.European Journal of Pharmacology 387 2000 4753www.elsevier.nlrlocaterejphar
Effects of the cannabinoid CB receptor antagonist, SR141716A, after1D9-tetrahydrocannabinol withdrawalq
Patrick M. Beardsley ), Billy R. MartinDepartment of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth Uniersity, Box 980613, 410 N. 12th Street, Smith
Building a756A, Richmond, VA 23298-0613, USA
Received 19 August 1999; received in revised form 1 November 1999; accepted 5 November 1999
Rats were trained to lever press according to variable interval 10 s schedules during daily experimental sessions composed of six 3min food reinforcement periods and were treated twice daily for 6 days with either vehicle or escalating regimens of D9-tetrahydrocanna-
. binol. On days 7 and 8, the rats were challenged with vehicle and cumulative doses of SR141716A N- piperidin-1-yl -5- 4-. . .chlorophenyl -1- 2,4,-dichlorophenyl -4-methyl-1H-pyrazole-3-carboxyamide hydrochloride , a cannabinoid CB receptor antagonist, up1
to 3 and 9 mgrkg, respectively. Response rates increased during D9-tetrahydrocannabinol withdrawal and towards those of the vehicletreatment group suggesting a waning of the direct effects of D9-tetrahydrocannabinol. SR141716A reduced response rates but only in ratspre-treated with D9-tetrahydrocannabinol. These data suggest that dependence upon D9-tetrahydrocannabinol was induced and SR141716Aprecipitated withdrawal. q 2000 Elsevier Science B.V. All rights reserved.
. .Keywords: THC tetrahydrocannabinol ; Dependence; Withdrawal; Rat ; Operant behavior; Behavioral dependence; SR141716A
Marijuana is amongst the most widely used recreationalsubstances, and it has been promoted by some for a varietyof medical applications as well. Repetitive use of behav-iorally active substances can sometimes induce depen-dence, as evidence by physiological or behavioral disrup-tions upon abstinence. There have been reports of with-drawal effects in humans upon abstinence from marijuanaand other cannabis products, and these effects have in-cluded hyperirritability, nervousness, tremor, dysphoria,
and sleep disturbance Jones and Benowitz, 1976; Jones et.al., 1981; Mendelson et al., 1984; Wiesbeck et al., 1996 .
When reports of these withdrawal effects were criticallyreviewed, however, reviewers have concluded that evenunder the most intense exposure regimens the effects aretypically mild in most subjects and are not of major
q This work was supported by NIDA grant DA03672. A preliminaryreport of this study was presented at the June 1997 College on Problemsof Drug Dependence Meetings held in Nashville, TN.
) Corresponding author. Tel.: q1-804-828-5185; fax: q1-804-828-2117.
.E-mail address: email@example.com P.M. Beardsley
.medical consequence Compton et al., 1990 . Attempts toexperimentally induce dependence in laboratory animalsupon D9-tetrahydrocannabinol, the main behaviorally ac-tive constituent of marijuana, have led to conflicting re-sults. When relying upon spontaneous withdrawal effectsto infer dependence, several attempts have been unable to
demonstrate dependence McMillan et al., 1970; Dewey et.al., 1972; Harris et al., 1974; Leite and Carlini, 1974 ,
while other attempts were reported as successful Deneauand Kaymakcalan, 1971; Fredericks and Benowitz, 1980;
.Beardsley et al., 1986 . The failure to consistently observewithdrawal effects following D9-tetrahydrocannabinol ad-ministration may, in part, be due to the mild nature of thedependence syndrome. Maximizing the sensitivity of de-pendent measures to potential withdrawal-induced pertur-bations maximizes the likelihood of detecting mild statesof dependence. Two general methods have been used tomaximize the likelihood of detecting states of dependence.These methods have included using antagonists to precipi-
tate withdrawal and by using baselines of operant i.e.,.schedule-maintained behavior for revealing withdrawal-
induced perturbations.Antagonist challenge to animals treated with depen-
dence-producing drugs typically elicits more intense with-drawal reactions than does non-precipitated withdrawal
0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. .PII: S0014-2999 99 00792-X
( )P.M. Beardsley, B.R. MartinrEuropean Journal of Pharmacology 387 2000 475348
. . Aceto, 1990 . SR141716A N- piperidin-1-yl -5- 4-chlo-. .rophenyl - 1- 2,4,-dichlorophenyl -4-methyl-1H-pyrazole-
.3-carboxyamide hydrochloride has been identified as acentral cannabinoid CB receptor antagonist Rinaldi-1
.Carmona et al., 1994 and is effective in antagonizingcannabinoid agonist effects in vivo Rinaldi-Carmona et
.al., 1994; Compton et al., 1996; Mansbach et al., 1996 .SR141716A has also been reported to precipitate with-drawal effects in D9-tetrahydrocannabinol-treated rats in-
dicative of physical dependence Aceto et al., 1995,1996;.Tsou et al., 1995 . Precipitated withdrawal effects during
these studies included disturbed locomotor activity includ-ing retropulsion, wet-dog shakes, forepaw fluttering andptosis. The precipitated withdrawal effects reported inthese studies are amongst the most intense ever reportedduring withdrawal from D9-tetrahydrocannabinol in rats,and suggest that challenging D9-tetrahydrocannabinol-treated animals with a cannabinoid antagonist may effec-tively reveal the dependence state.
