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

Yasumasa Otori, Ji Ye Wei, Colin Barnstable

Research output: Contribution to journalArticle

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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 μM) 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 α-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.

Original languageEnglish (US)
Pages (from-to)972-981
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume39
Issue number6
StatePublished - May 1998

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Retinal Ganglion Cells
Glutamic Acid
Calcium
Kainic Acid Receptors
Cell Death
2-Amino-5-phosphonovalerate
Excitatory Amino Acid Antagonists
Serum-Free Culture Media
Nerve Growth Factors
Patch-Clamp Techniques
Colforsin
N-Methyl-D-Aspartate Receptors
Cultured Cells
Cell Survival
Esters
Lasers
Coloring Agents
Fluorescence
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Ophthalmology

Cite this

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title = "Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells",
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 μM) 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 α-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.",
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Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells. / Otori, Yasumasa; Wei, Ji Ye; Barnstable, Colin.

In: Investigative Ophthalmology and Visual Science, Vol. 39, No. 6, 05.1998, p. 972-981.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells

AU - Otori, Yasumasa

AU - Wei, Ji Ye

AU - Barnstable, Colin

PY - 1998/5

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N2 - 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 μM) 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 α-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.

AB - 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 μM) 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 α-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.

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