Effects of CB1 cannabinoid receptor blockade on ethanol preference after chronic ethanol administration

BACKGROUND: Chronic ethanol administration results in neurobiological alterations similar to those observed after chronic cannabinoid exposure. The purpose of this study was to investigate alcohol drinking and the withdrawal responses after pulmonary chronic alcoholization with intraperitoneal or oral administration of a cannabinoid CB1 receptor antagonist. METHODS: The cannabinoid receptor antagonist SR141716A, 1, 3 or 10 mg/kg/day intraperitoneally or orally, was administered to Wistar rats either during a 30-day chronic ethanol exposure or at the cessation of this procedure. Motility was recorded during 18 hr after the cessation of chronic alcoholization just before the beginning of the free-choice paradigm (water versus alcohol 10% v/v). RESULTS: A significant increase in ethanol preference was observed during the free-choice paradigm after chronic alcoholization with concurrent SR141716A administration (3 or 10 mg/kg/day). A significant decrease in withdrawal motility after administration of SR141716A was observed with only the highest dose (10 mg/kg/day). The administration of SR141716A, 3 or 10 mg/kg/day, after chronic pulmonary alcoholization significantly decreased the preference for alcohol. Finally, a significant decrease in ethanol preference was seen during the free-choice paradigm of nonalcoholized rats treated with SR141716A, 3 or 10 mg/kg/day, during 30 days before the free-choice paradigm. CONCLUSIONS: The concurrent administration of the CB1 antagonist together with the chronic alcoholization increases the preference for ethanol. Also, the administration of the CB1 antagonist after the chronic alcoholization or at the time of withdrawal drastically diminishes the ethanol preference.     

Blockade of the leptin-sensitive pathway markedly reduces alcohol consumption in mice

BACKGROUND: The neuropeptide leptin links adipose stores with hypothalamic centers and serves as an endocrine signal involved in the regulation of appetite (and possibly in the endorphinergic modulation of the drug reward system). Increased plasma leptin has been observed at the onset of alcohol withdrawal in humans, and ethanol consumption after withdrawal was increased by injection of leptin in mice. We addressed the role of leptin in alcohol-related behaviors by studying ethanol consumption in two strains of spontaneously mutant mice that lack leptin (ob/ob) or the leptin receptor (db/db). METHODS: Two strains of mutant leptin-deficient (ob/ob) or leptin-resistant (db/db) mice were tested in a two-bottle-choice paradigm and were compared with wild-type (C57BL/6 inbred strain) mice. The effects of leptin injection on voluntary ethanol intake have been investigated in ob/ob and C57BL/6 mice. RESULTS: Males and females of both mutant strains showed a significantly lower preference for alcohol in a two-bottle-choice paradigm compared with wild-type mice. Male ob/ob mice demonstrated slightly higher avoidance of bitter taste, and females of the both mutant strains showed a reduced preference for saccharin solutions. Administration of leptin (1 mg/kg intraperitoneally, daily for 8 days) altered body weight but failed to increase the preference for ethanol in ob/ob mice; i.e., we could not correct the effects of leptin deficiency on alcohol consumption by the injection of leptin. Also, there were no differences between the effects of leptin (1 mg/kg intraperitoneally, daily for 8 days) and saline injections on alcohol consumption in C57BL/6 mice. CONCLUSIONS: These data show that blockade of the leptin pathway markedly decreases the preference for alcohol intake, but this decrease may be the result of compensatory or developmental changes in other systems rather than a more direct effect of leptin on alcohol consumption.     

Voluntary ethanol drinking during the first three postnatal weeks in lines of rats selectively bred for divergent ethanol preference

BACKGROUND: Using a procedure first developed by Hall (1979), we examined ethanol self-administration in preweanling pups from Wistar rats and in lines of rats selectively bred for divergent ethanol preference (alcohol-preferring P, alcohol-nonpreferring NP, high-alcohol-drinking HAD-1 and -2, and low-alcohol-drinking LAD-2) to determine if factors contributing to high and low alcohol intakes are present early in development. METHODS: From postnatal days 5 to 20, nondeprived male and female rat pups received 30 min daily access to either water or a 15% (v/v) ethanol solution. In each daily session, pups were placed in a heated chamber containing Kimwipes soaked with a water or ethanol solution. Pups were weighed before and after each session, and intake levels were calculated as a percentage of body weight change. RESULTS: Similar to previous reports, Wistar pups exhibited over a 2-fold higher level of ethanol ingestion than water on postnatal days 9 through 14, with ethanol intakes approaching 3 g/kg body weight. When the drinking patterns of the selected lines were examined, only the HAD replicate lines showed a comparable preference for ethanol versus water during the preweanling period. The ethanol consumption of P, NP, and LAD lines was not consistently distinguishable from water intake levels. To reveal whether early ethanol exposure would influence later ethanol drinking behavior, a subset of HAD and LAD rat pups received free-choice ethanol access upon weaning. Although the divergent ethanol preference between high- and low-alcohol-drinking lines was evident within the first 4 days of access (>4 g/kg/day for HAD; <2 g/kg/day for LAD), preweanling ethanol exposure did not alter the acquisition or maintenance of ethanol drinking in either line. CONCLUSIONS: Overall, these results suggest that (a) the enhanced ethanol ingestion observed during the middle portion of the preweanling period is a robust phenomenon and generalizes across nonselected strains of rats, (b) selective breeding for divergent alcohol preference has arrested this age-specific effect in all but the HAD lines of rats, and (c) early ethanol exposure does not alter genetic dispositions for later high or low alcohol preference.     

Chronic daily ethanol and withdrawal: 3. Forebrain pro-opiomelanocortin gene expression and implications for dependence, relapse, and deprivation effect

BACKGROUND: Although forebrain pro-opiomelanocortin (POMC)-producing neurons seem to mediate or modulate many responses to ethanol consumption, changes in activity of this opiomelanocortinergic system in response to chronic ethanol consumption, withdrawal, and subsequent abstinence remain unresolved. METHODS: We investigated the effects of chronic daily ethanol consumption, withdrawal, and subsequent abstinence on adult male Sprague-Dawley rat forebrain opiomelanocortinergic activity as reflected by changes in hypothalamic POMC messenger RNA (mRNA) content by using a well characterized liquid diet model that we have previously demonstrated to accurately simulate not only daily oral ethanol consumption quantity and pattern, but also both neuroendocrine and behavioral changes characteristic of actively drinking and subsequently abstinent alcoholics. RESULTS: After 7 weeks of daily ethanol consumption at night and withdrawal during the day, evening mediobasal hypothalamus POMC mRNA concentrations were suppressed versus both ad libitum-fed and pair-fed controls. Morning POMC mRNA concentrations were also suppressed versus ad libitum-fed controls and tended to be decreased versus pair-fed controls. Three weeks after gradual removal of ethanol from the diet, mediobasal hypothalamus POMC mRNA concentrations were increased relative to ad libitum-fed and pair-fed controls. Plasma concentrations of corticosterone, testosterone, and leptin were also altered by the daily ethanol/withdrawal treatment and by subsequent abstinence. CONCLUSIONS: Because each of these hormones has been demonstrated to modify forebrain POMC gene expression under some conditions, the overall changes in forebrain opiomelanocortinergic regulation in response to chronic daily ethanol/withdrawal and subsequent abstinence probably reflect, at least in part, regulation by multiple endocrine mechanisms, together with responses to stress, development of tolerance during chronic daily ethanol consumption, and rebound of function after termination of this consumption. Overall, the demonstrated changes in forebrain POMC gene expression are consistent with significant roles for forebrain opiomelanocortinergic regulation in mediating alcohol dependence, propensity to relapse, and the alcohol deprivation effect.     

Effects of topiramate on ethanol and saccharin consumption and preferences in C57BL/6J mice

BACKGROUND: Topiramate has recently been found to be more effective than placebo as an adjunct treatment for alcohol dependence, but it has not yet been investigated in animal models of ethanol consumption. The current experiment examined the effects of topiramate on ethanol drinking in mice using a continuous access, two-bottle choice procedure. METHOD: C57BL/6J male mice were offered a 10% v/v ethanol solution versus tap water over 4 consecutive days per week. Mice were assigned to topiramate (1-50 mg/kg) or saline groups and received injections before the beginning of the dark phase of the light cycle. Topiramate dose increased over 5 successive weeks (1, 5, 10, 25, and 50 mg/kg). Fluid intake was measured 2, 4, and 23 hr after injection. Body weight and food intake were measured at the time of injection. In a second phase, mice were offered saccharin solutions (0.2 and 2.5% w/v) versus tap water after topiramate (50 mg/kg) or saline injections. RESULTS: Results revealed that high topiramate doses (25 and 50 mg/kg) increased water intake and decreased ethanol preference. Compared with saline controls, topiramate produced dose-dependent, bidirectional effects on ethanol dose, with 25 mg/kg of topiramate increasing ethanol dose at 4 and 23 hr after injection but 50 mg/kg topiramate decreasing ethanol dose at 2 hr after injection. During saccharin exposure, topiramate decreased saccharin preference (for 2.5% w/v saccharin solution) and marginally increased water intake but did not directly alter intake of the saccharin solutions. Topiramate had no effects on body weight or daily food intakes. CONCLUSIONS: Topiramate reduced ethanol preference in C57BL/6J mice, but this effect was primarily attributable to elevated water intake. Topiramate also reduced saccharin preference, likely through marginally significant increases in water intake. Increases in water intake and bidirectional effects of topiramate on ethanol dose complicate conclusions with regard to the effects of topiramate on ethanol reward.     

P rats develop physical dependence on alcohol via voluntary drinking: changes in seizure thresholds, anxiety, and patterns of alcohol drinking

BACKGROUND: It has been proposed that the alcohol-preferring P rat meets many of the criteria for an animal model of alcoholism. However, the development of alcohol dependence has not been explored in rats that self-administer ethanol for less than 15-20 weeks. The present study investigated the development of physical dependence upon alcohol after 2-6 weeks of voluntary alcohol intake. Changes in bicuculline-induced seizure thresholds, microstructure of alcohol drinking, and anxiety-related behavior were used as indices of alcohol dependence. In addition, we evaluated the microstructure of alcohol drinking associated with the development of physical dependence upon alcohol. METHODS: Alcohol (10% ethanol solution) was measured in graduated drinking tubes with both alcohol and water available continuously. Microstructure of alcohol intake was monitored by a computerized drinkometer. Physical dependence upon alcohol was determined by measuring bicuculline-induced seizure thresholds after alcohol withdrawal. Anxiety-related behavior of P rats after alcohol withdrawal was determined by the social interaction and elevated plus maze tests. RESULTS: Initial alcohol intake in the alcohol-preferring P rat was relatively modest (3.9 +/- 0.4 g/kg/day). Four days of forced alcohol exposure (initiation) followed by 6 weeks of voluntary drinking resulted in an increase of alcohol intake to 5.5 +/- 0.2 g/kg/day. Ethanol self-administration for 6 weeks, but not for 2 or 4 weeks, produced a significant reduction (30%; p < 0.05) in bicuculline-induced seizure thresholds during alcohol withdrawal. Alterations in the microstructure of alcohol intake (i.e., 90% increase in the size of alcohol drinking bouts compared to the baseline [p < 0.001] with no change in bout frequency) were associated with the development of alcohol dependence. Termination of alcohol intake after 6 weeks of voluntary alcohol consumption resulted in increased anxiety according to both the social interaction and elevated plus maze tests. CONCLUSIONS: The results of this study indicate that 6 weeks of voluntary alcohol intake are sufficient for the development of physical dependence upon alcohol in the alcohol-preferring P rats as measured by susceptibility to bicuculline-induced seizures. This time is much shorter than the 15-20 weeks reported earlier. Development of physical dependence to alcohol was associated with an increase in daily alcohol intake (40% over the baseline), an increase in alcohol intake during each drinking bout (90% over the baseline), and elevated anxiety during alcohol withdrawal.     

