Blue Native-Polyacrylamide Gel Electrophoresis (BN-PAGE)

Six hours after the last session of RAM training on day 10, the animals were sacrificed after anaesthetizing them with CO₂. The brain was quickly extracted and was kept on a cold plate and the FC was cut 3 mm from the anterior end after removing olfactory bulb by a chilled razor blade. The tissues were immediately frozen in dry ice and were stored at –80°C. Only the 10 mg/kg dose group was used for biochemical studies.

Crude synaptosome fractions were prepared from the FC as described previously (Shanmugasundaram et al., 2015b). The samples were maintained at 4°C throughout the procedure. In brief, the FC tissues were homogenized in 5 ml of ice cold homogenization buffer containing 10 mM HEPES, pH 7.7, 300 mM sucrose, 1 mM EDTA, protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) using Ultra-Turrax^® (IKA, Staufen, Germany). The homogenate was centrifuged for 10 min at 1000 × g and the pellet was discarded. The supernatant was centrifuged at 50,000 × g for 30 min using an ultracentrifuge (Beckman Coulter Optima-L-90K). The pellet was re-suspended in washing buffer (homogenization buffer without sucrose), kept on ice for 1 h and centrifuged at 50,000 × g for 30 min to obtain membrane fractions of crude synaptosome extracts.

The synaptosome membrane pellets were solubilized in extraction buffer (1.5 M 6-aminocaproic acid, 300 mM Bis-Tris, pH 7.0) containing 1% Triton X-100 with vortexing every 10 min for 1 h. Following solubilization, samples were cleared by centrifugation at 20,000 × g for 60 min at 4°C. The protein content of the supernatant was estimated using the BCA protein assay kit (Pierce, Rockford, IL, USA). Extracted proteins were then aliquoted and stored at –80°C.

Equal amounts of proteins (70 μg) from all groups were loaded in the wells and the RCs were separated on 5–13% of blue native PAGE gels and the Western blot procedure was carried out using the procedure described previously (Kang et al., 2009). The details of antibodies used are as follows; D1R (1:5000, Abcam-ab81296, Cambridge, UK), D2R (1:5000, Abcam-ab21218, Cambridge, UK), D3R (1:5000, Abcam-ab42114, Cambridge, UK), pDAT (1:5000, DAT Thr53, Phosphosolutions-p435-53, Aurora, CO, USA) and DAT (1:5000, Abcam- ab111468, Cambridge, UK) and detected with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10000, Abcam-ab6721, Cambridge, UK). Immunoreactive bands were quantified by the software Image J (NIH). Coomassie blue R-350 stained membranes were used as loading control and normalized with the Western blot densitometric values (Welinder and Ekblad, 2011; Sase et al., 2012).