Animals and Surgery
Surgery for voltammetric measurements of dopamine were performed as previously described.
25 Briefly, male Sprague–Dawley rats aged 90–120 days (275–350 g) (Charles River Laboratories., Raleigh, NC) were anesthetized with urethane (1.5 g kg
–1 rat weight) and affixed into a stereotaxic frame (Kofp instruments). Flat-skull surgical techniques were employed using coordinates from a stereotaxic atlas.
34 A guide cannula (Bioanalytical Systems, West Lafayette, IL) was implanted above the nucleus accumbens (NAc) (stereotaxic coordinates relative to bregma: 2.2 mm anterior, 1.7 mm lateral), and a bipolar stimulating electrode (Plastics One, Roanoke, VA) was lowered into the medial forebrain bundle (MFB) (stereotaxic coordinates relative to bregma: 1.8 mm posterior, 1.7 mm lateral, 8.5 mm ventral). Reference electrodes (prepared as described below) were implanted in the contralateral hemisphere. Electrodes were stabilized with skull screws and cranioplastic cement. After voltammetry and impedance experiments, acetone was applied to the cement around the reference electrode in order to soften it. A small crater was drilled around the reference electrode pin so that it could be removed carefully from the tissue, minimizing tissue damage. All surgical procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee and in concordance with the NIH Guide for the Care and Use of Animals.
Fast-Scan Cyclic Voltammetry
Carbon-fiber microelectrodes were used for voltammetric recordings of dopamine. T-650 carbon fibers (Thornel, Amoco Corp., Greenville, SC) were aspirated into glass capillaries (AM Systems, Sequim, WA), pulled on a vertical pipet puller (model PE-22, Narishige Group, Tokyo, Japan), and cut to a length of 50–100 μm. An electrochemical waveform was applied to the electrode that scanned from −0.4 to 1.3 V at a rate of 400 V s–1 at 10 Hz. The holding potential of −0.4 V between voltammetric scans allows for adsorption of dopamine to the electrode. Waveform application, current monitoring, and stimulus application were performed using a customized version of the TH-1 software (ESA, Chelmsford, MA) written in LabVIEW (National Instruments, Austin, TX) with a custom built potentiostat (UEI, University of North Carolina Chemistry Department Electronics Facility) for waveform application. A DAC/ADC card (NI 6251 M, National Instruments) and a triggering card (NI 6711, National Instruments) were used to interface between the software and the instrument and the timing of the electrical stimulation with the waveform application.
Flow Injection Analysis
Flow injection analysis was used for in vitro calibration experiments.
35 The carbon-fiber microelectrode was placed in the output of a six-port HPLC loop injector mounted on a two-position actuator (Rheodyne model 7010 valve and 5701 actuator), operated by a 12 V DC solenoid valve kit (Rheodyne, Rohnert Park, CA). The apparatus enabled the introduction of a rectangular pulse of analyte to the microelectrode surface using a syringe infusion pump (Harvard Apparatus model 940, Hollison, MA) at a flow rate of 2 mL/min. The flow injection buffer was constituted with the following: Trizma hydrochloride (15 mM), NaCl (140 mM), KCl (3.25 mM), 2H
2O·CaCl (1.2 mM), H
2O· NaH
2PO
4 (1.25 mM), MgCl
2 (1.2 mM), and Na
2SO
4 (2.0 mM), all purchased from Sigma-Aldrich (St. Louis, MO).
Nafion Coatings
Reference electrodes were fabricated using a modified procedure originally described by Moussy and Harrison.
24 Sintered Ag/AgCl reference electrodes (E255A, In Vivo Metric, Healdsburg, CA) were mounted and soldered into conductive pins. The entirety of the electrode was dipped in 5% Nafion solution (Liquion-1105-MeOH, Ion Power, DE) for 10 s using a slow agitating motion. This coating was allowed to dry for 30 min, and the process was repeated for a total of five coatings. The electrodes were air-dried overnight and then cured at 120 °C for 1 h. Because of the toxic nature of the methanol solvent, the curing process at 120 °C is essential to ensure full evaporation of the methanol from the thick Nafion membrane.
Immunohistochemistry
After completion of voltammetric and impedance experiments, rats were transcardially perfused with ice-cold 0.1 M PBS, pH 7.4, followed by ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains were stored in 4% paraformaldehyde for 7 days and then sliced into 40 μM sections using a vibratome (VT1000S, Leica, Bannockburn, IL). Slices were washed three times for 10 min each in 0.1 M PBS and then incubated in Citra Antigen Retrieval Buffer (Biogenex, Freemont, CA) for 1 h at 70 °C. Slices were again washed in 0.1 M PBS and then incubated in 3% hydrogen peroxide for 10 min. Slices were then washed three additional times for 10 min each before being incubated in 10% goat serum with 0.075% Triton X-100 for 1 h at room temperature. Primary antibody (glial fibrillary acidic protein, GFAP; DAKO Chemical, Carpentieria, CA) was then applied to the slices at 1:1000 at 4 °C overnight. Primary antibody was removed from the slices by three washes of 0.1 M PBS. For light microscopy, secondary antibody (biotinylated goat anti-rabbit; Vector Laboratories, Burlingame, CA) was then applied at 1:200 at room temperature for 1 h. Slices were washed three times in PBS, and avidin–biotin-horseradish peroxidase complex (ABC Elite kit; Vector Laboratories) was applied for 1 h at room temperature. Slices were then washed three more times in PBS and exposed to a solution of 3,3′-diaminobenzidine (DAB; 0.5 mg/mL in PBS; Sigma-Aldrich, St. Louis, MO) for 1 min. DAB was removed by three additional washes with PBS. Slices were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), dried overnight, subjected to dehydration in graded ethanol followed by clearing in CitriSolv (Fisher Scientific), and finally coverslipped using Cytoseal (Richard Allen Scientific, Kalamazoo, MI). Micrographs were collected using an Olympus microscope attached to a Q-Imaging camera and controlled using BioQuant imaging software (Nashville, TN).
For fluorescence microscopy, hydrogen peroxide exposure was omitted and secondary antibody (AlexaFluor 600; Invitrogen, Carlsbad, CA) was applied at 1:200 at room temperature for 1 h in the dark. Secondary antibody was removed by three washes of PBS, and slices were wet-mounted on Superfrost Plus slides using Cytoseal. Fluorescence micrographs were obtained using the same setup as above.
SEM-EDS
Scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS) were performed on an S-4700 cold cathode field emission scanning electron microscope (Hitachi, Pleasanton, CA). SEM images were collected under high vacuum, using an excitation voltage of 20 kV. EDS data were collected using a Si (Li) detector and quantified using the INCA PentaFET -x3 software (Oxford Instruments, Concord, MA), and calibrated using 99% Cu. EDS spectra were collected at three distinct 300 × 300 μm2 locations on each electrode, and the values for atom % fluorine and chlorine were averaged.
Impedance Measurements
Impedance measurements were performed in anesthetized animals immediately after voltammetric measurements using a Hewlett-Packard 4284A precision LCR meter with Agilent 16048A test leads (Santa Clara, CA). The instrument was PC operated via a USB-GPIB interface with in-house LabView based (National Instruments, Austin, TX) software (UNC Chemistry Electronics Facility). Impedance for Nafion-coated and bare Ag/AgCl reference electrodes was measured versus an acutely implanted 1 cm long Ag/AgCl electrode. Impedance spectra for perturbation of 50 mV for frequencies from 20 Hz to 200 kHz were recorded.
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