The applicant's research background and previous training involved characterization of two of the tyrosine kinases known to be present in the brain, the insulin and IGF-1 receptors. These and other tyrosine kinases, for example the proto-oncogene products pp6O and pp6l(c-src), are abundant in post-mitotic neurons, however, their function is poorly understood. The studies proposed in this application represent an initial inquiry into the role of brain tyrosine kinases in neuronal signalling and plasticity The kindling model of epilepsy will be employed because it permits the investigation of biochemical changes occurring with seizure activity and with the development of permanent seizure susceptibility. In preliminary studies, tyrosine kinase activation was detected in the rat hippocampus immediately following generalized 'seizure activity. Tyrosine kinase activity was also increased in the hippocampus of kindled rats that had not seized for 10-14 days. The studies in this proposal will determine the time course of tyrosine kinase activation and will characterize the phosphotyrosines regionally and biochemically. The studies will examine how long this activation persists. Implanted electrodes will be used to induce seizures, produce kindling, and record brain electrical activity. Phosphotyrosines will be examined in experimental animal and controls by immunocytochemistry, protein autophosphorylation, Western analysis, phosphoamino acid analysis. Antiphosphotyrosine antibody-precipitable phosphatidylinositol 3-kinase activity will be used as a measure of tyrosine kinase activity. Kinase renaturation will be performed to determine which of the phosphotyrosines are autophosphorylated kinases. These data will contribute to the understanding of the biochemical changes associated with seizure activity and the kindled state, and to our knowledge of neuronal tyrosine kinases.
