Manwell & Mallet 2014 Comparative Effects of Pulmonary and Parenteral Tetrahydrocannabinol Exposure on Extinction of Opiate Induced Conditioned Aversion in Rats

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Psychopharmacology ISSN 0033-3158 PsychopharmacologyDOI 10.1007/s00213-014-3798-5

Comparative effects of pulmonary andparenteral Δ9-tetrahydrocannabinolexposure on extinction of opiate-inducedconditioned aversion in rats

Laurie A. Manwell & Paul E. Mallet

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ORIGINAL INVESTIGATION

Comparative effects of pulmonary and parenteralΔ9-tetrahydrocannabinol exposure on extinctionof opiate-induced conditioned aversion in rats

Laurie A. Manwell & Paul E. Mallet

Received: 18 February 2014 /Accepted: 31 October 2014# Springer-Verlag Berlin Heidelberg 2014

AbstractRationale Evidence suggesting that the endogenous cannabi-noid (eCB) system can be manipulated to facilitate or impairextinction of learned behaviours has important consequencesfor opiate withdrawal and abstinence. We demonstrated thatthe fatty acid amide hydrolase (FAAH) inhibitor URB597,which increases eCB levels, facilitates extinction of analoxone-precipitated morphine withdrawal-induced condi-tioned place aversion (CPA).Objectives The potential of the exogenous CB1 ligand, Δ9-tetrahydrocannabinol (Δ9-THC), to facilitate extinction of thisCPAwas tested. Effects of both pulmonary and parenteralΔ9-THC exposure were evaluated using comparable doses previ-ously determined.Methods Rats trained to associate a naloxone-precipitatedmorphine withdrawal with a floor cue were administeredΔ9-THC—pulmonary (1, 5, 10 mg vapour inhalation) orparenteral (0.5, 1.0, 1.5 mg/kg intraperitoneal injection)—prior to each of 20 to 28 extinction/testing trials.Results VapourizedΔ9-THC facilitated extinction of the CPAin a dose- and time-dependent manner: 5 and 10mg facilitated

extinction compared to vehicle and 1 mg Δ9-THC. InjectedΔ9-THC significantly impaired extinction only for the 1.0-mg/kg dose: it prolonged the CPA fourfold longer than thevehicle and 0.5- and 1.5-mg/kg doses.Conclusions These data suggest that both dose and routeof Δ9-THC administration have important consequencesfor its pharmacokinetic and behavioural effects; specifi-cally, pulmonary exposure at higher doses facilitates,whereas pulmonary and parenteral exposure at lowerdoses impairs, rates of extinction learning for CPA.Pulmonary-administered Δ9-THC may prove beneficialfor potentiation of extinction learning for aversive mem-ories, such as those supporting drug-craving/seeking inopiate withdrawal syndrome, and other causes of condi-tioned aversions, such as illness and stress.

Keywords Δ9-Tetrahydrocannabinol (THC) .

11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) .

Pulmonary administration . Parenteral administration .

Extinction learning . Opiate withdrawal . Conditioned placeaversion (CPA) . Conditioned place preference (CPP) .

Volcano vapourizer . Marijuana

AbbreviationsΔ9-THC Δ9-Tetrahydrocannabinol11-OH-Δ9-THC 11-Hydroxy-Δ9-tetrahydrocannabinolAEA Arachidonoyl ethanolamide2-AG 2-ArachidonoylglycerolCPA Conditioned place aversionCPP Conditioned place preferenceeCB EndocannabinoidFAAH Fatty acid amide hydrolaseip Intraperitonealsc Subcutaneous

L. A. Manwell : P. E. MalletDepartment of Psychology, Wilfrid Laurier University, Waterloo,ON N2L3C5, Canada

L. A. ManwellDepartment of Psychology, University of Guelph, Guelph,ON N1G2W1, Canada

L. A. Manwell (*)Centre for Addiction and Mental Health, Social Aetiology of MentalIllness Training Program, University of Toronto, Unit 1111, 33Russell Street, Toronto, ON M5S2S1, Canadae-mail: [email protected]

