Belgian Society for Neuroscience

The 5th bi-annual meeting 2003

Abstract list

ANALYSIS OF TAU m RNA IN ALZHEIMER'S DISEASE

A. Boutajangout, A. Boom and J. P. Brion
Laboratory of Histology, Neuropathology and Neuroanatomy, Universit Libre de Bruxelles, Brussels, Belgium

Neurofibrillary tangles (NFT) are bundles of abnormal filaments accumulating in neurons in Alzheimers disease (AD), and composed of aggregated forms of the MAP-tau. Missense mutations in the tau gene have been described in familial forms of FTD-17 associated with formation of NFT, leading to increased expression of selected tau isoforms. To investigate if a similar mechanism might be operating in AD, we have analyzed the expression of tau mRNA transcripts in brain tissue from sporadic AD patients and from control subjects. Total RNA was extracted from human temporal cortex (affected area) and from cerebellar cortex.. The yield of total RNA was the same control subjects and in AD patients both in temporal and cerebellar cortex. Three RT-PCR products corresponding to the tau mRNAs with 0, 1 or 2N isoforms and 2 RT-PCR products corresponding to the tau mRNAs with 3 or 4 repeat domains were analyzed. The relative ratio of the different tau mRNAs were estimated by densitometry. A significant increase in the proportion of 0N tau mRNA and a decrease in the 1N tau mRNA was observed in the temporal cortex in AD, by comparison with control subjects. These results suggest that a relative imbalance in the levels of selected tau mRNAs might play a role in the formation of NFT in AD.

Dopamine D4 receptor-mediated presynaptic inhibition of GABAergic transmission in the rat supraoptic nucleus

Karima Azdad, Richard Piet, Dominique A. Poulain and Stphane H. R. Oliet
INSERM U.378, Universit Victor Segalen, Institut Franois Magendie, 33077 Bordeaux, France

Because oxytocin- and vasopressin-secreting supraoptic neurons are under the control of a prominent GABAergic inhibition, we investigated the possibility that dopamine exerts its action by modulating GABA-mediated transmission. Whole-cell voltage-clamp recordings of supraoptic neurons were carried out in hypothalamic slices to determine the action of dopamine on miniature inhibitory postsynaptic currents. Application of dopamine caused a reversible reduction in the frequency, but not the amplitude, of unitary synaptic events indicating that dopamine was acting presynaptically to reduce GABAergic transmission. Dopamine inhibitory action was greatly reduced by two highly selective D4 receptor antagonists and to a lower extent by clozapine but was unaffected by, D1/D5 and D2/D3 receptor antagonists. In agreement with these results, the action of dopamine was mimicked by a potent D4 receptor agonist but not by D1/D5 and D2/D3 receptor agonists. These data clearly indicate that D4 receptors are located on GABA terminals in the supraoptic nucleus and that their activation reduces GABA release. Dopaminergic facilitation of neurohypophysial hormone release appears to result, at least in part, from disinhibition of magnocellular neurons caused by the depression of GABAergic transmission.

The ArKO mouse provides new evidence that estradiol is required for the development of the female brain

J. Bakker1, S. Honda2, N. Harada2, J. Balthazart1
1.Center for Cellular and Molecular Neurobiology, University of Liege, Belgium. 2. Division of Molecular Genetics, Fujita Health University, Toyoake, Japan.

The classic theory of sexual differentiation is that testosterone secreted by the testes promotes the development of a male brain whereas a female brain develops in the absence of any sex steroids. The recent introduction of the aromatase knockout (ArKO) mouse which is deficient in estradiol due to a targeted mutation in the aromatase gene has allowed us to re-evaluate this theory. Female mice (wildtype (WT), heterozygous (HET) and homozygous (ArKO)), were ovariectomized in adulthood and subsequently tested for odor preferences (choice intact male versus estrous female) in a Y-maze. When treated with testosterone, ArKO females spent less time sniffing odors (both volatile and non-volatile) from either male or female stimuli compared to WT and HET. These defects in olfactory investigation were partly corrected with estradiol treatment in adulthood. Estradiol-treated ArKO females no longer differed from WT and HET in the time spent investigating non-volatile odors. However, ArKO females still investigated volatile odors less than WT and HET. Sexual receptivity was severely impaired in ArKO females following treatments with estradiol and progesterone that induced receptivity in WT and HET. These findings demonstrate that estrogen is required for a normal female development.

ESTRADIOL RAPIDLY INCREASES CREB PHOSPHORYLATION IN THE QUAIL BRAIN

J. Balthazart, C. Dejace & G.F. Ball
Ctr. Cell. Molec. Neurobiol., Univ. Liege, Belgium & Dept Psychol. Brain Sciences , Johns Hopkins Univ., Baltimore, MD USA

In quail, the preoptic aromatase activity is rapidly modulated by the calcium-dependent activation of kinases C and A. Estrogen bioavailability could thus rapidly change in specific aromatase-expressing brain areas. Besides their action as transcription factors, estrogens stimulate adenylyl cyclase in rats leading to an increased phosphorylation of cyclic AMP response element binding protein (pCREB). Castrated male quail were injected with estradiol (E2) in oil (100 g/bird, n=12) or with the solvent (n=8). 15 or 30 min later, half of the subjects in each group were perfused and coronal brain sections were stained by immunocytochemistry for pCREB. Basal levels of pCREB expression were detected in control birds. In contrast, significantly higher numbers of pCREB immunoreactive cell nuclei were observed after E2 injections in the medial preoptic area, hippocampus and nucleus taeniae (avian homologue of the mammalian amygdala) where they largely overlapped with aromatase cell groups. Rapid changes of estrogen bioavailability could thus rapidly lead to localized variations in CREB phosphorylation in brain areas implicated in the control of reproductive behavior. Their behavioral significance should now be investigated.
Supported by MH50388, ARC99/04-241, FRFC 2.4555.01.

Transdifferentiation of mesenchymal stem cells into neural cells in vitro and in vivo studies

K. Bantubungi*, D.Blum, S.Wislet-Gendebien, T.Tondreau, A.Delforges, L.Lagneaux, B.Rogister, S.Schiffmann*
*Laboratoire de Neurophysiologie, ULB-Erasme, Brussels Laboratoire de Neurochirurgie Exprimentale, ULB-Erasme, Brussels CNCM, ULg,Lige Institut Bordet, ULB, Brussels

After brain transplants, intraperitoneal or intraveneous injections, mesenchymal stem cells (MSC) are particularly able to differentiate into neurons and astrocytes. These observations suggest that MSC could be use therapeutically in neurodegenerative diseases. Our project aims to characterize human MSC (hMSC) in vitro following differentiation protocol by immunocytochemical, biochemical, molecular and electrophysiological techniques. We also aim to evaluate the phenotypic plasticity of MSC cells in vivo as well as their therapeutical potential in models of Parkinsons and Huntingtons diseases. Our RT-PCR results showed that human MSC express GFAP in absence of differentiation protocol. MAP-2 expression is observed in differentiated cells but also in non-differentiated cells. We evaluate in vivo plasticity using rat MSC (rMSC) grafted into the striatum of nave rats or rats previously lesioned stereotaxically with quinolinic-acid, a compound which allow to generate a phenotypic model of Huntingtons disease. This allows us to evaluate (1) the phenotypic plasticity of MSC but also (2) their therapeutical potential. The first point is currently studied using immunohistofluorescence and confocal microscopy and the second point by rotametry.

Selectively rescued expression of calretinin in the cerebellar granule cells of calretinin knock-out mice

Bertrand Bearzatto, Fawzia Baba-Assa, Laurent Servais, Alban de Kerchove dExaerde, Guy Cheronb, Stephane Schurmansa and Serge N. Schiffmann
Laboratoire de Neurophysiologie and aIRIBHM, Faculte de Medecine, Universite Libre de Bruxelles, Brussels, Belgium. bLaboratoire de Neurophysiologie, Universit Mons-Hainaut, Mons, Belgium

The control of [Ca2+]i is crucial for the neuron and is performed among others by cytoplasmic Ca2+ binding proteins like calretinin (Cr). Cr is enriched in the cerebellum in granule, unipolar brush and lugaro cells. Changes in [Ca2+]i in cerebellar neurons would disturb the entire cerebellar physiology. Analysis of Cr-deficient (Cr-/-) mice showed impaired coordination and abnormalities in the Purkinje cell firing in vivo. The absence of Cr in cerebellar granule cells constitutes an hypothesis consistent with all these perturbations. We specifically rescued the expression of Cr in the cerebellar granule cells of Cr-/- mice. The Cr expression was targeted to cerebellar granule cells by using a fragment of the gene coding for the GABAA alpha 6 subunit. We obtained several lines of transgenic Cr-/- mice exhibiting a selective and restricted re-expression of Cr in granule cells as demonstrated by in situ hybridization and imunohistochemistry. These mice will allow us to assess for the precise role(s) of Cr in the cerebellar physiology and to elucidate the mechanisms leading to its dysfunction in the Cr-/- mice. We also believe that, together with other mutants, our model, will unmask new and unknown aspects of the cerebellar circuitry.

NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional GABAergic neurons in the adult dentate gyrus

Shibeshih Belachew, Ramesh Chittajallu, Xiaoqing Yuan, Adan A. Aguirre, Stacie Anderson, and Vittorio Gallo
Center for Neuroscience Research, Childrens National Medical Center, Washington, D.C., 20010-2970, USA.
Center for Cellular and Molecular Neurobiology, Department of Neurology, University of Lige, 4020 Lige, Belgium

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial, but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the CNP gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABA-ergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Effects of modulation of the extrasynaptic GABAA conductance in cerebellar granule cells a computational study

Michiel Berends, Reinoud Maex, Erik De Schutter
Laboratory of theoretical neurobiology, Born-Bunge Foundation, University of Antwerp, PhD student

GABAA receptors in cerebellar granule cells are located either at the Golgi cell (GoC) to granule cell (GrC) synapse or extrasynaptically. The GoC spike-evoked inhibition of the GrC contains a direct component, mediated by the synaptically located receptors, and a spillover component, caused by activation of the extrasynaptical receptors. The extrasynaptic receptors transfer most charge and are tonically activated even in the absence of GoC spikes (Hamann et al., Neuron, 2002). ACh increases their conductance (Rossi et al., J.Physiol., 2003).
The effect of modulation of this conductance is investigated on a model of a single granule cell and a network model.
Upon increasing the extrasynaptic conductance, the f-f-curve, which portrays the response of the GrC frequency to mossy fiber input frequency, shows a linear shift towards higher input frequencies. Furthermore the effective duration of GoC spike-evoked inhibition is prolonged in the case of increased extrasynaptic conductance.
In a network model of the granular layer with highly synchronously spiking GoCs, an increase of the extrasynaptic conductance of a subpopulation of GrCs, leads to reduced and slightly delayed spiking of these GrCs.

Thiamine triphosphatase is predomminantly expressed in hippocampal pyramidal neurons and cerebellar Purkinje cells

Grazyna CHANAS, Jan CZERNIECKI, Pierre LEPRINCE, Myriam VERLAET Bernard LAKAYE, Thierry GRISAR and Lucien BETTENDORFF
Center for Cellular and Molecular Neurobiology, University of Liege, 17, place Delcour, 4020 Liege, Belgium

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but its role remains unknown. In the nervous system, ThTP may act as a phosphate donor for protein phosphorylation and may be part of a new signal transduction pathway [1]. We have recently characterized a highly specific thiamine triphosphatase (ThTPase) from mammalian brain [2]. The role of this enzyme may be to control the intracellular ThTP concentrations. In order to assess the regional and subcellular localization of ThTPase in rodent brain, we prepared a chicken polyclonal antibody directed against recombinant human ThTPase. Using mouse brain sections, we show that ThTPase immunoreactivity is essentially localized in the pyramidal cell bodies of the CA1, CA2 and CA3 regions of the hippocampus and in cerebellar Purkinje cells. The 25-kDa ThTPase form is specific of mammalian cells and accordingly, no specific labeling was observed on sections made from quail cerebellum. This selective ThTPase expression was confirmed using in situ hybridization, but its significance remains unknown. It might be related to either specific signaling mechanisms or metabolic states in these cells.

