Fig. 3: 5-HT4R activation increases cofilin phosphorylation and induces spine maturation in hippocampal neurons. | Communications Biology

Fig. 3: 5-HT4R activation increases cofilin phosphorylation and induces spine maturation in hippocampal neurons.

From: Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

Fig. 3

a, b Hippocampal neurons (DIV12) isolated from WT (a) or 5-HT4R-deficient mice (KO) (b) were treated with the 5-HT4R agonist BIMU8 and/or antagonist GR with or without pre-treatment with the specific ROCK inhibitor Y-27632 (50 µM), followed by the western blot with antibodies against phosphorylated (upper row, p-Cof), total cofilin (middle, Cof) and GAPDH as a loading control (bottom). (Left) Representative western blot showing cofilin phosphorylation in WT neurons (a) and in 5-HT4R KO neurons (b). (Right) Quantification of the relative cofilin phosphorylation in WT neurons (a) and in 5-HT4R KO neurons (b, N = 5). c Representative time-lapse images (left) of cerulean-expressing hippocampal neurons from WT and 5-HT4R KO mice in control (vehicle, veh) and after treatment with BIMU8, GR, GR with BIMU8, Y-27632, and Y-27632 with BIMU8. The asterisk indicates the location of spine that underwent structural plasticity (maturation). Scale bar, 2 μm. (Right) Quantification of changes in the head width/length ratio of dendritic spines at 10 min after treatment of neurons with vehicle (WT: n = 425 spines, 10 cells; KO: n = 238 spines, 8 cells), BIMU8 (WT: n = 435 spines, 11 cells; KO: n = 425 spines, 6 cells), GR (WT: n = 418 spines, 10 cells), GR with BIMU8 (WT: n = 422 spines, 8 cells), Y-27632 (n = 248 spines, 6 cells), and Y-27632 with BIMU8 (n = 381 spines, 7 cells) *p < 0.05, **p < 0.01, ***p < 0.001 (nested ANOVA with Newman–Keuls test). d Representative images of secondary dendrites (left) in hippocampal neurons (DIV12) treated for 4 days with vehicle (veh) or BIMU8. Scale bar: 10 μm. (Right) Quantification for different types of dendritic protrusions, including stubby spines, thin spines, mushroom (mush) spines, and filopodia (N = 5 cultures, n = 90 spines, at least 4 neurons per condition/experiment). *p < 0.05, **p < 0.01 (one-way ANOVA, post hoc Sidak test). See also Supplementary Fig. 4. All data are presented as mean ± SEM.

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