Alcohol, Clinical and Experimental Research

IntroductionBinge drinking is common among adolescents and young adults and is associated with an increased risk of developing alcohol use disorder (AUD) and long-term cognitive deficits. We analyzed RNA-seq data from male Sprague Dawley rats to identify candidate genes that may play a role in the acute and chronic changes in cognitive function during binge-like adolescent alcohol/EtOH exposure and after a period of abstinence. MethodsAt postnatal day (PND) 30, male rats received chronic intermittent EtOH across 16 days. RNA was extracted from hippocampal tissue and sequenced at two acute timepoints, PND 35 and PND 46, and after 24 days forced abstinence (PND 70). We processed RNA-seq data, compiled gene counts, and performed normalization and differential expression analysis (DESeq2). Gene set enrichment analysis was performed through the R package fgsea. Gene sets of the Molecular Signatures Database (MSigDB) collections were used to identify gene pathways that were dysregulated following EtOH exposure. We also evaluated overlapping gene pathways that were affected across all timepoints. ResultsMultiple gene pathway analyses revealed that EtOH has robust effects on neuroinflammation, cellular remodeling, sleep, and bioenergetics. Changes were heavily dependent on whether gene expression was assessed during acute EtOH exposure or after abstinence. Genes involved in sleep regulation were selectively impacted during the acute timepoints, whereas dysregulation of genes involved in bioenergetics were only impacted after abstinence. The most striking changes occurred in genes that regulate neuroinflammatory processes and cellular remodeling. ConclusionThese data reveal acute and chronic effects of EtOH on multiple gene pathways that persist across analytic approaches and identify genes that have increased sensitivity to EtOH. These findings contribute to our understanding of the temporal effects of adolescent EtOH exposure and how gene pathway dysregulation contributes to the protracted emergence of neuronal remodeling in the hippocampus during a critical period of brain maturation.

BackgroundSex differences in alcohol use behaviors are well-established: males typically engage in heavier and more frequent drinking and exhibit more externalizing behaviors (e.g., other substance use), while females often transition to dependence more rapidly and present more internalizing psychopathology (e.g., depression). The biological mechanisms underpinning these differences are relatively unknown. MethodsIn this study, we investigated the sex-differentiated genetic architecture of 11 alcohol use phenotypes pertaining to frequency, quantity and problematic use by leveraging sex-stratified genome-wide association studies (Ns 40,335 to 613,148). Specifically, we compared SNP-based heritability (h2SNP) estimates, individual genetic locus effects, genetic correlations (rg) across alcohol phenotypes and with comorbid traits from independent GWAS, and polygenic score (PGS) associations with medical outcomes from clinical populations. Resultsh2SNP was broadly similar between sexes, except for higher estimates in males for beer quantity and problematic alcohol use (PAU). We identified four sex-differentiated top loci (psex-diff < 5 x 10-8), including a female-specific association in IZUMO1 for drinking frequency and quantity, and three male-specific associations in ADH1B, KLB and FTO for beer quantity and/or PAU. Between-sex genetic correlations ranged from 0.68{+/-}0.07 to 0.89{+/-}0.04, these estimates were lowest for quantity measures and varied by beverage type, indicating partially distinct polygenic architecture. In males, we identified stronger positive genetic correlations with several externalizing traits (e.g., general addiction) compared to females. In females, we identified a specific positive genetic correlation with a single internalizing trait, self-harm. PGS analyses revealed sex-specific medical associations (e.g., bone/musculoskeletal conditions in females; hepatic/respiratory/infectious sequelae in males) that were obscured in sex-combined analyses; however, sex-specific PGS did not outperform combined-sex PGS for predicting alcohol use disorder diagnosis. ConclusionsSex-aware analyses of alcohol use behaviors can improve our understanding of the genetic etiology of alcohol use and related health outcomes, and future studies should consider cultural variation (e.g., drinking attitudes, social norms) in the relationship between behavior and genetics.

BackgroundAlcohol use behaviors (AUBs) manifest in a variety of normative and problematic ways across the life course, all of which are heritable. Twin studies show that genetic influences on AUBs change across development, but this is usually not considered in research identifying and investigating the genes linked to AUBs. AimsUnderstanding the dynamics of how genes shape AUBs could point to critical periods in which interventions may be most effective and provide insight into the mechanisms behind AUB-related genes. In this project, we investigate how genetic associations with AUBs unfold across development using longitudinal modelling of polygenic scores (PGSs). DesignUsing results from genome-wide association studies (GWASs), we created PGSs to index individual-level genetic risk for multiple AUB-related dimensions: Consumption, Problems, a variable pattern of drinking associated with a preference for beer (BeerPref), and externalizing behavior (EXT). We created latent growth curve models and tested PGSs as predictors of latent growth factors (intercept, slope, quadratic) underlying trajectories of AUBs. SettingPGSs were derived in six longitudinal epidemiological cohorts from the US, UK, and Finland. ParticipantsParticipant data were obtained from AddHealth, ALSPAC, COGA, FinnTwin12, the older Finnish Twin Cohort, and Spit for Science (total N = 19,194). These cohorts included individuals aged 14 to 67, with repeated measures collected over a span of 4 to 36 years. MeasurementsPrimary measures included monthly frequency of typical alcohol consumption (CON) and heavy episodic drinking (HED). FindingsResults indicated that higher PGSs for all AUBs are robustly associated with higher mean levels of CON and/or HED (B = 0.064-0.333, p < 3.09E-04). However, these same genetic indices were largely not associated with drinking trajectories across cohorts. In the meta-analysis, only PGSs for chronic alcohol Problems consistently predicted a steeper slope (increasing trajectory) of CON across time (B = 0.470, p = 4.20E-06). ConclusionsThe results indicate that genetic associations with AUBs not only differ between behaviors, but also across developmental time points and across cohorts. Genetic studies that take such heterogeneity into account are needed to better represent the underlying etiology of AUBs. Individual-level genetic profiles may be useful to point to personalized intervention timelines, particularly for individuals with high alcohol Problems genetic risk scores.

ImportanceAlcohol genome-wide association studies (GWAS) have generally focused on alcohol consumption and alcohol use disorder (AUD); few have examined habitual drinking behaviors like maximum habitual alcohol intake (MaxAlc). ObjectiveIdentify MaxAlc loci and elucidate the genetic architecture across alcohol traits. DesignThe MaxAlc GWAS was performed in Million Veteran Program (MVP) participants enrolled from January 10, 2011 to September 30, 2020. Ancestry-specific GWAS were conducted in European (EUR) (n=218,623) and African (AFR) (n=29,132) ancestry subjects, then meta-analyzed (N=247,755). Linkage-disequilibrium score regression (LDSC) was used to estimate SNP-heritability and genetic correlations (rg) with other alcohol and psychiatric traits. Genomic structural equation modeling (gSEM) was used to evaluate genetic relationships between MaxAlc and other alcohol traits. Mendelian randomization (MR) was used to examine causal relationships. MTAG (multi-trait analysis of GWAS) was used to analyze MaxAlc and problematic alcohol use (PAU) jointly. SettingThe study was performed in a sample of U.S military Veterans. ParticipantsParticipants were 92.68% male and had mean age=65.92 (SD=11.70). 36.92% reported MaxAlc [&ge;] the binge-drinking threshold. Main Outcomes(s) and Measure(s)MaxAlc was defined from survey item: "in a typical month, what is/was the largest number of drinks of alcohol you may have had in one day?" with ordinal responses from 0 [&ge;] 15 drinks. ResultsThe MaxAlc GWAS resulted in 15 genome-wide significant (GWS) loci. Top associations in EUR and AFR were with known functional variants ADH1B*rs1229984 (p=3.12x10-104) and rs2066702 (p=6.30x10-17), respectively. Multiple novel associations were found. The SNP-heritability was 6.65% (s.e.=0.41%) in EUR and 3.42% (s.e.=1.46%) in AFR. MaxAlc was positively correlated with PAU (rg=0.79; p=3.95x10-149) and AUD (rg=0.76; p=1.26x10-127), and had negative rg with "alcohol usually taken with meals" (rg=-0.53; p=1.40x10-50). For psychiatric traits, MaxAlc had the strongest rg with suicide attempt (rg=0.40; p=3.02x10-21). gSEM supported a two-factor model with MaxAlc loading on a factor with PAU and AUD, and other alcohol consumption measures loading a separate factor. MR supported a small causal effect of MaxAlc on the liver enzyme gamma-glutamyltransferase ({beta}=0.012; p=2.66x10-10). MaxAlc MTAG resulted in 31 GWS loci. Conclusions and RelevanceMaxAlc closely aligns genetically with the etiology of problematic alcohol use traits. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the genetic etiology of maximum habitual alcohol intake (MaxAlc) and how does it compare to other alcohol consumption measures. FindingsThis MaxAlc study in 247,455 European and African ancestry individuals identified 15 genome-wide significant loci, including multiple novel associations. MaxAlc was strongly genetically correlated (rg) with measures of alcohol-related problems, demonstrated significantly different rg with psychiatric traits compared to other alcohol consumption traits, and loaded on a factor with alcohol problem traits while alcohol consumption state measures loaded on a separate factor. MeaningMaxAlc is genetically different from trait consumption measures in relation to problematic alcohol use.

Alcohol use disorders (AUDs) and related electrophysiological endophenotypes have been associated with the GABRA2 gene. However, the causal variants in GABRA2 and their mechanisms of influence on AUD and its correlates have not been established. Here we investigate the phenotypic spectrum of a GABRA2 coding variant (rs279858) through a phenome-wide association study (PheWAS) in two open-source datasets. We applied the PheWAS approach to identify a broad range of phenotypes associated with rs279858 in the MRC IEU OpenGWAS PheWAS and the Open Targets Genetics Portal. These databases extend the array of phenotypes beyond those available in electronic health records (EHR) to include numerous non-medical phenotypes and traits. We then followed up the results from those exploratory associations by examining the genetic correlations between our "top hits" and alcohol- and smoking-related phenotypes. In both data sources, rs279858 (C effect allele) was associated with anxiety-related phenotypes, including reduced risk-taking behavior and an increase in nervous feelings, as well as reduced number of lifetime sexual partners. Follow-up analyses revealed that these phenotypes were genetically correlated with each other and with alcohol- and smoking-related phenotypes. This work illustrates the utility of the PheWAS approach, particularly for phenotypes that extend beyond those that are typically captured in EHR data. In fact, the associations described here are all behavioral rather than clinical phenotypes. We postulate that these traits may be related to anxiety or behavioral inhibition that has been identified as a risk factor for AUD, and may represent pathophysiological intermediaries between GABRA2 and AUD.

Background and AimsSubstance use disorders (SUDs) are heritable and share genetic variance with externalizing and internalizing psychopathology. Although recent gene identification efforts have demonstrated the value of modeling the shared genetic architecture among SUDs and externalizing, most research has thus far failed to account for overlap with internalizing. In this study, we aim to characterize the genetic relationships of both externalizing and internalizing with SUDs. Design and settingWe used genome-wide association study (GWAS) summary statistics derived from previously published studies of externalizing, internalizing, and SUD outcomes to quantify the genetic overlap between these phenotypes. We characterize this overlap using omnibus, partial, and local genetic correlations, estimates of their shared polygenic effects, genetic causality models, polygenic score (PGS) analyses, and estimates of each SUDs residual variance derived from models in Genomic SEM. ParticipantsWe used GWAS summary statistics from individuals whose genomes were most similar to those from reference panels sampled from Europe (Ns ranged from 45,395 to 1,565,618) and Africa (Ns ranged from 30,000 to 122,571). For polygenic scores analyses, we used data from individuals of European and African ancestry groups available in the Collaborative Study on the Genetics of Alcoholism (COGA) sample (NMaximum = 7,394 for European-like genomes and 3,238 for African-like genomes) MeasurementsMeasurements in this study include GWAS summary statistics for externalizing, internalizing, and four substance use disorders: problematic alcohol use (PAU), cannabis use disorder (CUD), opioid use disorder (OUD), and tobacco use disorder (TUD). SUD outcomes in COGA were DSM-IV symptom counts of AUD, CUD, and OUD and scores on the Fagerstrom Test for Nicotine Dependence. FindingsWe found strong genetic relationships of externalizing and, to a lesser extent, internalizing with all SUDs across methods. Despite their more modest associations, internalizing emerged as an important genetic correlate of SUDs. After accounting for variance shared with externalizing, partial genetic correlations between internalizing and SUDs were attenuated but, with the exception of TUD, still significant. Similarly, the PGSINT accounted for a statistically significant increase in variance over and above PGSEXT. Two SUD specific patterns emerged such that TUD was least associated with both psychopathology spectra and OUD was most strongly related to internalizing relative to other SUDs. ConclusionsFrom these findings we conclude that shared genetic influences may explain comorbidity observed between SUDs and internalizing disorders and suggest that genetic risk for internalizing should be incorporated into SUD identification and prevention efforts. Future gene identification efforts should study SUDs in the context of both externalizing and internalizing psychopathology.

Integrated multi-omics and extracellular vesicle (EV) analysis are emerging as powerful, complementary strategies for biomarker discovery. These approaches offer promising tools to enhance early detection, diagnosis, and treatment of alcohol use disorder (AUD). Here we applied an integrated miRNomic and lipidomic approach to analyze plasma EVs from AUD patients and controls of both sexes to gain a comprehensive understanding of the underlying molecular mechanisms. We identified an AUD signature with predictive potential for diagnostic applications. Individual features (e.g., hsa-miR-99b-3p, hsa-miR-556-5p, Cer_NDS-d39:1, and PI18:0_18:2) represented important components; however, the strength of this signature lay in the combined profile rather than isolated markers. We also revealed an AUD-sex signature that provided insight into how biological responses to alcohol differ between females and males (including features such as hsa-miR-1301-3p and PC39:4), which also underscored the power of multi-omic integration. The individual miRNome approach also revealed an opposite functional alteration by sex in various alcohol related systems, such as pathways associated with immunity, oxidative stress, and autophagy. An open-access Shiny web application (https://carpercle.shinyapps.io/SexEVEthOmics/) accompanies this study, providing interactive access to the complete dataset and additional analyses for customized exploration. Together, our findings underscore the added value of multi-omics integration in identifying clinically relevant molecular signatures of disease; this sex-informed approach offers a promising path toward more personalized diagnostic tools and therapeutic strategies in AUD.

BackgroundRates of binge drinking have converged significantly between the sexes over recent decades, driven by increased rates of alcohol misuse in women. However, understanding of fundamental circuitry and neurobiology driving alcohol use in females, or how this may differ from male subjects remains underexplored. MethodsWe quantified c-Fos expression across 40 brain regions in alcohol naive, alcohol anticipating and binge drinking male and female mice. In vivo fiber photometry examined sex differences in basolateral amygdala (BLA) activity changes to alcohol intake. Chemogenetic BLA inhibition investigated a functional role in binge drinking. We then assessed sex differences in BLA efferent projection activation following binge drinking. Finally, we functionally interrogated the BLA to nucleus accumbens core (AcbC) projection in binge drinking. ResultsBinge drinking reduced network modularity (number of communities with similar activation patterns) in both sexes relative to alcohol naive and anticipating same-sex counterparts. Female binge drinking mice had increased BLA c-Fos expression compared to female naive and male binge drinking counterparts. In vivo fiber photometry revealed greater and more prolonged BLA responsivity at the onset of alcohol intake in females. Global BLA inhibition reduced reward intake in both sexes. However, the BLA to AcbC projection was preferentially activated in female binge drinking mice, and inhibition of this pathway reduced binge alcohol intake exclusively in females. ConclusionsWe identified sex differences in the neural circuits engaged in binge drinking, highlighting the BLA to AcbC projection may in part underpin sex differences in alcohol misuse. This provides further evidence of distinct neurobiological drivers of alcohol-related behaviors between the sexes.

Alcohol dependence and cirrhosis are key outcomes of excessive alcohol use. We studied the interaction between genetics and epigenetics at the aldehyde dehydrogenase (ALDH2) locus to understand differences in vulnerability to cirrhosis. Individuals were selected according to ICD 10 criteria for Alcohol dependence with Cirrhosis (AUDC+ve, N=116) and Alcohol dependence but without Cirrhosis; (AUDC-ve, N=123) from the clinical services of Gastroenterology and Psychiatry at the St Johns Medical College Hospital (SJMCH). Fibroscan/sonographic evidence was used to rule out fibrosis for the AUDC-ve group. Genomic DNA from blood was used for genotyping at ALDH2 (rs2238151) locus. A subset of the samples was assessed for DNA methylation (AUDC+ve, N=50; AUDC-ve, N=50) at the LINE-1 and ALDH2 CpG loci by pyrosequencing on a PyroMark Q24. LINE1 DNA methylation did not differ between the groups. ALDH2 DNA methylation was significantly lower in AUDC+ve group compared to AUDC-ve group (P <0.001). Lower methylation in T-allele carriers compared to T-allele non-carriers of the ALDH2 locus (rs2238151) was observed in AUDC+ve subjects (P=0.009). Compromised methylation in blood DNA at candidate loci, in those with liver disease in the context of prolonged severe alcohol abuse, could be explored as a biomarker for current pathology, and further progression.

BackgroundCharting the clinical course of substance use disorders (SUDs) to identify etiologic contributors to milestone onset and progression could inform intervention efforts. MethodsWe calculated polygenic risk scores (PRS) in 5,692 European-ancestry individuals (EUR) (56.2% male) and 4,918 African-ancestry (AFR) individuals (54.9% male) using genome-wide association studies (GWAS) of alcohol use disorder (AUD), opioid use disorder (OUD), and smoking trajectory (SMK). Using Cox regression, we examined the association of polygenic risk with age of first substance use, regular use, reported problems, and dependence diagnosis and with progression from regular use to onset of problems and dependence. ResultsEUR and males reported earlier onset and shorter progression times than AFR and females, respectively. Among EUR, higher AUD PRS predicted earlier onset and more rapid progression to alcohol-related milestones (ps<0.0001) and although a stronger moderator of problem onset among females (p=0.0165), it was more predictive of the progression to problems among males (p=0.0054). OUD and SMK PRS in EUR also predicted earlier onset of the respective milestones (ps=0.0002). Among AFR, where power is lower, AUD PRS predicted age of regular alcohol use (p=0.039) and dependence (p=0.001) and progression from regular use to diagnosis (p=0.045), while SMK PRS predicted earlier age of initiation (p=0.036). ConclusionsGenetic risk for SUDs predicts milestones and symptom progression in EUR and, to a lesser extent, among AFR. Larger, diverse discovery GWAS and target samples are needed to enhance the power of PRS to personalize interventions for individuals at genetic risk of serious substance-related outcomes. DisclosureDr. Kranzler is a member of advisory boards for Dicerna Pharmaceuticals, Sophrosyne Pharmaceuticals, and Enthion Pharmaceuticals; a consultant to Sobrera Pharmaceuticals; the recipient of research funding and medication supplies for an investigator-initiated study from Alkermes; and a member of the American Society of Clinical Psychopharmacologys Alcohol Clinical Trials Initiative, which was supported in the last three years by Alkermes, Dicerna, Ethypharm, Lundbeck, Mitsubishi, and Otsuka. Drs. Gelernter and Kranzler hold U.S. Patent 10,900,082: Genotype-guided Dosing of Opioid Receptor Agonists, 26 Jan. 2021. The other authors have no disclosures to make.

BACKGROUNDOne major hallmark of alcohol use disorder (AUD) is the persistence of alcohol drinking despite negative consequences. Among the indicators of AUD vulnerability, binge drinking is a strong risk factor. Although the lifetime prevalence of binge and AUD has been historically higher in men than women, this gap dramatically narrowed in the last decade. Additionally, sex differences in AUD and binge drinking have been shown in clinical and preclinical studies, respectively. The insular cortex plays an important role in AUD, and the anterior (aIC) and posterior (pIC) divisions have dimorphic functions. However, the contributions of the aIC and pIC sections in alcohol binge drinking and alcohol persistent drinking despite aversion, as well as the sexual dimorphism of these contributions, remained to be uncovered. METHODSFirst, by combining the drinking in the dark model with chemogenetics, we studied the causal role of aIC and pIC excitatory neurons in binge and persistent ethanol drinking in C57BL6/J male (n=49) and female (n=49) mice. Second, using calcium fiber photometry, we investigated pIC neuronal activity in both sexes (male n=14, female n=11) during both binge and persistent ethanol drinking. RESULTSWe identified a higher binge and persistent ethanol consumption in females compared to males. Chemogenetic inhibition of aIC glutamatergic neurons reduced bitter solutions intake independently of the solvent (ethanol or water), in both sexes. In contrast, inhibition of pIC glutamatergic neurons exclusively reduced persistent ethanol drinking in female mice. Finally, using fiber photometry recordings, we uncovered that pIC glutamatergic neuron activity was selectivity increased during ethanol persistent drinking in female mice. CONCLUSIONSThese findings suggest a sex-dependent function of the pIC in persistent ethanol drinking, providing a starting point in our understanding of the insular cortex function in the neurobiology of AUD in both sexes.

Individuals with alcohol use disorder continue to drink in excess despite the health and societal consequences, and the rate of problematic drinking and alcohol-related harms is increased in women. Clinical imaging studies report widespread adaptations in brain structure after chronic, heavy drinking, and alcohol-related cues enhance brain reactivity in reward-related regions. In rodents, alcohol drinking induces expression of the immediate early gene c-Fos, which can be a marker of cellular activity, across multiple brain regions. Recent evidence also suggests that abstinence from chronic intermittent alcohol exposure can produce mesoscale changes in c-Fos expression. However, there is a substantial gap in our understanding of how excessive drinking affects functional connectivity networks to influence alcohol-seeking behaviors. For this study, male and female C57BL/6J mice were given access to either water or a choice between water and ethanol in the intermittent access drinking model for 4 weeks. After a short-access drinking session, whole brains from high alcohol drinking male and female mice and water drinking controls were then subjected to c-Fos immunolabeling, iDISCO+ clearing, light sheet imaging, and whole-brain c-Fos mapping. Correlation matrices were then generated and graph theoretical statistical approaches were used to determine changes in functional connectivity across sex and drinking condition. We observed robust sex differences in the network of c-Fos+ cells in water drinking mice, and excessive alcohol drinking produce divergent and robust changes in functional network connectivity in male and female mice. In addition, these analyses identified novel hub regions in excessively drinking mice that were unique for each sex. In conclusion, the whole-brain c-Fos mapping analysis identified sex difference in functional network connectivity and unique and understudied regions that may play a critical role in controlling excessive ethanol drinking in male and female mice.

Inefficient aldehyde metabolism by an aldehyde dehydrogenase 2 (ALDH2) genetic variant, ALDH2*2 (rs671), increases the risk of esophageal cancer with alcohol consumption. Here we tested the hypothesis that additional genetic differences in ALDH2 besides ALDH2*2 exist resulting in inefficient acetaldehyde metabolism after alcohol consumption. Human volunteers were recruited who self-reported flushing after alcohol. The first stage recruited East Asians and the second stage non-East Asians. After phone screening and ALDH2 sequencing, volunteers were subjected to an alcohol challenge (0.25g/kg). Physiological parameters and breath acetaldehyde levels were assessed. Twenty-six participants were given an alcohol challenge. In the first stage, when comparing the ALDH2*1/*2 genotype to ALDH2*1/*1 genotype, tachycardia (104{+/-}3* versus 73{+/-}4 beats per minute), increases in facial skin temperature (99.6{+/-}0.4* versus 95.9{+/-}0.50F), and increases in breath acetaldehyde (peak: 2.1{+/-}0.4* versus 0.2{+/-}0.3ppm, n=8/group, *p<0.01) occurred after alcohol consumption. In the second stage, heterozygotes for an ALDH2 intron variant (rs4646777, G>A) caused increases in facial skin temperature (98{+/-}1* versus 94{+/-}10F, n=4, *p<0.01) without tachycardia or acetaldehyde accumulation after alcohol consumption. Subjects self-identifying as non-East Asian genotyped with an ALDH2*1/*2 variant also displayed a characteristic ALDH2*1/*2 phenotype after alcohol consumption. Further, ALDH2 point mutations rs747096195 (R101G) and rs190764869 (R114W) showed reduced acetaldehyde metabolism and increases in facial skin temperature after an alcohol challenge relative to wild type ALDH2 subjects. Taken together, we developed a method to non-invasively phenotype genetic differences for ALDH2 in humans including quantifying aldehyde metabolism in single subjects. We also discovered genetic differences besides ALDH2*2 that cause inefficient aldehyde metabolism. Brief SummaryWe developed a method to phenotype genetic differences in ALDH2 and we discovered novel ALDH2 mutations that result in inefficient acetaldehyde metabolism after alcohol consumption.

BackgroundLoss of control over drinking is a hallmark feature of alcohol use disorder (AUD) that is modeled preclinically through escalation of ethanol consumption and aversion-resistant drinking. Prior work with other reinforcers suggests that within-session unpredictable, intermittent access (uIntA) promotes loss of control over intake. However, the effect of uIntA on voluntary ethanol consumption is unknown. MethodsMale and female Long-Evans rats (n=9-10/group) underwent seven weeks of daily voluntary ethanol (20% v/v) drinking sessions under either a continuous access (ContA) or uIntA schedule. Following four weeks of baseline, rats were rendered dependent using a two-week chronic intermittent ethanol vapor exposure procedure. Daily testing was maintained through one week into withdrawal from vapor exposure. On the final day of testing, ethanol was adulterated with quinine (30 mg/L) to assess aversion-resistant drinking. ResultsRats drinking under ContA and uIntA exhibited similar levels of average daily ethanol consumption at baseline. However, uIntA elicited a more robust dependence-induced escalation of ethanol consumption compared to ContA, with uIntA sustaining escalation through early protracted withdrawal. Additionally, while rats with ContA to ethanol remained sensitive to quinine even after chronic ethanol vapor exposure, uIntA promoted aversion-resistant drinking in ethanol dependent rats. ConclusionsThese results demonstrate that, compared to ContA, uIntA maintains ethanol drinking and exacerbates AUD-related symptomatology while also providing researchers with the ability to capture additional measures of motivation and drinking patterns without increasing experimental burden. This work positions uIntA as a powerful tool to assess psychological and neurobiological factors underlying loss of control over drinking.

ImportanceEarly alcohol initiation (before age 15) is associated with adverse outcomes. Understanding mechanisms behind early alcohol initiation is essential for informing prevention efforts. ObjectiveTo examine whether structural covariance network properties at ages 9-10 years predict early alcohol initiation. DesignCase-control, population-based study design. SettingData from the Adolescent Brain Cognitive Development study were used. Baseline structural brain imaging data (ages 9-10) were used for generation and comparison of structural covariance networks. Data from baseline to 4-year follow-up ([&le;]age 15) assessments were used to determine alcohol initiation. ParticipantsParticipants were excluded if they reported consuming a full drink of alcohol at baseline, or did not meet imaging inclusion criteria. Controls were excluded if they had not yet been assessed or were missing substance use data at 4-year follow-up. In total, 3,878 participants met study criteria, of which 182 participants initiated alcohol. Structural covariance network properties were compared between the full sample and a 1:1 propensity-matched sample based on age, sex, race, ethnicity, religion, parental education, prenatal alcohol exposure, and baseline alcohol sipping. Main Outcomes and MeasuresStructural covariance networks were estimated using regional cortical thickness and volume measurements. Measures of network segregation (modularity, clustering coefficient), integration (characteristic path length, global efficiency), and resilience (degree assortativity) were compared between groups. Early alcohol initiation was defined as consuming a full drink between baseline and 4-year follow-up ResultsAlcohol initiators (n=182, median[IQR] age, 10.3[9.9-10.8]; 101 female[55.5%]) demonstrated lower network segregation (modularity: area-under-the-curve[AUC] difference[95%CI]=-0.017[-0.017,-0.007], p=0.030; clustering coefficient: AUC[95%CI]=-0.026[-0.027,-0.012], p=0.0495) and higher network integration (characteristic path length: AUC[95%CI]=-0.106[-0.099,-0.046], p=0.020; global efficiency: AUC[95%CI]=0.011[0.005,0.011], p=0.010), compared to non-initiators (n=3,696, median[IQR] age, 9.9[9.4-10.4]; 1750 female[47.4%]) when controlling for age, sex, and mean cortical thickness. Within the matched sample, only differences in network integration were preserved (characteristic path length: AUC[95%CI]=-0.044[-0.032,0.035], p=0.010; global efficiency: AUC[95%CI]=0.003[-0.003,0.003], p=0.040). There were no differences between full or matched samples when comparing cortical volume structural covariance networks. Conclusions and RelevanceDifferences in cortical thickness structural covariance network properties at ages 9-10 predicted alcohol initiation before age 15. These findings suggest cortical thickness network topology may reflect a neuroanatomical risk marker for early alcohol initiation. Key pointsO_ST_ABSQuestionC_ST_ABSDo structural covariance network properties at age 9-10 years predict alcohol initiation prior to age 15? FindingsIn this case-control study of 3,878 participants, early adolescent alcohol initiators demonstrated differences in cortical thickness network integration and segregation compared to their non-initiating peers. MeaningAlcohol-naive adolescents who initiate alcohol use early in life demonstrate differences in structural brain network organization compared to their abstinent peers, which may reflect a neuroanatomical risk marker for early alcohol use.

