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

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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.