The excitatory, ionotropic glutamatergic AMPA receptor is the most common membrane-bound receptor in the central nervous system. AMPARs and the NMDA receptors are central to synaptic plasticity, memory, and mechanisms of neurodegeneration. The AMPAR is an obligate heterotetramer, composed of subunits GluA1-4. Subunit permutation determines ion conductance, trafficking and other functional characteristics. Few available antibodies are subunit-specific, disabling researchers from accurately visualizing differential AMPAR subunit distribution in the nervous system. This study sought to visualize a novel monoclonal GluA1/2/3 antibody with functional avidity for three of four receptor subunits and to characterize the ultrastructural localization of these receptors using confocal and electron microscopy.

<p>Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive. </p><p>Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity. </p><p>We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.</p> / Dissertation

La navigation spatiale peut être subdivisée en deux processus: la construction d’une représentation mentale de l’espace à partir de l’exploration de l’environnement d'une part, et l’utilisation de cette représentation de façon à produire le trajet le plus adapté pour rejoindre le lieu souhaité d'autre part. Lors de l’exploration de l’environnement, des informations externes et des informations de mouvement propre (i.e. vestibulaires et proprioceptives) sont combinées pour former la carte cognitive. Depuis longtemps des études suggèrent que le cervelet participe à la navigation spatiale mais son rôle a souvent été confiné à l’exécution motrice. Notre équipe a étudié des souris mutantes L7-PKCI présentant un déficit de plasticité synaptique de type dépression à long terme (DLT) au niveau des synapses entre fibres parallèles et cellules de Purkinje du cortex cérébelleux. Ces travaux ont montré que les souris présentent à la fois un déficit dans l'optimisation de la trajectoire mais également dans le maintien de la carte cognitive formée dans l'hippocampe. En effet, les propriétés de décharge des cellules de lieu de l'hippocampe sont affectées chez ces souris exclusivement lorsque celles-ci doivent naviguer en se reposant sur les informations provenant de leur mouvement propre, c'est à dire quand elles explorent l'environnement dans le noir. A ces mêmes synapses, une plasticité de type potentialisation à long terme (PLT) a été observée et permet (avec la DLT) la modulation bidirectionelle de l’efficacité synaptique. La plasticité bidirectionnelle est un processus clé dans les modèles théoriques de type « filtre adaptatif » de traitement de l’information par le cervelet. Selon ces modèles, l’absence de PLT ou DLT devrait affecter de façon similaire la plasticité bidirectionnelle et conduire ainsi à des déficits comparables. Pour tester cette hypothèse, nous avons étudié les conséquences fonctionnelles d’un déficit de type PLT au niveau de la même synapse entre fibre parallèle et cellule de Purkinje. Nous avons utilisé la lignée transgénique L7-PP2B, spécifiquement déficitaire pour cette plasticité.Malgré un léger déficit moteur révélé exclusivement sur le rotarod, les capacités de navigation des souris L7-PP2B ne sont pas affectées dans une tâche de navigation en labyrinthe aquatique de type piscine de Morris. Les propriétés des cellules de lieu de l’hippocampe des souris L7-PP2B ont ensuite été caractérisées lors de l’exploration d’une arène circulaire dans différentes conditions environnementales. Contrairement à celles des souris L7-PKCI, les propriétés des cellules de lieux des souris L7-PP2B ne sont pas affectées lorsque les souris ne peuvent utiliser que les informations de mouvement propre pour s’orienter, c'est à dire dans le noir. Par contre, les cellules de lieux des souris L7-PP2B présentent une instabilité en l’absence de toute manipulation d’indice environnemental, dans 23% des sessions d’enregistrement. Cette instabilité, absente chez les souris contrôles, se manifeste de façon imprévisible dans un environnement familier et est caractérisée par une rotation angulaire cohérente de l’ensemble des cellules de lieux enregistrées. Ces données suggèrent qu’en l’absence de PLT cérébelleuse la représentation spatiale de l’hippocampe n’est pas ancrée de façon stable aux indices externes proximaux. Ces résultats, associés à ceux des souris L7-PKCI indiquent que le cervelet contribue de manière complexe à la fois à la représentation spatiale hippocampique et aux capacités de navigation et que DLT et PLT jouent probablement des rôles différents dans ces processus. / Spatial navigation can be divided into two processes: building a spatial representation from the environment exploration and using this representation to produce an adapted trajectory toward a goal. During the environment exploration, external and self-motion information (i.e. vestibular and proprioceptive) are combined to form the spatial map. It has long been suggested that the cerebellum participates in spatial navigation but its role has often been confined to motor execution. Our team has studied L7-PKCI mice which lack a plasticity mechanism (long term depression (LTD)) at parallel fiber-Purkinje cell synapses in the cerebellar cortex. These works have shown that L7-PKCI mice present a deficit in trajectory optimization as well as in the maintenance of the cognitive map in the hippocampus. Indeed in these mice, the firing properties of hippocampal place cells are affected specifically when mice have to rely on self-motion information, i.e. when exploring the environment in the dark.A these same synapses, another type of plasticity (long term potentiation (LTP)) has been described, and allows (with LTD) the bidirectional modulation of the synaptic efficiency. Bidirectional plasticity is a key element of the ‘adaptive filter’ theoretical models of cerebellar information processing. According to these models, a lack of LTP or LTD should similarly affect bidirectional plasticity and result in comparable deficits. To test this prediction, we investigated the functional consequences of a deficit of LTP at parallel fiber-Purkinje cell synapses using the L7-PP2B mice model, specifically impaired for this plasticity.In spite of a mild motor adaptation deficit, revealed on the rotarod task, spatial learning of L7-PP2B mice was not impaired in the watermaze task. Hippocampal place cell properties of L7-PP2B mice were characterized during exploration of a circular arena, following different experimental manipulations. In contrast to mice lacking cerebellar LTD, place cells properties of L7-PP2B mice were not impaired when mice had to rely on self-motion cues, i.e. in the dark. Surprisingly, L7-PP2B place cells displayed instability in the absence of any proximal cue manipulation in 23 % of the recording sessions. This instability occurred in an unpredictable way in a familiar environment and was characterized each time by a coherent angular rotation of the whole set of recorded place cells. These data suggest that, in the absence of cerebellar LTP, hippocampal spatial representation cannot be reliably anchored to the proximal cue. These results along with those from L7­PKCI mice, indicate that the cerebellum contributes to both hippocampal representation and subsequent navigation abilities and that LTP and LTD are likely to play different roles in these processes.

This thesis examines the functional connectivity of the hippocampus with the rest of the brain, with a focus on the neocortex. The hypothesis explored, in an animal model, is whether the frequency-dependent behaviour of certain brain connectivity relationships applies to hippocampal-neocortical connections. To encompass the temporal and spatial resolution necessary to do this, two main techniques are used in combination in most of the experimental work hereby presented: (1) electrophysiological recordings of local field potentials (LFPs), and (2) functional activity recordings of blood oxygenation level dependent (BOLD) signal using functional magnetic resonance imaging (fMRI). The main hypothesis is that the frequency-dependent behaviour of specific hippocampal synapses imposes the rules of extra-hippocampal activity propagation and hippocampal-neocortical interactions. The main discovery is that short and long-term plasticity modulates network activation, a finding suggesting a possible mechanism that could mediate the encoding and consolidation of memory traces. Chapters 1 to 3 introduce the vast literature review in which this project lies, and the general methods utilised. Chapter 4 (first experimental chapter) describes, using electrophysiology in rats, the evoked response of the main hippocampal output (CA1 neurons) when its major input (CA3 pyramidal cells) is activated at frequencies that in subsequent experiments were used to build brain-wide functional maps. CA1 spiking activity is found to be optimal