Stress-enhanced ethanol drinking does not increase sensitivity to the effects of a CRF-R1 antagonist on ethanol intake in male and female mice.

Research confirms that stress is associated with alcohol drinking and relapse in males and females and that there are sex differences in the alcohol-related adaptations of stress pathways. The predator stress (PS) model of traumatic stress produces an increase in alcohol drinking or self-administration in a subpopulation of rodents, so it is utilized as an animal model of comorbid alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD). Previous work determined that sensitivity to PS-enhanced drinking produced sex differences in proteins related to stress-regulating systems in the medial prefrontal cortex and hippocampus. The present studies examined whether male and female C57BL/6J mice differ in sensitivity to the ability of the corticotropin releasing factor receptor 1 antagonist CP-376395 to decrease PS-enhanced drinking. In control studies, CP-376395 doses of 5, 10, and 20 mg/kg dose-dependently decreased 4-hour ethanol drinking. Next, CP-376395 doses of 5 and 10 mg/kg were tested for effects on ethanol drinking in mice with differential sensitivity to PS-enhanced drinking. Subgroups of "Sensitive" and "Resilient" male and female mice were identified based on changes in ethanol intake in an unrestricted access ethanol drinking procedure following four exposures to PS (dirty rat bedding). During the first 2 hours post-injection of CP-376395, both doses significantly decreased ethanol licks versus vehicle in the females, with no significant interaction between subgroups, whereas the 10 mg/kg dose significantly decreased ethanol licks versus vehicle in the "Resilient" males. Thus, sensitivity to the suppressive effect of CP-376395 on stress-induced ethanol intake was greater in females versus males, whereas sensitivity and resilience to PS-enhanced drinking produced differential sensitivity to the ability of CP-376395 to decrease ethanol drinking only in male mice. Our results argue against greater efficacy of CRF-R1's ability to decrease ethanol intake in subjects with traumatic stress-enhanced ethanol drinking.

Differential c-Fos Response in Neurocircuits Activated by Repeated Predator Stress in Male and Female C57BL/6J Mice with Stress Sensitive or Resilient Alcohol Intake Phenotypes.

Comorbidity of post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) worsens the prognosis for each of these individual disorders. The current study aimed to identify neurocircuits potentially involved in regulation of PTSD-AUD comorbidity by mapping expression of c-Fos in male and female C57BL/6J mice following repeated predator stress (PS), modeled by exposure to dirty rat bedding. In experiment 1, the levels of c-Fos in the paraventricular nucleus of the hypothalamus (PVH) and the nucleus accumbens shell were higher after the second PS vs the first PS, indicating a sensitized response to this stressor. Additional brain regions showed varied sex-dependent and independent regulation by the two consecutive PS exposures. In experiment 2, mice that increased voluntary alcohol consumption following four exposures to PS (Sensitive subgroup) showed higher c-Fos induction in the PVH, piriform cortex and ventromedial hypothalamus than mice that decreased consumption following these exposures (Resilient subgroup). In contrast to these brain regions, c-Fos was higher in the anterior olfactory nucleus of Resilient vs Sensitive mice. Taken together, these data demonstrate that repeated PS exposure and voluntary alcohol consumption increase neuronal activity across neurocircuits in which specific components depend on the vulnerability of individual mice to these stressors. Increased PVH activity observed across both experiments suggests this brain area as a potential mediator of PS-induced increases in alcohol consumption. Future investigations of specific neuronal populations within the PVH activated by PS, and manipulation of these specific neuronal populations, could improve our understanding of the mechanisms leading to PTSD-AUD comorbidity.

Sensitivity and Resilience to Predator Stress-Enhanced Ethanol Drinking Is Associated With Sex-Dependent Differences in Stress-Regulating Systems.

Stress can increase ethanol drinking, and evidence confirms an association between post-traumatic stress disorder (PTSD) and the development of alcohol use disorder (AUD). Exposure to predator odor is considered a traumatic stressor, and predator stress (PS) has been used extensively as an animal model of PTSD. Our prior work determined that repeated exposure to intermittent PS significantly increased anxiety-related behavior, corticosterone levels, and neuronal activation in the hippocampus and prefrontal cortex in naïve male and female C57BL/6J mice. Intermittent PS exposure also increased subsequent ethanol drinking in a subgroup of animals, with heterogeneity of responses as seen with comorbid PTSD and AUD. The present studies built upon this prior work and began to characterize "sensitivity" and "resilience" to PS-enhanced drinking. Ethanol drinking was measured during baseline, intermittent PS exposure, and post-stress; mice were euthanized after 24-h abstinence. Calculation of median and interquartile ranges identified "sensitive" (>20% increase in drinking over baseline) and "resilient" (no change or decrease in drinking from baseline) subgroups. Intermittent PS significantly increased subsequent ethanol intake in 24% of male (↑60%) and in 20% of female (↑71%) C57BL/6J mice in the "sensitive" subgroup. Plasma corticosterone levels were increased significantly after PS in both sexes, but levels were lower in the "sensitive" vs. "resilient" subgroups. In representative mice from "sensitive" and "resilient" subgroups, prefrontal cortex and hippocampus were analyzed by Western Blotting for levels of corticotropin releasing factor (CRF) receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor, vs. separate naïve age-matched mice. In prefrontal cortex, CRF receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor levels were significantly higher in "sensitive" vs. naïve and "resilient" mice only in females. In hippocampus, CRF receptor 1, CRF receptor 2 and glucocorticoid receptor levels were significantly lower in "resilient" vs. naïve and "sensitive" mice across both sexes. These results indicate that sex strongly influences the effects of ethanol drinking and stress on proteins regulating stress and anxiety responses. They further suggest that targeting the CRF system and glucocorticoid receptors in AUD needs to consider the comorbidity of PTSD with AUD and sex of treated individuals.

Sexually divergent changes in select brain proteins and neurosteroid levels after a history of ethanol drinking and intermittent PTSD-like stress exposure in adult C57BL/6J mice.

Human studies reported that the number of past-year stressors was positively related to current drinking patterns, including binge drinking. In animal models, exposure to predator odor stress (PS), considered a model of traumatic stress, consistently increased ethanol intake. Recently, we reported that repeated PS significantly increased ethanol intake and had a synergistic interaction with prior binge drinking (binge group) in male but not in female C57BL/6J mice, when compared to mice without prior binge exposure (control group). The current studies utilized plasma and dissected prefrontal cortex (PFC) and hippocampal tissue from these animals and from age-matched naïve mice (naïve group). Western blots assessed relative protein levels of P450scc (an enzyme involved in the first step of steroidogenesis), of GABAA receptor α2 and α4 subunits, and of two proteins involved in synaptic plasticity - ARC (activity-regulated cytoskeletal protein) and synaptophysin. Gas chromatography-mass spectrometry simultaneously quantified 10 neurosteroid levels in plasma. A history of ethanol drinking and PS exposure produced brain regional and sex differences in the changes in proteins examined as well as in the pattern of neurosteroid levels versus (vs.) values in naïve mice. For instance, P450scc levels were significantly increased only in binge and control female PFC and hippocampus vs. naïve mice. Some neurosteroid levels were significantly altered by binge treatment in both males and females, whereas others were only significantly altered in males. These sexually divergent changes in neurosteroid and protein levels add to evidence for sex differences in the neurochemical systems influenced by traumatic stress and a history of ethanol drinking.

Genotype Differences in Sensitivity to the Anticonvulsant Effect of the Synthetic Neurosteroid Ganaxolone during Chronic Ethanol Withdrawal.

Sensitivity to anticonvulsant effects of the γ-aminobutyric acidA receptor-active neurosteroid allopregnanolone (ALLO) during ethanol withdrawal varies across genotypes, with high sensitivity in genotypes with mild withdrawal and low sensitivity in genotypes with high withdrawal. The present studies determined whether the resistance to ALLO during withdrawal in mouse genotypes with high handling-induced convulsions (HICs) during withdrawal could be overcome with use of ganaxolone (GAN), the metabolically stable derivative of ALLO. In separate studies, male and female Withdrawal Seizure-Prone (WSP-1) and DBA/2J (D2) mice were exposed to air (controls) or 72-h ethanol vapor and then were scored for HICs during withdrawal (hourly for the first 12 h, then at hours 24 and 25). After the HIC scoring at hours 5 and 9, mice were injected with 10 mg/kg GAN or vehicle. Area under the HIC curve (AUC) for hours 5-12 was analyzed. In control WSP-1 mice, GAN significantly reduced AUC by 52% (males) and 63% (females), with effects that were absent or substantially reduced during withdrawal. In contrast, GAN significantly reduced AUC in both control and ethanol-withdrawing male and female D2 mice. AUC was decreased by 81% (males) and 70% (females) in controls and by 35% (males) and 21% (females) during withdrawal. The significant anticonvulsant effect of GAN during withdrawal in D2 but not WSP-1 mice suggests that different mechanisms may contribute to ALLO insensitivity during withdrawal in these two genotypes. Importantly, the results in D2 mice suggest that GAN may be a useful treatment for ethanol withdrawal-induced seizures.

