Drosophila clock cells use multiple mechanisms to transmit time-of-day signals in the brain.

Regulation of circadian behavior and physiology by the Drosophila brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. We show here that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. We determined that morning and evening clock neurons have time-of-day–dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. Our findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior.