Les interneurones GABAergiques constituent une population mineure de cellules par rapport aux neurones glutamatergiques dans le néocortex. Cependant ils contrôlent fortement l'excitabilité neuronale, la dynamique des réseaux neuronaux et la plasticité synaptique. L'importance des circuits GABAergiques dans le processus fonctionnel et la plasticité des réseaux corticaux est soulignée par des résultats récents qui montrent que des modifications très précises et fiables des circuits GABAergiques sont associées à divers troubles du développement neurologique et à des défauts dans les fonctions cérébrales. De ce fait, la compréhension des mécanismes cellulaires et moléculaires impliquant le développement des circuits GABAergiques est la première étape vers une meilleure compréhension de la façon dont les anomalies de ces processus peuvent se produire. La molécule d’adhésion cellulaire neurale (NCAM) appartient à la super-famille des immunoglobulines de reconnaissance cellulaire et est impliquée dans des interactions homophiliques et hétérophiliques avec d’autres molécules. Même si plusieurs rôles de NCAM ont été démontrés dans la croissance neuronale, la fasciculation axonale, la formation et la maturation de synapses, de même que dans la plasticité cellulaire de plusieurs systèmes, le rôle de NCAM dans la formation des synapses GABAergiques reste inconnu. Ce projet visait donc à déterminer le rôle précis de NCAM dans le processus de maturation des synapses GABAergiques dans le néocortex, en modulant son expression à différentes étapes du développement. L’approche choisie a été de supprimer NCAM dans des cellules GABAergiques à paniers avant la maturation des synapses (EP12-18), pendant la maturation (EP16-24), ou durant le maintien de celles-ci (EP24-32). Les méthodes utilisées ont été le clonage moléculaire, l’imagerie confocale, la culture de coupes organotypiques et des techniques morphométriques de quantification de l’innervation GABAergique. Nos résultats montrent que l’inactivation de NCAM durant la phase de maturation des synapses périsomatiques (EP16-24) cause une réduction du nombre de synapses GABAergiques périsomatiques et du branchement de ces axones. En revanche, durant la phase de maintien (EP26-32), l’inactivation de NCAM n’a pas affecté ces paramètres des synapses GABAergiques. Or, il existe trois isoformes de NCAM (NCAM120, 140 et 180) qui pourraient jouer des rôles différents dans les divers types cellulaires ou à des stades développementaux différents. Nos données montrent que NCAM120 et 140 sont nécessaires à la maturation des synapses périsomatiques GABAergiques. Cependant, NCAM180, qui est l’isoforme la plus étudiée et caractérisée, ne semble pas être impliquée dans ce processus. De plus, l’inactivation de NCAM n’a pas affecté la densité des épines dendritiques ou leur longueur. Elle est donc spécifique aux synapses périsomatiques GABAeriques. Finalement, nos résultats suggèrent que le domaine conservé C-terminal KENESKA est essentiel à la maturation des synapses périsomatiques GABAergiques. Des expériences futures nous aiderons à mieux comprendre la mécanistique et les différentes voies de signalisation impliquées. / GABAergic interneurons, though a minor population in the neocortex, play an important role in cortical function and plasticity. Alterations in GABAergic circuits are implicated in various neurodevelopmental disorders. The GABAergic network comprises diverse interneuron subtypes that have different morphological and physiological characteristics, and localize their synapses onto distinct subcellular locations on the postsynaptic targets. Precisely how activity and molecularly driven mechanisms conspire to achieve the remarkable specificity of GABAergic synapse localization and formation is unknown. Therefore, unravelling the cellular and molecular mechanisms involved in this process is crucial for a better understanding of both cortical function and the basis of various neurological disorders. Here we focus our study on a subtype of GABAergic neurons - the basket interneurons which localize synapses, called perisomatic synapses, onto the soma and proximal dendrites of the postsynaptic targets, and tightly regulate their firing patterns. Although recent studies have shown the activity dependence of basket synapse formation, the molecular mechanisms implicated in the perisomatic synapse formation process are poorly understood. NCAM, the neural cell adhesion molecule, is a prime molecular player implicated both in early synaptogenesis events, and during maturation of glutamatergic synapses in the hippocampus. Recent studies have implicated the polysialylated form of NCAM (PSA-NCAM) in basket synapse formation. However, whether and how NCAM per se plays a role in the formation of GABAergic synapses is unknown. Using single cell genetics to knock down NCAM in individual basket interneurons at specific developmental time periods, we characterized the role of NCAM during perisomatic synapse formation and maintenance. Here we show that loss of NCAM during perisomatic synapse formation from equivalent postnatal day (EP) 16 to EP24, in organotypic slices from mouse visual cortex, significantly retards the process of basket cell axonal branching and bouton formation. However, loss of NCAM at a later stage (EP26 to EP32), when the synapses are already formed, did not affect the number or intricacy of perisomatic synapses. NCAM is therefore implicated in perisomatic synapse formation but not in its maintenance. Further studies also show that isoforms of NCAM, such as NCAM140 and NCAM120 are involved in perisomatic GABAergic synapse maturation. However, NCAM180 is not implicated in this process. Also, NCAM does not affect dendritic spine density and length during maturation and maintenance phases, therefore its action is specific only to GABAergic perisomatic synapses. Finally, the highly conserved C-terminal domain KENESKA is essential for GABAergic perisomatic synapse maturation. Future experiments will help us clarify this mechanism and the involved signalling pathways related to NCAM.

