Figure 1.

High-frequency field stimulation elicits parallel elevations of cytosolic calcium and superoxide radicals. A, B, In cultured rat CA1/CA3 hippocampal neurons, electrically evoked [Ca²⁺]_i elevations were measured by means of confocal fluorescence microscopy of fluo-3-loaded cells. Simultaneously, the oxidation of ethidine to the ethidium cation, which is specific for the superoxide anion, was used to determine O₂^- elevations (Bindokas et al., 1996). Micrographs are representative examples in which the right panel of a pair illustrates the typical general increase in fluorescence evoked by high-frequency stimulation. C, D, Weak, low-frequency stimuli (5 Hz, 18 or 180 sec) elicited only low, sustained [Ca²⁺]_i plateaus (C, bottom traces) and did not promote O₂^- generation (D, bottom traces). In contrast, strong, high-frequency stimulation (50 Hz/18 sec or 100 Hz/18 sec) evoked high-amplitude Ca²⁺ spikes (C, top traces), which returned to baseline within 1-2 min, and remained at basal levels for at least 45 min. It also evoked a sustained increase in cytosolic O₂^- (D, top traces). The sharp, transient rise in ethidium fluorescence occurring at the onset of stimulation and persisting for ∼2 min is an artifact arising, at least in part, because temporary, stimulus-induced collapse of the mitochondrial membrane potential retards mitochondrial uptake and subsequent fluorescence quenching of the ethidium cation (Budd et al., 1997).