Tissue plasminogen activator (tPA), a serine protease with limited proteolytic activity, is present in high concentrations in the adult nervous system. The CNS of mice with the tPA gene inactivated (tPA -/-) appear anatomically normal; however, these mice have a high threshold for drug-induced seizures and are highly resistant to excitotoxic death of hippocampal neurons (Tsirka et al., 1995). These observations suggest that tPA plays a critical role in seizures and in excitotoxic neuronal death which has considerable clinical implications for diseases and pathological conditions ranging from epilepsy and head trauma to stroke and various neurodegenerative diseases. Although implications of data obtained with tPA -/- mice are considerable, there is no information on mechanisms responsible for tPA-enhanced excitotoxic neuronal death or seizures. This information is necessary to define basic mechanisms involved in these pathologies as well as providing a rational approach for clinical intervention. The overall goal of the proposed studies is to characterize the mechanism of tPA-enhanced glutamate neurotoxicity with the long term objective to use this information to help develop therapeutic regimens to decrease pathologies associated with glutamate excitotoxicity. Preliminary studies indicate the tPA enhances glutamate toxicity in cultured hippocampal neurons and the proposed studies are designed to investigate cellular and biochemical sites of action and underlying mechanisms of tPA-enhanced glutamate induced neurotoxicity using primary cultures of hippocampal neurons from rats and from mice homozygous for tPA inactivation. Studies will: (1) provide a detailed analysis of the temporal aspects of tPA-enhanced glutamate toxicity and cellular components responsible, (2) characterize the biochemical focus and mechanism of this effect, (3) identify physiological and pathological regulators that release tPA from neurons and microglia, and (4) characterize the CNS tPA receptor. It is anticipated this information will improve our understanding of basic pathophysiological mechanisms associated with stroke, head injury, and epilepsy and other conditions associated with glutamate induced excitotoxicity and should impact on therapeutic regimens designed to decrease pathology associated with these conditions.