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.

CCL2/CCR2 system in neuroepithelial radial glia progenitor cells: involvement in stimulatory, sexually dimorphic effects of maternal ethanol on embryonic development of hypothalamic peptide neurons.

BACKGROUND: Clinical and animal studies show that alcohol consumption during pregnancy produces lasting behavioral disturbances in offspring, including increased alcohol drinking, which are linked to inflammation in the brain and disturbances in neurochemical systems that promote these behaviors. These include the neuropeptide, melanin-concentrating hormone (MCH), which is mostly expressed in the lateral hypothalamus (LH). Maternal ethanol administration at low-to-moderate doses, while stimulating MCH neurons without affecting apoptosis or gliogenesis, increases in LH the density of neurons expressing the inflammatory chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 and their colocalization with MCH. These neural effects associated with behavioral changes are reproduced by maternal CCL2 administration, reversed by a CCR2 antagonist, and consistently stronger in females than males. The present study investigates in the embryo the developmental origins of this CCL2/CCR2-mediated stimulatory effect of maternal ethanol exposure on MCH neurons. METHODS: Pregnant rats from embryonic day 10 (E10) to E15 during peak neurogenesis were orally administered ethanol at a moderate dose (2 g/kg/day) or peripherally injected with CCL2 or CCR2 antagonist to test this neuroimmune system's role in ethanol's actions. Using real-time quantitative PCR, immunofluorescence histochemistry, in situ hybridization, and confocal microscopy, we examined in embryos at E19 the CCL2/CCR2 system and MCH neurons in relation to radial glia progenitor cells in the hypothalamic neuroepithelium where neurons are born and radial glia processes projecting laterally through the medial hypothalamus that provide scaffolds for neuronal migration into LH. RESULTS: We demonstrate that maternal ethanol increases radial glia cell density and their processes while stimulating the CCL2/CCR2 system and these effects are mimicked by maternal administration of CCL2 and blocked by a CCR2 antagonist. While stimulating CCL2 colocalization with radial glia and neurons but not microglia, ethanol increases MCH neuronal number near radial glia cells and making contact along their processes projecting into LH. Further tests identify the CCL2/CCR2 system in NEP as a primary source of ethanol's sexually dimorphic actions. CONCLUSIONS: These findings provide new evidence for how an inflammatory chemokine pathway functions within neuroprogenitor cells to mediate ethanol's long-lasting, stimulatory effects on peptide neurons linked to adolescent drinking behavior.

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.

Hypothalamic CCL2/CCR2 Chemokine System: Role in Sexually Dimorphic Effects of Maternal Ethanol Exposure on Melanin-Concentrating Hormone and Behavior in Adolescent Offspring.

Clinical and animal studies show that ethanol exposure and inflammation during pregnancy cause similar behavioral disturbances in the offspring. While ethanol is shown to stimulate both neuroimmune and neurochemical systems in adults, little is known about their anatomical relationship in response to ethanol in utero and whether neuroimmune factors mediate ethanol's effects on neuronal development and behavior in offspring. Here we examined in female and male adolescent rats a specific population of neurons concentrated in lateral hypothalamus, which coexpress the inflammatory chemokine C-C motif ligand 2 (CCL2) or its receptor CCR2 with the orexigenic neuropeptide, melanin-concentrating hormone (MCH), that promotes ethanol drinking behavior. We demonstrate that maternal administration of ethanol (2 g/kg/d) from embryonic day 10 (E10) to E15, while having little impact on glia, stimulates expression of neuronal CCL2 and CCR2, increases density of both large CCL2 neurons colocalizing MCH and small CCL2 neurons surrounding MCH neurons, and stimulates ethanol drinking and anxiety in adolescent offspring. We show that these neuronal and behavioral changes are similarly produced by maternal administration of CCL2 (4 or 8 µg/kg/d, E10-E15) and blocked by maternal administration of a CCR2 antagonist INCB3344 (1 mg/kg/d, E10-E15), and these effects of ethanol and CCL2 are sexually dimorphic, consistently stronger in females. These results suggest that this neuronal CCL2/CCR2 system closely linked to MCH neurons has a role in mediating the effects of maternal ethanol exposure on adolescent offspring and contributes to the higher levels of adolescent risk factors for alcohol use disorders described in women.SIGNIFICANCE STATEMENT Ethanol consumption and inflammatory agents during pregnancy similarly increase alcohol intake and anxiety in adolescent offspring. To investigate how neurochemical and neuroimmune systems interact to mediate these disturbances, we examined a specific population of hypothalamic neurons coexpressing the inflammatory chemokine CCL2 and its receptor CCR2 with the neuropeptide, melanin-concentrating hormone. We demonstrate in adolescent offspring that maternal administration of CCL2, like ethanol, stimulates these neurons and increases ethanol drinking and anxiety, and these effects of ethanol are blocked by maternal CCR2 antagonist and consistently stronger in females. This suggests that neuronal chemokine signaling linked to neuropeptides mediates effects of maternal ethanol exposure on adolescent offspring and contributes to higher levels of adolescent risk factors for alcohol use disorders in women.

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

BACKGROUND: Embryonic ethanol (EtOH) exposure is known to increase alcohol drinking later in life and have long-term effects on neurochemical systems in the brain. With zebrafish having marked advantages for elucidating neural mechanisms underlying brain disorders, we recently tested and showed in these fish, similar to rodents, that low-dose embryonic EtOH stimulates voluntary consumption of EtOH while increasing expression of hypocretin/orexin (hcrt) neurons, a neuropeptide that promotes consummatory and reward-related behaviors. The goal of the present study was to characterize how embryonic EtOH affects early development of the hcrt system and produces persistent changes at older ages that may contribute to this increase in EtOH consumption. METHODS: We utilized live imaging and Imaris software to investigate how low-dose embryonic EtOH (0.5%), administered from 22 to 24 hours postfertilization, affects specific properties of hcrt neurons in hcrt:EGFP transgenic zebrafish at different ages. RESULTS: Time-lapse imaging from 24 to 28 hpf showed that embryonic EtOH increased the number of hcrt neurons, reduced the speed, straightness, and displacement of their migratory paths, and altered their direction early in development. At older ages up to 6 dpf, the embryonic EtOH-induced increase in hcrt neurons was persistent, and the neurons became more widely dispersed. These effects of embryonic EtOH were found to be asymmetric, occurring predominantly on the left side of the brain, and at 6 dpf, they resulted in marked changes in the anatomical location of the hcrt neurons, with some detected outside their normal position in the anterior hypothalamus again primarily on the left side. CONCLUSIONS: Our findings demonstrate that low-dose embryonic EtOH has diverse, persistent, and asymmetric effects on the early development of hypothalamic hcrt neurons, which lead to abnormalities in their ultimate location that may contribute to behavioral disturbances, including an increase in EtOH consumption.

Neurotensin in the posterior thalamic paraventricular nucleus: inhibitor of pharmacologically relevant ethanol drinking.

Individuals prone to ethanol overconsumption may have preexisting neurochemical disturbances that contribute to their vulnerability. This study examined the paraventricular nucleus of the thalamus (PVT), a limbic structure recently shown to participate in ethanol intake. To identify individuals prone to ethanol overconsumption, we tested Long-Evans rats in behavioral paradigms and found high levels of vertical time (rearing behavior) in a novel activity chamber to be a consistent predictor of subsequent excessive 20 percent ethanol drinking under the intermittent access model. Examining neurochemicals in the PVT, we found before ethanol exposure that prone rats with high rearing, compared with non-prone rats, had significantly lower levels of neurotensin (NTS) mRNA and peptide in the posterior (pPVT) but not anterior (aPVT) subregion of the PVT. Our additional finding that ethanol intake has no significant impact on either rearing or NTS levels indicates that these measures, which are different in prone rats before ethanol consumption, remain stable after ethanol consumption. The possibility that NTS directly controls ethanol drinking is supported by our finding that NTS administration specifically suppresses ethanol drinking when injected into the pPVT but not aPVT, with this effect occurring exclusively in higher drinkers that presumably have lower endogenous levels of NTS. Further, an NTS antagonist in the pPVT augments intake in lower drinkers with presumably more endogenous NTS, while NTS in the pPVT inhibits novelty-induced rearing that predicts excessive drinking. Together, these results provide strong evidence that low endogenous levels of NTS in the pPVT contribute to an increased propensity toward excessive ethanol drinking.

Substance P in the anterior thalamic paraventricular nucleus: promotion of ethanol drinking in response to orexin from the hypothalamus.