1.NGHIEM et al. (2000) FASEB J. 14 543-554
2.LAKAYE et al. (2002) J. Biol. Chem. 277 13771-13777

Differential mechanisms underlie cell death induced by 3-nitropropionic acid in cultured striatal and cortical neurons

Marie-Christine Galas*, Nicolas Bizat , Laetitia Cuvelier*, Kadiombo Bantubungi*, Emmanuel Brouillet, Serge N. Schiffmann* and David Blum*
Laboratory of Neurophysiology, CP601, and IRIBHM, Universit Libre de Bruxelles, 1070 Brussels, Belgium.

3-nitropropionic acid (3NP) produces preferential striatal degeneration reminiscent of Huntington's disease (HD). Here, we aimed to determine whether factors intrinsic to striatal neurons might contribute to cell susceptibility to 3NP. We compared the effects of 3NP between primary striatal and cortical cultures. 3NP produced similar inhibition of succinate dehydrogenase in cortical and striatal neurons, leading to cell death of comparable extent in both types of cultures. However, death occurred through different mechanisms in each type of neurons. Up-regulation and translocation of Bad, release of mitochondrial apoptogenic factors and calpain, but not caspase activation were selectively found in striatal neurons exposed to 3NP. Accordingly, only the calpain inhibitor but not caspase inhibitor was able to protect striatal cells against 3NP toxicity. Conversely, cortical neurons degenerated through a caspase- and calpain-independent pathway but developed, unlike striatal cells, an Akt/PKB activation. In contrast to 3NP, staurosporine, a well-known apoptosis inducer, produced similar caspase-dependent cell death pathway in both neuronal types. These results provide the first evidence that striatal neurons develop specific cell death pathways in response to 3NP.

A dual role of adenosine A2A receptors in 3-nitropropionic acid-induced striatal lesions implications for the neuroprotective potential of A2A antagonists

David BLUM, Marie-Christine GALAS, Annita PINTOR, Emmanuel BROUILLET, Catherine LEDENT, Christa E. MULLER, Kadiombo BANTUBUNGI, Mariangela GALLUZZO, David GALL, Laetitia CUVELIER, Anne-Sophie ROLLAND, Patrizia POPOLI, Serge N. SCHIFFMANN
Laboratory of Neurophysiology, Universit Libre de Bruxelles, 1070 Brussels, Belgium.

Reduction of A2A receptor expression is one of the earliest events occurring both in Huntingtons disease patients and in mice overexpressing mutated huntingtin. Interestingly, increased activity of A2A receptors has been found in striatal cells prone to degenerate in experimental models of this neurodegenerative disease. However, the role of A2A receptors in the pathogenesis of Huntingtons Disease (HD) remains obscure. In the present study, using A2A-/- mice and pharmacological compounds in rat, we demonstrate that striatal neurodegeneration induced by the mitochondrial toxin 3-nitropropionic acid (3NP) is regulated by A2A receptors. Our results show that the striatal outcome induced by 3NP depends on a balance between the deleterious activity of presynaptic A2A receptors and the protective activity of postsynaptic A2A receptors. Moreover, microdyalysis data demonstrate that this balance is anatomically determined since the A2A presynaptic control on striatal glutamate release is absent within the posterior striatum. Therefore, since blockade of A2A receptors have differential effects on striatal cell death in vivo, therapeutic use of A2A antagonists in Huntingtons could exhibit a undesirable biphasic effects.

C15O2-Positron Emission Tomography activation study during vagus nerve stimulation in patients with refractory epilepsy.

Tommy Bosman*, Kristl Vonck*, Koen Van Laere, Stefanie Dedeurwaerdere*, Jacques De Reuck*, Paul Boon*
*Reference Center for refractory epilepsy, Department of Neurology, Ghent University Hospital; Department of Nuclear Medicine, Leuven University Hospital, Belgium

Functional imaging may reveal pathways involved in the mode of action of vagus nerve stimulation (VNS) as a treatment for epilepsy. In a previous SPECT study changes of thalamic cerebral blood flow (CBF) was the most significant finding. To further explore these changes and to evaluate temporal relationships between CBF changes and electrical stimulation, we performed dynamic C15O2-positron emission tomography (PET) during VNS.
13 patients (7M,6F) treated with VNS for refractory epilepsy at Ghent University Hospital underwent C15O2-PET during the initial 30-seconds stimulation train. Image acquisition was performed during 4 one-minute intervals starting from the onset of stimulation. Each patient underwent 8 sequential scans alternating the on and off mode of the pulse generator.
During the first minute, SPM analysis showed a significantly increased CBF in a right-thalamus/left-midbrain cluster and in the right vermis spreading towards the left anterior lobe of the cerebellum. This hyperperfusion extended radially in the second minute. The third and fourth minute yielded no CBF changes.
The thalamus, a structure known to play a key role in the pathophysiology of epilepsy, is most consistently involved when evaluating the mechanism of action of VNS in this way.

Delayed injection of GM-CSF improves functional recovery and promotes axonal regeneration after spinal cord injury.

Delphine Bouhy, Jean Schoenen, Rachelle Franzen
Research Center for Cellular and Molecular Neurobiology, Neuroanatomy, University of Lige, Belgium

Injured axons in the adult CNS fail to regenerate. We have previously shown that macrophage activation decreases 14 to 21 days post-injury, and that this decrease was associated to a regression of spontaneous axonal invasion of the lesion site. This shift of macrophage from activated to de-activated phenotype probably contributes to the regeneration failure of central axons.
The aim of this work was to study the effect of GM-CSF, a macrophage-activating cytokine, on axonal regrowth and locomotor recovery after a spinal cord lesion. 44 rats underwent an irreversible T8 spinal cord compression. They received a GM-CSF or a saline injection (control) 28 days post injury and were allowed to survive for 4 more weeks. The effects on functional recovery were evaluated with the BBB scale.
Our results demonstrate that GM-CSF promotes functional recovery in the severely injured spinal cord. Western Blot and immunohistochemical studies of neuronal (NF, Serotonin, CGRP, GAP-43, BDNF) and non-neuronal (OX-42, NGFr,) markers demonstrate that macrophage activation is maintained in GM-CSF treated rats and that a lot of neurofilaments and Schwann cells invade the lesion.
Our results have relevant implications for future pharmacological therapeutic strategies of the injured spinal cord.

Induction of Astroglial fate in Neural Stem Cell by co-cultured Mesenchymal Stem Cell.

Bruyre, F.., Wislet-Gendebien, S., Leprince, P., Moonen, G. and Rogister, B.
Center for Cellular and Molecular Neuroscience, University of Lige, Belgium

The fate determination of the neural precursors, which generate neuronal and glial cells, is regulated by cell-intrinsic programs and extrinsic cues. It has recently been shown that the mesenchymal stem cell (MSC) fraction of bone marrow cells grafted in central nervous system after a lesion, is able to differentiate into neural tissue. We have studied the influence of mesenchymal stem cells on neural stem cells (mNSC) proliferation and differentiation. We showed that adult rat MSC are able to direct mNSC toward an astroglial fate. This effect appears in co-cultures of rMSC with mNSC with a direct cell to cell contact or with rMSC conditioned medium, suggesting that this is due, at least partly, to soluble factors. The stimulation of astrocytic differentiation is combined with a decrease in neuronal cell number. We have recently reported that rMSC can express nestin, an intermediate filament protein, when they are cultivated in specific conditions. The medium conditioned by nestin(+) rMSC is more active in inducing the astroglial fate than the medium conditioned by nestin(-) rMSC. Using a quantified RT-PCR approach, we demonstrated that nestin(+) rMSC express BMP4 (an already known astroglial fate inducer during development) 10 times more than the nestin(-) rMSC.

THE DISTRIBUTION OF THE STEROID RECEPTOR COACTIVATOR SRC-1 IS SEXUALLY DIFFERENTIATED IN THE SONG CONTROL NUCLEI OF MALE AND FEMALE CANARIES

Thierry D. Charlier1, Jacques Balthazart1, Gregory F. Ball2
1 Univ. Lige, Center Cell. Mol Neurobiol., Res. Grp. Behav.Neuroendo., Lige B-4020, Belgium
2 Dept Psych. Brain Sciences, Johns Hopkins Univ., Baltimore, MD 21218, USA

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including receptors for sex steroid hormones. In vivo studies confirm the physiological significance of SRC-1 in mediating the effects of estrogens on the differentiation and activation of female behavior in rats. The distribution of SRC-1 transcripts was analyzed in the brain of male and female canaries by in situ hybridization. A heterogeneous sexually differentiated distribution was observed with high numbers of densely labeled cells being present in many steroid-sensitive areas including the medial preoptic nucleus, several hypothalamic nuclei, five song control nuclei and several catecholaminergic areas. The volume of the HVC defined by the dense SRC-1 expression was significantly larger in males than in females. Moreover, area X could be distinguished in males but not females by a higher SRC-1 expression compared to the surrounding lobus parolfactorius. The presence of SRC-1 in the telencephalic song control nuclei and in the catecholaminergic cell groups that innervate the song system supports the idea that SRC-1 expression could play an active role in the control of singing behavior by modulating estrogen and androgen receptors action at both locations.

A higher-order motion region in human inferior parietal lobule.

Kristl G. Claeys1,2, Delwin T. Lindsey3, Erik De Schutter2, Guy A. Orban1
1Laboratorium voor Neuro- en Psychofysiologie, K.U.Leuven, Belgium, 2Laboratorium voor Theoretische Neurobiologie, University of Antwerp, Belgium, 3Department of Psychology, Ohio State University, USA

We studied the human neural substrate of a higher-order motion system using fMRI. In a first experiment (n=6), we used isoluminant horizontal red/green sinewave gratings and a luminance-modulated achromatic control grating, that moved either up or down or were stationary. Relative salience in the stimulus was manipulated and isoluminance was determined by motion photometry. By contrasting the salience-based motion conditions with their stationary counterparts, we observed activation sites in inferior parietal lobule (IPL) and in bilateral area MT+. The functional profile of IPL reveals bilateral activity which is salience-specific, whereas that of MT+ shows contralateral activity. In a second experiment (n=12), we used the quartet apparent motion display, which was contrasted to a control condition in which four dots were synchronously presented. Again we observed activation in IPL and in bilateral MT+. The IPL site showed specificity for the apparent motion condition and represented the entire visual field, contrary to MT+ that predominantly processed contralateral motion. We conclude that there are two motion-processing systems in humans, a luminance-based contralateral lower-level system including area MT+, and a salience-based bilateral higher-level system represented in IPL.

