Drosophila core circadian clock neurons peptidergically regulate activity of insulin-producing cells

Central pacemaker neurons use a combination of external stimuli and neuropeptide signaling to synchronize molecular oscillations leading to circadian behaviors. The clock network structure and signaling between these pacemaker neuron groups have been well described, but how these pacemakers communicate with specific brain output regions remains poorly understood. Here, we identified how core clock neurons in Drosophila, the ventrolateral neurons (LNvs), signal to the proto-hypothalamic region, the pars intercerebralis (PI). We provide evidence to show that LNvs functionally modulate insulin-producing cells (IPCs) of the PI in a time-of-day-dependent manner. This functional connectivity relies on neuropeptidergic signaling of two classical clock neuropeptides: pigment dispersing factor (PDF) and short Neuropeptide F (sNPF). Connectomic analysis does not identify any direct synaptic inputs from clock neurons to IPCs. Small ventrolateral clock neurons, which secrete both PDF and sNPF are 15-20 mm away from IPCs, suggesting that volume transmission across these distances may be possible in the fly dorsal protocerebrum. Peptide application with functional imaging of IPCs provides insight into how these two neuropeptides may act synergistically via their receptors to signal to IPCs. Our findings indicate that LNvs can signal to IPCs by volume transmission and also form indirect multisynaptic circuits with IPCs. Thus, neuropeptides associated with intra-clock signaling also serve as circadian cues to non-clock neurons, which may model more broadly how clock peptides communicate to clock output regions.