Alcohol, Clinical and Experimental Research

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.

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.

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.

Excessive alcohol drinking is a leading cause of preventable death in the United States. High alcohol consumption and persistent drinking despite adverse events, also known as compulsive drinking, are key criteria that contribute to the development and progression of alcohol use disorder (AUD). There is a clear need to better understand the mechanisms that support these related but distinct behaviors. The serotonin (5-HT) system has been associated with alcohol consumption and risk of alcohol dependence, however given the complexity of this system, there remains much to discover regarding specific alcohol related phenotypes. The current study uses a combination of volitional home-cage drinking and operant conditioning to phenotype mice based on ethanol intake and persistence of alcohol drinking following quinine adulteration, a model to study compulsive drinking. Brain tissue of 10 regions known to be implicated in regulating executive function, reward, and stress was collected, and gene expression of serotonergic receptors, transporters, and enzymes was quantified. Three opioid receptors were included given their well-established roles in alcohol-related behaviors and interactions with the 5HT system. Region-specific gene expression patterns emerged, with serotonergic and opioid receptor expression differentially associated with alcohol drinking phenotype. 5-HT and opioid receptors displayed opposing directionality across regions, consistent with functional heterogeneity within the system. These findings identify region-specific molecular alterations following chronic alcohol that may contribute to individual differences in alcohol drinking phenotypes, highlighting candidate targets for biomarkers of increased alcohol use disorder susceptibility or as interventions aimed at preventing the progression of AUD.

Both prenatal alcohol exposure (PAE) and adolescent alcohol exposure (AAE) persistently impair executive function in humans and animal models. Executive function encompasses multiple interrelated domains including working memory, inhibitory control, and behavioral flexibility. We hypothesized that a developmental "double hit" of PAE and AAE would produce more severe behavioral deficits associated with these executive domains compared to alcohol-naive and single-exposed animals. We tested this hypothesis in rats by assessing disinhibition (low-light elevated plus maze; LL-EPM), behavioral flexibility (attentional set shift test; ASST), and working memory (spontaneous alternations in a T-maze); we also tested behavioral flexibility (ASST) in mice. Pregnant Sprague Dawley rats received water or 5 g/kg alcohol from gestational day (GD)13.5-GD20.5, and offspring received water or 5 g/kg alcohol on a 2-day-on, 2-day-off paradigm from postnatal day (PD)25 to PD54. Pregnant C57BL/6J mice received water or 4.5 g/kg alcohol from GD13.5-GD17.5, and offspring received water or 4.5 g/kg alcohol on a 2-day-on, 2-day-off paradigm from PD25 to PD42. Offspring underwent behavioral testing in young adulthood. Double hit rats showed more exploration in the LL-EPM than controls and fewer alternations in the T-maze than AAE-only rats, suggesting deficits in disinhibition and spatial working memory, respectively. Double hit rats and mice exhibited more errors and/or more trials to criterion in the ASST, indicative of decreased behavioral flexibility. Overall, double hit animals showed altered performance on tests related to executive function, suggesting that the combined exposure alters executive function in a manner distinct from single-exposure models.

Background: Alcohol use disorder is associated with dysregulated glutamatergic signaling within mesocorticolimbic circuits that govern reinforcement and excessive ethanol intake. Group II metabotropic glutamate receptors (mGlu2/3) act primarily as presynaptic autoreceptors that regulate glutamate release. However, how voluntary alcohol intake alters mGlu2/3 expression within reward circuitry remains unclear. Methods and Results: We examined the effects of operant alcohol self-administration on mGlu2/3 protein expression and assessed the functional impact of group II receptor modulation on binge-like ethanol intake. Male C57BL/6J mice self-administered sweetened ethanol or sucrose under behaviorally matched conditions for 35 days. Immediately after the final session, tissue punches from the nucleus accumbens (NAc), amygdala, and prefrontal cortex were collected for Western blot analysis. Operant ethanol self-administration selectively reduced mGlu2/3 protein expression in the NAc, with no changes detected in the amygdala or prefrontal cortex. Both monomeric and dimeric mGlu2/3 protein levels were reduced, and a composite index revealed coordinated downregulation of receptor expression. In separate cohorts, systemic administration of the mGlu2/3 agonist LY379268 dose-dependently reduced binge-like ethanol intake in a limited-access home-cage drinking model, whereas positive allosteric modulation of mGlu2 receptors with LY487379 was ineffective. Conclusions: These results show that low-dose operant ethanol self-administration produces an ethanol- and region-specific reduction of mGlu2/3 protein expression in the NAc and that pharmacological activation of group II receptors, potentially involving mGlu3-specific receptors, is sufficient to suppress binge-like ethanol consumption. These data identify presynaptic mGlu2/3 dysregulation as a mechanism contributing to ethanol-related behaviors and support group II metabotropic glutamate receptors as therapeutic targets for alcohol use disorder.

BackgroundThe prevalences of suicidal ideation (SI) and suicide attempt (SA) are influenced by genetic, behavioral, and environmental factors. Alcohol use disorder (AUD) and adverse childhood experiences (ACEs) may mediate or moderate genetic liability for suicidality. MethodsUsing data from 10,275 participants (43.8% female; 47.2% African-like genetic ancestry [AFR], 52.8% European-like genetic ancestry [EUR]), we tested whether polygenic scores (PGS) for SI and SA predicted lifetime SI or SA. We also evaluated whether alcohol use disorder (AUD) mediated these associations and whether adverse childhood experiences (ACEs) moderated the direct and indirect pathways. ResultsAlthough there were significant direct associations of the SA PGS with SA (AFR: b = 0.36, SE = 0.01; EUR: b = 0.17, SE = 0.01; both ps < 2e-16), the SI PGS did not predict SI (p > 0.55). AUD mediated SA genetic risk (average causal mediation effect (ACME): AFR = 0.01, 95% CI [0.01-0.01]; EUR = 0.02, 95% CI [0.01-0.02]; both ps < 2e-16). Moderation analyses indicated that indirect effects were attenuated by ACEs score ({Delta}ACME: AFR = 0.02, p < 2e-16; EUR = 0.01, p = 0.03). There was neither mediation nor moderated mediation for SI. ConclusionsGenetic liability to SA operates partly through AUD, particularly among individuals with lower childhood adversity. Under higher adversity, alternative pathways to SA likely predominate. These findings highlight the need to consider distinct etiological pathways to the development of suicidality and the relevance of AUD as a modifiable target for suicide prevention among individuals at high genetic liability.

Neuroimmune signaling is increased in postmortem brain tissue from individuals with alcohol use disorder (AUD), and growing evidence suggests that it contributes to persistent alcohol-related neuroadaptations. Interferon regulatory factor 7 (IRF7), a transcription factor downstream of endosomal Toll-like receptor signaling, is induced in alcohol-relevant brain regions and may contribute to escalated drinking. Here, we tested whether chronic intermittent ethanol (CIE) vapor exposure engages IRF7 signaling during subsequent alcohol self-administration and whether this is associated with altered molecular E/I balance in the aIC and altered functional E/I balance in aICnucleus accumbens projection neurons. Female Wistar rats (n=30) were trained to self-administer alcohol (15% v/v; FR2 vs inactive lever) during 30-minute sessions. After establishing baseline drinking, rats underwent 1-3 cycles of CIE, which increased alcohol self-administration at the 72 h post vapor test. This increase positively correlated with IRF7 levels in the anterior insular cortex (aIC) and nucleus accumbens, while molecular, and immunofluorescence showed that CIE shifted aIC excitatory/inhibitory (E/I) balance toward reduced excitation. Electrophysiological recordings further showed reduced functional E/I balance in aIC neurons projecting to the nucleus accumbens. Knockdown of IRF7 in the aIC attenuated CIE induced escalation of alcohol self-administration, supporting a role for insular IRF7 signaling in alcohol related neuroadaptations that promote escalated drinking.

