Conselho Nacional de Desenvolvimento Científico e Tecnológico / The synthesis of new compounds alkyl 3-azolyl propanoate e alkyl 3-azolyl propanoic acid structurally analogues to gamma-aminobutiric acid (GABA) is reported. One more time using the acetal acylation method, now the acetal derivative of 4-oxopentanoic acid (levulinic acid), the methyl 4,4- dimethoxypentanoate, were obtained the precursors methyl 7,7,7-trifluoro[chloro]-4- methoxy-6-oxo-heptenoates, 1,3-dieletrophilic precursor with alcanoate substituent. Are presented efficient and regioespecific synthetic routes from reactions of cyclocondensation [3+2] among 1,3-dieletrofilic precursors with hydroxylamine and different hydrazines, until training isoxazoles and pyrazoles, functionalized with the side chain alkyl propanoate or propanoic acid. Simultaneously to the formation of heterocycles, were studied the hydrolysis reaction of trichloromethyl group and transesterification reaction of methyl propanoate, in the same reactional medium. All products are novel, presenting a good to excellent yields, high purity and the structures were assigned by 1H NMR, 13C NMR and mass spectrometry. / A síntese de novos compostos 3-azolil-propanoatos de alquila e 3-azolilácidos propanóicos, análogos estruturalmente ao ácido gama-aminobutírico (GABA) é relatada. Novamente aplicando o método de acilação de acetais, agora ao acetal derivado do ácido 4-oxopentanóico (ácido levulínico), o 4,4-dimetoxipentanoato de metila, foram obtidos os precursores 7,7,7-triflúor[cloro]-4-metoxi-6-oxo-heptenoatos de metila, precursores 1,3-dieletrofílicos com o substituinte alcanoato. São apresentadas rotas sintéticas eficientes e regioespecíficas a partir de reações de ciclocondensação [3+2] entre os precursores 1,3-dieletrofílicos, com hidroxilamina e hidrazinas diferentes, até a formação de isoxazóis e pirazóis, funcionalizados com a cadeia lateral propanoato de alquila ou ácido propanóico. Simultaneamente à formação dos heterociclos, foram estudadas as reações de hidrólise do grupamento triclorometila e hidrólise ou transesterificação do propanoato de metila, no mesmo meio de reação. Todos os produtos obtidos são inéditos, apresentando rendimentos de bons a excelentes e pureza alta e suas estruturas foram atribuídas por RMN1H e 13C e por espectrometria de massas.

Made available in DSpace on 2016-06-02T19:22:08Z (GMT). No. of bitstreams: 1 4470.pdf: 1297687 bytes, checksum: f9b62c4b271de961a3a6053528edf8c2 (MD5) Previous issue date: 2012-02-10 / Universidade Federal de Sao Carlos / Animal models have often been used to investigate the neurobiology of emotional states (fear and anxiety). In this sense, the elevated plus maze (EPM) and the rat exposure test are effective to evaluate these states and EPM exposure (aversive stimulus) can result in activation of serotonergic pathways with projections to structures belonging to the defense system, such as , amygdala, septum, hypothalamus, periaqueductal gray (PAG) and hippocampus. The hippocampus has a large amount of serotonin receptors (5- HT) and gamma-aminobutyric acid (GABA). In the present study, we used male mice of Swiss Albino, received surgical implantation of guide cannula and subsequent administration of drugs in the ventral hippocampus. After recovery, the animals were tested in EPM (Experiment 1 and 2) or the test exposure to the rat (Experiment 3 and 4). In Experiment 1, administration of midazolam (3.0 and 30.0 nmol) produced anxiolytic effect characterized by increased percentages of entries and time spent in the open arms of the EPM reduction measures and ethological (risk assessment) as percentages of dives stretched and secured. In Experiment 2, mice received combined injections Saline + Saline, Saline + MDZ, ondansetron (OND) + Saline and MDZ (30.0 nmol) + OND (0.03 nmol, antagonist of 5-HT3). Combined treatment of Sal + MDZ produced anxiolytic effect and this effect was reversed by the combined administration of OND + MDZ. The porcentanges of entries and time spent in open arms were lower (P> 0.05) than those found in group Sal + MDZ. Experiment 3 showed the effects of exposure