Book of abstracts - British Neuroscience Association

1.0
Paths to recovery: Modulating Putative Depression Circuits using
Deep Brain Stimulation
Mayberg HS
Emory University School of Medicine, Atlanta GA USA
Critical to development of deep brain stimulation (DBS) as a novel
therapy for treatment resistant depression, has been the evolving
understanding of brain systems mediating normal and abnormal mood
states and the ongoing, systematic characterization of neural
substrates mediating successful and unsuccessful response to other
antidepressant interventions. Based on previous work implicating the
subgenual cingulate (Cg25) and its functional connections to specific
paralimbic, cortical and subcortical regions in the pathophysiology of
depression and antidepressant response mechanisms, we tested the
use of chronic high frequency DBS to modulate Cg25 connectivity in
patients with treatment resistant depression. The theoretical and datadriven
foundation for developing this new procedure as well as clinical,
neuropsychological and imaging findings from the first experimental
patient cohort will be presented.
2.0
Fearful brains in an anxious world.
LeDoux J
New York University, NY, USA
Fear and anxiety are normal adaptive functions of the brain. However,
about 20 million Americans (15%) suffer from disorders involving excess or
inappropriate fear and anxiety. Included are phobic, panic, posttraumatic
stress, generalized anxiety, and obsessive compulsive disorder. Research
on the brain mechanisms of fear and anxiety gives us clues about how
these normally function, what changes in the brain when they malfunction,
and how specific malfunctions might be most effectively treated. Better
control of inappropriate and excess fear and anxiety would improve health
and well-being, both for individuals and societies.
3.01
Regulation of de novo axonal growth
Burk K, Davies A
Cardiff School of Biosciences, Museum Avenue, CF10 3US , Cardiff
Virtually all studies on the molecular regulation of axonal growth have
been carried out on axotomised neurons that regenerate axons in
culture. The technical difficulty of studying newly differentiated
neurons that initiate axonal growth for the first time in culture has
meant that developmentally relevant de novo axonal growth has been
almost ignored. Numerous studies have shown that neurotrophic
factor-dependent axonal regeneration from embryonic and postnatal
neurons is regulated by a variety of intracellular signalling pathways,
including those involving MEK, PI3 kinase, and GSK3. To investigate
the involvement of these key signalling proteins in de novo axonal
growth we studied placode-derived sensory neurons of the chicken
embryo
which can be cultured from the earliest stages of their development.
De novo axonal growth from these neurons is independent of
neurotrophic factors and occurs at a rate that is correlated with target
distance. Nodose neurons have the most distant targets, fastest
axonal growth rate and survive longest before becoming dependent on
the neurotrophic factor BDNF for survival (Davies, Nature, 337, 553-
555, 1989; Vogel and Davies, Neuron, 7, 819-830, 1991). Here we
show that inhibiting MEK, PI3 kinase, and GSK3 has no effect on de
novo axonal growth from nodose neurons, but that PI3 kinase and
GSK3 become important for axonal growth as the neurons acquire
responsive to BDNF during development. These results suggest that
de novo axonal growth is regulated in a different manner to that of
neurotrophic factor-dependent axonal growth.
3.02
NF-kappa B activation via tyrosine phosphorylation of I-kappaB -
alpha, is crucial for CNTF-promoted neurite growth from developing
neurons
Gallagher D, Gutierrez H, O` Keeffe G, Gavalda N, Hay * R, Davies A
School of Biosciences, Museum Avenue, PO Box 911, Cardiff CF10 3US,
Wales, , *School of Life Sciences, University of Dundee, Dow Street,
Dundee DD1 5EH, Scotland
The cytokine CNTF (ciliary neurotrophic factor) promotes the growth of
neural processes from many kinds of neurons in the developing and
regenerating adult nervous system, but the intracellular signalling
mechanisms mediating this important function of CNTF are poorly
understood. Here we show that CNTF activates the NF-κB transcriptional
system in neonatal sensory neurons and that inhibiting NF-κB signalling by
either blocking nuclear translocation of NF-κB or blocking NF-κB -
dependent transcription virtually eliminates CNTF-promoted neurite growth.
Selectively inhibiting NF-κB activation by the non-canonical pathway that
requires tyrosine phosphorylation of IκB -α, but not by the canonical
pathway that requires serine phosphorylation of IκB -α, also effectively
eliminates CNTF-promoted neurite growth. CNTF activates the SYK
protein-tyrosine kinase whose substrates include IκB -α. CNTF-induced
SYK activation is followed by increased tyrosine phosphorylation of IκB -α,
and blocking SYK activation or tyrosine phosphorylation of IκB -α prevents
CNTF-induced NF-κB activation and CNTF-promoted neurite growth.
These findings demonstrate that NF-κB signalling by an unusual activation
mechanism is essential for the ability of CNTF to promote the growth of
neural processes in the developing nervous system.
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3.03
TNF-related protein LIGHT reduces BDNF-dependant neuronal
growth in sensory neurons
Nuria Gavalda, Alun M Davies
Cardiff School of Biosciences,, Biomedical Sciences Building 3,,
Museum Avenue,, Cardiff CF10 3US,, Wales, UK
LIGHT is a protein belonging to the TNF superfamily that was originally
identified as a weak inducer of apoptosis. LIGHT has been shown to
be involved in the costimulation and homeostasis of T-cells, however,
no data exist that describe a function for this ligand during the
development of the nervous system. Our present work reveals a novel
role for LIGHT in the regulation of neurite growth during the
development of mouse sensory neurons. LIGHT is capable of binding
to both the lymphotoxin-beta (LTβR) and HVEM receptors, both of
which were expressed by nodose neurons. Nodose neurons that were
transfected with a plasmid encoding LIGHT had significantly reduced
neurite growth, during a specific period of embryonic through to
postnatal development.
The activation of several signalling pathways have been shown to
mediate the effects of LIGHT in T-cells. These include NF-κB, JNK and
ERK/MAPK. In our cellular model, only ERK/MAPK is activated by
LIGHT, but not NF-κB nor JNK. Specifically blocking the LIGHT
induced ERK activation with the selective inhibitors, U0126 and
PD98059, inhibits the ability of LIGHT to affect neurite growth in
nodose neurons. In contast to this, inhibiting the NF-κB pathway using
specific NF-κB mutant proteins or the JNK pathway, using specific JNK
inhibitor, had no effect on the ability of LIGHT to affect neurite growth
in nodose neurons. All together, these results show a novel role of
LIGHT in sensory neuron development.
3.04
Competitive, intrinsic and stochastic determinants of neuromuscular
synapse elimination in transgenic YFP expressing mice
Teriakidis A, Jenkins N, Willshaw D J, Ribchester R R
Centres for Neuroscience Research and Neuroinformatics DTC, University
of Edinburgh
Competition at motor endplates strongly influences elimination of
polyneuronal innervation in neonatal muscles but it is still not known
whether intrinsic properties of motor neurones also determine ultimate
motor unit size. We are using transgenic mice expressing fluorescent
protein in motor neurones to measure and model the changes in motor unit
size as neuromuscular synapses form or become eliminated. First we
designed experiments to test whether motor unit size in adult lumbrical
muscles becomes reduced even when all other competing motor units are
removed, by partial denervation at birth (neonates anaesthetised by
chilling). We found that motor unit size was not reduced when these mice
reached adulthood, suggesting that competition is sufficient to account for
synapse elimination during development. Next we examined whether the
initial motor innervation pattern is random. Analysis of YFP-expressing
motor units in lumbrical muscles of thy1.2-YFPH mice indicated that all
connections made by a motor neurone are restricted to a single lumbrical
muscle with no divergence between muscles. Stochastic modeling
suggested that a key indicator of randomness is initial converge of
branches of the same axon on single motor endplates. To test this we
examined adult muscles in the earliest stages of reinnervation (12-14 days)
after nerve crush under halothane/N20 anaesthesia. We found several
compelling instances of within-unit convergence on motor endplates. Thus,
stochastic properties play an important role in establishing the divergent
and convergent innervation pattern within muscles but competitive
interactions at polyinnervated junctions are more important than intrinsic
properties of neurones during synapse elimination.
3.05
Investigation into the differentiation of motor neurons derived
from human pluripotent stem cells and assessment of their
function in vitro
Pan C, Przyborski S
School of Biological and Biomedical Science, Durham University,
South Road, Durham DH1 3LE
It is understood that retinoic acid (RA), sonic hedgehog (Shh) and
bone morphogenic proteins (BMPs) play an important role in cell fate
determination and the specification of inter-neurons and motor
neurons along the dorsal-ventral axis in the neural tube. In this study,
we propose to evaluate the function of these signalling molecules to
instruct the differentiation of human pluripotent stem cells.
TERA2.cl.SP12 embryonal carcinoma (EC) cells are a robust
caricature of human embryogenesis and an accepted model of neural
differentiation. Gene and protein expression analyses indicate that
human EC cells respond to RA, BMPs and Shh in a conserved
manner and regulate neural transcription factors and structural
proteins in a predicted way as cells commit toward the motor neuron
phenotype. To assess the function of these differentiated neurons, we
tested their ability to innervate skeletal muscle myotubes and induce
contraction. We showed that muscle contraction could be manipulated
pharmacologically: curare and atropine blocked myotube contraction,
whereas acetylcholine and carbachol increased the number of
contractile events. In other experiments, we have also shown that cells
exposed to RA and Shh in conjunction with other growth factors over
different time periods, preferentially form oligodendrocytes and/or
interneurons. These results indicate it is feasible to control and direct
the differentiation of human stem cells and produce specific neuron
subtypes in vitro as a model to investigate the molecular mechanisms
and signalling pathways that control these processes in man.
3.06
Clonally derived adult stem cell-derived soluble factors and their
instructive effects on neural stem and progenitor cells
Emmerson R, Croft A, Przyborski S A
School of Biological and Biomedical Sciences, University of Durham, South
Road, Durham, DH1 3LE; ReInnervate Ltd, Durham, DH1 3HP
Transplantation of both mesenchymal stem cells (MSCs) and dermal cells
has been shown to result in functional improvement in animal models of
neurological disease, however, the mechanism by which this occurs
remains unclear. We have shown that MSCs produce factors that instruct
neural stem cells (NSCs) to adopt neuronal and glial lineages, and this is
dependent on the developmental status of the MSC. MSCs induced to form
cellular aggregates, express neural antigens such as nestin and GFAP, and
in co-culture studies instruct NSCs to adopt a predominantly neuronal fate.
We have clonally derived MSC cell lines that express neural markers such
as Tuj-1 and GFAP without induction of cell aggregates. Therefore clonal
MSCs expressing neural antigens could have a similar instructive effect on
NSCs as their parent MSC population. Induction of neural antigen
expression such as nestin and Tuj-1 has also been demonstrated in dermal
populations of stem cells. The dermal papilla (DP) region is thought to be a
niche of dermal cells able to express neural proteins. Clonal cell lines
derived from the DP and the dermal sheath (DS) form aggregates that are
similar to both those derived from dermal stem cells and induced MSCs.
We are investigating whether dermal aggregates could have an instructive
effect on NSCs that is similar to MSCs. The study of clonally derived hair
follicle and MSC populations will enable a better understanding of the
effects of soluble factors produced by adult stem cell populations on
neurogenesis.
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3.07
Congenital hypothyroidism alters formalin-induced pain
response in neonate rats
Behzadi G, Mohamad R
Neuroscience Research Center and Physiology Department, Fac.
Med., Shaheed Beheshti Med Sci Univ. POBox 19835-181
It is known that postnatal maturation of the CNS is critically dependent
on the thyroid hormone levels. The present study attempted to
investigate the development of nociceptive response following
induction of the formalin pain in congenitally hypothyroid pups during
the first three postnatal weeks. Time pregnant rats received PTU from
16th day of gestation until 23rd postnatal day. Control and offspring
pups were received different formalin solution in right hind paw at 7,
15, and 23 days of age. Significant reduction in weight gain was
evident in PTU-treated offspring from postnatal days 15 up to 23
(P

3.11
Dynamic integration of subplate neurons into the barrel cortex
during postnatal development in the Golli-tau-eGFP (GTE) mouse
Jethwa A, Piñon MC, Jacobs E, Campagnoni A, Molnár Z
Dept Physiology, Anatomy & Genetics, Oxford University and Semel
Institute for Neuroscience & Human Behaviour, UCLA, California.
Understanding the early steps of cortical development requires
knowledge of the largely transient population of subplate neurons
(SPn). Much of the information about the role of SPn in
thalamocortical patterning is derived from SP specific ablation studies
in the visual cortex of carnivores (Ghosh and Shatz, 1992). The Gollitau-eGFP
transgenic mouse, where the reporter gene expression is
largely confined to layer VIb allows visualisation of SPn and their
projections (Jacobs et al., 2007). In this study we describe the
dynamic integration of SPn into the barrel cortex at postnatal (P) ages
P0-P14. Subplate projections innervate layer IV in the overlying
cortex. The patterning of these projections to the primary
somatosensory cortex (S1) was visualized by fluorescent optical
imaging of the GFP in coronal and tangential sections. GFP labelled
cells initially project diffusely into layer IV of the cortex, then by P6 to
the barrel hollow. By P10-14 this pattern gradually changes so that the
projections are predominantly in the barrel septa. To determine
whether this intracortical reorganisation is dependent on peripheral
pattern, whiskers of row A were removed at P0 and the subplate
projections to S1 were examined at P10. In the contralateral region
corresponding to row A, bisbenzimide counterstain revealed the lack
of septa and GFP distribution the lack of clustering of subplate
projections. Our findings suggest a dynamic integration of SPn within
the barrel cortex which is at least partly dependent on the sensory
periphery.
3.12
Role of FMRP and mGluR5 in the developing mouse primary
somatosensory cortex
Wijetunge L S, Till S M, Dolen G, Gillingwater T H, Bear M F, Kind P C
Authors 1,2,4 & 6, University of Edinburgh, Hugh Robson Building,
Edinburgh, EH8 9XD, UK, , Authors 3 & 5, The Picower Institute for
Learning and Memory, Massachusetts Institute of Technology, 77
Massachusetts Avenue, 46-3301, Cambridge, MA, 02139, USA
Fragile X syndrome (FXS) is the most common genetically inherited form of
mental retardation and results from the loss of the fragile X mental
retardation protein (FMRP). The mGluR theory of FXS postulates that
FMRP limits group 1 mGluR signalling and the exaggerated signalling of
mGluRs underlies some aspects of the FXS pathology (Bear et al., 2004).
We have previously shown that mGluR5-/- mice fail to form “barrels”, the
characteristic cytoarchitectural units found in layer 4 of the primary
somatosensory cortex (S1; Hannan et al., 2001). We now show that FMRP
also regulates barrel development; Fmr1-/- mice show a significant
decrease in barrel segregation compared to WT mice. To gain insight into
the cellular mechanisms regulated by FMRP, we examined its
spatiotemporal expression profile during barrel formation. FMRP was
present in cell soma of all cortical layers at all ages examined with peak
expression during the first postnatal week. A diffuse neuropilar staining is
also prominent at P4 and P7. Immuno-electron-microscopy of layer 4 at P7
reveals restriction of FMRP to soma and dendrites, including regions near
postsynaptic densities (PSDs). mGluR5 expression was restricted to
dendrites and PSDs at this age. Finally, layer 4 cells from mGluR5-/- and
mGluR5+/- show a decrease in spine density relative to wildtypes at P21,
the opposite effect in spine density to that observed in mice lacking FMRP
(Irwin et al., 2000). These findings demonstrate a role for FMRP in the
differentiation of S1 and are consistent with the mGluR theory of FXS.
3.13
A molecular neuroanatomical study of the developing human
neocortex from formation of the cortical plate to the arrival of
thalamocortical axons in th
Clowry G 1, Bayatti N 1, Moss J 1, Sun L 3, Ambrose P 1, Lindsay S
1,2
Institute of Neuroscience and the , School of Clinical Medical
Sciences1, Institute of Human Genetics2, School of Biology and
Psychology3, Newcastle University, Newcastle upon Tyne,
By studying multiple protein expression patterns by
immunohistochemistry we have correlated structure with function in
different compartments of the developing human cortical wall. The
marginal zone exhibited intense expression of markers that indicate a
dense network of neurites and synapses at all ages studied. During an
initial phase of development 8-13 postconceptional weeks (PCW) the
cortical plate, which expressed MAP2 and dense Nissl staining, grew
rapidly, then more slowly from 13-16 PCW, although a distinct layer V
expressing Er81 immunoreactivity was observed. A distinct subplate
region developed from 10.5-13 PCW, rich in synaptophysin, GAP43
and vesicular GABA transporter immunoreactivity and larger than
previously described at this stage. From 13 PCW the subplate rapidly
expanded, becoming the largest compartment by 16 PCW and clearly
visible on MRI scans of post mortem brains. It was differentiated in
structure, with neuropeptide Y positive neurones found near to the
cortical plate, and KCC2 immunoreactive neurones localised near to
the intermediate zone. Glutamate decarboxylase and calretinin
positive inhibitory neurones migrated both tangentially and radially
from 11.5 PCW, with tangential migration seen largely, but not
exclusively, in the subventricular zone. From 12.5 PCW the Pax6 and
MAP2 immunoreactive subventricular zone differentiated into inner
and outer cell rich compartments divided by a fibrous layer. The Pax6
positive, MAP2 negative ventricular zone maintained its size
throughout the period studied. A spontaneously active synaptic
network may exist in the subplate that guides development of
migrating neurones and the newly forming cortical plate prior to the
arrival of thalamocortical afferents.
3.14
A search for genes involved in specifying the early cortical map in
human development
Bayatti N (1), Sarma S (2), Eyre J A (1), Lindsay S (2), Clowry G J(1)
(1) Neural Development Plasticity and Repair group (2) Institute of Human
Genetics (IHG), Institute of Neuroscience, School of Clinical Medical
Sciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE1
4LP, United Kingdom.
The homeodomain transcription factor Emx2 and the paired box-containing
transcription factor Pax6 are expressed in reciprocal gradients in the
proliferating zones of the developing rodent neocortex and interact in setting
up the early cortical map. Using in situ hybridization and
immunohistochemistry we have analysed the expression of these two genes
in the developing human cortex between 8-12 gestational weeks (GW) and
using Affymetrix gene chips we have looked for additional candidates during
human development. At 8 GW, expression of EMX2 and PAX6 was limited
to the ventricular and subventricular zones (VZ, SVZ) and EMX2 exhibited a
high caudomedial to low rostrolateral gradient, with PAX6 exhibiting the
opposite gradient. From 9 GW EMX2 was also strongly expressed in the
developing cortical plate whilst maintaining a gradient. However, PAX6
expression remained in the proliferative zones and became uniform
throughout the extent of the cortical plate. Therefore at 8 GW, around the
time the CP first appears, both EMX2 and PAX6 are localised to the
proliferative zones and express prominent reciprocal gradients suggesting
they could interact to confer early areal identity. Thus these genes exhibit
only a transient co-localisation, and additional candidates that are
expressed in similar gradients throughout all stages analysed were
identified by gene chip analysis. For instance, two such candidates ZIC1
and NR2F1, were found to be expressed at high levels at the rostral and
caudal poles of the developing human cortex respectively and expression
was confirmed by in situ hybridisation (ZIC1) and immunocytochemistry
(NR2F1).
Supported by the Wellcome Trust.
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4.01
The effect of hippocampal slice orientation, on the GluK5 receptor
subunit dependency of NMDA receptor independent mossy fibre
LTP.
Sherwood J L, Nistico R, Lodge D, Collingridge G L, Bortolotto Z A
[1,2,3,4,5] MRC Centre for Synaptic Plasticity, Department of Anatomy,
University of Bristol. BS8 1TD. UK, [2] Department of Pharmacobiology
and University Centre for Adaptive Disorders and Headache (UCHAD),
University of Calabria, Rende, Italy,
Recently it has been agreed that pre-synaptic kainate receptors play a
role in the induction of NMDA receptor independent long-term
potentiation (LTP) at mossy fibre (MF) synapses.1,2 However,
controversy remains over the involvement of GluK5 receptor subunits.2
Our laboratory is interested in identifying sources of disparity.3 While
our lab use parasagittal slices, others use transverse.2 Here we
investigate the effect of hippocampal slice orientation.
Parasagittal: isolated cerebral hemispheres were fixed on the base of a
vibratome, lateral surface down, and sliced parallel to the brain midline.
Transverse: isolated hippocampi were mounted on an agar block and
sliced perpendicular to the septo-temporal axis in a vibratome.
Using standard conditions,1 CA3 field potentials were recorded in
stratum lucidum and evoked by constant current stimulation of the MF
pathway. Efficacy of synaptic transmission was sampled every 30 s.
Following 30 min baseline period, a potent and highly selective GluK5
receptor antagonist, ACET (David Jane, personal communication), was
washed on for 20 minutes and LTP induction tested by delivering 100
stimuli at 100 Hz (in the presence of D-AP5 to exclude NMDA
receptor-dependent LTP). We confirm that GluK5 receptor antagonism
blocks NMDA receptor-independent MF LTP in parasagittal slices,1 but
also that GluK5 receptor antagonism has no effect on NMDA receptorindependent
LTP in transverse slices.2
4.02
Role of PICK1 in AMPA receptor trafficking events following oxygenglucose-deprivation
Dixon R, Mellor J, Hanley J
Department of Anatomy, School of Medical Sciences, University of Bristol,
Tyndall Avenue, Bristol, BS8 1TD
Ischaemia causes a downregulation in expression of GluR2 a widely
expressed α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor
(AMPAR) subunit that renders AMPARs largely impermeable to calcium.
Changes in total GluR2 protein expression have been observed 24-48
hours post-ischaemia, but changes in AMPAR trafficking that occur straight
after an ischaemic episode have been less widely studied. Using oxygenglucose-deprivation
(OGD) to mimic ischaemia in vitro, we show a rapid
shift in surface AMPAR subunit composition following OGD in hippocampal
cultures. Surface protein levels of GluR2-containing-AMPARs are
significantly reduced immediately after a 30 minute period of OGD,
whereas total GluR2 and surface GluR1 levels remain the same as
controls. In addition, using whole-cell patch-clamp recordings from CA1
pyramidal cells we show a decrease in rectification index (RI: +40 EPSC/-
70 EPSC) following a 30 minute period of OGD. This indicates that GluR2-
containing-AMPARs are being replaced with GluR2-lacking AMPARs at
CA3-CA1 synapses. In order to investigate the molecular mechanism for
this trafficking event we postsynaptically infused peptides that interfere with
GluR2 C-terminal interactions with PDZ-domain proteins. Infusion of pep2-
EVKI which selectively blocks the PICK1-GluR2 interaction prevented the
OGD-mediated decrease in RI, whilst a control peptide pep2-SVKE had no
effect. This shows PICK1 to be a crucial mediator in initial trafficking events
that occur post-ischaemia. This PICK-mediated change in subunit
composition would lead to an increase in calcium influx through GluR2-
lacking AMPARs resulting in activation of downstream cell death pathways.
These results present the GluR2-PICK1 interaction as a potential novel
therapeutic target in stroke treatment.
4.03
What is the significance for synaptic plasticity of the
developmental change in N-methyl D-aspartate receptor subunit
expression
Bartlett T, Bannister N, Collett V, Massey P, Bashir Z, Fitzjohn S,
Collingridge G, Lodge D
MRC Centre For Synaptic Plasticity,, University Of Bristol,, School Of
Medical Sciences,, University Walk,, Bristol,, UK
Long term potentiation (LTP) and long term depression (LTD) are
forms of synaptic plasticity important for learning and memory. In area
CA1 of the hippocampus the NMDA receptor is crucial for the
induction of LTP and LTD induced by high and low frequency
stimulation respectively. The NMDA receptor comprises four subunits,
two NR1 and two NR2 from four possible types (NR2A-NR2D). The
expression of NR2 subunits is developmentally regulated, with NR2A
appearing at P7 while NR2B is already high at birth (Sheng,
Cummings et al. 1994).
To detect if different subtypes of the NR2 subunit are involved in the
induction of LTP and LTD at different stages in development we
studied the effect of subunit selective antagonists on the induction of
LTD induced by low frequency stimulation (1 Hz, 15 mins) and LTP
induced by high frequency stimulation (100 Hz, 1 s) in area CA1 of
hippocampal slices from P14 and P42-56 Wistar rats.
The NR2B-selective antagonist Ro 25-6981 (Ro, 5μM) did not block
LTD at either P14 or P42-P56, although the induction of LTD in the
older slices required the glutamate uptake blocker, threo-βbenzylaspartic
acid (TBOA). This suggests the induction of LTD is not
exclusively dependent on NR1/NR2B NMDARs at either
developmental stage under our experimental conditions.
4.04
Activation of muscarinic acetylcholine receptors induces LTD in the
CA1 region of the hippocampus: GluR2 AMPA receptor internalisation
Dickinson B, Jo J, Cho K
Henry Wellcome Laboratories & , The MRC Centre for Synaptic Plasticity ,
Dorothy Hodgkin Building , Faculty of Medicine , University of Bristol ,
Whitson Street , BRISTOL BS1 3NY
The cholinergic system is implicated in Alzheimer’s disease (Kasa et al,
1997). Muscarinic acetylcholine receptors (mAChRs), a subfamily of
cholinergic receptors, have a critical role in synaptic plasticity in the
perirhinal cortex (Massey et al, 2001) and the visual cortex (Kirkwood et al,
1999). However, the molecular mechanism of mAChR-dependent long-term
depression (LTD) in the hippocampus is unknown.
Using whole-cell patch recording we have shown that bath application of
carbachol (CCh; 50ƒÝM for 10min) induces LTD in the CA1 of the
hippocampus. Our study also confirmed that inclusion of the Ca2+ chelator
BAPTA (10mM) in the electrode solution blocked LTD. Thus, a postsynaptic
mechanism might be involved in CCh-induced LTD. It has been
hypothesised that changes in AMPA receptor-mediated synaptic
transmission is a molecular mechanism of LTD. Therefore, we next
determined if CCh-induced LTD is due to the internalization of AMPA
receptors. Postsynaptic inclusion of PEP2-SVKI (YNVYGIESVKI), a peptide
analogous to the interaction site of GRIP and PICK with the GluR2 subunit
of AMPA receptors (Daw et al., 2000), was shown to block LTD. However,
the addition of the control peptide PEP2-SVKE (NVYGIESVKE) has no
effect on CCh-induced LTD. In conclusion, the data suggest that AMPA
receptor internalisation has a role in CCh-induced LTD in the CA1 of the
hippocampus.
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4.05
Corticosterone blocks muscarinic acetylcholine receptordependent
long-term depression in perirhinal cortical synapses
Gilpin H, Jo J, Cho K
Department of Biomedical Science, Alfred Denny Building, University
of Sheffield, Western Bank, SHEFFIELD S10 2TN, UNITED
KINGDOM, , Dorothy Hodgkin Building, Faculty of Medicine, University
of Bristol, Whitson Street, BRISTOL BS1 3NY, UNITED KINGDOM
Corticosterone (CORT), the principal glucocorticoid synthesized by the
adrenal cortex in rodents, is secreted in response to stress. In
particular, layer II and III of the perirhinal cortex show a high density of
glucocorticoid receptor (GR) immunoreactivity (Morimoto et al. 1996).
The perirhinal cortex is a transitional cortex interposed between
neocortex and the hippocampal formation, and is essential for the
familiarity component of recognition memory. Loss of recognition
memory is a major symptom of amnesia and early stages of
Alzheimer’s disease. However, little is known about whether CORT
has a role in synaptic plasticity in the perirhinal cortex. Recently,
cholinergic muscarinic receptors (mAChRs) have been implicated in
recognition memory and long-term depression (LTD; Warburton et al.
2003; Jo et al. 2006). Therefore the present study tests whether CORT
has an important role in mAChR-mediated synaptic plasticity.
The current study shows that pre-incubation with 200nM CORT blocks
mAChR-dependant LTD (5Hz 900 pulses at the entorhinal input of the
perirhinal cortex). However, this CORT effect on LTD is abolished
upon co-incubation with the glucocorticoid receptor (GR) antagonist,
RU486 (500nM). Carbachol-induced LTD is also significantly reduced
by CORT application. Our data suggest that CORT occludes mAChRdependant
LTD in the perirhinal cortex, thus implicating an important
role for CORT in recognition memory.
4.06
Dynamic regulation of N-terminal SynGAP isoforms
Barnett M W, Till S, Stoney P, Papadia S, Parkinson D, Hardingham G,
Kind P C
University of Edinburgh, Hugh Robson Building, George Square,
Edinburgh, EH8 9XD, UK
Maintenance of early phase LTP is mediated by the activation of
posttranslational modification of existing molecules whilst late phase LTP is
dependent on new mRNA transcription and protein synthesis. Following
induction of LTP, expression of a number of immediate early genes is
increased (eg. Krox24, Krox20, c-fos, c-jun). We have previously shown
that the N-terminal isoforms of the neuronal RasGTPase Syngap (Syngap-
A, Syngap-B and Syngap-C) have different transcription start sites and that
consensus binding sites for Krox20 and Krox24 are found in close proximity
to the transcription start sites of Syngap-B and Syngap-C respectively. We
now provide evidence for a highly dynamic and unique spatiotemporal
regulation of mRNA encoding Syngap-A, Syngap-B and Syngap-C. First,
Syngap-B and Syngap-C are increased in response to activity whilst
Syngap-A is not; this increase is partially dependent on the activity of
NMDA receptors. Second, levels of Syngap-B and Syngap-C mRNA are
selectively decreased in the hippocampi of mice lacking PLC-β1, a key
signalling molecule downstream of glutamate receptors. Finally we now
show that Syngap-A, Syngap-B and Syngap-C have differential temporal
and spatial expression profiles during postnatal brain development. These
findings suggest that different Syngap isoforms may regulate distinct
neuronal processes in specific neuronal subpopulations. The activityindependent
expression of the more abundant Syngap-A mRNA suggests
that the protein it encodes may play a constitutive role in neuronal function
whilst the activity-dependent isoforms (SynGAP-B and SynGAP-C) may
participate in activity-dependent plasticity.
4.07
Synaptic Tagging & Capture: the search for the tag
Feruza Nuritova*, Redondo R, Morris R G M, Frenguelli B G*
*Neurosciences Institute, University of Dundee & Centre for Cognitive
and Neural Systems, The University of Edinburgh,
Protein synthesis is required for both late long-term potentiation (L-
LTP) and long-term memory (LTM). This necessitates a process by
which these newly-formed plasticity-related proteins (PRPs) can be
utilised exclusively by synapses at which significant sensory
experience has occurred. However, associative learning enables the
correlation of strong sensory experiences with weaker ones. The
Synaptic Tagging and Capture hypothesis (STC) reconciles the two by
proposing the creation of a lower threshold synaptic tag, which
interacts with homo- or heterosynaptically generated PRPs. Thus,
although the tag alone would not give rise to L-LTP/LTM, tag-PRP
interactions would.
We are performing dual/tri-pathway electrophysiological experiments
in hippocampal slices (31–32°C) from 7-8 week old rats to test
whether CaMKII is involved in tag-setting. The LTP on S0, in response
to strong stimulation (3 x 100Hz/1s), measured 172.4±13.6% of
baseline at 120 mins post-tetanus (n=9). Weak stimulation of S1 (1 x
100Hz/0.5s) resulted in no long-lasting potentiation (96.2±3.7%; n=3).
However, if the weak tetanus was delivered 30 min after the strong
tetanus, L-LTP was observed on S1 (158.3±11.6%; n=9) – ie synaptic
tagging had occured. Tagging on S1 also occurred (116.0±6.7%; n=7)
even if the CaMKII inhibitor KN 62 (1μM) had been present during
strong stimulation of S0, which substantially reduced S0 L-LTP
(115.9±3.7%; n=7). Thus, it would seem that a) parallel,
heterosynaptic (presumably dopaminergic) inputs generated CaMKIIindependent
PRPs on S0, which are b) recruited by the CaMKII (or
CaMKII-dependent) tag on S1.
4.08
Leptin depotentiates long-term potentiation at hippocampal CA1
synapses.
Milojkovic B, Harvey J
Neurosciences Institute, Ninewells Hospital & Medical School, University of
Dundee, Dundee, DD1 9SY
There is growing evidence that the hormone leptin plays an important
modulatory role in the synaptic plastic mechanisms underlying learning and
memory. Indeed leptin-insensitive rodents display impairments in
hippocampal LTP and LTD, whereas at the cellular level, leptin promotes
conversion of STP into LTP and it evokes a novel form of LTD. In this study
we have examined the effects of leptin on potentiated hippocampal CA1
synapses using standard whole cell recordings. Transverse hippocampal
slices were prepared from 14-18 day old rats, maintained at 31oC and
perfused with aCSF containing picrotoxin (50µM). EPSCs were evoked by
stimulation (0.033Hz) of the Schaffer collateral commissural pathway and
recorded using patch pipettes filled with Cs+-based solution. In order to
examine the effects of leptin on potentiated synapses, LTP was evoked by
3 trains, 250 ms apart, 20-40 stimulations at 200 Hz, which resulted in
45.79 ± 1.64% increase in synaptic transmission (n=10). Perfusion of slices
with leptin (25-50nM), 30 min after LTP induction resulted in a rapid and
concentration-dependent reversal of LTP (n=8). Application of 10nM leptin
had no effect on LTP expression (n=2), whereas addition of 25 nM leptin
reversed LTP to 97.6 ± 7.85 % of control (n=5). In contrast, perfusion with
25nM leptin failed to modify basal synaptic transmission (n=3). These data
indicate that leptin, at concentrations that fail to effect basal transmission,
has the capacity to reverse hippocampal LTP. These findings have
important implications for the role of leptin in modulating hippocampal
synaptic plasticity.
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4.09
ß-adrenergic modulation of in vitro hippocampal theta- and
gamma-frequency oscillations
Haggerty D C, Cox D J, Eden D C, Racca C, Whittington M A, LeBeau
FEN
University of Newcastle-upon-Tyne, SNNP, Framlington Place,
Newcastle, NE2 4HH
Hippocampal gamma (20-80Hz) and theta (4-15Hz) oscillations have
been proposed to be involved in memory processing and visuospatial
representation. The noradrenergic system has been implicated in a
range of cognitive functions including arousal, attention, learning and
memory. Therefore, activation of hippocampal noradrenergic receptors
may have important effects on memory and visuospatial information by
modulating oscillatory activity. Transverse hippocampal slices were
prepared from adult male Wistar rats. Both kainate (gamma) (22.8 +/-
7.1 p

4.13
Regulation of Egr1 and Egr3 following the induction of LTP in
CA1 of the hippocampus.
Cheval H, Laroche S, Davis S
NAMC, UMR 8620, CNRS, Univ Paris-Sud, 91405 Orsay, France
The gene encoding Egr1 (zif268) is regulated and necessary for late
phases of LTP in dentate gyrus and the consolidation of a number of
different forms of memory. Very little data exists about the potential
role of Egr1 and other members of this family of transcription factors in
plasticity in CA1. We have examined regulation of both Egr1 and Egr3
following the induction of LTP in CA1 in both rats and Egr1-/- and +/+
mice. Preliminary evidence shows LTP induced in +/+ mice is
associated with an increase in Egr1 protein levels. However,
regulation of the protein is not specific to LTP as pseudotetanus also
induces an increase in Egr1 protein level. In mutant mice, LTP is
attenuated and western blotting confirms the lack of protein,
suggesting that Egr1 is neither necessary nor specific to LTP in CA1.
Induction of LTP also induced a similar pattern of overexpression of
Egr3 in both wildtype and mutant mice with regulation of the protein
restricted to the Ą isoform, suggesting that Egr3 regulation may be
linked with regulation of Egr1. As a transcription factor, Egr1/Egr3
presumably bind to downstream gene targets with a consensus ERE
sequence on their promoter. We are conducting EMSA and supershift
assays in rat to determine whether Egr1 and Egr3 functional binding to
promoter regions sequences is increased following induction of LTP.
Preliminary evidence suggests this to be the case and supershift
experiments will be conduced to examine the specificity of Egr1 and
Egr3 binding to the ERE sequence.
4.14
Multielectrode array-based platform for analysis of the synaptic
transmission and plasticity phenotypes in mutant mice
Kopanitsa MV, Afinowi NO, Grant SGN
Genes to Cognition Programme, The Wellcome Trust Sanger Institute,
Hinxton, Cambridge CB10 1SA
The Genes to Cognition Programme (G2C, www.genes2cognition.org) is a
multidisciplinary initiative to study genes involved in brain functions and
behaviour. An important part of G2C is production and characterisation of
transgenic mice. We sought to employ multielectrode array (MEA)
technology for the high-throughput electrophysiological assessment of
mutant animals. We have shown previously (Kopanitsa et al., BMC
Neuroscience 7: 61) that MEAs allow for efficient recording of synaptic
potentials and facilitate LTP studies in hippocampal slices.
To further validate MEA-based approaches, we studied several mutants
where deficiencies in LTP had been documented. Synaptic responses were
recorded in the CA1 area of hippocampal slices upon stimulation of
Schäffer collaterals. LTP was induced in one of the two stimulated
pathways by high frequency tetani and/or by theta-burst stimulation. We
have observed deficient LTP phenotypes in GluR-A -/-; SynGAP +/- and
NR2A ΔC/ΔC mice, which was consistent with published reports. Also, we
found a decrease of LTP in the NR2B/ΔC mice, which suggested that
integrity of cytoplasmic tails of both NR2A and NR2B subunits is important
for plasticity. The MEA-based platform was also used to investigate
synaptic transmission and LTP in a number of novel strains created in our
lab. We conclude that MEA technology is a rapid and efficient platform for
analysis of synaptic transmission and plasticity in mutant mice. In addition
to the throughput, an advantage of MEAs is that they allow standardising
recording conditions during LTP experiment, which is extremely important
for comparing the severity of different mutant phenotypes.
5.01
Human bone marrow stromal cells promote nerve growth over
the major nerve-inhibitory molecules found in the injured spinal
cord.
Wright K T, El Masri W, Osman A, Roberts S, Chamberlain G, Ashton
BA, Johnson WE
Centre for Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic
Hospital, Oswestry, Shropshire SY10 7AG, UK; Institute for Science
and Technology in Medicine, Keele University, Keele, Staffordshire
ST5 5BG, UK.
Chondroitin sulphated proteoglycans (CSPG), myelin associated
glycoprotein (MAG) and Nogo inhibit nerve growth in vivo. Their
inhibitory influence in spinal cord injury (SCI) has been shown in
animal models wherein treatments that reduced their presence,
integrity or blocked their activity promoted axonal regeneration and
functional recovery. Transplantation of bone marrow stromal cells
(MSC) into the injured spinal cord induced similar results, but it is still
unclear how the improvements noted in these animals were achieved.
We have examined the potential for human MSC isolated from SCI
patients to promote nerve growth across inhibitory molecules using an
established in vitro model of nerve growth. As previously described
CSPG, MAG and Nogo were each found to inhibit neurite outgrowth in
a concentration dependant manner, this effect was diminished in the
presence of MSC. Time-lapse video microscopy demonstrated that
MSC acted as “cellular bridges” and also “towed” neurites over
inhibitory substrates. Whereas conditioned medium from MSC cultures
stimulated neurite outgrowth over type I collagen, it did not promote
outgrowth over CSPG, MAG or Nogo.
5.02
Mesenchymal stem cell- derived soluble factors and their instructive
effects on neural stem and progenitor cells
Hardy S A, Croft A P, Przyborski S A
1School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham, DH1 3LE; 2ReInnervate Ltd, Durham, DH1 3HP
Mesenchymal stem cell (MSC) transplantation in animal models of
neurological disease has resulted in neurological improvement, however,
the mechanism by which this occurs is under debate. Evidence suggests
that MSCs have neurogenic potential and undergo differentiation into
neural tissue to replace cells damaged in disease (trans-differentiation).
These data are conflicting as others argue that MSCs do not transdifferentate
but instead fuse with host cells adopting their phenotype.
However, both mechanisms are unlikely to fully account for the
improvements seen. We are currently investigating a third mechanism
whereby transplanted MSCs promote endogenous repair of neurologically
damaged areas by the release of trophic factors and cytokines e.g. NGF
and BDNF. We have shown that MSCs produce factors that instruct neural
stem cells (NSCs) to adopt neuronal and glial lineages, and this is
dependent on the developmental status of the MSC. Under standard
culture conditions MSCs are negative for neural antigens, and produce
factors which instruct NSCs to adopt a predominantly astrocytic fate.
Conversely, we have shown that MSCs induced to form neurosphere-like
structures, which are positive for neural antigens such as nestin and GFAP,
instruct NSCs to adopt a predominantly neuronal fate. Such events are
likely to contribute to the functional improvements observed in disease
models following MSC transplantation. We are currently in the preliminary
stages of identifying these unknown factors.
These findings suggest that MSC transplantation may promote axonal
regeneration by stimulating nerve growth via secreted factors and also
by reducing the nerve-inhibitory effects of extracellular molecules.
Further experimentation using this model will help identify how MSC
are able to migrate over nerve-inhibitory molecules and how they
stimulate co-localised nerve growth. This thereby may permit
appropriate molecular targeting to further enhance nerve growth in
vivo.
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5.03
Constitutively active Ras and dominant negative MEK1 improve
functional recovery following C4 dorsal hemisection in mice
Makwana M (1), Anderson P N (2), Hristova M(1), Zbarsky V (1), Miller
F D (3), Kaplan D (3), Heumann R (4), Raivich G (1,2)
1. Centre for Perinatal Brain Protection and Repair, Dept of Obstetrics
& Gynaecology, University College London, UK, 2. Dept of Anatomy,
University College London,UK, 3. Univ of Toronto, Sick Children
Hospital, Toronto, Canada, 4. Dept of Neurobiochemistry, Ruhr-
University Bochum, Germany,
The extracellular-signal-regulated-kinase cascade is important in
neuronal survival and plasticity. To determine the function of neuronal
Ras and MEK in axonal regeneration, we generated constitutively
active Ras (Ras+), dominant-negative MEK1 (MEK1dn) and
Ras+xMEK1dn double mutant (DM) transgenic mice. Studies in these
mutants have shown extensive collateral sprouting of corticospinal
tract (CST) axons, rostral to the lesion in the grey and white matter on
the ipsilateral side in Ras+, MEK1dn and DM animals compared with
wild-type (WT) controls. Rearing and grid-walk tests were used to test
functional recovery over a period of 28 days following a unilateral left
dorsal hemisection (DH) at C4, followed by labelling of the CST with
biotinylated dextran amine to assess the sprouting response. Ras+
and MEK1dn groups performed significantly better in left forepaw use
than WT in the rearing test at day 14 (35.0%±9.0%; 45.0%±7.0%
versus 9.0%±5.0%; mean±SEM respectively; **p

6.03
Transcription factor expression analysis in adult human brain
stem/progenitor cells
Baer K*1, Curtis M A *2, Eriksson P S 2, Stapley H 1, Faull R L3, Mark
I. Rees1
* equal contribution; 1Molecular Neuroscience, School of Medicine,
Swansea University, Swansea SA2 8PP, UK; 2Centre for Brain Repair
and Rehabilitation, Göteborg University, Göteborg, Sweden;
3Department of Anatomy with Radiology, The University of Auckland,
Auckland, New Zealand.,
Transcription factors (TF) are responsible for the specification and fate
determination of cells as they develop from progenitor cells into
specific types of cells in the brain. Sox-2 and Pax-6 are TFs with key
functional roles in the developing brain, although less is known about
TFs in the rudimentary germinal zones in the adult human brain.
Endogenous stem/progenitor cells have been demonstrated in two
neurogenic regions in the adult human brain, the subependymal layer
(SEL) of the basal ganglia and the subgranular layer (SGZ) of the
hippocampus (Curtis et al., PNAS 2003; Eriksson et al., Nat Med
1998). Before potential therapeutic applications, we require a thorough
understanding of TF localization and the processes involved in
directing stem cells toward desired neuronal phenotypes.
Our hypothesis is that specific TF are responsible for the fate
decisions of stem/progenitor cells in the neurogenic regions of the
adult human brain. We aim to identify the key TF that are present in
the neurogenic regions of the adult human brain. In this study we have
investigated the distribution and characterization of Sox-2 and Pax-6
in the human subventricular zone (SVZ). Sox-2 immunoreactivity
showed a nuclear labelling pattern and colocalised on GFAP
immunoreactive cells, whereas Pax-6 immunoreactivity was
detectable in the nucleus and the cytoplasm of SVZ cells and
colocalised with PSA-NCAM positive progenitor cells. Thus, our data
surprisingly reveal that these TFs are differentially expressed in the
adult human SVZ where Sox-2 and Pax-6 specify a glial and neuronal
fate, respectively.
6.04
mGluR5 is involved in dendrite differentiation and excitatory synaptic
transmission in NTERA2 human embryonic carcinoma cell derived
neurons
Park H, Andrews P W, Molnar E, Cho K
1. Biomedical Science, University of Sheffield, Sheffield, 2. MRC Centre for
Synaptic Plasticity, University of Bristol, 3. Henry Wellcome Laboratories for
Integrative Neuroscience and Endocrinology, MRC Centre for Synaptic
Plasticity, University of Bristol, Bristol BS1 3NY, UK
The pluripotent human embryonic carcinoma cell line NTERA2 readily
differentiates into neurons when exposed to retinoic acid in vitro. These
neurons show characteristic morphology with long processes and they
express neuronal markers TUJ-1 and NeuN. NTERA2-derived neurons can
regulate Ca2+ signalling through ionotropic glutamate (iGluR) and
muscarinic receptors (mAChRs). Little is known, however, about the role of
metabotropic glutamate receptors (mGluRs) in these neurons. Here we
show that NTERA2-derived neurons express functional mGluR5, which is
involved in Ca2+ signalling. Blocking mGluR5 activity at early stages of
differentiation leads to fewer dendrites and a reduction in miniature
excitatory postsynaptic currents (mEPSCs). Furthermore, cells cultured in
the presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine
(MPEP) show reduced N-methyl-D-aspartate (NMDA) receptor-mediated
Ca2+ mobilisation but increased alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) receptor Ca2+ permeability. During normal
neuronal development the edited GluR2 renders AMPARs Ca2+
impermeable. The increased Ca2+ permeability of AMPARs in MPEPtreated
neurons is due to the reduced expression of GluR2 subunit protein.
Thus, mGluR5 activity at early stages of differentiation is likely to play a role
in the development of multipotent cell-derived neurons.
6.05
The effect of pro-inflammatory cytokines, IL-1 beta and TNF alpha
on embryonic rat hippocampal neuronal precursor cells
Keohane A, Sullivan A, Nolan Y
Anatomy Department, Biosciences Institute, University College, Cork,
EIRE.
6.06
Dopaminergic differentiation of human embryonal carcinoma cells
Bramwell T W 1, Lakics V 2, Przyborski S A 1
1School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham, DH1 3LE; 2Eli Lilly & Co. Ltd. ,Erl Wood Manor,
Windlesham Surrey, GU20 6PH
Hippocampal neurogenesis occurs in the developing and adult brain
due to the presence of multipotent stem cells. These cells can be
cultured in vitro as spherical aggregates called “neurospheres” and
when given appropriate signals, can differentiate into neurons,
astrocytes and oligodendrocytes. Hippocampal neurogenesis is
impaired in Alzheimer’s disease. In post-mortem Alzheimer’s brains,
large numbers of activated microglia have a deleterious effect on
neurons through the action of the pro-inflammatory cytokines,
interleukin 1β (IL-1β) and tumour necrosis factor α (TNFα).
The aim of this study was to assess the effects of these two cytokines
on neuronal and astroglial differentiation in cultures of embryonic
hippocampal neurospheres. Hippocampus was isolated from
embryonic day (E)18 rat and cells were allowed to proliferate for 7
days in vitro (DIV) in the presence of appropriate growth factors. Cells
from the neurospheres were then differentiated for 7DIV in the
presence of either IL-1β or TNFα (10ng/ml - 100ng/ml) without growth
factors and stained immunocytochemically for βIII-tubulin (post-mitotic
neurons), doublecortin (newly-born neurons) and glial fibrillary acidic
protein (astrocytes).
Immunolabelling experiments demonstrated that the percentages of
post-mitotic and newly-born neurons were reduced significantly in the
presence of IL-1β or TNFα (p< 0.05; ANOVA, n=3). Conversely, the
percentage composition of astrocytes increased significantly after
incubation with IL-1β or TNFα (p< 0.01; ANOVA, n=3).
This study demonstrates that IL-1β and TNFα have a detrimental effect
on neuronal development by inhibiting the differentiation of precursor
cells to a neuronal phenotype, and by promoting their differentiation to
an astroglial phenotype.
Parkinson’s disease (PD) principally affects a discrete population of
mesencephalic dopamine producing neurons in the substantia nigra pars
compacta, representing an ideal candidate for potential cell replacement
therapies. Consequently, the development of methods to generate pure
populations of dopaminergic cells from various sources in vitro is of great
interest.
Human pluripotent embryonal carcinoma (EC) stem cells derived from
teratocarcinomas offer a robust model system to study neural differentiation.
Using the human EC cell line TERA2.cl.SP12 work is being undertaken to
elucidate the molecular mechanisms governing the production of
dopaminergic neurons. Currently our studies are focused on the effects of
the use of physiological oxygen culture conditions, previously shown to
increase the proportion of dopaminergic neurons derived from midbrain
precursor cells (Studer et al, J. Neurosci. 20, 2000) in combination with
retinoic acid-induced differentiation. To that end, real-time reverse
transcription PCR, flow cytometry and western blotting are being employed
to assess the expression of the key neuronal (e.g. Tuj1) and more
specifically, dopaminergic markers such as tyrosine-hydroxylase, Nurr1,
dopamine receptor 1 and 2 and dopamine transporter. Our preliminary gene
expression data show that these markers are present in differentiated EC
cells. We propose to further investigate the molecular mechanisms
controlling the development of dopaminergic neurons under a range of
alternative growth conditions, and assess markers of neuronal
differentiation, as outlined above. These studies will hopefully allow for
production of purer populations of this particular neuronal phenotype with a
range of potential applications in basic research and the development of
pharmaceuticals.
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6.07
Proteomic identification of biomarkers for neural stem cells
Maltman D J, Przyborski S A
School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham, DH1 3LE, , ReInnervate Ltd, Durham, DH1 3HP
Stem cell research suffers from a lack of protein markers which allow
precise definition of cellular identity. Of the few antigens used to define
neural stem/progenitor cells none are expressed exclusively in these
populations. This highlights a strong requirement for novel specific
biomarkers. Comparative proteomic approaches hold great promise
for the rapid detection of such molecules, and employment of
complementary sample preparation methods will enhance the
likelihood of their detection. High-throughput determination of cellular
status is now achievable through the combined detection of several
differentially expressed proteins. Such profiling employs time-of-flight
(TOF) mass spectrometry and circumvents any requirement for
individual protein identification. Previously, surface-enhanced laser
desorption ionization (SELDI)-TOF biomarker profiling has been
successfully used in our laboratory to distinguish between control and
differentiated cultures of a model stem cell system. Currently we are
applying high accuracy matrix-assisted laser desorption ionization
(MALDI)-TOF to profile a human neural stem cell line and its
differentiated derivatives. Profiles generated will not only enable the
rapid evaluation of sample status, but also provide a lead into specific
biomarkers whose further characterization will increase our
understanding of neurogenesis. These proteins will also form the basis
for the development of immunological tools which may be used for
improved cellular characterization and isolation.
6.08
Neural effects of novel synthetic retinoids.
Christie V.B., Cartmell E, Whiting A, Marder T.B., Przyborski S.A.
Durham University, Biological and Biomedical sciences, South Rd, Durham,
DH1 3LE
Vitamin A and its derivatives, collectively termed retinoids, are essential for
many biologically important processes and are used to modulate cell
proliferation and differentiation in vitro. The importance of optimal retinoid
function in embryonic neural development is well known, and is now being
realised in certain adult neural cell populations. The aim of this research is
to study the role of both natural and synthetic retinoids during neural
differentiation in adult verses embryonic model systems. It has been shown
that all-trans-retinoic acid (ATRA), a naturally occurring retinoid which
activates all retinoic acid receptor (RAR) subtypes, induces neural
differentiation in several cell model systems, including embryonic stem cells
and adult neural progenitors. The in vitro study of ATRA, however, is
complicated by its photo-isomerisation when used under standard
laboratory conditions. To try and overcome this, we have synthesised
ATRA retinoid analogues, EC23 and EC19, which unlike ATRA do not
isomerise in response to light or heat. These compounds could therefore be
potentially advantageous over ATRA for use in in-vitro investigations into
retinoid modes of action. Preliminary data show that EC23 elicits similar
cellular responses to ATRA when tested in vitro, whereas EC19 appears to
induce a higher percentage of glia in adult neural progenitor models. This
work will have the potential to aid research into pharmacological
manipulation of neural differentiation and its associated receptors for
potential therapeutic application.
6.09
Generic scaffolds for 3-dimensional in vitro neural cell co-culture
Bokhari M, Carnachan R, Cameron N, Przyborski S
School of Biological and Biomedical Science; Department of
Chemistry, University of Durham DH1 3LE; 3ReInnervate Limited, Old
Shire Hall, Old Elvet, Durham DH1 3HP
The structure and function of cultured cells are dramatically affected
by the micro-environment in which they are grown. Traditionally, two
dimensional (2-D) polystyrene surfaces are used to support cell
growth in vitro; however such surfaces do not enable the most
favourable cell growth and function. A more thorough understanding of
cell biology and cell-cell interactions requires three dimensional (3-D)
culture systems that more closely represent the natural structure and
function of tissues in vivo. Here we present a cell culture device that
provides a 3-D environment for routine cell culture. We have
developed a polystyrene scaffold which exhibits a well defined and
uniform porous micro-architecture and have adapted these threedimensional
scaffolds for cell culture and/or tissue engineering
applications. These scaffolds are readily adaptable to many different
types of tissue culture plastic-ware including 6- and 24-well plates.
These culture devices are pre-fabricated, sterile, easy to use and are
handled in a similar manner to standard 2-D plastic-ware. Our work
investigates the production of these polymers for routine 3-D cell
growth in-vitro, as well as neuron-glia co-culture. Synergistic effects of
scaffold micro-architecture combined with chemical and/or biological
stimuli on neuronal cell culture have not yet been explored in detail.
The aim of this work is to determine the synergistic effects of physical,
chemical and biological guidance cues on neuronal differentiation,
viability and function in an in vitro microenvironment which more
closely resembles the cellular microenvironment in vivo.
6.10
Neuritogenesis in adult hippocampal neurons in growth- permissive
versus inhibitory environments in vitro
Mellough C B 1, Wood A 2, Przyborski S A 1
1School of Biological and Biomedical Sciences, University of Durham,
South Rd, Durham, UK. 2 Wyeth Neuroscience, 865 Ridge Rd, Monmouth
Junction, New Jersey, USA.
The inability of the adult central nervous system to regenerate following
injury largely depends on the expression of myelin-associated inhibitors.
These ligands are present before the glial scar has formed and bind to the
p75NTR-NgR receptor complex on regenerating neurons, causing growth
cone collapse and axonal retraction. Progress is being made towards
elucidation of the downstream events which result in growth cone collapse
and retraction of the axonal cytoskeleton. The majority of studies that
investigate myelin inhibition employ neuronal populations derived from the
postnatal developmental period, or which lie in close apposition to the
regeneration-permissive peripheral nervous system in vivo. In this study,
the effects of myelin-associated glycoprotein (MAG) on neurite outgrowth
was assessed in a population of differentiating neurons derived from adult
hippocampal neural progenitor cells. We show that MAG does not alter
neural progenitor cell fate but, unlike their developmental counterparts,
neurite outgrowth from differentiating neurons was significantly attenuated
by MAG. We demonstrate that this effect can be partially overcome (by up
to 69%) by activation of the neurotrophin, cAMP and PKA pathways or by
Rho-kinase suppression. We also demonstrate that combining regeneration
promoting methods elicits enhanced neurite outgrowth from differentiating
neurons under myelin inhibitory conditions when compared with solitary
application. This work pertains especially to the facilitation of neural repair
in the compromised adult brain by endogenous mechanisms, such as the
mobilisation and appropriate integration of host stem cells for functional
replacement within depleted neuronal circuitry.
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7.01
Effect of NCAM-derived mimetic peptide, FGL, on aged-related
inflammatory changes in the rat hippocampus
Downer E J (1), Cowley T R(1), Berezin V A(2), Bock E(2), Lynch M
A(1)
(1) Trinity College Institute of Neuroscience, Trinity College, Dublin 2,
Ireland., (2) Protein Laboratory, Institute of Molecular Pathology,
Panum Institute, University of Copenhagen, Blegdamsvej 3C, 2200
Copenhagen N, Denmark.
Evidence suggests that age-related cognitive deficits are associated
with changes that are indicative of neuroinflammation, typified by an
increase in proinflammatory cytokine production and microglial cell
activation. Here we provide evidence that the neural cell adhesion
molecule (NCAM)-derived mimetic peptide, FG loop (FGL), exerts
anti-inflammatory effects in the hippocampus of aged rats. Male Wistar
rats (3-4 months and 22 months) were divided into control and FGLtreated
groups; FGL (8mg/kg) was administered subcutaneously on
alternate days for 3 weeks. Following treatment rats were
anaesthetized by intraperitoneal injection of urethane (1.5g/kg),
sacrificed humanely, the brains rapidly removed and the hippocampus
dissected free. Our data indicate that FGL attenuated the age-related
increase in expression of the proinflammatory cytokine interleukin-1ß
at protein and mRNA level. We also show that an age-related increase
in the expression of several markers of microglial activation, MHCII,
CD86, ICAM-1 and CD40, was reversed by FGL treatment. FGL was
shown to enhance the hippocampal concentration of insulin-like
growth factor-1 receptor (IGF-1R) and promote the transcription of the
IGF-1R ligand, insulin-like growth factor-1 (IGF-1), an event likely to
downregulate microglial activation. A modulatory role for FGL on IGF-
1R signalling is supported by evidence that FGL stimulated
extracellular-signal-related kinase (ERK) signalling in the rat
hippocampus. The inverse correlation between the markers of
microglial activation and the regulatory effect of FGL on IGF-1
signalling events suggests that FGL acts as an anti-inflammatory
agent in the rat hippocampus.
7.02
Pathological mutations of á-synuclein increase the rate at which it
forms isoaspartate protein damage and aggregates during in vitro
ageing.
Vigneswara V, Ray D E, Carter W G
MRC Applied Neuroscience Group,, School of Biomedical Sciences,,
University of Nottingham,, Queens Medical Centre,, Nottingham,, NG7
2UH., United Kingdom.,
The enzyme, protein isoaspartyl O-methyltransferase (PIMT), methylates
isoaspartate damage within proteins or peptides that accrues during protein
ageing and/or from stressful conditions, thereby triggering damage repair. A
proteomic strategy that involved detection of PIMT substrates which
accumulated within PIMT knockout brain tissue enabled us to identify novel
substrates for PIMT which included α-synuclein and β-synuclein
(Vigneswara et al., 2006). Formation of isoaspartate within α-synuclein and
β-synuclein could be mimicked by in vitro ageing of recombinant proteins.
α–Synuclein formed isoaspartate at approximately 1 % of molecules per
day, a rate approximately five times that of β–synuclein. Furthermore, after
20 days of in vitro ageing α-synuclein also formed isoaspartate-rich protein
aggregates. Inheritable mutant forms of α–synuclein that have been linked
to Parkinson’s disease pathology had both accelerated rates of isoaspartate
formation and protein aggregation during recombinant protein in vitro
ageing. Since protein aggregates of α–synuclein characterise (and are
presumably pathogenic for) Lewy body diseases such as Parkinson’s
disease, these results suggest that isoaspartate protein damage within α–
synuclein may contribute to its potential to aggregate and thereby disease
pathogenesis.
Vigneswara, V., Lowenson, J.D., Powell, C.D., Thakur, M., Bailey, K.,
Clarke, S, Ray, D.E., and Carter, W.G. (2006) Journal of Biological
Chemistry, 281, 32619-32629.
Acknowledgements: Financial support from the EU-supported
integrated project PROMEMORIA.
7.03
Aged animals show an increased threshold for LTP induction at
the perforant path synapses in vivo.
Cowley T R, Lynch M A
TCIN,, Trinity College,, Dublin 2,, Ireland
We investigated the effect of two high frequency stimulation (HFS)
protocols which are regularly used to induce LTP, 250 Hz and 400 Hz,
in young and aged rats. Rats were anaesthetised with urethane
(1.5g/kg) and recordings were made from the medial perforant path of
both hemispheres, applying the two HFS protocols contralaterally.
Analysis of fEPSP slope showed that 250 Hz-HFS in a sub-population
of aged rats failed to induce LTP, whereas these animals showed no
deficit in response to 400 Hz. We assessed pop-spike amplitude (PSA)
and found age related deficits even before titanic stimulation and
subsequently the magnitude of LTP induction was diminished after
either stimulation protocol.
Furthermore, analysis of input/output curves was used to assess the
excitability of the neurons in young compared to aged. fEPSP slope
showed no significant difference, but PSA amplitude and excitability
ratio showed significant impairments in aged compared to young rats.
These results suggest that there is an increased threshold required to
reach action potential and for the induction of LTP in a sub-population
of aged rats.
Paired pulse stimulation was performed before and after LTP induction
with both protocols and a significant increase in facilitation of the PSA
was evident only after 400 Hz-HFS at inter-stimulus intervals between
50-100 ms.
These findings indicate that whereas all aged rats exhibit LTP in
response to 400 Hz only a proportion similarly responded to 250Hz.
7.04
The effect of exercise on hippocampal function in young andaged
male wistar rats
O`Callaghan R M, Kelly A M
Trinity College Institute of Neuroscience and Department of Physiology,
Trinity College, Dublin 2, Ireland.
Cognitive impairment is a natural consequence of age. The hippocampus is
a highly plastic brain region that is particularly vulnerable to the aging
process. Neurodegenerative diseases such as Alzheimer’s disease and
other forms of dementia are accompanied by hippocampal dysfuntion and
cognitive alteration. Current research has shown that exercise may improve
neuronal function and possibly ameliorate the mental decline associated
with aging by conferring protection against neurodegenerative decline or
brain insult. Here we investigate the effect of a short period of forced
exercise on long-term potentiation (LTP) and spatial learning in young and
aged rats. We also explore the potential role of neurotrophin signalling in
mediating exercise-induced effects on the brain. All experiments conformed
with local and national guidelines.
Following an acclimatisation period and familiarization to the exercising
treadmill male Wistar rats (4 months and 22 months) were assigned to
control and exercising groups. The exercise protocol involved running
1km/day on a motorised treadmill for seven consecutive days. Control rats
were placed on a stationary treadmill for the same duration. A significant
enhancement in LTP in the dentate gyrus (evoked by high-frequency
stimulation of the perforant pathway in urethene-anaesthetized rats) was
recorded in both young and aged animals who had completed the exercise
protocol when compared with age-matched controls. In contrast, no
significant effect of exercise on spatial learning in the Morris watermaze was
observed in either young or aged populations. Parallel changes in
neurotrophin signalling pathways were analysed in samples of dentate
gyrus from all subject groups.
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8.01
Dietary flavonoids stimulate PI-3 kinase dependent antioxidant
response element activity in astrocytes
Bahia PK, Rattray M, Williams RJ
King`s College London, The Wolfson Centre for Age-Related
Diseases, Hodgkin Building, Guy`s Campus,, London SE1 1UL,
Flavonoids are a large family of plant derived polyphenolic compounds
with neuroprotective properties. Recent work suggests that in addition
to acting as hydrogen-donors, they may also activate several
protective signalling pathways to regulate gene expression. The
antioxidant response element (ARE) promotes the expression of
protective proteins including glutathione S-transferases. A luciferase
reporter (ARE-luc) assay was used to examine whether the dietary
flavan-3-ol (-)epicatechin activates this pathway in primary neurones
and astrocytes. In primary cortical astrocytes, but not in neurones, (-
)epicatechin (100 nM; 15min) and the human metabolite methylepicatechin,
stimulated ARE-luc activity. This response was inhibited
by wortmannin (150 nM) suggesting the involvement of a PI3-kinasedependent
pathway. Nrf2 is a transcription factor that binds to the ARE
to upregulate gene expression. The distribution of Nrf2 was examined
in neurones and astrocytes by immunocytochemistry. There was clear
Nrf2 immunoreactivity in cortical astrocytes but not in neurones. Nrf2
accumulated in the nucleus of astrocytes following exposure to the
positive ARE modulator tBHQ (100 ЧM) and, to a lesser degree, (-
)epicatechin (100 nM). Finally (-)epicatechin increased glutathione
levels in astrocytes consistent with an upregulation of ARE-mediated
gene expression. Together, this suggests that flavonoids may be
cytoprotective by increasing the levels of antioxidant genes in addition
to acting as classic free radical scavengers.
Supported by the BBSRC (D20463)
8.02
Does TNF alpha promote excitotoxic neuronal death through alteration
of AMPA receptor subunit composition
Rainey-Smith S R, Williams R J, Rattray M A N
King`s College London, Wolfson Centre for Age-Related Diseases, Guy`s
Campus, London,, SE1 1UL.
Amyotrophic Lateral Sclerosis (ALS), the most common motor neurone
disease in human adults, is characterised by progressive paralysis with
muscle wasting due to a selective degeneration of upper and lower motor
neurones. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)
receptor-mediated excitotoxicity has been strongly implicated as a
contributor to this selective motor neurone vulnerability.
This study aims to test whether the astrocyte-derived inflammatory cytokine
TNFα, produced normally in the spinal cord but upregulated in motor
neurone disease, sensitises cultured mouse motor and cortical neurones to
excitotoxic cell death by altering cell surface expression, subunit
composition, and hence, calcium permeability of AMPA glutamate
receptors.
Western blotting, immunocytochemistry and RT-PCR determined AMPA
receptor subunit GluR1-4 expression in 7 and 14 day old primary mouse
cortical neurones. In these neurones GluR1 and 2 were abundant, while
GluR3 and 4 were present at considerably lower levels. Exposure of 7 or 14
day old primary mouse cortical neurones to recombinant mouse TNFα (10
ng/ml; 15 min) increased GluR1, GluR2, and GluR4 levels in the membrane
fraction with an accompanying loss in cytosolic levels, determined by
immunoblotting. Cell-surface biotinylation experiments confirmed these
increases.
Immunocytochemistry confirmed SMI-32 positive motor neurones prepared
from embryonic mouse spinal cord express the GluR1, GluR2, GluR3, and
GluR4 subunits. Interestingly, exposure of these cultures to TNFα (10 ng/ml;
15 min) unmasked kainate-induced cell death.
Taken together, these findings, suggest that TNFα might confer vulnerability
of motor neurones to excitotoxicity by altering AMPA receptor subunit
composition.
8.03
Development of biomimetic substrates to control the attachment
and differentiation of neural cells
Cooke M J 1, Philips S 2, Shah D S 2, Athey D 2, Lakey J H 2,
Przyborski S A 1
1 School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham, DH1 3LE, UK, , 2 Nanotechnology Centre,
Herschel Building, Newcastle Upon Tyne, NE1 7RU, UK.
In situ cells are subject to many different signaling mechanisms,
including; cell-cell interactions, soluble molecules and their local
environmental geometry. In vitro model systems have been developed
to recreate the in vivo environment and control and regulate cellular
behavior using known signaling molecules. The extracellular matrix
(ECM) is composed of many such signaling molecules. Cell culture
substrates are often coated with ECM components such as laminin,
collagen and fibronectin to enhance cell adhesion and differentiation.
For example, these molecules have been shown to enhance neural
cell adhesion and increase process outgrowth. However the process
of coating substrates in this manner can be time consuming and has
limited control and reproducibility. In this study, we in collaboration
with Orla Protein Technologies, are developing novel biomimetic
substrates to control cell behavior in vitro. The effects elicited by ECM
molecules laminin, collagen and fibronectin can be attributed to motifs
within the molecules which can be presented to cells using a
biomimetic substrate. In this way we mimic the interaction between
cells and their local environment experienced in tissues. We have
evaluated the application of this technology on cell adhesion and
differentiation using the well established rat pheocytochroma (PC12)
cell line. Our data indicate that this technology can be used to control
cell attachment to glass substrates and demonstrate that this
approach can be used to control process outgrowth by PC12 cells
following nerve growth factor induced differentiation. We propose this
system as an alternative method for consistent and robust
differentiation of PC12 cells.
8.04
Physiological electric fields direct hippocampal neuron migration
cathodally
Yao L 1,2, McCaig C 1, Zhao M 1
1School of Medical Sciences, University of Aberdeen, Aberdeen, UK; 2
National Centre for Biomedical Engineering Science, National University of
Ireland, Galway, Ireland
Introduction
Initiation and effectiveness of neuron migration to the right places are critical
for the development and repair of central nervous system. Endogenous
electric fields (EFs) are widespread in developing and regenerating tissues.
Application of EFs directs cell migration of many types of cells. However,
whether applied EFs can direct neuron migration has not been
demonstrated.
Materials and methods
Dissociated rat hippocampal neurons and microexplants were cultured on
poly-L-lysine and laminin coated dishes and were identified with MAP-2
staining. The neurons were exposed to small applied electric fields and the
migration was recorded with an imaging system. Results
1. In a DC EF, dissociated rat hippocampal neurons migrate to the cathode.
The migration direction was reversed when the EF polarity was reversed.
2. The cathodal migration of hippocampal neurons is voltage and time
dependent. A DC EF does not have significant influence on neuronal
migration speed.
3. The guidance effect of EFs is also seen in neuronal migration from
hippocampal micro-explants. Neurons from micro-explants migrated to the
cathode.
4. EFs direct growth cone path finding and neurite orientation. Leading
process branching, turning and swapping over of leading and trailing
processes are the main types of neurite re-orientation.
Conclusion
This study demonstrates that EFs can direct rat hippocampal neuron
migration cathodally. This raises the possibilities that EFs may be used as a
potential cue to direct neuronal migration in repair of central nervous
system.
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8.05
Regulation of Neuronal Mitochondrial Transport by a GRIF-1 /
Miro1/ Kinesin Motor Protein Trafficking Complex
Macaskill A F, Kittler J T
Department of Physiology, University College London, Gower Street, ,
London, , WC1E 6BT
The transport of mitochondria to specific neuronal locations is
necessary to meet local cellular energy demands and for the buffering
of intracellular calcium. Although a critical role for kinesin motor
proteins in mitochondrial transport has been demonstrated, little is
known regarding the molecular mechanisms that regulate the specific
recruitment of motor proteins to mitochondrial cargo. Here we provide
evidence that the Mitochondrial Rho GTPase Miro1 acts as an
acceptor site for the recruitment of motor proteins to mitochondria in
nerve cells. Miro1 is highly expressed in hippocampal neurons and comigrates
with mitochondria along neuronal processes in live cells.
Furthermore, using immunofluorescence and co-immunoprecipitation
we demonstrate that Miro1 forms a protein complex with the adaptor
protein GRIF-1 (Trak2) and kinesin-1 in mammalian neurons. The
formation of this complex is dependent on a direct interaction between
Miro1 and the adaptor protein GRIF-1, which interacts directly with
kinesin family (KIF) 5 heavy chains. In agreement with this Miro1 can
recruit both GRIF-1 and kinesin-1 motors to mitochondria in both
neurons and HEK cells, dependent on its transmembrane domain. In
support of a critical role for this complex in regulating mitochondrial
transport, altering Miro1 function, or the Miro-dependent recruitment of
kinesin-1 motors, dramatically alters mitochondrial distribution along
neuronal processes. These results support an important role for Miro1
in regulating the kinesin motor protein dependent transport of
mitochondria in mammalian nerve cells.
9.01
Mitochondrial and plasma membrane potential of cultured cerebellar
neurons during glutamate induced necrosis, apoptosis and tolerance
Ward M W, Huber H J, Weisová P, Duessmann H, Prehn J H M
Department of Physiology and Medical Physics and RCSI Neuroscience
Research Centre, Royal College of Surgeons in Ireland, 123 St
Stephenâ€s Green, Dublin 2, Ireland. 2 Siemens Medical Division,
Siemens Ireland, Dublin 2, Ireland.
A failure of mitochondrial bioenergetics has been shown to be closely
associated with the onset of apoptotic and necrotic neuronal injury. Here,
we developed an automated computational model that interprets the single
cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a
consequence of changes in either the ΔΨm or ΔΨp, allowing for a
characterization of responses in populations of single cells and subsequent
statistical analysis. Necrotic injury triggered by prolonged glutamate
excitation resulted in a rapid monophasic or biphasic loss of ΔΨm that was
closely associated with a loss of ΔΨp, neuronal swelling and early
membrane lysis. Delayed apoptotic injury was induced by transient
glutamate excitation and resulted in a small, reversible decrease in TMRM
fluorescence followed by a sustained increase in TMRM fluorescence.
While the initial decrease was found to be caused by a ΔΨp depolarization,
the subsequent increase was due to a hyperpolarization of ΔΨm as
confirmed using the anionic probe DiBAC2(3). The hyperpolarization of
ΔΨm was sustained until a collapse of ΔΨm ensued (after 11.8 h ± 0.8h).
Statistical analysis identified two major correlations; neurons that displayed
a more pronounced depolarization of ΔΨp during the initial phase of
glutamate excitation entered apoptosis more rapidly, and those neurons
that displayed a more pronounced hyperpolarization of ΔΨm survived
longer. Indeed, neurons tolerant to transient glutamate excitation (18%)
showed the most significant increases in ΔΨm, indicating that mitochondrial
hyperpolarization is associated with survival responses during excitotoxic
injury.
9.02
Functional differences in GLT-1 splice variants
Peacey E, Rattray M, Lou Z, Kramer A, Dunlop J
Authors 1 & 2:, King`s College London, Wolfson Centre for Age-
Related Diseases, St. Thomas` St, London, SE1 1UL, , Authors 3, 4 &
5:, Wyeth Research, CN 8000, Princeton, NJ 08543
Glutamate transporter 1 (GLT-1) is expressed predominantly in
astrocytes and is responsible for up to 95% of glutamate uptake in the
mammalian CNS. Multiple N- and C-terminal splice variants of GLT-1
have been described, but are as yet functionally uncharacterised. It is
possible that the splice variants may show differences in expression,
multimerisation, uptake kinetics or degradation, and may play a
pathological role in conditions such as amyotrophic lateral sclerosis
(ALS), in which there is a significant down-regulation of GLT-1.
In order to address these questions, four GLT-1 splice variants
expressed in mouse cerebral cortex (termed MAST KREK, MAST
DIETCI, MPK KREK or MPK DIETCI according to amino acid
sequence) were cloned and epitope tagged. When transiently
expressed in COS-7 or HEK-293 cells, Western blots showed similar
expression of monomeric and trimeric bands for all splice variants. Coimmunoprecipitation
experiments using the tagged splice variants
indicated that they are capable of forming homomeric and heteromeric
trimers.
Pharmacological and kinetic characterisations of the splice variants
were carried out using stably transfected HEK-293 cells. The splice
variants were pharmacologically indistinguishable using any of the 12
glutamate transporter inhibitor compounds tested. [3H] glutamate
uptake studies showed that MPK DIETCI has significantly lower
affinity than the more common MAST KREK variant (Km = 46.5 ± 5.9
µM and 24.6 ± 0.6 µM respectively). This difference in affinity may be
of functional importance, especially if there is an alteration of
regulation of splice variant expression or degradation under
pathological conditions.
9.03
Histamine H4 receptor: the first evidence for CNS and PNS expression
and isoform hetero-oligomerisation
Shenton F C(1), Rijn Rv (2), Bakker R (2), Leurs R (2), Grandi D (3), Morini
G (3), Chazot P L (1)
1.Centre for Integrative Neuroscience, Durham University, UK;
2.Amsterdam/ Leiden University, The Netherlands; 3.Department of Human
Anatomy, Pharmacology and Forensic Medicine, University of Parma, Italy
The histamine H3R is found predominantly in the CNS. Herein, we provide
the first evidence that the H4R is also expressed on neuroendocrine cells,
and in selective structures within the rodent and human central and
autonomic nervous system. Both the H3R and H4R are GPCRs, which
undergo alternative splicing to yield shorter isoforms, with distinct
distribution patterns. The functional role of splicing is of great interest, and in
previous work, we have demonstrated the existence of rat and human H3
and human H4 homo-dimers using biophysical and immunobiochemical
techniques, with our novel selective anti-H3 and anti-H4 antibodies(1,2). We
have also reported the first evidence that the rat H3 isoforms act as activitydependent
dominant negative subunits in the brain(1). Using biophysical,
immunobiochemical and pharmacological experiments, we show that
human H4 isoforms homo- and hetero-dimerise in mammalian cells. The
hH4(302) and (67) isoforms alone did not bind [3H]histamine or transduce a
signal, and did not significantly affect the affinity of [3H]histamine binding to
the hH4(390), however both short isoforms reduced the level of binding by
55% and 30%, respectively. Surface biotinylation experiments showed that
all three isoforms can reach the cell surface when expressed alone, and
concur with the negative effects of the isoforms upon H4(390) surface
receptor number. Dominant negative action of splice isoforms appears to be
a general theme for both H3- and H4-histamine receptors, and may
represent a common regulatory system for GPCRs in the brain.
1. Mol Pharmacol. 69, 1194-1206.
2. Mol. Pharmacol. 70, 604-615
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9.04
Inducing neural differentiation of embryonal carcinoma stem
cells using natural and synthetic retinoids: screening,
characterisation and mode of action
Bridgens C E, Barnard J H, Collings J C, Cartmell E J, Goss H M,
Whiting A, Marder T B, Przyborski S A
School of Biological and Biomedical Sciences; Department of
Chemistry, Durham University, South Road, Durham, DH1 3LE;
ReInnervate Limited, Old Shire Hall, Durham, DH1 3HP.
Retinoids are a group of natural and synthetic molecules that are
structurally and/or functionally analogous to all-trans-retinoic acid
(ATRA), the major active metabolite of vitamin A. ATRA regulates a
broad range of essential processes during mammalian embryogenesis
and adult homeostasis, including vision and cellular differentiation,
proliferation and apoptosis. Consequently, retinoids have the potential
to be used in numerous clinical indications including treatment of
neurological tumours.
We have commenced a study directed at the synthesis of stable
alternatives to ATRA and investigation of the molecular pathways that
regulate cell development in response to natural and synthetic
retinoids. A small library of synthetic retinoids have been designed and
prepared, some of which are efficacious at inducing the differentiation
of human embryonal carcinoma (EC) stem cells into both neural and
non-neural cell types. The most potent compounds can up-regulate
antigens associated with neural phenotypes at comparable rates and
in similar patterns to that which is observed in response to ATRA.
These compounds are more stable than ATRA, which is susceptible to
photo- and thermal-isomerisation under laboratory conditions, and
thus provide a more convenient reagent to modulate reproducible
differentiation in cultured stem cells.
9.05
Investigating the role played by microtubule-associated protein 1–LC2
(MAP1-LC2) in AMPA receptor trafficking and targeting.
Hann V, Ives J H, Fung S, Payne H L, Thompson C L
School of Biological and Biomedical Sciences, Durham University, South
Road, Durham, DH1 3LE.
Stargazin is a member of the Transmembrane AMPA Receptor Regulatory
Protein (TARP) family. Stargazin has been shown to be involved in both cell
surface trafficking (extrasynaptic) and synaptic targeting of AMPA receptors.
The last six amino acids of stargazin, –RRTTPV interacts with PDZ domains
of the membrane-associated guanylate kinase family of proteins, in
particular PSD-95. This association is pivotal to stargazin’s ability to target
AMPA receptors to the synapse, disruption of this interaction by deletion of
the TTPV sequence eliminates synaptic targeting. However, cell surface
trafficking (extrasynaptic) of AMPA receptors is not affected thus this aspect
of stargazin-mediated processing is independent of interactions through the
TTPV domain and must therefore be governed by sequences up-stream of
the extreme C-terminus.
Using the entire intracellular C-terminus of stargazin as ‘bait’ in a yeast-2
hybrid screen of a mouse brain cDNA library we identified MAP1-LC2 as a
‘RRTTPV’ sequence-independent interactor. Anti-stargazin antibody
immunoaffinity purifications of Triton X-100 soluble cerebellar membranes
followed by MAP1A-LC2 and GluR2 pull-downs confirmed that these
proteins exist in a tripartite complex in vivo.
By yeast-mating assays using stargazin and MAP1A-LC2 deletion clones
we are narrowing down the interaction domains in order to use dominantnegative
approaches to interfere with this interaction in neurones to
evaluate its functional significance.
Funded by the BBSRC.
9.06
Evaluation of RNA preservation in the human post-mortem brain
tissue and its suitability for the application of molecular biological
techniques
Fernando S, Dexter D, Reynolds R
BrainNet Europe Consortium, Dept of Cellular & Molecular
Neuroscience, Imperial College Faculty of Medicine, Charing Cross
Hospital, London
Brain Net Europe is an FP6 Network of Excellence incorporating 19
established brain banks in Europe, whose main objective is to collect
and distribute well-characterised high quality post-mortem brain tissue
for basic research in neuroscience.
In this collaborative study we have evaluated the quality of RNA in
archived tissue samples of 155 human post-mortem brains obtained
from 8 different brain banks within the network. Brain tissue from
several neurological disorders and controls was included in the study
with post-mortem delays of up to 100 hrs and freezer intervals up to 7
years. The quality of isolated total RNA was analysed against age,
gender, post-mortem delay, freezer interval and CSF pH to establish
the effects of post-mortem variables. We have also examined regional
variation in RNA quality using tissue obtained from anatomically
defined areas of the brain. Additionally some of the samples were
analysed on different microarray platforms and a selected set of genes
studied by quantitative RT-PCR.
All samples yielded good quantities of RNA with varying quality and
>70% of samples were found to be of suitable quality for molecular
biological purposes. None of the above mentioned factors analysed
seemed to have a significant effect on the quality of RNA. We conclude
that intact RNA for molecular biology can be obtained from human
post-mortem brain tissue, even with long post-mortem delays and
freezer intervals. However, the successful expression of certain genes
from post-mortem brain tissue may require enhanced procurement
efforts to maximize RNA integrity.
9.07
Disrupted-In-Schizophrenia 1 (DISC1) and protein kinase A signalling
Nick Bradshaw, Shaun Mackie, Christie S, Porteous D, Millar K
Molecular Genetics, University of Edinburgh, Molecular Medicine Centre,
Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU
Disrupted-In-Schizophrenia 1 (DISC1) is a widely accepted risk factor for
schizophrenia and related psychiatric disorders. We have previously
demonstrated that DISC1 interacts with phosphodiesterase 4B (PDE4B),
an independently identified risk factor for psychiatric illness. Type 4
phosphodiesterases are of interest because they are homologous to the
Drosophila learning and memory mutant Dunce, consistent with the
cognitive deficits that characterise schizophrenia. Moreover PDE4s are
specifically inhibited by the prototypic antidepressant rolipram, and PDE4-
deficient mice behave as if on antidepressants. PDE4B hydrolyses cAMP, a
key signalling molecule in the Protein Kinase A (PKA) pathway. PDE4B
cAMP hydrolysing activity is regulated by PKA phosphorylation, forming a
negative feedback loop. Intriguingly, PDE4B binding to DISC1 is dynamic
and cAMP-dependent, suggesting that DISC1 sequesters PDE4B in a low
activity state until cAMP hydrolysing activity is required to switch off cAMP
signalling, at which time PDE4B is released. We now demonstrate that
DISC1 is also phosphorylated by PKA in vivo, suggesting that DISC1
function may be modulated indirectly by PDE4B. This interaction and
potential for regulation of two independently identified genetic risk factors
for psychiatric illness implies that they are key to psychosis-related
molecular pathways.
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9.08
Regulation of potassium channel genes in response to neuronal
activity
Mucha M, Mordaka P M, Dalle C, Wood I C
Institute of Membrane and Systems Biology, University of Leeds,
Leeds LS2 9JT, UK
Epilepsy is the second most common neurological disease and 1 in 20
individuals will suffer an epileptic event sometime during their lifetime.
Once a seizure has occurred an individual becomes more susceptible
to further seizures. Although it is known that this effect requires
changes in gene expression, the exact nature of those changes is not
understood. It is likely that changes in expression of genes that
regulate neuronal excitability would be important for this response to
seizure activity. To investigate how such genes are regulated we have
isolated the genes, KCNQ2 and KCNQ3, which encode two potassium
channel subunits of the M-channel and function to regulate neuronal
excitability. Mutations in either KCNQ2 or KCNQ3, resulting in
diminished function in humans are responsible for a form of epilepsy
indicating the important role of these subunits. We have used
bioinformatic analysis, DNase I sensitivity, gel retardation assays and
reporter gene assays to identify functional promoter elements within
the human KCNQ2 and KCNQ3 genes. We show that KCNQ2 and
KCNQ3 are regulated by at least some shared transcription factors
that respond to seizure activity and that expression levels of these
genes in cultured neurons are responsive to neuronal activity.
9.09
The phosphorylation status of the p65 NF-kB subunit determines
whether NF-kB promotes or inhibits neurite growth
Gutierrez H, O`Keefe G, Davies A
Humberto Gutierrez PhD , Research Associate., Cardiff School of
Biosciences,, Biomedical Sciences Building 3,, Museum Avenue,, PO Box
911,, Cardiff CF10 3US,, Wales, UK, e-mail:gutierrezh@cf.ac.uk, Tel:
+(44)29 20 87 51 76
NF-kB is a ubiquitously expressed transcription factor that has recently
been shown to promote the growth of neural processes in the developing
peripheral and central nervous systems. The NF-kB dimer is held in an
inactive form in the cytoplasm by a member of the IkB family of inhibitory
proteins, and NF-kB is normally activated by IkB kinase
(IKK)-mediated phosphorylation of IkB on serine residues 32 and 36,
resulting in ubiquitination and proteosome-mediated degradation of IkB and
nuclear translocation of the liberated NF-kB. Overexpressing the
common p65/p50 NF-kB dimer in nodose ganglion sensory neurons
cultured from newborn mice enhanced NF-kB transcriptional activity and
increased neurite growth. Paradoxically, overexpressing IKK2 (but not
IKK1) also
enhanced NF-kB transcriptional activity but strongly inhibited neurite
growth. Furthermore, both p65/p50 growth stimulation and IKK2 growth
inhibition were reversed by expressing an IkBa mutant that cannot be
phosphorylated on serine residues. In addition to phosphorylating IkBa,
IKK2 (but not IKK1) phosphorylated p65 on serine 536 in these neurons.
Overexpression of a p65 mutant (S536A) that cannot be phosphorylated at
this site restored growth in IKK2 overexpressing neurons. Overexpression
of a phosphomimetic p65 mutant (S536D) led to a strong inhibition of
neurite growth. Taken together these results suggest that NF-kB can both
stimulate and inhibit neurite growth depending on the phosphorylation
status of p65. The implications for other neural populations and
extracellular signals involving NFkB activation are discussed
9.10
A novel TNF-alpha autocrine loop activates the canonical NF-kB
pathway to limit NGF-promoted axonal growth in developing
neurons.
O’Keeffe G W, Gutierrez H, Gavalda N, Davies A M
Cardiff School of Biosciences, Museum Avenue, Cardiff CF10 3US,
Wales.
Multiple signals from various cells control the growth, guidance and
terminal sprouting of axons in the developing nervous system as they
grow towards and within their innervation targets. Here we report a
novel autocrine signalling loop involving TNF-alpha produced by the
neurons themselves. This autcrine signalling loop functions to
regulate NGF-dependant axonal growth during the postnatal
development of mouse sympathetic neurons.
We found that TNF-alpha, signalling through TNFR1 significantly
reduced NGF-dependant neurite growth in sympathetic neurons
during a "developmental window". In these neurons, TNF-alpha
activated NF-kB as measured by degradation and Ser32/36
phosphorylation of IkB-alpha and an increase in NF-kB dependant
gene transcription using a GFP-reporter NF-kB construct. Using
selective mutants of proteins involved in the canonical and noncanonical
NF-kB signalling pathways, we found that that selectively
inhibiting NF-kB activation through the canonical pathway prevented
the reduction in growth triggered by TNF-alpha in sympathetic
neurons.
These results describe a novel function and mechanism of action for
TNF-alpha during neuronal development and they reveal a
unsuspected autocrine signalling loop in developing neurons that acts
to limit the extent of NGF-dependent axonal growth.
10.01
Interleukin-1 exacerbates excitotoxic brain injury via a neutrophil
dependant mechanism
LM McCluskey, SM Allan
Faculty of Life Sciences, University of Manchester, UK
Interleukin-1 (IL-1) is a pro-inflammatory cytokine that exacerbates
neurodegeneration, including alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionate (AMPA) induced excitotoxic neuronal death. The aim of
this study was to test the hypothesis that neutrophils contribute to IL-1
mediated exacerbation of injury.
Male Sprague-Dawley rats were injected in the striatum with vehicle, AMPA,
IL-1 or AMPA plus IL-1 (A+IL-1), sacrificed 4 or 8h post-injection and serum,
brain and liver removed. Immunohistochemistry was performed to
investigate neutrophil recruitment and c-fos expression. Serum, brain and
liver cytokine-induced neutrophil chemo-attractant-1 (CINC-1) levels were
determined by ELISA. A separate cohort was pre-treated with neutrophil
depletion serum or normal rabbit serum (control) before intra-striatal
injection of A+IL-1. The animals were killed 24h later and whole blood and
brain removed. Brain lesion volume was calculated from cresyl-violet
stained sections. Immunohistochemistry was used to detect neutrophils
within the brain and circulating neutrophils were counted in the blood.
Injection of AMPA+IL-1 caused extensive neutrophil recruitment and c-fos
expression at 4 and 8h compared to injection of vehicle, AMPA or IL-1.
CINC-1 levels in the serum, brain and liver were induced to a similar extent
at 4h by IL-1 or A+IL-1, but not by vehicle or AMPA. Serum and brain CINC-
1 levels declined between 4 and 8h after IL-1 treatment, but remained
elevated at 8h after A+IL-1 treatment. Neutrophil depletion of animals prior
to injection of A+IL-1 reduced the resulting total lesion volume by 60%
compared to control.
These data suggest that IL-1 exacerbates excitotoxic brain injury by a
neutrophil dependant mechanism, possibly via CINC-1.
Page 16/101 - 10/05/2013 - 11:11:03

