D'après le postulat de Hebb, les réseaux neuronaux adaptent leur connectivité sous l'influence des activités pré- et post-synaptiques. La " spike-timing-dependent plasticity " (STDP) est une règle d'apprentissage synaptique de type Hebbien, qui repose sur la structure temporelle précise des patrons d'activités appariées de part et d'autre de la synapse. La plasticité cortico-striatale serait le substrat biologique de l'apprentissage procédural effectué par les ganglions de la base. Les neurones de sortie du striatum agissent comme des détecteurs de coïncidence des activités corticales et thalamiques. La STDP cortico-striatale pourrait donc jouer un rôle crucial dans les processus d'encodage de l'apprentissage et la mémoire procédurale. Nous avons exploré les conditions d'émergence et d'expression de la STDP cortico-striatale. / According to Hebbian theory, neural networks refine their connectivity by patterned firing of action potentials in pre- and postsynaptic neurons. Spike-timing-dependent plasticity (STDP) is a synaptic Hebbian learning rule relying on the precise order and the millisecond timing of the paired activities on either side of the synapse. Temporal coding via STDP may be essential for the role of the striatum in learning of motor sequences in which sensory and motor events are associated in a precise time sequence. Corticostriatal long-term plasticity provides a fundamental mechanism for the function of the basal ganglia in procedural learning. Striatal output neurons act as detectors of distributed patterns of cortical and thalamic activity. Thus, corticostriatal STDP should play a major role in information processing in the basal ganglia, which is based on a precise time-coding process. Here, we explored the conditions required for the emergence of corticostriatal STDP.

The purpose of this paper was to develop a single assay system for the enzymes which regulate GABA and glutamate concentrations in brain and nerve tissue. Since all the enzymes produce L-glutamate, their activities were measured by coupling them to L-glutamate decarboxylase. Enzymatic activity was determined by measuring the release of co2 from radioactive substrates. The glutamate decarboxylase was obtained from a commercial acetone powder by simplifying existing procedures. The glutamate decarboxylase produced was of sufficient purity to be used in the coupled assays, which were checked with commercial preparations of each enzyme, where available, and with crude brain homogenates. All of the assays were shown to be linear with respect to both time and enzyme concentration, thus assuring the feasibility of the technique.

Proteins are dynamic molecules capable of performing complex biological functions necessary for life. The impact of protein dynamics in the development of medicines is often understated. Science is only now beginning to unravel the numerous consequences of protein flexibility on structure and function. This thesis will encompass two case studies in developing small molecule inhibitors targeting flexible enzymes, and provide a thorough evaluation of their inhibitory mechanisms of action. The first case study focuses on caspases, a family of cysteine proteases responsible for executing the final steps of apoptosis. Consequently, they have been the subject of intense research due to the critical role they play in the pathogenesis of various cardiovascular and neurodegenerative diseases. A fragment-based screening campaign against human caspase-7 resulted in the identification of a novel series of allosteric inhibitors, which were characterized by numerous biophysical methods, including an X-ray co-crystal structure of an inhibitory fragment with caspase-7. The fragments described herein appear to have a significant impact on the substrate binding loop dynamics and the orientation of the catalytic Cys-His dyad, which appears to be the origin of their inhibition. This screening effort serves the dual purpose of laying the foundation for future medicinal chemistry efforts targeting caspase proteins, and for probing the allosteric regulation of this interesting class of hydrolases. The second case study focuses on glutamate racemase, another dynamic enzyme responsible for the stereoinversion of glutamate, providing the essential function of D-glutamate production for the crosslinking of peptidoglycan in all bacteria. Herein, I present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase. The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to reduce off-target liabilities. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small molecule inhibitor with the catalytic Cys74 of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal, with unprecedented detail, the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency comparable to β-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action.

