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.     

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.     

High- and low-alcohol-preferring mice show differences in conditioned taste aversion to alcohol

Background: Genetic differences in sensitivity to the aversive effects of alcohol may contribute to alcohol drinking behavior. The present study examined the development of conditioned taste aversion (CTA) to various doses of alcohol in two pairs of mouse lines selectively bred for high (HAP) and low (LAP) alcohol preference. Methods: Alcohol‐naïve, male and female HAP and LAP mice from both replicate 1 (HAP n= 29; LAP n= 28) and replicate 2 (HAP n= 34; LAP n= 35) were adapted to a 2‐hr per day water restriction regimen. During five conditioning trials at 48 hr intervals, mice received an intraperitoneal injection of saline or 2 g/kg or 4 g/kg alcohol immediately following 1 hr of access to a 0.20 M NaCl solution. Results: LAP mice of both replicates showed a significantly greater magnitude of CTA to both 2 g/kg and 4 g/kg alcohol compared with HAP mice of both replicates. There were no line differences in consumption of the NaCl solution in the saline control groups. Conclusions: These data suggest that mice selectively bred for low alcohol preference are more sensitive to the development of alcohol CTA than mice selectively bred for high alcohol preference. The present findings indicate that common genes mediate both alcohol preference and the aversive effects of alcohol as measured in the CTA paradigm.     

Schedule-induced ethanol self-administration in DBA/2J and C57BL/6J mice

BACKGROUND: The purpose of these experiments was to provide an initial investigation into ethanol self-administration elicited in the schedule-induced polydipsia (SIP) paradigm. METHODS: Mature male mice were food deprived to between 80 and 85% of their baseline weight and received 20 daily 1 hr SIP test sessions in which a food pellet (20 mg) was delivered on a fixed-time 60 sec schedule. In different groups, the acquisition of drinking 5% (v/v) ethanol solution (experiment 1) or water (experiment 2) was recorded along with other behaviors that occurred in the test chambers. RESULTS: Results indicated that C57BL/6J mice drank significantly more ethanol than DBA/2J mice and that C57 mice achieved blood alcohol concentrations as high as 300 mg/dl. Blood alcohol concentrations were consistently correlated with g/kg ethanol intake. The groups did not differ in consumption of water. SIP test sessions using higher concentrations of ethanol (10-20% v/v, experiment 1) or sucrose solutions (0.1-2% w/v, experiment 2) then were performed. Group differences in ethanol consumption were maintained at all ethanol concentrations. Although DBAs drank more of a low concentration of sucrose (0.1%), when expressed as g/kg, sucrose intake was equivalent in the two strains at all concentrations. Analysis of the time course of drinking clearly showed that this behavior was adjunctive in nature. CONCLUSION: These results demonstrate the effectiveness of this procedure in inducing ethanol self-administration and its utility for investigating the genetic bases of vulnerability toward excessive ethanol consumption.     

Initiating Ethanol Drinking in a Simian Social Group in a Naturalistic Setting

We examined in nine group-living, male Japanese Snow monkeys the initiation of alcohol-drinking behavior in an enriched environment where both social and nonsocial stimuli could influence drinking. The monkeys usually could move freely between an indoor shelter and a large outdoor corral, which contained three drinkometers. During daily 2-hr sessions in pre- and post-training periods (with food and water available ad libitum), the drinkometers held (on different days) Koolaid-saccharin, water, water with 5% ethanol, Koolaid-saccharin with 5% ethanol, or Koolaid-sucrose with acetic acid (matched in calories and palatability to Koolaid-saccharin with 5% ethanol). Between pre- and post-training periods was a long training period, in which Koolaid-saccharin with various ethanol concentrations was presented with the daily food ration in 2-hr drinking sessions. On other selected training days, water with 5% ethanol was presented. During training sessions some monkeys drank high doses; others did not. During the period of peak drinking, the daily mean ethanol consumption ranged among animals from 0.54-1.99 ml/kg. Blood ethanol concentrations then sometimes exceeded 100 mg/dl. During the post-training period, with return to ad libitum food and water, consumption declined from these peaks, but remained significantly higher than pretraining consumption; the ethanol solution was established as a reinforcer. Monkeys differed significantly in the extent of this pre/post increase. In both pre- and post-training periods, consumption was significantly greater when all three drinkometers operated, compared with only one. On pretraining days when only one drinkometer operated, more dominant animals drank significantly more than less dominant animals; this difference disappeared by the post-training period, as less dominant animals learned to use the drinkometers at times when the dominant animals eschewed the machines. Pretraining consumption did not predict (among animals) post-training consumption. Before, during, and after training, these animals drank less of both alcohol and control solutions than did members of another species, which we had studied in smaller indoor pens. We discuss possible explanations and implications.     

