Les neurones établissent entre eux de nombreux contacts synaptiques, et l'on estime qu’en moyenne un neurone peut avoir dix mille contacts avec les neurones de son voisinage. L'une des synapses les plus importantes et les plus étudiées, dont les dysfonctionnements conduisent à des pathologies du cerveau, est la synapse excitatrice glutamatergique. Dans l’hippocampe, les synapses excitatrices présentent une structure postsynaptique particulière, sous la forme d’un renflement de la dendrite appelé épine dendritique. Cette épine possède un domaine particulier, la densité postsynaptique, concentrant de nombreux récepteurs aux glutamates, des protéines d’adhésion ainsi que des protéines d’échafaudage faisant le lien avec les cascades moléculaires intracellulaires et le cytosquelette d’actine. La morphologie de l’épine dendritique ainsi que le nombre de récepteurs présents dans la PSD sont des éléments clés dans la transmission synaptique et les phénomènes de potentiation et de dépression à long terme (LTP & LTD). Lors de ma thèse, j’ai identifié Scribble1 comme une nouvelle protéine régulant le trafic des récepteurs NMDA. Scribble1 est surtout connue pour son implication dans des processus de polarité, division et migration cellulaire. En modulant le taux de Scribble1, j’ai montré que je pouvais affecter le nombre et la morphologie des épines des neurones hippocampaux, ainsi que la polymérisation de l’actine. Ensuite, j’ai démontré que Scribble1 interagissait directement avec les récepteurs NMDA et permettait leur recyclage à la membrane. Enfin, chez le neurone immature, Scribble1 est impliqué dans la migration du cône axonal. Chez un animal mutant, qui n’exprime que 50% de la protéine (circletail) les performances mnésiques et sociales de l’animal sont perturbées, validant le rôle de la protéine au niveau du système nerveux. / One of the most studied and more important synapse is the glutamatergic excitatory synapse, which dysfunctions lead to brain pathologies. In the hippocampus, the most represented synapses are glutamatergic synapses using glutamate as neurotransmitter. Postsynaptic structures, such as dendritic spines, concentrate many glutamate receptors, adhesion proteins and scaffold proteins bridging receptors to molecular cascades and intracellular actin cytoskeleton. The morphology of the dendritic spine and the number of glutamate receptors at the surface of the spine are key-elements in synaptic transmission, such as of long-term potentiation (LTP). In this study, I identify Scribble1 as an important regulator of NMDA receptors trafficking. Scribble1 is well known for its roles in cell polarity, division and migration processes. First, I show that Scribble1 gain- and loss-of-function affect the number and morphology of spines, as well as the actin polymerization. Next, I showed that Scribble1 interacts directly with the NMDA receptor and stimulates its recycling to the membrane. Finally, in immature neuron, Scribble1 is involved in axon growth cone migration. In a Scribble1 mutant animal model, circletail, we observed disruption of synaptic transmission and memory and social performance defects, compatible with a role of the protein in central nervous system function.

Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate

L’évolution du Syndrome Douloureux Régional Complexe (SDRC) peut aboutir à un état de douleur chronique invalidant et difficile à traiter. Parmi les stratégies thérapeutiques utilisables, la kétamine, administrée sur 5 jours à des doses infra-anesthésiques, a une place de choix. Cependant, il s'agit d'un traitement invasif et dont le mécanisme d'action antalgique n'est pas connu. Notre travail a porté sur deux aspects du traitement du SDRC par la kétamine: la prédiction de l’efficacité thérapeutique et la compréhension des mécanismes d'action. Dans un premier temps, chez 105 patients SDRC, nous avons évalué l'intérêt de la scintigraphie osseuse au Technétium 99 réalisée avant le traitement pour prédire l’efficacité thérapeutique de la kétamine. Cette efficacité était corrélée à l’augmentation relative de l'activité inflammatoire et du remodelage osseux objectivée par scintigraphie. Dans une deuxième étude, portant sur l’évaluation de l’excitabilité corticale par stimulation magnétique transcrânienne et incluant 19 patients SDRC, nous avons observé que la kétamine réprimait fortement et de façon bilatérale la facilitation intracorticale, paramètre de transmission glutamatergique, et en revanche restaurait l’inhibition intracorticale correspondant au côté douloureux, paramètre gabaergique qui était très altéré avant le traitement. L'effet antalgique de la kétamine était corrélé à cette restauration d'inhibition ainsi qu'à la réduction de la facilitation correspondant au côté sain. Ainsi, la kétamine semble jouer un rôle dans la balance d'influences gabaergiques et glutamatergiques transcalleuses. Ce travail a permis de caractériser de nouveaux mécanismes physiopathologiques ainsi qu’une nouvelle justification de l’efficacité thérapeutique de la kétamine dans le SDRC. / Regional Complex Pain Syndrome (CRPS) can result in a chronic pain condition that is disabling and difficult to treat. Among the therapeutic strategies that can be used, ketamine, administered over 5 days at infra-anesthetic doses, has a place of choice. However, it is an invasive treatment and the mechanism of analgesic action is not known. Our work focused on two aspects of the treatment of CRPS by ketamine: predicting therapeutic efficacy and understanding mechanisms of action. Initially, in 105 CRPS patients, we assessed the benefit of pre-treatment Technetium 99 bone scintigraphy to predict the therapeutic efficacy of ketamine. This efficacy was correlated with the relative increase in inflammatory activity and bone remodeling detected by scintigraphy. In a second study, evaluating cortical excitability by transcranial magnetic stimulation and including 19 CRPS patients, we observed that ketamine strongly and bilaterally repressed intracortical facilitation, a glutamatergic transmission parameter, and on the other hand restored the intracortical inhibition corresponding to the pain side, a gabaergic parameter which was very altered before the treatment. The analgesic effect of ketamine was correlated with this restoration of inhibition as well as the reduction of the facilitation corresponding to the healthy side. Thus, ketamine seems to play a role in the balance of gabaergic and glutamatergic transcallosal influences. This work allowed new physiopathological mechanisms to be characterized as well as a new justification of the therapeutic efficacy of the ketamine in the CRPS.

Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.

La maladie d’Alzheimer (MA) se caractérise par une perte des fonctions cognitives liée à une dégénérescence neuronale induite par l’accumulation de peptides amyloïdes-β (Aβs) dans des régions vulnérables du cerveau comme l’hippocampe. Au niveau moléculaire, les peptides Aβs se lient préférentiellement à la densité post-synaptique des synapses excitatrices, espace au niveau duquel la protéine d’échafaudage PSD-95 organise l’ancrage des récepteurs NMDA (RNMDAs) et régule leur mobilité membranaire. A l’aide d’une stratégie intégrative qui favorise des niveaux d’analyse verticaux (du phénotype aux événements moléculaires) et qui combine un ensemble d’approches corrélatives et invasives chez des souris double transgéniques APPswe/PS1dE9 modèles de la MA, nous avons mis en évidence que les peptides Aβs déstabilisent l’organisation synaptique (altération de l’expression de la PSD-95) et augmentent le pool extrasynaptique de sous-unités GluN2B des RNMDAs dans l’hippocampe. Cette réorganisation se traduit par une perturbation des fonctions mnésiques. Par ailleurs, il a été montré que certaines oscillations de l’activité hippocampique, comme les « sharp-wave ripples » (SWRs) générées pendant les périodes de sommeil, jouent un rôle crucial dans la formation de la mémoire. De façon surprenante, l’accumulation des peptides Aβs semble épargner la dynamique d’expression des SWRs durant les comportements de routine. Afin d’examiner l’effet potentiel des Aβs sur les SWRs chez des animaux confrontés à des challenges cognitifs, nous avons soumis des souris adultes injectées intracérébralement avec une solution d’Aβs à un test de reconnaissance spatiale. Alors qu’elles sont capables de former une mémoire à court terme, les souris Aβs montrent un oubli plus rapide, suggérant qu’elles encodent avec succès, mais qu’elles sont incapables de stabiliser et de rappeler une information acquise antérieurement. En l’absence d’une demande cognitive préalable, les propriétés des SWRs ne sont pas altérées par les Aβs. En revanche, lorsqu’elles doivent résoudre un test cognitif, les pics de SWRs normalement observés après encodage ou reconnaissance chez les souris témoins sont abolis chez les souris Aβs, indiquant une perturbation du traitement hippocampique de l’information spatiale. Pris dans leur ensemble, ces résultats identifient deux nouveaux mécanismes délétères sous-tendant les déficits de mémoire spatiale associés à la MA. / Cognitive impairments in Alzheimer’s disease (AD) are thought to be related to degenerative synaptic changes caused by the accumulation of amyloid-β peptides (Aβs) in vulnerable brain regions such as the hippocampus. At the molecular level, Aβs bind preferentially to the postsynaptic density of neuronal excitatory synapses, where the scaffolding post-synaptic protein-95 (PSD-95) organizes NMDA receptor (NMDAR) location as well as its downstream signaling. By using an integrative strategy which favoured vertical levels of analyses (from phenotype to molecular events) and combined a set of interrelated correlative and invasive approaches in a double transgenic mouse model of AD (APPswe/PS1dE9 mice), we were successful in establishing that Aβs destabilize the synaptic organization (reduction of expression of PSD-95) and increase the extrasynaptic pool of GluN2B-containing NMDAR in the hippocampus, a reorganization which translates into impaired memory functions. It is also well-known that hippocampal sharp wave-ripples (SWRs) generated during sleep periods are crucial for memory formation but accumulation of soluble Aβs, surprisingly seems to spare SWR dynamics during routine behavior. To unravel a potential effect of Aβs on SWRs in cognitively-challenged animals, we submitted vehicle- and Aβ-injected mice to spatial recognition memory testing. While capable of forming short-term memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. Altogether these results identify two new disruptive mechanisms for the spatial memory deficits associated with AD.

