Figure 3

RIM1α levels correlate with synaptic efficiency. A) Post-hoc immunocytochemical images of FM1-43 loaded boutons in green and anti-RIM1α 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), RIM1α 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 RIM1α). E) Mean FM1-43 unloading values of the whole population of synaptic boutons (black), the subpopulation of synaptic boutons whose RIM1α content is higher than 2 (red) and the subpopulation of those ROIs which RIM1α content is lower than 0.5 (blue). F) Normalized FM1-43 unloading according to RIM1α 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 3666 synaptic boutons from 4 covers (whole population), 502 boutons from 4 covers (>2) and 1300 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.