Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. Here we demonstrate that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. We show that mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, we also demonstrate that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, we identify the perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output.

果蝇昼夜节律的行为依赖于时钟神经元异类组的网络。起搏器电路中的短程和远程信号协调时钟神经元的分子和神经节律，以产生连贯的行为输出。昼夜节律行为的神经化学是复杂的，并且尚未完全了解。在这里，我们证明气体信使一氧化氮（NO）是一个信号分子，将昼夜节律起搏器与节律性运动活动联系起来。我们显示，尽管主要起搏器神经元中的分子时钟不受影响，但缺乏一氧化氮合酶（NOS）的突变体在持续的黑暗中具有行为性心律失常。突变体的行为表型部分归因于主要起搏器神经元s-LNvs的神经突畸形。使用NOS的细胞类型选择性和阶段特定的功能获得和丧失，我们还证明了从不同细胞簇分泌的NO影响行为节律。此外，我们将神经胶质亚型（形成血脑屏障的两种神经胶质亚型之一）确定为调节昼夜运动能力输出的NO的主要来源。这些结果首次揭示了NO信号在肝癌中的关键作用。果蝇昼夜节律系统并突出神经胶质相互作用在神经回路输出中的重要性。