Mitochondrial Respiration Complex Assay

Mitochondrial respiration complex activity was measured in hippocampi or in SK cell homogenates as previously described (37 (link),39 (link)). Briefly, hippocampi or cultured cells were homogenized and sonicated in the isolation buffer containing 250 mmol/L sucrose; 20 mmol/L HEPES, pH 7.2; and 1 mmol/L EDTA. NADH-ubiquinone oxidoreductase (COXI) enzyme activity was determined in 25 mmol/L potassium buffer containing KCl, Tris-HCl ,and EDTA (pH 7.4). The change in absorbance was monitored at 340 nm wavelength every 20 s for 6 min using an Amersham Biosciences Ultrospect 3100 pro spectrophotometer. For homogenized samples (50 μg protein), the oxidation of NADH was recorded for 3 min after the addition of 2 μg/mL antimycin, 5 mmol/L MgCl₂, 2 mmol/L KCN, and 65 μmol/L coenzymes Q1 to the assay mixture, and then 2 μg/mL rotenone was added to the mixture. The absorbance of samples was measured for another 3 min. Enzyme activities in complex II, complex III, complex IV, and citrate synthase activity were determined as previously described (37 (link)). We also determined ATP content in hippocampus using an ATP Bioluminescence Assay Kit (Roche) according to manufacturer’s instructions with a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.