L'hippocampe est une région du cerveau située dans le lobe temporal médian. Avec d'autres structures limbiques, l'hippocampe est impliqué dans des processus d'apprentissage et de mémorisation et possède un rôle crucial dans le traitement spatial de l'information. Les synapses de l'hippocampe formées entre les fibres moussues (fm) originaires du gyrus denté et les neurones pyramidaux de CA3 ont reçu une attention particulière, compte tenu de la position stratégique occupée par le gyrus denté à l'entrée de l'hippocampe. En outre les synapses fm- CA3 sont distinctes de la plupart des autres synapses excitatrices du système nerveux central par leurs propriétés morphologiques et physiologiques uniques. Cela soulève la question de savoir si ces propriétés uniques reflètent aussi un rôle fonctionnel unique dans le traitement de l'information effectué par cette synapse au sein du microcircuit de l'hippocampe. Malheureusement nous ne savons que peu de choses sur la façon dont les cellules granulaires modulent l'activité des neurones de CA3 dans le réseau intact in vivo (Henze et al, 2002 ; Hagena et Manahan - Vaughan, 2010, 2011). Le manque d'information est dû au fait que la manipulation classique des circuits neuronaux par des approches électriques, pharmacologiques et génétiques manque de précisions spatiale et temporelle in vivo. L'utilisation de la stimulation extracellulaire de fibres moussues peut conduire à l'activation polysynaptique de cellules pyramidales de CA3, qui peuvent ensuite contaminer les réponses enregistrées. Par ailleurs, l'utilisation de critères trop conservateurs peut conduire à l'exclusion des réponses provenant des fibres moussues «purs» aux propriétés méconnues (Henze et al., 2000). Toutefois, le développement récent et rapide de l’optogénétique dans les neurosciences a fourni de nouveaux outils offrant une sélectivité spatiale élevée (activation optique spécifique de la cellule), et une grande précision temporelle (à l'échelle de la milliseconde), permettant la dissection et l'étude des circuits neuronaux in vivo. L'objectif de ma thèse était de mieux comprendre les mécanismes et les conséquences physiologiques de la plasticité synaptique à court terme se produisant à la synapse formée entre les fibres moussues et les neurones pyramidaux de CA3 dans le cerveau de souris intact. La présente thèse se compose de deux parties principales. Dans la première partie, j'ai exploré de nouveaux outils optogénétiques dans le but de contrôler l'activité des cellules granulaires à l’aide d’impulsions de lumière. La stimulation optogénétique repose sur l'activation du canal ionique channelrhodopsin - 2 - lumière fermée ( ChR2 ) par une lumière bleue et induit des potentiels d'action sur une large gamme de fréquences de stimulation. J'ai aussi observé que la stimulation optique peut être utilisée pour déclencher la plasticité à court terme au niveau des synapses fm-CA3.Dans la deuxième partie j'ai affiné la méthodologie de stimulation optogénétique in vivo pour la caractérisation non invasive du fonctionnement synaptique des synapses fm- CA3. La fiabilité de la stimulation optogénétique d'une population neuronale génétiquement ciblée ainsi que la résolution d'une seule cellule obtenue en utilisant des enregistrements de cellules entières sont des étapes importantes vers une meilleure compréhension du rôle fonctionnel des fibres moussues dans le réseau de l'hippocampe in vivo. / The hippocampus is a brain region located in the medial temporal lobe. Along with other limbic structures, the hippocampus is involved in learning and memory processes and has a crucial role in spatial information processing. Within the hippocampus synapses made between mossy fibers (mf) originating from the dentate gyrus and CA3 pyramidal neurons have received particular attention, given the strategic position occupied by the dentate gyrus at the entrance of the hippocampus. Moreover mf-CA3 synapses are distinct from most of other excitatory synapses in the central nervous system for their unusual morphological and physiological properties. This raises the question if these unique properties reflect a unique functional role in information processing carried out by this synapse within the microcircuit of the hippocampus. Unfortunately very little is known on how granule cells modulate the activity of CA3 neurons in the intact network in vivo (Henze et al., 2002; Hagena and Manahan-Vaughan, 2010, 2011). The paucity of information is due to the fact that classical manipulation of neuronal circuits using electrical, pharmacological and genetic approaches lack spatial and temporal precision in vivo. The use of bulk extracellular stimulation may lead to polysynaptic activation of CA3 pyramidal cells, which can subsequently contaminate putative mossy fibers synaptic responses measured in CA3 pyramidal cells. The use of overly conservative criteria on the other side may lead to the exclusion of “pure” mossy fibers responses with unexpected properties (Henze et al., 2000).However the recent and fast growth of optogenetics in neuroscience has provided new tools with high spatial selectivity (cell specific optical activation) and temporal precision (at the millisecond scale), allowing the dissection and investigation of neuronal circuits in vivo. The aim of my thesis was to gain insight into the mechanisms and the physiological consequences of short-term synaptic plasticity occurring at mossy fibers to CA3 pyramidal neurons synapses in the intact mouse brain. The present thesis consists of two main parts. In the first part I explored new optogenetic tools to control the activity of granule cells with pulses of light. Optogenetic stimulation, which relies on the activation of the light-gated ion channel channelrhodopsin-2 (ChR2) by blue light reliably induced action potentials over a wide range of frequencies of stimulation. I also found that optical stimulation can be used to trigger short term plasticity at mf-CA3 synapses. In the second part I refined optogenetic stimulation methodology in vivo for non-invasive characterization of synaptic functioning of the mf-CA3 synapses. The reliability of optogenetic stimulation of a genetically targeted neuronal population together with the single cell resolution obtained using whole-cell recordings are important steps towards a better understanding of the functional role of the mossy fibers in the hippocampal network in vivo.

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Ho, Tsz Wan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 125-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 67-80).

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-24T03:31:01Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-27T16:14:10Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) / Made available in DSpace on 2017-11-27T16:15:20Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) Previous issue date: 2005 / As epilepsias do lobo temporal s?o as que apresentam maior refratariedade ao tratamento farmacol?gico e perfazem 2/3 das interven??es cir?rgicas de epilepsia, sendo portanto de grande custo social, econ?mico e psicol?gico. Assim, modelos animais de epilepsia do lobo temporal s?o de grande relev?ncia n?o s? para o entendimento das bases neurais dessa patologia, mas tamb?m para o desenvolvimento de abordagens terap?uticas capazes de evitar a instala??o da doen?a. Esses s?o os objetivos da presente disserta??o de doutorado. Ap?s um evento traum?tico (no caso deste trabalho, um estado de mal epil?ptico), diversas altera??es morfol?gicas e fisiol?gicas acontecem, caracterizando a g?nese da s?ndrome epil?ptico (epileptog?nese). Dentre as altera??es podemos destacar a intensa fosforila??o de prote?nas em res?duos de tirosina e a ativa??o de diferentes segundos mensageiros. Os dois primeiros cap?tulos desta tese descrevem a tentativa de bloquear os processos de epileptog?nese por meio da inibi??o da fosforila??o de res?duos de tirosina atrav?s do tratamento farmacol?gico com inibidores das tirosina cinases, a herbimicina A e o K-252a. O terceiro cap?tulo analisa eletrofisiologicamente o circuito neural do giro denteado em animais que apresentavam uma muta??o em um s?tio inibit?rio da prote?na c?lcio/calmodulina cinase do tipo II (CaMKII). No primeiro cap?tulo, mostramos que o tratamento agudo com herbimicina A (348?M, 5?L, icv), ? capaz de bloquear a potencia??o duradoura (LTP) induzida por um est?mulo tet?nico bem como de atenuar (~40%) a ativa??o neuronal (express?o de c-Fos) decorrente de um estado de mal epil?ptico induzido pela administra??o sist?mica de pilocarpina (SE). Apesar dos significativos efeitos agudos, este tratamento mostrou-se incapaz de atenuar a freq??ncia de crises espont?neas, bem como o padr?o de morte neuronal observado ap?s o estado de mal epil?ptico induzido pela pilocarpina. Entretanto, o tratamento com herbimicina A alterou o padr?o de marca??o de metais pesados (Zn+2) no hilo do giro denteado e na regi?o de CA3 do hipocampo, por?m n?o apresentou efeito sobre o padr?o de brotamento das fibras musgosas observado na camada molecular do giro denteado. No segundo cap?tulo, mostramos que a herbimicina e o K- 252a modificam a atividade epileptiforme induzida pela administra??o intra-hipocampal de ?cido ca?nico, sem alterar o padr?o de morte neuronal. Esses resultados sugerem que o tratamento com inibidores de prote?nas tirosina cinases ? capaz de modificar o padr?o de ativa??o agudo do hipocampo ap?s um est?mulo (i.e., o estado de mal epil?ptico ou a LTP), por?m sem qualquer efeito sobre o processo de epileptog?nese. No terceiro cap?tulo, estudamos a excitabilidade e a plasticidade do giro denteado ? estimula??o da via perfurante (principal afer?ncia da forma??o hipocampal) em animais que apresentam uma CaMKII geneticamente modificada. Essa prote?na uma vez ativada n?o pode ser inibida. A caracteriza??o eletrofisiol?gica demonstrou que esses animais apresentam potenciais de campo evocados no giro denteado aparentemente semelhante aos animais controle (wild-type), por?m sua responsividade a padr?es de estimula??o em salvas e sua plasticidade apresentaram clara altera??o. Essa modifica??o foi caracterizada por uma maior variabilidade nas respostas ? trens de estimula??o (freq??ncias de 1 e 2 Hz) e maior inibi??o do pulso pareado em trens de estimula??o (para pulsos pareados aplicados a freq??ncia de 5 Hz). Al?m disso, conforme j? descrito na literatura, mostramos que a susceptibilidade a atividade epileptiforme depende do padr?o de estimula??o utilizado para os diferentes animais (mutantes vs. wild-type). Assim, utilizando o modelo cl?ssico do abrasamento demonstramos que a muta??o n?o altera a evolu??o da epileptog?nese. Entretanto, ao utilizarmos duas variantes de um padr?o de estimula??o similar ? freq??ncia teta (5Hz, Intermittent vs. Continuous theta-burst stimulations), demonstramos a import?ncia