Belgian Society for Neuroscience

The 4th bi-annual meeting 2001

Abstract list

DISTRIBUTION OF REELIN IMMUNOREACTIVITY IN THE BRAIN OF ADULT MALE EUROPEAN STARLING (Sturnus vulgaris)

P. Absil (1), R. Pinxten (1), J. Balthazart (2) and M. Eens (1)
1. Dept. Biol., Univ. Antwerp UIA, B-2610 Wilrijk, Belgium, and 2. Univ.Liège, Ctr. Cell. Molec. Neurobiol., B-4020 Liège, Belgium.

In the adult songbird brain, the nucleus HVc incorporates new neurons mainly in autumn. Steroids, and more specifically estradiol (E2), facilitate this process through post-mitotic mechanisms. However, as no E2 receptor can be detected neither in migrating nor in newly differentiated HVc cells, E2 is supposed to act on inter-neurons.
Reelin (Re) is involved in the positioning of young cortical neurons during development. Re is also expressed in the adult brain, but its role is unknown. This study represents a first step in investigating the potential involvement of Re in the neurogenesis process observed in the adult songbird brain.
Re and the division marker BrdU were immunocytochemically visualized in the brains of control and T-treated young adult male starlings. Both antigens were detected in the telencephalon, and in particular in HVc. Re was also present in di-, mes- and metencephalic regions where no adult neurogenesis has been reported. Exogenous T dramatically decreased Re expression in specific brain regions, like HVc.
Taken together, these data are consistent with the idea that Re may play a role in determining the final localization of newborn neurons in the adult songbird brain.
Supported by WO.007.96N, G.0075.98, NS35467, FRFC2.4555.01; P.A. is FWO postdoc.

MOLECULAR EVIDENCE FOR TRANSLATIONAL REGULATION OF TPH mRNA in RAT DRG NEURONS

Alvarez Gonzalez M-L (1,2) , Antoine M. (1), Rogister B. (2), Malgrange (2), Schoenen J. (2), Moonen G. (2) and Delre P. (1,2)
1. Inst. Path. Gen., B6280 Loverval, Belgium and 2. Ctr. Molec. Neurobiol., B4020 Liège, Belgium.

We previously demonstrated that culturing adult rat dorsal root ganglia (DRG) neurons modifies their neurotransmitter phenotype. In particular, serotonin (5-HT) and its rate-limiting enzyme tryptophan-hydroxylase (TPH) were identified in culture but not in vivo or in freshly dissociated cells.
In the present study, we adapted a semi-quantitative PCR technique (TaqMan) to measure TPH mRNA. Surprisingly, the amount of TPH mRNA expressed in DRG in vivo (i.e after two hours of dissection) or after 48 hours in vitro was similar. In other words, TPH mRNA is present at the same level in vivo and in vitro in DRG neurons, but TPH protein is only expressed in vitro. Taken together, these results suggest that the discrepancy between TPH mRNA and TPH or 5-HT expression may result, at least in part, from a difference in the efficiency of TPH mRNA translation between these two conditions.
These results raise different questions

What are the molecular mechanisms underlying such a regulation ?
Does such a regulation of TPH occur also in the raphe or epiphysis ?
Is TPH also expressed in Human DRG neurons in vitro ?
Does this in vitro TPH expression have in vivo equivalence, especially in development or in pathological conditions such as pain regulation (b.e. in fibromyalgy) ?

THE DISTRIBUTION OF TYROSINE HYDROXYLASE IN THE CANARY BRAIN REVEALS A HIGH, SPECIFIC AND SEXUAL DIMORPHIC CATECHOLAMINERGIC INNERVATION OF THE TELENCEPHALIC NUCLEI INVOLVED IN THE LEARNING AND PRODUCTION OF SONG.

D. Appeltants (1), G. F. Ball (2), J. Balthazart (1)
1. University of Liège, Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, 17 place Delcour, 4020 Liège. 2. Department of Psychology, The Johns Hopkins University, Baltimore, USA

Catecholamines have a wide range of actions in the brain, including the modulation of cognitive processes. To assess their possible implication in the control of song, we investigated here the distribution of tyrosine hydroxylase-immunoreactive (TH-ir) structures in the brain of male and female canaries. The major catecholaminergic cell groups identified in the brain of mammals were also observed in canaries and, TH-ir fibers were particularly abundant in the lobus parolfactorius, paleostriatum primitivum and nucleus septalis lateralis. In both sexes, a high density of TH-ir basket-like structures was found in the caudal neostriatum, an area involved in song perception and recognition. A high density of TH-ir fibers outlined the telencephalic motor song control nuclei HVc and RA in most males but not in females. In contrast, area X, a nucleus mainly involved in song learning and recognition, was defined by a high density of TH-ir fibers in both sexes. This high, specific and sexually dimorphic catecholaminergic innervation supports the notion that the morphological evolution of the song control nuclei was accompanied by a neurochemical specialization and, suggests that dopamine and/or norepinephrine play important roles in the modulation of song learning and production.

MOLECULAR MECHANISMS OF ADULT BRAIN PLASTICITY: GLUTAMATE AND BEYOND

L. Arckens, E. Vandergucht, I. Leysen, G. Vandenbergh, A. Massie, S. Clerens, K. Vandamme, F. Vandesande
Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Belgium

The mammalian central nervous system possesses the well-known capacity for activity-dependent circuitry rearrangement - or plasticity - even in adult life. In the sensory systems, loss of sensory input due to restricted deafferentations or injury to the sensory surface results in a dramatic topographic reorganization of the cortical maps.
Possible structural mechanisms for injury-induced cortical reorganization are the potentiation of previously existing sub-threshold inputs, axonal and dendritic growth and synapse turnover. Unfortunately, the molecular cascade responsible for representational plasticity remains illusive. Recent investigations using a battery of complementary techniques namely differential display, in situ hybridization, (S)Q-PCR, immunocytochemistry, western blotting, high performance liquid chromatography, 2-D electrophoresis and mass-spectrometry have lead to the implication of the neurotransmitter glutamate, its transporters and receptors, the housekeeping gene cyclophilin A, the anti-oxydant enzyme SOD1 and the phosphorylating enzyme CaM kinase II alfa in cortical plasticity. The identification of cellular mediators of cortical plasticity may lead to the development of new pharmaceuticals useful in the treatments for sensory loss and brain damage.

THE EFFECTS OF A CHANGE IN GRAVITY ON THE DYNAMICS OF PREHENSION

A-S. Augurelle, M. Penta, N. Heglund, P. Willems, O. White, J-L. Thonnard
Unit de Radaptation et de Mdecine Physique, Universit catholique de Louvain, Brussels, Belgium.

We have studied how humans modulated their grip force (GF) when the gravitational and the inertial components of the load were varied independently during parabolic flight. Eight subjects participated to this study; four had already experienced parabolic flights (ES) and four were inexperienced (NES). They had to move continuously a manipulandum up and down throughout the gravitational conditions (1g, 1.8g, 0g). In 1g, GF precisely anticipated the fluctuations of the load which was maximum at the bottom and minimum at the top of the trajectory. When the gravity changed (0 g and 1.8 g), the grip-load co-ordination remained for all the subjects from the first parabola. In 0g, GF was accurately adjusted to the two peaks of load occurring at the two trajectory's extremities due to the absence of weight. While the level of GF modulation was immediately adapted to the new force field for ES, the NES dramatically increased their grip when faced for the first time to altered gravity. A progressive release of the grip occurred and a continuous grip-load force relationship with regard to 1g was established after the fifth parabola. New gravitational field was rapidly integrated in internal models by the CNS which had some ability to reuse them as called by the occasion.

GENOMIC ORGANIZATION OF THE HUMAN AND MOUSE DAB1 GENES AND PRELIMINARY CHARACTERIZATION OF PROMOTER REGIONS.

I. Bar, T. Dauzo, C. de Raikem, A.M. Goffinet
Neurobiology Unit, Univ. Namur Med. School. Namur.

The Dab1 gene plays a key role in brain development, as Dab1 -/- mice have a reeler-like malformation. Dab1 is an intracellular adaptor expressed in cortical plate neurons. It interacts with the cytoplasmic sequence of two lipoprotein receptors, VLDLR and ApoER2, which bind extracellular Reelin secreted by Cajal-Retzius cells. The mouse Dab1 gene encodes transcripts corresponding to proteins of 555, 217 and 271 amino-acids. To identify regulatory elements responsible for the cell specific expression of Dab1, we defined the genomic organization of the human and mouse genes. The organization is conserved in both species. The coding regions (from exon 2 containing the ATG, to exon 14 containing the STOP) are spread over 320 kb of genomic DNA. Exon 15 contains a 3' UTR and is 3350 bp long. With the exception of exon 15 and exon 12 (546 bp,) exons are relatively small (39 to 140 bp). Introns sizes range from 85 bp to 146 kbp. Using 5'RACE, four alternative exons-1 were identified in human and mouse, suggesting the presence of multiple promoters. Three exons-1 are clustered in a 3 kb fragment, separated from exon 2 by an intron of more than 110 kb. A fourth exon-1 is localized more than 100 kb upstream from this 3 kb block, so that the whole Dab1 gene extends on more than 500 kb.

CONSTRUCTION OF TRANSGENIC MICE EXPRESSING A PROTEIN KINASE A INHIBITOR SPECIFICALLY IN THE CEREBELLAR GRANULE CELLS

B. Bearzatto, F. Baba-Assa, M. Verslijpe, A. de Kerchove dExaerde and S. N. Schiffmann
Laboratoire de Neurophysiologie et Physiopathologie du Systeme Nerveux, Faculte de Medecine, Universite Libre de Bruxelles, Brussels, Belgium.

Long-term changes in synaptic strength are believed to underlie some forms of learning and memory in the mammalian brain. In the cerebellum long-term potentiation (LTP) and long-term depression (LTD) have been revealed at the parallel fiber-Purkinje cell synapse. LTD occurs when parallel fiber and climbing fiber inputs are coactivated at moderate frequencies and its molecular mechanisms were extensively characterized. Cerebellar LTP is elicited by parallel fiber low frequency stimulation and its underlying mechanisms are mostly unknown, although in-vitro studies suggested the requirement of protein kinase A (PKA).
To explore in vivo the involvement of PKA in the induction of this LTP and the role of this LTP in the cerebellar physiology, we generated transgenic mice that express an active fragment of the PKA inhibitory protein (PKIa 1-31) specifically in cerebellar granule cells. We directed the PKIa 1-31 expression to cerebellar granule cells using a promoter of the gene coding for the GABAAa6 subunit. This promoter was previously shown to allow expression of a transgene solely in cerebellar granule cells.
Transgenic mice were produced by pronuclear microinjection of the construction. Four founders were identified and were crossed with C57Bl6 mice. The F1 were analysed.

EFFECTS OF S100A4 AND S100A6 ON ASTROCYTIC TUMOR CELL MIGRATION AND ON REGULATORS OF ACTIN CYTOSKELETTON ORGANIZATION

Nathalie Belot (1), Isabelle Salmon (2), Roland Pochet (1), Claus W. Heizmann (3), Robert Kiss (1), Christine Decaestecker (1)
1. Laboratory of Histopathology, Faculty of Medicine, and 2. Department of Pathology, Erasmus University Hospital; 3. Universit

Previously we showed a differential expression of various S100 proteins in astrocytic tumors with increasing malignancy levels. Of these proteins, the S100A4 and S100A6 proteins are suspected to be involved in cell migration because of their possible interaction with actin-binding proteins.
The present work aims to investigate the potential role of extracellular S100A4 and S100A6 on the migration of astrocytic tumor cells. Cell motility was investigated by means of computer-assisted videomicroscopy able to quantify speed and migration rate on living cells. In parallel we analyzed possible changes in the actin cytoskeleton and in the expression of a number of its molecular regulators (by means of RT-PCR, Western blotting and immunofluorescence). The regulators so investigated included small Rho-GTPases (i.e. RhoA, Rac1 and Cdc42) and actin-binding proteins (i.e. gelsolin, profilin and -actinin). Our results on motility features revealed that, in contrast to S100A6, externally added S100A4 is able to significantly stimulate the migration rate of astrocytic tumor cells. This effect is paralleled by a decrease of the F-actin levels as well as a decrease in expression of various potential actors in the regulation of the actin cytoskeletton (e.g., RhoA, Cdc42, gelsolin and profilin).

DIFFERENTIAL STRIATAL ALTERATIONS INDUCED BY3-NITROPROPIONIC ACID IN THE LEWIS RAT BINDING AND IN SITU HYBRIDIZATION DATA

D. Blum (1), D. Gall (1), L. Cuvelier (1), D. Brouillet (2), D. N. Schiffmann (1).
1. Laboratoire de Neurophysiologie,ULB-Erasme, Bruxelles,Belgium; 2. Département de Recherche Médicale, Orsay,France

Administration of 3-nitropropionic acid (3NP), a succinate dehydrogenase inhibitor, is able to reproduce motor, lesional and cognitive characteristics of Huntingtons disease in both rodents and primates. Chronic intoxication of Lewis rats, at a dose of 56mg/kg/j delivered by osmotic minipumps, induces behavioural manifestations 3 and 4 days after the beginning of the infusion without any histological alterations. At day 5, highly reproducible localized striatal lesions are observed. Using binding autoradiography and in situ hybridization, we have studied the kinetics of the expression of various striatal receptors (D1, D2, A2A, NMDA) and peptides (Enk, SP). We particularly observed a general and strong decrease of mRNA expression in the lateral part of the striatum where the lesion takes place whilst the densities in D1 and A2A receptors binding sites were only moderately decreased. In the perilesional area of the striatum as well as in the cortex, we interestingly observed upregulation of various markers. These results thus show that the 3NP-mediated specific striatal injury is associated with late alterations in gene expression and receptor binding sites density and that perilesional modifications could be related to a mechanism of functional compensation.

VAGAL NERVE STIMULATION (VNS) REDUCES FORMALIN-INDUCED FOS IN THE CAUDAL SPINAL TRIGEMINAL NUCLEUS (CSTN).

C. Bohotin, M.Scholsem, G.Brook, S.Multon, D.Martin, V.Bohotin, J.Mosen, J.Schoenen.
University of Liège, Center for Cellular and Molecular Neurobiology

To determine in rats the effects on trigeminal nociception of left cervical VNS with an implanted stimulator (NCP Cyberonics).
The vagal nerve was stimulated (20 Hz; 1mA; 1 msec pulse width; 20 sec on-18 sec off) for 24 h before and for 90 min after a s.c. injection of formalin 5% in the lip (n=5). Control animals were either not stimulated (n=2) or not implanted (n=4). Fos immunoreactivity (Fos-IR) was used as a marker for neuronal activity. The number of Fos-IR neurons was counted in 5 sections of cSTN and the difference (D) between injected and non-injected sides was calculated.
VNS induced a non-significant increase of D Fos-IR in cSTN. By contrast, VNS significantly reduced the peak levels of D Fos-IR which had been induced by the formalin injection (p<0.03).
Our data indicate that high frequency left cervical VNS is able to reduce the activation of 2nd order cSTN nociceptors in the acute formalin pain model. Whether this effect is due to interactions between vagal afferents and trigeminal brain stem nociceptors or mediated via descending pain control systems remains to be determined.VNS, already used in humans as a treatment for epilepsy and depression, might also be useful for resistant head and facial pains.
Study supported by NFSR grant 3452300.

VAGAL NERVE STIMULATION (VNS) LOWERS THERMAL PAIN TOLERANCE IN RATS

C.Bohotin, M.Scholsem, S.Multon, G.Brook, V.Bohotin, D.Martin, M.Mozin, J.Schoenen.
University of Liège, Center for Cellular and Molecular Neurobiology

VNS might be effective in pain. It is not known if the same stimulation parameters can be used as in epilepsy. We compared in rats the effect on heat tolerance of 2 patterns of left VNS with an implanted stimulator (NCP-Cyberonics).
We measured the time a hindpaw was maintained in 50C water before and after VNS with 2 patterns. Pattern 1 (n=4) 5 Hz, 2 mA, 0.5 msec width, 30 sec on /5 min off ; pattern 2 (n=7) 20 Hz, 1 mA, 1 msec width, 20 sec on /18 sec off. VNS was started 24h after implantation and continued for 2-6 days. Thermal tolerance was measured after 2h and then dayly until values stabilized.
Pattern 1 induced a tolerance decrease on day 1, which reversed to reach levels above baseline at day 4 (221 %, p >0.05). With pattern 2, heat tolerance increased by 2 h (235.2 %, p<0.05) and remained stable afterwards.
This study demonstrates that NCP-VNS modifies thermal pain tolerance in rats. A stimulation pattern similar to that used in epilepsy treatment (pattern 1) has a biphasic effect and requires several days to obtain a modest tolerance increase. By contrast, an increase in stimulation frequency and duty cycle (pattern 2) induces a rapid and stable increase in thermal pain tolerance, which may have clinical implication.
Study supported by NFSR grant 3452300.

THE EFFECTS OF LOW AND HIGH FREQUENCIES REPETITIVE TRANSCRANIAL MAGNETIC STIMULATION ON HABITUATION IN HEALTHY VOLUNTEERS

Bohotin V., Fumal A., Bohotin C., Schoenen J.
Univerity of Medicine, Department of Neurology, Liege, Belgium

To study the effects of repetitive transcranial magnetic stimulation (rTMS) on excitability of the cerebral cortex in healthy human volunteers by recording the visual evoked potential during sustained pattern-reversal stimulation (PR-VEPs).
We performed rTMS on visual cortex in normal subjects (n=24) with a 8-shape magnetic coil placed over the occipital scalp. 900 pulses were delivered at two different frequencies 1 Hz rTMS and 10 Hz rTMS. 1 Hz rTMS decreased N1-P1 and P1-N2 amplitudes in the first block in 80 % of subjects. A significant reduction of habituation or even a potentiation were also observed after 1 Hz. rTMS. 10 Hz rTMS produced an increase of the first block amplitude in 56 % of subjects, but no significant change of habituation.
As 1 Hz rTMS is known to inhibit the underlying cerebral cortex, the loss of habituation of PR-VEPs after repetitive stimuli observed here is likely to be due to a decrease of cortical excitability. The increase of cortical excitability, which, did not produce significant modifications of PR-VEPs. Our findings suggest that VEP habituation is an active process, that can be reduced by inhibiting cortical structures, which may be relevant for the interpretation of the habituation deficit of PR-VEPs reported in disorders such as migraine.

ANALYSIS OF TAU PHOSPHORYLATION IN TRANSGENIC MICE EXPRESSING ALZHEIMER DISEASE MUTANT APP AND PS1 PROTEINS

A. Boutajangout (1), M. Authelet (1), N. Touchet (2), Blanchard (2), G.Tremp (2), L. Pradier (2), J. P. Brion (1)
1. Laboratory of Histology and Neuropathology, Universit Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Brussels, Belgium. 2. Aventis Pharma, Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine, France.

Neurofibrillary tangles made of tau proteins and amyloid deposits made of the A peptide are neuropathological lesions observed in sporadic forms of Alzheimer disease (AD) and in familial forms due to mutations of presenilin 1 (PS1) and of amyloid peptide precursor (APP) genes. We have analyzed a transgenic mouse line expressing a human tau isoform, a mutant form of APP (751SL) and a mutant form of PS1 (M146L). Transgenic proteins were co-expressed in the same neurons. As in tau transgenic mice, a somatodendritic accumulation of tau was found in this transgenic line. These mice developped cerebral extracellular deposits of A, starting at 2.5 months. The A deposits were surrounded by a neuritic and a glial reaction. As in AD, the neuritic reaction was composed of dytrophic processes either globular (APP positive) or fusiform (tau positive). The latter processes were labelled by several tau phosphoantibodies characteristic of PHF-tau proteins. Animals observed up to 12 months of age did no develop neurofibrillary tangles. These results indicate that overexpression of mutated forms of PS1 and APP was not by itself sufficient to induce the formation of neurofibrillary tangles over this period of time, although local changes in tau phosphorylation were observed around A deposits.

PHOTOLYTIC RELEASE OF IP3 BUT NOT OF CALCIUM INITIATES THE RELEASE OF A PURINERGIC MESSENGER IN ENDOTHELIAL CELLS

K. Braet, W. Vandamme and L. Leybaert
Dept. Physiology and Pathophysiology, Ghent University, Ghent, Belgium

Calcium signals can be communicated between the cells of the blood-brain barrier by an intracellular/gap junctional and an extracellular/purinergic pathway. Evidence for the latter pathway largely derives from experiments in which mechanical cell stimulation was used. In the present study we investigated involvement of the two pathways in calcium signal propagation initiated by photolytically releasing IP3.
The experiments were performed on the cell lines SV-ARBEC, RBE4 (two brain endothelium cell lines) and ECV304. Releasing IP3 in a single cell initiated an intercellular calcium wave that was significantly reduced in size by the gap junction blocker a-GA and a connexin blocking peptide. IP3-triggered calcium waves could overpass cell-free lanes and were significantly inhibited by the purinergic inhibitors suramin, PPADS and apyrase, indicating involvement of the extracellular/purinergic pathway. FRAP experiments excluded the possibility of side effects of the purinergic inhibitors on gap junctional coupling. Photolytically releasing calcium did not produce any propagating calcium wave.
These results demonstrate that IP3 and the downstream calcium signal are able to trigger the release of a purinergic messenger, while an applied calcium signal alone is not sufficient.

INVESTIGATION OF THE INFLAMMATORY RESPONSE DURING ATTEMPTED TISSUE REPAIR AFTER EXPERIMENTAL SPINAL CORD INJURY

GA Brook (1), D Martin (2), F Scholtes (1), J Schoenen (1)
1. Department of Neuroanatomy, University of Liège; 2. Department of Neurosurgery, CHU Sart-Tilman, University of Liège

Post-traumatic inflammation plays an major role in the events taking place after spinal cord injury (SCI). Both beneficial and detrimental effects of inflammatory cells have been reported. The relationship between the activation status of macrophages and their effects on CNS tissue repair remain poorly understood. Here, we describe an apparent correlation between attempted spontaneous tissue repair and the phenotype of phagocytic macrophages which invade the lesion site of a clinically relevant model of spinal cord compression injury. The spinal cord of anaesthetised adult female Wistar rats was lesioned using a sub-durally positioned inflatable balloon. Morphological analysis showed a rapid invasion of activated macrophages (ED1-positive/OX42-positive) into the lesion site at 7 days post injury (d.p.i.). At this time, increased numbers of Schwann cells and axons also began to invade the lesion. Between 14 and 28 d.p.i., macrophages became ED1-positive/OX42-negative. This shift was accompanied by a reduction in the number of axons within the lesion. It is possible that the adoption of a more quiescent phenotype by the macrophages may have influenced the eventual failure of regenerative sprouting seen in this model of SCI.

INDUCTION OF EXPRESSION OF THE IMMEDIATE EARLY GENES C-FOS AND ZENK IN CATECHOLAMINERGIC NEURONS OF MALE QUAIL FOLLOWING COPULATORY BEHAVIOR

T. Charlier (1), A. Foidart (1), G.F.Ball (2) and J. Balthazart (1).
1. Center for Cell. and Molec. Neurobiol., Res. Grp Behav. Neuroendo., Univ. Liège, Belgium; 2. Dept Psychol., Johns Hopkins Univ., Baltimore, MD.

The expression of c-Fos and Zenk was analyzed by immunocytochemistry in the brain of male quail that were gonodally intact (I) or castrated (CX) or castrated and treated with testosterone (CX T). Birds were exposed for 60 min to a sexually mature female (F), an empty arena (EA) or left in their home cage (HC). These treatments identified 5 groups of 5 subjects. c-Fos and Zenk expression was statistically increased in 6 brain areas of sexually active birds (I F CX T F) as compared with controls (CX F, CX T EA, CX T HC) the preoptic area (POA), bed nucleus striae terminalis (BST), archistriatum, nucleus intercollicularis (ICo), substantia grisea centralis (GCt) and area ventralis of Tsai (AVT). Interestingly c-Fos and zenk expression were high in the midbrain vocal control nucleus (ICo) of I F and CX T F birds that displayed copulatory behavior but emitted few crows but not in the ICo of CX T EA birds that crowed frequently. An increased c-Fos expression in TH-immunoreactive cells was observed in GCt and AVT of I F and CX T F birds indicating the participation of dopaminergic neurons in sexual behavior. These data confirm the implication of the steroid-sensitive POA and BST in the control of copulation and support the notion that dopamine is directly implicated in this control.

EMERGENCE OF HIGH-FREQUENCY OSCILLATION IN THE CEREBELLUM OF MICE LACKING CALBINDIN

Cheron G. (1), Gall D. (2), Schurmans, S., Meyer, M. and Schiffmann, S. (2)
1. Lab of Electrophysiology, Univ. of Mons, and Lab of Movement Biomechanics (ULB), 2. Lab of Neurophysiology, Univ. of Brussels (ULB), Belgium; IRIBHN, Univ. of Brussels (ULB) ,Dep.of Neurochemistry, Max Planck Institute for Psychiatry;

Genetic suppression of Ca2 -binding proteins such as calretinin (Cr) in mice induces modifications of the Purkinje cells (PC) behavior. The absence of Cr enhances the simple spike (SS) firing and reduces both the duration of complex spike (CS) and the SS firing pause. Here we show the existence of 160 Hz oscillations in the cerebellar cortex of deficient calbindin (Cb) and Cr-Cb mice. Oscillation in local field potentials (LFP) characterized by spindle episodes were found throughout the explored cerebellar regions. Such oscillation was never recorded in wild type nor in Cr-/- and never reported previously in cerebellum. The amplitude of LFP oscillations increased when approaching a PC layer, reaching maximum just beneath the PC body. Cross-correlation of LFP recorded at different sites indicated that oscillations were synchronized in the frontal plane, but not along a rostro-caudal extent. In presence of oscillation, the increase in SS firing rate was stronger than in absence of oscillation suggesting that oscillation can be considered to result from internally produced synchronization in higher SS firing. Microinjection of bicuculine reversibly produced significant reduction of the oscillation suggesting that inhibitory synapses have a crucial role in the oscillation.

HUMAN BRAIN ACTIVITY RELATED TO A SUCCESSIVE COLOR DISCRIMINATION TASK

Kristl Claeys (1,2), Patrick Dupont (3), Stefan Sunaert (4), Paul Van Hecke (4), Erik De Schutter (2), Guy A. Orban (1)
1. Laboratorium voor Neuro- en Psychofysiologie, K.U.Leuven, Leuven, Belgium 2. Lab. Theoret. Neurobiology, Born Bunge Stichting, U.I.A, Antwerp, Belgium 3. PET Centre, UZGasthuisberg, Leuven, Belgium 4MR Research Centre, UZGasthuisberg, Leuven, Belgium

Previous studies (Cornette et al'99; Faillenot et al'01) showed involvement of both ventral and dorsal stream visual areas and several non-motor cerebellar regions in a successive visual orientation discrimination task. Here we chart the network using the same task but with the attribute color, processed in the ventral stream. The successive color discrimination task (TSD) was contrasted to a detection of dimming control task (DIM) in a PET (n=12) and fMRI study (n=4) over a range of rates. Motor output in DIM was twice that of TSD. The main effect of task (TSD-DIM) yielded bilateral activation in V2, VP and the ventral color region, intraparietal sulcus, inferior parietal lobule and cerebellar lateral hemispheres. Also the left superior parietal lobule and several frontal working memory related areas were activated. The opposite subtraction (DIM-TSD) yielded bilateral activation in V3A, primary and supplementary motor areas and in paramedially cerebellar regions, reflecting the larger motor output in DIM. Thus, even with color as the attribute, successive discrimination involved both ventral and dorsal stream visual areas. Also a clear distinction between non-motor and motor cerebellar regions could be made.(Supported by IUAP/P4-22,FWO-Flanders,fellowship University of Antwerp)

INFLUENCE OF THE MODALITY OF ITEMS PRESENTATION ON THE NEURAL SUBSTRATES OF THE PHONOLOGICAL LOOP

F. Collette, S. Majerus, M. Van der Linden, C. Degueldre, A. Luxen, E. Salmon
Neuropsychology Unit and Cyclotron Research Centre, University of Liège, Liège, Belgium

Studies exploring the phonological loop of working memory (WM) have related the phonological store to the left supramarginal gyrus and the articulatory rehearsal process to Brocas area. However, less attention has been given to other processes of verbal WM, such as those linked to the modality of item presentation. Consequently, the aim of this PET study was to explore the neural substrates of WM for visual or auditory verbal information. Four cognitive tasks were administered recall of (1) series of 5 consonants perceived auditorily; (2) series of 5 consonants perceived visually; (3) one consonant perceived auditorily; (4) one consonant perceived visually. Changes in cerebral metabolism were analysed with SPM99. Results indicate that cerebral areas involved in WM and common to both types of presentation are the cingulate gyrus (BA 32), middle frontal gyrus bilaterally (BA 9/10/46), left superior parietal (BA 7), brainstem and cerebellum bilaterally. Moreover, although interaction analysis found no specific cerebral area linked to the modality of presentation, plots of rCBF suggested that BA 7 is preferentially involved in visual WM. These results suggest that the modality of item presentation does not influence much the cerebral network underlying the phonological loop.