Disruptions of operant baselines during withdrawal, thatis, deviations from vehicle control rates, have also beenused as effective measures for detecting dependence upon
a variety of drugs e.g., Thompson and Schuster, 1964;Holtzman and Villarreal, 1973; Slifer et al., 1984; Beards-ley and Balster, 1987; Beardsley et al., 1986; Carroll et al.,
.1989 . At times, these withdrawal effects are observed inthe absence, or in minimal presence of the effects on
.physiological autonomically-mediated responses, andthey have been used to infer a type of dependence referred
to as behavioral dependence Holtzman and Villarreal,1973; Slifer et al., 1984; Beardsley and Balster, 1987;
.Beardsley et al., 1986 . We had previously observedmarked disruptions of operant performance during with-drawal from chronic i.v. infusions of D9-tetrahydrocanna-binol in rhesus monkeys, from which we inferred theability of D9-tetrahydrocannabinol to induce behavioral
.dependence Beardsley et al., 1986 . Combining antagonistchallenges with using operant baselines for monitoringwithdrawal effects, and thereby maximizing the likelihoodof detecting a mild dependence state, has not been previ-ously reported in D9-tetrahydrocannabinol-treated animals.
The purpose of the present study was to evaluate whetherbehavioral dependence could be induced in rats following
. 9several days of twice daily b.i.d. dosings with D -tetra-hydrocannabinol. We hypothesized that a change inresponse rates away from vehicle control levels duringD9-tetrahydrocannabinol withdrawal would be indicative ofbehavioral dependence. Three different D9-tetrahydro-cannabinol regimens were used in an attempt to maximizethe D9-tetrahydrocannabinol doses administered whileavoiding the total suppression of response rates by D9-tetrahydrocannabinol. These regimens included either acuteor cumulative dosages exceeding those reported to induceprofound physical dependence upon D9-tetrahydrocanna-
.binol in rats Tsou et al., 1995 . In this study, SR141716Adid not affect response rates in rats given chronic vehicle
regimens. SR141716A, but not D9-tetrahydrocannabinol .discontinuation i.e., non-precipitated withdrawal , pro-
duced changes in response rates away from vehicle controllevels in rats with recent D9-tetrahydrocannabinol exposureconsistent with a precipitated withdrawal effect.
2. Materials and methods
2.1. SubjectsTwenty-four adult male Long-Evans Hooded rats
.Harlan, Dublin, VA were individually housed in ananimal room maintained at 208C illuminated under a 12 hlightrdark cycle with continuous access to water. The ratswere maintained at 85% of their ad libitum body weight bysupplements with rodent chow following each experimen-tal session, and during the day on those days in whichexperimental sessions were not scheduled. All animalsreceived care according to the Guide for the Care andUse of Laboratory Animals, DHHS Publication, Revised,1985. The facilities are certified by the American Associa-tion for the Accreditation of Laboratory Care. These stud-ies were approved by the Institutional Animal Care andUse Committee at the Virginia Commonwealth University.
Eight identical two-lever operant chambers Lafayette.Instrument, Lafayette, IN were used. Each chamber was
enclosed in a sound- and light-attenuating chamber andventilated by an exhaust fan that produced a constantmasking noise. The two response levers were located onthe front panel of these chambers and were separated by a
food hopper into which 45 mg food pellets Bio-Serve,.Frenchtown, NJ could be delivered by activation of an
automated dispenser. A 4-W stimulus lamp was locatedabove each lever. The recording of lever presses, activa-tion of lights, and dispensing of food pellets were automat-ically controlled by a microcomputer operating MED-PC
.software MED Associates, St. Albans, VT .
