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 Ca²⁺ _i imaging and investigated SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca²⁺ _i spikes superimposed upon slow changes in baseline Ca²⁺ _i levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca²⁺ _i 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 Ca²⁺ _i spikes and slow changes in baseline Ca²⁺ _i 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.