Adolescent onset of volitional ethanol intake normalizes sex differences observed with adult-onset ethanol intake and negative affective behaviors during protracted forced abstinence.

Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST) and Crh+ neurons in this region play a key role in chronic ethanol-induced increases in volitional intake, hypothesized to be driven by emergent negative affective behaviors. Excitatory N-methyl-d-aspartate receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. Specifically, GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. Male and female mice underwent a home cage chronic drinking forced abstinence model (CDFA) to assess the impact of 1 day or 2 weeks of ethanol abstinence on BNST synaptic transmission and BNST Grin gene expression. Constitutive and conditional BNST GluN2D knockout mice were used to assess the impact of GluN2D deletion on continuous access ethanol intake as well as negative affect behaviors, using the open field, elevated zero maze, and forced swim tasks. We report here that excitatory transmission undergoes time-dependent upregulation in BNST Crh+ cells. Further, knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in the dBNST or dBNST Crh+ neurons. Finally, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. These data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking but not ethanol abstinence, highlighting sex differences and behavioral specificity. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

Opioids initiate dynamic maladaptation in brain reward and affect circuits that occur throughout chronic exposure and withdrawal that persist beyond cessation. Protracted abstinence is characterized by negative affective behaviors such as heightened anxiety, irritability, dysphoria, and anhedonia, which pose a significant risk factor for relapse. While the ventral tegmental area (VTA) and μ-opioid receptors (MORs) are critical for opioid reinforcement, the specific contributions of VTAMOR neurons in mediating protracted abstinence-induced negative affect is not fully understood. In our study, we elucidate the role of VTAMOR neurons in mediating negative affect and altered brain-wide neuronal activities following forced opioid exposure and abstinence in male and female mice. Utilizing a chronic oral morphine administration model, we observe increased social deficit, anxiety-related, and despair-like behaviors during protracted forced abstinence. VTAMOR neurons show heightened neuronal FOS activation at the onset of withdrawal and connect to an array of brain regions that mediate reward and affective processes. Viral re-expression of MORs selectively within the VTA of MOR knock-out mice demonstrates that the disrupted social interaction observed during protracted abstinence is facilitated by this neural population, without affecting other protracted abstinence behaviors. Lastly, VTAMORs contribute to heightened neuronal FOS activation in the anterior cingulate cortex (ACC) in response to an acute morphine challenge, suggesting their unique role in modulating ACC-specific neuronal activity. These findings identify VTAMOR neurons as critical modulators of low sociability during protracted abstinence and highlight their potential as a mechanistic target to alleviate negative affective behaviors associated with opioid abstinence.

Role and Regulation of BNST GluN2D-containing NMDARs in a Continuous Access Ethanol Task

Consumption of ethanol solutions by rodents in two-bottle choice tests is a model to study human alcohol intake. Mice of the C57BL/6ByJ strain have higher ethanol preferences and intakes than do mice of the 129P3/J strain. F2 hybrids between these two strains were phenotyped using two-bottle tests involving a choice between water and either 3% or 10% ethanol. High ethanol preferences and intakes of the B6 mice were inherited as additive or dominant traits in the F2 generation. A genome screen using these F2 mice identified three significant linkages. Two loci, on distal chromosome 4 (Ap3q) and proximal chromosome 7 (Ap7q), strongly affected 10% ethanol intake and weakly affected 3% ethanol intake. A male-specific locus on proximal chromosome 8 (Ap8q) affected 3% ethanol preference, but not indexes of 10% ethanol consumption. In addition, six suggestive linkages (on chromosomes 2, 9, 12, 13, 17, and 18) affecting indexes of 3% and/or 10% ethanol consumption were detected. The loci with significant and suggestive linkages accounted for 35-44% of the genetic variation in ethanol consumption phenotypes. No additive-by-additive epistatic interactions were detected for the primary loci with significant and suggestive linkages. However, there were a few modifiers of the primary linkages and a number of interactions among unlinked loci. This demonstrates a significant role of the genetic background in the variation of ethanol consumption.

