昼夜节律和睡眠的关系是近年来的研究热点之一。相对于哺乳动物的上万个神经元组成的中枢节律脑区-视交叉上核，果蝇只有150个节律神经元组成的神经回路，但能产生各种节律性行为例如睡眠和觉醒等，因此果蝇是研究节律和睡眠的较佳动物模型。我们前期研究发现，果蝇的不同等级的节律神经元DN1-DN3组成的神经回路，能将节律时间信息输出到中央脑区，并参与了对睡眠深度的维持。本项目将通过构建果蝇的转基因品系库，来精确的标记并分类果蝇脑中功能未知的下游DN3节律神经元，并利用跨突触追踪技术、膨胀显微技术等手段来描绘DN3和上下游神经元的连接回路。以及使用光遗传学偶联钙成像技术来鉴定DN1-DN3神经回路的功能性连接。最后将结合光遗传行为学工具和在体神经元活性记录方法，在行为上阐明这一节律神经回路对睡眠和觉醒阈值的调节功能。该研究将有助于我们理解节律系统对睡眠的调控机制。

The relationship between circadian rhythm and sleep is one of the core questions in the research field. Compared with the mammalian master clock located in the suprachiasmatic nuclei (SCN) of hypothalamus, which contains more than 10 thousand circadian neurons，Drosophila has only 150 circadian neurons, but can give rise to various rhythmic behaviors such as sleep and locomotion. Therefore, fly is an ideal model organism for investigating the interaction between circadian rhythm and sleep. Our preliminary study revealed a circadian output circuit which composed of hierarchical organized circadian neuron network including DN1s (Dorsal Neuron 1) and DN3s (Dorsal Neuron 3). Activation of this circuit can output circadian information to the central brain region and induce a deep sleep-like states in Drosophila with increased arousal threshold. .In this proposal, to better clarity the sleep-regulating DN3s, we plan to construct transgenic Drosophila GAL4 lines which can specifically label different subsets of DN3s, and accurately map and classify the morphologies and function of those DN3s. We will use multiple tools such as trans-synaptic tracing and expansion microscopy to demonstrate the synaptic connectivity between DN3s and upstream as well as the downstream neurons. We will also combine optogenetic tool with two-photo calcium imaging to identify the functional connection of DN1,DN3 and downstream neurons. The in vivo function of this circuit will be further tested by using optogenetic stimulation and video-recording device. Moreover, Tric-Luc method will be used to clarify the function of this circadian neuronal circuit on regulation of sleep and arousal thresholds. This research will help us better understand the regulation mechanism of the circadian system on sleep.