Bioluminescence/Fluorescence Imaging System for Long-Term In Vivo Recording

描述（由申请人提供）：许多影响人类健康和疾病的生理参数，包括睡眠/觉醒周期、核心体温、激素分泌、心血管功能、呼吸和新陈代谢，都表现出由内部生物钟产生的日常节律。对产生这些节律的机制的研究可能有助于制定考虑到昼夜节律变化的疾病诊断和治疗的改进方案。此外，生物钟缺陷与内在的昼夜节律睡眠障碍、癌症和代谢障碍直接相关。人类和其他真核生物的生物钟是基因表达反馈回路，可以通过监测昼夜基因表达节律直接研究。昼夜荧光素酶报告基因的使用与极其敏感的生物发光检测相结合，可以对活细胞和组织中的分子钟功能进行高频监测。此外，最近开发的系统能够成像非常微弱，但可靠的荧光素酶介导的生物发光，使得在单个细胞和细胞网络中的分子钟功能的高频和高分辨率分析成为可能。提出的3D生物发光/荧光成像系统将用于在培养的昆虫和哺乳动物细胞和组织中成像昼夜荧光素酶报告，参考与特定细胞类型或生物活性相关的共表达荧光标记。这些研究将集中在果蝇和哺乳动物的生物钟功能的细胞自主和网络特性上。3D生物发光/荧光成像将与强大的遗传工具和果蝇行为分析相结合，以剖析支配行为的生物钟控制基因表达节律，以及在这种情况下血清素能途径所起的作用。哺乳动物的生物钟功能将在促性腺激素释放激素神经元和脑结节部以及卵巢中成像，并测试对不同光周期、激素、神经肽和/或神经递质的反应。此外，将对哺乳动物视网膜进行成像，以验证多巴胺能无突细胞中是否存在生物钟，并测试内在光敏性视网膜神经节细胞间隙连接的形成是否受到生物钟控制。最后，所提出的仪器将有助于描述哺乳动物核心时钟组件CRYPTOCHROME 1和2的功能，通过允许时钟控制荧光素酶的表达和表达水平和亚细胞定位，荧光标记转染的CRY可以在平行培养的细胞中进行监测。公共卫生相关性：拟议的仪器和实验将提供对人类和动物内部日常时间保持机制的见解。拟议研究的结果不仅有助于阐明人体为什么以及如何随着日常时间的推移而表现出功能变化，而且还揭示了与时间保持缺陷有关的疾病的基础。

DESCRIPTION (provided by applicant): Many physiological parameters affecting human health and disease, including the sleep/wake cycle, core body temperature, hormonal secretion, cardiovascular function, respiration, and metabolism exhibit daily rhythms that are generated by an internal circadian clock. Studies of the mechanisms producing these rhythms may enable the development of improved protocols for diagnosis and treatment of disease that take circadian variation into account. In addition, circadian clock defects have been directly associated with intrinsic, circadian rhythm sleep disorders, cancer, and metabolic disorders. The circadian clocks of humans and other eukaryotes are gene expression feedback circuits that can be studied directly by monitoring circadian gene expression rhythms. The use of circadian luciferase reporter genes in combination with extremely sensitive bioluminescence detection has enabled high frequency monitoring of the molecular clock function in living cells and tissues. Moreover, the recent development of systems capable of imaging the very weak, but reliable luciferase-mediated bioluminescence has allowed high frequency and high resolution analysis of molecular clock function in individual cells and across cellular networks. The proposed 3D bioluminescence/fluorescence imaging system will be used to image circadian luciferase reporters in cultured insect and mammalian cells and tissues in reference to co-expressed fluorescent markers associated with specific cell types or biological activities. These studies will focus on the cell-autonomous and network properties of circadian clock function in Drosophila and mammals. 3D bioluminescence/fluorescence imaging will be used in combination with the powerful genetic tools and behavioral assays available in Drosophila to dissect the clock-controlled gene expression rhythms that govern behavior and the role that serotonergic pathways play in this context. Mammalian clock function will be imaged in the Gonadotrophin-releasing hormone neurons and Pars Tuberalis of the brain as well as the ovaries and tested for responses to different photoperiods, hormones, neuropeptides and/or neurotransmitters. In addition, mammalian retinas will be imaged to verify the presence of circadian clocks in dopaminergic amacrine cells and test if the formation of gap junctions at intrinsically photosensitive retinal ganglion cells is clock-gated. Finally, the proposed instruments will help delineate the function of the mammalian core clock components CRYPTOCHROME 1 and 2 by allowing both clock-controlled luciferase expression and the expression level and subcellular localization of fluorescently tagged transfected CRY to be monitored in parallel cultured cells. PUBLIC HEALTH RELEVANCE: The proposed instrumentation and experiments will provide insight in the internal daily time keeping mechanisms of humans and animals. Results from the proposed studies will not only help clarify why and how the human body shows functional changes with daily time, but also shed light on the basis for diseases linked to time keeping defects.