Alcohol

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.

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).

BackgroundThe dorsolateral striatum (DLS) is a key site for coordinating the alcohol-induced stimulant response, a behavioral marker predictive of future alcohol use disorder. Although ethanol (EtOH) affects all brain cells, little is known about the contribution of non-neuronal DLS cell types to EtOH-induced stimulation. MethodsWe used ex vivo two photon calcium imaging, in vivo fiber photometry of astrocyte and neuronal GCaMP, and astrocyte-specific manipulations in mice to determine DLS astrocyte contributions to EtOH-induced stimulation and voluntary EtOH drinking behavior. Using fiber photometry of GRAB-ACh sensors and cell-type specific chemogenetics, we also assessed the role of cholinergic signaling in observed astrocyte EtOH effects. ResultsAs expected, intraperitoneal EtOH injections (0.5-2g/kg) evoked a stimulant response, evidenced by increased locomotion compared to saline. In parallel, EtOH dose-dependently decreased astrocyte calcium activity but had minimal effects on direct and indirect pathway neuronal activity. Mimicking this reduction with astrocyte-specific expression of CalEX, a calcium extruding pump, facilitated EtOH stimulation compared to mice expressing a control fluorophore. Hence, EtOH-induced suppression of DLS astrocyte activity contributes to stimulation. Astrocyte calcium signaling is a well-known target of neuromodulation. Fiber photometry recordings of extracellular acetylcholine (ACh) levels via GRAB-ACh imaging showed inhibition of ACh release by acute EtOH. We virally expressed the excitatory chemogenetic actuator hM3Dq in striatal cholinergic interneurons to assess whether artificially increasing ACh release blocks EtOH-induced inhibition of astrocytic calcium activity. Despite facilitating ACh release, this manipulation did not impact astrocyte calcium activity under control (saline) or EtOH conditions. Together, this work identifies DLS astrocytes as key contributors to EtOH-induced stimulation and highlights the importance of considering astrocyte-neuron interactions in evaluating alcohol effects.

The gut microbiome has been implicated in alcohol use disorder (AUD), but its relationship to drinking intensity and treatment response remains poorly understood. We conducted a longitudinal multi-omics analysis of stool samples collected at baseline and endpoint (after 12 weeks) from 122 participants enrolled in a double-blind, placebo-controlled trial of dutasteride for AUD. Gut microbiome composition was characterized using 16S rRNA gene sequencing, and fecal metabolites were measured by LC-MS-based metabolomics. At baseline, drinking intensity was associated with increasingly lower microbial richness. Genera in the class Clostridia emerged as key microbial hubs associated with drinking intensity in an age- and sex-dependent manner. Drinking intensity promoted co-enrichment of [Ruminococcus] gnavus group and [Clostridium] inocuum group with amino acid catabolites, as well as the co-depletion of diverse Clostridia taxa and lipid metabolites. Dutasteride treatment and drinking reduction had minimal impact on gut microbiome composition. Random forest models integrating baseline clinical, microbiome, and metabolome data improved the classification of clinically meaningful drinking reduction compared to models using clinical data alone. These findings show that a coupled baseline gut microbiome-metabolome signature is associated with drinking intensity and future treatment response in AUD, highlighting the potential for multi-omics integration to inform precision treatment approaches.

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.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

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.

BackgroundAlcohol use disorder is a chronic relapsing condition characterized by excessive drinking and withdrawal symptoms. Alcohol dependence disrupts function across multiple brain regions, and recent evidence implicates the cortical amygdala (CoA) as a critical node in alcohol-related circuits. However, how CoA activity influences alcohol intake and brain-wide network function during withdrawal remains unclear. MethodsAlcohol dependence was induced using chronic intermittent ethanol vapor (CIE). In one cohort, electrophysiological activity of CoA neurons was assessed during withdrawal. In a second cohort, mice underwent CIE paired with two-bottle choice drinking, and inhibitory DREADDs (hM4Di) were used to suppress CoA activity during drinking and withdrawal while behavioral outcomes were measured. Brains were then collected for Fos immunolabeling and iDISCO+ based whole-brain activity mapping to determine how CoA inhibition during withdrawal altered network organization. ResultsRepeated CIE increased alcohol sensitivity in CoA neurons during withdrawal. Chemogenetic inhibition of the CoA reduced alcohol intake in dependent mice without affecting withdrawal-related behaviors. Whole-brain Fos mapping showed that CoA inhibition reduced activity within the CoA while enhancing functional connectivity across multiple brain regions, particularly in the isocortex, thalamus, and anterior hypothalamic nucleus. During withdrawal without CoA inhibition, thalamic regions exhibited negative connectivity, consistent with disrupted network function; CoA inhibition reversed this pattern, producing strongly positive thalamic and medial prefrontal cortex connectivity. ConclusionsThese findings demonstrate that alcohol dependence alters CoA sensitivity, alcohol dependence-induced drinking and brain-wide network organization during withdrawal. The CoA appears to selectively regulate withdrawal-associated alcohol drinking, and its inhibition may reduce intake by restoring thalamic and cortical connectivity. HighlightsO_LIThis study identifies the cortical amygdala as a previously underexplored brain region involved in alcohol-related behaviors. C_LIO_LIBy integrating chemogenetic inhibition with brain-wide network analysis, the study reveals candidate circuit connections through which the CoA may regulate alcohol dependence-related brain activity. C_LIO_LIThis study establishes the CoA as a potential driver of excessive alcohol drinking and alcohol-related network dysfunction. C_LI

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.

