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.

Proton circuits across the inner mitochondrial membrane link the primary energy generators, namely the complexes of the electron transport chain, to multiple energy utilizing processes, including the ATP synthase, inherent proton leak pathways, metabolite transport and linked circuits of sodium and calcium. These mitochondrial circuits can be monitored in both isolated preparations and intact cells and, for the primary proton circuit techniques, exist to follow both the proton current and proton electrochemical potential components of the circuit in parallel experiments, providing a quantitative means of assessing mitochondrial function and, equally importantly, dysfunction.