of mice in the presence of mouse toy (RB = neutral stimulus) or mouse real (VR = aversive stimulus, Long Evans rats), under the administration of midazolam (3.0 14 and 30.0nmol). The animals were exposed to RV shortening the holding box (model effect) compared to animals exposed to RB. Animals treated with MDZ in two doses, there was an increase in transitions between the sides of the apparatus, increased time in the area of operation and increases the latency of escape to the protected area and contact time with the grid. In Experiment 4, we evaluated the effect of combined injection of midazolam and ondansetron protocol (Experiment 2). The MDZ 30.0 nmol produced anxiolytic effects and the blockade of this effect when the mice were combined administration of ondansetron and midazolam in the ventral hippocampus. Data from this study suggest that, first, control over emotional reactions and defense of the ventral hippocampus of mice exposed to EPM test or exposure to the rat are mediated via GABABenzodiazepines. Furthermore, there is a likely cross-modulation between GABAergic interneurons and 5-HT3, for blocking 5-HT3 via ondansetron can decrease the GABA release. / Modelos animais têm sido frequentemente utilizados para a investigação da neurobiologia dos estados emocionais (ansiedade e medo). Neste sentido, o labirinto em cruz elevado (LCE) e teste de exposição ao rato são eficazes para avaliar esses estados e a exposição ao LCE (situação aversiva) pode resultar em ativação das vias serotonérgicas com projeções para estruturas pertencentes ao sistema de defesa, tais como, amídala, septo, hipotálamo, substância cinzenta periaquedutal (SCP) e hipocampo. O hipocampo apresenta grande quantidade de receptores de serotonina (5-HT) e do ácido gama-aminobutírico (GABA). No presente estudo, foram utilizados camundongos machos, da cepa Suíco Albino, que receberam implantação cirúrgica de cânulas guia e posterior administração de drogas no hipocampo ventral. Após recuperação, os animais foram avaliados nos testes LCE (Experimento 1 e 2) ou exposição ao rato (Experimento 3 e 4). No Experimento 1, a administração de midazolam (3,0 e 30 nmol) produziu efeito ansiolítico caracterizado pelo aumento das porcentagens de entradas e tempo gasto nos braços abertos do LCE e redução de medidas etológicas (avaliação de risco) como porcentagens de mergulhos e esticadas protegidas. No Experimento 2, os camundongos receberam injeção combinada de Salina+Salina, Salina+MDZ, ondansetron (OND)+Salina e MDZ (30 nmol) + OND (0,03 nmol, antagonista dos receptores 5-HT3). O tratamento combinado de Sal+MDZ produziu, efeito ansiolítico sendo este revertido pela administração combinada de OND+MDZ. As porcentagens de entradas e tempo gasto nos braços abertos foram menores (P>0,05) do que àqueles encontrados no grupo Sal+MDZ. O Experimento 3 mostrou os efeitos da exposição dos camundongos na presença 12 do rato de brinquedo (RB= estímulo neutro) ou rato de verdade (RV= estímulo aversivo, rato Long Evans), sob a administração de Midazolam (3,0 e 30 nmol). Os animais expostos ao RV apresentaram diminuição do tempo de exploração da caixa (efeito do modelo) quando comparados aos animais expostos ao RB. Para os animais tratados com MDZ nas duas doses, houve aumento nas transições entre os lados do aparato, aumento do tempo na área de exploração e aumentos da latência de fuga para a área protegida e tempo de contato com a grade. No Experimento 4, foi avaliado o efeito da injeção combinada de midazolam e ondansetron (conforme descrito no Experimento 2). O MDZ 30 nmol produziu efeito ansiolítico e a administração combinada de ondansetron e midazolam no hipocampo ventral, reverteu este efeito. Os dados deste trabalho sugerem que, o controle sobre as reações emocionais e de defesa do hipocampo ventral de camundongos expostos ao LCE ou ao teste de exposição ao rato são mediados via receptores GABA-Benzodiazepínicos. Além disso, provavelmente ocorre modulação cruzada entre os interneurônios GABAérgicos e os serotoninérgicos do tipo 5-HT3, pois o bloqueio desses receptores com o ondansetron, pode diminuir a liberação de GABA.

