Figure 7.

Long-term treatment of TSC2^−/− neurons with rapamycin changed Ca²⁺ dynamics from synchronous to sporadic patterns. A, Schematic illustration of rapamycin treatment and Ca²⁺ imaging after plating NPCs for differentiation. B, Ca²⁺ dynamics of DMSO-treated (left) or rapamycin-treated (right) TSC2^−/− neurons. The ΔF/F₀ changes of the Fluo-8 signals for 36 frames are shown. Neurons that were treated with DMSO or 10 nm rapamycin for >18 d from day 2 of neuronal differentiation were used for the analysis. C, Raster plots of Ca²⁺ spikes from B. D, Ca²⁺ spike frequencies of TSC2^+/+, TSC2^+/−, and TSC2^−/− neurons with or without rapamycin treatment. Data are mean ± SD. One-way ANOVA, F_(5,29) = 26.88, p < 0.0001, Dunnett's multiple comparisons test compared with TSC2^−/−, ****p < 0.00001. The number of experiments was 6 for each group except for TSC2^−/− + Rapa (n = 5). E, Ca²⁺ spike frequencies of TSC2^−/− neurons after treatment with 50 nm rapamycin for 5, 10, or 30 min. We used 50 nm rapamycin to completely and rapidly suppress the mTOR activity. The experiments were performed 3 times. One-way ANOVA, F_(3,8) = 0.009871, p = 0.999.