Access To Ethanol Research Articles

Identification of novel genetic loci and candidate genes for progressive ethanol consumption in diversity outbred mice.

Mouse behavioral genetic mapping studies can identify genomic intervals modulating complex traits under well-controlled environmental conditions and have been used to study ethanol behaviors to aid in understanding genetic risk and the neurobiology of alcohol use disorder (AUD). However, historically such studies have produced large confidence intervals, thus complicating identification of potential causal candidate genes. Diversity Outbred (DO) mice offer the ability to perform high-resolution quantitative trait loci (QTL) mapping on a very genetically diverse background, thus facilitating identification of candidate genes. Here, we studied a population of 636 male DO mice with four weeks of intermittent ethanol access via a three-bottle choice procedure, producing a progressive ethanol consumption phenotype. QTL analysis identified 3 significant (Chrs 3, 4, and 12) and 13 suggestive loci for ethanol-drinking behaviors with narrow confidence intervals (1-4 Mbp for significant QTLs). Results suggested that genetic influences on initial versus progressive ethanol consumption were localized to different genomic intervals. A defined set of positional candidate genes were prioritized using haplotype analysis, identified coding polymorphisms, prefrontal cortex transcriptomics data, human GWAS data and prior rodent gene set data for ethanol or other misused substances. These candidates included Car8, the lone gene with a significant cis-eQTL within a Chr 4 QTL for week four ethanol consumption. These results represent the highest-resolution genetic mapping of ethanol consumption behaviors in mice to date, providing identification of novel loci and candidate genes for study in relation to the neurobiology of AUD.

Impact of heavy alcohol consumption on cortical bone mechanical properties in male rhesus macaques

Chronic heavy alcohol consumption may influence the skeleton by suppressing intracortical bone remodeling which may impact the quality of bone and its mechanical properties. However, this aspect has not been thoroughly assessed in either humans or animal models whose cortical bone microstructure resembles the microstructure of human cortical bone. The current study is the first to investigate the effects of chronic heavy alcohol consumption on various mechanical properties of bone in a non-human primate model with intracortical remodeling. Male rhesus macaques (5.3 years old at the initiation of treatment) were induced to drink alcohol and then given the choice to voluntarily self-administer water or ethanol (4 % w/v) for approximately 14 months, followed by three abstinence phases (lasting 34, 41, and 39–46 days) with approximately 3 months of ethanol access in between. During the initial 14 months of open-access, monkeys in the alcohol group consumed an average of 2.9 ± 0.8 g/kg/d ethanol (mean ± SD) resulting in a blood ethanol concentration of 89 ± 47 mg/dl in longitudinal samples taken at 7 h after the daily sessions began. To understand the impact of alcohol consumption on material properties, various mechanical tests were conducted on the distal tibia diaphysis of 2–5 monkeys per test group, including dynamic mechanical analysis (DMA) testing, nano-indentation, microhardness testing, compression testing, and fracture resistance curve (R-curve) testing. Additionally, compositional analyses were performed using Fourier-transform infrared (FTIR) spectroscopy. Significant differences in microhardness, compressive stress-strain response, and composition were not observed with alcohol consumption, and only minor differences were detected in hardness and elastic modulus of the matrix and osteons from nanoindentation. Furthermore, the R-curves of both groups overlapped, with similar crack initiation toughness, despite a significant decrease in crack growth toughness (p = 0.032) with alcohol consumption. However, storage modulus (p = 0.029) and loss factor (p = 0.015) from DMA testing were significantly increased in the alcohol group compared to the control group, while loss modulus remained unchanged. These results indicate that heavy alcohol consumption may have only a minor influence on the material properties and the composition of cortical bone in young adult male rhesus macaques.

The neutral CB1 antagonist AM6527 reduces ethanol seeking, binge-like consumption, reinforcing, and withdrawal effects in male and female mice.

