Afin de mieux comprendre l’évolution des fonctions du récepteur EphA4 pendant le développement du système nerveux central (SNC), nous avons étudié sa localisation cellulaire et subcellulaire dans l’hippocampe du rat, d’abord chez l’adulte, puis pendant le développement postnatal, ainsi que ses rôles potentiels dans la genèse, la migration ou la maturation des cellules granulaires dans l’hippocampe adulte. Pour ce faire, nous avons utilisé la méthode d’immunocytochimie en microscopie photonique, électronique et confocale. En microscopie photonique, une forte immunoréactivité (peroxydase/DAB) pour EphA4 est observée aux jours 1 et 7 suivant la naissance (P1 et P7) dans les couches de corps cellulaires, avec un marquage notamment associé à la surface des corps cellulaires des cellules granulaires et pyramidales, ainsi que dans les couches de neuropile du gyrus dentelé et des secteurs CA3 et CA1. L’intensité du marquage diminue progressivement dans les couches de corps cellulaires, entre P7 et P14, pour devenir faible à P21 et chez l’adulte, tandis qu’elle persiste dans les couches de neuropile, sauf celles qui reçoivent des afférences du cortex entorhinal. En microscopie électronique, après marquage à la peroxydase/DAB, EphA4 décore toute la surface des cellules pyramidales et granulaires, du corps cellulaire jusqu’aux extrémités distales, entre P1 et P14, pour devenir confiné aux extrémités synaptiques, c’est-à-dire les terminaisons axonales et les épines dendritiques, à P21 et chez l’adulte. À la membrane plasmique des astrocytes, EphA4 est redistribué comme dans les neurones, marquant le corps cellulaire et ses prolongements proximaux à distaux, à P1 et P7, pour devenir restreint aux prolongements périsynaptiques distaux, à partir de P14. D’autre part, des axones en cours de myélinisation présentent souvent une forte immunoréactivité punctiforme à leur membrane plasmique, à P14 et P21. En outre, dans les neurones et les astrocytes, le réticulum endoplasmique, l’appareil de Golgi et les vésicules de transport, organelles impliquées dans la synthèse, la modification posttraductionnelle et le transport des protéines glycosylées, sont aussi marqués, et plus intensément chez les jeunes animaux. Enfin, EphA4 est aussi localisé dans le corps cellulaire et les dendrites des cellules granulaires générées chez l’adulte, au stade de maturation où elles expriment la doublecortine (DCX). De plus, des souris adultes knockouts pour EphA4 présentent des cellules granulaires DCX-positives ectopiques, c’est-à-dire positionnées en dehors de la zone sous-granulaire, ce qui suggère un rôle d’EphA4 dans la régulation de leur migration. Ces travaux révèlent ainsi une redistribution d’EphA4 dans les cellules neuronales et gliales en maturation, suivant les sites cellulaires où un remodelage morphologique s’effectue : les corps cellulaires lorsqu’ils s’organisent en couches, les prolongements dendritiques et axonaux pendant leur croissance, guidage et maturation, puis les épines dendritiques, les terminaisons axonales et les prolongements astrocytaires distaux associés aux synapses excitatrices, jusque chez l’adulte, où la formation de nouvelles synapses et le renforcement des connexions synaptiques existantes sont exercés. Ces localisations pourraient ainsi correspondre à différents rôles d’EphA4, par lesquels il contribuerait à la régulation des capacités plastiques du SNC, selon le stade développemental, la région, l’état de santé, ou l’expérience comportementale de l’animal. / To gain more insight into the various functions of EphA4 receptor during the development of the central nervous system (CNS), we have characterized its cellular and subcellular localization in the rat hippocampus, first in the adult, and second during the postnatal development. We have also examined its potential roles in the genesis, migration, or maturation of the granule cells in the adult hippocampus. For that purpose, we have used immunocytochemistry in light, electron, and confocal microscopy. At the light microsocpic level, a strong EphA4 immunoreactivity (peroxidase/DAB) is observed at postnatal days 1 and 7 (P1 and P7) in the cell body layers, with a labeling notably associated with the surface of pyramidal and granule cell bodies, as well as in the neuropil layers of CA3, CA1, and dentate gyrus regions. The intensity of the labeling diminishes progressively in the cell body layers, between P7 and P14, to become weak at P21 and in the adult, while it persists in the neuropil layers, except in those receiving inputs from the entorhinal cortex. At the electron microscopic level, after peroxidase/DAB labeling, EphA4 covers the entire surface of pyramidal and granule cells, from the cell body to the distal extremities, between P1 and P14, but becomes restricted to the synaptic extremities, i.e. the axon terminals and dendritic spines, at P21 and in the adult. At the plasma membrane of astrocytes, EphA4 is redistributed as in neurons, from the cell body and proximal to distal processes, at P1 and P7, to the distal perisynaptic processes, at P14 and older ages. In addition, axons in the process of myelination present strong punctiform immunoreactivity at their plasma membrane, at P14 and P21. Moreover, in neurons and astrocytes, the endoplamic reticulum, Golgi apparatus, and transport vesicles, organelles involved in the synthesis, post-translational modifications, and transport of glycosylated proteins, are also labeled, and also more intensely in younger animals. Lastly, EphA4 is located in the cell body and dendrites of adult-generated granule cells, at the stage of maturation where they express doublecortin (DCX). In addition, EphA4 adult knockout mice display DCX-positive granule cells in an ectopic position, outside of the subgranular zone, suggesting a role for EphA4 in the regulation of their migration. This work thus reveals a redistribution of EphA4 in neuronal and glial cells, in the cellular sites where cellular motility occurs during their maturation: the cell bodies when they position and organize themselves into layers, the dendritic and axonal processes during their growth, guidance, and maturation, and the dendritic spines, axon terminals, and distal astrocytic processes when synapses are formed or strengthened. These locations could thus reflect different roles for EphA4, similarly associated with the regulation of plasticity in the CNS, according to the stage of development, the region, the CNS integrity, or the behavioural experience of an animal.

