Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca2+]i increases and NMDA receptor mRNA levels
Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca2+]i increases and NMDA receptor mRNA levels
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DOI：
10.1016/s0006-8993(99)01995-2
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发表时间：
1999-12-04
期刊：
影响因子：
2.9
通讯作者：
Reynolds, IJ
中科院分区：
文献类型：
--
作者：
Cheng, CL;Fass, DM;Reynolds, IJ
我们研究了几个与胚胎前脑神经元在培养过程中随时间发生的兴奋性毒性敏感性增加有关的因素。神经元培养在体外5天(DIV)对100 mU谷氨酸/10 mU甘氨酸(DIV)5min耐受，但在14DIV时对同样的刺激变得脆弱。我们使用对钙离子具有高亲和力和低亲和力的荧光指示剂Fura-2和MagFura-2来测量[Ca~(2+)](I)的变化。谷氨酸刺激的Fura-2和MagFura-2比率在10DIV时达到最大值。Fura-2报告了类似的[Ca~(2+)](I)随暴露于3或100亩M谷氨酸5分钟的变化，而MagFura-2报告了更大的[Ca~(2+)](I)在暴露于100亩M谷氨酸5分钟时高于暴露于3亩M谷氨酸、100亩M海人藻酸盐或50 mM K+时。这表明，当使用MagFura-2而不是Fura-2来测量Ca~(2+)时，[Ca~(2+)](I)变化的幅度与刺激的兴奋毒性潜力相关。用核糖核酸酶保护实验检测NMDA受体亚单位的mRNA水平。随着培养时间的延长，NR1和NR2AmRNA持续增加，而NR2BmRNA在培养的前10d急剧增加，随后保持稳定。NR2B基因表达增加的时间进程与谷氨酸刺激的MagFura-2信号变化和神经元损伤的增加密切相关。因此，谷氨酸刺激的[Ca~(2+)](I)反应和NMDA受体亚单位(尤其是NR2B)mRNA水平的升高可能参与了培养的大鼠前脑神经元对兴奋性毒性敏感性的形成。(C)1999 Elsevier Science B.V.保留所有权利。
We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 mu M glutamate/10 mu M glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca2+, respectively, to measure changes in [Ca2+](i). Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca2+](i) changes with exposure to 3 or 100 mu M glutamate for 5 min, whereas magfura-2 reported larger [Ca2+](i) increases with 5-min exposure to 100 mu M glutamate than with exposure to 3 mu M glutamate, 100 mu M kainate or 50 mM K+ from 10 DIV onward. This suggests that the magnitude of the [Ca2+](i) changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca2+. We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca2+](i) responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons. (C) 1999 Elsevier Science B.V. All rights reserved.