Pathological chronic stress is stress exceeding the organisms ability to cope physiologically, which may act as a risk factor in the onset and relapse of alcohol use disorder. Chronic- restraint stress (CRS) and ethanol intake are independently known to induce changes in brain structure and function, however, their combined effects on neurodevelopment over long periods of time remains largely unexplored. We conducted an in vivo longitudinal rat model with three main goals. 1) to determine if chronic stress increases ethanol intake; 2) to determine the effect of chronic- stress and ethanol intake in behavioral measures, brain structure, and function; and 3) to investigate the effect of sex. This observational study included Wistar rats assigned to four groups: 1) ethanol consumption (EtOH+/CRS-), 2) stress exposure (EtOH-/CRS+), 3) both ethanol and stress exposure (EtOH+/CRS+), and 4) control group (EtOH-/CRS-). Our results showed that chronic stress did not affect ethanol intake but led to reduced body weight gain, elevated corticosterone levels, and impaired recognition memory. Structural MRI revealed that both exposures produced additive brain volume changes in regions such as the olfactory bulb, orbitofrontal cortex, caudate-putamen, hippocampus, and cerebellum. Functional connectivity analysis using network-based statistics identified disrupted cortical-subcortical connections. Results found here were sex-dependent in terms of volumetric changes (higher effects on males) and functional connectivity (higher effects on females). Findings suggest sex-dependent mechanisms where both chronic- ethanol intake and stress affect brain plasticity during neurodevelopment. Understanding these region-specific vulnerabilities is crucial for addressing alcohol use disorders and stress-related neuropathology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/638122v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@27094dorg.highwire.dtl.DTLVardef@d3b8faorg.highwire.dtl.DTLVardef@155deeorg.highwire.dtl.DTLVardef@c9bb7b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Created with bioRender C_FIG

RationaleIn humans, alcohol drinking is a significant driver of violent behaviors such as assaults and homicides. While acute intoxication is known to produce heightened aggression, little is known about alcohols long-term effects. Emerging evidence, however, suggests that chronic alcohol intake can promote heightened aggression, including during abstinence and may also sensitize individuals to alcohols acute aggression-heightening effects. ObjectivesThe goal of this study was to test the effects of chronic binge-like ethanol drinking on both alcohol-involved and alcohol-uninvolved aggression in male CFW mice. We aimed to model individual differences in binge drinking and assess changes in aggression during both acute and protracted abstinence. ResultsAfter 5 weeks of Drinking in the Dark (DID), CFW mice that showed higher levels of EtOH drinking ( high drinkers, 1.33 g/kg/h) became more aggressive than low drinkers (0.45 g/kg/h) and H2O controls, as measured via frequency of attack bites during resident-intruder fighting. In the first aggressive encounter following 1 week of abstinence, animals with an alcohol drinking history initiate a fight more rapidly and with greater consistency than H2O controls. We also found that a single session of binge-like alcohol drinking acutely heightened aggression regardless of drinking history. ConclusionsThese results suggest that repeated binge-like alcohol drinking causes escalations in alcohol-uninvolved aggression during acute (in high drinkers) and protracted abstinence (in all alcohol drinkers). However, chronic alcohol intake does not appear to sensitize animals to alcohol-involved aggression. These findings support the utility of genetically heterogeneous CFW mice for modeling individual variability in alcohol-related aggression.

Free-choice paradigms such as two-bottle choice (2BC) are commonly used to characterize ethanol consumption and preference of rodent models used to study alcohol use disorder (AUD). However, these assays are limited by low temporal resolution that misses finer patterns of drinking behavior, including circadian drinking patterns that are known to vary with age and sex and are affected in AUD pathogenesis. Modern, cost-effective tools are becoming widely available that could elucidate these patterns, including open-source, Arduino-based home-cage sipper devices. We hypothesized that adaptation of these home-cage sipper devices would uncover distinct age- and sex-related differences in temporal drinking patterns. To test this hypothesis, we used the sipper devices in a continuous 2BC paradigm using water and ethanol (10%; v/v) for 14 days to measure drinking patterns of male and female adolescent (3-week), young adult (6-week), and mature adult (18-week) C57BL/6J mice. Daily grams of fluid consumption were manually recorded at the beginning of the dark cycle, while home-cage sipper devices continuously recorded the number of sips. Consistent with prior studies, females consumed more ethanol than males, and adolescent mice consumed the most out of any age group. Correlation analyses of manually recorded fluid consumption versus home-cage sipper activity revealed a statistically significant prediction of fluid consumption across all experimental groups. Sipper activity was able to capture subtle circadian differences between experimental groups, as well as distinct individual variation in drinking behavior among animals. Blood ethanol concentrations were significantly correlated with sipper data, suggesting that home-cage sipper devices can accurately determine individual timing of ethanol consumption. Overall, our studies show that augmenting the 2BC drinking paradigm with automated home-cage sipper devices can accurately measure ethanol consumption across sexes and age groups, revealing individual differences and temporal patterns of ethanol drinking behavior. Future studies utilizing these home-cage sipper devices will further dissect circadian patterns for age and sex relevant to the pathogenesis of AUD, as well as underlying molecular mechanisms for patterns in ethanol consumption. HighlightsO_LIFemale mice consume more ethanol than males in a continuous access paradigm C_LIO_LIAdolescent male and female mice consume more ethanol than young or mature adult mice C_LIO_LIAutomated home-cage sipper devices accurately measure ethanol consumption C_LIO_LIDevices reveal sex- and age-dependent differences in circadian drinking patterns C_LIO_LIDevices reveal distinct individual variation in circadian drinking patterns C_LI