La sclérose en plaques (SEP) est une maladie inflammatoire démyélinisante et neurodégénérative du système nerveux central (SNC). Les cellules T activées qui expriment le PD-1 sont inhibées via l’interaction avec l’un des ligands: PD-L1 ou PD-L2. Des études effectuées chez le modèle murin de la SEP, l’encéphalomyélite auto-immune expérimentale (EAE), ont démontré que l’interaction du PD-1 avec ses ligands contribue à atténuer la maladie. Toutefois, le rôle du PD-1 et de ses ligands dans la pathogenèse de la SEP chez l’humain et dans le modèle murin n’a pas été complètement élucidé. Nous avons déterminé que plusieurs cellules du SNC humain peuvent exprimer les ligands du PD-1. Les astrocytes, les microglies, les oligodendrocytes et les neurones expriment faiblement le PD-L1 dans des conditions basales mais augmentent de façon significative cette expression en réponse à des cytokines inflammatoires. Le blocage de l’expression du PD-L1 par les astrocytes à l’aide de siRNA spécifiques mène à l’augmentation significative des réponses des cellules T CD8+ (prolifération, cytokines, enzymes lytiques). Nos résultats établissent ainsi que les cellules gliales humaines peuvent exprimer des niveaux suffisants de PD-L1 en milieu inflammatoire pour inhiber les réponses des cellules T CD8+. Notre analyse de tissus cérébraux post-mortem par immunohistochimie démontre que dans les lésions de la SEP les niveaux de PD-L1 sont significativement plus élevés que dans les tissus de témoins; les astrocytes et les microglies/macrophages expriment le PD-L1. Cependant, plus de la moitié des lymphocytes T CD8+ ayant infiltré des lésions de SEP n’expriment pas le récepteur PD-1. Au cours du développement de l’EAE, les cellules du SNC augmentent leur niveau de PD-L1. Le PD-1 est fortement exprimé par les cellules T dès le début des symptômes, mais son intensité diminue au cours de la maladie, rendant les cellules T insensibles au signal inhibiteur envoyé par le PD-L1. Nous avons observé que les cellules endothéliales humaines formant la barrière hémato-encéphalique (BHE) expriment de façon constitutive le PD-L2 mais pas le PD-L1 et que l’expression des deux ligands augmente dans des conditions inflammatoires. Les ligands PD-L1 et PD-L2 exprimés par les cellules endothéliales ont la capacité de freiner l’activation des cellules T CD8+ et CD4+, ainsi que leur migration à travers la BHE. L’endothélium du cerveau des tissus normaux et des lésions SEP n’exprime pas des taux détectables de PD-L1. En revanche, tous les vaisseaux sanguins des tissus de cerveaux normaux sont positifs pour le PD-L2, alors que seulement la moitié de ceux-ci expriment le PD-L2 dans des lésions SEP. Nos travaux démontrent que l’entrée des cellules T activées est contrôlée dans des conditions physiologiques grâce à la présence du PD-L2 sur la BHE. Cependant, l’expression plus faible du PD-L2 sur une partie des vaisseaux sanguins dans les lésions SEP nuit au contrôle de la migration des cellules immunes. De plus, une fois dans le SNC, les cellules T CD8+ étant dépourvues du PD-1 ne peuvent recevoir le signal inhibiteur fourni par le PD-L1 fortement exprimé par les cellules du SNC, leur permettant ainsi de rester activées. / Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Responses of activated T cells are suppressed upon engagement of the receptor programmed cell death-1 (PD-1) with its ligands (PD-L1 and PD-L2). Experiments using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have demonstrated that the PD-1/PD-Ls interaction contributes to attenuate disease severity. However, the expression and the role of PD-1 and PD-Ls have been partially documented in inflammatory murine models and human CNS data are still incomplete. We determined that primary cultures of human astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 levels under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA in co-culture led to significantly increased CD8 T cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of post-mortem brain tissues demonstrated a significantly greater PD-L1 expression in MS lesions compared to control tissues, which co-localized with astrocyte and microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Similar results were obtained in EAE mice. Even though CNS cells expressed PD-L1 at the peak of the disease, PD-1 intensity on infiltrating T cells decreased throughout EAE disease development. This reduction of PD-1 level on activated T cells prevented these cells to receive PD-L1 inhibitory signal. We also investigated whether human brain endothelial cells (HBECs), which form the blood brain barrier (BBB), can express PD-L1 or PD-L2 and thereby modulate T cells. HBECs expressed PD-L2 under basal conditions, whilst PD-L1 was not detected. Both ligands were up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 led to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increased CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions did not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues were PD-L2-positive, while only about 50% expressed PD-L2 in MS lesions. Therefore, our results demonstrate that under basal conditions, PD-L2 expression by HBECs impedes the migration of activated immune T cells through the BBB, and inhibits their activation. However, such impact is impaired in MS lesions due to down-regulation of PD-L2 levels on the endothelium. The majority of infiltrating CD8 T cells is devoid of PD-1, thus insensitive to PD-L1 inhibitory signal providing by CNS cells once they have entered the CNS.