Made available in DSpace on 2016-06-02T19:22:13Z (GMT). No. of bitstreams: 1 6708.pdf: 2113975 bytes, checksum: 30511a391fdfda439671c285b41c07c1 (MD5) Previous issue date: 2015-02-27 / Financiadora de Estudos e Projetos / The central nucleus of the amygdala (CeA) and the lateral parabrachial nucleus (LPBN) are important areas for the control of sodium appetite. The functional integrity of the CeA is critical to sodium and water intake when LPBN the inhibitory mechanisms are deactivated. Therefore, the aims of this study were to investigate the role of different neurotransmitters of the CeA in the control of sodium and water intake induced by a) sodium depletion and b) after blockade of LPBN inhibitory mechanisms. Male Holtzman rats with stainless steel guide cannula implanted bilaterally only in CeA or both into the CeA and LPBN were used. Sodium (0.3 M NaCl) intake was evaluated in satiated, hyperosmotic and sodium-depleted rats. In sodium-depleted animals, bilateral administration of α2- adrenergic/imidazoline receptor agonist moxonidine (10 nmol) into CeA reduced 0.3 M NaCl and water intake. Moreover, bilateral injections of muscimol (0.25 nmol) into CeA reduced sodium intake without change water intake. Oxytocin receptors activation or its blockade in the CeA, blockade of muscarinic cholinergic receptor or activation of 5HT2A/2C serotonergic receptor into the CeA did not change 0.3 M NaCl or water intake in sodium-depleted animals. Bilateral injections of opioid receptor antagonist naloxone (40 μg) into the CeA did not significantly change 0.3 M NaCl and water intake in sodium-depleted animals. However, sodium and water intake induced by bilateral injections of muscimol (0.5 nmol) into the LPBN in satiated animals were completely abolished after bilateral injections of naloxone (40 μg) into CeA. Furthermore, paradoxical sodium intake observed in rats that received oral gavage with 2 M NaCl (2 ml/rat) combined with bilateral injections of moxonidine (0.5 nmol) in LPBN was also blocked by bilateral naloxone (40 μg) into the CeA. 0.3 M NaCl and water intake induced by bilateral muscimol injections (0.5 nmol) into LPBN in satiated animals were abolished by blocking AT1 angiotensin receptors (losartan - 20 μg) in CeA. In sodium-depleted animals, bilateral injections of losartan (20 μg) into the CeA significantly reduced water intake but did not affect sodium intake. Bilateral injections of the aldosterone antagonist RU 28318 (50 ng) did not change sodium and water intake induced by sodium depletion. Present results suggest that ocitocinergic, cholinergic muscarinic, 5-HT2A/2C serotonergic receptors and aldosterone receptors of the CeA do not participate in the control of 0.3 M NaCl intake induced by sodium depletion. Moreover, present results suggest that GABAergic and α2-adrenergic receptors of the CeA have an inhibitory role for sodium appetite in this situation. Although opioids and angiotensinergic mechanisms of the CeA apparently do not contribute to sodium depletion-induced sodium intake, opioidergic and angiotensinergic mechanisms in CeA are essential for sodium intake when the LPBN inhibitory mechanisms are blockade by LPBN muscimol injection. In addition, opioidergic mechanisms in CeA are also essential for the paradoxical sodium intake by hyperosmotic animals when the inhibitory mechanisms are attenuated by LPBN moxonidine. Therefore, the activation of opioidergic and angiotensinergic receptors of the CeA is required for sodium intake observed after removal or attenuation of LPBN inhibitory mechanisms. / O núcleo central da amígdala (CeA) e o núcleo parabraquial lateral (NPBL) são regiões importantíssimas para o controle da ingestão sódio e água. A integridade funcional do CeA é fundamental para a ingestão de sódio e água quando ocorre redução da atividade dos mecanismos inibitórios do NPBL. Portanto, os objetivos do presente estudo foram investigar a participação de alguns neurotransmissores no CeA no controle da ingestão de sódio e água induzida a) por desidratação extracelular e b) após o bloqueio dos mecanismos inibitórios do NPBL. Para tanto foram utilizados ratos Holtzman com cânulas guia de aço inoxidável implantadas bilateralmente apenas no CeA ou bilateralmente no CeA e no NPBL. A ingestão de NaCl 1,8% foi avaliada em animais saciados, hiperosmóticos ou com depleção de sódio. Em animais depletados de sódio, a administração bilateral do agonista de receptores adrenérgicos α2/imidazólicos moxonidina (10 nmol), assim como a de muscimol (0,25 nmol) no CeA reduziram a ingestão de NaCl 1,8%. A ativação ou bloqueio dos receptores de ocitocina, o bloqueio de receptores muscarínicos, ativação de receptores serotenérgicos 5-HT2A/2C, ou ainda o bloqueio de receptores de aldosterona no CeA não modificaram a ingestão de sódio e água. Contudo, injeções bilaterais de losartan (20 μg) no CeA reduziram a ingestão de água, mas não modificaram a ingestão de sódio em animais depletados. Já a ingestão de sódio e água induzidas por muscimol (0,5 nmol) no NPBL em animais saciados foram abolidas após bloqueio de receptores AT1 de angiotensina com administração de losartan no CeA. Administração bilateral de naloxona (40 μg) no CeA não modificou a ingestão de NaCl 1,8% e de água em animais desidratados. No entanto, a ingestão de sódio e água induzidas por injeções bilaterais de muscimol (0,5 nmol) no NPBL em animais saciados foram completamente bloqueadas após injeções bilaterais de naloxona no CeA. Além disso, a ingestão paradoxal de NaCl 0,3 M observada em ratos hiperosmóticos após o tratamento bilateral de moxonidina no NPBL também foi bloqueada pelas injeções de naloxona no CeA. Os presentes resultados sugerem que receptores ocitocinérgicos, colinérgicos muscarínicos, serotoninérgicos 5-HT2 e receptores de aldosterona no CeA não participam do controle da ingestão de NaCl 1,8% induzida por depleção de sódio. Por outro lado, os presentes resultados sugerem que receptores GABAérgicos e receptores adrenérgicos α2 no CeA apresentam um papel inibitório para o apetite ao sódio nessa situação. Embora os mecanismos opióides e angiotensinérgicos no CeA aparentemente não contribuam para a ingestão de sódio induzida pela depleção de sódio, os mecanismos opióides e angiotensinérgicos no CeA são essenciais para a ingestão de sódio observada quando os mecanismos inibitórios do NPBL são desativados pela ação do muscimol nessa área. Além disso, os mecanismos opióides no CeA também são essenciais para a ingestão paradoxal de sódio em animais hiperosmóticos quando os mecanismos inibitórios são atenuados pela ação da moxonidina no NPBL. Portanto, a ativação de receptores opióides e de receptores angiotensinérgicos no CeA é necessária para a ingestão de sódio observada após a remoção ou atenuação dos mecanismos inibitórios NPBL.

