Cannabinoids exacerbate alcohol-induced eye and brain malformations in zebrafish. (a,b) The incidence of small eyes (a) and midbrain/hindbrain boundary defects (b) following exposure to fish water vehicle (n = 30 for eyes and MHB), 0.5% alcohol (n = 30 for eyes and MHB), 2.0% alcohol (n = 30 for eyes and MHB), CP 55,940 1.0 mg/L (n = 30 for eyes and MHB), CP 55,940 2.5 mg/L (n = 32 for eye, 34 for MHB), CP 55,940 3.8 mg/L (n = 41 for eye, 44 for MHB), CP 55,940 5.0 mg/L (n = 40 for eye, 43 for MHB), 0.5% alcohol + CP 55,940 1.0 mg/L (n = 32 for eye, 35 for MHB), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 35 for eye, 45 for MHB), 0.5% alcohol + CP 55,940 3.8 mg/L (n = 44 for eye, 49 for MHB). MHB defects were measured following exposure from 5.25 to 24 hours post fertilization (hpf), and small eyes were measured following exposure from 5.25 to 48 hpf. **p < 0.01, ***p < 0.001, vs fish water vehicle, ^^p < 0.01, ^^^p < 0.001 vs. respective doses of CP 55,940 alone using a Chi-square or Fischer’s exact test, depending on sample size. Data are expressed as the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100), observed in a single experiment. Dotted lines reference the zero incidence of spontaneous eye and MHB defects. Dashed lines within the bars corresponding to the alcohol and CP 55,940 simultaneous treatment indicate the predicted additive effects. (c–h) Representative photographs of zebrafish eyes and midbrain/hindbrain boundaries (i–n) following vehicle (c,i), 0.5% alcohol (d,j), 2% alcohol (e,k), CP 55,940 2.5 mg/L (f,l), CP 55,940 5.0 mg/L (g and m), and 0.5% alcohol + CP 55,940 2.5 mg/L (h and n). The black line in (c–h) represents a 50 μm scale bar. The white arrows in (i,j,l) indicate the midbrain/hindbrain boundary.
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