Yes / The activity-regulated cytoskeleton associated protein (Arc/Arg3.1) plays a key role in determining synaptic strength through facilitation of AMPA receptor (AMPAR) endocytosis. Although there is considerable data on the mechanism by which Arc induction controls synaptic plasticity and learning behaviours, several key mechanistic questions remain. Here we review data on the link between Arc expression and the clathrin-mediated endocytic pathway which internalises AMPARs and discuss the significance of Arc binding to the clathrin adaptor protein 2 (AP-2) and to endophilin/dynamin. We consider which AMPAR subunits are selected for Arc-mediated internalisation, implications for synaptic function and consider Arc as a therapeutic target. / The work in S.A.L.C. laboratory is supported by the BBSRC (BB/H018344/1 and BB/J02127X/1) and Wellcome Trust 200646/Z/16/Z. The work in M.J.W. Laboratory is supported by ERUK.

Dans le système nerveux central, la synaptogenèse est un processus complexe multi-étapes qui se déroule aux contacts axones/dendrites. Les molécules d’adhérence neurexines/neuroligines jouent un rôle essentiel dans ce processus, en créant un lien physique entre les compartiments pré- et post-synaptiques et en participant au recrutement des complexes macromoléculaires essentiels à la fonction synaptique. Plus spécifiquement, le complexe neurexine-1β/neuroligine-1 induit la formation de post-synapses excitatrices, en recrutant des molécules d’échafaudage telles que PSD-95 et des récepteurs du glutamate.Mon travail de thèse a consisté à étudier les mécanismes moléculaires mis en jeu par les adhésions neurexines/neuroligines lors de la formation des post-synapses glutamatergiques. En utilisant des systèmes biomimétiques (neurexine purifiée fixée sur des billes, ou agrégée par des anticorps réticulés), nous avons induit des adhésions spécifiques neurexine-1β/neuroligine-1 sur des neurones d’hippocampe en culture. Nous avons ainsi étudié la distribution dynamique des composants post-synaptiques (récepteurs AMPA, PSD-95) endogènes ou étiquetés avec des protéines fluorescentes, par vidéo-microscopie. Dans un premier article, nous avons montré que la formation de ces contacts induisait un recrutement rapide de PSD-95 ainsi que des récepteurs NMDA et AMPA fonctionnels. En utilisant des récepteurs AMPA recombinants, j’ai montré que ce recrutement était dicté par la sous-unité GluA2. Dans une deuxième étude, en comparant le recrutement de PSD-95 induit par la neurexine avec des anticorps non–activants, nous avons mis en évidence un mécanisme d’activation spécifique de neuroligine-1 induit par la liaison de neurexine-1β. L’utilisation de mutants ponctuels de neuroligine-1 a permis de montrer que cette activation passe probablement par la déphosphorylation d’une tyrosine unique située dans le domaine C-terminal de la neuroligine-1.Enfin, en étudiant la diffusion latérale des rAMPA de surface par suivi de particules uniques fluorescentes (Quantum dots), ainsi qu’une batterie d’outils moléculaires pour moduler les adhésions neurexine/neuroligine (sur-expression, siRNA, souris KO), nous avons montré que les rAMPA sont recrutés aux adhésions neurexine-1β/neuroligine-1 via l’échafaudage PSD-95 et que ce recrutement nécessite la diffusion des récepteurs dans la membrane plasmique. Nous proposons ainsi que les récepteurs AMPA soient recrutés aux contacts naissants via un mécanisme original de diffusion/piégeage. / In the central nervous system, synaptogenesis is a multi step process occuring at axo-dendritic contacts. Neurexins/neuroligins adhesions are particularly involved in this process, making a bridge between the pre- and the post-synapse, and participating to the recruitment of macromolecular complexes essential for synaptic function. More precisely neurexin-1β/neuroligin-1 complex is specifically involved in the formation of excitatory synapses, inducing the recruitment of glutamatergic post-synapses components, such as PSD-95, and glutamate receptors.During my PhD, I focused on the molecular mechanisms involved in glutamatergic post-synapses formation triggered by neurexin-1β/neuroligin-1 adhesions. Using biomimetic models (beads coated with purified neurexin, or purified neurexin cross-linked with aggregated antibodies) we induced specific neurexin-1β/neuroligin-1 adhesions on cultured hippocampal neurons. We then studied the dynamic distribution of either endogenous or recombinant post-synaptic components (PSD-95, AMPARs) with live-imaging techniques. First, we showed that the formation of these contacts induced a rapid recruitment of PSD-95 and functional NMDA and AMPA receptors. Using recombinant AMPA receptors, I showed that this recruitment was mediated by GluA2 subunit.In a second study, using systematic comparison between the recruitment of PSD-95 induced either by neurexin-1β or by “non activating” antibody binding on neuroligin-1, we revealed a specific activation mechanism of neuroligin-1 induced by neurexin-1β binding. Using point mutations on neuroligin-1, we showed that this activation mechanism is mediated by a tyrosine dephosphorylation on neuroligin-1 intracellular tail.Finally, we studied AMPA receptor surface diffusion with single particle tracking experiments, using different molecular tools to perturb neurexin-1β/neuroligin-1 adhesions (overexpression, RNA interference, KO mice). We showed that AMPA receptors recruitment at new-formed neurexin-1β/neuroligin-1 adhesions occurs through PSD-95, and involves surface diffusion of AMPA receptors. We proposed an original diffusion/trap mechanism of AMPA receptors at nascent contacts.

The IEM 1460 is a potential age-specific anticonvulsant and an indicator of the distribution of AMPA receptor subtypes among rat brain cells. It is a derivative of adamantane, that was tested in previous studies on models of human myoclonic and generalized tonic-clonic seizures with promising results. In this thesis we evaluated its effect on the motor activity of rat offspring in the age of 12, 18 and 25 days, we used 90 animals in total . The effect was evaluated 30 minutes after intraperitoneal administration of IEM 1460 in two doses, 10 mg/kg or 20 mg/kg, and was compared to the control animals with physiological solution applied intraperitoneally in amount of 20 mg/kg. To test the animals we used Open field test, righting reflex, negative geotaxis, horizontal bar test, rope climbing test, regular and irregular horizontal ladder test. The tests were applied to animals in mentioned order. There were found significant changes influencing motor behaviour, primarily in the 12 days old animals with the dose of 20 mg/kg IEM 1460 and in the 25 days old animals with both doses of IEM 1460, 10 and 20 mg/kg. In the 18 days old animals the results were less significant. Powered by TCPDF (www.tcpdf.org)

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

A perda de neurônios dopaminérgicos na substância negra é típica na doença de Parkinson (DP) e resulta em hiperexcitabilidade dos neurônios espinais médios advinda de uma neurotransmissão glutamatérgica corticoestriatal anormal, que pode provocar dentre outros danos, déficits motores característicos da doença. Considerando os efeitos neuroprotetores promovidos pelo exercício físico, o objetivo deste estudo foi observar o impacto do exercício realizado a curto e a longo prazo durante a evolução da lesão por 6-OHDA em ratos. As modificações encontradas nesse estudo podem ser relevantes para o circuito corticoestriatal, uma vez que a plasticidade dependente do exercício é capaz de modular a excitabilidade neuronal, reduzindo a hiperexcitabilidade glutamatérgica encontrada na DP. De forma geral, estes resultados suportam os potenciais efeitos do exercício físico em alterar a conectividade sináptica dos circuitos corticoestriatal e nigroestriatal em uma situação de depleção dopaminérgica, e assim, possivelmente modificar a progressão da doença em indivíduos com DP. / The loss of nigral dopaminergic neurons characteristic of Parkinson\'s disease (PD) is responsible for hyperexcitability of medium spinal neurons resulting in abnormal corticostriatal glutamatergic neurotransmission, which can cause, among other alterations, motor deficits typical of that disease. Considering the neuroprotective effects of exercise, the aim of this study was to observe the impact of short and long-term exercise during evolution of the 6-hydroxy-dopamine (6-OHDA) animal model of PD. The modifications found in this study may be relevant for corticostriatal circuits, since the exercise-dependent plasticity can modulate neuronal excitability by reducing glutamatergic hyperexcitability found in PD. Overall, these results reinforce the potential effects of exercise to change synaptic connectivity of corticostriatal and nigrostriatal circuits in a dopaminergic depletion state, and to possibly modify the progression of the disease in patients with PD.

No presente trabalho, foram desenvolvidos e validados dois métodos com o objetivo de determinar os herbicidas paraquat e glifosato, bem como o AMPA, principal metabólito do glifosato, em amostras de maconha apreendidas pela polícia de Campinas, São Paulo. A detecção e quantificação de herbicidas na maconha são necessárias e importantes para alertar o real risco que a droga pode oferecer aos usuários. Existem várias razões que explicam a presença de herbicidas na maconha em vários países, incluindo o Brasil. A eletroforese capilar foi utilizada para determinação dos herbicidas. Um método de detecção direta foi usado para determinar o paraquat e outro, com detecção indireta, para determinar o glifosato e AMPA. Os métodos desenvolvidos mostraram boa linearidade, precisão, exatidão e recuperação. Os dados da validação atestam que os métodos podem ser utilizados em laboratórios Forense no Brasil. Cento e trinta amostras foram analisadas, sendo que em doze amostras foram detectadas a presença de paraquat em várias concentrações e ainda três amostras forneceram resultados positivos apenas para o glifosato sendo uma delas, detectado a presença concomitante do AMPA. Os valores dos contaminantes encontrados podem representar um risco ao usuário, fazendo-se necessário novos estudos para delineamento sobre os reais efeitos que esses contaminantes podem apresentar aos usuários de Cannabis. / In the present work, two methods were developed and validate, aiming to determinate the herbicides paraquat and glyphosate and his major metabolite AMPA in seizured marijuana samples by the police in Campinas, São Paulo. The determination of herbicides in confiscated samples is necessary and important to alert the real risk of marijuana can offer to the users. There are many reasons that explain the presence of herbicides in marijuana in several countries, including Brazil. Capillary electrophoresis was used to determinate the studied herbicides. A method with direct detection was used to determinate paraquat and indirect detection to determinate glyphosate and AMPA. The developed methods showed good linearity, precision, accuracy, and recovery. Therefore, it can be applied in Forensics labs in Brazil. One hundred and thirty samples were analyzed, and twelve of them result positive for paraquat in several concentrations and three samples showed positive to glyphosate and one of them, detected the presence of AMPA. The values of the contaminants found, can offer a risk to the users, making it necessary new studies to know the real effects that such contaminants can offer to the Cannabis users.

