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Abstract
PURPOSE: To determine whether low concentrations of glutamate induce cell death in purified rat retinal ganglion cells (RGCs). METHODS: Rat retinal ganglion cells were purified from dissociated retinal cells by a modified two-step panning method and were cultured in serum-free medium containing neurotrophic factors and forskolin. Survival of RGCs after exposure to glutamate, with or without glutamate receptor antagonists, was measured by calcein-acetoxymethyl ester staining after 3 days in culture. To visualize calcium signals, RGCs were loaded with the calcium indicator dye, fluo-3 acetoxymethyl ester, and fluorescence was measured by laser scanning confocal microscope. Electrophysiological properties of RGCs were examined by using the whole-cell, patch-clamp technique. RESULTS: The application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days in culture. Neurotoxic effects of low doses of glutamate were totally blocked by a specific alpha-amino-3-dihydro-5-methyl-isoxazol-4-propionic acid-kainate (AMPA-KA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not by a specific N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate (APV). In addition, calcium imaging and patch-clamp recordings showed that intracellular calcium accumulation and glutamate-evoked inward currents were completely blocked by DNQX but not by APV. CONCLUSIONS: Low doses of glutamate can activate AMPA-KA receptors in RGCs, which causes increases in intracellular calcium and decreases in cell survival. This is the first report to show the functional role of calcium-permeable AMPA-KA receptors in cultured RGCs.