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.     

The CRF-1 receptor antagonist, CP-154,526, attenuates stress-induced increases in ethanol consumption by BALB/cJ mice

Background: Corticotropin‐releasing factor (CRF) signaling modulates neurobiological responses to stress and ethanol, and may modulate observed increases in ethanol consumption following exposure to stressful events. The current experiment was conducted to further characterize the role of CRF₁ receptor (CRF₁R) signaling in stress‐induced increases in ethanol consumption in BALB/cJ and C57BL/6N mice. Methods: Male BALB/cJ and C57BL/6N mice were given continuous access to 8% (v/v) ethanol and water for the duration of the experiment. When a baseline of ethanol consumption was established, animals were exposed to 5 minutes of forced swim stress on each of 5 consecutive days. Thirty minutes before each forced swim session, animals were given an intraperitoneal injection of a 10 mg/kg dose of CP‐154,526, a selective CRF₁R antagonist, or an equal volume of vehicle. The effect of forced swim stress exposure on consumption of a 1% (w/v) sucrose solution was also investigated in an ethanol‐naïve group of BALB/cJ mice. Results: Exposure to forced swim stress significantly increased ethanol consumption by the BALB/cJ, but not of the C57BL/6N, mice. Stress‐induced increases in ethanol consumption were delayed and became evident approximately 3 weeks after the first stressor. Additionally, forced swim stress did not cause increases of food or water intake and did not promote delayed increases of sucrose consumption. Importantly, BALB/cJ mice pretreated with the CRF₁R antagonist showed blunted stress‐induced increases in ethanol intake, and the CRF₁R antagonist did not influence the ethanol drinking of non‐stressed mice. Conclusions: The present results provide evidence that CRF₁R signaling modulates the delayed increase of ethanol consumption stemming from repeated exposure to a stressful event in BALB/cJ mice.     

The Alcohol Deprivation Effect in C57BL/6J Mice is Observed Using Operant Self-Administration Procedures and is Modulated by CRF-1 Receptor Signaling

Background: The alcohol deprivation effect (ADE) is characterized by transient excessive alcohol consumption upon reinstatement of ethanol following a period of ethanol deprivation. While this phenomenon has been observed in rats using both bottle drinking (consummatory behavior) and operant self‐administration (consummatory and appetitive “ethanol‐seeking” behavior) procedures, ADE studies in mice have primarily relied on bottle drinking measures. Furthermore, the neurochemical pathways that modulate the ADE are not well understood. Therefore, we determined whether the ADE can be observed in C57BL/6J mice using operant self‐administration procedures and if expression of the ADE is modulated by the corticotropin releasing factor‐1 (CRF‐1) receptor. Methods: C57BL/6J mice were trained in a 2‐hour operant self‐administration paradigm to lever press for 10% ethanol or water on separate response keys. Between operant sessions, mice had access to ethanol in their homecage. Once stable responding occurred, mice were deprived of ethanol for 4 days and were then retested with ethanol in the operant paradigm for 3 consecutive days. Next, to assess the role of the CRF‐1 receptor, mice were given intraperitoneal (i.p.) injection (0, 10, or 20 mg/kg) of the CRF‐1 receptor antagonist CP‐154,526 30 minutes before ADE testing. Additional experiments assessed (i) ADE responding in which the alternate response lever was inactive, (ii) the effects of CP‐154,526 on self‐administration of a 1% sucrose solution following 4 days of deprivation, and (iii) ADE responding in which mice did not received i.p. injections throughout the experiment. Results: Mice exhibited a significant increase in postdeprivation lever responding for ethanol with either a water reinforced or inactive alternate lever. Interestingly, i.p. injection of a 10 mg/kg dose of CP‐154,526 protected against the ADE while not affecting lever responding for a sucrose solution. Finally, baseline and deprivation‐induced increases of ethanol reinforced lever responding were greater in mice not given i.p. injections. Conclusions: The ADE in C57BL/6J mice can be modeled using the operant self‐administration paradigm and increased ethanol self‐administration associated with the ADE is modulated by CRF‐1 receptor signaling.     

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.     

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.     

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.     

