Alcohol

Understanding the neurobiological systems that regulate alcohol cue-induced craving is of utmost importance for the development of novel intervention strategies for alcohol use disorders (AUDs). However, although a human experimenter is required to conduct alcohol self-administration studies in the lab, the cues associated with the experimenter are seldom if ever factored into the experimental design. Thus, although we have learned much to date about alcohol cue-induced behavior and neurobiology, and in particular about discrete cues presented many times throughout a single daily alcohol self-administration session, we know relatively little about how responses to alcohol availability cues might predict subsequent alcohol consumption. For the current experiment, mice were exposed daily to auditory cues that preceded 2 hours of alcohol or water access using drinking-in-the-dark (DID) methods. An additional control group experienced cues but were not otherwise manipulated. Importantly, cues were initiated remotely from outside the animal facility, avoiding the experimenter being the first cue predicting ethanol availability. Head direction, location in the home cage, and movement velocity were the primary variables on interest. Surprisingly, during the cue period, there were no significant differences between groups in any of these measures, despite meaningful alterations over days. However, we observed many significant correlations between behaviors and drinking variables. First, we observed significant positive associations between ambulatory velocity during cues and subsequent total alcohol (R2=0.14; p<0.0001) and total water (R2=0.12; p=0.0002) consumption, but only in females. We also observed a significant positive relationship (R2=0.25; p<0.0001) between the amount of time oriented toward the sipper port during the auditory cues and the average rate of subsequent alcohol consumption (i.e. front-loading), but only in females. In males, head direction was found to be positively associated with subsequent total water consumption (R2=-0.21; p<0.0001), but not alcohol (R2=-0.01; p=0.2267). We also observed a significant negative relationship (R2=-0.15; p<0.0001) between proximity to the sipper during the cue period and subsequent total 2-hour alcohol intake in males. Although these associations were modest in strength, they suggest potential sex-specific behavioral predictors of alcohol consumption that are regulated by different neural dynamics.

Despite growing evidence that neuroimmune dysfunction contributes to Alcohol Use Disorder (AUD) pathology, the underlying neuroinflammatory mechanisms that may promote alcohol consumption are not as clear. We recently report that specific knockdown of interferon regulatory factor 7 (IRF7) in the anterior insula (aIC) mitigates escalation in ethanol self-administration in rats. In addition, we find pro-inflammatory activation of microglia contributes to other AUD-related behavioral impairments. Here, we sought to determine if pro-inflammatory activation of microglia from ethanol contributes to elevations in IRF7 and ethanol self-administration in rats. Male Wistar rats were trained under our ethanol self-administration paradigm (15% v/v; FR2 vs inactive lever) followed by 1-4 cycles of chronic intermittent ethanol exposure (CIE). To inhibit microglia, rats were treated with minocycline (30mg/kg, i.p.) before and after each ethanol vapor session. Escalation in self-administration and biochemical markers were assessed 72 hours into abstinence. We found CIE increased ethanol self-administration, which was positively correlated with aIC IRF7 levels. Minocycline treatment blunted IRF7 expression and alleviated ethanol self-administration following CIE. These data suggest a role for microglia in driving both IRF7 levels and escalation in ethanol self-administration in early abstinence.

Alcohol use disorder (AUD) is a widespread psychiatric condition, yet the molecular mechanisms underlying its development remain poorly understood. While prior studies have largely focused on protein-coding genes, long non-coding RNAs (lncRNAs) remain underexplored in AUD. Malat1, a highly abundant and evolutionarily conserved lncRNA, is elevated in post-mortem brain tissue of human AUD subjects and rodents chronically exposed to ethanol; however, its causal contribution to AUD-relevant behaviors remains unknown. Using CRISPR/Cas9 genome editing, we generated two complementary global Malat1 knockout models to assess its role in alcohol intake and related phenotypes. Constitutive knockout selectively attenuated acute functional tolerance rate and every-other-day two-bottle-choice alcohol intake in females. These results were supported by an inducible adult conditional global knockout model, which reduced ethanol consumption in females without altering taste preference. Together, our findings provide the first causal evidence that Malat1 regulates alcohol consumption in a sex-specific manner, supporting further investigation into its underlying mechanisms in AUD.

It is becoming increasingly clear that chronic exposure to lower levels of ethanol impact learning and behavior. To determine the impact of chronic low-dose ethanol exposure on sensitivity to changes in stimulus value, a conditioned taste aversion procedure was used. Adult male and female mice underwent a sucrose two bottle-choice drinking paradigm. Each day, mice received an injection of either low-dose ethanol (0.5g/kg) or saline two hours after sucrose access for 20 days. This was followed by a lithium chloride (LiCl)-induced conditioned taste aversion (CTA) paradigm in which 0.15M LiCl or vehicle injection was administered immediately after sucrose consumption for three days. On the fourth day, changes in sucrose consumption were analyzed. Chronic exposure to low-dose ethanol did not affect sucrose consumption in either female of male mice during two-bottle choice. In female mice, a history of chronic low-dose ethanol exposure blocked the development of LiCl-induced CTA. A history of chronic low-dose ethanol did not impact LiCl-induced CTA in male mice as both ethanol-naive and -exposed male mice who underwent LiCl pairing reduced sucrose consumption. This suggests that low-dose ethanol alters aversion-related learning in female mice which may have implication for development of aberrant behavior and risk for alcohol use disorder (AUD).

BackgroundAlcohol use disorder is characterized by continued alcohol use despite negative consequences, also known as aversion-resistant drinking. Alcohol related cues can invigorate motivation to seek and consume alcohol. It is currently unknown whether alcohol related cues can invigorate drinking despite negative consequences. Materials and MethodsLong-Evans rats were trained in a discriminative stimulus (DS) task with cues predicting the available of alcohol reward. They were then tested in the task for aversion-resistant drinking by measuring consumption of alcohol adulterated with increasing concentrations of quinine. As a control for an environment free of reward-related cues, rats were also tested for aversion-resistant drinking in the home cage. ResultsWe found that rats displayed robust aversion-resistant drinking in the DS task. When we compared alcohol consumption in the task with home cage consumption, we found that rats were more aversion-resistant in the task than in the home cage. We also found that individual differences in aversion-resistant drinking were correlated within behavioral context (i.e. home cage or DS task) but not between the home cage and the DS task. ConclusionsWe found that aversion-resistant drinking is invigorated during cue-induced alcohol seeking relative to free drinking without explicit cues. Home cage quinine-sensitivity is unrelated to quinine-sensitivity in the presence of cues. This suggests that cues motivate drinking despite negative consequences in a way that is unique from aversion-resistance driven by drinking history. While many behavioral measures use cues and ultimately test aversion-resistant drinking, this is the first explicit test of cue-evoked aversion-resistant drinking.

Binge alcohol drinking is a public health concern that can dramatically increase the risk for development of alcohol use disorder (AUD). Continued alcohol drinking in the face of negative consequences is another key feature of AUD. A better understanding of the neural circuitry that regulates these behaviors could provide insight as to novel treatments for AUD. Serotonin is a neurotransmitter that has been implicated in alcohol consumption in both human studies and animal models. The orbitofrontal cortex (OFC) is a brain region that both receives serotonergic input from the dorsal raphe and has been implicated in AUD. However, how volitional alcohol consumption impacts serotonin signaling within the OFC and how this contributes to alcohol related behaviors is unknown. Here, we show that a history of alcohol consumption alters the ability of 5-HT to hyperpolarize OFC pyramidal neurons in mice and monkeys. Consistent with this, a history of binge alcohol consumption decreases the expression of the 5-HT1A but not 5-HT2A receptor in the OFC from mice. Next, we show that deletion of the 5-HT1A receptor from the OFC increased alcohol intake and preference in male, but not female mice. Finally, we found that 5-HT1A receptor deletion led to increased quinine-adulterated alcohol intake, a measure of aversion-resistant drinking, in both male and female mice. Altogether, we identified serotonin signaling in the OFC as key target for modulation of binge and compulsive alcohol consumption.

