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Alcohol 48 (2014) 277e286

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Alcohol

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Operant alcohol self-administration in dependent rats: Focus on the vapor model

Leandro F. Vendruscolo a, Amanda J. Roberts b,*

aCommittee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USAbMolecular and Cellular Neuroscience Department, Mouse Behavioral Assessment Core, The Scripps Research Institute, 10550 North Torrey Pines Road, MB18, La Jolla, CA 92037, USA

a r t i c l e i n f o

Article history:Received 3 June 2013Received in revised form2 August 2013Accepted 5 August 2013

Keywords:AlcoholismAddictionAlcohol dependenceAlcohol (ethanol) vaporOperant self-administrationCompulsive behaviorRatReview

* Corresponding author. Tel.: þ1 858 784 9802.E-mail address: aroberts@scripps.edu (A.J. Roberts

0741-8329/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.alcohol.2013.08.006

a b s t r a c t

Alcoholism (alcohol dependence) is characterized by a compulsion to seek and ingest alcohol (ethanol),loss of control over intake, and the emergence of a negative emotional state during withdrawal. Animalmodels are critical in promoting our knowledge of the neurobiological mechanisms underlying alcoholdependence. Here, we review the studies involving operant alcohol self-administration in rat models ofalcohol dependence and withdrawal with the focus on the alcohol vapor model. In 1996, the first articleswere published reporting that rats made dependent on alcohol by exposure to alcohol vapors displayedincreased operant alcohol self-administration during acute withdrawal compared with nondependentrats (i.e., not exposed to alcohol vapors). Since then, it has been repeatedly demonstrated that this modelreliably produces physical and motivational symptoms of alcohol dependence. The functional roles ofvarious systems implicated in stress and reward, including opioids, dopamine, corticotropin-releasingfactor (CRF), glucocorticoids, neuropeptide Y (NPY), g-aminobutyric acid (GABA), norepinephrine, andcannabinoids, have been investigated in the context of alcohol dependence. The combination of modelsof alcohol withdrawal and dependence with operant self-administration constitutes an excellent tool toinvestigate the neurobiology of alcoholism. In fact, this work has helped lay the groundwork for severalongoing clinical trials for alcohol dependence. Advantages and limitations of this model are discussed,with an emphasis on what future directions of great importance could be.

� 2014 Elsevier Inc. All rights reserved.

Introduction

Increased operant alcohol (ethanol) self-administration in ratsassociated with alcohol dependence and withdrawal produced byalcohol vapor exposure was first demonstrated in 1996 (Roberts,Cole, & Koob, 1996). However, there was a very important body ofwork published prior to this that was critical in the development ofthis rat/ethanol-vapor/operant model. This history will be sum-marized in a manner that will highlight aspects of this model thatengender excessive alcohol intake. We will also review what hasbeen discovered using the vapor/operant model with respect toboth environmental and biological factors. Finally, we will discussadvantages and limitations of this model with an emphasis onwhatfuture directions we believe could be of great importance. But first,what was the motivation to develop such a model? Why drinkingsubsequent to dependence? Why operant self-administration?Why rats?

Alcohol was involved in 3.5% of deaths in the United States in2000, making it the third-leading cause of preventable death in thiscountry (Mokdad, Marks, Stroup, & Gerberding, 2004). Alcohol

).

ll rights reserved.

abusers drink perhaps partly for its euphorigenic effects, but pro-gressively more in order to avoid or reverse the negative symptomsassociated with withdrawal (Cappell & LeBlanc, 1981; Edwards,1990). Indeed, the Diagnostic and Statistical Manual of MentalDisorders, 4th edition (DSM-IV) criteria for substance dependenceon alcohol include awithdrawal syndrome and taking the substance(or a closely related substance) to relieve or avoid withdrawalsymptoms (American Psychiatric Association, 2000), similar to theDSM-V criteria for moderate to severe substance use disorder(O’Brien, 2011; Peer et al., 2013). The affective components ofwithdrawal, such as anxiety, dysphoria, and depressedmood, createa motivational drive that leads to compulsive ethanol drinkingbehavior and relapse even after long periods of abstinence(Hershon, 1977). These affective symptoms begin as blood alcohollevels drop and can continue for weeks tomonths to years followingwithdrawal (Alling et al., 1982; Mossberg, Liljeberg, & Borg, 1985;Parsons, Sinha, & Williams, 1990). Alcohol dependence is associ-ated with high rates of relapse, which is characterized by a return todrinking after a period of abstinence and involves the consumptionof excessive amounts of alcohol (U.S. Department of Health andHuman Services, 1990). Therefore, alcohol dependence is a disor-der with chronic relapses, with serious consequences to the indi-vidual, family, and society. Therefore, having a model of ethanol

L.F. Vendruscolo, A.J. Roberts / Alcohol 48 (2014) 277e286278

self-administration in animals experiencing withdrawal and inabstinent animals is important for the advancement of better pre-vention and treatment approaches.

Free-choice bottle drinking models capture consummatory as-pects, whereas operant self-administration is more versatile inmodeling different behavioral aspects of alcohol drinking. Both theappetitive/motivational (e.g., pressing a lever [workload] to receivea dose of alcohol) and consummatory (e.g., drinking the alcohol)components of ethanol consumption can be studied in operantmodels (Cunningham, Fidler, & Hill, 2000; Tabakoff & Hoffman,2000). Appetitive behaviors become compulsive as dependenceprogresses (Koob, 2013), in that they become persistent and re-petitive without leading to actual reward or pleasure. Compulsivityis described in the DSM: continued use despite knowledge of havinghad a persistent or recurrent physical or psychological problem anda great deal of time spent in activities necessary to obtain thesubstance. Thus, assessing operant ethanol self-administration independent animals allows the appetitive and consummatory pro-cesses and, in particular, the compulsive nature of addiction to bestudied.

Finally, there is a rich history of using rats in behavioral brainresearch. Issues of reliability and validity are critical in developingand utilizing animal models of complex neuropsychiatric disordersand must always be considered (Edwards & Koob, 2012; Geyer &Markou, 1995). As discussed by Bell et al. (2012), an effective ani-mal model of alcoholism should include both positive (euphoric)and negative (eliminating negative aspects of withdrawal) rein-forcement aspects. Several such models, including the one de-scribed in this review, have been developed, and are invaluable forstudies of neuropharmacological mechanisms of alcoholism thatwould be impossible to do in humans. For example, significant un-derstanding of the neurocircuitry involved in drug-seeking behaviorin the addicted state has come from rat studies, and, indeed, ratneuropharmacological studies continue to drive the development ofnew medication targets (Koob, 2010).

History and highlights in the development of the rat operant/vapormodel

Previous studies examined ethanol-drinking behavior independent animals. Both increases (Deutsch & Koopmans, 1973;Deutsch & Walton, 1977; Hunter, Walker, & Riley, 1974; Samson &Falk, 1974; Schulteis, Hyytiä, Heinrichs, & Koob, 1996; Veale &Myers, 1969; Wolffgramm & Heyne, 1991) and decreases (Begleiter,1975; Myers, Stoltman, & Martin, 1972; Winger, 1988) in ethanolintake were observed. In examining these studies, it became clearthat there were two general concepts that likely played a role inthese differential results that would require attention prior to beingable to produce a robust and reliable model. These were issues ofethanol’s palatability and reinforcing properties prior to induction ofdependence and concerns regarding the post-dependence with-drawal spectrum. Specifically, how should researchers producedependence while minimizing the potential of excessive physicalsymptoms that would compete with appetitive and/or consumma-tory behaviors, thus allowing the animals to learn the associationbetween drinking ethanol and the alleviation of withdrawal symp-toms?

Deutsch and Walton (1977) used a procedure in which the ratsdrank a flavored solution to receive infusions of ethanol directly intothe stomach and showed that dependence enhanced preference forthe ethanol-paired flavor. This model bypassed the aversive tasteproperties of ethanol and allowed ethanol to become a reinforcer.Historically, low levels of intake hampered rat models of oralethanol self-administration unless the animals were food- or fluid-deprived. Consumption, therefore, could be motivated by thirst or

the need for the calories in the ethanol solution, and not by etha-nol’s pharmacological effects. This changed with the breeding ofethanol-preferring rat strains (reviewed byMcBride, this issue) and,in outbred strains, the development of the sweetened solutionfading procedure by Samson and colleagues (Samson, 1986). In thelatter model, ethanol is initially sweetened, and ethanol concen-trations are gradually increased such that non-deprived rats willmaintain lever pressing for high concentrations of ethanol thatresult in pharmacologically relevant blood alcohol levels. Thesweetener is then removed gradually so that by the end of theprocedure, the rats are drinking unsweetened alcohol solutions.This development paved theway for subsequent studies by partiallysolving both the palatability and physiological need issues. None-theless, these procedures do not result in levels of alcohol intoxi-cation to the point of dependence.

The problem of making the rats dependent was an outstandingissue. It was known that most rats would not voluntarily consumeenough ethanol to induce dependence (Myers & Veale, 1972;Samson, Pfeffer, & Tolliver, 1988; Veale & Myers, 1969). Rats canbe made dependent on alcohol by repeated exposure to high dosesof alcohol via gastric intubation, oral gavage, mixing ethanol into aliquid diet, and systemic injections. While these techniques havebeen successfully used to produce dependence and subsequentincreases in ethanol intake (Deutsch & Walton, 1977; Hunter et al.,1974; Schulteis et al, 1996), intubation or injections require eitherstressful repeated administration or surgical intragastric cannula-tion. Ethanol-containing liquid diet-induced dependence has beenshown to produce escalated alcohol self-administration duringacute withdrawal compared with control rats (Schulteis et al, 1996).However, this response pattern depends on high blood alcohollevels at the time of withdrawal, and intake during the dependenceinduction phase can be difficult to control in this model. In addition,the liquid diet approach can have the caveat of potential mal-nourishment in both the ethanol- and pair-fed groups (Rogers,Wiener, & Bloom, 1979). Several laboratories began employingvaporized ethanol exposure to induce dependence (i.e., Karanianet al., 1986; Rigter, Dortmans, & Crabbe, 1980; Rogers et al., 1979).Thismethod has the benefit of more precise control of blood alcohollevels across varying periods of time and therefore allows for theexamination of known exposure patterns on behavior, physiology,and biochemistry.

The second challenge with dependence is capitalizing on theaffective symptoms without the excessive physical symptomsrendering the rats incapable of appetitive or consummatory be-havior. In all three of the studies that showed decreased ethanolintake following dependence, significant physical withdrawalsymptoms were observed. The majority of the rats in the Begleiter(1975) study had convulsions, and all of the monkeys in both theMyers et al. (1972) and Winger (1988) studies showed tremorduring withdrawal.

Finally, a critical component of the DSM criteria for substancedependence on alcohol or moderate to severe substance usedisorder must be established, namely taking the substance (or aclosely related substance) to relieve or avoid withdrawal symptoms(American Psychiatric Association, 2000; O’Brien, 2011; Peer et al.,2013). This requires the animal to experience symptoms of with-drawal or abstinence with ethanol available and then to associateethanol intake with the alleviation of these symptoms. Hunter et al.(1974) showed that rats did not voluntarily consume ethanolfollowing a single 20-day period of forced liquid diet despite thepresence of withdrawal symptoms. However, following a series of3 liquid diet exposures followed by free choice testing, the ratstransiently increased ethanol-drinking behavior, suggesting thatthe rats needed to learn the association between drinking ethanoland the alleviation of withdrawal symptoms.

Fig. 1. (A) Operant responding for alcohol across the second 12-h test period bydependent (alcohol vapor) and nondependent (air) rats. (B) Blood alcohol levels takenduring this second test (left) as well as at the same time points in a third test in whichrats were not able to lever-press for ethanol (right). (C) Alcohol withdrawal severityobtained during test 2 (while rats were allowed access to alcohol in the operant boxes)and test 3 (while in home cages). Taken from Roberts et al. (1996), with permission.

L.F. Vendruscolo, A.J. Roberts / Alcohol 48 (2014) 277e286 279

Fig. 1 (Roberts et al., 1996) illustrates several of the aspectsdescribed above, that are believed to be critical in developing asuccessful model of increased operant ethanol self-administrationin rats associated with alcohol dependence and withdrawal. First,operant ethanol self-administration was initially established innon-deprived animals, such that lever pressing for 10% ethanol wasstable. Second, operant responding for 10% ethanol was enhanced

following dependence induction by 14 days of continuous ethanolvapor exposure, maintaining blood alcohol levels of 150e200 mg%.This blood alcohol level range is associated with mild-to-moderatewithdrawal symptoms (Macey, Schulteis, Heinrichs, & Koob, 1996);therefore, it appeared that there was no interference in leverpressing and drinking behavior by physical symptoms. The ratswere allowed access to ethanol immediately following removalfrom the vapor chambers for 12 h across several withdrawalperiods. Therefore, the rats were given the opportunity to associateethanol consumption with the alleviation (and apparently avoid-ance by the second test) of symptoms as they arose.

This model has evolved over the years to include operantethanol self-administration testing after several weeks of with-drawal (Gilpin, Richardson, Cole, & Koob, 2008; Gilpin, Richardson,Lumeng, & Koob, 2008; Roberts, Heyser, Cole, Griffin, & Koob, 2000;Sommer et al., 2008), the use of intermittent ethanol vapor expo-sure to produce more rapid increases in the self-administration ofethanol relative to continuous exposure, and operant testing 6e8 hinto withdrawal during these intermittent vapor exposure periods(O’Dell, Roberts, Smith, & Koob, 2004). The next section describesthe rat/ethanol-vapor/operant model used currently and its use inelucidating neuropharmacological correlates of the increased op-erant ethanol self-administration following dependence induction.The focus of this review is on the alcohol vapor model, but a fewstudies involving liquid diet, which produces blood alcohol levelscomparable to vapor exposure, will be discussed.

Significant environmental and biological factors associated with thismodel

Operant self-administration is typically conducted in standardoperant conditioning chambers fitted with retractable levers andfluid receptacles. As mentioned above, because of the aversivepalatable properties of alcohol, rats will normally not readily press alever to drink alcohol. To overcome this issue, a method based onthat developed by Samson (1986) is used to facilitate the acquisitionof alcohol self-administration. Briefly, rats are trained to press alever to receive a highly palatable sweet solution (e.g., 10% sucrose).Alcohol is gradually added to the solution, and sucrose is graduallyeliminated. At the end of the training (a few weeks), rats will leverpress to receive an unsweetened alcohol solution. Some variationsof this method have been described (Walker & Koob, 2007). Alter-natively, rats are trained to lever press for water in long operantsessions (e.g., overnight). Subsequently, alcohol solution is providedinstead of water in 2-h, 1-h, and then 30-min sessions (Edwards,Guerrero, Ghoneim, Roberts, & Koob, 2012; Vendruscolo et al.,2012). Operant training is typically performed on a fixed-ratio 1(FR1) schedule of reinforcement, inwhich every operant response isreinforced with the delivery of alcohol solution.

Upon the completion of operant self-administration training, therats are exposed to continuous or intermittent alcohol vapor expo-sure (blood and brain alcohol levels between 150 and 250 mg/dL;Gilpin et al., 2009) to produce alcohol dependence (see Gilpin,Richardson, Cole, et al., 2008, for review). Operant alcohol self-administration sessions are performed during acute (2e10 h intowithdrawal from alcohol vapor) or protracted (one or more weeksafter removal from vapor) withdrawal. In this model, rats exhibitphysical withdrawal symptoms (e.g., tail stiffness, abnormal gait/posture and tremor; Macey et al., 1996) and emotional symptoms,reflected by increased anxiety-like behavior, hyperalgesia, increased22 kHz ultrasonic vocalizations, and elevated brain reward thresh-olds (Edwards et al., 2012; O’Dell et al., 2004; Rimondini, Sommer, &Heilig, 2003; Roberts et al., 2000; Schulteis, Markou, Cole, & Koob,1995; Sommer et al., 2008; Valdez et al., 2002; Williams et al.,2012; Zhao, Weiss, & Zorilla, 2007). Nondependent rats are

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maintained and tested under similar operant conditions but are notexposed to alcohol vapors. Intermittent alcohol vapor exposure hasbeen shown to produce a more rapid escalation of alcohol intakecompared with continuous vapor exposure (O’Dell et al., 2004),indicating that repeated cycles of intoxication and withdrawal aremore effective in producing dependence.

As illustrated in Fig. 2, rats exposed to chronic, intermittentalcohol vapor and tested during acute withdrawal (6e8 h afterremoval from vapor) display increased alcohol self-administrationcompared with nondependent rats in an FR1 schedule of rein-forcement (Fig. 2A). Responding on an FR1 schedule requires min-imal effort by the animal to obtain alcohol and is sometimesconsidered a consummatory measure. Dependent rats also displayincreased response levels for alcohol on a progressive-ratio (PR)schedule of reinforcement, under which the number of leverpresses necessary to obtain the next availability of alcohol increasesprogressively (Fig. 2B). In this test, the workload (“price”) for thenext alcohol reinforcer increases progressively until the rat reachesa “breakpoint” (i.e., a measure of compulsivity) beyond which it nolonger performs for alcohol. Additionally, dependent rats displaymore persistent consumption of alcohol than nondependent rats asquinine concentrations that are added to the solution are increased(Fig. 2C; i.e., they maintain ethanol consumption despite the aver-sive bitter taste of quinine). This is considered another measure ofcompulsive-like alcohol intake. Finally, dependent rats did notdiffer from nondependent rats in relation to the self-administrationof a sweet solution (without alcohol), indicating that the escalationof alcohol intake is specific for alcohol (Fig. 2D; O’Dell et al., 2004).These findings suggest that alcohol vapor exposure produces

Fig. 2. Specific increase in alcohol intake and compulsive-like drinking in alcohol vapor-exbefore (pre-vapor) and during alcohol vapor exposure on an FR1 schedule of reinforcement.drinking despite the aversive bitter taste of quinine (added to the alcohol solution). (D) Insignificant difference between dependent and nondependent; #p < 0.05, significant differe

escalation of alcohol intake and compulsive-like drinking that isspecific for alcohol (Vendruscolo et al., 2012).

The positive reinforcing properties of alcohol initially motivatealcohol intake. Alcohol self-administration increases the levels ofdopamine and serotonin (5-HT) in the nucleus accumbens (NAc;Weiss et al., 1996). However, following a prolonged history ofalcohol dependence, negative reinforcement becomes a dominantmotivational factor for continued alcohol use (i.e., alcohol is used toalleviate or prevent negative emotional states, such as anxiety,dysphoria, and hypohedonia, that emerge in the absence of thedrug). During the development of dependence, neurotransmissionin reward-/stress-related brain regions is changed. In fact, in addi-tion to changes in the systems involved in the initial reinforcingeffects of ethanol, other systems are recruited as these negativemotivational factors become critically important.

For example, the suppression of dopamine levels in the NAcoccurs with chronic ethanol liquid diet exposure, and these levelsare normalized after alcohol self-administration, suggesting that adeficit in dopamine release in the NAcmaymotivate dependent ratsto more vigorously lever press for alcohol to normalize theirdopamine levels (Weiss et al., 1996).

Differences in 5-HT and dopamine systems with respect toethanol withdrawal and ethanol self-administration have also beenreported in rats with left vs. right functional brain asymmetry.Similar to humans, rats also exhibit patterns of hemisphericspecialization, and turning behavior is used as an index of the de-gree and direction of asymmetry. Animals with right turning pref-erences displayed lower 5-HT and dopamine concentrations in theright side of the prefrontal cortex comparedwith the left side during

posed rats during acute alcohol withdrawal. (A) Alcohol intake in g/kg of body weight(B) Breakpoint for alcohol achieved in a progressive-ratio test. (C) Persistence of alcoholtake (in mL/kg of body weight) of a saccharin (0.004%, w/v) solution (FR1). *p < 0.05,nce from 0. Modified from Vendruscolo et al. (2012), with permission.

Table 1Drug effects on operant alcohol self-administration and reinstatement of alcohol seeking in rats exposed to alcohol vapor (dependent) and control (nondependent) rats.

Author(s) Year Compound/action Nondependent Dependent Withdrawal time

Liu and Weiss 2002 SCH23390 (D1 antagonist) Y(R) Y(R) 21 daysLiu and Weiss 2002 Eticlopride (D2 antagonist) Y(R) Y(R) 21 daysCiccocioppo et al. 2003 Naltrexone (m opioid antagonist) Y(R) Y(R) 1 weekKufahl et al. 2011 LY379268 (mGluR 2/3 agonist) Y(R) Y(R) 1 weekRichardson et al. 2008 MPZP (CRF1 antagonist) __ Y 6e8 hValdez et al. 2002 D-Phe-CRF12e41 ICV (CRF antagonist) __ Y 2 hFunk et al. 2006 D-Phe-CRF12e41 (CRF antagonist) in CeA __ Y 2 hFunk et al. 2006 D-Phe-CRF12e41 (CRF antagonist) in NAc or BNST __ __ 2 hRoberto et al. 2010 R121919 (CRF antagonist) Y Y 6e8 hFunk and Koob 2007 Urocortin 3 (CRF2 agonist) __ Y 2 hVendruscolo et al. 2012 Mifepristone (GR antagonist) __ Y 6e8 hVendruscolo et al. 2012 Mifepristone (GR antagonist) __ Y 3e6 weeksThorsell et al. 2005 Neuropeptide Y (ICV) NT Y 2 weeksGilpin et al. 2011 NPY (ICV) Y Y 6 hKallupi et al. 2013 JNJ-31020028 (systemic) and BIIE0246 (intra-CeA): NPY Y2

antagonists

__ __ 6e8 h

Roberts et al. 1996 Muscimol (GABAA agonist) __ Y 0e12 hRoberto et al. 2008 Gabapentin __ Y 2 hWalker and Koob 2007 Baclofen (GABAB agonist) Y Y 6 hWalker et al. 2011 nor-binaltorphimine (k opioid antagonist) __ Y 6 hKissler et al. 2013 nor-BNI (k opioid antagonist) in the CeA __ Y 6e10 hKissler et al. 2013 CTOP þ Naltrindole (m and d opioid antagonist) in the CeA Y __ 6e10 hKissler et al. 2013 Nalmefene (m and k opioid antagonist) in the CeA Y Y 6e10 hNealey et al. 2011 Nalmefene (m and k opioid antagonist) and CTOP þ Naltrindole

