A direct SCN-to-DMH output pathway organizes circadian behavioral timing

Circadian rhythms in behavior depend on the suprachiasmatic nucleus (SCN), but how SCN timekeeping is transmitted to downstream circuits that organize daily behavioral rhythms remains poorly defined. The dorsomedial hypothalamus (DMH) has been implicated in circadian behavioral output, but lesion studies cannot determine whether the DMH contributes through local molecular timekeeping or through intact neurons that relay SCN-derived timing signals. Here, we combined DMH neuronal ablation, local molecular clock disruption, retrograde and intersectional tracing, single-cell RNA sequencing, and intersectional optogenetics to define and test a direct SCN-to-DMH output pathway. DMH neuronal ablation disrupted locomotor activity rhythms, whereas DMH Cry1/2 disruption did not, indicating that intact DMH neurons, but not local molecular timekeeping, are required for locomotor rhythmicity. Retrograde tracing identified a sparse population of DMH-projecting SCN neurons concentrated in the dorsal SCN. These neurons showed minimal overlap with canonical AVP- and VIP-expressing SCN populations and were most strongly represented in a Prokr2/Vipr2-expressing transcriptional cluster. Repeated activation of DMH-projecting SCN neurons entrained locomotor rhythms, produced persistent phase shifts after stimulation ended, and compressed the temporal distribution of activity during entrainment. Together, these findings identify sparse, molecularly distinct DMH-projecting SCN neurons capable of organizing circadian locomotor timing.