Dysregulation of the dynorphin (DYN) / kappa-opioid receptor (KOR) system is heavily implicated in symptoms of alcohol use disorder (AUD) including negative affective-like states that can drive maladaptive behavioral regulation. Substantial efforts have been made towards understanding the neurobiology of DYN / KOR dysregulation; however, the role of dynorphinergic islands of Calleja within the ventral striatum remain poorly understood. Presently, adult male Wistar rats were trained to self-administer 10% alcohol, exposed to either air or alcohol vapor for eight weeks, and alcohol self-administration and 22-kHz ultrasonic vocalizations (USVs) assessed during acute withdrawal. Subsequently, brains were extracted during acute withdrawal and DYN A-like immunoreactivity was measured in the ventral striatum. Alcohol vapor-exposed rats demonstrated increased alcohol consumption and 22-kHz USVs compared to air-exposed controls. Vapor-exposed rats additionally demonstrated increased DYN A-like immunoreactivity in the islands of Calleja. Moreover, the average DYN A neuron size positively correlated with the number of 22-kHz USVs in vapor exposed animals, but not in air-exposed controls. The present findings identify the islands of Calleja as a novel DYN-associated region that may be recruited during alcohol dependence with enhanced DYN plasticity in the islands of Calleja contributing to affective dysregulation in AUD and other neuropsychiatric conditions.

Alcohol misuse leads to a range of health complications and induces various metabolic perturbations that impacts multiple physiological systems, including the cardiovascular system, liver, and gut microbiota. However, limited research has been reported on these metabolic profile changes, particularly using models of alcohol dependence such as after chronic intermittent ethanol (CIE) vapor exposure. This study investigated CIE-induced metabolomic alterations of CIE were investigated using fecal, liver, and serum samples of adult male and female C57BL/6J mice following 72 hr withdrawal. Significant metabolite changes were observed in both fecal and liver extracts and these changes were sex-specific. Both liver and fecal metabolites had systematic changes, while blood serum influences were limited after CIE. Female fecal samples showed higher metabolite perturbations than male samples according to PCA studies. The female samples showed significant butyrate downregulation and acetate upregulation, which are critical microbial products as beneficial microbe cell energy sources and influence intestinal absorption in the host. In addition, the female fecal samples showed significant downregulation of branched-chain amino acids including leucine, isoleucine, and valine, while male samples showed downregulation of glucose and taurine, with upregulated phenylalanine and tyrosine. In contrast, in the liver study, phenylalanine and tyrosine were upregulated while taurine was downregulated in females. Both sexes showed downregulation of liver glycine and glucose. These data indicate that CIE induces sex-specific metabolic perturbations in the mouse liver and fecal metabolome, and have implications for guy disturbances and liver damage observed following alcohol dependence. This study provides potential targets for future examination of mechanisms and treatment approaches for alcohol dependence.

The lateral habenula (LHb) encodes aversive states and negative affect, positioning it as a candidate region for the negative reinforcement that drives alcohol withdrawal. However, little is known about how chronic ethanol exposure affects LHb neuronal function and glial biology during withdrawal. Here, we used chronic intermittent ethanol (CIE) vapor exposure, a well-established model of alcohol dependence that reliably produces somatic and affective signs of withdrawal, to examine LHb physiology and astrocytic markers during acute withdrawal in male and female rats. Whole-cell and cell-attached recordings revealed that withdrawal reduced evoked and spontaneous firing in LHb neurons, with rebound firing following a crossover pattern between males and females. Despite these excitability changes, the overall distribution of firing phenotypes was unchanged, suggesting a shift in gain rather than a reorganization of cell types. Immunofluorescence revealed increased Sox9+ and GFAP labeling in the LHb during withdrawal at the same time point when electrophysiology experiments uncovered impaired astrocytic regulation of glutamate clearance. Together, these findings reveal that withdrawal from chronic ethanol exposure produces neuronal and glial adaptations in the LHb, pointing to impaired glutamate regulation as a candidate mechanism relevant to the negative affective state of alcohol withdrawal. These findings position the LHb as a potential node linking astrocyte-neuron dynamics to withdrawal symptoms and relapse vulnerability in alcohol use disorder.

Food addiction (FA) has been proposed as a phenotype sharing features with substance use disorders. Despite increasing recognition of food addiction as a behavioral phenotype with features overlapping substance use disorders, little is known about its prevalence or clinical significance among individuals with alcohol use disorder (AUD). Objective: To examine the prevalence of FA and to evaluate demographic, psychological, and alcohol-related correlates in individuals with AUD. Design, Setting, and Participants: This cross-sectional analysis included 743 adults with AUD who were either treatment seeking (Tx) (n = 534) for AUD and were enrolled in an inpatient program at the National Institutes of Health Clinical Center or not treatment-seeking (non Tx) (n = 209). Main Outcomes and Measures: FA symptoms were assessed using the Yale Food Addiction Scale, with >=2 symptoms categorized as FA in this report. Multivariable logistic regression models adjusted for age, education, and income were conducted separately within each cohort. Results: Among 743 adults with AUD, 238 (32.1%) met criteria for FA symptoms, with similar prevalence among Tx (32.6%) and nonTx (30.6%) participants despite marked differences in clinical characteristics. Across both cohorts, FA was independently associated with higher body mass index, greater psychological distress, and greater alcohol dependence severity. Childhood trauma and poorer sleep quality were additionally associated with FA among treatment-seeking participants, whereas alcohol-related measures differed according to treatment status. Conclusions and Relevance: FA was common among adults with AUD and was associated with greater psychological, behavioral, and metabolic burden regardless of treatment-seeking status. These findings suggest that FA identifies a clinically meaningful subgroup of individuals with AUD who may benefit from more comprehensive assessment and integrated treatment approaches.

Astrocytes play an important role in neuronal health. A critical function of astrocytes is to clear excess extracellular glutamate and prevent excitotoxicity. STAT3 is a transcription factor that promotes astrocyte development and astrocyte reactivity in neurodegenerative diseases and following central nervous system injury. To determine the innate molecular and behavioral functions of adult astrocyte-expressed STAT3 in a non-pathological state, we created conditional Stat3 astrocyte knockout mice (Stat3 aKO) using Stat3flox and the tamoxifen-activated Cre line, Aldh1l1-Cre/ERT2. We measured transcript levels of Gfap, a known STAT3 target gene, and glutamate transporter genes in the medial prefrontal cortex (PFC) of Stat3 aKO. Gfap, Slc1a2 and Slc17a8 transcripts were decreased in the PFC of Stat3 aKO of both sexes. GLT-1 protein, encoded by Slc1a2, was also reduced in the PFC of male Stat3 aKO. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) in male Stat3 aKO and control prelimbic pyramidal neurons and found increased sEPSC amplitude, consistent with a hyper-glutamatergic state due to impaired glutamate clearance. To determine the behavioral consequences of STAT3 depletion in astrocytes, Stat3 aKO were tested for locomotor activity, anxiety-like behavior and binge ethanol consumption, behaviors linked to dysregulation of glutamate homeostasis. Stat3 aKO mice did not differ in locomotor activity or anxiety-like behavior; however, male Stat3 aKO mice consumed significantly less ethanol than controls. These results indicate that STAT3 in adult astrocytes is crucial for maintaining glutamate transporter levels in the adult brain and that astrocytic STAT3 promotes ethanol consumption in male mice. Main pointsO_LIGfap, Slc1a2 and Slc17a8 expression are lower in the cortex of Stat3 astrocyte knockout mice (Stat3 aKO) C_LIO_LIGLT-1 protein is decreased and glutamate neurotransmission is elevated in the cortex of male Stat3 aKO C_LIO_LIMale Stat3 aKO consume less ethanol C_LI

Background: Compulsive alcohol use despite negative outcomes is a defining characteristic of alcohol use disorder. Rats exposed to long-term intermittent alcohol access (IAA) demonstrate sustained motivation for ethanol despite presence of the bitter additive quinine, offering a useful preclinical model of compulsive alcohol use. However, little is known about the role of habenular circuitry in the development of this phenotype. Here, we employed chemogenetic techniques targeting basal forebrain (BF) input to the lateral habenula (LHb) to probe the involvement of this neural circuitry in aversion-resistant alcohol consumption. Methods: Following long-term IAA or control conditions, male and female Long-Evans rats underwent surgery for the expression of designer receptors in BF-to-LHb projections. We then excited this pathway in rats with IAA history, or inhibited this pathway in rats with more limited ethanol history, before testing consumption of unadulterated and quinine-adulterated ethanol as well as unadulterated and quinine-adulterated sucrose. Results: Long-term IAA elevated ethanol drinking in all rats and aversion-resistant ethanol preference in males. Chemogenetic activation of BF-to-LHb neurons in rats with IAA history produced different effects in males and females: excitation enhanced ethanol intake in females, but reduced ethanol preference in males, regardless of quinine adulteration. Activation also led to a relative insensitivity to quinine-adulteration of sucrose when compared to controls, particularly in females. Chemogenetic inhibition in rats with limited prior ethanol exposure did not alter either ethanol or sucrose consumption with or without quinine. Conclusions: Our results suggest a differential role for BF-to-LHb circuitry in ethanol drinking based on sex, and a potential role for this circuitry in the sensitivity to quinine in the context of natural reward consumption.

Alcohol use disorder (AUD) is marked by substantial heterogeneity in drinking behaviors and health outcomes, underscoring the need for preclinical models that capture interindividual variability. We recently developed open-source capacitive lickometer systems for high-resolution monitoring of mouse fluid intake. Using LIQ PARTI and LIQ HD, we found substantial individual differences in alcohol intake that varied across sex and housing status in C57Bl6/J mice. Here, we conducted a secondary analysis of this continuous access ethanol drinking data to quantify behavioral variability in group and singly housed mice. We introduce a fluid "meal" pattern analysis that integrates drinking across ethanol and water sippers to define discrete drinking episodes. Using this approach, we observed sex- and housing-dependent reorganization of drinking structure across group and single-housed settings, with group-housed male mice exhibiting fewer but faster liquid meals. To further characterize multidimensional drinking patterns, we applied principal component analysis to meal variables and identified a "distributed meal" phenotype defined by increased meal number, reduced meal size, earlier onset of drinking, and higher ethanol preference. Considering factors that influence behaviors in a social environment, we next examined whether social hierarchy was associated with these patterns using a tube test dominance assay. Social rank was unrelated to ethanol and meal measures; however, offensive dominance behavior positively correlated with principal component scores in males. Together, these findings demonstrate that high-resolution, longitudinal analysis of ethanol drinking reveals distinct behavioral phenotypes that are associated with key components of social behaviors, providing a potential framework for understanding heterogeneity in AUD-related drinking. HighlightsO_LILIQ PARTI and HD enable high-resolution analysis of ethanol drinking patterns. C_LIO_LIFluid meal analysis captures sex- and housing-dependent drinking structures. C_LIO_LIBehavioral phenotyping reveals individual differences beyond total ethanol intake. C_LIO_LIPCA identifies a meal phenotype associated with male offensive dominance behavior. C_LI

Neuroimmune signaling is increasingly implicated in alcohol use disorder (AUD). Microglia, the brains resident immune cells, signal in part through the adaptor protein myeloid differentiation primary response 88 (MyD88), a key mediator of innate immune responses. Here, we investigated whether microglial-specific MyD88 signaling regulates voluntary alcohol consumption in adulthood, as whole-body loss of MyD88 was previously shown to increase drinking. We further determined if alcohol altered parvalbumin-expressing interneurons (PVIs) and microglia within the pre-frontal cortex, based on our previously described role for MyD88 signaling on perineuronal net (PNN) deposition on PVIs in several brain regions, and the well characterized role of inhibitory signaling in alcohol use disorders. Loss of microglial-MyD88 had minimal effects on voluntary alcohol intake and anxiety-like behaviors. Alcohol exposure did not modify observed MyD88-dependent changes in PVIs/PNNs, despite altering microglial morphology in the male prefrontal cortex independent of genotype. The addition of an early life endotoxin challenge was sufficient to induce an increase in adult alcohol consumption in both MyD88-deficient and control males. However, injection of saline alone also induced an increase in adult drinking in MyD88-deficient males. These findings suggest that microglial-MyD88 signaling does not strongly regulate alcohol intake under baseline conditions in a one-bottle, voluntary binge-drinking paradigm, however there may be a role for microglial-MyD88 signaling in modulating the impact of developmental environmental contexts, such as stress, in later-life male drinking behavior. This work highlights the importance of developmental context, such as stress or inflammatory history, in understanding underlying microglia signaling mechanisms in conferring AUD risk.

Alcohol use disorder (AUD) disrupts the gut-liver-brain axis, yet mechanistically grounded and therapeutically actionable targets within this network remain poorly defined. To identify microbial modulators of alcohol-induced tissue pathology, longitudinal advanced diffusion MRI and fecal 16S rRNA profiling were integrated across Marchigian Sardinian alcohol-preferring rats evaluated at baseline, after four weeks of voluntary alcohol intake, and following six weeks of abstinence. Machine learning, specifically random forest models combining neuroimaging and microbiota data, improved phase classification and identified Akkermansia as the microbial feature most strongly associated with alcohol-related white matter microstructural abnormalities. Alcohol exposure induced widespread white matter alterations alongside gut dysbiosis characterized by reduced microbial diversity. To evaluate functional relevance, Akkermansia muciniphila was administered during the abstinence phase. Supplementation with A. muciniphila restored intestinal mucus, reduced liver injury markers, and elevated myelin basic protein levels within affected white matter regions. Collectively, these findings highlight Akkermansia as a critical modulator of alcohol-induced gut-liver-brain pathology and provide experimental support for a causal contribution of specific gut bacteria to persistent white matter damage in AUD. More broadly, this work establishes a robust multimodal framework for microbiome-based target discovery with clear translational relevance for disorders characterized by dysfunction along the gut-liver-brain axis. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAlcohol use disorder (AUD) is associated with gut dysbiosis, impaired intestinal barrier function, liver injury, and persistent white matter abnormalities. Previous studies in patients and animal models have linked alcohol exposure to reduced microbial diversity, altered gut permeability, and white matter microstructural damage, particularly during abstinence. Other work has shown that microbiota-derived interventions can ameliorate peripheral consequences of alcohol exposure, especially in the gut and liver. However, the specific microbial features linked to alcohol-induced brain pathology remain poorly defined, and no prior study has integrated longitudinal microbiota and neuroimaging data to identify candidate microbial modulators of alcohol-related white matter damage and then functionally test them in vivo across the gut-liver-brain axis. Added value of this studyWe developed a multimodal framework that integrates longitudinal advanced diffusion MRI with fecal microbiota profiling and machine learning in alcohol-preferring rats. This approach identified Akkermansia as the microbial feature most strongly associated with alcohol-induced white matter abnormalities. Guided by this result, we administered Akkermansia muciniphila during abstinence and observed coordinated beneficial effects across multiple organs, including restoration of intestinal mucus, reduction of liver injury markers, and recovery of myelin basic protein in affected white matter regions. To our knowledge, this is the first study to combine longitudinal microbiota-MRI integration with experimental validation of a microbiota-based intervention that mitigates alcohol-induced pathology across the gut-liver-brain axis while restoring central white matter integrity. Implications of all the available evidenceOur findings support a mechanistic contribution of specific gut bacteria to persistent alcohol-induced tissue damage and identify Akkermansia as a candidate modulator of gut-liver-brain axis dysfunction in AUD. More broadly, this study establishes a generalizable strategy for integrating microbiota and neuroimaging data to discover biologically meaningful and therapeutically actionable targets in complex disorders involving coordinated peripheral and central pathology.

Alcohol use disorder demonstrates ~50% heritability, much of which remains unexplained by genetic sequence alone. Chronic alcohol exposure before conception changes offspring phenotypes through epigenetic mechanisms that are still being elucidated. Preconception ethanol exposure studies have focused on paternal exposure, neglecting maternal and biparental exposure. To address this, we exposed adult male and female mice to five cycles of chronic intermittent ethanol vapor interleaved with two bottle choice ethanol drinking and mated them to produce male and female F1 offspring with paternal, maternal, or biparental preconception ethanol exposure or controls. Whole blood and medial prefrontal cortex from adult, ethanol-naive offspring underwent RNA-sequencing. We also analyzed previously unpublished RNA-sequencing data from male and female preimplantation embryos derived from preconception ethanol-exposed sires. Here, we report transcriptomic patterns of preconception ethanol exposure that depend on the exposed parent, offspring sex, and tissue which suggest metabolic and immune dysfunction in offspring.

Alcohol craving and consumption fluctuate across the reproductive cycle in females with alcohol use disorder (AUD), suggesting that estrogen signaling contributes to disease vulnerability. Here, we investigated the role of estrogen receptor alpha (ER; Esr1; ESR1) in alcohol seeking using complementary mouse and human approaches, as this receptor was previously identified as a risk factor for AUD. Female mice were characterized in an IntelliCage-based multidimensional AUD paradigm that stratifies individuals into AUD-prone and AUD-resistant phenotypes. Transcriptomic profiling of the amygdala revealed that Esr1 is a top transcription factor for differentially expressed genes in mice drinking alcohol, and the estrogen signaling pathway was deregulated specifically in AUD-prone mice. Although alcohol exposure did not alter overall Esr1/ER mRNA or protein abundance, both transcript and protein levels positively correlated with cue-induced alcohol seeking, indicating that inter-individual variation in ER signaling predicts relapse-like behavior. Causal manipulations confirmed a functional role of ER. Local knockdown of Esr1 in the basolateral amygdala reduced excitatory synaptic transmission, attenuated alcohol motivation, cue-induced seeking, and relapse drinking, and impaired cue-associated memory recall without affecting anxiety-like behavior. Similarly, ovariectomy decreased amygdala ER expression, altered synaptic protein markers, and reduced alcohol-seeking behaviors, supporting regulation by endogenous ovarian hormones. Extending these findings to humans, ESR1 gene polymorphisms (rs6902771, rs11155819 and rs6557171) were associated with the probability of alcohol binge drinking and alcohol consumption days as well as craving and loss of control in real world in a longitudinal clinical cohort, while ESR1 mRNA blood levels were increased in women with AUD diagnosis. Together, these convergent molecular, circuit, behavioral, and genetic data identify ER signaling in the amygdala as an important modulator of alcohol-seeking behavior induced by alcohol cue and relapse vulnerability, highlighting estrogen pathways as potential therapeutic targets and markers for AUD.

BackgroundCircadian disruption is strongly associated with alcohol use disorder (AUD), but insight into the underlying brain-region and sex-specific mechanisms is limited. The function of the circadian clock gene Bmal1 within the striatum has been linked to alcohol drinking, yet its role within functionally distinct striatal subregions has not been systematically examined. MethodsWe deleted Bmal1 in medium spiny neurons of the dorsomedial striatum (DMS) or dorsolateral striatum (DLS). Male and female mice were tested for anxiety-like behavior, depressive-like behavior, and motor coordination. Voluntary alcohol intake was measured with an intermittent two-bottle choice paradigm, followed by sucrose preference and quinine-adulterated alcohol tests. To assess hormonal contributions, a subset of female mice underwent ovariectomy before behavioral testing. ResultsDeletion of Bmal1 in the DLS did not alter alcohol intake, alcohol preference, or quinine-adulterated alcohol intake in either sex. In contrast, DMS Bmal1 deletion significantly reduced alcohol consumption and alcohol preference in female mice, with no effect in males. These effects were not accompanied by changes in depressive-like behavior or motor coordination and were not explained by generalized reward changes, as sucrose preference was unaffected. Ovariectomy eliminated the effect of DMS Bmal1 deletion on alcohol intake, indicating dependence on ovarian hormones. ConclusionsThe DMS is a critical site at which Bmal1 regulates alcohol consumption in a sex-specific manner. These findings support an interaction between local circadian mechanisms and ovarian hormones in controlling alcohol drinking and highlight a potential target for sex-specific therapeutics in AUD.

RationaleAlcohol use disorder is defined by drinking alcohol despite knowledge of negative consequences, often referred to as aversion-resistant drinking (ARD). The dorsomedial (DMS) and dorsolateral striatum (DLS) are necessary for goal-directed and habitual action selection, respectively. Leading hypotheses posit that once drug use becomes compulsive, DMS dependence degrades while DLS dependence increases. This shift may be mediated by changes in synaptic weights from glutamatergic inputs. ObjectivesUsing a combination of western-blot, micro-injections, and ex-vivo electrophysiology, we investigated the role of -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors AMPAR, which drive glutamatergic transmission, during quinine-adulterated alcohol (QuA) drinking in the DMS and DLS across the development of ARD. ResultsWe found that AMPAR subunit composition and function change in the DMS across the development of ARD whereby, calcium permeable (CP) - AMPARs drive behavior. Western blots revealed a negative relationship between DMS GluA1 and QuA drinking in aversion-sensitive mice and positive relationships between DMS or DLS GluA1/A2 ratios and QuA drinking in ARD mice. DMS CP-AMPAR antagonism caused an increase in QuA drinking suggesting that CP-AMPARs in the DMS prevent ARD. Ex-vivo electrophysiology of DMS spiny projection neurons (SPNs) revealed that ARD mice had a greater rectification index than aversion-sensitive mice indicating that SPNs in the DMS express more CP-AMPARs following the development of ARD. ConclusionsThese data provide evidence that repeated alcohol binges alter DMS CP-AMPAR activity, where initial DMS activity acts to prevent ARD but after repeated binges that result in ARD, DMS SPNs recruit CP-AMPARs.

Background: Alcohol use and physical activity are common, interrelated behaviors with opposing associations to mental health, yet little is known about their joint relationship with anxiety and depression. This study evaluated whether the association between alcohol use and anxiety and/or depression symptoms differed by physical activity level in a large U.S. longitudinal cohort. Methods: Using data from 5,152 adults in the CHASING COVID Cohort (2021-2023), alcohol use was categorized as low-, moderate-, or high-risk based on repeated AUDIT-C assessments, and physical activity was classified as active versus sedentary using items adapted from the 2023 Behavioral Risk Factor Surveillance System. Moderate to severe symptoms of anxiety/depression, assessed subsequent to the exposure assessment period, was defined as a PHQ-8 or GAD-7 score [&ge;]10. Log-binomial regression estimated adjusted risk ratios (aRRs) for associations between alcohol use and anxiety/depression symptoms within strata of physical activity. Effect modification by physical activity was evaluated on the additive and multiplicative scales. Analyses were repeated among healthier participants without chronic conditions or poor self-rated health to reduce sick-quitter bias (N=3,141). Results: Most participants had low-risk or no alcohol use (70.3%), followed by moderate-risk (21.9%) and high-risk use (7.8%); 29.5% were sedentary. Sedentary participants had higher risk of moderate to severe anxiety/depression symptoms than active participants across alcohol use levels overall and in the healthier subsample. In the overall sample, active participants with high-risk alcohol use had elevated risk compared with active participants with low-risk or no alcohol use (aRR = 1.44, 95% CI: 1.15-1.80) and physical activity did not modify the association between alcohol use and anxiety/depression symptoms on either scale. Among healthier participants, risk was highest among sedentary participants with moderate-risk alcohol use compared with active participants with low-risk or no alcohol use (aRR = 1.92, 95% CI: 1.50-2.45); physical activity modified the association between moderate-risk alcohol use and moderate to severe anxiety/depression symptoms on the additive scale (RERI = 0.61, p=0.013) and multiplicative scales (ratio of aRRs = 1.49, p=0.001). Conclusions: Among healthier adults, physical activity modified the association between moderate alcohol use and symptoms of anxiety or depression on additive and multiplicative scales. Findings suggest sedentary behavior may amplify mental health vulnerability associated with moderate drinking; future studies are warranted to clarify these relationships, including those with larger numbers of high-risk drinkers.