 once with KHB buffer and incubated with compounds either 5 min for HEK-DAT and HEK-SERT cells or 8 min for HEK-NET cells. Subsequently, substrates were added and the reactions were stopped with ice-cold KHB buffer after either 1 min for HEK-DAT and HEK-SERT cells or 3 min for HEK-NET cells. Cells were lysed with 1% SDS and released radioactivity was measured by a liquid scintillation counter (Tri-carb-2300TR, Perkin Elmer). All the experiments were repeated in triplicate.
The substrate/efflux experiments were performed as described before (Sucic et al., 2010). Briefly, HEK-DAT cells were grown in 5 mm diameter PDL-coated coverslips. Cells were incubated with 0.1 μM 3H-MPP+ at 37°C for 20 min. The coverslips were transferred onto superfusion chambers (0.2 ml) and excess radioactivity was washed out with KHB buffer for 40 min (0.7 ml/min) at 25°C to obtain stable baselines. Thereafter, modafinil or D-amphetamine was added as depicted in Figure 1 and the experiment was started with the collection of fractions (2 min). During the experiments with enhanced intracellular sodium concentration, the buffer was switched either to monensin or remained at control buffer after the collection of three baseline fractions for another five fractions. Subsequently, modafinil or D-amphetamine was added for another five fractions as indicated in Figure 1. Finally, the remaining radioactivity was collected by treatment with 1% SDS.
FIGURE 1. Working memory error (WME) assessment of modafinil in the radial maze for 10 days. Rats in groups of 10 and 5 mg/kg body weight modafinil performed at a comparable level in the radial arm maze (RAM) training up to day 7, but from day 8 trained drug animals performed better as compared with their counterparts injected with vehicle whereas rats in groups of 1 mg/kg body weight performed better on day 9 and 10. Data analyzed using ANOVA and Bonferroni post hoc test. Data represented as mean ± SD.
Radial Arm Maze (RAM)
Animals
Male Sprague–Dawley rats, aged between 12 and 14 weeks, were used in all experiments. They were bred and maintained in cages made of Makrolon and filled with autoclaved woodchips in the Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna. Food and water in bottles was available ad libitum. The room was illuminated with artificial light at an intensity of about 200 l× in 2 m from 5 am to 7 pm. Experiments were carried out between 8 am and 2 pm All procedures were carried out according to the guidelines of the Ethics committee, Medical University of Vienna, and were approved by Federal Ministry of Education, Science and Culture, Austria (BMWFW-66.009/0114-WF/II/3b/2014). All efforts were made to minimize animal suffering and to reduce the number of animals used.
Sixty animals were injected intraperitoneally 1, 5, or 10 mg/kg body weight modafinil dissolved in DMSO or DMSO as vehicle and injected 30 min prior to training on each day and were trained or untrained in the RAM. Low dose treatment for CE was published previously (Eagle et al., 2007). The six groups were: three different doses of modafinil (1, 5, and 10 mg/kg) for trained group, one 10 mg/kg modafinil untrained group and two vehicle trained and untrained group. Animals in the vehicle group were injected with DMSO at a dose of 1 ml/kg of body weight (Bartsch et al., 1976).
Apparatus
The maze was made out of black plastic and kept at an elevation of 80 cm above the floor in a room with numerous visual cues. The central platform had a diameter of 50 cm with 12 arms (12 cm × 60 cm) projecting radially outward. A plastic cylinder was used to restrict the movement of rats in the center before the start of training. Lifting of the cylinder was controlled by a pulley system from the far end of the room.
