Journal of Pharmacological and Toxicological Methods 70 (2014) 112–119

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Journal of Pharmacological and Toxicological Methods

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Original article

A vapourized Δ9-tetrahydrocannabinol (Δ9-THC) delivery system part II:Comparison of behavioural effects of pulmonary versus parenteralcannabinoid exposure in rodents

Laurie A. Manwell a,b,c,⁎, Brittany Ford a, Brittany A. Matthews a, Heather Heipel a, Paul E. Mallet a

a Department of Psychology, Wilfrid Laurier University, Waterloo, ON N2L3C5, Canadab Department of Psychology, University of Guelph, Guelph, ON N1G2W1, Canadac Centre for Addiction and Mental Health, Social Aetiology of Mental Illness Program, University of Toronto, ON M5T1R8, Canada

Abbreviations:Δ9-THC,Δ9-tetrahydrocannabinol; 11-Orahydrocannabinol; IP, intraperitoneal; IV, intravenous;subcutaneous.⁎ Unit 1111, 33 Russell Street, Toronto, ON, M5S 2

8501x77632.E-mail addresses: laurie.manwell@camh.ca, laurieman

http://dx.doi.org/10.1016/j.vascn.2014.06.0041056-8719/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 25 February 2014Accepted 4 June 2014Available online 21 June 2014

Keywords:Δ9-Tetrahydrocannabinol (Δ9-THC)Pulmonary administrationLocomotionCross-sensitizationFood consumptionRat

Introduction: Studies of the rewarding and addictive properties of cannabinoids using rodents as animal modelsof human behaviour often fail to replicate findings from human studies. Animal studies typically employ paren-teral routes of administration, whereas humans typically smoke cannabis, thus discrepancies may be related todifferent pharmacokinetics of parenteral and pulmonary routes of administration. Accordingly, a novel deliverysystem of vapourized Δ9-tetrahydrocannabinol (Δ9-THC) was developed and assessed for its pharmacokinetic,pharmacodynamic, and behavioural effects in rodents. A commercially available vapourizer was used to assessthe effects of pulmonary (vapourized) administration of Δ9-THC and directly compared to parenteral (intraper-itoneal, IP) administration of Δ9-THC.Methods: Sprague–Dawley rats were exposed to pure Δ9-THC vapour (1, 2, 5, 10, and 20 mg/pad), using a Volca-no® vapourizing device (Storz and Bickel, Germany) or IP-administered Δ9-THC (0.1, 0.3, 0.5, 1.0 mg/kg), anddrug effects on locomotor activity, food and water consumption, and cross-sensitization to morphine (5 mg/kg)

were measured.Results: Vapourized Δ9-THC significantly increased feeding during the first hour following exposure, whereas IP-administered Δ9-THC failed to produce a reliable increase in feeding at all doses tested. Acute administration of10 mg of vapourized Δ9-THC induced a short-lasting stimulation in locomotor activity compared to control inthe first of four hours of testing over 7 days of repeated exposure; this chronic exposure to 10 mg of vapourizedΔ9-THC did not induce behavioural sensitization to morphine.Discussion: These results suggest vapourizedΔ9-THC administration produces behavioural effects qualitatively dif-ferent from those induced by IP administration in rodents. Furthermore, vapourized Δ9-THC delivery in rodentsmay produce behavioural effects more comparable to those observed in humans. We conclude that some of theconflicting findings in animal and human cannabinoid studies may be related to pharmacokinetic differences as-sociated with route of administration.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

Studies in both humans and rodents report significant variability inthe pharmacokinetic, pharmacodynamic and behavioural effects of can-nabinoids, particularly for Δ9-tetrahydrocannabinol (Δ9-THC), acrossdifferent routes of administration, and only a very few number of stud-ies have directly compared the effects of pulmonary and parenteral

H-Δ9-THC, 11-hydroxy-Δ9-tet-MDI, metered dose inhaler; SC,

S1, Canada. Tel.: +1 416 535

well@gmail.com (L.A. Manwell).

administration of Δ9-THC in humans (e.g., Naef, Russman, Petersen-Felix, & Brenneisen, 2004) or rodents (e.g., Fried, 1976; Fried &Neiman, 1973; Niyuhire et al., 2007; Wilson, Varvel, Harloe, Martin, &Lichtman, 2006; Wilson et al., 2002). In human trials, Naef et al.(2004) reported that pulmonary administration of a Δ9-THC liquidaerosol (0.053 mg/kg) produced peak plasma levels of Δ9-THC inthe range of 18.7 ± 7.4 ng/ml for approximately 10 to 20 min afterinhalation, and then rapidly decreased; however, plasma levelsafter intravenous (IV) administration of Δ9-THC (0.053 mg/kg)were more variable, ranging from 81.6 to 640.6 ng/ml (mean of271.5 ± 61.1 ng/ml) within 5 min after injection (Naef et al.,2004). More importantly, Naef et al. (2004) reported that the psy-chological and somatic side effects of Δ9-THC differed according tothe route of administration: although parenteral administration of

113L.A. Manwell et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 112–119

Δ9-THC produced greater euphoria, it also produced greater anxiety,irritation, confusion and disorientation, hallucination, nausea, andheadache compared to pulmonary administration, which only re-sulted in transient respiratory irritation.

Early studies on rodents, by Fried and Neiman (1973), reported thatrats exposed to inhaled cannabis smoke showed electrical brain activity(via electroencephalographic recordings) in both cortical and hippo-campal regions which was similar, but less pronounced, than that ob-served in rats administered intraperitoneal (IP) injections of Δ9-THC.Fried and Neiman (1973) also compared the effects of oral and IP ad-ministrated Δ9-THC, versus exposure to cannabis smoke, on rats in theopen-field test; all conditions significantly reduced exploratory behav-iour, with injected Δ9-THC having the greatest impact. However, ratsadministered IP or oral Δ9-THC, but not cannabis smoke, were reportedto be hypersensitive, exhibiting greater vocalizations when handledafter testing. In studies of chronic exposure to inhaled cannabis, Fried(1976) showed that male, but not female, rats developed cross-tolerance to acute exposure to IP administeredΔ9-THC.Male and femalerats exposed to cannabis smoke every other day for 32 days showed re-duced locomotor activity on the initial trials (1 to 10) which thenreturned to baseline levels near the last trials (13 to 16); however, re-peated exposure to cannabis smoke significantly increased locomotoractivity after IP injection of Δ9-THC, indicating tolerance in male butnot female rats (Fried, 1976).

More recent studies in rodents, by Wilson et al. (2002), used ametered dose inhaler (MDI) that aerosolized Δ9-THC to provide asystematic route of exposure in mice. They found that in malemice, intravenous (IV) administered and inhaledΔ9-THC produced sim-ilar levels of Δ9-THC in blood and brain tissue at the antinociceptiveED50 dose (median effective dose of 30mg) and that the behavioural ef-fects of inhaled Δ9-THC— antinociception, hypomotility, catalepsy, andhypothermia—were all significantly antagonized by co-administrationof the CB1 antagonist/inverse agonist SR141716. Subsequently, Wilsonet al. (2006) reported that although SR141716 induces withdrawalsigns (e.g., paw tremors) in mice chronically exposed to either inhaledmarijuana smoke or IV administered Δ9-THC, only co-administrationof IV administered Δ9-THC reversed these effects; inhaled marijuanasmoke did not prevent precipitated withdrawal induced by SR141716.To account for this unexpected finding, Wilson et al. (2006) suggestedthat levels of Δ9-THC from the marijuana smoke in the brain afterinhalation were insufficient to reverse the effects of SR141716 andthus themechanism of action was still likely CB1-mediated. This con-clusion was supported by additional findings of a dissociation be-tween Δ9-THC levels found in blood and brain that were dependentupon the route of administration; although blood and brain levelswere roughly equivalent following inhalation, brain levels were200–300% greater in brain than in blood after IV administration(Wilson et al., 2006). In mice, Niyuhire et al. (2007) showed thatthe effects of Δ9-THC on learning and memory also differed accordingto the route of administration: IP administration of Δ9-THC (1, 3,10 mg/kg) dose-dependently disrupted both acquisition and recall ofplatform location in the Morris water maze task, whereas inhaledsmoke from marijuana (50, 100, and 200 mg) only impaired perfor-mance at the highest dose (estimated to have 4.2 mg Δ9-THC beforeburning). Co-administrationwith SR141716 reversed these effects, sug-gesting that the mechanism of action was also CB1 mediated (Niyuhireet al., 2007).

All of these studies, which directly compared the effects of paren-teral versus pulmonary Δ9-THC demonstrate that the route of ad-ministration produces qualitatively different results, which mayaccount for some of the many conflicting findings in cannabinoid re-search in animal models of human behaviour. Since the combustionof cannabis releases many compounds in addition to Δ9-THC, includingother cannabinoids (e.g., cannabidiol) andmany toxins and carcinogens(e.g., anthrocyclines, nitrosamines, polycyclic aromatic hydrocarbons,terpenes, and vinyl chloride) (Turner, Bouwsma, Billets, & Elsohly,

1980; Turner, Elsohly, & Boeren, 1980; Sarafian et al., 1999; Zhanget al., 1999; Roth et al., 2001; Hashibe et al., 2005; Voirin et al., 2006;Aldington et al., 2008; Berthiller et al., 2008; also reviewed in Reece,2009) which may exert their own effects, vapourization of pure Δ9-THC provides a more accurate assessment of the direct effects of Δ9-THC and its primary psychoactive metabolite, 11-hydroxy-Δ9-THC(11-OH-Δ9-THC). In the current study, a novel delivery system ofvapourized Δ9-THCwas developed and assessed for its pharmacokinet-ic, pharmacodynamic, and behavioural effects in rodents. In addition,studies in animals need to account for actual bioavailable levels of Δ9-THC in blood after injection and inhalation of pure Δ9-THC, not justmarijuana smoke containing unknown quantities of Δ9-THC and nu-merous other toxicants. Previously, we have directly compared canna-binoid recovery levels of Δ9-THC and 11-OH-Δ9-THC in whole bloodafter IP and vapourization exposure to Δ9-THC in rodents (Manwellet al., 2014-in this issue); for the applications to animal behaviour stud-ies, we were only interested in the psychoactive cannabinoids Δ9-THCand itsmetabolite 11-OH-Δ9-THC. A commercially available vapourizer,commonly used by cannabis smokers, was used to assess the effects ofpulmonary administration of Δ9-THC and directly compared to paren-teral administration of Δ9-THC; drug effects on locomotor activity,food and water consumption, and cross-sensitization to morphinewere measured.

