Munc18-1 binding to the SNARE complex is critical for release readiness of synaptic vesicles. (A) Representative traces of synaptic excitatory responses to hypertonic solution (+500 mM sucrose to the bath) in WT neurons from littermate controls and Munc18-1 KO neurons rescued with lentivirus-expressing WT Munc18-1–24-cerulean or Munc18-1–24-cerulean with the E59K, K63E, or E66A mutations. (B) Bar diagram depicting the amplitudes of responses to hypertonic sucrose solution for WT cultures or Munc18-1 KO cultures rescued with the WT and mutant Munc18-1s (WT, n = 5; Munc18-1, K63E, and E66A, n = 8; E59K, n = 5). (C) Readily releasable synaptic excitatory transmission characterized as the amount of charge transfer induced by hypertonic sucrose. Asterisks in the bar diagrams mark statistical significance of the difference between the WT and mutant rescues (*, P < 0.05; **, P < 0.01). (B and C) Data are shown as means ± SEMs. Dashed lines indicate WT values.