The cellular redox status is determined by various extra- and intracellular factors, and contributes to cytosolic signaling and oxidative stress. Especially mitochondria modulate the cytosolic redox status by oxidizing NADH and FADH(2) and generating reactive oxygen species (ROS). Whereas cellular NADH and FAD levels are reliably detectable as autofluorescence, quantifying cellular ROS production is more demanding, because the various redox-sensitive dyes share major disadvantages including irreversible oxidation, autooxidation and photosensitivity. As an alternative, we took advantage of a genetically engineered redox-sensitive green fluorescent protein (roGFP1), carefully evaluated its response properties, and succeeded to monitor ROS dynamics in cultured rat hippocampal neurons and organotypic slices. The ratiometric properties and reversible oxidation/reduction of roGFP1 enable reliable, semi-quantitative analyses of cytosolic ROS levels and redox status. Cytosolically expressed roGFP1 readily responded to hydrogen peroxide, superoxide and hydroxyl radicals, and was only negligibly affected by intracellular pH or Cl(-) content. Furthermore, roGFP1 was well suited for two-photon excitation, reliably detected changes in endogenous ROS production during impaired mitochondrial respiration or neuronal stimulation, and was even capable of visualizing perimitochondrial ROS microdomains. Modulation of cellular scavenging systems confirmed the functional integration of roGFP1 into the cellular ROS and redox balance. We conclude that roGFP1 is well suited for dynamic, compartment specific, subcellular analyses even in complex neuronal networks. The ability to correlate dynamic changes in cellular ROS levels with mitochondrial metabolism and neuronal network activity is a promising step towards a detailed mechanistic understanding of redox- and ROS-mediated signaling in normal and diseased brain function.
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