THC) Alone Or Combined with Cannabidiol (CBD) on Cognition-Based Behavior and Activity in Adolescent Nonhuman Primates

Drug and Alcohol Dependence 221 (2021) 108629

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Drug and Alcohol Dependence

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Effects of daily Δ9-Tetrahydrocannabinol (THC) alone or combined with cannabidiol (CBD) on cognition-based behavior and activity in adolescent nonhuman primates

Sarah L. Withey a,b,c, Brian D. Kangas b,c, Sophia Charles a, Andrew B. Gumbert a, Jessica E. Eisold a, Susan R. George d, Jack Bergman b,c, Bertha K. Madras a,c,e,* a Laboratory of Addiction Neurobiology, McLean Hospital, 115 Mill St, Belmont, MA 02478, USA b Behavioral Biology Program, McLean Hospital, 115 Mill St, Belmont, MA 02478, USA c Department of Psychiatry, Harvard Medical School, 25 Shattuck St, Boston, MA 02115, USA d Department of Pharmacology, University of Toronto, Toronto, ON, M5S 1A8, Canada e Department of Medicine, University of Toronto, Toronto, ON, M5S 1A8, Canada

ARTICLE INFO ABSTRACT

Keywords: Background: Daily use of marijuana is rising in adolescents, along with consumption of high potency marijuana Marijuana products (high % Δ-9-tetrahydrocannabinol or THC). These dual, related trends have opened gaps in under Cannabis standing the long-term effects of daily consumption of a high dose of THC in adolescents and whether a ther Δ9-Tetrahydrocannabinol apeutic dose of cannabidiol (CBD) modulates THC effects. THC Methods: Adolescent squirrel monkeys (Saimiri boliviensis) were treated daily for four months with vehicle (n = 4), Cannabidiol = + + = CBD a high THC dose (1 mg/kg i.m.; n 4), or THC CBD (1 mg/kg 3 mg/kg i.m.; n 4), to investigate whether: Adolescent monkeys (1) a daily high THC dose affects performance in tasks of cognition (repeated acquisition, discrimination Nonhuman primates reversal); (2) a daily high THC dose affects spontaneous behavior and day/night activity (3) tolerance develops Cognition to the behavioral effects of THC; (4) whether CBD modulates THC effects. Unconditioned behavior Results: THC impaired performance of adolescent monkeys in a cognitive test initially, but not performance on a Sleep task of cognitive flexibility. THC reduced motor activity and increased sedentary behavior, with tolerance Emesis developing after weeks of daily treatment. Co-administered with THC, CBD did not modulate THC effects on cognitive performance, activity or tolerance, but prevented THC-induced emesis on the first day of daily treatment. Conclusions: Daily high dosing with THC compromised performance on a task of cognition, and reduced activity in adolescent primates, with tolerance developing within weeks. Whether our observations are relevant to a broader range of cognitive tasks vital for daily function in human adolescents is uncertain.

1. Introduction over the past 20 years, based on the concentration of its principal psy choactive cannabinoid Δ9-tetrahydrocannabinol (THC), while levels of Marijuana use among youth has risen globally, paralleling increased non-psychoactive cannabidiol (CBD) and CBD:THC ratios have fluctu access to legal marijuana and normalization of use (United Nations Of ated (ElSohly et al., 2021; Freeman et al., 2020a; Madras, 2019; Raber ficeon Drugs and Crime, 2019). Concurrent with declining perception of et al., 2015). THC engenders the positive reinforcing effects of mari marijuana harms (Hamilton et al., 2019; Johnston et al., 2020), more juana in adult humans and nonhuman primates via the CB1 cannabinoid adolescents are using marijuana daily in the United States, (e.g., 6.9 % of receptor (Huestis et al., 2001; Justinova et al., 2008). Marijuana or THC 12th graders, Miech et al., 2019; Monitoring the Future, 2020) and dose-dependently also impair cognitive functions in adults essential for consuming high potency strains of marijuana or marijuana-based con problem solving and regulating behavior, such as working memory, centrates (Fataar and Hammond, 2019; Meier, 2017; Meier et al., 2019). verbal learning, and attention (Adam et al., 2020; Broyd et al., 2016; The potency of marijuana has risen significantly in marijuana products Curran et al., 2002; Lovell et al., 2020; Sherif et al., 2016; Schindler

* Corresponding author at: Laboratory of Addiction Neurobiology, McLean Hospital, 115 Mill St, Belmont, MA 02478, USA. E-mail addresses: [email protected], [email protected] (B.K. Madras). https://doi.org/10.1016/j.drugalcdep.2021.108629 Received 23 September 2020; Received in revised form 6 February 2021; Accepted 8 February 2021 Available online 17 February 2021 0376-8716/© 2021 Elsevier B.V. All rights reserved.

Downloaded for Anonymous User (n/a) at Harvard University from ClinicalKey.com by Elsevier on February 27, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. S.L. Withey et al. Drug and Alcohol Dependence 221 (2021) 108629 et al., 2020; Schlienz et al., 2020; Woelfl et al., 2020). conducted 7 days a week. The experimental protocol was approved by In adolescents, marijuana use is associated with cognitive impair the Institutional Animal Care and Use Committee at McLean Hospital, ment, neuropsychiatric symptoms, diminished academic achievement, with subjects maintained in a facility licensed by the U.S. Department of addiction, and other adverse effects which may persist over time (Chan Agriculture, in accord with the Guidelines for the Care and Use of et al., 2021; Fontes et al., 2011; Gobbi et al., 2019; Ladegard, 2020; Mammals in Neuroscience and Behavioral Research Committee of the Lorenzetti et al., 2020; Mashhoon et al., 2019; Mason et al., 2020; Morin Institute of Laboratory Animals Resources, Commission on Life Sciences, et al., 2019; Scott et al., 2018; Wright et al., 2020). High potency National Research Council, 2011. marijuana, frequent or daily use by adolescents amplifies the risks of ongoing use or dependence, of poor cognitive function, and more severe 2.2. Dosing regimen psychological symptoms, which conceivably can disrupt educational and occupational goals (Barrington-Trimis et al., 2020; Fontes et al., Subjects were divided randomly into three groups and initially 2011; Hall et al., 2020; Meier, 2017; Silins et al., 2014; Wilson et al., treated with vehicle (control), THC, or THC + CBD (1:3) over a three- 2019). week period, with THC or THC + CBD doses, at identical ratios, but These dual, related trends in adolescent marijuana use have opened progressively increasing; week 1: one dose of THC (0.1 mg/kg) or THC + gaps in understanding the long-term effects of daily consumption of a CBD (0.1 mg/kg; 0.3 mg/kg, respectively); week 2: one dose of THC (0.3 regulated, high dose of THC in adolescents. To discern the direct effects mg/kg) or THC + CBD (0.3 mg/kg; 1 mg/kg, respectively); week 3: one of a daily high THC dose on cognition and behavior and avoid the dose THC (1 mg/kg) or THC + CBD (1 mg/kg; 3 mg/kg, respectively, confounds of human variables (uncertain potency and frequency of Withey et al., 2020). During this three-week dose-escalation period, marijuana use, polysubstance use, genetics, predisposing psychiatric co- subjects underwent initial touchscreen training (described below) but morbidities, environment), we administered THC daily to adolescent weekly dosing was arranged to minimize interference with touchscreen nonhuman primates and measured multiple behavioral endpoints. We training (i.e., drugs were administered on Friday, and the subsequent also interrogated whether a therapeutic dose of CBD modulates THC training session was initiated on Monday). On day 1 of week 4, the effects, as CBD reportedly mitigates, improves, attenuates, or is without subjects began daily treatment with the high dose of THC (1 mg/kg) or effect on THC-induced memory impairment, anxiety, psychosis, or THC (1 mg/kg) + CBD (3 mg/kg). One hour after dosing, they were neuroadaptation (Arndt and de Wit, 2017; Boggs et al., 2018; Englund tested in a repeated acquisition task for the firsttime and this procedure et al., 2013; Freeman et al., 2019, 2020b; Ghabrash et al., 2020; Hasbi of a daily injection 1 h before initiation of the touchscreen session was et al., 2020; Morgan et al., 2010; Schubart et al., 2011; Solowij et al., continued for 4 months. On days of behavioral observations, subjects 2018, 2019; Spindle et al., 2020). To the best of our knowledge, no were transferred from the vivarium to the observation arena 30 min. previous reports simultaneously determined whether THC administered after injection and behavior was taped for 30 min. Thereafter, subjects for 4 months: 1) affected performance of a cognitive measure of learning were transferred to the touchscreen chamber for daily cognitive testing, (repeated acquisition) and