9604841 Koenig The proposed study centers on novel, specialized domains located at intermittent intervals in the outer boundary of axons, called "periaxoplasmic plaques." The plaques were discovered in isolated axoplasm from the Mauthner neuron located in the goldfish central nervous system, and their presence has now been confirmed in axons of rabbit spinal nerve roots; therefore, they are likely to be specializations common to long axons in general. Identification of ribosomes in plaque domains suggests that the latter are potential local centers of protein synthesis concerned with local turnover of axoplasmic proteins. This premise is a significant departure from prevailing views on how the axon's vitality and mass are maintained in a steady state. One major experimental aim is to analyze the principal structural components located within plaque domains of goldfish and rabbit axons at an electron microscopic level in order to define the spatial organization of the plaque domain, and to characterize common features and distinctive differences between older and more recently evolved vertebrate axons. Another major experimental aim is to test the hypothesis that plaque domains represent local centers of protein synthesis, and are targeted endstations for RNA trafficking from the neuronal cell body. For this purpose, immunostaining, and molecular biological techniques will be used to evaluate localization in relationship to plaque domains in goldfish and rabbit axons of elongation factor 1a, an essential cofactor of protein synthesis, and localization of messenger RNA that codes for specific gene products, respectively. Labeled RNA constructs will also be microinjectied into the Mauthner cell to evaluate targeting and transport of RNA to plaque domains from the cell body. Finally, sites of de novo protein synthesis, assayed by metabolic radiolabeling in goldfish and rabbit axons, will also be evaluated by autoradiography in order to determ ine the spatial relationship between the localization of nascent peptide chains to plaque domains. Information from the proposed study should contribute to a greatly improved understanding of how axoplasmic proteins, and vitality of long axons are maintained on a long term basis.
