The mammalian brain contains a regulatory center in the diencephalic region known as the hypothalamus that plays a critical role in physiological homeostasis, and contains a variety of centers for behavioral drives, such as hunger and thirst. Located deep within the hypothalamus is the suprachiasmatic nucleus (SCN), or the master biological clock, that organizes rhythmic physiology and behavior, such that critical events take place at the most appropriate time of the day or night and in the most appropriate temporal, phase relationships. Cell-to-cell communication is essential for conveying inputs to and outputs from the SCN. The goal of the present study was to use an immortalized neural cell line (SCN2.2), derived from the presumptive anlage of the rat suprachiasmatic nucleus, as an in vitro model system to study intercellular communication among SCN cells. I tested whether the pineal neurohormone melatonin could modulate cell-to-cell signaling, via both dye coupling (gap junctional communication) and calcium waves (ATP-dependent gliotransmission). I also tested whether extracellular ATP could influence the spread of calcium waves in SCN2.2 cells. Lastly, the ability of extracellular ATP to modulate SCN physiological responses to melatonin in SCN2.2 cells was examined. I show that melatonin at a physiological concentration (nM) reduced dye coupling (gap junctional communication) in SCN2.2 cells, as determined by a scrape loading procedure employing the fluorescent dye lucifer yellow. Melatonin caused a significant reduction in the spread of calcium waves in cycling SCN2.2 cultures as determined by ratiometric calcium imaging with Fura-2 AM, a calcium sensitive indicator dye. This reduction was greatest when an endogenous circadian rhythm in extracellular ATP accumulation, determined by luciferase assay, was at its trough or lowest extracellular concentration. In addition, melatonin and ATP interacted in the regulation of gliotransmission (calcium waves), and this interaction was also specific to particular phases of the endogenous SCN physiological rhythmicity. Thus, I have established that a complex interaction exists between established melatonin signaling pathways and this newly discovered ATP accumulation rhythm, with the mechanisms underlying this relationship linked to endogenous cycling of SCN cellular physiology.

No description available.

The hypothalamus is an integral part of the brain's regulation of mammalian physiology and behavior. Among many functions, this regulatory center activates the sympathetic nervous system, maintains appropriate body temperature, controls food intake, and controls release of hormones from the pituitary gland. Deep within the hypothalamus lie a paired cluster of cells, the suprachiasmatic nuclei (SCN), which function as the chief circadian pacemaker. The goal of the present thesis research was to study rhythmically controlled ATP gliotransmission. I used an immortalized SCN2.2 hypothalamic cell line to determine the mechanism by which ATP signaling is regulated in this context. Additionally, this research aimed to elucidate if clock-controlled ATP gliotransmission is fundamentally distinct from stimulus-evoked calcium-dependent mechanisms that regulate intercellular ATP signaling among astrocytes. In this thesis, I show that there are multiple ATP signaling mechanisms present among SCN2.2 cells. cAMP-dependent signaling mediates clock-controlled ATP accumulation but not stimulus-evoked ATP signaling. In addition, pharmacological studies suggest that disparate purinergic receptor-mediated mechanisms are involved in the regulation of clock-controlled versus stimulus-evoked ATP signaling. Rhythmic accumulation of ATP in SCN2.2 cultures is modulated by calcium-dependent processes. Peaks in ATP accumulation coincide with elevated mitochondrial calcium levels, while troughs in ATP accumulation coincide with periods of high cytosolic calcium levels, suggesting a possible mechanistic link between circadian shifts in intracellular calcium handling and ATP handling in SCN2.2 cells. Clock-controlled ATP accumulation in SCN2.2 cells is not a by-product of rhythmic cell cycle or rhythmic cell death. Overall, my research suggests that the ATP accumulation rhythm in SCN2.2 cells is likely an output of the biological clock, mediated by astrocytic calcium signaling processes, and not an output of cell division or cell death. Estimation of ATP accumulation in SCN2.2 cultures at peak time points suggests that clock-controlled ATP release is critical to the function of astrocytes in the mammalian brain, perhaps in the regulation of brain metabolism, the regulation of sleep/wake physiology, or the integration of both.

