Embryonic exposure to ethanol increases the risk for alcohol use disorder in humans and stimulates alcohol‐related behaviours in different animal models. Evidence in rats and zebrafish suggests that this phenomenon induced by ethanol at low‐moderate concentrations involves a stimulatory effect on neurogenesis and density of hypothalamic neurons expressing the peptides, hypocretin/orexin (Hcrt) and melanin‐concentrating hormone (MCH), known to promote alcohol consumption. Building on our report in zebrafish showing that ethanol induces ectopic expression of Hcrt neurons outside the hypothalamus, we investigated here whether embryonic ethanol exposure also induces ectopic peptide neurons in rats similar to zebrafish and affects their morphological characteristics and if these ectopic neurons are functional and have a role in the ethanol‐induced disturbances in behaviour. We demonstrate in rats that ethanol at a low‐moderate dose, in addition to increasing Hcrt and MCH neurons in the lateral hypothalamus where they are normally concentrated, induces ectopic expression of these peptide neurons further anterior in the nucleus accumbens core and ventromedial caudate putamen where they have not been previously observed and causes morphological changes relative to normally located hypothalamic neurons. Similar to rats, embryonic ethanol exposure at a low‐moderate dose in zebrafish induces ectopic Hcrt neurons anterior to the hypothalamus and alters their morphology. Notably, laser ablation of these ectopic Hcrt neurons blocks the behavioural effects induced by ethanol exposure, including increased anxiety and locomotor activity. These findings suggest that the ectopic peptide neurons are functional and contribute to the ethanol‐induced behavioural disturbances related to the overconsumption of alcohol.

Embryonic ethanol exposure in zebrafish and rats, while stimulating hypothalamic hypocretin/orexin (Hcrt) neurons along with alcohol consumption and related behaviors, increases the chemokine receptor Cxcr4 that promotes neuronal migration and may mediate ethanol’s effects on neuronal development. Here we performed a more detailed anatomical analysis in zebrafish of ethanol’s effects on the Cxcl12a/Cxcr4b system throughout the entire brain as it relates to Hcrt neurons developing within the anterior hypothalamus (AH) where they are normally located. We found that ethanol increased these Hcrt neurons only in the anterior part of the AH and induced ectopic Hcrt neurons further anterior in the preoptic area, and these effects along with ethanol-induced behaviors were completely blocked by a Cxcr4 antagonist. Analysis of cxcl12a transcripts and internalized Cxcr4b receptors throughout the brain showed they both exhibited natural posterior-to-anterior concentration gradients, with levels lowest in the posterior AH and highest in the anterior telencephalon. While stimulating their density in all areas and maintaining these gradients, ethanol increased chemokine expression only in the more anterior and ectopic Hcrt neurons, effects blocked by the Cxcr4 antagonist. These findings demonstrate how increased chemokine expression acting along natural gradients mediates ethanol-induced anterior migration of ectopic Hcrt neurons and behavioral disturbances.

Numerous studies in animals demonstrate that embryonic exposure to ethanol (EtOH) at low-moderate doses stimulates neurogenesis and increases the number of hypothalamic neurons expressing the peptide, hypocretin/orexin (Hcrt). A recent study in zebrafish showed that this effect on the Hcrt neurons in the anterior hypothalamus (AH) is area specific, evident in the anterior (aAH) but not posterior (pAH) part of this region. To understand specific factors that may determine the differential sensitivity to EtOH of these Hcrt subpopulations, we performed additional measures in zebrafish of their cell proliferation, co-expression of the opioid dynorphin (Dyn), and neuronal projections. In association with the increase in Hcrt neurons in the aAH but not pAH, EtOH significantly increased only in the aAH the proliferation of Hcrt neurons and their number lacking Dyn co-expression. The projections of these subpopulations differed markedly in their directionality, with those from the pAH primarily descending to the locus coeruleus and those from the aAH ascending to the subpallium, and they were both stimulated by EtOH, which induced specifically the most anterior subpallium-projecting Hcrt neurons to become ectopically expressed beyond the aAH. These differences between the Hcrt subpopulations suggest they are functionally distinct in their regulation of behavior.

Studies in zebrafish and rats show that embryonic ethanol exposure at low-moderate concentrations stimulates hypothalamic neurons expressing hypocretin/orexin (Hcrt) that promote alcohol consumption, effects possibly involving the chemokine Cxcl12 and its receptor Cxcr4. Our recent studies in zebrafish of Hcrt neurons in the anterior hypothalamus (AH) demonstrate that ethanol exposure has anatomically specific effects on Hcrt subpopulations, increasing their number in the anterior AH (aAH) but not posterior AH (pAH), and causes the most anterior aAH neurons to become ectopically expressed further anterior in the preoptic area (POA). Using tools of genetic overexpression and knockdown, our goal here was to determine whether Cxcl12a has an important function in mediating the specific effects of ethanol on these Hcrt subpopulations and their projections. The results demonstrate that the overexpression of Cxcl12a has stimulatory effects similar to ethanol on the number of aAH and ectopic POA Hcrt neurons and the long anterior projections from ectopic POA neurons and posterior projections from pAH neurons. They also demonstrate that knockdown of Cxcl12a blocks these effects of ethanol on the Hcrt subpopulations and projections, providing evidence supporting a direct role of this specific chemokine in mediating ethanol’s stimulatory effects on embryonic development of the Hcrt system.