Quantification of acetylcholine (ACh) and measurement of AChE activity

The hippocampus (n=10/group) was homogenized in ice-cold 0.01 M Tris-HCl (pH 7.4) using bead beater (Taco™ Prep; GeneResearch Biotechnology Corp.) and ultrasonic cell disruptor (KS-750; Madell Technology Corp.), and then centrifuged at 12,000 × g for 15 min. ACh concentration and AChE activity in the hippocampal homogenates were measured using an Amplex Red ACh/AChE assay kit (Invitrogen), according to manufacturer's instruction. Fluorescence intensities at 560 nm (excitation wavelength) and 590 nm (emission wavelength) were detected using an automated microplate reader (Infinite 200 Pro; Tecan).