(m and d antagonist) in the NAc shellY Y 6 h

Nealey et al. 2011 nor-binaltorphimine (k opioid antagonist) in the NAc shell __ Y 6 hWalker and Koob 2008 nor-binaltorphimine (k opioid antagonist) (ICV) __ Y 6 hWalker and Koob 2008 Nalmefene (m and k opioid antagonist) and naltrexone

(m opioid antagonist)Y Y 6 h

Sabino et al. 2009 BD-1063 (s antagonist) __ Y 6 hGilpin and Koob 2010 Propranolol (b antagonist) Y Y 6 hWalker et al. 2008 Prazocin (a1 antagonist) Y Y 6 hEdwards et al. 2012 SSR149415 (vasopressin V1b antagonist) __ Y 6 hRodríguez de Fonseca et al. 1999 SR141716A (CB1 antagonist) __ Y 6e8 hSmith et al. 2011 FN-439 (matrix metalloproteinases inhibitor: ICV) NT Y 6 h

NT, not tested; R, cue-induced reinstatement.

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withdrawal from alcohol exposure via liquid diet. Animals with leftturning preferences displayed opposite results with regard todopamine concentrations in the prefrontal cortex during with-drawal. Interestingly, exposure to alcohol vapor increased alcoholself-administration in right-turners only, indicating that functionalbrain asymmetry and differences in dopamine and 5-HT neuro-transmission modulate escalated alcohol intake (Carlson & DrewStevens, 2006).

A critical goal of pharmacological studies is to reverse theallostatic changes in the brain that occur during dependence.Allostasis, literally “stability with change,” is an alternative tohomeostasis that allows dynamic adjustments to maintain stabil-ity in the face of predictable and unpredictable change. Whileallostasis is a critical mechanism, it requires more energy and ismore subject to perturbation than homeostatic regulation, andtherefore can be deleterious in the long run, leading to allostaticload/overload (McEwen & Stellar, 1993). The transition fromcontrolled to excessive alcohol intake has been hypothesized toinvolve a change in reward set point caused by an allostatic process(Koob & Le Moal, 1997; Roberts et al., 2000). Conceptually, theneuroadaptive changes caused by chronic intermittent ethanolmay contribute to allostatic load in the brain, thus disruptingreward circuitry as well as other brain functions (e.g., emotionalregulation), physiological processes (e.g., body temperature regu-lation and sleep), and cognitive processes (Koob, 2008). Therefore,it is important to study systems that are recruited in this allostaticprocess.

An “ideal” treatment for alcoholism would decrease alcoholdrinking in dependent individuals without changing alcohol drink-ing in nondependent individuals. The alcohol vapor/operant self-

administration model possesses good predictive validity, such thatacamprosate and naltrexone, two FDA-approved drugs to treatalcoholism, decrease alcohol self-administration in dependent rats(Morse & Koob, 2002; Walker & Koob, 2008). However, these drugsalso decreased nondependent drinking, suggesting that theymay beinterfering with the positive reinforcing properties of alcohol. In-vestigationsof other systemsare thereforeessential for thediscoveryof better drugs to effectively and specifically treat alcohol depen-dence. Table 1 summarizes the results of pharmacological manipu-lation of several systems (e.g., opioids, corticotropin-releasing factor[CRF], neuropeptide Y [NPY], g-aminobutyric acid [GABA], norepi-nephrine, and cannabinoids) on operant self-administration independent and nondependent rats.

Systemic, acute administration of MPZP, a small-molecule,nonpeptide, brain-penetrant antagonist with high affinity forCRF1 receptors, decreased alcohol self-administration in dependentrats (Richardson, Zhao, et al., 2008). Injections of the CRF receptorantagonist D-Phe-CRF12-41 into the cerebroventricular (CV) andcentral nucleus of the amygdala (CeA) also decreased alcohol self-administration in dependent rats, but did not change alcohol self-administration in nondependent rats; this effect was not seen inthe bed nucleus of the stria terminalis (BNST) or NAc (Funk, O’Dell,Crawford, & Koob, 2006; Valdez et al., 2002). Repeated treatment(every other day) with another CRF1 receptor antagonist, R121919,reduced alcohol self-administration in both dependent andnondependent rats (Roberto et al., 2010). The CRF2 receptor agonisturocortin 3, when acutely injected ICV (Valdez, Sabino, & Koob,2004) or intra-CeA (Funk & Koob, 2007) reduced alcohol self-administration specifically in rats made dependent on alcohol vialiquid diet or vapor exposure. These findings suggest that decreased

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CRF1 receptor activity and increased CRF2 receptor activity specif-ically reduce alcohol drinking in dependent rats.

Acute ethanol exposure activates the HPA axis to release CRFfrom the paraventricular nucleus of the hypothalamus (PVN), ad-renocorticotropic hormone (ACTH) from the pituitary, and cortico-sterone from the adrenal glands in the rat (Ellis, 1966; Richardson,Lee, O’Dell, Koob, & Rivier, 2008). Chronic alcohol exposure ap-pears to produce excessive activation of the HPA axis, ultimatelyleading to a dampening of HPA axis stimulation (Richardson, Lee,et al., 2008). These effects may be partially due to changes in CRFlevels and sensitivity in the PVN and pituitary (Richardson, Lee,et al., 2008). Recently, it has been reported that corticosteroid-dependent plasticity is important in the escalated alcohol intakeassociated with dependence. Chronic glucocorticoid receptor (GR)antagonism by mifepristone blocked the enhancement of alcoholself-administration in vapor-exposed rats during acute withdrawalwithout altering nondependent drinking (Vendruscolo et al., 2012).These findings suggest that preventing corticosterone-inducedexcessive activation of GRs during alcohol intoxication and with-drawal via chronic GR blockade may prevent allostatic changes inbrain stress systems and block escalated alcohol drinking.

