The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways.

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

Orexinergic lateral hypothalamus (LH) projections to medial septum (MS) modulate ethanol-induced sedation in male and female mice and binge-like ethanol drinking in male mice only.

Orexin in both the lateral hypothalamus (LH) and medial septum (MS) is involved in sleep- and consciousness-related conditions. Since orexin modulates the intoxicating as well as rewarding effects of ethanol, this study focused on the role of orexin-projecting neurons from the LH to the MS, and this neurocircuit's role in mediating the sedative effects of alcohol. Drinking-in-the-Dark (DID) behavior was also assessed as a measure of the role of the LH-MS pathway in modulating binge-like ethanol intake, with a particular focus on sex differences in both behavioral paradigms. Male and female Hcrt-ires-cre mice received cannulation in the MS, while the LH was injected bilaterally with cre-dependent excitatory (Gq) Designer Receptor Exclusively Activated by Designer Drug (DREADD), inhibitory (Gi) DREADD or control virus. All subjects received a 3.75 g/kg dose of 20 % ethanol intraperitoneally and the sedative effect was assessed by the loss of righting reflex (LORR). After behavioral testing, brains were used for c-Fos immunohistochemistry analyses. A separate cohort of mice was used for a 2-week DID protocol using excitatory (Gq) DREADD and control virus. Gq DREADD-induced activation of the orexin neurocircuitry from the LH to the MS significantly reduced sedation time in both female and male mice. Furthermore, CNO treatment failed to alter ethanol sedation times in both animals expressing Gi DREADDs and control virus. There were no significant differences in blood ethanol concentrations (BECs) in any experimental group, suggesting that changes in sedation were not due to treatment-induced alterations of ethanol metabolism. Interestingly, in the DID study, only male mice decreased their ethanol consumption when Gq DREADDs were activated. These results provide novel evidence on the role played by this orexinergic LH to MS circuit on the sedative effects of ethanol and ethanol consumption in a sex-dependent manner. Thus, the MS should be considered further as a novel sexually dimorphic target.

Modeling Brain Gene Expression in Alcohol Use Disorder with Genetic Animal Models.

Animal genetic models have and will continue to provide important new information about the behavioral and physiological adaptations associated with alcohol use disorder (AUD). This chapter focuses on two models, ethanol preference and drinking in the dark (DID), their usefulness in interrogating brain gene expression data and the relevance of the data obtained to interpret AUD-related GWAS and TWAS studies. Both the animal and human data point to the importance for AUD of changes in synaptic transmission (particularly glutamate and GABA transmission), of changes in the extracellular matrix (specifically including collagens, cadherins and protocadherins) and of changes in neuroimmune processes. The implementation of new technologies (e.g., cell type-specific gene expression) is expected to further enhance the value of genetic animal models in understanding AUD.

Removal of the ovaries suppresses ethanol drinking and promotes aversion-resistance in C57BL/6J female mice.

RATIONALE: Female rodents consume more ethanol (EtOH) than males and exhibit greater aversion-resistant drinking in some paradigms. Ovarian hormones promote EtOH drinking but the contribution of ovarian hormones to aversion-resistant drinking has not been assessed. OBJECTIVES: We aimed to investigate the role of ovarian hormones to aversion-resistant drinking in female mice in a drinking in the dark (DID) task. METHODS: Female C57BL/6 J mice first underwent an ovariectomy (OVX, n = 16) or sham (SHAM, n = 16) surgery. Four weeks following surgery, mice underwent a DID paradigm where they were given access to water and 15% EtOH 3 h into the dark cycle for up to 4 h across 15 drinking sessions. To assess frontloading behavior, bottles were weighed at 30 min, 2 h, and 4 h. Aversion-resistance was tested by adding escalating concentrations of quinine (0, 100, 250, and 500 µM) to the 15% EtOH bottle on sessions 16 - 19. RESULTS: Removal of the ovaries reduced EtOH consumption in OVX subjects. When assessing aversion-resistant EtOH drinking, mice with ovarian hormones (SHAM) reduced consumption of 250 and 500 µM quinine in EtOH, while OVX subjects exhibited aversion-resistance at all quinine concentrations. OVX mice had greater frontloading for quinine + EtOH at higher concentrations of quinine. CONCLUSIONS: These results indicate that circulating ovarian hormones may be protective against the development of aversion-resistant EtOH drinking and call for further investigation of the role of ovarian hormones in models of addictive behavior.

Somatostatin signaling modulates binge drinking behavior via the central nucleus of the amygdala.

