Les mécanismes cellulaires activés par la mélatonine pour synchroniser l’alternance entre la saison dereproduction et l’anoestrus sont encore mal compris. Les niveaux et l’importance fonctionnelle de la plasticitéinduite par une transition photopériodique ont été étudiés dans des régions du cerveau impliquées dans lasynchronisation saisonnière de la reproduction chez les ovins. Nos résultats suggèrent qu’un changement dephotopériode seule serait capable d’induire, dans plusieurs régions hypothalamiques, des variations d’unmarqueur de la plasticité, la PSA-NCAM, qui participeraient à la régulation de la reproduction saisonnée enparticulier dans l’aire préoptique. Des populations neuronales affectées par des réarrangementsmorphologiques ont également été identifiées. La PSA-NCAM est plus présente au contact des neurones àGnRH et à β-endorphine au moment le plus opportun pour l’activité du neurone suggérant que la plasticitéaffectant ces deux populations cellulaires serait essentielle au déroulement de la reproduction saisonnée. Lesneurones à kisspeptine sont également plus nombreux chez des animaux ayant un axe gonadotrope actifsuggérant que cette population serait un des derniers éléments activés par l’augmentation de la durée desécrétion de la mélatonine.Nos travaux montrent qu’un changement de photopériode est suffisant pour induire des remaniementsmorphologiques dans des régions hypothalamiques nécessaires à la synchronisation saisonnière de lareproduction chez les ovins. Ces remaniements affectent des populations neuronales spécifiques. / Cellular mechanisms induced by melatonin to synchronize seasonal reproduction are still unclear. Thescale and functional significance of neuronal plasticity induced by a photoperiodic transition have been studiedin brain regions involved in the seasonal synchronization of sheep reproduction. Our results suggested that achange of photoperiod alone seems able to induce variations of PSA-NCAM, a plasticity marker, in severalhypothalamic areas that may participate to the regulation of seasonal reproduction particularly in the preopticarea. Neuronal populations affected by morphological rearrangements have been identified. PSA-NCAM waspresent in contact with GnRH and β-endorphin neurons when the neuropeptide secreted by that population isrequired for the timing of seasonal breeding activity. Kisspeptin neurons were more numerous in ewes havingan active gonadotrope axis suggesting that kisspeptine is one of the last element activated by the increase of theduration of melatonin secretion.All together, our results suggest that a change of photoperiod alone is essential to induce morphologicalreorganizations in hypothalamic regions necessary for the seasonal synchronization of sheep reproduction.Plasticity affects specific neuronal populations.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The neuromuscular junction (NMJ) is a synapse formed between a motoneuron and a muscle fiber which transmits the signals required to initiate muscle contraction. The functional state of the NMJ is intimately tied to the structure and function of the motoneuron, such that reductions in postsynaptic activity retrogradely stimulate sustained reorganization of presynaptic motor terminals in an attempt to maintain normal contractile output. In the adult, these plastic changes occur most notably as regenerative responses following traumatic injury and during the progression of motoneuron diseases (MNDs), and can contribute to a considerable amount of functional repair. However, limitations to the regeneration capacity of motoneurons place an upper limit on the effectiveness of endogenous repair mechanisms and can restrict the extent of functional recovery. Using a combination of immunofluorescence, sharp electrode electrophysiology and live labeling of synaptic vesicle recycling during various forms of synaptic growth and regeneration in vivo and in vitro, I have identified that the neural cell adhesion molecule (NCAM) is a key regulator of the regenerative capacity of motoneurons. In vivo experiments revealed that NCAM influences the maturation and stabilization of regenerated synapses via the recruitment and recycling of synaptic vesicles necessary for effective synaptic transmission. The presence of both pre- and post-synaptic NCAM were necessary to maintain the abundance of recycling synaptic vesicles at regenerated synapses, demonstrating a coordinated influence of these molecules in regenerative synaptic plasticity in vivo. To accurately assess the regenerative potential of motoneurons in vitro, it was necessary to develop a system which could reliably and consistently generate mature NMJs amenable to experimental investigation. Motoneurons differentiated from embryonic stem cells were grown for 3-5 weeks in co-culture with muscle fibers and generated mature NMJs which possessed morphological and functional criteria consistent with NMJs formed in vivo. NMJs formed by NCAM-/- motoneurons did not mature and were found to exhibit deficits consistent with their in vivo counterparts. These studies have revealed that NCAM is a key mediator of regenerative plasticity at the NMJ and may be a target for efforts to enhance endogenous repair following traumatic injury or during the progression of neurodegenerative disease.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.

