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

用于长期体内记录的生物发光/荧光成像系统

• 批准号: 7595613
• 负责人: HERMAN WIJNEN
• 金额: $ 38.57万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7595613
• 关键词: Accounting; Affect; Amacrine Cells; Animals; Behavior; Behavioral Assay; Biological; Bioluminescence; Body Temperature; Brain; Cardiovascular Physiology; Cells; Circadian Rhythm Sleep Disorders; Circadian Rhythms; Computer Systems Development; Crying; Cultured Cells; Defect; Detection; Development; Diagnosis; Disease; Drosophila genus; Eukaryota; Exhibits; Feedback; Frequencies; Gap Junctions; Gene Expression; Genetic; Gonadotropin Hormone Releasing Hormone; Health; Hormonal; Hormones; Human; Human body; Image; Individual; Insecta; Life; Light; Link; Luciferases; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Metabolic Diseases; Metabolism; Molecular; Molecular Analysis; Monitor; Neurons; Neuropeptides; Neurotransmitters; Ovary; Pathway interactions; Photoperiod; Physiological; Play; Property; Protocols documentation; Reporter; Reporter Genes; Resolution; Respiration; Retina; Retinal Ganglion Cells; Role; Sleep Wake Cycle; System; Testing; Time; Tissues; Variant; base; cell type; circadian pacemaker; cryptochrome 1; fluorescence imaging; improved; in vivo; insight; instrument; instrumentation; public health relevance; research study; response; tool

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.

描述（由申请人提供）：影响人类健康和疾病的许多生理参数，包括睡眠/觉醒周期、核心体温、激素分泌、心血管功能、呼吸和代谢，都表现出由内部昼夜节律钟产生的每日节律。对产生这些节律的机制的研究可能有助于开发考虑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、癌症和代谢紊乱直接相关。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来直接研究。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，使得能够高频率监测活细胞和组织中的分子时钟功能。此外，最近开发的系统能够成像非常弱，但可靠的生物发光酶介导的生物发光允许高频率和高分辨率的分析分子时钟功能在单个细胞和跨细胞网络。所提出的3D生物发光/荧光成像系统将被用来在培养的昆虫和哺乳动物细胞和组织中对与特定细胞类型或生物活性相关的共表达荧光标记物进行昼夜荧光素酶报告子成像。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性和网络特性。3D生物发光/荧光成像将与果蝇中可用的强大遗传工具和行为测定相结合，以剖析控制行为的时钟控制的基因表达节律以及多巴胺能通路在这种情况下发挥的作用。将在促性腺激素释放激素神经元和大脑垂体部以及卵巢中对哺乳动物生物钟功能进行成像，并检测对不同光周期、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，以验证多巴胺能无长突细胞中是否存在昼夜节律钟，并测试内在光敏视网膜神经节细胞处间隙连接的形成是否是时钟门控的。最后，拟议的仪器将有助于描绘的功能，哺乳动物的核心时钟组件CRYPTOCHROME 1和2，允许时钟控制的荧光素酶的表达和表达水平和亚细胞定位的荧光标记转染CRY进行监测，在平行培养的细胞。公共卫生关系：拟议的仪器和实验将提供深入了解人类和动物的内部日常时间保持机制。这些研究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，还有助于阐明与时间保持缺陷相关的疾病的基础。