Regulation of circadian behavioral and molecular functioning by chronic social isolation stress

有相当多的证据表明，身体或情绪上的压力事件会扰乱昼夜行为和生理节律。重要的是，动物研究表明，主生物钟，即视交叉上核（SCN），不直接受到压力的影响，而大脑中其他地方的生物钟可能参与了压力和昼夜节律功能之间的界面。利用核心时钟蛋白PER2的节律性表达作为时钟标记，我们在多个前脑区域发现了在认知、动机和情绪调节中发挥重要作用的生物钟细胞。此外，我们发现SCN外的PER2振荡以区域特异性的方式被各种激素（皮质酮、雌激素）和行为（限制进食）扰动所改变，这些扰动直接影响动机和情绪状态。重要的是，我们还发现核心时钟蛋白PER1在这些区域的表达对不同形式的应激和脑糖皮质激素信号传导的操纵具有敏锐和不同的敏感性。社会隔离对人类的行为和生理功能有着深远的影响，并已被广泛用作实验室啮齿动物慢性非侵入性应激的模型。目前，社会隔离压力（SIS）对昼夜节律功能的影响仍未得到充分研究。最近一项对雄性大鼠的研究表明，长时间的断奶后SIS会改变下丘脑背内侧的时钟蛋白clock的表达。然而，SIS对大脑核心分子昼夜节律基因网络以及昼夜节律行为和内分泌功能的影响在很大程度上仍然未知。本研究的目的是阐明SIS对昼夜行为和生理功能的影响以及对大脑昼夜基因网络功能的影响。为此，我将首先描述在不同光照条件下单独饲养与分组饲养的雄性和雌性小鼠的昼夜行为（运动活动）和激素（褪黑激素、皮质酮）表型。接下来，我将分析SIS对涉及社会、动机和情绪调节的前脑结构中核心时钟基因Per1、Per2和Bmal1以及时钟控制基因Dbp的mRNA （qPCR）和蛋白质（Western blotting，免疫组织化学）节律性表达的影响。最后，为了确定SIS与昼夜节律功能之间的联系，我将使用药理学和遗传学方法来研究糖皮质激素和哺乳动物/雷帕霉素机制靶点（mTOR）两种信号机制的参与。总之，这些研究有望确定社会隔离对行为和分子昼夜节律功能的影响，并阐明在大脑生物钟基因表达调节中发挥关键作用的两种关键信号机制的参与。

There is considerable evidence that physically or emotionally stressful events disrupt circadian behavioral and physiological rhythms. Importantly, animal studies have shown that the master circadian clock, the suprachiasmatic nucleus (SCN), is not directly affected by stress and that circadian clocks elsewhere in the brain are likely involved in the interface between stress and circadian functioning. Using the rhythmic expression of the core clock protein, PER2 as a clock marker, we identified circadian clock cells in multiple forebrain regions that are known to play important roles in the regulation of cognition, motivation, and emotion. Furthermore, we found that the PER2 oscillations outside the SCN are altered, in a region-specific manner by various hormonal (corticosterone, estrogen) and behavioral (restricted feeding) perturbations that directly affect motivational and emotional state. Importantly, we also found that the expression of the core clock protein, PER1 in some of these regions is acutely and differentially sensitive to different forms of stress and to manipulations of brain glucocorticoid signaling. Social isolation has profound effects on behavioral and physiological functioning in humans and has been used extensively as a model of chronic non-invasive stress in laboratory rodents. Currently, the effect of social isolation stress (SIS) on circadian functioning remains poorly studied. A recent study in male rats has shown that prolonged post-weaning SIS alters the expression of the clock protein, CLOCK in the dorsomedial hypothalamus. However, the effect of SIS on the core molecular circadian gene network of the brain and on circadian behavioral and endocrine functioning remains largely unknown. The aim of the proposed research is to elucidate the effect of SIS on circadian behavioral and physiological functioning and on the functioning of the brain circadian gene network. To this end, I will first characterize the circadian behavioral (locomotor activity) and hormonal (melatonin, corticosterone) phenotypes of isolated vs. grouped-housed male and female mice kept under different lighting conditions. Next, I will analyze the effect of SIS on the rhythmic expression of the mRNA (qPCR) and proteins (Western blotting, immunohistochemistry) of the core clock genes, Per1, Per2 and Bmal1, and the clock-controlled gene, Dbp, in forebrain structures implicated in social, motivation and emotion regulation. Lastly, to identify links between SIS and circadian functioning I will use pharmacological and genetic approaches to investigate the involvement of two signaling mechanisms, glucocorticoids and mammalian/mechanistic target of rapamycin (mTOR). Together, these lines of research are expected to define the impact of social isolation on behavioral and molecular circadian functioning and to elucidate the involvement of two key signaling mechanisms shown to play a key role in the regulation of circadian clock gene expression in the brain.