In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca2+...

The relationship between changes in mitochondrial membrane potential (Δψ_m) and the failure of cytoplasmic Ca²⁺ homeostasis, delayed Ca²⁺deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM⁺) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Δψ_m and plasma membrane potential (Δψ_P). Glutamate usually caused an immediate decrease in Δψ_m of <10 mV, attributable to Ca²⁺ accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca²⁺ that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca²⁺([Ca²⁺]_c) has risen to the set point at which mitochondria retain Ca²⁺. In the presence of oligomycin no increase in [Ca²⁺]_c occurs during this depolarization. We conclude that transient Ca²⁺loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca²⁺ extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.