Adolescent C57BL/6J mice show elevated alcohol intake, but reduced taste aversion, as compared to adult mice: a potential behavioral mechanism for binge drinking

Background: Binge alcohol drinking during adolescence is a serious health problem that may increase future risk of an alcohol use disorder. Although there are several different procedures by which to preclinically model binge‐like alcohol intake, limited‐access procedures offer the advantage of achieving high voluntary alcohol intake and pharmacologically relevant blood alcohol concentrations (BACs). Therefore, in the current study, developmental differences in binge‐like alcohol drinking using a limited‐access cycling procedure were examined. In addition, as alcohol drinking has been negatively correlated with sensitivity to the aversive properties of alcohol, we examined developmental differences in sensitivity to an alcohol‐induced conditioned taste aversion (CTA). Methods: Binge‐like alcohol consumption was investigated in adolescent (4 weeks) and adult (10 weeks) male C57BL/6J mice for 2 to 4 h/d for 16 days. Developmental differences in sensitivity to an alcohol‐induced CTA were examined in adolescent and adult mice, with saline or alcohol (3 or 4 g/kg) repeatedly paired with the intake of a novel tastant (NaCl). Results: Adolescent mice showed a significant increase in alcohol intake as compared to adults, with adolescents achieving higher BACs and increasing alcohol consumption over successive cycles of the binge procedure. Conversely, adolescent mice exhibited a dose‐dependent reduction in sensitivity to the aversive properties of alcohol, as compared to adult mice, with adolescent mice failing to develop a CTA to 3 g/kg alcohol. Finally, extinction of an alcohol CTA was observed following conditioning with a higher dose of alcohol in adolescent, versus adult, mice. Conclusions: These results indicate that adolescent mice consume more alcohol, per kilogram body weight, than adults in a binge‐like model of alcohol drinking and demonstrate a blunted sensitivity to the conditioned aversive effects of alcohol. Overall, this supports a behavioral framework by which heightened binge alcohol intake during adolescence occurs, in part, via a reduced sensitivity to the aversive properties of alcohol.     

Sensitivity and Tolerance to the Hypnotic and Ataxic Effects of Ethanol in Adolescent and Adult C57BL/6J and DBA/2J Mice

Background: There is considerable research examining differences in adolescent and adult sensitivity and tolerance to ethanol related behavioral phenotypes. However, the available published data has almost exclusively assessed these behaviors in outbred rats. The present study was conducted using the alcohol preferring inbred mouse strain C57BL/6J (B6) and the alcohol nonpreferring inbred mouse strain DBA/2J (D2) to determine if differences in the sedative and ataxic effects of ethanol exist between adolescents and adults, and to determine whether there are any genetic influences involved therein. Methods: Adolescent and adult mice of each sex and genotype were given intraperitoneal (i.p.) injections of ethanol (1.5, 1.75, or 4.0 g/kg) or saline and assessed for the loss of righting reflex (LORR) or hind footslips on the balance beam apparatus. These animals were then tested for the development of tolerance to these behaviors on subsequent days. Results: Despite evident pharmacokinetic differences, D2 adolescents were found to be relatively less sensitive to ethanol’s hypnotic actions than their adult D2 counterparts. Adolescent and adult B6 animals did not differ. Furthermore, although adult animals appeared to develop significantly greater degrees of tolerance to ethanol‐induced hypnosis compared with adolescents, these effects were likely in part related to differences in ethanol absorption/metabolism across time. Taking into account pharmacokinetic differences and the overall poor performance of male adults, adolescent animals were found to be equally if not more sensitive to the motor incoordinating (ataxic) effects of ethanol. Overall, tolerance to these effects varied by age and genotype but appeared to be related to changes in ethanol pharmacokinetics rather than strict behavioral sensitivity. Conclusion: The current work suggests that adolescent B6 and D2 inbred mice exhibit ontogenetic differences in sensitivity to ethanol’s hypnotic and ataxic effects. Importantly, in some cases age differences emerge as a function of differential ethanol pharmacokinetics. These results extend the current literature examining this critical developmental period in mice and illustrate the benefits of comparing ethanol related developmental differences in different genetic mouse populations.     

