Using this SCN coupling assay, we found that SCN neurons are coupled by both VIP and GABAA signaling, and that these SCN factors operate in a cooperative or antagonistic manner depending on the state of the network. Male PER2::LUC mice (Yoo et al., 2004) were bred and raised under a 24 hr light:dark cycle with 12 hr light and 12 hr darkness (LD12:12). At 7–9 weeks of age, the mice either remained under LD12:12 or were transferred to a long-day-length condition

with 20 hr of light (LD20:4). As expected, LD20:4 produced a rapid decrease in the duration of the nocturnal active phase (Figure 1A; Figures S1A and S1B available online). In addition, LD20:4 mice displayed a stable phase angle of entrainment and free-running rhythms that derived from the predicted phase (Figures

selleck chemical 1C and S1A), both of which are measures of true entrainment. Lastly, LD20:4 decreased the free-running period by ∼30 min (Figure S1D), similar to previous Ku-0059436 chemical structure results obtained in this species (Pittendrigh and Daan, 1976a). Collectively, these results indicate that PER2::LUC mice entrain to this long-day-length condition. To investigate photoperiodic changes in pacemaker organization, coronal SCN slices were collected from PER2::LUC mice held under LD12:12 or LD20:4 (Figure 1B). Real-time bioluminescence imaging of PER2::LUC expression was conducted

in vitro and SCN spatiotemporal organization was mapped (see Experimental Procedures). Consistent with previous work (Evans et al., 2011), SCN slices from LD12:12 mice showed regional PER2::LUC peak time differences ranging from 2 to 4 hr on the first L-NAME HCl cycle in vitro (Figures 1C and S1E; Movie S1). In contrast, LD20:4 slices displayed a much larger range of PER2::LUC peak times, with reorganization of two spatially distinct subpopulations (Figures 1C and S1E; Movie S2). In particular, LD20:4 slices were characterized by a central region that phase-led a surrounding semiconcentric region by ∼6 hr on the first cycle in vitro (Figures 1C–1E, p < 0.0001). This organizational pattern resembles the functionally distinct SCN compartments that are often referred to as the “core” and “shell” (Abrahamson and Moore, 2001 and Antle et al., 2003). Indeed, the dense population of arginine vasopressin neurons that demarcates the SCN shell compartment was in spatial registry with the late-peaking shell-like region, but not the early-peaking core-like region (Figure 2). In addition to changing the spatiotemporal organization of the SCN network, LD20:4 increased the level of PER2::LUC expression within the central SCN on the first cycle in vitro (Figure 1F, p < 0.0001).