Les Sémaphorines de classe 3 modulent de nombreux comportement neuritiques lors de la formation du système nerveux central. Leur liaison sur des complexes récepteurs composés de la sous unité obligatoire de liaison au ligand, les Neuropilines1,2 (Nrp1,2) et de la sous unité de signalisation, les PléxinesA1-4, permet d'assurer la spécificité fonctionnelle. Cependant, les mécanismes moléculaires contrôlant la formation de ces complexes récepteurs ne sont pas connus. Au cours de ma thèse, j'ai étudié l'assemblage des complexes récepteurs des Sémaphorines de classe 3 par une approche FRET. J'ai pu montrer que Nrp1,2 forment des homo et hétéro-oligomères à la membrane plasmique. Nrp1 peut former des hétero-oligomères avec les PléxinesA1,2 et 4 mais apparemment pas avec PléxineA3. La délétion du domaine Séma de PléxineA3 (PléxinesA3∆sem) suffit pour induire la formation d'hétéro-complexes Nrp1-PléxinesA3∆sem. Ces résultats suggèrent que le domaine Séma de PléxineA3 adopte une conformation différente des autres PléxinesA et empêche l'interaction entre PléxineA3 et Nrp1. De plus, toutes les PléxinesA peuvent former des homo-oligomères de manière indépendante du domaine Séma. Ces résultats suggèrent que les récepteurs des Sémaphorines de classe 3 pourraient former des complexes oligomériques plus important, composés d'homo-dimères de Nrps et de PléxinesA. Dans la deuxième partie de ma thèse, j'ai étudié la spécificité de signalisation des PléxinesA dans les interneurones GABAergiques du cervelet. Au laboratoire, nous avons montré que Séma3A induit la formation de branches des interneurones GABAergiques du cervelet par l'intermédiaire de Fyn. Mais l'identité des complexes récepteurs Séma3A impliquée dans ce processus n'était pas connue. Nous avons montré que les interneurones GABAergiques expriment PléxineA1,A2 A3 mais pas A4. Alors que les PléxinesA1,2 et 3 interagissent avec la protéine Fyn, seule l'activation des voies de signalisation de PléxineA1 et A2 induit la formation de branches in vitro. De manière intéressante, Fyn interagit avec les PléxinesA1 et A2 via son domaine SH3 alors qu'elle utilise son domaine SH2 pour l'interaction avec PléxineA3, suggérant l'importance du mode d'interaction de Fyn avec les PléxinesA pour sa fonction. Enfin nous avons pu montrer que PléxineA1 est la seule Pléxine in vivo capable d'induire un défaut de formation de branches des interneurones GABAergiques du cervelet. / Secreted class 3 Semaphorin (Sema3) modulates a wide variety of axon behavior during central nervous system formation. Sema3 family functions are triggered through binding to specific receptor complexes that include the obligate binding subunit Neuropilins-1 and 2 (Nrp1-2) and the signalling subunit plexin-A1-4. Yet, the exact mechanism controlling Sema3 receptor complex formation is not known. Here, we investigate Sema3 cell surface receptor formation using a time resolved FRET approach. We showed that Nrp-1 and 2 formed homo and hetero-oligomers at the cell surface of leaving cells. Nrp-1 can form hetero-oligomers with PlexinA1, A2 and A4 but not with PlexinA3. Deletion of the Plexin-Sema domain of PlexinA3 (PlexinA3∆sem) induced the formation of Nrp1 and PexinA3∆sem hetero-complexes. These results showed that PlexinA3-sema domain adopts a different conformation from the other PlexinAs and inhibited the interaction with Nrp1. Furthermore, PlexinAs can form homo-oligomers independently of the Plexin-Sema domain. These results further suggest that Sema3 receptors could form higher oligomeric complex that include homodimers of both neuropilins and PlexinAs. Understanding the basic principles of Sema3 receptor assembly will be pivotal to decipher how ligand binding translates into specific pathways of cellular signaling. In the second part of my thesis we investigated PlexinA specific signaling in cerebellar GABAergic interneurons. Sema3A induced GABAergic axonal branching in cerebellar cortex in a Fyn dependent manner (Cioni et al.,2013). However the identity of Sema3A receptor complexes involved in this process needed further investigation. We showed that PlexinA1, A2 and A3, but not PlexinA4, are expressed in GABAergic interneurons. While PlexinA1-3 are associated with FYN, only PlexinA1 and A2 induced axon branching. Interestingly, Fyn interaction with PlexinA1 and A2 is mediated via the SH3 domain while the interaction with PlexinA3 is through the SH2 domain, suggesting that the binding mode of Fyn to PlexinA is important. These results were further supported by in vivo characterization of PlexinA1-4 deficient mice that showed GABAergic axon branch deficit only in PlexinA1 mutant.

Die kortikale spreading depression (SD), wie sie von Leão 1944 zuerst beschrieben wurde, ist ein elektrophysiologisches Phänomen, das in der Pathophysiologie der Aurasymptomatik einer Mi-gräneattacke und Ischämie-induzierter Zellschäden diskutiert wird. Während der akuten fokalen zerebralen Ischämie treten eine Reihe von Ereignissen wie eine massive Entzündungsreaktion und die allmähliche Einbeziehung einer zunächst viablen ischämischen Randzone - der Penum-bra - in das infarzierte Hirngewebe auf. Da an diesen Ereignissen SD-ähnliche Depolarisationen kausal beteiligt sind, ist die pharmakologische Verringerung von SD-Episoden bzw. eine Ver-kleinerung ihrer Amplitude und Dauer unter in vitro als auch tierexperimentellen in vivo Bedin-gungen eine mögliche neuroprotektive Strategie. In der vorliegenden Arbeit wurde ein in vitro Modell beschrieben, das am Hirnschnittpräparat des Neokortex der adulten Ratte eine reproduzierbare Auslösung von SD-Wellen unter normoxi-schen Bedingungen gestattet. Anhand von charakteristischen elektrophysiologischen Parametern einer SD wie Amplitude, Dauer und Ausbreitungsgeschwindigkeit wurden die gute Überein-stimmungen dieses in vitro Modells mit in vivo Modellen gezeigt. Obwohl SD Wellen am nicht-ischämischen Kortex keine morphologischen Schäden verursachen, zeigte sich in den hier vorge-stellten Experimenten eine funktionelle Unterdrückung der GABAergen hemmenden Mechanis-men des Neokortex nach repetitiven SDs auch bei ausreichender Energie- und Sauerstoffversor-gung. Die hier diskutierten Ergebnisse demonstrierten, daß unter in vitro Bedingungen der AMPA-Glutamatrezeptor für die Auslösung und Ausbreitung einer SD eine untergeordnete Rolle spielt. Demgegenüber erwies sich die NMDA-Rezeptoraktivierung als herausragend für eine SD, da die Blockade dieses Rezeptors mit dem nicht-kompetitiven Antagonisten Ketamin die SD-Amplitude und SD-Dauer signifikant verringerte. Die Anwendung der selektiven 5-HT1A-Agonisten 8-OH-DPAT und BAY x 3702 erwies sich als eine neue Möglichkeit, die Zeitdauer einer SD zu verringern. Die aufgezeigte SD-induzierte neuronale Hyperexzitabilität kann unter normoxischen Bedingun-gen zelluläre Dysfunktionen verursachen und auch an einer Generierung der Aura eines Migrä-neanfalls beteiligt sein. Unter hypoxisch-ischämischen Bedingungen könnte eine SD-induzierte Dysfunktion GABAerger Kontrollmechanismen die Ausweitung ischämischer Zellschäden be-wirken. Die Hoffnungen auf eine effektive Schlaganfalltherapie haben sich mit den bisherigen NMDA-Antagonisten trotz ihrer hier bestätigten guten in vitro Wirksamkeit aufgrund der Interferenz mit physiologischen Glutamatfunktionen im Kortex nicht erfüllt. Die hier gezeigte konzentrationsab-hängige Verkürzung der SD-Dauer durch die Aktivierung des 5-HT1A-Serotoninrezeptors unter in vitro Bedingungen kann bei der bekannten hohen 5-HT1A-Rezeptor-mRNA-dichte an beson-ders ischämievulnerablen Neuronen einen neuen neuroprotektiven Ansatz auch beim Menschen darstellen. Weitere Untersuchungen müssen zeigen, ob die hier beschriebene enge Verflechtung des serotonergen Systems mit der glutamatergen Neurotransmission eventuell auch zu uner-wünschte Wirkungen unter in vivo Bedingungen führt. / Repetitive cortical spreading depression (SD) and SD-like events, associated with a massive de-polarization of neuronal and glial cells, is thought to play a key role in the induction of neuronal damage in the peri-infarct zone following experimental focal cerebral ischemia. In addition, ex-perimental and clinical data suggest that SD is the underlying mechanism of neurological distur-bances during migraine auras as well. However, detailed analyses on the consequences of repeti-tive SDs on cortical function and involved receptors are lacking. Using an in vitro rat model of SD I investigated in this thesis the electrophysiological properties of repetitive potassium chloride (KCl)-induced SDs, their influence on synaptic neurotransmis-sion and the effects of ionotropic glutamate antagonists and 5-HT1A agonists in neocortical slices obtained from adult rats. Whereas repetitive SDs revealed only non-significant variations in du-ration, amplitude and integral when elicited at intervals of 30 min, paired-pulse inhibition of ex-tracellularly recorded field potential responses was significantly affected by repetitive SD even under normoxic conditions. Compared to the control recordings, each SD episode caused a sig-nificant decrease in the efficacy of intracortical GABAergic inhibition by approximately 10%. Since excitatory synaptic transmission was unaffected, these data indicate that repetitive SDs cause a selective suppression of GABAergic function even in the non-ischemic brain. None of the compounds tested prevented the SD-induced cortical disinhibition. However, the SD-associated negative shift in the extracellular DC potential was reduced by ketamine, a selective N-methyl-D-aspartic acid (NMDA-) receptor antagonist. Ketamine significantly (p < 0.01) re-duced the amplitude of the first SD peak and blocked the second SD peak. Ketamine also de-creased the SD duration at half maximal amplitude (p < 0.05). NBQX, a selective a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist did not affect the SD-accompanied cortical depolarization, whereas selective 5-hydroxytryptamine (5-HT)1A receptor agonists 8-OH-DPAT and BAY x 3702 shortened concentration-dependently the duration of the SD up to 50 %. Nevertheless, both 5-HT1A receptor agonists caused a strong disinhibition of neu-ronal function with a tendency towards paired-pulse facilitation as well. Thus, repetitive SD and SD-like events may induce neuronal hyperexcitability due to a selective suppression of intrinsic inhibitory GABAergic function. Under normoxic conditions, SD-induced disinhibition may be involved in the generation and maintenance of migraine or associated neurological disturbances. Under hypoxic-ischemic conditions, neuronal hyperexcitability may contribute to the gradual expansion of the ischemic core and the metabolic deterioration of the penumbral tissue after SD episodes. This underlines the deleterious effect of SD to the outcome of focal cerebral ischemia. Although the precise mecha-nisms of SD generation and propagation remains far from established, the present pharmacologi-cal profile of KCl-induced SD in vitro links the induction and propagation of SD in rat neocorti-cal slices mainly to a local increase of [K + ] e and a subsequent activation of NMDA- receptors. This corroborates the neuroprotective effect of a NMDA- receptor blockade observed in various in vitro and in vivo models. However, as it has been demonstrated in clinical trials, NMDA- re-ceptor antagonists in use today cause psychomimetic and cardiovascular side effects in humans and are therefore currently of low clinical benefit. The activation of 5-HT1A receptors by selective agonists represents a new pharmacological strategy in the treatment of acute ischemic stroke, since shortened SD waves may represent a less energy-consuming process under conditions of limited energy supply and are probably associated with an efflux of excitatory neurotransmitters to a lesser extent. The potential clinical benefit of 5-HT 1A receptor agonists remains to be investi-gated in clinical trials, since systemic administration of these compounds after the onset of acute focal cerebral ischemia might interfere with normal functions of glutamatergic neurotransmission in the intact, non-ischemic brain.