Intermittent (every-other-day) drinking induces rapid escalation of ethanol intake and preference in adolescent and adult C57BL/6J mice

Background: Using adult C57BL/6J (B6) mice, we previously developed a procedure that causes a progressive increase in ethanol intake and preference (i.e., alcohol escalation effect) following weekly (intermittent) access to ethanol ( Melendez et al., 2006 ). A limitation of this procedure is that it requires many weeks of testing, which limits its use to study ethanol escalation (i.e., binge‐like drinking) during adolescence. Previous studies have shown that intermittent every‐other‐day (EOD) access to ethanol is sufficient to induce ethanol escalation in rats. The objective of this study was to verify whether EOD access is sufficient to induce escalated levels of ethanol intake and preference in adult and adolescent B6 mice. Methods: Male B6 mice received free‐choice 24‐hour access to 15% ethanol and water on an EOD or daily basis for 2 weeks. Food and water were available at all times. Using adult mice, Experiment 1 characterized the induction of ethanol escalation following EOD access at 6 (i.e., drinking in the dark) and 24‐hour intervals, whereas Experiment 2 determined whether daily drinking reverses escalation induced by EOD drinking. Experiment 3 compared ethanol‐drinking capacity following daily versus EOD drinking in adolescent (P30‐45) and adult (P70‐85) mice. Results: Experiment 1 revealed that EOD drinking leads to a significant (nearly 2‐fold) increase in ethanol intake and preference over mice given daily access. Experiment 2 demonstrated that EOD‐elicited escalation is blocked and subsequently reversed following daily drinking. Experiment 3 revealed that ethanol drinking was greater in adolescent mice compared with adults following daily drinking and EOD (escalated) drinking. Although the escalated levels of ethanol intake were greater in adolescent mice, the rate or onset of escalation was comparable between both age‐groups. Conclusions: This study is the first to demonstrate that EOD drinking leads to escalation of ethanol intake and preference in adolescent and adult mice. Moreover, our results indicate that daily ethanol reverses ethanol escalation induced by intermittent drinking. The study also revealed that adolescent mice have a greater capacity to drink ethanol under both daily (controlled) and EOD (escalated) conditions, which further supports the notion of adolescent’s susceptibility to heavy drinking.     

Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol

Background: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling‐type transition from moderate to escalated use, leading to dependence. Intermittent 24‐hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats. Methods: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short‐term, limited access “binge” drinking procedure, similar to drinking‐in‐the‐dark (DID). B6 mice were also assessed for ethanol dependence with handling‐induced convulsion, a reliable measure of withdrawal severity. Results: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2‐hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h). Discussion: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.     

Aversive Effects of Ethanol in Adolescent Versus Adult Rats: Potential Causes and Implication for Future Drinking

Background: Many people experiment with alcohol and other drugs of abuse during their teenage years. Epidemiological evidence suggests that younger initiates into drug taking are more likely to develop problematic drug seeking behavior, including binge and other high‐intake behaviors. The level of drug intake for any individual depends on the balance of rewarding and aversive effects of the drug in that individual. Multiple rodent studies have demonstrated that aversive effects of drugs of abuse are reduced in adolescent compared to adult animals. In this study, we addressed 2 key questions: First, do reduced aversive effects of ethanol in younger rats correlate with increased ethanol consumption? Second, are the reduced aversive effects in adolescents attributable to reduced sensitivity to ethanol’s physiologic effects? Methods: Adolescent and adult rats were tested for ethanol conditioned taste aversion (CTA) followed by a voluntary drinking period, including postdeprivation consumption. Multivariate regression was used to assess correlations. In separate experiments, adolescent and adult rats were tested for their sensitivity to the hypothermic and sedative effects of ethanol, and for blood ethanol concentrations (BECs). Results: We observed that in adolescent rats but not adults, taste aversion was inversely correlated with postdeprivation consumption. Adolescents also exhibited a greater increase in consumption after deprivation than adults. Furthermore, the age difference in ethanol CTA was not attributable to differences in hypothermia, sedation, or BECs. Conclusions: These results suggest that during adolescence, individuals that are insensitive to aversive effects are most likely to develop problem drinking behaviors. These results underscore the importance of the interaction between developmental stage and individual variation in sensitivity to alcohol.     

