Figure 4

Munc13-1 levels do not correlate with synaptic efficiency. A) Post-hoc immunocytochemical images of FM1-43 loaded boutons in green and anti-Munc13-1 in red. B) binary mask of the field imaged in A) obtained using IgorPro software for processing the experiments. C) Higher magnification detail of the boxed area in A) showing FM1-43 staining (a), Munc13-1 labelling (b), the merged image (c) and phase contrast image (d). Arrowheads indicate different synaptic boutons. Scale bar = 5 μm. D) Area boxed in A after superimposition of the detail boxed in B. Note the absence of “dead” pixels in the different ROIs in both channels (FM1-43 and Munc13-1). E) Unloading fraction values of the whole population of synaptic boutons (black), the subpopulation of synaptic boutons whose Munc13-1 content is higher than 2 (red) and the subpopulation of those ROIs which Munc13-1 content is lower than 0.5 (blue). F) Normalized FM1-43 unloading according to Munc13-1 IR values: whole population in black, subpopulation of synaptic boutons with IR > 2 in red and subpopulation of synaptic boutons with IR value lower than 0.5 in blue. Unloading fractions in E and traces in F are means of 4461 synaptic boutons from 4 covers (whole population), 761 boutons from 4 covers (>2) and 2261 boutons from 4 covers (<0.5). Scale bar = 25 μm. One way ANOVA followed by Bonferroni’s test for means comparison was performed. Significance was considered when p < 0.05.