Mécanismes trans-synaptiques du trafic de récepteurs du glutamate in vivo – MossyGlu

The present project addresses the general question of the fine specification of properties of glutamatergic synapses which is essential for proper synaptic network function and plasticity. Our objective is to examine whether trans-synaptic mechanisms confer molecular and functional specificity to an identified glutamatergic synapse in the hippocampus, and to provide molecular insights into these mechanisms. We will examine these mechanisms in experimental conditions that approximate the in vivo situation of maturation and function of synaptic networks. The recent development of an array of methods to express mutated genes in vivo, as for instance the use of lentiviral delivery of genes, biolistic methods in slice cultures, or single cell electroporation allow for the genetic manipulation of selected brain populations. The readout will consist in the recording of synaptic currents selectively activated in CA3 pyramidal cells by stimulation of mossy fibers. In addition glutamate receptors will be mapped along the dendritic tree by glutamate uncaging using a two-photon confocal microscope coupled to electrophysiology. The project will be divided in three tasks. In task 1, we will test the hypothesis that afferent hippocampal mossy fibers may play a role in the restricted targeting and stabilization of ionotropic glutamate receptors in CA3 pyramidal cells, as well as in the specific equipment of these synapses with NMDA receptor subunits. For this we will examine the subcellular and synaptic distribution of ionotropic glutamate receptors in existing mouse models and experimental situations where mossy fibers are mis-targeted. Although this will not fully validate the hypothesis that Mfs induce the recruitment and stabilize iGluRs postsynaptically, it should provide important correlative data between the presence of Mfs and the expression of a given subset of iGluRs in CA3 pyramidal neurons. The feasibility of the experiments, the availability of the various models and the expertise of the groups in the combination of techniques give this task good chances of success. In task 2 we will examine the molecular determinants for trans-synaptic mechanisms underlying the restricted targeting and stabilization of KARs at Mf synapses. For this we have implemented a functional molecular replacement assay whereby wild-type and mutated GluR6 are re-expressed in a GluR6 ko background, followed by synaptic electrophysiology and functional imaging. This is an extension of studies performed in our lab and others on the trafficking of KARs in cell culture systems. It will however bring highly original results in two aspects. Firstly, it will provide data on structural elements of KARs in the in vivo situation, an important leap from earlier work because KAR trafficking and function appears to be very dependent on the extracellular environment. Secondly, we will determine the role of the extracellular N-terminal domain of KARs in synaptic stabilization at specific synapses, a question that has not yet been addressed. In task 3 we will explore the role of two adhesion protein complexes as trans-synaptic actors in the specification of iGluR distribution at Mf synapses. This task should capitalize on existing tools and knowledge on two adhesion molecule systems, neuroligins/neurexins, and N-cadherins. In this task we will test whether regulation or disruption or these two distinct adhesion protein complexes leads to impairment of glutamate receptor composition at Mf synapses. For these experiments, we will use conditional gene knock-out or knock-down, and selected mutant mice. Peptides known to affect N-Cadherin homophilic interactions, postsynaptic expression of dominant negative constructs, as well as molecular replacement strategies will be used. These experimental manipulations will be subjected to functional readout. Altogether this project proposes an original set of experiments to decipher the long sought mechanisms for the target specific trafficking of glutamate receptors in vivo. Technological improvements and validation of the mossy fiber-CA3 model for studies with molecular replacement open fruitful perspectives that go well beyond the present project. This will pave the way for many experiments on the molecular and cellular mechanism of trans-synaptic regulation of synaptic function and plasticity, in physiological as well as pathological situations.