cognitive flexibility (discrimination 1 h after dosing. Pretreatment times of 30 min. for unconditioned reversal); 2) affected unconditioned behavior and day/night activity; 3) behavior observations (Wooldridge et al., 2020) and 60 min. for resulted in tolerance; and 4) whether CBD modulated the effects of THC. touchscreen-based cognitive tasks were selected based on peak drug The preferential selection of adolescent nonhuman primates (NHP) over effects of cannabinoid agonists in previous studies in squirrel monkeys rodent species was decided by the comparable THC pharmacokinetics, (Kangas et al., 2016; Wooldridge et al., 2020). metabolism, and behaviors in NHP and humans (Murray and Wise, 2010; Watt et al., 2020). The THC dose (1 mg/kg) was based on % THC in current marijuana joint equivalent (Manthey et al., 2020). Notwith 2.3. Touchscreen-based cognitive tests standing variable THC bioavailability, a high potency THC cigarette currently contains approximately 60 mg THC, equivalent to 0.9 mg/kg 2.3.1. Apparatus (70 kg person). Vaping, a common mode of marijuana consumption, Details, schematics, and photographs of the touch-sensitive experi delivers high quantities of THC more efficiently (Davenport, 2019). mental chamber are shown in Kangas and Bergman (2012). Briefly, a Vaping cartridges can contain 366 mg of THC, equivalent to 5 mg/kg. custom-built Plexiglass chamber (38 × 40 × 60 cm) was situated in a Accordingly, the THC dose of this study, which at one time might have sound- and light-attenuating enclosure (50 × 60 × 70 cm). An accessible been considered high, falls within the range of current retail THC 17-inch touch-sensitive screen (1739 L, ELO Touch Systems, Menlo Park, products. The CBD dose (3 mg/kg) was guided by prior research in CA) was mounted on an inside wall of the enclosure and enclosed by a nonhuman primates (Hasbi et al., 2020; Jacobs et al., 2016; Withey frame in the chamber’s front wall (34 × 27 cm). An infusion pump et al., 2020), by an initial pediatric dose of cannabidiol, 2.5 mg/kg, in (PHM-100 10, Med Associates, St. Albans, VT) outside the enclosure ®, Epidiolex a therapeutic formulation of cannabidiol (Miller et al., was used to deliver 0.15 mL of 30 % sweetened condensed milk solution 2020), and within the range of CBD doses reportedly attenuating some, into an accessible shallow reservoir (diameter: 2.5 cm) of a but not all THC effects (Hindocha et al., 2015; Morgan et al., 2018). custom-designed Plexiglas receptacle (5 × 3.5 × 1.27 cm). A speaker (NQ576AT, Hewlett-Packard, Palo Alto, CA) mounted above the 2. Materials and methods touchscreen inside the enclosure was used to emit an audible feedback click each time the subject touched a stimulus presented on the screen. 2.1. Subjects All experimental events and data collection were programmed in E-Prime Professional 2.0 (Psychology Software Tools, Inc., Sharpsburg, Twelve adolescent (2–2.5 years; Brady, 2000) male squirrel monkeys PA). Subjects were placed in the experimental chamber immediately (Saimiri boliviensis), drug- and experimentally-naïve, were divided into prior to the daily session, without movement restraint or restrictions. three treatment groups receiving vehicle, or THC or THC + CBD. Sub jects were individually housed in a temperature- and humidity- 2.3.2. Initial training controlled vivarium with a 12 -h light/12 -h dark cycle (7AM to 7 P M), Each subject was firsttrained to drink milk from the sipper and then with daily environmental enrichment. They had unlimited access to trained to touch the screen with its hand using response shaping as water in the home cage, and were maintained at approximately described previously in Kangas and Bergman (2012). Subjects were free-feeding weights by post session feedings of a nutritionally balanced required to complete 90-training trials during at least 5 daily sessions diet of high protein chow (Purina Monkey Chow, St Louis, MO), sup before initiation of daily cannabinoid dosing and exposure to the plemented with fresh fruit and vitamins. Experimental sessions were repeated acquisition procedure.

2.3.3. Repeated acquisition the observational arena at least 48 h after an injection for 30-minute Sessions were conducted as outlined previously (Kangas and Berg observational sessions. Animals were videotaped continuously and man, 2014). In this phase, the blue box training stimulus was replaced rated by general methods adapted previously for different species and by photographs on the screen randomly selected from a laboratory bank for specificdrug-induced effects (Madras et al., 2006; Platt et al., 2000; of >10,000 images. Each session began with concurrent presentation of Saka et al., 2004). The videotapes were subsequently scored by inde two different 7 × 7 cm digital photographs, each in a different randomly pendent observers. Weekly observation sessions continued throughout + selected quadrant of the screen. A touch response on one photo (S ) the duration of the protocol. initiated the delivery of milk into the sipper paired with an 880-milli second yellow screen flash followed by a 10-second intertrial interval 2.5.2. Behavioral scoring (ITI) blackout; a touch response to the other photo (S ) immediately Videotape scoring was conducted by 3 observers (2 of 3 blinded to initiated the 10-second ITI, without a reward. The same two stimuli drug treatment) trained in the behavioral scoring system (Platt et al., were presented during each of the 200 trials comprising the daily ses 2000). To ensure inter-rater reliability in scoring, observers underwent + sion. Following discrimination mastery (described below), a new S /S training for at least 3 h until an interobserver reliability criterion of 90 % + pair was introduced. Thus, the subject was required to learn a new S /S- was reached. The behavioral scoring system included 4 categories discrimination based on distinguishing features of two visual stimuli (Table 1) scored by the presence or absence of each behavior (i.e., the that had not been previously viewed (i.e., repeated acquisition). If a absolute frequency) in 10-second intervals during the 30-minute subject did not reach mastery in a single session (200 trials), the same observational period. stimulus pair was presented on subsequent days until mastery was reached. 2.5.3. Data analysis The total number of counts over 30 min. for each of the 4 behavioral 2.3.4. Discrimination reversal categories was calculated for each subject at each time point. As an Prior to introduction of the discrimination reversal task, subjects observation was recorded every 10 s, the maximum number of counts were exposed to the repeated acquisition task until 30 novel discrimi was 180 for each behavior. Data for THC and THC + CBD treated sub nations were mastered. In the discrimination reversal task, a variant of jects for each behavior at each time point were normalized and plotted discrimination learning to examine cognitive flexibility (Easton, 2005; as a percent of the drug-free control counts. Mackintosh et al., 1968; Verrico et al., 2008), the programmed conse + _ quences of responding to the S and S stimulus were reversed after the 2.6. Activity monitoring subject learned the initial discrimination. From stimulus pair 31 on ward, the first 100 trials in the daily session were conducted exactly as 2.6.1. Day-night activity described previously, i.e., subjects learned a novel discrimination each Activity was monitored 24 h a day for the duration of the study using + session. However, on trial 101 the relationship between S and S was Fitbit Wireless Activity Trackers (Fitbit Zip, San Francisco, CA), secured reversed without signal, i.e., during trials 101–200 the stimulus that was in an inaccessible inside pocket of a jacket. Data for 4 parameters (total + initially S was made S- and vice versa. Subjects were exposed to daytime activity, total night-time activity, night-time fragmented ac discrimination reversal conditions until 30 novel discrimination re tivity and night-time latency to inactivity) were collected during the versals were mastered. weekly treatment and daily treatment phases, and averaged for three- week periods (weeks 1–3, weeks 10–12 and weeks 14–16). 2.4. Data analysis 2.6.2. Data analysis The primary dependent measure used to evaluate repeated acquisi Total daytime (7AM to 7 P M) and night-time activity (7 P M to 7AM) tion and discrimination reversal performance was trials-to-mastery. was calculated as total activity counts for each period, with average Mastery was defined as the number of trials needed until 9 out of 10 daily counts over a three-week period graphed analyzed. Night-time trials correct were observed, a criterion quantified as the number of fragmented activity was defined as total activity counts after each sub trials required either to learn to discriminate between a novel pair of ject was inactive (at least one hour with zero activity counts). Once images (repeated acquisition) or to inhibit the previously reinforced inactive, we counted subsequent activity counts that occurred between response and reliably respond to the stimulus that was previously S the last hour of inactivity and before the lights were illuminated at 7AM. (discrimination reversal). Median reaction times were calculated to The average fragmented activity over a three-week period was graphed reduce the influence of significantly longer pauses in the subjects’ response times. Statistical differences in the number of trials to mastery Table 1 and median reaction times were analyzed using two-way ANOVA. Categories of observed and measured behavior. Cannabinoid treated subjects were further subdivided into two groups: Category Description of behavior day one responders attempted one or more trials on day one, and delayed responders did not respond (did not touch the screen) on day one. For Motor activity Combined score of the following two categories: Locomotion - two or more directed steps in the horizontal and/ these two subdivisions, data were analyzed for significantdifferences by or vertical plane calculating mean trials to mastery for day one or for delayed responders Environmental manipulation - any tactile or oral manipulation and analyzing mean area under the curves. Significant differences be of any features of the observational arena tween the mean trials to mastery for the three groups were calculated Self-directed Combined score of the following two categories: behavior using a one-way ANOVA for each stimulus pair, followed by Tukey’s Self-grooming - picking, scraping, spreading or licking of fur multiple comparison test. Significantdifferences in area under the curve Scratching - movement of digits through fur in rhythmic, for the three groups were calculated using a one-way ANOVA followed repeated motion by Tukey’s multiple comparison test. Rest posture Combined score of the following two categories: Sleep posture - species-typical position: eyes closed, head tucked to chest, tail wrapped around upper body 2.5. Unconditioned behavioral observations Static posture - species-typical position: subject appears alert (i.e., not asleep) but immobile 2.5.1. Observations Emesis Combined score of the following two categories: All subjects initially were habituated to the observational arena, Emesis - the forceful expulsion of gastric contents Retching - abdominal contractions accompanied by oral gaping handling and injection procedures for one month and were placed into