ANATOMICAL RELATIONSHIPS BETWEEN AROMATASE AND IONOTROPIC GLUTAMATE RECEPTORS IN THE QUAIL PREOPTIC AREA AN IMMUNOCYTOCHEMICAL AND ELECTROPHYSIOLOGICAL STUDY

C. Cornil (1), V. Seutin (2) and J. Balthazart (1)
1. University of Liège, Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology and 2. Laboratory of Pharmacology, Liège, Belgium

Aromatase converts androgen to estrogen (E2) and controls many behavioral and physiological brain processes. Recently, we showed that the addition of glutamate agonists markedly depresses aromatase activity (AA) in hypothalamic explants but the anatomical relationships between ionotropic glutamate receptors (GluR) and aromatase are poorly understood. Immunocytochemistry (ICC) for the 3 main subtypes of GluR (AMPA, kainate, NMDA) in the quail brain revealed a dense and specific signal broadly distributed in the brain. In particular, immunoreactive cells were located within all major areas containing aromatase. Double-label staining revealed that GluR5-7 and NMDAR1 are partially colocalized with aromatase. Intracellular recordings performed in preoptic slices of adult male quail revealed spontaneous post-synaptic potentials suppressed by SR95531 and/or CNQX. We thus confirm here that preoptic neurons have functional GABAA and AMPA and/or kainate receptors. Together with ICC data, these results supports the idea that GluR co-exist and are sometimes colocalized with aromatase so that glutamate could regulate AA in a direct manner. Rapid changes in AA could modify E2 availability and thus control rapid estrogen effects in the brain.

INACTIVATION OF ADENOSINE A2A RECEPTOR IMPAIRS LONG TERM POTENTIATION IN THE ACCUMBENS NUCLEUS WITHOUT ALTERING BASAL SYNAPTIC TRANSMISSION.

P. dAlcantara (1,2), C. Ledent (2), S. Swillens (2) and S. N. Schiffmann (1)
1. Laboratory of Neurophysiology, Department of Neuroscience, and 2. Institut de Recherche Interdisciplinaire en Biologie Humaine et Nuclaire, School of Medicine, Universit Libre de Bruxelles, B-1070 Brussels, Belgium.

The nucleus accumbens is considered to be critically involved in the control of complex motivated behaviours. By modulating its glutamatergic excitatory input, mesolimbic dopaminergic afferences have been implicated in the reinforcing properties of drugs of abuse. The aim of this study was to investigate possible modulation of synaptic transmission at this glutamatergic synapse by adenosine receptors. The standard field potential recording technique was used on brain slices from wild-type and A2A receptor-deficient mice. Long term potentiation of the AMPA receptor-mediated synaptic transmission could be elicited in both wild-type and A2A receptor-deficient mice. However, LTP appeared to be quantitatively modulated by the A2A receptor pathway since the level of potentiation was reduced in A2A receptor-deficient mice as well as in slices of wild-type mice in which the A2A receptor pathway was blocked. The involvement of the cyclic AMP-dependent protein kinase was supported by the reduction in potentiation level in slices of wild-type mice treated an inhibitor of this enzyme. These data provide evidences that the adenosine acting at the A2A receptor is implicated in events directly or indirectly related to LTP induction in the accumbens.

FUNCTIONAL STRIATAL HYPODOPAMINERGIC ACTIVITY IN MICE LACKING ADENOSINE A2A RECEPTORS

D. Dassesse (1), A. Massie (3), R. Ferrari (4), C. Ledent (2), M. Parmentier (2), L. Arckens (3), M. Zoli (4), and S.N. Schiffmann (1)
1. Lab. Neurophysiology, ULB, Brussels, Belgium, 2. IRIBHN, ULB, Brussels, Belgium, 3. Lab. Neuroendocrinology, KUL, Leuven, Belgium, 4. Sezione di Fisiologia, Univ. Modena, Italy.

Adenosine modulates locomotor activity and striatal gene expression, partially through activation of striatal A2A receptors (A2A-R). A2A-R-/- mice presented unexpected locomotor alterations regarding the specific expression of A2A-R in striatopallidal neurons. To clarify the functions of A2A-R in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal gene expression as well as dopamine neurotransmission in the striatum.
Basal zif-268 and arc mRNAs expression was reduced in mutant mice by 60-80%, in the striatum but also in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by 50% and 30%, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45%-decrease in the extracellular dopamine concentration. This was associated with an up-regulation of D1 and D2 dopamine receptors expression. Altogether these results pointed out that the lack of A2A-R leads to a functional hypodopaminergic state and demonstrated that A2A-R are necessary to maintain a basal level in striatal and cortical gene expression, possibly through a dopaminergic control.
Supported by FMRE and FRSM, Hoguet Foundation.

MOLECULAR AND PHYSIOLOGICAL DIVERSITY OF NICOTINIC ACETYLCHOLINE RECEPTORS IN THE MIDBRAIN DOPAMINERGIC NUCLEI

Alban de Kerchove d'Exaerde, Ruby Klink, Michele Zoli and Jean-Pierre Changeux
Neurobiologie Moleculaire, Institut Pasteur, Paris, France; Lab. Neurophysiology, ULB, Brussels, Belgium; Department of Biomedical Sciences, Section of Physiology, Universtiy of Modena, Italy.

Nicotinic acetylcholine receptors (nAChRs) on dopaminergic (DA) and GABAergic (Gaba) projection neurons of the Substantia Nigra (SN) and Ventral Tegmental area (VTA) are characterized by single-cell RT-PCR and patch clamp recordings in slices of rat and wild type, b2-/-, a4-/- and a7-/- mice. The eight nAChR subunits expressed in these nuclei, a3-a7 and b2-b4, contribute to four different types of nAChR-mediated currents. The majority of DA neurons in the SN and VTA express two nAChR subtypes one is inhibited by methyllycaconitine (1 nM) but does not contain the a7 subunit and possesses a putative a4a6a5(b2)2 composition; the other subtype is inhibited by dihydro-b-erythroidine (2 M) and has a putative a4a5(b2)2 composition. Gaba neurons in the VTA exhibit a third subtype with a putative (a4)2(b2)3 composition, while Gaba neurons in the SN have either the putative (a4)2(b2)3 oligomer or the putative a4a6a5(b2)2 oligomer. The fourth subtype, a putative (a7)5 homomer, is encountered in less than half of DA and Gaba neurons, in the SN as well as in the VTA. Neurons in the DA nuclei thus exhibit a diversity of nAChRs which might differentially modulate reinforcement and motor behavior.

HUMAN FMRI-DATA ANALYZED BY LOOKING AT THEIR SPATIO-TEMPORAL DYNAMICS

P.A. De Maziere and M. Van Hulle
Laboratorium voor Neuro- and Psychofysiologie, K.U.Leuven, Belgium

The great majority of the available statistical tools for analyzing the relationship between the experimental paradigm and local changes in the cerebral blood flow, and to which the functional Magnetic Resonance Imaging (fMRI) signal is related, are based on univariate statistical techniques. However, since cognitive functions result from interactions between brain areas, new concepts and multivariate tools are needed.
We have developed a tool which considers the fMRI-data set as one originating from a multivariate, attractor-based system, and select the "prototypical" fMRI signals that best approximate the attractor's dynamics. These signals then serve as the basis for identifying the active brain regions. The experimental paradigm is invoked by regressing the fMRI signals of the identified brain regions with the design matrix which describes the experimental paradigm. Finally, by means of our new statistical technique, the significantly active voxels are identified and t- and corrected p-values attached to them. We will compare our results for a number of case studies with those obtained with SPM, the most widely used univariate statistical tool for analyzing fMRI-data.

CLONING OF CANDIDATE PARTNERS OF THE REELIN SIGNALLING PATHWAY, BY USING REPRESENTATIONAL DIFFERENCE ANALYSIS AND DIFFERENTIAL DISPLAY

A. Demirov (1), D. Demirov (1), M. Simonneau (2), A.M. Goffinet (1)
1. Neurobiology Unit, Univ. Namur Med. School, 5000 Namur and 2. INSERM E9935, Hopital Robert Debre, 75019 Paris France

Reelin, the extracellular matrix protein defective in reeler mutant mice, is secreted by pioneer neurons and activates a signaling cascade that instructs the architectonic organization of postmigratory neurons such as Purkinje cells and cortical plate cells. Reelin binds to lipoprotein receptors and activates the intracellular adaptor Dab1. Other partners of the reelin signaling pathway remain to be identified. In order to find potential partners of reelin, we applied two differential cloning techniques, representational differential analysis of cDNAs (cDNA-RDA) and differential display (DD), to isolate partial sequences corresponding to mRNA that might be differentially expressed in normal, reelin- and Dab1-deficient newborn mice. DD yielded 77 clones and cDNA-RDA 15 clones. Sequence analysis revealed that 8 clones were derived from known genes. Some of them, such as Sfrp1, DCX, PP2A, ROBO1, PITP and TrkB, are implicated in brain development. Of the remaining clones, 7 were selected based on similarity to mouse or human EST and presence of transcript in embryonic brain using RT-PCR and Northern blots. These candidates are being studied further, with characterization of full-length clones, definition of open reading frames and pattern of expression with in situ hybridization.

LOCALIZATION OF ESTROGEN RECEPTOR, AROMATASE, AND NOCICEPTION-RELATED COMPOUNDS IN THE QUAIL SPINAL DORSAL HORNS

H. Evrard, E. Willems, and J. Balthazart
Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Liège, B-4020 Liège, Belgium.

Estrogens modulate nociception presumably by activating estrogen receptors (ER) distributed all along the spinal dorsal horns (DH). The presence of estrogen-synthase (aromatase) in the DH indicates that these estrogens may be produced locally. Here, we used immunocytochemistry to analyze the anatomical relations of aromatase neurons with ER alpha nuclei, and with spinal compounds involved in nociception. Small numbers of ER alpha were found in DH at the boundary of laminae II and III while aromatase neurons were mostly located at the edges of these laminae suggesting a lack of co-localization. AMPA receptors, substance P (SP), and substance P receptors (SPR) as well as the opiate met-enkephalin were broadly distributed in the areas containing aromatase. In double-labeled sections, SP fibers and punctate structures were in close apposition to aromatase neurons. Moreover aromatase and SPR were co-localized in the same cells. These data indicate that the spinal aromatase may be influenced by nociceptive and anti-nociceptive inputs and conversely, that locally produced estrogens could modulate nociception by processes that may be independent of ER alpha.
Supported by NIMH 50388, ARC99/04-241, FRFC 2.4555.01, Crdits spciaux ULg

NEUROMODULATORY EFFECTS OF HYPNOTIC STATE ON PAIN PERCEPTION

M.-E. Faymonville, S. Laureys, N. Janssens, G. Del Fiore, C. Degueldre, M. Lamy, A. Luxen, P. Maquet
Cyclotron Research Center, Dept. of Anesthesiology and Int. Care Med. and Dept. of Neurology, University of Liege, Liege, Belgium

Using PET, we recently showed that the midcingulate cortex mediates the hypnosis-induced reduction of affective and sensory responses to noxious thermal stimulation (Faymonville, Laureys et al, Anesthesiology 2000 92 1257-67). Here, we studied changes in cerebral functional connectivity related to the hypnotic state, compared to simple distraction and the resting state. Using a psychophysiological interaction analysis, we identified brain areas that would respond to noxious stimulations under the modulatory action of the midcingulate cortex in, and only in, the hypnotic state. In nineteen healthy volunteers, we observed a significant increase in the functional relationships between midcingulate cortex and left insula, pregenual anterior cingulate cortex and supplementary motor area. These data point to a critical role for the midcingulate cortex in the modulation of insular and pregenual anterior cingulate regions underlying its influence on both sensory and affective noxious aspects of the stimuli, specifically during hypnosis. Functional relationships with nearby supplementary motor areas might also allow the midcingulate cortex to organize the most appropriate behavioral response, taking into account the affective component of stimuli to the pain perception.
Supported by FNRS

AREAS INVOLVED IN MOTION PROCESSING USING CONTRAST-AGENT ENHANCED FMRI IN AWAKE FIXATING MACAQUES

D. Fize, W. Vanduffel, J.B. Mandeville, K. Nelissen, P. Van Hecke, R.B.H. Tootell, G.A. Orban
Laboratorium voor Neuro- en Psychofysiologie, K.U.Leuven Medical School; Radiologie UZ-Gasthuisberg K.U.Leuven; MGH-NMR ctr., Boston, USA

To reduce the information gap between human neuroimaging and macaque physiology and anatomy, several fMRI experiments were conducted to map motion sensitive regions of two awake monkeys. Using blood oxygen level dependent (BOLD) as well as MION (monocrystalline iron oxide nanoparticles) contrast-agent enhanced imaging, we compared activity between moving and stationary random dot and line stimuli while the monkeys were fixating. During each scan session, the eye-movements were directly controlled. The injection of MION increased sensitivity in visual cortex by 1 log unit relative to the BOLD technique. Moreover, the use of the MION led to a better spatial resolution due to its optimal sensitivity for signals originating from parenchyma. Motion-sensitivity was observed using both techniques in areas V2, V3, VP, MT, vMST, FST, PIP, VIP, LIP, and FEF. Activations following line stimuli were observed in the same regions compared to dot stimuli, extended into area V4, anterior STS and IP. These regions were in close agreement with human fMRI results and with the concept of motion processing in the dorsal stream of primate visual cortex. Unlike human V3A, macaque V3A was not motion-sensitive. Supported by GOA, GSKE, and NFWO.

