Animals were killed by decapitation, brains were removed immediately, and regions of interest were dissected, frozen on dry ice, and stored at −80 °C until use. Tissues were thawed, homogenized in 25 mM Tris–HCl (pH 7.4) containing 1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride on ice, and then centrifuged at 100,000×g for 30 min. Pellets were twice rinsed with 25 mM Tris–HCl buffer and re-suspended in 0.32 M sucrose in 50 mM Tris–HCl (pH 7.0). Suspended membranes were passed through a 26.5 G needle five times and then frozen at −80 °C. Protein contents of membranes were determined by the BCA method (Smith et al., 1985).
