Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells

The relationship between changes in mitochondrial membrane potential (Delta psi(m)) and the failure of cytoplasmic Ca2+ homeostasis, delayed Ca2+ dere gulation (DCD), is investigated for cultured rat cerebellar granule cells e xposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM+) or rhodamine-12 3, we devised and validated a mathematical simulation with well characteriz ed effectors of Delta psi(m) and plasma membrane potential (Delta psi(P)). Glutamate usually caused an immediate decrease in Delta psi(m) of <10 mV, a ttributable to Ca2+ accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Ce lls for which the mitochondria showed a larger initial depolarization dereg ulated more rapidly. The mitochondria in a subpopulation of glutamate-expos ed cells that failed to extrude Ca2+ that was released from the matrix afte r protonophore addition were bioenergetically competent. The onset of DCD d uring continuous glutamate exposure in the presence or absence of oligomyci n 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 acti vation 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 t ransient Ca2+ loading of mitochondria as a consequence of NMDA receptor act ivation initiates oxidative damage to both plasma membrane Ca2+ extrusion p athways and the inhibition of mitochondrial respiration. Depending on exper imental conditions, one of these factors becomes rate-limiting and precipit ates DCD.