Additive Reduction of Alcohol Drinking by 5-HT1A Antagonist WAY 100635 and Serotonin Uptake Blocker Fluoxetine in Alcohol-Preferring P Rats

We found previously that alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons and fibers in key brain regions than alcoholnonpreferring (NP) rats. Because 5-HT uptake blockers increase synaptic 5-HT content and 5-HT_1A receptor antagonists increase 5-HT release by disinhibiting 5-HT autoinnervation, in the present study, our intent was to determine whether increased synaptic 5-HT content and/or 5-HT release in P rats would effectively reduce alcohol consumption. In experiment 1, the 5-HT antagonist WAY 100635 (WAY) was tested on adult female P rats maintained on 24-hr free-choice access to ethanol (10% v/v) and water. Twice daily doses of WAY (0.05, 0.1, 0.5, and 1.0 mg/kg, subcutaneously) were administered to each rat in a counterbalanced order. Baseline ethanol intake, derived from the mean ethanol intakes of the three previous non-drug days, was approximately 8 g/kg/day. Results indicated that 0.05,0.1, and 0.5 mg/kg doses of WAY reduced 24-hr ethanol drinking by 25-30% ( p < 0.01) without affecting 24-hr water intake or body weight In the second experiment, the effects of WAY (0.5 mg/kg), fluoxetine (1.0 mg/kg), or a combination of both were tested in another group of female P rats. WAY and fluoxetine, each alone, reduced ethanol drinking by around 20% and, when combined, decreased ethanol intake by 50%, whereas the body weight and the total fluid intake were not significantly affected. Taken together, these results indicate that both fluoxetine and WAY preferentially reduce ethanol drinking in the P line of rats and, when administered together, reduce ethanol intake in an additive manner. It is proposed that coadministration of these two compounds with distinct mechanisms of action may be a new strategy for reducing alcohol intake.     

Effect of ethanol on hypothalamic opioid peptides, enkephalin, and dynorphin: relationship with circulating triglycerides

BACKGROUND: Recent evidence has demonstrated that ethanol intake can stimulate the expression and production of the feeding-stimulatory peptide, galanin (GAL), in the hypothalamic paraventricular nucleus (PVN), and that PVN injection of this peptide, in turn, can increase the consumption of ethanol. To test the hypothesis that other feeding-related systems are involved in ethanol intake, this study examined the effect of ethanol on the hypothalamic opioid peptides, enkephalin (ENK), and dynorphin (DYN). METHOD: Adult, male Sprague-Dawley rats were trained to voluntarily drink increasing concentrations of ethanol, up to 9% v/v, on a 12-hour access schedule or were given a single injection of ethanol (10% v/v) versus saline vehicle. The effect of ethanol on GAL, ENK, and DYN mRNA was measured using real-time quantitative polymerase chain reaction and radiolabeled in situ hybridization, while radioimmunoassay was used to measure peptide levels. In addition to blood alcohol, circulating levels of triglycerides (TG), leptin, and insulin were also measured. RESULTS: The data demonstrated that: (1) rats voluntarily drinking 9% v/v ethanol (approximately 2.0 g/kg/d) show a significant increase in GAL, ENK, and DYN mRNA in the PVN compared with water-drinking rats; (2) voluntary consumption of ethanol also increases peptide levels of ENK and DYN in the PVN; (3) acute injection of 10% ethanol (1.0 g/kg of 10% v/v) similarly increases the expression of GAL, ENK, and DYN in the PVN; and (4) ethanol consumption and injection, while having little effect on leptin and insulin, consistently increase circulating levels of TG as well as alcohol, both of which are strongly, positively correlated with peptide expression in the PVN. CONCLUSIONS: These findings, together with published studies, suggest a possible role for hypothalamic opioid peptides in the drinking of ethanol. Based on evidence that dietary fat and lipid injections stimulate the PVN peptides and injection of the opiates and GAL increase ethanol intake, it is proposed that both TG and alcohol in the circulation, which are elevated by the ingestion or injection of ethanol, are involved in stimulating these peptides in the PVN, which in turn promote further consumption of ethanol.     