Tolerance to ethanol's ataxic effects and alterations in ethanol-induced locomotion following repeated binge-like ethanol intake using the DID model

Background: Tolerance to the behavioral and subjective effects of alcohol (ethanol) is thought to be a major predictive factor for the development of alcoholism. Evidence from rodent models has supported this view with those animals most likely to develop tolerance generally drinking and preferring ethanol more so than those resistant to it. Despite this evidence, very little is known about the behavioral relationships between ethanol‐induced tolerance and consumption. The goal of this study was to evaluate the development of tolerance to the ataxic effects of ethanol using a mouse model of binge‐like intake dubbed “Drinking in the Dark” (DID; Physiol Behav 2005, 84:53–63). We hypothesized that mice would become tolerant to the ataxic effects of ethanol as this behavior is known to be altered at the blood ethanol concentrations reached using this model (≥80 mg/dl). Methods: To evaluate this, we gave daily DID ethanol or water access sessions to male C57BL/6J (B6) mice and monitored ataxia (and in some cases locomotion) at various time points. Results: In general, mice given 14 consecutive days of ethanol access displayed tolerance to the ataxic effects of ethanol compared to water‐drinking controls. These effects were coupled with alterations in locomotor behavior and in some cases differences in ethanol pharmacokinetics. Conclusions: Thus, we can conclude that tolerance to the behavioral effects of binge‐like ethanol intake might play a key role in the daily maintenance of this behavior and that these effects may be evidence of important neuroadaptations involved in the development of alcoholism.     

Sensitization, Duration, and Pharmacological Blockade of Anxiety-Like Behavior Following Repeated Ethanol Withdrawal in Adolescent and Adult Rats

Background: Repeated ethanol withdrawal sensitizes anxiety‐like behavior in adult rats and causes anxiety‐like behavior and decreased seizure thresholds in adolescent rats. Current experiments determined if adolescent rats exhibit sensitized anxiety‐like behavior, the duration of this effect, if drug pretreatments blocked these effects, and if these effects differed from those seen in adults. Methods: Male adolescent rats received three 5‐day cycles of 2.5% ethanol diet (ED) separated by two 2‐day withdrawal periods, continuous 15 days of 2.5%ED, or a single 5‐day cycle of 2.5%ED. Male adult rats received three 5‐day cycles of either 2.5% or 3.5%ED. These groups were tested 5 hours into the final withdrawal for social interaction (SI) deficits (an index of anxiety‐like behavior). Ethanol intake was monitored throughout and blood concentrations were obtained from separate groups of rats. Additionally, adolescent rats were tested for SI 1, 2, 7, 14, and 18 days and adults 1 and 2 days after the final withdrawal. Some adolescent rats were also pretreated with the CRF₁ antagonist CP‐154,526, the 5‐HT_1A agonist buspirone, or the benzodiazepine receptor antagonist flumazenil during the first 2 withdrawals. Results: SI was reduced in adolescent rats following repeated withdrawals of 2.5%ED while neither a continuous or single cycle ED exposure caused this effect. Adult rats also had reduced SI following repeated withdrawals from both 2.5% and 3.5%ED. This effect was present up to 1 week following the final withdrawal in adolescents but returned to baseline by 1 day in adults. CP‐154,526, buspirone, or flumazenil prevented this reduction in SI in adolescent rats. Conclusions: Adolescent rats exhibit sensitized anxiety‐like behavior following repeated withdrawals at ED concentrations similar to those used in adults. However, this effect is longer lasting in adolescent rats. Drugs modulating CRF, 5‐HT, or GABA systems during initial withdrawals prevent the development of anxiety‐like behavior otherwise manifest during a final withdrawal in adolescent rats.     

Adolescent binge drinking alters adult brain neurotransmitter gene expression, behavior, brain regional volumes, and neurochemistry in mice