Ghrelin Receptor Antagonism Decreases Alcohol Consumption and Activation of Perioculomotor Urocortin‐Containing Neurons

Background: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut‐derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6. Methods: In Experiment 1 , male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2‐bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2 , the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c‐Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D‐Lys3‐GHRP‐6 affects alcohol intake, in Experiment 3 , the effect of D‐Lys3‐GHRP‐6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c‐Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4 , to test if blood ethanol concentrations were affected by D‐Lys3‐GHRP‐6, blood samples were taken at 2 time‐points after D‐Lys3‐GHRP‐6 pretreatment and systemic ethanol administration. Results: In Experiment 1 , D‐Lys3‐GHRP‐6 reduced preference to alcohol and in a follow‐up experiment ( Experiment 2 ) also dramatically reduced alcohol intake when compared to saline‐treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c‐Fos showed fewer immunopositive cells in the pIIIu of the antagonist‐treated mice but no difference was seen in the VTA or Arc. In Experiment 3 , D‐Lys3‐GHRP‐6 reduced the induction of c‐Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c‐Fos immunopositive cells after D‐Lys3‐GHRP‐6 administration, but the antagonist had no effect on ethanol‐induced expression of c‐Fos. D‐Lys3‐GHRP‐6‐pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline‐pretreated mice ( Experiment 4 ). Conclusions: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.     

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.     

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.     

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.     

Ethanol intake in the juvenile, adolescent, and adult rat: effects of age and prior exposure to ethanol

Background: Initial ingestion of ethanol by naïve rats has seemed to decrease dramatically with age. During the preweanling period, infant rats consume large quantities of high concentrations of ethanol without initiating procedures, in some instances exceeding doses required for severe motor incoordination. During adulthood, however, initial ingestion of ethanol without initiation procedures is low and infrequent. In the present study, the ontogeny of ethanol intake was measured in juvenile, adolescent and adult rats using a technique [consume off the floor (COF)] similar to that used to study intake during infancy. How this initial experience with ethanol affected subsequent affinity for ethanol intake was later assessed using 2‐bottle choice preference tests. Methods: Independent ingestion of ethanol was measured at 3 developmental periods, the juvenile period (P22–P28), adolescence (P30–P34) and adulthood (P60–P64), with systematic variation in ethanol concentration (15 or 30% v/v) and palatability (sweetness) of ethanol. Blood ethanol concentrations (BECs) were determined in all animals. This dependent variable served as an estimate of absolute ethanol ingestion. Three COF sessions were conducted for each age group. Following these sessions animals' ethanol consumption was also assessed using a 2‐bottle choice test (water vs 15% v/v unsweetened ethanol). Results: In all experiments, groups consuming 30% v/v ethanol exhibited significantly higher BECs than those exposed to 15% v/v ethanol. Adding saccharin to the ethanol increased absolute ethanol ingestion in only the oldest animals. During the pre‐exposure phase (COF sessions) of each experiment, absolute ethanol intake was found to decline with repeated exposures. Sex effects were particularly evident during later stages of ontogeny (adolescents and adults). The overall pattern of results indicated that juveniles relative to adults show a marked predisposition to consume highly concentrated ethanol solutions and that BECs derived from the COF sessions influenced ethanol acceptance patterns in the subsequent 2‐bottle test. Conclusions: Using the (COF) technique with BECs as an estimate of intake, absolute ethanol consumption seems to be quite high early in ontogeny and decline gradually into adulthood. Adding saccharin to ethanol solutions at the concentration used in the present study (0.1%) was generally not sufficient to increase absolute ethanol intake from the floor, except during adulthood. The experimental strategy employed in this study represents a novel approach for examining ethanol acceptance patterns across ontogeny and how experience with the process of intoxication affects subsequent ethanol preferences.     

Restraint stress and ethanol consumption in two mouse strains

Background: This study examined the interaction between restraint stress and ethanol drinking in mice that consume low and high amounts of ethanol. Methods: Two strains of mice (129SVEV and C57BL/6J) underwent 1 hour of restraint stress twice per day for 4 days in the presence of a CRF‐1 receptor antagonist, a glucocorticoid receptor antagonist or vehicle. Ethanol preference and consumption were assessed using a two bottle choice design. In another study, mice were implanted with pellets containing corticosterone; ethanol preference and consumption were assessed using a two bottle choice design. Results: Restraint stress significantly increased ethanol preference and consumption in 129SVEV mice but not in C57BL/6J mice. Then 129SVEV mice underwent the identical stress procedure; however, mice received either the CRF‐1 receptor antagonist, R121919 (15 or 20 mg/kg, ip) or vehicle 30 minutes prior to stress. R121919 did not block the stress‐induced change in ethanol preference despite causing a significant blunting in the HPA axis. Negative results were also obtained using the CRF‐1 receptor antagonist, Antalarmin (20 mg/kg, ip). In another study, 129SVEV mice were administered either the glucocorticoid receptor antagonist Mifepristone (25, 50 or 100 μg/kg, ip) or vehicle under the same procedure. Mifepristone did not alter ethanol preference. Moreover, the three receptor antagonist did not alter nonstress ethanol consumption either. In the last study, both mouse strains underwent active or sham adrenalectomy, then pellets containing corticosterone or placebo were implanted and preference for ethanol versus water was tested. Corticosterone administration decreased ethanol consumption in a strain‐dependent manner. Conclusion: These data show the restraint model for stress can modestly increase ethanol consumption in 129SVEV mice but not in C57BL/6J mice. Pharmacologic manipulation of CRF and corticosterone did not blunt baseline or stress‐induced change in ethanol preference nor did administration of corticosterone mimic the effects of restraint stress on ethanol consumption. These findings suggest the mechanism responsible for increasing ethanol consumption in this model is independent of the HPA axis and extra‐hypothalamic CRF.     

