Animal use and care

Subjects were Male Wistar rats (Charles River, Wilmington, MA), pair-housed on a 12 h (Lights off at 20:00) light/dark cycle in a temperature and humidity controlled vivarium with ad libitum availability of food and water, except where noted otherwise. All procedures were conducted in strict adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.

Drugs

CBD was administered transdermally to enhance translational relevance regarding future therapeutic applications. The clinical potential of CBD when administered orally is constrained to some extent by low (~6%) bioavailability [28] and potential for conversion into psychoactive cannabinoids in gastric fluid [29]. However, we have identified the transdermal route of administration as an effective delivery method that eliminates both these limitations and produces stable and sustained plasma CBD levels [26, 30]. Transdermal CBD was prepared as a fast-drying (<5 min) 2.5% hydroalcoholic proprietary gel formulation (2.5 g CBD/100 g gel) containing a non-toxic skin biocompatible permeation enhancer (Transcutol® HP, Gattefosse, USA). The gel was applied to a shaved area of skin outside the animals’ reach (shoulder blades). Following application, rats were single-housed for 4 h before behavioral testing or returned to group housing on non-test days.

Ethyl alcohol was diluted in tap water. Cocaine hydrochloride (National Institute on Drug Abuse, Bethesda, MD) and yohimbine hydrochloride (Sigma) were dissolved in sterile physiological saline.

Equipment

Standard behavioral equipment was used as previously described (alcohol self-administration and reinstatement [31], cocaine self-administration and reinstatement [32], experimental anxiety/elevated plus maze (EPM) [33], locomotor activity [32], impulsive choice [34]). Drug self-administration and reinstatement tests were conducted in sound-attenuated operant conditioning chambers equipped with two retractable levers, house and cue lights, and a speaker for presentation of auditory stimuli. A syringe pump, activated by responses at a designated lever, delivered 0.1 ml of liquid reinforcers into a 0.15 ml drinking reservoir or produced intravenous infusion of cocaine in a volume of 0.1 ml over 4 s. For delay-discounting tests, operant conditioning chambers were equipped with two nose-poke operanda that delivered 45 mg food pellets [34]. Locomotor activity was measured in metal wire hanging cages equipped with infrared emitter-detector photocells [32]. Experimental anxiety was measured on the EPM as previously described [33]. The EPM was located in a sound-insulated room with 1.5–2.0 lux of illumination for the open arms and <1 lux for the enclosed arms.

General experimental protocol

All rats (except subjects designated for delay-discounting tests, below) were trained to self-administer oral ethyl alcohol (10% w/v) or intravenous cocaine (0.25 mg/infusion) on a fixed ratio 1 (FR 1) schedule in daily 30 min (alcohol) or 2 h (cocaine) sessions using established procedures adapted from [31, 35]. In preparation for environmentally induced reinstatement testing, alcohol or cocaine availability was conditioned to a compound contextual stimulus, consisting of an olfactory component (banana extract; McCormick, Hunt Valley, MD) generated by depositing five drops of extract into the chamber’s bedding) plus an auditory (70 dB white noise) component. [As a technical note, the environmentally induced reinstatement procedure was identical to the discriminative stimulus (S D – S+/S−) model we have used in the past to study cocaine-seeking [35,36,37,38,39,40] and alcohol-seeking [31, 41,42,43,44,45] behaviors. Although, historically, we termed this methodology a “discriminative stimulus” procedure (i.e., before the concept of “context” received attention in the addiction field), the S D stimulus context serves as an occasion setter like the A/B stimulus environments in context studies. As well, operationally, the S D procedure is identical to ABA renewal/context-induced procedures [46], with only technical differences that have little bearing on the operational definition of reinstatement as behavior induced by context. Therefore, in the interest of terminological consistency across the literature, we define the behavioral effects of the S D procedure as context-induced]. Daily training under these conditions continued for a total of 10 sessions. During this phase, rats designated for stress-induced reinstatement continued drug self-administration without contextual conditioning. Drug-reinforced responding then was extinguished in daily 30-min sessions in the absence of the contextual stimulus until rats reached a criterion of ≦ 5 responses over three sessions. Following extinction, rats entered a 4-day VEH gel treatment phase (PreTx:VEH) for habituation and recording of baseline reinstatement performance induced by re-exposure to the contextual stimulus or the pharmacological stressor yohimbine. Rats then were matched based on their baseline reinstatement performance and divided into a CBD treatment and CBD vehicle control group. This matched-pair assignment assured an identical distribution of higher, average, and lower reinstatement responders in both groups. Rats in the respective groups then received either vehicle gel or CBD (in a gel volume of ≈640 µl/kg corresponding to ≈15 mg/kg CBD) at 24 h intervals over a 7-day treatment phase (Tx:CBD/VEH). Following application, rats were single-housed for 4 h before testing or returned to group housing on non-test days. Effects of CBD on reinstatement of drug seeking were established in 30-min session (in separate groups for the context and stress manipulations) on treatment days 1, 4, and 7 and on locomotor activity on day 6. Testing then continued without further CBD or vehicle treatment at early (day 3), intermediate (days 18, 48) and, in the alcohol group, late (day 138) post-treatment (PostTx) stages. Additionally, one test of experimental anxiety (EPM) was conducted on PostTx day 2. In separate groups of drug-naive rats, CBD was probed for nonspecific amotivational effects (i.e., context and stress-induced reinstatement motivated by a palatable natural reinforcer). The treatment design and sequence of behavioral tests are illustrated in Fig. 1 (see supplementary online material for additional procedural details).

