Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells.
Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells.
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发表时间：
1998-05
影响因子：
4.4
通讯作者：
Y. Otori;Ji-ye Wei;C. Barnstable
中科院分区：
文献类型：
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作者：
Y. Otori;Ji-ye Wei;C. Barnstable
目的 确定低浓度的谷氨酸是否会诱导纯化的大鼠视网膜神经节细胞 (RGC) 细胞死亡。方法采用改良的两步淘选法从离解的视网膜细胞中纯化大鼠视网膜神经节细胞，并在含有神经营养因子和毛喉素的无血清培养基中培养。在培养3天后，通过钙黄绿素-乙酰氧基甲酯染色来测量暴露于谷氨酸（有或没有谷氨酸受体拮抗剂）后RGC的存活率。为了可视化钙信号，RGC 加载了钙指示剂染料 Fluo-3 乙酰氧基甲酯，并通过激光扫描共聚焦显微镜测量荧光。使用全细胞膜片钳技术检查 RGC 的电生理特性。结果培养3天后，增加谷氨酸浓度（5-500μM）会导致RGC死亡呈剂量依赖性增加。低剂量谷氨酸的神经毒性作用可被特定的 α-氨基-3-二氢-5-甲基-异恶唑-4-丙酸-红藻氨酸 (AMPA-KA) 受体拮抗剂 6,7-二硝基喹喔啉-2,3-二酮 (DNQX) 完全阻断，但不能被特定的 N-甲基-D-天冬氨酸受体拮抗剂 2-氨基-5-磷酸戊酸 (APV) 阻断。此外，钙成像和膜片钳记录表明，细胞内钙积累和谷氨酸诱发的内向电流被 DNQX 完全阻断，但不被 APV 阻断。结论低剂量的谷氨酸可以激活 RGC 中的 AMPA-KA 受体，从而导致细胞内钙含量增加并降低细胞存活率。这是第一份显示钙渗透性 AMPA-KA 受体在培养的 RGC 中的功能作用的报告。
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 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.