Aβ-induced deficits in axonal transport depend on neuronal activity, but are not associated with obvious rises in [Ca²⁺]_i. (A) Primary hippocampal neurons from wild-type mice were treated with TTX (1 µM) to silence neuronal activity or with vehicle (Veh), followed by exposure to Aβ_1–42 oligomers. The percentage of moving mitochondria in the axons of Tau^+/+ neurons was measured before (baseline) and during these treatments. n = 22–26 axons per treatment combination from three independent sessions at DIV 12–14. ##, P < 0.01 as indicated by bracket (unpaired t test); ***, P < 0.001 versus corresponding baseline (repeated measures ANOVA, Dunnett’s test). (B–E) [Ca²⁺]_i levels in primary hippocampal neurons from Tau^+/+ and Tau^−/− mice were measured with Fura-2 (340:380 ratio) by live fluorescence microscopy at baseline (2 min) and after adding Aβ_1–42 oligomers (for 40 min) or KCl (50 mM final concentration for 5 min) to the medium. (B) Representative images of the cultures. Bars, 100 µm. (C–E) Quantitation of [Ca²⁺]_i relative to baseline levels (arbitrarily defined as 1.0). Aβ increased the variance in [Ca²⁺]_i in both genotypes compared with vehicle-treated Tau^+/+ neurons. **, P < 0.01 (Levene’s test). Kruskal-Wallis ANOVA, followed by Dunn’s post hoc test confirmed that the sevenfold rise in [Ca²⁺]_i induced by KCl in both genotypes was significant compared with vehicle-treated Tau^+/+ neurons (**, P < 0.01) and revealed no differences between genotypes for Aβ and KCl. (C and D) n = 356–1,002 neurons per genotype from 5–11 independent sessions at DIV 12–14. (E) n = 63–356 neurons per genotype from two to five independent sessions at DIV 12–14. Data in A are means ± SEM. Data in C are means ± SD. Plots in D and E show medians, quartiles, and ranges.