Chronic intermittent ethanol exposure in early adolescent and adult male rats: effects on tolerance, social behavior, and ethanol intake

Background: Given the prevalence of alcohol use in adolescence, it is important to understand the consequences of chronic ethanol exposure during this critical period in development. The purpose of this study was to assess possible age‐related differences in susceptibility to tolerance development to ethanol‐induced sedation and withdrawal‐related anxiety, as well as voluntary ethanol intake after chronic exposure to relatively high doses of ethanol during adolescence or adulthood. Methods: Juvenile/adolescent and adult male Sprague‐Dawley rats were assigned to one of five 10‐day exposure conditions: chronic ethanol (4 g/kg every 48 hours), chronic saline (equivalent volume every 24 hours), chronic saline/acutely challenged with ethanol (4 g/kg on day 10), nonmanipulated/acutely challenged with ethanol (4 g/kg on day 10), or nonmanipulated. For assessment of tolerance development, duration of the loss of righting reflex (LORR) and blood ethanol concentrations (BECs) upon regaining of righting reflex (RORR) were tested on the first and last ethanol exposure days in the chronic ethanol group, with both saline and nonmanipulated animals likewise challenged on the last exposure day. Withdrawal‐induced anxiety was indexed in a social interaction test 24 hours after the last ethanol exposure, with ethanol‐naïve chronic saline and nonmanipulated animals serving as controls. Voluntary intake was assessed 48 hours after the chronic exposure period in chronic ethanol, chronic saline and nonmanipulated animals using an 8‐day 2 bottle choice, limited‐access ethanol intake procedure. Results: In general, adolescent animals showed shorter durations of LORR and higher BECs upon RORR than adults on the first and last ethanol exposure days, regardless of chronic exposure condition. Adults, but not adolescents, developed chronic tolerance to the sedative effects of ethanol, tolerance that appeared to be metabolic in nature. Social deficits were observed after chronic ethanol in both adolescents and adults. Adolescents drank significantly more ethanol than adults on a gram per kilogram basis, with intake uninfluenced by prior ethanol exposure at both ages. Conclusions: Adolescents and adults may differ in their ability and/or propensity to adapt to chronic ethanol exposure, with adults, but not adolescents, developing chronic metabolic tolerance. However, this chronic exposure regimen was sufficient to disrupt baseline levels of social behavior at both ages. Taken together, these results suggest that, despite the age‐related differences in tolerance development, adolescents are as susceptible as adults to consequences of chronic ethanol exposure, particularly in terms of disruptions in social behavior. Whether these effects would last into adulthood remains to be determined.     

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.     

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.     

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.     

Lesions of the extended amygdala in C57BL/6J mice do not block the intermittent ethanol vapor-induced increase in ethanol consumption

Background: The central extended amygdala (cEA) which includes the central nucleus of the amygdala (CeA) and the lateral posterior bed nucleus of the stria terminalis (BNSTLP), has been proposed to play a key role in excessive ethanol consumption in humans (Koob and Le Moal, 2005 Nat Neurosci 8:1442). To examine this relationship, we used a murine model of ethanol dependence (Becker and Lopez, 2004 Alcohol Clin Exp Res 28:1829; Lopez and Becker, 2005 Psychopharmacology (Berl) 181:688) and compared animals with sham lesions and electrolytic lesions of the CeA and BNSTLP. Methods: Male C57BL/6J (B6) mice were first acclimated to a limited‐access 2‐bottle‐choice preference procedure. The access period began 3 hours into the dark phase of the light‐dark cycle and continued for 2 hours. Once acclimated (1 week), mice underwent chronic exposure to and intermittent withdrawal from ethanol vapor. The animals were then retested in the limited‐access 2‐bottle‐choice preference procedure. In some experiments, electrolytic and sham lesions of the CeA or BNSTLP were performed prior to initiating the 2‐bottle choice procedure. Results: In a series of 5 preliminary experiments, mice were randomly assigned either to the standard intermittent ethanol vapor procedure or to the standard procedure but with air in the vapor chamber (control). The air‐control procedure produced no change in ethanol intake when compared to baseline consumption. In contrast, intermittent ethanol vapor exposure increased ethanol consumption by almost 50%. The increase in consumption was associated with an increase in total fluid volume consumed and no change in ethanol preference. Lesions of both the BNSTLP and CeA significantly decreased baseline ethanol consumption, the former by decreasing fluid consumption and the latter by decreasing ethanol preference. Intermittent ethanol vapor exposure significantly increased consumption in both the BNSTLP‐ and CeA‐lesioned animals, largely by increasing the total volume of fluid consumed. Conclusions: The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited‐access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor‐induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.     