Subjects were initially trained to press the right lever .under a fixed-ratio 1 FR 1 schedule of reinforcement in
which each right side lever press resulted in a pelletdelivery. Subsequently, the rats were trained daily for
78-min experimental sessions usually MondayFriday,however, if the testing protocol required it for longer than
.5 consecutive days under a multiple timeout of 10 min .and variable interval of 10 s for 3 min schedule of
reinforcement reinforced with food pellet delivery. Eachexperimental session consisted of six 10-min timeout peri-ods alternating with six 3-min food availability periods. Anexperimental session would always begin with a 10-mintimeout period during which the test chamber was dark-ened and food pellets were unavailable although lever
( )P.M. Beardsley, B.R. MartinrEuropean Journal of Pharmacology 387 2000 4753 49
presses were recorded. During the 3-min food availabilityperiods, the right stimulus lamp was illuminated and foodpellets were delivered according to the variable interval of10 s schedules. During food pellet deliveries, the rightstimulus lamp was extinguished for 0.2 s and then re-il-luminated. According to the variable interval of 10 sschedules, the first press of the right lever, following anaverage of 10 s since the last pellet delivery or thebeginning of the food period resulted in a pellet delivery.Thirty-nine intervals ranging from 0.5 to 19.5 s in 0.5 sincrements were used for the random selection of variableinterval values. The variable interval schedule was chosenbecause its contingencies permitted reinforcement to con-tinue even under low rate conditions expected duringD9-tetrahydrocannabinol administration.
Each rat was trained until five consecutive experimentalsessions occurred in which its overall response rates right
.lever pressesrs during the first and fifth sessions did notexclusively represent the highest and lowest rates for thosefive sessions, and the rate during each individual sessionwas "20% of the average rate across these sessions. Aftera rats performance satisfied these training criteria, it washabituated to receiving within- and post-session vehicleinjections until five consecutive sessions occurred duringwhich overall response rates during sessions 4 and 5 wereG75% of the average response rates across sessions 13.Once a rat meet these habituation criteria, subsequenttesting begin.
Six rats were randomly distributed into four groups forthe dependence tests. All rats received 6 consecutive daysof b.i.d. D9-tetrahydrocannabinol or vehicle injection fol-lowed by tests with vehicle and SR141716A on days 7 and8. The conditions for these groups differed only withrespect to the D9-tetrahydrocannabinol doses administeredon days 16 as conditions were identical for all groups ondays 7 and 8. During the first 6 days of treatment, all ratsreceived an injection at approximately 0800 h, and anotherimmediately after the experimental sessions at approxi-
.mately 1600 h. VEH group i.e., vehicle group rats re-ceived injections of vehicle during these 6 days. TheD9-tetrahydrocannabinol treatment groups received D9-tetrahydrocannabinol injections on these 6 days and dif-fered by how rapidly the D9-tetrahydrocannabinol dose wasincreased and on the highest dose of D9-tetrahydrocanna-
binol administered 30, 40, or 50 mgrkg for groups 30. 9THC, 40 THC, and 50 THC, respectively . These D -tetra-
.hydrocannabinol regimens were chosen because: 1 pilotstudies had found administering 10 mgrkg D9-tetrahydro-cannabinol for 3 days neither suppressed operant respond-
.ing nor induced dependence; 2 given these pilot studyresults, we hoped that gradually increasing the D9-tetra-hydrocannabinol regimen would induce tolerance andwould permit, at least at the lowest dosage regimen,
.continued expression of operant responding; 3 we wantedto exceed in acute and in the cumulative b.i.d. dosage ofi.p. regimens reported to induce profound physical depen-
.dence in rats Tsou et al., 1995 in order to maximize thelikelihood of establishing dependence. It was not an objec-tive to parametrically manipulate the D9-tetrahydrocanna-binol dosage regimen in these groups but to bracket a6-day D9-tetrahydrocannabinol dosing condition whichwould permit tolerance to develop, permitting the expres-sion of operant behavior while maximizing the likelihoodof dependence. The 30 THC group received 10, 20, and 30mgrkg b.i.d. D9-tetrahydrocannabinol on days 12, 34,and 56, respectively; the 40 THC group received 10, 20,30, and 40 mgrkg b.i.d. D9-tetrahydrocannabinol on days12, 3, 45, and 6, respectively; and the 50 THC groupreceived 10, 20, 30, 40, and 50 mgrkg b.i.d. D9-tetra-hydrocannabinol on days 1, 2, 3, 45, and 6, respectively.All rats received the same test regimens during the experi-mental sessions on days 7 and 8 which consisted ofinjections given at the beginning of the first, third, andfifth timeout periods. On day 7, these injections consistedof vehicle, 1 and 2 mgrkg SR141716A; on day 8, theseinjections were vehicle, 3 and 6 mgrkg SR141716A.Injections on day 7 and 8 thus began approximately 23 and47 h following the last injection of D9-tetrahydrocanna-binol, respectively.
D9-Tetrahydrocannabinol was obtained from the Na-tional Institute on Drug Abuse and SR141716A was pro-vided by Pfizer Central Research, Groton, CT. Both drugswere dissolved in a solvent consisting of 1:1:18 of absolute
.ethanolremulphor EL-620, GAF, Linden, NJ rsterile0.9% saline. All injections were given s.c. in a volumeequivalent to 3 mlrkg. When tests with vehicle are indi-cated, the 1:1:18 mixture of ethanolremulphorrsaline wasused.
2.5. Data analysis
Lever pressing counts during food components 1 and 2,3 and...