Alteration of ethanol drinking in mice via modulation of the GABA(A) receptor with ganaxolone, finasteride, and gaboxadol

Background: Neurosteroids and other γ‐aminobutyric acid_A (GABA_A) receptor–modulating compounds have been shown to affect ethanol intake, although their mechanism remains unclear. This study examined how patterns of 24‐hour ethanol drinking in mice were altered with the synthetic GABAergic neurosteroid ganaxolone (GAN), with an inhibitor of neurosteroid synthesis (finasteride [FIN]), or a GABA_A receptor agonist with some selectivity at extrasynaptic receptors (gaboxadol HCL [THIP]). Methods: Male C57BL/6J mice had continuous access to a 10% v/v ethanol solution (10E) or water. Using lickometer chambers, drinking patterns were analyzed among mice treated in succession to GAN (0, 5, and 10 mg/kg), FIN (0 or 100 mg/kg), and THIP (0, 2, 4, 8, and 16 mg/kg). Results: GAN shifted drinking in a similar but extended manner to previous reports using low doses of the neurosteroid allopregnanolone (ALLO); drinking was increased in hour 1, decreased in hours 2 and 3, and increased in hours 4 and 5 postinjection. THIP (8 mg/kg) and FIN both decreased 10E drinking during the first 5 hours postinjection by 30 and 53%, respectively, while having no effect on or increasing water drinking, respectively. All 3 drugs altered the initiation of drinking sessions in a dose‐dependent fashion. FIN increased and GAN decreased time to first lick and first bout. THIP (8 mg/kg) decreased time to first lick but increased time to first bout and attenuated first bout size. Conclusions: The present findings support a role for the modulation of ethanol intake by neurosteroids and GABA_A receptor–acting compounds and provide hints as to how drinking patterns are shifted. The ability of THIP to alter 10E drinking suggests that extrasynaptic GABA_A receptors may be involved in the modulation of ethanol intake. Further, the consistent results with THIP to that seen previously with high doses of ALLO suggest that future studies should further examine the relationship between neurosteroids and extrasynaptic GABA_A receptors, which could provide a better understanding of the mechanism by which neurosteroids influence ethanol intake.     

Drinking typography established by scheduled induction predicts chronic heavy drinking in a monkey model of ethanol self-administration

Background: We have developed an animal model of alcohol self‐administration that initially employs schedule‐induced polydipsia (SIP) to establish reliable ethanol consumption under open access (22 h/d) conditions with food and water concurrently available. SIP is an adjunctive behavior that is generated by constraining access to an important commodity (e.g., flavored food). The induction schedule and ethanol polydipsia generated under these conditions affords the opportunity to investigate the development of drinking typologies that lead to chronic, excessive alcohol consumption. Methods: Adult male cynomolgus monkeys (Macaca fascicularis) were induced to drink water and 4% (w/v in water) ethanol by a Fixed‐Time 300 seconds (FT‐300 seconds) schedule of banana‐flavored pellet delivery. The FT‐300 seconds schedule was in effect for 120 consecutive sessions, with daily induction doses increasing from 0.0 to 0.5 g/kg to 1.0 g/kg to 1.5 g/kg every 30 days. Following induction, the monkeys were allowed concurrent access to 4% (w/v) ethanol and water for 22 h/day for 12 months. Results: Drinking typographies during the induction of drinking 1.5 g/kg ethanol emerged that were highly predictive of the daily ethanol intake over the next 12 months. Specifically, the frequency in which monkeys ingested 1.5 g/kg ethanol without a 5‐minute lapse in drinking (defined as a bout of drinking) during induction strongly predicted (correlation 0.91) subsequent ethanol intake over the next 12 months of open access to ethanol. Blood ethanol during induction were highly correlated with intake and with drinking typography and ranged from 100 to 160 mg% when the monkeys drank their 1.5 g/kg dose in a single bout. Forty percent of the population became heavy drinkers (mean daily intakes >3.0 g/kg for 12 months) characterized by frequent “spree” drinking (intakes >4.0 g/kg/d). Conclusion: This model of ethanol self‐administration identifies early alcohol drinking typographies (gulping the equivalent of 6 drinks) that evolve into chronic heavy alcohol consumption in primates (drinking the equivalent of 16 to 20 drinks per day). The model may aid in identifying biological risks for establishing harmful alcohol drinking.     

