The long term goal of this work is to better define the patterning of the developing mammalian brain. In previous experiments we have performed in vivo analyses of the growth and differentiation of optic nerve axons in the frog Xenopus laevis through the use of recently developed optical imaging techniques. This ability to perform direct microscopic examinations of optic nerve fibers as they form their connections permitted unprecedented longitudinal studies of the factors important in neuronal connectivity. The ability to observe identified axons in vivo offers the opportunity to experimentally dissect the signals that guide axonal growth in a biologically relevant setting. Here, we wish to extend this style of analysis to the developing mammalian brain through the use of MRI microscopy. The ability of MRI microscopy to obtain full three dimensional images at resolutions approaching 10 um make it well suited to following neuronal development. Small groups of cells will be labeled with the cell- autonomous contrast agents being developed in the MRI Contrast Agent Core to render their axonal projections distinct and recognizable. We will then: * image the brains as fixed preparations at high resolution to permit the construction of detailed three dimensional atlases of neuronal connectivity in developing and adult animals; * image the labeled cells and their axons over time in living animals so that the intermediate states in the development and refinement of neuronal connections can be mapped; * image the number and position of the labeled cells over time to obtain better estimates of the timing and magnitude of cell death during normal development. The results of these studies will better define the three dimensional map of axonal connections in the mammalian brain and offer insights into the interactions critical in shaping these connections, and permit us to add the key cellular processes of axonal growth, refinement, and cell death to our brain atlas.