Le trafic membranaire est essentiel dans les neurones pour la morphogénèse, le recyclage des vésicules synaptiques et des récepteurs. L’exocytose des récepteurs AMPA contenus dans les endosomes de recyclage (ER) est nécessaire pour l’expression de la plasticité synaptique à long terme (PLT). Pour étudier ce mécanisme, nous avons visualisé l’exocytose par microscopie de fluorescence sur des neurones en culture transfectés avec le récepteur de la transferrine (TfR), un marqueur des ER, fusionné à la phluorine. Un examen systématique des événements d'exocytose a révélé des différences de comportement. Dans la plupart des cas, les récepteurs diffusent rapidement dans la membrane plasmique après exocytose (discharge), mais dans environ 25% des cas, les récepteurs restent concentrés (display). L’utilisation de changements rapides de pH extracellulaire autour de la cellule montre que l’exocytose est transitoire : après quelques secondes (médiane 2.6s) les récepteurs sont réinternalisés. Ce mécanisme a pu être étendu aux récepteurs AMPA et β2-adrenergique, pour lesquels l’exocytose de type display avait déjà été décrite. L’imagerie deux couleurs montre que Rab11, un marqueur des ER, est enrichie au site d’exocytose. L’expression d’un dominant négatif de Rab11 connu pour inhiber la PLT provoque une diminution spécifique de la fréquence des évènements discharge. Dans nos recherches sur le mécanisme de l’exocytose, nous avons testé l’implication des protéines SNARE dans la fusion membranaire. Ainsi VAMP4 est enrichie avec le TfR dans les ER qui sont exocytés à une fréquence équivalente. De plus, elle est requise pour le recyclage du TfR. / Membrane trafficking is essential for neuronal function: from growth of neurons and synapse formation to recycling of synaptic vesicles and receptors, questions concerning exocytosis and endocytosis are stimulating neurobiology research. In particular, trafficking of glutamate receptors present in recycling endosomes (REs) is necessary for the expression of long term potentiation (LTP). To investigate the mechanism of exocytosis in dendrites, we have imaged cultured rat hippocampal neurons transfected with transferrin receptor, a classical marker of REs, tagged with phluorin. As for AMPA receptors or β2-adrenegric receptors, single exocytic events has revealed two main behaviors: in most cases, receptors diffuse quickly in the plasma membrane after exocytosis (discharge events), but receptors can also remain clustered (display events). Using fast extracellular pH changes around the recorded cell, we show that for display events exocytosis is transient: after a few seconds (median 2.6 s) receptors are internalized. Moreover, using two color imaging of single exocytosis events with markers of neuronal compartments, we found that Rab11 is enriched at the exocytosis site, confirming the endosomal origin of the vesicles. Overexpression of a dominant negative form of Rab11 known to impair LTP decreases selectively the frequency of discharge events. As SNARE proteins are involved in virtually all membrane fusion processes, we investigated the role of Vamp proteins in somatodendritic exocytosis events. We found that Vamp4, unlike Vamp2 or Vamp7, is enriched in TfR containing compartments and can undergo exocytosis at high frequency and is required for TfR exocytosis.

