurons, glia and vascular cells form a metabolic and homeostatic network to sustain 

brain activity. Therefore, it is important to examine the neuronal, glial, and vascular activity 

simultaneously in vivo. Two-photon laser scanning microscopy (TPLSM), combined with the 

recent developments in bulk-loading of fluorescent indicators and intravenous labeling of blood 

lumen, has opened an unprecedented possibility for in vivo imaging of neurovascular networks. 

Here, we investigate the feasibility of inferring single vessel diameter changes in vivo from 

movement of fluorescently labeled astrocytic endfoot and compare these measurements to direct 

labeling of the blood lumen. 

We used TPLSM in conjunction with the multi-cell bolus loading of calcium indicator Oregon 

Green 488 BAPTA-1 (OGB1) and astrocyte-specific marker sulforhodamine 101 (S101). Inplane 

and penetrating vessels appeared as dark bands and dark circles respectively and were 

surrounded by bright astrocytic endfeet. Both sides of the astrocytic membrane (facing the vessel 

and facing the tissue) were used to infer the vascular diameter during somatosensory stimulation. 

Frame and line scans were employed to measure diameter changes of pial and penetrating vessels 

together with the neuronal and astrocytic calcium signals as revealed by the fluorescence 

changes in OGB1. 

Our data show that 1) the astrocytic endfoot membrane can move outward or inward with the 

stimuli. The timecourse of this movement is consistent with the vascular diameter change 

measured by fluorescent labeling of blood lumen. The amplitude of the percentage change of 

astrocytic displacement is smaller than that obtained by labeling the blood lumen directly. 2) 

Both OGB1 and SR101 can be used to detect the displacement of the astrocytic endfoot. 

Inferring vascular diameter from the displacement of the astrocytic 

endfoot enables simultaneous imaging of the neuronal and astrocytic calcium signals together 

with measurements of the vascular diameter changes with only two different dyes and two PMT 

detectors. 

Disclosures: I.C. Teng, None; A. Devor , None; P. Tian, None; A.M. Dale, None. 

Poster 




Topic: E.09.a. Energy metabolism

Title: Inhibition of somatosensory afferent synaptic and vascular responses by preceding 

transcallosal stimulation in mouse sensory cortex 

Authors: B. L. LIND, *M. J. LAURITZEN; 

Dept Clin. Neurophysiol, Univ. Copenhagen, DK-2600 Glostrup, Denmark 

Abstract: Paired neural stimuli applied to neuronal networks targeting the same cortical 

networks, may enhance or inhibit the following synaptic or vascular responses, dependent on the 

inter-stimulus interval and the sequence of stimulations. This is the basis for using vascular 

signals to probe neural events on a millisecond time scale. The aim in this study was to 

investigate in mouse sensory cortex the mechanisms of interaction of 2 networks that target the 

same small cortical area with regard to synaptic, vasculary and metabolic responses in relation to 

changes in intracellular astrocytic and dendritic Ca 2+ . 

An area in the sensory barrel motor cortex was activated by stimulation of the infraorbitalis 

nerve (Io) and transcallosal (tc) fibers. 8 weeks old male fvb mice was anaesthetized with 

ketamin and xylazin while the animal was prepared surgically and given α-chloralose during the 

experiment. Both end-inspiratory CO2 and blood pressure were measured continuously. Two 

cranial windows were drilled over the cortex in each side and dura was removed. MK801 was 

applied to the left cortical hemisphere before the stimulation electrode. Laser-Doppler probe was 

placed just above and recording electrodes inserted 300 µm (layer 2/3) down in to the right 

cortex. Io and tc stimulation was done both separate and paired. Stimulation of either of the two 

networks preceded the other with 75 ms time interval. 

Our preliminary data show that tc stimulation may inhibit the normal Io response with regards to 

local field potential and blood flow, while the Io stimulation seems to leave the tc response 

intact. This example of paired stimulation can give a further understanding of how neuronal 

activity can be probed with vascular signals as discussed by Ogawa et al. PNAS 2000 in rats. 

The ability to perform paired stimulation in mice enable us to use transgenetic animals in twofoton 

microscopy. This will allow us to distinguish between responses in the different cell types 

and Ca 2+ imaging. 

Disclosures: B.L. Lind, full-time employment in Martin Lauritzens lab, A. Employment (full 

or part-time); M.J. Lauritzen , Lundbeck Foundation, B. Research Grant (principal investigator, 


Poster 




Topic: E.09.b. Blood flow



Title: Characterization of the spatiotemporal responses of surface and penetrating cerebral 

arterioles using two-photon microscopy 

Authors: *P. TIAN 1 , I. C. TENG 1 , A. M. DALE 2 , A. DEVOR 2,3 ; 

1 Neurosciences, 2 Neurosciences and Radiology, UCSD, La Jolla, CA; 3 MGH, Charlestown, MA 

Abstract: A complete mechanistic understanding of vascular-based functional imaging signals, 

such as those of functional MRI, requires knowledge of the vascular responses on the level of 

individual vessels within a large volume. Two-photon laser scanning microscopy (TPLSM) has 

been widely employed to image cerebral cortex about 500 microns below pial surface with 1 

micron resolution. Hence, we map the three-dimensional stimulus-evoked spatiotemporal 

diameter and velocity changes of single vessels in the rat somatosensory cortex in relation to the 

map of the underlying neural activity in vivo using TPLSM. 

Electrical forepaw stimuli are presented to rats. The center of neuronal activity is mapped using 

recordings of evoked surface potentials. Single-vessel vascular diameter and/or velocity changes 

are characterized across multiple cortical columns up to a volume of 4 x 4 x 0.5 mm 3. We 

measure both surface vessels (including arteries and veins) and penetrating arterioles/venues and 

capillaries that lie in layer II/III. The responses (timing, shape, amplitude) are examined as a 

function of cortical depths and the distance from the center of neuronal activity. 

Our data show that 1) Similar to our previous findings on the surface arteries/arterioles, 

penetrating arterioles (100 to 300 microns deep in the cortex) exhibit biphasic behavior in 

response to the stimulus. Namely, vasodilatation is followed by vasoconstriction. 2) There are 

temporal laminar specificities of the vascular responses. For example, dilation onset is earlier in 

penetrating arterioles in comparison with the parent pial arteries. 3) Both surface and penetrating 

arterioles show more vasodilatation near the center of the neuronal activity and more 

vasoconstriction further away from the center. 4) Veins show either no diameter change or 

diameter decrease. 

Disclosures: P. Tian, None; I.C. Teng, None; A.M. Dale, None; A. Devor, None. 

Poster 




Topic: E.09.a. Energy metabolism

Support: NIH RO1 NS051586 

VAMC Merit Review 

Title: NADH imaging and glycogen stores during hypoglycemia in rat hippocampal slices 

Authors: *M. P. SADGROVE 1,2 , F. GALEFFI 1,2 , D. A. TURNER 1,2 ; 

1 Neurosurg. and Neurobio., Duke Univ. Med. Ctr., Durham, NC; 2 Durham VAMC, Durham, NC 

Abstract: Glycogen stores, in astrocytes, have been proposed as a source of energy during brief 

periods of hypoglycemia. Glycogen within astrocytes may be released as either glucose or 

lactate, and subsequently utilized by neurons, providing an additional energy buffer when 

glucose supply is compromised, as occurs during hypoglycemia. 

We sought to determine the role of glycogen during aglycemia. Hippocampal slices (400κm, 

95%O2, 36°C, interface) were prepared from young adult (5-8 wks) F344 rats. Field excitatory 

post-synaptic potentials (fEPSP) and NADH fluorescence images were recorded from the CA1 

region. To block glycogenolysis in the treated group the glucosidase inhibitor 1- 

Deoxynojirimycin, and the glycogen phosphorylase inhibitor 1,4-Dideoxy-1,4-imino-Darabinitol 

were added to artificial cerebrospinal fluid (ACSF). After a baseline recording in 

ACSF (10 mins) the inhibitor cocktail was added to the ACSF (10 mins), and then slices were 

transferred to glucose free ACSF with inhibitors (30 mins). Time matched controls received no 

inhibitor cocktail. 

We found that these two inhibitors did not alter the NADH fluorescence under control, 10mM 

glucose conditions, and had no effect on control fEPSP amplitude. Thirty min exposure to 0mM 

glucose resulted in a significant decrease of NADH fluorescence, compared with the baseline 

recorded in 10mM glucose (2.0 ± 0.6% vs. 16.8 ± 3.3 %; P < 0.001). However, when slices were 

exposed to 0mM glucose in the presence of the glycogenolysis inhibitors the decrease in NADH 

fluorescence was more dramatic, being significantly lower than both baseline and the uninhibited 

controls (35 ± 1.7 %, both comparisons: P < 0.001, one way ANOVA with Tukey‟s post hoc 

test). 

The fEPSP amplitude was also affected by the presence of glyogenolysis inhibitors during the 

aglycemic insult, but not under control conditions (10mM glucose). The mean time for the 

fEPSP to fall below 50% of initial amplitude following onset of aglycemia was significantly 

reduced (without inhibitors 977 s vs. with inhibitors 702 s; P < 0.05 unpaired two tailed t-test ). 

In both groups fEPSP amplitudes fell to zero within 30 mins of the switch to aglycemia. The 

glycogen reserve appears to offer over 4 min of residual slice function. 

These observations suggest that metabolic substrates derived from glycogen break down may 

provide reducing equivalents for the mitochondria, and slow down the oxidation of the 

NAD/NADH pair, for a short reserve window. But, glycogen reserves are rapidly exhausted and 

are not sufficient to prevent the eventual loss of synaptic transmission. 

Disclosures: M.P. Sadgrove, None; F. Galeffi, None; D.A. Turner, None.

Poster 





Support: NIH grant R01-445671-01 

MRC New Investigator Award GO601581 

MRC project grant G0100538 

Title: Prolonged negative BOLD represents neural inhibition in the deeper cortical layers 

Authors: *L. W. BOORMAN, A. KENNERLEY, M. JONES, D. JOHNSTON, Y. ZHENG, J. 

MAYHEW, J. BERWICK; 

Univ. Sheffield, Sheffield, United Kingdom 

Abstract: Blood Oxygen Level Dependent (BOLD) functional magnetic resonance imaging 

(fMRI) is used to infer levels of neural activity by examining signal changes associated with 

changes in deoxyhemoglobin (Hbr) concentration. The most commonly used mapping signal, 

„the positive BOLD response‟, is due to a „washout‟ of Hbr produced by a large increase in blood 

flow into an active brain region. However, recently a prolonged negative BOLD signal has been 

described in both the primate (Shmuel et al 2006) and human (Smith et al 2004) cerebral cortex. 

The cause of this negative BOLD signal is unclear, but it is proposed to be one or a combination 

of two mechanisms: (i) a vascular blood „steal effect‟ in which active regions of cortex increase 

their blood flow by reducing blood flow to surrounding regions, and ii) inhibitory neural 

connections to adjacent regions that lower activity and therefore decrease metabolic demand and 

thus decrease blood flow. The current study examines the presence of prolonged negative BOLD 

in rat somatosensory cortex. 16s duration electrical stimuli (5Hz, 1.2mA) were presented to the 

contra-lateral whisker pad. Cortical hemodynamic responses were measured using 2-dimensional 

optical imaging spectroscopy (2D-OIS) and laser Doppler flowmetry (LDF). Neural responses 

were measured with multi-channel electrophysiology. Large increases in blood volume, 

saturation and flow were seen in the cortical whisker barrel region during the period of 

stimulation. Negative changes of a smaller magnitude were seen in regions surrounding the 

whisker barrel region. Multi-channel electrodes were placed into both the barrel cortex and the 

adjacent regions that displayed „negative‟ hemodynamic responses. Electrophysiological 

recordings from the „whisker‟ region displayed large evoked spiking responses with very small 

decreases in interim baseline multi-unit activity. Electrophysiological recordings from the 

„negative hemodynamic‟ region showed smaller evoked spiking responses, but displayed a far 

larger decrease in interim baseline firing. These decreases in baseline spiking were found 

predominantly in the deeper layers of the cortex. These data suggest that negative hemodynamic

changes observed in regions adjacent to active whisker barrel cortex may be related to neural 

interactions. 

Disclosures: L.W. Boorman , None; A. Kennerley, None; M. Jones, None; Y. Zheng, 

None; J. Mayhew, None; J. Berwick, None; D. Johnston, None. 

Poster 





Support: MRC NIA G050112 

Title: Cortical oxygenation changes evoked by sensory stimuli during hypercapnia 

Authors: M. SAKA, K. BARTLETT, J. MAYHEW, J. BERWICK, *M. O. JONES; 

Dept Psychol, Univ. Sheffield, Sheffield, United Kingdom 

Abstract: The changes in cerebral blood flow, volume and oxygenation that accompany focal 

increases in neural activity are collectively referred to as the hemodynamic response, and form 

the basis of non-invasive brain imaging techniques such as fMRI. However, the role of the 

hemodynamic response in terms of oxygen delivery to brain tissue is still not fully understood. 

Although neural activity is typically accompanied by hyperemia, direct measurements with 

polarographic electrodes have detected decreases in oxygen tension at the focus of sensoryevoked 

activity (e.g. Thompson et al., 2005). In an attempt to further understand the role of 

cerebral blood flow in oxygen delivery, we investigated whether this measure of sensory-evoked 

oxidative metabolism could still be detected during elevations in baseline CBF produced by 

hypercapnia. The well defined topography of the cortical somatosensory system of the 

aneasthetised rodent was a sufficient model to address this issue. Cortical somatosensory whisker 

„barrel‟ representations were first localised using optical imaging of intrinsic signals imaging 

following presentation of sensory stimuli to an individual whisker on the contra-lateral face. The 

resulting spatial maps were used to guide insertion of a polarographic glass electrode (Unisense) 

to a depth of 300-400µm orthogonal to the cortical surface. The glass electrode was used to make 

subsequent measurements of cortical brain tissue oxygenation. At these cortical depths changes 

in tissue oxygenation produced by sensory stimuli were predominantly negative and monophasic. 

The fraction of inspired Co2 was subsequently elevated to 5% and 10% to produce differing 

levels of hypercapnia. Hypercapnia resulted in monotonic increases in the „baseline‟ availability 

of brain tissue oxygen. Despite these increases in oxygen availability, similar stimulus-evoked

esponses were observed to those measured during normocapnia. These data provide evidence 

for increases in cerebral oxygen consumption even when „sub-naturalistic‟ sensory stimuli are 

presented to aneasthetised animals. They also suggest preserved neuro-metabolic coupling 

during elevations in baseline oxygen availability and that previously reported changes in 

stimulus evoked hemodynamics during hypercapnia (e.g. Sicard and Duong, 2005) may be due 

to vascular rather than metabolic effects. 

Disclosures: M. Saka, None; M.O. Jones , None; K. Bartlett, None; J. Berwick, None; J. 

Mayhew, None. 

Poster 





Support: NIH Grant ROI-NS44567-01 

MRC Grant 9825307 

EPSRC Grant EP/D001218 

Title: Sensory adaptation, latency shift and the recovery of evoked post-synaptic potentials in the 

cortex 

Authors: S. HARRIS 1 , *Y. ZHENG 3 , J. BERWICK 1 , A. KENNERLEY 1 , V. LEFEBVRE 1 , C. 

MARTIN 1 , S. BILLINGS 2 , D. COCA 2 , J. MAYHEW 1 ; 

1 Dept Psychology, 2 Dept. of Automatic Control and Systems Engin., Univ. of Sheffield, 

Sheffield, United Kingdom; 3 Dept Psychol, Univ. Sheffield, SHeffield, United Kingdom 

Abstract: Using stimulation of the rat whisker pad and multi-electrode recording of responses 

from the barrel cortex, we have investigated the effects that a preceding (conditioning) stimulus 

of different durations have on the amplitude and latency of the neural response to a subsequent 

probe stimulus presented two seconds after the cessation of the conditioning stimulus. Current 

source density (CSD) analysis of laminar local field potential (LFP) recordings are used as a 

measure of neural responses to stimulation at different cortical depths. The stimulation frequency 

is fixed at 5Hz, the stimulation intensity at 1.2mA, and stimulus pulse width is 0.3ms. In 

addition, concurrent measurements of regional cerebral blood flow are made using laser Doppler 

flowmetry.

We have found that the conditioning stimulation produced a marked attenuation in the 

amplitudes of EPSP responses to the probing stimulation. The longer the duration of the 

conditioning stimulus, the more pronounced the attenuation (Zheng et al 2007). Furthermore, the 

latency in the neural responses to probing stimulus is delayed compared to the latency of the 

neural responses to the conditioning stimulus. In order to investigate the timescale of this 

attenuation and the effect on latency, experiments with varying inter-stimulus interval (ISI) 

between the conditioning and probing stimulation are being conducted. ISI of 0.6, 1, 2, 4, 6 or 8s 

are used and the duration of the conditioning block of stimulation is varied at 2, 8 and 16s, whilst 

the duration of the probing block of stimulation is fixed at 1s. 

The implications of our recent findings for a model of the neural response to stimulation and the 

coupling between neural activity and the hemodynamic response will be discussed. 

References 

Zheng Y et al (2007). Neuroscience 2007 San Diego, USA. Abs. 89.2. 

Disclosures: S. Harris, None; Y. Zheng , None; J. Berwick, None; A. Kennerley, None; V. 

Lefebvre, None; C. Martin, None; S. Billings, None; D. Coca, None; J. Mayhew, None. 

Poster 





Support: Deutsche Forschungsgemeinschaft 

Hermann und Lilly Schilling Stiftung 

Title: Neurovascular coupling during combined local inhibition of nNOS, COX, adenosine 

receptors, CYP450-expoxygenase and Kir-channels 

Authors: *C. LEITHNER 1 , G. ROYL 1 , N. OFFENHAUSER 1 , M. FÜCHTEMEIER 1 , M. 

KOHL-BAREIS 2 , A. VILLRINGER 1 , U. DIRNAGL 1 , U. LINDAUER 1 ; 

1 Charite Berlin, Berlin, Germany; 2 Univ. of Applied Sci. Koblenz, Remagen, Germany 

Abstract: Neurovascular coupling provides the basis for functional neuroimaging techniques 

such as BOLD-fMRI. Many biochemical pathways have been implicated in dilating local 

arterioles during increased neuronal activity. Among these nitric oxide (NO), adenosine, 

cyclooxygenase products, CYP450 epoxygenase products and potassium are center stage. The 

authors used combined local inhibition of NO synthase, cyclooxygenase, adenosine receptors,

CYP450 epoxygenase and inward rectifier potassium channels to test whether these pathways, 

acting partly in parallel, could fully account for the blood flow response to neuronal activation. 

Cerebral blood flow (CBF) and oxygenation over the somatosensory cortex were measured 

during electrical forepaw stimulation in rats using a combined laser doppler/spectroscopy probe 

through a superfused cranial window. Somatosensory evoked potentials (SEP) were recorded as 

a correlate of neuronal activity. The CBF response was largely reduced with stable SEP during 

combined inhibition of NO synthase, cyclooxygenase, adenosine receptors and CYP450 

epoxygenase. Additional inhibition of inward rectifier potassium channels did not lead to a 

significant further reduction of the CBF response. Despite significant reduction of the CBF 

response, no deoxygenation could be detected. In summary, the NO, adenosine, cyclooxygenase, 

CYP450 epoxygenase and inward rectifier potassium channel pathways together account for 

approximately two third of functional hyperemia in the rat somatosensory cortex. As yet 

unidentified additional pathways might be involved in coupling neuronal activity to regional 

cerebral blood flow. In rat somatosensory cortex, only one third of the blood flow increase is 

required to prevent relative hypoxia during increased neuronal activity. 

Disclosures: C. Leithner , None; G. Royl, None; N. Offenhauser, None; M. Füchtemeier, 

None; M. Kohl-Bareis, None; A. Villringer, None; U. Dirnagl, None; U. Lindauer, None. 

Poster 




Topic: E.09.c. Blood brain barrier 

Support: DOD BC050006 

Title: Permeability measurements at the blood-brain and blood-tumor barrier using different 

molecular weight fluorescent markers 

Authors: *R. MITTAPALLI 1 , K. A. BOHN 2 , J. M. EGBERT 2 , V. K. MANDA 2 , V. 

RUDRARAJU 2 , K. S. TASKAR 2 , J. A. GAASCH 2 , Q. R. SMITH 2 , P. R. LOCKMAN 2 ; 

1 TTU HSC, Amarillo,, TX; 2 Pharmaceut. Sci., Texas Tech. Univ. HSC, Sch. of Pharm., 

Amarillo, TX 

Abstract: Introduction: The unique anatomical structure of the brain neurovasculature limits 

chemotherapeutic distribution into normal brain and to a lesser extent, brain tumors. While there 

are documented vascular permeability differences between tumor and normal brain, less is 

known about permeability changes in various tumor regions. Therefore, we developed a novel

quantitative fluorescent microscopy method to evaluate the regional transfer coefficient (Kin) at a 

sub-micron level within the tumor, tumor core (inner 50% of the tumor) and the BAT (100 

microns surrounding tumor). Methods: A stably transfected eGFP glioma cell line (RG-2) was 

implanted intracranially in Fisher 344 rats and tumors were allowed to develop for seven days. 

Similarly, a stably eGFP transfected metastatic tumor line (MDA-MB-231-Br) over expressing 

Her-2 was injected into the peripheral circulation via the left cardiac ventricle and brain 

metastases were allowed to develop for ~28 days. After tumor development, free Texas Red 

(TR), 625Da or Texas Red Dextran (TRD) (3kDa and 70kDa) was injected I.V. in separate 

animals and allowed to circulate for differing times. Fluorescent probe concentrations in blood 

and brain were determined by quantitative fluorescent microscopy (Olympus MVX-10 stereo 

microscope) with matching tissue and blood standards. Regional Kin values were calculated from 

the slope of the uptake plot (Patlak, 1983). Results: In control animals and contra-lateral brain 

the Kin for TRD-3kD was 0.4 ± 0.1 x 10 -5 mL/s/g . The glioma tumor Kin was increased 5-fold to 

2 ± 0.3 x 10 -5 mL/s/g, whereas tumor core was increased 7-fold to 2.8 ± 0.2 x 10 -5 mL/s/g and the 

BAT was increased 4-fold to 1.6 ± 0.2 x 10 -5 mL/s/g. In contrast, permeability changes were 

heterogeneous in metastatic lesions. For metastases which were permeable, we observed a 4-fold 

increase (TRD-3kD) in tumor, a 4 fold increase in tumor core and a 2 fold increase in BAT. 

Lastly, both models showed differential permeability changes dependent upon the molecular 

weight of the permeability marker. Conclusions: Using our novel method we have measured 

size-dependent permeability changes within tumor, tumor core and BAT in two separate tumor 

models. Our method is superior to autoradiography in development time, spatial resolution, and 

the ability to identify tumor boundaries. Of clinical significance, there is an increased 

fluorophore accumulation in BAT that decreases with distance from tumor edge. Two 

hypotheses can be drawn from this data; 1) the BAT increase is due to altered flux across the 

vasculature of the BAT or 2) the increased BAT accumulation is secondary to diffusion from 

tumor to BAT down the concentration gradient. 

Disclosures: R. Mittapalli , None; K.A. Bohn, None; J.M. Egbert, None; V.K. Manda, 

None; V. Rudraraju, None; K.S. Taskar, None; J.A. Gaasch, None; Q.R. Smith, None; P.R. 

Lockman, DOD: BC050006, B. Research Grant (principal investigator, collaborator or 


Poster 




Topic: B.04.g. Other 

Support: NIH intramural research training award

Title: Discovery of a neuronal population in deep layers of the entorhinal cortex sensitive to 

nicotine using voltage sensitive dye imaging 

Authors: *B. TU, J. SHEN, J. L. YAKEL; 

Lab. Neurobiol, NIEHS, RTP, NC 

Abstract: Hippocampal neurons mainly expressing α7 and/or α4ß2 subtypes of nicotinic 

acetylcholine receptors (nAChRs) are responsible for cognition and memory effects by tobacco 

smoking. At blood levels of nicotine in cigarette smokers (0.06~0.31 µM), α7 nAChRs are 

minimally activated and partially desensitized, while α4ß2 nAChRs are completely desensitized. 

Since non-α7 nAChRs are responsible for nicotine effects on hippocampal memory function in 

vivo, it is postulated that a subtype of non-α7 nAChR not desensitized by nicotine at blood levels 

of smokers may function in certain hippocampal neurons. However, the location and nature of 

these neurons are not known. In this study, we established a functional imaging method using a 

voltage sensitive dye (Di-4-ANEPPS) and a MiCAM02 camera in order to locate these neurons 

in hippocampal slices from 14-28 days-old rats. Imaging on slices was conducted in an interface 

chamber perfused with ACSF containing TTX (1 µM) at room temperature. We found that deep 

layers of entorhinal cortex (EC) and stratum oriens of subiculum (SbSO) are depolarized by bath 

applied nicotine (10µM) for over 10 min through activating non-α7 nAChRs. Whole-cell current 

clamping revealed that EC layer VI (ECLayVI) neurons were most depolarized (16.5±4.3 mV, 

Mean±SD, N=21) by 10 µM nicotine when compared to those in other regions. The EC50 

concentration for nicotine of ECLayVI neurons was 0.6 µM, which was lower than those of 

SbSO (1.2 µM) neurons and was closer to the average blood level of nicotine in smokers. A low 

concentration of nicotine (0.1 µM) depolarized all 6 ECLayVI neurons examined with current 

clamping (2.9±1.5 mV) but only affected 3 in 5 SbSO neurons (1.5±1.6 mV). ECLayVI neurons 

showed much less desensitization (15±16%, N=18) by nicotine (10 µM; over 5 min of bath 

perfusion) than SbSO neurons (57±18%, N=9). They also showed typical firing properties 

resembling interneurons, i.e., prominent after-hyperpolarization with no accommodation, but 

exhibit almost no h-current. Intracellular labeling with a fluorescent dye (Alexa488) revealed 

that nicotine-sensitive ECLayVI neurons are mid to large size multipolar oval neurons. 

Activation of non-α7 AChRs in these neurons through local acetylcholine application (while 

blocking other types of AChRs) significantly increased frequency of spontaneous GABAergic 

inhibitory postsynaptic events on EC layer V neurons. We conclude that ECLayVI neurons are 

inhibitory GABAergic interneurons most sensitive to nicotine and most resistant to 

desensitization within hippocampal formation. They may be critical in mediating nicotine effects 

on memory during smoking. 

Disclosures: B. Tu , None; J. Shen, None; J.L. Yakel, None. 

Poster 

286. Imaging the Nervous System




Support: NIH Grant 013759 

Gatsby Fellowship 

NIH NRSA F31 NS056834-01 

Title: Neuroimaging signals in the awake animal are mediated by stimulus-driven spiking and 

trial-related ongoing activity 

Authors: Y. B. SIROTIN 1 , *A. DAS 2,1 ; 

1 Neurosci., 2 Ctr. Neurobiol & Behavior, Columbia Univ., New York, NY 

Abstract: Interpretation of neuroimaging data (notably, fMRI) assumes a common linear 

relationship with all underlying driving components. Typical analyses are based on separating 

the overall signal into contributions from all putative components, each mediated by the same 

hemodynamic transfer function. Much of the evidence for this hypothesis comes from work in 

anesthetized preparations, however. 

Using optical imaging in alert monkey we showed, last year, that expectation of trial onsets 

evokes a robust, trial-related hemodynamic (neuroimaging) signal in V1. This signal is as strong 

as visually evoked responses, but present even in the absence of visual input. This suggested that 

the net hemodynamic signal in the awake animal contains components that are conventionally 

neuronally driven as well as those not explicable by underlying neuronal activity. 

To determine the relationship between hemodynamic and neural signals in the alert animal we 

made concurrent electrode and optical recordings while the animal performed cued fixation, in 

either the presence or absence of visual stimulation. 

Results: 

1. We failed to find any V1 neuronal activity that could explain the trial-related signals reported 

last year. 

2. The visual stimulus-evoked hemodynamic responses, on the other hand, were very well 

predictable by simultaneously recorded neuronal activity. 

3. Hemodynamic responses evoked by visual stimuli were linearly related to the simultaneously 

recorded multi-unit (MUA) responses. Contrast tuning (with full-field gratings) gave a linear 

relationship between MUA and hemodynamic responses, over 5 log units of stimulus contrast. 

4. For all stimuli, subtracting away the linear hemodynamic prediction left a residue that was 

independent of neuronal activity, but had consistent trial-related temporal structure. 

5. Thus in the mean, the stimulus-evoked hemodynamic response in the alert animal is a linear 

sum of a predictor derived from the stimulus-evoked MUA and a trial-related signal independent 

of local spiking. 

6. With local field potentials as with MUA we failed to find predictors that explained the full

hemodynamic response. Further, different frequency bands showed differential contributions 

from stimulus vs. non-stimulus related signals. 

Disclosures: Y.B. Sirotin, None; A. Das , None. 

Poster 







Title: Cortical laminar differences in neurometabolic coupling 

Authors: *A. VISWANATHAN, R. D. FREEMAN; 

Vision Sci., Univ. California-Berkeley, Berkeley, CA 

Abstract: Functional magnetic resonance imaging (fMRI) studies commonly assume 

homogeneity of neurometabolic coupling through different cortical layers. This is partly because 

averaging the blood oxygen level-dependent (BOLD) signal across cortical lamina has the 

intrinsic advantage of increasing the signal-to-noise ratio. However, capillary bed distribution is 

not uniform across cortex, and recent work suggests that capillary density is proportional to the 

metabolic demands of local neurons. Here, we used a unique procedure to examine 

neurometabolic coupling across cortical lamina at high spatial resolution. We simultaneously 

recorded metabolic (tissue oxygen concentration) and neural (multi-unit spike discharge and 

local field potentials) activity in cat primary visual cortex (area 17) using a 32-channel silicone 

probe (NeuroNexus Technologies, Inc.). Three channels were converted into polarographic 

oxygen sensors using a Ag/AgCl reference electrode. Neural activity was measured from three 

neighboring channels, each one 50κm above its corresponding oxygen sensing channel. These 

three neural-oxygen pairs were each in a different cortical layer as confirmed via histological 

analysis. We examined neural and tissue oxygen responses to sine wave gratings. Results to date 

indicate that tissue oxygen signal magnitude is greatest in the middle cortical layers, where 

capillary density is maximal. This suggests that cerebral vasculature plays a role in determining 

the strength of the hemodynamic response. Therefore, local vascular architecture should be 

considered when interpreting BOLD signal magnitudes.

Disclosures: A. Viswanathan , None; R.D. Freeman, None. 

Poster 

287. Blood Brain Barrier and CSF 




Support: DOD BC050006 

Title: Nicotine and cotinine inhibit P-glycoprotein at the Blood Brain Barrier 

Authors: *V. MANDA, R. K. MITTAPALLI, Q. R. SMITH, P. R. LOCKMAN; 

Texas Tech. Univ., Amarillo, TX 

Abstract: Introduction: P-glycoprotein (P-gp) at the blood brain barrier (BBB) alters the brain 

accumulation of several classes of drugs (e.g., anti-cancer, anti-epileptic and HIV-protease 

inhibitors). Recent studies have shown nicotine and its glucuronide metabolites inhibit organic 

anion efflux by MRP1 and MRP2, though at supra-physiologic concentrations. However, there 

are no studies indicating whether nicotine and its major metabolite cotinine interact with P-gp at 

the BBB at pharmacologic concentrations and thus influence CNS drug accumulation. The 

current study utilizes our novel quantitative fluorescent microscopy methodology to investigate 

whether nicotine inhibits P-gp at the BBB, and if this interaction increases the brain 

accumulation of the P-gp substrate rhodamine123 (R123). Methods: The in situ rat brain 

perfusion was used to determine the blood-to-brain transfer coefficient (Kin) of R123, in the 

absence and acute presence of pharmacologic concentrations of nicotine and or cotinine as well 

as known inhibitors of P-gp. Fluorescent probe concentrations in brain were determined by 

quantitative fluorescent microscopy with tissue standards. Regional Kin values were calculated 

from the quantity of R123 taken up by brain divided by the perfusion exposure (Cpf x T) Results: 

The blood-to-brain accumulation of rhodamine 123 was linear over 120 seconds with an 

apparent Kin of 1.5 ± 0.08 x 10 -4 mL/s/g. Addition of P-gp inhibitors to the perfusate increased 

R123 Kin by αππποξηκαηειψ 10 fold, suggestive of a significant component of active efflux. 

Similarly, perfusion with nicotine or cotinine at physiological concentrations increased BBB Kin 

of R123 in a concentration dependent manner by up to 3.5 fold. The nicotine-induced increase in 

R123 uptake was not reversed by addition of cholinergic receptor antagonists to the perfusion 

fluid. Conclusions: The blood-to-brain uptake of rhodamine R123 was characterized using our 

novel fluorescent microscopy method coupled to in-situ brain perfusion. The data suggest that 

nicotine and cotinine inhibit P-gp mediated efflux of R123 at the BBB and imply that the 

permeability of other P-gp drugs may be elevated in chronic smokers as well.

Disclosures: V. Manda , None; R.K. Mittapalli, None; P.R. Lockman, DOD: BC050006, B. 


grants already received); Q.R. Smith, None. 

Poster 





Support: Myelin Repair Foundation 


Title: Molecular mechansims regulating blood-brain barrier formation during development 

Authors: *R. DANEMAN, D. AGALLIU, L. ZHOU, B. A. BARRES; 

Stanford Univ., Stanford, CA 

Abstract: The vasculature of the central nervous system forms a barrier, not found in other 

tissues, that limits the flow of molecules and ions from the blood into the brain. This blood-brain 

barrier (BBB) is crucial for maintaining brain homeostasis and for blocking the flow of toxins, 

pathogens, and even the bodies own immune system into the brain. Despite its importance, little 

is known about the molecular mechanisms regulating the development of this crucial barrier. In 

this study, we perform comparative microarray analysis to determine the blood-brain barrier 

specific transcriptome. To accomplish this we utilized fluorescent associated cell sorting to 

purify endothelial cells from the brain, liver and lungs of Tie2GFP transgenic mice. Using cDNA 

microarrays to analyze the mRNA expressed in different cell populations we are able to make 

several important comparisons. First, we have compared the transcriptional profile of endothelial 

cells (GFP+) with CNS parenchymal cells (GFP-), to generate a data set of vascular specific 

genes. Second, comparison of transcriptional profile of brain GFP+ cells, with liver and lung 

GFP+ cells has allowed us to generate a comprehensive list of transcripts that are specific to the 

BBB. Finally, by isolating GFP+ cells from the brains of Tie2GFP mice of different ages, we are 

able to elucidate the transcriptional changes that occur during the development of the BBB. This 

analysis has identified that Wnt/beta-catenin signaling is enriched in CNS endothelial cells 

compared with the endothelial cells from the liver and lung. We further demonstrate that 

Wnt/beta-catenin signaling is necessary for proper CNS angiogenesis and also regulates the 

expression of BBB specific transporters in CNS endothelial cells. Taken together these data

provide a molecular link between CNS angiogenesis and BBB formation, two processes 

previously thought to be independent 

Disclosures: R. Daneman , None; D. Agalliu, None; L. Zhou, None; B.A. Barres, None. 

Poster 





Support: NIH GRANT RO-1 NS41405 

Title: Transferrin mediated transport of iron across rat brain microvascular endothelial cells: an 

EM study 

Authors: M. E. KLINGER 1 , K. DEVRAJ 1 , J. R. CONNOR 2 , A. CARRUTHERS 3 , *I. A. 

SIMPSON 1 ; 

1 Neural & Behav Sci., 2 Neurosurg., Coll Med, Penn State Univ., Hershey, PA; 3 Biochem. and 

Mol. Pharmacol., Univ. of Massachusetts Med. Sch., Worcester, MA 

Abstract: Iron is an essential element for neural cell survival and is involved in numerous 

catalytic reactions and regulatory processes, such as ATP production, myelin and dopamine 

synthesis. The initial uptake of iron at the blood-brain barrier (BBB) is mediated by the 

transferrin receptor in the luminal membrane, which upon binding two molecules of transferrin 

with two molecules of Fe to each, undergoes endocytosis and the iron transferrin complex is 

internalized. The subsequent fate of the receptor, transferrin and Fe within the endothelial cell 

then becomes controversial. In this study, we have monitored the distribution of the transferrin 

receptors in the luminial, abluminal, and intracellular membranes of brain miscrovascular 

endothelial cells in control and Belgrade rats by immunogold electron microscopy. The receptor 

was detected using a mouse monoclonal antibody (Zymed) and visualized with silver enhanced 

1.4 nm gold- labeled goat anti-mouse Fab fragments. In control cells, the ratio of receptors 

between luminal: abluminal: intracellular (L:A:I) is 1:1.2:3.6. The Belgrade rat has autosomal 

recessive hypochromic microcystic anemia associated with a mutation in the metal ion 

transporter DMT-1, which results in a decreased uptake of iron from the gut and impaired release 

of iron from the endocytic vesicles in most other cells. Western blotting of the brain 

miscrovascular endothelial cells from control and Belgrade rats revealed a 50% increase in total 

transferrin receptors in Belgrade rat cells. The EM study demonstrated an L:A:I ratio of 1:1.6:3.4 

in Belgrade rats indicating that the predominant increase in receptors occurs at the abluminal

membrane. This represents the first unequivocal demonstration of abluminal transferrin receptors 

at the BBB and computer modeling of this data suggests that the receptor distribution within the 

cell is closely dependent on the interstitial transferrin concentration. 

Disclosures: M.E. Klinger, None; K. Devraj, None; J.R. Connor, None; A. Carruthers, 

None; I.A. Simpson , None. 

Poster 





Support: Webster Family 

Title: Vascular endothelial growth factor in cerebrospinal fluid of children and adults with 

hydrocephalus 

Authors: *J. R. MADSEN 1 , M. Q. HAMEED 1 , S. CONNORS 2 , S. R. SMITH 2 , G. ABAZI 1 , L. 

FLEMING 1 , P. M. BLACK 3 , J. W. SHIM 1 ; 

1 Dept. of Neurosurg., 2 Vascular Biol. Program, Children's Hosp., Boston, MA; 3 Dept. of 

Neurosurg., Brigham and Women's Hosp., Boston, MA 

Abstract: Background: 

Vascular endothelial growth factor A (VEGF-A) belongs to a sub-family of signalling proteins 

that mediate both vasculogenesis and angiogenesis, and vascular permeability. Although most 

work on VEGF-A activity has looked at its actions on endothelial cell mitosis, migration, and 

permeability, VEGF-A has also been seen to influence a number of other cell types including 

macrophages, kidney epithelial cells, cancer cells and neurons. 

There are over 2500 new cases of hydrocephalus each year in the United States. Current study of 

communicating hydrocephalus focuses on abnormalities in intracranial vascular pulsatility, but 

the underlying pathophysiological mechanisms remain poorly understood. We hypothesized that 

alterations in microvascular pulsatile shear stress might result in increased CSF VEGF-A, which 

would suggest a novel therapeutic strategy not dependent on CSF diversion. 

Methods and Results: 

68 CSF samples were tested for VEGF-A levels using ELISA, and the differences between 

groups analysed for statistical significance using the Mann-Whitney U and Wilcoxon tests. 

VEGF-A levels in 21 CSF aliquots obtained from 15 pediatric hydrocephalus patients 

undergoing surgery were significantly elevated (median: 82 pg/ml; range:

compared to those in 26 CSF aliquots obtained from non-hydrocephalic pediatric patients 

undergoing surgery for unrelated reasons (median:

to wide-band noise at the level of 120 dB for 3 hours/day for two consecutive days, we found 

that the CLB was collapsed. Serum proteins such as H and L chains of IgG and albumin become 

present in the stria vascularis. The instrastrial space was enlarged. Basement membrane between 

the pericyte and the endothelial cell was discontinuous at some points. Pericytes also showed 

multiple abnormalities: (1) Pericytes had an abnormally loose association with endothelial cells, 

with wide spaces separating some regions of two types of cells. In some cases, pericytes‟ long 

processes extended away from the vessel wall; (2) The amount of pericytes structural protein, 

desmin, was substantially increased by 39 % (~12% in normal condition) in the stria vascularis; 

its mRNA was increased by 6.79 ± 2.6 fold; (3) Infiltration of GFP + bone marrow-derived cells 

(GFP + -BMDCs) was observed in the areas of spiral ligament. Some of GFP + -MBDCs expressed 

the pericyte marker protein, desmin, at the sites on or close to vessels. These observations 

indicate that the abnormal relationship of pericytes with endothelial cells may influence the 

endothelium and contribute to the leakness after sound trauma. The increase of pricytes structural 

protein and pericyte recruitment may suggest an early response for maintaining CBL. This work 

was supported by NIH R03 DC 008888. 

Disclosures: X. Shi , Oregon Hearing Research Center (NRC04), A. Employment (full or parttime); 

Department of Otolaryngology and Head & Neck Surgery, Oregon Health & Science 

University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. USA, A. Employment 

(full or part-time). 

Poster 





Title: Assessment of blood-brain barrier damage by microdialysis probe implantation 

Authors: *R. K. SUMBRIA, U. BICKEL; 

TTUHSC, Amarillo, TX 

Abstract: Intracerebral microdialysis is increasingly used as a technique to determine the 

pharmacokinetics of drug uptake by brain. However, it is an invasive technique, and it is crucial 

to answer the question whether blood-brain barrier (BBB) integrity is maintained after probe 

implantation before applying microdialysis as a quantitative tool. Studies done to date gave 

contradicting results, which may in part be due to technical variation in probe implantation and 

in the lag time of the dialysis sampling. We are using a microdialysis technique, which should 

result in the lowest possible BBB damage, because dialysate measurements are performed 24hr

after direct probe implantation, i.e without guide cannula. At this time point, acute tissue damage 

caused by the stereotaxic implantation has declined, while potential long term tissue alteration, 

such as gliosis around the probe, has not yet evolved. A suitable test substance to measure barrier 

breakdown should have a very low permeability at the healthy BBB and not be a substrate of any 

influx or efflux transport system. Hence, to determine the effect of probe implantation on BBB 

integrity, we measured the permeability of a low molecular hydrophilic solute, [ 14 C]sucrose. For 

this, 24 hr after probe implantation, [ 14 C]sucrose (40µCi/animal) was injected intravenously 

followed by collection of plasma samples and brain extracellular fluid (ECF) dialysate samples 

for different time periods. The permeability surface area product (PS) of [ 14 C]sucrose at the BBB 

was calculated based on dialysate values, and compared to values determined by the „classical‟ 

method of whole tissue sampling. Within the time period covered in our study (30 min), we 

found that the PS product of [ 14 C]sucrose determined by microdialysis (0.37 ± 0.1 µl/min/gm) 

was not significantly different from that determined by the classical method (0.34 ± 0.02 

µl/min/gm), suggesting that microdialysis probe implantation does not result in significant BBB 

leakage to a poorly permeable drug-like substance. 

Disclosures: R.K. Sumbria , None; U. Bickel, None. 

Poster 



Program#/Poster#: 287.7/RR1 


Support: CONACyT SEP-2004-C01-48002 

Title: Ultrastructural vascular permeability changes in the brain circumventricular organs of 

adult rats, in response to electromagnetic fields (120Hz harmonic waves and 0.66mT rms) 

Authors: *Y. K. GUTIERREZ-MERCADO 1 , L. CAÑEDO-DORANTES 3 , J. BAÑUELOS- 

PINEDA 2 , G. SERRANO 3 , A. FERIA-VELASCO 1 ; 

1 Cell Biol. and Mol., 2 Vet., Univ. Guadalajara, Zapopan Jalisco, Mexico; 3 Medicin, Autonomous 

Univ. of Morelos, Cuernavaca Morelos, Mexico 

Abstract: A limited number of studies dealing with the effects of electromagnetic fields (EMF) 

have been reported, mainly referring to microcirculation and microvasculature related to 

pressure. There are few reports on the effects of EMF on vascular permeability of brain blood 

vessels and no studies have been published as the evaluation of the effects of EMF on vascular 

permeability to non-liposoluble substances of some CVO of the adult rat, at light microscopy and

transmission electron microscopy (TEM) level. 

EMFs have been used for years at the clinic as an adjunct in the treatment of diseases or in the 

presence of ulcers well in bones, acting as a promoter of angiogenesis and this facilitates the 

recovery of ulcers, bones or injured tissues with little blood circulation. 

Male Wistar adult rats were maintained under controlled conditions with free access to food and 

water. Three groups of 32 animals each were carefully handled according the Mexican Health 

Laws and the National Institutes of Health (USA) Guidelines for the Care and Use of Laboratory 

Animals. 

In the experimental group (E) the animals were exposed to an electromagnetic field of 0.66mT 

rms, a frequency of 120 Hz harmonic waves. The Helmholtz coils where the unanesthetized 

animals were placed in a specially designed plastic cage. Exposure was for 2h daily for 7 days, 

after 3 days habituation with no connection of the coils. In the sham exposed group (S) the 

animals were treated similarly to those of the E group, but with no connection of the coils 

throughout the experiments. In the control group (C) the animals were intact. All animals were 

injected with 2 ml/Kg (v/w) colloidal carbon (CC) solution through the femoral vein and killed 

by intraperitoneal injection of 50 mg/Kg (w/w) pentobarbital at 0, 1, 5 and 10 min after the CC 

injection. The brain was fixed by immersion in 0.1M phosphate buffered-4% paraformaldehyde 

solution. Circumventricular organs, sensori-motor cerebral cortex (SMCC) and hippocampus (H) 

were dissected out to be embedded in epoxy resins. 0.5 κm-thick sections were stained with 

toloudin blue to be examined under a light microscope equipped with a professional image 

analysis system and 0.1κm-thick sections were stained with uranyl acetate-lead citrate to be 

examined under TEM. 

EMF-120Hz increased the CC-extravasation to parenchyma-intercellular space, also, cc was 

observed in the intercellular unions and vesicles, which join to each other, forming a system of 

pores or channels in endothelial cells, in fenestrae cells, in basal membrane, pericapillary space 

and intercellular spaces of CVO, this probably due to morphological changes in capillaries or 

another mechanism not even described. 

Disclosures: Y.K. Gutierrez-Mercado, None; J. Bañuelos-Pineda, None; G. Serrano, 

None; L. Cañedo-Dorantes, None; A. Feria-Velasco, None. 

Poster 




Topic: C.07.b. Neurovascular unit 

Title: Is there a relationship between neuronal and vascular protection in experimental 

glaucoma?

Authors: *M. ALMASIEH 1 , N. ZABOURI 2 , C. CASANOVA 2 , M. KELLY 3 , A. DI POLO 1 ; 

1 Pathol & Cell Biol, Univ. Montreal, Montreal, QC, Canada; 2 Sch. of optometry, Univ. of 

Montreal, Montreal, QC, Canada; 3 Dept. of Pharmacol., Dalhousie Univ., Halifax, QC, Canada 

Abstract: Purpose: A correlation between high intraocular pressure (IOP) and retinal ischemia 

has been established, but little is known about the relationship between neuronal and vasculature 

degeneration in glaucoma. We previously demonstrated that the acetylcholinesterase inhibitor 

galantamine protects retinal ganglion cells (RGCs) in glaucoma. Here, we asked whether 

galantamine-induced neuroprotection correlates with changes in the retinal microvasculature. 

Methods: Chronic elevation of IOP was induced in Brown Norway rats by injection of a 

hypertonic saline solution (1.85 M NaCl) into an episcleral vein. Treatment began on the first 

day of IOP increase by daily dose (3.5 mg/kg, i.p.) of galantamine. Structural neuroprotection 

was carried out by quantification of RGC soma and axons. The retinal vasculature was visualized 

by lectin immunohistochemistry or dextran, and the density of retinal capillaries was quantified. 

In addition, we measured the electroretinogram response in intact and glaucomatous eyes 

following galantamine or vehicle treatment. 

Results: At 5 weeks after ocular hypertension surgery, we observed RGC death accompanied by 

a dramatic reduction in the density of the retinal microvasculature. Galantamine-mediated 

protection of RGCs (69%, n=16) correlated with marked preservation of capillaries in the retinal 

fiber layer (70%, n=8) compared to vehicle (37% RGCs, n=14; 40% vessels, n=6). No changes 

in the choroidal vasculature were observed between intact and glaucomatous eyes in the presence 

or absence of galantamine. Consistent with this, the a-wave component of the ERG was not 

affected by high IOP. In vitro experiments using isolated retinal microvasculature demonstrated 

that galantamine did not induce vessel contraction. When the vasculature was pre-treated with 

galantamine (1-5 κM) and endothelin, no significant change in endothelin-mediated contraction 

was observed. 

Conclusions: Our data indicate that galantamine-mediated neuroprotection in glaucoma 

correlates with vasculature preservation. Our findings that galantamine by itself did not have an 

effect on isolated vessels suggests that neuroprotection is required for the preservation of the 

retinal vasculature or that the retinal microvasculature is an indirect target for galantamine‟s 

protective effects. 

Disclosures: M. Almasieh , None; N. Zabouri, None; C. Casanova, Canadian Institutes of 

Health Research, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); A. Di Polo, Canadian Institutes of Health 

Research, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); M. Kelly, None. 

Poster 




Topic: C.07.b. Neurovascular unit 

Title: Tissue inhibitor of metalloproteinases protect blood brain barrier disruption in focal 

cerebral ischemia 

Authors: *M. FUJIMOTO, Y. TAKAGI, N. HASHIMOTO, K. NOZAKI; 

Kyoto Univ., kyoto, Japan 

Abstract: Enhanced matrix metalloproteinases (MMPs) can cause vasogenic edema and 

hemorrhagic transformation after cerebral ischemia, and affect the extent of ischemic injury. We 

hypothesized that the endogenous MMP inhibitors, tissue inhibitor of MMPs (TIMPs), were 

essential to protect against blood brain barrier (BBB) disruption after ischemia by regulating the 

activities of MMPs. We confirmed the transition of MMP-2 and -9, and the TIMPs family after 

30 min middle cerebral artery occlusion, and elucidated the function of TIMP-1 and -2 in focal 

ischemia, using TIMP-1 -/- and TIMP2 -/- mice. TIMP-1 mRNA expression was gradually 

increased until 24 h after reperfusion. In TIMP-1 -/- mice, MMP-9 protein expression and 

gelatinolytic activity after cerebral ischemia were significantly more augmented than those in 

WT mice, and were accompanied by exacerbated BBB disruption, neuronal apoptosis, and 

ischemic injury. In contrast, TIMP-2 gene deletion mice exhibited no significant difference in 

MMPs expression and degree of ischemic injury, despite an increased Evans blue leakage. These 

results suggest that TIMP-1 inhibits MMP-9 activity and can play a neuroprotective role in 

cerebral ischemia. 

Disclosures: M. Fujimoto , None; Y. Takagi, None; N. Hashimoto, None; K. Nozaki, None. 

Poster 





Title: Critical role of brain temperature in regulating blood-brain barrier permeability 

Authors: *E. A. KIYATKIN 1 , H. S. SHARMA 2 ; 

1 Behavioral Neurosci Br., NIDA-IRP, NIH, DHHS, Baltimore, MD; 2 Univ. Hosp., Uppsala 

University, Sweden

Abstract: Previous studies have shown that various conditions associated with brain 

hyperthermia (environmental warming, stress or intense physical exercise, methamphetamine 

intoxication, opiate withdrawal, etc.) are accompanied by an increased blood-brain barrier (BBB) 

permeability. Although these data implicate hyperthermia as a leading factor affecting trans-BBB 

transport, its exact role remains hypothetical due to many other contributors (i.e., metabolic 

activation, oxidative stress, alterations in cerebral blood flow, hypoxia, etc.). The goal of this 

study was to clarify the role of brain temperature in regulating BBB permeability by delineating 

this physical factor from other potential contributors. To reach this goal, all rats were equally 

anesthetized (sodium pentobarbital 50 mg/kg) and then their bodies were passively warmed, 

resulting in graded differences in brain and body temperatures (range, 32-42°C with no to intense 

warming) when brains were taken for subsequent evaluations. These evaluations included 

albumin- and glial fibrillary acidic protein (GFAP) immunoreactivity, water and ion (K+, Na+, 

Cl-) contents, and cellular abnormalities (Nissl staining) that were quantified in the cortex, 

hippocampus, thalamus and hypothalamus as well as in different cortical areas. We found that 

the number of albumin-positive cells strongly correlates with brain temperature, gradually 

increasing from ~38.5°C and becoming saturated at 41-42°C. A similar dependence was found 

for GFAP, an index of acute glial activation. While evident in each tested structure, alterations in 

both parameters showed some structural specificity. Brains maintained at hyperthermia also 

showed morphological cell abnormalities; these alterations were seen at ~39°C, gradually 

progressed with further temperature increase, and peaked at high hyperthermia. Temperaturedependent 

changes in various brain parameters, moreover, were tightly correlated (albumin- 

GFAP, albumin-water, albumin-structural abnormalities). Therefore, brain hyperthermia per se is 

an important factor in BBB breakdown, development of brain edema, and subsequent structural 

abnormalities of brain cells. 

Supported by NIDA-IRP and DISCA-2007 award (SHS). 

Disclosures: E.A. Kiyatkin, NIDA-IRP, NIH, DHHS, A. Employment (full or part-time); H.S. 

Sharma, None. 

Poster 





Support: NIH GRANT RO-1 NS41405 

Title: Polarization of glucose transporters at the bovine blood-brain barrier: a proteomics 

approach

Authors: *K. DEVRAJ 1 , A. MOKASHI 2 , R. A. HAWKINS 2 , I. A. SIMPSON 1 ; 

1 Neural & Behavioral Sci., Penn State Univ., Hershey, PA; 2 Dept. of Physiol. and Biophysics, 

The Chicago Med. School, Rosalind Franklin Univ. of Med. and Sci., North Chicago, IL 

Abstract: Glucose is the primary metabolic fuel for the adult brain. It is known to be transported 

across the blood-brain barrier (BBB) by the action of facilitative GLUT-1 glucose transporters. 

The blood-brain barrier is a physiological barrier made up of the brain capillary endothelial cells 

joined by tight junctions with adjacent cells with support from astroglia, pericytes, and neurons 

of the central nervous system. Under the current project, we have isolated the individual 

membranes of the bovine BBB plasma membranes viz. the luminal (blood-side) and abluminal 

(brain-side) membranes. Earlier studies revealed a differential sensitivity to two distinct 

antibodies to GLUT-1. H-sera raised against entire human erythrocyte GLUT-1 glucose 

transporter recognized luminal and abluminal equally whereas the C-terminal antibody raised 

against 14 amino acid C-terminal sequence recognized abluminal with a three fold greater 

efficiency. We hypothesized this as resulting from a masking of the luminal GLUT-1 in the Cterminus. 

To this end, we performed 2-D gel electrophoretic separations of both luminal and 

abluminal membranes and eventually blotted with GLUT-1 H-sera and C-terminal antibodies. 

The objective was to identify the GLUT-1 species that were responsible for differential 

recognition in luminal and abluminal membranes. Our results indicate that luminal and 

abluminal membrane GLUT-1 have different isoelectric points (P.I) possibly resulting from 

differential phosphorylation and or o-glycosyaltion. We ruled out N-glycosylation as these 

luminal and abluminal membranes even after treatment with PNGaseF enzyme for Ndeglycosylation 

still preserved differences in P.I. for GLUT-1. We are currently utilizing the 

approach of Western blotting to locate various species followed by on-membrane digestion with 

chymotrypsin, and finally removal of nitrocellulose before performing MALDI MS/MS Mass 

Spectrometric identification of the peptide isoforms/potential post-translational modification(s). 

Disclosures: K. Devraj, None; I.A. Simpson, None; A. Mokashi, None; R.A. Hawkins, None. 

Poster 





Support: POCTI/SAU-NEU/56618/2004 from Fundação para a Ciência e Tecnologia (Portugal). 

Title: Altered iron metabolism is part of the choroid plexus response to peripheral inflammation

Authors: F. MARQUES 1 , A. L. FALCÃO 1 , J. C. SOUSA 1 , G. COPPOLA 3 , D. GESCHWIND 3 , 

N. SOUSA 1 , M. CORREIA-NEVES 1 , *J. A. PALHA 2 ; 

1 Life and Hlth. Sci. Res. Inst. (ICVS), Sch. of Hlth. Sci., 2 Hlth. Sci. Sch., Univ. of Minho, Braga, 

Portugal; 3 Dept. of Neurology, David Geffen Sch. of Med., UCLA, Los Angeles, CA 

Abstract: Iron is essential for normal mammalian cellular homeostasis but, in excess, promotes 

free radical formation and its accumulation has been associated with some neurodegenerative 

disorders. Iron is also required for microbial growth and in case of infection the host activates 

several mechanisms to prevent iron availability for bacteria. We have recently shown that the 

choroid plexus can contribute to the brain innate immune response by synthesizing and secreting 

lipocalin 2 into the cerebrospinal fluid (CSF). Here, we show the involvement of other genes in 

regulating iron homeostasis in the brain. We now show that hepcidin (HAMP) mRNA levels are 

up-regulated in the choroid plexus after intraperitoneal administration of lipopolysaccharide. 

HAMP is known to be a liver-derived hormone that regulates iron availability by decreasing 

ferroportin (FPN)-mediated iron export from the enterocyte into the bloodstream. Other genes 

also involved in the regulation of iron metabolism are up-regulated by the peripheral 

inflammatory stimulus. We propose that upon an acute peripheral inflammatory stimulus HAMP 

decreases FPN-mediated iron delivery into the CSF, therefore decreasing iron availability for 

bacterial growth and dissemination within the brain. 

Disclosures: F. Marques, None; J.A. Palha , None; A.L. Falcão, None; J.C. Sousa, None; M. 

Correia-Neves, None; N. Sousa, None; D. Geschwind, None; G. Coppola, None. 

Poster 





Support: The Roy J. Carver Charitable Trust 

Title: Localization of gamma-protocadherin proteins to the apical surface of choroid plexus and 

ependymal epithelial cells suggests novel functions distinct from cell-cell adhesion 

Authors: *J. A. WEINER, L. F. HELSPER; 

Dept Biolog Sci., Univ. Iowa, Iowa City, IA 

Abstract: Protocadherins (Pcdhs) comprise a large family of putative adhesion molecules related 

to the well-studied, homophilic classical cadherins. Genes encoding ~60 Pcdh molecules are

arranged in three clusters (Pcdh-α, -β, and -γ) at a single chromosomal locus in mammals. 

Protocadherins derived from each of these clusters are combinatorially expressed by developing 

neurons, in which they localize preferentially to synaptic and perisynaptic sites. Although they 

contain 6 extracellular cadherin domains, there is little evidence that the clustered Pcdhs mediate 

strong cell-cell adhesion in the nervous system, suggesting that they may have other cellular 

functions. In the course of our analysis of the 22-gene Pcdh-γ cluster, we found that γ-Pcdh 

proteins are expressed by epithelial cells of the choroid plexus and ependyma at levels higher 

than those in neurons or astrocytes. Surprisingly, γ-Pcdh proteins do not localize to the adherens 

junctions that are formed basolaterally between adjacent epithelial cells, but are instead tightly 

restricted to the apical membrane. Whole-mount immunostaining reveals that γ-Pcdh proteins 

localize throughout most of the apical membrane of developing and adult choroid plexus 

epithelia, but are excluded both from the tufts of cilia that project from this membrane, as well as 

from the tight junctions that separate it from the basolateral membrane. RT-PCR analysis of 

isolated choroid plexus RNA detects transcripts encoding many, but not all, of the 22 Pcdh-γ 

genes, as well as members of several other protocadherin families, including Fat, Dachsous, and 

Celsr, that have been implicated in planar cell polarity. Because the apical surface of ependymal 

and choroid plexus epithelial cells is exposed only to cerebrospinal fluid, and not to other cells, 

the restricted localization we observe suggests that the γ-Pcdhs have novel, non-adhesive 

functions in these cell types. Because Pcdh-γ null mutants die at birth, precluding many 

experiments, we are currently testing this hypothesis by utilizing a new, loxP-flanked conditional 

mutant allele (Pcdh-γ con ) and a transgenic mouse line expressing Cre specifically in ciliated 

epithelial cells. 

Disclosures: J.A. Weiner , None; L.F. Helsper, None. 

Poster 

288. Working Memory: Disorders, Genes and Connectivity 



Topic: F.01.f. Working memory 

Support: University Research Foundation 

Mind and Life Varela Award 

Title: Mindfulness training improves working memory performance in adults with ADHD 

Authors: Z. B. ROSEN 1 , M. J. BAIME 2 , J. R. RAMSAY 3 , A. ROSTAIN 4 , K. K. 

SREENIVASAN 1 , *A. P. JHA 5 ;

1 Dept. of Psychology, 2 Penn Program for Stress Mgmt., 3 Ctr. for Cognitive Therapy, 4 Dept. of 

Psychiatry, Univ. of Pennsylvania, Philadelphia, PA; 5 Ctr. Cognitive Neurosci, Univ. 

Pennsylvania, Philadelphia, PA 

Abstract: Mindfulness training (MT) emphasizes the development and cultivation of attention 

and affective regulation skills. We have recently demonstrated that MT improves adults‟ ability 

to attentionally select perceptual information (Jha, Krompinger, and Baime, 2007). Our previous 

studies of healthy adults suggest that attentional selection during perceptual and mnemonic tasks 

share common mechanisms (Sreenivasan and Jha, 2007) and that the flexible recruitment of 

attention to high selection demands results in improved selection on the subsequent trial. In the 

current study we investigate whether MT may improve the ability to 1) select mnemonic 

information during a working memory task and 2) flexibly allocate attention to aid mnemonic 

selection. Specifically, our interest was in determining if patients with attention deficit 

hyperactivity disorder (ADHD) who have dysfunction in both perceptual and mnemonic 

selection may benefit from MT. Two groups of patients were screened for ADHD and enrolled in 

an 8-week MT course, and tested twice at an 8 week interval on a delayed-recognition working 

memory task. The wait list control group (n = 11) received the training following the second 

testing session while the experimental group (n = 9) received the training between the two 

sessions. The task required patients to remember two faces or scenes over a brief delay and to 

indicate whether a probe image matched the memorandum. During the delay, two distractor 

images were presented that either matched the category of the memorandum (congruent trial) or 

did not match the category of the memorandum (incongruent trial). We predicted that MT would 

improve mnemonic selection ability in the experimental group and therefore improve 

performance on congruent trials. We further predicted that MT would improve the flexible 

recruitment of attention, and that this would result in working memory performance 

improvements following congruent high demand trials in the experimental group relative to the 

control group. Performance on congruent trials did not improve for either group. However, 

performance did improve on trials following congruent high demand trials for the experimental 

group relative to the control group (group x session x previous congruency interaction). Thus, 

our results suggest that MT may improve the ability to flexibly recruit attention during working 

memory, in a manner similar to the improvements in perceptual-level attentional selection 

observed previously. Further, these pilot results suggest that MT may be a useful 

nonpharmacologic treatment for patients with ADHD to improve working memory. 

Disclosures: Z.B. Rosen, None; M.J. Baime, None; J.R. Ramsay, None; A. Rostain, 

None; K.K. Sreenivasan, None; A.P. Jha , None. 

Poster 





Title: Effects of subthalamic nucleus stimulation on motor disability and cognition in 

Parkinson‟s disease 

Authors: *M. E. MAPSTONE 1 , K. SELZLER 2 , F. MARSHALL 1 , C. ZIMMERMAN 1 , J. 

SCHWALB 3 ; 

1 Neurol., 2 Neurobio. and Anat., 3 Neurosurg., Univ. of Rochester Sch. of Med., Rochester, NY 

Abstract: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) significantly 

ameliorates Parkinson‟s disease (PD)-related motor disability. Relatively little is known about 

DBS‟s effect on non-motor functions including cognition and mood. These non-motor effects 

may have great clinical relevance as evidenced by recent reports of increased impulsive 

behaviors including suicide and compulsive gambling following STN stimulation for PD. The 

STN is a critical node for basal ganglia output to multiple cortical regions including the frontal 

cortex, which is involved in cognitive functions that might also be affected by STN DBS. We 

tested the hypothesis that STN stimulation for PD-related motor disability also affects cognitive 

functions supported by these frontal cortical regions. We chose to focus on working memory 

which relies on dorsolateral prefrontal cortical regions. We tested 13 PD patients (mean age= 

61.2 years, SD=9.6) with bilateral STN DBS electrodes on a measure of nonverbal working 

memory and measures of motor speed (Finger Tapping) and motor disability (Unified 

Parkinson‟s Disease Motor Score). Our working memory measure was the Self-Ordered Pointing 

Test in which subjects select an object from an array of line drawings on a computer monitor . 

On each subsequent trial, the spatial locations of the items are rearranged and the subject must 

pick an item not picked previously. All subjects were tested with stimulator “on” and “off” on 

the same day, at least 1 month post-op, at optimal stimulation parameters for motor benefit, and 

off L-dopa treatment for at least 12 hours. We used the Wilcoxon signed-rank test to examine 

effects of stimulation (on vs off) on the dependent measures of working memory, motor speed 

and motor disability. As expected, STN stimulation significantly ameliorated motor disability 

(Z= -3.1, p=.002) and there was a trend for significant increase in motor speed in the nondominant 

hand (Z = -1.82, p = .069). In contrast, STN stimulation interfered with working 

memory performance, but only in the more demanding task condition (Z = -2.481, p = .013). Our 

results suggest that STN stimulation affects functional capacities reliant on both motor and nonmotor 

fronto-striatal circuits, however the effects on cognitive functions may be observed only 

when the cognitive task demands are high. 

Disclosures: M.E. Mapstone , None; K. Selzler, None; F. Marshall, Prestwick 

Pharmaceuticals, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); Teva Pharmaceuticals, D. Speakers 

Bureau/Honoraria (speakers bureau, symposia, and expert witness); Medtronics, Other; ANS 

Microsystems, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); C. Zimmerman, None; J. Schwalb, None.

Poster 





Support: NIH T32 MH019879 (AK) 

NARSAD Young Investigator Award (JWB) 

Title: The influence of working memory on error-likelihood prediction in the anterior cingulate 

cortex and its disturbance in schizophrenia 

Authors: *A. KRAWITZ 1 , T. S. BRAVER 2 , D. M. BARCH 2 , J. W. BROWN 1 ; 

1 Psychological & Brain Sci., Indiana Univ., Bloomington, IN; 2 Psychology, Washington Univ., 

St. Louis, MO 

Abstract: Recent research has shown that anterior cingulate cortex (ACC) modulates the degree 

to which top-down cognitive control systems, including working memory (WM) in dorsolateral 

prefrontal cortex (DLPFC), are brought to bear in particular task contexts. Specifically, 

according to the error-likelihood model (Brown & Braver, 2005), the magnitude of ACC output 

in a given situation is proportional to the perceived probability of making an error. It has 

remained unknown whether ACC may use WM contents to guide error-likelihood prediction and 

thus complete a reciprocal loop between DLPFC and ACC, as predicted by recent work with the 

error-likelihood model. Furthermore, past studies suggest that the cognitive impairment found in 

schizophrenia involves WM-related deficits in DLPFC and performance monitoring deficits in 

ACC, but the impact of these deficits on the reciprocal interaction between the two areas remains 

unclear. 

We investigated whether information in WM is used as context by ACC to inform its evaluation 

of error likelihood in schizophrenia patients and in non-psychiatric controls. In a modified 

version of the change-signal task of Brown and Braver (2005), a cue predicting error likelihood 

was incidentally encoded into WM by participants in order to perform a secondary delayed 

match-to-sample (DMTS) task. Following the cue, a blank-screen delay period occurred prior to 

the change-signal trial. Using a rapid event-related fMRI design with partial trial decomposition, 

we looked for error-likelihood related activity at the time of cue presentation and at the time of 

response to the change-signal target. 

In controls, we found an area of ACC whose activation at the response predicted error likelihood 

as indicated by the initial cue, even for trials without errors or response conflict. This area did 

not show error-likelihood related activity at the cue presentation. This illustrates that, in controls,

ecent but currently unavailable contextual information, presumably maintained in WM, is used 

by ACC in evaluating error likelihood during task performance. However, schizophrenia patients 

showed significantly less error-likelihood related activity in ACC at the response. In fact, in 

schizophrenia patients, we did not find ACC error-likelihood activity at the target response or at 

the cue presentation. This was the case even for a subset of patients matched with controls on 

DMTS performance and WM-related activation in DLPFC. This suggests a disturbance in the 

use of contextual information stored in WM for the evaluation of error likelihood in ACC. It 

further suggests that this disturbance is specific to ACC and not simply due to a deficit in WM 

maintenance. 

Disclosures: A. Krawitz , None; T.S. Braver, None; D.M. Barch, None; J.W. Brown, None. 

Poster 





Support: NIH R01 MH61625 

National Science Foundation Graduate Research Fellowship 

National MS Society SF1752-A-1 

Title: Neural plasticity resulting from task repetition is reduced in multiple sclerosis 

Authors: *C. A. MONTOJO 1 , S. SAYALA 1 , S. M. COURTNEY 1,2,3 ; 

1 Psychological & Brain Sci., 2 Dept. of Neurosci., Johns Hopkins Univ., Baltimore, MD; 3 FM 

Kirby Res. Ctr. for Functional Brain Imaging, Kennedy Krieger Inst., Baltimore, MD 

Abstract: Neural plasticity following repeated performance of a working memory (WM) task 

has been shown previously in healthy young adults, with observed decreases in fMRI activation 

interpreted as reflecting increased neural efficiency resulting from short-term practice. Both 

older adults and individuals with multiple sclerosis (MS) often show increased fMRI activation 

compared to controls when the data is averaged across a scanning session. This overall greater 

activation has been interpreted alternately as reflecting either compensatory neural plasticity or 

inefficient neural processing. In order to better understand the nature of such examples of neural 

plasticity and changes in efficiency, we examined whether individuals with MS and age-matched 

controls show the same pattern as young healthy adults of decreasing fMRI activation during

epetition of a WM task. Participants performed a series of runs that included either spatial WM 

task repetition (1 run object, 5 spatial, 2 object, 1 spatial) or object WM task repetition (1 run 

spatial, 5 object, 2 spatial, 1 object). 

MS patients show similar decreases in neural activity as healthy adults within an inferior frontal 

region during object task repetition. During spatial repetition in the superior frontal region, 

however, MS patients show no change in activity while healthy adults again show decreased 

activity. There were no regions in healthy adults that showed increasing activation with task 

repetition, but the MS patients showed increasing activation in a posterior parietal region. 

Behavioral results show no change in performance with task repetition for either patients or 

controls during the 30 minute repetition period, suggesting that decreases in neural activity may 

be interpreted as increases in neural efficiency. 

These preliminary data suggest that at least in some cortical regions, MS results in a decreased 

ability to alter activation efficiency with task repetition. Short-term neural plasticity may require 

precisely timed interactions within orderly networks. The demyelination caused by MS may 

disrupt these mechanisms. These results also suggest that previous results showing greater 

activation (averaged across the entire scan session) for MS patients compared to healthy adults 

may be due to a lack of decrease during repeated performance of a task rather than an overall 

increase in activation. 

Disclosures: C.A. Montojo, None; S.M. Courtney, None; S. Sayala, None. 

Poster 





Title: Effects of childhood lead exposure on working memory: a functional MRI study 

Authors: *J. S. SCHNEIDER 1 , J. LACKEY 2 , J. SHI 2 , S. LAI 2 ; 

1 Dept Pathol, Anat. & Cell Biol, 2 Radiology, Thomas Jefferson Univ., Philadelphia, PA 

Abstract: The neurotoxic effects of environmental lead exposure in children have been 

recognized for over a century, but the mechanism of lead‟s toxicity on the developing central 

nervous system remains poorly understood. While lead‟s effects on neurocognitive development 

are obviously mediated by its neurotoxic effects on the developing brain, there has been no study 

of how childhood lead exposure may alter brain function, manifested as brain activation levels 

and/or patterns, during performance of tasks that involve working memory. Working memory or 

the ability to hold and manipulate information on-line is an essential aspect of higher cognitive

processes such as language, planning, and problem solving. Additionally, the ability to represent 

and manipulate spatial and non-spatial information is a key requirement of everyday cognition 

and is an important component of a variety of cognitive skills, including reading and logical 

reasoning. The aim of this project is to obtain information concerning the effect of childhood 

lead poisoning on the integrity of neural circuits involved in spatial and non-spatial working 

memory and to correlate these data with extent of childhood lead exposure. In the present 

preliminary study, using functional MRI at 3T, we begin to examine the effect of childhood lead 

exposure on working memory by studying patterns and extents of working memory-related 

neural activation in adolescents with a documented history of different levels of childhood lead 

exposure. Brain fMRI scans were obtained on adolescents between the ages of 13 and 17 years, 

with documented histories of childhood blood lead testing prior to the age of 3 years, during 

performance of 1-back and 2-back spatial and non-spatial working memory tasks. A number of 

brain regions showed significant activation during performance of the 1-back and 2-back tasks. 

Preliminary analyses indicate negative correlations between level of lead exposure and activation 

in several frontal and parietal regions in both hemispheres in the 2-back working memory 

conditions, while no significant negative correlation was observed between level of lead 

exposure and activation in the 1-back working memory conditions. In addition, negative 

correlations between activation and lead exposure were found more frequently in the 2-back 

condition for spatial working memory. These preliminary findings suggest that prior childhood 

lead exposure has persistent effects on the brain and alters brain activation during working 

memory performance. 

Disclosures: J.S. Schneider, None; J. Lackey, None; J. Shi, None; S. Lai, None. 

Poster 





Title: The hippocampus and short-term memory for auditory sequences in children 

Authors: *E. CASTRO-SIERRA 1 , F. CHICO-PONCE DE LEON 2 , A. FERNANDEZ- 

STEFANO 3 , A. VARGAS-RODRIGUEZ 3 , S. MARTINEZ-RODIGUEZ 4 ; 

1 Lab. of Psychoacoustics, Hosp. Infantil de Mexico FG, Mexico DF, Mexico; 2 Dept. of 

Neurosurg., 3 Dept. of Psychiatry, 4 Dept. of Social Work, Hosp. Infantil de Mexico Federico 

Gomez, Mexico, DF, Mexico

Abstract: Many experimental memory tasks which are sensitive to hippocampal dysfunction 

show a lack of temporal contiguity lasting for more than 100 mseg between event presentations. 

At hippocampal CA3 region there is a pattern of recurrent excitatory connections between 

pyramidal cells which provides for potential associations within this structure and with the 

auditory cortex. Given connections between A1 and hippocampus, it is important to know which 

types of auditory short-term mnemonic associations are more appropriate for analysis at CA3 

and other hippocampal regions. 

Seven patients (5 F, 2 M; ages 10:6 to 16:9) with tumors or vascular malformations of different 

temporal regions and their controls (36 F, 29 M; ages 10:2 to 16:9) submitted to a Test of 

Frequency Discrimination between tones, a Test of Tonal Memory of 5-tone sequences and a 

Test of Pitch Perception of (Spanish) Language segments. Simultaneous cognitive evaluation of 

two patients with right or left hippocampal pathology and their personal controls using WISC-R 

(Spanish) and Rey‟s test was carried out. 

Data obtained point at an involvement of area around A1 in frequency discrimination between 

tones. Thus, a subject with a tumor affecting left A1 area connections had marked deficits in 

frequency discrimination. Moreover, subjects with tumoral or vascular lesions further away from 

A1, or the left operculum and basal nuclei, also manifested low frequency discrimination. On the 

other hand, a subject with a right fronto-temporal cyst, a subject with a right posterior cerebral 

artery malformation and a subject with a left hippocampal astrocytoma were not affected in 

frequency discrimination. However, the two latter subjects, with differing cognitive evaluations 

based on results from their psychological testing, were affected in tonal sequence memory. 

Interestingly, the younger subjects also showed deficits in perceiving the continuous pitch 

deflections of language. 

In conclusion, results point at an involvement of the hippocampal area in the association of 

complex auditory events in short-term memory of children. Subjects with other temporal 

pathology manifesting low or very low performance in frequency discrimination also had low 

performance in short-term tonal memory. Hannula & Ranganath (2008) have provided evidence 

regarding the importance of the activity of hippocampal structures in short-term relational 

memory binding in vision. From the data of the present study, it would appear that similar 

relational processes binding pitches in short-term auditory memory may take place in the 

hippocampus.. 

Disclosures: E. Castro-Sierra, None; F. Chico-Ponce de Leon, None; A. Fernandez-Stefano, 

None; A. Vargas-Rodriguez, None; S. Martinez-Rodiguez, None. 

Poster 



Program#/Poster#: 288.7/RR14


Support: Alzheimers and Related Diseases Research Award Fund, Virginia Center on Aging 

NIH grants (AG 19653 and others) 

Title: The effect of COMT Val158Met on human working memory depends on age, task 

difficulty and memory load 

Authors: *M. LIN 1 , R. SUNDARARAJAN 2 , P. M. GREENWOOD 3 , R. PARASURAMAN 3 , 

K. J. FRYXELL 2 ; 

1 Bioinformatics, 2 Mol. and Microbiol, George Mason Univ., Manassas, VA; 3 Psychology, 

George Mason Univ., Fairfax, VA 

Abstract: The age-related decline in working memory performance has been attributed to 

decline in dopaminergic functioning (Braver & Barch, 2002). Normal variation in genes 

controlling enzymes degrading dopamine in the synaptic cleft could affect enzyme efficiency, 

thereby exerting greater effects in the older brain. The COMT Val158Met polymorphism shown 

to influence rate of dopamine methylation in prefrontal cortex (Tunbridge et al., 2004) and has 

been found to affect working memory and executive performance (Weinberger, 2005). We asked 

whether normal aging would alter effects of the COMT SNP on spatial working memory 

performance. The task required retention of up to 3 target dot locations for 3 sec. Memory was 

assessed in the accuracy of a same/different decision indicating whether the probe dot and the 

target dot were in the same location. There were 4 target-probe distances (differing in difficulty, 

one match, and 3 non-match based on different target-probe distance) and 3 levels of memory 

load (number of target locations). Factor analysis applied to working memory accuracy identified 

three latent factors correlated with different experimental conditions: (1) match trials; (2) nonmatch 

hard discrimination trials; (3) non-match easy discrimination trials. Mixed repeatedmeasures 

ANOVAs assessed the influence of target-probe distance and memory load on 

accuracy, as well as their interactions with COMT genotype and age. The influence of 

discrimination difficulty on accuracy varied with COMT genotype (p < 0.001) and age group (p 

< 0.001). The influence of memory load on accuracy varied with COMT genotype (p = 0.01) but 

not with age group (p = 0.22). 

Follow up mixed repeated-measures ANOVA on each factor assessed the influence of memory 

load, COMT genotype, and age on accuracy. On match trials, the trend of decreasing accuracy 

with increasing memory load was observed in young adults across COMT genotypes but not 

observed in older adults with COMT Val/Val and Met/Met genotypes. On non-match hard 

discrimination trials, COMT genotype and age did not change the effect of memory load on 

accuracy. On non-match easy discrimination trials, COMT Val/Val performed better than the 

Val/Met and Met/Met in both young and older adults across three memory loads. These results 

confirm and extend our understanding of the role for genetic variation in dopamine availability 

on working memory performance in humans. 

References 

Braver, T.S., Barch, D.M., 2002. Neurosci. Biobehav. Rev. 26, 809-817. 

Tunbridge, E.M. et al. 2004. J Neurosci. 24, 5331-5335. 

Weinberger, D.R. 2005. Clin Ther. 27 Suppl A, S8-S15.

Disclosures: M. Lin, None; R. Sundararajan, None; P.M. Greenwood, None; R. 

Parasuraman, None; K.J. Fryxell, None. 

Poster 





Title: Brain-derived neurotrophic factor val 66 met polymorphism differentially affects regional 

cerebral blood flow during working memory and rest 

Authors: *S.-M. WEI, A. PADMANABHAN, K. V. ROE, P. D. KOHN, B. S. KOLACHANA, 

D. R. WEINBERGER, K. F. BERMAN; 

NIH, Bethesda, MD 

Abstract: The Val 66 Met single nucleotide polymorphism (SNP) in the human brain-derived 

neurotrophic factor (BDNF) gene influences hippocampal function, with the met allele being 

associated with abnormal hippocampal recruitment during episodic and working memory 1,2 . To 

further investigate the effects of this BDNF SNP on hippocampal and cortical function, we used 

H2 15 O positron emission tomography to assess regional cerebral blood flow (rCBF) as a function 

of BDNF genotype during rest as well as during a working memory task and a matched 

sensorimotor control. 

Seventy-one healthy right-handed Caucasians (44 val/val, 27 val/met, 5 met/met) performed a 0- 

and 2-back working memory task (7 scans of each task, 10 mCi oxygen-15 water/scan). Sixtynine 

of these participants (49 val/val , 20 val/met) also underwent eyes-closed resting PET scans 

(two scans each). Genotype groups were matched for age, sex, and performance. For the working 

memory paradigm, 2-back vs. 0-back contrast maps were generated for each participant and 

entered into a second-level random effects analysis to compare val homozygotes to met carriers. 

Additionally, rCBF values extracted from a sphere centered on the between-groups difference in 

the right hippocampus were entered into a whole-brain cross-correlation analysis to assess 

hippocampal connectivity. For the resting rCBF data, we used an a priori anatomical mask 

(bilateral hippocampus and parahippocampal cortices) to perform a small volume correction at 

p=0.01 to assess group differences. 

During rest, BDNF met carriers showed increased rCBF in bilateral hippocampal and 

parahippocampal regions compared to val homozygotes (p

indicated that during working memory, met individuals exhibited positive functional 

connectivity between right hippocampus and a DLPFC/parietal network, rather than the expected 

negative relationships observed in val homozygotes (p

present study we tested the hypothesis that striatal dopamine is particularly important for the 

manipulation of information in working memory in a pharmacological, event-related fMRI 

study. Twenty healthy human subjects were scanned twice, once after placebo and once after 

sulpiride 400 mg, a selective DA D2 receptor antagonist, while performing a working memory 

task requiring different levels of manipulation. We found a significant correlation between 

sulpiride plasma levels and process-specific BOLD signal bilaterally in the striatum, suggesting a 

dose-dependent effect of D2 antagonism on a striatally-based manipulation process. When 

subjects with high sulpiride plasma levels were examined separately we observed the predicted 

interaction between drug and condition, whereby striatal BOLD signal was lower during 

manipulation trials than during simple retrieval trials after sulpiride but not after placebo. No 

significant drug effects were observed in the PFC. These results support models of dopamine 

function which posit a „gating‟ function for dopamine D2 receptors in the striatum, which 

enables the flexible updating and manipulation of information in working memory. 

Disclosures: C.M. Dodds , None; U. Muller, None; L. Clark, None; T.W. Robbins, None. 

Poster 





Support: DAAD research scholarship 

Title: An fMRI study of transcranial direct current stimulation effects on planning performance 

and associative learning 

Authors: *C. DOCKERY, T. GABER, B. VARKUTI, R. HÜCKEL-WENG, N. 

BIRBAUMER; 

Inst. Med. Psychology, Univ. Tuebingen, Tuebingen, Germany 

Abstract: Transcranial direct current stimulation (tDCS) applied over the cortex transiently 

modifies cortical excitability via altering the membrane potential of underlying neurons. 

Different cortical areas can be targeted by tDCS, and it‟s pairing with fMRI imaging supports 

further understanding of the effect by providing a tool to evaluate neuropathologies, 

pathophysiology of disease and tDCS mechanisms of action. Baudewig et al. (2001) reported 

reduced motor cortex activity after cathodal tDCS on sensorimotor activity shown by fMRI 

BOLD signal changes post-stimulation. Combined fMRI and tDCS to study cognitive function 

can expand knowledge about the efficacy of tDCS.

Executive function impairment is a common feature in patient populations with frontal lobe 

pathologic abnormalities, such as schizophrenic, depressed and frontal lobe lesion patients. The 

Tower of London test (TOL) is a well-known neuropsychological tool to evaluate executive 

function, as it is sensitive in revealing deficits in patient planning performance compared to 

healthy controls. Neuroimaging studies have shown that brain activation during TOL 

performance involves a distributed network that relies markedly on the integrity of the 

dorsolateral prefrontal cortex. 

This study was designed to examine the neuronal bases of planning performance and associative 

learning in the TOL test and to assess the functional changes associated with the effects of tDCS 

administration over the left dorsolateral prefrontal cortex on BOLD signal. Over three sessions 

we studied the TOL performance of 17 healthy participants (7 men and 10 women) during 15 

min. of active anodal, cathodal and sham tDCS. A post-tDCS block of the TOL test was 

administered for each session, however measurement in the magnetic resonance scanner 

occurred during sessions one and three only. 

As reported by Dockery et al. (2008 submitted), it was proposed that performance gains would 

result from phase-specific tDCS application with cathodal preceding anodal tDCS, while sham 

tDCS would result in no change. No influence on performance accuracy was predicted; however 

significant improvement in planning would be indexed by reaction time. An accelerated learning 

effect across sessions was also predicted. tDCS may provide a tool to improve the cognitive 

function of patients with frontal lobe pathologies or learning disabilities by strengthening the 

connections in pathways associated with the damaged hemisphere. 

References 

Baudewig, J., Nitsche., M.A., Paulus, W. & Frahm, J. (2001). Regional modulation of BOLD 

MRI responses to human sensorimotor activation by transcranial direct current stimulation. 

Magn Reson Med. 45, 196-201. 

Disclosures: C. Dockery , None; T. Gaber, None; B. Varkuti, None; R. Hückel-Weng, 

None; N. Birbaumer, None. 

Poster 






MSFHR Research Scholar Award

NIH MH064498 

Title: A TMS “ping” during fMRI reveals physiological consequences of functional connectivity 

and dissociates multivariate from univariate maps of working memory storage 

Authors: *E. FEREDOES 1 , A. HELLER 2 , G. TONONI 3 , T. S. WOODWARD 1 , B. R. 

POSTLE 2,3 ; 

1 Psychiatry, Univ. of British Columbia, Vancouver, BC, Canada; 2 Psychology, 3 Psychiatry, 

Univ. of Wisconsin-Madison, Madison, WI 

Abstract: It is widely accepted that multivariate approaches to fMRI data analysis can, by 

measuring functional connectivity, provide information about distributed functional networks 

that cannot be inferred from traditional “massively univariate” analyses. However, most 

multivariate methods are nonetheless subject to the same inferential constraints fundamental to 

all correlational methods (i.e., neither necessity nor causality can be inferred). We used 

transcranial magnetic stimulation (TMS) to evaluate causally a verbal storage network suggested 

by a constrained principal components analysis (cPCA) of working memory data. The logic was 

that a physiological consequence of functional connectivity should be reflected in the manner in 

which a TMS-triggered impulse propagates through the putative functional network. (An analogy 

is to “ping” a network of computers by sending a signal from one computer to determine which 

other computers are actively connected to the network). In scanning session 1, subjects 

performed short-term delayed recognition (DR) of letters at two loads: 2 or 5 items. cPCA 

identified a (functionally connected) network of voxels whose delay-period activity was sensitive 

to variation of load (an operationalization of “short-term storage”). Scanning session 2 featured a 

2 (TMS present; TMS absent) x 3 (Letter DR; Location DR; no task) factorial design. A single 

pulse of TMS was delivered in the middle of the delay period to either a node in the cPCAdefined 

load-sensitive network, or to a control area. The cPCA map from session 1 was 

coregistered into session 2 space and within it the TMS “ping” increased Letter delay-period 

activity (i.e., Letter-DR/TMS-present > Letter-DR/TMS-absent), whereas it decreased Location 

delay-period activity. Univariate delay-activity maps were also created for Letter-DR/TMSabsent 

and Location-DR/TMS-absent trials. Within these univariate masks, TMS had the 

uniform effect of decreasing delay-period activity. Notably, within the left and right dorsolateral 

prefrontal cortex foci identified by the GLM for Letter-DR/TMS-absent trials, delay-period 

activity was greater during Letter-DR/TMS-absent than Letter-DR/TMS-present trials. These 

results suggest that a physiological consequence of functional connectivity is facilitation of 

synaptic transmission between nodes in the network. They also provide a stark demonstration 

that delay-period activity revealed by univariate analyses cannot be assumed a priori to 

correspond to the short-term storage of information. 

Disclosures: E. Feredoes , None; T.S. Woodward, None; G. Tononi, None; B.R. Postle, 

None; A. Heller, None. 

Poster





Support: NSERC Canadian Graduate Scholarship 

Natural Sciences and Engineering Research Council (NSERC) 

Michael Smith Foundation for Health Research (MSFHR) 

Canada Research Chairs (CRC) Program 

Title: Spatiotemporal brain dynamics underlying online maintenance 

Authors: L. RIGGS 1,2 , *A. T. HERDMAN 3 , J. RYAN 1,2 ; 

1 The Rotman Res. Inst., Toronto, ON, Canada; 2 Univ. of Toronto, Toronto, ON, Canada; 3 Simon 

Fraser Univ., Burnaby, BC, Canada 

Abstract: The spatiotemporal dynamics underlying online maintenance of relations among 

objects were outlined using magnetoencephalography (MEG). Subjects were presented with an 

initial scene of three abstract objects overlaid on a real-world background context. Following a 

delay of two seconds, a repeated or manipulated version of the original scene was presented. 

Performance accuracy was above 90%. MEG single trial data from 12 subjects were averaged 

with respect to stimulus onset. Neural activity during the two second delay was assessed. Online 

maintenance, relative to a baseline period before the onset of the first scene, was associated with 

sustained activity in many of the same regions implicated for processing of the scene such as the 

lingual gyrus, cuneus, precuneus, inferior parietal gyrus and regions in the prefrontal cortex. 

Activity in these regions peaked during the 500 ms immediately following first scene offset, and 

remained significantly above baseline for the remaining 1500 ms during the delay period. These 

findings suggest active maintenance of visual representations even in the absence of external 

stimuli. 

Disclosures: L. Riggs, None; J. Ryan, None; A.T. Herdman , None. 

Poster 





Support: JSPS Grant #19203032 

JSPS Grant #18330156 

Title: Individual differences of working memory capacity reflected as the different pattern of 

functional connectivity during visual working memory task 

Authors: *T. MINAMOTO 1 , M. OSAKA 2 , N. OSAKA 1 ; 

1 Dept Psychol, Kyoto Univ., Kyoto, Japan; 2 Dept Human Sci., Osaka Univ., Suita, Japan 

Abstract: Individual differences of working memory capacity (WMC) are known to predict 

several cognitive abilities. One of such abilities is distracter processing; subjects in high WMC 

group is less attracted by irrelevant information while ones in low WMC group are easily 

distracted by such information. This difference of distracter processing is implicated to be due to 

the different role of the prefrontal cortex (PFC); however, the interaction between the PFC and 

other brain areas are less known. This study aimed to reveal functional connectivity between the 

right PFC and the right visual association cortex (VAC) while high and low WMC participants 

were engaged in a visual working memory task. Eighteen healthy adult participants were divided 

into two groups in accordance with the score of the operation span test. In the MR scanner, a face 

delayed matching task was administered and two different distracters (face or scrambled face) 

were presented between encoding and recognition phase pseudo-randomly. Functional 

connectivity across the trial were obtained and compared with mixed ANOVA. Behavioral result 

showed the superior performance in high WMC subjects to low WMC subjects. Connectivity 

analysis showed the different pattern of functional connectivity depending on WMC. High WMC 

subjects showed the increased functional connectivity toward the recognition phase, reflecting 

the goal-directed enhancement of cortical interaction. On the other hand, low WMC subjects 

showed the decreased functional connectivity with the trial progression, implicating the natural 

decay of the interaction of those cortical regions. These results suggest that individual 

differences of WMC are reflected as the transitional change of functional connectivity. 

Disclosures: T. Minamoto, None; M. Osaka, None; N. Osaka, None. 

Poster 





Support: Drexel University Critical Research Fellowship 

Title: Coherent oscillatory networks supporting short-term memory retention 

Authors: *L. PAYNE, J. KOUNIOS; 

Drexel Univ., Philadelphia, PA 

Abstract: Recent research suggests that short-term memory (STM) is not simply a result of 

neural activity within prefrontal, parietal, and temporal regions, but that it is an emergent 

property of synchronous oscillations between these distant foci of activity. According to this 

scenario, top-down activation, reflected by frontal-midline theta-band (4-8 Hz) 

electroencephalogram (EEG) oscillations, is thought to strengthen the activation of a memory set 

during STM retention. In addition, the amplitude of posterior alpha-band (8-13 Hz) oscillations 

during STM retention is thought to reflect a sensory-gating mechanism that protects fragile STM 

activations from interference by bottom-up visual inputs. The present study examined large-scale 

network connectivity during STM retention by computing EEG wavelet coherence during the 

retention period of a modified Sternberg task using visually-presented letters as stimuli. In each 

trial, healthy, adult, participants judged whether a probe was one of the memory set which 

consisted of 2, 4, or 6 consonants. Spectral analysis of the 2.8 s retention period revealed a peak 

in alpha frequency over parietal-occipital regions that systematically increased with the number 

of items held in STM. Alpha coherence increased between posterior-midline electrode PZ and 

left-lateralized parietal-temporal sites only between set sizes 2 and 4. Although there was no 

change in theta power, coherence in the theta band between frontal-midline electrode FZ and left 

left-lateralized parietal-temporal sites strengthened as set size increased from 2 to 4 to 6 

consonants. Interestingly, alpha coherence between PZ and left temporal electrode T7 increased 

from 2 to 4 consonants, and then FZ/T7 theta coherence increased between 4 and 6 consonants. 

Analysis of T7 theta coherence revealed enhancement of a frontal-temporal network with 

simultaneous attenuation of temporal-posterior interactions. These findings support the view that 

interactions between frontal-midline cortex and left temporal/parietal cortex serve to strengthen 

the memory set. Furthermore, these results suggest that direct communication between posterior 

midline cortex and left temporal/parietal cortex coordinates sensory gating which prevents 

potential interference from bottom-up processes. 

Disclosures: L. Payne , None; J. Kounios, None. 

Poster 





Support: NIH grant R01 EB000473 

Title: Influence of physiologic noise on fMRI measures of functional connectivity between task+ 

and task- brain areas 

Authors: M. HAMPSON, *R. T. CONSTABLE; 

Dept Diagnos. Radiol, Yale Univ., New Haven, CT 

Abstract: Background: There has been great interest recently in a set of brain areas that 

deactivates during many cognitive tasks. This set of brain areas has been referred to as the 

default mode network, but we prefer the term task-negative brain areas. The functional 

relationships between these task-negative brain areas and classic working memory areas, 

otherwise known as task-positive brain areas, have been investigated in several studies. Negative 

correlations, or anticorrelations have been reported between task+ and task- brain areas in resting 

subjects and have been interpreted to reflect competition between cognitive and default mode 

networks, although they could also reflect cooperation. The analytic methods used in typical 

functional connectivity analyses include steps to normalize for the global signal (either by 

dividing at each timepoint by the global mean, or regressing out the global timecourse). These 

normalization steps help reduce the effects of physiologic noise, but can also distort correlational 

patterns. 

Methods: In this study, we collected simultaneous recordings of cardiac and respiratory cycles 

during the imaging session. The influence of physiologic noise (including cardiac and respiratory 

noise) on correlation patterns (particularly, on the anticorrelations between task- and task+ brain 

areas) is examined by comparing functional connectivity patterns after removal of physiologic 

noise sources (using RETROICORR) to those seen without physiologic noise removal. Results 

are also compared to those obtained using global regression/normalization rather than 

RETROICORR. 

Conclusions: The direct recording and removal of cardiac and respiratory noise is recommended 

in functional connectivity studies, particularly when examining the functional connectivity 

patterns between task+ and task- brain areas. 

Disclosures: M. Hampson, None; R.T. Constable , None. 

Poster 





Support: Grant-in-Aid for Young Scientists (S) from the JSPS (19670001) 

Title: Effective connectivity during task preparation reflects carry-over from a previous task 

Authors: *R. AKAISHI, Y. MORISHIMA, V. RAJESWAREN, K. SAKAI; 

Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan 

Abstract: We tend to repeat a same action knowing it is inappropriate in a new context. Task set 

inertia, carry-over from a previous task, is thought to contribute to the tendency of perseveration, 

such as slower and less accurate responses when we change from one task to another. Task set 

inertia can not be eliminated even with a long preparation time for the next task. In the present 

study we investigated the neural basis of task set inertia. Because task preparation was associated 

with interregional connectivity, we hypothesized that task set inertia is also based on 

interregional connectivity. In order to assess the connectivity at a specific timing during task 

preparation, we used a combination of transcranial magnetic stimulation (TMS) and EEG. This 

technique allows us to examine cortical effective connectivity, the efficiency of impulse 

transmission from an activated area to other areas. We asked normal human subjects to perform 

pro- or anti-saccade task based on a cue with a cue-target interval of 500 ms. We delivered a subthreshold 

single-pulse TMS on the right frontal eye field (rFEF) at 400 ms after the cue. We 

calculated the TMS evoked potentials by subtracting the EEG waveforms on no TMS trials from 

those on TMS trials. On the TMS evoked potentials at 20-40 ms after the TMS, we conducted 

two-way ANOVA with factors of previous and current task types. We found that the main effect 

of previous task was significant but that of current task was not significant. We also found that 

the TMS-evoked potential spread more broadly to posterior sites when saccadic responses were 

slower on trials preceded by antisaccade tasks. These results might indicate that inhibitory 

control required for antisaccade on a previous trial remains as effective connectivity. In contrast, 

the ERP on trials without TMS at the corresponding time window exhibited significant main 

effect of current task but non-significant effect of previous task. The time course of the ERP 

during the whole preparation period confirmed the major effect of preparation for a current task. 

Our results suggest that the control exerted during the execution of an antisaccade task on a 

previous trial creates the effective connectivity, which, consistent with the properties of task set 

inertia, persists during task preparation and modulates the task execution on the current trial. The 

effective connectivity reflecting previous tasks might also underlie the perseveration in human 

behavior. 

Disclosures: R. Akaishi, None; Y. Morishima, None; V. Rajeswaren, None; K. Sakai, None. 

Poster






MSFHR Scholar Award 

Title: Constrained principal component analysis reveals functionally connected load-dependent 

neural systems involved multiple phases of working memory 

Authors: S. WEINSTEIN, E. FEREDOES, P. METZAK, E. T. C. NGAN, *T. S. 

WOODWARD; 

Psychiatry, Univ. British Columbia, Vancouver, BC, Canada 

Abstract: Recent cognitive neuroscience models of working memory have posited that working 

memory is an "emergent" behavior in which, when behaviorally necessary, a set of "top-down" 

control processes are recruited to maintain or manipulate information in the same areas that 

represent that information under non-memory conditions. As a result, multivariate approaches to 

statistical analyses of fMRI data are becoming increasingly necessary in order to visualize 

connectivity between anatomically separate control and perceptual/sensory/action brain regions. 

Moreover, to test a central hypothesis of the model -- the same regions which encode the 

information also maintain the information -- requires consideration of multiple task stages 

together rather than modeling each separately. 

We introduce a novel application of constrained principal component analysis (cPCA) based on 

finite impulse responses (FIRs) which can provide functionally connected neural networks that 

can be temporally mapped onto different task stages. We applied this cPCA approach to a verbal 

delayed recognition task that varied the factor of memory load. Of the three extracted 

components, two were involved in both the encoding and delay stage, and the third was involved 

in both the delay and response stages. The encoding/delay functional networks were dominated 

by activation in occipital, dorsal anterior cingulate, and sensori-motor cortical regions, and 

deactivation in bilateral primary auditory cortex and "default network" regions. The 

delay/response functional network was dominated by activations in dorsal anterior cingulate, 

dorsolateral prefrontal cortex and left parietal cortex, with deactivations in the "default network" 

regions and visual cortex. We also observed sensitivity of all components for load, although only 

those involved in the encoding and delay phases reached significance. 

Our novel approach provides maps of functionally connected neural networks in addition to

demonstrating how they span the different stages of working memory and differ across task 

conditions. 

Disclosures: S. Weinstein, None; E. Feredoes, None; P. Metzak, None; E.T.C. Ngan, None; 

T.S. Woodward , CIHR MOP - 64431, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received). 

Poster 





Title: Genetic variance in the proline metabolism pathway is associated with schizophrenia and 

impact on striatal structure and function 

Authors: *I. J. LENT 1 , R. VAKKALANKA 2 , B. KOLACHANA 2 , R. STRAUB 2 , V. 

MATTAY 1 , J. CALLICOTT 2 , D. WEINBERGER 2 , L. KEMPF 1 ; 

1 Genes, Cognition and Psychosis, 2 Clin. Brain Disorders Br., NIMH, NIH, Bethesda, MD 

Abstract: Introduction: We have previously shown that genetic variation in proline 

dehydrogenase (PRODH) on chromosome 22q11.2 is associated with increased susceptibility to 

schizophrenia. PRODH is the rate limiting step of a metabolic pathway that converts proline to 

glutamate. These findings suggest that proline metabolism may be implicated in schizophrenia

susceptibility. To further dissect this pathway, four additional metabolic genes (ALDH4A1, 

ALDH18A1, OAT and PRODH2) were examined. In addition, SNPs in the SLC36A1, SLC6A7, 

VGLUT1 and VGLUT2 genes, which are involved in proline and glutamate transport, and the 

TP53 gene, a PRODH promoter, were analyzed. We staged our approach from clinical 

association to imaging genetics to assess the proline pathway phenotype. 

Methods: Tag SNPs were chosen via HaploView using 2-3 SNP aggressive tagging and forcing 

known functional SNPs into the model. Genotyping was performed using ABI Taqman and 

Illumina custom chips. Imaging was performed in a multimodal fashion using functional, 

structural and connectivity data. Haplotypes were constructed with Phase 2.1 and a second level 

imaging analysis was conducted using a regression approach with probabilistic haplotypes in the 

design matrix. Gene-Gene interactions were tested for additive and multiplicative interactions. 

Results: Imaging results showed that genetic variance in PRODH2 produced a similar striatal 

structure to that of PRODH. SLC6A7 haplotypes also exhibited altered working memory 

function in the striatum. 

Discussion and conclusion: Our data suggests that both genetic variation in PRODH2 and 

SLC6A7 produces changes in striatal structure and function. These results support previous 

findings linking proline metabolism and schizophrenia susceptibility. While intriguing, our 

findings are preliminary and require expanded genotyping and larger sample sizes for 

confirmation and refinement. 

Disclosures: I.J. Lent , None; R. Vakkalanka, None; B. Kolachana, None; R. Straub, 

None; V. Mattay, None; J. Callicott, None; D. Weinberger, None; L. Kempf, None. 

Poster 





Support: NIH Intramural Program 

Title: Contributions of genetic variation in dopamine clearance proteins to neural networks 

during n-back performance 

Authors: *D. P. EISENBERG 1 , P. D. KOHN 1 , A. MEYER-LINDENBERG 2 , J. APUD 2 , B. 

KOLACHANA 2 , D. R. WEINBERGER 2 , K. F. BERMAN 1 ; 

1 Sect Integrat Neuroimag, Clin. Brain Disord Brch -GCAP, 2 Clin. Brain Disord Brch -GCAP, 

NIMH, NIH, DHHS, Bethesda, MD

Abstract: Central synaptic dopamine is regulated by catechol-O-methyltransferase (COMT) and 

dopamine transporter (DAT) proteins, though regional cerebral differences in the relative impact 

of these proteins exist. In COMT, a single-nucleotide polymorphism (SNP) at codon 158 (val to 

met) results in diminished enzymatic activity and greater synaptic dopaminergic tone. In DAT1, a 

40 base pair, variable number of tandem repeats polymorphism (VNTR) regulates gene 

expression, with the 9-repeat allele conferring less DAT availability in vivo. Both 

polymorphisms have been associated with differential patterns of prefrontal activation in fMRI 

working memory paradigms, but much remains unclear about the interactions between these two 

genotypes across distributed neural networks. To examine 1) whether these genotypes or their 

interactions significantly contribute unique variance to regional brain activity during working 

memory, and if so, 2) how these contributions map topographically across the brain, we studied 

26 healthy, Caucasian volunteers, each of whom had genotyping for both polymorphisms as well 

as with seven, 60-second, 10 mCi 15 O-H2O PET scans during a 2-back task. Subjects were wellmatched 

for age (mean 35 + 9 years), sex (14 female), and 2-back performance (mean percent 

correct 79 + 14) across genotype groups. A multivariate linear model (MLM) analysis (Worsley 

et al 1997) implemented with multivariate modeling (MM) software (Kherif et al 2002) was 

applied to whole-brain data, constrained by genotype regressors. The analysis yielded three 

statistically significant eigenimages demonstrating distinct widespread cortical networks, 

representing DAT effects (first eigenimage, explaining over 40% of the model‟s variance) and 

COMT by DAT interaction effects (second and third eigenimages). These data confirm 

significant contributions of allelic variation in both COMT and DAT genotype to brain activity 

during working memory, including uncorrelated and interacting effects that manifest in a 

coordinated fashion throughout distributed brain networks. 

Kherif et al. (2002) Neuroimage 16:1068-83. 

Worsley et al. (1997) Neuroimage 6:305-19. 

Disclosures: D.P. Eisenberg , None; P.D. Kohn, None; A. Meyer-Lindenberg, None; B. 

Kolachana, None; D.R. Weinberger, None; K.F. Berman, None; J. Apud, None. 

Poster 





Support: NARSAD Young Investigator's Award 

Calhoun College Mellon Research Grant

Title: Visual perception, working memory, and binding in schizophrenia 

Authors: *J. E. PODELL 1 , P. C. DEGEORGE 2 , C. TEK 2 ; 

1 Cognitive Sci., Yale Col., New Haven, CT; 2 Psychiatry, Yale Univ. Sch. of Med., New Haven, 

CT 

Abstract: Background: Working memory impairments are consistently reported in 

schizophrenia, and they are believed to underlie a number of cognitive deficits associated with 

the disorder. Previous findings suggest that long-term, episodic memory binding is impaired in 

schizophrenia, but the extent to which abnormal binding might play a role in working memory 

impairments is relatively unknown. Disturbances have been identified in both “what” and 

“where” visual processing systems, but the specificity of these disturbances is not well 

characterized. The purpose of this study was to investigate visual perception and working 

memory binding of “what” and “where” features in schizophrenia using computerized forcedchoice 

delayed-response tasks. Methods: Subjects included 12 patients with schizophrenia or 

schizoaffective disorder and 9 healthy controls. Tasks designed to assess binding involved two 

colors (“what” features) and two spatial locations (“where” features) presented simultaneously 

either separated (unbound condition) or in an integrated fashion (bound condition) such that 

colors appeared co-localized with spatial locations. In addition, single feature tasks, including 

either two colors or two locations, were used to assess “what” and “where” system processing, 

respectively. Three delay conditions (side-by-side scene comparison, 250 ms delay control, and 

3000 ms delay working memory condition) were used in all tasks in order to identify the specific 

effects of perception, encoding, and retention. Results: Single feature task results revealed 

differential visual system impairments in patients compared to controls; we found evidence for a 

specific “where” system impairment in working memory retention (3000 ms), and a specific 

“what” system impairment in perceptual encoding (250 ms delay). However, there was no 

evidence for a specific feature binding impairment associated with the disorder, suggesting that 

early visual perception and working memory disruptions may disorganize cognition from the 

bottom-up, in the absence of a specific binding impairment. 

Disclosures: J.E. Podell, None; P.C. DeGeorge, None; C. Tek, None. 

Poster 





Support: Bio-Imaging Research Center

Title: Connectivity of the human working memory system 

Authors: *C. C. FARACO 1 , J. E. MACKEY 2 , N. YANASAK 3 , Q. ZHAO 1 , L. S. MILLER 1 ; 

1 Psychol, Neurosci, Univ. Georgia, Athens, GA; 2 Univ. of Georgia, Athens, GA; 3 Radiology, 

Med. Col. of Georgia, Augusta, GA 

Abstract: Working memory (WM) is a critical cognitive component required for normal 

performance in activities of daily living. Many of the cortical components of the WM system 

have been mapped through lesion and neuroimaging studies. Neuroimaging studies have used 

tasks such as the N-back to elicit activity in these areas, but many of these tasks have not been 

shown to correlate with measures of everyday functioning. The operation span (O-SPAN) task, 

on the other hand, is a robust measure of WM and has been shown to be highly correlated with 

measures of daily living. The current study has a dual purpose, the first is to asses the OSPAN 

task's ability to elicit proper activation of the WM system using functional magnetic resonance 

imaging (fMRI). The second is to develop a structural connectivity (white matter) map of the 

working memory system using diffusion tensor imaging (DTI). Participants performed a 

modified version of the OSPAN task, as per Kondo, et al. 2004, while fMRI data were acquired. 

Additionally, a DTI scan was acquired for each participant and fieldmaps were developed to 

correct for the inherent distortion in fMRI and DTI scans. Areas of fMRI activation will be used 

as seeding and tracking points for the DTI data in order to develop the white matter connectivity 

map. Whole brain fMRI and DTI scans were acquired on a 3T GE scanner (fMRI: 64x64 matrix, 

2.5mm slice thickness, 220mm FOV, 57 slices, TR=3s, TE=25ms, ASSET=2; DTI: 128x128 

matrix, 2.5mm slice thickness, 220mm FOV, 57 slices, TR=15600ms, TE=76.2ms, ASSET=2, 

25 directions, b-value=1000). Preliminary analysis of the fMRI data, using the FMRIB Software 

Library (FSL), indicate robust activity in areas usually implicated in different aspects of working 

memory. These include cingulate gyrus (BA32), granular frontal (dorsolateral prefrontal cortex; 

BA9), superior parietal (BA7), supramarginal (BA40), and insular (BA13) areas. The robust 

activity for each participant should provide sufficient highly activated voxels to be used as 

reference points for seeding and tracking of each participant's DTI data (i.e., cortical voxels will 

not be directly used as seeding or tracking points due to the significantly lower anisotropy values 

in gray matter). Based on previous neuroanatomical findings, direct connections are expected 

between frontal, parietal, and cingulate areas/netowrks; connections are also expected to pass 

through different thalamic regions. A group averaged fractional anisotropy map will be used to 

show the anatomical "strength" of these connections. 

Disclosures: C.C. Faraco, None; L.S. Miller, None; Q. Zhao, None; N. Yanasak, None; J.E. 

Mackey, None. 

Poster 





Support: Swartz Foundation 

Israeli Science Foundation 

Title: Working memory traces in neuronal networks 

Authors: S. GANGULI 1 , B. D. HUH 2 , *H. I. SOMPOLINSKY 3 ; 

1 Physiol., UCSF, San Francisco, CA; 2 Computat. Neurobio. Program, UCSD, San Diego, CA; 

3 Interdisciplinary Ctr. for Neural Computation, Hebrew Univ., Jerusalem 91904, Israel 

Abstract: Critical cognitive phenomena such as planning and decision making rely on the ability 

of the brain to hold information in working memory. Many proposals exist for the maintenance 

of such memories in persistent activity that arises from stable fixed point attractors in the 

dynamics of recurrent neural networks. However such fixed points are incapable of storing 

temporal sequences of recent events. An alternate, and relatively less explored paradigm, is the 

storage of arbitrary temporal input sequences in the transient responses of a recurrent neural 

network. Such a paradigm raises a host of important questions. Are there any fundamental limits 

on the duration of such transient memory traces? How do these limits depend on the size of the 

network? What patterns of synaptic connections yield good performance on generic working 

memory tasks? To what extent do these traces degrade in the presence of noise? Previous 

theoretical work has addressed some of these questions under restricted assumptions about input 

statistics and network architectures 1 . To study a more general setting, we use Fisher information 

to construct a novel measure of memory traces in neuronal networks. 

By combining Fisher information with dynamical systems theory, we find precise answers to the 

above questions for a class of linear neuronal networks. We prove that the temporal duration of a 

memory trace in any network is at most proportional to the number of neurons in the network. 

However, memory traces in generic recurrent networks have a short duration even when the 

number of neurons in the network is large. Networks that exhibit good working memory 

performance must have a (possibly hidden) feedforward architecture, such that the signal 

entering at the first layer is amplified as it propagates from one layer to the next. Such 

amplification may not be possible for neurons that operate within a limited dynamic range. We 

prove that networks subject to a saturating nonlinearity can achieve memory traces whose 

duration is proportional to the square root of the number of neurons. These networks have a 

feedforward architecture with divergent connectivity. By spreading excitation across many 

neurons in each layer, such networks achieve signal amplification without saturating single 

neurons. 

Overall this work reveals underlying limits on the duration of memory traces in neuronal 

networks, and elucidates the nature of synaptic connectivity patterns required for networks to 

achieve good working memory performance. More generally, these results provide a framework 

for the study of memory traces in more biologically realistic models of cortical networks. 

1 O.White, D. Lee and H.S., PRL 92 (14), 2004.

Disclosures: S. Ganguli, None; B.D. Huh, None; H.I. Sompolinsky , None. 

Poster 





Title: Allelic variation in NOS1AP is associated with altered prefrontal cortex function and 

functional connectivity during working memory 

Authors: *L. A. LIBBY 1 , K. K. NICODEMUS 2 , R. G. HIGIER 1 , M. J. PRUST 1 , H. TAN 1 , J. 

W. BUCKHOLTZ 1 , B. KOLACHANA 2 , R. E. STRAUB 2 , D. R. WEINBERGER 2 , J. H. 

CALLICOTT 1 ; 

1 UDIG, 2 CBDB, GCAP, NIMH IRP, NIH, Bethesda, MD 

Abstract: Schizophrenia is characterized by executive dysfunction, including a marked deficit in 

working memory (WM). NOS1AP (CAPON; 1q23.3), the gene for carboxyl-terminal PDZ 

ligand of neuronal nitric oxide synthase, has been identified as a potential susceptibility gene for 

schizophrenia, although association studies have yielded mixed results (Brzustowicz et al. 2004; 

Zheng et al. 2005). We used an imaging genetics approach based on the PFC efficiency 

phenotype (Callicott et al. 2003) to identify in vivo physiological ramifications of a NOS1AP 

SNP (rs347273) showing suggestive association in our family-based data. We have broadened 

the PFC phenotype to incorporate measures of WM capacity (Cowan‟s k-score) and functional 

connectivity. Fifty-six Caucasian healthy volunteers with no history of psychiatric illness were 

genotyped for 31 SNPs in NOS1AP; rs347273 was selected for further analysis within our WM 

fMRI dataset with T as the presumptive risk allele. Subjects (T/T=43 vs C Carriers=13) 

performed the 2back task during 3T fMRI scanning. Genotype groups did not differ significantly 

by 2back performance, reaction time, age, gender, or WAIS IQ score. Data were analyzed in 

SPM5. Multiple regression was used to identify main effects and interactions between genotype 

and WM activity. Functional connectivity analyses were based on a seed region in right DLPFC 

based on prior work. There was significantly greater right DLPFC activation in the T/T group 

than in the C carriers (p

the WM network. Furthermore, individuals homozygous for the risk allele exaggerated the 

previously found relationship between high capacity and increased activation in the right 

DLPFC. Variation in activation of the WM network associated with information processing can 

be accounted for in part by WM capacity and NOS1AP genotype. These data indicate that neural 

function associated with WM deficits in schizophrenia may be modulated by NOS1AP and that 

efficiency regions and capacity regions of the WM network are affected. 

Disclosures: L.A. Libby , None; K.K. Nicodemus, None; R.G. Higier, None; M.J. Prust, 

None; H. Tan, None; J.W. Buckholtz, None; B. Kolachana, None; R.E. Straub, None; D.R. 

Weinberger, None; J.H. Callicott, None. 

Poster 





Support: University of Cincinnati, Center for Imaging Research 

NIH Grant NIDA K01 DA020485 

NIH Grant NIDA R01 DA022221-02S2 

University of Cincinnati, Dean's Discovery Fund 

Title: fMRI brain activation in recreational ecstasy (MDMA) users during working memory 

Authors: *J. B. ALLENDORFER 1,2 , M. LAMY 1,2 , J. C. ELIASSEN 3,2 ; 

1 Neurosci Grad Prog, 2 Ctr. for Imaging Res., 3 Dept. of Psychiatry, Univ. Cincinnati, Cincinnati, 

OH 

Abstract: Ecstasy is a widely abused street drug, with a lifetime prevalence of about 10-14% for 

college students. MDMA, or 3,4-methelenedioxy-methamphetamine, is the primary psychoactive 

substance in ecstasy, and has been shown in animal studies to damage serotonergic neurons and 

to impair memory performance. However, due to the variability of drug exposure in humans, the 

cognitive and functional consequences of recreational ecstasy (MDMA) use remain unclear. To 

investigate the effects of ecstasy use on memory, we studied three groups of subjects: current 

ecstasy users (n=12), non-ecstasy drug users (n=10) matched to the ecstasy users by cannabis 

use, and non-drug users (n=10). During fMRI, participants performed an N-back task (0-back, 1-

ack and 2-back) in which they pressed the button corresponding to the number either on the 

screen (0-back), one screen prior (1-back), or two screens prior (2-back). Since neurotoxic 

effects of MDMA are thought to underlie memory impairments in ecstasy users, we 

hypothesized that ecstasy users will exhibit abnormal activation during a working memory task 

in brain regions that receive dense serotonergic innervation. Although all three groups showed a 

consistent decline in accuracy as the task increased in difficulty, there were no significant 

differences in performance (accuracy and response times) between groups for each task 

condition. Compared to non-drug users, drug users overall exhibit a relative increase in active 

brain regions and in the degree of activation during the working memory task. Compared to the 

cannabis comparison subjects, ecstasy users showed increased activation in temporal and frontal 

brain regions, including the left inferior and middle temporal gyrus and the right superior frontal 

gyrus, during the working memory conditions (1- and 2-back). These regions receive heavy 

serotonergic innervation, and the differences in activation may be attributed to ecstasy use. 

Disclosures: J.B. Allendorfer , None; M. Lamy, None; J.C. Eliassen, None. 

Poster 





Support: National MS Society SF1752-A-1 

Title: Object working memory performance depends on integrity of prefrontal-to-sensory whitematter 

tracts 

Authors: *M. K. WALSH 1 , C. A. MONTOJO 1 , D. S. REICH 2 , S. M. COURTNEY 1 ; 

1 Psychological & Brain Sci., 2 Radiology & Neurol., Johns Hopkins Univ., Baltimore, MD 

Abstract: Interactions between the prefrontal cortex and other brain regions, including 

secondary sensory cortices, are believed to be necessary for working memory. If true, damage 

within fiber bundles connecting these regions should correlate negatively with performance, even 

when the cortical regions themselves are intact. To address this question, we used functional 

MRI during delayed-recognition working memory tasks (without distraction), diffusion tensor 

imaging, and voxel-based morphometry (VBM) in 16 individuals with relapsing-remitting 

multiple sclerosis (MS). MS is an immune-mediated demyelinating disease of the central 

nervous system. Symptoms are highly variable across patients, dependent on disease severity and 

lesion locations, with about half exhibiting cognitive deficits. In the current study, diffusion

tensor imaging (DTI) was used to examine specific white matter tracts connecting areas showing 

fMRI activation for spatial or object working memory. Performance on these same tasks was 

tested for correlation with the amount of damage within those tracts. Additionally, VBM was 

used to test for correlations between working memory performance and localized cortical 

atrophy. Three tracts were identified within individual participants connecting areas involved in 

the task: Inferior Frontal Juction (IFJ) to Fusiform Gyrus (FG), Superior Frontal Sulcus (SFS) to 

Intraparietal Sulcus (IPS), and Middle Frontal Gyrus (MFG) to Temporal-Occipital Junction 

(TOJ). Damage to these tracts was quantified using the deviation from the mean Fractional 

Anisotropy (FA) at each slice along the posterior-anterior extent of each tract. Regression 

analyses revealed that damage to the IFJ-FG tract was the best predictor of object task accuracy 

(R2=.603, F(1,14)=21.287, p

Authors: *M. K. VAN VUGT 1 , M. J. KAHANA 2 , A. SCHULZE-BONHAGE 3 , B. LITT 4 , A. 

BRANDT 3 ; 

1 Neurosci, 2 Psychology, Univ. Pennsylvania, Philadelphia, PA; 3 Epilepsie Zentrum, Freiburg 

Univ., Freiburg, Germany; 4 Hosp. of the Univ. of Pennsylvania, Philadelphia, PA 

Abstract: Proactive interference is an important cause of human forgetting. It occurs when 

recently presented items interfere with memory judgements,such as in the recent negatives 

paradigm (Monsell, 1978). To assess how proactive interference is reflected in brain oscillations, 

we recorded intracranial EEG from a set of 16 patients while they were 

performing a Sternberg task. We compared the oscillatory correlates of proactive interference for 

letters and non-verbalizable face stimuli. Neuroimaging studies have found that proactive 

interference correlates most reliably with BOLD activity in the left inferior prefrontal cortex. 

Here we show that left dorsolateral prefrontal 90--128 Hz gamma oscillations increase, and right 

frontal 2--4 Hz delta oscillations decrease with proactive interference for faces. For letters, right 

frontopolar 2--4 Hz delta oscillations also decrease with proactive interference, but 2--14 Hz 

theta--alpha oscillations increase with proactive interference in the hippocampus. In summary, 

verbalizable and non-verbalizable stimuli show oscillatory correlates of proactive interference in 

different brain areas. 

Disclosures: M.K. van Vugt, None; M.J. Kahana, None; A. Schulze-Bonhage, None; B. Litt, 

None; A. Brandt, None. 

Poster 





Support: Swartz Foundation (Old Field NY) 

NSF 0613595 

Title: Relations between sensory and behavioral context and EEG spectra in a working memory 

task 

Authors: *S. MAKEIG 1 , J. ONTON 2 ; 

1 UCSD/Inc/SCCN, La Jolla, CA; 2 Inst. for Neural Computation, UCSD, La Jolla, CA

Abstract: The dominant approach to modeling relationships between experience, behavior, and 

brain dynamics is to acquire brain imaging data during presentation of a sequence of stereotyped 

classes of stimuli that elicit a small repertoire of participant behavioral responses, usually small 

finger movements. Researchers then average brain dynamics time locked to these stimulus or 

response classes and compare them by computing differences -- the method of planned 

comparisons. However, there is no guarantee that the dynamics produced by the brain during the 

experiment are solely or chiefly evoked by these event classes, or that each event class elicits 

only one type of brain response. The size, latency, and type of brain activity time locked to 

sensory and behavioral events may be linked to the detailed nature of the event, its relevance to 

the participant task, and/or to the exact context in which the event occurs. The brain may be said 

to continually „respond to the challenge of the moment‟ so as to maximize participant rewards 

and minimize penalties. But what is the nature of the brain‟s „challenge of the moment? What 

aspects of the event and its context determine the nature and size of the observed brain 

dynamics? Here we decomposed high-density scalp EEG data recorded during performance of a 

difficult continuous performance working memory task with auditory feedback („two-back with 

feedback‟) using independent component analysis (ICA), then converted the separated signals 

around feedback events of components associated with probable brain sources into normalized 

log spectrograms. To each spectrogram we appended a vector of context indicator variables, each 

the (yes/no) answer to a question about the trial itself or about preceding or succeeding trials. 

Decomposing this fused data by ICA produced a number of „independent context factors‟ 

combining a time/frequency log power template, plus a vector of context factor loadings, with a 

vector of multiplicative weights giving the relative polarity and strength of the factor on 

component spectral changes during the trial. The method revealed overlapping EEG spectral 

changes in many cortical source components that were associated with current or past reward, 

penalty, and neutral feedback, or with future performance. 

Disclosures: S. Makeig , None; J. Onton, None. 

Poster 





Support: Johnson O'Connor Research Support Corporation 

Title: Gender differences in correlations of regional white matter integrity with intelligence 

factor scores

Authors: *J. NG 1 , C. Y. TANG 1,2 , E. L. EAVES 1 , I. KANELLOPOULOU 2 , X. MAI 1 , D. 

CARPENTER 1 , P. R. HOF 3 , D. H. SCHROEDER 4 , C. A. CONDON 4 , R. J. HAIER 5 ; 

1 Dept. Radiology, 2 Dept. Psychiatry, 3 Dept. Neurosci., Mt. Sinai Sch. of Med., New York, NY; 

4 Johnson O'Connor Res. Fndn., Chicago, IL; 5 Univ. of California Irvine Sch. of Med., Irvine, CA 

Abstract: We investigated the correlations between white matter integrity and intelligence factor 

scores using diffusion tensor imaging (DTI) in 40 normals (19F, 21M; Mean age 27F, 26M). 

Imaging used a Siemens 3T Allegra. DTI data were acquired using a pulsed-gradient spin-echo 

sequence with EPI-acquisition (TR=4100ms, TE=80ms, FOV=21cm, matrix =128x128, 28 

slices, thickness=3mm skip 1mm, b-factor=1250 s/mm2, 12 gradient directions, 5 averages). FA 

maps were computed using in-house software written in Matlab v7.1. The voxels for the FA 

images measured 1.6x1.6x3 mm3. ROIs for the FA images were placed in corpus callosum (CC) 

(body, genu and splenium), forceps minor and major, anterior horn of the internal capsule, 

cingulum bundle (body, anterior and posterior), and superior corona radiata. Two raters defined 

the ROI locations on the MRIs and the resulting voxel values were averaged. The FA voxel 

values for each ROI were transferred to Statistica and merged with intelligence factor scores for 

correlation analysis. 

The body of CC and the right forceps minor were correlated to the general intelligence factor (g). 

The correlations of the FA values in the body of the CC reached significance with g factor scores 

(r=-0.335, p




Support: Johnson O'Connor Research Support Corporation 

Title: FMRI activations during the n-back test correlate with intelligence factor scores 

differently in males and females 

Authors: *E. L. EAVES 1 , C. Y. TANG 1,2 , J. NG 1 , D. M. CARPENTER 1 , P. R. HOF 3 , D. H. 

SCHROEDER 4 , C. A. CONDON 4 , R. J. HAIER 5 ; 

1 Radiology, 2 Psychiatry, 3 Neurosci., Mount Sinai Sch. of Med., New York, NY; 4 Johnson 

O'Connor Res. Fndn., Chicago, IL; 5 Pediatric Neurol. (Emeritus), Univ. of California, Sch. of 

Med., Irvine, CA 

Abstract: Previous research indicates that brain activation during a memory task in specific 

frontal and parietal areas is correlated to fluid intelligence scores, but these results may be 

confounded by sex. Here we used an n-back task during fMRI in forty normal subjects (19F, 

21M; Mean age 27F, 26M) who also completed a battery of psychometric and cognitive 

intelligence tests from which several factor scores were derived. 

Imaging was performed using a Siemens 3T Allegra scanner. After anatomical T1 and T2 images 

were acquired for co-registration, a gradient echo EPI sequence was used to acquire BOLD 

images were analyzed using SPM5. A group analysis used the general linear model with a one 

sample t-test (p < 10-9 uncorrected). Results included areas in the anterior cingulate, dorsal 

lateral prefrontal cortex (DLPFC), parietal and insular cortex (Fig 1). Individual percent 

activations for each subject were extracted from these clusters and correlated to the factor scores. 

Results: Females showed correlations in parietal regions whereas males showed correlations in 

frontal regions. All significant correlations between percent activations and factor scores were 

negative. For females, the parietal lobes correlated with the general intelligence factor (g) scores 

(Left r= -.52, p

Disclosures: E.L. Eaves, None; C.Y. Tang, None; J. Ng, None; D.M. Carpenter, None; P.R. 

Hof, None; D.H. Schroeder, Johnson O'Connor Research Foundation, A. Employment (full or 

part-time); C.A. Condon, Johnson O'Connor Research Foundation, A. Employment (full or parttime); 

R.J. Haier, Johnson O'Connor Research Foundation, F. Consultant/Advisory Board. 

Poster 

289. Human Decision Making 



Topic: F.01.g. Decision making and reasoning 

Support: NIH-DIRP 

Title: Neural correlates of reward-biased perceptual decision-making

Authors: *S. JAPEE, A. RAMOS, S. MARRETT, L. G. UNGERLEIDER; 

Lab. Brain & Cognition, NIH, Bethesda, MD 

Abstract: Studies suggest that higher-level cortical areas may compute perceptual decisions by 

comparing outputs of different pools of selectively tuned lower-level sensory neurons. A factor 

that may influence this decision-making process is the reward value associated with making a 

particular response choice. To study the effect of incentive on the process of perceptual decisionmaking, 

we used a face-house categorization task involving noise-degraded stimuli and multiple 

levels of monetary reward. 

Twenty subjects participated in an fMRI study involving categorization of noise-degraded 

images of faces and houses. Two levels of noise: 46% and 58% were used for degradation of 

images. To study the effect of incentive, four reward levels were used: No Reward, Equal 

Reward, High Face Reward (higher reward for correct face than house), and High House Reward 

(higher reward for correct house than face). On No Reward trials, subjects received simple 

feedback, i.e., correct or incorrect. On Equal Reward trials, subjects won 10¢ for a correct face or 

house response. On High Face Reward trials, subjects won 20¢ for a correct face response and 

10¢ for a correct house response. On High House Reward trials, subjects won 20¢ for a correct 

house response and10¢ for a correct face response. All incorrect responses resulted in a loss of 

2¢ while missed or late responses resulted in a loss of 5¢. On each trial, prior to stimulus 

presentation, subjects were cued to the level of reward for the upcoming trial. 

Behavioral data showed that subjects shifted their decision criterion (based on signal detection 

theory) towards the more profitable response choice, especially for noisy stimuli. fMRI data 

showed that, during the cue phase, relative to a no reward cue, a cue indicating higher reward for 

face elicited a greater response in fusiform gyrus, while a cue indicating a higher reward for 

house elicited a greater response in parahippocampal gyrus. Areas associated with reward, and 

higher-level cortical areas associated with decision-making, showed a differential response to 

reward-predictive cues compared to no-reward cues. During the stimulus-decision phase, house 

processing regions were more active when a noisy face was miscategorized as a house (Miss 

compared to Hit), and even more so when that trial was associated with a higher reward for face. 

Further, differential activity in a region in inferior frontal gyrus (BA 44) for Misses relative to 

Hits was greater for the high face reward condition compared to no reward. These results 

indicate that higher-level frontal regions may weigh sensory evidence with reward-induced 

response bias prior to arriving at a perceptual decision. 

Disclosures: S. Japee, None; A. Ramos, None; S. Marrett, None; L.G. Ungerleider, None. 

Poster 





Title: Sensory-motor mechanisms in human parietal cortex underlie arbitrary visual decisions 

Authors: A. TOSONI 1 , G. GALATI 2 , G. ROMANI 1 , *S. ASTAFIEV 3 , M. CORBETTA 4 ; 

1 Dept. of Clin. Sci. and Bioimaging, G. D‟Annunzio University, Inst. for Advanced Biomed. 

Technologies, G. D‟Annunzio Fndn., Chieti, Italy; 2 Inst. for Advanced Biomed. Technologies, 

G. D‟Annunzio Foundation, Dept. of Psychology, Sapienza University, Neuroimaging 

Laboratory, Santa Lucia Fndn., Roma, Italy; 3 Dept Radiol, Washington Univ. Sch. of Med., St 

Louis, MO; 4 Dept. of Clin. Sci. and Bioimaging, Inst. for Advanced Biomed. Technologies, G. 

D‟Annunzio Foundation, Departments of Neurology, Radiology, Anat. & Neurobiology, 

Washington Univ. Sch. of Med., St.Louis, MO 

Abstract: The neural mechanism underlying perceptual decision-making has been recently 

conceptualized as an integrative process in which sensory evidence supporting different response 

options accumulates gradually over time. In the critical neurophysiological experiments, oculomotor 

neurons in the lateral intraparietal (LIP) area, responsive to visual motion, accumulate 

over time motion information in favour of the specific oculomotor choice they are selective for 

(Shadlen et al., 1996; 2001). It is unclear, however, whether this mechanism generalizes to more 

complex humans decisions based on arbitrary stimulus-response associations. Here we tested 

with fMRI 12 subjects while performing a decision task requiring to arbitrarily associate visual 

stimuli (faces or places) to different actions (eye or hand pointing movements). On each trial the 

amount of sensory evidence available was varied by adding noise to the images. By using a ROIbased 

approach, we showed that activity of two hand pointing-selective regions in posterior 

parietal cortex was modulated, at a moment in which decisions were being formed, by the level 

of sensory evidence in favour of a manual response, the position of the action target, and even 

the outcome of the decision in the absence of helpful sensory information. Critically, the 

modulation by sensory evidence did not reflect greater sensory selectivity of parietal cortex to 

place stimuli that were associated with pointing responses. Therefore, human posterior parietal 

cortex contains a sensory-motor mechanisms, completely triggered by contextual stimulusresponse 

associations, that accumulates sensory evidence toward the behavioural outcome of an 

arbitrary decision. This mechanism is specific to posterior parietal cortex as pointing-selective 

regions in frontal cortex were not modulated either by sensory evidence levels or outcome. We 

conclude that visual decisions in human subjects do not necessarily involve high-level 

representations, independent of motor systems (Heekeren et al., 2006). Rather, decision 

processes seem to be embodied in the direct transformations between relevant sensory and motor 

representations, with motor circuitries querying sensory systems for evidence toward learned 

behavioural choices. More generally, these findings support the emerging idea of “embodied 

cognition”(Wilson 2002) according to which abstract cognitive functions do not depend on 

specialized modules, but are built on simpler sensory-motor mechanisms. 

Disclosures: A. Tosoni, None; G. Galati, None; G. Romani, None; S. Astafiev , None; M. 

Corbetta, None.

Poster 





Support: P50 MH062196 

P30 EY001583 

McKnight Foundation 

R01 EY015260 

Sloan Foundation 

Title: Computational, behavioral and physiological correlates of the value of uncertain 

information in a dynamic parameter estimation task 

Authors: *M. R. NASSAR, B. HEASLY, J. I. GOLD; 

Neurosci., Univ. Pennsylvania, Philadelphia, PA 

Abstract: The deliberative process of decision-making often requires updating beliefs that must 

take into account the uncertainty associated with possible interpretations or outcomes. Such 

belief updating is particularly challenging when the uncertainty itself takes various forms. 

Among the many forms of uncertainty are noise, which reflects the variance of a parameter, and 

volatility, which represents the instability of the parameter over time. Both forms of uncertainty 

are apparent in new evidence that deviates from a prior belief, but they prescribe opposite 

courses of action. Noise implies that new evidence should be de-emphasized, whereas volatility 

implies that new evidence should weigh heavily into the updated belief. We studied belief 

updating in human subjects using a dynamic parameter estimation task that includes both noise 

and volatility. 

The task required subjects to estimate the expected value of a set of sequentially presented 

numbers. The numbers were generated at random from a normal distribution. Within blocks of 

sequential trials, the mean and variance (noise) remained fixed. However, at random intervals the 

mean changed (volatility). We used a Bayesian model to determine the ability of an optimal 

agent to distinguish volatility from noise and generate appropriate estimates. The model, like the 

subjects, placed more value on information acquired immediately after a volatile change, and this 

effect was greater when the variance of the distribution was small. Subjects appeared to use the 

size of their errors as a heuristic to achieve this near-optimal behavior. 

A volatility signal has been reported previously in the anterior cingulate cortex (ACC) of 

subjects performing a two-choice reward probability tracking task (Behrens et al., 2007). The

ACC projects to locus coeruleus (LC), an area that sends diffuse noradrenergic projections 

throughout the brain and is thought to influence pupil diameter. To test for a role of the LC in 

belief updating we developed an isoluminent version of the task and measured pupil diameter 

after each presentation of new information. We found that pupillary response correlated with 

relative errors made by subjects. This pupillary response was also predictive of the extent to 

which subjects would use new information in a subsequent opportunity to update their belief. 

These data suggest that human subjects approximate optimal belief updating by using error size 

as a heuristic for volatility, and that a reflection of this measurement is seen in the pupillary 

response. We propose that LC may mediate an increased weighting of sensory information 

relative to previously established beliefs in response to volatile changes. 

Disclosures: M.R. Nassar, None; J.I. Gold, None; B. Heasly, None. 

Poster 





Support: AFRL Grant FA9550-07-1-0537 

Title: Integrating reward and stimulus information in a perceptual decision making task 

Authors: *J. GAO, R. TORTELL, J. L. MCCLELLAND; 

Dept. of Psychology, Stanford Univ., Stanford, CA 

Abstract: When observers must identify ambiguous stimuli, they adjust their decision toward 

the alternative offering a larger reward. Working in the context of dynamical models of decision 

making (Ratcliff, R., 1978, Psychological Review; Usher, M. & McClelland, J. L., 2001, 

Psychological Review), we consider how this reward bias effect might be incorporated. Previous 

investigations have considered the possibility that reward may affect the starting point of an 

evidence accumulation process, or that it may act like a constant input to this process. However, 

neither of these policies is necessarily optimal, especially if the time that will be available for 

evidence accumulation is uncertain. We investigated this situation. Human observers saw a 

rectangle offset 1, 3, or 5 pixels to the left or right of fixation, and were required to indicate the 

direction of shift within 250 msec of a response signal that could occur at any one of 10 different 

lags (0 to 2000 msec) relative to stimulus onset. 750 msec prior to stimulus onset, a cue indicated 

which response would, if correct, receive a larger reward (2 points vs 1 point). Previous research 

with similar stimuli has shown that accuracy identifying the direction of shift is at chance at early

signal lags, then follows a shifted exponential, reaching asymptote for lags of about 1 second. 

The trajectory is consistent with the Leaky, Competing Accumulator Model of Usher and 

McClelland (2001). Under these conditions, the optimal policy is to choose the response 

associated with the larger reward 100% of the time at the earliest signal lags, where stimulus 

identification accuracy is at chance. As evidence accumulates, the reliance on reward relative to 

stimulus information should gradually decrease, leveling off as accuracy approaches asymptote. 

We examine results from five participants. One showed no reward bias. For the others, the 

reward bias is very strong initially and then tapers off to a fixed level, in general agreement with 

the optimal policy. However, the initial bias is never as strong as would be optimal. The final 

bias is greater than optimal for some participants and less than optimal for others. Quantitative 

optimality analysis is carried out based on one dimensional stochastic accumulating models and 

signal detection theory. We also found that participants‟ fastest responses at each of the 10 

response lags tended to be more extreme than their slower responses (at short lags, faster 

responses were more biased toward the higher reward; at long lags, they were more accurate). 

We propose a model in which participants may maintain graded evidence and reward values until 

the arrival of the response signal, which then drives a race to threshold. 

Disclosures: J. Gao, None; R. Tortell, None; J.L. McClelland, None. 

Poster 





Support: EU IST 027198 "Decisions in Motion" 

Title: Neural correlates of stimulus independent decisions in motion in depth 

Authors: *G. KOVÁCS 1,2 , C. CZIRÁKI 1,2 , M. W. GREENLEE 2 ; 

1 Dept Cognitive Sci., Tech. Univ. Budapest, Budapest, Hungary; 2 Inst. of Psychology, Univ. of 

Regensburg, Regensburg, Germany 

Abstract: Perceptual decision making is a complicated, multi-stage process. Currently human 

neuroimaging studies implicated a set of regions, extending from the medial frontal cortex to the 

inferior parietal lobule in various steps of perceptual judgments. However, relatively little is 

known about the dependence of perceptual decisions on the visual stimulus itself. In the current 

study, we used functional magnetic resonance imaging during a demanding 3D heading 

estimation task. Subjects (n=13) were presented a constantly expanding optic-flow stimulus,

composed of disparate red-blue spheres, viewed through red-blue glasses. To systematically vary 

the difficulty of the task we either changed the ratio of coherently moving spheres or changed the 

lateral offset of the focus of the radial expansion, corresponding to the observer‟s direction of 

motion. We hypothesized that areas involved in perceptual decisions in motion should show 

similar activities, independent of the visual stimulus. In periods of sensory evidence 

accumulation the left dorsolateral prefrontal cortex, posterior cingulate and inferior parietal 

cortex showed such a pattern. During response selection the bilateral precuneus, posterior 

cingulate and inferior parietal cortex, as well as the right superior medial gyrus was found to 

fulfill the above condition. Our results suggest that a large, non-overlapping network of areas is 

involved in various steps of decisions in 3D motion. 

