Past research has demonstrated that the motivational effects of opiates causes a change in ventral tegmental area (VTA) γ-amino butyric acid (GABA) subtype A receptor [GABA(A)R] complexes in opiate-dependent animals, which switch from a GABA-induced hyperpolarization of VTA GABA neurons to a GABA-induced depolarization. Previously shown in naïve animals, superfusion of ethanol (IC50 = 30 mM) and the GABA(A)R agonist muscimol (IC50 = 100 nM) decreased VTA GABA neuron firing rate in a dose-dependent manner. The aim of this study was to evaluate VTA GABA neuron excitability, GABA synaptic transmission to VTA GABA neurons, and a potential switch in GABA(A)R functionality produced by alcohol dependence. To accomplish these studies, we used standard whole-cell, perforated patch, and attached-cell mode electrophysiological techniques to evaluate chronic ethanol effects on VTA GABA neurons in CD-1 GAD GFP mice, which enable the visual identification of GABA neurons in the slice preparation. In order to more conclusively demonstrate synaptic plasticity in VTA neurons associated with alcohol dependence, three studies were proposed to elucidate the mechanism underlying the switch in GABA synaptic function with dependence. First, we evaluated the effects of withdrawal from chronic ethanol exposure on muscimol-induced inhibition of VTA GABA neuron firing rate. Second, we evaluated the effects of withdrawal from chronic ethanol exposure on GABA(A)R-mediated synaptic responses in VTA GABA neurons by looking at eIPSCs, and corresponding changes in VTA DA neuron firing rate. Third, we evaluated chloride reversal potentials in VTA GABA neurons using perforated patch recordings in VTA GABA neurons.Through these studies, we found that there was less sensitivity to muscimol in animals treated with ethanol versus air-exposed controls. However, it is yet to be shown more conclusively if VTA GABA neurons undergo a switch in GABA(A)R function with chronic ethanol.

Orientadora: Profª Drª Raquel Vecchio Fornari / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2015. / O cortex insular (CI), ou insula, conquistou nos ultimos anos um lugar de destaque na area cientifica por seu suposto envolvimento em processos emocionais e cognitivos. No rato, como nos seres humanos, o CI pode ser dividido em duas sub-regioes funcionalmente heterogeneas. Embora a maioria dos estudos realizados em animais tenha mostrado um envolvimento da regiao mais rostral (CI anterior) na memoria gustativa, outros sugerem um papel mais amplo, abrangendo desde o reconhecimento de objetos ate o processamento de memorias espaciais e aversivas. A regiao mais caudal (CI posterior), por sua vez, recebe aferencias multissensoriais e supoe-se que esteja envolvida em processamento multissensorial e nociceptivo. Entretanto, pouquissimos trabalhos avaliaram a participacao dessa sub-regiao posterior em tarefas de memoria, com resultados inconclusivos. Nao havia, ate o momento, nenhum trabalho que tivesse investigado isoladamente o papel de ambas as sub-regioes do CI na consolidacao da memoria emocional. Portanto, o objetivo deste estudo foi investigar os efeitos da inativacao temporaria do CI anterior e posterior sobre a consolidacao da memoria de medo de tarefas de condicionamento de medo ao contexto e ao som. Ratos Wistar de 3 meses de idade passaram por cirurgia estereotaxica para implante de canulas-guia bilaterais no CI anterior ou posterior. Os animais tiveram pelo menos 7 dias de recuperacao e foram manipulados por 3 dias antes do inicio do procedimento comportamental. Para o treino de condicionamento de medo ao contexto e ao som, os ratos foram colocados individualmente em um caixa de condicionamento. Apos 120 segundos de livre exploracao, um som (90 decibeis, 2 kHz) foi emitido por 30 segundos, co-terminando com um choque nas patas (0,7 mA, 1s). Imediatamente apos, cada rato recebeu uma microinfusao bilateral de muscimol (agonista gabaergico, 0,5¿Êg/0,5¿ÊL por hemisferio) ou salina (grupo controle). O teste de condicionamento de medo ao contexto (CMC) ocorreu 48 horas apos o treino e consistiu na re-exposicao a caixa de condicionamento por 5 minutos, sem apresentacao de som ou choque. 24 horas depois, os mesmos animais foram submetidos ao teste de condicionamento de medo ao som (CMS), o qual ocorreu em uma caixa modificada, com duracao de 5 minutos. Ao final do segundo e terceiro minutos, o mesmo estimulo sonoro apresentado no treino foi emitido por 30 segundos. O tempo de congelamento e o comportamento motor foram utilizados como medidas de condicionamento. No CMS, os ratos que receberam a microinfusao de muscimol no CI anterior e posterior apresentaram uma media de tempo de congelamento menor durante o periodo pos-som. Entretanto, no CMC nao houve diferencas entre grupos para ambas as subregioes do CI. Portanto, os resultados deste estudo indicam que a inativacao pos-treino do CI como um todo prejudica exclusivamente o CMS. Entretanto, o prejuizo deste tipo