In mammals, tectal and tegmental divisions of the brainstem are involved in attentional mechanisms and responses to threatening stimuli such as predators. These centers are regulated by ascending connections, but the anatomical and neurochemical details of this drive are not fully known. The nucleus incertus (NI) in the pontine tegmentum is the source of ascending GABA projections to forebrain cognitive/emotional centers and NI neurons contain a number of neuropeptides, including relaxin-3 (RLN3). Tract-tracing studies have described NI projections within the tectum; and in this study we describe the distribution of relaxin-3 fibers within tectal and tegmental areas/nuclei of rat brain. RLN3-immunostained sections were also reacted with antisera against other neurochemical markers, as synaptophysin, nitric oxyde synthetase, tyrosine hydroxylase, calbindin, calretinin and 5-HT, to assist in demarcation of the area. RLN3-containing fibers were concentrated in the medial, olivary and ventrolateral pretectal nuclei; the medial intermediate grey layer of superior colliculus; and the pericentral area of inferior colliculus. Some labeled fibers were also detected in the cuneiform, parabigeminal and sagulum nuclei. RLN3 fibers were concentrated around the commissural bundles along the midline of the tectum, in the dorsal columns of the periaqueductal gray and in the dorsal raphe. In all areas, RLN3 and synaptophysin staining co-existed, indicating an association of the peptide with synapses. RLN3 projections target structures within the tectum and tegmentum that comprise the defensive system involved in detection of and response to unexpected threatening stimuli. NI neurons, which are a major source of RLN3 fibers and express corticotrophin-releasing factor receptors, may contribute to these responses following activation by stress-related stimuli. / Nos mamíferos, as divisões tectal e tegmental do tronco cerebral estão envolvidas em mecanismos de atenção e de respostas a estímulos ameaçadores, como os predadores. Esses centros são regulados por conexões ascendentes, mas os detalhes anatômicos e neuroquímicos desta unidade não são totalmente conhecidos. O núcleo incertus (NI) no tegmento pontino é a fonte de projeções ascendentes de GABA para prosencéfalo cognitivo/centros emocionais, e os neurônios do NI contêm alguns neuropeptídeos, incluindo relaxina-3 (RLN3). Estudos com traçadores descreveram projeções do NI para o tectum, e neste estudo, descrevemos a distribuição de fibras relaxina-3 nas áreas tectal e tegmental. Foram feitas imunocitoquímica para RLN3 conjugadas com outros marcadores neuroquímicos, tais como, sinaptofisina, óxido nítrico sintase neuronal, tirosina hidroxilase, calbindina, calretinina e 5-HT, para ajudar na demarcação da área. Fibras contendo RLN3 estavam concentradas na nucleos pretectais ventrolaterais, olivar e medial; na camada intermediária medial cinzenta do colículo superior; e na área pericentral de colículo inferior. Algumas fibras marcadas também foram detectadas nos núcleos cuneiforme, parabigeminal e sagulum. Fibras RLN3 foram concentradas em torno da feixes comissurais ao longo da linha mediana do tectum, nas colunas dorsais da substância cinzenta periaquedutal e na rafe dorsal. Em todas as áreas, a marcação para RLN3 e sinaptofisina co-existiu, indicando uma associação do péptideo com as sinapses. Estruturasalvo para as projeções de RLN3 do tecto e tegmento compõem o "sistema defensivo" envolvidos na detecção e resposta a estímulos ameaçadores. Neurônios do NI, são uma importante fonte de fibras RLN3 e expressam fatores de liberação de receptores para corticotropina, que podem contribuir para a respostas ao estímulos de estresse.