MRI CORRELATE OF CONGENITAL VISUAL PERCEPTUAL IMPAIRMENT IN THREE CHILDREN WITH MULTIPLE DISABILITIES BUT WITH AN OBJECT CATEGORIZATION DEFICIT ESTABLISHED WITH THE VISUAL PERCEPTION BATTERY L94 RELATIVE TO THEIR NON-VERBAL INTELLIÈGENCE LEVEL.

A. Fonteyne (1), P. Stiers (1), Ph. Demaerel (2), I. Casteels (3), L. Lagae (4), and E. Vandenbussche (1)*.
1. Laboratory of Neuropsychology, 2. Departments of Radiology, 3. Ophtalmology, and 4. Pediatrics, K.U.Leuven, Medical School, B-3000 Leuven.

Evaluation of visual perception against non-verbal intelligence level to single out visual perceptual impairment in children with multiple disabilities, revealed impaired visual categorization of sub-optimally presented objects in children with early hypoxic-ischaemic brain damage, but not in a control group of mentally retarded children. To validate the conclusion that these impairments are perceptual in nature, structural MRI scans of 3 children (7-10 yr.) with divers clinical features but an object categorization deficit, were used to ascertain whether their brain damage converged into lesions of visual neural structures.
All 3 were diplegic following preterm birth. 2 of them had periventricular leukomalacia, 1 with normal intelligence but learning disabilities, the other with mild verbal but moderate non-verbal mental retardation. The 3rd child had non-ischaemic hydrocephalus with mild mental retardation.
An uninformed neuroradiologist analyzed the MRI scans evaluating 53 relevant parameters. Bilateral extensive white matter reduction in parietal and occipital lobes with mild reduction of the posterior corpus callosum emerged as common site of damage. This result affirms attribution of the children's reduced performance on perceptual tasks to visual perceptual deficits.

ALTERED ELECTRORESPONSIVENESS OF CEREBELLAR GRANULE CELLS IN MICE LACKING CALRETININ.

D. Gall (1), I. Susa (2), E. D'Angelo (4), P. Rossi (4), S. Schurmans (3) and S.N. Schiffmann (1)
1. Lab. de Neurophysiologie, 2. Unite de Chronobiologie Theorique, 3. Institut de Biologie et de Medecine Moleculaire, Universite Libre de Bruxelles, Brussels, Belgium and 4. Department of Molecular/Cellular Physiology, University of Pavia, Italy.

Cytosolic calcium (Ca) regulates a large variety of neuronal fonctions and hence must be tighly regulated. Cytoplasmic Ca binding proteins play a key role in this regulation. Among these, calretinin is particularly enriched in cerebellar granule cells. Current clamp recording have been performed from single granule cells in cerebellar slices of control mices and knock-out mice deficient in calretinin (Cr -/-) using the perforated patch whole-cell configuration of the patch-clamp technique. We have measured the voltage response obtained at increasing injected current intensities. The slope of the linear part of current-frequency plots reflects the intrinsic granule cell excitability. Our results show a significant (p<0.05) increase in excitablity of granule cells of Cr -/- mice (6.60 Hz/pA, SEM=0.7 Hz/pA, n=8) vs. control mice (4.90 Hz/pA, SEM=0.2 Hz/pA, n=5). In addition we observe a significant (p<0.05) decrease in the action potential half-width from 1.020 ms, SEM=0.06 ms (n=5), in control mices to 0.780 ms, SEM=0.06 ms (n=7), in Cr-/- mice. We also propose a mathematical model providing a link between the observed alterations in the excitability of the granule cells and a decreased Ca buffering capacity of the cytosol.

IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN THE FRAGILE X KNOCKOUT MOUSE

I. Gantois, E. Reyniers, F. Kooy
Department of medical genetics, University of Antwerp, Antwerp, Belgium

Fragile X syndrome is the most common form of hereditary mental retardation. Affected individuals suffer from mild to severe mental retardation and macroorchidism in combination with behavioral and physical abnormalities. The fragile X syndrome is caused by a CGG repeat expansion in the FMR1 gene that is no longer transcribed. The FMR1 protein, FMRP, is expressed in most celltypes and shows high expression in neurons and Sertoli cells. FMRP shuttles between nucleus and cytoplasm, perhaps transporting a specific subset of the RNA's to the active translating ribosomes near the synapses in neurons.
A fragile X knockout mouse has been constructed. The knockout mouse shows some characteristics compatible with the symptoms of human patients, including macroorchidism, spatial learning deficit and dendritic spine abnormalities. Despite intense research over the last decade, the relationship between the lack of FMRP and mental retardation is still unknown. Brain mRNA's of which the expression is influenced by absence of the FMR1 gene are isolated using the differential display method. Differential expression of these sequences is confirmed using micro-arrays. Analysis of the differentially expressed genes may reveal some of the molecular pathways involved in fragile X mental retardation.

BIASED ATTENTIONAL COMPETITION IN NEGLECT PATIENTS

S. Geeraerts, C. Lafosse, K. Michiels, E. Vandenbussche
Laboratorium voor Neuropsychologie, K.U.Leuven, Belgium

Attentional competition in neglect patients was studied by means of threshold measurements. We determined the effect of an irrelevant contralateral distracter grating on left and right hemifield grating orientation discrimination (OD). OD thresholds were determined in brain damaged patients with or without neglect and in controls without brain damage. A constant stimuli paradigm was used to dissociate between orientation sensitivity and possible biases in the perception of verticality. In the neglect group, there was a significant effect of right distracters on thresholds obtained in the left hemifield, but no effect from left distracters on right hemifield thresholds. Moreover, the presentation of the right hemifield distracter caused a clockwise displacement of the subjective visual vertical. In the control groups, only a small, symmetric effect on orientation sensitivity was observed. We were able to show strong extinction effects in the neglect patients, although they were instructed to direct their attention towards the target stimulus position for the whole block and to disregard the irrelevant distracter. Competition from ipsilesional distracters not only increased the contralesional orientation thresholds but also distorted the perception of verticality.

RAT CEREBELLAR GOLGI CELLS, PURKINJE CELLS AND MOLECULAR LAYER INTERNEURONS EXPRESS 5-HT5A RECEPTOR IMMUNOREACTIVITY

F. J. Geurts (1,2), J.-P. Timmermans (1) and E. De Schutter (2)
1. Laboratory of Cell Biology and Histology, 2. Laboratory for Theoretical Neurobiology, Born-Bunge Foundation, University of Antwerp, Belgium; Supported by IWT grant SB981250 and UA-GOA 99/2/33

Although serotonin (5-HT) is known to exert a modulatory action on cerebellar function, our current knowledge of receptor subtypes mediating serotonergic activity in this part of the brain remains fragmentary. Here we describe the distribution of 5-HT5A receptors in the rat cerebellum using immunohistochemistry. Purkinje cells, evident as a monolayer of parvalbumin-positive cell bodies in the cerebellar cortex, clearly showed 5-HT5A immunostaining. In addition, 5-HT5A immunoreactivity (IR) was found in numerous small cells in the molecular layer. These cells expressed parvalbumin IR and were considered to be stellate/basket cells. In the granular layer, 5-HT5A IR was observed in large polygonal cell bodies with radiating dendritic arborizations. They represented Golgi cells and were distinguished from Lugaro cells and unipolar brush cells by their neurochemical content. Golgi cells expressed somatostatin and glutamate receptor mGluR2 IR, and were calretinin-negative. Finally, deep cerebellar nuclei neurons, identified by the cytoplasmic antigen Rat-303, did not show 5-HT5A IR. These results clearly demonstrate the presence of 5-HT5A receptor proteins in specific neuronal cell types and may consequently contribute to revealing the modulatory actions of 5-HT on cerebellar function.

MITOCHONDRION-TRIGGERED CASPASES ACTIVATION IN CARBOLINES-INDUCED NEURONAL APOPTOSIS

G. Hans, B. Malgrange, G. Moonen and J.-M. Rigo
CNCM, University of Liège, Belgium

Previous reports on the mechanism(s) by which -carbolines (Cs), mainly known as GABAA receptor negative allosteric modulators, induce the apoptosis of cultured neurons had suggested that the trigger was mitochondrial. We had hypothesized that Cs induce the formation or the opening of the permeability transition pore (PTP) leading to mitochondrial membrane depolarization and subsequent leakage of cytochrome C (Cyt C) initiating the caspase-9 apoptotic pathway.
Here we show, using cerebellar granule cells in culture, that 1) DMCM, a C, as well as Ro 5-4864, a specific ligand of the peripheral benzodiazepine receptor (PBR) which is likely to participate in PTP formation, first induce an increase in caspase activity that is followed by cell death. These effects are blocked by a pancaspase inhibitor. 2) Both compounds also induce a cytosolic release of Cyt C which is also antagonized by the pancaspase inhibitor. 3) A mitochondrial depolarization was observed upon treatment with Ro 5-4864 and, to a lesser extent, with DMCM. This depolarization was blocked by cyclosporin A, an inhibitor of the PTP.
These data suggest that the activation of the PBR could trigger the apoptosis of cultured neurons.

BINDING OF REELIN TO LIPOPROTEIN RECEPTORS IN VIVO AND IN VITRO.

N. Ignatova, Y. Jossin, A.M. Goffinet, C. Lambert de Rouvroit
Neurobiology Unit, University of Namur Medical School, 61 rue de Bruxelles, 5000 Namur

Reelin, the protein defective in reeler mice, is a large extracellular protein secreted by Cajal-Retzius neurons in the marginal zone. Reelin binds to two lipoprotein receptors named VLDLR and ApoER2, and signals in cortical plate neurons via phosphorylation of the adaptor Dab1. Receptors and Dab1 are expressed in reelin target cells. In vitro, ApoER2 and VLDLR bind reelin and interact with Dab1 via a cytoplasmic NPXY sequence. Using partial reelin constructs, we showed that the N-terminal region of Reelin containing repeats 1 and 2, and the C-terminal repeats 7 and 8 are not involved in receptor binding. The central repeats 3-6 bind to the receptor, but binding of shorter constructs could not be demonstrated, suggesting that Reelin binding to lipoprotein receptors requires cooperative interaction of at least 4 reelin repeats. To study Reelin receptors, we generated monoclonal antibodies against a polypeptide corresponding to the 355 N-terminal residues of mouse ApoER2, by immunized ApoER2 -/- mice. Twelve antibodies were obtained. They were used to demonstrate that ApoER2 co-immunoprecipitates with reelin in whole brain extracts, demonstrating for the first time an interaction between Reelin and ApoER2 in vivo.

MACAQUE INFERIOR TEMPORAL NEURONS ARE NOT SELECTIVE FOR ANTICORRELATED RANDOM DOT STEREOGRAMS

P. Janssen, R. Vogels, Y. Liu, G. A. Orban.
Lab. Neuro-en Psychofysiologie, KU Leuven Medical School, Herestraat 49, B-3000 Leuven, Belgium.

Random dot stereograms containing binocular disparity gradients are perceived as concave or convex if the dots are correlated between the eyes, i.e. corresponding dots have the same contrast polarity. When corresponding dots have opposite contrast polarity, i.e. anticorrelated stereograms, no depth can be perceived. The part of the inferior temporal cortex in the lower bank of the Superior Temporal Sulcus, TEs, contains neurons selective for disparity-defined 3D shapes. To determine whether the responses of TEs neurons correlate with stereoscopic depth perception, we recorded the responses of single neurons to correlated and anticorrelated surfaces. The disparity was maximal in the center and smoothly approached zero towards the boundaries along both the vertical and the horizontal axis. After a test with monocular presentations, concave and convex surfaces were presented at different positions in depth, demonstrating higher-order disparity selectivity.The vast majority of the neurons tested was not selective for the anticorrelated patterns only 4 out of 87 neurons were weakly modulated by the disparity in the anticorrelated stimuli. These results demonstrate that the responses of TEs neurons correlate with stereoscopic depth perception.

REELIN DOES NOT DIRECTLY INFLUENCE AXONAL GROWTH

Y. Jossin, A.M. Goffinet
Neurobiology Unit, University of Namur Medical School, 61 rue de Bruxelles, 5000 Namur

Reelin is a large extracellular glycoprotein involved in the development of the cerebral cortex and hippocampus. In the hippocampus, Reelin regulates the growth and/or distribution of afferent axons, and this was taken as evidence that it might affect growth cone navigation. The action of Reelin on axonal growth was studied using the 3D collagen-gel assay to measure axonal elongation from reeler cortical explants in the presence of Reelin. As Reelin is processed in vivo, normal cortical explants and pellets of reelin-transfected cells were used, respectively, as sources of processed and full-length protein. The control consisted in cultures of reeler explants. The reliability of the assay was tested using Semaphorin 3A and Semaphorin 3F (cDNA provided by M. Tessier-Lavigne). We analyzed 36 experiments with reeler versus normal explants, 29 with reeler versus transfected cells, 15 controls with reeler explants, and 29 controls with Sema 3A or Sema3F. There was a repulsive action of Sema3F on cortical axons (p < 0.0001). However, neither full-length nor processed Reelin exerted any attraction or repulsion on cortical axons. These results suggest that the effects of Reelin on axonal pathways are indirect, secondary to the disturbances that result from Reelin deficiency.

