DEVELOPMENT OF HIPPOCAMPAL NEURONS IN CULTURE

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.