Mechanisms of Ethanol's Influence on Sleep and Circadian Rhythms

描述(申请人提供)：大脑的两个区域对睡眠的启动和维持特别重要。视交叉上核(SON)是睡眠/清醒周期的主要发起者，丘脑参与睡眠期间大脑节律的产生和维持。以下具体目标将作为培训工具，并将寻求确定乙醇对睡眠和昼夜节律的细胞和分子影响。目标1：慢性饮酒者的行为改变。小鼠将被长期给予乙醇饮食(T通道阻滞剂乙磺胺)。将使用车轮运行活动和视频记录来确定睡眠/唤醒状态。我假设长期饮酒者和服用乙硫胺的动物总睡眠时间减少，昼夜节律改变(延迟)。目的2：慢性饮酒者的分子改变。RT-PCR和Western印迹技术将被用来检测长期饮酒对T通道和分子时钟基因和蛋白的影响。我推测，在目标1中观察到的睡眠和昼夜节律的变化，将与丘脑和SCN T通道亚型的基因(增加)和蛋白质(降低)表达的变化，以及SCN分子时钟基因和蛋白质(基因和蛋白质表达的显著节律性的减弱和丧失)的变化有关。目的3：慢性饮酒小鼠T通道的电生理特性。长期给予乙醇饮食的小鼠将被用来产生丘脑的体外脑片。在使用适当的电压方案激发T电流时，将进行全单元贴片记录。我推测T-电流的神经适应性减少可能是酒精中毒患者纺锤波振荡减少的原因。酒精对睡眠的干扰在酒精中毒的发展过程中起着关键作用。持续滥用乙醇会导致睡眠回路的长期变化，这种变化远远持续到停止使用乙醇之后。这项研究将让我有机会了解长期饮酒是如何改变睡眠电路中断的细胞和分子机制的，希望找到治疗乙醇扰乱正常睡眠的新药物靶点。

DESCRIPTION (provided by applicant): Two brain regions are particularly important for the initiation and maintenance of sleep. The suprachiasmatic nucleus (SON) is the primary initiator of sleep/wake cycles and the thalamus is involved in the generation and maintenance of brain rhythms that occur during sleep. The following specific aims will serve as the training vehicle and will seek to determine the cellular and molecular influence of ethanol onsleep and circadian rhythms. Aim 1: Behavioral alterations in chronic drinkers. Mice will be chronically administered an ethanol diet ( the T channel blocker ethosuximide). Wheel running activity and videographic records will be used to determine sleep/waking states. I hypothesize a decrease in total sleep time and shifts in circadian phase (delays) in chronic drinkers and animals administered ethosuximide. Aim 2: Molecular alterations in chronic drinkers. RTPCR and Western blot techniques will be used to examine the effect of chronic alcohol consumption on T channel and molecular clock genes and proteins. I hypothesize that the changes in sleep and circadian rhythms observed in Aim 1, will be correlated with changes in gene (increased) and protein (decreased) expression in both thalamic and SCN T channel isoforms, as well as in SCN molecular clock genes and proteins (attenuation and loss of pronounced rhythmicity of gene and protein expression). Aim 3: Electrophysiological characterization of the T-channel in chronic drinking mice. Mice chronically administered an ethanol diet will be used to generate thalamic in vitro brain slices. Whole cell patch recordings will be made while evoking T-current with appropriate voltage protocols. I hypothesize a neuroadaptational decrease in T-current that may underlie the decrease in spindle wave oscillations reported in alcoholic patients. Sleep perturbations by ethanol play a key role in the progression of alcoholism. Continued abuse of ethanol leads to long-term changes in sleep circuitry that last well beyond the cessation of ethanol administration. This research will afford me the opportunity to understand how chronic ethanol consumption alters the cellular and molecular mechanisms underlying the reported disruptions in sleep circuitry, with the hope of identifying novel drug targets for treatment of ethanol's disruption of normal sleep.