Background: Binge drinking is common in human adolescents. The adolescent brain is undergoing structural maturation and has a unique sensitivity to alcohol neurotoxicity. Therefore, adolescent binge ethanol may have long‐term effects on the adult brain that alter brain structure and behaviors that are relevant to alcohol‐use disorders. Methods: To determine whether adolescent ethanol (AE) binge drinking alters the adult brain, male C57BL/6 mice were treated with either water or ethanol during adolescence (5 g/kg/d, i.g., postnatal days P28 to P37) and assessed during adulthood (P60 to P88). An array of neurotransmitter‐specific genes, behavioral tests (i.e., reversal learning, prepulse inhibition, and open field), and postmortem brain structure using magnetic resonance imaging (MRI) and immunohistochemistry, were employed to assess persistent alterations in adult brain. Results: At P38, 24 hours after AE binge, many neurotransmitter genes, particularly cholinergic and dopaminergic, were reduced by ethanol treatment. Interestingly, dopamine receptor type 4 mRNA was reduced and confirmed using immunohistochemistry. Normal control maturation (P38 to P88) resulted in decreased neurotransmitter mRNA, e.g., an average decrease of 56%. Following AE treatment, adults showed greater gene expression reductions than controls, averaging 73%. Adult spatial learning assessed in the Morris water maze was not changed by AE treatment, but reversal learning experiments revealed deficits. Assessment of adult brain region volumes using MRI indicated that the olfactory bulb and basal forebrain were smaller in adults following AE. Immunohistochemical analyses found reduced basal forebrain area and fewer basal forebrain cholinergic neurons. Conclusions: Adolescent binge ethanol treatment reduces adult neurotransmitter gene expression, particularly cholinergic genes, reduces basal forebrain and olfactory bulb volumes, and causes a reduction in the density of basal forebrain acetylcholine neurons. Loss of cholinergic neurons and forebrain structure could underlie adult reversal learning deficits following adolescent binge drinking.     

Role of novelty and ethanol history in locomotor stimulation induced by binge-like ethanol intake

Background: The acute locomotor effects of voluntary ethanol (EtOH) intake in mice (stimulation/sedation) might be important behavioral indicators of an animals’ propensity to engage in EtOH consumption and/or EtOH seeking behaviors. Using a binge‐like EtOH intake model dubbed “Drinking‐in‐the‐Dark (DID),” we recently observed home cage locomotor stimulation in C57BL/6J mice during an acute EtOH intake session, but acute home cage locomotor sedation following repeated EtOH exposures. To determine the role of novelty and/or EtOH history on these previously described locomotor effects, and to determine the relationship between these variables on locomotor activity immediately following DID intake, we conducted 2 separate experiments. Methods: In experiment 1, mice were given access to either EtOH or water, and locomotor activity was monitored immediately afterwards. In experiment 2, mice were given 13 days access to EtOH or water solution while home cage locomotor activity was monitored. On the 14th day, half of the water consuming animals received EtOH access for the first time. On the 15th day, all animals received EtOH access, and locomotion was assessed afterwards in locomotor activity testing chambers. Results: In experiment 1, locomotor activity following DID was positively associated with EtOH intake and blood EtOH concentrations (BECs). In experiment 2, the group that received EtOH for the first time on the 14th day did not display locomotor stimulation. Locomotor activity following DID EtOH intake was positively associated with BECs in all groups regardless of EtOH history. Conclusions: These results suggest that (i) DID‐induced locomotor stimulation in the home cage may involve relative familiarity with the DID procedures, and (ii) locomotor stimulation immediately following DID is directly related to the relative concentration of EtOH in blood; an effect that is not altered by prior EtOH history. These data add new evidence of the pharmacological actions of binge‐like EtOH intake, and provide a basis by which we may explore the motivation and consequences of such binge consumption.     

Role of genotype in the development of locomotor sensitization to alcohol in adult and adolescent mice: comparison of the DBA/2J and C57BL/6J inbred mouse strains

Background: Animal models that explore differential sensitivity to the effects of acute and repeated exposure of alcohol (ethanol) may be influenced by both the developmental and genetic profile of the population. Therefore, we sought to compare the influence of ontogeny on sensitivity to ethanol‐induced locomotor stimulation and on the induction of locomotor sensitization to this effect across 2 inbred strains of mice; the ethanol consuming C57BL/6J and the ethanol avoiding DBA/2J strains. Methods: C57BL/6J and DBA/2J adults (postnatal day [PD] 60 to 80) and adolescents (PD 30 ± 2) were assessed for basal activity, acute response to 2.0 g/kg ethanol, and the expression of locomotor sensitization following repeated administration of 2.5, 3.0, or 3.5 g/kg ethanol. Results: Basal activity was different across development for the C57BL/6J, but not DBA/2J, with adult B6 mice showing persistently greater baseline activity. Adolescents of both strains were more sensitive than adults to acute ethanol‐induced locomotor stimulation; adults exhibited a decrease in their acute response across the testing session. Adolescent DBA/2J mice developed less ethanol sensitization compared to adults, with significant sensitization observed only following repeated administration of the lowest ethanol dose (2.5 g/kg), whereas DBA/2J adults sensitized to all doses. Age did not influence the development of ethanol sensitization for the C57BL/6J strain, as both adults and adolescents displayed a sensitized response following all ethanol doses. Conclusions: These results suggest that the developmental pattern of locomotor sensitivity to ethanol is unique to the genotypic profile of the animal model.     