Modulation of Brain Endocannabinoid Levels by Voluntary Alcohol Consumption in Alcohol-Preferring AA Rats

Background: The central nervous system cannabinoid CB1 receptors have been implicated in regulation of alcohol consumption. Less data are available on the role of the endogenous ligands for these receptors, anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), in alcohol‐related behaviors. The purpose of this study was to assess the effects of voluntary alcohol consumption on the levels of these endocannabinoids in key brain areas mediating alcohol reinforcement. Methods: Female and male alcohol‐preferring AA (Alko, Alcohol) rats were trained to drink 10% (v/v) alcohol during 90‐min limited access sessions every second day. Following establishment of stable alcohol drinking, half of the subjects were killed immediately before the daily alcohol access (“pre‐session” group), while the other half was killed after the drinking session (“post‐session” group). A separate control group consisted of water‐drinking rats. AEA and 2‐AG levels were measured from prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), amygdala, and hippocampus using liquid chromatography–tandem mass spectrometry (LC/MS/MS). Results: Voluntary alcohol drinking caused widespread alterations in the levels of both AEA and 2‐AG. Compared to the water group, increased AEA levels were seen in the pre‐session group, but they were decreased immediately following limited access drinking in the female AA rats. Also 2‐AG levels were significantly elevated after long alcohol exposure, and an additional increase was found after limited access drinking in PFC. In males, however, the only alterations caused by alcohol drinking were significantly elevated AEA levels in NAc and CPu in the post‐session group. No changes were seen in the levels of 2‐AG. Conclusions: These results demonstrate that voluntary alcohol drinking modulates the levels of endocannabinoids in several brain areas implicated in alcohol reinforcement. AEA and 2‐AG were differentially affected, suggesting that they could have partially separate modulatory roles. Alterations were more widespread in females than males, possibly reflecting their higher alcohol intake. Taken together, alcohol‐induced release of endocannabinoids may have an important role in alcohol reinforcement and development of alcohol addiction.     

High-Alcohol Preferring Mice Are More Impulsive Than Low-Alcohol Preferring Mice as Measured in the Delay Discounting Task

Background: Repeated studies have shown that high impulsivity, when defined as the tendency to choose small immediate rewards over larger delayed rewards, is more prevalent in drug addicts and alcoholics when compared with nonaddicts. Assessing whether impulsivity precedes and potentially causes addiction disorders is difficult in humans because they all share a history of drug use. In this study, we address this question by testing alcohol‐naïve mice from lines showing heritable differences in alcohol intake. Methods: Replicated selected lines of outbred high‐alcohol preferring (HAP) mice were compared to a low‐alcohol preferring (LAP) line as well as the low‐drinking progenitor line (HS/Ibg) on an adjusting amount delay discounting (DD) task. The DD task employs 2 levers to present subjects with a choice between a small, immediate and a large, delayed saccharin reward. By adjusting the quantity of the immediate reward up and down based on choice behavior, the task allows an estimate of how the subjective value of the delayed reinforcer decreases as delays increase. Latency to respond was also measured for each trial. Results: Both HAP2 and HAP1 lines of mice were more impulsive than the LAP2 and HS/Ibg lines, respectively. Hyperbolic curve‐fitting confirmed steeper discounting in the high‐alcohol drinking lines. In addition, the high‐alcohol drinking lines demonstrated greater within‐session increases in reaction times relative to the low‐alcohol drinking lines. No other differences (consumption of saccharin, total trials completed) consistently mapped onto genetic differences in alcohol drinking. Conclusions: Alcohol‐naïve outbred mice selected for high‐alcohol drinking were more impulsive with saccharin reinforcers than low‐alcohol drinkers. These data are consistent with results seen using inbred strain descendents of high‐alcohol drinking and low‐alcohol drinking rat lines, and suggest that impulsivity is a heritable difference that precedes alcoholism.     