The paraventricular nucleus of the thalamus (PVT) appears to participate in drug addiction. Recent evidence in rats shows that ethanol drinking is increased by orexin/hypocretin (OX) afferents from the hypothalamus, acting specifically in the anterior (aPVT) rather than posterior (pPVT) PVT subregion. The present study sought to identify neuropeptides transcribed within the PVT, which themselves might contribute to ethanol drinking and possibly mediate the actions of OX. We discovered that substance P (SP) in the aPVT can stimulate intermittent-access ethanol drinking, similar to OX, and that SP receptor [neurokinin 1 receptor/tachykinin receptor 1 (NK1R)] antagonists in this subregion reduce ethanol drinking. As with OX, this effect is site specific, with SP in the pPVT or dorsal third ventricle having no effect on ethanol drinking, and it is behaviorally specific, with SP in the aPVT reducing the drinking of sucrose and stimulating it in the pPVT. A close relationship between SP and OX was demonstrated by a stimulatory effect of local OX injection on SP mRNA and peptide levels, specifically in the aPVT but not pPVT, and a stimulatory effect of OX on SP expression in isolated thalamic neurons, reflecting postsynaptic actions. A functional relationship between OX and SP in the aPVT is suggested by our additional finding that ethanol drinking induced by OX is blocked by a local NK1R antagonist administered at a sub-threshold dose. These results, suggesting that SP in the aPVT mediates the stimulatory effect of OX on ethanol drinking, identify a new role for SP in the control of this behavior.

Regulation of the orexigenic neuropeptide, enkephalin, by PPARδ and fatty acids in neurons of the hypothalamus and forebrain.

Ingestion of a high-fat diet composed mainly of the saturated fatty acid, palmitic (PA), and the unsaturated fatty acid, oleic (OA), stimulates transcription in the brain of the opioid neuropeptide, enkephalin (ENK), which promotes intake of substances of abuse. To understand possible underlying mechanisms, this study examined the nuclear receptors, peroxisome proliferator-activated receptors (PPARs), and tested in hypothalamic and forebrain neurons from rat embryos whether PPARs regulate endogenous ENK and the fatty acids themselves affect these PPARs and ENK. The first set of experiments demonstrated that knocking down PPARδ, but not PPARα or PPARγ, increased ENK transcription, activation of PPARδ by an agonist decreased ENK levels, and PPARδ neurons coexpressed ENK, suggesting that PPARδ negatively regulates ENK. In the second set of experiments, PA treatment of hypothalamic and forebrain neurons had no effect on PPARδ protein while stimulating ENK mRNA and protein, whereas OA increased both mRNA and protein levels of PPARδ in forebrain neurons while having no effect on ENK mRNA and increasing ENK levels. These findings show that PA has a strong, stimulatory effect on ENK and weak effect on PPARδ protein, whereas OA has a strong stimulatory effect on PPARδ and weak effect on ENK, consistent with the inhibitory effect of PPARδ on ENK. They suggest a function for PPARδ, perhaps protective in nature, in embryonic neurons exposed to fatty acids from a fat-rich diet and provide evidence for a mechanism contributing to differential effects of saturated and monounsaturated fatty acids on neurochemical systems involved in consummatory behavior. Our findings show that PPARδ in forebrain and hypothalamic neurons negatively regulates enkephalin (ENK), a peptide known to promote ingestive behavior. This inverse relationship is consistent with our additional findings, that a saturated (palmitic; PA) compared to a monounsaturated fatty acid (oleic; OA) has a strong stimulatory effect on ENK and weak effect on PPARδ. These results suggest that PPARδ protects against the neuronal effects of fatty acids, which differentially affect neurochemical systems involved in ingestive behavior.

Anterior thalamic paraventricular nucleus is involved in intermittent access ethanol drinking: role of orexin receptor 2.

The paraventricular nucleus of the thalamus (PVT) has been shown to participate in hedonic feeding and is thought to influence drug seeking. This understudied nucleus contains anterior (aPVT) and posterior (pPVT) subregions, which receive dense projections from hypothalamic orexin/hypocretin (OX) but exhibit anatomical and functional differences. This study sought to characterize in Long-Evans rats the involvement of these PVT subregions and their OX receptor activity in consumption of the drug, ethanol. Compared with those maintained on water and chow only (water group), rats trained to drink pharmacologically relevant levels of ethanol (ethanol group) showed increased neuronal activation in the PVT, specifically the aPVT but not pPVT, as indicated by c-Fos immunoreactivity. Similar results were obtained in rats administered ethanol via oral gavage, indicating that this site-specific effect was due to ethanol exposure. In support of the involvement of OX, the ethanol group also showed increased mRNA levels of this neuropeptide in the hypothalamus and of OX 2 receptor (OX2R) but not OX 1 receptor (OX1R), again in the aPVT but not pPVT. Similarly, ethanol gavage increased double labeling of c-Fos with OX2R but not OX1R, specifically in the aPVT. Evidence directly supporting a role for aPVT OX2R in ethanol consumption was provided by results with local injections, showing ethanol intake to be enhanced by OX-A or OX-B in the aPVT but not pPVT and reduced by a local antagonist of OX2R but not OX1R. These results focus attention on the aPVT and specifically its OX2R in mediating a positive feedback relationship with ethanol intake.