2. Material and methods

2.1. Materials, standards, and chemicals

Δ9-THC (Dronabinol, N98% purity) was obtained from THC PharmGmbH (Frankfurt, Germany). For experiments involving IP drug ad-ministration, Δ9-THC was first dissolved in a small volume of ethanoland then mixed with TWEEN-80 (polyoxyethylene sorbitanmonooleate; ICN Biomedicals). The ethanol was evaporated undera stream of nitrogen gas, and the dispersion was then mixed withphysiological saline. The final vehicle contained 15 μl TWEEN-80per 2 ml saline. Δ9-THC was prepared in concentrations of 0.1, 0.30.5, 1.0, 1.5, and 2.0 mg/ml and injected in a volume of 1 ml/kg. Forexperiments involving pulmonary (inhaled) drug administration,Δ9-THC was prepared in ethanol at concentrations of 4, 8, 20, 40and 80 mg/ml, and 250 μl of each was applied to small steel woolpads (liquid pads, Storz & Bickel, Tuttlingen, Germany), yieldingfinal amounts of 1, 2, 5,10, and 20 mg/pad. The ethanol was thenallowed to evaporate completely before vapourization. Morphinehydrochloride (CDMV, St. Hyacinth, Quebec) was dissolved in 0.9%saline and administered subcutaneously (SC) at a dose of 5 mg/kgin a volume of 1 ml/kg body weight. Equivalent injections of vehiclewere given for the saline probe.

2.2. Animals

Male Sprague–Dawley rats (Charles River, Canada) (n = 120),weighing approximately 200–300 g, were used in experiments. Ratswere pair-housed in standard plastic shoebox cages (45 × 25 ×20 cm3) maintained at 21–22 °C in a colony room on a 12-h reversedlight–dark cycle (lights off at 7 AM) and fed standard rat chow (Harlan8640) and water ad libitum. Testing was conducted during the darkcycle. Experimental procedures followed Canadian Council on AnimalCare guidelines and were approved by the Wilfrid Laurier UniversityAnimal Care Committee. Rats were acclimatized to the colony and han-dling procedures prior to experimentation.

2.3. Apparatus

2.3.1. Vapourization apparatusA Volcano® Vapourization device (Storz and Bickel, GmbH and

Co., Tuttlingen, Germany) was used as described by Hazekamp,

114 L.A. Manwell et al. / Journal of Pharmacological an

Ruhaak, Zuurman, van Gerven, and Verpoorte (2006). Briefly, Δ9-THC(1, 2, 5, 10 or 20mg) was vapourized (at heat setting 9, approximate-ly 226 °C) and collected into detachable plastic balloons (approxi-mately 8 l), which were then fitted with a release valve and thevapour immediately expelled (over 10 s) into enclosed plasticboxes (32 × 16 × 12 cm3) containing two rats that inhaled the va-pour for 5 min. After 5 min, the lids were opened for ventilation.This device has been previously reported by Hazekamp et al.(2006) to deliver N50% of the loaded Δ9-THC into the balloon in a re-produciblemannerwith a pulmonary uptake comparable to smokingcannabis.

2.3.2. Behavioural testing apparatusSix enclosures (292 × 610 × 292mm high) equipped with a video

tracking system were used to detect locomotor activity and con-sumption testing. The chambers had ultra-high molecular weight(UHMA) polyethylene sides and acrylonitrile butadiene styrene(ABS) plastic floors, while the tops were made of clear acrylic. Activ-ity under dim red light (0.04 lx) was recorded by three video cam-eras mounted 110.5 cm above the chambers and images weretransmitted to a computer in the neighbouring room running ANY-maze Video Tracking System software (Stoelting Co., Wood Dale, IL,USA). During testing, food was presented in cylindrical glass dishes(120 mm diameter × 20 mm high) placed in one corner of the cham-ber, and plastic drinking bottles containing tap water were attachedto one side of the chamber. Dishes were washed daily at the comple-tion of the session with a detergent solution. Each rat received thesame dish for the duration of the experiment. Measurements offood and water were obtained using a digital scale. For all experi-ments, locomotor activity was recorded by ANY-maze using threeoverhead video cameras (110.5 cm above the chambers) and definedby total distance travelled (m), total time mobile (s), and absoluteturn angle (°) as quantified by ANY-maze.

2.4. Experimental procedures

2.4.1. Effects of vapourized Δ9-THC on locomotor activity andcross-sensitization to morphine

Rats were tested in 2 equal groups (n = 6) as follows: vapourizedΔ9-THC (10 mg) or vehicle. During testing, rats were exposed to eithervapourizedΔ9-THC (10mg) or vehicle, for 5min before being placed in-dividually in the locomotor test chambers for 4 h. Vehicle and Δ9-THC-treated ratswere counterbalanced across the experimental periods. Thiswas repeated 7 times every 48–72h for 15 days. Two days after thefinalvapour exposure, rats were injected subcutaneously (SC) with salineand placed in the test chambers for 4 h. Two days later, animals wereinjected with morphine (5 mg/kg SC) and following a 14-day drug-free interval, rats received a second morphine challenge (5 mg/kg SC).Rats were placed in the locomotor test chambers for 4 h followingboth morphine challenges.