10.02
The effect of PAT (thymulin related peptide) on pain related
behavior and cytokine production in the brain in a rat model of
neuroinflammation.
Safieh-Garabedian B, Poole S, Mazih B, Saade N E
Neuroscience Program, American University of Beirut, Bliss Street,
Beirut, Lebanon. Parenterals Section, NIBSC,, Blanche lane, South
Mimms, Potters Bar, Herts EN6 3QG, UK, ,
Despite the relative protection of the brain, intracerebral inflammation
can be detrimental for neurons. Our aim in this study, was to
investigate the possible protective role of PAT against inflammation.
Brain inflammation was induced in Sprague-Dawley rats by
intracerebroventricular (ICV) injection of endotoxin (1µg/5µl). Separate
groups of rats (n= 5-6 each) were pretreated with different doses of
PAT (ICV; 0.2µg, 1µg and 5µg) 30 minutes before ET. One group of
rats was treated with PAT only and sham operated animals served as
controls. The effects of pretreatment with PAT were assessed on pain
related behavior and on the increased intracerebral levels of cytokines
induced by ICV injection of ET. Under deep anesthesia, tissue samples
were isolated from different brain areas (hippocampus, diencephalon
and brainstem) and were processed for the determination of
interleukins (IL)-6, IL-8, IL-10 and NGF concentrations by ELISA. ET
injection produced sustained thermal and cold hyperalgesia and
significant upregulation of the levels of cytokines and NGF.
Pretreatment with PAT prevented in a dose-dependent manner the ETinduced
hyperalgesia and altered the cytokine levels in different areas
of the brain. The results suggest that PAT might have an important
protective role of the brain against inflammation.
(Supported by grants from URB and Lebanese National Scientific
Research Council)
10.03
Systemic inflammation alters expression of the kynurenine pathway
enzymes indolamine 2,3-dioxygenase and kynurenine 3-
monooxygenase in rat brain
Connor T J, Starr N, Harkin A
Trinity College Institute of Neuroscience, University of Dublin, Trinity
College, Dublin 2, Ireland
Indoleamine 2,3-dioxygenase (IDO) is the rate limiting enzyme in the
kynurenine pathway; the main pathway for tryptophan metabolism in the
brain. Here we demonstrate that systemic administration of the
inflammagen bacterial lipopolysaccharide (LPS) to rats induces IDO mRNA
expression in cortex and hippocampus 4hr post administration. Induction of
IDO was associated with a large increase in expression of the proinflammatory
cytokines TNF-alpha and IL-6, with a modest increase in IFNgamma
mRNA expression. Following its synthesis by IDO, kynurenine can
be further metabolised down one of two pathways. Specifically, the enzyme
kynurenine-3-monooxygenase (KMO) metabolises kynurenine into the free
radical generator 3-hydroxykynurenine; a compound the can be further
metabolized to form the excitotoxic N-methyl-D-aspartate receptor agonist
quinolinic acid. Alternatively, the enzyme kynurenine aminotransferase
(KAT II) metabolises kynurenine into kynurenic acid; a neuroprotective
compound with NMDA receptor antagonist properties. Consequently,
maintaining an appropriate balance between these two arms of the
kynurenine pathway is critical for a healthy brain. Here we demonstrate that
LPS suppressed KMO mRNA expression 4hr post administration, with a
rebound increase observed at 24hr post LPS. In contrast, LPS failed to
alter KAT II expression. This is the first demonstration that an inflammatory
stimulus impacts upon expression of KMO; an enzyme that drives
kynurenine metabolism in a neurotoxic direction. We speculate that the
reduction in KMO expression observed 4hr post LPS may represent a
physiological mechanism to limit production of neurotoxic products
following IDO induction in response to an inflammatory insult.
10.04
Noradrenaline reuptake inhibitors inhibit neuroinflammation
induced by a systemic inflammatory challenge
O`Sullivan J B, Harkin A, Connor T J
Trinity College Institute of Neuroscience, University of Dublin, Trinity
College, Dublin 2, Ireland.
Evidence suggests that the monoamine neurotransmitter
noradrenaline elicits anti-inflammatory actions in the central nervous
system (CNS), and consequently may play an endogenous
neuroprotective role in CNS disorders where inflammatory events
contribute to pathology. In line with this hypothesis, we demonstrate
that noradrenaline suppresses expression of the pro-inflammatory
cytokines IL-1beta and TNF-alpha and induction of iNOS/nitric oxide
production from mixed glial cultures prepared from rat cortex, in
response to the inflammagen bacterial lipopolysaccharide (LPS). As
previous studies indicate that the noradrenaline reuptake inhibitor
(NRI) desipramine has anti-inflammatory properties, we examined the
ability of desipramine and more selective NRI’s to alter glial proinflammatory
cytokine production. However, treatment of mixed glial
cells with NRI’s largely failed to alter inflammatory events induced by
LPS. In contrast to the in vitro situation, acute in vivo treatment of rats
with NRI’s elicited an anti-inflammatory effect in the CNS
characterised by reduced mRNA expression of the pro-inflammatory
cytokines IL-1beta and TNF-alpha and iNOS in cortex in response to
systemic LPS administration. The data also suggest that in vivo
treatment with NRI’s inhibited microglial activation in the cortex
indicated by reduced expression of the microglial activation makers
CD40 and CD11b. These data indicate that NRI’s do not have a direct
modulatory effect on the inflammatory response in glial cells, however
when administered in vivo can limit inflammatory events in the brain.
Overall, this study has yielded significant insights into the ability of
noradrenaline augmentation strategies to limit neuroinflammation.
10.05
The PPARã agonist, rosiglitazone, attenuates LPS-induced changes in
vitro in an IL-4-dependent manner.
Griffin R J, Loane D J, Deighan B F, Lynch M A
Trinity College Institute of Neuroscience, Physiology Department, Trinity
College, Dublin 2, Ireland.
Inflammatory changes in the brain contribute to neuronal deficits associated
with age and neurodegenerative conditions. These changes include
activation of microglia and the associated release of proinflammatory
cytokines. Evidence from this and other laboratories suggest that
rosiglitazone, a selective peroxisome proliferator-activated receptor gamma
(PPARγ) agonist, exerts an anti-inflammatory action in brain.
We investigated the modulatory effect of rosiglitazone on LPS-induced
changes in cultured rat cortical glia and report that it significantly reduced
the LPS-triggered increases in interleukin-1β (IL-1β) and tumour necrosis
factor-α (TNFα) (p < 0.001). The mechanisms underlying the antiinflammatory
effects of rosiglitazone are not thoroughly understood but our
data show that treatment with rosiglitazone increases hippocampal
concentration of the anti-inflammatory cytokine IL-4. To address this further,
we investigated the effect of rosiglitazone on LPS-induced changes in
primary glial cells prepared from 1-day old wild-type and IL-4-/- mice. The
data show that rosiglitazone attenuates the LPS-induced increase in IL-1β
concentration (p < 0.05) and the expression of major histocomaptabilty
complex class (MHC) II mRNA (p < 0.05) in cells prepared from in wild-type
but not IL-4-/- mice. In contrast, rosiglitazone attenuated the LPS-induced
increase to a similar extent in both preparations and had no effect on the
LPS-induced increase on IL-6. Our findings confirm an anti-inflammatory
role for rosiglitazone and suggest that the action may be mediated by IL-4.
Acknowledgements: Science Foundation Ireland, The Higher Education
Authority Ireland (PRTLI), G03007 8AA.
Page 17/101 - 10/05/2013 - 11:11:03

10.06
IL-4 increases neuronal expression of CD200 and thereby inhibits
microglial activation.
Lyons A, Downer E, Lynch M
Trinity College Institute of Neuroscience, Trinity College, Dublin 2.
Ireland
Several neurodegenerative disorders are associated with evidence of
inflammation, one feature of which is an increase in activation of
microglia, the most likely cell source of inflammatory cytokines like
interleukin-1β (IL-1β). It has been proposed that interaction of the
microglial cell surface receptor, CD200R, with its ligand, CD200, which
is expressed on several cell types including neurons, maintains
microglia in a quiescent state. Data from several studies have shown
that Aβ increases microglial activation and we have previously reported
that IL-4 attenuates this change. Here we assessed the role of CD200
ligand-receptor interaction in modulating inflammatory changes
induced by Aβ or lipopolysaccharide (LPS) and investigated the
possible contribution of IL-4 in this. Incubation of glia in the presence of
Aβ and LPS increased MHCII mRNA expression and the concentration
of proinflammatory cytokines. These changes were coupled with a
significant reduction on CD200 protein, although CD200 mRNA
expression was unchanged. The addition of neurons to Aβ- or LPSstimulated
glia inhibited these changes; significantly we observed that
the effect of neurons was attenuated by addition of a CD200
neutralizing antibody, highlighting a role for CD200 ligand-receptor
interaction in modulating microglial function. We observed that IL-4
increased CD200 expression. However, CD200 expression was
decreased in neurons prepared from IL-4 knockout mice in which there
was an inflammatory phenotype. These data suggest the mechanism
by which IL-4 attenuates inflammatory changes in the brain may be a
consequence of its ability to upregulate CD200 which, in turn acts to
maintain microglia in a quiescent state.
10.07
Modulation of glia by interaction with T cells
Murphy A, Lalor S, Lynch M, Mills K
Institute of Neuroscience, Lloyd Building, Trinity College Dublin, Dublin 2,
Ireland
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of
the central nervous system characterised by a persistently activated
microglial state. Studies in experimental autoimmune encephalomyelitis
(EAE), an animal model of MS, have shown that IFN-γ-secreting Th1 or IL-
17-secreting Th17 cells may mediate pathology. Furthermore, there is
evidence that microglia may become activated by invading peripheral T
cells.
In this study we assessed the effect of distinct T cells subtypes on
microglial activation. We compared the ability of antigen-specific Th1 and
Th17 cells to activate microglia via cell: cell contact or by secretion of
soluble factors. Microglial activation was assessed by measuring cell
surface expression of MHC class II, CD80 and CD86 (using flow cytometry)
and production of proinflammatory cytokines TNF-α and IL-6.
Th1 cells were found to be more potent activators of microglial cells than
Th17 cells, inducing greater upregulation of MHC class II, CD80 and CD86.
This activation required T cell:glial cell contact as soluble factors secreted
by T cells were not sufficient to upregulate cell surface marker expression.
Th1 cell contact with the glial cells resulted in greater TNF-α and IL-6
production than Th17 contact. Data will be presented indicating that Th1
cell: glial cell contact may play a significant role in microglial activation in
EAE.
10.08
Altered fractalkine expression in hippocampus of LPS-treated
aged rats
Lynch A M, Lynch M A
Trinity College Institute of Neuroscience,, Department of Physiology,,
Trinity College,, Dublin 2,, Ireland.,
It is well documented that microglial-driven inflammation is a feature
of the aged brain. There is also evidence which indicates that an
additional insult to aged animals, can exacerbate the inflammatory
status. We demonstrate that age-related neuroinflammation is further
exacerbated in lipopolysaccharide-treated (100ug/kg) aged Wistar
rats; increased expression of cell surface markers of activated
microglia (major histocompatibility complex II and CD40) and
increased pro-inflammatory cytokine production (IL-1beta), are
evident.
One way that CNS homeostasis is maintained is by cell-cell
interactions between neural cells. The chemokine fractalkine which is
expressed in neurones, binds with its microglial receptor and
maintains microglial functionality in a resting state. We present data
which indicates that fractalkine can dose-dependently modulate
microglial activation, and provide evidence which demonstrates that
fractalkine concentration is altered in the aged hippocampus.
Financial Support: Science Foundation Ireland
10.09
Meningeal B-cell follicles are associated with increased cortical
pathology and disease severity in secondary progressive multiple
sclerosis.
Magliozzi R (1, 2), Howell O (1), Nicholas R (1), Vora A (1), Fernando S (1),
Puopolo M (2), Serafini B (2), Aloisi F (2), Reynolds R (1)
(1)Department of Cellular and Molecular Neuroscience, Imperial College
London, London, UK, (2)Department of Cell Biology and Neuroscience,
Istituto Superiore di Sanità, Rome, Italy
Although multiple sclerosis (MS) is widely regarded as a white matter
disease, there is also evidence of extensive grey matter (GM) pathology. In
contrast to white matter lesions, purely cortical lesions have very few
inflammatory infiltrates, indicating that the mechanisms mediating GM
damage might differ qualitatively and/or quantitatively. During a
comprehensive immunohistochemical and morphometric analysis on postmortem
cerebral tissue from 29 secondary progressive (SP), 4 progressive
relapsing (PR) and 7 primary progressive (PP) MS cases, we noted that 12
out of 29 SPMS patients exhibited aberrant lymphoid-like structures in the
inflamed meninges. These ectopic B-cell follicles, resembling secondary
lymphoid organs, comprised proliferating B cells, plasma cells, fewer T cells
and a well organized network of follicular dendritic cells. Cortical GM from
SPMS cases had an increased number and extent of demyelinated lesions
and increased microglial activity and axonal damage compared to cases
without follicles. We have also detected significant differences in cortical
thickness and cell density in the cerebral cortex between the SP MS cases
with and without follicles. Interestingly, the SPMS cases with follicles
differed from SPMS cases without follicles with respect to gender (3:1
female: male), having a younger age at onset of MS (24 vs 34yrs) and at
death (42 vs 55yrs) and shorter time to wheelchair dependence (33 vs
47yrs). These data show a strong association between the presence of
meningeal follicles and increased cortical pathology in a subset of MS
patients and suggest a role for these abnormal structures in MS
pathogenesis and clinical progression.
Page 18/101 - 10/05/2013 - 11:11:03

10.10
Immuno-regulatory T cell function in patients with multiple
sclerosis undergoing autologous hematopoietic stem cell
transplantation
Abrahamsson S, Packer A, Oh U, Burt R K, Muraro P A
Department of Cellular and Molecular Neuroscience, , Division of
Neuroscience and Mental Health, , Imperial College Faculty of
Medicine, , Charing Cross Hospital, , St. Dunstan`s Road,, London W6
8RP, UK, and , , Neuroimmunology Branch, NINDS, National Institutes
of Health, Bethesda, MD 20892 USA
Autologous hematopoietic stem cell transplantation (HSCT) is a novel
experimental approach to treat multiple sclerosis (MS). Clinical trials
have shown promising results, with prolonged suppression of brain
inflammation. The positive clinical outcome of HSCT is likely to be due
to the reprogramming of the immune system and its substitution with a
new repertoire of cells, originated from precursors that repopulate the
bone marrow. However, since autoreactive cells may also be
regenerated in susceptible individuals, we hypothesize that
reconstitution of immune regulatory circuits could be an important
contributing factor to the positive clinical outcome of HSCT. Regulatory
cells that keep self-reactive cells under control to prevent autoimmune
reactions include a T cell subset identified by a CD4+CD25+
phenotype and intracellular expression of FoxP3. Regulatory
properties have also been suggested for CD8+ T cell subpopulations.
We assessed numerical recovery of immune cells in the peripheral
blood of patients with severe MS who underwent HSCT and observed
significant increases in the frequency of CD4+FoxP3+ and of
CD8+CD28-CD57+ cells, with demonstrated or suggested regulatory
properties. FoxP3-expressing CD4+ T cells were transiently upregulated
in patients at 6 months post-transplant. CD28-CD57+ T cells
were massively increased, constituting up to >80% of the CD8+ T cell
repertoire and persisting at high frequency for the entire duration of a
2-year follow-up post-transplantation. This suggests that these cells
may have a role in determining the positive clinical outcome of HSCT
therapy. We are examining the suppressive functions of CD8+CD28-
CD57+ T cells by in vitro co-culture and cytotoxicity assays.
10.11
Small molecule VLA-4 antagonist suppresses MOG-EAE in the DA rat
Papadopoulos D, Rundle J L, Patel R, Gonzalez I M, Reynolds R
Cellular & Molecular Neuroscience, Imperial College London, Charing
Cross Hospital, W6 8RF
Interaction of VLA-4 (α4β1 integrin) with its ligand vascular cell adhesion
molecule-1 (VCAM-1) is required for CNS migration of encephalitogenic T-
cells in EAE. Blockade of VLA-4 has been shown to be a beneficial target
for therapeutic intervention in Multiple Sclerosis (MS) both in preclinical
trials in EAE and in clinical trials in MS. This study sought to investigate the
efficacy of a small molecule VLA-4 antagonist (BIO5192) in a relapsingremitting
model of antibody-mediated inflammatory demyelination.
Chronic-relapsing EAE was induced in female DA rats by immunization with
recombinant mouse MOG. A group of 25 rats was treated from day 7 postimmunization
with BIO5192 and a control group of equal size was treated
with vehicle. Demyelination was assessed with LFB staining and
inflammatory activity, neuronal and axonal loss were assessed with CD45,
OX-42, NeuN and neurofilament immunostaining respectively.
BIO5192 ameliorated EAE reducing disease severity, relapse rate and
cumulative disease index. Pathological examination revealed that BIO5192
treatment prevented demyelination and CD45+ and OX-42+
immunoreactivity was reduced by 81% and 58% respectively in BIO5192-
treated rats. In addition, BIO5192 treatment decreased the extent of axonal
loss in the medial dorsal funiculus by 35% and neuronal loss in the upper
lumbar cord by 16.8%. No post-treatment exacerbation was observed
following BIO5192 withdrawal.
Our data suggests that WBC trafficking in MOG-EAE in the DA rat is VLA-4
dependent. Along with all other similarities with MS in pathology, this
common immunopathogenetic mechanism renders MOG-EAE in the DA rat
a valid and useful model for testing potential MS therapies.
10.12
TNF-alpha blockade by etanercept reduces neutrophil
recruitment into the injured brain through attenuation of the
acute phase response
Jiang Y, Campbell S J, Farrands R, Anthony D C
Experimental Neuropathology, Department of Pharmacology,
University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK
The release of inflammatory mediators by recruited leukocytes after
head injury is a significant cause of secondary damage to neurons.
An important local mediator of the host response to injury is the
cytokine TNF-alpha, which has proven to be a good target for
treatment of peripheral inflammatory disease. However, the blood
brain barrier restricts the access of high molecular weight TNF-alpha
inhibitors and limits their use for CNS pathologies. Our recent data has
shown that the liver produces TNF-alpha as part of the acute phase
response to CNS injury, and it may overcome the need for inhibitors to
cross the blood brain barrier. Here we show that microinjection of IL-
1beta into the brain results in the acute upregulation of TNF-alpha in
the liver, but not in the brain. The subcutaneous administration of
etanercept, a TNF-alpha antagonist, inhibited the production of hepatic
TNF-alpha and of TNF-alpha- induced genes, including chemokines
CCL-2, CXCL-5 and CXCL-10. There was also a significant reduction
in the number of neutrophils in the liver and in the circulation. As a
consequence, the number of neutrophils recruited to the liver, and
more importantly to the IL-1beta- challenged brain were markedly
reduced in our brain injury model. In summary, our findings suggest
that therapeutics which target hepatic TNF-alpha have a potential to
inhibit CNS inflammation without the necessity to cross the blood brain
barrier.
10.13
Inflammatory stimuli exert a more profound effect in tissue prepared
from IL-4 knockout mice
Mc Quillan K, Lyons A, O`Connell F, Lynch M A
Trinity College Institute of Neuroscience, Department of Physiology, Trinity
College Dublin, Dublin 2, Ireland
Among the changes that occur in the brain in response to stressors is an
increase in microglial activation, resulting in the release of proinflammatory
cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α
(TNFα). IL-1β is the most studied cytokine in the brain and its actions are
known to be mediated through the signaling receptor IL-1R1. Recent
investigations have indicated that IL-4 down-regulates microglial activation
and therefore decreases release of proinflammatory cytokines. Previous
data from this laboratory have indicated that IL-4 acts as a negative
regulator of IL-1 and lipopolysaccharide (LPS) signaling. In this study, we
prepared mixed glial cultures from wild-type IL-4-/- mice to establish
whether the absence of IL-4 resulted in a more pronounced inflammatory
response to lipopolysaccharide (LPS). We report that LPS increased
expression of several markers of microglial activation including TNF-α and
CD86 and that this activation was more pronounced in mixed glia prepared
from IL-4-/- compared with wild-type mice. The evidence suggests that a
similar inflammatory phenotype exists in vivo. Data will be presented which
indicates that, at least in the case of some measures, amyloid β also
induces a more profound response in tissue prepared from IL-4 knockout
animals.
Page 19/101 - 10/05/2013 - 11:11:03

10.14
The PPARgamma agonist, rosiglitazone attenuates microglial
activation in vitro by increasing IL-4.
Deighan B F, Loane D J, Lynch A M, Lynch M A
Department of Physiology, Trinity College Dublin, Dublin 2, Ireland
Microglia, the immune cells of the brain, are activated in the aged
brain and are the source of pro-inflammatory cytokines that contribute
to the change in the inflammatory profile of the brain with age. Recent
data from this lab have shown that rosiglitazone, a peroxisome
proliferator activated receptor gamma (PPARγ) agonist, modulates
inflammation in the hippocampus of aged rats.
In this study the effect of rosiglitazone treatment on mixed glial cells
that had been treated in the presence or absence of
lipopolysaccharide (LPS) was assessed. LPS significantly increased
expression of major histocompatabilty complex class (MHC) II mRNA
which is an indicator of microglial activation and this was accompanied
by a significant increase in pro-inflammatory cytokines such as IL-β.
The LPS induced increase in MHC II expression and IL-1β was
significantly attenuated by pre-treatment of cells with rosiglitazone (p

11.04
Expression profile of mesenchymal stem cells differentiated into
schwann cells
D Mahay, Shawcross S, Terenghi G
Blond McIndoe Research Laboratories,, The University of Manchester,
UK
Schwann cells (SC) are essential facilitators of peripheral nerve
regeneration following injury as they provide physical support and
guidance. In vitro these supporting cells are slow growing, hence not
well suited to a tissue engineering approach to nerve repair. We
differentiated rat bone marrow mesenchymal stem cells (MSC) into
SC-like cells using an established cocktail of growth factors. Semiquantitative
RT-PCR, Western blotting and immunocytochemistry
were used to detect glial cell marker as well as neurotrophic factors
transcripts and assess protein expression in MSC in comparison with
SC (positive control). The presence of the transcripts and proteins:
glial fibrillary acidic protein, low affinity neurotrophin receptor p75,
calcium binding protein S100 and the intermediate filament protein
nestin were investigated. Also investigated were transcripts of
neurotrophic factors: brain derived neurotrophic factor, glial derived
neurotrophic factor, ciliary neurotrophic factor, leukaemia inhibitory
factor, neurotrophin-3 and neurotrophin-4. Differentiated MSC
expressed transcripts of S100, nestin, glial derived neurotrophic factor,
leukaemia inhibitory and higher levels of brain derived neurotrophic
factor compared to SC. Likewise, MSC expressed the S100 protein
and higher protein levels of brain derived neurotrophic factor in
comparison to SC. Also, MSC and SC immunostained positively for
S100, glial fibrillary acidic protein, brain derived neurotrophic factor
and glial derived neurotrophic factor. Thus, the results indicate MSC
differentiated into SC-like cells appear to have similar morphological
and phenotypic characteristics to SC.
11.05
Adipose-derived stem cells differentiated into a schwann cell
phenotype promote neurite outgrowth in vitro
Kingham P J, Kalbermatten D F, Terenghi G
(1,2,3) Blond McIndoe Research Laboratory, The University of Manchester,
UK, (2) Department of Plastic, Reconstructive and Aesthetic Surgery,
University Hospital Basel, Switzerland
Peripheral nerve repair can be enhanced experimentally by transplanting
Schwann cells at the site of injury. The clinical application of this therapy is
however limited by donor site morbidity and the inability to generate a
sufficient number of cells in a short space of time. As an alternative, we
have investigated whether adult stem cells, isolated from adipose tissue,
can be differentiated into functional Schwann cells. Rat visceral fat was
enzymatically digested to yield rapidly proliferating fibroblast-like cells, of
which approximately 10% were positive for the stem cell markers, stro-1
and nestin. When the cells were treated with a mixture of glial growth
factors (GGF-2, bFGF, PDGF and forskolin) they adopted a bipolar
morphology similar to Schwann cells. Immunocytochemical staining
indicated that these cells also expressed the glial markers, S100 and
GFAP. In order to assess the function of these cells they were co-cultured
with the NG108-15 motor neuron-like cell line. Measurements of neurite
outgrowth were made using a computerised image analysis programme.
Differentiated stem cells released soluble factors which significantly
increased 3-fold the number of neurites expressed per NG108-15 cell
(p

11.08
Chloride intracellular channel 1 (clic1) plays a central role in
regulating free radical generation by microglia in response to
Beta-amyloid
Milton R H, Abeti R, Duchen M R, Mazzanti M
Deaprtment of Phsyiology, University College London, , Gower Street,
WC1E 6BT, , Dipartimento di Scienze Biomolecolari e Biotecnologie,
Universita` degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy,
β-Amyloid (Aβ) peptides accumulate in the brain in Alzheimer’s
Disease (AD) and are implicated in its pathogenesis. Microglia, the
immune cells of the CNS, are activated by Aβ and in response,
produce reactive oxygen species (ROS) through activation of the
plasmalemmal NADPH oxidase. Generation of ROS by microglia is
thought to contribute to the cell death seen in AD. We have previously
shown that Aβ activates a chloride current mediated by the protein
CLIC1 (Novarino et al, 2004). We now show that Aβ-induced
microglial ROS production is contingent upon the channel activity of
CLIC1. Rates of ROS generation were measured using hydroethidium
fluorescence from BV2 and primary microglial cells. Suppression of
CLIC1 protein expression using siRNA, inhibition of the CLIC1 chloride
current using IAA-94 or using an antibody to the channel protein, and
replacement of extracellular Cl- with impermeant anions, all
significantly reduced the ROS response to Aβ (p

12.01
Use of capillary chip based patch-clamp platform to study
molecular details of Kir channel gating
Kurata H T, Nichols C G
Dept of Cell Biology and Physiology,, Washington University School of
Medicine, , 660 South Euclid Avenue, Saint Louis, MO 63110, USA
Intracellularly applied blockers can be ‘trapped’ by voltage-dependent
channel closure of voltage-gated (Kv) potassium channels; seminal
evidence that the voltage-operated gate lies near the intracellular
opening of the channel, and closes an inner cavity around the
blockers. Similar ‘blocker-trapping’ experiments on ligand-gated Kir
channels are less straightforward, due to difficulties in achieving rapid
control of channel gating. To overcome these difficulties we used the
rapid solution-switching capabilities of the Dynaflow system
(Cellectricon Inc.), which requires very small solution volumes, and
allows full temporal control of both solution switching and voltage. We
have demonstrated that ATP-induced closure of KATP channels
prevents the dissociation of spermine from the pore, showing that
closure of the ligand-operated gate similarly isolates an inner cavity
that can accommodate a spermine molecule. We have examined the
trapping of a panel of compounds of differing lengths, and now report
that longer ‘extended’ synthetic polyamines, which share the same
voltage-dependence of block as spermine, are not trapped by ATPinduced
channel closure. These data suggest that extended
polyamines prevent channel closure in the presence of ATP. The
demonstrated length-dependence for trapping allows us to use
extended polyamines as coarse-scale molecular calipers of channel
dimensions. Computational simulations of polyamine blockade, in
models of inward rectifier channels based on crystal structures of
KirBac1.1, are consistent with the constraints on the inner cavity
dimensions imposed by this analysis. These findings illustrate the
essential similarity of ligand-dependent and voltage-dependent gating
in Kir and Kv channels, respectively.
12.02
Two-pore domain potassium channels enable sustained highfrequency
firing.
Brickley S G, Aller M I, Veale E L, Sandu C, Alder F G
Biophysics Group, Division of Cell and Molecular Biology, Imperial College
London, UK.
The ability of neurons to fire at high frequencies during sustained
depolarization is generally explained in terms of the properties of voltagegated
ion channels. By contrast, two-pore domain potassium (K2P)
channels impart a voltage-independent, non-inactivating, leak conductance
that hyperpolarizes the resting membrane potential (RMP) by increasing
the membranes potassium permeability. In addition to a depolarised resting
membrane potential, we find that cerebellar granule neurons (CGNs)
lacking the TASK-3 type K2P channel exhibit marked accommodation of
action potential firing. The accommodation phenotype was not associated
with any change in the functional properties of the underlying sodium
channels, nor could it simply be explained by the more depolarized resting
membrane potential that resulted from this K2P channel deletion. A
functional rescue experiment utilizing a dynamic current clamp approach to
mimic a non-linear conductance of the type lost in these mice was able to
restore wild-type firing properties to adult TASK-3 KO CGNs. We propose
that the TASK-3 conductance enables more sodium channels to be
available for activation, and so helps CGNs to fire at high frequencies in
response to sustained depolarization. This represents an unappreciated
contribution of K2P channels to neuronal function as TASK-3 channels
serve to both select and support high frequency firing.
12.03
Modulation of functional L-Type calcium channels by the
microtubule associated protein tau.
Montgomery J R, Marrion N V
Department of Pharmacology, School of Medical Sciences, University
Walk, Bristol, BS8 1TD
L-type Ca2+ channels are a subtype of voltage-gated Ca2+ channels
characterised by pharmacological sensitivity to dihydropyridines.
Cav1.2 and Cav1.3 pore forming subunits produce L-type currents
when expressed with auxiliary subunits. Both channel isoforms are
present in a number of neurons, including hippocampal neurons,
where they regulate many processes including activation of Ca2+activated
channels and gene transcription.
The cytoskeletal protein α-actinin2 is involved in association of L-type
channels with the Ca2+-activated K+ channel SK2 in cardiac
myocytes. It is proposed that functional coupling of L and SK channels
in hippocampus may also require interaction with a cytoskeletal
protein. One such protein is the microtubule associated protein tau,
which is abundant in hippocampus, with Cav1.3 and phosphorylated
tau sharing somatic subcellular localisation in hippocampal neurons.
Full length human tau (4R2N) altered function of L-type channels
composed of Cav1.3, Cavß3 and Caα2δ1subunits. The activation
curve for this channel complex was shifted to more depolarised
potentials in the presence of hTau4R2N, with no effect on current
amplitude. The effect of hTau4R2N was subunit specific because no
effect was observed on currents composed of Cav1.3, Cavß2A and
Cavα2δ1 or Cav1.2, Cavß3 and Cavα2δ1. A second human tau
isoform, 4R1N, which lacks an amino-terminal insert, was found to
have no effect on activity of any of the subunit combinations above.
13.01
Changes in protein phosphorylation reveal that different pools of
synaptic vesicles can release by distinct exocytotic modes
Green J E (1), Sihra T S (2), Ashton A C (1)
(1) Biological Sciences, University of Central Lancashire, Preston, UNITED
KINGDOM, (2) Pharmacology, University College London, London,
UNITED KINGDOM.
Exocytosis was studied in rat cerebrocortical synaptosomes maximally
loaded with FM2-10 styryl dye under conditions that labelled all releasable
synaptic vesicles (SVs). Greater FM2-10 release was induced upon
stimulation with various stimuli after pre-treatment with the protein
phosphatase inhibitor okadaic acid (OA) than in untreated controls. FM dye
release from individual synaptosomes was measured by confocal
microscopy, which demonstrated that OA changes the release kinetics of
individual terminals, and does not merely induce the exocytosis of some
non-releasing synaptosomes. However, release of ATP (a neurotransmitter
present in many terminal types) evoked by these same stimuli showed no
enhancement by OA. This indicates that FM2-10 dye release does not
accurately reflect the true amount of neurotransmitter secretion, as
maximum dye release relies on exocytosis occurring by full fusion (FF),
rather than “kiss-and-run” (KR) in which SVs release via a transiently open
fusion pore. Bromophenol blue (BPB) quenching of styryl dyes also reveals
the KR component of release; with BPB, evoked release was quantitatively
similar in control and OA pre-treated terminals. Intriguingly, studying the
release of the readily releasable pool (RRP) and reserve pool (RP) of
vesicles revealed that only the former pool normally undergo KR
exocytosis. Furthermore, the use of the above paradigms has revealed that
the RRP can be spontaneously released at 37°C. However, this RRP was
distinct from a spontaneously loaded pool that showed different
characteristics from both the RRP and RP.
These data suggest that tau is not acting as a chaperone to aid
surface expression, but interacts with Cav1.3 and/or Cavß3 subunits
to affect function by utilising the first amino-terminal insert of tau.
Page 23/101 - 10/05/2013 - 11:11:03

13.02
A vesicular dependent tonic GABAA receptor-mediated
conductance modifies thalamic relay neuron burst firing in the
mouse dLGN
Bright D P, BrickleyS G
Biophysics Section, Blackett Laboratory, Imperial College, South
Kensington, London, SW7 2AZ, UK.
Many studies have suggested that low ambient GABA concentrations
can persistently activate certain GABAA receptor subtypes, often
remote from synapses, to generate a “tonic” inhibition. An as yet
unidentified non-vesicular release mechanism is thought to be
responsible for generating these low ambient GABA concentrations in
cerebellar granule cells calling into question the function of tonic
inhibition in the dynamic control of neuronal excitability. In this study
we have examined the source of the GABA giving rise to a tonic
inhibition which has recently been reported in thalamocortical neurons
within the dorsal lateral geniculate nucleus (dLGN) in juvenile rats. At
physiological temperatures (37-38°C), whole-cell patch-clamp
recording in an acute slice preparation from adult C57Bl/6J mice also
shows a tonic GABAA conductance present within the mature dLGN.
When normalised to cell capacitance, this conductance has a
magnitude of 75.2 ± 20.3 pS/pF (mean ± sem; n=33). Reduction of the
extracellular Ca2+ concentration from 2 mM to 1 mM, results in a
decrease in the magnitude of this conductance to 6.7 ± 2.9 pS/pF
(n=7). Furthermore, this conductance is also significantly reduced by
the application of 500 nM tetrodotoxin (5.6 ± 2.3 pS/pF, n=7).
Therefore, in thalamic relay neurons of the dLGN, the tonic GABAA
conductance does appear to be generated by the vesicular release of
GABA.
13.03
Capabilities of the WinLTP data acquisition program that extend
beyond traditional LTP experimental functions
Anderson W W, Collingridge G L, Fitzjohn S M
MRC Centre for Synaptic Plasticity, Department of Anatomy, University of
Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK
WinLTP is a Windows data acquisition program designed for the
investigation of long-term potentiation (LTP), long-term depression (LTD),
and synaptic responses in general. The capabilities required for basic
LTP/LTD experiments include alternating two-input extracellular pathway
stimulation, LTP induction by single train, theta burst, and primed burst
stimulation, and LTD induction by low frequency stimulation. On-line
analyses of synaptic waveforms include slope, peak amplitude, populationspike
amplitude, average amplitude, area, rise time, decay time, duration,
cell input resistance, and patch electrode series resistance. WinLTP runs
on Molecular Devices Digidata 132x boards, and now also on National
Instruments M-Series boards.
WinLTP also has many advanced capabilities that extend beyond basic
LTP experimental functions: 1) Analysis of all the evoked synaptic
potentials individually within a sweep, and the analysis of the entire trainevoked
synaptic response as a single entity. 2) Dynamic Protocol Scripting
can be used to make more complicated protocols involving nested Loops
(with counters), Delays, Sweeps (with various stimulations), Run functions
(which execute a block of functions), and Run/ElseRun constructs. Protocol
flow can be changed while the experiment is running. 3) Multitasking can
be used to run a Continuous Acquisition task (saving data to a gap-free
Axon Binary File), while concurrently running the Stimulation/Acquisition
Sweeps task. Because circular buffers are used, sweeps can be
continuously output with no delay between them.
Future WinLTP capabilities will include stimulating up to five extracellular
pathways, and capturing and analyzing Spontaneous Events using a third
concurrent task. WinLTP is available at www.winltp.com.
13.04
Activity-driven transmitter release contributes more to global
background inhibition than to excitation in pyramidal neurones of
rat entorhinal cortex
Greenhill S D, Jones R S G
Department of Pharmacy and Pharmacology, University of Bath, Bath,
UK
We have previously examined glutamate and GABA release in the
entorhinal cortex (EC) in vitro using patch clamp recordings of
spontaneous synaptic currents. To determine the effect of this activity
on cellular excitability we have begun using an approach (Rudolph et
al, 2004) to mathematically estimate global background excitatory
(gBE) and inhibitory (gBI) conductances from membrane potential
fluctuations recorded with conventional intracellular techniques in layer
III neurones in rat EC slices. Here, we determined the contribution of
action potential-driven release to gBE and gBI using TTX, and
examined the effects of a general increase in background release by
increasing [K+]o.
TTX (1µM, n=7) reduced gBI from 18.3±3.8 to 6.9±1.5nS, and gBE
from 4.4±1.0 to 3.6±1.1nS, changing the ratio in favour of excitation
(4.1±0.3 to 2.2±0.5). Subsequent addition of bicuculline (10µM, n=3)
caused a further decrease in gBI to 2.3±1.0nS with little change in
gBE (3.5±1.2nS; ratio 0.6±0.1). In contrast, addition of NBQX (10µM)
to TTX treated slices (n=3) decreased gBE to 1.3±0.7nS, whilst gBI
was unchanged (5.8±4.8nS), giving an inhibition:excitation ratio of
4.0±2.7.
Elevating [K+]o from 3.75 to 7.5 mM (n=7) dramatically increased gBI
from 7.1±1.6 to 34.0±12.7nS. Excitation rose from 2.4±0.5 to
4.4±1.3nS, increasing the ratio of inhibition to excitation from 3.1±0.2
to 8.6±2.6.
Thus, activity-dependent release appears to contribute more to
background inhibition than to background excitation in quiescent
slices. Increasing excitability towards more in vivo-like levels favours
an increase in background activity of interneurones over principal
cells.
13.05
Positive feedback control of glutamate release by kainate receptors in
layer III of the entorhinal cortex
Chamberlain S E, Jones R S G
Department of Pharmacy and Pharmacology, University of Bath, Claverton
Down, Bath, BA2 7AY
Little is known about the role of kainate receptors (KAr) in regulating
glutamatergic transmission in the entorhinal cortex (EC). We have
examined the effect of a GluR5 selective KAr antagonist (UBP302) and
agonist (ATPA) on glutamate release in layer III in rat EC slices.
Whole-cell patch clamp recordings of evoked and spontaneous EPSCs
were used to monitor presynaptic glutamate release. eEPSCs, elicited by
trains of stimuli (5Hz, 1s), showed frequency-dependent facilitation in 60%
of cells. The paired-pulse ratio (PPR) between the first and second events
was 1.4±0.1 in control conditions. UBP302 (20µM) decreased this to
1.1±0.1 (n=6; P

13.06
Contribution of synaptic conductance to the action potential
waveform at the calyx of Held/MNTB synapse
Postlethwaite M 1,2, Johnston J 1,2, Forsythe I D 2
1 Department of Cell Physiology and Pharmacology,, 2 MRC
Toxicology Unit,, University of Leicester,, University Road,, Leicester,,
LE1 9HN
The calyx of Held projects onto neurones of the medial nucleus of
the trapezoid body (MNTB). This pathway is involved in brainstem
auditory processing for sound localisation. The calyx synapse has a
very strong safety factor, with an EPSC being 31 times the current
required for action potential (AP) generation so that the postsynaptic
cell always fires an AP.
Traditionally, direct injection of square current pulses are used to
trigger AP firing and elucidate the roles of voltage-gated channels.
However we show that APs generated by such stimuli are distinctly
different from those triggered by orthodromic synaptic currents.
Physiological synaptic stimulation (at 37°C) generates an AP with an
apparent after-depolarisation which is never observed when using
square current pulse injections. This after-depolarisation was
unaffected by NMDAR antagonists. We demonstrate that the afterdepolarisation
is the result of a slower component of the AMPAR
mediated EPSC. Furthermore the amplitudes of orthodromicallyinduced
APs were smaller than those evoked by current injection, and
rarely overshot 0mV. This finding is explained by shunting of the AP
by the EPSC.
Our findings demonstrate that synaptic conductances can influence
the AP waveform, and highlight the importance of considering the
actual physiological stimuli in studying AP initiation.
13.07
The small heat shock protein family: Physiological expression in the
mouse cns.
Quraishe S, Wyttenbach A, Holden-Dye L, O`Connor V
Neuroscience Research Group, School of Biological Sciences, University of
Southampton, UK, SO16 7PX
The small heat shock protein (sHsp) family comprises 10 members of low
molecular weight (15-30kDa). These proteins contain a characteristic C-
terminal α-crystallin domain that supports their function as molecular
chaperone. They are thought to play a role in protein misfolding diseases,
such as neurodegenerative disorders, cataract, and desmin related
myopathy.
The 10 members are believed to have a unique expression profile in
different tissues. Heart and muscle are the two tissues in which up to seven
sHsps are expressed. Little is known about the physiological role of the
sHsps in the CNS. We have analyzed expression of all 10 family members
in various tissues including the brain. We have confirmed the tissue specific
expression of the sHsps in the various tissues of the body by RT-PCR and
have found 7 to be expressed in the brain, 3 of which (B3, B7 and B9) have
previously not been reported in the brain. In-situ hybridization using naïve
animals evidenced a white matter specific expression pattern for HspB5.
HspB1 and HspB8 are expressed in the spinal cord. HspB8 is also
expressed in the cerebellum. Antibody characterization has confirmed
protein expression of HspB1, HspB5, HspB6, HspB8 and potentially HspB9
in the brain, highlighting a potential role for these sHsps as components of
the chaperone machinery in the CNS.
Sponsored by the MRC and BBSRC
13.08
GABAA receptor beta3 subunit N265M mutation introduces
heterogeneity in GABA sensitivity into cultured posterior
hypothalamic neurons
Sergeeva O, Hatt H, Haas H
Institute of Neurophysiology, Heinrich-Heine-University, Duesseldorf
and Department of Cell Biology, Ruhr University Bochum, Germany
The histaminergic tuberomamillary nucleus in the posterior
hypothalamus controls arousal and attention. Acutely isolated neurons
from this region show a remarkable range of GABA sensitivities
(EC50s 2 to 100 µM). This heterogeneity was not found in primary
cultures of posterior hypothalamus indicating that it is not genetically
programmed. Also co-cultures of dissociated posterior hypothalamus
with explants either from cortex or lateral hypothalamus did not
develop such heterogeneity. As the GABAAR beta3-subunit is
expressed transiently during embryogenesis at a high level in many
brain structures it may be involved in shaping the GABAAR. To
discriminate between neurons expressing only the beta3-subunit and
neurons expressing a mixture of beta-subunits we took advantage of
the mutation b3N265M (Jurd et al. 2003) which introduces propofolresistance
to the GABAAR. In mainly beta3-expressing cells propofol
enhanced GABA responses by

14.02
Characterising neuronal subtype vulnerability to HIV-1 infection
as ascertained by neurotransmitter expression in a human
neuronal model of HIV-1 dise
Palser A, Benson L, Jauniaux E, Miller D, Weiss R, Trillo-Pazos G
University College London, Institute of Neurology and Windeyer
Institute
HIV-1 associated dementia (HAD) is associated with microgliosis,
gliosis and neuronal damage. Clinically, it manifests with motor and
cognitive impairments 10-25 years after initial HIV-1 infection. HIV-1
infection is correlated with latent and lytic infection in different brain
cells. HIV-1 infection of neurones has been demonstrated ex-vivo and
in-vitro. We have shown that HIV-1 infection of primary human neural
cultures (70-80% neurones, 10-20% astrocytes, 5% oligodendrocytes,
no microglia) results in cellular functional damage at the tripartite
synapse similar to that observed in cases with HAD. We know that 10-
20% of cells are infected within this neural culture model of grey
matter. We are currently characterising specific neuronal subtype
vulnerability to HIV-1 infection (3 to 21 days post-infection) by
immunoblotting and RT-PCR to neurotransmitter synthetic enzymes
(dopamine, serotonin, acetylcholine, GABA, glutamate, epinephrine,
norepinephrine) and calcium binding proteins in these neural cultures.
Expression of neurotransmitter synthetic enzymes for dopamine and
serotonin are downregulated as a result of HIV-1 infection at 3-21
days post-infection. Moreover, infectious virus is produced at these
time points within this neural culture system as assessed in
chemokine transfected NP2 cells. These results suggest that there is
low level release of infectious viral particles in culture and that
Dopamine and serotonin producing neuronal subtypes are targeted by
HIV-1 within neural culture system. These findings suggest that
disruption of neurotransmitter synthetic pathways might be implicated
in neuronal damage in HIV-1 disease in the brain.
14.03
Effects of anaesthetics on basal and stimulated dopamine release,
measured by microdialysis
Young A M J, Dixon A L
School of Psychology,, University of Leicester,, Lancaster Road,, Leicester,
LE1 9HN
Many experiments investigating brain function, using neurochemical,
electrophysiological and imaging techniques, are performed in
anaesthetised animals for practical or ethical reasons. However, despite
the widespread use of anaesthetised animals in these types of study, very
little is known about the effect of anaesthetics on brain function, and in
particular on neurotransmitter release.
The present study measured the effect of anaesthesia on basal and
stimulated (potassium, amphetamine or mild footshock) levels of dopamine
and its metabolites, DOPAC and HVA, in rat nucleus accumbens using
brain microdialysis. The anaesthetics employed were halothane, urethane
and α-chloralose, all of which are commonly used for experiments in
animals.
Results showed that with halothane, relatively stable baseline levels of
dopamine and its metabolites were achieved, which were similar to levels
measured in unanaesthetised controls. With urethane normal levels of
dopamine were measured, but metabolite levels were significantly
decreased, while under α-chloralose dopamine was significantly raised, and
metabolites were lowered compared to controls. Under halothane and
urethane, changes in dopamine and its metabolites during stimulation were
similar to those seen in unanaesthetised animals, whereas under α-
chloralose the effects were markedly attenuated.
Thus, mesolimbic dopamine mechanisms remain relatively intact under
halothane and urethane anaesthesia. Under α-chloralose, however, both
basal and stimulated levels of dopamine are severely affected. We suggest,
therefore, that halothane is the most appropriate anaesthetic for studying
mesolimbic dopamine function in anaesthetised animals, although urethane
may be suitable under certain circumstances: α-chloralose, however is not
suitable, as the dopamine system is severely compromised under this
anaesthetic.
14.04
Effect of NMDA receptor activation in the lateral entorhinal area
on the left dorsal hippocampal glutama, and aspartate release in
two animal models
Brady A T, De Souza I E J, McCabe O M, Moran M P, O'Shea S D,
O'Connor W T
Applied Neurotherapeutics Research Group, UCD School of
Biomolecular and Biomedical Science, UCD Conway Institute,
University College Dublin, Belfield, Dublin 4, Ireland
We employed dual probe microdialysis in the lateral entorhinal area
(LEA) and dorsal hippocampus (DH) in the 82 day old adult rat (P82)
to investigate the effects of intra-LEA perfusion with (NMDA 300μM,
20 min) on dialysate glutamate and aspartate levels in the DH of
socially isolated and maternally deprived rats. Isolated rats were
weaned on P25 and housed individually for 55 days while maternally
deprived rat pups experienced a single 24-hour period of maternal
deprivation on P9. Socially reared rats acted as control.
Basal dialysate glutamate levels were stable over the duration of the
experiment. DH glutamate levels (чM) were similar in the social
control, isolated and maternally deprived rat (0.263*0.072),
(0.192*0.038) and (0.208*0.025) respectively. Aspartate levels (чM)
were similar in the social control (0.036*0.004) isolated (0.039*0.008)
and maternally deprived (0.059*0.010) groups. Intra-LEA NMDA was
associated with a 39%, 20% and 36% increase in DH glutamate
release in social control, isolated and maternally deprived rats. In
contrast DH aspartate levels were significantly increased in the
maternally deprived group (p=0.0.0042 v’s social control). A maximal
increase of 42% (p=0.0129) and (p=0.0038) in the isolated group and
maternally deprived group respectively.
Thus DH aspartate appears to be more responsive than glutamate to
LEA NMDA receptor activation. It is currently believed that glutamate
is the main neurotransmitter carried by the perforant pathway from the
LEA to the DH. The findings from the present study suggest that
aspartate also plays a role in perforant pathway transmission.
14.05
Novel mutations in the human GlyT2 gene in hyperekplexia
Carta E, Chung SK, Remy N, Vanbellinghen JF, Bours V, Sameer M.
Zuberi, John B.P. Stephenson, Marina A.J. Tijssen, Arn M.J.M. van den
Maagdenberg, Brian R. Pearce, Kirsten Harvey, Stéphane Supplisson,
Mark I. Rees, Robert J. Harvey
The School of Pharmacy, London; Univ. Wales Swansea; Liège University
Hospital, Belgium; Royal Hospital for Sick Children, Glasgow; Academic
Medical Centre, Amsterdam and Leiden Univ. Medical Centre, The
Netherlands; Laboratoire de Neurobiologie Moléculaire et Cellulaire, Paris
The glycine transporter GlyT2 has an essential role in maintaining a high
presynaptic pool of neurotransmitter at glycinergic synapses. We have
recently shown that missense, nonsense and frameshift mutations in the
GlyT2 gene (SLC6A5) can cause hyperekplexia. Since our initial study, we
have discovered further mutations in SLC6A5 in individuals from the UK,
Holland, France, Spain and Italy. In some of these additional hyperekplexia
cases, only one coding mutation has been found. Since most mutations in
the GlyT2 gene are inherited as compound heterozygotes (i.e. one
mutation on each allele) it is possible that: i) there is a deletion affecting
one or more exons in the second allele, which would not be easily
detectable by PCR and sequencing; ii) that the single ‘orphan’ mutations
exert dominant-negative effects (as previously described for mutation
S510R) or iii) that uncharacterised alternative splicing of the GlyT2 gene
has excluded some exons from genetic screening. To assess the latter
possibility, we performed 5’ and 3’ RACE on human spinal cord cDNA. This
revealed a number of additional splice variants of GlyT2 specifying novel N-
and C- termini. In particular, a new C-terminal GlyT2 variant (denoted
hGlyT2B) lacks the C-terminal class III PDZ binding motif (TQC) found in
GlyT2A that is crucial for interactions with the PDZ domain-containing
protein syntenin-1. Since syntenin-1 has been proposed to regulate the
trafficking and/or presynaptic localization of GlyT2, hGlyT2B is likely to be
distinct in terms of subcellular location and/or interacting partners.
Page 26/101 - 10/05/2013 - 11:11:03

