EtOH Consumption Research Articles

Habitual Ethanol Seeking and Licking Microstructure of Enhanced Ethanol Self-Administration in Ethanol-Dependent Mice.

A significant component of ethanol (EtOH) dependence is the disruption to decision-making processes. Prior work has shown EtOH dependence biases habitual seeking of EtOH and disrupts neural mechanisms supporting decision-making. This has contributed to the hypothesis that habitual EtOH seeking in EtOH dependence may promote excessive habitual or compulsive EtOH consumption. However, decision-making and behavioral processes underlying seeking and consummatory behaviors differ. Here, we examine the microstructure of EtOH consummatory behavior in the context of habitual EtOH seeking. Following home cage pre-exposure to EtOH, C57Bl/6J mice underwent 4 rounds of chronic intermittent EtOH (CIE) or air exposure. Following acute withdrawal, mice began training for operant self-administration of 15% EtOH. Training consisted of 16-hour sessions in which mice were trained in a random ratio (RR) schedule of reinforcement for 30-second access to the EtOH sipper. To test for CIE-induced changes in action control, we used sensory-specific satiation and assessed the effect of outcome devaluation on EtOH seeking. Importantly, the use of a lickometer during operant training allowed us to measure the microstructure of lick behavior. Prior induction of EtOH dependence led to increased EtOH seeking, consumption, and an insensitivity to outcome devaluation, the latter indicative of habitual EtOH seeking. We also found altered consummatory lick patterns in CIE-exposed mice compared to Air controls. While CIE mice had significantly more licks in a burst and a longer burst duration, there were no differences in the total number of bursts compared to Air controls. Furthermore, these EtOH consummatory behaviors correlated with blood EtOH concentrations (BECs), while EtOH-seeking responses did not. Our results confirm that EtOH dependence can produce habitual EtOH seeking and suggests the increased EtOH consummatory behaviors following EtOH dependence are separable from decision-making processes controlling EtOH seeking.

Intermittent binge-like ethanol exposure during adolescence attenuates the febrile response by reducing brown adipose tissue thermogenesis in rats

Ethanol (EtOH) consumption is a primary health risk worldwide, which generally starts during adolescence in a binge pattern (i.e., the episodic consumption of high amounts). Binge EtOH consumption can lead to modifications of the innate and adaptive immune responses, including fever. The present study evaluated the febrile response that was induced by lipopolysaccharide (LPS) and prostaglandins E2 (PGE2) and the mechanisms of thermoregulation in adolescent rats that were exposed to EtOH in a binge-like pattern. Male Wistar rats were treated with an intraperitoneal (i.p.) injection of EtOH or saline on postnatal days (PND) 25, 26, 29, 30, 33, 34, 37, and 38. On PND 51, they received a pyrogenic challenge with LPS (i.p.) or PGE2 (intracerebroventricular) to induce a febrile response. Interscapular brown adipose tissue (BAT) mass and uncoupling protein (UCP) activity in isolated mitochondria were evaluated on PND 51. The rats were then subjected to cold challenges to analyze adaptive thermogenesis. Intermittent EtOH exposure during adolescence impaired the LPS- and PGE2-induced febrile response 12 days after the end of EtOH exposure. Ethanol exposure decreased interscapular BAT mass, oxygen consumption, and UCP activity in isolated mitochondria, resulting in an impairment in thermogenesis at 5 °C. No morphological changes in BAT were observed. These findings indicate that binge-like EtOH exposure during adolescence impairs thermoregulation by reducing BAT mass and function. This reduction may last for a prolonged period of time after the cessation of EtOH exposure and may affect both cold defenses and the febrile response during the development of infectious diseases.

Chronic Voluntary Ethanol Drinking in Cynomolgus Macaques Elicits Gene Expression Changes in Prefrontal Cortical Area 46.

BackgroundGenome‐wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross‐sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within‐subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model.MethodsTwo cohorts of cynomolgus macaques (n = 23) underwent a schedule‐induced polydipsia procedure to establish EtOH self‐administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH‐naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA‐Seq paired analysis, which allowed for correction of individual baseline differences in gene expression.ResultsA total of 675 genes were significantly down‐regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up‐regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll‐like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites.ConclusionsThe data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro‐ and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.

Knock-in Mice Expressing an Ethanol-Resistant GluN2A NMDA Receptor Subunit Show Altered Responses to Ethanol.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-activated, heterotetrameric ligand-gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol (EtOH) inhibition of NMDARs is thought to mediate specific actions of EtOH during acute and chronic exposure. Studies from our laboratory, and others, identified EtOH-sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in subdomains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of EtOH on knock-in mice expressing a GluN2A subunit that shows reduced sensitivity to EtOH. A battery of tests evaluating locomotion, anxiety, sedation, motor coordination, and voluntary alcohol intake were performed in wild-type mice and those expressing the GluN2A A825W knock-in mutation. Whole-cell patch-clamp electrophysiological recordings were used to confirm reduced EtOH sensitivity of NMDAR-mediated currents in 2 separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR-mediated responses. Male and female mice homozygous for the GluN2A(A825W) knock-in mutation showed reduced EtOH inhibition of NMDAR-mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild-type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor-incoordinating effects of EtOH and showed a rightward shift in locomotor-stimulating effects of EtOH. There was no effect of the mutation on EtOH-induced anxiolysis or voluntary EtOH consumption in either male or female mice. These findings show that expression of EtOH-resistant GluN2A NMDARs results in selective and sex-specific changes in the behavioral sensitivity to EtOH.

Decreased ω-6:ω-3 PUFA ratio attenuates ethanol-induced alterations in intestinal homeostasis, microbiota, and liver injury

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies, including liver injury. ω-6 PUFAs exert pro-inflammatory activity, while ω-3 PUFAs promote anti-inflammatory activity that is mediated, in part, through specialized pro-resolving mediators [e.g., resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the ω-6:ω-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. ω-3 FA desaturase-1 (fat-1) mice, which endogenously increase ω-3 PUFA levels, were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-γ, Il-6, and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation, stem cell renewal, and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further, gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally, these data correlated with a significant reduction in liver injury. Our study suggests that ω-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury.

F654A and K558Q Mutations in NMDA Receptor 1 Affect Ethanol-Induced Behaviors in Drosophila.

N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH. Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects. We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant. Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila.

Social Interaction With an Alcohol-Intoxicated or Cocaine-Injected Peer Selectively Alters Social Behaviors and Drinking in Adolescent Male and Female Rats.

Drinking alcohol is facilitated by social interactions with peers, especially during adolescence. The importance of peer social influences during adolescence on alcohol and substance use has recently received more attention. We have shown that social interaction with an alcohol-intoxicated peer influences adolescent alcohol drinking differently in male and female rats using the demonstrator-observer paradigm. The present set of experiments analyzed the social interaction session to determine changes in social behaviors and subsequent alcohol drinking in adolescent male and female rats. Specifically, in Experiment 1, we determined whether specific social behaviors were altered during interaction with an alcohol-intoxicated demonstrator administered 1.5g/kg ethanol (EtOH) and assessed changes in EtOH intake in adolescent observers. Experiment 2 examined changes in voluntary saccharin consumption to determine whether social interaction with an alcohol-intoxicated demonstrator administered 1.5g/kg EtOH altered consumption of a palatable solution. In Experiment 3, we administered saline, and a low (5mg/kg) or high (20mg/kg) dose of cocaine to the demonstrator and assessed changes in the adolescent observers to determine whether social interaction with a "drugged" peer altered social behaviors and voluntary EtOH intake. We showed that social interaction with an alcohol-intoxicated demonstrator administered 1.5g/kg EtOH (i) decreased social play and increased social investigation and social contact in adolescent male and female observers, (ii) did not alter nonsocial behaviors, (iii) did not alter saccharin consumption, and (iv) increased voluntary EtOH intake in adolescent female but not male observers. When the peer was injected with cocaine, (i) social play was dose-dependently decreased, (ii) there were no changes in other social or nonsocial behaviors, and (iii) voluntary EtOH intake in adolescent male and female observers was unaffected. The present results are consistent and extend our previous work, showing that social interaction with an alcohol-intoxicated peer selectively alters social behaviors and alcohol drinking in adolescent rats. Females appear to be more sensitive to the elevating effects of social interaction on voluntary EtOH consumption.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n=13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22h/d, 7 d/wk, for a total of 12months. Control animals (n=9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. Monkeys in the EtOH group consumed an average of 2.8±0.2 (mean±SE) g/kg/d of EtOH (30±2% of total calories), resulting in an average blood EtOH concentration of 88.3±8.8mg/dl 7hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Changes in brain kynurenine levels via gut microbiota and gut-barrier disruption induced by chronic ethanol exposure in mice.

