Sleep promotion in zebrafish by hypocretin neuronal networks

描述（由申请人提供）：睡眠是整个动物界保守的基本生物过程。斑马鱼，一种在发育生物学中广泛使用的小型昼行硬骨鱼，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，这种物种存在一种真正的类似睡眠的状态，用行为标准来定义（昼夜节律、可逆的不动期、地点偏好、特征姿势、觉醒阈值增加、睡眠反弹）。此外，在哺乳动物中发现的睡眠-觉醒分子因子以及催眠药物靶点在哺乳动物中也是保守的。最后，斑马鱼体内也存在下丘脑分泌素（hcrt，又名食欲素）系统，这一系统与睡眠障碍嗜睡症的病理生理学有关。hcrt是一种参与哺乳动物睡眠和能量平衡调节的神经肽。我们发现成年斑马鱼下丘脑中有不到50个下丘脑分泌素神经元，并克隆了一个非常紧凑的斑马鱼hcrt启动子（1kb），能够准确地模仿天然的下丘脑分泌素模式。此外，我们还在斑马鱼唯一的下丘脑分泌素受体（hcrtr168）中发现了一个零突变（hcrtr168）。缺乏这种受体的鱼睡眠时间不稳定，在黑暗中睡眠时间减少30%。在本提案中，我们建议通过酵母单杂交试验鉴定能够结合核心13碱基对启动子元件的转录因子，这些元件对下丘脑分泌素的表达至关重要（第一个特定目标）。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（第二个特定目标）。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:GCaMP2和hcrtr:GCaMP2转基因系的钙成像试验研究其活性，以及（iii）通过分析神经递质共表达和hcrtr:GCaMP2阳性神经元（hcrtr:GCaMP2）在hcrt神经元沉默（hcrt:Kir2.1）或hcrtr缺失（hcrtr168）时的活性来评估下丘脑分泌素信号是兴奋性还是抑制性。最后，在第三个具体目标中，我们将通过对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的即时早期基因（c- fos）表达分析，确定该物种中新的睡眠和清醒活跃核。

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.