Synaptic release depends on Munc18-1 binding to the SNARE complex. (A) Analysis of spontaneous synaptic release upon rescue with WT and mutant Munc18-1s. Representative 10-s segments from 10-min-long traces of spontaneous excitatory synaptic activity, which was recorded at a holding potential of −70 mV in the presence of 1 µM tetrodotoxin and 50 µM picrotoxin. (B) Bar diagram describing the frequency (top) and amplitude (bottom) of spontaneous release (WT, n = 13; Munc18, n = 19; E59K, n = 10; K63E and E66A, n = 9). (C) Representative traces of field stimulation (at 0.4 Hz) evoked excitatory responses from neurons of WT (n = 14) or Munc18-1 KO infected with WT (n = 9), E59K (n = 7), E66A (n = 16), or K63E (n = 11) Munc18-1–24-cerulean. Note that only the first 400 ms of the traces are shown for clarity. (D) Bar diagram summarizing the amplitudes of evoked responses for cultures rescued with the WT Munc18-1 and different Munc18-1 mutants. (E) Synaptic responses characterized as the amount of transferred charge. Asterisks in the bar diagrams mark statistical significance of the difference between the WT and mutant rescues (*, P < 0.05; ***, P < 0.005). (B, D, and E) Data are shown as means ± SEMs. Dashed lines indicate WT values.