in maintaining the amplitude of the population spike (PS) at beta frequencies (10-20 Hz), whereas lower (< 10 Hz) and higher (> 20 Hz) frequencies are normally less effective. Chapter 5 describes, using fMRI, how these intra-hippocampal activity patterns relate to long-range activity propagation in fMRI experiments. Hippocampal activation exhibits a linear monotonic increase with evoked frequency, whilst a network of selected structures is activated preferentially when beta frequencies are applied (mainly neocortical structures like the prefrontal and parietal cortices, motor and sensory cortices, and some subcortical structures like the nucleus accumbens and the striatum). This data is highly correlated with the PS recorded in CA1 and with multi-unit activity (MUA) and single-unit activity (SUA) simultaneously recorded in the medial prefrontal cortex (mPFC), one of the structures receiving propagated activity at beta frequencies, as described in Chapter 6. As mPFC also receives hippocampal input at a restricted beta frequency range stimulation of the dorsal hippocampus, Chapter 7 describes the use of a combined electrophysiology/fMRI approach to identify the pathway responsible for activity propagation. We performed microsurgery lesions to investigate the pathway responsible for the polysynaptic propagation of activity. Findings indicate that the septo-temporal longitudinal pathway is the one leading information transfer from dorsal to ventral hippocampus in the rat, and from there directly to the ventral subiculum, apparently by-passing entorhinal cortex. Last, in Chapter 8 the effect of durable modifications of synaptic weights by long-term potentiation (LTP) in the previously described frequency-dependent activity propagation is also described and contextualized in the memory trace consolidation framework, both electrophysiologically (Chapter 5) and with fMRI (Chapter 6). LTP is a long-lasting change in synaptic weights that, at the CA3-CA1 synapse, is capable of modifying hippocampal-neocortical connections such as to open the opportunity for higher frequency patterns (> 40 Hz) to propagate to neocortical structures. These results suggest that, by means of frequency-coding, the hippocampus normally regulates propagation of selected information to the neocortex, but that at specific moments (e.g. when the hippocampus undergoes LTP) this regulation broadens to permit high-frequency information to pass through and affect neural activity in the cortex. It is a beautifully simple mechanism that merits further detailed examination in a multi-disciplinary manner as outlined in Chapters 9 and 10.

Submitted by Maurílio Figueiredo (maurilioafigueiredo@yahoo.com.br) on 2014-07-02T18:25:21Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO_EstudoComparativoMétodos.pdf: 2292877 bytes, checksum: 835505b7bbaee9d0eecceaf24630beaa (MD5) / Approved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2014-07-17T18:38:52Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO_EstudoComparativoMétodos.pdf: 2292877 bytes, checksum: 835505b7bbaee9d0eecceaf24630beaa (MD5) / Made available in DSpace on 2014-07-17T18:38:52Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO_EstudoComparativoMétodos.pdf: 2292877 bytes, checksum: 835505b7bbaee9d0eecceaf24630beaa (MD5) Previous issue date: 2012 / Produzidos desde a década de 40, os organofosforados foram os primeiros a substituírem os organoclorados, aos quais os insetos já apresentavam resistência. Dentre estes o clorpirifós apresenta-se como um inseticida altamente tóxico. Devido à sua elevada lipossolubilidade é absorvido pelo organismo humano, especialmente pela pele. Os organofosforadossão conhecidos por induzirem ou agravarem certos problemas de saúde; assim é importante seu monitoramento. Para a determinação de clorpirifós em amostras aquosas geralmente utiliza-se etapas de extração/pré-concentração antes de se efetuar a quantificação. Neste trabalho, foramavaliadas as técnicasextração líquido-líquido com partição a baixa temperatura (LLE-LTP) e microextração em fase sólida com headspace (HS-SPME) e as técnicas de cromatografia gasosa com detecção por captura de elétrons (GC-ECD) e cromatografia gasosa acoplada à espectrometria de massas (GC-MS) para definição do método analítico mais sensível, utilizando planejamentos experimentais multivariados para a otimização dos métodos de preparo de amostra. Após a otimização, o método HS-SPME/GC-ECD foi validado para o monitoramento de