Inflammatory processes have been shown to modify tryptophan (Trp) metabolism. Gut microbiota appears to play a significant role in the induction of peripheral and central inflammation. Ethanol (EtOH) exposure alters gut permeability, but its effects on Trp metabolism and the involvement of gut microbiota have not been studied. We analyzed several parameters of gut-barrier and of peripheral and central Trp metabolism following 2 different EtOH consumption patterns in mice, the binge model, drinking in the dark (DID), and the chronic intermittent (CI) consumption paradigm. Antibiotic treatment was used to evaluate gut microbiota involvement in the CI model. Mice exposed to CI EtOH intake, but not DID, show bacterial translocation and increased plasma LPS immediately after EtOH removal. Gut-barrier permeability to FITC-dextran is increased by CI, and, furthermore, intestinal epithelial tight-junction (TJ) disruption is observed (decreased expression of zonula occludens 1 and occludin) associated with increased matrix metalloproteinase (MMP)-9 activity and iNOS expression. CI EtOH, but not DID, increases kynurenine (Kyn) levels in plasma and limbic forebrain. Intestinal bacterial decontamination prevents the LPS increase but not the permeability to FITC-dextran, TJ disruption, or the increase in MMP-9 activity and iNOS expression. Although plasma Kyn levels are not affected by antibiotic treatment, the elevation of Kyn in brain is prevented, pointing to an involvement of microbiota in CI EtOH-induced changes in brain Trp metabolism. Additionally, CI EtOH produces depressive-like symptoms of anhedonia, which are prevented by the antibiotic treatment thus pointing to an association between anhedonia and the increase in brain Kyn and to the involvement of gut microbiota.-Giménez-Gómez, P., Pérez-Hernández, M., O'Shea, E., Caso, J. R., Martín-Hernández, D., Cervera, L. A., Centelles. M. L. G.-L., Gutiérrez-Lopez, M. D., Colado, M. I. Changes in brain kynurenine levels via gut microbiota and gut-barrier disruption induced by chronic ethanol exposure in mice.

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

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. We utilized live imaging and Imaris software to investigate how low-dose embryonic EtOH (0.5%), administered from 22 to 24hours postfertilization, affects specific properties of hcrt neurons in hcrt:EGFP transgenic zebrafish at different ages. 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. 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.

Selenium (Se) Deficiency After Orthotopic Heart Transplantation (OHT) (P12-056-19)

ObjectivesSe and selenoproteins play critical metabolic roles, as in glutathione peroxidase (antioxidant defense) and iodo-thyronine deiodinase 2 (thyroid hormone production). Other Se-dependent pathways can promote apoptosis and decrease oncogene expression. Se deficiency (SeD) is rare in the US, given its abundance in food and soil. SeD is linked to tobacco use, excessive ETOH consumption, birth control pills, and inflammatory bowel disease. SeD can increase the risk of prostate cancer and Burkitt’s lymphoma. Severe SeD can cause skeletal muscle dysfunction and endemic cardiomyopathy (Keshan disease). We present herein a case of SeD 7 + years after heart transplantation, in whom prostate cancer and Burkitt’s post transplant lymphoproliferative disorder (PTLD) developed. MethodsA 77-year-old male with hypertension, dyslipidemia, diabetes mellitus and non-ischemic cardiomyopathy underwent OHT in 2011. He developed prostate cancer in 2015, treated with hormonal and radiation therapy. In January, 2019 he had progressive dyspnea, muscle weakness, abdominal pain, weight loss and diarrhea x 1 week. An ascending colonic mass revealed Burkitt’s PTLD. A metabolic evaluation uncovered a serum Se of 31 mcg/L (normal range 63–160).The patient received TPN with additional Se. He underwent chemotherapy and developed tumor lysis syndrome, requiring renal replacement therapy. He remained on single drug immunosuppression with Tacrolimus. Renal function and oral intake returned after 3 weeks. ResultsOur patient presented with a severe SeD without apparent predisposing factors. He has developed 2 SeD-related neoplasms: prostate cancer and Burkitt’s PTLD. The congruence of his clinical course and biochemical results support the diagnosis of a true SeD. However, the etiology of the SeD state remains unknown. We hypothesize that his immunosuppression therapy with Tacrolimus may have played a role. If so, it could be a previously unidentified drug effect. ConclusionsSerum Se monitoring should be considered in post OHT patients who develop prostate cancer or Burkitt’s PTLD. Given the increased risk of prostate cancer in OHT patients, routine screening of Se levels and Se supplementation of deficiency states should be considered after OHT. Funding Sourcesnone.

1908-P: Factors Related to Hepatic Fat and Fibrosis in American Indians

American Indians (AI) experience high rates of both liver disease and type 2 diabetes (T2D), yet few studies have investigated the contributions of T2D and other known risk factors to liver disease in AI. We used magnetic resonance imaging (MRI)-estimated proton density fat fraction (PDFF) and MR elastography (MRE)-estimated stiffness to assess hepatic fat and fibrosis, respectively, and assessed whether obesity, T2D, and heavy episodic alcohol (ETOH) consumption are associated with these liver measures in AI. The Strong Heart Family Study (SHFS) is a population-based family study of AI recruited from 12 communities located within Arizona (AZ), the Dakotas, and Oklahoma (OK). Metabolic measures were assessed during a clinic visit in 2006-2009. A re-exam including MRI and MRE has been conducted on 109 participants to date from AZ and OK (mean age 50.6 ± 11.3 years, 72% women, 71% obese (BMI ≥ 30 kg/m2), 57% had T2D, and 28% reported ETOH consumption). Median liver PDFF was 8.9% (1rst and 3rd quartiles: 4.3%, 14.6%), with 71% of participants having elevated liver fat (PDFF ≥ 5%). Median liver stiffness was 2.6 kPa, with 23% showing mild liver fibrosis (stiffness 2.8 - 3.63 kPa) and 8.3% advanced liver fibrosis (stiffness > 3.63 kPa). In cross-sectional analyses, liver PDFF was significantly higher in obese vs. non-obese participants (Wilcoxon rank sum test, p<0.0001). In longitudinal analyses using a generalized linear model, after adjusting for age, sex, and study site, obesity measures (waist p=0.005; BMI p=0.01), HbA1c (p=0.001), liver enzyme levels (ALT p=0.03) and serum uric acid (p=0.0002) were associated with elevated PDFF. Obesity (waist p=0.001; BMI p=0.02) and cigarette smoking (p=0.002) were associated with elevated liver stiffness. In our at-risk subsample of AI participants of the SHFS, participants commonly had elevated liver fat and liver fibrosis, with obesity being the major, common, associated risk factor. Disclosure S.A. Cole: None. W.C. Henderson: None. T. Ali: None. C. West: None. M.S. Middleton: Consultant; Self; Kowa Pharmaceutical Europe Co. Ltd., Median, Novo Nordisk Inc. Stock/Shareholder; Self; General Electric, Pfizer Inc. A. Silva: None. D.N. Batakis: None. A.L. Louie: None. B.V. Howard: None. J.D. Dvorak: None. R. Devereux: Consultant; Self; Acer. J.G. Umans: None. C. Sirlin: Advisory Panel; Self; AMRA Medical, Bristol-Myers Squibb Company, VirtualScopics. Consultant; Self; Fulcrum Therapeutics. Other Relationship; Self; Celgene Corporation, Enanta Pharmaceuticals, Inc., Genzyme, Gilead Sciences, Inc., Gilead Sciences, Inc., ICON plc., Intercept Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., NuSirt Biopharma, Inc., Shire, Synageva, Takeda Pharmaceutical Company Limited, VirtualScopics. Y. Zhang: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK110096)

Impact of Genetic Variation on Stress-Related Ethanol Consumption.