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.     

Alcohol Self-Administration in a Nonrestricted Access Situation with Alcohol-Preferring (P) Rats

Genetic variables have been implicated as contributing factors in the development of alcoholic behavior. Rats bred selectively for alcohol preference have been used in laboratory studies to investigate the role of such variables. In the present study, rats from the alcohol preferring (P) line were placed in operant chambers in which food pellets, water, and 10% ethanol (v/v) were available continuously for 23 hr/day. Food pellets (45 mg) were presented on an FR 1 schedule of reinforcement, while ethanol was presented in a 0.1 ml dipper on an FR 4 schedule of reinforcement. Water was available in a drinking tube with licks monitored by a drinkometer. Data were analyzed in terms of both total daily intakes and computer defined bouts. The P rats showed greater daily ethanol intakes compared with Long-Evans (LE) animals previously studied under similar access conditions. The major difference in intake was a result of the P rats having a greater number of daily ethanol drinking bouts, while having only a slight increase in individual bout size. These data indicate that genetic selection for ethanol preference may result in the regulation of ethanol intake by means of changes in the frequency of ethanol drinking bouts but not by changes in bout size.     

Selected Line Difference in the Effects of Ethanol Dependence and Withdrawal on Allopregnanolone Levels and 5α-Reductase Enzyme Activity and Expression

Background: Allopregnanolone (ALLO) is a progesterone derivative that rapidly potentiates γ‐aminobutyric acid_A (GABA_A) receptor‐mediated inhibition and modulates symptoms of ethanol withdrawal. Because clinical and preclinical data indicate that ALLO levels are inversely related to symptoms of withdrawal, the present studies determined whether ethanol dependence and withdrawal differentially altered plasma and cortical ALLO levels in mice selectively bred for differences in ethanol withdrawal severity and determined whether the alterations in ALLO levels corresponded to a concomitant change in activity and expression of the biosynthetic enzyme 5α‐reductase. Methods: Male Withdrawal Seizure‐Prone (WSP) and ‐Resistant (WSR) mice were exposed to 72 hours ethanol vapor or air and euthanized at select times following removal from the inhalation chambers. Blood was collected for analysis of ALLO and corticosterone levels by radioimmunoassay. Dissected amygdala, hippocampus, midbrain, and cortex as well as adrenals were examined for 5α‐reductase enzyme activity and expression levels. Results: Plasma ALLO was decreased significantly only in WSP mice, and this corresponded to a decrease in adrenal 5α‐reductase expression. Cortical ALLO was decreased up to 54% in WSP mice and up to 46% in WSR mice, with a similar decrease in cortical 5α‐reductase activity during withdrawal in the lines. While cortical gene expression was significantly decreased during withdrawal in WSP mice, there was a 4‐fold increase in expression in the WSR line during withdrawal. Hippocampal 5α‐reductase activity and gene expression was decreased only in dependent WSP mice. Conclusions: These results suggest that there are line and brain regional differences in the regulation of the neurosteroid biosynthetic enzyme 5α‐reductase during ethanol dependence and withdrawal. In conjunction with the finding that WSP mice exhibit reduced sensitivity to ALLO during withdrawal, the present results are consistent with the hypothesis that genetic differences in ethanol withdrawal severity are due, in part, to modulatory effects of GABAergic neurosteroids such as ALLO.     

Opposite Effects of Ro 15-4513 on Acquisition and Maintenance of Ethanol Drinking Behavior in Male Wistar Rats

Acute treatment with Ro 15-4513, a benzodiazepine receptor inverse agonist, has been consistently shown to suppress ethanol self administration behavior by rats. The aim of the present study was to examine the effect of Ro 15-4513 on the acquisition and maintenance of ethanol drinking behavior in male Wistar rats. In rats maintained on a limited access procedure with a choice of 12% w/v ethanol solution and water, acute treatment with Ro 15-4513 (0.1 to 3.0 mg/kg) suppressed ethanol intake in a selective and dose-related manner ( p < 0.01). However, by the fourth day of chronic Ro 15-4513 treatment, ethanol intake had returned to baseline levels. In contrast, chronic administration of Ro 15-4513 during the acquisition phase increased ethanol drinking behavior, compared with vehicle ( p < 0.05). To assess whether the effects of Ro 15-4513 on ethanol intake were due to an alteration in ethanol kinetics or on behavior, blood ethanol levels and rat social interaction behavior were also assessed. Neither acute nor chronic Ro 15-4513 treatment significantly altered ethanol kinetics after oral administration of ethanol (1.0 g/kg), but did suppress locomotor activity and time spent engaged in active social interaction at the 1.0 and 3.0 mg/kg doses. These results show that Ro 15-4513 has opposite effects on ethanol consumption during the acquisition and maintenance phases of ethanol drinking behavior, suggesting that different mechanisms regulate Ro 15-4513's effects on acquisition and maintenance of ethanol intake. Ro 15-4513's reported anxiogenic or memory enhancing properties may be responsible for its effects on acquisition.     