Differential dietary ethanol intake and blood ethanol levels in adolescent and adult rats: effects on anxiety-like behavior and seizure thresholds

Background: Adult rats exhibit increased anxiety‐like behavior after exposure to repeated cycles of chronic ethanol and withdrawal. While adolescent rats have differential responses to both acute and chronic ethanol treatments, the potential differences in the effects of repeated withdrawals in this population have yet to be determined. Methods: Male adult and adolescent rats received three 5‐day cycles of either a 4.5% or 7% ethanol diet (ED) separated by two 2‐day withdrawal periods. Five hours into the final withdrawal, rats were tested for social interaction (SI) deficits (an index of anxiety‐like behavior) and then assessed for seizure thresholds (audiogenic and bicuculline‐induced). Ethanol intake was monitored throughout, and blood ethanol concentrations (BEC) were obtained from a separate group of rats. Results: Adolescent rats have reduced SI during the final withdrawal from either ED and exhibit a greater reduction in SI compared to adult rats when exposed to a 7%ED. Audiogenic seizures were not increased during withdrawal from either ED in adult rats, but adolescent rats that received 7%ED displayed increased seizures. The bicuculline seizure thresholds were decreased in both ages exposed to a 7%ED, but only adolescent rats showed this decreased threshold after 4.5%ED. Ethanol intakes and BECs were higher in adolescent rats compared to similarly treated adults. However, ethanol intakes and BECs were comparable between 4.5%ED‐treated adolescent and 7%ED‐treated adult rats. Conclusions: Behavioral results from the 7%ED‐treated groups suggested that adolescent rats may be more vulnerable to repeated withdrawals from ethanol than adults; however, differences in ethanol intake and BECs may be at least in part responsible. When ethanol intakes and BECs were similar between 4.5%ED‐treated adolescent and 7%ED‐treated adult rats, behavioral effects were not different. Importantly, these data illustrated that adolescent rats can exhibit anxiety and reduced seizure thresholds following this repeated withdrawal paradigm.     

Acute effects of acamprosate and MPEP on ethanol Drinking-in-the-Dark in male C57BL/6J mice

Background: Recently, a simple procedure in mice, Drinking‐in‐the‐Dark (DID), was hypothesized to have value for medication development for human alcoholism. In DID, mice are offered intermittent, limited access to ethanol over a series of days during the dark phase that results in rapid drinking to intoxication in predisposed genotypes. Methods: We measured the effects of acamprosate or MPEP, metabotropic glutamate 5 receptor (mGluR5) antagonist, on intake of 20% ethanol, plain tap water or 10% sugar water using the DID procedure in male C57BL/6J mice. Results: Acamprosate (100, 200, 300, or 400 mg/kg) dose dependently decreased ethanol drinking with 300 mg/kg reducing ethanol intake by approximately 20% without affecting intake of plain water or 10% sugar water. MPEP (1, 3, 5, 10, 20, or 40 mg/kg) was more potent than acamprosate with 20 mg/kg reducing ethanol intake by approximately 20% and for longer duration without affecting intake of plain water or 10% sugar water. Conclusions: These results support the hypothesis that mGluR5 signaling plays a role in excessive ethanol intake in DID and suggest DID may have value for screening novel compounds that reduce overactive glutamate signaling for potential pharmaceutical treatment of excessive ethanol drinking behavior.     

Gender and age at drinking onset affect voluntary alcohol consumption but neither the alcohol deprivation effect nor the response to stress in mice

Background: Epidemiological studies suggest that initiation of alcohol drinking at an early age is associated with an increased risk of developing an alcohol use disorder later in life. Nevertheless, relatively few studies using animal models have investigated the relationship between age of onset of drinking and ethanol drinking patterns in adulthood. Besides age at drinking onset, other factors such as gender could also affect the pattern of development of alcohol consumption. In rodents, many studies have shown that females drink more than males. However, even if it is assumed that hormonal changes occurring at puberty could explain these differences, only one study performed in rats has investigated the emergence of sex‐specific alcohol drinking patterns in adolescence and the transition from adolescence to adulthood. The aim of the present study was to compare the acquisition of voluntary alcohol consumption, relapse‐like drinking (the Alcohol Deprivation Effect—ADE) and stress‐induced alcohol drinking in male and female outbred mice that acquired alcohol consumption during adolescence or adulthood. Methods: Separate groups of naïve female and male WSC‐1 mice aged ± 28 days (adolescents) or ±70 days (adults) were given ad libitum access to water and 6% ethanol solution for 8 weeks (1st to 8th week) before undergoing a 2‐week deprivation phase (9th and 10th week). After the deprivation period, 2‐bottle preference testing (ethanol vs. water) resumed for 3 weeks (11th to 13th). During the 13th week, all animals were subjected to restraint stress for 2 consecutive days. Results: Over the entire time course of the experiment, ethanol intake and preference increased in females (both adults and adolescents). Adolescent animals (both females and males) showed a transient increase in alcohol consumption and preference compared to adults. However, by the end of continuous alcohol exposure (when all mice were adults), ethanol intake was not affected by age at drinking onset. A deprivation phase was followed by a rise in ethanol intake (ADE) that was not affected by sex or age. Finally, stress did not alter alcohol self‐administration either during or after its occurrence. Conclusions: Emergence of greater alcohol consumption in adult females does not seem to be limited to a specific developmental period (i.e., puberty). Age of voluntary drinking onset (adolescence vs. adulthood) does not affect eventual alcohol intake in adult WSC‐1 mice and does not modify the transient increase in ethanol consumption after alcohol deprivation.     

Effects of Pregnanolone and Dehydroepiandrosterone on Ethanol Intake in Rats Administered Ethanol or Saline during Adolescence

Background: 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. Methods: 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. Results: 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. Conclusions: 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.     

Corticotropin-releasing factor acting on corticotropin-releasing factor receptor type 1 is critical for binge alcohol drinking in mice

Background: The corticotropin‐releasing factor (CRF) system has been implicated in the regulation of alcohol consumption. However, previous mouse knockout (KO) studies using continuous ethanol access have failed to conclusively confirm this. Recent studies have shown that CRF receptor type 1 (CRFR1) antagonists attenuate alcohol intake in the limited access “drinking in the dark” (DID) model of binge drinking. To avoid the potential nonspecific effects of antagonists, in this study, we tested alcohol drinking in CRFR1, CRFR2, CRF, and urocortin 1 (Ucn1) KO and corresponding wild‐type (WT) littermates using the DID paradigm. Methods: On days 1 to 3, the CRFR1, CRFR2, Ucn1, and CRF KO mice and their respective WT littermates were provided with 20% ethanol or 10% sucrose for 2 hours with water available at all other times. On day 4, access to ethanol or sucrose was increased to 4 hours. At the end of each drinking session, the volume of ethanol consumed was recorded, and at the conclusion of the last session, blood was also collected for blood ethanol concentration (BEC) analysis. Results: CRFR1 KO mice had lower alcohol intakes and BECs and higher intakes of sucrose compared with WTs. In contrast, CRFR2 KO mice, while having reduced intakes initially, had similar alcohol intakes on days 2 to 4 and similar BECs as the WTs. To determine the ligand responsible, Ucn1 and CRF KO and WT mice were tested next. While Ucn1 KOs had similar alcohol intakes and BECs to their WTs, CRF KO mice showed reduced alcohol consumption and lower BECs compared with WTs. Conclusions: Our results confirm that CRFR1 plays a key role in binge drinking and identify CRF as the ligand critically involved in excessive alcohol consumption.     

