ABSTRACT Alcohol use disorder (AUD) is a complex brain disease with high morbidity, mortality, and limited treatment options, causing significant societal burden. Minocycline, well known for its anti-inflammatory and immunomodulatory properties, could potentially treat AUD but its antimicrobial activity would be inessential, and even a liability, for long-term treatment in AUD patients. 10-Butyl Ether Minocycline (BEM) was synthesized to retain minocycline’s off-target actions and eliminate antimicrobial properties. We hypothesized that BEM would reduce high ethanol consumption in common murine models of binge drinking without affecting ethanol elimination. The Drinking-In-the-Dark (DID) paradigm was used to assess BEM’s efficacy in reducing binge-like ethanol consumption. BEM dose-responsively reduced ethanol consumption in both female and male mice. Ethanol elimination following administration of both BEM and ethanol was evaluated using gas chromatography. BEM had no effect on ethanol metabolism in either sex, suggesting that its mechanism is independent and, thus, should not exacerbate ethanol-related toxicity when the two are taken together. Previous findings showed BEM effectively reduced chronic ethanol consumption. Future studies are underway to evaluate BEM’s potential as an AUD treatment, which so far have shown BEM to be safer than minocycline and inexpensive to produce.

Nest building is a natural behavior that can readily be analyzed in mice in the home cage environment. Nest building is involved in thermoregulation, positive motivational states, and motor function, and alterations in this behavior have been proposed as an index for ethanol withdrawal severity in mice. However, nest building outcomes after voluntary ethanol consumption have not been examined. Here, we tested male and female C57BL/6J mice on a 4-day drinking in the dark (DID) paradigm of binge-like drinking with either ethanol or a water control and analyzed nest scores at two timepoints (48 h and 7 days) after the last DID session. At 48 h after the last DID session, there were no differences between the two groups in nest quality. At 7 days after DID, ethanol-drinking animals showed significantly lower nest scores than the water group (z = -2.369, p = 0.030). No differences were found between the ethanol- and water-drinking groups in locomotor activity or anxiety-like behavior at this timepoint in an open field test, indicating that nest building deficits in the ethanol group were likely not due to underlying differences in these behaviors. Together, these results validate the use of nest building as a naturalistic assessment of post-ethanol behavioral changes following voluntary binge-like drinking.

Sphingosine-1-phosphate (S1P) is a lipid mediator signaling through broadly expressed G protein-coupled receptors. We found that S1P is regulated by alcohol and that S1P receptor agonists reduce alcohol drinking in rodent models. Specifically, we observed that two S1P receptor agonists FDA-approved for multiple sclerosis, fingolimod and ozanimod, and the more brain penetrant S1P 1 receptor agonist CYM5442, reduced binge alcohol drinking in the drinking in the dark (DID) paradigm in mice. CYM5442 also reduced drinking in dependent mice in the chronic intermittent ethanol vapor paradigm of dependence-induced increased drinking paired with 2 bottle-choice (CIE-2BC) as well as in non-dependent mice. CYM5442 reduced operant oral alcohol self-administration in both non-dependent and dependent rats made dependent by vapor exposure, and reduced motivation for alcohol in dependent rats tested in a progressive ratio schedule of reinforcement. CYM5442 significantly prevented cue-induced reinstatement in alcohol-dependent rats, a model of relapse to alcohol seeking. CYM5442 also reduced intake of non-drug reinforcers, including sucrose, food, water and, to a lesser extent, saccharine. Notably, CYM5442 was less aversive than naltrexone, an FDA-approved medication for the treatment of alcohol use disorder that shares a similar broad reducing action on alcohol intake and consummatory behavior. CYM5442 had no effect on loss of righting reflex, alcohol metabolism, motor coordination or spontaneous locomotor activity in rodents. Lastly, gene expression analysis by RNA-Seq revealed that S1P regulates a complex set of genes in the transition to alcohol dependence. Overall, our results establish S1P signaling as a novel therapeutic target for alcohol use disorder.

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 (ERRγ) 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 ERRγ 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 ERRγ as a regulator of excessive alcohol drinking and seeking, and a therapeutic target for AUD.

Binge alcohol drinking increases the risk of developing an alcohol use disorder (AUD) and comorbid psychopathology. The lateral hypothalamus (LH) is a brain structure that integrates cognitive and sensory information to tightly regulate motivated behavior, including binge drinking. Importantly, LH function is vulnerable to modulation by the pro-stress neuropeptide corticotropin-releasing factor (CRF), and acute antagonism of CRF receptor 1 (CRFR1) in the LH blunts binge drinking. However, the role of LH CRFR1 in chronic binge drinking is unknown. We used genetically targeted knockdown (KD) of CRFR1 in the LH of male and female mice followed by three weeks of binge drinking using the “Drinking in the Dark” (DID) model. CRFR1 KD in the posterior LH increased alcohol consumption, independent of sex, with no effect of KD in the anterior LH. Consistent with this, total alcohol consumption was negatively correlated with the location of CRFR1 KD in the LH along the anterior-posterior axis. CRFR1 KD did not alter water consumption or body weight, suggesting the effects of CRFR1 KD on alcohol consumption were not due to broad disruption of fluid intake or homeostatic function. In contrast to the observed effects on binge drinking, CRFR1 KD increased anxiety-like behavior and blunted sucrose preference, independent of KD location in the LH. Our findings provide foundational insight into LH function in the context of AUD and prompt further investigation into the divergent roles that distinct circuitry or cell populations along the anterior-posterior axis of the LH may play in binge drinking.

BackgroundAlcohol use disorder is characterized by maladaptive patterns of alcohol consumption, with emerging evidence suggesting that neuropeptide Y (NPY) signaling through Y1 and Y2 receptors (Y1R and Y2R) within the central amygdala (CeA) plays a critical role in modulating ethanol intake. The current experiments investigate the neural mechanisms underlying binge‐like ethanol drinking, focusing on the involvement of Y1R+ CeA‐to‐lateral hypothalamus (LH) projections, dynamic interactions between Y1R and Y2R within the CeA, and the impact of chronic ethanol exposure on Y1R protein expression.MethodsNPY1R‐ires‐cre mice received LH cannulation, were infused with cre‐dependent inhibitory (Gi) Designer Receptor Exclusively Activated by Designer Drug (DREADD) or control virus into the CeA, and went through drinking in the dark (DID). Other animals were treated intra‐CeA with an NPY overexpression vector (FIB‐NPY) or control, and went through DID, intermittent access to ethanol (IAE), and open‐field testing. Viral placements and receptor targets were assessed via qPCR. Finally, mice went through six cycles of DID, and Y1R immunohistochemical (IHC) labeling on neurons was assessed for animals sacrificed after the final DID session or after a 24‐h period of abstinence.ResultsChemogenetic inhibition of Y1R+ CeA‐LH projections selectively reduced binge‐like ethanol drinking in male mice without affecting female mice. Viral NPY overexpression revealed behavioral effects and predictive relationships between receptor mRNA expression and intake patterns. Although no significant differences were found in Y1R/NeuN colocalization across sex and treatment groups, correlational analyses revealed that Y1R expression varied with individual ethanol consumption.ConclusionsCollectively, these results support a model wherein Y1R signaling within the CeA regulates ethanol consumption through circuit‐specific mechanisms and broader neuroadaptive changes influenced by sex and individual drinking patterns. This research advances our understanding of the neurobiological mechanisms underlying binge‐like ethanol intake and highlights the complex, sex‐dependent roles of NPY‐Y1R and Y2R signaling in the CeA.

Excessive alcohol use has emerged as the strongest modifiable risk factor for the development of Alzheimer's disease (AD), but the underlying neural mechanisms are only beginning to be understood. Recent preclinical work suggests that alcohol consumption may have an impact on many pathologies and phenomena crucial to the development and pathogenesis of AD. However, little attention has been focused on pure tauopathy models to closely examine tau pathogenesis and neuroinflammation within a voluntary alcohol exposure paradigm. We exposed a mouse model of pathological tau (pTau), P301S, to a voluntary alcohol paradigm known as drinking-in-the-dark (DID) for 21 days of voluntary daily alcohol consumption. In P301S mice, moderate alcohol consumption contributed to gait disruptions, acceleration of pTau spread, and enhancement of damage-associated microglia. This work identifies key interactions between alcohol and AD-related phenotypes which set the stage for future investigation into the neurobiological mechanisms behind these interactions.

Developing a reverse translational model of low-intensity rTMS in alcohol use disorder: The influence of theta burst stimulation protocols on binge alcohol drinking in mice.

Voluntary wheel-running reduces harmful drinking in a genetic risk model for drinking to intoxication.