Le cervelet est un petit cerveau dans le cerveau. Il contient plus de la moitié du nombre total de neurones du cerveau. Sa structure très régulière est bien connue, toutefois son rôle demeure mystérieux. Le développement essentiellement postnatal du cervelet chez les rongeurs permet d’y étudier la formation activité-dépendante du réseau de neurones. C’est aussi le siège où s’opèrent diverses formes de plasticité synaptique, ce qui en fait un modèle d’étude idéal pour la plasticité synaptique développementale et adulte. Au cours de cette thèse, à l’aide d’enregistrements électophysiologiques en patch-clamp et en extracellulaire sur des tranches aigües de cervelet de souris et grâce aux techniques immunohistochimiques, j’ai étudié trois acteurs importants de la plasticité synaptique et du développement des cellules de Purkinje, les neurones centraux du cortex cérébelleux. Nous avons démontré que l’activation du récepteur métabotropique glutamatergique de type 1 (mGlu1) déclenche l’activation et l’ouverture de GluD2, un récepteur nécessaire au développement et à la plasticité des synapse des cellules de Purkinje (CPs). Nous avons également mis en évidence que les Pannexines 1, des canaux potentiellement impliqué dans la synchronisation neuronale récemment découverts et encore mal caractérisés, sont exprimées par les cellules de Purkinje Zebrine II –immunopositives, suivant les bandes parasagittales que délimitent les microdomaines du cervelet. Enfin, nous avons étudié la physiologie du cortex cérébelleux des souris néonatales, cherchant à caractériser les différents acteurs essentiels à l’activité neuronale de ce cortex en développement très particulier et peu étudié. L’activation du récepteur GluD2 médiée par mGlu1 dans la synapse entre Fibre Parallèle et cellule de Purkinje (synapse PF-PC). GluD2 est classifié parmi les récepteurs ionotropiques glutamatergique, pourtant aucun ligand n’est capable d’induire l’ouverture de son canal. Nous avons identifié pour la première fois un mécanisme physiologique d’ouverture du canal de GluD2 en démontrant que l’activation de mGlu1 déclenche l’ouverture du canal de GluD2 pour une voie intracellulaire, aussi bien dans un système d’expression en culture que dans les tranches aigues de cervelet murin. Cela nous permettra d’étudier la contribution du courant médié à travers GluD2 dans la plasticité à long terme, avec des perspectives totalement nouvelles. L’expression de Pannexine 1 par les CPs se superpose aux stries Zebrine II- immunopositives du cervelet. Les CPs adultes constituent une population hétérogènes, les différents sous-types étant organisés sur le plan parasagittal. Nous avons montré que l’expression des protéines Pannexine 1 (Panx1) We have shown that Pannexin1 (Panx1) déssine un gradient rostrocaudal discontinu dans les lobules de tranches parasagittales. Sur les coupes coronales, leur distribution forme une série de bandes parasagittales. Les canaux Panx1 médient la libération d’ATP en réponse à divers stimuli et pourrais de cette façon contribuer à une activité neuronale orientée sur le plan parasagittal en réponse aux signaux des fibres parallèles. Caractérisation de l’activité GABAergique des CPs immatures dans les souris néonatales. Le cortex cérébelleux entre les jours postnataux P0 et P4 consistent principalement en une multicouche dense de CPs fortement interconnectées. A cet âge, les CPs sont remplies de GABA extrasynapstique qui est libéré dans le milieu extracellulaire par un mécanisme qui n’est pas clairement identifié. Nos recherches préliminaires sur la première semaine de développment postnatal, nous montrons que l’activation de récepteur au GABA de type A induit une réponse excitatrice chez les CPs. Avec notre préparation, cet effet est indépendant de la présence de corps cétoniques ou de lactate comme substrats énergétiques dans le milieu extracellulaire. (...) / The cerebellum is a little brain in the brain. It houses more than half the total number of neurons in the brain. Its crystalline structure is very well known but, still, its function remain unclear to date. Its mainly postnatal development in rodents allows the study of the physiology of activity-dependent neuronal wiring. It is also the place of many types of neuronal plasticity, making it an ideal model to study both developmental and adult synaptic plasticity. In this thesis, using mainly patch-clamp and extracellular recordings in cerebellar slices as well as immunohistochemistry in mice, I have studied three important actors of synaptic plasticity and development in the Purkinje cells, the principal neurons of the cerebellar cortex. We have established that the type 1metabotropic glutamate receptor (mGlu1) triggers the gating of GluD2, a receptor necessary for Purkinje cells (PCs) synapses development and synaptic plasticity. We have also shown that the Pannexins 1, some channels likely involved in neuronal synchronization that have been recently discovered but yet remain poorly characterized, are expressed by Zebrin II immmunopositive Purkinje cells in the classical Zebra stripes formed by microdomains of the cerebellum. Last, we have studied the physiology of the primitive cerebellar cortex in neonatal mice, establishing the first elements of the neuronal activity of this very particular developing cortex at a stage still very poorly characterized. The mGlu1-mediated gating of Glu2D receptors at Parallel Fiber to PC (PF-PC) synapse. GluD2 are classified among ionotropic glutamate receptors, but no ligand has proved capable of gating their channel. We have identified for the first time a physiological mechanism of gating GluD2 channels by demonstrating that the activation of