Subjects and General Procedure

Male, Sprague-Dawley rats (350–375 g; Harlan Laboratories) were used as subjects. All animals were maintained according to the National Institutes of Health guidelines in accredited facilities. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine.

Animals were implanted with an intravenous jugular (i.v.) catheter and were single-housed in cages that served as both a home cage and their self-administration chamber. In the initial set of studies, a noncontingent infusion of cocaine (1.5 mg/kg in 0.1 ml saline), amphetamine (0.56 mg/kg in 0.1 ml saline), or saline (0.1 ml) was infused through the i.v. catheter 24 h following the start of their final self-administration session. This design yielded five groups: naive rats with saline infusion through i.v. catheters (Naive+Saline), cocaine self-administration rats with saline infusion (Coc SA+Saline), naive rats with amphetamine infusion (Naive+AMPH), cocaine self-administration rats with amphetamine infusion (Coc SA+AMPH), and cocaine self-administration rats with cocaine infusion (Coc SA+Coc). Animals were then killed for voltammetry studies 1 h after the noncontingent infusion of drug. In a follow-up study, a noncontingent infusion of amphetamine (0.56 mg/kg in 0.1 ml saline) was infused through the i.v. catheter exactly as described above, but animals were killed 24 h after the drug infusion. We selected this dose of amphetamine to be consistent with our earlier studies on amphetamine and cocaine (Ferris et al, 2011) and to match the amount of extracellular dopamine elicited in response to amphetamine to the amount of dopamine elicited by the dose of cocaine used in self-administration. In other words, our goal was to match each drug’s ability to elicit similar amounts of dopamine overflow.

Self-Administration Procedures

The procedures for cocaine self-administration have been described previously (Ferris et al, 2011, 2012) and are repeated herein for clarity. Each animal was maintained on a reverse light cycle (0300 h lights off; 1500 h lights on), and all self-administration procedures occurred during the active/dark cycle (0900 to 1500 h). Each lever press resulted in the delivery of 1.5 mg/kg cocaine over 4 s. This dose was chosen because it is the most reinforcing dose, at the top of the dose–response curve in rats responding under a progressive-ratio schedule of reinforcement, and preferred over lower doses in choice studies (Richardson and Roberts, 1996). Concurrent with the start of each injection, the lever retracted and a stimulus light located on the lever was activated for 20 s to signal a time-out period. The session was terminated after 40 injections or after 6 h, whichever occurred first. Once the animals reached the maximum number of injections allowed in a single session (ie, 40), they were required to self-administer 40 injections per day for five consecutive days before administration of the single, noncontingent drug infusion and subsequent voltammetry studies.

Fast-Scan Cyclic Voltammetry

All voltammetry experiments were conducted in the morning (active/dark cycle) following the final self-administration session (24 h from the start of the final session). Multiple coronal slices (400 μM) containing the nucleus accumbens (NAc) were prepared from each animal with a vibrating tissue slicer while immersed in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl (126), KCl (2.5), NaH 2 PO 4 (1.2), CaCl 2 (2.4), MgCl 2 (1.2), NaHCO 3 (25), glucose (11), L-ascorbic acid (0.4), and pH adjusted to 7.4. Once sliced, tissue was transferred to the testing chambers containing bath aCSF (32 °C) that flowed at 1 ml/min. After a 30-min equilibration period, a cylindrical carbon fiber microelectrode (100–200 μM length, 7 μM radius) and a bipolar stimulating electrode were placed into the core of the NAc. We chose the NAc because it is rich in dopamine nerve terminals and heavily implicated in goal-directed behavior, reinforcement, reward, drug addiction, and important for cocaine self-administration. Note that the effects of cocaine self-administration in our previous work are similar between the NAc core and NAc shell (Mateo et al, 2005). Dopamine was evoked by a single, rectangular, electrical pulse (300 μA, 4 ms), applied every 5 min. Extracellular dopamine was monitored at the carbon-fiber electrode every 100 ms by applying a triangular voltage waveform (−0.4 to +1.2 to −0.4 V vs Ag/AgCl, 400 V/s). Once the extracellular dopamine response was stable, cocaine (0.3–30 μM) was applied cumulatively to the brain slice. Immediately following the completion of each concentration–response curve, recording electrodes were calibrated by recording their response (in electrical current; nA) to a known concentration of dopamine in aCSF (3 μM). All voltammetry data were collected and modeled using Demon Voltammetry and Analysis Software (Yorgason et al, 2011) using Michaelis–Menten kinetics. The three voltammetry parameters used in the current investigation were the peak height of the signal (in μM concentration), the maximal rate of dopamine uptake (V max ), and apparent K m (app K m ) to study the effects of release, uptake, and cocaine-induced uptake inhibition, respectively. These parameters are described in detail in previous work (Ferris et al, 2013b), and described briefly here for clarity. V max is a rate term (measured as [dopamine] per second) that is inversely related to the time it takes dopamine to be cleared when no drug is present, and is measured on the descending slope of the drug-free dopamine curve. App K m measures the rightward shift/slowing of the descending limb of the dopamine trace following equilibration of each concentration of cocaine. Data were collected for 15 s during baseline collections and for 30 s following administration of cocaine in order to capture the entire descending limb of the curve. Two-way ANOVA with experimental group and concentration of the drug as factors was performed. When significant main effects were obtained (p<0.05), differences between groups at each dose were tested using Bonferroni or Tukey’s post hoc tests.

Western Blot Analyses of Fractionated Striatal Samples

Striatal tissues were processed by centrifugation to separate the cytosolic and the membrane fractions as described previously (Calipari et al, 2014c). Tissue was homogenized in 50 mM Tris Cl buffer (pH 7.4) containing 150 mM NaCl, 10% sucrose, and a cocktail of protease inhibitors (Sigma-Aldrich). A portion of the homogenates was saved, lysed in RIPA buffer, and used as the total homogenate. The remaining homogenates were centrifuged at 20 000 g for 1 h. The supernatant was collected and stored at −80 °C for further analyses of the cytosol-associated protein levels. The pellets were collected and resuspended by sonication in 20 mM Tris buffer (pH 8, containing 1 mM EDTA, 100 mM NaCl, 1% sodium deoxycholate, 1 mM dithiothreitol, and a cocktail of protease and phosphatase inhibitors) and lysed for 1 h at 4 °C. The lysate was centrifuged at 100 000 g for 60 min and the supernatants were collected for analyses of membrane-associated protein levels. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). Samples containing 10 μg protein were loaded onto 8% SDS-polyacrylamide gel. Gels were transferred electrophoretically onto nitrocellulose membranes. After incubation with the blocking buffer containing 5% nonfat milk in phosphate-buffered saline (PBS), the membranes were probed overnight at 4 °C with rabbit anti-DAT antibody (Millipore). The membranes were then incubated with goat anti-rabbit secondary antibodies conjugated with peroxidase. Actin was probed with the primary antibody goat anti-actin (Santa Cruz) followed by incubation with donkey anti-goat (Sigma-Aldrich). The enhanced chemiluminescence substrate solution (Pierce Chemical) was applied to the membrane and then exposed to X-ray film. Films were analyzed densitometrically using ImageJ (NIH). The band density was normalized to its respective actin level and then each group normalized to the control group (Naive+Saline). Data were represented relative to the control group.