title: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses.
abstract: Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.
authors.affiliation: Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health; junzhang@ninds.nih.gov.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Advanced Imaging Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Petralia
authors.affiliation: Advanced Imaging Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
authors.forename: Ya-Xian
authors.initials: YX
authors.lastname: Wang
authors.affiliation: Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Diamond
mesh_terms: D000818:Animals; D007150:Immunohistochemistry / Q000379:methods; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000378:metabolism; D017470:Receptors, Glutamate / Q000032:analysis*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000502:physiology; D013569:Synapses / Q000378:metabolism* / Q000502:physiology
chemical_list: D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate