Download pdf - Discriminative stimulus effects of the cannabinoid CB1 antagonist SR 141716A in rhesus monkeys pretreated with Δ9-tetrahydrocannabinol

ORIGINAL INVESTIGATION

Discriminative stimulus effects of the cannabinoid CB1

antagonist SR 141716A in rhesus monkeys pretreatedwith Δ9-tetrahydrocannabinol

Lance R. McMahon

Received: 22 February 2006 /Accepted: 29 June 2006 / Published online: 5 September 2006# Springer-Verlag 2006

AbstractRationale Drug discrimination can be used to examinetolerance and dependence in agonist-treated animals byestablishing an appropriate antagonist as a discriminativestimulus.Objective Establish intravenous SR 141716A as a discrim-inative stimulus in four rhesus monkeys pretreated with arelatively small dose ofΔ9-tetrahydrocannabinol (Δ9-THC).Methods Rhesus monkeys received i.v. Δ9-THC(0.32 mg/kg) and discriminated i.v. SR 141716A (1 mg/kg) from vehicle while responding under a fixed ratio (FR) 5schedule of stimulus-shock termination.Results The discriminative stimulus effects of SR 141716Awere dose-dependent (ED50=0.33 mg/kg) and were mim-icked by the CB1 antagonist AM 251 (ED50=0.98 mg/kg),but not by a benzodiazepine (midazolam) or an N-methyl-D-aspartate antagonist (ketamine). An additional dose(0.32 mg/kg in addition to 0.32 mg/kg administered beforethe session) of Δ9-THC shifted the SR 141716A dose–effect curve 3-fold rightward. Omitting Δ9-THC before testsessions resulted in responding on the SR 141716A leverthat was attenuated by subsequent administration of Δ9-THC (ED50=0.13 mg/kg), CP 55940 (ED50=0.013 mg/kg),and WIN 55212-2 (ED50=0.35 mg/kg); midazolam andketamine did not attenuate responding on the SR 141716Alever. SR 141716A (1 mg/kg) shifted the Δ9-THC and CP55940 dose–effect curves 3.4-fold rightward; the WIN55212-2 dose–effect curve was not significantly modifiedby a dose of 1 mg/kg of SR 141716A.

Conclusions SR 141716A can be established as a discrim-inative stimulus in animals pretreated with Δ9-THC, andthis assay is selective for cannabinoid activity. Differentialantagonism of cannabinoids by SR 141716A might indicatethat the mechanism of action of WIN 55212-2 is notidentical to other cannabinoids. This study demonstratesthat, under the appropriate conditions, drug discriminationhas utility for examining cannabinoid dependence andwithdrawal.

Keywords Antagonist . Cannabinoid . Cannabis .

Δ9-tetrahydrocannabinol . Drug discrimination .

Rhesus monkey . Rimonabant . SR 141716A

Introduction

The CB1 antagonist SR 141716A (rimonabant) is a usefulpharmacologic probe for examining CB1 receptors thatmediate the effects of naturally occurring cannabinoids[Δ9-tetrahydrocannabinol (Δ9-THC) and other cannabi-noids in Cannabis sativa] and synthetic cannabinoidagonists (e.g., CP 55940 and WIN 55212-2). Cannabinoidagonists have a variety of effects in animals, e.g., theydecrease body temperature, induce catalepsy, increase heartrate, and disrupt short-term memory, and these effects areattenuated by SR 141716A (Compton et al. 1996; Huestiset al. 2001; Lichtman and Martin 1996). Moreover,cannabinoid agonists have discriminative stimulus effectsthat provide a pharmacologically selective measure ofcannabinoid activity in vivo inasmuch as cannabinoidagonists share discriminative stimulus effects with eachother and not with noncannabinoids (Balster and Prescott1992), and these effects are attenuated by SR 141716A

Psychopharmacology (2006) 188:306–314DOI 10.1007/s00213-006-0500-6

L. R. McMahon (*)Department of Pharmacology,The University of Texas Health Science Center at San Antonio,7703 Floyd Curl Drive,San Antonio, TX 78229-3900, USAe-mail: [email protected]

(Järbe et al. 2001; McMahon et al. 2005; Wiley et al.1995b). That SR 141716A attenuates the discriminativestimulus effects of cannabinoid agonists is consistent withclinical studies reporting that SR 141716A attenuates thesubjective effects of smoked marijuana (Huestis et al.2001). Collectively, most studies with SR 141716A showit to be an appropriate antagonist of the behavioral effectsof Δ9-THC and provide ample pharmacologic evidence tosupport the hypothesis that CB1 receptors mediate thebehavioral effects of cannabinoid agonists.

