MECHANISMS OF EXCITOTOXIC DAMAGE IN CENTRAL NEURONS

近年来，已经清楚的是，细胞内Ca 2+的升高 参与神经元的突触可塑性，当过度时， 神经元细胞死亡。谷氨酸诱导的“兴奋毒性”神经元损伤是 可能与中风、头部创伤和癫痫持续状态有关。 这种延迟的损害似乎提供了干预的机会， 事实上，一些实验室已经提供了证据，表明封锁 Ca ~（2+）内流可限制神经元细胞死亡。 关于Ca ~（2+）进入的重要途径已经有了很多的了解。 关注 主要研究N-甲基-D-天冬氨酸（NMDA）类谷氨酸盐 受体，因为它们的高Ca 2+渗透性。 最近已经 发现一些非NMDDA谷氨酸受体也可渗透 Ca 2+和克隆的非NMDA谷氨酸受体亚单位的组合 其是Ca 2+可渗透的以及Ca 2+不可渗透的。 然而，非-NMDA的功能生理学之间的关系， 神经元中的受体和亚单位表达的潜在模式 并没有被确定。 关于这一点，我们知之甚少。 Ca 2+诱导的结构变化的次级介质的生理学， 例如Ca 2+激活的蛋白酶、脂肪酶和激酶。 这个项目的目的是在分子方面定义的作用， NMDDA谷氨酸受体亚单位在中枢神经元Ca 2+内流中的作用， 此外，开始探索钙离子诱导损伤的介质， 钙激活蛋白酶calpain I。 对于第一项任务， 神经元活动的生理测量，例如整个细胞 电压钳或[Ca 2 +]微荧光测量，必须采用 结合在相同细胞中亚基表达模式的测定， 细胞，如亚单位特异性免疫细胞化学，原位杂交， 或单细胞聚合酶链反应。 这些方法可以将 特定细胞中的功能性受体与受体亚单位的集合 表达，并照亮适当的分子靶点进行阻断 有毒的Ca 2+流入。 承担第二项任务，钙蛋白酶的研究 I在正常和兴奋性毒性细胞过程中的激活，需要 一种监测单细胞中钙蛋白酶活性的方法;这可以是 通过使用细胞内荧光底物， 荧光和成像实验。 通过对兴奋性毒性机制的研究， 更有效地指导治疗干预的过程， 造成不可逆的神经元损伤

In recent years it has become clear that elevations of intracellular Ca2+ are involved both in synaptic plasticity in neurons and, when excessive, in neuronal cell death. Glutamate-induced "excitotoxic" neuronal damage is likely to be involved in stroke, head trauma, and status epilepticus. This delayed damage seems to offer an opportunity for intervention, and indeed a number of laboratories have offered evidence that blockade of Ca2+ entry can limit neuronal cell death. Much has been learned of the important routes of Ca2+ entry. Attention has focused on the N-methyl-D-aspartate (NMDA) class of glutamate receptors because of their high Ca2+ permeability. Recently it has been discovered that some non-NMDDA glutamate receptors are also permeable to Ca2+, and combinations of the cloned non-NMDA glutamate receptor subunits which are Ca2+-permeable as well as Ca2+-impermeable have been found. However, the relationships between the functional physiology of non--NMDA receptors in neurons and the underlying patterns of subunit expression have not been established with certainty. Little is known of the physiology of the secondary mediators of Ca2+-induced structural changes, such as Ca2+-activated proteases, lipases, and kinases. This project is intended to define in molecular terms the role of non- NMDDA glutamate receptor subunits in Ca2+ entry in central neurons, and further, to begin to explore the mediators of Ca2+-induced injury such as the Ca2+-activated protease calpain I. For the first of these tasks, physiological measurements of a neuron's activity, such as whole cell voltage clamp or [Ca2+] microfluorimetric measurements, must be employed in conjunction with assays of the subunit expression pattern in the same cell, such as subunit specific immunocytochemistry, in situ hybridization, or single cell polymerase chain reaction. Such methods can relate the functional receptors in a given cell to the set of receptor subunits expressed, and illuminate the appropriate molecular targets for blockade of toxic Ca2+ influx. To undertake the second task, the study of calpain I activation in normal as well as excitotoxic cellular processes, requires a means to monitor calpain activity in single cells; this can be accomplished through the use of intracellular fluorogenic substrates in fluorimetric and imaging experiments. By the study of the mechanisms of excitotoxicity, it will be possible to more effectively direct therapeutic interventions to the processes which cause irreversible neuronal injury.