Psychopharmacology (2006) 189:175–186 DOI 10.1007/s00213-006-0546-5

ORIGINAL INVESTIGATION

Dissociation between opioid and CRF1 antagonist sensitive drinking in Sardinian alcohol-preferring rats Valentina Sabino & Pietro Cottone & George F. Koob & Luca Steardo & Mei J. Lee & Kenner C. Rice & Eric P. Zorrilla

Received: 19 June 2006 / Accepted: 29 July 2006 / Published online: 18 October 2006 # Springer-Verlag 2006

Abstract Rationale The role of positive vs negative ethanol reinforcement in ethanol intake of Sardinian alcohol-preferring (sP) rats is unclear. Objectives To test the hypothesis that spontaneous ethanol self-administration of sP rats was sensitive to the opioid receptor antagonist naltrexone, whereas withdrawalinduced, but not spontaneous, ethanol self-administration would be sensitive to corticotropin-releasing factor1 (CRF1) V.S. and P.C. contributed equally to this work. Supported by NIAAA Alcohol Research Center Grant P60 AA0006420-21 (G.F.K., E.P.Z.), NIDDK 64871 (E.P.Z.), by the Intramural Research Program of the NIH, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Institute on Drug Abuse (M.J.L. and K.C.R.), by the Pearson Center for Alcoholism and Addiction Research (G.F.K., E.P.Z.), by a merit fellowship award from the University of Palermo (V.S.), and by a merit fellowship award from the University of Rome La Sapienza (P.C.). V. Sabino (*) : P. Cottone : G. F. Koob : E. P. Zorrilla (*) Molecular and Integrative Neurosciences Department, SP30-2400, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA e-mail: [email protected] e-mail: [email protected] P. Cottone : L. Steardo Department of Human Physiology and Pharmacology, University of Rome La Sapienza, 00185 Rome, Italy M. J. Lee : K. C. Rice Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA V. Sabino : P. Cottone : E. P. Zorrilla Harold L. Dorris Neurological Research Institute, The Scripps Research Institute, La Jolla, CA 92037, USA

antagonists, implicating differential roles for positive and negative reinforcement, respectively. Methods Male sP rats operantly (FR1, 30 min/day) selfadministered ethanol (10% v/v) until responding stabilized. One group (n=11) was made ethanol dependent through intermittent ethanol vapor exposure. Both nondependent (n=10) and dependent rats received the CRF1 antagonist LWH-63 (5, 10, and 20 mg/kg, s.c.). Separate nondependent sP rats (n = 10) received the opioid antagonist naltrexone (16, 50, 150, and 450 μg/kg, s.c.). Finally, CRF1 antagonists (MJL-1-109-2, LWH-63, and R121919) were studied for their actions on home-cage ethanol drinking in nondependent sP rats (n=6–8/group) under continuous, limited-access, or stressed conditions. Results Naltrexone potently reduced ethanol self-administration in nondependent sP rats. LWH-63 reduced heightened ethanol self-administration of vapor-sensitive, dependent sP rats. CRF1 antagonists did not reduce ethanol intake in nondependent sP rats. R121919 (10 mg/ kg, s.c.) retained antistress activity in sP rats, blunting novelty stress-induced suppression of ethanol intake. Conclusions Spontaneous ethanol self-administration of sP rats was opioid dependent with CRF1 receptors implicated in withdrawal-induced drinking. Opioid and CRF1 receptors play different roles in ethanol reinforcement and perhaps the ethanol addiction cycle. Such distinctions may apply to subtypes of alcoholic patients who differ in their motivation to drink and ultimately treatment response. Keywords Sardinian alcohol-preferring or sP rat . Ethanol or alcohol intake . Genetic or selectively bred animal model . Anxiety or stress . Corticotropin-releasing factor or Corticotropin-releasing hormone or CRF or CRH . CRF1 receptor antagonist or CRH1 receptor antagonist . Opioids . Naltrexone . Withdrawal or abstinence . Dependence

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Introduction Two factors believed to contribute to the ethanol addictive process are positive and negative reinforcement. Positive reinforcing effects of ethanol, which are directly related to its acute rewarding properties, may be critical for establishing the addictive behavior. Negative reinforcing effects of ethanol include its ability to relieve negative emotional states, which can become more frequent and severe during repeated withdrawals from ethanol use. For this reason, negative reinforcement is also hypothesized to be a critical component for the maintenance of addictive behavior. Animal models have been developed not only for the acute positive reinforcing effects of ethanol but also for its negative reinforcing effects. Several widely used drug-taking paradigms are thought to reflect positive reinforcing effects of ethanol. These include limited access ethanol self-administration sessions in nondependent outbred animals, a methodology that usually involves a “sweetener→ethanol” fade procedure (Koob 2003; Samson 1986) to circumvent the acute aversion of rats to ethanol. Opioid receptors are thought to mediate the rewarding actions of several drugs of abuse, including ethanol, heroin, and morphine, as well as of nondrug reinforcers (Levine and Billington 2004) due to the ability of opioid receptor antagonists to reduce motivating actions of such substances in limited access sessions (Koob et al. 2003; Martin et al. 2002; Olmstead and Burns 2005; Carrera et al. 1999; Walker et al. 2000) or conditioned place preference models (Delamater et al. 2000). For example, naltrexone, a preferential (but not selective) mu-opioid receptor antagonist, reduces ethanol self-administration in saccharin-faded outbred Wistar rats (Koob 2003), and mu-opioid receptor deficient mice will not readily acquire oral ethanol selfadministration behavior (Roberts et al. 2000). Providing some predictive validity, opioid receptor antagonists are especially effective in reducing episodes of binge drinking in patients with alcoholism (Mason 2003; Mason et al. 1999). In contrast, a proposed animal model of the transition from “recreational” drug taking to compulsive, negatively reinforced use involves the induction of dependence through the chronic exposure of animals to ethanol vapor or ethanol liquid diets. This procedure results in reliably increased ethanol selfadministration in dependent animals during withdrawal (Koob 2003; Roberts et al. 2000) and is accelerated by intermittent, as opposed to continuous, exposure to ethanol vapor (O’Dell et al. 2004). In these negative reinforcement models, ethanol is hypothesized to be drunk excessively to remove the negative emotional state produced by ethanol abstinence. The neuropeptide corticotropin-releasing factor (CRF), a major stress-regulatory molecule, is thought to mediate stress- or withdrawal-induced ethanol-seeking and consummatory behavior (Sarnyai et al. 2001; Valdez et al.

