Effects of Oxidants and Glutamate Receptor Activation on Mitochondrial Membrane Potential in Rat Forebrain Neurons

Abstract

Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamatereceptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (z~W). We measured z~I'in rat forebrain neurons with the ratiometric fluorescent dye JO-i by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation-sensitive dyes 2',7'-dichlorodihydrofluorescein (DCFH 2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3-3 mM) or 1 mM xanthine/0.06 U/mi xanthine oxidase decreased L~.'I' in a way that was independent of the presence of extracellular Ca 2ãnd was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin Ainsensitive form of permeability transition, or a separate mechanism. tert-Butylhydroperoxide (730 1sM) had less of an effect on z~Wthan hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide-, tert-butylhydroperoxide-, and superoxideenhanced glutamate, but not kainate, induced decreases in z~W.The combined effect of peroxide or superoxide plus glutamate was Ca 2~dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate-mediated mitochondrial depolarization.

Spontaneous Changes in Mitochondrial Membrane Potential in Cultured Neurons

“…However, treatment with the uncoupler FCCP at the end of the high-light experiments led to an increase in monomer fluorescence of the same magnitude in both the exposed and unexposed regions (data not shown). This supports the hypothesis that JC-1 aggregate fluorescence responds to more than just changes in ⌬⌿ m (Scanlon and Reynolds, 1998;Chinopoulos et al, 1999) but indicates that light-induced changes in aggregate fluorescence do not change the ability of the JC-1 monomer signal to respond to changes in ⌬⌿ m .…”

“…We saw, however, a decrease in the number of fluctuations under high-light conditions, suggesting that dye-loaded mitochondria exhibit spontaneous fluctuations in ⌬⌿ m , which were not a consequence of illumination. These highlight conditions led to a decrease in the JC-1 aggregate signal without a concurrent change in the monomer signal (data not shown), similar to what is observed with oxidant treatments, such as hydrogen peroxide (Scanlon and Reynolds, 1998;Chinopoulos et al, 1999). However, treatment with the uncoupler FCCP at the end of the high-light experiments led to an increase in monomer fluorescence of the same magnitude in both the exposed and unexposed regions (data not shown).…”

“…Our previous measurements of ⌬⌿ m were limited in both spatial and temporal resolution (White and Reynolds, 1996;Scanlon and Reynolds, 1998), and thus these phenomena were undetected. Using the wide-field imaging system described here, we captured images at 0.2 Hz with resolution adequate to monitor signals from neuronal processes.…”

Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress

“…Nerve terminals, with an impaired a-KGDH activity, produce less ATP by ca 30% in the early phase of oxidative stress (Tretter et al 1997), but this is still adequate to maintain the resting function of the ATP-driven ion pumps in the plasma membrane. When oxidative stress is superimposed on some other burden such as a [Na C ] i load occurring during excessive stimulation of glutamate receptors or in ischaemia/reperfusion, the inability of mitochondria to meet an additional ATP demand, mainly due to inhibition of a-KGDH, becomes apparent and the [Na C ] and [Ca 2C ] gradients across the plasma membrane (Chinopoulos et al 2000) as well as the mitochondrial membrane potential rapidly collapse (Scanlon & Reynolds 1998). …”

Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice

“…Two hours prior to sacrifice, the mice were injected intraperitoneally with 1 mg/ml dihydroethidium (DHE) (Molecular Probes). In the presence of oxidative stress, DHE becomes oxidized to ethidium (Melendez et al, 1999;Scanlon and Reynolds, 1998), binds to DNA, and emits a red fluorescent signal. Brains were sectioned and immediately visualized using a Nikon Eclipse E1600W microscope.…”