Role of Prokineticin 2 in Regulating Sleep in Zebrafish

光通过影响生物钟间接影响睡眠，但也通过一种现象直接和迅速地影响睡眠。 被称为光对行为的掩蔽或直接影响。掩蔽是指观察到光暴露 在夜间诱导夜行动物的睡眠并抑制昼行动物的睡眠，包括人类， 而白天的黑暗暴露会抑制夜行动物的睡眠。虽然面具在世界上很普遍， 在动物王国中，介导掩蔽的机制在很大程度上是未知的。了解机制， 调节睡眠很重要，因为超过10%的美国人患有慢性睡眠障碍， 治疗选择，并造成每年1000亿美元的经济负担。了解如何在 特别影响睡眠是重要的，因为至少在现代社会中观察到的一些睡眠中断 这是由于夜间广泛暴露于人造光而造成的遮蔽。我们的初步实验 斑马鱼表明，神经肽prokineticin 2（Prok 2）调节掩蔽。据我们所知，这是 第一个基因显示出在视网膜中通过黑视蛋白调节光检测的初始步骤之外的掩蔽，和 因此，它提供了一个立足点，探索遗传和神经机制，通过光直接 影响睡眠。虽然在啮齿类动物中观察到类似的表型，但未探索掩蔽作用， prok 2突变小鼠的发育缺陷混淆了睡眠研究。斑马鱼是脊椎动物的模型 它展示了哺乳动物睡眠的行为、解剖学、遗传学和药理学保护。而 斑马鱼作为睡眠模型存在一定的局限性，其对遗传学、光遗传学和药理学的适应性 方法，以及它的透明度和相对简单而保守的脊椎动物大脑，以及缺乏 prok2突变动物的发育缺陷，为我们在本研究中利用的睡眠研究提供了优势。 提议斑马鱼特别适合于研究可能是掩蔽机制的基础， 因为它是一种白天活动的脊椎动物。在具体目标1中，我们还使用了功能获得和功能丧失遗传学， 作为光遗传学和化学遗传学工具，以测试Prok 2和prok 2表达神经元 规范掩蔽。我们还测试了我们对Prok2的发现是否适用于其他已经提出的肽 以类似于Prok2的方式调节行为。在具体目标2中，我们通过以下方式探索潜在机制： 其中Prok2调节掩蔽，例如通过调节去甲肾上腺素能蓝斑的活性（Aim 图2A）、下丘脑中促进睡眠的神经肽甘丙肽的表达（Aim 2B）或其他睡眠调节剂。 调控途径。这些研究将首次确定大脑中调节大脑活动的基因和神经元。 掩盖，从而提供了一个基础，研究遗传和神经机制的基础，这 现象因为Prok2通过G蛋白偶联受体起作用，而G蛋白偶联受体适合于小分子 这项工作可能最终导致睡眠障碍和神经精神疾病的新疗法。 因睡眠中断而加重的疾病。

Light affects sleep indirectly by entraining the circadian clock, but also directly and rapidly via a phenomenon known as the masking or direct effect of light on behavior. Masking refers to the observation that light exposure at night induces sleep in nocturnal animals and suppresses sleep in diurnal animals, including humans, whereas dark exposure during the day inhibits sleep in nocturnal animals. While masking is widespread in the animal kingdom, the mechanisms that mediate masking are largely unknown. Understanding mechanisms that regulate sleep is important because over 10% of Americans suffer from chronic sleep disorders that have poor therapeutic options and cause an annual economic burden of $100 billion. Understanding how masking in particular affects sleep is important because at least some of the sleep disruption observed in modern societies results from masking due to widespread exposure to artificial light at night. Our preliminary experiments using zebrafish suggest that the neuropeptide prokineticin 2 (Prok2) regulates masking. To our knowledge, this is the first gene shown to regulate masking beyond the initial step of light detection by melanopsin in the retina, and thus it provides a foothold to explore the genetic and neurological mechanisms through which light directly affects sleep. Although a similar phenotype was observed in rodents, a role in masking was not explored, and the developmental defects of prok2 mutant mice confound sleep studies. The zebrafish is a vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep. 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, and the lack of developmental defects in prok2 mutant animals, provide advantages for sleep studies that we exploit in this proposal. The zebrafish is particularly appropriate for studying mechanisms that may underlie masking in humans because it is a diurnal vertebrate. In Specific Aim 1 we use gain- and loss-of-function genetics, as well as optogenetic and chemogenetic tools, to test the hypotheses that Prok2 and prok2-expressing neurons regulate masking. We also test whether our findings for Prok2 apply to other peptides that have been proposed to regulate behavior in a manner similar to Prok2. In Specific Aim 2, we explore potential mechanisms through which Prok2 regulates masking, such as by regulating the activity of the noradrenergic locus coeruleus (Aim 2A), the expression of the sleep-promoting neuropeptide galanin in the hypothalamus (Aim 2B) or other sleep regulatory pathways. These studies will for the first time identify genes and neurons in the brain that regulate masking, and thus provide a basis to study the genetic and neurological mechanisms that underlie this phenomenon. Because Prok2 acts via G-protein coupled receptors, which are amenable to small molecule modulation, this work may eventually lead to novel therapies for sleep disorders and for neuropsychiatric disorders that are exacerbated by disrupted sleep.