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

有相当多的证据表明，身体或情感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，视交叉上核（SCN），不直接受到压力的影响，大脑其他地方的生物钟可能参与压力和昼夜节律功能之间的界面。使用核心时钟蛋白PER 2作为时钟标记的节律表达，我们确定了在多个前脑区域中的生物钟细胞，这些区域已知在认知，动机和情绪的调节中发挥重要作用。此外，我们发现，SCN外的PER 2振荡改变，在特定区域的方式由各种激素（皮质酮，雌激素）和行为（限制喂养）的扰动，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PER 1在这些区域中的一些区域中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是急性和差异敏感的。 社会隔离对人类的行为和生理功能有深远的影响，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然研究不足。最近一项对雄性大鼠的研究表明，断奶后长时间的SIS改变了背内侧下丘脑中时钟蛋白CLOCK的表达。然而，SIS对大脑的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。该研究的目的是阐明SIS对昼夜节律行为和生理功能以及大脑昼夜节律基因网络功能的影响。为此，我将首先描述昼夜行为（自发活动）和激素（褪黑激素，皮质酮）表型的隔离与分组圈养的雄性和雌性小鼠保持在不同的照明条件下。接下来，我将分析SIS对核心时钟基因Per 1，Per 2和Bmal 1的mRNA（qPCR）和蛋白质（蛋白质印迹，免疫组织化学）的节律表达的影响，以及与社会，动机和情绪调节有关的前脑结构中的时钟控制基因Dbp。最后，为了确定SIS和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两种信号传导机制的参与，糖皮质激素和哺乳动物/雷帕霉素机制靶点（mTOR）。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，并阐明两种关键信号传导机制的参与，这两种机制在调节大脑中的昼夜节律钟基因表达中起着关键作用。

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.