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生理、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)，不会直接受到压力的影响，而且大脑其他地方的生物钟可能参与了应激和昼夜功能之间的接口。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鉴定了在认知、动机和情绪调节中发挥重要作用的昼夜节律时钟细胞。此外，我们发现，SCN外的PER2振荡会受到各种激素(皮质酮、雌激素)和行为(限制进食)的干扰，以一种区域特有的方式改变，这些干扰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在其中一些区域的表达对不同形式的应激和脑糖皮质激素信号的操纵具有敏感度和差异性。*社会隔离对人类的行为和生理功能有深远的影响，并已被广泛用于实验室啮齿动物的慢性非侵入性应激模型。目前，关于社会隔离应激(SIS)对昼夜节律功能的影响的研究还很少。最近对雄性大鼠的一项研究表明，断奶后长时间的SIS改变了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的影响以及对昼夜节律行为和内分泌功能的影响在很大程度上仍不清楚。*本研究的目的是阐明SIS对昼夜节律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隔离和分组饲养的雄性和雌性小鼠的昼夜行为(运动活动)和荷尔蒙(褪黑激素、皮质酮)表型。接下来，我将分析SIS对参与社会、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，免疫组化)节律性表达的影响。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的方法来研究两种信号机制的参与，即糖皮质激素和哺乳动物/雷帕霉素机械靶点(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，并阐明两个关键信号机制的参与，这两个关键信号机制被证明在调节大脑中的昼夜节律基因表达方面发挥关键作用。**

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. **