This project studies cellular and physiological aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration ([Ca2+]i) that is strongly modulated by the transport activity of intracellular calcium stores. The latter activity plays an important role in spatio-temporally shaping the Ca2+ signals that regulate processes like gene expression and long-term potentiation. We and others had earlier shown that stimulus-induced increases in [Ca2+]i in a variety of neurons induce large, reversible elevations in the concentration of calcium within mitochondria, which in turn has important effects on physiological and pathophysiological processes, examples being regulatory kinase cascades and excitotoxic cell death. This year we have further explored the consequences of mitochondrial Ca2+ uptake, showing that in hippocampal pyramidal neurons this activity profoundly affects the prototypical signal transduction kinase ERK, as well as neuronal susceptibility to excitotoxic injury. In hippocampal neurons large [Ca2+]i increases activate several important kinases, whereas small increases enhance protein phosphatase activity. This Ca2+-dependent rebalancing of the phosphorylation status of certain key enzymes, e.g., Ca/calmodulin-dependent kinases (CaMKs), is important because it regulates pathways central to neuronal plasticity. Our laboratory previously reported that mitochondrial calcium accumulation mediated by strong Ca2+ entry leads to an increase in the production of superoxide radicals (O2-), which up-regulates the activity of several important kinases, including PKA, PKC, CaMKII, nuclear CaMKIV (and therefore the transcription factor CREB), and ERK 1/2. Up-regulation of all but PKC occurs by a common mechanism, namely, stabilization of the active, phosphorylated form of the kinase by O2- inhibition of the deactivating serine/threonine protein phosphatases PP1, PP2A and/or PP2B (collectively, PPs). In all these cases, mitochondria are the main source of active O2-, but ERK 1/2 is unique in that NADPH oxidase-derived O2- also plays a significant role. This year we have extended earlier studies showing that the more complex regulation of ERK arises because key steps in the Ras/Raf/MEK/ERK cascade, particularly the activation of Raf-1, an up-stream mediator of ERK, depend on both PP and protein tyrosine phosphatase (PTP) activities, but these enzyme classes are differentially regulated by O2- produced by NADPH oxidase as compared to that produced by mitochondria. By examining various time points in the ERK cascade, we have teased out the mechanisms underlying the differential effects of these two sources of O2-. Immediately after stimulation, both mitochondrial and NADPH oxidase-derived O2- are up-regulated and inhibit PTPs, which in turn enhances the non-receptor tyrosine kinases Pyk-2 and Src that activate Ras. At 1-min post-stimulation, NADPH oxidase activity has already decayed, but sustained mitochondrial O2- production inhibits PP2A and therefore delays the PP2A-dependent activation of Raf-1 and downstream MEK. Shortly thereafter, this block is overcome because mitochondrial O2- suppression of PPs (and PTPs) also promotes kinase-dependent pathways (CaMKII, for example) that lead to full activation of Raf-1. Essentially, mitochondrial O2-, because it is sustained, switches from inhibiting Raf-1 to activating it. Increased Raf-1 activity leads to elevated MEK at 3-min post-stimulus, and elicits maximal ERK1/2 phosphorylation at 10 min. These results differentiate NADPH oxidase from mitochondria in that the former briefly generates a spike of O2- that promotes the initial activation of Ras, while the latter produces a sustained O2- elevation that inhibits both PP- and PTP-dependent steps of the Ras/Raf/MEK/ERK cascade. Consequently, both pathways ultimately promote Raf-1-dependent ERK activation, but they regulate different steps, and therefore influence the timing, of this signaling cascade. We also continued to investigate mechanisms of mitochondrial involvement in excitotoxic death of cultured hippocampal neurons. We previously showed that NMDA overstimulation of hippocampal neurons leads to mitochondrial calcium accumulation that is spatially heterogeneous, and that cell vulnerability is correlated with the aggregate size of the mitochondrial Ca load and the number of Ca-overloaded, damaged mitochondria. There is increasing evidence that mitochondria mediate the neuroprotective effect of ischemic preconditioning, although the mechanisms involved are not well understood. Therefore, recent studies tested the hypothesis that neuronal preconditioning reduces mitochondrial damage caused by Ca overload during excitotoxic stimuli. Using a preconditioning protocol analogous to 'chemical ischemia', namely, exposure to 2 mM cyanide (CN) for 30 min, it was found that at the cellular level mild CN treatment mimics the effect of NMDA by inducing the reversible redistribution of intracellular cations (Na, K, and Ca) and the transient depolarization and Ca loading of mitochondria. These effects were fully reversible, such that CN exposure did not lead to cell death. Exposure to lethal concentrations of NMDA 24 h after CN preconditioning, however, reduced NMDA-induced cell death by >30%. In addition, the fraction of NMDA-treated neurons containing Ca-overloaded, swollen, and damaged mitochondria decreased approximately twofold, while the fraction of such mitochondria within affected neurons was reduced as compared with non-preconditioned cells. In contrast to these changes, cyanide pretreatment abolished neither the NMDA-induced elevation of cytosolic and mitochondrial Ca nor the formation of spatially heterogeneous Ca-rich inclusions within mitochondria. These results support the working hypothesis that CN preconditioning exerts a protective effect by increasing mitochondrial tolerance for large Ca loads, thereby attenuating mitochondrial dysfunction triggered by Ca overload.