Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations.

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCN) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCN neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCN neurons may regulate LMA rhythms. In vivo photometry revealed that while SCN neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCN neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCN neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCN cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCN subtypes.