Disclosures: G. Kovács, None; M.W. Greenlee, None; C. Cziráki, None. 

Poster 





Support: We acknowledge funding from the Mathers Foundation 

Title: Using single-trial EEG data to predict laterality of voluntary motor decisions 

Authors: U. MAOZ 1 , A. ARIELI 2 , S. ULLMAN 1 , *C. KOCH 3 ; 

1 2 

Computer Sci. and Applied Mathematics, Neurobio., Weizmann Inst. of Sci., Rehovot, Israel; 

3 

Div. Biol 216-76, Caltech, Pasadena, CA 

Abstract: Over the last decades, EEG techniques have rendered the study of internal deliberation 

processes empirically tractable, with the output of these decisions manifested in action (e.g. 

moving the hand). The investigations of these free choices demonstrated that the urge to move 

occurred 200-350 milliseconds (msec) before movement onset on average. However, these 

studies were conducted by aligning the brain signals of many trials according to action-time and 

then averaging over these trials, furthermore, the signal was often averaged over different 

choices and therefore could not be used to predict a particular outcome. We wanted to study free 

selection processes on a single-trial basis in order to be able to better understand the neural 

correlates of these processes. Electroencephalographic (EEG) signals were therefore recorded 

from 64 electrodes placed over five subjects, which were instructed to freely choose left- or 

right-hand movements at spontaneously selected points in time. We then attempted to predict 

movement laterality on a single-trial basis, using signals from only 2 of the electrodes, placed

over the motor areas of both brain hemispheres (C3 and C4 according to the 10-20 electrode 

system), after time-locking the signals to movement onset. 

Making use of the difference in signal strength between these two electrodes only up to 500 msec 

before movement onset, we managed to correctly predict movement laterality for 72-90% of the 

trials for which the EEG signal was strong enough for prediction (p

which the timing of the response was self-generated (the reaction time, or "RT", experiment) and 

one in which they were cued to respond at a variable time (the response signal, or "RS", 

experiment). In keeping with our predictions, subjects' behavioral data could be well-fit by a 

diffusion model. Moreover, in the RT experiment we found a broad set of cortical regions whose 

BOLD signal varied inversely with motion coherence, including presumptive MT, posterior and 

medial intraparietal sulcus, inferior frontal sulcus, and anterior insula, in a manner consistent 

with our predictions for an accumulation process. In the RS experiment we found that the 

evidence for decisions at different motion coherences continued to increase as the response 

signal came at progressively longer times. These data provide initial support for a network of 

areas involved in even a simple decision-making task, and establish a framework for further 

studies based on variations of this task design. 

Disclosures: D.T. Erickson , None; B. Buchsbaum, None; A. Kayser, None; M. D'Esposito, 

None. 

Poster 





Support: NIH Intramural Research Training Award 

Title: Using prior probability to bias decisions in noisy conditions 

Authors: S. F. HILLENBRAND, K. A. HANSEN, *L. G. UNGERLEIDER; 

Lab. Brain & Cog, NIMH-NIH, Bethesda, MD 

Abstract: In this study we sought to identify cortical activation associated with the use of prior 

probabilities (PP) to bias perceptual decisions under noisy visual conditions. We acquired fMRI 

data while subjects made decisions about abstract parametric shapes under two PP conditions. 

Task difficulty was manipulated by varying the amount of noise overlaid on the target. In a 

previous fMRI study, we examined decision-making with the same abstract shape set under 

noise-free visual conditions, manipulating task difficulty by varying the ambiguity of the targets 

themselves. In other fMRI studies of decision-making from our laboratory, stimuli were faces 

and houses, and task difficulty was manipulated by varying the amount of noise overlaid on the 

target. The current study sought to differentiate between the effects of visual target type and of 

visual noise on decision-making. Preliminary results are consistent with the pattern presented by

Heekeren et al. as supporting an evidence integrator mechanism in posterior superior frontal 

sulcus. 

Disclosures: S.F. Hillenbrand, None; L.G. Ungerleider , None; K.A. Hansen, None. 

Poster 





Title: Neural systems underlying decisions about affective odors 

Authors: *E. T. ROLLS, F. GRABENHORST, B. PARRIS; 

Dept Exptl. Psychology, Univ. of Oxford, Oxford, United Kingdom 

Abstract: Decision-making about affective value may occur after the reward value of a stimulus 

is represented, and may involve different brain areas to those involved in decision-making about 

the physical properties of stimuli, such as intensity (Rolls, 2008). In an fMRI study, we delivered 

two odors separated by a delay, with instructions on some trials to decide which odor was more 

pleasant, or more intense; and on other trials to rate the pleasantness and intensity of the second 

odor without making a decision. The four odors, citral, vanillin, hexanoic acid and isovaleric 

acid, were identical on the two types of trial. Activations in the medial prefrontal cortex area 10, 

and in regions to which it projects including the anterior cingulate cortex and insula, were higher 

when decisions were being made compared to ratings, implicating these regions in decisionmaking. 

Decision-making about affective value produced larger effects in the dorsal part of 

medial area 10 and the agranular insula, whereas decisions about intensity produced larger 

effects in the dorsolateral prefrontal cortex, ventral premotor cortex, and anterior insula. For 

comparison, the mid-orbitofrontal cortex had activations related to subjective pleasantness 

ratings, providing a continuous representation of affective value. Thus the orbitofrontal cortex 

represents continuous affective value, and medial prefrontal cortex area 10, and the anterior 

cingulate cortex, are implicated in reaching a decision in which a binary outcome is produced. 

Rolls,ET (2008) Memory, Attention and Decision-Making. Oxford University Press. 

Disclosures: E.T. Rolls , None; F. Grabenhorst, None; B. Parris, None.

Poster 





Support: NHMRC 

Prince of Wales Medical Research Institute 

Title: Risk taking and postural control in older people 

Authors: *A. A. BUTLER 1,2 , J. L. TAYLOR 1 , S. R. LORD 1 , R. C. FITZPATRICK 1 ; 

1 Prince Wales Med., Randwick, Australia; 2 Sch. of Med. Sciences, Univ. of New South Wales, 

Sydney, Australia 

Abstract: Introduction: It is common sense that taking undue risks causes mishap, falling and 

injury for the elderly, just as in youth. Understanding “undue risk” is critical here as risk depends 

on ability. Physical ability and sensorimotor performance are the best predictors of falling among 

older people. Here, we attempt to identify those subjects who endanger themselves by taking 

risks beyond their abilities. 

Methods: Subjects (n=294, aged over 70 years) undertook a challenge to walk a plank. They 

could choose one of six footpaths to reach the same goal but in each was a plank that had to be 

crossed. The shortest path had the narrowest and tallest plank and the longest path had the widest 

and lowest plank. Thus, they could walk a shorter but riskier path or take a longer but safer path 

to reach their goal as quickly as possible. Tests of physiological performance that predicted the 

likelihood of falling from their chosen plank were administered and a questionnaire related to 

perceived risk taking behavior was completed. 

Results: Subjects were rated on a timid-bold scale based on the probability of falling from their 

chosen path. Older people and people with poor physiological performance chose safer planks. 

However, despite this they also had the highest probability of falling from their chosen plank. 

Men were more likely to choose more difficult planks and had a higher probability of falling 

from their chosen plank than women. Overall, subjects with good physiological performance 

made appropriate choices while poor performers made either risky choices or overly safe 

choices. 

Discussion and Conclusions: While older people with poor physiological performance tend to 

choose safer plank paths to compensate for their reduced physiological abilities, they do not 

adequately compensate and still have a high probability of falling from their chosen plank. Some 

subjects adopt a timid or safe strategy whereas others take excessive risks that would be likely to 

cause falling over or injury in everyday life. Subjects with the poorest physiological performance 

reported risk-avoiding behaviour during daily activities on questionnaire but our test indicates 

that this group takes the greatest risks.

Disclosures: A.A. Butler , None; J.L. Taylor, None; S.R. Lord, None; R.C. Fitzpatrick, 

None. 

Poster 





Title: Reward processes in medicated and never medicated patients with Parkinson‟s disease 

Authors: *A. A. MOUSTAFA 1 , S. KERI 4 , C. MYERS 2 , M. GLUCK 3 ; 

2 Psychology, 3 Neurosci., 1 Rutgers, Newark, NJ; 4 Dept. of Psychiatry and Psychotherapy,, 

Semmelweis University,, Budapest,, Hungary 

Abstract: Here we are studying the effects of Parkinson‟s disease (PD) and anti-PD medications 

on a cognitive task in which subjects learn either from reward or punishment. The study has 

three groups: never-medicated patients, medicated patients, and healthy controls. The results 

show that never-medicated PD patients show impairment at learning from reward, in agreement 

with existing literature. Medicated patients however did not show any impairment on the task. In 

addition, all subjects were administered the Tridimensional Personality Questionnaire (TPQ) 

which tests different personality traits including novelty seeking and harm avoidance among 

others. We found a medication effect on the novelty seeking segment of the TPQ: Novelty 

seeking was lower in unmedicated than in medicated patients (and also controls). This confirms 

earlier results regarding the dopaminergic basis of novelty seeking. We discuss the results in 

light of recent computational models of basal ganglia function and also of how dopamine 

medications (agonists or precursors) might affect cognitive processes in Parkinson‟s disease. 

Disclosures: A.A. Moustafa , None; S. Keri, None; C. Myers, None; M. Gluck, None. 

Poster 





Support: Wellcome Project Grant 082535/Z/07/Z 

Title: Increased sensitivity to positive feedback in stochastic sequence learning in medicated 

patients with parkinson's disease 

Authors: *M. SEO, M. BEIGI, M. JAHANSHAHI, B. B. AVERBECK; 

Inst. of Neurol., Univ. Col. London, London, United Kingdom 

Abstract: OBJECTIVES: A growing body of evidence suggests that dopamine plays a key role 

in reinforcement learning. Animal studies have shown that phasic bursts of dopamine neurons 

are observed after reward, whereas the firing of dopamine neurons drops below baseline after 

choices that do not result in reward. Therefore, disruption of the midbrain dopamine system as in 

Parkinson's disease (PD) may lead to deficits on tasks that require learning from positive and 

negative feedback. Previous studies have shown that in a probabilistic selection task and a 

transitive inference task, learning from positive or negative feedback is differentially affected by 

PD and the dopaminergic medication used to treat it. However, previous studies have not directly 

examined these effects during learning, but rather have shown deficits after learning during 

transfer.The aim of the present study was to examine stochastic sequence learning in PD patients. 

METHODS: A group of medicated PD patients and age-matched healthy controls were assessed. 

Participants were required to learn sequences of four button presses, where each button press was 

performed either with the left or right index fingers. We used six sequences in the task, each of 

which had two left and two right button presses. After each button press, participants received 

feedback whether or not it had been correct. Importantly, the feedback was noisy (85% correct 

feedback). Thus, on 15% of the trials, even if the participants had pressed the correct buttons, the 

feedback informed them that they were incorrect. Their task was to use this 'noisy' feedback to 

learn the correct sequence of button presses and then repeat it eight times. After participants had 

executed the sequence correctly eight times, a new sequence was introduced and the participants 

once again had to learn the new sequence by trial and error. We used a randomized block design 

with eight blocks in total. 

RESULTS: Both medicated PD patients and age-matched controls were biased to preferentially 

learn from positive feedback relative to negative feedback. Although the medicated PD group 

required more trials to learn the sequences they showed an enhanced learning effect from 

positive feedback relative to the control group. 

CONCLUSION: Our finding that medicated patients with PD showed increased sensitivity to 

positive feedback in stochastic sequence learning, may reflect a dopamine 'overdosing' effect in 

the fronto-striatal circuits which are less affected in PD. Future assessment of stochastic 

sequence learning in PD patients in the "off" medication state would be of interest. 

Disclosures: M. Seo , UCL Institute of Neurology, A. Employment (full or part-time); 

Wellcome Project Grant 082535/Z/07/Z, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received); M. Beigi, UCL Institute of 

Neurology, A. Employment (full or part-time); M. Jahanshahi, UCL Institute of Neurology, A.

Employment (full or part-time); B.B. Averbeck, UCL Institute of Neurology, A. Employment 

(full or part-time); Wellcome Project Grant 082535/Z/07/Z, B. Research Grant (principal 


Poster 





Support: NIDA R01 DA022549 

R01 DA11779 

Title: Decision-making and executive control deficits in substance-dependent individuals: a 

comparison of alcohol, cocaine, and methamphetamine, and of men and women 

Authors: *E. A. A. VAN DER PLAS 1 , E. A. CRONE 2 , W. VAN DEN WILDENBERG 3 , D. 

TRANEL 1 , A. BECHARA 4 ; 

1 Dept. of Neurol., Univ. of Iowa, Iowa City, IA; 2 Dept. of Psychology, Univ. of Leiden, Leiden, 

Netherlands; 3 Dept. of Psychology, Univ. of Amsterdam, Amsterdam, Netherlands; 4 Brain and 

Creativity Inst. and Dept. of Psychology, USC, Los Angeles, CA 

Abstract: Substance dependence is associated with executive function deficits, but the nature of 

these defects has not been fully clarified. In the study reported here, we compared patterns of 

performance of alcohol- (n = 33; 18 women), cocaine- (n= 27; 14 women) and 

methamphetamine-dependent individuals (n= 38; 25 women) with sex-matched healthy 

comparisons (n= 36; 17 women) in different executive domains, including decision-making, 

working memory, cognitive flexibility, and response inhibition (capacities which may be 

subserved by different regions in the prefrontal cortex). Based on previous work, we 

hypothesized that cocaine and methamphetamine abuse, but not alcohol abuse, would result in 

deficits in these domains of executive functioning. In addition, we explored the possibility that 

deficits in decision-making in substance addicted men and women may be more severe for 

women. Age and years of education were entered as covariates in all analyses. Cocaine- and 

methamphetamine-dependent individuals were indeed found to be impaired in decision-making 

as measured with the Iowa Gambling Task (IGT). These two groups also performed significantly 

lower than healthy comparison participants on the working memory task and the task-switching 

paradigm, but not on the response inhibition measure. In line with earlier findings, the deficits in 

working memory and cognitive flexibility were mild and did not change as a function of memory

load or task switching, suggesting that the deficits may lie in the „executive‟ process of both 

cognitive areas. Thus, the problems related to working memory and switching in substance 

dependent individuals may arise when a strategy should be efficiently implemented in order to 

perform the operation. Finally, women addicted to either cocaine or methamphetamine 

performed significantly worse on the IGT than men addicted to the same drug types. This novel 

finding regarding sex-related differences raises the possibility that the frontal lobes and related 

decision-making abilities, are affected differently in men and women addicted to cocaine and 

methamphetamine. These results further elucidate the effects of different types of substance 

abuse and sex on separate domains of executive functioning. 

Disclosures: E.A.A. Van der Plas, None; E.A. Crone, None; W. van den Wildenberg, 

None; D. Tranel, None; A. Bechara, None. 

Poster 






Title: The neural correlates of value and probability in decision under risk and in an equivalent 

motor task 

Authors: *S.-W. WU 1 , M. R. DELGADO 3 , L. T. MALONEY 2 ; 

1 Psychology, 2 Psychology and Ctr. for Neural Sci., New York Univ., New York, NY; 

3 Psychology, Rutgers Univ., Newark, NJ 

Abstract: There is considerable evidence that human economic decision-making deviates from 

the predictions of Expected Utility Theory (EUT) and considerable evidence from research on 

perception and action, indicating that human performance is close to optimal. This discrepancy 

might suggest that humans are better at making perceptual and motor decisions than economic 

decisions. However, given the large differences in task design between classical economic and 

perceptual/motor paradigms, no conclusion can be drawn. The goals of this study were to (1) 

develop a novel method to translate classical economic tasks to mathematically equivalent 

visuomotor tasks to allow comparison of choice behavior in the two modalities, and (2) 

investigate the neural correlates of value and probability in an economic decision task and an 

equivalent motor task. 

The experiment consisted of 3 sessions. First, subjects (N=14) participated in a motor training

session (outside the scanner) in which they performed rapid pointing to a rectangular target for 

monetary incentives. For each subject, we estimated the subject‟s own motor uncertainty for the 

purpose of creating motor configurations mathematically equivalent to a single lottery. In the 

second (practice) and third (fMRI) session, subjects faced both economic lottery decision tasks 

and equivalent motor lottery tasks for monetary incentives revealed at the end of the experiment. 

We modeled each subject‟s motor performance and economic performance using Cumulative 

Prospect Theory (Tversky & Kahnrman, 1992), estimating value functions and probability 

weighting functions separately for the motor and economic conditions. While no differences in 

the value function between motor and economic tasks were observed, different patterns in the 

probability weight function between motor and economic tasks were apparent. For the economic 

task, we replicated the typical shape of the weighting function, i.e. overweighting of small 

probabilities and underweighting for moderate to large probabilities. In contrast, subjects 

underweighted small probabilities and overweighted large during the motor task. Our fMRI 

results revealed (1) common, overlapped activations between economic and motor tasks but (2) 

distinct, modality-specific encoding of probability information. The inferior parietal and the 

ventrolateral premotor area, previously proposed to be involved in motor imagery, were 

selectively correlated with probability in the motor task. Our results suggest that while there 

might be a global decision making system independent of task modality, the emergence of 

probability representation is highly modality specific. 

Disclosures: S. Wu, None; M.R. Delgado, None; L.T. Maloney, None. 

Poster 




Topic: F.01.c. Attention 

Support: NIMH grant 1R01MH076282-01 

DOE grant DE-FG02-99ER6274 

Function BIRN (U24-RR021992) 

the John Templeton Foundation 

Title: A dynamic Bayesian network analysis of functional network differences during the 

auditory oddball task, related to general intelligence

Authors: *M. S. SCULLY 1,3 , B. ANDERSON 1 , T. LANE 1 , H. J. BOCKHOLT 3 , V. P. 

CLARK 3,2 , V. CALHOUN 3,2 , R. GOLLUB 4 , B. HO 5 , J. LAURIELLO 2 , T. WHITE 6 , R. JUNG 3,2 ; 

1 Computer Sci., 2 Univ. New Mexico, Albuquerque, NM; 3 The Mind Res. Network, 

Albuquerque, NM; 4 Massachusetts Gen. Hosp., Boston, MA; 5 Unniversity of Iowa, Iowa City, 

IA; 6 Univ. Minnesota, Minneapolis, MN 

Abstract: Purpose: To evaluate the parietal-frontal integration theory (P-FIT) proposed by 

Jung, Hair et al (Behav Brain Sci. 2007 Apr), using a dynamic Bayesian network analysis of 

brain function data collected during a neurocognitive task. 

Methods: fMRI scans obtained during four runs of the auditory oddball task. This task used rare 

pure tone target (9% of stimuli, 1500 Hz, requiring a button press), frequent standard (1000Hz, 

92%) and rare novel stimuli (9%, random computer generated sounds) presented for 200 ms 

each, with ISIs varied from 550 to 2050 ms. Data were collected from 94 healthy normal human 

subjects (47 males, 47 females, matched on generalized intelligence/g score). The g score was 

calculated as the unrotated first component of the principle component analysis of a general 

neuropsychological battery. The subjects were split into low (17 subjects), medium (47 subjects), 

and high (30 subjects) groups based on their g score, and a region-based analysis was performed 

using the AAL atlas. SBNet (http://www.cs.unm.edu/~lawnguy/sbnet/index.html) was then used 

to extract the dynamic Bayesian networks representing the functional network of activity present 

in each group. The functional networks extracted were then compared between groups by 

analyzing network level differences and the nonlinear models represented by the conditional 

probability tables (CPTs) at each region. 

Results:Many differences exist between groups at both the network and CPT level. Initial 

findings indicate a functional network consistent with the structural network identified in the P- 

FIT model. 

Conclusions: We found that different parts of the P-FIT network have different levels of 

influence depending on whether the subject falls into the low, mid, or high g group. Specifically, 

Broca's region was found to be statistically significant in the high g group, but not the low g 

group, and Wernicke's region was found to be statistically significant in the low g group, but not 

the high g group. 

Disclosures: M.S. Scully , None; B. Anderson, None; T. Lane, None; H.J. Bockholt, 

None; V.P. Clark, None; V. Calhoun, None; R. Gollub, None; B. Ho, None; J. Lauriello, 

None; T. White, None; R. Jung, None. 

Poster 

290. Animal Cognition and Behavior: Learning and Memory: Pharmacology I 



Topic: F.02.j. Learning and memory: Pharmacology 


National Alliance for Research on Schizophrenia and Depression (NARSAD) 

Title: Suppressing effects of dopamine D1 and D2 receptor interaction on hippocampal synaptic 

plasticity and spatial learning/memory 

Authors: *E. DREMENCOV, M. ZENKO, J. HAN, X. ZHANG; 

Inst. Mental Hlth. Res., Univ. Ottawa, Ottawa, ON, Canada 

Abstract: Accumulating evidence suggests that the dopamine D1 and D2 receptors interact to 

each other at both molecular and functional levels. We recently demonstrated that coadministration 

of the D1 agonist SKF38393 and the D2 agonist quinpirole increased the firing 

rate of pyramidal neurons in the piriform cortex and impaired olfactory memory, which were not 

observed when SKF38393 and quinpirole were given alone (Zenko et al., SfN Meeting 2007, 

P710). We show the first evidence here that co-administration of SKF38393 and quinpirole not 

only blocked the in vivo induction of both long-term potentiation (LTP) and long-term 

depression (LTD) in the CA1 region of the rat hippocampus, but also prominently impaired both 

the requisition and retrieval of hippocampus-dependent spatial memory. When given alone, 

neither SKF38393 nor quinpirole produced significant effects on the in vivo induction of LTP 

and LTD in the hippocampal CA1 region, or the requisition and retrieval of spatial memory. 

SKF38393, co-administered with quinpirole, failed to attenuate the firing activity of basket cells 

of CA1, indicating that the suppressing effects of co-administered SKF38393 and quinpirole on 

synaptic plasticity of hippocampal pyramidal neurons are not mediated by GABAergic 

neurotransmission. These findings support the idea that the dopamine D1 and D2 receptor 

interaction plays an important role in hippocampal synaptic plasticity and spatial learning and 

memory. 

Disclosures: E. Dremencov , None; M. Zenko, None; J. Han, None; X. Zhang, None. 

Poster 





Support: Funded by Abbott

Title: Dopamine D3 receptor blockade reverses effects of extended habit training on conditioned 

approach responding in rats 

Authors: *A. Y. BESPALOV, M. M. VAN GAALEN, L. V. UNGER, H. GENESTE, A. 

HAUPT, K. WICKE, K. DRESCHER, H. SCHOEMAKER, G. GROSS; 

Neurosci. Res., Abbott, Ludwigshafen, Germany 

Abstract: Dopamine D1 receptor stimulation is critically involved in early appetitive phases of 

learning in various behavioral paradigms. Accordingly, extended habit training was previously 

shown to attenuate behavioral flexibility as suggested by reduced ability of dopamine D1 

antagonists such as SCH-23390 to disrupt behavioral performance. The present study aimed to 

evaluate whether co-administration of dopamine D3 receptor antagonists restores sensitivity to 

acute blockade of D1 receptors. Adult male Wistar rats were presented with 45-mg food pellets 

(unconditioned stimulus, US) delivered to the food tray and immediately preceded by the 400-ms 

tone (2.8 kHz, 78 dB; conditioned stimulus, CS). During each training and test session, there 

were 28 CS-US presentations with an average inter-trial interval of 70 sec (30-110 sec) and each 

head entry into the food tray was recorded. Drug tests were conducted on either Day 3 or Day 9 

of training using independent groups of animals. On day 3, acute administration of SCH-23390 

(0.04-0.16 mg/kg) dose-dependently reduced the number of trials during which rats made head 

entries into the food tray within 10 seconds after the food delivery (omitted trials). On day 9, the 

effect of SCH-23390 on the number of omitted trials was greatly reduced. Selective D3 receptor 

antagonists, A-767119 (1-10 mg/kg), or A-932156 (1-10 mg/kg), had no effects on their own and 

significantly enhanced the disrupting effects of SCH-23390 (0.08 or 0.16 mg/kg) when given on 

day 9 but not day 3 of training. In contrast, preferential dopamine D2 receptor antagonist L- 

741626 (1 and 3 mg/kg) enhanced effects of SCH-23390 to a similar extent on days 3 and 9. 

These results indicate that D3 receptor blockade restores sensitivity to appetitive behaviordisrupting 

effects of SCH-23390 that was lost due to extended habit training. Thus, D3 receptor 

antagonists may be capable of enhancing behavioral flexibility and attenuating habit-like 

behaviors 

Disclosures: A.Y. Bespalov , Abbott, A. Employment (full or part-time); M.M. van Gaalen, 

Abbott, A. Employment (full or part-time); L.V. Unger, Abbott, A. Employment (full or parttime); 

H. Geneste, Abbott, A. Employment (full or part-time); A. Haupt, Abbott, A. 

Employment (full or part-time); K. Wicke, Abbott, A. Employment (full or part-time); K. 

Drescher, Abbott, A. Employment (full or part-time); H. Schoemaker, Abbott, A. Employment 

(full or part-time); G. Gross, Abbott, A. Employment (full or part-time). 

Poster 





Title: Intra-amygdala administration of 5-HT receptor agonists and antagonists during memory 

formation 

Authors: *D. FRES-GVEZ, G. LIY-SALMERON, A. MENESES; 

CINVESTAV, Mexico, Mexico 

Abstract: Serotonin (5-hydroxytyptamine, 5-HT) receptors have been localized in brain areas 

involved in learning and memory, including cortex, hippocampus and amygdala. Previous 

evidence indicates systemic, intrahippocampal or intraprefrontal cortex administration of 

selective 5-HT receptor agonists and antagonists modulate memory formation. Nonetheless, the 

5-HT receptors role localized in the amygdala has been slightly explored in memory tasks. 

Hence, in this work bilateral (basolateral) intra-amygdala post-training administration of 5-HT1A 

(8-OHDPAT, WAY100635), 5-HT1B (SB-224289), 5-HT2A (MDL100907), 5-HT6 (SB-399885) 

and 5-HT7 (SB-269970) receptor agonists and antagonists was explored during short- (STM) and 

long-term memory (LTM) in an autoshaping learning task. Results show that 8-OHDPAT at 1 

but not 0.1 κg impaired STM without affecting LTM. Although, WAY100635 (20 ng or 1 κg) 

had not effects on STM however its higher dose impaired LTM. SB-399885 (1 κg) had no effect 

in STM but impaired LTM. SB-224289, MDL100907 or SB-269970 (1 κg) neither affected STM 

nor LTM. In the interaction experiments, coadministration of 8-OHDPAT and WAY100635 or 

8-OHDPAT and SB-269970 showed that both antagonists reversed the STM-impairment effect 

induced by 8-OHDPAT and LTM was unaffected. These results indicate that the stimulation of 

either amygdalar 5-HT1A or 5-HT7 receptors impaired STM, while blocking either 5-HT6 or 5- 

HT7 receptors impaired LTM. Apparently, amygdalar 5-HT1B, 5-HT2A and 5-HT7 receptors have 

no influence in STM or LTM. Further data will be presented with the STM ketamine amnesiclike 

effects and intra-amygdalar infusion of 5-HT drugs. 

Disclosures: D. Fres-Gvez , None; G. Liy-Salmeron, None; A. Meneses, None. 

Poster 





Support: EU-supported FP6 project PROMEMORIA-LSHM-CT-2005-512012

Title: NCAM signaling through the FGF receptor in health and disease 

Authors: *V. BEREZIN 1 , S. LI 2 , D. K. DITLEVSEN 2 , E. BOCK 1 ; 

1 Neurosci& Pharmacol, Protein Lab., Copenhagen 2200, Denmark; 2 ENKAM Pharmaceuticals 

A/S, Copenhagen, Denmark 

Abstract: Stimulation of the neural cell adhesion molecule (NCAM) by homophilic interactions 

induces activation of a number of signaling molecules, including the fibroblast growth factor 

(FGF) receptor, non-receptor tyrosine kinases Fyn and FAK, growth associated protein GAP-43, 

the MAP kinase pathway, protein kinases A, C and G, and intracellular calcium. The role of 

NCAM signaling through FGF receptor has been addressed employing a variety of experimental 

approaches. A direct interaction between the fibronectin type III (FN3) modules of NCAM has 

recently been demonstrated by nuclear magnetic resonance (NMR) and surface plasmon 

resonance (SPR) analyses. Using structural data, bioinformatics and SPR binding analysis six 

sequence motifs, four in the first and two in the second FN3 modules of NCAM, involved in 

interactions with the FGF receptor have been identified. Synthetic peptides corresponding to 

these motifs constitute valuable pharmacological tools for the study of NCAM and FGF receptor 

signaling properties. All six peptides are agonists of the FGF receptor. In vitro, they all induce 

receptor phosphorylation and promote neurite outgrowth of primary neurons. One of the 

peptides, termed FGL, and a cognate ligand, FGF1, are shown to have different kinetics of 

receptor activation and activation of downstream signaling molecules such as phospholipase C 

(PLC)-gamma, FGF receptor substrate (FRS)-2 and Src homology and collagen (Shc). This 

suggests that NCAM signaling through the FGF receptor differs from signaling events induced 

by FGFs. In vivo, the FGL peptide enhances synaptic plasticity and improves cognition under 

normal and pathological conditions. 

Disclosures: V. Berezin , ENKAM Pharmaceuticals, E. Ownership Interest (stock, stock 

options, patent or other intellectual property); S. Li, None; D.K. Ditlevsen, None; E. Bock, 

ENKAM Pharmaceuticals, E. Ownership Interest (stock, stock options, patent or other 

intellectual property). 

Poster 





Support: NIA Grant AG009525

Title: Differential sensitivity of hippocampal spatial memory and plasticity to dietary choline 

supply in adulthood as a function of prenatal choline availability 

Authors: *S. J. WONG-GOODRICH 1 , M. J. GLENN 1 , T. J. MELLOTT 2 , J. K. 

BLUSZTAJN 2 , W. H. MECK 1 , C. L. WILLIAMS 1 ; 

1 Psychol& Neurosci, Duke Univ., Durham, NC; 2 Pathology and Lab. Med., Boston Univ., 

Boston, MA 

Abstract: Altered dietary choline early in life leads to persistent changes in memory precision 

and capacity and hippocampal plasticity in adulthood. Early choline availability also appears to 

metabolically imprint the developing brain, resulting in long-term changes in synthesis, storage 

and release of acetylcholine, and reuptake and recycling of choline. Metabolic imprinting by 

early choline availability may make a brain differentially sensitive to choline supply in adulthood 

via determining the range of adult choline intake that is optimal for cognitive function and 

hippocampal plasticity. To test this, we conducted two experiments that investigated the 

interactive effects of prenatal choline and adult choline availability on 1) hippocampal dependent 

tasks of spatial memory, and 2) adult dentate cell proliferation/neurogenesis as a measure of 

hippocampal plasticity. Pregnant rats received either a choline-supplemented (SUP), -control 

(CON), or -deficient (DEF) diet on embryonic days 12-17. Adult male offspring from each diet 

group were used as subjects and were maintained on the CON (1.1 g/kg) diet. In Exp. 1, 

postnatal day 70 rats were first trained on a radial arm maze (RAM) for 14 d and then switched 

to a SUP or DEF diet for 24 d (10 d with no training followed by 14 d of RAM retraining). When 

maintained on a CON diet, prenatal SUP and DEF rats showed enhanced RAM performance 

compared to prenatal CON rats. When switched to a DEF diet, prenatal SUP rats were slightly 

impaired while CON and DEF rats were relatively unaffected. In contrast, when switched to a 

SUP (5.0 g/kg) diet, prenatal CON and DEF rats were significantly impaired, with DEF rats 

showing the greatest impairment, and prenatal SUP rats were relatively unaffected. Exp. 2 

examined whether extended adult supplementation in older rats would affect hippocampal 

function and plasticity as a function of prenatal choline treatment. Twelve mo. old prenatal SUP, 

CON, and DEF rats either remained on a CON diet or were put on a SUP diet for 16 weeks. 

After 12 weeks, rats were trained on a spatial working memory water maze task, then given 10 

daily injections of BrdU to label newly dividing cells in the hippocampus, and then sacrificed 24 

hrs following the last injection. Consistent with Exp.1, long-term adult SUP impairs performance 

of prenatal DEF rats while prenatal CON and SUP rats are less affected. This impairment in 

water maze performance was also accompanied by a decrease in levels of dentate cell 

proliferation in the hippocampus. Taken together, these findings demonstrate that the in utero 

availability of an essential nutrient, choline, causes differential behavioral and neuroplastic 

sensitivity to the adult choline supply. 

Disclosures: S.J. Wong-Goodrich, None; M.J. Glenn, None; T.J. Mellott, None; J.K. 

Blusztajn, None; W.H. Meck, None; C.L. Williams, None. 

Poster





Support: Connecticut College Psychology Department 

Title: Just say "nootropic": the effects of nicotine on memory and learning in adolescent and 

adult rats 

Authors: *E. N. GOVEIA, R. E. GRAHN; 

Psychology, Connecticut Col., New London, CT 

Abstract: This study investigated the effects of nicotine on memory and learning in adolescent 

and adult male Fischer-344 rats. Rats were given 0.2 mg/kg/day of either nicotine or saline 

chronically for 2 weeks and were tested in the Morris water maze as adolescents (Phase 1) and 

then again 4 months later as adults (Phase 2). There were 4 main groups: nicotine/nicotine, 

nicotine/saline, saline/nicotine, and saline/saline. In Phase 2 rats were tested for c-Fos and BrdU 

expression in the dentate gyrus of the hippocampus. Behavioral data indicated that as adults, rats 

given nicotine were significantly improved at the water maze task compared to rats given saline. 

Impairments of the S/N group suggest performance in the maze is state dependent upon the 

nicotine. An increase in c-Fos expression was seen in the saline rats, and no BrdU expression 

was seen in either group. The behavioral results imply that low doses of nicotine improve 

learning and memory in adult rats. This provides support for studies investigating nicotine as a 

therapeutic agent for diseases affecting cognition, such as Alzheimer‟s. 

Disclosures: E.N. Goveia, None; R.E. Grahn, None. 

Poster 





Support: 45943M

Title: Influence of memory formation and amnesia over serotonergic transporter in brain 

Authors: *R. TELLEZ, L. ROCHA, A. MENESES; 


Abstract: Memory disorders represent a major and growing problem and the understanding of 

their pharmacological and molecular mechanisms is crucial. Serotonin (5-hydroxytryptamine, 5- 

HT) is involved in memory formation, amnesia states and the treatment of memory disorders. 

Indeed, serotonergic transporter (SERT) is implicated in amnesia nonetheless it is unknown if it 

is modified by memory formation or amnesia states and where such changes may occur. Hence, 

herein autoradiographic and pharmacological were evaluated changes in 5-HT transporter (5- 

HTT) during memory formation, pharmacological induced amnesia and reversed amnesia in 

autoshaping and novel object recognition (NOR) learning tasks. Acute post-training 

administration (I.P.) of saline, the selective serotonin reuptake inhibitor (SSRI) fluoxetine (10 

mg/kg), the stimulant and higher abuse liability d-methamphetamine (d-mAMPH; 1-10 mg/kg) 

or fluoxetine plus the d-mAMPH effective dose were tested. In order to compare 5-HTT 

expression autoshaping saline, fluoxetine, d-mAMPH or fluoxetine-d-mAMPH treated trained 

and untrained animals were used. Results show that d-mAMPH at 1-10 mg/kg impaired shortterm 

memory (STM) in autoshaping and NOR and the dose of 1 mg/kg produced an amnesic-like 

effects affecting autoshaping long-term memory (LTM). In contrast, fluoxetine facilitated both 

STM and LTM and reversed the d-mAMPH. Autoradiographic data of d-mAMPH treated 

untrained animals relative to control rats showed reduced expression of 5-HT in 13 of 28 brain 

areas analyzed, including lateral septum, hippocampus (CA2), raphe nuclei. The resting 15 areas 

showed increased 5-HTT expression mainly in the left brain. Further autoradiographic data will 

be presented. 

Disclosures: R. Tellez , None; L. Rocha, None; A. Meneses, None. 

Poster 





Support: NSERC Grant # 2027-06 

Title: Elucidating the neural substrates of methylphenidate-induced memory impairment

Authors: *E. E. BIGNEY, H. K. TAUKULIS, D. LEBLANC-DUCHIN, J. A. WILSON; 

Univ. New Brunswick, SJ, Saint John, NB, Canada 


Methylphenidate (MPD) is a psychostimulant that increases extracellular dopamine (DA) and 

norepinephrine (NE). Recent studies have shown that chronic oral MPD treatment results in 

impairment of working memory. An optimal level of DA at the D1 receptor is required for 

cognitive function; either too much or too little can result in memory deficits. Studies also show 

that pre-treatment with a very selective DA D1 antagonist, SCH 23390, can block stimulantinduced 

memory impairment. The present study investigated whether it is the over-stimulation of 

the D1 receptors that is the critical factor in MPD-induced memory impairment. Sixty 

periadolescent male Long-Evans rats were randomly assigned to one of four treatment groups: 

Saline/NoMPD, Saline/MPD, SCH 23390/MPD or SCH 23390/NoMPD. SCH23390 was 

administered intraperitoneally at a dose of 0.03mg/kg, 15 minutes prior to a 10mg/kg oral dose 

of MPD for 21 days. After an 11- day washout period, all animals were tested in Open Field, 

Object Recognition, and Object Placement tests. These tests measure locomotor activity, 

recognition memory, and spatial memory, respectively. Those animals treated with Saline/MPD 

showed impairment in both recognition memory and spatial memory. However, animals in the 

SCH/MPD group showed no memory impairment, exhibiting behaviour similar to the two 

groups that did not receive MPD. These results replicated previous studies and show that 

repeated treatment with MPD causes working memory impairment. The novel discovery was that 

it is the over-activation of the dopamine D1 receptors that appears responsible for the MPDinduced 

memory deficit. 

Disclosures: E.E. Bigney , None; H.K. Taukulis, None; D. LeBlanc-Duchin, None; J.A. 

Wilson, None. 

Poster 





Support: Japan Canada Joint Health Research Program and Japan France Joint Health Research 

Program (Joint Project from Japan Society for the Promotion of Science 

Comprehensive Research on Aging and Health from the Ministry of Health, Labor and 

Welfare of Japan

Japan-China Sasakawa Medical fellowship 

Grant-in-Aid for the 21st Century Center of Excellence Program “Integrated Molecular 

Medicine for Neuronal and Neoplastic Disorders” and “Academic Frontier Project for Private 

Universities (2007-2011) from the Ministry of Education, Culture, Sports 

Uehara Memorial Foundation fellowship for Foreign Researchers in Japan 

Title: The extensive nitration of neurofilament light chain in the hippocampus contributes to the 

cognitive impairment in mice 

Authors: *T. ALKAM 1,2 , A. NITTA 3 , H. MIZOGUCHI 3 , A. ITOH 3 , K. YAMADA 3 , T. 

NABESHIMA 3,4 ; 

1 Neuropsychopharmacol, Nagoya Univ. Grad Sch. Med., Nagoya, Japan; 2 Dept. of Basic Med., 

Col. of Traditional Uighur Med., Hotan, China; 3 Nagoya Univ. Grad Sch. of Med., Nagoya, 

Japan; 4 Meijo Univ. Grad Sch. of Pharmaceut Sci., Nagoya, Japan 

Abstract: Tyrosine nitration of proteins at an extensive level is widely associated with cognitive 

pathology induced by Amyloid beta (Aβ). The precise identity and the explicit consequences of 

the protein nitration, however, have scarcely been addressed. In this study, we examined the 

detectable nitration of proteins in the hippocampus of mice with cognitive impairment (Day 5) 

induced by the i.c.v.-injection of Aβ (Day 0), the most toxic Aβ species detected in human brain. 

The intensity of nitration of proteins was inversely associated with the level of recognition 

memory in mice. The detectable tyrosine nitrations were revealed in proteins with a single size of 

approximately 70 kDa. The specific nitrated proteins at this size were identified utilizing the 

liquid chromatography / mass spectrometry / mass spectrometry analysis and immunodetection 

methods. Neurofilament light chain (NFL) was found intensely nitrated. Increased nitration of 

NFL was associated with its serine hyperphosphorylation and weak interaction with the nuclear 

distribution element-like (NUDEL), an essential protein for the stable assembly of 

neurofilaments. No changes in cell numbers in the hippocampus were found (Day 5) in Aβinjected 

mice. These findings suggested that extensive nitration of NFL contributes to the Aβinduced 

impairment of recognition memory in mice. 

Disclosures: T. Alkam, None; A. Nitta, None; H. Mizoguchi, None; A. Itoh, None; K. 

Yamada, None; T. Nabeshima, None. 

Poster 





Support: AG009525 

Title: Prenatal choline supplementation enhances depolarization-evoked expression of genes 

implicated in neural plasticity 

Authors: *T. J. MELLOTT, J. M. DAVISON, V. P. KOVACHEVA, J. K. BLUSZTAJN; 

Dept Pathol, Boston Univ. Sch. Med., Boston, MA 

Abstract: Normal development and function of the brain requires the availability of fundamental 

nutrients, such as choline, during the period of embryonic and fetal growth. Choline is an 

essential nutrient whose intake during the second half of fetal development can produce longlasting 

cognitive effects. The mechanism by which choline influences learning and memory 

remains unclear; however, it may involve changes in the levels of factors responsible for 

neuronal growth and survival within the brain. We have previously shown that prenatal choline 

supplementation increases levels of brain derived neurotrophic factor (BNDF) in the 

hippocampus and enhances activation of signaling molecules, specifically those induced during 

learning and memory (MAPK, CREB). Depolarization-evoked BDNF expression has been 

shown to involve methyl-CpG-binding protein 2 (MeCP2) phosphorylation and its dissociation 

from the methylated binding site on the BDNF promoter. In this study, we found a timedependent 

increase in MeCP2 phosphorylation, demethylation of a specific CpG in the BDNF 

promoter, and BDNF mRNA expression following stimulation of hippocampal slices from 

control animals with a depolarizing concentration of potassium (45 mM KCl). Using 

hippocampal slices derived from rats that were exposed to choline supplementation, a control 

amount of choline, or choline deficiency via the maternal diet on embryonic days 12-17, we 

measured the mRNA levels of BDNF, as well as NMDA receptor subunit 2B (NR2B), in 

hippocampal slices following stimulation with either a depolarizing concentration of potassium 

or insulin-like growth factor 2 (IGF2, 0.1 nM). Hippocampal slices from prenatally cholinesupplemented 

rats had the highest basal levels of BDNF mRNA and were most responsive to 

depolarization and IGF2 stimulation. Basal levels and depolarization-evoked NR2B mRNA 

levels were the lowest in hippocampal slices from prenatally choline-deficient animals. IGF2 

stimulation increased NR2B mRNA expression only in hippocampal slices from prenatally 

choline-supplemented rats. Therefore, we are currently examining MeCP2 phosphorylation in 

response to depolarization and the methylation status of the respective promoters to possibly 

explain differences in BDNF expression, as well as NR2B expression, between the groups of 

animals whose mothers consumed different amounts of choline during pregnancy. Elevated 

levels of BDNF and NR2B, both implicated in learning and memory processes, in prenatally 

choline supplemented animals may be in part responsible for the cognitive enhancement 

observed in these animals. 

Disclosures: T.J. Mellott , None; J.M. Davison, None; J.K. Blusztajn, None; V.P. 

Kovacheva, None.

Poster 






Title: Choline supplementation during adolescence reduces the severity of working memory 

deficits in rats exposed to alcohol during development 

Authors: R. D. SCHNEIDER 1 , H. D. DOMINGUEZ 1 , D. FELLMAN 1 , *J. D. THOMAS 2 ; 

2 Dept Psychol, 1 San Diego State Univ., San Diego, CA 

Abstract: Prenatal alcohol exposure can adversely influence the development of the fetus, 

leading to a range of physical, neuropathological and behavioral alterations. Given that women 

continue to consume alcohol during pregnancy, there is a need to identify effective treatments to 

reduce the severity of fetal alcohol spectrum disorders (FASD). We have previously shown that 

pre- and/or early postnatal choline supplementation can attenuate ethanol‟s adverse effects on 

learning and memory, as well as activity level. However, it is not known if choline administered 

later in life, during late adolescence or early adulthood, would have similar beneficial effects. 

Sprague-Dawley rats were exposed to binge-like alcohol (6.0 g/kg/day) via intubation from 

postnatal days (PD) 4-9, a period of brain development that is equivalent to the human third 

trimester. Sham intubated and non-intubated controls were included. On PD 40-60, a period of 

development equivalent to adolescence/young adulthood, subjects were treated with 100 

mg/kg/day choline or saline vehicle via sc injection. Beginning on PD 61, after choline treatment 

was complete, behavior was examined in the open field, on the Morris water maze spatial 

learning task, and on a working memory version of the water maze. Ethanol exposure 

significantly increased open field activity, impaired performance on both the spatial learning and 

working memory versions of the Morris water maze. Choline administration did not significantly 

alter activity levels or improve performance of ethanol-treated subjects on the spatial learning 

version of the Morris maze, but it did significantly improve performance on the working memory 

version. In fact, ethanol-exposed subjects treated with choline performed at control levels on the 

working memory task. These data suggest that choline supplementation may effectively mitigate 

some of ethanol‟s effects cognitive performance, even when administered later in life. These data 

have important implications for the treatment of individuals exposed to prenatal alcohol 

exposure. 

Disclosures: R.D. Schneider, None; H.D. Dominguez, None; J.D. Thomas , None; D. 

Fellman, None.

Poster 





Title: Effects of the 5-HT1F receptor agonist LY344864 during memory formation, amnesia and 

cAMP 

Authors: *T. PONCE-LOPEZ, A. MENESES; 


Abstract: Short-term memory involves functional modulation of the synapses, while long-term 

memory requires gene activation and protein synthesis to allow the development of new synaptic 

connections. In vitro evidence indicates that, 5-HT1F receptors coupled negatively to cAMP 

production. Notably, 5-HT1F receptors are localized in brain areas mediating memory, including 

cortex, hippocampus and raphe nuclei. Hence, the aim of this work was to determine the effects 

of 5-HT1F receptor agonist LY344864 (LY) on short- (STM) and long-term memory (LTM) and 

cAMP productions. Rats received autoshaping training and immediately afterwards, were treated 

with saline (mg/kg), LY (0.01-10), methiothepin (no selective 5-HT antagonist 5-HT), el 

GR127935 (5-HT1D antagonist) or SB-224289 (5-HT1B antagonist) and tested for STM and LTM, 

at 1.5 h and 24-48 h respectively. Following STM and LTM rats were euthanized, and prefrontal 

cortex, hippocampus and raphe nuclei were extracted for the ELISA immunoassay. Results show 

that LY did not affect STM; nonetheless, 0.01 and 0.3-10.0 mg/kg impaired LTM (24 h). The 

amnesic-like effects induced by 0.3 mg/kg of LY344864 was partially reversed by methiothepin 

(0.03), GR127935 (5) or SB-224289 (1). Methiothepin (0.01-0.1 mg/kg), GR127935 or SB- 

224289 alone had not effects. These memory results will be discussed with changes in cAMP 

production in hippocampus, prefrontal cortex and raphe nuclei. 

Disclosures: T. Ponce-Lopez , None; A. Meneses, None. 

Poster 





Title: Behavioral and neural responses to social isolation rearing in the rat as a function of 

prenatal choline supplementation 

Authors: R. S. ADAMS, *M. J. GLENN; 

Psychology, Colby Col., Waterville, ME 

Abstract: Choline is a crucial nutrient that contributes to several biological functions and serves 

as a precursor molecule to the neurotransmitter acetylcholine. Choline is integral to the 

development and function of the central nervous system, and its availability during the prenatal 

period has lasting and protective effects on neural function. Researchers have found that prenatal 

choline supplementation in the rat enhances learning and memory processes later in life, 

especially those involving spatial memory. Researchers have also demonstrated that choline 

protects against a number of physical stressors to the neural environment, such as prenatal 

alcohol exposure, induced seizures, and chronic stress-induced exposure to corticosteroids. 

Compared to the study of these types of physical stressors, relatively little research has examined 

the influence of prenatal choline exposure on psychological stress later in life. To contribute to 

this field, the present study examined the effects of prenatal choline supplementation on 

behavioral and neural reactions to social isolation rearing, which typically produces a number of 

behavioral and cognitive deficits in the rat. Pregnant rats were given either a choline-sufficient 

(CON; 1.1 mg/kg choline chloride in AIN76A) or a choline-supplemented (SUP; 5 mg/kg 

choline chloride) diet during embryonic days 10-22. On postnatal day (PD) 24, the male 

offspring were weaned into standard pair-housing (PH) or social isolation (SI) and remained in 

these conditions for the duration of the study. In adulthood (PD 60), behavioral measures of 

anxiety, exploration, object and place recognition memory, and spatial reference and working 

memory were taken. As our neural measure we assessed levels of adult neurogenesis by 

quantifying numbers of doublecortin+ neurons in the dentate gyrus. In analyzing behavioral 

measures we found several effects of prenatal choline supplementation. SUP rats demonstrated 

superior abilities in object and place recognition memory, and spatial reference and working 

memory, but were not less anxious than CON rats. Neural measures also revealed higher levels 

of adult neurogenesis in some groups of SUP rats. However, our failure to detect consistent 

effects of housing in CON rats prevented us from drawing conclusions about the potential for 

prenatal choline supplementation to protect against the psychological stress of social isolation. 

Even so, these novel findings suggest that the benefits of prenatal choline supplementation may 

be contingent upon experiences such as social rearing, and that choline supplementation may 

impact an animal‟s sensitivity to environmental conditions. 

Disclosures: R.S. Adams, None; M.J. Glenn , None. 

Poster





Title: The effects of three antidepressants on aggressive and locomotor behaviors in Betta 

splendens 

Authors: *J. G. KOHLERT 1 , B. P. MANGAN 2 , L. B. MANGAN 2 ; 

1 Dept Psychol, 2 Envrn. Program, King's Col., Wilkes-Barre, PA 

Abstract: Although selective serotonin reuptake inhibitors (SSRIs) have been widely studied, 

both behaviorally and physiologically in a wide variety of animal species, the behavioral effects 

of these compounds via a natural assimilation are not common. This study investigated the 

effects of three SSRIs, fluoxetine (Prozac), sertraline (Zoloft), and paroxetine (Paxil), dissolved 

in water of fish. Aggressive and locomotor behaviors of 600 male Betta splendens consisting 40 

fish in each of four treatment groups (44, 87, 175, and 350 κg/L for each SSRI) plus a control 

group for each SSRIs were observed and recorded. Fish exposed to fluoxetine demonstrated 

significant decreases in baseline movement, number of aggressive attacks, movement in the 

presence of intruders, and an increase in aggression-response time at all treatment concentrations 

(P0.05). 

Disclosures: J.G. Kohlert , None; B.P. Mangan, None; L.B. Mangan, None. 

Poster 





Support: NIA Grant AG09525

Title: Prenatal choline supplementation enhances the amplitude of hippocampal ripple/high 

frequency events and increases the percentage of their occurrence during SWS and REM sleep 

states 

Authors: *W. H. MECK, C. L. WILLIAMS, R. K. CHENG; 

Psychol & Neurosci, Duke Univ., Durham, NC 

Abstract: Prenatal choline availability has been shown to induce organizational changes in the 

hippocampus that contribute to changes in cognition across the lifespan depending upon the 

prenatal choline treatment (e.g., deprivation or supplementation). To determine the neural 

mechanism(s) for these cognitive effects, we studied hippocampal local field potentials (LFPs) in 

the range from 1-250 Hz as a function of prenatal choline availability and sex. Adult male and 

female rats (14 mo) exposed during embryonic days 12-17 to choline deficiency (DEF n=6+6: 0 

mmol/kg choline), choline sufficiency/control (CON n=6+6: 7.9 mmol/kg choline), or choline 

supplementation (SUP n=6+6: 35.6 mmol/kg choline) were implanted unilaterally with a 

stainless-steel wire array in the dentate gyrus of the hippocampus (A: -4.8 mm, L: +2.5 mm, and 

V: -4.0 mm to Bregma). An EMG patch was sutured in the nuchal muscle for chronic recording. 

LFP data were recorded on multiple days that included 5 baseline sleep recording sessions as 

well as one additional short session that contained exploration of novel objects for 1 hr followed 

by 2 hours of sleep recording. By filtering the raw LFP traces in the frequency range between 

100-250 Hz, we were able to identify ripple events (5 SD above the average root-mean square, 

RMS) and high frequency events (HFE, 3 SD above the average RMS) from all treatment 

groups. Results showed that the amplitude of the ripple/HFE (defined as the distance between the 

peak and the trough of the filtered LFP traces) in the SUP rats is higher than both CON and DEF 

groups. Furthermore, the occurrence of ripple/HFE is higher during REM sleep and SWS states 

in the SUP rats. Finally, after the exploration of novel objects in the recording environment, all 6 

groups showed an increased percentage of SWS and REM sleep that contain ripple/HFE events 

with an even stronger increase for the SUP rats during REM sleep. Recent evidence suggests that 

the ripple/HFE might be the neural substrate that reflects the process and/or provides the time 

window for inducing synaptic plasticity and information transfer from/to the hippocampus 

following new experiences. In vitro studies have shown that ripple events can produce long-term 

potentiation (LTP) while prenatal choline supplementation can reduce the threshold for inducing 

LTP. In addition, sleep is also hypothesized to provide a preferential state for memory 

consolidation, a process that co-occurs with hippocampal ripple events. In conclusion, we found 

that the enhanced high-frequency oscillations in the SUP rats provide a plausible neural 

mechanism that can account for the cognitive benefits of prenatal choline supplementation. 

Disclosures: W.H. Meck , None; C.L. Williams, None; R.K. Cheng, None. 

Poster 

290. Animal Cognition and Behavior: Learning and Memory: Pharmacology I




Support: Duke University Superfund Basic Research Center (NIH ES10356). 

Title: Persistent neurobehavioral impairments in rats exposed to low doses of the 

organophosphate pesticide, parathion 

Authors: *O. A. TIMOFEEVA 1 , D. SANDERS 1 , K. SEEMANN 1 , L. YANG 1 , D. 

HERMANSON 1 , S. REGENBOGEN 1 , S. AGOOS 1 , A. KALLEPALLI 1 , A. RASTOGI 1 , D. 

BRADDY 1 , C. WELLS 1 , C. PERRAUT 1 , F. J. SEIDLER 2 , T. A. SLOTKIN 2 , E. D. LEVIN 1 ; 

1 Dept Psychiatry & Behav. Sci. Box 3412, 2 Dept Pharmacol. and Cancer Biol., Duke Univ. Med. 

Ctr., Durham, NC 

Abstract: Developmental exposure of rats to the organophosphate (OP) pesticides, chlorpyrifos 

and diazinon, causes persistent neurobehavioral effects. The current study extended the OP series 

to parathion. On postnatal days (PND) 1-4, we administered parathion to Sprague-Dawley rats at 

doses below or at the threshold for overt signs of systemic toxicity and spanning the threshold for 

barely-detectable cholinesterase inhibition (0, 0.1 or 0.2 mg/kg/d). Starting on postnatal day 

(PND) 35 through PND 182 animals were given a battery of tests for emotional/cognitive 

function, including T-maze spontaneous alternation, elevated plus maze, figure-8 locomotor 

activity, novelty suppressed feeding, chocolate milk anhedonia test, prepulse inhibition and 

radial-arm maze (RAM). The higher dose (0.2 mg/kg) caused a significant (p

None; S. Agoos, None; A. Kallepalli, None; A. Rastogi, None; D. Braddy, None; C. Wells, 

None; C. Perraut, None; F.J. Seidler, None; T.A. Slotkin, None; E.D. Levin, None. 

Poster 





Title: Effects of 5-HT drugs in prefrontal cortex during memory formation and the ketamine 

amnesia-model 

Authors: *G. LIY-SALMERON, A. MENESES; 


Abstract: In previous works we reported that the glutamatergic antagonist ketamine (at 10, but 

not 30-100 mg/kg) impaired short- (STM) with affecting long-term memory (LTM). This work 

describes series of experiments investigating the effects of systemic or intra-prefrontal 

administration of serotonergic agents in the ketamine model of induced memory deficits. First, 

rats were trained on an autoshaping learning task and immediately after training were injected 

with saline or drug. Following drug administration, rats were tested after 1.5 hours for STM and 

24 hours for LTM. An increased or decrease in number of conditioned responses were an index 

of retention. Results show that ketamine impaired STM and this confirmed previous evidence. 

Notably, the ketamine STM-impairment effect was reversed, by either systemic or intraprefrontal 

cortex administration of the agonist 5-HT1A/7 8-OH-DPAT, the 5-HT receptor 

antagonists MDL100907 (5-HT2A), SB-399885 (5-HT6) and SB-269970 (5-HT7). The ketamine 

STM-impairment effect was not altered by the 5-HT1A antagonist WAY 100635 or the 5-HT1B 

antagonist SB-224289. Notably, prefrontal cortex inhibition of translation or transcription 

interrupted STM without affecting LTM suggesting different signaling mechanisms. The 

interacting effect of NMDA and serotonin agents in memory function is an interesting and 

important area of study; both receptors are considered to be important targets for the 

development of antipsychotic medication. Particularly, 5-HT1A/7, 5-HT2A 5-HT6 and 5-HT7 

receptors present in prefrontal cortex, represent important targets for development of drugs for 

the treatment of SMT-deficits. 

Disclosures: G. Liy-Salmeron, None; A. Meneses, None.

Poster 





Support: NIA Grant AG009525 

NIA Grant AG025052 

Title: Memory proficiency in old age is uniquely related to neurotrophin levels and immune 

factors in the hippocampus of male and female rats as a function of prenatal choline availability 

Authors: M. J. GLENN 1 , S. D. BILBO 2 , T. J. MELLOTT 3 , J. K. BLUSZTAJN 3 , *C. L. 

WILLIAMS 2 ; 

1 Psychology, Colby Col., Waterville, ME; 2 Dept of Psych & Neurosci, Duke Univ., Durham, 

NC; 3 Pathology and Lab. Med., Boston Univ. Sch. of Med., Boston, MA 

Abstract: Adult rats that are supplemented with the nutrient choline during prenatal 

development show a remarkable resistance to age-related memory decline. We have 

hypothesized that this enhanced cognition in old age, as demonstrated using hippocampallydependent 

spatial memory tasks, may occur, at least in part, from a lifelong increase in or 

preservation of hippocampal plasticity. In support of our hypothesis we recently reported (Glenn 

et al., 2007, Eur. J. Neurosci. 25, 2473) that adult rats treated with prenatal choline 

supplementation, compared to prenatally choline sufficient- and deficient-diet fed rats, showed a 

marked increase in numbers of new neurons born in the hippocampus, which was accompanied 

by elevated basal levels of BDNF. In the present study, we examined the relationship between 

hippocampal growth factors and memory proficiency in aged male and female rats that were 

treated on embryonic days 12-17 with choline supplementation (SUP; 5 mg/kg choline chloride 

in AIN76A diet), sufficiency (SFF; 1.1 mg/kg), or deficiency (DEF; 0 mg/kg). At 24 months of 

age, working memory in a water maze was assessed with 1-, 15-, and 120-min retention delays. 

Hippocampus was rapidly dissected from one hemisphere to assay growth factor proteins and 

inflammatory markers. Consistent with our past work, both male and female SUP, compared to 

SFF or DEF rats, showed enhanced spatial working memory. Of the growth factors examined, 

NGF and NT-3 showed significant negative correlations with long-term working memory errors 

in SUP female rats only. This pattern was not present in CON or DEF females or in any of the 

male groups. However, both male and female SUP rats had significant reductions in 

inflammatory markers (MHC-II, IL-1β) compared to CON and DEF, suggestive of a protective 

glial environment that could buffer them from some features of the aging process to preserve 

cognition. Taken together, these findings indicate that prenatal choline supplementation leads to 

lifelong alterations in several processes that make important contributions to plasticity in the 

hippocampus. We also have increasing evidence that there are sex differences in these effects

that will require further investigation. The observed changes in neurotrophic and inflammatory 

hippocampal milieu are compelling candidate mechanisms for prenatal choline 

supplementation‟s preservation of cognitive performance with old age. 

Disclosures: M.J. Glenn, None; S.D. Bilbo, None; C.L. Williams , None; T.J. Mellott, 

None; J.K. Blusztajn, None. 

Poster 





Support: Abbott Laboratories 

NIH Grant KO2 MH01072 


Title: nAChR agonist-evoked glutamatergic and cholinergic transients in the prefrontal cortex of 

mice lacking the beta2- or alpha7-nAChR receptor subunit 

Authors: *J. JI 1 , V. PARIKH 1 , M. W. DECKER 2 , M. SARTER 1 ; 

1 Dept Psychol, Univ. of Michigan, Ann Arbor, MI; 2 Neurosci. Drug Discovery, Abbott Labs., 

Abbott Parik, IL 

Abstract: Increases in cortical cholinergic activity mediate the pro-cognitive effects of nAChR 

agonists. Recently, we demonstrated in rats that the amplitudes of cholinergic transients that are 

evoked by nAChR agonists are mediated via glutamatergic transients and NMDA and AMPA 

receptor stimulation. Furthermore, compared to the effects of nicotine, the α4β2-selective 

nAChR partial agonist ABT-089, which has demonstrated efficacy in a small ADHD trial, was 

more potent in eliciting glutamatergic and cholinergic transients. Additionally, the cholinergic 

transients evoked by ABT-089 lacked the slow decay rate of nicotine-evoked transients. To 

substantiate the neuropharmacological model derived from these experiments, cholinergic and 

glutamatergic transients were measured following local pressure ejections of nicotine and ABT- 

089 into the prefrontal cortex of mice lacking the β2- or α7-subunit of the nAChR. Similar to 

results in rats, in wild-type (WT) mice, cholinergic signal amplitudes following ABT-089 (10 

pmol-2.5 nmol) were characterized by more potent signal amplitudes and faster decay rates when 

compared with the effects of nicotine (10 pmol-2.5 nmol). In β2 knock-out (KO) mice, nicotine-

evoked cholinergic signal amplitudes were significantly smaller (WT: 3.05±0.16 κM; β2 KO: 

1.78±0.64 κM). The decay rate (t50) of nicotine-evoked signals did not differ between the strains 

(WT: 14.47±2.42s; β2 KO: 14.67±0.97s). ABT-089 did not elicit cholinergic transients in β2 KO 

mice. In α7 KO mice, the amplitudes of nicotine and ABT-089 evoked cholinergic transients 

were identical to those recorded in WT mice. Nicotine-evoked cholinergic signals decayed faster 

in α7 KO mice (10.61±1.09s). These results are consistent with the hypothesis that the effects of 

ABT-089 are selectively mediated via β2-, but not α7-, containing nAChRs. Furthermore, α7containing 

nAChRs contribute to the regulation of cholinergic signal decay rate following 

nicotine administration. Ongoing experiments will determine if the amplitudes of glutamatergic 

signals in WT and KO mice predict the amplitudes of cholinergic signals. These experiments 

will test a neuropharmacological model suggesting that α4β2 nAChRs on glutamatergic 

(thalamic) afferents increase prefrontal glutamate release which in turn evokes cholinergic 

signals via NMDA and AMPA heteroreceptors located on cholinergic terminals. The continuing 

and slowly decaying release of acetylcholine evoked by nicotine is mediated in part via α7 

nAChRs and is predicted to be associated with less efficacious cognitive enhancement than the 

“sharp” signals evoked by α4β2-selective nAChR agonists. 

Disclosures: J. Ji, None; V. Parikh, None; M.W. Decker, Abbott Laboratories, A. 

Employment (full or part-time); Abbott Laboratories, E. Ownership Interest (stock, stock 

options, patent or other intellectual property); M. Sarter, Abbott Laboratories., B. Research 


already received); Abbott Laboratories., C. Other Research Support (receipt of drugs, supplies, 

equipment or other in-kind support); Abbott Laboratories., D. Speakers Bureau/Honoraria 

(speakers bureau, symposia, and expert witness). 

Poster 





Support: CONACYT Scholarship 18533 

Title: Memory formation and camp production: 5-ht1a and 5-ht7 receptors 

Authors: *G. P. GARCIA, A. MENESES; 

Dept Pharmacobiology, CINVESTAV, México, DF, Mexico

Abstract: Serotonin (5-hydroxytrypyamune, 5-HT) modulates positively and negatively through 

a G-protein-coupled cAMP-mediated pathway. Notably, cAMP production has been linked to 

memory formation and multiple 5-HT receptors have been implicated in memory. Hence, 

memory formation and cAMP production were investigated in an autoshaping task. The 

autoshaping time-course showed that there was a significant increment in the %CR at 48 h 

relative to 1.5 and 24 h testing times. Therefore, 24 and 48 h were selected for further memory, 

pharmacological and cAMP production experimental analysis. Notably, 24 and 48 h allow 

detecting upwards or downwards changes about cAMP and memory, thus providing an excellent 

window to determine how they are related. Even to further test this link, an overtraining group 

showing 100% of CR was included. The post-training administration of the 5-HT1A/7 receptor 

agonist 8-OH-DPAT or 5-HT7 receptor agonist AS19 improved memory, which were blocked by 

the 5-HT1A receptor antagonist WAY100635 or 5-HT7 receptor antagonist SB-269970. Changes 

in cAMP production in prefrontal cortex, hippocampus and raphe nuclei were associated to 

memory formation, pharmacological manipulation and amnesia. Indeed, untrained animals 

treated with saline, 8-OH-DPAT, AS19, scopolamine and dizocilpine showed changes in cAMP 

production ranging between 100 to 2000%, and similar but trained animals had changes varying 

between 100 to 500%. Together these data show for the first time, serotonergic modulation of 

memory formation and cAMP production. And this evidence is consistent with the notion that 

modulation of cAMP-dependent pathways is viable therapeutic tool in of cognitive impairment. 

Disclosures: G.P. Garcia , None; A. Meneses, None. 

Poster 





Support: NARSAD Toulmin Independent Investigator Award 


Title: Perinatal choline supplementation improves hippocampal-based learning and memory in 

the DBA/2 mouse 

Authors: *K. E. STEVENS 1,2 , R. C. BATES 2 , J. GAULT 3 ; 

1 Med. Rsrch, Veterans Affairs Med. Ctr., Denver, CO; 2 Psychiatry, 3 Neurosurg., Univ. of 

Colorado-Denver, Sch. of Med., Aurora, CO

Abstract: Deficient sensory inhibition is a cardinal symptom of schizophrenia which has been 

linked to reduced hippocampal α7 nicotinic receptors, poor attention and subsequent poor 

learning and memory. These functional deficits are improved with nicotinic agonist 

administration. The DBA/2 inbred mouse models the sensory inhibition and learning memory 

deficits associated with schizophrenia and also exhibits reduced hippocampal α7 nicotinic 

numbers. Previous studies showed that increased perinatal choline availability in DBA/2 mice 

permanently improves sensory inhibition in adulthood and also increases hippocampal α7 

nicotinic receptor numbers. The present studies assessed learning and memory using 2 paradigms 

in adult DBA/2 mice gestated on normal (1.1 gm/kg) or supplemented (5 gm/kg) choline diets. 

Dams consumed the specific diet throughout gestation and lactation. All pups were placed on 

normal choline diet at weaning. Initially, mice were trained in a contextual fear conditioning 

paradigm. This paradigm tests both hippocampal-based and non-hippocampal-based learning and 

memory by testing for memory of a context and for classical conditioning. The second learning 

and memory test was novel object recognition, which is also hippocampal-based. This paradigm 

entailed a habituation day (no objects), a training day (2 identical objects) and a test day (1 old 

object and 1 new object). In the contextual fear conditioning experiment, mice treated with 

supplemented choline during the perinatal period showed significantly better memory for the 

context (greater amount of freezing) than did mice treated with normal choline levels during the 

perinatal period. There were no differences in the amount of tone-cued freezing (classical 

conditioning) between the groups. During the habituation phase of the novel object recognition 

study, supplemented mice showed less anxiety (more time spent in the center of the open field) 

than the normal-level choline mice with no overall increase in activity level. On test day, the 

choline supplemented mice spent more time with the novel object than did the normal-choline 

level mice, indicating improved memory for the old object. These studies suggest that 

hippocampal-based learning and memory is improved by perinatal increases in choline 

availability while non-hippocampal learning and memory is not changed. This improvement may 

be related to the increase in hippocampal α7 nicotinic receptor numbers produced by increased 

perinatal choline. Coupled with the improvement previously seen in sensory inhibition, these 

data suggest that increased developmental choline availability may improve one of the risk 

factors for schizophrenia. 

Disclosures: K.E. Stevens , None; R.C. Bates, None; J. Gault, None. 

Poster 




Topic: F.02.j. Learning and memory: Pharmacology

Title: Effects of 5-HT6 and 5-HT7 receptors agonists and antagonists during memory formation 

Authors: *A. MENESES; 

Dept Pharmacobiol, Cinvestav - IPN, Coapa, Mexico 

Abstract: Growing evidence indicates that 5-hydrohytryptamine (5-HT) receptors mediate 

learning and memory. Particularly interesting have become 5-HT6 and 5-HT7 receptors, which 

are localized in brain areas involved in memory formation. Interestingly, recently selective 5- 

HT6 and 5-HT7 receptor agonists and antagonists have become available. Previous evidence 

indicates that 5-HT6 or 5-HT7 receptors antagonists had no effects, improved memory formation 

and/or reversed amnesia. Herein, the effects of EMD (a 5-HT6 receptor agonist) and AS19 (a 5- 

HT7 receptor agonist) in the associative learning task of autoshaping were studied. Post-training 

systemic administration of EMD (1-10 mg/kg) or AS19 (1-10 mg/kg) were tested in short- 

(STM) and long-term memory (LTM). Results showed that only EMD 5.0 mg/kg impaired both 

STM and LTM. AS19 at 1-10 mg/kg significantly impaired STM but not LTM. In those groups 

used to only test LTM, EMD impaired it; while AS19 improved LTM. In interaction 

experiments, the STM EMD-impairment effect was partially reversed by the selective 5-HT6 

receptor antagonist SB-399885 (10 mg/kg). The STM AS19-impairment effect (5.0 mg/kg) was 

not altered by the selective 5-HT1A antagonist WAY 100635 (0.3 mg/kg) but reversed by the 

selective 5-HT7 receptor antagonist SB-269970 (10.0 mg/kg). The AS19-SB-269970 

combination impaired LTM. Taken together these data suggest that the stimulation of 5-HT6 

impaired both STM and LTM. 5-HT7 receptors stimulation impaired STM but improved LTM. 