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.     

Assessment of voluntary ethanol consumption and the effects of a melanocortin (MC) receptor agonist on ethanol intake in mutant C57BL/6J mice lacking the MC-4 receptor

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent evidence shows that chronic exposure to ethanol significantly blunts central MC peptide immunoreactivity and MC receptor (MCR) agonists protect against high ethanol intake characteristic of C57BL/6J mice. Here, we assessed the role of the MC‐4 receptor (MC4R) in voluntary ethanol intake and in modulating the effects of the nonselective MCR agonist melanotan‐II (MTII) on ethanol consumption. Methods: To assess the role of the MC4R, MC4R knockout (Mc4r^?/?) and littermate wild‐type (Mc4r^+/+) mice on a C57BL/6J background were used. Voluntary ethanol (3, 5, 8, 10, 15, and 20%, v/v) and water intake were assessed using standard two‐bottle procedures. In separate experiments, Mc4r^?/? and Mc4r^+/+ mice were given intracerebroventricular (i.c.v.) infusion of MTII (0, 0.5, or 1.0 μg/1 μl) or intraperitoneal (i.p.) injection of MTII (0 or 5 mg/kg/5 ml). The effects of MTII (0 or 0.5 μg/1 μl, i.c.v.) on 10% sucrose and 0.15% saccharin intake were assessed in C57BL/6J mice. Results: Mc4r^?/? mice showed normal consumption of ethanol over all concentrations tested. I.c.v. infusion of MTII significantly reduced ethanol drinking in Mc4r^+/+ mice, but failed to influence ethanol intake in Mc4r^?/? mice. When administered in an i.p. injection, MTII significantly reduced ethanol drinking in both Mc4r^?/? and Mc4r^+/+ mice. MTII attenuated consumption of caloric (ethanol, sucrose, and food) and noncaloric (saccharin) reinforcers. Conclusions: When given centrally, the MCR agonist MTII reduced ethanol drinking by signaling through the MC4R. On the other hand, MTII‐induced reduction of ethanol drinking did not require the MC4R when administered peripherally. Together, the present observations show that the MC4R is necessary for the central actions of MCR agonists on ethanol drinking and that MTII blunts the consumption natural reinforcers, regardless of caloric content, in addition to ethanol.     

Development of ethanol withdrawal-related sensitization and relapse drinking in mice selected for high- or low-ethanol preference

Background: Previous studies have shown that high alcohol consumption is associated with low withdrawal susceptibility, while at the same time, other studies have shown that exposure to ethanol vapor increases alcohol drinking in rats and mice. In the present studies, we sought to shed light on this seeming contradiction using mice selectively bred for High‐ (HAP) and Low‐ (LAP) Alcohol Preference, first, assessing these lines for differences in signs of ethanol withdrawal and second, for differences in the efficacy of intermittent alcohol vapor exposure on elevating subsequent ethanol intake. Methods: Experiment 1 examined whether these lines of mice differed in ethanol withdrawal‐induced CNS hyperexcitability and the development of sensitization to this effect following intermittent ethanol vapor exposure. Adult HAP and LAP lines (replicates 1 and 2), and the C3H/HeNcr inbred strain (included as a control genotype for comparison purposes) received intermittent exposure to ethanol vapor and were evaluated for ethanol withdrawal‐induced seizures assessed by scoring handling‐induced convulsions (HIC). Experiment 2 examined the influence of chronic intermittent ethanol exposure on voluntary ethanol drinking. Adult male and female HAP‐2 and LAP‐2 mice, along with male C57BL/6J (included as comparative controls) were trained to drink 10% ethanol using a limited access (2 h/d) 2‐bottle choice paradigm. After stable baseline daily intake was established, mice received chronic intermittent ethanol vapor exposure in inhalation chambers. Ethanol intake sessions resumed 72 hours after final ethanol (or air) exposure for 5 consecutive days. Results: Following chronic ethanol treatment, LAP mice exhibited overall greater withdrawal seizure activity compared with HAP mice. In Experiment 2, chronic ethanol exposure/withdrawal resulted in a significant increase in ethanol intake in male C57BL/6J, and modestly elevated intake in HAP‐2 male mice. Ethanol intake for male control mice did not change from baseline levels of intake. In contrast, HAP‐2 female and LAP‐2 mice of both sexes did not show changes in ethanol intake as a consequence of intermittent ethanol exposure. Conclusions: Overall, these results indicate that the magnitude of ethanol withdrawal‐related seizures is inversely related to inherited ethanol intake preference. Additionally, intermittent ethanol vapor exposure appears more likely to affect high‐drinking mice (C57BL/6J and HAP‐2) than low drinkers, although these animals are less affected by ethanol withdrawal.     

