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). Previously thought to communicate with the PI only indirectly, 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 m 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 directly to IPCs by volume transmission and also form indirect multisynaptic circuits with IPCs, which may model more broadly how circadian clock peptides communicate with other clock output regions. Author SummaryCircadian clocks in the brains of animals from flies to humans allow animals to anticipate daily environmental changes and temporally coordinate internal processes. The central clock in the brain serves as a master pacemaker that sets the pace of circadian clocks in all other tissues. Fruit flies have been an essential model system for discovering the genetic and cellular underpinnings of circadian rhythms, with work in flies being awarded the 2017 Nobel Prize in Physiology or Medicine. Brain clocks in flies and mammals use classical synaptic communication and neuromodulatory peptides to signal between the neurons that make up the brain clock. Our study asks whether the neuropeptides that signal between clock neurons also signal from clock to non-clock neurons. We found that the signature fly clock neuropeptide pigment dispersing factor signals outside the brain clock to insulin producing cells of the brain. There are no synaptic connections from clock neurons to insulin producing cells, instead signaling occurs by diffusion of peptide signals across tens of microns, termed volume transmission. Our findings point to the possibility that neuropeptide volume transmission may be a general feature not only of intra-clock signaling but also of brain clock output signaling to non-clock neurons.