2.4.2. Effects of vapourized — Δ9-THC or IP-Δ9-THC on food and waterconsumption and locomotor activity

2.4.2.1. Effects of vapourized Δ9-THC. Rats were habituated to vapourexposure chambers and activity monitors for 5 days prior to experi-mentation. Non-deprived rats received one drug test session every48 h at the same time each day, that is, 1 h following the onset ofthe dark cycle. Rats were tested in 4 equal groups (n = 12) as fol-lows: vapourized Δ9-THC (1, 5, or 10 mg) or vehicle. During testdays, 2 rats were exposed to 8 l of vapourized Δ9-THC (1, 5, or10 mg) or vehicle for 10 min per vapour exposure chamber, with 4l being administered at 0 min and the remaining 4 l at 5 min. The an-imals were then placed immediately into activity monitors for a 4-htest period. Food (laboratory chow) andwater intake weremeasuredby weight each hour (spilled food recovered prior to weighing). Rats

received all treatments in a counterbalanced order. On days betweendrug testing, animals were exposed to no-drug vapour and the ex-periment was conducted as usual.

d Toxicological Methods 70 (2014) 112–119

2.4.2.2. Effects of IP-Δ9-THC. Rats were habituated to activitymonitors for5 days, and received saline injections (1 ml/kg) 1 day prior to experi-mentation. Rats were tested in 5 equal groups (n = 12) as follows: Δ9-THC (0.1, 0.3, 0.5, 1.0 mg/kg) or vehicle. Rats received one drug test ses-sion every 48 h at the same timeeach day, that is, 1 h following the onsetof the dark cycle. On test days, animals were injected IPwith one dose ofΔ9-THC (0.1, 0.3, 0.5, 1.0 mg/kg) or vehicle 30min prior to a 4-h test pe-riod. Food andwater intakeweremeasured byweight eachhour (spilledfood recovered prior to weighing). Rats received all treatments in acounterbalanced order. On days between drug testing, animals receivedsaline injections and the experiment was conducted as usual.

3. Data analysis

3.1. Effects of vapourized Δ9-THC on locomotor activity andcross-sensitization to morphine

For the vapour phase data, total distance travelled (m), total timemo-bile (s), and absolute turn angle (°) were grouped into four 1 h bins andwere analyzed separately using a three-factor (day × hour × treatment)analysis of variance (ANOVA), with repeated measures on day and hour.Pairwise comparisons were conducted using Bonferroni-adjusted t-tests(α= 0.05). Significant two-way interactions were followed by one-waytests of the simple main effects, and Bonferroni-adjusted t-tests (α =0.05) when these were significant. Four planned two-way (day ×treatment) ANOVAs with repeated measures on day were conductedseparately at each hour. Significant day by treatment interactions werefollowed by t-tests comparing the Δ9-THC- and vehicle-treated groupsat each day. For the cross-sensitization phase data, a three-factor(probe × hour × treatment) ANOVA with repeated measures on probeand hour was conducted separately for total distance travelled (m),total time mobile (s), and absolute turn angle (°). Pairwise comparisonswere conducted using Bonferroni-adjusted t-tests (α = 0.05). Signifi-cant two-way interactions were followed by one-way tests of the simplemain effects and Bonferroni-adjusted t-tests (α= 0.05). Four planned t-tests comparing the Δ9-THC- and vehicle-treated groups at each probewere conducted separately for each hour. Some tests did not meet thesphericity assumption. However, all F-tests reported remained signifi-cant when the Huynh–Feldt adjustment was applied. Corrected degreesof freedom are reported where relevant. Analyses were conducted usingSPSS 13.0 for Macintosh (SPSS, Chicago, IL).

3.2. Effects of vapourized — Δ9-THC or IP-Δ9-THC on food and waterconsumption and locomotor activity

Experimental food intake (g), water intake (ml), and locomotor ac-tivity were analyzed separately for each route ofΔ9-THC administration(IP or vapourized). Drug datawere grouped into four 1-h bins and treat-ed as a factor. Separate two-factor repeated measures (dose × hour)ANOVAs were conducted for each dependent variable. Bonferroni-adjusted t-tests were used to compare vehicle to each dose of Δ9-THCand to compare eachhour to all other hours.Mauchly'sWwas also com-puted in order to determine if the assumption of sphericitywas violated.Since it was expected that the dose used would yield different effectsacross hours, planned tests of the simplemain effectswere used to com-pare drug treatments at each level of hour. Post hoc tests were used tofurther compare each day, with planned comparisons of Day 1 vs.each of the other days. Analyses were conducted using SPSS 13.0 forWindows (SPSS Inc., Chicago, IL) (α = 0.05).

