The specific aim of the present proposal is an ultrastructural and biochemical characterization of the synapse to determine the relationship of subsynaptic structures to each other, specifically, membranous and cytoskeletal elements. The two membranous cytoskeletal associations of interest are 1) between the synaptic membranes and its underlying postsynaptic cytoskeletal network and 2) between the synaptic vesicle membrane and the cytoskeletal network in which it is suspended. The following paradigms will be used. A) The intact squid optic lobe and the synaptosome and synaptosomal plasma membrane (SPM) fractions derived from the optic lobe will be characterized and used to identify cholinergic endings and membranous and cytoskeletal elements of cholinergic synapses by 1) cytochemical and immunocytochemical techniques, 2) subcellular fractionation of synaptosomes into distinct subpopulations, 3) biochemical characterization of these subpopulations by enzyme assays, 4) preparation of SPMs from these subpopulations, 5) characterization of their proteins by gel electrophoresis, 6) preparation of antibodies and immunocytochemical localization of synaptic proteins on whole optic lobe, and 7) identification of actin at synapses by HMM decoration. B) The developing mouse olfactory bulb and the developing squid optic lobe will be used to examine the sequence of events in the formation of the postsynaptic membrane-cytoskeletal relationship. C) The squid optic lobe synaptosome fraction will be used to characterize the synaptic vesicle - cytoskeletal association by examining the localization of calcium binding sites in the presynaptic terminal in relation to the disposition (i.e., formation or deformation) of the cytoskeletal network using two probes: 1) calcium precipation procedures and 2) electron probe microanalysis. These studies will contribute to the understanding of the morphological and biochemical basis of cholinergic transmission and abberations of synapse development that may accompany metal retardation, as well as other studies of the pathology of the nervous system.
