CALCIUM HOMEOSTASIS IN HYPOXIC NEURONS--ROLE OF AMPA/KAINATE RECEPTOR ACTIVATION

越来越多的证据表明，AMPA/海人藻酸盐(AMP/KA)以及NMDA 受体参与了脑缺氧缺血的发病机制。 尽管NMDA受体过度激活与缺氧性神经元 死亡被广泛研究，AMPA/KA受体的细胞机制 介导的缺氧性神经元损伤仍有待建立。项目III 检验AMPA/KA受体过度激活有助于 通过破坏细胞内钙离子对缺氧性神经元损伤的影响 动态平衡。这个项目中的实验将集中在测量上。 体外培养的大脑皮层神经元细胞内钙离子浓度 细胞内钙离子的数字荧光和共聚焦显微镜 指标，富拉-2和INDO-1。钙稳态的改变将会 与神经元存活的实验终点进行比较。至 分离AMPA/KA受体的作用，培养神经元 在NMDA存在的情况下暴露于长期的缺氧-葡萄糖剥夺 受体拮抗剂；以前的实验已经确定 所致神经元损伤主要由AMPA/KA受体介导 激活。这个项目的具体目标是(1)描述 AMPA/KA受体的时间分布和亚细胞分布 缺氧缺糖时介导的钙内流，(2) 检查AMPA/KA受体激动剂和拮抗剂的药理作用， 包括改变受体脱敏的药物，以及(3) 确定AMPA/KA受体触发的钙内流途径 过度激活。

Growing evidence suggests that AMPA/kainate (AMP/KA) as well as NMDA receptors participate in the pathogenesis of cerebral hypoxia-ischemia. Although links between NMDA receptor overactivation and hypoxic neuronal death are extensively studied, cellular mechanisms of AMPA/KA-receptor mediated hypoxic neuronal injury remain to be established. Project III examines the hypothesis that AMPA/KA receptor overactivation contributes to hypoxic neuronal injury by disrupting intracellular calcium homeostasis. The experiments in this project will focus on measurements of intracellular calcium ([Ca2+]i) in cultured cortical neurons using digital fluorescence and confocal microscopy with the cytosolic calcium indicators, fura-2 and indo-1. Alterations in calcium homeostasis will be compared with the experimental endpoint of neuronal survival. To isolate the contribution of AMPA/KA receptors, cultured neurons will be exposed to prolonged oxygen-glucose deprivation in the presence of NMDA receptor antagonists; previous experiments have established that the resulting neuronal injury is mediated largely by AMPA/KA receptor activation. The specific aims of this project are (1) to characterize the temporal profile and subcellular distribution of AMPA/KA receptor- mediated calcium entry during oxygen-glucose deprivation, (2) to examine the pharmacology of AMPA/KA receptor agonists and antagonists, including agents which alter receptor desensitization, and (3) to determine the routes of calcium entry triggered by AMPA/KA receptor overactivation.