2.9. Ex Vivo Measurements of Acetylcholinesterase (AChE) Activity in the Brain

Ex vivo AChE activity in the cerebral cortex and hippocampus was determined with the colorimetric method as previously described by Ellman et al. [34]. Briefly, the assay mixture consisted of 50 μL of brain homogenates, 25 μL of 0.1 M phosphate buffer (pH 7.4), 125 μL of 0.1 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and 25 μL of 1 mM acetylthiocholine iodide (ATCI) which were mixed well. The absorbance of the assay mixture was measured by Benchmark Plus Microplate Spectrophotometer (Benchmark Plus, Bio-Rad, Japan). The spectrophotometric absorption at 405 nm during a 6 min incubation period at 25°C was quantitatively measured and was expressed as nmol ACh hydrolyzed (mg tissue)⁻¹ min⁻¹. The experiment was repeated six times.