clorpirifós em cursos d’água superficiais próximos às principais plantações de batata na zona rural de Ouro Branco/MG. Apresença de agrotóxicos, como o clorpirifós nesses cursos d’água, foi relacionada em pesquisas anteriores aos altos índices dos casos de hipertensão e aborto da população da zona rural. A definição dos pontos de coleta foi feita após visitas de campo e levantamento de informações obtidas junto aos órgãos e pessoas competentes. O planejamento fatorial completo apontou as variáveis tempo (1)e temperatura de extração (2), volume de amostra (3) e concentração de eletrólito (4) como importantes para o sistema;logo as mesmas foram avaliadas em mais níveis pela matriz Doehlert.As variáveis 1, 2 e 3 foram apontadas como as que influenciam o sistema. O modelo quadrático do planejamento Doehlert foi significativo não apresentando falta de ajuste e a condição ótima foi definida no tempo e temperatura de extração de 60 min e 85 °C, respectivamente, com um volume de amostra de 11 mL e 0,04 mol/L de concentração de eletrólito. O método otimizado foi validado considerando os parâmetros seletividade, linearidade, precisão (repetitividade e precisão intermediária), exatidão e os limites de detecção (LOD) e quantificação (LOQ). O LOQ e LOD foram 0,76 e 2,28 μg/L, respectivamente. O método se mostrou linear com R2 igual a 0,992. Os resultados indicaram que o método HS-SPME/GC-ECD proposto é eficiente para a determinação de clorpirifós em água e as análises das amostras coletadas indicaram a presença de clorpirifós em concentrações entre osvalores de LOD e LOQ. Os resultados desta pesquisa foram informados à Prefeitura Municipal de Ouro Branco e órgãos competentes. __________________________________________________________________________________________ / ABSTRACT: Produced since1940, organophosphates were the first to replace the organochlorines, which already had bug resistance. Among these chlorpyrifos presents itself as a highly toxic insecticide. Due to its high lipossolubility it is absorbed by the human body, especially the skin. Organophosphates are known to induce or worsen certain health problems; so it is important its monitoring. For the determination of chlorpyrifos in aqueous samples generally are used steps of extraction/pre-concentration before performing the measurement. In this study it was investigated the liquid-liquid extraction technique withlow temperature partition (LLE-LTP) and solid phase microextraction with headspace (HS-SPME) and the techniques of gas chromatography with electron capture detection (GC- ECD) and gas chromatography coupled to mass spectrometry (GC-MS) to define the most sensitive analytical method, using multivariate experimental designs for the optimization of the sample preparation techniques. After optimization, the HS-SPME/GC-ECD method was validated for the monitoring of chlorpyrifosin surface water bodies close to the main potato fields in countryside of OuroBranco/MG; the presence of pesticides, such as chlorpyrifos, in these streams has been linked in previous research to the high rates of hypertension and abortion cases of the population in rural areas. The definition of the sampling was made after field visits and information survey obtained from the competent agencies and people. The full factorial design showed the variables time (1) and temperature of extraction (2), sample volume (3) and concentration of electrolyte (4) as important to the system; in this way the same ones were evaluated in more levels by Doehlert matrix. Variables 1, 2 and 3 were identified as those that affect the system, the quadratic modelof Doehlert design was significant not showing lack of fit and the optimal condition was set in the time and temperature of extraction of 60 min and 85 °C, respectively, with a volume of 11 mL and 0.04 mol/L of electrolyte concentration. The optimized method was validated considering the parameters selectivity, linearity, precision (repeatability and intermediate precision), accuracy and limits of detection (LOD) and quantitation (LOQ). The LOD and LOQ were 0.76 and 2.28 μg/L, respectively. The method was linear with R2 equal to 0.992. The results indicate that the method HS-SPME/ECD-GC proposed is efficient for the determination of chlorpyrifos in water and the analysis of the samples indicated the presence of chlorpyrifos in concentrations between the values of LOD and LOQ. These results were reported to the Ouro Branco City Hall and competent agencies.