No presente trabalho, foram desenvolvidos e validados dois métodos com o objetivo de determinar os herbicidas paraquat e glifosato, bem como o AMPA, principal metabólito do glifosato, em amostras de maconha apreendidas pela polícia de Campinas, São Paulo. A detecção e quantificação de herbicidas na maconha são necessárias e importantes para alertar o real risco que a droga pode oferecer aos usuários. Existem várias razões que explicam a presença de herbicidas na maconha em vários países, incluindo o Brasil. A eletroforese capilar foi utilizada para determinação dos herbicidas. Um método de detecção direta foi usado para determinar o paraquat e outro, com detecção indireta, para determinar o glifosato e AMPA. Os métodos desenvolvidos mostraram boa linearidade, precisão, exatidão e recuperação. Os dados da validação atestam que os métodos podem ser utilizados em laboratórios Forense no Brasil. Cento e trinta amostras foram analisadas, sendo que em doze amostras foram detectadas a presença de paraquat em várias concentrações e ainda três amostras forneceram resultados positivos apenas para o glifosato sendo uma delas, detectado a presença concomitante do AMPA. Os valores dos contaminantes encontrados podem representar um risco ao usuário, fazendo-se necessário novos estudos para delineamento sobre os reais efeitos que esses contaminantes podem apresentar aos usuários de Cannabis. / In the present work, two methods were developed and validate, aiming to determinate the herbicides paraquat and glyphosate and his major metabolite AMPA in seizured marijuana samples by the police in Campinas, São Paulo. The determination of herbicides in confiscated samples is necessary and important to alert the real risk of marijuana can offer to the users. There are many reasons that explain the presence of herbicides in marijuana in several countries, including Brazil. Capillary electrophoresis was used to determinate the studied herbicides. A method with direct detection was used to determinate paraquat and indirect detection to determinate glyphosate and AMPA. The developed methods showed good linearity, precision, accuracy, and recovery. Therefore, it can be applied in Forensics labs in Brazil. One hundred and thirty samples were analyzed, and twelve of them result positive for paraquat in several concentrations and three samples showed positive to glyphosate and one of them, detected the presence of AMPA. The values of the contaminants found, can offer a risk to the users, making it necessary new studies to know the real effects that such contaminants can offer to the Cannabis users.

Ο νεογνικός χειρισμός, ένα πειραματικό μοντέλο πρώιμων εμπειριών, είναι γνωστό οτι επηρεάζει τη λειτουργία του άξονα υποθαλάμου-υπόφυσης-επινεφριδίων, βελτιώνοντας έτσι την προσαρμοστικότητα, την αντιμετώπιση του στρες, τις διανοητικές ικανότητες και γενικά τις διεργασίες του εγκεφάλου που σχετίζονται με την πλαστικότητα. Προηγούμενη μελέτη έχει δείξει οτι ο νεογνικός χειρισμός παρουσιάζει εκλεκτικές επιδράσεις στους υποδοχείς του N-μεθυλο-D-ασπαρτικού οξέος (NMDA) του διεγερτικού νευροδιαβιβαστή γλουταμινικό στον εγκέφαλο των επίμυων. Οι υποδοχείς του γλουταμινικού που διαμεσολαβούν την ταχεία συναπτική νευροδιαβίβαση στις διεγερτικές συνάψεις του κεντρικού νευρικού συστήματος είναι οι υποδοχείς του α-αμινο-3-υδροξυ-5-μεθυλο-ισοξαζολο-προπιονικού οξέος (AMPA) και είναι κρίσιμοι κατά τη νευρωνική ανάπτυξη, τη συναπτική πλαστικότητα και τη δομική αναδιαμόρφωση του εγκεφάλου. Συντίθενται από τέσσερις υπομονάδες, τις GluRA, GluRB, GluRC και GluRD, οι οποίες συνδυάζονται για να σχηματίσουν τετραμερή. Οι περισσότεροι υποδοχείς AMPA είναι