Aβ and Aβ-AChE complexes induce morphological changes, apoptosis and intracellular Ca2+ increase in rat hippocampal neurons. (A) Immunofluorescence for MAP-2 protein of 14 DIV hippocampal neurons treated with: (a) control, (b) 5 μM Aβ-AChEo, (c) 5 μM Aβo, (d) 5 μM Aβ-AChEf, (e) 5 μM Aβf, (f) 5 nM AChE and (g) 5 μM Aβ42-1 for 1 h. Scale bar, 10 μm. (B) Cell viability was performed with the MTT reduction assay in hippocampal neurons treated for 12 h with the indicated μM concentrations. *p ≤ 0.01 and **p ≤ 0.001 compared with the control condition. (C) Hippoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 30 min at 37°C) to measure changes in free intracellular Ca2+. The graph shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβf (White circle); 5 μM Aβ-AChEf (Black circle); 5 nM AChE (Grey circle). Inset shows the final normalized fluorescence intensities reached at the end of 1 h of recording. *p ≤ 0.001 compared to control; #p ≤ 0.001 compared to the Aβ-AChEf treatment. (D) Detection of Caspase-3 activity in hippocampal neurons. The graph shows the activity of Caspase-3 under effect of different preparation of Aβ and Aβ-AChE peptide. The cells were treated by 1 h at 37°C. Results are the mean ± S.E.M; *p < 0.05.