da muta??o na manuten??o da excitabilidade do giro denteado. Esses resultados destacam a import?ncia da CaMKII na atividade epileptiforme al?m de sugerir novas abordagens experimentais (i.e., sensibilidade ? padr?es de estimula??o eletrofisiol?gica) no estudo da epileptog?nese. Em resumo, os resultados apresentados nessa tese contribuem para um melhor entendimento dos fen?menos subjacentes aos processos de plasticidade neuronal e da contribui??o destes para o fen?meno de epileptog?nese, al?m de sugerir / Temporal lobe epilepsies are highly refractory to pharmacological treatment. Up to 70% of these patients undergo chirurgical resection of temporal region, procedure with important consequences for the social, economic and psychological spheres. Experimental animal models that mimic temporal lobe epilepsy provide an insightful approach to study the neural basis of epilepsy as well as create opportunities to test promising therapeutic drugs. The present thesis tests the antiepileptogenic activity of two protein tyrosine kinase inhibitors and the relevance of Ca+2/calmodulin kinase type II (CaMKII) mutants. Multiples morphological and physiological alterations take place after a traumatic brain injury (in this thesis, the status epilepticus) leading the animal to an epileptic conditions. During this period, the epileptogenesis process, there is strong tyrsine phosphorylation with the activation of many second messengers. The first two chapters of the thesis describe experiments in which herbimycin A and K-252a, two protein tyrosine kinase inhibitors, were used to attenuate synaptic plasticity and epileptogenesis. The third chapter, the dentate gyrus network was studied after angular bundle stimulation in animals presenting one punctual mutation at the autoinhibitory phosphorylation site of the CaMKII. In the first chapter, we showed that one single herbimycin A injection (348?M, 5?L, icv) was able to attenuate long-term potentiation (LTP) in the commissural CA3 neurons and also, to decrease status epilepticus- (SE-) induced neuronal activation (c-Fos expression) in almost 40%. Although markedly acute effects, the present herbimycin A treatment was not able to diminish spontaneous seizure frequency, cell death or aberrant mossy fiber sprouting observed after the pilocarpine-induced SE. Curiously, herbimycin-treated animals presented decreased neo-Timm staining in the hilus and CA3 region despite the epileptic condition. In the second chapter, we confirmed the ability of protein tyrosine kinase inhibitors to decrease SE-induced neuronal activation. Herbimycin A icv treatment altered the kainic acid-induced epileptiform profile in EEG recordings. Cell death pattern was not altered by any pharmacological treatment. These results suggest that protein tyrosine kinase inhibitiors are able to modify the acute neuronal activation and plasticity (ictogenesis or LTP) but is ineffective in attenuating the epileptogenesis process. In the third chapter, we studied the dentate gyrus excitability and plasticity after angular bundle stimulation in CaMKII mutant animals. Once in its self-sustained mode, this mutation does not allow the reduction of the catalytic activity of the kinase. These animals present normal electrophysiological profiles (similar to wild-type animals) but with reduced amplitude. Shortterm plasticity was clearly altered. Mutant animals presented increased variability in the responses to trains of stimulation at 1 and 2 Hz, and at at 5Hz stronger paired-pulse inhibition. Accordingly to the literature, we also showed that the epileptiform susceptibility depends on the stimulation pattern used in both animals (mutants vs. wild-type). Thus, although the mutation did not altered the behavior and the electrographic kindling evolution, we showed that mutant animals were prone to afterdischarges when stimulate by an intermittent theta-burst stimulation. On the other hand, the same animals needed more bursts to induce afterdischarges when the stimulation was set in the continuos mode. Taken together, the present results contribute to a better understanding of the protein tyrosine kinase and CaMKII function in neuronal plasticity underlying the epileptogenesis process and sum efforts in searching for a clinic antiepileptogenic drug.

No / Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of these events are necessary for the internalization of GluK2-containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.