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.

CB1 receptors mediate CB teratogenesis. (a) Effects of pretreatment with a CB1 receptor antagonist, SR 141716A, on CP 55,940-induced eye defects in mice. Mice received CB vehicle (n = 43/6 litters) SR 141716A 5.0 mg/kg + the CB vehicle (n = 62/8 litters), the CB vehicle + CP 55,940 0.5 mg/kg (n = 51/8 litters), SR 141716A 2.0 mg/kg + CP 55,940 0.5 mg/kg (n = 45/6 litters), or SR 141716A 5.0 mg/kg + CP 55,940 0.5 mg/kg (n = 88/10 litters). SR 141716A or CB vehicle was given 10 min before the second injection. **p < 0.01, ***p < 0.001 vs. CB vehicle, ^^p < 0.01 vs CP 0.5, using a Chi-square test. Data are the percent of mice with defects (number of affected mice/total number of mice *100). See Supplementary Table 4 for eye defect severity, body weight and length, and the number of live offspring/litter. See Supplementary Fig. 2a–f. for separate analysis of left and right eye defects. (b) Small eyes in zebrafish embryos receiving exposure to fish water vehicle (n = 39), SR 141716A 3.0 mg/L (n = 40), CP 55,940 5.0 mg/L alone (n = 54), SR 141716A 3.0 mg/L + CP 55,940 5.0 mg/L alone (n = 49), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 42), SR 141716A 3.0 mg/L + 0.5% alcohol + CP 55,940 2.5 mg/L (n = 49). **p < 0.01, ***p < 0.001 vs. vehicle. ^^p < 0.01, ^^^p < 0.001 vs. corresponding treatment without SR 141716A using a Chi-square test. Data are the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100). (c,d) CB1-Smo co-localization in the mouse neural tube (GD 9.5) using a proximity ligation assay (Duolink) to visualize sites (fluorescent red) where anti-CB1 and anti-Smo antibodies bind within 40 nm of one another. (e,f). CB1-GPR 161 co-localization in the mouse neural tube (GD 9.5). Nuclei of neural tube cells are labeled with DAPI in blue. Sections (7 µm) were imaged on a confocal microscope using a 40× (c,e) and 63× (d,f) oil lens.