In addition to providing a highly quantitative andpharmacologically selective measure of acute drug actionin vivo, drug discrimination can be used to examine theconsequences of daily drug administration (e.g., toleranceand dependence). For example, in animals that discriminatean agonist from vehicle, the discriminative stimulus effectsof drugs can be compared before and after a period inwhich discrimination training is suspended and the trainingdrug, or a pharmacologic equivalent, is administeredchronically. This approach has been used to examinetolerance to opioids (Sannerud and Young 1987), benzo-diazepines (Pugh et al. 1992), and cannabinoids (Wiley etal. 1993). Alternatively, animals can be treated chronicallywith an agonist and trained to discriminate an antagonist;the consequences of agonist treatment are assessed bycomparing the effects of drugs in agonist-treated animals(that discriminate an antagonist) to the effects of drugs inuntreated animals that discriminate the same agonist or apharmacologic equivalent. For example, the discriminativestimulus effects of a benzodiazepine antagonist in benzo-diazepine-treated animals have been used to show thatbenzodiazepine treatment produces differential cross-toler-ance among drugs with benzodiazepine-like discriminativestimulus effects (McMahon et al. 2001). Moreover, underthe appropriate conditions, the discriminative stimuluseffects of an antagonist in agonist-treated animals can bepredictive of dependence and withdrawal.

In a previous study, SR 141716A (1 mg/kg) wasestablished as a discriminative stimulus in rhesus monkeysthat were treated daily with Δ9-THC, and the discrimina-tion appeared to be related to antagonism of Δ9-THC(McMahon and France 2003). However, there were markedindividual differences in the effects of cannabinoids,perhaps due to individual differences in the absorptionand metabolism of cannabinoids administered intramuscu-larly or subcutaneously, and limited information wascollected regarding the pharmacologic profile of the SR141716A discriminative stimulus in that study. Relative tothe intramuscular route, the time required for i.v. Δ9-THCto be absorbed and metabolized is more consistent acrossindividual rhesus monkeys (Perlin et al. 1985). To increasehomogeneity in the behavioral effects of cannabinoidsacross individuals, monkeys in this study received i.v. Δ9-

THC (0.32 mg/kg) immediately before sessions in whichthey discriminated i.v. SR 141716A (1 mg/kg) fromvehicle. A relatively small dose of Δ9-THC was chosenfor study so that the effects of Δ9-THC would beminimized on days when Δ9-THC was not administeredbefore test sessions. Relative to the previous study(McMahon and France 2003), discriminative stimuluseffects were more consistent among monkeys in the presentstudy, and a more comprehensive pharmacologic profilewas established by studying the CB1-selective antagonistAM 251 and the cannabinoid agonists CP 55940 and WIN55212-2, alone and in combination with SR 141716A.

Materials and methods

Subjects Four adult (two female and two male) rhesusmonkeys (Macaca mulatta) were housed individually on a14-h light/10-h dark schedule; were maintained at 95% free-feeding weight (range 5.5–9.4 kg) with a diet consisting ofprimate chow (High Protein Monkey Diet; Harlan Teklad,Madison, WI), fresh fruit, and peanuts; and were providedwater in the home cage. All monkeys had been previouslytrained to discriminate i.m. SR 141716A (1 mg/kg) whilereceiving s.c. Δ9-THC (1.12 mg/kg/day; McMahon andFrance 2003). Monkeys were maintained in accordance withthe Institutional Animal Care and Use Committee, TheUniversity of Texas Health Science Center at San Antonio,and the Guide for the Care and Use of Laboratory Animals(National Research Council 1996).

Surgery Using aseptic surgical procedures, monkeys re-ceived chronic indwelling catheters (heparin coated poly-urethane, OD=1.68 mm, ID=1.02 mm; Instech Solomon,Plymouth Meeting, PA). Upon anesthesia with ketamine(10 mg/kg i.m.) and isoflurane (1.5–3.0%, inhaled via facemask), a catheter was inserted into a subclavian or femoralvein and advanced 5 cm. Suture silk (coated vicryl, EthiconInc., Somerville, New Jersey) was used to anchor thecatheter to the vessel and to ligate the section of the vesselproximal to the catheter insertion. The other end of thecatheter, which passed subcutaneously and exited at themidscapular region of the back, was protected by a custom-made nylon-mesh jacket (Lomir Biomedical, Toronto, ON).

Apparatus During experimental sessions, monkeys wereseated in chairs (Model R001; Primate Products, Miami,FL) that provided restraint and were placed in ventilated,sound-attenuating chambers equipped with two responselevers and stimulus lights. Feet were placed in shoescontaining brass electrodes through which a brief electricstimulus (3 mA, 250 ms) could be delivered from an A/Cgenerator. The operant conditioning equipment was

Psychopharmacology (2006) 188:306–314 307

connected via an interface (MedAssociates, St. Albans, VT)to a computer that controlled and recorded experimentalevents.

Drug discrimination procedure Monkeys received i.v. Δ9-THC (0.32 mg/kg) 30 min before sessions and discriminat-ed i.v. SR 141716A (1 mg/kg) from vehicle whileresponding under a multiple-cycle procedure. Each cyclebegan with a 15-min time-out, during which responses hadno programmed consequence, followed by a 5-minresponse period, during which illumination of two redlights (one positioned above each of the two levers)signaled a pending electric stimulus (every 40 s). Thecorrect lever was determined by an infusion of vehicle orSR 141716A; determination of correct levers (e.g., left,vehicle; right, SR 141716A) varied among monkeys andremained the same for an individual throughout the study.Five consecutive responses (FR5) on the correct leverextinguished the red lights and postponed the schedule for30 s. Responding on the incorrect lever reset the responserequirement on the correct lever. Response periods endedafter 5 min or after the delivery of four electric stimuli,whichever occurred first.

During training, vehicle or SR 141716A was adminis-tered 15 min prior to sessions, during which additionalvehicle infusions or sham (catheter accessed from withinthe jacket only) were administered nonsystematically at thebeginning of subsequent cycles. SR 141716A trainingconsisted of 2–4 cycles and vehicle training consisted of2–6 cycles. Completion of the FR on the correct lever wasrequired for a reinforcer during each training cycle. Thefirst test was conducted when, for 5 consecutive or for 6 of7 days, at least 80% of the total responses occurred on thecorrect lever and fewer than five responses (one FR)occurred on the incorrect lever prior to completion of theFR on the correct lever; criteria had to be satisfied in everycycle. During test sessions, five consecutive responses oneither lever postponed the shock schedule. Tests wereconducted at least 3 days apart and only when performancefor consecutive training sessions, including both vehicleand SR 141716A training sessions, satisfied the samecriteria described above. The type of training sessionpreceding test sessions varied nonsystematically. Oncetesting began, monkeys received Δ9-THC (0.32 mg/kg) atleast 5 days per week.

For tests conducted 30 min after i.v. Δ9-THC(0.32 mg/kg), vehicle was administered 15 min before—or at the beginning of—the first cycle, followed bycumulative doses of SR 141716A, AM 251, midazolam,or ketamine in subsequent cycles, with doses increasing by0.25 or 0.5 log unit per cycle. Midazolam and ketaminewere chosen as noncannabinoid controls because benzodia-zepines and noncompetitive N-methyl-D-aspartate (NMDA)

antagonists (1) have discriminative stimulus effects thattypically differ from those of cannabinoid agonists (Browneand Weissman 1981; Wiley et al. 1995a) and (2) can besafely studied in rhesus monkeys up to doses that disruptoperant responding (e.g., McMahon and France 2002). Anadditional test was conducted by administering i.v. Δ9-THC(0.32 mg/kg) in the first cycle, in addition to i.v. Δ9-THC(0.32 mg/kg) administered 30 min before the session,followed by cumulative doses of SR 141716A. For testsconducted in the absence of Δ9-THC before sessions (i.e.,30 min after i.v. vehicle), i.v. vehicle or SR 141716A(1 mg/kg) was administered 15 min before sessions, inwhich vehicle was administered in the first cycle followedby cumulative doses of Δ9-THC, CP 55940, or WIN55212-2 in subsequent cycles. Additional tests wereconducted by administering i.v. vehicle before sessionsfollowed by cumulative doses of midazolam or ketamine.Tests conducted after i.v. Δ9-THC ended when greater than80% of the total responses occurred on the SR 141716Alever, and tests conducted in the absence of Δ9-THC beforesessions (i.e., 30 min after i.v. vehicle) ended when lessthan 20% of the total responses occurred on the SR141716A lever. Tests with midazolam and ketamine endedwhen response rate decreased sufficiently to result in thedelivery of electric stimuli. The duration of action of Δ9-THC (0.32 mg/kg) administered intravenously or subcuta-neously was determined by administering Δ9-THC onseparate days at a fixed time prior to 6-cycle test sessionsthat were conducted in 2-h increments thereafter. Tomaintain catheter patency, up to 5 ml of heparinized saline(100 μ/ml, Baxter Healthcare Corp., Deerfield, IL) wasadministered after each i.v. drug infusion.

Drugs SR 141716A base and Δ9-THC (The ResearchTechnology Branch, National Institute on Drug Abuse,Rockville, MD); AM 251 and CP 55940 (Tocris, Ellisville,MO); and WIN 55212-2 (Sigma, St. Louis, MO) wereadministered in a volume of 0.03–3 ml/kg and weredissolved in a 1:1:18 mixture of absolute ethanol, Emul-phor-620 (Rhone-Poulenc Inc., Princeton, NJ) and sterilewater. Midazolam hydrochloride (Roche Pharma Inc.,Manati, Puerto Rico) and ketamine hydrochloride (FortDodge Laboratories, Fort Dodge, IA) were purchased ascommercially prepared solutions and were diluted withsterile saline. Doses (in milligrams per kilogram) wereexpressed as the forms listed above.

Data analyses Discrimination data were expressed as apercentage of the total responses on the SR 141716A leveraveraged among monkeys (±SEM) and plotted as a functionof dose. Doses of a compound required to produce 50% ofresponses (ED50) on the SR 141716A lever and the 95%confidence limits (CL) were estimated with linear regres-

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sion on doses producing 20–80% of responses on the SR141716A lever, including not more than one dose produc-ing less than 20% of responses on the SR 141716A leverand not more than one dose producing more than 80% ofresponses on the SR 141716A lever. ED50s were deter-mined for individual animals, and the 95% CL wasdetermined from the t statistic. ED50s were considered tobe significantly different when the 95% CL of their potencyratio did not include 1 (Tallarida 2000).

Differences in absolute rate of responding betweentraining conditions among individual monkeys wereassessed by examining whether the response rate from SR141716A training sessions were outside the 95% CL of themean response rate from vehicle training sessions (teneach). Response rate during tests was compared to controlresponse rate, defined as the average of the five vehicletraining sessions before the test, with separate one-wayanalyses of variance (ANOVAs) for repeated measures(main effect of dose) for each treatment; Tukey–Kramermultiple comparison tests were used to examine significantdifferences among treatments (p<0.05). Response rate wascalculated as a percentage of control for individual animals,then averaged among subjects (±SEM) and plotted as afunction of dose. Discrimination data were not included foranalysis when response rate for an individual was less than20% of control; however, response rate data were includedin the group average.

Results

Control performance After changing the dosing parametersof SR 141716A and Δ9-THC from those described inMcMahon and France (2003) to those used in the currentstudy (see “Materials and methods”), individual monkeyssatisfied the criteria for testing in 5, 7, 23, and 45 trainingsessions (including both vehicle and SR 141716A trainingsessions). Over ten vehicle training sessions in which thecriteria for testing were satisfied, average (95% CL) rates oflever pressing for individual monkeys were 0.56 (0.44–0.68), 0.66 (0.57–0.75), 1.26 (0.99–1.53), and 1.53 (1.34–1.72) responses per second. Average response rates over thesame number of SR 141716A training sessions were 0.69,0.67, 1.80, and 1.88 responses per second for the samemonkeys, respectively. Response rates were higher in threemonkeys after SR 141716A relative to vehicle, andresponse rates were not different across the two trainingconditions in a fourth monkey.

Discriminative stimulus and rate effects when Δ9-THC wasadministered before sessions In tests conducted 30 minafter i.v. Δ9-THC (0.32 mg/kg), administration of vehicle

resulted in little or no responding on the SR 141716A lever(Fig. 1, top, V). SR 141716A dose-dependently increasedresponding on the drug lever, with the training dose (1mg/kg)producing high levels of responding on the drug lever (Fig. 1,top, open circles); the ED50 (95% CL) of SR 141716A was0.33 (0.18–0.62) mg/kg (Table 1). SR 141716A did notsignificantly alter response rate relative to control (Fig. 1,bottom, open circles). The cannabinoid antagonist AM 251also dose-dependently increased responding on the SR141716A lever, with doses of 1.78 and 3.2 mg/kgproducing high levels of responding on the drug lever(Fig. 1, top left, squares); the ED50 (95% CL) of AM 251was 0.98 (0.55–1.77) mg/kg (Table 1). AM 251 did notsignificantly alter response rate relative to control (Fig. 1,bottom left, squares). In contrast, midazolam and ketamineproduced little or no responding on the drug lever up to dosesof midazolam (0.56 mg/kg) and ketamine (5.6 mg/kg) thatsignificantly decreased responding (Fig. 1, left, diamondsand triangles, respectively; p<0.05).

Thirty minutes after 0.32 mg/kg of Δ9-THC wasadministered, an additional dose (0.32 mg/kg) of Δ9-THC resulted in predominantly vehicle-lever responding(Fig. 1, top right, closed circle above V) and attenuatedthe discriminative stimulus effects of SR 141716A suchthat a dose of 3.2 mg/kg was required for SR 141716A toproduce greater than 80% of responses on the drug lever(Fig. 2, top right, closed circles). The ED50 (95%) of SR141716A was significantly increased to 0.99 (0.51–1.92)mg/kg (i.e., 3.0-fold), as evidenced by the 95% CL (1.6–5.6) of the potency ratio not including 1 (Table 1). Theadditional dose (0.32 mg/kg) of Δ9-THC significantlydecreased response rate (Fig. 2, bottom right, closedcircle; p<0.05).

Sessions were conducted at various times after adminis-tration of Δ9-THC to compare its time course uponintravenous or subcutaneous administration. Followingsubcutaneous administration, the effects of Δ9-THC(0.32 mg/kg) were not evident until 2 h after administration,i.e., monkeys responded predominantly (70–78%) on theSR 141716A lever for up to 2 h, and then responded lessthan 25% on the drug lever from 2 to 8 h later (data notshown). As compared to the subcutaneous route, intrave-nous administration resulted in a more rapid onset and asomewhat shorter duration of action, i.e., monkeysresponded less than 5% on the SR 141716A lever within15 min and for up to 4 h, and then responded 25–45% onthe drug lever from 4 to 8 h later (data not shown). For boththe subcutaneous and intravenous routes, monkeysresponded predominantly (greater than 87%) on the druglever 24 h after administration of Δ9-THC.

Discriminative stimulus and rate effects when Δ9-THC wasnot administered before sessions When vehicle was admin-


istered instead of Δ9-THC 30 min before test sessions,monkeys responded predominantly on the SR 141716Alever (Figs. 2 and 3, top, V). In a cumulative dose–effecttest, i.v. Δ9-THC dose-dependently attenuated respondingon the drug lever, with monkeys responding predominantlyon the vehicle lever at a dose of 0.32 mg/kg of Δ9-THC(Fig. 2, top left, circles). Similarly, CP 55940 and WIN55212-2 dose-dependently attenuated responding on thedrug lever, with monkeys responding predominantly on thevehicle lever at doses of 0.032 mg/kg of CP 55940 and1 mg/kg of WIN 55212-2 (Fig. 2, top middle and right,respectively, circles). The ED50s (95% CL) were 0.13(0.10–0.18) mg/kg for Δ9-THC, 0.013 (0.0087–0.021)mg/kg for CP 55940, and 0.35 (0.14–0.86) mg/kg forWIN 55212-2 (Table 1).

SR 141716A (1 mg/kg i.v.) significantly attenuated theeffects of Δ9-THC and CP 55940 (Fig. 2, top left andmiddle, triangles; Table 1). Thus, the ED50 (95%) of Δ9-THC was increased 3.4-fold to 0.46 (0.25–0.86), and the95% CL (2.4–4.9) of the potency ratio did not include 1.The ED50 (95%) of CP 55940 also was increased 3.4-foldto 0.046 (0.035–0.059) mg/kg, and the 95% CL (2.6–4.3)of the potency ratio did not include 1. In contrast, the ED50

(0.58 mg/kg; 95% CL, 0.55–0.60) of WIN 55212-2determined after pretreatment with SR 141716A (1 mg/kg)was not significantly different from the control ED50 ofWIN 55212-2 (i.e., the 95% CL (0.7–4.0) of the potencyratio (1.7) included 1) (Table 1). At the doses studied,response rate was not altered by the agonists, either alone orin combination with SR 141716A (Fig. 2, bottom).

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Fig. 1 Discriminative and rate effects of cannabinoid antagonistsand noncannabinoids in rhesus monkeys discriminating SR141716A after treatment with Δ9-THC (left) and attenuation ofdiscriminative stimulus effects of SR 141716A by supplemental Δ9-THC (right). Abscissae: dose in milligrams per kilogram bodyweight; V vehicle. Ordinates: mean (±SEM) percentage of respondingon the SR 141716A lever (top) and mean (±SEM) response rate

expressed as a percentage of control (V training days) rates [Rate(percentage of control), bottom]. Monkeys received Δ9-THC(0.32 mg/kg) 30 min before sessions, and V or an additional injectionof Δ9-THC (0.32 mg/kg; right panel, closed circles) at the beginningof the time-out of the first cycle. Data represent average values of fourmonkeys. Asterisks denote rate of responding that was significantlydifferent from control (p<0.05)


In contrast to cannabinoid agonists, responding onthe SR 141716A lever that occurred in the absence ofΔ9-THC was not attenuated by midazolam or ketamineup to doses of midazolam (0.056 mg/kg) and ketamine

(5.6 mg/kg) that significantly decreased response rate(Fig. 3; p<0.05).

Discussion

SR 141716A was established as a discriminative stimulusfollowing pretreatment with Δ9-THC (0.32 mg/kg) in fourrhesus monkeys responding under an FR schedule ofstimulus-shock termination. Relative to a previous studyin which i.m. SR 141716A was established as a discrim-inative stimulus in Δ9-THC-treated monkeys (McMahonand France 2003), the experimental parameters of thecurrent study, including intravenous administration ofdrugs, resulted in more consistent discriminative stimuluseffects among subjects. The current study was morecomprehensive than the previous study in its pharmaco-logic assessment of discriminative stimulus effects, toinclude tests with additional cannabinoid agonists (CP55940 and WIN 55212-2) and a selective CB1 antagonist(AM 251). The discriminative stimulus effects of SR

Table 1 ED50s (95% CL) in milligrams per kilogram for discrimina-tive stimulus effects of antagonists (after Δ9-THC) and agonists (aftervehicle), and potency ratios (95% CL) for combinations of SR141716A with cannabinoid agonists

ED50 (95% CL) Potency ratio(95% CL)

After Δ9-THC (0.32mg/kg)SR 141716A 0.33 (0.18–0.62)+ Δ9-THC (0.32mg/kg) 0.99 (0.51–1.92)a 3.0 (1.6–5.6)AM 251 0.98 (0.55–1.77)After VehicleΔ9-THC 0.13 (0.10–0.18)+ SR 141716A (1mg/kg) 0.46 (0.25–0.86)a 3.4 (2.4–4.9)CP 55940 0.013 (0.0087–0.021)+ SR 141716A (1mg/kg) 0.046 (0.035–0.059)a 3.4 (2.6–4.3)WIN 55212-2 0.35 (0.14–0.86)+ SR 141716A (1mg/kg) 0.58 (0.55–0.60) 1.7 (0.7–4.0)

a Significantly different from control ED50

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sessions, V (circles) or SR 141716A (1 mg/kg; triangles) 15 minbefore sessions, and V at the beginning of the time-out of the firstcycle. See Figure 1 for other details