Fig. 1. Total distance travelled (m) following vapourized exposure to vehicle or 10mgΔ9-THC. Data aremeans (±SEM) at four consecutive 1 hmeasurement intervals for the 7-dayvapour exposure phase and 3-day cross-sensitization test phase. *p b 0.05 vehicle vs. Δ9-THC, **p b 0.05, significantly different from day 7, ***p b 0.01, significantly different fromsaline; ****p b 0.05, significantly different from hour 1.

115L.A. Manwell et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 112–119

4. Results

4.1. Effects of vapourized Δ9-THC on locomotor activity andcross-sensitization to morphine

Fig. 1 shows the total distance travelled for both the vapourizedΔ9-THC andmorphine cross-sensitization phases. The twomost interest-ing findings were that 1) acute administration of 10 mg of vapourizedΔ9-THC induced a short-lasting stimulation in locomotor activity com-pared to control in the first of four hours of testing over 7 days of repeat-ed exposure, and 2) this chronic exposure to 10 mg of vapourized Δ9-THC did not induce behavioural sensitization to morphine.

First, the vapourized Δ9-THC phase three-way (day × hour ×treatment) ANOVA for total distance travelled showed a main effectof day [F(6, 60) = 40.08, p b 0.001], main effect of hour [F(3, 30) =20.74, p b 0.001], day by hour interaction [F(18,180) = 3.56, p b 0.001],and hour by treatment interaction [F(3, 30) = 6.19, p b 0.005]. Forall rats, locomotor activity decreased across consecutive days (e.g., day 7was less than days 1, 2, 3, and 6 across all four hours (p b 0.001;p b 0.01; p b 0.05; and p b 0.05, respectively)) and across hours(e.g., greater during hour 1 than hours 2, 3, and 4 (p b 0.001; p b 0.01;and p b 0.05, respectively)). Locomotor activity also decreased acrossday by hour (e.g., day 7 was significantly less than day 1 during hour1, 2, and 3 (p b 0.001; p b 0.05; and p= 0.001, respectively)). Most im-portantly, Δ9-THC-treated rats showed greater locomotor activity thanvehicle-treated rats only during hour 1 [t(10) = 2.79, p b 0.05].

Second, the morphine cross-sensitization phase three-way(probe ×hour× treatment) ANOVA for total distance showed amain ef-fect of probe [F(corrected df = 1.592,15.920) = 14.50, p b 0.001] and aprobe by hour interaction [F(6,60)= 4.09, p b 0.01]. For all rats, bothmorphine probes increased locomotor activity compared to the sa-line probe (p b 0.01 for both) and across hours 1, 2, and 3 ((pb 0.05). p b 0.05, p b 0.01, respectively) but not hour 4. This finding isconsistent with previous work showing that increases in morphine-induced locomotor activity are the greatest between hours 2 and 3post-injection (e.g., Singh, Verty, Prince, McGregor, & Mallet, 2004). Inaddition, locomotor activity was greater after the second morphineprobe (p b 0.05) but there were no differences between the threeprobes during hour 4. Most importantly, there were no significant dif-ferences in locomotor activity between Δ9-THC-treated rats andvehicle-treated rats. All remaining data and analyses for total time mo-bile and absolute turn angle were almost identical to distance travelled(data not shown).

4.2. Effects of vapourized — Δ9-THC or IP-Δ9-THC on food and waterconsumption and locomotor activity

4.2.1. Habituation dataIn the 5 days prior to experimentation, the mean values of food and

water intake for the vapourized and injectedΔ9-THC groups did not dif-fer greatly across routes of administration; results revealed food andwater consumption increased from Day 1 to Day 5 (data not shown).

4.2.2. Effects of vapourized — Δ9-THCFig. 2 (top) shows the amount of food consumed during each of four

1-h bins following administration of vehicle and 1, 5, and 10 mg ofvapourized Δ9-THC. A two-factor repeated measures (dose × hour)ANOVA showed only a dose × hour interaction [F(9, 11) = 3.05,p b .01]. Within the first hour, vapourized Δ9-THC dose-dependentlyincreased feeding [F(3, 11) = 4.26, p b .05], while a decrease wasseen in hour 2 for the highest dose [F(3, 11) = 3.80, p b .05]; by hour4, food consumption was consistent across groups. Post-hoc testsshowed the 10 mg dose of Δ9-THC significantly increased feeding rela-tive to vehicle at hour 1 (p b 0.05), but none of the doses were signifi-cantly different from vehicle at hour 2. Fig. 2 (bottom) shows thevolume of water consumed during each of four 1-h bins following

Fig. 2. Total laboratory chow consumed (total 4-h intake) (top) and total water consumed(bottom) in non-deprived rats (n=12) following vapourized exposure to vehicle, 1, 5, or10 mg Δ9-THC. Data are means (+SEM) at four consecutive 1-h measurement intervals.*p b 0.05, significantly different from vehicle at hour 1.

Fig. 3. Total distance travelled (top), time in motion (middle), and absolute turn angle(bottom) in non-deprived rats (n = 12) following vapourized exposure to vehicle, 1, 5,or 10mgΔ9-THC. Data aremeans (+SEM)at four consecutive 1-hmeasurement intervals.*p b 0.05, significantly different from vehicle at hour 1; **p b 0.05, significantly differentfrom vehicle at hour 2; ***p b 0.05, significantly different from vehicle at hour 3.

