HORMONAL INFLUENCES ON INSECT MOTOR NEURONS DEVELOPING IN CELL CULTURE

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Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS during postembryonic development. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential components of efforts to understand how the CNS is modified postembryonically to accommodate normal neural function and behavioral maturation, yet these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will take advantage of a well-characterized insect model system, where identified neurons undergo predictable phases of structural and functional modifications in response to defined steroid hormonal cues. The thoracic leg motoneurons of the moth, Manduca sexta, undergo dramatic dendritic and axonal regression and regrowth during metamorphosis to accommodate changes in their synaptic inputs an tar get muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20-HE ), acts directly on the motoneurons to regulate neurite outgrowth. The principal goal of the proposed experiments is to characterize the cellular consequences of steroid hormone action in vitro and relate them to the cellular mechanisms that are involved in the structural and functional remodeling of the leg motoneurons in vivo. In the first specific aim, video analysis will be employed to determine whether 20-HE enhances the growth of cultured leg motoneurons by stimulating branch formation, or promoting the retention of existing branches. Motoneurons placed in vitro just prior to the phase of neurite regression, rather than growth, will also be analyzed in comparison. Laser-scanning confocal microscopy images of dye-injected motoneurons will be used to examine similar questions in vivo. The second specific aim is to correlate steroid hormone effects on neurite growth and regression with modifications in the organization and stability of the cytoskeleton. Immunofluorescence, coupled with confocal microscopy, will be used to obtain high-resolution images of microtubules and actin filaments in cultured leg motoneurons that are undergoing 20-HE induced neurite growth or regression. Complementary experiments will employ electron microscopy to reveal cytoskeletal structures within identified motoneurons at crucial stages of development in vivo. The final aim is to test the hypothesis that 20-HE induced changes in voltage-gated Ca2+ and K+ channels, and consequent changes i Ca2+ influx, are essential elements of the intracellular pathway through which 20-HE regulates the remodeling of the leg motoneurons. We will determine whether changes in the levels of Ca2+ and k+ currents, that have already been described, are mediated by 20- HE and whether Ca2+-induced release of Ca2+ and K= currents, that have already been described, are mediated by 20-HE and whether Ca2+ -induced release of Ca2+ from internal stories is important for regulating structural remodeling. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.