Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse

Figure 2

Comparing NADPH oxidase and mitochondrial activities in isolated synaptosomes using high resolution respirometry and fluorescence spectroscopy. (a, b) Representative traces of NADPH oxidase and synaptosomal mitochondria activity assessed by simultaneous measurement of rates of oxygen consumption (a) and H₂O₂ production (b) using high resolution respirometry combined with HRP/Amplex Red, in the same sample under identical conditions except that NOX substrate is substituted by mitochondria ones. Activities were monitored as described in Figure 1’s legend. Mitochondrial resting (state 4) respiration was triggered by the addition of 10 mM pyruvate + 10 mM malate + 10 mM glutamate. Active phosphorylating (state 3) respiration was induced by adding 1 mM ADP (OXPHOS I) followed by 10 mM succinate for (OXPHOS I+II). (c) Representative traces depicting ETC substrate-specific O₂ utilization by mitochondria in saponin-permeabilized synaptosomes during substrate-uncoupler inhibitor-titration. (d, e) Quantifications of the sum of the absolute values of O₂ (d) and H₂O₂ (e) fluxes following NOX and mitochondria activations. (f) Percept ratios of the H₂O₂-produced to the O₂-consumed. Values are given as mean ± SEM, paired Student -test was used to determine statistical significance in comparison with NADPH-induced collective activity, and . .