Effect of Aβ-AChE oligomers in hippocampal neurons. (A) Aβ (5 μM) in the absence (a, c, e, g) and presence of 50 nM AChE (b, d, f, h) was aggregated at 37°C without stirring. 5 μl aliquot was obtained at 0 (a, b); 1 (c, d); 2 (e, f) and 4 hr (g, h), stained with 2% uranyl acetate and photographed with an electron microscope. More Aβ oligomers were formed in the presence of AChE, corresponding to protofibrils (arrow) (f), or amylospheroids (head-arrow) (h). Scale bar, 100 nm. (B) Western blot for 50 μM Aβ aggregated alone or in the presence of 50 nM AChE, an aliquot of supernatant obtained after centrifugation was used (a). Hipoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 20 min at 37°C) to measure changes in free intracellular Ca2+ every 1 min. The graphic shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβ-AChEf (black circle); 5 μM Aβ-AChEo (white circle) (b).