3.3. Unconditioned behavioral observations

The three groups displayed comparable motor activity prior to the daily drug treatment phase (Fig. 4). On Day 1 of daily treatment, THC or THC + CBD treated subjects displayed significantly fewer episodes of motor activity between 30 60 min after drug administration compared with the control group (THC: F(9,45) = 3.90, p < 0.01; THC + CBD: F (9,45) = 3.90, p < 0.05). Two weeks after daily cannabinoid treatment, tolerance to reduced motor activity was observed and THC or THC + Fig. 1. Performance in repeated acquisition task for control (circle), and THC- CBD treated subjects did not differ significantlyfrom drug-free controls. + (square) or THC + CBD-treated (triangle) subjects. Upper panel, average number Periods of resting posture increased on Day 1 of daily THC or THC of trials (mean ± SEM) to master 30 novel discriminations. Lower panel, median CBD treated subjects compared with drug-free controls (F(9,45) = 3.71, reaction time in seconds for each stimulus pair. Ordinate: Upper panel, trials-to- p < 0.05 and F(9,45) = 3.71, p < 0.05, respectively), but tolerance mastery. Lower panel, median reaction time (s). Abscissa: Stimulus pair. developed and thereafter, the three groups were not statistically

Fig. 2. Acquisition of a learning task (discrim ination) by adolescent monkeys treated daily with vehicle (n = 4) or THC (1 mg/kg; n = 4) or THC + CBD (1 mg/kg+3 mg/kg, respectively, n = 4). Performance in the task is quantified by trials-to-mastery. Individual data is shown for vehicle-treated controls (●), THC- (◼) and THC + CBD-treated (▴) subjects. The left panel shows trials-to-mastery of vehicle-treated con trols which all responded to and mastered the task on Day one of chronic treatment. The middle panel shows cannabinoid-treated sub jects, which responded on Day one, but required more trials-to-mastery than controls. The right panel shows trials-to-mastery of cannabinod-treated subjects which did not respond at all on Day one of chronic treatment. When they eventually responded, they required fewer trials to mastery than cannabinoid- treated Day one responders. Significant differ ences between trials to mastery at each stimulus pair for day one responders and controls are denoted by * (p < 0.05) or ** (p < 0.01). Sig nificantdifferences between trials to mastery at each stimulus pair for day one responders and delayed responders are denoted by # (p < 0.05) or ## (p < 0.01). Ordinate: Trials-to-mastery. Abscissa: Stimulus pair.

Fig. 3. Performance in a discrimination reversal task quantified as trials-to-mastery by control (circles, left panel), THC- (squares, middle panel), and THC + CBD- treated subjects (triangles, right panel). Filled symbols are average trials-to-mastery during the repeated acquisition (RA) phase (trials 1-100) and unfilled symbols are average trials-to-mastery during the discrimination reversal (DR) phase (trials 101-200). Ordinate: trials-to-mastery. Abscissa: stimulus pair.

Fig. 4. Observer-rated behavior of control, THC-, and THC + CBD-treated subjects. Thirty mins after daily treatment with vehicle, THC or THC + CBD, motor activity, self-directed behavior and rest posture were quantifiedfor 30 min. on Day 1 and periodically until week 16. Data are expressed as mean ± SEM for either the THC- or THC + CBD- treated groups (n = 4/group) normalized to the average of vehicle-treated controls (n = 4) at each time point, represented as 100 % baseline and shown as a dotted line. Significantdifferences between vehicle-treated controls and THC-treated subjects are denoted by * (p < 0.05) or ** (p < 0.01). Significant differences between vehicle-treated controls and THC + CBD-treated subjects are denoted by # (p < 0.05).