Binge alcohol drinking is a risk factor linked to numerous disease states including alcohol use disorder (AUD). While men binge drink more alcohol than women, this demographic gap is quickly shrinking, and preclinical studies demonstrate that females consistently consume more alcohol than males. Further, women are at increased risk for the co-expression of AUD with neuropsychiatric diseases such as anxiety and mood disorders. However, little is understood about chronic voluntary alcohol drinking and its long-term effects on behavior. Here, we sought to characterize sex differences in chronic binge drinking and the effects of protracted alcohol abstinence on anxiety- and affective-related behaviors in males and females. We assessed binge alcohol drinking patterns in male and female C57BL/6J mice using a modified Drinking in the Dark (DID) paradigm in which mice received home cage access to one bottle of 10% or 20% alcohol (EtOH) or water for 2h per day on Days 1-3 and to two bottles (EtOH/H2O + H2O) for 24h on Day 4 for 8 weekly cycles. Mice were then tested for the effects of protracted abstinence on avoidance, affective, and compulsive behaviors. Female mice consumed more alcohol than males consistently across cycles of DID and at 2, 4, and 24-h timepoints within the day, with a more robust sex difference for 20% than 10% EtOH. Females also consumed more water than males, an effect that emerged at the later time points; this water consumption bias diminished when alcohol was available. Further, while increased alcohol consumption was correlated with decreased water consumption in males, there was no relationship between these two measures in females. Alcohol preference was higher in 10% vs. 20% EtOH for both sexes. During protracted abstinence following chronic binge drinking, mice displayed decreased avoidance behavior (elevated plus maze, open field, novelty suppressed feeding) and increased compulsive behavior (marble burying) that was especially robust in females. There was no effect of alcohol history on stress coping and negative affective behaviors (sucrose preference, forced swim test, tail suspension) in either sex. Female mice engaged in higher volume binge drinking than their male counterparts. Although females also consumed more water than males, their higher alcohol consumption was not driven by increased total fluid intake. Further, the effects of protracted abstinence following chronic binge drinking was driven by behavioral disinhibition that was more pronounced in females. Given the reciprocal relationship between risk-taking and alcohol use in neuropsychiatric disease states, these results have implications for sex-dependent alcohol drinking patterns and their long-term negative neuropsychiatric/physiological health outcomes in humans.

With alcohol readily accessible to adolescents, its consumption leads to many adverse effects, including impaired learning, attention, and behavior. Adolescents report higher rates of binge drinking compared to adults. They are also more prone to substance use disorder in adulthood due to physiological changes during the adolescent developmental period. We used C57BL/6J male and female mice to investigate the long-lasting impact of binge ethanol exposure during adolescence on voluntary ethanol intake and open field behavior during later adolescence (Experiment 1) and during emerging adulthood (Experiment 2). The present set of experiments were divided into four stages: (1) adolescent intermittent vapor inhalation exposure, (2) abstinence, (3) voluntary ethanol intake, and (4) open field behavioral testing. During adolescence, male and female mice were exposed to air or ethanol using intermittent vapor inhalation from postnatal day (PND) 28-42. Following this, mice underwent short-term abstinence from PND 43-49 (Experiment 1) or protracted abstinence from PND 43-69 (Experiment 2). Beginning on PND 50-76 or PND 70-97, mice were assessed for intermittent voluntary ethanol consumption using a two-bottle choice drinking procedure over 28days. Male adolescent ethanol-exposed mice showed increased ethanol consumption following short-term abstinence and following protracted abstinence. In contrast, female mice showed no changes in ethanol consumption following short-term abstinence and decreased ethanol consumption following protracted abstinence. There were modest changes in open field behavior following voluntary ethanol consumption in both experiments. These data demonstrate a sexually divergent shift in ethanol consumption following binge ethanol exposure during adolescence and differences in open field behavior. These results highlight sex-dependent vulnerability to developing substance use disorders in adulthood.

Adolescent alcohol use may contribute to long-term changes in the receptors and neuroactive steroids that may mediate its effects and to subsequent alcohol abuse and dependence as an adult. Therefore, in this study, ethanol preference and intake as an adult were examined after adolescent ethanol or saline administration. In addition, ethanol intake in the same groups was examined after administration of 2 neuroactive steroids with modulatory effects at GABA(A) receptors. Two groups of male Long-Evans rats were administered 15 intraperitoneal (i.p.) injections of either ethanol (2 g/kg, 20% v/v) or saline between postnatal days 35 and 63. Starting on postnatal day 75, both groups were trained to consume 10% ethanol using a saccharin-fading procedure, and ethanol intake and preference were measured after a series of manipulations involving food deprivation, changes in the duration of access to ethanol, and changes in the concentrations of ethanol presented. Following these manipulations, pregnanolone (1 to 10 mg/kg) and dehydroepiandrosterone (DHEA, 1 to 100 mg/kg) were administered prior to preference sessions with an 18% ethanol solution. Adult ethanol preference and intake did not differ significantly in subjects treated with either saline or ethanol as adolescents during training, the substitution of other ethanol concentrations (3.2 to 32%), ad-lib feeding, or moderate food deprivation. Pregnanolone administration altered the intake of both adolescent-treated groups after the first injection of 3.2 mg/kg and after repeated injections with 10 mg/kg, a dose that produced sedation. In contrast, multiple doses of DHEA consistently decreased intake of an 18% ethanol concentration in both groups after repeated injections and 3 doses of DHEA (10, 32, and 56 mg/kg) administered with various ethanol concentrations dose-dependently shifted the ethanol-concentration curves for the volume and dosage of ethanol consumed downward. These results indicate that chronic intermittent ethanol (CIE) administration of 2 g/kg during adolescence did not alter preference or overall consumption of ethanol in outbred rats trained to drink ethanol as an adult under the conditions tested, and that DHEA may be more effective than pregnanolone at significantly decreasing ethanol consumption.

