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C. Guo, A. A. Hirano, S. L. Stella, Jr., M. Bitzer, N. C. Brecha; Expression of Gaba, Gad65 and Vgat in Guinea Pig Horizontal Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5795.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the expression of GABA, its rate-limiting synthetic enzymes GAD (glutamate acid decarboxylase) 65 and 67, the vesicular GABA transporter (VGAT), and GABA plasma membrane transporter-1 (GAT-1) and GAT-3 in guinea pig outer retina. These studies are focused on testing the hypothesis that GABA, GAD and VGAT are expressed in horizontal cells of the guinea pig retina.
Immunohistochemistry with antibodies to GABA, GAD65, GAD67, VGAT, GAT-1 and GAT-3 were used to evaluate adult guinea pig retina in vertical sections and after acute dissociation. Retinal whole-mounts were evaluated with antibodies to GAD65 and the horizontal cell marker calbindin. Horizontal cells were collected using laser capture microdissection (LCM). Total RNA was extracted from LCM microdissected horizontal cells using a RNA Nanoprep kit (Stratagene, CA). Degenerate RT-PCR was performed to detect GAD65 mRNA in horizontal cells.
Horizontal cells contained GABA, GAD65 and VGAT immunoreactivity, and they did not contain GAD67, GAT-1 and GAT-3 immunoreactivity, indicating the presence of GABA synthesis in horizontal cells. GABA, GAD65 and VGAT immunoreactivity was distributed to all horizontal cell bodies, as well as their processes and tips. GAD65, and GAD67 immunostaining in enzymatically dissociated horizontal cells and in retinal whole-mounts showed that GAD65 immunoreactivity was present in both A-type and B-type horizontal cells. Degenerate RT-PCR using primers specific to GAD65 detected the presence of GAD65 mRNA in horizontal cells. Sequencing of the amplified GAD65 fragment (126 bp) showed an 87% identity of the GAD65 mRNAs between guinea pig and mouse horizontal cells.
These studies provide evidence for GABA synthesis in horizontal cells of the guinea pig retina and support the idea that vesicular transmitter release machinery is present in the mammalian horizontal cells.
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