ετεροτετραμερή, αποτελούμενα από τουλάχιστον δύο από τις αναφερθείσες υπομονάδες. Οι υποδοχείς AMPA που είναι διαβατοί στο ασβέστιο δεν περιέχουν την υπομονάδα GluRB, ενώ τόσο η υπομονάδα GluRA, όσο και η GluRB, παίζουν σημαντικό ρόλο στη συναπτική διακίνηση των υποδοχέων AMPA. Ο σκοπός της παρούσας εργασίας ήταν να ερευνηθεί κατά πόσο ο νεογνικός χειρισμός μπορεί να επιδρά στους υποδοχείς AMPA, εφόσον έχει δειχθεί οτι η σύνθεση των υπομονάδων και κατά συνέπεια και οι ιδιότητες των υποδοχέων AMPA στη σύναψη μεταβάλλονται από την αισθητική εμπειρία και κατά πόσο αυτή η επίδραση είναι εξαρτώμενη από το φύλο. Σύμφωνα με το παρόν πρωτόκολλο νεογνικού χειρισμού, κάθε νεογνό απομακρυνόταν από τη φωλιά για 15 λεπτά καθημερινά από την πρώτη μεταγεννητική ημέρα μέχρι τον απογαλακτισμό του τρεις εβδομάδες μετά. Η τεχνική του in situ υβριδισμού χρησιμοποιήθηκε για την εντόπιση και ποσοτικοποίηση της έκφρασης του mRNA της κάθε υπομονάδας. Η έκφραση των υπομονάδων του υποδοχέα AMPA μελετήθηκε σε συγκεκριμένες εγκεφαλικές περιοχές που εμπλέκονται στο συναίσθημα, στη μάθηση, στη μνήμη και στην αισθητική αντίληψη, όπως ο ιππόκαμπος, η αμυγδαλή και ο εγκεφαλικός φλοιός ενήλικων αρσενικών και θηλυκών επίμυων. Τα αποτελέσματα της έρευνας έδειξαν οτι ο νεογνικός χειρισμός προκάλεσε διαφορετικές και σε κάποιες περιπτώσεις φυλετικά διμορφικές μεταβολές στην έκφραση των υπομονάδων του υποδοχέα AMPA, με τρόπο ειδικό ανά υπομονάδα και περιοχή. Πιο συγκεκριμένα, τα ζώα που είχαν υποστεί νεογνικό χειρισμό, αρσενικά και θηλυκά, είχαν μειωμένα επίπεδα mRNA της GluRB και της GluRC στο ραχιαίο ιππόκαμπο και στο σωματαισθητικό φλοιό, συγκριτικά με τα ζώα χωρίς χειρισμό. Επιπλέον, τα επίπεδα mRNA της GluRC ήταν μειωμένα στην αμυγδαλή και στον προμετωπιαίο φλοιό και τα επίπεδα mRNA της GluRD ήταν μειωμένα στον κοιλιακό ιππόκαμπο αρσενικών και θηλυκών ζώων που είχαν υποστεί χειρισμό. Επιπροσθέτως, ο νεογνικός χειρισμός είχε φυλετικά διμορφικές επιδράσεις, αυξάνοντας τα επίπεδα mRNA της GluRA στο ραχιαίο ιππόκαμπο των αρσενικών ζώων και μειώνοντάς τα στα θηλυκά, καθώς και μειώνοντας τα επίπεδα mRNA της GluRΒ στον κοιλιακό ιππόκαμπο και στην αμυγδαλή μόνο των θηλυκών επίμυων. Οι μεταβολές στο mRNA των υποδοχέων AMPA θεωρούμε οτι συντελούνται μέσω επιγενετικής ρύθμισης λόγω του νεογνικού χειρισμού. Τα αποτελέσματα μας στο ραχιαίο ιππόκαμπο υποδεικνύουν ότι ο νεογνικός χειρισμός είναι πιθανό να προκαλεί επαγόμενη από την εμπειρία συναπτική ενδυνάμωση και αποδυνάμωση αρσενικών και θηλυκών ζώων αντίστοιχα, δεδομένων των αντίθετων μεταβολών που παρατηρούνται στα επίπεδα του mRNA της υπομονάδας GluRA στα δύο φύλα. Επιπλέον, η παρατηρούμενη μείωση της έκφρασης του mRNA της GluRB πιθανόν να οδηγεί σε μειωμένο αριθμό αδιάβατων στο ασβέστιο υποδοχέων AMPA που περιέχουν την υπομονάδα GluRB, οπότε ο νεογνικός χειρισμός μπορεί να προκαλεί μεταβολή στο φαινότυπο του υποδοχέα AMPA από αδιάβατο σε διαβατό στο ασβέστιο και τελικά αύξηση της πλαστικότητας της σύναψης στις περιοχές όπου παρατηρούνται οι μεταβολές. Συμπερασματικά, στην παρούσα μελέτη δείξαμε οτι οι υπομονάδες του υποδοχέα AMPA μεταβάλλονται από το νεογνικό χειρισμό, ένα μοντέλο πρώιμης εμπειρίας, στον εγκέφαλο ενήλικων επίμυων, με τρόπο ειδικό ανά υπομονάδα και ανά περιοχή και σε κάποιες περιπτώσεις φυλετικά διμορφικό. Αυτό σημαίνει οτι η γλουταμινεργική νευροδιαβίβαση μεταβάλλεται από μια πρώιμη εμπειρία. Αυτή η επίδραση του νεογνικού χειρισμού μπορεί να είναι ένας από τους παράγοντες που υπόκεινται της αυξημένης πλαστικότητας του εγκεφάλου των ζώων που έχουν υποστεί νεογνικό χειρισμό, η οποία εκδηλώνεται τόσο σε κυτταρικό επίπεδο όσο και στο συμπεριφορικό επίπεδο. / Neonatal handling, an experimental model of early