Thus, serotonin via 5-HT6 and 5-HT7 receptors might function in serial or parallel manner, 

respectively, during memory formation. 

Disclosures: A. Meneses, None. 

Poster 





Title: Behavioral phenotyping of heterozygous acetylcholinesterase knockout (AChE+/-) mice 

showed no memory enhancement but hyposensitivity to amnesic drugs 

Authors: *J. ESPALLERGUES 1 , L. GALVAN 1 , L. LEPOURRY 2 , B. BONNAFOS 2 , T. 

MAURICE 1 , A. CHATONNET 2 ;

1 EPHE, Univ. Montpellier II, INSERM U710, Montpellier, France; 2 INRA UMR 866, 

Montpellier, France 

Abstract: Decrease in acetylcholinesterase (AChE) expression or activity results in increased 

cholinergic tonus in the brain or periphery, with concomittant regulations of nicotinic and 

muscarinic receptors expression. We generated AChE knockout mice and characterized the 

behavioral phenotype of heterozygous animals, particularly focusing on learning and memory 

functions. Male and female, AChE+/- and AChE+/+ littermate controls (129/Sv strain), tested at 

5-9 weeks of age, failed to show any difference in terms of locomotion, exploration and anxiety 

parameters in the open-field test. Animals were then tested for place learning in the water-maze. 

They were trained using a 'sustained acquisition' protocol (3 swims/day during 5 days) or a 'mild 

acquisition' protocol (2 swims/day during 9 days) to locate an invisible platform in fixed position 

(reference memory procedure). Then, during 3 days, they were trained to locate the platform in a 

variable position (working memory procedure). Learning profiles and probe test performances 

were unchanged in AChE+/- mice as compared with AChE+/+. Mice were then treated with the 

muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg sc) 20 min before each training 

session (3 swims/day during 5 days). Scopolamine impaired learning at both doses in AChE+/+ 

mice, but only at the highest dose in AChE+/- mice. Moreover, the central injection of amyloid 

beta25-35 peptide (9 nmol) 7 days before water-maze acquisition failed to induce learning 

deficits in AChE+/- mice, contrarily to AChE+/+ controls. These behavioral study shows that the 

increase in cholinergic tonus did not result in increased memory abilities in these heterozygous 

AChE+/- mice, but allowed a significant prevention of the deleterious effects of muscarinic 

blockade or amyloid toxicity. Major adaptations in the expression and activity of nicotinic and 

muscarinic receptors have been observed in response to the chronic hypercholinergy in AChE-/- 

mice, that have been suggested to contribute the limited efficacy of AChE inhibitors in 

Alzheimer's disease. Our behavioral study suggest that such adaptations do not occur in 

heterozygous mice. 

This work is a project (#03) of the CompAn behavioral phenotyping facility (Montpellier, 

France). 

Disclosures: J. Espallergues , None; L. Galvan, None; L. lepourry, None; B. Bonnafos, 

None; A. Chatonnet, None; T. Maurice, None. 

Poster 






FRAXA Research Foundation 

Title: Initial characterization of a site-specific Rac deficient mouse 

Authors: O. Y. N. BONGMBA, L. A. MARTINEZ, *M. TEJADA-SIMON; 

Dept Pharmacol & Pharma Sci., Univ. Houston, Houston, TX 

Abstract: The role of the small GTP-binding proteins as signaling molecules has been studied 

extensively in many cell types, but their importance has been somewhat overlooked in the field 

of neuroscience. Until recently members of the Rho subfamily of GTPases were believed to be 

involved primarily in the regulation of cytoskeletal organization in response to extracellular 

growth factors. Specifically, one of the members of the Rho family of small GTPases, Rac, has 

been implicated in regulation of the mitogenic response, superoxide generation, regulation of 

transcription, morphological changes necessary for migration, and specific cytoskeleton 

rearrangements that play a crucial role in cell motility, cytokinesis and cell shape changes. 

Related to the nervous system, its organization relies on the subtle morphological complexity of 

neurons. Rac is known to participate in neuronal morphogenesis, presumably leading to synaptic 

plasticity and synapse formation. Therefore, we hypothesize that in the brain, Rac is an important 

molecule involved in synaptic plasticity, as well as learning and memory functions. Our 

laboratory has reported that Rac is highly expressed in the adult mouse hippocampus, a brain 

area that is crucial for the attainment of associative memories; and that translocation and 

activation of Rac occurs during associative contextual fear learning in the hippocampus of the 

adult animal. Thus, to begin testing our hypothesis, in this study we investigate a newly 

developed site-specific Rac deficient mouse created under the Cre-LoxP system. We carried out 

a preliminary behavioral characterization of these mice to determine performance on learning 

tasks as well as non-cognitive related functions and compared Rac-deficient mice to parental and 

wild type animals. Experimenters were blind to the mouse genotype at the time of the task. We 

have found that Rac-deficient mice showed no significant differences in non-cognitive related 

functions as compared to parental and wild type animals, while presenting deficits on learning 

tasks. Together, our preliminary data suggest that Rac is important in brain and its function is 

associated with hippocampus-dependent learning. Supported by NIH Grant NS48037 (M.V.T.S), 

and FRAXA Research Foundation (M.V.T.S). 

Disclosures: O.Y.N. Bongmba, None; M. Tejada-Simon , None; L.A. Martinez, None. 

Poster 

291. Animal Cognition and Behavior: Learning and Memory: Pharmacology II 




Support: NIMH Z01-MH-002498-17 

Title: Differential gene expression profiling in Avpr1b knockout mouse brain 

Authors: *H.-J. LEE 1 , G. KANNAN 1 , M. PALKOVITS 2 , S. YOUNG 1 ; 

1 SNGE, NIMH/NIH/DHHS, Bethesda, MD; 2 Lab. of Neuromorphology, Hungarian Acad. of 

Sci. and Semmelweis Univ., Budapest, Hungary 

Abstract: The neuropeptide vasopressin (Avp) has a crucial role in behaviors such as aggression 

and social motivation via the vasopressin 1b receptor (Avpr1b). Expression of Avpr1b was 

initially described in the pituitary and thereafter in various brain tissues including the cortex, 

septum, amygdala, and hippocampus. In order to elucidate the role of Avp, we used microarray 

technology to examine gene expression profiles of five brain regions (entorhinal cortex, septum, 

amygdala, ventral hippocampus and dorsal hippocampus) from Avpr1b deficient mice 

(Wersinger S.R., et al., 2002). RNA was extracted from mice of three age groups (average 70d, 

108d, and 159d). cDNA labeled with Cy3 for WT and Cy5 for KO samples (and vice versa), was 

then hybridized to 2 microarray chips (Ilumina and Qiagen). The expression patterns of 

candidate genes were verified by RT-qPCR. Interestingly, some changed genes varied among the 

age groups (e.g., some decreasing in expression with age and some increasing), suggesting that 

different genes are influenced by the Avp/Avpr1b system during postnatal development. Several 

genes located on the same chromosome within 1-3 Mb of the deleted region of the KO mice 

showed a strong reduction in expression, suggesting that the 5‟ region of the Avpr1b gene 

contains one or more regulatory elements of other genes. We expect that the results of our study 

will provide valuable information about the actions of Avp in the brain. 

Disclosures: H. Lee, None; G. Kannan, None; S. Young, None; M. Palkovits, None. 

Poster 





Support: CAPES/GRICES Grant 176/2007

FCT Grant PTDC/SAU-NEU/81064/2006 

Fundo Fundação Oriente/Johnson & Johnson 

FCT Grant PTDC/SAU-NEU/74318/2006 

Title: Chronic caffeine consumption prevents memory deficits in animal models of diabetic 

neuropathy, chronic unpredictable stress or juvenile convulsions 

Authors: *R. A. CUNHA, G. P. COGNATO, M. P. KASTER, J. M. N. DUARTE, P. M. 

CANAS; 

Ctr. for NeuroSci of Coimbra, Inst. Biochem, Fac Med, Univ. Coimbra, Coimbra, Portugal 

Abstract: Caffeine (an adenosine receptor antagonist at non-toxic doses) is the most widely 

consumed psychoactive drug. Its chronic consumption prevents memory deficits upon aging or 

Alzheimer‟s disease, an effect mimicked by adenosine A2A receptor antagonists in animal 

models. We now tested if caffeine also affords protection in other diseases also leading to 

memory dysfunction, such as stress, childhood convulsions or diabetes. A protocol of chronic 

unpredictable stress for 3 weeks in c57Bl6 mice increased immobility in the forced swim test and 

decreased both spontaneous alternation and time spent in a novel arm in a Y-maze test; chronic 

caffeine consumption (1 g/L in the drinking water starting 4 weeks before beginning and 

throughout the stress paradigm) prevented this stress-induced memory deficit, as well as the 

stress-induced astrogliosis and loss of immunoreactivity of presynaptic markers (SNAP-25, 

syntaxin) in the hippocampus. The induction of a convulsive episode in post-natal day 7 (PN7) 

Wistar rats by kainate ip injection lead to a reduction of the number of entrances in a novel arm 

of a Y-maze apparatus, determined to evaluate response to novelty (2 minutes inter-trial interval) 

and spatial recognition memory (2 hours inter-trial interval) at PN90 (but not PN30 or PN60); 

caffeine consumption (1 g/L in the drinking water starting at P21) abrogated this 

developmentally-related memory dysfunction at PN90 caused by a convulsive period in infancy. 

Since diabetes is associated with an increased risk of cognitive dysfunction mainly in older 

adults, we studied the effect of caffeine treatment (1 g/L caffeine in drinking water during 4 

months) in a mouse model of type 2 diabetes associated with obesity (NONcNZO10/Ltj, 12 

months-old). These diabetic mice displayed a reduced spontaneous alternation in the Y-maze test 

and an increased astrogliosis and reduced density of synaptic proteins (SNAP25 and 

synaptophysin) in the hippocampus; caffeine consumption reduced body weight and glycaemia 

of diabetic mice, abrogated memory deficits and attenuated both astrogliosis and synaptotoxicity. 

These results support the notion that the chronic consumption of caffeine, possibly acting 

through A2A receptor blockade, confers a robust protection against memory deficits in different 

noxious brain conditions. 

Disclosures: R.A. Cunha, FCT, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); Fundação Oriente, B. Research 


already received); G.P. Cognato, CAPES, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); FCT, B. 

Research Grant (principal investigator, collaborator or consultant and pending grants as well as

grants already received); M.P. Kaster, CAPES, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); FCT, B. 


grants already received); J.M.N. Duarte, FCT, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); Fundação 

Oriente, B. Research Grant (principal investigator, collaborator or consultant and pending grants 

as well as grants already received); P.M. Canas, FCT, B. Research Grant (principal investigator, 


Poster 





Support: National Institute on Drug Abuse DA017949 TG 

National Cancer Institute/National Institute on Drug Transdisciplinary Tobacco 

Research Center Grant P5084718 

National Institute on Alcohol and Alcohol Abuse AA015515 TG 

Title: The effects of nicotine administration on the extinction and renewal of a conditioned fear 

response in mice 

Authors: *T. J. GOULD, G. A. ELIAS; 

Dept Psychol, Temple Univ., Philadelphia, PA 

Abstract: Within the population of individuals diagnosed with Post-Traumatic Stress Disorder 

(PTSD) there is a significantly higher rate of nicotine dependence and significantly lower quit 

rate compared to the population of individuals with no history of mental illness. A recent review 

suggested that the connection between PTSD and smoking might be bidirectional in that 

smoking prior to trauma increases the odds that PTSD will develop and alternatively that the 

development of PTSD increases an individual‟s susceptibility to nicotine dependence. PTSD is 

thought to result from dysfunctions in emotional regulation. Fear conditioning and subsequent 

extinction are animal models used to examine emotional learning and regulation. In this study we 

examined the effects of nicotine administration on fear extinction in an attempt to understand the 

relationship between PTSD and nicotine dependence. Mice underwent cued fear conditioning 

and 24hrs later received the first of six, daily extinction sessions in either the conditioning

context (Context A) or an altered context (Context B). Those mice extinguished in Context B 

were returned to Context A on day 6 for a final extinction session to examine the effect of 

nicotine administration on renewal. Two experimental conditions were utilized in an attempt to 

address the proposed bidirectional relationship of PTSD and nicotine dependence. In one 

condition mice received either saline or nicotine injections prior to daily extinction sessions and 

in the other, mice received injections prior to conditioning as well as extinction. Our results show 

that when nicotine is given only prior to extinction it enhances extinction and attenuates renewal 

compared to saline controls. When administered at both conditioning and extinction, nicotine 

was found to inhibit extinction when conditioning and extinction occurred in the same context 

and showed no enhancement or deficit when extinction occurred in an altered context. These 

results show that depending upon when nicotine is administered it is capable of enhancing or 

hampering fear extinction and suggests that prior smoking may sensitize an individual to 

contextual fear-related memories that may interfere with extinction of these memories while 

nicotinic drugs administered during treatment of PTSD may facilitate reduction of fear-related 

memories. 

Disclosures: T.J. Gould , None; G.A. Elias, None. 

Poster 





Title: Potentiation of acetylcholine-mediated facilitation of synaptic transmission by an 

azaindolizinone derivative, ZSET1446 (ST101), in the rat hippocampus 

Authors: K. TAKEDA 1 , *Y. YAMAGUCHI 1 , M. HINO 1 , F. KATO 2 ; 

1 Zenyaku Kogyo Co, Ltd, Tokyo, Japan; 2 Dept. of Neurosci., Jikei Univ. Sch. of Med., Tokyo, 

Japan 

Abstract: Enhancement of acetylcholine (ACh)-mediated signaling has been a promising 

strategy against cognitive impairments such as those observed in Alzheimer‟s disease (AD). We 

have demonstrated that ZSET1446 (ST101), an azaindolizinone derivative, significantly 

improves the learning deficits in rats treated with amyloid-beta or scopolamine (Yamaguchi et 

al., J. Pharmacol. Exp. Ther. 2006). As ZSET1446 also recovers the amyloid-beta-induced 

reduction in nicotine-induced ACh release in the hippocampus of freely moving rats (ibid.), we 

analyzed the effect of ZSET1446 on modulatory effects of nicotinic ACh receptor (nAChR) 

activation on spontaneous synaptic transmission in the hippocampus of the brain slices prepared

from young rats in order to clarify the mechanism underlying its procognitive effect. 

Application of ACh in the presence of atropine and physostigmine immediately increased the 

frequency of spontaneous postsynaptic currents (sPSCs) recorded in CA1 pyramidal neurons. 

ZSET1446 significantly potentiated this facilitatory effect of ACh with a maximum effect at a 

concentration of 100 pM (range tested, 10 pM - 1 nM), suggesting a bell-shaped dose-response 

relation. Such potentiation with ZSET1446 was observed both in the pharmacological isolation 

of inhibitory and excitatory sPSCs and also either in the pharmacological blockade of alpha7 or 

alpha4beta2 nAChRs. It is unlikely that this potentiation resulted from the effect on spontaneous 

transmission because the sPSC frequency before ACh application was not significantly affected 

by ZSET1446. ACh-activated inward current and ACh-induced depolarization of interneurons in 

the stratum radiatum and lucunosum-moleculare were not significantly affected by ZSET1446, 

suggesting modulation of the ACh-mediated excitation of interneurons does not underlie the 

potentiation by ZSET1446. These results indicate that ZSET1446 potentiates the nicotinemediated 

enhancement of synaptic transmission in the hippocampal neurons, without affecting 

nAChRs themselves, providing a novel possible mechanism of procognitive action that might 

partly underlie the improvement of the learning deficit in animal models of AD. 

Disclosures: K. Takeda, Zenyaku Kogyo Co, Ltd, A. Employment (full or part-time); Y. 

Yamaguchi , Zenyaku Kogyo Co, Ltd, A. Employment (full or part-time); M. Hino, Zenyaku 

Kogyo Co, Ltd, A. Employment (full or part-time); F. Kato, None. 

Poster 





Title: Behavioral phenotyping of mice knockout for the sigma-1 chaperone protein (Oprs1-/-) 

revealed sex-related memory deficits 

Authors: *T. MAURICE, F. MALHAIRE, N. CHEVALLIER; 

INSERM U.710, Montpellier, France 

Abstract: The sigma-1 receptor (S1R) is an intracellular neuromodulatory protein newly 

identified as an endoplasmic reticulum (ER)/mitochondrion interface-associated chaperone (Su 

& Hayashi, 2007, Cell 131:596). It regulates Ca2+ mobilization through IP3 receptors and cross 

activates other ER resident chaperones like Grp78/Bip. S1R therefore facilitates activitydependent 

signaling and several compounds selectively target S1R, thus appearing as a unique 

ligand-operated chaperone. S1R ligands are anti-amnesic, antidepressant, anti-addictive and

neuroprotective (Monnet & Maurice, 2006, J Pharmacol Sci 100:93). The observations that S1R 

is expressed from embryonic stage to late aging, and that its activation in control animals or 

physiological preparations does not lead to major changes drive us to characterize the behavioral 

phenotype of S1R knockout mice (Oprs1 KO). Oprs1 mice were available through the Texas 

Institute of Genomic Medicine. Male and female, wild-type (Oprs1+/+), heterozygous (Oprs1+/-) 

and nullizygous (Oprs1-/-) littermates were tested. Delivery and offspring outcomes were similar 

under homozygous or heterozygous breeding, in terms of viability scores of the litters. At 7-8 

weeks of age, mice were tested for general activity in the open-field. Parameters assessing 

locomotion, exploration and anxiety were unchanged among animal groups. The antidepressantlike 

response was tested in the forced swimming test. No difference was observed among groups, 

in terms of total immobility duration or time-course profiles. The sensitivity to classical 

antidepressants of Oprs1-/- animals has now to be examined. Learning and memory abilities 

were then examined. Spatial working memory was assessed using spontaneous alternation, 

contextual long-term memory using passive avoidance and spatial reference memory using 

water-maze learning. It appeared that male and female Oprs1+/- or male Oprs1-/- mice did not 

show significant difference in learning, as compared with wild-type controls. Female Oprs1-/-, 

however, showed significant deficits in all procedures. S1R thus appears not to be intrinsically 

necessary to memory processes, but since it is a known target for neuroactive steroids, regulation 

by sex-related steroids has to be characterized in these animals. 


France). 

Disclosures: T. Maurice, None; F. Malhaire, None; N. Chevallier, None. 

Poster 






Title: The H3 receptor antagonist ABT-239 blocks the sedative and amnesic effects of 

diphenhydramine: in vivo demonstration of a histaminergic mechanism of action 

Authors: *R. J. RADEK 1 , K. E. BROWMAN 1 , M. D. COWART 1 , M. CHENG 1 , T. A. 

ESBENSHADE 1 , T. A. FEY 1 , G. B. FOX 2 , V. A. KOMATER 1 , N. R. RUSTAY 1 , M. J. 

VOORBACH 1 , J. B. WETTER 1 , J. D. BRIONI 1 , R. S. BITNER 1 ; 

1 Neurosci. Res., 2 Integrative Pharmacol., Abbott Labs, Abbott Park, IL

Abstract: Enhanced vigilance and attention are observed with histamine H3 receptor antagonists 

in preclinical models of cognition, which may result from increased release of neurotransmitters 

that include acetylcholine, norepinephrine, and histamine. In the later case, studies in H1 receptor 

knockout mice have shown a lack of arousal normally produced by H3 antagonists, suggesting 

that the pro-vigilant effects of these agents may involve increasing extracellular histamine and 

subsequent H1 receptor stimulation. We hypothesize here that this mechanism of H3 antagonist - 

evoked histamine release would block the sedative and amnesic effects of the H1 antagonist 

diphenhydramine by increasing the amount of histamine available to H1 receptors. To test this 

hypothesis, the selective H3 receptor antagonist ABT-239 (0.3 mg/kg) was administered to rats 

and was found to block the sedative-like increases of slow-wave EEG produced by 

diphenhydramine (10.0 mg/kg). Furthermore, the amnesic effects of diphenhydramine (10.0 

mg/kg) were also blocked by ABT-239 (0.1-1.0 mg/kg) in a mouse 24-hour inhibitory avoidance 

(IA) assay. As previously reported, ABT-239 improves 5-trial IA learning in spontaneously 

hypertensive rat (SHR) pups, an indication of potential therapeutic efficacy in Attention Deficit / 

Hyperactivity Disorder (Fox et. al., 2005). In the current study, diphenhydramine (10.0 mg/kg) 

blocked the ability of ABT-239 to improve IA learning in SHR pups, suggesting that the 

histaminergic system contributes to efficacy observed in this model. The doses of ABT-239 that 

either reverse the effects of diphenhydramine in rat EEG or mouse IA, or were blocked by 

diphenhydramine in SHR pups, were within a range that increased extracellular histamine in the 

rat pre-frontal cortex as measured by in vivo microdialysis. Together, these data support a 

histaminergic mechanism for ABT-239 to enhance vigilance and attention. 

Disclosures: R.J. Radek, Abbott Laboratories, A. Employment (full or part-time); K.E. 

Browman, Abbott Laboratories, A. Employment (full or part-time); M.D. Cowart, Abbott 

Laboratories, A. Employment (full or part-time); M. Cheng, Abbott Laboratories, A. 

Employment (full or part-time); T.A. Esbenshade, Abbott Laboratories, A. Employment (full or 

part-time); T.A. Fey, Abbott Laboratories, A. Employment (full or part-time); G.B. Fox, Abbott 

Laboratories, A. Employment (full or part-time); V.A. Komater, Abbott Laboratories, A. 

Employment (full or part-time); N.R. Rustay, Abbott Laboratories, A. Employment (full or parttime); 

M.J. Voorbach, Abbott Laboratories, A. Employment (full or part-time); J.B. Wetter, 

Abbott Laboratories, A. Employment (full or part-time); J.D. Brioni, Abbott Laboratories, A. 

Employment (full or part-time); R.S. Bitner, Abbott Laboratories, A. Employment (full or parttime). 

Poster 





Support: Cephalon, Inc. 

Title: Armodafinil improves short term/working memory in a rat social recognition test 

Authors: *A. M. DICAMILLO, J. R. MATHIASEN, M. GASIOR, M. J. MARINO; 

CNS Biol., Cephalon, Inc., West Chester, PA 

Abstract: Armodafinil is the R-enantiomer of modafinil, a novel wake-promoting agent 

indicated to improve wakefulness in adult patients with excessive sleepiness associated with 

narcolepsy, obstructive sleep apnea/hypopnea syndrome, and shift work sleep disorder. There is 

evidence from both rodent and human studies (Minzenberg and Carter, Neuropsychopharmacol; 

doi: 10.1038/sj.npp.1301534) indicating that modafinil improves working memory and cognitive 

function. The rat social recognition model is a facile assay of short term/working memory where 

the social memory of an adult rat for a juvenile rat decreases as the time interval between 

presentations of the same juvenile rat is increased, e.g., an inter-trial interval (ITI) of 120 min. A 

ratio of investigational duration (trial 2/trial 1) was used as a measure of memory retention or 

loss. Robust memory loss was demonstrated in controls at a 120-min ITI. Armodafinil 

administered 30 min prior to trial 2 improved the short term/working memory of adult rats at 3, 

10 and 30 mg/kg, i.p. (30 min pretreatment; ANOVA, Dunnett‟s post hoc test p < 0.05). A 

separate group of control adult rats treated with 3 or 30 mg/kg, i.p. armodafinil and then exposed 

to novel juvenile rats in trial 2 did not exhibit any change in investigational behavior, indicating 

that the effects observed with familiar juvenile rats reflected an enhancement of shortterm/working 

memory. The effect of armodafinil contrasts to that of the psychostimulant damphetamine, 

which improved memory at low doses (0.3 and 1.0 mg/kg, i.p., 30 min 

pretreatment; ANOVA, Dunnett‟s post hoc test p < 0.05) but disrupted investigational behavior 

at 3.0 mg/kg i.p. as reflected in reduced exploration of novel juvenile rats. These data suggest 

that armodafinil produces a dose-dependent enhancement of short term/working memory in rats 

without producing a psychostimulant-like disruption of behavior. 

Disclosures: A.M. DiCamillo, Cephalon, Inc., A. Employment (full or part-time); Cephalon, 

Inc. (Armodafinil), C. Other Research Support (receipt of drugs, supplies, equipment or other inkind 

support); Cephalon, Inc., E. Ownership Interest (stock, stock options, patent or other 

intellectual property); J.R. Mathiasen, Cephalon, Inc., A. Employment (full or part-time); 

Cephalon, Inc., E. Ownership Interest (stock, stock options, patent or other intellectual property); 

M. Gasior, Cephalon, Inc., A. Employment (full or part-time); M.J. Marino, Cephalon, Inc., A. 


Poster 





Support: National Institute of Mental Health grant # MH 64494. 

Title: Blockade of alpha4beta2 nicotinic innervation of the mediodorsal thalamic nucleus 

improves working memory in rats 

Authors: R. D. CANNADY, R. WEIR, B. WEE, E. GOTSCHLICH, N. KOLIA, *E. D. 

LEVIN; 

Neurobehav Res. Lab. Box 3412, Duke Univ. Med. Ctr., Durham, NC 

Abstract: Abnormalities of nicotinic acetylcholine receptors have been linked with cognitive 

deficits seen in disorders such as schizophrenia and Alzheimer‟s disease. Nicotinic receptors are 

widely distributed in the brain. The differential roles of nicotinic receptors in different brain 

areas are still being determined. Nicotinic receptors in the hippocampus and the frontal cortex 

have been shown to play key roles in memory. Nicotinic receptors are present in high 

concentrations in the thalamus. The thalamic nucleus most closely connected to the frontal cortex 

is the mediodorsal (MD) thalamic nucleus. The involvement of MD nicotinic receptors with 

working memory was investigated in female Sprague-Dawley rats. We evaluated interactions 

between locally infused nicotinic antagonists, and systemic nicotine and the antipsychotic drug 

clozapine. The 16-arm radial maze was used to test working memory. After training on the maze 

for 18 sessions cannulae were implanted into the MD to locally deliver chronic doses of the 

alpha4beta2 nicotinic receptor antagonist DH-beta-E (100 ug/side/day), the alpha7 antagonist 

MLA (83 ug/side/day) or vehicle. The rats also received systemic nicotine (5 mg/kg/day) or 

saline, delivered sc via minipumps and acute injections of clozapine (0, 1.25, or 2.5 mg/kg, sc). 

Blockade of alpha4beta2 nicotinic receptors significantly improved working memory. Treatment 

with nicotine reversed this cognitive enhancement. MLA was not seen to significantly affect 

working memory. Clozapine did not have an effect on working memory, however there was a 

dose dependent effect on response latency. The effect of alpha4beta2 blockade in the MD 

thalamic nucleus was opposite of that previously seen in the hippocampus. The differential roles 

of nicotinic receptors in different brain areas may explain the complex effects of systemic 

nicotinic drug effects on memory. 

Disclosures: R.D. Cannady, None; R. Weir, None; B. Wee, None; E. Gotschlich, None; N. 

Kolia, None; E.D. Levin , None. 

Poster 





Title: Phencyclidine-induced reversal learning deficits in rats; role of 5-HT2C and 5-HT1A 

receptors 

Authors: *S. L. MCLEAN 1 , M. L. WOOLLEY 2 , J. C. NEILL 1 ; 

1 Sch. of Pharm., Univ. Bradford, Bradford, United Kingdom; 2 GlaxoSmithKline, Harlow, United 

Kingdom 

Abstract: Introduction: Phencyclidine (PCP) produces robust deficits in models of relevance to 

cognition in schizophrenia including reversal learning, novel object recognition, and attentional 

set-shifting in our laboratory in the rat. There is evidence for involvement of 5-HT receptor 

mechanisms, specifically 5-HT1A and 5-HT2C receptors in improving cognition in schizophrenia. 

Aim: To investigate the involvement of 5-HT receptor mechanisms in sub-chronic PCP induced 

reversal learning deficits in female rats. 

Methods: Adult female hooded-Lister rats were trained to perform an operant reversal learning 

task. Rats received sub-chronic PCP (2 mg/kg) or vehicle (1 ml/kg) i.p. twice daily for seven 

days, followed by 7-days washout. In experiment 1, PCP-treated rats received the selective 5- 

HT2C receptor antagonist, SB-243213A (1.0, 3.0, 10.0; i.p.) or vehicle and were tested 30 

minutes later. In experiment 2, PCP-treated rats received buspirone (0.15625, 0.3125, 0.625 

mg/kg, i.p.) in combination with the 5-HT1A antagonist WAY100635 0.3mg/kg or vehicle 30 

min prior to buspirone and were tested 30 minutes later. 

Results: Data are expressed as mean ± SEM and analysed by ANOVA followed by post-hoc 

Dunnett‟s t-test. In all experiments sub-chronic PCP significantly impaired reversal phase 

performance (p





HD02528 

Title: Amphetamine disrupts differential-reinforcement-of-low-rate (DRL) timing behavior by 

interfering with spatial processing in rats 

Authors: *J. W. PINKSTON 1 , E. VORONTSOVA 2 , S. LATIF 2 , S. C. FOWLER 3 ; 

1 Schiefelbush Inst. Life Span Studies, Univ. Kansas, Lawrence, KS; 3 Pharmacol., 2 Univ. of 

Kansas, Lawrence, KS 

Abstract: The DRL schedule of reinforcement has been a popular method for investigating 

pharmacological (e.g., amphetamine) or neural (e.g., hippocampal lesion) variables that affect 

multi-second timing behavior. Both types of interventions reduce the time between responses 

and decrease the number of reinforcers (rewards) earned per hr. However, little is known about 

the contribution of “off-lever” behavior to DRL timing performance in the intact or 

physiologically challenged rat. Accordingly, we used an operant chamber with a force-plate 

actometer as its floor to measure rats‟ location, locomotion, and force of movements while 

performing on a water-rewarded DRL 72-s schedule of reinforcement (reward was delivered 

only if 72 s or more separated operant responses). Rats were male Sprague Dawleys from Harlan 

(Indianopolis). Under no-drug conditions rats consistently located themselves away from the 

operandum and remained nearly motionless during the wait-time before reinforcement, with 

amount of motion decreasing significantly as the time of reinforcement availability drew near. 

Compared to the no-drug condition, amphetamine 1) induced increased locomotion, 2) abolished 

the pre-reinforcement loss of motion gradient, 3) shifted the peak of the inter-response time 

distribution down to 24 s from a no-drug value of 64 s, and 4) increased by 6.5 fold the percent 

of time that rats stayed close to the operandum. These data suggest a substantial involvement of 

spatial control in successful DRL timing performance and raise the possibility that previously 

reported hippocampal-lesion-related degradation of timing performance is the indirect result of 

disrupted spatial processing. Supported by MH43429 and HD02528. 

Disclosures: S. Latif, None; E. Vorontsova, None; S.C. Fowler, None; J.W. Pinkston , None. 

Poster 

291. Animal Cognition and Behavior: Learning and Memory: Pharmacology II




Support: Virginia Tobacco Settlement Foundation 

Title: Acute nicotine administration disrupts fear conditioning measured by potentiated startle 

but not by freezing 

Authors: *J. A. BURK, P. S. HUNT, R. C. BARNET, A. C. FEGHALI, J. M. BESHO, A. A. 

KEMPSELL, J. D. MOORE; 

Dept Psychol, Col. William & Mary, Williamsburg, VA 

Abstract: The effects of nicotine can depend upon the particular cognitive processes being 

examined (e.g. attention, working memory), the dose administered, and whether or not the 

subject has any prior experience with the drug. The present experiment addressed whether acute 

nicotine administration would affect fear conditioning, and whether this might interact with the 

manner in which this learning was assessed. Subjects were young adult Sprague-Dawley male 

and female rats (ns = 10-12/group). In the first experiment subjects were assigned to receive one 

of three doses of nicotine (0, 0.2 or 0.4 mg/kg, i.p.) immediately prior to training. For delay 

conditioning all animals were given 10 pairings of a 10-s flashing light conditioned stimulus 

(CS) with a 0.6 mA footshock unconditioned stimulus (US). In Experiment 1 the animals were 

tested for CS-elicited freezing beginning approximately 24 h after training. In Experiment 2 

subjects received nicotine (0, 0.15 or 0.4 mg/kg) immediately prior to a single training session. 

Approximately 24 h after training the animals were tested for fear-potentiated startle (FPS). Fearpotentiated 

startle was defined as a greater magnitude startle response on CS+noise trials 

compared with that recorded on noise-alone trials. The results indicated that pre-conditioning 

administration of nicotine had no observable effect on CS-elicited freezing. In Experiment 2, 

pre-conditioning administration of nicotine significantly impaired conditioned fear. Subjects 

administered the highest dose of nicotine (0.4 mg/kg) immediately prior to training failed to 

exhibit fear-potentiated startle 24 h later. Animals administered the intermediate dose (0.15 

mg/kg) exhibited some FPS, but levels were significantly lower than those observed in salinetreated 

subjects. Collectively the results indicate that nicotine can exert different effects on 

associative learning when this learning is measured via these two commonly used measures of 

fear. One possible explanation for these results is that potentiated startle is sensitive to specific 

temporal information relevant to the timing of US delivery, which the freezing response is not, 

and this timing is affected by pre-training nicotine injection. 

Disclosures: J.A. Burk, None; P.S. Hunt, None; R.C. Barnet, None; A.C. Feghali, 

None; J.M. Besho, None; A.A. Kempsell, None; J.D. Moore, None.

Poster 





Support: NIDA Grant DA017949 

NIDA Grant T32DA07237 

NIDA Grant P5084718 

Title: Effects of direct hippocampal nicotine infusion on trace fear conditioning 

Authors: *J. D. RAYBUCK, T. J. GOULD; 

Psychol/CSAR, Temple Univ., Philadelphia, PA 

Abstract: Nicotine enhances certain forms of cognition, such as attention and working memory. 

Effects of nicotine on these cognitive processes may be important during both the formation of 

nicotine addiction and during smoking cessation. Trace fear conditioning is one task thought to 

depend upon both attention and working memory. Acute, systemic nicotine administration doseresponsively 

enhances trace fear conditioning, a task shown to be hippocampus dependent. To 

determine if nicotine‟s action within the hippocampus is sufficient to enhance trace fear 

conditioning, we infused multiple doses of nicotine (0 - 0.35κg/side, freebase) bilaterally into the 

hippocampus of C57BL/6 mice 2-5 minutes prior to trace fear conditioning. Mice were trained 

with 5 CS-US pairings. The CS (30s, 85 dB white noise) and US (2s, 0.57 mA footshock) were 

temporally separated with a 30 second trace interval. Training lasted approximately 15 minutes. 

Hippocampal nicotine infusion dose-dependently enhanced trace fear conditioning over saline 

controls. These results suggest that nicotine‟s action in the hippocampus may be sufficient to 

enhance trace fear conditioning. To determine if activation of high-affinity nicotinic 

acetylcholine receptors (nAChRs) within the hippocampus is critical to nicotine-induced 

enhancement of trace fear conditioning, we administered systemic nicotine (0.09mg/kg, ip) to 

mice that received intra-hippocampal DHβE infusions (18κg/side) prior to both training and 

testing of trace fear conditioning. DHβE blocked the enhancement of trace fear conditioning, 

suggesting that activation of high-affinity nAChRs in the hippocampus is critical to nicotineinduced 

enhancement of trace fear conditioning. Collectively, these results suggest that 

activation of high-affinity nAChRs within the hippocampus may be sufficient to enhance trace 

fear conditioning. 

The authors would like to acknowledge grant support from the National Institute on Drug Abuse 

(DA017949 TG), the National Cancer Institute and National Institute on Drug Abuse (P5084718 

PI: Caryn Lerman Ph.D), additionally, Jonathan D. Raybuck was supported by a NIH/NIDA 

training grant (T32DA07237).

Disclosures: J.D. Raybuck , None; T.J. Gould, None. 

Poster 





Support: NIDA 

U.S. Department of Energy 

NIAAA 

Title: High fat food self-administration behavior in rats prone or resistant to diet-induced 

obesity: effects of bromocriptine 

Authors: *J. CHO 1 , R. KIM 1,2 , M. MICHAELIDES 1,2 , V. GOPEZ 1 , S. PRIMEAUX 3 , G. 

BRAY 3 , G. J. WANG 1 , N. D. VOLKOW 1,2 , P. K. THANOS 1,2 ; 

1 Brookhaven Natl. Lab., Upton, NY; 2 Psychology, Stony Brook Univ., Stony Brook, NY; 

3 Pennington Biomed. Res. Ctr., Baton Rogue, LA 

Abstract: Background: Dopamine (DA) and the DA D2 receptor (DR2) have been implicated 

in obesity and drug addiction. Osborne Mendel (OM) rats are obesity susceptible and S5B/PI 

(S5B) rats are obesity resistant when fed a high fat diet. We have previously shown that OM and 

S5B rats do not differ in D2R binding when fed a normal diet but there was a 60% decrease in 

striatal D2R binding in OM rats compared to S5B rats when both are fed a high fat diet for 6 

weeks. Bromocriptine (BC) is a D2R agonist and previous studies have shown that BC may 

useful in the treating obesity. The effects of BC are thought to be due to its action on central 

D2R. We hypothesized that the two strains will differ in high fat food self-administration (FSA) 

behavior and BC would differentially affect this behavior between the two strains. 

Methods: Ad-libitum fed OM and S5B rats were placed in a FSA operant chamber and were 

trained to lever press for food pellets under a Fixed-Ratio 1 (FR1) schedule. The schedule was 

progressively increased to an FR4 schedule, at which operant responding for high-fat pellets was 

maintained for 7 days. Afterwards, the schedule was changed to a progressive schedule (PR) to 

determine the lever pressing threshold for high fat food pellets. After 16 days of the PR trial, rats 

were administered three BC doses (1, 10 and 20 mg/kg). 

Results: Surprisingly, our results show no significant difference between the two strains in 

regards to the number of lever presses. Furthermore, 1mg/kg BC did not have a significant effect

on lever presses. However, 10mg/kg and 20mg/kg BC did increase in the number of lever 

presses in both strains, with 10mg/kg having the most profound effect. Although BC appeared to 

increase the number of lever presses, BC also decreased rat chow intake. 1mg/kg and 10mg/kg 

BC reduced chow intake in the OM rats. In the S5B rats, 10mg/kg and 20mg/kg BC decreased 

chow intake. Interestingly, these results were not observed on the day of BC treatment but rather 

on the following day. There was no effect on locomotor activity associated with BC treatment 

but S5B rats showed greater locomotor activity prior to BC treatment. 

Conclusion: Although OM and S5B rats differ in susceptibility to obesity and studies have 

documented distinct high fat food preference between the two strains, this study shows these rats 

do not differ in operant responding for high-fat food. BC increases operant response for high-fat 

food but decreases chow intake in both strains. The increased operant response for high fat food 

may potentially be attributed to a reduction in chow intake. Our findings may suggest D2R 

agonism from BC affects neural systems associated with homeostatic control of food intake and 

not food palatability. 

Disclosures: J. Cho, None; R. Kim, None; M. Michaelides, None; V. Gopez, None; S. 

Primeaux, Pennington Biomedical Research Center, A. Employment (full or part-time); G. 

Bray, Pennington Biomedical Research Center, A. Employment (full or part-time); G.J. Wang, 

Brookhaven National Laboratory, A. Employment (full or part-time); N.D. Volkow, National 

Institutes of Health, A. Employment (full or part-time); Brookhaven National Laboratory, A. 

Employment (full or part-time); P.K. Thanos, Brookhaven National Laboratory, A. Employment 

(full or part-time). 

Poster 






NIDA Grant DA017949 

Title: Interactive effects of ethanol and nicotine on memory, anxiety, and locomotion in 

C57BL/6 mice in the plus-maze discriminative avoidance task 

Authors: *D. GULICK, T. J. GOULD; 

Psychology, Temple Univ., Philadelphia, PA

Abstract: Introduction: The co-abuse abuse of alcohol and nicotine represents a major public 

health crisis. The National Institute of Health (2007) reports that nicotine-addicted smokers are 

four times more likely than non-smokers to be addicted to alcohol and that alcoholics are three 

times more likely than the rest of the population to smoke cigarettes. Understanding how alcohol 

and nicotine interact to modulate behavior is an essential step in treating addiction to these drugs, 

and the plus-maze discriminative avoidance task (PMDAT) allows measurement of learning and 

memory, anxiety, and locomotion at both training and testing. Methods: Male C57BL/6J mice 

were tested at 8-12 weeks of age in a modified elevated plus-maze consisting of grey Plexiglas 

floors and walls with no top. A 75-watt lamp was placed directly over one enclosed arm and a 

speaker connected to a noise generator (85 dB) was placed directly below the same arm. For the 

training session, each mouse was placed in the center of the apparatus and, for five minutes, the 

time spent in each arm or in the center area was recorded by the researcher. Each time that the 

mouse entered the aversive arm, the light and the white noise were turned on. Both cues were 

turned off when the mouse exited the aversive arm. For the testing session, each mouse was 

returned to the center of the apparatus and, for three minutes, the time of each entry into an arm 

or into the center area was recorded. No cues were turned on during the testing session. Results: 

Ethanol dose-dependently decreased anxiety, increased locomotion, and impaired memory. 

Nicotine dose-dependently increased anxiety and locomotion and impaired memory. 

Furthermore, a moderate dose of nicotine reversed decreases in memory retention due to 1.0 and 

1.4 g/kg ethanol as well as the decrease in anxiety due to 1.4 g/kg ethanol, but this dose of 

nicotine did not reverse changes due to 2.6 g/kg ethanol. A dose of ethanol (1.0 g/kg) that did not 

alter anxiety or locomotion still impaired memory, and a dose of nicotine (0.09 mg/kg) that 

decreased anxiety did not impair memory. Conclusions: There are dissociable effects of ethanol 

and nicotine, alone and in combination, on memory, anxiety, and locomotion in the PMDAT. 

This suggests that these drugs act on multiple neural systems. Furthermore, ethanol-induced 

anxiolysis may be one factor underlying alcohol consumption, while nicotine-induced reversal of 

ethanol-induced cognitive deficits may be a factor underlying co-abuse. Finally, the failure of 

nicotine to reverse the cognitive deficit associated with a high dose of ethanol suggests that there 

is a ceiling effect for mitigation of the effects of ethanol by nicotine. 

Disclosures: D. Gulick , None; T.J. Gould, None. 

Poster 





Support: NIDA Grant DA017949, TJG

NIDA Training Grant DA07237, JWK 

Title: Towards an understanding of how nicotinic acetylcholine receptors modulate 

hippocampus dependent learning and memory 

Authors: *J. W. KENNEY, T. J. GOULD; 

Dept Psychol/Neurosci, Temple Univ., Philadelphia, PA 

Abstract: Nicotine is known to enhance learning and memory in a variety of hippocampus 

dependent tasks. In particular, the enhancement of contextual fear conditioning and related tasks 

by nicotine has proven useful in delineating the neurobiological substrates that underlie the effect 

of nicotine on learning and memory. For nicotine to enhance contextual fear memories the drug 

must be administered prior to both training and recall in these tasks. In an effort to understand 

why nicotine must be administered at both of these time points we have developed a hypothesis 

as to how nicotine alters hippocampal-cortical-amygdala interactions that stems from the work of 

Rudy and colleagues (2004) and Fanselow (1999, 2000). We hypothesize that training in 

contextual fear tasks in the presence of nicotine results in the alteration of hippocampal-corticalamygdala 

connectivity such that a pathway that is not critically involved in the formation of a 

short-term contextual fear memory is recruited. This additional pathway then summates with the 

usual hippocampal-amygdala pathway that normally underlies the formation of the contextual 

fear memory resulting in the enhanced memory. For the alteration in hippocampal-corticalamygdala 

connective strength to occur we hypothesize that nicotine 1) increases hippocampalcortical 

connectivity during context learning and recall and 2) reduces hippocampal inhibition of 

the cortical input to the amygdala, an inhibition that is postulated to exist based on experimental 

data discussed by Rudy et al (2004). This modified model is capable of explaining why nicotine 

must be administered prior to both learning and recall to enhance contextual fear memories and 

makes various novel predictions. If accurate, this model will further our understanding of how 

nicotine alters contextual information processing that may play an important role in a number of 

cognitive disorders such as addiction. 

Disclosures: J.W. Kenney , None; T.J. Gould, None. 

Poster 





Support: NIDA Grant DA017949

NCI/NIDTTURC Grant P5084718 

NIAAA Grant AA015515 

Title: The effect of different time points of withdrawal from chronic nicotine on contextual fear 

in C57BL/6 mice 

Authors: *J. M. ANDRE, T. J. GOULD; 

Psych/Neuroscience Program, Temple Univ., Philadelphia, PA 

Abstract: Nicotine withdrawal is associated with a variety of symptoms that include anxiety, 

increased appetite, agitation, changes in affect, and disrupted cognition in humans. These 

symptoms may be critical factors in the high relapse rates that occur in smokers. Although 

animal models have provided useful insights into the somatic and affective symptoms of nicotine 

withdrawal, less research has focused on the effects of nicotine withdrawal on cognitive 

processes. Previous research found that withdrawal from chronic nicotine disrupted contextual 

fear conditioning in C57BL/6 mice; however, the duration of these deficits was unknown. In the 

present study, we investigated the duration of nicotine withdrawal deficits in contextual fear 

conditioning in mice chronically administered 6.3 mg/kg/day (freebase) nicotine for 12 days. 

Mice were trained in contextual fear conditioning between 24 hours to 192 hours (2-8 days) after 

cessation of nicotine treatment. Deficits were seen up to 4 days after withdrawal but then 

dissipated. 

Disclosures: J.M. Andre , None; T.J. Gould, None. 

Poster 





Title: The effects of a dopamine antagonist, SCH 23390, on learning and memory in planarians 

Authors: R. E. RUPSLAUKYTE, L. MANZA, *M. NICULESCU; 

Dept. of Psychology, Lebanon Valley Col., Annville, PA 

Abstract: Introduction: The treatment of mental health disorders have been the cause of much 

debate among professionals in the field. Many clinical disorders such as schizophrenia, bipolar, 

and depression are treated with therapeutic or pharmacological interventions. The development

of pharmacological treatments has progressed through time, but patients often complain about 

the negative side effects. One of the side effects patients often report when taking dopamine 

antagonists in the form of antipsychotics is cognitive impairment. As of now, research is lacking 

in determining how much of an effect a dopamine antagonist has on cognitive ability and 

requires further investigation. Thus, the current study‟s purpose aims to investigate how the D1 

dopamine antagonist, SCH 23390, affects learning and memory in a simple planarian model. 

Method: Twenty planarians were used in two conditions to assess learning and memory on maze 

performance: a control group (water) and an experimental group (SCH 23390). Each planarian 

was placed in a resting pool contained within the apparatus that had either water or a 1 κM SCH 

23390 for thirty minutes. Next, the planarians were placed in one end of the Y-shaped training 

maze and allowed to swim for ten trials in order to determine if the planarian had a side 

preference, left or right. After the side preference was determined, a biased design was 

employed; when choosing the preferred side, they were administered a 6V shock in order to train 

them to go to their learned side, which is the opposite of their preferred side, for ten training 

sessions. After the ten training sessions, the planarian entered the testing session by allowing 

them to go where they chose without shocks for three trials. The number of times the planarians 

chose the learned side out of three trials determined the effectiveness of their learning of the task. 

In addition, other measures were taken such as how many times the planarian chose the preferred 

side and how fast they completed the testing session. 

Results: The current study showed that SCH 23390 decreased learning and memory in maze 

performance of planarians. Specifically, the drug group had a higher error rate and moved at a 

slower rate than the control group. In addition, the drug group selected the trained side 

significantly less than the control group which suggested a decreased ability to learn the maze. 

Discussion: The current study showed that a dopamine antagonist has the potential to decrease 

cognitive behavior, specifically learning and memory behavior. Thus, since dopamine 

antagonists are contained in psychotropic drugs that humans consume, it is important to look at 

the implications it has for higher animal and human models. 

Disclosures: R.E. Rupslaukyte, None; M. Niculescu, None; L. Manza, None. 

Poster 



Program#/Poster#: 291.18/SS1 



Title: Dizocilpine (MK-801) impairs olfactory memory span in rats

Authors: *D. A. MACQUEEN 1 , L. A. BULLARD 2 , T. GUZE 2 , M. GALIZIO 2 ; 

2 Psychology, 1 UNCW, Wilmington, NC 

Abstract: The lack of procedures suitable to assess working memory capacity in non-humans 

has limited neurobiological research in this area. The olfactory memory span procedure (OMS) 

recently developed by Dudchenko, Wood & Eichenbaum (2000), appears a promising candidate 

to study working memory capacity in rodents. The present study sought to evaluate a variation of 

the OMS and used it to investigate the effects of the non-competitive NMDAr antagonist, 

dizocilpine, and the opioid agonist, morphine in rats. The OMS task was designed as an animal 

analogue to the human digit span task. Rats were placed in a large arena with 18 food locations. 

In the initial trial of each session, one food cup marked with a distinct olfactory stimulus was 

present and responding to it was reinforced. Each subsequent trial added a new olfactory 

stimulus and responding to the new stimulus was always reinforced (non-matching). The 

dependent measures were number of stimuli that incremented without error (span) and overall 

percent correct responses. Sessions were conducted five days per week and each session included 

24 trials of the OMS task as well as a performance control task involving a simple olfactory 

discrimination. Five male Sprague-Dawley rats received 30 or more sessions of training on the 

OMS task and drug administration began when performances met criteria for accuracy and 

consistency over sessions. When the drug studies began, rats were exposed to i.p. injections prior 

to the testing session twice per week with testing conducted under baseline conditions the other 

three days. Dose effect functions were determined for morphine (saline, 1.0, 3.0, 5.6, 10.0 and 

17.0 mg/kg) and dizocilpine (saline, .03, .10, .17, and .30 mg/kg). Baseline performance was 

characterized by mean spans (M=8.7) that were similar to those reported by Dudchenko et al. and 

decreasing percent correct as a function of memory set size. Both drugs produced significant 

impairments on memory span and overall accuracy at the highest doses, but in the case of 

morphine, these effects occurred only at high doses that also disrupted the performance control. 

In contrast, dizocilpine produced significant impairments on the OMS task at doses (.10 and .17 

mg/kg) that did not impair performance of the control. Thus, the effects of this NMDAr 

antagonist can be interpreted in terms of selective impairment of working memory capacity. 

Disclosures: D.A. MacQueen , None; L.A. Bullard, None; T. Guze, None; M. Galizio, None. 

Poster 





Support: NARSAD Young Investigator Award

Title: Contextual fear memory is impaired by administration of l-kynurenine prior to training 

Authors: *A. M. LANDERS 1 , A. C. CHESS 2 , D. J. BUCCI 1 ; 

1 Psychological & Brain Sci., Dartmouth Col., Hanover, NH; 2 Psychology, Univ. of Vermont, 

Burlington, VT 

Abstract: Kynurenic acid (KYNA) is synthesized and released exclusively by astrocytes and 

acts as an antagonist at α7-nicotinic acetylcholine receptors and at higher non-physiological 

concentrations, also antagonizes the glycine site of the NMDA receptor. KYNA is increased in 

the brain tissue and cerebral spinal fluid of individuals with schizophrenia or Alzheimer‟s 

disease. Both of these disorders are characterized by acetylcholine-mediated cognitive 

impairments, suggesting the possibility that increases in cerebral KYNA may be associated with 

specific cognitive dysfunction. Contextual learning, for example, has been shown to be 

compromised in individuals with schizophrenia. Recent evidence indicates that nicotine 

administration in rats enhances contextual conditioning but does not affect conditioning to a 

discrete cue. In this study, we tested the hypothesis that elevations in KYNA concentration in 

rats would selectively disrupt contextual conditioning while leaving conditioning to a discrete 

conditioned stimulus (CS) unaffected. Endogenous KYNA concentration was increased by 

administering l-kynurenine (LKYN), the precursor of KYNA. Previous studies have shown that a 

100 mg/kg dose of LKYN is sufficient to produce a 4 fold increase in KYNA concentration. Rats 

received i.p. injections of a vehicle solution or 100 mg/kg LKYN 2hr prior to training, which 

consisted of 2 presentations of a 10-sec auditory CS (a 1500 Hz, 86 dB tone) paired with a 

0.75mA, 500msec footshock. Twenty-four hours later, rats were returned to the training context 

and were tested for freezing to the context. Twenty-four hours after the context test, rats were 

placed in a novel context and were tested for freezing to the tone. Consistent with our hypothesis, 

injections of LKYN did not affect acquisition of the conditioned freezing response during 

training or the expression of conditioned freezing to the tone during the tone test session. 

However, freezing during the context test session was significantly reduced in L-KYN-treated 

rats. These data suggest that elevated levels of KYNA may contribute to contextual memory 

deficits associated with certain forms of psychopathology. 

Disclosures: A.M. Landers , None; A.C. Chess, None; D.J. Bucci, None. 

Poster 





Title: Effects of jasmine, lavender, and ylang-ylang essential oils on spatial learning in mice 

Authors: *N. PRACHANTASENA 1 , T. PIRIYAPUNYAPORN 2 , N. WANNASILP 1 , K. 

WATTANASIRMKIT 3 , N. KOTCHABHAKDI 1 ; 

1 Neuro Behav Biol. Ctr., Mahidol Univ., Nakorn Pathom, Thailand; 2 Fac. of Sci., Mahidol Univ., 

Bangkok, Thailand; 3 Dept. of Biol. Fac. of Sci. Chulalongkorn Univ., Bangkok, Thailand 

Abstract: Essential oils, secondary metabolic product of some plants, have been widely used for 

various medicinal purposes, including relaxation and memory enhancement. A number of studies 

have been shown that they have various effects on the nervous system, such as lavender 

fragrance is used to improve sleep patterns, and reduce aggression. Other kinds of essential oil 

frequently used in aromatherapy are jasmine oil. It was found to promote the EEG beta waves 

that may show the stimulating effect of this fragrance. Moreover, ylang-ylang oil is also used as 

antidepressant and is shown to increase attention. However, the importance of essential oils for 

learning and memory is still less documented. The aim of this study is to investigate the effects 

of jasmine, lavender, and ylang-ylang essential oils on spatial learning in mice. ICR Mice were 

randomly divided into three essential oil treated groups and one control group, eight mice each. 

Housing and testing were carried out at different places to avoid the odorant contamination on 

control group. 15 µl of pure essential oil were sprayed to the cotton ball, which was refreshed 

every 2 days, and put in to the cotton ball attached to the cage-covered. ICR mice were exposed 

to essential oils for 20 days, and then their spatial learning capability in the Morris water maze 

was analyzed by using video tracking system Ethovision (Noldus Information Technology, 

Netherlands). Following the behavioral study, animals were perfused and their brains were 

collected. Brain serial sections were reconstructed, and analyzed by SGI O2 running software 

Micro3D (version 2004; NeSys, Oslo, Norway). In the behavioral study, jasmine oil-treated mice 

took significantly shorter path length and time to find hidden platform in the water maze as 

compared to control group. Lavender and ylang-ylang treated mice also used slightly shorter 

time to find platform compared to controlled mice. Jasmine treated group has significantly larger 

hippocampal volume than the control group. In contrast, lavender treated group showed 

significantly decreased as compared to non-treated group. In addition, volumes of left and right 

hippocampus were not significantly different in all conditions. This study suggests that jasmine 

and perhaps ylang-ylang oil might enhance spatial learning in mice. 

Disclosures: N. Prachantasena, None; T. Piriyapunyaporn, None; N. Wannasilp, None; N. 

Kotchabhakdi, None; K. Wattanasirmkit, None. 

Poster 



Program#/Poster#: 291.21/SS4



Title: Club drugs, hippocampal neurogenesis and spatial learning in rats 

Authors: *L. A. BULLARD, E. BLACKWELL, B. BYRD, B. GRIFFIN, M. HULICK, E. 

KIDDER, P. MCKINNEY, L. POERSTAL, C. PRIESTER, B. RAYBURNE-REEVES, A. 

ROBINSON, J. KEITH, M. GALIZIO; 

UNCW, Wilmington, NC 

Abstract: Several studies have demonstrated that repeated high doses of MDMA and 

methamphetamine can be neurotoxic, producing long term depletion of 5-HT or dopamine, 

respectively. Some studies have found enduring cognitive deficits after neurotoxic doses and we 

hypothesized that these might be linked to reduced hippocampal neurogenesis. The present study 

sought to determine the effects of neurotoxic doses of MDMA and methamphetamine on 

hippocampal neurogenesis and spatial learning in rats. Subjects were first trained under a 

repeated acquisition and performance procedure adapted to the Morris Swim Task. In the 

performance component, subjects were required to swim to a hidden platform that stayed in the 

same position in the pool across sessions. In the acquisition component, subjects were required to 

swim to a hidden platform that remained in the same position within a session, but was moved to 

a different location across sessions. After training, subjects were separated into three groups 

receiving either 1) injections of 20 mg/kg MDMA every 12 hours for 4 days; 2) 4 injections of 

10 mg/kg methamphetamine every 2 hours for 1 day; 3) saline injections based on the MDMA 

regimen. Subjects were then exposed to ten additional test sessions in the Morris Swim Task. 

After completion of the final swim task session, subjects were injected with 120 mg/kg BrdU for 

4 days and were perfused 7 days later. Brains were sectioned with cryocut at 35µm and every 

sixth section was collected for immunohistochemical staining. Sections were stained with 

Doublecortin, an immature neuron marker, Ki67, a cell division marker, and BrdU/NeuN, a 

neurogenesis marker. BrdU-, Ki67-, and DCX-labeled cells in the granule cell layer and 

subgranular zone of the dentate gyrus were counted in both hemispheres from each section that 

corresponded to plates 31, 35 and 44 of Paxinos and Watson‟s atlas of the rat brain (Paxinos and 

Watson, 1998). MDMA and methamphetamine both decreased neurogenesis, but in different 

ways. MDMA reduced the production of new neurons as indicated by reduced BrdU/NeuN 

colabeling whereas methamphetamine reduced the population of immature neurons as indicated 

by a decrease in doublecortin labeling. No differences in Ki67 labeling were observed in either 

group, suggesting that at the time of perfusion cell proliferation rates both in MDMA and 

methamphetamine treated animals had recovered to control levels. Despite large MDMA- and 

methamphetamine-induced changes in hippocampal neurogenesis, the three groups did not differ 

in terms of their spatial learning or performance abilities in the Morris Swim Task. 

Disclosures: L.A. Bullard , None; E. Blackwell, None; B. Byrd, None; B. Griffin, None; M. 

Hulick, None; E. Kidder, None; P. McKinney, None; L. Poerstal, None; C. Priester, None; B. 

Rayburne-Reeves, None; A. Robinson, None; J. Keith, None; M. Galizio, None.

Poster 





Support: SENACYT Grant FID06-001 

Title: Long-term exposure to clinically relevant doses of oral methylphenidate has enduring 

effects on emotional behaviors in rats 

Authors: J. A. BETHANCOURT 1,2 , Z. Z. CAMARENA 1,2 , C. B. SALAZAR 3 , G. C. 

QUINTERO 1 , *G. B. BRITTON 1 ; 

1 Cognición, Cerebro y Conducta, INDICASAT, Panama, Panama; 2 Facultad de Psicología, Univ. 

de Panamá, Panamá, Panama; 3 Facultad de Psicología, Univ. Católica Santa María la Antigua, 

Panamá, Panama 

Abstract: Methylphenidate (MPH) is the most commonly prescribed psychostimulant used in 

the treatment of symptoms associated with attention-deficit/hyperactivity disorder (ADHD) in 

children, adolescents and adults. Despite its widespread use, general safety and effectiveness, a 

rising concern involves its potential to modify neural developmental processes that take place 

during pre- and peri-adolescence and to produce enduring changes in neural structure and 

function. Behavioral studies have shown that rats treated with MPH during early developmental 

periods exhibit cognitive and emotional deficits in adulthood, presumably due to long-lasting 

effects of MPH on maturing dopaminergic systems. Whether the therapeutic benefits of MPH 

outweigh its effects on neurobehavioral processes remains unknown. Because a growing 

proportion of adolescents continue MPH use into young adulthood, the present study examined 

the long-term effects of MPH administration from early developmental periods through 

adulthood on emotional and cognitive behaviors, and spontaneous locomotion and exploration. 

Peri-adolescent Wistar rats (beginning at post-natal day 28) were treated twice daily with MPH 

(2, 3 or 5 mg/kg) for seven weeks using an oral administration procedure that parallels clinical 

practice (weekdays only). Two weeks following the last drug treatment, animals were tested over 

a five-week period on a battery of procedures including the open field, novel object recognition, 

light-dark transition, elevated plus maze, and fear conditioning paradigms. We found that rats 

exposed to 3 or 5 mg/kg exhibited increases in anxiety-related behaviors measured in the open 

field, light-dark transition, and fear conditioning tests, but no deficits were observed in 

locomotion or exploration, or in the object recognition test at short or long retention intervals. 

These results are a first demonstration that MPH administration from juvenile periods through 

adulthood at clinically relevant doses has enduring effects on emotional responding without

affecting cognitive, motor, or exploratory behaviors. Parallel studies examining the underlying 

brain plasticity produced by exposure to psychostimulants aimed at pediatric populations are 

merited. 

Disclosures: J.A. Bethancourt, None; G.B. Britton , None; Z.Z. Camarena, None; G.C. 

Quintero, None; C.B. Salazar, None. 

Poster 






Title: Long-term effects of adolescent nicotine exposure on context conditioning and the 

character of fear expression 

Authors: *R. C. BARNET, J. A. BURK, P. S. HUNT, J. C. SMYTH; 

Col. of William & Mary, Williamsburg, VA 

Abstract: Adolescence is a critical period of brain development during which increased 

susceptibility to the detrimental effects of drugs has been observed. Interest in the impact of 

nicotine exposure during this period is considerable because of nicotine‟s known effects on 

memory and brain function (e.g., Gould, 2006; Slotkin, 2002) and also because it is one of the 

most commonly used drugs by human adolescents. An important missing gap in nicotine 

research concerns how long lasting the consequences are of adolescent nicotine exposure on later 

brain and cognitive function (cf. Smith et al., 2006). Our research examined long-term effects of 

adolescent nicotine exposure in rats on context conditioning, a hippocampus-dependent memory 

task. Rats were exposed to nicotine as adolescents via osmotic minipump (0, 3, or 6 mg/kg/d) on 

postnatal days (PD) 28-42 and later trained and tested for context conditioning as adults 

beginning PD60. During the context conditioning session, rats were exposed to 10 unsignaled 

shocks. Minute-by-minute freezing in a later shock-free context test was taken as an indicator of 

contextual fear learning. When freezing was assessed during the minute of the test session which 

corresponded to shock onset on the prior training day, a dose-dependent impairment in context 

conditioning was observed. When freezing was assessed across the entire span of the test session, 

a dose-dependent decrease in latency to peak freezing was observed. That is, drug exposed rats 

displayed maximal fear responses at earlier points in the test session than controls. In context 

conditioning used here with rats, outcomes suggest (a) the effects of exposure to nicotine during

adolescence last well into adulthood, (b) nicotine and non-nicotine exposed adolescents do not 

regulate fear in the same way later as adults, and (c) adults previously exposed to nicotine as 

adolescents display a more immediate vulnerability to the effects of environmental stressors, 

when those effects are measured during real-time exposure to the stressor. Supported by a grant 

from the Virginia Tobacco Settlement Foundation. 

Disclosures: R.C. Barnet , None; J.A. Burk, None; P.S. Hunt, None; J.C. Smyth, None. 

Poster 





Support: Plan de Promoción de la Investigación en la UNED 

Agencia Antidroga de la Comunida de Madrid 

ISCIII RD06/0001/0029 

SAF2007-064890 

S-SAL/0261/200 

Title: Hippocampal glutamatergic systems in the hippocampus of adult animals chronically 

exposed to a cannabinoid agonist during adolescence 

Authors: *A. HIGUERA MATAS, M. MIGUENS, C. GARCÍA-LECUMBERRI, E. 

AMBROSIO; 

Psychobiology, UNED, Madrid, Spain 

Abstract: Marihuana is the illicit drug most widely consumed during adolescence. This early 

cannabinoid exposure has been linked to long lasting alterations in learning and memory 

processes; however, as yet, the neural bases of these alterations remain largely unknown. Longterm 

potentiation is the form of synaptic plasticity most directly related to information storage in 

the brain and it heavily relies on glutamatergic neurotransmission. Given the relevance of this 

amino acid transmitter in synaptic plasticity and learning and memory, we devised this study to 

evaluate the long term changes in this system in the hippocampus of adult animals exposed to 

cannabinoids during adolescence. We used Wistar albino rats of both sexes. The chronic

treatment consisted on 11 injections (from postnatal day 28 to postnatal day 38; 0.4mg/2ml/kg) 

of the cannabinoid agonist CP 55,940 (CP) or its corresponding vehicle 

(ethanol:cremophor:saline; 1:1:18; VH). Microdialysis was carried out when these animals 

reached adulthood (beginning on postnatal day 100) in the CA1 region of the hippocampus and 

glutamate content in the samples was analyzed by means of capillary electrophoresis with laserinduced 

fluorescence detection. Additionally, in situ hybridization was performed to analyze the 

gene expression glutamate transporters in the hippocampus of animals subjected to the same 

cannabinoid treatment. Our results shown that male had higher levels of extracellular glutamate 

than females. There was a trend in CP-males to have lower glutamate levels than their VHtreated 

controls while the reverse happened in the females. However, no changes in glutamate 

transport were evident given that the gene expression of the glutamate transporter was similar in 

all the groups. 

Our results point to long-lasting alteration in hippocampal glutamate systems as a result of a 

chronic cannabinoid exposure during adolescence. This data might have some relevance to 

explain the enduring cognitive alterations induced by adolescent marihuana chronic 

consumption. 

Disclosures: A. Higuera Matas , None; M. Miguens, None; C. García-Lecumberri, None; E. 

Ambrosio, None. 

Poster 





Title: Chronic caffeine administration prevents stress-induced impairment of hippocampusdependent 

memory 

Authors: *K. H. ALZOUBI 1 , K. ABDUL-RAZZAK 1 , O. KHABOUR 2 , G. AL-TUWEIQ 3 , K. 

ALKADHI 4 ; 

1 Dept Clin. Pharm., 2 Dept Med. Lab. Sci., 3 Dept Legal Med., Jordan Univ. of Sci. & Technol., 

Irbid, Jordan; 4 Univ. of Houston, Houston, TX 

Abstract: Psychosocial stress induces cognitive and memory impairment in experimental 

animals and humans. Caffeine has been shown to be effective in alleviating memory impairment 

associated with certain mental disorders. It is unknown whether chronic caffeine treatment 

prevents stress-induced memory impairment. Therefore, we investigated the combined effects of 

chronic caffeine treatment and psychosocial stress on hippocampus-dependent learning and

memory measured in the radial arm water maze (RAWM) task. Chronic stress was induced using 

an intruder psychosocial stress model for 2.5-3 months. Caffeine was administered in drinking 

water (0.35mg/ml) concurrently with stress. In the RAWM, rats were allowed the following: an 

acquisition phase of two blocks of 6 consecutive trials separated by a 5 min rest period, 15 

minutes short-term memory, 5 hours and 24 hours long-term memory tests per day for 5 

consecutive days or until the animal reaches days to criterion (DTC) in the 12 th acquisition trial 

and in all memory tests. DTC is the number of days that animal takes to make zero error in two 

consecutive days. Groups were compared based on the number of errors per trial or test as well 

as on the DTC. On days 4 and 5, caffeine treated stressed animals made significantly fewer 

errors than untreated stressed animals in short-term memory and long-term memory tests. In 

addition, the number of errors made by the caffeine treated stressed animals was not different 

from that of the control or the caffeine only groups. Furthermore, DTC value for caffeine treated 

stressed animals is significantly lower than that of untreated stressed animals and not different 

than those of control or caffeine only groups. Therefore, our study shows that chronic caffeine 

administration prevents or attenuates chronic stress-induced impairment of hippocampusdependent 

short-term and long-term memory. 

Disclosures: K.H. Alzoubi , None; O. khabour, None; K. Abdul-Razzak, None; G. Al- 

Tuweiq, None; K. Alkadhi, None. 

Poster 





Support: Support Contributed By: NIDA, NIAAA (Intramural Research Program, LNI), by the 

U.S. Department of Energy under contract DEAC02-98CH10886. http://www.bnl.gov/thanoslab 

Title: Differential expression and disruption of cocaine conditioned place preference in rats 

prone or resistant to diet induced obesity 

Authors: *R. KIM 1,2 , J. CHO 1 , D. SHUSTAROVICH 1,2 , M. MICHAELIDES 1,2 , B. J. 

ANDERSON 2 , J. K. ROBINSON 2 , S. D. PRIMEAUX 3 , G. A. BRAY 3 , G.-J. WANG 1 , N. D. 

