The long-term goal of this research is to understand from a cell biological perspective how neurons establish and maintain distinct axonal and dendritic domains that differ in structure and function. This feature of nerve cells -- referred to as neuronal polarity -- is essential to normal neural function and a disruption of neuronal polarity is thought to contribute to the pathophysiology of many neurologic diseases. An understanding of the cellular mechanisms underlying neuronal polarity will be necessary for the development of molecular therapies for neurologic diseases. The development and maintenance of polarity will be studied in cell cultures of embryonic rat hippocampal neurons, using techniques that range from observation and manipulation of living cells to investigations at the cellular and molecular levels. The particular questions to be addressed derive from previous work that has identified the key stages in the development of hippocampal neurons that determine their polarity. The initial emergence of the axon, the defining event in the establishment of polarity, will be analyzed by high resolution time-lapse microscopy under normal and experimental conditions. The response of more mature cells to axonal transection will be used to determine when polarity becomes irreversibly specified. Dendritic growth and the behavior of dendritic growth cones will be analyzed by time-lapse video microscopy. Electron microscopy will be used to identify ultrastructural features that distinguish dendritic from axonal growth cones. Sorting signals that direct integral membrane proteins to axons or dendrites will be identified. Defective Herpes viral vectors will be used to introduce cDNA constructs into cultured cells in order to assess how deletions in the endo-and ectodomains of polarized membrane proteins affect their distribution. Exocytic vesicle traffic between the Golgi complex and sites of incorporation into the plasma membrane will be analyzed at each stage of neuronal development using fluorescent lipid analogs.
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