Binge Ethanol Drinking Produces Sexually Divergent and Distinct Changes in Nucleus Accumbens Signaling Cascades and Pathways in Adult C57BL/6J Mice.

We previously determined that repeated binge ethanol drinking produced sex differences in the regulation of signaling downstream of Group 1 metabotropic glutamate receptors in the nucleus accumbens (NAc) of adult C57BL/6J mice. The purpose of the present study was to characterize RNA expression differences in the NAc of adult male and female C57BL/6J mice following 7 binge ethanol drinking sessions, when compared with controls consuming water. This binge drinking procedure produced high intakes (average >2.2 g/kg/30 min) and blood ethanol concentrations (average >1.3 mg/ml). Mice were euthanized at 24 h after the 7th binge session, and focused qPCR array analysis was employed on NAc tissue to quantify expression levels of 384 genes in a customized Mouse Mood Disorder array, with a focus on glutamatergic signaling (3 arrays/group). We identified significant regulation of 50 genes in male mice and 70 genes in female mice after 7 ethanol binges. Notably, 14 genes were regulated in both males and females, representing common targets to binge ethanol drinking. However, expression of 10 of these 14 genes was strongly dimorphic (e.g., opposite regulation for genes such as Crhr2, Fos, Nos1, and Star), and only 4 of the 14 genes were regulated in the same direction (Drd5, Grm4, Ranbp9, and Reln). Interestingly, the top 30 regulated genes by binge ethanol drinking for each sex differed markedly in the male and female mice, and this divergent neuroadaptive response in the NAc could result in dysregulation of distinct biological pathways between the sexes. Characterization of the expression differences with Ingenuity Pathway Analysis was used to identify Canonical Pathways, Upstream Regulators, and significant Biological Functions. Expression differences suggested that hormone signaling and immune function were altered by binge drinking in female mice, whereas neurotransmitter metabolism was a central target of binge ethanol drinking in male mice. Thus, these results indicate that the transcriptional response to repeated binge ethanol drinking was strongly influenced by sex, and they emphasize the importance of considering sex in the development of potential pharmacotherapeutic targets for the treatment of alcohol use disorder.

Sex differences in the synergistic effect of prior binge drinking and traumatic stress on subsequent ethanol intake and neurochemical responses in adult C57BL/6J mice.

Alcohol-use disorders (AUDs) are characterized by repeated episodes of binge drinking. Based on reports that exposure to predator odor stress (PS) consistently increases ethanol intake, the present studies examined whether prior binge drinking differentially altered responsivity to PS and subsequent ethanol intake in male and female mice, when compared to mice without prior binge exposure. Initial studies in naïve male and female C57BL/6J mice confirmed that 30-min exposure to dirty rat bedding significantly increased plasma corticosterone (CORT) levels and anxiety-related behavior, justifying the use of dirty rat bedding as PS in the subsequent drinking studies. Next, separate groups of male and female C57BL/6J mice received seven binge ethanol sessions (binge) or drank water (controls), followed by a 1-month period of abstinence. Then, 2-bottle choice ethanol intake (10% or 10E vs. water, 23 h/day) was measured in lickometer chambers for 4 weeks. After baseline intake stabilized, exposure to intermittent PS (2×/week × 2 weeks) significantly enhanced ethanol intake after the 2nd PS in male, but not female, binge mice vs. baseline and vs. the increase in controls. However, in a subgroup of females (with low baselines), PS produced a similar increase in 10E intake in control and binge mice vs. baseline. Analysis of lick behavior determined that the enhanced 10E intake in binge male mice and in the female low baseline subgroup was associated with a significant increase in 10E bout frequency and 10E licks throughout the circadian dark phase. Thus, PS significantly increased 10E intake and had a synergistic interaction with prior binge drinking in males, whereas PS produced a similar significant increase in 10E intake in the low baseline subgroup of binge and control females. Plasma CORT levels were increased significantly in both binge and control animals after PS. CORT levels at 24-h withdrawal from daily 10E intake were highest in the groups with elevated 10E licks (i.e., binge males and control females). At 24-h withdrawal, protein levels of GABAA receptor α1 subunit, corticotropin releasing factor receptor 1, and glucocorticoid receptor in prefrontal cortex (PFC) and hippocampus (HC) were differentially altered in the male and female mice vs. levels in separate groups of age-matched naïve mice, with more changes in HC than in PFC and in females than in males. Importantly, the sexually divergent changes in protein levels in PFC and HC add to evidence for sex differences in the neurochemical systems influenced by stress and binge drinking, and argue for sex-specific pharmacological strategies to treat AUD.

