Figure 1.
PI(3)P is present at dendrites and its synthesis increases upon cLTP. (A) Rat hippocampal neurons were transfected at DIV16 with dsRed-EEA1-FYVE and eGFP as a filler. 24 h later, neurons were treated with either DMSO, 1 μM VPS34-IN1, or 1 μM SAR405 for 30 min before fixation. Scale bar, 5 µm. (B) The number of dsRed-EEA1-FYVE–positive puncta in the first 30 μm of secondary dendrites was quantified. DMSO: 0.247 ± 0.016, N = 30 neurons; VPS34-IN1: 0.070 ± 0.011, N = 30 neurons; SAR405: 0.037 ± 0.007, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) Example confocal images taken from DIV17 neurons expressing dsRed-EEA1-FYVE and eGFP (filler) under conditions of cLTP or HBS control (mock). Images of live neurons in an environmental chamber were captured before cLTP (baseline); during cLTP; and at 0, 5, 10, 15, 20, 25, and 30 min after the cLTP stimulus. Representative images of baseline, t = 10, and t = 30 are shown. Intensity presented in the “fire” LUT color scheme. Scale bar, 5 µm. (D) The number of dsRed-EEA1-FYVE–positive puncta in 30 µm of dendritic spines at the different time points following cLTP (or mock) was quantified and normalized to the baseline for each dendrite. Mock: N = 15 dendrites (baseline: 1.00 ± 0.071, cLTP: 1.04 ± 0.077, 0: 1.06 ± 0.076, 5: 1.05 ± 0.072, 10: 1.10 ± 0.069, 15: 1.08 ± 0.080, 20: 1.09 ± 0.083, 25: 1.14 ± 0.079, 30: 1.15 ± 0.079); cLTP: N = 15 dendrites (baseline: 1.00 ± 0.051, cLTP: 1.20 ± 0.070, 0: 1.38 ± 0.072, 5: 1.46 ± 0.076, 10: 1.51 ± 0.075, 15: 1.63 ± 0.089, 20: 1.69 ± 0.082, 25: 1.72 ± 0.072, 30: 1.75 ± 0.076). Three independent experiments. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001. Error bars are SEM. (E) The size of dsRed-EEA1-FYVE–positive puncta in 30 µm of dendritic spines at the indicated time points following cLTP (or mock) was quantified. Mock: N = 15 dendrites (baseline: 0.184 ± 0.013, 10: 0.187 ± 0.011, 30: 0.186 ± 0.014); cLTP: N = 15 dendrites (baseline: 0.184 ± 0.023, 10: 0.188 ± 0.023, 30: 0.181 ± 0.021). Three independent experiments. Data were analyzed using one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. (F) Hippocampal neurons were co-transfected at DIV16 with dsRed-EEA1-FYVE and either an empty pRK5 vector or pRK5-HA-CaMKII-T286D. 24 h later, neurons were fixed, permeabilized, and incubated with an anti-HA antibody. The number of dsRed-EEA1-FYVE–positive puncta in the first 30 μm of secondary dendrites was quantified. Ctrl: 0.201 ± 0.010, N = 30 neurons; CaMKII-T286D: 0.311 ± 0.017, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

PI(3)P is present at dendrites and its synthesis increases upon cLTP. (A) Rat hippocampal neurons were transfected at DIV16 with dsRed-EEA1-FYVE and eGFP as a filler. 24 h later, neurons were treated with either DMSO, 1 μM VPS34-IN1, or 1 μM SAR405 for 30 min before fixation. Scale bar, 5 µm. (B) The number of dsRed-EEA1-FYVE–positive puncta in the first 30 μm of secondary dendrites was quantified. DMSO: 0.247 ± 0.016, N = 30 neurons; VPS34-IN1: 0.070 ± 0.011, N = 30 neurons; SAR405: 0.037 ± 0.007, N = 30 neurons. Three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (C) Example confocal images taken from DIV17 neurons expressing dsRed-EEA1-FYVE and eGFP (filler) under conditions of cLTP or HBS control (mock). Images of live neurons in an environmental chamber were captured before cLTP (baseline); during cLTP; and at 0, 5, 10, 15, 20, 25, and 30 min after the cLTP stimulus. Representative images of baseline, t = 10, and t = 30 are shown. Intensity presented in the “fire” LUT color scheme. Scale bar, 5 µm. (D) The number of dsRed-EEA1-FYVE–positive puncta in 30 µm of dendritic spines at the different time points following cLTP (or mock) was quantified and normalized to the baseline for each dendrite. Mock: N = 15 dendrites (baseline: 1.00 ± 0.071, cLTP: 1.04 ± 0.077, 0: 1.06 ± 0.076, 5: 1.05 ± 0.072, 10: 1.10 ± 0.069, 15: 1.08 ± 0.080, 20: 1.09 ± 0.083, 25: 1.14 ± 0.079, 30: 1.15 ± 0.079); cLTP: N = 15 dendrites (baseline: 1.00 ± 0.051, cLTP: 1.20 ± 0.070, 0: 1.38 ± 0.072, 5: 1.46 ± 0.076, 10: 1.51 ± 0.075, 15: 1.63 ± 0.089, 20: 1.69 ± 0.082, 25: 1.72 ± 0.072, 30: 1.75 ± 0.076). Three independent experiments. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001. Error bars are SEM. (E) The size of dsRed-EEA1-FYVE–positive puncta in 30 µm of dendritic spines at the indicated time points following cLTP (or mock) was quantified. Mock: N = 15 dendrites (baseline: 0.184 ± 0.013, 10: 0.187 ± 0.011, 30: 0.186 ± 0.014); cLTP: N = 15 dendrites (baseline: 0.184 ± 0.023, 10: 0.188 ± 0.023, 30: 0.181 ± 0.021). Three independent experiments. Data were analyzed using one-way ANOVA with Tukey’s post hoc test. Error bars are SEM. (F) Hippocampal neurons were co-transfected at DIV16 with dsRed-EEA1-FYVE and either an empty pRK5 vector or pRK5-HA-CaMKII-T286D. 24 h later, neurons were fixed, permeabilized, and incubated with an anti-HA antibody. The number of dsRed-EEA1-FYVE–positive puncta in the first 30 μm of secondary dendrites was quantified. Ctrl: 0.201 ± 0.010, N = 30 neurons; CaMKII-T286D: 0.311 ± 0.017, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

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