Regulation of Zebrafish Sleep by Neuromedin U

超过10%的美国人患有慢性睡眠障碍，估计每年花费1000亿美元 并且几乎没有可用的治疗方法。尽管睡眠障碍的影响，事实上，我们花了三分之一的时间， 我们的睡眠生活，以及睡眠状态的进化保护，调节睡眠的机制是 不太了解。在脊椎动物中理解这些机制的进展部分受到以下因素的阻碍： 哺乳动物大脑的复杂性以及在哺乳动物中进行基因筛选的挑战。到 克服这些限制，我们和其他人最近证明了行为，解剖，遗传和 药理学保护斑马鱼和哺乳动物之间的睡眠，建立斑马鱼作为一个简单的 和廉价的脊椎动物睡眠模型。我们最近用斑马鱼进行了第一次大规模的筛选 调节脊椎动物睡眠的基因我们发现神经肽神经介肽U的过度表达 (Nmu)促进自发活动并抑制睡眠。大多数关于哺乳动物Nmu的研究都集中在其 调节食欲的作用，尽管急性Nmu给药已显示短暂延长 失眠和睡眠中断。然而，Nmu在调节睡眠中的作用尚未得到广泛研究 并且还没有探索nMU表达神经元在睡眠中的潜在作用。的目的 这项研究的目的是利用斑马鱼来确定Nmu和 nmu表达神经元调节睡眠。在具体目标1中，我们将测试nmu及其受体 通过使用CRISPR/Cas9在每个基因中产生突变， 介导的基因组编辑和使用高通量行为测定监测对睡眠的影响。我们将 还研究了通过交配Nmu功能获得性介导Nmu对睡眠影响的遗传机制。 和功能丧失的斑马鱼，我们已经产生了一系列突变和转基因品系， 允许影响斑马鱼和哺乳动物睡眠的神经调质和神经肽的过表达。 这些实验将Nmu信号传导置于已知的睡眠调节机制的背景下。在特定 目的2：我们将通过以下方法检验nmu表达神经元促进觉醒和抑制睡眠的假设： 使用高通量光遗传学和化学遗传学方法刺激、抑制和消融这些神经元， 我们最近开发的方法。在具体目标3中，我们将测试Nmu促进 通过刺激促肾上腺皮质激素释放激素（crh）表达的脑干神经元， 信号，蓝斑和去甲肾上腺素是Nmu诱导的觉醒所需的。验证该 这一假设将确定一种新的唤醒促进神经元回路。通过描述基因和神经元 我们的研究结果可能最终会导致睡眠障碍的新疗法。因为 不正常的睡眠与几种神经系统疾病有关，在某些情况下可能是因果关系， 该项目还可能最终导致一些神经系统疾病的治疗方法得到改进。

More than 10% of Americans suffer from chronic sleep disorders, with an estimated annual cost of $100 billion and for which few therapies are available. Despite the impact of sleep disorders, the fact that we spend a third of our lives asleep, and the evolutionary conservation of sleep-like states, mechanisms that regulate sleep are poorly understood. Progress in understanding these mechanisms in vertebrates has been hindered in part by the complexity of mammalian brains and the challenge of performing genetic screens in mammals. To overcome these limitations, we and others recently demonstrated behavioral, anatomical, genetic and pharmacological conservation of sleep between zebrafish and mammals, establishing zebrafish as a simple and inexpensive vertebrate model for sleep. We recently used zebrafish to perform the first large-scale screen for genes that regulate vertebrate sleep. We found that overexpression of the neuropeptide neuromedin U (Nmu) promotes locomotor activity and inhibits sleep. Most studies of Nmu in mammals have focused on its role in regulating appetite, although acute Nmu administration has been shown to transiently prolong wakefulness and disrupt sleep. However, a role for Nmu in regulating sleep has not been extensively studied and the potential role of nmu-expressing neurons in sleep has not been explored. The objective of this proposal is to use zebrafish to determine the genetic and neurological mechanisms through which Nmu and nmu-expressing neurons regulate sleep. In Specific Aim 1 we will test the hypothesis the nmu and its receptors are required for normal levels of wakefulness by generating mutations in each gene using CRISPR/Cas9- mediated genome editing and monitoring effects on sleep using high-throughput behavioral assays. We will also investigate genetic mechanisms that mediate the effects of Nmu on sleep by mating Nmu gain-of-function and loss-of-function zebrafish to a collection of mutant and transgenic lines that we have generated that lack or allow overexpression of neuromodulators and neuropeptides that affect sleep in zebrafish and mammals. These experiments will place Nmu signaling in the context of known sleep regulatory mechanisms. In Specific Aim 2 we will test the hypothesis that nmu-expressing neurons promote wakefulness and inhibit sleep by stimulating, inhibiting and ablating these neurons using high-throughput optogenetic and chemical-genetic methods that we recently developed. In Specific Aim 3 we will test the hypothesis that Nmu promotes wakefulness by stimulating corticotropin releasing hormone (crh)-expressing brainstem neurons, and that Crh signaling, the locus coeruleus and noradrenaline are required for Nmu-induced arousal. Validation of this hypothesis would identify a novel arousal promoting neuronal circuit. By characterizing a gene and neuronal circuit that regulate sleep, our results may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neurological disorders, and may be causal in some cases, this project may also eventually lead to improved therapies for some neurological disorders.