Les récepteurs N-méthyl-D-aspartate (NMDA) sont des récepteurs ionotropiques du glutamate jouant un rôle clé dans la plasticité synaptique et les fonctions cognitives. En conséquence, la perturbation de leur activité est impliquée dans de nombreux troubles neurologiques et psychiatriques tels que l'épilepsie et la schizophrénie. La particularité de ces récepteurs est qu'ils nécessitent pour être activés la liaison simultanée de leur agoniste, le glutamate, et d'un co-agoniste. La glycine fut le premier co-agoniste identifié mais plus récemment, de nombreuses études ont révélé que la D-sérine joue également ce rôle dans de nombreuses régions cérébrales, notamment dans l'hippocampe. Toutefois il restait à définir si les fonctions de ces deux co-agonistes étaient régulées au cours du développement ou si elles étaient spécifiques à certaines synapses. Dans la première partie de mon travail, j'ai montré que la D-sérine est le co-agoniste préférentiel des synapses SC-CA1 matures alors que la glycine est le co-agoniste préférentiel des synapses mPP-DG. De plus, le remplacement des récepteurs NMDA composés de sous-unités GluN2B par des récepteurs contenant GluN2A au cours du développement post-natal survient au même moment qu'un changement dans l'identité du co-agoniste préférentiel des synapses SC-CA1. Dans la seconde partie de mon travail, je me suis intéressée à la contribution de la D-sérine en conditions pathologiques sur un modèle murin d'épilepsie chimio-induite par la pilocarpine. J'ai ainsi montré l'implication de la D-sérine dans l'activité épileptique initiée par la pilocarpine. / N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ionotropic receptors which play a crucial role in synaptic plasticity and cognitive functions. As a consequence, disturbance in their activity is correlated with a broad range of neurological and psychiatric disorders including epilepsy and schizophrenia. The major particularity of NMDA receptors is the requirement of simultaneous binding of their agonist, glutamate, and a co-agonist to be activated. Glycine was the first co-agonist identified but more recently several studies showed that D-serine is also playing this role in many brain areas including the hippocampus. Whether the identity of the co-agonist is synapse specific or developmentally regulated remains unexplored. In the first part of my work I showed that D-serine is the preferred co-agonist at SC-CA1 mature synapses while glycine is the preferred one at mPP-DG synapse. Moreover, we showed that during postnatal development, the replacement of GluN2B by GluN2A-containing NMDA receptors at SC-CA1 synapses parallels a change in the co-agonist identity from glycine to D-serine. In the second part of my work I investigated the contribution of D-serine in pathological conditions. By using a model of acute intoxication of pilocarpine, I demonstrated that D-serine is implicated in epileptiform activity initiated after pilocarpine perfusion.

