GANGLIOSIDES AND CNS EFFECTS OF ACUTE ETHANOL

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 并揭示其作用机制。一种方法 对酒精中毒的治疗或避免策略是发现生化 混乱的组成部分，设计出改变这些组成部分的方法， 并测试它们在改变酗酒方面的有效性。临床测试是 与这项研究相去甚远，但实现了具体目标 将提供一类临床安全的化合物的信息， 神经节苷脂，可以改变中枢神经系统的乙醇敏感性。LS和SS小鼠是 来自异质HS系的品系在双向遗传 只对差异进行选择的一种选择试验 中枢神经系统对乙醇催眠的敏感性。HS株系、LS系、SS系和LS系 本研究将使用X个SS重组自交系(RIS)。这个 本研究的具体目的和方法是：1.确定 神经节苷脂在体内调节乙醇敏感性。外源性神经节苷脂 脑室注射微量渗透压泵给药 将使用SS小鼠。乙醇作用所需的官能团 也将被确定。2.确定神经节苷脂的调节作用 体外乙醇敏感性试验。新生小脑的原代培养 HS小鼠颗粒细胞和出生前浦肯野细胞的富集化培养 将用于确定外源性神经节苷脂对乙醇的影响 抑制NMDA受体门控钙通道。来自大脑的突触体 将用来确定LS和SS小鼠的B亚单位的作用 霍乱毒素(与GM1结合)或外源性神经节苷脂，对基因 乙醇增强GABA门控氯离子通量的差异。3.至 确定GM1增加了乙醇在表面的相互作用 突触体质膜(SPM)及其相互关系 GM1浓度与中枢神经系统对乙醇的敏感性。这些研究将使用 添加和不添加外源GM1和傅里叶变换核磁共振的SS SPM 确定GM1对膜-乙醇相互作用的影响，以及LS和 SS RIS确定酒精敏感性与GM1的相关性 集中精神。4.测定乙醇对神经节苷脂的影响 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出生前 蒲肯野细胞富集培养以确定乙醇对细胞生长的影响 神经节苷脂的浓度和组成。乙醇对这些指标的影响 参数将被表征为在同一 LS和SS小鼠的细胞培养。5.确定钙诱导的变化 在体外暴露于神经节苷脂表面的缺失和 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浓缩培养 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。

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.