Neuralgia trigeminal (NT) é um tipo de dor neuropática orofacial intensa e debilitante sentida ao longo do nervo trigêmeo. Seu diagnóstico é meramente clínico, sem exames específicos, o que dificulta a presteza e eficácia do tratamento. O sintoma mais característico é alodínia mecânica relacionada à sensibilização central. Seu tratamento é farmacológico e/ou cirúrgico, no entanto pode trazer poucos benefícios e/ou importantes efeitos colaterais sem a garantia de remissão total dos sintomas. No tratamento farmacológico, os anticonvulsivantes são fármacos de primeira linha de tratamento, mas também são obtidas respostas de melhora com o uso de fármacos ansiolíticos e opioides. A dificuldade no tratamento da NT se deve, em parte, à falta de compreensão dos mecanismos envolvidos na geração da dor; sendo, desta forma, imprescindível a busca de um melhor entendimento da fisiopatologia da NT buscando identificar as vias envolvidas no processo de dor. Sendo assim, o objetivo deste estudo foi avaliar o possível envolvimento das vias gabaérgica, glutamatérgica e opioidérgica na fisiopatogenia de um modelo de neuralgia trigeminal em ratos. A resposta nociceptiva dos animais foi avaliada por meio do teste de von Frey eletrônicoantes e após a administração de agonistas gabaérgicos e opioidérgico e de antagonista glutamatérgico. Além da a resposta nociceptiva, foram avaliados os níveis de fator neurotrófico derivado do cérebro (BDNF) em gânglio trigeminal, tronco encefálico e córtex pré-frontal. Todos os procedimentos experimentais foram realizados na Unidade de Experimentação Animal (UEA), Unidade de Análises Moleculares e de Proteínas (UAMP) do Hospital de Clínicas de Porto Alegre (HCPA). A indução da dor neuropática foi por constrição crônica do nervo infraorbitário (CCI-ION) proposto por Imamura em 1997 adaptado (Imamura Y., Kawamoto H., Nakanishi O. Charactherization of heat-hiperalgesia in experimental trigeminal neuropathy in rats. Exp Brain Res, 1997; 116: 97-103.). Para isso foram utilizados 112 ratos Wistar, machos divididos para o procedimento cirúrgico em 3 grupos: Controle Total, Dor e Sham. Quatorze dias após o procedimento cirúrgico, tempo necessário para o desenvolvimento da dor neuropática, realizamos dois protocolos e dividimos os grupos em: protocolo 1: controle veículo, cirurgia sham veículo, cirurgia sham agonista benzodiazepínico, cirurgia sham antagonista glutamatérgico, dor veículo, dor agonista benzodiazepínico, dor antagonista glutamatérgico; protocolo 2: controle veículo, cirurgia sham veículo, cirurgia sham agonista gabaérgico, cirurgia sham agonista opioidérgico, dor veículo, dor agonista gabaérgico, dor agonista opioidérgico. Os animais dos grupos sham sofreram apenas incisão cirúrgica, sem constrição do nervo. Durante o procedimento cirúrgico a indução anestésica foi feita com cetamina (50mg/kg) e xilazina (5mg/kg) e a manutenção, com isoflurano 2-3%. O controle da dor pós-operatória foi obtido com cloridrato de tramadol (10mg/kg) no intervalo de 12h por 48h. A resposta hiperalgésica mecânica (von Frey) foi avaliada antes da indução do modelo (medida basal), 7 e 14 dias após cirurgia, pré-administração dos fármacos, 15,30 e 60 minutos após administração dos fármacos: veículo - solução salina; agonista benzodiazepínico – diazepam (2 mg/kg); antagonista glutamatérgico - MK- 801 (0,25mg/Kg), agonista opioidérgico – morfina (5,0 mg/kg) e agonista gabaérgico – fenobarbital (100mg/kg). Os dados foram analisados por Equações Estimativas Generalizadas e expressos em média+EPM. Todos os procedimentos foram aprovados pelo CEUA/HCPA e CEUA/UFRGS sob número: 14-0604 e 29310, respectivamente. Inicialmente foram realizados testes para verificação do modelo de dor, foi observado que 14 dias após cirurgia de constrição do nervo infraorbitário, os animais do grupo dor apresentaram redução nos limiares de retirada da face no teste de von Frey facial comparado aos grupos controle e sham [GEE: interação grupo x tempo (Waldχ2=15,81; 2, P<0,05)]. Após o estabelecimento do modelo, foram realizados testes nociceptivos para avaliação das vias envolvidas na neuralgia trigeminal, observou-se interação grupo x tempo (Wald _2=175,74;18, P<0,01). No protocolo 1, os animais do grupo dor que receberam agonista benzodiazepínico (diazepam) apresentaram aumento no limiar nociceptivo a partir de 15 minutos após administração e este resultado permaneceu por até 60 minutos. Os animais do grupo dor que receberam antagonista glutamatérgico (MK-801) apresentaram aumento no limiar de retirada da face somente na avaliação realizada 60 minutos após a administração do fármaco. No protocolo 2, análise estatística mostrou interação entre as variáveis tempo x grupo (Wald χ2=657,53;18, P<0,01), os animais do grupo dor que receberam fenobarbital apresentaram aumento no limiar nociceptivo a partir de 15 minutos após administração e este resultado permaneceu por até 60 minutos. Por outro lado, os animais que receberam morfina apresentaram um aumento no limiar nociceptivo significativamente maior que os animais que receberam fenobarbital, no entanto ambos os fármacos reverteram a hiperalgesia induzida pela exposição ao modelo de dor. Na análise bioquímica, em córtex e tronco encefálico foi observado aumento nos níveis de BDNF tanto nos animais submetidos ao modelo de NT quanto aos que foram expostos a cirurgia sham (ANOVA de uma via/SNK, F(2,22)=13,46; F(2,25)=6,08, P<0,05, respectivamente. Em nível periférico foi observado aumento nos níveis de BDNF em gânglio trigeminal nos animais submetidos ao modelo de NT (ANOVA de uma via/SNK,F(2,22)=4,09; P<0,05). Em soro não foi observada diferença nos níveis de BDNF entre os grupos (ANOVA de uma via, P>0,05). Com base nos resultados encontrados, este estudo sugere que há o envolvimento das vias gabaérgica, glutamatérgica e opioidergica no processamento da dor neuropática orofacial (neuralgia trigeminal) sugerindo que há funcionalidade dos receptores testados. No entanto, estes agonistas gabaérgicos não induziram analgesia nos animais controles e cirurgia sham sugerindo uma alteração na liberação ou na síntese de GABA nos animais com NT. Neste estudo, não foram realizadas análises de expressão e quantificação de receptores, apenas avaliou-se a resposta nociceptiva e os níveis de BDNF centrais e periféricos. De acordo com estes achados a via gabaérgica teve uma resposta mais rápida que a via glutamatérgica. Alguns estudos demonstram que os receptores glutamatérgicos estão localizados em fibras não mielinizadas na periferia isto pode justificar o atraso na resposta dos animais que receberam antagonista glutamatérgico em nosso estudo. Quanto aos níveis de BDNF observamos que há um aumento desta neurotrofina nos animais submetidos ao modelo de NT sugerindo um envolvimento deste neuromodulador em eventos neuroplásticos da NT. / Trigeminal neuralgia (TN) is a type of intense and debilitating orofacial neuropathic pain felt along the trigeminal nerve. The diagnosis is purely clinical, without specific tests, which makes the promptness and effectiveness of treatment. The most characteristic symptom is mechanical allodynia related to central sensitization. Its treatment is pharmacological and / or surgical treatment, however can bring few benefits and / or significant side effects without the guarantee of complete remission of symptoms. In drug treatment, the anticonvulsant drug are first-line treatment, but also enhances responses are obtained with the use of anxiolytic drugs and opioids. The difficulty in treating NT is due in part to the lack of understanding of the mechanisms involved in the generation of pain; It is thus essential to search for a better understanding of the pathophysiology NT seeking to identify the pathways involved in pain process. Thus, the aim of this study was to evaluate the possible involvement of GABAergic pathways, glutamatergic and opioidergic in the pathogenesis of a model of trigeminal neuralgia in rats. The nociceptive response of the animals was assessed using the von Frey test eletrônicoantes and after administration of opioid agonists and GABAergic and glutamatergic antagonist. In the nociceptive response was evaluated derived neurotrophic factor levels in the brain (BDNF) in the trigeminal ganglia, brainstem, and prefrontal cortex. All experimental procedures were performed in the Animal Experimentation Unit (UEA), Unit of Analysis and Molecular Protein (UAMP) Porto Alegre Clinical Hospital (HCPA). The induction of neuropathic pain was by chronic constriction of the infraorbital nerve (CCI-ION) proposed by Imamura in 1997 adapted. For that they were used 112 male Wistar rats divided into the surgical procedure into 3 groups: Full Control, Pain and Sham. Fourteen days after surgery, time required for the development of neuropathic pain, performed two protocols and divided groups: Protocol 1: vehicle control, sham surgery vehicle, sham surgery benzodiazepine agonist, sham surgery glutamatergic antagonist, vehicle pain, agonist benzodiazepine pain and antagonist glutamatergic pain. Protocol 2: vehicle control, vehicle sham surgery, sham surgery gabaergic agonist, sham surgery opioid agonist, vehicle pain, gabaergic agonist pain, opioid agonist pain. The animals of the sham group underwent only surgical incision without nerve constriction. During surgery anesthesia was induced with ketamine (50 mg / kg) and xylazine (5 mg / kg) and maintained with isoflurane 2-3%. The control of postoperative pain has been obtained with tramadol hydrochloride (10mg / kg) at 12h intervals for 48 hours. The mechanical hyperalgesic response (von Frey) was assessed before induction model (baseline measurement), 7 and 14 days after surgery, pre-administration of drugs, 15,30 and 60 minutes after drug administration: Vehicle - saline; benzodiazepine agonist - diazepam (2 mg / kg); glutamatergic antagonist - MK-801 (0.25mg / kg), opioid agonist - Morphine (5.0 mg / kg) and gabaergic agonist - phenobarbital (100 mg / kg). Data were analyzed by Equation Generalised Estimates and expressed as mean ± SEM. All procedures were approved by CEUA / HCPA and CEUA / UFRGS under number: 14-0604 and 29310, respectively. Initially tests were carried out to verify the pain model, it was observed that 14 days after infraorbital nerve constriction surgery, animals pain group