Melatonin, behavior and neuronal activity in zebrafish

描述（由申请人提供）：我们研究的长期目标是确定褪黑激素对人类睡眠和昼夜节律的生理影响的机制，并将这些知识应用于设计新的失眠治疗策略。我们最近的研究已经确定，斑马鱼的昼夜休息状态与睡眠有关键的行为相似性，褪黑激素可以通过特定的褪黑激素受体促进斑马鱼的睡眠状态。该项目的目标是确定内源性褪黑激素夜间褪黑激素受体激活对斑马鱼夜间睡眠样状态和昼夜节律活动的定量参数的贡献，确定负责睡眠和昼夜节律调节的褪黑激素受体亚型，并确定网状脊髓神经元介导褪黑激素治疗的运动效应。褪黑激素对野生型和褪黑激素受体敲除的斑马鱼幼虫的睡眠状态和昼夜节律参数的影响将通过以下方法进行评估：i)用于运动活动记录的高通量自动图像分析系统；Ii)褪黑激素受体配体对褪黑激素受体亚型的不同亲和力及对褪黑激素合成的抑制作用；Iii)用反义寡核苷酸敲除褪黑素受体亚型；Iv)体内共聚焦钙成像用于监测幼虫运动行为高速记录过程中单个神经元的自发和诱发活动；V)共聚焦显微镜下激光消融候选神经元，然后进行行为评价。这些研究将阐明内源性褪黑激素在昼行性脊椎动物睡眠调节中的作用，表征褪黑激素受体的功能特异性，以及网状脊髓神经元在褪黑激素运动效应中的作用。它们也将为识别神经元网络和细胞内信号通路介导褪黑激素对睡眠样状态和昼夜节律的影响提供背景。此外，这些关于斑马鱼睡眠状态生理调节的研究的继续，结合多种斑马鱼突变体的可用性和斑马鱼基因组完整序列的可用性，可能会更好地理解脊椎动物睡眠调节和睡眠功能的遗传基础。

DESCRIPTION (provided by applicant): The long-term goal of our studies is to identify the mechanisms mediating the physiological effects of melatonin on human sleep and circadian rhythms, and to apply this knowledge to designing new therapeutic strategies for insomnia. Our recent studies have established that rest state in diurnal genetically well-characterized lower vertebrate, zebrafish, has critical behavioral similarities with sleep and that melatonin can promote a sleep-like state in zebrafish via specific melatonin receptors. The goals of the proposed project are to determine the contribution of overnight melatonin receptor activation by endogenous melatonin to quantitative parameters of nighttime sleep-like state and the circadian rhythm of activity in zebrafish, to identify melatonin receptor subtypes responsible for sleep and circadian modulation, and to identify the reticulospinal neurons mediating locomotor effects of melatonin treatment. The effects of melatonin on sleep-like state and circadian rhythm parameters will be assessed in wild-type and melatonin receptor knockdown larval zebrafish using: i) high throughput automatic image analysis system for locomotor activity recordings; ii) melatonin receptor ligands with different affinity to melatonin receptor subtypes and inhibition of melatonin synthesis; iii) knockdown of melatonin receptor subtypes with antisense morpholino oligonucleotides; iv) in vivo confocal calcium imaging for monitoring spontaneous and evoked activity of individual neurons during high-speed recordings of larval locomotor behavior; v) laser ablation of candidate neurons under confocal microscope, followed by behavioral evaluation. These studies would clarify the role of endogenous melatonin in sleep regulation in the diurnal vertebrate, characterize functional specificity of melatonin receptors and role of reticulospinal neurons in locomotor effects of melatonin. They would also constitute a background for the identification of neuronal networks and intracellular signaling pathways mediating the effects of melatonin on sleep-like state and circadian rhythmicity. Furthermore, a continuation of these studies on the physiological regulation of the sleep-like state in zebrafish, in conjunction with the availability of multiple zebrafish mutants and a complete sequence of zebrafish genome soon to be available, could potentially lead to a better understanding of the genetic basis of sleep regulation and sleep function in vertebrates.