Hypocretin and Locus Ceruleus Interactions in Zebrafish Development and Behavior

下丘脑分泌素和蓝斑在斑马鱼发育和行为中的相互作用

• 批准号: 8454987
• 负责人: Grigorios Oikonomou
• 金额: $ 4.92万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454987
• 关键词: Ablation; Address; Affect; Alzheimer' s Disease; Anatomy; Animal Model; Animals; Anxiety Disorders; Arousal; Attention deficit hyperactivity disorder; Bathing; Behavior; Behavioral; Brain; Brain Stem; Bypass; Cells; Confocal Microscopy; Cues; Development; Down Syndrome; Drug Addiction; Electrophysiology (science); Exhibits; Exposure to; Health; Human; Hypothalamic structure; Image; In Vitro; Individual; Injection of therapeutic agent; Larva; Life; Location; Mammals; Mediating; Microscopy; Monitor; Morphogenesis; Motor Activity; Mus; Mutation; Narcolepsy; Neurons; Parkinson Disease; Patients; Pattern; Peptides; Pharmaceutical Preparations; Phenotype; Probability; Process; Rattus; Resolution; Rett Syndrome; Signal Transduction; Sleep; Sleep Disorders; System; Systems Development; Techniques; Testing; Time; Wakefulness; Zebrafish; hypocretin; in vivo; insight; nerve supply; nervous system disorder; neural circuit; neuron development; noradrenergic; optogenetics; overexpression; receptor; relating to nervous system; research study; sleep regulation; teleost; time use; zebrafish development

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. !

描述（由申请人提供）：下丘脑的下丘脑分泌素（Hcrt）表达神经元和脑干蓝斑（LC）的去甲肾上腺素能（NA）神经元形成睡眠调节的两个主要中心。这些中心的活动与觉醒相关，刺激任一簇都会增加睡眠小鼠醒来的可能性。重要的是，任何一个簇的破坏都会导致神经系统衰弱。 hcrt 基因座的突变或表达 hcrt 的神经元的缺失会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、唐氏综合症和雷特综合症、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）并且Hcrt可以在体外激活LC。然而，我们对体内这两个系统之间的发育、神经元和行为相互作用的理解是有限的，因为哺乳动物中这些神经元数量众多、它们位于哺乳动物大脑深处、它们投射的复杂性、神经成像的难度。活体哺乳动物的电路以及以高通量方式分析哺乳动物行为的困难。斑马鱼幼虫是光学透明的，每个半球只有约 10 个 Hcrt 和约 6 个 LC 神经元。 hcrt 表达的哺乳动物模式以及 Hcrt 神经元解剖结构、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。该提案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 的发展以及这些投射的树枝化将使用延时显微镜在活体动物中以单神经元分辨率进行表征。为了检验 LC 神经元为 Hcrt 投射的正常延伸和树状化提供必要线索的假设，LC 将被消融和沉默。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，将生成异位 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。斑马鱼幼虫中 Hcrt 的过度表达已被证明可以增加运动活动，减少处于类睡眠状态的时间并诱导过度警觉。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。这些体内研究应该能够深入了解对睡眠调节和人类健康至关重要的两个神经元回路之间的发育和功能相互作用。 公共卫生相关性：下丘脑的下丘脑分泌素神经元和脑干的蓝斑神经元形成睡眠调节的两个主要中心。任一中枢的破坏都与使人衰弱的神经系统疾病有关：发作性睡病患者的下丘脑分泌素神经元受到破坏，而蓝斑在帕金森病和阿尔茨海默病、唐氏综合症和雷特综合症、药物成瘾、注意力缺陷多动障碍和焦虑症的发展中发挥着重要作用。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。 ！