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他部分仍然是一个不明确但关键的研究领域。SCN的不同区域投射到大量重叠的大脑区域；然而，每个区域的输出路径是否传递冗余或不同的信号是未知的。为了评估区域相互作用和不同SCN区域的功能贡献，我使用体内光周期操作分离了SCN背侧和腹侧的节律。为了跟踪SCN内单个神经元的阶段，我们采用PER2:LUC小鼠SCN切片的实时生物发光成像，PER2:LUC小鼠是一种转基因小鼠模型，其中使用萤火虫荧光素酶报告基因监测单个细胞内PER2的产生。在初步研究中，我在体外使用超长光周期（20小时光照：4小时黑暗，LD 20:4）将SCN重组为在第一个周期中表达游离节律的背侧和腹侧区域。在随后的体外循环中，背侧和腹侧SCN之间的相位差减小，表明分离区域在体外相互作用。我建议验证以下假设：1)体外相关系随时间的变化取决于SCN区域之间的耦合；2)背侧和腹侧SCN向下游组织传递功能不同的时序信号。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、成像数据的先进计算分析、药理操作和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，形成一个能够调节行为和生理节奏的功能性起搏器。

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.