Inhalants are a loosely defined diverse group of volatile substances which people abuse. Despite widespread misuse of inhalants, there are limited preclinical methods available to study the reinforcement-like properties of inhalants. One procedure which has demonstrated substantial promise as a tool to investigate inhalant pharmacology is the intracranial self-stimulation (ICSS) procedure. ICSS utilizes pulses of electrical stimulation to the mesolimbic reward pathway to serve as a temporally defined and controlled operant reinforcer with a highly adjustable efficacy. The first aim of the project was to characterize the effects of commonly abused inhalants: including toluene, trichloroethane, nitrous oxide, isoflurane and R134a in ICSS. The second aim was to attenuate inhalant-facilitated ICSS by utilization of compounds which would attenuate the pharmacological actions of toluene on GABAA receptors. The low efficacy benzodiazepine negative modulator Ro15-4513 significantly attenuated the ability of toluene to facilitate ICSS without itself significantly altering baseline ICSS responding. Pretreatment with Ro15-4513 also attenuated methamphetamine ICSS even though there is no evidence of methamphetamine interacting with GABAA receptors. Given these unexpected results, I employed a microdialysis procedure to examine the effect of Ro15-4513 on methamphetamine stimulated dopamine release in the nucleus accumbens. Pretreatment with Ro15-4513 significantly attenuated methamphetamine stimulated dopamine release while having a negligible effect on dopamine release when administered alone. These results suggest that a modest level of benzodiazepine-site negative modulation can reduce the reinforcement enhancing effects of abused drugs regardless of their primary mechanism of action through allosteric modulation of GABAergic neurons within the mesolimbic pathway. Further, these results may have implications for expanding the examination of GABAA negative modulator medications beyond those trials currently being conducted with alcohol. Finally, the effects of chronic intermittent toluene exposure on ICSS and nesting behaviors were examined. Subjects were systemically exposed to air, chronic intermittent toluene (CIT), or escalating chronic intermittent (ECIT) toluene for 15 min at 3300 PPM toluene vapor per exposure. The results show that ECIT resulted in decreased overall responding in ICSS relative to air control and showed a tolerance-like effect to facilitatory effects of 3300 ppm toluene during ICSS compared to CIT group. These results indicate that escalating use of toluene produces reductions in its reward-like effects and may contribute to escalation to other drugs of abuse.