141716A appeared to be selective for cannabinoid activityinsofar as the cannabinoid antagonist AM 251 substitutedfor the SR 141716A discriminative stimulus and non-cannabinoids (midazolam and ketamine) did not. Immedi-ate pharmacologic history was an important determinantof discriminative stimulus effects, i.e., monkeys respondedon the SR 141716A lever when Δ9-THC was notadministered before sessions. Under these conditions,responding on the SR 141716A lever was dose-depen-dently attenuated by Δ9-THC, CP 55940, and WIN55212-2; in contrast, midazolam and ketamine did notattenuate responding on the SR 141716A lever. Theseresults suggest that the discriminative stimulus effects ofSR 141716A in Δ9-THC-treated monkeys provide apharmacologically selective measure of cannabinoid ac-tivity and show this assay to be sensitive to bothcannabinoid agonists and antagonists.

SR 141716A is a CB1 antagonist that attenuates many ofthe behavioral effects of Δ9-THC including its hypother-mic, cataleptic, antinociceptive, discriminative stimulus,positive reinforcing, and subjective effects (Compton et al.1996; Huestis et al. 2001; Järbe et al. 2001; McMahon et al.2005; Rinaldi-Carmona et al. 1995; Tanda et al. 2000;Wiley et al. 1995b). Consistent with these findings, theresults of the current study suggested that antagonism ofΔ9-THC was responsible for the discriminative stimuluseffects of SR 141716A in monkeys pretreated with Δ9-THC. For example, another selective CB1 antagonist (AM251) substituted for the SR 141716A discriminativestimulus (i.e., antagonized Δ9-THC). By increasing thedose of Δ9-THC administered before sessions, the SR141716A dose–effect curve for discriminative stimuluseffects was shifted to the right (i.e., Δ9-THC attenuatedSR 141716A). When vehicle was administered instead ofΔ9-THC before sessions, monkeys responded predominant-ly on the SR 141716A lever, and these effects wereattenuated by Δ9-THC. Under these conditions, adminis-tration of the training dose (1 mg/kg) of SR 141716Ashifted the Δ9-THC dose–effect curve 3.4-fold to the right.Collectively, these results suggested that the discriminativestimulus effects of SR 141716A in Δ9-THC-treatedmonkeys were related to the attenuation—or blockade—ofthe effects of Δ9-THC.

In addition to having cannabinoid antagonist activity, SR141716A alone induces a variety of behavioral effectsincluding increases in locomotor activity and scratching(Aceto et al. 1996; Bass et al. 2002; Lichtman et al. 2001),and these effects could be related to inverse agonist activityor blockade of endogenous cannabinoid tone at cannabi-noid receptors or to mechanisms unrelated to cannabinoidreceptors (Pertwee 2006 for review). Although the phar-macologic profile of the SR 141716A discriminativestimulus was consistent with antagonism of Δ9-THC, it is

possible that the direct effects of SR 141716A contributedto its discriminative stimulus effects in the current study.However, the training dose of SR 141716A was relativelysmall in terms of its cannabinoid antagonist activity(present results; McMahon et al. 2005), and it appears thatthis dose of SR 141716A cannot be readily established as adiscriminative stimulus in the absence of Δ9-THC treat-ment. For example, i.v. SR 1417167A up to a dose of1.78 mg/kg has not been established as a discriminativestimulus in two untreated rhesus monkeys after approxi-mately 200 training sessions. In contrast, i.v. SR 141716A(1 mg/kg) was established as a discriminative stimulus in43 sessions in an experimentally naïve rhesus monkeyreceiving daily Δ9-THC treatment (unpublished observa-tions). Thus, pretreatment with Δ9-THC appears to haveincreased the sensitivity of monkeys to the discriminativestimulus effects of SR 141716A (1 mg/kg).

