<p>Cyclooxygenase-2 (COX-2) catalyses prostaglandin synthesis from arachidonic acid during inflammation. COX-2 is expressed in the normal brain and is induced in neurological disorders. There is evidence that COX-2 is involved in secondary events leading to cell death in the brain. The first objective was to study the expression of COX-2 in the brain after kainate (KA)-induced limbic seizures and brain trauma caused by controlled cortical contusion (CCC) and fluid percussion injury (FPI). COX-2 mRNA and protein were strongly induced by limbic seizures in the hippocampus, amygdala and piriform cortex. CCC and FPI resulted in an upregulation of COX-2 mainly in the dentate gyrus and cortex, with differences in expression levels in these regions between the models. The second objective was to evaluate the effects of selective COX-2 inhibitors on delayed cell death. Limbic seizures induced cell death in parts of the hippocampus, amygdala and functionally connected regions. Treatment with the selective COX-2 inhibitor rofecoxib 8 h after KA injection significantly reduced hippocampal cell death. Pre-treatment with the COX-2 inhibitor nimesulide augmented acute seizures with increased mortality and thus the effect of nimesulide on delayed cell death could not be evaluated. Effects of rofecoxib on trauma-induced cell death were studied in the FPI model. FPI induced delayed cell death mainly in the ipsilateral cortex and bilaterally in the dentate gyrus. Rofecoxib treatment, starting directly after injury was caused, had no protective effect against cell death. </p><p>The results suggest that COX-2 inhibition may be both detrimental and beneficial and largely dependent on the time schedule of treatment. COX-2 inhibitors might thus be of value as a neuroprotective treatment approach, provided that the role of COX-2 and the time course of effects of its metabolites in the brain are elucidated.</p>

Cyclooxygenase-2 (COX-2) catalyses prostaglandin synthesis from arachidonic acid during inflammation. COX-2 is expressed in the normal brain and is induced in neurological disorders. There is evidence that COX-2 is involved in secondary events leading to cell death in the brain. The first objective was to study the expression of COX-2 in the brain after kainate (KA)-induced limbic seizures and brain trauma caused by controlled cortical contusion (CCC) and fluid percussion injury (FPI). COX-2 mRNA and protein were strongly induced by limbic seizures in the hippocampus, amygdala and piriform cortex. CCC and FPI resulted in an upregulation of COX-2 mainly in the dentate gyrus and cortex, with differences in expression levels in these regions between the models. The second objective was to evaluate the effects of selective COX-2 inhibitors on delayed cell death. Limbic seizures induced cell death in parts of the hippocampus, amygdala and functionally connected regions. Treatment with the selective COX-2 inhibitor rofecoxib 8 h after KA injection significantly reduced hippocampal cell death. Pre-treatment with the COX-2 inhibitor nimesulide augmented acute seizures with increased mortality and thus the effect of nimesulide on delayed cell death could not be evaluated. Effects of rofecoxib on trauma-induced cell death were studied in the FPI model. FPI induced delayed cell death mainly in the ipsilateral cortex and bilaterally in the dentate gyrus. Rofecoxib treatment, starting directly after injury was caused, had no protective effect against cell death. The results suggest that COX-2 inhibition may be both detrimental and beneficial and largely dependent on the time schedule of treatment. COX-2 inhibitors might thus be of value as a neuroprotective treatment approach, provided that the role of COX-2 and the time course of effects of its metabolites in the brain are elucidated.

