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.     

Increased ethanol drinking after repeated chronic ethanol exposure and withdrawal experience in C57BL/6 mice

BACKGROUND: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self-administer ethanol. METHODS: With the use of a modified sucrose-fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8-hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5-day test period for ethanol drinking. RESULTS: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air-exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels. CONCLUSIONS: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2-fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).     

Hybrid C57BL/6J x FVB/NJ mice drink more alcohol than do C57BL/6J mice

BACKGROUND: From several recent strain surveys (28 strains: Bachmanov et al., personal communication; 22 strains: Finn et al., unpublished), and from data in >100 other published studies of 24-hr two-bottle ethanol preference, it is known that male C57BL/6 (B6) mice self-administer about 10-14 g/kg/day and that female B6 mice self-administer about 12-18 g/kg/day. No strain has been found to consume more ethanol than B6. In one of our laboratories (Texas), we noted a markedly greater intake of ethanol in an F1 hybrid of B6 and FVB/NJ (FVB) mice. METHODS: To confirm and extend this finding, we repeated the study at another site (Portland) using concentrations up to 30% ethanol and also tested B6xFVB F1 mice in restricted access drinking procedures that produce high levels of alcohol intake. RESULTS: At both sites, we found that B6xFVB F1 mice self-administered high levels of ethanol during two-bottle preference tests (females averaging from 20 to 35 g/kg/day, males 7-25 g/kg/day, depending on concentration). F1 hybrids of both sexes drank significantly more 20% ethanol than both the B6 and FVB strains. Female F1 hybrids also drank more 30% ethanol. In the restricted access tests, ethanol consumption in the F1 hybrids was equivalent to that in B6 mice. CONCLUSIONS: These data show that this new genetic model has some significant advantages when compared to existing inbred strains, and could be used to explore the genetic basis of high ethanol drinking in mice.     

Operant Self-Administration of Sweetened Versus Unsweetened Ethanol: Effects on Blood Alcohol Levels

BACKGROUND: Sweeteners are often added to ethanol solutions to increase ethanol intake. However, literature on studies that use human subjects and laboratory animals suggests that sucrose, other sugars, and carbohydrate-rich foods alter ethanol absorption and metabolism, which leads to lower blood alcohol levels (BAL) relative to ethanol absorbed alone. This experiment was designed to test whether the addition of the nutritive sweetener sucrose, or the nonnutritive sweetener saccharin, to a 10% ethanol solution, self-administered in an oral operant paradigm, affected BAL in rats relative to self-administration of an unsweetened 10% ethanol solution. METHODS: All rats were trained to lever press for ethanol by use of a saccharin fading procedure. Half of the rats then received 30-min sessions in which ethanol + 2% sucrose and water were available and were alternated daily with sessions in which ethanol + 0.2% saccharin and water were available. The other half of the rats went on to receive daily sessions of unsweetened ethanol and water. BAL were taken after these standard daily sessions as well as after a 1-week period of alcohol deprivation (to enhance ethanol intake). RESULTS: Rats responded for more ethanol + sucrose than unsweetened ethanol, but had lower BAL per gram ethanol consumed in both the baseline test and alcohol deprivation effect test. No effect of saccharin on BAL was detected. An additional experiment that examined the effects of four concentrations of both sucrose and saccharin on self-administration of ethanol and BAL showed that, whereas rats consumed more ethanol + sucrose than ethanol + saccharin, BAL were significantly lower per gram ethanol consumed in the sucrose group. CONCLUSIONS: These results confirm previous reports and suggest that the addition of sucrose to an ethanol solution can result in lower BAL relative to unsweetened ethanol in an oral operant self-administration paradigm.     

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.     

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.     

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.     

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.     

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.     

Intra-amygdala infusion of the NPY Y1 receptor antagonist BIBP 3226 attenuates operant ethanol self-administration

BACKGROUND: Neuropeptide Y (NPY) is the most abundant and widely distributed peptide in the mammalian central nervous system. Evidence suggests that NPY transmission at Y1 receptors may regulate alcohol self-administration in rodent models. The purpose of the present study was to test the involvement of NPY Y1 receptors in the amygdala in the reinforcing effects of alcohol. METHODS: Long-Evans rats were trained to self-administer ethanol (10% v/v) vs. water on a concurrent FR-1 schedule of reinforcement using a sucrose fading procedure. After a 1 month baseline period, bilateral injector cannulae were surgically implanted to terminate 1 mm dorsal to the central nucleus of the amygdala. Daily (Monday through Friday) operant self-administration sessions were conducted for 6 months after surgery. Then, the effects of intra-amygdala infusion of the high-affinity nonpeptide NPY Y1 receptor antagonist BIBP 3226 (1, 10, or 20 microMg) were determined on parameters of operant alcohol self-administration. RESULTS: Intra-amygdala administration of 10 microM or 20 microM BIBP 3226 decreased total alcohol-reinforced responding and dose of self-administered ethanol (g/kg) without significantly altering total water responses or intake compared with vehicle control. Response onset was unaffected. Analysis of the temporal pattern of ethanol- and water-reinforced responding showed that BIBP 3226 decreased cumulative ethanol-reinforced responding during the 30 to 60 min period of the sessions. Water-reinforced responses were increased by the low dose of BIBP 3226 (1 microM) during the 50 to 60 min period. CONCLUSIONS: Results from this study indicate that alcohol-reinforced responding is reduced by acute blockade of NPY Y1 receptors in the amygdala of rats with a long-term history of alcohol self-administration. These data are consistent with the hypothesis that alcohol self-administration is maintained by NPY neurotransmission at Y1 receptors in the central nucleus of the amygdala.     

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.     

Effects of Acute and Chronic Doses of Naltrexone on Ethanol Self-Administration in Rhesus Monkeys

The effects of acute and chronic administration of intramuscular naltrexone (0.1, 0.3, 1.0, and 3.0 mg/kg) on oral ethanol (8%) self-administration were examined. Naltrexone (1.0 mg/kg) effects on the self-administration of ethanol concentrations ranging from 0.5 to 8% (w/v) were also investigated. Rhesus monkeys with substantial histories of drug and ethanol drinking served as subjects. During daily 3-hr sessions, monkeys were presented with ethanol solutions, concurrently available with water, under fixed-ratio reinforcement schedules. Naltrexone decreased the consumption of ethanol (g/kg). Biphasic temporal effects were observed within sessions. Naltrexone dose-dependently decreased the number of ethanol deliveries by a maximum of 56% ( n = 18; 3 monkeys × 6 sessions) during the first hour of the session. During the second and third hours, however, ethanol intake recovered such that maximum decreases over the 3-hr session were ∼27% ( n = 18), and the mean decrease was 16% ( n = 18). Often marked tolerance was observed, such that the effects of acute naltrexone administration were greater than effects after chronic administration. The self-administration of low ethanol concentrations (≤2% w/v) was increased in several monkeys, by up to 340%, after naltrexone pretreatment. In summary, the effects of naltrexone on ethanol self-administration, in drug- and alcohol-experienced rhesus monkeys, are not characterized by unitary decreases in measures of ethanol self-administration. Rather, differential naltrexone effects were a function of experimental parameters, including the dose and number of naltrexone injections, the ethanol concentration, and the time point of measurement.     

Oral Ethanol Self-Administration in Free-Feeding Rhesus Monkeys

The ability of a conditioning procedure to establish oral ethanol self-administration in free-feeding rhesus monkeys was assessed. The conditioning procedure required the monkey to drink an ethanol solution in order to have access to a sweet orange-flavored solution. Following an average of 14 sessions under these conditions, the orange solution was no longer delivered and ethanol solution alone was made available in the sessions. During both the conditioning and the ethanol self-administration portions of the experiment each monkey was required to drink an average of 0.5 g/kg ethanol per session in order to continue in the experiment. Of the nine monkeys exposed to these contingencies, five monkeys continued to self-administer ethanol after the presentation of the orange drink was discontinued. However, two of these five monkeys decreased their ethanol solution intake below 0.5 g/kg within 3 weeks after the conditioning sessions had terminated. The three monkeys that sustained high ethanol intake were male and had histories of drug self-administration, suggesting that gender and drug history may influence the initiation of ethanol self-administration. Once ethanol self-administration was established, concentrations of ethanol from 4 to 15% (v/v) were made available. The monkeys consumed intoxicating amounts of ethanol, as indicated by average intakes ranging from 0.5 to 0.9 g/kg and blood ethanol levels over 100 mg/dl. These results demonstrate that ethanol self-administration can be established and maintained through the initial reinforcement of ethanol consumption by the contingent presentation of another reinforcing stimulus. However, the results of this study also indicate that individual differences may be an important determinant of animals initiating ethanol self-administration.     

Increased drinking during withdrawal from intermittent ethanol exposure is blocked by the CRF receptor antagonist D-Phe-CRF(12-41)

BACKGROUND: Studies in rodents have determined that intermittent exposure to alcohol vapor can increase subsequent ethanol self-administration, measured with operant and 2-bottle choice procedures. Two key procedural factors in demonstrating increased alcohol intake are the establishment of stable alcohol self-administration before alcohol vapor exposure and the number of bouts of intermittent vapor exposure. The present studies provide additional behavioral validation and initial pharmacological validation of this withdrawal-associated drinking procedure. METHODS: Studies at 2 different sites (Portland and Scripps) examined the effect of intermittent ethanol vapor exposure (3 cycles of 16 hours of ethanol vapor+8 hours air) on 2-hour limited access ethanol preference drinking in male C57BL/6 mice. Separate studies tested 10 or 15% (v/v) ethanol concentrations, and measured intake during the circadian dark. In one study, before measuring ethanol intake after the second bout of intermittent vapor exposure, mice were tested for handling-induced convulsions (HICs) indicative of physical dependence on ethanol. In a second study, the effect of bilateral infusions of the corticotropin-releasing factor (CRF) receptor antagonist D-Phe-CRF(12-41) (0.25 microg/0.5 microL) into the central nucleus of the amygdala (CeA) on ethanol intake was compared in vapor-exposed animals and air controls. RESULTS: Intermittent ethanol vapor exposure significantly increased ethanol intake by 30 to 40%, and the mice had higher blood ethanol concentrations than controls. Intra-amygdala infusions of D-Phe-CRF(12-41) significantly decreased the withdrawal-associated increase in ethanol intake without altering ethanol consumption in controls. Following the second bout of intermittent vapor exposure, mice exhibited an increase in HICs, when compared with their own baseline scores or the air controls. CONCLUSIONS: Intermittent alcohol vapor exposure significantly increased alcohol intake and produced signs of physical dependence. Initial pharmacological studies suggest that manipulation of the CRF system in the CeA can block this increased alcohol intake.     

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.     

Blood Ethanol Concentrations in Rats Drinking Sucrose/Ethanol Solutions

BACKGROUND: The addition of sucrose to ethanol solutions results in a substantial increase in ethanol self-administration by rats that are deprived of neither food nor water. However, if sucrose alters ethanol absorption or metabolism, resulting in blood ethanol concentrations (BECs) not different from those resulting from lower intakes of ethanol/water solutions, then the usefulness of sucrose/ethanol mixtures in increasing ethanol consumption is questionable. The present study was conducted to determine whether the addition of sucrose to ethanol solutions altered BECs in an operant self-administration paradigm. METHODS: Tail blood (from male Long-Evans rats) was collected 30 min after the intake of four different solutions, i.e., 5% sucrose/20% ethanol, 5% sucrose/10% ethanol, 2% sucrose/10% ethanol, and 10% ethanol. RESULTS: Ethanol intakes (mean, 1.57+/-0.21 g/kg) and BECs (mean, 78.4+/-9.3 mg/100 ml) were highest when 5% sucrose was added to the ethanol solution. Moreover, the ratios between ethanol intakes and resulting BECs were approximately the same for all solutions. CONCLUSIONS: These findings indicate that, under the conditions of this procedure, the BEC reached is dependent on the amount of ethanol consumed and is not influenced by the addition of sucrose to the solution.     

