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.     

Individual differences in ethanol self-administration following withdrawal are associated with asymmetric changes in dopamine and serotonin in the medial prefrontal cortex and amygdala

BACKGROUND: Ethanol withdrawal alters brain neurochemistry, causes asymmetric activation of neurons in the medial prefrontal cortex (mPFC) and amygdala (AMY), and increases ethanol craving and drinking. Rats with intrinsic rightward-turning preferences drink more ethanol than those with left or no preferences; they also exhibit an ethanol-induced neurochemical activation that favors the right side of the mPFC. Our experiments used rats with different turning preferences to assess differences in withdrawal effects on mPFC and AMY neurochemistry as well as ethanol self-administration. METHODS AND RESULTS: Rats with left-turning, right-turning, and nonturning preferences were fed a 6% ethanol-containing liquid diet (WD) or a pair-fed control diet for 14 days. Differences in dopamine (DA), serotonin (5HT), norepinephrine (NE), and metabolite [3,4-dihydroxphenylacetic acid, homovanillic acid (HVA), and 5-hydroxyindoleacetic acid) concentrations were assessed in each side of the mPFC and AMY during acute withdrawal. Similar groups were fed the same diets and tested for consumption of 10% ethanol versus water and 1% sucrose versus water. WD increased HVA/DA in the mPFC and caused depletions of DA and 5HT in the mPFC and 5HT in the AMY. These effects were greater in the right than in the left side of these structures in rats with right-turning preferences. WD reduced ethanol drinking but right turners drank significantly more than left turners on day 2 of testing and drank more on days 2 and 3 than on day 1. No effects were observed on sucrose drinking. Similar groups were also trained to self-administer ethanol using a sucrose-fade sipper tube procedure that separated measures of ethanol seeking (bar pressing) and consumption. Following 14 days of vapor chamber exposure to ethanol, rats of all turning preferences had a lower rate of bar pressing on the first postwithdrawal day and shorter latencies to begin bar pressing on the third withdrawal day versus prewithdrawal baseline. Only right-turning-preference rats consumed more ethanol following withdrawal. CONCLUSIONS: These studies show that individual rats differ in postwithdrawal brain neurochemistry and ethanol consumption and that these differences are associated with differences in functional brain asymmetry.     

Effects of ethanol and ethanol withdrawal on nociception in rats

The effect of acute and chronic administration of ethanol and ethanol withdrawal on a radiant heat tail-flick assay of nociception was examined in rats. Acute administration of ethanol (2.0 g/kg, i.p.) produced peak antinociception (68% of maximum) by 30 min, and effects were gone by 120 min. Cumulative doses of ethanol (0.5-2.0 g/kg, i.p.) produced dose-dependent increases in latencies to 49% of maximum. During chronic administration, a liquid diet containing ethanol (6.5%) was given for 10 days. Tail-flick latencies were measured on day 0 (baseline), day 2, 4, 6, 8, and 10 of chronic ethanol and at 3, 6, 12, and 36 hr after removal of ethanol. To test for behavioral tolerance, both between- and within-group designs were used. In both between- and within-group experiments, the antinociceptive effects of chronic ethanol peaked by day 4 of exposure to the liquid diet, and tolerance developed by day 10. When the liquid diet was removed, hyperalgesia was detected at 6 and 12 hr after withdrawal, and was gone by 36 hr after withdrawal. When cumulative doses of ethanol (0.5-2.0 g/kg) were administered starting 12 hr after withdrawal, ethanol (0.5 g/kg) fully reversed the hyperalgesia induced by ethanol withdrawal, even though this dose was without antinociceptive effect in the absence of withdrawal. Higher doses of ethanol during ethanol withdrawal did not increase tail-flick latencies over baseline. In summary: (1) ethanol produces antinociception when administered acutely or chronically; (2) tolerance to the antinociceptive effects develops during chronic administration; (3) ethanol withdrawal induced hyperalgesia, which was reversed by ethanol; and (4) repeated testing did not produce behavioral tolerance.     

