Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

Dopamine is a wakefulness promoting neuromodulator in mammals and fruit flies. In , the network of clock neurons that drives sleep/activity cycles comprises both wake and sleep promoting cell types, indicating that the sleep-wake circuitry is intimately linked to the circadian clock. The large and small ventrolateral neurons (l-LNs and 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. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l-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. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LNs does not affect sleep. Unexpectedly, we find a moderate decrease of daytime sleep with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LNs do not utilize Dop1R2 receptors and the s-LNs respond also 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.