Inhibition of 5alpha-reduced steroid biosynthesis impedes acquisition of ethanol drinking in male C57BL/6J mice

Background: Allopregnanolone (ALLO) is a physiologically relevant neurosteroid modulator of GABA_A receptors, and it exhibits a psychopharmacological profile that closely resembles the post‐ingestive effects of ethanol. The 5α‐reductase inhibitor finasteride (FIN), which inhibits biosynthesis of ALLO and structurally related neurosteroids, was previously demonstrated to reduce the maintenance of limited‐access ethanol consumption. The primary aim of the current work was to determine whether FIN would reduce the acquisition of drinking in ethanol‐naïve mice. Methods: Male C57BL/6J (B6) mice were acclimated to a reverse light/dark schedule, and were provided ad libitum access to chow and water. Following habituation to vehicle injections (VEH; 20% w/v β‐cyclodextrin; i.p.) administered 22‐hour prior to drinking sessions with water only, mice were divided into 3 treatment groups: vehicle control (VEH), 50 mg/kg FIN (FIN‐50), and 100 mg/kg FIN (FIN‐100). Twenty‐two hours after the first treatment, mice were permitted the inaugural 2‐hour limited access to a 10% v/v ethanol solution (10E) and water. The acquisition of 10E consumption and underlying drinking patterns were assessed during FIN treatment (7 days) and subsequent FIN withdrawal (13 days) phases. Results: FIN dose‐dependently blocked the acquisition of 10E drinking and prevented the development of ethanol preference, thereby suggesting that the GABAergic neurosteroids may be important in the establishment of stable drinking patterns. FIN‐elicited reductions in 10E intake were primarily attributable to selective and marked reductions in bout frequency, as no changes were observed in bout size, duration, or lick rates following FIN treatment. FIN‐treated mice continued to exhibit attenuated ethanol consumption after 2 weeks post‐treatment, despite a full recovery in brain ALLO levels. A second study confirmed the rightward and downward shift in the acquisition of ethanol intake following 7 daily FIN injections. While there were no significant group differences in brain ALLO levels following the seventh day of ethanol drinking, ALLO levels were decreased by 28% in the FIN‐50 group. Conclusions: Although the exact mechanism is unclear, FIN and other pharmacological interventions that modulate the GABAergic system may prove useful in curbing ethanol intake acquisition in at‐risk individuals.     

Ataxia and c-Fos expression in mice drinking ethanol in a limited access session

BACKGROUND: Although previous murine studies have demonstrated ethanol self-administration resulting in blood ethanol concentrations (BECs) believed to be pharmacologically relevant, to our knowledge, no study reported to date has demonstrated intoxication via ataxia after self-administration. Thus, the goal of this study was to demonstrate ataxia and to examine changes in c-Fos expression in mice after self-administration of intoxicating doses of ethanol. METHODS: Male C57BL/6J mice were trained to drink a 10% ethanol solution during daily 30-min limited access sessions. Mice were exposed to increasing concentrations of ethanol until a 10% ethanol solution was reached. BEC and ataxia, measured as foot slips off of a balance beam, were examined after the limited access self-administration session. In a separate experiment, various brain structures from mice drinking water or ethanol were examined for changes in c-Fos expression two hr after the limited access session. RESULTS: Mice drank between 1.5 and 2 g/kg of 10% ethanol during the daily 30-min session. BECs for these mice 15 min after the limited access session ranged between 0.52 and 2.13 mg/ml. A significant increase in foot slips off a balance beam was seen immediately after ethanol consumption during the limited access session. Among mice drinking ethanol, an increase in c-Fos expression was seen in the Edinger-Westphal nucleus, and a decrease in c-Fos expression was seen in the cingulate cortex, ventral tegmental area, lateral and medial septum, CA1 region of the hippocampus, and basolateral amygdala. CONCLUSIONS: After this procedure in mice, BECs are achieved that are in a range considered pharmacologically relevant and intoxicating. Significant ataxia was observed after ethanol self-administration. Brain regions showing changes in c-Fos expression after voluntary intoxication were similar to those previously reported, suggesting that these brain regions are involved in regulating behavioral effects of alcohol intoxication.