15.01
L-serine enhances the inhibition of calcium currents by
anandamide in cultured sensory neurones from neonatal rats
Khairy H, Ross R, Pertwee R, Scott R
School of Medical Sciences, University of Aberdeen, Institute of
Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.
The endocannabinoid anandamide (AEA) has previously been found to
inhibit high voltage-activated Ca2+ currents in cultured dorsal root
ganglion (DRG) neurones (Evans et al., 2004 Br. J. Pharmacol. 141:
1223-1233). Using the whole cell recording patch clamp technique with
choline chloride-based extracellular solution and CsCl-based patch
pipette solution we have investigated the modulation of AEA responses
by 1 µM L-serine in cultured DRG neurones. High voltage-activated
Ca2+ currents were evoked from a holding potential of -90 mV by
voltage step commands to 0mV. Application of 1µM AEA for 3-5
minutes inhibited the mean peak Ca2+ current by 34 ± 12 % (n=7;
P

15.05
Influence of sodium valproate on the EEG characteristics in
epileptic children
Khachidze I, Gugushvili M, Maloletnev V
14 Gotua str, I. Beritashvili Institute of Physiology, Tbilisi,
0160,GEORGIA
The study was aimed to investigate the alteration of EEG
characteristics in epileptic children during the treatment with
anticonvulsant - depakine.146 patients aged 3 to 9 years were
examined. The duration of their disease ranged from 3 months to 9
years. Absolute and Relative values of the power spectra (AVP and
RVP) of standard EEG were analyzed. Expressed decrease of total
AVP was observed in frontal, temporal and occipital areas. This
indicates that D reduces the signs of excessive EEG. Synchronization
pointing to the status of readiness to seizure activity in the CNS. This
inference is confirmed by the analysis of the dynamics of the selected
ranges of activity, since observation during the treatment course
showed a significant decrease in AVP of Low-frequency range in
temporal, parietal and occipital areas. The dynamics of high frequency
fraction EEG activity requires a special consideration. It appeared that
the action of D produces an advantageous decrease within the range
of beta-1, and especially, beta2 activity in the parietal and occipital
areas, i.e exactly in the regions where its presence is conventionally
accounted for the CNS regulatory mechanisms dysfunction. The
analysis of D to influence on epileptiform graphoelements shows that
its effect reveals primarily in the reduction of typical epileptiform
complexes peak-wave, sharp waves. The influence of D on grouped,
polyphasic sharp waves, as well as on paroxysmal bursts provoked by
functional tests was expressed to a less extent: these graphoelements
continued to be recorded after 6-8 months after the commencement of
the treatment.
15.06
Influence of sodium valproate on the EEG characteristics in epileptic
children
Khachidze I, Gugushvili M, Maloletnev V
14 Gotua str, I. Beritashvili Institute of Physiology, Tbilisi, 0160,GEORGIA
The study was aimed to investigate the alteration of EEG characteristics in
epileptic children during the treatment with anticonvulsant - depakine.146
patients aged 3 to 9 years were examined. The duration of their disease
ranged from 3 months to 9 years. Absolute and Relative values of the
power spectra (AVP and RVP) of standard EEG were analyzed. Expressed
decrease of total AVP was observed in frontal, temporal and occipital
areas. This indicates that D reduces the signs of excessive EEG.
Synchronization pointing to the status of readiness to seizure activity in the
CNS. This inference is confirmed by the analysis of the dynamics of the
selected ranges of activity, since observation during the treatment course
showed a significant decrease in AVP of Low-frequency range in temporal,
parietal and occipital areas. The dynamics of high frequency fraction EEG
activity requires a special consideration. It appeared that the action of D
produces an advantageous decrease within the range of beta-1, and
especially, beta2 activity in the parietal and occipital areas, i.e exactly in the
regions where its presence is conventionally accounted for the CNS
regulatory mechanisms dysfunction. The analysis of D to influence on
epileptiform graphoelements shows that its effect reveals primarily in the
reduction of typical epileptiform complexes peak-wave, sharp waves. The
influence of D on grouped, polyphasic sharp waves, as well as on
paroxysmal bursts provoked by functional tests was expressed to a less
extent: these graphoelements continued to be recorded after 6-8 months
after the commencement of the treatment.
16.01
Posttranslational modifications and signaling in postsynaptic
densities.
Schoepfer R, Trinidad J C, Thalhammer A, Burlingame A L
1. Laboratory for Molecular Pharmacology, University College London,
London, UK, 2. Department of Pharmaceutical Chemistry, UCSF, San
Francisco, CA, USA ,
Postsynaptic densities (PSDs) form a highly structured environment of
signal transmission and exquisite signal processing in the nervous
system. Glutamate serves as the main excitatory neurotransmitter in
the mammalian central nervous system, and glutamate receptors are
an integral part of excitatory PSDs.
PSDs combine the receptors and channels, their downstream
signaling machinery (such as kinases and phosphatases), and a
cytoskeletal network into a structured environment of signaling
pathways. The phosphorylation state of components of the PSD is
central to the regulation of synaptic transmission and is known to play
a role in synaptic plasticity, learning and memory.
We purified PSDs from mouse brain on sucrose density gradients, and
analyzed its protein components and phosphorylation state by mass
spectrometry, following two-dimensional liquid chromatography plus
immobilized metal affinity chromatography of tryptic digests.
Using PSDs isolated from four different brain regions, hippocampus,
cortex, midbrain and cerebellum, we examined region specific
variations at both the protein and phosphorylation level. The tryptic
digests of the four different PSD preparations were labeled individually
with one of the four iTRAQ isobaric labels, pooled, and subjected to
the same workflow as previously established for single PSD
preparations. This workflow allowed us to perform relative quantitation
and bioinformatic analysis of over 1000 proteins and 1376 unique
phosphorylated peptides.
16.02
Synapse evolution is linked to neuroanatomical diversity
Grant S G N, Anderson C N G, Emes R D, Pocklington A J, Vickers C A,
Croning M D R, Armstrong J D A
† The Wellcome Trust Sanger Institute, Genes to Cognition Program,
Hinxton, Cambridge CB10 1SA UK, Present address: Department of
Biology, University College London, Darwin Building, Gower Street,
London, WC1E 6BT UK, ‡ University of Edinburgh, Institute for Adaptive
and Neural Computation, 5 F
Increasing brain size and number of synaptic connections is thought to be
the primary mechanism underlying evolution of intelligence. Here we
examine the molecular evolution of the synapse in 19 species and focus on
postsynaptic signaling complexes essential for learning and memory.
Striking increases in number of synapse components preceded the
evolution of large vertebrate brains. Key synapse components predate the
origins of nerve cells and later molecular innovations at the metazoan and
chordate boundaries contribute novel signaling functions and greater
molecular and signaling complexity. mRNA and protein expression patterns
in mouse brain show recently evolved synapse proteins preferentially
contribute to differences between brain regions. Progressive elaboration
upon an ancestral synapse signaling structure leading to increased
synapse diversity and signaling complexity was further supported by
analyses of protein function and interactions. We propose that evolution of
synapses and brain size are linked and synergistically important for species
differences in behaviour.
Support Contributed By: NIH NCRR grants RR12961, RR14606, and
RR01614 to ALB and grants from the Wellcome Trust (UK) to RS
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16.03
Quantitative analysis of membrane microdomains-associated
proteins in the DLPFC in the schizophrenic and bipolar disorder
brain
Áine T. Behan*, Kieran Wynne#, Connie Byrne#, Patricia Maguire#,
Niaobh O`Donoghue$, Michael J. Dunn$, Gerard Cagney#, David R.
Cotter*
*Department of Psychiatry, Royal College of Surgeons in Ireland ERC,
Beaumont Hospital, Dublin 9., $Proteome Research Centre, UCD
Conway Institute of Biomolecular and Biomedical Research, UCD,
#School of Biomolecular, and Biomedical Science, Conway Institute,
UCD.
Membrane microdomains (MM) are defined by their cholesterol and
sphingolipid-rich nature with numerous roles been ascribed them such
as their involvement as vehicles of endocytosis and trafficking during
signaling. Studies analysing protein changes potentially contributing to
human brain diseases have identified numerous MM associated
proteins as candidate players. We reason that by identifying and
quantifying MMs in the dorsolateral prefrontal cortex (DLPFC), an area
implicated in schizophrenia (SCZ), we may better understand the
molecular mechanisms underlying dysfunction in the SCZ and bipolar
disorder (BPD) brain.
A 10/10/10 series of the best matched brains (pH. PMI etc.) were
chosen from the 35/35/35 Stanley Foundation frozen post-mortem
brain tissue from DLPFC grey matter. Ten samples were pooled for
each of the groups (control, SCZ, BPD) and MMs were isolated and
analysed using two proteomic methods, (i) 2D-DIGE and MALDI-TOF
and (ii) 1D SDS PAGE and ion-trap MS. Proteins of interest were
validated in whole cell lysates (10/10/10 series – not pooled) using
Western blotting.
21 proteins were found to be differentially expressed by both proteomic
methods in our pooled samples. Further validation by Western blotting
demonstrated this differential expression for 3 synaptic/neuroplasticity
proteins, namely brain acid soluble protein 1(BASP1), limbic system
associated membrane protein(LAMP) and syntaxin binding protein
1(STXBP1).
BASP1, STXBP1 and LAMP are increased in the SCZ and BPD brain.
This suggests a dysfunction in synaptic function and/or neuroplasticity
in these psychiatric disorders.
16.04
Cellular and proteomics approaches to protein synthesis in axons.
Minnen Jv
Department of Molecular and Cellular Neurobiology, Center for
Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit, De
Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Recent studies have begun to focus on axonal protein synthesis and the
functional significance of localized protein synthesis. However, identification
of proteins that are synthesized in mammalian axons has not been
rigorously addressed. Here, we used axons purified from cultures of injuryconditioned
adult dorsal root ganglion (DRG) neurons and proteomics
methodology to identify axonally synthesized proteins. Proteins and the
encoding mRNAs for cytoskeletal proteins were identified in glial cell free
axonal preparations. In addition to the cytoskeletal elements, several heat
shock proteins, resident endoplasmic reticulum (ER) proteins, proteins
associated with neurodegenerative diseases (ubiquitin C-terminal
hydrolase L1, rat ortholog of human DJ-1/Park7, -synuclein, superoxide
dismutase 1), anti-oxidant proteins and metabolic proteins were identified
among the axonally synthesized proteins. Detection of the mRNAs
encoding each of the axonally synthesized proteins identified by mass
spectrometry in the axonal compartment indicates that the DRG axons
have the potential to synthesize a complex population of proteins.
Ultrastructural studies on in vivo injured and regenerating axons
demonstrated that these axons harbor high numbers of polyribosomes,
indicating active protein synthesis. Surprisingly, by creating GFP-tagged
ribosomes in Schwann cells, we demonstrated that the axonal
polyribosomes have originated in the Schwann cells. These data indicate
that mammalian axons have the ability to synthesize a large variety of
proteins, of which part of the encoding mRNAs and synthetic machinery
have originated in Schwann cells.
17.01
Molecular regulation of thalamocortical axonal navigation
Price D
Centres for Integrative Physiology and Neuroscience Research,
Edinburgh University, Hugh Robson Building, George Square,
Edinburgh EH8 9XD
The cerebral cortex receives most of its sensory innervation via
thalamocortical axons, whose development requires interactions
between advancing thalamic growth cones and guidance cues that
they encounter, attracting them towards or repelling them from specific
regions. We have been studying the regulation of guidance molecules
in thalamic neurons and in the environment through which their growth
cones navigate. Regarding control of navigation in thalamic cells, we
found that thalamic growth cones and neurons contain β-catenin
mRNA and that manipulations predicted to affect β-catenin levels
cause major disruption to thalamic axonal navigation. (1) In vivo, loss
of adenomatous polyposis coli (APC) in the forebrain blocks
completely thalamic axonal navigation from ventral to dorsal
telencephalon. (2) In vitro, application of lithium blocks thalamic axonal
growth in thalamocortical co-cultures. Regarding control of the
environment that thalamic axons navigate, we found direct evidence
that telencephalic expression of the transcription factor Pax6 is
required for thalamic axon guidance. We made mice with loxP sites
flanking exons encoding Pax6’s paired DNA binding domain
(Pax6loxP), so that exposure to Cre recombinase excises this crucial
domain and knocks the remainder of the protein out of frame, creating
a null-allele. We removed Pax6 from some cells in ventral
telencephalon using a Six3Cre allele that drives Cre recombinase
expression in cells around the developing internal capsule but neither
in diencephalon nor in cortex. In Pax6loxP/loxP;Six3Cre embryos, a
60-70% depletion of Pax6-expressing cells around the internal capsule
correlates with a ventral misrouting of a significant proportion of
thalamic axons.
17.02
Tangential neuronal migration controls axon guidance: a role for
neuregulin-1 in thalamocortical axon navigation
López-Bendito G
Instituto de Neurociencias de Alicante, CSIC & Universidad Miguel
Hernández, 03550 San Joan d’Alacant, Spain
Neuronal migration and axon guidance constitute fundamental processes in
brain development that are generally studied independently. Although both
share common mechanisms of cell biology and biochemistry, little is known
about their coordinated integration in the formation of neural circuits. Using
several in vitro coculture assays, in situ hybridation and
immunohistochemistry, here we show that the development of the
thalamocortical projection, one of the most prominent tracts in the
mammalian brain, depends on the early tangential migration of a population
of neurons derived from the ventral telencephalon. This tangential migration
contributes to the establishment of a permissive corridor that is essential for
thalamocortical axon pathfinding. Our results also demonstrate that in this
process two different products of the Neuregulin-1 gene, CRD-NRG1 and
Ig-NRG1, mediate the guidance of thalamocortical axons. These results
have been demonstrated in part by the analysis of specific mutant mice
deficient on either or all isoforms of Ngr1. TCAs fail to extend normally
through the telencephalon in mice with a loss of function mutation in the
Nrg1 gene. We have also demonstrated that this function seems to be
mediated by the tyrosine kinase receptor ErbB4. In sum, these results show
that neuronal tangential migration constitutes a novel mechanism to control
the timely arrangement of guidance cues required for axonal tract formation
in the mammalian brain
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17.03
Activity-independent and dependent mechanisms for the
formation of the thalamocortical projection
Yamamoto N, Uesaka N, Hayano Y, Yamada A
Cellular and Molecular Neurobiology, Graduate School of Frontier
Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-
0871
During development, thalamocortical (TC) axons form elaborate
branches in the specific layers of the neocortex. The target- and
activity-dependent mechanisms of TC axon branching were
investigated in organotypic coculture preparations of the thalamus and
cortex. Axon tracing with enhanced yellow fluorescent protein
demonstrated that TC axons formed extensive branches during the
second week in culture. Simultaneously, spontaneous firing activity
emerged in both thalamic and cortical neurons. To study the role of
the spontaneous activity in branch formation, TC axon branching was
examined in the presence of the drugs that block synaptic or firing
activities. The result demonstrated that lamina-specific branch
formation was suppressed considerably by addition of these blockers.
What aspect of TC axon branching is affected by neural activity was
further investigated by time-lapse imaging of individual axons. TC
axon branching was found to be generated dynamically by addition
and elimination, with a preference toward branch accumulation in the
target layer. Blockade of firing or synaptic activity reduced the
remodeling process, in particular, branch addition in the target layer.
Together, these findings suggest that TC axon branching is regulated
by the remodeling process with branch-inducing and inhibiting
molecules and that neural activity can modify the molecular
mechanisms.
17.04
Specificity and plasticity in thalamocortical connectivity: lessons from
Semaphorin-6A mutant mice
Little G, López-Bendito G, Rünker A E, Molnár Z, Mitchell K J
Smurfit Institute of Genetics, Trinity College Dublin, Ireland; (2) Department
of Human Anatomy and Genetics, University of Oxford, UK; 3Instituto de
Neurociencias, San Juan de Alicante, Spain.
The mechanisms controlling the specificity of connectivity between thalamic
nuclei and cortical areas are not well understood. There is good evidence
to support targeting of thalamic axons to specific regions of the cortex but
also to suggest that the “identity” of cortical areas may be defined by their
thalamic innervation. We have observed in Semaphorin-6A mutant mice a
defect in connectivity from thalamus to cortex. This is specific to the visual
axons from the thalamus that would normally project to the visual part of
the cortex. In Sema6A mutants these axons project inappropriately into the
amygdala. In their absence occipital cortex is partially invaded by
somatosensory axons leading to a shift of cortical domains, which persists
in adults. The Sema6A mutant mice are the first animals that show this type
of phenotype that survive to adulthood. As such they are a unique model in
which to explore the targeting of thalamocortical axons and the
specification of cortical areas.
18.01
Are there stem cells in the olfactory system
Barnett S, Tome M, Toft A, Riddell J
1Division of Clinical Neurosciences. Beatson Labs, Glasgow
University, Glasgow G61 1BD. ^IBLS, University of Glasgow G12 8QQ
The peripheral olfactory system is thought to contain stem cells. To
assess the stem cell properties of these cells, we compared their
ability to form neurospheres and to differentiate in vitro with CNS
neural stem cells. Brain and olfactory mucosa (OM) of embryonic rats
were processed using a standard methodology for CNS neurosphere
culture. Primary spheres were formed by both tissues but OM-spheres
rarely formed secondary spheres. OM-spheres exhibited two main
morphologies, one which resembled CNS neurospheres, (type-I) the
second which was smaller and more tightly formed (type-II). As
expected brain neurospheres were strongly positive for the stem cell
marker nestin and differentiated into typical neural cells. The type-I
OM-spheres, although expressing nestin also expressed markers for
peripheral glia and connective tissue. The type-II spheres were mainly
cytokeratin positive, a marker of sustentacular and horizontal basal
cells. To assess the potential of olfactory cells in CNS repair we used
a partial transection model of SC injury. We compared the
regenerative capacity of a mixed population of OM cells with OMspheres.
Both types of transplant survived and filled the injury site.
Mixed mucosa grafts produced an extensive graft environment that
supported axonal ingrowth and myelination. OM-sphere grafts
produced less extensive graft areas in general. These were
characterised by the presence of large spheres into which there was
little axon ingrowth or consisted of a more diffuse cellular environment
into which some axonal ingrowth occurred. Thus neurospheres from
the olfactory mucosa have no particular advantage over candidate
cells for CNS repair.
18.02
Embryonic solutions for adult problems
Chandran S
Cambridge Centre for Brain Repair, UK
Repair of the damaged brain has long been regarded as the holy grail of
regenerative medicine. The emergence of stem cells as an experimental
and therapeutic resource represents a major opportunity for brain repair.
Although repair need not necessarily recapitulate development, insights
rooted in an understanding of developmental principles are central to the
design of novel neural repair strategies. Human embryonic stem cells
(hESCs) are an attractive source of defined tissue for cell-based therapies
and drug screening in regenerative neurology. In order for their therapeutic
potential to be realised, there is a need for controlled and large-scale
differentiation of hESCs into defined neural precursors without the use of
animal products or genetic manipulation. Derivation of hESC-NSCs under
defined conditions will be discussed.
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18.03
Finding cells for replacement therapy in Huntington’s disease
Rosser AE, Kelly CM , Allen ND , Zietlow R , Jeyasingham J , Pekarik
V , Dunnett SB
1. Cardiff University School of Biosciences, PO BOX 911, Museum Av,
Cardiff CF10 3US , , 2. Dept Neurology,, University of Wales College
of Medicine, Heath Park, Cardiff CF14 4XN,
The most striking pathology in Huntington’s disease (HD) in the early
to moderate stages is the loss of medium spiny neurons (MSNs) from
the striatum. One treatment strategy is to implant developing MSNs in
an attempt to reconstruct damaged neuronal circuits. To date, the
success of this approach has depended on harvesting cells from the
developing striatum during a specific development window. A small
number of ongoing clinical studies world-wide have demonstrated the
safety of this procedure and have provided preliminary evidence of
efficacy. However, the practical difficulties associated with the
collection of human fetal tissue make it imperative to identify an
alternative source of donor cells.
Stem cells from various sources may provide a renewable source of
donor cells, but functional benefit will depend on neurons differentiating
from such populations being of an MSN phenotype. It is this
requirement that currently presents the greatest challenge for the
clinical application of such cells.
We focus on neural precursor cells isolated from either human fetal
CNS or embryonic stem (ES) cells. We show that differentiation
phenotype is influenced by expansion in vitro and that MSN phenotype
may be retained after limited expansion. We undertook a systematic
Affymetrix analysis of developing striatal neurons in order to identify
factors important for normal MSN differentiation, and are currently
validating significantly up-regulated genes as markers of the MSN
phenotype and for their role in MSN differentiation. Ultimately this
information will be used to make rational manipulations of the culture
conditions to promote MSN differentiation.
18.04
Neural stem cells for the treatment of Parkinsons disease.
Caldwell M
, Henry Wellcome Laboratory for Integrative Neuroscience and
Endocrinology, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY
Neural stem cells can be isolated from the developing and adult brain and
can be expanded in culture with the mitogens epidermal growth factor
(EGF) and fibroblast growth factor (FGF2). However, as they expand in
culture they lose their neurogenic potential and become more gliogenic.
With that in mind these cells would have limited usefulness for the
treatment of neurodegenerative diseases such as Parkinsons Disease
which affects more than one percent of the population over the age of 65.
However, recent studies have shown that overexpression of transcription
factors such as Nurr1 or pitx3 (both important during dopamine neuron
development and maintenance) result in generation of dopamine neurons.
Here we show that overexpression of Pitx3 in neural stem cells results in
larger numbers of dopamine neurons when cocultured with developing
ventral mesencephalon. This has a significant effect on behavioural
recovery in a variety of motor tasks and greater survival of dopamine
neurons in transplants. This could have implications for the treatment of
Parkinsons Disease.
19.01
Neuroanotomical substrates and disorders of frontal-lobe related
cognitive processes
Garavan H
School of Psychology, Trinity College, Dublin 2, Ireland
While there is a consensus that the frontal lobes play a critical role in
regulating behaviour, the particulars of how this is accomplished are
still far from clear. This talk will describe a number of recent studies
from my lab that attempt to describe the neurobiology of regulatory
processes such as inhibitory and attentional control, performance
monitoring and control over automatic behaviours. Our results suggest
that the right PFC, especially right inferior frontal gyrus, is central to
inhibitory control. The anterior cingulate appears to monitor behaviour
and serves a central role in intervening when ongoing behaviour
should be interrupted either to avoid an error or attain a large reward.
Left PFC may have a central role in maintaining task goals and
interactions between it and other regions involved in implementing
control will be described. In addition to understanding the neural
substrates of these functions, we will also describe evidence of their
dysfunction in substance dependent individuals, a group characterised
by impoverished control over behaviour. Drug users show reduced
activity in the anterior cingulate and have a poorer awareness of their
performance failures. In addition, reduced PFC activity associated with
inhibitory control appears to be accompanied by reliance on suboptimal
cerebellar networks. These results suggest that impairments in
cognitive control may be an important element of drug dependence,
either in its maintenance or in increasing risk of relapse.
19.02
Novel Therapeutic Targets for Stress-Related Disorders
Cryan J F
School of Pharmacy, Dept Pharmacology & Therapeutics, University
College Cork
The complex interaction between stress and genetics that leads to the
manifestation of disorders such as depression, anxiety, drug dependence,
cognitive dysfunction and irritable bowel syndrome in certain susceptible
individuals is one of prime interest in neuroscience. This is paralleled with
efforts to develop pharmacological strategies to counteract the deleterious
effects of stress. It is largely thought that an imbalance of the
neurotransmitters GABA and glutamate are responsible at least in part for
the manifestation of many stress-related disorders. Their fast actions are
mediated by ionotropic receptors. In addition, metabotropic receptors
mediate slower modulatory actions of GABA and glutamate on
neurotransmitter release and cell excitability. Of all glutamate receptors, the
role of group III metabotropic glutamate receptors (mGluR4; mGluR6;
mGluR7; mGluR8) and in particular mGluR7 in stress-related disorders is
the least investigated because of the lack of specific tools. Likewise a
definitive role of metabotropic GABAB receptors in such disease states has
remained elusive. In this presentation, recent data highlighting the role of
these two receptors in stress related disorders will be highlighted. The
development of novel pharmacological (allosteric modulators and
antagonists) and genetic tools (knockout, knockin and siRNA-induced
knockdown) for GABAB and mGluR7 has allowed the investigation of their
role in the manifestation of the molecular, neurochemical and behavioural
responses induced by stress; aversive fear learning and drugs of abuse. In
parallel we are investigating the contribution of cortical regions such as the
anterior cingulated cortex and infralimbic cortex to the manifestation of
anxiety and depression-like behaviour.
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19.03
Effects of early adversity on hemispheric functional connectivity
of the medial prefrontal cortex
(1, 3)Stevenson C W, (2) Halliday D M, (3) Marsden C A, (3) Mason R
(1) Leicester School of Pharmacy, De Montfort University, (2)
Department of Electronics, University of York, (3) School of
Biomedical Sciences, University of Nottingham
Early adversity increases the predisposition to develop mental illness.
Maternal separation (MS) models the enhanced behavioural and
neuroendocrine stress responses caused by early adversity. These
alterations may involve functional changes in medial prefrontal cortex
(mPFC), a region which modulates various stress responses.
Moreover, hemispheric specialisation may play a role in mPFC
function, such that the right hemisphere is preferentially involved in
mediating adaptive coping responses to stress. In the present study,
mPFC activity was examined in left and right hemispheres of adult
male Lister hooded rats subjected to MS (6 hrs/day on postnatal days
2-14), handling (H; 15 min/day) or animal facility rearing (AFR) as
pups. In vivo electrophysiology was used to record neuronal activity
under basal conditions and in response to FG-7142, a benzodiazepine
receptor inverse agonist which mimics behavioural and
neuroendocrine stress responses. Basal activity was attenuated by
MS compared to H and AFR, an effect which was lateralised to the
right hemisphere. Moreover, increased activity induced by FG-7142
was lateralised to different hemispheres in the early rearing groups.
FG-7142 increased left hemisphere activity in AFR and MS animals
(AFR>MS). Conversely, FG-7142 increased right hemisphere activity
in H animals. Thus, MS induces a lateralised deficit in mPFC function
which may model certain cognitive and affective disturbances
observed in psychiatric disorders associated with early adversity. The
effects of FG-7142 reported in H animals also add to evidence
suggesting increased resilience to the effects of stress in this group.
Supported by a Marie Curie Fellowship from the EU.
19.04
Basolateral amygdala aminergic transmission as a target of atypical
antipsychotic drug action
Glennon J C, Lewis L, Van der Kooij M A, Kleefstra A J, Tros R, Leguit, N,
Hamelink, R, McCreary, AC, Kruse, CK
1) Department of Chemistry, National University of Ireland Maynooth, Co.
Kildare, Ireland. 2) Solvay Pharmaceuticals Research Laboratories, Weesp,
1381CP, The Netherlands.
The basolateral amygdala (BLA) is part of a mesolimbic dopaminergic (DA)
circuit which may be hyperactive in psychosis. Changes in BLA DA and
noradrenaline (NA) transmission remain unstudied in animal models of
psychosis which encompass emotional and cognitive disturbance. Here,
the effect of the psychomimetic phencyclidine (PCP) alone and together
with the antipsychotics aripiprazole (ARI), clozapine (CLO) and haloperidol
(HAL), the partial D2 receptor agonist (-)-3PPP and the 5-HT1A receptor
agonist, 8-OH-DPAT were examined on BLA dialysate DA and NA levels.
Administration of PCP is associated with large sustained increases in
dialysate BLA DA and NA levels. Co-administration of ARI with PCP
reversed the PCP-induced increase in BLA dialysate DA and NA levels. In
contrast, co-administration with CLO or HAL with PCP failed to do this.
Interestingly, co-administration of (-)-3PPP with PCP could reverse the
PCP-mediated increase in NA but not DA levels while co-administration of
8-OH-DPAT with PCP could antagonise both the PCP mediated increase in
BLA DA and NA dialysate levels. Taken together, the data suggests that
BLA DA levels are elevated by PCP consistent with the DA hypothesis of
schizophrenia and the ability of ARI to reverse the PCP-induced increase in
BLA DA and NA may be 5-HT1A receptor mediated. The significance of the
PCP mediated increase in BLA NA levels and their reversal by both (-)-
3PPP and 8-OH-DPAT is deserving of further investigation but suggests
that both D2 partial and 5-HT1A agonists can regulate BLA NAergic tone
which may underlie emotion-related memory consolidation.
19.05
Comparing transient and persistent gamma oscillations in the rat
basolateral amygdala in vitro
Randall F E, Whittington M A, Cunningham M O
Newcastle University, School of Neurology, Neurobiology and
Psychiatry, Medical School, Framlington Place, Newcastle Upon Tyne
NE2 4HH
Oscillatory activity in the basolateral amygdala (BLA) is thought to
have an important role in the consolidation of emotional memories
(Pelletier and Paré, 2004). During emotional arousal the BLA has
been shown to produce gamma frequency oscillations (30-60 Hz) in
vivo (Pagano and Gault, 1964; Feschenko and Chilingaryan, 1990).
Understanding mechanisms by which oscillatory activity is generated
in the amygdala could identify how the amygdala communicates with
other brain areas in emotional situations. We previously reported a
model of transient gamma oscillations in the BLA in vitro evoked by
brief applications of L-glutamate (10 mM) in coronal slices (450μm) of
BLA from adult male Wistar rats. Here we report a persistent gamma
oscillation model evoked by bath application of Kainic acid (400nM).
The mean frequency of the persistent oscillation was 35 ± 1 Hz and
the mean power was 2030 ± 288 μV2/Hz. Pharmacological
investigation showed that, like the transient model, the network activity
underlying these oscillations was GABAA receptor-dependent but not
NMDA receptor-dependent. The persistent oscillations also showed
differential responses to AMPA/Kainate receptor antagonists different
to those previously seen in other brain areas including hippocampus.
Unlike the transient oscillations, the persistent oscillations were
abolished by gap junction blocker Octanol (1mM). The
pharmacological characteristics of the two models are similar and both
provide useful tools for characterizing the cell networks involved in
generating this activity.
Feschenko and Chilingaryan (1990) Neurosci. Behav. Physiol. 20:506-
13
Pagano and Gault (1964) Electroencephalogr. Clin. Neurophysiol.
17:255-60
Pelletier and Paré (2004) Biol. Psychiatry 55:559-62
19.06
Emotional responses to novelty and open spaces in a 3D maze: the
role of GABA-A and histamine H3 receptors
Ennaceur A (1), Michalikova S (1), Rensburg Rv (2), Curruthers N I (4),
Leurs R (3), Esch I (3), Chazot P L (2)
1.Sunderland Pharmacy School,Sunderland University,UK;Centre for
Integrative Neuroscience, Durham University;3Leiden-Amsterdam Center
for Drug Research, The Netherlands; 4.4Johnson & Johnson
Pharmaceutical Research and Development, San Diego, USA.
In the present study, we examined the behaviour of mice in our novel 3D
maze, composed of eight flexible arms radiating from a central platform.
Each arm can be manipulated independently and presented at the same,
below or above the level of a central platform. Mice need to cross a bridge
to reach flat, raised or elevated arms. Naïve mice were tested for the first
time in the raised arm configurations of the maze. This maze can also be
used to assess anxiety, learning and memory behaviours in an all-in-one
continuous system (see Ennaceur et al, this meeting). The effects of
different doses of GABA-A receptor selective compounds, chlordiazepoxide
and THIP, and H3R selective compounds methimepip (agonist) and JNJ-
5207852 (antagonist) have been explored on the behaviour of Balb/c mice.
This strain of mice has been previously shown to be more anxious than
C57 mice and less anxious than C3H mice2. An anxiolytic effect of the drug
is expected to align the behaviour of Balb/c to that of C57 mice, and an
anxiogenic effect of the drug would align the behaviour of Balb/c to that of
C3H mice. This model will be an ideal system to determine, for the first
time, the true role of the GABA-A and H3 receptors in bone fide ‘normal’
anxiety. We will present our early findings.
1. Ennaceur, A, Michalikova S, Chazot PL (2006a) Behav. Brain Res.171:
26–49.
2. Ennaceur, A, Michalikova S, van Rensburg, R, Chazot PL (2006b)
Behav. Brain Res 174: 9-38.
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20.01
Cannabinoid neuropharmacology: recent developments
Ross R
Institute of Medical Sciences, University of Aberdeen. Aberdeen. AB25
2ZD. Scotland.
The endocannabinoid system comprises two known receptors (CB1
and CB2); a family of endogenous ligands; and specific molecular
machinery for the synthesis, transport, and inactivation of CB ligands.
CB1 receptors are highly expressed throughout the CNS and, more
recently, CB2 receptors have been identified and functionally
characterised in the brainstem; however, CB2 receptor expression on
neurones remains the subject of controversy. There is considerable
pharmacological evidence that the endocannabinoid system may
encompass additional targets. For example, recent patent applications
provide the first evidence that certain synthetic and endogenous
cannabinoids interact with orphan receptors, a prominent example
being GPR55. Evidence has also emerged that the cannabinoid CB1
receptor contains an allosteric binding site. Novel compounds
targeting this site thereby herald a new generation of therapeutics to
be used, for example, as analgesics (allosteric enhancers) or antiobesity
agents (allosteric inhibitors). The endocannabinoids,
anandamide and 2-arachidonoylglycerol (2-AG) are rapidly hydrolysed
by the microsomal enzyme, fatty acid amide hydrolase (FAAH). They
can also be metabolized by a range of oxygenase enzymes that are
already known to convert arachidonic acid to potent biologically active
compounds. These include cyclooxygenase, lipoxygenase and P450
enzymes. Inhibition of COX-2 potentiates retrograde endocannabinoid
effects in the hippocampus and PGE2 glycerol ester, a COX-2
oxidative metabolite of 2-AG, modulates inhibitory synaptic
transmission in hippocampal neurones. Oxygenation of anandamide
and 2-AG may therefore lead to the production of a range of novel lipid
products that may have an important physiological or
pathophysiological role.
20.02
Influence of cannabinoids on neural fate
Campbell V, Gowran A, Downer E
Department of Physiology and Trinity College Institute of Neuroscience,
Trinity College, Dublin 2, Ireland
The cannabinoid system has been demonstrated to exert an influence on
neuronal viability with neuroprotective and neurotoxic properties being
reported. The neuroprotective effects are mediated through antioxidant
properties whilst we have found that the neurotoxic effects are mediated
through activation of the CB1 cannabinoid receptor, induction of stressactivated
protein kinases and subsequent commitment to apoptosis.
Exposure of cultured cortical neurons to tetrahydrocannabinol (THC, ЧM),
the principal psychoactive moeity of marijuana, evokes apoptosis (P

21.01
GAD levels in cerebellar Purkinje cells, interneurons and deep
cerebellar neurons in autism: pathophysiological implications
Yip J, Soghomonian J-J, Blatt G J
Department of Anatomy and Neuobiology, Boston University School of
Medicine, Boston, MA 02118 USA,
Purkinje cell loss is one of the most consistent findings in autism
neuropathology but little is known about their functional status.
Purkinje cells are the output neurons from the cerebellar cortex
sending inhibitory GABAergic projections to the cerebellar nuclei that
connect to the thalamus and higher association cortices. Purkinje cell
outputs are crucial for sensorimotor and cognitive functions. Purkinje
cells are vulnerable due to high metabolic demand, but the status of
GABAergic functions in the remaining PCs in the autistic brain remains
to be elucidated. In the current study, we used in situ hybridization
tecnhniques to investigate the mRNA levels of two isoforms of GABA
key synthesizing enzymes, glutamic acid decarboxylase (GAD)
GAD67 and GAD65. We demonstrated a 40% reduction in GAD67
mRNA levels Purkinje cells and a 28% increase in cerebellar basket
cells in the autistic cerebellar posterolateral lobe, a region previously
demonstrated to have the most robust Purkinje cells decrease. The
levels of GAD65 mRNA in the dentate nuclei were significantly
reduced, indicating potential adverse effects in their output projections
to higher centers. These results suggest that there is a deficit in
GABAergic function that may have contributed to the decreased
Purkinje cell numbers in autism. Reduced GABA transmitter release
from Purkinje cells in autism is likely to enhance neuronal excitability
consequently resulting in widespread changes to motor and/ cognitive
output from higher cortical centers in autism. Tissue was provided by
the Autism Research Foundation (TARF) and The Autism Tissue
Program. Supported by NICHD HD39459-04 (GJB, P.I.).
21.02
Neurochemical alterations in the autistic cerebellum: a model of
cerebellar dysfunction of the GABA/glutamate systems
Blatt G J, Yip J, Thevarkunnel S
Department of Anatomy and Neurobiology, Boston University School of
Medicine, Boston, MA 02118 USA
In the normal human cerebellar cortex, olivocerebellar climbing fibers
(OCFs) innervate up to ten Purkinje cells (PCs) and each PC receives a
single OCF input. In autism, a neurologic disorder characterized by delayed
and disordered language, stereotypical repetitive behavior and abnormal
socio-emotional interactions, many OCFs are abnormally thicker in caliber
width than age-matched adult controls, and some PCs receive multiple
OCF inputs. This suggests possible increased glutamate/aspartate
stimulation of PCs that potentially triggers off a cascade of neurochemical
and neurophysiological events that could adversely affect cerebellar output
via the deep cerebellar nuclei. Consequently, there is a down regulation of
AMPA receptors in the autistic cerebellar cortex that may also be the result
of abnormal mossy or parallel fiber innervation. Increased GAD67 in basket
cells (see Yip et al. abstract) may translate to increased GABA innervation
to PCs, and to support this, there is also a down regulation of GABA-A
receptors and benzodiazepine binding sites in the cerebellar cortex of adult
autistic subjects. These cerebellar cortical changes may directly affect the
functioning of the deep cerebellar nuclei leading to abnormal regulation of
high order cortical association areas that participate in the motor and/or
cognitive behaviors associated with autism. Tissue was provided by the
Autism Tissue Program (ATP) and The Autism Tissue Foundation (TARF).
Grant support was provided by NIH NICHD HD39459, Cure Autism Now
Foundation, the Institute for Brain Potential and the Hussman Foundation
(all GB, P.I).
21.03
Cholinergic systems in autism
Perry E, Maddick M, Court J
Cholinergic systems may be involved in attentional dysfunction in
autism. Our previous autopsy investigations indicated nicotinic and
muscarinic acetylcholine receptor deficits in some brain areas in young
adults with autism. Major reductions ( 50-70%) in α4β2 containing
nicotinic receptors were evident in the neocortex and cerebellum,
whereas less severe deficits were observed in muscarinic receptors
(M1, 30% in parietal cortex).
Anatomically, nicotinic receptor deficits were extensive, being evident
in many neocortical regions (BA 7, 9, 17 and 39), the thalamus and
striatum as well as cerebellum. A consistent and significant reduction
in M1 receptors was also demonstrated in the striatum but not the
thalamus.
Since the cholinergic system may have a role in neurogenesis and the
establishment of cytoarchitecture during brain development, we
examined cholinergic markers (choline acetyltransferase activity,
muscarinic M1 receptors and α4β2 containing receptor binding in
children with autism (2-9 years). In contrast to the adult, high affinity
nicotinic receptor binding (α4β2) was not reduced in the frontal cortex
of children with autism. There were also no significant changes in α-
bungarotoxin (α7) or pirenzepine (M1) binding in this brain region.
However a proportion of children with autism in common with some
adults exhibited very low M1 receptor binding in the frontal cortex
suggestive of this being a feature in a subset of autistic individuals.
Since the earlier pathological studies, pilot clinical trials have indicated
the possible benefit of cholinergic therapy in both adults and children
with autism.
21.04
Glial cells within the cerebral cortex in autism
Rezaie P, Landau S, Mason S, Schmitz C
Neuropathology Research Laboratory, Department of Biological Sciences,
The Open University, Milton Keynes MK7 6AA, and Department of
Neuroscience, Institute of Psychiatry, King’s College London, DecRespigny
Park, London SE5 8AF
The underlying neurobiology of autism remains obscure. However, subtle
disturbances in the development of neurons within the brain, and certain
immune system abnormalities are believed to occur in patients with autism.
Neuro-immune interactions are governed locally by networks of resident
glial cells- microglia and astrocytes, whose activation parallels ongoing
pathology within this tissue. Disturbances in the function of glial cells may
have adverse consequences on the activity of neurons and vice versa.
Neuroglial activation and neuroinflammation have been proposed to
contribute towards the neuropathology of autism. Previously, we reported
differential activation of microglia and astrocytes within the frontal lobe of a
cohort of cases with autism (Neuropathol. Appl. Neurobiol. 2006; 32:226-
227) - glial cell activation was largely restricted to astrocytes within cortical
white matter (WM) in all autism cases examined. Considering that such WM
astrocyte activation was not associated with diffuse axonal injury, postmortem
delay, age or history of seizures in autism, and taken together with
the lack of lymphocytic/monocytic infiltration, or of significant microglial
activation, we proposed that glial cell alterations do not necessarily reflect
‘neuroinflammation’ as recently suggested. Instead they may be a
consequence of acute hypoxic-ischaemic injury associated with the agonal
state of the cases examined, or they could represent a specific dysfunction
targeting astrocytes in autism. We have now systematically investigated
glial cell responses within several regions of the cerebral cortex known to
be affected in autism, in a separate cohort of cases derived from the Autism
Tissue Program (USA).
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21.05
Spatial arrangement of cortical neurons and glia in autism.
Luthert PJ 1, McDermott CJ 1, Dean AF 2, Bailey A 3
1 Division of Pathology, Institute of Ophthalmology, London, UK, 2
Neuropathology, Addenbrookes Hospital, Cambridge, UK, 3
Department of Psychiatry, University of Oxford, UK,
Previously we identified relatively focal qualitative disturbances of
neuronal organisation in the cortex of individuals with autism.
Subsequently, Casanova and colleagues drew attention to
abnormalities of minicolumn arrangment in autism. In this study we
tested the hypothesis that there were distributed abnormalities of
spatial arrangement of neurons or glia in autism.
Using a photo-microscope with a stepping stage, sections from the
anterior cingulate gyrus, at the level of the genu of the corpus
callosum, and the occipito-temporal gyrus at the level of the pes of the
hippocampus were examined. X and Y coordinates of neurons and
glia were recorded. Probability density maps were then constructed by
taking each individual cell in turn and plotting the positions of its
neighbours. Neurons and glia were plotted separately. In this way it
was possible to generate a probability map giving the relative
likelihood of finding cells at a given distance and angle from any cell.
The analysis was carried out for each lamina.
Density maps generally had a central peak with smaller, approximately
symmetrical peaks either side. This pattern is consistent with a
minicolumnar organisation. The width of the central peak and the
distance from the central peak to those either side was measured. The
main finding was of a narrowing of the glial, but not neuronal, central
cluster width in individuals with autism in both areas examined.
These findings suggest that there may be a disturbance of glial,
neuronal interaction as part of the the neuroanatomical substrate of
autism.
22.01
Noradrenergic control of inflammatory processes in the CNS
Connor T J, McNamee E N
Neuroimmunology Research Group, School of Medicine & Trinity College
Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2.
Evidence suggests that inflammation is a significant contributor to
pathology in a number of neurodegenerative disease states. In this regard,
the pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in
initiating an immune response within the central nervous system (CNS).
The actions of IL-1β can be regulated by interleukin-1 receptor antagonist
(IL-1ra), which prevents IL-1β from acting on the IL-1type I receptor.
Consequently, the balance between IL-1ra/IL-1β is of pathological
importance, and pharmacological strategies that tip the balance in favour of
IL-1ra may be of therapeutic benefit. Evidence is emerging to suggest that
the neurotransmitter noradrenaline elicits anti-inflammatory actions in the
CNS, and consequently may play an endogenous neuroprotective role.
Here we report that noradrenaline induces production of secreted IL-1ra
from primary rat mixed glial cells. This noradrenaline-induced increase in
IL-1ra production is mediated via β-adrenoceptor activation, and
downstream signaling via the cAMP-Protein Kinase A pathway. In addition
to increasing IL-1ra, noradrenaline increased expression of the IL-1type II
receptor; a decoy receptor that serves to sequester IL-1β. The ability
noradrenaline to induce IL-1type II receptor expression was also mediated
via β-adrenoceptor activation. Importantly, in parallel with its ability to
increase IL-1ra and IL-1type II receptor expression, noradrenaline
attenuated functional responsiveness to IL-1β in mixed glial cells.
Considering that the pivotal role played by IL-1β in neuroinflammation, the
ability of noradrenaline to negatively regulate the IL-1 system in glial cells
may of therapeutic relevance in neurodegenerative disorders where
inflammation contributes to pathology.
The author acknowledge grant support from Science Foundation Ireland
22.02
The systemic control of acute and chronic inflammation in the
brain
Campbell S, Mann D, Deacon R, Jiang Y, Pitossi F, G³¹biñski A,
Anthony DC
Experimental Neuropathology, Department of Pharmacology,
University of Oxford, Oxfordshire, OX1 3QT, UK.,
Acute brain injury induces NFkB-dependent early and transient
hepatic expression of chemokines, which amplify the injury response
and give rise to movement of leukocytes into the blood and,
subsequently, the brain and liver. We have now discovered that an
ongoing injury stimulus within the brain continues to drive the hepatic
chemokine response, which impacts both on behaviour and on CNS
integrity. We generated chronic IL-1β expression in rat brain by
adenoviral-mediated gene transfer, which resulted in chronic leukocyte
recruitment, axonal injury, and prolonged depression of spontaneous
behaviour. IL-1β could not be detected in circulating blood, but a
chronic systemic response was established, including extended
production of hepatic and circulating chemokines, leukocytosis, liver
damage, weight loss, decreased serum albumin, and marked liver
leukocyte recruitment. Similarly, we have also discovered that an
extended hepatic chemokine response is a feature of chronic immunemediated
(PLP-EAE) CNS disease; increased hepatic chemokine
expression accompanies the onset of clinical signs. Thus hepatic
chemokine synthesis is a feature of active chronic CNS disease and
provides an accessible target for the suppression of CNS
inflammation.
22.03
Modulating microglial activation in the brain of aged rats impacts on
synaptic function
Lynch M A
Trinity College Institute of Neuroscience, Trinity College, Dublin
It is now accepted that several neurodegenerative conditions are
associated with evidence of neuroinflammatory changes and, similarly,
inflammatory changes are evident in brains of animal models of
neurodegenerative diseases. Among the features of inflammation is an
increase in microglial activation and this is likely to account for the
increased expression of proinflammatory cytokines like interleukin-1β (IL-
1β) which has also been reported in these conditions. Both increased
microglial activation and increased expression of several proinflammatory
cytokines, coupled with decreased expression of anti-inflammatory
cytokines like IL-4, have been shown to accompany ageing and these
changes are associated with deficits in cognitive function. This deficit in
cognitive function is typified by a decrease in one form of synaptic plasticity,
long-term potentiation (LTP), which is restored when the age-related
increase in microglial activation is attenuated. We have established that
reversing the age-related decrease in hippocampal IL-4 concentration is a
key factor leading to restoration of LTP and have found that IL-4 may
mediate its effects by modulating interaction between neurons and
microglia. Evidence will be presented which indicates that IL-4 increases
expression of the glycoprotein, CD200, on neurons and that interaction of
CD200 with its cognate receptor, CD200R, which is expressed on
microglia, is an important factor in maintaining microglia in a quiescent
state. Significantly CD200 expression is decreased with age, but at least
some measures which attenuate the age-related microglial activation
increase IL-4 and subsequently increase CD200 expression.
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22.04
Inflammation and CNS remyelination
Franklin R J M
, Cambridge Centre for Brain Repair, University of Cambridge,
Cambridge CB3 OES, UK,
Remyelination, the process by which new myelin sheaths are restored
to demyelinated axons, represents one of the most compelling
examples of adult multipotent stem/precursor cells contributing to
regeneration of the injured CNS. This process can occur with
remarkable efficiency in both clinical disease, such as multiple
sclerosis, and in experimental models, revealing an impressive ability
of the adult CNS to repair itself. However, the inconsistency of
remyelination in multiple sclerosis, and the loss of axonal integrity that
results from its failure, makes enhancement of remyelination an
important therapeutic objective. Identifying potential targets will
depend on a detailed understanding of the cellular and molecular
mechanisms of remyelination. This talk will review 1) the nature of the
cell or cells that respond to demyelination and generate new
oligodendrocytes, 2) the concept of inflammation-induced adult
progenitor activation, 3) how an environment favourable to
remyelination is generated by inflammation, and 4) will introduce the
concept of a matrix of signalling events critical for the successful
completion of remyelination.
23.01
Mechanisms of adenosine release in the basal forebrain and control of
sleep
Dale N
Department of Biological Sciences, University of Warwick, Coventry, CV4
7AL
Adenosine, an important homoeostatic regulator of sleep, accumulates in
the brain during wakefulness and dissipates during sleep. In the basal
forebrain and preoptic areas these increased levels of adenosine lead to
slow wave sleep. The mechanisms by which adenosine accumulates during
wakefulness, and disappears during sleep remain opaque. I have examined
the mechanisms of adenosine release in the basal forebrain in vitro using
patch clamp recordings and novel real-time biosensor measurements.
Although depolarization is a sufficient stimulus for adenosine release, only
selective depolarizing stimuli (activation of NMDA, AMPA or mGlu
receptors) are able to evoke adenosine release. Other powerful
depolarizing stimuli of basal forebrain neurons such as orexin, neurotensin
and histamine do not evoke adenosine release. Adenosine release evoked
by high K+, AMPA, NMDA and mGlu receptors does not require
extracellular Ca2+, suggesting that neuronal exocytosis cannot underlie its
production. This glutamate receptor mediated adenosine release exhibits
diurnal variation and can be significantly enhanced by prior sleep
deprivation. Some aspects of adenosine accumulation related to sleep
homoeostasis can therefore be studied in vitro. I suggest that activitydependent
release of adenosine (mediated via glutamate receptors)
coupled with diurnal variation in the capacity to release adenosine could
explain the sleep-wake cycle of adenosine accumulation and removal.
Identification of the adenosine-releasing cells will greatly assist the
understanding of adenosine-dependent mechanisms of sleep homeostasis.
23.02
The role of ATP in the brain mechanisms controlling breathing
Gourine A V, Dale N, Spyer K M
Department of Physiology, University College London, Gower Street,
London WC1E 6BT and *Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, United Kingdom
There has been increasing interest in the role of purines in the
nervous system. In addition to its known role as an intracellular energy
source, ATP also functions as an extracellular signalling molecule in
the brain and many peripheral tissues. Immunohistochemical studies
demonstrate the presence of ATP receptors throughout the brainstem
structures involved in respiratory control. Results obtained in our
laboratory indicate that ATP-mediated purinergic signalling indeed
plays an important role in the brain mechanisms controlling breathing.
Using transgenic animal models we have demonstrated that ATP
acting via ionotropic P2X2 receptors is essential for the development
of normal ventilatory response to hypoxia. It was also found that ATP
mediates central respiratory CO2 chemosensory transduction in the
medulla oblongata. Recently we obtained evidence suggesting that
ATP is also involved in central afferent processing – ATP is released
with glutamate from the central terminals of slowly adapting lung
stretch receptors to activate second-order relay neurones. Thus, it
emerges that ATP acts as a common mediator of peripheral and
central chemosensory transduction and also contributes to
neurotransmission at a first central synapse in the Breuer-Hering
reflex pathway. These data reveal the importance of purinergic
signalling in the brainstem mechanisms underlying respiratory control
(Supported by the BBSRC and the Wellcome Trust).
23.03
Astrocytic purinergic signaling coordinates synaptic networks
Haydon P G
Department of Neuroscience, University of Pennsylvania School of
Medicine,
Many recent studies have demonstrated that astrocytes release chemical
transmitters in a process termed gliotransmission. Though gliotransmission
can modulate synaptic transmission and neuronal excitability, it is less clear
how this process regulates neuronal networks. We have generated
inducible, astrocyte-specific transgenic mice which express a dominant
negative SNARE domain that suppresses vesicular exocytosis and
consequently reduces the release of gliotransmitters from astrocytes. Using
brain slices from this animal we demonstrate that excitatory synaptic
transmission at the Schaffer collateral CA1 synapse is augmented when
gliotransmission is suppressed, a result which arises from a reduction in
extracellular adenosine. These studies demonstrate that astrocytes
regulate adenosine which causes a presynaptic inhibition of synaptic
transmission. Neighboring synaptic pathways exhibit adenosine-mediated
heterosynaptic depression. Using dnSNARE transgenic animals our results
demonstrate that activation of astrocytes leads to ATP release, which
following hydrolysis to adenosine, results in the depression of neighboring
synapses. Since each astrocyte contacts tens of thousands of synapses,
our results indicate that astrocytes are able to coordinate groups of
synapses in the hippocampus. Because of the potential for adenosine in
the control of sleep and seizures, current studies are examining neural
networks in vivo to test the hypothesis that astrocytes are critically involved
in these two processes.
Supported by funds from the NINDS and NIMH
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23.04
Adenosine, astrogliosis and epilepsy: a rational approach for
novel cell and gene therapies
Boison D
R.S. Dow Neurobiology Laboratories, , Legacy Research, , Portland
OR, , USA
Adenosine is an inhibitory modulator of brain activity with
neuroprotective and anticonvulsant properties. In adult brain,
extracellular levels of adenosine are mainly regulated by intracellular
metabolism via the astrocyte-based enzyme adenosine kinase (ADK),
which removes adenosine via phosphorylation to AMP. Recent
evidence suggests that ADK expression undergoes rapid and
coordinated changes during brain development and following brain
injury, such as after status epilepticus or stroke. Thus, after acute
brain injury, transient downregulation of ADK initially protects the brain
from seizures and cell death. However, astrogliosis as a chronic
response to brain injury leads to overexpression of ADK, which can
cause seizures and promote cell death in epilepsy. To address the
role of ADK in epileptogenesis, we created a panel of transgenic mice
with either elevated levels of brain ADK (160%, Adk-tg), or reduced
levels of forebrain ADK (60%, fb-Adk-def). Subjecting these animals to
intraamygdaloid kainic acid injections revealed that even subtle
changes in ADK expression render the brain more vulnerable (Adk-tg)
or more resistant (fb-Adk-def) to subsequent epileptogenesis. In line
with these findings intrahippocampal implants of ADK-deficient – and
thus adenosine releasing – ES cell derived neural progenitor cells
retard the development of kindling epileptogenesis in rats. ES cell
derived brain implants showed integration into the CA1 region of the
hippocampus and neuronal differentiation. We conclude that tight
regulation of ambient levels of adenosine by ADK controls the
vulnerability of the brain but also offers a rationale for therapeutic
intervention.
24.01
Behavioural feedback and circadian rhythms
Piggins H D
Faculty of Life Sciences, University of Manchester, Manchester UK M13
9PT
Daily rhythms in mammalian physiology and behaviour are a product of the
activities of the master circadian clock in the suprachiasmatic nuclei (SCN)
and the entrainment of this clock to photic cues (the light-dark cycle) and
arousal-promoting, non-photic cues such as social interactions, food
availability, etc. The SCN is composed of thousands of autonomous cellular
clocks and the past 5 years has seen considerable progress in
understanding the mechanisms via which these clock cells become
synchronized to one another. Vasoactive intestinal polypeptide (VIP) acting
via the VPAC2 receptor has emerged as a key SCN intercellular signalling
pathway involved in such processes. Transgenic mice deficient in VIP (VIP-
/-) or the VPAC2 receptor (Vipr2-/-) manifest profoundly disrupted circadian
competence, reduced SCN neuronal excitability, and do not respond
properly to photic cues. It is unknown if behavioural rhythms in these mice
can be altered by non-photic cues. Using a schedule of daily locomotor
activity whereby mice voluntarily exercised in a running wheel for 6h/day,
we found that this non-photic stimulus reorganizes and sculpts locomotor
rhythms in VIP-/- and Vipr2-/- mice, such that they can sustain robust near
24h rhythms in behaviour. These findings indicate that behavioural
feedback is more effective than light in resynchronizing rhythms in mice
with impaired neuropeptide signalling, raising the possibility that scheduled
exercise can be used as rescue circadian competence in organisms with
impaired circadian clocks. Supported by the BBSRC.
24.02
Melatonin in the avian pineal gland and retina – photic, circadian,
and neurochemical regulations
Zawilska J B
Centre for Medical Biology, Polish Academy of Sciences and
Department of Pharmacodynamics, Medical University of Lodz,
Poland
The avian pineal gland and retina synthesize melatonin in a lightdependent
circadian rhythm with high levels at night. The rate of
melatonin formation is regulated primarily by serotonin N-
acetyltransferase (AANAT). Circadian oscillations in AANAT activity
were found in pineal glands and retinas of chickens and turkeys kept
under constant darkness, or continuous light (pineals only). Exposure
to white light and near-ultraviolet radiation (UV-A) acutely suppressed
nocturnal AANAT activity and melatonin. This light action is mediated
by D4-dopamine receptors in the retina and alpha2-adrenergic
receptors in the pineal, and involves decrease in cAMP, ultimately
leading to the proteosomal destruction of AANAT protein. Light also
resets the circadian pacemaker generating melatonin rhythm. Pulses
of light applied during the first and second half of the night produced
phase delay and phase advance, respectively, of the circadian rhythm
of AANAT activity in the pineal gland and retina. In galliforms, pineal
activity, in addition to being directly photosensitive, is regulated by
retinally perceived light. White light and UV-A, acting on the eyes only,
suppressed nocturnal melatonin synthesis in the pineal gland. Both
light signals were also capable of resetting the phase of the circadian
rhythm of pineal AANAT activity. It is suggested that regulation of
melatonin synthesis in the chicken pineal by retinally perceived white
light and UV-A might involve input from different photoreceptors. The
cascade of events triggered by white light and UV-A includes
stimulation of retinal D1-dopamine and NMDA-glutamate receptors,
respectively.
24.03
Circadian plasticity of neurons and glial cells
Pyza E, Gorska-Andrzejak J, Weber P, Radowska A
Department of Cytology and Histology, Institute of Zoology, Jagiellonian
University,, Ingardena 6, 30-060 Krakow, Poland,
In the visual system of flies, the first order interneurons, the lamina
monopolar cells L1 and L2 and epithelial glial cells show circadian rhythms
in morphological changes. These rhythms have been detected in three
species; Musca domestica, Calliphora vicina and Drosophila melanogaster.
In all species the cross-sectional area of L1 and L2 axons changes during
the day and night (LD) and also under constant darkness (DD) and
continuous light (LL) conditions, indicating on their endogenous generation
by a circadian clock. In the housefly both neurons swell during the day and
shrink during night while the epithelial glial cells show the opposite pattern
of morphological changes, swelling during the night. Using Drosophila
transgenic line in which L2 cells are labelled with green fluorescent protein
(GFP) we showed that beside L2 axons, dendrites and nuclei, but not
somata, change their sizes. These changes are under control of clock
genes since in per01 mutants, L2 morphology do not change in LD and DD.
Daily changes of glial cell morphology were detected in another Drosophila
transgenic line expressing GFP under control of glia specific gene repo.
Like in M. domestica, glial cells in Drosophila are larger when L2 are
shrank. The changes in morphology of L2 and glial cells seem to correlate
with daily changes of synaptic contacts between the photoreceptors and
monopolar cells in the lamina and with expression of certain genes.
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24.04
Membranes, ions and clocks: cellular physiology of pacemaker
neurons
Nitabach M N
Department of Cellular & Molecular Physiology Interdepartmental
Neuroscience Program Yale University School of Medicine
Canonical views of circadian pacemaker function rely on intracellular
transcriptional/translational feedback loops. However, work in a
number of model systems has over the last few years led to the
development of a richer view of cellular timekeeping that involves
depolarization-dependent events at the plasma membrane of
pacemaker neurons. In this presentation, we will discuss recent
findings using the genetically tractable fruit fly, Drosophila
melanogaster, as a system for analyzing the role of intracellular
calcium signaling in pacemaker neurons for circadian timekeeping,
and the roles of particular voltage-gated ion channels in regulating
pacemaker membrane properties and timekeeping.
25.01
Structural biology of neuronal cell adhesion molecules and their
counter receptors
Bock E, Berezin V
Panum Insitute, University of Copenhagan, Denmark
We have studied the structure of NCAM in order to identify in which way
NCAM interacts with itself (so-called homophilic binding) and with other
ligands/counter receptors, e.g. the FGF-receptor (so-called heterophilic
binding). Our results indicate that NCAM binds to itself both in a cis- and in
a trans- manner forming a cis-dimer on the cell surface which can interact
with NCAM-dimers on opposing cells in trans, establishing two types of
zippers, a flat and a dense zipper. In combination the two types of zippers
are capable of establishing a two-dimensional NCAM patch. NCAM
interaction with the FGF-receptor involves at least five binding sites in
NCAM. To get a more clear impression of the NCAM-FGF-receptor
interaction, we have also identified the binding site for NCAM in the FGFreceptor
itself and determined in which manner it overlaps with the binding
sites for FGF and heparan sulphate. Recently, we have presented the first
determination of the N-terminal Ig-module of the FGF-receptor and shown
that this module is a regulator of the function of this receptor. The many
identified binding sites have been prepared as peptides, and their binding
capacity has been studied by means of surface plasmon resonance, and
subsequently they have been characterized with various in vitro and in vivo
biological tests. It turns out that these peptides have individual functional
profiles. In vitro some affect neuronal differentiation and survival, and in
vivo, learning and memory. Moreover, some have beneficial effects in
models of Alzheimer’s disease and traumatic brain injury.
25.02
The role of cell adhesion molecules (CAMS) and cam mimetics in
synaptic plasticity: ultrastructural studies
Stewart M G
Dept. of Biological Sciences, , The Open University,, Milton Keynes,
MK7 6AA,
The Neural Cell Adhesion molecule (NCAM) is a member of the Ig
superfamily expressed on the surface of neural cells and is involved in
cell–cell interactions and synaptic plasticity. FGL (fibroblast growth
loop) is an NCAM mimetic consisting of a 15 amino acid peptide
derived from the FGF binding site of NCAM. FGL (icv) facilitates
spatial memory consolidation, and can reduce the β-amyloid load in
rats.
We have examined how FGL affects synaptic and dendritic
morphology, focusing initially on aged rats (22 months, ~560g). Rats
(from M. Lynch Trinity, Dublin) were injected subcutaneously with FGL
(8mg/kg) at 2-day intervals until 19 days after the experiment start;
control rats were injected with sterile water. Animals were perfused
with fixative, brains removed and coronal vibratome sections
containing the hippocampus cut at 100um. Tissue was embedded and
ultra-thin sections viewed in a JEOL 1010 electron microscope and
digitised images captured with a GATAN camera. Analyses were
made of synaptic and dendritic parameters following 3D reconstruction
via images from up to 150 serial sections
(http://synapses.bu.edu/index.htm).
FGL altered neither spine nor synaptic density in medial molecular
layer, but increased the ratio of mushroom to thin spines, the number
endosomes, and the abundance of smooth endoplasmic reticulum and
spine apparatus, whilst it decreased synaptic and spine curvature.
These data indicate that FGL induces rapid and large-scale changes
in synapse and dendritic spines in the hippocampus of aged rats
complimenting data showing its marked effect on cognitive processes.
25.03
Role of the neural cell adhesion molecule in memory consolidation
and cognitive flexibility
Sandi C
Brain Mind Institute, Swiss Federal Institute of Technology Lausanne
(EPFL), Switzerland
The neural cell adhesion molecule (NCAM) plays critical roles in synaptic
plasticity and learning and memory. We showed that NCAM is increased in
the hippocampus 24h after training rats in hippocampus-dependent tasks
(water maze, contextual fear conditioning). To evaluate the functional
contribution of NCAM to the mechanisms of memory consolidation, we
tested the effects of a intracerebroventricular infusions of a number of
peptides (developed by E. Bock and V. Berezin, Copenhagen) addressed
to either interfere or enhance NCAM function. First, we tested a synthetic
peptide, C3d, which through the binding to the first, N-terminal
immunoglobulin-like module, interferes with NCAM homophilic adhesion.
This peptide impaired the consolidation of both contextual fear conditioning
and water maze learning. Conversely, administration of FGL, a synthetic
peptide corresponding to the binding site of NCAM for the fibroblast growth
factor receptor-1 (FGFR1), induced a strong potentiation of memory for
both tasks, as well as a long-lasting facilitation of reversal learning. In
addition, a combination of behavioral, biochemical, genetic and
pharmacological experiments showed that NCAM polysialylation (PSA-
NCAM) also plays critical roles in memory consolidation and behavioral
flexibility. These and novel findings will be discussed to highlight NCAM as
a learning-modulated molecule critically involved in the hippocampal
remodeling processes underlying memory formation and cognitive
flexibility.
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25.04
The L1 cell adhesion family and their interaction with the 4.1
superfamily
Lissa Herron(1), Davey F(1), Maria Hill(1), Aviva Tolkvosky(2), Diane
Sherman (3), Peter Brophy (3), Frank Gunn-Moore(1)
(1) School of Biology, Bute Medical Building, University of St Andrews,
Scotland, UK. KY16 9TS, (2) Dept Biochemistry,, University of
Cambridge, Building 0,, The Downing Site, Cambridge CB2 1QW, UK,
(3)Division of Veterinary Biomedical Sciences, University of
Edinburgh, Summerhall, Edinburgh EH9 1QH
The L1 immunoglobulin (Ig) subfamily of cell adhesion molecules
includes L1, NrCAM, (Neuron glial-related cell adhesion molecule) and
neurofascin. Their fundamental importance in mammalian
development is highlighted by their constituting 1% of all membrane
proteins in mature brains; their involvement in growth cone and
synapse formation, and cancer development. Mutations can lead to
human retardation and knockout studies show phenotypic changes.
We have shown that these receptors bind to differing cytoplasmic
proteins and so elicit differing signals. From these studies we found
that both neurofascin and L1 but not NrCAM, can bind to the 4.1
superfamily protein member, Ezrin, but by different binding motifs.
Physiologically the interaction of Neurofascin and Ezrin appears to
occur in the microvilli of interdigitating Schwann cells over the node of
Ranvier, whilst L1 and Ezrin interaction is important for neuronal
growth. This interaction between Neurofascin and Ezrin was via the
FERM (4.1 Ezrin-Radixin-Moesin) domain of Ezrin and 28 amino acid
sequence at the cytoplasmic C-terminus of Neurofascin. As part of
these studies, we identified a novel FERM containing protein, “Willin”.
Willin has a recognizable N-terminal FERM domain, which is able to
bind both phospholipids and proteins. Recently Willin has been
identified as the human homologue to the Drosophila protein,
Expanded, which associates with Merlin, a tumour suppressor protein
responsible for neurofibromatosis. We have shown that Willin is
expressed in the peripheral nervous system in Schwann cells and we
are currently investigating its association with Neurofascin and Merlin.
25.05
Theta frequency-induced bursting of dentate gyrus cells correlates
with long-term synaptic plasticity
Tsanov M, Manahan-Vaughan D
International Graduate School of Neuroscience, Ruhr University Bochum,
FNO 1/116, Universitaetsstr 150 44780 Bochum, Germany
Neocortico-hippocampal transfer of new spatial information occurs during
exploratory behaviour. The entorhinal cortex encodes, in a theta rhythmassociated
manner, particular representations in subsets of hippocampal
neurons where memories are temporarily held. However, it remains
unclear, how neuronal cooperativity during theta oscillations is powerful
enough to bring about the nondecremental synaptic change required to
achieve this. We report that long-term potentiation occurs in the dentate
gyrus after phasic activation of entorhinal afferents in the theta-frequency
range in freely moving rats. This plasticity is proportional to the bursting
ability of granule cells during the stimulation, and may comprise a key step
in spatial information transfer. Long-term potentiation of the synaptic
component results only after temporal proximity between the afferent
stimulus and the evoked population burst. Our findings confirm
synchronization-dependent memory models and gap the transition between
functional and pathological patterns of network activity in hippocampus.
26.0
Nitric oxide, bioenergetics and cell signalling
Moncada S
Wolfson Institute for Biomedical Research, University College London,
Gower Street, London WC1E 6BT
At physiological concentrations nitric oxide (NO) inhibits mitochondrial
complex IV (cytochrome c oxidase) in competition with oxygen. This
action allows NO to act not only as a physiological regulator of cell
respiration but also as a signalling agent in the mitochondria. Using a
technique that we have developed based on visible light spectroscopy
we have recently demonstrated that endogenous NO enhances the
reduction of the mitochondrial electron transport chain, thus
contributing to a mechanism whereby cells maintain their VO2 at low
[O2]. This favours the release of superoxide anion, which initiates the
transcriptional activation of NF-ÜB as an early signalling stress
response.
Many cells respond to a decrease in oxygen availability via
transcriptional activation of hypoxia-inducible genes. Activation of
these genes requires the stabilisation of hypoxia-inducible factor-1Ü
(HIF-1Ü) whose accumulation is normally prevented by the action of
prolyl hydroxylases. We have found that inhibition of mitochondrial
respiration by low concentrations of NO leads to inhibition of HIF-1Ü|
stabilisation. This prevents the cell from registering a state of hypoxia
at low oxygen concentrations, which would normally lead to the
upregulation of defensive genes associated with, for example,
glycolysis and angiogenesis. Furthermore, upon inhibition of
mitochondrial respiration in hypoxia, oxygen is redistributed toward
non-respiratory oxygen-dependent targets. The relevance of these
mechanisms to the survival/death of cells from the nervous system will
be discussed.
27.0
Insights into the molecular basis of memory
Collingridge G L
MRC Centre for Synaptic Plasticity, Department of Anatomy, School of
Medical Sciences, University Walk, Bristol, BS8 1TD, UK
Understanding the molecular basis of information storage in the brain
requires a concerted multidisciplinary approach involving all areas of
neuroscience. My group has focussed on one aspect of the topic, namely
the identification of molecular events that are involved in the modification of
synaptic strength. Using long-term potentiation (LTP) and long-term
depression (LTD) in the hippocampus as our experimental model we have
identified the roles of glutamate receptors (AMPA, NMDA, kainate and
mGluR), some proteins that interact with glutamate receptors (e.g., NSF,
PICK1) and some downstream signalling molecules (protein tyrosine
phosphatase, glycogen synthase kinase (GSK3)) in these plastic
processes. In addition to using electrophysiology we have exploited
imaging techniques to study the behaviour of native and recombinant
glutamate receptors and their associated calcium signals during synaptic
plasticity.
I will present an overview of our work which has helped establish these
molecules in synaptic plasticity and will discuss some of our recent work, in
which we have identified a role for GSK3 in cross-talk between LTP and
LTD.
Page 39/101 - 10/05/2013 - 11:11:03