SENSITIVITY OF MACAQUE INFERIOR TEMPORAL NEURONS TO PARAMETRICALLY VARIED SHAPE CHANGES.

G Kayaert, R Vogels* and I Biederman.
Lab. Neuro- en Psychofysiologie, KULeuven Medical School, Leuven, Belgium and University of Southern California

We recorded the responses of 75 IT neurons to rendered volumes that were parametrically varied along several dimensions.
Given an original shape, e.g., a cylinder with a curved axis, values on each dimension were selected to produce a change in a nonaccidental property (NAP), such as a straight axis, or changes in a metric property (MP), such as the degree of axis curvature.
The physical differences between the shapes were calibrated by pixel and wavelet measures. The absolute difference in response increased monotonically with increasing physical difference between the original shape and its metric variations. The size of the response difference between shapes depended on the particular dimension varied. 69% of the neurons responded as well to silhouettes as to the original 3D objects. The silhouettes allowed a test of the relative sensitivity of metric and nonaccidental shape differences, unconfounded by variations in shading and local features.
Neurons showed a larger sensitivity to NAP changes compared to physically equal MP changes. The metric change had to be 2 to 3 times larger than the non-accidental variation to produce the same neuronal response change. Rotating the stimuli produced response modulations that were equivalent to approximately equal NAP changes.

FUNCTIONAL CHARACTERIZATION OF A HUMAN RECEPTOR FOR NEUROPEPTIDE FF AND RELATED PEPTIDES.

M. Kotani, C. Mollereau, M. Detheux, E. Le Poul, S. Brzillon, J. Vakili, H. Mazarguil, G. Vassart, J.-M. Zajac, and M. Parmentier
IRIBHN, Universit Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium; Euroscreen S.A., Brussels, Belgium, and Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.

Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR is a functional high affinity receptor for NPFF and related peptides. CHO cell membranes expressing NPFFR bound [125I]-EYF with a Kd of 0.06 nM. Various NPFF analogs and related peptides inhibited [125I]-EYF specific binding with a rank order consistent with previous pharmacological data. In a functional assay based on the co-expression of NPFFR, Ga16 and apoaequorin, the rank order of potency was of the peptides was consistent with the binding assay results. Membranes from NPFFR expressing CHO cells bound GTPg[35S] in the presence of SQA-NPFF. This response was prevented by pertussis toxin, demonstrating the involvement of Gi family members. SQA-NPFF inhibited forskolin induced cAMP accumulation in CHO cells, and this response was abolished as well by pertussis toxin. RT-PCR revealed expression of NPFFR in human placenta, thymus and at lower levels in pituitary gland, spleen and testis. The availability of the cloned receptor will lead to a better understanding of the physiological and pathophysiological roles of NPFF and related peptides in the central nervous system.

HEMISPATIAL GRAVICEPTIVE AGNOSIA IN UNILATERAL NEGLECT

C. Lafosse (1,3), P. Santens (2), E. Vandenbussche (1)
1. Laboratory of Neuropsychology, K.U.Leuven 2. Departement of Neurology, University of Gent 3. Scientific Unit Rehabilitation Center Hof ter Schelde, Antwerp

It is hypothesised that in unilateral neglect a mismatch develops between the disturbed egocentric body-centred representation of space and the point where they perceive themselves to be aligned with the gravitational vector. The aim of this study was (1) to investigate the location of the center-of-gravity (COG) in relation to the subjective postural vertical (SPV) and straight forward pointing (SFP). (2) In order to compensate the misalignment it should be possible to reduce this bias by transcutaneous electrical nerve stimulation (TENS). We compared four groups of RBD neglect patients (N=34) and presented a longitudinal follow-up of patient with a progressieve posterior parietal atrophy who gradually developed a severe hemispatial neglect. The results point to the existence of an hemispatial graviceptive agnosia indicating a decreased sensitivity to the gravitational vector with a directional bias of the spatial axis in alignment with an altered internal reference of the body vertical (increased deviation of the COG, SPV and SFP) in gravitational space. These shifts can be reduced by TENS indicating that they are integrated in a high order egocentric boby-centred dsturbance of the representation of space.

GENERATION AND CHARACTERISATION OF KNOCK-OUT MICE FOR THE PROLACTIN-RELEASING PEPTIDE (PRRP) RECEPTOR.

P. Laurent, C.Ledent and M. Parmentier
I.R.I.B.H.N. Campus Erasme (ULB) 808 route de lennik 1070 Bruxelles

Recently, Hinuma et al. isolated a novel peptide using the method of reverse pharmacology. This peptide stimulated cells transfected with the orphan G-Protein Coupled Receptor GPR10 and had Prolactin-releasing properties; hence, it was named Prolactin Releasing Peptide (PrRP).
However, the pattern of expression suggests that this ligand and receptor may control several additional aspect of physiology in brain and other tissue. Interestingly, intracerebroventricular injection of PrRP suggests a role in regulating the release of hormones from both anterior and posterior pituitary Null mutant mice for GPR10 receptor will allow us to determine the physiological functions of the receptor. The gene encoding GPR10 receptor was disrupted by insertion of a cassette containing Tau-LacZ fusion protein and neomycin resistance in the coding exon of GPR10. We generated mice heterozygous for the mutant allele and used these to establish all possible genotype.
Knock-out mice are viable and show no apparent morphological abnormality. The Tau-LacZ reporter gene will allow us to see the axonal projections of GPR10 neurones to know in which mechanism it is involved.

ASSESSING RESIDUAL BRAIN FUNCTION FOLLOWING ACUTE BRAIN INJURY. A PET STUDY OF COMATOSE, VEGETATIVE, MINIMALLY CONSCIOUS AND LOCKED-IN PATIENTS

S Laureys, J Berre, S Antoine, M-E Faymonville, C Phillips, S Elincx, P Van Bogaert, X Detiege, J Aerts, D Wikler, P Damhaut, F Damas, A Luxen, G Moonen, M Lamy, J-L Vincent, S Goldman, P Maquet
Cyclotron Research Centre, Dept. of Neurology and Anesthesiology and Intensive Care Medicine, University of Liege; PET/Biomedical Cyclotron Unit, Dept. of Intensive Care Medicine, ULB Erasme, Brussels; Dept. of Neurology, AZ-VUB, Brussels, Belgium.

The clinical evaluation of possible cognitive function in comatose, vegetative, minimally conscious or locked-in victims remains extremely difficult because behavioral responses may be very small and inconsistent. We used PET to measure their residual brain metabolism.
Compared to controls, mean gray matter metabolism was 50% lower in coma, 60% lower in vegetative state, 40% lower in minimally conscious state and within normal limits in locked-in syndrome. Both comatose and vegetative patients showed dysfunctional associative cortices (bilateral prefrontal, parieto-temporal, posterior parietal and precuneal areas). Interestingly, the brain area that differentiated comatose or vegetative from minimally conscious patients was the precuneus. In locked-in patients, there were no voxels that showed a significant decrease in metabolism.
Within the neural network of polymodal associative cortices, the precuneus seems to play a critical role it is the most active region of the brain in conscious waking (i.e., controls or locked-in) and one of the least active in unconscious states (i.e., coma, vegetative state, sleep or general anesthesia). In minimally conscious state, its activity was significantly above that observed in coma or vegetative patients. Supported by FNRS and FMRE.

PHENOTYPIC PLASTICITY OF CEREBELLAR RADIAL GLIA

Leprince P., Chanas-Sacr G., Nguyen L, Thiry M., Rogister, B. and Moonen G.
Center for Cellular and Molecular Neurobiology, University of Liège, 17 Place Delcour, B-4020 Liège, Belgium

Radial glia cells are among the earliest cells to differentiate in the early CNS. Radial glial cells disappear or transform into astrocytes when neuronal migration is completed. Phenotypic changes occurring during the transition between radial glia and astrocytes include the replacement of early forms of intermediate filaments components (Vimentin, Nestin, Radialin), by GFAP. Radialin, the antigen recognized by the RC2 antibody, has been identified as an intermediate filament-associated protein (IFAP) of 300 kDa which is also present in developping skeletal muscle. In glial cells cultures, RC2 immunoreactivity is strongly dependent on the presence of serum. Thrombin and Lysophosphatidic acid, also control the phenotypic transition between radial glia and astrocytes, including their expression of Radialin. Nestin is another IF/IFAP which exhibits an in vivo distribution similar to that of Radialin. The two proteins could in fact be related or even be two forms of the same protein, suggesting a relationship between radial glia and multipotential progenitor cells. Indeed, analysis of the differentiation of progenitor cells migrating out of striatal neurospheres reveals a prominent staining by RC2 that is progressively replaced by neuronal and glial differentiation markers.

THE ACTIVE FORM OF GLYCOGEN SYNTHASE KINASE-3 IS ASSOCIATED WITH GRANULOVACUOLAR DEGENERATION IN NEURONS IN ALZHEIMERS DISEASE

Leroy K. (1), Boutajangout A. (1), Authelet M. (1), Woodgett J.R. (2), Anderton B.H. (3), Brion J.P. (1)
1. Lab of histology, neuroanatomy and neuropathology, universit libre de Bruxelles, Faculty of medicine, Brussels, Belgium; 2. Ontario cancer institute, Toronto, Canada; 3. Dep of neuroscience, Institute of psychiatry, Kings college London, London, UK

We have investigated in Alzheimers disease (AD) the immunocytochemical localization of glycogen synthase kinase-3b (GSK-3b), a protein kinase involved in the hyperphosphorylation of tau present in PHF-tau of neurofibrillary tangles (NFT). Neurons exhibiting strongly GSK-3a and b-immunoreactive granules co-localizing with the granules of the granulovacuolar degeneration (GVD) were observed in AD, with a much higher frequency than in control subjects. In addition, the GVD labelling was observed with an antibody against the tyrosine phosphorylated and active form of GSK-3. Some GVD granules were intensely labeled with antibodies specifics for tau phosphorylated on ser262 and on Thr212/Ser214, two phosphorylation sites generated in vitro by GSK-3a and. GSK-3 was expressed in neurons containing NFT but only a small proportion of intracellular NFT were GSK-3 immunoreactive and immunoblotting analysis of fractions enriched in PHF-tau did not show any GSK-3 immunoreactivity. These results suggest that neurons developing GVD sequester an active, potentially deleterious, form of GSK-3 in this compartment.

PATTERNING THE EMBRYONIC BRAIN

A. Lumsden
MRC Centre for Developmental Neurobiology, King's College London, Guy's Hospital, London, United Kingdom

The brain contains a very large number of different kinds of neurons arranged within structurally and functionally distinct regions and subregions. Correct early regionalization of the embryonic brain is crucial to later events in development when the different cell types appear and interconnect to form functional networks. For the nervous system, as for the entire body, our understanding of patterning processes has advanced rapidly through the discovery of genes that control development, and the elucidation of mechanisms that regulate their spatial and temporal expression. Deciphering the genetic instructions for regional neural pattern may be a key to understanding the complexities of the adult brain.

PERIPHERAL STIMULI EXCITE CORONAL BEAMS OF GOLGI CELLS IN RAT CEREBELLAR CORTEX

R. Maex, A. Volny-Luraghi, B. Vos and E. De Schutter
Laboratory for Theoretical Neurobiology, Born-Bunge Foundation, Universiteit Antwerpen

Owing to the abundance of their source granule cells, parallel fibers (PFs) in the cerebellar cortex constitute the largest axonal pathway of the brain. Nevertheless, the contribution of PFs to the excitation of interneurons, and particularly of Purkinje cells, remains unclear. Previously, we showed that PFs synchronize the spontaneous activity of Golgi interneurons (Gocs) aligned along the length axis of CrusII in anaesthetized rats. Here, we compared the responses of Gocs to punctate and brush stimulation of the facial skin, and matched these experimental responses to the responses of model Gocs in a realistic network. This way, the response components evoked by PF input could be distinguished from monosynaptic mossy fiber input. Differences in response strength and latency between simultaneously recorded Gocs could be completely explained by assuming that mossy fibers have small receptive fields from which only a small patch of Gocs and granule cells is monosynaptically excited. The long PFs (granule cell axons) convey this input to distant Gocs. The slow PF conduction speed hereby causes a temporal low-pass filtering. Our results confirm the importance of the parallel fiber pathway for Goc excitation in particular and for cerebellar functioning in general.

LEXICALITY AND PHONOTACTIC FREQUENCY IN ORAL LANGUAGE PROCESSING A POSITRON EMISSION TOMOGRAPHY(PET)STUDY

S. Majerus (1), F. Collette (1), M. Van der Linden (1), Philippe Peigneux (2), G. Delfiore (2), C. Degueldre (2), A. Luxen (2) , E. Salmon (2)
1. Department of Neuropsychology, 2. Cyclotron Research Centre, University of Liège, Belgium

Lexicality and phonotactic frequency effects are observed in many cognitive studies on language processing, but little is known on their underlying neural substrates, especially with regard to phonotactic frequency effects. We conducted a PET study in which 11 right-handed volunteers had either to repeat or to listen lists of words, high phonotactic frequency nonwords and low phonotactic frequency nonwords. The comparison of repetition and passive listening of words versus nonwords confirmed previous activation findings in the left temporal and frontal regions, classically ascribed to lexico-semantic processing. The comparison between high and low phonotactic frequency nonwords processing did not reveal significant differences. However, higher activation in the right posterior superior temporal gyrus was observed when comparing high phonotactic frequency nonwords to words, but not for low phonotactic frequency nonwords. We propose that the right posterior superior temporal gyrus region might be implicated in the construction of new phonological representations, and that behavioral differences in processing high and low phonotactic frequency nonwords might be related to the ease with which new phonological representations can be constructed in the right hemisphere.