La migration cellulaire est un processus complexe. Lors du développement, elle permet aux cellules de rejoindre leur destination finale, puis à l'âge adulte elle intervient dans de nombreux processus tels la réponse immunitaire ou le développement de pathologies.La migration est modulée par l'action de molécules d'adhérence. Nous nous sommes intéressés à la molécule NCAM (Neural Cellular Adhesion Molecule) dont les effets sont modulés par l'acide polysalique (PSA). Lors de précédentes études, il a été montré que le peptide mimétique de PSA-NCAM, PR-21 stimulait la migration issus de la zone sous-ventriculaire (SVZ).Nous avons synthétisés des analogues non-peptidiques de PR-21 en nous appuyant sur des analogies structurales. ces analogues ont été testés dans divers modèles de migration cellulaire : explants de SVZ et cellules C6 sur-exprimant PSA. Nous avons mis en évidence la présence de fonctions structurales clés dans la modulation de la migration cellulaire. / Cell migration is a complex process. During development, it contributes to cell reaching their final target. during adulthood, cell migration is involved in immune response or pathological processes.Migration is modulated by adhesion molecules. We concentrated on the Neural Cellular Adhesion Molecule (NCAM) which action is regulated by the post traductional addition of polysialic acid (PSA). PR-21 is a mimetic peptide of PSA-NCAM. In previous studies, PR-21 has been shown to stimulate in the migration of meuroblasts from sub-ventricular zone (SVZ) to the olfactory bulb.We designed non-peptidic analogues of PR-21 on the basis of structural analogies. these analogues were tested on various cell migration models : SVZ explants and C6 over-expressing PSA. We then established the need of key structural functions to modulate cell migration.

Les interneurones GABAergiques constituent une population mineure de cellules par rapport aux neurones glutamatergiques dans le néocortex. Cependant ils contrôlent fortement l'excitabilité neuronale, la dynamique des réseaux neuronaux et la plasticité synaptique. L'importance des circuits GABAergiques dans le processus fonctionnel et la plasticité des réseaux corticaux est soulignée par des résultats récents qui montrent que des modifications très précises et fiables des circuits GABAergiques sont associées à divers troubles du développement neurologique et à des défauts dans les fonctions cérébrales. De ce fait, la compréhension des mécanismes cellulaires et moléculaires impliquant le développement des circuits GABAergiques est la première étape vers une meilleure compréhension de la façon dont les anomalies de ces processus peuvent se produire. La molécule d’adhésion cellulaire neurale (NCAM) appartient à la super-famille des immunoglobulines de reconnaissance cellulaire et est impliquée dans des interactions homophiliques et hétérophiliques avec d’autres molécules. Même si plusieurs rôles de NCAM ont été démontrés dans la croissance neuronale, la fasciculation axonale, la formation et la maturation de synapses, de même que dans la plasticité cellulaire de plusieurs systèmes, le rôle de NCAM dans la formation des synapses GABAergiques reste inconnu. Ce projet visait donc à déterminer le rôle précis de NCAM dans le processus de maturation des synapses GABAergiques dans le néocortex, en modulant son expression à différentes étapes du développement. L’approche choisie a été de supprimer NCAM dans des cellules GABAergiques à paniers avant la maturation des synapses (EP12-18), pendant la maturation (EP16-24), ou durant le maintien de celles-ci (EP24-32). Les méthodes utilisées ont été le clonage moléculaire, l’imagerie confocale, la culture de coupes organotypiques et des techniques morphométriques de quantification de l’innervation GABAergique. Nos résultats montrent que l’inactivation de NCAM durant la phase de maturation des synapses périsomatiques (EP16-24) cause une réduction du nombre de synapses GABAergiques