title: Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells.
abstract: 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.
authors.affiliation: Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06520-8061, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Otori
authors.forename: J Y
authors.initials: JY
authors.lastname: Wei
authors.forename: C J
authors.initials: CJ
authors.lastname: Barnstable
mesh_terms: 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
chemical_list: 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