GANGLIOSIDES AND CNS EFFECTS OF ACUTE ETHANOL

急性乙醇对神经节苷脂和中枢神经系统的影响

• 批准号: 2045721
• 负责人: M D ULLMAN
• 金额: $ 20.42万
• 依托单位: EUNICE KENNEDY SHRIVER CTR MTL RETARDATN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2045721
• 关键词: NMDA receptors; alcoholism /alcohol abuse; calcium flux; centrally acting drug; chemical group; chemical structure; chloride channels; ethanol; gangliosides; genetic strain; laboratory mouse; nervous system disorder; synaptosomes; tissue /cell culture

The objectives of this research are to ascertain the gangliosides and their molecular structures that modulate central nervous system (CNS) ethanol sensitivity and to reveal their mechanisms of action. One approach to alcoholism therapy or avoidance strategies is to discover biochemical components of the disorder, devise methods for altering those components, and testing their efficacy in altering alcohol abuse. Clinical testing is far removed from this research, but accomplishment of the specific aims will provide information on a clinically safe class of compounds, gangliosides, that can modify CNS ethanol sensitivity. LS and SS mice are lines derived from the heterogeneous HS stock in a bidirectional genetic selection experiment in which the selection was made only for differential CNS sensitivity to ethanol hypnosis. The HS strain, LS and SS lines and LS x SS recombinant inbred strains (RIS) will be used in this research. The specific aims and methods of this research are: 1. To determine which gangliosides modulate ethanol sensitivity in vivo. Exogenous gangliosides administered intracerebroventricularly with micro-osmotic pumps in LS and SS mice will be used. The functional groups required for ethanol action will also be ascertained. 2. To determine which gangliosides modulate ethanol sensitivity in vitro. Primary cultures of neo-natal cerebellar granule cells and pre-natal Purkinje cell enriched cultures from HS mice will be used to determine the effect of exogenous gangliosides on ethanol inhibition of NMDA receptor-gated calcium flux. Synaptosomes from brains of LS and SS mice will be used to determine the effect of the B subunit of cholera toxin (which binds GM1) or exogenous gangliosides, on the genetic differences in ethanol enhancement of GABA-gated chloride flux. 3. To determine that GM1 increases the interaction of ethanol at the surface of synaptosomal plasma membranes (SPM) and to ascertain the correlation of GM1 concentration with CNS sensitivity to ethanol. These studies will use SS SPM with and without exogenously added GM1 and Fourier Transform NMR to determine the effects of GM1 on membrane-ethanol interactions, and LS and SS RIS to determine the correlation of ethanol sensitivity with GM1 concentration. 4. To determine ethanol-induced changes in ganglioside concentration and composition in specific cell types in vitro. These studies will use HS neo-natal cerebellar granule cells and pre-natal Purkinje cell enriched cultures to ascertain the effects of ethanol on ganglioside concentration and composition. The effects of ethanol on these parameters will be characterized for differential responses in the same cell cultures from LS and SS mice. 5. To determine calcium-induced changes in the surface exposure of gangliosides in vitro in the absence and presence of ethanol. These studies will use LS and SS synaptosomes, cerebellar granule cells and Purkinje cell enriched cultures which have been exposed to galactose oxidase as an enzyme probe of plasma membrane ganglioside surface exposure.

本研究的目的是确定神经节苷脂， 调节中枢神经系统（CNS）的分子结构 乙醇敏感性，并揭示其作用机制。一种方法 酒精中毒治疗或避免策略是发现生化 疾病的组成部分，设计改变这些组成部分的方法， 并测试它们在改变酒精滥用方面的功效。的临床试验 远离这项研究，但具体目标的实现， 将提供关于临床安全的化合物类别的信息， 神经节苷脂，可以改变CNS乙醇敏感性。LS和SS小鼠是 来自异质HS股票在一个双向遗传 一种选择实验，其中只对差异进行选择 中枢神经系统对酒精催眠的敏感性。HS株、LS和SS系以及LS 本研究将使用x SS重组近交系（RIS）。的 本研究的具体目的和方法是：1.以确定哪些 神经节苷脂调节体内乙醇敏感性。外源性神经节苷脂 在LS中用微渗透泵脑室内给药， 将使用SS小鼠。乙醇作用所需的官能团 也将得到确认。2.为了确定哪些神经节苷脂调节 体外乙醇敏感性。 新生大鼠小脑原代培养 来自HS小鼠的颗粒细胞和出生前浦肯野细胞富集培养物 将用于确定外源性神经节苷脂对乙醇的影响 抑制NMDA受体门控钙流。大脑中的突触体 将使用LS和SS小鼠的B亚基来确定 霍乱毒素（结合GM 1）或外源性神经节苷脂，在遗传上 乙醇增强GABA门控氯离子通量的差异。 3.到 确定GM 1增加了乙醇在表面的相互作用， 突触体质膜（SPM），并确定相关性 GM 1浓度与CNS对乙醇的敏感性。 这些研究将使用 具有和不具有外源添加的GM 1的SS SPM和傅立叶变换NMR， 确定GM 1对膜-乙醇相互作用的影响，以及LS和 SS RIS用于确定乙醇敏感性与GM 1的相关性 浓度. 4.确定乙醇诱导的神经节苷脂变化 浓度和组成在特定细胞类型中的体外研究。 这些 研究将使用HS新生小脑颗粒细胞和产前 浦肯野细胞富集培养物，以确定乙醇对 神经节苷脂浓度和组成。乙醇对这些的影响 参数将被表征为相同的差分响应， 来自LS和SS小鼠的细胞培养物。5.确定钙诱导的变化 在体外神经节苷脂的表面暴露中， 乙醇的存在。这些研究将使用LS和SS突触体， 小脑颗粒细胞和浦肯野细胞富集培养物，具有 半乳糖氧化酶作为质膜的酶探针 神经节苷脂表面暴露。