Craving - the powerful urge to seek and consume alcohol in response to alcohol-associated cues does not diminish after drinking cessation but rather is magnified throughout abstinence. This phenomenon, termed "incubation of craving", contributes to the relapsing nature of alcohol use disorder. Despite its occurrence in human populations and being well-studied in rodent models of psychostimulant drug relapse, the underlying neural mechanisms and potential treatments remain largely unexplored for alcohol-related incubation of craving. Our research seeks to meet this gap, and this particular study investigated the neural correlates of the incubation of craving for alcohol-associated cues and assessed whether exercise could prevent increased relapse propensity in rats. Male Long Evans rats were trained to lever press for an alcohol reward delivered with simultaneous presentation of a discrete cue. This response was then extinguished and reinstated by presenting the discrete cue alone when rats pressed the lever. Cue-induced reinstatement occurred either on day 1 following extinction (No Abstinence) or on day 29 (Abstinence). A third group was tested on day 29 and had 4-hour daily voluntary running wheel access throughout this abstinence period (Exercise). All rats were perfused 90 minutes following test, and relative activation across the brain was estimated by quantifying c-Fos protein immunoreactivity. The brain-wide coordination of neural activity was also mapped. We found a robust incubation of craving effect for alcohol-associated cues, which was mitigated by exercise. Immunohistochemistry revealed that the Abstinence group demonstrated higher c-Fos immunoreactivity compared to the No Abstinence group in multiple reinstatement-related brain regions. This effect was reversed in the Exercise group. Brain-wide neural mapping demonstrated that the Abstinence group had decreased modularity (groups of coordinating brain regions) compared to the no-abstinence group. Although network connectivity profile in the exercise group was different from no abstinence, we found that overall neural activation returned to a similar modularity profile of clustered regions as this condition, indicating that exercise does not attenuate the incubation of craving effect by reversing all the neural effects of abstinence. Rather, exercise may be acting upon select brain regions or pathways to exert relapse protective effects by restoring widespread interconnectivity. This is the first study to investigate neural activation in incubated alcohol-seeking, and provides supporting evidence for promoting voluntary exercise as an adjunctive treatment for relapse prevention in alcohol-use disorder.

RationalePrenatal alcohol exposure (PAE) can result in Fetal Alcohol Spectrum Disorder (FASD), which consists of a group of diagnosable medical conditions that can include an increased risk for anxiety disorders and/or alcohol misuse, and sensory issues, such as increased mechanical sensitivity. ObjectiveThis study investigated how a single moderate PAE on gestational day 12 (G12) alters anxiety-like behavior, ethanol (EtOH) intake, and mechanical sensitivity across the lifespan of Sprague Dawley rats. MethodsPregnant dams were exposed to vaporized EtOH or room air (control) for 6 hours (BECs [~]108 mg/dL). Testing in male and female offspring began at three different ages: juveniles ([~]postnatal day (P) 25), adolescents ([~]P45) and adults ([~]P80). ResultsThe greatest PAE effects were observed in adolescent animals, with alterations in anxiety-like behaviors demonstrated in the light-dark box and elevated plus maze. Additionally, adolescent female animals consumed more sweetened EtOH compared to males. However, PAE adolescent animals consuming less sweetened EtOH compared to their counterparts, which was also observed in adult PAE females. Interestingly, this effect is reversed in juvenile and adolescent males when tested with unsweetened EtOH, with juvenile females consuming more EtOH also. Finally, PAE and air animals exhibited increased mechanical sensitivity following post-natal EtOH consumption across all ages. ConclusionThese data demonstrate that there are age- and sex-specific effects of PAE on anxiety-like behaviors, EtOH intake, and mechanical sensitivity that are more distinct in adolescent animals.