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.     

Naltrexone Blocks Acquisition of Voluntary Ethanol Intake in Rats

The effects of naltrexone (NTX) on the acquisition of ethanol drinking was assessed in rats. NTX (0, 2.5, 5.0, or 10.0 mg/kg) was administered to rats presented with an ascending series of ethanol concentrations (2%, 4%, 6%, and 8% v/v) and water. The 2.5 and 10 mg/kg doses of NTX attenuated the acquisition of voluntary drinking of 8% ethanol, but the 5.0 mg/kg dose of NTX had no effect on ethanol intake. The acquisition paradigm was repeated in experiment 2 with naïve animals that received 0, 5.0, or 7.5 mg/kg of NTX. Neither dose of NTX affected ethanol intake, preference for alcohol, or water intake. Total fluid intake was suppressed in the NTX groups, but only on the second presentations of the 2% and 6% concentrations of ethanol. We suggest that the 2.5 and 10 mg/kg doses of NTX may have attenuated the acquisition of ethanol drinking by at least two different behavioral mechanisms.     

Blockade of the corticotropin releasing factor type 1 receptor attenuates elevated ethanol drinking associated with drinking in the dark procedures

Background: Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations (BECs) reaching levels that have measurable affects on physiology and/or behavior. The present experiments determined whether the increased ethanol drinking caused by DID procedures can be attenuated by pretreatment with CP‐154,526; a corticotropin releasing factor type‐1 (CRF₁) receptor antagonist. Methods: In Experiment 1, male C57BL/6J mice received ethanol (20% v/v) in place of water for 4 hours, beginning with 3 hours into the dark cycle. On the fourth day, mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle. In Experiment 2, C57BL/6J mice had 2 hours of access to the 20% ethanol solution, beginning with 3 hours into the dark cycle on days 1 to 3, and 4 hours of access to the ethanol bottle on day 4 of DID procedures. Mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle on day 4. Tail blood samples were collected immediately after the 4‐hour ethanol access period on the fourth day of each experiment. Additional control experiments assessed the effects of CP‐154,526 on 4‐hour consumption of a 10% (w/v) sucrose solution and open‐field locomotor activity. Results: In Experiment 1, the vehicle‐treated group consumed approximately 4.0 g/kg/4 h of ethanol and achieved BECs of approximately 30 mg%. Furthermore, pretreatment with the CRF₁ receptor antagonist did not alter ethanol consumption. On the other hand, procedures used in Experiment 2 resulted in vehicle‐treated mice consuming approximately 6.0 g/kg/4 h of ethanol with BECs of about 80 mg%. Additionally, the 10 mg/kg dose of CP‐154,526 significantly reduced ethanol consumption and BECs to approximately 3.0 g/kg/4 h and 27 mg%, respectively, relative to vehicle‐treated mice. Importantly, the 10 mg/kg dose of the CRF₁R antagonist did not significantly alter 4‐hour sucrose consumption or locomotor activity. Conclusions: These data indicate that CRF₁R signaling modulates high, but not moderate, levels of ethanol drinking associated with DID procedures.     

Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats

Background: There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent‐access to 20% ethanol in a 2‐bottle‐choice setting [ Wise, Psychopharmacologia 29 (1973), 203 ]. In this study, we have further characterized this drinking model. Methods: Ethanol‐naïve Long–Evans rats were given intermittent‐access to 20% ethanol (three 24‐hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long‐term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent‐access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous‐access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out‐bred alcohol preferring (P) rats following intermittent‐access to 20% ethanol. Results: The intermittent‐access 20% ethanol 2‐bottle‐choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long–Evans: 5.1 ± 0.6; Wistar: 5.8 ± 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long–Evans rats. P‐rats initiate drinking at a higher level than both Long–Evans and Wistar rats using the intermittent‐access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 ± 0.8 g/kg/24 h). Conclusion: Standard laboratory rats will voluntarily consume ethanol using the intermittent‐access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.     

Effects of long-term episodic access to ethanol on the expression of an alcohol deprivation effect in low alcohol-consuming rats

Background: The alcohol‐preferring (P) and ‐nonpreferring (NP) and high alcohol–drinking (HAD) and low alcohol–drinking (LAD) rats have been selectively bred for divergent preference for ethanol over water. In addition, both P and HAD rats display an alcohol deprivation effect (ADE). This study was undertaken to test whether the NP, LAD‐1, and LAD‐2 lines of rats could display an ADE as well. Method: Adult female NP, LAD‐1, and LAD‐2 rats were given concurrent access to multiple concentrations of ethanol [5, 10, 15% (v/v)] and water in an ADE paradigm involving an initial 6 weeks of 24‐hr access to ethanol, followed by four cycles of 2 weeks of deprivation from and 2 weeks of re‐exposure to ethanol (5, 10, and 15%). A control group had continuous access to the ethanol concentrations (5, 10, and 15%) and water through the end of the fourth re‐exposure period. Results: For NP rats, a preference for the highest ethanol concentration (15%) was evident by the end of the fifth week of access (～60% of total ethanol fluid intake). Contrarily, LAD rats did not display a marked preference for any one concentration of ethanol. All three lines displayed an ADE after repeated cycles of re‐exposure to ethanol, with the general ranking of intake being LAD‐1 > NP > LAD‐2 (e.g., for the first day of reinstatement of the third re‐exposure cycle, intakes were 6.5, 2.9, and 2.4 g/kg/day compared with baseline values of 3.1, 2.0, and 1.3 g/kg/day for each line, respectively). By the 13th week, rats from all three lines, with a ranking of LAD‐1 > NP > LAD‐2, were drinking more ethanol (3.3, 2.2, and 2.0 g/kg/day, respectively) compared with their consumption during the first week of access (～1.1 g/kg/day for all three lines). Conclusion: These data indicate that access to multiple concentrations of ethanol and exposure to multiple deprivation cycles can partially overcome a genetic predisposition of NP, LAD‐1, and LAD‐2 rats for low alcohol consumption. In addition, the findings suggest that genetic control of low alcohol consumption in rats is not associated with the inability to display an ADE.     

Effects of food availability and administration of orexigenic and anorectic agents on elevated ethanol drinking associated with drinking in the dark procedures

Background: Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations reaching levels that have measurable affects on physiology and/or behavior. The present study determined if increased ethanol drinking associated with DID procedures may be motivated by caloric need rather than by the postingestive pharmacological effects of ethanol. To this end, food availability was manipulated or mice were given peripheral administration of orexigenic or anorectic agents during DID procedures. Methods: C57BL/6J had 2‐hours of access to the 20% (v/v) ethanol solution beginning 3‐hours into the dark cycle on days 1 to 3, and 4‐hours of access to the ethanol bottle on day 4 of DID procedures. In Experiment 1, the effects of food deprivation on ethanol consumption during DID procedures was assessed. In Experiments 2 and 3, mice were given intraperitoneal (i.p.) injection of the orexigenic peptide ghrelin (0, 10 or 30 mg/kg) or the anorectic protein leptin (0 or 20 μg/g), respectively, before access to ethanol on day 4 of DID procedures. In Experiment 4, hourly consumption of food and a 0.05% saccharin solution were assessed over a period of hours that included those used with DID procedures. Results: Consistent with previous research, mice achieved blood ethanol concentrations (BECs) that ranged between 100 and 150 mg% on day 4 of DID experiments. Neither food deprivation nor administration of orexigenic or anorectic compounds significantly altered ethanol drinking with DID procedures. Interestingly, mice exhibited their highest level of food and saccharin solution consumption during hours that overlapped with DID procedures. Conclusions: The present observations are inconsistent with the hypothesis that C57BL/6J mice consume large amounts of ethanol during DID procedures in order to satisfy a caloric need.     