L. A. Manwelle-mail: [email protected]

PsychopharmacologyDOI 10.1007/s00213-014-3798-5

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Introduction

Several lines of evidence suggest that the endogenous canna-binoid (eCB) system plays a critical role in the extinction ofaversively motivated behaviours and a nonessential role in theextinction of reward-based and appetitively motivated behav-iours (Chhatwal et al. 2005; Harloe et al. 2008; Hölter et al.2005; Lutz 2007; Manwell et al. 2009; Marsicano et al. 2002;Niyuhire et al. 2007a, b; Pamplona et al. 2006; Varvel andLichtman 2002; Varvel et al. 2007). First, Marsicano et al.(2002) showed a central role for the CB1 receptor and itsendogenous ligands, arachidonoyl ethanolamide (AEA) and2-arachidonoylglycerol (2-AG), in the extinction of auditory-fear conditioning. CB1-deficient and wild-type mice adminis-tered the CB1 antagonist/inverse agonist, SR141716,displayed impairments in both short- and long-term extinctionof fear-conditioned freezing while suffering no deficits inacquisition and consolidation; these effects were related toelevated levels of AEA and 2-AG in the basolateral amygdala(Marsicano et al. 2002). Second, memory deficits observed inthe aversively motivatedMorris water maze task (e.g. inabilityto learn a new platform location) may be due to interferencearising from the failure to extinguish a previously establishedmemory (e.g. old platform location) (Varvel and Lichtman2002; Varvel et al. 2005). In addition to direct CB1 manipula-tion, the fatty acid amide hydrolase (FAAH), which degradeseCB ligands AEA and 2-AG, can be manipulated by geneticdeletion or pharmacological inhibition (with OL-135 orURB597) to increase natural levels of eCBs. Compared tountreated wild-type (FAAH +/+) mice, both FAAH-deficient(FAAH −/−) and OL-135 (FAAH inhibitor)-treated miceshowed enhanced extinction rates and increased rates of ac-quisition; the effects of OL-135 were blocked by co-administration of SR141716 (Varvel et al. 2007).Interestingly, the exogenous CB1 ligand Δ9-tetrahydrocan-nabinol (Δ9-THC) did not affect extinction rates when admin-istered at doses of 0.1, 0.3, 1, or 10 mg/kg intraperitoneally(ip) 30 min prior testing (Varvel et al. 2007). Third, Manwellet al. (2009) demonstrated that the FAAH inhibitor URB597significantly facilitated extinction of conditioned place aver-sion (CPA) but not conditioned place preference (CPP) learn-ing in rats. Specifically, URB597 promoted extinction of analoxone-precipitated morphine withdrawal-induced CPA,whereas the CB1 antagonist/inverse agonist SR141716significantly impaired extinction. In comparison, extinc-tion rates of morphine-induced CPP and operantresponding for sucrose reward in rats were not affectedby URB597, SR141716, or the CB1 antagonist/inverseagonist AM251 (Manwell et al. 2009). However, whetherpulmonary-administered cannabinoids would similarly fa-cilitate opioid-induced withdrawal-induced CPA is cur-rently unknown. This can have important implicationsfor research on opiate withdrawal and abstinence and for

our understanding of the effects of exogenous cannabi-noids on cognitive-behavioural functions.

Evidence suggests thatΔ9-THC, the primary psychoactivecomponent in marijuana, may produce differential effects onbehaviour and memory depending upon the route of adminis-tration (Ford et al. 2009; Manwell et al. 2014a, b; Naef et al.2004; Niyuhire et al. 2007a). Injected Δ9-THC (1, 3,10 mg/kg) dose-dependently disrupted both acquisition andrecall of the platform location in the Morris water maze task,whereas inhaled smoke frommarijuana (50, 100, and 200 mg)only impaired performance at the highest dose (4.2 mg Δ9-THC) (Niyuhire et al. 2007a). Although Niyuhire et al.(2007a) suggested that their previous pharmacokinetic studies(e.g. Wilson et al. 2006) indicated that brain levels ofΔ9-THCfrom 1 mg/kg intravenous exposure may be roughly equiva-lent to smoke from 200 mg marijuana, they did not directlyquantify bioavailable levels of Δ9-THC. Our investigationssuggest there are different concentrations of Δ9-THC and itsprimary psychoactive metabolite 11-hydroxy-Δ9-tetrahydro-cannabinol (11-OH-Δ9-THC) in whole blood after pulmonaryor parenteral administration of Δ9-THC (Manwell et al.2014a, b). Using a commercially available vapourizer com-monly used by cannabis smokers, we directly compared thepharmacokinetic and behavioural effects of effects of pulmo-nary (inhaled) versus parenteral (ip) administration of Δ9-THC. First, these experiments demonstrated different dose-dependent and time-dependent concentration changes in Δ9-THC and its metabolite 11-OH-Δ9-THC in whole blood afterinhaled and injectedΔ9-THC (Manwell et al. 2014a). Second,these experiments demonstrated different drug effects on be-havioural measures including locomotor activity, food andwater consumption, place conditioning, and cross-sensitization to morphine (Manwell et al. 2014a, b). We con-clude that some of the conflicting findings in animal andhuman cannabinoid studies may be related to pharmacokineticdifferences associated with route of administration (Manwellet al. 2014a, b).