Age-dependent expression of collapsin response mediator proteins (CRMPs) in cat visual cortex

L. Cnops, G. Van den Bergh, K. Burnat and L. Arckens
Lab. Neuroendocrinology and Immunological Biotechnology, K.U.Leuven, Belgium
Lab. Visual Perception, Nencki Institute, Poland

The visual cortex of the cat is immature at birth and develops gradually during the first postnatal weeks. In previous 2D-DIGE experiments we identified 16 proteins with an age-dependent expression in kitten visual cortex, including four members of the collapsin response mediator protein family (Van den Bergh et al., 2003). The CRMPs are known to be involved in growth cone collapse and neuronal differentiation. We therefore initiated a detailed investigation of their distribution and expression in cat visual cortex to reveal their possible role in the development of the visual system. In situ hybridization revealed a specific distribution for CRMP1,2,4 and 5 mRNA in kitten visual cortex, and a comparable pattern but clearly of lower intensity in adult cat. CRMP3 could not be detected at all. Upon determining the specificity of the respective antibodies for cat CRMPs, Western blotting allowed quantification of the protein expression level of CRMP2, 4 and 5. CRMP4 and 5 showed a high expression level in kitten with a down regulation in the adult state. For CRMP2 the differences between young and adult animals were less clear and may possibly result from posttranslational modifications. These experiments prove that CRMPs are important in the development of cat visual cortex.

ALPHA NORADRENERGIC RECEPTORS MEDIATE DOPAMINERGIC EFFECTS IN THE QUAIL PREOPTIC AREA

WCornil C.A., Seutin V., Motte P., Balthazart J.
Ctr.Molec.Cell.Neurobiol.,Univ. Liege, Belgium. Dept. Life Sciences, Univ.Liege, Belgium.

The enzyme aromatase catalyzes the conversion of androgens into estrogens and is critical for the activation of male sexual behavior. Anatomical and biochemical data suggest that dopamine (DA) may directly control aromatase activity in the medial preoptic nucleus (POM). Intracellular recordings indeed showed that DA hyperpolarises most POM neurons (76%) but depolarizes a small minority (5%) and some biocytin-injected cells that were hyperpolarised by DA are actually immunoreactive for aromatase. These effects are not blocked by D1 or D2 antagonists (SCH-23390, sulpiride). In extracellular recordings, DA inhibits the firing of most cells (52%) while 24% are excited. These effects also are not blocked by DA antagonists, they are inhibited by alpha2-(yohimbine, Yo) and alpha1-(prazosin) noradrenergic antagonists respectively. The ability of Yo to displace the inhibitory concentration-response curves is similar for DA and NE indicating interactions with a same receptor. Together, these data demonstrate that the effects of DA in the POM are mediated mainly by the activation of alpha2 or alpha1 receptors and that DA may act directly on aromatase cells.
Supported by MH50388, ARC99/04-241, FRFC 2.4555.01, FRFC 2.4542.00 and les Fonds Speciaux pour la Recherche. CC is FNRS Research Fellow

Acute vagus nerve stimulation (VNS) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

S. Dedeurwaerdere1, K. Vonck1 , P. Vanhese1, T. Grisar2, P. Boon1.
Clinical and Experimental Neurophysiology Laboratory, Ghent University Hospital, Ghent, Belgium; Laboratory for Biochemistry, University of Liege, Liege, Belgium.

VNS is a treatment for refractory epilepsy. GAERS are a validated model for absence epilepsy. The absence seizures are accompanied by Spike and Wave Discharges (SWDs) on the electroencephalogram (EEG). These discharges can be stopped by unexpected stimuli. The aim of this work was to evaluate if acute VNS could interrupt or shorten SWDs.
Twelve GAERS were implanted with 5 EEG electrodes and a stimulation electrode around the left vagus nerve. A randomised crossover design was used. The control group (n=5) was not stimulated. Stimuli (2V, 30Hz and 500s pulses) were delivered when a SWD occurred on the EEG. The experiment was repeated on a consecutive day.
The duration of SWDs was significantly (p<0.05) prolonged by VNS when compared with baseline conditions and the control group. The mean duration of SWDs without VNS was 13.8s (SD=5.3) and became 18.1s (SD=4.9) with VNS. This effect was most pronounced (p<0.01)the first time the experiment was performed. The difference in SWD duration was not significant on the second day of the study.
In a previous study when a chronic set-up (7 days of VNS) was used, no significant changes in number and duration of the SWDs were found. The results found in this study seem related to an acute effect of VNS and disappear in a chronic setting.

The processing of 2D shape in the cerebral cortex of the monkey and the human

K. Denys(1), W. Vanduffel(1,2), D. Fize(1), H. Peuskens(1), K. Nelissen(1), D. Van Essen(3), G.A. Orban(1)
(1)Neuro- and Psychophysiology Laboratory, K.U.Leuven, Medical School.
(2)MGH/MIT/HMS Athinoula A. Martino's Center for Biomedical Imaging ctr., Boston, USA.
(3)Washington University St. Louis, School of Medicine, Washington, USA

Neurons in inferior temporal cortex of the monkey brain are selective for two dimensional shape (Gross et al. 1972, Vogels 1999). In humans, fMRI studies show that the lateral occipital region (LO) exhibits a high degree of sensitivity to image scrambling (Mallach et al. 1995, Kourtzi and Kanwisher 2000). In this fMRI study we compared the brain regions activated in macaques and humans by presenting intact and scrambled images of objects. We scanned 21 human subjects and 4 monkeys, using contrast-agent enhanced fMRI (Vanduffel et al. 2001), while they fixated grayscale photographs, line drawings and their scrambled counterparts (Kourtzi and Kanwisher 2000).
Human areas more sensitive to intact compared to scrambled images include LOS, posterior ITG and middle FG in ventral cortex and V3A, VIPS, POIPS, DIPSM/L and DIPSA in dorsal cortex. In monkeys V3, V4, inferotemporal cortex, pIPS, LIP and frontal cortex were shape sensitive.
In both species, the LO complex consists of multiple extrastriate visual areas. While the occipito-temporal part of the LO complex is relatively similar in humans and monkeys (human regions are more cue invariant), the IPS part and frontal component is more extensive in monkeys compared to humans.
Supported by GOA2000/11, FWO, GSKE, MAPAWAMO

Determination by a gain of fonction approach of the roles of ephrins in the development of neuronal connections of the cerebral cortex.

Vanessa Depaepe, Catherine Ledent and Pierre Vanderhaeghen.
I.R.I.B.H.M (U.L.B).Route de Lennik 808, 1070 Brussels. Belgium

The Eph family constitutes the largest family of receptor tyrosine kinases, with at least 14 members in mammals. Their ligands, the ephrins, constitute a family of 8 membrane-anchored polypeptides that mainly act on cell motility and guidance, either through adhesion or repulsion. The ephrin/Eph family has been implicated in an array of important developmental processes, from gastrulation to axon guidance. Our laboratory has recently started the characterization of this family during the development of pattern and neuronal connectivity in the cerebral cortex. Using the cre/lox technology and a combination of forebrain-specific promoters and constructs allowing generation of BAC(bacterial artificial chromosomes) transgenes, we are currently generating transgenic models that display a gain of ephrin function (generation of ectopic gradients of expression), specifically in the developing forebrain. Here we present the making-of of the construct implicated in this project, and the preliminary results obtained in the course of generation of the transgenic founders. The combined anatomical and functional analysis of the mutants obtained will enable us to determine how the function and distribution of cortical ephrins can influence the patterning and connectivity of the forebrain

The effect of levetiracetam in rapid kindling

T. De Smedt, R. Raedt, S. Dedeurwaerdere, K. Vonck, P. Boon
Clinical and Experimental Neurophysiology Laboratory Ghent University Hospital, De Pintelaan 185 B-9000 Ghent, Belgium

Rationale Levetiracetam (LEV) is a currently available antiepileptic drug. Rapid Kindling with Recurrent Hippocampal Seizures (RKRHS) provides a fast and reliable rat kindling model for temporal lobe epilepsy. The aim of the study is to demonstrate in the RKRHS model the anticonvulsive effect of LEV.
Methods Male wistar rats (n= 9, 300g) were implanted with both a bipolar stimulation electrode and a monopolar recording electrode in the right hippocampus. The rats were stimulated according to the RKRHS protocol (10s trains at 20Hz every 30 min, 12 times a day).
Fully kindled rats (n=6) were injected with LEV (54 mg/kg, i.p.). Three control animals were injected with saline (2 ml/kg, i.p.). One hour after injection, all animals received 4 kindling stimulations. Afterdischarge duration (ADD) and behavioural manifestations were measured.
Results A highly significant reduction in ADD was seen after injection of LEV (p<0,01). Average ADD was reduced from 50,58s (SD=19,15s) to 28,75s (SD=21,07s) (p<0,01). Mean behavioural seizure score (Racine) was reduced from 4,60 (SD=0,75) to 2,75 (SD=1,30) (p<0,01), meaning a seizure reduction from tonic-clonic convulsions to myoclonic jerks and jaw movement.
Conclusion LEV displayed strong anticonvulsive properties in the RKRHS model

Area-Specificity and Topography of Thalamocortical Projections is Controlled by Ephrin/Eph Genes.

Audrey Dufour1, Julie Seibt2, Lara Passante1, Vanessa Depaepe1, Franck Polleux2, and Pierre Vanderhaeghen1.
1 IRIBHM, University of Brussels(U.L.B.), 808 Route de Lennik, B-1070 Brussels, Belgium. 2 INSERM U371, 69675 Bron, France.

The neocortex is organized into different areas, each characterized by a specific pattern of connectivity. The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Based on their role in topographic mapping in lower systems, ephrin/Eph genes were previously proposed to control several aspects of TC mapping. We performed an axon tracing study of the thalamocortical (TC) projections in ephrin/Eph compound mutant mice. This approach first allowed us to demonstrate in vivo that ephrin-A5/EphA4 interaction is required for the topographic mapping of TC axons within the somatosensory (S1)area. Analysis of ephrin-A5/EphA4 mutants unexpectedly revealed defects in the area-specificity of TC projections, with axons from the motor nucleus VL projecting aberrantly to the S1 area. A novel organotypic co-culture assay enabled us to show that ephrins control area-specificity of TC projections through the topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.

ADENOSINE RECEPTOR MODULATION OF NMDA CURRENTS IN THE DORSAL HORN OF RAT SPINAL CORD.

E. Guntz1; C. Roussel 1; B.K. Rycroft 2; S. Schiffmann 1; M. Sosnowski1; A.J. Gibb2.
1.Neurophysiology, ULB, Brussels, Belgium
2. Pharmacology, UCL, London, United Kingdom

Dorsal horn adenosine receptors modulate sensory information and adenosine and synthetic adenosine receptor agonists are known to be analgesic in a number of animal models of pain. In these experiments the effects of the adenosine A1 receptor agonist CPA and A2a receptor agonist CGS 21680 were investigated on background and NMDA activated currents during whole-cell patch-clamp recordings from lamina II neurones from transverse slices of 10 days old rat lumbar spinal cord. At a membrane potential of -60 mV the average holding current was 59. 9 pA and 10 M NMDA plus 10 M glycine evoked 77.14 pA of inward current (n = 18 cells). Application of CPA (0.01 - 1.0 M) produced a dose-dependent inhibition of the whole-cell current with an IC50 = 0.06 M. However, CPA (0.1 M) also inhibited the whole cell current by 22 .12 % (n= 6 cells) in the absence of NMDA. Voltage ramp experiments demonstrated that the CPA - sensitive current in both presence and absence of NMDA had a reversal potential close to the Cl-reversal potential suggesting that activation of adenosine A1 receptors in these cells may inhibit a Cl- conductance.
Preliminary results indicate that the A2a receptor activation (CGS 21680) may also reduce the NMDA receptor current through a calcium intra-cellular pathway.

Estrogen synthesis in the spinal cord rapid effects on responsiveness to noxious stimuli

Henry C. Evrard and Jacques Balthazart
Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Lige, 17 Place Delcour, B-4020 Lige, Belgium

Estradiol (E2) exert rapid non-genomic effects paralleled and potentially controlled by rapid changes in E2 synthesis by aromatase. The presence of aromatase in the spinal dorsal horn suggests that E2 produced locally could regulate rapidly nociception. This study assessed the effect of an acute aromatase inhibition on nociception in quail, a useful model for the study of neural aromatase. The latency of foot withdrawal from 54C water was recorded in males before and 1, 5, and 30 min after an intrathecal injection of a non-steroidal aromatase inhibitor (Vorozole) or its vehicle. The specificity of the effect of Vorozole on aromatase and on E2 depletion was assessed by repeating the experiment with a steroidal aromatase inhibitor (ATD) and with a co-injection of E2 with Vorozole, respectively. Vehicle injections resulted in latencies of about 2.5 s. Vorozole and ATD increased the latency 1 and 5 min after injection to about 15 and 20 s, respectively. This increase vanished after 30 min and was not observed when E2 was co-injected with Vorozole. Inhibition of the physiological spinal estrogen synthesis rapidly alters nociception. This suggests that non-genomic E2 effects depend on a rapid control of aromatase. MH50388, ARC99/04-241, FRFC 2.4555.01.

FMRI evidences of kinetic boundaries processing in monkeys

D. Fize, W. Vanduffel, K. Denys, K. Nelissen, G.A. Orban
Laboratorium voor Neuro- en Psychofysiologie, K.U.Leuven Medical School; MGH-NMR ctr., Boston, USA

Kinetic boudaries are powerfull cues to extract shape information from a moving environment. In humans, the processing of kinetic boundaries involve selectively a region (KO) located in between areas hV3 and hMT/V5 (Van Oostende et al., 1997). To locate the KO-homologue in the macaque, we examined the effect of kinetic boundaries as well as motion and luminance patterns in monkeys trained to fixate in a MR scanner. We presented kinetic gratings of varying diameter (3-28 degrees) and position (central, upper, and lower VF). In addition, we studied the effects of the direction of motion that define the kinetic boundaries, as well as the spatial frequencies of the gratings. All the results confirmed the kinetic-specific activation in V4. Although kinetic related activity was also observed in V3 and FST, the MR response changes of these regions to size, spatial frequency and boundary types show clearly that these responses are less specific to the presence of such boundaries. Furthermore, foveal V4 and its dorsal part give significant more activity than ventral V4, when manipulating the position of the stimuli. These results suggest a good match between human and macaque visual cortical regions sensitive for kinetic boundaries (V4/KO). Supported by GOA, GSKE, and NFWO.

A Knock-out mice for the study of the MCH/MCHR1 axis

Antoine ADAMANTIDIS, Bernard LAKAYE, Thierry GRISAR, Agnes FOIDART
University of Liege, Research Center for Cellular and Molecular Neurobiology, 17, Place Delcour, 4020 Liege, Belgium.

Melanin-concentrating hormone (MCH) is a cyclic hypothalamic neuropeptide derived from a larger prohormone precursor (pMCH), which also encodes neuropeptide EI (NEI) and neuropeptide GE (NGE). pmch-deficient mice (pMCH-/-) are lean, hypophagic, and have an increased metabolic rate. Transgenic mice overexpressing pMCH are hyperphagic and develop mild obesity. Consequently, MCH but also NEI and/or NGE has been implicated in the regulation of energy homeostasis. We cloned an orphan metabotropic receptor which was identified as the MCH receptor 1 (MCHR1), one of two recently isolated G protein-coupled receptors known to interact with MCH in mammals.
Therefore, to specifically analyse the MCH/MCHR1 axis, we have generated by blastocyst microinjection a MCHR1 knock-out mice.
We used a positive/negative replacement vector strategy to genetically modify embryonic stem cells (ES cells). Two independent recombinant ES cells lines were microinjected into host blastocysts which were then reimplanted into pseudopregnant foster mothers giving rise to seven founders for the subsequent generation of heterozygotes animals. Finally, Cre-loxP strategy allowed us to remove (by breeding with Cre-deleter mice) the neomycine resistance gene cassette which could interfere with the observed phenotype.

Involvement of GABAA receptor subunit d in the fragile X syndrome

Ilse Gantois1, Jo Vandesompele2, Frank Speleman2, Edwin Reyniers1, Rudi D'Hooge3, Lies-Anne Severijnen4, Rob Willemsen4, Flora Tassone5 and R. Frank Kooy1
1Medical Genetics, Antwerp
2Medical Genetics, Gent
3Neurochemistry and behavior, Antwerp
4Clinical Genetics, Rotterdam
5Biological Chemistry, Davis, California

FMRP, the protein missing in fragile X patients, is an RNA binding protein that shuttles between the nucleus and cytoplasm. In neurons, the protein may transport certain brain mRNAs towards the actively translating ribosomes near the synapses.
However, despite intense research it is still unclear why absence of the fragile X protein leads to the mental retardation, macroorchidism and specific behavior problems observed in fragile X patients. In an attempt to unravel this mechanism, we performed gene expression analysis by means of the differential display method using the fragile X mouse model. In analogy to human patients, the fragile X knockout mouse shows a learning deficit and macroorchidism. Using real-time PCR we confirmed the differential expression of a GABAA receptor subunit d in hippocampus and cortex. Immunohistochemistry data did not observe changes in protein localization for this receptor subunit. Preliminary data on human fragile X brains confirm differential expression of this subunit in the cortex. Very likely the differential expression of the GABAA receptor subunit d in fragile X knockout mouse plays an important role in the behavioral problems, like epilepsy and hyperactivity, observed in the mouse model as well as in fragile X patients.

Cortical Regions Involved in Extracting 3D Shape from Shading

Georgieva S.*, Todd J.**, Peeters R.*** and Orban G.*
*Laboratorium voor Neuro- en Psychofysiologie, KULeuven Medical School, Campus Gasthuisberg, B-3000 Leuven, Belgium,**Department of Psychology, The Ohio State University, OH 43210, ***Afdeling Radiologie, UZ Gasthuisberg, 3000 Leuven, Belgium

The present study was designed to determine which human cortical areas are involved in extracting of 3D shape from shading and highlights, using fMRI (n=16). The 3D visual stimuli depicted roughly spherical objects with surfaces shaded with a reflectance model containing both diffuse and specular components (3D shading). A set of matched control stimuli was created, which appeared perceptually as 2D luminance patterns (2D shading), whose image luminance histogram and amplitude spectra were matched to those of the 3D surfaces. Additional 2D control stimuli included the outlines of the 3D objects filled with a uniform luminance levels (2D uniform) and patterns obtained by scrambling the 3D pattern (2D scrambled). The comparison of 3D shading to all 2D conditions revealed significant activation in lateral occipital complex and in intraparietal sulcus. Lambertian shading produces stronger cortical activation comparing to highlights, mainly in the parietal cortex.
Supported by FWO, GOA and Insight 2+.

EXPRESSION OF CXCR4 CHEMOKINE RECEPTOR mRNA DURING MOUSE BRAIN DEVELOPMENT

C.E. Wang, A.M. Goffinet, F. Tissir
Developmental Genetics Unit, University of Louvain Medical School, B1200 Brussels, Belgium

In addition to its role in the immune system and as coreceptor for HIV, the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) are implicated in hippocampal and cerebellar development. In the present work, the expression of CXCR4 mRNA was studied during mouse brain development, using in situ hybridization, from embryonic day (E)9.5 to maturity. From the earliest stage, CXCR4 mRNA is expressed in germinative zones around the ventricles. At later stages, expression is also detected in some neuronal differentiation fields. At E12.5, there is prominent expression in the external granular layer (EGL) of the cerebellum, the brainstem reticular formation and Cajal-Retzius (CR) cells. At E14.5, strong expression is seen in CR cells, ganglionic eminences and retinal ganglion cells. A strong signal is also found in the trigeminal ganglion and olfacroty receptors. At late embryonic stages and perinatal stages CXCR4 mRNA expression is evident in olfactory mitral, neurons in the cortical marginal zone, the cerebellar EGL and the dentate gyrus. CXCR4 transcript levels are gradually downregulated and, in the mature brain, low expression persists in the olfactory bulb and periepidendymal zones in the cortex and hippocampus.

EXPRESSION OF CXCR4 CHEMOKINE RECEPTOR mRNA DURING MOUSE BRAIN DEVELOPMENT

C.E. Wang, A.M. Goffinet, F. Tissir
Developmental Genetics Unit, University of Louvain Medical School, B1200 Brussels, Belgium

SEXUAL PAVLOVIAN CONDITIONING OF RHYTHMIC CLOACAL SPHINCTER MUSCLE MOVEMENT IN MALE JAPANESE QUAIL

Kevin S. Holloway, Charlotte C. Cornil & Jacques Balthazart
Vassar College, Poughkeepsie, NY 12604 USA & Ctr. Cell. Molec. Neurobiol., Univ. Liege, Belgium

Adult male Japanese quail produce a thick foam from a sexually dimorphic, external swelling of the dorsal cloaca. This foam has been demonstrated to increase fertility that results from copulation, to prolong sperm motility in vitro, and to enhance sperm competition. Foam production is dependent on innervation of the sphincter cloacae muscle. Rhythmic activity of this muscle is responsible for foam production and occurs during copulation and voiding. Social stimulation, the ability to view but not copulate with a quail hen, also elicits rhythmic contractions of the cloacal sphincter muscle. The present experiment was conducted to explore whether exposure to an arbitrary stimulus that predicts visual access to a female conspecific would also elicit rhythmic cloacal sphincter muscle movements. Male subjects were exposed to a terry-cloth covered pillow for 30 s followed by either 2 min visual access to a quail hen (Group Paired) or 2 min visual access to an empty presentation chamber (Group Unpaired). Subjects in Group Paired demonstrated a rapid and orderly acquisition of rhythmic cloacal sphincter muscle movements in response to the arbitrary stimulus. Thus, exposure to an arbitrary stimulus may enhance fertility by increasing muscle movements necessary for foam production.

Modulation of the voltage-gated sodium channel in rat striatal neurons by the phospholipase C pathway

Raphal Hourez, Karima Azdad, Celine Roussel, Emmanuel Guntz, David Gall and Serge Schiffmann
Laboratoire de Neurophysiologie, Universite Libre de Bruxelles, Brussels, Belgium

The link between neurotransmitters involved in the physiopathology of movement disorders and the electroresponsiveness of striatal neurons is only partly clarified. If adenylyl cyclase is known to be involved in the coupling between neurotransmitters and voltage-gated Na+ channels in striatal neurons, other intracellular signalling pathways, such as the phospholipase C (PLC) pathway, have to be further investigated. Indeed, PKC is able to phosphorylate the voltage-gated Na+ channel in other neuronal populations and neurotransmitters such as dopamine, adenosine or acetylcholine are likely to act via PLC in the striatum. The effects of a selective agonist of the IP3 receptor and of a selective activator of PKC are tested on the voltage-gated Na+ current in rat striatal neurons in culture using the voltage-clamp whole-cell technique. The activation of IP3 receptor with adenophostin A increases the amplitude of the voltage-gated Na+ current by 11.5+/-2.9% while the activation of PKC with TPA decreases the amplitude of the voltage-gated Na+ current by 31+/-5.5%. We propose a model where the voltage-gated Na+ channel in rat striatal neurons can be modulated by the PLC pathway in two different ways an inhibiting way mediated via PKC and an activating one via IP3, Ca++ and calcineurin.