Discriminative stimulus effects of ethanol in C57BL/6J and DBA/2J inbred mice

BACKGROUND: Two of the most widely used mouse strains for studying the behavioral effects of ethanol are C57BL/6J (B6) and DBA/2J (D2) mice. These strains exhibit marked differences in behavioral and physiological responses to ethanol. The subjective discriminative stimulus effects of ethanol may play a role in ethanol abuse, but the discriminative stimulus profile of ethanol has not been compared in B6 and D2 mice. Examination of the discriminative stimulus effects of ethanol in B6 and D2 mouse strains may enhance our understanding of the relationship between the subjective effects of ethanol and other ethanol-induced behavioral effects. METHODS: Twelve adult male C57BL/6J mice and 12 male DBA/2J mice were trained to discriminate 1.5 g/kg ethanol from saline in daily 15 min, milk-reinforced operant sessions. After training, ethanol substitution and response-rate suppression dose response curves were determined for ethanol, midazolam, diazepam, pentobarbital, pregnanolone, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), dizocilpine, and morphine. RESULTS: D2 mice learned the ethanol discrimination significantly more quickly than did B6 mice. Ethanol, midazolam, pregnanolone, and dizocilpine fully substituted for ethanol in both strains. Pentobarbital was more potent in producing ethanol-like discriminative stimulus effects in D2 than B6 mice. Midazolam and diazepam were significantly more potent in suppressing response rates in D2 than B6 mice. Morphine failed to substitute for ethanol in either strain, but the ED50 for morphine suppression of responding was significantly lower in B6 than D2 mice. CONCLUSIONS: The initial stimulus effects of 1.5 g/kg ethanol may be more salient in D2 than B6 mice. This does not appear to result from differences in the neurotransmitter systems that mediate ethanol's discriminative stimulus effects. In both strains, gamma-aminobutyric acid-positive modulators and a noncompetitive NMDA antagonist substituted for ethanol. However, strain differences did exist in the potency of gamma-aminobutyric acid-positive modulators and morphine for suppressing operant responding.     

Ethanol‐Induced Increase of Agouti‐Related Protein (AgRP) Immunoreactivity in the Arcuate Nucleus of the Hypothalamus of C57BL/6J,but not 129/SvJ,Inbred Mice

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor, pro‐opiomelanocortin (POMC). Previous research has shown that MC receptor (MCR) agonists reduce, and MCR antagonists increase, ethanol consumption in rats and mice. Consistently, genetic deletion of the endogenous MCR antagonist, agouti‐related protein (AgRP), causes reductions of ethanol‐reinforced lever pressing and binge‐like ethanol drinking in C57BL/6J mice. Ethanol also has direct effects on the central MC system, as chronic exposure to an ethanol‐containing diet causes significant reductions of α‐melanocyte stimulating hormone (α‐MSH) immunoreactivity in specific brain regions of Sprague‐Dawley rats. Together, these observations suggest that the central MC system modulates neurobiological responses to ethanol. To further characterize the role of the MC system in responses to ethanol, here we compared AgRP and α‐MSH immunoreactivity in response to an acute injection of saline or ethanol between high ethanol drinking C57BL/6J mice and moderate ethanol drinking 129/SvJ mice. Methods: Mice received an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg or 3.5 g/kg; mixed in 0.9% saline) or an equivolume of 0.9% saline. Two hours after injection, animals were sacrificed and their brains were processed for AgRP and α‐MSH immunoreactivity. Results: Results indicated that acute ethanol administration triggered a dose‐dependent increase in AgRP immunoreactivity in the arcuate (ARC) of C57BL/6J mice, an effect that was not evident in the 129/SvJ strain. Although acute administration of ethanol did not influence α‐MSH immunoreactivity, C57BL/6J mice had significantly greater overall α‐MSH immunoreactivity in the ARC, dorsomedial, and lateral regions of the hypothalamus relative to the 129/SvJ strain. In contrast, C57BL/6J mice displayed significantly lower α‐MSH immunoreactivity in the medial amygdala. Conclusions: The results show that acute ethanol exposure has direct effects on endogenous AgRP activity in ethanol preferring C57BL/6J mice. It is suggested that ethanol‐induced increases in AgRP may be part of a positive feedback system that stimulates excessive binge‐like ethanol drinking in C57BL/6J mice. Inherent differences in α‐MSH immunoreactivity may contribute to differences in neurobiological responses to ethanol that are characteristically observed between the C57BL/6J and 129/SvJ inbred strains of mice.     

Ethanol-induced behavioral sensitization in adolescent and adult mice: role of the nNOS gene

