Fig. 4

Loss of VAPB or PTPIP51 disrupts synaptic activity. a Kinetics of FM 4–64 release from synaptic boutons in hippocampal neurons either untreated (UT) or treated with control (Ctrl), VAPB or PTPIP51 siRNAs. Neurons were loaded with FM 4–64 and synaptic activity induced by electrical field stimulation. Periods of electrical field stimulation are indicated by shaded regions. FM 4–64 signals were determined from images acquired by time-lapse microscopy. F/F0 represents the ratio of the FM 4–64 fluorescent signals at each time point to signals at time 0. Error bars are mean ± SEM. Bar chart shows F/F0 FM 4–64 fluorescent signals at time 330 s. Data were analysed by one-way ANOVA with Tukey’s post hoc test. N = 24 boutons UT, N = 40 boutons Ctrl siRNA, N = 24 boutons VAPB siRNA, N = 35 boutons PTPIP51 siRNA from 3 independent experiments. Error bars are SEM, ***p ≤ 0.001. b Representative images of transfected SypHy-RGECO axonal signals in neurons prior to and following electrical field stimulation to induce synaptic activity. Scale bar = 1.5 μm. Graphs show changes in SypHy (ΔG) and RGECO (ΔR) fluorescent signals; shading shows time of electrical field stimulation. N = 18 synapses; error bars are SEM. c SypHy-RGECO reporter reveals reduced synaptic responses to electrical field stimulation in VAPB and PTPIP51 siRNA treated hippocampal neurons. Bar-graph shows the ratio between SypHy (ΔG) and RGECO (ΔR) fluorescence signals in response to electrical field stimulation. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N = 39 synapses UT, N = 48 synapses Ctrl siRNA, N = 59 synapses VAPB siRNA, N = 41 synapses PTPIP51 siRNA from 3 to 4 independent experiments. Error bars are SEM; ***p ≤ 0.001