28.01
Electrophysiological responsiveness of lateral habenular
neurones to arginine vasopressin
Guilding C, Hughes A T L, Piggins H D
Faculty of Life Sciences, University of Manchester, UK
The suprachiasmatic nuclei (SCN) of the hypothalamus contain the
master circadian pacemaker in the mammalian brain. The intrinsic
timekeeping signal generated in the SCN is propagated throughout the
brain by means of output factors such as arginine vasopressin (AVP)
and prokineticin 2, thus coordinating daily behavioural and
physiological rhythms. However, important recent findings have
indicated that other brain regions, including the lateral habenular
(LHb), express endogenous daily rhythms in cellular excitability and
immediate early gene expression. The LHb is linked to a number of
regions involved in circadian function, and may receive a direct
vasopressinergic input from the SCN. We investigated the
electrophysiological properties of LHb neurones and their
responsiveness to AVP in both rat and mouse using in vitro brain slice
preparations. LHb neurones displayed spontaneous firing of action
potentials; cells were generally active in mid to late day, and often only
for a short duration. Both regular firing (at frequencies of between 0.1-
10 Hz) and bursting type firing patterns were observed; bursts
commonly containing 2-6 spikes in a 20ms period. Following bath
application of AVP both excitatory and inhibitory responses were
observed. These data demonstrate that LHb neurones are responsive
to a known SCN output factor, AVP, and suggest that this brain region
may integrate and relay SCN circadian information to downstream
oscillators controlling physiology and behaviour. Supported by the
BBSRC.
28.02
The acute response to ketamine is modified by lamotrigine pretreatment
in healthy volunteers: a pharmacological challenge-fMRI
study
Lees J (1), McKie S (1), Hallak J(2), Dursun S (1), Deakin B (1), Williams S
(3)
(1) Neuroscience and Psychiatry Unit, University of Manchester (2)
Department of Neurology, Psychiatry and Psychological Medicine,
University of Sao Paulo (3) Imaging Science and Biomedical Engineering,
University of Manchester
Ketamine (a N-methyl-D-aspartate (NMDA) receptor channel blocker) has
been shown to mimic some schizophrenic symptoms. Pharmacomodulation
studies have shown that agents, such as lamotrigine (an
inhibitor of pre-synaptic glutamate release), block some of the ketamineinduced
effects in healthy subjects. Using direct pharmacological challenge
functional MRI (phMRI), we examined whether ketamine-induced changes
in blood oxygenation level dependent (BOLD) signal were mediated by
increased glutamate release or directly by NMDA blockade.
19 healthy volunteers were tested on two occasions receiving either
placebo or lamotrigine (300mg) in tablet form 2 hours prior to a ketamine
infusion (bolus 0.26mg/kg over 1 minute, maintenance infusion
0.25mg/kg/hr for rest of scan) in a randomised, balanced order, doubleblind
fashion. Each subject underwent a 16 minute fMRI scan 8 minutes
into which they received the infusion. Images were acquired on a 1.5T
Philips scanner with a multi-slice, single shot EPI sequence to achieve
whole brain coverage. Data were analysed using SPM2. Data analysis
identified voxels showing significant changes in successive 1 minute timebins
compared to pre-ketamine baseline and compared to placebo pretreatment
responses.
Several brain areas, which in a previous study had shown significant
ketamine-induced BOLD signal changes, showed a significant attenuation
of the ketamine-induced BOLD signal due to pre-treatment with lamotrigine.
We suggest ketamine phMRI reveals two mechanisms relevant to
psychosis – aberrant sensory processing through enhanced glutamate
function and dissociation through cognitive-emotional processing
suppression.
28.03
Altered dopamine D2 receptor function in VTA DA cells in
adenosine A2A knockout mice
Al-Hasani R, Hourani S M O, Kitchen I, Chen Y
School of Biomedical and Molecular Sciences, University of Surrey,
Guildford, UK
The adenosine A2A receptor knockout mice exhibit altered addictive
behaviours, which include reduced cocaine self–administration,
abolition of cannabinoid reward , but increased signs of morphine
withdrawal. A2A receptors are highly expressed in the dopaminergic
(DA) pathways in the brain and co-localized with dopamine D2
receptors. Direct interactions between the two receptors may be an
underlying mechanism for the involvement of adenosine A2A
receptors in the reward pathway. The VTA DA cells are critically
involved in the sensitization stage of the reward process. We
hypothesised that in the adenosine A2A receptor knockout mice, VTA
DA cell firing and dopamine D2 receptor function may be altered and
could account for altered addictive behaviours. Midbrain slices
containing the VTA were prepared from wildtype and adenosine A2A
knockout mice and the firing of single VTA DA cells was recorded.
There was no difference in the basal firing rate between the wildtype
and adenosine A2A knockout mice. The dopamine D2 receptor
agonist quinpirole caused a concentration-dependent decrease in DA
cell firing rate in both genotypes. However, DA cell firing was
completely inhibited in the wildtype mice, but not in the adenosine A2A
knockout mice. In addition, the adenosine A2A agonist CGS 21680
produced no significant response in either the wildtype or the
adenosine A2A knockout mice. These results showed a significant
reduction in dopamine D2 receptor function in the VTA DA cells in the
adenosine A2A knockout mice, even though functional A2A receptors
were not detected in these DA cells.
28.04
3-Carboxy-1-methylpyridinium reduces population spike amplitude
but has no effect on fibre volley amplitude
Woolley D R (1), Al-Hayani A (2), Davies S N (1), Scott R H(1)
(1) School of Medical Sciences, Institute of Medical Sciences, University of
Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, U.K. (2)
Department of Anatomy, Faculty of Medicine, King Abdul-Aziz University,
Jeddah, Kingdom of Saudi Arabia.
Marine natural products are of great interest, both therapeutically and as
scientific tools. It has previously been shown that 3-carboxy-1-
methylpyridininum (CMP), found in the soft corals Sarcophyton glaucum
and Lobophyton evoked multiple spike firing in isolated rat dorsal root
ganglion neurons and inhibited an outward K+ conductance (Temraz et al.,
2006 BMC Pharmacology 6: 10).
Following on from these experiments, the effects of CMP on synaptically
evoked responses recorded in murine hippocampal slices were
investigated. A bi-polar stimulating electrode was placed in the stratum
radiatum of the CA1 region, whilst a glass recording electrode, filled with
artificial cerebrospinal fluid (aCSF), was placed in either the cell body or the
dendritic layer of the same region. Perfusion of aCSF containing CMP
(100μM) for 10 min reduced population spike amplitude to 13 ± 5% of
control (n= 6; P< 0.05). At higher concentrations (300μM) the effect of CMP
reduced population spike amplitude to 30 ± 10% of control (n= 8; P

28.05
The effects of the FAAH inhibitor URB597 on kainate-evoked
neuronal ensemble activity in the rat medial prefrontal cortex.
Coomber B, Owen M, O`Donoghue M, Mason R
Schools of Biomedical Sciences & Mathematical Sciences, University
of Nottingham, Nottingham, and Department of Neurology, QMC,
Nottingham University Hospital, UK
The endocannabinoid system is pivotal in regulating neuronal
excitability and plasticity in the brain. We present data on the effects of
blockade of endocannabinoid metabolism using the FAAH inhibitor
URB597 on kainate (KA)-evoked changes in neuronal ensemble
activity, in medial prefrontal cortex (mPFC) in the anaesthetised rat.
Multiple single-unit and local field potentials (LFPs) were recorded
simultaneously from mPFC and hippocampus of isofluraneanaesthetised
Lister hooded rats using electrode arrays and a Plexon
MAP recording system. Rats were co-administered KA (10 mg kg-1;
i.p.) and either URB597 (1 mg kg-1; i.p.) or vehicle (5% EtOH/5%
Cremaphor EL/90% saline). Neuroexplorer and Matlab were used for
time-series cross-correlation analysis of unit-pair spike trains and
spike-triggered averaging (STA) of LFP activity.
Putative pyramidal mPFC neurones (firing rate

28.09
Neuronal ensemble dynamics between medial prefrontal cortex
and the ventral tegmental area in the rat: Effects of haloperidol
and olanzapine
Gupta R, Suckling J, Owen M, Mason R
Schools of Biomedical Sciences & Mathematical Sciences, University
of Nottingham, Nottingham, UK.
Anatomical and electrophysiological evidence support the view of
reciprocal projections between ventral tegmental area (VTA) and
prefrontal cortex (mPFC). We present data in the anaesthetised rat on
the directionality of VTA-mPFC interactions using cross-correlation
and partial directed coherence (PDC) analysis.
Multiple single-unit and local field potentials (LFPs) were recorded
simultaneously from VTA and mPFC of isoflurane anaesthetised male
Sprague-Dawley rats using electrode arrays and a Plexon recording
system. Neuroexplorer and Matlab were used for time series crosscorrelation
analysis of unit pairs and PDC analysis of LFP activity.
Cross-correlation analysis of single-unit data revealed highly
synchronised firing within mPFC ensembles, and between mPFC and
VTA neurones under basal conditions, with a VTA ‘lag’ indicating
mPFC cells fired before VTA cells. LFP power spectral density
analysis indicated that the majority of both LFP signals power was
slow oscillatory activity (

28.13
Genetic determinants of ethanol effects in a simple nervous
system, C. elegans
Mitchell P H, Bull K, Hopper N A, Glautier S(1), Holden-Dye L,
O`Connor V
Neurosciences Research Group, School of Biological Sciences,
(1)School of Psychology, University of Southampton, Bassett Crescent
East, Southampton, SO16 7PX
Ethanol interacts with multiple extra- and intra-cellular sites including
neurotransmitter receptors, transporters, metabolizing enzymes and
cellular scaffolding or supporting downstream signalling molecules.
These pleiotropic targets can adapt and differentially modify their
individual molecular outputs in response to prolonged ethanol
exposure, addin nce of the animal`s behavioural response to ethanol.
We have established that ethanol rapidly equilibriates across the
animal`s cuticle. Therefore populations of animals can be exposed to
precisely defined concentrations and durations of ethanol facilitating
the design of protocols that can monitor the behavioural
consequences of acute, chronic and episodic exposure to ethanol and
that serve as paradigms for human tolerance and withdrawal.
Currently we are optimizing these protocols in studies on wild-type
animals. We have developed a protocol for making agar plates
containing a defined concentration of ethanol and have shown that
these inhibit the behaviour of wild type animals to the same extent as
liquid ethanol solution. We are currently using these plates to maintain
animals in culture with ethanol for several days in order to investigate
ethanol conditioning.
Funded by the BBSRC, UK.g a further layer of complexity that is
particularly relevant during the establishment of behaviours associated
with ethanol dependence. Here we are using use one of the simplest
biological and behavioural model systems, C. elegans, which allows
an analysis at all levels of organization from gene, molecule, and
neurone through to neuronal circuit and behaviour. In the first instance
we have established the concentration-depende
28.14
Evidence for tonically active CB2 type cannabinoid receptors at
inhibitory synaptic terminals in the medial entorhinal cortex of the rat.
Morgan N H, Stanford I M, Woodhall G L
School of Life and Health Sciences, Aston University, Aston Triangle,
Birmingham B47ET UK
Although the role of type 1 cannabinoid receptors (CB1Rs) in modulation of
GABAergic signalling in temporal lobe structures such as the hippocampus
is well documented, much less is known about their role in the
parahippocampal region, or if CB2Rs are present at central inhibitory
synapses. In the current study, spontaneous inhibitory postsynaptic
currents (sIPSCs) were recorded from combined entorhinal cortex (EC)-
hippocampal slices under conditions that preserved excitatory network
activity. Experiments using specific agonists indicated that CB1Rs
modulate GABA release in EC layers II and V. However, the synthetic
CB1R antagonist/inverse agonist LY320135 failed to block marked effects
of the non-specific endogenous agonist 2-AG, suggesting the presence of
non-CB1 receptors. Further experiments showed that whilst 500 nM
LY320315 increased normalised IPSC charge transfer (CT) in layer II by 61
± 22%, this did not prevent the specific CB2R agonist JWH133 (66 nM)
reducing mean CT to -27 ± 8% of control. Subsequent application of the
CB2R specific antagonist AM-630 (50 nM) reversed this effect, increasing
CT to 108 ± 43 % of control; P

28.17
Synergic effect of cannabinoids and terpenes from Cannabis
sativa against PTZ-induced seizures
Utan* A, Speroni* E, Grassi** G
*University of Bologna, Department of Pharmacology, Via Irnerio, 48,
40126 Bologna, Italy, , **Research Institute for Industrial Crops (ISCI),
Via Amendola, 82, 45100 - Rovigo, Italy
The psychoactive effects of Cannabis preparations have been
attributed largely to the presence of delta-9-tetrahydrocannabinol, but
the non-psychoactive plant cannabinoid cannabidiol is assumed to
contribute to the attenuation of its side effects. Terpenoids may also be
involved in the clinical effects of Cannabis; in fact, its essential oil
possesses complex pharmacological activity.
The aims of the present project were to assess the potential
anticonvulsant activity of the cannabinoid-free essential oil of Cannabis
sativa and of the single major terpenes present in its mixture, and to
compare three different ethanol crude extracts of hemp aerial parts,
harvested by genetically selected genotypes (one rich in THC, one
CBD-rich, and one devoid of cannabinoids). The pentylentetrazol
(PTZ)-induced seizure model was used, with animals treated i.p. with
the compounds 60 minutes prior to injections with PTZ (85 mg/kg i.p.).
Animals were observed in individual cages for 30 minutes and
behavioural responses recorded to evaluate onset time of generalized
convulsions (1st end-point) and lethality (2nd end-point). THC-rich and
CBD-rich extracts prolonged the latency to the 1st end-point ten-times,
whereas the cannabinoid-free extract, essential oil and terpenes did
not show any significant effect. THC and CBD extracts, essential oil,
and terpenes prolonged the latency to the 2nd end point (seven-times,
four-times, and five-times, respectively), whereas cannabinoids-free
extract did not show any significant effect.
These data suggest that Cannabis-based terpenoids may contribute to
its anti-seizure activity. The extract results indicate that cannabinoids
are an essential anti-convulsant component of the phytocomplex.
28.18
Binding characteristics of selective NPY Y2 Receptor Antagonists
BIIE0246 and JNJ-5207787
Nepomuceno D, Bonaventure P, Jablonowski J, Rudolph D, Chai W,
Motley T, Carruthers N, Lovenberg T
3210 Merryfield Row, San Diego, CA 92121, USA
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant
neuropeptide in the central nervous system. NPY peptides bind to and
activate five G protein-coupled receptors, Y1, Y2, Y4, Y5 and Y6. The NPY
Y2 receptor is an autoinhibitory pre-synaptic GPCR and is expressed in
brain areas that are implicated in the development of psychiatric and eating
disorders. The role of hypothalamic NPY Y2 receptors in bone formation
has also been suggested.
Recent studies have demonstrated unique irreversible binding kinetics of
NPY ligands to Y2 but not to Y1 and Y5 receptors (Dautzenberg, 2005).
BIIE0246 was shown to behave as a competitive antagonist when coapplied
with agonist. However, when pre-incubated with the Y2 receptor,
BIIE0246 behaved as an insurmountable antagonist. Here we confirm the
slow/irreversible binding of BIIE0246 in radioligand binding studies as well
as in functional experiments. Pre-incubation of BIIE0246 with theY2
receptor resulted in a >20-fold shift in the Ki from 15 nM (no preincubation)
to 0.5 nM (with preincubation). Our functional experiments also confirm the
insurmountable antagonism of BIIE0246 when applied before agonist
addition. In addition to BIIE0246, we demonstrate the same binding and
functional characteristic with JNJ-5207787. Pre-incubation of JNJ-5207787
with the Y2 receptor caused a >20 fold shift in the Ki from 210 nM to 10 nM
in radioligand binding studies and an insurmountable antagonist profile in
our functional assay.
These findings will be an important aspect to monitor in the future
development of Y2-specific small molecule antagonists.
28.19
In vitro and in vivo pharmacological characterization of sedativehypnotic
targets in Zebrafish.
Motley T, Renier C, Faraco J H, Bourgin P, Bonaventure P, Rosa F,
Mignot E
Johnson&Johnson Pharmaceutical Research & Development, L.L.C.,
San Diego, CA, USA;Department of Psychiatry and Behavioral
Sciences, Stanford University School of Medicine, Palo Alto, CA,
94304;Unité 368 INSERM, École Normale Supérieure, Paris,
France;Howard Hughes Medical Institute, Stanford, CA
The Zebrafish model is increasingly being used for pharmacological
and behavioral research. Here we describe investigations utilizing
zebrafish to examine the effects on locomotor activity of human
hypnotics. We have identified the corresponding genomic and receptor
binding targets for GABA-A, GABA-B and H1. We identified the
Zebrafish homologs of histamine receptor H1, GABA A (alpha subunit)
and GABA B (1 and 2) receptor genes through translating queries of
the Zebrafish Zv4 database using human receptor protein sequences
as probes. We studied both radioreceptor binding and behavioral
responses to compounds with known sedative hypnotic properties.
These compounds represented multiple pharmacological classes. An
automated system was used to quantify behavioral effects. Immersion
of 5-7 day old larvae in drug resulted in reduced mobility. In some
cases, the drug produced a complete state of unresponsive immobility
similar to anesthesia. These effects were dose-dependent and rapidly
reversible in water. As established in mammals, (R)-baclofen was
more active behaviorally and had higher affinity in binding studies
when compared to (S)-baclofen. Radioreceptor binding studies
revealed high affinity binding sites for known GABA-A, GABA-B and
histaminergic ligands. These results demonstrate conservation of
gene, protein and function for many established sedative hypnotic
pathways and the utility of the zebrafish as a relevant vertebrate
model for examining compounds targeting these genes of
pharmacological interest.
29.01
Role of the hydrophobic core in the activation of glycine receptors
Miller P S, Smart T G
Pharmacology Department, Medical Sciences Building, UCL, Gower Street,
London, WC1E 6BT
Glycine receptors (GlyR) are fast activating inhibitory neuronal receptors
that are highly expressed in the mammalian spinal cord and hindbrain.
Defects in the gene encoding GlyRa1 can result in Startle Syndrome, a
disorder where patients experience an exaggerated response to stimuli. It
is important therefore, to ascertain the molecular mechanism of operation
of GlyRs, not only because of their physiological relevance, but also
because of their homology to the g-aminobutyric acid (GABAAR), nicotinic
acetylcholine (nAChR) and serotonin (5-HT3) receptors, which all share a
conserved general topology. Part of this conserved topology includes the
hydrophobic core in the ligand-binding, extracellular domain (ECD). This
sits between the agonist binding loops and the gating domain which
connects the ECD to the transmembrane helices forming the channel. The
hydrophobic core therefore represents an ideal conduit between these two
locations. Here we identify a key set of residues which contribute to a
hydrophobic ring around the ECDs of the GlyR a1 pentamer, and which are
required for retaining the GlyR in its closed conformation -- removing them
results in the GlyR opening spontaneously. We propose that receptor
activation in response to glycine binding, induces a reorganisation, via its
binding loops, of the hydrophobic core through the residues identified here,
and this conformational change is communicated downstream to the Cys
loop gating domain, so allowing the channel to open in response to glycine
binding.
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29.02
A HAP1-kinesin protein trafficking complex directs kinesin motor
protein mediated transport of inhibitory ligand-gated ion
channels
Twelvetrees A, Arancibia-Carcamo L, Lumb M J, Kittler J T
Department of Physiology, University College London, Gower Street,
London, WC1E 6BT, UK, Department of Pharmacology, University
College London, Gower Street, London, WC1E 6BT, UK
The microtubule motor proteins important for the regulated membrane
trafficking and specific sorting of inhibitory gamma-aminobutyric acid
type A receptors (GABAARs) have yet to be identified. We have
recently reported that inhibitory synaptic transmission is regulated by a
direct interaction of the huntingtin associated protein 1 (HAP1) with
GABAAR beta-subunits. However, the mechanisms of HAP1-
dependent regulation of GABAAR function remain unknown. Here we
show that HAP1 interacts with Kinesin-1 family member motor proteins
via a direct interaction with the C-terminal domain of KIF5 heavy
chains. In agreement with this, HAP1 can cause the redistribution of
KIF5 proteins in a heterologous expression assay and HAP1 / KIF5
complexes can be detected in vivo. Furthermore, GABAARs can also
be shown to interact with the kinesin family proteins in vivo. This
suggests that HAP1 regulates GABAAR function by acting as an
adaptor / scaffold protein that can associate with both GABAAR
containing transport vesicles and cytoskeletal motors. In agreement
with a critical role for KIF5 motor proteins in GABAAR transport,
disruption of kinesin-1 based microtubule transport in cultured neurons
affects the number of surface and synaptic GABAARs. These results
support a critical role for HAP1 as an adaptor linking GABAARs to
motor protein dependent receptor sorting mechanisms and in addition
provide the first evidence that kinesin family member proteins play a
critical role in regulating GABAAR trafficking and the strength of
inhibitory synaptic transmission.
29.03
Activation of AMPA and kainate receptor signalling modulates GABA-
A receptor expression in cultured mouse cerebellar granule cells
Payne H L 1, Ives J H 1, Sieghart W 2, Thompson C L1
1 School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham DH1 3LE, UK, 2 Centre for Brain Research, Medical
University Vienna, Spitalgasse 4, A-1090 Vienna, Austria and Section for
Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna,
Austria
Studies on animal models of epilepsy and cerebellar ataxia, e.g. stargazer
mice, have identified abnormalities in the GABAergic properties of neurons
associated with the defective brain loci. Whether these aberrant inhibitory
profiles contribute to, or constitute homeostatic adaptations to neuronal
hyperexcitability has yet to be resolved.
Cultured cerebellar granule cells (CGCs) from control (+/+:+/stg; AMPA
receptor-competent, kainate receptor (KAR)-competent) and stargazer
mice (AMPAR-incompetent, KAR-competent) were employed to investigate
whether non-NMDA receptor activity regulates GABAA receptor (GABAR)
expression. CGCs were cultured in 5 mM KCl-containing media or
depolarising media containing either 25 mM KCl (25K) or AMPAR/KAR
agonist, kainic acid (KA, 100 uM). KCl and KA-mediated depolarization,
down-regulated GABAR α1, α6 and β2 subunit proteins while GABAR α4,
β3 and δ were up-regulated in control CGCs. The KCl but not KA-evoked
effects were reciprocated in stargazer CGCs, indicating that the KA-evoked
effects on GABAR expression in control CGCs were AMPAR-mediated.
Conversely, GABAR γ2 expression was insensitive to KCl-mediated
depolarization but was down-regulated by KA treatment in control and
stargazer CGCs, compatible with a KAR–mediated response.
KA-mediated up-regulation of GABAR α4, β3 and δ was reversed by L-type
calcium channel (L-VGCC) blockers and the Ca2+/calmodulin-dependent
protein kinase inhibitor, KN-62. Up-regulation of GABAR α4 and β3 was
also partially reversed by the calcineurin (CaN) inhibitors, FK506 and
cyclosporin A. Down-regulation of GABAR α1, α6 and β2 was independent
of L-VGCC activity but reversed by inhibitors of CaN indicating that at least
three mutually exclusive AMPAR-mediated signalling mechanisms were
involved.
29.04
Generation of a novel anti-TARP gamma8-specific antibody: CNS
localisation and immunopurification of TARP gamma8-protein
complexes.
Donoghue P S, Spedding M, Thompson C L
1 & 3) Centre for Integrative Neurosciences, School of Biological and
Biomedical Sciences, University of Durham. 2) Institute de
Recherches Servier, Neuilly sur Seine, France
The stargazer mutant mouse, an animal model of absence epilepsy
and cerebellar ataxia, has lead to the identification of, and verified the
importance of a family of transmembrane AMPA receptor regulatory
proteins (TARPs), of which there are 4 known isoforms – γ2, γ3, γ4,
and γ8. The stargazer mouse arose through a spontaneous mutation
of the TARPγ2 isoform gene, rendering it TARPγ2 protein deficient,
leading to its subsequent phenotype. TARPγ2 was subsequently
shown to play a crucial role in the cell-surface trafficking and synaptic
targeting of AMPA receptors, however very little is known about the
functional roles of the other TARP isoforms.
Using peptides corresponding to specific domains of TARPγ8, we
have raised and purified TARPγ8-specific antibodies. Here we
describe their characterization and subsequent use as probes to map
TARPγ8 distribution throughout the mouse CNS using immunoblotting
techniques. Previous reports have identified TARPγ8 as being
exclusively located within the hippocampal formation. Here we show
that TARPγ8 is indeed enriched in the hippocampal formation, but is
also expressed in other brain regions at lower levels, e.g. spinal cord,
frontal cortex, and cerebellum.
We have shown that these antibodies are also excellent probes for
immunohistochemical mapping and will provide data showing the
cellular distribution within the CNS.
Furthermore, we have begun to use these antibodies for
immunopurification of TARPγ8 interacting protein complexes, such as
AMPA receptor subunits and the previously identified TARPγ2
interacting protein MAP-1A. By a proteomics route we hope to build a
proteomic map of TARPγ8 interacting partners.
29.05
Differential cytoprotective effects of NR3A and NR3B upon cell death
mediated by NMDA receptor subtypes in vitro: implications for Motor
Neuron disease
Chaffey H E (1), Gunthorpe M (2), Chazot P(1)
1.Centre for Integrative Neuroscience, Durham University; Neurology and
GI CEDD, GSK, Harlow,UK
Motor neuron disease(MND)/Amyotrophic lateral sclerosis(ALS) is a
devastating set of neurodegenerative diseases, characterized by selective
loss of spinal motor neurons (MN). No treatment is currently available to
halt the loss of MN and, consequently, the disease process. NMDA
receptors are fast-acting ligand-gated ion channels constituting a major
subclass of excitatory glutamate receptors, which play an essential role in
normal neurological development and function, and may be critically
involved in many neuropathological processes, including MND. It has been
previously shown that the NR3 subunit has a dominant negative effect upon
the calcium permeability of NR1/NR2 receptors in vitro. Therefore,
disregulation of the subunit composition of NMDA receptors, specifically the
NR3B, may account for the selective loss of motor neurons in MND. We
have immunological evidence, using our panel of selective NMDA receptor
subunit antibodies1, including a new anti-rNR3B antibody, that NR1, NR2A,
NR2B and NR3B subunits are all co-expressed in rodent and human spinal
MN. We have investigated the potential cytoprotective effects of the rNR3A
and rNR3B subunits when co-expressed with rNR1/NR2B and rNR1/NR2A
receptor subtypes in HEK 293 cells. Co-expression of NR3A and NR3B
with NR1/NR2B resulted in a respective 23 + 10% and 52 + 18% (mean +
SD, n = 6 individual transfections) reduction in cell death. In contrast, coexpression
of NR3A and NR3B with NR1/NR2A receptor subtype displayed
no significant cytoprotection. We have preliminary radioligand binding
evidence that NR3B subunit reduces the number of available NR1/NR2B
receptor complexes. HEC is a BBSRC/GSK (CASE) doctoral student.
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29.06
Functional and radioligand binding properties of alternate
stoichometries of human α4β2 nAChRs
Carbone A L, Moretti M, Moroni M, Zwart R, Gotti C, Bermudez I
1,3,6 School of Life Sciences, Oxford Brookes University, Gipsy Lane,
Oxford OX3 OBD, UK, 2,5 CNR, University of Milan, 20129, Milan, 4
Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor,
Sunninghill Road, Windlesham, Surrey GU20 6PH, UK
α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are ligandgated
ion channels belonging to the Cys-loop superfamily of ligandgated
ion channels. Several lines of evidence have shown that
heterologously expressed α4β2 nAChRs exist in at least two different
stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, that differ considerably in
their functional and pharmacological properties. By using radioligand
binding assays, site-direct mutagenesis and the voltage-clamp
technique we have investigated the contribution of the ligand binding
sites of the α4β2 nAChR to the functional differences between the two
receptor stoichiometries expressed heterologously in Xenopus
oocytes. The two stoichiometries can be expressed in Xenopus
oocytes by manipulating the ratio of α4 and β2 subunit cDNAs
injected. The functional potency and efficacy of a range of nicotinic
ligands at both receptor forms were significantly different. In contrast,
radioligand binding studies showed no differences in the ligand
binding profile of the α4β2 stoichiometries. Single-point mutants of the
amino acids that form the hydrophobic ring of the binding site of the
α4β2 nAChR showed that the mutations affect the function of the two
receptors in a different manner. In contrast, radioligand binding studies
showed that the mutations affect the ligand binding properties of both
receptors in the same manner and to the same extent. These results
suggest that the differences between the two alternate stoichiometries
of the α4β2 nACh receptor may lie at the gating level. Thus, nicotinic
agonists and competitive antagonists may bind the two stoichiometries
with the same affinity but activate the channels in a different manner.
29.07
Controlling desensitized states in ligand-receptor interaction studies
with cyclic scanning patch-clamp protocols
Granfeldt D, Sinclair J, Millingen M, Lincoln P, Orwar O
Department of Chemical and Biological Engineering, Physical Chemistry,
Chalmers University of Technology, SE-412 96 Göteborg, Sweden, ,
Cellectricon AB, Fabriksgatan 7, SE-412 50 Göteborg, Sweden
Ligand-gated ion channels are important control elements in regulation of
cellular activities, and increasing evidence demonstrates their role as
therapeutic targets. The receptors display complex desensitization kinetics,
occurring on vastly different time scales. This is not only important in
biology and pharmacology but might also be of technological significance
since populations of receptors under microfluidic control can function
analogously to DRAM memory circuits. Using a novel microfluidic method,
and computer modeling of the receptor state distributions, we here
demonstrate that GABAA receptor populations can be controlled to display
high or low EC50 values, depending on input function (i.e., the exact
pattern of agonist application). The sensitivity of the receptors can be tuned
up to 40-fold (β-alanine) by the particular agonist exposure pattern. By
combining patch-clamp experiments with computer modeling of receptor
state distributions, we can control the assembly of receptors in desensitized
states. The technique described can be used as an analytical tool to study
the effect of desensitization on the activity of ion channel effectors. We
describe the differential blocking effect of the competitive antagonist
bicuculline on the high- and low-EC50 GABAA receptor preparations and
conclude that the inhibition is dramatically dependent on how the different
desensitized states are populated. Furthermore, we show that both GABA
and β-alanine, two agonists with different affinity but similar efficacy, induce
the same type of desensitization behavior and memory effects in GABAA
receptor populations.
29.08
Impaired hippocampal network function in mice expressing a
hypomorphic NMDA receptor variant: ultrastructural correlates
Peddie C J 1, Schoepfer R 2, Davies H A 1, Kraev I 3, Tigaret C 2,
Popov V 1,3, Stewart M G 1
1The Open University, Department of Biological Sciences, Walton
Hall, Milton Keynes MK7 6AA, 2University College London, Laboratory
for Molecular Pharmacology, Gower Street, London NW7 1AA,
3Institute of Cell Biophysics, Pushchino, Russia,
NMDA receptors (NMDARs) mediate a form of coincidence detection
crucial to the refinement of activity dependent synaptic connectivity, an
important mechanism in putative learning and memory processes,
such as long-term potentiation (LTP). Expression of NR1 subunit
mRNA containing the N598R mutation (NR1R) generates NMDARs
with altered Mg2+ sensitivity and Ca2+ permeability, thereby impaired
in coincidence detection ability. Mice expressing the NR1Rneo allele,
a hypomorphic variant of the NR1R allele, generate both wild-type
NR1 and NR1R mRNAs, in a 95:5 ratio. Consequently, NR1Rneo/+
NMDARs are approximately 90% wild-type,

29.10
Identification of molecular determinants that mediate zinc effects
on alternate stoichiometries of the human α4β2 Nicotinic
Acetylcholine Receptors
Moroni M, Vijayan+ R, Biggin+ P, Carbone* A L, Bermudez I*
*Oxford Brookes University, School of Life Sciences.+Department of
Biochemistry, University of Oxford
Chelatable zinc is present in synaptic vesicles and is co-released with
a variety of neurotransmitters during synaptic signalling to most likely
modulate the activity of pre- and post-synaptic ligand-gated ion
channels. Although the effects of Zn2+ on NMDA, GABA-A, Glycine,
P2X and α7 nicotinic acetylcholine (nACh) receptors has been
characterised in detail, the effects of Zn2+ on α4β2 nACh receptors
have not been examined. In heterologous system, such as Xenopus
oocytes, the α4β2 nAChR assembles in two alternate stoichiometries,
(α4)2(β2)3 and (α)3(β2)2, that differ in functional sensitivity to the
neurotransmitter ACh and sensitivity to up-regulation of receptors
number by chronic exposure to nicotine. Here we report the molecular
determinants that mediate the Zinc effects on the two α4β2
arrangements. The responses elicited by agonists at the (α)3(β2)2
stoichiometry are potentiated by micromolar zinc concentrations and
are inhibited by millimolar zinc concentrations, whereas the (α4)2(β2)3
stoichiometry is only inhibited by zinc. The effects of Zn2+ at both
receptors were sensitive to external pH and were abolished by DEPC,
suggesting that histidines played a crucial role in chelating Zn2+.
Modelling and disruption of putative Zn2+ binding sites by alanine
substitution suggest the presence of a potentiating site at the αα
interface at the (α)3(β2)2 stoichiometry. Two inhibitory sites lie
presumably at the interface between the α
and the β subunits at both receptors. The (α4)2(β2)3 stoichiometry is
also subject to a voltage dependent inhibition that can be eliminated
by mutation of histidine and glutamates on the beta subunits.
29.11
Development of the first anti-mRIC-3 specific antibody: identification
of RIC-3 protein in the mammalian brain
Rensburg Rv (1), Ennaceur A (2), Chazot P L (1)
1. Centre for Integrative Neuroscience, Durham University; 2. Sunderland
Pharmacy School, UK
The α7-nACh receptor clearly plays a significant role in human emotional
and cognitive behaviours, and is currently a therapeutic target for a range
of acute and chronic neuropathologies. Therefore, regulation of functional
expression of the α7-nAChR has significant implications. Ric-3 has been
recently identified as an obligatory trafficking protein for the α7-nAChR in
heterologous expression studies, yet to be confirmed in vivo. In order to
address this issue, we have developed a new anti-mRic-3 polyclonal
antibody, which we have validated and used to define the anatomy of ric-3
expression in the mammalian brain.
Our new anti-mRic-3 antibody has identified a major Mr ~55,000 species
based on immunoblotting, using murine brain membranes, which is absent
in liver and kidney membranes. This immunoreactive species migrates
close to the recombinant mRic-3 expressed in HEK 293 cells and is
consistent with a potential dimeric species. Two protein species, Mr
~26,000 and ~50,000 were identified when mRic-3 was expressed alone in
HEK293 cells, but when co-expressed in a bicystronic IRES-mediated
vector together with α7 nAChR, a specific single Mr ~26,000 species was
detected. This may indicate that the α7 nAChR disrupts the putative dimeric
Ric-3 structure as part of the interaction and/or trafficking process. This
requires further study. Our initial immunohistochemistry findings on mouse
brain slices will be reported using this novel immunological probe, and
compared in parallel, to FITC α-bungarotoxin binding as a measure of α7-
AChR complexes.
Funded by the BBSRC (UK), Durham School of Health, ORS and NRF
(South Africa).
29.12
Clustering of inhibitory GABA-A receptors by a
gephyrin/collybistin complex
Saiepour L, Harvey R J, Harvey K
Department of Pharmacology, The School of Pharmacy, 29-39
Brunswick Square, London WC1N 1AX
Gephyrin, a peripheral tubulin-linker protein, is now well established as
a key molecule in GABA-A receptor clustering and localization at
synapses. Curiously, however, no-one has been able to demonstrate
either a direct or indirect interaction between any single GABA-AR
subunit (α1-6, β1-3, γ1-3, δ, ε, θ and π) and gephyrin in recombinant
systems or in vivo. We have recently reported that a mutation in the
src homology 3 (SH3) domain of the RhoGEF collybistin results in the
mislocalisation of gephyrin and GABA-ARs in a model of human
epilepsy (Harvey et al 2004, J. Neurosci. 24:5816-5826). This led us to
consider whether the correct membrane apposition of gephyrin via
collybistin activity could be crucial for gephyrin-GABA-AR interactions.
To examine this possibility, we fused the GABA-AR α2, β2, β3, γ2S
and γ2L subunit intracellular loops to various fluorescent proteins.
Surprisingly, when expressed alone in HEK293 cells, HcRed-γ2S and
HcRed-γ2L targeted to the submembrane compartment. This property
was not shown by HcRed alone or EYFP-α2, ECFP-β2, ECFP-β3,
HcRed-β2 or HcRed-β3. This targeting does not appear to be
dependent on the γ2S/L splicing, nor palmitoylation. When coexpressed
with EGFP-gephyrin alone, none of the GABA-AR fusion
proteins showed targeting to large intracellular gephyrin aggregates.
However, co-expression of collybistin, EGFP-gephyrin and HcRed-γ2L
resulted in a re-distribution of HcRed-γ2L to submembrane areas
enriched in EGFP-gephyrin. Our results suggest that correct
membrane apposition of the γ2L subunit, gephyrin and collybistin are
likely to be vital for GABA-AR-gephyrin interactions.
29.13
Identification of a Maf1 / Macoco myosin-like protein complex
important for regulating the number of surface GABAA receptors.
Smith K, Lumb M J, Arancibia-Carcamo L, Oliver P, Brandon N J, Moss S
J, Kittler J T
Department of Physiology and Department of Pharmacology University
College London, London, WC1E 6BT, UK, Department of Neuroscience,
University of Pennsylvania School of Medicine, MRC Functional Genetics
Unit, Department of Physiology, Anatomy and Genetics, University of
Oxford, Oxford, UK
Appropriate membrane transport and clustering of gamma amino-butyric
acid type A receptors (GABAARs) to cell surface and synaptic sites is
critical for neuronal inhibition. These processes are in part controlled by
interaction of receptor intracellular domains with proteins in the cytosol.
Here we have cloned and characterised a novel myosin like protein
complex that associates with GABAAR β-subunit intracellular domains and
which consists of two previously uncharacterised mammalian proteins,
which we have called Maf1 (Membrane protein associated factor 1) and
Macoco (Maf1 associated coiled coil protein). Maf1, a 29kDa protein,
interacts directly with GABAAR β-subunits and can, in addition, interact with
Macoco, a Maf1 binding protein with a large open reading frame of 913
amino acids. Macoco is rich in coiled coil domains and in addition has a
myosin tail and intermediate filament domains suggesting a possible role
for Macoco in cytoskeletal transport processes. Using in situ hybridisation
and antibody labelling approaches we find both Maf1 and Macoco to be
expressed in brain and also show Macoco to be enriched at inhibitory
postsynaptic sites. In cultured cortical neurones disruption of the interaction
between Maf1 and Macoco by overexpression of the Macoco interacting
domain of Maf1 results in a decrease in GABAAR cell surface number, as
revealed by surface biotinylation. This suggests that a Maf1/Macoco protein
complex may regulate GABAAR localisation and surface stability by linking
these receptors to vesicular transport or clustering machinery.
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29.14
Presynaptic modulation of nociceptive primary afferent terminals
by the endocannabinoids 2-AG in the outer dorsal horn of the rat.
Belle M, Maxwell D, Morris R
Department of Veterinary Preclinical Sciences, University of Liverpool,
Liverpool, L69 7ZJ, UK
Systemic administration of cannabinoid receptor agonists to rats
reduces allodynia and thermal hyperalgesia. CB1 receptor has been
localised in both LI /LII of the dorsal spinal cord, in regions that also
contain TRPV1 receptor. Several isoforms of diacylglycerol lipase
(DAG L) have been cloned and antibodies have been raised. In this
study DAG L has been localised, and the effects of cannabinoid
receptor activation tested on the rat outer dorsal horn.
Adult rats were terminally anaesthetised and perfuse-fixed.
Transverse sections from the spinal cord were subjected to
immunohistochemistry for DAG L, CGRP, SP, and IB4. Parasagittal
lumbar spinal cord slices were also prepared from neonatal rats and
whole-cell current-clamp recordings were made from LII(o) neurones
which were then morphological reconstructed.
Immunoreaction for DAG lipase (DAGL-ir) was seen in the primary
afferent terminals throughout LI and in LII(o) of the spinal cord. DAGLir
showed colocalisation with CGRP, SP and IB4. Immunoreaction for
DAG-L, CGRP and SP were significantly reduced, whilst IB4 staining
was almost absent in adults that were subcutaneously injected with
capsaicin as neonates (P0). In the spinal slice preparation capsaicin
significantly increased the frequency of EPSPs and action potentials.
CP55940 application significantly inhibited this capsaicin-evoked
excitation in a reversible manner.
This data reveals a mechanism by which pain sensation from thermal
and/or chemical sources can be modulated by presynaptic
cannabinoid receptors. The distribution of DAG L suggests that 2-AG
is produced locally in the terminals of primary afferents where it could
act on CB1 receptors to modulate synaptic transmission.
29.15
Differential control of NMDA receptors by calmodulin
Michiko Takahashi, Alasdair J. Gibb
Department of Pharmacology, University College London,, Gower Street,
London, WC1E 6BT
It has been previously shown that calmodulin reduces NMDA receptor
single channel open time and open probability (Ehlers et al., 1996; Rycroft
and Gibb, 2002). To investigate the influence of calmodulin on macroscopic
whole-cell NMDA currents, we have recorded NMDA responses with
intracellular calmodulin appliedvia the whole-cell patch pipette solution
(active concentration 1 µM) from medium spiny neurones of striatal slices
from humanely killed 7 day old rats. Application of the membranepermeable
calmodulin antagonist calmidazolium (20 µM) reduced the
whole-cell NMDA current from 350±39 pA to 250±43 pA; 62.8±10.5% of
control (mean±SE, n=9, P=0.01). Inclusion of calmodulin inhibitory peptide
(10µM) in the patch pipette in addition to calmodulin reduced the control
NMDA response to 262±67 pA (n=11) and further application of
calmidazolium (20 µM) reduced the size of the response to NMDA to
a lesser degree (81.8±9.1% of control, 206±60 pA, P=0.02). When
calmodulin was not added, calmidazolium had no significant effect. There
was no significant effect of calmidazolium on NMDA receptor single
channel currents recorded from outside-out patches. Inclusion of the
CaMKII inhibitor KN-93 (5 µM) in the patch pipette did not occlude, but
reduced the effect of calmidazolium. These results suggest that calmodulin
does not only modulate NMDARs by direct binding but also via CaMKII and
thus regulates NMDA receptor density.
Supported by the Wellcome Trust.
Ehlers et al., (1996). Cell. 84:745-755.
Rycroft and Gibb, (2002). J Neurosci. 22:8860-8868.
29.16
Investigation of NR2B and NR2D-containing NMDA receptors in
dopamine cells and GABAergic cells of rat substantia nigra
F. Suarez, D.T. Monaghan, D. Jane, S. Jones, A.J. Gibb
Department of Pharmacology, University College London, Gower
Street, London, WC1E 6BT. **Department of Physiology,
Development & Neuroscience, Anatomy Building, Downing Street,
Cambridge, CB2 3DY., *Department of Pharmacology & MRC Centre
for Synaptic Plasticity, University of Bristol.
NMDA receptors may contribute to excitotoxic neurodegeneration of
dopamine (DA) neurones in Parkinsonand#8217;s disease (PD). PD
patients and animal models of PD show a dramatic reduction in the
number of DA cells of the Substantia nigra pars compacta (SNc). In
parkinsonian models, the cells of the Substantia nigra pars reticulate
(SNr) neighbouring SNc cells, are much less affected. We have used
patch-clamp whole-cell recording methods to quantify the proportions
of NR2B and NR2D NMDA receptors in both cellular groups using 300
um coronal slices of substantia nigra from humanely killed 7 day old
rats.
Dopamine cells in SNc were identified by the presence of a
hyperpolarisation-activated slow inward current (Ih current) on
stepping the membrane potential from-60 mV to -120 mV. Responses
to 50 uM NMDA and 10 uM glycine in the presence of TTX (100 nM)
were recorded in control (SNc; -2352 ± 343 pA, SNr; -978 ±
113 pA) and following 5 min in presence of the NR2B selective
antagonist, ifenprodil (10 uM) (SNc; -1233 ± 252 pA, SNr; -534 ± 132
pA, n=10) or in control (SNc; -2127 ± 386 pA, SNr; -916 ± 133 pA) and
following 5 min in the presence of the partially NR2D selective
antagonist, UBP141 (3 uM) (Morley et al., 2005) (SNc; -1612 ± 127
pA, SNr; -653 ± 110 pA, n=9). These results suggest that SNc and
SNr cells both express a mixed population of NR2B and NR2D type
NMDA receptors.
29.17
Magnesium block of NMDA receptors in dopaminergic neurons of
neonatal rat substantia nigra pars compacta
Huang Z , Gibb A
Department of Pharmacology, University College London,, London WC1E
6BT, U.K.
In SNc, NMDA receptors may be triheteromers of NR1, NR2B and NR2D
subunits (Jones and Gibb, 2005). We have used patch-clamp whole-cell
recording to quantify magnesium block of NMDA receptors in 300 um
coronal slices of SNc from humanely killed 7 day old rats. NMDA currents
at -60mV, evoked by 0.1mM NMDA and 0.01mM glycine in the presence of
100nM of TTX were blocked by 59±3.0% (n=9) and 88.2±1.8% (n=9) in
0.1mM and 1.0mM magnesium respectively. Voltage ramps
(-100mV to +40mV) were used to quantify the voltage-dependence,
and#948;, and equilibrium constant, Kb (0mV), of magnesium block.
Magnesium IC50 values at -100mV, -80mV and -60mV were 5.98uM,
11.9uM and 55uM, respectively (n=9), similar to NR2A- and NR2Bcontaining
receptors. The residual NMDA current in 10 µM ifenprodil had a
lower magnesium sensitivity (IC50 = 25.1uM at -100mV, 73.2uM at -80mV
and 229uM at -60mV); similar to NR2C- and NR2D-containing receptors
(Kuner and Schoepfer, 1995; Qian et al., 2005). To analyse the data we
developed two new models that include trapping block of NMDA channels,
magnesium potentiation on the extracellular site of NR2B receptors and
proton block. The results suggest that ifenprodil-sensitive (NR2B) and lowmagnesium
sensitivity NR2D subunits are expressed by dopaminergic
neurons in SNc.
Supported by the Wellcome Trust and the BBSRC. Z.H. is funded by an
Overseas
Research Studentship and UCL Old Students Association
Trust Scholarship.
Supported by the Wellcome Trust and the BBSRC.
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29.18
Supporting study skills: workshops for undergraduates in
pharmacology
Harkness P C, Hayes N A, McKenna C, Crème P
Writing and Learning Mentors Project, Centre for the Advancement of
Learning and Teaching, University College London, Gower Street,
LONDON WC1E 6BT
Writing and Learning Mentors Project, Centre for the Advancement of
Learning and Teaching, University College London, Gower Street,
LONDON WC1E 6BT
Science students often struggle with the demands of university written
work, whether in essays completed for coursework or examinations, or
in laboratory reports for practical classes or research projects. The
Centre for the Advancement of Teaching and Learning at UCL
contributes to scholarship in the field of pedagogy, and is involved in
many activities which seek to assist students` learning. The "Writing
and Learning Mentors (WLM)" project facilitates peer-supported
discussion and development of skills appropriate to writing in specific
academic disciplines. Now in its third year, it consists of a series of
multi-disciplinary workshops designed to furnish participants with both
the theoretical basis of "writing in the disciplines" and the practical
resources to enable students (graduates and undergraduates) to
develop their writing skills. Writing is seen as an activity to be used not
only as an assessment tool but also as an aid to learnng. Having
taken part in the workshops, WLM participants then establish a
programme of study support for students within their own
departments. In the Pharmacology Department, we have hosted
workshops for first and second year undergraduates, giving advice on
data-mining, critical reading and structured writing, and designed to
offer practical exercises and examples of good practice to facilitate
their study of pharmacology.
30.01
Pineal proteins and its influence on adrenal cortex to ameliorate
thermal stress in goats
Sejian. V, Srivastava. R. S., Varshney. V.P, Raviprakash. V, Dandapat. S
Neurophysiology Laboratory, Centre for Advanced Studies in Animal
Physiology and Climatology, Indian Veterinary Research Institute,
Izatnagar, Bareilly – 243122, UP, India,
The present study was conducted to establish the anti thermal-stress
properties of pineal proteins. Pineal proteins were isolated as per standard
protocol followed in the laboratory. Six female goats weighing around 15 kg
were used for the present study. These animals themselves served as self
controls prior to start of experiment. The entire study was conducted for a
period of seventeen days in the psychrometric chamber at 40°C and 60 %
relative humidity. Blood samples were drawn to establish thermal-stress
effects on various parameters studied. Chemical adrenalectomy was
induced using metyrapone followed by exogenous pineal protein treatment.
Blood sampling was done twice daily after each treatment to record the
effects of pineal proteins on plasma sodium, potassium, glucose, total
protein, cortisol, insulin, aldosterone, melatonin, corticosterone and
phagocytosis index. Chemical adrenalectomy aggravated the thermal
stress however the condition was reversed by exogenous administration of
pineal proteins. All the parameters were estimated as per standard
methods. These parameters showed significant (P ≤ 0.05) changes in
plasma levels when compared to both, control as well as thermal-stress
levels. The data obtained from the study clearly established anti thermalstress
effects of pineal proteins in goats. The results obtained from the
experiment suggest that apart from melatonin,other pineal proteins also
possess properties that relieve thermal-stress in goats.
31.01
Functionalized micropatterned scaffold influence neurite growth
architecture
Yao L, Cui W, O'Connell C, Damodaran G, Sherlock R, Wang S,
Pandit A
National Center for Biomedical Engineering Science, Natiional
University of Ireland, Galway.
Introduction: Guided neurite growth is critical in neurogenesis. Poly-
(D,L lactic-co-glycolic acid) (PLGA) has been widely investigated for
neural tissue engineering for its mechanical strength. In order to guide
neurite growth in appropriate target reinnervation, we have fabricated
micropatterns on PLGA scaffold. The objective of study is to
investigate the influence of micropatterns and surface coating proteins
on neurite growth pattern.
Methods: Coupons of PLGA structured with parallel arrays of straight
micro-grooves were produced by laser energy (ATL Atlex® in
conjunction with an Optec MicroMaster® machining centre). Two
group of microgrooves were performed on PLGA scaffold (5 µm wide
grooves, 5 µm wide spacing, 2-3 µm deapth; 10 µm wide grooves, 10
µm wide spaceing, 2-3um depth). PLGA scaffold were pre-coated with
collagen or laminin peptide. The morphology of PC12 cells was
studied by SEM and Rhodamine Phalloidin staining after 3days or
7days growth on PLGA scaffold with NGF treatment (50 ng/ml).
Results: 1. Micropatterned PLGA scaffold impacts guidance effect on
both neurite outgrowth and neurite extension.
2. Small size (5µm) of grooves showed stronger effect on neurite
parallel growth than larger size grooves (10µm).
3. Laminin peptide coating enhanced the parallel growth of neurites on
micropatterned scaffold. Neurite branching and total neurite length
decreased on micropatterned PLGA scaffold.
4. Neurite showed a preference growth in microgrooves rather than on
spaces.
Conclusion: This study provides direct evidence to show the guidance
effect of microchannels on neurite growth neurite growth and
implicates its application in nerve regeneration strategy.
31.02
Very extensive remyelination of spinal cord by schwann cells in the
rat MOG-EAE model of multiple sclerosis
McIntosh P, Norman A, Reynolds R
Dept. of Cellular & Molecular Neuroscience, Division of Neuroscience &
Mental Health, Charing Cross Campus, Imperial College School of
Medicine, London W6 8RP, UK
Experimental allergic encephalomyelitis induced in female Dark Agouti rats
by administration of myelin oligodendrocyte glycoprotein (MOG) provides a
good model of multiple sclerosis (MS) and its opticospinal variant,
neuromyelitis optica (NMO). We found a surprising degree of remyelination
of CNS axons in spinal cord by Schwann cells, which normally provide
myelin sheaths only on peripheral axons. We have used
immunofluorescent histochemistry and image analysis to explore this
phenomenon further. Schwann cell remyelination was assessed using P0
immunostaining for peripheral-type myelin, and very extensive in the spinal
cord of most rats 3-4 months after MOG injection. Such regions extended
continuously throughout the length of the entire cord in some animals, and
reached levels approaching 50% of total stained myelin (P0 cf. PLP
staining) in some transverse sections. Although in some cases Schwann
cell remyelination commenced in areas lacking oligodendrocyte precursor
cells (OPCs), NG2 immunostaining showed OPCs present in many other
similar regions. The pattern of remyelination was consistent with initial
Schwann cell migration from pial surfaces and associated vascular
invaginations via breaches in the astrocytic glia limitans, similar to the
pattern of prior demyelination by invasive monocytes. We conclude that
since astrocytic GFAP+ processes penetrated all remyelinated regions, it is
unlikely that local astrocytic reactivity is capable of preventing remyelinating
activity by invading Schwann cells. We further conclude that the present
model is suitable for investigating why Schwann cells can prevail over
oligodendrocytes in the remyelination of some spinal cord regions in MS
and NMO patients.
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31.03
Dopaminergic modulation of spinal neuronal excitability
Han P, Michelle A. Tran, Whelan P J
Rm 2068 Hotchkiss Brain Institute, University of Calgary, 3330
Hospital Drive,NW,Calgary, AB T2N4N1,CANADA
Although dopamine is well known to play a role in motor control it is
generally assumed that dopamine does not directly modulate spinal
motor circuits. However, in recent years several studies have
demonstrated a modulatory action of dopamine on spinal networks
and reflexes. DA fibres and receptors are present in the spinal cord
and evidence for DA release within the spinal cord has been
published. A critical gap is our lack of data regarding dopaminergic
modulation of intrinsic and synaptic properties of motoneurons and
ventral interneurons in the mammalian spinal cord. In this study, we
address this issue by examining the intracellular mechanisms
underlying DA’s excitatory effect on motor systems. We examine DA’s
effects on two classes of cells important for motor control,
motoneurons and Hb9 interneurons located in lamina VIII. We show
that DA can boost excitability in spinal motor systems by decreasing
the first spike latency and the AHP. Collectively, this leads to an
increase in the F-I slope. Our data suggest that these changes are
due to modulation of IA and SKCa currents. We also demonstrate that
DA modulates glutamatergic transmission by increasing both the
amplitude and frequency of miniature and spontaneous excitatory
postsynaptic currents suggesting that DA acts both pre and
postsynaptically. Collectively these data suggest that DA has
widespread actions on intrinsic and synaptic properties of spinal
neurons consistent with its ability to modulate and stabilize spinal
networks. In addition, dopaminergic therapy may provide promising
perspectives on spinal cord rehabilitation.
32.01
Stimulus timing-dependent plasticity in the adult auditory system
Dahmen J, Ahmed B, Hartley D E H, King A J
Department of Physiology, Anatomy and Genetics, Sherrington Building,
University of Oxford, Parks Road, Oxford OX1 3PT
Adult cortical circuits possess considerable plasticity over different time
scales, which can be induced by conditioning or task-specific training. For
instance, it has been shown that repetitive pairing of stimuli that differ either
in their orientation (Yao and Dan, 2001, Neuron 32:315-323) or location in
space (Fu et al., 2002, Science 296:1999-2003) can induce rapid shifts in
the orientation tuning and receptive field location, respectively, of neurons
in the primary visual cortex. The direction of those shifts was found to
depend on the order of stimulus presentation, while their magnitude
depended on the interval between the stimuli in each pair, with intervals
larger than about 20 ms being ineffective. This is consistent with the
temporal window of spike timing-dependent synaptic plasticity. To examine
whether stimulus timing-dependent plasticity also exists in the auditory
system, we recorded from neurons in primary auditory cortex of
anaesthetized adult ferrets. Presentation of ≥600 pairs of tones that differed
in frequency resulted in shifts in the best frequencies of the neurons. These
shifts were observed when stimulus onset asynchronies of up to 12 ms
were used and their direction depended on the order in which the tones in a
pair were presented. The shifts in frequency tuning persisted for several
minutes and appeared to be most pronounced in the supragranular layers
of the cortex. These data show that stimulus timing-dependent plasticity is
a property of the auditory cortex and exhibits a temporal specificity similar
to that observed in the visual system.
32.02
Regulation of CSPG expression in the developing barrel cortex
A Petrie, Matthews R L, Kind P C
1 & 3, University of Edinburgh,, Hugh Robson Building,, George
Square,, Edinburgh,, EH8 9XD,, 2, SUNY Upstate medical University,
New York
Chondroitin-sulphate proteoglycans (CSPGs) are a principle
component of the extracellular matrix (ECM) of the CNS. In adult
animals, CSPGs appears to restrict plasticity induced by monocular
deprivation (Pizzorusso et al 2002) and the expression of aggrecan, a
principle CSPG in adult brain, correlates well with the termination of
developmental plasticity (Sur et al 1988). Aggrecan localises to
perineuronal nets, a specialised ECM that surrounds soma and
proximal dendrites. We have used Cat-315, an antibody that labels an
epitope on aggrecan (Lander et al, 1997), to examine the role of this
molecule in cortical development. In mouse neocortex, staining
increases through development and, in adults, is highest in primary
somatosensory cortex (S1), retrosplenial cortex and motor cortex. In
S1, staining appears as two distinct bands of labelled soma, one
through layer 4 and the other through lower layer 5 and layer 6.
Staining with Cat-315 of tangential sections shows an intense staining
in a barrel-like pattern in S1.
We have previously shown that mGluR5 signalling via PLC-b1
regulates barrel formation. Aggrecan expression is known to be
dependent on synaptic activity (Sur et al., 1988; Kind et al., 1995)
raising the possibility that mGluR5 may regulate aggrecan expression.
To determine whether mGluR5 signalling via PLC- b1 also regulates
aggrecan expression we investigated Cat-315 expression in Mglur5-/-
and Plc-b1-/- mice. Qualitative assessment of Mglur5-/- show fewer
Cat-315 immunopositive cells and staining at a lower intensity than
Mglur5+/+, indicating mGluR5 may regulate the formation of
perineuronal nets in barrel cortex.
33.01
Space-Time Spike triggered covariance analysis of fly motion
selective neuron
Saleem A, Schultz S R
Department of Bioengineering,, Imperial College London,, UK SW7 2AZ
The H1 neuron of the fly lobula plate has been widely studied with an
interest to understand the motion detection systems of the visual system.
Most properties of H1 have been studied in the steady state through tuning
curve functions of the cells, which assume separability of the response
properties along the different dimensions. The spike triggered covariance
(STC) analysis of these cells avoids this. STC in the velocity-time domain is
also limited as it assumes velocity tuning of a neuron that is known to be
temporal frequency tuned. We carry out STC in the space-time domain,
thus making fewer assumptions about the properties of the cells.
We recorded extracellular activity of the H1 cells of blowflies while
presenting a bar stimulus: the contrasts of the bars changing as ternary
noise. We carry out STC analysis on this data. We recovered the spacetime
receptive fields of the cell. These show a temporal frequency tuning of
about 120 cycles/deg, which matches previous studies. We also observe
that the cell responds to changes is position that are within 8msec. We
recovered spatially separated excitatory and suppressive filters. We also
recovered the non-linear stage of a stochastic linear-nonlinear model of the
cell. We present a complete model of the cell recovered from the data.
The space-time STC analysis has been used to build models of cells in the
MT/V5 of the primate visual cortex, and hence we can directly compare our
results between the two systems.
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33.02
Noradrenaline reuptake inhibitors inhibit neuroinflammation
induced by a systemic inflammatory challenge
O`Sullivan J B, Harkin A, Connor T J
Trinity College Institute of Neuroscience, University of Dublin, Trinity
College, Dublin 2, Ireland.
Evidence suggests that the monoamine neurotransmitter
noradrenaline elicits anti-inflammatory actions in the central nervous
system (CNS), and consequently may play an endogenous
neuroprotective role in CNS disorders where inflammatory events
contribute to pathology. In line with this hypothesis, we demonstrate
that noradrenaline suppresses expression of the pro-inflammatory
cytokines IL-1beta and TNF-alpha and induction of iNOS/nitric oxide
production from mixed glial cultures prepared from rat cortex, in
response to the inflammagen bacterial lipopolysaccharide (LPS). As
previous studies indicate that the noradrenaline reuptake inhibitor
(NRI) desipramine has anti-inflammatory properties, we examined the
ability of desipramine and more selective NRI’s to alter glial proinflammatory
cytokine production. However, treatment of mixed glial
cells with NRI’s largely failed to alter inflammatory events induced by
LPS. In contrast to the in vitro situation, acute in vivo treatment of rats
with NRI’s elicited an anti-inflammatory effect in the CNS
characterised by reduced mRNA expression of the pro-inflammatory
cytokines IL-1beta and TNF-alpha and iNOS in cortex in response to
systemic LPS administration. The data also suggest that in vivo
treatment with NRI’s inhibited microglial activation in the cortex
indicated by reduced expression of the microglial activation makers
CD40 and CD11b. These data indicate that NRI’s do not have a direct
modulatory effect on the inflammatory response in glial cells, however
when administered in vivo can limit inflammatory events in the brain.
Overall, this study has yielded significant insights into the ability of
noradrenaline augmentation strategies to limit neuroinflammation.
33.03
Functional segregation of synaptic GABA(A) and GABA(C) receptors
in retinal bipolar cell terminals
Palmer M J
Institute for Science and Technology in Medicine, Keele University
The transmission of light responses to retinal ganglion cells is regulated by
inhibitory input from amacrine cells to bipolar cell (BC) synaptic terminals.
GABAA and GABAC receptors in BC terminals mediate currents with
different kinetics and are likely to have distinct functions in limiting BC
output but the synaptic properties and localisation of the receptors are
currently poorly understood. By recording endogenous GABA receptor
currents directly from BC terminals in goldfish retinal slices, I show that
spontaneous GABA release activates rapid GABAA receptor mIPSCs in
addition to a tonic GABAC receptor current. The GABAC receptor
antagonist TPMPA has no effect on the amplitude or kinetics of the rapid
GABAA mIPSCs. In addition, inhibition of the GAT-1 GABA transporter,
which strongly regulates GABAC receptor currents in BC terminals, fails to
reveal a GABAC component in the mIPSCs. These data suggest that
GABAA and GABAC receptors are highly unlikely to be synaptically
colocalised. Using non-stationary noise analysis of the mIPSCs, I estimate
that GABAA receptors in BC terminals have a single-channel conductance
of 17 pS and that an average of just seven receptors mediates a quantal
event. From noise analysis of the tonic current, GABAC receptor singlechannel
conductance is estimated to be 4 pS. Identified GABAC receptor
mIPSCs exhibit a slow decay and are mediated by approximately 42
receptors. The distinct properties and localisation of synaptic GABAA and
GABAC receptors in BC terminals are likely to facilitate their specific roles
in regulating the transmission of light responses in the retina.
33.04
Immunocytochemical studies of GABA receptors and
transporters at amacrine cell to bipolar cell terminal synapses in
the goldfish retina.
Jones S, Palmer M, Furness D
ISTM, Huxley Building, Keele University, Keele, Staffordshire, ST5
5BG
The vertebrate retina transduces light and carries out the initial stages
of visual signal processing. The visual signal is transmitted from
photoreceptors to bipolar cells and then to ganglion cells, the output
cells of the retina. Transmission between bipolar cells and ganglion
cells is modulated by a class of lateral inhibitory interneurones,
amacrine cells, which form both reciprocal and conventional
GABAergic synapses with bipolar cell terminals. GABA transporters
are likely to be associated with these synapses to control receptor
activation properties and recycling of GABA.
We have therefore attempted to determine the ultrastructural
distribution of GABA transporters on and around the bipolar cell
terminal using postembedding immunogold labelling of freezesubstituted,
Lowicryl embedded goldfish retina. Preliminary results
show that labelling for GAT-1 is found at relatively high density in
putative amacrine cell processes, some distance from their contact
with bipolar cell terminals, and at a lower density around the periphery
of GABAergic synapses with bipolar cell terminals. Labelling for GAT-3
is weaker and more diffuse.
33.05
Using SSVEPs to investigate the half-life of attentional lapses
Dockree P M, O’Connell R G, Shalgi S, Kelly S P, Bellgrove M A,
Robertson I H
The Lloyd Building, Trinity College Institute of Neuroscience, Trinity College
Dublin , Dublin 2, Ireland
Traditional measures of vigilance have shown that time-on-task decrements
emerge after several tens of minutes; however more recent research
suggests that the half-life of sustained attention failures is apparent over
shorter periods of minutes and seconds (Pardo et al., 1991; Robertson et
al., 1997; Weissman et al., 2006). The present study utilises a steady-state
visual evoked potential (SSVEP) measure of sensory facilitation in the
visual cortical pathways and behavioral data of momentary lapses of
attention to examine the temporal dynamics of sustained attention
modulation. Importantly, SSVEPs are ongoing oscillatory waveforms that
provide a continuous record of cortical facilitation suited to capturing
fluctuations in sustained attention over time. Ten neurologically healthy
participants were presented with flickering (25Hz) pattern-reversal stimuli.
Participants monitored standard stimuli presented for 800ms and
responded with a key press to target stimuli presented for the longer
duration of 1040ms. Temporal Spectral Evolution (TSE) analysis was
conducted for the time window prior to detected versus undetected targets
and transient ERP analysis of targets and standards and relationship to
SSVEP modulation was investigated. Results are discussed in the context
of fronto-parietal modulation of visual cortical pathways.
Both GABAA and GABAC receptors can be identified physiologically
in bipolar cell terminals and the terminals exhibit response properties
which suggest that these receptor subtypes may be located at
different sites in the postsynaptic membrane. The above technique will
be applied to enable us to determine where the receptor subtypes are
located in relation to the synapse and the GABA transporters.
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33.06
FMR-Adaptation reveals a view-invariant representation for
familiar faces in the fusiform face area
Andrews T, Ewbank M
Department of Psychology, University of York, York UK, MRC
Cognition and Brain Sciences Unit, Cambridge UK
Recognising complex objects, such as faces, is a simple and effortless
process for most human observers. However, as we move about or as
gaze or expression change, the size and shape of the face image on
the retina also changes. To facilitate recognition, the visual system
must take into account these sources of variation. The aim of this
study was to explore whether face recognition is dependent on a
viewpoint-dependent or viewpoint-invariant neural representation.
Using the technique of fMR-adaptation, we measured the MR
response to repeated images of the same face. We report that activity
in the face-selective FFA was reduced following repeated
presentations of the same face. This adaptation was similar for both
familiar and unfamiliar faces. To establish if the neural representation
of faces in the FFA was invariant to changes in viewpoint, we varied
the viewing angle of the face between successive presentations. We
found that adaptation to familiar faces was apparent across all
changes in viewpoint. In contrast, we found signficant adaptation in
the FFA to unfamiliar faces only occurred when the viewing angle
between successive images was 2 degrees or less. Face-selective
regions in the superior temporal lobe failed to adapt to repeated
presentations of the same face. These results are consistent with
cognitive models of face perception that predict a view-invariant neural
representation underlies the recognition of familiar faces.
34.01
3-D alterations in ultrastructure of dendritic spines and postsynaptic
densities after LTP and LTD induction in dentate gyrus of awake rats.
Medvedev N I 1, Doyere V 2, Davies H A 1, Rodriguez J J 1, Dallérac G 2,
Gabbott P L 1, Laroche S 2, Kraev I V 1,3, Popov V I 1,3, Stewart M G 1
1The Open University, Milton Keynes, UK; 2 NAMC, CNRS-UMR8620,
Univ. Paris Sud, Orsay, France; 3Institute of Cell Biophysics, Russian
Academy of Sciences, Pushchino, RF
Using serial ultrathin sections, 3-D reconstructions of synapses and
dendritic spines in dentate gyrus (DG) were prepared following induction of
long-term potentiation (LTP) or heterosynaptic long-term depression (LTD)
in awake rats. Control rats were pseudo-tetanized, or LTP was blocked by
i.p. injection of the NMDA-receptor antagonist D,L-3[(+/-)-2-
carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Animals were
perfused intracardially 24h later and brains prepared for electron
microscopy. 3-D reconstructions enabled quantitative structural changes in
dendritic spines and postsynaptic densities (PSDs) to be defined in inner,
middle and outer molecular layers of the DG. A novel method enabled
calculation of curvature of PSDs in each serial section. No significant
changes were found in either synapse density or proportion of 4 synapse
classes (thin, mushroom, stubby and shaft). However significant increases
in both volume and surface area of thin spines and PSDs were found in
LTP compared to control and CPP-treated animals. There was a significant
reduction in volume of mushroom spines in CPP-treated animals compared
to LTP and control rats. A significant reduction in concavity of spine PSD
area was observed in all layers of the DG for thin and mushroom spines in
LTP and LTD animals, but not CPP treatment. In LTD animals, there was a
more marked reduction in concavities. Both LTP and LTD animals showed
spinule growth on spine heads in all three layers examined (blocked with
CPP). These data suggest substantial remodelling of large dendritic spines
24h after induction of LTP and/or LTD. Supported by EU FPVI Promemoria
programme # 512012
34.02
Neuronal network oscillations in primary motor cortex
Naoki Yamawaki, Gavin L. Woodhall, Ian M. Stanford
School of Life and Health Sciences, Aston University, Birmingham
Parkinson’s disease (PD) is associated with abnormal synchronization
of neuronal network oscillations at theta (4-10Hz) and beta (15-30Hz)
frequency bands across nuclei of the basal ganglia (BG) (Brown et al.,
2001 J. Neurosci. 21(3):1033-8). Deep brain stimulation of specific BG
nuclei appears to reduce these pathological activities, thereby
alleviating PD symptoms. Recently, direct stimulation of primary motor
cortex (M1) has also been shown to be effective in reducing symptoms
in an animal model of PD, indicating that the cortex may pattern these
pathological rhythms through its connections with BG (Drouot et al.,
2004 Neuron 44(5):769-78). Here we examined properties of the M1
network oscillations in vitro. Co-application of the glutamate receptor
agonist kainic acid (400nM) and muscarinic receptor agonist carbachol
(50μM) induced oscillatory activity at beta frequencies in all layers
(e.g. layer 5, 27.8 ± 1.1Hz, n=6).
Dual extracellular recordings, local application of TTX and microsections
indicate that the activity in superficial layers is strongly
influenced by that within deep layers V/VI. All oscillations were
abolished by the GABAA receptor antagonist picrotoxin and
modulated by pentobarbital and zolpidem indicating dependence on
networks of GABA interneurons. High frequency stimulation (125Hz) in
superficial layers tended to generate gamma oscillations (incidence
95%, 62.2 ± 5.6Hz, n=19) whereas stimulation at 4Hz led to a higher
incidence of theta oscillation (62.5%, 5.4 ± 0.5Hz, n=12). Often
evoked oscillations were observed within the same recording and at
the same time as the pharmacologically induced beta oscillation
suggesting that they are generated by different interneuron
populations.
34.03
The electrode-brain interface in deep brain stimulation is modulated
by systemic and local physiological factors: A 3-dimensional
computational model
Yousif N, Richard Bayford, Xuguang Liu
1. Movement Disorder and Neurostimulation Unit, Department of Clinical
Neuroscience, Division of Neuroscience and Mental Health, Imperial
College London;, 2. Bio-modelling/Bio-informatics Group, Department of
Biomedical Science, Institute of Social and Health Research, Middlesex
University
Deep brain stimulation (DBS) is a widely used clinical treatment for various
neurological disorders, and particularly movement disorders. However, the
mechanism by which these high frequency electrical pulses act on
underlying neuronal activity is not understood. Once the stimulating
electrode is placed in situ, an electrode-brain interface (EBI) is created. We
used computational modelling to construct a three-dimensional model of
the EBI using the finite element method to compensate for the clinical
restrictions on the study of EBI in situ. In this way both the structural details
and the biophysical properties of the EBI are retained. We focussed on
systemic and local, and dynamic physiological and pathological modulation
of the EBI, specifically by brain pulsation and giant cell formation. Our
results show that the EBI is influenced by both the systemic and the local
physiological factors. We find that there is a linear correlation between
cerebral blood perfusion and the magnitude of the potential distribution
induced in the tissue surrounding the electrode. Furthermore, giant cell
growth at the EBI creates a ‘shielding effect’, which impedes the flow of
current to the surrounding tissue. These results provide a quantitative
assessment of the current flow in the brain tissue surrounding the
implanted DBS electrode, the effects of the EBI on the current spreading
under physiological conditions, and consequently on the therapeutic effect
of DBS.
The project was supported by MRC grants (id 71766 and 78512)
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35.01
Synchrony and sensory coding in the olivocerebellar pathway in
vivo
Schultz S R, Kitamura K, Hausser M
1 Dept of Bioengineering, Imperial College London, 2 Dept of
Neuroscience, Osaka University, 3 Wolfson Institute for Biomedical
Research, University College London
The climbing fibre pathway from the inferior olive to the cerebellum
has been hypothesized to provide information about non-anticipated
sensory signals for the modification of motor programmes. However,
the nature of the neural coding, and particularly the population coding,
of this sensory information is poorly understood. To investigate this,
we used two-photon imaging of calcium signals in multiple Purkinje
neurons loaded with membrane-permeant dye. Rats (P18-25) were
anaesthetized using urethane or ketamine/xylazine, and Purkinje cells
in the Crus IIa area of the cerebellum were bulk-loaded with AM-ester
calcium dye (Oregon Green BAPTA-1 AM) through a pipette inserted
into the molecular layer. As previously reported, calcium transients
triggered by spontaneous climbing fibre inputs were observed in the
Purkinje cell dendritic tree. These signals showed fine-scale spatial
structure, with synchronization between neighbouring neurons falling
off over hundreds of microns transversely. We investigated the role of
this synchronization in coding sensory information, by stimulating the
upper or lower lip with a brief airpuff. Using the two-photon
microscope, we were able to search in three dimensions for regions of
tissue in which sensory evoked calcium signals were to be found.
Sensory stimuli which evoked strong fluorescence responses
compared to baseline accentuated synchrony between nearby cells.
Periodic sensory stimuli were observed to result in the locking of
calcium signals to stimulus onset. These findings suggest that sensory
input modulates the fine structure of spatiotemporal patterns of
synchrony in the cerebellar cortex, perhaps by changing the dynamics
of oscillatory synchrony in the inferior olive.
36.01
Cholinergic neurons in human midbrain labelled with 125I Urotensin II
in post-mortem tissue in Progressive Supranuclear Palsy and normal
elderly
Piggott M A, Muelas M W, Burn D J
Dementia and Brain Ageing Group , Wolfson Research Centre , Newcastle
University Institute for Ageing and Health , Newcastle General Hospital ,
Westgate Road , Newcastle-upon-Tyne NE4 6BE
Progressive supranuclear palsy (PSP) is a disorder often misdiagnosed as
Parkinson’s disease, however patients do not respond well to levodopa.
Other clinical characteristics include postural instability resulting in frequent
falls, and symmetric signs with trunk more than limbs affected. These
features may relate to a greater pathological burden in nuclei that have
bilateral influence on the basal ganglia motor loop, including the
pedunculopontine nucleus (PPN). Degeneration of the PPN could also be
implicated in the absence of REM sleep in PSP. The PPN is severely
affected in PSP, with approximately 60% neuronal loss.
The PPN is located in the rostral midbrain and has connections with the
basal ganglia, thalamus, lower brainstem and spinal cord. Urotensin II
receptor transcripts and radioligand binding have been detected in
cholinergic neurones of rat midbrain, especially in PPN and the laterodorsal
tegmentum (LDTg) (Clark SD et al, 2001) where their presence may relate
to sensory-motor integration.
We have taken frozen sections from midbrain in PSP and normal elderly
controls at levels incorporating the PPN and LDTg. Sections have been
stained for acetylcholinesterase, with relative density of staining quantified.
Areas with strong staining include the PPN, LDTg, substantia nigra, raphe
nuclei, pontine nuclei, and cuneiform nucleus. Radioligand receptor
autoradiography has been carried out to visualise urotensin receptors (125I
urotensin II) showing binding localised to acetylcholinesterase-rich areas.
Specific binding was demonstrated by displacement with the antagonist
palosuran (gift from Actelion Pharmaceuticals Ltd), and unlabelled
urotensin. Urotensin II binding in PSP compared to control cases is
presented.
36.02
Computational Modelling of Neostriatal Neurons
Hoyland D, Wood R B, Overton P G, Gurney K
Department of Psychology, University of Sheffield, Sheffield, , United
Kingdom
Computational modelling at the biophysical level often encounters a
severe limitation in that the modelling enterprise is divorced from the
gathering of physiological data, and hence generic parameters, and
membrane behaviour gathered from the work of others, have to be
used. Here we present the results of an exercise to model certain
neuronal types from the neostriatum - medium spiny neurons (MSNs)
and fast spiking interneurons (FSNs) - using data gathered in parallel
with the modelling.
Brain slices (250-400µm) were prepared from rats at P14-19,
containing the neostriatum in coronal section. Electrophysiological
data were acquired under current clamp in the whole cell patch
configuration. The acquired data consisted of measurements of
voltage responses to short current pulses (to determine passive
membrane properties) and responses to longer duration subthreshold
and suprathreshold pulses (to activate voltage gated conductances).
Medium spiny neurons (N = 11) were identified by their hyperpolarised
membrane potential (-81.95mV ± 2.78) and long delay to first spike
(216.45ms ± 42.77) at rheobase. Fast spiking interneurons (N = 4)
were identified by high maximum firing frequency (typically around 75
Hz) and faster spike responses at threshold (132.50ms ± 58.10).
Passive membrane and voltage response data were used to drive a
parameter search algorithm developed previously by our group, which
refines models of the cell concerned until target voltage behaviour is
achieved. These models of MSNs and FSNs, and refinements that
take into account neuronal morphology, will be used to build large
scale striatal models for the investigation of striatal functionality.
36.03
How visual stimuli activate subthalamic neurones at short latency.
Graham J H, Coizet V, Overton P G, Redgrave P
Biology Dept., King College, Bristol, TN, USA, Dept. Psychology, University
of Sheffield, S10 2TP
The midbrain superior colliculus (SC) is one of a number of brainstem
sensorimotor structures that provides input to and receives output from the
basal ganglia (BG). Recently, the status of the subthalamic nucleus (STN)
as a major input station of the BG has been firmly established. Although the
STN is known to receive many afferents from the cerebral cortex,
comparatively little is known about inputs from subcortical sensorimotor
structures. We have recently described a pronounced projection to the STN
from the SC (Redgrave et al., 2005); however, the functional implications of
this connectivity are unknown. The purpose of the present
electrophysiological investigation was, therefore, to determine whether
short-latency visual signals are relayed to the STN from the visually
responsive SC. In the anaesthetised rat, cells in the intermediate and deep
layers of the SC, as well as cells in the STN, were found to be
unresponsive to a bright wholefield light flash. However, following a local
disinhibitory injection of the GABAA antagonist bicuculline into the SC, both
SC and STN neurons exhibited phasic, short latency responses to the
flash. These results demonstrate the SC is a major source of short latency
visual signals to the STN. This route could enable unexpected events to
interrupt ongoing behaviour. (Supported by BBSRC and Wellcome Trust)
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36.04
Characterisation of neurons in the pedunculopontine nucleus in
vitro.
Sehirli U S, Stanford I M
University of Aston, Birmingham
The pedunculopontine nucleus (PPN) is located in mesopontine
tegmentum, extending from the posterior pole of substantia nigra back
to lateral tip of the superior cerebellar peduncle. Having connections
with the spinal cord, brain stem, thalamus and basal ganglia, the PPN
has a role in sleep and wakefulness, arousal and locomotion. Indeed,
it may be an alternative site of deep brain stimulation in relieving
symptoms of Parkinson’s disease. The PPN contains morphologically,
neurochemically and electrophysiologically diverse neurons including
cholinergic, GABA, glutamate and dopamine containing cells. In this
study we have used extracellular single-unit recordings in brain slices
obtained form 50g male Wistar rats. Neurons were initially classified
on the basis of firing rate (range 1.4 - 30.7Hz), firing pattern assessed
by coefficient of variation (range 5.8 - 92% ) and action potental (AP)
duration (range 0.46 - 4.22ms) . Group 1 exhibited low firing rates, a
low coefficient of variation and long AP duration consistent with ChAT
positive cells. Group 2 exhibited low firing rates, a low coefficient of
varition and short AP duration and Group 3 exhibited high firing rates,
a low coefficient of variation and short AP duration. In addition the
effect of dopamine, 5-HT and carbachol on firing rate was assessed.
Picrotoxin was found to increase firing rate in some cells indicating the
presence of GABA tone. Future juxtacellular labeling revealing
dendritic morphology may shed futher light of neuronal subtypes and
allow neurochemical charaterisation.
36.05
Dopamine D1-like receptor activation of a non-specific cation
conductance in the mouse subthalamic nucleus.
Ian M. Stanford, Alexandre J.C. Loucif, Gavin L. Woodhall
Aston University, Birmingham.
The postsynaptic effects of dopamine and associated receptor
pharmacology were studied using extracellular single-unit and whole-cell
recordings from STN neurons in vitro. Application of dopamine (30µM)
induced membrane depolarization accompanied by an increase in firing
rate of 12 ± 0.6Hz in all 69 cells independent of GABA or glutamate
synaptic transmission. This action was mimicked by the dopamine D1-like
receptor agonist SKF38393 (10µM, 35 cells) and the D2-like receptor
agonist quinpirole (10µM, 17 cells). Using voltage ramps, dopamine
induced an inward current of 69 ± 9.4pA at a holding potential of -60mV
(n=17). This current was accompanied by an increase in input conductance
of 1.55 ± 0.35nS which reversed at -30.6 ± 2.3mV, an effect mimicked by
SKF38393 (10µM, 9/10cells). Similar responses were observed in the
presence of ZD7288 and when measuring instantaneous current evoked by
voltage steps, indicating effects independent of Ih. The increase in
conductance was blocked by SCH23390 (2µM, n=4), mimicked by forskolin
(10µM, n=7) and blocked by H-89 (10 µM, n=6) indicating the D1-like
receptor activation of cAMP dependent protein kinase A. Less frequently,
dopamine induced inward currents accompanied by a decrease in input
conductance which reversed around Ek, an effect mimicked by the D2
agonist quinpirole (10µM, 5/5cells). These results indicate that dopamineinduced
depolarization of STN neurons is due to a D1 receptor-mediated
activation of a non-specific cation conductance as well as D2 receptor
decrease in a potassium conductance.
36.06
The spatial relationship between thalamostriatal terminals and
dopaminergic nigrostriatal terminals
Moss J, Boyes J, Bolam J P
MRC Anatomical Neuropharmacology Unit, Mansfield Road, Oxford
OX1 3TH
The glutamatergic corticostriatal projection has long been considered
the major excitatory input to the basal ganglia. Corticostriatal terminals
mainly contact the dendritic spines of medium-sized spiny projection
neurons. These also receive input from dopaminergic terminals
derived from the substantia nigra and, thus, represent a major site of
interaction between these two afferents. The thalamus also provides a
glutamatergic projection to the striatum and gives rise to terminals
contacting both dendritic shafts and spines. We tested the hypothesis
that thalamostriatal terminals, like corticostriatal terminals, have a
precise spatial relationship with dopaminergic terminals at the level of
individual dendritic spines. We addressed this by double
immunocytochemistry in rat striatum for the vesicular glutamate
transporter 2 (VGluT2), a marker of thalamostriatal terminals, and
tyrosine hydroxlyase (TH), a marker of dopaminergic terminals.
Consistent with previous observations, electron microscopic analyses
revealed that VGluT2-positive terminals formed asymmetric synapses
with both dendritic spines and dendritic shafts, and TH-positive
terminals formed symmetric synapses mainly with spine necks and
dendritic shafts. In double-immunolabelled tissue, individual spines
were often postsynaptic to both VGluT2-positive and TH-positive
terminals. In addition, dendritic shafts were also seen to receive inputs
from both types of terminals.
This suggests that glutamatergic input from the thalamus is likely to be
modulated by the dopamine input in a similar manner to the
modulation of the cortical input. Furthermore, since plasticity at
corticostriatal synapses is often dependent on the dopamine input,
thalamostriatal synapses are predicted to also exhibit dopaminedependent
plasticity.
36.07
Immediate early gene expression in the mesopontine tegmentum and
midbrain after acute or chronic systemic nicotine
Porter A, Alderson H L, Latimer M P, Winn P
School of Psychology, University of St Andrews, St Andrews, KY16 9JP
The reinforcing properties of nicotine depend partly on cholinergic
projections from the pedunculopontine tegmental (PPTg) and laterodorsal
tegmental (LDTg) nuclei to midbrain dopamine (DA) neurons in the ventral
tegmental area (VTA) and substantia nigra pars compacta (SNc). We
investigated neuronal activation using c-fos expression in these areas
following acute or chronic systemic nicotine (0, 0.1, 0.4, 0.8 mg/kg given
either once, or once per day for 5 days). We examined co-localization of c-
fos expression in bNOS and TH positive neurons.
Acute nicotine resulted in dose related c-fos expression, with the biggest
increase seen after 0.4 mg/kg nicotine. Despite large increases in c-fos
expression there was no co-localization with bNOS in the PPTg/LDTg. This
confirms the results of Lanca et al (Neuropharmacology [2000] 39: 2808-
2817): nicotine appears preferentially to activate non-cholinergic neurons in
the PPTg/LDTg. Surprisingly, nicotine also failed to activate midbrain DA
neurons. After animals were sensitized to nicotine there was a dosedependent
increase in c-fos expression, with the biggest increase seen
after 0.8 mg/kg nicotine. Increases in LDTg and VTA, regions associated
with reinforcement, were much larger than increases found in the PPTg and
SNc. Chronic nicotine, like acute, also preferentially activated noncholinergic
neurons in the mesopontine tegmentum and non-DA neurons in
the midbrain. The data suggest that the mechanisms by which DA is
involved in the pharmacological actions of passively administered nicotine
are more complex than was first thought and that the role of non-DA
neurons in the VTA (possibly GABA or glutamate containing) are also
important..
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37.01
Nitric oxide inhibition increases activity dependent
neuroprotective protein expression and neurogenesis in the
dentate gyrus during epileptogenesis
Cosgrave A S, McKay J S 2, Quinn J P 1, Thippeswamy T
Department of Preclinical Veterinary Sciences, University of Liverpool,
1Department of Physiology, University of Liverpool and 2AstraZeneca,
Macclesfield, UK.
Neurogenesis is an ongoing process in several areas of the adult
mammalian brain, the dentate gyrus (DG) being one such area.
Postnatal neurogenesis occurs after injury such as stroke and
seizures implying its importance in network homeostasis.
Epileptogenesis refers to the cellular and molecular mechanisms
involved in the development of epilepsy. Nitric oxide (NO) produced
from L-Arginine via nitric oxide synthase (NOS) has been shown to
negatively regulate neurogenesis; however, the precise mechanism is
not yet understood. Activity dependent neuroprotective protein (ADNP)
has recently been shown to regulate embryogenesis. We have
investigated this novel protein in relation to neurogenesis and NO
synthesis during epilepsy in the kainic acid (KA, 10 mg/kg i.p.) rodent
model of epilepsy. NOS inhibitor, N-nitro-L-arginine methyl ester (L-
NAME, 50 mg/kg i.p.) pre-treatment combined with (+)-5-Bromo-2’-
deoxyuridine (BrdU) (100 mg/kg, i.p.) were used to inhibit NO
production and label newly dividing cells, respectively. All experiments
were carried out in accordance with UK Home Office regulations. RT-
PCR/QPCR analysis of RNA from the DG of L-NAME treatment prior
to KA showed a significant increase in ADNP mRNA expression as
early as 3h compared to vehicle control. Immunostaining of DG from
similar treatments also showed an increase in ADNP and BrdU
immunopositive cells in the subgranular zone of the DG compared
with appropriate control. These observations suggest a relationship
between ADNP expression and NO regulation of neurogenesis during
epilepsy.
37.02
Oscillatory activity in hippocampal slices from GAD67-GFP (delta-neo)
mice
1Lu C, 1,2Janzsó G, 3Yanagawa Y, 4Obata K, 2Halasy K, 1Henderson Z
1Univ of Leeds, LS2 9JT, UK; 2Szent István University, H-1078 Budapest,
Hungary; 3Genetic and Behavioral Neuroscience, Gunma University
Graduate School of Medicine, Maebashi 371-8511, Japan; 4Brain
Research Institute, RIKEN, Wako 351-0198, Japan
The hippocampus exhibits theta frequency rhythms in vivo but gamma
oscillatory activity in vitro due to presence of the septo-hippocampal
pathway in vivo. Our long term aim is to examine modulation of oscillatory
activity in hippocampal interneurons by the septo-hippocampal pathway;
this would be facilitated by use of GAD67-GFP (delta-neo) mice (termed
GAD67-GFP mice for simplicity). We have examined the generation of
oscillatory in hippocampal slices from heterozygous GAD67-GFP mice and
wild type litter mates, bred and processed as authorized by the UK Animals
(Scientific Procedures) Act 1986. Confocal microscopy of tissue from
GAD67-GFP mice showed presence of GFP fluorescence in interneurons
but not principal cells in CA1 and CA3, except for GFP found in granule
cells of the dentate gyrus. In hippocampal slices prepared blind, oscillatory
activity was induced in CA3 by bath application of 300 nm kainate and
allowed to stabilise; presence of GFP fluorescence was determined after
the experiment. No significant difference was found in the oscillations
between slices from GAD67-GFP (n = 8) and wild type mice (n = 8) with
regards area power (range 20 - 60 Hz, t test, P > 0.5), but there was a
significant difference in peak frequency (GAD67-GFP: 28.4 ± 2.2 Hz, mean
and S.D; wild type: 25 ±1.6 Hz, n=8, P

