Sleep promotion in zebrafish by hypocretin neuronal networks

睡眠是整个动物界保守的一个基本的生物过程。斑马鱼，一种昼夜活动的小鱼 硬骨鱼广泛应用于发育生物学，将被用作研究睡眠和睡眠调节的模型 网络。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，正如使用 行为标准(昼夜节律、可逆性不动周期、位置偏好、特征姿势、 提高觉醒阈值，睡眠反弹)。此外，在哺乳动物中发现的睡眠-觉醒分子因素是 在本物种以及催眠药物靶标中也保守。最后，下丘脑肌素(hcrt，又名增食欲素)系统， 与睡眠障碍发作性睡病的病理生理学有关的系统也存在于斑马鱼中。HCRT是 神经肽参与调节哺乳动物的睡眠和能量平衡。我们发现有更少的 在一条成年斑马鱼的下丘脑中克隆了一个非常紧凑的hcrt 启动子(1kb)能够准确地模拟天然的下丘脑克汀型式。此外，我们还标识了一个空 斑马鱼中唯一的下丘脑素受体(Hcrtr)突变(Hcrtr168)。缺乏这种受体的鱼类 零碎的睡眠，在黑暗中睡眠减少30%。在这项提案中，我们建议用一种酵母来鉴定 单杂交试验，转录因子(S)能够结合核心13bb的启动子元件必需的和 足够表达下丘脑分泌素(第一个特定目标)。此外，我们计划研究斑马鱼hcrt神经回路。 了解其促进睡眠的功能(第二个特定目的)。为此，我们将：(I)研究其与 Hcrt：GFP和hcrtr：mCherry转基因株系，(Ii)用钙显像法研究其活性 Hcrt：GCaMP2和hcrtr：GCaMP2转基因株系，以及(Iii)评价下丘脑分泌素信号是兴奋性的还是 通过分析神经递质表型共表达和hcrtr阳性神经元的活性进行抑制 (hcrtr：GCaMP2)当hcrt神经元静音(hcrt：Kir2.1)或hcrtr缺失时(Hcrtr168)。最终在第三局 具体地说，我们将利用即刻早期基因(c-基因)在该物种中鉴定新的睡眠和觉醒活性核团。 成年斑马鱼大脑在夜间和白天、睡眠剥夺后和睡眠剥夺后的FOS)表达分析 催眠药物治疗。15%的人口患有睡眠障碍。然而，睡眠仍然很差 理解现象。我们实验室使用了一种简单的模型动物--斑马鱼 了解神经元下丘脑系统的发育、组织和功能， 睡眠障碍发作时负责发作性睡病。作为一种发展模式， 斑马鱼将帮助我们了解人类下丘脑泌素的表达是如何丢失的 而且，作为神经生物学/睡眠模型，它将帮助我们破译潜在的 睡眠的分子和细胞机制，并产生基本知识 对于未来的有效治疗来说是不可或缺的。

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 coexpression 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. 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.