Hypocretin and Locus Ceruleus Interactions in Zebrafish Development and Behavior

描述(申请人提供)：下丘脑的下丘脑(Hcrt)表达神经元和脑干蓝斑(LC)的去甲肾上腺素(NA)神经元形成两个主要的睡眠调节中心。这些中枢的活动与觉醒相关，刺激任一簇都会增加睡眠小鼠醒来的可能性。重要的是，任一簇的破坏都可能导致衰弱的神经紊乱。Hcrt基因突变或hcrt表达神经元的丢失会导致睡眠障碍发作性睡病。LC的中断被认为与帕金森氏症和阿尔茨海默病、唐氏综合症和雷特综合征、药物成瘾、注意力缺陷多动障碍和焦虑症有关。体外研究已经为Hcrt神经元和LC之间的相互作用提供了强有力的证据。Hcrt神经元密集分布于LC，LC神经元表达Hcrt受体(HcrtR)，Hcrt在体外可激活LC。然而，由于哺乳动物中这两个神经元的数量众多，它们位于哺乳动物大脑深处，它们的投射复杂，活体哺乳动物的神经回路成像困难，以及高通量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、神经元和行为相互作用的了解是有限的。斑马鱼幼虫是光学透明的，每个半球只有~10个Hcrt神经元和~6个LC神经元。Hcrt在哺乳动物中的表达模式和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/清醒行为，这表明LC的功能是保守的。这项建议利用斑马鱼幼虫的优势特征来研究Hcrt-LC在发育、神经元活动和行为方面的相互作用。Hcrt神经元投射到LC的发展和这些投射的树枝状分布将在活体动物的单个神经元分辨率下使用时间推移显微镜进行表征。为了验证LC神经元为HCRT投射的正常延伸和树枝形成提供必要线索的假设，LC将被消融和沉默。为了验证LC提供的线索足以指导Hcrt神经元形态发生的假设，将产生异位LC样神经元。Hcrt的过表达，以及Hcrt神经元的光遗传刺激，将被用来评估Hcrt神经元是否可以在体内驱动LC激活。Hcrt在斑马鱼幼体中的过表达已被证明可以增加运动活动，减少处于睡眠状态的时间，并诱导高度唤醒。细胞消融和神经沉默技术将被用来评估LC在多大程度上调节这些表型。这些活体研究应该能深入了解两个神经元回路之间的发育和功能相互作用，这两个回路对睡眠调节和人类健康至关重要。

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.