The proper fusion of the adult nervous system depends on a precise pattern of neuronal connections established during development. In higher vertebrates, neuronal plasticity in the central nervous system is limited to modifications of the morphology and efficacy of synapses. In contrast, songbirds exhibit a tremendous degree of neuronal plasticity in the adult CNS. Several brain nuclei, associated with learning and production of song, undergo dramatic, seasonal changes and correlated with fluctuating plasma testosterone levels. High plasma testosterone levels induce increases in neuronal cell number in the hyperstriatum ventrale pars caudale (HVc) by as much as 50% with a daily rate of addition of new neurons reaching 0.5%. The majority of new HVc-neurons project axons through densely myelinated brain regions to their target neurons in the robust nucleus of the archistriatum (RA). There is strong evidence that testosterone regulates post-mitotic events rather than cellular proliferation. This proposal will investigate the role of testosterone in stimulating axon outgrowth in the presence of myelin resulting in th formation of functional neuronal connections. This in vitro study will utilize HVc-neurons in dissociated cultures and seeded onto cryosections of adult songbirds brain. Furthermore, axon guidance mechanisms of HVc- neurons will be addressed in living, long-term brain slice preparations. HVc-neurons to be studied will be labeled with DiI and by adenoviral- mediated expression of green fluorescent protein and/or beta- galactosidase. Expression of these reporter genes is driven by various promoters to achieve exclusive expression in HV/c-neurons or other neuronal population associated with learning and production of song. Key methods employed in the proposed research include live video microscopy under phase and DIC optics, fluorescence microscopy, immunocytochemical and histochemical staining. Understanding the principal of testosterone- dependent axon outgrowth in the presence of myelin could improve our knowledge to address functional regeneration in the adult mammalian CNS.