Racional: A epilepsia do lobo temporal está comumente associada à farmacorresistência e tem a esclerose hipocampal como achado neuropatológico em mais da metade dos casos. Histologicamente, a esclerose hipocampal está associada à perda neuronal diferencial e gliose, além de alterações nos níveis de moléculas associadas à homeostase da água tecidual, como a aquaporina 4 e a molécula de matriz sulfato de condroitina. Em imagens de ressonância nuclear magnética, a esclerose é caracterizada por redução de volume em sequências ponderadas em T1, aumento de sinal e tempo de relaxamento em sequências ponderadas em T2 e redução na transferência de magnetização. Justificativa e Objetivos: Uma vez que tanto o sinal T2 quando a transferência de magnetização são dependentes da água tecidual, nosso objetivo é avaliar, na formação hipocampal de pacientes com epilepsia do lobo temporal, as correlações entre populações celulares e moléculas ligadas à homeostase da água e as imagens ponderadas em T2 e transferência de magnetização. Visamos ainda definir, na formação hipocampal de indivíduos sem alterações neuropatológicas, o volume de cada um dos subcampos hipocampais. Metodologia: Pacientes com epilepsia do lobo temporal farmacorresistente (ELT, n = 43), bem como voluntários sadios (controle radiológico, CH, n = 20), foram submetidos a exames de ressonância magnética em máquina de 3T para mensuração da volumetria hipocampal, tempo de relaxamento T2 e transferência de magnetização hipocampal (exames in vivo). Após o tratamento cirúrgico para o controle das crises, os hipocampos dos pacientes com ELT foram fixados por 8 dias e submetidos aos exames ex vivo em máquina de 3T para cálculo do tempo de relaxamento T2 de cada subcampo hipocampal. Hipocampos controle (Controle historadiológico, CHR, n = 14), foram obtidos de autópsias de pacientes sem histórico ante-mortem de doença neurológica ou presença de patologia no exame do encéfalo pos mortem. Ambos os grupos controle foram pareados para idade em relação ao grupo ELT. Alguns dos casos CHR (n = 6) foram também submetidos à imagem 3D T2 em máquina de 4,7T para cálculo de volumetria dos subcampos hipocampais. Após emblocamento em parafina, secções coronais hipocampais dos casos CHR e ELT foram submetidas às técnicas de histoquímica básica Hematoxilina e Eosina e Luxol Fast Blue, e às imuno-histoquímicas para avaliação das populações neuronais (NeuN), astrócitos reativos (GFAP), micróglias ativadas (HLA-DR) e para a expressão de aquaporina 4 (AQP4) e níveis de sulfato de condroitina (CS-56). Para a comparação entre os grupos, foram realizados testes t para dados paramétricos e Mann-Whitney para dados não-paramétricos. Testes de correlação foram empregados para análise da associação entre as avaliações histológicas e os exames de ressonância magnética. Resultados: Pacientes com ELT apresentaram menor volume hipocampal, maior tempo de relaxamento T2 e menor transferência de magnetização no exame in vivo, quando comparados com o CR. O exame ex vivo para a volumetria dos subcampos hipocampais em casos do grupo CHR indicou que a fascia dentata, a região CA1 e o subículo correspondem à 85 % do volume hipocampal total. Quanto ao tempo de relaxamento T2 ex vivo, foi observado aumento em todos os subcampos hipocampais do grupo ELT, à exceção da fascia dentata, quando comparados ao CHR. A avaliação da densidade neuronal indicou redução significativa em todos os subcampos dos casos ELT, à exceção do subículo, quando comparados ao CHR. Em relação aos valores do grupo CHR, foi observada astrogliose em quase todos subcampos da formação hipocampal (a exceção da zona subgranular e do hilo) e microgliose em todos os subcampos (exceto pelo subículo) dos casos com ELT. Pacientes com ELT apresentaram redução na expressão de aquaporina 4 perivascular em todos os subcampos do hipocampo, comparados ao CHR. Aumento nos níveis de sulfato de condroitina foi observado em todos os subcampos da formação hipocampal, à exceção da camada granular, nos pacientes com ELT. O volume hipocampal e a transferência de magnetização in vivo dos pacientes com ELT correlacionaram-se tanto com a população neuronal como com os níveis de sulfato de condroitina, enquanto que o tempo de relaxamento in vivo correlacionou-se com a população astroglial e os níveis de sulfato de condroitina. O exame ex vivo corroborou a correlação entre a população glial e o tempo de relaxamento observado nos pacientes com ELT. A diferença entre o tempo de relaxamento in vivo e ex vivo correlacionou-se tanto com a difusibilidade da água no tecido como com os níveis de sulfato de condroitina. Conclusões: Nossos dados indicam correlação entre a patologia hipocampal e as imagens de ressonância nuclear magnética, sendo que a maior qualidade das imagens ex vivo permitiu uma avaliação mais direta entre o sinal de ressonância e a patologia, indicando importância da população celular e matriz extracelular para o volume hipocampal e a transferência de magnetização, e da astrogliose para o tempo de relaxamento T2. Finalmente, nossos dados mostraram que CA1, subículo e fascia dentata tem grande participação no volume hipocampal, sendo que alterações nestas regiões tem um papel mais relevante nas alterações observadas na ressonância magnética, como indicado por nossas correlações. / Rationale: Drug resistant temporal lobe epilepsy is often associated with hippocampal sclerosis. Histological evaluation reveals differential neuronal loss, gliosis and changes in molecules associated with water homeostasis, such as aquaporin 4 and chondroitin sulfate. Magnetic resonance imaging in these cases often reveals hippocampal atrophy, increased T2 signal and T2 relaxation and reduced magnetization transfer ratio in the hippocampus. Aims: Once both T2 signal and magnetization transfer are affected by tissue water, our goal was to evaluate, in the hippocampus of drug-resistant temporal lobe epilepsy patients who underwent surgery for seizure control, the associations between cellular populations, aquaporin 4 and chondroitin sulfate with T2 relaxation time and magnetization transfer. Additionally, we intended to measure the individual volume of each hippocampal subfield in hippocampus from patients without neurological disease. Methods: Patients with drug-resistant temporal lobe epilepsy (TLE, n = 43) and age-matched health volunteers (radiological control, RC, n = 20) were submitted to magnetic resonance in a 3T machine for hippocampal volumetry measure, T2 relaxation and magnetization transfer (in vivo examination). After surgical treatment for seizure control, hippocampi from the TLE patients were fixed in formalin for 8 days and then submitted to ex vivo imaging