Background: In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) has a role in synaptic plasticity. Recent evidence suggests the role of NO in a variety of effects produced by alcohol in the central nervous system. The current study investigated the role of the nNOS gene in the development of behavioral sensitization to ethanol in adolescent and adult mice. Methods: Adolescent and adult wild type (WT; B6;129SF2) and nNOS knockout (KO; B6;129S4‐Nos1) mice of both sexes received saline or ethanol (1.5 g/kg; intraperitoneally) for 5 consecutive days, and locomotor activity was recorded daily. The locomotor response to challenge ethanol and saline injections was investigated at various time points following withdrawal from ethanol. Results: Adolescent WT but not nNOS KO mice developed a long‐lasting sensitized response to ethanol as well as context‐dependent hyperlocomotion (in response to saline) from adolescence through adulthood; sex‐dependent differences were not observed. Compared to adolescent WT mice, adult WT males developed a short‐term sensitized response to ethanol and context‐dependent hyperlocomotion; adult WT females showed only short‐term context‐dependent hyperlocomotion. Adult nNOS KO males (like their adolescent counterparts) did not develop behavioral sensitization; no significant differences between adult nNOS KO and WT females were observed. Blood ethanol concentrations did not show genotype‐ or sex‐dependent differences. Conclusions: (1) The nNOS gene is required for the development of behavioral sensitization to ethanol in adolescent male and female mice. (2) Adolescent exposure to ethanol results in long‐lasting behavioral sensitization through adulthood, while adult exposure to ethanol results in a shorter behavioral sensitization. (3) Sex‐dependent differences are observed when ethanol exposure begins in adulthood but not in adolescence. (4) Ethanol‐induced behavioral sensitization in adulthood is nNOS‐dependent in males but not in females. Taken together, results suggest genotype‐, ontogeny‐, and sex‐dependent differences in the development of behavioral sensitization to ethanol.     

Factors influencing elevated ethanol consumption in adolescent relative to adult rats

BACKGROUND: Many teenagers report frequent alcohol use during the adolescent period. Animal models may be an important tool for exploring factors that contribute to alcohol consumption during this critical period. METHODS: Using a 24-hour, free-access, two-bottle-choice procedure between water and a sweetened solution with or without ethanol in nondeprived rats, the present series of experiments examined the contribution of a variety of contextual and experimental variables (i.e., isolate-housing versus pair-housing, type of sipper tube, caloric value of solution, prior experimental perturbations) on alcohol consumption in both adolescent and adult Sprague-Dawley rats. RESULTS: Ethanol consumption was particularly magnified among adolescent rats using ball bearing-containing ball-point (BP) sipper tubes, with this exacerbated intake not due to caloric content of the ethanol solution. Isolation housing for 12 days did not alter ethanol consumption of adolescents relative to their socially housed counterparts while suppressing consumption of isolated adults. An examination of differences in the relative magnitude of adolescent ethanol consumption across experiments in this series revealed that ethanol intake among adolescents was elevated not only by the inclusion of BP sipper tubes but also by staggering the timing of isolate housing relative to the presentation of the novel ethanol solution. CONCLUSIONS: The results of this experimental series demonstrate that adolescent animals consume significantly more ethanol than adult animals under a variety of home cage continuous-access circumstances, with the relatively greater intake of adolescents further magnified by a number of test conditions. Subtle experimental details often thought to be innocuous can have a substantial impact on overall amount of voluntary ethanol consumption observed in both adolescent and adult animals.     

Enhanced prepulse inhibition following adolescent ethanol exposure in Sprague-Dawley rats

OBJECTIVES: Recent studies have demonstrated that ethanol exposure differentially affects adolescents and adults. The current studies were designed to compare the effects of 2-week exposure to ethanol during adolescence or adulthood on the acoustic startle response (ASR) and prepulse inhibition (PPI) METHODS: Male Sprague-Dawley rats were exposed to ethanol vapor 12 hr/d (on from 6 pm to 6 am) for 14 days during adolescence or adulthood. Six days after the cessation of ethanol vapor exposure, the ASR and PPI were assessed. RESULTS: During ethanol treatment, overall blood alcohol levels averaged 230 to 250 mg/dl in the adolescent and adult treatment groups. Assessment of the ASR revealed that latency to startle was more rapid in adolescents than in adults, but ASR latency was not altered by ethanol exposure. In addition, ASR magnitude was lower in adolescents and was decreased in ethanol-exposed rats on startle trials. Ethanol exposure significantly enhanced PPI, but only after adolescent exposure CONCLUSIONS: These data further demonstrate a differential sensitivity of adolescents and adults to the effects of ethanol exposure. Specifically, a 2-week period of ethanol exposure during adolescence selectively enhanced PPI, a neurobehavioral index of sensorimotor gating. However, ASR magnitude was decreased by ethanol exposure regardless of age. On the basis of previous studies, the effects of ethanol exposure on PPI data could indicate that adolescent rats exposed to ethanol are more likely to exhibit behavioral inflexibility and that ethanol exposure acts as a more potent physical stressor in adolescent rats.