VOLKOW 4 , P. K. THANOS 1,2,4 ; 

1 Behav Neuropharm,Neuroimag Lab., Brookhaven Natl. Lab., Upton, NY; 2 Psychology, Stony 

Brook Univ., Stony Brook, NY; 3 Dietary Obesity Lab., Pennington Biomed. Res. Ctr., Baton 

Rouge, LA; 4 Niaa, nih, Lab. of Neuroimaging, Bethesda, MD

Abstract: Background: Dopamine (DA) and the DA D2 receptor (D2R) have been implicated in 

obesity as well as drug addiction and are specifically thought to be involved in the rewarding 

properties of natural reinforcers (food) and unnatural reinforcers (drug reward) (Volkow and 

Wise, 2006). Osborne Mendel (OM) rats are genetically prone to obesity and S5B rats are 

genetically resistant to obesity when fed a high fat diet. We have previously shown that normal 

ad-libitum fed OM and S5B/P rats do not differ in D2R binding but after given a high-fat diet (35 

%) for 6 weeks OM rats showed a 60 % decrease in D2R binding in the striatum compared to 

S5B/P rats (Primeaux et al., 2007). Bromocriptine (BC) is a D2R agonist and previous studies 

have shown that BC may help in the treatment for both cocaine abuse and obesity (Hoffman, 

2007). These effects of BC are thought to be primarily due to its action on central D2R. Based on 

our findings of a differential high-fat diet preference and D2R binding profile in these rats, we 

hypothesized that OM and S5B/P rats will show a differential preference to cocaine as assessed 

by using a conditioned place preference (CPP) procedure. Furthermore, we hypothesize that BC 

may differentially affect preference for cocaine in the two strains. 

Methods: OM and S5B/P rats were conditioned with cocaine (5 or 10 mg/kg) in one chamber 

and saline in the other for 8 days. Rats were then tested for cocaine preference. The time spent in 

the cocaine paired chamber was measured after different doses of BC (0.5, 1, 5, 10 & 20 mg/kg) 

and vehicle treatments. 4 reconditioning sessions separated each test session. Results: Our results 

demonstrated that OM rats showed less of a preference for the cocaine-paired chamber relative to 

S5B/P rats. Furthermore, high doses of BC (10mg/kg and 20mg/kg) greatly reduced the 

preference for the cocaine-paired chamber in OM rats but only slightly reduced the time S5B rats 

spent in the cocaine-paired chamber. Conclusion: Rats that are genetically prone to diet-induced 

obesity may be less susceptible to the reinforcing effects of cocaine and more susceptible to a 

reduction in the reinforcing effects of cocaine upon treatment with BC. Finally, this study raises 

the question of whether people that differ in susceptibility to obesity also differ in susceptibility 

to cocaine abuse and our findings suggest that individuals who are resistant to diet-induced 

obesity may be more vulnerable to abusing cocaine. 

Disclosures: R. Kim, None; J. Cho, None; D. Shustarovich, None; M. Michaelides, 

None; B.J. Anderson, None; J.K. Robinson, None; G. Wang, None; S.D. Primeaux, 

None; G.A. Bray, None; N.D. Volkow, None; P.K. Thanos, None. 

Poster 





Support: NIDA Grant DA017949 TG

NCI Grant P5084718 PI: Caryn Lerman Ph.D 

NIAA Grant AA015515 TG 

Title: The effects of nicotine withdrawal on contextual learning depend upon whether 

acquisition occurs before or after withdrawal 

Authors: *G. S. PORTUGAL, T. J. GOULD; 

Psychology, Temple Univ., Philadelphia, PA 

Abstract: The present study investigated whether nicotine withdrawal disrupted recall of prior 

contextual memories or acquisition of contextual memories by examining the effects of nicotine 

withdrawal on nicotine conditioned place preference (CPP; a measure of appetitive contextual 

learning) and contextual fear conditioning (a measure of aversive contextual learning). Nicotine 

CPP was evaluated in a two-chamber apparatus. Following seven conditioning trials, C57BL/6J 

mice treated with .35 mg/kg nicotine exhibited a significantly greater preference for an initially 

non-preferred chamber that was paired with nicotine. After initial tests for nicotine CPP, mice 

were implanted with mini-osmotic pumps containing 6.3 mg/kg/d nicotine or saline. Twelve days 

later, all pumps were removed and nicotine CPP was tested 24 hours later. Mice withdrawn from 

chronic nicotine or saline exhibited a CPP for the nicotine-paired chamber, suggesting that older 

drug-context associations are not disrupted by nicotine withdrawal. Next, the same mice were 

trained and tested in contextual and cued fear conditioning; nicotine withdrawal disrupted 

contextual but not cued fear conditioning. A subsequent experiment investigated the effects of 

nicotine withdrawal on fear conditioning when acquisition occurred before nicotine withdrawal. 

Nicotine withdrawal had no effect on the recall of contextual or cued fear conditioning. Taken 

together, these data suggest that nicotine withdrawal disrupts new contextual learning, but does 

not alter contextual learning that occurred before withdrawal. 

Disclosures: G.S. Portugal, None; T.J. Gould, None. 

Poster 





Support: NIMH Z01-MH-002498-17

Title: Triple knockouts of the mouse vasopressin 1a, vasopressin 1b and oxytocin receptors are 

fertile and useful reagents for antibody controls 

Authors: *S. YOUNG 1 , H.-J. LEE 1 , G. KANNAN 1 , S. J. LOLAIT 2 , A. IACANGELO 1 , E. 

SHEPARD 1 , A.-M. O'CARROLL 2 ; 

1 SNGE, NIMH, NIH, DHHS, Bethesda, MD; 2 LINE, DHB, Univ. of Bristol, Bristol, United 

Kingdom 

Abstract: The nonapetides vasopressin (Avp) and oxytocin (Oxt) are essential for normal social 

behavior in mammals. They exert their actions in the brain through three receptors - the 

vasopressin 1a (Avpr1a), vasopressin 1b (Avpr1b) and oxytocin (Oxtr) receptors (1). The 

development of mouse knockouts (KOs) of these receptors has greatly facilitated our 

understanding of their respective roles in regulating the behaviors, as well as peripheral actions, 

generated by Avp and Oxt. As we have KOs for each of the receptors (2-4), we investigated the 

effects of eliminating all 3 receptors in single mice (3KOs). These 3KOs were viable with no 

apparent gross physical abnormalities. Furthermore, both male and female 3KOs were fertile 

when mated with C57Bl/6 mates. A 3KO female had litters at 30, 21 and 21d and a 3KO male at 

20 and 28 days after matings (continuous pairing). The females were, as expected, unable to 

support their pups through lactation. Also, double KOs-Single Hets (2KO1Het, Avpr1a/Avpr1b - 

Oxtr) were fertile, although slightly less than normal (6.2 pups per 1st and 2nd litters at an 

average of 30 days after mating with continuous pairing, range of 22-48d). Although there are 

practical difficulties in obtaining enough 3KO and control mice for behavioral studies (crossfostering 

C57Bl/6 pups with the 2KO1Het dams may be a suitable compromise), these mice 

might be quite useful for studies of stress responses or residual CNS responses to Avp or Oxt, for 

example. In addition, as the Oxtr KO is conditional, it could be selectively inactivated at a later 

time. Finally, these single and multiple KOs are useful to examine the specificity of receptor 

antibodies. We are in the process of screening available Avpr1b and Oxtr antibodies by western 

blotting and, to date, none is effective in the mouse. We will make this data available on our 

website (http://intramural.nimh.nih.gov/lcmr/snge/). 

1. Caldwell, HK, Young, WS 3rd (2006) In: Handbook of Neurochemistry and Molecular 

Neurobiology. pp. 573-607. Ed. R. Lim. Springer: New York. 

2. Wersinger, SR, et al. (2002). Mol. Psychiat. 7, 975-984. 

3. Caldwell, HK, et al. (2006) Front. Neuroendocrinol. 27, 126. 

4. Lee, H-J, et al. (2008) Endocrinology Epub Mar 20. 

Disclosures: S. Young , None; H. Lee, None; G. Kannan, None; S.J. Lolait, None; A. 

Iacangelo, None; E. Shepard, None; A. O'Carroll, None. 

Poster 






Title: Improvement of sensory gating by the histamine H3 antagonist ABT-239 is blocked by the 

muscarinic antagonist scopolamine 

Authors: *H. ROBB, R. S. BITNER, J. D. BRIONI, R. J. RADEK; 

Neurosci. Res., Abbott Labs, Abbott Park, IL 

Abstract: ABT-239 is an H3 receptor antagonist that improves sensory gating in DBA/2 mice. 

Gating is a processing function of the CNS that filters redundant sensory information and is 

deficient in schizophrenic patients. H3 antagonists increase extracellular histamine and 

acetylcholine (ACh), which may mediate the effects of ABT-239 on sensory gating. To 

investigate this, we attempted to block the effects of ABT-239 on sensory gating in mice with 

antagonists of histamine and ACh receptors. Sensory gating was assessed by recording 

hippocampal evoked potential (EP) EEG in freely moving DBA/2 mice. EPs were elicited with 

pairs of auditory clicks, and gating ratios were calculated by dividing the amplitude of the second 

EP (click 2) by the amplitude of the first EP (click 1). In the first study, the histamine H1 receptor 

antagonist diphenhydramine (10.0 mg base/kg) failed to block the effect of ABT-239 (3.0 mg/kg 

i.p.) to lower gating ratio and improve gating. An ACh receptor mechanism was investigated by 

pretreating animals with either the nicotinic ACh receptor antagonist mecamylamine (MEC, 5.0 

κmol/kg), or the muscarinic ACh receptor antagonist scopolamine (SCOP, 1.0 mg base/kg). 

SCOP, but not MEC, blocked the effects of ABT-239 to improve gating. In summary, these 

studies suggest that the effect of ABT-239 on sensory gating is mediated by muscarinic ACh 

receptors, but not nicotinic ACh or histamine H1 receptors. 

Disclosures: H. Robb, Abbott Laboratories, A. Employment (full or part-time); R.S. Bitner, 

Abbott Laboratories, A. Employment (full or part-time); J.D. Brioni, Abbott Laboratories, A. 

Employment (full or part-time); R.J. Radek, Abbott Laboratories, A. Employment (full or parttime). 

Poster 






Title: Adolescent nicotine exposure disrupts context conditioning in adulthood 

Authors: *A. M. SPAETH, J. C. SMYTH, P. S. HUNT, J. A. BURK, R. C. BARNET; 

Dept Psychol, Col. of William & Mary, Williamsburg, VA 

Abstract: In humans, adolescence is a time when nicotine administration is most likely to begin. 

The long-term effects of adolescent nicotine exposure on learning remain unclear. The current 

experiment tested the effects of adolescent nicotine exposure on context conditioning in 

adulthood in rats. Sprague-Dawley rats were exposed to saline or nicotine (6.0 mg/kg/day) via 

osmotic minipump during adolescence (P 28-42) and learning was assessed during adulthood (P 

65). After inducing water restriction, rats were acclimated to a chamber with an available sipping 

tube. After the acclimation sessions, half of the rats received a mild electric footshock (1.0 s, 1.0 

mA) and half did not. When the animals were placed back in the same chamber during the test 

session, the latency to lick from the sipping tube was recorded. For rats previously exposed to 

footshock, nicotine decreased the latency to lick compared with saline-treated rats. Compared to 

saline treatment, nicotine had no effects on performance of rats that had not been previously 

exposed to footshock. Collectively, these data suggest that adolescent nicotine exposure 

decreases context conditioning in adulthood, at least when measured by lick suppression. The 

lack of effects of adolescent nicotine treatment on rats unexposed to footshock suggests that the 

effects of nicotine on context conditioning are not due to nonspecific drug effects, such as 

motivation to approach the sipping tube. These findings suggest that adolescent nicotine 

exposure can have long-term effects on the neural circuitry responsible for context conditioning. 

Disclosures: A.M. Spaeth, None; J.C. Smyth, None; P.S. Hunt, None; J.A. Burk, None; R.C. 

Barnet, None. 

Poster 

292. Acetylcholine, Neurotrophins, and Cognition 



Topic: F.02.d. Cognitive learning and memory systems 

Support: CIHR 

Title: Evidence for a possible role for Quinone Reductase 2 in neuroprotection and memory in 

rats

Authors: *C.-E. BENOIT 1,2 , J. BROUILLETTE 2 , S. BASTIANETTO 2 , J. A. BOUTIN 3 , P. 

DELAGRANGE 3 , M. WIERZBICKI 3 , P. SARRET 1 , R. QUIRION 2 ; 

1 Univ. Sherbrooke, Sherbrooke, QC, Canada; 2 Douglas Mental Hlth. Univ. Inst., Montreal, QC, 

Canada; 3 Inst. de Recherches Servier, Croissy-sur-Seine, France 

Abstract: Scopolamine is a well known amnesic drug that blocks muscarinic receptors. Animals 

treated with this cholinergic blocker develop memory impairments. It thus represents a 

psychopharmacological model to investigate molecular mechanisms associated with cognitive 

deficits. Using a gene microarray approach and confirmed with RT-PCR, we have recently 

demonstrated the differential expression of quinone reductase 2 (QR2), a cytosolic flavoprotein, 

in rats treated with scopolamine as well as in aged memory-impaired animals following spatial 

memory stimulation in the Morris water maze (MWM) task. Immunohistochemistry was used to 

demonstrate the expression of QR2 in the hippocampal formation. In the present study, we 

investigated the potential functional significance of these findings using both in vitro and in vivo 

approaches. A specific QR2 enzyme inhibitor was investigated for its potential neuroprotective 

effect against cell death induced by growth factor deprivation in primary rat hippocampal cell 

cultures. Co-treatment with the specific QR2 enzyme inhibitor protected, in a concentrationdependent 

manner, against cell death in this model. The compound was then investigated in 

scopolamine (2 mg/kg) treated adult rats. ICV infusion was performed over 7 days using osmotic 

mini-pumps. It reversed in a dose-dependent manner cognitive deficits induced by scopolamine. 

A pilot study suggests that this specific QR2 enzyme inhibitor decreased latency time in the 

MWM and reversed aged associated memory impairments. Taken together, these results suggest 

that the inhibition of QR2 can facilitate learning behaviours and can be neuroprotective in our 

models. 

Disclosures: C. Benoit , None; J. Brouillette, None; S. Bastianetto, None; J.A. Boutin, 

None; P. Delagrange, None; M. Wierzbicki, None; P. Sarret, None; R. Quirion, None. 

Poster 





Title: The activity of cholineacetyl transferase (ChAT) is increased in HCNP precursor 

transgenic mouse 

Authors: N. UEMATSU 1 , N. MATSUKAWA 1 , T. TOYODA 1 , T. SAGISAKA 1 , T. 

KANAMORI 1 , Y. HASHIZUME 2 , *K. OJIKA 1 ;

1 Dept. of Neurol., Nagoya City Univ. Med. Sch., Nagoya, Japan; 2 Inst. for Med. Sci. of Aging, 

Nagakute, Japan 

Abstract: The cholinergic neuronal system of septo-hippocampus is known to play a important 

role in memory. The reduction of acethylcholine, due to the inactivation of 

cholineacethyltransferase (ChAT), was ascertained in this cholinergic system in animal models 

of dementia and in patients with Alzheimer‟s disease. 

Hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat 

hippocampus, stimulates the synthesis of acetylcholine in the septal nucleus, inducing the 

activity of ChAT in vitro, and can be released from the neurons of hippocampus by the 

stimulation of N-methyl-D-asparate (NMDA)-type receptor in vitro. 

To examine directly the involvement of HCNP and HCNP-precursor protein(HCNP-pp) in 

memory formation, we generated the conditional transgenic mouse of HCNP-pp, driven by 

αCaMKII promoter. In transgenic mouse, we confirmed the amelioration of learning ability, and 

the increase of ChAT positive neurons in medial septal nucleus in vivo. These results suggest 

that HCNP and HCNP-pp can be one of key molecules to play a crucial role in memory in vivo. 

Disclosures: N. Uematsu, None; N. Matsukawa, None; T. Toyoda, None; T. Sagisaka, 

None; T. Kanamori, None; K. Ojika , None; Y. Hashizume, None. 

Poster 





Support: NINDS Grant RO1NS054272 

Title: The effects of tacrine on spontaneous alternation performance and hippocampal 

acetylcholine release in an animal model of diencephalic amnesia 

Authors: *J. J. ROLAND, M. LEVINSON, J. PATEL, L. M. SAVAGE; 

Psychology, Univ. Binghamton, Binghamton, NY 

Abstract: Tacrine, an acetylcholinesterase inhibitor (AChE-I), has been used to treat the 

memory deficits associated with amnesia and dementia. However, AChE-Is have had mixed 

efficacy in human patients as well as in a variety of animal models of cognitive dysfunction. In 

the present study, pyrithiamine-induced thiamine deficiency (PTD) was used to create a rodent 

model of diencephalic amnesia. PTD treatment results in diencephalic lesions, septohippocampal

cholinergic deficiencies, and learning impairments and is positively affected by hippocampal 

administration of AChE-Is. Different delivery methods (systemic vs. intraseptal) of tacrine were 

behaviorally tested in PTD and pair-fed control animals. In all animals, behaviorally relevant 

hippocampal ACh release was assessed with in vivo microdialysis during performance of a 

spontaneous alternation task. When tacrine was administered systemically or infused directly 

into the medial septum, it reduced but did not eliminate the behavioral impairment. This data 

should be contrasted with previous results that demonstrated that when the AChE-I 

physostigmine was administered to the hippocampus, the behavioral impairment seen in PTD 

animals was completely eliminated. Thus, although the medial septum has been suggested as the 

critical site of action for AChE-Is, such drugs may be more efficacious if they can be selectively 

targeted at the hippocampus. 

Disclosures: J.J. Roland , None; M. Levinson, None; J. Patel, None; L.M. Savage, None. 

Poster 





Support: NINDS RO1NS054272 

Title: Cholinergic cortical dysfunction in an animal model of diencephalic amnesia 

Authors: *S. J. ANZALONE 1 , R. P. VETRENO 2 , L. M. SAVAGE 3 ; 

1 Binghamton univ, Vestal, NY; 2 Psychology, 3 Binghamton univ, Binghamton, NY 

Abstract: Pyrithiamine-induced thiamine deficiency (PTD) is used to create a functional rodent 

paradigm of Wernicke-Korsakoff syndrome. The PTD model has been associated with various 

cholinergic deficits, including impairments in hippocampal acetylcholine (ACh) efflux, medial 

septal cholinergic cell loss and degeneration of select cholinergic fibers. However, it is unknown 

though whether these cholinergic deficits are limited to subcortical areas or extend to cortical 

regions as well. The current study used in-vivo microdialysis to assess ACh levels during 

spontaneous alternation in two cortical regions, the frontal and retrosplenial cortices, as well as 

the ventral hippocampus in PTD and control (PF) rats. As in previous studies from our 

laboratory, ACh efflux in the hippocampus of PTD-treated rats was significantly lower than 

those observed in the PF group during spontaneous alternation. Cortical assessment of ACh 

efflux showed a marked decrease in the frontal cortex and a moderate decrease in the 

retrosplenial cortex during spatial exploration as a function of PTD treatment. These results

suggest that decreased activity in cortical regions-particularly the frontal cortex contributes to 

cognitive dysfunction in WKS and potentially diencephalic amnesia. 

Disclosures: S.J. Anzalone , None; R.P. Vetreno, None; L.M. Savage, None. 

Poster 





Support: NINDS Grant RO1NS054272 

Title: The role of anterior thalamic nuclei and mammillary bodies in spatial exploration and 

hippocampal acetylcholine release: relation to diencephalic amnesia 

Authors: *R. P. VETRENO 1 , L. M. SAVAGE 2 ; 

1 Dept Psychology, 2 Binghamton Univ. - SUNY, Binghamton, NY 

Abstract: Several nuclei of the diencephalon are important for normal learning and memory 

function. However, the specific diencephalic nuclei and/or neural pathways that must undergo 

damage to result in memory impairment have not yet fully been determined. Damage to certain 

nuclei and fiber systems within the diencephalon interrupt the flow of information between key 

memory structures. Thus, the diencephalon may serve as a link between limbic and cortical 

structures, and damage to the diencephalon can contribute to amnesia through what has been 

called a "disconnection syndrome". 

In the current investigation using male Sprague-Dawley rats, N-methyl-D-aspartate (NMDA) 

was used to produce lesions of either: (1) the anterior thalamic nuclei (ATH), or (2) the 

mammillary bodies (MMB). In vivo acetylcholine (ACh) efflux in the ventral hippocampus was 

measured as the subjects were tested on a spontaneous alternation task. Statistically, ATH and 

sham-lesioned (SHAM) control rats did not differ in the number of arms entered during testing, 

but the ATH animals demonstrated significantly impaired alteration rates relative to the SHAM 

subjects. The ATH animals also demonstrated a statistically significant increase in perseveration 

rates. The preliminary data on MMB lesioning was suggestive of impaired spontaneous 

alternation and increased perseveration. We will assess whether lesioning to the anterior thalamic 

nuclei and mammillary bodies impair ACh efflux and functioning in the hippocampus. The goal 

of this neurochemical analysis was to assess the “disconnection syndrome” hypothesis. 

Disclosures: R.P. Vetreno , None; L.M. Savage, None.

Poster 





Title: Transcriptional changes accompanying the long lasting increase in Alpha-7 receptor 

binding and efficacy in novel object recognition after acute, low dose AZD0328 administration 

Authors: *A. WILLIAMS, J. WERKHEISER, S. SYDSERFF, J. SMITH; 

AstraZeneca, Wilmington, DE 

Abstract: AZD0328 is a selective Alpha-7 neuronal nicotinic receptor agonist. Previously we 

have shown acute administration of low dose AZD0328 (0.0001 mg/kg) induces a significant 

(p






MRC UK 

Title: Cholinergic depletion of the inferior temporal cortex interferes with recovery from 

episodic memory deficits 

Authors: *P. L. CROXSON, P. G. F. BROWNING, D. GAFFAN, M. G. BAXTER; 

Dept Exptl. Psychol, Univ. of Oxford, Oxford, United Kingdom 

Abstract: Cholinergic innervation of the temporal lobe has been suggested to have a role in 

episodic memory, a function which is also disrupted by lesions or disconnections of the medial 

temporal lobe circuit. Acetylcholine may be necessary for the specific function of some brain 

regions. Alternatively, it may be necessary for cortical plasticity and remodeling in those 

conditions in which the animal has to adapt following new task demands or injury. 

To investigate the role of cholinergic projections to inferotemporal cortex in episodic memory, 

and how loss of these projections might interact with damage to other brain structures necessary 

for normal memory function, we trained monkeys preoperatively on object-in-place scene 

discrimination problems until they could rapidly learn many problems within a testing session. 

Because learning occurs rapidly, mostly in a single trial, and depends on the presentation of 

discrimination problems in unique background scenes, this task models key features of human 

episodic memory. For the first stage of the experiment, the monkeys then received either a fornix 

transection or mammillary body ablation, both of which are known to impair learning in this 

task. All of the monkeys were impaired at scene learning after fornix or mammillary body 

lesions compared to their preoperative performance, consistent with previous results. 

In the second stage of the experiment, the monkeys underwent a second surgery in which we 

used the immunotoxin ME20.4-saporin to selectively deplete cholinergic inputs to the 

inferotemporal cortex. We then re-tested the monkeys on scene learning, and they were no more 

impaired than they were after their first surgery. This result is in striking contrast to an earlier 

finding by our laboratory that the effect of fornix transection is greatly exacerbated by prior 

depletion of acetylcholine from inferotemporal cortex (Browning et al. 2008, in press). The key 

difference between these two experiments is the order in which the lesions were placed: 

cholinergic depletion of inferotemporal cortex before fornix transection results in severe 

amnesia, whereas severe amnesia does not occur if the lesions are sustained in the opposite 

order. 

This finding suggests that monkeys require acetylcholine in inferotemporal cortex in order to 

adjust to the effects of a fornix lesion on episodic memory. This is consistent with a role for

cholinergic input to neocortex in cortical plasticity and remodelling, rather than a specific role in 

certain brain functions such as episodic memory. 

Disclosures: P.L. Croxson , None; P.G.F. Browning, None; D. Gaffan, None; M.G. Baxter, 

None. 

Poster 





Support: Medical Research Council UK 

Christopher Welch Trust 

Title: A novel method for infrared eyetracking of macaque monkeys without head fixation is 

behaviourally confirmed by demonstrating degraded transfer of memories between the visual 

hemifields in normal macaques 

Authors: *C. R. WILSON, M. J. BUCKLEY, D. GAFFAN; 

Exptl. Psychology, Univ. of Oxford, Oxford, United Kingdom 

Abstract: Investigations of the effects of unilateral lesions in the human brain often show 

lateralized effects in the contra-lesional hemifield (e.g. Hornak et al., 1997; Vuilleumier et al., 

2007). To understand the mechanisms involved experiments in macaque monkeys may be used 

to test specific hypotheses, because circumscribed and experimentally produced lesions can be 

made in monkeys. However, existing methods for measuring the direction of gaze in the monkey 

require the surgical implantation of a head post for head restraint, and often the implantation of 

scleral search coils. The presence of a head post, however, impedes surgical access to the brain, 

and ideally, in a lesion experiment with monkeys, one should measure the pre-operative ability 

of each animal in the task of interest before making any lesion. Here we present a novel method 

for measuring gaze direction which allows us to present visual stimuli at known positions in the 

visual field in normal monkeys with no head post or other surgical preparation. The method 

provides online measurements in real time, thus allowing stimuli to be presented in known 

positions in the visual field. 

In order to provide a behavioural validation of this method, we replicated in monkeys a recent 

finding in normal human subjects (Hornak et al., 2002). Normal human subjects show degraded 

recognition memory for visual objects when the objects are presented for memorization in one

hemifield and tested in the opposite hemifield (horizontal shift), by comparison with a control 

condition in which the objects are shifted by an equal displacement between presentation and 

test, but within a single hemifield (vertical shift). In the present experiment monkeys showed a 

substantial memory decrement in horizontal shift, as compared with vertical shift, in the transfer 

of object-reward association learning. Thus, in monkeys as well as in humans, and in association 

learning as well as in recognition memory, visual memories can be to a large extent hemifieldspecific. 

The presence of hemifield-specific impairments following unilateral lesions (Hornak et 

al., 1997; Vuilleumier et al., 2007) is supported by our result, and further study of these 

impairments will reveal important information about the nature of the storage of visual 

information and the way in which such information is integrated across hemifields. 

Disclosures: C.R. Wilson , None; M.J. Buckley, None; D. Gaffan, None. 

Poster 






Title: Cholinergic depletion of prefrontal cortex impairs acquisition of the delayed response task 

in rhesus monkeys 

Authors: *M. G. BAXTER, D. A. KYRIAZIS, P. L. CROXSON; 

Dept Exptl. Psychol, Oxford Univ., Oxford, United Kingdom 

Abstract: The involvement of corticopetal cholinergic projections in cognition remains difficult 

to define. Some investigators have suggested that normal cortical function requires an intact 

cholinergic input, whereas others emphasize a selective role of acetylcholine in attentional 

function or plasticity. Because of the anatomical and functional homology of human and 

macaque cortical structures, studies of the effects of selective ablation of cholinergic projections 

to cortical regions in the macaque would clarify the functions for which these projections are 

essential. 

We have tested 3 male rhesus monkeys with multiple bilateral injections of the immunotoxin 

ME20.4-saporin into lateral and orbital prefrontal cortex on a suite of cognitive tasks dependent 

on the integrity of orbital and ventrolateral prefrontal cortex, on which they were unimpaired. 

These tasks included new object-in-place scene learning, strategy implementation, and reinforcer 

devaluation.

To determine the involvement of acetylcholine in dorsolateral prefrontal cortex function, we then 

trained these monkeys on the spatial delayed response task (Goldman, 1970; Bachevalier and 

Mishkin, 1986) in a manual testing apparatus. In this task the monkey watches as an 

experimenter places a small food reward in one of two wells of a test tray and then covers both 

wells with identical gray plaques. After a brief delay (1-5 sec) during which an opaque screen is 

interposed between the monkey and experimenter, the monkey is allowed to obtain the reward by 

displacing the plaque covering the well that was baited by the experimenter. Thus, the monkey 

must maintain the baited location (left or right) in memory during the brief delay interval in order 

to choose correctly. Performance of this task is devastated by ablation of dorsolateral prefrontal 

cortex. The monkeys with cholinergic depletion of lateral and orbital prefrontal cortex were also 

unable to learn the task to criterion, which four unoperated control monkeys learned readily. 

This finding suggests that acetylcholine, although not critical for functions of ventrolateral and 

orbital prefrontal cortex, is essential for dorsolateral prefrontal cortex function. An alternative 

explanation, which we are currently investigating, is that acetylcholine is necessary for the 

prefrontal cortex to adapt to the different task demands of delayed response, relative to the tests 

of discrimination learning with which these monkeys had extensive experience. This would be 

consistent with a role for cholinergic input to neocortex in cortical plasticity and remodeling. 

Disclosures: M.G. Baxter, None; D.A. Kyriazis, None; P.L. Croxson, None. 

Poster 





Support: NIDA Grant DA023209 to AM 

Title: Mice lacking the β4 subunit of the nicotinic acetylcholine receptor show long-term 

memory deficits in hippocampus-dependent tasks 

Authors: H. M. JARRELL, S. SEMENOVA, *A. MARKOU; 

Psychiatry, Univ. of California, La Jolla, CA 

Abstract: Nicotine binds to nicotinic acetylcholine receptors (nAChRs) throughout the brain and 

elicits a range of behavioral responses related to cognition, including enhancement of learning 

and memory. However, the role of specific nAChRs subtypes in regulating behavior in the 

absence of nicotine is still not clear. The present work investigated the role of β4-containing 

nAChRs in learning and memory using wildtype (WT, β4 +/+ ) and null mutant (KO, β4 -/- ) mice for

the β4 nAChR subunit. Mice were tested in a battery of cognitive tasks including the Y-maze 

(spontaneous alternations), the novel object recognition task, the Barnes circular maze, and 

contextual and cued fear conditioning. There were no genotype differences in mouse 

performance in any of the spatial and working memory tasks used. However, there were 

interactive gender- and genotype-dependent differences in long-term memory retention assessed 

in the Barnes maze and the contextual fear conditioning tests 71 and 73 days, respectively, after 

training. In the Barnes maze memory retention test, male β4 -/- mice made more working memory 

errors and showed reduced use of the spatial search strategy (i.e., directly approaching the target 

hole based on spatial cues), indicating potential spatial memory deficits compared to WT mice. 

Instead, male β4 -/- mice were using random or serial hole by hole search strategies. In the 

contextual fear conditioning memory retention test, male β4 -/- mice exhibited reduced freezing 

time in the context previously paired with foot-shock compared to WT mice. Interestingly, these 

same deficits were not observed in female β4 -/- or WT mice. In conclusion, the present findings 

indicate that lack of the β4 nAChR subunit resulted in deficits in hippocampus dependent longterm 

memory retention tests in male, but not female, mice. Thus, long-term retention of spatial 

memories in males appears to be at least partially dependent on β4-containing nAChRs in the 

hippocampus. 

Disclosures: H.M. Jarrell, None; A. Markou , None; S. Semenova, None. 

Poster 




Topic: F.02.f. Fear and aversive learning and memory 

Support: NIH grant P20 RR15567 

Title: Brain derived neurotrophic factor mRNA in the hippocampus (dorsolateral pallium) of 

Rainbow trout is differentially regulated by coping strategy, revealed in a new model of fear 

conditioning 

Authors: *R. E. CARPENTER 1 , D. H. ARENDT 1 , J. P. SMITH 1 , I. SABIRZHANOV 2 , T. G. 

CLARK 2 , C. H. SUMMERS 1 ; 

1 Dept Biol, Univ. South Dakota, Vermillion, SD; 2 Neurosci. Group, Basic Biomed. Sci., Sanford 

School of Medicine, SD 

Abstract: Brain derived neurotrophic factor (BDNF) is an important factor in the molecular 

machinery of synaptic plasticity, learning, and memory formation. Recently we have developed a

new paradigm for rainbow trout that combines elements of fear conditioning and spatial learning. 

Utilizing social aggression from a large conspecific as our unconditioned stimulus (US), paired 

with cessation of water inflow to the tank as our neutral conditioned stimulus (CS), we can 

reliably induce approximately half of the test fish to escape into an empty tank chamber, a spatial 

learning task that may be dependent on BDNF in the hippocampus. Those fish that do not learn 

to escape from the US remain in their home tank and are subjected to social aggression for the 

duration of the 15 minute daily interaction. Following 7 days of CS+US pairings, presentation of 

the CS alone on Day 8 induced a significant increase in plasma cortisol in non-escapers, but had 

no effect on fish that reliably escaped. This is a conditioned response and not an artifact of the 

paradigm, as control groups exposed to both CS and US alone for 7 days did not show an 

increase in plasma cortisol compared to basal levels. In escaping fish, over 7 days of CS+US 

pairings, latency to escape decreases drastically. Presumably, this decrease in escape latency is 

mediated not only by fear conditioning to the aggressive US, but also through circuits related to 

spatial learning, which includes not only important contextual information but also exactly where 

the escape hole is located. The dorsolateral pallium (hippocampus) was microdissected from 

brains of escaping, non-escaping, and control fish, and BDNF mRNA levels were measured by 

quantitative rtPCR and normalized to GAPDH. Hippocampal BDNF mRNA expression in trout 

was significantly up-regulated by escaping, a 3-D spatial learning task. 

Disclosures: R.E. Carpenter , None; D.H. Arendt, None; J.P. Smith, None; I. Sabirzhanov, 

None; T.G. Clark, None; C.H. Summers, None. 

Poster 





Support: NIH/NCRR (P51RR000165) 

NSF (IBN-987675) 

NIH (MH069884, DA019624, NS055077 

Kirschstein NRSA (MH77420) 

NSF (GRFP DGE-0234618)

Title: Cortical brain derived neurotrophic factor modulates expression of appetitive and aversive 

learning in mice 

Authors: *D. C. CHOI 1 , K. M. MYERS 1 , K. A. MAGUSCHAK 1 , K. J. RESSLER 2 ; 

1 Dept Psychiatry, Emory Univ., Atlanta, GA; 2 Psychiatry, Emory University, Howard Hughes 

Med. Inst., Atlanta, GA 

Abstract: Considerable evidence suggests the medial prefrontal cortex (mPFC) is a major 

modulator of fear conditioning and extinction by directly influencing the amygdala, but the 

neural mechanisms are still unclear. One potential candidate may be brain derived neurotrophic 

factor (BDNF), a neurotrophin that is well known to be involved in neuronal plasticity and has 

been shown to be involved in learning, including fear conditioning. BDNF is highly expressed in 

the medial prefrontal cortex. Therefore, we hypothesize that BDNF in the mPFC is a prime 

candidate for mediating the learning and/or extinction of fear. To investigate this, a novel cortexspecific 

BDNF knockout mouse line was generated by crossing homozygous BDNF-floxed mice 

(loxP sites both upstream and downstream of BDNF exon 5), with a transgenic line that has 

neocortex-specific expression of Cre-recombinase, to selectively knockout BDNF in select areas 

of the neocortex. In normal mice, BDNF mRNA is abundantly expressed in the prefrontal cortex, 

including the prelimbic and infralimbic cortices. In contrast, the cortical BDNF knockout mice 

have pronounced loss of BDNF expression in the prelimbic cortex, while BDNF expression is 

completely spared in the more ventrally located infralimbic cortex. The cortical BDNF knockout 

mice were found to be no different than controls for various behavioral tests, including open 

field, elevated plus maze, and novel object recognition, suggesting these knockout mice have 

normal motor functioning, and unconditioned fear. However, with cue-dependent fear 

conditioning we found that although cortical BDNF knockout mice had normal acquisition of 

fear, they displayed a profound deficit in the expression of this previously learned fear behavior 

(freezing) when tested the next day. A second set of BDNF knockout mice were trained on 

cocaine conditioned place preference. In contrast to the fear expression deficit, the BDNF 

knockout mice appear to have enhanced learning of cocaine preference. We propose that the 

knockout of BDNF mRNA in the prelimbic cortex is the primary candidate responsible for a 

deficit in expression of aversive learning and enhancement of appetitive learning. 

Disclosures: D.C. Choi , None; K.M. Myers, None; K.A. Maguschak, None; K.J. Ressler, 

None. 

Poster 





Support: Grant from Japan Cardiovascular Research Foundation 

Title: Intake of pre-germinated brown rice increases brain-derived neurotrophic factor levels in 

the brain, and enhances spatial learning in mice 

Authors: *H. YANAMOTO 1,2 , S. MIYAMOTO 3 , Y. NAKAJO 1,2 , T. HORI 1 , Y. NAKANO 1,4 ; 

1 Lab. Cerebrovasc Disorders, Res. Inst. of Natl. Cardiovasc. Ctr., Suita, Japan; 2 Cardiovasc. Sci., 

Osaka Univ. Grad. Sch. of Med., Suita, Japan; 3 Neurosurg., Natl. Cardiovasc. Ctr., Suita, Japan; 

4 Rakuwakai Otowa Hosp., Kyoto, Japan 

Abstract: Brain-derived neurotrophic factor (BDNF), a nerve growth factor widely 

distributed in the brain, has been known to modulate synaptic plasticity, contributing to 

learning and memory consolidation. BDNF also participates in the regulation of appetite 

and glucose metabolism. Based on these mechanisms of BDNF, development of a safe 

method to increase BDNF in the brain is expected to have utility in the enhancement of 

learning and memory, and in improving impaired glucose metabolism and obesity. It has 

been suggested that the intake of pre-germinated brown rice, called "Hatsuga-genmai", 

improves memory and reduces obesity. Here, we investigated the effects of daily intake of 

pre-germinated brown rice on BDNF levels in the brain, spatial learning, and body weight 

gain. Adult male mice (C57BL/6J), 7 weeks old, were fed pre-germinated brown rice, or 

conventional food for mice (CE-2, CLEA) as a control, over 6 weeks. At the end of the 

period, BDNF levels in the brain were analyzed using ELISA. The protein concentration in 

each sample was measured to standardize the BDNF levels. Alterations in spatial learning 

were analyzed by the Morris water maze test (MWM). This analysis consisted of 5 sessions 

over consecutive 5 days. Feeding with pre-germinated brown rice significantly increased 

BDNF levels in the brain compared with control. The levels were 0.85 ± 0.22 ng/mg protein 

and 0.74 ± 0.13 ng/mg protein, respectively (mean ± s.d., n = 25, P < 0.05, unpaired t-test). 

Pre-germinated brown rice also improved escape latency, i.e. the time needed to reach the 

hidden platform in the MWM compared with control; 31.2 ± 6.4 sec or 69.5 ± 16.9 sec, 

respectively (mean ± s.e.m. in the 2-5 session, n = 25, P < 0.05, unpaired t-test). It also 

suppressed body weight gain compared with control; 23.4 ± 1.4 g or 27.5 ± 1.1 g, 

respectively (mean ± s.d., n = 25, P < 0.001, unpaired t-test). In conclusion, constant daily 

intake of pre-germinated brown rice was found to increase BDNF levels in the brain, 

improve spatial learning, and suppress age-dependent natural body weight gain. 

Disclosures: Y. Nakajo, None; S. Miyamoto, None; H. Yanamoto , None; T. Hori, None; Y. 

Nakano, None. 

Poster 

292. Acetylcholine, Neurotrophins, and Cognition




Support: NSC96-2752-B-006-001-PAE 

Title: A late expression of brain-derived neurotrophic factor in the amygdala is required for the 

maintenance of long-term fear memory 

Authors: L.-C. OU 1 , S.-H. YEH 2 , *P.-W. GEAN 2 ; 

1 Inst. Basic Med. Sci., 2 Dept Pharmacol, Natl. Cheng-Kung Univ., Tainan 70101, Taiwan 

Abstract: In many instances, increase in neuronal activity can elicit a biphasic secretion of a 

modulator. The initial release of the modulator triggers the induction of synaptic plasticity 

whereas the second phase reinforces the efficacy of synaptic transmission and the growth of 

dendrites and axons. Here we show that fear conditioning induces a second peak of brain-derived 

neurotrophic factor (BDNF) expression at 12 h post-conditioning. Fluorescent 

immunohistostaining confirmed that BDNF expression was increased at 1 and 12 h after 

conditioning and returned to baseline at 30 h after conditioning. Mature BDNF level was 

similarly increased at 1 and 12 h after fear conditioning. Pre-training administration of TrkB IgG 

or K252a impaired fear memory in a test carried out 1 day after training. By contrast, TrkB IgG 

or K252a infusion at 9 h after fear conditioning, which did not affect memory retention measured 

at 1 day after training, impaired memory when the rats were tested 7 days post-training. Fear 

conditioning induced a significant increase in Zif268 expression in the amygdala 12 h after fear 

training and the increase was completely blocked by TrkB-IgG infused 9 h post-training. 

Growth-associated protein 43 (GAP-43), a marker of newly formed synapses, in the amygdala 

was increased 7 days after fear conditioning. The change in GAP-43 was completely blocked by 

pre-training or 9 h post-training infusion of TrkB-IgG. These results suggest that the second peak 

of BDNF expression in the amygdala is responsible for the persistence of fear memory. 

Disclosures: L. Ou, None; P. Gean , None; S. Yeh, None. 

Poster 




Topic: F.02.f. Fear and aversive learning and memory


MH068850 

NX052819 

Title: BDNF and the developmental onset of contextual and cue-dependent fear in mice 

Authors: *S. S. PATTWELL 1 , K. G. BATH 2 , F. S. LEE 2 ; 

1 Neurosci., 2 Psychiatry, Weill Grad Sch. Biomed Sci., New York, NY 

Abstract: Many psychiatric disorders originating in childhood and adolescence involve 

improper regulation of fear. Complimentary human and animal studies have uncovered key brain 

regions in the acquisition and maintenance of fear learning including the amygdala, hippocampus 

and prefrontal cortex (PFC). Many of these regions undergo protracted development in humans 

and are not fully mature until the early twenties. Thus, different brain circuits and mechanisms 

may be involved in regulating fear responses across development. Brain derived neurotrophic 

factor (BDNF), a molecule involved in neuronal survival and differentiation, is important for 

long-term potentiation and memory consolidation. Due its role in learning and memory, BDNF 

signaling is also important for fear conditioning. In addition to maturational changes in fear 

circuitry, BDNF levels change dynamically during postnatal development. Male, C57BL/6J mice 

underwent fear conditioning during several important developmental periods. P29, P39, and P49 

mice were used, as these ages have been shown to best approximate human pre-, mid-, and postadolescence, 

respectively. A developmental dissociation between contextual and cue-dependent 

fear was observed in mice, replicating results of previous studies using rats. A delayed 

developmental onset of contextual fear in mice may be indicative of an immature hippocampal 

network. BDNF levels, in addition to protracted regional brain maturation, may prove to be a key 

factor in regulating age- dependent differences in fear expression. 

Disclosures: S.S. Pattwell , None; K.G. Bath, None; F.S. Lee, None. 

Poster 

293. Startle and Modulation of Startle 




Support: NIMH Grants MH069056, MH47840, MH57250, and MH 59906

Center for Behavioral Neuroscience NSF agreement #IBN-9876754 

FACES NSF Award #0450303 Subaward # I-66-606-63 

Title: Systemic rapamycin disrupts the consolidation of context but not cued fear memory 

Authors: *E. M. GLOVER 1 , M. DAVIS 2 ; 

1 Dept Psychol, Ctr. Behav Neurosci, Emory Univ., Atlanta, GA; 2 Dept Psychiat & Behavioral 

Sci., Emory Univ. Sch. Med., Atlanta, GA 

Abstract: The mammalian target of rapamycin (mTOR) kinase regulates protein synthesis. 

Rapamycin, an mTOR kinase inhibitor, was recently shown to disrupt the consolidation of toneshock 

and context-shock fear memories (assessed with freezing) when infused into the amygdala 

(Parsons et al., 2006) immediately after training, and also context-shock memories (also assessed 

with freezing) when injected systemically either before or after training (Blundell et al., 2008). 

Because rapamycin is centrally active following systemic administration and is FDA-approved 

for use in humans, it has recently attracted interest as a possible prophylactic treatment for 

PTSD-associated fear memories. To evaluate the generality of rapamycin effects on fear memory 

consolidation, we assess here the effects of systemic rapamycin injections on the consolidation of 

odor-shock and context-shock fear memories using fear-potentiated startle (FPS) as an 

alternative fear measure. On each of 2 consecutive days, rats received a pre-conditioning test 

during which their startle response to 30 95-dB noise bursts (noise alone test trial) was measured. 

Twenty-four hours later, they were returned to the chamber where they received a single 4-sec 

odor stimulus (5% amyl acetate) and co-terminating footshock (0.4 mA, 0.5-sec) and, 

immediately afterwards, an i.p. injection of either rapamycin (40 mg/kg) or vehicle. 7 days later, 

they were returned to test cage where they received 70 startle-eliciting noise bursts, of which 10 

of the final 40 were preceded by the odor conditioned stimulus. For some rats, the training and 

test contexts were identical. For others, the test context was altered (i.e., sandpaper inserts over 

the shock bars, Velcro on the sides, and 2 chains suspended from the top). Rapamycin 

significantly reduced FPS to the context CS (calculated as the percent change in startle from the 

pre-training test to the initial 30 noise alone trials of the post-conditioning test) but, in contrast to 

Parsons et al. (2006), did not reduce FPS to the discrete cue CS (calculated as the percent change 

in startle amplitude from noise alone to odor-noise test trials). This may reflect the different 

modalities used in these two studies (i.e., tone vs. odor) or, alternatively, a greater sensitivity of 

discrete cue fear conditioning to intra-amygdala vs. systemic rapamycin administration - a result 

similar to that seen with many other systemically administered compounds which selectively 

disrupt hippocampal-dependent (i.e., context fear or inhibitory avoidance) but not hippocampalindependent 

fear memories. If the latter, then this selectivity may limit the effectiveness of 

mTOR inhibitors as a PTSD treatment. 

Disclosures: E.M. Glover, None; M. Davis, None. 

Poster





Support: NIH/NCRR base grant P51RR000165 

Burroughs Wellcome Fund 


NSF agreement IBN-987675 


Title: Rapid internalization of GABAA receptor α1 subunits in cultured mouse hippocampal 

neurons following treatment with benzodiazepines 

Authors: *L. MOU, S. HELDT, K. RESSLER; 

Behav. Neuroscience, Yerkes, Emory Univ., Atlanta, GA 

Abstract: Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the 

mammalian brain, performs most of its functions via its actions at GABAA receptors. These 

receptors are a complex of heterogeneous polypeptide subunits (α, β, γ, δ, ζ). The most abundant 

conformation in the brain is α1β2γ2. Benzodiazepines, which are commonly used in anxiety and 

seizure disorders, bind to recognition sites on GABAA receptors and can produce tolerance and 

dependence, although the mechanisms by which these effects occur remain controversial. The 

goal of the present study was to examine changes in GABAA receptor α1 subunit expression in 

cultured mouse hippocampal neurons following administration of drugs that bind to the 

diazepam binding site. The cells were exposed to GABA, diazepam, zolpidem, or picrotoxin for 

1, 2, 6, or 16 hours, and then were probed with a specific antibody against α1 to detect surface 

and intracellular α1 subunit expression. We found that diazepam treatment resulted in 40% lower 

surface expression of α1 relative to non-treated cells, and that this decrease appeared following 

just 1 hour of exposure to the drug and persisted through 16 hours of exposure. A similar effect 

was noted following exposure to zolpidem, an α1-specific benzodiazepine. Both diazepam and 

zolpidem also increased intracellular α1 expression following 6 hours of exposure and decreased 

intracellular α1 expression following 16 hours of exposure. We conclude that binding at the 

diazepam binding site of the GABAA receptor can cause rapid internalization of α1 subunits and 

possibly induce their degradation following prolonged treatment. 

Disclosures: L. Mou , None; S. Heldt, None; K. Ressler, None.

Poster 





Support: Intramural Program of the National Institute of Mental Health to J.T.W. 

Natural Sciences and Engineering Research Council of Canada Grant to E.A.A. 

NIH Grant R37 MH057502, RR00169, and MH 41479 to D.G.A. 

NIH Grants R37 MH47840 and 2P50 MH58922 to M.D. 

McDonnell Foundation Grant to D.G.A. 

Title: The primate amygdala is necessary for the acquisition but not the retention of fearpotentiated 

startle: Effects of chronic lesions in the rhesus monkey (Macaca mulatta) 

Authors: *E. A. ANTONIADIS 1,2 , J. T. WINSLOW 3 , M. DAVIS 4,5 , D. G. AMARAL 1,2,6 ; 

1 Psychiatry and Behavioral Sc., Univ. of Cal, Davis, Davis, CA; 2 California Natl. Primate Res. 

Center, Univ. of California, Davis, CA; 3 Natl. Inst. of Mental Hlth. (N.I.M.H), Bethesda, MD; 

4 Dept. of Psychiatry and Behavioral Sci. and Ctr. for Behavioral Neuroscience, Emory Univ., 

Atlanta, GA; 5 Yerkes Natl. Primate Res. Ctr., Atlanta, GA; 6 Med. Investigation of 

Neurodevelopmental Disorders, Inst. (M.I.N.D), Univ. of California, Davis, CA 

Abstract: We continue to use a non-human primate version of the fear-potentiated startle 

paradigm to investigate the role of the macaque monkey amydala in affective learning and the 

expression of learned fear behaviors. In a previous study, we found that rhesus monkeys 

prepared with bilateral lesions of the amygdala failed to acquire fear-potentiated startle to a 

visual cue. However, a second group of monkeys, that received the lesion after training, 

successfully demonstrated fear-potentiated startle learned prior to the lesion. In the current 

experiment, the eight monkeys used in the second part of the original study, four of whom had 

bilateral amygdala lesions and their four controls, were trained using an auditory cue and tested 

in the fear-potentiated startle paradigm. This test was performed to determine whether they could 

acquire fear-potentiated startle to a new cue. Monkeys with essentially complete damage to the 

amygdala (based on histological analysis), who had retained and expressed fear-potentiated 

startle to a visual cue learned before the lesion, failed to acquire fear-potentiated startle to an 

auditory cue, when training occurred after the lesion. The results suggest that while the non-

human primate amygdala is essential for the initial acquisition of fear conditioning, it does not 

appear to be necessary for the memory and expression of conditioned fear. These findings are 

discussed in relation to a network of connections between the amygdala and the orbitofrontal 

cortex that may subserve different component processes of fear conditioning. 

Disclosures: E.A. Antoniadis, None; J.T. Winslow, None; M. Davis, None; D.G. Amaral, 

None. 

Poster 





Support: NIH -F32 MH073389-01 

NIH-DA-019624 

NIH -MH069056 

NIH -MH57250 

NIH -MH47840 

Title: Anxiolytic effects of the neurokinin 1 receptor antagonist gr-205171 in the elevated plus 

maze and contextual fear-potentiated startle model of anxiety 

Authors: *S. A. HELDT 1 , M. DAVIS 1 , D. TRIST 2 , E. RATTI 2 , M. CORSI 2 , K. J. RESSLER 1 ; 

1 Psychiatry & Behav Sci., Emory Univ,Ctr Behav Neurosci, Atlanta, GA; 2 GlaxoSmithKline 

SpA, Verona, Italy 

Abstract: Because the NK1 receptor in the gerbil, compared to rats or mice, is more like the 

NK1 receptor in humans, a number of different behavioral models have been developed to assess 

the anxiolytic-like properties of NK1 receptor antagonists in gerbil. The aim of the present study 

was to determine whether systemic administration of the NK1 receptor antagonist GR-205171 

produced anxiolytic-like effects in the elevated plus-maze and in a novel contextual conditioned 

fear test using fear-potentiated startle (FPS). On the elevated plus-maze, treatment of GR-205171 

at 0, 0.3, 1.0 and 5.0 mg/kg doses 30 min before testing produced anxiolytic-like effects in an 

increasing dose-response fashion as measured by the percentage of open arm time and

percentage of open arm entries. For contextual FPS, gerbils were first matched into four 

equivalent groups, based on their baseline startle. During training animals received 10-0.6 mA 

unsignaled footshocks at a 2 min variable interstimulus interval in distinctive training context 

(A). Twenty-four hours after training gerbils received treatment of GR-205171 at 0, 0.3, 1.0 and 

5.0 mg/kg doses 30 min before testing in which startle was elicited in the same context in which 

they were trained (A-A). Contextual FPS was defined as an increase in startle over pre-training 

baseline values. All drug doses levels (0.3, 1.0 and 5.0 mg/kg) significantly attenuated contextual 

FPS when compared to the vehicle control group. A control group which received testing in a 

different context, show little FPS. These findings support other evidence for anxiolytic activity 

of NK1 receptor antagonists and provide a novel Pavlovian conditioned fear test which may be 

an appropriate model to test other NK1 antagonists for preclinical anxiolytic activity in gerbils. 

Disclosures: S.A. Heldt, GlaxoSmithKline SpA3, C. Other Research Support (receipt of drugs, 

supplies, equipment or other in-kind support); M. Davis, None; K.J. Ressler, None; E. Ratti, 

GlaxoSmithKline, B. Research Grant (principal investigator, collaborator or consultant and 

pending grants as well as grants already received); M. Corsi, GlaxoSmithKline SpA, B. 


grants already received); D. Trist, GlaxoSmithKline SpA, B. Research Grant (principal 


Poster 





Support: The Science and Technology Center of the NSF under agreement No. IBN-9876754 

MH59906 

MH069056 

MH57250 

MH47840 

Title: Corticotropin-releasing factor (CRF) type 1 receptors in the bed nucleus of the stria 

terminalis (BNST) mediate long- (minutes) but not short- (seconds) duration startle increases to 

shock-predicting cues

Authors: *L. A. MILES 1 , D. WALKER 2 , M. DAVIS 2 ; 

1 Dept Pharmacol, 2 Psychiatry & Behavioral Sci., Emory Univ., Atlanta, GA 

Abstract: Oral administration of the CRF-R1 antagonist GSK008 disrupts sustained startle 

increases produced by intra-ventricular CRF infusions and sustained startle increases that occur 

when rats are tested in an illuminated environment (an innate anxiety response), but not startle 

increases produced by short-duration (i.e., 3.7-sec) lights that predict shock. These and other 

results suggest a preferential role of CRF in sustained (vs. short) or innate (vs. conditioned) fear 

reactions, or in fear reactions to vague (vs. imminent and well-defined) threats. 

To evaluate these alternatives, the effects of orally administered GSK008 was evaluated using 

several different fear conditioning and potentiated startle test procedures in which we attempted 

to isolate the contribution of these different variables. For experiment 1, rats received variableduration 

(3 sec to 8 min) 60-Hz clicker stimuli and co-terminating footshock and were later 

tested for FPS to 8-min clicker presentations (long, conditioned, and unpredictable since the CS 

onset to shock interval was variable). For experiment 2, rats received 3.7-sec clicker stimuli and 

co-terminating footshock, and were later tested for FPS to 3.7-sec clicker presentations (i.e., 

short, conditioned, predictable). For experiment 3, rats were trained with variable duration 

clicker stimuli, but were tested with 3.7-sec clicker presentations (short, conditioned, 

unpredictable). FPS to 8-min (Experiment 1) but not 3.7-sec (Experiments 2 and 3) CS 

presentations was blocked by GSK008. Together, these results indicate that CRF mediates longduration 

fear responses irrespective of conditioning or predictability. 

Previous findings indicate a similar role for the bed nucleus of the stria terminalis (BNST) - a 

structure that also mediates the sustained startle increases produced by intra-ventricular CRF 

infusions. To determine if the effects of oral GSK008 administration might have involved the 

BNST, we next compared the effect of intra-BNST GSK008 infusions on FPS to 3.7-sec vs. 8min 

CS presentations. As with oral infusions, intra-BNST GSK008 blocked FPS to the 8-min but 

not 3.7-sec shock-associated CS. 

We then evaluated the effect of systemic buspirone (a 5HT1A partial agonist and dopamine D2 

antagonist) administration - a treatment previously shown to block FPS to 3.7-sec light and tone 

CSs. Buspirone also blocked FPS to the 3.7-sec clicker CS, but had no effect on FPS to the 8min 

clicker CS. Together, these results indicate a double pharmacological dissociation between 

the neural substrates of short- and long-duration startle increases. 

Disclosures: L.A. Miles , None; D. Walker, None; M. Davis, None. 

Poster 





Support: Science and Technology Center (The Center for Behavioral Neuroscience of the NSF 

under Agreement No. IBN-9876754) and the Yerkes Base Grant 





Title: Differential involvement of the bed nucleus of the stria terminalis (BNST) and 

corticotropin releasing factor (CRF) receptors in short- versus long-duration fear responses 

Authors: *D. L. WALKER 1 , L. A. MILES 1 , Y. YANG 1 , E. RATTI 3 , M. CORSI 3 , D. TRIST 3 , 

M. DAVIS 2 ; 

1 Dept Psychiat & Behavioral Sci., 2 Dept Psychiat & Behavioral Sci. and Cntr for Behavioral 

Neurosci, Emory Univ. Sch. Med., Atlanta, GA; 3 Medicines Res. Ctr., GlaxoSmithKline, 

Verona, Italy 

Abstract: Startle amplitude is increased by short-duration (3.7-sec) conditioned fear stimuli that 

predict shock (fear-potentiated startle, conditioned), and by sustained exposure (20-min test) to 

bright lights that don‟t (light-enhanced startle, innate). Although both effects are disrupted by 

AMPA antagonist (NBQX) infusions into the basolateral amygdala (BLA), fear-potentiated but 

not light-enhanced startle is disrupted by NBQX infusions into the central amygdala nucleus 

(CeA), and light- but not fear-potentiated startle is disrupted by NBQX infusions into the 

closely-related BNST (double dissociation). The differential involvement of the BNST vs. CeA 

in light-enhanced vs. fear-potentiatoned startle may reflect differences in stimulus/response 

duration or in conditioning history. To disambiguate these variables, the effects of pre-test 

NBQX infusions were evaluated in rats trained and tested for conditioned fear to a long-duration 

stimulus (i.e., an 8-min tone CS). Although conditioned, intra-BNST NBQX infusions still 

disrupted potentiation to the CS, but only during the 2 nd block of startle stimuli (i.e., sustained 

fear during the final 4-min of the CS). Potentiation during the early phase was actually greater 

than in ACSF controls, suggesting that the BNST participates preferentially in sustained fear 

responses. NBQX infusions into the BLA disrupted potentiation during both phases. Infusions 

into the CeA disrupted potentiation during neither. 

CRF infusions into the lateral ventricle or BNST, but not into the amygdala, also produce a 

sustained startle increase and this effect is dependent on the BLA (based on the effect of intra- 

BLA infusions of NBQX + muscimol) and BNST but not on the CeA. To determine if sustained 

BNST-dependent fear in general (i.e., innate or conditioned) requires CRF receptor activation, 

the novel potent and selective CRF-R1 antagonist GSK008 was administered orally prior to fearpotentiated 

(3.7-sec vs. 8-min tone CS), light-enhanced, or CRF-enhanced startle testing. 

GSK008 dose-dependently disrupted CRF-enhanced startle (long, unconditioned), fearpotentited 

startle to the 8-min CS (long, conditioned), and light-enhanced startle (long,

unconditioned) but did not disrupt fear-potentiated startle to the 3.7-sec CS (short, conditioned). 

Thus, CRF like the BNST seems to participate preferentially in sustained fear responses. 

Based on these and related findings, we present a provisional model in which sustained fear 

responses are mediated by a potentiation of glutamate release onto BNST neurons by presynaptic 

CRF receptors located n BLA terminals. Preliminary data in support of this model are 

presented. 

Disclosures: D.L. Walker, None; L.A. Miles, None; Y. Yang, None; E. Ratti, 

GlaxoSmithKline, A. Employment (full or part-time); M. Corsi, GlaxoSmithKline, A. 

Employment (full or part-time); D. Trist, GlaxoSmithKline, A. Employment (full or parttime); 

M. Davis, None. 

Poster 




Topic: F.03.a. Sexual behavior 

Support: German Research Foundation, IRTG 1389/1 

Title: Direct gaze of photographs of female nudes influences startle in men 

Authors: *J. LASS-HENNEMANN 1 , A. SCHULZ 1 , F. NEES 1 , T. D. BLUMENTHAL 2 , H. 

SCHAECHINGER 1 ; 

1 Univ. Trier, Trier, Germany; 2 Dept. of Psychology, Wake Forest Univ., Winston-Salem, NC 

Abstract: Foreground presentation of photographs of opposite sex nudes lowers startle elicited 

by sudden acoustic stimuli. However, the impact of gaze direction of the presented nudes on this 

startle modulation has not been investigated. Theoretically, direct gaze of photographs of female 

nudes could either lead to a larger inhibition of the startle reaction due to a summating valence 

and arousal effect of direct eye contact, or lead to a smaller inhibition due to an attention 

capturing effect of the eyes. Two subsets of erotic photographs of female nudes (women looking 

directly at the observer vs. gazing away) and standard IAPS neutral pictures were viewed by 26 

male volunteers, while startle eye blink responses to binaural bursts of white noise (50 ms, 105 

db) were recorded by EMG. Erotic pictures reduced startle eyeblink magnitudes as compared to 

neutral pictures. Furthermore, erotic stimuli without direct gaze at the observer showed a greater 

startle eyeblink inhibition than erotic stimuli with direct gaze at the observer. Our data suggest 

that direct gaze of opposite sex nudes may direct attention to the face, thereby reducing the 

appetitive impact of an attractive body.

Disclosures: J. Lass-Hennemann, None; A. Schulz, None; F. Nees, None; T.D. Blumenthal, 

None; H. Schaechinger, None. 

Poster 





Title: Modulation of acoustic startle by appetitive and aversive conditioned olfactory cues 

Authors: *G. A. COUSENS, A. BLUMENTHAL, C. R. BOSKA, M. GREGER-MOSER, L. H. 

HULL-RAWSON, F. MIELE; 

Dept of Psychol, Drew Univ., Madison, NJ 

Abstract: The acoustic startle response is known to be modulated by a wide variety of stimulus 

manipulations and has been used extensively as a probe to assess emotional state. This response 

is sensitive to associative learning mechanisms as conditional stimuli have been shown to either 

potentiate or attenuate startle amplitude, depending in part on unconditional stimulus valence. 

Stimuli associated with aversive outcomes such as footshock typically enhance startle (fearpotentiation 

of startle; FPS), whereas those associated with appetitive outcomes have been 

shown to diminish the response (pleasure-attenuation of startle; PAS). Although key components 

of the neural circuitry underlying FPS have been identified, the extent to which this circuitry 

supports PAS is largely unknown. Determination of the neural mechanisms by which associative 

learning can bidirectionally modulate startle would be facilitated by a rodent behavioral model in 

which the behavioral effects of appetitive and aversive conditional stimuli could be assessed 

within-subjects. Here, we report preliminary data that startle amplitude was unaffected by reexposure 

to diffuse background odor previously trained as an effective discriminative stimulus 

for consumption of palatable liquid Ensure ® diet, relative to that observed during re-exposure to 

a neutral odor. However, startle amplitude was enhanced during re-exposure to an odor 

previously paired with footshock (0.5sec; 0.8mA). Current efforts are determining whether 

manipulation of training and testing parameters can elicit PAS without significant disruption of 

FPS. 

Disclosures: G.A. Cousens , None; A. Blumenthal, None; C.R. Boska, None; L.H. Hull- 

Rawson, None; M. Greger-Moser, None; F. Miele, None.

Poster 





Title: Exercise reduces the anxiogenic effect of intra-bed nucleus of the stria terminalis 

injections of mCPP 

Authors: *J. H. FOX, S. HAMMACK, W. FALLS; 

Dept Psychology, Univ. Vermont, Burlington, VT 

Abstract: We have shown that voluntary exercise is associated with reduced anxiety in mice 

across several putative models of anxiety. The most widely prescribed pharmacological agents in 

the treatment of anxiety disorders are the selective serotonin reuptake inhibitors (SSRIs), which 

target brain serotonin (5-HT) systems, and numerous studies have shown that 5-HT 

manipulations can alter anxiety-like behaviors. In rats, voluntary exercise has been shown to 

increase mRNA for 5-HT1A autoreceptors in the dorsal raphe. Such changes might result in 

decreased release of 5-HT in target areas controlling anxiety behaviors. We have shown a 

decreased sensitivity to the anxiogenic drug mCPP, a serotonin 2B/C agonist, in mice that had an 

opportunity to exercise for 2 weeks. These findings suggest that exercise may help to reduce 

anxiety by altering 5-HT systems, perhaps by down regulating post synaptic 5HT 2B/2C 

receptors. One area that has been shown to be involved in anxiety-like behaviors is the bed 

nucleus of the stria terminalis (BNST). Intra-BNST infusion of mCPP facilitates acoustic startle 

indicative of anxiety. Therefore, we assessed whether voluntary exercise would alter the 

anxiogenic response to intra-BNST mCPP. Male C57BL/6J mice were given 2 weeks of free 

access to either a functioning or non-functioning running wheel in their home cage. Mice were 

then given intra-BNST injections of either 1ug/ul of mCPP or vehicle through indwelling 

cannula. mCPP facilitated acoustic startle in non-exercising mice. This effect was blunted in 

exercising mice suggesting that voluntary exercise alters 5-HT function in the BNST. Studies are 

underway to examine the nature of the putative changes in the BNST following exercise. 

Disclosures: J.H. Fox , None; S. Hammack, None; W. Falls, None. 

Poster 





Support: Grants-in-Aid from MECSST 

Health Sciences Research Grants from MHLW 

Title: Prefrontal NET blockade ameliorates prepulse inhibition deficits in dopamine transporter 

knockout mice: involvement of cortical-subcortical pathways 

Authors: *Y. ARIME 1 , F. S. HALL 2 , G. R. UHL 2 , I. SORA 1 ; 

1 Dept of Biol. Psychiatry, Tohoku Univ., Sendai, Japan; 2 NIDA-IRP, Baltimore, MD 

Abstract: Dopamine transporter knockout (DAT KO) mice exhibit hyperdopaminergic tone in 

striatum (CPu) and nucleus accumbens (NAc) and normal dopamine levels in medial prefrontal 

cortex (mPFc). DAT KO mice have deficits in prepulse inhibition (PPI) that are also 

reproducibly identified in patients with neuropsychiatric disorders that include schizophrenia. 

Selective norepinephrine transporter (NET) blockers are approved for treating ADHD and are 

being considered for treating cognitive dysfunction in schizophrenics. We have recently reported 

that the selective NET blocker nisoxetine reverses PPI deficits in DAT KO mice. To elucidate 

the anatomical substrates that might underlie this recovery of PPI in these mice, we have 

examined the region-specific effects of nisoxetine on PPI deficits in DAT KO mice. We have 

compared results of systemic and local injections of nisoxetine on PPI, extracellular dopamine 

levels and on expression of the activation marker c-Fos in neuronal populations labeled by 

neuronal markers and the retrograde neuron tracer fluorogold. Systemic administration of 

nisoxetine doses that normalize PPI defects in DAT KO mice increase the number of c-Fos 

positive cells in mPFc, orbitofrontal cortex, NAc shell, basolateral amygdala and ventral 

tegmental area. mPFc infusions of nisoxetine increased PPI in DAT KO mice. By contrast, NAc 

infusions had no effect on PPI in either wildtype (WT) or DAT KO mice. Studies using labeling 

with c-Fos antibodies, neuronal markers and fluorogold revealed that nisoxetine selectively 

activates mPFc glutamatergic pyramidal neurons that project to NAc. Administration of 

nisoxetine also increased extracellular dopamine levels in mPFc, consistent with a substantial 

role for NET in accumulating dopamine in this brain region. These results suggest that alteration 

of glutamatergic neurotransmission from mPFc to NAc underlies at least a portion of the ability 

of nisoxetine to ameliorate PPI defects found in DAT KO mice. 

Disclosures: Y. Arime, Health Sciences Research Grants from MHLW, B. Research Grant 


received); Grants-in-Aid from MECSST, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received); F.S. Hall, NIDA-IRP, 

NIH/DHHS, B. Research Grant (principal investigator, collaborator or consultant and pending 

grants as well as grants already received); G.R. Uhl, NIDA-IRP, NIH/DHHS, B. Research Grant 


received); I. Sora, Grants-in-Aid from MECSST, B. Research Grant (principal investigator, 

collaborator or consultant and pending grants as well as grants already received); Health

Sciences Research Grants from MHLW, B. Research Grant (principal investigator, collaborator 

or consultant and pending grants as well as grants already received). 

Poster 





Support: HKU7327/03M 

Title: Transgenic mice with over-expression of endothelin-1 in endothelial cells have increased 

locomotor activity, impaired startle response and mild cognitive deficit 

Authors: *X. ZHANG 1 , Y. ZHOU 2 , J. F. CHIU 2 , V. K. L. HUNG 2 , S. M. CHUNG 3 , S. K. 

CHUNG 2,4 ; 

1 LiKa Shing Fac Med., Hong Kong, Hong Kong; 2 Anat., Li Ka Shing Fac Med., Hong Kong, 

China; 3 Physiol., Li Ka Shing Fac. of Medicine, The Univ. of Hong Kong, Hong Kong, China; 

4 Res. centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Fac. of Med., Hong 

Kong, China 

Abstract: Intracerebroventricular administration of low doses of endothelin-1 (ET-1), which is a 

potent vasoconstrictor, has been shown to markedly increase the spontaneous locomotor activity 

of rats. Previously, we have generated transgenic mice with over-expressing ET-1 in endothelial 

cells (TET-1) and showed that these TET-1 mice have hypertension and left ventricular 

hypertrophy. Here, we investigated the behavioral changes and cognitive function in adult (5~6 

months) and aged (21~23 months) TET-1 mice under normal condition. Adult TET-1 mice 

showed significantly increased spontaneous locomotor activity in Figure 8 maze than nontransgenic 

mice. The difference was even more obvious in aged mice. On the other hand, TET-1 

mice showed significantly lower response to acoustic startle stimulus than non-transgenic mice. 

These behavioral changes are similar to those of spontaneously hypertensive rat (SHR), which is 

used as an attention-deficit hyperactivity disorder (ADHD) model. In addition, TET-1 mice 

showed a trend of cognitive deficit in spatial learning and memory, though not significant. By 

two-dimensional electrophoresis, altered antioxidant protein was found in the cerebral cortex of 

aged TET-1 mice and immunohistochemistry analysis showed increased heat-shock protein 70 

(Hsp-70) expression in the cerebral blood vessels in the brain of aged TET-1 mice compared to 

non-transgenic mice. Thus, it is possible that the behavioral changes in aged TET-1 mice might 

be the consequence of oxidative stress.