Submitted by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-03-09T14:45:09Z No. of bitstreams: 2 dissertacao Andre Costa.pdf: 10722387 bytes, checksum: bff242b1a21e34e27f228538f8f5d6b1 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-09T14:45:10Z (GMT). No. of bitstreams: 2 dissertacao Andre Costa.pdf: 10722387 bytes, checksum: bff242b1a21e34e27f228538f8f5d6b1 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-11-25 / Capes / Um detector de pedestres básico geralmente possui dois componentes principais: um que seleciona regiões da imagem que possivelmente contêm um pedestre (gerador de candidatos) e outro que classifica estas regiões em grupos de pedestres e não-pedestres (classificador). Estes classificadores normalmente baseiam-se em extratores de características, que são transformações que alteram a intensidade ou cor original dos pixels de uma imagem em uma nova representação, para ressaltar algum tipo de conhecimento sobre o conteúdo da imagem. Quando o ambiente é não-controlado, fatores externos podem influenciar negativamente no desempenho do classificador. Baixa resolução, ruído, desfoque e oclusão são alguns efeitos que podem ser gerados por estes fatores, degradando a qualidade das imagens obtidas e, consequentemente, das características extraídas. Esta dissertação propõe-se a avaliar como extratores de características comportam-se nesse tipo de ambiente. Estes cinco tipos de degradação foram simulados nas bases de imagem usadas nos experimentos: INRIA Person e Caltech Pedestrian. Como estamos interessados apenas na etapa de classificação, as imagens foram transformadas em janelas de tamanho fixo na etapa de pré-processamento. Os experimentos usam uma combinação de extratores de características (HOG, LBP, CSS, LGIP e LTP) e modelos de aprendizagem (AdaBoost e SVM linear) para formar classificadores. Os classificadores foram treinados com as imagens intactas e testados com imagens em diversos níveis de degradação. O HOG (42%) e LTP (54%) foram superiores aos demais em aproximadamente metade dos pontos de teste na INRIA Person e Caltech Pedestrian, respectivamente. Foi confirmada a queda de desempenho do LBP quando exposto a ruído, mostrando que o LGIP e o LTP amenizam isso. Também observou-se que o CSS é robusto a ruído, mas gera características fracas no geral. Por fim, notou-se que classificadores que combinam mais de um extrator de características foram superiores aos individuais em boa parte dos pontos de teste. Combinando-se todos os extratores, tem-se um classificador superior em 95,8% das situações ao criado somente com o melhor extrator no geral (HOG, na base da INRIA, e LTP, na base da Caltech).

Le régulateur endogène de la calcineurine 1 (RCAN1) est exprimé dans les neurones, cependant son rôle dans la régulation de la transmission et de la plasticité synaptique est mal connu. De manière intéressante, plusieurs études dans les cellules cardiaques et les levures montrent une double régulation de la calcineurine 1 (CaN1) par RCAN1. Il est décrit qu'en fonction de son état de phosphorylation par la kinase glycogène synthase 3β (GSK3β), RCAN1 réprime la CaN1 à l'état déphosphorylé, mais il faciliterait son activité à l'état phosphorylé. La régulation de la CaN1 par RCAN1 phosphorylé n'a encore jamais été étudiée dans les neurones et pourrait mettre en relation deux acteurs majeurs de la dépression à long terme (LTD), à savoir la CaN1 et la GSK3β. Par ailleurs une étude récente a montré que RCAN1 peut également être phosphorylé par la protéine kinase A (PKA), une kinase essentielle dans la mise en place de la potentialisation à long terme (LTP), entrainant ainsi une augmentation de l’inhibition de la CaN1 par RCAN1. Dans les neurones, RCAN1 pourrait donc potentiellement réguler à la fois la LTP et la LTD dépendamment de son état de phosphorylation. Mes travaux visent à élucider si RCAN1 est capable de réguler la transmission et de la plasticité synaptique en fonction de son état de phosphorylation et si son action dépend de la CaN1. Afin de déterminer le rôle de RCAN1 dans ces processus, une combinaison de techniques de biologie moléculaire, d’électrophysiologie et d'imagerie a été employée. Nous avons généré des mutations ponctuelles de RCAN1 sauvage de manière à rendre RCAN1 non phosphorylable par la GSK3β ou la PKA. L’expression virale de RCAN1 et de ses différents mutants dans des cultures primaires de neurones d’hippocampe a révélé que RCAN1, dans ses versions sauvage et mutées, est localisé au niveau des épines dendritiques, suggérant une possible fonction de RCAN1 à la synapse. De manière à déterminer les effets de RCAN1 sur la transmission et la plasticité synaptique, j’ai exprimé de manière virale RCAN1 et ses différents mutants dans des tranches organotypiques d'hippocampes de rat et analysé leurs effets par enregistrement en ‘‘patch-clamp’’ en configuration de cellule entière. J’ai pu observer que le blocage du site de phosphorylation de RCAN1 par la GSK3β entraînait une augmentation de la transmission synaptique ainsi qu’un blocage de la LTD. De plus j’ai démontré que la LTP été bloquée lorsque la PKA ne pouvait pas phosphoryler RCAN1. Enfin nous avons pu déterminer que ces différents effets de RCAN1 sur la transmission et la plasticité synaptique étaient dépendants de la CaN1. Nous avons donc démontré une cascade d’évènements et mis en évidence le rôle clé de RCAN1 dans la régulation de la LTP et de la LTD. Nous proposons donc que RCAN1 permet de moduler la transmission et la plasticité synaptique en fonction de son état de phosphorylation par la GSK3β et la PKA en agissant sur la CaN1, en étant un effecteur de la GSK3β lors de l’induction de la LTD ainsi qu’un effecteur de la PKA lors de l’induction de la LTP. / The endogenous regulator of calcineurin 1 (RCAN1) is expressed in neurons, nevertheless its role in the regulation of synaptic transmission and plasticity is not well understood. Interestingly, several studies in cardiac cells and yeasts show that RCAN1 is able to inhibit or activate CaN1 depending on its phosphorylation state by glycogen synthase kinase 3β (GSK3β). RCAN1 is able to inhibit CaN1 when it is not phosphorylated by GSK3β and able to activate it in its phosphorylated state. The regulation of CaN1 by phosphorylated RCAN1 has never been studied in neurons although it could provide a critical link between two major actors of long-term depression (LTD), CaN1 and GSK3β. Furthermore, a recent study revealed that RCAN1 can also be phosphorylated by protein kinase A (PKA), a kinase involved in regulating long-term potentiation (LTP), leading to an increase of CaN1 inhibition by RCAN1. Thus, in neurons, the differential phosphorylation of RCAN1 could potentially regulate both LTP and LTD. My work therefore investigates how, depending on its phosphorylation state, RCAN1 affects synaptic transmission and plasticity and if this occurs via a direct action on CaN1. In order to determine the role of RCAN1 phosphorylation in synaptic plasticity, a combination of molecular biology, imaging and electrophysiology was used. We generated point mutations of wild type RCAN1 in order to obtain two non-phosphorylable forms of RCAN1: one that couldn’t be phosphorylated by GSK3β, and another one that PKA could not phosphorylate. Viral expression of RCAN1 and its phosphorylation deficient mutants in dissociated hippocampal cultures revealed that they are localized within dendritic spines, hinting at a synaptic function of RCAN1. To determine the effects of RCAN1 on synaptic transmission and plasticity, I virally expressed RCAN1 and the phosphorylation deficient mutants of RCAN1 in rat organotypic hippocampal slice cultures and analyzed their effects on synaptic plasticity by whole cell ‘‘patch-clamp’’ recordings. I observed that the blockade of the GSK3β phosphorylation site in RCAN1 increased synaptic transmission and blocked LTD induction. Furthermore, I demonstrate that LTP was blocked when PKA was unable to phosphorylate RCAN1. Finally, I determined that these distinct effects of RCAN1 on synaptic transmission and plasticity were directly dependent on CaN1. I thus define a cascade of events as well as demonstrate the key role of RCAN1 in the regulation of both LTP and LTD. Based on my results, I propose that iv RCAN1 modulates synaptic transmission and plasticity according to its phosphorylation states by GSK3β and PKA, via its direct action on CaN1, being an effector of both GSK3β during LTD and PKA during LTP induction.

