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Helen E. Vuong, Helen L. Kornmann, Salvatore L. Stella, Jr., Nicholas Brecha; Gabaergic Synaptic Vesicles In Guinea Pig Horizontal Cells Participate In Ca2+ Dependent Recycling. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4110.
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© ARVO (1962-2015); The Authors (2016-present)
Mammalian horizontal cells (HC) contain the presynaptic molecular components necessary to synthesize, package and release GABA via synaptic vesicles. The goal of this study is to determine if GABA-containing vesicles undergo depolarization- and Ca2+-dependent recycling in horizontal cells by determining the presence of VGAT, the transporter responsible for packaging GABA into synaptic vesicles.
Guinea pig eyes were processed for immunohistochemistry or living retinal slices. Slices were incubated in a solution containing anti-VGAT-N or anti-VGAT-C conjugated to Oyster-550 in 60 mM K+ at 37° C for 10 minutes. Antibody uptake was evaluated using confocal microscopy. The Ca2+ dependence of anti-VGAT-C uptake was evaluated by incubating slices in 0, 2 and 10 mM Ca2+, and the voltage gated Ca2+ channel antagonists, Cd2+ (200 µM), nifedipine (Nif, 10 µM) and ω-conotoxin (CTX, 1 µM). Endocytotic activity was tested using sequential incubations of anti-VGAT-C Oyster-550 followed by anti-rabbit IgG conjugated to Alexa 488.
Consistent with previous studies, anti-VGAT-C and anti-VGAT-N were colocalized with calbindin, a marker for horizontal cells. In slices, anti-VGAT-C, which targets the luminal C-terminus of the synaptic vesicle VGAT protein, heavily labeled the IPL and OPL only in the presence of 60 mM K+, whereas, anti-VGAT-N, directed against the N-terminus of VGAT located on the cytosolic surface of synaptic vesicles, did not. Anti-VGAT-C labeling of horizontal cell processes in the OPL was produced by stimulation with high K+ in 2 and 10 mM Ca2+, but not 0 mM Ca2+. In the presence of CTX, Nif, and Cd2+, there was a ~79%, ~85%, and ~90% reduction of anti-VGAT-C fluorescence in the OPL, respectively (n=3, each drug). Finally, sequential high K+ depolarization of living slices with anti-VGAT-C Oyster 550 followed by a second depolarization with anti-rabbit IgG-Alexa 488 ab resulted in co-localization of fluorescence throughout the OPL and IPL.
VGAT immunoreactive synaptic vesicles are present in guinea pig HC processes. HC synaptic vesicle fusion is depolarization- and Ca2+-dependent, and mediated by N- and L-type Ca2+ channels. Finally, sequential labeling of HC processes with anti-VGAT-C/anti-IgG suggests that a vesicular recycling mechanism mediates vesicle fusion and uptake in horizontal cells.
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