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Assessing negative affect in mice during abstinence from alcohol drinking: Limitations and future challenges.

Alcohol use disorders emerge from a complex interaction between environmental and genetic factors. Stress and dopamine D2 receptor levels (DRD2) have been shown to play a central role in alcoholism. To better understand the interactions between DRD2 and stress in ethanol intake behavior, we subjected Drd2 wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice to 4 weeks of chronic mild stress (CMS) and to an ethanol two-bottle choice during CMS weeks 2-4. Prior to and at the end of the experiment, the animals were tested in the forced swim and open field tests. We measured ethanol intake and preference, immobility in the force swim test, and activity in the open field. We show that under no CMS, Drd2+/- and Drd2-/- mice had lower ethanol intake and preference compared with Drd2+/+. Exposure to CMS decreased ethanol intake and preference in Drd2+/+ and increased them in Drd2+/- and Drd2-/- mice. At baseline, Drd2+/- and Drd2-/- mice had significantly lower activity in the open field than Drd2+/+, whereas no genotype differences were observed in the forced swim test. Exposure to CMS increased immobility during the forced swim test in Drd2+/- mice, but not in Drd2+/+ or Drd2-/- mice, and ethanol intake reversed this behavior. No changes were observed in open field test measures. These findings suggest that in the presence of a stressful environment, low DRD2 levels are associated with increased ethanol intake and preference and that under this condition, increased ethanol consumption could be used as a strategy to alleviate negative mood.

Background: Operant experiments which indicate that ethanol can serve as a reinforcer to maintain lever responding during limited periods of access have not been conducted on non-food-deprived mice, as they have for rats and monkeys. Furthermore, there are no reports of the effects of chronic ethanol and subsequent abstinence on ethanol reward in mice. Finally, although naltrexone reduces responding for ethanol in food-deprived mice, the effects of the drug on ethanol reward for non-food-deprived mice have not been reported. Methods: In three experiments, lever responding for ethanol (10–12%) was established in C57BL/6 (B6) mice by using either sucrose or saccharin fading procedures commonly used for rats. Experiment 1 examined both appetitive and consummatory responses while sucrose was faded from the ethanol solutions. Experiment 2 examined lever responding and ethanol intake (1) during saccharin fading; (2) when reinforcement schedules, reward availability, and primary conditioned reinforcers were manipulated; and (3) when mice were allowed chronic ethanol consumption followed by forced abstinence. Experiment 3 examined the effects of low doses of naltrexone on ethanol reward. Results: Lever responding for ethanol can be established in non-food-deprived mice with the sucrose and saccharin fading procedures commonly used for rats. Lever responses increased with decreases in the reinforcer and increases in schedule demand, which indicated the reward value of the ethanol solution. Removal of ethanol from the solution reduced consumption with no change in the appetitive, instrumental response, which indicated that the two responses were under control of different stimuli, perhaps mediated by different neural mechanisms. Forced abstinence after chronic ethanol exposure increased responding for the drug, which suggested increased reward value. Naltrexone reduced responding as previously reported for food-deprived B6 mice. Conclusions: Ethanol appears to serve as a reinforcer for non-food-deprived or non-water-deprived B6 mice. Its reinforcing effects are increased by forced abstinence after chronic exposure and are decreased by naltrexone.

