Synaptic impairments in haploinsufficient and heterozygous G544D neurons and differential partial rescue with Doc2A/B. (A–E) Analysis of spontaneous neuronal activity. Munc18-1 wild-type (WT), heterozygous knockout (with and without overexpression of Doc2A/B) and heterozygous knockout + G544D (with and without overexpression of Doc2A/B) were cultured on a multi-electrode array with 16 electrodes per well (A) and analysed at 14 days in vitro (DIV14) for mean firing rate (A and B), inter-burst interval (A and C), network burst frequency (A and D) and network burst duration (A and E). Purple boxes in A indicate network burst activity. One-way ANOVA with Šídák’s post hoc test or Kruskal–Wallis with Dunn’s post hoc test;*P < 0.05, **P < 0.01; n = 70–244 independent experiments. (F and G) Neurons cultured as above were stimulated by high potassium and subjected to an antibody uptake assay. Endocytosed anti-synaptotagmin-1 (Syt-1) antibody was quantified by measuring immunostaining under a synaptic mask derived from the SV2 signal. One-way ANOVA with Šídák’s post hoc test; ^#P < 0.05, **P < 0.01, ***^,###P < 0.001; n = 4–7 independent experiments. Scale bar = 5 µm. (H and I) Munc18-1 WT, heterozygous knockout (with and without overexpression of Doc2A/B), and heterozygous knockout + G544D (with and without overexpression of Doc2A/B) neurons lentivirally expressing synaptophysin-pHluorin (SypHy) were stimulated by treatment with 55 mM potassium chloride (KCl) and the change in SypHy fluorescence over baseline (ΔF/F_i) normalized to the total vesicle pool (revealed by treatment with NH₄Cl) was analysed. Two-way ANOVA with Bonferroni’s post hoc test; ****P < 0.0001; n = 6–11 independent experiments. All data are presented as mean ± standard error of the mean.