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起作用， 神经调质和神经肽。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仍有待发现。事实上，我们最近发现了一个 足以在C. elegans，并发现 斑马鱼FMRFamide神经肽NPVF足以诱导两种C. elegans 还有斑马鱼这些观察结果表明，NPVF在一个古老的中央机制中起作用， 睡吧虽然啮齿动物通常用于脊椎动物睡眠研究，但研究NPVF所需的遗传工具， 啮齿动物尚未被描述。斑马鱼是另一种脊椎动物模型， 哺乳动物睡眠的解剖学、遗传学和药理学保护，以及使用这种简单和 一个廉价的脊椎动物模型将为投入所需的大量时间、劳动力和费用提供理论基础 来研究小鼠的NPVF。虽然斑马鱼作为睡眠模型有一些局限性， 对遗传学、光遗传学和药理学方法的顺从性，以及其透明度和相对 简单而保守的脊椎动物大脑，为我们在这个提议中利用的睡眠研究提供了优势。 NPVF的蛋白质序列和下丘脑表达在斑马鱼、啮齿动物和哺乳动物中是保守的。 这表明在斑马鱼中的发现将与人类有关。在具体目标1中，我们使用增益， 功能丧失遗传学，以确定是否有一个或多个的三个成熟肽产生的 NPVF前原蛋白是促进睡眠所必需和足够的。我们还将确定哪些受体 调节NPVF在睡眠中的作用在具体目标2中，我们将检验以下假设： 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的 光遗传学和化学遗传学测定以刺激、抑制和消除自由行为的幼虫中的NPVF神经元。 在具体目标3中，我们将识别在刺激和抑制反应中被激活或抑制的神经元 使用双光子单平面照明显微镜和全脑钙成像的NPVF神经元。的 斑马鱼是目前唯一可行的全脑钙成像的脊椎动物模型。这 这种方法将以公正和全面的方式识别可能介导以下影响的神经元： NPVF对睡眠的影响，我们将使用遗传学和光遗传学进行功能测试。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， 睡眠障碍因为不正常的睡眠与几种神经精神疾病有关， 在某些情况下，该项目也可能最终导致某些形式的疾病的治疗方法得到改善。 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.