Animals, 6-OHDA lesioning, and microdialysis surgery
Procedures involving animals were approved by the University of British Columbia's ethics committee and followed the guidelines of the Canadian Council on Animal Care. We report data from three normal control (age-matched, not sham-operated) and eight unilaterally 6-OHDA-lesioned [21, 22] rats (Sprague Dawley, males, from Charles River, St. Constant, Quebec, Canada). These rats underwent multiple PET imaging sessions, as reported in Walker et al. , before undergoing two microdialysis sessions. The animals had free access to standard diet and tap water. They were housed at 21°C with a 12-h light cycle (light from 7 a.m. to 7 p.m.). Dopaminergic denervation was produced by injection of 10 μg of 6-OHDA hydrobromide (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 4 μL of 0.05% ascorbic acid solution. The injection co-ordinates (SNc) were as follows: anteroposterior (AP) −4.7 mm (from the bregma), mediolateral (ML) −1.5 mm (from the midline), and dorsoventral (DV) −7.9 mm (from the skull surface) . To protect noradrenergic nerve terminals, desipramine (Sigma-Aldrich) was given at least 30 min prior to surgery (25 mg/kg intraperitoneally (i.p.)). This ensured selectivity for dopaminergic neurons . Rats were 2.4 ± 0.5 months (average and standard deviation) old at the time of lesioning. Denervation severity was determined by [11C]DTBZ PET imaging, which was performed at least 1 month after lesioning (2.1 ± 0.8 months) at which time 6-OHDA-induced denervation is considered relatively stable. The [18 F]FDOPA PET imaging sessions were also performed at least 1 month after lesioning (average of 3.5 months, maximum of 8.5 months post lesioning). Microdialysis was performed after completion of all PET imaging at 12 ± 3.6 months post lesioning. Surgery was performed for implantation of a microdialysis guide cannula (CMA 7, Harvard Apparatus, Holliston, MA, USA) into the left and right striata at co-ordinates AP +1.0 from the bregma, ML ±2.6 from the bregma, and DV −3.0 from the skull. A headcap was built up around the cannula using dental cement (Jet Acrylic, Lang Dental, Wheeling, IL, USA) and secured to the skull with five self-tapping bone screws (shaft diameter 1.17 mm, shaft length 4.7 mm, Fine Science Tools, North Vancouver, Canada). All surgeries were performed under isoflurane anesthesia. Ketoprofen (5 mg/kg sc.) was given for pain relief, and a local analgesic (bupivacaine, 2.5 mg/kg) was applied at the incision site.
Following headcap surgery, rats were allowed 36 h of recovery. Microdialysis was then performed under isoflurane anesthesia twice in each rat within the next 4 days, with at least 1 day of recovery between microdialysis sessions. The use of anesthesia replicated the conditions during PET scanning and simplified the microdialysis procedure. Each session began with the insertion of a microdialysis probe (CMA 7, Harvard Apparatus) into each guide cannula. These probes have a shaft length of 7 mm with a 2-mm-long cuprophane membrane (cutoff of 6,000 Da) of 0.24-mm outer diameter. Probes had been previously prepared by flowing sterile artificial cerebrospinal fluid (aCSF) through them (CMA, Harvard Apparatus). The aCSF flow rate was 0.5 μL/min, but 0.9 μL/min in a few rats where extra dialysate was required (for other testing not reported here). Dialysate collection began 2 h after insertion of the probe to allow some recovery from potential tissue damage caused by probe insertion. In both sessions, 1 h of baseline data was first collected. This was followed by injection (i.p.) of 40 mg/kg entacapone (which inhibits peripheral COMT) in one session and of benserazide/LDOPA (15 and 50 mg/kg, respectively, with the LDOPA given 30 min after the peripheral AADC inhibitor) in the other session. Entacapone was obtained from a local pharmacy in pill form and ground to a fine powder. Benserazide (Sigma-Aldrich), LDOPA (Sigma-Aldrich), and entacapone were mixed in distilled water, 0.05% ascorbic acid, and saline, respectively, for injected volumes of 1 mL/kg.
Dialysates were collected from each probe at intervals of 24 min and immediately injected (9 μL) onto a high-performance liquid chromatography system consisting of a pump (Series IV, Rose Scientific, Edmonton, Canada), pulse damper (Scientific Systems Inc., State College, PA, USA), injector with 20-μL injection loop (Rheodyne, IDEX Health & Science, Oak Harbor, WA, USA), reverse-phase column (TSKgel super-ODS, 2-μm silica particles, 110-Å pores, 2-mm i.d. × 10-cm length, Tosoh Bioscience, San Francisco, CA, USA), and electrochemical detector (Decade II, Antec, Fremont, CA, USA). The flow rate was 0.18 mL/min, with a pressure of approximately 1,600 psi. Each liter of mobile phase contained 100 mL methanol, 900 mL water, 6 g sodium acetate, 40 mg ethylenediaminetetraacetic acid (EDTA), 4 mg sodium dodecyl sulfate (SDS), and glacial acetic acid (to pH 4.0). The electrochemical cell (VT-03, Antec) had a 2-mm glassy carbon working electrode with a salt bridge reference electrode. A 25-μm spacer was used. The applied voltage was +650 mV; the cell and column were maintained at 38°C. Data were collected and analyzed using EZChrom Elite (Agilent, Santa Clara, CA, USA).