Nous avons démontré la présence d'apoptose dans le système limbique suivant un infarctus du myocarde. Cette mort cellulaire serait partiellement reliée à l'augmentation de cytokines pro-inflammatoires. Des études démontrent que certains probiotiques ont des effets bénéfiques en diminuant le ratio de cytokines pro/anti-inflammatoires. La prise de probiotiques en prévention, avant l’occlusion d’une artère coronarienne, pourrait-elle diminuer l’apoptose dans le système limbique? Méthodes : La combinaison de probiotiques Lactobacillus helveticus R0052 et Bifidobacterium longum R0175 ou son véhicule fut additionné dans l’eau des rats pendant 28 jours consécutifs. Un infarctus du myocarde fut provoqué par l’occlusion de l’artère coronaire gauche. Après 40 minutes d'occlusion, les régions ischémiques ont été reperfusées pour 72 heures. Les animaux furent sacrifiés et la taille de l'infarctus mesurée. L'amygdale et l'hippocampe furent prélevés pour déterminer l'activité de la caspase-3 (pro-apoptotique), le ratio Bax/Bcl2(proapoptotique/ anti-apoptotique) et l'activité d'Akt (survie cellulaire). Résultats : La taille de l’infarctus n'est pas diminuée dans le groupe probiotique (45% de la région à risque)comparé au groupe placebo. Nos marqueurs d’apoptose démontrent une diminution dans les régions du gyrus denté, de l’amygdale latérale et médiane dans le groupe probiotique par rapport au placebo. L’activité de la caspase-3 et le ratio Bax:Bcl2 furent réduits dans le groupe probiotique de 50% et 40% respectivement (p < 0.05) et phosphorylation d’Akt fut augmentée de 35% (p<0.05). Aucune différence fut observée pour les régions Ca1 et Ca3. Conclusion : La combinaison de probiotiques utilisée réduit l’apoptose dans différentes régions du système limbique 72 heures après un IM. / Apoptosis is observed in limbic system after a myocardial infarction (MI). This cell death is due to the release of pro-inflammatory cytokines. Since probiotics reduce the pro/anti-inflammatory cytokine ratio, we hypothesise that probiotics will lessen apoptosis in the limbic system following MI. Methods: Rats were given probiotics or placebo for 4 consecutive weeks. Rat in the probiotic group received a daily dose of over 1 billion live bacterial cells of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 in combination. A MI was then induced in anesthetised rats by a 40-minute occlusion of the left anterior coronary artery followed by a 72 hours of reperfusion. Infarct size was measured and apoptosis was determined in the amygdala and hippocampus in both groups. Results: Infarct size was not diminished in the probiotic group (45% of the risk area), apoptosis was lessened in the dentate gyrus (DG), the lateral (LA) and medial (MA)amygdala compared to the placebo group. Caspase-3 and Bax/Bcl2 ratio were reduced in the probiotic group by about 50% and 40% respectively. Akt activity was increased by 35% in these regions. No difference was observed in the hippocampus Ca1 and Ca3 regions. Conclusion: This probiotic combination can reduce the apoptosis found in specific regions of the limbic system following a MI, which may have significance for post-MI depression.

