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C. Guo, M. Bitzer, A.A. Hirano, N.C. Brecha; Expression of GAD67 in Horizontal Cells of a GAD67–GFP BAC Transgenic Mouse Line . Invest. Ophthalmol. Vis. Sci. 2006;47(13):394.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the expression of GABA, its rate–limiting synthetic enzymes GAD (glutamic acid decarboxylase) 65 and 67 and the vesicular GABA transporter (VGAT) in a GAD67–GFP BAC transgenic (tg) mouse line, that carries a GFP reporter under the control of the GAD67 promoter. These studies are focused on testing the hypothesis that GABA, GAD65 and GAD67 are expressed in horizontal cells of the mouse retina.
GAD67–GFP–fluorescent horizontal cells (HCs) were evaluated in vertical sections and whole mounts of developing (postnatal day 5 to 20) and adult GAD67–GFP BAC tg mice. The distribution of GAD67–GFP–containing HCs was determined in 450x450 µm2 areas in superior, temporal, inferior, and nasal retina at 2 eccentricities: 700 µm and 2 mm from the optic nerve at postnatal day (PD) 20, PD25 and PD54. The number of GFP–fluorescent HCs, relative to the total number of HCs containing calbindin–28k (CaBP), a horizontal cell marker, was determined in these regions. Immunohistochemistry with antibodies to GABA, VGAT, GAD65, GAD67, and GABA plasma membrane transporter–1 (GAT–1) and GAT–3 were used to evaluate HCs in the GAD67–GFP BAC tg and C57Bl/6 lines.
Vertical sections of the GAD67–GFP BAC tg mouse retina showed GAD67–GFP–fluorescent HCs at PD5, PD10 and in adults (PD60 and PD136), implying the presence of GABA synthesis in HCs. GAD67–GFP–fluorescent HCs were present in all retinal regions, although there was an uneven distribution of fluorescent cells in the retina. The greatest number of GAD67–GFP–fluorescent HCs was in temporal retina at 700 µm (mean: 63.4±5.3%) and the lowest number of cells was in nasal retina at 2 mm (mean: 4.2± 4.1%). The total number of CaBP–containing HCs in these regions was similar, demonstrating large differences in the number of GAD67–GFP–fluorescent HCs across the retina. GABA, GAD65 and GAD67 immunostaining in HCs was strongest at P5 and P10, and immunostaining was weak to absent at late postnatal ages and the adult retina of both the GAD67–GFP BAC tg and C57Bl/6 lines. In contrast, strong VGAT immunostaining was in the tips of horizontal cell dendrites and axons in both the developing and adult GAD67–GFP BAC tg and C57Bl/6 retinas. GAT–1 and GAT–3 immunoreactivities were absent in HCs in the developing and adult retina.
These studies provide evidence for GABA synthesis in the horizontal cells of the developing and adult mouse retina. The GAD67–GFP BAC tg line may be a good model to perform functional analysis of transmitter release from horizontal cells.
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