NMDA RECEPTORS

NMDA受体

基本信息

批准号：
6409939
负责人：
MICHAEL D BROWNING
金额：
$ 18.46万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-12-01 至 2001-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6409939
关键词：
NMDA receptors behavioral /social science research tag biological signal transduction ethanol laboratory mouse neuropharmacology nitric oxide posttranslational modifications protein structure function psychopharmacology receptor expression sleep disorders

项目摘要

Component 6 will focus on the role of one type of glutamate receptor, the N-methyl-D-aspartate receptor (NMDAR), in initial sensitivity to ethanol. Ethanol inhibits NMDAR function, and NMDARs play an important role in brain function. We have observed that long-Sleep (LS) and Short-Sleep (SS) mice, which differ in their initial sensitivity to the hypnotic effects of ethanol, show marked behavioral differences to the NMDAR antagonists MK- 801 and CPP. SS mice exhibited greater locomotor activation than LS mice at lower drug doses; and LS, but not SS, mice were sedated at higher drug doses. We have also found line differences in the density of [3H]MK-801 binding sites and ratio of NMDAR subunits (NR2A:NR2B) in LS and SS mouse brains. Therefore, this proposal focuses on the NMDAR as a candidate gene for differential ethanol sensitivity in the inbred lines of LS (ILS) and SS (ISS) mice. They hypothesis being tested is that: Differences between ILS/ISS in initial sensitivity to ethanol may be due to differences in NMDAR expression, composition, post-translational processing and/or signal transduction. Quantitative autoradiographic analyses of [3H]MK-801 and [3H]CPP binding and quantitative western analysis of NR1, NR2A, NR2B and NR2C subunits will be used to determine whether differences in NMDAR expression are localized to specific regions of the ILS and ISS mouse brains, particularly those involved in locomotor activity. In order to assess whether ILS and ISS mouse brain NMDARs contribute to the differential ethanol sensitivity of these mice, behavioral studies with MK-801 and CPP will be conducted using the LSxSS recombinant inbred strains. Next, in vivo electrochemical measures of NMDA-induced nitric oxide generation in intact brain and in vitro measures of NMDA induced electrophysiological activity in brain slices will be used to determine whether differences also exist in NMDAR signal transduction and in ethanol's inhibition of NMDAR activity in ILS and ISS mouse brain. Lastly, the contribution of potential differences in NMDAR subunit composition and/or phosphorylation state in specific regions of ILS and ISS brain will be investigated using co-immunoprecipitation assays, quantitative western analyses and front phosphorylation assays. The results of these experiments will increase our understanding of how excitatory glutamate systems contribute to genetic differences in initial sensitivity to ethanol. Understanding the factors mediating initial ethanol sensitivity is important because low initial sensitivity has been found to be associated with high risk for alcoholism.

第 6 部分将重点关注一种谷氨酸受体的作用，即 N-甲基-D-天冬氨酸受体（NMDAR），最初对乙醇敏感。乙醇抑制NMDAR功能，NMDARs在大脑功能。我们观察到长睡眠（LS）和短睡眠（SS）小鼠对催眠作用的初始敏感性不同乙醇，表现出与 NMDAR 拮抗剂 MK- 显着的行为差异 801 和 CPP。 SS 小鼠比 LS 小鼠表现出更强的运动激活较低的药物剂量；和 LS，但不是 SS，小鼠以更高的药物镇静剂量。我们还发现 [3H]MK-801 的密度存在线差异 LS 和 SS 小鼠中 NMDAR 亚基 (NR2A:NR2B) 的结合位点和比例大脑。因此，本提案重点关注NMDAR作为候选基因 LS（ILS）自交系中乙醇敏感性的差异 SS (ISS) 小鼠。他们正在测试的假设是： ILS/ISS 对乙醇的初始敏感性可能是由于 NMDAR 表达、组成、翻译后处理和/或信号转导。 [3H]MK-801 和的定量放射自显影分析 [3H]CPP 结合以及 NR1、NR2A、NR2B 和 NR2A 的定量蛋白质分析 NR2C 亚基将用于确定 NMDAR 是否存在差异表达定位于 ILS 和 ISS 小鼠的特定区域大脑，特别是那些参与运动活动的大脑。为了评估 ILS 和 ISS 小鼠大脑 NMDAR 是否有助于这些小鼠的乙醇敏感性差异，行为研究 MK-801 和 CPP 将使用 LSxSS 重组近交系进行菌株。接下来，NMDA 诱导的硝酸的体内电化学测量完整大脑中氧化物的生成和 NMDA 诱导的体外测量脑切片中的电生理活动将用于确定 NMDAR 信号转导和乙醇对 ILS 和 ISS 小鼠脑中 NMDAR 活性的抑制作用。最后， NMDAR 亚基组成中电位差异的贡献和/或 ILS 和 ISS 大脑特定区域的磷酸化状态使用免疫共沉淀分析、定量蛋白质印迹分析等进行研究分析和前沿磷酸化测定。这些结果实验将加深我们对兴奋性谷氨酸如何发挥作用的理解系统导致初始敏感性的遗传差异乙醇。了解介导初始乙醇敏感性的因素很重要，因为已发现初始灵敏度较低与酗酒的高风险有关。