The purpose of this application is to obtain a photon-counting camera and accessories to allow high temporal and spatial resolution of luminescence within cultured cells and in whole animals; accessories include both multiwell plate and single tube luminometers required for optimization of expression and activation of the photoproteins in vitro and an imaging station in which whole animal imaging will be performed. There is no such high sensitivity camera or whole animal imager currently at Einstein College of Medicine, and there is currently neither type of luminometer in the nine-floor Kennedy Center housing the Department of Neuroscience, where the proposed studies will be performed. The studies that are proposed are all natural extensions of NIH-funded projects of nine investigators that will use the relatively new technique of spatially resolved luminescence recording that has become more accessible due to new generations of vector constructs in which the photoproteins have been optimized with regard to intensity and wavelengths of light output and destabilized with regard to gene expression reporting. The studies of users outlined in this proposal are diverse, including bioluminescent resonance energy transfer to determine dimerization and binding partners for connexin molecules, luminescent reporter molecules to localize ATP release from within astrocytes and astrocytoma cells, luminescence-tagged bone marrow cells in order to characterize their tissue distribution following transplantation into mice, and gene promoters fused to luminescent reporter molecules to identify time course of gene activation in cytokine-stimulated astrocytes and to develop a moderately high throughput assay by which to identify connexin-specific gap junction channel blockers. The proposal also includes the plan for a bimonthly user group meeting, through which new users will be attracted to the instrument and new analytical tools and protocols will be discussed, as well as problems that may arise. It also includes plans for training as well as inclusion of relevant methods in a graduate student course. ? ?