mGlu1 triggers the opening of GluD2 channels through intracellular pathways, both in expression systems and in acute murine cerebellar slices. This will allow us to study the contribution of GluD2-mediated current in long-term plasticity in a totally new way. Expression of Pannexin1 by PCs matches with adult Zebrin II immunopositive cerebellar stripes. Adult PCs constitute an heterogeneous population, the different subtypes being parasagittaly organized. We have shown that Pannexin1 (Panx1) proteins expression by PCs, draw a rostrocaudal discontinuous gradient in lobules of parasagittal slices. In transverse slices, their distribution forms an array of parasagittal stripes. Panx1 channels mediate ATP release in response to various stimuli and may in this way contribute to parasagittally oriented response to PF inputs. Characterization of GABAergic activity of immature Purkinje cells of newborn mice. The cerebellar cortex during postnatal days P0 to P4 essentially consists in a dense multilayer and highly interconnected network of PCs. At this age, PCs are filled with extrasynaptic GABA which is released in the extracellular space by a mechanism that is not clear. In our preliminary investigation of first week postnatal development, we show that activation of GABA-A receptors leads to excitatory responses in PCs. In our preparation, this effect is independent of the presence of keton bodies or lactate as energetic substrates in the extracellular medium. The complete inhibition of spontaneous discharge of PCs by Panx1 channel blockers, suggests that they mediate ion fluxes or release of neuromediators, such as ATP or GABA.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

La stimulation magnétique transcrânienne (SMT) et la spectroscopie par résonance magnétique (SRM) sont des techniques non-invasives permettant de quantifier l’activité GABAergique et glutamatergique du cerveau. La SMT et la SRM ont plusieurs applications en clinique et en recherche. En effet, ces outils peuvent être utilisés afin de déterminer l’efficacité d’un traitement ou la progression d’un processus pathologique. Cependant, malgré leur utilisation croissante dans le domaine médical, une certaine incertitude demeure quant aux substrats neurochimiques de ces techniques et à la stabilité à long terme des données acquises par SMT et SRM. Donc, dans un premier temps, la stabilité à long terme de plusieurs mesures prises par SMT et par SRM a été étudiée. En second lieu, afin de mieux comprendre quelles composantes du système GABAergique sont ciblées par ces deux techniques, des mesures de SRM et de SMT ont été obtenues après l’administration d’une benzodiazépine, le lorazépam, selon un devis expérimental randomisé, croisé, à double-aveugle et contrôlé par placébo. Deux articles composent cette thèse. Le premier article fait état d’une étude longitudinale, auprès d’adultes en santé, ayant pour but de déterminer la stabilité à long terme des concentrations de GABA et de Glx (glutamate + glutamine) obtenues par SRM ainsi que la stabilité des mesures d’inhibition et de facilitation corticale obtenues par SMT (rMT : seuil moteur au repos, %MSO : pourcentage d’intensité maximale du stimulateur, SICI : inhibition intra-corticale courte, LICI : inhibition intra-corticale longue, ICF : facilitation intra-corticale). Il a été démontré que les niveaux de GABA et de Glx sont stables au cours d’une période de trois mois. Alors que les mesures SMT de seuil moteur au repos, d’excitabilité corticale et de période corticale silencieuse sont stables à travers le temps, l’inhibition corticale à court intervalle et à long intervalle ainsi que la facilitation corticale sont beaucoup plus variables. Le deuxième article vise à comprendre la dissociation dans la sensibilité des mesures de SMT et SRM à refléter différentes facettes de l’activité GABAergique du cortex moteur. L’article porte sur une étude dans laquelle du lorazépam a été administré à des participants adultes en santé selon un devis randomisé, croisé, à double-aveugle et contrôlé par placébo. Des données SRM (GABA et Glx; cortex sensorimoteur et occipital) ainsi que des mesures SMT (cortex moteur) ont été obtenues suivant l’administration de lorazépam (ou de placébo). Il a été démontré que la prise de lorazépam réduisait les niveaux de GABA occipitaux, augmentait l’inhibition corticale et réduisait l’excitabilité du cortex moteur. La prise de médicament n’avait pas d’effet sur les autres mesures obtenues. De plus, il a été trouvé que l’effet du traitement sur l’inhibition corticale dépendait des concentrations endogènes de GABA dans le cortex sensorimoteur; une plus grande concentration de GABA étant prédictive d’une plus grande inhibition corticale suivant la prise de lorazépam. Dans leur ensemble, les résultats provenant des deux articles présentés dans cette thèse permettent de conclure que les mesures SRM des divers neurométabolites sont stables à long terme dans le cortex moteur et pourraient potentiellement servir de marqueurs dans l’évaluation de l’efficacité d’un traitement ou de l’évolution de processus pathologiques. Par contre, bien que certaines mesures SMT soient stables à long terme (rMT, %MSO, CSP), d’autres sont beaucoup plus variables (SICI, LICI, ICF); ainsi, la prudence est conseillée dans l’interprétation de ces mesures lors d’études cliniques. De plus, les effets différents que produit la prise de lorazépam sur les mesures SRM et SMT supportent la théorie selon laquelle les deux techniques n’ont pas les mêmes substrats neurochimiques. En effet, alors que les mesures TMS d’inhibition corticale refléteraient l’activité phasique des récepteurs GABAA, le signal SRM de GABA serait