de memoria, provocado pela inativacao do CI posterior, foi maior, evidenciando, portanto, que esta subregiao esta mais importantemente envolvida na circuitaria neural responsavel pela consolidacao do medo condicionado a um estimulo sonoro discreto. / The insular cortex (IC), or insula, has achieved over the last years an eminent position in the scientific literature due to its involvement in emotional and cognitive processes. In the rat, as in humans, the IC can be divided into two functionally heterogeneous sub-regions. Although most animal studies have shown an involvement of the rostral subregion (anterior IC) in gustatory memory, others suggest a broader role in memory, ranging from object recognition to the processing of spatial and aversive memories. In addition, even though the most caudal area (posterior IC) seems to be involved in multisensory and nociceptive processing, very few studies have evaluated its role in mnemonic processes and the results so far are unclear. Nevertheless, no work, to the best of our knowledge, had investigated the specific role of both sub-regions of the IC on consolidation of fear conditioning tasks. Thus, the aim of the present study was to investigate the effects of temporary inactivation of the anterior and posterior IC on memory consolidation of contextual and tone fear conditioning tasks. 3-month-old male Wistar rats underwent stereotaxic surgery for implantation of bilateral guide cannulae aimed directly above the anterior or posterior IC. The animals were allowed at least 7 days of recovery and were handled once a day for 3 days prior to behavioral sessions. For the contextual and tone fear conditioning training session, the rats were individually placed in the conditioning box. After 120 seconds of free exploration, a tone (90 decibels, 2 kHz) was delivered for 30 seconds, coterminating with a footshock (0.7 mA, 1 s). Immediately after, each rat received a bilateral microinjection of muscimol (GABAergic agonist, 0.5 ìg/0.5ìL by hemisphere) or saline (control group) into the intended IC subregion. The contextual fear conditioning test (CFC) was performed 48 hours after training and consisted in the re-exposure to the conditioning box for 5 minutes, without delivery of tone and shock. After 24 hours, the same animals were submitted to tone fear conditioning test (TFC), which occurred in a modified chamber, for 5 minutes. At the end of the second and third minutes, the same tone stimulus presented in the training session was delivered for 30 seconds. Freezing time and motion behavior were used as measures of conditioning. In TFC, the rats that had received muscimol microinfusion into the anterior and posterior IC displayed a lower freezing time during the post-tone period. However, for both IC subregions, there were no differences between groups in the CFC. Thus, our findings indicate that the posttraining inactivation of both IC subregions impaired the TFC. However, the impairment in this kind of memory, caused by the the inactivation of the posterior IC, was higher, thus, highlighting that this subregion is more importantly involved in the neural circuitry related to the consolidation of the discrete tone conditioned fear.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2018-01-30T17:09:56Z No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-02-01T17:39:56Z (GMT) No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-02-01T17:40:05Z (GMT) No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Made available in DSpace on 2018-02-01T17:44:38Z (GMT). No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) Previous issue date: 2017-07-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Water and sodium intake are fundamental behaviors for body fluid homeostasis. These behaviors are controlled by a neural circuitry involving facilitatory and inhibitory mechanisms, which are modulated by signals activated by changes in body fluid volume and composition. Important inhibitory mechanisms for water intake and particularly for sodium intake are located in the lateral parabrachial nucleus (LPBN), a pontine structure. It is known that LPBN inhibitory mechanisms are controlled by several neurotransmitters, some increasing, others reducing the action of this nucleus on sodium intake... / A ingestão de água e a ingestão de sódio são comportamentos fundamentais para a homeostase dos líquidos corporais. A manifestação destes comportamentos é controlada por um circuito neural constituído por mecanismos facilitatórios e inibitórios os quais são constantemente modulados por informações relativas aos líquidos corporais. Importantes mecanismos inibitórios para a ingestão de água e especialmente para a ingestão de sódio localizam-se no núcleo parabraquial lateral (NPBL), uma estrutura pontina. Sabe-se que os mecanismos inibitórios do NPBL são controlados por diversos neurotransmissores, alguns aumentando e outros diminuindo a ação deste núcleo sobre a ingestão de sódio .....