Disclosures: X. Zhang , None; Y. Zhou, None; J.F. Chiu, None; V.K.L. Hung, None; S.M. 

Chung, None; S.K. Chung, None. 

Poster 





Support: Grant-in-Aid for Young Scientists (#16730376) from the MEXT of Japan to TS 

Title: Startle response in spontaneously hypertensive rats (SHR): Effects of two different 

hypotensive drugs on fear-potentiated startle 

Authors: *T. SATO; 

Hlth. and Social Services, Tohoku Bunka Gakuen Univ., sendai, Japan 

Abstract: Spontaneously hypertensive rats (SHR) show some marked differences in behavioral 

reactivity to emotional stimuli. The present study focuses on fear-potentiated startle (FPS). To 

determine the relationship between FPS and blood pressure level, the author examined the effects 

of blood pressure reduction on the magnitude of FPS, using two hypotensive drugs: vasodilator 

hydralazine and ganglionic blocker hexamethonium. FPS training sessions were conducted on 2 

consecutive days. Each rat was given 10 light stimuli (conditioned stimulus, CS) for 3.7 s paired 

with electric shocks of 0.7 mA for 0.5 s. The rats received a short test for FPS 24 h after the 

second session. In this test, the rats were presented with 10 startle-alone trials (5 at 95 dB and 5 

at 105 dB, each startle stimulus lasting 50 ms) followed by 8 startle-alone and 8 CS-paired trials 

(4 at 95 dB and 4 at 105 dB for both types of trials). The rats were divided into three groups 

based on the percentage index of FPS (%FPS) in the test. There was no significant difference in 

%FPS in the short test. The rats were intravenously treated with hydralazine (0.6 mg/kg), 

hexamethonium (20 mg/kg), or vehicle (saline) 24 h after the short test; blood pressure was 

measured 20 min after the treatment, soon after which the post-treatment FPS measurement was 

obtained. For this, 4 startle-alone trials were administered, followed by 16 startle-alone and 16 

startle-CS paired trials (8 at 95 dB and 8 at 105 dB). An ANOVA and post hoc tests revealed that 

systolic blood pressure in the hydralazine- and hexamethonium-treated groups was significantly 

lower than that in the saline-treated group; systolic blood pressure in the hydralazine-treated 

group was significantly lower than that in the hexamethonium-treated group. Nevertheless, the 

hydralazine-treated group also showed higher values with regard to heart rate than the other two 

groups, whereas no significant differences were found between the hexamethonium-treated and 

saline-treated groups. Further, the %FPS for the 95 dB startle stimuli in the hydralazine- and

hexamethonium-treated groups was significantly smaller than that in the saline-treated group. 

There was no significant difference in %FPS for the 95 dB trials between the hydralazine- and 

hexamethonium-treated groups. No significant differences were found in the %FPS for the 105 

dB trials. This study confirmed that the %FPS of the SHRs became relatively smaller when their 

blood pressure was reduced in the hydralazine- and hexamethonium-treated groups, in which the 

rats‟ heart rate remained unchanged. It is possible that blood pressure level and not heart rate 

contributes to the expression of fear-related behavioral responses. 

Disclosures: T. Sato , Novartis Pharma Inc., C. Other Research Support (receipt of drugs, 

supplies, equipment or other in-kind support). 

Poster 

294. Gene Expression and Fear Learning 




Support: 2682/POI/2006/31 

Title: Searching for mechanism of learning-induced plasticity of cerebral cortex: focus on 

immediate early genes, c-Fos and Arc 

Authors: A. RADWANSKA, R. K. FILIPKOWSKI, *M. M. KOSSUT; 

Dept Neurophysiol., Nencki Inst. Expmtl Biol, Warsaw 02093, Poland 

Abstract: The objective of this study is to learn whether immediate early genes (IEG) are 

involved in plastic modification of the somatosensory cortex in mice. Each whisker on mouse 

snout is represented in contralateral somatosensory cortex by a group of neurons called barrel. 

The pattern of barrels mimics the arrangement of whiskers on the muzzle and cortical 

representation of peripheral receptors can be easily localized. Sensory conditioning, in which 

association between stimuli is formed by pairing whisker stroking with a tail shock (10 minutes a 

day for three consecutive days) evokes enlargement of a functional representation of trained 

whiskers, as shown by [14C]2-deoxyglucose uptake. We want to approach molecular 

underpinning of the observed learning-dependent plastic change. Immediate early genes are 

detectors of neuronal activation, novelty and plasticity, so we hypothesize that increase of 

neuronal activity and induction of plastic remodeling within the trained barrel can be reflected by 

IEG expression. Preliminary data indicate that with the vibrissa and tail stimulation applied 

during the training, labeling with c-Fos antibody in the barrel cortex is weak, while the cingulate 

cortex , the SII/ insular cortex region and the amygdaloid complex show high c-Fos expression.

Also, there is a gradient of c-Fos protein expression in the barrel cortex, with barrels of rows E 

and D showing more labeled cells than B and A, and this gradient is not changed by the training. 

Intensive stimulation of selected whiskers by stroking produces augmentation of the number of 

cells expressing c-Fos protein in corresponding barrels. In further course of the experiment we 

will compare effects of stimulation and training using c-Fos immunohistochemistry, c-Fos and 

Arc in situ hybridization. 

Disclosures: A. Radwanska, None; M.M. Kossut, None; R.K. Filipkowski, None. 

Poster 





Support: NIDA RO1 DA019107 

Lois Pope LIFE Fellowship 

Title: The nNOS gene plays a role in long-term memory formation of contextual fear learning 

but not cued fear learning 

Authors: *J. B. KELLEY 1 , K. L. ANDERSON 2 , Y. ITZHAK 2 ; 

1 Neurosci. Program, 2 Psychiatry & Behavioral Sci., Univ. of Miami Sch. of Med., Miami, FL 

Abstract: Activation of the N-methyl-D-aspartate receptor (NMDAR) causes increase in 

calcium influx and subsequent activation of neuronal nitric oxide synthase (nNOS). In brain, NO 

produced by nNOS is a retrograde neuronal messenger involved in synaptic plasticity including 

late phase of long-term potentiation (LTP) and formation of long-term memory (LTM). Yet, 

little is known about the role of NO signaling in classical Pavlovian conditioning. The fear 

conditioning paradigm has been successfully used to investigate the role of various genes, 

neurotransmitters and substrates in the formation of contextual and cued LTM. Using knockout 

(KO) mice with targeted mutation of the NOS gene and their wild type (WT) counterparts, the 

role of NO signaling in fear conditioning was investigated. Male and female nNOS KO mice 

show a deficiency in LTM memory of contextual but not cued fear learning compared to WT 

counterparts. Sex-dependent differences in both contextual and cued fear learning were not 

observed in either genotype. Deficits in contextual fear learning in nNOS KO mice were 

improved by multiple training sessions with a short intertrial time of 12-15 minutes. A 

relationship between increase in plasma corticosterone levels and the magnitude of contextual,

ut not cued, freezing was also observed. A reduction in the phosphorylated form of cyclic 

adenosine monophosphate response element binding protein (CREB) was observed in the dentate 

gyrus region of naïve nNOS KO mice compared to WT mice. Results suggest that nNOS KO 

mice exhibit a selective disruption of contextual hippocampus-mediated, but not cued amygdalamediated, 

fear learning, which may be overcome by different training strategies. Since CREB is 

a transcription factor responsible for regulating numerous genes related to learning and memory, 

this deficiency may contribute to the contextual learning deficits observed in nNOS KO mice. 

Disclosures: J.B. Kelley, None; K.L. Anderson, None; Y. Itzhak, None. 

Poster 





Support: NIH Neuroscience Blueprint funding 


NIMH 

NARSAD 

NIA 

Title: Regulation of MeCP2 and MBD1 in learning and memory 

Authors: *L. A. QADRI, D. SWEATT; 

UAB, Birmingham, AL 

Abstract: Ninety-five percent of females with Rett Syndrome have mutations in the methyl- 

CpG-binding domain protein MeCP2. Mutations in both MeCP2 and the related protein MBD1 

cause learning deficits in mice. However, while these proteins are known to be essential for 

development, their role in normal learning and memory has not yet been investigated. Therefore, 

we are examining the regulation of these proteins in response to a classical model of learning, 

fear conditioning. C57/bl6 mice were trained for contextual fear conditioning, a hippocampusdependent 

learning task. Following training, hippocampal area CA1 was removed for 

biochemistry. Sprague-Dawley rats received cued fear conditioning, an amygdala-dependent

learning task, after which the basolateral amygdala was removed. Tissue was removed at three 

time points during the memory consolidation phase, thirty minutes to two hours following 

training, and RNA or protein was extracted. Mecp2 and mbd1 mRNA levels were assessed using 

real time PCR, and protein levels were examined using Western blotting. Associative training 

did not significantly alter the transcriptional regulation of mecp2 or mbd1 at the time points 

examined. MeCP2 and MBD1 can also be regulated through protein modifications. MeCP2 is 

inactivated through phosphorylation by CDKL5, and PIAS1 and PIAS3 inactivate MBD1 

through sumoylation. Changes in levels of the inactive forms of both proteins, phospho-MeCP2 

and sumo-MBD1, are currently being examined, as are CDKL5 and PIAS proteins 1 and 3. 

These studies help elucidate the role of MeCP2 and MBD1 in normal learning and memory. 

Disclosures: L.A. Qadri, None; D. Sweatt, None. 

Poster 





Support: Philip Morris Extramural Research 

NINDS 

Evelyn F. McKnight Brain Research Foundation 

NIMH 

Title: Persisting changes in DNA methylation with learning 

Authors: *C. A. MILLER 1 , R. PARRISH 2 , J. D. SWEATT 2 ; 

1 Dept Neurobiol, Univ. Alabama-Birmingham, Birmingham, AL; 2 Neurobio., Univ. of Alabama 

at Birmingham, Birmingham, AL 

Abstract: DNA methylation is a covalent chemical modification of DNA catalyzed by DNA 

methyltransferases. DNA methylation is associated with transcriptional silencing and has been 

studied extensively as a lifelong molecular information storage mechanism put in place during 

development. Recently, we have begun investigating the hypothesis that the adult central nervous 

system utilizes this covalent modification of DNA to aid in the formation and storage of longterm 

memories. Accumulating evidence indicates that hippocampal methylation is a critical

participant in the transcriptional regulation of memory-associated genes during consolidation. 

But interestingly, within the hippocampus, the alterations in methylation produced by learning 

return to baseline within 24 hours after training, at least in the subset of genes examined to date. 

To investigate the possibility that the CNS employs methylation as a more permanent storage 

mechanism, methylation was examined in the medial prefrontal cortex (mPFC) or basolateral 

amygdala (BLA) 7 days after training for contextual and cued fear conditioning. There were no 

differences between trained animals and their control counterparts (context only or shock only) 

in the promoters of the immediate early gene zif or the negative regulator of memory, 

calcineurin, in the mPFC 7 days after contextual fear conditioning. However, the promoter 

region of reelin was hypermethylated in animals that had received associative training a week 

earlier. Interestingly, this effect was NMDA receptor-dependent, as treatment with MK-801 at 

the time of training prevented the reelin hypermethylation. In contrast, both reelin and 

calcineurin were hypomethylated in the BLA one week after cued fear conditioning relative to 

controls, while zif was hypermethylated. Taken together, these results demonstrate that a single 

trial of fear conditioning is sufficient to produce lasting, gene-specific changes in DNA 

methylation. In addition, the rapid methylation changes observed in the hippocampus versus 

sustained methylation in the cortex are consistent with the notion that associative memories may 

shift from the hippocampus where they are initially consolidated to a more permanent storage 

site in the cortex. 

Disclosures: C.A. Miller , None; R. Parrish, None; J.D. Sweatt, None. 

Poster 





Support: NSF 0444632 

New Zealand Tertiary Education Commission 

NIH 073949 

Title: Auditory thalamic neurons that express early growth response gene 1 (EGR-1) following 

fear conditioning project to the lateral amygdala

Authors: K. OVEREEM 1,2 , *G. E. SCHAFE 2,3 ; 

1 Psychology, The Univ. of Canterbury, Christchurch, New Zealand; 2 Dept Psychol, 

3 Interdepartmental Neurosci. Program, Yale Univ., New Haven, CT 

Abstract: We have recently shown that auditory fear conditioning regulates the expression of 

early growth response gene 1 (EGR-1) mRNA and protein in the auditory thalamus (MGm/PIN), 

and that intra-MGm/PIN infusion of EGR-1 antisense impairs fear memory consolidation. We 

have also observed that synaptic plasticity and nitric oxide (NO) signaling in the lateral 

amygdala (LA) are required for training-induced elevations in EGR-1 in the MGm/PIN. These 

findings are consistent with an emerging hypothesis that LA-driven alterations in intracellular 

signaling pathways in MGm/PIN neurons is critical for promoting fear memory consolidation, 

perhaps by influencing presynaptic aspects of plasticity at thalamo-LA synapses (Apergis- 

Schoute et al, 2005). In the present study, we have used anatomical tract tracing techniques in 

combination with immunohistochemical labeling to determine whether auditory thalamic 

neurons that express EGR-1 after fear conditioning project to the LA. Rats received intra-LA 

infusion of the retrograde tracer fluorogold (FG; 2%; 0.3 κl/side). After 10-14 days, rats were 

conditioned with 3 pairings of a tone (5 KHz, 75 dB, 20 sec) with footshock (1.0 mA, 1 sec). 

Control rats received intra-LA FG infusions followed 10-14 days later by 3 immediate shocks 

(1.0 mA, 1 sec), a procedure that does not support fear conditioning. Two hours following 

training, animals were sacrificed by perfusion and thalamic tissue was processed for 

immunohistochemical localization of EGR-1 and FG. We focused our analysis on two regions of 

the auditory thalamus that exhibit the most robust EGR-1 labeling after fear learning: the 

peripeduncular (PP) nucleus, and the adjacent posterior intralaminar nucleus (PIN). Preliminary 

data has revealed a significant overall difference between the percentage of double labeled cells 

in the paired group (14.4 ± 1.2%) compared to the group receiving immediate shock (8.8 ± 

1.2%), while the number of fluorogold-positive projection cells between the two groups does not 

significantly differ. Closer examination of individual thalamic nuclei has revealed that the 

difference between the paired and immediate shock groups is significantly higher in the PP 

(paired: 14.6 ± 2.3%; immediate shock: 6.6 ± 0.7%) compared to the adjacent PIN (paired: 14.2 

± 0.7% and immediate shock: 11.0 ± 2.3%). These results extend those of our previous work and 

provide anatomical evidence for LA modulation of thalamic gene expression during fear memory 

consolidation. 

Disclosures: K. Overeem, None; G.E. Schafe , None. 

Poster 





Support: NIH 073949 

NSF Graduate Research Fellowship 

NSF 0444632 

Title: The NO-cGMP-PKG signaling pathway in the lateral amygdala (LA) bidirectionally 

regulates immediate early gene expression in the LA and in the auditory thalamus 

Authors: *K. T. OTA 1 , G. J. YOUNG 1 , G. E. SCHAFE 1,2 ; 

1 Dept. of Psychology, 2 Interdepartmental Neurosci. Program, Yale Univ., New Haven, CT 

Abstract: We have recently shown that blockade or facilitation of the NO-cGMP-PKG signaling 

pathway in the lateral amygdala (LA) impairs or enhances memory consolidation of Pavlovian 

fear conditioning and synaptic plasticity in the LA by activating the ERK/MAPK signaling 

pathway. Previous work in our lab, however, has pointed to a critical role of ERK-mediated 

transcription in fear memory consolidation not only in the LA, but also in the auditory thalamus 

(MGm/PIN; Apergis-Schoute et al, 2005). In the present series of experiments, we ask whether 

the NO-cGMP-PKG signaling pathway in the LA regulates the expression of different ERKdriven 

immediate early genes (IEGs) in the LA and the MGm/PIN. Rats received intra-LA 

infusion of either vehicle (ACSF), the PKG inhibitor Rp-8-Br-PET-cGMPS (1 κg/side), or the 

PKG activator 8-Br-cGMP (10 κg/side). Rats were infused 60 min prior to fear conditioning and 

trained with either 3 or 2 pairings of a 5 kHz, 75 dB, 20s tone with a 1 sec, 1 or 0.5 mA 

footshock. All rats were sacrificed 2 hr after training, a time point that we have previously shown 

to be optimal for observing training-induced increases in IEG expression in the LA and 

MGm/PIN. A combination of Western blotting and immunohistochemistry was performed on LA 

and MGm/PIN tissue to determine whether training induced expression of the IEGs Arc/Arg3.1, 

c-Fos, and EGR-1 is impaired or further enhanced when animals are trained under the influence 

of the PKG inhibitor or activator in the LA, respectively. Relative to vehicle-infused controls, 

rats infused with the PKG inhibitor prior to training exhibited decreases in IEG 

immunoreactivity in the LA and MGm/PIN. Conversely, those rats infused with the PKG 

activator prior to training exhibited increases in IEG immunoreactivity in the LA and MGm/PIN. 

Together with our previous experiments, these results suggest that the NO-cGMP-PKG signaling 

pathway bidirectionally regulates fear memory consolidation, in part, by promoting ERK-driven 

transcriptional regulation in the LA. These findings further suggest that LA-driven gene 

expression in the auditory thalamus might be equally important for fear memory consolidation. 

Disclosures: K.T. Ota, None; G.J. Young, None; G.E. Schafe, None. 

Poster





Support: NIH Grant 073949 

Title: Identification of genes induced by fear conditioning in the lateral amygdala 

Authors: *J. E. PLOSKI, K. PARK, G. E. SCHAFE; 

Psychology, Yale, New Haven, CT 

Abstract: The amygdala is critical for emotional learning and memory, including Pavlovian fear 

conditioning (FC), which is thought to play a critical role in the development of post-traumatic 

stress disorder. In recent years, a considerable amount of progress has been made in defining the 

cellular and molecular mechanisms of FC. Despite this progress, there still remains a paucity of 

information regarding the genes required for long term fear memory formation. This lack of 

progress is due, in part, to the relatively small size of the lateral nucleus of the amygdala (LA) 

and to relatively small changes in gene expression induced by FC, which leads to difficulties in 

unambiguously identifying gene expression changes following fear learning. To circumvent this 

limitation, we have adopted a strategy of using microarray technology to initially identify genes 

induced by long-term potentiation (LTP) and then to ask whether these genes are regulated in an 

associative manner by FC in the LA. LTP is an activity-dependent form of synaptic plasticity 

that is the leading candidate for the encoding and storage of long-term memory and, unlike 

learning, has the advantage of robustly and unambiguously driving gene expression in learningrelated 

regions of the brain. In our experiments, we have chosen to focus on LTP-driven changes 

in gene expression in the dentate gyrus, a hippocampal region that exhibits a high cell body 

density and in which LTP can be readily and robustly induced. Urethane anesthetized rats were 

implanted with a bipolar stimulation electrode into the angular bundle of the perforant path. 

Fifteen mins after implantation, rats were given high frequency stimulation (HFS) consisting of 

six trains of pulses (400 Hz, 20 msec), delivered at a 10 sec interval and repeated six times at an 

interval of 2 min. Low-frequency stimulation (LFS) consisted of six pulses, delivered at a 10 sec 

interval, repeated six times with an interval of 2 min, to match the tetanus without inducing LTP. 

Thirty minutes following LTP induction, the dentate gyrus was microdissected and global gene 

expression analysis was performed using DNA microarrays. We unambiguously identified ~40 

genes that were 3 fold or more induced upon LTP induction as compared to controls that 

received LFS. To determine if these identified genes were also induced in the LA by FC, we 

exposed an additional group of rats to FC consisting of 3 tone-shock pairings. Additional groups 

of rats received either three immediate shocks, a procedure that does not support fear learning, or 

no stimulation. We then performed qrtPCR on mRNA samples from microdissected LA samples 

from each group. This approach has allowed us to identify novel genes induced by FC. 

Disclosures: J.E. Ploski , None; K. Park, None; G.E. Schafe, None.

Poster 





Support: NSF Grant IBN-0129809 

Title: State anxiety in the rat: gene expression in the amygdala, hippocampus and prefrontal 

cortex 

Authors: *M. DONLEY, J. B. ROSEN; 

Dept Psychology, Univ. Delaware, Newark, DE 

Abstract: Several areas of the brain are known to be important in the learning and memory of 

fear, including the amygdala, the hippocampus, and the prefrontal cortex. Although the amygdala 

is critical for fear conditioning, it has been suggested that the amygdala is not only active during 

times of overt fear, but also during times of uncertainty and ambiguity, when state anxiety levels 

are higher than normal. Using a behavioral test and the immediate-early gene, egr-1, we find that 

during high or low levels of state anxiety, activity in the lateral nucleus of the amygdala (LA) 

responds differently when experiencing a novel (uncertain) environment. High and low levels of 

state anxiety were induced by handling rats in either a quiet or noisy room for 7 days. 

Behaviorally, when tested in an open field test on the eighth day, rats handled in a quiet 

environment show higher levels of anxiety (high state anxiety), whereas rats handled in an 

intermittently noisy environment show lower levels of anxiety (low state anxiety). Another set of 

quiet and noisy handled rats were divided into 3 groups: handled control, exposure to a novel 

chamber without footshock (context-no-shock), or contextually fear conditioned. Behaviorally, 

high and low state anxiety rats were indistinguishable, those fear conditioned froze after shock, 

while context-no-shock rats explored, but did not freeze. Thirty minutes after context exposure 

or fear conditioning, rats were sacrificed and in situ hybridization for egr-1 was performed. Both 

fear-conditioned high and low state anxiety rats had significantly increased egr-1 in the LA. 

Interestingly, in the context-no-shock condition, low state anxiety rats had egr-1 levels in the LA 

similar to handled rats, whereas high state anxiety rats had increased egr-1 levels similar to fear 

conditioned rats. The data suggest that high state anxiety rats experience more uncertainty in a 

novel environment, and therefore have higher amygdala activation than low state anxiety rats. 

Egr-1 expression in the amygdala is contrasted with that in the hippocampus. Levels of egr-1 in 

CA1 of the hippocampus were similarly increased in high and low state anxiety rats compared to 

handled controls, whether they were fear conditioned or in the context-no-shock condition. The 

findings indicate that the amygdala is involved in emotional information processing, even if it

does not reach the level of overt fear, and that the hippocampus processes novel spatial 

information regardless of its emotional content. Current experiments are examining the response 

of egr-1 in the medial prefrontal cortex during high and low states of anxiety. 

Disclosures: M. Donley , None; J.B. Rosen, None. 

Poster 






NRSA 

VA Merit Review 

Title: Expressing ASIC1a in the BLA of ASIC1a-null mice rescues deficits in fear memory but 

not fear learning 

Authors: *M. W. CORYELL 1 , A. M. WUNSCH 2 , J. HAENFLER 3 , J. E. ALLEN 3 , M. J. 

WELSH 4,5 , J. A. WEMMIE 3,6 ; 

1 Neurosci. Prog, 2 Psychiatry, Univ. Iowa, Iowa City, IA; 3 Psychiatry, 4 Intrnl. Med., Univ. of 

Iowa, Iowa City, IA; 5 Howard Hughes Med. Inst., Iowa City, IA; 6 Vetrans Affairs Med. Ctr., 

Iowa City, IA 

Abstract: The acid-sensing ion channel-1a (ASIC1a) contributes to fear-related behavior 

including conditioned fear and unconditioned fear. ASIC1a is expressed widely in the brain, but 

is most abundant in the basolateral amygdala (BLA) and other fear circuit structures. However, 

where in the brain ASIC1a acts to increase fear is not known. Here, we tested the effects of 

restricting ASIC1a expression to the BLA. We used an adeno-associated virus 1 (AAV1) 

encoding ASIC1a to restore ASIC1a expression to the BLA of ASIC1a-null mice. We then tested 

the behavioral effects in response to the predator odor TMT, a model of unconditioned fear. We 

also tested the acquisition of context fear conditioning and conditioned fear memory. We found 

that restoring ASIC1a expression to the BLA did not affect TMT-evoked freezing, suggesting 

that ASIC1a in the BLA is not sufficient to affect this behavior. In addition, ASIC1a expression 

in the BLA did not affect the acquisition of freezing during context fear conditioning; the AAV1- 

ASIC1a-injected mice remained impaired and froze no more than AAV1-GFP-injected controls.

In contrast, expressing ASIC1a in the BLA increased the recollection and expression of fear 

memory, and restored context evoked freezing in the ASIC1a-null mice to normal levels. 

Together, these studies demonstrate that AAV1-mediated gene transfer combined with 

traditional knockout technology can be a valuable tool in probing the complex relationship 

between genes, neural circuits, and behavior. Furthermore they suggest that ASIC1a expression 

in the BLA is sufficient to rescue some fear behaviors in the ASIC1a-null mice, but not all. 

Finally, these results demonstrate an unusual dissociation, the unexpected restoration of memory 

in the face of impaired acquisition. 

Disclosures: M.W. Coryell , NRSA, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); A.M. Wunsch, None; J. 

Haenfler, None; J.E. Allen, None; M.J. Welsh, None; J.A. Wemmie, NARSAD, B. Research 


already received). 

Poster 





Title: Transcriptional co-regulators and memory processes: behavioral phenotyping of RIP140 

knock-out mice 

Authors: *F. DUCLOT 1 , S. FRITSCH 2 , M. G. PARKER 3 , V. CAVAILLÈS 2 , T. MAURICE 1 ; 

1 INSERM U710, UM II / EPHE, Montpellier, France; 2 Montpellier Cancer Res. Inst., 

Montpellier, France; 3 Inst. for Reproductive and Developmental Biol., London, United Kingdom 

Abstract: Receptor-interacting protein 140 (RIP140) is a negative transcriptional co-regulator of 

gene expression, which was first described as recruited by estrogen receptor alpha (Cavailles et 

al., 1995, EMBO J. 14:3741). In fact, RIP140 interacts with many nuclear receptors, including 

retinoic acid receptors or glucocorticoid receptors. Recruitment of RIP140 results in inhibition of 

nuclear receptor target gene expression through different repressive domains interacting with 

histone deacetylase (HDAC) or C-terminal binding proteins. Chen et al. (2004, Mol Cell 

Endocrinol. 226:43) provided evidences for a competition between RIP140 and the 

transcriptional co-activator p300/CBP-associated factor (PCAF) for recruitment by nuclear 

receptor. PCAF bears an intrinsic histone acetyltransferase activity, remodeling chromatin 

structure towards a more decompacted state associated to transcriptional activation. We recently 

analyzed the role of PCAF in learning and memory process (Maurice et al., 2008,

Neuropsychopharmacol. in press). In order to determine whether RIP140 activity is also involved 

in learning and memory process, we analyzed the performances of male RIP140 knock-out (RIP- 

/-) mice, at two months of age, using several behavioral paradigms. We first tested general 

mobility of RIP140-/- mice in the open-field and rotarod tests and no difference was observed 

between wild-type (RIP140+/+), heterozygous (RIP140+/-) and RIP140-/- animals. To evaluate 

learning and memory procedures, we used spontaneous alternation behavior in the Y-maze for 

spatial working memory and place learning in the Morris water maze (MWM) for spatial longterm 

memory (reference and working memory components). Contextual long-term memory was 

also evaluated using a step-down type passive avoidance procedure. As compared with wild-type 

or heterozygous animals, RIP140-/- mice showed spatial working memory impairments (in terms 

of decreased alternation performance and higher escape latencies in the variable platform 

position procedure in the MWM) and drastic long-term memory deficits (no decrease of escape 

latencies in MWM and passive avoidance latencies). These observations reveal that RIP140 gene 

invalidation results in marked memory deficits suggesting that this transcriptional co-regulator 

plays an important role in the neurophysiological mechanisms underlying learning and memory. 


France). 

Disclosures: F. Duclot, None; S. Fritsch, None; M.G. Parker, None; V. Cavaillès, None; T. 

Maurice, None. 

Poster 

295. Memory Consolidation, Reconsolidation, Anatomy, and Mechanisms 



Topic: F.02.h. Memory modulation, consolidation, and reconsolidation 



Title: Reconsolidation of a cocaine-paired stimulus requires amygdalar protein kinase a 

Authors: *H. SANCHEZ 1 , J. J. QUINN 1,2 , M. M. TORREGROSSA 1 , J. R. TAYLOR 1 ; 

1 Div. Molec Psychiatry, Yale Univ., New Haven, CT; 2 Psychology, Miami Univ., Oxford, OH 

Abstract: Cued fear memory re-stabilization (or reconsolidation) following retrieval requires 

protein kinase A (PKA). Intra-amygdalar infusion of the PKA inhibitor, Rp-cAMPs, following 

retrieval of a discrete fear stimulus disrupts freezing to that stimulus in a subsequent test.

Conversely, intra-amygdalar infusion of the PKA activator, 6-BNZ, following retrieval enhances 

subsequent freezing to the fear stimulus. Here we assessed whether PKA is critical for the 

reconsolidation of an appetitive, cocaine-paired stimulus. Rats were trained to lever press for 

cocaine across 12 days. Active lever presses were reinforced with a cocaine infusion on a FR1 or 

FR3 schedule. Every cocaine infusion was paired with a tone/light conditioned stimulus (CS). 

Inactive lever presses were never reinforced. Following the last day of acquisition, rats were 

placed into a novel context and received 3 discrete presentations of the tone/light CS. 

Immediately following this reactivation session, rats received bilateral intra-amygdala infusions 

of Rp-cAMPs or vehicle. Rats in each group were then split into two testing conditions: 

conditioned reinforcement test and reinstatement test. The conditioned reinforcement test was 

used to assess the ability of the tone/light CS to serve as a reinforcer in the acquisition of a new 

response (nosepoke) in a novel context. Rats in the reinstatement test condition underwent 8 days 

of lever-press extinction in the original self-administration context. No CSs or drug was 

presented during these extinction sessions. One day following the last day of extinction, rats 

were tested for cue-induced reinstatement of lever-pressing during which active lever presses 

were followed by presentation of the tone/light CS. Rats then received two more days of leverpress 

extinction. The following day, rats were tested for cocaine-induced reinstatement in which 

they were given a systemic injection of cocaine (15mg/kg i.p.) and then lever-pressing was 

assessed. Rats that had received Rp-cAMPs into the amygdala following the tone/light CS 

reactivation showed lower levels of both conditioned reinforcement and cue-induced 

reinstatement compared to controls. Nevertheless, there were no differences in the magnitude of 

cocaine-induced reinstatement. These data show that PKA is necessary for reconsolidation of a 

cocaine-paired stimulus and the effect of PKA is specific to cue-induced responding. 

Disclosures: H. Sanchez , None; J.J. Quinn, None; M.M. Torregrossa, None; J.R. Taylor, 

None. 

Poster 






DGAPA-UNAM Grant IN220706-3 

Title: Memory retrieval is not necessary for memory reconsolidation

Authors: *P. GARCÍA-DELATORRE, C. J. RODRIGUEZ-ORTIZ, J. L. ARREGUIN- 

MARTINEZ, P. CRUZ-CASTAÑEDA, C. ZAMORANO-NGTEAJAN, C. PÉREZ-SÁNCHEZ, 

F. BERMÚDEZ-RATTONI; 

UNAM-IFC, Mexico city, Mexico 

Abstract: The memory consolidation theory postulates that through the synthesis of new 

proteins, recently acquired memories are strengthened over time into a stable long-term memory 

trace. However, it has been proposed that reactivated memory requires protein synthesis once 

again to be retained in long-term storage; this process is known as reconsolidation. We have 

provided evidence for reconsolidation as part of an updating process that integrates information 

to a previously consolidated memory trace. Until now, reconsolidation has been thought to 

depend on retrieval. Here we used the conditioned taste aversion (CTA) task to asses the 

hypothesis to whether reconsolidation depends on retrieval or not. On the first acquisition day, 

three groups of rats received the conditioned stimulus saccharin (0.1%) followed by the 

unconditioned stimulus LiCl injection (0.15M, 10mL/kg) and as a result, aversion to saccharin 

was obtained. On a second CTA trial, one group of animals was injected with anisomycin (a 

protein synthesis inhibitor) in both IC and amygdala, another group was infused with NBQX 

only in the amygdala (AMPA receptor antagonist that prevents CTA memory retrieval). The last 

group was injected with anisomycin in IC and amygdala and NBQX in amygdala. Twenty four 

hours later animals were tested. As expected, when tested, anisomycin infusions in both the IC 

and amygdala, impaired incorporation of new information and the previously consolidated trace 

was affected. Interestingly, animals injected with NBQX, despite they did not retrieve, acquired 

strong taste aversion. The group injected with NBQX and anisomycin submitted to a probe test 

showed that the previously stored memory was impaired even though retrieval did not take place. 

Our results indicate that retrieval is not a necessary process for reconsolidation to occur. 

Disclosures: P. García-delaTorre , None; C.J. Rodriguez-Ortiz, None; J.L. Arreguin- 

Martinez, None; P. Cruz-Castañeda, None; C. Zamorano-NgTeajan, None; C. Pérez- 

Sánchez, None; F. Bermúdez-Rattoni, None. 

Poster 





Support: NSF GRF

Title: Cannabinoid administration impairs acquisition, consolidation and reconsolidation of 

rodent spatial memory 

Authors: *F. T. CANDELARIA 1 , D. A. HAMILTON 1,2 ; 

1 Dept Psychol, Univ. New Mexico, Albuquerque, NM; 2 Neurosciences, Univ. of New Mexico, 

Albuquerque, NM 

Abstract: Memory reconsolidation is proposed to stabilize labile, reactivated memory in ways 

similar to those involved in initial consolidation. In principle, any treatment that disrupts initial 

consolidation could also disrupt reconsolidation. Cannabinoids have been shown to interfere with 

learning, consolidation, and retrieval, thus, it is possible that cannabinoids also disrupt 

reconsolidation. The effect of cannabinoids on memory acquisition, consolidation and 

reconsolidation of spatial memory in the Morris water task (MWT) were evaluated in 3 

experiments. Experiment 1 examined learning versus performance deficiencies using the 

Repeated Acquisition Procedure (Keith & Galizio 1997). During each daily session (10 sessions 

total), rats were given 6 trials in environment 1 in which the platform always remained in a fixed 

location, and 6 trials in environment 2 where the platform was repositioned at the beginning of 

each daily session. Rats were given injections of cannabinoid agonist WIN 55, 212-2 (i.p., 3.7 

mg/kg) or vehicle 30 min prior to training. An initial deficit in the fixed platform condition was 

observed in rats that received the agonist, however, following 4-5 sessions agonist and vehicle 

animals performed similarly. Rats given the agonist showed modest learning deficits in the 

moving platform condition, but always reached comparable levels of performance to that of 

controls within each session. Experiment 2 examined effects of the agonist on initial 

consolidation. Rats were trained in the fixed platform MWT and given agonist or vehicle 

injections immediately following training; memory was assessed using a single non-reinforced 

probe trial the next day. Rats who received the agonist showed poorer memory compared to 

vehicle controls. Experiment 3 examined the effects of the agonist on reconsolidation. Rats were 

trained in the fixed platform MWT and the next day memory was reactivated via a single nonreinforced 

probe trial. Immediately afterward rats were given agonist or vehicle injections while 

a separate group of rats received injections but no memory reactivation. Memory was assessed 

24hrs later by a single non- reinforced probe trial. Rats who received the agonist plus 

reactivation showed poorer memory compared to vehicle controls and animals that received the 

drug in the absence of memory reactivation, indicating that reactivation was crucial for the 

cannabinoid-induced spatial memory impairment. In summary, agonism of central cannabinoid 

receptors resulted in no disturbances in performance, a modest deficit in acquisition, and 

significant impairments in consolidation and reconsolidation of spatial memories. 

Disclosures: F.T. Candelaria, None; D.A. Hamilton, None. 

Poster 

295. Memory Consolidation, Reconsolidation, Anatomy, and Mechanisms






Title: Fear reconsolidation following an unexpected retrieval event requires amygdalar protein 

synthesis 

Authors: *J. J. QUINN 1,2 , H. SANCHEZ 1 , S. S. PARIKH 1 , J. R. TAYLOR 1 ; 

1 Div. Mol. Psychiatry, Yale Univ., New Haven, CT; 2 Psychology, Miami Univ., Oxford, OH 

Abstract: Memory re-stabilization (or reconsolidation) following retrieval usually requires de 

novo protein synthesis. In particular, fear memory reconsolidation can be disrupted using postretrieval 

intra-amygdalar protein synthesis inhibitors. The specific encoding and retrieval 

conditions under which fear memory reconsolidation occurs, and is dependent upon protein 

synthesis, are not fully understood. We examined a role for protein synthesis-dependent 

amygdalar reconsolidation of an auditory fear discrimination memory. Rats were trained to 

discriminate two auditory stimuli. The CS+ was consistently paired with footshock while the CS- 

was never paired with footshock. Another group was trained with partial reinforcement of both 

stimuli in which each stimulus was paired with footshock on half of its presentations. Thus, both 

discrimination training and partial reinforcement training involved equivalent numbers of 

conditional and unconditional stimulus presentations. Three presentations of each CS were given 

per session across five consecutive daily sessions. Reactivation of the CS+ (discrimination 

trained group) or one of the two CSs (partial reinforcement trained group) consisted of a single 

presentation in a novel context. Bilateral intra-amygdalar infusions of the protein synthesis 

inhibitor, cyclohexamide, or vehicle were made immediately after the reactivation session. 

Subsequently, freezing during each CS was assessed in separate sessions on two consecutive 

days. Cyclohexamide infused following a "non-reinforced" reactivation of the CS+ in 

discrimination-trained rats attenuated freezing to that CS in a subsequent test. However, 

cyclohexamide had no effect in rats trained with two-cue partial reinforcement. Post-training 

lesions of the amygdala disrupted cued fear expression following both discrimination and partial 

reinforcement training, confirming that the amygdala is critical for both types of fear memories. 

Finally, intra-amygdalar cyclohexamide following a "reinforced" reactivation trial had no effect 

on subsequent freezing to the cue in either discrimination- or partial reinforcement-trained rats. 

These data suggest that the degree to which cue reactivation leads to an unexpected outcome 

determines whether or not reconsolidation of that cue memory requires protein synthesis. 

Disclosures: J.J. Quinn , None; H. Sanchez, None; S.S. Parikh, None; J.R. Taylor, None.

Poster 





Support: NIMH Grant R01 MH074736 

NIMH Grant R01 MH65635 

NARSAD Independent Investigator Award 

Title: Memory reactivations strengthen memory retention and persistence 

Authors: *J. S. RICEBERG, D. Y. CHEN, S. M. TAUBENFELD, C. M. ALBERINI; 

Neurosci., Mount Sinai Sch. of Med., New York, NY 

Abstract: Newly learned information is initially in a labile state and over time stabilizes into 

long-term memory by a consolidation process requiring RNA and protein synthesis. Previously 

established memories can also become transiently labile if reactivated, for example, by retrieval. 

This post-reactivation phase again depends on new protein synthesis and is known as 

reconsolidation. Why should a stabilized memory return to this vulnerable state? One hypothesis 

proposes that the labile phase of reconsolidation mediates memory strengthening. Using a singletrial 

inhibitory avoidance (IA) paradigm in rat, we tested whether reactivation exposures affect 

memory strength. During IA, animals form an association between a CS (context) and a US 

(foot-shock): reactivation of this memory typically consists of an exposure to the CS alone. 

However, exposure to a non-reinforced CS can also elicit extinction learning, which results in a 

decrease of the conditioned response. We used protocols that do not induce behavioral extinction 

to test whether the strength and/or persistence of IA memory is affected by reactivation 

exposures and whether these potential changes critically involve the labile phase of 

reconsolidation. We found that the retention of IA memory is a graded function of the number of 

reactivations. Results from experiments using both short (2-day) and long (7-day) interreactivation 

intervals indicate that the incremental changes in memory retention contribute to 

both strengthening and preventing the decay of memory. These findings support the hypothesis 

that single-trial fear memories are strengthened and maintained through reactivation/s of the 

original experience. 

Disclosures: J.S. Riceberg , None; D.Y. Chen, None; S.M. Taubenfeld, None; C.M. Alberini, 

None.

Poster 





Support: JUST grant 67-2007 

Title: The combined effect of diet and exercise on memory formation and expression of BDNF 

Authors: *O. F. KHABOUR 1 , K. ALZOUBI 2 , M. ALOMARI 3 , M. A. Y. AL-ZU'BI 4 ; 

1 Dept Med. Lab. Sci., 2 Dept Clin. Pharm., 3 Dept of Physical Therapy, 4 Dept of Biotech., Jordan 

Univ. Sci. & Technol., Irbid, Jordan 

Abstract: Substantial data suggests that cognitive function can be influenced by many lifestyle 

activities associated with changes in energy metabolism such as exercise and diet. Exercise is a 

physical activity that increases energy expenditure. Studies on humans and animals have shown 

that exercise seams to improve learning and memory formation and to counteract the effect of 

depression. Dietary restriction (DR) enhances memory formation and reduces aging associated 

deficits in cognitive function. Brain-derived neurotrophic factor (BDNF) is implicated in the 

effects of exercise on cognitive function and its level is also modulated by dietary restriction and 

body metabolism. In spite of the positive effects of these lifestyles activities on general body 

health, cognitive functions and the possible cross interactions in the pathways at the molecular 

level, no single research has studied the effects of changes in energy metabolism associated with 

exercise and DR on cognitive function or molecular measures. 

This study investigated the combined effects of exercise and DR on spatial memory formation in 

rats using the radial arm water maze paradigm and the levels of expression of BDNF in the brain. 

Disclosures: O.F. Khabour, None; K. Alzoubi, None; M. Alomari, None; M.A.Y. Al-Zu'bi, 

None. 

Poster 





Support: NIH 

Title: Genetic inducible blockade of the temporoammonic pathway: spatial coding and 

acquisition of spatial and contextual information in the absence of direct cortical input 

Authors: *J. SUH 1,2,3 , A. J. RIVEST 1,2 , S. TONEGAWA 1,2,3 ; 

1 Picower Inst. Learning & Memory, MIT, Cambridge, MA; 2 RIKEN-MIT Ctr. for Neural Circuit 

Genet., Cambridge, MA; 3 Howard Hughes Med. Inst., Cambridge, MA 

Abstract: Hippocampal CA1 pyramidal neurons, place cells, receive converging inputs from 

both the associative network in CA3 via the Schaffer collaterals and entorhinal cortex (EC) layer 

III via the temporoammonic (TA) pathway. Previous studies reported that lesions in layer III in 

the EC resulted in larger and more dispersed place fields in CA1, and lesions in the TA pathway 

resulted in deficits in finding platform location four weeks after training in the Morris water 

maze. Although suggestive, these lesions do not discount incompleteness of lesions and damage 

to adjacent anatomical structures and pathways. To address this issue rigorously, we created a 

triple transgenic mouse line (ECIII-TeTX), in which synaptic transmission can be selectively 

blocked at the TA-CA1 synapses in an inducible and reversible manner, while leaving the 

hippocampal tri-synaptic pathway input to CA1 intact. Tetrode recording in CA1 of freely 

behaving mutant mice demonstrated confined place fields and indistinguishable spatial 

information density in both novel and familiar environments. The mutant mice also displayed no 

deficits in learning and short term memory recall in the Morris water maze task and contextual 

fear conditioning. These results suggest that direct cortical input from the EC layer III to the 

hippocampus may not play a crucial role in spatial coding of CA1 or in spatial and contextual 

learning. 

Disclosures: J. Suh , None; A.J. Rivest, None; S. Tonegawa, None. 

Poster 





Title: Temporal duration of cocaine-enhanced spatial memory in C57BL/6 mice

Authors: S. A. BAELLA 1 , S. D. IÑIGUEZ 2 , S. CHARNTIKOV 1 , M. S. HERBERT 1 , *C. A. 

CRAWFORD 1 ; 

1 Dept Psych, California State Univ., San Bernardino, CA; 2 Dept Psych, Florida State Univ., 

Tallahassee, FL 

Abstract: Cocaine affects memory consolidation in a complex manner, because studies have 

reported that this indirect dopamine agonist both enhances and inhibits performance on memory 

tasks. These divergent results may be due to differences in the dose and time-course of cocaine 

administration. For example, administering low-to-moderate doses of cocaine improve 

performance on avoidance tasks, while higher doses of cocaine decrease performance. Moreover, 

cocaine is often administered prior to training, which can impact acquisition by inducing 

nonspecific effects (e.g., by increasing locomotion). In the present study, we examined the ability 

of post-training injections of cocaine to alter the consolidation of spatial memory using the 

Morris water maze. In the first experiment, 109 male and female C57BL/6 mice were given eight 

training trials to learn the location of a submerged escape platform. Immediately following the 

eighth trial, mice were given a single injection of cocaine (0, 1.25, 2.5, 5, or 20 mg/kg, ip) and 

returned to their home cage. After 24 or 48 hr, mice were given a single trial to assess spatial 

memory. Cocaine had a dose-dependent effect on spatial memory performance, because only 2.5 

mg/kg cocaine caused a significant reduction in the latency to find the platform. The cocaineinduced 

enhancement in spatial memory was observed 24 hr, but not 48 hr, after training. There 

were no sex differences in water maze performance. In the second experiment, 40 male C57BL/6 

mice were trained as in the first experiment except they received an injection of cocaine (0 or 2.5 

mg/kg) 0, 1, 2, or 4 hr after the last training trial and were then tested 24 hr later. We again found 

that 2.5 mg/kg cocaine enhanced memory performance, but only when the drug was 

administered 1 or 2 hr, but not 4 hr, after the last training trial. Overall, these data show that a 

moderate dose of cocaine, given up to 2 hr after training, can enhance the spatial memory of 

adult mice on the Morris water maze. 

Disclosures: S.A. Baella, None; S.D. Iñiguez, None; C.A. Crawford, None; S. Charntikov, 

None; M.S. Herbert, None. 

Poster 





Support: HSFC

NIMH (MH61460) 

AHFMR 

NSERC 

Title: Learning parameters mitigate retrograde amnesia after hippocampal damage 

Authors: *H. LEHMANN 1 , F. T. SPARKS 2 , S. C. SPANSWICK 2 , R. J. SUTHERLAND 2 ; 

1 Dept Psychol, Trent Univ., Peterborough, ON, Canada; 2 Neurosci., Univ. Lethbridge, 

Lethbridge, AB, Canada 

Abstract: Damage to the hippocampus (HPC) causes retrograde, but not anterograde, amnesia 

for several types of memories. With similar learning parameters, HPC damage impairs memories 

that were acquired before damage, but does not impair the ability to establish new memories. 

Thus, neural systems outside the HPC are capable of acquiring and storing memories, but 

normally the HPC competes with and prevents these systems from fully doing so. We examined, 

in contextual fear conditioning and object recognition, whether repeated learning sessions would 

engage the other systems and mitigate the effects of post-training HPC damage. In the contextual 

fear-conditioning task, rats were given 1 or 11 fear conditioning sessions, which were matched 

for number of shocks and amount of time in the context. One week after the initial learning 

session, the rats received sham or neurotoxic-induced damage to the entire hippocampus. 

Following recovery, rats were returned to the context for a retention test during which the 

amount of behavioural freezing was measured as an index of memory. The rats were also tested 

in a novel context to determine whether the freezing was specific to the learning context. In the 

object recognition task, rats received either 10 or 30 learning sessions. Approximately 45 days 

after the first learning session, rats received sham or complete HPC damage. Results indicate 

that, in the contextual fear conditioning task, sham rats, regardless of the number of learning 

sessions, expressed robust freezing in the context that was paired with shock and very little 

freezing in the novel context. Rats with HPC damage that previously received a single learning 

session expressed minimal and significantly less freezing than their respective control group. In 

contrast, rats with HPC damage that received repeated conditioning sessions expressed high 

levels of freezing and did not significantly differ from rats in their respective control group. 

Moreover, this freezing was specific to the conditioning context because the same rats did not 

freeze in the novel context. In the object recognition task, sham rats remembered which of two 

objects they had previously encountered, regardless of the number of learning sessions they 

received. However, only the HPC rats that received 30 learning sessions showed intact object 

recognition memory. Combined, these findings demonstrate that changing learning parameters 

can create a memory that is independent of the HPC. 

Disclosures: H. Lehmann , None; F.T. Sparks, None; S.C. Spanswick, None; R.J. 

Sutherland, None.

Poster 





Support: R01-AG13622 

R01-NS38480 

P50 MH077972 

Title: Following structural dynamics of neurons in mammalian brain during spatial learning task 

Authors: *J. BALAJI 1 , R. CZAJKOWSKI 2 , A. J. SILVA 2 ; 

1 Departments of Neurobiology,Ps, 2 Departments of Neurobiology, Psychiatry and Biobehavioral 

Sciences, Psychology and Brain Res. In, UCLA, Los angeles, CA 

Abstract: Neurons involved in memory formation have long been postulated to undergo 

structural changes during memory acquisition and consolidation. However there is little direct 

evidence that synaptic level structural changes occur following behavioral training. This is 

mostly due to technical limitations in imaging neurons that are involved in well established 

behavioral paradigms such as the Morris water maze. Emerging optical technologies, such as the 

use of long working distance objectives for multi-photon excitation (following craniotomy) or 

imaging through Gradient Index (GRIN) lenses, have helped to overcome some of these 

limitations. Using a relatively small, simple to implement and chronically sustainable optical 

probe together with state of the art in vivo imaging technology, we show that it is possible to 

follow structural changes occurring in the mammalian brain over a prolonged period of time. We 

further characterize our method in vitro, in tissue and in live brain to arrive at the limits of our 

approach. 

Disclosures: J. Balaji, None; R. Czajkowski, None; A.J. Silva, None. 

Poster 





Support: CONACyT 60478 

DGAPA-UNAM IN220706 

Title: Dorsal hippocampus is differentially involved in consolidation of extinction but not 

consolidation of aversive taste memory 

Authors: *I. BALDERAS, C. RODRIGUEZ-ORTIZ, A. ISLAS, J. CHAVEZ-HURTADO, F. 

BERMUDEZ-RATTONI; 

Neurociencias, IFC-UNAM, Mexico city, Mexico 

Abstract: Taste recognition memory is an important ability that animals have developed 

throughout evolution in order to respond efficiently to the consumption of edibles. In laboratory 

conditions when a novel taste is presented and has adverse consequences (e.g. induced gastric 

malaise), the animal develops a long-lasting taste aversion (CTA). Afterward, an increased 

consumption is observed in subsequent presentations when the taste is not followed by gastric 

malaise, this behavior is called extinction. The participation of the hippocampus in taste 

recognition memory formation has been slightly studied and results are controversial. Some 

studies showed lack of effects in CTA after lesions of the hippocampus; while others reported an 

enhancement in CTA. We studied if protein synthesis in the dorsal hippocampus is required for 

long-term aversive taste memory and its extinction. The protein synthesis inhibitor anisomycin 

was locally injected immediately after acquisition and either long-term CTA or extinction taste 

memory was tested. The results showed that anisomycin applied into the hippocampus disrupted 

consolidation of extinction memory. Interestingly, anisomycin infusions into the hippocampus 

did not affect CTA memory consolidation. These results suggest that the hippocampus has a 

differential involvement in the consolidation of taste recognition memories, depending on the 

taste association to appetitive or aversive signals. 

Disclosures: I. Balderas , None; C. Rodriguez-Ortiz, None; A. Islas, None; J. Chavez- 

Hurtado, None; F. Bermudez-Rattoni, None. 

Poster 






National Institute of Mental Health F31 MH 081621-A1 

National Institute of Mental Health R01 MH 74736 

National Institute of Mental Health R01 MH 65635 

Title: Temporal requirement of hippocampal BDNF for long-term memory consolidation 

Authors: *D. Y. CHEN, G. POLLONINI, C. M. ALBERINI; 

Neurosci., Mount Sinai Sch. Med., New York, NY 

Abstract: Newly learned information becomes long-term memory through a process known as 

consolidation, which depends on an initial phase of transcription and translation. However, little 

is known about the temporal progression of the molecular requirements of consolidation. The 

neurotrophin, brain-derived neurotrophic factor (BDNF) is known to play an important role in 

memory consolidation. Recently, BDNF has been also reported to mediate memory maintenance 

as dorsal hippocampal injection of function-blocking anti-BDNF antibodies 12 hours after 

inhibitory avoidance (IA) training disrupted memory retention at 7 days, but not at 2 days after 

training. To determine the role of hippocampal BDNF in long-term memory consolidation, using 

2 distinct strategies, we disrupted hippocampal BDNF functions before or at several times points 

after IA training. We found that dorsal hippocampal injection of either function-blocking anti- 

BDNF antibody or BDNF scavenger (TrkB-Fc) 15 minutes before training blocked the 

consolidation of long-term memory as the animals were amnesic when tested at 2 days after 

training. The effect was persistent at 7 days after training and the memory did not recover 

following a reminder foot-shock. Furthermore, dorsal hippocampal injection of the same anti- 

BDNF antibody or TrkB-Fc immediately after training only modestly decreased long-term 

memory retention at 2 days after training, but significantly disrupted the memory retention at 7 

days after training. This memory impairment was not restored after a subsequent foot-shock 

reminder. Conversely, disruption of hippocampal BDNF function at 2 and 7 days after IA 

training did not affect memory retention. These results suggest that IA memory consolidation 

critically requires hippocampal BDNF not only during training, but also after training when it is 

needed to complete the consolidation process. Potential molecular mechanism underlying the 

hippocampal BDNF-mediated long-term memory consolidation will be discussed. 

Disclosures: D.Y. Chen , None; G. Pollonini, None; C.M. Alberini, None. 

Poster 





Title: Retrograde effect of hippocampal LTP induction on spatial memory: influence of memory 

strength and memory reactivation 

Authors: *K. G. AKERS, D. A. HAMILTON; 

Psychology Dept., Univ. of New Mexico, Albuquerque, NM 

Abstract: It is generally believed that memories depend on specific patterns of synaptic 

strengths across a neural network. If this is the case, then interfering with synaptic strength after 

learning should disturb memory for what was learned. Here, we examined whether interfering 

with hippocampal synaptic strength via LTP induction disrupts memory for a previouslyacquired 

spatial task. First, rats were implanted with stimulating electrodes in the perforant path 

and recording electrodes in the dentate gyrus. Next, rats received either 12 or 48 training trials in 

the Morris water task, in which a hidden escape platform could be located based on its spatial 

relationship to available distal cues. After training, bilateral high-frequency stimulation (HFS) 

was administered (ten 400Hz trains, 10 pulses/train, 30s inter-train interval). Following HFS, rats 

were given a memory retention test comprised of two consecutive no-platform probe trials. We 

found that, among rats that received 12 training trials, the HFS group exhibited a significant 

decline in performance across the two probe trials compared to the control group, evidenced by 

an increase in latency to reach the former platform location. Among rats that received 48 training 

trials, however, there was no difference between the HFS and control groups. A memory 

reactivation treatment given immediately before HFS (i.e. brief placement of rats on the hidden 

platform at its trained location) did not modulate the effect of HFS on probe trial performance. 

These results support the hypothesis that memories depend on changes in synaptic strength. 

Furthermore, they suggest that stronger memories are more resistant to disruption by LTP than 

weaker memories, and that the disruptive effect of LTP is independent of whether the memory is 

in an active or inactive state at the time of HFS. 

Disclosures: K.G. Akers, None; D.A. Hamilton, None. 

Poster 






Title: De novo translation initiation in CA1 pyramidal cells is critical for hippocampal memory 

consolidation 

Authors: *Z. JIANG, J. BELFORTE, Y. LU, Y. YABE, J. PICKEL, C. SMITH, B. LU, K. 

NAKAZAWA; 

NIMH, Bethesda, MD 

Abstract: An essential role for new protein synthesis in synaptic plasticity and memory 

formation has been proposed based on previous studies using protein synthesis inhibitors (PSIs). 

However, PSIs are known to have significant side effects. It has been questioned whether 

memory impairments induced by PSIs are due to global protein synthesis inhibition or other 

cellular actions caused by PSIs. In addition, brain region or cell-type specific inhibition of 

protein synthesis by these drugs is hard to achieve. Therefore, the inducible genetic manipulation 

of translational machinery in particular brain regions and/or cell types has been desired. It is 

known that double-strand RNA-dependent protein kinase R (PKR) inhibits protein synthesis by 

phosphorylating eIF2α, a key factor in translation initiation. Previous studies indicated that 

dimerization of the PKR kinase domain is necessary and sufficient for its kinase activity. To 

establish an inducible genetic manipulation of the protein translation system, we fused FKBP12, 

a chemically induced dimerization cassette, to the PKR kinase domain to generate floxed-PKR 

transgenic strains. After crossing one floxed-PKR line, fPKR#6, with a forebrain specific Cre 

line, T29-1, expression of PKR was targeted to the hippocampus, primarily CA1 and DG 

excitatory neurons. Activation of PKR with AP20187 infusion induced a selective increase of 

phospho-eIF2α and ATF4 in CA1 pyramidal cells. L-LTP, but not E-LTP, at CA1 synapses was 

impaired in hippocampal slices following activation of PKR. Mutant mice with the drug infusion 

were impaired in habituation to a new open field, context discrimination in a step-through 

avoidance task, and formation of contextual, but not auditory, fear memory 24 hr after the 

trainings. However, retrieval of contextual fear memory 30 min after the training was not 

affected by the manipulation. These results suggest that manipulation of protein synthesis in CA1 

pyramidal cells is able to impair hippocampal-dependent memory consolidation. Despite these 

behavioral deficits, overall levels of protein synthesis in CA1 were not changed after PKR 

activation. In contrast, significant decreases in protein synthesis mediated by anisomycin were 

not sufficient to induce memory impairments. This double dissociation suggests that global de 

novo protein synthesis inhibition per se does not contribute to impairments in memory 

consolidation. It rather suggests a critical role of particular protein pathways in CA1, such as 

ATF4 and its downstream targets, in the consolidation of hippocampal-dependent memories. 

Disclosures: Z. Jiang, None; J. Belforte, None; Y. Lu, None; Y. Yabe, None; J. Pickel, 

None; C. Smith, None; B. Lu, None; K. Nakazawa, None.

Poster 





Support: Research supported by the School of Behavioral and Brain Sciences at the University 

of Texas at Dallas 

Title: Environmentally induced modification of the long term consolidation process of 

contextual fear memory 

Authors: *D. F. PENA, V. C. ROBERTS, K. CHANG, M. P. KILGARD, C. K. MCINTYRE; 

Univ. of Texas at Dallas, Richardson, TX 

Abstract: Environmental enrichment (EE) refers to a housing regimen that, relative to standard 

housing, has larger cages, activity-related toys, more conditions or states in which to learn, and 

greater social complexity. Early findings showing marked increases in cortical thickness in the 

occipital cortices and increased dendritic spine densities were the first to demonstrate that an 

enriched environment could affect plastic changes in the brain. Current research suggests that EE 

leads to behavioral, cellular and molecular changes. Recent work by Fischer and colleagues 

suggests that prolonged enrichment can recover previously unrecoverable memories in CK-p25 

transgenic mice. However, little is known about the effects of enrichment on loss and recovery of 

memories in normally functioning animals. Here we examined the effects of enrichment on 

acquisition and memory retention performance 2 days and 2 weeks after a single training 

experience. Female Sprague-Dawley rats were housed on a reverse night/day cycle in one of two 

paradigms for 30 days. Enriched animals were communally housed (6 rats/cage) in a large (76 L 

x 45 W x 90 H cm) cage with a running wheel and several forms of auditory stimulation. Control 

animals were individually housed (25 L x 15 W x18 H) in a 30 cage colony rack. All animals 

were subsequently trained on a continuous multi-trial inhibitory avoidance task. Memory 

retention was tested 2 days and again 2 weeks after training. Enriched animals did not 

differentially acquire the task (p>0.9), nor did they perform significantly better in a retention test 

2 days post training (p > 0.5). However, retention latency was reduced after 2 weeks in nonenriched 

animals, but it was increased in enriched animals. A within animal retention 

comparison (2 wks/2 days) showed a significant difference in performance between enriched and 

non-enriched animals (p

Poster 





Support: Centennial Research Scholars Grant 

Title: Spatial working memory formation is associated with changes in hippocampal cortactin 

Authors: *M. L. OLSON, J. R. BEYER, T. M. FOM, C. R. HENDRICKS, K. M. 

RANDOLPH; 

Psychology, Concordia Col., Moorhead, MN 

Abstract: The cellular mechanisms that cause neurons to undergo structural changes following 

memory formation are largely unknown. We set out to determine if cortactin, an actin-binding 

protein and known regulator of dendritic spine structure, is involved in the cellular changes that 

underlie spatial working memory formation. Spatial working memory is the ability of an 

organism to temporarily record and manipulate information in its spatial environment. We used 

the spatial win-shift task in the eight-arm radial maze to determine if the level of hippocampal 

cortactin is altered following spatial working memory formation. Male, Sprague-Dawley rats 

were trained on the task for 25 days, until all animals met the learning criterion of 1 or fewer 

errors. On the testing day animals were again trained then humanely sacrificed 1 hr post-training. 

Hippocampi were dissected out and cortactin levels were assessed via Western blot. Our results 

indicate that hippocampal cortactin levels are significantly decreased in rats trained on the spatial 

win-shift task compared with control animals. These results suggest a relationship between 

hippocampal cortactin expression and spatial working memory formation. We speculate that 

cortactin may mediate the activity-induced structural plasticity thought to underlie memory 

formation. 

Disclosures: M.L. Olson, None; J.R. Beyer, None; T.M. Fom, None; C.R. Hendricks, 

None; K.M. Randolph, None. 

Poster 





Support: School of Behavioral and Brain Sciences at The University of Texas at Dallas 

Title: Training induced arc expression in the rat anterior cingulate cortex 

Authors: *C. HOLLOWAY, J. R. MCREYNOLDS, C. K. MCINTYRE; 

Sch. Behav and Brain Sci., Univ. Texas, Dallas, Richardson, TX 

Abstract: The amygdala, particularly the basolateral area (BLA), has been shown in previous 

research to modulate long-term memory for emotional experiences through its projections to 

other regions in the brain. The anterior cingulate cortex (ACC) has been shown to be important 

for memory of inhibitory avoidance (IA) training, and this effect is modulated by the BLA. The 

transcript for the immediate early gene named activity-regulated-cytoskeletal-associated protein 

(Arc) has been localized specifically to stimulated dendrites and is important for learning, thus 

making it a candidate marker for neuronal plasticity. Our previous research suggests that the 

BLA modulates hippocampal plasticity by mediating expression of Arc. In the present 

experiment we examined Arc protein expression after IA training in areas of the brain that are 

involved in memory processing, including the ACC. If the BLA is modulating long-term 

memory by influencing plasticity in other areas of the brain in the same way it is influencing 

plasticity in the hippocampus, we would expect to see an increase in Arc protein expression due 

to IA training in those areas that have interactions with the BLA. Male Spraque-Dawley rats 

were trained on an IA task, and sacrificed thirty minutes later. Arc protein expression was 

visualized using immunohistochemistry. Preliminary data suggest that IA training increases Arc 

expression in the ACC. These results support the growing evidence that Arc expression in areas 

other than the hippocampus plays a role in long-term memory. Furthermore, these results lay the 

groundwork for examining the interaction of the BLA and the ACC in memory consolidation. 

Research supported by The School of Behavioral and Brain Sciences, The University of Texas at 

Dallas. 

Disclosures: C. Holloway, None; J.R. McReynolds, None; C.K. McIntyre, None. 

Poster 






CIHR 

Title: The relationship between auditory and contextual memory in Pavlovian fear conditioning 

Authors: *M. J. HONSBERGER, K. NADER; 

Psychology, McGill Univ., Montreal, QC, Canada 

Abstract: In Pavlovian auditory fear conditioning shock paired with a tone can lead to 

conditioned responses, such as freezing, being elicited by both the training context and tone. 

Conditioned responses elicited by the tone are often assessed by presenting the conditioned tone 

in a novel context in order to minimize confounds due to contextual freezing induced by the 

training context. Therefore, the training of auditory fear conditioning results in two fear 

memories, auditory and contextual. It remains unclear whether the context and the tone are part 

of the same fear memory or have their own fear representation. We have begun to address this by 

exploiting the fact that fear memories have been shown to undergo reconsolidation in the 

basolateral amygdala (BLA) following reactivation. Previously we showed that blocking 

reconsolidation of contextual fear conditioning led to an impairment in freezing to the tone, even 

though the tone was not reactivated. Conversely, blocking reconsolidation of auditory fear 

conditioning had no effect on freezing induced by the context. We reasoned that one possible 

explanation of these results is that in order for the tone to normally express freezing, it requires 

an intact context-shock association. For example, perhaps the single pairing protocol used led to 

a serial tone-context-shock association. In the present experiment, we tested this hypothesis by 

extinguishing either the auditory or contextual fear memories. If these two memories are serially 

associated then only the extinction of the CS directly associated with the shock should result in 

both memories showing reduced freezing. Preliminary results suggest that similar to our previous 

experiment, extinction of the tone only effects the auditory fear memory. However, preliminary 

results indicate that extinction of the context results in reduced freezing to both the context and 

tone. This suggests that the auditory fear memories acquired during Pavlovian fear conditioning 

are not independently acquired. 

Disclosures: M.J. Honsberger , None; K. Nader, None. 

Poster 






CIHR 

Title: Short and long term expression of extinction memory 

Authors: *G. E. ARCHBOLD, K. NADER; 

McGill Univ., Montreal, QC, Canada 

Abstract: Evidence suggests that behavioral extinction, a process whereby a conditioned 

response is diminished after presentations of the conditioned stimulus in the absence of 

reinforcement, is new learning rather than unlearning of the original association. In the initial 

acquisition of fear, there is both a short term memory (STM) and long term memory (LTM) 

component. However, few studies have examined early and late phases of an extinction trace. In 

conditioned taste aversion (CTA) Berman et al. (2003) reported that expression of an 

extinguished CTA is only observed 2 hours after extinction and this latency for expression not 

mediated by -adrenergic receptors, muscarinic cholinergic receptors, or protein synthesis. The 

present study examines the expression of behavioral extinction of an auditory fear memory at 

various time points after extinction training. Animals were conditioned via tone-shock pairings 

to induce freezing behavior and later received non-reinforced tone presentations to extinguish 

freezing. Testing for expression of extinction was conducted 1, 4, 8 or 24 hours after training. 

Freezing remained high at both the 1 and 4 hour time points but was significantly reduced by 24 

hours. We tested whether the absence of short term extinction was masked by reinstatement of 

the original trace as result of the aversive experience of extinction itself. To address this issue a 

second experiment was performed where animals were trained to fear two different auditory 

stimuli, CS1 and CS2, on successive training sessions. Three days later CS1 was extinguished. 

The following day CS2 was extinguished and animals were presented with a test for CS1 either 1 

or 24 hours after this second extinction session (i.e. 25 or 48 hours after extinction of CS1). Only 

animals tested 1 hour after CS2 extinction exhibited recovered freezing to CS1. Recently, 

Westbrook et al. (2005) found that the β-adrenergic system mediated the reinstatement of 

freezing to an extinguished CS by pre-exposure to a feared context 2 min before presentation of 

the CS. We hypothesized that a similar mechanism may be mediating the return of fear to CS1 if 

it is delivered shortly after CS2 extinction. To test this, 10mg/kg propranolol, a β-adrenergic 

receptor antagonist, was injected 20 min prior to testing. Propranolol did not restore expression 

of extinction 1 hour after CS2 extinction. However, in a renewal test conducted after a delay of 4 

days, propranolol treated groups demonstrated reduced responding to CS1. Overall, the lack of 

short term extinction expression is consistent with Berman et al. (2005). There may be no STM 

mechanisms mediating extinction or extinction STM may be masked by other factors not tested 

for. 

Disclosures: G.E. Archbold, None; K. Nader, None.

Poster 





Support: VW 

HFS 

NSERC 

EJLB 

CIHR 

Title: The dynamic nature of memory retrieval: active NMDAR-regulated trafficking of 

AMPARs in the amygdala, during expression of emotional memory 

Authors: *C. BEN MAMOU 1,2 , K. NADER 1 ; 

1 Dept Psychol, McGill Univ., Montreal, QC, Canada; 2 Groupe de recherche FRSQ sur le système 

nerveux central, Fac. of medicine, Univ. de Montréal, Montreal, QC, Canada 

Abstract: In some experimental behavioral paradigms, anisomycin blocks memory acquisition, 

consolidation or reconsolidation when administered before training, after training or after 

retrieval, respectively. Like acquisition, retrieval is an active experience, though often considered 

a simple readout of a memory trace. We evaluated the dynamic nature of auditory fear memory 

retrieval, by testing its sensitivity to anisomycin. We also analyzed synaptic dynamics of 

NMDARs and AMPARs during the expression of memory. Previous studies showed that postsynaptic 

insertion of GluR1-containing AMPARs in the basolateral amygdala (BLA) was 

necessary for acquisition and consolidation of auditory fear memory. Also, BLA AMPAR 

activation during retrieval mediates the expression of the fear response. Sprague Dawley rats 

implanted with cannulas aiming at the BLA were trained with 5 pairings of a tone and a footshock. 

Twenty-four hours later, we bilaterally infused anisomycin into the BLA, then 10 min 

later, we measured the fear response_freezing_to the tone. We sacrificed rats immediately, to 

analyze GluR1 protein in Western blots of BLA synaptoneurosomes and post-synaptic density 95 

co-immunoprecipitates (PSD95 co-IP). Anisomycin induced an impairment in freezing to the 

tone and a decrease of GluR1 protein level in PSD95 co-IP. This suggested that retrieval might 

be more than a simple readout of AMPARs: it might activate trafficking of AMPARs. The 

analysis of BLA GluR1 immediately after retrieval in the absence of drug showed an increase in 

Ser845 phosphorylation only in tone-tested rats, confirming that memory retrieval might affect 

AMPAR trafficking. Since NMDARs are plasticity triggers that initiate molecular cascades

leading to GluR1 phosphorylation, we infused the NMDAR antagonist ifenprodil into the BLA 

before the retrieval test. Ifenprodil abolished Ser845 phosphorylation during retrieval, without 

changing GluR1 post-synaptic amounts or freezing. Thus GluR1-Ser845 phosphorylation was 

NMDAR-dependent, suggesting that in normal conditions, AMPAR trafficking might be 

regulated by NMDARs during retrieval. This raised the possibility that administering ifenprodil 

before retrieval might reduce the NMDAR-activated trafficking of AMPARs and transform 

memory expression into a simple readout of post-synaptic AMPARs. We co-infused ifenprodil 

and anisomycin before tone-test and we found that ifenprodil rescued anisomycin‟s effect on 

behavior and on GuR1 level in PSD95 co-IP. We concluded that retrieval of auditory fear 

memory was a dynamic process that engages anisomycin-sensitive mechanisms involving active 

AMPAR trafficking controlled by NMDARs. 