GENERATION OF SUPPORTING AND HAIR CELLS FROM PROGENITORS ISOLATED FROM A NEWBORN MAMMALIAN ORGAN OF CORTI

B. Malgrange, S. Belachew, M. Thiry, B. Rogister, M.-L. Alvarez, J.-M. Rigo, L. Nguyen, T.R. Van De Water, G. Moonen and P.P. Lefebvre
CNCM, University of Liège, B-4020 Liège, Belgium; Laboratory of Cell and Tissue Biology, University of Liège, B-4020, Liège, Belgium; Departments of Otolaryngology, AECOM, NY 10461, USA.

Production of sensory cells in the mammalian cochlea is completed by the end of the first half of gestation. Immunolabeling of the rat organ of Corti suggests that many progenitor cells (nestin) are present at the level of the inner sulcus from embryonic day 19 up to postnatal day 15. In this study, cells isolated from the newborn rat organ of Corti cultured on a non-adherent surface were expanded by treatment with either epidermal growth factor or fibroblast growth factor type 2. These cells formed proliferative spheres which initially expressed nestin and subsequently developed the morphology and antigenic properties of either a hair cell or a supporting cell. Here, we report for the first time that progenitors of both hair cells and supporting cells are present in the mammalian auditory epithelium after the period of production of the sensory cells. Such progenitors open up new prospects for replacement of hair cells in vivo. If HC regeneration is to become a possibility in the mammalian auditory receptor, a progenitor cell must be identified that, after cell division, can give rise to new HCs and SCs.
This work was supported by the Fonds National de la Recherche Scientifique

ALTERED EXPRESSION OF HIGH-AFFINITY GLUTAMATE TRANSPORTERS IS RELATED TO CHANGES IN EXCITATORY NEUROTRANSMITTER LEVELS DURING CORTICAL REORGANIZATION.

A. Massie (1), L. Cnops (1), U.T. Eysel (2), E. Vandenbussche (3), F. Vandesande (1) and L. Arckens (1)
1. Lab. Neuroendocr. and Immunol. Biotechnol., Katholieke Universiteit Leuven, Belgium; 2. Dept Neurophysiol., Ruhr-Universitt Bochum, Germany; 3. Lab. Psychophysiol., Katholieke Universiteit Leuven, Belgium

Previous microdialysis experiments revealed a role for Glu and Asp in cortical reorganization as induced by central retinal lesions. Asp and Glu concentrations were significantly lower in lesion-affected (LA) area 17 compared to non-affected (NA) visual cortex. To determine whether these differences were a consequence of a change in the removal of Glu and Asp from the synaptic cleft, we perfused the microdialysis probe with an inhibitor of Glu transporters, PDC. This resulted in increased Asp (745%) and Glu (438%) concentrations in area 17 of control animals. In retinal lesion cats, Asp and Glu levels increased 924% and 714% in LA cortex compared to 476% and 337% in NA visual cortex. Blocking the transporters induced almost equal Asp and Glu concentrations in both brain regions. Western blotting revealed higher concentrations for the glial Glu transporters, GLAST and GLT-1, in LA compared to NA visual cortex.
In conclusion, the altered cortical extracellular Glu and Asp concentrations following retinal lesions are, at least partially, a consequence of altered re-uptake from the synaptic cleft. This difference in re-uptake results from altered expression of the glial Glu transporters and possibly also from a difference in activity of the high-affinity Glu transporters.

MECHANISM OF ESTRADIOL POTENTIATION OF GLUTAMATE-INDUCED GONADOTROPIN-RELEASING HORMONE (GNRH) SECRETION IN VITRO.

Matagne V., Lebrethon M-C., Gerard A., Bourguignon J-P.
Developmental Neuroendocrinology Unit, University of Liege, Liege, Belgium.

Using explants of retrochiasmatic hypothalamus from 50-day-old male rats, the secretory response of GnRH induced by glutamate (10mM for 7.5min) was studied. That response (meanSD, % of controls) was significantly and rapidly (within 7.5 min) increased by 17beta-estradiol (E2) in a dose dependent manner (100nM 14314%, 1M 16823%) while no effect was observed using 17alpha-estradiol or progesterone. Using specific agonists (kainate or NMDA, 10mM) or antagonists (DNQX or AP5, 1M) of ionotropic glutamate receptor subtypes, only the kainate receptors were found to be involved in E2 effects. The involvement of estrogen receptor (ER) or an estrogen binding element was suggested by the inhibitory effect of ER antagonists (ICI 182.780, 100nM or Tamoxifen, 1M) on the E2 potentiated secretory response to glutamate. Using E2 coupled to BSA, no effect was observed. Using specific antagonists of PKA, PKC or MAPK, complete inhibition of the E2 effect indicated the possible role of those intracellular transducers. In summary, E2 has rapid potentiating effects on amplitude of GnRH secretion in vitro which involve the kainate subtype of glutamate receptors. Supported by grants from the ARC (99/04-241), the FRSM (3.4515.01), the Faculty of Medicine at the

IS THE AGGRESOME INVOLVED IN POLYGLUTAMINE AGGREGATION?

A. Michalik, S. Serneels and C. Van Broeckhoven
Department of Molecular Genetics, Flanders Institute for Biotechnology (VIB), Born-Bunge Foundation (BBS), University of Antwerp (UIA), B-2610 Antwerpen, Belgium

At least 9 neurodegenerative disorders are caused by expansion of a polyglutamine tract within the respective proteins. Expanded polyglutamine assumes a novel conformation which leads to its aggregation in cytoplasm and nuclei of neurons. Proteins with non-native conformations are rapidly degraded by the ubiquitin-proteasome pathway. However, when the capacity of this pathway is exceeded, misfolded proteins aggregate. Recently, a novel structure termed aggresome was described, which forms in the proximity of the nucleus by active sequestration of protein aggregates from the cell's periphery by retrograde transport on microtubules. In this way aggregates are concentrated into a structure that can be disposed of by autophagy. To examine whether cytoplasmic polyglutamine aggregates are concentrated into an aggresome, we overexpressed expanded polyglutamine (103Q) fused to EGFP in COS-1 cells. Although the majority of aggregates were perinuclear, their localization or number did not change upon treatment of the cells with microtubule-disrupting chemicals nocodazole and vinblastine, nor upon overexpression of p50/dynamitin, which selectively inhibits retrograde transport along microtubules. These data suggest that aggregated polyglutamine is not actively accumulated into an aggresome.

SYSTEMIC NITROGLYCERIN INCREASES FOS EXPRESSION IN MOUSE TRIGEMINAL NUCLEUS CAUDALIS.

Multon S, Mosen J, Halleux S, Hua M.T., Defays C, Schoenen J.
Research Center for Cellular and Molecular Neurobiology. Neuroanatomy laboratory, University of Liège. Belgium.

Nitroglycerin (NTG), a NO donor, is able to trigger attacks in migraineurs several hours after sytemic administration. In rats NTG produces neuronal activation visualized by Fos immunoreactivity in trigeminal nucleus caudalis as well as a number of structures involved in pain and blood pressure control.
Our aim was to study the possibility of reproducing these results in mice. A "model" of trigeminovascular activation could be used in transgenic or knock-out strains, e.g. ArKO mice lacking aromatase, in order to better understand the influence of estrogens on migraine.
NTG (10 mg/kg) was administrated subcutaneously. Animals were sacrificed 4 hours later and the cervical portion of the trigeminal nucleus caudalis, which receives most of the nociceptive afferents from the dural vasculature, was examined.
The results show a significant increase of Fos-positive neurons in laminae I and II of the cervical part of the trigeminal nucleus caudalis (p<0.01) in mice injected with NTG in comparison with those injected with placebo. Our data confirm that systemic NTG is able to induce activation of second order nociceptors in trigeminal nucleus caudalis which may be relevant for sensitization in the trigeminovascular system and used as a model in ArKO mice.

THE SH2 DOMAIN CONTAINING 5-PHOPHATASE SHIP2 IS EXPRESSED IN THE GERMINAL LAYERS OF EMBRYO AND ADULT MOUSE BRAIN INCREASED EXPRESSION IN N-CAM-DEFICIENT MICE

Muraille, E (1), Bantunbungi K (2), Dassesse, D (2), Vanderwinden, JM (2), Cremer, H (3)., Rogister, B (4), Erneux, C (1),, and Schiffmann, SN (2)
1. IRIBHN, 2. Laboratory of Neurophysiology, Universite Libre de Bruxelles, 3. Laboratoire de Genetique et Physiologie du Developpement, CNRS/INSERM, Marseille, 4. Center for Cellular and Molecular Neurobiology, University of Liege.

In the germinative ventricular zone (VZ), growth factors exert their effects by activating phosphatidylinositol-3 kinase and Ras/MAP kinase pathways. SHIP2, an inositol polyphosphate 5-phosphatase, modulate these pathways and therefore potentially regulates neuronal development. SHIP2 mRNA was highly expressed in the VZ at early embryonic stages and subventricular zones (SVZ) at latter stages as revealed by in situ hybridization. No significant expression was seen in differentiated fields. This restricted expression was maintained from E11.5 to birth. In adult brain, SHIP2 was mainly restricted in structures containing neural stem cells the anterior SVZ, the rostral migratory stream (RMS) and the olfactory tubercle. An increase in SHIP2 mRNA signal was detected in N-CAM-deficient mice, which present an accumulation of progenitor cells in the aSVZ and RMS. Western blotting revealed an abundant expression of SHIP2 in proliferating neurospheres and its downregulation in non proliferating differentiated neurospheres. In conclusion, the closely correlation between the pattern of SHIP2 expression in neural stem cell in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.

GLYCINE RECEPTORS ARE EXPRESSED BY POSTNATAL MULTIPOTENT NEURAL PROGENITORS

Nguyen, L., Malgrange, B., Belachew, S., Rogister, B., Rocher, V., Moonen, G. and Rigo, J.-M.
CNCM, University of Liège, Belgium

Multipotent neural progenitors (MNPs) are a well established cell subopulation occuring in the developing but also in the mature mammalian nervous system. Trophic and transcription factors are currently the main extracellular signals known to influence the development and the commitment of MNPs and their progeny. However, several recent works suggest that neurotransmitters could contribute also to neural development. In that respect, rodents cultured embryonic MNPs have been reported to express functional neurotransmitter receptors. Meanwhile, no similar investigation has been made in postnatal and/or in adult rodent brains.
In this study, using immunocytochemical and RT-PCR methods, we show that glycine ionotropic receptors (GlyR) are expressed by postnatal rat striatum-derived nestin positive cells within neurospheres in vitro cultures. By means of the whole-cell patch-clamp technique, we moreover report that glycine triggers in these cells strychnine-sensitive currents in a dose-dependent and reversible way, demonstrating that these GlyRs are functional. Our next effort will be to search for the biological role(s) of GlyRs in MNPs.

FINE STRUCTURAL ANALYSIS OF HISTOPATHOLOGICAL CHANGES, INCLUDING SPONTANEOUS REGENERATION AND REMYELINATION OCCURRING AFTER COMPRESSION INJURY OF THE ADULT RAT SPINAL CORD.

Y. Nyssen, M. Mozin, C.Oliveira, F. Scholtes, G. Brook, J. Schoenen.
Neuroanatomy laboratory, University of Liège, Belgium, Research Center for Cellular and Molecular Neurobiology.

Maceration of the adult rat spinal cord (SC) is followed by a rapid, but transient, in-growth of axons and Schwann cells, which the mechanims remain poorly understood. Here, we perform a fine structural analysis to identify the cellular events involved in tissue repair, regeneration and remyelination.
A sub-dural inflatable balloon compression model was used. Rats were sacrificed at 4, 7, 14 and 28 days post-injury (dpi; n = 10). SC were studied at both light- and EM level. By 4 dpi, the lesion was occupied by many macrophages, degenerating axons and myelin debris.Between 7 and 14 dpi, many small calibre, unmyelinated axons were found in close association with capillaries and epithelial-like cells, which migth be leptomeningeal. From 14-28 dpi, both myelinated - by Schwann cells as myelin periodicity indicate - and unmyelinated axons could be found in bundles, which were encircled by thin fibroblast-like processes. This data shows an early in-growth of regenerating axons and myelination by Schwann cells. We believe that all Schwann cell-associated axons within these lesions were derived from either intrinsic or peripheral neurons. Immuno-EM studies are required to determine their origin. Further studies are needed to determine the role of these findings, in SC plasticity.

EFFECT OF TREADMILL TRAINING ON REGENERATION AFTER A SPINAL CORD INJURY (SCI) IN ADULT RATS

Oliveira C, Multon S, Scholtes F, Poirrier A, Collin H, Martin D, Brook G, Schoenen J
Institut A. Swaen, Rue de Pitteurs 20, 4020 Liège

This study examines motor recovery and tissue repair processes caused by treadmill training of adult Wistar rats after compression injury caused by a sub-durally positioned microballoon (20ml, 5 min). Rats were trained on a treadmill (30min/day, 5days/wk, for 20 wks). Motor skills were evaluated by the BBB score after which electrophysiological and neuroanatomical investigations were performed.
The treadmill-trained rats showed an increase of post-SCI motor performance when compared to non-trained rats (p<0.001). In the latter, the threshold for motor responses induced by transcranial magnetic stimulations tended to be higher (p<0.06). There was no correlation between the sparing or sprouting of 5-HT and CGRP-positive fibres and the training induced improvement of locomotor scores. However, rats with a better BBB score generally demonstrated more Schwann cells and axonal regeneration and fewer arachnoid cells.
More detailed studies are required to determine if the improved BBB scores are due to sprouting or regenerative mechanisms. Furthermore, the potential role of arachnoid cells in reducing axon regeneration needs further attention.

