Central and peripheral clocks; Mechanisms of temporal integration

我们研究了交叉上核（SCN）中的多个振荡细胞如何相互同步并携带到外部光线索以协调外围时钟的机制。我们还研究了SCN中心时钟对外周时钟的同步机制。本研究建立了两种荧光素酶报告基因转基因小鼠。转基因小鼠的建立：我们建立了在Per1启动子（Per1-luc）控制下表达萤火虫荧光素酶的转基因小鼠和在Per1启动子（ppvb1）下携带分泌荧光素酶（VL）和不分泌荧光素酶（FL）的双报告基因转基因小鼠。这些转基因小鼠的行为节律和对光的反应与野生型对照无明显差异。中心时钟的多振荡结构：携带LD12:12的Per1-luc小鼠暴露于LD18:6或LD6:18。在监测行为节律后，连续2张冠状切片，连续5天监测生物发光。在检查的所有SCN切片中，在主观中午前后检测到强烈的Pen表达节律，但在LD18:6下的前SCN中检测到双峰Pen峰值。无论光周期如何，SCN前部的Pen峰与活动开始相关，而SCN后部的Pen峰与活动结束相关，这表明E和M振荡器分别位于SCN前部和后部。外周时钟的自主振荡和相位调节：测量PVBF小鼠的各种外周组织和Bmal1的表达节律。在所有检测的外周组织中，检测到强劲的Bmal1节律，其周期和峰值阶段都是特定于组织的。肝切片长期培养显示出自主节律延续和由介质交换设定的相位依赖相位，提示单细胞振荡耦合。

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.