BackgroundPrenatal stress (PNS) is a well-established risk factor for neuropsychiatric vulnerability, yet its sex-specific behavioral consequences remain incompletely defined. Because males and females follow distinct neurodevelopmental trajectories, clarifying how early-life stress differentially shapes behavior is essential for developing targeted interventions. However, few preclinical studies directly compare male and female offspring within the same experimental framework, limiting the ability to identify true sex-dependent effects. MethodsUsing a validated mouse model of gestational restraint stress, we conducted a comprehensive, within-study assessment of sex-dependent behavioral outcomes in adult offspring. Behavioral domains included locomotor activity, anxiety-like behavior, sociability, fear learning and extinction, recognition memory, and alcohol-related responses (ethanol preference and behavioral sensitivity), all measured using identical paradigms across sexes. ResultsPNS broadly disrupted behavior and cognition in both sexes, increasing locomotor activity and anxiety-like behavior, impairing fear extinction and recognition memory, and altering behavioral sensitivity to ethanols sedative effects. Direct comparison revealed distinct sex-dependent vulnerabilities: males showed reduced social interaction, whereas females exhibited numerically greater impairment in fear extinction and a significantly stronger ethanol preference. Baseline fear responses, total fluid intake, and sucrose consumption were unaffected. ConclusionPrenatal stress programs neurobehavioral trajectories in a sex-dependent manner, conferring vulnerability to anxiety-related behavior, cognitive disruption, and alcohol use. By directly comparing males and females within the same experimental design, this study provides one of the most integrated evaluations of sex-specific PNS outcomes to date and offers a robust framework for investigating the biological mechanisms underlying divergent pathways to stress-related psychopathology.

Prenatal alcohol exposure (PAE) disrupts embryonic development and gives rise to a variable fetal alcohol spectrum disorder (FASD) phenotype characterized by neurodevelopmental and dysmorphological defects. We investigated the effects of PAE on placental gene regulation by performing genome-wide DNA methylation (DNAm) microarray and gene expression (mRNA sequencing) analyses in 87 PAE, 77 unexposed control, and 11 smoking-exposed-only placentas. Significant alterations were identified in genes involved in synaptic function including both excitatory and inhibitory neurotransmission, and in genes previously associated with attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder, schizophrenia, and addiction. When placental molecular alterations were compared with neuropsychological and dysmorphological phenotypes of the same children assessed at six years of age, numerous correlations were observed between DNAm and gene expression, as well as head circumference, cognitive performances, ADHD traits, and dysmorphology. These included associations between DAOA methylation and verbal intellectual abilities and language development, GPHN methylation and working memory index, and GLI3 expression and both working memory index and midfacial hypoplasia. As these alterations were observed in the placenta, this tissue not only enables the identification of phenotype-specific FASD candidate genes but also represents a valuable tool for studying complex developmental disorders in human.

Epitranscriptomic mechanisms dynamically regulate neuronal function through gene expression, but their precise roles in neuropsychiatric and neurological disorders remain to be fully elucidated. A major obstacle to advancing such studies is the absence of a methodology for precise, cell-type and brain-region-specific quantification of critical epitranscriptomic regulators under these complex brain conditions. To overcome this challenge, we developed a super-resolved, three-dimensional spatial transcriptomics method to quantify key epitranscriptomic switches in intact brains. Using this method, we quantified the expression of Mettl3, an N6-methyladenosine (m6A) methyltransferase enzyme recently shown to be upregulated in the amygdala after adolescent intermittent ethanol (AIE) exposure in rats. We observed a significant increase in cytoplasmic Mettl3 mRNA in neurons, but not in astrocytes or microglia, within the adult central amygdala and the CA1 and dentate gyrus of hippocampus following AIE. In contrast, no significant changes were observed across neurons, astrocytes, or microglia within the basolateral amygdala or the hippocampal CA3. Additionally, we found both the cytoplasmic density and subcellular localization of Mettl3 mRNA were dependent on the specific cell types and brain subregions examined. These results suggest that AIE increases Mettl3 expression in a highly cell-type-specific and spatially heterogeneous manner, underscoring the necessity of high-resolution spatial transcriptomics methods for studying transcriptomic and epitranscriptomic regulations under neurological conditions. Significance StatementEpitranscriptomics plays a crucial role in neuronal functions by influencing the splicing, stability, and translation of genes. However, the exact role of epitranscriptomic mechanisms, such as m6A RNA methylation, in brain disorders remains unclear, particularly in a cell-type and circuitry-specific manner. Here we developed a super-resolved, three-dimensional spatial transcriptomics method and applied it to a model of alcohol exposure. We found differential cell-type- and brain-region-specific modulation of Mettl3, a key m6A enzymatic switch, across major brain regions following adolescent intermittent ethanol exposure in adulthood. Our findings, coupled with our pipeline, are expected to address existing methodological limitations and knowledge gaps, thereby accelerating brain transcriptomic and epitranscriptomic studies under various psychiatric and neurological conditions.

