Circadian rhythms of PER2::LUC expression are revived by forskolin treatment. (A) EM-CCD images from the MBH slice depicted in Fig. 2 showing one and a half circadian cycles of PER2::LUC bioluminescence expression following addition of 10 μM forskolin to the culture medium. Single cells can be discriminated in the DMH (inset) and Arc. Calibration bar 250 μm. (B) Plots of relative PER2::LUC expression integrated across delineated DMH, Arc, ependymal cell layer, ME/PT and VMH. Circadian rhythms in all regions except the VMH are revived, and in the Arc, ME/PT and ependymal cell layer are potentiated with respect to initial amplitude. (C) Circadian rhythms in six representative individual cells in the DMHc, ArcD and ArcL are resynchronized. (D) Rayleigh vector plots showing phase clustering of cells in the DMHc, ArcD and ArcL at 2 days and 5 days following forskolin treatment (days indicated in panel C). Circadian rhythms are initially resynchronized in all areas (day 2: DMHc, p < 0.05; ArcD, p < 0.00001; ArcL p < 0.00001). Five days after forskolin treatment cells in the ArcD and ArcL are still synchronized (ArcD, p < 0.00001; ArcL p < 0.001). This continued synchrony is reflected in the sustained circadian rhythm in the Arc at day 5 after forskolin, when signal is integrated across the whole Arc (B). In contrast, individual cells in the DMHc become desynchronized by 5 days after forskolin (p > 0.05), which is reflected in the arrhythmicity in the whole DMH (B) at this time.