One of the defining characteristics of the brain is its plasticity, which is the ability to alter and reorganize neuronal circuits. The brain is constantly being shaped and moulded by the external world through endogenous factors like neurotransmitters, growth factors and circulating hormones. 17β-estradiol, which is the most potent estrogen among the group of ovarian steroid hormones, has widespread effects throughout the central nervous system. Apart from its actions on regions of the brain concerned with reproduction, estradiol has profound effects on brain areas not classically associated with reproductive function like cerebral cortex, midbrain, brainstem, hippocampus and spinal cord. This enables the hormone to influence learning and memory, emotions, affective state, cognition, motor coordination and pain sensitivity. Estradiol exerts these effects by regulating gene expression via intracellular estrogen receptors. In addition to this, the hormone interacts with receptors at the cell membrane to rapidly alter the electrical activity of neurons and astrocytes, and regulate second messenger systems. The aim of this study was to investigate the cellular and functional effects of estradiol on neuronal networks and on signaling between neurons and astrocytes in primary mixed hippocampal cultures. Estradiol is proconvulsant; it increases neuronal excitability and decreases the threshold for seizures. This property of estradiol is instrumental in precipitating catamenial seizures in women with epilepsy. These are epileptic seizures influenced by cyclical hormone changes and occur in over one-third to half of women with epilepsy. In the first part of the work, the effects of 24-hour estradiol treatment on hippocampal neurons were investigated using fluorescence imaging and electrophysiological techniques. Further, the ability of gabapentin, an antiepileptic drug sometimes used to treat hormone sensitive seizures, to counteract the effects of estradiol was studied. Synaptic vesicles were labeled by uptake of FM 1-43, and high K+- triggered exocytotic release was monitored by fluorescence imaging. The reduction in intensity of FM 1-43 fluorescence, which is a measure of vesicular release, was enhanced by estradiol, suggesting that estradiol upregulates the exocytotic machinery. The high K+-evoked intracellular Ca2+ rise in neurons, studied by loading the neurons with the Ca2+ indicator dye fluo-3 AM, was potentiated following estradiol treatment. Electrophysiological recordings from neurons following estradiol treatment showed an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a larger number of mEPSC events with a predominant NMDA component. Many of the estradiol-induced excitatory effects on the neuronal network were abolished by incubating the cultures with a combination of estradiol and gabapentin suggesting a mechanism of action for the drug in the treatment of hormone sensitive seizures. Glial cells were once regarded as passive, supportive elements in the nervous system. This view of glial cells has drastically changed over the past decade and it is now known that glial cells are dynamic signaling elements in the brain. In view of the emerging importance of glia in the physiology of the nervous system and accumulating evidence of direct effects of steroid hormones on these cells, the subsequent part of the work delves into the consequences of 24-hour estradiol treatment on astrocytes and neuron-to-astrocyte signaling. Estrogen receptors have been described on both neurons and astrocytes in the hippocampus suggesting a complex interplay between the two cell types in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration, ([Ca2+]i). Astrocyte ([Ca2+]i) transients can modulate neuronal activity, indicating a bi-directional form of communication between neurons and astrocytes. Using simultaneous electrophysiology and calcium imaging techniques, neuronal activity-evoked ([Ca2+]i) changes in fluo-3 AM loaded astrocytes were monitored. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca2+]i) elevations in adjacent astrocytes which could be blocked by 200 µM MCPG and also 1 µM TTX. Comparison of astrocytic ([Ca2+]i) transients in control and estradiol treated cultures revealed that the amplitude of the ([Ca2+]i) transient, the number of responsive astrocytes and the ([Ca2+]i) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca2+]i) rise in astrocytes in response to local application of the metabotropic glutamate receptor agonist t-ACPD was attenuated in estradiol treated cultures suggesting functional changes in the astrocyte metabotropic glutamate receptor following 24-hour treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca2+]i) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling. The acute effects of estradiol on astrocyte-to-astrocyte and astrocyte-to-neuron signaling have been addressed in the next part of the study. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes, and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. In addition to its rapid actions on neuronal electrical activity, estradiol can rapidly alter astrocyte ([Ca2+]i) levels through a plasma membrane-associated estrogen receptor. The functional consequences of acute estradiol treatment (5 min) on astrocyte-astrocyte and astrocyte-neuron communication were investigated using calcium imaging and electrophysiological techniques. Mechanical stimulation of an astrocyte evoked a ([Ca2+]i) rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a ([Ca2+]i) wave. Following acute treatment with estradiol, the amplitude of the ([Ca2+]i) elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the ([Ca2+]i) rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, t-ACPD. Mechanical stimulation of astrocytes induced ([Ca2+]i) elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated ([Ca2+]i) rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol mediated hormonal control.

