Michael E. Geusz - US grants

Description (applicant's abstract): Gene expression can be followed continuously in living neural tissue by imaging bioluminescence produced from reporter genes that are based on the firefly enzyme luciferase and its substrate luciferin. This powerful, non-invasive technique can be modified to track gene activation in response to neural activity by combining the promoter for the immediate early gene c-fos with the luciferase gene. To help establish luminescence imaging in brain slice cultures as a standard neuroscience technique, an optical and tissue culture system is presented that is specifically designed for luciferase-based imaging. The penetration of luciferin into the brain slice and its influence on luminescence signals will be examined for the first time in transgenic mice that contain luciferase reporter genes. Software for providing confocal fluorescence images will be applied to bioluminescence image data. Finally, the imaging system will be validated using the c-fos/luciferase transgenic mouse and hypothalamic brain slices containing the circadian pacemaker that controls sleep and daily rhythms in behavior. Experiments will focus on imaging the interaction of the circadian pacemaker with adjacent areas. This approach has the potential of providing unparalleled views of interactions between brain regions which will complement established medical imaging techniques performed on the brain of intact subjects. Ultimately, the organization of the brain and temporal changes in brain function associated with several diseases states and behavioral disorders could be examined in animal models through in vitro bioluminescence imaging.

Abstract Bullerjahn

To enhance ongoing research activities in the departments of Biological Sciences and Chemistry at Bowling Green State University, a molecular imaging system, and a multifunction
plate reader will be placed in common lab facilities shared by the two departments. The laboratories of seven major users will use the instruments. Molecular imagers analyze radioactive, fluorescent and luminescent samples, yielding linear data over a wide dynamic range. The imager will be used to visualize and quantify electrophoretic mobility shift assays, western blots, RNase protection assays, northern blots, sequencing gels, and DNA footprints. A multifunction plate reader capable of photometric, fluorescence and luminescence modes with adequate software for data analysis and storage will be used on similar projects.

The molecular imaging system will be used in several very diverse projects, such as quantitation of ferredoxin and flavodoxin levels in metal-limited phytoplankton by western blotting; functional analyses of Fur-type repressor-operator interactions; northern/western analyses of mRNA and protein levels in brain cells comprising the circadian pacemaker;
quantitiation of western blots of hybrid Nif proteins involved in nitrogenase expression and assembly; assessing protein phosphorylation of ErbB3 receptors in; characterizing transcription of genes involved in crayfish monoamine synthesis; and quantifying by gel electrophoresis RNA-RNA interactions in model systems optimized for determining binding constants. The projects employing the plate reader are studies parallel to those employing the molecular imager, and such work includes screening cyanobacterial promoter fusions by luciferase-dependent luminescence; using such promoter fusions in a cyanobacterial-based metal biosensor assay; determining phosphate nutrient status in phytoplankton by assaying alkaline phosphatase activity; measuring interacting Nif proteins by a beta-galactosidase assay; and quantifying cAMP and cGMP levels following activation of oocytes bearing the AT2 receptor by angiotensin II.

Given the diversity of the research projects described above, the award will provide direct benefits to the widest possible spectrum of research projects at BGSU. The instrumentation will provide analytical tools currently not available on campus, thus the immediate impact will be
Improved data acquisition for the PI, co-PIs and major users. Such benefits will allow these faculty to make more rapid progress on their funded research projects. The instrumentation will also be available to other scientists on campus, thus yielding an improved environment for
research and training of graduate and undergraduate students.