Alcohol

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

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.

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.

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

Previous preclinical and human studies have shown that high-fat ketogenic diet and ketone supplements (KS) are efficacious in reducing alcohol craving, alcohol consumption, and signs of alcohol withdrawal. However, the effects of KS on alcohol sensitivity are unknown. In this single-blind, cross-over study, 10 healthy participants (3 females) were administered a single, oral dose of a KS (25 g of ketones from D-{beta}-hydroxybutyric acid and R-1,3-butanediol) or placebo 30 min prior to an oral alcohol dose (0.25 g/kg for women; 0.31 g/kg for men). Assessments of breath alcohol concentration (BrAC) and blood alcohol levels (BAL) and responses on the Drug Effect Questionnaire were repeatedly obtained over 180 min after alcohol consumption. In a parallel preclinical study, 8 Wistar rats (4 females) received an oral gavage of KS (0.42 g ketones/kg), water, or the sweetener allulose (0.58 g/kg) followed 15 min later by an oral alcohol dose (0.8 g/kg). BAL were monitored for 240 min after alcohol exposure. In humans, the intake of KS prior to alcohol significantly blunted BrAC and BAL, reduced ratings of alcohol liking and wanting, and increased disliking for alcohol. In rats, KS reduced BAL more than either allulose or water. In conclusion, KS altered physiological and subjective responses to alcohol in both humans and rats and the effects were likely not mediated by the sweetener allulose present in the KS drink. Therefore, KS could potentially reduce the intoxicating and rewarding effects of alcohol and thus be a novel intervention for treating alcohol use disorder.

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

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.

BackgroundAlcohol is the most widely consumed psychoactive substance worldwide. In Portugal, alcohol consumption is deeply embedded in social and cultural practices, contributing to high prevalence rates among university students, with binge drinking emerging as a predominant consumption pattern. Despite the associations between this drinking behaviour and numerous social, physical, and psychological problems, research on alcohol consumption in Portuguese university populations remains limited. Thus, this study aimed to provide a comprehensive description of alcohol use patterns among a large sample of Portuguese university students, focusing on adolescents and young adults. MethodsA total of 1,746 students, aged 17-24 years, were surveyed using the Alcohol Use Disorders Identification Test (AUDIT) and additional questionnaires regarding socio-demographic information, alcohol and illicit drug use, smoking habits, and alcohol cravings. Students were classified into five drinking groups: Abstainers (16.8%), Moderate Drinkers (35.1%), Hazardous Drinkers (25.8%), Binge Drinkers (20.8%), and Dependent Drinkers (1.5%). Statistical analyses included descriptive statistics, group comparisons, and multinomial logistic regressions to obtain Odds Ratios (ORs) for group membership. ResultsAlcohol consumption was reported by 83.2% of students over the past year. Nearly 47% of students revealed harmful drinking patterns, and 1.5% exhibited symptoms of alcohol dependence. A progressive increase in the severity of alcohol consumption characteristics was observed across the groups, with Dependent Drinkers reporting the highest levels overall. Significant predictors of group membership included polydrug use, standard weekly consumption, earlier drinking onset, and higher levels of alcohol craving. Polydrug use, reported by 27.3% of students, was the strongest predictor for being a Hazardous Drinker (OR = 10.75), Binge Drinker (OR = 13.20), and Dependent Drinker (OR = 21.40). Binge Drinkers displayed standard weekly consumption and craving levels comparable to Dependent Drinkers, while Moderate Drinkers exhibited the least risky patterns, including a later age of onset of drinking. Male students reported significantly greater consumption and craving levels than their female peers. ConclusionsThis study highlights the prevalence of harmful drinking behaviours among Portuguese university students and identifies critical risk factors, such as polydrug use and early drinking onset. These findings underscore the need for prevention programmes focused on delaying the onset of alcohol use, reducing polydrug use, and promoting healthier behaviours within academic settings.

Adolescence is a developmental period characterized by significant changes in brain architecture and behavior. The immaturity of the adolescent brain is associated with heightened vulnerability to exogenous agents, including alcohol. Alcohol is the most consumed drug among teenagers, and binge-drinking during adolescence is a major public health concern. Studies have suggested that adolescent alcohol exposure (AAE) may interfere with the maturation of frontal brain regions and lead to long-lasting behavioral consequences. In this study, we used a mouse model of AAE in which adolescent mice reach high blood alcohol concentration after voluntary binge-drinking. In order to assess short- and long-term consequences of AAE, a battery of behavioral tests was performed during late adolescence and during adulthood. We showed that AAE had no short-term effect on young mice behavior but rather increased anxiety- and depressive-like behaviors, as well as alcohol consumption during adulthood. Moreover, alcohol binge-drinking during adolescence dramatically decreased recognition memory performances and behavioral flexibility in both adult males and females. Furthermore, we showed that voluntary consumption of alcohol during adolescence did not trigger any major activation of the innate immune system in the prefrontal cortex (PFC). Together, our data suggest that voluntary alcohol binge-drinking in adolescent mice induces a delayed appearance of behavioral impairments in adulthood.

Pavlovian conditioned contextual cues have been suggested to modulate instrumental action and might explain maladaptive behavior such as relapse in patients suffering from alcohol use disorder (AUD). Pavlovian-to Instrumental transfer (PIT) experimentally assesses the magnitude of this context-dependent effect and studies have shown a larger PIT effect in AUD populations. Taken this into account, a reduction of the influence of cues on behavior seems warranted and one approach that could alter such cue reactivity is mindfulness. Mindfulness-based interventions have been shown to be efficient in the treatment of AUD, but underlying mechanisms are yet to be elucidated. Therefore, we aim at investigating the effect of a brief mindful body scan meditation on the magnitude of the PIT effect in AUD subjects and matched controls. Using a randomized within-subjects design, we compared the effect of a short audio guided body scan meditation against a control condition (audio of nature sounds) on PIT in healthy (n = 35) and AUD (n = 27) participants. We found no differences in PIT effect between healthy and AUD participants as well as between conditions. However, a significant interaction effect points to a decreased PIT effect after body scan meditation in AUD subjects only. These results suggest that AUD might be susceptible to mindfulness-induced changes in PIT, with these findings contributing to entangling the underlying mechanisms of the efficacy of mindfulness-based interventions in AUD.

Binge drinking is a relatively common pattern of alcohol use among youth with normative frequency trajectories peaking in emerging and early adulthood. Frequent binge drinking is a critical risk factor for not only the development of alcohol use disorders (AUDs) but also increased odds of alcohol-related injury and death, and thus constitutes a significant public health concern. Changes in binge drinking across development are strongly associated with changes in impulsive personality traits (IPTs) which have been hypothesized as intermediate phenotypes associated with genetic risk for heavy alcohol use and AUD. The current study sought to examine the extent to which longitudinal changes in binge drinking and intoxication frequency across adolescence and early adulthood are associated with genetic influences underlying dual-systems IPTs (i.e., top-down [lack of self-control] and bottom-up [sensation seeking and urgency] constructs) alongside genetic risk for alcohol consumption and AUD. Associations were tested using conditional latent growth curve polygenic score (PGS) models in three independent longitudinal samples (N=10,554). Results suggested consistent significant and independent associations across all samples between sensation seeking PGSs and model intercepts (i.e., higher frequency of binge drinking at first measurement occasion) and alcohol consumption PGSs and model slopes (i.e., steeper increases toward peak binge drinking frequency). Urgency PGSs were not significantly associated with changes in binge drinking or intoxication frequency. Collectively, these findings highlight the role of unique but correlated IPT and alcohol-specific genetic factors in the emergence and escalation of binge drinking during adolescence and early adulthood.

IntroductionConsidering sex as a biological variable (SABV) in preclinical research can enhance understanding of the neurobiology of alcohol use disorder (AUD). However, the behavioral and neural mechanisms underlying sex-specific differences remain unclear. This study aims to elucidate SABV in ethanol (EtOH) consumption by evaluating its reinforcing effects and regulation by glutamate AMPA receptor activity in male and female mice. MethodsC57BL/6J mice (male and female) were assessed for EtOH intake under continuous and limited access conditions in the home cage. Acute sensitivity to EtOH sedation and blood clearance were evaluated as potential modifying factors. Motivation to consume EtOH was measured using operant self-administration procedures. Sex-specific differences in neural regulation of EtOH reinforcement were examined by testing the effects of a glutamate AMPA receptor antagonist on operant EtOH self-administration. ResultsFemale C57BL/6J mice exhibited a time-dependent escalation in EtOH intake under both continuous and limited access conditions. They were less sensitive to EtOH sedation and had lower blood levels post-EtOH administration (4 g/kg) despite similar clearance rates. Females also showed increased operant EtOH self-administration and progressive ratio performance over a 30-day baseline period compared to males. The AMPAR antagonist GYKI 52466 (0-10 mg/kg, IP) dose-dependently reduced EtOH-reinforced lever pressing in both sexes, with no differences in potency or efficacy. DiscussionThese findings confirm that female C57BL/6J mice consume more EtOH than males in home-cage conditions and exhibit reduced acute sedation, potentially contributing to higher EtOH intake. Females demonstrated increased operant EtOH self-administration and motivation, indicating higher reinforcing efficacy. The lack of sex differences in the relative effects of GYKI 52466 suggests that AMPAR activity is equally required for EtOH reinforcement in both sexes.

