7. Project Summary/Abstract Therapy for ischemic brain injury is poor in part because of our limited understanding of mechanisms leading to neuronal loss. While roles of excessive glutamate release and neuronal Ca2+ accumulation have been much studied, recent evidence implicates critical contributions of another divalent cation, Zn2+. After ischemia or prolonged seizures, free Zn2+ accumulates in neurons and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria, powerfully disrupting their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. Using acute hippocampal slices subjected to oxygen glucose deprivation (OGD) to model ischemia, we recently made the first simultaneous measurements of cytosolic Zn2+ and Ca2+ changes, and found that Zn2+ accumulation is an early event in hippocampal pyramidal neurons that precedes and contributes to a subsequent sharp and terminal Ca2+ deregulation event, causatively linked to loss of membrane integrity. We have further found that the acute deleterious effects of Zn2+ seem to result specifically from its uptake into mitochondria via the mitochondrial Ca2+ uniporter (MCU). In ongoing slice studies, we find evidence for major differences between sources of the Zn2+ that accumulates in hippocampal CA1 and CA3 pyramidal neurons contributing to acute OGD induced damage, with considerable Zn2+ accumulation in mitochondria of CA1 but not of CA3 neurons at delayed time points after a sublethal episode of OGD. These differences in Zn2+ contributions may bear upon the differential vulnerabilities of CA1 vs CA3 neurons in disease conditions, with CA1 preferentially degenerating after transient global ischemia, and CA3 after recurrent limbic seizures. This proposal continues ongoing studies, generally organized around a Hypothesis: Mitochondrial Zn2+ accumulation is an early event after transient ischemia, which causes disruption of mitochondrial function and contributes to delayed cell death. Aim I applies imaging techniques to acute hippocampal slices to further clarify mitochondrial effects of Zn2+ in hippocampal neurons in the hours after transient oxygen glucose deprivation (as a model of ischemia), and to study events occurring after restoration of O2/glucose (“reperfusion”) that may be amenable to beneficial therapeutic interventions. Aim II seeks to make initial test of principle studies of our hypothesis in an in vivo rat global ischemia model. These studies will provide mechanistic insights that will aid the development of new and effective therapeutic interventions to be delivered after an episode of transient ischemia, that will disrupt the pathological cascade, enabling improved outcomes.