Experimental design of the short-term zebrafish birefringence assay. (A) Heterozygous sapje or sapje-like pairs were mated and their respective embryos were collected and pooled. Drug treatment was initiated on 1 dpf and continued through 4 dpf when birefringence was analyzed. (B) Representative images of the patchy muscle birefringence pattern characteristic of sapje and sapje-like homozygous mutants compared to the highly organized sarcomere structure of (+/+) and (+/−) siblings. Given that the sapje and sapje-like dystrophin mutations are recessive, 25% of untreated offspring are expected to exhibit the affected muscle phenotype.

Short-term assay of serotonin, serotonin precursors, products and receptor agonists. (A–E) Treatment with serotonin, 5-hydroxy-L-tryptophan (5-HTP), tryptophan, melatonin and cisapride did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug. (F) Both affected and unaffected zebrafish treated with ≤16.5 μM cisapride exhibited abnormal body morphology.

Short-term assay of SSRIs. (A–F) Treatment with citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. (G) Zebrafish treated with fluoxetine exhibited dose-dependent toxicity. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug.

Short-term assay of tricyclic antidepressants and RIMAs. (A–F) Treatment with amitriptyline, clomipramine, imipramine, moclobemide, pirlindole and toloxatone did not significantly decrease the percentage of zebrafish exhibiting the affected muscle phenotype detected by birefringence. Treatment with 2.5 μg/ml aminophylline significantly decreased the percentage of affected fish. Data represent means±s.e.m.; *P<0.05 versus paired control by one-way ANOVA and Bonferroni post-hoc test. Values above each column indicate the number of sapje (N) and sapje-like (n) fish treated with the respective drug.

Long-term zebrafish survival assay. (A) Experimental design of the long-term survival assay. Cohorts of sapje or sapje-like offspring were screened as affected or unaffected on 4 dpf, at which time drug treatment was initiated and continued through 30 dpf. The water was changed and surviving fish were counted every other day. (B–G) Treatment with 33 μM serotonin, 66 μM 5-HTP, 16.5 μM tryptophan, 33 μM melatonin, 8.25 μM cisapride or 33 μM moclobemine did not significantly improve the survival of affected fish. 8.25 μM cisapride was toxic to both affected and unaffected fish beginning on 14 dpf. For each condition, 30–40 fish were tested in three replicate experiments. Data represent means±s.e.m. †P<0.05 affected versus respective unaffected, *P<0.05 drug-treated versus respective control by two-way ANOVA and Bonferroni post-hoc test. AF, affected; UA, unaffected. (H) Affected fish treated with 2.5 μg/ml aminophylline significantly increased survival versus affected controls. †P<0.05 affected versus respective unaffected, *P-values are for the closed blue circles and indicate significant difference between drug-treated AF versus control AF by two-way ANOVA and Bonferroni post-hoc test.