Somatostatin (SST) is a neuropeptide widely expressed in the central nervous system with dense expression in limbic regions such as the extended amygdala. It has recently gained attention for playing a role in modulating alcohol use disorders and co-morbid neuropsychiatric disorders. However, the role of SST in the central nucleus of the amygdala (CeA), a key region for neuropeptide regulation of alcohol and anxiety related behaviors, in alcohol consumption has not been assessed. In this work we perform an initial examination of the interaction between the CeA SST system and binge ethanol intake. Binge intake is a dangerous pattern of excessive ethanol consumption associated with health complications and the transition into alcohol dependence. We use the Drinking in the Dark (DID) model of binge intake in C57BL/6J male and female mice to examine: 1) the impact of 3 DID cycles on CeA SST expression; 2) the effect of intra-CeA SST injection on binge-like ethanol consumption; and 3) if the SST receptor 2 or 4 (SST2R or SST4R) mediate any effect on consumption. Our results show binge-like ethanol intake decreases SST expression in the CeA, but not neighboring basolateral amygdala. We further found intra-SST CeA administration reduces binge ethanol intake. This decrease was replicated by the administration of an SST4R agonist. These effects were not sex-dependent. Overall, this work lends further support for SST playing a role in alcohol related behaviors and as a potential therapeutic target.

Sex chromosome and gonadal hormone contributions to binge-like and aversion-resistant ethanol drinking behaviors in Four Core Genotypes mice.

Introduction: While substantial research has focused on the contribution of sex hormones to driving elevated levels of alcohol drinking in female rodents, fewer studies have investigated how genetic influences may underlie sex differences in this behavior. Methods: We used the Four Core Genotypes (FCG) mouse model to explore the contribution of sex chromosome complement (XX/XY) and gonad type [ovaries (Sry-)/testes (Sry+)] to ethanol (EtOH) consumption and quinine-resistant drinking across two voluntary self-administration tasks: limited access consumption in the home cage and an operant response task. Results: For limited access drinking in the dark, XY/Sry + (vs. XX/Sry +) mice consumed more 15% EtOH across sessions while preference for 15% EtOH vs. water was higher in XY vs. XX mice regardless of gonad type. XY chromosomes promoted quinine-resistant drinking in mice with ovaries (Sry-) and the estrous cycle did not affect the results. In the operant response task, responding for EtOH was concentration dependent in all genotypes except XX/Sry + mice, which maintained consistent response levels across all concentrations (5-20%) of EtOH. When increasing concentrations of quinine (100-500 µM) were added to the solution, FCG mice were insensitive to quinine-punished EtOH responding, regardless of sex chromosome complement. Sry + mice were further found to be insensitive to quinine when presented in water. Importantly, these effects were not influenced by sensitivity to EtOH's sedative effect, as no differences were observed in the time to lose the righting reflex or the time to regain the righting reflex between genotypes. Additionally, no differences in EtOH concentration in the blood were observed between any of the genotypes once the righting reflex was regained. Discussion: These results provide evidence that sex chromosome complement regulates EtOH consumption, preference, and aversion resistance and add to a growing body of literature suggesting that chromosomal sex may be an important contributor to alcohol drinking behaviors. Examination of sex-specific genetic differences may uncover promising new therapeutic targets for high-risk drinking.

Effects of chronic psychosocial stress on 'binge-like' sucrose intake in mice.

Binge eating episodes are persistent and are essential features of numerous eating disorders (EDs). Susceptibility to EDs is largely presumed to be associated with early life stress. In fact, converging evidence from preclinical animal studies have implicated stress as a driver of binge eating. Still, literature examination indicates that vulnerability to EDs may depend on factors such as severity, time, and the type of stressor. Therefore, we aimed at exploring the link between chronic psychosocial stress and 'binge-like' sucrose intake in adolescent mice. To this aim, intruders' experimental mice were exposed to the chronic subordinate colony (CSC) housing, in the presence of a resident aggressive mouse for 2 weeks. At the end of the stress period, mice were tested for anxiety-like behavior then assessed for 'binge-like' intake of sucrose using a long-term drinking in the dark (DID) method that successfully replicates binge eating in humans. As expected, and compared to single housed colony controls (SHC), CSC exposure elicited an anxiogenic-like response in the open field (OF) and elevated-plus maze (EPM) tests and reduced weight gain. Most importantly, we report here for the first time, that mice exposed to chronic psychosocial stress displayed a 'binge-like' consumption of sucrose. However, neither quinine (bitter) nor saccharin (sweet) intakes were affected by CSC exposure. Finally, using Pearson's correlation, results showed a strong correlation between anxiety-like behavior parameters and sucrose intake. Overall these findings support the validity of our chronic psychosocial stress to model binge EDs and establish the long-term consequences of stress on 'binge-like' eating in male mice. These data suggest that chronic psychosocial stress is a risk factor for developing anxiety-associated EDs.

