Cocktail 2

Mice were euthanized by cervical dislocation one day after the behavioral test finished. Brains were immediately removed from the skull. The hippocampus was then isolated and frozen in powdered dry ice. They were maintained at −80°C for further use. Tissue samples were homogenized in lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma). Total protein levels were obtained and protein concentration was determined by the method of Bradford.

Three days after the behavioural and cognitive tests, mice were euthanized by cervical dislocation. Brains were immediately removed from the skull. The hippocampus and cortex were then isolated and frozen in powdered dry ice. They were maintained at −80 °C for further use. Tissue samples were homogenized in lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma). Total protein levels were obtained, and protein concentration was determined by the method of Bradford.

For WB, brain tissues (n = 6 mice per group) were homogenized with lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma, St. Louis, MI, USA), and Bradford’s method was used to determine the protein concentration. Aliquots of 15 µg of protein were separated by SDS-PAGE (10–16% gels) and transferred into a PVDF membrane (Millipore, Burlington, MA). Afterwards, the membranes were blocked with 5% BSA for 1 h at room temperature. The blockage was followed by overnight incubation at 4 °C with the primary antibodies (Table S2). The next day, the membranes were washed and incubated with secondary anti-mouse or anti-rabbit antibody for 1 h at room temperature (Table S2). The proteins were revealed using chemiluminescence-based detection kits (Thermofisher and ECL kit, Millipore, Burlington, MA, USA) and images were obtained using an Amersham Imager 680 (BioRad, Hercules, CA, USA). Then, quantitative analysis was carried out using ImageLab Software (Bioke, CA, USA) and the results were expressed in arbitrary units (AU), considering the control group as 100%. To ensure that the differences between the samples were not a result of inaccurate sample preparation, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein charge control.

Brain regions (PFC, striatum, hippocampus) were homogenized in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors (complete EDTA free, Roche) and phosphatase inhibitors (Cocktail II, Sigma-Aldrich) using a motor pestle (Kimble). Protein extracts were quantified by the BCA Protein Assay Kit (Pierce) and separated by SDS-PAGE in 4-12% polyacrylamide gels (Invitrogen). Samples were then blotted onto polyvinylidene difluoride membranes, blocked with 5% milk in 0.1% Tween 20 in TBS (TBS-T) and probed with primary antibodies overnight at 4°C. After three TBS-T washes, membranes were probed with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 1 h at room temperature. Detection was performed by chemiluminescence (Pierce). The following primary antibodies were used: rabbit anti-tyrosine hydroxylase (Sigma-Aldrich, SAB2701683, 1:1000); rat anti-DAT (Santa Cruz Biotechnology, sc-32259, 1:1000); and mouse-anti β-actin (Sigma-Aldrich, A5316, 1:1000).

After 3 days of the cognitive and memory tests, all mice groups were euthanized by cervical dislocation and brains were immediately removed from the skull. For molecular experiments, the hippocampi were isolated and frozen in powdered dry ice. They were maintained at − 80 °C for further use for protein extraction and RNA isolation. For protein extraction, tissue samples were homogenized in lysis buffer (50-mM Tris–HCl pH 7.4, 150-mM NaCl, 5-mM EDTA, and 1% Triton X-100) containing phosphatase and protease inhibitors cocktails (Cocktail II, Sigma-Aldrich, St. Louis, MO, USA). Total protein levels were obtained, and protein concentration was determined by the method of Bradford. For thioflavin-S staining, mice were anesthetized (ketamine 100 mg/kg and xylazine 10 mg/kg, intraperitoneally) and then perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer solution intracardially. Brains were removed and postfixed in 4% PFA overnight at 4 °C. Afterward, brains were changed to PFA + 15% sucrose. Finally, the brains were frozen on powdered dry ice and stored at -80 °C until sectioning. Brain coronal sections of 30 μm were obtained (Leica Microsystems CM 3050S cryostat, Wetzlar, Germany) and kept in a cryoprotectant solution at − 20 °C until use.

Mice were euthanised by cervical dislocation 3 days after the behavioural and cognitive tests were finished. Brains were immediately dissected out from the skull. The hippocampus of each animal was separated, snap frozen in dry ice and kept at −80 °C.
For Western Blot (WB) and immunodetection, hippocampus samples were thawed and mixed in lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma-Aldrich). Once mixed, samples were maintained on ice for 30 min. Samples were centrifugated at 10,000× g for 30 min at 4 °C, and the supernatants were collected and saved at −80 °C. Total protein amounts were obtained, and the Bradford method was used to determine protein concentration.
For ELISA evaluation, samples were processed following the instructions provided by the kit manufacturer (Biosensis, Thebarton, Australia). In brief, hippocampus samples were thawed and mixed through sonication at 4 °C in 200 volumes of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) NP-40 and 0.5% (w/v) sodium deoxycholate, pH = 7.5–8) containing a protease and phosphatase inhibitor cocktail. Once mixed, samples were maintained on ice for 30 min. Sample sonication and cooling with ice were performed. Samples were centrifuged at 14,000× g for 30 min at 4 °C, and the supernatants were obtained and maintained at −80 °C. Protein amount was quantified through the Bradford method.