Les synapses se forment selon plusieurs étapes comprenant la synaptogenèse, la maturation et la plasticité synaptique. Les molécules d’adhésion et les récepteurs ionotropiques du glutamate ont des rôles clés dans ces processus. Lors de ma thèse, je me suis intéressée à la dynamique des interactions impliquant deux protéines membranaires, la N-cadhérine et le récepteur NMDA. La N-cadhérine joue un rôle important dans l’induction de la croissance axonale mais les mécanismes moléculaires sous-jacents sont peu connus. Par des approches de vidéo-microscopie et de pinces optiques, j’ai démontré que la transmission directe des forces générées par le flux rétrograde d’actine aux adhésions N-cadhérines, via les caténines, induit l’avancée du cône de croissance. Les récepteurs NMDA synaptiques ont un rôle crucial dans la maturation et la plasticité synaptique, néanmoins, les mécanismes moléculaires régulant la distribution des récepteurs NMDA synaptiques sont peu connus. En combinant le développement de peptides compétiteurs divalents et des approches d’imagerie haute résolution, nous avons étudié la dynamique de surface des récepteurs NMDA endogènes. Mes résultats montrent que l’interaction dynamique entre les protéines d’échafaudage à domaine PDZ et les récepteurs NMDA contenant la sous-unité NR2A régule leur rétention synaptique et leur distribution de surface. Le déplacement des récepteurs NMDA contenant la sous-unité NR2A en dehors des synapses est compensé par une insertion synaptique de récepteurs contenant la sous-unité NR2B, indiquant que l’ancrage synaptique des différents sous-types de récepteurs NMDA est différentiellement régulé. De plus, cette redistribution des récepteurs NMDA affecte la maturation et la plasticité synaptique. L’ensemble de ces résultats révèle une régulation rapide et spécifique des récepteurs NMDA synaptiques par les protéines à domaine PDZ suggérant un rôle de l’organisation de la densité postsynaptique dans la stabilisation synaptique des récepteurs et les processus adaptatifs. / The formation of synapses follows different steps including synaptogenesis, maturation and plasticity. Adhesion molecules and ionotropic receptors play key roles in these processes. During my thesis, I have been interested in the dynamics of the interactions mediated by two membrane proteins, N-cadherin and the NMDA receptor N-cadherin plays important roles in axon outgrowth, but the molecular mechanisms underlying this effect are mostly unknown. Using live imaging and optical trapping, I demonstrated that the direct transmission of actin-based traction forces to N-cadherin adhesions, through catenin partners, drives growth cone advance. Synaptic NMDA receptors (NMDARs) play key roles during synaptic refinement and plasticity, however the molecular mechanisms that govern the distribution of the synaptic surface NMDARs are largely unknown. We investigated the dynamics of endogenous NMDARs using high-resolution single particle imaging and a newly-developed biomimetic divalent competing ligand. My results show that the dynamic interaction between PDZ domain-containing scaffold proteins and NR2A-NMDARs regulates their synaptic retention and surface distribution. Interestingly, a rapid displacement of NR2A-NMDARs out of synapses is paralleled by a compensatory increase in NR2B-NMDARs, providing functional evidence that the sites of synaptic anchoring of native surface NR2-NMDARs are different. Furthermore, such redistribution of surface NR2-NMDARs strongly impairs synaptic maturation and plasticity. Together, these data reveal a rapid and specific regulation of surface NR2-NMDARs by PDZ domain-containing scaffolds in synapses, supporting a role of the postsynaptic density architecture in regulating specific NR2-NMDAR retention and synaptic adaptation.

N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...

Im Rahmen dieser Arbeit wurde das Motilitätsverhalten von Blut-Hirn Schranken-Endothelzellen unter ischämischen Bedingungen an Hand der cerebEND-Zelllinie untersucht. Da es bisher noch kein Modell für diese Fragestellung gab, wurde zunächst ein solches mit Hilfe des kommerziellen Motilitätsassay der Firma ibidi® etabliert. Danach konnte der Einfluss von ischämischen Bedingungen, von Astrozyten konditioniertem Medium (C6-Zelllinie) und letztendlich der therapeutische Ansatz durch Modulation des NMDA-Rezeptors untersucht werden. Dabei zeigte sich durch das C6-konditionierte Medium eine deutliche Zunahme der Motilität. Diese verstärkte Motilität konnte durch den NMDA-Rezeptor-Antagonisten MK801 verhindert werden. Trotz Analyse einiger an der Proliferation und Migration beteiligter Botenstoffe wie VEGF und MMPs konnte keine Regulation dieser durch MK801 nachgewiesen werden. / In this work, the motility behavior of blood-brain barrier endothelial cells under ischemic conditions was investigated using the cerebEND cell line. As there was no model for this question available until now, a model was first established using the commercial motility assay of the company ibidi®. Subsequently, the influence of ischemic conditions, astrocyte conditioned medium (C6-cell line) and finally the therapeutic approach by modulation of the NMDA receptor could be investigated. The C6-conditioned medium showed a significant increase in motility. This increased motility could be prevented by the NMDA receptor antagonist MK801. Despite analysis of some messenger substances involved in the proliferation and migration such as VEGF and MMPs, no regulation of these substances by MK801 could be detected.

No description available.

The neurotransmitter dopamine (DA) represents a neural substrate for positive motivation as its spatiotemporal distribution across the brain is responsible for goaldirected behavior and learning reward associations. The critical determinant of DA release throughout the brain is the firing pattern of DA-producing neurons. Synchronized bursts of spikes can be triggered by sensory stimuli in these neurons, evoking phasic release of DA in target brain areas to drive reward-based reinforcement learning and behavior. These bursts are generated by NMDA-type glutamate receptors (NMDARs). This dissertation reports a novel form of long-term potentiation (LTP) of NMDARmediated excitatory transmission at DA neurons as a putative cellular substrate for changes in DA neuron firing during reward learning. Patch-clamp electrophysiological recording from DA neurons in acute brain slices from young adult rats demonstrated that synaptic NMDARs exhibit LTP in an associative manner, requiring coordinated pre- and postsynaptic burst firing. Ca2+ signals produced by postsynaptic burst firing needed to be amplified by preceding metabotropic neurotransmitter inputs to effectively drive plasticity. Activation of NMDARs themselves was also necessary. These two coincidence detectors governed the timingdependence of NMDAR plasticity in a manner analogous to the timing rule for cuereward learning paradigms in behaving animals. Further mechanistic study revealed that PKA, but not PKC, activity gated LTP induction by regulating the magnitude of Ca2+ signal amplification via the inositol 1,4,5-triphospate (IP3) receptor and release of Ca2+ from intracellular stores. Plasticity of NMDARs was input specific and appeared to be expressed postsynaptically, but was not associated with a change in NMDAR subunit stoichiometry. LTP of NDMARs was DA-independent, and was specific for NMDARs: the same induction protocol produced long-term depression of AMPA receptors. NMDARs that had undergone LTP could be depotentiated in a spike-conditional manner, consistent with active unlearning. Finally, repeated, in vivo amphetamine experience dramatically increased facilitation of spike-evoked Ca2+ signals, which in turn drove enhanced plasticity. NMDAR plasticity thus represents a potential neural substrate for conditioned DA neuron burst responses to environmental stimuli acquired during reward-based learning as well a novel therapeutic target for intervention-based therapy of addictive disorders. / text

<p> </p><p>In previous studies the carboxyl-terminus of microtubule-associated protein 1S was shown to interact with the <em>N</em>-methyl-D-aspartate receptor subunit NR3A (Eriksson <em>et. al.</em>)¹.  In this study, interactions between three truncations of the microtubule-associated proteins 1B and one truncation of the microtubule-associated protein 1S carboxyl-terminus and the <em>N</em>-methyl-D-aspartate receptor subunits NR1 and NR3A were examined. The study showed that an interaction occurred between amino acids 2167 to 2365 of the microtubule-associated protein 1B and NR3A.  That region of microtubule associated protein 1B corresponds to a microtubule-binding region in the light chain. It has been shown in earlier studies (Reviewed in Halpain S. <em>et a1²</em>, Riederer, BM<em>.  et.al³</em>.) that the light chain is a active part of the protein that have been post translational cleaved. The MAP 1 proteins are present in all tissue but has higher concentrations in the Post Synaptic Density of neurons in the central nervous system.  The <em>N</em>-methyl-D-aspartate receptors are present in glial cells and in the dendritic shafts of the central nervous system neurons (Eriksson <em>et. al.</em>)¹. The diseases were these proteins may play a part is mainly memory destructive diseases such as Alzheimers disease and in muscular dystrophy, but these assumptions are still being speculated.</p><p> </p>

Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.

Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of disease process. Our exploratory studies showed that oligodendrocytes (OLs) are direct targets of HIV-1 Tat (transactivator of transcription). Tat induces a dose-dependent increase of intracellular Ca2+ level ([Ca2+]i) in cultured murine OLs, which can be attenuated by ionotropic glutamate receptor (iGluR) antagonists MK801 and CNQX. The Tat-induced [Ca2+]i increase leads to increased death in immature (O4+, MBP-), but not mature (O4+, MBP+) OLs, over 96 h. In addition, Tat-induced [Ca2+]i increase also reduced myelin-like membrane production by mature OLs. Calcium/Calmodulin dependent kinase IIβ (CaMKIIβ) and glycogen synthase kinase 3β (GSK3β) have been known to regulate differentiation, myelination, and apoptosis in OLs. Since both CaMKIIβ and GSK3β are important downstream modulators of [Ca2+]i change, we hypothesized that the detrimental effects of Tat on immature/mature OL viability and function are mediated via CaMKIIβ and GSK3β activation. Our results showed that Tat activates both CaMKIIβ and GSK3β in immature OLs, but only activates CaMKIIβ in mature OLs. MK801 completely blocks Tat-induced CaMKIIβ and GSK3β activation in both immature and mature OLs, while CNQX blocks GSK3β activation, but has only a partial effect on CaMKIIβ activity. Blocking iGluRs or inhibiting GSK3β both rescue Tat-induced immature OL death, but only MK801 reverses the membrane injury in mature OLs. Together, these data strongly suggest that 1) activity of CaMKIIβ and GSK3β in OLs can be regulated by Tat-induced iGluRs activation and 2) OLs at different developmental stages show different responses to Tat, possibly due to activation of different signaling pathways.

La reelin est une glycoprotéine sécrétée de la matrice extracellulaire essentielle pour le développement embryonnaire des structures laminaires du système nerveux central (SNC): cortex, hippocampe et cervelet. Dans le cerveau postnatal et adulte, la reelin potentialise la plasticité synaptique, exerce une action trophique sur la croissance neuritique dans l’hippocampe et contrôle la maturation des récepteurs NMDA. Le but de ma thèse a été d’étudier les mécanismes cellulaires à l’origine des fonctions de la reelin dans la plasticité postnatale des structures stratifiées du SNC. Nous avons utilisé une stratégie intégrant des approches d’électrophysiologie, d’imagerie calcique, d’immunocytochimie, de biochimie et de pharmacologie, sur des modèles in vitro (culture primaires de neurones d’hippocampe et de cervelet) et ex vivo (tranches aigues de cortex frontal). Dans les neurones d’hippocampe in vitro, nous avons mis en évidence que la reelin est synthétisée et sécrétée par des neurones GABAergiques montrant un marquage reelin intense alors que les neurones cibles de la reelin sont caractérisés par une expression ponctiforme et de faible intensité. En revanche, dans le cervelet in vitro, les 2 fonctions, sécrétion et liaison de la reelin, sont assurées par la quasi totalité des cellules granulaires glutamatergiques. Nous avons finalement examiné les conséquences physiologiques de l’absence ou de la diminution de reelin endogène dans l’hippocampe et dans le cortex frontal. Nous avons mis en évidence que dans l’hippocampe in vitro la sécrétion continue de reelin régule l’homéostasie des récepteurs NMDA. Nous montrons également que dans le cortex frontal ex vivo, la reelin facilite la maturation des fonctions synaptiques glutamatergiques. Nos résultats démontrent donc que la reelin joue un rôle majeur dans la plasticité neuronale du SNC postnatal. / Reelin is an extracellular matrix protein essential for the correct formation of laminated structures during embryonic brain development. In the postnatal and adult brain, reelin promotes hippocampal dendrite development, enhances long term potentiation (LTP) at hippocampal synapses and favors the maturation of glutamatergic transmission. During my thesis, I studied the cellular mechanisms underlying the functions of reelin in laminated structures of the postnatal central nervous system: hippocampus, cerebellum and cortex. By combining immunocytochemical, biochemical and pharmacological approches, we first characterized the expression profile of reelin in primary cultures of hippocampal and cerebellar neurons. Our results showed that in the hippocampus reelin is synthesized and secreted by a population of GABAergic neurons expressing an intense reelin immunoreactivity (IR). We also showed that secreted reelin binds lipoprotein receptors present on a different neuronal population characterized by a punctate and light reelin IR. In contrast, in cerebellar cultures, we observed that reelin is synthesized and secreted by glutamatergic cells expressing a single type of reelin punctate and light staining. Using calcium imaging, we demonstrated that the continuous secretion of reelin is necessary to regulate glutamate receptor homeostasis and maintain the subunit composition of NMDARs in the hippocampus in vitro. We next examined the effect of decreased levels of reelin in the postnatal development of prefrontal cortex (PFC) glutamatergic synapses using electrophysiology on heterozygotes reeler mice (HRM) slices. Our data revealed that reelin facilitates the maturation of glutamatergic synaptic functions in the PFC and plays a central role in neuronal plasticity in the central nervous system.