Assay of Ach, ChAT, and AchE

Cholinergic markers were measured as described previously study (Ruan et al., 2010). Briefly, the hippocampal supernatant was further diluted with a buffer solution appropriate for the relevant biochemical index. The level of Ach in the supernatant was detected with an ELISA kit (A105-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Each reaction contained a 100 μL sample, 0.5 U/mL AchE, 200 μM Amplex Red reagent, 0.1 U/mL choline oxidase, and 1 U/mL HRP in reaction buffer. The reaction systems were incubated at room temperature for 30 min with protection from light, and then fluorescence intensity was measured at an excitation wavelength of 560 nm and an emission wavelength of 580 nm. Similarly, the activities of ChAT and AchE were detected spectrophotometrically with commercial assay kits (A079-1 and A024, Nanjing Jiancheng Bioengineering Institute) and are described in units per milligram fresh tissue protein.