Embryonic ethanol exposure induces ectopic Hcrt and MCH neurons outside hypothalamus in rats and zebrafish: Role in ethanol-induced behavioural disturbances.

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.

Sexually dimorphic and asymmetric effects of embryonic ethanol exposure on hypocretin/orexin neurons as related to behavioral changes in zebrafish.

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote reward-related behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH). We used here in zebrafish three-dimensional analyses of the entire population of Hcrt neurons to examine how embryonic EtOH exposure at low-moderate concentrations (0.1% or 0.5% v/v) alters these neurons in relation to behavior. We found that EtOH in the water for 2 h (22-24 h post fertilization) increases the number of Hcrt neurons on the left but not right side of the brain through a stimulation of cell proliferation, this is accompanied by a decrease in locomotor activity under novel conditions but not after habituation, and these effects are evident in both larvae and adults indicating they are long lasting. Our analyses in adults revealed sexually dimorphic effects, with females consuming more EtOH-gelatin and exhibiting more freezing behavior along with an asymmetric increase in Hcrt neurons and males exhibiting increased aggression with no change in Hcrt. These findings suggest that a long lasting, asymmetric increase in Hcrt neurons induced by EtOH results from an asymmetric increase in proliferation specific to Hcrt and contributes to behavioral changes in females.

Involvement of Cxcl12a/Cxcr4b Chemokine System in Mediating the Stimulatory Effect of Embryonic Ethanol Exposure on Neuronal Density in Zebrafish Hypothalamus.

BACKGROUND: Embryonic exposure to ethanol (EtOH) produces marked disturbances in neuronal development and alcohol-related behaviors, with low-moderate EtOH doses stimulating neurogenesis without producing apoptosis and high doses having major cytotoxic effects while causing gross morphological abnormalities. With the pro-inflammatory chemokine system, Cxcl12, and its main receptor Cxcr4, known to promote processes of neurogenesis, we examined here this neuroimmune system in the embryonic hypothalamus to test directly if it mediates the stimulatory effects low-moderate EtOH doses have on neuronal development. METHODS: We used the zebrafish (Danio rerio) model, which develops externally and allows one to investigate the developing brain in vivo with precise control of dose and timing of EtOH delivery in the absence of maternal influence. Zebrafish were exposed to low-moderate EtOH doses (0.1, 0.25, 0.5% v/v), specifically during a period of peak hypothalamic development from 22 to 24 hours postfertilization, and in some tests were pretreated from 2 to 22 hpf with the Cxcr4 receptor antagonist, AMD3100. Measurements in the hypothalamus at 26 hpf were taken of cxcl12a and cxcr4b transcription, signaling, and neuronal density using qRT-PCR, RNAscope, and live imaging of transgenic zebrafish. RESULTS: Embryonic EtOH exposure, particularly at the 0.5% dose, significantly increased levels of cxcl12a and cxcr4b mRNA in whole embryos, number of cxcl12a and cxcr4b transcripts in developing hypothalamus, and internalization of Cxcr4b receptors in hypothalamic cells. Embryonic EtOH also caused an increase in the number of hypothalamic neurons and coexpression of cxcl12a and cxcr4b transcripts within these neurons. Each of these stimulatory effects of EtOH in the embryo was blocked by pretreatment with the Cxcr4 antagonist AMD3100. CONCLUSIONS: These results provide clear evidence that EtOH's stimulatory effects at low-moderate doses on the number of hypothalamic neurons early in development are mediated, in part, by increased transcription and intracellular activation of this chemokine system, likely due to autocrine signaling of Cxcl12a at its Cxcr4b receptor within the neurons.

Moderate Prenatal Ethanol Exposure Stimulates CXCL12/CXCR4 Chemokine System in Radial Glia Progenitor Cells in Hypothalamic Neuroepithelium and Peptide Neurons in Lateral Hypothalamus of the Embryo and Postnatal Offspring.

BACKGROUND: Prenatal exposure to ethanol (EtOH) has lasting effects on neuropeptide and neuroimmune systems in the brain alongside detrimental alcohol-related behaviors. At low-to-moderate doses, prenatal EtOH stimulates neurogenesis in lateral hypothalamus (LH) and increases neurons that express the orexigenic peptides hypocretin/orexin (Hcrt/OX) and melanin-concentrating hormone (MCH), and the proinflammatory chemokine CCL2, which through its receptor CCR2 stimulates cell differentiation and movement. Our recent studies demonstrated that CCL2 and CCR2 colocalize with MCH neurons and are involved in EtOH's stimulatory effect on their development but show no relation to Hcrt/OX. Here, we investigated another chemokine, CXCL12, and its receptor, CXCR4, which promote neurogenesis and neuroprogenitor cell proliferation, to determine if they also exhibit peptide specificity in their response to EtOH exposure. METHODS: Pregnant rats were intraorally administered a moderate dose of EtOH (2 g/kg/d) from embryonic day 10 (E10) to E15. Their embryos and postnatal offspring were examined using real-time quantitative PCR and immunofluorescence histochemistry, to determine if EtOH affects CXCL12 and CXCR4 and the colocalization of CXCR4 with Hcrt/OX and MCH neurons in the LH and with radial glia neuroprogenitor cells in the hypothalamic neuroepithelium (NEP). RESULTS: Prenatal EtOH strongly stimulated CXCL12 and CXCR4 in LH neurons of embryos and postnatal offspring. This stimulation was significantly stronger in Hcrt/OX than MCH neurons in LH and also occurred in radial glia neuroprogenitor cells dense in the NEP. These effects were sexually dimorphic, consistently stronger in females than males. CONCLUSIONS: While showing prenatal EtOH exposure to have a sexually dimorphic, stimulatory effect on CXCL12 and CXCR4 in LH similar to CCL2 and its receptor, these results reveal their distinct relationship to the peptide neurons, with the former closely related to Hcrt/OX and the latter to MCH, and they link EtOH's actions in LH to a stimulatory effect on neuroprogenitor cells in the NEP.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets.