Supplementary Material for: Disrupted Circadian Control of Hormonal Rhythms and Anticipatory Thirst by Dim Light at Night

Aims: Our study addresses underlying mechanisms of disruption of the circadian timing system by low-intensity artificial light at night (ALAN), which is a growing global problem, associated with serious health consequences. Methods: Rats were exposed to low-intensity (~2 lx) ALAN for 2 weeks. Using in situ hybridization, we assessed 24-h profiles of clock and clock-controlled genes in the suprachiasmatic nuclei (SCN) and other hypothalamic regions, which receive input from the master clock. Moreover, we measured daily rhythms of hormones within the main neuroendocrine axes as well as the detailed daily pattern of feeding and drinking behavior in metabolic cages. Results: ALAN strongly suppressed the molecular clockwork in the SCN, as indicated by the suppressed rhythmicity in the clock (Per1, Per2 and Nr1d1) and clock output (arginine vasopressin) genes. ALAN disturbed rhythmic Per1 expression in the paraventricular and dorsomedial hypothalamic nuclei, which convey the circadian signals from the master clock to endocrine and behavioral rhythms. Disruption of hormonal output pathways was manifested by the suppressed and phase-advanced corticosterone rhythm and lost daily variations in plasma melatonin, testosterone, and vasopressin. Importantly, ALAN altered the daily profile in food and water intake and eliminated the clock-controlled surge of drinking two hours prior to the onset of the rest period, indicating disturbed circadian control of anticipatory thirst and fluid balance during sleep. Conclusion: Our findings highlight compromised time-keeping function of the central clock and multiple circadian outputs, through which ALAN disturbs the temporal organization of physiology and behavior.

研究目标：本研究旨在阐明夜间低强度人工光（ALAN, artificial light at night）对昼夜节律计时系统的干扰机制。该问题已成为日益严峻的全球性问题，并可引发严重健康危害。
实验方法：将大鼠暴露于强度约2勒克斯（lx）的夜间人工光环境中，持续2周。采用原位杂交技术（in situ hybridization），我们检测了视交叉上核（SCN, suprachiasmatic nuclei）及其他接收主时钟信号输入的下丘脑区域内，时钟基因与时钟调控基因的24小时表达谱。此外，我们通过代谢笼检测了主要神经内分泌轴内激素的日节律，以及摄食与饮水行为的详细每日模式。
实验结果：夜间人工光显著抑制视交叉上核内的分子生物钟运作，这一效应体现为时钟基因（Per1、Per2与Nr1d1）及时钟输出基因（精氨酸加压素（arginine vasopressin））的节律性表达被抑制。夜间人工光还干扰了室旁核与背内侧下丘脑核内Per1基因的节律性表达，而这两个核团负责将主时钟的昼夜节律信号传递至内分泌与行为节律通路。内分泌输出通路的紊乱表现为皮质酮节律被抑制且时相提前，血浆中褪黑素、睾酮与精氨酸加压素的日波动完全消失。尤为重要的是，夜间人工光改变了摄食与饮水的每日模式，并消除了休息期开始前2小时由时钟调控的饮水激增现象，表明睡眠期间预见性口渴与体液平衡的昼夜调控受到破坏。
研究结论：本研究结果表明，夜间人工光会损害中枢时钟的计时功能以及多条昼夜节律输出通路，进而干扰生理与行为的时间组织模式。