*2.4. Brain Homogenate Preparation and Mitochondria Isolation*

Fasted (12 h) rats were anesthetized with an overdose of sodium thiopental (100 mg/kg) and the brain immediately removed, freeze-clamped and stored in liquid nitrogen. The tissue was homogenized in a van Potter–Elvehjem homogenizer with nine volumes of ice-cold 0.1 M potassium phosphate buffer (pH 7.4) and an aliquot was separated as total homogenate. The remaining homogenate was centrifuged (11,000*g*/15 min) and the supernatant separated as homogenate supernatant. Brain mitochondria were isolated by differential centrifugation as previously described [33] with some modifications. The fresh brain was placed in ice-cold solution containing 200 mM mannitol, 76 mM sucrose, 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N- ,N- -tetraacetic acid (EGTA), 0.1 mM PMSF (phenylmethylsulfonyl fluoride) and 10 mM 2-amino-2-hydroxymethyl- propane-1,3-diol (TRIS; pH 7.4). The brain was cut into small pieces and homogenized in a van Potter–Elvehjem homogenizer. The homogenate was then first centrifuged at 650*g* for 10 min and the mitochondria in the supernatant were sedimented by centrifuging at 8000*g* (10 min). The resulting mitochondria were washed and resuspended in the same buffer. An aliquot of this preparation was used to prepare disrupted mitochondria by a repeated freeze-thawing procedure in liquid nitrogen.
