Animals

Male C57BL/6J mice from Harlan (Barcelona, Spain) weighing 20–25 g were housed in groups of six per cage (40 × 25 × 22 cm) under controlled conditions (temperature, 21 ± 2 °C; relative humidity, 60 ± 10 %; 12 h light/dark cycle, lights on from 08:00 to 20:00). Behavioral analyses were initiated 1 week after acclimatization to the animal room and were performed by placement of the home cage in the operant-task room during the development of conditioning experiments. All studies were conducted in compliance with the Spanish Royal Decree 1201/2005, the Spanish Law 32/2007, and the European Union Directive of 22 September, 2010 (2010/63/UE), regulating the care of experimental animals.

Drugs

For CPP and the evaluation of CBD withdrawal syndrome, CBD was obtained from STI Pharmaceuticals (Essex, UK), dissolved in ethanol:cremophor:saline (1:1:18), and injected intraperitoneally (i.p.) at the appropriated doses (CPP: 15, 30, and 60 mg/kg; CBD withdrawal syndrome: 30 mg/kg/12 h, 6 days).

For the oral CBD self-administration procedure, water-soluble CBD (purified CBD organic plant extract, 99.9% purity (high-performance liquid chromatography (HPLC)), total THC content (%) < 0.01%; CBD Pur US, Hilton Head Island, SC, USA) was purchased. This formulation is a water-soluble powder with certain excipients that increase the oral bioavailability of CBD. The water-soluble CBD contains 99.9% pure CBD, arabic gum, maltodextrin, and polyphosphates, and is a nano-sized formulation to avoid the stickiness-related problems of CBD. CBD was immediately dissolved in distilled water (37 °C) before its use, following the instructions of the manufacturer, at the desired concentration (50 mg/kg; CBD water-soluble solution (1.2 mg/mL)). This concentration was calculated considering the average number of active lever presses (50), the volume released in each lever’s activation (25 µL), and the weight of the mice to provide a final dose of 50 mg/kg.

Dose–response effects of CBD on CPP

CPP was performed following a protocol previously described [50]. The CPP apparatus consisted of two compartments (30 × 20 × 20 cm), one black with a smooth plastic floor and the other one white with a stainless-steel grid floor, separated by a sliding door. The CPP procedure consisted of three phases as follows:

(A) Pre-conditioning (Pre-C) phase: on day 1, mice were placed between the two compartments and allowed free access to the entire apparatus for a period of 20 min. Mice that spent more than 75% of time in any of the two compartments were discarded from the experiment.

(B) Conditioning phase: on days 2–11, the animals were randomized and assigned to the vehicle (VEH) or CBD groups (15, 30, 60 mg/kg; i.p.) (n = 8/group). CBD groups were counterbalanced for chamber type (context A/B) associated with VEH and CBD. Animals remained in the conditioned compartment for 15 min.

(C) Test day: on day 12, the door was opened and mice were placed in the center to allow free exploration of the two compartments. The time that mice spent in each compartment was recorded during the 15 min session using the SMART (Spontaneous Motor Activity Recording and Tracking) software system (Panlab, Barcelona, Spain).

Determination of plasma CBD concentrations

Before the evaluation of the CBD withdrawal syndrome, plasma concentrations of CBD were measured to confirm that there was no significant amount of CBD remaining in the blood that could disturb the interpretation of the results.

Briefly, after a single administration of CBD (30 mg/kg; i.p.), trunk blood was collected from mice at different time points (2, 4, 8, 12, and 24 h). The extraction and analysis of plasma was performed using a method previously described with some modifications [51]. Aliquots of 0.1 mL of plasma were transferred to 12 mL glass tubes and spiked with CBD-d3 (Sigma-Aldrich, Madrid, Spain). After dilution with 0.1 M ammonium acetate buffer (pH 4.0), samples were extracted with tert-butyl methyl ether. The dry organic extracts were reconstituted in 75 µL of a mixture of water:acetonitrile (10:90, v/v) with 0.1% formic acid (v/v). Detection of CBD in plasma was performed using an Agilent 1200 series HPLC system (Agilent Technologies, CA, USA) coupled to a 6410 Triple Quadrupole LC-MS (Agilent Technologies) mass spectrometer with an electrospray interface. Chromatographic separation was achieved on a C18-CSH column (3.1 × 100 mm, 1.8 μm particle size) (Waters Corp., Milford, MA, USA) at 40 °C. The electrospray ion source was set on the positive ionization mode. The mass spectrometry detection was done by single-reaction monitoring. Quantification was calculated with the slope (s), intercept, and correlation coefficient (r) by weighting the (1/x) least-squares linear regression of the peak area ratio (analyte/IS) vs. the concentration of the standard. The detection limit and quantification limit of the analytical procedure were 0.15 and 0.45 ng/mL, respectively.

CBD withdrawal syndrome

The analysis of the CBD withdrawal syndrome was carried out according to a protocol previously described by our group for cannabinoid spontaneous withdrawal [52, 53]. Briefly, CBD (30 mg/kg; i.p.) or VEH were administered twice a day for 6 days (n = 8/group). On day 7, 12 h after the last administration of CBD or the corresponding VEH, motor activity and behavioral withdrawal signs (rearing, grooming, and rubbing) were evaluated during a 15 min period (see schematic representation, Fig. 3a).

Motor activity was recorded using SMART software and withdrawal signs were recorded using video cameras, and further analyzed by a blind observer.

Oral CBD self-administration paradigm

Oral CBD self-administration paradigm was based on a method previously described by our group with some modifications [54]. Oral CBD self-administration tests were carried out in 12 modular operant chambers (Panlab, Harvard Apparatus) equipped with a chamber light, two levers, one receptacle to receive a drop of liquid solution, one syringe pump, one stimulus light, and one buzzer. Packwin software (Panlab) controlled the stimulus and fluid delivery, and recorded the operant responses. Pressing one lever did not produce any action (inactive lever), whereas pressing the other lever delivered 25 µL of fluid combined with the delivery of a 0.5 s light stimulus and a 0.5 s, 2850 Hz, 85 dB buzzer (active lever), followed by a 6 s timeout period. The experiment was divided into three phases as follows (see schematic representation, Fig. 4):

(A) Training phase: mice were given 1 h daily saccharin (0.2%) self-administration sessions during days 1–10. During the first 5 days of the training phase, food was provided 1 h before the beginning of each session to increase the motivation for the active lever (postprandial). The following 5 days of the training phase and during the rest of the experiment, food access was provided after the end of each daily session (preprandial).

(B) Saccharin substitution phase: during this phase, the saccharin concentration was reduced and the CBD concentration was increased. Mice were given 1 h daily saccharin 0.15% + 50 mg/kg CBD (CBD water-solution 1.2 mg/mL) or VEH (days 11–13), saccharin 0.1% + 50 mg/kg CBD (CBD water-solution 1.2 mg/mL) or VEH (days 14–16), and saccharin 0.05% + 50 mg/kg (CBD water-solution 1.2 mg/mL) or VEH (days 17–19) sessions. Mice that maintained a stable response with < 30% deviation from the mean of the last three consecutive sessions and gave at least 75% of their responses on the active lever were selected for the following phase.

(C) CBD (50 mg/kg, 1.2 mg/mL) consumption phase: for days 20–25, saccharin was removed from the solution and mice were given 50 mg/kg CBD (CBD water-solution 1.2 mg/mL) or VEH for 1 h a day under a fixed ratio 1 (FR1) reinforcement schedule.

All solutions used in this paradigm were prepared daily, immediately before the beginning of the experimental procedure.

Statistical analysis

Statistical analyses were performed using one-way analysis of variance (ANOVA) with repeated measures (RM) followed by the Student–Newman–Keul test to compare CBD plasma concentrations at different time points. The CPP and oral CBD self-administration paradigms data were analyzed using two-way ANOVA with RM followed by the Student–Newman–Keul test. Motor activity and somatic withdrawal signs (rearing, grooming, and rubbing) were analyzed using Student’s t-test. Statistical analyses were performed with SigmaStat (Systat Software Inc., Chicago, IL, USA) software. Differences were considered significant if the probability of error was less than 0.05.