本研究以連續性負向對比（successive negative contrast, SNC）動物行為模式進行比較行為所涉及的心理歷程及神經生理系統之探究。實驗首先建立以大白鼠在舔舐不同濃度蔗糖液所引發的連續性負向對比效果之模式，繼而探討飢餓動機在此模式中所扮演的角色，並且以benzodiazepines受體促進劑diazepam進行週邊及中樞注射，期望對此模式之神經行為機制有進一步的發現，並釐清benzodiazepines在當中所扮演的角色。實驗一是為了建立本實驗室大白鼠在舔舐蔗糖液由較高濃度（32﹪）降為較低濃度（4﹪）時產生的連續性負向對比行為的表現，從結果發現剝奪吃食的大白鼠或自由吃食的大白鼠皆有連續性負向對比行為的效果產生，剝奪吃食的大白鼠在減抑負向對比的行為效果比自由吃食的大白鼠還快，而自由吃食的大白鼠在連續性負向對比的行為保持上會持續較久。實驗二是操弄吃食狀態的調換，以檢視大白鼠在負向對比效果表現是否隨飢餓驅力的高低而變化，就結果而言，剝奪吃食改為自由吃食組大白鼠之連續性負向對比行為的產生只發生在蔗糖液濃度變化後的第一、二天，而自由吃食改為剝奪吃食組大白鼠會發生在蔗糖液濃度變化後的四天。實驗三大白鼠進行diazepam腹腔注射，結果發現能有效減抑負向對比效果，但只發生在蔗糖液濃度改變後的第二天。實驗四進行大白鼠腹腔注射diazepam 5 mg/kg以檢視其是否因增加對蔗糖液的喜好因素而減抑了連續性負向對比的效果，結果顯示大白鼠在第一、二天負向對比的效果就不明顯，不過受藥物作用的影響，可以發現舔水次數或舔水量等指標都有增加趨勢，此可解釋為diazepam增加對蔗糖液的喜好得影響。實驗五進行了內側杏仁體及背側海馬體的diazepam微量注射，結果發現蔗糖液濃度改變後第一天，上述兩部位的藥物注射後皆產生負向對比效果，但第二天只有注射內側杏仁體大白鼠減抑了負向對比效果，而背側海馬體大白鼠則繼續保持負向對比效果。綜觀上述結果顯示以舔舐蔗糖液濃度差異所引發連續性負向對比效果所涉及的心理歷程及神經生理系統有其複雜性，benzodiazepines受體促進劑diazepam的藥物測試結果發現會影響此行為模式。 / The present study successive negative contrast (SNC) investigated what psychological processes and neural systems were involved in the comparison behavior. The SNC effect induced by rat’s licking different concentrations of sucrose solution was established and the effect in diazepam (a benzodiazepine agonist) as well as experimental manipulation of food deprivation were observed. In Experiment 1, the SNC effect was induced when the sucrose solution shifted from 32% down to 4%.This effect was observed across the consecutive 4 post-shift days in the free-feeding subjects; however, such effect was gradually diminished in the food-deprived subjects. Experiment 2 manipulated the food deprivation states to study how the hunger drive would affect the SNC. The results revealed that the food-deprived subjects in the pre-shift session show the SNC effect only on the second day of post-shift session with food supplied freely. However, the SNC effects were observed in the consecutive four post-shift days in the subject with free-feeding in the pre-shift session but was then altered into the state of food-deprivation. In Experiment 3, the SNC effect was attenuated by systemic injection of diazepam with the observation of the reduced licking suppression on the second post-shift day. In Experiment 4, with similar manipulation of food supply, diazepam was found to enhance the sucrose licking in addition to its reduction of the SNC effect. The central loci for diazepam to attenuate the SNC effect were investigated in Experiment 5. Although the SNC effect was attenuated by diazepam infused into the medial amygdala or the dorsal hippocampus, the time courses to observe such reduction were different for drug infused into both sites. The study indicates that（a）the SNC effect on licking can be reliably induced by decreasing the sucrose concentration,（b）such effect is attenuated by diazepam via central neural mechanisms. However, further research is needed to determine whether the attenuation of SNC by diazepam is based on the anxiety suppression or appetite enhancement process.

Bc. Žaneta Jakubová, Specialist in laboratory methods Signs of epilepsy spectrum disorder in persons with psychostimulants addiction Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences, Background: The aim of this diploma thesis is to study the occurrence of symptoms of epileptic spectrum disorder in psychostimulant subjects and to demonstrate the effect of psychostimulants on the occurrence of this disorder. Methods: For questionnaire survey was used to collect the data, in which probands submitted a total of 3 questionnaires. Input anamnestic questionnaire to obtain general information, the remaining two are focused on symptoms of epileptic spectrum disorder - Complex Partial Seizure-like Symptoms Inventory (CPSI) and Limbic System Checklist-33 (LSCL-33). Statistical methods of percentage comparison, chi-square test and unpaired t-test were used for evaluation. Results: Unusual scores and scores for epileptic spectrum disorder reached a total of 60 % of respondents in the CPSI. In the LSCL-33 questionnaire, a suspect and abnormal score reached 100 % of respondents. Conclusions: Both hypotheses have been demonstrated, namely that psychostimulants influence the occurrence of symptoms of epileptic spectrum disorder and that...