A moxidectina (MOX) é um antiparasitário utilizado na clínica veterinária. Em mamíferos seu mecanismo de ação envolve o ácido ?-aminobutírico (GABA), um neurotransmissor que tem papel relevante na regulação dos comportamentos sexual e motor. Dados anteriores por nós obtidos mostraram que a MOX prejudicou o comportamento sexual e a coordenação motora de ratos machos avaliados na trave elevada. Assim, dando continuidade a esse estudo, o objetivo deste trabalho foi avaliar os efeitos da administração da dose terapêutica de MOX (0,2 mg/kg) na motivação sexual e ereção peniana de ratos machos, bem como estudar seu envolvimento em diferentes sistemas de neurotransmissão central. Em todos os experimentos os ratos do grupo experimental receberam a MOX por via subcutânea (SC); e os ratos do grupo controle receberam 1 ml/kg de óleo de amêndoas pela mesma via, e foram avaliados após 72 h. A motivação sexual foi avaliada em um aparelho constituído de uma arena e dois compartimentos separados desta por tela de arame; num compartimento foi colocado um rato macho experiente e no outro uma fêmea sexualmente receptiva. Neste aparelho foi medido o tempo que o rato permaneceu nas proximidades de cada compartimento. Os resultados obtidos neste experimento não mostraram diferenças significantes entre os grupos. A ereção peniana foi induzida pela administração SC de 80 ?g/kg de apomorfina, sendo avaliadas a latência e a freqüência de ereção. Os resultados mostraram aumento da latência e redução da freqüência de ereção peniana dos animais tratados com MOX, enquanto que a administração dos antagonistas GABAérgicos (biculina e faclofen) não alterou estes parâmetros. Por outro lado, observou-se que a biculina (antagonista GABAA) reverteu os efeitos da MOX na ereção peniana, enquanto o faclofen aumentou a freqüência de ereção peniana em ratos tratados com a MOX. Quanto aos níveis hipotalâmicos e estriatais de neurotransmissores e metabólitos, observou-se que a MOX reduziu os níveis estriatais de dopamina e de seu metabólito ácido homovanílico (HVA) e também os níveis hipotalâmicos de GABA. Estes dados sugerem que a MOX embora não interfira na motivação sexual, prejudica o desempenho sexual avaliado pela ereção peniana. Esse efeito da MOX pode ser atribuído a sua ação em receptores GABAA, os quais modulam receptores tipo B, aumentando a liberação de GABA, e 72 h depois, conseqüente redução dos níveis deste neurotransmissor no hipotálamo (uma das áreas centrais envolvidas com o comportamento sexual) e também dos níveis de dopamina e seu metabólito HVA no estriado, área do sistema nervoso central relacionada com a função motora e na qual neurônios GABAérgicos modulam a atividade de neurônios dopaminérgicos. / The moxidectina (MOX) is an antiparasitic drug used in veterinary clinic. In mammals its mechanism of action involves GABA, neurotransmitter that has an important role in the regulation of the sexual and motor behaviors. Previous data showed that MOX impair male rat\'s sexual behavior and motor coordination observed at wooden dowel. The objective of the present work was to evaluate the effects of therapeutic dose of MOX (0.2 mg/kg) in sexual motivation and penile erection of male rats, as well as to study its involvement in different central systems of neurotransmission. In all experiments the rats of experimental groups received MOX subcutaneous (SC), and the rats of control groups received 1.0 mL/kg of almonds oil (SC), and were observed after 72h. Sexual motivation test was performed in an arena with two cages, separate from the arena with a wall of wire screen; in one cage was put an intact male rat and in the other one, a sexually receptive female. In this test was measured the time that the rats stayed near of each cage. The data obtained in this experiment didn\'t show any significant differences among the groups. The penile erection (PE) was induced by 80 ?g/kg of Apomorphine (SC), being evaluated the latency to and frequency of PE. The results showed increased latency and reduction of the frequency of PE of animals treated with MOX, while the GABAergic antagonists\' administration (Biculline and Phaclofen) didn\'t change these parameters. On the other hand, it was observed that the Biculline (GABAA antagonist) reversed the effects of MOX in PE, while the Phaclofen increased the frequency of PE in rats treated with MOX. About Hypothalamic and Striatal neurotransmitters levels and their metabolites, was observed that MOX reduced Dopamine (DA) and its metabolite homovanillic acid (HVA) striatal levels and hypothalamic GABA levels. These data suggest that MOX although doesn\'t interfere in sexual motivation, impair sexual performance evaluated by penile erection. This effect of MOX can be attributed to its action in GABAA receptors, which modulate type B receptors, increasing GABA release, and consequent reduction of its levels in the Hypothalamus (one of the central areas involved with sexual behavior) and also, reduction of the DA and its metabolite HVA striatal levels. Striatum is a central nervous system area related with motor function in which GABAergic neurons modulate the activity of dopaminergic neurons.

In the field of drug addiction, relapse back to drug seeking and taking is the major unmet clinical need. The rate of relapse back to drug-taking is ~70-80% within a year of drug abstinence. Gaining a better understanding of the prolonged neuronal changes that have taken place during drug addiction may lead to the design of better anti-relapse therapies. It is now widely believed that one component of drug addiction is by aberrant learning and memory processes. To study this, we investigated synaptic changes caused by the development of drug-seeking behaviour in C57BL/6J mice. Mice were treated either with non-contingent morphine or trained to exhibit drug-seeking behaviour following morphine-induced conditioned place preference (CPP) training, hippocampal slices were taken from these animals and synaptic changes examined at the CA3-CA1 synapse using electrophysiological methods. Mice that underwent morphine CPP were demonstrated to exhibit a significant preference for the morphine paired compartment before ex vivo electrophysiological analysis. Using field recordings, both non-contingent morphine and morphine CPP treatments resulted in a reduced ability to undergo stimulus-induced LTP compared to their respective controls. Whole cell patch clamp was then utilised to further investigate these effects. Non-contingent morphine treatment resulted in both pre- and post-synaptic changes with an increased AMPA:NMDA receptor ratio, concurrent increases in cell size, and reductions in the release probability of both glutamate and GABA. Morphine CPP treatment resulted in a more variable increase in AMPA:NMDA receptor ratio (presumably by the same mechanism but in a more specific group of neurones) and GABA release probability was also decreased. There were no detected increases in cell size however, or any detected changes in glutamate release probability. These findings therefore reveal a set of synaptic adaptations in the hippocampus unique to morphine-induced behavioural change, and may provide targets for future intervention in drug addiction.

