Microglia are a class of resident brain cells that play key roles in the tissue response to injury or infection of the mammalian brain. The long-term goal of this research program is to understand the cellular and molecular bases of microglial function following brain tissue injury. The present application will use dynamic imaging of cells in live mammalian brain tissues to investigate the molecular basis, regulation, and function of microglial motility. Changes in cellular motility are incorporated into most models of microglial activation following brain injury, but little is known about the motile behaviors of microglia within native brain tissue. Our working hypothesis is that parenchymal microglia represent a heterogeneous population of cells, and that differences in the motile behaviors and intercellular interactions of activated microglial cells are determined by dynamic patterns of cell adhesion molecule expression, providing a basis for functional diversity within the microglial population. Our primary goals here are to elucidate the diversity in cellular response to activation, to define and characterize distinct moti' phenotypes and intercellular interactions of activated microglia, and to determine whether these are regulated by the cytokine, tumor necrosis factor (TNF)-a, acting via the transcription factor, NF-KB. Microglial movements in live rat and mouse brain tissue slices will be analyzed by vital fluorescent staining, 3-D time-lapse confocal imaging, and computer-assisted quantitative image analysis. Retrospective antibody staining following time-lapse observation will enable a quantitative determination of immunophenotypes in relation to the stages of activation and motile phenotypes. Multi-channel time-lapse imaging will be used to characterize functional interactions among microglial cells, and between microglia and dead/dying neurons. Finally, microglial behaviors in slices from TNF receptor knockout mice will be assessed to determine whether TNF regulates motility-based microglial functions. A more complete understanding of the molecular mechanisms of microglial function should yield insight into strategies for regulating the microglial response under a variety of CNS injury conditions. including trauma, epilepsy, stroke, Alzheimer's Disease, and AIDS.