The present study extends the work of Manwell et al.(2009, experiment (Exp.) 3) by evaluating the effects of in-haled and injected Δ9-THC on extinction rates of naloxone-precipitated morphine withdrawal-induced CPA. Althoughmost studies evaluating the effects of Δ9-THC on memoryin animal models employ various routes of injection, humanusers typically smoke cannabis. Indeed, the vast majority ofanimal research on the pharmacological and behavioural ef-fects of Δ9-THC and its metabolites administer Δ9-THCparenterally (intraperitoneal or intravenous injection), where-as the most common route of administration for human usersis pulmonary (smoking or vapourizing marijuana).Combustion of cannabis releases many compounds in addi-tion to Δ9-THC, including other phytocannabinoids, toxins,and carcinogens (Turner et al. 1980a, b; Reece 2009).Vapourization of pure Δ9-THC provides a more accurate

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assessment of the direct effects of Δ9-THC and 11-OH-Δ9-THC. Here, we describe two behavioural experimentsassessing the effects of inhaled and injected Δ9-THC onextinction of a CPA using a vapourized Δ9-THC exposureprocedure that produces neither a CPP nor a CPA (Manwellet al. 2014a, b).

Materials and method

Animals

Male Sprague–Dawley rats (Charles River, Canada) (N=88),weighing 200–300 g, were pair-housed in standard plasticshoebox cages (45×25×20 cm3), maintained at 21–22 °C ina colony room on a 12-h reversed light–dark cycle (lights offat 0700 h), and fed standard rat chow (Harlan 8640) and waterad libitum. Testing was conducted during the dark cycle.Experimental procedures followed Canadian Council onAnimal Care guidelines and were approved by the WilfridLaurier University Animal Care Committee. Rats were accli-matized to the colony and handling procedures prior toexperimentation.

Drugs and analytical chemicals

Morphine (Medisca, St-Laurent, QC) was prepared in physi-ological saline at a concentration of 20 mg/ml and adminis-tered subcutaneously (sc) in a volume of 1 ml/kg. Naloxone(Sigma) was prepared in physiological saline at a concentra-tion of 1 mg/ml and administered (sc) in a volume of 1 ml/kg.Δ9-THC (Dronabinol, >98 % purity) was obtained from THCPharmGmbH (Frankfurt, Germany). For pulmonary (inhaled)drug administration, Δ9-THC was prepared in ethanol atconcentrations of 4, 20, and 40 mg/ml, and 250 μl of eachwas applied to small steel wool pads (liquid pads, Storz &Bickel, Tuttlingen, Germany), yielding final amounts of 1, 5,or 10 mg/pad. Ethanol was then allowed to evaporate beforevapourization. For parenteral (ip) administration, Δ9-THCwas dissolved in a small volume of ethanol and then mixedwith Tween-80 (polyoxyethylene sorbitan monooleate; ICNBiomedicals). Ethanol was evaporated under a stream ofnitrogen gas, and the suspension was then mixed with phys-iological saline. The final vehicle contained 15 μl Tween-80per 2 ml saline. Δ9-THC was administered in a volume of1 ml/kg body weight.

Apparatus

The conditioning apparatus consisted of a black acrylic rect-angular box (60×25×25 cm3) with a wire mesh lid. Duringconditioning trials, tactile cues on both sides of the box wereidentical. During pretest and choice tests, one side of the

chamber had a plastic floor with raised bumps and the otherside had a plastic floor with holes (counterbalanced); a smallplastic smooth floor, defined as a neutral zone (9×25 cm),separated the two-cued floors. The amount of time (s) each ratspent on each of the floors was recorded and subsequentlyanalysed by the ANY-maze behavioural video-tracking soft-ware (Stoelting, Wood Dale, IL). Pretests did not indicate asignificant difference between time spent on the plastic bumpsor holes floors indicating that the apparatus provides an unbi-ased test of conditioned preference/aversion. A Volcano®vapourizing device (Storz & Bickel, GmbH & Co.,Tuttlingen, Germany) was used as described by Hazekampet al. (2006). Briefly, Δ9-THC (1, 5, or 10 mg/pad) wasvapourized (at heat setting 9, approximately 226 °C) andcollected into detachable plastic balloons (approximately8 L), which were then fitted with a release valve that expelledthe vapour over 10 s into enclosed plastic boxes (45×25×20 cm3) containing two rats that inhaled the vapour for 5 min.This specific Δ9-THC vapourization administration proce-dure produces neither a CPP nor a CPA in rats (Manwellet al. 2014a, b). Animals were removed 5 min later and testedin the conditioning apparatus (Exp. 1). This device has beenpreviously reported to deliver >50 % of the loaded Δ9-THCinto the balloon in a reproducible manner with a pulmonaryuptake comparable to smoking of cannabis in humans(Hazekamp et al. 2006).