Disclosures: C. Ben Mamou , None; K. Nader, None. 

Poster 






DGAPA-UNAM IN220706-3 

Title: Off-line transient glutamatergic activity in the insular cortex is necessary for memory long 

term consolidation 

Authors: *K. R. GUZMAN-RAMOS, V. DIAZ-MEDINA, R. BAUTISTA, F. BERMÚDEZ- 

RATTONI; 

UNAM IFC, Mexico, Mexico 

Abstract: Glutamatergic activity is important for associative learning consolidation by means of 

its N-Methyl-D-aspartate (NMDA) receptors activation. We used conditioned taste aversion 

(CTA) task, where animals learn to avoid a saccharin solution followed by gastric malaise 

induced by a lithium chloride (LiCl) injection. The aim of this work was to monitor glutamate 

extracellular concentration in the insular cortex (IC) by microdialysis during the presentation of a 

new taste (sodium saccharin 0.1% as conditioned stimulus, CS) followed by an i.p. injection of 

LiCl 0.4 M (unconditioned stimulus, US) and during an hour after the association. Male Wistar 

rats were implanted with a guide cannula aimed to the IC, after 10 days animals were submitted

to in vivo microdialysis to measure glutamate release using a capillary electrophoresis method 

based on micellar electrokinetic chromatography (MECK). The results showed a transient 200% 

extracellular glutamate increase immediately after LiCl injection but not during the CS 

presentation. To our surprise a significant off-line transient release of glutamate occurred 50 

minutes after pairing saccharin with LiCl but not after saccharin followed by NaCl. Furthermore, 

animals trained with backward conditioning (CS presentation after the US, that is ineffective for 

CTA memory formation) did not present any off-line glutamate release in post-training fractions. 

To evaluate the further role of the off-line glutamate release in memory consolidation, we 

infused an NMDA receptor antagonist (DL-2-amino-5-phosphonovaleric acid, APV) just before 

the second transient glutamate release. The results showed a significant impairment in long, but 

not in short-term memory. Altogether this evidence points out that off-line NMDA receptor reactivation 

seems to be necessary for CTA memory consolidation. 

Disclosures: K.R. Guzman-Ramos , None; V. Diaz-Medina, None; R. Bautista, None; F. 

Bermúdez-Rattoni, None. 

Poster 





Support: NIMH Grant MH069558 

NIMH Grant MH060668 

Title: Acute corticosterone treatment following fear memory retrieval has divergent results 

dependent upon the length of the retrieval session 

Authors: *M. E. LONERGAN, F. J. HELMSTETTER; 

Psychology, Univ. Wisconsin-Milw, Milwaukee, WI 

Abstract: Acute administration of corticosterone has previously been shown to enhance memory 

consolidation of conditional fear via noradrenergic signaling in the amygdala (Cordero & Sandi, 

1998; Hui et al., 2004; reviewed in McGaugh & Roozendaal, 2002). More recent studies have 

explored the effect of corticosterone given at the time of memory retrieval. Corticosterone given 

prior to retrieval results in impaired memory recall, whereas, post-retrieval corticosterone 

appears to enhance extinction learning (Cai et al., 2006). The post-retrieval effects of 

corticosterone hold promise in developing therapeutic treatments for individuals suffering from

fear and anxiety related disorders. However, the length of the retrieval session may impact the 

outcome. Some research suggests that shorter retrieval sessions lead to the induction of processes 

underlying reconsolidation, whereas, longer retrieval sessions initiate extinction learning 

(Pedreira & Maldonado, 2003; Suzuki et al., 2004). The current study investigated the effect of 

post-retrieval corticosterone following short or long retrieval sessions. Male Long Evans rats 

were trained in a 10-min session of contextual fear conditioning, in which they received three 

unsignaled footshocks (1.0mA/1sec). Twenty-four hours later the animals were given either a 2min 

or a 10-min retrieval session in the training context. Immediately following retrieval, the rats 

were given i.p. injections of either corticosterone (3mg/kg in 2ml/kg vehicle) or vehicle (5% 

ethanol in sterile saline). All rats were tested for fear memory retention during a 10-min context 

test session given 1 and 6 days after retrieval. No significant differences in fear behavior (i.e., 

freezing) were detected between groups during training or retrieval sessions. However, by the 

second context test, a drug treatment X retrieval length interaction was evident. Specifically, rats 

given corticosterone after a 10-min retrieval session exhibited reduced freezing behavior relative 

to vehicle-treated controls, which is indicative of enhanced extinction. In contrast, rats injected 

with corticosterone following a 2-min retrieval session show higher levels of freezing compared 

to controls, which is suggestive of enhanced reconsolidation of the fear memory. The cellular 

and systems level processes underlying these effects are currently under investigation in our 

laboratory. 

Disclosures: M.E. Lonergan , None; F.J. Helmstetter, None. 

Poster 



Program#/Poster#: 295.23/TT1 


Support: NIH Grant SOGGMO 8102-26S1 

NIH Grant 5R25GM061151 

NIH Grant 5P20-RR015565 

Title: Behavioral, cellular, and molecular effects of DNA ligase inhibition during context fear 

conditioning

Authors: E. CASTRO, J. Y. LAGUNA-TORRES, I. J. SANTOS SOTO, Y. GOENAGA, C. 

LAGO, H. G. ORTIZ ZUAZAGA, *S. PENA DE ORTIZ; 

Dept Biol, Univ. Puerto Rico, San Juan, Puerto Rico 

Abstract: We assess here the importance of genomic rearrangement mechanisms in 

consolidation of hippocampal dependent learning. Our overarching hypothesis is that long-term 

memory formation involves a regulatory mechanism of DNA recombination in the hippocampus 

that includes the activation of effector molecules, such as DNA ligases, and the rearrangement of 

specific target genes. Using context fear conditioning as a model of learning in C57BL/6 mice, 

our main goal is to study genetic changes associated to DNA rearrangements and their impact on 

hippocampal dependent memory formation and synaptogenesis. Our previous studies using 

pharmacological blockade of DNA ligase function, showed that this treatment specifically 

blocked consolidation of context fear conditioning without interfering short-term memory or 

reconsolidation (Colón-Cesario et al., 2006). The present studies have determined that similar 

treatments block remote memory of context fear conditioning and impair hippocampal 

synaptogenesis. These studies are being followed up by experiments aimed at identifying the 

genes that are subject to DNA recombination as a result of conditioning. Our results so far 

suggest that context fear conditioning results in associative learning-specific changes in 

hippocampal gene expression, some of which may be associated to genomic diversification 

processes in the brain. 

Disclosures: E. Castro, None; J.Y. Laguna-Torres, None; I.J. Santos Soto, None; Y. 

Goenaga, None; C. Lago, None; H.G. Ortiz Zuazaga, None; S. Pena de Ortiz , None. 

Poster 





Support: NIH 

RIKEN 

Title: Creation of a transgenic mouse line lacking NMDA receptor subunit 1 in layer III 

pyramidal neurons of the entorhinal cortex 

Authors: A. RIVEST 1 , J. SUH 1,3,4 , *S. TONEGAWA 2,3,4 ; 

1 Picower Inst. Memory & Learning, 2 Dir, Picower Inst. Memory & Learning, Massachusetts Inst.

Technol., Cambridge, MA; 3 RIKEN-MIT Ctr. for Neural Circuit Genet., Cambridge, MA; 

4 Howard Hughes Med. Inst., Cambridge, MA 

Abstract: The entorhinal cortex (EC) is a prominent component of the parahippocampal region, 

providing and receiving the majority of synaptic inputs to and from the hippocampus. The 

superficial layers of the EC are particularly interesting from the perspective of synaptic plasticity 

since neurons in these layers receive multi-modal information from the various cortical areas and 

also receive input from the deeper layer EC neurons, which receive inputs processed through the 

hippocampal circuit. Hence, synaptic plasticity in the superficial layers may play a role in 

abstracting cortical information and integrating it with hippocampal feedback. To investigate the 

role of NMDA receptor-dependent synaptic plasticity, we generated a transgenic mouse, in 

which the expression of Cre recombinase is restricted to neurons in the superficial layer III. 

Double immunohistochemistry with antibodies against NeuN (neuronal marker), and 

parvalbumin or GAD-67 (inhibitory neuronal markers) revealed that the recombination is 

restricted to the excitatory neurons. By breeding this transgenic mouse with a floxed NMDA 

receptor subunit 1 (NR1) mouse, we generated a new conditional knockout mouse, in which NR1 

mRNA is absent in layer III of the EC at 6 month. Spatial and temporal patterns of NR1 deletion 

with young animals are currently being assessed. 

Disclosures: A. Rivest, None; S. Tonegawa , None; J. Suh, None. 

Poster 





Support: R01-MH078821 

Title: The role of Schaffer collateral-CA1 transmission in systems consolidation of contextual 

fear memory 

Authors: *T. NAKASHIBA 1,2,3 , D. L. BUHL 1,2,3,4 , T. J. MCHUGH 1,2,3 , S. TONEGAWA 1,2,3,4 ; 

1 Dept.Brain & Cognitive Sci., MIT, Cambridge, MA; 2 The Picower Inst. for Learning and 

Memory, Cambridge, MA; 3 RIKEN-MIT Ctr. for Neural Circuit Genet., Cambridge, MA; 

4 Howard Hughes Med. Inst., Cambridge, MA 

Abstract: Retrograde amnesia following the medial temporal lobe damage has suggested that the 

hippocampus and related structures are crucial for declarative memory formation, yet have a

time-limited role in the transfer to permanent memory. Although lesion studies in animals have 

provided further support for the transient requirement of the hippocampus in system 

consolidation, little is known about how the hippocampus is involved in this phenomenon. 

Neurophysiological data has suggested that the sharp-wave/ripple and/or reactivation, which are 

thought to originate from CA3, may be involved in the transfer of hippocampal memory to the 

neocortex during consolidation. To test this hypothesis, we have generated “circuit” mutant mice 

in which synaptic transmission from CA3 to CA1 can be selectively inhibited in a temporally 

controllable manner (See Nakashiba et al., Science 2008). The advantage of this temporally 

controlled circuit inhibition is that we can test the requirement of CA3 output during system 

consolidation or during memory recall of hippocampal-dependent memory. Preliminary 

behavioral studies suggest that synaptic transmission from CA3 to CA1 is necessary in the 

consolidation of certain types of hippocampal-dependent memory. 

Disclosures: T. Nakashiba , The Picower Institute for Learning and Memory, A. Employment 

(full or part-time); RIKEN-MIT Center for Neural Circuit Genetics, A. Employment (full or parttime); 

Howard Hughes Medical Institute, A. Employment (full or part-time); Massachusetts 

Institute of Technology, A. Employment (full or part-time); R01-MH078821, B. Research Grant 


received); D.L. Buhl, None; T.J. McHugh, None; S. Tonegawa, None. 

Poster 






Title: Genetic dissection of CA3 inputs: spatial encoding in area CA3 in the absence of recurrent 

or mossy fiber transmission 

Authors: *T. J. MCHUGH, T. NAKASHIBA, D. L. BUHL, S. TONEGAWA; 

Picower Inst. for Learning & Memory, MIT, Cambridge, MA 

Abstract: Hippocampal CA3 pyramidal cells receive three major excitatory inputs: the Mossy 

Fibers (MF) from the DG, recurrent collateral (RC) input from other CA3 pyramidal cells, and 

the perforant pathway (PP) from the entorhinal cortex (EC). Although spatial and contextual 

information can be transmitted through all three of these pathways, it is not known how these 

inputs are integrated to guide the encoding, storage and recall of spatial information in CA3. To

directly examine the specific contributions of the MF and RC inputs in the establishment of 

spatial and contextual encoding we generated two lines of transgenic mice, in the first synaptic 

transmission was specifically blocked in CA3 pyramidal cells, resulting in a loss of RC input to 

CA3, and in the second transmission was blocked specifically in DG granule cells, resulting in a 

loss of MF inputs. Tetrode recording in CA3 of freely behaving mice suggests MF input is 

important in the encoding of novel space, while RC input is required for the maintenance of a 

stable representation. 

Disclosures: T.J. McHugh , The Picower Institute for Learning and Memory, A. Employment 

(full or part-time); RIKEN-MIT Center for Neural Circuit Genetics, A. Employment (full or parttime); 

Howard Hughes Medical Institute, A. Employment (full or part-time); Massachusetts 

Institute of Technology, A. Employment (full or part-time); T. Nakashiba, None; D.L. Buhl, 

None; S. Tonegawa, None. 

Poster 






Title: The role of CA3 output in CA1 ripples and reactivation during slow wave sleep 

Authors: *D. BUHL, T. NAKASHIBA, T. J. MCHUGH, F. KLOOSTERMAN, M. A. 

WILSON, S. TONEGAWA; 

Picower Inst. Learning and Mem, MIT, Cambridge, MA 

Abstract: The hippocampus has long been thought to be necessary for the consolidation and/or 

recall of long term episodic memories. How the hippocampus encodes this information remains 

to be elucidated. Numerous studies have shown that spike patterns occurring during the 

exploration of a novel environment “reactivate” during the sharp-wave ripple complex, both 

during brief non-theta periods in the environment as well as during the subsequent sleep (Foster 

and Wilson, 2006). The ripple oscillation, a 140-200 Hz field oscillation restricted to the CA1 

pyramidal cell layer of the hippocampus, is thought to be triggered by a synchronous discharge 

of CA3 pyramidal cells (Buzsaki et al., 1992; Csicsvari et al., 1999). The ripple is reflected 30- 

100 ms later downstream from CA1 in entorhinal cortex (EC) layers V and VI as a much slower 

large-amplitude field potential, followed by activity in at least two cortical targets, the perirhinal 

cortex and the medial prefrontal cortex. It is this chain of activity that has been hypothesized to

allow large ensembles of hippocampal neurons via sharp-wave ripple events to alter the synaptic 

connectivity of neocortical circuitry, thereby providing a possible mechanism for consolidating 

episodic memories to long term storage. To directly test this hypothesis we generated mice in 

which synaptic transmission was specifically blocked in CA3 pyramidal cells, resulting in a loss 

of the tri-synaptic pathway input to CA1 while leaving the temporoammonic (TA) pathway 

intact (See Nakashiba et al., Science 2008). Tetrodes were used to simultaneously record local 

field potential and putative neurons from the CA1 layer of the hippocampus during exposure to a 

novel linear track and slow wave sleep. We previously reported a significant impairment of CA1 

ripple oscillations and spike phase correlations (Buhl et al., SFN 2007). Here we focus on testing 

consolidation hypothesis, correlating the observed deficiency in ripples, “reactivation” of cells 

that fire in the novel environment and behavior (See Nakashibi SFN poster 2008) in the absence 

of CA3 input to CA1. 

Disclosures: D. Buhl , The Picower Institute for Learning & Memory, A. Employment (full or 

part-time); RIKEN-MIT Center for Neural Circuit Genetics, A. Employment (full or part-time); 

Howard Hughes Medical Institute, A. Employment (full or part-time); Massachusetts Institute of 

Technology, A. Employment (full or part-time); T. Nakashiba, None; T.J. McHugh, None; F. 

Kloosterman, None; M.A. Wilson, None; S. Tonegawa, None. 

Poster 





Support: UVA Dissertation Year Fellowship awarded to ECK 

NSF Grant 0720170 to CLW 

Title: The Nucleus Accumbens Shell: A repository for highly processed information from either 

the amygdala or hippocampus 

Authors: *E. C. KERFOOT, C. L. WILLIAMS; 

Psychology, Univ. Virginia, Charlottesville, VA 

Abstract: The nucleus accumbens shell (NAC) is a site of converging inputs during memory 

processing for emotional events. The NAC receives input from the nucleus of the solitary tract 

(NTS) regarding changes in peripheral autonomic functioning following emotional arousal. The 

NAC also receives input from the amygdala and hippocampus regarding affective and contextual

attributes of new learning experiences. The successful encoding of affect or context is facilitated 

by activating noradrenergic systems in either the amygdala or hippocampus. Recent findings 

indicate that memory enhancement produced by activating NTS neurons, is attenuated by 

suppressing NAC functioning after learning. This finding shows the significance of the NAC in 

integrating information from the periphery to modulate memory for arousing events. However, it 

is not known if the NAC plays an equally important role in consolidating information that is 

initially processed in the amygdala and hippocampus. The present study determined if the 

convergence of inputs from these limbic regions within the NAC contributes to successful 

encoding of emotional events into memory. To assess this relationship, we assessed whether the 

mnemonic consequences of activating either the amygdala or hippocampus is compromised by 

functionally inactivating the NAC after an extended period of time following learning. Male 

Sprague-Dawley rats received bilateral cannula implants 2mm above the accumbens shell and a 

second bilateral implant 2mm above either the amygdala or hippocampus. The subjects were 

trained for 6 days to drink from a water spout. On day 7, a 0.35mA footshock was initiated as the 

rat approached the spout and was terminated once the rat escaped into a neutral compartment. 

Subjects were then given intra-amygdala or hippocampal infusions of PBS or a dose of 

norepinephrine (0.2κg) previously shown to enhance memory. One hour later, all subjects were 

removed from their home cages and given an intra-accumbens infusion of either muscimol 

(100ng) or PBS. Muscimol inactivation of the NAC was delayed to allow sufficient time for 

norepinephrine to activate intracellular cascades that lead to long-term synaptic modifications 

involved in forming new memories. Results show that blocking accumbens functioning with 

muscimol an hour following amygdala or hippocampus activation attenuates the improvement in 

memory seen following noradrenergic activation of the amygdala or hippocampus alone. These 

findings suggest that the accumbens shell plays an integral role in consolidating converging 

information that is initially processed by the amygdala and hippocampus following exposure to 

emotionally arousing events. 

Disclosures: E.C. Kerfoot , None; C.L. Williams, None. 

Poster 

296. Learning and Memory: Physiology and Imaging III 



Topic: F.02.i. Learning and memory: Physiology and imaging 


NIH Grant MH071847

Title: Further characterization of trial outcome cells in the primate MTL cortex during a 

location-scene association task 

Authors: *E. L. HARGREAVES, W. A. SUZUKI; 

Ctr. for Neural Sci., New York Univ., New York, NY 

Abstract: We have previously reported that entorhinal neurons differentiate correct from error 

trials during the performance of a location-scene associative learning task (outcome-selective 

response; Hargreaves et al., 2007). Here, we ask how stable outcome-selective responses of 

entorhinal and parahippocampal cells are by examining their activity during the initial learning 

of new location-scene associations and during reversals. In this task, animals initiated each trial 

by fixating on a central spot for 500 ms. Four identical targets superimposed on a complex visual 

scene were then presented for 500 ms followed by a 700 ms delay period, during which only the 

fixation spot and targets remained on the screen. The disappearance of the fixation spot cued the 

animal to make an eye movement to one of the four targets, of which only one was rewarded. On 

correct trials, animals were given a random number of juice reward drops (2-4) 43 ms after a 

correct response that lasted an average of 900 ms followed by a 1000 ms inter-trial-interval (ITI). 

Following an error trial, no juice was given and the trial progressed immediately to a 2000 ms 

ITI. Animals typically learned 2-4 new location-scene associations and for a subset of the 

sessions, 2 of the location-scene associations were reversed after the initial learning. We 

analyzed 116 units from the entorhinal and parahippocampal cortices of two monkeys. We found 

that the activity of 67% (78/116) of the cells were significantly responsive during either the 

reward, ITI or both periods relative to baseline (t; p




Title: Neuronal activity encoding temporal-order memory of visual objects in the macaque 

entorhinal and perirhinal cortices 

Authors: *Y. NAYA, W. A. SUZUKI; 

Ctr. Neural Sci., New York Univ., New York, NY 

Abstract: The ability to recall specific temporal sequences of events in an episode is severely 

impaired in humans with memory impairment following medial temporal lobe damage. In the 

present study, we characterize neural activity in the macaque entorhinal and perirhinal (ER & 

PR) cortices as animals processed temporal order of items in memory. The task includes both an 

encoding and a retrieval phase. The encoding phase starts with the animal fixating a fixation 

point on a video monitor for 0.4 s. Then, a sequence of two cue stimuli (0.3 s for each) was 

presented with a blank delay interval (0.9 s) between them. Cue stimuli were pseudo-randomly 

chosen out of a pool of eight well-learned visual items. Following the second cue stimulus, 

another 0.5 s of fixation was required for the animal and a single drop of water was given as a 

reward. Then a blank inter-phase interval of 0.7-1.5 s was presented during which time no 

fixation was required. The retrieval phase started with the animal fixating a fixation for 1.0 s. 

Then, three choice stimuli were presented together; two of them were the items that had been 

presented as cue stimuli in the encoding phase, and another was chosen from the other six items. 

If the animal touched the two items in the same order as they were presented in the encoding 

phase, six drops of water was given as a reward. We recorded 125 neurons from the ER and PR 

cortices: 71 from Monkey G and 54 from Monkey B. Their responses during the cue periods 

were characterized at the single neuron level using a two-way ANOVA with temporal rank order 

and stimulus identity as main factors. We found 19.2 % (n = 24) of the recorded neurons showed 

temporal-order-selective responses (P < 0.05) and 36.8 % (n = 46) showed stimulus-selective 

responses (P < 0.05). Moreover, 19.6 % (n = 9) of stimulus-selective neurons showed significant 

interaction between temporal order and stimulus identity (P < 0.05). These results suggest that 

ER and PR neurons as a whole represent rank order information as well as visual object 

information during the temporal order task and that some neurons can encode a particular 

temporal order of specific items at the single neuron level. This latter finding cannot be 

explained by a simple linear summation of temporal-order-selective responses and stimulusselective 

responses. Taken together, our present study suggests that ER and PR cortices 

participate in the encoding of stimulus-selective information for a temporal order memory task. 

Disclosures: Y. Naya, None; W.A. Suzuki, None. 

Poster







Title: Modulation of correlated activity in primate entorhinal cortex cell pairs for well-learned 

associations compared to new associations 

Authors: *A. TAMBINI 1 , E. L. HARGREAVES 2 , W. A. SUZUKI 2 ; 

1 Ctr. for Neural Sci., 2 New York Univ., New York, NY 

Abstract: Several studies have shown that measures of correlated activity across cells can reflect 

complex cognitive operations, such as engaging working memory (Jones & Wilson, 2005) or 

goal selection and maintenance (Tsujimoto et al., 2008). Here we compared correlated activity in 

response to novel vs. very well-learned associations during the performance of a location-scene 

association task (Wirth et al., 2003). Animals initiated each trial by fixating on a central spot for 

500 ms. Four identical targets superimposed on a complex visual scene were then presented for 

500 ms followed by a 700 ms delay period, during which only the fixation spot and targets 

remained on the screen. The disappearance of the fixation spot cued the animal to make an eye 

movement to one of the four targets, of which only one was rewarded. Reward was followed by 

a 2000-3000 ms inter-trial interval period. Animals typically learned 2-4 new location-scene 

associations (new trials) together with 4 highly familiar “reference” associations (reference 

trials). We analyzed data from 210 entorhinal cortex cell pairs recorded from two monkeys. In 

order to determine the correlated activity across cell pairs, we computed the cross-correlation 

coefficient for different trial types and task periods for each pair. Since no a priori direction of 

spiking between cells was expected, we computed the bias corrected cross-correlation between 

cells with a window of ±25 ms. First, we asked if there were any differences in the overall 

correlations across the various trial periods for correctly executed new trials compared to 

correctly executed reference trials. We found significantly larger correlations in entorhinal cortex 

cell pairs in the scene, delay, and inter-trial interval periods of the task for reference trials 

compared to new trials (p

Disclosures: A. Tambini, None; E.L. Hargreaves, None; W.A. Suzuki, None. 

Poster 





Title: Functional brain mapping in transverse patterning task; PET study with conscious 

monkeys 

Authors: *S. YAMAMOTO 1 , H. TSUKADA 2 , C. YOKOYAMA 3 , Y. WATANABE 3 , H. 

ONOE 3 ; 

1 Hamamatsu Univ. Sch. Med., Hamamatsu, Japan; 2 Central Res. Laboratory, Hamamatsu 

Photonics KK, Hamamatsu, Japan; 3 RIKEN Frontier Res. Syst. Mol. Imaging Res. Program, 

Kobe, Japan 

Abstract: Monkeys were trained to perform the transverse patterning (TP) task which has the 

same rule as a childhood game “rock-paper-scissors”, and changes in regional cerebral blood 

flow (rCBF) were measured as an index of neuronal activity, while the monkeys were 

performing the TP task by positron emission tomography (PET) with H2 15 O. The simple 

visuomotor task was used as control task, in which a white square was presented randomly on 

left or right side of a display, and the subject was required to press a corresponding lever to get a 

reward. Monkeys were introduced to perform the TP task which is composed with three different 

geometrical figures (A, B, and C) as visual stimuli. In each trial, two different stimuli are 

presented in pairs, and the subject is rewarded by choosing (+) in one case (e.g., A+ vs. B-, B+ 

vs. C-, C+ vs. A-). Monkeys were trained in three steps, and reaction time and number of eye 

sampling were measured. In step 1, a single pair (A+ vs. B-) was presented, where A is always 

correct, and B is always incorrect. In step 2, two kinds of pairs, (A+ vs. B-) and (C+ vs. A-) pairs 

are presented in pseudorandom order. In step 3, all three kinds of pairs (A+ vs. B-) (B+ vs. C-) 

(C+ vs. A-) are presented and all stimuli are equally rewarded during experimental session. 

Behavioral results showed that reaction time and number of eye sampling in step 2 and 3 were 

increased compared to those in step 1. The correlation between the RT and eye sampling became 

strong along the training progression. PET results showed that rCBF in the right hippocampus 

and the anterior cingulate cortex were increased with progression of the training. In step 3, a 

positive correlation of rCBF between the right hippocampus and the anterior cingulate cortex 

was observed. These results demonstrate that, in addition to the hippocampus, which has been 

already suggested by the lesion study (Alvarado MC et al., Hippocampus 15:118-131, 2005), the 

anterior cingulate cortex is required to solve the TP task.

Disclosures: S. Yamamoto, None; H. Tsukada, None; C. Yokoyama, None; Y. Watanabe, 

None; H. Onoe, None. 

Poster 





Support: BMBF 

Title: Tetrode recording of local neuronal ensembles provides insight into coding mechanisms of 

short-term memory in macaque prefrontal cortex 

Authors: *M. WAIZEL 1,2 , E. STAEDTLER 1 , G. PIPA 1,3 , N.-H. CHEN 4 , M. H. J. MUNK 2,5 ; 

1 Max-Planck-Inst, Frankfurt am Main, Germany; 2 Dept. Physiol. of Cognitive Processes, Max 

Planck Inst. for Biol. Cybernetics, Tuebingen, Germany; 3 Frankfurt Inst. for Advanced Studies, 

Johann Wolfgang Goethe Univ., Frankfurt, Germany; 4 KunMing Inst. of Zoology, Chinese 

Acad. of Sci., KunMing, China; 5 Neurophysiol., Max Planck Inst. for Brain Res., Frankfurt am 

Main, Germany 

Abstract: As most cortical neurons are broadly tuned to various stimulus parameters, it is 

inevitable that individual neurons participate in the representation of more than one visual object. 

Vice versa, accurate representations of individual objects for example in short-term memory that 

can support reliable decisions require the participation of large neuronal populations. To provide 

evidence in favor of population codes, we have recently analyzed simultaneously recorded multi- 

and single-unit signals derived from arrays of single-ended microelectrodes (Waizel et al., SfN 

2007). Multi-contact electrodes like tetrodes (tts) which have a real 3D-structure provide signals 

that allow for estimating the position of the recorded neurons by triangulation. Here we set out to 

study whether recording from 3D-tts would improve the quality of sorting and hence allow for 

the extraction of more information about the stimulus. Based on single trial firing rate values, we 

calculated one-way ANOVAs at 1% significance thresholds and performed subsequent posthoc 

comparison (Scheffé) in order to detect stimulus selectivity and determine stimulus specificity 

for the activity at each single site, respectively. In order to investigate the coding of distributed 

neuronal ensembles, we computed binary activity patterns for all active electrodes in the array 

and determined their stimulus selectivity and specificity. Compared to what we found previously 

in single microelectrode recordings, the number of object selective or even specific recording 

sites increased up to 3 times which provides highly specific sites in 3 out of 4 sessions (3000 

trials, 13.5 million spikes). Given that our monkeys always performed the memory task with a

set of twenty visual stimuli, we found highly specific sites coding for only one object which 

revealed up to 18 of 19 possible pairwise comparisons. According to the proposal that single 

neurons participate in more than one specific object representation we also found bi- or even triobject-dependant 

sites (average 27 significant pairwise comparisons per session) and never nonsystematic 

object specificity. As clusters of triangulation-reconstructed spikes tend to have intercluster 

regions with smooth transitions which could potentially reflect synchronous spikes, we 

wanted to know how much information could be carried by these signals. After removing spikes 

between clusters, we found object specificity highly decreased (in one session only 6 out of 

previous 38 significant pairwise comparisons remained). These results suggest that the use of 

tetrodes with a real 3D-structure provides more information about neuronal object 

representations. 

Disclosures: M. Waizel, None; E. Staedtler, None; G. Pipa, None; N. Chen, None; M.H.J. 

Munk, None. 

Poster 





Support: Emory University Alzheimer‟s Disease Research Center Pilot Grant 

Yerkes NCRR Base Grant P51 RR00165 

NIGMS Grant 5T32GM008605-10 

Yerkes NCRR Venture Grant P51 RR000165-46 

Title: Hippocampal activity reflects recognition memory on a trial-by-trial basis 

Authors: *M. J. JUTRAS 1,2 , P. FRIES 3 , E. A. BUFFALO 2,4 ; 

1 Neurosci. Program, Emory Univ., Atlanta, GA; 2 Yerkes Natl. Primate Res. Ctr., Atlanta, GA; 

3 F.C. Donders Ctr. for Cognitive Neuroimaging, Radboud Univ. Nijmegen, Nijmegen, 

Netherlands; 4 Neurol., Emory Univ. Sch. of Med., Atlanta, GA 

Abstract: Recognition memory, the ability to perceive recently encountered items as familiar, 

depends on the integrity of the medial temporal lobe (MTL). Although previous 

neurophysiological studies have reported recognition memory signals in the monkey MTL, it has

een difficult to examine whether these signals correlate with performance because monkeys are 

typically overtrained to perform at high levels, resulting in very few error trials. To address this 

issue, we examined hippocampal activity as two monkeys performed the Visual Preferential 

Looking Task (VPLT). This task relies on the monkey‟s innate preference for novelty, and 

therefore requires no specific training. Additionally, this task elicits variations in performance 

that allow for an examination of the relationship between trial-to-trial fluctuations in recognition 

memory and neural signals recorded in the hippocampus. 

Monkeys freely viewed large stimuli (11°), which were presented twice each and remained on 

the screen as long as the monkey continued to look at them, up to a maximum looking time of 5 

seconds. Successful recognition is evidenced by a decrease in looking time for the repeated 

stimulus presentation, i.e., a preference for novelty. Overall, both monkeys demonstrated 

significant recognition memory (p

Title: Neuronal activity of SI cortex of untrained monkeys in haptic-haptic unimodal and visuohaptic 

crossmodal working memory tasks 

Authors: X. LI 1 , L. WANG 1 , S. HSIAO 2 , M. BODNER 1,3 , F. LENZ 1 , Y. KU 1 , J. FUSTER 4 , *Y. 

ZHOU 1 ; 

1 Dept Neurosurg., 2 Dept Neurosci., Johns Hopkins Univ. Sch. Med., Baltimore, MD; 3 MIND 

Res. Inst., Santa Ana, CA; 4 Semel Inst. for Neurosci. and Human Behavior, UCLA, Los Aneles, 

CA 

Abstract: Previous studies have shown that somatosensory cortical neurons in well-trained 

monkeys participate in both haptic-haptic (HH) unimodal and visuo-haptic (VH) cross-modal 

delayed matching-to-sample tasks. In the present study, we recorded from single units in SI 

cortex from monkeys “naïve” to those tasks. During unit recording, the naïve animals performed 

tasks identical to those for trained monkeys but without the requirements (untrained) to 

discriminate and memorize sensory cues. In the HH trials, the naïve animal had to touch 

("sample") an object and after a delay, grasp and pull another object that was the same or 

different from the first (the animal was given the same reward in either case). In the VH trials, 

the animal was first given a visual stimulus (presented at the animal‟s eye level), and after a 

delay, the animal was required to grasp and pull an object as it did in the second part of the HH 

task to get a reward. In HH, the majority of cells showed significant firing rate change when the 

monkey touched the first tactile object in the sample period; some of those touch-responsive cells 

showed differential activity depending on which object was touched. These results were similar 

to those from well-trained monkeys in the first-stimulus sample period, suggesting a similar 

neuronal process of perception in the trained and the naïve monkeys. However, the percentage of 

delay-active cells, including differential delay cells, was significantly lower in naïve monkeys 

than that in well-trained monkeys. In the VH task, significantly lower percentages of active cells- 

-in both sample and delay periods--were found in naïve than in trained monkeys. Present data, in 

conclusion, indicate that a population of SI neurons in naïve monkeys responds to tactile or 

visual stimuli even though their retention is not necessary for task performance. Our data also 

indicate that, through training, unimodal and cross-modal networks in SI cortex increase their 

involvement in working memory and cross-modal association. 

Disclosures: X. Li, None; L. Wang, None; S. Hsiao, None; M. Bodner, None; F. Lenz, 

None; Y. Ku, None; J. Fuster, None; Y. Zhou, None. 

Poster 



Program#/Poster#: 296.8/TT14


Support: Startup funds from the Univ. of Iowa 

NIDCD grant DC0007156 

Title: Neuronal activity in primate prefrontal cortex during performance of an auditory delayed 

matching-to-sample task 

Authors: *A. L. POREMBA, B. PLAKKE, C. W. NG, R. OPHEIM; 

Psychology, Div. Beh Cog Neuros, Univ. of Iowa, Iowa City, IA 

Abstract: Both imaging and neurophysiological recording studies have demonstrated that the 

prefrontal cortex (PFC) is responsive to auditory stimuli (e.g., Poremba et al., 2003; Romanski & 

Goldman-Rakic, 2002). Additional work has shown the PFC is involved in tasks requiring 

working or short-term memory (e.g., Bodner et al., 1996; Fuster, 2000; Warden & Miller 2007). 

Here, we examine rhesus macaques on an auditory go/no go delayed matching-to-sample 

(DMTS) task wherein two acoustic stimuli (500 ms), separated by a fixed memory delay (5000 

ms), were either identical sound presentations, or two different sound presentations. A small 

candy reward was delivered after a correct button press on match trials. Each training session 

consisted of 200 trials, where half the trials were match trials and half were nonmatch trials. The 

sound stimuli for each recording day were 8-10 sounds chosen from a larger sound set. Each 

sound was repeated at least 8 times on both match and nonmatch trials within a recording 

session. The sound stimuli included pure tones, frequency sweeps, man-made environmental 

sounds, monkey vocalizations, natural sounds, synthesized sounds, and other animal sounds. 

After reaching a criterion of 80% correct or better on the behavioral task recording wells were 

implanted over the left lateral bank of the principal sulcus in prefrontal cortex and recording 

sessions began. Assessment of neuronal firing rates and waveforms was completed for task 

related activity, as well as for the same sound stimuli during passive listening. Preliminary 

results from one female monkey indicate that cells in this area are responsive to complex 

auditory stimuli in a selective manner. Additionally, there are some single-units that fire more 

during the delay period correlated with behavioral performance. Examination of individual 

sounds presented during match trials associated with correct versus incorrect behavioral 

performance revealed single units with higher firing rates during correct match performance to 

the sound stimuli than on incorrect match trials. Some of these same single-units displayed a 

higher frequency of spike firing during the delay period for incorrect match trials compared to 

correct match trials for the same sound stimulus. Preliminary data indicate that this region, which 

receives auditory input from the superior temporal gyrus, is encoding complex auditory stimuli 

and may also be active during the delay memory period under specific conditions. 

Disclosures: A.L. Poremba, None; B. Plakke, None; C.W. Ng, None; R. Opheim, None. 

Poster





Support: Yale-Pfizer Bioimaging Alliance 

Pfizer Global Research and Development 

Title: Functional imaging of D1 agonist reversal of ketamine-induced cognitive deficits in the 

nonhuman primate as shown by regional cerebral glucose metabolism 

Authors: *G. V. WILLIAMS 1,3 , B. M. ROBERTS 1,3,4 , D. E. HOLDEN 1,3 , D. W. 

CAMPBELL 1,3 , D. P. WEINZIMMER 2 , C. M. SANDIEGO 2 , R. E. CARSON 2 , S. A. 

CASTNER 1,3 ; 

1 Psychiatry, 2 Diagnos. Radiology, Yale, New Haven, CT; 3 VA Connecticut Healthcare Syst., 

West Haven, CT; 4 Psychology, Univ. of Connecticut, Storrs, CT 

Abstract: Reversal of deficits in spatial working memory induced by acute ketamine in the 

nonhuman primate is a major platform for investigating the impact of particular receptor targets 

for cognition. We have previously shown that both full and partial dopamine D1 receptor 

agonists provide substantial protection against these deficits. Here, we sought to test whether the 

site of action or interaction of a D1 agonist and ketamine includes known aspects of working 

memory circuitry, particularly prefrontal cortex. Moreover, we wondered whether it was possible 

to see for the first time in a primate if a full D1 agonist had the ability to tune cortical activity in 

the task and to circumscribe the activity induced by ketamine. Eight rhesus macaques were 

acclimated and trained to test on the spatial delayed response task in a specialized chair with the 

placement of a saphenous venous catheter. The D1 agonist A77636 (0.01 µg/kg) or vehicle was 

administered 1 hr prior to injection of 18F-fluorodeoxyglucose (FDG; 2 - 3 mCi). Ketamine (1.0 

- 1.7 mg/kg, IM) or vehicle was administered ~15 min prior to FDG. Cognitive testing 

commenced 1 - 2 min prior to FDG injection and continued for up to 25 min. Animals were then 

anesthetized with propofol and transported to an ECAT HRRT scanner (Siemens) for positron 

emission tomography. The 20 min emission scan commenced ~1 hr post FDG injection and was 

immediately followed by a 6 min transmission scan. Blood was drawn for measures of plasma 

ketamine concentration and glucose. An additional arm of the study was added to include the 

dose of 0.1 µg/kg A77636 vs. ketamine. Each animal had an MRI from which we constructed a 

template for the analysis of the data which currently includes statistical parametric mapping. 

Ketamine alone induced a dramatic elevation in metabolism in anterior cingulate cortex, caudate 

n. and dorsolateral prefrontal cortex (dlPFC). The low dose of A77636 on its own induced a 

significant elevation of activity in discrete regions of dlPFC, including principal sulcus. When 

administered together, the agonist selectively reduced the elevated metabolism in discrete 

regions of dlPFC in a dose-dependent fashion. These findings demonstrate disruption of working

memory circuitry by ketamine that is consistent with human data and a critical interaction 

between a D1 agonist and ketamine which is targeted towards dlPFC in the primate. 

Disclosures: G.V. Williams , Pfizer Global Research and Development, B. Research Grant 


received); B.M. Roberts, None; D.E. Holden, None; D.W. Campbell, None; D.P. 

Weinzimmer, None; C.M. Sandiego, None; R.E. Carson, None; S.A. Castner, Pfizer Global 

Research and Development, B. Research Grant (principal investigator, collaborator or consultant 

and pending grants as well as grants already received). 

Poster 





Support: FNRS grant 3100AO-108336/1 and /2 to PJM 

Title: JULIDE : a new software for the unbiased analysis and 3D reconstruction of brain 

autoradiograms 

Authors: *J. PARAFITA 1 , F. MAGARA 3 , R. CHARRIER 2 , D. RIBES 2 , J.-P. THIRAN 2 , P. 

MAGISTRETTI 1,3 ; 

1 SV-BMI, 2 STI-LTS5, EPFL, Lausanne, Switzerland; 3 Ctr. for Psychiatric Neurosciences, 

CHUV, Lausanne, Switzerland 

Abstract: The 14 C-2 Deoxy-D-Glucose (2DG) autoradiographic technique affords a valuable 

means to assess in laboratory animals the engagement of brain regions and circuits in a given 

behavioural task or following a pharmacological intervention, by indirectly determining glucose 

utilization (CMRGlu) associated with neuronal activity (Sokoloff et al 1977). One of the 

drawbacks of the technique is that it implies an a priori definition of regions of interest (ROI); in 

addition 3D reconstructions of the whole brain with unbiased identification of activated (or 

deactivated) regions would be highly desirable, as currently achieved in human functional 

imaging studies. Recent attempts in this direction have been proposed (Dubois, A et al 2008). 

Here we report the development of a novel approach to achieve such goals, based on merging 

and warping of brain autoradiograms prepared from mice belonging to the same experimental 

group, and aimed at the unbiased identification of regions of interest combined with 3D 

reconstruction. 

JULIDE is a software that allows users to detect activated areas in the brain by performing a

voxel-based statistical analysis. It is fully written in ITK (National Library of Medicine Insight 

Segmentation and Registration Toolkit) and presents a user-friendly interface. 

JULIDE is based on a four-step approach which includes: (1) an automated pre-processing to 

select the desired section, (2) a 3D reconstruction of autoradiographic volumes, (3) a spatial 

normalization to compensate all the differences in position, orientation and shape and finally (4) 

a 3D voxel-based statistical analysis based on t-test. 

Outputs of JULIDE are two 3D activation maps allowing to identify the significant differences 

(positive and negative) in CMRGlu between two groups. The comparison of CMRGlu between 

two groups is represented by color-coded statistical maps of p values defined by the user. 

We have applied this approach to the study of the regional variations in CMRGlu in mice 

undergoing a spatial learning task. Significant differences in the areas engaged during the 

behavioural task at day 1 vs day 9, when animals are highly performing, have been identified . 

Results obtained with JULIDE are comparable to those obtained manually by optical 

densitometry of pre-selected ROI such as the hippocampus, the most activated region at day 1. 

This metabolic activation is localized to the cingulate and retrosplenial cortices at day 9. 

Softwares such as JULIDE and other recently proposed (Dubois, A et al 2008) are likely to 

provide useful tools to exploit to its full extent the use of 2DG autoradiography to complement 

behavioural and pharmacological studies in laboratory animals. 

Disclosures: J. Parafita , None; F. Magara, None; R. Charrier, None; D. Ribes, None; J. 

Thiran, None; P. Magistretti, None. 

Poster 





Support: NIMH Grant MH-072641 

Sigma Xi Grants-in-Aid of Research 

NASA GSRP Grant NNX06AH25H-S01 

Title: Hemodynamic and electrophysiological evidence of resting-state network activity in the 

primate

Authors: *A. ARDESTANI 1 , F. DARVAS 3 , A. TOGA 2 , J. FUSTER 1 ; 

1 UCLA Dept of Psychiatry, 2 Lab. of NeuroImaging, UCLA, Los Angeles, CA; 3 Dept of 

Neurolog. Surgery, Univ. of Washington, Seattle, WA 

Abstract: An expanding body of literature describes the existence of concerted brain activations 

in the absence of any external stimuli. Resting-state networks have been identified and 

demonstrated to be modulated during the performance of specific cognitive operations. However, 

despite mounting evidence the possibility still remains that those correlated signal fluctuations 

reflect non-neural phenomena. In order to isolate functionally relevant spontaneous 

coactivations, we utilized a multi-level sampling approach to obtain co-registered brain signals 

across a range of sampling resolution and sensitivity. Surface and local field potentials, 

hemodynamic signals (near-infrared spectroscopy, NIRS), and cell spiking were recorded from 

dorsolateral prefrontal and posterior parietal cortices in four monkeys trained to remain 

motionless in a primate chair. The use of an optical recording technique (NIRS) allows 

measurement of a signal that is physiologically equivalent to that obtained using BOLD fMRI, 

though with millisecond temporal resolution and minimal technical or environmental constraints. 

The different signal types exhibited correlations between the two regions of interest in both the 

frequency and time domains. This evidence suggests that the resting-state network activations 

detected by fMRI do in fact reflect functional coactivations of areas across multiple levels of 

network communication. 

Disclosures: A. Ardestani, None; F. Darvas, None; A. Toga, None; J. Fuster, None. 

Poster 





Support: RIKEN BSI 

MRC 

Title: Structural changes in cortex associated with tool-use in non-human primates 

Authors: M. QUALLO 1,4 , C. J. PRICE 2 , J. T. DEVLIN 3 , K. UENO 5 , T. ASAMIZUYA 5 , R. A. 

WAGGONER 6 , K. CHENG 5,6 , K. TANAKA 6 , F. KHERIF 2 , R. N. LEMON 1,4 , *A. IRIKI 4,1 ; 

1 Sobell Dept., 2 Wellcome Ctr. for Neuroimaging, 3 Cognitive, Perceptual and Brain Sci., UCL

Inst. of Neurol., London, United Kingdom; 4 Symbolic Cognitive Develop., 5 FMRI Support Unit, 

6 Cognitive Brain Mapping, RIKEN Brain Sci. Inst., Wako-shi, Saitama, Japan 

Abstract: There are few reports of macaques using tools in the wild. However macaque 

monkeys can be trained to use a rake to retrieve out-of-reach food (Ishibashi et al, 2000). It takes 

approximately 15 days for the monkeys to reach a level where the food can be retrieved 

efficiently. Neurophysiological and anatomical studies suggest that training to use a rake induces 

changes in macaque cortex: receptive fields of neurons in the intraparietal sulcus (IPS) adapt to 

include the tool in the body schema (Maravita & Iriki, 2004), and expression of BDNF, its 

receptor trkB and NT-3 is increased during tool-use training (Ishibashi et al, 2002). Following 

training, cortico-cortical projections are revealed from the higher visual centres to the 

intraparietal area (Hihara et al, 2006). We used non-invasive structural MRI and voxel based 

morphometry (VBM) to confirm whether learning to use a tool causes long-term structural 

changes in the cortex. Three tool-use naive young Japanese macaques (4.1, 5.1, 5.6 kg) were 

trained to use the rake. Each monkey was scanned on 6 occasions over a period of 6 weeks with 

3 scans taken during each session, using a 4T Varian MRI scanner. During training, which began 

in the third week, there was a timed test period of 50 trials, each trial was assigned a score of 1-3 

(3: food retrieved on first attempt; 2: food obtained on the second attempt and 1: food obtained 

only after multiple attempts). A VBM analysis revealed structural increases in a number of 

cortical regions that were significantly correlated (p



Topic: F.03.c. Social behavior 

Support: NIH grant HD48462 

Title: Pair-bond formation alters reward responses 

Authors: *J. CURTIS; 

Pharmacol & Physiol, Oklahoma State Univ., Tulsa, OK 

Abstract: Voles, especially prairie voles, have been used extensively in studies of the neural and 

endocrine mechanisms underlying monogamous pair-bonds between adults. Reports from the 

past several years suggest that the mesolimbic dopamine system plays an important role in the 

formation of monogamous pair-bonds in prairie voles. Further, up-regulation of the D1 family of 

dopamine receptors within the nucleus accumbens appears to play an important role in the switch 

from sociability to the aversion toward strangers that serves to maintain the monogamous 

relationship in prairie voles (Aragona et al, 2006, Nat Neurosci 9:133). Since the mesolimbic 

dopamine system also is implicated in reward processing, changes associated with pair bonding 

may alter subsequent responses to rewarding stimuli. To test this possibility, we compared 

ingestion of a sucrose solution in sexually naïve male prairie voles and in males that were paired 

with a female for two weeks. Subjects were paired with sexually naïve, reproductively intact 

females between 1700 and 1800h. The following morning, males were separated from their 

partners and were presented with a bottle containing a 0.5M sucrose solution for 6h hours, 

followed by reunion with the partner. This pattern was repeated on each of the subsequent 12 

days but the males received a bottle containing water instead of sucrose. Finally, the sucrose 

solution was presented again on the 14th day of cohabitation. Control males received the same 

sucrose access pattern, but remained with their respective siblings when not being tested for 

ingestion. The amounts of sucrose ingested during each of the sucrose exposures then were 

compared. Paired and control males did not differ in the amounts of sucrose ingested during their 

respective first exposures, and the amounts of sucrose ingested during the first and second 

exposures did not differ for the males that remained with their respective siblings. In contrast, 

males that were allowed to pair with a female for two weeks significantly increased their sucrose 

intakes from the first and to the second exposures to sucrose. These data suggest that the central 

changes associated with the formation of a pair-bond can alter responses to rewarding stimuli. 

Disclosures: J. Curtis, None. 

Poster 

297. Social Recognition and Partner Preference




Support: NIH grant DAR01-19627 

NIH grant MHR01-58616 

NIH grant DAK02-23048 

Title: Repeated amphetamine exposure blocks social bonding in monogamous female prairie 

voles; the involvement of mesolimbic dopamine 

Authors: *K. A. YOUNG, Y. LIU, Z. WANG; 

Psychology, Florida State Univ., Tallahassee, FL 

Abstract: Prairie voles are monogamous rodents that form mating-induced partner preferences, a 

behavior mediated in part by dopamine (DA) in the nucleus accumbens (NAcc). Recently, we 

have established the prairie vole as an animal model of drug reward, and demonstrated that 

amphetamine (AMPH) induced conditioned place preferences in this species (Aragona et al., 

2007, Neurosci Lett, 418:190-194). The current experiments tested the hypothesis that AMPH 

and social reward interact in the female prairie vole and that this interaction is mediated by NAcc 

DA. In the first experiment, estrogen primed female subjects received repeated saline (control) or 

AMPH (0.2, 1.0 or 5.0mg/kg) injections (1 injection per day for 3 days), were then allowed to 

mate with a male for 24 hours and were then tested for partner preferences. While females 

injected with saline or higher doses of AMPH (1.0 or 5.0mg/kg) displayed partner preferences 

after mating, females treated with 0.2mg/kg AMPH did not show mating-induced partner 

preferences. These results demonstrate a dose-specific effect of AMPH on the inhibition of 

partner preference formation in female prairie voles. A second experiment investigated the 

mechanisms underlying this behavioral interaction. Female prairie voles received estrogen 

treatment alone (control) or estrogen treatment plus repeated 0.2 mg/kg AMPH injections (1 

injection per day for 3 days). Using Western blotting, AMPH treated females were found to have 

significantly lower levels of DA D2 receptors in the NAcc than females treated with estrogen 

alone. In a third experiment, three repeated AMPH (0.2mg/kg) injections were found to 

significantly increase DA D1 receptor protein levels in the NAcc of female prairie voles as 

compared to saline injected controls. As D2R activation facilitates, and D1R activation prevents 

partner preference formation, these data, taken together, suggest a potential mechanism by which 

AMPH interferes with social bonding in the female prairie vole. 

Disclosures: K.A. Young , None; Y. Liu, None; Z. Wang, None.

Poster 

297. Social Recognition and Partner Preference 




Support: NSF IBN-9876754 

NIH RR00165 

NIMH56897 and MH064692 (L.J.Y.) 

Title: Enhanced oxytocin receptor expression in the nucleus accumbens by viral vector gene 

transfer accelerates partner preference formation in female prairie voles 

Authors: *H. E. ROSS 1,4 , L. L. SPIEGEL 2 , L. J. YOUNG 3,4 ; 

1 Neurosci., 3 Dept. of Psychiatr. and Yerkes Ntl. Primate Cntr, 2 Emory Univ., Atlanta, GA; 4 Cntr. 

Behav. Neurosci., Atlanta, GA 

Abstract: Oxytocin receptors (OTR) in the nucleus accumbens (NAcc) have been implicated in 

the regulation of spontaneous maternal care and social bonding in prairie voles. There is 

significant individual and species variation in the density of OTR in the NAcc, which may 

contribute to variation in social behaviors. In juvenile and virgin female prairie voles, individual 

variation in NAcc OTR expression positively correlates with the propensity for displaying 

spontaneous maternal care. Furthermore, blocking OTR in the NAcc impairs spontaneous 

maternal behavior as well as inhibits the ability to form a partner preference in this socially 

monogamous species. Using viral vector mediated gene transfer, we tested the hypothesis that 

variation in OTR density in the NAcc in the adult can contribute to variation in maternal care and 

partner preference formation. Adult, sexually naïve female prairie voles were injected bilaterally 

into the NAcc with either an adeno-associated viral vector (AAV) containing the prairie vole 

OTR gene under the control of the CMV promoter (AAV-OTR), a control GFP expressing 

vector (AAV-GFP), or were sham operated. Four weeks later they were tested for spontaneous 

maternal behavior. The AAV-OTR injection resulted in a significant increase in OTR expression 

in the NAcc as determined by receptor autoradiography. We observed no significant difference 

between groups in the amount of time spent licking/grooming or hovering over the pups (p > 

0.05). The voles were then tested for a partner preference following 18 hours of cohabitation 

with a sexually experienced male. AAV-OTR treated voles, but not control or sham animals, 

spent significantly more time with their mating partner compared to the stranger (p < 0.05). 80% 

of AAV-OTR injected voles developed a partner preference, compared to only 31% of AAV- 

GFP injected animals. There was, however, no difference in mating behavior between groups. 

Thus, we hypothesize that variation in OTR density in the NAcc may have an organizational

effect on spontaneous maternal care, while having activational effects on partner preference 

formation. 

Disclosures: H.E. Ross, None; L.L. Spiegel, None; L.J. Young, None. 

Poster 






NIH RR00165 

NSF IBN-9876754 

Title: A role for the opiate system in partner preference formation in female prairie voles 

Authors: J. P. BURKETT 1 , L. L. SPIEGEL 1 , *L. J. YOUNG 2,1 ; 

1 Cntr Behav Neurosci, 2 Yerkes Res. Ctr., Emory Univ., Atlanta, GA 

Abstract: Socially monogamous prairie voles (Microtus ochrogaster) form enduring pair bonds 

between mates, and have become an informative animal model for exploring the neurobiology of 

social attachment. The opioid system has long been implicated in the regulation of infant-mother 

attachment, but its role in adult pair bonding has not been explored. Mu-opioid receptors in the 

nucleus accumbens (NAcc) mediate the reinforcing properties of natural rewarding stimuli. The 

NAcc plays a critical role in partner preference formation, the laboratory proxy for pair bonding. 

We hypothesize that endogenous opioids play a role in partner preference formation in prairie 

voles via activation of mu-opioid receptors in the NAcc. To test this hypothesis, we first 

administered the opioid antagonist naltrexone (NTX; IP, 7.5 mg/kg) or saline to adult female 

prairie voles during an 18-hour mating period with a male partner. Females received either three 

NTX injections at 6-hour intervals, a single NTX injection followed by two saline injections, or 

three saline injections. 8 out of 10 saline controls, 4 out of 8 single NTX injected and 0 of 8 triple 

NTX injected females displayed a partner preference following co-habitation, suggesting that the 

non-selective antagonist effectively blocks partner preference formation ( p < 0.01). Triple NTX 

injected females displayed a significant preference for the stranger (p < 0.01), and both drugtreated 

groups mated significantly less during the first 4 hours of cohabitation (p < 0.01). We 

then explored the role of mu-opioid receptors in the NAcc more specifically by site-specific

administration of the mu-opioid selective antagonist CTAP in the NAcc (3 κg/side) at 12-hour 

intervals during a 24-hour mating period. Control females receiving vehicle in the NAcc 

displayed a significant partner preference (p = 0.01), while females receiving CTAP displayed no 

preference. Furthermore, the antagonist treatment did not affect mating behavior during the 

cohabitation. We are in the process of performing anatomical control experiments to confirm the 

site-specificity of these effects. The data strongly implicate the endogenous opioid system in pair 

bond formation in female prairie voles, and further suggest that mu-opioid receptors in the NAcc 

may mediate these effects. 

Disclosures: J.P. Burkett, None; L.L. Spiegel, None; L.J. Young, None. 

Poster 





Support: NIMH Z01-MH-002498-17 

Title: Development and validation of a reliable social recognition/discrimination paradigm 

Authors: *A. H. MACBETH, J. STEPP, H.-J. LEE, W. S. YOUNG 3RD; 

NIMH, NIH, Bethesda, MD 

Abstract: Social recognition, in which an animal identifies and recognizes another individual of 

the same species, is critical for appropriate display of social behaviors. Unfortunately, tests for 

social recognition vary in the literature, making reliable and replicable testing of social 

recognition difficult. Therefore, we investigated various components of social recognition testing 

to design a robust paradigm where male mice are capable of consistently discriminating between 

familiar and novel female stimulus animals every time the task is administered. 

We first determined that males could discriminate between familiar and novel females when 

stimulus females are isolated in corrals, eliminating the need to extinguish mounting behavior 

prior to social recognition testing. The social recognition task was then administered a total of six 

times to two cohorts of C57Bl/6J males, manipulating housing conditions (group or singly 

housed) and numbers of corrals present during each trial (1 or 2 corrals) with each test. 

Ultimately, social recognition reliably occurred using group housed males and singly housed 

stimulus females (to avoid odor transmission between females). Furthermore, we used one corral 

during the first trial (containing one female), and two corrals during the second trial, 30 minutes 

later (one corral containing first, familiar female; one corral containing novel female).

We are now using this paradigm to further examine social recognition in four lines of knockout 

(KO) mice: vasopressin 1a receptor (Avpr1a), vasopressin 1b receptor (Avpr1b), whole body 

oxytocin receptor (Oxtr -/-), and partial forebrain-specific oxytocin receptor (FB/FB). We have 

previously shown that both Avpr1b and Oxtr -/- KO males investigate all stimulus females as if 

they were less familiar (Wersinger et al., Molecular Psychiatry, 2002; Lee et al., Endocrinology, 

2008, respectively), whereas FB/FB males investigate all stimulus females of the same strain as 

if they were familiar (Lee et al., Endocrinology, 2008). Studies of Avpr1a KO males are 

conflicting, but we have previously found no social recognition deficits (Wersinger et al., Genes, 

Brain & Behavior, 2006). Using the new social recognition paradigm developed in our lab, we 

have confirmed in both Oxtr lines that wildtype (WT) males can discriminate between familiar 

and novel females regardless of strain (C57Bl/6J, BalbC, or Swiss-Webster), whereas KO males 

present a more complicated, strain-dependent situation. Furthermore, we are examining the 

performance of both Avpr1a and Avpr1b KOs to better understand those receptors' contributions 

to social recognition. 

Disclosures: A.H. Macbeth, None; J. Stepp, None; H. Lee, None; W.S. Young 3rd, None. 

Poster 





Support: CAPES 

FAPESP 

CNPQ 

Title: Long-term social recognition memory and the main behaviors to be analysed in rats 

Authors: *P. J. MOURA, S. T. MEIRELLES, G. F. XAVIER; 

Dept. of Physiol., Univ. of Sao Paulo, Sao Paulo, Brazil 

Abstract: In the intruder-resident paradigm (IRP) an intruder rat is exposed to a resident 

conspecific for 5 min (1st trial). Then, the resident rat is exposed, for another 5 min, either to the 

same or to a novel intruder (2nd trial). When the intertrial interval (ITI) is 30 min, there is a 

reduction of the resident´s social behaviors towards the familiar intruder as compared to the 

corresponding parameter for the novel intruder; this is taken as an index of social recognition

memory (SRM). If the ITI is 60 min, SRM vanishes being thus considered as a type of shortterm 

memory. This study shows that SRM may last at least 24h when the 1st trial-time-duration 

is increased for 2h or longer. One hundred and forty-six adult male Wistar rats, exposed to a 

12/12h light/dark schedule, were used. In the 1st trial, independent groups of resident rats were 

exposed to intruder rats for either 0.083, 0.5, 2, 24 or 168h. Twenty-four hours later the resident 

rats were exposed, along 10 min, either to the familiar or to a novel intruder rat; specific social 

and non-social behaviors were scored in this 2nd trial, both for the resident and the intruder rats, 

along 4 consecutive time bins, each of them with 150s. A Principal Component Analysis (PCA) 

included behavioral scores associated with familiarity (exposure to the same or to a novel 

intruder), social role (intruder or resident), duration of the first meeting (0.083, 0.5, 2, 24 or 

168h), and time bin, followed by a traditional ANOVA. The results showed that both the resident 

and the intruder rats exhibited SRM when the 1st trial lasted 2h or longer; in addition, 

confirming previous observations, no SRM was observed when the 1st trial duration was 0.083 

or 0.5 h. Furthermore, most of critical behaviors indicating SRM occurred in the first time bin. 

The PCA revealed that two components allow explanation of more than 50% of data variability. 

The first main component, characterized as investigatory activity, originated from an opposition 

between social investigation of the novel intruders by the resident rats (including anogenital, 

head and body sniffing, and following behaviors) and an environmental investigation by the 

intruder rats (rearing and environmental sniffing). The second main component seems to involve 

agonistic behavior originated from an opposition between self-grooming occurring in subjects 

which exposure to the conspecific was short, and dominance and aggression occurring in 

subjects which exposure was longer. These results show that SRM may last for at least 24h and 

also shows that the parameters choice may influence the type of behavioral outcome seen in the 

intruder-resident paradigm. 

Disclosures: P.J. Moura, None; S.T. Meirelles, None; G.F. Xavier, None. 

Poster 





Support: University of Tromsø (to TS and AÅ) 

NIH grants MH 38273 and HD 05751 (to DWP) 

Title: Social recognition in female rats depends on estrogen receptor alpha in the amygdala

Authors: T. SPITERI 1 , S. MUSATOV 2 , A. RIBEIRO 3 , S. OGAWA 4 , D. W. PFAFF 3 , *A. 

AGMO 1 ; 

1 Inst. Psykologi, Univ. Tromsoe, N-9037 Tromso, Norway; 2 Lab. of Mol. Neurosurg., Weill 

Med. Col. of Cornell Univ., New York, 10021, NY; 3 Lab. of Neurobio. and Behavior, The 

Rockefeller Univ., New York, 10021, NY; 4 Lab. of Behavioral Neuroendocrinology, Univ. of 

Tsukuba, Tsukuba, Ibaraki 305-8577, Japan 

Abstract: Social recognition manifests itself in decreased investigation of a previously 

encountered individual. There is evidence suggesting that oxytocin (OT) in the medial amygdala 

regulates both social recognition and anxiety in rodents. OT action is mediated by OT receptors 

and estrogens drive this receptor expression, especially through the ERα in various brain areas 

including the amygdala. Studies of female mice, whose genes for either OT, ERα or ERβ had 

been knocked out, showed that they were deficient in social recognition and they were less 

anxious. These data suggest that ERα is implicated in these behaviors. However, the effects of a 

selective reduction of ERα in the medial amygdala on social recognition and anxiety have not 

been evaluated. This was made in the present experiment. The effects of silencing ERα in the 

ventromedial nucleus of the hypothalamus (VMN) were also determined because this nucleus is 

implicated in neural circuits regulating maternal behaviors. For these, the recognition of pups is 

believed to be crucial. A shRNA encoded within an adeno-associated viral (AAV) vector 

directed against the ERα receptor gene (or containing luciferase control), was infused bilaterally 

into the medial posterodorsal amygdala (MePDA) or the VMN of female rats. About 3 weeks 

later, the females were given an sc injection of EB followed by P. Starting 6 hr thereafter they 

were repeatedly exposed to the same juvenile rat. After 4 exposures, a novel juvenile was 

presented. Social interactions, including aggressive, were recorded. Anxiety-like behaviors were 

evaluated in the light/dark choice test. Finally, aggression towards an adult intruder was 

evaluated. An 80 % reduction of ERα expression in the MePDA eliminated social recognition. 

There was no sign of habituation to the juvenile, and consequently no sign of dishabituation 

when the familiar juvenile was replaced by a novel juvenile. No change in aggression towards 

the juvenile was observed. Social recognition was unaffected after ERα knockdown in the VMN. 

Females presented a normal habituation to the juvenile and a restoration of investigation when a 

novel juvenile replaced the familiar. To the contrary, ERα knockdown in the VMN enhanced the 

aggressiveness against the juvenile conspecific at the first exposure and at presentation of the 

novel juvenile. The light/dark choice test as well as the resident-intruder test did not reveal any 

significant effect after infusion of ERα AAV into the MePDA or into the VMN. In conclusion, 

social recognition in female rats depends on the ERα in the MePDA. Moreover, aggression 

against juveniles but not against adults could, at least partly, depend on the ERα in the VMN. 

Disclosures: T. Spiteri, None; A. Agmo , None; S. Musatov, None; A. Ribeiro, None; S. 

Ogawa, None; D.W. Pfaff, None. 

Poster 

297. Social Recognition and Partner Preference




Support: NSF grant IBN0130986 to EAR 

Title: The effects of a dopamine receptor antagonist on courtship behaviors and pair formation in 

male and female zebra finches 

Authors: *S. B. BANERJEE, E. ADKINS-REGAN; 

Dept. of Psychology, Cornell Univ., Ithaca, NY 

Abstract: The neural mechanisms that underlie the formation and maintenance of a pair bond in 

avian species have not been elucidated to date although almost 90% of avian species are socially 

monogamous. The zebra finch, Taeniopygia guttata is one such species in which individuals 

form long term pair bonds that last for life. The neuromodulator dopamine could be hypothesized 

to play a role in pair formation, as it has been implicated in the mediation of reward in response 

to food, sex and drugs of abuse. Hence it is possible that if pair formation is considered as a 

pleasure seeking behavior, dopamine mediates reward in response to pair formation. Indeed, 

studies performed by other groups have demonstrated that dopamine plays a key role in the 

formation and maintenance of a pair bond in a mammalian rodent species- the prairie vole. 

To begin to address the role of dopaminergic neurotransmission via D1-like and D2-like 

receptors in pair formation, we administered the nonselective dopamine receptor antagonist 

haloperidol systemically to sexually inexperienced male and female zebra finches for 5 days. 

Two doses of drug were used, a high dose of 0.1mg/kg and a low dose of 0.05mg/kg. The birds 

were observed for 15 minutes after injection of the drug on days 1,3 and 5. Courtship, pairing 

and aggressive behaviors were scored on all 3 days and on day 5 the pairing status of each bird 

was noted and categorized as either paired or unpaired. We observed significant differences in 

behavior between the high dose and the saline injected control group on day 1 of observation. 

The high dose males sang far fewer bouts of direct song to females and performed fewer beak 

wipes suggesting that dopaminergic signaling via the D1 and/or D2 like receptors is involved in 

the expression of courtship behaviors. We did not observe any significant differences in pairing 

behaviors (clumping, allopreening or time in a nest box with another bird), in either males or 

females. In addition, at the end of 5 days, there were no significant differences in the numbers of 

paired birds in the low dose, high dose and saline control groups. These results are interesting, as 

although high dose males perform fewer courtship behaviors due to the administration of a 

dopaminergic antagonist, they are able to form pairs with stimulus females, suggesting that 

pairing behavior might not involve signaling via the D1/D2-like receptors. There remains the 

possibility that D1-like or D2-like receptor specific antagonists would interfere with the pairing 

process and studies are ongoing to address this question. 

Disclosures: S.B. Banerjee, None; E. Adkins-Regan, None.

Poster 





Support: NIH Grant RO1-MH55488 

NIH Grant K02-MH65907 

Title: The effect of pairing on behavioral responses in female zebra finches 

Authors: *L. A. SVEC, K. M. LICHT, J. WADE; 

Prog in Neurosci, Michigan State Univ., East Lansing, MI 

Abstract: Zebra finches form strong pair bonds prior to breeding. These pair bonds are signified 

by affiliative behaviors such as clumping (perching in close contact) and preening. In order to 

determine the role of pairing in female behavioral responses to interactions with male zebra 

finches, females were either paired with a male or individually housed for 14 days. The male was 

then removed from the shared cages and housed within visual and acoustic contact for two days. 

Next, a one-hour social interaction test was conducted in which either a familiar male (n= 7) or a 

new male (n= 9) was introduced to the paired females, and a new male was introduced to the 

females that had not been paired (n= 10). A variety of social, including affiliative and 

reproductive, behaviors of both the male and female were quantified. Significant effects (with 

Kruskal-Wallis tests) were observed in five female behaviors directed toward males, including 

beak wipes, clumping (both frequency and duration), and preening (frequency and duration). In 

all cases, these behaviors were increased in response to a familiar male compared to a new male 

in the paired females (all Mann-Whitney U, p< 0.017). In addition, among females interacting 

with new males, clumping (duration, and to a lesser extent frequency) was increased in unpaired 

individuals compared those who had been housed with males (Mann-Whitney U, p= 0.04, p= 

0.053, respectively). Finally, males attempted a greater number of mounts when introduced to 

females that had been individually housed compared to those previously paired (Kruskal-Wallis, 

p= 0.032, both Mann-Whitney U, p< 0.068). Collectively, the results indicate that housing a 

female with a male decreases her affiliative behaviors toward novel males, but this diminished 

response to new males is not observed in females that have recently lived alone. The data also 

suggest that males likely receive some signal from females indicating whether or not they have 

been paired with a male. The neural correlates of the behavioral effects will be investigated in 

the females by examining the expression of the immediate early gene ZENK following these

social interactions. 

Supported by NIH grants R01-MH55488 and K02-MH65907. 

Disclosures: L.A. Svec , None; K.M. Licht, None; J. Wade, None. 

