Alcohol

Compulsive alcohol drinking is a key symptom of alcohol use disorder (AUD) that is particularly resistant to treatment. An understanding of the biological factors that underly compulsive drinking will allow for the development of new therapeutic targets for AUD. One animal model of compulsive alcohol drinking involves the addition of bitter-tasting quinine to an ethanol solution and measuring the willingness of the animal to consume ethanol despite the aversive taste. Previous studies have demonstrated that this type of aversion-resistant drinking is modulated in the insular cortex of male mice by specialized condensed extracellular matrix known as perineuronal nets (PNNs), which form a lattice-like structure around parvalbumin-expressing neurons in the cortex. Several laboratories have shown that female mice exhibit higher levels of aversion-resistant ethanol intake but the role of PNNs in females in this behavior has not been examined. Here we compared PNNs in the insula of male and female mice and determined if disrupting PNNs in female mice would alter aversion-resistant ethanol intake. PNNs were visualized in the insula by fluorescent labeling with Wisteria floribunda agglutinin (WFA) and disrupted in the insula by microinjecting chondroitinase ABC, an enzyme that digests the chondroitin sulfate glycosaminoglycan component of PNNs. Mice were tested for aversion-resistant ethanol consumption by the addition of sequentially increasing concentrations of quinine to the ethanol in a two-bottle choice drinking in the dark procedure. PNN staining intensity was higher in the insula of female compared to male mice, suggesting that PNNs in females might contribute to elevated aversion-resistant drinking. However, disruption of PNNs had limited effect on aversion-resistant drinking in females. In addition, activation of the insula during aversion-resistant drinking, as measured by c-fos immunohistochemistry, was lower in female mice than in males. Taken together, these results suggest that neural mechanisms underlying aversion-resistant ethanol consumption differ in males and females.

Any increase in alcohol use is associated with an increase in risk of illness and mortality and consequences of chronic alcohol use include cancer, hypertension, heart and liver disease, and Alcohol Use Disorder. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective anti-glycemic and weight-loss medications with a strong safety record. There is substantial preclinical evidence and mounting retrospective and prospective randomized controlled trial evidence that GLP-1RAs could be effective for reducing alcohol consumption. However, the mechanism by which GLP-1RAs reduce alcohol intake remains unclear. While medications that reduce alcohol intake such as naltrexone and acamprosate have central nervous system action, disulfiram reduces alcohol intake through peripheral mechanisms. Here, we test whether GLP- 1RAs alter alcohols peripheral pharmacokinetics as a potential mechanism of action for their alcohol intake suppressive effects. In this pilot study, twenty participants with obesity in the GLP-1RA or control group consumed a challenge dose of alcohol, and we measured breath alcohol (BrAC) and the subjective effects of alcohol. We observed a delayed rise in BrAC and subjective effects in the GLP-1RA group as compared to controls, that was not explained by nausea. These data provide preliminary evidence that GLP-1RAs could act through peripheral mechanisms to suppress alcohol intake.

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

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

Alcohol use disorder (AUD) imposes a significant global health burden, yet effective treatments remain limited due to the scarcity of well-characterized biological sample repositories. To address this gap, we established the UCSD Alcohol BioBank, a comprehensive resource containing thousands of samples from over 700 genetically diverse heterogeneous stock (HS) rats. Modeled after successful cocaine and oxycodone biobanks, this repository utilizes the chronic intermittent ethanol vapor exposure (CIE) model, paired with oral self-administration, to characterize AUD-like behaviors, including ethanol consumption, preference, motivation, and withdrawal symptoms such as allodynia and anxiety-like behavior. Longitudinal samples (blood, urine, and feces) are collected before, during, and after ethanol exposure, while terminal samples (brain, heart, liver, kidneys, cecum, reproductive organs, adrenal glands, peripheral blood mononuclear cells) are obtained at intoxication, acute withdrawal, protracted abstinence, or from naive controls. Samples are preserved via snap-freezing or paraformaldehyde fixation to support diverse applications, including genomics, transcriptomics, proteomics, and neuroanatomy. The genetic diversity of HS rats enables genome-wide association studies (GWAS) to identify AUD-related genetic variants. Freely available to non-profit organizations at www.alcoholbiobank.org, with genetic and behavioral data deposited in public repositories, the Alcohol BioBank facilitates collaborative research to uncover biomarkers and develop novel therapies for AUD, addressing a critical need in addiction science.

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.

ObjectiveFetal alcohol spectrum disorders affect approximately 1 in 20 school age children in the United States of America. To study fetal alcohol spectrum disorders, mouse models are commonly used. Of the many approaches of gestational exposure, voluntary drinking paradigms represent the most similar mechanism of drinking as human exposure. These exposures can be done through low-tech solutions such as test tubes (TT), or more high-tech methods such as a volumetric drinking monitor (VDM). Here were compare the TT method and the VDM directly, to evaluate their effect on female mouse drinking. MethodWe adapted a drinking in the dark, active cycle, limited access (4 hr.) voluntary drinking paradigm first described by Brady et al. (2012) to test tubes and the volumetric drinking monitor. 8 mice were placed in either drinking method and we evaluated their drinking volume and blood alcohol concentrations (BACs). We compared the values for each group using t-tests. ResultsAfter 2 weeks of drinking 10% ethanol with 0.4% saccharine, BACs were not significantly different [t(14)=0.2681, p=0.7935] between the VDM (81.56 {+/-} 21.16 mg/dL) vs.TT (73.14 {+/-} 23.20 mg/dL) groups. Calculated intake of ethanol (g/kg) on the day of blood draw for BAC analysis was also not significantly different [t(14)=0.4308, p=0.6732] between VDM (2.985 {+/-} 0.4127) vs.TT (3.260 {+/-} 0.4863; Fig 1B) groups. O_FIG O_LINKSMALLFIG WIDTH=139 HEIGHT=200 SRC="FIGDIR/small/658718v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@5f3ad4org.highwire.dtl.DTLVardef@f45edorg.highwire.dtl.DTLVardef@b7d7f8org.highwire.dtl.DTLVardef@1c40c63_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig 1.C_FLOATNO C_FIG ConclusionsTest tube or VDM resulted in similar average daily ethanol consumption and resultant BACs in female mice

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.

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.

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

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.

Alcohol intake progressively increases after prolonged consumption of alcohol, but relatively few new therapeutics targeting development of alcohol use disorder (AUD) have been validated. Here, we conducted a genome-wide RNA-sequencing (RNA-seq) analysis in mice exposed to different modes (acute vs chronic) of ethanol drinking. We focused on transcriptional profiles in the amygdala including the central and basolateral subnuclei, a brain area previously implicated in alcohol drinking and seeking, demonstrating distinct gene expression patterns and canonical pathways induced by both acute and chronic intake. Surprisingly, both drinking modes triggered similar transcriptional changes, including up-regulation of ribosome-related/translational pathways and myelination pathways, and down-regulation of chromatin binding and histone modification. Notably, multiple genes that were significantly regulated in mouse amygdala with alcohol drinking, including Atp2b1, Slc4a7, Nfkb1, Nts, and Hdac2, among others had previously been associated with human AUD via GWAS or other genomic studies. In addition, analyses of hub genes and upstream regulatory pathways predicted that voluntary ethanol consumption affects epigenetic changes via histone deacetylation pathways, oligodendrocyte and myelin function, and oligodendrocyte-related transcriptional factor, Sox17. Overall, our results suggest that the transcriptional landscape in the central and basolateral subnuclei of the amygdala is sensitive to voluntary alcohol drinking. They provide a unique resource of gene expression data for future translational studies examining transcriptional mechanisms underlying the development of AUD due to alcohol consumption.

