Regulation of Zebrafish Sleep by Neuromedin U

超过10%的美国人患有慢性睡眠障碍，估计每年的成本为1000亿美元 而且几乎没有可用的治疗方法。尽管有睡眠障碍的影响，但事实上我们花了三分之一的时间 在我们的睡眠生活中，以及类似睡眠状态的进化保守中，调节睡眠的机制是 人们对此知之甚少。对脊椎动物这些机制的理解在一定程度上受到了以下因素的阻碍 哺乳动物大脑的复杂性以及在哺乳动物身上进行基因筛选的挑战。至 克服这些限制，我们和其他人最近展示了行为、解剖学、遗传学和 斑马鱼和哺乳动物之间睡眠的药理保护，使斑马鱼成为一种简单的 和廉价的睡眠脊椎动物模型。我们最近用斑马鱼表演了第一个大型银幕 寻找调节脊椎动物睡眠的基因。我们发现神经肽Neuromedin的过度表达 (NMU)促进运动活动，抑制睡眠。大多数对哺乳动物NMU的研究都集中在它的 在调节食欲方面的作用，尽管急性NMU给药被证明是暂时延长的 失眠和打乱睡眠。然而，NMU在调节睡眠中的作用还没有得到广泛的研究 而表达NMU的神经元在睡眠中的潜在作用还没有被探索过。这样做的目的是 建议使用斑马鱼来确定NMU和NMU通过 表达NMU的神经元调节睡眠。在特定的目标1中，我们将检验NMU及其受体的假设 是正常清醒水平所必需的，通过使用CRISPR/Cas9在每个基因中产生突变- 使用高通量行为分析来调节基因组编辑和监测对睡眠的影响。我们会 还研究了NMU通过交配功能增益调节NMU对睡眠影响的遗传机制 和丧失功能的斑马鱼转移到我们已经产生的缺乏或 允许影响斑马鱼和哺乳动物睡眠的神经调节剂和神经肽的过度表达。 这些实验将把NMU信号放在已知睡眠调节机制的背景下。具体而言 目的2我们将验证NMU表达的神经元通过以下方式促进觉醒和抑制睡眠的假设 利用高通量光遗传和化学遗传刺激、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，我们将检验NMU促进的假设 通过刺激表达促肾上腺皮质激素释放激素(CRH)的脑干神经元来唤醒，并且CRH 信号传递、蓝斑和去甲肾上腺素是NMU诱导觉醒所必需的。对此进行验证 假说将确定一种新的促进觉醒的神经元回路。通过描述一个基因和神经元 我们的研究结果可能最终会导致睡眠障碍的新疗法。因为 睡眠异常与几种神经疾病有关，在某些情况下可能是因果关系，这 该项目还可能最终导致一些神经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.