Figure 6. Disynaptic circuits connect ipRGCs to mood-regulating centers.

(A-D) A three-virus retrograde transynaptic tracing system was used to identify the retino-PHb-vmPFC pathway. EnvA-GΔRabies-mCherry expresses mCherry in PHb neurons expressing both Cre-EYFP and the Cre-dependent helper AAV (starter neurons) and presynaptic inputs to these neurons. A retrolabeled RGC expressing mCherry counterstained for melanopsin is shown (D). (E) The PHb was transfected with a fluorescent Ca²⁺ sensor by injecting a cre-dependent GCaMP6m vector in the PHb region and a retrogradely transported ere virus in the vmPFC. (F) Average response to a 2 s light pulse delivered at zeitgeber time (ZT) 18 to a mouse kept under the T24 cycle. The Ca²⁺ transient comprises a fast-rising component plateauing for 1.1 s before a second phase supervenes to generate a higher peak 3 s after light presentation. A biphasic response could bespeak either the transfection in our experimental design of two distinct (sub)populations that respond to light with different time courses, or a functional property of a single neuronal population. (G) A map of the 14 individual trials averaged in (F) is shown. (H) PHb activity following regularly-scheduled lights-on event in T24 cycle. (I) In T7 cycle, the PHb responds to dark-to-light transitions with a biphasic Ca²⁺ transient analogous to the one observed in T24 cycle. (J) Comparison of peak fluorescence in T24 vs. T7-housed mice recorded over 72 hours. Peak fluorescence in T7 cycle is significantly higher (**p<0.001; by Student’s t test). (K-L) Time course of peak fluorescence (calculated as the difference between maximum and median for each sample recording) recorded over multiple days in T24 or T7 cycles. Dashed orange lines indicate light transitions. Data are mean±SEM; n=3 mice. Scale bars: (D) l00μm; (C) 200μm; (B) 500μm. See also Figure S6.