Genetic and Neuronal Mechanisms that Regulate Zebrafish Sleep

摘要 睡眠障碍很普遍，会导致几种精神障碍的发病率，并导致每年 1000亿美元的经济负担。然而，尽管睡眠对健康很重要，但调节睡眠的机制 人们对此了解甚少。我们正在通过开发斑马鱼的有用特征来解决这个问题 回答一个重要而基本的问题：是什么遗传和神经机制调节睡眠？睡眠是 由反映睡眠需要的内部线索的动态平衡过程和反映睡眠需求的昼夜节律过程调节 受到环境提示的影响，并将睡眠限制在适当的时间。睡眠也是直接和快速的 受一种被称为掩蔽的现象的调节，在这种现象中，光诱导觉醒，黑暗诱导白天睡眠 动物。调节动态平衡过程的因素已经确定，包括我们最近发现的 5-羟色胺能中缝促进斑马鱼和小鼠的睡眠稳态。我们还表明，褪黑素是 对斑马鱼睡眠的昼夜调节是必不可少的，并确定了大脑中调节睡眠的一条路径 蒙面。在这里，我们以这些发现为基础，阐明构成动态平衡、昼夜节律、 以及对睡眠的光依赖调节。我们将使用斑马鱼来研究这些机制，斑马鱼是一种昼夜活动的 脊椎动物，具有补充啮齿动物模型的几个优势，使用遗传、光遗传和 与高通量行为分析和全脑神经元相结合的化学发生扰动 使用单细胞分辨率进行活动监测。在项目1中，我们将识别促进睡眠的中缝大小的子系统 动态平衡，并识别作用于这些子系统上下游的遗传和神经元回路 在睡眠控制方面。在项目2中，我们将确定褪黑素受体介导的促进睡眠的功能 褪黑激素，并进行筛选，以识别褪黑素执行昼夜节律的神经元 对睡眠的调节。项目3建立在我们最近发现的下丘脑神经肽原运动素2的基础上 抑制由光和暗诱导的遮罩行为。与项目1类似，我们将确定基因和 神经回路作用于原激动素2的上游和下游以调节掩蔽。在项目4中，我们将 验证全基因组鉴定的大量人类睡眠障碍候选基因 协会研究。我们将通过利用斑马鱼高效和廉价地产生和测试许多 睡眠表型突变系。我们将确定确认候选基因的机制 调节睡眠，并将这些基因整合到项目1-3中确定的途径中。动态平衡(项目 1)、昼夜节律(项目2)和光依赖(项目3)调节睡眠以及睡眠的机制 在人类中识别并在斑马鱼中验证的紊乱基因(项目4)很可能在多个 产生睡眠或清醒状态的水平。这一研究计划提供了一个统一的平台来探索 在每个项目中确定的基因和神经元之间的相互作用。这将使我们能够推导出一个全面的 了解调节睡眠的机制，并将为睡眠障碍的新疗法奠定基础。

ABSTRACT Sleep disorders are pervasive, contribute to morbidity in several psychiatric disorders, and cause an annual economic burden of $100 billion. However, despite its importance for health, the mechanisms that regulate sleep are poorly understood. We are taking a new approach to this problem by exploiting useful features of zebrafish to answer an important and basic question: What genetic and neuronal mechanisms regulate sleep? Sleep is regulated by a homeostatic process that reflects internal cues of sleep need and a circadian process that is entrained by environmental cues and restricts sleep to the appropriate time. Sleep is also directly and rapidly regulated by a phenomenon known as masking, in which light induces wake and dark induces sleep in diurnal animals. Factors that regulate the homeostatic process have been identified, including our recent finding that the serotonergic raphe promote sleep homeostasis in zebrafish and mice. We also showed that melatonin is essential for circadian regulation of sleep in zebrafish, and identified a pathway in the brain that regulates masking. Here we build upon these discoveries to elucidate mechanisms that underlie homeostatic, circadian, and light-dependent regulation of sleep. We will investigate these mechanisms using zebrafish, a diurnal vertebrate with several advantages that complement rodent models, using a combination of genetic, optogenetic, and chemogenetic perturbations coupled with high-throughput behavioral assays and whole-brain neuronal activity monitoring with single cell resolution. In Project 1, we will identify raphe subsystems that promote sleep homeostasis, and identify genetic and neuronal circuits that act upstream and downstream of these subsystems in sleep control. In Project 2, we will identify melatonin receptors that mediate the sleep-promoting function of melatonin, and also perform a screen to identify neurons through which melatonin implements circadian regulation of sleep. Project 3 builds on our recent discovery that the hypothalamic neuropeptide prokineticin 2 suppresses both light- and dark-induced masking behavior. Similar to Project 1, we will identify genetic and neuronal circuits that act upstream and downstream of prokineticin 2 to regulate masking. In Project 4, we will validate a large number of human sleep disorder candidate genes that were identified by genome-wide association studies. We will do so by leveraging zebrafish to efficiently and inexpensively generate and test many mutant lines for sleep phenotypes. We will determine the mechanisms through which validated candidate genes regulate sleep, and integrate these genes into the pathways identified in Projects 1-3. The homeostatic (Project 1), circadian (Project 2) and light-dependent (Project 3) mechanisms that regulate sleep, as well as the sleep disorder genes identified in humans and validated in zebrafish (Project 4), are likely to be integrated at multiple levels to produce either sleep or wakefulness. This research program provides a unified platform to explore interactions between genes and neurons identified in each project. This will allow us to derive a comprehensive understanding of mechanisms that regulate sleep, and will set the stage for novel therapies for sleep disorders.