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

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

Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats

Background: There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent‐access to 20% ethanol in a 2‐bottle‐choice setting [ Wise, Psychopharmacologia 29 (1973), 203 ]. In this study, we have further characterized this drinking model. Methods: Ethanol‐naïve Long–Evans rats were given intermittent‐access to 20% ethanol (three 24‐hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long‐term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent‐access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous‐access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out‐bred alcohol preferring (P) rats following intermittent‐access to 20% ethanol. Results: The intermittent‐access 20% ethanol 2‐bottle‐choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long–Evans: 5.1 ± 0.6; Wistar: 5.8 ± 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long–Evans rats. P‐rats initiate drinking at a higher level than both Long–Evans and Wistar rats using the intermittent‐access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 ± 0.8 g/kg/24 h). Conclusion: Standard laboratory rats will voluntarily consume ethanol using the intermittent‐access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.     

Overexpression of neuropeptide Y in the central nucleus of the amygdala decreases ethanol self-administration in "anxious" rats

BACKGROUND: Neuropeptide Y (NPY) has been implicated in a variety of behaviors including those associated with anxiety and ethanol administration. The current experiment investigated the predictive role of anxiety-like behaviors in ethanol self-administration and the relationship of NPY in the central nucleus of the amygdala (CeA) with anxiety and ethanol self-administration. METHODS: Rats were divided into anxious and nonanxious groups based on behavior in the elevated plus maze. Following elevated plus maze testing, rats were allowed to consume increasing concentrations of ethanol (2, 4, and 6%) in a 2-bottle choice procedure over a period of 31 days. anxious rats showed an increased preference for 4% ethanol and 6% ethanol compared with non-anxious rats. Following 20-day access to 6% ethanol, rats underwent gene transfer surgery with replication-defective recombinant herpes simplex 1 vectors encoding prepro-NPY, an antisense NPY RNA, or LacZ (control) into the CeA. RESULTS: In anxious rats, bilateral injections into the CeA with the NPY-antisense vector increased 6% ethanol preference, while the vector encoding NPY decreased 6% ethanol preference. Herpes simplex viral-mediated alterations in CeA NPY expression did not alter ethanol preference in nonanxious rats. CONCLUSIONS: These results suggest that virally mediated alterations in NPY levels in the CeA differentially affect ethanol consumption in rats with low and high basal levels of anxiety.     

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.     

Relationship between ethanol's acute locomotor effects and ethanol self-administration in male Long-Evans rats

Background: Human studies have suggested an important relationship between ethanol sensitivity and risk of alcoholism. These studies have led some to hypothesize that a low initial sensitivity to ethanol’s depressant effects and/or an elevated response to ethanol’s stimulant effects may represent important risk factors associated with the development of abusive drinking behavior. Unfortunately, elucidating neurobiologic mechanisms that may underlie these relationships between ethanol sensitivity and ethanol drinking have been hampered by difficulties in modeling some of these interactions in animals. In this study, we re‐examined some of these relationships in an outbred strain of rats using continuous access two‐bottle choice drinking and a limited‐access operant procedure that engenders pharmacologically relevant levels of ethanol intake and permits the discrete assessment of appetitive and consummatory measures of ethanol drinking behavior. Methods: Twenty‐three male Long‐Evans rats were habituated to a locomotor activity box and then tested for their response to a stimulant (0.5 g/kg) and depressant (1.5 g/kg) ethanol dose. Rats were then trained to complete a lever pressing requirement to gain access to 10% ethanol for 20‐minute sessions conducted 5 d/wk for 5 weeks. Appetitive behavior was assessed after 2.5 and 4.5 weeks using 20‐minute extinction trials in which ethanol was not presented and lever responses were recorded. Home‐cage ethanol preference was also assessed prior to and immediately following the 5‐week self‐administration regimen using a continuous access, two‐bottle choice procedure. Results: A significant increase in home‐cage ethanol preference was observed following the self‐administration procedure, however, neither measure of ethanol preference correlated with average daily ethanol intake during the operant self‐administration sessions or with initial sensitivity to ethanol’s stimulant or depressant effects. Notably, a significant negative correlation was observed between sensitivity to ethanol’s locomotor depressant effect and daily intake during the operant self‐administration sessions. No significant relationships were noted between sensitivity to ethanol’s locomotor effects and extinction responding. Conclusions: The results of these studies suggest that the well‐established relationship between a low level of response to ethanol and increased ethanol consumption reported in human studies can be observed in an outbred rodent strain using a limited‐access operant self‐administration procedure, but not with home‐cage ethanol drinking.     