37.05
Hippocampal maps are true neural objects: Evidence from
combined place cell recordings and the cellular distribution of
immediate early gene Mrna
Roberts* L, Habibian-Dehkordi S, Masih N, Rivard B, Muller R
MRC Centre for Synaptic Plasticity, Department of Anatomy,
University of Bristol, BS8 1TD., *current address: St Georges,
University of London, Cranmer Terrace, London, SW17 0RE
From electrophysiological evidence, the rat hippocampus is believed
to form a map-like representation of each different environment. Each
map is a randomly selected subset of hippocampal pyramidal cells
called “place cells” each discharging only within a cell- and
environment-specific region - its “firing field”. Each subset is a unit
since its cells fire together in only one environment but perhaps the
cells in a map are also linked to each other more directly. To test this
possibility we recorded place cells twice from individual rats, in a
cylinder and later in half the cylinder; recordings were made in the
reverse order in other rats. In all cases cells had the same fields in the
half as in the whole cylinder; the map was preserved. We then used
“cellular analysis of temporal activity by fluorescent in situ
hybridisation” (catFISH) to visualize mRNA from the immediate early
gene ARC. Cytoplasmic fluorescence indicates that ARC was
activated during the earlier exposure; nuclear fluorescence indicates
activation during the later exposure. The key finding is that the same
number of cells was labelled at the early and late time points,
regardless of the order of exposure. Thus, even though only half the
place cells discharge in the half cylinder, by another measure the
entire subset is activated! We conclude that the map has its own
structural identity – it is a neural entity and not merely a collection of
independent place cells. All procedures were performed in accordance
with the Animals (Scientific Procedures) Act 1986.
37.06
Depth profiles of antidromic and orthodromic responses evoked in
hippocampal subfield CA1 by entorhinal stimulation
Vorobyov N A, Brown M W
MRC Centre for Synaptic Plasticity, Department of Anatomy, University of
Bristol, Bristol, BS8 1TD, UK
Transmission of activity between the entorhinal cortex and hippocampus is
central to operation of the hippocampal memory system. Previously we
reported that electrical stimulation of entorhinal cortex evoked spatially
separated antidromic and orthodromic responses in the pyramidal layer of
CA1, indicating that entorhinal cortex could successively transfer activity
from one part of CA1 to another (Vorobyov and Brown, FENS 2004, p.402).
Here we establish that the differences between antidromic and orthodromic
responses do not arise from the reversal of potentials within CA1 or from
volume conduction from the underlying dentate gyrus.
We recorded simultaneously using multiple electrodes responses from
layers at different depths within CA1 and from the underlying molecular
layer of dentate gyrus in rats. The findings established that there was no
reversal of the potentials for either antidromic or orthodromic responses
throughout the depth of CA1. Both the negative potential of orthodromic
responses and the positive potential of antidromic responses were larger in
deeper compared to superficial layers. Increasing the stimulation intensity
resulted in an increase in the amplitude of responses but did not change
their shape and depth profile. Responses evoked in CA1 differed in shape
from those in the underlying dentate gyrus and changed size independently
of them.
Thus neither antidromic or orthodromic responses reversed through the
depth of CA1 and these CA1 responses were independent of potentials in
the underlying dentate gyrus. Accordingly, the previously described
differences between antidromic and orthodromic responses reflect
differences in the origin of these responses.
Supported by The Wellcome Trust.
37.07
The effect of 5-Fluorouracil and environmental enrichment on
recognition memory and cell proliferation in the adult rat
hippocampus.
Mustafa S, Wigmore P, Bennett G
Institute of Neuroscience, School of Biomedical Sciences, University
of Nottingham, Queen’s Medical Centre, , Nottingham, NG7 2UH
‘Chemobrain’ describes the cognitive deficits in adults which are
prevalent with systemic chemotherapy using agents such as 5-
Fluorouracil (5-Fu) that disrupts cell proliferation by interfering with
DNA synthesis. Proliferating cells are found within the adult dentate
gyrus (DG) of the hippocampus and give rise to new neurons that are
involved in memory and learning. A significant proportion of these
proliferating cells are associated with vascular endothelial cells. We
hypothesise that 5-Fu may affect proliferating cells in the adult DG and
cause deficits in hippocampal-mediated memory and this may be
modulated by environmental enrichment.
In this study, ‘chemobrain’ is modelled in rats by chronic systemic 5-Fu
administration. The effects of 5-Fu treatment and environmental
enrichment on hippocampal recognition and location memory were
observed using novel object and object location recognition memory
tasks, respectively. Immunohistochemistry with Ki67 and RECA-1
antibodies was used to measure the numbers of vascular-associated
(VA) and non-vascular associated (NVA) proliferating cells.
5-Fu treatment disrupted long-term recognition memory as indicated
by decreased novel object recognition, which was enhanced by
environmental enrichment, and also affected location recognition
memory. On-going measurement of proliferating cells in the DG is
being correlated with these behavioural changes to determine if 5-Fu
treatment caused decreases in VA and NVA proliferating cells.
37.08
Phase Precession as a Temporal Code for Trajectory
Huxter J, Allen K, Senior T, O`Neill J, Csicsvari J
MRC Anatomical Neuropharmacology Unit, Mansfield Road, Oxford, OX1
3TH, United Kingdom,
Pyramidal cells ("place cells") in the dorsal hippocampus have spatially
selective firing fields. In addition, as a rat traverses a given cell’s place field,
the cell fires at progressively earlier phases of each successive theta cycle.
This “phase precession” improves location prediction on tasks where rats
are trained to run in stereotyped trajectories. It has also been proposed as
a mechanism by which sequences of locations along a trajectory may be
learned, and a means of facilitating LTP. However, phase precession has
been poorly characterised in tasks where animals can move more freely,
and it is not clear what type of information this temporal code may carry
during non-stereotyped running. To address these questions, we
characterized phase precession in a large number of pyramidal cells in a
random foraging task. We identified robust monotonic phase precession,
regardless of the direction in which place fields were crossed. In agreement
with previous findings, the distance travelled through the place field was the
best predictor of firing phase. Moreover, using a Bayesian maximum
likelihood method, we were able to reconstruct movement trajectory from
the firing phases of a population of place cells. This represents the first
direct evidence of a hippocampal signal for the direction in which the animal
is moving. Taken together with the existence of place cells and the
modulation of their firing rates by running speed, this has important
implications for models of the hippocampus as a path integrator, and
demonstrates the utility of simultaneous rate and temporal coding.
Further studies are required to confirm if 5-Fu-induced changes in DG
proliferating cells are associated with disruption of hippocampusmediated
memory. This animal model of ‘chemobrain’ will help
achieve a better understanding of the mechanisms that cause
chemotherapy-associated decline in memory.
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37.09
Behavioural and electrophysiological correlates of local versus
global contextual processing in episodic memory
Moore J L, Cassidy S, Joyce E, Boll S, Roche R A P
Department of Psychology, , 2nd Floor John Hume Building, NUI
Maynooth, , Maynooth,, Co. Kildare,, Ireland.,
Here we report three sets of experiments which investigate the
influence of both local and global context upon episodic memory and
retrieval. Experiment 1 (n=120) investigated the impact of
contextually-sparse versus contextually-rich environments on memory
retrieval. Results suggest that re-instantiation of the learning context,
even when contextually-sparse, can elicit facilitation of recall for
episodic information learned in that context. Experiment 2 investigated
local context by varying the backgrounds in a standard Visual Paired-
Associates task. Forty participants completed a Visual Paired
Associates task with a local contextual background to each pair. All
pairs were learned and tested in a block wherein contextual cues were
either congruent or incongruent. Participants showed improved
accuracy and faster reaction times in the congruent context condition
compared to the incongruent context. Experiment 3 (n=20)
investigated the electrophysiological correlates of local context by
varying the backgrounds in a standard Visual Paired-Associates task.
The resultant ERP waveforms reflected contextually-based
processing, and were enhanced in context-congruent trials. Taken
together, the findings of these experiments suggest that measures of
hippocampal-based episodic memory may be particularly susceptible
to facilitation following reinstantiation of context.
37.10
Synaptically induced mGluR-LTD can be replicated by concurrent
activation of p38 MAPK and inhibition of Src family tyrosine kinases.
Moult PR, Fitzjohn SM, Collingridge GL, Bashir ZI
MRC Centre for Synaptic Plasticity, Department of Anatomy, School of
Medical Sciences, University of Bristol, University Walk, Bristol
As previously demonstrated, mGluR dependent LTD can be induced
synaptically in the CA1 region of acute, adult, hippocampal slices by
applying a paired pulse low frequency stimulation protocol (PP-LFS; 900
paired stimuli @ 1Hz, 50ms paired pulse interval). PP-LFS induced LTD of
68 &plusmn; 5% of baseline (n = 12). We show this form of LTD to be
inhibited by either of two structurally distinct protein tyrosine phosphatase
(PTP) inhibitors, orthovanadate (99 &plusmn; 10 % of baseline; n = 6 and P
&lt; 0.05 compared to control LTD) or PAO (100 &plusmn; 10 % of
baseline; n = 6; P &lt; 0.05 compared to control LTD). PP-LFS induced LTD
is also sensitive to inhibition of p38 MAPK by SB203580 (98 &plusmn; 16
% of baseline; n = 6; P &lt; 0.05 compared to control LTD). Crucially, this
form of LTD can be replicated by concurrently activating p38 MAPK and
inhibiting protein tyrosine kinases (PTKs) with anisomycin and PP2
respectively (depression was 63 &plusmn; 3 % of baseline; n = 3 and P &lt;
0.05 compared to baseline). Subsequent PP-LFS elicited no LTD (99
&plusmn; 3 % of baseline; n = 3 and P &lt; 0.05 compared to control LTD)
suggesting the mechanism is already saturated. These results suggest that
PP-LFS LTD is mediated by activation of both p38 MAPK and tyrosine
dephosphorylation of a residue that is usually phosphorylated under basal
conditions by constitutive activity of a Src family PTK.
38.01
IPSCs in hypothalamic TMN and Pef neurons are insensitive to
the general anaesthetic propofol in the N265M knock-in mouse
Zecharia A, Schumacher M, Jurd R, Rudolph U, Maze M, Franks, NP
1. Biophysics Section, Imperial College London, London, United
Kingdom. , 2. Institute of Pharmacology and Toxicology, University of
Zurich, Zurich, Switzerland. ,
How general anaesthetics produce a loss of consciousness is
currently unknown. However, the EEG suggests similarities between
this state and that of NREM sleep. We are investigating the hypothesis
that anaesthetics interact with natural sleep pathways. Lesions to the
sleep-active, GABAergic ventrolateral preoptic area (VLPO) produce
insomnia. These neurons are believed to cause sleep through the
inhibition of wake-active nuclei, including the histaminergic
tuberomammillary nucleus (TMN) and the orexinergic neurons of the
perifornical area (Pef). The GABAergic nature of the VLPO is critical to
this hypothesis of anaesthetic action because these drugs potentiate
the GABAA receptor in vitro. Furthermore, mice with a point-mutation
(N265M) in the β3 subunit of this receptor are markedly less sensitive
to the hypnotic action of propofol, but retain sensitivity to the steroid
anaesthetic alphaxalone. Previous work from this laboratory suggests
the TMN is an important target for general anaesthetics and c-fos data
(unpublished) implies orexinergic neurons may also be inhibited.
39.01
Involvement of PKA and CREB in single-trial reward learning in
Lymnaea
Michel M, Daniels M, Mueller U, Kemenes G
Sussex Centre for Neuroscience, Life Sciences, University of Sussex
Invertebrate and vertebrate work has outlined conserved molecular
components of long-term memory including the A kinase and the
constitutive transcription factor CREB. In this study, the role of these
proteins was looked at in associative memory stored in the well identified
feeding circuit of the pond-snail Lymnaea stagnalis.
We found an evolutionarily conserved PKA-like protein that is up-regulated
after learning and required for the consolidation of long-term memory. The
transcriptional activator CREB is selectively phosphorylated in trained
animals compared to all the possible control groups. Preliminary data show
that this phosphorylation may be due to the conditioning-induced activation
of PKA in vivo.
Funded by the MRC
We are using coronal slices of the TMN and Pef to measure the
prolongation by propofol and alphaxalone of GABAergic IPSC decay
time in wild-type and N265M knock-in mice.
IPSCs recorded from wild-type histaminergic and orexinergic neurons
were prolonged by both propofol and alphaxalone. Strikingly, in both
neuronal types, IPSCs from the N265M knock-in were insensitive to
propofol. However, mirroring in vivo findings, alphaxalone sensitivity
was unaltered in the knock-in mouse.
These findings are consistent with the hypothesis that enhancement of
GABAergic input into these nuclei is integral to propofol`s mechanism
of action in vivo.
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39.02
Protein synthesis inhibition by puromycin and long-term
associative memory after single-trial classical conditioning in
Lymnaea
Parker J, Michel M, Kemenes I, Kemenes G
Sussex Centre for Neuroscience, School of Life Sciences, Department
of Biology and Environmental Sciences, Brighton, UK, BN1 9QG
mRNA translation and the consequent production of new proteins are
fundamental to both long-term synaptic plasticity and long term
memory (LTM) in vertebrates and invertebrates. It has been
suggested that anisomycin, a protein synthesis inhibitor routinely
utilised in studies of synaptic plasticity and memory, additionally has
MAP-kinase activating properties, a crucial molecule in the induction
of gene expression and de novo protein synthesis. The protein
synthesis inhibitors emetine, cycloheximide and puromycin are
frequently utilised in memory research in both mammalian and
invertebrate models. This study explores the potential of these protein
synthesis inhibitors to block LTM after single-trial appetitive classical
conditioning in the pond snail Lymnaea stagnalis, a significant
invertebrate model with evolutionarily conserved molecular
mechanisms of LTM (including a role for MAP-kinase). Puromycin
(0.1mM, 1mM) but not emetine or cycloheximide, was found to inhibit
the incorporation of radioactively labelled methionine into the brain
within a range comparable to that of anisomycin (~60% and ~15%,
respectively of control radioactivity). Furthermore, behavioural learning
tests demonstrated that puromycin applied at 10 min post-training
inhibited the feeding response to the CS 24 hr following CS+US
training (0.1 mM, partial inhibition; 0.2 mM, full inhibition). Importantly,
low concentrations of puromycin, unlike emetine and cycloheximide,
did not have any adverse effects on the snails’ unconditioned feeding
response tested 24 hr after treatment. These results demonstrate the
suitability of puromycin as an alternative to anisomycin in learning and
LTM studies in the Lymnaea stagnalis feeding behaviour and the
underlying identified neuronal network.
39.03
The role of extracellular- signal regulated kinases, ERK, in recognition
memory.
Narduzzo K E, Brown M W, Warburton E C
School of Medical Sciences, University Walk, Clifton, Bristol, BS8 1TD,
The perirhinal cortex (Prh) has been shown to be the key neural structure
responsible for familiarity discrimination: a component of recognition
memory, in which a stimulus can be identified as previously encountered or
novel (Brown and Aggleton, 2001).
ERK extracellular signal regulated kinase is a member of the MAP- kinase
family of signalling cascades, identified in a number of studies to be
important in synaptic plasticity and long- term memory formation (LTM).
The MAPK cascade has been shown to be required for several forms of
hippocampus- dependent LTM, as demonstrated in spatial and fearconditioning
paradigms. Evidence also suggests a role for ERK in
entorhinal cortex and insular cortex- dependent LTM (reviewed in Adams
and Sweatt, 2002).
The aim of this study was to assess the role of ERK specifically in the Prh,
and its role in recognition memory.
Bilateral infusions of U0126, a specific MEK 1/2 inhibitor, were made
directly into the Prh, via chronically implanted guide cannula. The
spontaneous preferential exploration task was used to examine the effect of
ERK inhibition on familiarity discrimination; using delays of 20 min, 1 h, 3 h
and 24 h between the sample and test phase.
Animals were able to discriminate between novel and familiar stimuli
following delays of 20 min, 1h and 3h. Following a 24 h delay, U0126 was
found to significantly impair long- term familiarity discrimination.
These results suggest a critical role for ERK in long- term but not shorterterm
recognition memory formation.
Supported by the MRC
39.04
Effects of Type 2 diabetes on recognition memory
Brierley R, Molnar E, Bashir Z, Warburton C
Department of Anatomy, School of Medical Sciences, University of
Bristol, University Walk, Bristol, BS8 1TD
Many epidemiological and clinical studies show that ageing sufferers
of type 2 diabetes are more likely to be affected by cognitive
impairments (Ott et al, 1999; Beeri et al, 2004; Grodstein et al, 2001).
These cognitive deficits are paralleled by neurophysiological and
structural changes in the brain, possibly involving changes in
glutamate receptor neurotransmission and plasticity. The Goto-
Kakizaki (GK) rat is a non-obese spontaneous animal model of type 2
diabetes which was developed by selective breeding of
hyperglycaemic Wistar rats. The aim of the present study was to
investigate whether GK rats demonstrated age-related impairments in
visual or olfactory recognition memory, compared to age-matched
Wistar control rats, using two variants of the spontaneous novel object
preference task (Ennaceur et al, 2005). To investigate visual
recognition memory, the standard object recognition procedure,
involving the presentation of novel and familiar objects was used. To
investigate olfactory recognition memory, novel and familiar odours
were presented in place of the objects. While the GK rats showed
impaired glucose tolerance by 3 months of age, no significant
impairments in either visual or olfactory recognition memory were
evident at 3, 6 and 9 months of age. Further experiments will be
conducted at 3 month intervals to investigate whether there is a
decrease in the cognitive abilities of aged GK rats.
39.05
Interfering with Fos expression impairs recognition memory in rats
Seoane A, Brown M W
Department of Anatomy, School of Medical Sciences, University of Bristol,
UK
The aim of this study was to determine whether interference with Fos
expression in the perirhinal cortex resulted in an impairment of recognition
memory. Fos is an immediate-early gene product and its production can be
blocked by administering an antisense oligodeoxynucleotide (ODN). Such
treatment produces a deficit in long-term aversive taste memory, spatial
memory and fear-conditioning (e.g. Yasoshima et al, PNAS 103: 7106-
7111, 2006). Fos expression provides a marker for recognition memory
processes: compared to familiar visual stimuli, novel visual stimuli cause an
increase of Fos-stained neurones in perirhinal cortex, a region known to be
essential for the familiarity discrimination component of recognition memory
(Brown and Aggleton, Nature Rev. Neurosci. 2: 51-61, 2001). Familiarity
discrimination was assessed by using a task which evaluates the
spontaneous preference of rats to explore a novel rather than a familiar
object (Ennaceur and Delacour, Behav. Brain Res. 31: 47-59, 1988). One
hour after local perirhinal administration of either antisense or sense ODN,
rats were presented with two identical objects and, following a delay of
20min, 3h or 24h, the time spent exploring the familiar and a novel object
was measured. Both sense and antisense ODNs were infused into the
perirhinal cortex bilaterally via implanted cannulae (1nmol per side in 1μl
saline over 2min). Familiarity discrimination was unimpaired at the 20min
delay but impaired at delays of 3h and 24h by antisense but not sense
ODN infusion. These results indicate that Fos expression is involved in
long-term but not shorter-term recognition memory processes.
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39.06
The role of glutamate receptor subtypes within the prefrontal and
perirhinal cortices in associative recognition memory.
Barker G R I, Warburton E C
Department of Anatomy, School of Medical Sciences, University Walk,
Bristol, BS8 1TD
Experimental evidence has suggested that the perirhinal cortex (PRH)
and the medial prefrontal cortex (mPFC) play a crucial role in
recognition memory based upon in object-place associative
discriminations, and that these two cortical regions operate within a
functional neural network (Barker et al 2007). The present study
sought to examine the importance of glutamatergic and cholinergic
receptor neurotransmission in these cortical regions for performance
of an object-in-place discrimination task.
Selective AMPA/kainite, NMDA or muscarinic receptor antagonists
were infused into the PRH or mPFC via chronically implanted bilateral
guide cannulae prior to acquisition in an object-in-place task. The
behavioural effects of such infusions were compared with the effects
of unilateral infusions into both PRH and mPFC in either the same or
contralateral hemispheres.
Infusions of CNQX (an AMPA/kainate receptor antagonist) into the
PRH, mPFC or both regions in contralateral hemispheres impaired
short-term object-in place performance.
Infusions of scopolamine (a muscarinic receptor antagonist) into the
mPFC, PRH or both regions in contralateral hemispheres impaired
short-term and long-term object in place performance.
Infusions of AP5 (an NMDA receptor antagonist) into the mPFC
impaired long-term and short-term object-in-place memory, while
infusions of AP5 into the PRH, or into both the PRH and mPFC in
contralateral hemispheres impaired short-term but not long term
object-in place-memory. These results indicate the importance of both
glutamatergic and cholinergic neurotransmission for discriminations
based upon object-place associations. Further these results support
the hypothesis that the PRH and mPFC interact within the same
hemisphere to process associational recognition memory information.
39.07
The role of pCAMKII-alpha in visual memory processing
Tinsley C J, Brown M W, Warburton E C
MRC Centre for Synaptic Plasticity, Department of Anatomy, Bristol
University, Bristol BS8 1TD.
We investigated the role of the activated form of Ca2+-Calmodulin
dependent kinase II alpha (pCAMKII-α) in visual processing of novel and
familiar images. Using a paired viewing procedure (Zhu et al., 1996), we
presented novel or familiar images on computer monitors positioned in the
left or right-hand visual hemifield of rats, thus restricting initial visual
processing to the opposite hemisphere of the brain. Dark Agouti male rats
saw familiar and novel images over 11 sessions. On the final session novel
images were presented to one hemisphere and familiar images to the other
hemisphere. The rats were transcardially perfused at one of three time
points following the last image: 10 minutes, 40 minutes or 70 minutes. The
brains were stained with an antibody for pCAMKII-α. Stained neurons were
counted in different brain regions (V1, TE, perirhinal, entorhinal and
auditory cortex). Normalised counts were analysed with repeated measures
ANOVA with factors novelty, time and brain area. There was a significant
interaction between novelty, time and area (F(9.14,64.0) = 2.314, p< 0.05
with Huyn-Feldt correction). Viewing novel compared to familiar stimuli
produced a significant increase in stained cells limited to the perirhinal
region below the rhinal sulcus in rats of the 70 minute group and (F(1,5) =
18.6, p< 0.01). These results indicate that pCAMKIIα has a differential role
in the visual processing of novel and familiar images within a region of the
perirhinal cortex.
39.08
Use of super ecliptic pHluorin tagged GluR2 to study DHPGinduced
LTD
Sanderson T M, Molnar E, Collingridge G L, Fitzjohn S M
MRC Centre for Synaptic Plasticity, Department of Anatomy, School of
Medical Sciences, University of Bristol, University Walk, Bristol, BS8
1TD, UK,
A distinct form of hippocampal long term depression (LTD) is induced
by activation of group I metabotropic glutamate receptors (mGluRs) by
the specific agonist dihydroxyphenylglycine (DHPG)1.
AMPA receptor redistribution has been implicated in this form of LTD.
Also the AMPA receptor subunit GluR2 is transiently
dephosphorylated in response to DHPG via a protein tyrosine
phosphatase (PTP)2. The mechanism of AMPA receptor redistribution
is not fully understood however. Here we have used a super ecliptic
pHluorin tagged version of GluR2 (SEP-GluR2) to study this
phenomenon3. 14 day old postnatal hippocampal cultured neurones
were infected with SEP-GluR2 using the sindbis virus. 17-24 hours
later these neurones were used in experiments.
SEP-GluR2 infected neurones show a reduction in mEPSC frequency
upon DHPG application that is comparable to that seen in non-infected
neurones (76±2% of pre-DHPG frequency). The mEPSC amplitude
was unaffected. In addition we found that 30 minutes after DHPG
application the fluorescence from surface expressed SEP-GluR2 was
significantly decreased to 83±6% of pre-DHPG values. The DHPG
induced decrease in surface SEP-GluR2 was prevented when DHPG
was applied in the presence of either the mGluR5 antagonist MPEP
(93±10% of control) or the PTP inhibitor orthovanadate (100±7% of
control).
In summary, by using live cell imaging we have found that DHPG
causes a reduction in surface SEP-GluR2 containing AMPA receptors.
39.09
Involvement of myosin VI and the synapse-associated protein 97 in
membrane trafficking of glutamate receptors
Corrêa S A L, Nash J E, Collingridge G L, Molnar E, Fitzjohn S M
Medical Research Council Centre for Synaptic Plasticity; Department of
Anatomy, University of Bristol, School of Medical Sciences, Bristol, BS8
1TD, United Kingdom
Cell surface expression of plasma membrane proteins is regulated by
different mechanisms that control insertion and removal of proteins from the
membrane. Synapse-associated protein 97 (SAP97) is a member of a
protein family involved in the trafficking of glutamate receptors and is
known to interact with the C terminal of the AMPA receptor subunit GluR1.
Furthermore, SAP97 interacts with myosin VI (MyoVI), an actin-based
motor protein. In the current study we aimed to investigate the role of
MyoVI and SAP97 in the surface expression of the AMPA receptor subunits
GluR1 and GluR2.
Transfection of cultured hippocampal neurons with Myo VI C-terminal
dominant negative (MyoVIC) resulted in a significant decrease in the
surface expression of both GluR1 and GluR2 subunits when compared with
non-transfected or EGFP-transfected cells, as measured using
immunocytochemistry. However, when a truncated form of SAP97
(SAP97d1-65) lacking both the MyoVI binding domain and the
phosphorylation site for CaMKII was expressed, no significant differences
in the number of GluR1 and GluR2 surface puncta were observed when
compared with EGFP or non-transfected cells. In contrast, increased
surface expression of both GluR1 and GluR2 was observed when neurons
were transfected with a truncated form of SAP97 (SAP97d66-126) lacking
only the MyoVI binding domain. These data are consistent with a role for
both MyoVI and SAP97 in AMPA receptor trafficking at synapses.
1. Palmer MJ, et al (1997), Neuropharmacology, 36(11-12):1517-
1532.
2. Moult PR, et al (2006), J Neurosci, 43(2):175-180.
3. Ashby MC, et al (2004), J Neurosci, 24(22): 5172-5176.
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39.10
Metabotropic glutamate receptor mediated long term depression
involves AMPA receptor redistribution triggered by protein
tyrosine phosphatases
Gladding C M, Fitzjohn S M, Bashir Z I, Collingridge G L, Molnar E
Medical Research Council Centre for Synaptic Plasticity; Department
of Anatomy, University of Bristol, School of Medical Sciences, Bristol,
BS8 1TD, United Kingdom
Long-term depression (LTD) can be induced at hippocampal CA1
synapses, by activation of either N-methyl-D-aspartate receptors
(NMDARs) or metabotropic glutamate receptors (mGluRs). Although
NMDAR-LTD has been relatively well characterised, the molecular
mechanisms underlying mGluR-LTD are still unknown. However,
these can be investigated by bath application of the group 1 mGluR
agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Previous studies
have indicated that DHPG-LTD is calcium independent and does not
require activation of CaMKII, protein kinases A or C or
serine/threonine phosphatases. It does however require protein
tyrosine phosphatases (PTPs), GĄq activation and involve
internalisation of AMPA receptors (AMPARs) and NMDARs. In this
study, 400 μm thick acute hippocampal slices were obtained from
female Wistar rats (10-12 weeks of age), and incubated in 100 μM
DHPG for 10 minutes, followed by immunoprecipitation of AMPARs
and quantitative immunoblot analysis using an anti-phosphotyrosine
antibody at different time-points. We have established that DHPG-LTD
involves a transient tyrosine dephosphorylation of the GluR2 AMPAR
subunit, and that this is blocked by prior application of the PTP
inhibitor, orthovanadate. The total AMPAR content in the slices is
unaffected in DHPG-LTD. AMPAR phosphotyrosine levels do not
change significantly upon activation of NMDARs implying that the
mechanism is specific to mGluR-LTD. The combination of cell surface
biotin-labelling and immunoprecipitation revealed that tyrosine
dephosphorylation is required for the trafficking of AMPARs away from
the synapse. This study has therefore established that AMPAR
tyrosine dephosphorylation by PTPs is an important postsynaptic
component of mGluR-LTD which does not seem to be involved in
NMDAR-LTD.
39.11
Learning-specific changes in long-term depression in rat perirhinal
cortex.
Massey P V, Pythian D, Narduzzo K E, Warburton E C, Brown M W, Bashir
Z I
MRC Centre for Synaptic Plasticity, Dept of Anatomy, University of Bristol,
BS8 1TD, UK.
Learning is widely believed to involve mechanisms of synaptic plasticity but
their precise relationship remains poorly understood. The perirhinal cortex
is required for both single exposure learning of recognition memory
(familiarity discrimination) and multi-trial perceptual and reinforcement
learning. We exploited the involvement of rat perirhinal cortex in these
different forms of learning to compare within the same brain their effects on
synaptic plasticity. Every effort was made to minimise pain and discomfort
in accordance with the Animals (Scientific Procedures) Act 1986. A paired
viewing apparatus allowed novel pictures to be presented to the monocular
field of one eye, and familiar pictures to the other under the same
behavioural conditions. Picture presentations were accompanied by a juice
reward. 60 min after the last session perirhinal slices were prepared from
‘novel’ and ‘familiar’ hemispheres and extracellular field potential recording
used to assess synaptic plasticity. Repeated exposure to familiar pictures,
but not many novel pictures, occluded, in a muscarinic-dependent
manner, subsequent induction of both depotentiation and de novo LTD in
perirhinal cortex in vitro whilst having no effect on LTP. The contrast in the
effects of the two types of learning on LTD indicates that the change cannot
be due to synapse-specific plastic changes registering precise details of the
individual learned associations. Instead, we conclude that the occlusions of
LTD arise from a learning-related generalised change in plasticity gain. The
existence of this additional mechanism has important implications for
interpretations of how plasticity relates to learning. Supported by Wellcome
Trust.
40.01
Effects of melatonin on neuronal activity in the rat
suprachiasmatic nuclei (SCN) in vitro.
Scott F, Brown T, Delagrange P, Piggins H
(1,2,4) University of Manchester, UK, (3) Institut de Recherches
Internationales Servier (IRIS), France
The suprachiasmatic nuclei (SCN) act as the master circadian
pacemaker in mammals, controlling daily rhythms in many aspects of
physiology, including secretion of the pineal gland hormone melatonin.
Melatonin levels peak during the early night and exogenous
application of melatonin during the evening alters the activity of rodent
SCN neurons and the timing of rodent behavioural rhythms via high
affinity G protein-coupled receptors. Using a novel recording
technique, we investigated the acute and long-term effects of
exogenous melatonin on single and multiple unit (SUA and MUA) SCN
cellular activity in vitro.
We used suction electrodes to record SCN MUA from coronal, 400µm
thick rat SCN slices maintained in an interface-style tissue chamber.
SUA firing patterns were discriminated from these MUA recordings
offline using Spike2 software (CED, UK). For acute studies, melatonin
(10fM to 1μM) was applied via the perfusion line for at least 10min
between zeitgeber times (ZT) 3 and ZT 8.5 where ZT12=projected
lights-off.
Consistent with previous reports, melatonin evoked activations or
suppressions in SCN SUA, but contrasting with these earlier studies,
we found that melatonin-induced predominantly activational effects on
both MUA and SUA firing rates. Late day application (ZT 10-11) of
1pM melatonin also produced a phase advance in the timing of peak
firing in a subset of single SCN neurons. These findings indicate
heterogeneity in the responses of individual SCN neurons, in vitro, to
exogenous melatonin.
40.02
Scheduled exercise stabilises behavioural rhythms of mice with
deficient neuropeptide signalling
Power A, Hughes A T, Namvar S, Brown T M, Piggins H D
Faculty of Life Sciences, University of Manchester, Manchester, UK
The principal mammalian circadian pacemaker, located within the
hypothalamic suprachiasmatic nuclei (SCN), is reset by both light (photic)
and arousal-promoting non-photic cues (eg.scheduled exercise).
Vasoactive intestinal polypeptide and its VPAC2 receptor are abundant
within the SCN. Mice lacking the VPAC2 receptor (Vipr2-/-) exhibit altered
behavioural and neuronal rhythms and lack normal responses to photic
stimuli, suggesting a completely dysfunctional SCN pacemaker. It is
unknown if Vipr2-/- mice show resynchronisation to non-photic stimuli. Here
we assessed the effects of scheduled exercise on locomotor and drinking
rhythms in wild-type (WT) and Vipr2-/- mice. Mice were individually housed
in running wheel equipped cages under a light-dark cycle and then
released into constant darkness (DD). After 14 days (DD1), access to the
running wheel was restricted to 6h a day. This scheduled exercise
continued for 21 days, during which both genotypes showed apparent
entrainment of drinking rhythms. Subsequently mice freely exercised for a
further 21-35 days (DD2). In DD1, all WT mice were strongly rhythmic
(period=23.54±0.05h;n=20), 23/26 Vipr2-/- animals exhibited very weak
rhythms (period=22.91±0.29h) and 3 were arrhythmic. In DD2, all WT mice
were rhythmic (period=23.55±0.08h) while 25/26 Vipr2-/- mice were
rhythmic (period=24.01±0.19h). Two previously arrhythmic Vipr2-/- mice
now expressed robust ~24h behavioural rhythms. These results show that,
unlike light, scheduled exercise, can stabilise behavioural rhythmicity in
Vipr2-/- mice and reveal surprising plasticity in the circadian system to
reorganise to a non-photic stimulus.
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40.03
Live imaging of Per1 driven GFP expression in mice lacking
VPAC2 receptors.
Hughes A T L 1, Lennox L 1, McMahon D G 2, Piggins H D 1
1 Faculty of Life Sciences, University of Manchester, UK ;, , 2
Department of Biological Sciences, Vanderbilt University, Nashville,
Tennessee, USA
Vasoactive intestinal polypeptide and its receptor, VPAC2, play
important roles in the functioning of the mammalian circadian clock
located in the hypothalamic suprachiasmatic nuclei (SCN). Mice
lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms in
locomotor behaviour, neuronal firing rate and clock gene expression,
however the nature of molecular oscillations at the individual cell level
are unclear. In this study we used real-time confocal imaging of a
destabilised green fluorescent protein (dGFP) reporter to track the
expression profile of the core clock gene Per1 in live SCN-containing
brain slices taken from wildtype (WT) and Vipr2-/- mice. Significantly
more dGFP expressing cells were visible in WT slices than in Vipr2-/-
slices. Rhythmic oscillations in Per1-driven dGFP were detected in
both WT and Vipr2-/- cells, though a significantly greater proportion of
cells imaged in WT slices expressed detectable rhythms. WT cells
expressed a mean period of ~24h and Vipr2-/- cells expressed a mean
period of ~22.5h. Vipr2-/- cells expressed significantly lower amplitude
oscillations than WT cells, with significantly lower average and peak
fluorescence. Within each slice, the phases of WT cells were
synchronised whereas cells in Vipr2-/- slices where poorly
synchronised. These data demonstrate that intercellular
communication via the VPAC2 receptor is important for SCN neurones
to sustain robust, synchronous oscillations in clock gene expression.
Supported by the BBSRC.
40.04
Uncertainty coding neurons in the primate orbitofrontal cortex
O`Neill M, Schultz W
Physiology, Development and Neuroscience,, University of Cambridge,,
Downing Street,, Cambridge,, CB2 3DY
The orbitofrontal cortex plays an important role in decision-making under
conditions of uncertainty. Human patients and orbitofrontal-lesioned
animals show alterations in tasks involving uncertainty, including loss of risk
sensitivity, increased risk-seeking, and increased risk aversion. However,
there has been no clear evidence of the involvement of orbitofrontal
neurons in uncertainty processing. We recorded single unit neuronal
activity from the orbitofrontal cortex from 2 rhesus macaques presented
with cues with varying degrees of reward uncertainty.
Reward uncertainty was defined by the variance of a high or low
equiprobable (p=0.5) reward following cue presentation. Preliminary results
show that 15% (29/196) of orbitofrontal neurons that responded to a task
event selectively responded to the degree of reward uncertainty
represented by the visual cues. We identified neurons that were selective
for high (18/29) or low (6/29) reward uncertainty and neurons with activity
that reflected a gradient of uncertainty (low, medium, high: 5/29).
These findings suggest that neurons in the orbitofrontal cortex are
involved in encoding the degree of reward uncertainty signalled by visual
cues and this coding may be involved in decision making under conditions
of uncertainty.
40.05
Deficits in emotional and social behaviours in the complexin 2
knockout mouse
Glynn D, Morton A J
Department of Pharmacology, University of Cambridge, Cambridge,
CB2 1PD
Complexins are presynaptic proteins that bind to the SNARE complex
and modulate neurotransmitter release. Complexin II levels are
reduced in neurodegenerative diseases such as Huntington’s disease
(HD) as well as in schizophrenia and depression. All of these are
diseases in which motor, cognitive and psychiatric disturbances
coexist. Previously, we showed that adult complexin 2 knockout
(Cplx2-/-) mice exhibit progressive deficits of motor and cognitive
function (Glynn et al., 2003). We wondered whether or not Cplx2-/-
mice might also show abnormalities in emotional and social behaviour,
since these are prominent in patients with HD, schizophrenia and
depression.
We used a battery of tests to investigate information processing
(prepulse inhibition of acoustic startle) anxiety-type behaviour (the
zero-maze), depression (Porsolt forced swim test) and social
behaviour (a resident-intruder test, a social recognition task and a test
of sociability and preference for social novelty).
Both the acoustic startle response and prepulse inhibition of startle
were decreased in Cplx2-/- mice. Further, Cplx2-/- mice exhibited
early deficits in other emotional and social behaviours. In particular,
they showed deficits in social recognition and social memory,
sociability and preference for social novelty.
Our results show that in addition to the motor and cognitive deficits we
have already described, Cplx2-/- mice have abnormalities in
information processing and emotional-type behaviour. Since all of the
disorders in which complexin II levels are decreased include deficits in
socialisation and cognition, Cplx2-/- mice potentially provide a model
in which to study these aspects of those disorders.
40.06
Object-centred neglect in rats with unilateral 6-hydroxydopamine
lesions of the medial forebrain bundle
Annett L E, Lord K L, Watt C A, Thiemann G, Hasenöhrl R U
School of Psychology, University of Hertfordshire, College Lane, Hatfield,
Hertfordshire, AL10 9AB, UK
Neglect of contralateral stimuli is well documented in animals with unilateral
dopamine loss, for example responses are slow or absent to stimuli
presented on the side contralateral to a unilateral 6-hydroxydopamine
(6OHDA) lesion and ipsilateral biases are evident when food is retrieved
from different spatial locations. Neglect in humans can result from right
parietal cortex damage and a mild form of neglect has been reported in
people with asymmetric symptoms of Parkinson’s disease1. One interesting
feature of neglect in humans is that it may be object-centred, i.e. the
contralateral side of an object is ignored irrespective of the location of that
object in space. The present study used a ‘line bisection’ task devised for
rats to investigate whether the neglect produced by unilateral 6-OHDA
lesions of the medial forebrain bundle includes an object-centred
component. The experiment was conducted in accordance with the UK
Animals (Scientific Procedures) Act 1986. The lesions were confirmed at
the end of the study by tyrosine-hydroxylase immunohistochemistry. Rats
were trained to retrieve a single coco pop (chocolate flavoured cereal)
hidden in sawdust from the exact centre of a rectangular tray. Following a
unilateral 6OHDA lesion, rats showed neglect in that their initial head
response over the tray was displaced ipsilaterally. As the tray width
increased (6, 10, 14, 18 and 22 cm) the extent of the ipsilateral
displacement also increased, an indication of object-centred neglect.
1Lee AC et al. (2001) Vision Research 41:2677-2686
The support of the Parkinson’s Disease Society, UK, is gratefully
acknowledged.
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40.07
Neurotransmitter interaction and the adaptive response to the
absence of food in the pharynx of C. elegans
Luedtke S, Walker R, Hopper N, O`Connor V, Holden-Dye L
University of Southampton; School of Biological Sciences, Bassett
Crescent East,, Southampton,, SO16 7PX
We are interested in peptidergic and classical neurotransmitter
interaction and how these modulate behaviour as a response to
internal and external signals. As a model behaviour we are using the
pharyngeal pumping in the nematode C. elegans. The pharyngeal
nervous system regulates feeding behaviour in C. elegans. This neural
network regulates muscle pumping via a number of different classical
transmitters and neuropeptides. We are trying to delineate a
mechanism that controls pharyngeal pumping in the intact worm under
differing environmental conditions (on food and increasing time off
food). Placing worms off food identified 3 distinct behavioural phases
of pharyngeal pumping with increasing time. Subsequent analyses of
several mutants have shown modified responses to the absence of
food. In particular a number of mutants in classical and peptidergic
inhibitors support the notion that pharynx is under negative regulation
that is lessened with increasing time of food. We are currently trying to
correlate the distinct phases of pharyngeal behaviour with the
metabolic status of the worm (measuring fat storage) and switches in
the locomotory behaviours worms use when foraging for food. In doing
so we hope to develop an integrated view of cellular and behavioural
adaptive responses underlying feeding behaviour.
Funded by the Gerald Kerkut Charitable Trust. We are grateful to the
C. elegans Genetics Centre for providing strains.
40.08
Distinguishing between error detection and error awareness:
implications for clinical research
O’Connell R G, Dockree P M, Bellgrove M A, Lau A, Fitzgerald M, Foxe J J,
Robertson I H
School of Psychology and Trinity College Institute of Neuroscience, Trinity
College Dublin
In everyday life our ability to detect errors is critical for smooth and dynamic
interaction with our environment, providing us with the opportunity to realign
our behaviour with prevailing goals and to learn the consequences of
different behaviours. Error processing is reliant, in part, on communication
between anterior cingulate and lateral prefrontal cortices and is impaired in
a number of putatively frontal conditions including schizophrenia, ADHD
and OCD. A common limitation of error processing studies, and particularly
those involving clinical groups, has been a failure to distinguish between
error detection and conscious error awareness. Damage to the frontal lobes
has been associated with decreased awareness of one’s deficits including
a tendency to ‘miss’ errors during neuropsychological tasks. The present
study constitutes a systematic electrophysiological investigation of error
processing networks in a group of adults diagnosed with ADHD. Eighteen
patients and 21 matched controls performed a Go/No-go response
inhibition paradigm that was specially designed to ascertain levels of error
awareness. This experiment reveals that adults with ADHD are significantly
less likely to consciously detect their errors and identifies a number of ERP
abnormalities relating to aspects of performance monitoring and conscious
error processing. The Error Positivity (Pe) is identified as a key marker of
conscious error processing and is found to be attenuated in the patient
group even following consciously detected errors. In addition, the results of
source analysis indicate de-activation of midline frontal regions during
conscious error processing in the ADHD group.
40.09
Effects of early life deprivation and fluoxetine treatment on
central dopamine D2 receptors in adult Wistar rats
Leventopoulos M (1), Lukito S (1),, Russig H (2), Feldon J (2), Pryce C
R (2), Opacka–Juffry J. (1)
(1)Roehampton University, London, UK., (2)Behavioural Neurobiology
Laboratory, Swiss Federal Institute of Technology, Zurich,
Switzerland.,
Studies on animal models of depression have shown that early life
stress affects monoamine status in adult rats. The present study on
adult Wistar rats exposed to early life deprivation (ED) assessed the
level of postsynaptic dopamine D2 receptor binding in the subregions
of cortex, striatum and nucleus accumbens (NAc).
Male Wistar rat P1–14 pups were isolated for 4h/day (ED) or were
handled for 1 minute (CON). They were weaned at P21 and left
undisturbed until 4-6 months old. The ED and CON groups were
halved to receive either vehicle or fluoxetine (FLX, 10 mg/kg, 31 days).
Thus gave four treatment groups: CON-VEH, CON-FLX, ED-VEH and
ED-FLX, n=8 each. ED-VEH rats showed significantly reduced
motivation to obtain sucrose in a progressive ratio schedule (vs CON-
VEH); FLX reversed this deficit.
Quantitative receptor autoradiography was used to determine D2
receptor binding with [3H]raclopride. D2 binding in dorsolateral
striatum and NAc, both core and shell, was significantly reduced (by
16-20%) in ED vs CON. Although fluoxetine treatment did not
significantly affect these changes, it caused a significant reduction in
D2 binding in the prelimbic cortex, dorsolateral and ventromedial
striatum and NAc core and shell in CON-FLX vs CON-VEH.
Correlation analysis of D2 binding between NAc core and shell in each
of the treatment groups indicated a different response to fluoxetine
between NAc core and NAc shell in ED rats when compared with
CON. This is of interest, considering the putative differential
involvement of NAc core versus shell in reward-related behaviour
40.10
Changes in 5HT1A and 5HT2C receptor binding in response to early
life deprivation and fluoxetine treatment in adult Wistar rats
Leventopoulos M (1), Opacka–Juffry J (1), Russig H (2), Feldon J (2), Pryce
C R (2)
(1)Roehampton University, London, UK., (2)Behavioural Neurobiology
Laboratory, Swiss Federal Institute of Technology, Zurich, Switzerland.,
Early life deprivation (ED) can act as a risk factor in aetiology of
depression; in rats, it leads to depression-like behaviour in adulthood. The
central serotonergic system is a putative player in such long-term
responses. The present study hypothesised that ED would reduce
postsynaptic 5HT receptor binding in the brain regions that regulate
depression-like behaviour, and that the antidepressant fluoxetine would
correct receptor responses in adult rats.
Male Wistar rat P1–14 pups were isolated for 4h/day (ED) or were handled
for 1 minute (CON). They were weaned at P21 and left undisturbed until 4-
6 months old. The ED and CON groups were halved to receive either
vehicle or fluoxetine (FLX, 10 mg/kg, 31 days). Thus, four treatment groups
were studied: CON-VEH, CON-FLX, ED-VEH and ED-FLX, n=8 each. ED-
VEH rats showed significantly reduced motivation to obtain sucrose in a
progressive ratio schedule (vs CON-VEH); FLX reversed this deficit.
Quantitative receptor autoradiography was used to determine 5HT1A and
5HT2C receptor binding with [3H]WAY100635 and [3H]mesulergine (added
spiperone and 8-OH-DPAT), respectively. 5HT1A binding was significantly
reduced in anterior cingulate, premotor cortex and ventral hippocampal
CA1 in ED-VEH vs CON-VEH specifically. Same regions and, additionally,
prelimbic and frontal cortices, and accumbens had significantly reduced
5HT2C binding. Whilst fluoxetine reversed the ED-dependent changes in
5HT1A binding in cortices, it did not correct those of 5HT2C.
These results indicate that the depression-like behavioural phenotype
studied here implicates abnormal serotonergic regulation. 5HT1A receptor
acts as a selective therapeutic target for behavioural improvement achieved
with antidepressant treatment.
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41.01
A polymorphism at the 3’ untranslated region of the human clock
gene is associated with adult Attention-Deficit Hyperactivity
Disorder
Kissling C, Retz W, Wieman S, Hunnerkopf R, Conner A C, Freitag C,
Rosler M, Coogan A N, Thome J
The Institute of Life Science and School of Medicine, Swansea
University, United Kingdom. Institute for Forensic Psychology and
Psychiatry, University of the Saarland, Germany. , Division of
Molecular Genome Analysis, German Cancer Research Centre,
Heidelberg, Germany
Attention-Deficit Hyperactivity Disorder (ADHD) is commonly found in
subjects with antisocial personality disorders and predicts criminal
activity in adulthood. Several studies demonstrate a relationship
between ADHD and sleep problems indicating an increased nocturnal
activity and significant daytime somnolence in unmedicated ADHD
patients. Since ADHD is a very complex disease with a high genetic
load involving multiple genes of moderate effect we hypothesized a
link of between adult ADHD to and genes involved in the circadian
timekeeping system.
We performed an association of this polymorphism with ADHD in 238
male adult detainees with German background suffering of from
clinically defined ADHD. We examined a previously characterised C/T
SNP in the 3’ UTR of the Clock gene, a polymorphism postulated to be
associated with evening preference. Our results reveal a strong
association of adult ADHD with a genotype of the SNP rs1801260,
with the C mutation being the risk allele. To our knowledge, this is the
first study to link adult ADHD to polymorphisms of a biological clock
gene. This finding confirms that the Clock gene represents a potential
candidate gene and susceptibility factor for disturbed circadian
rhythmicity and sleep disorders, both of which are frequently observed
in patients with ADHD.
41.02
Characterising SH-SY5Y cells as a model to study the molecular
effects of ethanol on the brain.
Hodder E J M, Rulten S L, King S L, Mayne L V
Trafford Centre for Medical Research, University of Sussex, Falmer,
Brighton, UK., ,
Neuronal cell culture provides a powerful system to study the molecular
actions of drugs of abuse in the brain. This study aimed to characterize and
exploit SH-SY5Y cells as an in vitro model to explore the effects of ethanol
on the NMDA receptor and genomic integrity. SH-SY5Y cells are derived
from a human neuroblastoma cell line and can be differentiated to a mature
neuronal phenotype in a novel modified method involving growth in low
serum and addition of retinoic acid. Behavioural changes resulting from
ethanol intoxication have been linked to changes in NMDA receptor
function and expression in the brain. Here we show that SH-SY5Y cells
express functional NMDA receptors with NR1, NR2B, NR2C and NR2D
subunit expression measured by qualitative real time PCR. We have
examined expression patterns of specific subtypes of the NR1 subunit in
response to ethanol exposure, using primers designed to splice-variants of
the C-terminal region and examined whether ethanol exposure has an
effect on the expression of any one splice variant. We have also explored
whether ethanol toxicity was associated with DNA strand breaks in SH-
SY5Y cells. Using the alkaline comet assay, we demonstrated that ethanol
itself caused negligible DNA damage. However, acetaldehyde, the
metabolic by-product of ethanol, caused significant dose and time
dependent DNA damage in SH-SY5Y cells consistent with DNA damage
arising through the metabolic break down of ethanol and the actions of
acetaldehyde.
41.03
Cyclin D1 protein downregulation in high grade brain tumors
Farizan A 1, Abdullah J 1, Jaafar H 2, Asmarina K 1, Aini I 3, Manaf A
3, Rahman A O 3, Khatijah Y 3, Mohd Azmi M L 3, Fauziah O 3
1Department of Neurosciences, 2Department of Pathology, School of
Medical Sciences, Universiti Sains Malaysia, Kubang Kerian,
Kelantan, Malaysia. , 3Universiti Putra Malaysia, Serdang, Selangor,
Malaysia.
Overexpression of cyclin D1 has been observed in various
malignancies including breast cancer, colorectal cancer, parathyroid
adenoma and prostate cancer. However, there is no systematic
immunohistochemical study of the protein expression in brain tumors.
This study was thus performed to better understand the expression
pattern of cyclin D1 in brain tumors, particularly in Malaysian patients.
For the purposes, 24 meningiomas and 23 gliomas samples were
collected. All samples were analyzed by immunohistochemistry
analysis to determine cyclin D1 protein expression. Our results
revealed equal expression of cyclin D1 in low grades of gliomas, and
the expression decreased in higher grades of gliomas with 76.9%
(10/13) were low expressers (