116 L.A. Manwell et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 112–119

administration of vehicle, or one of three doses of vapourized Δ9-THC.The two-factor repeatedmeasures (dose × hour) ANOVA did not revealany significant main effects or interaction.

Fig. 3 shows locomotor activity (e.g., distance travelled, time inmotion, and absolute turn angle) during each of four 1-h bins follow-ing administration of vehicle and 1, 5, and 10 mg of vapourized Δ9-THC. For total distance travelled, a two-factor repeated measures(dose × hour) ANOVA showed a main effect of hour [F(3, 11) =6.16, p b .01] and dose × hour interaction [F(9, 11) = 4.85, p b .01].Vapourized Δ9-THC increased locomotor activity at hour 1 [F(3, 11) =5.21, p b .05], hour 2, [F(3, 11) = 3.76, p b .05], hour 3 [F(3, 11) =5.20,p b .05], but not hour 4. Only rats administered 10 mg of vapourizedΔ9-THC showed an initial increase in locomotor activity in hour 1 com-pared to subsequent hours 3 and 4 (p b 0.05).

For total time in motion, a two-factor repeated measures (dose× hour) ANOVA showed a main effect of hour [F(3, 11) =3.42, pb .05] and dose × hour interaction [F(9, 11) = 3.44, p b .01].Vapourized Δ9-THC increased locomotor activity at hour 2 [F(3,11) =6.59, p b .01], hour 3 [F(3,11) = 4.62, p b .05], but not hours 1 or 4.However, rats administered 10 mg of vapourized Δ9-THC significantlydecreased time inmotion relative to vehicle at hours 2 and 3 (p b 0.05).

For absolute turn angle, a two-factor repeatedmeasures (dose ×hour)ANOVA showed a main effect of hour [F(3, 11) = 5.46, p b .05] and dose× hour interaction [F(9, 11) = 3.38, p b .01]. Vapourized Δ9-THC in-creased locomotor activity hour 2 [F(3,11) = 5.76, p b .01], hour 3[F(3,11) = 4.61, p b .01], but not hours 1 or 4. Rats administered 10 mgof vapourized Δ9-THC significantly decreased locomotor activity relativeto vehicle at hour 2 [p b 0.05], whereas 5 mg Δ9-THC significantly de-creased activity relative to vehicle at hour 3 (p b 0.05).

Carryover effects of vapourized Δ9-THC on feeding 24 h after ini-tial exposure were assessed using two-factor repeated measures(dose × hour) ANOVAs (data not shown). Although feeding behaviourwas not significantly affected by any dose of vapourized Δ9-THC 24 hafter administration, analysis ofwater consumption showed an increase

in drinking behaviour (main effect of hour [F(3, 11) = 3.44, p b 0.05]).The later finding was expected as rats typically increase drinking anddecrease activity over time when placed in a novel environment. Loco-motor activity 24 h after administrationwas not affected by any dose ofvapourized Δ9-THC.

4.2.3. Effects of IP-Δ9-THCFig. 4 shows the amount of food andwater consumed during each of

four 1-h bins following administration of vehicle or 0.1, 0.3, 0.5, or1.0 mg/kg IP Δ9-THC. Neither food nor water consumption were foundto be significantly affected by any dose of injected Δ9-THC.

Fig. 5 shows locomotor activity during each of 4 1-h bins followingadministration of vehicle, or one of four doses of IPΔ9-THC. For distancetravelled and absolute turn angle, two-factor repeated measures (dose× hour) ANOVAs did not show anymain effects or interactions; neithermeasure of locomotor activitywas significantly affected by any dose. Fortime in motion, the two-factor ANOVA showed only a main effect of

Fig. 4. Total laboratory chow consumed (total 4-h intake) (top) and total water consumed(bottom) in non-deprived rats (n=12) following IP administration of vehicle, 0.1, 0.3, 0.5,or 1.0 mg/kg Δ9-THC. Data are means (+SEM) at four consecutive 1-h measurementintervals.

Fig. 5. Total distance travelled (top), time in motion (middle), and absolute turn angle(bottom) in non-deprived rats (n=12) following intraperitoneal administration of vehi-cle, 0.1, 0.3, 0.5, or 1.0 mg/kg Δ9-THC. Data are means (+SEM) at four consecutive 1-hmeasurement intervals. ***p b 0.05, significantly different from vehicle at hour 3.

117L.A. Manwell et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 112–119

hour [F(3, 11) = 6.21, p b .05]. Post-hoc tests showed that only the1.0 mg/kg dose of IP Δ9-THC significantly decreased locomotor activityrelative to vehicle at hour 3 (p b 0.01).

