Mitochondrial Respiration Profiling

The oxygen consumption rate of mitochondria within synaptosomes was determined in units of picomoles of O₂ · minute⁻¹ · 10 μg of protein⁻¹ using a microplate-based respirometer (XF24; Seahorse Bioscience) as previously described (Gerencser et al., 2009 (link)). The assay buffer (S buffer) comprised 3.5 mM KCl, 120 mM NaCl, 1.3 mM CaCl₂, 0.4 mM KH₂PO₄, 1.2 mM Na₂SO₄, 15 mM D-glucose, 10 mM pyruvate, 0.4% (w/v) fatty acid-free bovine serum albumin, and 10 mM TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), pH 7.4. Nonmitochondrial respiration was defined as the average of three measurements after addition of 2 μM rotenone plus 2 μM myxothiazol (respiratory chain inhibitors), and this value for each well was subtracted from all other values for that well before calculation of the following respiration parameters: basal respiration (average value of the first three time points before any treatment); maximum respiration [first measurement value after addition of 4 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation]; respiration driving proton leak (average value of three time points after addition of 4 μg/ml oligomycin, an inhibitor of ATP synthase); respiration driving ATP synthesis (basal respiration minus respiration driving proton leak); coupling efficiency (100 × respiration driving ATP synthesis/basal respiration); and spare respiratory capacity (100 × maximum respiration/basal respiration). The calculated values for each well were averaged for 3–10 technical replicate wells on one plate to give n = 1 biological replicate.