périsomatiques et du branchement de ces axones. En revanche, durant la phase de maintien (EP26-32), l’inactivation de NCAM n’a pas affecté ces paramètres des synapses GABAergiques. Or, il existe trois isoformes de NCAM (NCAM120, 140 et 180) qui pourraient jouer des rôles différents dans les divers types cellulaires ou à des stades développementaux différents. Nos données montrent que NCAM120 et 140 sont nécessaires à la maturation des synapses périsomatiques GABAergiques. Cependant, NCAM180, qui est l’isoforme la plus étudiée et caractérisée, ne semble pas être impliquée dans ce processus. De plus, l’inactivation de NCAM n’a pas affecté la densité des épines dendritiques ou leur longueur. Elle est donc spécifique aux synapses périsomatiques GABAeriques. Finalement, nos résultats suggèrent que le domaine conservé C-terminal KENESKA est essentiel à la maturation des synapses périsomatiques GABAergiques. Des expériences futures nous aiderons à mieux comprendre la mécanistique et les différentes voies de signalisation impliquées. / GABAergic interneurons, though a minor population in the neocortex, play an important role in cortical function and plasticity. Alterations in GABAergic circuits are implicated in various neurodevelopmental disorders. The GABAergic network comprises diverse interneuron subtypes that have different morphological and physiological characteristics, and localize their synapses onto distinct subcellular locations on the postsynaptic targets. Precisely how activity and molecularly driven mechanisms conspire to achieve the remarkable specificity of GABAergic synapse localization and formation is unknown. Therefore, unravelling the cellular and molecular mechanisms involved in this process is crucial for a better understanding of both cortical function and the basis of various neurological disorders. Here we focus our study on a subtype of GABAergic neurons - the basket interneurons which localize synapses, called perisomatic synapses, onto the soma and proximal dendrites of the postsynaptic targets, and tightly regulate their firing patterns. Although recent studies have shown the activity dependence of basket synapse formation, the molecular mechanisms implicated in the perisomatic synapse formation process are poorly understood. NCAM, the neural cell adhesion molecule, is a prime molecular player implicated both in early synaptogenesis events, and during maturation of glutamatergic synapses in the hippocampus. Recent studies have implicated the polysialylated form of NCAM (PSA-NCAM) in basket synapse formation. However, whether and how NCAM per se plays a role in the formation of GABAergic synapses is unknown. Using single cell genetics to knock down NCAM in individual basket interneurons at specific developmental time periods, we characterized the role of NCAM during perisomatic synapse formation and maintenance. Here we show that loss of NCAM during perisomatic synapse formation from equivalent postnatal day (EP) 16 to EP24, in organotypic slices from mouse visual cortex, significantly retards the process of basket cell axonal branching and bouton formation. However, loss of NCAM at a later stage (EP26 to EP32), when the synapses are already formed, did not affect the number or intricacy of perisomatic synapses. NCAM is therefore implicated in perisomatic synapse formation but not in its maintenance. Further studies also show that isoforms of NCAM, such as NCAM140 and NCAM120 are involved in perisomatic GABAergic synapse maturation. However, NCAM180 is not implicated in this process. Also, NCAM does not affect dendritic spine density and length during maturation and maintenance phases, therefore its action is specific only to GABAergic perisomatic synapses. Finally, the highly conserved C-terminal domain KENESKA is essential for GABAergic perisomatic synapse maturation. Future experiments will help us clarify this mechanism and the involved signalling pathways related to NCAM.

