The formation of specific chemical synapses during development involves a multistep process that includes a) neurogenesis of the specific presynaptic and postsynaptic populations, b) axonogenesis and guidance of axons towards the appropriate target region, c) target recognition and formation of synapses with appropriate cells within the target region, and d) fine tuning the number and distribution of synapses by activity- dependent stabilization/elimination of specific connections. Local inductive interactions via diffusible factors and/or the selective expression of cell surface or extracellular matrix molecules facilitate each step in this process. One member of the immunoglobulin-like family of cell adhesion molecules, NCAM, appears to play important roles at several stages in development. The major objective of this proposal is to study the role of different isoforms of a recently characterized member of the NCAM-like family of cell adhesion molecules in the formation and stabilization of specific chemical synapses using an in vitro model system consisting of identified neurons isolated from the ganglia of the marine mollusk Aplysia californica. Individual Aplysia neurons in cell culture reliably form stable chemical connections. Some cells appear to form connections only with their appropriate targets while others are more permissive and form connections indiscriminately. Manipulating the level of expression of these NCAM-like molecules, called apCAM, on the surface of the cells can significantly influence the pattern of outgrowth and synapse formation. The proposed studies will combine electrophysiological methods for detecting the presence and modulation of chemical connections and for intracellular injection of pharmacological agents, with a variety of light microscope techniques to monitor live growth cone behavior and to detect by immunofluorescent methods the distribution or level of expression of different isoforms of apCAM, synapse-specific antigens, cytoskeletal elements and kinase activities, and with electron microscope techniques for characterizing newly formed transmitter release sites.
The specific aims of the proposal are to test the hypotheses: 1) Synapse formation is initiated when a presynaptic growth cone slows upon contact of apCAM 'hot spot' on the surface of the postsynaptic cell and is mediated by the activation of presynaptic protein kinase C. 2) Synapse stabilization involves asymmetric distribution of apCAM isoforms such that transmembrane forms are concentrated on the postsynaptic surface and GPI- linked forms are concentrated on the presynaptic surface. 3) Local modulation of apCAM isoforms on presynaptic or postsynaptic membrane is critical for local cell- and site-specific changes in the number of synaptic contacts. 4) Branch-specific target contact influences cellular changes at other branches of the same cell via signals first triggered by apCAM-apCAM interaction. These studies will begin to identify the critical cellular and molecular events during the formation of synapses that permit neurons to establish mature neural circuits that control normal behavior.
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