life experiences, is known to affect the hypo¬thalamic-pituitary-adrenal axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related pro¬cesses. Previous study has shown selective effects of neonatal handling on rat brain NMDA receptors. AMPA receptors (AMPARs) mediate fast synaptic trans¬mission at excitatory synapses in the CNS and are crucial during neuro¬nal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as GluRA, GluRB, GluRC and GluRD, which combine to form tetramers. Most AMPARs are heterotetramerics, made of at least two of the four pro¬per subunits GluRA-D. AMPA receptors that are permeable to Ca2+ lack the GluRB subunit, while both GluRA and GluRB subunits have an important role in AMPAR trafficking towards the synapse. The present study addressed the question of whether neonatal handling might have an effect on AMRARs, since it has been shown that the subunit composition and thus the synaptic properties of AMPARs, changes in response to sensory experience, and whether this effect is gender-specific. According to the current neonatal handling pro¬tocol, each pup of a litter was removed from the nest for 15 min daily from the first postnatal day 1 (PND1) until weaning (PND22). In situ hybridization was used in order to localize and quantify subunit mRNA expression, with specific cDNA oligo¬nucleotides. AMPAR subunit expression was studied in specific brain regions that are involved in emotions, learning, memory and sensory perception, such as the hippo¬campus, cerebral cortex and amydgala of adult male and female rats. We found that neonatal handling caused differential and in cases sexually dimorphic changes in AMPA receptor subunit expression, depending on the brain region and the subunit. More specifically, neonatally handled animals, both males and females, had lower GluRB and GluRC mRNA levels in the dorsal hippocampus and the somatosensory cortex, compared to the non-handled. Moreover, GluRC mRNA levels were decreased in the amygdala and the prefrontal cortex and GluRD mRNA levels were decreased in the ventral hippocampus of handled animals of both sexes. Furthermore, neonatal handling had sexually dimorphic effects, increasing GluRA mRNA levels in the dorsal hippocampus of males while decreasing them in females, as well as decreasing GluRB mRNA levels in the ventral hippocampus and the amygdala only of the females. The observed changes in AMPA receptor mRNA levels are thought to occur via epigenetic regulation in response to neonatal handling. Our results in dorsal hippocampus suggest that neonatal handling may induce experience-dependent synaptic strengthening in males and weakening in females, due to the opposite changes observed in GluRA mRNA levels of the two sexes. Moreover, the observed decrease in GluRB mRNA expression most likely leads to reduced GluRB-containing calcium-impermeable AMPA receptors, therefore neonatal handling may cause a switch in AMPA receptor phenotype from calcium-impermeable to calcium-permeable AMPA receptors, and thus induce a higher synaptic plasticity in the regions where the changes are observed. In the present study we show that neonatal handling, an experimental model of early life experiences, induces changes in the AMPA receptor subunits expression in the adult rat brain that are region- and subunit-specific and in cases sexually dimorphic. These results demonstrate that glutamatergic transmission changes in response to an early experience. This effect of handling could be one of the factors underlying the increased plasticity of the brain of neonatally handled animals, which is manifested both at the cellular and at the behavioural/systemic level.

