Fig. 3.

IL-33 regulates homeostatic plasticity through the activation of its ST2/IL-1RAcP neuronal receptor complex. (A–E) Knockdown of ST2 (sh-ST2) or IL-1RAcP (sh-IL-1RAcP) in cultured hippocampal neurons abolished the IL-33-induced increase in excitatory synaptic transmission and the number of synaptic PSD-95 clusters. (A) Representative traces of mEPSCs. (B) Cumulative probability distributions of mEPSC interevent intervals. (C) Quantification of mean mEPSC frequency (***P < 0.001, two-way ANOVA; sh-Con, Con: n = 55 neurons; sh-Con, IL-33: n = 49 neurons; sh-ST2, Con: n = 23 neurons; sh-ST2, IL-33: n = 22 neurons; sh-IL-1RAcP, Con: n = 22 neurons; sh-IL-1RAcP, IL-33: n = 24 neurons). (D) Representative images of endogenous PSD-95 clusters (labeled with GFP-tagged PSD-95 intrabody) in tdTomato-expressing hippocampal neurons. White arrowheads indicate endogenous PSD-95 clusters in dendritic spine protrusions. (Scale bar, 5 μm.) (E) Quantification of synaptic PSD-95 clusters (***P < 0.001, two-way ANOVA; sh-Con, Con: n = 23 neurons; sh-Con, IL-33: n = 22 neurons; sh-ST2, Con: n = 19 neurons; sh-ST2, IL-33: n = 18 neurons; sh-IL-1RAcP, Con or IL-33: n = 15 neurons). (F–J) Cultured hippocampal neural cells were transfected with sh-ST2 or sh-Con and treated with TTX for 48 h before mEPSC analysis (F–H) or immunocytochemical analysis (I and J). (F) Representative traces of mEPSCs. (G) Cumulative probability distributions of mEPSC interevent intervals. (H) Quantification of mean mEPSC frequency (***P < 0.001, two-way ANOVA; sh-Con, Con: n = 25 neurons; sh-Con, TTX: n = 22 neurons; sh-ST2, Con: n = 20 neurons; sh-ST2, TTX: n = 22 neurons). Representative images (I) and quantification (J) of synaptic PSD-95 clusters (***P < 0.001, two-way ANOVA; sh-Con, Con: n = 23 neurons; sh-Con, TTX: n = 20 neurons; sh-ST2, Con or TTX: n = 19 neurons). (Scale bar, 5 μm.) All experiments were performed in triplicates. Values are the mean ± SEM.