in 3T for relaxation time of every hippocampal subfield. Control hippocampi were obtained from autopsies of age-matched patients without ante mortem history of neurological disease or post mortem neurological pathology, and underwent the same ex vivo imaging (histo-radiological control, HRC, n = 14). Six cases from the HRC underwent 3D T2 imaging in a 4.7T machine, in order to measure the volumes of the hippocampal subfields. Paraffin embedded hippocampal sections from TLE and HRC were submitted to Hematoxilin-Eosin and Luxol Fast Blue histochemistries, and to immunohistochemistries for the evaluation of neurons (NeuN), reactive astrocytes (GFAP), activated microglia (HLA-DR), for aquaporin 4 (AQP4) and for chondroitin sulfate (CS-56). Students t-test or Mann-Whitneys test were performed for comparison between groups, and correlation tests were performed for the comparison between histological and magnetic resonance measures. Results: Patients with TLE presented reduced hippocampal volume, increased T2 relaxation time and reduced magnetization transfer, when compared to RC. The ex vivo volumetry of the hippocampal subfields revealed that fascia dentata, CA1 and subiculum together correspond to 85 % of the total hippocampal volume. Ex vivo relaxation time, as the in vivo, were increased in the subfields of TLE patients, when compared to HRC. Compared to HRC, TLE patients presented neuron loss and microgliosis in all hippocampal subfields but the subiculum, and astrogliosis in all hippocampal subfields but the subgranule zone and the hilus. Reduced perivascular aquaporin 4 was observed in all hippocampal subfields of TLE patients, and increased chondroitin sulfate was observed in all hippocampal subfields, with the exception of granule cell layer, of TLE patients, when compared to HRC. In TLE, both in vivo hippocampal volume and magnetization transfer correlated with the levels of chondroitin sulfate and the neuronal population, whereas the in vivo relaxation time correlated with the astroglial population and the levels of chondroitin sulfate. Ex vivo relaxation time also correlated with the astroglial population in TLE patients. The difference between in vivo and ex vivo relaxation values correlated with water difusibility and the levels of chondroitin sulfate. Conclusion: Our data indicate the importance of neuron population and extracellular matrix to both hippocampal volume and magnetization transfer, and of the reactive astrocytes for T2 relaxation. Ex vivo relaxation time allowed a more detailed evaluation, and indicated more robust correlations between reactive astrocytes and T2 relaxation. Finally, Our data indicated that CA1, the subiculum and fascia dentata are the major contributors to hippocampal volume, so changes in these subfields most likely will affect magnetic resonance imaging.

As ações cardiovasculares decorrentes do tabagismo devem-se principalmente à nicotina. O alcalóide exerce suas funções quando na corrente sangüínea, mas também atravessa a barreira hemato-encefálica onde pode participar da regulação de sistemas de neurotransmissão importantes para o controle central da pressão arterial e, eventualmente, desenvolvimento da hipertensão. Portanto, o abuso à nicotina pode ser especialmente relevante para indivíduos com predisposição genética à hipertensão. Desta forma, os objetivos do presente trabalho foram o de estudar os sistemas de neurotransmissão em núcleos encefálicos envolvidos no controle cardiovascular após tratamento crônico periférico com nicotina, assim como avaliar a influência da nicotina sobre o desenvolvimento da hipertensão essencial em ratos espontaneamente hipertensos (SHR) e compará-los a ratos normotensos (WKY). Para isso, utilizaram-se técnicas como a imunohistoquímica, análise da ligação de receptores, hibridização in situ, cultura de células neuronais e gliais, PCR em tempo Real e Western Blotting. Nossos resultados demonstraram que o tratamento crônico com nicotina não só antecipou o desenvolvimento como também intensificou a hipertensão nos animais SHR. Os ratos WKY não tiveram a pressão arterial alterada. De modo geral, o efeito do alcalóide sobre os sistemas catecolaminérgico e do neuropeptídeo Y não parece ter relação com a antecipação e a intensificação da hipertensão nos ratos SHR. O sistema glutamatérgico está, pelo menos em parte, relacionado à antecipação e intensificação da hipertensão em ratos SHR após exposição crônica à nicotina. O tratamento com nicotina gerou evidências de que o alcalóide interage com o sistema angiotensinérgico a fim de promover a hipertensão em ratos SHR. Por fim, os resultados apresentados aqui indicam que a nicotina modula diferentes sistemas de neurotransmissão, os quais podem estar envolvidos na antecipação e intensificação da hipertensão em ratos SHR submetidos ao tratamento com nicotina. / Nicotine is one of the most important agents for cardiovascular diseases in tobacco smoking. This alkaloid acts in the blood stream, but it also crosses the blood-brain-barrier and participate in the regulation of pivotal neurotransmitter systems for the blood pressure control and, eventually, for hypertension development. In this context, nicotine abuse could be very relevant to individuals carrying genetic factors to hypertension. The objectives of the present work were to study neurotransmitter system in brain cardiovascular areas involved in the control of blood pressure after chronic peripheral nicotine exposure, as well as to evaluate nicotine influence on essential hypertension development in spontaneously hypertensive (SHR) and normotensive rats (WKY). By means of immunohistochemistry, binding, in situ hybridization, neuron and glial culture, real time PCR and Western Blotting, we have demonstrated that chronic treatment with nicotine not only anticipated but also intensified hypertension on SHR. WKY rats did not showed any change on blood pressure. We observed no evidences of the involvement of neuropeptide Y and catecolamines systems in the development of hypertension after nicotine treatment. However, it seems that the glutamatergic system is, at least in part, responsible for the hypertension development after chronic nicotine exposure. To study the angiotensinergic system we cultivated neuron and glial cells from SHR and WKY rats and treated them with nicotine. The responses of this system agree with the hypothesis that nicotine interacts with angiotensin to promote hypertension only in the hypertensive strain. In conclusion, results presented herein support the hypothesis that nicotine modulates neurotransmitter systems that might have relevant functions in the development and intensification of hypertension in SHR.