Background:Chronic pain is a leading cause of disability, significantly decreases quality of life, and is highly comorbid with substance use disorders, including alcohol use disorder (AUD). This is due, in part, to the pain-relieving effects of alcohol acting as a potential driving force for the progression and maintenance of AUD. Despite a substantial body of historic, anecdotal, clinical, and epidemiological evidence supporting the analgesic efficacy of alcohol, few preclinical studies have investigated the effects of pain on volitional alcohol drinking. Further, no studies to date have investigated aversion-resistant drinking in the context of persistent pain.Methods:To address this gap in the literature, the current study combined quinine adulteration with the drinking in the dark (DID) model of binge-like alcohol drinking to assess the effects of complete Freund’s adjuvant (CFA)-induced persistent inflammation on aversion-resistant binge-like alcohol drinking in female and male Long Evans rats.Results:Consistent with previous findings from our laboratory, CFA did not affect binge-like alcohol drinking in either sex, although female rats did consume greater levels of alcohol during baseline and post-CFA DID sessions. Similarly, CFA did not affect quinine adulterated binge-like alcohol drinking in either sex.Conclusions:This study is the first to investigate the impact of persistent inflammation on aversion-resistant alcohol drinking. Although we found no effects of CFA on quinine adulterated binge-like alcohol drinking, these findings provide the groundwork for future investigations into this otherwise unstudied aspect of the pain-alcohol relationship.

Altered c-Fos expression following alcohol intake in discrete brain regions of galanin 3 receptor knockout mice.

Objective:Fetal 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.Method:We 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.Results:After 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.Conclusions:Test tube or VDM resulted in similar average daily ethanol consumption and resultant BACs in female mice

The prefrontal cortex (PFC) is one of the last brain regions to fully mature, making it particularly sensitive to stress and drug use early in life. Both human and rodent studies find long-lasting behavioral changes after adolescent alcohol exposure that implicate underlying disruptions in PFC development, including structural abnormalities and altered brain functional connectivity. Few rodent studies have been conducted to understand the network-level implications of these disruptions. We assessed how adolescent binge-like alcohol consumption in a drinking in the dark (DID) model affected adult aversion-resistant alcohol consumption, exploration, and brain-wide functional connectivity in mice. Approximately one month after the conclusion of DID, only female mice exposed to alcohol during adolescence exhibited aversion-resistant alcohol preference in adulthood. Adult females exhibited additional sex-specific changes in exploratory behavior in the elevated plus maze after adolescent alcohol consumption. Resting state neuroimaging revealed changes in prefrontal cortical connections with sensory motor, hippocampal, striatal, and other networks, providing insights into the putative systems underlying deficits caused by adolescent alcohol exposure. Critically, our data corroborate a growing body of literature in human and rodent studies demonstrating that adolescent alcohol use may increase risk for adult alcohol use more strongly in females. Finally, we identify neural correlates of this effect that include both known and novel networks and tie these back to human datasets, allowing biological and mechanistic targets to be further explored for future study and interventions.

Nest building is a natural behavior that can readily be analyzed in mice in the home cage environment. Nest building is involved in thermoregulation, positive motivational states, and motor function, and has been proposed as an index for ethanol withdrawal severity in mice. However, nest building outcomes after voluntary ethanol consumption have not been examined. Here, we tested male and female C57BL/6J mice on a 4-day drinking in the dark (DID) paradigm of binge-like drinking with either ethanol or a water control and analyzed nest scores at two time points (48 hours and 7 days) after the last DID session. At 48 hours after the last DID session, there were no differences between the two groups in nest quality. At 7 days after DID, ethanol-drinking animals showed significantly lower nest scores than the water group. No differences were found between the ethanol- and water-drinking groups in locomotor activity or anxiety-like behavior at this timepoint, indicating that nest building deficits in the ethanol group were likely not due to underlying differences in these behaviors. Together, these results validate the use of nest building as a naturalistic assessment of withdrawal-associated behavioral changes following voluntary binge-like ethanol consumption.

Prolonged drinking in the dark in adult female mice attenuates the central and peripheral cytokine response to lipopolysaccharide.

Industrial workers and active military personnel within combat roles face heightened risk for blast pressure wave traumatic brain injury (bTBI). Previous studies have shown that experiencing TBI is associated with increased alcohol (EtOH) consumption and illicit substance use. Notably, alcohol use typically begins during late adolescence or early adulthood, a period that precedes many TBI incidents; moreover, early-onset drinking is further associated with heightened risk of developing an alcohol use disorder (AUD) even in the absence of TBI. Adolescent binge drinking can induce lasting cognitive and astrocyte changes, impacting brain recovery and repair. However, the impact of adolescent drinking history on behavioral recovery after bTBI and its role in the subsequent escalation of alcohol consumption remain unexplored. Here, we used a mouse model to investigate how adolescent (PND28-42) and young adult (PND60-90) EtOH consumption affects behavioral outcomes following bTBI. We aim to determine whether the history of adolescent binge drinking contributes to bTBI-induced escalation in EtOH intake, preference, or worsened fear memory and anxiety. Adolescent mice were subjected to drinking in the dark (DID) EtOH paradigm for 4 weeks, then randomly assigned to sham, mild-bTBI, or severe-bTBI. Behavioral testing was conducted, followed by a second DID. Both EtOH and bTBI independently induced hyperlocomotor activity in a sex-dependent manner. These findings reflect an increase in risk-taking rather than generalized anxiety. Importantly, a history of adolescent EtOH consumption synergistically worsened bTBI-induced impaired fear extinction in both sexes. Changes in EtOH preference post-bTBI are context-dependent, with male mice showing a significant decrease in preference following mild-bTBI and prior EtOH exposure, while females exhibited a trend toward increased preference post-bTBI, with significant increases in preference observed only when comparing pre- to post-bTBI drinking behavior. Both males and females exhibited vulnerability to the combined effects of adolescent EtOH consumption and bTBI on fear extinction, while female mice showed a unique vulnerability to the escalation in EtOH preference.

Ethanol metabolism is intimately linked with the physiological and behavioral aspects of ethanol consumption. Ethanol is mainly oxidized by alcohol dehydrogenase (ADH) to acetaldehyde and further to acetate via aldehyde dehydrogenases (ALDHs). Understanding how ethanol and its metabolites work together to initiate and drive continued ethanol consumption is crucial for identifying interventions for alcohol use disorder (AUD). Therefore, the goal of our study was to determine how ADH1, which is mainly peripherally expressed and metabolizes >90% of ingested ethanol, modulates ethanol metabolite distribution and downstream behaviors. Ethanol consumption in drinking-in-the-dark (DID) and two-bottle choice (2BC) drinking paradigms, ethanol metabolite concentrations, and lickometry were assessed after ADH1 inhibition and/or in Adh1-knockout (Adh1 KO) mice. We found that Adh1 KO mice of both sexes exhibited decreased ethanol consumption and preference compared with wild-type (WT) mice in DID and 2BC. ADH1 inhibitor fomepizole (4-MP) also significantly decreased normal and sweetened ethanol consumption in DID studies. Measurement of ethanol and its metabolites revealed that ethanol was increased at 1 h but not 15 min, peripheral acetaldehyde was slightly decreased at both timepoints, and ethanol-induced increases in acetate were abolished after ethanol administration in Adh1 KO mice compared with controls. Similarly, ethanol accumulation as a function of consumption was 2-fold higher in Adh1 KO or 4-MP-treated mice compared with controls. We then used lickometry to determine how this perturbation in ethanol metabolism affects drinking microstructure. Adh1 KO mice consume most of their ethanol in the first 30 min, like WT mice, but display altered temporal shifts in drinking behaviors and do not form normal bout structures, resulting in lower ethanol consumption. Our study demonstrates that ADH1-mediated ethanol metabolism is a key determinant of ethanol consumption, highlighting a fundamental knowledge gap regarding how ethanol and its metabolites drive ethanol consumption.

IntroductionAlcohol use disorder (AUD) is a major problem that causes significant health and social problem. Pharmacological treatments for AUD are limited in their effectiveness, and new drugs are required (ref). Recently, the potential use of psychedelics as novel therapies in treating mental diseases has come to the forefront in psychiatry. For instance, lysergic acid diethylamide (LSD) is a psychedelic acting as partial agonist of 5-HT2A receptors and exerts its mechanism of action through the modulation of the dopaminergic system in the ventral tegmental area (VTA). For instance, VTA plays a pivotal role in mediating the neurophysiology of AUD. Indeed, chronic alcohol consumption produces a disruption on the dopaminergic (DA) neurotransmission in the VTA. However, the mechanism of action and pre-clinical data about the effectiveness of psychedelic -based therapies for AUD remain limited.Aims and ObjectivesThis translational study aims at determining whether the LSD can reduce alcohol consumption in an animal model of AUD as well as its ability to restore the impaired VTA DA neurotransmission induced by the AUD. To do so, we employed a mouse model of self-ethanol administration with a paradigm DID (Drinking in the dark) to mimic binge-like drinking in humans and we performed behavioral paradigms and in vivo single-unit extracellular recordings.MethodC57BL6/J 8-week-old male mice underwent 6 cycles of DID. In each cycle, the water bottles were removed from all cages and replaced with bottles containing 20% ethanol solution for 2 hours per day, for 4 consecutive days in one experimental group (AUD mice). In another group of mice (control, CTL) the water bottles were not replaced with ethanol. After 6 cycles of DID, mice received a single intraperitoneal injection of vehicle or LSD (150 mcg/kg, a dose that has been demonstrated to exert antidepressant effects. The day after the injections, animals were assessed for the alcohol consumption with the 40 days two-bottle choice test. Twenty-four hours from the last drinking session in the two- bottle choice test, locomotion was assessed by using the open field test (OFT) and the rotarod test. Finally, in vivo single unit extracellular recordings of VTA DA neurons were performed.ResultsAUD mice did not show difference in locomotion in the OFT, compared to CTL (p=0.012). However, AUD mice displayed decreased latency to fall (p<0.01) in the rotarod test. These effects were coupled to an increased DA VTA firing rate activity (p<0.05) and reduced intra-burst frequency (p<0.001) compared to CTL mice. LSD significantly reduced the alcohol intake (p<0.05) and normalized the locomotor impairments in the AUD mice (p<0.001). Moreover, LSD did not revert the enhanced DA VTA firing rate activity but it increased the intra-burst frequency (p<0.001) which was reduced by the chronic alcohol consumption.Discussion and ConclusionThis work show that single administration of LSD reduces alcohol consumption and reverts locomotor impairment. Moreover, LSD partially restores the VTA DA deficits induced by chronic alcohol consumption. Overall, this study will broaden our understanding of the potential therapeutic effects of psychedelics in treating AUD.