Fig. 1 Experimental design. Effects of CBD were established on recovery of extinguished drug seeking on treatment days 1, 4, and 7. Testing then continued without further CBD or vehicle (VEH) treatment at early (day 3), intermediate (days 18, 48), and late (day 138) post-treatment stages. Additionally, one test of experimental anxiety (EPM) was conducted on post-treatment day 2. The diagram illustrates the design and sequence of CBD vs vehicle (VEH) treatments and behavioral tests across experimental phases. PreTx:VEH daily VEH gel.T:CBD/VEHdaily CBD or VEH gel. To evaluate the drug specificity of its effects, CBD was probed at some time points for interference with context and stress-induced reinstatement motivated by a sweet solution in separate groups of drug-naive rats. PostTx no treatment. Symbols denote ethanol (●), cocaine (○), or sweet solution (□) self-administration history, and sampling days for plasma (∆) and brain (▲) CBD levels Full size image

Reinstatement—drugs of abuse

Context-induced and stress-induced reinstatement tests were conducted using within-subject designs. For context-induced reinstatement, the drug-associated compound stimulus was re-introduced 30 s before onset of testing (Fig. 1), and remained present until session termination. Stress-induced reinstatement, studied in a separate group of rats, was induced by yohimbine (1.25 mg/kg; IP), administered 30 min before session onset. To establish the generality of CBD effects on stress-induced drug seeking, a separate group of rats with an alcohol self-administration and CBD treatment history identical to that of the yohimbine group was tested for reinstatement induced by 15 min of variable intermittent electric footshock (current intensity 0.5 mA, as previously described [45] on treatment day 7).

Reinstatement—palatable natural reinforcer (sweet solution)

To establish that reduction of drug seeking by CBD is not the result of general “amotivational” actions, we examined CBD for effects on reinstatement motivated by a sweet solution [(SSC) 3% glucose, 0.125% saccharin]. Drug-naive rats with a history of SSC self-administration and subjected to the 7-day CBD treatment regimen were tested for context-induced reinstatement of SSC seeking induced by a SSC stimulus context on treatment day 1, and for yohimbine stress-induced reinstatement on PostTx days 3 and 18.

Locomotor activity

To examine effects of CBD on spontaneous locomotor activity, rats of the alcohol and cocaine contextual reinstatement groups were placed into locomotor activity cages 4 h after CBD application on treatment day 6.

Experimental anxiety

Anti-anxiety effects of CBD were evaluated on the EPM test as previously described [47]. On PostTx day 2, rats of the context reinstatement group were habituated for 2 h to the EPM procedure in the presence of white noise (70 dB) and then placed individually onto the center of the EPM facing a closed arm, with white noise (70 dB) present. During 5-min tests, behavior was recorded by video camera and scored blind for time spent in the open and closed arms. EPM performance was expressed as % time in open arms/(% time in open arms + % time in closed arms). To determine whether the 15 mg/kg dose used for CBD effects on experimental anxiety lies on the ascending limb of a possibly U-shaped CBD dose-response function, a second EPM study was conducted in a different group of drug-naive rats comparing two doses of CBD (15 and 30 mg/kg) vs. vehicle.

