Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus host the circadian pacemaker that synchronizes mammalian rhythms with the day-night cycle. SCN neurons are intrinsically rhythmic, thanks to a conserved cell-autonomous clock mechanism. In addition, circuit-level emergent properties confer a unique degree of precision and robustness to SCN neuronal rhythmicity. However, the multicellular functional organization of the SCN is not yet fully understood. Indeed, although SCN neurons are well-coordinated, experimental evidences indicate that some neurons oscillate out of phase in SCN explants, and possibly to a larger extent in vivo. Here, to tackle this issue we used microendoscopic Ca imaging and investigated SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca spikes superimposed upon slow changes in baseline Ca levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca were highly coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent coactivity in neuron subsets. Hence, fast Ca spikes and slow changes in baseline Ca levels highlighted how multiple individual activity patterns could articulate within the temporal unity of the SCN cell network in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.