Background:Ethanol metabolism is intimately linked with the physiological and behavioral aspects of ethanol consumption. Ethanol is mainly oxidized by alcohol dehydrogenase (ADH) to acetaldehyde and further to acetate via aldehyde dehydrogenases (ALDHs). Understanding how ethanol and its metabolites work together to initiate and drive continued ethanol consumption is crucial for identifying interventions for alcohol use disorder (AUD). Therefore, the goal of our study was to determine how ADH1, which is mainly peripherally-expressed and metabolizes >90% of ingested ethanol, modulates ethanol metabolite distribution and downstream behaviors.Methods:Ethanol consumption in drinking-in-the-dark (DID) and two-bottle choice (2BC) drinking paradigms, ethanol metabolite concentrations, and lickometry were assessed after ADH1 inhibition and/or in Adh1-knockout (Adh1 KO) mice.Results:We found that Adh1 KO mice of both sexes exhibited decreased ethanol consumption and preference compared to wild-type (WT) mice in DID and 2BC. ADH1 inhibitor fomepizole (4-MP) also significantly decreased normal and sweetened ethanol consumption in DID studies. Measurement of ethanol and its metabolites revealed that ethanol was increased at 1h but not 15 min, peripheral acetaldehyde was slightly decreased at both time points, and ethanol-induced increases in acetate were abolished after ethanol administration in Adh1 KO mice compared to controls. Similarly, ethanol accumulation as a function of consumption was 2-fold higher in Adh1 KO or 4-MP treated mice compared to controls. We then used lickometry to determine how this perturbation in ethanol metabolism affects drinking microstructure. Adh1 KO mice consume most of their ethanol in the first 30 min like WT mice but display altered temporal shifts in drinking behaviors and do not form normal bout structures, resulting in lower ethanol consumption.Conclusions:Our study demonstrates that ADH1-mediated ethanol metabolism is a key determinant of ethanol consumption, highlighting a fundamental knowledge gap around how ethanol and its metabolites drive ethanol consumption.

Social stress is widely recognized for increasing ethanol consumption, an effect mediated by an enhanced neuroinflammatory response. High-fat diets (HFDs) alleviate stress and have been shown to reduce cocaine-seeking behavior, likely by serving as an alternative reinforcer. This study examines whether intermittent HFD administration following social defeat (SD) mitigates long-term increases in ethanol (EtOH) consumption and neuroinflammatory responses in adult male mice. Two intermittent HFD protocols were tested: HFD1 × 2 (one-hour access twice a week), and HFD2 × 3 (two-hour access three times a week), initiated immediately after the last SD episode and maintained through the drinking-in-the-dark (DID) protocol. Defeated mice were categorized as resilient or susceptible using the social interaction test (SIT). At the end of the procedure, gene expression analysis of neuroinflammatory markers was conducted in the hippocampus and striatum. Intermittent HFD did not increase body weight, despite promoting binge-eating behavior. As expected, only susceptible SD mice on a standard diet (STD) showed increased ethanol consumption, whereas those on either HFD protocol consumed ethanol at levels comparable to non-stressed controls. Elevated Il-6 and Tnfα levels, along with mitochondrial dysfunction, were observed in the hippocampus and striatum of STD-fed defeated mice but were entirely absent in HFD-fed mice. These findings suggest that intermittent HFD access effectively prevents stress-induced ethanol consumption and neuroinflammatory alterations. Limited HFD access may represent a promising nutritional strategy to counteract stress-related alcohol use disorders.