Dissertations / Theses on the topic 'Synapse activity'

Les astrocytes périsynaptiques participent activement, au côté des neurones, dans le traitement de l’information cérébrale. Une propriété essentielle des astrocytes est d’exprimer un niveau élevé de protéines appelées connexines (Cxs), et formant les sous-unités des jonctions communicantes. Étonnamment, bien qu’il ait été suggéré très tôt que la Cx30 astrocytaire soit impliquée dans des processus cognitifs, son rôle exact dans la neurophysiologie demeure cependant encore mal connu. Nous avons récemment révélé que la Cx30, via une fonction non-canal inédite, contrôle la force et la plasticité de la transmission synaptique glutamatergique de l’hippocampe en régulant les niveaux synaptiques de glutamate par le biais du transport astrocytaire du glutamate. Cependant, les mécanismes moléculaire et cellulaire impliqués dans ce contrôle, ainsi que sa régulation dynamique par l’activité neuronale et son impact in vivo dans un contexte physiologique restaient inconnus. Dans le cadre de cette problématique, j’ai démontré durant ma thèse que: 1) La Cx30 induit la maturation morphologique des astrocytes de l’hippocampe par l’intermédiaire de la modulation d’une voie de signalisation dépendante de la laminine et régulant la polarisation cellulaire ; 2) l’expression de la Cx30, sa localisation perisynaptique, ainsi que ses fonctions sont modulées par l’activité neuronale ; 3) Le contrôle de la couverture astrocytaire des synapses du noyau supraoptique de l’hypothalamus par la Cx30 fixe les niveaux plasmatiques de base de la neurohormone ocytocine et ainsi favorise la mise en place de comportements sociaux adaptés. Dans l’ensemble, ces résultats éclairent les régulations des Cxs astrocytaires par l’activité neuronale et leur rôle dans le développement postnatal des réseaux neurogliaux, ainsi que dans le contrôle des interactions structurelles astrocytes-synapses à l’origine de processus comportementaux
Perisynaptic astrocytes are active partners of neurons in cerebral information processing. A key property of astrocytes is to express high levels of the gap junction forming proteins, the connexins (Cxs). Strikingly, astroglial Cx30 was suggested early on to be involved in cognitive processes; however, its specific role in neurophysiology has yet been unexplored. We recently reveal that Cx30, through an unconventional non-channel function, controls hippocampal glutamatergic synaptic strength and plasticity by directly setting synaptic glutamate levels through astroglial glutamate clearance. Yet the cellular and molecular mechanisms involved in such control, its dynamic regulation by activity and its impact in vivo in a physiological context were unknown. To answer these questions, I demonstrated during my PhD that: 1) Cx30 drives the morphological maturation of hippocampal astrocytes via the modulation of a laminin signaling pathway regulating cell polarization; 2) Cx30 expression, perisynaptic localization and functions are modulated by neuronal activity; 3) Cx30-mediated control of astrocyte synapse coverage in the supraoptic nucleus of the hypothalamus sets basal plasmatic level of the neurohormone oxytocin and hence promotes appropriate oxytocin-based social abilities. Taken together, these data shed new light on astroglial Cxs activity-dependent regulations and roles in the postnatal development of neuroglial networks, as well as in astrocyte-synapse structural interactions mediating behavioral processes

Neuronal activity and subsequent calcium influx activates a signaling cascade that causes transcription factors in the nucleus to rapidly induce an early-response program of gene expression. This early-response program is composed of transcriptional regulators that in turn induce transcription of late-response genes, which are enriched for regulators of synaptic development and plasticity that act locally at the synapse.

The nervous system is a dynamic structure. Both during development and in the adult, synapses display activity-dependent plasticity which can modify their structure and function. In the neonate, activity influences the stability of functional connections between the muscle and nerve. In adults, the process of neurotransmitter release and the structure of the postsynaptic muscle can also be altered by external stimuli such as exercise. It is important to understand this plasticity of the neuromuscular system, the ways in which it can be modified, and its relationship to the maintenance or degeneration of synapses. After injury, peripheral nerve undergoes Wallerian Degeneration, during which the connections between axons and muscle fibres are lost, followed by the fragmentation of the nerve itself. The primary goal of this thesis was to determine whether activity modulates this process; that is, whether enhancing or reducing neuromuscular activity creates a susceptibility to degeneration or alternatively provides any protection against it. Developing greater understanding of this process is essential in relation to neurodegenerative disorders in which the benefits of activity, in the form of exercise, are controversial. Using Wlds mice, in which synaptic degeneration occurs approximately ten times more slowly than normal after nerve injury, I investigated the influence of both decreased (tetrodotoxin induced paralysis) and increased (voluntary wheel running) activity in vivo on this process. Paralysis prior to axotomy resulted in a significant increase in the rate of synapse degeneration. Using a novel method of repeatedly visualising degenerating synapses and axons in vivo I also established that this effect was specific to the synapse, as it did not affect the degeneration of axons. In contrast, voluntary wheel running had no effect on the rate of either axonal or neuromuscular synapse degeneration, but induced a slight modification of neuromuscular transmission. To provide a more stringent test I developed a novel assay based on overnight, ex vivo incubation of nerve-muscle preparations at 32°C. I first demonstrated that this procedure separates the different degeneration time courses for neuromuscular synapse degeneration in wild-type and Wlds preparations. I then extended the study to investigate further ways of modulating synaptic degeneration. First, I tested the effects of electrical stimulation. Intermittent high frequency (100Hz) stimulation reduced the level of protection. Finally, I tested the effects of NAMPT enzymatic inhibitor FK866 on synaptic degeneration. Interestingly, the synaptic protection observed in Wlds muscles was enhanced in the presence of FK866. The results of my findings are relevant to understanding the plasticity of synapses and its relationship to degeneration. Together, these studies highlight the potential of genetic and epigenetic factors, including activity, to regulate neuromuscular synapse degeneration. My study also provides proof of concept for a novel organotypic culture system in which to identify pharmacological modulators of synaptic degeneration that could form part of a second-line screen for neuroprotective compounds or phenotypes. My findings may be viewed in the wide context of neurodegenerative disease, since synaptic use or disuse is widely thought to influence susceptibility, onset and progression in such disorders.

Although autism spectrum disorders (ASDs) have long been known to have a strong heritability, the genetic basis of these disorders has remained largely elusive. Hundreds of genes have been linked to ASDs, but most of them only contribute a small increase in risk. In 2009, a genome-wide association study identified Semaphorin 5A (SEMA5A) as a novel autism susceptibility gene. Sema5A is a member of the Semaphorin family consisting of secreted and membrane-associated proteins characterized by the Sema domain. Although initially identified as axon guidance cues, Semaphorins have been found to play numerous key roles in the development and function of the nervous system. Here, I provide evidence that Sema5A, along with Sema5B, regulates dendritic morphology and excitatory synaptic elimination in hippocampal neurons. The overexpression of Sema5A/Sema5B negatively impacted dendrite complexity and reduced excitatory synapse density without affecting inhibitory synapses, in contrast the knockdown of Sema5A/Sema5B increased excitatory synapse density. I also investigated the relationship between Sema5A/Sema5B and activity-dependent plasticity including long-term potentiation (LTP) and long-term depression (LTD), which are cellular models of learning and memory. It was demonstrated that the overexpression of Sema5A/Sema5B attenuated the LTP-mediated increase of synapse density, whereas the knockdown of Sema5A/Sema5B blocked the LTD-mediated decrease of synapse density. Furthermore, soluble Sema5A treatment altered the surface expression of the AMPA receptor subunit GluA1 with total level of GluA1 unchanged. Finally, I examined the signaling mechanisms of Sema5A-mediated synapse elimination and plasticity. I found that in vitro Sema5A signalled through two members (Plexin A1 and Plexin A2) of the Plexin family, which are known as the neuronal receptors for the Semaphorin family. Moreover, TAG-1, a cell adhesion molecule also known as Contactin-2, was necessary for the function of Sema5A and Sema5B. Lastly I found that ALLN, an inhibitor of protease calpain, significantly rescued Sema5A-mediated synapse elimination, suggesting that calpain was downstream of Sema5A signaling in hippocampal neurons. Thus, my data revealed a new role for class 5 Semaphorins in synapse density and plasticity, and may therefore provide insights into the critical roles of Sema5A in the general mechanisms of circuit formation and the specific etiology of ASDs.
Medicine, Faculty of
Graduate

Synaptic plasticity, the ability of synapses to change in strength, underlies complex brain functions such as learning and memory, yet little is known about the precise molecular mechanisms and downstream signaling pathways involved. The major goal of my doctoral thesis was to understand these molecular mechanisms and cellular processes underlying synaptic plasticity using the Drosophilalarval neuromuscular junction (NMJ) as a model system. My work centered on a signaling pathway, the Wg/Wnt signaling pathway, which was found to be crucial for activity-driven synapse formation. The Wg/Wnt family of secreted proteins, besides its well-characterized roles in embryonic patterning, cell growth and cancer, is beginning to be recognized as a pivotal player during synaptic differentiation and plasticity in the brain. At the DrosophilaNMJ, the Wnt-1 homolog Wingless (Wg) is secreted from presynaptic terminals and binds to Frizzled-2 (DFz2) receptors in the postsynaptic muscle. Perturbations in Wg signaling lead to poorly differentiated NMJs, containing synaptic sites that lack both neurotransmitter release sites and postsynaptic structures. In collaboration with other members of the Budnik lab, I set out to unravel the mechanisms by which Wg regulates synapse differentiation. We identified a novel transduction pathway that provides communication between the postsynaptic membrane and the nucleus, and which is responsible for proper synapse development. In this novel Frizzled Nuclear Import (FNI) pathway, the DFz2 receptor is internalized and transported towards the nucleus. The C-terminus of DFz2 is subsequently cleaved and imported into the postsynaptic nucleus for potential transcriptional regulation of synapse development (Mathews, Ataman, et al. Science (2005) 310:1344). My studies also centered on the genetic analysis of Glutamate Receptor (GluR) Interacting Protein (dGRIP), which in mammals has been suggested to regulate the localization of GluRs and more recently, synapse development. I generated mutations in the gene, transgenic strains carrying a dGRIP-RNAi and fluorescently tagged dGRIP, and antibodies against the protein. Remarkably, I found dgrip mutants had synaptic phenotypes that closely resembled those in mutations altering the FNI pathway. Through the genetic analysis of dgrip and components of the FNI pathway, immunoprecipitation studies, electron microscopy, in vivotrafficking assays, time-lapse imaging, and yeast two-hybrid assays, I demonstrated that dGRIP had a hitherto unknown role as an essential component of the FNI pathway. dGRIP was found in trafficking vesicles that contain internalized DFz2. Further, DFz2 and dGRIP likely interact directly. Through the use of pulse chase experiments I found that dGRIP is required for the transport of DFz2 from the synapse to the nucleus. These studies thus provided a molecular mechanism by which the Wnt receptor, DFz2, is trafficked from the postsynaptic membrane to the nucleus during synapse development and implicated dGRIP as an essential component of the FNI pathway (Ataman et al. PNAS (2006) 103:7841). In the final part of my dissertation, I concentrated on understanding the mechanisms by which neuronal activity regulates synapse formation, and the role of the Wnt pathway in this process. I found that acute changes in patterned activity lead to rapid modifications in synaptic structure and function, resulting in the formation of undifferentiated synaptic sites and to the potentiation of spontaneous neurotransmitter release. I also found that these rapid modifications required a bidirectional Wg transduction pathway. Evoked activity induced Wg release from synaptic sites, which stimulated both the postsynaptic FNI pathway, as well as an alternative presynaptic Wg pathway involving GSK-3ß/Shaggy. I suggest that the concurrent activation of these alternative pathways by the same ligand is employed as a mechanism for the simultaneous and coordinated assembly of the pre- and postsynaptic apparatus during activity-dependent synapse remodeling (Ataman et al. Neuron (2008) in press). In summary, my thesis work identified and characterized a previously unrecognized synaptic Wg/Wnt transduction pathway. Further, it established a mechanistic link between activity-dependent synaptic plasticity and bidirectional Wg/Wnt signaling. These findings provide novel mechanistic insight into synaptic plasticity.

La maladie de Huntington (MH) est une maladie neurodégénérative causée par la mutation de la Huntingtine (HTT). La réduction de l'expression de la HTT mutante est une piste thérapeutique évidente en cours d’exploration chez les patients. Le ciblage de la HTT mutante s’accompagne cependant le plus souvent d’une réduction concomitante de la HTT non mutée. Les conséquences de la perte de cette protéine sur la santé des neurones restent mal connues.Mon travail de thèse traite cette question en utilisant des modèles in vitro de réseaux neuronaux humains différenciés à partir de cellules souches induites à la pluripotence. Mes travaux démontrent que la perte de HTT induit des anomalies de développement et d’homéostasie de ces réseaux. Mes résultats suggèrent que les thérapies ciblant indifféremment la HTT mutante et non mutante pourraient compromettre la santé des circuits neuronaux ciblés
Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the Huntingtin gene (HTT). Reducing the expression of mutant HTT is an obvious therapeutic approach explored in patients. However, targeting mutant HTT often leads to a simultaneous reduction in non-mutant HTT. The consequences of losing this protein on neuronal health remain poorly understood.My doctoral work addresses this question using in vitro models of human neuronal networks differentiated from induced pluripotent stem cells. My research demonstrates that HTT loss induces developmental and homeostatic abnormalities in these networks. My results suggest that therapies targeting both mutant and non-mutant HTT indiscriminately could compromise the health of targeted neuronal circuits

[Truncated abstract] One of the problems in researching tinnitus is that it has often been assumed that the physiological mechanisms underlying the tinnitus percept cannot be objectively measured. Nonetheless, it is generally accepted that the percept results from altered spontaneous neural activity at some site along the auditory pathway, although it is still debated whether it is produced by: synchronisation of activity of adjacent neurones; a change in the temporal pattern of activity of individual neurones; or an increase in the spontaneous firing rate per se. Similarly, it is possible that the recently coined “auditory neuropathy” is produced by under-firing of the primary afferent synapse, although several other mechanisms can also produce the symptoms described by this disorder (normal cochlear mechanical function but absent, or abnormal, synchronous neural firing arising from the cochlea and auditory brainstem, known as the auditory brainstem response, or ABR). Despite an absent ABR, some subjects can detect pure tones at near-normal levels, although their ability to integrate complex sounds, such as speech, is severely degraded in comparison with the pure-tone audiogram. The aim of the following study was to investigate the normal mechanisms underlying neural firing at the primary afferent synapse, and its regulation, to determine the possible mechanisms underlying over-firing (tinnitus) or under-firing (auditory neuropathy) of primary afferent neurones.

Le striatum a pour rôle de sélectionner et d'intégrer les informations provenant du cortex et ainsi construire et transmettre une réponse adaptée aux stimuli environnementaux. Nous avons caractérisé les propriétés électrophysiologiques des différents neurones du striatum (neurones de sortie, NETM, et interneurones) dans des conditions normales, et lors d'une déplétion de dopamine striatale. Grâce à un modèle de tranche de cerveau de rat dans laquelle les afférences cortico-striatales sont conservées intactes, nous avons mis en évidence une plasticité synaptique bidirectionnelle dans les NETM ainsi qu'une homéostasie puissante au niveau des synapses cortico-striatales. Nous avons ensuite observé que, outre les NETM, le cortex contacte également les interneurones striataux, avec une séquence d'activation particulière et qu'il existe une spécificité cellulaire de la " spike-timing dependent plasticity " (STDP) dans le striatum. Enfin, nous avons mis en évidence que, au niveau des NETM, des signaux sous-liminaires, en coïncidence avec une activité corticale, sont capables d'induire des phénomènes de plasticité synaptique à long-terme.

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders.

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders.

In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release -aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein (Prnp0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABAA-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP S still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD at immature MF-CA3 synapses have been investigated in detail in the second paper. STD-plasticity is a Hebbian form of learning which consists in bi-directional modifications of synaptic strength according to the temporal order of pre and postsynaptic spiking. Interestingly, we found that at immature mossy fibers (MF)-CA3 synapses, STD-LTD occurs regardless of the temporal order of stimulation (pre versus post or viceversa). However, as already mentioned, while STD-LTD induced by positive pairing (pre before post) could be shifted into STD-LTP after blocking presynaptic GluK1 receptors, STD-LTD induced by negative pairing (post before pre) relied on the activation of CB1 receptors. At P3 but not at P21, endocannabinoids released by the postsynaptic cell during spiking-induced membrane depolarization retrogradely activated CB1 receptors, probably expressed on MF terminals and persistently depressed GABA release in the rat hippocampus. Thus, bath application of selective CB1 receptor antagonists prevented STD-LTD. Pharmacological tools allow identifying anandamide as the endogenous ligand responsible of activity-dependent depressant effect. To further assess whether STD-LTD is dependent on the activation of CB1 receptors, similar experiments were performed on WT-littermates and CB1-KO mice. While in WT mice the pairing protocol produced a persistent depression of MF-GPSCs as in rats, in CB1-KO mice failed to induce LTD. Consistent with these data, in situ hybridization experiments revealed detectable levels of CB1 mRNA in the granule cell layer of P3 but not of P21mice. These experiments strongly suggest that at immature MF-CA3 synapses STD-LTD is mediated by CB1 receptors, probably transiently expressed, during a critical time window, on MF terminals. In the third paper, I studied synaptic transmission and activity dependent synaptic plasticity at immature MF-CA3 synapses in mice devoid of the prion protein (Prnp0/0). The prion protein (PrPC) is a conserved glycoprotein widely expressed in the brain and involved in several neuronal processes including neurotransmission. If converted to a conformationally altered form, PrPSc can cause neurodegenerative diseases, such as Creutzfeldt-Jakob disease in humans. Previous studies aimed at characterizing Prnp0/0 mice have revealed only mild behavioral changes, including an impaired spatial learning, accompanied by electrophysiological and biochemical alterations. Interestingly, PrPC is developmentally regulated and in the hippocampus its expression parallels the maturation of MF. Here, we tested the hypothesis that at immature (P3-P7) MF-CA3 synapses, PrPC interferes with synaptic plasticity processes. To this aim, the rising phase of Giant Depolarizing Potentials (GDPs), a hallmark of developmental networks, was used to stimulate granule cells in the dentate gyrus in such a way that GDPs were coincident with afferent inputs. In WT animals, the pairing procedure induced a persistent increase in amplitude of MF-GPSCs. In contrast, in Prnp0/0 mice, the same protocol produced a long-term depression (LTD). LTP was postsynaptic in origin and required the activation of cAMP-dependent PKA signaling while LTD was presynaptic and was reliant on G protein-coupled GluK1 receptor and protein lipase C downstream to G protein activation. In addition, at emerging CA3-CA1 synapses of PrPC-deficient mice, stimulation of Schaffer collateral failed to induce LTP, known to be PKA-dependent. Finally, we also found that LTD in Prnp0/0 mice was mediated by GluK1 receptors, since UBP 302 blocked its induction. These data suggest that in the immature hippocampus PrPC controls the direction of synaptic plasticity.

Rhythmic activity is a ubiquitous feature of sympathetic nerve discharge and has been postulated to be involved in sympathetic reflex control, coordinating the responses of anatomically separated sympathetic nerves and enhancing the responsiveness of the viscera to sympathetic activation. Several studies indicate that some sympathetic rhythms are generated in the spinal cord. This activity has primarily been studied in vivo or in isolated nervous system preparations. Whilst these techniques are useful for elucidating the functional consequences of such activity, a detailed description of the underlying rhythmogenic networks has not been . forthcoming. Therefore, a neonatal (10-12 day old) rat transverse spinal cord slice preparation was developed to study rhythmic network activity in the sympathetic intermediolateral cell column (IML). This thesis presents data which validate the slice preparation as a useful tool for investigating oscillations in the IML. Extracellular recordings were made from the IML, and spontaneous oscillations at 7.5-22 Hz were observed in 29% of slices. The power of these oscillations was enhanced by 5~HT and agonists of5-HT2A and 5-HT2C receptors without affecting the frequency. Furthermore, the same drugs induced similar oscillations in previously nonoscillating slices. Occasionally, action potentials could be observed occurring in phase with the oscillation. The oscillations were abolished by TTX and gap junction blockers, and were partially sensitive to blockade ofGABAA receptors. To enhance understanding of the cellular mechanisms underlying IML oscillations and their modulation by 5-HT, patch clamp recordings ofIML neurones were made.SPNs in the IML were depolarised bY5-HT28 and 5-HT2C (but not 5-HT2A) receptor activation, whereas IML intemeurones were hyperpolarised by 5-HT and the 5-HT1AI5A17 agonist 8-0H-DPAT and depolarised by agonists of5-HT2C receptors. 5-HT also modulated synaptic inputs to both types of neurone. More than 50% of SPNs displayed ongoing gap junction-mediated potentials ('spikelets') which were increased in frequency and amplitude by 5-HT2 receptor agonists. The effects of gap junctions on the firing behaviour of SPNs were significant and might explain the effects of gap junction blockade on IML oscillations. It was concluded that rhythmic IML activity depends on transmission of action potentials between SPNs via gap junctions, and is modulated by GABAergic .inputs. IML oscillations may represent a novel sympathetic rhythm, or may be analogous to the 10Hz rhythm observed in intact animals, or may represent a spinal substrate for. synchronisation ofSPN activity in response to supraspinal activity.

I- Les astrocytes contribuent à la neurotransmission par une variété de mécanismes, allant de l'isolation de la synapse à la signalisation dynamique. Ces cellules ont une signalisation dynamique qui leur permet à la fois de détecter l'activité neuronale grâce à des canaux ioniques, des récepteurs ou des transporteurs aux différents neurotransmetteurs, et de répondre de manière appropriée par une signalisation calcique élaborée, par une plasticité morphologique ou bien par le relargage de nombreuses substances neuroactives. Cependant la nature, la plasticité, et l'impact des courants induits par l'activité neuronale sur la plasticité synaptique à court-terme est encore élusive dans les astrocytes de l'hippocampe. Nous avons montré qu'une stimulation unique des collatérales de Schaeffer dans des tranches d'hippocampe induit dans les astrocytes de stratum radiatum un courant entrant, complexe et prolongé, lui-même synchronisé à la transmission synaptique rapide des cellules pyramidales de la région CAl. Ce courant est constitué de trois composantes: un courant potassique lent, sous-tendu par les canaux Kir4. 1, un courant de transporteur au glutamate transitoire et un courant résiduel lent, partiellement sous-tendu par des transporteurs au GABA et d'autres canaux potassiques que le Kir4. 1. Tous les courants présentent une plasticité à court-termeactivité dépendante, cependant seul le courant astrocytaire de transporteur au glutamate présente une plasticité de type neuronale. Étant donné que 80% du courant astrocytaire évoqué synaptiquement est sous-tendu par les canaux potassiques de type Kir4. 1, nous avons investigué son effet sur la plasticité synaptique à court-terme de l'hippocampe, en combinant l'électrophysiologie à la modélisation mathématique. En utilisant la souris invalidée pour le gène Kir 4. 1 sélectivement dans les cellules gliales, nous avons trouvé que les canaux Kir4. 1 diminuent les réponses synaptiques aux stimulations répétitives ainsi que la potentialisation post-tétanique. Afin de d'identifier la voie de signalisation et la dynamique du cycle potassique entre les astrocytes et les neurones pendant l'activité neuronale, nous avons construit un modèle mathématique de trois compartiments qui quantifie les échanges de potassium entre les neurones, les cellules gliales et l'espace extracellulaire. Nous avons trouvé que les canaux Kir4. 1 sont responsables de la lente capture de potassium pendant l'activité neuronale, ce qui régule l'excitabilité neuronale dans des conditions tant physiologiques que pathologiques. Finalement, nous avons investigué la signalisation calcique activité-dépendante dans les astrocytes des régions CA1 et CA3 de l'hippocampe ainsi que son impact sur la transmission et la plasticité synaptique à court terme Nous avons trouvé que bien que de tels signaux régulent l'activité synaptique des synapses des collatérales de Schaeffer dans la région CA1, son effet n'est que modeste sur la synapse de la fibre moussue dans la région CA3. Cela est probablement dû à la différence de couverture astrocytaire des synapses dans ces deux régions de l'hippocampe, comme le suggère les études de microscopie électronique ainsi que le patron différentiel d'expression de la connexine 30, une protéine astrocytaire qui détermine la localisation synaptique des prolongements astrocytaires. De façon remarquable, la connexine 30 dans les astrocytes régule fortement la plasticité à court terme des synapses des fibres moussues dans la région CA3 de l'hippocampe, révélant ainsi le rôle important des astrocytes dans la régulation synaptique des niveaux de glutamate.

The main purpose of the study was to answer how body-positive movement on Instagram is portrayed by female influences. The aim was also to understand how young women relate to body ideals and how they challenge the normative female body. To answer these questions, the study has been inspired by a narrative analysis method. My intention for this essay was to draw attention to the women's story about their experiences. To understand and be able to carry out an analysis of the empiric, previous research was used about social media and Instagram, the term influences, body ideal on social media, what impact social media can have on women's self-image, what body positivity means and fat phobia in society. The starting point for the literature search has been to map the research field and to strengthen my research question. To analyze the extracted empire, different theories were chosen. The theories I considered most suitable were feminist theory, the dramaturgical perspective, the mirror self and the social comparison theory. The result was divided into 7 themes which were: imagery of body positivity, feminism and body activism, influencers´ responsibilities to their followers, society's view of fat people, Instagram vs. reality, the concept of taking care of oneself as well as sisterhood and role models. The result shows that the female influences, with their body activism, wants to challenge the normative assumptions about how a woman should look and be. They want to create a platform to help in the normalization process of women with a larger body shape, as well as highlight the complications you as a woman with a non-normative body can encounter in society. The influencers hope that with their body activism they can strengthen the self-esteem of fat women.
Studien hade som huvudsyfte att besvara hur kroppspositiva rörelsen på Instagram framställs av kvinnliga influenser. Syftet var även att förstå hur unga kvinnor förhåller sig till kroppsideal och hur de utmanar den normativa kvinnokroppen. För att besvara studiens frågeställningar har studien inspirerats av en narrativ analysmetod. Min avsikt för arbetet var att rikta uppmärksamheten på kvinnornas berättelse om deras upplevelser och erfarenheter. För att förstå och kunna föra en analys kring empirin användes tidigare forskning av sociala media och Instagram, termen influenser, kroppsideal på sociala medier, vilken påverkan sociala medier kan ha på kvinnors självbild, vad kroppspositivitet innebär samt fettfobi i samhället. Utgångspunkten för litteratursökningen har varit att kartlägga forskningsfältet samt att styrka min forskningsfråga. För att analysera den utvunna empirin valdes olika teorier. De teorier jag ansåg vara bäst lämpade var feministisk teori, det dramaturgiska perspektivet, spegeljaget samt social jämförelseteorin. Resultatet delades upp i 7 teman som var: bildframställning av kroppspositivitet, feminism och kroppsaktivism, samhällets syn på tjocka människor, influensers ansvar gentemot sina följare, Instagram vs. reality, begreppet ta hand om sig själv samt systerskap och förebilder. I resultatet framkommer att de kvinnliga influenserna vill med sin kroppsaktivism utmana de normativa antagandena om hur en kvinna ska se ut och vara. De vill skapa en plattform för att hjälpa till i normaliseringsprocessen av kvinnor med större kroppsform, samt belysa vilka komplikationer man som kvinna med en icke normativ kropp kan stöta på i samhället. Influenserna hoppas att med sin kroppsaktivism kunna styrka tjocka kvinnors självkänsla.

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique
Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity

Pendant le développement, les projections axonales à longue distance se ramifient pour se connecter à leurs cibles. L’établissement et le remodelage de ces connexions est notamment régulé par l’activité neuronale. L’adaptation de la morphologie de l’axone nécessite alors des quantités importantes de matériel sécrétoire et de facteur trophiques comme le BDNF (brain derived neurotrophic factor). Ce matériel est transporté dans des vésicules le long de l’axone depuis le corps cellulaire où il est synthétisé, vers les sites actifs à l’extrémité de l’axone. Si le relargage de vésicules sécrétoires à la synapse est bien étudié, les mécanismes régulant le transport axonal par l’activité sont encore méconnus.Dans ce travail de thèse, nous avons dans un premier temps développé des outils permettant d’étudier les dynamiques intracellulaires dans des réseaux neuronaux. Nous avons ainsi développé une chambre microfluidique permettant de reconstruire in vitro des réseaux neuronaux physiologiques et compatibles avec de la vidéomicroscopie à haute résolution. Nous avons caractérisé l’établissement et la maturation du réseau et validé l’intérêt de ce dispositif microfluidique dans le contexte de la maladie de Huntington. Nous avons ensuite étudié l’évolution des dynamiques intracellulaires avec la maturation du réseau. Nous avons notamment observé une augmentation du transport axonal de vésicules sécrétoires en fonction de l'état de maturation du réseau neuronal. Ces premières observations ont renforcé l’hypothèse d’une régulation directe du transport axonal de vésicules sécrétoires par l’activité neuronale au cours du développement du réseau.Nous avons ainsi fait évoluer la plateforme microfluidique par l’ajout d’un réseau d’électrodes (MEA) qui permet d'étudier les dynamiques intracellulaires tout en contrôlant l’activité neuronale. A l’aide de ce système, nous avons identifié un groupe de vésicules sécrétoires ancré le long de l’axone et recruté en réponse à une haute activité neuronale en direction des sites présynaptiques actifs. Nous avons alors identifié les acteurs impliqués dans ce mécanisme dépendant de l’activité. Nous avons montré que la myosine Va permettait l’attachement des vésicules le long de l’axone dans des structures d’actine dynamique. L’activité neuronale induit une augmentation de calcium le long de l’axone, via l’activation des canaux calciques dépendant du voltage, qui régule la myosine Va et entraine le recrutement des vésicules stockées dans l’axone sur les microtubules. Une fois les acteurs identifiés, nous avons pu mettre en évidence le rôle de ce mécanisme dépendant de l’activité dans la formation de branches axonales pendant le développement. Enfin, nous avons confirmé l’existence de ce groupe de vésicules dépendant de l’activité et résidant dans l’axone in vivo grâce à la mise au point d'un système d’étude du transport axonal sur tranches aigües de cerveau en microscopie biphotonique.L’ensemble de ce travail propose de nouveaux outils in vitro et in vivo pour comprendre les régulations des dynamiques intracellulaires dans des réseaux neuronaux physiologiques. Grâce à ces outils, nous avons identifié un mécanisme de régulation local qui permet l'adressage rapide de facteurs trophiques vers les branches en développement en réponse à l’activité neuronale
During postnatal development, long-distance axonal projections form branches to connect with their targets. Establishment and remodeling of these projections are tightly regulated by neuronal activity and require a large amount of secretory material and trophic factors, such as brain derived neurotrophic factor (BDNF). Axonal transport is responsible for addressing trophic factors packed into vesicles to high demand sites where mechanisms of secretion are well-known. However, mechanisms controlling the preferential targeting of axonal vesicles to active sites in response to neuronal activity are unknown.In this work, we first developed tools to study intracellular dynamics in neuronal networks. We thus developed a microfluidic chamber to reconstruct physiologically-relevant networks in vitro which is compatible with high resolution videomicroscopy. We characterized the formation and maturation of reconstructed networks and we validated the relevance of the microfluidic platform in the context of Huntington’s disease. We then studied the evolution of intracellular dynamics with the maturation of reconstructed neuronal networks in microfluidic chambers. We observed an increase of anterograde axonal transport of secretory vesicles during maturation. These first results lead us to think that neuronal activity could regulate axonal transport of secretory vesicles over maturation of the network.Therefore, we improved the in vitro microfluidic system with a designed microelectrode array (MEA) substrate allowing us to record intracellular dynamics while controlling neuronal activity. Using this system, we identified an axon-resident reserve pool of secretory vesicles recruited upon neuronal activity to rapidly distribute secretory materials to presynaptic sites. We identified the activity-dependent mechanism of recruitment of this axonal pool of vesicles along the axon shaft. We showed that Myosin Va ensures the tethering of vesicles in the axon shaft in axonal actin structures. Specifically, neuronal activity induces a calcium increase after activation of Voltage Gated Calcium Channels along the axon, which regulates Myosin Va and triggers the recruitment of tethered vesicles on microtubules. We then showed the involvement of this activity-dependent pool for axon branches formation during axon development. By developing 2-photon live microscopy of axonal transport in acute slices, we finally confirmed that a pool of axon-resident static vesicles is recruited by neuronal activity in vivo with a similar kinetic.Altogether, this work provides new in vitro and in vivo tools to study intracellular dynamics in physiological networks. Using these tools, we identified the existence of a local mechanism of axonal transport regulation along the axon shaft, allowing rapid supply of trophic factors to developing branches

Le cerveau antérieur est l’aire cérébrale supportant les fonctions biologiques les plus complexes. Certaines altérations de son développement peuvent entraîner des maladies psychiatriques comme l’autisme ou encore la schizophrénie. Ainsi, les cellules du cerveau, appelées neurones, doivent être correctement positionnés au sein du cerveau et établir des connexions (appelées synapses) avec les autres neurones. Ce travail de thèse vise à mieux comprendre comment le positionnement des neurones et la formation des synapses sont régulés dans le cerveau antérieur. En premier lieu, nous avons étudié l’impact de l’activité neuronale sur le positionnement des différents types de neurones du bulbe olfactif. Dans un second temps, nous avons identifié le gène NeuroD2 comme régulateur majeur de la formation synaptique dans le cortex, l’absence de ce gène dans le cortex provoquant également l’altération du comportement social des souris
The forebrain is the brain area that supports the most complex biological functions. Any alteration during its development can provoke psychiatric disorders such as autism or schizophrenia. The cells composing the brain, called neurons, must be adequately positioned and must establish functional connections (named synapses) with other neurons. This thesis work aims at understanding how neuronal positioning and synapse formation are controlled in the forebrain. In a first instance, we explored the impact of neuronal activity on the positioning of the different subtypes of olfactory bulb neurons. In a second instance, we identified the gene NeuroD2 as a major regulator of synapse formation in the cortex, the absence of this gene leading to social behavior deficits as well

Angelman syndrome (AS) is a genetic disorder characterized by paternal imprinting and maternal deletion of Ube3a. Therefore AS patients have reduced levels of expression of Ube3a in several regions of the brain including the hippocampus and cerebellum. AS patients have motor impairment, mental retardation and absence of speech. Ube3a is an E3 ligase responsible for the ubiquitination of protein leading to the proteasomal degradation and the lack of function as been associated with loss of synaptic plasticity. Although there’s been the identification of several Ube3a substrates with important role in the postsynapse the role of presynaptic Ube3a and the symptoms found in AS patients is still not clear. Ube3a transcription is induced by synaptic activity and glutamate release during early stages of development, indicating that neuronal excitability is important to regulate Ube3a activity. Nonetheless, Ube3a role in excitatory synapse formation and maturation is still not clear. In this work we did a subcellular characterization of Ube3a expression in several regions of Rat and Mouse hippocampal neurons. We observed that Ube3a is expressed at high levels within the nucleus of hippocampal neurons but is also present in the cytoplasm and along the axon. Our results show that Ube3a is highly expressed in the presynaptic compartments of neurons in early stages of development followed by a decrease in the later stages of development. Furthermore, we show that expressing a catalytic inactive form of Ube3a in rat embryonic hippocampal neurons disrupts synapse formation and maturation. Our data suggests that Ube3a catalytic function is necessary for promoting excitatory synapse formation. Collectively, these data contributes to a deeper understanding of the cognitive alterations found in patients with Angelman Syndrome.
Síndrome de Angelman é uma doença genética caracterizada por imprinting paternal e deleção maternal da Ube3a. Deste modo, pacientes com Síndrome de Angelman têm níveis reduzidos de expressão da Ube3a em várias regiões do cérebro, incluindo o hipocampo e o cerebelo. Pacientes com Síndrome de Angelman apresentam dificuldades motoras, retardação mental e ausência de fala. Ube3a é uma E3 ligase responsável pela ubiquitinação de proteínas levando a degradação proteasomal dessas proteínas e a perda de função tem sido associada com perda de plasticidade sináptica. Ainda que tenham sido identificados vários substratos desta proteína com um papel pós sináptico importante o seu papel a nível pré sináptico e a correlação com os sintomas encontrados ainda não é clara. A transcrição da Ube3a é induzida por atividade sináptica e libertação de glutamato durante os primeiros estágios de desenvolvimento indicando que a atividade da Ube3a é importante para regular a excitabilidade neuronal. Apesar disso, o papel desta proteína na formação e maturação das sinapses excitatórias é ainda desconhecido. Neste trabalho realizamos uma caracterização da expressão desta proteína em várias regiões dos neurónios do hipocampo de duas espécies de rato (Mus musculus) e (Rattus norvegicus). Nós observamos que a Ube3a está expressa em elevados níveis nos núcleos dos neurónios em estágio de desenvolvimento iniciais, mas está também expressa no citoplasma e axónios. Os nossos resultados mostram que esta proteína está altamente expressa pré-sinapticamente tendo maior presença em estágios de desenvolvimento inicias seguido de um decréscimo em estágios de desenvolvimento tardios. Além disso, demonstramos que expressar neurónios do hipocampo de Rato com uma proteína cataliticamente inativa perturba a formação e maturação de sinapses. Estes dados indicam que a função catalítica da Ube3a é necessária para promover a formação de sinapses excitatórias. Coletivamente, estes dados podem explicar as alterações cognitivas encontradas em pacientes com Síndrome de Angelman.
Mestrado em Biologia Molecular e Celular

Conductive hearing loss (otitis media) in young children can effect speech and language development. However, little is known about the effects of conductive loss on neural activity in the auditory system. Hypothesis: Conductive hearing loss will change resting activity levels at the inner hair cell synapse, and lead to auditory deprivation of central auditory pathways. A conductive loss was produced by blocking the ear canals in mice. Resting neural activity patterns were quantified in brainstem and midbrain using c-fos immuno-labelling. Experimental subjects were compared to normal hearing controls and subjects with cochlear ablation. Conductive loss subjects showed a trend in reduction in c-fos labelled cells in cochlear nucleus and the central nucleus of inferior colliculus compared to normal controls. Results seen in this study may indicate the influence of conductive hearing loss on the developing auditory brain during early postnatal years when the system is highly plastic.

This thesis describes efforts at the interface of chemistry and neuroscience to design and characterize fluorescent probes capable of tracing neurotransmitters from individual release sites in brain tissue. As part of the Fluorescent False Neurotransmitters (FFNs) program, small organic fluorophores have been developed that undergo uptake into specific presynaptic release sites and synaptic vesicles by utilizing the native protein machinery, which can then be released during neuronal firing. The most advanced generation of FFNs are pH-sensitive, and display an increase in fluorescence when released from the acidic vesicular lumen into the extracellular space, called a “FFN Flash.” In Chapter 2, the utility of the dopamine-selective and pH-sensitive functionality of FFN102 to study the mechanisms that regulate changes in pre-synaptic plasticity, a critical component of neurotransmission was explored. This included using the FFN flash to quantitatively trace dopamine release, changes in the release probability of individual release sites, and changes in vesicular loading that can affect quantal size. The second goal of this thesis research, as detailed in Chapters 3 and 4, sought to expand the substrate scope of the FFN program to neurotransmitter systems other than dopamine. Described in Chapter 3, is the identification of a fluorescent phenylpyridinium, APP+, with excellent labeling for dopamine, norepinephrine, and serotonin neurons, however, the properties of the probe were found to be ill-suited for measuring neurotransmitter release. As a result, it was concluded that this class of compounds was not suitable for generating viable FFN leads. In contrast, Chapter 4 highlights the design, synthesis, and screening towards generating the novel noradrenergic-specific FFN, FFN270. This probe was further tested for application in acute murine brain slices where it labeled noradrenergic neurons, and was demonstrated to release upon stimulation. This chapter also describes the application of this compound in a series of in vivo experiments, where the ability to measure norepinephrine release from individual release sites was demonstrated in a living animal for the first time. This work opens the possibility for many exciting future FFN experiments studying the presynaptic regulation of neurotransmission in vivo.

Le fonctionnement du cortex cérébral nécessite l’action coordonnée de deux des sous-types majeurs de neurones, soient les neurones à projections glutamatergiques et les interneurones GABAergiques. Les interneurones GABAergiques ne constituent que 20 à 30% des cellules corticales par rapport au grand nombre de neurones glutamatergiques. Leur rôle est toutefois prépondérant puisqu’ils modulent fortement la dynamique et la plasticité des réseaux néocorticaux. Il n’est donc pas surprenant que les altérations de développement des circuits GABAergiques soient associées à plusieurs maladies du cerveau, incluant l’épilepsie, le syndrome de Rett et la schizophrénie. La compréhension des mécanismes moléculaires régissant le développement des circuits GABAergiques est une étape essentielle menant vers une meilleure compréhension de la façon dont les anormalités se produisent. Conséquemment, nous nous intéressons au rôle de l’acide polysialique (PSA) dans le développement des synapses GABAergiques. PSA est un homopolymère de chaînons polysialylés en α-2,8, et est exclusivement lié à la molécule d’adhésion aux cellules neuronales (NCAM) dans les cerveaux de mammifères. PSA est impliqué dans plusieurs processus développementaux, y compris la formation et la plasticité des synapses glutamatergiques, mais son rôle dans les réseaux GABAergiques reste à préciser. Les données générées dans le laboratoire du Dr. Di Cristo démontrent que PSA est fortement exprimé post- natalement dans le néocortex des rongeurs, que son abondance diminue au cours du développement, et, faits importants, que son expression dépend de l’activité visuelle i et est inversement corrélée à la maturation des synapses GABAergiques. La présente propose de caractériser les mécanismes moléculaires régulant l’expression de PSA dans le néocortex visuel de la souris. Les enzymes polysialyltransférases ST8SiaII (STX) et ST8SiaIV (PST) sont responsables de la formation de la chaîne de PSA sur NCAM. En contrôlant ainsi la quantité de PSA sur NCAM, ils influenceraient le développement des synapses GABAergiques. Mon projet consiste à déterminer comment l’expression des polysialyltransférases est régulée dans le néocortex visuel des souris durant la période post-natale; ces données sont à la fois inconnues, et cruciales. Nous utilisons un système de cultures organotypiques dont la maturation des synapses GABAergiques est comparable au modèle in vivo. L’analyse de l’expression génique par qPCR a démontré que l’expression des polysialyltransférases diminue au cours du développement; une baisse majeure corrélant avec l’ouverture des yeux chez la souris. Nous avons de plus illustré pour la première fois que l’expression de STX, et non celle de PST, est activité-dépendante, et que ce processus requiert l’activation du récepteur NMDA, une augmentation du niveau de calcium intracellulaire et la protéine kinase C (PKC). Ces données démontrent que STX est l’enzyme régulant préférentiellement le niveau de PSA sur NCAM au cours de la période post-natale dans le cortex visuel des souris. Des données préliminaires d’un second volet de notre investigation suggèrent que l’acétylation des histones et la méthylation de l’ADN pourraient également contribuer à la régulation de la transcription de cette enzyme durant le développement. Plus d’investigations seront toutefois nécessaires afin de confirmer cette hypothèse. En somme, la connaissance des mécanismes par lesquels l’expression des ii polysialyltransférases est modulée est essentielle à la compréhension du processus de maturation des synapses GABAergiques. Ceci permettrait de moduler pharmacologiquement l’expression de ces enzymes; la sur-expression de STX et/ou PST pourrait produire une plus grande quantité de PSA, déstabiliser les synapses GABAergiques, et conséquemment, ré-induire la plasticité cérébrale.
The functioning of the cerebral cortex requires coordinated action of two major neuronal subtypes - the glutamatergic projection neurons and the GABAergic interneurons. GABAergic interneurons represent 20 to 30% of all cortical cells. Even though they are a minor cell population in the cerebral cortex compared to glutamatergic neurons, they are key modulators of network dynamics and plasticity of neocortical circuits. It is therefore not surprising that aberrant development of GABAergic circuits is implicated in many neurodevelopmental disorders including epilepsy, Rett syndrome and schizophrenia. Understanding the molecular mechanisms governing the development of GABAergic inhibitory synapses in neocortex is important towards a better comprehension of how abnormalities in this developmental process can occur. Therefore, we focus specifically on the role of polysialic acid (PSA) in the development of GABAergic synapses. PSA is a α-2,8 polysialylated homopolymer, which is exclusively linked to the Neural Cell Adhesion Molecule (NCAM) in the mammalian brain. It is involved in several developmental processes including formation and plasticity of glutamatergic synapses; however its role in GABAergic circuit formation has not been explored so far. Previously in Dr Di Cristo’s lab, we showed that PSA is strongly expressed post-natally and its expression steadily declines during development in mice neocortex. We also showed that the developmental and activity-dependant regulation of PSA expression is inversely correlated with the maturation of perisomatic GABAergic innervation. Our aim is to characterize the molecular mechanisms regulating PSA expression in mouse iv visual cortex during post-natal development. Two polysialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), are responsible for PSA attachment to NCAM. By controlling the amount of PSA on NCAM, they can influence GABAergic synapses development. The mechanisms regulating STX and PST expression is crucial but remain still unknown. My research project focused on the mechanisms regulating STX and PST transcription in the mouse postnatal cortex. We used an organotypic culture system, which recapitulates many aspects of GABAergic synapse maturation as observed in vivo. Polysialyltransferases transcript levels were measured by qPCR and showed that STX and PST mRNA levels steadily decline during post-natal development in the mouse cortex; the sharpest reduction in the expression of both enzymes correlate with eye opening. We further demonstrate for the first time that STX mRNA levels is activity-dependant, requires the activation of NMDA receptors, an increase in intracellular Calcium levels and is PKC-dependent. Altogether, we show that the regulation of the expression of STX is the main mechanism responsible for PSA expression levels in the cortex around eyes opening. We next investigated whether epigenetic mechanisms regulate STX transcription and preliminary data suggest that histone acetylation and DNA methylation may contribute to STX expression during development. However, further experiments are required to confirm this hypothesis. In summary, understanding the mechanisms modulating STX and PST expression in the neocortex is essential for the comprehension of their precise role in GABAergic synapse maturation. This knowledge could allow us to modulate pharmacologically the expression of these enzymes; in turn overexpression of STX and PST may re-induce PSA expression, thereby destabilizing GABAergic synapses, and ultimately facilitating cortical plasticity in the adult.

Dans le cortex visuel des mammifères, une cellule à panier (BC) qui représente un sous-type majoritaire d’interneurones GABAergiques, innerve une centaine de neurones par une multitude de synapses localisées sur le soma et sur les dendrites proximales de chacune de ses cibles. De plus, ces cellules sont importantes pour la génération des rythmes gammas, qui régulent de nombreuses fonctions cognitives, et pour la régulation de la plasticité corticale. Bien que la fonction des BC au sein des réseaux corticaux est à l'étude, les mécanismes qui contrôlent le développement de leur arborisation complexe ainsi que de leurs nombreux contacts synaptiques n’ont pas été entièrement déterminés. En utilisant les récepteurs allatostatines couplés aux protéines G de la drosophile (AlstR), nous démontrons in vitro que la réduction de l'excitation ainsi que la réduction de la libération des neurotransmetteurs par les BCs corticales individuelles des souris, diminuent le nombre de cellules innervées sans modifier le patron d'innervation périsomatique, durant et après la phase de prolifération des synapses périsomatiques. Inversement, lors de la suppression complète de la libération des neurotransmetteurs par les BCs individuelles avec l’utilisation de la chaîne légère de la toxine tétanus, nous observons des effets contraires selon le stade de développement. Les BCs exprimant TeNT-Lc pendant la phase de prolifération sont caractérisées par des arborisations axonales plus denses et un nombre accru de petits boutons homogènes autour des somas innervés. Toutefois, les cellules transfectées avec TeNT-Lc après la phase de la prolifération forment une innervation périsomatique avec moins de branchements terminaux d’axones et un nombre réduit de boutons avec une taille irrégulière autour des somas innervés. Nos résultats révèlent le rôle spécifique des niveaux de l’activité neuronale et de la neurotransmission dans l'établissement du territoire synaptique des cellules GABAergiques corticaux. Le facteur neurotrophique dérivé du cerveau (BDNF) est un modulateur puissant de la maturation activité-dépendante des synapses GABAergiques. Grâce à l'activation et à la signalisation de son récepteur tyrosine kinase B (TrkB), la liaison de mBDNF module fortement la prolifération des synapses périsomatiques GABAergiques formés par les BCs. Par contre, le rôle du récepteur neurotrophique de faible affinité, p75NTR, dans le développement du territoire synaptique des cellules reste encore inconnu. Dans ce projet, nous démontrons que la suppression de p75NTR au niveau des BCs individuelles in vitro provenant de souris p75NTRlox induit la formation d'une innervation périsomatique exubérante. BDNF est synthétisé sous une forme précurseur, proBDNF, qui est par la suite clivée par des enzymes, y compris la plasmine activée par tPA, pour produire une forme mature de BDNF (m)BDNF. mBDNF et proBDNF se lient avec une forte affinité à TrkB et p75NTR, respectivement. Nos résultats démontrent qu’un traitement des cultures organotypiques avec la forme résistante au clivage de proBDNF (mut-proBDNF) réduit fortement le territoire synaptique des BCs. Les cultures traitées avec le peptide PPACK, qui inactive tPA, ou avec tPA altèrent et favorisent respectivement la maturation de l’innervation synaptique des BCs. Nous démontrons aussi que l’innervation exubérante formée par les BCs p75NTR-/- n’est pas affectée par un traitement avec mut-proBDNF. L’ensemble de ces résultats suggère que l'activation de p75NTR via proBDNF régule négativement le territoire synaptique des BCs corticaux. Nous avons ensuite examiné si mut-proBDNF affecte l’innervation périsomatique formée par les BCs in vivo, chez la souris adulte. Nous avons constaté que les boutons GABAergiques périsomatiques sont significativement diminués dans le cortex infusé avec mut-proBDNF par rapport à l’hémisphère non-infusé ou traité avec de la saline. En outre, la plasticité de la dominance oculaire (OD) est rétablie par ce traitement chez la souris adulte. Enfin, en utilisant des souris qui ne possèdent pas le récepteur p75NTR dans leurs BCs spécifiquement, nous avons démontré que l'activation de p75NTR via proBDNF est nécessaire pour induire la plasticité de la OD chez les souris adultes. L’ensemble de ces résultats démontre un rôle critique de l'activation de p75NTR dans la régulation et le maintien de la connectivité des circuits GABAergiques, qui commencent lors du développement postnatal précoce jusqu’à l'âge adulte. De plus, nous suggérons que l'activation contrôlée de p75NTR pourrait être un outil utile pour restaurer la plasticité dans le cortex adulte.
Cortical GABAergic basket cells (BC) innervate hundreds of postsynaptic targets with synapses clustered around the soma and proximal dendrites. They are important for gamma oscillation generation, which in turn regulate many cognitive functions, and for the regulation of developmental cortical plasticity. Although the function of BC within cortical networks is being explored, the mechanisms that control the development of their extensive arborisation and synaptic contacts have not been entirely resolved. By using the Drosophila allatostatin G-protein-coupled receptors (AlstR), we show that reducing excitation, and thus neurotransmitter release, in mouse cortical single BC in slice cultures decreases the number of innervated cells without changing the pattern of perisomatic innervation, both at the peak and after the proliferation phase of perisomatic synapse formation. Conversely, suppressing neurotransmitter release in single BCs by using the tetanus toxin light-chain can have completely opposite effects depending on the developmental stage. Basket cells expressing TeNT-Lc during the peak of the proliferation were characterized by denser axonal arbors and an increased number of smaller, homogenous boutons around the innervated somatas compared with control cells. However, after the peak of the synapse proliferation, TeNT-Lc transfected BCs formed perisomatic innervation with fewer terminal axon branches and fewer irregular-sized boutons around innervated somatas. Our results reveal a remarkably specific and age-dependent role of neural activity and neurotransmission levels in the establishment of the synaptic territory of cortical GABAergic cells. Brain derived neurotrophic factor (BDNF) has been shown to be a strong modulator of activity-dependent-maturation of GABAergic synapses. Through the activation and signaling of their receptor Tropomyosin-related kinase B (TrkB), mBDNF binding strongly modulates the proliferation of GABAergic perisomatic synapses formed by BCs. Whether the low-affinity neurotrophin-receptor p75NTR also play a role in the development of basket cell synaptic territory is unknown. Here, we show that single-cell deletion of p75NTR in BCs in cortical organotypic cultures from p75NTRlox mice induce the formation of exuberant perisomatic innervations by the mutant basket cells, in a cell-autonomous fashion. BDNF is synthesized as a precursor, proBDNF, which is cleaved by enzymes, including tPA-activated plasmin, to produce mature (m)BDNF. mBDNF and proBDNF bind with high-affinity to TrkB and p75NTR, respectively. Our results show that treating organotypic cultures with cleavage-resistant proBDNF (mut-proBDNF) strongly reduces the synaptic territory of BCs. Treating cultures with the tPA-inactivating peptide PPACK or with tPA impairs and promotes the maturation of BC synaptic innervations, respectively. We further show that the exuberant innervations formed by p75NTR-/- basket cells are not affected by mut-proBDNF treatment. All together, these results suggest that proBDNF-mediated p75NTR activation negatively regulates the synaptic territory of BCs. We next examined if mut-proBDNF affects perisomatic innervation formed by BCs in vivo, in the adult mouse. We found that perisomatic GABAergic boutons are significantly decreased in the cortex infused with mut-proBDNF as compared to non-infused or saline-treated hemispheres. Further, ocular dominance (OD) plasticity is restored by this treatment in adult mice. Finally, we found that proBDNF-mediated activation of p75NTR is necessary to induce OD plasticity in the adult mice, by using mice that lack p75NTR specifically in BCs. All together, these results demonstrate a critical role of p75NTR activation in regulating and maintaining GABAeric circuit connectivity from early postnatal development to adulthood. Further, we suggest that controlled activation of p75NTR could be a useful tool to restore plasticity in adult cortex.