Le cortex préfrontal (PFC) est le principal locus des perturbations dans l’activité des réseaux de neurones chez les schizophrènes. Ces perturbations résultent d’une dérégulation des interactions entre le système dopaminergique et le système glutamatergique dont l’origine demeure inconnue. Il est acquis que les cellules gliales détectent et intègrent les signaux synaptiques, et libèrent différentes substances neuroactives comme la D-sérine. Cet acide aminé est aujourd’hui reconnu comme le coagoniste endogène des récepteurs au glutamate de type NMDA dans de nombreuses aires cérébrales. Mon travail de thèse est centré sur le rôle de la d-sérine dans la transmission synaptique excitatrice glutamatergique dans le PFC du rongeur adulte et dans la gouvernance des interactions entre systèmes glutamatergique et dopaminergiques J’ai tout d’abord montré en utilisant des enregistrements électrophysiologiques sur tranches que la d-sérine est le coagoniste des récepteurs NMDA synaptiques dans les couches V/VI du PFC. Cet acide aminé est synthétisé par les astrocytes et contrôle l’induction de la potentialisation à long terme. D’autre part, j’ai montré que la dopamine exerce un effet biphasique sur l’activité des récepteurs NMDA synaptiques et sur l’excitabilité des neurones pyramidaux des couches V/VI du PFC et ce en contrôlant la libération de d-sérine. Une approche pharmacologique sélective a permis de mettre en évidence le rôle des récepteurs D1 dans les effets potentialisateurs et le rôle des récepteurs D2/D3 dans les effets inhibiteurs de la dopamine. Mon travail démontre que les astrocytes arborent des récepteurs à la dopamine qui contrôlent la libération de la d-sérine. / The prefontal cortex (PFC) is the main locus where dysfunctions of neuronal networks are evident in schizophrenia. These dysfunctions are caused by an impairment of cross-talk between dopaminergic and glutamatergic systems whose origin is unknown. It is now accepted that glia detect and integrate synaptic signals and then release many neuroactive substances such as D-serine. This amino acid is now considered to be the endogenous coagonist of the NMDA subtype receptors for glutamate in many brain areas. My PhD work focuses on the functions of d-serine in glutamatergic excitatory synaptic transmission in the PFC of adult rodent and in governing the interactions between dopaminergic and glutamatergic systems. First, using electrophysiological recordings on brain slices, I have shown that d-serine is the coagonist of synaptic NMDA receptors in layers V/VI of PFC. This amino acid is synthesized by glia and is crucial for the induction of long term potentiation. In addition, I have shown that dopamine has a bell-shape effect on the activity of synaptic NMDA receptors and on the excitability of excitatory pyramidal neurons by controlling the release of d-serine. The use of specific pharmacological tools allowed me to show the potentiating effects of dopamine are mediated by D1 receptors whereas the inhibitory effects are due to the activation of D2/D3 receptors. Finally, my work highlights the presence of functional dopaminergic receptors on astrocytes that modulate the release of d-serine in the PFC, thus impacting NMDA receptor activity.

Increased airway smooth muscle (ASM) mass contributes to airway hyperresponsiveness (AHR) in asthma and is orchestrated by growth factors, cytokines and chemokines. Airway contractile responses are influenced by neuromediators, such as acetylcholine, and glutamate released by parasympathetic and sympathetic airway nerves. Hyperactivity of these neural elements, termed neurogenic inflammation, is linked with hypercontractility and AHR. Glutamate is a non-essential amino acid derivative, and its physiological role is traditionally considered with respect to its being the primary excitatory neurotransmitter in brain, and regulation of neuronal development and memory. In allergic inflammation, immune cells including dendritic cells, neutrophils and eosinophils, constitutively synthesize and release glutamate, which signals through activation of glutamate receptors, most important among which are ionotrophic N-methyl D-aspartate receptors (NMDA-R). We hypothesized that glutamatergic signaling mediated through NMDA-Rs plays an important role in inducing functional Ca2+ responses in human (H) ASM cells that can underpin airway hypercontractility. We investigated the expression and function of NMDA-Rs in HASM cells, and assessed the effects of pro-inflammatory cytokines on NMDA-R expression and functional responses. Moreover, we measured airway responses to NMDA in mice, murine thin cut lung slice preparations, and floating collagen gels seeded with HASMs. Our data reveal that airway myocytes express multi-subunit NMDA-R complexes that function as receptor-operated calcium channels (ROCCs), mobilizing intracellular Ca2+ in ASM in vitro and airway contraction ex vivo. Individual airway myocytes treated with NMDA-R agonist exhibit disparate temporal patterns of intercellular Ca2+ flux that can be partitioned into four discrete function sub-groups. Further we show that tumor necrosis factor (TNF) exposure modulates NMDA-R subunit expression, and these changes are associated with a shift in the distribution of myocytes in individual Ca2+-mobilization sub-groups in vitro. Further, post-TNF exposure, NMDA-R agonists’ treatment induced Ca2+-dependent airway dilation in murine lung slice preparations, an effect that was prevented by co-treatment with inhibitors of nitric oxide synthase (NOS) or cyclooxygenase (COX). Taken together, we conclude that NMDA-R regulate HASM-mediated airway contraction and their role can be affected upon exposure to asthma-associated inflammatory mediators. Thus, NMDA-Rs are of relevance to mechanisms that determine airway narrowing and AHR associated with chronic respiratory diseases. / October 2015