A côté de la triade motrice symptomatologique de la maladie de Parkinson (MP), des troubles comportementaux et cognitifs sont également fréquemment observés, incluant notamment l'apathie, définie comme une importante diminution des comportements motivés et qui est souvent associée à de l'anxiété et de la dépression. De façon intéressante, ces troubles neuropsychiatriques sont souvent observés chez des patients parkinsoniens traités par la stimulation cérébrale à haute fréquence du noyau sous-thalamique. Bien que les mécanismes physiopathologiques à l'origine de ces troubles restent aujourd'hui encore inconnus, il a été récemment suggéré qu'il pouvait exister un lien entre la diminution du traitement dopaminergique (DA) (rendue possible par les effets bénéfiques sur le plan moteur de la stimulation cérébrale profonde) et la résurgence du syndrome d'apathie chez ces patients parkinsoniens stimulés. Dans une première partie expérimentale, nous avons voulu déterminer le rôle du système DA dans l'émergence de ces troubles neuropsychiatriques d'identifier les projections DA ascendantes qui pourraient préférentiellement être impliquées. Notre modèle animal chez le rat, obtenu par des lésions neurotoxiques sélectives, bilatérales et partielles des différentes régions du continuum DA mésencéphalique nous a permis de mettre en évidence un rôle déterminant de la voie DA nigrostriatale dans les processus motivationnels et dans la régulation des comportements de type dépressif et anxieux, tout en s'affranchissant d'une altération des fonctions motrices. Dans une deuxième partie, une approche pharmacologique par des traitements chroniques à la L-DOPA ou au Ropinirole (agoniste D2/D3) nous a permis de vérifier de la bonne valeur prédictive de notre modèle animal vis-à-vis des traitements utilisés en clinique. L'administration de différents agonistes DA sélectifs des récepteurs de type D1, D2 et D3 nous a permis par la suite de montrer une implication majeure des récepteurs D3 dans la capacité à corriger le phénotype induit par la lésion de la voie nigrostriatale, ce qui ouvre ainsi la voie à de nouvelle cible thérapeutique. Au final, les données obtenues au cours de ce travail doctoral apportent de nouveaux arguments en faveur d'une implication de la voie DA nigrostriée dans l'émergence des troubles apathiques. Le modèle lésionnel mis en place pourra permettre de mieux appréhender le rôle de la dopamine dans les processus qui sous-tendent ces troubles motivationnels et thymiques dans la MP et identifier de nouvelles stratégies thérapeutiques mieux adaptées pour leur traitement. / Beyond the classical triad of motor symptoms observed in Parkinson's disease (PD), behavioural and cognitive disturbances are also commonly observed, including apathy, defined as a decrease in motivated directed behaviours, and which is often associated with anxiety and depression. Interestingly, these neuropsychiatric symptoms are frequently observed in parkinsonian patients with high frequency stimulation of the subthalamic nucleus. Although the pathophysiological mechanisms behind these troubles are still unknown, a link has been recently suggested between the reduction of dopaminergic drugs (DA) (made possible by the benefits on the motor ability of deep brain stimulation) and the resurgence of the apathetic syndrome in these stimulated patients. In a first set of experiments, we set out to determine the role of the DA system in the emergence of such neuropsychiatric symptoms and which part of the DA ascending pathway is more specifically involved. Our lesion model in rats, by neurotoxic partial, selective and bilateral lesions of different regions of the DA midbrain continuum were performed allowed us to highlight a key role of the nigrostriatal DA pathway in the motivational processes and in regulating depressive- and anxiety-like behaviours, while avoiding motor impairments. In the second part, a pharmacological approach by chronic treatment with L-DOPA or Ropinirole (D2/D3 agonist) allowed us to verify the predictive value of our animal model in regard to treatment classically used in clinic. The administration of different selective agonists of the D1, D2 and D3 DA receptors has allowed us to show the major involvement of the D3 receptors in the efficiency to reverse the phenotype induced by the nigrostriatal pathway lesion, which opens the way for new therapeutic targets. Finally, the data obtained during this doctoral work provides new arguments for an involvement of the nigrostriatal DA pathway in the emergence of the apathetic syndrome in PD. The lesion model developed can lead to a better understanding of the role of DA in the processes underlying these motivational and mood disorders in PD and to identify new therapeutic strategies, more suitable for further treatment.