THE REPRESENTATION OF SIMILARITY BETWEEN COMPLEX SHAPES IN MACAQUE MONKEYS

H. Op de Beeck (1,2), J. Wagemans (2), R. Vogels (1)
1. Laboratorium voor Neuro- en Psychofysiologie, K.U.Leuven, Belgium, 2. Department of Psychology, K.U.Leuven, Belgium

Stimulus similarity is an important factor in recognition and categorization, but the computation of similarity is not straightforward in the case of complex, multidimensional stimuli. This problem can be solved by representing these stimuli in a low-dimensional representation space in which the proximity between stimuli is related to their similarity. Studying the neural basis of this capacity, we found a striking isomorphism between the perceptual low-dimensional representation of shape similarity and the way in which shape similarity was represented at the neuronal level in macaque inferotemporal cortex. The responses of single neurons in this population depended on the distance between shapes within these low-dimensional representation spaces. Although all ordinal aspects of the representation spaces could be captured by the physical similarities between the shapes, some systematic metric biases were observed. The neural implementation of shape similarity as found here confirms recent computational models, but these models need to be augmented to capture these metric biases.

FUNCTIONAL PARCELLATION OF GESTURE-SPECIFIC VISUAL PROCESSES IN THE OCCIPITO-TEMPORAL CORTEX

P Peigneux (1,3), E Salmon (1,2), G Garraux (1,2), S Laureys (1,2), J Aerts (1), G Del Fiore (1), C Degueldre (1), A Luxen (1), M Van Der Linden (3)
1. Cyclotron Research Centre, University of Liege, Belgium, 2. Department of Neurology, CHU Sart Tilman, Belgium, 3. Department of Neuropsychology, University of Liege, Belgium

We investigated with PET the neural bases of two stages in the visual perception of human gestures. First, the elaboration of a structural description of the gesture is processed through high-level perceptual analysis. Next, the end-product of this analysis is matched to stored gestural representations, enabling further recognition or imitation. Regional cerebral blood flow comparisons were conducted between four pairs of matched conditions in two populations (a) gesture [GN] vs. object [ON] naming, (b) gesture [GO] vs. object [OO] orientation decision [n=12, from Peigneux et al. (2000) NeuroImage], (c) actual imitation [IMS] vs. production to verbal command [CO] of meaningful gestures, (d) meaningless gestures imitation [IMNS] vs. [CO] (n=18; original study). Random effect conjunction analyses (SPM99) between contrasts [1] confirmed lateral occipito-temporal/V5 involvement in visuo-gestural analysis [conjunction (GO vs. OO) and (IMNS vs. CO)], and [2] disclosed a bilateral middle occipital activation located nearby but inferior [conjunction ([GN vs. ON] X [GO vs. OO]) and (IMS vs. CO)], subtending the storage and/or matching of meaningful gestural representations. These results suggest a functional segregation of gesture-related processes in the occipito-temporal region.

HUMAN BRAIN AREAS INVOLVED IN THE PERCEPTION OF BIOLOGICAL MOTION

H Peuskens (1), S Sunaert (2), J Vanrie (3), K Verfaillie (3), P Vanhecke (2), GA Orban (1)
1. Lab. Neuro- en Psychofysiologie, K.U.Leuven Medical School, 2. Radiology, UZ Gasthuisberg, 3. Dept. Psychology, K.U.Leuven, B-3000 Leuven, Belgium

Perception of biological motion from point-light displays is based on higher order motion processing, temporally and spatially summating motion of only a few dots. Brain areas specifically involved in processing of such stimuli were delineated using block design fMRI experiments in which subjects were presented with a point-light figure performing several actions, alternated with scrambled versions of this stimulus. This design yielded significant differential activation in bilateral hMT/V5, bilateral inferior temporal gyrus and right posterior superior temporal gyrus. Further functional segregation was investigated with a fMRI study testing stimuli ranging from stationary patterns over simple coherent translation, to complex 'jointed' motion patterns without any figure percept. Also, 3D rotation of stationary point light figures without jointed motion, upside-down biological motion and written verbs denoting human actions were added. ITG sites were more strongly activated both in the verb condition and in motion conditions with a clear figure percept than in motion conditions without. By contrast, hMT/V5 , and to a lesser degree, STG was activated by several complex structured motion patterns, regardless of a clear human figure percept.
Supported by GSKE, FWO and IUAP 4/22.

PLASTICITY OF VOLTAGE-GATED CHANNELS IN THE VESTIBULAR NEURONS IN RESPONSE TO A SUPPRESSION OF THEIR LABYRINTHINE SYNAPTIC INPUT.

L. Ris and E. Godaux
Department of Neurosciences, University of Mons-Hainaut, B-7000 Mons.

Labyrinthectomy induces a severe depression of the spontaneous activity of vestibular neurons which completely restores over one week. Here, we explored the underlying mechanisms of this model of brain plasticity by experiments carried out on slices prepared from control guinea pigs and from animals labyrinthectomized one week before, and perfused with artificial cerebrospinal fluid containing a cocktail of synaptic blockers. (1) We found an increase in the pacemaker activity, assessed by the number of spontaneously active neurons per slice (recorded extracellularly) (9.5 in controls and 26.3 after labyrinthectomy). (2) We found an increase in the " dynamic " excitability, assessed by a short ramp current followed by a steady state current (injected intracellularly). In 39 % of the control neurons and in 73 % of the neurons from labyrinthectomized animals, the firing rate increased to a maximum which was higher than the value measured at the end of the plateau. (3) We found a twofold increase in the number of neurons presenting a low-threshold calcium current able to greatly influence neuronal excitability (65 % after labyrinthectomy instead of 29 % in controls). These three phenomena could probably play a role in the restoration of activity observed in vivo.

EFFECT(S) OF SEROTONIN ON THE DEVELOPMENT OF OLIGODENDROGLIAL LINEAGE CELLS

V. Rocher, L. Nguyen, E. Willems, J.-M. Rigo, B. Rogister, G. Moonen and S. Belachew
CNCM, University of Liège, Belgium

As already demonstrated for glycine, neurotransmitters released by neurons may serve as physiological signals triggering oligodendrogliogenesis events that could be of interest for remyelination strategies in some neurodegenerative pathologies.
In a previous report, we had demonstrated, by electrophysiological means, that oligodendrocytes precursors cells (OPCs), as well as mature oligodendrocytes, in culture express 5-HT3 receptors. Since the amplitude of serotonin-elicited currents seemed to increase with in vitro maturation, we hypothesized a role of serotonin in the late stages of oligodendrocytes development, e.g. differentiation and/or myelinisation.
Here we report that serotonin 1) is not toxic for OPCs or mature oligodendrocytes, 2) does not seem to influence OPCs proliferation and 3) tends to favor oligodendrocytes differentiation, even if these latest results deserve confirmation. Finally, RT-PCR experiments revealed that oligodendrocytes in culture express the 5-HT3A isoform of 5-HT3 receptor subunits. Future experiments include the determination of the 5-HT receptor responsible for the observed biological effects and of the effect of 5-HT on the myelinisation process.
This work was supported by the Fonds National de la Recherche Scientifique.

NEUREGULIN SIGNALING REGULATES NEURAL PRECURSOR GROWTH AND THE GENERATION OF OLIGODENDROCYTES IN VITRO

B. Rogister (1), V. Calaora (2), K. Bismuth (2), K. Murray (2), H. Brandt (1), P. Leprince (1), M. Marchionni (3) and M. Dubois-Dalcq (2)
1. Centre de Recherches en Neurobiologie Cellulaire et Molculaire, Université de Liège, Belgium, 2. Neurovirologie et Régénration du Système Nerveux, Institut Pasteur, Paris, France, 3. Cambridge NeuroScience, Inc. Norwood, MA, USA

Neuregulin 1 (Nrg-1) isoforms have been shown to influence the emergence and growth of oligodendrocytes. We have investigated how Nrg-1 signaling of ErbB receptors specifically controls the early stages of oligodendrocyte generation from multipotential neural precursors (NP). We show here that embryonic striatal NP express multiple Nrg-1 transcripts and proteins, as well as their specific receptors, ErbB2 and ErbB4, but not ErbB3. The major isoform synthesized by striatal NP is a transmembrane Type III isoform called Cystein-rich domain Nrg-1. To examine the biological effect of Nrg-1, we added soluble ErbB3 (sErbB3) to growing neurospheres. This inhibitor of Nrg-1 bioactivity decreased NP mitosis and increased their apoptosis, resulting in a significant reduction in neurosphere size and number. When NP were induced to migrate and differentiate by adhesion of neurospheres, the level of type III isoforms detected by RT/PCR and Western blot decreased in parallel with a reduction in Nrg-1 fluorescence intensity. Pretreatment of growing neurospheres with sErbB3 induced a three-fold increase in the proportion of oligodendrocytes generated from NP. We propose that Type III Nrg-1 signaling modulate oligodendrocyte development from NP

LONGITUDINAL STUDY OF HISTOLOGICAL AND LOCOMOTOR CHANGES IN PARAPLEGIC RATS AFTER SPINAL CORD INJURY

Scholtes F., Multon S., Franzen R., Collin H., Brook G., Martin D., Moonen G., Schoenen J.
Centre de Recherches en Neurobiologie Cellulaire et Molculaire (CNCM), Universit de Liège

Introduction. The consequences of spinal cord injury (SCI) can be devastating. SCI is responsible for paralysis and loss of autonomy, urinary tract infections, pressure sores, pain and psychological distress. It is a problem of considerable impact in the European Community alone, there are more than 200,000 para- and tetraplegic patients. At present, there is no cure. The objective of our study is to assess the pathophysiological consequences of experimental SCI, special attention being directed to spontaneous endogenous tissue repair processes and the effects of treadmill training on motor recovery from SCI in the rat.

A QUANTITATIVE EVALUATION OF THE EFFECT OF VARIOUS SK CHANNEL BLOCKERS ON THE APAMIN-SENSITIVE AFTERHYPERPOLARIZATION OF RAT DOPAMINERGIC NEURONS

J. Scuvee-Moreau (1), J-F. Liègeois (2) and V. Seutin (1)
1. Lab of Pharmacology, 2. Medicinal Chemistry, University of Liège, B-4000 Sart-Tilman

Mesencephalic DA neurons play an important role in the control of motivational and motor behaviours. Agents modulating their activity are widely used clinically. Besides neurotransmitter receptors, several types of ionic channels are involved in the modulation of cell excitability. SK-type Ca2 -activated K channels control firing frequency and pattern in tonically spiking neurons. These channels are responsible for the prolonged AHP observed in DA neurons. In order to assess the potential therapeutic interest of drugs acting on these channels, it is critical to identify specific and reversible modulators.
Previous work has shown that apamin and bicuculline salts block these channels in DA neurons (Seutin et al, 1997). However, the effect of the former is irreversible whereas the latter are not specific. We therefore sought to identify better blockers. Preliminary results were obtained with gallamine, laudanosine and methyl-laudanosine. Intracellular recordings in rat brain slices showed that the three compounds reversibly block the apamin-sensitive slow AHP of DA neurons. IC50s were estimated at respectively 77, 152 and 14 microM. None of the drugs modified the decrease in input resistance induced by the GABAA agonist muscimol.
Seutin et al., Neuropharmacology 36, 1653, 1997

FEED-FORWARD INHIBITION TO PURKINJE CELLS IN RAT CEREBELLAR CORTEX BIOLOGICALLY REALISTIC SIMULATIONS.

S. Solinas, R. Maex* and E. De Schutter.
Lab. of Theoretical Neurobiology, Born-Bunge Foundation, Univ. of Antwerp, Antwerp (BELGIUM).

Isolated model Purkinje cells have been shown to reproduce the in-vivo firing patterns characteristic of real PCs, when their inhibitory and excitatory synapses were randomly activated (De Schutter and Bower, J. N.Physiol,1994). In the present study, the model PC was placed in a realistic network of interconnected inhibitory interneurons, out of which 150 stellate cells and 50 basket cells made a synapse on the PC. The PC and the inhibitory network were driven by the same set of randomly firing PFs. In this configuration, the model PC followed fast modulations of the PF firing rate (from 4 to 12 spks/s), with a CV of the interspike intervals between 0.2 and 1 over its entire output range (from 0 to 120 spks/s).
The global activity of molecular layer interneurons was not very coherent because of the sparse network connectivity and the strong effectiveness of parallel fiber spikes in firing iterneurons. Preliminary simulation data show that PFs synchronize pairs of PCs more easily by common disynaptic inhibition than by common monosynaptic excitation.
To conclude, feed-forward inhibition controls the gain of PCs, stabilizes their firing pattern under rapidly varying input conditions, and can represent a disynaptic pathway for synchronization of PCs by PFs.

LONG-TERM DEPRESSION AND PATTERN RECOGNITION IN A MULTI-COMPARTMENTAL MODEL OF A CEREBELLAR PURKINJE CELL

V. Steuber and E. De Schutter
Laboratory for Theoretical Neurobiology, Born-Bunge Foundation, University of Antwerp

A contentious issue in cerebellar research is if long-term depression (LTD) of parallel fibre (PF) synapses is the basis of learning in cerebellar Purkinje cells (PCs). It has been suggested that the storage of PF patterns by LTD leads to a reduced PC response, disinhibition of neurons in the cerebellar nuclei and increased output from the cerebellum. Here, we study the recognition of PF patterns that have been stored by PF LTD in a multi-compartmental PC model with active dendrites. In PC models where PF background input leads to simple spike firing, the only criterion that can be used to distinguish stored and novel patterns is the length of the simple spike pause that occurs shortly after presentation of a PF pattern. Contrary to the classic view, stored PF patterns result in shorter pauses and an increased PC output. The PC model performs two orders of magnitude worse than a corresponding artificial neural network. The performance of the model improves with an increasing frequency of PF background input, and is insensitive to different kinds of intrinsic noise and to temporal jitter in the PF input patterns. Our results cast some doubt on classic theories of cerebellar learning. Supported by FWO (Flanders) and an HFSPO long-term fellowship to VS.

