Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells

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 Δπ(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 n t a 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.