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S. L. Stella, Jr., M. E. Rome, A. Vila, N. C. Brecha; YFP as a Novel Biosensor for Imaging GABA-Induced Chloride Gradients in Cone Bipolar Cells of the Mammalian Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4588.
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© ARVO (1962-2015); The Authors (2016-present)
Little is known about GABA’s action at cone bipolar cells, and the ability of GABA to regulate their excitability through intracellular chloride ion (Cl-) changes. Ionotropic GABA receptor expression is well documented in rod bipolar cells, but the spatial distribution and pharmacological profile is not known in cone bipolar cells. The goal of this project was to characterize GABA-mediated [Cl-]i changes in cone bipolar cells of the mouse retina using yellow fluorescent protein (YFP) as a Cl- biosensor.
Mice expressing YFP under the Thy-1.2 promoter were used for this study. YFP fluorescence is sensitive to quenching by many anions, of which Cl- is the most physiologically relevant. This provides a way to monitor [Cl-]i changes in real-time. To address ionotropic GABA receptor function on cone bipolar cells, two techniques were employed: real-time Cl- imaging using YFP fluorescence confocal microscopy and immunocytochemistry.
Currently, we have identified both ON and OFF cone bipolar cell types expressing YFP. We discovered that changes in YFP fluorescence were correlated with increasing GABA concentrations (0.001 to 1 mM). Changes in YFP fluorescence were observed at the dendrites and soma. These YFP fluorescence changes to GABA were suppressed by replacing [Cl-]o with gluconate. In addition, the YFP fluorescence changes were independent of changes in pH, suggesting [Cl-]i changes were induced by GABA. We also observed different responses in the soma and dendrite to both GABA and the Cl- transport inhibitor, furosemide, suggesting that there is a unique spatial Cl- gradient in some cone bipolar cells.
GABAA and GABAC receptors were expressed at the dendrites, soma and terminals of cone bipolar cells. This distribution of GABA receptors on cone bipolar cells is in agreement with the Cl- imaging of GABA-induced changes in [Cl-]i, suggesting that YFP expressing neurons provide an elegant way to monitor spatial and temporal Cl-i gradients in real time.
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