Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling

Hydrogen peroxide (H₂O₂) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H₂O₂ signaling, which activates ATP-sensitive potassium (K_ATP) channels to inhibit dopamine release. However, the origin of this modulatory H₂O ₂ has been elusive. Here we addressed three possible sources of H₂O₂ produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H₂O₂ and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H ₂O₂ production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H₂O₂ in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H₂O₂ production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H₂O₂ generation for dynamic neuronal signaling.