GROWTH CONE SURFACE PROTEINS THAT MEDIATE TARGET CONTACT

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During embryonic development, neurons extend growth cones that seek out and synapse on the appropriate target. The culmination of such contacts is the neuronal circuitry that underlies behaviors. The goal of our research is to determine how neurons recognize, and are recognized, by their targets. Any attempt to define recognition at the molecular level requires the ability to isolate and characterize molecules on the growth cone surface and the means to assess the function of putative recognition ligands isolated from the growth cones. These prerequisites are difficult to attain in the complex and heterogenous vertebrate system. They can be attained, however, by using the nervous system of Aplysia california where many neurons can be recognized as individuals and are large enough to study using biochemical, immunological and electrophysio- logical approaches. Growth cones, for example, can be isolated in pure form RUQ neurons growing in vitro and their polypeptide composition can be examined. Also, neuron L7 synapses on auricle and gill vein muscles to elicit initiation of the heartbeat and gill withdrawl, respectively. This simple circuit has been assembled in vitro and synaptogenesis between L7 and its target muscles has been quantitated, thereby providing a statistically meaningful way to assay components of the growth cone that block synapse formation. One of the constituents enriched at RUQ growth cones is a 75kd membrane glycoprotein that is exposed on the surface. This glycoprotein is one member of a class of similar glycoproteins (GP-75) that are rapidly transported to the terminals of many Aplysia neurons, including L7. GP-75 is heterogenous since it contains a glycopeptide composed of several oligosaccharides that bind to wheat germ agglutinin. It is significant, therefore, that one or more of these oligosacch-arides bind specifically to the muscle cells that are targets of L7. WE will test two hypotheses: first, that the GP-75 found ont eh growth cones of motor neurons has oligosaccharides that participate in recognition of muscle cells during synaptogenesis. Second, that muscle cells have receptors on their surface that recognize GP-75 oligosaccharide. These ideas will be investigated by: 1) isolating and characterizing the GP-75 oligosaccharides and identifying the constituents that block the binding of GP-75 to isolated muscle membranes; 2) generating monoclonal antibodies against the various species of GP-75 to determine their distribution among identified neurons; 3) identifying the antibodies and GP-75 oligosaccharides that prevent synaptogenesis between L7 and its targets; and 4), using the oligosaccharides as affinity ligands to isolate the receptors from each muscle type. In this way we will define the ligands and receptors involved in the formation of simple circuit.