Peripheral blood transcriptomic profiling of molecular mechanisms commonly regulated by binge drinking and placebo effects.

Molecular responses to alcohol consumption are dynamic, context-dependent, and arise from a complex interplay of biological and external factors. While many have studied genetic risk associated with drinking patterns, comprehensive studies identifying dynamic responses to pharmacologic and psychological/placebo effects underlying binge drinking are lacking. We investigated transcriptome-wide response to binge, medium, and placebo alcohol consumption by 17 healthy heavy social drinkers enrolled in a controlled, in-house, longitudinal study of up to 12 days. Using RNA-seq, we identified 251 and 13 differentially expressed genes (DEGs) in response to binge drinking and placebo, respectively. Eleven protein-coding DEGs had very large effect sizes in response to binge drinking (Cohen's d > 1). Furthermore, binge dose significantly impacted the Cytokine-cytokine receptor interaction pathway (KEGG: hsa04060) across all experimental sequences. Placebo also impacted hsa04060, but only when administered following regular alcohol drinking sessions. Similarly, medium-dose and placebo commonly impacted KEGG pathways of Systemic lupus erythematosus, Neutrophil extracellular trap formation, and Alcoholism based on the sequence of drinking sessions. These findings together indicate the "dose-extending effects" of placebo at a molecular level. Furthermore, besides supporting alcohol dose-specific molecular changes, results suggest that the placebo effects may induce molecular responses within the same pathways regulated by alcohol.

Cocaine and amphetamine regulated transcript (CART) mediates sex differences in binge drinking through central taste circuits.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) has been implicated in alcohol consumption and reward behaviours, yet mechanisms mediating these effects have yet to be identified. Using a transgenic CART knockout (KO) mouse line we uncovered a sexually dimorphic effect of CART in binge drinking, with male CART KO mice increasing intake, whilst female CART KO mice decreased their alcohol intake compared to controls. Female CART KO mice show greater sensitivity to bitter solutions that can be overshadowed through addition of a sweetener, implicating taste as a factor. Further we identify that this is not driven through peripherally circulating sex hormones, but the central nucleus of the amygdala (CeA) is a locus where CART contributes to the regulation of alcohol consumption, with CeA CART neutralisation specifically reducing plain alcohol, but not sweetened alcohol consumption in female mice. These findings may have implications for the development of sex-specific treatment options for alcohol use disorders through targeting the CART system.

Binge-pattern alcohol consumption and genetic risk as determinants of alcohol-related liver disease.

Alcohol-related liver disease (ARLD) represents a major public health burden. Identification of high-risk individuals would allow efficient targeting of public health interventions. Here, we show significant interactions between pattern of drinking, genetic predisposition (polygenic risk score, PRS) and diabetes mellitus, and risk of incident ARLD, in 312,599 actively drinking adults in UK Biobank. Binge and heavy binge drinking significantly increase the risk of alcohol-related cirrhosis (ARC), with higher genetic predisposition further amplifying the risk. Further, we demonstrate a pronounced interaction between heavy binge drinking and high PRS, resulting in a relative excess risk due to interaction (RERI) of 6.07. Diabetes consistently elevates ARC risk across all drinking and PRS categories, and showed significant interaction with both binge patterns and genetic risk. Overall, we demonstrate synergistic effects of binge drinking, genetics, and diabetes on ARC, with potential to identify high-risk individuals for targeted interventions.

Multi-omics analysis of alcohol effects on the liver in young and aged mice.

Excessive alcohol consumption has detrimental effects on the entire organism, especially on the liver. The toxicity is partly dependent on age, as older individuals metabolize alcohol more slowly leading to increased cellular injury. This study aimed to investigate the effects of moderate binge drinking on the liver of young and aged mice in a genome-wide multi-omics approach. We determined DNA methylation (DNAm) using the Illumina MouseMethylation array and gene expression by RNA sequencing in 18 female Balb/c mice in a 2 × 2 design. The animals underwent three moderate binge drinking cycles (ethanol vs. vehicle) and liver tissue was harvested at 4 or 19 months of age. We tested differential gene expression (DE) and DNAm associated with ethanol intake in linear models separately in young and aged mice, performed enrichment analyses for pathways and GWAS signatures of problematic alcohol use, and analysed the overlap of DNAm and gene expression. We observed DE in young and aged animals and substantial overlap in genes such as Bhlhe40, Klf10, and Frmd8. DE genes in aged animals were enriched for biological processes related to alcohol metabolism, inflammation, liver fibrosis, and GWAS signatures of problematic alcohol use. We identified overlapping signatures from DNAm and gene expression, for example, Frmd8 in aged and St6galnac4 in young mice. This study offers converging evidence of novel age-related targets in a moderate alcohol consumption model highlighting dysregulations in genes related to alcohol metabolism, inflammation, and liver fibrosis. Future studies are needed to confirm these results and elucidate the underlying mechanisms.