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

Differential gene expression is often inadequate to predict the activity of transcription factors and their contribution to the phenotypes associated with specific gene expression states. Here we used a systems biology approach based on gene network inference and master regulator analysis (MRA) to identify candidate drivers of the gene network dysregulations in the prefrontal cortex (PFC) of human subjects with a history of alcohol dependence. The estrogen-related receptor gamma (ERR{gamma}) gene ESRRG, an orphan nuclear receptor protein that acts as a transcription activator, emerged as a high-ranking Master Regulator (MR) based on the expression of its targets and was selected for functional validation due to its translational and druggability potential. The ERR{gamma} agonist, GSK4716, reduced alcohol drinking in the mouse binge drinking paradigm of drinking in the dark (DID) and in both non-dependent mice as well as in mice made dependent by chronic intermittent vapor exposure (CIE). GSK4716 also prevented alcohol-conditioned place preference without affecting saccharin intake or mouse locomotion. Similarly, in rats, GSK4716 reduced operant oral alcohol self-administration in non-dependent and dependent (by CIE) rats under fixed and progressive ratio schedules of reinforcement. Overall, these results support the efficacy of transcriptome-wide gene regulatory network approaches for the identification of key druggable regulators of long-term transcriptional adaptations that sustain the molecular and behavioral pathology of alcohol dependence and identify ERR{gamma} as a regulator of excessive alcohol drinking and seeking, and a therapeutic target for AUD.

Excessive alcohol (ethanol) consumption is a hallmark of alcohol use disorder (AUD). Activation of innate immune system and proinflammatory signaling in the brain may play a key role in promoting alcohol consumption and development of AUD in humans. Innate immune activation by toll-like receptor (TLR) agonists in rodents is associated with release of proinflammatory cytokines and changes in alcohol consumption, and these effects are genotype- and sex-dependent. For example, C57BL/6J male, but not female mice increase alcohol intake after TLR3 activation. In order to better understand the interactions between neuroimmune signaling, genotype, and sex and their effects on ethanol drinking, males and females of more genotypes need to be tested. The goal of this study was to test the effects of innate immune activation on ethanol consumption and neuroimmune molecular profiles of F1 hybrid mice from reciprocal crosses between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains, which are animals with high levels of ethanol intake. Animals were randomly assigned to receive intraperitoneal injections of either saline, Poly(I:C) (PIC, 2 or 10 mg/kg), a TLR3 agonist, or lipopolysaccharide, (LPS, 0.1 mg/kg), a TLR4 agonist, administered every 4 days for a total of 10 injections and subjected to a 2-bottle choice every-other-day ethanol drinking paradigm for a total of 18 dinking sessions, which generated high levels of voluntary ethanol consumption. Six and 24 hours after the last injection, brains were removed, frontal cortex dissected, and levels of 3 proinflammatory cytokines (Tnfa, Il1b, Ccl5), as well as Tlr3, and Tlr4 were measured using qPCR. Immune activation by PIC produced escalation of ethanol drinking, while LPS resulted in a reduction of ethanol consumption or a trend to reduce drinking in males but not females of both FVB/B6 and B6/FVB crosses. Furthermore, activation of TLR3 by PIC produced sex-specific time course responses of pro-inflammatory cytokines, which may, at least in part, explain behavioral differences. Taken together, these results validate previous findings that the effects of immune activation on ethanol consumption depend on genotype, sex, and mode of activation (TLR3 vs TLR4) and suggest that FVB/B6J and B6J/FVB F1 males are a suitable model to study TLR3-dependent escalation of alcohol drinking. HighlightsO_LIImmune activation by Toll-like receptor 3 (TLR3) agonist, Poly(I:C), produced an escalation of ethanol drinking, while immune activation by TLR4 agonist, LPS, reduced ethanol intake in male but not female FVB/NJ x C57BL/6J hybrid mice. C_LIO_LIPoly(I:C)-induced escalation of alcohol consumption in males was reproducible and consistent across different Poly(I:C) doses. C_LIO_LIActivation of TLR3 by Poly(I:C) produced sex-specific time course responses of pro-inflammatory cytokines, which may, at least in part, explain sex differences in alcohol consumption. C_LIO_LIOur data suggest that FVB/NJ x C57BL/6J hybrid male mice are a suitable model to study TLR3-dependent escalation of alcohol drinking. C_LI

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).

This study investigated the relationship between stress exposure and subsequent ethanol use, focusing on individual differences among male rats. We combined operant self-administration with behavioral economics to assess how intermittent swim stress affects ethanol consumption. This approach allowed for a nuanced analysis of the transition from regular ethanol intake to stress-induced escalation in economic demand. Results showed a consistent rise in ethanol demand post-stress among subjects, irrespective of exposure to actual swim stress or a sham procedure. This increase may result from a two-week abstinence or an inherent rise in demand over time. Significantly, we identified a direct link between post-stress corticosterone levels and the demand for ethanol, considering baseline levels. This correlation was particularly pronounced when examining the shifts in both corticosterone levels and demand for ethanol post-stress. However, neither post-stress corticosterone levels nor their change over time correlated significantly with changes in ethanol demand following a forced swim test that was administered 24 hours after the intermittent swim stress test. This suggests potential context-specific or stressor-specific effects. Importantly, pre-stress ethanol demand did not significantly predict the corticosterone response to stress, indicating that high ethanol-demand rats do not inherently exhibit heightened stress sensitivity. Our research brings to light the complex interplay between stress and ethanol consumption, highlighting the critical role of individual differences in this relationship. This research introduces a nuanced perspective, underscoring the need for future studies in the realm of stress and substance use to give greater consideration to individual variability.

BackgroundDopamine and serotonin are key regulators of reward sensitivity, yet their distinct roles in motivating natural (e.g., sucrose) versus drug (e.g., ethanol) rewards remain unclear. Understanding these mechanisms could help explain individual variability in reward processing relevant to substance use vulnerability. MethodsWe assessed reward sensitivity in dopamine transporter (DAT) and serotonin transporter (SERT) knockout (KO) rats using both home cage (two-bottle choice for sucrose and ethanol) and operant paradigms (Pavlovian and instrumental learning). ResultsDAT KO rats showed lower sucrose preference (-27% for 2%, -13% for 4%) and intake (-42% for 4%), diminished Pavlovian responding for sucrose (-68%), and slower acquisition of sucrose-taking behavior ([~]+30 days) compared to WT rats. DAT KO rats also showed reduced ethanol preference in the home cage (-16%) despite an unchanged intake. Furthermore, operant performed was markedly reduced operant performance after the sucrose-to-ethanol transition (-83%), with no increase in ethanol-taking following sucrose exposure (0% change), unlike WT controls (+41%). SERT KO rats presented reduced sucrose preference (-5%) and intake (-46%) for the 4% solution only. In addition, SERT KO rats also showed reduced Pavlovian sucrose responding (- 28%) and slower acquisition of sucrose-taking ([~]+30 days) but intact responding and learning for ethanol. In the home cage, they displayed lower ethanol preference (-35%) without significant change in operant ethanol performance. A modest overall increase in ethanol-taking was seen post-sucrose in both SERT KO and WT, but without genotype-specific effects. Conclusion and ImplicationsDAT deletion broadly impaired sensitivity for both natural sucrose and ethanol rewards, particularly under effortful or devalued conditions. In contrast, SERT deletion produced more selective impairments by disrupting sucrose operant responding and moderately reducing ethanol reward preference. These findings reveal distinct but overlapping roles of DAT and SERT in regulating reward sensitivity, with implications for understanding individual vulnerability to substance use.

Voluntary alcohol consumption is influenced by a variety of environmental and genetic factors, including circadian clock genes. Even though their sex-specific role in alcohol drinking was identified through selective ablation of Bmal1 and Per2 from neurons of the mouse striatum, the contribution of specific striatal subregions to the observed drinking behavior remains unclear. Thus, alcohol intake and preference was investigated in male and female mice with a conditional knockout of Bmal1 and Per2 from cells in the nucleus accumbens (Nac). Mood- and anxiety-related behaviors were assessed prior to alcohol drinking to exclude potential confounding effects of the animals behavioral state on alcohol consumption. Alcohol consumption and preference were increased in male and female mice with a conditional knockout of Bmal1, whereas the same effect was only found in males with a deletion of Per2. Because affective behaviors were only mildly influenced by the conditional gene knockouts, observed alcohol-drinking phenotypes can be directly associated with the Nac-specific clock gene deletion. The results thus suggest an inhibitory role of Bmal1 and Per2 in the Nac on alcohol consumption in male mice. In females, the inhibitory effect of Bmal1 is strictly localized to the Nac, because striatal-wide deletion of Bmal1 caused a suppression of alcohol consumption. This sex-dependent stimulatory effect of Bmal1 on alcohol drinking is probably mediated through other striatal subregions such as the dorsal striatum.

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.