ALTERATIONS OF PETIT MAL SEIZURES VIA EFFECTS OF DOPAMINE ON THE LGN

E. Thomas, T. Grisar and V. Seutin
Research Center for Cellular and Molecular Neurobiology, University of Liege, Belgium

The Genetic Absence Epilepsy Rat from Strasbourg (GAERS) is an animal model of genetic absence epilepsy. Dopamine effects the spike and wave discharges (SWD) in the GAERS. Since the thalamus has a central role in absence epilepsy, we studied the effects of dopamine on neurons of the lateral geniculate nucleus (LGN). Intracellular recordings were made in slices. Recorded neurons had the properties classically described for relay neurons of the dorsal thalamus including a low-threshold Ca spike and Ih current. Bath application of dopamine (100microM) resulted in a mean depolarization of 7 mV. The depolarization was not blocked by the D1 antagonist SCH23390 (1microM) or by the beta-adrenoreceptor antagonist propronolol (1microM). It was very slightly reduced by the D2 antagonist sulpiride (1microM). In the case of 3 out of 9 cells tested, the depolarization was greatly reduced by the alpha1-adrenoreceptor antagonist, prazosin (1microM). The influence of dopaminergic agents on SWD may therefore be mediated by their direct effect on the thalamus.
Supported by the ARC 99/04-241, University of Liege, Belgium

DIFFERENTIAL EXPRESSION OF THE AMPA2-SUBUNIT IN THE PRIMARY VISUAL CORTEX OF ADULT CATS AFTER INDUCTION OF CENTRAL BINOCULAR RETINAL LESIONS

K. Van Damme (1), L. Vercammen (1), I. Leysen (1), U.T. Eysel (2), F. Vandesande (1) and L. Arckens (1)
1. Lab. Neuroendocr. and Immunol. Biotechnol., Katholieke Universiteit Leuven, Belgium, 2. Dept. Neurophysiol., Ruhr-Universitt Bochum, Germany

In cats with central retinal lesions, glutamate levels decreased in lesion-affected area 17 but increased in its border zone, implicating this neurotransmitter in adult cortical plasticity (Arckens et al., 2000). We investigated the involvement of ionotropic Glu-receptors in cortical reorganization with special emphasis on the AMPA2-subunit. Determination of the AMPA2 nucleotide sequence allowed the construction of a cat-specific probe for in situ hybridization studies (ISH). ISH could not reveal a change in AMPA2-mRNA expression in visual cortex of retinal lesion cats compared to normal cats. Using Western Blot analysis we found a higher expression of the AMPA2-protein in the central region of area 17 compared to the peripheral region of normal cats. In retinal lesion cats however we found the opposite, that is lower expression of AMPA2 in the central, visually deprived region versus the non-deprived counterpart. Thus, visual deprivation does affect the AMPA2-expression at the cortical level. Using immunocytochemistry we determined the distribution of AMPA2 in area 17 and 18. The staining is somato-dendritical. Labeled cells are of pyramidal shape and are found in layer II/III and layer V/VI. This pattern corresponds to the mRNA-distribution as found with ISH.

EFFECTS OF TNF-ALPHA ON ENDOTHELIAL CELL-TO-CELL CALCIUM SIGNAL COMMUNICATION

W. Vandamme, K. Braet and L. Leybaert.
Dept. Physiology and Pathophysiology, Ghent University, Ghent, Belgium.

Blood-brain barrier (BBB) opening is an important aspect in the pathogenesis of multiple sclerosis. TNF-a is thought to increase the BBB permeability but the mechanisms leading to this action are not fully elucidated. Endothelial intracellular calcium is a major factor controlling BBB permeability, and we therefore investigated effects of TNF-a on calcium signal communication.
The experiments were performed on two brain endothelium cell lines SV-ARBEC and RBE4. Intercellular calcium waves triggered by mechanical stimulation were significantly reduced in size following 2h TNF-a pretreatment and this effect was more pronounced after 24h pretreatment. Intercellular calcium waves triggered by photolytically releasing IP3 were also inhibited by TNF-a but this effect was only observed after 2h pretreatment. Calcium signal propagation between cells is mediated by an intracellular/gap junctional pathway and an extracellular/purinergic pathway. To investigate whether TNF-a affects the latter, we tested its effect on calcium transients induced by ATP exposure. In RBE4 cells TNF-a did not influence calcium transients and in SV-ARBEC the effect differed between 2h and 24h pretreatment. Taken together, these data indicate that endothelial calcium signal communication is suppressed by TNF-a.

INDUCTION OF BINOCULAR RETINAL LESIONS IN ADULT CATS DOES NOT ALTER THE TYROSINE PHOSPHORYLATION STATE OF PRIMARY VISUAL CORTEX PROTEINS LYING WITHIN A PI-RANGE OF 4 TO 7

G. Van den Bergh (1), A. Lanckriet (1), U. T. Eysel (2), F. Vandesande (1), L. Arckens (1)
1. Lab. Neuroendocr. and Immunol. Biotechnol., Katholieke Universiteit Leuven, Belgium, 2. Dept. Neurophysiol., Ruhr-Universitt Bochum, Germany

In adult cats, the induction of binocular retinal lesions causes a dramatic retinotopic reorganization of the visual cortex. The molecular mechanisms underlying this phenomenon are unknown, but are thought to rely on changes in mRNA and protein expression and on changes of the phosphorylation-state of these proteins. Therefore, a proteomics approach was used to study whether changes in the levels of tyrosine phosphorylation occur in our model of adult cortical plasticity using a monoclonal anti-phosphotyrosine antibody to detect the phosphoproteins. However, comparison of the two-dimensional phosphotyrosine maps of the central, lesion-affected zone of cortical area 17 and the peripheral, non-deprived zone showed no differences in the tyrosine phosphorylation state of any of the visible proteins (pI between 4 and 7).
Some of these phosphoproteins were subsequently identified using mass spectrometry. Besides cytoskeletal proteins(actin, tubulin) and a few metabolic enzymes, proteins involved in signal transduction pathways were identified (transducin beta, 14-3-3 protein gamma).
Phosphorylation site prediction analysis of the identified proteins showed that all of them contain putative phosphotyrosine sites. However, only few of these sites have been experimentally determined.

EXPRESSION OF NEUROFILAMENT PROTEIN AS A MARKER FOR THE ANATOMICAL ORGANIZATION IN THE VISUAL SYSTEM OF CAT BRAIN.

E. Van der Gucht, F. Vandesande, L. Arckens
Lab. Neuroendocrin. and Immunol. Biotechnol., Katholieke Universiteit Leuven, Leuven, Belgium.

In this immunocytochemical study, we examined neurofilament protein staining profiles in cat visual system as revealed with the SMI-32 antibody. This monoclonal antibody labels primarily the cell body and dendrites of pyramidal neurons in cortical layers III, V and VI. Neurofilament protein-immunoreactive neurons were prominent in twenty visual areas areas 17, 18, 19, 20a, 20b, 21a, 21b, 7, PMLS, PLLS, AMLS, ALLS, DLS, VLS, PS, EVA, SVA, CVA, INSULAR and ALG. In addition, we have also found strong immunopositive cells in the lateral geniculate nucleus complex (dLGN, MIN), but no cells were present in the vLGN, the PGN and the parvocellular C (1-3) laminae. In each cortical area, the SMI-32 antibody against neurofilament protein revealed a characteristic pattern of immunostaining size, shape, intensity and density of neurofilament protein-immunoreactive neurons and their dendritic arborization differed substantially across all visual areas. Moreover, it was also obvious that several visual areas showed differences in laminar distribution. Those diverse profiles may be used to delineate various cortical areas. Therefore, the expression of neurofilament protein can be used as a specific marker to define areal patterns and topographic boundaries in cat visual system.

EPHRINS ARE REQUIRED FOR THE PATTERNING OF NEURONAL CONNECTIONS IN THE DEVELOPING NEOCORTEX

P. Vanderhaeghen, V. Depaepe, A. Dufour, L. Passante, S. Wart, Vassart G.
Institute of Interdisciplinary Research, University of Brussels (U.L.B.).

The neocortex contains numerous maps of sensory and motor information. The amount of cortical surface devoted to different regions within these maps varies between species and even between individuals, in a manner that reflects functional importance and neural abilities. The molecular mechanisms responsible for the generation of sensory maps in the neocortex remain poorly understood. Here we show that ephrin-A5 and its receptor EphA4 display complementary gradients of expression and binding in the developing somatosensory cortex and thalamus. In vitro assays show that ephrin-A5 can act as topographically-specific repellent for thalamocortical axons. Strikingly, ephrin-A5 gene disruption causes a graded, topographically specific distortion in the cortical anatomical and functional somatosensory maps, with medial regions contracted and lateral regions expanded, giving changes of up to 50% in relative area. Distortions were not seen at thalamic or brainstem levels. Our results provide evidence for within-area thalamocortical mapping labels. Moreover, they indicate that a genetic difference in mapping labels can change the relative scale of different regions within a cortical map.

INTERSTITIAL CELLS OF CAJAL AND AUTONOMIC NERVES IN THE STRIATED MUSCULATURE OF THE MOUSE ESOPHAGUS.

J.-M. Vanderwinden, J.J. Rumessen, A. de Kerchove dExaerde, S. Mignon, J.-J. Panthier, S.N. Schiffmann
Laboratoire de Neurophysiologie, Facult de Mdecine, Universit Libre de Bruxelles; Brussels, Belgium

Interstitial cells of Cajal (ICC) are specialized cells intercalated between nerves and smooth muscle of the gastrointestinal (GI) tract. Some ICC generate the slow waves of depolarization of the smooth muscle while others are involved in nitrergic and cholinergic neurotransmission.
Here we used Kit immunoreactivity to study their distribution and development in the striated musculature of the mouse esophagus. Kit W-lacZ transgenic mice (lacZ inserted in place of the first exon of Kit), were also processed for quantitative and ultrastructural studies.
Spindle-shaped ICC were scarce in the thoracic esophagus, while their number increased toward the cardia. They did not form networks and had no relationship with intrinsic ganglia and motor end-plates. They were close to nerves immunoreactive for nNOS, VIP or NPY but not for SP, CGRP, ENK or the capsaicin receptor VR1. They were present in fetus but absent in adult ICC deficient Kit W-lacZ / Kit Wv mice. ICC were identified by electron microscopy in the striated muscle of the esophagus by their ultrastructure and by lacZ expression, while other cells were unlabeled.
ICC are scattered between striated muscle cells in the mouse esophagus. They could represent specialized esophageal spindle proprioceptors for intrinsic reflexes.

DISPARITY-SENSITIVE FMRI SIGNALS IN THE AWAKE FIXATING MONKEY

W. Vanduffel, D.Y. Tsao, D. Fize, K. Nelissen, P. Van Hecke, J.B. Mandeville, R.B.H. Tootell, G.A. Orban.
Neuro- en Psychofysiologie, K.U.Leuven Medical School; Radiologie UZ-Gasthuisberg K.U.Leuven; MGH-NMR ctr., Boston, USA.

We used functional magnetic resonance imaging (fMRI) to study the representation of binocular disparity in the visual cortex of the awake monkey. To this end, we trained two monkeys to fixate while they viewed stereoscopic stimuli through anaglyph goggles. Prior scanning (1.5T, Siemens, Vision), we injected a contrast agent (dextran-coated iron oxide agents; MION; 4-7mg/kg) with a long blood half-live. MR-signals evoked during the presentation of a random dot stereogram (annuli and several shapes) were compared with signals evoked during the viewing of a full-field zero-disparity random dot stereogram. The disparity defined annulus changed in depth every 66-4000ms. This comparison revealed that zero-order stereo-sensitivity (for the annulus) was mainly restricted to dorsal visual areas V3, V3A, posterior parietal areas, regions within the posterior portion of the intraparietal sulcus and the dorsal-most portion of the superior temporal sulcus (abutting area MT). Activity evoked by the disparity-defined shapes was also compared with that of shapes defined by illusory contours and kinetic boundaries. Several mid-level visual areas were involved in the processing of shapes (or parts of shapes) irrespective the cue. Supported by GOA 2000/11, GSKE, and NFWO G0112.00.

IN-VIVO FMRI STUDY OF HYPOTHALAMUS ACTIVATION DURING THERMOREGULATION IN THE RAT

G. Vanhoutte (1), M. Verhoye (1), E. Raman (2), A. Van der Linden (1)
1. Bio-Imaging Lab, RUCA, University of Antwerp, Antwerp, Belgium, 2. Visie Lab, RUCA, University of Antwerp, Antwerp Belgium

We studied hypothalamic activation during increasing rectal temperature (Tr) using fMRI applying both, blood oxygenation level dependent BOLD and cerebral blood volume CBV sensitive MRI protocols, using an intravascular contrast agens.
The study was performed using a 7T MR-system on rats anaesthetised with alfa-chloralose and catheterised in the tail vein for contrast injection of NC100150. Mean intensity was calculated from control images at Tr 37C and subtracted from the consecutive data obtained during thermal stimulation. The negative BOLD signal change of more than 5 to 10% was superimposed on high resolution images.
In the hypothalamus, a gradual decrease in the BOLD signal intensity is seen while CBV remains unchanged. As a consequence we can conclude that the negative BOLD signal in hypothalamus is due to a rise in O2 consumption (activation) in absence of any regulatory changes in local CBV. A possible explanation is that the blood vessels in the hypothalamus are always dilated. The subtracted CBV images failing to show any signal change in the hypothalamus confirm this.

VISUAL AND AUDITORY TEMPORAL PROCESSING IN CHILDREN WITH DYSLEXIA

M. Van Ingelghem (1), P. Ghesquiere (1), A. van Wieringen (2), J. Wouters (2), P. Onghena (3), E. Vandenbussche (4)
K.U. Leuven 1. Section of Orthopedagogics, 2. Laboratory for Experimental ORL, 3. Center for Methodology of Empirical Educational Research, 4. Laboratory for Neuropsychology

In this study the hypothesis of a general (i.e. cross-modal) temporal processing deficit is tested by examining the function of the subsystems that are responsible for temporal processing in either the visual and the auditory system in the same children with dyslexia. Subjects were dyslexi

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