Neuronal death following cerebral ischemia constitutes the basis of stroke. The cellular mechanisms of irreversible damage remain largely unexplained, however, due in part to the complex array of events triggered by ischemia in vivo. I propose to study neuronal death in forebrain culture, utilizing a well-characterized model of acid-induced lethal injury which we have established. These experiments will allow the definition and mechanistic analysis of those necessary, causal events leading to cellular death. In particular, I will focus upon those properties of already injured neurons which may permit their selective rescue and repair. This work will target the already damaged neuron as a subject of study in its own right, distinguishing this approach from those whose aim is the prevention of neuronal injury following external insult. Through an integrated series of physiological and biochemical experiments, the following issues will be addressed: 1. Does the absolute increase in cytosolic calcium observed with lethal injury in vitro correlate with the irreversibility of cell injury? Does there exist a threshold level of calcium, whether cytosolic or subcellular, which mandates neuronal death? A normal cell is fully capable of handling large and even long-lasting increases in cytosolic calcium without lethal injury. These experiments will test the twin hypotheses that increments in cytosolic calcium constitute neither necessary nor sufficient conditions for neuronal death, and that only when combined with a more proximal stressor, such as receptor activation, physical membrane damage, or insufficient energy production, does a threshold calcium increment necessarily forebode cell death. Also these experiments will explore the observation that a loss of potassium induced increase in cytosolic calcium is an early sign of cell injury. 2. Do repeated, transient waves of depolarization in energy deprived brain cells, analogous to in vivo spreading depression, convert reversible to irreversible damage? These experiments will test the hypothesis that repeated, transient waves of depolarization in energy deprived brain cells may cause selective neuronal injury. With these experiments, I will attempt to model the ischemic penumbra in vitro by which means the mechanism of selective neuronal necrosis can be explored. In examining these issues, I will concentrate upon defining the potential for recovery of injured cells. Such studies of the cellular biology of neuronal death may provide the rationale for a new generation of experimental and clinical therapeutics, whose goal will be the directed rescue of damaged cells following ischemic injury.