Functional Analysis of Sleep Homeostasis in Drosophila

描述(申请人提供)：睡眠由两个过程控制：一个是在醒着时增加而在睡眠中消散的动态平衡驱动力，另一个是控制其时间的昼夜节律起搏器。尽管这两个系统可以独立运行，但最近的研究表明，这两个系统之间的关系更为密切。事实上，没有一个像在典型的功能丧失时钟突变周期(Cyc01)发现的那样戏剧性。Cyc01突变体表现出不成比例的睡眠反弹，并在睡眠不足10小时后死亡，尽管他们比其他时钟突变体更能抵抗各种压力。我们的数据表明，这种病理的特点是醒着的有害影响加速，而且表明这些过程随后增加了对睡眠的需求(Shaw等人，2002年)。通过对这些特别敏感的动物进行基因组研究，我们已经开始确定睡眠稳态的功能靶点及其分子机制。因此，我们知道有100个基因受长时间清醒状态的影响。为了更充分地了解这些基因所起的作用，我们将确定它们随着清醒时间的增加而变化的时间动态。此外，由于我们已经开发了独立的遗传、药理学和行为分析，这些分析产生了不同地激活动态平衡反应的清醒时期，因此我们将确定这些基因与动态平衡的具体关联程度。更重要的是，我们已经获得了110多个突变系，代表了100个感兴趣的遗传位点中的大约60个，并已经开始评估它们的睡眠参数和对睡眠剥夺的反应。我们建议通过定位mRNA和蛋白质的表达来进一步确定选择基因的特征，并通过使用UAS、GAL4和GAL80载体创建各种有用的转基因系来操纵这些基因和表达它们的细胞的活性。

DESCRIPTION (provided by applicant): Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep and a circadian pacemaker that controls its timing. Although these two systems can operate independently recent studies suggest a more intimate relationship. Indeed, none has been as dramatic as that found for the canonical loss-of-function clock mutant cycle (cyc01). cyc01 mutants showed a disproportionately large sleep rebound and died following 10 hours of sleep deprivation, although they were more resistant than other clock mutants to various stressors. Our data indicate that the pathology is characterized by an acceleration of the detrimental effects of waking and furthermore, suggests that these processes subsequently increase the need for sleep (Shaw et al., 2002). Using genomic studies in these uniquely sensitive animals, we have begun to identify functional targets of sleep homeostasis and its molecular mechanisms. Thus we know of 100 genes that are modulated by prolonged wakefulness. In order to more fully understand the role these genes play, we will determine their temporal dynamics in response to increasing amounts of waking. Moreover, because we have developed independent genetic, pharmacological and behavioral assays that produce periods of waking that differentially activate homeostatic responses, we will determine the extent to which these genes are specifically associated with homeostasis. More importantly, we have acquired over 110 mutant lines representing approximately 60 of the 100 genetic loci of interest and have begun to evaluate their sleep parameters and responses to sleep deprivation. We propose to characterize select genes further by localizing mRNA and protein expression and to manipulate the activity of these genes and the cells that express them by creating a variety of useful transgenic lines using UAS, GAL4 and GAL80 vectors.