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Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase–ERK pathway.The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5–1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen–glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3β (in situ immunostaining and Western blots).Preconditioning with 0.5–1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen–glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7–15%, which involved Ca2+ release from inositol triphosphate–sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3β. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin.Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.