Ethanol withdrawal-induced dysregulation of neurosteroid levels in plasma, cortex, and hippocampus in genetic animal models of high and low withdrawal.

RATIONALE: Endogenous γ-aminobutyric acidA receptor (GABAAR)-active neurosteroids (e.g., allopregnanolone) regulate central nervous system excitability and many physiological functions, so fluctuations are implicated in several neuropsychiatric disorders. Pertinently, evidence supports an inverse relationship between endogenous GABAAR-active neurosteroid levels and behavioral changes in excitability during ethanol withdrawal (WD). OBJECTIVES: The present studies determined mouse genotype differences in ten neurosteroid levels in plasma, cortex, and hippocampus over the time course of ethanol WD in the WD Seizure-Prone (WSP) and WD Seizure-Resistant (WSR) selected lines and in the DBA/2J (DBA) inbred strain. METHODS: Gas chromatography-mass spectrometry was utilized to simultaneously quantify neurosteroid levels from control-treated male WSP-1, WSR-1, and DBA mice and during 8 and 48 h of WD. RESULTS: Combined with our prior work, there was a consistent decrease in plasma allopregnanolone levels at 8 h WD in all three genotypes, an effect that persisted at 48 h WD only in DBA mice. WSR-1 and WSP-1 mice exhibited unexpected divergent changes in cortical neurosteroids at 8 h WD, with the majority of neurosteroids (including allopregnanolone) being significantly decreased in WSR-1 mice, but unaffected or significantly increased in WSP-1 mice. In DBA mice, hippocampal allopregnanolone and tetrahydrodeoxycorticosterone were significantly decreased at 8 h WD. The pattern of significant correlations between allopregnanolone and other GABAAR-active neurosteroid levels differed between controls and withdrawing mice. CONCLUSIONS: Ethanol WD dysregulated neurosteroid synthesis. Results in WSP-1 mice suggest that diminished GABAAR function is more important for their high WD phenotype than fluctuations in neurosteroid levels.

Tigecycline Reduces Ethanol Intake in Dependent and Nondependent Male and Female C57BL/6J Mice.

BACKGROUND: The chronic intermittent ethanol (CIE) paradigm is valuable for screening compounds for efficacy to reduce drinking traits related to alcohol use disorder (AUD), as it measures alcohol consumption and preference under physical dependence conditions. Air control-treated animals allow simultaneous testing of similarly treated, nondependent animals. As a consequence, we used CIE to test the hypothesis that tigecycline, a semisynthetic tetracycline similar to minocycline and doxycycline, would reduce alcohol consumption regardless of dependence status. METHODS: Adult C57BL/6J female and male mice were tested for tigecycline efficacy to reduce ethanol (EtOH) consumption using a standard CIE paradigm. The ability of tigecycline to decrease 2-bottle choice of 15% EtOH (15E) versus water intake in dependent (CIE vapor) and nondependent (air-treated) male and female mice was tested after 4 cycles of CIE vapor or air exposure using a within-subjects design and a dose-response. Drug doses of 0, 40, 60, 80, and 100 mg/kg in saline were administered intraperitoneally (0.01 ml/g body weight) and in random order, with a 1-hour pretreatment time. Baseline 15E intake was re-established prior to administration of subsequent injections, with a maximum of 2 drug injections tested per week. RESULTS: Tigecycline was found to effectively reduce high alcohol consumption in both dependent and nondependent female and male mice. CONCLUSIONS: Our data suggest that tigecycline may be a promising drug with novel pharmacotherapeutic characteristics for the treatment of mild-to-severe AUD in both sexes.

Pharmacologically Counteracting a Phenotypic Difference in Cerebellar GABAA Receptor Response to Alcohol Prevents Excessive Alcohol Consumption in a High Alcohol-Consuming Rodent Genotype.

