Retinal bipolar cells are second-order neurons in the retina that relay signals from photoreceptors to third-order neurons, ganglion cells and amacrine cells. Bipolar cells are divided into two major classes: ON and OFF bipolar cells. In mammalian retina, rods contact a single class of rod ON bipolar cells, whereas cones contact both ON and OFF cone bipolar cells. The functional types of mammalian bipolar cells are well characterized, with rod bipolar cells being the best studied in the mammalian retina; however, little is known about cone bipolar cells, and the ability of GABA to regulate their excitability. 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 receptors in cone bipolar cells of the mouse retina. The model organism used for these studies was the transgenic mouse expressing yellow fluorescent protein (YFP) under the Thy-1 promoter. YFP is uniquely sensitive to halides, like chloride ions, and can quench YFP fluorescence when Cl- enters the cell and interacts with YFP. This provides a way to monitor [Cl-]i changes in real time. To address which ionotropic GABA receptors (GABAA and GABAC) are expressed on cone bipolar cells, two techniques were employed: real-time Cl- imaging using YFP fluorescence microscopy and immunohistochemistry with antibodies raised against both GABAA and GABAC receptors. We discovered that fluorescence changes correlated with increasing drug concentrations; thus, the higher the concentration of GABA (0.001 to 1 mM), the greater the decrease in fluorescence observed. These data also demonstrate that GABA hyperpolarizes cone bipolar cells as previously reported. These observed fluorescence changes corroborate the evidence that GABA is an inhibitory signaling molecule in the retina. GABAA receptors were expressed at the dendrites, soma, and terminals of cone bipolar cells and GABAC expression was most intense in the terminals and dendrites of cone bipolar cells. This differential distribution of GABA receptors on cone bipolar cells is in agreement with the Cl- imaging, suggesting that GABA tightly regulates cone bipolar excitability, which will have a profound impact on light-modulated input from cones in the retina.
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