Marini A M, Ueda Y, June C H
Department of Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Ann N Y Acad Sci. 1999;890:421-37. doi: 10.1111/j.1749-6632.1999.tb08021.x.
Cultured rat cerebellar granule cells are resistant to the excitotoxic effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor agonists under three conditions: 1) prior to day seven in vitro when cultured in depolarizing concentrations of potassium [25 mM]; 2) at any time in vitro when cultured in non-depolarizing concentrations of potassium 5 mM[; and 3) when neurons, cultured in depolarizing concentrations of potassium 25 mM[ for eight days in vitro, are pretreated with a subtoxic concentration of NMDA. The focus of this paper is to determine: a) whether the resistance to excitotoxicity by NMDA and non-NMDA receptor agonists is due to a decreased intracellular calcium Ca++[i response to glutamate receptor agonists in cultured rat cerebellar granule cells; or b) whether Ca++[i levels induced by the agonists are similar to those observed under excitotoxic conditions. Granule cells, matured in non-depolarizing growth medium, treated with glutamate resulted in an increase in Ca++[i followed by a plateau that remained above baseline in virtually all neurons that responded to glutamate. The response was rapid in onset (< 10 sec) and the pattern of response heterogeneous in that cells responsive to glutamate increased their Ca++[i to different extents; some cells did not respond to glutamate. Kainate also produced significant elevations in Ca++[i. The Ca++[i response to glutamate in neurons matured in depolarizing (25 mM K+) growth medium for three days was rapid, transient and heterogeneous, which reached a plateau that was elevated above baseline levels; removing the glutamate markedly reduced the Ca++[i concentration. Activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors by kainic acid produced similar changes in Ca++[i responses. At a time when cultured cerebellar granule cells become susceptible to the excitotoxic effects of glutamate acting at NMDA receptors (day in vitro (DIV) 8) in depolarizing growth medium, glutamate elicited Ca++[i responses similar to those observed at a culture time when the neurons are not susceptible to the excitotoxic effects of glutamate (DIV 3). Pretreatment of the cultured neurons with a subtoxic concentration of NMDA, which protects all neurons against the excitotoxic effects of glutamate, did not alter the maximal Ca++[i elicited by an excitotoxic concentration of glutamate.
培养的大鼠小脑颗粒细胞在三种情况下对N-甲基-D-天冬氨酸（NMDA）和非NMDA受体激动剂的兴奋毒性作用具有抗性：1）在体外培养至第7天之前，当在去极化浓度的钾[25 mM]中培养时；2）在体外的任何时间，当在非去极化浓度的钾5 mM中培养时；3）当在体外以去极化浓度的钾25 mM培养8天的神经元用亚毒性浓度的NMDA预处理时。本文的重点是确定：a）对NMDA和非NMDA受体激动剂的兴奋毒性抗性是否是由于培养的大鼠小脑颗粒细胞中细胞内钙Ca++[i]对谷氨酸受体激动剂的反应降低；或者b）激动剂诱导的Ca++[i]水平是否与在兴奋毒性条件下观察到的水平相似。在非去极化生长培养基中成熟的颗粒细胞，用谷氨酸处理导致Ca++[i]增加，随后达到一个平台期，几乎所有对谷氨酸有反应的神经元都保持在基线以上。反应开始迅速（<10秒），反应模式异质性，即对谷氨酸有反应的细胞将其Ca++[i]增加到不同程度；一些细胞对谷氨酸没有反应。海人酸也使Ca++[i]显著升高。在去极化（25 mM K+）生长培养基中成熟三天的神经元中，对谷氨酸的Ca++[i]反应迅速、短暂且异质性，达到高于基线水平的平台期；去除谷氨酸显著降低了Ca++[i]浓度。海人酸激活α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）/海人酸受体在Ca++[i]反应中产生类似变化。在培养的小脑颗粒细胞在去极化生长培养基中对作用于NMDA受体的谷氨酸的兴奋毒性作用变得敏感的时间点（体外培养天数（DIV）8），谷氨酸引发的Ca++[i]反应与在神经元对谷氨酸的兴奋毒性作用不敏感的培养时间（DIV 3）观察到的反应相似。用亚毒性浓度的NMDA预处理培养的神经元，可保护所有神经元免受谷氨酸的兴奋毒性作用，但不会改变兴奋毒性浓度的谷氨酸引发的最大Ca++[i]。
