Ethanol and cannabinoids regulate zebrafish GABAergic neuron development and behavior in a sonic hedgehog and fibroblast growth factor dependent mechanism

Background: Ethanol has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that ethanol and cannabinoids may interact to broadly disrupt neuronal function during brain development. Methods: Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk taking behavior. Results: Combined acute subthreshold ethanol and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the ethanol and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the ethanol- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by ethanol and cannabinoids. Conclusions: These studies provide evidence that forebrain GABAergic neuron development, and zebrafish risk-taking behavior, are sensitive to both ethanol and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of ethanol and cannabinoids in FASD.