Prenatal exposure to nicotine stimulates neurogenesis of orexigenic peptide-expressing neurons in hypothalamus and amygdala.

Animal and clinical studies show that gestational exposure to nicotine increases the propensity of offspring to consume nicotine, but the precise mechanism mediating this behavioral phenomenon is unclear. The present study in Sprague Dawley rats examined the possibility that the orexigenic peptide systems, enkephalin (ENK) and orexin (OX), which are stimulated by nicotine in adult animals and promote consummatory behavior, may be similarly responsive to nicotine's stimulatory effect in utero while having long-term behavioral consequences. The results demonstrated that nicotine exposure during gestation at low doses (0.75 or 1.5 mg/kg/d) significantly increased mRNA levels and density of neurons that express ENK in the hypothalamic paraventricular nucleus and central nucleus of the amygdala, OX, and another orexigenic peptide, melanin-concentrating hormone, in the perifornical lateral hypothalamus in preweanling offspring. These effects persisted in the absence of nicotine, at least until puberty. Colabeling of the cell proliferation marker BrdU with the neuronal marker NeuN and peptides revealed a marked stimulatory effect of prenatal nicotine on neurogenesis, but not gliogenesis, and also on the number of newly generated neurons expressing ENK, OX, or melanin-concentrating hormone. During adolescence, offspring also exhibited significant behavioral changes, increased consumption of nicotine and other substances of abuse, ethanol and a fat-rich diet, with no changes in chow and water intake or body weight. These findings reveal a marked sensitivity during gestation of the orexigenic peptide neurons to low nicotine doses that may increase the offspring's propensity to overconsume substances of abuse during adolescence.

Stimulatory role of the chemokine CCL2 in the migration and peptide expression of embryonic hypothalamic neurons.

Neuroinflammation is a feedback mechanism against infection, with recent studies suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2), and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and migration in response to damage. Although CCL2 co-localizes with neuropeptides in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2 is unknown. This led us to consider the possibility that CCL2 acting through CCR2, under natural conditions, may affect the migration and peptide levels of hypothalamic neurons that control voluntary behavior. This study used primary embryonic hypothalamic neurons to examine the effect of CCL2 on migratory behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment with CCL2 led to a significant, dose-dependent increase in the number of migrated neurons and an increase in the velocity and distance traveled. CCL2 also significantly increased the number of ENK-expressing and CCR2/ENK co-expressing neurons and the percentage of neurons that contain higher levels of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and galanin. These results provide evidence for a stimulatory effect of CCL2 on embryonic hypothalamic neurons involving changes in migratory behavior, expression, and synthesis of neuropeptides that function in controlling behavior. Our results demonstrate that the chemokine, CCL2, functions through its receptor, CCR2, to stimulate the migration and expression of the orexigenic peptides, enkephalin (ENK) and galanin (GAL), in developing embryonic hypothalamic neurons that are important for controlling ingestive behavior. This evidence reveals broad effects of CCL2 in the developing hypothalamus, showing this chemokine system to be tightly linked to the hypothalamic peptide neurons.

Differential role of D1 and D2 receptors in the perifornical lateral hypothalamus in controlling ethanol drinking and food intake: possible interaction with local orexin neurons.