42.02
In vivo dopamine release in adenosine A2A receptor knockout
mice during acute cocaine administration
Wells L A 1, Opacka-Juffry J 2, Hourani S M O 1, Kitchen I 1
1-SBMS, University of Surrey, Guildford, UK., 2-SHLS, Roehampton
University, London, Uk.
Adenosine A2A receptor knockout mice show an altered addiction
phenotype with respect to psychostimulants. A significant reduction in
self-administration of cocaine in A2A receptor knockout mice has been
reported. To determine whether this is associated with altered
accumbal dopamine (DA), we have studied the effects of acute
cocaine administration on extracellular DA in the nucleus accumbens
(NAc), using in vivo microdialysis in A2A receptor knockout mice. In
addition, we have monitored locomotor behaviour and stereotypy
during the dialysis procedures.
A significant enhancement in locomotor activity was seen in cocainetreated
knockout mice compared to cocaine-treated wild-type mice (3-
way ANOVA p

43.02
Neuroleptic treatment worsens cognitive function and Alzheimertype
pathology in dementia patients
Rabai E, King E, Smith M Z, Nagy Z
Erzsebet Rabai, Zsuzsanna Nagy, Department of Clinical
Neuroscience, Neuroscience Division, The Medical School, University
of Birmingham, Vincent Drive, Birmingham, B15 2TT, , Elizabeth King,
Maria Z Smith, OPTIMA, Radcliffe Infirmary NHS Trust, Oxford OX2
6HE
Neuroleptics, often used in the management of psychotic symptoms in
Alzheimer’s disease patients might contribute to cognitive
deterioration.
In this study, we analysed the effect of neuroleptic treatment on
memory functions and the expression of AD-related proteins in 86 AD
patients. The cognitive status of the patients has been assessed not
more than 6 months prior to death. After necropsy, diagnoses were
confirmed using the CERAD protocol and the Braak staging.
Quantitative immunohistochemistry was used to measure
hyperphosphorylated tau, PHF tau, and beta-amyloid in the
hippocampus.
The cognitive scores of neuroleptic treated patients in the early stages
of AD were significantly lower than those of patients who received no
neuroleptic treatment. The recent memory and learning memory
scores show a similar effect. The remote memory scores are not
significantly different between the two groups.
The treatment had no significant effect on the expression of
hyperphosphorylated tau in any region of the hippocampus, except
CA4. In contrast, the expression of PHF tau in the CA2/3, CA4 and the
dentate gyrus is significantly higher in the neuroleptic treated patients
than in the matched controls. There was no difference in beta-amyloid
deposition.
We conclude that the worsening of memory functions induced by
neuroleptic therapy in demented patients is associated with increased
amounts of AD-type pathology in the hippocampus.
43.03
The effect of a fibroblast growth factor receptor agonist on the betaamyloid(25-35)
treated hippocampus.
Corbett N J, Gabbott P L, Stewart M G, Klementiev B, Davies H A, Colyer F
M, Novikova T, Berezin V, Bock E
The Open University,, Department of Biology,, Milton Keynes,, MK7 6AA
The accumulation of β-amyloid (Aβ) is thought to underlie the characteristic
neuropathology of Alzheimer’s disease (AD). Active glycogen synthase
kinase-3β (GSK-3β) phosphorylates amyloid precursor protein (APP)
causing Aβ accumulation. Fibroblast Growth Loop (FGL) acts as a
fibroblast growth factor receptor 1 (FGFR1) agonist that inactivates GSK-
3β. To demonstrate whether FGL reduces Aβ-induced hippocampal
neurodegeneration, a fragment of Aβ (25-35) was injected
intracerebroventricularly (icv) at 5mg/15μl into rats (n=12). Eight days after
Aβ injection, animals received either a subcutaneous injection of FGL
(10.8mg/kg) or distilled water (dw) every 3 days (Aβ-FGL, Aβ–dw groups
respectively). Another group (dw-FGL: n=6) had dw injected icv followed by
FGL treatment whilst the control group (dw-dw: n=6) received dw icv
followed by dw treatment.
On day 25, animals were transcardially perfused with 2%
paraformaldehyde and 3.75% acrolein in 0.1M phosphate buffer (pH 7.4).
Serial coronal sections were prepared from the hippocampi of each animal.
Using the Cavalieri method applied to a 1:4 series of 50μm Nissl sections,
the total hippocampal volumes of 4 animals per group were determined.
Results indicated that dw-FGL animals had smaller hippocampi
(57.43±6.32mm3) than dw-dw controls (68.64±3.35mm3) and Aβ-dw
animals (66.45±2.27mm3), whilst the Aβ-FGL group (62.05±1.81mm3) was
not different to any group.
The results demonstrate the effect of Aβ and FGL on total hippocampal
volume. Ongoing studies aim to interpret these volume changes in terms of
quantitative alterations in the neuronal and synaptic circuitry of the
hippocampus in this model of AD.
43.04
Aggregation of beta amyloid (1-28) and ultrastructural effects
following intracerebral injection into the avian brain
Evans D S, Gabbott P L, Davies H A, Colyer F M, Stewart M G
Department of Biological Sciences, , The Open University, , Walton
Hall, , Milton Keynes, , MK7 6AA
Beta amyloid (Aβ) accumulation is implicated in the synaptic and
neuronal pathology underlying the cognitive deficits found in
Alzheimer’s disease. Self-aggregation of Aβ leads to the formation of
plaques in the brains of affected individuals. By using fluorescence
microscopy, scanning and transmission electron microscopy we have
investigated: (i) the temporal aggregation profile of Aβ fragment 1-28
tagged with Hi-Lyte Fluor 555 (Aβfluor) over a period of 7 days in vitro,
and (ii) the ultrastructural effects of Aβfluor injected intracerebrally into
the avian brain.
Optimal aggregation of Aβfluor (assessed by the presence of fibres
and smaller aggregated structures) occurred after 3 days.
Intracerebral injections of Aβfluor were made into the intermediate
medial mesopallium (IMM) of day old chicks (n=4). Following a 48
hour survival period, the chicks were either transcardially perfused
with aldehyde fixatives for ultrastructural observation or decapitated
and the brains rapidly excised and frozen in isopentane, and serial
tissue sections (50µm thick) cut through each injection site. The lateral
diffusion of Aβfluor from the injection site was defined quantitatively at
7 injection sites. The mean maximum lateral diffusion distance from
the centre of the injection sites was 113µm (±40.6 SD) with a range of
53-197µm. Compared with double distilled water injected controls,
qualitative ultrastructural observations immediately adjacent to the
Aβfluor IMM injection sites revealed extensive apoptosis of neuronal
and glial cell types and widespread synaptic and dendritic dystrophy.
Current studies are investigating the effect of Aβ injection on synapse
density in chick brain regions involved in cognitive behaviours.
43.05
Deprivatives of rer peptide as potential theraputic agents in
Alzheimer's disease
Lancashire C L, Mileusnic R, Rose S R
The Open University, , Milton Keynes, MK7 6AA, UK
The discovery of an effective agent to alleviate the cognitive and memory
deficits in Alzheimer’s disease (AD) is a high priority. As the amyloidogenic
processing of APP is crucial for the development of AD, focussing on APP
and endeavouring to find ways of restoring its function is a rational
approach for development of new neuroprotective strategies. Thus,
peptidomimetics derived from the amyloid precursor protein (APP) might
become of considerable interest as potential therapeutic agent in the early
stages of Alzheimer’s disease.
We reported that in animals rendered amnestic by Aβ memory was rescued
by the tripeptide Arg-Glu-Arg (RER), part of the growth-promoting domain
of APP. A protected form of RER, when injected peripherally, is rapidly
transported across the blood-brain-barrier, and protects against memory
loss induced by Aβ1-42 and Aβ25-32.
In efforts both to cast light on the mode of action of the peptide, and to
increase its efficacy as a potential cognitive enhancer, we have now
studied the effects of various D/L-forms of the peptide. Here we report that
rER (where the lower case indicates the D-isomeric form of the amino
acid), protects against Aβ induced memory loss when injected peripherally
up to 12hr prior to the training task, and thus becomes of considerable
interest as the basis for a potential therapeutic agent in the early stages of
Alzheimer’s Disease.
Conflict of Interest disclosure: RM and SPRR are named as inventors of the
tripeptides described (UK Patent Number GB2391548 owned by The Open
University). Research was partially funded by Talisker.
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43.06
Proteomics of axonal degeneration in a model of optic nerve
transection
Broom L J, Skipp P J, Perry V H
CNSIG, University of Southampton, Southampton , UK
Axonal injury is integral to the pathology of neurodegenerative
diseases such as multiple sclerosis, ALS and toxic neuropathy. Injury
triggers degeneration of the distal axon by an active process termed
`Wallerian degeneration`. The mechanisms underlying this
degenerative process are largely unknown so we are using a
proteomic approach to identify the pathways involved.
To model axonal degeneration a unilateral optic nerve crush was
performed on adult Wistar rats and the optic nerves collected 48 hour
post-injury. The uninjured nerve provides a suitable control. In order to
focus on axon-specific events we have optimised a novel protocol for
extraction of soluble axoplasmic proteins, which minimises sample
contamination by proteins from surrounding glial cell populations.
Western blotting results confirm that the polymer-based absorption
technique is able to enrich for axonal proteins while reducing
contributions from oligodendrocytes, astrocytes and the myelin sheath.
Axoplasm was extracted from normal and degenerating nerves and
duplicate samples were labelled using iTRAQ reagents. LC-MS/MS
analysis was performed and quantitative data used to compare the
protein expression profiles that were obtained. Analysis of axoplasm
from normal and degenerating nerves has identified 286 proteins, of
which 32 were found to exhibit a statistically significant, greater than
two-fold change in abundance. Further investigation is ongoing to
verify these results by alternative methods including Western blotting.
In addition we are performing a time course study to investigate earlier
protein changes.
43.07
Early stage behavioural deficits and accompanying pathology in three
strains of prion disease
Hilton K, Reynolds R, Cunningham C, Perry V H
School of Biological Sciences, University of Southampton, UK., Department
of Biochemistry, Trinity College Institute of Neuroscience, Rep. of Ireland.
Transmissible Spongiform Encephalopathies (TSEs) are fatal
neurodegenerative diseases. TSEs represent a class of protein misfolding
diseases identified by the accumulation of aggregated prion protein. The
circuitry of neurodegeneration in prion diseases has not been well
characterised and this is particularly true at pre-clinical stages. Previous
work in this laboratory has established that mice infected with different
strains of prion disease show the same sequence of behavioural deficits,
despite widely divergent end stage pathologies. This indicates that there
may be common pathways of dysfunction or degeneration in these strains
and that certain populations of neurons could be particularly susceptible.
To investigate the pathology underlying the behavioural deficits, C57BL/6
mice were inoculated bilaterally into the hippocampus with brain
homogenate infected with the ME7, 79A or 22L strain and euthanized after
13 weeks. At this time point there were significant behavioural impairments
but no overt symptoms. Control animals were inoculated with normal brain
homogenate. Coronal sections of brain were processed
immunohistochemically with a marker of pre-synaptic terminals
(synaptophysin), microglia (IBA1), astrocytes (GFAP), the protease
resistance form of prion protein (6H4) and for apoptotic cells using TUNEL.
At 13 weeks prion infected mice, regardless of strain, have structures that
show increased numbers of astrocytes and microglia and have decreased
expression of synaptophysin. There are also strain specific differences in
the expression of these markers. The localisation and implications of these
changes to the observed behavioural deficits and the possible vulnerability
of specific neuronal populations will be discussed.
43.08
How does cellular polyglutamine expanded huntingtin
aggregation cause oxidative stress
Hands S L, Ockert W, Cuttle M, Wyttenbach A
Southampton Neuroscience Group, School of Biological Sciences,
University of Southampton, Bassett Crescent East, Southampton,
SO16 7PX
Oxidative damage is evident in Huntington’s Disease (HD), suggesting
that reactive oxygen species (ROS) play an important role during
disease. Previous work on HD cellular models has shown that mutant
huntingtin Exon 1 (httEx1) induces the production of ROS in a polyQ
length-dependent manner and that ROS and ensuing death can be
suppressed by antioxidants. However, the mechanism responsible for
production of ROS due to the presence of a polyQ expanded protein is
unclear, although it is thought that mitochondrial dysfunction is likely to
participate in the process, as energetic defects and mitochondrial
dysfunction have been associated with the pathophysiology of HD. We
have developed assays to monitor ROS production using 2’,7’-
dichlorofluorescein diacetate, dihydroethidine and Mitosox as
fluorescent probes in single living cells expressing either wild-type or
mutant httEx1. Intracellular ROS levels have been compared between
cells transfected with wild-type or mutant httEx1 and the relationship
between production of ROS, inclusion body formation and cell death
has been explored. We are using this approach to define the origin of
neuronal/non-neuronal ROS production and explore its relation to
polyQ-oligomerisation, inclusion body formation and cell death. We
are also using our cellular models to investigate changes in
expression, localisation and activity of antioxidant enzymes.
43.09
Proteomic analysis of neurodegeneration in the ME7 model of prion
disease
Asuni A A, Gray B C, Siskova Z, Skipp P, O`Connor V, Perry V H
University of Southampton, School of Biological Sciences
Prion disease is characterized by the accumulation of misfolded protein,
astrogliosis, neurodegeneration and neuronal loss. We have used the
hippocampal injection of brain homogenates harbouring a murine form of
scrapie (ME7) to initiate a staged in vivo model of prion disease
(Cunningham et al., EJN 17. 2147). We have performed an initial proteomic
comparison of hippocampal extracts from control and ME7 treated animals
exhibiting end stage pathology (21 weeks post inoculation). We have used
isobaric Tags for Relative and Absolute Quantification (iTRAQ) and mass
sprectrometry to profile and quantify differences in the protein expression of
100 ƒÝg extracts from control and treated animals. This analysis produced
robust signals encompassing 1-9 peptides from >200 individual
hippocampal proteins. We used a 2 fold change as a cut off to score
changes in protein expression; identifying 3 proteins as down regulated and
8 proteins as up-regulated in ME7 animals. Three of the up-regulated
proteins (GFAP, high affinity glutamate transporter and peroxiredoxin) are
components of astrocytes, reinforcing previous observations that the
astrocyte proteome contributes significantly to total hippocampal extracts
across the ME7 model. Quantitative western blotting of proteins identified
as differentially regulated will independently verify changes and help clarify
if increases reflect proliferation of astrocytes and/or a selective activation of
biochemical pathways in these cells during prion disease. Additional
candidates identified, in this initial single time point analysis, function in cell
types other than astrocytes and their investigation may provide further
insight into the complex biology associated with chronic neurodegeneration.
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43.10
Biochemical estimates of SNARE complexes in an in vivo model
of prion disease.
Asuni A A, Cunningham C, Siskova Z, Vigneswaran P, Perry V H,
O`Connor V
University of Southampton, School of Biological Sciences.
+Biochemical Sciences Undergraduate Program.
Chronic neurodegeneration associated with protein misfolding
underlies many brain diseases including Alzheimer’s and prion
disease. We use the hippocampal injection of brain homogenates
harbouring a murine form of scrapie (ME7) to initiate a staged in vivo
model of prion disease. The accumulation of misfolded prion protein
during disease progression correlates with a synaptic dysfunction
(Cunningham et al.,2003). Previous studies using cell culture models
show the accumulation of the misfolded prion, associated with
disease, correlates with a decreased ability of three proteins,
synaptobrevin, syntaxin and SNAP-25 to form the biochemically
defined SNARE complex (Sandberg and Low 2005). As the formation
of the SNARE complex is an essential intermediate in releasing
neurotransmitter its inhibition might contribute to the synaptic
dysfunction seen in the ME7 model. SNARE complexes in
hippocampal tissue resist disassociation by the denaturing detergent
SDS when resolved by PAGE. Accordingly, the relative levels of
monomeric and complexed syntaxin were scored by western blotting
to measure SNARE complexes during the ontogeny disease. This
analysis showed no change in the relative levels of SNARE complexes
extracted from control animals or those inoculated for 12 and 21
weeks (stages associated with synaptic dysfunction). We are currently
estimating SNARE complexes from the same animals in which
hippocampi have been pre-extracted in the non-denaturing detergent
Triton-X 100, a procedure shown to promote SNARE complexes exvivo.
This should clarify how relevant the disappearance of SNARE
complexes, as reported in culture, is for the synaptic dysfunction that
we observe in an in vivo model of prion disease.
43.11
Systemic inflammation superimposed on chronic neurodegeneration
induces acute behavioural and cognitive changes and accelerates
neurological decline.
Cunningham C, Campion S, Lunnon K, Deacon R M J, Rawlins J N P,
Perry V H
Department of Biochemistry, Trinity College Institute of Neuroscience,
Trinity College Dublin, Ireland, *CNS Inflammation Group, School of
Biological Sciences, University of Southampton, SO16 7PX, UK, +
Department of Experimental Psychology, South Parks Road, University of
Oxford, UK
There is evidence that infections have more severe behavioural
consequences in aged and demented patients than in normals, and can
induce episodes of delirium. Chronic neurodegeneration results in
microglial activation and this may contribute to the progress of
neurodegeneration. We have previously shown that microglia activated by
prion disease-associated neurodegeneration show very low cytokine
synthesis, but are primed to produce a more pro-inflammatory cytokine
profile upon further activation by systemic challenge with bacterial
endotoxin (lipopolysaccharide, LPS). In the current study we investigated
whether systemic challenge with LPS in the early stages of prion disease
can a) produce exaggerated behavioural responses in diseased animals
compared with controls and b) whether this LPS challenge can accelerate
the progression of disease. Intra-peritoneal injection of prion-diseased and
normal animals with LPS (100µg/Kg) at 12 weeks post-inoculation, resulted
in exaggerated impairments in burrowing and locomotor activity, and bodytemperature
and cognitive changes in prion-diseased animals that were not
present in normal animals challenged with LPS. At 14-15 weeks LPS
(500µg/Kg) challenges produced altered motor coordination and muscle
strength in prion-diseased but not in normal animals. These animals also
showed poorer subsequent performance on motor coordination and muscle
strength tasks affected by the disease. These data demonstrate that a
single systemic inflammatory event in animals with prior neurodegenerative
disease is sufficient to produce exaggerated sickness behaviour, acute
exacerbations of cognitive and motor symptoms of disease and to induce
lasting neurological deficits. These data have consequences for the
treatment of patients with dementia and suffering episodes of delirium.
43.12
A novel phospholipid based drug formulation inhibits amyloid-ß
induced granzyme B, caspase-3 and sphingomyelinase activation
in rat cortex
Miller A-M (1), Piazza A (1), Walsh M (1, 2), Martin D S D (1, 2),
Mandel A (3), Bolton A. (2), Lynch M.A. (1)
1 Department of Physiology, Trinity Institute of Neuroscience, Dublin,
Ireland, 2 Vasogen Ireland Limited., Dublin, Ireland, 3 Vasogen Inc.,
Toronto, ON, Canada.
Background: VP025 (Vasogen Inc.), a drug based on synthetic lipid
technology; is composed of bilayered phospholipid particles containing
phosphatidylglycerol. It has been shown to abrogate
lipopolysaccharide- and age-induced up-regulation of IL-1ß and IL-1ß
-induced signalling (Martin et al., 2004). Methods: We assessed the
effect of VP025 on changes induced in the rat cortex by chronic
administration of amyloid-ß. Forty eight young male Wistar rats were
injected intramuscularly with either 150µl of saline or VP025 (1.2 x 107
particles/ml) 14, 13 and 1 days prior to surgery. On the day of surgery
these animals were anaesthetized (ketamine 75 mg/kg, xylazine 10
mg/kg) and implanted with Alzet minipumps (Model 2004) to allow
chronic intracerebroventricular delivery of amyloid-ß 1-40 (26.9µM)
and 1-42 (36.9µM) or control peptide, amyloid-ß 40-1 (63.8µM). One
group of 24 rats received amyloid-ß for 8 days and the second for 28
days prior to sacrifice when cortex and hippocampus were taken for
subsequent analysis. Results: Granzyme B, caspase-3 and
sphingomyelinase activation were significantly increased in cortical
tissue prepared from saline-treated rats that received amyloid-ß 1-40 /
1-42 for 8 and 28 days when compared with tissue prepared from
saline-treated rats that received the control peptide amyloid-ß 42-1.
Granzyme B, caspase-3 and sphingomyelinase activity were
significantly reduced in cortical tissue prepared from VP025-treated
rats that received amyloid-ß 1-40 / 1-42 for 8 and 28 days. Conclusion:
The findings support earlier observations which demonstrate an antiinflammatory
and neuroprotective effect of VP025.
43.13
The role of c-Jun in amyotrophic lateral sclerosis (ALS) Cu/Zn
superoxide dismutase 1 SODG93A transgenic mice.
Makwana M (1), Gilchrist A (1), Da Costa C (3), Behrens A (3), Raivich
G(1,2)
1. Centre for Perinatal Brain Protection and Repair, Department of
Obstetrics & Gynaecology, University College London, London, UK, 2.
Department of Anatomy, University College London, London UK, 3.
Mammalian Genetics Laboratory, Cancer Research UK, London Research
Institute, UK ,
Mutations in the ubiquitously expressed SOD gene are a cause of familial
ALS. Transgenic mice carrying SODG93A mutations develop an adult
onset form of lethal motorneuron disease, implicating programmed cell
death. c-Jun is involved in neuronal survival and regeneration and its
presence at increased levels in the surviving spinal motoneurons of ALS
patients suggests it may play a role in neurodegenerative disease. To study
the role of c-jun in ALS, we crossed conditional mouse mutants lacking c-
jun in the CNS (c-jun-deltaN mice) with transgenic SODG93A mice.
SODG93A mice lacking c-jun survived significantly longer (115±3days),
compared with SODG93A mice with c-jun (104+/-4, p

44.01
Functional-pharmacological MRI in drug discovery
James M F, Pohlmann A, Barjat H, Tilling L, Upton N, Schwarz AJ,
Bifone A
Neurology & GI and Psychiatry CEDDs, GlaxoSmithKline, Harlow UK,
Verona Italy
Pharmacological magnetic resonance imaging (phMRI) is used to
detect, most commonly, blood oxygenation level dependent (BOLD) or
blood volume (CBV) changes in the cerebral vasculature following the
administration of a brain-penetrating compound. The compound
evokes a metabolic response that is detected in repetitive images as a
change from the baseline condition that also differs from the response
to control vehicle. The technique builds on 2-deoxyglucose uptake
experiments that first localised drug effects in the conscious rat brain
(J McCulloch and colleagues). Three elements of the experiment are
crucial: 1) expert animal preparation to provide stable physiology – the
choice and dose of anaesthetic is very important; 2) sensitive MR
imaging to yield contrast changes without motion; 3) sufficiently
sensitive statistical techniques – with good understanding of their
functionality. From a drug discovery perspective, phMRI is potentially
valuable as an in vivo pharmacodynamic marker of central activity;
especially for agents whose effects otherwise require complex
behavioural assays. The BOLD-phMRI technique is also potentially
translatable to humans. However, despite its increasing use, the
question of how animal phMRI findings reflect activity in the human
brain remains relatively unexplored. In humans, the use of
pharmacological challenges with task-based functional MRI (fMRI) has
been preferred, particularly for cognition-enhancing agents. Brain
perfusion measurement with techniques such as arterial spin labelling
in conjunction with fMRI may provide insight into human phMRI
responses in future. This talk will use results from methodological
studies in GSK to highlight the problems and possibilities of phMRI for
drug discovery.
44.02
Probing serotonin and glutamate actions on the brain in vivo:
pharmacological challenge fMRI in animal models and human
volunteers
Williams S, McKie S, Stark J, Davies K, Luckman S, Bill Deakin
Imaging Science and Biomedical Engineering, 2Neuroscience and
Psychiatry Unit and 3Faculty of Life Sciences; Oxford Rd; University of
Manchester; Manchester M13 9PT
Functional magnetic resonance imaging (fMRI) is one of the most important
techniques used in cognitive neurosciences to relate brain structure to
function and to elucidate brain processing networks. Conventionally a task
(cognitive or motor) or sensory input (visual, auditory, somatic) is used to
stimulate regional brain activity, but in the last few years pharmacological
stimuli have also been used. We have used pharmacological-challenge
fMRI (pMRI) to investigate the direct effects on the brain of serotinergic and
glutamatergic drugs. In both rats and man, we have used the antidepressant
m-chlorophenylpiperazine (a 5HT2c agonist) as a challenge
and identified brain areas which respond to the drug. We showed that these
regions are related to post mortem receptor distribution in man and to c-Fos
expression in rats. Combined antagonist challenges were used to tease out
the receptor sub-types activated in the rat. Ketamine has been used as a
glutamatergic challenge and we have detected de-activations in the limbic
system which correlated with subjective ‘dissociative’ effects of ketamine,
as reported by the participants. Combining ketamine with pre-dosing by
lamotrigine, which blocks glutamate-activated Na+ channels, we were able
to distinguish brain areas where ketamine acts by inhibiting NMDA
receptors, and where it acts by increasing glutamate release leading to
stimulation of AMPA receptors. Most of the effects of ketamine were
antagonized by lamotrigine, suggesting that the drug acts mainly by
stimulating glutamate release.
44.03
Modulating neuronal and haemodynamic responses: coupling
and uncoupling
Sibson N
Head of Experimental Neuroimaging, Department of Physiology,
Anatomy & Genetics, Sherrington Building, Parks Rd, Oxford, OX1
3PT, , , Head of Experimental Neuroimaging, Department of
Physiology, Anatomy & Genetics, Sherrington Building, Parks Rd,
Oxford, OX1 3PT,
We have adapted a model described for somatotopically mapping the
hindpaw pathway from the cortex to the brainstem to enable direct
cortical stimulation (DCS) of the rodent brain within a high-field
magnetic resonance imaging (MRI) system. Unilateral electrical
stimulation of the rat hindpaw motor cortex yields BOLD (blood
oxygenation level dependent) and CBV (cerebral blood volume) fMRI
signal changes not only in the electrically stimulated motor cortex, but
also in the functionally connected homotypic contralateral motor
cortex, both the ipsilateral and contralateral secondary somatosensory
cortices and striatal areas in both hemispheres. Since activation is
observed in multiple brain regions with disparate neuronal
architecture, the DCS model offers a useful approach for investigating
regional differences in drug action. By combining the BOLD and CBV
fMRI approaches we can potentially obtain additional information on
metabolism, since the BOLD signal is a composite of both
haemodynamic and metabolic responses. We have used this
approach to determine the effect of a metabotropic glutamate type 5
receptor (mGluR5) antagonist, MPEP, on neuronal and
haemodynamic responses to stimulation. MPEP caused significant
reductions in both the BOLD and CBV fMRI responses to DCS across
all brain regions, although the magnitude of this effect varied between
cortical and striatal areas. Electrophysiological recordings
demonstrated no effect of MPEP on neuronal responses to DCS,
suggesting uncoupling of the haemodynamic response to neuronal
activation. We propose that this experimental approach provides a
means of dissecting the consequences of drugs on neuronal activity,
perfusion and metabolism across multiple brain regions
simultaneously.
44.04
Analysis of pMRI data with blind source separation
Schiessl I
Faculty of Life Sciences, Jacksons Mill, The University of Manchester,
Sackville St, Manchester M60 1QD,
Most Functional magnetic resonance imaging (fMRI) experiments involve
controlled delivery of stimuli (e.g. visual, auditory, somatosensory) or
require the subject to undertake specific cognitive tasks. More recently
fMRI methods have been used to monitor direct effects of neuroactive
substances on the brain, so-called pharmacological challenge fMRI (pMRI).
This approach brings the methodology into a more clinical domain with the
possibility to characterise effects of drugs, provide biomarkers of effect and
help to understand the neurochemical basis of diseases. However,
although these methods have considerable potential there is uncertainty as
to the most appropriate methods of analysis. The problem in the analysis of
pharmacological challenge pMRI arises from the fact that because of the
long time it takes to return to baseline often only one trial per session is
available for analysis. Standard hypothesis driven analysis methods of fMRI
data statistically test the time course of voxels against a model hypothesis.
An alternative approach is the exploratory analysis of data Independent
Component Analysis (ICA) to find common statistical features. We use an
ICA algorithm called Extended Spatial Decorrelation (ESD) to improve the
analysis of pMRI data. The ESD algorithm is based on the assumption that
(i) the original spatial prototype patterns are mutually uncorrelated but auto
correlated, and (ii) that correlations also vanish between sources that are
shifted by any non zero vector with respect to each other. Therefore the
ESD algorithm exploits the spatial structure within and between the original
sources that are underlying the recorded mixture to separate them.
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45.01
Mutant SOD1-PUMA knockout double mutants in relation to
amyotrophic lateral sclerosis
Kieran D, Koegel I, Villunger A, Strasser A, Prehn J H M
1Department of Physiology and Medical Physics and RCSI
Neuroscience Research Centre, Royal College of Surgeons in Ireland,
123 St. Stephen’s Green, Dublin 2, Ireland. 2 Division of
Developmental Immunology, Biocenter, Innsbruck Medical University,
A-6020 Innsbruck, Austria.
The accumulation of unfolded or misfolded proteins in the
endoplasmic reticulum activates evolutionary conserved stress
responses which if severe and persistent activate apoptosis.
Previously, we reported that activation of the BH3-only protein PUMA
is necessary and sufficient for ER-stress induced apoptosis, however
PUMA’s role in neurodegeneration in-vivo remained undetermined.
Defects in protein processing and transport are implicated in
Amyotrophic Lateral Sclerosis, a fatal neurodegenerative condition
characterized by motoneuron degeneration and paralysis. Here we
show activation of the ER-stress response and PUMA up-regulation in
SOD1G93A mice. We also show that genetic deletion of PUMA
protects cultured motoneurons against ER-stress, but not excitotoxininduced
apoptosis, and delays disease onset in SOD1G93A mice.
However, deletion of PUMA failed to prevent disease progression or
increase lifespan. Together, these findings show chronic
neurodegeneration associated with defects in protein quality control
are controlled in-vitro and in-vivo by PUMA, however in later stages of
disease other cell death mechanisms can compensate for a loss of
PUMA.
45.02
Semaphorin and Plexin mutants as models for disorders of
connectivity.
Rünker A E, Morris D W, O’Tuathaigh C M P, Dunleavy M, Henshall D,
Waddington, J.L, Gill, M, Corvin, A.P., Mitchell, K.J.
(1) Smurfit Institute of Genetics and (2) Neuropsychiatric Genetics
Research Group, Trinity College Dublin, Ireland, (3) Dept. of Neuroscience
and (4) Dept. of Clinical Pharmacology, Royal College of Surgeons in
Ireland
There is mounting anatomical, physiological and genetic evidence that
many psychiatric and neurological disorders have underlying defects in
neural connectivity. We have been investigating the functions of a
transmembrane semaphorin, Sema6A, and two interacting proteins, Plexin-
A2 and -A4, in cell migration and axon guidance in the developing mouse
brain. Sema6A mutants display a spectrum of defects in cell migration in
the cerebellum, hippocampus and piriform cortex and in axon guidance of
the anterior commissure, thalamocortical axons, fornix and corticospinal
tract. PlxnA2 and PlxnA4 mutants show overlapping subsets of defects,
and complex epistatic interactions with Sema6A.
Some of the anatomical regions affected have been implicated in the
pathology of schizophrenia (SZ) and we have found that Sema6A mutant
mice show a number of behavioural and physiological phenotypes thought
to model aspects of schizophrenia, several of which are completely
reversible by the antipsychotic clozapine. In addition, we have evidence of
an association of variants in the human SEMA6A gene with increased risk
of schizophrenia. These mutants may thus provide an excellent model to
investigate how neurodevelopmental defects can lead to altered function of
circuits and behaviour.
45.03
Dissecting the genetic contribution to psychosis: new evidence
from susceptibility gene mutants
O’TuathaighCMP, Babovic D, O’Sullivan GJ, O’Meara G, Croke DT,
Harvey R, Waddington JL
1Molecular & Cellular Therapeutics, Royal College of Surgeons in
Ireland, 123 St. Stephen’s Green, Dublin 2. 2 The Victor Chang
Cardiac Research Institute, St. Vincent’s Hospital, 384 Victoria St.,
Darlinghurst 2010, Australia
Neuregulin-1 (NRG1) has been identified as a candidate susceptibility
gene for schizophrenia. We have studied the functional role of the
NRG1 gene, as it relates to cognitive and social processes known to
be disrupted in schizophrenia, in mice with heterozygous deletion of
transmembrane (TM)-domain NRG1 in comparison with wildtypes
(WT). Social affiliative behaviour was assessed using the sociability
and preference for social novelty paradigm, while social dominance
and aggressive behaviour was examined in the resident-intruder task.
Spatial learning and memory was assessed using the Barnes maze
paradigm, while spatial working memory was measured using the
continuous variant of the spontaneous alternation task. We also
examined the effect of subchronic treatment with the NMDA receptor
antagonist phencyclidine (PCP) on the performance of NRG1 mutants
in a number of these behavioural paradigms.
45.04
An aneuploid mouse with a human chromosome modelling Down
syndrome.
Fisher E M C, Doherty A O, Ruf S, Mulligan C, Cooke S, Vanes L,
Hernandez D, Sharpe P T, Brandner S, Bliss T V P, Tybulewicz V*
Department of Neurodegenerative Disease, Institute of Neurology, Queen
Square, London WC1N 3BG: NIMR, London NW7
At least 5% of all human pregnancies are aneuploid, and ~1 in 700 children
are born with Down syndrome (DS) which results from having three copies
of human chromosome 21 (Hsa21). DS is the most common known genetic
cause of mental retardation and also results in increased susceptibility for
other disorders, including developmental deficits. It is a complex disorder
that involves multiple Hsa21 genes in interaction with the rest of the
genome. To gain insight into the biology of DS, we have generated a new
type of mouse model in which an almost complete human chromosome,
Hsa21, segregates through the germline. We present evidence that this
trans-species aneuploid mouse strain, ‘Tc1’, displays phenotypic alterations
in behaviour, synaptic plasticity, cerebellar neuronal number and heart
development that relate directly to human DS and to other partial trisomy
models of DS. Transchromosomic mouse lines such as Tc1 may represent
useful genetic tools to dissect other aneuploidies and complex human
genetic conditions.
Spatial learning and working memory processes appear intact in the
NRG1 mutant; any phenotypic changes in performance in these tasks
are proposed to be due to elevated baseline activity levels. However,
heterozygous deletion of TM-domain NRG1 was associated with
disruption to a number of elements of social functioning including
social dominance behaviour and response to social novelty.
Subchronic administration of PCP was found to significantly alter the
NRG1 mutant phenotype. These data inform at a novel phenotypic
level on the functional role of this gene in the context of its association
with risk for schizophrenia.
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46.01
Neurotransmitter regulation of the sleep-waking cycle
Haas H L
Institute of Neurophysiology, Heinrich-Heine-University, Düsseldorf,
Germany
Sleeping, waking, feeding and resting at the right time in the right
place is an advantage in Darwin’s sense. Our biological clock is
synchronized by light and creates signals for the daily rhythm which is
modulated by homoeostatic sleep pressure factors such as adenosine.
The ascending arousal system comprises cholinergic, serotonergic,
noradrenergic and dopaminergic cell groups in the brain stem as well
as histaminergic neurons located in the posterior hypothalamus
(tuberomamillary nucleus) and orexin/hypocretin - containing neurons
in the nearby perifornical area. The two latter major waking centers
project widely through the whole brain and are tonically active during
wakefulness but cease firing during sleep. Are the aminergic neurones
playing in a self-organizing orchestra or under a conductor like the
orexin nucleus They are all switched off through GABAergic inhibition
from the sleep active preoptic area. The orexin neurones integrate
circadian, metabolic and feeding signals and control the transitions
between slow wave sleep, REM sleep and waking. Degeneration of
these neurons results in a severe disorder of sleep architecture, called
narcolepsy with diurnal sleep attacks, inadequate transitions to a
REM-sleep like state, cataplexy, with loss of muscle tone, not
consciousness, out of the waking. Hypnagogig hallucinations, dreams
before the loss of consiousness occur at sleep onset. The orexin
neurones provide “flip-flop switches” (C. Saper) that prevent too
frequent oscillations between the sleep and waking states.
46.02
Mechanisms of general anaesthesia and the involvement of sleep
pathways
Franks N P
Biophysics Section, Blackett Laboratory, Imperial College of Science,
Technology and Medicine, South Kensington, London SW7 2AZ, U.K
Because the potencies of most anaesthetics can be accurately predicted by
lipid partitioning (the Meyer-Overton correlation), they have long been
considered to be archetypal “non-specific” drugs. However, this view has
now changed radically and it is recognised that even the simplest
anaesthetics (including the inert gas xenon) can be surprisingly selective in
their actions and exert their effects by binding directly to protein targets.
Identifying which protein targets are pharmacologically relevant, and which
are not, has been a major challenge, yet great progress has been made in
recent years. In this talk I will review the evidence on the nature and identity
of anaesthetic binding sites in the central nervous system and show that for
some commonly used agents, the relevant targets can be unambiguously
identified. The identification of the important anaesthetic targets has
facilitated investigations into the possible connections between the
mechanisms underlying general anaesthesia and those responsible for
natural sleep. Because the one behavioural feature that is common to all
general anaesthetics is their ability to induce a loss of consciousness that,
at least superficially, resembles natural non-REM sleep, it has long been
suspected that the neuronal pathways that are involved in NREM sleep
may also be relevant to the induction and maintenance of general
anaesthesia. Only recently, however, has evidence showing a causal link
been provided. I will describe experiments that show how certain key nuclei
in the brain, which are involved in the regulation of sleep, are also involved
in the sedative actions of general anaesthetics.
47.01
Synaptic function of the synaptic vesicle-associated CSP/Hsc70
chaperone
Zinsmaier K E
Arizona Research Laboratories Division of Neurobiology, Department
of Molecular and Cellular Biology, University of Arizona, Tucson, AZ
85721.
Synaptic terminals exhibit not only a remarkable speed and precision
as secretory machines but also an autonomy and durability that is
unusual. Not surprisingly, synaptic terminals contain special
mechanisms that protect them from detrimental effects of damaged,
aged or otherwise functionally impaired proteins. Accumulating
evidence suggests that molecular chaperones facilitate numerous
synaptic mechanisms and form a critical first line of defense against
diverse neurodegenerative diseases that might have a common cause
— the misfolding, aggregation and accumulation of toxic protein forms
in the brain. Genetic studies in flies and mice suggest that the synaptic
vesicle-associated cysteine string protein (CSP) is a key factor for the
maintenance of synaptic function. Specifically, our genetic studies
support four roles for CSP at synaptic terminals of Drosophila NMJs:
1) CSP protects synapses against use- and/or stress-induced damage
and prevents subsequent degeneration of nerve terminals. 2) CSP
facilitates a step close to SNARE-mediated synaptic vesicle fusion. 3)
CSP facilitates presynaptic Ca2+ homeostasis and may control Ca2+
channel activities. 4) CSP facilitates synaptic growth. The protective
role of CSP is at least in part based on its biochemical function as a
cofactor of the classical chaperone heat-shock cognate protein
(Hsc70). I will further discuss whether the cooperative synaptic action
of CSP and Hsc70 requires small glutamine-rich tetratricopeptide
repeat-containing protein (SGT).
47.02
Targeting cellular prion protein reverses early cognitive deficits and
neurophysiological dysfunction in prion-infected mice.
Mallucci GR 1, White MD 1, Farmer M 1, Dickinson A 1, Khatun H 2, Powell
AD 2, Brandner S 1, Jefferys JGR 2, Collinge J 1
1MRC Prion Unit and Department of Neurodegenerative Disease, Institute
of Neurology, Queen Square, London WC1N 3BG, UK
2Department of Neurophysiology, Division of Neuroscience, University of
Birmingham, Birmingham, B15 2TT.
Currently no treatment can prevent the cognitive and motor decline
associated with widespread neurodegeneration in prion disease. However,
we previously showed that targeting endogenous neuronal prion protein
(PrPC) (the precursor of its disease-associated isoform, PrPSc) in mice
with early prion infection, reversed spongiform change and prevented
clinical symptoms and neuronal loss. We now show that cognitive and
behavioral deficits and impaired neurophysiological function accompany
early hippocampal spongiform pathology. Remarkably, these behavioral
and synaptic impairments recover when neuronal PrPC is depleted, in
parallel with reversal of spongiosis. Thus early functional impairments
precede neuronal loss in prion disease and can be rescued. Further, they
occur before extensive PrPSc deposits accumulate and recover rapidly
after PrPC depletion, supporting the concept that they are caused by a
transient neurotoxic species, distinct from aggregated PrPSc. These data
suggest that early intervention in human prion disease may lead to
recovery of cognitive and behavioral symptoms.
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47.03
VAP proteins
Skehel P
Center for Neuroscience Research,, University of Edinburgh,,
Edinburgh., EH8 9JZ,
VAP proteins are intracellular membrane proteins enriched on the
cytoplasmic surface of the endoplasmic reticulum. They have been
shown to mediate interactions between the ER and both cytosolic and
cytoskeletal factors, and are implicated in membrane trafficking
events. The proteins contain three structural features: an N-terminal
domain homologous to the nematode Major Sperm Protein (MSP), a
central coiled coil region, and a hydrophobic C-terminal membrane
anchoring domain. Recently a mis-sense mutation in the vapb gene
was found to be linked to a familial form of motor neuron disease
classified as Amyotrophic Lateral Sclerosis type 8 (ALS8). The
mutation results in the substitution of a proline by a serine residue
within a highly conserved region of the MSP domain. When expressed
in cultured cells this structural change causes the protein to form small
intracellular aggregates, containing both the mutant and wild-type
proteins. Recent studies suggest that VAP proteins may perform both
structural and regulatory functions in the homeostatic regulation of
intracellular membrane systems, the mis-function of which may
underlie the degenerative disease ALS8.
47.04
Molecular chaperones as modulators of proteotoxicity in
neurodegenerative disease
Hartl F U
Max-Planck-Institute of Biochemistry, Department of Cellular Biochemistry,
Am Klopferspitz 18, D-82152 Martinsried, Germany;
The formation of insoluble protein aggregates in neurons is a hallmark of
neurodegenerative diseases including those caused by proteins with
expanded polyglutamine (polyQ) repeats (e.g., Huntington’s disease).
However, the mechanistic relationship between polyQ aggregation and its
toxic effects on neurons remains unclear. There are mainly two, nonexclusive,
hypotheses for how polyQ expansions may cause cellular
dysfunction. In one model neurotoxicity results from the ability of polyQexpanded
proteins to recruit other important cellular proteins with short
polyQ stretches into the aggregates. In the other model, aggregating polyQ
proteins cause a partial inhibition of the ubiquitin-proteasome system for
protein degradation. In general, protein misfolding and aggregation are
prevented by the machinery of molecular chaperones. Some chaperones,
such as the members of the Hsp70 family, also modulate polyQ
aggregation and suppress its toxicity. In a recent study, we could show that
the eukaryotic chaperonin TRiC acts synergistically with Hsp70 in this
process. Both chaperone systems appear to form an effective network
preventing the formation of aberrantly folded proteins with cellular toxicity.
Based on these findings, the chaperone pathways involved in de novo
protein folding and protein misfolding may be more similar than anticipated.
48.01
Drosophila as a genetic model for ethanol-induced
neurodegeneration
French R, Heberlein U
. University of California, San Francisco Department of Anatomy, Box
2822, 1550 4th Street, Rock Hall, Room 445, San Francisco, CA
94158.
It is well established that acute, or “binge” ethanol exposure causes
apoptosis of both adult and developing neurons. Further, it is clear that
the response of neurons to an ethanol insult is heavily influenced by
genetic background, but the mechanisms behind this effect are not
well understood. We have found that a single intoxicating exposure to
ethanol causes apoptosis of Drosophila olfactory receptor neurons
(ORNs), accompanied by blackening of the third antennal segment,
which is the primary Drosophila olfactory organ. In addition, we have
shown that shaggy, the Drosophila homolog of glycogen synthase
kinase 3ƒÒ (GSK-3ƒÒ), is required for ethanol-induced apoptosis.
GSK-3ƒÒ has previously been implicated in the mediation of cell
death under a wide variety of neurotoxic conditions, but its targets in
response to ethanol insult are not known. Finally, we have
demonstrated that the GSK-3 inhibitor lithium is protective against the
neurotoxic effects of ethanol, indicating the possibility for
pharmacological intervention in cases of alcohol-induced
neurodegeneration. The system we describe will allow us to
investigate the genetic and molecular basis of ethanol-induced
apoptosis in general, and specifically to identify targets of GSK-3ƒÒ in
programmed cell death.
48.02
Measuring motivation for moonshine in mutant mice; altered
responses to booze and drugs in mice with mutations of GABAA
receptor alpha subunits
Stephens D N
Psychology, University of Sussex, Brighton, BN1 9QG
A primary action of alcohol is facilitation of transmission at GABAA
receptors, pentameric structures, consisting of a,b, g subunits. Each
subunit occurs in several isoforms. We investigated the consequences of
deleting two a isoforms, a2 and a5, on behavioural effects of alcohol and
cocaine. a5 knockout mice consumed less ethanol at concentrations above
10%, but did not differ from wildtypes in performing an operant response to
obtain ethanol/sucrose mixtures. A novel benzodiazepine receptor ligand,
L-792782 (0.03 – 3 mg/kg) with inverse agonist activity selective for a5-
containing GABAA receptors, decreased lever-pressing rates for 10%
ethanol at doses giving rise to 90% receptor occupancy, but did not affect
lever-pressing for 4% sucrose. Furthermore, the non-selective inverse
agonist, R015-4513, which reduced lever-pressing for ethanol/sucrose in
wildtype mice, had less effect in a5 knockouts; lever-pressing for sucrose
was unaffected. Thus, although a5 subunits are not essential to signaling
ethanol reward, inverse agonists acting at a5-containing receptors can
reduce ethanol self-administration. Genetic variants of the GABAA receptor
a2 subunit gene (GABRA2) have been associated with human alcohol
dependence. At ethanol concentrations above 10%, a2 knockout mice
consumed more alcohol, and showed a greater preference for alcohol than
wildtype mice. However, there were no effects of the deletion on rates of
lever pressing (motivation) to obtain ethanol. Thus, although both a2 and
a5-containing GABAA receptors influence measures relevant to alcohol
abuse, their precise role in mediating alcohol reward remains unclear.
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48.03
Alcohol dependence; what happens when drinking stops
Little H
Alcohol dependence continues for months or even years after an
alcoholic manages to stop drinking, and the great majority of
alcoholics return to excess alcohol drinking. For the last few years, my
research group has been investigating changes in neuronal function
and behaviour that occur during the abstinence phase after withdrawal
from chronic alcohol consumption. We found that long term alcohol
intake and withdrawal caused a long lasting sensitisation to the effects
of other drugs of dependence, amphetamine, cocaine and nicotine.
We have also demonstrated changes in mesolimbic dopamine
transmission that last long after the end of the acute alcohol
withdrawal phase in rodents. Such neuronal changes may be involved
in the prolonged effects of chronic alcohol consumption that make the
dependence so difficult to treat. Changes were also found in the
concentrations of glucocorticoid in specific brain areas, particularly the
prefrontal cortex and hippocampus, even though the plasma
concentrations returned to control levels. In other brain areas, such as
the cerebellum, the corticosterone concentrations were not different
from controls. These alterations were seen for several weeks after
withdrawal from the chronic alcohol treatment. These glucocorticoid
changes may also be involved in the development of dependence or
other prolonged effects of alcohol.
49.01
Ubiquitin dependent downregulation of GABAA receptors
Arancibia-Carcamo I L (1), Michels G (2), Armstrong-Gold C(2), Lumb M J
(1), Moss S J (2), Kittler J T (3)
1. Pharmacology 3. Physiology, University College London. London. UK, 2.
Neuroscience. University of Pennsylvania. PA. USA
γ-amino-butyric acid type A (GABAA) receptors are the major sites of fast
synaptic transmission in the central nervous system and can be assembled
from 7 subunit classes: α1-6, β1-3, γ1-3, δ, ε, π, and θ. Although
expression of receptor α and β subunits can produce functional GABAgated
chloride channels the presence of the γ2 subunit within the GABAA
receptor complex has been shown to play a key role in clustering and
synaptic targeting. Here we investigate the molecular mechanisms for the
regulation of the endocytic sorting of GABAA receptors and their role in
synaptic inhibition. In the present study we show a role for the GABAA
receptor γ2 subunit in regulating the lysosomal targeting of internalized
GABAA receptors. Microscopy and biochemical experiments revealed that
the γ2 subunit of GABAA receptors confers an enhancement in the
targeting of GABAA receptors to a degradative pathway after internalization
from the plasma membrane. Using a chimeric system and site directed
mutagenesis together with antibody feeding and quantitative confocal
microscopy we have identified a specific lysosomal targeting amino acid
sequence within the γ2 subunit that can be regulated by ubiquitination.
Importantly, using a combination of electrophysiological, biochemical and
immunofluorescence techniques we were able to show how regulating the
endocytic sorting fate of GABAA receptors plays an important role in
modulating inhibitory synaptic strength.
49.02
SUMOylation regulates kainate receptor endocytosis and
synaptic transmission
Martin S, Nishimune A, Mellor J, Henley J M
MRC Centre for Synaptic Plasticity, Anatomy Department, University
Walk, University of Bristol, Bristol, BS8 1TD, UK.,
Small Ubiquitin-like MOdifier protein (SUMO) regulates transcriptional
activity and the translocation of proteins across the nuclear membrane
but little is known about the wider cellular role of protein SUMOylation.
We have found a completely new role for protein SUMOylation in the
regulation of KAR endocytosis that, in turn, modulates synaptic
transmission. GluR6 SUMOylation is necessary for agonist-dependent
internalisation but not for internalisation per se. We anticipate that, like
phosphorylation and ubiquitination, protein SUMOylation may have
complex, varied and crucial roles in determining the fate of modified
synaptic proteins and will have far reaching implications for the
understanding of synaptic function.
49.03
Pruning and loss of excitatory synapses by a postsynaptic ubiquitin
ligase
Ehlers M
Department of Neurobiology, Howard Hughes Medical Institute, Duke
University Medical Center, Durham, North Carolina, USA
E3 ubiquitin ligases mediates the transfer of ubiquitin onto diverse
substrate proteins, thereby tagging proteins for proteosomal degradation.
Here we identify a RING domain E3 ligase (REL) that resides near the
postsynaptic membrane of glutamatergic synapses and regulates synaptic
function. In hippocampal neurons, postsynaptic expression of REL
dampens excitatory synaptic transmission and causes a marked loss of
excitatory synapses. Conversely, expression of mutant forms of REL or
reduced expression of endogenous postsynaptic REL, profoundly
enhances synaptic efficacy, triggers a proliferation of glutamatergic
synapses, and increases vulnerability to synaptic excitotoxicity. By
regulating the number and strength of excitatory synapses, REL controls
the normal elaboration of synaptic circuitry. Further, increased excitatory
drive produced by disruption of REL function may contribute to neuronal
pathophysiology
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49.04
Regulation of synaptic function by ubiquitin-specific protease 14
Wilson S
Dept of Neurology, University of Alabama, Birmingham, Alabama USA
The ataxia mutation (axJ) is a recessive neurological mutation that
results in reduced growth, ataxia and hind-limb muscle wasting in
mice. The axJ gene encodes ubiquitin-specific protease 14 (Usp14), a
deubiquitinating enzyme (DUB) that associates with the proteasome.
Transgenic rescue of the axJ mice with neuronal-specific expression
of Usp14 demonstrated that the full-length form of Usp14 was
sufficient to restore viability and motor system function to the axJ
mice. Biochemical analysis showed that the ubiquitin-hydrolyase
activity of this form of Usp14 is dependent upon the presence of
proteasomes, and neuronal expression of full-length Usp14 was able
to restore the levels of monomeric ubiquitin in the brains of axJ mice.
However, axJ-rescued mice still displayed the Purkinje cell axonal
swellings that are seen in the axJ mice, indicating that this cerebellar
alteration is not the primary cause of the axJ movement disorders.
Examination of spinal cords from the axJ mice did reveal the presence
of pathological markers of motor neuron dysfunction. While the axJ
mice do not lose motor neurons, analysis of neuromuscular junction
demonstrated that the axJ mice have defects in vesicle recycling and
display increased synaptic rundown. These results show that the
motor defects observed in the axJ mice are due to a neuropathic
disease rather than a muscular disorder, and suggest that changes in
proteasomal function may contribute to neuromuscular junction
disease observed in the axJ mice.
50.01
Synaesthesia: a window into genetic and behavioural bases of
multisensory processing
Newell F
School of Psychology and Institute of Neuroscience, Trinity College Dublin
Synaesthesia is a rare condition where particular stimuli elicit additional
sensory experiences. Although there are many different subtypes of
synaesthesia we attempted to elucidate the processes common to these
various forms. First, we investigated the heritability of synaesthesia by
conducting a large-scale study of the nature of synaesthetic experiences
within familial settings. We recruited 53 synaesthetes and 42% of these
probands reported a first-degree relative with synaesthesia. Interestingly,
we found that different types of synaesthesia can occur within the same
family, suggesting that various types of synaesthesia are fundamentally
related at the genetic level. In a second series of studies we assessed the
multisensory nature of synaesthetic experiences. We found that colours
induced to letter stimuli are independent of the encoding modality in that
the visual, auditory and tactile versions of a letter all induced the same
colour. This finding suggested both that grapheme-colour synesthesia may
be mediated through vision and that synaesthesia is linked more to
perceptual decisions than sensory input. Consistent with these
suggestions, we found evidence that i) synaesthetes report more vivid
mental images than controls and, as revealed through the “McGurk effect”
ii), that synaesthetic experiences are driven by higher-level perceptual
output. Our data indicate that various forms of synaesthesia have a
common etiology and share basic processes of multisensory perception.
50.02
A Multisensory spin on self-motion perception
Smith S
School of Psychology, University College Dublin
Moving through one’s environment is a naturally multisensory task
involving a coordinated set of sensorimotor processes that encode
and compare information from visual, vestibular, proprioceptive,
motor-corollary, and cognitive inputs. Interaction between visual and
vestibular information in the perception of self-motion has been
reported in the literature for over 50 years [e.g. Battersby et at, 1956].
The importance of visual inputs for estimation of self-motion direction
(heading) was first recognised by Gibson (1950) who postulated that
heading could be recovered by locating the focus of expansion (FOE)
of the radially expanding optic flow field coincident with forward
translation. We have recently shown [Stone, Smith and Bush, 2004]
that humans with intact vestibular function can estimate their direction
of linear translation using vestibular cues alone with as much certainty
as they do using visual cues. Here we report the results of an ongoing
investigation of self-motion estimation that addresses whether visual
and vestibular information can be combined in a statistically optimal
fashion. We discuss our results from the perspective that successful
execution of self-motion behaviour requires the computation of one’s
own spatial orientation relative to the environment.
A combined intracranial EEG, MEG and fMRI look at audiovisual
speech integration.
50.03
The Case for Feedforward Multisensory Convergence during Early
Cortical Processing
Foxe J
Institute of Neuroscience, Trinity College Dublin; The Cognitive
Neurophysiology Laboratory, Nathan S. Kline Institute for Psychiatric
Research. 140 Old Orangeburg Road, Orangeburg, New York, USA;
The prevailing hierarchical model of sensory processing in the brain holds
that different modalities of sensory information emanating from a single
object are analyzed extensively during passage through their respective
unisensory processing streams before they are combined in higher-order
“multisensory” regions of cortex. Because of this view, multisensory
interactions that have been found at early, putatively “unisensory” cortical
processing stages during hemodynamic imaging studies have been
assumed to reflect feedback modulations that occur subsequent to
multisensory processing in the higher-order multisensory areas. In this talk,
I will consider findings that challenge an exclusively feedback interpretation
of early multisensory integration effects. First, high-density electrical
mapping studies in humans have shown that multisensory convergence
and integration effects can occur so early in the timecourse of sensory
processing, that purely feedback mediation becomes extremely unlikely.
Second, direct neural recordings in monkeys show that in some cases,
convergent inputs at early cortical stages have physiological profiles
characteristic of feedforward, rather than feedback inputs. Third, damage to
higher order integrative regions in humans often spares the ability to
integrate across sensory modalities. Finally, recent anatomic tracer studies
have reported direct anatomical connections between primary visual and
auditory cortex. These findings make it clear that multisensory convergence
at early stages of cortical processing results from feedforward, as well as
feedback and lateral connections, thus using the full range of anatomical
connections available in brain circuitry.
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50.04
A combined intracranial EEG, MEG and fMRI look at audiovisual
speech integration.
Calvert G
School of Psychology, University of Bath
Combining information from the different senses can dramatically
improve the detection and discrimination of external stimuli and speed
responsiveness. Given the ubiquitous nature of crossmodal
processing for human experience, knowledge of the underlying
neurophysiology seems vital for a complete understanding of human
sensory perception. Modern human brain imaging techniques now
provide a means of characterising the neural bases of these
intersensory interactions. In our laboratory, we have used a
combination of imaging techniques including functional magnetic
resonance imaging (fMRI), magnetoencephalography (MEG) and
electrocorticography (EcoG) to explore these phenomena. By
combining the information obtainable from these different
methodologies, in some instances in the same subjects and using the
same paradigms, we are beginning to elucidate many of the brain
areas involved in multisensory integration and the time course of
information flow through these emerging networks.
51.01
Alzheimer’s disease is a world-wide problem:the burden and current standards
of care
Mohs R
Eli Lilly, Indianapolis, USA
ABSTRACT NOT RECEIVED
51.02
What is the significance of tau pathology in Alzheimer’s disease
Davies P
Department of Pathology, Albert Einstein College of Medicine, 1300
Morris Park Ave, Bronx, NY 10461.
Changes in the phosphorylation state and the conformation of tau
occur early in the development of Alzheimer’s disease. These
changes appear to be reliable markers of the neurodegenerative
process in this condition, and distinguish the process of Alzheimer’s
disease from other neurological disorders (eg: Parkinson’s disease,
Huntington’s, cerebrovascular disease). At the least, changes in tau
appear to be useful as sensors of the process of Alzheimer’s disease.
Two phosphorylations, at serine 202 and at threonine 231 appear to
occur very early in the course of the disease, prior to formation of
filamentous inclusions or tangle formation, and prior to obvious signs
of neuronal degeneration. It is tempting to speculate that these
phosphorylations drive conformational changes in tau, but there is
currently no clear evidence that this is the case. Conformational
changes, recognized by the Alz50/MC1 antibodies, appear to occur as
early as these two phosphorylations, prior to filament or tangle
formation. It is possible that the increasing severity of these changes
in tau cause cellular dysfunction and eventually neuronal death in
Alzheimer’s disease. However, it is becoming increasingly clear that in
at least some human tau mutation cases, and in some tau transgenic
mice neuronal death may occur without the accumulation of abnormal
tau. Neuronal dysfunction and death may thus occur by different
mechanisms in Alzheimer’s disease and tau mutation cases. A better
understanding of these mechanisms will be essential to the
development of effective treatments for both types of disease.
51.03
Drug discovery approaches for the treatment of Alzheimer’s disease.
Pangalos M N
Wyeth Discovery Research, Prnceton, NJ, USA
Discovering and developing novel therapeutics for diseases of the central
nervous system is one of the most challenging, high cost and high-risk
areas for the pharmaceutical industry. Neurodegenerative diseases are
perhaps at the pinnacle of this challenge due in part to the incredible
complexity of the central nervous system, and the relative lack of scientific
understanding of key pathological processes.
Many of the diseases in question remain devoid of effective drugs with no
current therapies able to impact or modify disease pathophysiology. Thus
the promise of therapies which can stop or reverse neurodegenerative
processes in the brain are clear in terms of positive impact to patients, care
givers as well as the socioeconomic state of healthcare systems. Present
treatment standards for Alzheimer’s disease (AD) are primarily founded on
the replenishment of the neurotransmitter acetylcholine and are poorly
effective at best. I will present pre-clinical data for a number of drug
discovery programs aimed at enhancing dysfunctional neurotransmitter
systems in the degenerating brains of AD patients, in addition to data on a
number of disease modifying approaches. The ultimate goal is to develop
an array of complimentary and effective therapies for the treatment of AD.
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51.04
The use of MRI for tracking disease progression and assessment
of novel therapies in Alzheimer’s disease
Scahill R I
Dementia Research Centre, Institute of Neurology, London, WC1N
3BG
Brain atrophy is a macroscopic effect of the pathological hallmarks of
Alzheimer’s disease (AD). Whilst definitive diagnosis of the disease
requires neuropathological confirmation, brain atrophy can be
visualised in vivo using MRI. Numerous studies have demonstrated
whole brain and regional atrophy in individuals with AD compared with
age-matched controls. However, due to the large inter-individual
variation in brain size, there remains considerable overlap between
the two groups, limiting the clinical utility of this technique in the
earliest stages of the disease.
Examining change over time within an individual has proved a
powerful technique for separating individuals with AD from controls, as
well as a means for tracking disease progression. Positional matching
of serial scans allows quantification of brain atrophy1, a measure
which has been used widely in the assessment of novel therapies.
Using this technique individuals immunised with the AN1792 vaccine
were shown to have greater rates of atrophy than placebo patients,
raising the possibility that there may have been clearance of the
amyloid plaques in those undergoing treatment2. Non-linear matching
techniques e.g. fluid registration can give more specific information
about atrophy location. Voxel-based statistical analysis of fluid
parameters has proved useful in tracking disease progression from the
presymptomatic stages through to established disease3. MRI is a
powerful tool for revealing the natural history of AD as well as
assessing the effect of potential therapies on this disease course.
52.01
Brain plasticity and antidepressant treatments: new cells, new
connections
Reid I
Department of Mental Health, Institute of Medical Sciences, University Of
Aberdeen, Foresterhill, Aberdeen AB25 2ZH
The mechanisms that underpin brain plasticity are many and varied. The
field has been a focus of research for decades now, and current
conceptualisations have their roots in learning and memory research.
Studies have turned more recently to psychiatric disease generally and to
mood disorder in particular, with widespread interest in the reciprocal
effects of stress and antidepressant treatments on neurogenesis. Attention
has been drawn to the delayed onset of action of antidepressant activity
and the argument advanced that this time-course mirrors the timing of
antidepressant induced increases in neurogenesis rates. However, the
delayed onset notion is increasingly challenged, and some antidepressant
treatments, most notably electroconvulsive therapy, have clearly more rapid
effects than others. More rapidly modified forms of plasticity than the
production of new neurones may need to be considered in developing
hypotheses about the aetiology of mood disorder. This presentation will
focus on the role of changes in synaptic connectivity as it relates to the
effects of stressors and antidepressant treatments. Links with neurotrophic
hypotheses of the pathophysiology of mood disorder will be emphasised.
52.02
Genetic background influences neurogenesis and other
associated phenotypes in the NK1-/- mouse
McCutcheon J E, Morcuende S, Gadd C A, Lightman S L, Hunt S P
Hippocampal neurogenesis has been implicated in the
pathophysiology of depression due to the observation that most
antidepressants will increase the number of proliferating cells. Indeed,
neurogenesis may also be necessary for the therapeutic efficacy of
antidepressant treatment. Current antidepressants exert their effects
by targeting the neurotransmitter systems that are believed to regulate
mood and emotion. Most commonly drugs which act on the
serotonergic system are prescribed, however, the modulation of other
monoamines, neuropeptides and glucocorticoids may also provide
valid methods of treating these diseases. One of the most promising
classes of compound to emerge recently have been the NK1 receptor
antagonists. The NK1 receptor is the preferred receptor for the
neuropeptide substance P and is found in many areas of the limbic
system. Work using antagonists to this receptor and mice engineered
to lack a functional receptor (NK1-/- mice) found antidepressant-like
effects accompanied by increased neurogenesis, although this did not
affect measures of learning and memory. Early clinical studies using
NK1 receptor antagonists were generally positive, however, in later
studies the drugs failed to produce any significant improvement above
placebo. We have continued work using NK1-/- mice in our laboratory
and have found that transferring the mutation onto different genetic
backgrounds greatly influences the antidepressant-like phenotype,
including the levels of neurogenesis. These differences may be related
to genetically-conferred variation in HPA axis activity across
individuals. This work may help to explain the failure of NK1 receptor
antagonists, as well as many other drugs, to succeed in clinical trials.
52.03
Adult cytogenesis is susceptible to chronic stress
Fuchs E, Czéh B
Clinical Neurobiology Laboratory , German Primate Center, Göttingen,
Germany
As shown in various mammalian species, stress is among the most potent
inhibitor of neurogenesis in the adult dentate gyrus. Parallel to these
findings, numerous studies demonstrated that in chronic stress paradigms
and various animal models of depression, the suppressed adult
neurogenesis can be reversed by different types of antidepressant
treatments including electroconvulsive stimulation. These findings resulted
in much work on the concept that altered neurogenesis may contribute to
the pathogenesis of depression and may play a significant role in, for
example, the cognitive symptoms occurring in this disorder. In addition to
neurogenesis, there is experimental evidence indicating that stress and
antidepressant treatment can induce changes in gliogenesis (glial
morphology and cell numbers). In a chronic social stress model in the tree
shrew, we recently reported that in subordinate individuals, the number of
GFAP-positive astrocytes was significantly reduced, but concomitant
treatment with fluoxetine could block this effect. Furthermore, both stress
and drug treatment altered the somal volume of the astrocytes. Besides
their “housekeeping” functions, astrocytes are regarded as dynamic
regulators of synaptic strength, synaptogenesis and neuronal production in
the adult dentate gyrus. Astrocytes also possess receptors for
neurotransmitters and steroid hormones that, similarly to receptors in
neurons, can trigger electrical and biochemical events in the cell.
Therefore, structural changes of astrocytes are likely to have an important
functional significance for neuron–glia and neuron–neuron communication
and are also of particular relevance for hippocampal volume changes
observed in depressed patients.
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52.04
Stress and adult neurogenesis
Glasper E
Department of Psychology, Program in Neuroscience, Princeton
University, Princeton NJ 08544
The hippocampus of adult mammals continues to add new granule
neurons throughout life. Adult neurogenesis in the hippocampus is
modulated by hormones and experience. Several studies indicate that
stress, during development or in adulthood, decreases cell
proliferation and immature neuron in the dentate gyrus of adult rodents
and primates. Some evidence links these effects to stress-induced
elevations in glucocorticoids. However, other studies suggest that the
motivational valence of the stressor is important for mediating stress
effects on neurogenesis – positive stressors like running and sexual
experience do not inhibit adult neurogenesis and indeed, can increase
the number of new neurons. The mechanisms which serve to buffer
the brain from high levels of glucocorticoids under conditions of
“positive stress” remain unexplored.
53.01
Subcortical loops through the basal ganglia.
Redgrave P
The architecture of cortico-basal ganglia-cortical connections is
characterised by parallel, largely segregated, closed-loop projections.
Evidence will be considered suggesting that such loops involving the
neocortex are neither novel nor the first evolutionary example of closedloop
architecture involving the basal ganglia. The specific proposal will be
that a phylogenetically older, closed-loop series of subcortical connections
exist between the basal ganglia and brainstem sensorimotor structures, a
good example of which is the midbrain superior colliculus. Insofar as this
organisation represents a general feature of brain architecture, cortical and
subcortical inputs to the basal ganglia may act independently, cooperatively,
or competitively to influence the mechanisms of action
selection.
53.02
Behavioural roles of the pedunculopontine tegmental nucleus
Winn P
School of Psychology, St Andrews University, St Andrews, Fife KY16
9JP
The pedunculopontine tegmental nucleus (PPTg), in the mesopontine
tegmentum, has a structure and pattern of connectivity consistent
across vertebrate species. It has intimate connections with the basal
ganglia, both ascending and descending. It is a target of outflow from
pallidum, subthalamic nucleus (STn) and substantia nigra reticulata,
while PPTg neurons innervate pallidum, STn and provide excitatory
input to dopamine containing neurons in substantia nigra compacta
and the ventral tegmental area. Growing recognition that the PPTg
can be considered as part of the basal ganglia family has been
accompanied by re-assessment of its behavioural functions. An older
literature emphasizes roles for the PPTg in locomotion and
behavioural state control, but recent research has played down the
importance of the PPTg in regard to these and instead emphasized
roles in other processes. We have conducted research over the last
several years using excitotoxins to lesion the whole PPTg. In rats,
bilateral lesions of the entire PPTg produce no deficits in locomotion,
sleep regulation (in non-deprived conditions), or "emotional"
behaviour. There are however, profound disturbances in tests of
learning, memory and attention, higher-order functions associated with
corticostriatal systems. These studies show that the PPTg has more
complex functions than previously suspected. Having established this,
the key tasks now are (i) to better characterize the behavioural deficits
– are they all related to a core dysfunction, perhaps in association
learning (ii) To define better the internal structure of the PPTg such
that specific functions of component parts (identified neurochemically
and hodologically) can be identified.
53.03
Physiological analyses of cholinergic and non-cholinergic neurons in
the PPN that innervate the basal ganglia
Mena-Segovia J, Sims H M, Magill P J, Bolam J P.
MRC Anatomical Neuropharmacology Unit, Mansfield Road, , Oxford OX1
3TH, United Kingdom,
Previous studies have shown that neurons of the pedunculopontine nucleus
(PPN) are highly interconnected with the basal ganglia. The PPN, once
considered a predominantly cholinergic structure, is now recognised as a
neurochemically heterogeneous nucleus, with populations of GABAergic
and glutamatergic neurons. Electrophysiological studies show a high
degree of heterogeneity among PPN neurons. To define the characteristics
of the neurons that innervate the basal ganglia, we recorded the activity of
single cells in vivo and then labelled them by the juxtacellular method to
define their location within the PPN and define their morphological and
neurochemical properties. Identified cholinergic neurons fired in association
with the cortical gamma oscillations during both slow wave activity, and the
activated state. Their axons projected to the basal ganglia (substantia nigra
pars compacta and reticulata, and subthalamic nucleus), and they also had
ascending and descending collaterals (towards the thalamus and colliculi,
and lower brainstem, respectively). In contrast, identified non-cholinergic
neurons showed more heterogeneous electrophysiological characteristics:
their firing was correlated with cortical activity or independent of it, in which
case the pattern of firing was tonic, bursty or nearly silent. Non-cholinergic
neurons also have differences in their branching pattern: they have a single
collateral projecting towards the basal ganglia, a single ascending collateral
projecting towards the inferior and superior colliculi, or two collaterals
projecting to basal ganglia and thalamus. The PPN thus contains
neurochemically distinct populations of neurons that have distinct
electrophysiological properties and distinct connections with the basal
ganglia.
Supported by the Parkinson’s Disease Society and MRC.
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53.04
The role of the pedunculopontine nucleus in Parkinson’s disease
Nandi D, Jenkinson E, Stein J F, Aziz T Z
Oxford Functional Neurosurgery
Gait freezing and poor balance are two of the most crippling and drug
resistant symptoms of advanced Parkinson’s disease (PD) and also of
other untreatable disorders such as MSA and PSP. The
pedunculopontine nucleus (PPN) has been implicated in these
symptoms.
The PPN is in the upper brain stem. The major inhibitory input is from
the medial pallidum and SN reticulata and bilateral output is to the SN
compacta, thalamus and spinal cord.
Stimulation of the PPN in the decerebrate rat, cat and dog induced
gait-like movements. In autopsy studies in PD, MSA, PSP and the
DYT-1 dystonic brain, the PPN is degenerate. Autoradiography of the
MPTP-parkinsonian primate shows excessive inhibition in the PPN.
Lesions of the PPN in the normal primate induced PD-type
bradykinesia which was persistent with bilateral lesions. In the MPTPprimate
model, microinjections of the GABA antagonist bicuculine into
the PPN reversed Parkinsonian akinesia implying that stimulation of
this region might have a therapeutic role in drug resistant PD [1]. Low
frequency (5-10Hz) stimulation of the PPN in the same model
reversed akinesia [2] independently of L-Dopa; moreover, L-Dopa and
stimulation effects were additive [3,4], implying separate pathways.
Clinical reports in PD patients by Mazzone [5] and other groups
(Bristol, Toronto, Brisbane) showed that PPN stimulation did have the
beneficial effects that were predicted by the primate studies.
This research demonstrates the validity of the MPTP-primate model of
Parkinsonism and the role of the PPN in akinesia. Long term effects
PPN stimulation need to be monitored.
54.0
Neurotrophins in development and diseases
Barde Y-A
Biozentrum, University of Basel Switzerland
The neurotrophin gene family comprises 4 members with structural and
functional similarities. While nerve growth factor was the founding member
of the family, it is brain-derived neurotrophic factor (BDNF) that is
expressed at the highest levels in all areas in the adult brain. These levels
increase substantially post-natally and mouse models have been generated
allowing the role of BDNF to be examined in the adult nervous system.
Elaborate animal models of complete BDNF deprivation compatible with
survival of adult animals are all the more desirable that in humans, gene
polymorphisms and inactivation have been identified that significantly
impact the behaviour of affected individuals.
Most of the trophic actions of BDNF can be explained by its ability to
activate the tyrosine kinase receptor TrkB. However, it has been clear for
some time that the neurotrophin signalling system also comprises another
pathway activated by the neurotrophin receptor p75. Its recruitment
explains many of the “regressive” events associated with the neurotrophins
and their receptors, including cell death in their most radical manifestation,
as well as the limitation of axonal elongation and branching of dendrites. As
the signalling events downstream of this receptor are still poorly
understood, we recently begun to use neurons derived from embryonic
stem cells to explore novel pathways activated by p75. We recently found
that p75 transcriptionally increases the levels of an endogenous lectin
which causes the degeneration of processes and that interfering with the
activity of this lectin prevents the degeneration of neurons and of axons.
55.0
In vivo voltammetry: real-time monitoring of brain chemistry
Lowry J P
Sensors Development & Neurochemistry Research Units, BioAnalytics
Laboratory, Department of Chemistry, National University of Ireland,
Maynooth, Co. Kildare, Ireland.
The importance of chemical signalling between cells in the functioning
of neural networks is highlighted empirically by the use of drugs in the
treatment of neurological disorders, such as Parkinson’s disease,
schizophrenia and depression, as well as by mind-altering substances
of abuse, all of which have specific chemical actions on brain neurons.
A growing number of methodologies are being developed, including
sampling (e.g. brain microdialysis), spectroscopic (e.g. fMRI) and
electrochemical, to study neurochemical dynamics in the living brain.
With the electrochemical approach, a microvoltammetric electrode is
implanted in a specific brain region to monitor local changes in the
concentration of substances in the extracellular fluid with sub-second
time resolution over extended periods. This allows investigations of
the functions of specific chemicals in neuronal signalling, drug actions,
and well defined behaviours.
The development of new technologies for long-term in-vivo
electrochemistry (LIVE) in the conscious brain is now possible
following major advances in the fabrication of sensing devices using
polymer-enzyme composites (PECs) synthesised in situ on the
electrode surface. We have already demonstrated the feasibility of
using classical microelectrodes to monitor brain ascorbate, oxygen
and blood flow, and PEC-based biosensors to monitor brain glucose in
vivo. New in-vivo sensors for glutamate, hydrogen peroxide, NO and
lactate, based on both classical and PEC designs are presently at
various stages of development. In this presentation these LIVE
devices will be described along with their application in novel studies
of brain energy metabolism and animal models of psychiatric disease.
56.01
G2Cdb: The genes to cognition database.
Croning M D R, Elliott P T, Marshall M C, McLaren P A, Grant S G N
Genes to Cognition Programme, The Wellcome Trust Sanger Institute,
Hinxton, Cambridge, CB10 1SA.
Neuroscience databases linking genes, physiology, anatomy and
behaviour across species will be valuable in a broad range of studies of the
nervous system. G2Cdb is such a neuroscience database focused on
synaptic proteins and diseases of the nervous system, specifically those
affecting cognition, aiming to present a global view of the role of synapses
in physiology and disease. It integrates human and mouse genomic
information with available experimental resources including: synapse
proteomics, human gene mutation information in CNS disease, brain gene
expression resources, knockout mouse behavioural experiments, and
electrophysiological studies of synaptic plasticity.
It captures the experimental data output of the Wellcome Trust-funded
Genes to Cognition Programme (G2C), our systematic curation of the
relevant neuroscience literature, and data submitted directly to G2Cdb by
investigators worldwide. Currently G2Cdb contains data on more than 1300
genes, including: components of the NMDA receptor complex; postsynaptic
density (PSD; Collins et al, 2006); knockout and transgenic mice (ca. 200
lines) and complementary studies of human gene sequence variation
(Grant et al, 2005).
The databasL, Brandon, JM, Anderson, CN, Blackstock, WP, Choudhary
JS and Grant SGN. J. Neurochem. (2006) 97 Suppl. 1:16-23.
Grant, SG, Marshall, MC, Page KL, Cumiskey MA and Armstrong JD. Hum.
Mol. Genet. (2005) 14 Spec. No. 2:R225-34.
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56.02
The acquisition and consolidation of recognition memory are
neurotrophin-dependent
Callaghan C K, Kelly A M
Department of Physiology and Trinity College Institute of
Neuroscience, Trinity College, Dublin 2, Ireland.
Acquisition and consolidation of newly-acquired information is
essential for formation of long-lasting memories, and both these
processes may share common signaling pathways. The neurotrophins
NGF, BDNF, NT3 and NT4 have diverse functions in the adult brain,
including learning and memory. Their biological effects are elicited by
binding to Trk receptors. In this study we investigated the roles of
neurotrophins in both acquisition and consolidation of memory by
using neutralising antibodies against each neurotrophin isoform.
An object recognition task was used to test acquisition and
consolidation of memory; rats were tested 10min and 24hr after
training to discriminate experimentally between acquisition and
consolidation respectively. Male Wistar rats (n=12) were implanted
with cannulae for administration of pharmacological agents to the third
ventricle. Rats were injected with antiNGF, antiBDNF, antiNT3,
antiNT4 or control serum (5ƒÝl), or the pan-Trk inhibitor
TyrphostinAG879 or vehicle control, one hour before training in the
task.
Rats treated with TyrphostinAG879 were unable to discriminate
between the novel and the familiar object at both 10min and 24hr
when compared with controls, suggesting Trk receptor activation is
required for both acquisition and consolidation. In contrast, rats treated
with anti-BDNF, anti-NGF and anti-NT 4 displayed comparable
learning to control rats at the 10min timepoint, but displayed learning
impairments 24hr later. Treatment with anti-NT3 had no effect on
learning at either timepoint.
Neurotrophins appear to play an important role in both acquisition and
consolidation of recognition memory. Further experiments are required
to assess the precise signaling mechanisms involved in such plastic
events.
56.03
The role of NMDA receptors in learning and memory
Belsham A A G, Darlison M G, Freeman F M
School of Biomedical and Natural Sciences, Nottingham Trent University,
Clifton Campus,, Nottingham, NG11 8NS ,
The N-methyl-D-aspartate receptor (NMDAR) is involved in the early stages
of long-term memory formation (LTMF). NMDAR stimulation causes Ca2+
influx, resulting in plasticity. NMDARs in the chick (Gallus domesticus)
forebrain predominantly comprise of NR1 and either NR2A or NR2B
subunits. NR2B containing receptors demonstrate higher Ca2+
conductance compared to those containing NR2A. Increased neuronal
activity causes NR2A receptor subtypes to be internalised and replaced
with those containing NR2B. The current study investigates this activitydependent
switch on a molecular level using radioactive in situ
hybridisation to quantify levels of mRNA encoding the NR1, NR2A and
NR2B genes in the chick forebrain at significant time points during LTMF.
Both the intermediate medial mesopallium (IMM), a sensory information
sorting centre, and the medial striatum (MSt), which is involved in the
suppression of pecking behaviour, show changes in mRNA levels encoding
the NMDAR subunits at time points when they become activated during
LTMF. These changes may reflect the activity dependent switch between
NR2A and NR2B subtypes. In addition, this exchange of receptor subtypes
is investigated using behavioural pharmacology. Ligands specific for NR2A
and NR2B receptor subtypes were injected into specific regions of the
forebrain at relevant time points pre- and post-training. The effects of these
subtype specific ligands on task recall and discrimination are presented
here.
56.04
Motor dysfunction, and impairments in short-term memory and
synaptic plasticity in a mouse model (Tc1) of Down syndrome
Morice E. (1), Cooke S.F. (1), Vanes L. (2), Fisher E. M. C. (3),
Tybulewicz V. (2), Bliss T. V. P. (1)
(1) Division of Neurophysiology, NIMR, The Ridgeway, Mill Hill,
London NW7 1AA UK; (2) Division of Immune Cell Biology, NIMR, The
Ridgeway, Mill Hill, London NW7 1AA UK; (3) Department of
Neurodegenerative Disease, Institute of Neurology, Queen Square,
London WC1N 3BG UK
Down syndrome (DS) is a genetic disorder arising from the presence
of a third copy of human chromosome 21 (Hsa21). Recently,
O’Doherty et al (2005) generated a trans-species aneuploid mouse
line (Tc1) that carries an almost complete Hsa21 chromosome. The
Tc1 mouse is the most complete animal model for DS currently
available. Tc1 mice show many features that relate to human DS,
including alterations in learning, synaptic plasticity, cerebellar neuronal
number, heart development, and mandible size. Because motor
deficits and mental retardation are among the most frequently
occurring features of DS, we have undertaken a more detailed
analysis of motor and cognitive functions, and hippocampal long-term
potentiation (LTP) of Tc1 mice. Our results reveal that, compared to
controls, Tc1 mice exhibit a higher novelty-driven spontaneous activity
and a major deficit in motor coordination in a cerebellum-dependent
learning task. In the Morris water maze, Tc1 mice display intact spatial
long-term memory in a probe trial 20 days after the last training
session. However, Tc1 mice are severely impaired in a working
memory version of the task. In addition, we demonstrate reduced LTP
but normal maintenance over days in the dentate gyrus of freely
moving Tc1 mice. Thus, the deficits in synaptic plasticity are paralleled
by corresponding deficits in memory: short-term plasticity and shortterm
memory are impaired, whereas long-term plasticity and long-term
memory are unaffected.
56.05
Post-training intrahippocampal infusion of nicotine-bucladesine
combination causes a synergistic enhancement effect on spatial
memory retention in rat
Gholizadeh S, Sharifzadeh M, Zamanian A R, Zarrindast M R, Roghani A
1.Dept. of Toxicology and Pharmacology, School of Pharmacy,
Pharmaceutical Sciences and Medicinal plants research Centers, Tehran
University of Medical Sciences, Tehran, Iran, 2.Department of
Pharmacology and Neuroscience, Texas Tech University Health Sciences
Center,Lubbock, TX, USA
We previously had shown that bilateral intrahippocampal infusion of 1 µg
nicotine (but not 0.5 µg dose) led to an improvement in spatial memory
retention in the Morris water maze task in male rats. We also reported that
a similar type of bilateral infusion of H89, a protein kinase AII (PKA II)
inhibitor, caused a deficit in spatial memory retention. In the present study,
we wished to test the hypothesis that intrahippocampal infusion of dibutyryl
cyclic AMP (DB-cAMP also called bucladesine), a membrane permeable
selective activator of PKA, into the CA1 region can cause an improvement
in spatial memory in this maze task. Indeed, bilateral infusion of 10 and 100
µM bucladesine (but not 1 and 5 µM doses) led to a significant reduction in
escape latency and travel distance (showing an improvement in spatial
memory) compared to the control. Also, bilateral infusion of 0.5 µg nicotine
or 1 µM bucladesine alone did not lead to an improvement in spatial
memory. However, such bilateral infusion of bucladesine at 1 and 5 µM
concentrations infused within minutes after 0.5 µg nicotine infusion
improved spatial memory retention. Taken together, our data suggest that
intrahippocampal bucladesine infusions improve spatial memory retention
in male rats and that bucladesine can interact synergistically with nicotine
to improve spatial memory.
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56.06
Dissecting exploratory behaviour: Cue-based searching
strategies during Morris water maze acquisition.
Harvey D, Commins S
Department of Psychology,, National University of Ireland,, Maynooth,,
Co. Kildare,, Ireland.
The Morris water maze (MWM) was described over 25 years ago as a
simple and effective paradigm for examining spatial learning and
memory in laboratory animals. Since then, many elaborate studies
spanning several areas of neuroscience have employed this
straightforward task, showing its wide versatility and ease of
adaptation to many protocols. However, despite innumerable findings
since its introduction, the precise intricacies on how rodents acquire
information regarding the hidden platforms location remain
ambiguous. Often, overarching navigational strategies are reported
when documenting rodent performance in the pool. These include
those termed egocentric (defining the relation of an object, goal, or
location relative to the subject) or allocentric (defining the relation of
an object, goal, or location relative to another location, where this
object is independent of the subject). Indeed previous research has
strongly publicised the use of allocentric processes in solving the
MWM; particularly through the use of visual distal cues in forming
spatial relations with the platforms location. The current study
demonstrates how animals interact with these external visual cues
during acquisition of the task. It is shown that the use of several
identified swimming strategies, strongly associated with the cues, in
combination with initial view-matching and egocentric guidance,
ultimately allows animals infer the hidden platforms’ location (via
allocentric extrapolation) with training. Furthermore, hippocampal
BDNF expression is also shown to closely correlate with this
acquisition, elucidating its role in learning.
56.07
Neural correlates of source memory in young and old humans are
revealed by a high-density erps array.
Scanlon P, Commins S, Roche R
National University of Ireland, Maynooth, Maynooth, Co. Kildare, Ireland.
Failures of source memory (the ability to recall the specific context in which
events took place) have been associated with cognitive decline in the
elderly. Source memory deficits appear to be more indicative of age-related
memory impairment than disruption of item memory. An Opposition
Procedure developed by Jacoby and colleagues (Jennings and Jacoby,
1997) tests this source memory capacity by drawing on repetition errors in
a word recall task in which novel words are presented repeatedly at 3
differing lags (0, 4 and 16 trials). This task is sensitive to source memory
dysfunction, with older adults being found to produce significantly more
errors in repetition than healthy younger adults. In this study we recorded
128-channel EEG from normal healthy participants (N=10) (age 20-30) and
healthy older adults (N=10) (aged 60-70) while they executed the
Opposition Task. Behavioural results showed that accuracy decreased
significantly (p

