Differences in O2 availability resolve the apparent discrepancies in metabolic intrinsic optical signals in vivo and in vitro.

Monitoring changes in the fluorescence of metabolic chromophores, reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide, and the absorption of cytochromes, is useful to study neuronal activation and mitochondrial metabolism in the brain. However, these optical signals evoked by stimulation, seizures and spreading depression in intact brain differ from those observed in vitro. The responses in vivo consist of a persistent oxidized state during neuronal activity followed by mild reduction during recovery. In vitro, however, brief oxidation is followed by prolonged and heightened reduction, even during persistent neuronal activation. In normally perfused, oxygenated and activated brain tissue in vivo, partial pressure of oxygen (P(O2)) levels often undergo a brief 'dip' that is always followed by an overshoot above baseline, due to increased blood flow (neuronal-vascular coupling). By contrast, in the absence of blood circulation, tissue P(O2)in vitro decreases more markedly and recovers slowly to baseline without overshooting. Although oxygen is abundant in vivo, it is diffusion-limited in vitro. The disparities in mitochondrial and tissue oxygen availability account for the different redox responses.