Alternative polyadenylation (APA) is a common posttranscriptional mechanism to regulate gene expression. APA generates mRNAs with varying lengths of 3' UTRs or transcripts that encode distinct protein carboxy-terminal ends. APA is especially important in neurons, where different mRNA variants are often asymmetrically localized to dendrites and axons, and can be locally translated into proteins. Local protein synthesis is crucial for axon guidance, synaptic plasticity, and learning and memory, key processes associated with the development of alcohol use disorder (AUD). We investigated the role of APA in AUD using a mouse model of alcohol dependence characterized by increased voluntary drinking after chronic intermittent ethanol (CIE) exposure. We examined APA during protracted withdrawal from alcohol in three brain regions of male and female mice. Our analyses revealed hundreds of genes undergoing APA in males, but substantially fewer in females, suggesting sex-specific effects of CIE on APA. Notably, male and female mice displayed distinct APA signatures. APA genes were different from differentially expressed genes (DEGs), suggesting that these molecular processes are regulated independently. We also determined that the expression of APA genes was associated with neurons, while DEGs were associated with non-neuronal cells. Many of the APA genes were involved in synaptic integrity, neuroplasticity, and neuronal maintenance, which was consistent with their enrichment in neurons. Our study suggests that APA is a crucial sex- and cell type-specific mechanism in AUD with the potential to influence localized neuronal protein expression during protracted withdrawal and to modify alcohol consumption behavior. HIGHLIGHTSO_LIChronic ethanol exposure in mice results in profound changes of APA genes in brain. C_LIO_LICommonly regulated cleavage and polyadenylation sites and genes were identified in male but not in female mice. C_LIO_LIThere was a minimal overlap between APA and differentially expressed genes (DEGs). C_LIO_LIAPA genes were primarily associated with neurons, whereas DEGs were associated with non-neuronal cells. C_LI

ImportancePolysubstance use is common, but substance use associations with neuroimaging measures have largely been investigated within individual drug types. Whether effects are substance-specific or -general, and how predispositional risk and exposure contribute, remains unclear. ObjectiveIdentify shared and unique associations between substance use and brain structure, and characterize the contributions of predispositional risk and environmental exposure, in a large sample of young adults in the US. DesignThis cross-sectional family-based study used data from the Human Connectome Project (2017 release, collected from 2012-2015). SettingData were collected at Washington University in St. Louis, MO, USA. ParticipantsTwins, non-twin siblings, and singletons with magnetic resonance imaging (MRI) and substance use self-report were included in the analysis. Data were analyzed in 2025. ExposureHistory of substance use was assessed using the Semi-Structured Assessment for the Genetics of Alcoholism. Variables included lifetime use, heavy or past-year hazardous use, and age of use onset for alcohol, marijuana, tobacco, and illicit drugs. Additionally, alcohol and marijuana dependence were assessed. Main Outcomes and MeasuresLinear mixed-effect models examined associations between substance use and brain structure, with an initial focus on past-year hazardous alcohol use, as 95% of the sample endorsed lifetime alcohol use. Analyses then tested associations with other substance use variables, and whether effects were shared or substance-specific. Between-family, within-family, and genetic variance component analyses tested risk and exposure effects. Results1,113 participants (N = 445 families; ages 22 - 37; M=28.8, SD=3.7) had no missing data for the primary analyses. Hazardous alcohol use was negatively associated with global brain thickness ({beta} = -0.12, p < 0.001), which explained all other regional and global associations. Of the drugs with a shared-effect on global brain thickness, only lifetime marijuana use explained unique variance over alcohol ({beta} = -0.08, p = 0.013). Within-family analyses found evidence for unique putative exposure effects of both alcohol ({beta} = -0.11, p < 0.001) and marijuana use ({beta} = -0.07, p = 0.002) on global thickness. Marijuana use further showed a predispositional effect, both in between-family comparisons ({beta} = -0.11, p = 0.007) and genetic variance component analyses ({rho}G = -0.2, p = 0.004), which were not explained by alcohol use. Conclusions and RelevanceBrain structural associations with substance use reflect substance-general and -specific effects, as well as a combination of predispositional and exposure effects. Findings suggest that the negative consequences of polysubstance use may reflect the additive effects of multiple unique exposures.