Astrocyte expression in the extended amygdala of C57BL/6J mice is sex-dependently affected by chronic intermittent and binge-like ethanol exposure.

Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.

The effects of voluntary binge-patterned ethanol ingestion and daily wheel running on signaling of muscle protein synthesis and degradation in female mice.

Excessive ethanol ingestion can reduce skeletal muscle protein synthesis (MPS) through the disruption of signaling along the Akt-mTOR pathway and increase muscle protein degradation (MPD) through the Ubiquitin Proteasome Pathway (UPP) and autophagy. Identification of interventions that curb the disrupting effects of alcohol misuse on MPS and MPD are of central importance for the prevention of chronic health complications that arise from muscle loss. Physical activity is one potential strategy to combat the deleterious effects of alcohol on skeletal muscle. Therefore, the purpose of this study was to investigate the interaction between daily wheel running and binge-patterned ethanol consumption, through episodes of voluntary binge-patterned ethanol drinking, on signaling factors along the Akt-mTOR, Ubiquitin-Proteasome, and autophagy pathways. Adult female C57BL/6J mice received daily access to cages with or without running wheels for 2.5 h/day for five weeks. During the final five days of the study, mice received 2-4 h of daily access to sipper tubes containing water (n = 14 sedentary; n = 15 running) or 20% ethanol (n = 14 sedentary; n = 16 running) 30 min after running wheel access, using the "Drinking in the Dark" (DID) model of binge-patterned ethanol consumption. Immediately after the final episode of DID, gastrocnemius muscle was extracted. Western blotting was performed to measure proteins along Akt-mTOR, Ubiquitin-Proteasome, and autophagy pathways, and PCR was used to assess mRNA expression of atrogenes. Ethanol access increased expression of MAFbx by 82% (p = 0.048), but did not robustly influence Akt-mTOR or UPP signaling. Daily wheel access did not prevent alcohol-induced MAFbx expression; however, ethanol access decreased the phosphorylation of p70S6K by 45% in running mice (p = 0.020). These results suggest that physical activity may be insufficient to prevent alcohol-induced changes to signaling factors along pathways involved in muscle loss. Instead, binge-patterned ethanol ingestion may impair the benefits of physical activity on factors involved in MPS.

The development, but not expression, of alcohol front-loading in C57BL/6J mice maintained on LabDiet 5001 is abolished by maintenance on Teklad 2920x rodent diet.

BACKGROUND: Excessive alcohol (ethanol) consumption, such as binge drinking, is extremely commonplace and represents a major health concern. Through modeling excessive drinking in rodents, we are beginning to uncover the neurobiological and neurobehavioral causes and consequences of this pattern of ethanol intake. One important factor for modeling binge drinking in mice is that they reliably drink to blood ethanol concentrations (BECs) of 80 mg/dl or higher. Drinking-in-the-dark (DID) is a commonly used mouse model of binge drinking, and we have shown that this method reliably results in robust ethanol front-loading and binge-level BECs in C57BL/6J (B6) mice and other ethanol-preferring mouse strains/lines. However, establishing the DID model in a new vivarium space forced us to consider the use of rodent diet formulations that we had not previously used. METHODS: The current set of experiments were designed to investigate the role of two standard rodent diet formulations on binge drinking and the development of ethanol front-loading using DID. RESULTS: We found that BECs in animals maintained on LabDiet 5001 (LD01) were double those found in mice maintained on Teklad 2920x (TL20). Interestingly, this effect was paralleled by differences in the degree of front-loading, such that LD01-fed mice consumed approximately twice as much ethanol in the first 15 min of the 2-h DID sessions as the TL20-fed mice. Surprisingly, however, mice that developed front-loading during maintenance on the LD01 diet continued to display front-loading behavior after being switched to the TL20 diet. CONCLUSIONS: These data emphasize the importance of choosing and reporting diet formulations when conducting voluntary drinking studies and support the need for further investigation into the mechanisms behind diet-induced differences in binge drinking, particularly front-loading.

Regulation of alcohol drinking by ventral striatum and extended amygdala circuitry.