showed a reduction in the face withdrawal thresholds to von Frey facial test compared to control and sham groups [ GEE: group x time interaction (Wald 2 = 15.81; 2, P <0.05)]. After the model category, nociceptive tests were performed to assess the pathways involved in the trigeminal neuralgia, observed group x time interaction (Wald _2 = 175.74; 18 P <0.01). In protocol 1, the animals in the group who received pain benzodiazepine agonist (diazepam) showed an increase in nociceptive threshold from 15 minutes after administration and this result remained for up to 60 minutes. Animals of group pain that received glutamate antagonist (MK-801) showed an increase in the withdrawal threshold of the face only in the assessment was performed 60 minutes after drug administration. In protocol 2, statistical analysis showed interaction between the variables time x group (Wald χ2 = 657.53; 18, P <0.01), the animals showed increased nociceptive threshold from 15 minutes after administration of phenobarbital and morphine and this result remained for up to 60 minutes. On the other hand, animals receiving morphine showed an increased nociceptive threshold significantly higher in the animals receiving phenobarbital, however both drugs reversed hyperalgesia induced by exposure to pain model. In biochemical analysis, cortex and brainstem was observed increase in BDNF levels in both animals subjected to NT model and those who were exposed to sham surgery (one-way ANOVA / SNK, F (2,22) = 13.46 ;. F (2,25) = 6.08, P <0.05, respectively, at the peripheral level increase was observed in BDNF levels in trigeminal ganglion of animals subjected to the NT model (one-way ANOVA / SNK F ( 2,22) = 4.09; P <. 0.05). In serum was no difference in BDNF levels between groups (one-way ANOVA, P> 0.05) based on the results, this study. suggests that there is involvement of gabaergic pathways, glutamatergic and opioidergic processing orofacial neuropathic pain (trigeminal neuralgia) suggesting that there is functionality of the tested receptors. However, these GABAergic agonists did not induce analgesia in control animals and sham surgery suggesting a change in release or GABA synthesis in animals with NT. in this study, were not realized expression analysis and quantification of receptors, only evaluated the nociceptive response and the central and peripheral levels of BDNF. According to these findings, gabaergic pathways had a faster response than glutamatergic pathways. Some studies have shown that glutamate receptors are located in non-myelinated fibers in the periphery that can justify the delay in the animals that received glutamatergic antagonist in our study. As for BDNF levels we observed that there is an increase of this neurotrophin in animals subjected to NT model suggesting an involvement of this neuromodulator in neuroplastic events NT.

No description available.

No description available.

Le fonctionnement du cortex cérébral nécessite l’action coordonnée de deux des sous-types majeurs de neurones, soient les neurones à projections glutamatergiques et les interneurones GABAergiques. Les interneurones GABAergiques ne constituent que 20 à 30% des cellules corticales par rapport au grand nombre de neurones glutamatergiques. Leur rôle est toutefois prépondérant puisqu’ils modulent fortement la dynamique et la plasticité des réseaux néocorticaux. Il n’est donc pas surprenant que les altérations de développement des circuits GABAergiques soient associées à plusieurs maladies du cerveau, incluant l’épilepsie, le syndrome de Rett et la schizophrénie. La compréhension des mécanismes moléculaires régissant le développement des circuits GABAergiques est une étape essentielle menant vers une meilleure compréhension de la façon dont les anormalités se produisent. Conséquemment, nous nous intéressons au rôle de l’acide polysialique (PSA) dans le développement des synapses GABAergiques. PSA est un homopolymère de chaînons polysialylés en α-2,8, et est exclusivement lié à la molécule d’adhésion aux cellules neuronales (NCAM) dans les cerveaux de mammifères. PSA est impliqué dans plusieurs processus développementaux, y compris la formation et la plasticité des synapses glutamatergiques, mais son rôle dans les réseaux GABAergiques reste à préciser. Les données générées dans le laboratoire du Dr. Di Cristo démontrent que PSA est fortement exprimé post- natalement dans le néocortex des rongeurs, que son abondance diminue au cours du développement, et, faits importants, que son expression dépend de l’activité visuelle i et est inversement corrélée à la maturation des synapses GABAergiques. La présente propose de caractériser les mécanismes moléculaires régulant l’expression de PSA dans le néocortex visuel de la souris. Les enzymes polysialyltransférases ST8SiaII (STX) et ST8SiaIV (PST) sont responsables de la formation de la chaîne de PSA sur NCAM. En contrôlant ainsi la quantité de PSA sur NCAM, ils influenceraient le développement des synapses GABAergiques. Mon projet consiste à déterminer comment l’expression des polysialyltransférases est régulée dans le néocortex visuel des souris durant la période post-natale; ces données sont à