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.     

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.     

A limited access mouse model of prenatal alcohol exposure that produces long-lasting deficits in hippocampal-dependent learning and memory

Background: It has been estimated that approximately 12% of women consume alcohol at some time during their pregnancy, and as many as 5% of children born in the United States are impacted by prenatal alcohol exposure (PAE). The range of physical, behavioral, emotional, and social dysfunctions that are associated with PAE are collectively termed fetal alcohol spectrum disorder (FASD). Methods: Using a saccharin‐sweetened ethanol solution, we developed a limited access model of PAE. C57BL/6J mice were provided access to a solution of either 10% (w/v) ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin (control) for 4 h/d. After establishing consistent drinking, mice were mated and continued drinking during gestation. Following parturition, solutions were decreased to 0% in a stepwise fashion over a period of 6 days. Characterization of the model included measurements of maternal consumption patterns, blood ethanol levels, litter size, pup weight, maternal care, and the effects of PAE on fear‐conditioned and spatial learning, and locomotor activity. Results: Mothers had mean daily ethanol intake of 7.17 ± 0.17 g ethanol/kg body weight per day, with average blood ethanol concentrations of 68.5 ± 9.2 mg/dl after 2 hours of drinking and 88.3 ± 11.5 mg/dl after 4 hours of drinking. Food and water consumption, maternal weight gain, litter size, pup weight, pup retrieval times, and time on nest did not differ between the alcohol‐exposed and control animals. Compared with control offspring, mice that were exposed to ethanol prenatally displayed no difference in spontaneous locomotor activity but demonstrated learning deficits in 3 hippocampal‐dependent tasks: delay fear conditioning, trace fear conditioning, and the delay nonmatch to place radial‐arm maze task. Conclusions: These results indicate that this model appropriately mimics the human condition of PAE and will be a useful tool in studying the learning deficits seen in FASD.     

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.     

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

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

Free-choice ethanol consumption under the influence of GABAergic drugs in rats

BACKGROUND: Neurobiological mechanisms leading from controlled alcohol consumption to addiction are poorly understood. Among multiple neurotransmitters gamma-amino-butyric acid (GABA) is suggested to play a role. The present investigation studied effects of drugs interacting with the GABAergic system on the motivation of ethanol consumption. METHODS: Fifty male PVG/OlaHsd rats were analyzed for free-choice ethanol drinking behavior without and with pre-exposure to drugs acting on the GABAergic system. For pretreatment, animals received the benzodiazepine agonists or antagonists diazepam, flumazenil, or Ro15-4513, or the GABA uptake inhibitor tiagabine via the drinking water for 4 weeks (day -21 until day 7). On day 0, two bottles containing 5% and 12% ethanol were added. On day 7, GABAergic drug exposure was discontinued and drug solutions were replaced by water. Between days 8 and 35, three alcohol deprivation periods of 1 to 3 days were randomly implemented. RESULTS: The animals ingested substantial amounts of ethanol that was differentially affected by the GABAergic drugs. Diazepam increased and flumazenil decreased ethanol consumption significantly by about 30%. Without GABAergic drug pretreatment, a significant alcohol deprivation effect indicated by enhanced ethanol consumption after re-exposure to alcohol was observed after the third deprivation phase. The deprivation effect was prevented by pretreatment with diazepam or flumazenil, unaffected by Ro15-4513, and advanced by tiagabine. CONCLUSIONS: Modulation of GABAergic neurotransmission affects subsequent ethanol consumption and deprivation effects. Because enhancing of the GABAergic tone by the GABA uptake inhibitor tiagabine or by the benzodiazepine diazepam had different behavioral consequences, it seemed likely that the two drugs induce differential adaptive changes leading to distinct alterations in the motivation to consume alcohol.     

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).     

Alcohol consumption promotes body weight loss in melanoma-bearing mice

BACKGROUND: Alcohol consumption is an important risk factor for cancer. Little is known about its effects on cancer progression. Previously, we showed that high ethanol consumption inhibited metastasis of B16BL6 melanoma-bearing mice without affecting primary tumor growth. On the other hand, ethanol-consuming tumor-bearing (TE) mice exhibited decreased survival and decreased body weight as compared to water-drinking, tumor-bearing (TW) mice. The focus of this study was to determine how alcohol promotes weight loss in melanoma-bearing mice. METHODS: Female, C57BL/6 mice were given water or 20% w/v ethanol in the drinking water for 3 weeks to 6 months before subcutaneous inoculation of 1 x 10(6) B16BL6 melanoma cells. Mice continued to receive the same fluids. Biochemical parameters were evaluated at various times after tumor inoculation. Body weight, water content, tumor weight and carcass fat content were determined at necropsy. RESULTS: TW mice elicted a modest weight loss. This response was magnified 2-fold by alcohol consumption. The weight loss in TE mice is not caused by dehydration, decreased energy intake, or loss of skeletal muscle mass. It resulted specifically from loss in body fat. Other alterations associated with the fat loss in TE mice were: (1) decreased glucose, (2) elevated fatty acids, (3) elevated beta-hydroxybutyrate, (4) elevated glucagon, and (5) increased leptin levels in plasma. Body temperature decreased about 2.9 degrees C in TE mice. Metabolic rate increased in TW mice. The fat loss due to alcohol consumption in tumor-bearing mice was not due to increased metabolic rate. CONCLUSIONS: The response elicited by alcohol consumption in tumor-bearing mice is complex and associated with alterations in metabolism and hormones. These findings suggest that alcohol abuse could be a risk factor for cancer patients because it invokes a strong depletion of body fat. This could facilitate wasting and shorten survival time.     

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.     

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.     

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.     

A mouse model of prenatal ethanol exposure using a voluntary drinking paradigm

BACKGROUND: The incidence of fetal alcohol spectrum disorders is estimated to be as high as 1 in 100 births. Efforts to better understand the basis of prenatal ethanol-induced impairments in brain functioning, and the mechanisms by which ethanol produces these defects, will rely on the use of animal models of fetal alcohol exposure (FAE). METHODS: Using a saccharin-sweetened alcohol solution, we developed a free-choice, moderate alcohol access model of prenatal alcohol exposure. Stable drinking of a saccharin solution (0.066%) was established in female mice. Ethanol then was added to the saccharin in increasing concentrations (2%, 5%, 10% w/v) every 2 days. Water was always available, and mice consumed standard pellet chow. Control mice drank saccharin solution without ethanol. After a stable baseline of ethanol consumption (14 g/kg/day) was obtained, females were impregnated. Ethanol consumption continued throughout pregnancy and then was decreased to 0% in a step-wise fashion over a period of 6 days after pups were delivered. Characterization of the model included measurements of maternal drinking patterns, blood alcohol levels, food consumption, litter size, pup weight, pup retrieval times for the dams, and effects of FAE on performance in fear-conditioned learning and novelty exploration. RESULTS: Maternal food consumption, maternal care, and litter size and number were all found to be similar for the alcohol-exposed and saccharin control animals. FAE did not alter locomotor activity in an open field but did increase the time spent inspecting a novel object introduced into the open field. FAE mice displayed reduced contextual fear when trained using a delay fear conditioning procedure. CONCLUSIONS: The mouse model should be a useful tool in testing hypotheses about the neural mechanisms underlying the learning deficits present in fetal alcohol spectrum disorders. Moreover, a mouse prenatal ethanol model should increase the opportunity to use the power of genetically defined and genetically altered mouse populations.