Neuropeptide Y is a stress-related peptide that has been shownto participate in alcohol dependence. Thorsell, Slawecki, Khoury,Mathe, and Ehlers (2005) trained rats to press a lever 20 times toreceive access to alcohol for 25 min. After alcohol vapor exposure,the rats were tested with a fixed time schedule in which access toalcohol was provided 10 min after the session began, regardless ofthe number of lever presses. This schedule allowed for the inde-pendent evaluation of both the motivation to obtain alcohol (leverpresses) and alcohol intake. Dependent rats displayed increasedalcohol intake, an effect that was blocked by acute ICV infusions ofNPY. Interestingly, dependent rats did not show increased lever-pressing behavior using this paradigm. Using an FR1 schedule ofreinforcement, Gilpin et al. (2011) reported that chronic/repeatedICV NPY infusions reduced alcohol self-administration in bothdependent and nondependent rats. However, NPY Y2 receptor an-tagonists acutely injected, systemically or intra-CeA, produced noeffect on alcohol self-administration in dependent or nondepen-dent rats (Kallupi et al., 2013). The actions that NPY has on alcoholdependence have been suggested to involve the modulation ofGABA neurotransmission (Gilpin et al., 2011).

Acute intra-CeA injection of the potent, selective agonist for theGABAA receptor muscimol decreased responding for alcohol spe-cifically in dependent rats (Roberts et al., 1996). Other studies haveinvestigated the role of GABAB receptors on alcohol dependence.The GABAB receptor agonist baclofen acutely decreased respondingfor alcohol in both dependent and nondependent rats in FR1 and PRschedules of reinforcement, with increased sensitivity in depen-dent rats (Walker & Koob, 2007). Gabapentin produces distinctelectrophysiological changes in the CeA in dependent comparedwith nondependent rats via GABAB receptors. Consistent with theseresults, acute systemic injection of gabapentin reduced escalatedalcohol self-administration in dependent rats, with no effect onnondependent drinking (reviewed by Clemmens & Vendruscolo,2008; Roberto et al., 2008). These results suggest that while NPYand GABA systems play an important role in alcohol dependence,they might also affect the positive reinforcing properties of alcohol.

The opioid system, especially the k-opioid system, has beenshown to play a specific role in alcohol dependence. Acute nal-trexone, a preferential m-opioid receptor antagonist, and nalmefene,a m- and k-opioid receptor antagonist, blocked alcohol self-administration in both dependent and nondependent rats (Kissleret al., 2013), with nalmefene being more effective than naloxonein reducing intake in dependent rats. Persistent k-opioid receptorantagonism by nor-Binaltorphimine (nor-BNI) injected systemically

(Walker & Koob, 2008; Walker, Zorilla, & Koob, 2011), intra-NAcshell (Nealey, Smith, Davis, Smith, & Walker, 2011), or intra-CeAreduced alcohol self-administration exclusively in dependent rats.Interestingly, the s-opioid receptor antagonist BD-1063 dose-dependently reduced alcohol self-administration in dependent butnot nondependent rats (Sabino et al., 2009).

Acute administration of the b-adrenergic receptor blocker pro-pranolol but not nadolol, which does not cross the bloodebrainbarrier, reduced alcohol self-administration in dependent rats. Ahigh dose of propranolol reduced responding for alcohol (FR1 andPR tests) in both dependent and nondependent rats (Gilpin & Koob,2010). The a1-noradrenergic receptor antagonist prazosin acutelydecreased alcohol self-administration in dependent and nonde-pendent rats, with higher sensitivity in dependent rats (Walker,Rasmussen, Raskind, & Koob, 2008).

Cannabinoid and vasopressin systems have also been tested onescalated alcohol drinking in the vapor model. The vasopressin V1breceptor antagonist SSR149415 (Edwards et al., 2012) and canna-binoid receptor antagonist SR141716A (Rodríguez de Fonseca,Roberts, Bilbao, Koob, & Navarro, 1999) both injected acutelyreduced alcohol self-administration in dependent but not nonde-pendent rats.

Additionally, alcohol vapor exposure reduces cell proliferationand survival in the mPFC (Richardson et al., 2009). Inhibition ofmatrix metalloproteinases, which degrade the extracellular matrixand modulate learning processes, reduced the development ofescalation of alcohol-self-administration produced by vapor expo-sure but had no effect once escalated levels of alcohol self-administration were already reached (Smith, Nealey, Wright, &Walker, 2011). These findings suggest that vapor-induced escala-tion of alcohol self-administration is dependent on learning pro-cesses and neuroadaptation in brain areas related to cognition.

Several systems stand out as potential medication targets foralcohol dependence, including CRF1, GRs, k opioids, and thoseaffected by gabapentin (GABA and glutamate). Indeed, the worksummarized above and in Table 1 has been important in guidingclinical trials for alcohol dependence (clinicaltrials.com). There aretwo ongoing clinical trials testing CRF1 antagonists (GSK561679 andPexacerfont) led by NIAAA Clinical Director Marcus Heilig. Dr. Bar-bara Mason at The Scripps Research Institute is leading a clinicaltrial of Korlym (i.e., mifepristone, a GR antagonist). Dr. SuchitraKrishnan-Sarin from Yale is leading a study examining whethernaltrexone’s ability to bind k-opioid receptors is related to itseffectiveness. Finally, there are several clinical trials involvingbaclofen, including one led by Dr. Lorenzo Leggio at NIH examiningthe link between anxiety and craving in alcoholics. This model ofincreased alcohol self-administration in rats following chronic va-por exposure has helped to uncover important biological factorsthat can be directly tested in the clinic.