La science a avancé depuis le XIX ème siècle. Des nouveaux outils sont apparus : la microscopie optique nous donne la vision des cellules, la microscopie électronique nous entraine au cœur de celles-ci. L’imagerie par résonance magnétique est apparue dans les années soixante-dix. Depuis son évolution, l’IRM nous entraine de plus en plus loin dans les profondeurs secrètes de nos cerveaux. La possibilité d’observer l’activité neuronale à l’aide de l’imagerie fonctionnelle est une grande révolution. Cette thèse montrera la possibilité que l’on a d’observer l’activité d’un neurone individuel sans modification de son réseau grâce à l’imagerie rehaussée au manganèse. L’étude sera effectuée sur des Aplysies à très haut champ (17T). Ces animaux sont des mollusques marins gastropodes qui possèdent une particularité : leurs neurones sont de tailles importantes, ils peuvent atteindre 1 mm de diamètre. Leurs neurones sont regroupés en plusieurs ganglions. Mon étude portera sur le ganglion buccal, qui est le ganglion le plus étudié dans les recherches en électrophysiologie. Avant de réaliser les acquisitions, j’ai dû concevoir plusieurs antennes de tailles microscopiques adaptées à la taille des ganglions. En réduisant la taille des antennes, le rapport signal sur bruit augmente. Dans un deuxième temps, une double antenne a été développée permettant l’acquisition de deux échantillons simultanément. Cette antenne a nécessité de créer des préamplificateurs fonctionnant à 730 MHz. La première série d’expériences a permis d’observer l’évolution de l’activité neuronale selon différents stimulus liés au comportement alimentaire des aplysies in-vivo. J’ai montré grâce à la technique mise en place que l’on peut distinguer par IRM l’activité de chaque neurone face à un stimulus. Par la suite, pour continuer ce travail, une deuxième série d’expériences a été effectuée in-vitro. J’ai étudié le comportement des neurones selon les neuro-stimulateurs perfusés : la dopamine et la sérotonine, tous les deux présents naturellement dans l’aplysie. Globalement les neurones ont été activés mais après les avoir observés individuellement, j’ai remarqué quelques différences selon les neurotransmetteurs. Cette technique peut maintenant être utilisée pour étudier d’autres conduites de l’aplysie comme le comportement compulsif. L’étude sur la mémoire peut être aussi envisagée. Les origines comportementales ont probablement des mécanismes identiques entre les différentes espèces animales et donc avec l’Homme comme l’a démontré les études d’Eric Kandel sur la mémoire. / Science has evolved since the 19th century. New tools have appeared such as optical microscopy which gives us the vision of cells and electronic microscopy which leads us into their hearts. The magnetic resonance imaging appeared in the seventies. Evolving over time, the MRI has taken us farther and farther into the secret depths of our brains. The possibility of observing the neuronal activity thanks to the functional imaging is a major evolution. This thesis will show the possibility we have to observe the activity of a single neuron without modification of its network thanks to the manganese enhanced magnetic resonance imaging technique. The study was done on the Aplysia at very high field magnet (17T). These animals are marine gastropod mollusks with a peculiarity: their neurons are of important size and can reach 1 mm in diameter. Their neurons are grouped into several ganglia. My study concerns the buccal ganglion which is the most studied ganglia in the research in electrophysiology. Before making any acquisitions, I had to conceive several microscopic coils adapted to the size of the ganglions. By reducing the size of the coils, the signal of the noise ratio increases. Then, a double coil allowing the simultaneous acquisition of two samples was built. This antenna required the construction of pre-amplifiers operating at 730 MHz. The first series of experiments helped observe the evolution of the neuronal activity according to different stimuli linked to the eating habits of the Aplysia in vivo. Thanks to the technique implemented, I shall show that, using MRI, it is possible to distinguish the activity of each neuron with respect to a stimulus. Afterwards, to continue this work, a second series of experiments was made in vitro. I studied the behavior of neurons when perfused with neural stimulators: dopamine and serotonin, both naturally present in the Aplysia. Generally, all neurons were activated but when observing them individually, I noticed some differences. Studies in electrophysiology will allow us to get a better understanding and a confirmation of the results of this study. The MEMRI technique can be used in the future to study various disorders such as compulsive behaviors, which are present in the Aplysia, and probably have the same origins as in humans, given that many fundamental processes (such as memory studied by Eric Kandel who he demonstrated that human and Aplysia memories works with the same mechanism) are similar between the two species.