5. Discussion

We report here the development of a novel Δ9-THC vapourizationmethod for rodent behavioural neuropharmacology and comparisonof the behavioural effects of vapourized and IP-administered Δ9-THCon locomotor activity, feeding behaviour and cross-sensitization tomorphine in rats. The results of the present study demonstrate im-portant differences in the effects of pulmonary versus parenteral ad-ministration of Δ9-THC in rodents, which are summarized as follows:(1) vapourized Δ9-THC increased feeding behaviour compared to con-trol and IP-administered Δ9-THC, (2) vapourized Δ9-THC induced ashort-lasting stimulation in locomotor activity compared to control,(3) chronic exposure to vapourized Δ9-THC did not induce behaviouralsensitization to morphine compared to control, and (4) the findingsfrom vapourized Δ9-THC contrast with previously reported findingsfor parenteral administration of Δ9-THC.

In the locomotor activity and cross-sensitization to morphine study,repeated exposure to vapourized Δ9-THC significantly increased loco-motor activity (e.g., distance travelled, total time mobile, and absoluteturn angle) compared to the vehicle control for only hour 1 of eachday's testingduring the vapour phase; both groups showed similar loco-motor activity for hours 2, 3 and 4. In the cross-sensitization to mor-phine phase, acute exposure to vapourized Δ9-THC did not have asignificant effect on any of the three measures of locomotor activitycompared to the vehicle control for the saline and twomorphineprobes.Results showed an increase in locomotor activity after both morphineprobes for the vapourizedΔ9-THC and vehicle control groups comparedto the saline probe. The saline and morphine probes demonstrated that

vapourized Δ9-THC, unlike IP-administered Δ9-THC, does not appear topromote the behavioural sensitization to opioid-induced hyperactivity.We demonstrated that a high dose of 10 mg of vapourized Δ9-THC didnot induce cross-sensitization, thus future studies should evaluatepotential dose–response effects of vapourized Δ9-THC on cross-sensitization to opioids and other drugs of abuse. This is not likely aneffect due to reaching subthreshold levels after pulmonary administra-tion. We have previously shown similar dose-dependent and time-dependent concentration changes in Δ9-THC and its metabolite 11-OH-THC after pulmonary and parenteral administration of Δ9-THC andreported similar levels of cannabinoid recovery after low andhigh levelsofΔ9-THC administration (Manwell et al., 2014-in this issue). For exam-ple, 20 min after exposure to 10 mg/pad vapourized Δ9-THC and1.5 mg/kg injected Δ9-THC, 402.31 and 349.33 ng/ml/kg of Δ9-THCwere recovered respectively.

These results are novel and in contrast to the findings of many stud-ies on parenterally-administered Δ9-THC in the literature indicating

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cross-sensitization between cannabinoid agonists and opioids in rats(e.g., Cadoni, Pisanu, Solinas, Acquas, & Di Chiara, 2001; Lamarque,Taghzouti, & Simon, 2001; Norwood, Cornish, Mallet, & McGregor,2003; Singh, McGregor, & Mallet, 2005). In fact, previous findings thatparenterally-administered Δ9-THC induced cross-sensitization toopioidswas argued to be (1) a potential neurobiologicalmechanismun-derlying poly-drug abuse and (2) in support of the “gateway hypothesis”which suggests that cannabis use facilitates progression to the use ofopiates and other illicit drugs in individuals vulnerable to the effects ofdrugs of abuse (e.g., Cadoni et al., 2001; Lamarque et al., 2001;Norwood et al., 2003; Singh et al., 2005). However, the novel, albeit pre-liminary, findings in the current study indicate that the route of admin-istration may mediate cross-sensitization to opioids which is criticallyimportant because humans do not inject Δ9-THC but rather inhale or in-gest cannabis (for amore in-depth discussion seeManwell et al., 2014-inthis issue).

In the acute exposure to vapourized Δ9-THC (1, 5, 10 mg) feedingstudy, a dose-dependent increase in feeding behaviour was found forhour 1 compared to the vehicle control group and to the 5 d habituationbaseline; a decrease in feeding was observed for only the highest doseof Δ9-THC for hour 2. In comparison, vapourized Δ9-THC did not affectwater consumption rates for any of the doses in comparison to thevehicle control or 5 d habituation baseline. In addition, vapourized Δ9-THC increased locomotor activity only at the highest dose for hour 1and for the two highest doses for hour 2; locomotor activity was similarto vehicle control for the lowest dose and for hours 3 and 4. In compar-ison, the IPΔ9-THC (0.1, 0.3, 0.5, 1.0 mg/kg) feeding study did not showany effect on feeding behaviour in comparison to vehicle control or tothe 5 day habituation baseline; similar to the vapourization study, IPΔ9-THC also did not affect water consumption rates for any of thedoses. However, IP Δ9-THC did not affect locomotor behaviour at anyof the four doses administered. The initial stimulation in locomotor ac-tivity for vapourized Δ9-THC is once again at odds with the injectedroute of administration. Importantly, injected Δ9-THC often results ininconsistent findings. For example, in contrast to our study, which dem-onstrated that four doses of IPΔ9-THC (0.1–1.0 mg/kg) did not affect lo-comotor activity, Sañudo-Peña, Romero, Seale, Fernandez-Ruiz, andWalker (2000) found a triphasic effect of IP Δ9-THC, also in maleSprague–Dawley rats, in which low doses (0.2 mg/kg) decreased loco-motor activity, moderate doses (1–2 mg/kg) increased activity, andhigh doses (2.5–5 mg/kg) induced catalepsy and decreased activity.Even higher doses of IP Δ9-THC (10 mg/kg) significantly decrease loco-motor activity (Whitlow, Freedland, & Porrino, 2003). However, atten-uation of locomotor activity after 1 and 7 days of exposure, but notafter 21 days, indicates that behavioural sensitization and toleranceemerge even after repeated exposure to very high doses in Sprague–Dawley rats (Whitlow et al., 2003). Our findings, that repeated expo-sure to a high dose of vapourized Δ9-THC (10 mg) did not have similarinhibitory effects on locomotor activity and behavioural sensitization,are in contrast to previous findings demonstrated in Sañudo-Peñaet al. (2000) and Whitlow et al. (2003).

