MECHANISMS REGULATING EXCITATORY AMINO ACID STIMULATION OF CHICK SPINAL NEURONS

调节鸡脊髓神经元兴奋性氨基酸刺激的机制

• 批准号: 3760486
• 负责人: BURGESS N. CHRISTENSEN
• 金额: --
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760486
• 关键词: NMDA receptors; acidity /alkalinity; calcium flux; chemical group; chick embryo; cyclic AMP; electrophysiology; excitatory aminoacid; fluorescence spectrometry; glutamate receptor; glutamates; glycine; immunocytochemistry; inhibitor /antagonist; inositol phosphates; kainate; magnesium; membrane channels; neurons; protein engineering; receptor expression; second messengers; stimulant /agonist; ventral column; voltage /patch clamp; zinc

The long term goals of this proposed research project are to understand the mechanisms of action on ventral horn spinal cord neurons of candidate excitatory amino acid neurotransmitters. The putative neurotransmitters will include the glutamate analogs quisqualic acid, kainic acid and n- methyl-d-aspartate. These studies encompass both an electrophysiological analysis of cell membrane currents recorded under voltage clamp conditions in the presence of specific agonists and antagonists as well as the sue of fluorescent probes to monitor receptor/channel distribution and changes in intracellular ion concentration. Agonist and antagonist application will employ a rapid superfusion technique in which the cell is repositioned following electrode insertion into a small chamber of approximate volume 100 mul. Solution changes occur within 20-30 msec thus achieving an effective concentration clamp of the cell environment. These studies will be done on cells acutely isolated from the ventral horns of 7 day old chick embryos using enzymatic dissociation methods. The cells will not be maintained in a tissue culture system to avoid the possibility that receptor alterations may occur with cell growth. Apart from identifying the complement of amino acid receptors on isolated neurons, and to positively identify the number of receptor types associated with different ion channels, we plan to examine the types of functional groups on the receptor/channel protein by using pH titration and protein modifying reagents. In addition, the possibility than any of the agonists can alter intracellular calcium either by calcium permeation through an agonist gated channel or by an agonist-receptor interaction that triggers a second messenger system will be investigated using fluorescent probes and immunohistochemistry. The focus will be one the inositol phospholipid and cyclic nucleotide pathways. Fluorescent probes will also be used to determine the distribution of receptor/channel proteins on the cell surface in particular the n-methyl-d-aspartate receptor. It is expected that this research will supply basic knowledge about the physiological importance and role that excitatory amino acid receptors play in the intact nervous system.

这项拟议研究项目的长期目标是了解 候选大鼠脊髓腹角神经元的作用机制 兴奋性氨基酸类神经递质。推定的神经递质 将包括谷氨酸类似物准品质酸、红藻氨酸和n-谷氨酸。 甲基-D-天冬氨酸。这些研究包括电生理学和 电压钳条件下记录的细胞膜电流分析 在特定激动剂和拮抗剂以及诉讼的情况下 用于监测受体/通道分布和变化的荧光探针 细胞内离子浓度。应用激动剂和拮抗剂将 采用快速灌流技术，其中细胞被重新定位 在将电极插入到体积大致相同的小腔室中之后 100-100。解决方案更改在20-30毫秒内发生，从而实现 细胞环境的有效浓度钳制。这些研究将 对7日龄雏鸡腹角细胞进行急性分离 使用酶解离方法的胚胎。这些细胞将不会 在组织培养系统中保持，以避免 随着细胞的生长，受体可能会发生变化。除了识别 分离的神经元上氨基酸受体的补体，并 积极识别与不同类型的受体相关的数量 离子通道，我们计划研究在离子通道上 用pH滴定和蛋白质修饰研究受体/通道蛋白 试剂。此外，这种可能性比任何激动剂都无法改变的 通过钙离子通过激动剂门控渗透细胞内钙 或通过激动剂-受体相互作用触发第二个 信使系统将使用荧光探针和 免疫组织化学。焦点将是肌醇磷脂和 环核苷酸途径。荧光探头还将用于 确定受体/通道蛋白在细胞表面的分布 尤其是N-甲基-D-天冬氨酸受体。预计这将是一次 研究将提供有关生理重要性的基本知识和 兴奋性氨基酸受体在完整神经中的作用 系统。