*2.6. LC–MS/MS Analysis*

HPLC was performed through an Exion LCTM AC System (AB Sciex, Foster City, CA, USA) composed of a vacuum degasser, a double plunger pump, a cooled autosampler, and a temperature-controlled column oven. The MS/MS analysis was carried out with a Triple QuadTM 3500 system (AB Sciex, Foster City, CA, USA). The analytes were separated on a Synergi 4 μm Hydro-RP 80 A LC Colum 150 × 4.6 mm (Phenomenex, Torrance, CA, USA) with a mobile phase composed of 0.1% formic acid in water (A) and methanol (B) at a rate flow of 0.800 mL/min. The chromatographic gradient is described below in Table 1.

**Table 1.** Chromatographic gradient used for analysis by LC-MS/MS. Phase A: 0.1% formic acid in water; phase B: methanol.


The injection volume was 10 μL for each sample. Mass spectrometric detection was done in negative ionization (ESI) mode, and the parameters were set as follows: curtain gas at 30 psi, ionization voltage at −4500 V, source temperature at 500 ◦C, and nebulization gas 1 and nebulization gas 2 at 50 psi. The optimized compound-dependent MS/MS parameters (declustering potential, entrance potential, collision energy, and collision cell exit potential) were obtained, in multiple-reaction-monitoring (MRM) mode, by a separate infusion of the analyte (CBD) and the internal standard (CBG). The analyte and the internal standard were analyzed by using the following mass transitions: 313/245 (CBD), 315/136 (CBG). The LC–MS/MS system was controlled by AB Sciex Analyst (version 1.7) software.

The Limit of Detection (LOD) of CBD was detected by multiple injections in LC-MS of serial dilutions of the sample. LOD was found 1.2 pg. This can be considered the lowest concentration of CBD that the instrument is able to detect.

#### *2.7. RNA Preparation and Real-Time Polymerase Chain Reaction*

Total RNA from mPFC was isolated by single-step guanidinium isothiocyanate/phenol extraction using PureZol RNA isolation reagen<sup>t</sup> (Bio-Rad Laboratories, Segrate, Milan, Italy) according to the manufacturer's instructions and quantified by spectrophotometric analysis. Following total RNA extraction, the samples were processed for real-time reverse transcription polymerase chain reaction (real time RT-PCR) to assess mRNA levels. Briefly, an aliquot of each sample was treated with DNase to avoid DNA contamination. RNA was analyzed by TaqMan qRT-PCR instrument (CFX384 real time system, Bio-Rad Laboratories) using the iScriptTM one-step RT-PCR kit for probes (Bio-Rad Laboratories). Samples were run in 384-well formats in triplicate as multiplexed reactions. Data were analyzed with the comparative threshold cycle (ΔΔCt) method using *36B4* as reference gene [33]. Primers and probe for *Bdnf exon IV* and *VI* were purchased from Applied Biosystems (*Bdnf exon IV*: ID Rn01484927\_m1 and *Bdnf exon VI*: ID Rn01484928\_m1). Primers and probe for total *Bdnf* and *36B4* were purchased from Eurofins MWG-Operon. Their sequences are shown below:


#### *2.8. Protein Extracts Preparation and Western Blot Analyses*

Proteins from mPFC and striatum were homogenized in a glass −glass potter in cold 0.32 M sucrose buffer pH 7.4 containing 1 mM HEPES, 0.1 mM PMSF, in presence of com-

mercial cocktails of protease and phosphatase (Sigma-Aldrich, Milan, Italy) inhibitors and then sonicated. Total proteins were measured in the whole homogenate and quantified according to the Bradford Protein Assay procedure (Bio-Rad, Milan, Italy), using bovine serum albumin as calibration standard, and stored at −20 ◦C for subsequent molecular analysis. Western blots (WB) were run using sodium dodecyl sulfate −8% polyacrylamide gel under reducing conditions as previously described [34] on the whole homogenate lysate (10 μg) of mPFC and striatum and then electrophoretically transferred (dry transfer) onto nitrocellulose membranes (GE Healthcare). The strips of nitrocellulose membrane close to the molecular weights at which the bands of the protein of interest were expected were cut from the entire squared blot (full areas) as suggested by their specific molecular weight and the information present in the datasheet of the antibody. Blots were blocked for 1 h at room temperature (25 ± 2 ◦C) with I-Block solution (Life Technologies, Monza, Italy) in TBS + 0.1% Tween-20 buffer and washed with TBS + 0.1% Tween-20 buffer. The conditions of the primary antibodies were the following: anti mBDNF (1:1000, Icosagen, Tartu, Estonia, cod: 327-100), anti phospho-TrkB Tyr706 (1:500, Novus Biologicals, Littleton, CO, USA, cod: NBP2-54764), anti phospho-Akt Ser473 (1:1000, Cell Signaling Technology, Danvers, MA, USA, cod: 9271), anti phospho-ERK2 Thr185/Tyr187 (1:1000, Cell Signaling Technology, cod: 4370), anti-total TrkB (1:500, Cell Signaling Technology, cod: 4603), Akt (1:1000, Cell Signaling Technology, cod: 9272), ERK2 (1:5000, Cell Signaling Technology, cod: 4695), and anti β-actin (1:10.000, Sigma-Aldrich, cod: A5441). Results were standardized to β-actin control protein, which was detected by evaluating the band density at 43 kDa. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories) after 2–3 min of enhanced chemiluminescence substrate (ECL) exposure (Cyanagen Srl, Bologna, Italy). Activation of the proteins investigated were expressed as a ratio between the phosphorylated and the respective total forms and analyzed. Gels were run two times each, and the results represent the average from two different runs. We used a correction factor to average the different gels: correction factor gel B = average of (OD protein of interest/OD β-actin for each sample loaded in gel A)/(OD protein of interest/OD β-actin for the same sample loaded in gel B) [35].
