Animal Subjects

Male C57BL/6 mice (PND 30−120) and Wistar rats (PND 80−120) were bred and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. At weaning (PND 21), animals were housed on a reverse light/dark cycle (lights on from 2000 hours to 0800 hours) in groups of 2−5 per cage and given ad libitum access to food and water. Experimental protocols were approved by the Brigham Young University Institutional Animal Care and Use Committee according to NIH guidelines.

Brain Slice Preparation

Coronal brain slices were obtained as previously described (Steffensen et al, 2008). Animals were anesthetized with isoflurane (5%), decapitated, and brains were dissected and sectioned into 400 μm slices in ice-cold cutting solution consisting of (in mM): 220 sucrose, 3 KCl, 1.25 NaH 2 PO 4 , 25 NaH 2 CO 3 , 12 MgSO 4 , 10 glucose, and 0.2 CaCl 2 . Slices were then transferred to room temperature artificial cerebrospinal fluid (ACSF) consisting of (in mM): 124 NaCl, 2 KCl, 1.25 NaH 2 PO 4 , 24 NaHCO 3 , 12 glucose, 1.2 MgSO 4 , 2 CaCl 2 , pH 7.3, which was bubbled with 95% O 2 /5% CO 2 . Slices were then transferred to a recording chamber with continuous ACSF flow (2.0 ml/min) maintained at 34−36 °C. Unless otherwise specified, all drugs were purchased from Sigma Aldrich (St. Louis, Missouri, USA). For slice preparation experiments, METH, lidocaine, GBR-12909 (Cayman Chemical, Ann Arbor, MI, USA), BD-1063 (Cayman Chemical), CPA (Cayman Chemical), TEMPOL, and GSH were dissolved in stock solutions and then diluted into ACSF at specified concentrations.

Fast-Scan Cyclic Voltammetry Recordings

Carbon fiber electrodes (CFEs) were positioned at an angle, ~75 μm below the surface of the slice in the NAc. DA release was evoked every 2 min by biphasic electrical stimulation (4 ms pulses, 10 pulse, 350 μA, 20 Hz) from an ACSF filled micropipette (5−10 μm tip diameter), placed 100−200 μm from the CFE. The CFE potential was linearly scanned from−0.4 to 1.2 V and back to−0.4 V vs Ag/AgCl (scan rate=400 V/s). Cyclic voltammograms were recorded every 100 msec (10 Hz) with ChemClamp potentiometers (Dagan Corporation, Minneapolis, MN, USA). Recordings were performed and analyzed using Demon Voltammetry software (Yorgason et al, 2011).

For DA efflux experiments, stimulated DA release was detected first to verify CFE placement. When electrically evoked DA release did not vary by more than 5% for five successive collections, the electrical stimulation was turned off and voltammograms were recorded at 0.5 Hz for 3 h. Unless otherwise specified, all other recordings were performed with the pharmacological agent added at 30 min and METH at 60 min from the start of the recording. DA release and efflux is expressed as a change in μM DA, not absolute extracellular DA concentration, due to the background subtracted nature of voltammetric recordings.

VMAT2 Co-Immunoprecipitation Assay and Immunoblot

Aged-matched subjects were divided into three groups that were then double-injected intraperitoneally with saline/saline, saline/METH (10 mg/kg), and BD-1063 (30 mg/kg)/METH (10 mg/kg), respectively. The injections were ~10 min apart. NAc tissue was immediately harvested and rapidly chilled 30 min after the second injection. The tissue samples were lysed in ice-cold TNTE buffer (in mM: 50 Tris pH 7.4, 150 NaCl, and 1 EDTA in addition to 0.5% Triton X-100) for 10 min with a rotation at 4 °C. Tissue lysates were triterated 10 times using a 23 gauge needle and cleared by centrifugation at 14 800 g for 10 min. Anti-VMAT2 antibody (EMD Millipore, Billerica, MA, USA) was coupled to magnetic dynabeads (ThermoFisher, Waltham, MA, USA) and immunoprecipitated VMAT2 from tissue lysates according to the manufacturer’s instructions. Immunoprecipitated VMAT2 was run on a 10% SDS-PAGE and immunoblotted for anti-GSH (Virogen, Watertown, MA, USA) and anti-VMAT2 antibodies. After immunoblotting, samples were analyzed/quantified using fluorescent secondary antibodies and the Li-Cor Odyssey imaging system.

Circling Behavior Assay

Adult male Wistar rats (n=10, >400 g at the start of the experiment) were implanted with a guide cannula (MD-2250, Basi Instruments, West Lafayette, IN, USA) in the NAc (+1.2 to +1.7 AP,−0.8 to−1.6ML, +6.0 to +7.0 DV), which was fixed in place using dental cement. Subjects were maintained under isoflurane anesthesia (1.5−2.0%) throughout the surgical procedure and allowed a minimum of 1 week of recovery time. Circling experiments were performed in a 16 × 16 × 16 inch plexiglass compartment. Rats were briefly anesthetized using isoflurane (4.0%) during injections to mitigate injection stress. On test days, rats were injected intraperitoneally with either 5 mg/kg METH dissolved at 5 mg/ml in 0.9% NaCl saline solution or 1 ml/kg 0.9% NaCl saline solution. This injection was followed 5 min later by either a unilateral 0.5 μl injection of 5 μg BD-1063 dissolved in ACSF or a 0.5 μl unilateral injection of ACSF into the NAc. Microinjections were accomplished using a microinjection pump (A-99, Razel Scientific Instruments, Fairfax, VT, USA) and a 25 μl glass syringe (Hamilton Robotics, Reno, NV, USA) connected by silastic tubing to an injection needle that extended 0.5−1.0 mm past the end of the guide cannula. The injection occurred over 30 s, with the needle left in place for an additional 30 s prior to removal. After injections, one hour of behavior in the circling chamber was recorded using a camera mounted on the ceiling above the apparatus connected to a Windows 7 PC running Pinnacle Studio 16 (Corel, Menlo Park, CA, USA). Injection order was counterbalanced across rats to avoid order effects. At the end of the experiment, rats were injected with 0.5 μl of 100 mM pontamine sky blue (Avocado Research Chemicals Limited, Lancashire, UK) dissolved in distilled water. Rats were then killed and their brains were removed for sectioning using a vibratome. Sectioned brain slices were examined to determine microinjection location. Two rats were removed from the experiment, one due to chronic distress and a second due to improper injection location. In the distressed rat an autopsy revealed that the lower portion of the guide cannula had disconnected from the implant resulting in an extensive striatal lesion. Two additional rats did not complete the saline+BD-1063 experimental condition despite completing all other conditions.

Statistical Analyses

Immediately following METH administration, the amplitude of electrically evoked DA release increased, but dropped after several collection sweeps. For the sake of directly comparing against DA efflux, we averaged the maximal three peak DA amplitude measurements. For statistical measure of DA efflux, the results were determined from maximum oxidation peak values for evoked DA experiments. As the DA efflux evoked by METH generally decreased after 30−60 min, the area under the curve (AUC) between the initial DA increase and the return to baseline was calculated using Igor Pro (WaveMetrics, Lake Oswego, OR, USA). The AUC was then divided by the Δt of the DA efflux for the averaged data plots. This standardized experiments for average DA release per second. These values were then grand-averaged across animals. Values were expressed as means±SEM for cumulated data.

Statistics were performed using IBM SPSS Statistics 21 (Armonk, NY, USA) or STATA 14.2 (College Station, TX, USA). A two-tailed Student’s t test was used for comparisons with only two variables. To determine significance between more than two variables, a one-way analysis of variance (ANOVA) was used with Tukey’s post hoc analysis. Circling and locomotor activity was analyzed using repeated measures ANOVAs with a Greenhouse–Geisser correction for sphericity. Following analysis using repeated measures ANOVAs, significant effects were further characterized by comparing specific treatment conditions using Tukey’s HSD test. Prior to analysis, data were assessed for normality and outliers. Circling data were found to be normally distributed using the Shapiro–Wilk test for normality. In checking for outliers, a data point was considered to be an outlier if it fell more than three interquartile ranges (IQR) above or below the median. Using this criterion, 2 points across two rats were identified as outliers in the circling data. These points were fenced to the outer limit (median±3 IQR), as there was no reason to believe that the data for these subjects was incorrect. For all experiments, the criterion of significance was set at p<0.05 (*), p<0.01 (**), and p<0.001 (***).

Supplementary Materials and Methods

For details relating to CFE construction, mass spectrometry and the VMAT2 activity assay methods, please see Supplementary Materials and Methods.