Developmental ethanol exposure is able to induce Fetal Alcohol Spectrum Disorder (FASD) phenotypes in Japanese rice fish (Oryzias latipes) embryogenesis although the mechanisms yet unknown. The present study was designed to investigate the probable roles played by cannabinoid (CB) receptors in FASD induction. Searching of public databases (GenBank, Ensembl) indicated that Japanese rice fish genome includes three human ortholog CB receptor genes (cnr1a, cnr1b and cnr2) with substantial amino acid identities. A quantitative real-time PCR (qPCR) and whole mount in situ hybridization (WMISH) techniques were used to analyze the expression of these cnr genes during Japanese rice fish embryogenesis and also in response to developmental ethanol exposure. qPCR analyses showed that the expression of all three CB receptor genes were developmentally regulated and only cnr2 mRNA showed maternal expression. The mRNA concentrations of these cnr genes were found to be enhanced after 3 dpf and attained maximal levels either prior to or after hatching. Analysis by WMISH technique indicated that all three cnr genes were expressed in head region of hatchlings. During development, ethanol selectively attenuated the expression of cnr1a mRNA only; other two (cnr1b and cnr2) remained unaffected. Blocking of cnr1a by CB1 receptor antagonists rimonabant (10-20μM) or AM251 (0.2-1μM) 0-2 dpf were unable to induce any FASD-related phenotypic features in embryos or in hatchlings. However, continuous exposure of the embryos (0-6 dpf) to AM251 (1μM) was able to reduce the hatching efficiency of the embryos. Our data indicated that in Japanese rice fish, among these three cnrs, ethanol disrupted the expression of only cnr1a in a concentration-dependent manner that induced delay in hatching and might be responsible for the development of neurological disorders as observed in FASD phenotypes.
Copyright © 2014. Published by Elsevier Inc.