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2002). Acute restraint stress and ethanol withdrawal increase extracellular CRF levels in the central nucleus of the amygdala (CeA) of outbred rats (Merlo-Pich et al. 1995; Richter et al. 2000). Conversely, anxiogenic-like behavior associated with stress or ethanol withdrawal is reduced by intracranial administration of subtype nonselective, peptide CRF receptor antagonists, systemic administration of small molecule CRF1 antagonists, or deletion of the CRF1 receptor gene (Baldwin et al. 1991; Overstreet et al. 2004; Timpl et al. 1998; Valdez et al. 2002; Zorrilla and Koob 2004). In parallel, withdrawal-induced increases in ethanol self-administration of rats rendered dependent by chronic ethanol vapor inhalation were reduced by central administration of anxiolytic-like doses of the CRF receptor antagonist D-PheCRF(12–41) (Valdez et al. 2002) or, as members of our group very recently found, systemic administration of anxiolyticlike doses of small molecule selective CRF1 receptor antagonists (Funk et al. 2006). In contrast, CRF receptor antagonists do not alter anxiety-like behavior in relatively unstressed animals (Zorrilla and Koob 2004) or decrease limited access ethanol self-administration in nondependent outbred rats (Funk et al. 2006; Valdez et al. 2002). Accordingly, dysregulation of CRF–CRF1 signaling via experience of negative affect such as anxiety has been hypothesized to underlie the maintenance or reinstatement of excessive drinking in certain forms of ethanol dependence. The nature of ethanol reinforcement is less clear in genetic models, in which animals selectively bred for high alcohol consumption and preference voluntarily drink ethanol in high quantity and preference over water in their home cages. Among alcohol-preferring rats, the Sardinian alcohol-preferring (sP) rat is one well-characterized line (Colombo 1997) that provides an animal model of some of the signs and symptoms associated with alcoholism (for a review, see McBride and Li 1998). When provided continuous two-bottle choice access to water versus 10% v/v ethanol, sP rats voluntarily drink 5–7 g of ethanol/kg body weight per day and achieve moderate and consistent blood alcohol levels (BALs) at each drinking bout (Colombo et al. 1998). They will also work to obtain ethanol (Vacca et al. 2002) and maintain a constant intake of ethanol when ethanol concentrations are varied. It has been hypothesized that sP rats spontaneously drink ethanol in part to relieve constitutive anxiety (Colombo et al. 1995), a negative reinforcement mechanism analogous to a postulated subtype of negative affect “self-medicating” alcoholism (Lesch and Walter 1996; Merikangas et al. 1998). For example, ethanol-naïve sP rats show increased anxiogenic-like behavior in the elevated plus maze test relative to Sardinian alcohol-nonpreferring rats (sNP), and recent ethanol access reduces anxiogenic-like behavior of sP rats (Colombo et al. 1995). In addition, consistent with this hypothesis, ethanol-naïve sP rats exhibit significantly elevated basal extracellular CRF levels in the

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CeA relative to sNP rats (Richter et al. 2000). These data suggest that perturbations in the regulation of limbic CRF, perhaps via an anxiety-related negative reinforcement mechanism, might also be involved in the constitutively increased ethanol intake of genetically selected sP rats. However, the hypothesis that excessive ethanol drinking of sP rats is driven by a CRF-dependent anxiety-like state is challenged by several findings. For example, sP rats do not show increased anxiety-like behavior when tested in models of “active” anxiety-like behavior, such as defensive burying (Richter et al. 2000), and their pattern of alcohol consumption under continuous access conditions is mainly prandial (Colombo 1997). In addition, anxiolytic agents have not been shown to reduce ethanol intake of sP rats. Rather, bicuculline, which potentially blocks GABAA mediated anxiolyticlike effects of ethanol, did not attenuate ethanol intake of Marchigian–Sardinian alcohol-preferring rats (Perfumi et al. 2002). Furthermore, Ro 19-4603, an anxiogenic-like benzodiazepine receptor inverse agonist, reduced ethanol intake in sP rats (Balakleevsky et al. 1990). These results suggest the alternate hypothesis that ethanol drinking in sP rats is mediated by positive reinforcing effects of ethanol, perhaps via an opioid receptor-dependent mechanism. The present studies therefore tested the primary hypothesis that either opioid antagonists or antistress-like small molecule CRF1 receptor antagonists would reduce the excessive spontaneous oral ethanol self-administration or voluntary drinking of sP rats. In addition, the effects of CRF1 receptor antagonists on withdrawal-induced ethanol self-administration in sP rats exposed to chronic ethanol vapor were also explored. This comparison tested the alternate hypothesis that spontaneous self-administration and withdrawal-induced self-administration in sP rats were differentially subserved. Spontaneous excessive self-administration was postulated to be sensitive to opioid receptor antagonists and withdrawalinduced “excess” self-administration to be sensitive to CRF1 antagonists, putatively reflecting the roles for positive and negative reinforcement, respectively.