56.10
The use of NMDA antagonists as cognitive disrupters on first and
repeated exposure in the delayed (non) matching to position task
Childs K, Foss J, Smith J W
Eli Lilly, Windlesham, Surrey, UK
NMDA receptor hypofunction has been suggested to underlie aspects
of the symptomatology of schizophrenia and is associated with
disruptions in learning and memory mechanisms in animals. We
assessed the effects of first exposure and subsequent exposure of
animals to NMDA receptor antagonists in the delayed-(non)-matchingto-position
(D(N)MTP) task that measures working memory in rodents.
Male Lister Hooded rats were required to respond on a lever when
presented with a stimulus. A delay period of between 1 and 32
seconds then began. At the end of the delay, a head entry initiated the
choice phase when the rats were required to match (DMTP) or not
match (DNMTP) to the sample lever to earn a food pellet and a 5 s
inter-trial interval (ITI) began. If animals failed to make the appropriate
responses, the trial was recorded as an omission, the house-light was
switched off, and the ITI proceeded in darkness. Phencyclidine (1-5
mg/kg) or SDZ 220-581 (1-3 mg/kg) were each given SC in 5%
glucose, 30 minutes before the start of the session.
Both SDZ-220581 and phencyclidine produced delay-independent
deficits in the DMTP task on first exposure. However on repeated
exposure, the disruptive effects of the compounds diminished.
Furthermore when given using a Latin-square design with testing done
every 3-4 days, all effects of both SDZ 220-581 and PCP decreased
across time. The data indicate that acute challenge with NMDA
receptor antagonists is of limited use in the investigation of the
modulation of cognitive performance in these tasks.
56.11
A unique all-in-one behavioural test for assessing emotional
responses and cognitive performance in rodents:Mice strains, age
and sex differences
Michalikova S (1), Rensburg Rv (2), Chazot P L (2), Ennaceur A(1)
1.Sunderland Pharmacy School, Sunderland University, Tyne and Wear,
UK; 2. Centre for Integrative Neuroscience, Durham University, UK
The 3D maze is composed of eight flexible arms radiating from a central
platform. Each arm can be manipulated independently and presented at the
same, below or above the level of a central platform. Mice need to cross a
bridge to reach flat, raised or elevated arms. When exposed for the first
time to the maze, emotional responses are reflected by number of entries
to bridges and to arms. C57 and CD1 mice visit significantly more arms
than Balb/c and C3H mice. In C57 and CD1 the number of entries to
bridges decreases when the number of entries to arms increases which
means that every time they enter a bridge, C57 and CD1 would continue to
arms and this becomes obvious from third session. It takes a much longer
time to reach this stage for Balb/c and C3H. The behaviour of C57 and
Balb/c is confirmed when compared to that of CBA and DBA mice. C57 and
DBA mice appear less anxious than Balb/c and CBA mice. All these strains
were able to perform a working memory task in the 3D maze with Balb/c
performing better than C57 mice after their emotional responses subsided.
Significant differences between young (3months) and adult (12 months)
CD1 mice as well as between sexes were observed on some behavioural
responses of animals. We also observed significant differences between
groups in the working memory test, with young female mice performing
better than young males and than both adult male and female.
56.12
Molecular mechanisms of long-term associative memory in a
defined neuronal network
Kemenes G
Sussex Centre for Neuroscience, School of Life Sciences, Department
of Biology & Environmental Science, Brighton, UK, BN1 9QG
Work using the cellular reductionist approach to the analysis of
learning and memory has been very successful in mapping out the
signalling pathways that play a role in the formation of long-term
synaptic plasticity. However, it remained largely unknown how the
same signalling cascades contribute to the formation of long-term
memory (LTM) in intact animals. The pond snail Lymnaea provides
valuable experimental models for top-down analyses of associative
learning and memory, enabling us to gain new insights into the
molecular mechanisms of long-term behavioural plasticity. LTM forms
after multi-trial aversive classical or operant conditioning but
remarkably, also after a single pairing of a conditioned stimulus (amyl
acetate) with food (‘flash-bulb memory’). To date, the most detailed
information on the molecular mechanisms of associative LTM in
Lymnaea has been gained from experiments using this single-trial
food-reward classical conditioning paradigm. Molecular mechanisms
of single-trial conditioning involve the activation of highly conserved
signalling pathways (NO/cGMP, cAMP/PKA, CaMKII and MAPK),
transcriptional activation by CREB and de novo protein synthesis. A
key new finding emerging from work in Lymnaea is that depending on
how recent or remote consolidated memories are relative to the time
of learning, different molecular pathways are activated by memory
retrieval and contribute differentially to memory reconsolidation.
Importantly, a number of molecular processes involved in associative
LTM have now been traced from the behavioral level to neuronal
networks and single identified neurons, consolidating Lymnaea as a
useful model of learning and memory research using the top-down
approach.
Funded by the MRC
56.13
Time-dependent differential roles for the alpha isoform of calmodulin
kinase II (alphaCaMKII) in associative memory in the snail Lymnaea
Stagnalis
Wan H, Iqbal H, Kemenes G
Sussex Centre for Neuroscience, School of Life Sciences, Department of
Biology & Environmental Science, University of Sussex, Brighton, BN1
9QG, UK
Electrophysiological, pharmacological and gene knockout studies in
mammals have demonstrated a critical role for αCaMKII in the induction of
long term potentiation (LTP) and for both acquisition and consolidation of
long-term memory (LTM) in a variety of learning paradigms. We found that
in the snail Lymnaea stagnalis, a major invertebrate model successfully
used in the analysis of evolutionarily conserved molecular mechanisms of
memory function and dysfunction, administration of KN-62 (100-200 μM), a
specific CaMKII inhibitor, 30 min before, but not 10 or 15 min after, a singletrial
food-reward classical conditioning, significantly reduced the feeding
responses to the conditioned stimulus (CS) 24 hr post-training. Moreover,
administration of KN-62 24 hr post-training significantly decreased
responses to the CS when tested 18 hr after treatment. These results
demonstrate for the first time that αCaMKII plays differential roles in the
acquisition or early consolidation of LTM versus late consolidation or
maintenance processes. In order to construct an in situ RNA probe and a
double stranded RNA (dsRNA) for RNAi experiments we cloned an
αCaMKII-like gene in Lymnaea (Lym-αCaMKII). Preliminary data from in
situ hybridization and PCR studies suggest that injection of 5 μg of LymαCaMKII
dsRNA leads to a significant silencing of the Lym-αCaMKII gene.
Further experiments using Lym-αCaMKII dsRNA, in situ RNA probes and
pharmacological inhibitors are being performed to explore the precise
temporal involvements of αCaMKII in different stages of memory formation
and maintenance and the underlying molecular mechanisms.
Supported by MRC
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57.01
Opioid Receptor Agonists’- Induced changes during hypoxia in
B50 Neuronal cells.
Ibegbu* A O, McBean D*, Fyfe L*, Mullaney I
*Department of Dietetics,Nutrition & Biological Sciences, School of
Health Sciences,Queen Margaret University,Edinburgh Scotland.,
+Department of Pharmacology, School of Pharmacy, Murdoch
University, Pert Australia
Opioid receptor agonists (µ, δ and κ) have been shown to have some
positive benefits on the nervous system (NS), while the action of G-
protein coupled receptors like MOR, DOR and KOR are involved in the
activation of G-proteins necessary for several neural activities.The aim
of this work was to investigate the role of Opioid receptor agonists
during neuronal hypoxia using cortical cells in culture. Cortical B50
cells were cultured in normal incubator (21%O2; 5% CO2) and
hypoxic incubator (5%O2; 5% CO2) and three Opioid receptor
agonists namely Damgo(µ) DSLET(δ) and ICI (κ) were selected and
administered to the cells as treatment against hypoxia at a dose of
10µM, 50µM and 100µM concentration respectively.
Neuronal viability, Proliferation, differentiation and second messenger
activity were assessed using lactate dehydrogenase (LDH) leakage,
cellular proliferation assay, second messenger (cAMP) assay and
dbcAMP induced differentiation. The levels of G-protein coupled
receptors (MOR), mRNAs were assessed using RT-PCR. The results
showed some significant changes(P

57.05
Prolonged Ser/Thr phosphatase inhibition reduces NMDA
responses and surface expression of NR1 subunit in neonatal rat
hippocampal cultures.
Koss D, Riedel G, Platt B
Institute of Medical Sciences, Foresterhill, University of Aberdeen,
Aberdeen, AB25 2ZD, UK
In Alzheimer’s disease (AD), key cytoskeleton components express
elevated phosphorylation, possibly an early stage towards protein
aggregation and/or cell death. Evidence for the reduction of major
serine/threonine phosphatases has been demonstrated in AD brains.
Such an imbalance of phosphorylation may have immediate
consequences for neuronal signalling, leading to neuron dysfunction
prior to protein aggregation and cell death. In the present study, the
consequences of prolonged protein phosphatases inhibition on
neuronal signalling (NMDA and KCl) have been investigated using
Fura-2-AM Ca2+ imaging in hippocampal cultures treated with the
protein phosphatase inhibitor Okadaic acid (OA). We observed that
prolonged (2hr 100nM / 24hr 1nM OA) protein phosphatase inhibition
reduces neuronal excitability towards both NMDA and KCl challenges.
The reduction in NMDA signalling corresponded to reduced surface
expression of the NMDA receptor NR1 subunit, together with
significant elevation in the phosphorylation of key cytoskeletal
components including Tau and neurofilament. It was determined that
protein kinase C is involved in phosphorylation pathways responsible
for OA mediated reductions in neuronal excitation and surface
expression of NR1, since the PKC inhibitor GÖ8369 (1µM) prevented
both Ca2+ signalling and immunocytochemical changes, and PMA
(500nM) mirrored OA mediated Ca2+ signalling changes.
Taken together, our data suggests that suppression of neuronal
excitability is a result of prolonged phosphatase inhibition prior to
major protein aggregation, which may resemble early synaptic
changes in AD.
57.06
The effects of memantine on hippocampal signalling
B. Drever1, Anderson W 1, Seo S W 1,2, Choi D Y 2, Riedel G1, Platt B 1
1School of Life Sciences, College of Life Sciences and Medicine, University
of Aberdeen, AB25 2ZD, UK. , 2 LG Life Sciences Research Park, LG Life
Sciences, Ltd.; 104-1 Moonji-dong, Yusong-gu, Taejon, 305-380 South
Korea.,
Tonic activation of NMDA receptors and subsequent calcium overload has
been implicated in neurodegenerative processes, including Alzheimer’s
disease (AD). The NMDA receptor non-competitive antagonist memantine
is a drug currently prescribed for the treatment of AD.
Here, we investigated the effect of acute application of memantine to
mouse hippocampal slices, by recording evoked population spikes (POP
spikes) in the CA1 region. At 10 and 100 μM, memantine caused a
significant upward shift in the amplitude I-O curve and a corresponding
downward shift in the latency curve, indicating enhanced synaptic
transmission. As anticipated for an NMDA antagonist, 100μM memantine
was found to inhibit long-term potentiation (LTP) in addition to a cholinergic
form of LTP induced by carbachol (50nM).
The pharmacology of memantine was further investigated using bicuculline
(20μM), scopolamine (10μM) and MK-801 (10μM). Enhanced synaptic
transmission was blocked in scopolamine treated slices but maintained in
slices treated with bicuculline and MK-801, revealing cholinergic effects of
memantine.
Secondly, Fura-2 calcium imaging established acute memantine effects on
NMDA-, carbachol- and caffeine-induced calcium responses in primary
hippocampal neurones. Memantine almost completely blocked NMDA
calcium responses at 10uM. The NMDA response fully recovered after
memantine washout. Carbachol and caffeine calcium responses were also
significantly reduced.
Overall, our data suggests actions of memantine beyond NMDA receptor
antagonism, which may include effects on muscarinic receptors, possibly
downstream from receptor activation. The facilitation of synaptic
transmission as well as interactions with cholinergic signalling is likely to
have therapeutic implications, and should thus be characterised further.
57.07
Excitatory neurotransmission in the rat superior colliculus (SC)
in aging and in glaucoma
Georgiou A L, Guo L, Cordeiro M F, Salt T E
Authors 1. & 4. Visual Science,, Authors 2. & 3. Glaucoma and Retinal
Neurodegeneration Research Group, UCL Institute of Ophthalmology,
London, UK., Author 3. Western Eye Hospital, London, UK.
We assessed the effect of experimental glaucoma on
neurotransmission from retinal ganglion cells to their major target the
SC using the sensitivity of responses to the NMDA receptor (NMDAR)
antagonist D-AP5 to evaluate the contribution of NMDARs to synaptic
transmission. Chronic ocular hypertension (OHT) was induced in one
eye (n=4). We also looked at a group of age matched (22weeks, n=5)
and a group of younger (12weeks, n=6) untreated controls. 16 weeks
after surgery recordings of field excitatory postsynaptic potentials
(fEPSPs) were made in vitro in SC slices in response to optic tract
stimulation in the presence of antagonists to block GABAa,b,c
receptors. We found a significant age related decrease in the
contribution of the excitatory fEPSP revealed by blockade of NMDARs
(12weeks mean 64 ± 1.5% SEM, n=17 slices, 22weeks 54.5 ± 1.9%,
n=16, P=0.002) as seen previously (Pothecary et al., 2005). There
was no significant difference in the contribution of the excitatory
fEPSP revealed by blockade of NMDARs between slices receiving
input from OHT treated eyes (56.4 ± 2.3%, n=7) and those receiving
input from untreated eyes (59 ± 3.8%, n=6, P=0.9), or their age
matched untreated controls (P=0.9). Pothecary et al. (2005) have
shown in dystrophic rats with progressive retinal degeneration there
were changes in NMDAR contribution to the fEPSP compared to
control animals. We did not find the same trend in the glaucoma
model: this may be due to differences in the time course and severity
of the two models.
57.08
Assessing motor performance in an animal model of Huntington`s
disease: influence of other demands.
Pallier P N, Drew C, Morton A J
University of Cambridge, Dpt. of Pharmacology, Tennis Court Road,
CAMBRIDGE CB2 1PD
Protocols aimed at testing coordination and balance in animal models of
Huntington’s disease (HD) rarely control for potentially confounding factors
like fatigue, level of arousal and amount of training involved. Here, motor
function was assessed in the R6/2 transgenic mouse model of HD using
three protocols that differed from each other in physical and/or cognitive
demand.
Two groups of 10 R6/2 and 10 wild type (WT) female mice each were
trained on the rotarod at 24 rpm. At 11 and 16 weeks of age, one group
was tested on a fixed speed rotarod (two trials at 8 speeds from 4 to 40
rpm), the other group on an accelerating rotarod (3 trials with the rod
accelerating from 5 to 40 rpm within 5 min). At 17 weeks, all mice were
tested for gait analysis on DigigaitTM (Mouse Specifics) for 3 sec (belt
speed: 14.6 cm/sec).
Results showed that R6/2 mice were significantly impaired on both versions
of the rotarod compared to WT mice. Impairments in performance
worsened with age. Accelerating rotarod was more sensitive for detecting
early deficits in R6/2 mice. Surprisingly however, although they had rotarod
deficits, when R6/2 mice were tested on the Digigait treadmill (that forced
them to walk), they showed none of the major deficits expected.
We propose that the differences seen between the tasks are due to
different levels of arousal required for each. This should be taken into
account when assessing pharmacological treatments aimed at restoring
coordination and balance in HD.
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57.09
Cognitive rigidity and altered interactions between memory
systems in a transgenic mouse model of Huntington’s disease
Ciamei A, Morton A J
Department of Pharmacology, University of Cambridge, Tennis Court
road, Cambridge CB2 1PD
In Huntington’s disease (HD), progressive cognitive deficits can
become as debilitating as motor impairment. Previous studies showed
that R6/2 mice have deficits in spatial and reversal learning. Here we
have tested R6/2 mice in different behavioural paradigms of learning
and memory that should allow us to study the functionality of
hippocampus- and striatum-based memory systems.
In the social transmission of food preference, R6/2 mice chose
flavours that had been eaten previously by a demonstrator mouse
(cued food) but were impaired in shifting their preference when a new
cued food was introduced in the task. Deficits were also seen in R6/2
mice in the active avoidance task where a mild footshock was paired
with a light. In the passive avoidance task, R6/2 mice learned to avoid
a place where they had previously received a footshock, but their
avoidance responses were not modulated by the intensity of the
footshock, as was observed with WT mice. Moreover, when ‘place
learning’ (hippocampus-dependent) and ‘response learning’ (dorsal
striatum-dependent) were studied in a modified version of the water
maze, R6/2 mice behaved mainly as ‘response learners’ while WT
mice adopted both place- and response-driven strategies to escape
the maze.
Our data show that R6/2 mice retain rigid cognitive abilities that allow
learning of socially and emotionally relevant experiences. Moreover,
they suggest an alteration in the interaction between striatum- and
hippocampus-based systems in tasks where these two systems act in
a competitive way to achieve learning and memory consolidation.
57.10
Seeking a mechanism of action for Mellissa Officinalis essential oil
treatment of agitation in human dementia
Chazot P L (1), Abhuhamdah S (1), Huang L(2), Ennaceur A(3), French D
(1), Elliott M S J(4), Perry E K (5), Ballard C (6), Francis P T (4), Lees G(2)
1. Centre for Integrative Neuroscience, Durham University,UK;2.Otago
Medical School, NZ;3.Sunderland Pharmacy School, UK;4.Wolfson Centre
for Age Related Diseases,King`s College, London,UK;5.Institute of Ageing
and Health, Newcastle University, UK;6.University of Northumbria,
Newcastle
Agitation is a severe and persistent feature of advanced dementia, which
affects both sufferers and carers alike. Atypical neuroleptic drugs have
frequently been used as treatments but often cause oversedation, social
withdrawal, enhanced risk of stroke and may exacebate cognitive decline.
Melissa officinalis (Mo) has been used historically for its calming and
attention maintenance properties and a large multi-centre trial to assess Mo
in late-stage AD patients will be commencing this year. In order to support
this trial, we have used radioligand binding and electrophysiology
techniques to address the hypothesis that the Mo may be a GABA-A
receptor modulator. Increasing concentrations of Mo (0.001-1 mg/ml) were
incubated with rat cortical brain membrane homogenates or the rat α1β2γ2
GABA-A receptor subtype expressed in HEK 293 cells, and a fixed
concentration of radioligand. Mo (IC50 = 0.03mg/ml) significantly displaced
[35S]-TBPS binding in both preparations, but had no effect upon
[3H]flunitrazepam, MK801, AMPA or nicotine binding to native membranes
up to 1mg/ml. Patch-clamp experiments on primary rat cortical cultures
demonstrated that Mo (0.1mg/ml) reduced currents through the GABA-A
channel but concurrently blocked the spontaneous synaptic traffic in the
cultured networks. We conclude that Mo essential oil does exert depressant
effects on neural activity, but that this is not a reflection of its disinhibitory
effect on the GABA-A complex. We are currently attempting to delineate
the active constituents within this oil, and assess the behavioural effects of
Mo in our unique all-in-one animal behavioural model .
Funded by the Alzheimer’s Disease Association (UK).
57.11
Characterisation of the binding properties of anti-beta amyloid
antibodies using optical biosensors
Chapman T D, Soden P E, Burbidge S A
Neurodegeneration Research Dept., Neurology and GI CEDD,
GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow,
Essex CM19 5AW, U.K.
In Alzheimer’s disease, the Beta-amyloid peptide has become a major
therapeutic target. Monoclonal antibodies generated against Betaamyloid
are often used to characterise the location and concentration
of Beta-amyloid in vivo, by IHC and ELISA respectively, but also have
potential in immunotherapeutic approaches. Optical biosensors can be
used to characterise the binding of these antibodies to Beta-amyloid,
in order to select the most advantageous clones. In these instruments
the mass of one binding partner is measured as it interacts with the
other partner, the later having already been immobilised on a sensor
surface. Data is presented that demonstrates the use of the SRU Bind
biosensor to map the epitopes of various antibodies along the Betaamyloid
peptide sequence. Antibodies directed towards the N-
terminus (6E10), mid-region (4G8) and C-termini (G210 and 5G5)
were observed. Data derived from the Biacore 3000 biosensor is also
shown that confirms the avidity, affinity and specificity of these
antibodies. The data shows how important it is for their respective
epitopes to be fully presented for binding, and not be masked by
incorrect immobilisation strategies, or possibly hidden within the
hydrophobic regions of fibrillar forms of the Beta-amyloid peptide.
57.12
The Effects of the Amyloid Peptide (Aβ1-42) and Fragments on
Recombinant Human Neuronal Nicotinic Acetylcholine Receptors
Craig M, Pym L, Buckingham S, Sattelle D
MRC Functional Genetics Unit, University of Oxford, Oxford, OX1 3QX
Alzheimer’s disease (AD) is the most prevalent form of dementia affecting
the elderly. It is widely believed that the amyloid peptide Aβ1-42 plays an
important role in AD progression, as its accumulation is toxic to neurons
both in vitro and in vivo. Aβ1-42 exerts subtype-selective actions on
nicotinic acetylcholine receptors (nAChRs) but it is not known what role is
played by an M35 residue in Aβ
be important in Aβ
Two-electrode voltage-clamp electrophysiology has been used to study the
actions on α7, α4β2 α3β4 recombinant human neuronal nAChRs
heterologously expressed in Xenopus laevis oocytes of full length Aβ 1-42
and Aβ peptide fragments, scrambled peptides, and Aβ 1-42 peptide
containing mutations of the methionine in position 35. Whereas Aβ 1-42 with a
M35C mutation had similar subtype specificity to wild-type Aβ 1-42, Aβ 1-42 with
a M35V substitution reduced the peak amplitude of ACh-induced currents
recorded from α4β2 nAChRs, but did not affect those recorded from α7 or
α3β4 receptors. Interestingly, the Aβ 25-35 fragment, which is often used as
an experimental mimetic of the full length peptide, did not display subunit
specificity. Our results suggest that that the amino acid in position 35 of
Aβ 1-42 is an important determinant of the subtype-specificity of the actions of
this peptide on human recombinant α7, α4β2 α3β4 nAChRs. We are
also exploring the actions of full length Aβ peptide and the neuroprotective
properties of nicotinic ligands using a human neuroblastoma cell line (SH-
SY5Y).
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57.13
Characterization of a focal eae-mog model for the study of
reactivation of CNS lesions by a systemic inflammatory response
Serres S 1, Jiang Y 2, Amor S 3, Anthony D C 2, Sibson N R 1
1Experimental Neuroimaging Group, Department of Physiology,
Anatomy & Genetics, and, 2Experimental Neuropathology Lab.,
Department of Pharmacology, University of Oxford, UK, 3Department
of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The
Netherlands,
Multiple sclerosis (MS) is a chronic inflammatory disorder which can
display a relapsing-remitting form of disease. It is thought that
bacterial and viral infections may trigger relapses and progression of
MS. Our aim was to characterise a focal EAE-MOG model targeted in
the brain by intracerebral injection of cytokines, and to determine
whether this lesion could be reactivated by a systemic inflammatory
response.
MOG model was induced in Male Lewis rats by injection of MOG
peptide (35-55) or MOG protein following three weeks later by
injection of cytokines in the left hemisphere. Four weeks later, the
animals were challenged by i.p injection of lipoplysaccharide (LPS;
100 μg/kg). We caracterized the lesion by both MRI and
Immunohistochemistry.
T2-weighted images shows a disruption of the signal in the injection
site (corpus callosum). The longitudinal study reveals the presence of
a chronic hypointense signal in the striatum below the injection site,
the increase in ventricle size and incomplete recovery of the signal
from the corpus callosum.
The immunostaining correlated with the hypointense signal and the
disruption of the corpus callosum structure, suggesting involvement of
both activated macrophage and microglia.
Induction of a systemmic inflammatory response with LPS challenge,
resulted in an increase in the left/right rCBV ratio with recruitment of
macrophages as revealed by ED1 staining. These findings show that
the reactivation of inflammatory processes within a previously
quiescent CNS lesion can be obtained after a systemmic inflammatory
response to a bacterial endotoxin in a clinically-relevent model of MS.
57.14
Localisation of the PARK8 protein kinase LRRK2 in cellular models
and human tissue
Sancho R M, Kingsbury A E*, Bandopadhyay R*, Harvey R J, Harvey K
Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick
Square, London WC1N 1AX; *Reta Lila Weston, Institute, University
College London, London WC1N 1PJ
Missense mutations in the protein kinase LRRK2 are the most common
known cause of Parkinson’s disease (PD), found in ~ 5% and 1.6% of
patients with familial and idiopathic PD, respectively. LRRK2 contains
leucine-rich repeats and GTPase, COR (C-terminal of Roc), kinase and
WD40 domains. Mutations are found in all of these domains, but it is
unclear how these changes cause PD. To enable us to investigate LRRK2
expression, we raised two polyclonal antibodies (pAbs) against LRRK2. In
HEK cells, endogenous and exogenous LRRK2 (encoded by various
mammalian expression constructs) was clearly recognised by antibody
BC300-267. In Western blots from human brain lysates BC300-267
recognised a specific band of ~260 kDa that was completely abolished in
competition experiments using an excess of the peptide used for
immunization. BC300-267 was further tested on post-mortem tissue from
neurologically normal and sporadic PD cases, revealing diffuse staining of
cell bodies and processes of midbrain melanized neurons and selected
brainstem and midbrain nuclei. In nine separate pathologically-verified
sporadic PD cases, LRRK2 immunoreactivity was detected in the inner part
of the halo of cerebellar and midbrain LBs, with some variation in staining
intensity between individual LBs. Interestingly, no LRRK2 immunopositive
cortical LBs were seen in these cases. Lastly, using the yeast two-hybrid
system and mammalian cellular models, we demonstrate that LRRK2
interacts with synphilin-1. Confocal imaging showed colocalisation of
LRRK2 and synphilin-1 in the cytoplasm and perinuclear aggregates with a
central core of synphilin-1 and an LRRK2 halo.
57.15
Anosmia in subjects with dementia is associated with Lewy
pathology in the cortical olfactory pathway.
Hubbard P, Esiri M M, Smith A D, King E, Reading M, Nagy Z,
McShane R
1,6 University of Birmingham, Birmingham, UK., 2,5 Neuropath. Dept,
John Radcliffe Hospital, Oxford, UK, 3, Oxford Centre for Gene
Function University of Oxford,Oxford, UK, 4, OPTIMA Radcliffe
Infirmary NHS Trust Oxford, UK , 7,Dept Old Age Dept Dept Old Age
Psychiatry, Churchill Hospital, Oxford,UK
Neurodegenerative diseases currently affect over 750,000 people in
the UK. The most common disorders leading to dementia are
Alzheimer’s disease and dementia with Lewy bodies. So far the
definitive diagnosis of these dementias can only be confirmed postmortem.
However, loss of smell (anosmia) is an early symptom in
patients who develop dementia. The use of a smell test has been
proposed as an early diagnostic procedure and distinguishes those
with early dementia with Lewy bodies (DLB) from those with early
Alzheimer’s disease.
The present study aimed to examine the relationship between
anosmia and the presence of Lewy body pathology in the olfactory
pathways.
Participants in the Oxford Project to Investigate Memory and Ageing
were tested for basic olfactory function during life. Full ethical
permission was obtained for use of tissue. Tissue was taken from 5
areas of the olfactory pathway; the Olfactory Tract/Bulb, the insertion
of the Olfactory Tract, the orbito-frontal cortex, the hippocampus and
the amygdala. Lewy bodies were detected using
immunohistochemistry to alpha-synuclein.
Results show that the quantity of Lewy Body pathology in the olfactory
system varied, but may follow a particular pattern of development. The
presence of pathology in the cortical pathway but not the limbic
pathway is associated with anosmia, and is further evidence towards
the use of a smell test to aid diagnosis of neurodegenerative diseases.
57.16
A fibroblast growth factor receptor 1 agonist reduces the loss of
hippocampal dendritic spines caused by beta-amyloid(25-35)
Corbett N J, Stewart M G, Gabbott P L, Klementiev B, Davies H A, Colyer F
M, Berezin V, Bock E
The Open University, , Department of Biological Sciences, Walton Hall,
Milton Keynes, Buckinghamshire, MK7 6AA,
In Alzheimer’s disease (AD), β-amyloid (Aβ) affects neural circuits
underlying learning and memory. Fibroblast Growth Loop (FGL), a
pentadecapeptide which mimicks the heterophilic binding of neural cell
adhesion molecule (NCAM) to Fibroblast Growth Factor Receptor 1
(FGFR1), has the potential to significantly reduce the neurodegeneration
seen in AD.
Aβ25-35 was injected intracerebroventricularly (icv) into the right
hemisphere of 2 groups of rats (5mg/15μl; n=6/group). Eight days after Aβ
injection, animals received either a subcutaneous injection of FGL
(10.8mg/kg) or distilled water (dw) every 3 days (Aβ-FGL, Aβ–dw groups
respectively). Another group (dw-FGL: n=6) had dw injected icv followed by
FGL treatment whilst the control group (dw-dw: n=6) received both forms of
dw.
On day 25, animals were transcardially perfused with 2%
paraformaldehyde and 3.75% acrolein in 0.1M phosphate buffer (pH 7.4).
Using the ‘Single-section’ Golgi method on 100μm thick coronal sections,
the density of dendritic-spines along CA1 pyramidal apical dendrites in the
stratum radiatum were obtained (Neurolucida). This area of spiny dendrites
was chosen due to the excitatory interactions with the Schaffer axon
collaterals that are notably damaged in AD.
Results indicated that the Aβ-dw group had a significantly lower average
spine density at 1.5 ± 0.1μm-1 (P

57.17
An analysis of DJ-1 (PARK7) and tau isoform expression in
neurodegenerative disorders
Bandopadhyay R, Kumaran R, Kingsbury A, de Silva R, Mann D,
Holton J, Revesz T, Lees A
Reta Lila Weston Institute, Institute of Neurology, UCL, 1 Wakefield
Street, WC1N 1PJ
Homozygous mutations in the DJ-1 gene are associated with
autosomal recessive PD but its link to disease pathogenesis is
unknown. This study examines DJ-1 expression in a variety of
neurodegenerative disorders, AD, Pick’s (PiD), PSP, CBD, FTLD-with
tau mutations in which hyperphosphorylated-tau deposits are a major
pathological signature, and its co-existence with the two major
isoforms of tau, namely 3R and 4R tau by immunohistochemistry and
double confocal fluorescence microscopy. DJ-1 immunoreactivity (DJ-
1-IR) was present in neurofibrillary tangles (NFTs), neuropil threads
(NTs), and extracelluar plaques in AD. Tau inclusions in AD are
composed of 3R and 4R tau. DJ-1 -IR was present in a subset of Pick
bodies which are 3R tau positive. In PSP, a few DJ-1 positive NFT’s,
NTs and glial inclusions were present in all cases examined.
Interestingly, in cases of FTLD with tau mutations that are 3R tau
negative, DJ-1 was present mostly in glial inclusions. The FTLD-
R406W tau mutation cases which are both 3R and 4R tau positive,
DJ-1 IR was associated mainly with NFTs and NTs. Thus DJ-1 is
associated with both 3R and 4R tau-isoform inclusions and in both
neuronal and glial inclusions. Furthermore, DJ-1 is enriched in
sarkosyl-insoluble fractions in AD and PiD brains, which also contain
hyperphosphorylated tau. DJ-1-IR was not associated with ubiquitin
inclusions in FTLD-U cases. DJ-1 deposition in tau inclusions is likely
to be a specific phenomenon and may contribute to disease
pathogenesis.
57.18
Lower brain stem neurons in Parkinson’s disease
Kingsbury A, Hussein K, Bandopadhyay R, Lees A
Reta Lila Weston Institute, Institute of Neurology, UCL, 1 Wakefield Street
WC1N 1PJ
It is well-established that deposition of the pre-synaptic protein alpha
synuclein in neuronal perikarya and processes is a major component of
pathological change in Parkinsons’s disease (PD), although its precise role
in the disease process is unclear. A recent model of progression of PD
pathology proposes that alpha synuclein deposition begins in the lower
brain stem before the motor systems are affected and the clinical signs of
the disease appear. It has also been proposed that alpha synuclein
deposition in neurons and processes may reflect altered axonal transport in
affected nuclei. We report here preliminary findings on the involvement of
lower brain stem neurons in PD. Twelve neuropathologically-evaluated
sporadic PD cases, with nigral neurodegeneration and Lewy body
formation in nigra and cortex were studied. All cases had alpha synuclein
deposition in brain stem neurons and neuronal processes, as predicted by
the model, varying between light and intense. Protein markers of axonal
transport, kinesin and dynein, were examined in lower brain stem by
immunohistochemistry and immunoblotting. Both proteins were expressed
in lower brain stem neurons, including cranial nerve nuclei, inferior olive,
neurons of the central nucleus of the medulla and neuronal processes.
Dynein appeared to stain processes more strongly than kinesin.
Immunoblotting showed that levels of expression of kinesin and dynein
extracted from lower brain stem were unaffected by the disease and
subjective assessment of the immunochemical findings indicated that the
expression of both proteins was similar in PD cases and controls.
57.19
The relationship between oxidative DNA damage and somatic
mutations in Alzheimer’s disease
Timms S C, Nagy Z
Division of Neuroscience, Medical School, University of Birmingham,
B15 2TT
The cell cycle hypothesis for the pathogenesis of Alzheimer’s disease
(AD) postulates that the disease-related pathology is due to aberrant
cell-cycle re-entry of neurones followed by a regulatory failure at the
G1/S transition point. There is direct evidence that neuronal DNA
replication precedes the development of Alzheimer-type pathology.
Although many hypotheses have been formulated, the causes of this
cell cycle deregulation and subsequent neuronal damage are still
elusive. Oxidative stress is one of the factors thought to contribute to
cellular dysfunction and death following cell cycle activation.
We investigated the hypothesis that oxidative stress might contribute
to the accumulation of somatic mutations (genomic instability) in
neurones which, if left unrepaired, could further hinder the functional
integrity of these cells.
In order to investigate genomic instability we carried out comparative
RAPD analysis from two different parts of the brain in AD patients and
controls. 8-hydroxi guanesin, a marker of oxidative DNA damage, was
quantified by ELISA. The expression of neuronal markers, Map-2 and
synaptophysin, were also quantified by ELISA.
Results showed significant decreases in overall neuronal and synaptic
densities in AD brains compared to controls. There was a significant
positive correlation between genomic instability and oxidative stress.
However, there was no significant difference in oxidative damage (8-
HG) per neurone or genomic instability between AD brains and
controls.
Our data indicate that while oxidative DNA damage might contribute to
neuronal dysfunction and loss in AD, the process is not necessarily
disease-specific.
57.20
Neurotoxicity due to overexpression of drosophila tau is comparable
to that seen with human tau
Ubhi K, Shaibah H, Newman T, Shepherd D, Mudher A
School Of Biological Sciences, University Of Southampton, Bassett
Crescent East, Southampton, SO16 7PX
Neurofibrillary tangles containing the microtubule-associated protein tau,
together with extracellular amyloid plaques comprise the two characteristic
neuropathological features of Alzheimer’s disease. Tau in these tangles is
abnormal in a number of aspects including phosphorylation status and
structure; similar abnormalities are also associated with a group of
disorders collectively known as tauopathies.
To model these disorders, human tau has been overexpressed in neuronal
populations in Drosophila melanogaster resulting in neuronal degeneration,
diminished locomotor activity, disrupted axonal transport and impaired
synaptic function. These effects are seen prior to neuronal death and in the
absence of tangle formation.
Although the Drosophila tau protein shares a large degree of homology with
human tau, is unknown how the effects of overexpression of human tau in
Drosophila compare to overexpression of Drosophila tau.
Drosophila tau was overexpressed in larval motorneurones to compare its
effects with that of human tau. The locomotor abilities and axonal transport
characteristics of these larvae were assessed as previously described
(Mudher et al 2004)
Overexpression of Drosophila tau led to a similar decrease in locomotor
activity as seen with overexpression of human 3RT. The effects of
overexpression of Drosophila tau on axonal transport were also
investigated. These results show that overexpression of Drosophila tau
leads to neurotoxicity that is comparable to that seen with overexpression
of human tau. This indicates that it is overexpression of tau per se that is
detrimental to neuronal function and that neuronal degeneration is not a
feature of the specific overexpression of human tau in these models.
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58.01
Electrophysiological characterization of Oligophrenin-1 null
mouse, a mouse model of X-linked mental retardation
Saintot P-P, Powell A D, Jefferys J G R
Department of Neurophysiology, The Medical School, Division of
Neuroscience, University of Birmingham, Birmingham B15 2TT, UK.
Mental retardation (MR) is defined by an overall intellectual quotient
less than 70. It is the most common brain disease, with prevalence in
developed countries of approximately 2-3%. One of the first genes
identified in X-linked mental retardation (XLMR) was the OPHN-1
gene, which normally encodes the protein oligophrenin-1. Mutation of
this gene has been described in patients with moderate to severe
cognitive impairment. The underlying mechanisms of mental
retardation are yet to be elucidated, although recent evidence
suggests that it is associated with abnormalities in dendritic spines.
The aim of this study is to determine how oligophrenin-1 disrupts
cognitive function with perspectives to developing treatment and to
understanding normal brain function
Oligophrenin-1 is a RhoGTPase Activating Protein, which negatively
modulates RhoGTPase signalling pathways, and is implicated in
neuronal morphogenesis by regulating the actin cytoskeleton. We
have hypothesised that mutations in the OPHN-1 gene would produce
alterations in synaptic properties. We have used an OPHN-1 null
mouse model to examine the functional role of Oligophrenin-1. A first
approach was to investigate the functioning of a neuronal population
using extracellular recording. We have found that OPHN-1 null mice
displayed significantly smaller kainate-induced gamma oscillations
(20-80Hz), in CA3 area, than wild type littermates (OPHN-1-/y: 103.3
µV2 ± SE 36.9; OPHN-1+/y: 271.8 µV2 ± SE 72.7). Furthermore, we
observed a significant reduced facilitation of synaptic strength in
response to paired pulse stimulation of the mossy fibres onto CA3
neurons. We are now investigating differences in synaptic currents in
voltage-clamped CA3 pyramidal cells
58.02
Reduced inhibitory neurotransmission in the dentate gyrus of a
mouse model of mental retardation.
Powell A D, Saintot P-P, Jefferys J G R
Department of Neurophysiology, Division of Neuroscience, The Medical
School, University of Birmingham, Birmingham, B15 2TT.
Mental retardation (MR) is defined by an intelligent quotient of less than 70,
and is the most common brain disorder (prevalence 2-3%). The
mechanisms underlying MR are not fully understood; although recent
identification of putative genes responsible for MR has facilitated research.
The gene OPHN-1, which encodes for the protein Oligophrenin-1, has been
identified as malfunctioning in some mentally retarded individuals.
Oligophrenin-1 is a RhoGTPase activating protein, which negatively
modulates Rho signalling pathways. Oligophrenin-1 has been implicated in
regulating neuronal morphology, particularly at the level of the dendritic
spines.
We have examined the effect of deletion of OPHN-1 gene on the
neurophysiology of the dentate gyrus, with the prediction that OPHN-1 null
mice (OPHN1-/y) would display alterations in synaptic properties. We have
used the whole-cell voltage clamp technique to study inhibitory
neurotransmission onto granule cells in the dentate gyrus. We found that
evoked GABAergic neurotransmission is significantly smaller in OPHN-1-/y
mice (OPHN-1+/y – 908.3 ± 116.9 pA, OPHN-1-y – 381.3 ± 79.9 pA; p <
0.001). Furthermore, synaptic properties such as paired pulse and
frequency following were also significantly reduced in OPHN-1-/y neurons.
Spontaneous release events were significantly less frequent in OPHN-1-/y
neurons than in OPHN-1+/y neurons (Interevent interval - 294.5 ± 34.5 ms
and 180.7 ± 19.0 ms, respectively; p < 0.001). This reduction appeared to
be a result of less synaptic vesicles in the Readily Releasable Pool (OPHN-
1+/y - 1179 ± 270, OPHN-1-/y – 471 ± 153; p < 0.05).
58.03
Effects of clozapine withdrawal on dialysate lactate levels in two
animal models of Schizophrenia
Moran M P, De Souza I E J, Brady A T, McCabe O M, O’Shea S D,
O’Connor W T
Applied Neurotherapeutics Research Group, UCD School of
Biomolecular and Biomedical Science, UCD Conway Institute,
University College Dublin, Belfield, Dublin 4, Ireland.
Sudden discontinuation of clozapine causes rebound psychosis in
39% of patients but in only 6-11% of those taking typical
antipsychotics. The present study investigated the effect of abrupt
clozapine withdrawal on dialysate lactate levels in the mPFC of the
isolated and the maternally deprived animal models of schizophrenia.
Microdialysis was employed in the mPFC to monitor dialysate lactate
levels (µM) in vehicle and clozapine-treated (5mg/kg i.p. daily, 10
days) isolated and maternally deprived rats over a 4-day withdrawal
period. In the isolated group, young adult rats were weaned on
postnatal day 25 (P25) and individually housed while the maternally
deprived group experienced a single 24-hour period of maternal
deprivation on P9. Socially reared rats acted as controls. ANOVA was
employed for significance (n=5-8 animals per group).
Clozapine withdrawal was associated with a reducing effect on lactate
levels in the clozapine treated social control, isolated and maternally
deprived rat by -42 ±12% (p=0.0010 v’s vehicle-treated control), -28
±8% (p=0.0002 v’s vehicle treated isolated) and -45 ±8% (p=0.0001
v’s vehicle treated maternally deprived) respectively over a 4-day
withdrawal period.
The reduction in mPFC lactate in the control and in both animal
models of schizophrenia is associated with clozapine withdrawal and
may reflect a switch towards an altered glucose metabolism. Thus the
clozapine-withdrawal induced reduction in lactate mPFC levels may
play a role in rebound psychosis. The exact mechanism by which an
antipsychotic drug causes disturbances in glucose metabolism
remains to be determined.
58.04
Pharmacological manipulation of sensorimotor gating. A dual probe
microdialysis study in an animal model of schizophrenia
O’Shea S D, De Souza I E J, Brady A T, McCabe O M, Moran M P,
O’Connor W T
Applied Neurotherapeutics Research Group, UCD School of Biomolecular
and Biomedical Science, UCD Conway Institute, University College Dublin,
Belfield, Dublin 4, Ireland
The AMPA/kianate receptor antagonist CNQX (0.5mg/kg, i.p.) and the
GABAA receptor antagonist flumazenil (10mg/kg, i.p.) were combined with
dual probe microdialysis in rat medial prefrontal cortex (mPFC) and ventral
tegmental area (VTA) to compare the effects of an early postnatal stressor
(maternal deprivation) on prepulse inhibition (PPI) and basal dialysate
mPFC and VTA GABA and glutamate levels. PPI was performed on P82,
P84 and P86. Drugs were acutely administered on P84. The maternally
deprived rat experienced a 24-hour period of maternal deprivation on P9.
Socially reared rats acted as controls. Protocols were approved by UCD
Animal Research Ethics Committee and the Department of Health and
Children in accordance with European Community Directive 86/609/EC.
Basal dialysate mPFC GABA and glutamate levels were elevated by +79%
(p=0.011 v’s social control) and reduced by -93% (p

58.05
Differential effects of Interleukin-1alpha and Interleukin-1beta on
kainate seizures
Rutter J, Allan S
Faculty of Life Sciences, , University of Manchester, , Manchester, ,
England
Interleukin-1 (IL-1) is a pro-inflammatory cytokine, which is an
important mediator of neuroinflammation in various neurological
disorders including epilepsy. There are two closely related IL-1
proteins, IL-1alpha and IL-1beta, which are assumed to exert identical
biological actions. IL-1 levels are increased in human epileptic
conditions and experimental paradigms(1). Exogenous application of
IL-1beta into the mouse hippocampus can prolong experimental
seizures(2) and the naturally occurring IL-1 receptor antagonist (IL-
1RA) has anti-convulsant properties in experimental seizure
models(3). The main aim is to further investigate the role of IL-1 in
kainic-acid induced seizures in mice.
Mice were injected with either IL-1alpha or IL-1beta followed by kainic
acid into the dorsal hippocampus. Seizure activity was recorded by
EEG radiotelemetry for 3 hours to determine seizure onset, seizure
duration and the number of seizure episodes. Results indicated that
unlike IL-1beta, IL-1alpha did not increase seizure activity in this
paradigm. Furthermore, western blot analysis of hippocampal tissue
showed that IL-1alpha and IL-1beta did not have differential effects on
the classical transcription-dependent signalling pathways NF-kB and
MAP kinase. Both isoforms significantly activated ERK1/2, JNK1/2 and
p38 compared to vehicle-treated mice.
These data suggest that IL-1beta acts via an alternative mechanism,
which results in seizure exacerbation. Consequently, current studies
are focusing on the possible differential signalling pathways elicited by
IL-1alpha and IL-1beta.
References
58.06
Body weight changes in two animal models of Schizorphrenia: effects
of pathway- specific lesions and abrupt clozapine withdrawal
McCabe O M, Brady A T, O’Shea S D, Moran M P, De Souza I E J,
O’Connor W T
Applied Neurotherapeutics Research Group, UCD School of Biomolecular
and Biomedical Science, UCD Conway Institute, University College Dublin,
Belfield, Dublin 4, Ireland
We employed implantation of microdialysis probes to investigate the effects
of three pathway-specific lesions namely (1) the medial prefrontal cortex
(mPFC) and ventral tegmental area (VTA), (2) the lateral entorhinal cortex
(LEA) and dorsal hippocampus (DH) and (3) the nucleus accumbens (NUC)
and ventral pallidus (VP) on body weight in the isolated rearing and
maternal deprivation rat models of schizophrenia. In the isolated group,
young adult rats were weaned on postnatal day 25 (P25) and individually
housed while the maternally deprived group experienced a single 24-hour
period of maternal deprivation on P9. A socially reared group acted as
controls. The effect of abrupt clozapine withdrawal on body weight was also
investigated. Protocols were approved by UCD Animal Research Ethics
Committee and the Department of Health and Children in accordance with
European Community Directive 86/609/EC.
While lesions in the mPFC-VTA, LEA-DH and NUC-VP pathways resulted
in weight loss in some animals one day post-implantation, body weight in all
groups had recovered to pre-implantation weights 4-5 days postimplantation,
indicating that rats had recovered from the adverse effects of
these pathway-specific lesions on body weight.
Withdrawal of clozapine for 5-6 days did not affect body weight in either of
the two animal models, suggesting that the increase in weight often
associated with this drug has a more long term effect. These findings have
important implications for our understanding of the role of discrete nerve
pathways in the regulation of body weight and on the effects of withdrawal
of clozapine and related antipsychotic drugs.
(1) Eriksson et al., (2000) J. Neurosci. Res. 60, 266-279.
(2) Vezzani et al., (1999) J. Neurosci. 19, 5054-5065.
(3) Vezzani et al, (2000) Proc. Natl. Acad. Sci. U. S. A 97, 11534-
11539
58.07
Variation in blood vessel density in multiple sclerosis lesions
implies a role in scar formation
Holley J E, P. Eggleton, J. Whatmore, N.J. Gutowski
Peninsula Medical School (Exeter) and Royal Devon and Exeter
Foundation Hospital
The dynamic processes involved in the development of multiple
sclerosis (MS) lesions culminate with chronic glial scarring which
inhibits repair. Astrocytes, which constitute the main cell type in the
glial scar, have a characteristic scar phenotype which is different from
quiescent or reactive astrocytes. During normal wound healing
angiogenesis (the formation of new blood vessels from pre-existing
vessels) takes place to restore vascular integrity in areas undergoing
repair. However in diseases where damage and repair occur
concomitantly, erratic angiogenic signals might influence scar
deposition. Previously we showed preliminary data suggesting a
change in blood vessel density in MS. This study extends our former
work by investigating blood vessel density in post-mortem sections of
cerebral white matter from normal control (NC), MS normal appearing
white matter (NAWM), acute, subacute and chronic lesions by triple
label immunohistochemistry. Blood vessel density was quantified by
counting vessels in three random fields from NC, NAWM and MS
lesions. Statistical analysis of the results confirmed alterations in the
numbers of blood vessels in the different stages of lesion
development. This might indicate that angiogenesis could be an
important factor in scar development in MS.
58.08
The electrode-brain interface in deep brain stimulation is modulated
by systemic and local physiological factors: A 3-dimensional
computational model
Yousif N, Richard Bayford, Xuguang Liu
1. Movement Disorder and Neurostimulation Unit, Department of Clinical
Neuroscience, Division of Neuroscience and Mental Health, Imperial
College London;, 2. Bio-modelling/Bio-informatics Group, Department of
Biomedical Science, Institute of Social and Health Research, Middlesex
University
Deep brain stimulation (DBS) is a widely used clinical treatment for various
neurological disorders, and particularly movement disorders. However, the
mechanism by which these high frequency electrical pulses act on
underlying neuronal activity is not understood. Once the stimulating
electrode is placed in situ, an electrode-brain interface (EBI) is created. We
used computational modelling to construct a three-dimensional model of
the EBI using the finite element method to compensate for the clinical
restrictions on the study of EBI in situ. In this way both the structural details
and the biophysical properties of the EBI are retained. We focussed on
systemic and local, and dynamic physiological and pathological modulation
of the EBI, specifically by brain pulsation and giant cell formation. Our
results show that the EBI is influenced by both the systemic and the local
physiological factors. We find that there is a linear correlation between
cerebral blood perfusion and the magnitude of the potential distribution
induced in the tissue surrounding the electrode. Furthermore, giant cell
growth at the EBI creates a ‘shielding effect’, which impedes the flow of
current to the surrounding tissue. These results provide a quantitative
assessment of the current flow in the brain tissue surrounding the
implanted DBS electrode, the effects of the EBI on the current spreading
under physiological conditions, and consequently on the therapeutic effect
of DBS.
The project was supported by MRC grants (id 71766 and 78512)
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59.01
Characteristics of epidermal primary afferents expressing GFP
under the control of thy1.2 in the mouse.
Bruce G, Belle M D C, Morris R
Department of Veterinary Preclinical Sciences, University of Liverpool,
Liverpool, L69 7ZJ.
Epidermal primary afferents were found to be brightly labelled with
enhanced green fluorescent protein (eGFP), in a mouse expressing
the transgene thy1.2-eGFP. The eGFP was also expressed in their
cell bodies in the dorsal root ganglia (DRG) and central terminals
permitting both anatomical and electrophysiological characterisation.
Conventional immunostaining and lectin histochemistry was used to
investigate the expression of a range of molecules in relation to the
eGFP fluorescence in the skin, DRG and dorsal horn. Acutely
dissociated DRG neurones were used for whole cell patch studies.
In the DRG small and medium sized neurones that did not express
CGRP or Substance P, but did typically bind IB4 lectin expressed
eGFP. A small population of medium sized neurones were eGFP
positive, IB4 negative, but contained calbindin. In the skin this
correlated with the eGFP staining of club-shaped endings in hair
follicles. The smaller eGFP +ve DRG neurones expressed P2X3
receptors but not TRPV1 ion channels. Preliminary
electrophysiological characterisation revealed that the small eGFP +ve
DRG neurones typically produced a single spike to depolarising pulses
or showed strong spike frequency adaptation. The ones producing
single spikes also showed delayed rectification to hyperpolarising
pulses and occasional rebound spikes.
This transgenic mouse should permit a detailed analysis of the
properties of a single tissue specific group of primary afferents.
59.02
Network-based rhythmic activity induced by 4-Aminopyridine in rat
dorsal horn in vitro: contribution of gap junction-mediated
connectivity.
Chapman R J, Cilia La Corte P F, King A E
Institute for Membrane and Systems Biology, University of Leeds, Leeds,
LS2 9JT, UK
Network behaviour within the 6-13 Hz frequency range is manifest within
spinal dorsal horn (DH) in vivo (Sandkuhler and Eblen-Zajjur, 1994,
Neuroscience 61:991-1006). Previously, we characterized rhythmic network
behavior in rat DH in vitro that had a dominant frequency of 7-12 Hz
(Asghar et al, 2002, J. Physiol., 562:183-198) and was partly maintained
through gap junction (GJ) connectivity. The potassium channel blocker 4-
aminopyridine (4-AP) triggers widespread oscillatory activity in spinal DH
(Ruscheweyh and Sandkuhler, 2003, Pain, 105: 327-328) in vitro but it is
not known if GJs contribute to this behaviour. Here, we determined whether
GJs localized to glia or neurones may be involved in 4-AP-induced
enhanced excitation in DH in vitro. Extracellular field recordings of 4-AP (50
µM)-induced oscillations were made from rat substantia gelatinosa (SG) in
vitro. Data values for peak amplitude and area of power spectra were
quantified before and after application of the non-specific GJ uncoupler
Carbenoxolone (100 µM, n=6) and the selective neuronal GJ uncoupler
quinine (250 µM). Carbenoxolone significantly reduced the peak amplitude
(64%, P