UNLABELLED: Cerebellar granule cell GABAA receptor responses to alcohol vary as a function of alcohol consumption phenotype, representing a potential neural mechanism for genetic predilection for alcohol abuse (Kaplan et al., 2013; Mohr et al., 2013). However, there are numerous molecular targets of alcohol in the cerebellum, and it is not known how they interact to affect cerebellar processing during consumption of socially relevant amounts of alcohol. Importantly, direct evidence for a causative role of the cerebellum in alcohol consumption phenotype is lacking. Here we determined that concentrations of alcohol that would be achieved in the blood after consumption of 1-2 standard units (9 mm) suppresses transmission through the cerebellar cortex in low, but not high, alcohol consuming rodent genotypes (DBA/2J and C57BL/6J mice, respectively). This genotype-selective suppression is mediated exclusively by enhancement of granule cell GABAA receptor currents, which only occurs in DBA/2J mice. Simulating the DBA/2J cellular phenotype in C57BL/6J mice by infusing the GABAA receptor agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol hydrochloride, into cerebellar lobules IV-VI, in vivo, significantly reduced their alcohol consumption and blood alcohol concentrations achieved. 4,5,6,7-Tetrahydroisoxazolo-[5,4-c]pyridine-3-ol hydrochloride infusions also significantly decreased sucrose consumption, but they did not affect consumption of water or general locomotion. Thus, genetic differences in cerebellar response to alcohol contributes to alcohol consumption phenotype, and targeting the cerebellar GABAA receptor system may be a clinically viable therapeutic strategy for reducing excessive alcohol consumption. SIGNIFICANCE STATEMENT: Alcohol abuse is a leading cause of preventable death and illness; and although alcohol use disorders are 50%-60% genetically determined, the cellular and molecular mechanisms of such genetic influences are largely unknown. Here we demonstrate that genetic differences in cerebellar granule cell GABAA receptor responses to recreational concentrations of alcohol are the primary determinant of alcohol's impact on cerebellar processing and that pharmacologically modifying such responses alters alcohol consumption. These data highlight the cerebellum as an important neuroanatomical region in alcohol consumption phenotype and as a target for pharmacological treatment of alcohol use disorders. The results also add to the growing list of cognitive/emotional roles of the cerebellum in psychiatric disease and drug abuse.

Quantification of ten neuroactive steroids in plasma in Withdrawal Seizure-Prone and -Resistant mice during chronic ethanol withdrawal.

RATIONALE: The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. OBJECTIVES: Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABAAR sensitivity to ALLO in Withdrawal Seizure-Prone (WSP) but not in Withdrawal Seizure-Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABAAR-active steroids is not known. METHODS: After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of ten neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. RESULTS: We quantified levels of nine neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of five neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed five neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. CONCLUSIONS: Due to lower basal levels of some GABAAR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.

A method for mapping intralocus interactions influencing excessive alcohol drinking.

Excessive alcohol (ethanol) consumption is the hallmark of alcohol use disorders. The F1 hybrid cross between the C57BL/6J (B6) and FVB/NJ (FVB) inbred mouse strains consumes more ethanol than either progenitor strain. The purpose of this study was to utilize ethanol-drinking data and genetic information to map genes that result in overdominant (or heterotic) ethanol drinking. About 600 B6 x FVB F2 mice, half of each sex, were tested for ethanol intake and preference in a 24-h, two-bottle water versus ethanol choice procedure, with ascending ethanol concentrations. They were then tested for ethanol intake in a Drinking in the Dark (DID) procedure, first when there was no water choice and then when ethanol was offered versus water. DNA samples were obtained and genome-wide QTL analyses were performed to search for single QTLs (both additive and dominance effects) and interactions between pairs of QTLs, or epistasis. On average, F2 mice consumed excessive amounts of ethanol in the 24-h choice procedure, consistent with high levels of consumption seen in the F1 cross. Consumption in the DID procedure was similar or higher than amounts reported previously for the B6 progenitor. QTLs resulting in heightened consumption in heterozygous compared to homozygous animals were found on Chrs 11, 15, and 16 for 24-h choice 30% ethanol consumption, and on Chr 11 for DID. No evidence was found for epistasis between any pair of significant or suggestive QTLs. This indicates that the hybrid overdominance is due to intralocus interactions at the level of individual QTL.