Role of the intracellularcalcium increase in the toxicity of Aβ-AChE complexes. (A) Hippocampal neurons were loaded with Calcein-AM to analyze the neuronal integrity and with FuraRed as an indicator of intracellular free calcium. The confocal photographs correspond to neurons before treatment (a, c and e) and after 1 h treatment with 5 μM Aβ-AChEf (b, d and f). (B) The figure shows a representative SDS-PAGE of AChE immunoblot to detect Aβ-AChEo complexes on neuronal surface by biotinylation. The graph shows a quantification of two independent experiments, results are the mean ± S.E.M. (C) Hippocampal neurons were loaded with Fluo-3 AM and treated with increasing Aβ-AChEf concentrations (μM), indicated by the black bars on top of the graph. The complexes were not washed out in between increasing concentrations. Fluorescence changes were recorded at 1 min intervals and are shown as ratio ΔF/Fo. (D) Final normalized fluorescence intensities reached at the end of experiments are expressed over the control situation for the following treatments: 5 μM Aβ-AChEf; 2 mM EGTA; 30 μM BAPTA-AM; * p ≤ 0.001. (E) Cell viability was evaluated by MTT assay. Hippocampal neurons were treated for 24 h as follow: 5 μM Aβ-AChEf; 2 mM EGTA. Mean MTT reduction value was expressed as % of control situation; *p ≤ 0.001 compared with the control condition; #p ≤ 0.005 compared to the Aβ-AChEf treatment.