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Studies have shown that ethanol altered expression of an astrocyte marker, i.e., glial fibrillary acidic protein (GFAP), in two key corticolimbic regions, the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These regions comprise anatomically and functionally different subregions, i.e., the prelimbic (PL) and infralimbic (IL) cortex of the mPFC, the shell and core subregions of the NAc. However, ethanol effects on GFAP expression within these subregions remain largely unknown. In addition, effects of pharmacological manipulation of astrocytes on alcohol drinking have been understudied. Western blot was conducted to determine GFAP expression in subregions of the mPFC and NAc after chronic ethanol drinking. Fluorocitrate, an astrocyte-specific metabolic inhibitor, was administered to inhibit astrocytes and was tested on ethanol drinking. Ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell. Intra-ventricular administration of fluorocitrate reduced ethanol intake and preference, but increased water consumption during choice ethanol drinking. In addition, fluorocitrate did not affect total fluid consumption or basal locomotor activity. These results indicate that chronic ethanol drinking induced GFAP elevation in a subregion-specific manner within the mPFC and NAc, and that metabolic inhibition of astrocytes selectively attenuated ethanol drinking without non-specific effects on water drinking or general activity. Together, these results suggest that astrocytes may play an important role in ethanol drinking. HighlightsO_LIEthanol drinking enhanced GFAP levels in the PL cortex and NAc core. C_LIO_LIFluorocitrate inhibition of astrocytes reduced intermittent ethanol drinking. C_LIO_LIFluorocitrate did not alter total fluid consumption or basal locomotor activity. C_LI

BackgroundGrowing evidence suggests regional and network-level brain imaging features in late childhood are predictive of alcohol use in adolescence. However, the directionality of these effects (i.e. whether they reflect accelerated or delayed neuromaturation) are mixed. We applied a Brain Age Gap Estimation (BrainAGE) model to examine whether overall deviations from typical brain aging trajectories are predictive of (1) alcohol initiation and (2) use behaviour (experimentation versus binge drinking) in adolescence. MethodsData from the Adolescent Brain Cognitive Development study release 6.0 were used. Baseline (ages 9-11) structural imaging features (cortical volume, area, and subcortical volume) were used to estimate BrainAGE. Alcohol use was determined using self-report data from the Substance Use Interview and Timeline Follow-Back across follow-ups (waves 1-6; ages 10-17). Logistic generalized mixed effects models examined whether BrainAGE predicted group status between (1) non-initiators (n=3,639) and initiators (n=1,176), and; (2) experimentation (at least one full drink, no binge episodes; n=461) and binge drinking (at least one episode; n=438). ResultsWhen adjusting for age, sex, and pubertal status, a one-standard-deviation decrease in BrainAGE (equivalent to 1.64 years) at baseline was associated with a 9.5% increase in odds of alcohol initiation in adolescence. However, this effect did not survive adjustment for sociodemographic and prior alcohol exposure covariates. Further, BrainAGE did not discriminate between experimentation and binge drinking. ConclusionsFindings suggest BrainAGE in late childhood may reflect potential risk for alcohol initiation, but not behaviours, in adolescence. However, this association likely reflects complex interactions between brain structure and contextual factors, warranting further investigation.

Astrocytes play essential roles in maintaining brain homeostasis and in contributing to synaptic functions, but, in response to injury, infection, or disease, astrocytes can downregulate their homeostatic and physiological functions while increasing neuroinflammatory responses. The central amygdala (CeA) is important for stress responsivity and the development of alcohol (ethanol) dependence. Using a multi-omics approach in Aldh1l1-EGFP/Rpl10a mice and the chronic intermittent ethanol two-bottle choice (CIE-2BC) model, we have characterized the translational response of CeA astrocytes, as well as the proteomic and phosphoproteomic changes in ethanol dependent, non-dependent, and naive mice. We identified astrocyte-specific alterations in neuroimmune functions and antioxidant/oxidative stress pathways in ethanol dependent mice as well as cytoskeletal plasticity related pathways in non-dependent mice. Proteomic analysis showed down-regulation of astrocyte physiological functions in dependent animals while phosphoproteomic analysis identified pathways associated with cytoskeleton remodeling in both dependent and non-dependent mice. Reconstructions of astrocyte morphologies demonstrated increased CeA astrocyte complexity in dependent and non-dependent groups compared to naive mice. The astrocyte-specific activation of neuroimmune and antioxidant pathways, down-regulation of homeostatic functions, alteration in protein phosphorylation-mediated cytoskeleton remodeling, and increased astrocyte morphological complexity demonstrate that ethanol dependence induces astrocyte reactivity in the CeA consistent with both adaptive and maladaptive changes. These findings highlight the role of CeA astrocytes in the progression from alcohol intake to dependence and represent a first step toward identifying astrocyte-specific therapeutic strategies to treat Alcohol Use Disorder (AUD) aimed at potentiating reactive astrocyte adaptive changes and inhibiting maladaptive responses.

Alcohol use disorder is a major public health concern worldwide and there is a high comorbidity with psychiatric disorders. The basolateral amygdala (BLA) has been implicated in both mood and alcohol use disorders; however, the mechanisms contributing to the shared pathophysiology remain unknown. Extensive evidence indicates that ethanol modulates GABAergic signaling in the BLA, including actions on neurosteroid-sensitive, extrasynaptic {delta} subunit-containing GABAA receptors (GABAARs), which has been suggested to mediate many of the behavioral effects. In fact, several studies have suggested that 5-reduced neurosteroids, such as allopregnanolone, may mediate some of the behavioral effects of alcohol. Here we demonstrate that chronic intermittent ethanol (CIE) exposure impairs endogenous neurosteroidogenesis via downregulation of key neurosteroidogenic enzymes, 5-reductase type 1 and type 2. To examine the impact of impaired endogenous neurosteroidogenesis of the behavioral consequences of chronic alcohol exposure, including withdrawal-induced anxiety and increased alcohol consumption, we used CRISPR/Cas9 mediated knockdown of 5-reductase in the BLA. Reduced expression of 5-reductase in the BLA did not impact post-CIE alcohol intake or anxiety-like behaviors during withdrawal, perhaps because endogenous neurosteroidogenesis is already impaired following CIE. Therefore, we examined the impact of enhancing neurosteroid levels, treating mice post-CIE with SGE-516, a synthetic GABAAR positive allosteric modulator, which increased voluntary alcohol intake. These findings implicate endogenous neurosteroidogenesis in behavioral outcomes associated with withdrawal from chronic alcohol exposure. Further, this study suggests that targeting endogenous neurosteroidogenesis may be a novel and useful therapeutic target.