Behavioral traits predicting alcohol drinking in outbred rats: an investigation of anxiety, novelty seeking, and cognitive flexibility

Background: Most adults in Western society consume alcohol regularly without negative consequences. For a small subpopulation, however, drinking can quickly progress to excessive and chronic intake. Given the dangers associated with alcohol abuse, it is critical to identify traits that may place an individual at risk for developing these behaviors. To that end, we used a rat model to determine whether anxiety‐related behaviors, novelty seeking, or cognitive flexibility predict excessive alcohol drinking under both limited and continuous access conditions. Methods: Adult male rats were assessed in a series of behavioral tasks (elevated plus maze [EPM], locomotor activity, and discrimination/reversal learning in a Y‐maze) followed by 6 weeks of daily, 1‐hour access to alcohol in a free‐choice, 2‐bottle paradigm (10% alcohol vs. tap water). Next, subjects were given the opportunity to consume alcohol for 72 hours in drinking chambers that permit separate measures of each drinking bout. Half of the animals experienced a 2‐week deprivation period between the limited and continuous access sessions. Results: Time spent on the open arms of the EPM, but not novelty seeking or discrimination/reversal learning, predicted alcohol consumption during limited, 1‐h/d access sessions to alcohol. Anxiety‐related behavior also predicted the escalation of intake when animals were given 72 hours of continuous access to alcohol. Bout size, but not frequency, was responsible for the increased consumption by high‐anxiety subjects during this period. Finally, intake during limited access sessions predicted intake during continuous access, but only in subjects with low intake during limited access. Conclusions: These findings confirm that preexisting anxiety‐related behavior predicts alcohol intake under several schedules of alcohol access. Moreover, when access is unlimited, the high‐anxiety‐related group exhibited an increase in bout size, but not frequency, of drinking. In addition, we show that modest intake when alcohol is restricted may or may not progress to excessive intake when the drug is freely available.     

Enhanced morphine-induced ethanol drinking in alcohol-preferring alko alcohol rats sensitized to morphine

BACKGROUND: Alcohol-preferring alko alcohol (AA) rats are more susceptible to morphine-induced behavioral and neurochemical sensitization than alcohol nonpreferring alko nonalcohol (ANA) rats. Alko alcohol rats sensitized to morphine, however, do not show enhanced acquisition of ethanol drinking. The purpose of the present study was to clarify further interactions between morphine-induced behavioral sensitization and voluntary ethanol drinking in the AA rats. METHODS: Alko alcohol rats drinking ethanol in a limited 6-hour access paradigm were sensitized to morphine with repeated injections of morphine (5-15 mg/kg). Injection days alternated with days of ethanol access. Controls had access only to water and/or were given injections of saline. After a 5-day washout period from ethanol and morphine, the rats were challenged with morphine or saline and subsequent ethanol drinking or locomotor activity was recorded. RESULTS: Ethanol intake was suppressed during the repeated treatment with morphine, and the morphine-treated rats did not differ in ethanol intake from the controls when given access to ethanol after the washout. Intake of ethanol was, however, increased when the rats were challenged with morphine [1 or 10 mg/kg, subcutaneously (s.c.)], while in the controls an increase in ethanol intake was seen only after 1 mg/kg morphine. Sensitization to the locomotor stimulating effects of morphine was revealed in the morphine-treated rats after a challenge with morphine (3 or 10 mg/kg, s.c.). The controls that had been drinking ethanol also showed a sensitized response after morphine (3 mg/kg). CONCLUSIONS: Ethanol did not interfere with the development of sensitization to morphine. Furthermore, the neuroadaptations induced by repeated exposure to ethanol were sufficient to cause behavioral cross-sensitization to morphine. Sensitization to the behavioral effects of morphine alone, however, neither enhances the reinforcing properties of voluntarily consumed ethanol nor contributes to increase in its intake. The increase in ethanol intake found after an acute dose of morphine was augmented in rats withdrawn from repeated treatment with morphine. The data suggest that the neuronal mechanisms underlying behavioral sensitization to morphine probably are distinct from those mediating reinforcement from ethanol and that the morphine-induced neuroadaptations contribute to the enhancement of increase in ethanol intake by morphine.     