Restraint stress and ethanol consumption in two mouse strains

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

Differential Effects of Chronic Ethanol Consumption and Withdrawal on Homer/Glutamate Receptor Expression in Subregions of the Accumbens and Amygdala of P Rats

Background: Homer proteins are constituents of scaffolding complexes that regulate the trafficking and function of central Group1 metabotropic glutamate receptors (mGluRs) and N‐methyl‐d ‐aspartate (NMDA) receptors. Research supports the involvement of these proteins in ethanol‐induced neuroplasticity in mouse. In this study, we examined the effects of short versus long‐term withdrawal from chronic ethanol consumption on Homer and glutamate receptor protein expression within striatal and amygdala subregions of selectively bred, alcohol‐preferring P rats. Methods: For 6 months, male P rats had concurrent access to 15% and 30% ethanol solutions under intermittent (IA: 4 d/wk) or continuous (CA: 7 d/wk) access conditions in their home cage. Rats were killed 24 hours (short withdrawal: SW) or 4 weeks (long withdrawal: LW) after termination of ethanol access, subregions of interest were micropunched and tissue processed for detection of Group1 mGluRs, NR2 subunits of the NMDA receptor and Homer protein expression. Results: Within the nucleus accumbens (NAC), limited changes in NR2a and NR2b expression were detected in the shell (NACsh), whereas substantial changes were observed for Homer2a/b, mGluRs as well as NR2a and NR2b subunits in the core (NACc). Within the amygdala, no changes were detected in the basolateral subregion, whereas substantial changes, many paralleling those observed in the NACc, were detected in the central nucleus (CeA) subregion. In addition, most of the changes observed in the CeA, but not NACc, were present in both SW and LW rats. Conclusions: Overall, these subregion specific, ethanol‐induced increases in mGluR/Homer2/NR2 expression within the NAC and amygdala suggest changes in glutamatergic plasticity had taken place. This may be a result of learning and subsequent memory formation of ethanol’s rewarding effects in these brain structures, which may, in part, mediate the chronic relapsing nature of alcohol abuse.     

Increased ethanol consumption and preference in mice lacking neurotensin receptor type 2

Background: Neurotensin receptors (NTS) regulate a variety of the biological functions of neurotensin (NT) in the central nervous system. Although NT and neurotensin receptors type 1 (NTS1) are implicated in some of the behavioral effects of ethanol, the functional roles of neurotensin receptors type 2 (NTS2) in ethanol intoxication and consumption remain unknown. Here, we investigated behavioral effects mediated by NTS2 in response to ethanol, which are implicated in ethanol consumption and preference, using NTS2 null mice. Method: First, we examined ethanol‐induced locomotion, ataxia, hypnosis, and hypothermia in NTS2 null mice. Next, we measured ethanol consumption and preference in NTS2 null mice by giving them free choice between ethanol‐ and tap water‐containing bottles. Then using a brain‐permeable NT analog, NT69L, we examined the role of NTS2 in locomotor activity and ataxia. Finally, we examined the effect of NT69L on ethanol consumption and preference in NTS2 null mice. Results: We found that NTS2 null mice appear less sensitive to the acute hypnotic effects of ethanol and consumed more ethanol compared to wild‐type littermates in a 2‐bottle choice experiment, even though ethanol‐induced locomotion, ataxia, and hypothermia were similar between genotypes. Interestingly, the administration of NT69L for 4 consecutive days significantly reduced alcohol consumption and preference in wild‐type littermates as well as in NTS2 null mice. Conclusions: Our findings suggest that NTS2 regulates ethanol‐induced hypnosis and ethanol consumption.     

Histone deacetylases (HDAC)-induced histone modifications in the amygdala: a role in rapid tolerance to the anxiolytic effects of ethanol

Background: Rapid tolerance to the anxiolytic effects of ethanol appears to be an important factor in the development of alcoholism. Here, we investigated the involvement of amygdaloid histone deacetylases (HDAC)‐induced epigenetic changes in rapid ethanol tolerance (RET). Methods: RET in rats was induced by 2 ethanol injections administered 24 hours apart. Both ethanol‐tolerant and control rats were treated with the HDAC inhibitor, trichostatin A (TSA), and anxiety‐like behaviors were measured. HDAC activity, histone (H3 and H4) acetylation, and neuropeptide Y (NPY) expression in the amygdala of these rats were also measured. Results: A single ethanol exposure was able to produce an anxiolytic response, inhibit amygdaloid HDAC activity, and increase both histone acetylation and NPY expression (mRNA and protein levels) in the central nucleus of amygdala (CeA) and medial nucleus of amygdala (MeA) of rats. In contrast, 2 exposures of the same dose of ethanol (24 hours apart) neither elicited a similar anxiolytic response nor modulated HDAC activity, histone acetylation, or NPY expression in the amygdala. However, exposure to a higher dose of ethanol on the second day was able to produce an anxiolytic response and also inhibit amygdaloid HDAC activity. TSA treatment caused the reversal of RET by inhibiting HDAC activity, thereby increasing histone acetylation and NPY expression in the CeA and MeA. Conclusions: Cellular tolerance to the initial acute ethanol‐induced inhibition of HDAC activity and the subsequent upregulation of histone acetylation and NPY expression in the amygdala may be involved in the mechanisms underlying rapid tolerance to the anxiolytic effects of ethanol.     