Our results indicate that acute exposure to vapourized Δ9-THCproduces a short-lasting stimulation of feeding behaviour and locomo-tor activity whereas IP-administered Δ9-THC produces inconsistent re-sults. It has been suggested that parenteral administration of Δ9-THCproduces a biphasic dose response, with low doses stimulating feedingand higher doses inhibiting feeding. For example in rats, doses of 0.5, 1.0and 2.0 mg/kg IP-administered Δ9-THC have been shown to increasefeeding (e.g., Glick & Milloy, 1972; Järbe & DiPatrizio, 2005; Koch,2001; Williams, Rogers, & Kirkham, 1998), whereas doses in excess of2.5 mg/kg have been found to decrease feeding (e.g., Sofia & Barry,1974); other studies have failed to demonstrate any effect of IP Δ9-THC (e.g., 1.0 and 2.0 mg/kg) on feeding (e.g., Graceffo & Robinson,1998) (also reviewed in Abel, 1975 and Kirkham & Williams, 2001).The dose range employed in the IP-administered Δ9-THC (0.1 to1.0 mg/kg) consumption experiment reported here was chosen to

reflect doses typically found to increase feeding; however, none ofthe doses significantly affected food or water consumption. Thesefindings support the notion that parenteral administration of Δ9-THC produces inconsistent effects on feeding. These findings arealso inconsistent with studies in humans which typically show in-creased feeding following exposure to cannabis smoke (Abel, 1971;Greenberg, Kuehnle, Mendelson, & Bernstein, 1976; reviewed inKirkham & Williams, 2001). It is therefore noteworthy that inhalationof 10 mg of vapourized Δ9-THC increased feeding during the first hourof testing. These results suggest that the rate of absorption, which israpid for inhaledΔ9-THC but slower for IP-administration ofΔ9-THC, af-fects the behavioural outcome of the drug on feeding. Because inhaledadministration replicates the type of absorption in humans whosmoke cannabis, produces similar effects of feeding, it serves to improveanimal models in the field of cannabinoid research.

Results from the present study are important to the study of can-nabinoid behavioural neuropharmacology, particularly regardingthe effects of Δ9-THC in animal models currently used: the route ofadministration of Δ9-THC needs to be considered to ensure findingsare relevant to those obtained in human studies of cannabis use. Past re-search has shown that cannabis smoke exposure produces similar ef-fects to those obtained using IV administration of THC, such as adepression of locomotor activity and increased immobility (Fried &Neiman, 1973; Lichtman, Sheikh, Loh, & Martin, 2001). Yet in thesestudies, animals were exposed to smoke in an immobilization tube,hence this could have affected spontaneous locomotor activity by in-ducing stress. The present study avoided this problem by exposingrats to vapourizedΔ9-THC in a receptaclewhere they couldmove freely,and had been habituated to previously. Studies have also shown thatnon-cannabinoid compounds present in smoke can produce effects ontheir own and alter the pharmacological effects of pure Δ9-THC(Truitt, Kinzer, & Berlo, 1976). It is therefore suggested that pulmonaryexposure to burnt Δ9-THC produces behavioural effects, yet methodsthat deliver purified Δ9-THC may demonstrate the sole effects of thisdrug more clearly. Hence the current study is the first of its kind to ex-amine the effects of vapourizedΔ9-THC on feeding and place condition-ing, and utilizes a less stressful procedure in terms of measuringlocomotor activity.

In conclusion, results from the present study suggest that varyingthe route of administration of Δ9-THC can yield different patternsof behavioural effects. This would appear to result from differencesin pharmacodynamics. Taken together, the present study offers pre-liminary data suggesting that some of the shortcomings of commonanimal models used for examining the behavioural effects of canna-binoids may be solved by replacing an injected route of administra-tion with a novel vapourized Δ9-THC mode of drug delivery. This inturn will serve to provide a clearer understanding of the short- andlong-term effects of cannabis use in humans.

Competing interests

The authors declare that they have no competing interests.

Author contributions

LMwrote themanuscript; BF, BM andHH performed the behaviouralexperiments (locomotor activity, cross-sensitization behaviour, and con-sumption testing) under the supervision of PM; PM supervised all exper-iments and co-wrote the manuscript.

Acknowledgments

This work was performed with grants from Wilfrid Laurier (to PM)and the Natural Sciences and Engineering Research Council of Canada(to LM, BM, and PM) and from the Wilfrid Laurier University Scienceand Technology Endowment Program (to BM).

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