Downloaded for Anonymous User (n/a) at Harvard University from ClinicalKey.com by Elsevier on February 27, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. S.L. Withey et al. Drug and Alcohol Dependence 221 (2021) 108629 distinguishable (p > 0.05). behavior after initial exposure, but two weeks after daily treatment, Scratching and self-grooming (self-directed behavior) was signifi activity resumed pre-treatment levels. Third, over the course of four cantly lower in THC + CBD treated subjects than in controls during the months, daily THC treatment increased night-time activity and sleep pre-chronic phase of testing (F(9,45) = 1.90, p < 0.05) and this differ fragmentation in some subjects. Fourth, daily co-administration of CBD ence re-emerged in week 14 of daily treatment (F(9,45) = 1.90, p < with THC did not modulate the effects of THC on cognitive performance 0.05). No significantdifferences were measured at any other time point, or unconditioned behavior, but blocked THC-induced emesis on the first nor did the THC and THC + CBD treated subjects differ at any time point. day of daily treatment; thereafter no emesis was observed in On Day 1 of the chronic treatment regimen, emesis was observed in THC-treated animals. all four THC-treated subjects, in 1 of 4 THC + CBD treated subject, but in THC alone or THC + CBD disrupted the ability to repeatedly no control subjects (Fig. 5) (Control vs THC: p=0.0014; Control vs discriminate between two novel visual stimuli in some subjects, THC+CBD: ns; p=8702; THC vs THC+CBD: p=0.0028;one-way ANOVA consistent with impaired cognition-based performance in rodents, followed by Tukey’s multiple comparisons test). At later time periods, nonhuman primates and humans (D’Souza et al., 2004; Harte-Hargrove emesis was not detectable in any subject. and Dow-Edwards, 2012; Kangas et al., 2016; Taffe, 2012; Taurisano et al., 2016; Weed et al., 2016; Winsauer et al., 1999, 2011). Three 3.4. Daily electronic monitoring of activity cannabinoid-treated animals attempted the task on day one but per formed poorly compared with controls. The remaining five At each time point, daytime activity of controls was higher than THC cannabinoid-treated subjects delayed engaging in the cognitive task for or THC + CBD treated subjects, but differences were not statistically 2–17 days. These variable effects may be attributable to motor, cogni significant (Fig. 6). From weeks 12–16, but not in weeks 1–3, count tive, perceptual or motivational deficits. Reduced motor impairment averages of nighttime activity, sleep latency and sleep fragmentation was an unlikely cause of poor performance, because on days animals were higher in THC treated subjects, but less so in THC + CBD subjects, responded, they completed all 200 trials at the same pace as the control compared with controls, but differences did not reach statistical subjects. By the 7th novel stimulus pairings, tolerance to cannabinoids significance. developed. These findings contrast with analogous research in adoles cent rhesus monkeys, which manifest impairment in a spatial working 4. Discussion memory task for more than 30 weeks of THC exposure (Verrico et al., 2012, 2014; 2020). These discrepancies conceivably are attributable to Daily consumption of high potency marijuana products by adoles differences in dose and dosing protocols as Verrico and colleagues used a cents is rising, a pattern of use associated with heightened risks of lower dose of THC (up to 0.240 mg/kg, IV) and did not administer THC compromised cognition and other adverse consequences (Barrington- for two days each week. Other possible explanations for the contrasting Trimis et al., 2020; Fontes et al., 2011; Hall et al., 2020; Leventhal, 2020; rates at which tolerance developed include differences in species, or in Meier, 2017; 2019; Wilson et al., 2019). To avoid individual and envi cognitive load of the two tasks. Improvements in cognitive performance ronmental confounds prevalent in human studies, we investigated the reported in month 12 (Verrico et al., 2020) may be attributable to effects of high daily THC doses administered to adolescent primates on additional intensive training rather than tolerance to THC. Finally, several behavioral endpoints. Foremost, we found that daily THC discrepant rates in development of tolerance may also be attributable to impaired the performance of adolescent subjects during the initial phase task-dependent regional differences in prefrontal development and or of learning a task which assesses discrimination learning, but not of a ganization (Conklin et al., 2007) associated with working memory and subsequent task of cognitive flexibility. Tolerance to the detrimental discrimination learning (Kangas and Bergman, 2014; Kangas et al., effects of daily THC on cognitive performance developed over time. 2016). The ventral prefrontal cortex (vPFC) is implicated in remem Second, THC reduced activity and concomitantly increased sedentary bering a novel stimulus (current study), whereas the dorsal prefrontal cortex (dPFC) is associated with facilitating spatial memory or object location (Wilson et al., 1993). If, as reported for the human adolescent brain (Gogtay et al., 2004), the vPFC matures relatively early during nonhuman primate adolescence, the faster rate of recovery from THC-induced impaired memory of a novel stimulus (this study) conceivably is a function of earlier development and greater plasticity of the vPFC than the dPFC (Verrico et al., 2020). The therapeutic potential of CBD to attenuate THC-mediated cognitive impairment was not borne out. In adult rhesus monkeys, CBD failed to modulate performance on some, but not all cognitive tasks compromised by THC (Jacobs et al., 2016; Wright et al., 2013). In rodent models of a neuropsychiatric disorder CBD enhanced cognition (Watt et al., 2020). CBD reportedly protects against memory-impairing effects and psychosis-like experiences associated with human marijuana use (Colizzi et al., 2017; Morgan et al., 2010; Solowij et al., 2018). Consis tent with our findings, a randomized, double-blind crossover study of adults found no evidence that CBD attenuated THC-induced impairment of episodic and working memory (Morgan et al., 2018). Fig. 5. Observer-rated emesis of control, THC-, and THC + CBD-treated sub Daily cannabinoid treatment did not impair reversal learning (a jects. Emesis (including vomiting and retching) observations counted over a 30- measure of cognitive flexibility), and may have accelerated the pace of minute observation session monitored prior to (prechronic), on Day 1 and mastering reversals, though this difference did not reach significance periodically during daily treatment for 16 weeks. Data are presented as mean ± due to small sample size and high inter-subject variability. The apparent SEM for control (●; n = 4), THC- (◼; n = 4), and THC + CBD- (▴; n = 4) treated enhanced performance of cannabinoid-treated subjects may be a subjects. Ordinate: Counts of emetic behavior over 30 min observation period. Abscissa: Treatment day. Significant differences between vehicle-treated con consequence of learning the repeated acquisition task under more trols and THC-treated subjects are denoted by ** (p < 0.01; Control vs THC: demanding conditions, i.e., THC intoxication. Based on previous ob p=0.0014; Control vs THC+CBD: ns; p=8702; THC vs THC+CBD: p=0.0028; servations that THC impairs memory (Taffe, 2012), it is also conceivable one-way ANOVA followed by Tukey’s multiple comparisons test). that THC-treated subjects may not have learned the initial

