2 Material and methods

2.1 Animals

Twelve adult male Sprague-Dawley rats (Charles River, Italy) 275-300g were single-housed in temperature and humidity-controlled environment (19−23 °C, 60 ± 20 %) on a 12-h light/dark cycle, with light ON at 7:30 pm. Water and food were available ad libitum . All animal care and experimental procedures are reported in compliance with the European Union regulations and Directive 2010/63/EU and were approved by the ethical committee (OPBA) of the University of Verona and by the Ministry of Health (authorization n. 780/2019-PR).

2.2 Brief Environmental Enrichment exposure

After two weeks of acclimatisation period in the animal facility, half of the rats were exposed for 22 h to Environmental Enrichment (EE group). EE consisted of a novel housing cage (35.6 × 48.5 × 21.8 cm, Optirat Gen II, Animal Care System), where rats were single-housed (i.e., no social component) with various objects (toys with different materials, shapes, and colours, i.e., plastic balls and ladders, wood bricks) for sensory stimulation. The second half of the rats did not receive Environmental Enrichment exposure (NoEE group).

2.3 Western Blot Assays

Two hours after the end of EE exposure, rats were sacrificed, the brains were rapidly removed, and following the brain atlas of Paxinos and Watson (Paxinos & Watson, 2007), 1-mm thick slices containing the medial prefrontal cortex (mPFC, bregma +3.20 mm), the nucleus accumbens (NAc, bregma +1.70 mm), and the hippocampus (Hipp, bregma -3.30 mm) were dissected by using a 1-mm Coronal Brain Matrix (SouthPointe Surgical Supply, Florida, USA), frozen on dry ice and stored at −80 °C. After the dissection of brain areas, proteins were isolated and analyzed in the whole homogenate and PSD as previously described with minor modifications (Piva et al. , 2020). Briefly, mPFC, NAc, and Hipp were homogenized in a Teflon-glass potter in cold 0.32 M sucrose buffer pH 7.4 containing 1 mM HEPES, 1 mM MgCl2, 1 mM NaHCO3, and 0.1 mM PMSF, in presence of commercial cocktails of protease (Roche, Monza, Italy) and phosphatase (Sigma-Aldrich, Milan, Italy). An aliquot of each homogenate was then sonicated and stored at -20°C. The remaining homogenate was centrifuged at 800 g for 5 min; the obtained supernatant was then centrifuged at 13000 g for 15 min, obtaining a pellet. This pellet was resuspended in a buffer containing 75 mM KCl and 1% Triton X-100 and centrifuged at 100000 g for 1 h. The resulting supernatant, referred as Triton X-100 soluble fraction (TSF, extra-synaptic fraction), was stored at -20°C; the pellet, referred as PSD or Triton X-100 insoluble fraction (TIF, post-synaptic density), was homogenized in a glass–glass potter in 20 mM HEPES, protease and phosphatase inhibitors and stored at -20°C in presence of glycerol 30%. Total proteins have been measured in the homogenate and TIF fractions according to the Bradford Protein Assay procedure (Bio-Rad, Milan, Italy), using bovine serum albumin as calibration standard.
Western blots were run as previously described (Mottarlini et al. , 2022). Briefly, equal amounts of proteins of the homogenate (10 μg) and TIF fraction (8 μg) were run on a sodium dodecyl sulfate-8% polyacrylamide gel under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were blocked for 1 h at room temperature with I-Block solution (Life Technologies Italia, Italy) in TBS 0.1% Tween-20 buffer and incubated with antibodies against the proteins of interest. The conditions of the primary antibodies were the following: anti-vGLUT-1 (1:1000, Cell Signaling Technology Inc. RRID: AB_2797887), anti-GLT-1 (1:5000, AbCam. RRID: AB_1566262), anti-GluN1 (1:1000, Cell Signaling Technology Inc. RRID: AB_659874), anti-GluN2B (1:1000, Cell Signaling Technology Inc; RRID: AB_1264223), anti-GluN2A (1:1000, Cell Signaling Technology Inc. RRID: AB_2112295), anti-SAP102 (1:1000, AbCam; RRID: AB_1860292), anti-GluA1 (1:1000, Cell Signaling Technology Inc. RRID: AB_2732897), anti-GluA2 (1:2000, Cell Signaling Technology Inc. RRID: AB_10622024), anti-SAP97 (1:1000, AbCam), anti-PSD95 (1:2000, Cell Signaling Technology Inc. RRID: AB_2292883), anti-GRIP (1:1000, Synaptic System. RRID: AB_887728), anti-Arc/Arg3.1 (1:500, BD Transduction Lab. RRID: AB_399886), and anti-β-Actin (1:10000, Sigma-Aldrich. RRID: AB_476744). Results were standardized using β-actin as the control protein, which was detected by evaluating the band density at 43 kDa. Each set of proteins shown in Figures 1-2-3 was run in the same WB assay, thus only one band of β-Actin is presented. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories). Gels were run 3 times each and the results represent the average from 3 different western blots, averaged and normalized by using a specific correction factor (Caffino et al. , 2020). Cropped immunoblots are reported in Supplementary Information (SI) (Fig.S1-6).

2.4 Statistical analysis

All the numerical data are given as mean ± SEM. Data were tested for normal distribution using Shapiro–Wilk’s test. Data were collected in individual animals (independent determinations) and molecular changes produced by Environmental Enrichment exposure were analyzed separately for each brain area using unpaired Student’s t-test. Non-parametric test (i.e., Mann-Whitney U-test) was chosen when data were found not to be normally distributed, while t-test with Welch’s correction was chosen when there was significant variance in homogeneity. Differences were considered significant at p < 0.05. All analyses were performed using the GraphPad software package (Prism, version 9; GraphPad, San Diego, California, USA).