Hypocretin and Locus Ceruleus Interactions in Zebrafish Development and Behavior

描述（由申请人提供）：下丘脑的下丘脑泌素（Hcrt）表达神经元和脑干中蓝斑（LC）的去甲肾上腺素能（NA）神经元形成两个主要的睡眠调节中心。这些中心的活动与觉醒有关，刺激任何一个集群都会增加睡眠小鼠醒来的可能性。重要的是，任何一个集群的破坏都可能导致衰弱性神经系统疾病。hcrt基因位点突变，或表达hcrt的神经元缺失，导致睡眠障碍发作性睡病。LC的破坏与帕金森病和阿尔茨海默病、唐氏综合征和雷特综合征、药物成瘾、注意力缺陷多动障碍和焦虑症有关。体外研究为Hcrt神经元和LC之间的相互作用提供了强有力的证据。Hcrt神经元密集地支配LC，LC神经元表达Hcrt受体（HcrtR），Hcrt在体外可激活LC。然而，由于哺乳动物中大量的这些神经元，它们在哺乳动物大脑深处的位置，它们的投射的复杂性，在活的哺乳动物中成像神经回路的困难和以高通量方式分析哺乳动物行为的困难，我们对这两个系统在体内的发育，神经元和行为之间的相互作用的理解是有限的。斑马鱼（Danio rerio）幼虫是光学透明的，每个半球只有~10个Hcrt和~6个LC神经元。哺乳动物的hcrt表达模式和Hcrt神经元的解剖、活性和功能在斑马鱼中是保守的。此外，影响NA系统的药物同样会影响斑马鱼和哺乳动物的睡眠/觉醒行为，这表明LC的功能是保守的。该建议利用斑马鱼幼虫的有利特征来研究Hcrt-LC在发育、神经元活动和行为中的相互作用。Hcrt神经元投射到LC的发展和这些投射的分支将使用延时显微镜在活体动物中以单个神经元分辨率表征。为了检验LC神经元提供Hcrt投射的正常延伸和分支所必需的线索的假设，将消融LC并使其沉默。为了检验LC提供足以指导Hcrt神经元形态发生的线索的假设，将产生异位LC样神经元。Hcrt过表达以及Hcrt神经元的光遗传学刺激将用于评估Hcrt神经元是否可以在体内驱动LC激活。在斑马鱼幼鱼中Hcrt的过度表达已经被证明可以增加自发活动，减少在睡眠状态中花费的时间，并诱导过度觉醒。细胞消融和神经沉默技术将用于评估LC介导这些表型的程度。这些在体研究应该产生的见解，在睡眠调节和人类健康的根本重要性的两个神经元回路之间的发育和功能的相互作用。 公共卫生相关性：下丘脑的下丘脑泌素神经元和脑干的蓝斑神经元形成两个主要的睡眠调节中心。任一中心的破坏都涉及使人衰弱的神经障碍：下丘脑泌素神经元在发作性睡病患者中被破坏，并且蓝斑在帕金森病和阿尔茨海默病、唐氏综合征和雷特综合征、药物成瘾、注意力缺陷多动障碍和焦虑症的发展中是重要的。该提案旨在研究这两个中心之间的物理连接，这些连接在发育过程中如何形成，以及这些系统如何相互作用以调节睡眠/觉醒行为。!

DESCRIPTION (provided by applicant): The hypocretin (Hcrt)-expressing neurons of the hypothalamus and the noradrenergic (NA) neurons of the locus ceruleus (LC) in the brainstem form two major centers of sleep regulation. Activity in these centers is correlated with wakefulness and stimulation of either cluster increases the probability of waking in sleeping mice. Importantly, disruption of either cluster can give rise to debilitating neurological disorder. Mutation of the hcrt locus, or loss of hcrt expressing neurons, results in the sleep disorder narcolepsy. Disruption of the LC has been implicated in Parkinson's and Alzheimer's diseases, Down and Rett syndromes, drug addiction, attention- deficit hyperactivity disorder and anxiety disorders. In vitro studies have provided strong evidence for interactions between Hcrt neurons and the LC. Hcrt neurons densely innervate the LC, LC neurons express the Hcrt receptor (HcrtR) and Hcrt can activate the LC in vitro. However, our understanding of the developmental, neuronal and behavioral interactions between these two systems in vivo is limited due to the large number of these neurons in mammals, their location deep within the mammalian brain, the complexity of their projections, the difficulty of imaging neural circuits in live mammals and the difficulty of analyzing mammalian behavior in a high-throughput fashion. Danio rerio (zebrafish) larvae are optically transparent and have only ~10 Hcrt and ~6 LC neurons per hemisphere. The mammalian pattern of hcrt expression and Hcrt neuron anatomy, activity and function are conserved in zebrafish. Furthermore, drugs that impinge upon the NA system similarly affect sleep/wake behaviors in zebrafish and mammals, suggesting that the function of the LC is conserved. This proposal exploits the advantageous features of zebrafish larvae to investigate Hcrt-LC interactions in development, neuronal activity and behavior. The development of Hcrt neuron projections to the LC and the arborization of these projections will be characterized at single neuron resolution in live animals using time-lapse microscopy. To test the hypothesis that LC neurons provide cues necessary for the normal extension and arborization of Hcrt projections, the LC will be ablated and silenced. To test the hypothesis that the LC provides cues that are sufficient to instruct Hcrt neuron morphogenesis, ectopic LC-like neurons will be generated. Hcrt overexpression, as well as optogenetic stimulation of Hcrt neurons, will be used to assess whether Hcrt neurons can drive LC activation in vivo. Hcrt overexpression in zebrafish larvae has been shown to increase locomotor activity, decrease the amount of time spent in a sleep-like state and induce hyperarousal. Cell ablation and neural silencing techniques will be used to assess the extent to which the LC mediates these phenotypes. These in vivo studies should yield insights into the developmental and functional interactions between two neuronal circuits of fundamental importance in sleep regulation and human health. PUBLIC HEALTH RELEVANCE: The hypocretin neurons of the hypothalamus and the locus ceruleus neurons of the brainstem form two major centers of sleep regulation. Disruption of either center is implicated in debilitating neurological disorders: hypocretin neurons are disrupted in narcoleptic patients, and the locus ceruleus is important in the development of Parkinson's and Alzheimer's diseases, Down and Rett syndromes, drug addiction, attention- deficit hyperactivity disorder and anxiety disorders. This proposal aims to investigate the physical connections between these two centers, how these connections are formed during development and how these systems interact with each other to regulate sleep/wake behaviors. !