la fois inconnues, et cruciales. Nous utilisons un système de cultures organotypiques dont la maturation des synapses GABAergiques est comparable au modèle in vivo. L’analyse de l’expression génique par qPCR a démontré que l’expression des polysialyltransférases diminue au cours du développement; une baisse majeure corrélant avec l’ouverture des yeux chez la souris. Nous avons de plus illustré pour la première fois que l’expression de STX, et non celle de PST, est activité-dépendante, et que ce processus requiert l’activation du récepteur NMDA, une augmentation du niveau de calcium intracellulaire et la protéine kinase C (PKC). Ces données démontrent que STX est l’enzyme régulant préférentiellement le niveau de PSA sur NCAM au cours de la période post-natale dans le cortex visuel des souris. Des données préliminaires d’un second volet de notre investigation suggèrent que l’acétylation des histones et la méthylation de l’ADN pourraient également contribuer à la régulation de la transcription de cette enzyme durant le développement. Plus d’investigations seront toutefois nécessaires afin de confirmer cette hypothèse. En somme, la connaissance des mécanismes par lesquels l’expression des ii polysialyltransférases est modulée est essentielle à la compréhension du processus de maturation des synapses GABAergiques. Ceci permettrait de moduler pharmacologiquement l’expression de ces enzymes; la sur-expression de STX et/ou PST pourrait produire une plus grande quantité de PSA, déstabiliser les synapses GABAergiques, et conséquemment, ré-induire la plasticité cérébrale. / The functioning of the cerebral cortex requires coordinated action of two major neuronal subtypes - the glutamatergic projection neurons and the GABAergic interneurons. GABAergic interneurons represent 20 to 30% of all cortical cells. Even though they are a minor cell population in the cerebral cortex compared to glutamatergic neurons, they are key modulators of network dynamics and plasticity of neocortical circuits. It is therefore not surprising that aberrant development of GABAergic circuits is implicated in many neurodevelopmental disorders including epilepsy, Rett syndrome and schizophrenia. Understanding the molecular mechanisms governing the development of GABAergic inhibitory synapses in neocortex is important towards a better comprehension of how abnormalities in this developmental process can occur. Therefore, we focus specifically on the role of polysialic acid (PSA) in the development of GABAergic synapses. PSA is a α-2,8 polysialylated homopolymer, which is exclusively linked to the Neural Cell Adhesion Molecule (NCAM) in the mammalian brain. It is involved in several developmental processes including formation and plasticity of glutamatergic synapses; however its role in GABAergic circuit formation has not been explored so far. Previously in Dr Di Cristo’s lab, we showed that PSA is strongly expressed post-natally and its expression steadily declines during development in mice neocortex. We also showed that the developmental and activity-dependant regulation of PSA expression is inversely correlated with the maturation of perisomatic GABAergic innervation. Our aim is to characterize the molecular mechanisms regulating PSA expression in mouse iv visual cortex during post-natal development. Two polysialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), are responsible for PSA attachment to NCAM. By controlling the amount of PSA on NCAM, they can influence GABAergic synapses development. The mechanisms regulating STX and PST expression is crucial but remain still unknown. My research project focused on the mechanisms regulating STX and PST transcription in the mouse postnatal cortex. We used an organotypic culture system, which recapitulates many aspects of GABAergic synapse maturation as observed in vivo. Polysialyltransferases transcript levels were measured by qPCR and showed that STX and PST mRNA levels steadily decline during post-natal development in the mouse cortex; the sharpest reduction in the expression of both enzymes correlate with eye opening. We further demonstrate for the first time that STX mRNA levels is activity-dependant, requires the activation of NMDA receptors, an increase in intracellular Calcium levels and is PKC-dependent. Altogether, we show that the regulation of the expression of STX is the main mechanism responsible for PSA expression levels in the cortex around eyes opening. We next investigated whether epigenetic mechanisms regulate STX transcription and preliminary data suggest that histone acetylation and DNA methylation may contribute to STX expression during development. However, further experiments are required to confirm this hypothesis. In summary, understanding the mechanisms modulating STX and PST expression in the neocortex is essential for the comprehension of their precise role in GABAergic synapse maturation. This knowledge could allow us to modulate pharmacologically the expression of these enzymes; in turn overexpression of STX and PST may re-induce PSA expression, thereby destabilizing GABAergic synapses, and ultimately facilitating cortical plasticity in the adult.