Poster 





Title: Neural correlates of long-term pair-bonding in a sample of intensely in-love humans 

Authors: B. P. ACEVEDO 1 , A. ARON 1 , H. FISHER 2 , *L. L. BROWN 3 ; 

1 State Univ. of New York, Stony Brook, Stony Brook, NY; 2 Anthrop., Rutgers Univ., New 

Brunswick, NJ; 3 Dept Neurol., Albert Einstein Col. Med., Bronx, NY 

Abstract: This study examined neural correlates of romantic love among individuals in longterm 

pair-bonds using functional magnetic resonance imaging (fMRI; BOLD response). One 

question was how being “in love” in a long-term relationship (> 10 years) differs from the early 

stages of intense romantic love (1-17 mos.; Aron et al., 2005; Bartels & Zeki, 2000). Procedures 

used in Aron et al. (2005) were replicated and additional stimuli were used to control for 

closeness and familiarity. Ten females and 7 males (mean ages: 51 and 55 years, respectively) 

who reported being intensely in love with a long-term spouse (married mean of 21.4 ± 5.89 

years) underwent fMRI scanning while they viewed face images of their partner and controls, 

interspersed with a distraction-attention task. Control images were a high-familiar, neutral 

person; a low-familiar, neutral person; and a close friend or sibling. Group activation specific to 

the long-term partner compared to each of the control conditions occurred in dopamine rich 

regions associated with reward and motivation in mammals. Specifically, significant activations 

appeared in the right ventral tegmental area, and the ventral striatum/pallidum. Other activations 

occurred in serotonin-rich areas, the median and dorsal raphe nuclei, consistent with data from 

monogamous pair-bonding studies in voles (Lim, Nair, & Young, 2005). The results show that in 

some human individuals, being “in love” with a long-term partner is similar to early-stage 

romantic love because ventral midbrain areas were affected under the various control conditions. 

In addition, activation was found in brain areas implicated by animal studies to be involved in 

mammalian pair-bonding, or more generally, social affiliation. These additional areas may be 

part of a human attachment system. We conclude that this group of individuals revealed both 

early attraction and attachment brain systems whose activity is important for reproductive 

behavior and the establishment and maintenance of pair-bonds in humans.

Disclosures: B.P. Acevedo, None; A. Aron, None; H. Fisher, None; L.L. Brown , None. 

Poster 






NIMH 

NIH 

Title: "Trust" in mice: Oxytocin mediated mate choice copying by female mice 

Authors: *M. KAVALIERS 1 , E. CHOLERIS 2 , S. OGAWA 3 , D. W. PFAFF 4 ; 

1 Dept of Psychol, Univ. Western Ontario, London, ON, Canada; 2 Dept. Psychology, Univ. of 

Guelph, Guelph, ON, Canada; 3 Lab. of Behavioral Neuroendocrinology, Univ. of Tsukuba, 

Tsukuba, Japan; 4 Lab. of Neurobio. of Behavior, The Rockefeller Univ., New York, NY 

Abstract: Results of a number of studies have suggested that the mate choice decisions of 

females can be influenced by the mating choices of others. Such non-independent mate choice 

has been termed “mate choice copying”, whereby a female utilizes the mate choices of others. 

Odors guide the social behavior and mate responses of rodents. As such, female odors that are 

associated with that of a male may be used to guide the social interests and mate choice of other 

females. Here we show that female mice preferentially choose and respond to the odors of a male 

with which there is associated the odor of another female. This suggests that female mice may 

use olfactory based indicators of other females‟ interest in a male and engage in mate choice 

copying. The presence of the odors of another female are also able to reverse the aversion that 

females naturally show for males who are parasitized with lice. This results in a choice for the 

odors of an infected male suggesting that when choosing which male to associate with, sexually 

inexperienced females may rely upon social information more than their own innate aversions 

and, or preferences. The neuropeptide, oxytocin (OT), has been implicated in the mediation of 

odor guided social behavior in rodents as well as “trust” in humans. Here we further show the 

female mice with either deletions of the OT gene (OT knockout mice) or administered the 

oxytocin antagonist, L3668,899, were impaired in their use of the odors of other females to 

modulate their responses to male odors. This suggests that oxytocin is involved in the mediation

of mate choice copying and the “trust” that female mice have in the mate choice decisions of 

others. 

Disclosures: M. Kavaliers , None; E. Choleris, None; S. Ogawa, None; D.W. Pfaff, None. 

Poster 




Topic: F.03.a. Sexual behavior 

Title: The role of the medial amygdala in mate preference of female rats 

Authors: K. SICILIANO 1 , K. LEE 1 , K. VONDERHEIDE 1 , R. J. FROHARDT 2 , *F. A. 

GUARRACI 1 ; 

1 Psychology, Southwestern Univ., Georgetown, TX; 2 Psychology, St. Edward's Univ., Austin, 

TX 

Abstract: Female rats display a marked preference for one male rat over another when given the 

opportunity to mate with two male rats simultaneously (Lovell et al., 2007). Pheromonal 

information, processed by the vomeronasal organ and projected to the medial amygdala (MeA), 

may provide one source of sensory information that female rats use to discriminate between 

potential mates. To evaluate the role of the MeA in mate preference, ovariectomized, hormoneprimed, 

female Long-Evans rats received either ibotenic acid lesions of the MeA or sham 

lesions. One week following surgery, the female rats were given the opportunity to mate with 

two male rats simultaneously. Female rats with sham lesions preferred one male over another 

during the test and returned to their preferred mate faster than their nonpreferred mate following 

intromissions. Although female rats with MeA lesions also preferred one male over another, they 

returned to their preferred mate at the same rate as their nonpreferred mate after receiving 

intromissions. One week later, all of the female rats (MeA and sham lesioned) were given the 

opportunity to spend time in the vicinity of bedding soiled by two gonadally intact male rats. 

Female rats displayed a weak preference for the bedding of one male rat over another, 

independent of lesion condition. Although we hypothesized that mate preference would depend 

on an intact MeA, we found that the MeA may instead be critical for discriminating the quality 

of sexual stimulations that a female rat receives when mating with two males simultaneously. 

Disclosures: K. Siciliano, None; F.A. Guarraci , None; K. Lee, None; K. Vonderheide, 

None; R.J. Frohardt, None.

Poster 





Support: Division of Intramural Research, NICHD 

Title: EEG activity in response to facial gestures in 1-7 days old infant rhesus macaques 

Authors: *P. F. FERRARI 1,2 , R. VANDERWERT 3 , K. HERMAN 2 , A. PAUKNER 2 , N. A. 

FOX 3 , S. J. SUOMI 2 ; 

1 Neurosci., Univ. Parma, Parma 43100, Italy; 2 Nat. Inst. Child Hlth. and Human Dev., NIH, 

Poolesville, MD; 3 Dept. Human Dev., Univ. of Maryland, College Park, MD 

Abstract: Rhesus macaque infants are able to imitate facial gestures in their first week of life. 

However, the possible mechanisms underlying this phenomenon are presently not known. We 

investigated infant macaque cortical activity by electroencephalography (EEG) while observing 

macaque responses to human facial gestures or a moving object. We used an EEG cap 

specifically designed to record from 4 active electrodes over anterior and posterior scalp 

locations (f3, f4, p3 and p4). We tested 11 infant macaques from day 1 through day 7 of age in 

three different experimental conditions (two facial gestures and a non-biological stimulus): a) 

Lipsmacking (a high-frequency opening and closing of the mouth) b) Tongue protrusion and c) 

Disk (a rotating disk). Each trial started with a 40-second baseline with the presentation of a still 

stimulus. We then presented each moving stimulus condition for 20 seconds, followed by 20 

seconds of still face or disk. Results showed that rhythms between 5 to 8 Hz are sensitive to the 

moving stimuli. More specifically, we observed a decrease in these rhythms during the stimulus 

period compared to the baseline. It is possible that the changes in these rhythms reflect activity in 

the alpha band. These data also suggest that in newborn macaques cortical activity as indexed by 

the ongoing EEG is sensitive to social stimuli. They also provide important information 

regarding the development of cortical activity during processing of social information. 

Disclosures: P.F. Ferrari , None; R. Vanderwert, None; K. Herman, None; A. Paukner, 

None; N.A. Fox, None; S.J. Suomi, None. 

Poster





Support: NIH/NCRR RR-00165 (Yerkes National Primate Research Center) 

R01-MH068791 (Lisa A. Parr) 

NSF IBS#9876754 (Center for Behavioral Neuroscience) 

Title: Functional neuroimaging of the neural correlates of face recognition in chimpanzees 

Authors: *E. E. HECHT 1 , S. K. BARKS 2 , T. M. PREUSS 3 , J. K. RILLING 2 , J. R. VOTAW 3 , 

L. A. PARR 3 ; 

1 Neurosci Program, 2 Dept. of Anthrop., 3 Yerkes Natl. Primate Res. Ctr., Emory Univ., Atlanta, 

GA 

Abstract: Objective: The ability to recognize individuals is a crucial component of social 

cognition, enabling behavior which takes into account prior interactions and observations of that 

individual. Humans and chimpanzees show evidence of greater “face expertise” than macaque 

monkeys, including the ability to reliably and rapidly identify individuals from faces, secondorder 

configural processing of faces, and a consistent face inversion effect. Humans and 

macaques activate similar, but not identical, neural networks during face perception. The neural 

correlates of chimpanzee face recognition are unknown. To investigate the evolution of neural 

specializations for face recognition, the current study examined the neural correlates of face 

recognition in chimpanzees using PET neuroimaging. 

Methods: Five captive chimpanzees were administered radioactively labeled glucose 

(fluorodeoxyglucose), performed a match-to-sample task with photographs of unfamiliar 

conspecific faces during brain uptake, and were scanned afterwards using a high resolution 

research tomograph. In a control task, subjects matched images of clip art objects. The 

fluorodeoxyglucose tracer accumulates in brain regions of higher metabolism during the 

conscious uptake period. Regions with higher radioactivity in the scan thus represent regions that 

are active during the task. Individual PET scans were coregistered to anatomical MRI scans. 

Activity associated with face versus object matching tasks was compared using a full-factorial 

ANOVA in SPM5. 

Results: Face recognition-specific activation was observed in regions similar to those identified 

in humans, including the precuneus, temporoparietal junction, fusiform gyrus, lingual gyrus, and 

posterior superior temporal sulcus, using a whole brain analysis (p< 0.05). Interestingly, this 

network is similar to one identified by Gobbini and Haxby (2007) underlying the recognition of 

familiar or famous faces. The chimpanzees have repeatedly encountered photographs of the 

conspecifics used in this experiment over a period of years, possibly resulting in a type of 

exposure similar to human media exposure to famous individuals.

Conclusions: These preliminary results suggest that humans and chimpanzees employ a similar 

distributed neural system for face recognition. A second study will use these results as functional 

ROIs to further investigate chimpanzee face-selective regions, including a possible chimpanzee 

fusiform face area. This study represents an important development in the use of functional 

neuroimaging of our closest living relative towards the identification of human neural 

specializations for social cognition. 

Disclosures: E.E. Hecht , None; S.K. Barks, None; T.M. Preuss, None; J.K. Rilling, 

None; J.R. Votaw, None; L.A. Parr, None. 

Poster 





Support: CREST, JST 

JSPS Research Fellowships for Young Scientists 

Title: Simultaneous presentation of the facial and vocal emotion induces bimodal responses in 

the amygdala neurons 

Authors: *K. KURAOKA 1,2,3 , K. NAKAMURA 1,2 ; 

1 Dept Animal Models Human Dis, Natl. Inst. Neurosci, Kodaira, Japan; 2 CREST, JST, 

Kawaguchi, Japan; 3 JSPS, Chiyoda, Japan 

Abstract: The face and voice can independently convey the same information about people‟s 

emotional states. Therefore, when we see an angry face or hear an angry voice, we can perceive 

the person‟s anger. These two different sensory cues are interchangeable in this sense. The 

amygdala has been implicated in the processing of emotional expressions. In our previous study 

(Kuraoka & Nakamura, 2007), we recorded the activity of single neurons in the amygdala of the 

rhesus monkey while presenting video clips of species-specific emotional expressions and found 

that about one fifth of neurons, which responded to at least one of the monkey emotional 

expressions, maintained a good response when either the facial emotion or vocal emotion was 

presented alone. However, it remained unclear how those bimodal responses were generated. 

One possibility is that those bimodal responses were the result of simultaneous presentation of 

facial emotion and vocal emotion because the subject monkey had simultaneously seen facial 

emotion and heard vocal emotion many times during the experiment. Thus, in the present study,

we examined the neuronal activity in the monkey amygdala using the same stimulus set 

including 3 monkeys by 3 emotional expressions (aggressive threat, scream, and coo) as we used 

in the previous study. However, we presented facial emotion and vocal emotion separately and 

never presented both facial emotion and vocal emotion simultaneously in the present study. We 

found that 40 neurons of 101 amygdala neurons tested were monkey-responsive. Of these, only 

two neurons responded to both facial emotion and vocal emotion. The proportion (2/40, 5%) of 

neurons which showed the bimodal responses was significantly lower than that (16/79, 20%) in 

the previous study (chi-square test, P < 0.05). These results indicate that the bimodal responses 

of the neurons we found in the monkey amygdala are the result of the simultaneous presentation 

of facial emotion and vocal emotion. 

Disclosures: K. Kuraoka, None; K. Nakamura, None. 

Poster 

298. Neural Mechanisms of Reward: Self-Administration and Opioid Modulation 



Topic: F.03.d. Reward 


J.P. Bickell Foundation Grant 

Title: Cocaine self-administration changes D2-dopaminergic modulation of synaptic 

transmission in the bed nucleus of the stria terminalis 

Authors: *E. C. DUMONT, J. MACKENZIE-FEDER, N. J. BAILEY; 

Anesthesiol, Queen's Univ., Kingston, ON, Canada 

Abstract: Dopamine is a key neurotransmitter in goal-oriented behaviors and dysfunctions of 

dopaminergic systems contribute to pathologies such as addiction. The midbrain ventral 

tegmental area is rich in dopamine neurons and projects to several cortical and subcortical 

structures such as the prefrontal cortex, the amygdala, the striatum and the hippocampus. The 

bed nucleus of the stria terminalis (BST) also has a dense dopaminergic input that has received 

very little attention. Our laboratory recently demonstrated that dopamine in the BST is critical 

for operant behaviours towards natural (sucrose) or pharmacological (cocaine) rewards. The goal 

of the present study was to determine the synaptic mechanisms by which dopamine in the BST 

contributes to goal-directed behaviours. Brain slices were prepared from drug naïve Long-Evans 

rats or from rats trained to self-administer cocaine under a progressive ratio schedule of

einforcement. Whole-cell voltage clamp recordings were made from neurons located in the 

dorsolateral region of the anterior BST, site of the BST densest dopaminergic innervation. 

AMPA-mediated excitatory (EPSC) and GABAA-mediated inhibitory (IPSC) postsynaptic 

currents were electrically evoked by local fiber stimulation. Each electrical stimulus consisted of 

two pulses (50msec interval) at 0.1 Hz in order to obtain paired-pulse ratios (PPR) of the evoked 

PSCs. The effect of bath application of dopamine or dopaminergic agonists on the PSC 

amplitudes was measured. Dopamine produced a bidirectional modulation of EPSC and IPSC in 

both naïve and cocaine-dependent rats. Low concentration (10µM) of dopamine increased the 

amplitude of evoked PSCs through a post-synaptic mechanism (no change in PPR). In contrast, 

dopamine at high concentration (100µM) decreased PSCs by 45% on average, likely through a 

pre-synaptic mechanism (increase in PPR). The specific D1 dopamine receptor agonist SKF 

(1µM) and the D2 dopamine receptor agonist quinpirole (1µM) respectively replicated the 

effects of low and high concentrations of dopamine with the only exception that quinpirole no 

longer reduced EPSC in cocaine-dependent rats. Our study demonstrates that dopamine 

modulates synaptic transmission in the BST. The direction of the modulation depends both on 

the concentration of dopamine and whether dopamine activates D1 or D2 receptors in the BST. 

Although the effect of dopamine was maintained in cocaine-dependent rats, the D2 agonist 

quinpirole no longer reduced evoked EPSC suggesting modification in dopamine receptor 

function or expression. These observations might represent the mechanism by which dopamine 

in the BST contribute to reinforcement. 

Disclosures: E.C. Dumont , None; J. Mackenzie-Feder, None; N.J. Bailey, None. 

Poster 





Support: NIDA Grant DA021325 

Title: Hypocretin/orexin neurotransmission regulates dopamine responses to cocaine within the 

nucleus accumbens core 

Authors: J. L. LOCKE, R. A. ESPANA, D. C. S. ROBERTS, *S. R. JONES; 

Dept Physiol/Pharamacol, Wake Forest Univ. Sch. Med., Winston-Salem, NC 

Abstract: The hypocretins/orexins (HCRT) are comprised of two neuroexcitatory peptides, 

HCRT-1 and HCRT-2, which bind to two receptor subtypes, the HCRT 1 and HCRT 2 receptors.

Recent evidence indicates that the HCRT system modulates reinforcement and addiction 

processes, likely via actions on the mesolimbic dopamine (DA) system. For example, a highly 

selective HCRT-1 receptor antagonist, SB-334867, blocks reinstatement of extinguished 

cocaine-seeking and behavioral sensitization to cocaine. Further, HCRT increases DA cell firing 

and increases DA release in the nucleus accumbens (NAc). Finally, in self-administration and 

voltammetry studies from this lab, we have shown that SB-334867 reduces cocaine intake and 

attenuates cocaine-induced increases in voltammetry measures of phasic DA signaling in the 

NAc core. 

To further assess the relationship between the HCRT system and reinforcement processing, the 

current studies used microdialysis techniques to examine the extent to which SB-334867 

attenuates cocaine-induced increases in tonic DA signaling within the NAc core. Rats were 

pretreated with vehicle or SB-334867 (30 mg/kg) and then 40 minutes later given a single i.p. 

injection of cocaine (10mg/kg). Results indicate that in vehicle-pretreated rats cocaine produces 

an expected increase in extracellular DA levels that peak within 20 min of injection and return to 

near baseline levels within 1.5 hrs. In contrast, SB-334867 pretreatment reduces cocaine-induced 

elevations in extracellular DA throughout the experiment. These observations offer additional 

support to the hypothesis that HCRT neurotransmission participates in reinforcement and 

addiction processes through actions on the mesolimbic DA system. 

Disclosures: J.L. Locke, None; S.R. Jones , None; R.A. Espana, None; D.C.S. Roberts, 

None. 

Poster 





Support: R01 DA021325 

P50 DA06634 

Title: Hypocretin/Orexin neurotransmission is necessary for normal cocaine self-administration 

and dopamine signaling in the nucleus accumbens 

Authors: *R. A. ESPANA, J. L. LOCKE, D. C. S. ROBERTS, S. R. JONES; 

Dept Pharm & Physiol, Wake Forest Univ., Winston-Salem, NC

Abstract: The hypocretin/orexin (HCRT) system has recently been implicated in the regulation 

of reinforcement and addiction processes, likely via actions on the mesolimbic dopamine (DA) 

system. For example, HCRT infusions increase the firing rate of ventral tegmental DA neurons, 

and the HCRT1 receptor antagonist SB-334867 blocks reinstatement of extinguished cocaineseeking 

and locomotor sensitization to cocaine. In addition, we have shown that microdialysis 

measures of cocaine-induced elevations in tonic DA signaling are reduced in rats pretreated with 

SB-334867. 

To further assess the relationship between the HCRT system and reinforcement processing, the 

current studies sought to examine the extent to which HCRT neurotransmission regulates 

behavioral and neurochemical responses to cocaine. Specifically, these studies utilized: 1) a 

combination of discrete trials and progressive ratio self-administration procedures to assess 

whether the HCRT 1 receptor antagonist, SB-334867, reduces cocaine intake; and 2) in vivo fast 

scan cyclic voltammetry to test the extent to which SB-334867 infusions into the ventral 

tegmental area attenuate cocaine-induced changes in phasic DA signaling within the NAc core. 

Results indicate that SB-334867 reduces cocaine intake on both self-administration schedules 

and attenuates cocaine-induced increases in DA signaling when injected into the VTA. These 

observations offer additional support for the hypothesis that HCRT neurotransmission 

participates in reinforcement and addiction processes through actions on the mesolimbic DA 

system. 

Disclosures: R.A. Espana , None; J.L. Locke, None; D.C.S. Roberts, None; S.R. Jones, 

None. 

Poster 





Support: NIH grant DA14245 

NIH grant DA00436 

Title: Galanin modulates cocaine self-administration and locomotor activation 

Authors: *C. P. BRABANT 1,2 , A. S. KUSCHPEL 1 , R. C. STEINER 1 , M. R. PICCIOTTO 1 ; 

1 Yale Univ., New Haven, CT; 2 Univ. de Liège, Liège, Belgium

Abstract: Previous studies have found that endogenous galanin inhibits morphine reward. In the 

current studies, we determined whether galanin‟s effects were limited to opiates or would 

generalize to psychostimulants. We first characterized spontaneous locomotion and acute 

cocaine-induced hyperactivity (0, 5, 10 and 20 mg/kg, i.p.) in wildtype and galanin knock out 

(Gal-KO) mice. In a second experiment, we examined the acquisition of intravenous cocaine 

self-administration (0.75 mg/kg per infusion) in wildtype and Gal-KO mice under a fixed ratio 

(FR1). After acquisition, mice were tested with several doses of cocaine (0.03, 0.1, 0.3, 1 and 3 

mg/kg per infusion) to obtain a dose-response curve. The third experiment tested the effect of 

galnon, a non selective galanin receptor agonist that crosses the blood brain barrier, on cocaine 

self-administration behavior. Wildtype mice were trained to self-administer cocaine, as in the 

second experiment. Following acquisition of stable responding, mice were pretreated with 

galnon (2 mg/kg, ip) or vehicle prior to self-administration sessions. The first experiment 

demonstrated similar levels of spontaneous locomotor activity in wildtype and Gal-KO mice. 

However, Gal-KO mice were more stimulated by cocaine than control mice, suggesting that 

endogenous galanin inhibits cocaine-induced hyperlocomotion. Similarly, galanin appears to 

attenuate cocaine self-administration. Taken together, our results suggest that galanin inhibits the 

locomotor and rewarding effects of cocaine, demonstrating that this neuropeptide can affect 

responses to multiple categories of drugs of abuse, including both opiates and psychostimulants. 

This work was supported by grants DA14245, DA00436 from the National Institutes of Health. 

Christian Brabant is a research associate under contract with the Belgian National Funds for 

Scientific Research (FNRS, Belgium). 

Disclosures: C.P. Brabant , None; A.S. Kuschpel, None; R.C. Steiner, None; M.R. Picciotto, 

None. 

Poster 






Title: Effect of mesocorticolimbic microinjections of the kappa-opioid agonist U50,488 on 

intracranial self-stimulation in rats 

Authors: M. C. CHEN, A. PARSEGIAN, *W. A. CARLEZON; 

Dept Psychiat, Harvard Med. Sch., Belmont, MA

Abstract: Our group and others have shown that systemic administration of kappa-opioid 

receptor (KOR) agonists produces prodepressive-like effects in rats, including anhedonia 

(reduced sensitivity to reward). There is substantial evidence that this effect is mediated within 

the nucleus accumbens (NAc): KORs are located on the terminals of ventral tegmental area 

(VTA) dopamine neurons that innervate the NAc, and microinfusion of KOR agonists directly 

into this region reduces extracellular concentrations of dopamine. However, KOR stimulation in 

other regions may also contribute to these prodepressant effects. For example, KORs are located 

on the cell bodies of VTA dopamine neurons, and microinfusions of KOR agonists into the VTA 

decrease the firing rates of these neurons. KORs are also located on the terminals and cell bodies 

of prefrontal cortex (PFC) neurons that provide glutamate input to the mesolimbic system. Here, 

we examined the effects of microinjections of the selective KOR agonist U50,488 (U50) into the 

NAc, VTA, or PFC on brain stimulation reward using the intracranial self-stimulation (ICSS) 

test. We implanted rats with lateral hypothalamic (LH) stimulating electrodes and guide cannula 

aimed at the NAc, VTA, or PFC. The rats were trained and subsequently tested in a ratefrequency 

variant of the ICSS paradigm. Following assessment of daily baseline ICSS 

thresholds, rats received microinjections of U50 (1.0, 3.0, 10, and 30 µg/rat, or distilled water 

vehicle). Doses were administered in ascending order with at least 48 hr between drug tests. 

Microinjections of U50 into each of these regions elevated ICSS thresholds, suggesting that they 

decreased the rewarding impact of the LH stimulation. In the NAc and VTA, the thresholdelevating 

effects of U50 were prominent at low doses, whereas higher doses tended to have 

smaller effects. The threshold elevating effects were more dose-orderly in the PFC, although 

there appeared to be some variability related to the depth of the infusion, raising the possibility 

that stimulation of KORs in this region affects diverse populations of cells. Regardless, our data 

implicate all major elements of the mesocorticolimbic system in mediating the prodepressivelike 

effects of KOR agonists, consistent with previous work involving brain microinjections and 

place conditioning. 

Disclosures: M.C. Chen, None; W.A. Carlezon , None; A. Parsegian, None. 

Poster 





Support: Bradley University Special Emphasis Grant 

Title: The effects of social and environmental enrichment on the acquisition and reinstatement of 

cocaine self-administration in rats

Authors: *D. K. TAYLOR, K. KOTWICA, W. WAYMAN, G. RICHARD, C. WEIR, R. 

HENDERSON, T. E. KOELTZOW; 

Bradley Univ., Peoria, IL 

Abstract: Previous studies indicate that social or environmental enrichment is sufficient to 

influence spontaneous locomotor activity and various indices of dopamine function. The purpose 

of the present study is to assess the impact of social (SE) or environmental enrichment (EE) 

during development on the subsequent acquisition and reinstatement of operant cocaine selfadministration 

behavior. Preliminary findings indicate that SE rats exhibited a reduced locomotor 

response in an open field compared to rats in the EE and standard isolation (I) housing 

conditions. Similarly, SE rats were resistant to the reinforcing effects of cocaine as measured by 

the rate of cocaine self-administration acquisition, extinction and reinstatement. These findings 

indicate that social enrichment may serve as a protective risk factor against the vulnerability to 

addiction and relapse. On-going analysis aims to identify the neural correlates of SE that may 

account for these findings. 

Disclosures: D.K. Taylor , None; K. Kotwica, None; W. Wayman, None; G. Richard, 

None; C. Weir, None; R. Henderson, None; T.E. Koeltzow, None. 

Poster 





Support: Whitehall Foundation 

Title: Assessing the contributions of nucleus accumbens shell compartments to cocaine-seeking 

behavior in rats from amphetamine microinfusion 

Authors: *D. G. C. HILDEBRAND, A. TANG, K. M. MORIN, M. CHAN, J. R. STELLAR; 

Dept. of Psychology, Northeastern Univ., Boston, MA 

Abstract: The nucleus accumbens (NAc) is known to play a key role in brain reward processes. 

Most previous research has focused on two subregions of the NAc, the shell and the core. Recent 

anatomical and biochemical studies in rats have revealed distinct compartments of the NAc shell. 

Two particular areas of interest are the dorsal medial (DMS) and the intermediate zone (INT) 

compartments because they differ in expression of the immediate-early gene c-Fos and the longacting 

immediate-early gene ΓFosB after cocaine sensitization. These results suggest that each

compartment may play a different role in the reward and motivational factors underlying 

behavioral acts such as cocaine self-administration. The present study attempts to better 

understand the contributions of the DMS and the INT to producing drug-seeking behavioral 

responses. Rats are trained to intravenously self-administer cocaine, extinguished, and subjected 

to a reinstatement treatment consiting of an intracranial amphetamine (AMPH) microinfusion in 

either the DMS or the INT. This microinfusion is performed using a hands-free intracranial 

micropump system capable of reproducibly delivering nanoliter volumes while the subject is in 

the operant chamber. Initial results from low and high volume (300 nL and 900 nL of 0.1 

nmoles/nL AMPH, respectively) AMPH microinfusions show reinstatement behavior that 

respects the NAc shell anatomy compartmentalization. 

Disclosures: D.G.C. Hildebrand, None; A. Tang, None; K.M. Morin, None; M. Chan, 

None; J.R. Stellar, None. 

Poster 





Support: Robertson Fund of Emory University 

Title: Intraventricular serotonin does not affect the reward of ventral tegmental self-stimulation 

Authors: *D. B. NEILL 1 , A. COKER 2 ; 

1 Dept Psychology, 2 Neurosci. and Behavioral Biol. Program, Emory Univ., Atlanta, GA 

Abstract: Serotonin-selective reuptake inhibitors (SSRIs) are widely used in the treatment of 

depression. One of the hallmarks of major depression is anhedonia, a loss of the appreciation of 

pleasure. Pleasure in humans is often equated with reward in non-human animals. Paradoxically, 

both acute and chronic treatment with SSRIs appear to reduce reward, and thus perhaps pleasure, 

in tests with non-human animals. One of these tests is intracranial self-stimulation (ICSS), in 

which an animal performs a response to deliver electrical stimulation to a brain site via an 

implanted electrode. We examined the effect of a global increase in serotonin, via 

intraventricular administration, on autotitration ICSS in male Sprague-Dawley rats. Doses of 10 

and 20 µg serotonin HCl, dissolved in 10 µl artificial cerebrospinal fluid, were injected into the 

lateral ventricle contralateral to a bipolar ICSS electrode implanted in the ventral tegmental area. 

In our autotitration procedure, the intensity of brain stimulation declined 3 µa every 5th response 

on a “stimulation” lever; a single response on a second “reset” lever reset the intensity at the

stimulation lever back to the maximum for that rat. Serotonin administration lowered both reset 

intensity and responding on the stimulation lever. This pattern did not resemble that of cocaine 

(10 mg/kg, IP; lower reset intensity, increased responding), which increases ICSS reward, or the 

anxiogenic caffeine (40 mg/kg, IP; higher reset intensity; decreased responding), which 

decreases ICSS reward. We conclude that intraventricular serotonin neither increased nor 

decreased VTA ICSS reward as measured by autotitration. Rather, the serotonin seemed to result 

in “behavioral deactivation.” 

Disclosures: D.B. Neill , None; A. Coker, None. 

Poster 





Support: NIDA Grant 1F31DA024493-01 awarded to CJC 

Mellon Foundation Grant awarded to ALR 

Title: Essential value of food and cocaine in Fischer and Lewis rat strains: food is more 

reinforcing to Lewis rats, but cocaine is more reinforcing to Fischer rats 

Authors: C. J. CHRISTENSEN, S. J. KOHUT, S. HANDLER, A. SILBERBERG, *A. L. 

RILEY; 

Dept Psychology, American Univ., Washington, DC 

Abstract: F344 Fischer and Lewis inbred rat strains have been used as an animal model for a 

number of disorders, including drug abuse and addiction. In assessments with drugs of abuse, the 

two strains differ within a number of preparations used to measure the affective properties of 

these drugs, e.g., self administration, conditioned place preferences, conditioned taste aversions. 

In relation to self administration, rate of acquisition, total drug intake, response rate and 

progressive ratios have all been reported to vary by strain, suggesting genetic differences in drug 

reactivity and vulnerability. The purpose of the present study was to extend this analysis of the 

F344 and LEW rat strains by assessing their reactivity in a previously untested measure-demand 

analysis, which measures the degree to which individuals defend consumption at increasing 

costs. More specifically, we were interested in determining whether there was any strain by 

commodity interaction by assessing food and cocaine demand in both strains. In Experiment 1, 

Fischer and Lewis rats were trained to respond on a lever for food, following which a demand

curve was assessed by increasing the fixed-ratio (FR) requirement every three days according to 

a log scale. In Experiment 2, the conditions were the same as in Experiment 1 except separate 

rats were used and cocaine was used instead of food. Demand curves, consumption plotted as a 

function of FR, were normalized and fitted with the exponential model of demand. Results 

showed a commodity by strain interaction, i.e. food had a higher essential value in Lewis rats 

compared to Fischer rats, but the essential value of cocaine was lower in Lewis rats. These 

results suggest that Fischer strains might be the more vulnerable strain to drug abuse since food 

is less reinforcing and cocaine is more reinforcing (relative to Lewis rats). 

Disclosures: C.J. Christensen, None; A. Silberberg, None; A.L. Riley , None; S.J. Kohut, 

None; S. Handler, None. 

Poster 





Support: DA 09311 

Title: Glutamate and opioid systems in the nucleus accumbens regulate stress-induced 

suppression of feeding 

Authors: R. C. SPENCER, L. PASCAL, A. E. KELLEY, V. P. BAKSHI, *B. A. BALDO; 

Psychiat, Unvi Wisconsin Madison Sch. Med., Madison, WI 

Abstract: The nucleus accumbens (Acb) is a critical node in the forebrain network that regulates 

food intake and food-seeking behaviors. It has been shown that Acb opioid transmission 

augments measures of positive affect associated with palatable feeding, and preferentially 

increases intake of energy-dense sweetened foods and tastants. Moreover, repeated intra-Acb 

mu-opioid receptor stimulation sensitizes intake of standard rat chow. Blockade of AMPA-type 

glutamate receptors in the shell subregion of the Acb also augments feeding; however, in 

contrast to opioid effects, intra-Acb shell AMPA blockade does not display selectivity for 

palatable foods, and resembles stimulus-bound feeding elicited by electrical stimulation of the 

hypothalamus. A theory has been advanced that opioid transmission throughout the ventral 

striatum enhances the rewarding properties of consummatory feeding behaviors, while glutamate 

inputs to the Acb shell act as a switch to flexibly terminate feeding motor programs when 

necessitated by incompatible motivational states or external stimuli (such as stress or threat). The 

present studies examined the roles of Acb glutamate and opioid systems in the reduction of

feeding produced by predator stress. In the first set of experiments, hungry rats were tested in the 

presence of food and a cloth containing ferret odor (ferrets are natural predators of rats). Intra- 

Acb shell administration of the AMPA receptor antagonist, CNQX, nearly abolished exploratory 

behaviors directed at the cloth, greatly reduced latency to feed, and markedly increased food 

intake compared to saline-treated rats. Conversely, in hungry rats tested in the absence of ferret 

odor, intra-Acb shell infusions of AMPA (given remotely) immediately arrested feeding when 

administered at the onset of a feeding bout. In the second experiment, a sensitizing regimen of 

intra-Acb DAMGO (a mu-opioid agonist) pretreatments prevented the decrease in sweetened-fat 

intake produced by ferret exposure (DAMGO-pretreated rats did not receive DAMGO on the 

ferret-exposure day). Taken together, these results suggest that the Acb shell is a critical node for 

stress-related neural processing (as conveyed by glutamatergic inputs) to adaptively interrupt and 

diminish feeding behavior, and that general Acb opioid tone may modulate the ability of stress to 

impact food intake. 

Disclosures: R.C. Spencer, None; B.A. Baldo , None; L. Pascal, None; V.P. Bakshi, 

None; A.E. Kelley, None. 

Poster 





Support: NIH Grant MH09311 (B.A.B.) 

Title: Glutamate and opioid systems in the nucleus accumbens regulate stress-induced 

suppression of feeding 

Authors: *L. PASCAL 1 , V. P. BAKSHI 2 , B. A. BALDO 2 , R. C. SPENCER 1 , A. E. KELLEY 2 ; 

2 Dept. of Psychiatry, 1 Univ. Wisconsin, Madison, Madison, WI 

Abstract: The nucleus accumbens (Acb) is a ventral striatal region involved in reward processes 

including palatable feeding. Stimulation of the κ-opioid receptor in the Acb has been shown to 

increase the positive hedonic response to high-calorie palatable food, as well as increase intake 

of such foods in ad-libitum fed rats. Moreover, repeated intra-accumbens κ-opioid stimulation in 

rats sensitizes intake of standard rat chow. This experiment examined the effects of an acute 

psychological stress, predator exposure, on fatty food intake in rats after chronic opioid 

stimulation in the Acb core. Brief periods of ferret exposure (in which the rat is placed inside a 

protective cage that precludes physical contact but permits hearing, seeing, and smelling the

predator) have been shown previously to reduce feeding and increase defensive behaviors in the 

rat. The mu-opioid peptide agonist DAMGO (D-Ala2, NMe-Phe4, Glyol5-enkephalin) was 

chronically microinfused directly into the Acb core of male Sprague-Dawley rats. Separate 

groups of animals received 4 daily microinfusions of either DAMGO or saline into the Acb core 

10 minutes before being placed in a test chamber with 5 grams of sweetened fat. After 

completion of these four chronic treatment days, all rats received a sham injection and were 

allowed to eat sweetened-fat ad-libitum. Enhancement of baseline fat intake was not observed in 

DAMGO-pretreated rats. Two days later, sham-injected animals were exposed to the ferret 

predator for 5 minutes and given the opportunity to eat sweetened-fat. Control rats showed a 

marked decrease in ad-libitum sweetened-fat intake immediately after ferret exposure whereas 

the prior chronic κ-opioid receptor stimulation by DAMGO prevented this stress-induced 

anorexic effect. Ad-libitum fat intake returned to pre-ferret baseline levels in all rats on a 

subsequent test day. Hence, chronic intra-Acb DAMGO pretreatment fundamentally alters the 

behavioral response to predator stress. These results establish a role for the nucleus accumbens in 

regulating stress-induced anorexia and may imply that individual differences in the effects of 

stress on palatable food intake may relate to nucleus accumbens opioid tone. 

Disclosures: L. Pascal , None; V.P. Bakshi, None; B.A. Baldo, None; R.C. Spencer, 

None; A.E. Kelley, None. 

Poster 






Title: Hyperdopaminergic mice exhibit enhanced 'wanting' but normal modulation of 'liking' 

Authors: *G. M. SPROW 1 , S. V. MAHLER 1 , K. C. BERRIDGE 1 , J. A. BEELER 2 , X. 

ZHUANG 2 ; 

1 Psychology, Univ. Michigan, Ann Arbor, MI; 2 Neurobio., Univ. Chicago, Chicago, IL 

Abstract: Dopamine has long been thought to be a critical component of the natural reward 

pathway, although the exact role it plays in appetitive „wanting‟ versus hedonic „liking‟ remains 

undetermined. Mounting evidence points to a role of dopamine levels specifically in the 

„wanting‟ of rewards but neither the „liking‟ of them nor the „learning‟ about them. To further 

investigate this role, genetic and behavioral approaches were combined. Dopamine transporter

(DAT) knockdown mice, with only 10% normal DAT and thus chronically elevated extracellular 

dopamine levels, were subjected to a battery of tests to examine the role of dopamine in both the 

„wanting‟ and „liking‟ of sweet rewards. A limited access food intake paradigm using a highly 

palatable reward measured „wanting‟ and taste reactivity (TR) testing using intraoral cannulae, a 

novel procedure in mutant mice, was used to examine „liking.‟ For this general TR testing, three 

concentrations of sucrose and two concentrations of quinine were used to procure hedonic 

(pleasurable) and aversive (negative) reactions. Additionally, both a satiation (using soy milk, a 

pleasurable tastant) and a pharmacological manipulation (using diazepam, a benzodiazepine 

known to increase hedonic responding) were performed to examine whether DAT knockdown 

mice displayed normal modulation of hedonics. We found the hyperdopaminergic mice exhibited 

increased levels of „wanting‟, as measured by increased food intake and body weight over the 

course of the study. During TR testing, mutant mice showed normal (and in some cases 

decreased) hedonic reactions to a pleasurable sucrose taste as compared to wild-type mice; other 

trials showed normal modulation of hedonic reactions. These results suggest that dopamine 

selectively increases „wanting‟ without increasing „liking‟ or affecting either satiety- or 

diazepam-induced shifts in hedonics. 

Disclosures: G.M. Sprow, None; S.V. Mahler, None; K.C. Berridge, None; X. Zhuang, 

None; J.A. Beeler, None. 

Poster 







Title: Accumbens shell opioids enhance „wanting‟ for preferred and non-preferred reward cues 

Authors: *E. D. JACKSON, K. C. BERRIDGE; 

Psychology, Univ. of Michigan, Ann Arbor, MI 

Abstract: In the nucleus accumbens, dopamine appears to mediate the attribution of incentive 

salience „wanting‟ while mu-opioids modulate the attribution of hedonic „liking.‟ But can muopioids 

in nucleus accumbens also directly increase incentive salience „wanting,‟ similar to intraaccumbens 

dopamine? We report that accumbens shell MOR activation increased „wanting‟ in

two tests of incentive salience: autoshaping and conditioned reinforcement. 

During autoshaping, Sprague-Dawley rats received bilateral microinjection of the MOR agonist 

Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol-enkaphalin (DAMGO, 0.1ug per side) or vehicle 

immediately prior to each test day. Rats were exposed to six days of 25 non-contingent pairings 

of the insertions of a lit, retractable lever with a 45mg sucrose pellet, delivered into a recessed 

food cup immediately after lever retraction. DAMGO increased consummatory behaviors 

directed at both a preferred and non-preferred food conditioned stimuli (CS). 

In conditioned reinforcement testing, the retractable lever was moved to the opposite wall and 

infrared ports were placed on either side. Nose pokes in the active port initiated a brief insertion 

of the lever, while pokes in the inactive port were recorded but did not generate a response. 

DAMGO elevated the conditioned reinforcement value of lever presentation in all animals. 

Our findings indicate that accumbens shell DAMGO increased the motivational magnet 

properties of a CS paired with food. Additionally, DAMGO boosted the conditioned 

reinforcement value of a food CS in a novel instrumental task, even in animals that did not prefer 

this cue during autoshaping. This indicates that accumbens shell MOR activation can directly 

elevate 'wanting,' amplifying the incentive salience of both preferred and non-preferred reward 

cues. 

Disclosures: E.D. Jackson, None; K.C. Berridge, None. 

Poster 






NIDA Grant DA015188 

Title: Cannabinoid enhancement of sensory pleasure is blocked by an opioid antagonist in 

nucleus accumbens 

Authors: *M. R. MITCHELL, S. V. MAHLER, K. C. BERRIDGE; 

Psychology, Univ. of Michigan, Ann Arbor, MI 

Abstract: Cannabinoid drugs stimulate the intake of food, especially palatable foods. A recent 

study determined that stimulation of a dorsal medial accumbens shell „hedonic hotspot‟ with the 

endocannabinoid anandamide enhanced both food intake „wanting‟ and hedonic „liking‟ or

intraorally administered sucrose solution. Since mu (µ) opioid stimulation of a similar dorsal 

accumbens shell hotspot also enhances „liking,‟ and cannabinoids and opioids interact from the 

behavioral to the molecular level, we asked if the opioid antagonist naloxone would block 

„liking‟ enhancements by anandamide in the nucleus accumbens. We used a taste reactivity 

paradigm, which measures the effects of brain manipulations on characteristic hedonic or 

aversive orofacial reactions displayed by many species, from rats to humans, to intraorally 

administered tastes. Two groups of rats were tested: males (n=15), and ovariectomized females 

(n=6). All rats were given 0.2µl microinjections, to the medial accumbens shell, of vehicle, 

anandamide alone, naloxone alone, and anandamide mixed with naloxone in randomized order 

over four testing days. We examined taste reactivity to a 1mL of 1% sucrose solution orally 

infused at 30, 45, and 60 minutes after drug injection. We found that anandamide enhanced 

hedonic reactions to sucrose compared to vehicle, while this increase was totally abolished by 

simultaneous co-infusion of naloxone. Naloxone itself had no effect on hedonic reactivity to 

sucrose. These data support the idea that, within the nucleus accumbens, cannabinoid and opioid 

circuits interact to modulate hedonic „liking‟ responses to a sweet reward. These findings have 

implications for understanding the neural substrates of sensory pleasure involved in disorders 

such as addiction and obesity. 

Disclosures: M.R. Mitchell, None; S.V. Mahler, None; K.C. Berridge, None. 

Poster 





Title: Where in the ventral pallidum does neural inactivation cause aversive “disliking” and loss 

of reward? 

Authors: *C.-Y. HO, K. C. BERRIDGE; 

Univ. Michigan, Ann Arbor, MI 

Abstract: Ventral pallidum (VP) has been shown to be important for mediating „liking‟ and 

„wanting‟ of natural rewards, such as food, and of drugs. Lesions of VP or inactivation of VP by 

GABA agonist microinjections lead to a decrease in appetitive reward consumption or „wanting‟ 

along with a decrease of hedonic reward “liking” and an increase of aversive reward “disliking”. 

However, it is unclear what substrate causes the loss of hedonic reward “liking” and the 

replacement of aversive reward “disliking”. The goal of the present study is to identify the 

specific “disliking” site in basal forebrain for lesions or local inactivation, with special attention

to the VP. Here we began to functionally map the critical aversive sites for production of 

„disliking‟ induced by excitotoxin lesion, or by microinjection of inactivating agents: lidocaine 

or GABA agonists. In order to do so, we measured rats‟ aversive “disliking‟ response induced by 

sucrose and quinine after microinjection of muscimol/baclofen cocktail, lidocaine, or one of the 

two excitotoxins (quinolinic acid or ibotenic acid). We then functionally mapped the behavioral 

effects onto the corresponding localization of microinjection. We aimed to identify the most 

critical site at which the drug caused significant aversion enhancement. Our preliminary data 

show that VP and nearby regions are crucial to reward processing, and local inactivation there 

causes production of aversive „disliking‟. This finding may extend our understanding of the role 

of VP in reward processing and carry implications for understanding hedonic dysfunction in drug 

addiction, obesity and mood disorders. 

Disclosures: C. Ho , None; K.C. Berridge, None. 

Poster 





Support: NIH Grants MH63649 


Title: Feeding and sensorimotor suppression by metabotropic glutamate receptor blockade in 

medial shell of nucleus accumbens 

Authors: *J. M. RICHARD, K. C. BERRIDGE; 

Psychology, Univ. Michigan, Ann Arbor, MI 

Abstract: Metabotropic glutamate receptors are the target of many new drugs aimed at clinical 

problems relevant to both affect and motivational processes, including drug addiction and 

withdrawal, anxiety, and schizophrenia. Here we tested whether blockade of Group II 

metabotropic glutamate receptors in medial shell of nucleus accumbens alters affective and/or 

motivated behaviors. Microinjection of mglu2/3 antagonist LY341495 (10 κg per .5 κl) reduced 

eating behavior and food intake without reducing overall locomotion in the form of cage crosses 

and rears. Additionally, we tested the effect of LY341495 microinjections using the taste 

reactivity test. We found that mglu2/3 blockade mildly reduced both positive and negative

eactions to tastes, possibly implicating a sensorimotor effect, as also indicated by a reduction in 

grooming behavior. 

Disclosures: J.M. Richard, None; K.C. Berridge, None. 

Poster 






Title: Amphetamine effects on ventral pallidal coding of cue-triggered incentive salience and 

hedonia 

Authors: *C. A. ITOGA 1 , K. C. BERRIDGE 2 , J. W. ALDRIDGE 3 ; 

1 Dept Neurosci, 2 Psychology Dept., 3 Dept. of Neurol., Univ. Michigan, Ann Arbor, MI 

Abstract: Appetitive responses to drug cues are thought to be an important feature of drug 

addiction, and cues that act as motivational magnets can pose a risk for drug relapse. The ventral 

pallidum (VP) is responsive to reward-associated cues. Dopamine increases the “wanting” of 

rewards, as supported by the incentive-sensitization theory of addiction (Robinson & Berridge 

1993) while opiates enhance both the “wanting” and “liking” of rewards (Pecina & Berridge 

2005). In this study, our aim was to determine the effects of dopamine agonists on VP neural 

coding of cues for natural rewards, and correlated changes in “liking” and/or “wanting” taste 

reactions. 

Rats were trained in a Pavlovian association task in which all stimuli were taste infusions 

directly into the mouth. Two cues (water, 0.1 mL each) were followed by reward (9% sucrose 

solution, 0.1mL). The first cue provides predictive information of eventual reward delivery. The 

second cue is thought to have the most incentive value, as it is temporally closer to the reward. 

Changes in hedonic value were detected by behavioral taste reactions. Water on its own elicits 

primarily neutral taste reactions, but as a taste cue associated with reward, its hedonic value may 

be transformed to evoke positive taste reactions. 

Amphetamine (1 mg/kg; ip) was used to boost dopamine levels. We hypothesized that 

amphetamine would shift dominant VP responses to the incentive cue, reflecting a change in 

motivation without altering hedonic responses. In rewarded trials in recordings from 10 rats 

(total of 92 cells), amphetamine, compared to vehicle controls, increased the proportion of 

responsive cells to the incentive (second) cue (54% vs. 35%) without altering the size of the

neural population responsive to predictive cues (77% vs. 76%). Also, amphetamine, compared to 

vehicle controls, resulted in an increase in the proportion of cells responsive to rewards (74% vs. 

57%). Our behavioral analysis indicates that dopamine activation does not increase hedonic 

reactions. The increase in activation to the incentive cue, with no alteration in hedonic reactions, 

suggests that neurons in VP are encoding incentive salience and that dopaminergic mechanisms 

are involved in amplifying incentive value. 

Disclosures: C.A. Itoga, None; K.C. Berridge, None; J.W. Aldridge, None. 

Poster 





Support: NIDA R01 DA015188 (KCB) 

NIDA NRSA DA021481 (SVM) 

NIMH R01 MH63649 

Title: You can‟t always „like‟ what you „want‟: dissociation of „wanting‟ and „liking‟ with 

central amygdala opioid stimulation 

Authors: *S. V. MAHLER, K. C. BERRIDGE; 

Psychology, Univ. Michigan, Ann Arbor, MI 

Abstract: The amygdala is involved in appetitive emotion and learning, and stimulating µ opioid 

receptors in the central nucleus of amygdala (CeA), but not the nearby basolateral nucleus 

(BLA), enhances food intake, autoshaping, and Pavlovian to instrumental transfer measures of 

cue-triggered „wanting.‟ The hedonic impact of a received reward („liking‟) is processed 

separately from „wanting,‟ however, so here we asked whether opioid stimulation with the µ 

agonist DAMGO (0.1µg/0.2µl) would enhance „liking‟ as well as „wanting‟ in amygdala. The 

taste reactivity paradigm allows measurement of the hedonic impact of intraorally administered 

nice or nasty tastes by the quantification of evolutionarily conserved hedonic (eg. tongue 

protrusions) or aversive (eg. gapes) facial responses. Rats received 4 days of testing in which 

they received microinjections of either DAMGO or vehicle, followed by 15 and 30min later by 

intraoral infusions of either sucrose or quinine. By analyzing Fos plumes around microinjection 

sites, we also plotted functional drug spread to determine the localization of DAMGO effects in

the amygdala. Surprisingly, sucrose „liking‟ was consistently reduced by stimulating opioids in 

either the CeA or BLA, while aversive responding to quinine was not affected by DAMGO in 

either structure. Spontaneous food intake behavior was measured immediately following taste 

reactivity testing on the same days in the same rats, and as expected, DAMGO robustly enhanced 

food intake „wanting.‟ These data provide strong evidence for a dissociation of central amygdala 

opioid-induced „wanting‟ and „liking‟ for rewards. Therefore, like accumbens dopamine, CeA 

opioids specifically enhance „wanting‟ of rewards and their cues, even as they simultaneously 

make these rewards less „liked‟ once they are received. 

Disclosures: S.V. Mahler, None; K.C. Berridge, None. 

Poster 





Support: University of Missouri Research Board 

Title: Central opioid and cannabinoid interactions and their influence on palatability-driven 

feeding behavior 

Authors: *M. J. WILL 1 , K. E. PARKER 2 , J. G. MCCALL 2 , C. E. P. KELLEY 2 , A. E. 

GILLMAN 2 , D. K. MILLER 2 ; 

1 Dept Psych - Life Sci. Cntr, Univ. Missouri-Columbia, Columbia, MO; 2 Psychology, Univ. of 

Missouri, Columbia, MO 

Abstract: The neural system most clearly implicated in the rewarding actions of both drugs of 

abuse and food is the ventral striatum and its associated corticostriatal circuitry. In order to 

understand and successfully treat the diseases of addiction and obesity, it is necessary to 

determine the exact neurochemical nature of their underlying mechanisms. The present 

experiments investigated the central interactions of the cannabionoid and opioid systems within 

an established model of hedonically-driven binge eating of palatable food. Specifically, this 

model involves activation of the accumbens opioid system with the mu opioid agonist D-Ala2, 

NMe-Phe4, Glyol5-enkephalin (DAMGO), which produces a robust binge-like consumption of 

preferentially palatable diets such as those that are high in fat and/or sugar. Interestingly, it has 

also been demonstrated that the endocannabinoid system produces many of the same behavioral 

effects as the opioid system, such as increased feeding behavior, hypolocomotion, and 

reinforcement of drug self-administration behavior. Further evidence demonstrates that the

ehavioral influences produced by activation of the endocannabinoid system are partially 

dependent on co-activation of the opioid system. The present study explored the involvement of 

corticostriatal cannabinoids in mediating the palatability-driven feeding behavior, and 

characterized its interactions with corticostriatal opioids. 

Disclosures: M.J. Will , None; K.E. Parker, None; C.E.P. Kelley, None; J.G. McCall, 

None; A.E. Gillman, None; D.K. Miller, None. 

Poster 





Support: University of Missouri 

Title: Intra-accumbens adenosine and its role in mediating palatable food intake: interactions 

with striatal opioids 

Authors: *C. E. KELLEY 1,2 , M. M. THAKKAR 3 , M. J. WILL 1 ; 

1 Pscyhological Sci., Univ. of Missouri, Columbia, MO; 2 Neurosci., Pennsylvania State 

University, Hershey Med. Ctr., Hershey, PA; 3 Neurol., Univ. of Missouri, Columbia Harry S. 

Truman Mem. Veterans' Hosp., Columbia, MO 

Abstract: Adenosine receptors located within the nucleus accumbens are believed to have a 

significant modulatory role over a variety of neurotransmitters and their associated psychomotor 

and feeding behaviors. However, it is currently unknown to what extent A1 and A2a receptors 

differently contribute to the extensively studied intra-accumbens opiate-induced feeding 

behavior. The present study investigated the intra-accumbens effects of selective A1 and A2a 

receptor agonists and antagonists alone and when co-administered with the µ-opioid agonist D- 

Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO; 2 doses) or the non-selective opiate antagonist 

naltrexone (20 µg/0.25 µl/side). Adult male Sprague-Dawley rats were implanted with bilateral 

cannula aimed at the nucleus accumbens core. The influence of the A1 receptor agonist 2- 

Chloro-N 6 -cyclopentyladenosine (CCPA; 4 doses), the A2a receptor agonist 2-p-(2carboxyethyl)phenethylamino-5‟-N-ethylcarboxyamidoadenosine 

(CGS 21680; 3 doses), and the 

A2a receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1propargylxanthine 

phosphate disodium salt (MSX-3; 2 doses) on feeding behaviors. CCPA 

administration had no effect on baseline or DAMGO-induced feeding or activity however 

significant changes were observed in response to both the A2a agonist and antagonist. CGS

21680 administration decreased baseline activity, but not feeding, and significantly increased 

DAMGO-induced feeding. Alternatively, MSX-3 produced robust feeding and activity increases 

and facilitated DAMGO-induced behaviors and these were blocked by naltrexone. Overall, it 

was observed that A2a receptors exerted stronger modulatory effects than A1 receptors on 

consumption and activity, both alone and when co-administered with DAMGO. Indeed, it is 

expected that A2a receptors, being more densely localized throughout the accumbens, would 

have greater impact on behaviors mediated by the nucleus accumbens than A1 receptors. In 

summary, these results suggest an intriguing role for striatal adenosine in mediating baseline and 

striatal opioid-mediated behaviors. 

Disclosures: C.E. Kelley, None; M.M. Thakkar, None; M.J. Will, None. 

Poster 





Title: D2 receptor manipulations in the sublenticular central extended amygdala affect 

responding for brain stimulation reward 

Authors: B. FARRAL, *M. A. WARACZYNSKI; 

Dept Psychol, Univ. Wisconsin Whitewater, Whitewater, WI 

Abstract: Past work in our laboratory has shown that the central extended amygdala (EAc), 

especially its sublenticular aspect (SLEAc), is important to the reward effectiveness of medial 

forebrain bundle (MFB) stimulation. The medium spiny neurons abundant in this macrostructure 

receive dopaminergic input from the A8-A10 cell groups. Given the well-documented role of 

dopamine in reward function in the neighboring nucleus accumbens we examined whether 

dopamine is important to reward function in the SLEAc. Specifically, we investigated the effects 

on MFB self-stimulation of agonizing and antagonizing D1 and D2 dopamine receptors. Male 

Long Evans rats received stimulation electrodes in the rostral and caudal MFB and guide 

cannulae in the SLEAc both ipsi- and contralateral to the stimulation sites. The stimulation‟s 

reward efficacy was assessed using the rate-frequency curve shift technique. Relative to noninjection 

baseline conditions, 0.50 µl infusions of 5.0 κg of SKF38393 (D1 agonist) and of 2.0 

κg of SCH23390 (D1 antagonist) were as ineffective as saline in altering either the rats‟ 

maximum rate of responding for stimulation or the frequency required to maintain half-maximal 

response rates. The absence of any D1 drug effects may reflect the relative paucity of D1 

receptors in the EAc compared to their abundance in neighboring striatopallidum. On the other

hand injections of 3.0 κg of eticlopride (D2 receptor antagonist) modestly enhanced maximum 

rates while injections of 10 κg of quinpirole (D2 receptor agonist) did the opposite. Eticlopride 

also slightly decreased the frequency required to maintain half-maximal responding. Maximum 

rate shifts were more pronounced than required frequency shifts, suggesting a stronger role for 

SLEAc D2 receptors in governing responding for rewards than in evaluating the stimulation‟s 

reward efficacy. The D2 drugs were effective only when they were injected contralateral to the 

stimulation site, possibly because the MFB stimulation may have directly driven neural activity 

in the ipsilateral SLEAc that overcame any effects of manipulating D2 receptors. The modest 

nature of the D2 drugs‟ effects might indicate that dopaminergic input to the SLEAc is secondary 

to, and perhaps interacts with, a more primary input such as glutamate in controlling reward 

functions. 

Disclosures: B. Farral, None; M.A. Waraczynski , None. 

Poster 





Support: CIHR 

Title: OSU-6162, a dopamine stabilizer, acts at both pre- and post-synaptic receptor sites to alter 

brain stimulation reward and locomotor activity 

Authors: *F. BENALIOUAD 1,2 , P.-P. ROMPRÉ 2,3 , S. KAPUR 4 ; 

1 Ctr. Fernand-Seguin, Montreal, QC, Canada; 2 Physiol., 3 Psychiatry, Univ. of Montreal, 

Montreal, QC, Canada; 4 Psychiatry, King's Col. London - Univ. of London, London, United 

Kingdom 

Abstract: OSU-6162 belongs to a new class of drugs named dopamine stabilizers on the basis of 

their ability to maintain a certain homeostasis of the dopamine function. This molecule displays 

specificity for dopamine receptors and acts as a partial agonist at the type 2 (D2) receptor (Ki = 

447 nM for D2 receptors and Ki > 1µM for other receptors). Previous studies showed that OSU- 

6162 increases locomotor activity in rats habituated to the testing environment but reduces 

amphetamine-induced locomotion. In a previous study, we showed that OSU-6162 produced a 

dose-orderly attenuation of operant responding for brain stimulation reward, a dopamine 

sensitive behaviour. This attenuation effect suggests that OSU-6162 acts at both pre-synaptic and 

post-synaptic dopamine receptor sites. This study was aimed at testing this hypothesis.

We have studied the effect of OSU-6162 on quinpirole-induced changes in brain stimulation 

reward and in spontaneous locomotor activity. We also compared the effect of OSU-6162 to that 

of haloperidol, a prototypical dopamine antagonist. Male Sprague-Dawley rats were trained to 

produce an operant response (nose-poke) in order to obtain a medial forebrain bundle electrical 

stimulation. Once training was completed, we compared the effectiveness of OSU-6162 (10 

mg/kg sc) and haloperidol (0.010 and 0.025 mg/kg, sc) at blocking the alteration of reward 

induced by a dose of quinpirole (0.15 mg/kg sc) that produces a pre- and post-synaptic effect. 

The pharmacological treatment was also tested on other groups of naïve rats on locomotor 

activity. Results have shown that at the dose tested, quinpirole produced a time-dependent effect 

on reward which consists of a suppression followed by a slight facilitation. The facilitation, but 

not the suppression, effect was attenuated by OSU-6162 and by haloperidol. However, on 

spontaneous locomotor activity both drugs blocked the suppressant effect of quinpirole. Timecourse 

analysis of the changes in locomotor activity suggests that the lack of effect of OSU-6162 

and haloperidol on quinpirole induced suppression of reward was due to the poor time resolution 

of the curve-shift paradigm. These results demonstrate that OSU-6162 interacts at both pre- and 

post-synaptic receptor sites to alter dopamine dependent behaviors in a manner consistent with a 

functional antagonism. 

Disclosures: F. Benaliouad , None; P. Rompré, None; S. Kapur, None. 

Poster 





Support: CIHR 

NSERC 

Title: NMDA receptor transmission in the medial prefrontal cortex modulates the encoding of 

opiate-related reward information through amygdala and dopamine-dependent inputs 

Authors: *S. F. BISHOP 1 , S. R. LAVIOLETTE 2 ; 

1 Neurosci., Univ. Western Ontario, London, ON, Canada; 2 Anat. and Cell Biol., Univ. of 

Western Ontario, London, ON, Canada 

Abstract: Recent theories emphasize the significance of functional interactions between 

emotional associative learning and memory formation mechanisms involving the dopamine (DA)

and glutamate (GLUT) systems during the drug addiction process. It has been demonstrated that 

neurons in the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) encode 

emotionally significant conditioned learning and functionally interact with DA and GLUT 

systems to modulate emotional associative learning. Considerable evidence implicates both the 

NMDA and AMPA GLUT receptor subtypes in the processing of opiate-related motivational 

information. Furthermore, NMDA antagonist drugs such as ketamine and PCP strongly modulate 

emotional processing and lead to increased GLUT and DA release within the mPFC, presumably 

by modulating inhibitory versus excitatory network activity within the mPFC. To examine the 

potential role of GLUT receptor substrates during the encoding of morphine reward in rats, we 

performed a series of behavioural pharmacology experiments wherein we performed bilateral 

microinfusions of the NMDA receptor antagonist, AP-5, into the rat mPFC, prior to systemic 

administration of morphine during a conditioned place preference procedure (CPP). We found 

that blockade of mPFC NMDA with AP-5, dose-dependently potentiated morphine reward 

learning as demonstrated by potentiated morphine CPP over an order of magnitude morphine 

dose range (0.05-5.0 mg/kg). Similarly, intra-mPFC micro-infusions of the AMPA receptor 

antagonist, DNQX (0.1-1.0 κg/0.5 κl) induced a potentiation of morphine‟s rewarding properties. 

Although AP-5 potentiates morphine reward learning during the acquisition phase of training, it 

does not appear to affect the extinction of opiate addiction in these rats. We further examined the 

potential role of BLA GLUT inputs to the mPFC by pharmacologically inactivating the BLA 

with a GABA-A receptor agonist, muscimol (500 ng/0.5 κl), prior to mPFC AP-5 microinfusions 

and systemic injection of a sub-reward threshold dose of morphine (0.05 mg/kg). Blockade of 

BLA>mPFC input completely blocked the ability of mPFC NMDA receptor modulation to 

potentiate morphine reward, demonstrating that a BLA-dependent GLUT input to the mPFC acts 

upon an NMDA receptor population to modulate encoding of opiate-reward related associative 

learning and memory. Finally, intra-mPFC co-administration of a mixed DA D1/D2 antagonist 

(α-flupenthixol; 0.3-3.0 κg) with AP-5, blocked the reward potentiating effects of AP-5, 

demonstrating an important functional link between mPFC NMDA and DA receptor substrates 

during the encoding of opiate-related reward information. 

Disclosures: S.F. Bishop, None; S.R. Laviolette, None. 

Poster 





Support: NIDA

NIMH 

Title: The N-terminal domain of ΓFosB is functional in behavioral responses 

Authors: *Y. OHNISHI 1,2 , Y. H. OHNISHI 1,2 , A. GRAHAM 1 , C. COWAN 1 , C. STEFFEN 1 , S. 

BIRNBAUM 1 , Y. NAKABEPPU 3 , E. J. NESTLER 1,2 ; 

1 Psychiatry, UT southwestern Med. Ctr., Dallas, TX; 2 Dept of Neurosci., Mount Sinai Sch. of 

Med., New York, NY; 3 Div. of Neurofunctional Genomics, Med. Inst. of Bioregulation, 

Fukuoka, Japan 

Abstract: We have shown that ΓFosB accumulates in a region-specific manner in brain after 

chronic exposure to several types of stress, electroconvulsive seizures, or drugs of abuse, and 

that ΓFosB enhances drug reward and promotes antidepressant responses. In the present study, 

we investigated unique variants of ΓFosB, studies which will provide novel insight into the role 

of this transcription factor in molecular and behavioral plasticity. 

The fosB gene has an exonic intron in exon 4/5, which allows alternative splicing and the 

encoding of two distinct mRNAs, FosB and ΓFosB. The latter is a truncated form of FosB 

lacking 101 aa at its C-terminal transactivation domain. In addition, the fosB gene has three 

alternative translational initiation sites in exon 2. This means that, together, the fosB gene has the 

potential to express eight protein products, FosB and its 3 N-terminal truncations, termed 

Γ1FosB, Γ2FosB, Γ3FosB, plus ΓFosB and its 3 N-terminal truncations, Γ1ΓFosB, Γ2ΓFosB, 

and Γ3ΓFosB. 

Due to translational efficiency, the main products of the fosB gene in brain are FosB, ΓFosB, and 

Γ2ΓFosB. Importantly, both ΓFosB and Γ2ΓFosB accumulate in response to repeated or 

prolonged stimuli due to their extraordinary stability. While ΓFosB can act as a transcriptional 

activator or repressor both in vitro and in vivo, the transcriptional potential of Γ2ΓFosB remains 

unknown. 

We have established a new mouse line, in which the 3‟ UTR of the fosB gene has been truncated. 

3‟ UTRs can be very important for both mRNA stability and translation. For this reason, in these 

mutant mice (termed fosB nA/nA for non-accumulating), the fosB gene expresses no FosB, normal 

baseline levels of ΓFosB but no further induction of this variant, and higher levels of Γ2ΓFosB. 

In contrast, we have already established fosB d/d mice in which the fosB gene expresses no FosB 

and higher levels of both ΓFosB and Γ2ΓFosB. The main difference in fosB gene expression 

between these two mouse lines is whether ΓFosB is able to accumulate or not, suggesting that 

the molecular and behavioral differences between fosB d/d and fosB nA/nA mice will disclose the 

functional significance of the N-terminal domain of ΓFosB. Behavioral characterization of these 

mice, which will be reported at the meeting, suggests that the N-terminal domain of ΓFosB is 

functionally important and influences behavioral responses mediated by the protein. 

Supported by NIDA and NIMH. 

Disclosures: Y. Ohnishi, NIDA, B. Research Grant (principal investigator, collaborator or 

consultant and pending grants as well as grants already received); NIMH, B. Research Grant 


received); Y.H. Ohnishi, None; A. Graham, None; C. Cowan, None; C. Steffen, None; S. 

Birnbaum, None; Y. Nakabeppu, None; E.J. Nestler, None.

Poster 




Topic: F.02.a. Attention 

Support: ZonMw TOPgrant 91206148 

Title: Operant alcohol self-administration and five-choice reaction time task performance in 

Wistar rats exposed to chronic alcohol during adolescence 

Authors: J. A. WOUDA 1 , T. PATTIJ 1 , A. B. SMIT 2 , A. N. M. SCHOFFELMEER 1 , *T. J. DE 

VRIES 1 ; 

1 Anat. & Neurosci, CNCR VU&VUmc, Amsterdam, Netherlands; 2 Mol. and Cell. Neurobio., 

CNCR VU, Amsterdam, Netherlands 

Abstract: Adolescence is a transition phase that can be characterized by increased risk taking, 

increased novelty seeking and intensified peer relationships in both humans and rats. These 

behavioral changes are accompanied development of prefrontal cortex and hippocampus, 

extensive synaptic pruning throughout the brain, and maturation of several neurotransmitters and 

receptors towards adult levels and subunit composition. 

Adolescence is also often the age of onset of alcohol use. In the last two decades an alarming 

trend is seen of increased alcohol intake among youth in this unique developmental period. As 

alcohol is known to affect many of the changing neurological systems it is important to 

investigate the effect of adolescent alcohol use on adult behavior. The present study was 

designed to investigate the effects of adolescent alcohol exposure on disturbances in motivational 

and cognitive behavior in later life as assessed by operant alcohol self-administration (SA) and 

five-choice serial reaction time task (5CSRTT) performance. 

Male Wistar rats were exposed to either water/water, water/ 0.2% saccharin, or10%alcohol /0,2% 

saccharin in a two bottle free choice paradigm during either postnatal day (PND) 34-43 (periadolescents) 

or PND 60-69 (post-adolescents). Ethanol intake was between 3-6 g/kg/day. At 

adult age, 91 days after the last treatment day, rats were either trained to self administer a 10% 

alcohol solution (EXP1) or on 5CSRTT performance (EXP2). 

Exp1: All groups acquired SA at the same pace and no difference was found in performance 

during between session progressive ratio or extinction training. A cue-induced relapse test 

revealed a borderline significant reduction of reinstatement in alcohol treated peri group 

compared to water treated animals. 

Exp2: No treatment effects were found on baseline performance in the 5CSRTT. However, a 

reduced correct response latency in post-adolescent treated animals compared to peri-adolescent

animals was observed. 

A history of chronic alcohol exposure during adolescence had no effect on acquisition, 

motivation, extinction and reinstatement of operant self administration of adult Wistar rats or on 

performance in 5CSRTT. The age related effect on response latency might be attributed to 

differences in neurobiology at time of treatment. 

Adolescent binge-like alcohol exposure has been reported to have more detrimental behavioural 

effects than chronic exposure. Therefore, effects of adolescent binge-like exposure on SA and 

5CSRTT performance need to be assessed. 

Disclosures: J.A. Wouda, None; T.J. De Vries , None; T. Pattij, None; A.N.M. Schoffelmeer, 

None; A.B. Smit, None.

[Abstract Title]. - Society for Neuroscience

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