Long-term memory formation is accompanied by enduring changes in synaptic organization. Tissue plasminogen activator (tPA), a serine protease that is highly expressed in the hippocampus, is implicated in modifying synaptic efficacy during learning and memory. In models of tPA mediated-plasticity, tPA is postulated to undergo regulated secretion from synaptic sites. We developed a system that allows visualization of tPA-containing dense-core granules (DCGs) at postsynaptic structures. Using this system, secretion can be followed in real-time from individual postsynaptically localized DCGs. To better understand the molecular determinants of synaptic plasticity, we propose to characterize the properties and mechanisms of the synaptic secretion of tPA from DCGs in cultured hippocampal neurons. In Specific Aim 1, studies are proposed to evaluate how physiologically relevant patterns of electrical stimulation influence the postsynaptic secretion of tPA from DCGs. In Specific Aim 2, experiments are proposed to determine if tPA-containing DCGs localize to presynaptic sites and to discern if these DCGs undergo activity-dependent release from presynaptic terminals. If secretion from presynaptic sites is observed, the cellular determinants that favor either pre- or postysynaptic secretion of tPA from DCGs will be evaluated. Finally, in Specific Aim 3, we propose using a RNA interference approach to evaluate if one of the synaptotagmin isoforms serves as a modulator of calcium sensitivity for the regulated secretion of tPA from DCGs present at postsynaptic sites. The proposed studies rely on the use of tPA fluorescent chimeras, multi- wavelength, time-lapse fluorescence imaging and computational deblurring techniques. Findings derived from these studies are relevant not only to physiological processes in the nervous system such as learning and memory but also to neuropathological processes. Excitotoxic cell death and the neuronal degeneration associated with Alzheimer's disease are directly linked to the tPA/plasmin system. The proposed studies focus on the properties and cellular mechanisms of the synaptic secretion of tissue plasmingen activator. This enzyme has been implicated in physiological and pathological functions in the nervous system including learning and memory and the neurotoxicity associated with Alzheimer's disease. [unreadable] [unreadable]