Glycoproteins are thought to play an important role during the formation of synapses. The goal of this research proposal is to examine the hypothesis that glycoproteins are essential substrates for the formation and maintenance of synapses in the retina. The implication that glycoproteins are also involved in mechanisms underlying synaptic specificity will also be evaluated by searching for antibodies that bind to the different types of synapses present in the retina. The general approach to this problem will be to produce mouse and/or rabbit antibodies to specific chick retinal synaptic glycoproteins in order that they may be localized. In order to maximize the chances of obtaining antibodies to the antigens of interest such as: a) synapse-specific glycoproteins, b) retinal-synapse, specific glycoproteins and c) retinal cell class-specific, synaptic glycoproteins, several subfractions of the retina will be isolated and used a immunogens. The subfractions will include: 1) retinal synaptic plasma membranes (SPM), 2) retinal synaptic junction complexes (SJC), 3) lectin affinity isolated glycoproteins and 4) cone-specific subcellular fractions, all of which can be reliably isolated and characterized (progress report). Antibodies will be assayed for their abilities to bind to tissue sections of the retina and/or to Western blots of the subfractions. A combined lectin and immunoprobe analysis of Western blots of these fractions will allow identification of antibodies to specific, characterized glycoproteins. This screening will allow us to identify antibodies that will be useful for analysis in tissue localization studies. An electron microscope, immunocytochemical analysis of developing retinal synapses in embryonic and hatched chicks will be performed to determine the time of appearance and mode of insertion of specific glycoproteins into synapses. The data derived from this project will help elucidate mechanisms underlying synaptogenesis and will be most useful for characterizing development of patterns of specific classes of synapses within the retina. Evidence of accomplishment in each of the steps, necessary for the successful execution of this research proposal, is presented in the progress report.
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