Materials and methods

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chow (Harlan Teklad 7012) available ad libitum. Experiments were conducted during the rats’ dark cycle. All experimental procedures adhered to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute. Drugs Ethanol solution (10% v/v) was prepared using 95% ethyl alcohol and tap water. The nonpeptide “second-generation” CRF1 antagonists LWH-63, R121919 and MJL-1-109-2 were used in the present study (Fig. 1). LWH-63 (4-ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3d]pyrimidin-4-yl]amino-1-butanol), a structurally related analog of antalarmin, was synthesized as described in Hsin et al. (2002). At the pH of blood, LWH-63 exhibits the high, selective affinity of antalarmin for CRF1 receptors (Ki =0.7 nM) but is 10-fold more hydrophilic than the excessively lipophilic antalarmin, resulting in increased solubility and bioavailability and decreased toxicity potential (Zorrilla and Koob 2004). R121919 (originally NBI 30775), or 2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3yl)-7-dipropylaminopyrazolo[1,5-a]pyrimidine, was synthesized as described in Chen et al. (2004). R121919 is a high affinity (Ki = 3.5 nM) selective CRF1 antagonist with physiochemical properties superior to many other CRF1 antagonists (e.g., decreased logP and logD, increased water solubility) and which reduced anxious and depressive symptoms in an open-label clinical trial (Zobel et al. 2000; Kunzel et al. 2003; Zorrilla and Koob 2004). MJL1-109-2, or [8-(4-bromo-2-chlorophenyl)-2,7-dimethylpyrazolo[1,5-a][1,3,5]triazin-4-yl]-bis-(2-methoxyethyl) amine, a structural derivative of DMP696 (He et al. 2000), was synthesized as described in Jagoda et al. (2003). MJL1-109-2 penetrates the blood–brain barrier and has high, selective affinity for CRF1 receptors (Ki =1.9 nM) (Jagoda et al. 2003). The current lots of MJL-1-109-2 and R121919 dose-dependently selectively reduced excess ethanol selfadministration of ethanol vapor-dependent outbred Wistar

Animals Male genetically selected Sardinian alcohol-preferring rats (350–450 g at study onset) were subjects in this study. They were bred in the Molecular and Integrative Neurosciences Department of The Scripps Research Institute for 18–20 generations from sP rats at the 32nd generation, provided by Prof. G.L. Gessa, University of Cagliari. Unless stated otherwise, rats were housed in groups of two to three per cage in a humidity- and temperature (22°C)-controlled vivarium on a 12-h light–dark cycle with water and rodent

Fig. 1 Chemical structures of the three CRF1 antagonists used in this study: LWH-63, R121919, and MJL-1-109-2

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rats during acute withdrawal at the doses used herein without affecting self-administration of nondependent Wistar rats (Funk et al. 2006), and LWH-63 exhibited anxiolytic-like activity in the defensive burying test (Zorrilla et al. 2003). For testing, the compounds were first solubilized in 1 M HCl (10% of final volume), then diluted to a final vehicle of 20% (w/v) 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich), backtitrated with NaOH to pH 4.5. The MJL-1-109-2 suspension was administered intraperitoneally (i.p.) in a volume of 4 ml/kg body weight, whereas the R121919 solution and LWH-63 suspension were administered subcutaneously (s.c.) in a volume of 2 ml/kg. Apparatus for operant oral ethanol self-administration The test chambers used for operant oral ethanol selfadministration (Coulbourn Instruments, Allentown, PA) were located in sound-attenuating, ventilated environmental cubicles. Syringe pumps (Razel Scientific Instruments, Stamford, CT) dispensed ethanol or water into two stainless steel drinking cups mounted 4 cm above the grid floor in the middle of one side panel. Two retractable levers were located 4.5 cm to either side of the drinking cups. Fluid delivery and recording of operant responses were controlled by microcomputers. A continuous fixed ratio reinforcement (FR1) schedule was used, and each response resulted in delivery of 0.1 ml of fluid. Lever presses had no scheduled consequences for 2.01 s after the activation of the pumps to avoid double-lever hits. Experiment 1. Ethanol self-administration in sP rats: acquisition and maintenance The first experiment tested the hypothesis that sP rats will spontaneously acquire and maintain operant ethanol selfadministration behavior. For this purpose, sP rats (n=21) were first allowed continuous (24-h/day) two-bottle choice access to ethanol (10% v/v) and water in their home cages for 3 weeks. Intakes were determined daily immediately prior to the onset of the dark cycle by weighing bottles (0.1 g precision). Bottles were filled every 2 days with fresh solution, and their positions were alternated daily to avoid position preference. After ethanol intake and preference had increased to stable levels, rats were allowed limited daily access (1-h/day) for 6 days. Rats were then allowed overnight (16 h) two-choice operant access (0.1 ml volume reinforcer; FR1 schedule, lever press response) to ethanol or water in a test chamber with chow available ad libitum. Following this initial overnight session, all subsequent ethanol self-administration sessions were 30 min in duration and conducted during the first 2 h of the rats’ dark cycle.

