Fig. 7

In vivo and in vitro neuro-inhibitory effects of NA/EE-mixture exposure NA/EE-mixture effects on zebrafish nerve outgrowth and patterning. Embryos were treated at 24 hpf with either DMSO (A,C and E) or NA/EE-mixture (B,D and F) and fixed at 6 hours and 24 hours. Embryos incubated with DMSO present normal nerve outgrowth and patterning. Compared to DMSO controls, embryos treated with NA/EE-mixture presented fasciculation defects. White asterisk denotes end of yolk sac. White arrowheads denote position of major nerve tract in spinal cord; at 24 hr nerve tract is stunted and defasciculated in treated embryos, compare arrowheads in C and D. Nerves are missing in the head of treated embryos (F) compared with control embryos, grey arrowhead denotes axon projections (E). Direct effect of NA/EE-mixture on nerves was assessed through retinal explants culture exposed to DMSO and NA/EE-mixture over different concentrations; 0.05% DMSO (G), NA 3.125 μg/mL + EE 6.25 ng/mL (H), NA 6.25 μg/mL + EE 12.5 ng/mL (I) and NA 12.5 μg/mL + EE 25 ng/mL (J) treatment. (K) Ratio of nerve length to body length is reduced in treated embryos at 6 hpf and 24 hpf as is the ratio of nerve outgrowth (L) in the head following treatment at 24 hpf. Statistical significance was analysed through Mann-Whitney test. (M) Neurite outgrowth in retinal explants was decreased significantly following NA/EE-mixture exposure at NA 6.25 μg/mL + EE 12.5 ng/mL (H and M) and NA 12.5 μg/mL + EE 25 ng/mL (J and M). White arrow denotes an example of an axon projection. Statistical significance was analysed using Kruskal-Wallis test with Dunn’s post-hoc test. Graphs represent mean ± S.E.M. ns, p > 0.05; *p < 0.05; ***p < 0.001. Scale bars: 100 µm.