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Dentate microcircuitry is thought to be involved in filtering, integrating, and relaying extrinsic hippocampal inputs to the hippocampus proper, which contributes to memory formation and retrieval. The axons of granule cells are called mossy fibres (MFs), and contain multiple terminal types that form characteristic synaptic connections with their postsynaptic targets. This diversity of presynaptic release sites that exists on the same MF provides an extremely interesting axonal type to study the organizing principles of presynaptic release regulation. A remarkable set of neurotransmitters and receptors present at the MF synaptic complex allow diverse computational modification of information from the dentate gyrus to the hippocampus. There are several types of glutamate receptors expressed at MF, such as group II/III mGluRs and kainate receptors (KARs). Presynaptic KARs modulate transmission at MF-CA3 pyramidal cell synapses; however, it is not known whether presynaptic KARs affect other synapses made by MFs. The aim of the first part of this thesis was to establish the principles of synapse-specific actions of presynaptic KARs in MFs. Combining electrophysiology and calcium imaging, this study provides compelling evidence that presynaptic KARs and Ca²⁺ stores can be activated by glutamate release from a single action potential in a single MF axon. This contributes to short-term, use-dependent facilitation of presynaptic Ca²⁺ entry and glutamate release exclusively at MF-CA3 pyramidal cell synaps, but not at other MF synapses, on hilar mossy cells or interneurons. Thus, our findings indicate that the presynaptic KARs, coupled with intracellular stores, exist in a synapse-specific autoreceptor mechanism. Activation of KARs strengthened MF-CA3 pyramidal cell synapses by increasing the Ca²⁺ influx at giant boutons, which might also contribute to the KAR-dependent hyper-excitability of the MF circuitry related to the mechanisms of temporal lobe epilepsy (TLE). This makes KARs good potential targets for therapies in CNS disorders such as epilepsy and other neurological and psychiatric disorders. The second part of this thesis was to explore the actions on the hippocampus of purified antibodies from a limbic encephalitis (LE) patient. LE is a CNS disease characterized by subacute onset of memory loss and temporal lobe seizures. The serum of these patients strongly labels MFs apparently co-localizing with the VGKC. The patients improve with immunotherapies that reduce the VGKC antibody levels in the serum, thus, strongly suggesting that these antibodies cause the condition. We found that LE serum IgGs enhance CA3 pyramidal cell excitability by blocking α-DTX sensitive VGKCs, which results in the increased release of glutamate. This, in turn, strengthens and desynchronizes MF and CA3 pyramidal cells synaptic transmission. However, these effects were occluded by α-DTX, a Kv1.1, Kv1.2 and Kv1.6 antagonist which, when applied alone, mimicked the action of the LE IgG, suggesting that they may share similar mechanisms of action. In contrast serum taken from healthy control patients had no significant effect under same recording conditions. Thus, this study provides the first evidence that the LE IgG functionally affects VGKC containing Kv1.1, Kv1.2 and/or Kv1.6 at both presynaptic MF axon terminals as well as the postsynaptic somatodendritic domain of CA3 pyramidal cells. Whatever defines the exact nature of LE IgG action, our results suggest that drugs acting specifically as openers of VGKC might help to protect the hippocampus from immune-mediated damage. In conclusion my data is consistent with the increasingly documented idea that MFs play a critical role in regulating the excitability of the hippocampal circuits and the dysfunction of MF transmission profoundly impairs hippocampal function.

Les synapses se forment selon plusieurs étapes comprenant la stabilisation des contacts nouvellement formés et leur maturation. Ces différentes étapes dépendent d’une mise en place coordonnée entre la terminaison pré- et postsynaptique. Les protéines composant la présynapse et les récepteurs ionotropiques du glutamate ont des rôles clés dans ces processus. Lors de ma thèse, je me suis intéressé à l’implication de la protéine présynaptique Bassoon lors de la maturation des synapses glutamatergiques entre les fibres moussues et les cellules pyramidales de CA3 dans l’hippocampe. Cette synapse constitue un modèle attractif pour l’étude de la maturation synaptique car elle suit des étapes de maturation morphologique et fonctionnelle bien définies. Bassoon est une des premières protéines se mettant en place au niveau des contacts synaptiques nouvellement formés. Par des approches électrophysiologiques, nous avons montré que la protéine Bassoon était importante pour l’organisation du site de libération de neurotransmetteur durant les deux premières semaines de vie post-natale chez la souris.Les récepteurs kaïnate jouent un rôle important dans la régulation de l’activité de réseau au cours du développement post-natal. Cependant l’impact de l’activation de ces récepteurs sur la maturation synaptique est peu connu. J’ai pu mettre en évidence un délai dans la maturation fonctionnelle de la synapse fibre moussue/cellule pyramidale de CA3 chez les souris déficientes pour la sous-unité GluK2 des récepteurs kaïnate (GluK2-/-). Afin de comprendre si ce délai de maturation fonctionnelle est corrélé à un retard dans la maturation morphologique de cette synapse, nous avons mis en place des infections de lentivirus codant pour une protéine membranaire fluorescente (YFP) chez le souriceau nouveau-né (P1-P2). A l’aide de microscopie confocale et de reconstruction en 3D, nous avons ainsi pu décrire la maturation morphologique de la synapse fibre moussue/cellule pyramidale de CA3. Cela m’a également permis de corréler la maturation fonctionnelle à la maturation morphologique et mes résultats montrent également un retard dans la mise en place des synapses chez les souris GluK2-/-. L’ensemble de cette étude révèle l’importance de l’activité synaptique et de la coordination entre mise en place de la pré- et de la postsynapse au cours de la maturation synaptique. / The formation of synapses follows different steps including synaptogenesis and maturation. These different steps depend on coordinated pre- and post-synaptic assembly. Pre-synaptic proteins and ionotropic glutamate receptors play a central role in these processes. During my thesis, I have been interested in the implication of the presynaptic protein Bassoon in the maturation of the hippocampal mossy fiber to CA3 pyramidal cell glutamatergic synapses. This synapse constitutes an attractive model for the study of synaptic maturation because it follows several steps of defined morphological and functional maturation. Bassoon in one of the first protein present at newly formed synaptic contacts. By electrophysiological approaches, we showed that Bassoon is important for the organization of the active zone during the first two postnatal weeks.Kainate receptors play an important role in the regulation of network activity during postnatal development. However, the impact of kainate receptors activation on synaptic maturation is less known. I showed a delay in functional maturation of mossy fiber synapses in mice deficient for the GluK2 subunit of kainate receptors (GluK2-/-). To know if this delay is correlated to morphological alterations of this synapse, we setup in vivo lentiviral infections of membrane fluorescent protein (YFP) in mouse pups (P1-P2). Using confocal microscopy and 3D reconstruction, we described the morphological maturation of mossy fiber synapses. We were able to correlate functional and morphological maturation and our results also showed an impairment in the formation of mossy fiber synapses in GluK2-/-. Together, these data reveal the importance of synaptic activity and of the coordination of pre- and post-synaptic assembly during synaptic maturation.

Les récepteurs ionotropiques du glutamate peuvent être compartimentés de manière très spécifique au niveau des différentes afférences synaptiques d’un neurone. Dans les neurones pyramidaux de CA3, les récepteurs de type kaïnate (rKA) post-synaptiques sont localisés à la synapse formée entre les fibres moussues et les cellules pyramidales de CA3 (synapse FM-CA3) mais ils sont totalement absents des autres afférences glutamatergiques sur ce même neurone. Nous avons cherché à comprendre les mécanismes moléculaires de cette compartimentation subcellulaire. En réalisant une cartographie fonctionnelle des récepteurs du glutamate par décageage focalisé de glutamate dans les cellules pyramidales de CA3, nous avons montré que les rKA présentent une localisation subcellulaire strictement confinée dans les excroissances épineuses, éléments post-synaptiques des synapses FM-CA3, et sont exclus des compartiments somato-dendritiques, contrairement aux récepteurs AMPA. Nous avons identifié une séquence du domaine C-terminal de GluK2a nécessaire pour la stabilisation des rKA. Cette séquence est responsable d’une interaction avec la protéine d’adhérence N-cadhérine. L’altération de la fonction de la N-cadhérine dans les cellules pyramidales de CA3 entraine une déstabilisation des rKA à la synapse FM-CA3. Ces travaux suggèrent que plusieurs mécanismes participent à la compartimentation des rKA à la synapse FMCA3 impliquant le recrutement et la stabilisation des rKA par les N-cadhérines. / Distinct subtypes of ionotropic glutamate receptors can be segregate to specific synaptic inputs in a given neuron. In CA3 pyramidal cells (PCs), kainate receptors (KARs) are present at mossy fiber (mf) synapses and absent from other glutamatergic inputs. The mechanisms for such a constrained subcellular segregation is not known. We have investigated the molecular determinants responsible for the subcellular segregation of KARs at mf-CA3 synapses. Using functional mapping of glutamate receptors by focal glutamate uncaging we show that KARs display a strictly confined expression on thorny excrescences, the postsynaptic elements of mf-CA3 synapses, being excluded from extrasynaptic somatodendritic compartments, at variance with AMPA receptors. We have identified a sequence in the GluK2a C-terminal domain necessary for restricted expression of KARs which is responsible for GluK2a interaction with N-Cadherin. Targeted deletion of N-Cadherin or overexpression of a dominant negative N-Cadherin in CA3 PCs greatly induce a destabilization of KARs at the mf-CA3 synapses. Our findings suggest that multiple mechanisms combine to control the compartmentalization of KARs at mf-CA3 synapses, including a stringent control of the amount of GluK2 subunit in CA3 PCs, a limited number of slots for KARs, and the recruitment/stabilization of KARs by N-Cadherins.