Alcohol use disorder (AUD) is a debilitating physiological and psychiatric disorder which affects individuals globally. The current pharmacological interventions to treat AUD are limited, and hence there is an urgent need for a novel pharmacological therapy which can be effective and safe across the population. We aimed to investigate a novel neutral cannabinoid receptor-1 (CB1R) antagonist, AM6527, in several preclinical models of ethanol consumption using male and female C57BL6/J mice. Independent groups of male and female mice were subjected to repeated cycles of drinking in the dark (DID), or intermittent access to alcohol (IAA) procedures. Twenty minutes prior to ethanol access in each procedure, animals were treated with intraperitoneal injections of either 1, 3, and 10 mg/kg of AM6527 or its respective vehicle. Acamprosate (100, 200, 300, and 400 mg/kg) or its respective vehicle was used as a positive control. Separate groups of male mice were subjected to a chain schedule of ethanol reinforcement to gain access to ethanol wherein completion of a fixed interval (FI; 5 min) schedule (link 1: "Seeking") was reinforced with continuous access to ethanol (fixed ratio; FR1) for up to 1.8 g/kg (link 2: "consumption"). All the animals were treated with 1, 3, and 10 mg/kg of AM6527 or its respective vehicle 20 mins prior to the start of the FI chain of the procedure. Separately, AM6527 was also evaluated in male and female mice undergoing acute ethanol withdrawal following 8 weeks of intermittent or continuous access to 20% ethanol drinking. In both DID and IAA procedures, AM6527 reduced ethanol consumption in a dose-related manner in both male and female mice. AM6527 produced no tolerance in the DID procedure; mice treated with 3 mg/kg of AM6527 for 3 weeks continuously drank significantly smaller amounts of ethanol as compared to vehicle-treated mice over a period of three DID cycles. Moreover, in the IAA procedure, AM6527 caused an increase in water intake over the 24-h period. Acamprosate transiently reduced ethanol intake in male mice in both the DID and the IAA procedures but failed to produce any significant effect in female mice. AM6527 also produced a decrease in the FI responding ("ethanol seeking") in animals trained to self-administer ethanol. Lastly, AM6527 mitigated neurological withdrawal signs, i.e., handling induced convulsions(HIC) in mice undergoing acute ethanol withdrawal. Current findings support previous studies with CB1R neutral antagonist in reducing voluntary ethanol intake and seeking behavior. Based on results shown in this work, AM6527 can be developed as a first in class CB1R neutral antagonist to treat AUD in both males and females.

Ethanol alters the relationship between IGF-1 and bone turnover in male macaques.

Insulin-like growth factor 1 (IGF-1) influences bone turnover. Transient decreases in IGF-I levels and/or bioavailability may contribute to the detrimental effects of alcohol on bone. The goals of this non-human primate study were to i) evaluate the 20-h response of bone turnover markers to ethanol consumption and ii) assess how ethanol consumption influences the relationship between IGF-1 and these markers. Osteocalcin (bone formation), carboxyterminal cross-linking telopeptide of type 1 collagen (CTX, bone resorption), IGF-1, and IGF binding protein 1 (IGFBP-1) were measured in plasma from male rhesus macaques (N = 10, 8.4 ± 0.3 years) obtained at 12:00, 16:00, and 06:00 h during two phases: pre-ethanol (alcohol-naïve) and ethanol access. During the ethanol access phase, monkeys consumed 1.5 g/kg/day ethanol (4% w/v) beginning at 10:00 h. Osteocalcin and CTX were lower, and the ratio of osteocalcin to CTX was higher at each time point during ethanol access compared to the pre-ethanol phase. Pre-ethanol marker levels did not vary across time points, but markers varied during ethanol access. IGF-1 levels, but not IGFBP-1 levels, varied during the pre-ethanol phase. In contrast, IGF-1 levels were stable during ethanol access but IGFBP-1 levels varied. There were positive relationships between IGF-1 and turnover markers during the pre-ethanol phase, but not during ethanol access. In conclusion, chronic ethanol consumption reduces levels of bone turnover markers and blocks the normal positive relationship between IGF-1 and turnover markers and alters the normal relationship between IGF-1 and IGFBP-1. These findings support the hypothesis that chronic alcohol consumption leads to growth hormone/IGF-1 resistance.