Experimental procedure

Experiment 1: effect of inhaled Δ9-THCon naloxone-precipitated morphine withdrawal-inducedconditioned aversion

Forty rats (n=10/group) were assigned to extinctiongroups (0, 1, 5, and 10 mg Δ9-THC) matched on thebasis of a 20-min pretest score and pair housing. Ratshad two conditioning trial cycles (separated by 72 h),each consisting of a 3-day schedule separated by 24 h(previously described by Manwell et al. 2009, Exp. 3).On day 1, rats were injected (sc) with 1 ml/kg saline10 min prior to placement in the chamber with a dis-tinctive floor cue for 30 min. On day 2, rats wereinjected (sc) with 20 mg/kg morphine in their homecages. On day 3, rats were injected (sc) with 1 mg/kgnaloxone 10 min prior to placement in the chamber withthe opposite distinctive floor cue (as on day 1) for30 min. Extinction testing began 72 h after the finalconditioning cycle. For all extinction tests, rats (n=10/group) were administered vapourized Δ9-THC—(0, 1, 5,or 10 mg/pad) in groups of two rats per box (with theirpair-housed partner) for 5 min then injected (sc) with1 ml/kg saline 10 min prior to a choice test trial—withboth conditioned floors and the neutral floor—which

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lasted for 20 min. Extinction testing continued every24 h for 20 consecutive days with a 20-min drug-freespontaneous recovery (SR) test 1 week later.

Experiment 2: effect of injected Δ9-THCon naloxone-precipitated morphine withdrawal-inducedconditioned aversion

Forty-eight rats (n=12/group) were assigned to four extinctiongroups (0, 0.5, 1.0, and 1.5 mg/kg Δ9-THC) matched on thebasis of a 20-min pretest score and pair housing (two wereremoved prior to the end of testing). The procedure wasexactly the same as Exp. 2 except for the following: (a) 72 hafter the last conditioning trial, a 20 min drug-free choice testwas administered to probe for the strength of an aversion, and(b) for all extinction tests, rats were administeredΔ9-THC (0,0.5, 1.0, and 1.5 mg/kg) parenterally (ip) 20 min prior totesting, including an injection (sc) of 1 ml/kg saline 10 minprior to a choice test trial—with both conditioned floors andthe neutral floor—which lasted for 20 min. Extinction testingcontinued every 24 h for 28 consecutive days and a 20-mindrug-free SR test 1 week later. One week after the SR test, areinstatement test (RS) was performed to determine the po-tential of naloxone-precipitated morphine withdrawal to rein-state the conditioned aversion: On day 1, rats wereinjected (sc) with 1 ml/kg saline 10 min prior to a 20-min choice test; on day 2, rats were injected (sc) with20 mg/kg morphine in their home cage; and on day 3, ratswere injected (sc) with 1 mg/kg naloxone 10 min prior toa choice test to determine the effect of prior extinctiontreatments on reinstatement of the CPA.

Data analysis

Similar to Manwell et al. (2009, Exp. 3), data for the20 extinction trials in Exp. 1 were analysed in blocks offour trials in a 2×5 (floor × blocks of trials) repeatedmeasures ANOVA and a mixed factor ANOVA for thedrug-free choice test data 1 week after the final extinc-tion trial. In addition, the time spent on the neutral floor(intersecting zone) and overall locomotor activity wasalso evaluated. Data for the drug-free probe test and the28 extinction trials in Exp. 2 were analysed as blocks offour trials in a 2×8 (floor × blocks of trials) repeatedmeasures ANOVA and a mixed factor ANOVA for thedrug-free SR choice test data 1 week after the finalextinction trial and the RS test 2 weeks after the finalextinction trial. In addition, the time spent on the neu-tral floor (intersecting zone) was also evaluated.Statistical significance was defined as p<0.05.

Results

Experiment 1: effect of inhaled Δ9-THC vapouron naloxone-precipitated morphine withdrawal-inducedconditioned aversion

Pulmonary administration of Δ9-THC facilitated the rate ofextinction of a naloxone-precipitated morphine withdrawal-induced CPA and in a dose- and time-dependent manner.Compared to vehicle, both 10 and 5 mg/pad of inhaledvapourized Δ9-THC showed facilitated extinction rates ofthe conditioned aversion, whereas 1 mg/pad did not differsignificantly from vehicle. However, the CPAwas maintainedup to 1 week after extinction testing only for rats previouslyadministered 1 mg/pad inhaled Δ9-THC; thus, 1 mg/padinhaled Δ9-THC appears to have impaired extinction, com-pared to vehicle, as indicated by aversion maintenance up to1 week after extinction testing ended. Figure 1 and Table 1present the extinction data depicted as five blocks of four trialseach. There was a main effect of floor (F(1, 36)=21.75,p<0.001) and block of trials (F(4, 44)=5.55, p<0.001) butno interaction. Based on the results of Manwell et al. (2009,Exp. 3), it was hypothesized that there would be different ratesof extinction across trials for the different drug groups; thus,planned pairwise comparisons were conducted indicating thefollowing: during block 1 (extinction tests 1–4), all groupsshowed a significant aversion to the withdrawal-paired floorin comparison to the saline-paired floor (all ps<0.05). Duringblock 2 (extinction tests 5–8), only the 0-mg Δ9-THC groupshowed an aversion (p<0.05). In block 3 (extinction tests 9–12), the 0-, 1-, and 5-mgΔ9-THC groups showed an aversion(all ps<0.05) but not the 10-mg Δ9-THC group. In block 4(extinction tests 13–16), only the 1-mg Δ9-THC groupshowed an aversion (p<0.05). In block 5 (extinction tests17–20), only the 0- and 1-mg Δ9-THC groups maintainedan aversion (ps<0.05), whereas the 5- and 10-mg Δ9-THCgroups maintained extinction of the aversion. In the drug-freechoice test 1 week after the final extinction trial, there was asignificant floor-by-trial interaction (block 5 and drug-freetest) (F(1, 36)=8.80, p<0.01) wherein only the 1-mg Δ9-THC group maintained the aversion (p<0.05). Effects ofvapourized Δ9-THC on locomotor activity were assessedusing the total time mobile (in sec±sem) during pretest, adrug-free probe test, and each block of four trials of extinctiontests; results indicated locomotor activity decreased equallyacross all groups from the first to last trial.

Experiment 2: effect of injected Δ9-THCon naloxone-precipitated morphine withdrawal-inducedconditioned aversion

Parenteral administration of Δ9-THC impaired the rateof extinction of a naloxone-precipitated morphine

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withdrawal-induced CPA for 1.0 mg/kg Δ9-THC only.Injected Δ9-THC significantly impaired extinction onlyfor 1.0 mg/kg: it prolonged the CPA fourfold longer(16 days) than the vehicle, 0.5- and 1.5-mg/kg groups.There were no differences between groups administeredvehicle, 0.5 and 1.5 mg/kg. Figure 2 and Table 2 pres-ent the extinction data depicted as eight blocks of fourtrials each. There was a main effect of floor (F(1, 42)=18.54, p<0.001) and a trial-by-floor interaction (F(7,294)=4.20, p<0.001. Planned pairwise comparisons in-dicated the following: during the drug-free probe test,all groups showed a significant aversion to the

withdrawal-paired floor in comparison to the saline-paired floor (all ps<0.01). During block 1 (extinctiontests 1–4) and block 2 (extinction tests 5–8), the vehicleand all of the THC groups (0.5, 1.0, and 1.5 mg/kg)showed an aversion (all ps<0.05). However, for block 3(extinction tests 9–12), block 4 (extinction tests 13–16),block 5 (extinction tests 17–20), and block 6 (extinctiontests 21–24), only the 1.0-mg/kg THC group continuedto show an aversion (all ps<0.05); the vehicle and 0.5and 1.5 mg/kg THC groups all maintained extinction ofthe aversion (all ps n.s.). In block 7 (extinction tests25–28), none of the groups showed an aversion (all ps

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Fig. 1 Experiment 1. Mean (± sem) seconds spent on the naloxone-precipitated morphine withdrawal-paired floor and on the saline-pairedfloor during each block of four trials for rats exposed for 5 min tovapourized Δ9-THC: 0 mg (n=10), 1 mg (n=10), 5 mg (n=10), or

10 mg (n=10) in 8 L of vapour. Asterisks indicate that rats spent lesstime on the withdrawal-paired floor than the saline-paired floor duringeach block of trials (*p<0.05)

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n.s.). Effects of Δ9-THC on locomotor activity wereassessed using the total time mobile (in sec±sem) dur-ing pretest, a drug-free probe test, and each block offour trials of extinction tests; results indicated that lo-comotor activity decreased equally across all groupsfrom the first to last trial. For the drug-free SR choicetest 1 week after the final extinction trial, a 2×2 re-peated measures ANOVA comparing the last extinctionblock (tests 25–28) to the SR test showed maintenanceof the extinction (all ps n.s.). Approximately 1 weeklater, the RS test was conducted. On day 1, a 2×2repeated measures ANOVA comparing the day 1 ofthe RS test (the saline prime) to day 3 of the RS test(the naloxone prime) showed a main effect of trial (F(1,43)=66.46, p<0.001), a main effect of floor (F(1, 43)=8.10, p<0.01), and a trial-by-floor interaction (F(3,43)=4.12, p<0.05). All groups spent less time on thenaloxone-paired floor on both trials (days 1 and 2);however, all groups also spent more time on thesaline-paired floor in the presence of the naloxoneprime (on day 3).

Discussion

The present findings are consistent with our previous work(Manwell et al. 2009, Exp. 3) showing that increasing eCBlevels can facilitate extinction of a conditioned aversion toopiate withdrawal. First, rats showed an aversion to thewithdrawal-paired floor cue during the first block of extinc-tion trials, indicating that Δ9-THC administration did notinterfere with recall memory, similar to previous results withURB597 and SR141716 (Manwell et al. 2009, Exp. 3).Second, Δ9-THC began to facilitate or impair extinctionlearning only after repeated exposure, similar to previousfindings (Manwell et al. 2009), and as early as the secondextinction block, significant differences emerged for both doseand route of administration of Δ9-THC. Third, inhaled Δ9-THC vapour facilitated extinction learning in a dose-dependent manner: the highest dose of inhaled Δ9-THC va-pour rapidly extinguished the CPA compared to vehicle andthe lowest dose of inhaled Δ9-THC, which failed to extin-guish the CPA. A drug-free test 1 week later indicated thatonly the lowest dose ofΔ9-THC maintained the CPA. Fourth,injectedΔ9-THC impaired extinction rates: the middle dose ofinjected Δ9-THC prolonged the CPA up to fourfold longerthan vehicle and both the lower and higher doses.

Differences in extinction rates between the two routes ofadministration may be due to differential brain levels of Δ9-THC and 11-OH-Δ9-THC. InhaledΔ9-THC, which is rapidlyabsorbed from the lungs into the blood then circulated to thebrain, avoids hepatic first pass metabolism, which greatlyT

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EX1

307.14

(43.75)

713.01

(52.18)

352.53

(50.68)

627.17

(73.26)

409.24

(59.66)

653.21

(57.31)

337.56

(40.70)

658.94

(48.207)

EX2

316.57

(53.66)

690.86

(67.60)

362.92

(65.46)

641.77

(74.35)

410.39

(77.92)

624.84

(82.42)

460.51

(77.27)

590.03

(78.46)

EX3

337.01

(45.80)

667.23

(60.14)

345.25

(48.15)

628.27

(57.91)

375.45

(50.27)

642.90

(64.71)

446.45

(43.58)

537.17

(42.51)

EX4

373.43

(56.55)

593.750(66.25)

328.04

(38.98)

592.78

(60.21)

447.70

(83.1)

568.81

(75.67)

423.412(46.27)

571.06

(56.61)

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373.89

(40.73)

611.16

(57.86)

319.96

(49.71)

620.58

(58.69)

411.49

(69.37)

600.68

(77.14)

419.71

(68.20)

581.51

(73.76)

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reduces the amount ofΔ9-THC and its metabolite 11-OH-Δ9-THC available to bind to CB1 (Koob and Le Moal 2006;Manwell et al. 2014a; Matsunaga et al. 1995; Grotenhermen2003). InhaledΔ9-THC may have activated CB1 receptors ingreater density and distribution throughout the brain, afterrepeated exposure every 24 h, in comparison to injected Δ9-THC which may have produced more variable brain levels ofΔ9-THC and 11-OH-Δ9-THC resulting in no behaviouraleffect or a subjective aversive effect. Although injected andinhaled Δ9-THC produces similar blood levels of Δ9-THCand 11-OH-Δ9-THC, studies show brain levels are signifi-cantly higher after inhalation (Manwell et al. 2014a; Tsenget al. 2004;Wilson et al. 2002). Similar toWilson et al. (2002),we found comparable levels ofΔ9-THC and 11-OH-Δ9-THCin whole blood when there was an approximate sixfold in-crease in the amount ofΔ9-THC that was inhaled compared toinjected (Manwell et al. 2014a). However,Wilson et al. (2002,2006) reported a dissociation between Δ9-THC levels inblood and brain that depended upon route of administration;

other studies have reported either increased (Christensen et al.1971; Gill and Jones 1972) or decreased (Gill and Lawrence1974; Ohlsson et al. 1980a, b) Δ9-THC levels in brain com-pared to blood after parenteral administration. Although fur-ther experimentation is necessary to determine the specificbiological mechanisms mediating the dose- and route-dependent effects of Δ9-THC on extinction rates of CPA, afew related experiments may help explain our results.

First, narrowing the dose range of injected Δ9-THC canproduce significant differences in the acquisition of CPA orCPP in rats (reviewed in Murray and Bevins 2010). Forexample, 1.0 to 1.5 mg/kg Δ9-THC can produce a CPA,whereas minimal (0.5 mg/kg) or maximal (eight- to tenfold)increases or decreases outside of that range have no effect(Mallet and Beninger 1998). In Exp. 2 here, only 1.0 mg/kgΔ9-THC prolonged the CPA; neither 0.5 nor 1.5 mg/kg hadany effect. Second, in place conditioning studies usinginjected Δ9-THC, increasing the time between training ses-sions generally results in lower doses producing CPP and

Fig. 2 Experiment 2. Mean (± sem) seconds spent on the naloxone-precipitated morphine withdrawal-paired floor and on the saline-pairedfloor during each block of four trials for rats pretreated with injectedΔ9-THC: 0 mg/kg (n=11), 0.5 mg/kg (n=12), 1.0 mg/kg (n=11), or

1.5 mg/kg (n=11). Asterisks indicate that rats spent less time on thewithdrawal-paired floor than the saline-paired floor during each blockof trials (*p<0.05)

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higher doses producing CPA; these effects are typically aresult of biphasic effects of Δ9-THC metabolism (Murrayand Bevins 2010). For example, shifting training sessionintervals from 24 to 48 h can shift a dose effect curve suchthat higher doses (2 to 4 mg/kg) produce CPA rather than CPP,and lower doses (1 mg/kg), previously having no effect,produce CPP (Lepore et al. 1995). In Exp. 1 with injectedΔ9-THC, a 24-h washout period may have been sufficient toimpair extinction learning for 1 mg/kg and/or produced asubjective aversive effect. In comparison, 1.5 mg/kg Δ9-THC may have produced a greater level of CB1 activationthat was not aversive, but also not sufficient to facilitateextinction learning. In Exp. 2 with inhaled Δ9-THC, a 24washout period may have been insufficient to produce anyaversive rebound effects for high doses of Δ9-THC but mayhave had an aversive effect for the lowest dose which showeda CPA up to 1 week after extinction testing.

Third, chronic exposure to cannabinoids leads to rapiddevelopment of tolerance and withdrawal (Howlett et al.2004). High doses (10 mg/kg) of injected Δ9-THC every24 h for up to 21 days rapidly desensitize and downregulatehippocampal CB1 receptors (Breivogel et al. 1999; Romeroet al. 1997), whereas lower doses of Δ9-THC (3 mg/kg) orAEA (3 mg/kg) significantly increase CB1 binding (Romeroet al. 1995). In Exp. 1, repeated exposure every 24 h to 5 and10 mg/pad inhaled Δ9-THC could have gradually increasedCB1 receptor binding during the first block of trials (4 days)which facilitated extinction over subsequent trials. This wouldbe consistent with URB597 facilitating extinction learning byincreasing AEA levels in the brain and activating CB1 recep-tors (Manwell et al. 2009). It is also consistent with the failureto find any effects of URB597 or AM251 on extinction ofmorphine-induced CPP wherein morphine-induced CPP rap-idly extinguished within 2 to 3 days, whereas morphinewithdrawal-induced CPA required a minimum of 5 to 8 daysto extinguish (Manwell et al. 2009). These findings suggestthat repeated activation of CB1 may increase CB1 bindingactivity, which then facilitates extinction learning in rats ad-ministered URB597 and inhaledΔ9-THC; these findings alsosuggest that even the highest dose of injectedΔ9-THC used inExp. 2 may not have been sufficient to affect CB1 bindingacross repeated exposures.

Fourth, procedures for Exps. 1 and 2 were identical exceptfor the additional step required for inhalation ofΔ9-THC. Theimmediate effects of vapourized Δ9-THC on the brain couldhave mediated the effects of subsequent activation of thehypothalamic–pituitary–adrenal (HPA) axis and stress re-sponse induced by subsequent injections; some studies indi-cate there are synergistic effects of concurrent Δ9-THC andenvironmental stress exposure (Patel et al. 2005; Schramm-Sapyta et al. 2007). For example, injections of vehicle andΔ9-THC (5 mg/kg) can increase corticosterone (15 and 30 min),but levels only remain elevated (>90 min) with Δ9-THCT

able2

Experim

ent2.M

ean(±

sem)secondsspentonthenaloxone-precipitatedmorphinewith

draw

al-pairedfloorandon

thesalin

e-paired

floorduring

each

blockof

four

trialsforratspretreated

with

injected

Δ9-THC:0

mg/kg

(n=11),0.5mg/kg

(n=12),1.0mg/kg

(n=11),or

1.5mg/kg

(n=11)

Dose

0mg/kg

0.5mg/kg

1.0mg/kg

1.5mg/kg

Extinction

trial

Tim

eon

with

draw

al-

paired

floor(±

sem)

Tim

eon

salin

e-paired

floor(±

sem)

Tim

eon

with

draw

al-

paired

floor(±

sem)

Tim

eon

salin

e-paired

floor(±

sem)

Tim

eon

with

draw

al-

paired

floor(±

sem)

Tim

eon

salin

e-paired

floor(±

sem)

Tim

eon

with

draw

al-

floor(±

sem)

Tim

eon

salin

e-paired

floor(±

sem)

DRF

347.71

(31.58)

682.03

(41.55)

337.30

(39.99)

719.14

(45.93)

354.64

(56.55)

672.15

(58.33)

356.93

(46.66)

678.77

(43.14)

EX1

376.63

(59.58)

681.48

(64.89)

405.72

(49.48)

647.27

(52.03)

375.11

(45.31)

669.00

(46.87)

381.74

(29.51)

659.42

(32.43)

EX2

377.25

(71.34)

690.61

(71.18)

366.71

(48.42)

681.50

(58.55)

388.91

(59.21)

667.60

(62.52)

420.20

(47.90)

660.42

(52.63)

EX3

400.00

(74.60)

639.88

(73.23)

435.32

(58.06)

627.15

(59.46)

363.09

(52.01)

702.23

(61.75)

435.51

(66.87)

643.73

(71.75)

EX4

430.64

(80.10)

609.07

(78.81)

422.69

(57.71)

635.90

(60.16)

369.55

(55.50)

693.22

(57.11)

479.55

(68.84)

578.39

(74.14)

EX5

423.78

(73.11)

604.36

(68.37)

443.08

(52.60)

618.18

(59.41)

372.36

(50.44)

683.19

(62.50)

417.22

(80.68)

640.29

(87.37)

EX6

431.88

(78.88)

599.32

(75.92)

432.11

(53.01)

611.31

(55.88)

412.43

(48.64)

643.25

(56.20)

422.97

(68.54)

636.18

(76.17)

EX7

432.55

(73.10)

604.63

(77.38)

480.96

(69.56)

571.51

(75.58)

439.65

(58.56)

613.08

(61.22)

464.74

(78.71)

581.96

(78.04)

Psychopharmacology

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exposure, suggesting that injection initially increases stresshormone levels, and Δ9-THC prolongs their circulation inthe blood (Schramm-Sapyta et al. 2007). However, it is un-likely the effects of Δ9-THC on HPA function alone accountfor the difference between inhaled and injected Δ9-THCbecause the levels of stress that rats experienced in the twoprocedures did not vary significantly. To control for injectionstress (and ensure testing procedures matched conditioningprocedures), rats in Exps. 1 and 2 received injections 10 minprior to all CPA acquisition and extinction trials. Rats receiv-ing inhaled Δ9-THC were injected immediately after inhala-tion; therefore, the expected synergistic effects of Δ9-THCexposure and injection stress should be comparable. Thehighest dose ofΔ9-THC administered ip in Exp. 2 was three-fold less than that reported by Schramm-Sapyta et al. (2007)and thus may not have been sufficient to increase or prolongcorticosterone levels. None of the doses ofΔ9-THC in Exps. 1and 2 affected expression of the CPA in the first block of trials,and the lowest dose of inhaledΔ9-THC did not affect extinc-tion rates at all. Furthermore, injected URB597 had the sameeffect on extinction rates with the same injection procedures inManwell et al. (2009, Exp. 3).

Finally, an interaction between the eCB and opioid systemscould have mediated the effects reported here, which is con-sistent with research showing significant structural and func-tional overlap between the eCB and opioid systems (Corcheroet al. 2004; Ghozland et al. 2002; Lichtman et al. 2001; Mas-Nieto et al. 2001; Navarro et al. 1998, 2001; Yamaguchi et al.2001). Administration of the endogenous CB1 receptor li-gands AEA and 2-AG, and exogenous and the syntheticligandsΔ9-THC andHU-210, has been shown to significantlydecrease unconditioned somatic signs of naloxone-precipitated morphine withdrawal (Vela et al. 1995;Yamaguchi et al. 2001). This suggests that CB1 agonism inManwell et al. (2009, Exp. 3), and Exps. 1 and 2 here, mighthave also reduced the strength of the CPA. However, neitherΔ9-THC preexposure here nor pretreatment with URB597 inManwell et al. (2009, Exp. 3) reduced the strength of the CPAduring the first block of extinction trials. Only repeated expo-sure to Δ9-THC and URB597, which chronically activatedCB1 receptors, affected the CPA; this is consistent with theresults of Romero et al. (1995) demonstrating significantincreases in CB1 binding after chronic exposure to Δ9-THCand AEA (Romero et al. 1995).

Selectively targeting CB1 receptors may be important ininterventions aimed at extinguishing both the unconditioned(somatic signs) and conditioned (negative affect) effects ofmorphinewithdrawal syndrome, which have been proposed tocontribute to drug-craving/seeking behaviour in opioid addic-tion and relapse (Bechara et al. 1995, 1998; Koob 1996). If theeCB system can be manipulated to facilitate extinction ofpreviously learned responses, then it may serve as apharmacotherapeutic target for facilitating the extinction of

maladaptive associations between environmental context/cuesand the aversive effects of drugs of abuse. In animal models,stress induces significantly greater self-administration ofdrugs and, in clinical studies, human patients consistentlyreport links between stress and drug abuse (Gordon 2002).In summary, these experiments suggest that the route of ad-ministration of Δ9-THC has important consequences for itspharmacokinetic and behavioural effects, specifically, thatpulmonary exposure facilitates, whereas parenteral exposureimpairs, rates of extinction learning for conditioned aversions.Thus, inhaled Δ9-THC may prove more beneficial for phar-macological potentiation of extinction learning for aversivememories, such as those supporting drug-craving/seeking inopiate withdrawal syndrome. It will also be important forfuture research on withdrawal and relapse to other drugs ofabuse and exposure to factors which cause conditioned aver-sions, such as illness and stress.

Acknowledgments This work was performed with grants from theNatural Sciences and Engineering Research Council of Canada (to LMand PM) and grants from Wilfrid Laurier University (to PM).

Conflict of interest The authors declare that they have no competinginterests.

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