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: 3113553
• 负责人: M D ULLMAN
• 金额: $ 19.6万
• 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/3113553
• 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和 它们调节中枢神经系统（CNS）的分子结构 乙醇敏感性并揭示其作用机制。一种方法 酗酒治疗或避免策略的关键是发现生化 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。临床测试是 与这项研究相去甚远，但实现了具体目标 将提供有关临床安全类别化合物的信息， 神经节苷脂，可以改变中枢神经系统乙醇敏感性。 LS 和 SS 小鼠是 源自双向遗传中异质 HS 种群的品系 选择实验，其中仅针对差异进行选择 中枢神经系统对乙醇催眠的敏感性。 HS 菌株、LS 和 SS 菌株以及 LS x SS 重组近交系 (RIS) 将用于本研究。这 本研究的具体目的和方法是： 1. 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 和 LS 中用微渗透泵进行脑室内给药 将使用SS小鼠。乙醇作用所需的官能团 也将被确定。 2. 确定调节哪些神经节苷脂 体外乙醇敏感性。 新生儿小脑的原代培养 HS 小鼠的颗粒细胞和产前浦肯野细胞富集培养物 将用于确定外源神经节苷脂对乙醇的影响 抑制 NMDA 受体门控钙流。来自大脑的突触体 LS 和 SS 小鼠的 B 亚基将用于确定 霍乱毒素（结合 GM1）或外源性神经节苷脂，对遗传 乙醇增强 GABA 门控氯化物通量的差异。 3. 到 确定 GM1 增加了乙醇表面的相互作用 突触体质膜（SPM）并确定其相关性 GM1 浓度与 CNS 对乙醇的敏感性。 这些研究将使用 有和没有外源添加 GM1 的 SS SPM 和傅里叶变换 NMR 确定 GM1 对膜-乙醇相互作用的影响，以及 LS 和 SS RIS 确定乙醇敏感性与 GM1 的相关性 专注。 4. 确定乙醇诱导的神经节苷脂变化 体外特定细胞类型的浓度和组成。 这些 研究将使用 HS 新生儿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 神经节苷脂浓度和成分。乙醇对这些的影响 参数将被表征为相同的差异响应 LS 和 SS 小鼠的细胞培养物。 5. 确定钙引起的变化 在体外神经节苷脂的表面暴露中不存在和 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 暴露于半乳糖氧化酶作为质膜的酶探针 神经节苷脂表面暴露。