Flexible enzymes are notoriously a bane to structure-based drug design and discovery efforts. This is because no single structure can accurately capture the vast array of conformations that exist in solution and many are subject to ligand-associated structural changes that are difficult to predict. Glutamate racemase (GR) – an antibiotic drug discovery target involved in cell wall biosynthesis – is one such enzyme that has eluded basic structure-based drug design and discovery efforts due to these flexibility issues. In this study, our focus is on overcoming the impediment of unpredictable ligand-associated structural changes in GR drug discovery campaigns. The flexibility of the GR active site is such that it is capable of accommodating ligands with very different structures. Though these ligands may bind to the same pocket, they may associate with quite dissimilar conformations where some are more favorable for complexation than others. Knowledge of these changes is invaluable in guiding drug discovery efforts, indicating which compounds selectively associate with more favorable conformations and are therefore better suited for optimization and providing starting structures to guide structure-based drug design optimization efforts. In this study, we develop a mutant GR possessing a genetically encoded non-natural fluorescent amino acid in a region remote from the active site whose movement has been previously observed to correlate with active site changes. With this mutant GR, we observe a differential fluorescence pattern upon binding of two structurally distinct competitive inhibitors known to associate with unique GR conformations – one to a favorable conformation with a smaller, less solvated active site and the other to an unfavorable conformation with a larger, more solvated active site. A concomitant computational study ascribes the source of this differential fluorescence pattern to ligand-associated conformational changes resulting in changes to the local environment of the fluorescent residue. Therefore, this mutant permits the elucidation of valuable structural information with relative ease by simply monitoring the fluorescence pattern resulting from ligand binding, which indicates whether the ligand has bound to a favorable or unfavorable conformation and offers insight into the general structure of this conformation.

Repeated exposure to drugs of abuse conditions individuals to anticipate the behavioral consequences of drug use specifically in the presence of a drug-associated context. In rodents, preferences and aversions for alcohol and cocaine have been conditioned; however, the mechanisms underlying the expression of these conditioned effects remain unknown. Given that alcohol and cocaine polysubstance abuse is prevalent in young individuals, with more than 50% of these polysubstance abusers reporting to be under the age of 21, it is important to understand the mechanisms contributing to the behavioral effects of alcohol and cocaine co-dependency. Aim 1 determined if age differentially impacted the effects of repeated alcohol exposure on conditioned cocaine preferences. Adolescent [postnatal day (PND) 30) and adult (PND 60) male Sprague-Dawley rats were administered ethanol (0.5 or 1.75 g/kg, i.p.) immediately before each cocaine conditioned place preference (CPP) session (20 mg/kg, i.p.; 15 minutes). Aim 2, Experiments 1 and 2, identified the role of NMDA receptors within the nucleus accumbens septi (NAcc) in conditioned ethanol/cocaine behavior. Adolescent and adult rats in Experiment 1 were administered the NMDA antagonist MK-801 (0.1 or 0.2 m/kg, i.p.) 30 minutes prior to cocaine conditioning. Adolescent and adult rats within Experiment 2 underwent bilateral cannulation for chronic implantation of the cannulae into the NAcc of both hemispheres. Rats administered 1mM MK-801 or saline into the NAcc prior to cocaine (20.0 mg/kg, i.p.) conditioning, completed additional testing to determine the role of NAcc NMDA receptors in the consolidation, reconsolidation and expression of cocaine conditioned behavior in a drug-induced reactivation manner. Findings show adolescent and adult rats responded similarly to co-administration of ethanol/cocaine with both ages showing a decrease in the rewarding properties of cocaine. What differed between the age groups were the aversive properties of ethanol, with adolescents being less sensitive to the aversive properties of ethanol and its modulating effects on cocaine reward. A role for the NAcc NMDA receptors was observed in contributing to the modulating effects of ethanol on cocaine reward. Lastly, the reconsolidation of cocaine reward was more sensitive to disruption in adolescent rats, as compared to their adult counterparts. These results suggest an increased vulnerability for adolescents to continue engaging in polysubstance abuse. However, this at-risk age group also appeared to be more responsive to pharmacological treatment in decreasing addictive behavior.

