NMDA RECEPTORS

组件6将侧重于一种类型的谷氨酸受体的作用 N-甲基-D-天冬氨酸受体(NMDAR)，对乙醇的初始敏感性。 乙醇抑制NMDAR功能，NMDAR在 大脑功能。我们观察到长睡眠(LS)和短睡眠(SS) 小鼠，它们对催眠作用的初始敏感度不同 乙醇对NMDAR拮抗剂MK-1表现出明显的行为差异。 801和CPP。SS小鼠表现出比LS小鼠更强的运动激活 在较低的药物剂量下，LS而不是SS的小鼠在较高的药物剂量下被镇静 剂量。我们还发现[~3H]MK-801的密度存在线形差异 LS和SS小鼠NMDAR亚基(NR2A：NR2B)的结合部位和比例 大脑。因此，本提案将NMDAR作为候选基因 LS(ILS)和ILS自交系对乙醇的不同敏感性 SS(ISS)小鼠。他们正在检验的假设是：不同于 ILS/ISS对乙醇的初始敏感性可能是由于 NMDAR表达、合成、翻译后处理和/或信号 转导。[~3H]MK-801和[~3H]MK-801的放射自显影定量分析 NR1、NR2A、NR2B和NR2B的CPP结合及定量Western分析 NR2C亚基将被用来确定NMDAR的差异 表达定位于ILS和ISS小鼠的特定区域 大脑，尤其是那些参与运动活动的大脑。为了 评估ILS和ISS小鼠脑NMDAR是否有助于 这些小鼠对酒精的不同敏感性，行为研究 利用LSxSS重组自交系进行MK-801和CPP 菌株。接下来，NMDA诱导的一氧化氮的体内电化学测量 正常脑组织中氧化物的产生及NMDA诱导的体外检测 脑片的电生理活动将被用来确定 NMDAR信号转导是否也存在差异 乙醇对ILS和ISS小鼠脑内NMDAR活性的抑制。最后， 潜在差异对NMDAR亚基组成的贡献 和/或ILS和ISS脑Will特定区域的磷酸化状态 用免疫共沉淀法进行研究，定量Western 分析和前沿磷酸化分析。这些研究的结果 实验将增加我们对兴奋性谷氨酸是如何 系统导致初始敏感度的遗传差异 乙醇。了解影响初始酒精敏感性的因素 很重要，因为已发现低初始敏感度 与酗酒的高风险有关。

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.