Myopia is a refractive defect of the eye in which collimated light produces images focused in front of the retina. Myopia can be artificially induced in animal models by form deprivation (form deprivation myopia, FDM) or by application of negative lenses (lens induced myopia, LIM). In this study myopia was induced using diffusers. The project had two main aims: 1. To determine if there is an interaction between the GABAergic system and dopaminergic system in the retina in terms of myopia? 2. To determine if there is an interaction between the GABAergic system and cholinergic system in the retina in terms of myopia? Firstly, an experiment focusing on the interaction between dopaminergic receptors antagonists and GABAC receptor antagonist was developed. Comparison of the different drug treated eye with the control was found and the effects of combination injections were compared to individual drug injections. Use of different blockers for various subtype of receptors simplified the understandings the underlying pharmacological interventions for GABAC receptor antagonist TPMPA. The D1 subtype of receptors was found to be involved in transmission of signals from GABAC receptors. Our results showed that D1 receptor antagonist SCH-23390 antagonizes the actions of TPMPA. In addition to this it was also found that possibly 5HT receptor may also play an important role in modulation of signaling from GABA receptor to dopaminergic receptors in the retina. These results were consistent with the drug combination effects for agonists. GABA A/C receptor agonist muscimol negativate the efficacy of D1 receptor agonist SKF-38393 but the activity of D2/4 receptor agonist quinpirole was not affected by muscimol. Although dopaminergic receptors are found to interact with GABAergic signaling, but an alternative interaction with anticholinergic (most widely studied antimyopic agents) could not be ruled out. This problem led to a follow-up experiment, in which GABA receptors intervention in anticholinergic agents was studied. The GABAergic receptor agonist muscimol when injected with anticholinergics (atropine and pirenzepine) showed a moderate interaction. As muscimol interacted with atropine to a lesser extent a more specific M1/5 receptor antagonist pirenzepine (earlier found to inhibit myopia) was used under these circumstances. The second aim to study the interaction between muscimol and pirenzepine showed more interaction with GABAA/C receptor agonist. There were data suggesting that there is a muscarinic and GABAergic interaction in retina, such that each modulation of each receptor had an effect on FDM. However, a drug combination treatment helped in understanding the underlying mechanism. Several previous studies have indicated that there exist a strong interaction between excitatory neurotransmitter acetylcholine and inhibitory transmitter GABA in retina. The results of this study indicate a similar finding. Thus results of this study may be summarized as: 1. D1 antagonists and not D2 antagonists blocks the antimyopic effects of GABAC antagonist TPMPA 2. GABA A/C agonist muscimol partially blocks the antimyopic activity of anticholinergics (e.g. atropine and pirenzepine).

No description available.

Le traumatisme cérébral (TC) touche des millions de personnes chaque année dans le monde. Les premières conséquences peuvent être une perte de conscience, des hémorragies et l’apparition d’un œdème cérébral. Cependant les personnes qui subissent un TC peuvent présenter des séquelles importantes à plus long terme. Ainsi le traitement préventif des pathologies post-traumatiques est devenu un réel problème de santé publique. La dépression représente la pathologie post-traumatique dont l’occurrence est la plus fréquente. Les origines connues de son apparition s’orientent vers une altération de la neurogenèse adulte hippocampique ainsi que des changements dans la neurotransmission GABAergique, qui est dépendante de l’homéostasie des ions chlorures. Mon travail de thèse suggère que la phase critique, responsable de l’apparition des pathologies post-traumatiques, survient au cours de la première semaine suivant le TC. Pendant cette période, mes résultats montrent que l’hyperexcitabilité des réseaux neuronaux hippocampiques est due à une perturbation des transporteurs des ions chlorure entraînant notamment, une diminution de l’inhibition neuronale. J’ai aussi pu mettre en évidence une altération de la neurogenèse adulte hippocampique liée à la perte d’interneurones dans le gyrus denté. Consécutivement à ces changements, vont s’installer des troubles dépressifs majeurs. Mes travaux indiquent également que la restauration précoce, de l’homéostasie des ions chlorure par un agent pharmacologique, prévient la mort des interneurones ainsi que les changements dans la neurogenèse et permet sur le long terme de réduire très fortement les troubles dépressifs majeurs. / Traumatic brain injury (TBI) affects annually millions of people over the world. The first major consequences include loss of consciousness, haemorrhage and the appearance of cerebral edema. However, people who experience TBI may have significant long-term sequelae and in the majority of cases develop major depressive disorders. In addition, debilitating effects of TBI substantially impair health-related quality of life and are associated with high health care costs. Hence, preventive treatment against posttraumatic pathologies has become a real public health concern. Increasing evidence points to an association between depressive disorders and changes in GABAergic neurotransmission as well as alteration of adult hippocampal neurogenesis.My thesis suggests that the critical phase of posttraumatic pathology occurs over the first week following the trauma. During this period, my results show that hippocampal network hyperexcitability is induced by a disruption of the chloride ion transporters, leading notably to a decrease in neuronal inhibition. Then my work highlighted an alteration of hippocampal neurogenesis related to the loss of interneurons in the dentate gyrus. After some latency, these changes will trigger major depressive disorders. My work also indicates that the early restoration, during this first post-traumatic week, of chloride ion homeostasis by a pharmacological agent, prevents cell death of interneurons as well as changes in neurogenesis and allows significant long-term reduction of major depressive disorders. This therefore suggests the possibility of developing new therapeutic strategies to prevent the emergence of posttraumatic pathologies.

