Figure 1A Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P and Homer (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments. B Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001C Representative 3-color confocal images of cultured hippocampal neurons fixed and stained for VGlut1, VGAT and PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or VGAT+ presynaptic terminals. Scale bar, 10 µm. D PI(3)P intensity in VGlut1+ and VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory and inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Relative PI(3)P levels in the MAP2+ (Soma) and Syp+ (Synapse) areas in DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001F Cultured hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification.G Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001H Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) for 24 h. N = 3 independent experiments (≥ 65 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001I Cultured hippocampal neurons treated with AP5 (20 µM), CNQX (10 µM) or TTX (0.2 µM) for 24 h, fixed and stained for MAP2, Syp and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm.J Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001K Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001