Fig. 6. Automated monitoring of activity (Fit bit) of adolescent monkeys. Data are presented as three-week averages (weeks 1-3, weeks 10–12 and weeks 14-16) ± SEM for total day time activity (top left), total night-time activity (top right), nighttime fragmented activity (bottom left) and night-time latency to inac tivity (bottom right). Nighttime hours were from 19:00 – 7:00. Control (white bars; n = 4), THC- (black bars; n = 4) and THC + CBD- treated subjects (hatched bars; n = 4) are rep resented in each graph. Statistical analysis was conducted using a two- way ANOVA with Tukey’s multiple comparison test. Ordinate: Average activity counts (top left, top right, bottom left), number of hours (bottom right). Abscissa: Weeks of treatment.

discrimination as robustly as the control subjects or, when faced with the attenuation of THC-induced emesis by CBD (Parker et al., 2004) appears reversal condition, did not remember the initial discrimination. Such to be mediated by a specific target, possibly via the 5-HT1A receptor memory deficitsmight allow THC-treated subjects to readily align their (Rock et al., 2015), but not via CB1 receptor antagonism of response with the reversed stimulus-reward contingency. Accordingly, THC-mediated behaviors. Paradoxically, emesis is among the adverse the ‘improved’ reversal performance could be interpreted as a events produced by cannabidiol in clinical trials for management of THC-mediated learning deficit. Whether due to impaired memory or seizure activity (Devinsky et al., 2018), but in a self-selected online other mechanisms, altered reversal learning conceivably reflects a survey of a convenience sample, CBD reportedly is used as an THC-mediated weakening in the control of behavior by the previously anti-nausea remedy (Corroon and Phillips, 2018). learned discrimination compared with control subjects, an explanation The observed tolerance to THC-induced pharmacological effects to be viewed with caution in the absence of supporting data. On a more cannot be attributed to THC or CBD pharmacokinetic tolerance, as blood challenging task of discrimination reversal in adolescent rhesus mon levels of THC, CBD and THC metabolites of subjects in the current study keys, a lower THC dose severely compromised performance and inter remained relatively stable (Withey et al., 2020). Tolerance to marijuana fered with reversal learning in adult monkeys (Verrico et al., 2020). and THC appears to be domain-specific in humans and in nonhuman Reduced motor activity and increased resting posture on the firstday primates (Broyd et al., 2016; Freeman et al., 2021; Ginsburg et al., of daily treatment in both THC- and THC + CBD subjects corresponded 2014). In primates, tolerance develops to the response rate decreasing to prior observations of the acute sedative effects of THC in nonhuman and hypothermic effects of THC, but not to the pharmacokinetic profile primates (Beardsley et al., 1987; Meschler et al., 2000). Similarly, rapid of these drugs. Clearly, blood levels of cannabinoids do not necessarily tolerance to hypoactivity was in accord with previous observations of reflect physiological, behavioral effects of THC or the development of rapid tolerance to multiple effects of THC (McMahon, 2011), which tolerance (Grotenhermen et al., 2007; Hollister et al., 1981; Withey conceivably results from CB1 receptor and effector desensitization et al., 2020). For marijuana and other drugs of abuse, tolerance may (Selley et al., 2004). That CBD neither modulated the motor effects of portend dose escalation, problematic use and dependence. THC nor the development of tolerance to THC was consistent with some THC-treated, but not control or THC + CBD-treated subjects showed (Taffe et al., 2015) but not all rodent observations. CBD reportedly po a tendency to increased total night-time activity and fragmented night- tentiates THC-induced hypolocomotion in mice or rats (Britch et al., time activity over time, overlapping with some parameters of sleep 2017; Todd and Arnold, 2016). Such qualitatively dissimilar findingsin compromised by marijuana use in human adolescents (Maultsby et al., our nonhuman primate study and previous rodent studies may reflect 2021). The inter-subject variability in the present study, and the species-specific differences in response to THC or CBD, or doses, pre inconsistent data on sleep patterns of human exposed to cannabinoids treatment vs co-administration, THC:CBD ratios, metabolism, or dura (Babson et al., 2017; Kuhathasan et al., 2019; Nicholson et al., 2004), tion of monitoring activity across studies. highlight the need for well-designed trials in adolescents to objectively The unanticipated and novel findingthat THC-induced emesis on the measure the effects of long-term daily use marijuana with varying po first day of daily dosing is unlikely to model cannabis hyperemesis tencies and CBD on parameters of sleep. syndrome reported in some chronic high-intake marijuana users (Galli Overall, these results may reflect specific design features, including et al., 2011), as tolerance to THC-induced emesis developed within 2 the size and composition of the sample (drug-naïve males) as well as the weeks. Given that motor activity, self-directed behavior or resting study of a single THC or CBD dose or THC:CBD ratio in the absence of posture did not differ in the THC- or THC + CBD-treated subjects, other marijuana constituents, administered parenterally and daily for