The effect of stress on alcohol consumption in humans is highly variable, and the underlying processes are not yet understood. Attempts to model a positive relationship between stress and increased ethanol (EtOH) consumption in animals have been only modestly successful. Our hypothesis is that individual differences in stress effects on EtOH consumption are mediated by genetics. We measured alcohol consumption, using the drinking-in-the-dark (DID) paradigm in females from 2 inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), and 35 of their inbred progeny (the BXD family). A control group was maintained in normal housing and a stress group was exposed to chronic mild stress (CMS), consisting of unpredictable stressors over 7weeks. These included predator, social, and environmental perturbations. Alcohol intake was measured over 16weeks in both groups during baseline (preceding 5-week period), CMS (intervening 7-week period), and post-CMS (final 4-week period). We detected a strong effect of CMS on alcohol intake. A few strains demonstrated CMS-related increased alcohol consumption; however, most showed decreased intake. We identified 1 nearly significant quantitative trait locus on chromosome 5 that contains the neuronal nitric oxide synthase gene (Nos1). The expression of Nos1 is frequently changed following alcohol exposure, and variants in this gene segregating among the BXD population may modulate alcohol intake in response to stress. The results we present here represent the first study to combine chronic stress and alcohol consumption in a genetic reference population of mice. Differences in susceptibility to the effects of stressful environments vis-à-vis alcohol use disorders would suggest that the differences have at least some basis in genetic constitution. We have also nominated a likely candidate gene underlying the large individual differences in effects of stress on alcohol consumption.

Nicotine Produces a High-Approach, Low-Avoidance Phenotype in Response to Alcohol-Associated Cues in Male Rats.

Nicotine and alcohol use are highly comorbid. Modulation of drug-paired extrinsic and intrinsic cues likely plays a role in this interaction, as cues can acquire motivational properties and augment drug seeking. The motivational properties of cues can be measured through Pavlovian conditioning paradigms, in which cues either elicit approach following pairing with the reinforcing properties of alcohol or elicit avoidance following pairing with the aversive consequences of alcohol. The present experiments tested whether nicotine would enhance the incentive properties of an appetitive ethanol (EtOH) cue and diminish the avoidance of an aversive EtOH cue in Pavlovian paradigms. In experiment 1, male Long-Evans rats with or without prior chronic intermittent access to EtOH were administered nicotine or saline injections prior to Pavlovian conditioned approach (PavCA) sessions, during which conditioned approach to the cue ("sign-tracking") or the EtOH delivery location ("goal-tracking") was measured. In experiment 2, male Long-Evans rats were administered nicotine or saline injections prior to pairing a flavor cue with increasing doses of EtOH (i.p.) in an adaptation of the conditioned taste avoidance (CTA) paradigm. Results from PavCA indicate that, regardless of EtOH exposure, nicotine enhanced responding elicited by EtOH-paired cues with no effect on a similar cue not explicitly paired with EtOH. Furthermore, nicotine reduced sensitivity to EtOH-induced CTA, as indicated by a rightward shift in the dose-response curve of passively administered EtOH. The ED50 , or the dose of EtOH that produced a 50% reduction in intake relative to baseline, was significantly higher in nicotine-treated rats compared to saline-treated rats. We conclude that nicotine increases the approach and diminishes the avoidance elicited by Pavlovian cues paired, respectively, with the reinforcing and aversive properties of EtOH consumption in male rats. As such, nicotine may enhance alcoholism liability by engendering an attentional bias toward cues that predict the reinforcing outcomes of drinking.