A sinalização muscarínica exerce função modulatória sobre diferentes aspectos da cognição e emoções. Todos os cinco subtipos de receptores muscarínicos (mAChR), M1 a M5, são expressos no hipocampo de mamíferos e são ativados de forma sobreposta pela maioria dos fármacos, dificultando avanços significativos na compreensão da contribuição de cada componente desse sistema. A toxina muscarínica 3 (MT3) é um antagonista seletivo para o subtipo M4, permitindo a investigação das ações modulatórias deste receptor no aprendizado, memória e plasticidade sináptica. Os M4 são receptores acoplados à proteína G (GPCRs) que atuam via Gi/o desencadeando efeitos inibitórios sobre as células em que estão presentes. Estudos comportamentais anteriores indicam que a administração de MT3 imediatamente após o treino em uma tarefa aversiva produz efeito amnéstico, enquanto que a administração antes da evocação, causa facilitação. Uma explicação para estes resultados é que os circuitos locais envolvidos na consolidação e na evocação da memória diferem em sua natureza. Nesse contexto, sugere-se que o efeito amnéstico da MT3 sobre a consolidação seja consequência da supressão da inibição de interneurônios GABAérgicos; enquanto que na evocação, esse efeito se daria sobre as sinapses glutamatérgicas. Assim, no presente trabalho, com o objetivo de investigar como o receptor M4 modula a plasticidade sináptica de longa duração e interage com uma dessas sinalizações, no caso a GABAérgica, utilizou-se a técnica de eletrofisiologia in vivo de hipocampo de ratos anestesiados. Para tanto, foram realizados registros extracelulares do potencial excitatório pós-sináptico de campo (fEPSP) de CA1 evocados por estimulação contralateral da via Colateral de Schaffer com infusão dos fármacos 15 min antes ou depois da estimulação elétrica de alta ou baixa frequência (HFS: 10 trens 0,5 Hz, 20 pulsos 100 Hz; ou LFS: 600 pulsos 1 Hz, respectivamente). MT3 (4,0 μg/μl), bicuculina (0,06 μg/μl), baclofen (0,2 μg/μl) e veículo, isoladamente ou combinados, não alteraram a amplitude da resposta evocada basal ou a facilitação por pulso pareado (FPP) 15 min após a infusão. MT3 aparentemente atenuou, mas não de forma significativa, a potenciação de longa duração (LTP) em relação ao controle (potenciação 60 min após a HFS de 31,8% e 66,0%, respectivamente). Além disso, não houve diferença significativa entre a amplitude do fEPSP no período basal e 60 min após a HFS sob ação da MT3. Bicuculina, embora não tenha abolido a LTP e nem causado alteração na FPP, produziu uma potenciação de apenas 36,4%. Baclofen promoveu uma potenciação semelhante à dos controles. A administração de baclofen também reduziu significativamente a FPP em relação ao basal. A administração conjunta de MT3 com bicuculina ou baclofen promoveu uma potenciação semelhante ao controle. MT3 não apresentou efeito sobre a manutenção da LTP quando aplicada 15 min após a HFS. Por fim, não foi possível induzir a depressão de longa duração (LTD) com o protocolo de LFS utilizado. Embora não tenha ocorrido diferença estatisticamente significativa entre os grupos devido ao baixo número de animais utilizados, os dados sugerem a possibilidade de uma amplitude reduzida da LTP quando da injeção de bicuculina. Baclofen alterou a FPP em relação ao fEPSP basal, o mesmo não tendo sido observado no grupo controle. Com a administração concomitante de MT3, tais alterações deixam de ser identificadas. Ainda que os achados experimentais sejam inconclusivos e preliminares, este trabalho permitiu a padronização da técnica de eletrofisiologia in vivo em animal anestesiado o que abre portas para futuras investigações. / The cholinergic muscarinic system exerts modulatory function over different aspects of cognition and emotion. All five muscarinic receptors subtypes (mAChR), M1 to M5, are expressed at mammals hippocampus and at least two of them are simultaneously activated by most of the drugs, hindering significant advances on the role of each component of this system. The muscarinic toxin 3 (MT3) is a selective antagonist for the M4 subtype, allowing the investigation of the modulatory actions of this receptor over learning, memory and synaptic plasticity. The M4 are G protein coupled receptors (GPCRs) that act through Gi/o triggering inhibitory effects on which cells they are occur. Previous behavioral studies have shown that administration of MT3 soon after aversive task training exerts amnestic effects over memory, while administration prior to recall, leads to facilitation. A possible explanation to these results could be that the local circuits involved on memory consolidation and recall are different in nature. On this perspective, the amnestic effect of MT3 over memory consolidation should be consequence of GABAergic interneurons inhibition suppression; while the effect on recall, should be over glutamatergic synapses modulation. Thereby, the present work, with the objective to investigate how the M4 receptor modulates long-term synaptic plasticity and interacts with the GABAergic system, in vivo electrophysiological approach of anesthetized rats’ hippocampus was applied. Hence, field excitatory postsynaptic potentials (fEPSP) from CA1 were recorded after stimulation of contralateral Schaffer Collateral pathway with drugs infusion 15 min before or after high or low frequency electric stimulation (HFS: 10 trains 0.5 Hz, 20 pulses 100 Hz; LFS: 600 pulses 1 Hz, respectively). Neither MT3 (4.00 μg/μl), bicuculline (0.06 μg/μl), baclofen (0.20 μg/μl) nor vehicle, isolated or combined, changed the baseline evoked response amplitude 15 min after infusion nor the paired-pulse facilitation ratio (PPF). MT3 apparently attenuated, but not significantly, the long-term potentiation (LTP) compared to control (31.8% and 66.0% potentiation 60 min after HFS, respectively). In addition, there was no significant difference between baseline and 60 min after HFS fEPSP amplitude at MT3 group. Bicuculline, although did not abolish LTP neither changed PPF, it did produce a potentiation of only 36.4%. Baclofen induced a potentiation similar to control group. Baclofen administration also significantly reduced PPF compared to baseline. The simultaneous administration of MT3 and bicuculline or baclofen led to a potentiation similar to the control group. MT3 did not show any effect over LTP maintenance when applied 15 min after HFS. Lastly, it was not possible to induce long-term depression (LTD) with the used LFS protocol. Although there was no statistical significance between groups due to the low animal numbers used, data suggest that bicuculline had reduced LTP amplitude. Baclofen did alter PPF and the same was not observed on control group. When bicuculline or baclofen were injected with MT3, those alterations were not observed. These are inconclusive and preliminary results, notwithstanding this work allowed to set up the in vivo electrophysiology technique in anesthetized animals what will provide new tools for future research.