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).