La distractibilité est un syndrome qui se caractérise par une réactivité exagérée d'un individu vis-à-vis de son environnement. Il a été démontré que des atteintes limitées au cortex préfrontal dorsolatéral (CPFDL) sont responsables d'un tel syndrome chez l'homme. Ce trouble peut être quantifié par un test spécifique et sensible appelé " test des antisaccades ". Ce syndrome se rencontre dans de nombreuses pathologies neurodégénératives telles que la Paralysie supranucléaire progressive (PSP) ou encore psychiatriques telles que la schizophrénie. L'objectif de cette recherche est de tenter de mieux comprendre ce syndrome de distractibilité à la fois sur le plan anatomique et pharmacologique. Le projet anatomique débute par une étude chez l'homme de lésions au niveau sous cortical pour déterminer l'implication réelle du sous cortex dans ce syndrome. Afin d'apporter des données indiscutables sur le rôle du CPFDL dans la distractibilité, nous avons réalisé une étude chez le primate de microinjections intracérébrales de muscimol au niveau de cette aire cérébrale. L'approche pharmacologique a consisté à réaliser un modèle animal de distractibilité en reproduisant chez le primate ce syndrome par administration de kétamine à dose subanesthésique. Celle-ci est connue pour induire des symptômes " shizophréniques like " avec des troubles liés à un dysfonctionnement du cortex préfrontal. La seconde étude a pour but d'évaluer l'effet d'un inhibiteur de l'acétylcholine estérase sur le syndrome de distractibilité chez des patients atteints de PSP. Cette recherche, en particulier les études chez le primate devraient permettre à terme de développer une thérapeutique pour enrayer ce trouble.

One of the most common risk factors for stroke is diabetes. Diabetics are 2 to 4 times more likely to have a stroke and are also significantly more likely to show poor functional recovery. In order to determine why diabetes is associated with poor stroke recovery, we tested the hypotheses that diabetes either exacerbates initial stroke damage, or inhibits neuronal circuit plasticity in surviving brain regions that is crucial for successful recovery. Type 1 diabetes was chemically induced in mice four weeks before receiving a targeted photothrombotic stroke in the right forelimb somatosensory cortex to model a chronic diabetic condition. Following stroke, a subset of diabetic mice were treated with insulin to determine if controlling blood glucose levels could improve stroke recovery. Consistent with previous studies, one behavioural test revealed a progressive improvement in sensory function of the forepaw in non-diabetic mice after stroke. By contrast, diabetic mice treated with and without insulin showed persistent deficits in sensori-motor forepaw function. To determine whether these different patterns of stroke recovery correlated with changes in functional brain activation, forepaw evoked responses in the somatosensory cortex were imaged using voltage sensitive dyes at 1 and 14 weeks after stroke. In both diabetic and non-diabetic mice that did not have a stroke, brief mechanical stimulation of the forepaw evoked a robust and near simultaneous depolarization in the primary (FLS1) and secondary somatosensory (FLS2) cortex. One week after stroke, forepaw-evoked responses had not been remapped in the peri-infarct cortex in both diabetic and non-diabetic mice. Fourteen weeks after stroke, forepaw evoked responses in non-diabetic mice re-emerged in the peri-infarct cortex whereas diabetic mice showed very little activation, reminiscent of the 1 week recovery group. Moreover, controlling hyperglycemia using insulin therapy failed to restore sensory evoked responses in the peri-infarct cortex. In addition to these differences in peri-infarct responsiveness, we discovered that stroke was associated with increased responsiveness in FLS2 of non-diabetic, but not diabetic or insulin treated mice. To determine the importance of FLS2 in stroke recovery, we silenced