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E.–J. Lee, T. Gibo, N. Grzywacz; Dark–Rearing–Induced Falls of GAD65 and GABA and Reversion by BDNF in the Mouse Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):164.
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© ARVO (1962-2015); The Authors (2016-present)
Gamma–aminobutyric acid (GABA) has been reported to be an important neurotransmitter in the retina of many species. We studied the expression of GABA and GAD65, by immunocytochemistry and western blot, in the retinas of the control and dark–reared C57BL/6J black mouse. This study asked three questions: First, is visual input necessary for the normal expression of GABA and GAD65? Second, can the retina recover from the effects of dark rearing? Third, does BDNF reverse the influence of dark rearing on the expression of GABA and GAD65?
Fourteen litters of C57BL/6J black mice were used for control and dark–rearing studies at developmental stages (postnatal (P) days 7, 10, 14, 15, 21, and 30). Control animals were maintained on a daily 12–hour light/dark cycle. In turn, dark–reared animals were raised under completely dark conditions from P0. Retinas were immunostained with rabbit polyclonal antisera against GABA (1:3000; sigma), GAD65 (1:1000, Chemicon); mouse monoclonal antiserum against F480 (1:50; Serotec); Biotinylated isolectin B4 (1:25; Vector laboratories). For double labeling, we used GABA and goat polyclonal antiserum against ChAT (1:300; Chemicon). We injected 2 µg of human recombinant BDNF in 2µl of sterile saline at the level of the temporal peripheral of the P14 dark–reared retina. Sham injections, for the control, consisted of 2 µl sterile saline.
At postnatal Day 10 (P10), before eye opening, GABA and GAD65 immunoreactivities were present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL), and throughout the inner plexiform layer (IPL) of control and dark–reared retinas. In control P30, GABA and GAD65 immunoreactivities showed similar patterns as those in P10. However, in dark–reared P30, GABA immunoreactive cells had lower density in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis also showed corresponding differences in the levels of expression of GAD65 between control and dark–rearing conditions. In our study, dark–rearing effects were reversed upon returning the mice to cyclic light/dark conditions for 2 weeks. Moreover, dark–reared retinas treated with BDNF showed intact expression of both GABA and GAD65.
Our data indicates that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggests that BDNF controls this dependence.
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