Ethanol Self-Administration in Deprived Rats: Effects of Ro15-4513 Alone, and in Combination with Flumazenil (Ro15-1788)

Previous work in our laboratory demonstrated that Ro15-4513, a partial inverse benzodiazepine agonist, decreases self-administration of ethanol (ETOH) in rats maintained on a two-bottle regmine of a saccharin ethanol solution (ES) and water over a 35-day consumption period. The present study extended the consumption period to 60 days and examined the effects of Ro15-4513 (2.5 mg/kg), flumazenil (Ro15-1788) (8.0 mg/kg), and Ro15-4513 in combination with Ro15-1788 on the time course of ETOH self-administration. High initial intake of ES observed during the first 4 weeks declined significantly over subsequent weeks. Ro15-4513 pretreatment, however, resulted in significant reduction of ES, while significantly preventing the "normal" reduction of consumption as was observed under control conditions. The antagonistic actions of Ro15-4513 were blocked/attenuated by the benzodiazepine receptor antagonist, Ro15-1788, independent of whether consumption of the ES was low or high. Both Ro15-4513 and Ro15-1788 affected water intake differentially compared with vehicle-injected controls. The results suggest that GABA-benzodiazepine mechanisms may be important in altering chronic ETOH drinking patterns depending upon experience with ETOH, tolerance, or learning.     

Effects of Naltrexone and Ro 15-4513 on a Multiple Schedule of Ethanol and Tang Self-Administration

BACKGROUND: Both the opioid antagonist, naltrexone, and GABAA/benzodiazepine-site negative modulator, Ro 15-4513, decrease ethanol self-administration in rodents and nonhuman primates. However, the selectivity of these drugs for decreasing ethanol self-administration relative to reducing responding maintained by other reinforcers in primates is not clear. The present study used a multiple schedule self-administration procedure in cynomolgus monkeys to examine the selectivity of naltrexone and Ro 15-4513 for reducing ethanol self-administration relative to an orange flavored sugar-free sweetened solution (Sugar-free Tang). METHODS: Six adult cynomolgus monkeys were trained to self-administer 4% (w/v) ethanol and 4% or 6% (w/v) Tang under a multiple schedule of liquid access. The effect of acute administration of naltrexone (0.1, 0.3, 1, 3 mg/kg) was examined. The effect of 15 days of chronic, 1 mg/kg naltrexone on ethanol and Tang self-administration was then examined in four monkeys. Acute administration of Ro 15-4513 (0.01, 0.03, 0.1, 0.3 mg/kg) as well as 15 days of chronic administration of 0.1 mg/kg Ro 15-4513 was also examined in four monkeys. RESULTS: Ethanol and Tang were self-administered at similar volumes and patterns under baseline conditions. Acute naltrexone administration significantly decreased total session ethanol and Tang intake as well as the number and volume of ethanol and Tang drinks. Chronic naltrexone also significantly decreased ethanol and Tang intake. Ethanol, but not Tang, drink volume was significantly decreased by chronic 1 mg/kg naltrexone pretreatment. The number of ethanol and Tang drinks and drink duration were not significantly decreased by chronic naltrexone. Acute Ro 15-4513 pretreatment significantly decreased ethanol and Tang intake, mean drinks and median drink duration. Chronic 0.1 mg/kg Ro 15-4513 pretreatment significantly decreased total ethanol intake only during the first week of pretreatment, but it significantly decreased Tang intake for all 3 pretreatment weeks. CONCLUSIONS: Similar to rodent studies, acute and chronic naltrexone and Ro 15-4513 reduced ethanol and Tang intake in cynomolgus monkeys. However, unlike rodent studies, neither drug showed selectivity for reducing ethanol intake compared with a comparison reinforcer. These differences highlight the need for testing putative ethanol abuse treatment drugs under diverse conditions and multiple species before undertaking human clinical trials.     

Effect of Pentylenetetrazole on Ethanol Intake, Ethanol Kinetics, and Social Behavior in Male Wistar Rats