the FLS2 cortex and found that it re-instated behavioural impairments in stroke recovered mice, significantly more so than naïve mice that still had a functioning FLS1. Collectively, these results indicate that both diabetes and the secondary somatosensory cortex play an important role in determining the extent of functional recovery after ischemic cortical stroke. Furthermore, the fact that insulin therapy after stroke did not normalize functional recovery, suggests that prolonged hyperglycemia (before stroke) may induce pathological changes in the brain’s circulation or nervous system that cannot be easily reversed. / Graduate

Made available in DSpace on 2016-06-02T19:22:52Z (GMT). No. of bitstreams: 1 2885.pdf: 1014245 bytes, checksum: f9caee9a0cfd9bbc23af2760d02bb6a9 (MD5) Previous issue date: 2009-12-07 / Universidade Federal de Minas Gerais / Previous studies have shown the importance of serotonergic GABAergic and aadrenergic mechanisms of the lateral parabraquial nucleus (LBPN) in the control of sodium intake. The importance of the central nucleus of the amygdala (CeA) for sodium intake induced by different protocols was also demonstrated. Considering the studies showing reciprocal connections between these two structures, the objective of the present study was to investigate if the increase of sodium and water intake produced by the blockade of serotonergic mechanism, or the activation of GABAergic receptors or α2-adrenoceptors in the LPBN would depend on the CeA integrity. Male Holtzman rats with bilateral CeA lesions and bilateral stainless steel cannulas implanted in the LPBN were used to study the possible involvement of the CeA: 1) in water and 0.3 M NaCl intake produced by injections of the diuretic furosemide (FURO) combined with the angiotensin converting enzyme inhibitor captopril (CAP) subcutaneously (sc); 2) in the increase of 0.3 M NaCl intake induced by the blockade of serotonergic mechanisms or activation of the α2-adrenoceptors of the LPBN in rats treated with FURO + CAP sc; 3) in 0.3 M NaCl intake induced by the activation of GABAergic receptorss of the LPBN in satiated and normovolemic rats. Additionally, the pharmacological blockade of the CeA neurons with bilateral injections of GABAA receptor agonist muscimol was performed in order to test if the effects of CeA electrolytic lesions after the blockade of the inhibitory mechanisms of the NPBL were due to destruction of CeA neurons or destruction of fibers of passage. CeA lesionedrats had a decrease in daily water intake in comparison to sham-lesioned rats during the whole period of test, while the reduction of daily 0.3 M sodium intake occurred after the eighth day of lesions. Animals with bilateral lesions of the CeA also showed a reduction in body weight when compared to sham lesioned-rats. Bilateral lesions of the CeA did not affect FURO+CAP induced-water (9.2 1.6 ml/2 h vs. sham lesion: 12.8 0.7 ml/2 h) and 0.3 M NaCl intake (6.5 3.5 ml/2 h vs. sham lesion: 5.2 0.9 ml/2 h). Bilateral lesions of the CeA (3 days) completely abolished the ingestion of water (0.1 0.05 ml/4 h vs. sham lesion: 8.2 3.5 ml/4 h) and 0.3 M NaCl (0.1 0.1 ml/4 h vs. sham lesion: 16.1 5.4 ml/4 h) induced by bilateral injections of muscimol (0.5 nmol/0.2 μl) into the LPBN in satiated rats. Bilateral lesions of the CeA (5 to 18 days) also abolished the increase in 0.3 M NaCl (11,7 2,8 ml/2 h e 11,7 2,8 ml/2 h vs. sham lesion: 31,5 4,2 ml/2 h e 18,3 ± 3,1 ml/2 h) and water intake (6,7 1,8 ml/2 h e 13,8 2,7 ml/2 h vs. sham lesion: 19,9 3,2 ml/2 h e 22,4 2,5 ml/2 h) produced respectively by bilateral injections of moxonidine (0.5 nmol/0.2 μl) or methysergide (4 μg/0.2 μl) into the LPBN in FURO + CAP treated-rats. Bilateral injections of muscimol (0.5 nmol) into the CeA abolished water (0.1 0.02 ml/4 h vs. saline: 8.8 3.2 ml/4 h) and 0.3 M NaCl intake (0.1 0.04 ml/4h vs. saline: 19.1 6.4 ml/4 h) induced by bilateral injections of muscimol (0.5 nmol/0.2 μl) in the NPBL in satiated