Effects of lorazepam treatment for multiple ethanol withdrawals in mice

Background: Although many alcohol‐dependent patients present with a history of prior detoxifications, the efficacy and safety of pharmacotherapy in the context of multiple ethanol withdrawal experiences have not been extensively studied. The purpose of this study was to evaluate the ability of lorazepam treatment for multiple withdrawals to prevent or blunt the development/expression of sensitized central nervous system hyperexcitability during a subsequent untreated withdrawal episode. A mouse model of withdrawal sensitization involving repeated ethanol withdrawals was used. Methods: Adult male C3H/He mice were exposed to different patterns of chronic ethanol vapor in inhalation chambers. One group received four cycles of 16 hr of ethanol exposure separated by 8‐hr withdrawal periods, another group was tested after a single 16‐hr exposure period, and a final group served as ethanol‐naïve controls. These groups were further divided into lorazepam dosage (0.25–1.0 mg/kg) conditions. Lorazepam was administered 1 hr into each of the first three withdrawal cycles (or equivalent times); no drug injections were given during the final (fourth) withdrawal cycle. The ability of lorazepam treatment to alter development and expression of sensitized handling‐induced convulsions (HIC), as well as changes in pentylenetetrazol seizure threshold dosage during an untreated withdrawal episode, was examined. Separate animals were used to assess the effects of lorazepam treatment on blood ethanol clearance and plasma levels of the benzodiazepine during the test withdrawal cycle. Results: Lorazepam dose‐dependently reduced HIC activity during successive withdrawal cycles, and this resulted in attenuated expression of the sensitized HIC response during the acute phase of a subsequent untreated withdrawal episode. However, HIC activity was exacerbated at later time points during this final test withdrawal in mice that had received lorazepam treatment for earlier withdrawals. A similar pattern of results was obtained for changes in pentylenetetrazol seizure threshold dosage. These results do not seem to be due to pharmacokinetic factors, because peak blood ethanol levels, rate of ethanol elimination, and plasma levels of lorazepam did not significantly differ among groups during the final test withdrawal cycle. Conclusions: Blocking central nervous system hyperexcitability during repeated ethanol withdrawals with lorazepam effectively blunts the development and expression of sensitized seizure activity during the acute phase of a subsequent unmedicated withdrawal episode. At later time points, withdrawal‐related seizure activity was exacerbated, and this is possibly reflective of an interaction between protracted ethanol withdrawal and withdrawal from the benzodiazepine. The clinical implications of these findings suggest that repeated use of benzodiazepines for treatment of multiple ethanol withdrawals may have some initial beneficial effects, but such treatment may also place patients at increased risk of seizures at later time points.     

Effects of Chronic Ethanol Exposure on Oral Self-Administration of Ethanol or Saccharin by Wistar Rats

The study of alcohol abuse traditionally has placed great emphasis on the development of tolerance and dependence as key factors. However, animal models of ethanol self-administration in dependent rats have been difficult to establish, caused in part by ethanol's aversive taste cues and subsequent aversive effects (i.e., “hangover” malaise) that prevent substantial ethanol consumption. In this study, this problem was addressed in animals trained to self-administer ethanol (10% w/v) in a sweetened-solution fading procedure before induction of dependence and repeated exposure to withdrawal. Once stable rates of responding for ethanol were achieved, a palatable liquid diet containing 8.7% (v/v) ethanol was introduced as the sole source of calories and fluid for one group of rats [ethanol diet (ED) group]. A second group of rats received a control diet with sucrose isocalorically substituted for ethanol (CD group). After 14–17 days of liquid diet exposure, the rats were withdrawn once a week for 4 weeks and 8 hr into each withdrawal session were allowed to self-administer ethanol or water for 60 min. As compared with CD rats, ED rats showed significantly greater intake of ethanol, but not water. No significant differences were found when separate groups of ED/CD rats were allowed to self-administer an alternate reinforcer (0.0075% saccharin solution). Rats who consistently had blood alcohol levels (BALs) above 100 mg% at the time of withdrawal sustained high levels of ethanol self-administration throughout the four withdrawal sessions. In contrast, rats who had an average BAL at withdrawal below 100 mg% showed progressive decreases in ethanol self-administration during repeated withdrawal episodes. The results demonstrated that chronic exposure to ethanol and repeated periods of abstinence are accompanied by elevated rates of ethanol intake in certain animals, and the persistence of elevated self-administration behavior of individual rats is predicted by their BAL at the time of withdrawal.     

Accentuated decrease in social interaction in rats subjected to repeated ethanol withdrawals

BACKGROUND: Previous work has shown that repeated withdrawals from chronic ethanol exposure can kindle seizures in rodents. In this article, the effects of a three-cycle model of ethanol exposure and withdrawal on the social interaction test of anxiety are summarized. METHODS: Rats were exposed to ethanol (7% or 4.5%) diets over three periods of 5 days, with 2 days of withdrawal between cycles. Between 5 and 6 hr after the ethanol was removed, pairs of rats were placed in open field chambers for the assessment of social interaction behavior and locomotor activity. RESULTS: After the third cycle of ethanol (7%) presentation, both male and female rats exhibited lower social interaction behavior (more anxiety) and activity than after a single cycle. Rats exposed to a similar amount of ethanol but tested while ethanol was still available did not exhibit a reduction in social interaction. The decrease in social interaction was still present for up to 24 hr but had disappeared by 48 hr after ethanol was withdrawn. When rats were allowed 8 or 16 days to recover from the effects of the three-cycle protocol, a further exposure to 5 days of 7% ethanol diet resulted in a reduction in social interaction on withdrawal similar to that seen from the three-cycle protocol. In contrast, rats exposed continuously to 7% ethanol diet for 15 consecutive days exhibited higher levels of social interaction when maintained on control diet for 8 or 16 days and then reexposed to ethanol. Rats that were exposed to the three-cycle protocol and allowed 32 days to recover before being reexposed to ethanol still had a partial deficit in social interaction. Finally, animals subjected to repeated withdrawals from 4.5% ethanol exhibited a reduction in social interaction without a change in activity after the final withdrawal from ethanol, whereas rats exposed continuously to a 4.5% ethanol diet did not exhibit a reduction in social interaction or activity. Neither blood ethanol concentrations nor changes in body weight could account for these behavioral differences. CONCLUSION: Repeated withdrawal from ethanol can lead to accentuated or more persistent anxiety-like behavior in rats, as indicated by a decrease in social interaction. The withdrawal-induced decrease in locomotor activity is not accentuated by repeated withdrawals. This model of repeated withdrawals from ethanol may prove useful in defining the neurochemical basis of this accentuation.     

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.     

Repeated Ethanol Withdrawal Produces Site-Dependent Increases in EEG Spiking

Recent studies have suggested an important kindling-like exacerbation of ethanol withdrawal symptoms after repeated cycles of ethanol intoxication and withdrawal. Few studies, however, have evaluated the effect of multiple episodes of intoxication and withdrawal on spontaneous EEG activity after cessation of ethanol intake. In this study, electrographic activity in cortical and subcortical structures of male Sprague-Dawley rats was examined after multiple cycles of ethanol intoxication and withdrawal. After surgical implantation of electrodes, animals received repeated cycles of chronic ethanol exposure in vapor inhalation chambers for 10 or 20 days, with 4-day withdrawal periods between each. Upon removal from the inhalation chamber, spontaneous EEG activity was recorded intermittently for 72 hr. These data were then examined for the presence of spikes and sharp waves. Results indicate that the levels of spike and sharp wave activity observed vary with both length of ethanol exposure and with the number of withdrawal cycles, and that these effects varied with neuronal site. Changes in spike and sharp wave activity were first observed within hippocampal areas, with other subcortical and cortical sites showing increased activity after additional ethanol exposure or additional cycles of intoxication and withdrawal. Hippocampal areas CA1 and CA3 differed significantly from one another in their response to chronic ethanol exposure, with area CA1 most affected by changes in amount of ethanol exposure and area CA3 most affected by number of withdrawal cycles. These results indicate an increased severity of the ethanol withdrawal syndrome after repeated ethanol withdrawal episodes and suggest differential, site-specific changes in neuronal excitability.     

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.     

Effects of dexmedetomidine on rat locus coeruleus and ethanol withdrawal symptoms during intermittent ethanol exposure

In the present study, the neuroprotective effects of dexmedetomidine on rat locus coeruleus were studied during a 5-week intermittent ethanol exposure. Male Wistar rats (3 to 4 months old) were given ethanol or isocaloric sucrose by intragastric intubations three times a day for 4 days, which was followed by a 3-day withdrawal period. This 7-day cycle of ethanol exposure and withdrawal was repeated five times. Dexmedetomidine (at a dose decreasing from 30 microg/kg to 10 microg/kg, s.c.) was given to the treatment group during the withdrawal phase. The results showed that, during the 5-week experiment, dexmedetomidine significantly relieved the ethanol withdrawal syndrome, measured as the sum of the three most specific symptoms (rigidity, tremor, and irritability). The total neuron number of locus coeruleus (LC) decreased in the ethanol-treated group by 24%, compared with the nontreated control group and by 11%, compared with the sucrose-treated control group. Interestingly, the LC neuron numbers were found to decrease in the sucrose-intubated rats as well, compared with the nontreated control group. Dexmedetomidine was found to relieve ethanol-induced neuronal loss in the LC. Dexmedetomidine might be a new interesting alternative in the treatment of ethanol withdrawal syndrome, particularly due to its possible neuroprotective effects in the central nervous system.     

Effects of acamprosate on excitatory amino acids during multiple ethanol withdrawal periods

Background: Our previous studies on the effects of acamprosate on enhanced locomotion during repeated withdrawals are now extended to the effects of acamprosate on excitatory amino acids in the hippocampus during repeated ethanol withdrawals. Methods: In this study, Wistar rats were made ethanol dependent by 4 weeks of vapor inhalation. After this first cycle of chronic ethanol treatment, rats underwent repeated and alternate cycles of 24 hr withdrawals and 1 week of chronic ethanol treatment. The microdialysis technique was used together with high‐performance liquid chromatography and electrochemical detection to quantify different amino acids such as aspartate and glutamate. Results: An intraperitoneal administration of acamprosate (400 mg/kg) to naïve rats did not alter aspartate or glutamate levels compared with the saline groups. During the first cycle of ethanol withdrawal, the administration of acamprosate (400 mg/kg, intraperitoneally) 2 hr after the commencement of ethanol withdrawal decreased both aspartate and glutamate microdialysate levels when compared with their respective saline group. Acamprosate administration also significantly decreased glutamate levels during the third withdrawal compared with the saline group, whereas no changes were seen in aspartate levels. Conclusion: The results of this work demonstrate that acamprosate reduced the excitatory amino acid glutamate increase observed during repeated ethanol withdrawal. These effects of acamprosate may provide a protective mechanism against neurotoxicity by reducing excitatory amino acids, particularly glutamate.     

Effects of Naloxone on Limited-Access Ethanol Drinking in Rats

The hypothesis that naloxone (NAL) decreases oral ethanol intake in rats by inducing a conditioned taste aversion (CTA) to ethanol was investigated. Rats were trained to drink 8% ethanol (v/v) on a 1-hr limited-access schedule. They received 4 days of intraperitoneal injections of 10 mg/kg of NAL, 10 min before limited-access (–10MIN group), immediately after limited-access (1HR group), or 3 hr after limited-access (3HR). Ethanol intake decreased in the -10MIN and 1HR groups during the injection period and on the postinjection day. In experiment 2, rats received 4 days of NAL injections when ethanol was not available (pre-exposure), and then the paradigm was repeated. In this experiment, there was no suppression of ethanol intake for any group on the postinjection day. The decrease in ethanol intake during injections observed for the 1HR in experiment 1 and the sustained suppression postinjection was interpreted as a CTA. Pre-exposure in experiment 2 abolished the CTA. Differences in the pattern ethanol intake for the -10MIN and 3HR groups during the experiments, however, suggest that a CTA is not the sole mechanism underlying NAL's suppressant effects on ethanol intake. In conclusion, in rats both the dose of NAL and the relative timing of NAL injections and ethanol drinking effect subsequent NAL suppression of ethanol intake.     

Ethanol and sucrose seeking and consumption following repeated administration of the GABA(B) agonist baclofen in rats

BACKGROUND: Baclofen, a GABA(B) agonist, has been found to decrease alcohol craving in humans and to nonselectively decrease ethanol intake in some rodent models. This experiment assessed the effects of repeated administration of baclofen on reinforcer seeking and consumption using the sipper tube appetitive/consummatory model of ethanol access. METHODS: Subjects were divided into 2 groups and trained to make 30 lever press responses that resulted in access to either 10% ethanol or 2% sucrose in a sipper tube-drinking spout for 20 minutes. Three doses of baclofen were tested (0.3, 1.0, and 3.0 mg/kg) and each drug treatment was assessed using the following schedule: Monday, saline; Tuesday to Thursday, baclofen; and Friday, saline. RESULTS: The low dose of baclofen had no effect on the seeking or intake of either sucrose or ethanol, and the 1.0 mg/kg dose also had no effect on the appetitive, seeking response. However, the 1.0 mg/kg dose significantly decreased sucrose intake (from an average of 0.56 to 0.41 g/kg) and significantly increased ethanol intake (from an average of 0.77 to 1.00 g/kg). Similarly, the high dose (3.0 mg/kg) decreased sucrose intake and had a tendency to increase ethanol intake while decreasing both sucrose seeking and ethanol seeking. CONCLUSIONS: Overall, baclofen treatment affected reinforcer intake at doses that had no effect on reinforcer seeking, and effective doses decreased both sucrose seeking and ethanol seeking. Moreover, the effects on reinforcer intake were disparate, in that baclofen increased ethanol drinking and decreased sucrose drinking. The nonspecific effects of baclofen suggest that the GABA(B) system may be involved in general consummatory or drinking behaviors and does not appear to specifically regulate ethanol-motivated responding.     

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

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

Effect of neuropeptide Y (NPY) on oral ethanol intake in Wistar, alcohol-preferring (P), and -nonpreferring (NP) rats

BACKGROUND: Neuropeptide Y (NPY) deficient mice consume more ethanol than controls, whereas NPY over-expressing mice consume less ethanol than controls. Thus, ethanol drinking may be inversely associated with NPY activity. To determine whether exogenously administered NPY would alter ethanol intake, two experiments were conducted. METHODS: A within-subject design was used with intracerebroventricular (ICV) administration of NPY or artificial cerebral spinal fluid (aCSF) into the lateral ventricles. Infusions were separated by 2 to 7 days. In experiment 1, male Wistar rats (n = 10) were tested for the effects of NPY on an intake of 5% sucrose or 8% (w/v) ethanol during daily 2-hr testing periods with food and water available at all other times. In experiment 2, male alcohol-preferring (P) and alcohol-nonpreferring (NP) rats (n = 8/line) were tested for the effects of NPY on 8% (w/v) ethanol intake. RESULTS: In experiment 1, NPY (5, 10, 20 microg) significantly increased sucrose intake relative to aCSF baseline in Wistar rats, a finding consistent with previous observations of the orexigenic effects of the peptide. However, NPY (10 microg) did not alter ethanol intake in Wistar rats. In experiment 2, NPY (5 and 10 microg) significantly decreased ethanol intake in P rats, but not in NP rats. CONCLUSION: The reduction in ethanol intake seen with the P rats is consistent with the postulated negative relationship between NPY activity and ethanol intake. The lack of effect of NPY on ethanol intake in Wistar and NP rats may be related to the lower baseline levels of ethanol intake in these rats or to differential central nervous system basal NPY activity or sensitivity to the peptide.     

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.     

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.     

Effects of neuropeptide Y on sucrose and ethanol intake and on anxiety-like behavior in high alcohol drinking (HAD) and low alcohol drinking (LAD) rats

BACKGROUND: In a previous study, neuropeptide Y (NPY) administered into the lateral ventricles decreased ethanol intake in alcohol-preferring (P) rats but not in alcohol-nonpreferring (NP) or unselected Wistar rats. The purpose of the present investigation is to extend these findings in selectively-bred high-alcohol-drinking (HAD)1 and low-alcohol-drinking (LAD)1 rats by examining the effects of intracerebroventricularly administered NPY on the elevated plus maze test of anxiety and on ethanol and sucrose intake. METHODS: Female HAD1 and LAD1 rats were surgically implanted with cannula into the lateral ventricle. Following recovery, a test of anxiety was conducted in which the rats (n = 12-13/group) received either artificial cerebrospinal fluid (aCSF) or NPY (10 microg) 10 min prior to a 5-min test on an elevated plus maze. Following anxiety testing, 11 HAD and 11 LAD rats were trained to self-administer ethanol (8% w/v), and 5 HAD and 8 LAD rats were trained to self-administer sucrose (2.5%) during daily 2-hr sessions. A within-subject design was used in which the rats were pretreated once a week with aCSF, 5 microg NPY, or 10 microg NPY prior to the drinking sessions. RESULTS: HAD and LAD rats treated with aCSF did not differ in time spent in open arms of the plus maze. NPY increased time spent on the open arms to similar degrees in both rat lines. HAD rats consumed more ethanol and sucrose than LAD rats. NPY increased sucrose intake in both rat lines. However, the same doses of NPY reduced ethanol intake in HAD but not in LAD rats. CONCLUSION: The plus maze results indicated that selective breeding for high and low alcohol preference in the HAD1 and LAD1 rats, respectively, did not yield differences in anxiety-like behavior and in response to the anxiolytic effects of NPY. The increases in sucrose intake were consistent with the known orexigenic effects of NPY. The decreased ethanol intake following NPY administration in HAD rats was similar to previous observations with P rats and is consistent with the hypothesis that ethanol intake and NPY activity may be inversely related.     

Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: regulation by corticotropin-releasing factor

BACKGROUND: Animal models of alcohol dependence suggest that long-term alterations in brain corticotropin-releasing factor (CRF) systems, key mediators of the behavioral stress response, may be involved in the development and reinstatement of dependence on drugs of abuse. The objective of the present study was to investigate the role of CRF in the regulation of ethanol self-administration and to examine the behavioral stress response during acute withdrawal and protracted abstinence. METHODS: Male Wistar rats were made dependent on ethanol via chronic exposure to ethanol vapor. Ethanol self-administration and exploratory behavior in the elevated plus maze were measured at 2 hr and 3 to 5 weeks after exposure. The role of CRF in ethanol self-administration was examined via central injection of the CRF receptor antagonist D-Phe-CRF(12-41). RESULTS: Rats showed increased responding for ethanol 2 hr and 3 to 5 weeks after chronic ethanol exposure, which was attenuated by central injection of D-Phe-CRF(12-41). In addition, rats displayed a decrease in open-arm exploration in the elevated plus maze when tested 2 hr and 4 weeks after exposure. CONCLUSIONS: These results indicate that chronic ethanol exposure leads to increased ethanol self-administration and decreased open-arm exploration in the elevated plus maze during acute withdrawal and protracted abstinence. Attenuation of ethanol self-administration via central injection of D-Phe-CRF(12-41) implicates CRF as an underlying mechanism regulating long-term motivational effects associated with alcohol dependence.     

Selective Effects of Alcohol Drinking on Restraint-Induced Expression of Immediate Early Genes in Mouse Brain

BACKGROUND: Analysis of immediate early gene expression in brain is a common contemporary method for mapping changes in neuronal activation with cellular resolution. This method has been applied previously in models of involuntary alcohol exposure. In this study, immunohistochemical expression analysis of immediate early genes c-fos, fosB, and zif268 was performed in brain of C57BL/6J mice after voluntary alcohol consumption. METHODS: Mice were trained to consume 10% ethanol/10% sucrose, using a 30-min limited-access paradigm. Animals consumed approximately 1.5 g/kg of ethanol per session. Control animals consumed 10% sucrose solution. Gene expression was determined in half of the animals 1.5 hr after the drinking session. Gene expression in the remaining animals was determined after 0.5 hr of restraint stress, which is known to elevate expression of immediate early genes in many brain regions. Analysis of the stressed animals was also performed 1.5 hr after the drinking session. RESULTS: Blood alcohol concentrations were significantly reduced in animals exposed to restraint stress. However, stressed mice showed the greatest alcohol-induced changes in gene expression. Specifically, animals consuming ethanol/sucrose with subsequent exposure to restraint stress had lower c-Fos expression in the CA3 region of hippocampus, and higher c-Fos expression in nucleus accumbens than mice exposed to restraint stress after drinking the sucrose solution. Consumption of the ethanol/sucrose solution also significantly reduced FosB expression in the basolateral amygdala and lateral hypothalamus, and Zif268 expression in the CA1 region of the hippocampus of stressed animals. CONCLUSIONS: These data confirm previous observations showing selective effects of alcohol administration on immediate early gene expression. Furthermore, the effects of voluntary alcohol self-administration on immediate early gene expression differ from involuntary alcohol exposure and suggest several brain regions as substrates for alcohol consumption.