m4 & m5 muscarinic acetylcholine receptor subtypes in ethanol-induced locomotion

描述(由申请人提供)：我们的长期目标是确定影响对酒精急性运动刺激敏感性的基因，这是酒精中毒的内表型(行为刺激)的假定小鼠模型。这项建议的总体目标是确定M4和/或M5毒扁豆碱乙酰胆碱受体(MAChR)亚型基因是否在乙醇急性运动刺激中起作用。小鼠2号染色体(Chr2)上的基因影响对乙醇刺激的敏感性；在Chr2上的相关区域存在M4和M5mAChR亚型基因。这项拟议的研究旨在阐明M4和/或M5基因表达的差异是否与乙醇诱导的刺激易感性有关，以及它们是否直接参与了这一行为。小鼠的快系和慢系是有选择地培育的，对乙醇的运动刺激效应具有高和低的敏感性，因此是研究这些差异的敏感遗传动物模型。这项建议的具体目标是1.)确定是否存在M4和/或M5 mAChR亚型基因差异表达的特定脑区 快鼠和慢鼠，以及2.)确定M4和/或M5 mAChR亚型基因的选择性基因沉默是否影响对酒精的急性运动刺激。将使用qRT-PCR和Western印迹分析来评估激光捕获解剖的大脑区域中的基因表达。M4和M5受体亚型基因的沉默将通过将选择性RNA干扰(RNAi)转录本分别注入伏核(NAC)和腹侧被盖区(VTA)来完成。与慢速鼠相比，快鼠在NAC中的M4基因表达降低，而在VTA中的M5基因表达增强。我们还假设，在慢速小鼠的NAc内微量注射M4选择性RNAi将增强对乙醇的急性运动刺激反应，而在快小鼠的VTA内微量注射M5选择性RNAi将阻断乙醇诱导的刺激。了解酒精急性敏感性的药物遗传学基础是很重要的，因为它对预测未来酒精使用/滥用有一定的价值。这项建议的发现可能会为未来酒精中毒治疗的药物疗法的发展提供参考。

DESCRIPTION (provided by applicant): Our long-term goal is to identify genes that influence sensitivity to acute locomotor stimulation to ethanol, a putative mouse model of an endophenotype (behavioral stimulation) for alcoholism. The overall goal of this proposal is to determine if the m4 and/or m5 muscarinic acetylcholine receptor (mAChR) subtype genes have a role in acute locomotor stimulation to ethanol. Genes on mouse chromosome 2 (Chr 2) influence sensitivity to ethanol-induced stimulation; within the relevant region on Chr 2 reside the m4 and m5 mAChR subtype genes. The proposed studies are intended to elucidate if differences in m4 and/or m5 gene expression are associated with predisposition to ethanol-induced stimulation, and if they are directly involved in this behavior. The FAST and SLOW lines of mice were selectively bred for high and low sensitivity to the locomotor stimulant effects of ethanol, and are therefore a sensitive genetic animal model in which to study these differences. The specific aims of this proposal are to 1.) determine whether there are specific brain regions in which the m4 and/or m5 mAChR subtype genes are differentially expressed in FAST and SLOW mice, and 2.) determine whether selective gene silencing of the m4 and/or m5 mAChR subtype genes affects acute locomotor stimulation to ethanol. Gene expression will be assessed in lasercapture-dissected brain regions using qRT-PCR and Western blot analysis. Silencing of the m4 and m5 receptor subtype genes will be accomplished by microinjecting selective RNA interference (RNAi) transcripts into the nucleus accumbens (NAc) and ventral tegmental area (VTA), respectively. It is hypothesized that FAST mice will display decreased expression of the m4 gene in the NAc and enhanced expression of the m5 gene in the VTA as compared to SLOW mice. It is also hypothesized that microinjection of m4-selective RNAi into the NAc of SLOW mice will enhance the acute locomotor stimulant response to ethanol, while microinjection of m5-selective RNAi into the VTA of FAST mice will block ethanol-induced stimulation. It is important to understand the pharmacogenetic underpinnings of acute sensitivity to ethanol because it has some value for the prediction of future alcohol use/abuse. Findings from this proposal may inform future pharmacotherapies for development in the treatment of alcoholism.