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Experiment 2. Effect of chronic exposure to intermittent ethanol vapor on ethanol self-administration in sP rats Experiment 2 tested the hypothesis that a history of chronic exposure to intermittent ethanol vapor would increase ethanol self-administration of ethanol-withdrawn sP rats. After an initial 4-week training period consisting of self-administration sessions conducted 7 days per week, rats were divided into two groups, matched for body weight and ethanol intake during the last four ethanol self-administration sessions. One group received chronic, intermittent exposure to ethanol vapor (dependent group), and the other group served as controls (nondependent group) (n=10–11/group). For the induction of ethanol dependence by vapor exposure, rats were subjected to intermittent exposure to ethanol vapor (14 h/day beginning 2 h into light cycle) in specialized chambers, similar to those previously described (Rogers et al. 1979). Ethanol vapor concentrations were titered to result in target BALs of 150–200 mg% in sP rats across the exposure time. To achieve this, tail blood (0.5 ml) was repeatedly sampled during the ethanol vapor exposure period (twice during the first week, then once a week) and plasma alcohol levels were analyzed using the nicotinamide adenine dinucleotide (NAD)/alcohol dehydrogenase (ADH) enzyme spectrophotometric method (Sigma), as previously described (O’Dell et al. 2004). This paradigm induces physical dependence and increases operant ethanol self-administration during withdrawal from vapors in outbred Wistar rats (O’Dell et al. 2004; Funk et al. 2006). Control rats were kept under similar condition, but without ethanol vapor exposure, as previously described (O’Dell et al. 2004). Starting 3 weeks after initiation of vapor exposure, rats were removed from the chambers three times per week, 6 h after the offset of vapor (when BALs are back to 0 mg/dl, personal observations) and tested for operant ethanol self-administration for a total of eight 30-min test sessions. These sessions allowed subjects to experience the potential negative reinforcing effects of ethanol during acute withdrawal. Experiment 3. Effect of LWH-63 administration on ethanol self-administration in nondependent and acutely withdrawn dependent sP rats Experiment 3 tested the hypothesis that acute administration of the CRF1 antagonist LWH-63 would reduce operant oral ethanol self-administration of nondependent and acutely withdrawn ethanol-dependent sP rats. Rats were pretreated (−1 h) with LWH-63 (0, 5, 10, and 20 mg/kg, s.c.) using a within-subjects Latin square design prior to “withdrawal” test sessions (6 h after vapor offset for dependent rats, n=11, or control conditions for nondependent rats, n=10). Test sessions, 30 min in duration, took place every 2–3 days. After each test session, rats were

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returned to the vapor chambers until the next selfadministration session. Tests were performed after 5.5 to 7 weeks of vapor exposure. Experiment 4. Effect of naltrexone administration on ethanol self-administration in nondependent sP rats Experiment 4 tested the hypothesis that acute administration of the opioid receptor antagonist naltrexone would very potently reduce spontaneous operant oral ethanol selfadministration of nondependent sP rats. Rats (n=10) were pretreated (−30 min) with low doses of naltrexone (0, 16, 50, 150, and 450 μg/kg, s.c.) using a within-subjects Latin square design. Test sessions were 30 min in duration and took place every 2–3 days. Experiment 5. Effect of administration of CRF1 antagonists on home-cage ethanol drinking in sP rats Experiment 5 tested the hypothesis that CRF1 antagonists might be able to reduce voluntary intake, if not spontaneous ethanol self-administration, of nondependent sP rats. Several CRF1 antagonists were studied for their actions on home-cage drinking under different schedules of ethanol access. sP rats in the following experiment had previously received continuous, two-bottle choice access (24 h per day, 7 days per week) to ethanol (10% v/v) and tap water for four consecutive weeks. At the end of the 4 weeks, sP rats consumed about 5 g/kg of ethanol daily with a strong preference (>85%) for ethanol over water. Experiment 5A. Effect of MJL-1-109-2 or R121919 on home-cage ethanol drinking under continuous access conditions

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groups of eight rats each and pretreated (−1 h) with either LWH-63 (0, 5, 10, and 20 mg/kg, s.c.) or R121919 (0, 5, 10, and 20 mg/kg, s.c.) in a within-subjects design every 2 days. Ethanol, water, and food intake were measured hourly during the 2 h of ethanol access. Experiment 6. Effect of R121919 on stress-induced modulation of ethanol drinking Experiment 6 had the following two purposes: (1) to determine the effects of acute anxiogenic-like stimuli on ethanol intake in nondependent sP rats and (2) to determine the ability of a CRF1 antagonist to reverse the effects of such a stressor on ethanol intake in sP rats. The latter finding would indicate that sP rats remain sensitive to the antistress properties of CRF1 antagonists on ethanol intake. Ethanol-experienced sP rats were given limited (2 h/day), daily access to ethanol as in Experiments 5A and 5B. After obtaining a measure of stable baseline intake on prior days, rats were pretreated (1 h prior to ethanol access) with R121919 (0 or 10 mg/kg, s.c., between-subjects design, n= 8 per group) and then exposed to acute novelty stress, which consisted of being placed on an elevated plus maze for 5 min. Rats were then returned to their home-cages where they were allowed their daily 2-h access to ethanol. Intake was measured hourly. The elevated plus maze consisted of four arms made of black Plexiglas (50 cm long×10 cm wide), elevated 50 cm above the floor. Each arm was positioned at a 90° angle to the adjacent arm. Two arms were open, bordered by ledges 0.5 cm in height, and two arms were enclosed with black Plexiglas walls (height, 40 cm). Exposure to the plus maze took place under dim lighting (0.9–1.2 lx along the two open arms). Statistical analysis

In Experiment 5A, ethanol-experienced sP rats on 24-h access to ethanol were divided into four groups, matched for their 24-h ethanol intake and preference over the last 3 days. Rats were pretreated (−1 h) with either MJL-1-1092 (0, 1.25, 2.5, and 10 mg/kg, i.p.) or R121919 (0 and 10 mg/kg, s.c.) using a between-subject design (n=6–8/ dose). Preweighed solutions and food were presented immediately after the onset of the dark cycle. Intake during the first hour of the dark cycle was measured. Experiment 5B. Effect of LWH-63 or R121919 on home-cage drinking under limited access conditions In Experiment 5B, ethanol-experienced sP rats (n=16) were given daily access to ethanol (7 days a week), but only during the first 2 h of their dark cycle (limited access). Food and water were available ad libitum. Once rats had achieved stable ethanol intake, they were divided into two