INTRODUÇÃO: O processamento emocional pelo cérebro humano tem sido atualmente investigado através do uso de ressônancia magnética funcional (RMf). A RMf possibilita o estudo in vivo e não invasivo de mudanças na atividade cerebral regional em voluntários humanos saudáveis. O processamento emocional pode ser modulado através do uso de antidepressivos que influenciam sistemas neurais relacionados ao processamento emocional, através da modulação da ação de neurotransmissores como a serotonina e a noradrenalina. A clomipramina, um antidepressivo tricíclico, tem sido relacionada com efeitos de resposta clínica mesmo em voluntários saudáveis. Estudos utilizando a RMf permitem a investigação do efeito de antidepressivos nos sistemas neurais envolvidos no processamento emocional em indivíduos saudáveis que apresentam resposta ao uso destes medicamentos comparados a sujeitos que não apresentam resposta ao tratamento. MÉTODOS: Nesta tese, dezoito voluntários saudáveis foram investigados em relação a mudanças de atividade neural em resposta à indução emocional através da apresentação de fotografias do International Affective Pictures System (IAPS). Foram estudadas particularmente as emoções de raiva, felicidade e medo. Os voluntários foram submetidos ao tratamento prolongado com doses baixas de clomipramina por quatro semanas. A amostra foi subdividida em respondedores (n=6) e não respondedores (n=12) ao tratamento com clomipramina. A atividade neural foi estimada com o uso da RMf, através da mensuração do efeito blood oxygenation level dependent (BOLD). As imagens foram processadas e analisadas usando o programa Statistical Parametric Mapping (SPM). Indivíduos não respondedores foram comparados sob o efeito e na ausência de efeito da clomipramina, através de comparações planejadas utilizando t-teste pareado. Indivíduos respondedores foram comparados com os não respondedores sob o efeito da clomipramina através de t-teste não pareado. RESULTADOS: Nos voluntários não respondedores à clomipramina, a comparação entre os estados medicado versus não medicado evidenciou menor atividade neural na região da amídala quando sob efeito da clomipramina em resposta a estímulos de valência negativa. Demonstramos ainda, em paradigmas de valência positiva e negativa, diminuição da atividade neural no giro do cíngulo anterior, na ínsula e no putâmen na vigência da medicação. Quando foram comparados os indivíduos respondedores com os não respondedores sob efeito de clomipramina, um aumento consistente de atividade cerebral foi observado nos voluntários respondedores na região da ínsula. CONCLUSÕES: O uso prolongado de doses baixas de clomipramina apresentou ação em regiões cerebrais envolvidas com o processamento emocional. Quando indivíduos não respondedores foram comparados sob o efeito e sem o efeito da clomipramina, foi observada menor atividade amidalar durante o tratamento em resposta a estímulos de valência negativa, possivelmente devido à menor demanda neural na avaliação inicial do estímulo de valência negativa. Também foi observada menor ativação no giro do cingulo anterior, na ínsula e no putâmen na vigência do uso da clomipramina, possivelmente em associação a uma diminuição do mapeamento cortical de funções interoceptivas em resposta a estímulos emocionais positivos e negativos. Quando indivíduos respondedores foram comparados com os não respondedores ao tratamento prolongado com doses baixas de clomipramina, foi observada maior ativação insular nos indivíduos respondedores quando estavam sob efeito de clomipramina; estes resultados indicam que possivelmente os indivíduos que respondem ao tratamento antidepressivo são os que percebem mais as alterações de seu estado corporal durante o processamento emocional. / INTRODUCTION: The emotional processing by the human brain has now been investigated through the use of functional magnetic resonance imaging (fMRI). The fMRI technique allows the noninvasive study of in vivo changes in regional brain activity in healthy human volunteers. The emotional processing may be modulated through the use of antidepressants that influence neural systems linked to emotional processing, by modulating the action of neurotransmitters such as serotonin and norepinephrine. Clomipramine, a tricyclic antidepressant, has been reported to elicit clinical response even in healthy volunteers. Studies using fMRI allow the investigation of the effect of antidepressants on neural systems involved in emotional processing in healthy subjects showing response to the use of antidepressant drugs compared to subjects who do not respond to treatment. METHODS: In this thesis, eighteen healthy volunteers were investigated in relation to changes in neural activity in response to emotional induction through the presentation of photos of the International Affective Picture System (IAPS). We studied especially the emotions of anger, happiness and fear. The volunteers were subjected to prolonged