Typical probe recoveries as measured at room temperature in vitro were 24%, 26%, and 24% for DA and the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), respectively, at 0.5-μL/min flow rate. The recoveries were, on average, 14% at 0.9 μL/min. The in vitro measured probe recoveries were used as estimates of the in vivo probe recoveries, thus correcting for differences in recovery between probes and allowing the relative dialysate concentrations to be compared across rats. The procedure does not provide a highly accurate measurement of the absolute concentration in the ECF since the in vivo recoveries are known to differ from those measured in vitro. Recoveries were combined with the measured calibration factor for each compound to produce the concentrations reported here. The data include those from baseline (average of both sessions), the percentage change from baseline following injection of benserazide/LDOPA (one session), and the percentage change from baseline following injection of entacapone (one session). The post-drug concentrations were found by averaging data from 100 to 180 min post benserazide injection (70 to 150 min post LDOPA injection) and from 90 to 150 min post entacapone injection. Reported times are corrected for the time required for the sample to travel from the probe to the collection vial. In our microdialysis experiments using LDOPA, we did not pre-dose with a COMT inhibitor since the chromatographic analysis can separate DA and DA metabolites from 3-O-methyldopa and because a relatively high dose of LDOPA was used. The testing allowed assessment of the basal concentrations, the central effects (if any) of entacapone, and the increase in DA metabolite concentrations following LDOPA.
PET scanning and image analysis
Extensive details of the PET imaging and analysis procedures are reported in Walker et al.  and are summarized here. The results reported here are from one [11C]DTBZ scan and two [18 F]FDOPA PET scans. For the [18 F]FDOPA scans, a COMT inhibitor (40 mg/kg entacapone) and an AADC inhibitor (10 mg/kg benserazide) were given i.p. prior to the administration of the radiotracer (at 90 and 30 min, respectively). The scans are a subset of those reported previously in Walker et al. , namely the scans from rats that were subsequently available for microdialysis and which did not include administration of tolcapone prior to [18 F]FDOPA PET. Scanning was conducted under 2.5% isoflurane gas anesthesia using the MicroPET Focus120 small animal scanner (Concorde/Siemens, Knoxville, TN, USA) . Emission data were collected for 3 h ([18 F]FDOPA) or 1 h ([11C]DTBZ) and split into 26 or 17 frames, respectively. Frames had increasing durations, in the range 30 to 900 s ([18 F]FDOPA) and 30 to 480 s ([11C]DTBZ). Data were fully corrected for randoms, attenuation, scatter, normalization, and dead time. Images were reconstructed by filtered backprojection. The spatial resolution within the resulting images is <1.5-mm full width at half maximum . Blood was sampled from the tail during [18 F]FDOPA scanning and assayed for radiolabeled metabolites , following which a small fraction of scans were discarded due to high concentrations of the radiolabeled metabolite 3-O-methyl-6-[18 F]fluoro-l-dopa ([18 F]OMFD; >25%) or low concentrations of the parent compound (<40%) at 160 min post tracer injection (i.e., in scans for which peripheral COMT and/or AADC inhibition was insufficient). Despite this, all rats had at least one [18 F]FDOPA scan that was retained (most had two, in which case the average result is reported).
PET images were analyzed using the ASIPro software (CTI Concorde Microsystems). For each scan, three regions of interest (ROIs) were placed on summation radioactivity images, defining the left and right striata and the cerebellum. The ROI volumes were 0.022 cm3 (striatal) and 0.043 cm3 (cerebellum). In contrast to the data reported in , the ROI for the cerebellum used for PET data analysis was placed with the guidance of a co-registered atlas ; this improved the consistency of the data. Kinetic modeling and analysis was performed using Matlab (The Mathworks, Natick, MA, USA) with in-house software.
[11C]DTBZ was analyzed using Logan graphical analysis with a cerebellum reference region , which estimates the distribution volume ratio (DVR). The time after which data are fitted, t*, was set to 30 min, and the term was omitted . The binding potential of DTBZ (BPND) was calculated as BPND = DVR – 1.
[18 F]FDOPA was similarly analyzed using the Logan plot, with the modification that the time-activity curve (TAC) from the reference region was subtracted from the striatal TAC prior to running the analysis . The estimated slope equals the effective distribution volume ratio (EDVR), which informs on the distribution volume of the striatal 6-[18 F]fluorodopamine (FDA) compartment as compared to the reference region.
Extended Patlak graphical analysis [5, 30], which includes a term describing the loss of radiolabeled metabolites from the sequestered compartment, was also applied to the [18 F]FDOPA data. The analysis provided estimates of k
ref, the rate constant that models the decarboxylation of FDOPA to FDA and the sequestration of FDA in vesicles.
The asymmetry for results from both PET and microdialysis was calculated as 1 minus the ratio of the value from the ipsilateral side to the contralateral side; normal control rats were hence expected to have an asymmetry of 0 for all measures, with severely lesioned rats likely to have asymmetries closer to 1 (maximum asymmetry). The asymmetry in the DTBZ PET measure provides the assessment of denervation severity [11, 12].
Correlation analysis and statistical testing
Data from PET and microdialysis were subject to regression analysis using Matlab (The MathWorks). In each case, Pearson's product–moment correlation coefficient (r) was calculated, along with the probability of obtaining the measured correlation by chance if the true correlation was 0. Group comparisons between rats were also made, with group assignment for lesioned rats based on the DTBZ PET denervation severity measure. For these inter-group comparisons (three groups, n ≥ 3 in each group), statistical analysis followed Fisher's method of analysis of variance (ANOVA) followed by post hoc comparisons using the least significant difference technique when the omnibus test found a significant difference .