Investigating circadian communication within the brain and body

描述(由申请人提供)：SCN内的振荡器如何相互作用，以及大脑的其余部分如何相互作用仍然是一个定义模糊但关键的研究领域。SCN的不同区域投射到很大程度上重叠的大脑区域；然而，尚不清楚每个区域的输出路径传递的是冗余信号还是不同的信号。为了评估区域相互作用和不同SCN区域的功能贡献，我使用活体光周期操作分离了背侧和腹侧SCN的节律。为了跟踪SCN内单个神经元的相，我们使用了来自PER2：LUC小鼠的SCN切片的实时生物发光成像，这是一个转基因小鼠模型，其中单个细胞内的PER2产生使用萤火虫荧光素酶报告程序进行监测。在初步研究中，我使用超长的光周期(20小时光：4小时暗，LD 20：4)在体外将SCN重组为背侧和腹侧区域，表达第一个周期的解离节律。在随后的体外循环中，背侧和腹侧SCN之间的相位差减小，表明分离的区域在体外相互作用。我建议验证以下假设：1)体外时相关系随时间的变化取决于SCN区域之间的耦合；2)SCN背侧和腹侧SCN向下游组织传递功能上不同的定时信号。为了研究这些问题，我将使用多种组织类型的实时生物发光成像，成像数据的高级计算分析，药物操作，以及SCN靶点时钟基因表达的免疫组织化学。我的长期目标是了解SCN内的神经振荡器如何相互作用，形成能够调节行为和生理节奏的功能性起搏器。 与公共健康相关：昼夜节律组织长期以来一直是研究大脑功能和复杂行为之间联系的最强大的模型系统之一，研究提供了对多个层次分析的洞察。在系统水平上，SCN是第一批确定与复杂行为调控有关的离散大脑区域之一，并继续为研究支配哺乳动物大脑系统和神经功能的基本原理提供一个杰出的模型。对昼夜节律功能的细胞和分子研究揭示了昼夜节律系统、睡眠、代谢障碍、癌症、心血管疾病、情感障碍和其他健康功能障碍之间的新联系。这些发展标志着昼夜生物学对人类健康和病理的重要意义。在这里，我建议使用光周期操作来研究SCN振荡器如何相互作用以及身体其他部分的原理。在人类和啮齿动物中，光周期的变化与各种行为和生理系统的变化有关，包括生殖功能、免疫功能、代谢功能、认知功能和情感行为。由于目前的研究调查了光周期操作对昼夜节律组织的影响，结果与与日常强光暴露持续时间变化相关的人类健康问题高度相关，例如在季节变化和夜班工作中经历的变化。此外，通过研究昼夜节律组织的基本原理，这里提出的研究可能会导致新的诊断和治疗方法，以解决与生物钟功能障碍相关的其他病理状态。

DESCRIPTION (provided by applicant): How oscillators within the SCN interact with one another and the rest of the brain remains an ill-defined but critical area of research. Different regions of the SCN project to largely overlapping brain regions; however, it is unknown whether output pathways from each region convey redundant or distinct signals. To assess regional interactions and the functional contribution of different SCN regions, I have dissociated rhythms in the dorsal and ventral SCN using an in vivo photoperiodic manipulation. To track the phase of individual neurons within the SCN, we employ real-time bioluminescence imaging of SCN slices from the PER2:LUC mouse, a transgenic mouse model where a PER2 production within individual cells is monitored using a firefly luciferase reporter. In preliminary studies, I have used an ultra long photoperiod (20h light: 4h darkness, LD 20:4) to reorganize the SCN into dorsal and ventral regions that express dissociated rhythms on the first cycle in vitro. Over the subsequent cycles in vitro, the phase difference between dorsal and ventral SCN is reduced, suggesting that dissociated regions interact in vitro. I propose to test the hypotheses 1) that changes in phase relationships over time in vitro depend on coupling between SCN regions and 2) that dorsal and ventral SCN convey functionally distinct timing signals to downstream tissues. To investigate these questions, I will use real-time bioluminescence imaging of multiple tissue types, advanced computational analyses of the imaging data, pharmacological manipulations, and immunohistochemistry for clock gene expression in SCN targets. My long-term objective is to understand how neural oscillators within the SCN interact to form a functional pacemaker capable of regulating rhythms in behavior and physiology. PUBLIC HEALTH RELEVANCE: Circadian organization has long been one of the strongest model systems for investigating the links between brain function and complex behavior, with research providing insight into multiple levels of analysis. At the systems level, the SCN was among the first discrete brain regions to be conclusively linked to the regulation of complex behavior and continues to provide an outstanding model for investigating the fundamental principles that govern brain systems and neural function in mammals. Cellular and molecular studies of circadian function are revealing new links between the circadian system, sleep, metabolic disorder, cancer, cardiovascular disease, affective disorders, and other health dysfunctions. Such developments signal the import of circadian biology for human health and pathology. Herein I propose to use a photoperiodic manipulation to investigate the principles of how SCN oscillators interact with each other and the rest of the body. In humans and rodents, changes in photoperiod are associated with changes in a variety of behavioral and physiological systems, including reproductive function, immune function, metabolic function, cognitive function, and affective behavior. Since the present studies investigate the consequences of photoperiodic manipulations for circadian organization, the results are highly pertinent to human health problems associated with changes in the duration of daily bright light exposure, such as that experienced during changing seasons and night shift work. Moreover, by investigating the fundamental principles of circadian organization, the studies proposed here may lead to novel diagnostic and therapeutic approaches for addressing other pathological states related to circadian clock dysfunction.