Figure 2.
A decrease in PI(3)P levels blocks functional and structural plasticity during LTP. (A) Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 stratum radiatum using ACSF-filled glass pipettes (3–5 MΩ). Schaffer collateral fibers were stimulated every 30 s with 0.1-ms pulses (50–250 μA), and slices were pretreated with DMSO (n = 4 slices, 4 mice) or 5 μM VPS34-IN1 (n = 6 slices, 4 mice) for 30 min. After a 10-min stable baseline, LTP was induced with two high-frequency stimulations (HFS; 100 Hz, 1 s) separated by 30 s. Data were analyzed by two-way ANOVA with Sidak’s post hoc test, ****P < 0.0001. Error bars are SEM. (B) Summary data (55–60 min after HFS) corresponding to (A). DMSO: 136.441 ± 4.657; VPS34-IN1: 102.142 ± 4.388. Data were analyzed by Student’s unpaired t test, ***P < 0.001. (C) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. 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. Scale bar, 5 µm. (D) 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: 0.354 ± 0.007, N = 30 neurons; DMSO with cLTP: 0.435 ± 0.009, N = 30 neurons; VPS34-IN1: 0.359 ± 0.007, N = 30 neurons; VPS34-IN1 with cLTP: 0.375 ± 0.006, 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. (E) Neurons were transfected at DIV12 with eGFP (filler). At DIV16, neurons were treated with either DMSO or 1 μM SAR405 for 30 min, followed by a 5-min cLTP stimulus in the presence of compounds where indicated. Neurons were further incubated in the presence of DMSO or SAR405 for 50 min before fixation. 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: 0.373 ± 0.006, N = 30 neurons; DMSO with cLTP: 0.439 ± 0.011, N = 30 neurons; SAR405: 0.367 ± 0.008, N = 30 neurons; SAR405 with cLTP: 0.351 ± 0.006, 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. (F) Validation of an shRNA clone to knockdown rat VPS34. Rat cortical neurons were infected with lentiviruses carrying either VPS34-shRNA (TRCN0000025373; Millipore Sigma) or control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002, Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (G) The levels of VPS34 protein were quantified in neurons infected with VPS34-shRNA and normalized to VPS34 levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; VPS34-shRNA: 30.5 ± 8.50%. N = 3 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) DIV12 neurons were co-transfected with eGFP (filler) and either ctrl-shRNA or VPS34-shRNA. At DIV16, neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. 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: 0.351 ± 0.007, N = 30 neurons; ctrl-shRNA with cLTP: 0.436 ± 0.010, N = 30 neurons; VPS34-shRNA: 0.347 ± 0.008, N = 30 neurons; VPS34-shRNA with cLTP: 0.360 ± 0.007, 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. DIV, days in vitro. Source data are available for this figure: SourceData F2.

A decrease in PI(3)P levels blocks functional and structural plasticity during LTP. (A) Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 stratum radiatum using ACSF-filled glass pipettes (3–5 MΩ). Schaffer collateral fibers were stimulated every 30 s with 0.1-ms pulses (50–250 μA), and slices were pretreated with DMSO (n = 4 slices, 4 mice) or 5 μM VPS34-IN1 (n = 6 slices, 4 mice) for 30 min. After a 10-min stable baseline, LTP was induced with two high-frequency stimulations (HFS; 100 Hz, 1 s) separated by 30 s. Data were analyzed by two-way ANOVA with Sidak’s post hoc test, ****P < 0.0001. Error bars are SEM. (B) Summary data (55–60 min after HFS) corresponding to (A). DMSO: 136.441 ± 4.657; VPS34-IN1: 102.142 ± 4.388. Data were analyzed by Student’s unpaired t test, ***P < 0.001. (C) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. 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. Scale bar, 5 µm. (D) 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: 0.354 ± 0.007, N = 30 neurons; DMSO with cLTP: 0.435 ± 0.009, N = 30 neurons; VPS34-IN1: 0.359 ± 0.007, N = 30 neurons; VPS34-IN1 with cLTP: 0.375 ± 0.006, 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. (E) Neurons were transfected at DIV12 with eGFP (filler). At DIV16, neurons were treated with either DMSO or 1 μM SAR405 for 30 min, followed by a 5-min cLTP stimulus in the presence of compounds where indicated. Neurons were further incubated in the presence of DMSO or SAR405 for 50 min before fixation. 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: 0.373 ± 0.006, N = 30 neurons; DMSO with cLTP: 0.439 ± 0.011, N = 30 neurons; SAR405: 0.367 ± 0.008, N = 30 neurons; SAR405 with cLTP: 0.351 ± 0.006, 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. (F) Validation of an shRNA clone to knockdown rat VPS34. Rat cortical neurons were infected with lentiviruses carrying either VPS34-shRNA (TRCN0000025373; Millipore Sigma) or control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002, Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (G) The levels of VPS34 protein were quantified in neurons infected with VPS34-shRNA and normalized to VPS34 levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; VPS34-shRNA: 30.5 ± 8.50%. N = 3 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) DIV12 neurons were co-transfected with eGFP (filler) and either ctrl-shRNA or VPS34-shRNA. At DIV16, neurons were either treated with cLTP or left untreated and fixed 50 min after cLTP. 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: 0.351 ± 0.007, N = 30 neurons; ctrl-shRNA with cLTP: 0.436 ± 0.010, N = 30 neurons; VPS34-shRNA: 0.347 ± 0.008, N = 30 neurons; VPS34-shRNA with cLTP: 0.360 ± 0.007, 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. DIV, days in vitro. Source data are available for this figure: SourceData F2.

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