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.     

Acoustic startle at baseline and during acute alcohol withdrawal in replicate mouse lines selectively bred for high or low alcohol preference

BACKGROUND: Previous data in both rat and mouse genetic models suggest that there is a genetic relationship between acute alcohol withdrawal responses and innate alcohol drinking behavior. The purpose of the present study was to examine whether acute alcohol withdrawal responses, as measured by acoustic startle and prepulse inhibition (PPI) of acoustic startle, may be genetically related to innate differences in alcohol preference in 2 mouse lines selectively bred for high (HAP1 and HAP2) or low (LAP1 and LAP2) alcohol preference. Line differences in startle responses at baseline, prior to alcohol or saline treatment, were also measured. METHODS: Alcohol-naive, male and female HAP1 (n = 35) and LAP1 (n = 32) and HAP2 (n = 43) and LAP2 (n = 40) mice were tested under baseline conditions and during withdrawal from a single injection of 4.0 g/kg alcohol or equal volume of saline at 4, 8, and 12 hours post-injection. RESULTS: On most trial types, baseline startle responses and PPI were greater in both HAP lines than in both LAP lines, and startle responses were greater in males than in females. During acute alcohol withdrawal, both male LAP lines, and LAP1 females, showed reduced startle responses at the 4-hour time point during acute alcohol withdrawal. In contrast, both HAP1 males and females showed a trend toward enhanced startle at 4 hours in withdrawal. No clear differences in PPI during withdrawal were evident. CONCLUSIONS: These findings indicate good evidence for a genetic relationship between greater baseline acoustic startle responses and PPI and high alcohol preference. Modest support for a genetic correlation between low alcohol preference and reduced startle responses at 4 hours in withdrawal was found in male mice. The suppression in acoustic startle during acute alcohol withdrawal in male LAP lines but not in male HAP lines suggests that a genetic propensity toward low alcohol preference may be related to greater sensitivity to alcohol as measured by acoustic startle responses during acute alcohol withdrawal.     

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).     

Age- and sex-dependent effects of footshock stress on subsequent alcohol drinking and acoustic startle behavior in mice selectively bred for high-alcohol preference

Background: Exposure to stress during adolescence is known to be a risk factor for alcohol‐use and anxiety disorders. This study examined the effects of footshock stress during adolescence on subsequent alcohol drinking in male and female mice selectively bred for high‐alcohol preference (HAP1 lines). Acoustic startle responses and prepulse inhibition (PPI) were also assessed in the absence of, and immediately following, subsequent footshock stress exposures to determine whether a prior history of footshock stress during adolescence would produce enduring effects on anxiety‐related behavior and sensorimotor gating. Methods: Alcohol‐naïve, adolescent (male, n = 27; female, n = 23) and adult (male, n = 30; female, n = 30) HAP1 mice were randomly assigned to a stress or no stress group. The study consisted of 5 phases: (1) 10 consecutive days of exposure to a 30‐minute footshock session, (2) 1 startle test, (3) one 30‐minute footshock session immediately followed by 1 startle test, (4) 30 days of free‐choice alcohol consumption, and (5) one 30‐minute footshock session immediately followed by 1 startle test. Results: Footshock stress exposure during adolescence, but not adulthood, robustly increased alcohol drinking behavior in both male and female HAP1 mice. Before alcohol drinking, females in both the adolescent and adult stress groups showed greater startle in phases 2 and 3; whereas males in the adolescent stress group showed greater startle only in phase 3. After alcohol drinking, in phase 5, enhanced startle was no longer apparent in any stress group. Males in the adult stress group showed reduced startle in phases 2 and 5. PPI was generally unchanged, except that males in the adolescent stress group showed increased PPI in phase 3 and females in the adolescent stress group showed decreased PPI in phase 5. Conclusions: Adolescent HAP1 mice appear to be more vulnerable to the effects of footshock stress than adult mice, as manifested by increased alcohol drinking and anxiety‐related behavior in adulthood. These results in mice suggest that stress exposure during adolescence may increase the risk for developing an alcohol‐use and/or anxiety disorder in individuals with a genetic predisposition toward high alcohol 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.     

Effects of ethanol exposure on subsequent acquisition and extinction of ethanol self-administration and expression of alcohol-seeking behavior in adult alcohol-preferring (P) rats: I. Periadolescent exposure

Background The current study examined the effects of ethanol (EtOH) drinking during periadolescence on the subsequent acquisition and extinction of operant self‐administration of EtOH and expression of alcohol‐seeking behavior in adult alcohol‐preferring (P) rats to test the hypothesis that alcohol drinking during periadolescence produces enduring alterations that enhance the reinforcing properties of EtOH. Methods Periadolescent female P rats were given 24 hr free‐choice access to 15% (v/v) EtOH starting at postnatal day (PND) 30 and ending on PND 60 or were similarly housed and received water only. On PND 75, without any prior training, periadolescent alcohol‐drinking and periadolescent alcohol‐naïve rats were placed in standard two‐lever (15% EtOH and water) chambers to examine acquisition of EtOH self‐administration with a fixed ratio (FR) 1 schedule of reinforcement. After the acquisition phase and after stable responding was established on an FR5 for EtOH and FR1 for water, P rats underwent extinction training for both EtOH and water rewards. After extinction training and a 2 week home cage period, rats were returned to the operant chambers in the absence of reward for seven consecutive sessions (Pavlovian spontaneous recovery). After this testing period, animals were maintained in their home cage for a week before being returned to the operant chambers and allowed to respond for EtOH and water (reacquisition). Results Compared with periadolescent alcohol‐naïve rats, periadolescent alcohol‐drinking rats acquired EtOH responding sooner (i.e., in the first acquisition session), displayed a greater resistance to extinguish EtOH responding (i.e., higher levels of responding in sessions 4–6), had higher responding for more sessions on the EtOH lever in the absence of reward after a prolonged home cage rest period, and had a more prolonged elevated level of EtOH responding during reacquisition (four sessions versus one session). Conclusions Overall, the results suggest that periadolescent EtOH drinking by P rats produced long‐lasting alterations in the reinforcing effects of alcohol, which increased the likelihood that alcohol drinking would be initiated in adulthood, decreased the likelihood that once adult alcohol drinking began it could be extinguished easily, and increased the potential for relapse.     

Genetic Selection for High and Low Alcohol Consumption in a Limited-Access Paradigm

BACKGROUND: Several rat lines have been bred for their differences in alcohol consumption based on a continuous-access paradigm in which alcohol solution is available 24 hr/day. The limited-access paradigm (LAP), in which access to alcohol solution is restricted to a short period per day, however, has been used extensively to investigate the neurochemical mechanisms underlying alcohol consumption. There is evidence of possible differences in genetic determination of alcohol drinking in a continuous- versus limited-access condition. For these reasons, selective breeding for high- and low-alcohol consumption (HARF and LARF, respectively) based on a LAP was conducted. METHODS: N/Nih rats were used as the breeding stock. A within-family breeding procedure was used to develop HARF and LARF lines with 10 families per line. Access to alcohol solution was restricted to 20 min/day. Alcohol was provided as 3%, 6% and 12% w/v solutions. Average intake of alcohol during the 12% phase was used as the selection criterion. Inbreeding began in the seventh generation. RESULTS: After the sixth generation of selection, rats from the HARF line consumed an average of 1.2 g/kg, whereas rats from the LARF line consumed an average of 0.6 g/kg of alcohol during the 20-min access period. Alcohol consumption remained stable over the next eight generations of inbreeding. In the continuous-access-drinking paradigm, the HARF and LARF rats consumed an average of 5.5 to 7.0 g/kg and 1.0 to 2.0 g/kg of alcohol per day respectively. An estimated heritability of 0.25 was obtained. CONCLUSIONS: These findings indicate that alcohol drinking in the LAP is influenced by genetic factors. Differences in alcohol drinking in the LAP also generalize to continuous access drinking. These rat lines will be very useful for investigations into the genetic and neurochemical mechanisms underlying alcohol drinking.     

