Circadian rhythms of PER2::LUC expression in the MBH. (A) EM-CCD images from an MBH slice (representative of 12 independent experiments) showing one and a half circadian cycles of PER2::LUC bioluminescence expression. 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. Expression is circadian in all regions except the VMH. Note how tissue wide circadian expression damps at different rates in different tissues. (C) Plots of integrated bioluminescence for representative individual cells in the DMHc, ArcD and ArcL from the same slice. Individual cells are still rhythmic after 8 days in culture, yet the amplitude and synchrony of rhythms decreases over time. (D) Rayleigh vector plots showing phase clustering of cells in the DMHc, ArcD and ArcL at 2 days and 5 days following culture (days indicated in panel C). Circadian rhythms are initially synchronized in all areas (day 2: DMHc, p < 0.005; ArcD, p < 0.005; ArcL p < 0.05) but become desynchronized over the course of the experiment (day 5: all p > 0.05). Filled circles indicate the phase of individual cells. Direction of arrow indicates the mean phase vector, its length indicates the significance of phase clustering, with the surrounding box indicating the variance of phase. The inner broken line indicates the significance threshold of p = 0.05.