majoritairement intracellulaire et ne représenterait pas la neurotransmission GABAergique. / Transcranial magnetic stimulation (TMS) and magnetic resonance spectroscopy (MRS) are non-invasive techniques that allow the measurement of GABAergic and glutamatergic activity in the brain. TMS and MRS can be used to assess inhibitory and excitatory mechanisms, treatment response or disease presence and progression in vivo. However, despite their growing use in research and medical settings, ambiguity remains regarding their neurochemical substrates and long-term reproducibility. The goal of the present thesis is twofold. First, the long-term stability and reliability of various MRS and TMS measurements, obtained in the motor cortex, was investigated. Second, to better understand which aspects of the GABAergic network are targeted by the two techniques, TMS and MRS measures reflecting cortical inhibition and excitation were obtained following lorazepam administration using a placebo-controlled, double-blind, randomized, crossover design. Two articles comprise this thesis. The first article is a longitudinal assessment of the stability and reliability of MRS-GABA and Glx (glutamate + glutamine) and TMS measures of cortical inhibition and facilitation in the sensorimotor (SMC) cortex of healthy adults. It was determined that MRS-GABA and MRS-Glx are stable over a three-month interval. TMS measures of resting motor threshold (rMT), cortical excitability (% maximum stimulator output; MSO) and cortical silent period (CSP) were also found to be stable and reliable. However, paired-pulse TMS measures such as short-interval cortical inhibition (SICI), long-interval cortical inhibition (LICI) and intracortical facilitation (ICF) had greater variability. The second article aims to understand the differential sensitivity of TMS and MRS with respect to GABAergic activity in the primary motor cortex. It is based on the results and conclusions of a placebo-controlled, double-blind, randomized, crossover study, where benzodiazepine lorazepam was given to healthy adult volunteers. Magnetic resonance spectroscopy (GABA and Glx) was performed in the sensorimotor cortex and occipital cortex (OC). TMS measurements were acquired in the motor cortex only. MRS and TMS measures of cortical inhibition and excitability (rMT, input/output (I/O) curve, SICI, LICI, ICF, CSP) were obtained following lorazepam or placebo administration. Lorazepam was found to decrease occipital GABA concentration, increase motor cortical inhibition and decrease cortical excitability. Lorazepam administration had no effect on other neurometabolites or TMS measurements. The effect of Lorazepam on short-interval cortical inhibition was found to depend on endogenous GABA levels in the SMC; higher GABA concentrations predicted a greater increase in SICI following drug intake. Taken together, the studies presented in this thesis indicate that MRS neurometabolite levels are stable over time and may thus potentially serve as markers for the monitoring of disease progression and treatment response. However, while some TMS measures have good long-term stability (rMT, %MSO, CSP), others are not as reliable nor stable (SICI, LICI, ICF); care must be taken in clinical settings. Furthermore, the differential effects of lorazepam on MRS and TMS measures support the idea that the two techniques probe different aspects of the GABAergic system. Whereas TMS measures of cortical inhibition reflect phasic GABAA receptor activity, MRS-GABA primarily reflects intracellular, non-neurotransmitter metabolic GABA.

Indiana University-Purdue University Indianapolis (IUPUI) / Extrasynaptic GABAA receptors, often identified as those containing both α4 and δ subunits, appear to be a target for the actions of alcohol (ethanol) at relatively low concentrations, perhaps suppressing the activity of GABAergic interneurons which regulate activity in the mesolimbocortical circuit. Pharmacological studies in rodents using the δ-subunit selective agonist Gaboxadol (THIP) have found both promotional and inhibitory effects on alcohol consumption. The goal of this project was to determine the role of extrasynaptic GABAA receptors located in the infralimbic cortex (ILC) in the binge-like alcohol intake of male C57BL/6J (B6) mice. The ILC is of interest due to its demonstrated involvement in stress reactivity and alcohol exposure has been shown to interfere with extinction learning; impairments of which may be related to inflexible behavior (i.e. problematic alcohol consumption). Adult male B6 mice were bilaterally implanted with stainless steel guide cannulae aimed at the ILC and were offered limited access to 20% ethanol or 5% sucrose for 6 days. On day 7, mice were bilaterally injected with 50 or 100 ng THIP (25 or 50 ng per side respectively) or saline vehicle into the ILC. It was found that the highest dose of THIP (100 ng/mouse) increased alcohol intake relative to vehicle controls, although control animals consumed relatively little ethanol following infusion. Furthermore, THIP had no effect on sucrose consumption (p > 0.05), suggesting that the effect of THIP was selective for ethanol consumption. Together, these findings suggest that the mice that consumed ethanol may have been particularly reactive to the microinfusion process relative to animals that consumed sucrose, perhaps because ethanol consumption was not as reinforcing as sucrose consumption. In addition, the observation that THIP effectively prevented the decrease in ethanol intake on day 7 induced by the microinjection process may be related to a role for the ILC in adaptive learning processes, which in turn, promote behavioral flexibility.

Dans ce manuscrit est proposée une méthode originale pour élaborer des N-hétérocycles saturés possédant un groupe trifluorométhyl (Tfm) en position alpha. Ces composés fluoro-organiques sont des cibles attrayantes, dues à leur intérêt potentiel élevé pour l'industrie pharmaceutique ainsi que pour l'agrochimie. En employant une méthode développée au laboratoire, basée sur une réaction de type Mannich intramoléculaire des béta-aminocétals sur des dérivés carbonylés, nous avons préparé un éventail de structures pipéridiniques originales. Tout d'abord nous avons appliqué cette méthodologie pour la synthèse diastéréosélective des Tfm-pipéridines. Les béta-aminocétals nécessaires à cette étude ont été facilement obtenus par l'addition conjuguée du phtalimide sur les énones correspondantes, suivi d'une céto-protection et d'une hydrazinolyse. Ces intermédiaires ont été soumis aux réactions de Mannich intramoléculaires avec divers aldéhydes, donnant ainsi un accès stéréosélectif aux analogues trifluorés de la pipéridine naturelle. Des réactions chimiques sur ces composés ont également permis la synthèse de nouveaux substrats tels que les 4-aza et 4-hydroxy Tfm-piperidines. Une version énantiosélective de cette stratégie alors a été réalisée avec succès au départ de béta-aminocétals énantiomériquement enrichi, préparés à partir d'une oxazolidine fluoré (FOX) par une réaction de type Mukaiyama-Mannich. L'emploi de ces nouvelles amines énantio-enrichies dans des conditions identiques de cyclisation avec différents aldéhydes a fourni une série de Tfm-pipéridines enantiopure. Cette stratégie a été validée par la première synthèse asymétrique des analogues trifluorométhylés des acides pipécoliques, des analogues l'acide gamma-aminobutyrique (GABA) et des structures polycycliques comme les indolizidines, contenant un groupe trifluorométhyle.

Bâti sur des données ayant identifié le noyau suprachiasmatique de l'hypothalamus (NSC), composante centrale de l'horloge circadienne des mammifères, comme une structure à fortes potentialités de plasticité structurale, ce travail de thèse a visé à déterminer si la synchronisation photique de l’horloge pouvait mettre en jeu une telle plasticité. Par une analyse ultrastructurale, nous démontrons que le cycle jour/nuit s’accompagne de remaniements neurono-gliaux du NSC affectant différentiellement les neurones à VIP, principales cibles d'intégration des messages rétiniens, et les neurones à vasopressine. Ces remaniements seraient bien liés à la synchronisation photique de l’horloge dans la mesure où, dans le NSC, l’expression rythmique de la GFAP, une protéine du cytosquelette des astrocytes, est abolie sous obscurité constante, tandis que les fluctuations journalières des glucocorticoïdes circulants, connues pour moduler la synchronisation photique, sont apparues être impliquées dans la régulation rythmique de cette plasticité. Grâce au développement d’une méthode d’analyse quantitative en imagerie confocale, nous montrons une augmentation diurne de la densité d’innervation synaptique, glutamatergique et non glutamatergique, sélective des neurones à VIP. Des données en microscopie électronique indiquent que ce remodelage synaptique n’implique pas les synapses GABAergiques, au moins au niveau dendritique des neurones à VIP. Ce travail a permis d’identifier formellement la composante centrale de l’horloge circadienne, le NSC, comme une structure capable d’adapter, de manière rapide et réversible, son architecture gliale et synaptique aux exigences fonctionnelles. / This thesis work was based on data identifying the suprachiasmatic nucleus of the hypothalamus (SCN), the central component of the mammalian circadian clock, as a structure with a high potential for structural plasticity. It was aimed at determining whether the photic synchronization of the clock may involve such a plasticity.Using an exhaustive ultrastructural analysis, we showed that the SCN underwent day/night rearrangements of its neuronal-glial network, which affected differentially the VIP (vasoactive intestinal peptide)-synthesizing neurons, the main targets for retinal signals, and the vasopressin-synthesizing neurons. The rearrangements appeared to be linked with photic synchronization as the rhythmic expression of GFAP, an astrocytic cytosqueletal protein, was abolished in SCN under constant darkness. Moreover we found the daily fluctuations of circulating glucocorticoids, known as modulators of photic synchronization of the clock, to be involved in the rhythmic regulation of SCN structural plasticity.Thanks to the development of a quantitative analysis method in confocal imaging, we showed a selective increase at daytime in the glutamatergic and non-glutamatergic synapses made on the VIP neurons. Complementary electron-microscopic data indicated that the synaptic remodeling did not involve GABAergic synapses, at least at the dendritic level of VIP neurons.This work permitted to formally identify the central component of the circadian clock, the SCN, as a structure able to adapt, rapidly and reversibly, its glial and synaptic architecture to functional needs.