treatment with low doses of clomipramine for four weeks. The sample was divided into responders (n = 6) and non-responders (n = 12) to treatment with clomipramine. The neural activity was estimated by using fMRI, by measuring the blood oxygenation level dependent effect (BOLD). Images were processed and analyzed using the Statistical Parametric Mapping (SPM) program. Non-responders were compared under two conditions: when using clomipramine, and after drug washout, using paired t-tests. Individuals who responded to clomipramine treatment were compared with non-responders under the effect of the drug by independent t-test. RESULTS: In volunteers not responding to clomipramine, a comparison between the non-medicated versus medicated states showed less neural activity in the region of the amygdala when under effect of clomipramine in response to stimuli of negative valence. We also demonstrated, both in the paradigms of positive and negative valence, decreased neural activity in the anterior cingulate gyrus, insula and putamen during the medicated state. When responders were compared with non-responders under the effect of clomipramine, a consistent increase in brain activity was observed in the former group in the insula. CONCLUSIONS: The prolonged use of low doses of clomipramine induced activity changes in brain regions involved in emotional processing. When non-responders were compared under the influence and without the effect of clomipramine, the amygdala displayed lower activity during treatment in response to stimuli of negative valence, possibly due to lower demand in the initial evaluation of stimuli of negative valence. There was less activation in the anterior cingulate gyrus, insula and putamen during the use of clomipramine, possibly in association with a decrease in the cortical mapping of interoceptive changes in response to positive and negative emotional stimuli. When responders were compared with non-responders after prolonged treatment with low doses of clomipramine, insular activation was greater in responders when individuals were under the effect of clomipramine. These results indicate that individuals who respond to antidepressant treatment are those who perceive more changes in their bodily state during emotional processing.

Os estudos dos processos cognitivos propiciam um cenário promissor para a realização de pesquisas visando uma maior compreensão de como o funcionamento do cérebro pode favorecer a educação, possibilitando o desenvolvimento de novas teorias e abordagens que estimulem a aprendizagem. O presente trabalho abordará principalmente como se dá a aquisição, o armazenamento, processamento e a recuperação do conhecimento do ponto de vista da neurociência e de que forma o mapa conceitual (MC) pode mapear o conhecimento do indivíduo. O MC pode ser utilizado tanto como uma estratégia de aprendizagem para a compreensão de conceitos-chave bem como as relações entre estes quanto para promover o pensamento crítico do indivíduo. É uma representação gráfica bidimensional cuja estrutura permite organizar visualmente as relações entre conceitos que podem ser indicadas por palavras, frases e símbolos. É usado para facilitar o aprendizado ao hierarquizar os conceitos por meio de construções significativas para o indivíduo. Os conceitos aparecem nas caixas e as relações nas linhas que os unem: a dois conceitos conectados chamamos de proposição. Durante a construção de um MC o indivíduo exercita a sua capacidade de estabelecer relações entre o conhecimento que já tem e o adquirido no decorrer da aprendizagem ao representar graficamente os conceitos sobre um determinado assunto. / The study of cognitive processes provide a promising scenario to research aimed at better understanding of how the functioning of the brain may promote the education, enabling the development of new theories and approaches that encourage learning. This work will mainly occurs as the acquisition, storage, processing and retrieval of knowledge from the viewpoint of neuroscience, and how the conceptual map can map the knowledge of the individual. The conceptual map (CM) can be used both as a strategy of learning for the understanding of key concepts and relations between them and to promote critical thinking of the individual. Two-dimensional graphical representation, the CM allows visually organize the relationships between concepts. This structure from the wider concepts up to less comprehensive and relations between them can be indicated by words, phrases and symbols. It is used to facilitate the learning concepts ranking by building significant to the individual. The concepts appear in the boxes and lines that unite them: two concepts connected call proposition. During the construction of a CM, the individual exercises its ability to establish relationships between knowledge that he has already acquired in the course of learning to represent graphically the concepts of a particular subject.

Caspr2 est une molécule d'adhérence impliquée dans diverses pathologies neurologiques telles que l'autisme et l'encéphalite limbique (EL). Les mécanismes pathogéniques restent inconnus. Caspr2 est associé aux canaux potassiques Kv1.1/1.2 aux juxtaparanoeuds et au segment initial (SI). Dans un premier article publié dans Front. Cell. Neurosci. (2015), nous avons mis en évidence que les autoanticorps anti-Caspr2 issus de patients atteints d'EL ciblent majoritairement les neurones GABAergiques. Caspr2 est localisé au niveau des axones et des terminaisons présynaptiques inhibitrices dans les neurones d'hippocampe en culture. De plus, nous avons généré une chimère Caspr2-Fc soluble qui a permis d’identifier TAG-1 comme récepteur de Caspr2 localisé au niveau du compartiment somato-dendritique postsynaptique. Les neurones incubés avec des IgGs de patients, présentent une densité diminuée des clusters de Géphyrine marqueur des post-synapses inhibitrices. Ces anticorps sont d'isotype IgG4 et reconnaissent le plus communément des épitopes de la région Discoïdine-LaminineG1. Un blocage fonctionnel de Caspr2 au niveau synaptique permettrait de comprendre l'hyperexcitabilité associée à l'EL. Dans un second article en préparation, nous avons étudié la régulation de l’expression de Caspr2 au SI. Nous avons utilisé différentes constructions et identifié les domaines LamineG2-EGF1 extracellulaires de Caspr2 requis pour son expression axonale. De plus, les domaines cytoplasmiques de liaison aux protéines 4.1B et PDZ sont impliqués dans la rétention de Caspr2 et MPP2 au SI. Notablement, l'expression de TAG-1 ou ADAM22 induit des effets opposés sur l'expression de Caspr2 au SI. / Caspr2 is a cell adhesion molecule associated with neurologic diseases, such as autism spectrum disorders and limbic encephalitis. The underlying pathogenic mechanisms are still unknown. Caspr2 is associated with the voltage-gated potassium channels Kv1.1/1.2 localized at the axon initial segment (AIS) and the juxtaparanodes in myelinated axons. In a first paper published in Front. Cell. Neurosci. (2015), we characterized anti-Caspr2 autoantibodies from limbic encephalitis (LE) patients and showed that these autoantibodies preferentially targeted GABAergic neurons. Caspr2 was localized along axons and at the presynaptic terminals of inhibitory neurons in hippocampal cultures. Next, we generated a soluble Caspr2-Fc chimera to identify TAG-1 as a receptor for Caspr2 localized at the somato-dendritic compartment and post-synapses. We determined that neurons displayed decreased synaptic gephyrin clusters when incubated with anti-Caspr2 IgGs from LE patients. The autoantibodies mainly bound the N-terminal Discoidin-LamininG1 domains and were of the IgG4 isotype. They may exert functional blocking activity on inhibitory connections underlying the hyperexcitability linked with LE. In a second article in preparation, we examined the regulated expression of Caspr2 at the AIS using deletion and reporter constructs. We mapped the LamininG2 and EGF1 modules in the ectodomain as implicated in the axonal distribution of Caspr2 and the cytoplasmic motifs for binding to 4.1B and PDZ proteins as implicated in Caspr2 AIS retention together with MPP2. Strikingly, co-expression with TAG-1 and ADAM22 induced opposite effects on AIS Caspr2 distribution.