animals. Bilateral injections of muscimol (0.25 nmol/0.2 μl) in the CeA abolished the increase of water (3.3 2.3 ml/2 h vs. saline: 26.4 6.7 ml/2 h) and 0.3 M NaCl intake (2.8 1.6 ml/2 h vs. saline: 29.7 7.2 ml/2 h) produced by the bilateral injections of moxonidine (0.5 nmol/0.2 μl) into the NPBL. The present results show that CeA is essential for sodium and water intake after the blockade of LPBN inhibitory mechanisms. The suggestion is that CeA facilitatory mechanisms for sodium intake might be activated after the blockade of LPBN inhibitory mechanisms which might drive rats to ingest sodium. Therefore, if LPBN inhibitory mechanisms were acting normally, they may limit sodium intake because they inhibit CeA facilitatory signals for sodium intake. / Estudos anteriores demonstraram a importancia dos mecanismos serotoninergicos, GABAergicos e adrenergicos do nucleo parabraquial lateral (NPBL) na regulacao da ingestao de sodio hipertonico. Tambem ja foi demonstrada a importancia do nucleo central da amigdala (CeA) para a ingestao de sodio hipertonico induzida por diferentes protocolos. Considerando-se os estudos mostrando conexoes reciprocas entre essas duas estruturas, o objetivo do presente estudo foi investigar se o aumento da ingestao de sodio hipertonico produzido pelo bloqueio serotoninergico ou ativacao GABAergica ou adrenergica no NPBL dependeria da integridade do CeA. Em ratos com lesoes bilaterais do CeA e com canulas de aco inoxidavel implantadas bilateralmente no NPBL, foi estudado o possivel envolvimento do CeA: 1) na ingestao de agua e NaCl 0,3 M produzida pelo tratamento subcutaneo com o diuretico furosemida (FURO) combinado com o inibidor da enzima conversora de angiotensina captopril (CAP); 2) no aumento da ingestao de NaCl 0,3 M produzido pelo bloqueio de receptores serotoninergicos ou ativacao dos receptores adrenergicos α2 do NPBL em ratos tratados com FURO + CAP sc; 3) na ingestao de NaCl 0,3 M induzida pela ativacao de receptores GABAergicos do NPBL em ratos saciados e normovolemicos. Adicionalmente, foi realizado o bloqueio farmacologico dos neuronios do CeA com injecoes bilaterais de muscimol, agonista de receptor GABAA, para verificar se os efeitos das lesoes eletroliticas do CeA apos o bloqueio dos mecanismos inibitorios do NPBL eram devido a destruicao de neuronios do CeA ou destruicao de fibras de passagem. Em animais com lesoes bilaterais do CeA a ingestao diaria de agua foi menor quando comparada aos animais com lesoes ficticias ao longo de todo periodo experimental, enquanto que a ingestao diaria de NaCl 0,3 M foi reduzida a partir do oitavo dia apos as lesoes. Esses animais apresentaram uma reducao no peso corporal persistente por todo periodo experimental comparado com o grupo com lesoes ficticias. As lesoes bilaterais do CeA nao afetaram a ingestao de agua (9,2 1,6 ml/2 h vs. lesoes ficticias: 12,8 0,7 ml/2 h) e NaCl 0,3 M (6,5 3,5 ml/2 h vs. lesoes ficticias 5,2 0,9 ml/2 h) induzida por FURO + CAP sc. As lesoes bilaterais do CeA (3 dias) aboliram a ingestao de NaCl 0,3 M (0,1 0,1 ml/4 h vs. lesoes ficticias: 16,1 5,4 ml/4 h) e de agua (0,1 0,05 ml/4 h vs. lesoes ficticias: 8,2 3,5 ml/4 h) induzida pelas injecoes bilaterais de muscimol (0,5 nmol/0,2 μl) no NPBL de ratos saciados. As lesoes bilaterais do CeA (5 a 18 dias) tambem aboliram o aumento da ingestao de NaCl 0,3 M (11,7 2,8 ml/2 h e 11,7 2,8 ml/2 h vs. lesoes ficticias: 31,5 4,2 ml/2 h e 18,3 ± 3,1 ml/2 h) e agua (6,7 1,8 ml/2 h e 13,8 2,7 ml/2 h vs. lesoes ficticias: 19,9 3,2 ml/2 h e 22,4 2,5 ml/2 h) induzidos, respectivamente, pelas injecoes bilaterais de moxonidina (0,5 nmol/0,2 μl) ou metisergida (4 μg/0,2 μl) em ratos previamente tratados com FURO+CAP sc. Injecoes bilaterais de muscimol (0,5 nmol/0,2 μl) no CeA aboliram a ingestao de agua (0,1 0,02 ml/4 h vs. salina: 8,8 3,2 ml/4 h) e NaCl 0,3 M (0,1 0,04 ml/4h vs. salina: 19,1 6,4 ml/4 h) induzidas pela injecao bilateral de muscimol (0,5 nmol/0,2 μl) no NPBL em animais saciados, como