April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Gabaergic Synaptic Vesicles In Guinea Pig Horizontal Cells Participate In Ca2+ Dependent Recycling
Author Affiliations & Notes
  • Helen E. Vuong
    Neurobiology, UCLA, Los Angeles, California
  • Helen L. Kornmann
    Neurobiology, UCLA, Los Angeles, California
    Jules Stein Eye Institute, Los Angeles, California
  • Salvatore L. Stella, Jr.
    Ophthalmology, University of Kansas City, Missouri, Kansas City, Missouri
  • Nicholas Brecha
    Neurobiology, UCLA, Los Angeles, California
    VAGLAHS, Los Angeles, California
  • Footnotes
    Commercial Relationships  Helen E. Vuong, None; Helen L. Kornmann, None; Salvatore L. Stella, Jr., None; Nicholas Brecha, None
  • Footnotes
    Support  VA Career Scientist Award (NB), EY 15573, JSEI EyeSTAR (HLK)
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4110. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: calcium • horizontal cells • inhibitory neurotransmitters 
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