Figure 5.
PI(3)P synthesis is required for SNX17- and SNX27-dependent structural plasticity of dendritic spines. (A) Representative confocal images of dendritic spines in DIV16 hippocampal neurons co-transfected at DIV12 with eGFP (filler) and either ctrl-, SNX17-, or SNX27-shRNAs. Neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. Scale bar, 5 µm. (B) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. ctrl-shRNA: 0.374 ± 0.007, N = 30 neurons; ctrl-shRNA with cLTP: 0.475 ± 0.012, N = 30 neurons; SNX17-shRNA: 0.360 ± 0.008, N = 30 neurons; SNX17-shRNA with cLTP: 0.351 ± 0.008, N = 30 neurons; SNX27-shRNA: 0.354 ± 0.008, N = 30 neurons; SNX27-shRNA with cLTP: 0.358 ± 0.005, N = 30 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) DIV16 hippocampal neurons co-transfected at DIV12 with eGFP (filler) and either ctrl-, VPS26C-, or VPS26B-shRNAs. Neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. Dendritic spine width was calculated as in B. ctrl-shRNA: 0.376 ± 0.006, N = 30 neurons; ctrl-shRNA with cLTP: 0.472 ± 0.012, N = 30 neurons; VPS26C-shRNA: 0.371 ± 0.007, N = 30 neurons; VPS26C-shRNA with cLTP: 0.378 ± 0.007, N = 30 neurons; VPS26B-shRNA: 0.384 ± 0.009, N = 30 neurons; VPS26B-shRNA with cLTP: 0.381 ± 0.008, N = 30 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Rat hippocampal neurons were co-transfected at DIV12 with SNX17-shRNA and a shRNA-resistant SNX17 construct (SNX17-R). At DIV16, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min, followed by a 5-min cLTP stimulus in the presence of the compounds where indicated. Neurons were further incubated in the presence of DMSO or VPS34-IN1 for 50 min before fixation. The width of dendritic spines in the first 30 µm of secondary dendrites was quantified. DMSO: 0.359 ± 0.009, N = 30 neurons; DMSO with cLTP: 0.430 ± 0.010, N = 30 neurons; VPS34-IN1: 0.364 ± 0.009, N = 30 neurons; VPS34-IN1 with cLTP: 0.369 ± 0.007, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (E) Rat hippocampal neurons were co-transfected at DIV12 with SNX27-shRNA and a shRNA-resistant SNX27 construct (SNX27-R). At DIV16, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min, followed by a 5-min cLTP stimulus in the presence of the compounds where indicated. Neurons were further incubated in the presence of DMSO or VPS34-IN1 for 50 min before fixation. The width of dendritic spines in the first 30 µm of secondary dendrites was quantified. DMSO: 0.340 ± 0.006, N = 30 neurons; DMSO with cLTP: 0.420 ± 0.009, N = 30 neurons; VPS34-IN1: 0.347 ± 0.011, N = 30 neurons; VPS34-IN1 with cLTP: 0.338 ± 0.007, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

PI(3)P synthesis is required for SNX17- and SNX27-dependent structural plasticity of dendritic spines. (A) Representative confocal images of dendritic spines in DIV16 hippocampal neurons co-transfected at DIV12 with eGFP (filler) and either ctrl-, SNX17-, or SNX27-shRNAs. Neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. Scale bar, 5 µm. (B) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. ctrl-shRNA: 0.374 ± 0.007, N = 30 neurons; ctrl-shRNA with cLTP: 0.475 ± 0.012, N = 30 neurons; SNX17-shRNA: 0.360 ± 0.008, N = 30 neurons; SNX17-shRNA with cLTP: 0.351 ± 0.008, N = 30 neurons; SNX27-shRNA: 0.354 ± 0.008, N = 30 neurons; SNX27-shRNA with cLTP: 0.358 ± 0.005, N = 30 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) DIV16 hippocampal neurons co-transfected at DIV12 with eGFP (filler) and either ctrl-, VPS26C-, or VPS26B-shRNAs. Neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. Dendritic spine width was calculated as in B. ctrl-shRNA: 0.376 ± 0.006, N = 30 neurons; ctrl-shRNA with cLTP: 0.472 ± 0.012, N = 30 neurons; VPS26C-shRNA: 0.371 ± 0.007, N = 30 neurons; VPS26C-shRNA with cLTP: 0.378 ± 0.007, N = 30 neurons; VPS26B-shRNA: 0.384 ± 0.009, N = 30 neurons; VPS26B-shRNA with cLTP: 0.381 ± 0.008, N = 30 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Rat hippocampal neurons were co-transfected at DIV12 with SNX17-shRNA and a shRNA-resistant SNX17 construct (SNX17-R). At DIV16, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min, followed by a 5-min cLTP stimulus in the presence of the compounds where indicated. Neurons were further incubated in the presence of DMSO or VPS34-IN1 for 50 min before fixation. The width of dendritic spines in the first 30 µm of secondary dendrites was quantified. DMSO: 0.359 ± 0.009, N = 30 neurons; DMSO with cLTP: 0.430 ± 0.010, N = 30 neurons; VPS34-IN1: 0.364 ± 0.009, N = 30 neurons; VPS34-IN1 with cLTP: 0.369 ± 0.007, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (E) Rat hippocampal neurons were co-transfected at DIV12 with SNX27-shRNA and a shRNA-resistant SNX27 construct (SNX27-R). At DIV16, neurons were treated with either DMSO or 1 μM VPS34-IN1 for 30 min, followed by a 5-min cLTP stimulus in the presence of the compounds where indicated. Neurons were further incubated in the presence of DMSO or VPS34-IN1 for 50 min before fixation. The width of dendritic spines in the first 30 µm of secondary dendrites was quantified. DMSO: 0.340 ± 0.006, N = 30 neurons; DMSO with cLTP: 0.420 ± 0.009, N = 30 neurons; VPS34-IN1: 0.347 ± 0.011, N = 30 neurons; VPS34-IN1 with cLTP: 0.338 ± 0.007, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

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