Neuropeptidergic Regulation of Zebrafish Sleep

睡眠和清醒状态由几个大脑区域调节，这些区域通过多种神经递质发挥作用， 神经调节剂和神经肽。虽然已经确定了几个这样的大脑区域和神经肽， 调节睡眠的其他机制可能还有待发现。事实上，我们最近发现了一种 FMRFamide样神经肽家族，足以诱导线虫的睡眠状态，并发现 斑马鱼FMRFamide神经肽NPVF足以在两种线虫中诱导睡眠状态 还有斑马鱼。这些观察表明，NPVF在一种古老的中央机制中发挥作用，这种机制调节 睡吧。虽然啮齿动物通常用于脊椎动物睡眠研究，但需要遗传工具来研究NPVF在 啮齿动物还没有被描述过。斑马鱼是另一种脊椎动物模型，它表现出行为， 哺乳动物睡眠的解剖学、遗传学和药理学保护，以及使用这一简单和 廉价的脊椎动物模型将为投资所需的大量时间、劳动力和费用提供理论基础 开发研究小鼠NPVF的工具。虽然斑马鱼作为睡眠模型有一些局限性，但它的 对遗传、光遗传和药理学方法的适应性，以及其透明度和相对 简单但保守的脊椎动物大脑，为我们在这项提议中利用的睡眠研究提供了优势。 NPVF的蛋白序列和下丘脑表达在斑马鱼、啮齿动物和 这表明在斑马鱼身上的发现将与人类相关。在具体目标1中，我们使用增益-AND 功能丧失遗传学，以确定三个成熟肽中的一个或多个是否由 NPVF前蛋白是促进睡眠所必需的，也是足够的。我们还将确定哪些受体 调节NPVF在睡眠中的作用。在特定目标2中，我们将检验~15npvf表达的假设 神经元是促进睡眠所必需的，也是足够的。我们将使用非侵入性和高通量 光遗传学和化学遗传学分析刺激、抑制和消融自由行为幼虫的NPVF神经元。 在特定的目标3中，我们将识别在刺激和抑制反应中被激活或被抑制的神经元 用双光子单面照明显微镜和全脑钙成像对NPVF神经元进行研究。这个 斑马鱼是目前唯一可以进行全脑钙成像的脊椎动物模型。这 该方法将以公正和全面的方式识别可能介导 NPVF对睡眠的影响，我们将使用遗传学和光遗传学对其进行功能测试。该项目将建立一个 新的和进化上保守的睡眠调节系统，并可能最终导致新的治疗方法 睡眠障碍。因为睡眠异常与几种神经精神障碍有关，而且可能 在某些情况下，该项目还可能最终导致对某些形式的 神经精神障碍。

Sleep and wake states are regulated by several brain regions that act via multiple neurotransmitters, neuromodulators and neuropeptides. While several such brain regions and neuropeptides have been identified, additional mechanisms that regulate sleep likely remain to be discovered. Indeed, we recently identified a family of FMRFamide-like neuropeptides that are sufficient to induce a sleep-like state in C. elegans, and found that the zebrafish FMRFamide neuropeptide NPVF is sufficient to induce sleep-like states in both C. elegans and zebrafish. These observations suggest that NPVF acts in an ancient and central mechanism that regulates sleep. While rodents are commonly used for vertebrate sleep studies, genetic tools needed to study NPVF in rodents have not been described. The zebrafish is an alternative vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep, and studies using this simple and inexpensive vertebrate model will provide a rationale to invest the significant time, labor and expense required to generate tools to study NPVF in mice. While the zebrafish has some limitations as a sleep model, its amenability to genetic, optogenetic and pharmacological approaches, as well as its transparency and relatively simple yet conserved vertebrate brain, provide advantages for sleep studies that we exploit in this proposal. Both the protein sequence and hypothalamic expression of NPVF are conserved in zebrafish, rodents and humans, suggesting that findings in zebrafish will be relevant to humans. In Specific Aim 1 we use gain- and loss-of-function genetics to determine whether one or more of the three mature peptides produced by the NPVF preproprotein are necessary and sufficient to promote sleep. We will also determine which receptors mediate the role of NPVF in sleep. In Specific Aim 2 we will test the hypothesis that the ~15 npvf-expressing neurons are necessary and sufficient to promote sleep. We will use noninvasive and high-throughput optogenetic and chemogenetic assays to stimulate, inhibit and ablate NPVF neurons in freely behaving larvae. In Specific Aim 3 we will identify neurons that are activated or inhibited in response to stimulation and inhibition of NPVF neurons using two-photon single plane illumination microscopy and whole-brain calcium imaging. The zebrafish is the only vertebrate model in which whole-brain calcium imaging is currently feasible. This approach will in an unbiased and comprehensive manner identify neurons that may mediate the effects of NPVF on sleep, which we will functionally test using genetics and optogenetics. This project will establish a novel and evolutionarily conserved sleep-regulatory system, and may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neuropsychiatric disorders and may be causal in some cases, this project may also eventually lead to improved therapies for some forms of neuropsychiatric disorders.