59.05
Blockade of paw incision-induced hypersensitivity by the
selective metabotropic glutamate receptor 7 agonist AMN082 in
rats
Gunn M D, Nolan A M, Biddlestone L, Dolan S
Division of Cell Sciences, University of Glasgow, Glasgow G61 1QH;
Department of Biological & Biomedical Sciences, Glasgow Caledonian
University, Glasgow G4 0BA
The aim of this study was to characterise the contribution of
metabotropic glutamate receptor subtype 7 (mGlu7) activity to
alleviation of post-incisional pain in rats, using a novel systemically
active mGlu7 receptor agonist, N,N`-dibenzhydrylethane-1,2-diamine
dihydrochloride (AMN082).
Adult male Sprague-Dawley rats (n = 6/group; 250-300 g) were
anaesthetised with isoflurane (2%) and a longitudinal 1 cm incision
was made through the skin, fascia and plantaris muscle of the left
hindpaw. Control animals were submitted to anaesthesia only. The
effects of pre-(30 min) or post-(3 h) surgical treatment with AMN082 (1
or 5 mg kg-1; i.p), or drug-vehicle, on hindpaw withdrawal latency (in
secs) to thermal stimulation, and response threshold (in grams) to
mechanical stimulation, were measured before, 2, 4, 6, 24, 48 and 72
h post-surgery.
Paw incision induced significant hypersensitivity to mechanical
stimulation at 2 and 4 h (p < 0.01), and to thermal stimulation at 6 h (p
< 0.05), and 48 h (p < 0.01). Pre-surgical administration of AMN082 (1
and 5 mg kg-1) reversed paw incision-induced thermal hypersensitivity
at 6 h and 48 h (both p < 0.05). Low-dose AMN082 (1 mg kg-1)
delayed the onset of mechanical hypersensitivity (p < 0.05). Postsurgical
administration of AMN082 failed to reverse thermal and
mechanical hypersensitivity.
This is the first report of AMN082 blocking the onset of post-injury
thermal hypersensitivity. Activation of mGlu7 receptors may provide a
new therapeutic target for the prevention of post-injury pain.
Acknowledgements: BBSRC
59.06
Ginkgo biloba extract inhibits the nitric oxide signalling pathway in
vitro and in vivo
Biddlestone L, Goldie M, Dolan S
Department of Biological and Biomedical Sciences, Glasgow Caledonian
University, Glasgow, G4 0BA., ,
Oral Administration of Ginkgo biloba extract inhibits carrageenan-induced
hyperalgesia (Biddlestone et al., Br J Pharmacol., in press). This study
investigated the role of the nitric oxide (NO) signalling pathway in Ginkgo
biloba-mediated analgesia in vivo and inhibition of inflammation in vitro.
Raw 264.7 macrophages were stimulated with LPS (0.1 ug/ml)/PBS and
treated with Ginkgo biloba extract EGb-761 (1-100 ug/ml; gifted from
Schwabe Pharmaceuticals) or drug vehicle for 24 h (n = 6/group). Adult
male rats (n=6/group; 250 – 400g) were injected into the hindpaw with λ-
carrageenan (3%; i.d.)/saline and treated with EGb-761 (30 mg/kg,
p.o.)/drug-vehicle at 3 h. Animals were euthanased at 6 h (time of maximal
hyperalgesia) and lumbar spinal cord collected. RNA was extracted from
spinal cord and Raw 264.7 cells and processed for iNOS mRNA using realtime
PCR. Media supernatant was also collected from Raw 264.7 cells and
nitrite (measure of NO release) measured (Griess reaction).
A significant increase in iNOS mRNA and NO release was observed in
LPS-stimulated Raw 264.7. Treatment with EGb-761 dose-dependently
inhibited LPS-induced iNOS mRNA (p < 0.001) and NO release (p < 0.05).
A significant increase in iNOS mRNA was detected in ipsilateral spinal cord
6 h after carrageenan injection (6 fold increase vs. saline i.d; p < 0.05),
which was blocked by treatment with EGb-761 (p < 0.05).
Results demonstrate targeted inhibition of the NO pathway by EGb-761 in
vitro and in vivo, suggesting that blockade of NO-mediated inflammation
may underlie the analgesic effects of EGb-761 observed in carrageenaninduced
inflammatory pain.
60.01
Endogenous adenosine A1 receptor activation delays
electrocortical recovery following cerebral ischemia in vivo
Andrei Ilie 1, Dragos Ciocan 1, Tudor Vladoiu 1, Haradja F 1, Zagrean
A-M 1, Alexandru Nita D 2, Zagrean L 1, Moldovan M 3,1
1 - Center for Excellence in Neuroscience, "Carol Davila" University of
Medicine and Pharmacy, Bucharest, , 2 - Neurophysiology Laboratory,
Medical School, Laval University, Québec, , 3 - Department of
Neuroscience and Pharmacology, Panum Institute of Medical
Physiology, University of Copenhagen
Global cerebral ischemia (GCI) suppresses within seconds the
electrocortical (ECoG) activity partly due to adenosine release and
subsequent A1 receptor activation. Following restoration of cerebral
circulation, ECoG recovery is much slower, proportional with the
duration of ischemia. Recent in vitro studies, using highly sensitive
biosensors, suggested that the post hypoxic/ischemic purine efflux
(PPE) contributes to the delayed recovery of synaptic transmission
during reoxygenation. The aim of our study was to investigate in vivo
the effect of PPE on ECoG recovery following a brief GCI. All
procedures were carried out in adult male Wistar rats under chloral
hydrate anesthesia. Transient 10-minutes GCI was induced by 4-
vessel-occlusion. At the onset of reperfusion, the rats were
administered either 1.25 mg/Kg of adenosine A1 antagonist 8-
cyclopentyl-1,3-dipropylxanthine (DPCPX) dissolved in 2 ml/Kg of
dimethyl sulfoxide (DMSO) or the same volume of DMSO alone
(control group) by intraperitoneal injection. We monitored the recovery
of ECoG activity and cerebral blood during the first 2 hours of
reperfusion and at 24 hours. Both in control and DPCPX groups,
complete recovery was attained within the first day after GCI, however
the rate of ECoG recovery underestimated the restoration of blood
flow during the first hour of reperfusion. DPCPX administration
reduced the mismatch between blood flow and ECoG recovery. Our
data suggest that PPE, via adenosine A1 receptors, contributes to the
delayed ECoG recovery early during reperfusion.
60.02
Systemic inflammation exacerbates ischaemic brain injury via a
neutrophil- and MMP9-dependent mechanism that targets the
neurovascular unit
McColl B W, Allan S M, Rothwell N J
Neurobiology, Faculty of Life Sciences, University of Manchester
Growing evidence suggests that systemic inflammation modulates the
response to acute brain injury. Peripheral inflammatory stimuli, such as
infection, increase the risk of stroke and are associated with poorer
outcome but the mechanisms are poorly defined. We tested the hypothesis
that a systemic inflammatory challenge exacerbates brain damage after
experimental stroke by potentiating neutrophil responses and investigated
the role of matrix-metalloproteinase-9 (MMP9) in this paradigm. Focal
cerebral ischaemia was induced in C57Bl/6J mice by transient (30min)
middle cerebral artery occlusion. Systemic inflammation was induced by
recombinant IL-1β challenge (i.p.) and the volume of ischaemic damage
and neurological deficit determined. Cerebral neutrophil accumulation,
MMP9 immunoreactivity and gelatinolytic activity were assessed 8h and
24h after MCAo. Neutrophils were depleted prior to focal ischaemia and the
effects on IL-1β-mediated exacerbation of ischaemic brain damage
assessed. The role of MMP9 was tested by co-administration of an MMP9
inhibitor (SB-3CT) with IL-1β. Systemic administration of recombinant IL-1β
significantly exacerbated ischaemic brain damage and neurological deficit
and potentiated circulating neutrophil counts and cortical neutrophil
infiltration. Neutropenia attenuated the IL-1β-induced exacerbation of
ischaemic damage. IL-1β increased MMP9 immunoreactivity and
gelatinolytic activity. MMP9 immunoreactivity was observed in neutrophils
and blood vessels whereas gelatinolytic activity was vascular and neuronal.
Co-administration of SB-3CT attenuated the IL-1β-mediated exacerbation
of ischaemic damage. These data show the detrimental effects of a
systemic inflammatory challenge on experimental stroke outcome and
indicate key roles for neutrophils and MMP9 in this paradigm. These
mechanisms may underlie the poorer outcome in stroke patients presenting
with infection.
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60.03
Characterization of the effect of selective cyclooxygenase-2
inhibition in experimental cerebral hypoperfusion in rats
Farkas E, Institoris A, Sule Z, Bari F
Department of Anatomy, School of Medicine, University of Szeged,
Szeged, Hungary, Department of Physiology, School of Medicine,
University of Szeged, Szeged, Hungary, Pharmacy, University of
Bradford, Bradford, United Kingdom,
An experimental model to create cerebral hypoperfusion as it occurs in
aging and Alzheimer’s disease is the permanent, bilateral occlusion of
the common carotid arteries of rats (2VO). Ishemic brain injury
involves the upregulation of the cyclooxygenase-2 (COX-2) having
deleterious effects on the ischemic site. We have set out to identify the
role of COX-2 in the 2VO-induced hippocampus damage.
2VO was imposed on male Wistar rats (n=24), while controls were
sham-operated (SHAM, n=31). After surgery, the animals received
either the selective COX-2 inhibitor NS-398 (3 x 10 mg/kg daily, i.p.),
or vehicle. Animals were sacrificed 3 days (n=29) or 2 weeks after the
onset of 2VO (n=26). In the later group, the memory capacity was
assessed in the Morris water maze 1 week after 2VO initiation.
Hippocampal slices were stained with cresyl violet to examine
neuronal damage. The density of MAP-2-positive dendrites, the
proliferation of GFAP-labeled astrocytes and the level of OX-42-
labeled microglial activation were determined immunocytochemically.
2VO induced memory impairment, dendritic degeneration, astrocytic
proliferation and microglial activation. The neurodegenerative
processes were more pronounced at 2 weeks as compared with 3
days after 2VO onset. NS-398 improved spatial learning but also
augmented granule cell damage in the dentate gyrus. COX-2 inhibition
was ineffective on dendritic degeneration, astrocytic proliferation and
microglial activation.
The data demonstrate that COX-2 inhibition leads to paradoxical
effects on the hypoperfused hippocampus. Since COX-2 gives rise to
a number of prostanoids with various physiological roles, the inhibition
of their combined actions may result in complex effects.
60.04
Stimulation or lesioning of the dopaminergic A11 cell group affects
neuronal firing in the trigeminal nucleus caudalis
Charbit A, Holland P, Goadsby P
Headache Group, UCL Institute of Neurology, Queen Square, London,
WC1N 3BG
Introduction: The A11 nucleus, located in the posterior hypothalamus,
provides the only known source of descending dopaminergic innervation for
the spinal grey matter. The study aimed to investigate the effect of A11
stimulation and lesioning on trigeminovascular nociceptive transmission in
the rat.
Methods: Male Sprague-Dawley rats were anaesthetised with propofol (20-
25mg/kg-1/hr-1). Extracellular recordings were made in the trigeminal
nucleus caudalis (TNC), in response to electrical stimulation of the middle
meningeal artery (MMA, 8-16v, 0.2-1ms, 0.5-0.8Hz). Receptive fields were
characterised by mechanical noxious and innocuous stimulation of the
ipsilateral ophthalmic dermatome. After recording baseline firing evoked by
MMA stimulation (20 sweeps) and receptive field nociceptive or innocuous
stimulation (2s), the A11 was either stimulated (5-50μA, 0.5ms, 5-100Hz; n
= 14) or lesioned (200-1000μA, 0.5ms, 20-50Hz for 2-3 minutes; n = 8) and
the effect on TNC firing determined.
Results: Stimulation of the A11 significantly inhibited MMA (F4.4,48.5 =
2.59; P < 0.05) and noxious pinch (F9.0,108.0 = 4.58; P < 0.001) evoked
firing of neurons from the TNC. This inhibition was reversed by the D2
receptor antagonist, eticlopride (3 mg/kg i.v; n = 5) (MMA: F1.3,3.8 = 1.65;
P = 0.284; Noxious pinch: F1.3,2.6 = 2.12; P = 0.266). Lesioning of the A11
significantly facilitated evoked firing of neurons from the TNC (MMA:
F2.3,13.6 = 3.62; P < 0.05; Noxious Pinch: F3.0,14.9 = 4.60; P < 0.05;
Innocuous bush: F4.0,23.8 = 2.43; P < 0.05).
Conclusion: Neurons in the A11 may through a dopaminergic mechanism
modulate trigeminovascular nociceptive traffic.
60.05
Activation of GluR5 kainate receptors inhibits neurogenic dural
vasodilation in trigeminovascular nociception animal model
Andreou A, Holland P R, Goadsby P J
Headache Group, Institute of Neurology London, UK and University of
California, San Francisco, San Francisco CA USA
Objectives: To investigate the possible involvement of pre-juctional
kainate receptors which carry the glutamate receptor subunit 5
(GluR5) in a model of neurogenic dural vasodilation (NDV).
Methods: Rats were anaesthetised with pentobarbitone (60 mg/kg)
and cannulated for measurement of blood pressure, experimental drug
administration and maintenance of anaesthesia. Techniques were
carried out under a project licence issued by the Home Office under
the UK Animals (Scientific Procedures) Act, 1986. The effects of the
specific GluR5 antagonist (S)-1-(2-Amino-2-carboxyethyl)-3-(2-
carboxybenzyl)pyrimidine-2,4-dione (UBP302; 50 mg/kg) and the
specific GluR5 agonist (S)-(-)-5 Iodowillardiine (IWA; 10 mg/kg) were
investigated on neurogenic and CGRP induced dural vasodilation,
using intravital microscopy.
Results: Administration of IWA was able to inhibit MMA dilation
caused by electrical stimulation (F3,12 = 10.6; P < 0.001). This effect
was blocked by pre-treatment with UBP 302 (F2,11 = 0.59; P = 0.59).
Administration of the GluR5 antagonist UBP 302 alone had no
significant effect on NDV. CGRP (1 mg/kg) induced dural vasodilation,
was not inhibited by the GluR5 agonist IWA (F2,9 = 3.2; P = 0.08).
Conclusions: The current study demonstrates that activation of the
GluR5 kainate receptors with the selective agonist IWA is able to
inhibit neurogenic dural vasodilation. This effect is likely to result from
inhibition of pre-junctional release of CGRP from trigeminal neurons.
60.06
Pre- and post-synaptic involvement of GluR5 kainate receptors in
trigeminovascular nociceptive processing
A Andreou, Holland P R, Goadsby P J
Headache Group, Institute of Neurology London, UK and University of
California, San Francisco, San Francisco CA USA
Objectives: To investigate the possible involvement of kainate receptors
carrying the glutamate receptor subunit GluR5 in trigeminovascular
nociceptive processing in the trigeminocervical complex (TCC).
Methods: Rats were anaesthetised with pentobarbitone (60 mg/kg) and
cannulated for blood pressure measurement and anaesthesia maintenance
(UK Animals (Scientific Procedures) Act, 1986). Wide-dynamic-range
neurons (n = 30), responding to electrical stimulation of the middle
meningeal artery (MMA) and microiontophorised L-glutamate, (2S,4R)-4-
Methylglutamic acid (SYM2081; kainate receptor agonist), (S)-(-)-5
Iodowillardiine (IWA; specific GluR5 receptor agonist ) or the α-amino-3-
hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor agonist (S)-(-)-5-
Fluorowillardiine (FWA), were studied. The effect of (S)-1-(2-amino-2-
carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione (UBP302), a
specific GluR5 receptor, antagonist was also studied..
Results: Application of UBP302 significantly inhibited cell firing responses
to IWA (P < 0.05) and at higher currents the responses to L-glutamate and
SYM2081. Cells responding to IWA, demonstrated a significant facilitation
of the MMA activation during UBP302 application at higher doses (P <
0.001), while non-noxious and noxious (corneal) receptive fields (RF) were
inhibited at lower doses. In cells tested for the presence of FWA responses,
non-noxious responses were significantly reduced (P < 0.001) by UBP302,
while 50% of the cells displayed significant inhibition of the MMA (P <
0.001) and noxious RF responses (P < 0.005).
Conclusions: The data provides evidence for the presence of GluR5
carrying kainate receptors on second order neurons, particularly in
superficial laminae of the TCC. The significant facilitation observed with
MMA stimulation responses at high doses of UBP302, suggests the
possible involvement of pre-synaptic GluR5 kainate receptors in
trigeminovascular nociceptive processing.
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60.07
2-Dimensional gel electrophoresis reveals altered CRMP-2
expression in an in vitro model of ischaemia
Gatherer M, Pringle A K P, Sundstrom L E, Lillycrop K A*
Division of Clinical Neuroscience, University of Southampton,
Southampton General Hospital, Southampton, * School of Biological
Science , University of Southampton , Basset Cresent East ,
Southampton
Many putative neuroprotective therapies for ischaemic stroke have
failed in the clinic, despite showing significant promise in pre-clinical
testing, possibly because they target processes occurring too proximal
to the time of the insult. If targets further downstream of the ischaemic
episode can be identified, these may prove more promising
therapeutic targets.
Organotypic hippocampal slice cultures were exposed to 1 hour
glucose and oxygen deprivation followed by a 24-hour recovery
period, and protein changes determined by proteomic analysis.
CRMP-2 (collapsin response mediator protein-2) associated peptides
were recovered from four different spots on each gel which showed
significantly altered expression in response to ischaemia.
Three of the protein spots identified as CRMP-2 showed a significant
down regulation in response to ischaemia. This was ameliorated by
the NMDA receptor antagonist MK801. In addition to CRMP-2, α-
tubulin was also suppressed, and MK801 also abolished this effect.
Conversely, the fourth spot revealed an increase in CRMP-2,
(p=

62.01
Complication outcome and clinical evaluation for the isolated and
combined craniocerebral trauma
Lim L W, Volkodav O V, Molchanov V I
Dept. of Psychiatry and Neuropsychology, Division of Cellular
Neuroscience, Faculty of Health, Medicine and Life Sciences,
Universitietssingel 50, 6229ER Maastricht. The Netherlands.
Craniocerebral trauma (CCT) has become a major cause of death and
disability worldwide. It usually results from a variety of etiologic factors
that significantly affect the cognitive, physical, and psychological
functions. Our objective is to evaluate the clinical diagnostic and
therapeutic method, and the complication outcome in both isolated
and combined CCT. Seventy-eight patients, aged 16-54 with 8%
isolated and 60% combined CCT were investigated at the
neurosurgical intensive-care unit (ICU). Approximately 2% mortality
rates result from the excessive hemorrhage during the moment of
accidents and eventually lead to cerebral hypoxia/ischemia. It was
estimated that 30% victims suffered with fatal consequences during
the ambulatory transportation to the hospital. The mortality rate in
isolated CCT accounts for 3.3% and the combined CCT consists of
20.4-35% with Glasgow-coma-scale score of 3-5. The reasons of
death were mainly due to the severe complication in combined CCT
especially caused by profuse hemorrhage and multiple injuries
including thoracic and abdominal trauma and long-bone fractures. The
therapeutic approach covers a wide range of critical care measures
including oxygenation and blood pressure, intracranial pressure
monitoring, cerebral perfusion pressure management, nutritional
support, hypertonic solution &amp; mannitol infusion, decompressive
craniotomy, and pharmacological interventions for the severe CCT
patient in the neurosurgical ICU. The treatment of CCT requires a
fundamental understanding of its pathophysiological changes in the
primary impact and secondary insults that promote excitotoxic
secondary brain damage. It is an obligatory to conduct the initial
resuscitation, elimination of cerebral compression, neuro-rehabilitation
and the prevention of avoidable complications.
62.02
Complication outcome and clinical evaluation for the isolated and
combined craniocerebral trauma
Lim L W, Volkodav O V, Molchanov V I
Dept. of Psychiatry and Neuropsychology, Division of Cellular
Neuroscience, Faculty of Health, Medicine and Life Sciences,
Universitietssingel 50, 6229ER Maastricht. The Netherlands.
Craniocerebral trauma (CCT) has become a major cause of death and
disability worldwide. It usually results from a variety of etiologic factors that
significantly affect the cognitive, physical, and psychological functions. Our
objective is to evaluate the clinical diagnostic and therapeutic method, and
the complication outcome in both isolated and combined CCT. Seventyeight
patients, aged 16-54 with 8% isolated and 60% combined CCT were
investigated at the neurosurgical intensive-care unit (ICU). Approximately
2% mortality rates result from the excessive hemorrhage during the
moment of accidents and eventually lead to cerebral hypoxia/ischemia. It
was estimated that 30% victims suffered with fatal consequences during
the ambulatory transportation to the hospital. The mortality rate in isolated
CCT accounts for 3.3% and the combined CCT consists of 20.4-35% with
Glasgow-coma-scale score of 3-5. The reasons of death were mainly due
to the severe complication in combined CCT especially caused by profuse
hemorrhage and multiple injuries including thoracic and abdominal trauma
and long-bone fractures. The therapeutic approach covers a wide range of
critical care measures including oxygenation and blood pressure,
intracranial pressure monitoring, cerebral perfusion pressure management,
nutritional support, hypertonic solution and mannitol infusion,
decompressive craniotomy, and pharmacological interventions for the
severe CCT patient in the neurosurgical ICU. The treatment of CCT
requires a fundamental understanding of its pathophysiological changes in
the primary impact and secondary insults that promote excitotoxic
secondary brain damage. It is an obligatory to conduct the initial
resuscitation, elimination of cerebral compression, neuro-rehabilitation and
the prevention of avoidable complications.
62.03
Iatrogenic traumatic brain injury: Penetration of kirschner’s
knitting needle into the middle cranial cavity
Lim L W, Volkodav O V, Molchanov V I
Dept. of Psychiatry and Neuropsychology, Division of Cellular
Neuroscience, Faculty of Health, Medicine and Life Sciences,
Universiteitssingel 50, 6226ER Maastricht. The Netherlands.
Traumatic penetrations of foreign objects into the craniocerebral cavity
are often encountered in the department of emergency and
traumatology. A 5-year-old child who was brought to the department of
pediatric neurosurgery with complains of severe headache and
fatigue. On admission, the patient had initial neurological examinations
and radiological scans. The consciousness assessment by Glasgow
Coma Scale was thirteen. Neuroradiological studies revealed a long
hyperdensed object extending from extracranial cavity into the middle
cranial fossa. A thorough history was obtained with attention to how
and when the injury was sustained. Two weeks before the incident,
the child had a blunt trauma of mandibular fractures with dislocation of
the temporomandibular joint. Maxillomandibular surgery was
performed with a Kirschner’s knitting needle to fixate the
temporomandibular articulation and simple interdental ligatures for
mandibular fracture stabilization. The present radiological film
suggested that the mandibular fracture was not properly fixated which
allowing the mobilization of Kirschner’s needle moving either
externally or internally. A standard pterional access with Frontotemporo-shpenoidal
approach was performed according to the method
of M.G.Yasargil and Oikawa-Miyazawa; followed by an extradural
approach method of V.V.Dolenc to the middle cranial structure at the
skull base. Several stages of hemostasis were carried out with
electrohemocoagulation on the penetrated Kirschner’s needle during
the needle extracting process at the extradural space of middle cranial
fossa. Two weeks after post-operation, CT scan revealed the
supratentorial and middle-craniocerebral structures were in
symmetrical localization. The patient was free of neurological deficits
and no signs of excessive CSF volume formation.
63.01
Testing for the presence of viral infection in Multiple Sclerosis using a
new human panviral diagnostic array.
Trillo-Pazos G, Khan A, Kellam P, Reynolds R, Bell J, Weiss R, Miller D
University College London , Institute of Neurology, Dept Of
Neuroinflammation and Windeyer Institute, Division of Infection of
Immunity, London W1T 4JF
MS is a neuroinflammatory disorder characterised by microgliosis, gliosis,
neuronal damage and multifocal demyelinated plaques. Clinically, MS
manifests with motor and cognitive neurological impairments. MS has being
associated with a specific HLA-DR2 phenotype and a putative viral
aetiology based on epidemiological evidence. Over the last 30 years
numerous viruses have being associated with MS, currently EBV, HHV6
and endogenous retroviruses have being implicated in MS.
We have developed a Human Panviral Diagnostic Array (HVDA) to detect
viruses in primary and secondary progressive MS post-mortem tissue
lysates from the basal ganglia and cortical regions of the brain.
Histologically these cases have active and chronic plaques as
characterised by immunocytochemistry to different cellular antigens.
Panviral arrays designed to date are based on the detection of structural
viral genes that are predominantly expressed in lytic/acute viral infection. It
is likely that MS, if viral in aetiology, is associated with latent and chronic
infection. We designed the HVDA using a biology based approach with
probes to regulatory, replicator and structural genes corresponding to 210
Human viral genomes from 25 different viral families. These arrays were
validated by using cell culture lysates from known viral infections (HIV-1,
EBV, KSHV, HHV6). We detected different patterns of viral gene
expression in latent versus lytic infection in 10, 000 to 100 cells infected
with HIV-1 or EBV using our HVDA. To conclude, we have designed and
validated a Human Panviral array to test for the presence of viruses MS
and other brain diseases ex vivo.
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63.02
Subpyrogenic systemic inflammation impacts on brain and
behavior, independent of cytokines.
Teeling J L, Felton L M, Deacon R M J, Cunningham C, Rawlins J N
P, Perry V H
CNS Inflammation Group, School of Biological Sciences, University of
Southampton, Bassett Crescent East, SO16 7PX, UK., Experimental
Psychology, University of Oxford, South Parks Road, Oxford, OX1
3UD, UK.,
Introduction: Systemic inflammation impacts on the brain and gives
rise to behavioral changes, often referred to as `sickness behavior`.
These symtoms are thought to be mainly mediated by proinflammatory
cytokines. We have investigated the communication
pathways between the immune system and the brain following subpyrogenic
inflammation using a variety of anti-inflammatory drugs,
including high affinity monoclonal anti- cytokine antibodies and
indomethacin.
Experimental methods: Low grade systemic inflammation was induced
in mice using lipopolysaccharide (LPS, 1-100 µg/kg) to mimic aspects
of bacterial infection. Changes in fever, open-field activity, burrowing
and consumption of glucose solution were assessed and immune
activation was studied in the periphery and brain by measuring
cytokine production, and immunohistochemistry to study changes in
immune phenotype.
Results: Low grade, systemic inflammation resulted in a marked
change in burrowing behavior, a species-typical, untrained behavior
that depends on the integrity of the hippocampus, without induction of
a fever response. Increased expression of cytokines was observed in
the periphery and selected regions of the brain which coincided with
behavior changes. However, peripheral neutralization of LPS-induced
pro-inflammatory cytokines IL-1ß, IL-6 and TNF-a did not abrogate the
LPS-induced behavioral changes nor affect CNS cytokine synthesis.
In contrast, pre-treatment of mice with indomethacin completely
prevented LPS-induced behavior changes, without affecting peripheral
and central cytokine levels.
Conclusion: Our work, studying the effect of low grade, systemic
inflammation on sickness behavior in mice, suggest a key role for
prostaglandins, rather than cytokines, in communicating some
components of systemic inflammation to the brain.
64.01
Thinning in the cerebral cortex in schizophrenia - findings in the
temporal lobe.
Williams M R, Perera S, Pearce R K B, Hirsch S R, Maier M
Department of Neuropathology, Research Block, Charing Cross Hospital,
Hammersmith W6 8RP
Background. The temporal lobe contains structures that have been
implicated in loss of cortical grey matter in severe mental illness.
Postmortem and imaging studies have revealed the superior temporal
gyrus (STG) and fusiform gyrus (FG) to have decreased grey matter in
indivduals suffering schizophrenia (Lee et al, 2002; Onitsuka et al, 2003;
Kawasaki et al, 2004; Suzuki et al, 2005) but not the medial temporal gyrus
(MTG) or inferior temporal gyrus (ITG) (Takahashi et al, 2006).
Methods. Coronal sections of whole temporal lobe were obtained from the
Corsellis collection from cases of schizophrenia (Sch), depressive disorder
(DD) or cases with no psychiatric illness (NPI). Images of the stained
neuropathological sections from the crowns of the STG, MTG, ITG and FG
were taken throughout the depth of the grey matter at x40 and x100
magnification. Blinded measurements were taken of the thickness of the
grey matter through cortical layers I-VI at the STG crown.
Results. Our preliminary results show that cortical thickness is significantly
lower in the schizophrenic STG (2604um, n=5) than in NPI (2962um, n=5,
p=0.025). This appears to be a decrease in layer V thickness only, NPD
(1046um), Sch (811um, p=0.075 vs. NPD) and DD (798um, n=3, p=0.047).
Conclusion. Cortical thinning occurs in the superior temporal gyrus of
schizophrenics as compared to control cases, and there is a trend toward
STG cortical thinning in depressive disorder. Observed cortical thinning is
significant in layer V in depression, and layer V shows non-significant
thinning in the STG of schizophrenics.
64.02
Escape behavior induced by electrical brain stimulation of the
periaqueductal gray & ventromedial hypothalamus
Lim L W, Temel Y, Hameleers R, Sesia T, Vlamings R, Schruers K,
Steinbusch H, Blokland A, Griez E, Visser-Vandewalle V
Department of Psychiatry and Neuropsychology, Division of Cellular
Neuroscience, Faculty of Health, Medicine and Life Science,
Universiteitssingel 50, 6229ER Maastricht. The Netherlands.
Objective: Electrical stimulation of the dorsolateral Periaqueductal
gray (dlPAG) and ventromedial hypothalamus (VMH) have been
implicated to induce escape or “panic-like” behavior. It remains
unknown which parameters are responsible for this behavior, and also
whether this can be inhibited by high-frequency stimulation (HFS). For
instance, HFS has been considered very effective treatment in most
brain disorder diseases and is thought to inhibit the target structure.
Relying on the “inhibitory effect” of HFS, we hypothesized that escape
behavior would be inhibited by HFS when the dlPAG or VMH was
stimulated.
Material and Method: The present study examined the different
levels of frequency and amplitude response in the dlPAG and VMH
microelectrode implanted male albino-Wistar rats. All animals were
subjected to the stimulation parameters at: pulse width (0.1ms),
frequency (1- 300Hz) and amplitude (1- 650microampere).
Result: Our results showed that HFS (1-300Hz) of the dlPAG has no
inhibitory effect to the escape behavior; instead, the escape behavior
was intensified in term of face validity. Whereas, VMH stimulation
demonstrated that HFS elicited escape behavior without the significant
characteristic as compared to dlPAG stimulation. All stimulated
animals, on the subsequent day, decreased their mobility and
exploratory behavior when they were introduced again into the same
open-field arena. Besides, the escape threshold of stimulation was
raised when the animals were stimulated without any sufficient resting
period.
Conclusion: It was not possible to inhibit the escape behavior with
HFS in both PAG and VMH, suggesting that these structures are not
sensitive to HFS.
64.03
Effect of buspirone treatment on the behavioral performance in the
enclosed and exposed open-field
Lim L W, Temel Y, Schruers K, Hameleers R, Steinbusch H, Griez E,
Blokland A
Department of Psychiatry and Neuropsychology, Division of Cellular
Neuroscience, Faculty of Health, Medicine and Life Sciences,
Universiteitssingel 50, 6229ER, Maastricht. The Netherlands.
Introduction: Buspirone is a 5-HT1A partial agonist and is reported to
have an anxiolytic effect. However, it is unknown whether this is related to
the different levels of anxiety. In this study, we evaluated the effect of
buspirone in two different conditions of the open-field which distinguished
as low and high anxiety.
Material and Method: 28 albino-Wistar rats (350-400g) were tested in two
different arena settings, enclosed and exposed open-field. 14-animals were
initially injected with 1ml saline while the others n=14 received buspirone
3mg/kg. Each group of animals was divided into two subgroups (each, n=7)
and tested for 2 sessions in both conditions. After a week, these animals
were again subjected to the same protocol of experiment and the sequence
of testing was reshuffled.
Results: The multivariate tests revealed no behavioral learning effect
during the testing process. One-way ANOVA showed significantly
difference between the animals treated with saline and buspirone. Post hoc
analysis revealed that buspirone treatment significantly (p

64.04
Evidence for cellular and proteomic abnormalities in the insular
cortex in schizophrenia
Pennington K, Dicker P, Dunn M J, Cotter D R
1 and 4; Department of Psychiatry and 2; Molecular and Cellular
Therapeutics, Royal College of Surgeons in Ireland. 1 & 3 Proteome
Research Centre, UCD Conway Institute of Biomolecular and
Biomedical Research, University College Dublin, Ireland.
The insular cortex is a paralimbic area of the brain thought to have a
role in sensory integration, auditory hallucinations and language.
Structural and functional MRI studies have shown abnormalities in
size and activity in schizophrenic patients in comparison with control
cases. No study do date has investigated these abnormalities at the
cellular or molecular level.
In this study stereological examination of neuronal and glial size and
density was evaluated in lamina II and III of the posterior region of the
insular cortex in 15 schizophrenic, 15 bipolar, 15 major depressive and
15 control patients. Following statistical analysis using ANCOVA,
lamina II neuronal volume was shown to be significantly decreased in
schizophrenia. 2D difference gel electrophoresis (2D-DIGE) was
subsequently used to analyze differences between protein expression
in schizophrenia and control cases in laser-assisted microdissected
lamina II tissue. Seventy eight protein spots were found to be
significantly differentially expressed (p

64.08
Modulation of ketamine-induced blood-oxygen level dependent
(BOLD) responses by an AMPA antagonist’
de Groote C, McKie S, Deakin B, Williams S
Neuroscience and Psychiatry Unit, and Imaging Science and
Biomedical Engineering; University of Manchester, Manchester, M13
9PT, United Kingdom
The glutamate hypothesis of schizophrenia proposes an important role
for glutamate in the symptoms of the disease, which can be mimicked
experimentally by ketamine (KET). We previously established
localised changes in blood-oxygenation level dependent (BOLD)
contrast in the rat brain in regions relevant to schizophrenia using
direct pharmacoMRI. To investigate whether KET-induced BOLD
changes are due to glutamate release and subsequent stimulation of
post-synaptic glutamate receptors, we pretreated with GYKI52466, an
AMPA antagonist. Young adult male rats were anaesthetised with
isoflurane (1.5%) and placed in a 7T horizontal magnet. BOLD
sensitive T2*-weighted images were acquired using a gradient echo
sequence. Ten minutes before the start of functional imaging, vehicle
or GYKI52466 (10 mg/kg) was injected (i.p.). In total 72 volumes of 70
seconds were collected, with 18 volumes (20 minutes) of baseline
scans and 52 post-injection scans (63 minutes). KET (30 mg/kg s.c.)
was injected at the start of volume 19. Data were pre-processed and
analyzed using a general linear model in SPM2. Drug and time
interactions were investigated using a one way ANOVA (uncorrected,
p&lt;0.01). GYKI52466 pre-treatment reduced KET-induced activations
in the thalamus, hippocampus, auditory cortex, cingulate cortex and
retrosplenial cortex. GYKI52466 pre-treatment enhanced KET-induced
BOLD changes in the striatum, somatosensory cortex, colliculus, and
somatosensory cortex. Our study demonstrates that pre-treatment
with the AMPA antagonist GYKI52466 reduces KET-induced BOLD
changes in key areas of the rat brain. This supports recent findings for
a role of enhanced glutamatergic transmission in schizophrenia.
CdG was supported by a NARSAD YIA Award
65.01
Confocal microendoscopic analysis of neuromuscular phenotypes in
ethylnitrosourea (ENU)-mutagenised WldS mice.
Wong F, Fan L, Coleman M P, Blanco G, Ribchester R R
MRC Mary Lyon Centre, Harwell and Centre for Neuroscience Research, 1
George Square, Edinburgh.
Severing the motor nerve supply to skeletal muscle normally triggers rapid
Wallerian degeneration. In homozygous WldSmutant mice, axon
degeneration is actively delayed by expression of an Nmnat/Ube4b
chimeric gene: disconnected motor nerve terminals persist for several days;
and axons are preserved for up to three weeks, rather than 24-72 hours
characteristic of wild-type mice. However, in heterozygous WldS mice
axotomy-induced degeneration of presynaptic motor nerve terminals occurs
at a normal rate. This observation supports a compartmental model of
neurodegeneration, according to which cell bodies, axons and nerve
terminals degenerate in response to surgical or chemical trauma by
different sub-cellular mechanisms. Discovery of other gene mutations that
selectively protect synapses would validate this hypothesis. We are
attempting this in a high-throughput screen of mice mutagenised by
ethylnitrosourea (ENU), designed to reveal covert neuromuscular
phenotypes after axotomy in vivo. We perform a novel phenotypic assay:
650µm or 1500 µm tipped fibre-optic probes connected to a Cellvizio
confocal microendoscope (Mauna Kea Technologies, Paris). The
procedure is minimally invasive yet can resolve intact and degenerating
axons and synapses in living anaesthetised (halothane/N20) transgenic
mice that co-express yellow fluorescent protein (YFP) in motor neurones as
a biomarker. We use WldS mice as a sensitized background, examining for
either additive synaptic protection or block of axonal protection following
axotomy in the F1 offspring of the ENU x thy1.2:YFP16-WldS crossbred
mice. To date, we have studied more than 23 ENU lines but none has yet
shown evidence of interaction with the WldS/+ phenotype.
65.02
Strong protection of annulospiral Ia afferent axon terminals from
Wallerian degeneration in muscle spindles of WldS mutant mice.
Oyebode O R O, Singhota J, Gillingwater T H, Ribchester R R
Centre for Neuroscience Research, University of Edinburgh, EH8 9JZ
The Wld S mouse is a mutant in which axons survive several weeks
after transection, by virtue of expression of a chimeric Nmnat1/Ube4b
protein. The Wld S phenotype extends to axons in both CNS and
PNS. Wld S also protects presynaptic terminals but studies on this
have been limited to neuromuscular junctions and synapses in the
brain. There are no published data on the degeneration of sensory
axons and their terminals in these mice. Here we report that
annulospiral endings of Ia afferent axons are very strongly preserved
after axotomy in mice. Homozygous or heterozygous Wld S mice
crossbred with thy1.2-CFP transgenic mice were sacrificed 1-20 days
after sciatic nerve transection under halothane/N 2 O inhalation
anaesthesia. Fluorescence microscopy of whole mount preparations
of lumbrical muscles revealed excellent preservation of annulospiral
endings on muscle spindles for at least 10 days after axotomy. No
significant difference was detected in the protection with age or gene
dose, in contrast to the protection of motor nerve terminals, which
degenerated rapidly in heterozygous and &gt;4-month old
homozygous Wld S mice. However, Ia afferent axons were protected
for longer than their annulospiral endings. Quantitative image analysis
of reconstructions from confocal projections (z-series) also suggested
that slow degeneration of annulospiral endings in Wld S mice occurs
by intercalary loss of their intrafusal annuli, rather than either retraction
or fragmentation, as shown by axotomised motor terminals. Thus, like
motor terminals, sensory endings are less well protected by WldSthan
their parent axons, but sensory endings are protected better and
longer than motor nerve endings.
65.03
Neuroprotective properties of the non-peptidyl radical scavenger
IACVITA in rats following tMCAO.
Nurmi A, Puoliväli J, Pussinen R, Soleti A, Bagate K, Paolini M, Riccardino
F, Grundy R I, Yrjänheikki J
Cerebricon Ltd, Microkatu 1, FIN-70211 Kuopio, FINLAND, , Medestea
Research & Production S.p.A., Via Cernaia 31, 10121 Torino, , Forenap
Pharma, France., , University of Bologna,, Bologna,, Italy., , ,
Substantial evidence exists to suggest that reactive free radicals are
generated during brain ischemia. Anti-oxidant neuroprotective agents have
also been found to be effective in animal models of stroke. However,
clinical trials have proved inconsistent. Here we investigated the effect of a
novel radical scavenger, IACVITA, on cerebral infarct volume and sensorymotor
performance in a rat transient Middle Cerebral Artery Occlusion
model (tMCAO). Male Sprague-Dawley rats were subjected to 90 min
tMCAO and treated with i.p. or i.v. injections of vehicle or IACVITA after the
onset of tMCAO. Sensory-motor performance was evaluated daily by 7 and
28 point Neuroscore tests (NS). Cerebral infarct volume was evaluated at
72 h after tMCAO. Rats exhibited a significant decrease in 7 and 28 point
NS during the 3-day monitoring period. Rats treated with IACVITA i.p. (1 or
6 h after the onset of tMCAO) or i.v. (1 h after the onset of tMCAO) showed
significant improvement in 7 and 28 point NS after tMCAO during the 3-day
follow-up period when compared to vehicle treated rats. Cerebral infarct
volumes were significantly decreased compared to vehicle in rats receiving
IACVITA i.p. 1 or 6 h or i.v. 1 h after the onset of tMCAO, which supported
observations from the 7 and 28 point NS tests. These results demonstrate
that IACVITA has unique neuroprotective properties with a wide therapeutic
window in 90 min tMCAO model in rats, which is reflected in the improved
sensory-motor performance and reduced infarct volumes.
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65.04
Lipidomic analysis of phospholipids in mouse cerebral cortex:
investigating the molecular mechanism of PUFA neuroprotection.
Williams A, Nicolaou A, Obrenovitch T
School of Pharmacy,, University of Bradford,, Richmond Road,,
Bradford, BD7 1DP
Brain phospholipids have important structural and physiological
functions. They also constitute a pool of precursors for lipid mediators.
Polyunsaturated fatty acids (PUFA) influence the activity of many
membrane – bound proteins and receptors, regulate gene expression
and participate in cell signalling. Several PUFA were neuroprotective
when administered systemically to rats and mice prior to focal brain
ischaemia, but the molecular mechanisms accounting for
neuroprotection remain uncleart. The purpose of this study was to test
whether PUFA administration causes changes in brain phospholipid
composition that could account for its neuroprotective effects. Cerebral
cortex phospholipids were analysed in mice treated with alphalinolenic
acid (ALA) or vehicle. Following their extraction from cortical
tissue samples, lipidomic analysis of phospholipids was performed by
electrospray ionisation tandem mass spectrometry (ESI–MS/ MS). We
detected 14 species of phosphatidylcholine (PC) and sphingomyelin
(SM), 24 species of phosphatidylethanolamine (PE) species, 10
species of phosphatidylinositol (PI) and 13 species of
phosphatidylserine (PS). The ALA treatment did not alter significantly
the relative abundance of any of these species, suggesting that the
neuroprotective effect of this PUFA is not directly linked to changes in
membrane phospholipids occuring prior to the test insult.
65.05
Adenosine preconditions against ouabain but not glutamate in CA1
neurons
Ferguson A L, Stone T W
Institute of Biomedical and Life Sciences,, University of Glasgow, Glasgow,
G12 8QQ
Preconditioning is induced by exposing tissue to sublethal insults resulting
in a tolerant state within the tissue protecting against further damage. To
investigate the role of adenosine in excitotoxic preconditioning, we evoked
responses from rat hippocampal slices exposed to glutamate and ouabain.
A significant depression was observed in epsp slope, orthodromic
population spike and antidromic population spike amplitudes in response to
10mM glutamate and 100µM ouabain. Antidromic population spikes
showed a significantly greater recovery following ouabain exposure (82.6 ±
1.6%) (n = 6) compared with the orthodromic responses (epsps 58.6 ±
7.1%, n = 7; population spikes 56.6 ± 8.3%, n = 8) (p

65.08
The anti-inflammatory role of Interleukin-1F5 in the rat brain
Watson M, Lyons A, Costello C, Lynch M
Department of Physiology, Trinity College, Dublin 2, Ireland
It is well established that inflammatory changes contribute to the
deficits associated with the aged brain. Central to these changes is
increased expression of the pro-inflammatory cytokines, interleukin-1β
(IL-1β) and interleukin-6 (IL-6) and increased IL-1β-induced signaling.
Recent data show that interleukin-1F5 (IL-1F5), a newly characterised
member of the IL-1 ligand superfamily shares significant homologies
with Interleukin-1receptor antagonist (IL-1ra), suggesting a possible
anti-inflammatory action. In this study the effects of IL-1F5 on
lipopolysaccharide (LPS) treatment was assessed both in vivo and in
vitro. The data show that LPS increased hippocampal expression of
IL-1β mRNA and protein and downstream IL-1β-induced signaling,
including enhanced phosphorylation of the stress-activated protein
kinases, JNK and p38. Importantly IL-1F5 abrogated this LPS-induced
pro-inflammatory cytokine profile and downstream signaling.
Furthermore, IL-1F5 attenuated the LPS induced increases in IL-1β
and IL-6 in both mixed glial cells and astrocytes. These results
demonstrate that IL-1F5 has an anti-inflammatory role in the rat brain
acting to ameliorate some of the detrimental effects of LPS.
65.09
7β-hydroxyepiandrosterone (HF0220) induces neuroprotection
through increased production of 15-Deoxy- 12,14-prostaglandin J2
(PGJ2)
Wülfert E, Rotondo D
1. Hunter Fleming Ltd. Regus House, 1 Friary, Temple Quay, Bristol BS1
6EA, UK, 2. Strathclyde Institute of Pharmacy and Biomedical Sciences
University of Strathclyde, The John Arbuthnott Building 27 Taylor Street
Glasgow G4 0NR, UK,
7β-hydroxy-epiandrosterone (HF0220) protects against neuronal cell death
in vitro (organotypic hippocampal slice cultures, OTHSC, and PC12 cells)
and brain damage in vivo (Pringle A et al. (2003) Eur. J. Neurosci. 18: 117-
124; Dudas B et al. (2004) Neurobiology of Disease 15: 262-8). HF0220
also reduces the level of soluble amyloid peptide Abeta42 in the brain of 8-
months old TgAPP/717I mice, and facilitates neurite growth from mouse
sensory neurones and spinal cord motor neurones in culture.
Recent findings show that incubation of PC12 cells and mouse sensory
neurones with indomethacin, a cyclooxygenase (COX) inhibitor, abrogates
the effects of HF0220 suggesting that COX activity is required for HF0220-
induced neuroprotection and neurite out-growth. Moreover, incubation of
human monocytic blood cells (hMBC) with nanomolar concentrations of
HF0220 caused a 10-12-fold increase in the production of 15-deoxy- 12,14-
prostaglandin J2 (15d-PGJ2), but did not significantly alter the production of
prostaglandin E2 (PGE2). In contrast, incubation of hMBC with the proinflammatory
cytokine TNFα increased the production of both
prostaglandins approx. 3-fold. Addition of nanomolar concentrations of
HF0220 caused a further increase in TNF-α-induced 15d-PGJ2 similar to
the increase observed with HF0220 alone, but completely abolished the
increases in PGE2 by TNFα.
Our results suggest that HF0220 could have beneficial effects in numerous
inflammatory degenerative disorders such as stroke and AD and that the
effects of HF0220 are mediated by 15d-PGJ2. The clinical safety of
HF0220 is currently assessed in patients with mild-to-moderate Alzheimer’s
disease.
66.01
Excess porphyrin excretion in childhood autistic disorder, a
marker of environmental toxicity
Lathe R (1), Skorupka C (2), Springbett A (3), Lam A (4), Amet L (2),
Nataf, R. (4)
(1) Pieta Research, Edinburgh, UK; (2) Association ARIANE, Clichy,
France; (3) Dept. Statistics, Roslin Institute, Roslin, UK; (4)
Laboratoire Philippe Auguste,Paris, France
Urinary porphyrin levels increase in response to inhibition of heme
synthesis enzymes. To address possible environmental toxicity in
autism porphryin levels were studied in a 269 children referred to a
Paris clinic for neurodevelopmental disorders. Subjects with autistic
disorder had elevated levels of porphyrins relative to control groups
(p

66.03
The role of p53 in Д-Tetrahydrocannabinol-induced neuronal
apoptosis.
Gowran A M, Downer E J, Campbell V A
Trinity College Institute of Neuroscience,, Trinity College,, Dublin 2,,
Ireland.
Aoife M. Gowran, Eric J. Downer, Veronica A. Campbell.
Trinity College Institute of Neuroscience, Department of Physiology,
Trinity College, Dublin 2, Ireland.
We have previously shown that the plant-derived cannabinoid Д9-
Tetrahydrocannabinol (THC) induces apoptosis via the CB1 receptor
in rat cortical neurons1. In post-mitotic neurons, the tumour supressor
protein, p53 may mediate apoptosis induced by a range of insults
including DNA damage, hypoxia and oxidative stress. This study
investigated the role of p53 in THC-induced apoptosis in cultured
cortical neurons. Exposure to THC (5µM; 5 min) caused a rapid and
transient increase in the activated form of p53 (phospho-p53ser15) via
the CB1 receptor as determined by western immunoblot. THC caused
p53 to colocalise with lysosomes, an event which co-insided with
THC-induced destabilisation of the lysosomal membrane. Neurons
exposed to THC (5µM; 2 h) displayed a significant increase in
caspase-3 activity and DNA fragmentation (P

68.01
A comparison of carbachol and kainate-induced gamma and
betaII oscillations in the rodent auditory cortex
Roopun A K, Cunningham M O, Le Beau F E N, Traub R D,
Whittington M A
(1,2,3,5) School of Neurology, Neurobiology and Psychiatry, Medical
School, Newcastle University, NE2 4HH. UK. (3) Neurology, SUNY
Downstate, Med, Ctr., Brooklyn, NY, USA.
High-frequency oscillatory activity at betaII (20-30Hz) and gamma (30-
80Hz) frequencies is proposed to mediate cognitive processing in the
cortex. Oscillatory activity is mediated by a variety of neuromodulators,
including the cholinergic and glutamatergic system. Horizontal
neocortical slices (450µm) containing deafferented auditory cortex
were prepared from adult male Wistar rats and maintained at the
interface between artificial cerebrospinal fluid and carbogen gas at
34oC. Oscillatory activity was generated upon the application of the
cholinergic agonist carbachol [25µM] or the glutamatergic agonist
kainate [400nM]. Concurrent betaII and gamma oscillations were
induced by carbachol (n=25), whereas only gamma oscillations were
induced by kainate (n=25). Kainate-induced activity was resistant to
the muscarinic-receptor blocker atropinµe [10µM] (n=6), and
carbachol-induced oscillations was resistant to kainate-receptor
antagonist UBP302 [10µM] (n=5), indicating two independently-driven
networks producing a gamma oscillation. The similarities in gamma
oscillations induced by kainate and carbachol were many-fold: the
frequencies were similar, the peak power of oscillations were
generated in superficial layers of the cortex, and all oscillations were
abolished by carbenoxolone [100µM] (a gap-junction blocker),
SYM2206 [20µM] (an AMPA-receptor antagonist) and bicuculline
[2µM] (a GABAA-receptor antagonist) (n=6). Intracellular electrodes
recorded gamma frequency firing from interneurons and gamma
frequency IPSPs from pyramidal cells, suggesting an inhibitory-based
oscillatory network. BetaII oscillations were only generated by
carbachol, and may rely on intrinsically bursting (IB) cells interacting
with an interneuronal network. This study indicates that cholinergic
and glutamatergic systems operate independently in the auditory
cortex, with the cholinergic drive exclusively producing a betaII
oscillation.
68.02
Patterns of spontaneous activity in hippocampal cultures developing
on multi electrode arrays: the influence of the NMDA receptor
Charlesworth P, Eglen S J, Humphreys L, Morton A, Grant S G N
Genes to Cognition Programme, The Wellcome Trust Sanger Institute,
Hinxton, Cambridge CB10 1SA, Cambridge Computational Biology
Institute, University of Cambridge
We have used multi electrode arrays to record action potentials from
developing embryonic hippocampal cultures and a suite of software tools to
analyse the resulting network activity. Action potentials are first observed at
~ 4 days in vitro (DIV) and thereafter activity increases sigmoidally until a
plateau at maturity (~20+ DIV) with a period of very rapid increase between
~8 – 15 DIV. Concomitant with the rise in the number of spikes, a greater
number of recording sites detect activity and also an entrainment of spikes
into bursts is observed, such that by maturity most (~95+%) action
potentials occur within bursts. Furthermore, these bursts show a
progressively increasing degree of both temporal regularity and spatial
correlation. Treating cultures with APV at DIV 6-7 (just prior to the rapid
upsweep in network activity and synaptogensis) has a profound effect on
this developing spike pattern: burst patterns are less regular, both spatially
and temporally, and many more non-bursting, individual, spikes occur. The
overall number of spikes is however unaltered. We conclude that the
NMDA receptor plays an important role in the development of spike pattern
in culture, with implications for the transfer of information in neuronal
networks. It is the aim of the Genes to Cognition programme
(www.genes2cognition.org) to employ the MEA-culture platform and
analytical methods developed here to phenotype mutant mice generated
within the programme (see Kopanitsa et al, this meeting). We are also
currently developing statistical methods for the automated analysis of these
datasets as part of the CARMEN project.
68.03
Effects of DHEA administration on auditory evoked potentials
Alhaj H A, McAllister-Williams R H
Psychiatry, Leazes Wing, Royal Victoria Infirmary, Newcastle upon
Tyne NE1 4LP
Background: The adrenal steroid dehydroepiandrosterone (DHEA)
has been shown to improve memory performance and modulate
neural activity in rodents. Further, we have recently demonstrated
beneficial effects of DHEA on long-term episodic memory performance
in healthy humans. To further evaluate the effects of DHEA on
attention and short-term memory in man, a study of auditory eventrelated
potentials (ERPs) was conducted.
Methods: Double-blind placebo-controlled crossover study in 24
healthy men (aged 18 to 34). DHEA (300 mg/day) or placebo was
administered for one week. Following each treatment period, subjects
participated in an auditory oddball paradigm, during which subjects
heard high (frequent) and low (rare) tones and were instructed to
count the number of rare tones silently. Stimulus-locked ERPs were
recorded from 29 scalp electrodes. The amplitude of the N1/P2 ERP
components, and the latency and amplitude of the P3 component to
rare tones, were calculated.
Results: DHEA treatment led to a significant increase in the amplitude
of the N1 component of ERPs recorded from posterior electrodes
(p

69.02
Effects of AB42 deposition in Alzheimer`s APPxPS1 mice:
inflammatory response and regional MRI volumetry
James, M.F. (a), Maheswaran, S. (b), Barjat, H. (a), Rueckert, D. (b),
Bate, S.T. (e), Howlett, D.R. (a), Tilling, L. (a), Smart, S.C. (a),
Pohlmann, A. (a), Hill, D.L.G. (c), Hajnal, J.V. (d), Upton, N. (a)
a) Neurology & GI CEDD, GlaxoSmithKline, Harlow, UK., b) Dept. of
Computing, Imperial College, London, UK., c) IXICO Ltd, London, UK.,
d) Imaging Sciences Dept., Imperial College, London, UK., e)
Statistical Sciences, GlaxoSmithKline, Harlow, UK.
In Alzheimer`s disease (AD) progressive brain volume changes are
observed, which correlate with clinical status and amyloid (Aβ42)
deposition. TASTPM transgenic mice over-express the AD associated
human proteins APP(K670N,M671L) x PS1(M146V) resulting in
progressive deposition of cerebral Aβ42.
To investigate the deposition of Aβ and its effects on brain structure,
as well as inflammatory processes, immunohistochemistry and
regional MRI volumetry were utilized. An advanced non-linear image
registration technique in combination with the LONI mouse atlas
(UCLA) was used to calculate the volumes of the 14 largest atlas
regions. We studied 17 TASTPM and 14 wildtype (WT) mice in vivo
from the age of 6 months (repeatedly imaging at 6,9,11,14 m).
Abundant APP was found already in young TASTPM (WT none)
resulting in a dramatic and widespread increase in Aβ42. The
inflammatory response was observed as considerable astrogliosis and
microgliosis. Between 6-11 months whole brain volumes of WT
increased significantly, but levelled thereafter. This sustained growth
has been reported in the literature. In TASTPM however, brain volume
growth is similar up to 11 month but continues afterwards. The
majority of individual brain regions also grew significantly in both
strains, but different temporal trends were found in several regions
with some growing in TASTPM while changing little in WT, including
cerebral cortex, hippocampal formation, and thalamus. A rationale for
these observations is based on the sheer volume of Aβ deposited and
resulting astrogliosis.
Volumetric MRI detects structural brain changes, which are consistent
with immunohistochemical findings and assumed to result from
amyloid deposition.
69.04
MRI measurements of diffusion, perfusion and T2 with proteomic
analysis in the rat hippocampus following status epilepticus
Choy M (1), Scott R C (1), Thomas D L (2), Gadian D G (1), Greene N
D E (1), Wait R (3), Leung K-Y (4), Lythgoe M F (1)
(1) UCL Institute of Child Health, London; (2) Department of Medical
Physics and Bioengineering, University College London, London; (3)
Kennedy Institute of Rheumatology Division, Imperial College London,
London; (4) William Harvey Research Institute, Bart`s and the London,
London.
Introduction Status epilepticus (SE) in humans may be associated
with hippocampal injury, epileptogenesis and development of temporal
lobe epilepsy. Diffusion (ADC), perfusion (CBF) and T2 MRI changes
have been reported in both clinical and experimental settings following
SE. However, the temporal relationships between these changes
remain uncertain. The aim of this study was to characterise diffusion,
perfusion and T2 in the lithium-pilocarpine model of SE in the rat and
proteomic analysis was used to investigate the underlying tissue
status.
MethodsSprague-Dawley rats were injected with lithium chloride
(3mEq/kg i.p.) 18 to 20h prior to either pilocarpine (30mg/kg) (n=6) or
saline (n=6). Diazepam (10mg/kg i.p.) was administered 90 min after
the onset of SE. MRI was performed pre-injections and on days 0, 1,
2, 3, 7, 14 and 21 days after SE. For the proteomics study (n = 6),
animals were imaged and sacrificed on day 2 for proteome analysis
using 2D gels and mass spectrometry.
Results and Discussion We have demonstrated that time-dependent
hippocampal changes in ADC, CBF and T2 occur following SE. These
changes peaked on day 2 and returned to baseline by day 7. The
time-dependence of these changes may indicate an opportunity for
early intervention and therefore we conducted proteomic analysis on
the hippocampus on day 2. We identified changes in proteins related
to stress (HSP-27), the cytoskeleton (alpha-tubulin, ezrin), and
neurogenesis (CRMP-2). Further studies are necessary to elucidate
the mechanisms that underlie these changes and the role that they
may play in epileptogenesis.
69.03
Pathologies in the thalamus of TASTPM transgenic mice model of
Alzheimer’s disease - characterisation by MRI, micro-CT and histology
Evans S C, Barjat H, Pohlmann A, Tilling L, Vidgeon-Hart M, Hayes, B.P.,
Upton, N., James, M.F.
Neurology and GI CEDD, GlaxoSmithKline, Harlow, UK
TASTPM transgenic (Tg) mice over-express the Alzheimer’s disease (AD)-
associated human proteins APP(K670N, M671L) x PS1(M146V). Cerebral
Aβ is progressively deposited, and is widespread at 6 month. The Tg model
will be most useful if plaque deposition is accompanied by
neurodegeneration, as in AD patients.
We repeatedly carried out in-vivo MR imaging of TASTPM and wildtype
(WT) strains from 6 to 14 months of age to try and measure temporal
neurodegenerative changes non-invasively in vivo. Some animals were
imaged post-mortem using MRI and micro-CT, the brains were then
analysed using histology. All MR images obtained were T2*-weighted. All
observations reported below are seen in Tg, but not in WT animals.
Serial in vivo MR images reveal the presence of progressive pathologies in
the thalamus of animals from 6 months of age. Post-mortem CT images
show areas of X-ray dense material in the thalamus; the latter coincide with
the areas seen by post-mortem MRI.
Histology shows elevated levels of astrocytes and glial cells (GFAP and
iba-1 respectively) and presence of amyloid deposits (1E8) in all areas of
the brain. In the thalamus, it shows co-accumulation of calcium (von Kossa)
and ferrous iron (positive Schmeltzer and negative Perl) with some amyloid
plaques. The distribution of calcium plaques coincides with that of the
features observed in CT and MR images.
The thalamic pathologies described may be the equivalent of calcifications
or micro-bleeds seen in the brains of AD patients.
69.05
Pharmacological challenge magnetic resonance imaging in rat brain
following cannabinoid receptor agonist THC, or the antagonist,
rimonabant
Stark J A, Dodd G, Williams S R, Luckman S M
1,2,4: Faculty of Life Sciences, 3: Imaging Science and Biomedical
Engineering, University of Manchester, Manchester M13 9PT.
Delta9-tetrahydrocannabinol (THC) increases feeding in satiated rats by
acting on central reward systems. It was suggested that cannabinoid
receptor 1 (CB1) antagonists/inverse agonists could treat obesity, and this
has led to the CB1 inverse agonist rimonabant to be tested in phase 3
clinical trials, despite a lack of information on its site of action. We have
used pharmacological challenge MRI to compare brain blood oxygen level
dependent (BOLD) maps produced by 1mg/kg THC with an anorectic dose
of rimonabant (1mg/kg).
THC and rimonabant had strikingly opposite effects. Rimonabant increased
BOLD signal in sensory and motor, as well as in limbic regions of the brain.
THC produced either decreased or no signal in these same areas. THC
increased BOLD signal in olfactory cortical areas and the anterior
amygdala, but had no effect in the hypothalamus – areas that displayed
decreased signal with rimonabant.
It is difficult to attribute regional brain activity to specific effects of the drug,
but these results suggest that rimonabant may reduce food intake by acting
on the limbic forebrain. This is supported by the fact that THC had weak
though consistently opposite effects in these areas. In addition, changes in
BOLD signal were observed in motor systems. This is consistent with
known actions of cannabinoids, though no altered motor behaviour
following this dose of rimonabant is reported in the literature.
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69.06
Comparison of data analysis techniques for direct
pharmacological challenge fMRI (phMRI)
McKie S (1), Lees J (1), Richardson P(1), Elliott R(1), Anderson I (1),
Deakin B (1), Williams S(2)
(1) Neuroscience and Psychiatry Unit (2) Imaging Science and
Biomedical Engineering, , University of Manchester
Direct pharmacological challenge-fMRI (direct phMRI) is used to study
the dynamic effects of drugs on brain haemodynamics. A common
analysis approach is to use subjective-ratings (subjective effects
scales, SES) to measure subject-specific changes while a drug is
being infused. However, there are several problems with this method.
Therefore, we have developed an alternative based on the
conventional block analysis of fMRI data – the pseudo-block analysis
method (p-block).
The infused drugs were mCPP and ketamine. Each subject underwent
a 9 minute fMRI scan during which they self-assessed their mental
state using SES. After 1 minute the drug/saline was infused. Images
were acquired on a 1.5T Philips scanner with a multi-slice, single shot
EPI sequence to achieve whole brain coverage. Data were analysed
using SPM5. The SES analysis involved creating a regressor based
on significant ratings from the SES and a random effects two-sample
t-test. The p-block analysis involved dividing the 9 minute fMRI scan
into 9 one-minute time-bins (T0 to T8). The average signal for each
time-bin (Tn) was compared to the pre-infusion average (T0) and a
random effects ANOVA was used to investigate significant signal
changes.
Both analysis methods resulted in the detection of similar areas for
each drug, however more areas were present in the p-block analysis.
This is thought to be due to individual differences in drug-induced
BOLD signal time courses as well as latency effects between different
brain regions. The p-block method can also be applied when no
regressor is available such as animal studies.
69.07
Structural correlates of autistic features in young people with special
educational needs: a voxel-based morphometric analysis
Spencer M D, Moorhead T W J, Hoare P, Muir W J, Owens D G C, Lawrie
S M, Johnstone E C
Division of Psychiatry, Royal Edinburgh Hospital, Morningside Park,
Edinburgh, EH10 5HF, UK
Background: A strong association exists between intellectual disability
and autism spectrum disorders (ASD). Although previous studies have
investigated the neural correlates of ASD in intellectually unimpaired
subjects, the present study is the first to address these issues in
intellectually impaired subjects.
Methods: We used structural MRI and voxel-based morphometry to study
63 intellectually disabled adolescents receiving additional learning support,
recruited via their place of education. Participants comprised 34 males and
29 females with mean age 16.0 years (SD: 1.8 years) and mean IQ 59.7
(SD: 7.7). We used the Social Communication Questionnaire to classify
participants according to their parentally reported degree of autistic
features, as scoring 1) below the threshold for ASD (n=32); 2) within the
pervasive developmental disorder (PDD) range (n=16); and 3) above the
threshold for autism (n=15). Groups did not differ significantly in terms
of gender, age or IQ. We report tissue density differences at cluster level
with adjustment for underlying smoothness.
Results: We detected a reduction in grey matter density in the thalamus of
subjects with autistic features scoring within the PDD range as compared to
subjects below the threshold for ASD (p