Stress and anxiety are often implicated in excessive alcohol use. The nature of this interaction, however, is not understood. The aim of this study was to examine the effect of the anxiogenic agent, pentylenetetrazole (PTZ), on the acquisition and maintenance of ethanol drinking behavior in male Wistar rats. In rats maintained on a limited access procedure, with a choice between a 12% w/v ethanol (ETOH) solution and water available for 30 min each day, acute PTZ administration (1.5 to 15.0 mg/kg) did not modify ETOH intake. Chronic PTZ administration elicited a significant suppression in ETOH intake; however, this effect developed gradually over time. During the acquisition phase, chronic PTZ treatment also suppressed ETOH consumption. Chronic, but not acute, treatment with PTZ seemed to enhance water consumption. To assess whether the effect of PTZ on ETOH intake was due to either alterations in ETOH kinetics or behavior, blood ETOH levels and social interaction behaviour were examined. PTZ (15.0 mg/kg) produced a significant suppression in social interaction behavior, although tolerance developed to this effect on chronic PTZ administration. Both acute and chronic PTZ treatment (15 mg/kg) resulted in lower blood ETOH levels achieved after administration of 1.0 g/kg po of ETOH. Because the anxiogenic effect of PTZ was not maintained on repeated administration, yet the suppression of ETOH intake was only observed after chronic treatment, this suggests a dissociation between the processes regulating these behaviors.     

Ro15-4513 Attenuates the Consumption of Ethanol in Deprived Rats

This research investigated the effects of Ro15-4513 (Ro15), a partial inverse benzodiazepine agonist, on the drinking behavior of 23-1/2 hr fluid deprived rats. Water-deprived rats were maintained on a two-bottle regimen of a saccharin-ETOH solution along with tap water available for 30 min/day for several days. Following this acclimation period, animals were pretreated with either Ro15 (1.0, 2.5, and 5.0 mg/kg) or Tween-80 vehicle injections. Pretreatment with Ro15 at all doses tested resulted in a significant reduction of the saccharin-ETOH solution; however, Ro15 did not alter the rats' consumption of water. The effects of Ro15 on general fluid consumption was investigated in Experiment 2. Following acclimation to a two-bottle regimen of a saccharin-solution and tap water 30 min/day, naive animals were pretreated with Tween-80 vehicle or Ro15 injections. Ro15 failed to alter saccharin or water consumption. The results of this study support previous reports suggesting that Ro15 attenuates the oral consumption of ETOH; however, this effect does not appear to be due to a general suppression of fluid intake.     

Pregnenolone and ganaxolone reduce operant ethanol self-administration in alcohol-preferring p rats

Background: Neuroactive steroids modulate ethanol intake in several self‐administration models with variable effects. The purpose of this work was to examine the effects of the long‐acting synthetic GABAergic neurosteroid ganaxolone and the endogenous neurosteroid pregnenolone, a precursor of all GABAergic neuroactive steroids, on the maintenance of ethanol self‐administration in an animal model of elevated drinking—the alcohol‐preferring (P) rats. Methods: P rats were trained to self‐administer ethanol (15% v/v) versus water on a concurrent schedule of reinforcement, and the effects of ganaxolone (0 to 30 mg/kg, subcutaneous [SC]) and pregnenolone (0 to 75 mg/kg, intraperitoneal [IP]) were evaluated on the maintenance of ethanol self‐administration. After completion of self‐administration testing, doses of the neuroactive steroids that altered ethanol self‐administration were assessed on spontaneous locomotor activity. Finally, the effect of pregnenolone administration on cerebral cortical levels of the GABAergic neuroactive steroid (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone, 3α,5α‐THP) was determined in both ethanol‐experienced and ethanol‐inexperienced P rats because pregnenolone is a precursor of these steroids. Results: Ganaxolone produced a dose‐dependent biphasic effect on ethanol reinforcement, as the lowest dose (1 mg/kg) increased and the highest dose (30 mg/kg) decreased ethanol‐reinforced responding. However, the highest ganaxolone dose also produced a nonspecific reduction in locomotor activity. Pregnenolone treatment significantly reduced ethanol self‐administration (50 and 75 mg/kg), without altering locomotor activity. Pregnenolone (50 mg/kg) produced a significant increase in cerebral cortical allopregnanolone levels. This increase was observed in the self‐administration trained animals, but not in ethanol‐naïve P rats. Conclusions: These results indicate that pregnenolone dose‐dependently reduces operant ethanol self‐administration in P rats without locomotor impairment, suggesting that it may have potential as a novel therapeutic for reducing chronic alcohol drinking in individuals that abuse alcohol.     

GABA Antagonist and Benzodiazepine Partial Inverse Agonist Reduce Motivated Responding for Ethanol

Brain γ-aminobutyric acid (GABA) systems have long been associated with the behavioral actions of ethanol. This study investigated the effects of GABAergic agents on ethanol reinforcement. Rats were trained to orally self-administer ethanol in a 30-min, free-choice operant task. Responses at one of two levers produced contingent access to ethanol (10% w/v) or water. Pretreatment with RO 154513, a benzodlazepine inverse agonist (0.375 to 3.0 mg/kg ip), selectively reduced responses for ethanol, and a higher dose of RO 15-4513 (6.0 mg/kg) reduced both ethanol and water responses. Self-administration of saccharin in a free-choice task with access to saccharin (0.05%) and water was unaffected by RO 15-4513, suggesting that the effects of RO 15-4513 on ethanol reinforcement may not necessarily generalize to other reinforcers. Isopropylbicyclophosphate (IPPO), a picrotoxin ligand (5 and 10 μ g/kg ip), selectively reduced responses for ethanol in alcohol-preferring, nonpreferring and Wistar rats. However, the highest dose of IPPO (20 μ g/kg) reduced both ethanol and water responses. Chlordiazepoxide, a benzodiazepine, did not reduce responses for ethanol in the selectively bred animals, suggesting that this drug does not substitute for the reinforcing properties associated with acute ethanol intake. Together, these results suggest that compounds that act at the benzodiazepine inverse agonist and picrotoxin sites of the GABA/benzodiazepine receptor complex may decrease motivated responding for ethanol.     

Naltrexone Blocks Acquisition of Voluntary Ethanol Intake in Rats

The effects of naltrexone (NTX) on the acquisition of ethanol drinking was assessed in rats. NTX (0, 2.5, 5.0, or 10.0 mg/kg) was administered to rats presented with an ascending series of ethanol concentrations (2%, 4%, 6%, and 8% v/v) and water. The 2.5 and 10 mg/kg doses of NTX attenuated the acquisition of voluntary drinking of 8% ethanol, but the 5.0 mg/kg dose of NTX had no effect on ethanol intake. The acquisition paradigm was repeated in experiment 2 with naïve animals that received 0, 5.0, or 7.5 mg/kg of NTX. Neither dose of NTX affected ethanol intake, preference for alcohol, or water intake. Total fluid intake was suppressed in the NTX groups, but only on the second presentations of the 2% and 6% concentrations of ethanol. We suggest that the 2.5 and 10 mg/kg doses of NTX may have attenuated the acquisition of ethanol drinking by at least two different behavioral mechanisms.     

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.     

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.     

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.     

Zolpidem generalization and antagonism in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol

Background: The subtypes of γ‐aminobutyric acid (GABA)_A receptors mediating the discriminative stimulus effects of ethanol in nonhuman primates are not completely identified. The GABA_A receptor positive modulator zolpidem has high, intermediate, and low activity at receptors containing α₁, α_2/3, and α₅ subunits, respectively, and partially generalizes from ethanol in several species. The partial inverse agonist Ro15‐4513 has the greatest affinity for α_4/6‐containing receptors, higher affinity for α₅‐ and lower, but equal, affinity for α₁‐ and α_2/3‐, containing GABA_A receptors, and antagonizes the discriminative stimulus effects of ethanol. Methods: This study assessed Ro15‐4513 antagonism of the generalization of zolpidem from ethanol in male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) trained to discriminate 1.0 g/kg (n = 10) or 2.0 g/kg (n = 7) ethanol (i.g.) from water with a 30‐minute pretreatment interval. Results: Zolpidem (0.017 to 5.6 mg/kg, i.m.) completely generalized from ethanol (≥80% of total session responses on the ethanol‐appropriate lever) for 6/7 monkeys trained to discriminate 2.0 g/kg and 4/10 monkeys trained to discriminate 1.0 g/kg ethanol. Zolpidem partially generalized from 1.0 or 2.0 g/kg ethanol in 6/7 remaining monkeys. Ro15‐4513 (0.003 to 0.30 mg/kg, i.m., 5‐minute pretreatment) shifted the zolpidem dose–response curve to the right in all monkeys showing generalization. Analysis of apparent pK_B from antagonism tests suggested that the discriminative stimulus effects of ethanol common with zolpidem are mediated by low‐affinity Ro15‐4513 binding sites. Main effects of sex and training dose indicated greater potency of Ro15‐4513 in males and in monkeys trained to discriminate 1.0 g/kg ethanol. Conclusions: Ethanol and zolpidem share similar discriminative stimulus effects most likely through GABA_A receptors that contain α₁ subunits, however, antagonism by Ro15‐4513 of zolpidem generalization from the lower training dose of ethanol (1.0 g/kg) may involve additional zolpidem‐sensitive GABA_A receptor subtypes (e.g., α_2/3 and α₅).     

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.     

Sex and strain differences in ethanol drinking: effects of gonadectomy

BACKGROUND: Alcohol drinking behavior in rats is known to be sexually dimorphic and strain-dependent. METHODS: To test whether the gonadal steroid milieu exerts activational effects on ethanol intake and can modulate individual sensitivity toward alcohol use and misuse, we examined the effects of gonadectomy on oral self-administration (OSA) of ethanol in male and female rats from different strains. After castration, animals were given continuous free choice between water and ethanol solutions. The ethanol concentration was progressively increased from 2% to 10% and maintained at 6% (the preferred concentration) for 24 days. Ethanol solutions were then withdrawn for 9 days. During the second phase of free-choice drinking, the ethanol concentration was gradually increased every 4 days by the following amounts, in order as listed: 6%, 12%, and 24%. RESULTS: Our results confirm both gender and strain differences in ethanol drinking: females exhibited higher ethanol intake than males, and the WKHA strain drank more than the WKY and SHR strains. However, except for a small decrease in ethanol drinking during the acquisition of ethanol OSA in males after castration, no clear-cut difference was found between gonadectomized and sham-operated animals during the maintenance of ethanol OSA behavior. CONCLUSIONS: These data suggest that gender and strain differences observed are insensitive to gonadal steroids during adulthood, and that different sensitivities to the effect of gonadal steroids do not explain the sex x strain interaction observed in ethanol drinking.     

Low-dose alcohol actions on alpha4beta3delta GABAA receptors are reversed by the behavioral alcohol antagonist Ro15-4513

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant "extrasynaptic" alpha4/6beta3delta GABA(A) receptors at doses that do not reduce the GABA-induced Cl(-) current. At low ethanol concentrations (< or =30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (> or =100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the beta3 subunit (beta3N265M). Therefore, alpha4/6beta3delta GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in alpha4/6beta3delta receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and (ii) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of alpha4beta3N265Mdelta subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant alpha4beta3delta receptors is blocked by flumazenil and beta-carboline-ethyl ester (beta-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABA(A) receptors are important mediators of behavioral alcohol effects.     

Specific Alterations in the Cerebellar GABAA Receptors of an Alcohol-Sensitive ANT Rat Line

The AT (alcohol-tolerant) and ANT (alcohol-nontolerant) rat lines, selected for differential sensitivity to the acute motor-impairing effects of ethanol, have been shown to differ in the ligand binding characteristics of their cerebellar GABAA receptors. In the present study, we characterized these binding differences further and determined whether similar differences are present in other rodent line pairs produced by selective breeding for differences in ethanol sensitivity. The alcohol-insensitive AT rats had more high-affinity [3H]muscimol binding sites in the cerebellum than the alcohol-sensitive ANT rats. The cerebellar "diazepam-insensitive" [3H]Ro 15-4513 binding sites were displaced by several benzodiazepine agonists (diazepam, lorazepam, clonazepam, and midazolam) at micromolar concentrations with greater efficacy in the ANT than the AT rats. Analyses of the displacement curves indicated that the "diazepam-insensitive" [3H]Ro 15-4513 binding sites have 30 to 300 times higher affinity to benzodiazepine agonists in the ANT than AT rats. There was no difference between the rat lines in the displacing potency of Ro 15-1788, a weak partial agonist; Ro 15-4513, a partial inverse agonist; or Ro 5-4864, a peripheral-type benzodiazepine receptor ligand. Thus, the affinity difference seen in the cerebellar [3H]Ro 15-4513 binding sites seems to be specific for benzodiazepine agonists. This difference in affinity may explain the behavioral difference in sensitivity to lorazepam between the rat lines. No differences in [3H]muscimol binding or in the sensitivity of [3H]Ro 15-4513 binding to micromolar diazepam concentrations were found between other rodent line pairs tested (LS/SS, HAS/LAS, HOT/COLD, FAST/SLOW, AA/ANA).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

6-OHDA-Lesions of the Nucleus Accumbens Disrupt the Acquisition but not the Maintenance of Ethanol Consumption in the Alcohol-Preferring P Line of Rats

The aim of the present study was to determine whether reduction of dopamine (DA) innervation to the nucleus accumbens (ACB) alters the maintenance and/or acquisition of ethanol drinking in female alcohol-preferring P rats. Compared with sham-lesioned animals, bilateral microinjections of 6-OHDA (12 μg/2.4 μl/site) into the ACB did not alter the consumption of 10% ethanol in rats that had prior experience of ethanol drinking, with both sham- and 6-OHDA-lesioned groups recovering to presurgical consumption levels at similar rates. On the other hand, the identical lesion procedure disrupted the acquisition of ethanol intake in rats with no ethanol-drinking experience prior to the lesions. A sham-lesioned group attained an ethanol intake of approximately 7 g/kg/day in 1 week, which was maintained over the following 2-week period, while the ethanol intake of the 6-OHDA-lesioned group was approximately 60% lower after 1 week and 30% lower at the end of 3 weeks. DA content of the ACB was 60% lower in both groups of the 6-OHDA-treated rats compared with the controls. The results suggest that different neural mechanisms may underlie the acquisition and maintenance of ethanol drinking behavior; the ACB DA system appears to play an important role in the acquisition of ethanol drinking.     

Effects of Raclopride in the Nucleus Accumbens on Ethanol Seeking and Consumption

BACKGROUND: Nucleus accumbens dopamine has been shown to play a role in the processing of behaviorally relevant stimuli and to mediate ethanol-reinforced responding. Previous research that used a fixed-ratio schedule of responding maintained by the presentation of small dippers (0.1 ml) of ethanol demonstrated that the dopamine D2 antagonist, raclopride, decreased total responding for ethanol by both delaying the onset of responding and causing the early termination of lever-press behavior. Because these studies required animals to continuously respond to obtain access to small amounts of ethanol over a period of self-administration, this procedure assessed a combination of appetitive (seeking) and consummatory (drinking) behavior. The paradigm used in the present study separated the appetitive or seeking response from the consummatory response to assess the effects of raclopride on both types of ethanol-related behaviors. METHODS: Male Long-Evans rats were trained to emit a fixed number of lever-press responses that resulted in access to a drinking tube that contained 10% ethanol for 20 min, once each day. We measured the effects of microinjections of raclopride (1.0, 3.0, and 10.0 microg/subject) into the nucleus accumbens, before the sessions, on appetitive and consummatory responding. RESULTS: Raclopride delayed the onset of ethanol-seeking (appetitive responding) at all doses and decreased the number of responses made at the low and high doses. The rate of responding, however, was unaffected. Raclopride had no effect on the latency to begin consuming ethanol or on any of the characteristics of the initial bout of ethanol intake at all doses tested. Total ethanol intake was decreased, after an initially "normal" pattern of self-administration, following only the highest dose of raclopride. Mean ethanol intake (g/kg) was 0.54 (+/-0.03) after no injection, 0.51 (+/-0.04) after sham treatment, and 0.38 (+/-0.05) after 10 microg of raclopride. CONCLUSIONS: The procedural separation of the seeking and intake responses used in this experiment allowed us to assess the effects of dopamine receptor antagonism in the nucleus accumbens on these two different behaviors. Overall, appetitive responding that preceded the delivery of an ethanol solution was more sensitive to raclopride treatment than was consummatory responding. These findings are consistent with a stimulus-processing function of the mesolimbic dopamine system.     

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

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

Ethanol acceptance is high during early infancy and becomes still higher after previous ethanol ingestion

BACKGROUND: Infant (preweanling) rats readily accept ethanol without initiation procedures. During their second and third postnatal weeks, rats ingest large quantities of even high concentrations of ethanol. The present study tested the consequences of still earlier exposure to ethanol differing in concentration, mode of administration, and contextual circumstances. METHODS: Every 48 hours from postnatal day 2 (P2) to P10, pups were given access to 0, 5, 15, or 25% ethanol through an independent feeding procedure (consumption off the floor; COF) or by intragastric (i.g.) administration. The amount of ethanol consumed was matched for the 2 modes of ethanol delivery. On P12 pups were tested for intake of 15% ethanol through an intraoral infusion test or COF. RESULTS: Beginning on P6, pups ingested more ethanol solution than water, and by P8 and P10 they ingested large quantities of ethanol--1.5 to 2.0 g/kg ethanol from 15 or 25% ethanol solution within a 10-minute period. This early experience with ingestion of ethanol increased subsequent ethanol intake on P12, particularly when concentration and mode of ingestion were the same as before. CONCLUSIONS: Intake on P12 was increased by prior exposure to ethanol intragastrically as well as by the conventional oral route, suggesting pharmacological effects of prior ethanol exposure. Yet, the apparently greater influence of prior exposure by the oral route and the influence of prior ethanol concentration also implicate the importance of ethanol's chemosensory attributes for effects of prior ethanol exposure. The equivalent g/kg intake of ethanol in 15 or 25% solutions during the early part of the second postnatal week suggests that regulation of ethanol intake at P8 and P10 is similar to that observed previously in adults. Change in aspects of ethanol ingestion at about P6 may reflect the shift in function of the GABA system at about this age.