Les données accumulées au cours des deux dernières décennies ont montré que l’astrocyte joue un rôle clé dans la régulation de la transmission synaptique. Cela est dû à sa capacité à détecter, via des récepteurs, et réguler, via la libération de gliotransmetteurs, la transmission synaptique. Cependant, les astrocytes deviennent réactifs dans des conditions pathologiques comme la maladie d’Alzheimer et la régulation de l’activité neuronale par ces cellules est susceptible d’être altérée.L'objectif principal de cette thèse est d'étudier le rôle de l‘astrocyte en tant qu'intégrateur et modulateur de la transmission synaptique dans des conditions physiologique et pathologique.Premièrement, nous avons montré qu’un astrocyte est capable de détecter et d’augmenter la transmission synaptique de base au niveau d’une synapse chez des rats jeunes. On ignore si cette régulation est toujours présente chez les adultes et si elle est affectée par l'activité synaptique des synapses voisines présentes dans le même domaine astrocytaire. En utilisant l’imagerie STED et des enregistrements électrophysiologiques sur des tranches d’hippocampes de rats adultes, nous montrons ici que la voie de régulation décrite précédemment est également présente chez les adultes. En effet, les astrocytes détectent la transmission glutamatergique de base au niveau de synapses individuelles par le biais des mGluR5 et l’augmente en libérant des purines qui activent les récepteurs présynaptiques A2A. Plus important encore, nos données suggèrent fortement qu'un astrocyte est capable d'adapter sa régulation de la transmission glutamatergique en fonction du nombre de synapses activées dans son domaine. Lorsque le nombre d’afférences activées est faible, les astrocytes facilitent l'efficacité synaptique par un mécanisme dépendant des purines. Fait intéressant, ce processus n’est plus présent lorsqu’un plus grand nombre d’afférences est activé, ce qui suggère que les astrocytes sont capables d’intégrer différemment les informations entrantes et d’adapter leur réponse en termes de libération de purine.Deuxièmement, des études sur des modèles de la maladie d’Alzheimer ont rapporté que plusieurs fonctions astrocytaires, comme leur capacité à réguler la transmission synaptique, étaient perturbées. Cependant, la contribution de la réactivité astrocytaire dans cette pathologie reste méconnue et débattue du fait que les modifications rapportées dans ces études peuvent être bénéfiques et/ou néfastes pour les neurones. En effet, les astrocytes réactifs présentent une hétérogénéité morphologique, moléculaire et fonctionnelle qui peut expliquer leurs effets controversés dans cette pathologie. Pour comprendre comment la réactivité astrocytaire contribue à la maladie d'Alzheimer et trouver des voies thérapeutiques, il est essentiel de développer une nouvelle stratégie qui module efficacement tous les types d'astrocytes réactifs. Ici, nous avons utilisé des approches in vivo visant spécifiquement les astrocytes et identifié la voie JAK2/STAT3, comme étant nécessaire et suffisante pour l'induction et le maintien de la réactivité astrocytaire. La modulation de cette cascade par approche virale contrôle efficacement plusieurs caractéristiques morphologiques et moléculaires de la réactivité. De plus, son inhibition chez des modèles murins de la maladie d'Alzheimer améliore des caractéristiques pathologiques clés en réduisant les dépôts amyloïdes et en améliorant l'apprentissage spatial. En combinant l’approche virale avec des enregistrements électrophysiologiques, notre équipe a montré que réduire la réactivité astrocytaire en inhibant la voie JAK2/STAT3 rétablit les déficits de transmission synaptique et de plasticité observés chez un modèle 3xTg de la maladie d'Alzheimer. En conclusion, la cascade JAK2/STAT3 est un régulateur principal de la réactivité astrocytaire in vivo. Son inhibition offre de nouvelles opportunités thérapeutiques pour la maladie d'Alzheimer. / Data accumulated over the two last decades have demonstrated that astrocytes play key roles in the regulation of synaptic transmission and plasticity. This is due to their capability to detect and regulate synaptic transmission by expressing receptors and releasing gliotransmitters, respectively. However, astrocytes become reactive in pathological condition such as Alzheimer’s disease, and neuronal activity regulation by these glial cells is likely to be altered.The main objective of this thesis is to study the role of astrocytes as integrators and modulators of synaptic transmission under both physiological and pathological conditions.First, we have shown that a single astrocyte is able to detect and in turn up-regulate basal synaptic transmission at individual synapses in juvenile rats. Whether this upregulation is still present in adults and whether it is affected by the synaptic activity occurring at neighboring synapses present within the same astroglial domain is unknown. Using STED imaging on fixed tissue and electrophysiological recordings on acute hippocampal slices of adult male rats we here show that the upregulation pathway previously described in juvenile rats is also present in adults. Indeed, as in juvenile, astrocytes detect basal glutamatergic transmission at individual synapses through mGluR5 and in turn upregulate it by releasing purines and activating presynaptic A2A receptors. More importantly, our data suggest strongly that an individual astrocyte is able to adapt its purine-mediated regulation of glutamatergic transmission as a function of the number of synapses activated in its domain. When the number of afferent inputs activated is small, astrocytes facilitate synaptic efficacy through a purine-mediated process. Interestingly, this process is no longer present when a higher number of afferences is activated, suggesting the astrocytes integrate the incoming information and adapt its response in terms of purine release.Second, studies on Alzheimer’s disease models have reported changes in several astrocyte functions such as its ability to regulate synaptic transmission. However, the contribution of astrocytic reactivity in this pathology remains largely unknown and debated as the reported changes in these studies can have beneficial, deleterious or even mixed effects on neurons. Indeed, reactive astrocytes display a morphological, molecular and functional heterogeneity that could explain their controversial effects in this pathology. To understand how astrocyte reactivity contributes to Alzheimer's disease and to find therapeutic pathways, it is crucial to develop a new strategy that efficiently modulates all types of reactive astrocytes. Here, we used cell type-specific approaches in vivo and identified the JAK2/STAT3 pathway, as necessary and sufficient for the induction and maintenance of astrocyte reactivity. Modulation of this cascade by viral gene transfer in mouse astrocytes efficiently controlled several morphological and molecular features of reactivity. Inhibition of this pathway in mouse models of Alzheimer's disease improved key pathological hallmarks by reducing amyloid deposition and improving spatial learning. Combining this viral gene transfer with electrophysiological recordings, we specifically showed in our lab that reducing astrocyte reactivity by inhibiting JAK2/STAT3 cascade in astrocytes restores synaptic transmission and plasticity deficits observed in a 3xTg mouse model of Alzheimer's disease. In conclusion, the JAK2/STAT3 cascade operates as a master regulator of astrocyte reactivity in vivo. Its inhibition offers new therapeutic opportunities for Alzheimer's disease.

