Synchronous Drosophila circadian pacemakers display nonsynchronous Ca²⁺ rhythms in vivo

In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brainwide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca(2+) that were entrained by environmental cues and timed by molecular clocks. However, these rhythms were not synchronous, as each group exhibited its own phase of activation. Ca(2+) rhythms displayed by pacemaker groups that were associated with the morning or evening locomotor activities occurred ~4 hours before their respective behaviors. Loss of the receptor for the neuropeptide PDF promoted synchrony of Ca(2+) waves. Thus, neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers.