AXON TARGET INTERACTIONS IN THE DEVELOPING CEREBELLUM

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DESCRIPTION (Verbatim from the Applicant's Abstract): The interactions of afferents with their targets are essential to target cell dendritic development. During the formation of the cerebellar cortex, granule cells migrate inward, leaving behind a t-shape axon, the parallel fibers, which extend through emerging Purkinje cell dendritic trees. Purkinje dendrite branching, spine emergence, and synapse formation then ensue. With a model in vitro system based on purified cells, we previously confirmed that granule neurons are sufficient to instigate such morphological differentiation. The proposed studies are based on the hypothesis that granule neuron afferents provide molecular signals for Purkinje cell dendritic and spine differentiation. Experiments will study the dynamics of granule-Purkinje interactions, and the role of two molecular systems in this process. In Aim 1, the dynamic behaviors of the Purkinje cell surface during spine development will be chronicled. A preparation has been developed by which individual Purkinje cells within cerebellar slices are labeled with GFP constructs by biolistic particle delivery and imaged in the living state by two-photon microscopy. Spine emergence, motility and morphogenetic changes will be monitored. The extent to which different spine forms receive synapses will be studied. The interactions of granule cell axons with Purkinje cell dendrites during spine development will be analyzed by labeling both elements in slices and dynamic imaging. Aim 2 will investigate the role of two receptor systems during Purkinje cell differentiation. Base on our evidence that TrkB signaling regulates spine formation, the role of TrkB signaling in spine emergence, maintenance, and synapse formation will be studied by dynamic imaging, as in Aim 1. Experiments will analyze which cell, the granule or Purkinje cell, is important for this signaling, and will determine involvement of other signaling pathways implicated in neurotrophin-mediated functions. In addition, Aim 2 will focus on a role for DCC, a receptor expressed in granule cells, in stimulating Purkinje cell dendritogenesis. The extent to which this receptor system is important for granule-Purkinje interactions directly, or for granule-granule cell interactions that only indirectly lead to Purkinje cell development, will be assessed. These studies should suggest molecular mechanisms underlying synaptogenesis in the mammalian CNS, important for understanding neurological disease, and for devising approaches to ensure neural process regrowth after injury.