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H. Lee, N. C. Brecha; Evidence of Vesicular Exocytosis in Guinea Pig Retinal Horizontal Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1037.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor terminals, horizontal cell (HC) endings, and bipolar cells comprise a synaptic triad in the retina, and their interactions mark the first step in processing visual information in the outer plexiform layer of the of the retina. HCs integrate input from photoreceptors over a large retinal area via feedforward (bipolar cells) and feedback (cones) communication, thereby contributing to surround inhibition. However, the cellular mechanisms that underlie the transfer of information in mammalian HCs are poorly understood. Unlike the GABA plasma membrane transport mechanism described for non-mammalian retinas, the occurrence of clear core, spherical vesicles and the vesicular GABA transporter (VGAT) in mammalian HCs supports a vesicular transmitter release mechanism. In this study, we examine the localization of key synaptic proteins in guinea pig retina, and focus on the detection of the v-SNARE, vesicle associated membrane protein (VAMPs).
Frozen section of guinea pig retina were used for immunohistochemistry and double labeled with antibodies to calbindin, a horizontal cell marker, and the SNARE complex proteins SNAP-25, syntaxin-4, and VAMP1. Sections were also double-labeled for calbindin and synaptic vesicle associated proteins and imaged with confocal microscopy. For the identification of VAMP1 protein in guinea pig retina, the whole retina was extracted and PCR identified the presence of VAMP1 RNA using degenerate primers.
Syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, strongly labeled horizontal cells processes in a punctuate fashion, and was coexpressed with the vesicular GABA transporter, a marker of horizontal cells located at the tips of HC processes. SNAP-25, another SNARE protein that contributes two alpha-helices in the formation of the exocytotic fusion complex, labeled all parts of the horizontal cell, including the soma, processes, and tips. Synaptic associated proteins were also found in HCs. Complexin I/II labeled localized to the HC soma and processes, and two markers of conventional synapses, SV2A and synapsin I, labeled HC tips in a stippled manner. The vesicle associated membrane protein, VAMP1 isoform, was found in the outer plexiform layer, but light microscopy could not resolve the location of these proteins to the HC tips or photoreceptor terminals. PCR using degenerate primers for VAMP1 have revealed the presence of this protein in whole retina.
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