STUDIES OF REELIN RECEPTORS BY IN SITU HYBRIDIZATION AND NEW MONOCLONAL ANTIBODIES

Nina IGNATOVA, Yves JOSSIN, Fadel TISSIR, Andre M. GOFFINET
Developmental Genetics Unit, Univ. Louvain Med. School, B1200 Brussels, Belgium

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 neurons via phosphorylation of the adaptor Dab1. It is known that the mRNA of VLDLR is present in the brain and myocardium, whereas the mRNA of ApoER2 is present in a high level in brain, placenta and testis. We used in situ hybridization of P-33-labeled riboprobes to study VLDLR and ApoER2 mRNA expression in the brain and confirmed overlapping expression at most levels as predicted from the phenotypes of mutant mice. We undertook the production of monoclonal antibodies against the extracellular domains of receptors. Mutant VLDLR-/- and ApoER2-/- mice were immunized with recombinant proteins that correspond to part of the extracellular regions of receptors. Hybridomas were produced by splenocyte fusion. Supernatants from hybridomas were tested in western blot against tissue extracts and in immunohistochemistry on brain sections. Thus far, 12 and 6 IgG antibodies were obtained against VLDLR and ApoER2 respectively, some of which are able to bind to receptors and to trigger Dab1 phosphorylation in neuronal cultures.

Differential protein expression profiles across the layers in cat visual cortex as revealed by Laser MicroDissection

S. Jacobs, E. Van der Gucht, L. Arckens
Lab. Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Belgium

The 6 layers of the neocortex of mammals contribute differently to the functional diversity within the cortical circuitry. We compared the protein expression patterns between cortical layers within cat primary visual area 17. Up till now, manual collection of cortical gray matter seriously hampered such a detailed laminar analysis, since the neocortex is only 2 mm thick. Application of Laser MicroDissection (LMD) allowed the separate procurement of tissue samples from the granular (IV), infra- (V-VI) and supragranular (II-III) layers of cat area 17. To this end a correct demarcation of the six cortical layers was achieved by a histochemical methylene blue staining, revealing specific cell characteristics like size and morphology. The effects of tissue preparation and staining have been investigated and were rendered compatible with subsequent protein analysis. The first 1D-electrophoresis (PAGE) experiments demonstrated differential protein expression patterns between supra- and infragranular cortical layers of area 17. Future combination of LMD, 1D-electrophoresis, fluorescent 2D-differential gel electrophoresis and mass spectrometry should identify these layer-enriched expressed proteins in mammalian neocortex.

An in vitro model to study the radial migration in the cerebral cortex

Yves JOSSIN, Nina IGNATOVA, Andre-M. GOFFINET
University Louvain Med. School, Developmental Genetics Unit, Avenue E. Mounier, 73 Box 82, B1200 Brussels, Belgium

During cortical development, the majority of neurons generated in the telencephalon migrate to the cortical plate (CP) radially, whereas others originate in ganglionic eminences and migrate tangentially. CP development is abnormal in mice defective in Reelin. Reelin is secreted by Cajal-Retzius cells and binds to VLDLR and ApoER2 on the surface of CP cells, thereby inducing phosphorylation of the Dab1 adapter. The identification of Dab1 kinase(s) is hampered by genetic redundancy. Using an in vitro system we show that inhibition of Src family kinases generates a reeler-like malformation. Reelin is cleaved in vivo and we show that the region corresponding to the central processing product is necessary and sufficient for receptor binding in vitro and for Dab1 phosphorylation in primary neuronal cultures. This central Reelin composed of repeats 3 to 6 is able to rescue the reeler phenotype in the slice culture assay. Interestingly, Dab1 phosphorylation is also triggered by monoclonal antibodies against the extracellular domains of receptors, but these antibodies cannot rescue the reeler phenotype in slices. These observations show that the function of Reelin is critically dependent on the central repeat region, and presumably other coreceptor(s) that remain to be identified.

Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways

Lallemend F1, Lefebvre PP1,2, Hans G1,3,Rigo JM1, Moonen G1,3 and Malgrange B1
1CNCM, University of Lige, 17 Place Delcour, B-4020 Lige, Belgium
Departments of 2Otorhinolaryngology and 3Neurology, University of Lige, C.H.U. (B35) Sart-Tilman, B-4000 Lige, Belgium

We have investigated the ability of substance P (SP) to protect 3 days-old (P3) rat spiral ganglion neurons (SGNs) from trophic factor deprivation (TFD)-induced cell death. The presence of SP high affinity neurokinin-1 receptor (NK1) transcripts was detected in the spiral ganglion and the NK1 protein localized to SGNs both ex vivo and in vitro. Treatment with SP increased cytoplasmic Ca2+ in SGNs, further arguing for the presence of functional NK1 on these neurons. Both SP and the agonist [Sar9,Met(O2)11]-SP significantly decreased SGN cell death induced by TFD. This survival promoting effect of SP was blocked by the NK1 antagonist, WIN51708. Both pan-caspase inhibitor BOC-D-FMK and SP treatments markedly reduced activation of caspases and DNA fragmentation in TFD neurons. The neuroprotective action of SP was antagonised by specific inhibitors of second messengers, including BAPTA-AM, bisindolylmaleimide I and U0126. In contrast, nifedipine and LY294002 had no effect on SP trophic support. Moreover, activation of endogenous PKC by PMA also reduced the loss of trophic factor deprived-SGNs. Thus, NK1 expressed by SGNs transmit a survival-promoting regulatory signal during TFD-induced SGN cell death via pathways involving PKC activation, Ca2+ signaling and MAPK/ERK activation.

Short term plasticity of complex spike waveform in Purkinje cell.

Servais L (1,2),Bearzatto Bertrand (2), Dan Bernard (1) Schiffmann SN (2), Cheron G(1)
(1) Laboratory of electrophysiology, Universit Mons-Hainaut, Mons, Belgium
(2) Laboratory of neurophysiology, Erasmus hospital, University of Brussels, Belgium

Cerebellar Purkinje cell receives two different excitatory inputs from parallel and climbing fibers, causing simple and complex spike firing, respectively. The influence of complex spike firing on simple spike firing has been extensively studied. However, it is unclear whether and how simple spike firing may influence complex spike waveform and firing rate in physiological conditions. We studied complex spike firing during active and silent simple spike firing in alert wild type and calretinin deficient non-stimulated mice. We found that complex spikes undergo a paired pulse depression similar to that described in vitro. Complex spike firing rate was not influenced by simple spike firing, but its waveform was altered following high frequency simple spike firing or if complex spike closely follows a simple spike. This alteration appeared as a specific decrement of the second depolarizing component of the complex spike. As this peak is related to Ca++ entry during the complex spike, this form of short term plasticity may have important functional consequences on various Ca++-regulated metabolic and/or physiological pathways in Purkinje cells such as the rebound potentiation of inhibitory synaptic inputs or the long term depression at the parallel fiber-Purkinje cell synapse.

Lysophosphatidic acid regulation of cerebellar radial glia phenotype

Leprince, P.; Chanas, G.; Wislet, S.; Deroanne, C.; Rogister, B.
Center for Molecular and Cellular Neurobiology, University of Lige, 17 place Delcour, 4020 Lige, Belgium

Cerebellar glial cells from mouse embryo express in culture both astroglial markers (GFAP, Glast) and radial glia markers (RC2, Nestin). When grown in serum, most cells have an epitheliod shape and express largely RC2 and Nestin together with Glast and less frequently with GFAP. In serum-free medium, most cells assume stellate shapes and RC2 and Nestin stainings disappear totaly, while GFAP staining is maintained. As similar changes of phenotype occur in cortical astrocytes treated in vitro with lysophosphatidic acid (LPA), a component of serum, we have characterized LPA signaling mechanisms and effects in cerebellar glial cells.
LPA receptors were detected both in postnatal day 3 (P3) cerebellum (LPAR2, LPAR3) and in cerebellar glial cells cultures (LPAR1, LPAR2, LPAR3). LPA added in serum-free cultures was found to mimic the effect of serum on both the morphology of glial cells and their expression of the RC2 antigen. Half-maximal effect was observed with 2.5 M LPA and inhibition occured in the presence of the LPA receptor NASPA.
Thus LPA signaling which is known to control neural precursors behavior during cortical neuronogenesis is also able to modulate the phenotype of radial glial cells in the developping cerebellum.

Connexin channels, connexin mimetic peptides and ATP release in brain endothelial cells

Katleen Braet and Luc Leybaert
Dept. of Physiology, Ghent University, Ghent, Belgium

ATP is an important paracrine messenger of calcium signal communication but the mechanism of ATP release is still a matter of debate. We investigated the involvement of connexin hemichannels in ATP release in brain endothelial cells by making use of connexin mimetic peptides (CMP). Photoactivation of InsP3 or application of zero extracellular calcium (0Ca) triggered significant ATP release that was inhibited by intracellular BAPTA and is thus calcium dependent. ATP release triggered with both stimuli was blocked by a-glycyrrhetinic acid (a-GA), the CMPs gap 26 & 27, and the trivalent ions Gd and La, while 0Ca-triggered ATP release was in addition blocked by fenamates and NPPB. 0Ca application also triggered dye uptake into the cells which showed similar pharmacological sensitivity as 0Ca-triggered ATP release. Gap 27 selectively blocked 0Ca-triggered ATP release in connexin-43 transfected HeLa cells and had no effect on wild-type or connexin-32 transfected cells. CMP did not affect gap junctional coupling between cells. Our results suggest that InsP3- and 0Ca-triggered ATP release occurs through a similar mechanism, which is connexin-dependent, permeable in both directions, potently blocked by CMPs, and consistent with the opening of connexin hemichannels.

Resonant synchronization in networks of inhibitory neurons

Reinoud Maex and Erik De Schutter
Laboratorium voor Theoretische Neurobiologie, Born-Bunge Stichting, Universiteit Antwerpen UIA, Universiteitsplein 1, B-2610 Antwerpen

Brain rhythms arise through the synchronization of neurons and their entrainment in regular firing patterns. In this process, networks of reciprocally connected inhibitory neurons are often involved. We describe a new phenomenon of resonant synchronization in computer-simulated networks of inhibitory neurons. At the resonant level of network excitation, neurons fire synchronously and rhythmically with period about four times the mean delay over the inhibitory connections. By varying the delay, a network can be tuned to frequencies from 40 to >200 Hz. The amplitude and decay time constant of the inhibitory synaptic response have relatively minor effects on the emerging frequency band. This resonance phenomenon is a candidate mechanism for all oscillations that depend on intact inhibitory synaptic transmission.

Estrogens are able to modulate facial inflammatory pain a study in aromatase knock-out mice using the formalin model

Multon Sylvie, Estelle Rikir, Sandrine Halleux, Jeanine Mosen, Jean Schoenen
Neuroanatomy laboratory. Research Center for Cellular and Molecular Neurobiology. University of Lige. Belgium.

The mechanisms responsible for the higher prevalence of certain head and facial pains in females are not known. We studied the role of gender and estrogens on pain using the orofacial formalin test in mice. We measured the frequency of rubbing of the formalin-injected lip in male and female estrogen-deficient aromatase knock-out mice (ArKO) with or without pre-treatment with -estradiol and in their wild-type littermates (WT).
There was no difference in nociceptive behaviour between male and female WT mice neither in the first acute phase of pain nor in the second tonic phase. By contrast, lip rubbing was significantly more pronounced in ArKO than in WT females during the second pain phase. ArKO males did not differ significantly from their WT littermates regarding phases 1 and 2, but they displayed a third phase of pain 36 min. post-injection. In ArKO mice treated with estradiol the differences in phase 2 for females and phase 3 for males were not detectable anymore.
All together, these results indicate that estrogens have an antinociceptive effect in facial pain and that the different behavioural response in ArKO mice is not due to developmental changes. The differences between male and female ArKO mice suggest that underlying control mechanisms of pain differ between genders.

