Mechanisms determining specificity in synapse formation between cells of different types will be studied in low-density cultures of dissociated primary cells and in explant cultures of neural tissue on muscle cells. Microelectrode techniques will be used to monitor synaptic activity. Specificity of innervation will be examined by allowing neurons to choose among different cell types as targets for innervation, and by allowing different neuronal cell types to compete for innervation of the same target cells. Poly-innervated target cells will be sought as transitionary stages in such competition. Choline acetyltransferase (CAT) activity will be measured in these cultures as a possible biochemical correlate of innervation and as a potential signal for competition between pre-synaptic cell types. Some experiments will initially be performed with explants of spinal cord and ciliary ganglia on muscle cell cultures. However, a major effort will be made to fractionate suspensions of spinal cord cells into populations enriched in motoneurons to carry out these studies with identified cell types in low-density cultures of dissociated primary cells. Methods of fractionation will include (1) velocity sedimentation of cells through shallow ficoll gradients under gravity, (2) differential adherence of cells to muscle cultures, and (3) preferential survival and release of cells from 4-5 day spinal cord explants after 1-2 days in culture. Initial criteria for enrichment of motoneurons will be increases in CAT activity and in the incidence of synapse formation with muscle cells. Efforts will also be made to directly identify surface "Recognition" components on target cells which may play a role in synapse formation. Such components will be made radioactive (e.g. iodination of intact cells with I125 and lactoperoxidase; or growth in (H3) glucosamine) and recovered (e.g. by light trypsinization), and specific binding of these components to cultures of neurons assessed by autoradiography.