Intermittent (every-other-day) drinking induces rapid escalation of ethanol intake and preference in adolescent and adult C57BL/6J mice

Background: Using adult C57BL/6J (B6) mice, we previously developed a procedure that causes a progressive increase in ethanol intake and preference (i.e., alcohol escalation effect) following weekly (intermittent) access to ethanol ( Melendez et al., 2006 ). A limitation of this procedure is that it requires many weeks of testing, which limits its use to study ethanol escalation (i.e., binge‐like drinking) during adolescence. Previous studies have shown that intermittent every‐other‐day (EOD) access to ethanol is sufficient to induce ethanol escalation in rats. The objective of this study was to verify whether EOD access is sufficient to induce escalated levels of ethanol intake and preference in adult and adolescent B6 mice. Methods: Male B6 mice received free‐choice 24‐hour access to 15% ethanol and water on an EOD or daily basis for 2 weeks. Food and water were available at all times. Using adult mice, Experiment 1 characterized the induction of ethanol escalation following EOD access at 6 (i.e., drinking in the dark) and 24‐hour intervals, whereas Experiment 2 determined whether daily drinking reverses escalation induced by EOD drinking. Experiment 3 compared ethanol‐drinking capacity following daily versus EOD drinking in adolescent (P30‐45) and adult (P70‐85) mice. Results: Experiment 1 revealed that EOD drinking leads to a significant (nearly 2‐fold) increase in ethanol intake and preference over mice given daily access. Experiment 2 demonstrated that EOD‐elicited escalation is blocked and subsequently reversed following daily drinking. Experiment 3 revealed that ethanol drinking was greater in adolescent mice compared with adults following daily drinking and EOD (escalated) drinking. Although the escalated levels of ethanol intake were greater in adolescent mice, the rate or onset of escalation was comparable between both age‐groups. Conclusions: This study is the first to demonstrate that EOD drinking leads to escalation of ethanol intake and preference in adolescent and adult mice. Moreover, our results indicate that daily ethanol reverses ethanol escalation induced by intermittent drinking. The study also revealed that adolescent mice have a greater capacity to drink ethanol under both daily (controlled) and EOD (escalated) conditions, which further supports the notion of adolescent’s susceptibility to heavy drinking.     

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

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

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.     

The effects of continuous and intermittent ethanol exposure in adolesence on the aversive properties of ethanol during adulthood

Background:  Alcohol abuse among adolescents is prevalent. Epidemiological studies suggest that alcohol abuse during the adolescent developmental period may result in long-term changes such as an increased susceptibility to alcohol-related problems in adulthood. Laboratory findings suggest that alcohol exposure during the adolescent developmental period, as compared with adulthood, may differentially impact subsequent neurobehavioral responses to alcohol. The present study was designed to examine whether ethanol exposure, continuous versus intermittent, during the adolescent developmental period would alter the aversive properties of ethanol in adult C3H mice.
Methods:  Periadolescent (PD28) male C3H mice were exposed to 64 hours of continuous or intermittent ethanol vapor. As a comparison, adult (PD70) C3H mice were also exposed to 64 hours of continuous or intermittent ethanol vapor. Six weeks after ethanol exposure, taste aversion conditioning was carried out on both ethanol pre-exposed and ethanol-naive animals using a 1-trial, 1-flavor taste-conditioning procedure.
Results:  Ethanol exposure during the periadolescent period significantly attenuated a subsequent ethanol-induced conditioned taste aversion, as compared with control animals. Adult animals exposed to chronic ethanol vapor during adolescence showed less of an aversion to an ethanol-paired flavor than ethanol-naive adults. Intermittent exposure to ethanol vapor during periadolescence produced a greater attenuation.
Conclusion:  It is suggested that ethanol exposure during the periadolescent period results in long-term neurobehavioral changes, which lessen a conditioned aversion to ethanol in adulthood. It is suggested that this age-related effect may underlie the increased susceptibility to alcohol-related problems which is negatively correlated with the age of onset for alcohol abuse.     

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).     

Voluntary ethanol intake enhances excitatory synaptic strength in the ventral tegmental area

Background: Addiction has been considered a disorder of motivational control over behavior, and the ventral tegmental area (VTA), in conjunction with other limbic brain structures, is thought to play a critical role in the regulation of a number of motivated behaviors including seeking of addictive drugs such as alcohol. Of particular interest is the ability of prolonged exposure of addictive drugs to enhance the function of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamatergic receptors (AMPAR) in the VTA, as glutamate receptor activation can significantly regulate VTA neuron activity. Here, we examined whether voluntary ethanol intake altered VTA AMPAR function. Methods: We utilized in vitro electrophysiology to examine glutamatergic function in the VTA neurons 12 to 24 hours after the last self‐administration bout, which occurred 35 to 50 days after the initiation of ethanol self‐administration under a 2‐bottle intermittent access model. Results: Voluntary intermittent ethanol intake in a 2‐bottle paradigm enhanced postsynaptic AMPAR function, indicated by an increased ratio of evoked AMPAR to N‐methyl‐d ‐aspartic acid receptor currents, and by an increase in the amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) measured in the presence of tetrodotoxin to prevent action potential‐dependent release. In contrast, ethanol self‐administration did not alter evoked presynaptic glutamate release, indicated by no change in the paired‐pulse ratio of 2 AMPAR EPSCs evoked 50 ms apart, although spontaneous glutamate release was significantly enhanced, indicated by enhanced mEPSC frequency. Conclusions: Our results suggest that postsynaptic AMPAR function in VTA neurons was significantly enhanced after ethanol self‐administration. As increased VTA AMPAR function can significantly regulate firing and enhance the reinforcing and activating effects of drugs of abuse, the increased AMPAR activity observed here may facilitate the drive to consume ethanol.     

Intensity and Duration of Chronic Ethanol Exposure Is Critical for Subsequent Escalation of Voluntary Ethanol Drinking in Mice

Background: Excessive alcohol drinking continues to be an important health problem. Recent studies from our laboratory and others have demonstrated that animal models of ethanol dependence and relapse can contribute to understanding factors that contribute to excessive drinking. In this study, we tested the hypothesis that the amount and duration of ethanol exposure is critical for promoting the escalation in drinking by mice given access to ethanol in a limited access paradigm. Methods: We used several methods of chronic intermittent ethanol exposure in male C57BL/6J mice that would vary in the amount and duration of exposure to ethanol as indicated by blood ethanol concentrations (BEC). After establishing baseline drinking in the mice using a 2 hours, 2 bottle choice drinking paradigm, each study involved alternating between periods of ethanol exposure and periods of limited access to ethanol (1 cycle) for a total of 3 cycles. In Study 1, mice were allowed extended access (16 hours) to ethanol for oral consumption or remained in the home cage. In Study 2, the ethanol exposure consisted of intragastric gavage of increasing doses of ethanol or isocaloric sucrose as the control. Study 3 compared intragastric gavage combined with pyrazole, an alcohol dehydrogenase inhibitor, with vapor inhalation of ethanol using procedures known to lead to increased drinking in mice. Finally, Study 4 was a retrospective review of several studies conducted in our laboratory using inhalation procedures. The retrospective review encompassed a range of postvapor chamber BEC values and ethanol intakes that would allow a relationship between increased drinking and BEC to be examined. Results: Allowing mice to drink for longer periods of time did not cause increased drinking in subsequent limited access sessions. Likewise, gastric intubation of ethanol which produced high BEC (>300 mg/dl) with or without pyrazole did not increase drinking. Only the vapor inhalation procedure, which was associated with sustained BEC above 175 mg/dl for the entire exposure period resulted in increased drinking. The retrospective study provided further evidence that sustained BEC levels above 175 mg/dl was critical to the escalation in drinking. Conclusions: We found that the intensity (amount) and duration of ethanol exposure, indexed by BEC, is critical to produce increased drinking in mice. Specifically, BEC must regularly exceed 175 mg/dl for the escalation in drinking to occur. Future studies will examine neurobiological adaptations that may underlie the increased drinking behavior caused by chronic intermittent ethanol exposure.     

Neuropeptide Y Signaling in the Central Nucleus of Amygdala Regulates Alcohol‐Drinking and Anxiety‐Like Behaviors of Alcohol‐Preferring Rats

Background: The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol‐preferring (P) rats as an animal model. Methods: Alcohol‐preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety‐like behaviors were assessed by the 2‐bottle free‐choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT‐PCR. Double‐immunofluorescence labeling was performed to observe the colocalization of NPY and Ca²⁺/calmodulin‐dependent protein kinase IV (CaMK IV). Results: We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element‐binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic α‐subunit of protein kinase A (PKA‐Cα) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA‐Cα without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV‐positive cells were co‐localized with NPY in amygdaloid structures of P rats. Conclusions: These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV‐dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.     

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.     

Blockade of the corticotropin releasing factor type 1 receptor attenuates 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 (BECs) reaching levels that have measurable affects on physiology and/or behavior. The present experiments determined whether the increased ethanol drinking caused by DID procedures can be attenuated by pretreatment with CP‐154,526; a corticotropin releasing factor type‐1 (CRF₁) receptor antagonist. Methods: In Experiment 1, male C57BL/6J mice received ethanol (20% v/v) in place of water for 4 hours, beginning with 3 hours into the dark cycle. On the fourth day, mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle. In Experiment 2, C57BL/6J mice had 2 hours of access to the 20% ethanol solution, beginning with 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. Mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle on day 4. Tail blood samples were collected immediately after the 4‐hour ethanol access period on the fourth day of each experiment. Additional control experiments assessed the effects of CP‐154,526 on 4‐hour consumption of a 10% (w/v) sucrose solution and open‐field locomotor activity. Results: In Experiment 1, the vehicle‐treated group consumed approximately 4.0 g/kg/4 h of ethanol and achieved BECs of approximately 30 mg%. Furthermore, pretreatment with the CRF₁ receptor antagonist did not alter ethanol consumption. On the other hand, procedures used in Experiment 2 resulted in vehicle‐treated mice consuming approximately 6.0 g/kg/4 h of ethanol with BECs of about 80 mg%. Additionally, the 10 mg/kg dose of CP‐154,526 significantly reduced ethanol consumption and BECs to approximately 3.0 g/kg/4 h and 27 mg%, respectively, relative to vehicle‐treated mice. Importantly, the 10 mg/kg dose of the CRF₁R antagonist did not significantly alter 4‐hour sucrose consumption or locomotor activity. Conclusions: These data indicate that CRF₁R signaling modulates high, but not moderate, levels of ethanol drinking associated with DID procedures.     

Early ethanol consumption predicts relapse-like behavior in adolescent male rats

Background: Alcohol abuse disorders emerge over time with repeated consumption of ethanol, but not all ethanol drinkers develop these disorders. There are pre‐existing characteristics that indicate which drinkers are most likely to abuse alcohol. Adolescence, novelty seeking, and high stress reactivity are among the characteristics of the most vulnerable individuals. In addition, an individual’s response to his or her first exposure to the drug influences future consumption. We assessed an array of behavioral and hormonal characteristics in adolescent (28‐day‐old) male rats before exposure to ethanol, and then determined which rats were most prone to high levels of alcohol drinking. Methods: The assessments consisted of measures of anxiety (elevated plus maze), response to novelty (open field locomotion, novel object exploration), and circulating corticosterone levels after mild restraint and after the elevated plus maze task. After this test battery, the rats were placed in lickometer cages nightly (5 pm to 9 am ) for evaluation of fluid consumption. Rats were first habituated to the cages with water in the lickometer bottles, and then given 10% (v/v) ethanol for 3 nights as the only available fluid. After this forced ethanol exposure, the rats were allowed to choose between 8% ethanol and water for 10 consecutive nights. After 2 nights of abstinence, the rats were again placed in the lickometer cages and given a choice between 8% ethanol and water to assess ethanol consumption in response to alcohol deprivation, a measure of relapse‐like behavior. Results: Ethanol consumption on the third day of forced consumption was significantly correlated with ethanol consumption on days 8 to 10 of the choice phase, which in turn was significantly correlated to relapse‐like consumption. Preference for ethanol was also significantly correlated with early consumption. Novel object exploration, open field activity, open arm time in the elevated plus maze, initial water consumption, and circulating corticosterone levels did not significantly predict deprivation‐stimulated consumption. Conclusions: These results suggest that consumption during early exposure to ethanol establishes a pattern leading to development of increased alcohol consumption and preference in adolescent male rats. In addition, they represent an animal model of the well‐described observation that humans who consume large quantities of ethanol during early exposure are the most likely to repeat heave drinking behavior. Furthermore, early consumption is distinct from novelty seeking, anxiety, and stress hormone levels which are also thought to contribute to vulnerability to alcoholism.