Psychostimulant-induced reinstatement of place preferences have been used to investigate underlying physiological mechanisms mediating drug-seeking behavior in adolescent and adult rodents; however, it is still unclear how psychostimulant exposure during adolescence affects neuronal communication in the mesolimbic dopamine (DA) pathway and whether these changes would elicit enhanced drug-seeking behavior later in adulthood. The aim of the present study was to investigate the effects of intra-ventral tegmental area (VTA) or intra-nucleus accumbens septi (NAcc) DA D2 receptor antagonist infusions on cocaine-induced reinstatement of cocaine place conditioning in high and low responders for cocaine reward. Adolescent rats were exposed to cocaine place conditioning [postnatal day (PND 28-39)] and divided into high and low responders for cocaine reward based on their place preference expression score. Place preferences were extinguished and guide cannula were implanted into either the VTA or NAcc followed by one of the following: 1) intra-VTA or intra-NAcc infusion of the DA D2 receptor antagonist sulpiride (100 µM) during a cocaine-primed reinstatement test (10 mg/kg/ip cocaine) or 2) measurement of NAcc DA levels during intra-VTA or intra-NAcc infusion of sulpiride (100 µM), a cocaine prime (10 mg/kg cocaine) and re-exposure to the cocaine paired chamber. Infusion of sulpiride into the VTA but not the NAcc blocked reinstatement of cocaine place conditioning in rats exposed to cocaine during adolescence. Furthermore, re-exposure to cocaine-associated cues and simultaneous local infusion of sulpiride into either the VTA or NAcc attenuated cocaine-induced increases in accumbal DA levels for rats pretreated with cocaine during adolescence, regardless of phenotype. These data suggest intrinsic compensatory mechanisms in the mesolimbic DA pathway mediate adolescent behavioral responsivity to cocaine prime-induced reinstatement of cocaine place conditioning later on in adulthood.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Les progrès récents des techniques d'imagerie cérébrale ont permis d'identifier les défauts de migration neuronale comme cause majeure de retards mentaux, d'épilepsies et de nombreux syndromes neurologiques. La connaissance des mécanismes intervenant dans la modulation de la migration neuronale est donc capitale afin de prévenir de telles anomalies du développement cérébral foetal, dont le coût socioéconomique est élevé. Le travail réalisé au cours de cette thèse s'inscrit dans le cadre de cette thématique.<br />De nombreuses études ont identifié les neurotransmetteurs comme des molécules porteuses d'informations à un niveau plus large que celui de la seule transmission synaptique. En effet, avant même la formation de synapses, les neurotransmetteurs sont présents au sein du tissu cérébral embryonnaire et exercent des actions variées, influençant les étapes de genèse, migration, différentiation et de mort neuronale. Nous avons évalué les rôles joués par les neurotransmetteurs GABA et glutamate au cours de la migration neuronale, ainsi que les conséquences de la perturbation de leurs actions durant la construction du cerveau foetal.<br />Afin d'étudier la migration neuronale, nous avons mis au point des préparations originales permettant la visualisation directe de neurones fluorescents en migration. Grâce à elles, nous avons démontré l'existence d'une modulation de la migration neuronale par le GABA et glutamate, libérés selon un mode de sécrétion « non vésiculaire » et agissant par l'intermédiaire de récepteurs spécifiques. Nous avons montré que les mécanismes modulant la migration sont caractéristiques du type neuronal et du mode migratoire considéré. Ainsi, la migration radiale des futurs neurones glutamatergiques est modulée majoritairement par l'activation de récepteurs GABAA, alors que la migration tangentielle des futurs interneurones GABAergiques est modulée par l'activation de récepteurs glutamatergiques de type AMPA. Ces résultats suggèrent également l'existence d'une communication précoce entre neurones glutamatergiques et interneurones, pouvant contribuer à la construction cérébrale. Par la suite, nous avons évalué le risque de survenue de malformations cérébrales foetales, suite à l'administration de médicaments anti-épileptiques, qui potentialisent l'action du GABA, limitent l'action du glutamate et modulent l'activité des canaux ioniques. Nous avons observé une incidence accrue de micro-dysplasies cortico-hippocampiques, à rapprocher de défauts de migration, suite à l'administration chez la rate gestante de valproate et de vigabatrin à des doses compatibles avec celles employées pour le traitement de la femme enceinte épileptique.<br />Ces résultats soulignent le rôle central joué par les neurotransmetteurs GABA et glutamate, en tant que signaux informatifs majeurs du cerveau en développement. De plus, ils suggèrent le besoin d'évaluer l'impact de l'exposition à des composés pharmacologiques interférant avec les actions de ces neurotransmetteurs lors de la maturation cérébrale.