Hepatic Inactivation of the Type 2 Deiodinase Confers Resistance to Alcoholic Liver Steatosis.

A mouse with hepatocyte-specific deiodinase type II inactivation (Alb-D2KO) is resistant to diet-induced obesity, hepatic steatosis, and hypertriglyceridemia due to perinatal epigenetic modifications in the liver. This phenotype is linked to low levels of Zfp125, a hepatic transcriptional repressor that promotes liver steatosis by inhibiting genes involved in packaging and secretion of very-low-density lipoprotein. Here, we used chronic and binge ethanol (EtOH) in mice to cause liver steatosis. The EtOH treatment causes a 2.3-fold increase in hepatic triglyceride content; Zfp125 levels were approximately 50% higher in these animals. In contrast, Alb-D2KO mice did not develop EtOH-induced liver steatosis. They also failed to elevate Zfp125 to the same levels, despite being on the EtOH-containing diet for the same period of time. Their phenotype was associated with 1.3- to 2.9-fold up-regulation of hepatic genes involved in lipid transport and export that are normally repressed by Zfp125, that is, Mttp, Abca1, Ldlr, Apoc1, Apoc3, Apoe, Apoh, and Azgp1. Furthermore, genes involved in the EtOH metabolic pathway, that is, Aldh2 and Acss2, were also 1.6- to 3.1-fold up-regulated in Alb-D2KO EtOH mice compared with control animals kept on EtOH. EtOH consumption elevates expression of Zfp125. Alb-D2KO animals, which have lower levels of Zfp125, are much less susceptible to EtOH-induced liver steatosis.

Effects of Endogenous ω6:ω3 PUFA Ratio Reduction on Ileum Homeostasis and Liver Injury in Mice Chronically Fed Ethanol

The objective of the current study was to test the hypothesis that increasing endogenous levels of w3 PUFAs alters gene expression profiles in the intestine of mice chronically fed an ethanol (EtOH) diet, leading to improved gut barrier and liver function, in a mouse model of alcoholic liver disease (ALD). ALD is a significant human health issue leading to increased morbibity and mortality resulting from liver dysfunction. Chronic alcohol consumption causes changes in intestinal permeability resulting in translocation of the gut luminal contents into the circulation. Therefore, a complete understanding of the mechanism(s) by which alcohol consumption alters gut barrier function is essential in order to develop therapeutic strategies to combat these effects. To test the above hypothesis, wild‐type and fat‐1 transgenic mice (which express an w6 fatty acid desaturase and therefore can directly convert w6 to w3 PUFAs) were fed a liquid diet with or without EtOH (pair‐fed, PF) for 6 weeks. Prior to euthanasia, a subset of the mice were challenged with LPS, which induces endotoxemia and is a frequent comorbidity to ALD. At the end of the study, liver and intestine pathology was evaluated and an RNA‐seq analysis was performed on ileum tissue. We identified numerous, statistically significant changes in ileum gene expression in fat‐1 mice when compared to wild‐type controls. Significant changes in the expression of genes that encode enzymes important for the metabolism and detoxification of EtOH were found in both pair‐fed and EtOH‐fed, LPS‐challenged mice. For example, the expression of Adh1 and Adh6a, two members of the alcohol dehydrogenase family, were significantly increased in the ileums of fat‐1 mice. In addition, several isoforms of aldehyde dehydrogenase were increased in fat‐1 mice, including Aldh1a7 and Aldh3b1, in both pair‐fed and EtOH/LPS‐treated animals. The expression of catalase, which metabolizes hydrogen peroxide, was increased and the expression of Cyp2e1 was decreased in PF fat‐1 mice, suggesting that these mice are better positioned to resist tissue damage as a result of reactive oxygen species. In mice fed only EtOH and not challenged with LPS, there was a significant change in the expression of genes important for xenobiotic metabolism. These include several cytochrome p450s (Cyp2c65, Cyp2d10, and Cyp2d34). Lastly, we found that the expression many genes involved in the metabolism of purines was significantly altered in the ileums of fat‐1 mice. For example, Entpd8 (ectonucleoside triphosphate diphosphohydrolase 8), Enpp3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) and Ada (adenosine deaminase) were all increased in fat‐1 mice. The metabolism of adenosine is critically important for regulating immune function and therefore, altered purine metabolism may be one mechanism by which fat‐1 mice are protected from the toxic effects of chronic EtOH consumption. In summary, these data suggest that fat‐1 mice are much more efficient at metabolizing EtOH and the toxic metabolite, acetaldehyde, and have changes in purine metabolism that potentially have a beneficial effect on overall intestine function.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paternal Preconception Every-Other-Day Ethanol Drinking Alters Behavior and Ethanol Consumption in Offspring.

