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Neuronal connectivity in the circadian clock network is essential for robust endogenous timekeeping. In the Drosophila circadian clock network, the small ventral lateral neurons (sLNvs) serve as critical pacemakers. Peptidergic communication mediated by the neuropeptide Pigment Dispersing Factor (PDF), released by sLNvs, has been well characterized. In contrast, little is known about the role of the synaptic connections that sLNvs form with downstream neurons. Connectomic analyses revealed that the sLNvs form strong synaptic connections with previously uncharacterized neurons called superior lateral protocerebrum 316 (SLP316). Here, we show that silencing the synaptic output from the SLP316 neurons via tetanus toxin expression shortened the free-running period, whereas hyperexciting them by expressing the bacterial voltage-gated sodium channel resulted in period lengthening. Under light-dark cycles, silencing SLP316 neurons caused lower daytime activity and higher daytime sleep. Our results reveal that the main postsynaptic partners of key Drosophila pacemaker neurons are a nonclock neuronal cell type that regulates the timing of sleep and activity. 

The circadian system regulates the timing of multiple molecular, physiological, metabolic, and behavioral phenomena. In Drosophila, as in other species, most of the research on how the timekeeping system in the brain controls the timing of behavioral outputs has been conducted in males, or sex has not been included as a biological variable. A critical set of circadian pacemaker neurons in Drosophila release the neuropeptide pigment-dispersing factor (PDF), which functions as a key output factor in the network with complex effects on other clock neurons. Lack of Pdf or its receptor, PdfR, results in most flies displaying arrhythmicity in activity–rest cycles under constant conditions. However, our results show that female circadian rhythms are less affected by mutations in both Pdf and PdfR. CRISPR-Cas9-mediated mutagenesis of Pdf, specifically in ventral lateral neurons (LNvs), also has a greater effect on male rhythms. We tested the influence of M-cells on the circadian network and showed that speeding up the molecular clock specifically in M-cells led to sexually dimorphic phenotypes, with a more pronounced effect on male rhythmic behavior. Our results suggest that the female circadian system is more resilient to manipulations of M-cells and the PDF pathway, suggesting that circadian timekeeping is more distributed across the clock neuron network in females.