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 2.5h/day for five weeks. During the final five days of the study, mice received 2-4h of daily access to sipper tubes containing water (n=14 sedentary; n=15 running) or 20% ethanol (n=14 sedentary; n=16 running) 30min 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.

Benztropine Reduces Reacquisition of Alcohol Self‐Administration in Rats with Stress History: Role of FKBP5

Post‐traumatic stress disorder (PTSD), which can affect 1 in 11 adults during their lifetime, is often comorbid with chronic alcohol use problems and increases alcohol relapse risk. PTSD and alcohol‐use phenotypes are associated with variants in the FKBP propyl isomerase 5 (FKBP5) gene, which encodes FK506‐binding protein 5 (FKBP5), a chaperone modulator of glucocorticoid receptors (GR) that is functionally implicated in stress‐related psychiatric disorders and alcohol withdrawal severity. Data suggests that FKBP5 inhibition may decrease alcohol intake, but its role in the post‐traumatic recurrence of alcohol drinking is unknown. Here, we tested the hypotheses that amygdala Fkbp5 expression is increased in rats with a history of traumatic stress and is associated with greater reacquisition of ethanol self‐administration. Then, we tested the hypothesis that benztropine mesylate, an FDA‐approved drug that disrupts FKBP5‐GR binding, reduces reacquisition of ethanol self‐administration in rats with a history of traumatic stress. Separate cohorts (for mRNA vs. benztropine studies) of male (n = 43) and female (n = 20) Wistar rats received 3 sessions of light‐cued footshock stress (30 min session, 60 footshocks, 1‐s 0.4 mA) before acquiring operant ethanol (10% v/v) self‐administration (1‐h sessions, fixed ratio1‐3), followed by extinction (16 sessions), and then renewed alcohol access. Amygdala Fkbp5 expression correlated significantly with increased ethanol self‐administration (rs(23) = 0.463, p = 0.023) during renewal of ethanol access. Subchronic (3 days) benztropine pretreatment (‐2 h, i.p., 0, 5, and 10 mg/kg [n = 10, 14, and 14]) significantly reduced the reacquisition of ethanol self‐administration. On days 2 and 3 of reacquisition, self‐administration was significantly reduced in drug treated animals (Dose x Day: F(4,64)=2.83, p < 0.04), culminating in a 68% reduction in males (0.78±0.16 vs. 0.25±0.13 g/kg) and a 41% reduction in females (0.88±0.14 vs. 0.52±0.13 g/kg) at the 10 mg/kg dose. Overall, the results warrant further study of benztropine and its potential inhibition of FKBP5 to reduce post‐traumatic alcohol relapse risk.

From binge eating to binge drinking: A new and robust paradigm for assessing binge ethanol self-administration in male rats.

Animal models of alcohol (ethanol) self‐administration are crucial to dissect the neurobiological mechanisms underlying alcohol dependence, yet only a few of these induce pharmacologically relevant levels of alcohol consumption and rarely the alcohol self‐administration co‐occurs with other addictive behaviours. The present study aims to validate a novel model of voluntary ethanol consumption in male Wistar rats, in which ethanol access follows a binge eating experience. Over 10 sessions, Wistar rats were exposed to binge or control eating (i.e., the ingestion of 11.66 and 0.97 kcal/3 min, respectively, derived from a highly palatable food), immediately followed by two‐bottle choice intake tests (2%, 6%, 10% or 14% w/w ethanol vs. water). Rats exposed to binge eating drank significantly more 6% or 10% (w/w) ethanol than control peers, reaching up to 6.3 gEtOH/kg. Rats stimulated with 2%, 6%, 10% or 14% ethanol after binge eating, but not those given those ethanol concentrations after control eating, exhibited significant within‐group increases in ethanol drinking. This ethanol consumption was not altered by quinine adulteration (up to 0.1 g/L), and it was blocked by naltrexone (10 mg/kg), administered immediately before binge eating. Blood ethanol levels significantly correlated with ethanol consumption; and the more ethanol consumed, the greater the distance travelled in an open field test conducted after the two‐bottle choice test. Altogether, this self‐administration model seems a valid and robust alternative with remarkable potential for research on different stages of the alcohol addiction and, particularly, to assess interactions between alcohol consumption and others addictive‐like behaviours.

The impact of Drinking in the Dark (DID) procedural manipulations on ethanol intake in High Drinking in the Dark (HDID) mice.

The High Drinking in the Dark mouse lines (HDID-1 and HDID-2) were selectively bred to achieve high blood ethanol concentrations (BECs) in the Drinking in the Dark (DID) task, a widely used model of binge-like intake of 20% ethanol. There are several components that differentiate DID from other animal models of ethanol intake: time of day of testing, length of ethanol access, single-bottle access, and individual housing. Here, we sought to determine how some of these individual factors contribute to the high ethanol intake observed in HDID mice. HDID-1, HDID-2, and non-selected HS/NPT mice were tested in a series of DID experiments where one of the following factors was manipulated: length of ethanol access, fluid choice, number of ethanol bottles, and housing condition. We observed that 1) HDID mice achieve intoxicating BECs in DID, even when they are group-housed; 2) HDID mice continue to show elevated ethanol intake relative to HS/NPT mice during an extended access session, but this is most apparent during the first 4h of access; and 3) offering a water choice during DID prevents elevated intake in the HDID-1 mice, but not necessarily in HDID-2 mice. Together, these results suggest that the lack of choice in the DID paradigm, together with the length of ethanol access, are important factors contributing to elevated ethanol intake in the HDID mice. These results further suggest important differences between the HDID lines in response to procedural manipulations of housing condition and ethanol bottle number in the DID paradigm, highlighting the distinct characteristics that each of these lines possess, despite being selectively bred for the same phenotype.

Importance of sex and trauma context on circulating cytokines and amygdalar GABAergic signaling in a comorbid model of posttraumatic stress and alcohol use disorders.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based “2-hit” rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel context (NOV). Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-Vulnerable individuals (≥0.8g/kg average 2-hr ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin and IL-4 that discriminated between stress context (NOV>FAM>Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Non-genetic factors that influence methamphetamine intake in a genetic model of differential methamphetamine consumption.

Genetic and non-genetic factors influence substance use disorders. Our previous work in genetic mouse models focused on genetic factors that influence methamphetamine (MA) intake. The current research examined several non-genetic factors for their potential influence on this trait. We examined the impact on MA intake of several non-genetic factors, including MA access schedule, prior forced MA exposure, concomitant ethanol (EtOH) access, and gamma-aminobutyric acid type B (GABAB) receptor activation. Selectively bred MA high drinking(MAHDR) and low drinking (MALDR) mice participated in this research. MAHDR, but not MALDR, mice increased MA intake when given intermittent access, compared with continuous access, with a water choice under both schedules. MA intake was not altered by previous exposure to forced MA consumption. Male MAHDR mice given simultaneous access to MA, EtOH, and an EtOH+MA mixture exhibited a strong preference for MA over EtOH and EtOH+MA; MA intake was not affected by EtOH in female MAHDR mice. When independent MAHDR groups were given access to MA, EtOH, or EtOH+MA vs. water in each case, MA intake was reduced in the water vs. EtOH+MA group, compared with the water vs. MA group. The GABAB receptor agonist R(+)-baclofen (BAC) not only reduced MA intake but also reduced water intake and locomotor activity in MAHDR mice. There was a residual effect of BAC, such that MA intake was increased after termination of BAC treatment. These findings demonstrate that voluntary MA intakein MAHDR mice is influenced by non-genetic factors related to MA access schedule and co-morbid EtOH exposure.

Evaluation of the effect of doxasozin and zonisamide on voluntary ethanol intake in mice that experienced chronic intermittent ethanol exposure and stress.

The comorbidity between alcohol use disorder and post-traumatic stress disorder represents a serious health care burden with few effective treatment options. The current study was designed to evaluate the effect of an alpha 1 receptor antagonist (doxazosin) and a novel anticonvulsant (zonisamide) in a model of alcohol (ethanol) dependence and stress exposure. The main dependent variable was voluntary ethanol intake in mice that experienced chronic intermittent ethanol (CIE) exposure and forced swim stress (FSS) alone, and in combination. Adult male and female C57BL/6J mice had access to a single bottle of 15% (v/v) ethanol for 1-hr in the home cage, 3-hr into the dark phase of the light/dark cycle. Once stable ethanol intake was established (~4 weeks), mice were separated into four groups (CTL, CIE, FSS, CIE+FSS). Mice in the FSS condition received 10-min FSS exposure 4-hr prior to drinking sessions (remaining mice were not disturbed). During baseline and the first two test cycles, all mice received vehicle (saline) injections (IP) 30-min before ethanol access. As previously observed, FSS increased ethanol drinking in dependent (CIE-exposed) mice but not in nondependent control (CTL) mice. In the following test cycles mice were evaluated for ethanol intake after administration of doxazosin, zonisamide or their combination. Results indicated that the three doses of doxazosin evaluated significantly reduced voluntary ethanol intake in all mice. Zonisamide had a more modest effect and may require a more prolonged treatment regime. The combined administration of both compounds was not more effective than each drug alone. This study suggests that doxazosin is reliable at reducing voluntary ethanol intake in mice independently of their history of ethanol dependence and stress exposure.

Spontaneous Early Withdrawal Behaviors after Chronic 24-hour Free-Choice Access to Ethanol.

Abstinence after chronic alcohol consumption leads to withdrawal symptoms, which are exacerbated after repeated cycles of relapse. This study examined withdrawal-like behaviors after chronic ethanol drinking, with or without repeated cycles of deprivation. Male alcohol-preferring (P) rats had access to continuous ethanol (CE), chronic ethanol with repeated deprivation (RD), or remained ethanol naïve (EN). The RD group experienced seven cycles of 2weeks of deprivation and 2weeks of re-exposure to ethanol after an initial 6weeks of ethanol access. Withdrawal was measured after an initial 24h of ethanol re-exposure in the RD group, which coincided with the same day of ethanol access in the CE group. Withdrawal-like behavior was measured by (a) ethanol intake during the initial 24h of re-exposure, (b) locomotor activity (LMA) in a novel field 9-13h after removal of ethanol at the beginning of the fifth re-exposure cycle and (c) acoustic startle responding (ASR) 8-15h after removal of ethanol at the beginning of the sixth re-exposure cycle. The RD rats displayed a 1-h alcohol deprivation effect (ADE) (temporary ethanol increase), relative to CE rats, during the first to fourth and seventh re-exposure cycles. RD and CE rats displayed significant increases in LMA than EN rats. Regarding ASR, RD rats displayed significantly greater ASR relative to EN rats. This study confirms that P rats meet the animal model criterion for ethanol-associated dependence, without a reliance on either behavioral (limited fluid access) or pharmacological (seizure threshold manipulation) challenges.

Brain regional gene expression network analysis identifies unique interactions between chronic ethanol exposure and consumption.

Progressive increases in ethanol consumption is a hallmark of alcohol use disorder (AUD). Persistent changes in brain gene expression are hypothesized to underlie the altered neural signaling producing abusive consumption in AUD. To identify brain regional gene expression networks contributing to progressive ethanol consumption, we performed microarray and scale-free network analysis of expression responses in a C57BL/6J mouse model utilizing chronic intermittent ethanol by vapor chamber (CIE) in combination with limited access oral ethanol consumption. This model has previously been shown to produce long-lasting increased ethanol consumption, particularly when combining oral ethanol access with repeated cycles of intermittent vapor exposure. The interaction of CIE and oral consumption was studied by expression profiling and network analysis in medial prefrontal cortex, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, and central nucleus of the amygdala. Brain region expression networks were analyzed for ethanol-responsive gene expression, correlation with ethanol consumption and functional content using extensive bioinformatics studies. In all brain-regions studied the largest number of changes in gene expression were seen when comparing ethanol naïve mice to those exposed to CIE and drinking. In the prefrontal cortex, however, unique patterns of gene expression were seen compared to other brain-regions. Network analysis identified modules of co-expressed genes in all brain regions. The prefrontal cortex and nucleus accumbens showed the greatest number of modules with significant correlation to drinking behavior. Across brain-regions, however, many modules with strong correlations to drinking, both baseline intake and amount consumed after CIE, showed functional enrichment for synaptic transmission and synaptic plasticity.

Intermittent Ethanol Access Increases Sensitivity to Social Defeat Stress

Comorbidity between alcoholism and depression is extremely common. Recent evidence supports a relationship between alcohol exposure and stress sensitivity, an underlying factor in the development of depression. Our laboratory has recently shown that chronic alcohol gavage increases sensitivity to social defeat stress (SDS). However, the effects of voluntary alcohol consumption, resulting from protocols such as intermittent ethanol access (IEA), on defeat stress sensitivity have yet to be elucidated. We first assessed the effects of 4weeks of IEA to 20% alcohol on sensitivity to subthreshold SDS exposure. Next, to examine neuroinflammatory mechanisms, we analyzed gene expression of inhibitor of NFkB (IkB) following IEA or chronic alcohol exposure (10days of 3.0g/kg alcohol via intragastric gavage). Then, we quantified NFkB activation via β-galactosidase immunohistochemistry following IEA or chronic alcohol gavage in NFkB-LacZ mice. IEA-exposed mice displayed an increase in sensitivity to subthreshold SDS compared to water-drinking controls. We also found that IkB gene expression was decreased in the nucleus accumbens (NAC) and amygdala (AMY) following IEA but was not altered following chronic alcohol gavage. Finally, we observed increased NFkB activity in the central amygdala (CEA), basolateral amygdala (BLA), and medial amygdala (MEA) after IEA, and increased NFkB activity solely in the CEA following chronic alcohol gavage. These findings further corroborate that prior alcohol exposure, in this case intermittent voluntary consumption, can impact development of depressive-like behavior by altering stress sensitivity. Furthermore, our results suggest the CEA as a potential mediator of alcohol's effects on stress sensitivity, as NFkB was activated in this region following both IEA and chronic alcohol gavage. Thus, this study provides novel insight on alterations in the NFkB pathway and identifies specific regions to target in future experiments assessing the functional role of NFkB in these processes.

Chronic repeated predatory stress induces resistance to quinine adulteration of ethanol in male mice

BackgroundTrauma related psychiatric disorders, such as posttraumatic stress disorder (PTSD), and alcohol use disorder (AUD) are highly comorbid illnesses that separately present an opposing, sex-specific pattern, with increased prevalence of PTSD in females and increased prevalence of AUD diagnoses in males. Likewise, PTSD is a risk factor in the development of AUD, with conflicting data on the impact of sex in the comorbid development of both disorders. Because the likelihood of experiencing more than one traumatic event is high, we aim to utilize chronic repeated predatory stress (CRPS) to query the extent to which sex interacts with CRPS to influence alcohol consumption, or cessation of consumption. MethodsMale (n = 16) and female (n = 15) C57BL/6 J mice underwent CRPS or daily handling for two weeks during adolescence (P35-P49) and two weeks during adulthood (P65-P79). Following the conclusion of two rounds of repeated stress, behavior was assessed in the open field. Mice subsequently underwent a two-bottle choice intermittent ethanol access (IEA) assessment (P90-131) with the options of 20 % ethanol or water. After establishing drinking behavior, increasing concentrations of quinine were added to the ethanol to assess the drinking response to adulteration of the alcohol. ResultsCRPS increased fecal corticosterone concentrations and anxiety-like behaviors in the open field in both male and female mice as compared to control mice that had not been exposed to CRPS. Consistent with previous reports, we observed a sex difference in alcohol consumption such that females consumed more ethanol per gram of body mass than males. In addition, CRPS reduced alcohol aversion in male mice such that higher concentrations of quinine were necessary to reduce alcohol intake as compared to control mice. CRPS did not alter alcohol-related behaviors in female mice. ConclusionCollectively, we demonstrate that repeated CRPS can induce anxiety-like behavior in both sexes but selectively influences the response to ethanol adulteration in males.