Alcohol use disorder is a devastating disease with a complex etiology. Recent preclinical studies have revealed that paternal preconception chronic intermittent ethanol (EtOH) exposure via vaporized EtOH altered drinking behaviors and sensitivity to EtOH selectively in male offspring. In the current study, we used a voluntary oral route of paternal preconception EtOH exposure, i.e., intermittent every-other-day two-bottle choice drinking, and tested offspring for behavioral alterations. Fifteen EtOH drinking sires and 10 control sires were mated to EtOH naïve females to produce EtOH-sired and control-sired offspring. These offspring were tested using the elevated plus maze, open field, drinking in the dark, and unlimited access two-bottle choice assays. We found that paternal preconception every-other-day two-bottle choice drinking resulted in reduced EtOH consumption selectively in male offspring in the drinking in the dark assay compared to control-sired offspring. No differences were detected in either sex in the unlimited access two-bottle choice and elevated plus maze assays. Open field analysis revealed complex changes in basal behavior and EtOH-induced behaviors that were sex specific. We concluded that paternal preconception voluntary EtOH consumption has persistent effects that impact the next generation. This study adds to a growing appreciation that one’s behavioral response to EtOH and EtOH drinking behavior are impacted by EtOH exposure of the prior generation.

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

The cerebellum is sensitive to ethanol (EtOH) consumption. Chronic EtOH consumption impairs motor learning by modulating the cerebellar circuitry synaptic transmission and long-term plasticity. Under in vitro conditions, acute EtOH inhibits both parallel fiber (PF) and climbing fiber (CF) long-term depression (LTD). However, thus far it has not been investigated how chronic EtOH consumption affects sensory stimulation-evoked LTD at the molecular layer interneurons (MLIs) to the Purkinje cell (PC) synapses (MLI-PC LTD) in the cerebellar cortex of living animals. In this study, we investigated the effect of chronic EtOH consumption on facial stimulation-evoked MLI-PC LTD, using an electrophysiological technique as well as pharmacological methods, in urethane-anesthetized mice. Our results showed that facial stimulation induced MLI–PC LTD in the control mice, but it could not be induced in mice with chronic EtOH consumption (0.8 g/kg; 28 days). Blocking the cannabinoid type 1 (CB1) receptor activity with AM-251, prevented MLI-PC LTD in the control mice, but revealed a nitric oxide (NO)-dependent long-term potentiation (LTP) of MLI–PC synaptic transmission (MLI-PC LTP) in the EtOH consumption mice. Notably, with the application of a NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP) alone prevented the induction of MLI–PC LTD, but a mixture of SNAP and AM-251 revealed an MLI-PC LTP in control mice. In contrast, inhibiting NO synthase (NOS) revealed the facial stimulation-induced MLI-PC LTD in EtOH consumption mice. These results indicate that long-term EtOH consumption can impair the sensory stimulation-induced MLI–PC LTD via the activation of a NO signaling pathway in the cerebellar cortex in vivo in mice. Our results suggest that the chronic EtOH exposure causes a deficit in the cerebellar motor learning function and may be involved in the impaired MLI–PC GABAergic synaptic plasticity.

Detecting Neurodevelopmental Effects of Early-Gestation Ethanol Exposure: A Nonhuman Primate Model of Ethanol Drinking During Pregnancy.

Gestational ethanol (EtOH) exposure is associated with multiple developmental abnormalities, collectively termed fetal alcohol spectrum disorder (FASD). While the majority of women abstain from EtOH following knowledge of pregnancy, one contributing factor to the high FASD prevalence is that pregnancy is not detected until 4 to 6weeks. Thus, EtOH consumption continues during the initial stages of fetal development. An experimental protocol is described in which rhesus macaques self-administer 1.5g/kg/d EtOH (or isocaloric maltose dextrin) prior to pregnancy and through the first 60days of a 168-day gestation term. Menstrual cycles were monitored, including measurements of circulating estradiol and progesterone levels. The latency to consume 1.5g/kg EtOH and blood EtOH concentration (BEC) was measured. Twenty-eight fetuses (14 EtOH and 14 controls) were generated in this study. EtOH did not affect menstrual cycles or the probability of successful breeding. No EtOH-induced gross adverse effects on pregnancy were observed. Individual variability in latency to complete drinking translated into variability in BEC, measured 90minutes following session start. Drinking latencies in controls and EtOH drinkers were longer in the second gestational month than in the first. All pregnancies reached the planned experimental time point of G85, G110, or G135, when in utero MRIs were performed, fetuses were delivered by caesarean section, and brains were evaluated with exvivo procedures, including slice electrophysiology. Fetal tissues have been deposited to the Monkey Alcohol Tissue Research Resource. This FASD model takes advantage of the similarities between humans and rhesus macaques in gestational length relative to brain development, as well as similarities in EtOH self-administration and metabolism. The daily 1.5g/kg dose of EtOH through the first trimester does not influence pregnancy success rates. However, pregnancy influences drinking behavior during the second month of pregnancy. Future publications using this model will describe the effect of early-gestation EtOH exposure on anatomical and functional brain development at subsequent gestational ages.

Sex and β-Endorphin Influence the Effects of Ethanol on Limbic Gabra2 Expression in a Mouse Binge Drinking Model.

Binge drinking is a widespread problem linked to increased risk for alcohol-related complications, including development of alcohol use disorders. In the last decade, binge drinking has increased significantly, specifically in women. Clinically, sexually dimorphic effects of alcohol are well-characterized, however, the underlying mechanisms for these dimorphisms in the physiological and behavioral effects of alcohol are poorly understood. Among its many effects, alcohol consumption reduces anxiety via the inhibitory neurotransmitter GABA, most likely acting upon receptors containing the α-2 subunit (Gabra2). Previous research from our laboratory indicates that female mice lacking the endogenous opioid peptide β-endorphin (βE) have an overactive stress axis and enhanced anxiety-like phenotype, coupled with increased binge-like alcohol consumption. Because βE works via GABA signaling to reduce anxiety, we sought to determine whether sexually dimorphic binge drinking behavior in βE deficient mice is coupled with differences in CNS Gabra2 expression. To test this hypothesis, we used βE knock-out mice in a “drinking in the dark” model where adult male and female C57BL/6J controls (βE +/+) and βE deficient (βE -/-; B6.129S2-Pomctm1Low/J) mice were provided with one bottle of 20% ethanol (EtOH) and one of water (EtOH drinkers) or two bottles of water (water drinkers) 3 h into the dark cycle for four consecutive days. Following a binge test on day 4, limbic tissue was collected and frozen for subsequent qRT-PCR analysis of Gabra2 mRNA expression. Water-drinking βE +/+ females expressed more Gabra2 in central nucleus of the amygdala and the bed nucleus of the stria terminalis than males, but this sex difference was absent in the βE -/- mice. Genotype alone had no effect on alcohol consumption or drug-induced increase in Gabra2 expression. In contrast, βE expression had bi-directional effects in females: in wildtypes, Gabra2 mRNA was reduced by binge EtOH consumption, while EtOH increased expression in βE -/- females to levels commensurate with drug-naïve βE +/+ females. These results support the contention that βE plays a role in sexually dimorphic binge-like EtOH consumption, perhaps through differential expression of GABAA α2 subunits in limbic structures known to play key roles in the regulation of stress and anxiety.