Ce travail a eu comme objectif de mettre en place des techniques de spectroscopie RMN du proton optimisées pour la détection du glutamate cérébral afin d'étudier des changements éventuels du métabolisme glutamatergique dans la maladie de Parkinson. En vue d'une application chez le patient, une approche de filtrage à double quanta et une méthode liée à la spectroscopie bi-dimensionnelle ont été évaluées à 3 Tesla sur un imageur corps entier. Une étude incluant dix patients atteints par la maladie de Parkinson idiopathique et dix volontaires sains a ensuite été réalisée, utilisant une méthode liée à la spectroscopie bi-dimensionelle et ciblant le noyau lenticulaire (putamen et globus pallidum). Aucun changement significatif du glutamate total (intra- et extracellulaire) n'a pu être mis en évidence comparant le patient sous traitement par levodopa (L-DOPA) ou ayant temporairement interrompu son traitement et le volontaire sain. Une tendance à la diminution de la creatine/phosphocreatine a été observée chez le patient. En vue d'une étude complémentaire sur le modèle animal, la méthode mise en œuvre chez le patient a été évaluée chez le rat à 7 Tesla. Une étude sur le rat traité par 6-hydroxydopamine (6-OHDA) a ensuite été réalisée, utilisant une séquence PRESS standard à un temps d'écho de 136 ms et ciblant le striatum. Sur le même animal, des spectres avant traitement à la 6-OHDA, après traitement et suivant une injection de levodopa ont été acquis. Aucun changement de glutamate n'a pu être mis en évidence, alors qu'une tendance à la réduction de la choline suivant le traitement à la 6-OHDA était observée. Nos résultats indiquent que les changements du métabolisme glutamatergique, ayant lieu dans le cadre de la maladie de Parkinson qui ont été mis en évidence dans le modèle 6-OHDA par d'autres méthodes, ne sont pas reflétés par des changements des concentrations du glutamate total mesuré par RMN in vivo.

Prior work from our laboratory has shown that the spinal cord is capable of supporting a simple form of instrumental (response-outcome) learning. In a typical experiment, animals are given a spinal transection at the second thoracic vertebra, and tested 24 h after surgery. If animals are given shock when their leg is in a resting position (controllable shock), they quickly learn to maintain the leg in a flexed position, thereby minimizing shock exposure. Animals exposed to shock that is independent of leg position (uncontrollable shock) fail to learn. This learning deficit can be induced by as little as 6 minutes of shock to either limb or to the tail, and lasts for up to 48 h. The aim of this dissertation was to explore whether the deficit shares behavioral features and pharmacological mechanisms similar to those involved in the induction of neuropathic pain. Work within the pain literature has identified a spinal hyperexcitability that is induced by intense stimulation of pain fibers. This phenomenon, known as central sensitization, is characterized by an increase in tactile reactivity (allodynia) that can be induced by shock or peripheral inflammation. Pharmacological findings have revealed that central sensitization depends on the activation of the N-methyl-D-aspartate (NMDA) and group I metabotropic glutamate receptors (mGluRs). Experiment 1 showed that uncontrollable shock induces a tactile allodynia similar to that observed in central sensitization. Experiment 2 showed that peripheral inflammation caused by a subcutaneous injection of formalin generates a dose-dependent deficit. Experiment 3 indicated that the formalin-induced deficit was observed 24 h after delivery of the stimulus. Experiments 4-8 revealed that the NMDA and group I mGluRs are involved in the deficit. The NMDA receptor was found to be necessary (Experiment 4), but only sufficient to induce a deficit at neurotoxic doses (Experiment 5). Both of the group I mGluRs (subtypes, mGluR1 and mGluR5) were found to be necessary (Experiments 6 & 7). A general group I mGluR agonist summated with a subthreshold intensity of shock to produce a robust deficit (Experiment 8), suggesting shock and mGluR activation produce a deficit through a common mechanism.