ACR reduces presynaptic nerve terminals in zebrafish larvae. At the neuromuscular junctions (NMJ) of the trunk, ACR-exposed larvae exhibit an strong reduction in the labelling of synaptic vesicle glycoprotein 2a (marker of synaptic terminals of the spinal motor neurons), whereas the α-bungarotoxin labelling (post-synaptic marker at the NMJ) labelling remains unaltered. Detail of the trunk, in lateral view, of control (a–c) and ACR-treated (d–f) larvae after co-labelling with α -bungarotoxin Alexa Fluor 488 conjugate (a,d) and SV2 antibody. The co-localization of the pre-synaptic and post-synaptic markers of NMJ is also showed (c,f). Scale bar: 100 μm.

Determination of MTC for ACR systemic toxicity.

(a) Gross morphology assessment shows that whereas 1.25 mM ACR is the LOEC, 1 mM ACR is the NOEC for this endpoint. (b) Morphometric analysis shows that 0.5-1.0 mM ACR has no effect on total length of the larvae. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test. Results represent mean ± sem; NS: not significant (c) Exposure to 1 mM ACR has no effect on liver morphology. Scale bar: (a) 500 m; (c) 50 m.

Histopathological analysis of control and 1 mM ACR exposed larvae. Representative semithin sections of the brain (a,b), spinal cord (c,d) and the retina (e,f) of control (a,c,e) and ACR-treated (b,d,f) zebrafish larvae. No histopathological changes were identified using this methodology. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL; outer plexiform layer; RPE, retinal pigment epithelium; sc, spinal cord. Scale bar: (a,b,e,f) 100 μm, (c,d) 50 μm.

Axonal tracts and myelin sheets are not primary targets for acrylamide neurotoxicity in zebrafish larvae. Detail of the trunk, in lateral view, of control (a,c) and ACR-treated larvae (b,d) after double whole-mount immunofluorescence with 3A10 antibody labelling axonal tracts and MBP antibody labelling the myelin sheets. No clear differences in both structures were evident between the two groups. Scale bar: 100 m.