Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract

The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)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 Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) 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 Ca(2+) 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 Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…TMRM has been widely used to monitor changes in ⌬⌿ m in models of injury (36,37,45,46,59). However, TMRM is also sensitive to alterations in ⌬⌿ p ; therefore care has to be taken in the interpretation of TMRM fluorescent signals.…”

“…Single Cell Characterization of ⌬⌿ m and ⌬⌿ p Relative to O 2 Consumption Following K ϩ Stimulation-The potentiometric probe TMRM has been used extensively as a tool for the single cell characterization of ⌬⌿ m (36,37,(43)(44)(45)(46) in vitro. Here we have utilized a nonquenching concentration of TMRM (20 nM) to monitor mitochondrial bioenergetics in d PC12 cells relative to changes in i O 2 .…”

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

“…It is characterized by a dramatic increase in [Ca 2 þ ] i following the activation of Ca 2 þ -permeable receptors, particularly glutamate receptors, even after the stimulus has ended, thought to be due to loss of the mitochondrial ability to buffer intracellular Ca 2 þ . [28][29][30][31] More recently, it was demonstrated that, in cerebellar granule neurons, NCX also participates in this process of secondary Ca 2 þ overload, 14 due to its loss of the ability to pump Ca 2 þ out of the cell, following its proteolytic inactivation by calpains upon exposure to glutamate, providing a new hypothesis for the origin of delayed Ca 2 þ deregulation. Hoyt et al 32 have shown in cultured rat forebrain neurons that, although reversal of NCX contributes to Ca 2 þ transients and to the immediate rise in intracellular Ca 2 þ levels upon exposure to glutamate, it has no effect in the mean peak increase caused by a prolonged exposure to glutamate nor does it contribute to neuronal injury.…”

Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera)

“…This experiment was biologically replicated five times and used a total of five young and five old workers. In addition, ΔΨm was also assayed in the trophocytes and fat cells of one young and one old worker using the specific dye, tetramethylrhodamine methyl ester (TMRM) (T668; Invitrogen, Carlsbad, CA, USA) (Ward et al 2000). Trophocytes and fat cells were stained with 200 nM TMRM for 10 min, washed three times with PBS, and visualized under a confocal microscopy (Leica TCS SP2; Leica, Wetzlar, Germany) at an excitation wavelength of 550 nm and an emission wavelength of 610 nm.…”