NEUROTOXIC EFFECTS OF LOW-DOSES OF GLUTAMATE ON PURIFIED RAT RETINAL GANGLION-CELLS

PURPOSE. To determine whether low concentrations of glutamate induce c ell 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 me dium containing neurotrophic factors and forskolin. Survival of RGCs a fter exposure to glutamate, with or without glutamate receptor antagon ists, was measured by calcein-acetoxymethyl ester staining after 3 day s in culture. To visualize calcium signals, RGCs were loaded with the calcium indicator dye, fluo-3 acetoxymethyl ester, and fluorescence wa s 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 (5-5 00 mu M) of glutamate caused a dose-dependent increase in RGC death af ter 3 days in culture. Neurotoxic effects of low closes of glutamate w ere totally blocked by a specific lpha-amino-3-dihydro-5-methyl-isoxaz ol-4-propionic acid-kainate (AMPA-KA) receptor antagonist, 6,7-dinitro quinoxaline-2,3-dione (DNQX), but not by a specific N-methyl-D-asparta te 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 comple tely blocked by DNQX but not by APV. CONCLUSIONS. Low doses of glutama te can activate AMPA-KA receptors in RGCs, which causes increases in i ntracellular calcium and decreases in cell survival. This is the first report to shon the functional role of calcium-permeable AMPA-KA recep tors in cultured RGCs.