Fig 1: RIM-BP2 disruption does not seem to alter the molecular composition of the endbulb AZs. A, C, E, G, I, Representative maximal z-projections of confocal image stacks of BCs in RIM-BP2 WT shown on left and RIM-BP2 KO on right; 30-µm coronal brainstem slices were immunolabelled for RIM-BP2 (A), Munc13-1 (C), RIM2 (E), and CAST (G) and co-stained for VGlut1 to outline endbulbs and Gephyrin for inhibitory synapse labeling. For Bsn immunolabeling (I), we co-stained for Homer1 (excitatory postsynapses) and Gephyrin. B, D, F, H, J, Quantification of fluorescence intensity of CAZ proteins at endbulbs and inhibitory synapses of BCs (left), and the number of spots of the target CAZ protein at endbulbs (right). A, Representative z-projection shows loss of RIM-BP2 immunoreactivity in the RIM-BP2 KO brainstem slices. There was only a faint residual and likely unspecific signal remaining at excitatory and inhibitory synapses (B, left), RIM-BP2 immunofluorescent spots were nearly abolished (B, right). The immunofluorescence intensity of Munc13-1 (D), RIM2 (F), CAST (H), and Bsn (J) were unaltered at RIM-BP2 KO endbulbs. Right columns of D, F, H, J show similar number of CAZ protein spots localized at KO endbulb AZs as in the WT AZs. The numbers were calculated by subtracting the Gephyrin colocalized spots from the total number of spots per BC. The data are presented as box and whiskers plots (grand median of mean estimates for all BCs, lower/upper quartiles, 10–90th percentiles). Each data point represents the mean estimate of fluorescence intensity of all the AZs of each BC included in the analysis. Statistical significance of differences between groups was determined with unpaired Student's t test (with Welch's correction, when variances differed significantly), if the data's distribution did not differ from a normal distribution or with Mann–Whitney U test in case of non-normally distributed data. Normality of distribution was tested with Jarque–Bera test, and variances were compared with F test; *p = 0.05, **p = 0.01, ***p = 0.001, n.s. p = 0.05. Samples from RIM-BP2 WT and RIM-BP2 KO mice, aged p15–p21, were harvested and processed strictly in parallel, and images were acquired in parallel using the same laser power and gain settings at the same confocal microscope. Data information: N: number of animals, S: number of slices, n: number of BCs. All scale bars: 5 µm.