BackgroundEpidemiological studies suggest heavy adolescent binge drinking is strongly associated with later development of an alcohol use disorder (AUD). Alcohol tolerance (i.e., an acquired reduction in acute alcohol responsivity) is a universally recognized symptom of AUD, but the direct contribution of adolescent binge drinking to adult alcohol tolerance is poorly understood. Methods and MaterialsTo investigate the contributions of adolescent binge ethanol exposure to lasting acquisition of acute tolerance, we used our ethanol response battery (ERB) to assess intoxication rating, hypothermia, motor coordination, and balance across cumulative ethanol doses (i.e., 0.0, 0.5, 1.0, 2.0, and 3.0 g/kg) in adult female Wistar rats following adolescent intermittent ethanol (AIE), lipopolysaccharide (LPS), and glycyrrhizic acid treatment following AIE. ResultsWe report AIE, which models human adolescent binge drinking, confers lasting alcohol tolerance across cumulative ethanol doses and blunts ethanol-induced increases in proinflammatory HMGB1 plasma levels. Adolescent LPS (1.0 mg/kg, i.p.) treatment, which mimics AIE-induced HMGB1-mediated neuroinflammation, induces adult alcohol tolerance and blunts HMGB1 release across cumulative ethanol doses on the ERB. Assessment of proinflammatory HMGB1 involvement in AIE-induced acquisition of lasting alcohol tolerance revealed that post-AIE administration of the HMGB1 inhibitor glycyrrhizic acid reversed the AIE-induced acquisition of alcohol tolerance in adulthood. ConclusionsThese data reveal that (1) adolescent binge drinking confers long-lasting low ethanol responsivity, (2) proinflammatory neuroimmune activation contributes to the development of alcohol tolerance, and (3) blockade of proinflammatory HMGB1 signaling reverses AIE-induced acquisition of alcohol tolerance in adulthood. These findings suggest a potential mechanistic target for the development of novel therapeutics for the treatment of AUD.

BackgroundSex differences in ethanol consumption have been reported in both humans and laboratory rodents, but the independent/dependent contributions of genetic and hormonal sex{square}biasing mechanisms to these phenotypes have not yet been fully explored. MethodsTo examine the contributions of sex-chromosome complement (SCC) and gonadal sex (GS) to ethanol consumption, we studied adolescent (28-32 days old) four core genotypes (FCG) mice (C57BL/6J background; FCG model allows for independent assortment of GS and SCC) using a modified drinking-in-the-dark (DID) procedure. Mice were offered concurrent access to 20%, 10% and 0% ethanol (in water) in four daily 2-hour sessions. Consumption at the level of individual bouts was recorded. ResultsAlthough all four genotype groups preferred the 20% ethanol over 10% and 0%, and showed similar consumption of the 10% and 0% solutions, the group rankings for consumption of the 20% ethanol solution were XX+testes > XY+testes > XY+ovaries > XX+ovaries. Thus, an interaction was observed between SCC and GS for which the simple effect of SCC was greatest in mice with ovaries (XY > XX) and the simple effect of GS was greatest in XX mice (testes > ovaries). Moreover, these effects varied in magnitude across and within drinking sessions. The behavioral microstructure of ethanol consumption (i.e., parameterization of within-session discriminable drinking bouts) support the validity of our 3-bottle modification of the DID procedure as a model of binge-like consumption as: (1) the consumption rate of the 20% ethanol solution was ~80 g EtOH/kg/h within a bout (~12 s/bout, ~3 bouts/session), (2) most of this ethanol consumption was completed in a single bout and (3) within-session ethanol consumption was greater earlier than later, indicating "front loading." ConclusionsThese results indicate that SCC and GS interact on ethanol consumption in adolescent FCG mice on a C57BL/6J background to affect binge-like consumption from the very initiation of access and that these effects are dynamic as they varied both across and within sessions. HighlightsO_LIGonadal sex and sex-chromosome complement interact on ethanol consumption in adolescent four core genotypes mice C_LIO_LIIn adolescent four core genotypes mice, mice with testes drink more ethanol than mice with ovaries, particularly in the presence of an XX karyotype C_LIO_LIIn adolescent four core genotypes mice, XY mice drink more ethanol than XX mice, but only in mice with ovaries C_LIO_LIThe effects of sex-biasing biological factors on the patterns of ethanol consumption by adolescent four core genotypes mice that we observed in our 3-bottle Drinking-in-the-Dark procedure showed face validity with some of the sex/gender differences observed in human adolescents C_LI