Sustained inhibition of PI(3)P synthesis is necessary to block the cLTP-dependent structural plasticity of spines. (A) Diagram of experiment. DIV16 hippocampal neurons were transfected with eGFP as a filler to visualize dendritic morphology. In the long treatment condition, 24 h after transfection, cells were either treated with 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 then further incubated in the presence of DMSO or VPS34-IN1 for an additional 50 min before fixation. In the short treatment condition, treatment with DMSO or 1 μM VPS34-IN1 was initiated 5 min after the cLTP stimulus. (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. DMSO (short): 0.360 ± 0.007, N = 30 neurons; DMSO (short) with cLTP: 0.417 ± 0.012, N = 30 neurons; DMSO (long): 0.353 ± 0.006, N = 30 neurons; DMSO (long) with cLTP: 0.453 ± 0.013, N = 30 neurons; VPS34-IN1 (short): 0.345 ± 0.008, N = 30 neurons; VPS34-IN1 (short) with cLTP: 0.425 ± 0.011, N = 30 neurons; VPS34-IN1 (long): 0.355 ± 0.009, N = 30 neurons; VPS34-IN1 (long) with cLTP: 0.351 ± 0.008, N = 30 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***P < 0.005, ****P < 0.001. Error bars are SEM. DIV, days in vitro.