The regulation of RRP augmentation in the absence of RIM and Munc13-1. A, Sample traces represent RRP augmentation protocol in RIM1/2flox cultures infected with ΔCre + Scramble (Scr.) shRNA (black), ΔCre + 40 × 105 IU Munc13-1 KD shRNA (orange), Cre + Scramble shRNA (blue), and Cre + 40 × 105 Munc13-1 shRNA (gray). After initial sucrose-evoked charge measurement (Suc1), 50 APs at 10 Hz were applied followed by another sucrose-evoked charge measurement 2 s after (Suc2). B, Bar plot represents the mean RRP augmentation (ratio of Suc2 to Suc1) in neurons described in A. Graph labels indicate endogenous expression “+.” “KD” refers to the Munc13-1 KD. “cDKO” refers to RIM1/2-deficient neurons; nonparametric one-way ANOVA with Kruskal–Wallis test followed by Dunn's post hoc test. C, Sample traces of RRP augmentation protocol applied on Munc13-2 KO neurons with Munc13-1 KD (40 × 105 shRNA IU). D, Bar plot represents the measurement of RRP augmentations (Suc2/Suc1) in neurons as described in C. The nonparametric t test, followed by Mann–Whitney test, did not show differences between Munc13-2 KO with Scramble and Munc13-1 KD. Numbers in bar plots indicate the cell number/culture number. Data indicate mean ± SEM. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. For the statistical overview, see also the table in Extended Data Figure 7-1.