Sex differences in cognitive performance and alcohol consumption in High Alcohol-Drinking (HAD-1) rats

Excessive alcohol (ethanol) consumption negatively impacts social, emotional, as well as cognitive function and well-being. Thus, identifying behavioral and/or biological predictors of excessive ethanol consumption is important for developing prevention and treatment strategies against alcohol use disorders (AUDs). Sex differences in alcohol consumption patterns are observed in humans, primates, and rodents. Selectively bred high alcohol-drinking rat lines, such as the “HAD-1” lines are recognized animal models of alcoholism. The present work examined sex differences in alcohol consumption, object recognition, and exploratory behavior in male and female HAD-1 rats. Naïve male and female HAD-1 rats were tested in an object recognition test (ORT) prior to a chronic 24 h intermittent ethanol access procedure for five weeks. Object recognition parameters measured included exploratory behavior, object investigation, and time spent near objects. During the initial training trial, rearing, active object investigation and amount of time spent in the object-containing section was significantly greater in female HAD-1 rats compared to their male counterparts. During the subsequent testing trial, time spent in the object-containing section was greater in female, compared to male, rats; but active object investigation and rearing did not statistically differ between females and males. In addition, female HAD-1 rats consumed significantly more ethanol than their male counterparts, replicating previous findings. Moreover, across all animals there was a significant positive correlation between exploratory behavior in ORT and ethanol consumption level. These results indicate there are significant sex differences in cognitive performance and alcohol consumption in HAD-1 rats, which suggests neurobiological differences as well.

P.010 Dose increase of s-adenosyl-methionine and escitalopram in a randomized clinical trial for major depressive disorder

The mechanisms behind relapse to ethanol intake in recovering alcoholics are still unclear. The negative reinforcing effects contributing to ethanol addiction, including relapse, are considered to be partly driven by the κ-opioidergic system. As the κ-opioidergic system interacts with the mesolimbic reward pathway, the aim of the study was to clarify the role of nucleus accumbens shell κ-opioidergic mechanisms in relapse to ethanol intake by using the alcohol deprivation effect (ADE) paradigm. The ADE is defined as a transient increase in voluntary ethanol intake after a forced period of abstinence. Male Long-Evans rats were trained to voluntarily consume 10% (v/v) ethanol solution. Ethanol access and deprivation cycles were initiated after stable ethanol intake baselines had been reached and bilateral guide cannulas had been implanted above the nucleus accumbens shell. One cycle consisted of 10 days of 90 min access to ethanol followed by 6 days of ethanol deprivation. The ADE was measured in the beginning of a new cycle. Rats received JDTic, a selective κ-antagonist, either subcutaneously (10 mg/kg) or intra-accumbally (15 µg/site) or, as a reference substance, systemic naltrexone (0.3 mg/kg) before ethanol re-access, and the effects on the ADE were evaluated. Systemic and intra-accumbal JDTic significantly attenuated the ADE on the first day of ethanol re-access, as did systemic naltrexone. Additionally, naltrexone decreased ethanol intake levels. These results suggest that nucleus accumbens shell κ-opioidergic mechanisms may have a role in mediating relapse to ethanol intake. Additionally, κ-antagonism could be a valuable adjunct in ethanol relapse prevention.

The selective κ-opioid receptor antagonist JDTic attenuates the alcohol deprivation effect in rats

The mechanisms behind relapse to ethanol intake in recovering alcoholics are still unclear. The negative reinforcing effects contributing to ethanol addiction, including relapse, are considered to be partly driven by the κ-opioidergic system. As the κ-opioidergic system interacts with the mesolimbic reward pathway, the aim of the study was to clarify the role of nucleus accumbens shell κ-opioidergic mechanisms in relapse to ethanol intake by using the alcohol deprivation effect (ADE) paradigm. The ADE is defined as a transient increase in voluntary ethanol intake after a forced period of abstinence. Male Long-Evans rats were trained to voluntarily consume 10% (v/v) ethanol solution. Ethanol access and deprivation cycles were initiated after stable ethanol intake baselines had been reached and bilateral guide cannulas had been implanted above the nucleus accumbens shell. One cycle consisted of 10 days of 90 min access to ethanol followed by 6 days of ethanol deprivation. The ADE was measured in the beginning of a new cycle. Rats received JDTic, a selective κ-antagonist, either subcutaneously (10 mg/kg) or intra-accumbally (15 µg/site) or, as a reference substance, systemic naltrexone (0.3 mg/kg) before ethanol re-access, and the effects on the ADE were evaluated. Systemic and intra-accumbal JDTic significantly attenuated the ADE on the first day of ethanol re-access, as did systemic naltrexone. Additionally, naltrexone decreased ethanol intake levels. These results suggest that nucleus accumbens shell κ-opioidergic mechanisms may have a role in mediating relapse to ethanol intake. Additionally, κ-antagonism could be a valuable adjunct in ethanol relapse prevention.

