Dopamine Signaling in Wake-Promoting Clock Neurons Is Not Required for the Normal Regulation of Sleep in Drosophila

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LN(v)s) and small ventrolateral neurons (s-LN(v)s) have been identified as wake-promoting neurons within the clock neuron network. The 1-LN(v)s are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvS 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 I-LN(v)s, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D-1-like dopamine receptor (DopIR1) in the l-LNys and s-LN(v)s, but not of Dop1R2, abolished the depolarization of l-LNvvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LN(v)s. Downregulation of Dop1R1 or Dop1R2 in the I-LN(v)s and s-LN(v)s does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of DopIRI and of nighttime sleep with downregulation of Dop1R2. Since the I-LN(v)s do not use Dop1R2 receptors and the s-LN(v)s also respond to dopamine, we conclude that the s-LN(v)s are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LN(v)s is not required for daytime arousal, but likely promotes nighttime sleep via the s-LN(v)s.