Dipyridamole rescues presynaptic defects in the chodl−/−mutant. (A) Representative images of motor axons in control chodl+/+ (mnx1:EGFP) and chodl mutant fish (chodl−/−; mnx1:EGFP) after control (DMSO) or drug (dipyridamole, 10 µM) treatment. Motor axons are labelled in green, the presynaptic compartment was labelled for the synaptic marker synaptotagmin-2 (red, using Znp-1) and the postsynaptic compartment by antibodies against AChR (blue). Yellow squares indicate the horizontal myoseptum (HM). Scale bars: 10 µm. (B-D) Quantification of the presynaptic (B), postsynaptic (C) and total (D) area in zebrafish embryos as described in A. (B) The presynaptic compartment is enlarged in chodl mutants but 10 µM dipyridamole rescues this phenotype (one-way ANOVA ****P<0.0001 with Tukey's multiple comparison test: DMSO-control vs DMSO-chodl mutant ****P<0.0001, dipyridamole-control vs DMSO-chodl mutant ****P<0.0001, DMSO-chodl mutant vs dipyridamole-chodl mutant **P=0.0090, statistical power=0.9999). (C) Dipyridamole induces an increase in the total postsynaptic area in chodl mutants (one-way ANOVA *P=0.0269 with Tukey's multiple comparison test *P=0.0148, statistical power=0.7222). (D) The enlarged total synaptic area in chodl mutants is not fully rescued by dipyridamole (Kruskal–Wallis test ****P<0.0001 with Dunn's multiple comparison test: DMSO-control vs DMSO-chodl mutant ****P<0.0001, DMSO-control vs dipyridamole-chodl mutant *P=0.0194, dipyridamole-control vs DMSO-chodl mutant ***P=0.0004, DMSO-chodl mutant vs dipyridamole-chodl mutant *P=0.0382, statistical power=0.9999). (E-G) Quantification of the presynaptic (E), postsynaptic (F) and total (G) number of discernible puncta in zebrafish embryos as described in A. (E) The reduced number of presynaptic discernible puncta in chodl mutants is not fully rescued by dipyridamole (Kruskal–Wallis test ****P<0.0001 with Dunn's multiple comparison test: DMSO-control vs DMSO-chodl mutant ****P<0.0001, DMSO-control vs dipyridamole-chodl mutant **P=0.0034, dipyridamole-control vs DMSO-chodl mutant ****P<0.0001, statistical power=0.9999). (F) The reduced number of postsynaptic discernible puncta in chodl mutants is not rescued by dipyridamole (one-way ANOVA ****P<0.0001 with Tukey's multiple comparison test: DMSO-control vs DMSO-chodl mutant ****P<0.0001, DMSO-control vs dipyridamole-chodl mutant ****P<0.0001, dipyridamole-control vs DMSO-chodl mutant ***P=0.0002, dipyridamole-control vs dipyridamole-chodl mutant ***P=0.003, statistical power=0.9999). (G) The total number of discernible puncta in chodl mutants is not rescued by dipyridamole (one-way ANOVA ****P<0.0001 with Tukey's multiple comparison test: DMSO-control vs DMSO-chodl mutant ****P<0.0001, DMSO-control vs dipyridamole-chodl mutant **P=0.0039, dipyridamole-control vs DMSO-chodl mutant ****P<0.0001, statistical power=0.9995). (H,I) Labelling intensity of pre- (H) and postsynaptic (I) area in zebrafish embryos as described in A. (H) The increased presynaptic labelling intensity in chodl mutant is not rescued by dipyridamole (Kruskal–Wallis test *P=0.0249 with Dunn's multiple comparison test: DMSO-control vs DMSO-chodl mutant *P=0.0213, statistical power=0.9517). (I) Application of dipyridamole does not change the mean labelling intensity of the postsynaptic compartment in different treatment groups. chodl mutants, blue bars; wild-type embryos, white bars; with drug application (dipyridamole) and without drug application (DMSO). Each data point represents one animal, n-numbers are indicated within each bar. Error bars represent means±s.e.m.
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