Operant experiments which indicate that ethanol can serve as a reinforcer to maintain lever responding during limited periods of access have not been conducted on non-food-deprived mice, as they have for rats and monkeys. Furthermore, there are no reports of the effects of chronic ethanol and subsequent abstinence on ethanol reward in mice. Finally, although naltrexone reduces responding for ethanol in food-deprived mice, the effects of the drug on ethanol reward for non-food-deprived mice have not been reported. In three experiments, lever responding for ethanol (10-12%) was established in C57BL/6 (B6) mice by using either sucrose or saccharin fading procedures commonly used for rats. Experiment 1 examined both appetitive and consummatory responses while sucrose was faded from the ethanol solutions. Experiment 2 examined lever responding and ethanol intake (1) during saccharin fading; (2) when reinforcement schedules, reward availability, and primary conditioned reinforcers were manipulated; and (3) when mice were allowed chronic ethanol consumption followed by forced abstinence. Experiment 3 examined the effects of low doses of naltrexone on ethanol reward. Lever responding for ethanol can be established in non-food-deprived mice with the sucrose and saccharin fading procedures commonly used for rats. Lever responses increased with decreases in the reinforcer and increases in schedule demand, which indicated the reward value of the ethanol solution. Removal of ethanol from the solution reduced consumption with no change in the appetitive, instrumental response, which indicated that the two responses were under control of different stimuli, perhaps mediated by different neural mechanisms. Forced abstinence after chronic ethanol exposure increased responding for the drug, which suggested increased reward value. Naltrexone reduced responding as previously reported for food-deprived B6 mice. Ethanol appears to serve as a reinforcer for non-food-deprived or non-water-deprived B6 mice. Its reinforcing effects are increased by forced abstinence after chronic exposure and are decreased by naltrexone.

Increased ethanol consumption and preference and decreased ethanol sensitivity in female FAAH knockout mice

Previous studies using the maternal separation (MS) model have shown that environmental factors early in life affect adult ethanol consumption. Prolonged MS is related to enhanced propensity for high adult ethanol intake when compared to short MS. Less is known about the environmental impact on adolescent ethanol intake. In this study, the aim was to compare establishment of voluntary ethanol consumption in adolescent and adult rats subjected to different rearing conditions. Wistar rat pups were separated from their mother 0 minutes (MS0), 15 minutes (MS15), or 360 minutes (MS360) daily during postnatal days (PNDs) 1 to 20. After weaning, the male rats were divided into two groups; rats were given free access to water, 5 and 20% ethanol at either PND 26 or 68. Ethanol was provided in 24-hour sessions three times per week for 5 weeks. MS resulted in altered ethanol consumption patterns around the pubertal period but otherwise the rearing conditions had little impact on ethanol consumption in adolescents. In adults, the establishment of ethanol consumption was dependent on the rearing condition. The adult MS0 and MS15 rats had a stable ethanol intake, whereas the MS360 rats increased both their ethanol intake and preference over time. With the use of intermittent access to ethanol, new data were provided, which confirm the notion that MS360 represents a risk environment related to higher ethanol intake compared to MS15. The adolescent rats had higher ethanol intake than adult rats but the consumption was independent of rearing condition. Experiences during the first three postnatal weeks thus affect the establishment of voluntary ethanol consumption differently in adolescent and adult rats. Further studies are now warranted to examine the consequences of a combination of early environmental influence and high adolescent ethanol intake.

Ethanol intake levels characteristic of adult males and females emerge postpubertally. The present set of experiments examined the consequences of prepubertal and adult gonadectomies to explore whether the presence of gonadal hormones at puberty exerts organizational influences and/or plays an activational role in age- and sex-typical patterns of ethanol consumption. Male and female Sprague-Dawley rats were gonadectomized (GX), received sham gonadectomy (SH), or were left nonmanipulated (NM) at 1 of 2 ages, either prepubertally on postnatal day (P) 23 (early) or postpubertally in adulthood on P70 (late). Early surgery animals were tested for ethanol consumption either during adolescence (P28 to 39) or in adulthood at the same age that late surgery animals were tested (P75 to 86). Voluntary ethanol consumption was indexed using a 2-hour limited-access paradigm, with access to 2 bottles: one containing water and the other a sweetened ethanol solution. Age of GX did not impact patterns of ethanol consumption. Removal of testicular hormones in males, regardless of age of removal, elevated consumption levels in adulthood to female-typical levels. Ovariectomy did not have notable effects on ethanol drinking in females. Ethanol intake and preference of early SH males were significantly greater than those of both late SH and NM males. Removal of the gonads prior to puberty did not influence ethanol drinking or preference during adolescence in either males or females. These results suggest that testicular hormones play an activational role in lowering ethanol intake and preference of adult male rats. Pubertal hormones, in contrast, were found to exert little influence on ethanol drinking or preference during adolescence, although the effect of surgical manipulation itself during development was found to exert a long-lasting facilitatory effect on ethanol consumption in adulthood.