A basic feature of the nervous system is the high degree of specificity with which synaptic connections among neurons and between neurons and muscles are formed. It is of fundamental importance to learn how this specificity arises. How does a nerve cell know when and where to grow and what to innervate? The proposed experiments will explore these questions by determining 1) the patterns of axonal growth and target selection and 2) the patterns of intercellular communication of developing motoneurons. A newly developed technique will be used to observe living motoneurons, in vivo, as their axons grow and make synaptic contacts with muscle fibers. Primary motoneurons which are few in number and uniquely identifiable in zebrafish embryos will be labeled with flourescent dyes by direct injection or by injection of precursor cells. The labeled cells will be observed in the developing fish with a video intensification camera attached to a compound microscope. Individual neurons and their labeled processes will be followed as they grow in these rapidly developing, optically clear embryos. These data will, for the first time, enable us to provide a description of """"""""dynamic"""""""" aspects of neuronal development in vertebrates. To study intercellular communication, the restriction of gap-junctional communication pathways that occurs during embryonic development will be determined. Using lucifer yellow, a dye that passes between coupled cells, the pattern of dye-coupling among identified neurons will be determined at progressive developmental stages. These data will tell us if coupling persists among specific types of neurons. Then, the onset of electrical activity in the developing nervous system will be blocked to determine the possible role of electrical activity in the uncoupling process. This information may provide a functional explanation for coupling and its loss during development.
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