Caffeine modulates voluntary alcohol intake in mice depending on the access conditions: Involvement of adenosine receptors and the role of individual differences

Caffeine is the most consumed psychoactive stimulant and the main active ingredient of energy drinks. Epidemiology studies have shown a positive correlation between the consumption of energy drinks and that of ethanol. The popular belief is that caffeine antagonizes the intoxicating effects of alcohol. Both drugs act on the adenosine system but have opposite effects. Caffeine is a methylxanthine that acts as a nonselective adenosine receptor antagonist, binding to A1 and A2A receptor subtypes. In contrast, ethanol increases extracellular adenosinergic tone. The purpose of this study was to examine the impact of a broad range of doses of caffeine and of selective adenosine A1 and A2A receptor antagonists on voluntary ethanol intake under different ethanol access conditions. C57BL/6 J male mice had access to ethanol (10% w/v) under different conditions: restricted (2 h in the dark), unrestricted (24 h access), or after 4 days of alcohol removal following several periods of unrestricted access. Mice reduced ethanol intake in the restricted access condition after receiving caffeine (20.0 mg/kg), or theophylline (20.0 mg/kg), another methylxanthine. Selective A1 and A2A adenosine receptor antagonists, or their combination, did not have any effect. However, under unrestricted access conditions caffeine and the adenosine A2A receptor antagonist increased ethanol intake. After splitting animals into high, moderate and low ethanol consumers, caffeine (2.5–20.0 mg/kg) significantly increased ethanol consumption in moderate consumers with no effect on low or high consumers. In addition, after reintroducing ethanol access, caffeine (5.0 mg/kg) decreased ethanol consumption among moderate consumers. Thus, caffeine produced different effects on ethanol intake depending on the access condition and the baseline consumption of ethanol.

The effects of social instability stress and subsequent ethanol consumption in adolescence on brain and behavioral development in male rats.

Excessive drinking in adolescence continues to be a problem, and almost a quarter of young Canadians have reported consuming five or more alcoholic drinks in one occasion in recent surveys. The consequences of such drinking may be more pronounced when commenced in adolescence, given the ongoing brain development during this period of life. Here, we investigated the consequences of 3 weeks' intermittent access to ethanol in mid-adolescence to early adulthood in rats, and the extent to which a stress history moderated the negative consequences of ethanol access. In experiment 1, male rats that underwent adolescent social instability stress (SS; daily 1h isolation+return to unfamiliar cage partner every day from postnatal day [PND] 30-45) did not differ from control (CTL) rats in intake of 10% ethanol sweetened with 0.1% saccharin (access period; PND 47-66). Ethanol drinking reduced proteins relevant for synaptic plasticity (αCaMKII, βCaMKII, and PSD-95) in the dorsal hippocampus, and in CTL rats only in the prefrontal cortex (αCaMKII and PSD 95), attenuating the difference between CTL and SS rats in the water-drinking group. In experiment 2, ethanol also attenuated the difference between SS and CTL rats in a social interaction test by reducing social interaction in SS rats; CTL rats, however, had a higher intake of ethanol than did SS rats during the access period. Ethanol drinking reduced baseline and fear recall recovery concentrations of corticosterone relative to those exposed only to water, although there was no effect of either ethanol or stress history on fear conditioning. Ethanol drinking did not influence intake after 9 days of withdrawal; however, ethanol-naïve SS rats drank more than did CTL rats when given a 24-h access in adulthood. These results reveal a complex relationship between stress history and ethanol intake in adolescence on outcomes in adulthood.