Localizing GABA Receptors and Glutamate Transporters Using Conjugated Quantum Dots in Rat Cerebellum Slices
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Localizing GABA receptors and glutamate transporters using conjugated quantum dots in rat cerebellum slices 1 1,3 Abdel I. El Abed , Anne Baudot , Mireille Chat2 , Sanaa Ben Khalifa1 and Gérard Louis1. 1 Laboratoire de Neuro-Physique Cellulaire (LNPC), Université Paris Descartes, 45 rue des Saints-Pères, 75006 Paris, France. 2 Laboratoire de Physique Cérébrale (LPC), CNRS UMR8118, Université Paris Descartes, 45 rue des Saints-Pères, 75006 Paris, France. 3 Département MCBT, Institut Néel (CNRS-UJF), BP166, 38042 Grenoble, France. ABSTRACT GABA and glutamate are known as the principal inhibitory and excitatory neurotransmitters in the vertebrate central nervous system, respectively. However, recent electro-physiological and immunogold data reported by Stell et al. [1] indicate that GABA may undergo also an excitatory action on presynaptic varicosities of parallel fibers (PFs) in the molecular layer of the rat cerebellum. PFs are axonal extensions, with a cross section of about 0.1 µm, of the glutamatergic granule cells. Such an unexpected excitatory action of GABA indicates clearly the presence of GABA receptors in the PFs of granule cells. We show in this study that quantum dots may be used specifically and efficiently to label two endogenous synaptic proteins, namely R-GABAAα1 receptors (GABAA Rs) and glutamate transporters (VGLUT1) in order to target their localization in very small structures such as the presynaptic varicosities of the PFs, in agreement with the results recently reported by Stell et al.. INTRODUCTION Thanks to their unique optical properties, quantum dots (qdots) opened a new area in cellular imaging and single molecule detections [2-8]. For example, in the field of neuroscience, Dahan et al. [7] used conjugated qdots to track individual glycine receptors and analyzed their lateral dynamics in the synaptic region of living cultured neurons during periods of time ranging from milliseconds to few minutes. However, because of the ability of neurons to establish complex and wide networks in the central nervous system (CNS), results obtained generally from isolated cultured neurons do not meet very much attention among the neurobiologists community, for whom brain slice, cut either from living or fixed tissues, represent a more suitable material model. However, the relative big size of functionalized qdots by regards to organic fluorescents dyes size, i. e. 10 nm vs. 1 nm, the heterogeneous structure of tissue slices and their autofluorescence require different fixation and permeabilization conditions for optimum immunostaining of slices. Up to date there have been few reports concerning the immunostaining of brain slices with conjugated qdots. One may cite a report made by Giepmans et al. [9] where it has been shown that conjugated qdots can be used efficiently to identify and to localize different proteins in mouse cerebellum slices, such as IPR3 (inositol 1,4,5-triphosphate receptor) which is localized predominantly on the endoplasmic reticulum membrane and GFAP (glial fibrillary acidi
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