Processing of Kinetic boundaries in Macaque V4 A single cell study

Santosh M.G, Raiguel S, Vogels R and Orban G.A.
Laboratorium voor Neuro-en-Psychofysiologie, KULEUVEN Medical School, Herestraat, Leuven.

While the kinetic occipital (KO) region is involved in processing static kinetic contours in humans (Orban et al 1995; Van Oostende et al. 1997), its homologue has not yet been identified in lower primates. However, 2-DG and fMRI studies (Nellisson K et al 2000; Fize et al 2001) have suggested that dorsal V4 may be the macaque homologue of KO. We recorded single cells (n=257) in dorsal area V4 of an awake behaving monkey (Macaca mulatta). We used static square wave gratings defined by luminance contrast (lum), by random pixels (kin) moving in opposite directions while the boundaries remained stationary. The kin stimuli were of two categories, with pixels moving parallel or orthogonal to the boundary. Controls included transparent motion and uniform motion stimuli. The shape stimuli were defined by either luminance contrast or kinetic boundaries. 113 cells were selective for orientation to lum gratings, 29 for both kin gratings. The majority of the kin selective cells showed nearly identical tuning for both kin gratings. Out of the 86 cells selective for lum shapes, 34 were also selective for kin shape stimuli. Our study shows that single cells in dorsal V4 extract patterns defined by relative motion and results are comparable with the monkey fMRI data.
Supported by GOA & GSKE

In vitro models a shortcut to dissect molecular mechanisms underlying cell migration in brain

Passante l,Vanderhaeghen p
IRIBHM, University of Brussels (U.L.B.), 808 Route de Lennik, B-1070 Brussels, Belgium.

Neuronal migration is one of the major events occuring during brain development. The movement of the different neuronal subtypes generated in the telencephalon (the rostral-most part of the, embryonic brain) produces three main migratory streams radial migration of glutamatergic neurons in the cortex, radial migration of neurons in the striatum and tangential migration of GABAergic inter-neurons from the basal telencephalon to the cortex.
To dissect the molecular mechanisms underlying neuronal migration in the telencephalon we have developed an in vitro approach based on electroporation and organotypic co-culture of telencephalic brain slices. These techniques allowed us to directly visualize different migration events such as radial migration of projecting neurons in the cortex and striatum and tangential migration of inter-neurons from the basal telencephalon to the cortex. The versatility of our in vitro approach makes it a useful tool to test directly the role of candidate genes potentially involved in neuronal migration.

Vagus Nerve stimulation reduces body weight in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

S. Dedeurwaerdere, K. Vonck, R. Raedt, P. Claeys, P. Boon
Laboratory for Clinical and Experimental Neurophysiology Ghent University Hospital, De Pintelaan 185 B-9000 Ghent, Belgium

Vagus nerve stimulation is an adjunctive therapy for medically intractable partial seizures with or without secondary generalisation. The purpose of this study was to determine the effects of vagal nerve stimulation on weight regulation in a rat model for absence epilepsy.
GAERS (n=10) were implanted with 5 epidural electrodes and a stimulation electrode around the left vagal nerve. During four weeks after the implantation the rats were weighted every week for three consecutive days. From the second week on, the animals were continuously stimulated for two (n=2) or three (n=3) weeks with the following parameters 60 sec trains of 0.5 ms monophasic pulses at 30 Hz with a train interval of 12 sec. Control rats (n=5) received no stimulation.
During three weeks of continuous stimulation, stimulated rats showed a significant loss of body weight (p<0.001). Average weight at the end of the stimulation period was 242 37g. In the control group average weight was 294 21 g.
Chronic VNS has a significant influence on weight regulation in a rat model for absence epilepsy. Further studies are needed to determine the factors underlying the reduction of body weight in response to VNS.

ALTERED NEURONAL EXCITABILITY IN CEREBELLAR GRANULE CELLS OF MICE LACKING CALRETININ

Cline Roussel 1, David Gall 1, Isabella Susa 2,Egidio D 'Angelo 4, Paola Rossi 4, Bertrand Bearzatto 1, Marie Christine Galas 1, David Blum 1, Stphane Schurmans 3 and Serge N. Schiffmann 1
1 Laboratoire de Neurophysiologie, 2 Optique Nonlinaire Thorique, 3 Institut de Biologie et de Mdecine Molculaire, Universit Libre de Bruxelles, Brussels, 4 Dept of Cellular/Molecular Physiology and Pharmacology, University of Pavia, Pavia, Italy

Calcium binding proteins, such as calretinin, are abundantly expressed in the central nervous system but their physiological functions remains poorly understood. Calretinin is expressed in cerebellar granule cells and calretinin deficient mice exhibit dramatic alterations in motor coordination and in Purkinje cell firing recorded in vivo through unknown mechanisms. Granule cells provide the major excitatory input to Purkinje cells through parallel fibers. In this study, we have used patch clamp techniques in acute slice preparation to investigate the effect of a null mutation of the calretinin gene on the intrinsic electroresponsiveness of cerebellar granule cells. Calretinin deficient granule cells exhibit faster action potentials and generate repetitive spike discharge showing an enhanced frequency increase with injected currents. These alterations disappear when 0.15 mM of the exogenous fast calcium buffer BAPTA is infused in the cytosol to restore the calcium buffering capacity. A mathematical model is proposed demonstrating that the observed alterations of granule cell excitability can be explained by a decreased cytosolic calcium buffering capacity due to the absence of calretinin. This result suggests that calcium binding proteins modulate intrinsic neuronal excitability.

Neurogenin2 specifies the connectivity of thalamic neurons by controlling axon responsiveness to intermediate target cues

Julie SEIBT, Carol SCHUURMANS, Pierre VANDERHAEGHEN, Colette DEHAY, Franois GUILLEMOT and Franck POLLEUX
INSERM U371 18 avenue Doyen Lpine 69675 -Bron - FRANCE

Many lines of evidence indicate that in mammals some traits of neuronal phenotype such as cell-body position and neurotransmitter expression are specified through complex interactions between extrinsic and intrinsic genetic determinants. However, the molecular mechanisms specifying neuronal connectivity are less well understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 specifies cell-autonomously the projection of thalamic neurons to frontal cortical areas. Unexpectedly, Neurogenin2 exerts its role by specifying the responsiveness of thalamic axons to cues encountered at the level of an intermediate target, the ventral telencephalon, unraveling the mechanism whereby thalamic axons project to specific cortical domains. Our results demonstrate that, in parallel to their well-documented proneural function, bHLH transcription factors also specify neuronal connectivity in the mammalian brain

In vitro and in vivo characterization of the effects of the new SK channel blocker methyl-laudanosine

Vincent Seutin*, Fabienne Graulich-Lorge*, Jacqueline Scuvee-Moreau*, Laurent Massotte*, Elizabeth Thomas* and Jean-Francois Liegeois+
*Laboratory of Pharmacology and Research Center for Cellular and Molecular Neurobiology, University of Liege
+Laboratory of Medicinal Chemistry and Natural and Synthetic Drugs Research Center, University of Liege

SK channels are calcium-activated potassium channels that underlie some components of the afterhyperpolarization (AHP) which follows one or a train of action potentials in many neurons. They are subdivided in SK1, SK2 and SK3 channels. The latter are heavily expressed in brainstem monoaminergic cell groups. SK channel modulation in these neurons may be of therapeutic interest.
We have previously reported that methyl-laudanosine blocks SK channels with some specificity (Scuvee-Moreau et al., 2002).
In transfected cell lines (kindly provided by M. Stocker, University College London), we found that the compound is equipotent on SK1, SK2 and SK3 channels. In slices, the compound was found to block the apamin-sensitive component of the AHP with similar potency in dorsal raphe serotonergic and locus coeruleus noradrenergic (NA) neurons as compared to what we had found in dopaminergic (DA) neurons.
When applied iontophoretically in the brain of anaesthetized animals, methyl-laudanosine induced a very robust burst firing in DA neurons, but had little effect on the firing pattern of NA neurons. This is consistent with the hypothesis that the apamin-sensitive portion of the AHP, which is larger in DA neurons, has more influence on the firing of these cells than in the case of NA neurons.

Different roles for dendritic and somatic inhibition in a large neuron model

Sergio Solinas, Reinoud Maex, Erik De Schutter
Laboratory of Theoretical Neurobiology - Born-Bunge Foundation, UIA Antwerpen

We examined how dendritic and somatic inhibitory inputs affect the neuronal firing patterns by simulating a realistic computer model of a cerebellar Purkinje cell driven by random synaptic inputs. Spike triggered averages of inhibitory post-synaptic potentials were measured at the soma. Comparison of these IPSPs for different locations of activated synapses showed a significant attenuation of distal input. As a consequence dendritic inhibitory inputs had no direct effect on the somatic activity. However, inhibitory synapses onto the main dendrite and the soma acted as an interrupt signal, shutting down the generation of action potentials. In contrast, dendritic inhibition indirectly affected the model Purkinje cell firing pattern by preventing dendritic over-excitation and the consequent activation of Ca channels which would otherwise lead to an unrealistic rhythmic bursting pattern. Thus, dendritic inhibition was necessary to prevent the occurrence of calcium bursts and to generate the irregular firing pattern characteristic of Purkinje cells in vivo. Somatic inhibition could enhance the Purkinje cell firing pattern variability but failed to achieve control of calcium bursts.

Variability in the response patterns of Purkinje cells to tactile stimulation

S.-L. Shin; E. De Schutter
Theoretical Neurobiology, University of Antwerp-UIA, Antwerp, Belgium

Purkinje cells (PCs) are the only output neurons in the cerebellar cortex. Theoretical considerations suggest that background membrane potential in the dendrites and EPSP amplitude in the soma are variable and are negatively correlated (De Schutter, J Neurophysiol 1998). In anesthetized rats, we examined the stability of responses in 12 PCs to repetitive tactile stimulation (3 blocks of 250 trials each). Each block was used to construct a peristimulus time histogram (PSTH).
The 3 PSTHs obtained from a given PC often exhibited different patterns. Moreover, the mean firing rate of each PC varied during recordings. To test whether the variability of firing rate was due to stimulation, we recorded the spontaneous activity of PCs (3 epochs of 5 min each) the mean spontaneous firing rate of PCs also varied with time. Trial by trial analysis showed an inverse correlation between background firing rate and response amplitude (difference between firing rate for 1 s before stimulation and for 100 ms after the stimulation). This study shows that variability in background firing rate can account for that of the response in PCs.
Supported by FWO, GSKE and EC

THE FUNCTIONAL STATE OF SPECIFIC NEURONAL POPULATIONS IN THE SONG SYSTEM OF LIVING CANARIES UPON LISTENING TO CONSPECIFIC SONGS AS ASSESSED BY DYNAMIC MANGANESE ENHANCED MRI

I. Tindemans (1), M. Verhoye (1), J. Balthazart (2), A. Van der Linden (1)
(1) Bio-Imaging Lab, University of Antwerp, B-2020 Antwerp, Belgium
(2) Center for Cellular and Molecular Neurobiology, University of Lige, B-4020 Lige, Belgium

HVC, a key center for song control in oscines responds in a selective manner to conspecific songs as indicated by electrophysiology. However no Immediate Early Genes induction can be detected in this nucleus after song stimulation. HVC contains neurons projecting to RA (motor pathway) or to area X (anterior forebrain pathway) and both types respond to auditory stimuli. We analyzed these responses separately in the two types of HVC projection neurons of canaries by a new in vivo approach using manganese (Mn2+) as a calcium analogue that can be transported anterogradely and used as a paramagnetic contrast agent for Magnetic Resonance Imaging (MRI). Mn2+ was stereotaxically injected into HVC and taken up by HVC neurons. The time course of the anterograde axonal transport of Mn2+ from HVC to RA and area X was then followed by MRI during 8 hours. Data obtained within the same birds exposed or not to conspecific songs revealed that song stimulation differentially affects the activity of the two types of HVC projection neurons. Dynamic Manganese Enhanced-MRI thus allows assessing the functional state of specific neuronal populations in the song system of living canaries in a manner reminiscent of fMRI, 2 deoxyglucose autoradiography or electrophysiology but with specific advantages.