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Ketamine

∆9-THC

Midazolam

*

*

Fig. 3 Discriminative and rate effects of midazolam, Δ9-THC, andketamine in monkeys that received vehicle instead of Δ9-THC beforesessions. Monkeys received vehicle (V) instead of Δ9-THC(0.32 mg/kg) 30 min before sessions and V at the beginning of thetime-out of the first cycle. Data for Δ9-THC are replotted fromFigure 2. Asterisks denote rate of responding that was significantlydifferent from control (p<0.05). See Figure 1 for other details


SR 141716A alone has been trained as a discriminativestimulus in rats, although not with experimental parametersthat are commonly used in drug discrimination procedures.For example, in two previous studies, SR 141716A couldnot be trained as a discriminative stimulus in pigeons or ratsresponding for food (Mansbach et al. 1996; Pèrio et al.1996), whereas SR 141716A was established as a discrim-inative stimulus using another reinforcer (avoidance of anaversive taste) and procedure (discriminated taste aversion;Järbe et al. 2004). Under those conditions, the pharmaco-logic profile of the discriminative stimulus effects of SR141716A was strikingly similar to that obtained in thecurrent study. Discriminated taste aversion appears to bemore sensitive than operant procedures to the discrimina-tive stimulus effects of antagonists (also Smurthwaite et al.1992). Treatment with a cannabinoid agonist, therefore, isnot always necessary for SR 141716A to exert discrimina-tive control over behavior, and agonist treatment might notbe necessary to increase the pharmacologic specificity ofthe SR 141716A discriminative stimulus. Nevertheless, ifSR 141716A is to be established as a discriminativestimulus in the absence of Δ9-THC treatment in rhesusmonkeys responding under a schedule of stimulus-shocktermination, then a relatively large dose (e.g., greater than1.78 mg/kg) might be required.

When vehicle was administered instead of Δ9-THCbefore test sessions, Δ9-THC, CP 55940, and WIN 55212-2had qualitatively similar effects, results consistent withprevious studies showing that CP 55940 and WIN 55212-2substitute for a Δ9-THC discriminative stimulus in rhesusmonkeys (Gold et al. 1992; Wiley et al. 1995a). Collec-tively, these results are consistent with the hypothesis thatcannabinoid agonists act through a common mechanism toexert their behavioral effects. That SR 141716A similarlyantagonized the discriminative stimulus effects of Δ9-THCand CP 55940 is consistent with this view. In contrast, SR141716A appears to be less potent in antagonizing thediscriminative effects of WIN 55212-2 (also Pèrio et al.1996). The lesser potency of SR 141716A to antagonize theeffects of WIN 55212-2 is consistent with a study reportingthat WIN 55212-2 has agonist activity in the brains of micelacking CB1 receptors (Breivogel et al. 2001). In addition toCB1 receptors, it appears that CB2 receptors are present inbrain (Gong et al. 2006), and differential binding ofagonists at cannabinoid or other receptor subtypes mightbe responsible for differences in their antagonism by CB1-selective ligands.

Agonist-treated animals that discriminate an antago-nist have been used to examine dependence andwithdrawal associated with a number of drug classes(e.g., opioids and benzodiazepines; Emmett-Oglesby etal. 1990 for review), and this study demonstrates thefeasibility of using drug discrimination to examine Δ9-

THC dependence and withdrawal. However, because agoal of the current study was to establish conditions thatwere sensitive to not only cannabinoid antagonists butalso cannabinoid agonists, a relatively small dose(0.32 mg/kg) of Δ9-THC was chosen to minimize itsduration of action, thereby increasing the likelihood thatthe assay would be sensitive to cannabinoid agonists (i.e.,monkeys would respond on the SR 141716A lever) 24 hafter administration of Δ9-THC. Consequently, depen-dence on Δ9-THC did not likely develop under theseconditions, and consistent with this view, SR 141716Adid not induce observable signs of withdrawal afterpretreatment with 0.32 mg/kg of Δ9-THC in this study.By establishing SR 141716A as a discriminative stimulusat doses (e.g., 1.2–1.6 mg/kg/day) of Δ9-THC previouslyreported to produce dependence in rhesus monkeys(Beardsley et al. 1986; Deneau and Kaymakcalan 1971),drug discrimination might provide a reliable index ofcannabinoid dependence and withdrawal.

Acknowledgments Supported by US Public Health Service GrantsDA15468 and DA19222. The author thanks C. Cruz, M. Hernandez,and D. Logan for providing technical assistance.

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