Alcohol use disorder (AUD) is related to excessive binge alcohol consumption, and there is considerable interest in associated factors that promote intake. AUD has many behavioral facets that enhance inflexibility toward alcohol consumption, including impulsivity, motivation, and attention. Thus, it is important to understand how these factors might promote responding for alcohol and can change after protracted alcohol intake. Previous studies have explored such behavioral factors using responding for sugar in the 5-Choice Serial Reaction Time Task (5-CSRTT), which allows careful separation of impulsivity, attention, and motivation. Importantly, our studies uniquely focus on using alcohol as the reward throughout training and testing sessions, which is critical for beginning to answer central questions relating to behavioral engagement for alcohol. Alcohol preference and consumption in C57BL/6 mice were determined from the first 9 sessions of 2-hour alcohol drinking which were interspersed among 5-CSRTT training. Interestingly, alcohol preference but not consumption level significantly predicted 5-CSRTT responding for alcohol. In contrast, responding for strawberry milk was not related to alcohol preference. Moreover, high-preference (HP) mice made more correct alcohol-directed responses than low-preference (LP) during the first half of each session and had more longer reward latencies in the second half, with no differences when performing for strawberry milk, suggesting that HP motivation for alcohol may reflect "front-loading." Mice were then exposed to an Intermittent Access to alcohol paradigm and retested in 5-CSRTT. While both HP and LP mice increased 5-CSRTT responding for alcohol, but not strawberry milk, LP performance rose to HP levels, with a greater change in correct and premature responding in LP versus HP. Overall, this study provides three significant findings: 1) alcohol was a suitable reward in the 5-CSRTT, allowing dissection of impulsivity, attention, and motivation in relation to alcohol drinking, 2) alcohol preference was a more sensitive indicator of mouse 5-CSRTT performance than consumption, and 3) chronic alcohol drinking promoted behavioral engagement with alcohol, especially for individuals with less initial engagement.

Structured abstractO_ST_ABSBackgroundC_ST_ABSEven at lower doses, ethanol exposure impacts both the brain and behavior. Emerging work has shown that chronic exposure to lower doses of ethanol may lead to inflexible behaviors and promote aberrant reward seeking. This study investigated the impact of chronic, low-dose ethanol exposure on neural substrates of reward and on motivated behavior. MethodsAdult C57BL/6J mice were trained to self-administer sucrose. Throughout training, mice received an injection of low-dose ethanol (0.5g/kg) or saline, 1 hour after each session. Mice did not receive ethanol during testing. Mice were then tested in a PR task in which the reward magnitude of reinforcer was reduced (small or large) or increased (small or large). A subset of mice expressed a retrograde tracer in the nucleus accumbens (NAc), and cFos expression within NAc circuits was analyzed following a sucrose self-administration session. ResultsChronic low-dose ethanol exposure altered behavioral responding in female mice following small changes in reward magnitude. Female mice showed divergent response patterns when there was a small reduction in reward magnitude, with greater proportions of ethanol-exposed female mice either increasing or decreasing responding versus controls. Following a small increase, low-dose ethanol female mice significantly increased responding versus controls. Female - but not male - mice exposed to chronic low-dose ethanol shifted behavioral strategy with a reduction in magazine checking behavior. Low-dose ethanol exposure altered cFos expression within the prelimbic cortex and its projections to the NAc during reward seeking. ConclusionsChronic, low-dose ethanol altered behavioral responding and strategy in female mice in response to changes in reward value. Low-dose ethanol exposure impacted cFos induction in prelimbic cortex and its projections to NAc in both female and male mice. Future studies should investigate the consequences of chronic, low-dose ethanol on the brain and behavior to understand what underlying processes drive aberrant reward-seeking behaviors.

Oxytocin has been proposed to play a role in the development and maintenance of alcohol use disorder (AUD) through its interactions with stress pathways. Empirical evidence that indicates altered associations between endogenous oxytocin and stress reactivity in AUD is currently lacking. In this study, we investigated baseline plasmatic oxytocin concentrations of early-abstinent patients with AUD (N = 40) and matched healthy control participants (N = 37), who completed the Trier Social Stress Test (TSST) as well as a control task on two separate visits. We measured salivary cortisol and pulse rate as indicators of a physiological stress response, and anxiety ratings as an indicator of an affective stress response at multiple time points. Baseline oxytocin levels did not significantly differ between the groups. However, our results suggest an altered association of oxytocin and affective stress responses in participants with AUD: while participants with AUD showed a positive relationship between plasmatic oxytocin levels and stress-induced anxiety increase, the opposite relationship was found in control participants. We did not find evidence for an association of oxytocin with physiological stress responses. Assuming that affective stress responses mediate addiction-related behaviors like craving and relapse, our findings suggest that administering exogenous oxytocin might not be beneficial in treating AUD, at least during the early stages of abstinence.

Background and AimsIncreasing evidence suggests that a ketogenic (high-fat, low-carbohydrate) diet intervention reduces alcohol withdrawal severity and alcohol craving in individuals with alcohol use disorder (AUD) by shifting brain energetics from glucose to ketones. We hypothesized that the ketogenic diet would reduce a brain craving signature when individuals undergoing alcohol detoxification treatment were exposed to alcohol cues. MethodsWe performed a secondary analysis of functional magnetic resonance data of n=33 adults with an AUD were randomized to a ketogenic diet (n=19) or a standard American diet (n=14) and underwent three weeks of inpatient alcohol detoxification treatment. Once per week, participants performed an alcohol cue-reactivity paradigm with functional magnetic resonance imaging. We extracted brain responses to food and alcohol cues and quantified the degree to which each set of brain images shared a pattern of activation with a recently validated Neurobiological Craving Signature (NCS). We then performed a group-by-time repeated measures ANOVA to test for differences in craving signature expression between the dietary groups over the three-week treatment period. We also correlated these expression patterns with self-reported wanting ratings for alcohol cues. ResultsFor alcohol relative to food cues, there was a main effect of group, such that the ketogenic diet group showed lower NCS expression across all three weeks of treatment. The main effect of time and the group-by-time interaction were not significant. Self-reported wanting for alcohol cues reduced with KD compared to SA but did not correlate with the NCS score. ConclusionsA ketogenic diet reduces self-reported alcohol wanting, and induced lower brain craving signatures to alcohol cues during inpatient treatment for AUD.

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, for 24 hr/day with daily intake measured via change in bottle weight over the 24 hr period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lickometer systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD, has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs. SUMMARYThis protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lickometer system that enables microstructural analysis of drinking behavior.

BackgroundStudies show that Black and Hispanic Veterans have a higher prevalence of alcohol use disorder (AUD) than White Veterans. We examined whether the relationship between self-reported race/ethnicity and AUD diagnosis varies by self-reported alcohol consumption. MethodsThe sample included 700,013 Black, Hispanic, and White Veterans enrolled in the Million Veteran Program cohort. Alcohol consumption was defined as an individuals maximum score on the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) questionnaire, a screen for hazardous or harmful drinking. The primary outcome, AUD, was defined by the presence of ICD-9/10 codes in the electronic health record. We used logistic regression with interactions to assess the association between race/ethnicity and AUD by maximum AUDIT-C score. ResultsBlack and Hispanic Veterans were more likely to have an AUD diagnosis than White Veterans despite similar levels of alcohol consumption. The difference was greatest between Black and White men. At all but the lowest and highest levels of alcohol consumption, Black men had 24%-111% greater odds of an AUD diagnosis. The association between race/ethnicity and AUD diagnosis remained after adjustment for alcohol consumption, alcohol-related disorders, and other potential confounders. ConclusionsThe large discrepancy in AUD diagnosis across groups despite a similar distribution of alcohol consumption measures suggests that Veterans are differentially assigned an AUD diagnosis by race/ethnicity. Efforts are needed to examine the causes of the observed differences and to implement changes, such as structured diagnostic methods, to address a likely contributor to racial differences (i.e., bias) in AUD diagnosis.

Development and severity of alcohol use disorder (AUD) has been linked to variations in gut microbiota and their associated metabolites in both animal and human studies. However, the involvement of the gut microbiome in alcohol consumption of individuals with AUD undergoing treatment remains unclear. To address this, stool samples (n=48) were collected at screening (baseline) and trial completion from a single site of a multi-site double-blind, placebo-controlled trial of Zonisamide in individuals with AUD. Alcohol consumption, gamma-glutamyl transferase (GGT), and phosphatidylethanol (PEth)levels were measured both at baseline and endpoint of 16-week trial period. Fecal microbiome was analyzed via 16S rRNA sequencing and metabolome via untargeted LC-MS. Both sex (p = 0.003) and psychotropic medication usage (p = 0.025) are associated with baseline microbiome composition. The relative abundance of 12 genera at baseline was correlated with percent drinking reduction, baseline and endpoint alcohol consumption, and changes in GGT and PeTH over the course of treatment (p.adj < 0.05). Overall microbiome community structure at baseline differed between high and low responders (67-100% and 0-33% drinking reduction, respectively; p = 0.03). A positive relationship between baseline fecal GABA levels and percent drinking reduction (R=0.43, p < 0.05) was identified by microbiome function prediction and confirmed by ELISA and metabolomics. Predicted microbiome function and metabolomics analysis have found that tryptophan metabolic pathways are over-represented in low responders. These findings highlight importance of baseline microbiome and metabolites in alcohol consumption in AUD patients undergoing zonisamide treatment.

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