Helplessness in the tail suspension test is associated with an increase in ethanol intake and its rewarding effect in female mice

BACKGROUND: Depression is frequently observed in drug abusers. However, depression may be a primary factor of predisposition to drug abuse or a consequence of drug abuse. The aim of this study was to analyze the influence of a preexisting depressive-like state/helplessness on subsequent alcohol responsiveness in mice. METHODS: Male and female CD1 mice were selected according to their immobility time in the tail suspension test, and only mice with "high immobility" and "low immobility" time were retained. Using a two-bottle free-choice paradigm, these mice were given continuous access to tap water or solutions of ethanol (3-20% v/v), quinine (12.5-50 mg/liter), or sucrose (1-4% w/v). In female mice, rewarding and aversive effects of ethanol (1.5 and 3 g/kg, intraperitoneally) were also investigated using the conditioned place preference and the conditioned taste aversion paradigms. RESULTS: Female mice were more immobile and drank more ethanol than male mice. No striking sex difference was observed in quinine consumption. Sucrose intake was higher in female than in male mice, whatever the solution concentration. At the 4% concentrated solution, a sucrose-induced increase in daily fluid intake was observed only in female mice. Female mice with high immobility time (HI) consumed more ethanol at the highest concentration than female mice with low immobility time (LI), whereas no difference was observed between HI and LI male mice. Moreover, whereas LI female mice failed to express place conditioning induced by the 3-g/kg dose of ethanol, HI female mice were strongly responsive to the rewarding effect of this high ethanol dose. Ethanol dose-dependently induced a conditioned taste aversion with a similar magnitude in both LI and HI female mice. CONCLUSIONS: The findings indicate that female CD1 mice tend to drink greater amounts of ethanol or sucrose solutions than male CD1 mice, suggesting that female mice may be a better model of excessive alcohol intake. Furthermore, no relationship was found between immobility scores and ethanol consumption in male mice. On the contrary, within female mice, HI mice consumed higher amounts of ethanol than LI mice probably because they experienced greater rewarding effects of ethanol. The present results support the hypothesis that depressive-like responses may predispose to ethanol abuse in female mice.     

Patterns of Ethanol Consumption in a Continuous Access Situation: The Effect of Adding a Sweetener to the Ethanol Solution

The effect of the addition of sucrose to an ethanol solution upon daily intake patterns was examined in a continuous-access operant situation with Wistar rats. Rats were first initiated to self-administer orally a 10% ethanol (v/v) solution using the sucrose-substitution procedure in 30-min limited-access conditions. When then studied in a continuous-access operant situation (23 hr ethanol access), substantial increases in ethanol consumption were found when varying concentrations of sucrose were added to the ethanol solutions presented. This increased consumption was found to be a complex function of both an increase in the number of drinking occurrences each day and in the size of each drinking occurrence. When 2% sucrose was compared with 2% sucrose/10% ethanol, the consumption of the sweetened ethanol was greater than consumption of the sweetener alone, suggesting that the ethanol added to the ability of the solution to maintain behavior beyond that of the sucrose alone. This study supports the use of sweetened ethanol solutions for the study of ethanol drinking patterns, and as a model system for examining factors involved in the regulation of ethanol consumption.     

Circadian Timing of Ethanol Exposure Exerts Enduring Effects on Subsequent Ad Libitum Consumption in C57 Mice

Background:  There is a daily rhythm in the voluntary intake of ethanol in mice, with greatest consumption in the early night and lowest intake during the day. The role of daily timing of ethanol exposure on the development and control of long-term ethanol self-administration has been neglected. The present study examines these issues using C57BL/6J mice.
Methods:  Mice were repeatedly exposed to 10% ethanol for 2 hours early in the night or day for several weeks. Subsequently, ethanol was available at the opposite time (Expt 1) or 24 hours daily (Expts 1 and 2). Lick sensors recorded the patterns of drinking activity in Experiment 2.
Results:  Mice exposed to ethanol during the night drink more than mice exposed during the day. Prior history did not affect ethanol intake when the schedule was reversed. Under 24-hour exposure conditions, mice with a history of drinking during the night consumed significantly more than mice drinking during the day. The circadian patterns of drinking were not altered.
Conclusions:  These results demonstrate that the daily timing of ethanol exposure exerts enduring effects of self-administration of ethanol in mice. Understanding how circadian rhythms regulate ethanol consumption may be valuable for modifying subsequent intake.     

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.