Immediate Responses to Ambient Light in the Suprachiasmatic Nucleus Reveal Distinct Activity Profiles and Retinal Connectivity for SCN <sup>VIP</sup> Neurons

Light is essential for the entrainment of circadian rhythms through the hypothalamic suprachiasmatic nucleus (SCN), the circadian rhythm pacemaker. Vasoactive intestinal peptide (VIP)-expressing neurons in the SCN (SCNVIP) are known to respond immediately to light, resulting in regulation of the sleep-wake cycle. However, the effect of light properties, such as spectra and intensity, has not been assessed in freely behaving animals in vivo . In addition, the SCN is known to get input from intrinsically photosensitive retinal ganglion cells (ipRGC), expressing Opn4 (Melanopsin); however, the specific connectivity of SCNVIP neurons from RGCs and ipRGCs has not been determined. Using fiber-photometry recordings of GCaMP signal from SCNVIP neurons in behaving mice, we observed that SCNVIP neurons were highly sensitive to blue light (438 nm), and although they had lower responsiveness to red light (650 nm), it was enough to affect SCNVIP neuron activity at both short (15 sec) and long (10 min) intervals. Interestingly, the last caused a phase delay in locomotor activity when applied during dark-dark (DD) conditions, compared to a complete DD condition over three weeks. To better understand SCNVIP neuron light sensitivity and connectivity, we observed that high and low-intensity blue light caused two unique activity patterns. These distinct activity profiles were also identified under the influence of Opn4 antagonist and at the single-cell level, separating SCNVIP neurons into two subpopulations. The two activity profiles suggest distinct connectivity of SCNVIP neurons to RGCs, supported by monosynaptic tracing. These conclusions have important implications for basic science, animal and human health, and the design of light-related treatments.