L’encéphalite limbique à auto-anticorps anti-CASPR2 est une atteinte du système nerveux central, caractérisée par la présence des auto-anticorps (autoAcs) dirigé contre CASPR2 dans le sérum et fluide céphalorachidien. La pathologie affecte majoritairement des hommes âgés présentant l’épilepsie comme symptôme prédominant. CASPR2 est une molécule d’adhésion neuronale, connue pour son rôle d’assemblage des canaux Kv1, régulateurs de l’excitabilité neuronale, à la région juxtaparanodale du nœud de Ranvier, une organisation essentielle pour la conduction saltatoire des flux nerveux. Un nombre croissant de données dans la littérature suggère un rôle pour CASPR2 dans des fonctions synaptiques et l’activité neuronale. Ceci pourrait expliquer l’épilepsie, un symptôme neurologique qui trouve son origine dans la perturbation de l’activité neuronale, observée chez les patients avec de l’encéphalite limbique anti-CASPR2. Dans ce travail de thèse, j’ai utilisé des autoAcs de patients comme outil pour investiguer le rôle de CASPR2 dans des neurones normalement développés en culture, permettant aussi d’évaluer l’effet des autoAcs des patients sur les fonctions synaptiques et de révéler des mécanismes physiopathologiques possibles sous-jacents à la maladie. Je me suis d’abord intéressée aux effets des autoAcs des patients sur l’expression et la distribution en surface de CASPR2 et sur l’expression des canaux Kv1.2 dans des neurones hippocampiques matures in vitro. J’ai montré que les neurones inhibiteurs sont positifs pour les canaux Kv1.2 et CASPR2 en surface, et que les autoAcs de patients augmentent l’expression de Kv1.2 et n’induisent pas l’internalisation de CASPR2. Dans un second temps, j’ai analysé les effets des autoAcs de patients sur les synapses excitatrices et inhibitrices dans des neurones hippocampiques immatures et matures in vitro. Dans les neurones immatures, la densité des épines dendritiques et le contenu des récepteurs AMPA sont augmentés, tandis que dans les neurones matures l’altération de la géphyrin suggère une perturbation de la transmission neuronale après traitement avec des autoAcs de patients. Mes résultats permettent de mieux comprendre les fonctions de CASPR2 dans les processus synaptiques et révèlent des mécanismes pathologiques possibles des autoAcs anti-CASPR2 menant à la présentation clinique des patients atteints d’encéphalite limbique anti-CASPR2 / Anti-CASPR2 autoimmune limbic encephalitis is a central nervous system disorder, characterized by the presence of autoantibodies (autoAbs) directed against CASPR2 in the serum and cerebrospinal fluid. Elderly men are mostly affected, with epilepsy being the predominant symptom. CASPR2 is a neuronal cell adhesion molecule, known for its role in gathering Kv1 channels, regulators of neuronal excitability, at the juxtaparanodal region of the node of Ranvier, an essential organization for saltatory conduction of nervous influxes. Increasing sets of data in literature point out a role for CASPR2 in synaptic functions and neuronal activity. This could explain the observed epilepsy, a neurological symptom that finds its origin in disturbed neuronal activity, in patients with anti-CASPR2 autoimmune limbic encephalitis. In this work, I used patients’ autoAbs as a tool to investigate the role of CASPR2 in normally developed cultured neurons which also allowed me to assess the effects of patients’ autoAbs on synaptic functions and reveal possible physiopathological mechanisms underlying the disease. I first assessed the effects of patients’ autoAbs on CASPR2 surface expression and distribution and on Kv1.2 channel expression in mature in vitro hippocampal neurons. I provided evidence that inhibitory neurons are positive for both Kv1.2 channels and surface CASPR2, and that patients’ autoAbs increase Kv1.2 expression and do not induce CASPR2 internalization. Secondly, I analyzed effects of patients’ autoAbs on excitatory and inhibitory synapses in vitro, in immature and mature hippocampal neurons. In immature neurons, dendritic spine densities and AMPA receptor content are increased, while in mature neurons alteration of gephyrin suggests disturbed neuronal transmission after treatment with patients’ autoAbs. My results allow for a better understanding of CASPR2 functions in synaptic processes and unravel possible pathological mechanisms regarding how anti-CASPR2 autoAbs lead to the clinical presentation of patients with anti-CASPR2 autoimmune limbic encephalitis