Les interneurones GABAergiques constituent une population mineure de cellules par rapport aux neurones glutamatergiques dans le néocortex. Cependant ils contrôlent fortement l'excitabilité neuronale, la dynamique des réseaux neuronaux et la plasticité synaptique. L'importance des circuits GABAergiques dans le processus fonctionnel et la plasticité des réseaux corticaux est soulignée par des résultats récents qui montrent que des modifications très précises et fiables des circuits GABAergiques sont associées à divers troubles du développement neurologique et à des défauts dans les fonctions cérébrales. De ce fait, la compréhension des mécanismes cellulaires et moléculaires impliquant le développement des circuits GABAergiques est la première étape vers une meilleure compréhension de la façon dont les anomalies de ces processus peuvent se produire. La molécule d’adhésion cellulaire neurale (NCAM) appartient à la super-famille des immunoglobulines de reconnaissance cellulaire et est impliquée dans des interactions homophiliques et hétérophiliques avec d’autres molécules. Même si plusieurs rôles de NCAM ont été démontrés dans la croissance neuronale, la fasciculation axonale, la formation et la maturation de synapses, de même que dans la plasticité cellulaire de plusieurs systèmes, le rôle de NCAM dans la formation des synapses GABAergiques reste inconnu. Ce projet visait donc à déterminer le rôle précis de NCAM dans le processus de maturation des synapses GABAergiques dans le néocortex, en modulant son expression à différentes étapes du développement. L’approche choisie a été de supprimer NCAM dans des cellules GABAergiques à paniers avant la maturation des synapses (EP12-18), pendant la maturation (EP16-24), ou durant le maintien de celles-ci (EP24-32). Les méthodes utilisées ont été le clonage moléculaire, l’imagerie confocale, la culture de coupes organotypiques et des techniques morphométriques de quantification de l’innervation GABAergique. Nos résultats montrent que l’inactivation de NCAM durant la phase de maturation des synapses périsomatiques (EP16-24) cause une réduction du nombre de synapses GABAergiques périsomatiques et du branchement de ces axones. En revanche, durant la phase de maintien (EP26-32), l’inactivation de NCAM n’a pas affecté ces paramètres des synapses GABAergiques. Or, il existe trois isoformes de NCAM (NCAM120, 140 et 180) qui pourraient jouer des rôles différents dans les divers types cellulaires ou à des stades développementaux différents. Nos données montrent que NCAM120 et 140 sont nécessaires à la maturation des synapses périsomatiques GABAergiques. Cependant, NCAM180, qui est l’isoforme la plus étudiée et caractérisée, ne semble pas être impliquée dans ce processus. De plus, l’inactivation de NCAM n’a pas affecté la densité des épines dendritiques ou leur longueur. Elle est donc spécifique aux synapses périsomatiques GABAeriques. Finalement, nos résultats suggèrent que le domaine conservé C-terminal KENESKA est essentiel à la maturation des synapses périsomatiques GABAergiques. Des expériences futures nous aiderons à mieux comprendre la mécanistique et les différentes voies de signalisation impliquées. / GABAergic interneurons, though a minor population in the neocortex, play an important role in cortical function and plasticity. Alterations in GABAergic circuits are implicated in various neurodevelopmental disorders. The GABAergic network comprises diverse interneuron subtypes that have different morphological and physiological characteristics, and localize their synapses onto distinct subcellular locations on the postsynaptic targets. Precisely how activity and molecularly driven mechanisms conspire to achieve the remarkable specificity of GABAergic synapse localization and formation is unknown. Therefore, unravelling the cellular and molecular mechanisms involved in this process is crucial for a better understanding of both cortical function and the basis of various neurological disorders. Here we focus our study on a subtype of GABAergic neurons - the basket interneurons which localize synapses, called perisomatic synapses, onto the soma and proximal dendrites of the postsynaptic targets, and tightly regulate their firing patterns. Although recent studies have shown the activity dependence of basket synapse formation, the molecular mechanisms implicated in the perisomatic synapse formation process are poorly understood. NCAM, the neural cell adhesion molecule, is a prime molecular player implicated both in early synaptogenesis events, and during maturation of glutamatergic synapses in the hippocampus. Recent studies have implicated the polysialylated form of NCAM (PSA-NCAM) in basket synapse formation. However, whether and how NCAM per se plays a role in the formation of GABAergic synapses is unknown. Using single cell genetics