Induction and maintenance of ethanol self-administration in cynomolgus monkeys (Macaca fascicularis): long-term characterization of sex and individual differences

BACKGROUND: Investigations of oral ethanol self-administration in nonhuman primates have revealed important parallels with human alcohol use and abuse, yet many fundamental questions concerning the individual risk to, and the biological basis of, excessive ethanol consumption remain unanswered. Moreover, many conditions of access to ethanol in nonhuman primate research are largely unexplored. This set of experiments extends within- and across-session exposure to ethanol to more fully characterize individual differences in oral ethanol self-administration. METHODS: Eight male and eight female adult cynomolgus monkeys (Macaca fascicularis) were exposed to daily oral ethanol self-administration sessions for approximately 9 months. During the first 3 months, a fixed-time (FT) schedule of food delivery was used to induce the consumption of an allotted dose of ethanol in 16-hr sessions. Subsequently, the FT schedule was suspended, and ethanol was available ad libitum for 6 months in 16- or 22-hr sessions. RESULTS: Cynomolgus monkeys varied greatly in their propensity to self-administer ethanol, with sex and individual differences apparent within 10 days of ethanol exposure. Over the last 3 months of ethanol access, individual average ethanol intakes ranged from 0.6 to 4.0 g/kg/day, resulting in blood ethanol concentrations from 5 to 235 mg/dl. Males drank approximately 1.5-fold more than females. In addition, heavy-, moderate-, and light-drinking phenotypes were identified by using daily ethanol intake and the percentage of daily calories obtained from ethanol as criteria. CONCLUSIONS: Cynomolgus monkeys displayed a wide intersubject range of oral ethanol self-administration with a procedure that used a uniform and prolonged induction that restricted early exposure to ethanol and subsequently allowed unlimited access to ethanol. There were sex and stable individual differences in the propensity of monkeys to consume ethanol, indicating that this species will be important in characterizing risk factors associated with heavy-drinking phenotypes.     

Time Course of Acamprosate Action on Operant Ethanol Self-Administration after Ethanol Deprivation

The effects of the new alcohol anticraving compound acamprosate on the alcohol deprivation effect were tested in an operant two-lever free choice paradigm with concurrent water. Two groups of rats were tested after long-term voluntary ethanol self-administration: the "continuous access" group consisting of animals that had continuous access to ethanol before operant testing; and the "limited access" group that was tested only after ethanol deprivation. The limited access group exhibited a strong alcohol deprivation effect with immediate high ethanol consumption and preference. Acamprosate (100, 200, or 400 mg/kg) dose-dependently reduced lever pressing for ethanol and, accordingly, ethanol consumption in both groups in a 23-hr session. The consumption-reducing effect was still evident at the end of the session. Ethanol preference was dose-dependently reduced during the first hour of the session, but returned to basal levels before the end of the 23-hr session in both groups. Thus, the time course of preference reduction was not identical with that of the reduction of ethanol consumption. Surprisingly, preference reduction was observed only after a considerable amount of ethanol had been consumed. These results suggest that the specific effect of preference reduction depended on the simultaneous presence of sufficient levels of acamprosate and ethanol, and that the longer-lasting reduction of ethanol consumption was the consequence of this experience.     

Effects of Taste Aversion Training on the Acquisition of Alcohol Drinking in Adolescent P and HAD Rat Lines

Early alcohol drinking has been hypothesized to cause alcohol-related problems in adulthood. In addition, a potential role for genetic factors exist in the etiology of some types of alcoholism. The objective of the present study was to determine if taste aversion training to ethanol during adolescence in previously ethanol-naive, alcohol-preferring P and high-alcohol drinking HAD-1 lines of rats would retard or prevent the onset of high alcohol drinking. Taste aversion training began at 30 days of age. Male and female rat pups were fluid-deprived for 24 hr before 30 min access to a 10% (v/v) ethanol solution, followed by an intraperitoneal injection of either saline or 0.15 M LiCl (10 ml/kg). A total of five training sessions were administered every other day with unrestricted access to water on intervening training days. Twenty-four hours after the last training trial, rats were given continuous free-choice between water and 10% ethanol for 4 weeks with food available ad libitum. There were no obvious gender or line differences to the effects of taste aversion training. All LiCl-treated subjects avoided the usually preferred ethanol solution for the entire 4-week test period, whereas saline-treated rats steadily increased their alcohol intake to over 6.0 g/kg/day by week 4. Rats in the saline and LiCl-treated groups gained weight at comparable rates, and the groups did not differ in total fluid intake. The findings demonstrate that early environmental intervention can prevent the onset of high alcohol drinking in the selectively bred alcohol-preferring P and high-alcohol drinking HAD-1 lines of rats.     

Genomewide SNP Screen to Detect Quantitative Trait Loci for Alcohol Preference in the High Alcohol Preferring and Low Alcohol Preferring Mice

Background:  The high and low alcohol preferring (HAP1 and LAP1) mouse lines were selectively bred for differences in alcohol intake. The HAP1 and LAP1 mice are essentially noninbred lines that originated from the outbred colony of HS/Ibg mice, a heterogeneous stock developed from intercrossing 8 inbred strains of mice.
Methods:  A total of 867 informative SNPs were genotyped in 989 HAP1 × LAP1 F2, 68 F1s, 14 parents (6 LAP1, 8 HAP1), as well as the 8 inbred strains of mice crossed to generate the HS/Ibg colony. Multipoint genome wide analyses were performed to simultaneously detect linked QTLs and also fine map these regions using the ancestral haplotypes.
Results:  QTL analysis detected significant evidence of association on 4 chromosomes: 1, 3, 5, and 9. The region on chromosome 9 was previously found linked in a subset of these F2 animals using a whole genome microsatellite screen.
Conclusions:  We have detected strong evidence of association to multiple chromosomal regions in the mouse. Several of these regions include candidate genes previously associated with alcohol dependence in humans or other animal models.     

Thyrotropin Releasing Hormone Analog TA-0910 Suppresses Alcohol Intake in Alcohol Drinking African Green Monkeys

In previous studies, we found that single injections of the thyrotropin-releasing hormone analog TA-0910 dose-dependently reduced alcohol intake and preference in alcohol-preferring (P) and Fawn-Hooded (FH) rats over a 24-hr period of continuous access to alcohol and water. However, several consecutive daily injections of TA-0910 resulted in the development of tolerance to these effects. In the present study, we found that in a 5-hr limited-access schedule in which monkeys could select an aqueous alcohol solution (7.5% v/v) or tap water, single doses of TA-0910 (0.0625, 0.125, 0.25, 0.5, and 0.75 mg/kg), similar to those found effective in P and FH rats, reduced consumption of alcohol. In this protocol, tolerance to the attenuating effects of TA-0910 on alcohol intake was not evident after five consecutive once-daily doses of 0.5 mg/kg. Furthermore, it was shown that a single dose of 0.75 mg/kg TA-0910 did not significantly influence 24-hr water intake when water was the only available fluid, but did reduce the intake of a preferred solution of saccharin. These findings suggest that activation of brain thyrotropin-releasing hormone systems reduces alcohol intake in primates and that tolerance to this effect is not evident within 5 days under a limited access schedule.