Sleep promotion in zebrafish by hypocretin neuronal networks

下丘脑分泌素神经元网络促进斑马鱼的睡眠

• 批准号: 7620945
• 负责人: Emmanuel J Mignot
• 金额: $ 53.78万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7620945
• 关键词: Adult; Affect; Animal Model; Animals; Arousal; Base Pairing; Behavioral; Binding; Biological Assay; Biological Process; Brain; Calcium; Cell Nucleus; Cells; Characteristics; Circadian Rhythms; DNA; Development; Developmental Biology; Drosophila melanogaster; Drug Delivery Systems; Electrophoretic Mobility Shift Assay; Elements; Excessive Daytime Sleepiness; FOS gene; Fishes; Functional disorder; Future; Gene Expression; General Population; Genes; Human; Hybrids; Hypothalamic structure; Image; Immediate-Early Genes; In Situ Hybridization; Investigation; Knowledge; Laboratories; Location; Mammals; Mining; Modeling; Molecular; Molecular Biology; Monitor; Narcolepsy; Neurobiology; Neurons; Neuropeptides; Neurotransmitters; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Population; Posture; Regulation; Research; Role; Signal Transduction; Sleep; Sleep Deprivation; Sleep Disorders; Sleep Fragmentations; Sleep Wake Cycle; Structure; System; Testing; Time; Transgenic Organisms; Yeasts; Zebrafish; energy balance; excitatory neuron; gain of function; hypnotic; hypocretin; in vivo; interest; mutant; nervous system disorder; neural circuit; neurotransmission; novel; null mutation; postsynaptic; preference; promoter; public health relevance; receptor; research study; sleep regulation; teleost; transcription factor; zebrafish development

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.

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。斑马鱼是一种广泛用于发育生物学的小型昼夜硬骨鱼，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，根据行为标准（昼夜节律、可逆的不动期、位置偏好、特征姿势、觉醒阈值增加、睡眠反弹）的定义，该物种存在真正的类睡眠状态。此外，在哺乳动物中发现的睡眠-觉醒分子作用者以及催眠药物靶标在该物种中也是保守的。最后，斑马鱼中也存在下丘脑分泌素（hcrt，又名食欲素）系统，该系统参与睡眠障碍发作性睡病的病理生理学。 HCRT 是参与哺乳动物睡眠和能量平衡调节的神经肽。我们发现成年斑马鱼下丘脑中只有不到 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)，能够准确模仿天然下丘脑分泌素模式。此外，我们还在斑马鱼中唯一的下丘脑分泌素受体（hcrtr）中发现了无效突变（hcrtr168）。缺乏这种受体的鱼的睡眠会变得支离破碎，在黑暗中的睡眠会减少 30%。在本提案中，我们建议通过酵母单杂交测定来鉴定能够结合核心 13 碱基对启动子元件的转录因子，该元件对于下丘脑分泌素表达至关重要且足够（第一个具体目标）。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:GCaMP2 和 hcrtr:GCaMP2 转基因系通过钙成像测定研究它何时具有活性，以及​​ (iii) 通过分析神经递质表型共表达和下丘脑分泌素信号传导是兴奋性还是抑制性的 当 hcrt 神经元沉默 (hcrt:Kir2.1) 或 hcrtr 缺失 (hcrtr168) 时，hcrtr 阳性神经元 (hcrtr:GCaMP2)。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立即早期基因（c-fos）表达分析来识别该物种中新的睡眠和觉醒活跃核。 公共卫生相关性 15% 的人口患有睡眠障碍。然而，睡眠仍然是一个人们知之甚少的现象。我们的实验室使用一种简单的模型动物斑马鱼来了解神经元下丘脑分泌素系统的发育、组织和功能，该系统在睡眠障碍（发作性睡病）受到干扰时负责。作为一种发育模型，斑马鱼将帮助我们了解人类下丘脑分泌素的表达是如何丢失的；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，并为未来的有效治疗提供不可或缺的基础知识。