tambem as injecoes bilaterais de muscimol (0,25 nmol/0,2 μl) no CeA aboliram o aumento da ingestao de agua (3,3 2,3 ml/2 h vs. salina: 26,4 6,7 ml/2 h) e NaCl 0,3 M (2,8 1,6 ml/2 h vs. salina: 29,7 7,2 ml/2 h) produzido pela injecao bilateral de moxonidina (0,5 nmol/0,2 μl) no NPBL em animais tratados com FURO + CAP sc. Esses resultados demonstram que o CeA e essencial para a ingestao de sodio e agua que ocorre apos o bloqueio dos mecanismos inibitorios do NPBL. A sugestao e que mecanismos facilitatorios para a ingestao de sodio presentes no CeA seriam ativados apos o bloqueio dos mecanismos inibitorios do NPBL o que estimularia os animais a ingerirem sodio. Portanto, se estiverem atuando normalmente, os mecanismos inibitorios do NPBL limitariam a ingestao de sodio porque inibiriam os sinais facilitatorios para ingestao de sodio produzidos pelo CeA.

Behavioral adaptation to a continuously changing environment is critical for the survival of the animals, but also day-to-day interactions in the human world. The main components maintaining flexibility in cognition and behavior are well-established and depend mostly on proper intercommunication within the prefrontal cortex (PFC) and striatum. Some parts of the PFC are densely innervated by the ventral hippocampus (vHPC), which has a great impact on its functioning. Also, hippocampal-prefrontal circuit dysfunction has been shown to disrupt the integrity of flexible cognition in some neuropsychiatric diseases. Therefore, the exact functional role of this pathway is an indispensable part of the research. The aim of this study was to test the role of the vHPC and the medial PFC (mPFC) in an active place avoidance task on a rotating arena in 1) two flexibility task variants - reversal learning and set- shifting - and 2) the spatial memory retrieval. We inactivated these structures by muscimol (GABAA receptor agonist) in a variety of unilateral, bilateral, and combined local injections. Disrupted performance was apparent in reversal learning in vHPC-inactivated rats. No effect was seen in mPFC-inactivated rats. Impairments after the task acquisition were observed in bilateral vHPC and mPFC inactivations...

Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Ã ). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4-benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2-ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (ï ¡-methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the ï ¡-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the ï ¢-carbons. / Ph. D.

Monosialoganglioside (GM1) is a component of most cell membranes and is thought to play a role in development, recognition and cellular differentiation. Furthermore, GM1 is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. In the present study we investigate the effect of GM1 on the catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, on the spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cortex of rats ex vivo and in vitro. Systemic GM1 administration (50 mg/kg, i.p.; twice) reduced spontaneous chemiluminescence and increased CAT activity ex vivo. On the other hand, GM1 (103-104 nM) reduced CAT activity in vitro. The other parameters were not affected by GM1 administration. These findings agree with the view that the antioxidant action of GM1 is not due to an intrinsic antioxidant activity of this glycolipid, but due to a secondary decrease of reactive species generation and/or increase of antioxidant defenses. Moreover, we evaluated whether GM1 could have a neuroprotective action on the experimental model of glutaric acidemia, an inherited metabolic disorder characterized by glutaric acid (GA) accumulation and neurological dysfunction, as striatal degeneration and convulsion. The systemic GM1 administration (50 mg/kg, i.p. twice) protected against the convulsions, oxidative damage markers increase (total protein carbonylation and thiobarbituric acid-reactive substances - TBARS) production and