Advantages and limitations of this model

There are several important advantages of this model of in-creased ethanol self-administration in dependent rats. For example,in the vapor model, the experimenter controls alcohol exposureto generate the desired range of blood alcohol levels (Gilpin,Richardson, Cole, et al., 2008) to successfully produce alcoholdependence. Initially, alcohol vapor is set to low levels and thenprogressively increased over time. Different from other methods ofdependence, including the liquid diet, this gradual increase inalcohol vapor allows the animal to develop tolerance to alcoholwithout having any noticeable detrimental health effects (e.g.,weight loss or hypophagia). This model presents good reliabilityand predictive validity, arguably the only necessary and sufficientcriteria for the evaluation of an animal model (Edwards & Koob,

L.F. Vendruscolo, A.J. Roberts / Alcohol 48 (2014) 277e286 283

2012; Geyer & Markou, 1995). As described above, the vapor modelreliably leads to increases in alcohol intake and compulsive-likedrinking and produces many other physical and motivationalsymptoms reminiscent of human alcohol dependence. This modelhas predictive validity in that it allows one to make predictionsabout the human condition generally and more specifically toidentify drugs with potential therapeutic value. For example, drugsthat are used to treat alcohol dependence in humans also reducedependence symptoms in alcohol vapor-exposed rats. Probablymost importantly, the publications listed in Table 1 are cited asrationale for ongoing clinical trials with new potential medications(as described above).

Exposure to alcohol vapor is imposed and, therefore, a criticismof this model has limited face validity given that humans usually donot breathe alcohol to become dependent nor are they forced toingest alcohol. This disadvantage is partially balanced by the factthat rodents have a much shorter lifespan than humans, andtherefore the length of exposure to high blood alcohol levels needsto be compressed in rodents. Unless genetically selected alcohol-preferring rats are used, getting sustained high blood alcohollevels would be difficult without experimenter influence.

The operant alcohol self-administration procedure also possessesadvantages and limitations. Different from voluntary home-cagealcohol drinking, operant self-administration requires a relativelylong training period, and not all animals will display stable levels ofresponding for alcohol. The investigator must establish an operantbehavior (lever pressing) thatmost ratswill not readily perform for afluid that most rats will not readily consume. Therefore, the use ofhighly palatable sweet solutions or lengthy sessions is necessary totrain rats to lever press and consume the delivered ethanol solution.However, once stable responding levels are achieved, operant self-administration is very versatile in terms of behavioral assessment.Less demanding schedules of reinforcement, such as FR1, are used tomeasure alcohol intake in a condition of low motivational require-ment (“low price,” “low workload”). Progressive-ratio schedules ofreinforcement measure the willingness of an animal to work toobtain alcohol (“high price,” “high workload”), which is used as anindex of reward efficacy or compulsive-like behavior (Hodos, 1961).Continued alcohol use despite negative consequences is anotherimportant aspect of compulsive behavior in alcohol dependence thatcan be modeled with punishment schedules. For example, thealcohol solution can be adulterated with aversive substances (e.g.,quinine is a bitter substance disliked by rodents; Wolffgramm &Heyne, 1995), or lever presses for alcohol can be paired with mildfootshock to cause stress.

Another major advantage of this model is its utility in in-vestigating self-administration following periods of protractedwithdrawal. While most studies have investigated the effects ofbiological manipulations on alcohol self-administration duringacute alcohol withdrawal, it is critical to investigate the effects ofdrugs during protracted alcohol withdrawal when the acute with-drawal symptoms have dissipated and relapse risk is highest.Escalated alcohol self-administration (Gilpin, Richardson, Cole,et al., 2008; Gilpin, Richardson, Lumeng, et al, 2008; Roberts et al.,2000; Sommer et al., 2008; Valdez et al., 2002; Vendruscolo et al.,2012) and neuroadaptations in reward-/stress-related brain re-gions (Francesconi et al., 2009; Vendruscolo et al., 2012) have beenobserved in rats with a history of alcohol vapor exposure afterdetoxification (3e8 weeks post removal from alcohol vapor). Un-fortunately, as of yet, very few pharmacological studies havefocused on protracted withdrawal. The NPY Y2 receptor antagonistBIIE0246 reduced alcohol self-administration during protractedwithdrawal in rats with a history of alcohol vapor exposure(Rimondini, Thorsell, & Heilig, 2005). Recently, we reported thatchronic GR antagonismwithmifepristone blocked escalated alcohol

drinking (FR1) and compulsive-like responding (PR) during pro-tracted withdrawal (Vendruscolo et al., 2012). It is not clear at thistime how long lasting the craving and increased self-administrationis in this model and howwell this will model the human condition,but initial investigations have been promising.

Overall, alcohol vapor-exposed rats display increased intake ofalcohol (FR1 test) and compulsive-like behavior toward alcohol (PRand adulteration tests) compared with nondependent rats, and thisappears to be maintained following the acute withdrawal phase.Therefore, while there are a few areas of weakness, the combinationof the vapor model with operant self-administration in rats con-stitutes an excellent tool for the study of the neurobiological basisof alcohol dependence.

Future directions

The majority of studies examining alcohol self-administrationhave used nondependent animals. Effective targets and treatmentdevelopment ideally should be investigated using dependencemodels, which have better predictive validity for alcoholism. Theliterature reviewed above describes several biological systemsimportant in alcohol dependence and the way these have contrib-uted to ongoing clinical trials. However, numerous issues remain tobe addressed. These include genetics, gender, and age, as well ascraving and long-term impacts on behavior.

Most studies described above used outbred Wistar rats, whosebehavioral reactions could be considered adequate from the adap-tive point of view because they may represent those of the het-erogeneous human population. However, the use of geneticallydefined inbred rat strains (e.g., Vendruscolo et al., 2006) andselectively bred rat lines may facilitate the identification of geneticmechanisms underlying susceptibility or resistance/resilience toalcohol dependence. Additionally, the use of rat strains displayingcontrasting emotional and cognitive responses, such as high andlow anxiety-like behavior, depressive-like behavior, impulsivity,behavior inhibition, and working memory, constitutes a useful toolto gain knowledge regarding behavioral characteristics that pro-mote drinking and dependence.

While female rodents are beginning to make their way intoalcohol studies (discussed in other reviews in this special issue),every one of the studies listed in Table 1 only employed males.Although alcohol dependence is still more prevalent in men, someevidence suggests that women initiate use earlier and progressfaster to dependence and therefore represent a vulnerable popu-lation that has different and very understudied characteristics(Zilberman, Tavars, & el-Guebaly, 2003). For example, severalstudies showed that the female brainmight bemore sensitive to thedegenerative effects associated with alcohol dependence (i.e., brainarea volume reductions) and that women seek treatment earlier intheir drinking history than males (reviewed by Sharrett-Field,Butler, Reynolds, Berry, & Prendergast, 2013). Future studies usingvapor-induced increases in operant ethanol self-administrationneed to include female rats.

The ageof alcohol drinking onset (or alcohol exposure) is anothercritical issue to be studied. Few studies have investigated the con-sequences of adolescent alcohol exposure, which is a period ofintense brain maturation and the time when humans start bingedrinking, on subsequent alcohol-relatedmaladaptivebehavioral andpharmacological changes. Interestingly, Slawecki and Betancourt(2002) reported that exposure of adolescent rats (postnatal day30e40) to intermittent alcohol vapor exposure did not predisposethem to increased alcohol self-administration later in adulthood(>3 months old). Moreover, the effect of a stressor on alcoholself-administration was not different in animals with a history ofalcohol vapor exposure compared with control rats. Binge drinking

L.F. Vendruscolo, A.J. Roberts / Alcohol 48 (2014) 277e286284

of a sweetened alcohol solution during adolescence leads toincreased alcohol self-administration in adulthood, regardlessof alcohol vapor exposure in adulthood (Gilpin, Karanikas, &Richardson, 2012). Additionally, pharmacological and environ-mental manipulations (e.g., exposure to psychostimulant drugs oroverconsumption of palatable solutions) during adolescence havebeen shown to produce changes in alcohol intake later on in adult-hood (Vendruscolo, Izídio, Takahashi, & Ramos, 2008, 2011) innondependent rats. Whether similar effects would be observed independent rats remains to be investigated. Finally, the consequenceof prenatal alcohol exposure on the vulnerability to alcohol depen-dence in adulthood is an area still needing further investigation.

Alcohol craving and its role in relapse is a critical area of study(Martin-Fardon & Weiss, 2013). There are several models usedto investigate craving-like behavior in rodents, including condi-tioned reinstatement, stress-induced reinstatement, and condi-tioned place preference. These have begun to be combined withdependence models to better allow for the study of the chronicrelapsing nature of addiction. For example, Liu and Weiss (2002),Ciccocioppo, Economidou, Fedeli, and Massi (2003), and Kufahl,Martin-Fardon, and Weiss (2011) trained animals to associate ol-factory discriminative stimuli with the availability of alcohol. Oncestable responding was reached, the rats underwent extinctionsessions without the discriminative stimuli, in which lever presseswere no longer reinforced with alcohol. This procedure produced aprogressive reduction of operant responding. Upon presentation ofthe previously paired discriminative stimulus predictive of alcohol,the rats reinstated lever-press responding even in the absence ofalcohol. The dopamine D1 receptor antagonist SCH-23390 anddopamine D2 receptor antagonist eticlopride dose-dependentlyblocked this effect. The same animals were exposed to alcoholvapor and tested again for reinstatement of alcohol-seeking be-havior during protracted withdrawal. The induction of dependencedid not alter the magnitude of reinstatement, but the effect of theantagonists was increased during protracted alcohol withdrawal(Liu & Weiss, 2002). However, Ciccocioppo et al. (2003) reportedthat the preferential m-opioid receptor antagonist naltrexonereduced reinstatement in animals with a history of alcohol vaporexposure and control rats, but the effect was less pronounced inrats with a history of intermittent alcohol vapor exposure. Usingsimilar procedures, Kufahl et al. (2011) reported that the mGluR 2/3agonist LY379268 dose-dependently reduced cue-induced rein-statement in nondependent rats and in rats with a history ofalcohol dependence, but the effect was more pronounced in thelatter group.

To date, the majority of studies have been conducted duringacute/early alcohol withdrawal. In humans, the acute symptoms ofalcohol withdrawal (e.g., anxiety, agitation, shaking, tremor, head-ache, sweating, nausea, confusion, hallucinations, delirium tremens,seizures, high blood pressure, and fever) are often managed withbenzodiazepines or barbiturates until the symptoms have dissi-pated (Stehman & Mycyk, 2013). However, a major challenge in thealcohol field is to control the long-lasting symptoms of alcoholwithdrawal (e.g., alcohol craving, increased anxiety and depressivesymptoms, increased stress reactivity, hypohedonia, disorientation,nausea, headache, and insomnia), which lead to high rates of relapsein alcoholics after detoxification. Studies with models of alcoholdependence and withdrawal and operant self-administration havejust started to uncover the molecular and pharmacological mecha-nisms underlying protracted alcohol withdrawal.

Here, we described results obtained from studies of rats trainedto lever press for alcohol and made dependent, mostly throughvapor exposure. These studies have already provided critical infor-mation regarding alcohol dependence and are helping to improveour knowledge in the field, which will ultimately contribute to the

development of better strategies of prevention, diagnosis, andtreatment of alcoholism.

Acknowledgments

This is publication number 24056 from The Scripps ResearchInstitute. Research was financially supported by the IntegrativeNeuroscience Initiative on Alcoholism e West (INIA-West) andPearson Center for Alcoholism and Addiction Research. The authorsthank Michael Arends and Tali Nadav for editorial assistance.

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