Du fait de sa complexité, le fonctionnement du cerveau est exploré par des méthodes très diverses, telles que la neurophysiologie et les neurosciences cognitives, et à des échelles variées, allant de l'observation de l'organe dans son ensemble jusqu'aux molécules impliquées dans les processus biologiques. Ici, nous proposons une étude à l'échelle cellulaire qui s'intéresse à deux briques élémentaires du cerveau : les neurones et les cellules gliales. L'approche choisie est la biophysique, de part les outils utilisés et les questions abordées sous l'angle de la physique. L'originalité de ce travail est d'utiliser des cellules primaires du cerveau dans un souci de proximité avec l'in vivo, au sein de systèmes in vitro dont la structure chimique et physique est contrôlé à l'échelle micrométrique. Utilisant les outils de la microélectronique pour un contrôle robuste des paramètres physico-chimiques de l'environnement cellulaire, ce travail s'intéresse à deux aspects de la biologie du cerveau : la polarisation neuronale, et la sensibilité des cellules gliales aux propriétés mécaniques de leur environnement. A noter que ces deux questions sont étroitement imbriquées lors de la réparation d'une lésion. La première est cruciale pour la directionalité de la transmission de signaux électriques et chimiques et se traduit par une rupture de symétrie dans la morphologie du neurone. La seconde intervient dans les mécanismes de recolonisation des lésions, dont les propriétés mécaniques sont altérées., Les études quantitatives menées au cours de cette thèse portent essentiellement sur la phénoménologie de la croissance de ces deux types de cellules et leur réponse à des contraintes géométriques ou mécaniques. L'objectif in fine est d'élucider quelques mécanismes moléculaires associés aux modifications de la structure cellulaire et donc du cytosquelette. Un des résultats significatifs de ce travail est le contrôle de la polarisation neuronale par le simple contrôle de la morphologie cellulaire. Ce résultat ouvre la possibilité de développer des architectures neuronales contrôlées in vitro à l'échelle de la cellule individuelle. / The complex structure of the brain is explored by various methods, such as neurophysiology and cognitive neuroscience. This exploration occurs at different scales, from the observation of this organ as a whole entity to molecules involved in biological processes. Here, we propose a study at the cellular scale that focuses on two building elements of brain: neurons and glial cells. Our approach reachs biophysics field for two main reasons: tools that are used and the physical approach to the issues. The originality of our work is to keep close to the in vivo by using primary brain cells in in vitro systems, where chemical and physical environments are controled at micrometric scale. Microelectronic tools are employed to provide a reliable control of the physical and chemical cellular environment. This work focuses on two aspects of brain cell biology: neuronal polarization and glial cell sensitivity to mechanical properties of their environment. As an example, these two issues are involved in injured brains. The first is crucial for the directionality of the transmission of electrical and chemical signals and is associated to a break of symmetry in neuron morphology. The second occurs in recolonization mechanisms of lesions, whose mechanical properties are impaired. During this thesis, quantitative studies are performed on these two cell types, focusing on their growth and their response to geometrical and mechanical constraints. The final aim is to elucidate some molecular mechanisms underlying changes of the cellular structure, and therefore of the cytoskeleton. A significant outcome of this work is the control of the neuronal polarization by a simple control of cell morphology. This result opens the possibility to develop controlled neural architectures in vitro with a single cell precision.

Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1 Abstract Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) has been identified as an interacting partner of cannabinoid receptor 1 (CB1R). Their protein-protein interaction was confirmed by co-immunoprecipitation. SGIP1 hinders the internalization of activated CB1R and modulates its signaling in HEK293 cells. Employing whole-cell patch-clamp electrophysiology, we have shown that SGIP1 affects CB1R signaling in autaptic hippocampal neurons. Using a battery of behavioral tests in SGIP1 constitutive knock-out (SGIP1-/- ) and WT mice, we investigated the consequences of SGIP1 deletion on behavior regulated by the endocannabinoid system. In SGIP1-/- mice, exploratory levels, working memory and sensorimotor gating were unaltered. SGIP1-/- mice showed decreased anxiety-like and depressive-like behaviors. Fear extinction to tone was enhanced in SGIP1-/- females. Several cannabinoid tetrad behaviors were altered in the absence of SGIP1. SGIP1-/- males exhibited abnormal THC withdrawal behaviors. SGIP1 deletion also reduced acute nociception, and SGIP1-/- mice were more sensitive to antinociceptive effects of CB1R agonists and morphine. CB1R-SGIP1 interaction results in profound modification of CB1R...

Le cerveau contient environ cent milliards de cellules nerveuses, ou neurones. Ces neurones communiquent entre eux par des structures fonctionnellement distinctes – l’axone et la dendrite – capables d’émettre et recevoir des signaux électriques ou chimiques à partir d’un compartiment présynaptique vers un compartiment, dit post-synaptique. Nous avons focalisé notre étude sur les synapses des neurones hippocampiques, qu’on estime responsables de fonctions cérébrales dites supérieures, comme la mémoire et l’apprentissage. Plus particulièrement, on s’est intéressé au développement et au maintien des épines dendritiques, dont les changements morphologiques sont intimement liés à la plasticité synaptique, autrement dit, capacité de réponse à l’activité synaptique. Les épines dendritiques ont pour origine les filopodes qui évoluent en épines lors du contact axonal. La transition entre filopode et épine implique une myriade de molécules, dont des récepteurs glutamatergiques, des protéines d’échafaudage et du cytosquelette d’actine capables de recevoir, transmettre et intégrer le signal présynaptique. Cependant, la coordination spatiale et temporelle de tous ces composants moléculaires au long de la formation et maturation d’une synapse reste largement méconnue.Scribble1 (Scrib1) est une protéine de polarité cellulaire (PCP) classiquement impliquée dans l’homéostasie de tissues épithéliaux ainsi que dans la croissance et progression des tumeurs. Scrib1 est aussi une protéine d’échafaudage critique pour le développement et le bon fonctionnement du cerveau. L’objectif de cette étude a donc été d’étudier les mécanismes moléculaires sous-jacents à un rôle potentiel de Scrib1 dans la formation et le maintien des synapses. Dans un premier temps, on a décrit l’importance d’interactions dépendantes des domaines PDZ sur le trafic des récepteurs glutamatergiques ainsi que sur la voie de signalisation de plasticité synaptique sous-jacente à la mémoire spatiale. Dans un second temps, nous avons évalué les conséquences fonctionnelles d’une mutation de Scrib1 récemment identifiée chez un patient humain atteint des troubles du spectre autistique (TSA) dans la morphologie et fonction des neurones. On a démontré que Scrib1 régule l’arborisation dendritique ainsi que la formation et le maintien fonctionnel des épines dendritiques via un mécanisme dépendent du cytosquelette d’actine. Le dérèglement de ces mécanismes pourrait être à l’origine du phénotype TSA. L’ensemble de ce travail met en évidence que Scrib1, protéine d’échafaudage clé dans le développement et la fonction du cerveau, joue une multitude de rôle du niveau subcellulaire au niveau cognitif. / The brain is made up of billions of nerve cells, or neurons. Neurons communicate with each other through functionally distinct structures - the axon and the dendrite - which are able to release and receive an electrical or chemical signal from a pre- to a post-synaptic compartment, respectively. We focused our study on hippocampal neurons synapses, which ultimately underlie high-order brain functions, such as learning and memory. In particular, we studied the development and maintenance of dendritic spines, whose changes in morphology are intimately correlated with synaptic plasticity, or the ability to respond to synaptic activity. Dendritic spines originate from motile dendritic filopodia, which mature into spines following axonal contact. The filopodia-to-spine transition involves a plethora of molecular actors, including glutamate receptors, scaffold proteins and the actin cytoskeleton, able to receive, transmit and integrate the pre-synaptic signal. The spatial and temporal coordination of all these molecular components throughout the formation and maturation of a synapse remains, however, unclear. Scribble1 (Scrib1) is planar cell polarity protein (PCP) classically implicated in the homeostasis of epithelial tissues and tumour growth. In the mammalian brain, Scrib1 is a critical scaffold protein in brain development and function. The main goal of this work was, therefore, to investigate the molecular mechanisms underlying Scrib1 role in synapse formation and maintenance. In a first part, we depict the importance of Scrib1 PDZ-dependent interactions on glutamate receptors trafficking as well as bidirectional plasticity signalling pathway underying spatial memory. In a second part, we focus on the functional consequences of a recently identified autism spectrum disorder (ASD) mutation of Scrib1 on neuronal morpholgy and function. We demonstrated that Scrib1 regulates dendritic arborization as well as spine formation and functional maintenance via an actin-dependent mechanism, whose disruption might underlie the ASD phenotype. Taken altogether, this thesis highlights the PCP protein Scrib1 as key scaffold protein in brain development and function, playing a plethora of roles from the subcelular to the cognitive level.