MECHANISMS OF EXCITOTOXIC DAMAGE IN CENTRAL NEURONS

近年来，细胞内钙离子的升高已变得明显 既参与神经元的突触可塑性，当过量时， 导致神经细胞死亡。谷氨酸诱导的“兴奋性毒性”神经元损伤 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，而且 事实上，一些实验室已经提供了证据，证明封锁 Ca~(2+)内流可限制神经细胞死亡。 人们对钙离子进入的重要途径已经有了很多了解。注意 专注于N-甲基-D-天冬氨酸(NMDA)类谷氨酸 受体，因为它们具有很高的钙离子渗透性。最近，它一直在 发现一些非NMDDA谷氨酸受体也可以通透 Ca~(2+)和克隆的非NMDA谷氨酸受体亚基的组合 其中既有钙离子不透水的，也有钙离子不透水的。 然而，非NMDA的功能生理之间的关系 神经元中的受体和亚单位表达的潜在模式 还没有确定地建立起来。人们对此知之甚少 钙离子诱导的结构变化的次要介质的生理学， 如钙激活的蛋白水解酶、脂肪酶和激动酶。 这个项目的目的是用分子术语来定义非 中枢神经元钙内流中的NMDDA谷氨酸受体亚单位，以及 此外，为了开始探索钙离子诱导的损伤的介体，如 钙激活的蛋白水解酶Calain I.对于这些任务的第一个， 神经元活动的生理测量，如整个细胞 必须使用电压钳或[Ca~(2+)]微量荧光测量 结合同一组中的亚基表达模式的分析 细胞，如亚基特异性免疫细胞化学、原位杂交、 或单细胞聚合酶链式反应。这类方法可以将 特定细胞中受体亚基集合的功能性受体 表达并阐明要封锁的适当分子目标 有毒的钙离子内流。承担第二项任务，即研究钙调蛋白 在正常和兴奋性毒性的细胞过程中，i的激活需要 一种监视单个细胞中的钙蛋白酶活性的方法；这可以是 通过使用细胞内的荧光底物在 荧光和成像实验。 通过对兴奋性毒性机制的研究，将有可能 更有效地直接对以下过程进行治疗干预 造成不可逆转的神经元损伤。

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.