Brain-derived neurotrophic factor Val66Met polymorphism moderates the relationship between impulsivity, negative emotions, and binge drinking intensity in university students.

Studies on the genetic factors involved in binge drinking (BD) and its associated traits are very rare. The aim of this cross-sectional study was to investigate differences in the association between impulsivity, emotion regulation and BD in a sample of young adults according to the rs6265/Val66Met variant in the brain-derived neurotrophic factor (BDNF) gene, a well-known candidate gene in alcohol use disorders. We recruited 226 university students (112 women), aged between 18 and 25 years old, from two centers in France. The participants completed measures related to alcohol consumption, depression severity, state anxiety levels, impulsivity (UPPS-P), and difficulties in emotion regulation [Difficulty in Emotion Regulation Scale (DERS)]. The relationship between the BD score and the clinical characteristics in the BDNF genotype groups was assessed by partial correlation analyses and moderation analyses. The partial correlation analyses showed that, in the Val/Val genotype group, the BD score was positively related to UPPS-P Lack of Premeditation and Sensation Seeking scores. In the Met carriers group, the BD score was positively related to UPPS-P Positive Urgency, lack of Premeditation, lack of Perseverance and Sensation Seeking scores and to Clarity score of the DERS. Moreover, the BD score was positively associated with depression severity and state anxiety scores. The moderation analyses revealed that BDNF Val/Met genotype moderated the relationship between several clinical variables and BD. The results of the present study support the hypothesis of common and specific vulnerability factors regarding impulsivity and emotion regulation difficulties associated with BD according to this BDNF rs6265 polymorphism.

Risk Factors for Binge Drinking in Young Adulthood: The Roles of Aggregate Genetic Liability and Impulsivity-Related Processes.

OBJECTIVE: Binge drinking is characterized by excessive alcohol use and is widespread in youth. We explore the relationship between binge drinking's risk factors by considering (a) aggregate genetic liability (polygenic risk score [PGS]) for alcohol use and problems and (b) impulsivity-related processes. We examined whether the associations between PGS and binge drinking were mediated by impulsivity, with a possible shared genetic liability between alcohol phenotypes and impulsivity. METHOD: Participants were from the Avon Longitudinal Study of Parents and Children (N = 2,545). We evaluated PGS for alcohol use and problems and impulsivity-related processes (sensation seeking at age 18 and inhibition at age 24) and measured binge drinking frequency (24 years old) as the outcome. Correlations and structural equation models were used to test a hypothesized model of the relationships among these variables. RESULTS: Higher binge drinking frequency was related to higher aggregate genetic liability for alcohol use and problems in both models (standardized betas = .055-.064, all ps < .009). We found an association between binge drinking and sensation seeking (standardized beta = .224, p < .0001) but not inhibition (standardized beta = -.015, p = .437). Although the association between binge drinking and PGS for alcohol use and problems was mainly direct, a proportion of the association with alcohol problems was mediated by sensation seeking (14.61%). CONCLUSIONS: Targeting sensation seeking at the end of adolescence may be means to prevent binge drinking in adulthood, whereas considering the role of genetic factors may improve our understanding of at-risk youth.

Targeting Persistent Changes in Neuroimmune and Epigenetic Signaling in Adolescent Drinking to Treat Alcohol Use Disorder in Adulthood.