Alcohol use disorder is a complex psychiatric disorder that can be modeled in rodents using a number of drinking paradigms. Drinking-in-the-dark (DID) is widely used to model the binge/intoxication stage of addiction, and chronic intermittent ethanol vapor procedures (CIE) are used to induce dependence and model withdrawal/negative affect induced escalation of drinking. We discuss experiments showing the ventral striatum (vStr) and extended amygdala (EA) are engaged in response to ethanol in rodents through c-Fos/Fos immunoreactivity studies. We also discuss experiments in rodents that span a wide variety of techniques where the function of vStr and EA structures are changed following DID or CIE, and the role of neurotransmitter and neuropeptide systems studies in these ethanol-related outcomes. We note where signaling systems converge across regions and paradigms and where there are still gaps in the literature. Dynorphin/κ-opioid receptor (KOR) signaling, as well as corticotropin releasing factor (CRF)/CRF receptor signaling were found to be important regulators of drinking behaviors across brain regions and drinking paradigms. Future research will require that females and a variety of rodent strains are used in preclinical experiments in order to strengthen the generalizability of findings and improve the likelihood of success for testing potential therapeutics in human laboratory studies.

Adolescence alcohol exposure impairs fear extinction and alters medial prefrontal cortex plasticity.

After experiencing a traumatic event people often turn to alcohol to cope with symptoms. In those with post-traumatic stress disorder (PTSD) and a co-occurring alcohol use disorder (AUD), PTSD symptoms can worsen, suggesting that alcohol changes how traumatic memory is expressed. The objective of this series of experiments is to identify how alcohol drinking (EtOH), following cued fear conditioning and extinction, impacts fear expression in mice. Molecular (activity-regulated cytoskeleton-associated protein, Arc/arg3.1) and structural (dendrite and spine morphometry) markers of neuronal plasticity were measured following remote extinction retrieval. Mouse age (adolescent and adult) and sex were included as interacting variables in a full factorial design. Females drank more EtOH than males and adolescents drank more EtOH than adults. Adolescent females escalated EtOH intake across drinking days. Adolescent drinkers exhibited more conditioned freezing during extinction retrieval, an effect that persisted for at least 20 days. Heightened cued freezing in the adolescent group was associated with greater Arc/arg3.1 expression in layer (L) 2/3 prelimbic (PL) cortex, greater spine density, and reduced basal dendrite complexity. In adults, drinking was associated with reduced L2/3 infralimbic (IL) Arc expression but no behavioral differences. Few sex interactions were uncovered throughout. Overall, these data identify prolonged age-related differences in alcohol-induced fear extinction impairment and medial prefrontal cortex neuroadaptations.

Sex differences and the lack of effects of chemogenetic manipulation of pro-opiomelanocortin (POMC) neurons on alcohol consumption in male and female mice.

The endogenous opioid system has been implicated in the rewarding and reinforcing effects of alcohol. Pro-opiomelanocortin (POMC) neurons located within the arcuate nucleus of the hypothalamus (ArcN) secrete multiple peptides associated with alcohol consumption, including ß-endorphin (ß-END), α-melanocyte stimulating hormone (α-MSH), and adrenocorticotropic hormone (ACTH). In this study, we utilized chemogenetics to bidirectionally modulate ArcN POMC neurons to determine their role in alcohol and saccharin consumption and regional levels of POMC-derived peptides. Male and female POMC-cre mice were infused with viral vectors designed for cre-dependent expression of either excitatory and inhibitory DREADDs or a control vector into the ArcN. Following recovery, animals were allowed to consume alcohol or saccharin using the drinking-in-the-dark (DID) paradigm of binge-like intake for 4 consecutive days. Prior to the final test session, animals were injected with clozapine-N-oxide (2.5 mg/kg, i.p.) for DREADD activation. Following the last DID session, animals were euthanized and the ArcN, VTA, amygdala and NAc were dissected and assessed for POMC peptide expression utilizing western blotting. We found that female mice consumed more alcohol than males during DID sessions 2-4, and that chemogenetic activation had no effect on alcohol or saccharin consumption in either sex. We found that ß-END expression within the ArcN positively correlated with alcohol consumption. Given the molecular and functional heterogeneity of ArcN POMC neurons, future studies are needed to assess the effects of modulation of specific subpopulations of these neurons within the ArcN on consumption of rewarding substances such as alcohol and saccharin.

Long-term alcohol drinking in High Drinking in the Dark mice is stable for many months and does not show alcohol deprivation effects.