to knock down NCAM in individual basket interneurons at specific developmental time periods, we characterized the role of NCAM during perisomatic synapse formation and maintenance. Here we show that loss of NCAM during perisomatic synapse formation from equivalent postnatal day (EP) 16 to EP24, in organotypic slices from mouse visual cortex, significantly retards the process of basket cell axonal branching and bouton formation. However, loss of NCAM at a later stage (EP26 to EP32), when the synapses are already formed, did not affect the number or intricacy of perisomatic synapses. NCAM is therefore implicated in perisomatic synapse formation but not in its maintenance. Further studies also show that isoforms of NCAM, such as NCAM140 and NCAM120 are involved in perisomatic GABAergic synapse maturation. However, NCAM180 is not implicated in this process. Also, NCAM does not affect dendritic spine density and length during maturation and maintenance phases, therefore its action is specific only to GABAergic perisomatic synapses. Finally, the highly conserved C-terminal domain KENESKA is essential for GABAergic perisomatic synapse maturation. Future experiments will help us clarify this mechanism and the involved signalling pathways related to NCAM.

Skeletal muscle satellite cells located between the plasma membrane and the basal lamina of muscle fibres, could for many years, only be studied in situ by electron microscopy. The introduction of immunohistochemistry and the discovery of molecular markers of satellite cells then made them accessible for light microscopic studies and a wealth of information is today available. Satellite cells are myogenic stem cells that can be activated from a quiescent state to proliferate for self-renewal or differentiate into myogenic cells. The satellite cells are involved in muscle growth during fetal and postnatal development and play a key role in repair and regeneration of damaged muscle fibres. The satellite cells are also essential for muscle fibre hypertrophy and maintenance of muscle mass in the adult. When the present thesis was initiated, studies on satellite cells in human skeletal muscle relied on the neuronal cell adhesion molecule (NCAM) as a marker for satellite cell identification. The results from different studies varied markedly. Therefore the aims of the present thesis were i) to develop a highly reliable method using light microscopy for satellite cell identification and quantification in biopsies of human skeletal muscle in normal and pathological conditions. A molecular marker for the myofibre basal lamina or plasma membrane to enhance the reliability of myonuclei and satellite cell identification were to be included. Furthermore unbiased morphometric methods should be used in the quantification process. ii) to evaluate which molecular markers which had been described for satellite cell and stem cell identification in different cell states (quiescence, activated or differentiated) are the most useful for studies on human skeletal muscle. iii) to further explore the function and heterogeneity of satellite cells with respect to different markers in human skeletal muscle by studying the effects of strength-training, intake of anabolic substances and pathological conditions. A new immunofluorescence method was developed where in the same tissue section, two satellite cell markers, the basal lamina and nuclei were monitored. From the evaluation of different markers it was found that both NCAM and Pax7 identified the majority of satellite cells but that both markers were needed for reliable identification. The members of the myogenic regulatory family were evaluated and by using the new method MyoD and myogenin were found to be useful markers to identify activated and differentiated satellite cells. Upon re-examination of biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects it was observed that the new results on satellite cell frequency were significantly different from those obtained when using staining for NCAM and nuclei alone. In addition three subtypes of satellite cells (94.4% NCAM+/Pax7+, 4.2% NCAM+/Pax7– and 1.4% NCAM–/Pax7+) were observed. Thus the multiple marker method gave more information about satellite cells heterogeneity in human muscle and we propose that this is more reliable than previous methods. Low numbers of MyoD or myogenin stained satellite cells were observed in both untrained and strength trained subjects. Other markers such as DLK1/FA1, a member of the EGF-like family and c-Met, the receptor for hepatocyte growth factor showed that satellite cell heterogeneity in human muscle is far greater than previously shown. Furthermore, new evidence is presented for so called fibre splitting observed in hypertrophic muscle fibres to be due to defect regeneration of partially damaged fibres.