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 acamprosate on ethanol-seeking and self-administration in the rat

BACKGROUND: Acamprosate (calcium acetyl homotaurinate) has been used clinically to treat relapse in alcoholics. In rats, it has been shown to decrease ethanol, but not water, self-administration after ethanol deprivation. METHODS: To further investigate the effect of acamprosate on reinforced behaviors in rats, the present experiment used: (1) both ethanol and sucrose reinforcer solutions to better assess the distinct effects of acamprosate on ethanol-directed behaviors, and (2) an operant model that procedurally separates the "cost" to begin drinking from consuming the reinforcer solutions to dissociate the effects of acamprosate on appetitive versus consummatory processes. In daily sessions (5 days/week), rats (n = 6/group) were trained to make 30 lever-press responses to gain access for 20 min to a sipper tube containing either ethanol (10%) or sucrose (3%). After stable responding, acamprosate treatment was given. Three doses were tested (50, 100, and 200 mg/kg/injection, intraperitoneally), one dose per week. Each week, a total of four injections were given (21 and 2 hr before the operant sessions over 2 consecutive days). RESULTS: At these doses, acamprosate had no effect on the measures of appetitive responding for either solution. However, all doses reliably decreased ethanol consumption on the 2nd day of treatment (from an average of 0.83 to 0.63 g/kg). Analysis of the pattern of ethanol consumption showed that the effects of acamprosate occurred after the onset of a normal pattern of intake, as measured by lick rate and size of the initial bout of drinking, which suggested that acamprosate is most effective when combined with the pharmacological effects of ethanol. Sucrose intake was unaffected by all acamprosate treatments, which indicated that the treatment effects were specific to ethanol and not due to a general decrease in consummatory behavior. CONCLUSIONS: Overall, these results suggest that acamprosate is effective at reducing total ethanol intake, but may not reliably alter subjects propensity to begin a drinking bout as measured by this model. However, whether this applies to the clinical use of acamprosate, where other types of reinforcement may also precipitate relapse drinking, is not certain.     

Determination of an estradiol dose-response relationship in the modulation of ethanol intake

BACKGROUND: Estradiol (E2) potentiates the self-administration of numerous psychoactive drugs in female rodents. An analogous modulatory role of E2 on ethanol consumption remains unresolved because of examination of limited doses. The purpose of this study was to delineate a dose-response relationship for E2 on ethanol intake with an extended range and number of E2 doses. METHODS: Female Long-Evans rats had continuous access (22 hr/day) to both 10% ethanol (10E) solution and water. After the establishment of stable 10E intake baselines, rats were assigned to one of seven dose groups balanced for 10E intake [sham-operated (Shm) or ovariectomized (Ovx) plus E2 (microgram/kg)]: Shm + 0, Ovx + 0, Ovx + 0.05, Ovx + 0.15, Ovx + 0.5, Ovx + 1.5, and Ovx + 5. Ethanol preference drinking was evaluated during 25 consecutive days of E2 replacement treatment, and trunk blood was collected for the determination of plasma E2 and progesterone concentrations. RESULTS: Chronic E2 replacement regimens (0.05-1.5 micrograms/kg) dose-dependently augmented 10E intakes and ethanol preference ratios without concomitantly altering water consumption. Despite the maintenance of 2- to 3-fold greater plasma E2 levels, a supraphysiologic E2 dose (5 micrograms/kg) failed to precipitate ethanol intakes in excess of levels observed after treatment with a high physiologic E2 dose (1.5 micrograms/kg). Plasma progesterone concentrations were significantly increased in treatment groups (1.5 and 5 micrograms/kg E2) that exhibited corresponding significant increases in ethanol consumption. CONCLUSIONS: A positive dose-response relationship between E2 and ethanol intake (incremental increases in E2 dose corresponded to incremental increases in intake) was apparently limited to a physiologic concentration range, because a supraphysiologic dose failed to elicit an additional stepwise increase in ethanol intake. These findings stipulate a modulatory role for E2 in the regulation of moderate ethanol intake and suggest that endogenous fluctuations of E2 may alter the propensity toward consumption in women and in female animal models of ethanol self-administration.