Stress is a major trigger of relapse in alcohol use disorder (AUD), and dysfunction of prefrontal cortex (PFC) circuits has been implicated in this process. Epigenetic regulators may contribute to relapse by shaping transcriptional programs within these circuits. Here, we investigated the role of the histone methyltransferase PRDM2 in stress-induced alcohol seeking. Analysis of postmortem human tissue showed that PRDM2 expression in the PFC was reduced in both men and women with AUD compared with control individuals. To examine the functional significance of this reduction, we used viral-mediated knockdown of Prdm2 in the dorsomedial prefrontal cortex (dmPFC) of male and female rats. Prdm2 knockdown enhanced stress-induced reinstatement of alcohol seeking in both sexes, particularly at intermediate shock intensity, without altering pain sensitivity or being influenced by estrous cycle stage. To determine whether this effect was mediated through specific prefrontal output pathways, we selectively reduced Prdm2 expression in dmPFC neurons projecting to the nucleus accumbens (NAc). Projection-specific knockdown also increased stress-induced reinstatement of alcohol seeking in male and female rats in an intensity-dependent manner. Together, these findings suggest that reduced PRDM2 expression in the PFC may contribute to stress-induced relapse-like behavior and identify the dmPFC-NAc projection as a circuit through which PRDM2 can influence alcohol seeking.

Acute stress activates the immune system, leading to the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6). Chronic alcohol consumption alters the physiological stress systems and is associated with increased chronic inflammation. However, it remains unclear how IL-6 responds to acute stress in individuals with alcohol use disorder (AUD). Forty patients with AUD during early abstinence and 37 healthy controls (HC) completed two study visits. On one day, an acute stress induction task was performed, and on the other, a non-stressful control task, with the order of tasks being balanced. Plasma IL-6 and C-reactive protein (CRP) were measured as inflammatory markers at baseline and changes in IL-6 were assessed 90 minutes after the experimental manipulation. Patients with AUD showed significantly elevated baseline IL-6 and CRP compared to HC. In HC, inflammatory parameters were positively correlated with age and BMI, whereas in patients with AUD, they were correlated with the amount of consumed alcohol. IL-6 responses to the stress intervention did not differ between groups. Increases in IL-6 were observed on stress and control days and were larger when samples were collected via an indwelling catheter than with a butterfly needle. These findings suggest that heavy chronic alcohol use may mask the typical associations between inflammatory markers and physiological factors. However, IL-6 responses to acute stress do not differ between AUD and HC, despite increased baseline inflammation. Furthermore, the results indicate that blood collection methods can influence IL-6 measurements and highlight the importance of methodological considerations.

Alcohol use disorder (AUD) is a chronic relapsing condition with limited pharmacological treatments. Ethanol modulates neuronal excitability in part through activation of G-protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, which dampen neuronal activity in reward- and stress-related circuits implicated in AUD pathophysiology. In this study, we investigated the therapeutic potential of targeting activation of GIRK channels in mouse models of ethanol intoxication. GiGA1 (G protein-independent GIRK activator type 1) is a selective activator of GIRK1/GIRK2 channels and has good brain bioavailability. Systemic GiGA1 administration prevented acquisition of ethanol-induced conditioned place preference (CPP) in both male and female mice. GiGA1 also significantly reduced voluntary ethanol intake and decreased blood alcohol concentrations, when administered to mice after they developed high preference and consumption of ethanol. Similarly, Baclofen, a GABAB receptor agonist that leads to activation of GIRK channels also decreased ethanol consumption. However, systemic Baclofen did not prevent acquisition of ethanol-dependent CPP, suggesting a broader efficacy of direct GIRK1/GIRK2 activation by GiGA1. Whole-brain c-Fos mapping as a proxy for neuronal activity revealed that GiGA1 blunted ethanol-induced neuronal activation in several AUD-relevant brain regions, including the central amygdala, paraventricular thalamus, paraventricular hypothalamus, and Edinger-Westphal nucleus. These findings demonstrate that pharmacological activation of GIRK channels modulates key neural circuits involved in ethanol reward and intake, supporting GiGA1 as a promising lead compound for targeted AUD therapy.