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. 2020 Dec 28;118(1):e2020810118. doi: 10.1073/pnas.2020810118

Fig. 2.

Fig. 2.

IL-33 is required for TTX-induced homeostatic synaptic plasticity in cultured rat hippocampal neurons. (A–D) IL-33 knockdown abolished the stimulatory effect of TTX to induce homeostatic synaptic plasticity. (A) Schematic diagram of the experimental design. Cultured hippocampal cells at 15 to 16 DIV were transfected with IL-33 siRNA (si-IL-33) or control siRNA (Con) for 48 h and treated with TTX or vehicle (Con) for 3 h. The TTX from the conditioned media (CM) was removed by centrifugal filtration (3-kDa cutoff), and the medium was then reconstituted and transferred to naïve cultures of the same growth stage for 48 h before analysis. (B) Representative traces of mEPSCs. (C) Cumulative probability distributions of mEPSC interevent intervals. (D) Quantification of mean mEPSC frequency (***P < 0.001, two-way ANOVA; si-Con, Con: n = 23 neurons; si-Con, TTX: n = 21 neurons; si-IL-33, Con: n = 22 neurons; si-IL-33, TTX: n = 19 neurons; four independent experiments). (E–G) mEPSC analysis of cultured hippocampal cells treated with different doses of IL-33. (E) Representative traces of mEPSCs. (F) Cumulative probability distributions of mEPSC interevent intervals. (G) Quantification of mean mEPSC frequency (*P < 0.05, one-way ANOVA; IL-33 at 0, 1, 10, and 100 ng/mL: n = 29, 21, 23, and 29 neurons, respectively; three independent experiments). (H–K) Immunohistochemical analysis of excitatory synapses in cultured hippocampal neurons after IL-33 administration (100 ng/mL, 24 h). Immunostaining of PSD-95 (H) or surface GluA2 (sGluA2) (J) and VGluT1 together with MAP2 (blue, to indicate dendritic morphology). (Scale bar, 5 μm.) (I) Quantitative analysis of VGluT1-positive PSD-95 clusters (white arrowheads in H) (*P < 0.05, two-tailed unpaired t test; Con: n = 15 neurons; IL-33: n = 12 neurons; three independent experiments). (K) Quantitative analysis of sGluA2-positive VGluT1 clusters (white arrowheads in J) (*P < 0.05, two-tailed unpaired t test; Con: n = 16 neurons; IL-33: n = 17 neurons; three independent experiments). Values are the mean ± SEM. See also SI Appendix, Fig. S4.