Identifying targets in cluttered scenes is critical for our interactions in complex environments. Our visual system is challenged to both detect elusive targets that we may want to avoid or chase and discriminate between targets that are highly similar. These tasks require our visual system to become an expert at detecting distinctive features that help us differentiate between indistinguishable targets. As the human brain is trained on this type of visual tasks, we observe changes in its function that correspond to improved performance. We use functional brain imaging, to measure learning-dependent modulations of brain activation and investigate the processes that mediate functional brain plasticity. I propose that dissociable brain mechanisms are engaged when detecting targets in clutter vs. discriminating between highly similar targets: for the former, background clutter needs to be suppressed for the target to be recognised, whereas for the latter, neurons are tuned to respond to fine differences. Although GABAergic inhibition is known to suppress redundant neuronal populations and tune neuronal representations, its role in visual learning remains largely unexplored. Here, I propose that GABAergic inhibition plays an important role in visual plasticity through training on these tasks. The purpose of my PhD is to investigate the inhibitory mechanisms that mediate visual perceptual learning; in particular, learning to detect patterns in visual clutter and discriminate between highly similar patterns. I show that BOLD signals as measured by functional Magnetic Resonance Imaging (fMRI) do not differentiate between the two proposed mechanisms. In contrast, Magnetic Resonance Spectroscopy (MRS) provides strong evidence for the distinct involvement of GABAergic inhibition in visual plasticity. Further, my findings show GABA changes during the time-course of learning providing evidence for a distinct role of GABA in learning-dependent plasticity across different brain regions involved in visual learning. Finally, I test the causal link between inhibitory contributions and visual plasticity using a brain stimulation intervention that perturbs the excitation-inhibition balance in the visual cortex and facilitates learning.

La stimulation haute fréquence (SHF) du noyau sous-thalamique (NST) est un traitement efficace dans l'abolition des symptômes moteurs de la maladie de Parkinson. Cependant, les mécanismes cellulaires et moléculaires qui sous-tendent ces effets sont encore loin d'être élucidés. Le laboratoire avait précédemment montré que la SHF du NST entraîne une augmentation des taux extra cellulaires de glutamate et de GABA chez les animaux éveillés ou anesthésiés dans une des principales structures cibles du NST, la substance noire réticulée (SNr). Au niveau de la SNr, les données suggèrent que la régulation de l'activité des neurones nigraux est due à la mise en œuvre conjointe d'une excitation des neurones glutamatergiques du NST et une implication des fibres de passages GABAergiques provenant du GPe et passant au voisinage de la zone sous-thalamique stimulée. L'objectif de ce travail de thèse est d'étudier l'implication des cellules gliales dans les réponses cellulaires de la SNr, sous stimulation du NST, et repose sur la mise en place d'une technique d'imagerie sur tranches de cerveau de rat adultes. Les tranches horizontales de cerveau contiennent à la fois le NST et la SNr, avec un maintien des connexions glutamatergiques subthalamonigrales.<br />Dans un premier temps, nous avons montré par immunohistochimie que la SNr contient 32% de neurones pour 68% de cellules gliales. Par la suite, nous avons montré que la SHF du NST induit une réponse calcique dans environ 12% des cellules gliales de la SNr. Ces réponses calciques enregistrées dans les cellules gliales impliquent à la fois du glutamate, du GABA et de l'ATP libérés dans la SNr lors de la SHF du NST. Cette étude a donc permis de montrer que les transmetteurs libérés dans la SNr sous l'effet de la SHF du NST, comme le glutamate et le GABA, peuvent activer les cellules gliales de cette structure. Elle a également permis de mettre en évidence la libération au sein de la SNr, d'un des principaux gliotransmetteurs, l'ATP. Ainsi, les cellules gliales de la SNr répondent à la SHF du NST, et il est probable qu'elles soient impliquées dans la modulation de l'activité neuronale de cette structure.

In comparison to other Pseudomonas, P. aeruginosa grows poorly in L-lysine as a sole source of nutrient while fast growth mutants can be obtained. The proposed catabolic pathway involves lysine decarboxylation to cadaverine and its subsequent degradation through g-glutamylation pathway to d-aminovalerate and glutarate. The lysine decarboxylase A (ldcA) gene, previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism. The ldcA gene encodes a decarboxylase which takes L-lysine but not L-arginine as substrate. Contrarily, the ldcA expression was inducible by L-arginine but not by L-lysine. This peculiar arginine control on lysine utilization was also noted from uptake experiments. The lack of lysine-responsive control on lysine catabolism and its tight connection to arginine regulatory network provided an explanation of lysine as poor nutrient for P. aeruginosa. Catabolism of cadaverine, a product from lysine decarboxylation, was investigated and compared to that of putrescine, another diamine of similar biochemical properties that is derived from arginine and ornithine. While the g-glutamylation pathway was first reported in E. coli for putrescine utilization, an expanded version of this pathway was found in P. aeruginosa with redundant enzymes for polyamine degradation. The PauR protein was identified as a transcriptional repressor of genes for the catabolism of putrescine and cadaverine, as well as their corresponding downstream metabolites, g-aminobutyrate (GABA) and d-aminovalerate (AMV). PauR shows distinct dimer configuration after glutaraldehyde crosslinkage, and possible conformational changes could be triggered by the presence of putrescine and cadaverine, but not GABA. A newly identified ABC transport system, encoded by the agtABCD operon, was found important for the uptake of GABA and AMV; and expression of which is controlled by the AgtSR two-component system. The CbrAB two-component system was proposed to regulate the catabolite repression control protein Crc through a small RNA CrcZ. A consensus CbrB recognition sequence was proposed based on the conserved palindromic nucleotide sequence in the upstream activating sequence of the crcZ promoter. Genetic studies indicated utilization of arginine, lysine and diamines (but not histidine, GABA and AMV) might be under CbrAB regulation through the CbrAB/CrcZ/Crc system in P. aeruginosa.

Thèse de doctorat : Sciences du vivant. Neurosciences : Strasbourg 1 : 2007. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 17 p.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-112).

Οι ιπποκάμπιες συνάψεις επιδεικνύουν σημαντική ικανότητα για μακρόχρονη πλαστικότητα, η οποία θεωρείται ότι είναι η βάση της μνήμης και της μάθησης. Υπάρχουν ολοένα και αυξανόμενες αποδείξεις ότι αυτή η ικανότητα διαφέρει κατά μήκος του ιπποκάμπου, με τις συνάψεις της CA1 περιοχής του κοιλιακού ιπποκάμπου να επιδεικνύουν σημαντικά μικρότερη ικανότητα για μακρόχρονη ενίσχυση (LTP) σε σύγκριση με τις αντίστοιχες συνάψεις του ραχιαίου ιπποκάμπου, όταν ενεργοποιούνται με υψηλόσυχνο ερεθισμό. Στην παρούσα εργασία, δείχνουμε ότι μία μικρή συχνότητα ερεθισμού, των 10 Hz, επάγει μακρόχρονη ενίσχυση πιο αξιόπιστα στην περιοχή CA1 του ραχιαίου απ' ό,τι του κοιλιακού ιπποκάμπου. Προτείνουμε ότι η δραστηριότητα που επάγεται από του υποδοχείς α5GABAA παίζει έναν σημαντικό ρόλο στην ρύθμιση του κατωφλίου επαγωγής του LTP ειδικά στις συνάψεις της περιοχής CA1 του κοιλιακού ιπποκάμπου. Αυτό το γεγονός μπορεί να έχει σημαντικές συνέπειες για την λειτουργική εξειδίκευση κατά μήκος του ιπποκάμπου. / The hippocampal synapses display conspicuous ability for long-term plasticity which is thought to underlie learning and memory. Growing evidence shows that this ability differs along the long axis of the hippocampus, with the ventral CA1 hippocampal synapses displaying remarkably lower ability for long-term potentiation(LTP) compared with their dorsal counterpart when activated with high-frequency stimulation. Here, we show that low frequency, 10Hz stimulation induced LTP more reliably in DH than in VH CA1 field. Blockade of alpha5 subunit-containing GABAA receptors eliminated the difference between DH and VH. We propose that α5GABAA receptor-mediated activity plays a crucial role in regulating the threshold for induction of LTP especially at the ventral CA1 hippocampal synapses. This might have important implications for the functional specialization along the hippocampus.