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  Effect of the endocannabinoid receptor antagonists AM251 and AM630 on zebrafish embryo morphology. (A) Schematic showing the timeline for drug exposures and when experimental measurements were made. (A) Drug exposure during the first 24 h of development (0–24 hpf) and drug exposure during the second 24 h of development (24–48 hpf) are highlighted by the yellow bar. Embryos were allowed to develop in normal egg water after each treatment. Primary or secondary motor neuron axonal branching was investigated between 48 and 52 hpf, while locomotion was recorded at 120 hpf (5 dpf). Gross morphological observations occurred at 2 and 5 dpf. The treatments include vehicle control (0.1% DMSO), AM251 0.5 µmol l⁻¹, AM630 5 µmol l⁻¹ or AM251 0.5 µmol l⁻¹+AM630 5 µmol l⁻¹, either from 0 to 24 hpf or 24 to 48 hpf. (B,C) Representative images of treated embryos were taken at 48–52 hpf; scale bars: 0.5 mm. (D,E) Body length (mm) at 2 dpf development (n=33, 31, 40 and 35 for vehicle control, 0.5 µmol l⁻¹AM251, 5 µmol l⁻¹ AM630 and 0.5 µmol l⁻¹ AM251+5 µmol l⁻¹ AM630 treatments, respectively, from 0 to 24 hpf, and n=29, 27, 37 and 34 for vehicle control, 0.5 µmol l⁻¹ AM251, 5 µmol l⁻¹AM630 and 0.5 µmol l⁻¹ AM251+5 µmol l⁻¹ AM630 treatments, respectively, from 24 to 48 hpf). Data are presented as means±s.e.m. ^aSignificantly different from vehicle control, P<0.05; ^bsignificantly different from 0.5 µmol l⁻¹ AM251, P<0.05; ^csignificantly different from 5 µmol l⁻¹AM630, P<0.05 (one-way ANOVA followed by Tukey’s post hoc multiple comparisons test).

  
  

Effect of the endocannabinoid receptor antagonists, AM251 and AM630 on morphological development in 5 dpf zebrafish larva. (A) Incidence of pericardial edema and yolk sac edema in 5 dpf larvae when treated with AM251 and AM630 in the first and second 24 h of development. Scale bars: 0.7 mm. (B,D) Rates of pericardial and yolk sac edema and tail and body malformation in 5 dpf larvae treated with AM251 and AM630 in the first 24 h of development (n=62, 56, 51 and 58 for vehicle control, 0.5 µmol l⁻¹ AM251, 5 µmol l⁻¹ AM630 and 0.5 µmol l⁻¹ AM251+5 µmol l⁻¹AM630, respectively). (C,E) Rates of pericardial and yolk sac edema and tail and body malformation in 5 dpf larvae treated with AM251 and AM630 in the second 24 h of development (24–48 hpf) (n=74, 56, 62 and 64 for vehicle control, 0.5 µmol l⁻¹ AM251, 5 µmol l⁻¹ AM630 and 0.5 µmol l⁻¹ AM251+5 µmol l⁻¹ AM630, respectively). Data are presented as means±s.e.m. ^aSignificantly different from vehicle control, P<0.05; ^bsignificantly different from 0.5 µmol l⁻¹AM251, P<0.05; ^csignificantly different from 5 µmol l⁻¹ AM630, P<0.05 (one-way ANOVA followed by Tukey’s post hoc multiple comparisons test).

Effect of the endocannabinoid receptor antagonists AM251 and AM630 on development and inflation of 5 dpf larval zebrafish swim bladders. (A) AM251 and AM630 treatments at the early stage either from 0 to 24 hpf or 24 to 48 hpf showed fully inflated to partial or non-inflated swim bladder (SB; red arrow) development in zebrafish larvae observed at 5 dpf. Representative images of three different fish, labelled 1, 2 and 3, show fully inflated, partially inflated or non-inflated swim bladders in larvae. (B–G) Rates of swim bladder inflation at 0–24 hpf (B–D: n=73, 59, 57 and 58 for vehicle control, 0.5 µmol l⁻¹ AM251, 5 µmol l⁻¹ AM630 and 0.5 µmol l⁻¹AM251+5 µmol l⁻¹ AM630 treatments, respectively) and at 24–48 hpf (E–G: n=78, 58, 61 and 60 for vehicle control, 0.5 µmol l⁻¹ AM251, 5 µmol l⁻¹ AM630 and 0.5 µmol l⁻¹ AM251+5 µmol l⁻¹AM630 treatments, respectively). Data are presented as means±s.e.m. ^aSignificantly different from vehicle control, P<0.05; ^bsignificantly different from 0.5 µmol l⁻¹ AM251, P<0.05; ^csignificantly different from 5 µmol l⁻¹ AM630, P<0.05 (one-way ANOVA followed by Tukey’s post hoc multiple comparisons test).