Sleep promotion in zebrafish by hypocretin neuronal networks

描述（由申请人提供）：睡眠是动物王国中保存的基本生物过程。斑马鱼是一种在发育生物学中广泛使用的小型昼夜硬骨鱼，将被用作研究睡眠和睡眠调节网络的模型。先前的研究表明，这种物种存在真正的睡眠状态，如使用行为标准（昼夜节律，可逆的不动期，位置偏好，特征姿势，唤醒阈值增加，睡眠反弹）所定义的。此外，在哺乳动物中鉴定的睡眠-觉醒分子因子在该物种中也是保守的，以及催眠药物靶点。最后，下丘脑泌素（hcrt，又名食欲素）系统，一个参与睡眠障碍发作性睡病的病理生理学的系统，也存在于斑马鱼中。HCRT是参与哺乳动物睡眠和能量平衡调节的神经肽。我们发现在成年斑马鱼下丘脑中有少于50个下丘脑泌素神经元，并克隆了一个非常紧凑的斑马鱼hcrt启动子（1 kb），能够精确模拟天然下丘脑泌素模式。此外，我们还发现了斑马鱼中唯一的下视丘分泌素受体（hcrtr）中的无效突变（hcrtr 168）。缺乏这种受体的鱼会有不完整的睡眠，在黑暗中睡眠减少30%。在该提议中，我们提出用酵母单杂交测定法鉴定能够结合核心13个碱基对启动子元件的转录因子，所述核心13个碱基对启动子元件对于下丘脑泌素表达是必需的且足够的（第一特定目标）。此外，我们计划研究斑马鱼hcrt神经回路，以了解其促进睡眠的功能（第二个具体目标）。为此，我们将（i）研究其与hcrt：GFP和hcrtr：mCherry转基因系的联系，（ii）使用hcrt：GCaMP 2和hcrtr：GCaMP 2转基因系，通过钙成像试验研究其何时具有活性，以及（iii）通过分析神经递质表型共表达和hcrtr阳性神经元（hcrtr：GCaMP 2）的活性，评估下丘脑泌素信号传导在hcrt神经元沉默（hcrt：Kir2.1）或hcrtr缺失（hcrtr 168）时是兴奋性还是抑制性。最后，在第三个具体目标，我们将确定新的睡眠和觉醒活跃的核在这个物种中使用立即早期基因（c-fos）表达分析成年斑马鱼大脑在夜间和白天，睡眠剥夺后和催眠药物治疗后。 15%的人口患有睡眠障碍。然而，睡眠仍然是一个知之甚少的现象。我们的实验室使用一个简单的模型动物，斑马鱼，了解神经元下丘脑泌素系统的发展，组织和功能，负责睡眠障碍发作性睡病时中断。作为一种发育模型，斑马鱼将帮助我们了解下丘脑泌素表达在人类中是如何丢失的，作为一种神经生物学/睡眠模型，它将帮助我们破译睡眠的潜在分子和细胞机制，并产生未来有效治疗所不可或缺的基础知识。

DESCRIPTION (provided by applicant): Sleep is a fundamental biological process conserved across the animal kingdom. Zebrafish, a small diurnal teleost extensively used in developmental biology, will be used as a model to study sleep and sleep regulatory networks. Previous studies have shown that a genuine sleep-like state exists in this species, as defined using behavioral criteria (circadian rhythm, reversible periods of immobility, place preference, characteristic posture, increased arousal threshold, sleep rebound). Moreover, sleep-wake molecular actors identified in mammals are also conserved in this species as well as hypnotic drug targets. Finally, the hypocretin (hcrt, aka orexin) system, a system involved in the pathophysiology of the sleep disorder narcolepsy, also exists in zebrafish. HCRTs are neuropeptides involved in the regulation of sleep and energy balance in mammals. We found that there are less than 50 hypocretin neurons in an adult zebrafish hypothalamus and cloned a very compact zebrafish hcrt promoter (1kb) capable of accurately mimicking the native hypocretin pattern. Moreover, we also identify a null mutation (hcrtr168) in the sole hypocretin receptor (hcrtr) present in zebrafish. Fish lacking this receptor have fragmented sleep and a 30% sleep reduction in the dark. In this proposal, we propose to identify, with a yeast one-hybrid assay, transcription factor(s) able to bind a core 13 base pair promoter element essential and sufficient for hypocretin expression (first specific aim). Further, we plan to study the zebrafish hcrt neurocircuitry to understand its sleep-promoting function (second specific aim). To do so, we will, (i) study its connections with hcrt:GFP and hcrtr:mCherry transgenic lines, (ii) study when it is active with a calcium imaging assay using hcrt:GCaMP2 and hcrtr:GCaMP2 transgenic lines, and (iii) evaluate whether hypocretin signaling is excitatory or inhibitory by analyzing neurotransmitter phenotype co-expression and the activity of the hcrtr positive neurons (hcrtr:GCaMP2) when hcrt neurons are silenced (hcrt:Kir2.1) or the hcrtr is missing (hcrtr168). Finally in the third specific aim, we will identify novel sleep- and wake-active nuclei in this species using immediate early gene (c- fos) expression analysis of adult zebrafish brains during the night and the day, after sleep deprivation and after hypnotic drug treatments. PUBLIC HEALTH RELEVANCE 15% of the population suffers of sleep disorders. However, sleep is still a poorly understood phenomenon. Our laboratory uses a simple model animal, the zebrafish, to understand the development, organization and function of a neuronal hypocretin system, responsible when disrupted of the sleep disorder Narcolepsy. As a developmental model, zebrafish will help us to understand how hypocretin expression can be lost in humans and, and as a neurobiology/sleep model, it will help us to decipher the underlying molecular and cellular mechanisms of sleep, and to generate the basic knowledge indispensable for future efficient therapies.