We have modelled genetic risk for binge-like drinking by selectively breeding High Drinking in the Dark-1 and -2 (HDID-1 and HDID-2) mice for their propensity to reach intoxicating blood alcohol levels (BALs) after binge-like drinking in a single bottle, limited access paradigm. Interestingly, in standard two-bottle choice (2BC) tests for continuously available alcohol versus water, HDID mice show modest levels of preference. This indicates some degree of independence of the genetic contributions to risk for binge-like and sustained, continuous access drinking. We had few data where the drinking in the dark (DID) tests of binge-like drinking had been repeatedly performed, so we serially offered multiple DID tests to see whether binge-like drinking escalated. It did not. We also asked whether HDID mice would escalate their voluntary intake with prolonged exposure to alcohol 2BC. They did not. Lastly, we assessed whether an alcohol deprivation effect (ADE) developed. ADE is a temporary elevation in drinking typically observed after a period of abstinence from sustained access to alcohol choice. With repetition, these periods of ADE sometimes have led to more sustained elevations in drinking. We therefore asked whether repeated ADE episodes would elevate choice drinking in HDID mice. They did not. After nearly 500 days of alcohol access, the intake of HDID mice remained stable. We conclude that a genetically-enhanced high risk for binge-like drinking is not sufficient to yield alterations in long-term alcohol intake.

FACTORS CONTRIBUTING TO THE ESCALATION OF ALCOHOL CONSUMPTION.

Understanding factors that contribute to the escalation of alcohol consumption is key to understanding how an individual transitions from non/social drinking to AUD and to providing better treatment. In this review, we discuss how the way ethanol is consumed as well as individual and environmental factors contribute to the escalation of ethanol consumption from intermittent low levels to consistently high levels. Moreover, we discuss how these factors are modelled in animals. It is clear a vast array of complex, interacting factors influence changes in alcohol consumption. Some of these factors act early in the acquisition of ethanol consumption and initial escalation, while others contribute to escalation of ethanol consumption at a later stage and are involved in the development of alcohol dependence. There is considerable need for more studies examining escalation associated with the formation of dependence and other hallmark features of AUD, especially studies examining mechanisms, as it is of considerable relevance to understanding and treating AUD.

Orbitofrontal cortex subregion inhibition during binge-like and aversion-resistant alcohol drinking.

Two important contributors to alcohol-related problems and alcohol use disorder (AUD) are binge- and compulsive-like drinking. The orbitofrontal cortex (OFC), a brain region implicated in outcome valuation and behavioral flexibility, is functionally altered by alcohol exposure. Data from animal models also suggest that both the medial (mOFC) and lateral (lOFC) subregions of the OFC regulate alcohol-related behaviors. The current study was designed to examine the contributions of mOFC and lOFC using a model of binge-like and aversion-resistant ethanol drinking in C57BL/6J male and female mice. The inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM4Di were used to inhibit neurons in either the mOFC or the lOFC in mice drinking 15% ethanol in a two-bottle, limited-access, modified drinking in the dark paradigm. The effects of chemogenetic inhibition on consumption of quinine-adulterated ethanol, water, and water + quinine were also assessed. Inhibiting the mOFC did not alter consumption of ethanol or aversion-resistant drinking of ethanol + quinine. In contrast, inhibition of neurons in the lOFC increased consumption, but not preference, of ethanol alone. mOFC and lOFC inhibition did not alter water or quinine-adulterated water intake, indicating the effects shown here are specific to ethanol drinking. These data support the role of the lOFC in regulating alcohol consumption but fail to find a similar role for mOFC.

Reducing effect of the novel positive allosteric modulator of the GABAB receptor, COR659, on binge-like alcohol drinking in male mice and rats.

RATIONALE: Binge drinking (BD) is a widespread drinkingpattern that may contribute to promote the development of alcohol use disorder (AUD). The comprehension of its neurobiological basis and the identification of molecules that may prevent BD are critical. Preclinical studies demonstrated that positive allosteric modulators (PAMs) of the GABAB receptor effectively reduced, and occasionally suppressed, the reinforcing and motivational properties of alcohol in rodents, suggesting their potential use as pharmacotherapy for AUD, including BD. Recently, we demonstrated that COR659, a novel GABAB PAM, effectively reduced (i) alcohol drinking under the 2-bottle choice regimen, (ii) alcohol self-administration under both fixed and progressive ratio schedules of reinforcement, and (iii) cue-induced reinstatement of alcohol-seeking behavior in Sardinian alcohol-preferring (sP) rats. OBJECTIVES: The present study investigated whether the "anti-alcohol" properties of COR659 extend to binge-like drinking in rodents. METHODS: COR659 was tested on the "drinking in the dark" (DID) paradigm in C57BL/6J mice and the 4-bottle "alcohol [10%, 20%, 30% (v/v)] versus water" choice regimen with limited and unpredictable access to alcohol in sP rats. RESULTS: Acute administration of non-sedative doses of COR659 (10, 20, and 40 mg/kg; i.p.) effectively and selectively suppressed the intake of intoxicating amounts of alcohol (> 2 g/kg) consumed by C57BL/6J mice and sP rats exposed to these binge-like drinking experimental procedures. CONCLUSIONS: The present data demonstrate the ability of COR659 to suppress binge-like drinking in rodents and strengthen the hypothesis that GABAB PAMs may represent a potentially effective pharmacotherapy for alcohol misuse.

Corrigendum: The Influence of Moderate Physical Activity on Brain Monoaminergic Responses to Binge-Patterned Alcohol Ingestion in Female Mice.

[This corrects the article DOI: 10.3389/fnbeh.2021.639790.].

Effects of a Peripherally Restricted Hybrid Inhibitor of CB1 Receptors and iNOS on Alcohol Drinking Behavior and Alcohol-Induced Endotoxemia.

Alcohol consumption is associated with gut dysbiosis, increased intestinal permeability, endotoxemia, and a cascade that leads to persistent systemic inflammation, alcoholic liver disease, and other ailments. Craving for alcohol and its consequences depends, among other things, on the endocannabinoid system. We have analyzed the relative role of central vs. peripheral cannabinoid CB1 receptors (CB1R) using a "two-bottle" as well as a "drinking in the dark" paradigm in mice. The globally acting CB1R antagonist rimonabant and the non-brain penetrant CB1R antagonist JD5037 inhibited voluntary alcohol intake upon systemic but not upon intracerebroventricular administration in doses that elicited anxiogenic-like behavior and blocked CB1R-induced hypothermia and catalepsy. The peripherally restricted hybrid CB1R antagonist/iNOS inhibitor S-MRI-1867 was also effective in reducing alcohol consumption after oral gavage, while its R enantiomer (CB1R inactive/iNOS inhibitor) was not. The two MRI-1867 enantiomers were equally effective in inhibiting an alcohol-induced increase in portal blood endotoxin concentration that was caused by increased gut permeability. We conclude that (i) activation of peripheral CB1R plays a dominant role in promoting alcohol intake and (ii) the iNOS inhibitory function of MRI-1867 helps in mitigating the alcohol-induced increase in endotoxemia.

Lateral hypothalamus-projecting noradrenergic locus coeruleus pathway modulates binge-like ethanol drinking in male and female TH-ires-cre mice.

A growing body of literature implicates noradrenergic (NE) signaling in the modulation of ethanol consumption. However, relatively few studies have detailed specific brain pathways that mediate NE-associated binge-like ethanol consumption. To begin to fill this gap in the literature, male and female C57BL6/J and TH-ires-cre mice underwent pharmacological and chemogenetic testing, respectively, in combination with "drinking in the dark" procedures to model binge-like consumption of ethanol or sucrose solutions. First, we showed that intraperitoneal administration of the NE reuptake inhibitor, reboxetine, blunted binge-like ethanol intake in C57BL6/J mice. Chemogenetic activation of locus coeruleus (LC) tyrosine hydroxylase (TH)-expressing neurons blunted binge-like ethanol intake regardless of sex. Chemogenetic activation of LC projections to the lateral hypothalamus (LH), a region implicated in ethanol consumption, blunted binge-like ethanol drinking without altering sucrose intake in ethanol-experienced or ethanol-naïve mice. In C57BL/6 J mice, LH-targeted microinfusion of an α1-adrenergic receptor (AR) agonist blunted binge-like ethanol intake across both sexes, while LH infusion of a ß-AR agonist blunted binge-like ethanol intake in females exclusively. Finally, in mice with high baseline ethanol intake both an α1- AR agonist and an α-2 AR antagonist blunted binge-like ethanol intake. The present results provide novel evidence that increased NE tone in a circuit arising from the LC and projecting to the LH reduces binge-like ethanol drinking in mice, and may represent a novel approach to treating binge or heavy drinking prior to the development of dependence. This article is part of the special Issue on "Neurocircuitry Modulating Drug and Alcohol Abuse".