Downloaded for Anonymous User (n/a) at Harvard University from ClinicalKey.com by Elsevier on February 27, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. S.L. Withey et al. Drug and Alcohol Dependence 221 (2021) 108629 four months. To extend the applicability of these findings to human Declaration of Competing Interest adolescent users, additional research is needed with larger cohort sizes, male and female subjects, a wider range of doses, dosing regimens, The authors declare no conflict of interest. routes of administration, differing ratios of THC:CBD, CBD alone, and other tests of cognition. Extrapolating the current results on cognitive References impairment and tolerance to human adolescent daily marijuana users is tempered by caveats; tasks of repeated acquisition are but one element Adam, K.C.S., Doss, M.K., Pabon, E., Vogel, E.K., de Wit, H., 2020. 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Randomized, dose-ranging safety trial of cannabidiol in Dravet syndrome. Brian Kangas contributed to conceptualization, methodology, Neurology 90, e1204–e1211. https://doi.org/10.1212/WNL.0000000000005254. writing and editing. Easton, A., 2005. Behavioural flexibility, social learning, and the frontal cortex. In: Easton, A., Emery, N.J. (Eds.), The Cognitive Neuroscience of Social Behavior. Sophia Charles contributed to data curation, formal analysis, inves Psychology Press, New York, pp. 59–80. tigation, methodology, software. ElSohly, M.A., Chandra, S., Radwan, M., Gon, C., Church, J.C., 2021. A comprehensive Andrew B. Gumbert contributed to data curation, formal analysis, review of Cannabis potency in the USA in the last decade. Biol. Psychiatry Cogn. Neurosci. Neuroimaging. https://doi.org/10.1016/j.bpsc.2020.12.016. Jan 25: investigation, methodology, software. S2451-9022(21)00022-00027. Susan George contributed to conceptualization, funding acquisition. Englund, A., Morrison, P.D., Nottage, J., Hague, D., Kane, F., Bonaccorso, S., Stone, J.M., Jack Bergman contributed to conceptualization, methodology, Reichenberg, A., Brenneisen, R., Holt, D., Feilding, A., Walker, L., Murray, R.M., Kapur, S., 2013. Cannabidiol inhibits THC-elicited paranoid symptoms and project administration, resources, writing review and editing. hippocampal-dependent memory impairment. J Psychopharmacol. 27, 19–27. Bertha Madras contributed to conceptualization, data curation, https://doi.org/10.1177/0269881112460109. formal analysis, funding acquisition, methodology, project administra Fataar, F., Hammond, D., 2019. The prevalence of Vaping and smoking as modes of tion, resources, supervision, validation, writing original draft, reviewing delivery for nicotine and Cannabis among youth in Canada, England and the United States. Intern. J. Environ. Res. Public Health 16, 4111. https://doi.org/10.3390/ and editing. ijerph16214111. 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