Fig. 2

Synaptic activity regulates ER-mitochondria contacts and the VAPB-PTPIP51 interaction in hippocampal neurons. a Confocal z-stack of neuron transfected with SPLICS_s ER-mitochondria reporter plasmids and immunostained for NFH and MAP2 to show axons and dendrites; SPLICS_s fluorescent signal is present in cell bodies axons and dendrites. Scale bar = 10 μm. b SPLICS_s signals are stable in unstimulated neurons. SPLICS_s transfected neuron axons were imaged in time-lapse over a 10 min period and displayed no significant changes in SPLICS_s signals. Graph shows average fluorescence signal F/initial signal F0 as a %. Error bars are SEM; N = 14 neurons from 8 different cultures. (c and d), Electrical field stimulation of synaptic activity increases ER-mitochondria contacts including contacts close to synapses. c neurons transfected with SPLICS_s ER-mitochondria reporter plasmids were imaged in time-lapse prior to and after electrical field stimulation; image shows axon in transfected cell. d SPLICS_s transfected neurons were loaded with FM 4–64 and SPLICS_s (green) and FM 4–64 (red) signals imaged in time-lapse prior to and after electrical field stimulation. Arrows indicate increased SPLICS_s signals closely associated with active synapses identified by loss of FM 4–64 signal. Graph shows changes in fluorescence signal (fluorescence at each time-point (F)/average of all pre-stimulation frames (F0)) from 7 synapses. Error bars are SEM. Analyses of SPLICS_s signal reveals no changes prior to stimulation in agreement with data shown in (b) above but significant increases following stimulation (time 30–60 s; prior to stimulation v time 300–360; post stimulation p ≤ 0.0001, one way ANOVA with Tukey’s post hoc test). e Representative SIM image of VAPB-PTPIP51 PLA signals in synaptic regions of unstimulated neurons and in neurons after (60 s) electrical field stimulation. VAPB-PTPIP51 PLAs (red) were performed and neurons then immunostained for synaptophysin (green) and PSD95 (blue). Arrows indicate PLA signals close (less than 1 μm) to synapses as identified by apposition of synaptophysin and PSD95 signals. Bar chart shows normalised VAPB-PTPIP51 PLA signals (%) close to synapses in unstimulated neurons and in neurons after electrical field stimulation. Data were analysed by Student’s t test. N = 36 neurons unstimulation and 34 neurons post stimulation from 3 independent experiments. Error bars are SEM; ***p ≤ 0.001. Scale bars in c, d and e = 2 μm