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）：许多影响人类健康和疾病的生理参数，包括睡眠/唤醒周期，核心体温，荷尔蒙分泌，心血管功能，呼吸和代谢表现出每日节奏，这些节奏是由内部昼夜节律产生的。对产生这些节奏的机制的研究可能使得能够开发改进的诊断和治疗疾病的方案，这些方案考虑了昼夜节律变化。此外，昼夜节律缺陷与固有的，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昼夜节律是基因表达反馈电路，可以通过监测昼夜节律基因表达节奏直接研究。使用昼夜节律荧光素酶报告基因与极其敏感的生物发光检测结合使用，已使活细胞和组织中分子时钟功能的高频监测。此外，最近能够成像非常弱但可靠的荧光素酶介导的生物发光的系统的开发使单个细胞和跨细胞网络中分子时钟功能的高频分析和高分辨率分析。提出的3D生物发光/荧光成像系统将用于对培养的昆虫和哺乳动物细胞和组织中的昼夜节律荧光素酶报告，并参考与特定细胞类型或生物学活性相关的共表达荧光标记。这些研究将集中于果蝇和哺乳动物中昼夜节律钟功能的细胞自主和网络特性。 3D生物发光/荧光成像将与果蝇中强大的遗传工具和行为分析结合使用，以剖析控制行为的时钟控制基因表达节律以及在这种情况下血清素能途径起的作用。哺乳动物的时钟功能将在促性腺营养蛋白释放激素神经元和大脑的结核以及卵巢中成像，并测试对不同光疗，激素，神经肽和/或神经传递剂的反应。此外，将成像哺乳动物视网膜，以验证多巴胺能无花细胞中昼夜节律的存在，并测试在本质上具有光敏性视网膜神经节细胞处间隙连接的形成。最后，提出的仪器将通过允许时钟控制的荧光素酶表达以及荧光标记的转染的cry的表达水平和亚细胞定位，以在平行培养的细胞中监测荧光标记的转染的cry的表达水平和亚细胞定位，从而帮助描述哺乳动物核心时钟组分的功能。公共卫生相关性：拟议的仪器和实验将提供内部日常时间保持人类和动物机制的见解。拟议的研究的结果不仅将有助于阐明人体在日常时间内表现出功能变化的原因，而且还阐明了与时间保持缺陷相关的疾病的基础。