GENERATION OF MONOCLONAL ANTIBODIES AGAINST THE CENTRAL PART OF REELIN

Lanrun GUI, Nina IGNATOVA, Yves JOSSIN, Fadel TISSIR, Jacques VAN SNICK, Andre M. GOFFINET
Developmental Genetics Unit, Univ. Louvain Med. School, B1200 Brussels, Belgium

Reelin is an extracellular matrix protein that plays a key role during brain development. The structure is the following. A signal peptide is followed by a region with similarity to F-spondin, a unique region, and then by a succession of eight repeats of 300 to 350 residues. Reelin is processed in vivo at two sites between repeats 2 and 3, and between repeats 6 and 7. The central fragment that results from cleavage (repeats 3 to 6) is necessary and sufficient for signal activation. As antibodies were never developed against the central, active part of Reelin, we produced a His-tagged Reelin composed of subrepeats 4B and 5A that was used to immunize homozygous reeler mice deficient in Reelin. Hybridomas were produced by spleen fusion, screened by ELISA against the antigen, and validated by western blot of antigen, recombinant Reelin proteins composed of repeats 3 to 6 and 3 to 8, and mouse brain extracts. Six clones were obtained, two of which were IgG2b and selected for further studies. In immunohistochemistry, both antibodies stain Cajal-Retzius cells, further confirming their specificityfor Reelin. Further studies using these antibodies will focus on detection of the predicted processing fragments of reelin in tissues and their putative interference with reelin signaling.

TNF-alpha inhibits purinergic signaling in blood-brain barrier endothelial cells

Wouter Vandamme and Luc Leybaert
Dept. of Physiology, Ghent University, Ghent, Belgium

The breaching of the blood-brain barrier (BBB) is an essential aspect in the pathogenesis of neuroinflammatory diseases. TNF-alpha together with BBB endothelial calcium changes play a key role in the opening of the barrier, thereby allowing immune cells to enter the brain. We investigated the hypothesis that TNF-alpha has an influence on the cell-to-cell communication of calcium signals between BBB cells. Intercellular calcium waves triggered in GP8 or RBE4 brain endothelial cell lines were significantly reduced in size after TNF-alpha exposure (1000 U/ml, 2 or 24 h). This inhibitory effect was mediated in part by inhibition of gap junctional coupling between the cells but also in part by a blocking action of the cytokine on cellular ATP release triggered by either photoliberation of InsP3 or by zero extracellular calcium conditions (0Ca). TNF-alpha also blocked 0Ca-triggered uptake of fluorescent markers in the cells. We conclude that TNF-alpha depresses calcium signal communication by reducing gap junctional coupling and by inhibiting triggered cellular ATP release. This last effect might be expected to profoundly influence the communication between all the partners at the BBB including astrocytes, neurons and blood cells.

Neurochemical subdivision and 3D-reconstruction of cat and monkey ventral lateral geniculate nucleus

E. Van der Gucht and L. Arckens
Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Belgium.

There is an astonishing lack of information concerning the neuronal organization, including neuronal morphology and neurochemical phenotype, of the ventral LGN (vLGN) of cat and monkey, in contrast to the overwhelming information about the anatomical organization of the mammalian dorsal LGN. This immunocytochemical study revealed distinguished neurochemical reactivity for eight cellular markers, i.e. GABA, Calbindin, Calretinin, Parvalbumin, Neuropeptide Y, Somatostatin, Enkephaline and Neurofilament protein, within the vLGN of cat and monkey. A characteristic Neurofilament protein expression profile was apparent allowing the delineation of the vLGN into a two subzones in both species. Calretinin, Neuropeptide Y and Enkephaline are also considered efficient anatomical markers allowing the parcellation of the monkey vLGN in two subdivisions. In cat, Enkephaline immunoreactivity clearly demarcated the medial part of the vLGN. Three-dimensional reconstructions of cat and monkey vLGN based on such characteristic neurochemical marker expression profiles elucidate the true anatomical constellation of cat and monkey vLGN. These findings suggest a well-defined part of the vLGN of cat and monkey as homologue of the rodent intergeniculate leaflet.

The relation between glia and oxidative stress and their significance in the disease pathogenesis in Creutzfeldt-Jakob and Alzheimers Disease.

Van Everbroeck B, Dobbeleir I, De Waele M, De Leenheir E, Lbke U, Martin JJ, Cras P
Dept of Neurobiology & Neuropathology, Born Bunge Foundation, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium

Alzheimers disease (AD) and CreutzfeldtJakob disease (CJD) are characterized by the deposition of amyloid beta (Ab), neurofibrillary tangles and the prion protein (PrP), respectively. The relation of glia with oxidative stress and their influence on the disease pathogenesis was studied. Three cortical areas, the hippocampus, and the cerebellum of CJD, AD and control patients were examined. Astroglia were found in low numbers in the cortex whereas microglia and neurons suffering from oxidative stress were only found around Ab plaques in controls. These finding were duplicated in AD although the numbers in cortex and hippocampus were significantly higher. The morphology of the lesions was very different in CJD. Astroglia were less present whereas enormous amounts of microglia were observed although, maximal numbers were seen in patients with the fasted progression. The same result was found for the percentage of neurons suffering from oxidative stress.
Glia in the brain are though to be the main producers of oxidative stress in AD and cause secondary oxidative stress resulting in neuronal death. Our results not only confirm that this hypothesis is also valid in CJD but that the amount of glia and therefore oxidative stress have an influence on the disease progression.

Quantification of cerebral perfusion parameters in different mouse models upon neurodegeneration using in-vivo bolus tracking Magnetic Resonance Imaging (MRI)

Vanhoutte G", Storkebaum E', Carmeliet P', Van der Linden A"
"Bio-Imaging Lab Groenenborgerlaan 171, 2020 Antwerpen
'Centre for Transgene Technology & Gene Therapy, Herestraat 49, 3000 Leuven

Vegfd/d mice, with a deletion of the hypoxic response element of the Vascular Endothelial Growth Factor (VEGF) have late-onset progressive motor neuron degeneration, reminiscent of Amyotrophic Lateral Sclerosis (ALS).VEGF is a research topic in the field of vascularization and the impact of VEGF on the progression of ALS might be reflected in defect cerebral perfusion. 4 different genotypes(n=5, age=3m),including both the VEGF mutation and Super Oxidase Dismutase (SOD) mutation, which results in the inherited form of ALS (VEGF/SOD Wt/Wt, Ho/Wt, Wt/He, Ho/He), were studied with bolus tracking MRI. Quantitative cerebral blood flow (CBF) and cerebral blood volume (CBV) was determined by following the passage of an intravenous bolus injection of Gd-DTPA (0.2mmol/kg). Mice were anaesthetised using 0.4% isoflurane and monitored on end-tidal CO2 and breaths per minute using a micro-capnometer (Linton Instruments). The results show reduced CBF only in double transgenic mice (Ho/He), indicating a possible molecular interaction between SOD and VEGF mutations, while CBV remains unchanged. This approach of simultaneously measuring CBV and CBF makes it possible to determine genetically induced abnormalities at the level of the correlation between CBV and CBF, related to neurodegeneration.

Functional brain Imaging of Songbirds when Listening to Songs a Functional Magnetic Resonance Imaging (fMRI) study

Vincent Van Meir, Tiny Boumans, Geert De Groof, Marleen Verhoye, Johan van Audekerke and Annemie Van der Linden
Bio-Imaging Lab, University of Antwerp, Groenenborgerlaan 171, Antwerp, Belgium

Songbirds are famous for their complex vocalisations. Like in humans, this is a very important social behavior, which requires a learning process during childhood in order to produce socially relevant songs in adulthood. The brain networks involved in both hearing and singing songs are very well documented in songbirds, which make them an excellent model to study the functionality of brain regions during cognitive tasks and learning processes. fMRI data were obtained in anesthetized male European starlings upon listening consecutively to white noise, unfamiliar conspecific birdsong, and their own song. Field L, the birds analogue of the mammalian auditory cortex, was activated upon all three stimuli, while NCM, the region considered as substrate for auditory recognition memory, was exclusively activated upon hearing birdsong. The exposure to its own song elicited a response in the auditory regions as well in the motor regions for song production. The activity of the song control system was studied previously with the aid of invasive electrophysiological and molecular techniques. The non invasive and in-vivo nature of fMRI will allow us to visualize activation patterns during different stages of life or after artificially changing the song behavior of the bird during adulthood.

Results of long-term vagus nerve stimulation in patients with refractory epilepsy treated at the Reference Center for Refractory Epilepsy at Ghent University Hospital in Belgium

Kristl Vonck, Liesbeth De Groote, Erik Achten, Evert Thiery, Guy Vingerhoets, Luc Defreyne, Edward Baert, Geert Michielsen, Frank De Waele, Jacques Caemaert, Jacques De Reuck, Paul Boon
Reference Center for Refractory Epilepsy, Department of Neurology, Neuroradiology, Neuropsychology and Neurosurgery, Ghent University Hospital, Belgium

Objective Vagus nerve stimulation (VNS) is a treatment option for patients with medically or surgically refractory epilepsy. We evaluated the long-term efficacy and safety in 70 patients treated with VNS at Ghent University Hospital. Methods Since March 1995, 74 patients have been implanted with VNS. We prospectively assessed seizure frequency, seizure type, prescribed anti-epileptic drugs (AEDs) and side effects. Mean monthly complex partial seizure (CPS) frequency during the year before and the follow-up period after implantation were compared in patients with at least 3 months of follow-up (n=70). Results Patients (33F,37M) had a mean age of 30 years and a mean duration of refractory epilepsy of 20 years. Mean post-implantation follow-up was 3 years (range 0.25-8 years). Mean CPS frequency changed from 37 CPS/month (range 2-200/month) to 17 CPS/month (range 0-100) (p=0.004). No peri-operative morbidity was reported. Hoarseness during the stimulation on-time was the most frequent side effect. The mean number of AEDs remained unchanged. Discussion VNS has been successful in reducing seizure frequency in the majority of patients in this series. It is a useful alternative treatment with few side-effects that remains efficacious with longer follow-up

Acute and chronic cerebral blood flow changes induced by vagus nerve stimulation in patients with refractory epilepsy

Kristl Vonck (1), Tommy Bosman (1), Koen Van Laere (2), Rudi Dierckx (3), Stefanie Dedeurwaerdere (1), Jacques De Reuck (1), Paul Boon (1)
(1) Reference Center for Refractory Epilepsy and Department of Neurology, (3) Department of Nuclear Medicine; Ghent University Hospital; (2) Department of Nuclear Medicine, Leuven University Hospital, Belgium

Introduction Functional imaging is a useful non-invasive tool to investigate functional pathways involved in the mechanism of action of neurostimulation. We performed a SPECT study in patients treated with vagus nerve stimulation (VNS) for epilepsy. Patients and Methods 27 patients treated with VNS at Ghent University Hospital underwent an activation 99mTc-ECD SPECT at the time of the first stimulation (acute SPECT) and after 6 months of treatment (chronic SPECT, 12 patients). Cerebral blood flow (CBF) changes were correlated with prospectively assessed long-term clinical efficacy data. Results In the acute and chronic acitivation paradigms significant CBF changes were found in the thalamus, hippocampus and parahippocampal gyrus. An acute increase in left medial temporal inferior CBF was positively correlated with an increasing efficacy. Correlation between the chronic baseline condition and clinical efficacy revealed a positive correlation between a decreasing left thalamic CBF and an increasing clinical efficacy. Conclusion Neurostimulation induces acute and chronic CBF changes that can be measured by SPECT. The thalamus is estimated to play a key role in the mechanism of action of VNS. Increased left temporal lobe CBF is predictive for positive clinical outcome.

Long-term Amygdalohippocampal deep brain stimulation for refractory temporal lobe epilepsy

Paul Boon (1), Kristl Vonck (1), Pieter Claeys (1), Stefanie Dedeurwaerdere (1), Erik Achten (2), Jacques De Reuck (1), Jacques Caemaert (3)
Reference Center for Refractory Epilepsy, Department of Neurology(1), Neuroradiology(2), Neurosurgery(3), Ghent University Hospital, Belgium

Rationale Post-operative outcome is less favourable in refractory epilepsy patients with normal MRI undergoing epilepsy surgery. We evaluated the efficacy and safety of amygdalohippocampal deep brain stimulation (AHDBS) as a less invasive treatment alternative in these patients.
Patients and Methods Seven patients (6M,1F) with normal MRI underwent AHDBS. and were followed-up at the epilepsy clinic every 2-4 weeks.
Results The mean follow-up in the first 6 patients is 13 months (range 4.5-20). 1 patient is seizure free for 1.5 years and 2 AEDs tapered. 3/6 have a > 50% reduction in seizure frequency; 1/3 is seizure free during the day; 2/3 had tapering of 1 AED. 2/6 patients have a seizure frequency reduction of 25%and tapering of 2 AEDs. In one patient no change in seizure frequency occurred. None of the patients report side effects related to stimulation.
Conlcusion In this open pilot trial AHDBS significantly reduces seizure frequency during long-term follow-up without side effects. For patients who are less suitable candidates for epilepsy surgery, AHDBS may become a valuable alternative. Controlled studies in larger patient series are mandatory to identify the potential treatment population and optimal stimulation paradigms.

Functional magnetic resonance imaging during refractory simple partial seizures

Kristl Vonck (1), Erik Achten (2), Karel Deblaere (2), An Thieleman (2), Pieter Vandemaele (2), Jacques De Reuck (1), Paul Boon (1)
Reference Center for Refractory Epilepsy, Department of Neurology (1) and Neuroradiology (2), Ghent University Hospital, Belgium

Objective Functional magnetic resonance imaging (fMRI) allows non-invasive evaluation of cerebral blood flow (CBF) changes. In the context of epilepsy it may help to localize seizure onset prior to epilepsy surgery. We evaluated the feasibility and gain of fMRI for seizure onset localization during simple partial seizures (SPS).
Patients and methods In two patients (1M,1F) fMRI was performed during SPS. One patient with a right frontal dysplasia had motor SPS of the left hand; the second had autonomic SPS after a previous temporal lobectomy for complex partial seizures. fMRI acquisition was performed on a 1.5 Tesla machine in the presence of a neurologist. Results In the first patient a right interparietal activation was found. This patient is currently being investigated for subpial transsection of this region. In the second patient, a right temporal activation was found corresponding to residual temporal lobe tissue. This patient is rescheduled for more extensive surgery.
Conclusion fMRI acquisition is feasible in patients with a high seizure frequency experiencing seizures with minimal motor activity during simple partial seizures. Moreover in these patients it can reveal localizing information on the epileptic focus that proves useful during presurgical evaluation.

Nestin-positive mesenchymal stem cells can be differentiated into functional neurons.

WISLET-GENDEBIEN, S., HANS, G., LEPRINCE, P., RIGO, J-M., MOONEN, G.and ROGISTER, B.
Center for Cellular and Molecular Neurobiology, University of Lige

In neurological disorders, auto-graft of neural cells derived from mesenchymal stem cells offers the potential of replacing lost cells and recovering lost functions. In this study, we demonstrated that rat mesenchymal stem cells (rMSCs) can be differentiated into astrocytes and neurons when they are co-cultivated with granular cerebellum cells. Two factors contributed to the neural differentiation 1) the expression of nestin (intermediate filament protein) by rMSCs and 2) a direct cell-cell interaction between the two types of cells. Electrophysiological analysis demonstrated that mesenchyme-derived neuron-like cells respond to several neurotransmitters. Moreover, we demonstrated that those cells expressed functional voltage-gated potassic channels and acquired, after at least 8 days of co-culture, functional voltage-gated sodium channels. In order to characterized the molecular event allowing the neural phenotypic plasticity of rMSCs, we analyzed membranous proteomic profile of the nestin (+) and (-) rMSCs. We observed an overexpression of some spots in the nestin-positive rMSCs extracts and we are now running analysis by mass spectrometry to identify these proteins and their possible implications in the neural fate choice of rMSCs.

The Neuroinformatics Site.org

Belgian Society for Neuroscience

The 5th bi-annual meeting 2003

Abstract list

ANALYSIS OF TAU m RNA IN ALZHEIMER'S DISEASE

Dopamine D4 receptor-mediated presynaptic inhibition of GABAergic transmission in the rat supraoptic nucleus

The ArKO mouse provides new evidence that estradiol is required for the development of the female brain

ESTRADIOL RAPIDLY INCREASES CREB PHOSPHORYLATION IN THE QUAIL BRAIN

Transdifferentiation of mesenchymal stem cells into neural cells in vitro and in vivo studies

Selectively rescued expression of calretinin in the cerebellar granule cells of calretinin knock-out mice

NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional GABAergic neurons in the adult dentate gyrus

Effects of modulation of the extrasynaptic GABAA conductance in cerebellar granule cells a computational study

Thiamine triphosphatase is predomminantly expressed in hippocampal pyramidal neurons and cerebellar Purkinje cells

Differential mechanisms underlie cell death induced by 3-nitropropionic acid in cultured striatal and cortical neurons

A dual role of adenosine A2A receptors in 3-nitropropionic acid-induced striatal lesions implications for the neuroprotective potential of A2A antagonists

C15O2-Positron Emission Tomography activation study during vagus nerve stimulation in patients with refractory epilepsy.

Delayed injection of GM-CSF improves functional recovery and promotes axonal regeneration after spinal cord injury.

Induction of Astroglial fate in Neural Stem Cell by co-cultured Mesenchymal Stem Cell.

THE DISTRIBUTION OF THE STEROID RECEPTOR COACTIVATOR SRC-1 IS SEXUALLY DIFFERENTIATED IN THE SONG CONTROL NUCLEI OF MALE AND FEMALE CANARIES

A higher-order motion region in human inferior parietal lobule.

Age-dependent expression of collapsin response mediator proteins (CRMPs) in cat visual cortex

ALPHA NORADRENERGIC RECEPTORS MEDIATE DOPAMINERGIC EFFECTS IN THE QUAIL PREOPTIC AREA

Acute vagus nerve stimulation (VNS) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

The processing of 2D shape in the cerebral cortex of the monkey and the human

Determination by a gain of fonction approach of the roles of ephrins in the development of neuronal connections of the cerebral cortex.

The effect of levetiracetam in rapid kindling

Area-Specificity and Topography of Thalamocortical Projections is Controlled by Ephrin/Eph Genes.

ADENOSINE RECEPTOR MODULATION OF NMDA CURRENTS IN THE DORSAL HORN OF RAT SPINAL CORD.

Estrogen synthesis in the spinal cord rapid effects on responsiveness to noxious stimuli

FMRI evidences of kinetic boundaries processing in monkeys

A Knock-out mice for the study of the MCH/MCHR1 axis

Involvement of GABAA receptor subunit d in the fragile X syndrome

Cortical Regions Involved in Extracting 3D Shape from Shading

EXPRESSION OF CXCR4 CHEMOKINE RECEPTOR mRNA DURING MOUSE BRAIN DEVELOPMENT

EXPRESSION OF CXCR4 CHEMOKINE RECEPTOR mRNA DURING MOUSE BRAIN DEVELOPMENT

SEXUAL PAVLOVIAN CONDITIONING OF RHYTHMIC CLOACAL SPHINCTER MUSCLE MOVEMENT IN MALE JAPANESE QUAIL

Modulation of the voltage-gated sodium channel in rat striatal neurons by the phospholipase C pathway

STUDIES OF REELIN RECEPTORS BY IN SITU HYBRIDIZATION AND NEW MONOCLONAL ANTIBODIES

Differential protein expression profiles across the layers in cat visual cortex as revealed by Laser MicroDissection

An in vitro model to study the radial migration in the cerebral cortex

Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways

Short term plasticity of complex spike waveform in Purkinje cell.

Lysophosphatidic acid regulation of cerebellar radial glia phenotype

Connexin channels, connexin mimetic peptides and ATP release in brain endothelial cells

Resonant synchronization in networks of inhibitory neurons

Estrogens are able to modulate facial inflammatory pain a study in aromatase knock-out mice using the formalin model

Processing of Kinetic boundaries in Macaque V4 A single cell study

In vitro models a shortcut to dissect molecular mechanisms underlying cell migration in brain

Vagus Nerve stimulation reduces body weight in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

ALTERED NEURONAL EXCITABILITY IN CEREBELLAR GRANULE CELLS OF MICE LACKING CALRETININ

Neurogenin2 specifies the connectivity of thalamic neurons by controlling axon responsiveness to intermediate target cues

In vitro and in vivo characterization of the effects of the new SK channel blocker methyl-laudanosine

Different roles for dendritic and somatic inhibition in a large neuron model

Variability in the response patterns of Purkinje cells to tactile stimulation

THE FUNCTIONAL STATE OF SPECIFIC NEURONAL POPULATIONS IN THE SONG SYSTEM OF LIVING CANARIES UPON LISTENING TO CONSPECIFIC SONGS AS ASSESSED BY DYNAMIC MANGANESE ENHANCED MRI

GENERATION OF MONOCLONAL ANTIBODIES AGAINST THE CENTRAL PART OF REELIN

TNF-alpha inhibits purinergic signaling in blood-brain barrier endothelial cells

Neurochemical subdivision and 3D-reconstruction of cat and monkey ventral lateral geniculate nucleus

The relation between glia and oxidative stress and their significance in the disease pathogenesis in Creutzfeldt-Jakob and Alzheimers Disease.

Quantification of cerebral perfusion parameters in different mouse models upon neurodegeneration using in-vivo bolus tracking Magnetic Resonance Imaging (MRI)

Functional brain Imaging of Songbirds when Listening to Songs a Functional Magnetic Resonance Imaging (fMRI) study

Results of long-term vagus nerve stimulation in patients with refractory epilepsy treated at the Reference Center for Refractory Epilepsy at Ghent University Hospital in Belgium

Acute and chronic cerebral blood flow changes induced by vagus nerve stimulation in patients with refractory epilepsy

Long-term Amygdalohippocampal deep brain stimulation for refractory temporal lobe epilepsy

Functional magnetic resonance imaging during refractory simple partial seizures

Nestin-positive mesenchymal stem cells can be differentiated into functional neurons.