EXCITOTOXIC MECHANISMS OF ETOH--NMDA RECEPTOR FUNCTION

乙醇的兴奋性毒性机制--NMDA受体功能

• 批准号: 2894193
• 负责人: MARY LOU VALLANO
• 金额: $ 7.54万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2894193
• 关键词: NMDA receptors; biological signal transduction; brain mapping; calmodulin dependent protein kinase; dentate gyrus; drug withdrawal; enzyme activity; ethanol; glutamates; hippocampus; laboratory rat; mature animal; neurotoxins; polymerase chain reaction; tissue /cell culture; western blottings

DESCRIPTION: Evidence from several independent laboratories using different experimental approaches indicates that acute ethanol selectively inhibits N-methyl-D-aspartate receptor (NR) function at pharmacologically relevant concentrations, whereas chronic ethanol produces NR supersensitivity due, at least in part, to receptor upregulation. As a consequence, excessive activation of NRs during alcohol withdrawal is thought to contribute to the associated seizures, autonomic instability and neurotoxicity. Pharmacologic treatment with available NR antagonists is undesirable due to generalized inhibition of these ubiquitous receptors. However, NRs are functionally diverse due to differences in subunit composition and associated signal transduction systems. This diversity may account for the selective effects of ethanol on NRs in different brain regions, and in different neuronal populations within a particular region. By exploring the mechanistic basis for this regional selectivity, it should be possible to design and utilize in the alcoholic patient more selective antagonists that are targeted to vulnerable neuronal populations. To further define the role of NRs in chronic ethanol-induced neurotoxicity in hippocampus, the following hypotheses will be tested in adult rats: (I) increased levels of specific NR subunits, and possibly mRNAs, will be observed in hippocampal subfields (CA1, CA3, DG) of ethanol-treated rats, relative to pair-fed control rats, in a manner that corresponds to development of ethanol dependence. Moreover, increased levels of NR subunits will be positively correlated with increased expression or activity of a type II Ca2+/calmodulin-dependent protein kinase (CaM KII) that is co-localized with NRs at excitatory synapses and has been implicated in other forms of excitotoxicity; (ii) chronic ethanol ingestion will differentially sensitize CA1, CA3 and DG neurons to NR-mediated excitotoxicity, relative to pair-fed controls, in a reversible manner. This effect will be mediated by CaM KII. To test these hypotheses, ethanol will be administered to rats for 1-12 weeks and, in some cases, will include a withdrawal period. In ai-m 1, hippocampal subfields from ethanol-treated and control rats will be compared using NRI, NR2 and CaM KII immunoblotting and RT-PCR assays. In aim 2, hippocampal slices will be compared for regional differences in vulnerability to glutamate- or NMDA-mediated excitotoxic damage using a "live-dead" assay in conjunction with confocal imaging analysis.

描述：来自几个独立实验室的证据，使用不同的 实验方法表明，急性乙醇选择性抑制 N-甲基-D-天冬氨酸受体（NR）功能与 浓度，而慢性乙醇会产生NR超敏感性，因为在 至少部分地与受体上调有关。 因此，过度 在酒精戒断过程中，神经元受体的激活被认为有助于 相关的癫痫发作、自主神经不稳定和神经毒性。 药理学 用可用的NR拮抗剂治疗是不希望的， 抑制这些普遍存在的受体。 然而，NR在功能上 由于亚基组成和相关信号的差异， 转导系统 这种多样性可以解释选择性效应 乙醇对不同脑区和不同神经元的NRs的影响 特定区域内的人口。 通过探索机械基础 对于这种区域选择性，应该可以设计和利用 在酒精中毒患者中，靶向 脆弱的神经元群体。 进一步界定国家代表在以下方面的作用： 慢性乙醇诱导的海马神经毒性， 将在成年大鼠中测试假设：（I）增加的特异性 NR亚基，可能还有mRNA，将在海马亚区观察到 (CA1，CA 3，DG），相对于成对喂养的对照大鼠， 以与乙醇依赖性的发展相对应的方式。 此外，NR亚基水平的增加将与 II型Ca 2 +/钙调蛋白依赖性钙通道蛋白的表达或活性增加 蛋白激酶（CaM KII），与NR共定位于兴奋性 突触，并已牵连在其他形式的兴奋性毒性;（ii） 慢性乙醇摄入对CA 1、CA 3和DG的敏感性不同 神经元对NR介导的兴奋性毒性，相对于配对喂养的对照，在一个 可逆的方式。 这种作用将由CaM KII介导。 测试这些 假设将乙醇给予大鼠1-12周，并且在一些实施方案中， 将包括停药期。 在ai-m1中，海马亚区 将使用NRI、NR 2和 CaM KII免疫印迹和RT-PCR测定。 在aim 2中，海马切片将 比较对谷氨酸的脆弱性的区域差异-或 NMDA介导的兴奋性毒性损伤，使用“活-死”测定结合 通过共聚焦成像分析。