Mitochondria were freshly isolated from brains. Respiration of mitochondria was measured using a Seahorse XF24 analyzer as previously described (38). Mitochondria (10 μg of protein) were plated in each well of an XF24 plate in 50 μl of 1× MAS (pH 7.4; 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM Hepes, 1 mM EGTA, and 0.2% fatty acid–free bovine serum albumin). XF24 plates were centrifuged at 4°C for 20 min at 2000g, and then 450 μl of MAS containing 5 mM pyruvate/5 mM malate, 5 mM glutamate/5 mM malate, 40 μM palmitoyl-CoA/40 μM carnitine/5 mM malate, or 10 mM succinate was added to each well. Plates were incubated at 37°C in a non-CO2 incubator for 8 to 10 min. OCRs were measured under basal conditions in the presence of ADP (4 mM), oligomycin (1.5 μM), FCCP (4 μM), and rotenone (1 μM), as described above.

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