Impulsive choice—delay discounting

CBD effects on impulsive behavior in rats with a history of alcohol dependence were studied using the “delay discounting” model in which impulsivity is defined as the selection of a small immediate over a larger delayed food reward (impulsive choice). Rats, maintained at 85–90% of free-feeding weight to ensure reliable motivation to work for food pellet reinforcers, were trained on a delayed reward task using established procedures (adapted from [34]). Rats were initially trained under a forced-choice contingency to obtain immediate small (1 pellet) and delayed large (4 pellets) reward by nose-poke responses. Subsequently, free-choice trials began in which nose pokes in one hole produced immediate small reward and responses in the other hole produced large delayed reward, allowing for choice of the preferred reward. Failure to make a response within 10 s was counted as an omission and initiated a new intertrial interval. The respective nose-poke hole and food magazine position associated with the small and large reward was the same for individual rats, but counterbalanced across rats. Delays for the large reward increased progressively (0, 5, 10, 20, and 40 s) within each session per block of 12 trials. Daily trials continued until stable responding was established across a block of 5 sessions. Rats then were subjected to a 7-day dependence-inducing intragastric alcohol intoxication (vs. vehicle) procedure as previously described [48] during which they were treated at 24 h intervals with CBD (≈15.0 mg/kg) or vehicle gel. Following 5 days of recovery, testing resumed for 10 sessions to record performance in the delayed reward task. Data were expressed as the number of choices of large reward/(number choices large + small reward) × 100.

Plasma and brain CBD levels

For determination of plasma CBD, rats received two CBD doses (15 mg/kg or 30 mg/kg). Blood samples were obtained on Tx day 7, 4 h after CBD application, and three PostTx time points corresponding those of behavioral testing (Fig. 1). Blood (100 µl) was collected by the tail bleed method. Plasma was separated by centrifugation and stored at −80 °C. Samples were analyzed by electrospray LC-MS as previously described [26, 30]. The limit of detection (LOD) was 1 ng/ml. For measurement of brain CBD levels, rats received the 15 mg/kg CBD dose used in the behavioral studies. Brain tissue was obtained at the same time points used for plasma sampling (Fig. 1). CBD was extracted from brain tissue by homogenization in chloroform/methanol [49] containing 100 ng/ml of CBD-d 3 as internal standard (8:3:1) followed by centrifugation, decanting of the lower supernatant phase, evaporation and reconstitution in acetonitrile for analysis. Samples were analyzed for CBD (and CBD-d 3 ) by electrospray LC-MS. The LOD was 0.2 ng/ml.

Data analysis

Differences between CBD and VEH effects on reinstatement induced stimuli conditioned to alcohol, cocaine, natural reward, and yohimbine stress (including only the Tx and PostTx phases) were separately analyzed by mixed-factorial analyses of variance (ANOVA). Following confirmation of significant main effects in the overall ANOVA, differences between group means across time were verified by simple effects ANOVAs. Differences between extinction and baseline reinstatement, Tx, or PostTx days were analyzed by Student’s t-tests. CBD vs VEH effects on footshock-induced reinstatement were analyzed by Student’s t-tests (2-tailed). CBD vs VEH effects on experimental anxiety in the EPM test were separately analyzed for open arm time and crossings by Student’s t-tests. Dose-dependent effects of CBD vs VEH on experimental anxiety in drug-naive rats were analyzed by one-way ANOVA followed by Tukey’s post hoc tests. Effects of CBD vs VEH on locomotor activity were analyzed separately for the alcohol and cocaine groups by Student’s t-test. Effects of CBD vs VEH on the development of impulsive choice were analyzed by mixed-factorial ANOVA, followed by Tukey’s post hoc tests to ascertain differences among individual means. Plasma CBD levels produced by the two CBD doses and brain CBD levels in CBD-treated vs drug-naive controls were analyzed by mixed-factorial ANOVA, followed by simple effects ANOVAs to confirm differences among group means across time.