Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells

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 palming 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 mu M) of glutamate caused a dose-dependent increase in RGC death after 3 days in culture. Neurotoxic effects of low closes 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 shon the functional role of calcium-permeable AMPA-KA receptors in cultured RGCs.