Increased frequency of multi-synapse boutons in RIM4 KO_PCP2 mice. (A and B) Examples of scanning transmission electron micrographs of the molecular layer of the cerebellum (n = 4 RIM4 WT and n = 4 KO_PCP2 mice were analysed for this figure). Asterisks mark Purkinje cell dendrites in the overview images. Arrowheads (orange) mark the release sites in the presynaptic varicosities (orange). Regions for analysis were chosen ∼50 µm distant to the Purkinje cell layer. Varicosities formed on dendrites (and not on spines) were not included in this analysis. (C) Quantitative analysis of abundance of presynaptic varicosities of single and multi-varicosities. n = 4 mice per group, 2500 µm² region of interest analysed per mouse. Data are presented as sum ± 99% confidence interval of the proportions. Data were statistically compared using the Poisson means test with alpha = 1%, resulting in a significance level of ∼5% when taking into account a Bonferroni-type of correction for three simultaneous comparisons. For RIM4 wild-type (WT) and KO_PCP2 mice 243, 234, 215, 220 and 261, 260, 215, 186 synapses were analysed per region of interest, respectively. Proportions were calculated after summing the counts across all mice within each group. (D) Example climbing fibre (CF) synapses identified in scanning transmission electron micrographs. Dendrites are shaded green and were identified by long longitudinal structures typically filled with either filaments (smaller branches, wild-type example) or pronounced smooth endoplasmic reticulum and mitochondria (larger branches, KO_PCP2 example). Climbing fibre synapses are shaded blue and in some cases the axon (the CF) connecting individual release sites was visible (right). (E and F) Total counts of parallel fibre (PF) and CF synapse release sites, respectively. As CF synapses occur less frequently, a larger area was counted for this type of synapse. Counting regions of interest (ROIs, each 30 × 30 µm²) were equally distributed across mice and randomly placed in the proximal, medial and distal parts of the molecular layer. Release site counts were summed across mice within the wild-type and the KO_PCP2 groups and plotted as sum ± 99% confidence interval per region. The Poisson means test with alpha = 1%, resulting in a significance level of ∼5% when taking into account a Bonferroni-type of correction for three simultaneous comparisons, was used to assess statistical significance. KO = knockout.