Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides

Abstract

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24 h post-fertilization, zebrafish embryos were exposed for 2 h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol.

Embryonic Ethanol Exposure Affects the Early Development, Migration, and Location of Hypocretin/Orexin Neurons in Zebrafish

“…In addition, zebrafish have a comparable central nervous system (CNS) that contains neuropeptide systems (Berman et al., ; Faraco et al., ), including a well‐conserved hcrt system (Elbaz et al., ; Kaslin et al., ). Using a behavioral model developed in our laboratory, we have shown voluntary consumption of EtOH–gelatin by adult zebrafish to stimulate hcrt expression (Sterling et al., ) and central injection of hcrt/orexin‐A to increase EtOH consumption (Sterling et al., ), similar to our findings in rodents (Chang et al., , ). Although there are few reports of hcrt's reward‐related functions in zebrafish (Sterling et al., , ), there is evidence in this species that optogenetic activation (Singh et al., ) and overexpression (Prober et al., ) of hcrt neurons stimulate arousal behaviors, which are behaviors associated with and sometimes predictive of excess consumption of EtOH (Barson et al., ).…”

“…There is also clinical evidence showing even low levels of fetal alcohol exposure to increase alcohol use and produce behavioral dysfunction in the offspring (Goldschmidt et al., ; Murray et al., ), suggesting that no amount of alcohol during pregnancy is safe. This is supported by animal studies from our laboratory (Chang et al., ; Sterling et al., ) and others (Chotro et al., ) showing low‐dose ethanol (EtOH) during gestation in rats to increase intake of EtOH during adolescence. These alterations in behavior are persistent and occur in association with long‐term neuronal changes, including an increased expression of orexigenic peptides (Chang et al., , ).…”

Orexin/Hypocretin System: Role in Food and Drug Overconsumption

“…Prenatal exposure to low-to-moderate levels of alcohol leads to elevated alcohol drinking in adolescent and adult rodents and even zebrafish (Fabio, Macchione, Nizhnikov, & Pautassi, 2015; Nizhnikov, Popoola, & Cameron, 2016; Sterling, Chang, Karatayev, Chang, & Leibowitz, 2016). Notably, this exposure to alcohol also stimulates the proliferation of OX neurons, subsequently leading to their increase in gene expression and density (Chang, Karatayev, Liang, Barson, & Leibowitz, 2012; Sterling et al, 2016). This suggests that the greater number of neuropeptide neurons may contribute to elevated levels of alcohol intake.…”

Role of Lateral Hypothalamic Orexin (Hypocretin) Neurons in Alcohol Use and Abuse: Recent Advances

“…injection of OX-A stimulated novelty-induced locomotor behavior while also increasing intake of food and alcohol equally. These findings suggest alcohol-induced increases in neurogenesis and expression of OX peptides contribute to the behavioral changes induced by embryonic exposure to alcohol in zebrafish [31]. The similarity of findings in zebrafish and rodent models give validity for this model to be used as a screening tool to investigate genetic manipulations in controlling alcohol intake [30].…”

“…Intake of alcohol-gelatin increased locomotion, reduced anxiety and stimulated aggressive behavior while increasing expression of OX and GAL in hypothalamic areas [30]. Furthermore, using this model, they examined the impact of embryonic alcohol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, voluntary alcohol consumption, locomotor behaviors and effects of central OX-A injections on these alcohol-related behaviors [31]. Embryonic alcohol exposure (0.25 and 0.5 %, 2 h) dose-dependently increased hypothalamic neurogenesis of OX neurons and OX expression in the anterior hypothalamus.…”