Intake data were analyzed by analysis of variance (ANOVA) and expressed as M±SEM, normalized for body weight (i.e., ethanol, g/kg; water, ml/kg). In Experiment 1, acquisition of operant ethanol self-administration was analyzed using repeated measures ANOVAs. Rats were divided into subgroups of subjects with “fast” or “slow” acquisition of self-administration (“fast” acquisition being defined as >12 ethanol lever responses with >60% preference ratio over water during the first 2 h of the overnight session). Data were then subjected to separate mixed-design two-way ANOVAs with hour (for the overnight session) and day (for the subsequent daily limited access sessions) as within-subject factors. Rate of acquisition (fast vs slow) was a between-subject factor in both ANOVAs. In Experiment 2, postvapor ethanol responding was analyzed by a repeated measures one-way ANCOVA, with postvapor session as the within-subjects factor and

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prevapor baseline intake as a covariate. Pairwise post hoc comparisons were made using Fisher_s protected least significant difference (PLSD) test, where each session’s intake was compared to prevapor intake. In Experiment 3, the effect of LWH-63 on ethanol intake in nondependent sP rats was analyzed by a one-way ANOVA, with dose as a within-subjects factor; and the effect of LWH-63 on ethanol intake during withdrawal in dependent sP rats was analyzed by a two-way ANCOVA, with subgroup (dependent vaporsensitive and dependent vapor-resistant) as a betweensubjects factor, dose as a within-subjects factor, and prevapor ethanol responding as a covariate. Pairwise post hoc comparisons were made using Student’s t tests. In Experiment 4, the effect of naltrexone on ethanol and water responding was analyzed by one-way ANOVAs, with dose as a within-subjects factor. Pairwise post hoc comparisons were made using Fisher_s PLSD test. In Experiment 5A, intake was analyzed by separate one-way ANOVAs, with dose as the between-subjects factor. In Experiment 5B, intake was analyzed by a three-way ANOVA in which the specific antagonist treatment (R121919 vs LWH-63) was a between-subjects factor and dose and time (1 and 2 h) were within-subjects factors. Pairwise post hoc comparisons were made using Fisher_s PLSD test. In Experiment 6, the effect of R121919 pretreatment on stress-induced suppression of drinking was analyzed by Student’s t test. Potential order effects were evaluated by determining whether effects of CRF1 antagonists on mean ethanol self-administration or intake differed according to the cycle of the Latin square in which the dose was administered. No substantial differences were observed in relation to the cycle of administration. Statistical significance was set at p<0.05.

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Fig. 2 Acquisition of ethanol self-administration in sP rats during daily 30-min sessions. Data are expressed as mean±SEM of ethanol (Black dots) and water (White dots) responses (n=21)

sessions [Ethanol Responses: Day, F(25,500) = 8.54, p < 0.0001; Ethanol Preference: Day, F(25,500) = 5.66, p<0.0001], culminating in rats consuming about 0.8 g/kg during each 30-min session. Individual preference for ethanol versus water was between 92% and 100%. Effect of chronic exposure to intermittent ethanol vapor on ethanol self-administration in sP rats Exposure to intermittent ethanol vapor significantly increased ethanol self-administration in sP rats compared to the prevapor levels [F(8,72)=3.37, p<0.01] (Fig. 3). Post hoc analysis revealed that average ethanol intake signifi-

Results Acquisition and maintenance of ethanol self-administration in sP rats As shown in Fig. 2 (Session 1), TSRI sP rats rapidly acquired ethanol self-administration during the overnight (16-h) session without any need for sweeteners. Rats showed individual differences in the speed of acquisition; some rats (45%) acquired the behavior within the first 2 h of the session and others not until later (“fast” vs “slow” acquisition groups, respectively) [Rate of Acquisition× Hour, F(15,270)=3.15, p<0.0001]; however, the rate of acquisition during the initial overnight session appeared to be unrelated to the individual reinforcing efficacy of ethanol, as demonstrated by the comparable subsequent performance of the two groups in limited access sessions (not shown). Rats gradually increased their responding, and preference for ethanol across the daily limited access

Fig. 3 Effect of chronic exposure to intermittent ethanol vapor (14 h/ day) on 30-min ethanol self-administration of sP rats, 6 h after vapor offset. Acutely withdrawn sP rats drank significantly more ethanol than control rats on five of the eight sessions, conducted three times a week. The dashed line corresponds to prevapor intake level. Shading indicates prevapor (white) vs postvapor (grey) sessions. Data represent mean±SEM of n=8/group. *p<0.05, **p<0.01 vs prevapor intake level (Fisher_s PLSD test)

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Fig. 4 Effect of LWH-63, a CRF1 antagonist, on ethanol self-administration in nondependent sP rats and withdrawn dependent sP rats 6 h after ethanol vapor offset. LWH-63 administration did not affect ethanol self-administration in nondependent sP rats (left panel) (n= 11), but it significantly reduced the vapor-induced increase in ethanol

self-administration of “vapor sensitive” dependent sP rats (n=5); the treatment did not affect ethanol responding in “vapor resistant” sP rats (n=6) (right panel). Data represent mean±SEM. *p<0.05 vs vaporresistant responding, #p<0.05 vs prevapor responding (Fisher_s PLSD test)

cantly exceeded prevapor intake during five of eight sessions, as displayed in Fig. 3. No effects were detected on water responding (data not shown).

[F(3,27)=0.64, ns]. In dependent sP rats, exposure to ethanol vapor significantly increased ethanol responding during withdrawal only in a subgroup of rats, when compared to their prevapor responding, whereas it did not affect ethanol responding in another subgroup of rats [subgroup effect, F(1,8)=7.26, p<0.05]. These two subgroups, which did not differ reliably in age, body weight (prevapor, postvapor, or change), rate of acquisition of ethanol responding, prevapor level of ethanol self-administration, or cage membership, will hereafter be called “vapor sensitive” and “vapor resistant,” respectively. As shown in Fig. 4 (right panel), acute treatment with the CRF1 antagonist LWH-63 significantly reduced vapor-induced increases in ethanol self-administration only in “vapor sensitive” dependent sP rats (n=5); in contrast, the treatment did not affect ethanol responding in “vapor resistant” sP rats (n=6). This was shown by a significant Dose × Subgroup interaction [F(3,24)=3.12, p<0.05]. Water responding ranged from 0.10 to 0.30 ml/kg and was unaffected by vapor history or LWH-63 treatment (data not shown). To rule out the alternative hypothesis that the ability of LWH-63 to reduce ethanol self-administration of “vapor sensitive” rats simply reflected a rate-sensitive effect of LWH-63, the effects of LWH-63 were reanalyzed in those nondependent sP rats that exhibited the highest baseline levels of ethanol responding. In this subgroup of highdrinking nondependent rats, doses of LWH-63 that were effective in vapor-sensitive rats were still without effect (M±SEM: 0.89±0.10 and 0.88±0.07 g/kg, vehicle vs 20 mg/kg, respectively).

Effect of LWH-63 administration on ethanol self-administration in nondependent and dependent sP rats during acute withdrawal As shown in Fig. 4 (left panel), LWH-63 treatment did not reduce ethanol responding in nondependent sP rats (n=11)

Fig. 5 Effect of acute pretreatment with naltrexone on ethanol self-administration in Data represent mean±SEM. **p< 0.01 vs (Fisher_s PLSD test), ###p<0.001, significant

the opioid antagonist nondependent sP rats. vehicle-treated group linear trend of dose

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Table 1 Effect of acute MJL 1-109-2 and R121919 administration on 1-h ethanol and food intake in sP rats: continuous access (24 h/day) Treatment MJL-1-109-2 (mg/kg) 0 1.25 2.5 10 R121919 (mg/kg) 0 10

Ethanol (g/kg)

Food (g/kg)

0.63±0.07 0.35±0.10 0.54±0.17 0.39±0.22

4.85±1.77 4.50±1.39 5.22±2.26 1.93±0.46

0.51±0.03 0.48±0.03

6.46±0.53 6.10±0.38

Data represent mean±SEM intake during the first hour of renewed access. n=6–8 animals/dose.

Effect of naltrexone administration on ethanol self-administration in nondependent sP rats As shown in Fig. 5, naltrexone treatment significantly, dose-dependently, and potently affected ethanol responding in nondependent sP rats (n=10) [Dose: F(4,36)=19.96, p < 0.0001; log-linear trend for Dose: F(1,36) = 77.17, p<0.0001]. Post hoc analysis revealed that the doses of 50, 150, and 450 μg/kg significantly reduced ethanol intake. In contrast, water responding was not affected (data not shown). Effect of CRF1 antagonists on home-cage drinking Continuous access As shown in Table 1, systemic administration of MJL-1-109-2 to nondependent rats in Experiment 5A (0, 1.25, 5, and 10 mg/kg, i.p.) did not reliably

modify 1-h ethanol intake [F(3,23)=1.94, ns] or water intake (data not shown) under continuous access conditions. At the highest dose, MJL-1-109-2 tended to reduce food intake relative to vehicle treatment (60% reduction). Treatment with R121919 (0 and 10 mg/kg, s.c.) did not affect ethanol intake [t(14)=1.39, ns] or food or water intake. Limited access In Experiment 5B, acute s.c. administration of LWH-63 and R121919 (0, 5, 10, and 20 mg/kg) did not reliably decrease voluntary home-cage ethanol intake in nondependent sP rats accustomed to a 2-h limited access schedule. Rather, the antagonists very slightly increased voluntary home-cage ethanol intake [Dose, F(3,42)=3.07, p<0.05; Antagonist identity, F(1,14)=0.19, ns], but they also did so for water intake [Dose, F(3,42)=3.97, p<0.05; Antagonist identity, F(1,14)=0.87, ns], indicating a nonspecific facilitation of fluid intake under these particular experimental conditions (see Table 2). Food intake was unaffected by the CRF1 antagonist treatments (data not shown). Effect of R121919 on stress-induced modulation of ethanol drinking As shown in Fig. 6, novelty stress, as elicited by exposure of subjects to an elevated plus maze, significantly suppressed subsequent 2-h ethanol intake in vehicle-treated rats as compared to baseline consumption [F(1,7) = 8.77, p<0.05]. Rats pretreated with R121919 (10 mg/kg) prior to novelty stress drank significantly more ethanol in 1 h [t(14)=2.12, p<0.05] and 2 h [t(14)=1.85, p<0.05] than vehicle-treated rats, suggesting that CRF1 antagonist pretreatment attenuated novelty stress suppression of drinking.

Table 2 Effect of acute LWH-63 and R121919 administration on 1- and 2-h ethanol and water intake in sP rats: limited access (2 h/day) Ethanol (g/kg) Treatment LWH-63 (mg/kg) 0 5 10 20 R121919 (mg/kg) 0 5 10 20

Water (ml/kg)

1h

2h

1h

2h

0.87±0.11* 0.99±0.10* 1.07±0.06*,** 1.05±0.08 *,**

1.27±0.05* 1.40±0.13* 1.45±0.08* 1.46±0.09*

0.96±0.41 1.01±0.28 1.35±0.57 1.36±0.57

1.43±0.61* 1.40±0.36* 2.23±0.72* 2.51±0.58*

0.81±0.07* 1.05±0.12* 1.14±0.15* 0.93±0.07*

1.23±0.14 1.39±0.12 1.45±0.12 1.27±0.06

0.76±0.32 0.81±0.31 0.93±0.32 0.83±0.34

1.30±0.50 1.37±0.46 1.45±0.58 1.29±0.55

Data represent mean±SEM (n=8 per drug). *p<0.05, significant linear effect of dose **p<0.05 vs vehicle-treated condition (Fisher_s PLSD test)

Psychopharmacology (2006) 189:175–186

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Fig. 6 Effect of acute pretreatment with R121919, a CRF1 antagonist, on stress-induced modulation of ethanol intake under limited access conditions. Acute novelty stress, consisting of a 5-min session on an elevated plus maze, reduced 2-h ethanol drinking, but this suppression was attenuated by R121919 (10 mg/kg, s.c.),

administered 1 h prior to ethanol access. White bars represent basal intake; black bars represent poststress intakes. Data represent mean± SEM (n=8 per group). *p<0.05 vs vehicle-treated group or poststress R121919-treated group (Student_s t test)

Discussion

perhaps analogous to binges in alcoholics, whereas the greater self-administration of dependent, acutely withdrawn sP rats, may instrumentally relieve a CRF1-mediated negative affective state (Mason et al. 1999; Funk et al. 2006). A similar distinction between modes of action of pharmacotherapies for alcoholism also was proposed by Mason (2003). Specifically, naltrexone, which reduces heavy (“binge”) drinking, was proposed to attenuate positive reinforcing actions of ethanol by reducing mesolimbic dopaminergic activity. Acamprosate, which prolongs total abstinence, was proposed to do so by normalizing glutamatergic transmission during protracted withdrawal. Double dissociations in treatment response of patients with different alcoholism subtypes to particular pharmacological or behavioral treatments (Litt et al. 1992; Pettinati et al. 2000; Dundon et al. 2004) further support the hypothesis that different neurochemical systems subserve different stages or subtypes of alcoholism. Ethanol is hypothesized to be consumed by some human alcoholics for its negative reinforcing (e.g., self-medicating) effects, but perhaps by other subtypes for its positive reinforcing effects (e.g., euphoria). For example, the negative reinforcing properties of ethanol have been proposed to play a role in excessive intake of type 1 alcoholics, which is characterized by constitutive negative affect, or in the motivation to continue or resume drinking to oppose negative affective symptoms of abstinence, such as anxiety (American Psychiatric Association 2000). Consistent with this hypothesis, pharmacological agents that reverse the stress-like consequences of ethanol withdrawal, such as CRF1 receptor antagonists (Rassnick et al. 1993),

The primary finding of the present experiments is that spontaneous and withdrawal-induced ethanol reinforcement are subserved by different neurochemical mechanisms in Sardinian alcohol-preferring rats. Systemic administration of a CRF1 receptor antagonist eliminated the withdrawalassociated increment in ethanol self-administration of dependent sP rats. At the same time, none of three CRF1 antagonists tested under operant or free access conditions reduced the high basal levels of responding for ethanol or voluntary ethanol intake in nondependent sP rats. The inefficacy of CRF1 antagonists against ethanol intake in nondependent rats did not reflect a general insensitivity to CRF1 ligands, as R121919 (10 mg/kg) blunted the ability of novelty stress to suppress ethanol intake, confirming an “antistress”-like activity of CRF1 antagonists vis-à-vis ethanol consumption in sP rats. In contrast, acute treatment with naltrexone, an opioid receptor antagonist, very potently (16–450 μg/kg) and efficaciously reduced basal ethanol self-administration of nondependent sP rats. Thus, basal ethanol intake of nondependent sP rats is not motivated by self-medication of a CRF1-receptor mediated anxiety-like state. Rather, an opioid receptor-dependent mechanism underlies their spontaneous excessive ethanol intake. However, withdrawal-induced increments in ethanol self-administration by dependent sP rats, like that of Wistar rats (Funk et al. 2006), are amenable to CRF1 antagonist intervention. The results support the hypothesis that different aspects of the alcohol addiction process may be modeled in sP rats. Their spontaneously high intake may be positively reinforced by an opioid-dependent mechanism,

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also block withdrawal-associated increases in operant oral ethanol self-administration in alcohol-dependent rats as shown in the current study and elsewhere (Valdez et al. 2002; Funk et al. 2006). However, CRF1 antagonists do not decrease ethanol intake of nondependent genetically selected sP rats (present study) or outbred rats (Valdez et al. 2002; Funk et al. 2006). Thus, CRF stress-regulatory systems may uniquely be involved in excessive intake motivated by constitutive or withdrawal dysphoria (Valdez and Koob 2004). In this context, the lack of action of CRF1 receptor antagonists against the high basal ethanol intake of sP rats suggests that their spontaneously high ethanol selfadministration and the heightened ethanol self-administration of withdrawal are motivated and subserved by (partly) different mechanisms. The observed single dissociation between the efficacy of naltrexone and CRF1 antagonists in the current study of nondependent vs dependent sP rats is consistent with this perspective and provides further evidence for the hypothesis that opioid and CRF1 receptors play different roles in the ethanol addiction cycle. For example, in Wistar rats, naltrexone reduces both baseline and withdrawal-induced ethanol self-administration (Koob et al. 2003) (whether the latter effect of naltrexone also is present in sP rats was not tested in the present study, so a double dissociation remains possible). CRF antagonists, on the other hand, do not block baseline alcohol drinking in outbred Wistar rats, but they do block the excessive drinking associated with alcohol withdrawal in Wistar rats (Valdez et al. 2002). Furthermore, naltrexone also blocks cue-induced reinstatement of alcohol-seeking behavior, but not stress-induced reinstatement (Liu and Weiss 2002), whereas CRF antagonists block stress-induced relapse but not cue-induced relapse (Liu and Weiss 2002). The high potency of naltrexone (minimum effective dose ≤50 μg/kg) to reduce ethanol self-administration in nondependent sP rats might suggest a basal difference in opioid transmission of alcohol-preferring rats. For example, substantially higher naltrexone doses did not reduce ethanol self-administration in some studies of outbred rats (e.g., up to 3 mg/kg in Wistar rats) (Bienkowski et al. 1999). Consistent with this hypothesis, more binding sites for the μ-opioid receptor-selective radioligand [3H]DAMGO were seen in several discrete brain regions of Indiana P rats (McBride and Li 1998), another line of rats genetically selected from Wistar stock for high alcohol preference. Further study may better clarify the role of constitutive differences in μ-opioid system responsiveness in the ethanol preference of Sardinian alcohol-preferring rats. The high spontaneous ethanol consumption of sP rats was previously proposed by us (Richter et al. 2000) and others (Colombo 1997) to reflect “self-medication” of high innate anxiety, with the suggestion that sP rats consumed ethanol for its negative reinforcing, “tension-reducing”

Psychopharmacology (2006) 189:175–186

properties, analogous to the “tension-reduction” hypothesis of human ethanol intake (Conger 1956; Cappell and Herman 1972). If so, one would predict that biologically active doses of antistress drugs, such as CRF1 antagonists, would reduce the excessive ethanol drinking of sP rats. In the present experiments, sP rats received second-generation CRF1 antagonists that exhibit anxiolytic-like activity in rat models (Zorrilla and Koob 2004; Jagoda et al. 2003; Zorrilla et al. 2003; Heinrichs et al. 2002; Gutman et al. 2003), each of them with nanomolar affinity for CRF1 receptors and reasonable lipophilicity (clogP=3.1, 5.2, 4.8, respectively). The antagonists were the pyrazolotriazine MJL-1-109-2, a derivative of DMP-696 (He et al. 2000) first synthesized by Jagoda et al. (2003); LWH-63, a more hydrophilic hydroxy-derivative of antalarmin (Zorrilla et al. 2002; Hsin et al. 2002); and R121919, a pyridyl-pyrazolopyrimidine with good physiochemical properties that reduced anxious symptoms in humans in an open-label trial (Zobel et al. 2000; Kunzel et al. 2003; Zorrilla and Koob 2004). Under no condition did biologically active doses of the CRF1 antagonists reduce operant or nonoperant ethanol self-administration in nondependent sP rats. These findings did not reflect a lack of antistress activity by CRF1 antagonists in sP rats because R121919 blunted the action of novelty stress to suppress ethanol intake acutely. In contrast to this inefficacy against drinking in nondependent sP rats, acute treatment (5–20 mg/kg) with LWH-63, a selective nonpeptide CRF1 antagonist, inhibited ethanol self-administration in “vapor-sensitive” dependent sP rats—that is, rats that responded to acute withdrawal from chronic ethanol vapor by increasing their ethanol intake. The withdrawal-associated increased intake shown by this subset of rats was abolished by pretreatment with the CRF1 antagonist. In contrast, sP rats that did not respond to withdrawal by increasing their ethanol selfadministration (“vapor-resistant” rats) remained, like the spontaneously highest drinking nondependent rats, insensitive to LWH-63, further supporting the hypothesis that CRF1 receptors mediate acute withdrawal-induced excessive intake. The results are analogous to our recent findings in withdrawn outbred Wistar rats that CRF1 antagonists only reduce withdrawal-induced increases in ethanol selfadministration and do not appear to modulate basal, putatively positively reinforced, levels of operant responding (Funk et al. 2006). Selectively bred sP rats were heterogeneous in their vulnerability to develop increased ethanol self-administration in response to chronic intermittent ethanol vapor exposure. Because variability was present in genetically selected sP rats, which share genes that determine baseline 24-h voluntary intake and preference of 10% v/v ethanol but remain “unfixed” in other alleles, then either unselected (and still heterogeneous) alleles underlie the propensity to

Psychopharmacology (2006) 189:175–186

increase ethanol self-administration during withdrawal from chronic ethanol or environmental factors contribute importantly to variability in this response. Consistent with both possibilities, the increment in ethanol responding during withdrawal was unrelated to the level of ethanol selfadministration prior to induction of dependence and the speed of initial acquisition of ethanol responding. Vapor “sensitivity” also was unrelated to age, body weight, and cage membership. One question of interest is whether rats that increased their ethanol self-administration during withdrawal from vapor exposure also differentially showed increased anxiogenic-like behavior as compared to vaporinsensitive rats. Such a finding would strengthen further the proposed role of negative reinforcement in withdrawalinduced, CRF1 antagonist-mitigated drinking. Intraperitoneal administration of MJL-1-109-2 reduced food intake in this study. Similar anti-ingestive effects were not observed following subcutaneous administration of similar or higher doses of R121919 or LWH-63. Whether these findings reflect differences in the route of administration or antagonist is unclear. Antalarmin also reduces intake nonspecifically when administered intraperitoneally at high doses (e.g., antalarmin; Zorrilla et al. 2002; Hope et al. 2000), whereas the CRF1 antagonist CRA-1000 does not (Ohata et al. 2002). The results advocate caution in assuming the specificity of MJL-1109-2 and possibly other CRF1 antagonist effects on appetitive behavior. In summary, the present experiments found that withdrawal-induced but not spontaneous ethanol drinking of sP rats is decreased by CRF1 antagonists and that novelty stress reduces ethanol intake of sP rats in a CRF1 antagonist reversible manner. The results do not support the hypotheses that CRF1 receptors mediate spontaneous excess ethanol intake of sP rats or that sP rats drink ethanol to relieve an anxiety-like state. However, it cannot be ruled out that anxiety-related substrates unrelated to CRF are altered in sP rats. Alternatively, sP rats may model a form of excessive drinking that is not motivated by tension reduction, but rather by the acute rewarding properties of ethanol and sensitive to opioid receptor antagonist treatment. In a similar way, different subtypes of alcoholic patients can be distinguished (Lesch and Walter 1996), according to their motivation to drink; correlated biochemical, psychological, clinical and behavioral characteristics; and, ultimately, treatment response. The different aspects of alcoholism that each of these models might represent may be critical for translating findings of preclinical efficacy to the different stages or subtypes of ethanol dependence. Acknowledgments This is manuscript number 18046 from The Scripps Research Institute. The authors gratefully recognize the technical assistance of Molly Brennan, Maury Cole, Robert Lintz, and Maegan Mattock and the editorial assistance of Mike Arends.

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