Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNs) and small ventrolateral neurons (s-LNs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNs, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D-like dopamine receptor (Dop1R1) in the l-LNs and s-LNs, but not of Dop1R2, abolished the depolarization of l-LNs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNs. Downregulation of Dop1R1 or Dop1R2 in the l-LNs and s-LNs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNs do not use Dop1R2 receptors and the s-LNs also respond to dopamine, we conclude that the s-LNs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNs.In insect and mammalian brains, sleep-promoting networks are intimately linked to the circadian clock, and the mechanisms underlying sleep and circadian timekeeping are evolutionarily ancient and highly conserved. Here we show that dopamine, one important sleep modulator in flies and mammals, plays surprisingly complex roles in the regulation of sleep by clock-containing neurons. Dopamine inhibits neurons in a central brain sleep center to promote sleep and excites wake-promoting circadian clock neurons. It is therefore predicted to promote wakefulness through both of these networks. Nevertheless, our results reveal that dopamine acting on wake-promoting clock neurons promotes sleep, revealing a previously unappreciated complexity in the dopaminergic control of sleep. Drosophila melanogaster v v v v v1v v v v v v v v v v v SIGNIFICANCE STATEMENT