La dépression majeure constitue aujourd’hui une maladie dévastatrice qui affecte jusqu’a 20% de la population mondiale et qui est classifiée comme une des causes principales des années de vie en handicap. Depuis les années ’50 et avec la serendipité des composés antidépresseurs, la physiopathologie et le traitement actuel de la dépression sont dominés par l’hypothèse monoaminergique. Malgré leur utilisation répandue encore aujourd’hui, les antidépresseurs qui ciblent le système monoaminergique sont inefficaces chez une proportion significative des patients et présentent un délai du début d’action prolongé. Dans l’optique d’approfondir l’action antidépressive, nombreuses études récentes ont démontré différents mécanismes à travers les quels ces composés restaurent la plasticité neuronale et la neurotransmission, qui sont trouvées perturbées dans les troubles de l’humeur et dans des modèles animaux de la dépression. La transmission glutamatergique, notamment le récepteur AMPA (AMPA R), et des cascades intracellulaires de signalisation ont été impliqués dans la physiopathologie de la dépression et l’action antidépressive. Au cours de cette étude, des potentiateurs AMPA R (modulateurs positives allostériques) ont montré des effets antidépresseurs dans un modèle chronique de dépression et des résultats préliminaires suggèrent un délai du début d’action précoce de ces composés. La voie de signalisation d’ERK/MAPK a été aussi étudiée, comme un intégrateur des modifications de la plasticité synaptique qui constitue le lien entre les signaux extracellulaires et la régulation de l’expression génique. On a utilisé une nouvelle classe d’inhibiteurs de la voie de signalisation d’ERK. La construction de ces molécules est basée sur la propriété particulière d’ERK à s’attacher à ses cibles à travers des domaines de liaison spécifiques. Une batterie de tests comportementaux, aigus et chroniques, ont apporté une somme de données convaincants sur l’action antidépressive de l’inhibition sélective du complexe de signalisation ERK/Elk1. L’ensemble des résultats de cette étude apportent de nouveaux éléments sur la physiopathologie de la dépression et révèlent des pistes thérapeutiques prometteuses. / Major depression is a devastating disease that affects up to 20% of world population and is classified today as a leading cause of disability-adjusted life years. Since late 50s with the serendipitous discovery of the first antidepressant agents, pathophysiology and therapeutics of depression are governed by the monoaminergic hypothesis. Monoaminergic-based treatment, although still in use today, was proven inefficient to treat a significant proportion of cases and presents a delayed onset of action. Recent research has unveiled an array of new mechanisms through which antidepressant medication helps restore neuronal plasticity and neurotransmission that is disrupted in mood disorders and in animal models of depression. Glutamatergic transmission, in particular AMPA receptor, and signal transduction cascades have been implicated both in antidepressant action and the pathophysiology of depression, as here and now regulators that mediate persistent changes. In this study, AMPA receptors positive modulators demonstrated antidepressant-like effects in a chronic model of depression and preliminary data suggest a faster onset of action than conventional antidepressants. The ERK/MAPK signaling pathway was also studied as a major integrator of synaptic plasticity modifications that links extracellular signals to gene expression regulation via its downstream molecular partners. We used a new class of inhibitors of the ERK pathway, whose design was based on the particular property of ERK to bind to its downstream targets via specific docking domains. A considerable amount of data provided evidence for an antidepressant action of selective inhibition of the ERK/Elk1 signaling complex in multiple animal models of depression. Overall, the findings of this work reveal novel, promising targets for the treatment of depression.