Na+,K+-ATPase activity inhibition induced by GA (4 mol/ 2 l) in striatum of rats. Furthermore, convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum, but not with oxidative stress marker measures. In addition, GM1 (50-200 M) protected against Na+,K+-ATPase inhibition induced by GA (6 mM), but not against oxidative damage in vitro. Intrastriatal administration of muscimol (46 pmol/striatum), a GABAA receptor agonist, but not glutamatergic receptor antagonists MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum), prevented GA-induced convulsions and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance in the injected striatum with GA. Since GM1 and muscimol prevented neurotoxic effects induced by GA, we investigated the GM1 action after intrastriatal administration of pentylenetetrazole (PTZ), a GABAA receptor antagonist. GM1 treatment prevented seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl induced by PTZ (1.8 mol/striatum) in the rats striatum. Furthermore, these data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1. / O monossialogangliosídeo (GM1) é um componente natural de membrana plasmática que está envolvido no crescimento, reconhecimento e diferenciação celular, além de proteger o SNC da ação dos radicais livres. No presente estudo investigou-se o efeito do GM1 sobre a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GPx), assim como na quimiluminescência e capacidade antioxidante total (TRAP) em córtex cerebral de ratos machos adultos ex vivo e in vitro. A administração sistêmica de GM1 (50 mg/kg, i.p.; duas doses: 24 horas e 30 minutos antes do sacrifício) reduziu a quimiluminescência e aumentou significativamente a atividade da CAT ex vivo. A adição de GM1 (103-104 nM) ao meio de incubação diminuiu a atividade da CAT in vitro. Estes resultados sugerem que o efeito neuroprotetor do GM1 não é devido à ação antioxidante intrínseca deste glicoesfingolipídeo, mas devido ao aumento secundário das defesas antioxidantes e/ou uma redução da geração de radicais livres. Além disso, avaliamos se o GM1 tinha efeito neuroprotetor em um modelo experimental da acidemia glutárica, um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido glutárico (GA) e alterações neurológicas, como degeneração estriatal e convulsões. A administração de GM1 preveniu as convulsões, o aumento da produção dos marcadores do dano oxidativo (carbonilação protéica total e substâncias reativas do ácido tiobarbitúrico - TBARS) e a inibição da atividade da Na+,K+-ATPase induzidas pelo GA (4 mol/2 µl) em estriado de ratos. Além disso, os episódios convulsivos induzidos por GA apresentaram uma correlação significativa com a inibição da atividade da Na+,K+-ATPase no estriado injetado, mas não com os níveis dos marcadores do estresse oxidativo. A adição de GM1 (50 200  ao meio de incubação preveniu a inibição da Na+,K+-ATPase, mas não reduziu o dano oxidativo induzido por GA (6 mM) in vitro. A administração intraestriatal de muscimol (46 pmol/0,5 l), um agonista de receptor GABAA, mas não dos antagonistas de receptores glutamatérgicos, MK-801 (3 nmol/0,5 l) e DNQX (8 nmol/0,5 l), preveniu as convulsões e a inibição da atividade da Na+,K+-ATPase induzidas por GA. A proteção do GM1 e muscimol contra as convulsões induzidas por GA apresentou uma correlação significativa com a manutenção da atividade da Na+,K+-ATPase no estriado injetado com GA. Desde que o GM1 e o muscimol preveniram os efeitos neurotóxicos induzidos pelo GA, investigou-se a ação do GM1 após a administração intraestriatal de pentilenotetrazol (PTZ), um antagonista de receptores GABAA. O tratamento com GM1 preveniu as convulsões, o dano oxidativo e a inibição da atividade da Na+,K+-ATPase induzidas por PTZ (1,8 µmol/2 µl). Esses dados sugerem que a atividade da Na+,K+-ATPase e mecanismos mediados pela ativação de receptores GABAérgicos podem ser de grande importância para a atividade convulsiva induzida por GA, bem como nos mecanismos de neuroproteção induzidos pelo GM1.

Au cours des dernières années, des méthodes non-invasives de stimulations permettant de moduler l’excitabilité des neurones suivant des lésions du système nerveux central ont été développées. Ces méthodes sont maintenant couramment utilisées pour étudier l’effet de l’inhibition du cortex contralésionnel sur la récupération motrice à la suite d’un accident vasculocérébral (AVC). Bien que plusieurs de ces études rapportent des résultats prometteurs, les paramètres permettant une récupération optimale demeurent encore inconnus. Chez les patients victimes d'un AVC, il est difficile de débuter les traitements rapidement et d'initier l’inhibition dans les heures suivant la lésion. L'impact de ce délai est toujours inconnu. De plus, aucune étude n’a jusqu’à maintenant évalué l’effet de la durée de l’inhibition sur la récupération du membre parétique. Dans le laboratoire du Dr Numa Dancause, nous avons utilisé un modèle bien établi de lésion ischémique chez le rat pour explorer ces questions. Nos objectifs étaient d’évaluer 1) si une inactivation de l’hémisphère contralésionnel initiée dans les heures qui suivent la lésion peut favoriser la récupération et 2) l’effet de la durée de l’inactivation sur la récupération du membre parétique. Suite à une lésion dans le cortex moteur induite par injections d’un vasoconstricteur, nous avons inactivé l’hémisphère contralésionnel à l’aide d’une pompe osmotique assurant l’infusion continue d’un agoniste du GABA (Muscimol). Dans différents groupes expérimentaux, nous avons inactivé l’hémisphère contralésionnel pour une durée de 3, 7 et 14 jours suivant la lésion. Dans un autre groupe, le Muscimol a été infusé pour 14 jours mais à un débit moindre de façon à pouvoir étudier le lien entre la fonction du membre non-parétique et la récupération du membre parétique. Les données comportementales de ces groupes ont été comparées à celles d’animaux ayant récupéré de façon spontanée d'une lésion similaire. Nos résultats indiquent que l’augmentation de la durée de l’inactivation (de 3 à 14 jours) accélère la récupération du membre parétique. De plus, les deux groupes ayant reçu une inactivation d'une durée de 14 jours ont montré une plus grande récupération fonctionnelle que le groupe n’ayant pas reçu d’inactivation de l’hémisphère contralésionnel, le groupe contrôle. Nos résultats suggèrent donc que l’inactivation de l’hémisphère contralésionnel initiée dans les heures suivant la lésion favorise la récupération du membre parétique. La durée d’inhibition la plus efficace (14 jours) dans notre modèle animal est beaucoup plus longues que celles utilisées jusqu’à maintenant chez l’homme. Bien qu’il soit difficile d’extrapoler la durée idéale à utiliser chez les patients à partir de nos données, nos résultats suggèrent que des traitements de plus longue durée pourraient être bénéfiques. Finalement, un message clair ressort de nos études sur la récupération fonctionnelle après un AVC: dans le développement de traitements basés sur l’inhibition de l’hémisphère contralésionnel, la durée de l’inactivation est un facteur clef à considérer. / With the introduction of non-invasive brain stimulation methods aimed at modulating the excitability of cortical areas after stroke, many groups are intensively investigating the effects of inhibition of the contralesional hemisphere on functional recovery. Although the reported results of these studies are very promising, limitations of enrolling acute stroke patients as well as technical difficult of establishing continuous inhibition protocols have left several open ended questions regarding the treatment parameters and patient selection. For example, the efficacy of inhibition treatment in acute setting after stroke and the effect of treatment duration are two questions that are virtually unexplored. Therefore, in the laboratory of Prof. Numa Dancause, we took advantage of a well established rodent model of cortical ischemic lesion to gain direct and objective insight about the importance of contralesional inactivation on motor recovery of the paretic limb. Using an Endothelin-1 rodent model of ischemic cortical lesion, we pharmacologically inactivated the contralesional hemisphere with a GABA agonist (Muscimol). By doing so we were interested in the effect of early treatment when contralesional inactivation is initiated rapidly after the lesion. Early after induction of cortical ischemic lesion, the contralesional hemisphere was inactivated with continuous infusion of the Muscimol for 3, 7 or 14 days in three different groups of animals. In a fourth group, Muscimol was infused at slower rate for 14 days to provide additional insights on the relation between the effects of inactivation on the non-paretic forelimb behavior and the recovery of the paretic forelimb. We included a group of animals with spontaneous recovery that received no inactivation after lesion. Our results indicated that increasing inactivation duration (from 3 to 14 days) accelerated the recovery of grasping function. Both groups with 14 days of inactivation had similar recovery profiles and performed better than animals that spontaneously recovered. In fact, the duration of inactivation, not the intensity, correlated with the better functional outcomes. Our results support early contralesional inactivation to improve recovery of the paretic forelimb after cortical lesion. Moreover, based on our results, the duration of inactivation is the most important factor to correlate with the functional outcomes. Therefore, by providing precise temporal and behavioral evidence, our results provide a window of opportunity for the researchers in which the current gap in our understanding of the clinical efficacy of contralesional inhibition in acute phase after stroke can be approached with more confidence.