BACKGROUND: The neurotransmitter dopamine (DA), acting in various mesolimbic brain regions, is well known for its role in promoting motivated behaviors, including ethanol (EtOH) drinking. Indirect evidence, however, suggests that DA in the perifornical lateral hypothalamus (PF/LH) has differential effects on EtOH consumption, depending on whether it acts on the DA 1 (D1) or DA 2 (D2) receptor subtype, and that these effects are mediated in part by local peptide systems, such as orexin/hypocretin (OX) and melanin-concentrating hormone (MCH), known to stimulate the consumption of EtOH. METHODS: The present study in brain-cannulated Sprague-Dawley rats measured the effects of dopaminergic compounds in the PF/LH on drinking behavior in animals trained to consume 7% EtOH and also on local peptide mRNA expression using digoxigenin-labeled in situ hybridization in EtOH-naïve animals. RESULTS: Experiments 1 and 2 showed that the D1 agonist SKF81297 (10.8 nmol/side) in the PF/LH significantly increased food intake, while tending to increase EtOH intake, and the D1 antagonist SCH23390 significantly decreased EtOH intake without affecting food intake. In contrast, the D2 agonist quinelorane (6.2 nmol/side) in the PF/LH significantly reduced EtOH consumption, while the D2 antagonist sulpiride increased it. Experiments 3 and 4 revealed differential effects of PF/LH injection of the DA agonists on local OX mRNA, which was increased by the D1 agonist and decreased by the D2 agonist. These DA agonists had no impact on MCH expression. CONCLUSIONS: These results support a stimulatory role of the PF/LH D1 receptor in promoting the consumption of both EtOH and food, in contrast to a suppressive effect of the D2 receptor on EtOH drinking. They further suggest that these receptors affect consumption, in part, through local OX-expressing neurons. These findings provide new evidence for the function of PF/LH DA receptor subtypes in controlling EtOH and food intake.

Embryonic ethanol exposure and optogenetic activation of hypocretin neurons stimulate similar behaviors early in life associated with later alcohol consumption.

The initiation of alcohol use early in life is one of the strongest predictors of developing a future alcohol use disorder. Clinical studies have identified specific behaviors during early childhood that predict an increased risk for excess alcohol consumption later in life. These behaviors, including increased hyperactivity, anxiety, novelty-seeking, exploratory behavior, impulsivity, and alcohol-seeking, are similarly stimulated in children and adolescent offspring of mothers who drink alcohol during pregnancy. Here we tested larval zebrafish in addition to young pre-weanling rats and found this repertoire of early behaviors along with the overconsumption of alcohol during adolescence to be increased by embryonic ethanol exposure. With hypocretin/orexin (Hcrt) neurons known to be stimulated by ethanol and involved in mediating these alcohol-related behaviors, we tested their function in larval zebrafish and found optogenetic activation of Hcrt neurons to stimulate these same early alcohol-related behaviors and later alcohol intake, suggesting that these neurons have an important role in producing these behaviors. Together, these results show zebrafish to be an especially useful animal model for investigating the diverse neuronal systems mediating behavioral changes at young ages that are produced by embryonic ethanol exposure and predict an increased risk for developing alcohol use disorder.

Neurochemical heterogeneity of rats predicted by different measures to be high ethanol consumers.

BACKGROUND: Alcoholism is a heterogeneous disease, with subjects possibly differing both in the best measure that predicts their excess consumption and in their most effective pharmacotherapy. Two different measures, high novelty-induced activity and high-fat-induced triglycerides (TGs), are known to identify subgroups of animals prone to consuming higher amounts of ethanol (EtOH). The question investigated here is whether these subgroups are, in fact, similar in their neurochemical phenotype that may contribute to their overconsumption. METHODS: EtOH-naïve, Sprague-Dawley rats were subgrouped based on the 2 predictor measures of activity or TG levels, and then quantitative real-time polymerase chain reaction and digoxigenin-labeled in situ hybridization were used to measure their expression of hypothalamic peptides that affect EtOH intake. In additional subgroups subsequently trained to drink 9% EtOH, the opioid antagonist and alcoholism medication, naltrexone, was tested at a low dose (0.02 mg/kg, s.c.) to determine the rats' sensitivity to its effects. RESULTS: The 2 measures, while both effective in predicting amount of EtOH intake, were found to identify distinctive subgroups. Rats with high compared to low activity exhibited significantly greater expression of galanin and enkephalin in the paraventricular nucleus (PVN) and of orexin in the perifornical lateral hypothalamus (PFLH), but no difference in melanin-concentrating hormone in PFLH or neuropeptide Y in arcuate nucleus. This contrasts with rats having high TG, which exhibited greater expression only of PVN galanin, along with reduced PFLH orexin. The high-activity rats with elevated enkephalin, but not high-TG rats, were also unusually sensitive to naltrexone, which significantly reduced their alcohol intake. CONCLUSIONS: In addition to revealing differences in endogenous peptides and drug responsiveness in predicted high EtOH drinkers, this study demonstrates that these disturbances differ markedly between the 2 at-risk subgroups. This indicates that simple tests may be effective in identifying subjects most responsive to a specific pharmacotherapy.

Glutamatergic input to the lateral hypothalamus stimulates ethanol intake: role of orexin and melanin-concentrating hormone.