Il est maintenant établi que le sommeil paradoxal (SP) facilite certaines formes de mémoires dépendantes de l'hippocampe bien que les mécanismes impliqués restent mal compris. Des résultats de l'équipe et d'autres laboratoires suggèrent toutefois que cet effet facilitateur serait en partie sous-tendu par une modulation de la plasticité synaptique de longue durée dans l'hippocampe. Nos travaux se proposent donc d'étudier ces mécanismes chez la souris, à l'échelle synaptique et systémique. Pour cela nous nous avons privilégié trois approches : 1) La caractérisation comportementale et électrophysiologique du rôle des neurones à Hormone de Mélano-Concentration (MCH), une hormone hypothalamique libérée principalement pendant le SP, qui pourrait être un acteur associé au SP, impliqué dans la formation ou la consolidation de nouveaux apprentissage ; 2) L'étude de la neuromodulation de la transmission synaptique hippocampique induite par la MCH ; 3) Une étude du rôle du SP dans le remodelage des réseaux corticaux de la mémoire prenant place pendant les processus de consolidation à court et long terme d'un conditionnement contextuel à la peur. L'ensemble de nos résultats indique que le SP, grâce notamment à des effecteurs moléculaires comme la MCH, peut moduler la plasticité synaptique hippocampique et ainsi intervenir aussi bien dans la formation que la consolidation à long terme des souvenirs / It is now established that paradoxical sleep (PS) facilitates some hippocampaldependent memory forms although mechanisms implied are still not well understood. However, results from our lab and others suggest that part of this facilitating effect is underlined by hippocampal long term synaptic plasticity modulation. Our work investigates these mechanisms in mice, at a synaptic and systemic scale. We opted for three different approaches: 1) A behavioural and electrophysiological characterization of the role of Melanin-Concentrating Hormone (MCH) neurons. Indeed, as this hypothalamic hormone is mainly liberated during PS, it could be a PS associated effector implied in formation or consolidation of new memories ; 2) A study of neuromodulating effects on hippocampal synaptic transmission induced by MCH ; 3) A study of PS role in memory cortical networks remodeling happening during short and long-term consolidation of a contextual fear conditioning task. Altogether, our results point out that PS modulates hippocampal synaptic plasticity, notably through molecular effectors such as MCH, and thus takes part in both encoding and long-term consolidation of memories process

No description available.

Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq-linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq-linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks.