Projections from the Nucleus Accumbens (NAc) to the Ventral Pallidum (VP) play a critical role in motivation and reward. Rewards and reward-associated cues are thought to alter this pathway by suppressing GABA release to the VP, however, the role of the NAc to VP pathway has never been investigated with regard to nicotine self-administration. We hypothesized that increasing GABAergic signaling from the NAc to the VP would decrease the reinforcing effects of nicotine. To increase GABA release, CRE dependent DREADD was expressed in the NAc of male rats. Administration of low dose clozapine activated the DREADD receptor and showed a reduction in responding for nicotine suggesting that activation of the NAc to VP pathway reduced reinforcement. However, a nonspecific effect was observed leading to future investigation of locomotor activity. Immunohistochemistry and microdialysis were used to confirm DREADD expression in the NAc and increased extracellular GABA in the VP.

Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol's effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol’s effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

La proline est un acide aminé unique qui se caractérise par un cycle pyrrolidine qui lui confère des propriétés physiques et chimiques spécifiques comparé aux autres 20 acides aminés acycliques retrouvés de façon majoritaire dans le protéome humain. Cette différence se retrouve également dans les motifs polyproline et est utilisée par la Nature en de nombreuses façons comme le repliement des protéines, les mécanismes régulatoires liés à l’activité de certaines protéases, la formation de structures secondaires, le biomarquage de peptides substrats des oligopeptidases, et le clivage des motifs diproline par les proline-proline endopeptidases. La proline a par conséquent été un centre d’intérêt pour les chimistes médicinaux lors du développement d'agents thérapeutiques. Cette thèse se propose d’étudier plusieurs composés biologiquement actifs dérivés de la proline. La première étude relate les avancements liés au composé SH-BC-893, un azacycle contraint dérivé de FTY720 développé au sein du groupe Hanessian. SH-BC-893 est un agent anticancéreux qui permet l’affamement des cellules cancéreuses au travers de la disruption de l’apport des nutriments cellulaires externes et internes. Par son activation de la protéine phosphatase 2A, il permet d’internaliser les récepteurs transmembranaires d’acides aminés et de glucose et de réguler à la baisse les récepteurs de lipides à faible densité. Son action permet également la perturbation de la dégradation de nutriments internes provenant de la macropinocytose et de l’autophagie en prévenant la fusion entre le lysosome et les endosomes respectifs. Afin d’augmenter l’activité de SH-BC-893, deux séries de composés ont été synthétisés et testés en vue d’une activité double. La première série a eu pour but de cibler HDAC2 suite à une étude de Spiegel au sein de laquelle son inhibition a été rapportée pour FTY720. La seconde a visé à obtenir des composés activant d’autres portions ou d’autres homologues de PP2A via l’insertion de fragments tricycliques suite aux études rapportant l’activation de PP2A par des dérivés de composés neuroleptiques tricycliques du type de la perphénazine. La synthèse et les tests biologiques de ces composés sont également décrits. Le deuxième chapitre décrit la synthèse d’une série de morpholines pontées comportant le motif de l’acide gamma-aminobutyrique (GABA) contraint. GABA est un composant prépondérant de la régulation du système nerveux central au travers de son action inhibitrice sur les neurorécepteurs GABA-A, GABA-B, GABA-C. De nombreux agents thérapeutiques inspirés de GABA ont été développés avec les années, notamment baclofen. Les morpholines pontées rapportées ici possèdent un centre quaternaire stéréocontrôlé pour lequel un ensemble de substituants a été varié, incluant le groupement phényle para-chloré du baclofen ainsi que l’iso-butyle de la Pregabalin, autre composé actif dans le système nerveux central dérivé de GABA. La modélisation du dérivé du baclofen a été réalisée au sein du site actif de GABA-B. L’ensemble des composés ont également été modélisés sous forme d’acides aminés au sein de la base de données LLAMA afin de déterminer leur capacité à occuper l’espace de Lead-likeness et confirmer qu’ils satisfont les conditions de Lipinski. La troisième partie décrit la synthèse d’un nouveau mime de diproline, nommé ProCyp. Le dimère est composé d’un noyau pyrrolidine attaché à une unité cyclopentane à l’aide d’un pont méthylène hydroxylé. Les quatre isomères cyclopentanes trans dérivés de la (L)-proline ont été synthétisés et leur stéréochimie absolue a été déterminée par étude RMN et de cristallographie. Ces composés représentent la première forme de mimes de diproline de type ProCyp et peuvent être intégrés au sein de peptidomimétiques afin de mimer l’aspect structurel des diprolines ainsi que l’intermédiaire tétrahédral des ProPro endopeptidases. L’ensemble des isomères disponibles permet de choisir le plus opportun à l’appuis d’études de modélisation. Le dernier chapitre rapporte le développement de deux séries de composés incluant le dimère ProCyp. Les motifs polyproline sont de prime importance pour de nombreux mécanismes de reconnaissance par la Nature, en particulier dans les voies de signalisation dont le dérèglement peut entrainer une myriade de troubles de santé notamment inflammatoires, immunitaires et cancéreux. La première application est centrée sur la synthèse et les tests biologiques de peptidomimétiques du motif riche en proline du peptide p22phox, sous-unité de la NADPH oxidase 2 (NOX2) dont la reconnaissance par le domaine riche en proline de p47phox permet l’assemblage de NOX2 nécessaire à son activité. Lors de stress cellulaire externe, cet assemblage peut devenir excessif et engendrer un excès de production d’espèces réactives d’oxygènes, entrainant un ensemble de biomécanismes délétaires pour l’hôte. Les peptidomimétiques comportent un cœur triproline, dont deux des trois prolines ont été remplacées par le module ProCyp. Le meilleur isomère a été sélectionné sur la base de la modélisation moléculaire afin de mimer les angles de torsion de la triproline correspondant à une hélice de type polyproline II. La deuxième application concerne le développement de peptidomimétiques inhibiteurs de la ProPro endopeptidase (PPEP-1) de Clostridium difficile (C. diff). C. diff a été identifiée comme une des menaces nosocomiales majeures en raison de son caractère opportuniste lorsque la flore intestinale des patients est détruite suite à un traitement impliquant la prise soutenue d’antibiotiques. C. diff possède un arsenal de mécanismes d’évasion et de prolifération au sein de l’hôte incluant la formation de colonies recouvertes d’un biofilm protecteur sur la paroi epithéliale de l’hôte lors de la réponse immunitaire. Ces mécanismes sont complétés à l’aide de peptidases qui leurs permettent de couper les flagelles d’ancrages, dont PPEP-1. Le site de scission P1-P1’ de PPEP-1 implique deux prolines, flanquées par une séquence peptidique clairement identifiée. Une série de peptides basés sur cette séquence dont la diproline centrale a été remplacée par le dimère ProCyp a été synthétisée. L’isomère ProCyp a été choisi sur la base de son recouvrement avec l’heptapeptide cristallisé au sein d’un mutant de PPEP-1 par le Pr. Baumann. / Proline is a unique amino acid characterized by a cyclic side chain that gives it specific physical and chemical properties compared to the other 20 acyclic amino acids found in the human proteome. This difference is also found in polyproline motifs and is used by Nature in many ways such as protein folding, regulatory mechanisms related to the activity of certain proteases, formation of secondary structures, biomarking of peptide substrates of oligopeptidases, and cleavage of diproline motifs by proline-proline endopeptidases. Proline has therefore been a focus of interest for medicinal chemists in the development of therapeutic agents. This thesis proposes to study several biologically active compounds derived from proline. The first study reports on the advances related to the compound SH-BC-893, a constrained azacycle derived from FTY720 developed within the Hanessian group. SH-BC-893 is an anti-cancer agent that allows the starvation of cancer cells through the disruption of external and internal cellular nutrient supply. Through its activation of protein phosphatase 2A, it internalizes transmembrane amino acid and glucose receptors and downregulates low-density lipid receptors. Its action also allows the disruption of internal nutrient degradation from macropinocytosis and autophagy by preventing fusion between the lysosome and the respective endosomes. To enhance the activity of SH-BC-893, two series of compounds were synthesized and tested for dual activity. The first set aimed at targeting HDAC2 following a study by Spiegel in which its inhibition was reported for FTY720. The second set aimed at obtaining compounds activating other portions or homologs of PP2A via the insertion of tricyclic fragments following studies reporting PP2A activation by derivatives of tricyclic neuroleptic compounds of the perphenazine type. The synthesis and biological tests of these compounds are also described. The second chapter describes the synthesis of a series of bridged morpholines with a constrained gamma butyric amino acid (GABA) motif. GABA is a major component of central nervous system regulation through its inhibitory action on GABA-A, GABA-B, GABA-C neuroreceptors. Many therapeutic agents inspired by GABA have been developed over the years, notably baclofen. The bridged morpholines reported here have a stereocontrolled quaternary center for which a variety of substituents have been used, including the para-chlorophenyl of baclofen and the iso-butyl of Pregabalin, another GABA-derived compound active in the central nervous system. The baclofen derivative was modeled within the GABA-B active site. All compounds were also modeled as amino acids within the LLAMA database to determine their ability to occupy the lead-likeness space and confirm that they satisfy the Lipinski conditions. The third part describes the synthesis of a new diproline mime, named ProCyp. The dimer is composed of a pyrrolidine ring attached to a cyclopentane unit by means of a hydroxylated methylene bridge. The four trans cyclopentane isomers derived from (L)-proline were synthesized and their absolute stereochemistry was determined by NMR and crystallographic studies. These compounds represent the first form of ProCyp-type diproline mimes and can be incorporated into peptidomimetics to mimic the structural aspect of diprolines as well as the tetrahedral intermediate of ProPro endopeptidases. The set of available isomers allows to choose the most appropriate one to support modeling studies. The final chapter reports the development of two series of compounds including the ProCyp dimer. Polyproline motifs are of prime importance for many of Nature's recognition mechanisms, particularly in signaling pathways whose dysregulation can lead to a myriad of health disorders including inflammatory, immune and cancerous. The first application is focused on the synthesis and biological testing of peptidomimetics of the proline-rich motif of the peptide p22phox, a subunit of NADPH oxidase 2 (NOX2) whose recognition by the proline-rich domain of p47phox allows the assembly of NOX2 necessary for its activity. During external cellular stress, this assembly can become excessive and lead to an excess of reactive oxygen species production, resulting in a series of deleterious biomechanisms for the host. The peptidomimetics comprise a triproline core, of which two of the three prolines have been replaced by the ProCyp module. The best isomer was selected on the basis of molecular modeling to mimic the torsion angles of the triproline corresponding to a polyproline II helix. The second application concerns the development of peptidomimetics that inhibit the ProPro endopeptidase (PPEP-1) of Clostridium difficile (C. diff). C. diff has been identified as one of the major nosocomial threats due to its opportunistic nature when the intestinal flora of patients is destroyed following treatment with sustained antibiotics. C. diff has an arsenal of mechanisms of escape and proliferation within the host including the formation of colonies covered by a protective biofilm on the host epithelial wall during the immune response. These mechanisms are complemented by peptidases that allow them to cleave anchoring flagella, including PPEP-1. The P1-P1' cleavage site of PPEP-1 involves two prolines, flanked by a clearly identified peptide sequence. A series of peptides based on this sequence with the central diproline replaced by the ProCyp dimer was synthesized. The ProCyp isomer was chosen on the basis of its overlap with the heptapeptide crystallized in a PPEP-1 mutant by Prof. Baumann.

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The aim of the two studies presented here was to evaluate the effects of cocaine and mefloquine (MFQ) on γ-aminobutyric acid (GABA) and dopamine (DA) neurons in the ventral tegmental area (VTA). Cocaine: In vivo, lower doses of intravenous cocaine (0.25-0.5 mg/kg), or methamphetamine (METH), enhanced VTA GABA neuron firing rate via D2/D5 receptor activation. Higher cocaine doses (1.0-2.0 mg/kg) inhibited their firing rate. Cocaine and lidocaine inhibited the firing rate and spike discharges induced by stimulation of the internal capsule (ICPSDs) at dose levels 0.25-2 mg/kg (IC50 1.2 mg/kg), but neither DA nor METH reduced ICPSDs. In VTA GABA neurons in vitro, cocaine reduced (IC50 13 µM) current-evoked spikes and sodium currents in a use-dependent manner. In VTA DA neurons, cocaine reduced IPSCs (IC50 13 µM), increased IPSC paired-pulse facilitation, and decreased sIPSC frequency, without affecting mIPSC frequency or amplitude. These findings suggest cocaine reduces activity-dependent GABA release on DA neurons in the VTA, and that cocaine's use-dependent blockade of VTA GABA neuron voltage-sensitive sodium channels (VSSCs) may synergize with its DAT inhibiting properties to enhance mesolimbic DA transmission implicated in cocaine reinforcement. Mefloquine: Mefloquine (MFQ) is an anti-malarial agent, Connexin-36 (Cx36) gap junction blocker, 5-HT3 antagonist, and calcium ionophore. Mounting evidence of a Cx36-mediated VTA GABA neuron syncytium suggests MFQ-related dysphoria may attribute to its gap junction blocking effects on VTA synaptic homeostasis. We observed that MFQ (25 µM) increased DA neuron spontaneous IPSC frequency 6 fold, and mIPSC 3 fold. Carbenoxolone (CBX, 100 µM) only increased sIPSC frequency 2 fold, and did not affect DA mIPSC frequency. Ondansetron did not mimic MFQ. Additionally, MFQ did not affect VTA DA evoked IPSC paired pulse ratio (PPR). However, Mefloquine did induce a 3.5 fold increase in bath-applied GABA current. Remarkably, MFQ did not affect VTA GABA neuron inhibition. At VTA DA neuron excitatory synapses MFQ increased sEPSC frequency in-part due to an increase in the AMPA/NMDA ratio. These finding suggest MFQ alters VTA synapses differentially depending on neuron and synapse type, and that these alterations appear to involve MFQ's gap junction blocking and calcium ionophore actions.

Les mutations héréditaires causant une perte de fonction du gène CACNA1A, encodant la sous-unité α1 du canal CaV2.1, entraînent chez l’humain le développement d’une ataxie épisodique s’accompagnant parfois d’épilepsie et d’atteintes cognitives. Également, des mutations de novo de CACNA1A ont été rapportées chez près de 1 % des enfants souffrant d’encéphalopathies épileptogènes, ainsi que chez des enfants présentant un trouble du spectre de l’autisme isolé. Ensemble, ces données suggèrent que les altérations de CACNA1A peuvent jouer un rôle central dans la pathogenèse de divers troubles neurodéveloppementaux avec atteintes cognitives et développementales. D’ailleurs, notre évaluation de 16 patients, issus de quatre familles non consanguines, porteurs de différentes mutations induisant une perte de fonction de CACNA1A a révélé l’existence de déficits neurocognitifs modérés à sévères chez la majorité des individus atteints, allant de déficits d’attention avec difficultés d’apprentissage à une déficience intellectuelle avec ou sans trouble du spectre de l’autisme. Alors que les mécanismes pathologiques exacts par lesquels l’haploinsuffisance de CACNA1A induit de tels troubles cognitifs sont encore indéterminés, les mécanismes conduisant à l’épilepsie ont été mieux étudiés. La délétion embryonnaire du canal CaV2.1 dans les interneurones (IN) émanant de l’éminence ganglionnaire médiale (MGE), incluant les IN exprimant la parvalbumine (IN PV) et ceux exprimant la somatostatine (IN SOM), entraîne une épilepsie avec crises tonico-cloniques ainsi que des crises de type absences résultant en une mortalité précoce chez la souris Nkx2.1Cre; Cacna1ac/c. Cependant, la perte du canal dans les IN SOM, chez le modèle SOMCre; Cacna1ac/c, n’induit pas d’épilepsie et la perte ciblée aux IN PV, chez le modèle PVCre; Cacna1ac/c, entraîne une épilepsie caractérisée par des crises d’absence et de rares crises motrices. L’objectif de cette thèse consistait donc, dans un premier temps, de comprendre les mécanismes sous-jacents aux différences épileptiques entre les modèles Nkx2.1Cre; Cacna1ac/c et PVCre; Cacna1ac/c. Les techniques combinées d’imagerie immunohistochimique, d’imagerie 2-photon, d’électrophysiologie, d’analyse d’électroencéphalogramme et de croisement de modèles conditionnels nous ont permis d’identifier les conséquences cellulaires et électrophysiologiques de la délétion de Cacna1a de manière précoce ou tardive dans les IN PV. Elles ont dévoilé, chez le modèle PVCre; Cacna1ac/c, un gain d’inhibition dendritique dans les cellules pyramidales (CP) résultant d’une arborescence axonale accrue des IN SOM. Ce remodelage, dépendant de mTORC1, suffit à prévenir l’apparition de crises motrices et l’inhibition de cette croissance axonale à l’aide de rapamycine renverse l’effet protecteur observé chez la souris PVCre; Cacna1ac/c. Enfin, nous démontrons que l’activation chémogénétique des IN SOM corticaux prévient l’apparition de crises motrices dans un modèle d’épilepsie induite à l’acide kaïnique. Puisque les IN PV en panier du cortex sont essentiels à plusieurs processus cognitifs, telles la flexibilité cognitive et l’attention, qu’ils sont affectés par la perte de fonction homozygote de CaV2.1 et afin de reproduire une condition semblable à celle de nos patients, nous avons exploré dans un deuxième temps l’implication pathologique de ces neurones dans les troubles cognitifs associés à l’haploinsuffisance de Cacna1a. À l’aide du modèle murin portant une délétion hétérozygote de Cacna1a ciblée aux populations neuronales exprimant la PV (PVCre; Cacna1ac/+), nous démontrons par électrophysiologie que la perte du canal CaV2.1 dans ces neurones suffit à réduire l’inhibition corticale. Les tests comportementaux incluant l’Openfield, l’Elevated Plus Maze, le Morris Water Maze, une tâche testant la rigidité cognitive ainsi qu’une tâche évaluant l’attention, ont démontré que les mutants PVCre; Cacna1ac/+ présentent de l’impulsivité, de la rigidité cognitive ainsi qu’un déficit d’attention sélective. Bien que l’ablation homozygote du canal réduise la relâche synaptique des CP chez le mutant homozygote Emx1Cre; Cacna1ac/c, aucun déficit de relâche synaptique, comportemental ou cognitif n’a été observé chez les souris Emx1Cre; Cacna1ac/+ suggérant qu’au niveau cortical, la délétion hétérozygote de Cacna1a affecte sélectivement les IN PV. De plus, à l’aide de délétions ciblées au cortex orbito-frontal (OFC) et au cortex préfrontal médial (mPFC), nous démontrons que l’haploinsuffisance de Cacna1a dans ces régions entraîne de la rigidité cognitive et des troubles de l’attention, respectivement. Enfin, nous révélons que ces deux atteintes peuvent être corrigées via une activation chémogénétique locale des IN PV. Dans son ensemble, ce travail contribue au développement des connaissances portant sur les délétions de Cacna1a. Il présente également de nouvelles avenues pour le traitement de crises épileptiques motrices et pour la prise en charge des atteintes cognitives chez les patients souffrant d’haploinsuffisance de CACNA1A. / Loss-of-function mutations in the CACNA1A gene, encoding the α1 subunit of voltage-gated CaV2.1 channels, result in epilepsy and neurocognitive impairments, including attention deficits, intellectual deficiency and autism. Also, de novo mutations in CACNA1A have been reported in nearly 1% of children with epileptogenic encephalopathies, as well as in children with isolated autism spectrum problems. Taken together, these data suggest that alterations in CACNA1A may play a central role in the pathogenesis of various neurodevelopmental disorders with cognitive and developmental impairment. Moreover, our evaluation of 16 patients, from four non-consanguineous families, carriers of different mutations inducing a loss of function of CACNA1A have shown the existence of moderate to severe neurocognitive deficits in the majority of affected individuals, ranging from deficits from attention with learning difficulties to intellectual disabilities with or without an autism spectrum problem. While the exact pathological mechanisms by which CACNA1A haploinsufficiency induces such cognitive impairment are still unknown, the mechanisms leading to epilepsy have been better studied. Embryonic deletion of CaV2.1 in interneurons (IN) emanating from the medial ganglionic eminence (MGE), including INs expressing parvalbumin (PV IN) and those expressing somatostatin (SOM IN), causes epilepsy with tonic-clonic seizures and absence seizures resulting in early mortality in the Nkx2.1Cre; Cacna1ac/c mice model. However, loss of the channel in SOM IN (SOMCre; Cacna1ac/c) does not induce epilepsy whereas targeted loss in PV IN (PVCre; Cacna1ac/c) causes epilepsy with absence and rare motor seizures. The objective of this thesis was therefore, first of all, to understand the mechanisms underlying the epileptic differences between the Nkx2.1Cre ;Cacna1ac/c and the PVCre; Cacna1ac/c mice. The combined techniques of immunohistochemistry, 2-photon imaging, electrophysiology, electroencephalogram analysis and the crossing of different conditional models identified the cellular and electrophysiological consequences of the deletion of Cacna1a in the IN PV. Compared to Nkx2.1Cre; Cacna1ac/c mice, PVCre; Cacna1ac/c mice have a net increase in cortical inhibition, with a gain of dendritic inhibition through sprouting of SOM IN axons, largely preventing motor seizures. This beneficial compensatory remodeling of cortical GABAergic innervation is mTORC1-dependent and its inhibition with rapamycin leads to a striking increase in motor seizures. Furthermore, we show that a direct chemogenic activation of cortical SOM-INs prevents motor seizures in a model of kainate-induced seizures. Cortical PV IN basket cells are essential for several cognitive processes, such as cognitive flexibility and attention and they are affected by CaV2.1 knock-out. CACNA1A haploinsufficiency also causes cause epilepsy, ataxia, and a range of neurocognitive deficits, including inattention, impulsivity, intellectual deficiency and autism. Therefore, this thesis had for second objective to clarify the consequences of Cacna1a haploinsufficiency in PV IN. Using the mice model carrying a heterozygous deletion of Cacna1a targeted at neuronal populations expressing PV (PVCre; Cacna1ac/+), we demonstrated by electrophysiology that the loss of the CaV2.1 in this neuronal population is sufficient to reduce cortical inhibition. Behavioral tests including the OpenField, the Elevated Plus Maze, the Morris Water Maze, a cognitive rigidity task as well as an attention set-shifting task have shown that PVCre; Cacna1ac/+ exhibit impulsivity, cognitive rigidity, and selective attention deficit. Although Cacna1a homozygous ablation reduced synaptic release of PC in the Emx1Cre; Cacna1ac/c mice mutant, no synaptic, behavioural or cognitive relaxation deficits were observed in the Emx1Cre; Cacna1ac/+ mice suggesting that, at the cortical level, the heterozygous deletion of Cacna1a selectively affects PV IN. These findings have enabled us to determine, using targeted deletions within the orbitofrontal cortex (OFC) and the medial prefrontal cortex (mPFC), that the haploinsufficiency of Cacna1a in PV IN results in reversal learning deficits and impairs selective attention, respectively. These deficits can be rescued by the selective chemogenetic activation of cortical PV IN respectively in the OFC or mPFC of PVCre; Cacna1ac/+ mutants As a whole, this work contributes to the development of knowledge on Cacna1a deletions. It also presents new avenues for the treatment of motor epileptic seizures and for the management of cognitive impairment in patients with CACNA1A haploinsufficiency.

A holy grail in neuroscience is being able to control plasticity to facilitate recovery from insult in the adult brain. Despite success in animal models, few therapies have translated from bench to bedside. This thesis is aimed at addressing 2 major stumbling blocks in translation. The first gap is in our understanding of the mechanisms of plasticity-enhancing therapies, and the second is in our understanding the relevance of those mechanisms for human development. In chapters 2 and 3, I address the first gap by asking whether fluoxetine, a selective serotonin reuptake inhibitor, which reinstates juvenile-like plasticity in adult animals, reinstates a juvenile-like synaptic environment. We found evidence to suggest that fluoxetine is neuroprotective, as it rescued all of the MD-driven changes, but surprisingly we found no evidence that fluoxetine recreated a juvenile-like synaptic environment, with the exception of Ube3A. Ube3A is necessary for critical period plasticity, indicating that Ube3A may play a crucial in enhancing plasticity in the adult cortex. In chapter 4, I address whether D-serine, an amino acid that has similar effects to fluoxetine in terms of both plasticity and anti-depression, shares a common neurobiological signature with fluoxetine. I found that D-serine’s effects were strikingly similar to fluoxetine, with respect to markers of the E/I balance, indicating that it may be an effective alternative to fluoxetine. In chapter 5, I address the second gap by studying the development of 5 glutamatergic proteins in human V1. Some changes occurred early, as would be predicted from animals studies, while other changes were protracted, lasting into the 4th decade. These results will help guide the use of treatments, like fluoxetine, which effect glutamatergic proteins. iv Together the findings in this thesis significantly advances our understanding of the mechanisms involved in restating plasticity in the adult cortex, and their relevance to humans. / Dissertation / Doctor of Philosophy (PhD) / Neurons change to rewire, adapt, and recover. This plasticity is greatest early in development, so much research has focused on bringing it back in adults. There has been amazing progress in animal models, but this has not translated to humans. Two reasons for this are that we do not fully understand the mechanisms of these treatments in animals or whether those mechanisms are relevant for humans. My thesis addresses this by studying how 2 treatments, fluoxetine and D-serine, affect proteins that are important for plasticity, and how those proteins develop in the humans. I found that these treatments are neuroprotective, but do not recreate a younger state. One interesting standout is an increase in Ube3A, which is essential for juvenile plasticity. I also found that much of human development is similar to animals, but the time course for some proteins is uniquely prolonged in humans. These findings have implications for the use of plasticity-enhancing treatments at different ages.

Des variants récessifs touchant le gène PIGB, encodant une enzyme impliquée dans la biosynthèse des ancres GPI, ont récemment été décrits chez des patients présentant une déficience héritée des ancres GPI ainsi qu’une encéphalopathie épileptogène (EE), une forme d’épilepsie infantile sévère associée à des atteintes cognitives. Chez l’humain, plus de 150 protéines, dont certaines sont critiques pour la fonction neuronale, sont localisées à la membrane cellulaire grâce aux ancres GPI. Des données préliminaires du laboratoire Rossignol démontrent que la délétion embryonnaire du gène Pigb dans les interneurones GABAergiques (IN) dérivés de l’éminence ganglionnaire médiale (MGE) est suffisante pour induire des crises d’épilepsie spontanées et des déficits cognitifs chez la souris, suggérant un rôle critique de PIGB dans le développement de l’inhibition corticale. Toutefois, les mécanismes cellulaires et moléculaires sous-tendant les phénotypes cliniques associés aux délétions du gène PIGB sont inconnus. Compte tenu du rôle central joué par les molécules de guidage, dont certaines sont des protéines à ancrage GPI, lors de la migration des IN vers la plaque corticale, nous postulons que la perte sélective des ancres GPI, résultant d’une délétion conditionnelle de Pigb dans les IN, altère leur dynamique de migration, ce qui a pour conséquence de réduire leur nombre dans le cortex postnatal, menant à une désinhibition corticale et au développement de l’épilepsie. L’imagerie en temps réel d’explants cellulaires de MGE a révélé que la perte de fonction du gène Pigb dans les IN dérivés du MGE entraine un défaut de la migration tangentielle et des anomalies morphologiques se traduisant par une réduction de la densité des IN dans le cortex postnatal. Nous avons également démontré que la signalisation motogène EphA4-éphrineA2 est altérée dans les IN déficients en ancres GPI, contribuant au délai de migration observé. En somme, nos travaux ont permis de préciser les mécanismes physiopathologiques sous-tendant les EE associées à des variants pathogéniques du gène PIGB et d’approfondir notre compréhension du rôle des ancres GPI durant le neurodéveloppement et plus précisément, durant la migration des IN. / Recessive variants in the PIGB gene, encoding an enzyme involved in the biosynthesis pathway of GPI anchors, were recently described in children with an inherited GPI anchor defect and epileptic encephalopathy (EE), a neurodevelopmental disorder characterized by early-onset epilepsy with cognitive impairment. GPI anchors are critical for the membrane attachment of at least 150 human proteins, some of which are important for proper neuronal function. Preliminary data from the Rossignol group show that the embryonic deletion of Pigb in GABAergic interneurons (INs) emanating from the medial ganglionic eminence (MGE) causes spontaneous seizures and cognitive deficits in mice, suggesting a critical role of PIGB in the establishment of cortical inhibition. However, the cellular and molecular mechanisms leading to epilepsy remain unknown. Given the central role of guidance molecules, some of which are GPI-anchored proteins, during neuronal migration, we postulate that loss of GPI anchors following the conditional deletion of Pigb in MGE-derived INs disrupts chemotactic guidance and IN migration dynamics, leading to cortical disinhibition and epilepsy post-natally. Time-lapse live imaging of MGE explants revealed that the targeted deletion of Pigb impairs the tangential migration as well as the morphological development of MGE-derived INs, resulting in reduced IN densities in the postnatal cortex. We showed that the kinetic deficits are partly due to a loss of EphA4-ephrinA2 motogenic signaling in PigbcKO INs. In summary, our work helps clarify the physiopathology underlying PIGB associated-EE and deepens our understanding of the roles of GPI-anchor-related pathways in neurodevelopment and more specifically, in the migration of cortical INs.

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