Central and peripheral clocks; Mechanisms of temporal integration

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We examined the mechanisms how the multiple oscillator cells in the suprashicasmatic nucleus (SCN) synchronize to each other and entrain to the external light cue to orchestrate peripheral clocks. We also studied the synchronization mechanisms of the peripheral clocks by the central clock in the SCN. We established two types of luciferase reporter transgenic mice for this study.1.Establishment of transgenic mice: We established a transgenic mouse expressing firefly luciferase under the control of Per1 promoter (Per1-luc) and a dual reporter transgenic mouse bearing secreting luciferase (VL) under Per1 promoter and non-secreting luciferase (FL) under Bmal1 promoter (PVB1). Behavioral rhythms and responses to lights were not different in these transgenic mice from wild type controls.2.Multioscillatory structure in the central clock: Per1-luc mice entrained to LD12:12 were exposed to either LD18:6 or LD6:18. After monitoring behavioral rhythms, we made two serial coronal slices and monitored bioluminescence for 5 consecutive days. Robust Pen expression rhythms were detected with a peak around the subjective noon in all SCN slices examined but anterior SCN under LD18:6 in which bimodal Pen peaks were detected. Irrespective of photoperiods, Pen peaks in the anterior SCN correlated with activity onset, while that in the posterior SCN, with the activity end, suggesting the localization of E and M oscillator in the anterior and posterior SCN, respectively.3.Autonomous oscillation and phase-adjustment of the peripheral clocks: Various peripheral tissues of PVBF mice and Bmal1 expression rhythms were measured. In all peripheral tissues examined, robust Bmal1 rhythms were detected with the periods as well as the peak phases were specific to tissues. Long-term culture of liver slices showed autonomous rhythms continuation and phase-dependent phase set by medium exchange, suggesting coupling of single cell oscillators.