*2.3. Experiments*

Upon arrival, all the animals were habituated in the facility before starting any procedure for one week. Rats were left undisturbed for two days and then they were handled for 5 days before the injections; such manipulation was performed to avoid any potential bias due to stress-related effects. After this period of acclimation to the facility and the procedures, a total of 48 animals were divided into two separate sets of rats for acute (Experiment 1) and repeated (Experiment 2) treatments with CBD. In Experiment 1, a subset of 32 male Sprague Dawley rats was exposed to acute treatment of different CBD doses and was further subdivided into four experimental groups: (1) control group receiving a single i.p. injection of saline (*n* = 8), (2) single i.p. injection of CBD 5 mg/kg (*n* = 8), (3) single i.p. injection of CBD 15 mg/kg (*n* = 8), and (4) single i.p. injection of CBD 30 mg/kg (*n* = 8). The animals were sacrificed one hour after the acute treatment with saline or CBD. In Experiment 2, a subset of 32 male Sprague Dawley rats was subdivided into four experimental groups: rats exposed to (1) repeated saline (*n* = 8), or CBD treatments at the dose of (2) 5 mg/kg (*n* = 8), (3) 15 mg/kg (*n* = 8), or (4) 30 mg/kg (*n* = 8) for seven consecutive days. Animals were sacrificed twenty-four hours after the last treatment with saline or CBD. After decapitation, brains from Experiment 1 and 2 were rapidly removed, and the medial prefrontal cortices (mPFC, defined as Cg1, PL, and IL subregions, corresponding to plates 5–9) and striatum tissues (caudate putamen, corresponding to plates 10–25) were immediately dissected from 2 mm thick slices following the coordinates of the Rat Brain Atlas of Paxinos and Watson [31], then frozen on dry ice and stored at −80 ◦C for subsequent molecular analysis.

#### *2.4. Plasma Preparation for CBD Measurements*

Trunk blood from each rat was promptly collected after decapitation in tubes containing sodium citrate 3.8% (200 μL × 2 mL of blood collected) as anticoagulant agent. Plasma was separated by centrifugation (6500× *g* for 20 min) and stored at −20 ◦C for future molecular analysis. Plasma samples were prepared for CBD quantification as follows: 25 μL of the internal standard [cannabigerol (CBG) 3 ng/μL] was added to 150 μL of each plasma sample. Protein precipitation was performed by adding 100 μL of acetonitrile, while the extraction of CBD was performed with 1 mL of hexane. The samples were mixed by vortex and centrifuged at 10,000 rpm for 10 min, 900 μL of the supernatant was transferred into a new 1.5 mL tube, and the solvent was removed under nitrogen gas flow. The residue was reconstituted in 150 μL of methanol. Each sample was mixed and centrifuged at 10,000 rpm for 5 min. The final supernatant was used for the LC–MS/MS analysis.

#### *2.5. Brain Tissue Preparation for CBD Measurements*

The extraction of CBD from the mPFC was performed by using the whole protein homogenate extracted from each sample (see Protein Extracts preparation below). Ten μL of the internal standard (CBG 3 ng/μL) was added to 250 μL of each protein homogenate. The samples were mixed by vortex and centrifuged at 10,000 rpm for 10 min, 900 μL of the supernatant was transferred into a new 1.5 mL tube, and the solvent was removed under nitrogen gas flow. The residue was reconstituted in 150 μL of methanol. Each sample was mixed and centrifuged at 10,000 rpm for 5 min. The final supernatant was used for the LC–MS/MS analysis [32].