Doenças do neurônio motor (DNM) representam um grupo de doenças que cursam com fraqueza muscular progressiva e inexorável, e o manejo clínico é baseado no controle dos sintomas. Estes doentes sofrem de acometimentos motores e não motores intensos e de evolução progressiva. Entretanto, além dos sintomas motores, de humor e de déficits cognitivos, uma caracterização mais profunda de sintomas não- motores nesses doentes raramente foi relatada. Este estudo transversal objetivou descrever os sintomas não motores na DMN e seu impacto na qualidade de vida e no estado funcional, com foco na dor e alterações sensoriais. Oitenta doentes (31 mulheres, 55,7 ± 12,9 anos) com DNM foram submetidos a exame clínico extenso, avaliação de dor (questionário de dor McGill, Inventário breve de dor, questionário douleur neuropathique-4), avaliação psicofísica [teste quantitativo da sensibilidade (TQS) e modulação condicionada da dor (MCD)], avaliações de humor e catastrofismo, e foram comparados com controles saudáveis (CS) pareados por sexo e idade. Dor crônica (presente a maior parte dos dias por mais de três meses) foi presente em 46% dos doentes (escala numérica da dor = 5,18 ± 2,0). A dor de origem musculo- esquelética ocorreu em 40,5% e foi localizada principalmente na região da cabeça/pescoço (51%) e da região lombar (35%). A dor neuropática não presente nesta amostra. Comparado aos CS, os doentes com DNM apresentaram menor limiar de detecção de frio (p < 0,002) e valores de MCD significativamente menores (4,9 ± 0,2% vs. 22,1 ± 0,2%, p = 0,012). Os resultados do TQS/MCD não diferiram entre os doentes com DNM com e sem dor. A intensidade da dor foi correlacionada estatisticamente com ansiedade, depressão e catastrofismo, e os escores de espasticidade foram correlacionados inversamente com a MCD (rho = -0,30, p = 0,026). A dor é um sintoma frequentemente relatado por doentes com DNM. Alterações somatossensoriais e de MCD existem em DNM e podem estar relacionadas com a natureza neurodegenerativa da doença. Estudos adicionais devem investigar formas de melhor quantificar estas alterações e explorar estratégias de tratamento mais apropriadas para o seu controle / Motor neuron disorders (MNDs) represent a group of diseases that curse with inexorable muscle weakness and medical management is based on symptom control. These patients suffer from intense motor and non-motor progressive symptoms. However, apart from motor symptoms, mood and cognitive impairments, deeper characterization of non-motor symptoms in these patients have been rarely reported. This cross-sectional study aimed to describe non-motor symptoms in MND and their impact on quality of life and functional status, with a focus on clinical pain and sensory changes. Eighty patients (31 females, 55.7±12.9 years old) with MND underwent a extensive clinical examination, pain (McGill pain questionnaire, brief pain inventory, douleur neuropathique-4), psychophysics [quantitative sensory testing (QST) and conditioned pain modulation (CPM)], mood and catastrophizing assessments, and were compared to sex- and age-matched healthy controls (HC). Chronic pain (present on most days for more than three months) was present in 46% of patients (numerical visual scale=5.18±2.0). Pain of musculoskeletal origin occurred in 40.5% and was mainly located in the head/neck (51%) and lower back (35%). Neuropathic pain was not present in this sample. Compared to HC, MND patients had a lower cold detection threshold (p < 0.002), and significantly lower CPM scores (4.9±0.2% vs. 22.1±0.2%, p=0.012). QST/CPM results did not differ between MND patients with and without pain. Pain intensity was statistically correlated with anxiety, depression, and catastrophism, and spasticity scores were inversely correlated with CPM (rho=-0.30, p=0.026). Pain is frequently reported by patients with MNDs. Somatosensory and CPM changes exist in MNDs and may be related to the neurodegenerative nature of the disease. Further studies should investigate ways to better quantify these changes and explore the treatment strategies most appropriated for their control

Au cours du développement du système nerveux, l'activité des cibles post-synaptiques permet le raffinement du nombre et de la force des connexions neuronales. En employant la jonction neuromusculaire de Caenorhabditis elegans comme système modèle, nous avons étudié deux aspects de la mise en place de ces connexions. D'une part, nous montrons que le nombre de récepteurs présents à la jonction neuromusculaire est contrôlé par l'activité musculaire : une augmentation de l'activation synaptique entraîne une régulation différentielle des trois types de récepteurs présents à la jonction neuromusculaire. D'autre part, nous avons étudié les changements de la morphologie de certains motoneurones de la tête du ver, appelés neurones SAB, en fonction de l’activité musculaire. Une diminution de l’activité musculaire durant une période critique du développement entraîne une surcroissance axonale des neurones SAB. À travers différentes approches, nous avons pu identifier la suppression de la surcroissance axonale dans des mutants où la biosynthèse des neuropeptides est perturbée. Enfin, nous avons mis en évidence que la surcroissance axonale apparait également lors de perturbations plus générales de la physiologie cellulaire, telles qu'un choc thermique ou la surexpression d'un transgène, ce qui suggère que le système SAB est plastique et particulièrement sensible au cours du développement / Throughout nervous system development, activity of the post-synaptic targets can regulate the connectivity of neural networks, affecting both the number and strength of synapses. Using the neuromuscular junction of Caenorhabditis elegans as a model system, we studied two processes displaying such plasticity. First, we show that the number of receptors present at the neuromuscular synapse is regulated by muscle activity: an increase in synaptic activity can lead to a differential regulation of the three types of receptors present at the neuromuscular junction. Second, we studied the activity-dependent morphological changes of one type of motor neurons in the worm’s head, called the SAB neurons. A decrease of muscle activity during a critical developmental phase leads to SAB axonal overgrowth. Using several approaches, we were able to observe suppression of SAB axonal overgrowth in mutants with a disruption of neuropeptides biosynthesis. Finally, we give evidence that axonal overgrowth also occurs following more general disruptions of cell physiology, such as a heat-shock or transgene overexpression, which suggest that the SAB system is plastic and sensitive during development

No description available.

Doenças do neurônio motor (DNM) representam um grupo de doenças que cursam com fraqueza muscular progressiva e inexorável, e o manejo clínico é baseado no controle dos sintomas. Estes doentes sofrem de acometimentos motores e não motores intensos e de evolução progressiva. Entretanto, além dos sintomas motores, de humor e de déficits cognitivos, uma caracterização mais profunda de sintomas não- motores nesses doentes raramente foi relatada. Este estudo transversal objetivou descrever os sintomas não motores na DMN e seu impacto na qualidade de vida e no estado funcional, com foco na dor e alterações sensoriais. Oitenta doentes (31 mulheres, 55,7 ± 12,9 anos) com DNM foram submetidos a exame clínico extenso, avaliação de dor (questionário de dor McGill, Inventário breve de dor, questionário douleur neuropathique-4), avaliação psicofísica [teste quantitativo da sensibilidade (TQS) e modulação condicionada da dor (MCD)], avaliações de humor e catastrofismo, e foram comparados com controles saudáveis (CS) pareados por sexo e idade. Dor crônica (presente a maior parte dos dias por mais de três meses) foi presente em 46% dos doentes (escala numérica da dor = 5,18 ± 2,0). A dor de origem musculo- esquelética ocorreu em 40,5% e foi localizada principalmente na região da cabeça/pescoço (51%) e da região lombar (35%). A dor neuropática não presente nesta amostra. Comparado aos CS, os doentes com DNM apresentaram menor limiar de detecção de frio (p < 0,002) e valores de MCD significativamente menores (4,9 ± 0,2% vs. 22,1 ± 0,2%, p = 0,012). Os resultados do TQS/MCD não diferiram entre os doentes com DNM com e sem dor. A intensidade da dor foi correlacionada estatisticamente com ansiedade, depressão e catastrofismo, e os escores de espasticidade foram correlacionados inversamente com a MCD (rho = -0,30, p = 0,026). A dor é um sintoma frequentemente relatado por doentes com DNM. Alterações somatossensoriais e de MCD existem em DNM e podem estar relacionadas com a natureza neurodegenerativa da doença. Estudos adicionais devem investigar formas de melhor quantificar estas alterações e explorar estratégias de tratamento mais apropriadas para o seu controle / Motor neuron disorders (MNDs) represent a group of diseases that curse with inexorable muscle weakness and medical management is based on symptom control. These patients suffer from intense motor and non-motor progressive symptoms. However, apart from motor symptoms, mood and cognitive impairments, deeper characterization of non-motor symptoms in these patients have been rarely reported. This cross-sectional study aimed to describe non-motor symptoms in MND and their impact on quality of life and functional status, with a focus on clinical pain and sensory changes. Eighty patients (31 females, 55.7±12.9 years old) with MND underwent a extensive clinical examination, pain (McGill pain questionnaire, brief pain inventory, douleur neuropathique-4), psychophysics [quantitative sensory testing (QST) and conditioned pain modulation (CPM)], mood and catastrophizing assessments, and were compared to sex- and age-matched healthy controls (HC). Chronic pain (present on most days for more than three months) was present in 46% of patients (numerical visual scale=5.18±2.0). Pain of musculoskeletal origin occurred in 40.5% and was mainly located in the head/neck (51%) and lower back (35%). Neuropathic pain was not present in this sample. Compared to HC, MND patients had a lower cold detection threshold (p < 0.002), and significantly lower CPM scores (4.9±0.2% vs. 22.1±0.2%, p=0.012). QST/CPM results did not differ between MND patients with and without pain. Pain intensity was statistically correlated with anxiety, depression, and catastrophism, and spasticity scores were inversely correlated with CPM (rho=-0.30, p=0.026). Pain is frequently reported by patients with MNDs. Somatosensory and CPM changes exist in MNDs and may be related to the neurodegenerative nature of the disease. Further studies should investigate ways to better quantify these changes and explore the treatment strategies most appropriated for their control