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Grenoble Institut des Neurosciences Grenoble Institut des Neurosciences

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Immunogold labelling and correlative optical/electron microscopy to visualize synaptic modifications

Objectifs

Identiy the proteins that are localized in synaptic spinules.

Résumé

Excitatory synapses of the brain harbour dynamic cytoskeletal networks that drive structural modifications essential to long-term memory. Detailed images of these networks and their surrounding structure are necessary to understand the structural basis of synaptic plasticity. To obtain such images, Electron Microscopy (EM) remains unmatched in terms of resolution.
Here we will focus on a particular structure revealed by electron microscopy : the synaptic spinule which consists of a double-membrane invagination inside 70 % of synaptic terminals. If their morphology has been described in detail, their function remain undetermined. Therefore, we will try to identify the proteins that are localised in these structures both by immuno-gold labelling on cryosections as well as by expressing GFP proteins together with a genetically encoded tag (Apex) that could be visualised by EM after a revelation.

Méthodes

Samples will be frozen in liquid nitrogen, ultrathin sections will be done with an ultra-cryomicrotome and sections will be labelled with antibodies. Alternatively, neurons will be transfected with the GFP-protein of interest together with Apex-GBP, le neurons will be imaged by optical microscopy before embedding in epoxy resin. The same cell will be observed by EM

Références

  • Pernet-Gallay K, Jouneau PH, Bertrand A, Delaroche J, Farion R, Rémy C, Barbier EL. Vascular permeability in the RG2 glioma model can be mediated by macropinocytosis and be independent of the opening of the tight junction. J Cereb Blood Flow Metab. 2016 Jun 15.
  • Gory-Fauré S, Windscheid V, Brocard J, Montessuit S, Tsutsumi R, Denarier E, Fukata Y, Bosc C, Delaroche J, Collomb N, Fukata M, Martinou JC, Pernet-Gallay K, Andrieux A. Non-microtubular localizations of microtubule-associated protein 6 (MAP6). PLoS One. 2014 Dec 19;9(12)
  • De Rossi P, Harde E, Dupuis JP, Martin L, Chounlamountri N, Bardin M, Watrin C, Benetollo C, Pernet-Gallay K, Luhmann HJ, Honnorat J, Malleret G, Groc L, Acker-Palmer A, Salin PA, Meissirel C. A critical role for VEGF and VEGFR2 in NMDA receptor synaptic function and fear-related behavior. Mol Psychiatry. 2016 Jan 5.

Contacts

Karin Pernet Gallay, IR2 Inserm
Email : karin.pernet-gallay@univ-grenoble-alpes.fr

Mise à jour le 23 mai 2017

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