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 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

Alcohol use disorder (AUD) is a significant public health concern. In Canada, about 18% of individuals aged 15 or older will meet the clinical criteria for AUD at some point in their lives (CAMH, 2023). Treatment options for AUD are limited, and the high relapse rates highlight the urgent need for innovative methods to study and address AUD. Zebrafish (Danio rerio) is an emerging model for exploring the neurobiological impacts of alcohol. Previous studies have demonstrated that zebrafish respond to the rewarding effects of alcohol, but most research methods rely on passive administration, such as immersion, which does not reflect the typical routes of alcohol intake in humans. We previously showed that zebrafish can learn to self-administer drugs of abuse in small groups and conditioned animals are displaying key features of substance abuse disorders. However, group-based conditioning limits our understanding of individual drug preference and intake profile. In this study, we improved upon our previous design by establishing an individual self-administration protocol to measure voluntary alcohol intake and model alcohol use disorder. In this novel assay, individual adult fish learn to discriminate between two zones to self-administer a 5% ethanol solution. Moreover, animals conditioned in this assay can perform progressive ratio and display signs of withdrawal upon cessation of ethanol intake. These results suggest zebrafish can develop ethanol abuse-like behaviour, providing a powerful platform to study genetic predisposition and screen for therapeutic compounds.

Alcohol drinking motives vary among individuals and shape experiences and beliefs about alcohol, influencing the processing of alcohol-related cues. In real-life settings, these cues are contextually rich, amplifying the role of such individualized drinking motives on cue processing. However, previous literature has primarily relied on images of alcohol, which lack contexts and differ significantly from real-life. Here, aiming to investigate real-life craving, we examined the role of alcohol drinking motives in craving in response to naturalistic alcohol-drinking videos. We asked fifty-three problematic alcohol users to speak about their reasons for drinking alcohol to capture unique alcohol drinking motives of each individual. Participants also underwent functional MRI while watching fifteen alcohol-drinking videos, and reported their subjective level of craving and self-relatedness for each video. Behavioral data analysis revealed that individuals with greater alcohol use severity tended to report greater cue-induced craving, but only when they reported that a video was related to themselves. Inter-subject representational similarity analysis showed that participants with similar alcohol drinking motives, reflected in shared drinking reasons and similar self-relatedness to the videos, exhibited synchronized craving-related neural responses during video-watching. Notably, these shared neural processes mediated the link between similar drinking motives and similar self-reported craving levels across participants. Together, our findings highlight the crucial role of alcohol drinking motives in shaping cue-induced alcohol craving, and provide deeper insights into craving in real-world contexts.

Excessive alcohol consumption remains a major public health challenge with limited therapeutic options. Both glucagon-like peptide-1 (GLP-1) and fibroblast growth factor-21 (FGF21) independently regulate alcohol intake through complementary metabolic and reward pathways, but their combined potential has not been explored. Here, we report that a long-acting dual agonist, GLP1-ELP-FGF21 modulates behavioural, neurophysiological, and cognitive components of alcohol seeking in mice. A single GLP1-ELP-FGF21 dose reversibly reduces voluntary alcohol intake for at least 72 hours in male mice, has sustained effects in female mice, and markedly blunts nucleus accumbens dopamine transients aligned to the initiation and termination of lick bouts during alcohol consumption. To assess its effects on decision-making, we used a novel two-choice (alcohol versus food) decision task modelled with evidence-accumulation frameworks. Alcohol choice behaviour conformed to evidence accumulation decision models: Linear Ballistic Accumulator (LBM) and Racing diffusion models (RDM). Critically, GLP1-ELP-FGF21 selectively reduces choices for alcohol and slows the latent accumulation rate for alcohol options, without affecting food-directed choice or non-decision processes. Sensory-specific satiety devaluation confirms that reductions in reward value are explained by reductions in accumulation rates. Together, these results highlight GLP1-ELP-FGF21 as a therapeutic strategy for alcohol use disorder via modulation of central reward pathways and decision-making when confronted with alcohol rewards.

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.

Prenatal alcohol exposure (PAE) is associated with long-term neurodevelopmental risk, yet how neuronal progenitor metabolism responds to alcohol across developmental stages remains unclear. Here, we integrated brain-isoform-focused analysis of public datasets with targeted transcriptional, translational, and functional mitochondrial assessments in human iPSC-derived cortical progenitor models representing distinct bioenergetic states. Re-analysis of prior PAE studies revealed broad metabolic gene downregulation in embryonic systems, whereas neurodevelopmental models showed limited and non-coherent transcriptional signatures. In vitro, acute ethanol exposure (AEE) induced stage-dependent transcriptional remodeling in iPSC-derived neuronal progenitor cells. Early progenitors exhibited selective upregulation of mitochondrial-associated transcripts alongside increased neuronal lineage markers. In contrast, late progenitors showed broader increases in glycolytic, lipid, and mitochondrial gene expression. Despite these transcriptional changes, mitochondrial ATP production and mitochondrial protein abundance remained unchanged in both models with altered dynamics being restricted to late progenitors. These findings indicate that ethanol exposure is associated with developmental stage-dependent neuronal remodeling of bioenergetic genes. Measurements of mitochondrial-neural frameworks after AEE showed no major alterations in either model. Overall, our results reveal a dissociation between transcriptional, translational, and functional bioenergetic outputs in models of early human neurodevelopment and highlight that transcriptional alterations of mitochondria and cellular bioenergetics under AEE should be interpreted in the context of developmental stage rather than in isolation as evidence of energetic dysfunction.

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.

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.