Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations

Circadian pacemaker neurons in the Drosophila brain display daily rhythms in the levels of intracellular calcium. These calcium rhythms are driven by molecular clocks and are required for normal circadian behavior. To study their biological basis, we employed genetic manipulations in conjunction with improved methods of in vivo light-sheet microscopy to measure calcium dynamics in individual pacemaker neurons over complete 24-h durations at sampling frequencies as high as 5 Hz. This technological advance unexpectedly revealed cophasic daily rhythms in basal calcium levels and in high-frequency calcium fluctuations. Further, we found that the rhythms of basal calcium levels and of fast calcium fluctuations reflect the activities of two proteins that mediate distinct forms of calcium fluxes. One is the inositol trisphosphate receptor (ITPR), a channel that mediates calcium fluxes from internal endoplasmic reticulum calcium stores, and the other is a T-type voltage-gated calcium channel, which mediates extracellular calcium influx. These results suggest that Drosophila molecular clocks regulate ITPR and T-type channels to generate two distinct but coupled rhythms in basal calcium and in fast calcium fluctuations. We propose that both internal and external calcium fluxes are essential for circadian pacemaker neurons to provide rhythmic outputs and thereby, regulate the activities of downstream brain centers.