Studies universally find early age of drinking onset is linked to lifelong risks of alcohol problems and alcohol use disorder (AUD). Assessment of the lasting effect of drinking during adolescent development in humans is confounded by the diversity of environmental and genetic factors that affect adolescent development, including emerging personality disorders and progressive increases in drinking trajectories into adulthood. Preclinical studies using an adolescent intermittent ethanol (AIE) exposure rat model of underage binge drinking avoid the human confounds and support lifelong changes that increase risks. AIE increases adult alcohol drinking, risky decision-making, reward-seeking, and anxiety as well as reductions in executive function that all increase risks for the development of an AUD. AIE causes persistent increases in brain neuroimmune signaling high-mobility group box 1 (HMGB1), Toll-like receptor, receptor for advanced glycation end products, and innate immune genes that are also found to be increased in human AUD brain. HMGB1 is released from cells by ethanol, both free and within extracellular vesicles, that act on neurons and glia, shifting transcription and cellular phenotype. AIE-induced decreases in adult hippocampal neurogenesis and loss of basal forebrain cholinergic neurons are reviewed as examples of persistent AIE-induced pathology. Both are prevented and reversed by anti-inflammatory and epigenetic drugs. Findings suggest AIE-increased HMGB1 signaling induces the RE-1 silencing transcript blunting cholinergic gene expression, shifting neuronal phenotype. Inhibition of HMGB1 neuroimmune signaling, histone methylation enzymes, and galantamine, the cholinesterase inhibitor, both prevent and reverse AIE pathology. These findings provide new targets that may reverse AUD neuropathology as well as other brain diseases linked to neuroimmune signaling. SIGNIFICANCE STATEMENT: Adolescent underage binge drinking studies find that earlier adolescent drinking is associated with lifelong alcohol problems including high levels of lifetime alcohol use disorder (AUD). Preclinical studies find the underage binge drinking adolescent intermittent ethanol (AIE) model causes lasting changes in adults that increase risks of developing adult alcohol problems. Loss of hippocampal neurogenesis and loss of basal forebrain cholinergic neurons provide examples of how AIE-induced epigenetic and neuroimmune signaling provide novel therapeutic targets for adult AUD.

Differential Regulation of Tachykinin and Opioid System Gene Expression in Brain and Immune Cells of Chronic Binge Alcohol-Treated Simian Immunodeficiency Virus-Infected Macaques.

People living with HIV have a high likelihood of at-risk alcohol use and are at increased risk for neurocognitive decline. The underlying mechanisms involved in HIV-associated neurocognitive disorder (HAND) are not completely understood. Previously, we showed that chronic binge alcohol (CBA) administration produced behavioral deficits in non antiretroviral therapy (ART)-treated simian immunodeficiency virus (SIV)-infected macaques. Moreover, we observed that CBA/SIV enhanced neuroinflammatory gene expression and attenuated growth factor signaling in the frontal cortex (FC) and basal ganglia, effects that were partially ameliorated by ART. We hypothesized that the neuroinflammatory and growth factor changes observed could be associated with alterations in opioid, tachykinin, and endocannabinoid gene expression. Furthermore, we proposed that gene expression patterns in peripheral blood mononuclear cells (PBMCs) could serve as an indicator of expression changes in the brain (FC). We examined gene expression patterns of opioid, tachykinin, and endocannabinoid systems in FC and PBMCs isolated from CBA/SIV macaques. Expression of targeted genes as determined by reverse transcription-quantitative polymerase chain reaction was analyzed in relation to CBA, ART, plasma, and brain viral loads (PVL and BVL, respectively) and compared with baseline (PBMC) or FC from SIV- controls. FC expression of ORM1, POMC, and TACR1 was negatively associated with PVL (p = .03, .002, .05 respectively). FC expression of TAC1 was positively associated with CBA exposure (p = .05). PBMC expression of DAGLA was positively associated with CBA exposure; but negatively associated with combined CBA/ART exposure (p = .03). Our findings reflect the complex interactions of SIV, CBA, and ART in modulating opioid and tachykinin system gene expression. Contrary to our prediction, results did not reveal parallel changes (in magnitude or direction) in PBMC and FC gene expression. Further studies are warranted to determine the relevance of these transcriptional changes in modulating HAND-related behaviors resulting from at-risk alcohol use and HIV/SIV exposure.

Alcohol consumption and epigenetic age acceleration in young adults.

Alcohol is a widely consumed substance in the United States, however its effect on aging remains understudied. In this study of young adults, we examined whether cumulative alcohol consumption, i.e., alcohol years of beer, liquor, wine, and total alcohol, and recent binge drinking, were associated with four measures of age-related epigenetic changes via blood DNA methylation. A random subset of study participants in the Coronary Artery Risk Development in Young Adults Study underwent DNA methylation profiling using the Illumina MethylationEPIC Beadchip. Participants with alcohol consumption and methylation data at examination years 15 (n = 1,030) and 20 (n = 945) were included. Liquor and total alcohol consumption were associated with a 0.31-year (P = 0.002) and a 0.12-year (P = 0.013) greater GrimAge acceleration (GAA) per additional five alcohol years, while beer and wine consumption observed marginal (P = 0.075) and no associations (P = 0.359) with GAA, respectively. Any recent binge drinking and the number of days of binge drinking were associated with a 1.38-year (P < 0.001) and a 0.15-year (P < 0.001) higher GAA, respectively. We observed statistical interactions between cumulative beer (P < 0.001) and total alcohol (P = 0.004) consumption with chronological age, with younger participants exhibiting a higher average in GAA compared to older participants. No associations were observed with the other measures of epigenetic aging. These results suggest cumulative liquor and total alcohol consumption and recent binge drinking may alter age-related epigenetic changes as captured by GAA. With the increasing aging population and widespread consumption of alcohol, these findings may have potential implications for lifestyle modification to promote healthy aging.

Genetic variation in fatty acid amide hydrolase (FAAH): Associations with early drinking and smoking behaviors.

BACKGROUND: The endocannabinoid system is implicated in psychiatric disorders and drug dependence. Within this system, fatty acid amide hydrolase (FAAH) metabolizes endocannabinoids. Individuals with A-group genotypes (C/A or A/A) of a common FAAH variant (rs324420; C > A; Pro129Thr) have slower enzymatic activity compared to C-group individuals (C/C genotype). Slow FAAH activity is differentially associated with alcohol and nicotine use. METHODS: Among European-ancestry participants in the NDIT study (n = 249-607), genotype associations with past-year binge drinking in young adults were estimated in logistic regression models. In adolescents, hazard ratios (HR) were estimated from Cox proportional hazards models to assess the FAAH genotype group association with time to drinking initiation and attaining drinking frequency outcomes. HR were also used to assess genotype effect on time to smoking initiation and attaining early smoking milestones (e.g., first inhalation, ICD-10 dependence). RESULTS: Compared to those in the C-group, those in the A-group had higher odds of binge drinking at ages 20 (Odds ratio (OR) = 2.16, 95 % CI 1.36-3.42) and 30 (OR = 1.61, 95 % CI 1.10-2.36). Time to initiation of drinking and daily drinking was faster in adolescents in the A-group (HR = 1.39, 95 % CI 1.09-1.77 and HR = 2.24, 95 % CI 1.05-4.76, respectively). Time to smoking initiation was faster in the A-group (HR = 1.20, 95 % CI 1.04-1.39); however, time to smoking milestones among adolescent smokers was not consistently different for the A- versus C-groups (HR = 0.43 to 1.13). CONCLUSIONS: Slow FAAH activity (A-group) was associated with greater risks for binge drinking, drinking initiation and escalation, and cigarette smoking initiation, but had little impact on the escalation in cigarette smoking behaviors.

Overexpression of wild type glycine alpha 1 subunit rescues ethanol sensitivity in accumbal receptors and reduces binge drinking in mice.

The nucleus accumbens (nAc) is a critical region in the brain reward system since it integrates abundant synaptic inputs contributing to the control of neuronal excitability in the circuit. The presence of inhibitory α1 glycine receptor (GlyRs) subunits, sensitive to ethanol, has been recently reported in accumbal neurons suggesting that they are protective against excessive binge consumption. In the present study, we used viral vectors (AAV) to overexpress mutant and WT α1 subunits in accumbal neurons in D1 Cre and α1 KI mice. Injection of a Cre-inducible AAV carrying an ethanol insensitive α1 subunit in D1 Cre neurons was unable to affect sensitivity to ethanol in GlyRs or affect ethanol drinking. On the other hand, using an AAV that transduced WT α1 GlyRs in GABAergic neurons in the nAc of high-ethanol consuming mice caused a reduction in ethanol intake as reflected by lowered drinking in the dark and reduced blood ethanol concentration. As expected, the AAV increased the glycine current density by 5-fold without changing the expression of GABAA receptors. Examination of the ethanol sensitivity in isolated accumbal neurons indicated that the GlyRs phenotype changed from an ethanol resistant to an ethanol sensitive type. These results support the conclusion that increased inhibition in the nAc can control excessive ethanol consumption and that selective targeting of GlyRs by pharmacotherapy might provide a mechanistic procedure to reduce ethanol binge.

Targeting the Maladaptive Effects of Binge Drinking on Circadian Gene Expression.

Previous studies (1) support a role of circadian genes in regulating alcohol intake, and (2) reveal that harmful alcohol use alters circadian rhythms. However, there is minimal knowledge of the effects of chronic alcohol processes on rhythmic circadian gene expression across brain regions important for circadian biology and alcohol intake. Therefore, the present study sought to test the effects of chronic binge-like drinking on diurnal circadian gene expression patterns in the master circadian pacemaker (SCN), the ventral tegmental area (VTA), and the nucleus accumbens (NAc) in High Drinking in the Dark-1 (HDID-1) mice, a unique genetic risk model for drinking to intoxication. Consistent with earlier findings, we found that 8 weeks of binge-like drinking reduced the amplitude of several core circadian clock genes in the NAc and SCN, but not the VTA. To better inform the use of circadian-relevant pharmacotherapies in reducing harmful drinking and ameliorating alcohol's effects on circadian gene expression, we tested whether the casein kinase-1 inhibitor, PF-67046, or the phosphodiesterase type-4 (an upstream regulator of circadian signalling) inhibitor, apremilast, would reduce binge-like intake and mitigate circadian gene suppression. PF-67046 did not reduce intake but did have circadian gene effects. In contrast, apremilast reduced drinking, but had no effect on circadian expression patterns.

Maternal binge alcohol consumption leads to distinctive acute perturbations in embryonic cardiac gene expression profiles.

BACKGROUND: Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.5) causes perturbations in the expression patterns of specific genes in the developing heart in the acute period (1-3 days) following the binge episode. To test this hypothesis and identify strong candidate ethanol-sensitive target genes of interest, we adapted a mouse binge alcohol model that is associated with a high incidence of congenital heart defects as described below. METHODS/RESULTS: Pregnant mice were administered a single dose of alcohol (2.5 g/kg in saline) or control (saline alone) via oral gavage. To evaluate the impact of maternal binge alcohol on cardiac gene expression profiles, we isolated embryonic hearts from both groups (n = 5/group) at 24, 48, and 72 h post-gavage for transcriptomic analyses. RNA was extracted and evaluated using quantitative RNA-sequencing (RNA-Seq) methods. To identify a cohort of binge-altered cardiac genes, we set the threshold for change at >2.0-fold difference with adjusted p < 0.05 versus control.  RNA-Seq analysis of cardiac gene expression revealed that of the 17 genes that were altered within the first 48 h post-binge, with the largest category consisting of transcription factors (Alx1, Alx4, HoxB7, HoxD8, and Runx2), followed by signaling molecules (Adamts18, Dkk2, Rtl1, and Wnt7a). Furthermore, multiple comparative and pathway analyses suggested that several of the candidate genes identified through differential RNA-Seq analysis may interact through certain common pathways. To investigate this further, we performed gene-specific qPCR analyses for three representative candidate targets: Runx2, Wnt7a, and Mlxipl. Notably, only Wnt7a showed significantly (p < 0.05) decreased expression in response to maternal binge alcohol in the qPCR assays. CONCLUSIONS: These findings identify Wnt7a and a short list of potential other candidate genes and pathways for further study, which could provide mechanistic insights into how maternal binge alcohol consumption produces congenital cardiac malformations.

Long-term alcohol drinking in High Drinking in the Dark mice is stable for many months and does not show alcohol deprivation effects.

We have modelled genetic risk for binge-like drinking by selectively breeding High Drinking in the Dark-1 and -2 (HDID-1 and HDID-2) mice for their propensity to reach intoxicating blood alcohol levels (BALs) after binge-like drinking in a single bottle, limited access paradigm. Interestingly, in standard two-bottle choice (2BC) tests for continuously available alcohol versus water, HDID mice show modest levels of preference. This indicates some degree of independence of the genetic contributions to risk for binge-like and sustained, continuous access drinking. We had few data where the drinking in the dark (DID) tests of binge-like drinking had been repeatedly performed, so we serially offered multiple DID tests to see whether binge-like drinking escalated. It did not. We also asked whether HDID mice would escalate their voluntary intake with prolonged exposure to alcohol 2BC. They did not. Lastly, we assessed whether an alcohol deprivation effect (ADE) developed. ADE is a temporary elevation in drinking typically observed after a period of abstinence from sustained access to alcohol choice. With repetition, these periods of ADE sometimes have led to more sustained elevations in drinking. We therefore asked whether repeated ADE episodes would elevate choice drinking in HDID mice. They did not. After nearly 500 days of alcohol access, the intake of HDID mice remained stable. We conclude that a genetically-enhanced high risk for binge-like drinking is not sufficient to yield alterations in long-term alcohol intake.

The persistent impact of adolescent binge alcohol on adult brain structural, cellular, and behavioral pathology: A role for the neuroimmune system and epigenetics.

Adolescence is a critical neurodevelopmental window for maturation of brain structure, neurocircuitry, and glia. This development is sculpted by an individual's unique experiences and genetic background to establish adult level cognitive function and behavioral makeup. Alcohol abuse during adolescence is associated with an increased lifetime risk for developing an alcohol use disorder (AUD). Adolescents participate in heavy, episodic binge drinking that causes persistent changes in neurocircuitry and behavior. These changes may underlie the increased risk for AUD and might also promote cognitive deficits later in life. In this chapter, we have examined research on the persistent effects of adolescent binge-drinking both in humans and in rodent models. These studies implicate roles for neuroimmune signaling as well as epigenetic reprogramming of neurons and glia, which create a vulnerable neuroenvironment. Some of these changes are reversible, giving hope for future treatments to prevent many of the long-term consequences of adolescent alcohol abuse.

Effect of ketamine on binge drinking patterns in crossed high alcohol-preferring (cHAP) mice.

BACKGROUND: Previous research has demonstrated the utility of subanesthetic doses of ketamine in decreasing binge (Drinking-in-the-Dark, or DID) 20% alcohol intake in female inbred (C57BL/6J) mice when administered 12 hours prior to alcohol access (Crowley et al., 2019). In the current study, we assess the efficacy of a similar ketamine pretreatment using male and female selectively bred, crossed High Alcohol Preferring (cHAP) mice, which also drink to intoxication, but are not inbred. We hypothesized that ketamine would decrease binge alcohol intake without impacting locomotor activity. METHODS AND RESULTS: Subjects were 28 adult cHAP mice. Mice first received a 2-week DID drinking history using 2-h/day alcohol access. On day 12, prior to ketamine treatment, the average blood ethanol concentration (BEC) was 130 mg/dL, confirming that mice reliably reached intoxicating BECs. On day 15, mice were given 0, 3, or 10 mg/kg of ketamine 12 hours prior to the DID session. Ketamine did not decrease total (2-h) alcohol consumption or locomotion. Interestingly, the 10 mg/kg dose of ketamine did alter the drinking pattern in male mice, decreasing front-loading for a single day. We opted to then increase the doses to 32 or 100 mg/kg (i.e., an anesthetic dose) two days after the initial treatment, keeping the saline control. Mice of both sexes decreased total binge alcohol intake at the 100 mg/kg dose only, but again, the effect only lasted one day. CONCLUSIONS: The current study found that cHAP mice reached more than double the BECs observed in C57BL/6J mice during DID, but did not respond to subanesthetic ketamine. Modest efficacy was found for ketamine pretreatment at anesthetic doses. Differences in findings may be due to differential intake during DID, or genetic differences between C57Bl/6J mice and cHAP mice. Drug efficacy in multiple models is important for discovering reliable pharmacotherapies for alcoholism.

Alcohol drinking during early adolescence activates microglial cells and increases frontolimbic Interleukin-1 beta and Toll-like receptor 4 gene expression, with heightened sensitivity in male rats compared to females.

Adolescent drinking is risky because neural circuits in the frontal lobes are undergoing maturational processes important for cognitive function and behavioral control in adulthood. Previous studies have shown that myelinated axons in the medial prefrontal cortex (mPFC) are particularly sensitive to alcohol drinking, especially in males. Pro-inflammatory mediators like toll-like receptor 4 (TLR4) and interleukin-1 beta (IL1b) have been implicated in alcohol induced-inflammation and demyelination; thus, herein we test the hypothesis that voluntary alcohol drinking early in adolescence elicits a pro-inflammatory state that is more pronounced in the brain of males compared to females. Adolescent male and female Wistar rats self-administered sweetened alcohol or sweetened water from postnatal days 28-42 and separate sets of brains were processed for 1) immunolabeling for ionized calcium-binding adapter molecule 1 to analyze microglial cell morphology, or 2) qPCR analysis of gene expression of pro-inflammatory mediators. Binge drinking alcohol activated microglia in the mPFC and hippocampus of both males and females, suggesting that voluntary alcohol exposure initiates an inflammatory response. Il1b mRNA was upregulated in the mPFC of both sexes. Conversely, Tlr4 mRNA levels were elevated after drinking only in males, which could explain more robust effects of alcohol on myelin in this region in developing males compared to females. Il1b mRNA changes were not observed in the hippocampus, but alcohol elevated Tlr4 mRNA in both sexes, highlighting regional specificity in inflammatory responses to alcohol. Overall, these findings give insight into potential mechanisms by which low-to-moderate voluntary alcohol intake impacts the developing brain. This article is part of the special Issue on 'Vulnerabilities to Substance Abuse'.

Adolescent intermittent ethanol (AIE) produces sex specific alterations in adult neuroimmune gene expression and ethanol sensitivity that are independent of ethanol metabolism.

The goal of the present studies was to determine long-lasting effects of adolescent intermittent ethanol (AIE), a rodent model of binge patterns of ethanol consumption, on (i) behavioral sensitivity to ethanol challenge in adulthood using the Loss of Righting Reflex (LORR) test; (ii) ethanol pharmacokinetics and ethanol-metabolizing enzyme expression when re-challenged with ethanol as adults; and (iii) induction of neuroimmune gene expression during an adult binge-like ethanol challenge. To evaluate the impact of AIE on ethanol sensitivity in adulthood, adult rats received a sedative ethanol dose of 3.5 g/kg and were tested for the LORR. Sexually dimorphic effects were observed, with AIE males showing more rapid recovery than vehicle exposed controls, an effect that was completely absent in females. Rats exposed to the same AIE procedure were challenged with 0.75, 1.5, or 3.0 g/kg i.p. ethanol in adulthood. Female rats with a history of AIE displayed a small increase in ethanol clearance rate when challenged with 0.75 g/kg, however no other significant differences in ethanol pharmacokinetics were noted. To assess persistent AIE-associated changes in neuroimmune gene expression, rats were challenged with 0 or 2.5 g/kg ethanol. Both male and female adult rats with a history of AIE displayed sensitized hippocampal IL-6 and IκBα gene expression in response to ethanol challenge. Changes in cytokine gene expression as well as ethanol sensitivity assessed by LORR were not shown to be the result of changes in ethanol pharmacokinetics and point to AIE altering other mechanisms capable of significantly altering the neuroimmune and behavioral response to ethanol.

TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration.

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer's or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer's disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.

Antisense-Induced Downregulation of Clock Genes in the Shell Region of the Nucleus Accumbens Reduces Binge Drinking in Mice.

INTRODUCTIONS: Binge drinking is a deadly pattern of alcohol consumption. Evidence suggests that genetic variation in clock genes is strongly associated with alcohol misuse; however, the neuroanatomical basis for such a relationship is unknown. The shell region of the nucleus accumbens (NAcSh) is well known to play a role in binge drinking. Hence, we examined whether clock genes in the NAcSh regulate binge drinking. METHODS: To address this question, 2 experiments were performed on male C57BL/6J mice. In the first experiment, mice exposed to alcohol or sucrose under the 4-day drinking-in-the-dark (DID) paradigm were euthanized at 2 different time points on day 4 [7 hours after light (pre-binge drinking) or dark (post-binge drinking) onset]. The brains were processed for RT-PCR to examine the expression of circadian clock genes (Clock, Per1, and Per2) in the NAcSh and suprachiasmatic nucleus (SCN). In the second experiment, mice were exposed to alcohol, sucrose, or water as described above. On day 4, 1 hour prior to the onset of alcohol exposure, mice were bilaterally infused with either a mixture of circadian clock gene antisense oligodeoxynucleotides (AS-ODNs; antisense group) or nonsense/random ODNs (R-ODNs; control group) through surgically implanted cannulas above the NAcSh. Alcohol/sucrose/water consumption was measured for 4 hours. Blood alcohol concentration was measured to confirm binge drinking. Microinfusion sites were histologically verified using cresyl violet staining. RESULTS: As compared to sucrose, mice euthanized post-binge drinking (not pre-binge drinking) on day 4 displayed a greater expression of circadian genes in the NAcSh but not in the SCN. Knockdown of clock genes in the NAcSh caused a significantly lower volume of alcohol to be consumed on day 4 than in the control treatment. No differences were found in sucrose or water consumption. CONCLUSIONS: Our results suggest that clock genes in the NAcSh play a crucial role in binge drinking.