L'efficacité de la transmission synaptique GABAergique est influencée par la concentration intracellulaire en ions chlorure. Dans les neurones matures, l'extrusion de ces ions par le transporteur neuronal potassium chlore de type 2 (KCC2) permet l'influx d'ions chlorure lors de l'activation des récepteurs du GABA de type A. Néanmoins, KCC2 est aussi enrichi à proximité des synapses excitatrices portées par les épines dendritiques qui correspondent à des protrusions dendritiques enrichies en actine. Alors que l'effet d'une suppression de KCC2 sur l'homéostasie des ions chlorure et la transmission GABAergique est largement documenté, peu de choses sont connues sur l'impact qu'une telle suppression peut avoir sur la transmission glutamatergique. Lors de ma thèse, j'ai exploré le rôle de KCC2 dans la potentialisation à long terme (LTP) de la transmission glutamatergique à l'origine des phénomènes d'apprentissage et de mémorisation. Ce travail a révélé que la suppression de KCC2 compromet les modifications fonctionnelles et structurales sous-tendant la LTP. Cet effet est associé à une inhibition de la cofilin, protéine responsable de la dépolymérisation de l'actine, qui corrèle avec une augmentation de la quantité d'actine filamenteuse dans les épines dendritiques. En empêchant l'inhibition de la cofilin liée à l'absence de KCC2, il m'a alors été possible de restaurer la LTP suggérant que KCC2 pourrait influencer cette forme de plasticité en régulant la dynamique de polymérisation du cytosquelette d'actine. Mes résultats démontrent que la fonction de KCC2 va au-delà du contrôle de l'homéostasie des ions chlorure et influence les mécanismes de plasticité de la synapse excitatrice. / The polarity and efficacy of GABAergic synaptic transmission are both influenced by the intra-neuronal chloride concentration. In mature neurons, chloride extrusion through the neuronal K/Cl cotransporter KCC2 allows an inhibitory influx of chloride upon activation of GABAA receptors. Nevertheless, KCC2 is enriched in the vicinity of excitatory synapses within the dendritic spines that are actin-rich protrusions emerging from dendritic shafts. While it has become clear that KCC2 suppression alters chloride homeostasis and GABA signaling, little is known on its impact on glutamatergic transmission. In the laboratory, we have previously demonstrated that KCC2 suppression in mature neurons leads to decreased glutamatergic transmission efficacy through an ion-transport independent function of KCC2. During my PhD, I have explored how KCC2 may also impact LTP of glutamatergic synapses. My work reveals that KCC2 suppression compromises both functional and structural LTP at these synapses. This effect is associated with inhibition of the actin-severing protein cofilin and enhanced mobilization of F-actin in dendritic spines. Since LTP can be rescued by preventing cofilin inhibition upon KCC2 suppression, I suggest KCC2 might influence LTP through altered actin cytoskeleton dynamics. My results demonstrate that KCC2 function extends beyond the mere control of neuronal chloride homoeostasis and suggest regulation of KCC2 membrane stability may act as a metaplastic switch to gate long term plasticity at excitatory synapses in cortical neurons.

Learning and memory are important processes that occur in the brain. The brain is comprised of neurons that make connections with each other known as synapses. Synaptic plasticity is widely believed to be the physiologic mechanism by which learning and memory occur. Synapses can either be strengthened through a process known as long-term potentiation (LTP) or weakened through long-term depression (LTD). The area of the brain that is most studied for its role in learning and memory is the hippocampus, which has been shown to be involved in memory consolidation. The detection of endocannabinoids and their receptors has opened a whole new field of study in regards to synaptic plasticity. Cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) are among the commonly studied endocannabinoid receptors found in the central nervous system. In the brain, these receptors' natural ligands, anandamide and 2-arachidonylglycerol (2-AG), are found in abundance. Yet not all forms of observed plasticity are accounted for by just these two receptors, so studies into other G-protein coupled receptors (GPCRs) continues. One GPCR, GPR55 is found in many regions of the brain, as well as lysophosphatidylinositol (LPI), its specific ligand. Here we have researched the role of GPR55 in modulating synaptic plasticity in the hippocampus. Using quantitative reverse transcription PCR and immunohistochemistry, we have found GPR55 to be expressed in the hippocampus with highest expression in pyramidal cells, the main excitatory neurons in the hippocampus. Using field and whole cell electrophysiology, we have investigated its effects on synaptic plasticity, discovering that activation of GPR55 by LPI significantly enhances LTP. In memory behavioral assays there are no significant differences between GPR55 KO mice and wild type littermates, indicating that it may not be involved in endogenous memory processes. However, our electrophysiology data makes GPR55 a potential target for treating memory disorders such as dementia. We have also investigated GPR18 and GPR119 for their potential roles in synaptic plasticity. First, we confirmed their expression in the hippocampus and then investigated the effects of their agonists on plasticity. Another receptor, TRPV1 has been studied to alter plasticity. However, the study of how protein translation and RNA transcription involvement in TRPV1 plasticity in mammals has not been investigated. While translation and transcription are known to be important in many forms of LTP, it is unknown whether these processes are important for TRPV1-induced LTD. We are investigating their necessity via whole cell patching and using translation and transcription inhibitors Anisomycin and Actinomycin D, both previously used in slice electrophysiology.

No description available.