Procedure
Radial arm maze training was performed as described in Levin et al. (2010) and Timofeeva et al. (2010) with some modification. In brief: rats were handled for 5 days for adaptation (30 min/day/rat) and also to reduce the body weight to 85%. Water was provided ad libitum during the training. The amount of food (ssniff Spezialdiäten GmbH) was provided to maintain a lean, healthy body weight of approximately 85% of the free-feeding weight during training. Out of 12 arms, eight arms were baited with a small piece (40 mg) of same food during the training and four remained un-baited. Before the start of the training, rats were given two habituation sessions in which food was placed all over the maze and rats were allowed to explore the maze and eat the food for 5 min. During the training session, the same arms were baited for each rat once at the beginning of each session to assess WM, while the other four arms were always left un-baited in order to test reference memory. The pattern of baited and un-baited arms were consistent throughout testing for each rat but differed among rats. Each trial started by placing the rat onto the central platform, after 10 s the cylinder was lifted slowly and the rat was allowed to enter any arm. The session lasted 8 min or until all eight baited arms were entered-whatever occurred first. The maze was cleaned between the two trials by using 1% incidin (Ecolab GmbH, Austria). Arms were baited only once and a repeated entry into a baited arm was counted as a WME, whereas any entry into an un-baited arm was recorded as a reference memory error. The rats were given 10 training sessions, one training per day. Untrained group rats underwent similar handling, habituation, food restriction, and spent the same time in the RAM as their counterpart trained group rats except there was no food during the trials so that they do not form any memory. The training sessions were recorded with a computerized tracking video camcorder: 1/3 SSAM HR EX VIEW HAD. Six hours after the end of the tenth training animals were deeply anesthetized with CO2 and killed by neck dislocation. Brain tissues were quickly removed and hippocampi were rapidly dissected on a cold plate set at 4–6°C and stored at –80°C till further biochemical analysis.
Blue Native-Polyacrylamide Gel Electrophoresis (BN-PAGE)
Only the 10 mg/kg body weight dose group was used for proteomic studies. Ten whole hippocampi from each of the four groups were run on BN-PAGE followed by immunoblotting with antibodies against the DA receptor and transporter systems, namely D1R, D2R, D3R, pDAT, and DAT.
Homogenization, Sample Preparation and Extraction of Membrane Proteins
All procedures were performed at 4°C as published previously (Saroja et al., 2014). The extracted membrane receptor proteins were then aliquoted and stored at -80°C until use. The extracted fractions were used for BN-PAGE.
Blue Native-Polyacrylamide Gel Electrophoresis
Membrane pellets from the ultracentrifugation fraction were solubilized in extraction buffer (1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0) and 10% Triton X-100 (stock solution was added at a ratio of 1:4 to achieve final 2% Triton X-100 concentration) 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 was estimated using the BCA protein assay kit (Pierce, Rockford, IL, USA). Seventy microgram of the membrane protein preparation were applied onto gels. 16 mL of BN-PAGE loading buffer [5% (w/v) Coomassie G250 in 750 mM 6-aminocaproic acid] were mixed with 100 μL of the membrane protein preparation and loaded onto the gel. BN-PAGE was performed in a PROTEAN II xi Cell (Bio-Rad, Germany) using 4% stacking and 5–13% separating gel. The BN-PAGE gel buffer contained 500 mM 6-aminocaproic acid, 50 mM Bis–Tris, pH 7.0; the cathode buffer 50 mM Tricine, 15 mM Bis–Tris, 0.05% (w/v) Coomassie G250, pH 7.0 and the anode buffer 50 mM Bis–Tris, pH 7.0. The voltage was set to 50 V for 1 h, 75 V for 6 h, and was increased sequentially to 400 V (maximum current 15 mA/gel, maximum voltage 500 V) until the dye front reached the bottom of the gel (Kang et al., 2008). Native high molecular mass markers were obtained from Invitrogen (Carlsbad, CA, USA).
Immunoblotting
Membrane proteins were transferred from BN-PAGE to PVDF membranes. After blocking of membranes for 1 h with 10 % non-fat dry milk in 0.1% TBST (100 mM Tris–HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), membranes were incubated with primary antibodies D1R (diluted 1:5000, Abcam-ab78021, Cambridge, UK), D2R (diluted 1:5000, Abcam-ab21218, Cambridge, UK), D3R (diluted 1:5000, Abcam- ab42114,