Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells.
OBJECTIVE
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.
Otori
Y
Y
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06520-8061, USA.
Wei
J Y
JY
Barnstable
C J
CJ
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018698:Glutamic Acid / Q000008:administration & dosage / Q000633:toxicity*; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D018091:Receptors, AMPA / Q000037:antagonists & inhibitors / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D018691:Excitatory Amino Acid Antagonists; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011810:Quinoxalines; D018091:Receptors, AMPA; D014966:Xanthenes; C085925:calcein AM; C059715:Fluo-3; D018698:Glutamic Acid; C056723:FG 9041; D015763:2-Amino-5-phosphonovalerate; D002118:Calcium