BACKGROUND: Glutamate (GLUT) in the lateral hypothalamus (LH) has been suggested to mediate reward behaviors and may promote the ingestion of drugs of abuse. This study tested the hypothesis that GLUT in the LH stimulates consumption of ethanol ( EtOH ) and that this effect occurs, in part, via its interaction with local peptides, hypocretin/orexin (OX), and melanin-concentrating hormone (MCH). METHODS: In Experiments 1 and 2, male Sprague-Dawley rats, after being trained to drink 9% EtOH , were microinjected in the LH with N-methyl-d-aspartate (NMDA) or its antagonist, D-AP5, or with alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) or its antagonist, CNQX-ds. Consumption of EtOH , chow, and water was then measured. To provide an anatomical control, a separate set of rats was injected 2 mm dorsal to the LH. In Experiment 3, the effect of LH injection of NMDA and AMPA on the expression of OX and MCH was measured using radiolabeled in situ hybridization (ISH) and also using digoxigenin-labeled ISH, to distinguish effects on OX and MCH cells in the LH and the nearby perifornical area (PF) and zona incerta (ZI). RESULTS: When injected into the LH, NMDA and AMPA both significantly increased EtOH intake while having no effect on chow or water intake. The GLUT receptor antagonists had the opposite effect, significantly reducing EtOH consumption. No effects were observed with injections 2 mm dorsal to the LH. In addition to these behavioral effects, LH injection of NMDA significantly stimulated expression of OX in both the LH and PF while reducing MCH in the ZI, whereas AMPA increased OX only in the LH and had no effect on MCH. CONCLUSIONS: Glutamatergic inputs to the LH, acting through NMDA and AMPA receptors, appear to have a stimulatory effect on EtOH consumption, mediated in part by increased OX in LH and PF and reduced MCH in ZI.

Hypothalamic huntingtin-associated protein 1 as a mediator of feeding behavior.

The hypothalamus responds to circulating leptin and insulin in the control of food intake and body weight. A number of neurotransmitters in the hypothalamus, including gamma-aminobutyric acid (GABA), also have key roles in feeding. Huntingtin-associated protein 1 (Hap1) is expressed more abundantly in the hypothalamus than in other brain regions, and lack of Hap1 in mice leads to early postnatal death. Hap1 is also involved in intracellular trafficking of the GABA(A) receptor. Here, we report that fasting upregulates the expression of Hap1 in the rodent hypothalamus, whereas intracerebroventricular administration of insulin downregulates Hap1 by increasing its degradation through ubiquitination. Decreasing the expression of mouse hypothalamic Hap1 by siRNA reduces the level and activity of hypothalamic GABA(A) receptors and causes a decrease in food intake and body weight. These findings provide evidence linking hypothalamic Hap1 to GABA in the stimulation of feeding and suggest that this mechanism is involved in the feeding-inhibitory actions of insulin in the brain.

Utility of the Zebrafish Model for Studying Neuronal and Behavioral Disturbances Induced by Embryonic Exposure to Alcohol, Nicotine, and Cannabis.

It is estimated that 5% of pregnant women consume drugs of abuse during pregnancy. Clinical research suggests that intake of drugs during pregnancy, such as alcohol, nicotine and cannabis, disturbs the development of neuronal systems in the offspring, in association with behavioral disturbances early in life and an increased risk of developing drug use disorders. After briefly summarizing evidence in rodents, this review focuses on the zebrafish model and its inherent advantages for studying the effects of embryonic exposure to drugs of abuse on behavioral and neuronal development, with an emphasis on neuropeptides known to promote drug-related behaviors. In addition to stimulating the expression and density of peptide neurons, as in rodents, zebrafish studies demonstrate that embryonic drug exposure has marked effects on the migration, morphology, projections, anatomical location, and peptide co-expression of these neurons. We also describe studies using advanced methodologies that can be applied in vivo in zebrafish: first, to demonstrate a causal relationship between the drug-induced neuronal and behavioral disturbances and second, to discover underlying molecular mechanisms that mediate these effects. The zebrafish model has great potential for providing important information regarding the development of novel and efficacious therapies for ameliorating the effects of early drug exposure.

Role of Chemokine Cxcl12a in Mediating the Stimulatory Effects of Ethanol on Embryonic Development of Subpopulations of Hypocretin/Orexin Neurons and Their Projections.

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.

Subpopulations of hypocretin/orexin neurons differ in measures of their cell proliferation, dynorphin co-expression, projections, and response to embryonic ethanol exposure.

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.