It is proposed to study the role of protein phosphorylation in neurotransmitter release from presynaptic terminals in mammalian brain and the effects of psychoactive drugs on this system. This proposal will focus on two neuronal phosphoproteins: synapsin I, a synaptic vesicle-associated protein that is phosphorylated at specific sites by cAMP- and Ca/calmodulin-dependent protein kinases, and """"""""87k"""""""" protein, a cytosolic protein that is specificallly phosphorylated in nerve terminals by protein kinase C. The pharmacological regulation of the phosphorylation of synapsin I and 87k will be studied with standard endogenous ATP labelng and """"""""back-phosphorylation"""""""" techniques in brain slice and brain synaptosome preparations using agents that affect presynatpic receptors. We will sequence the regions on synapsin I and 87k containing the phosphoacceptor sites. We will then synthesize peptides of these sites and raise antibodies specific for either the phospho or de-phospho forms of these peptides. We will study the effect of these phosphosite peptides and antibodies (a) on the interaction of synapsin I with synaptic vesicles and (b) on neurotransmitter release from synaptosomes in """"""""microinjection"""""""" experiments. We will isolate and characterize the synaptic vesicle component to which synapsin I binds. A """"""""microinjection"""""""" technique that we have just developed will be used to introduce into brain synaptosomes molecules that modulate the phosphorylation of either synapsin I or 87k, and appropriate phosphosite peptides and antibodies. Neurotransmitter release will be studied in these same preparations under those conditions found to regulate the phosphorylation of these two phosphosubstrates. We will study the effects of several classes of psychoactive drugs on the phosphorylation of synapsin I and 87k and on neurotransmitter release. Also, brains and CSF from schizophrenic individuals and individuals with affective disorders will be examined for any changes in synapsin I or other phosphoproteins.
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