Fifty-three (n=8–10/group) male Sprague–Dawley rats (Harlan, Indianapolis, Indiana, USA) weighing 300 g at surgery were used. Rats were housed at 21°C on a 12-h light : dark cycle (lights on at 0.700) and ad lib food and water. Testing was conducted between 09.00 and 15.00. Procedures were in accordance with the Institutional Animal Care and Use Committee.
Drugs were dissolved in 0.9% saline, except BMY-14802, which was dissolved in distilled water. Drugs were administered at 1 ml/100 g of body weight via intraperitoneal (i.p.) injection 30 min before l-DOPA, except for mecamylamine, which was administered subcutaneously (s.c.) 20 min prior. l-DOPA methyl ester, benserazide hydrochloride, l-ascorbic acid, d-amphetamine sulfate, ( + )-MK-801 hydrogen maleate, dextromethorphan hydrobromide, and mecamylamine hydrochloride were obtained from Sigma-Aldrich (St Louis, Missouri, USA). (R)-( + )-HA-966 and BMY-14802 were obtained from Tocris (Ellisville, Missouri, USA). Doses were chosen based on efficacy to suppress behavioral effects of dopamine agonists (e.g. stereotypy, locomotion) without reducing basal activity levels. Rats were randomly assigned to receive up to three test compounds at least 1 week apart.
Rats underwent stereotaxic surgery under isoflurane anesthesia. A 30-gauge infusion cannula, attached via PE20 tubing to a 10 μl Hamilton gastight syringe and driven by a microinfusion pump (Harvard Apparatus, Holliston, Massachusetts, USA) was used to infuse 6-OHDA (22.8 μg/ 2 μl of 0.9% saline with 0.02% ascorbic acid, 0.5 μl/min) into each of two sites within the right medial forebrain bundle (AP −4.3 and −4.8, ML±1.2, DV −8.6 in mm). Rats then received an injection of buprenorphen analgesic (0.05 mg/ kg, s.c.; Reckitt Benckiser Pharmaceuticals, Richmond, Virginia, USA) and supplemental soft food until regaining their presurgery weight.
Amphetamine-evoked rotational behavior
An a priori
criterion of an average of ≥5 turns/min over 10 consecutive minutes in response to amphetamine (5 mg/kg, i.p.) was used to select rats with significant hemiparkinsonism [8
At least 3 days after rotational screening, all rats received l
-DOPA (7.5 mg/kg, i.p.), combined with benserazide (15 mg/kg, i.p.) and ascorbic acid (2.6 mg/kg, i.p.), once daily for 21 consecutive days, a regimen that induces dyskinesias [9
]. Thereafter, rats received a maintenance regimen of two injections/week. All test drugs were administered during the maintenance phase to examine their efficacy to suppress dyskinesia expression.
Dyskinesias were assessed by an investigator blind to treatment, using an adaptation of the Abnormal Involuntary Movement Scale described by Cenci et al
]. Limb, axial, and oral dyskinesias, as well as contraversive rotational behavior (an index of l
-DOPA's motor activating effects), were rated on a 0–4 severity scale every 20th min for 3 h, beginning 20 min after l
-DOPA. Total dyskinesia scores were computed as the sum of limb + axial + oral dyskinesias.
Lesion size quantification
Brains were exposed to immunohistochemistry or Western analysis, and average tyrosine hydroxylase loss was 92.50±4.19 and 92.95±2.63% of control, respectively.
For immunohistochemistry, rats were sacrificed under anesthesia by cardiac perfusion with 60 ml each of phosphate-buffered saline and 4% paraformaldehyde, then 40 μm coronal sections were cut. Free-floating sections were rinsed in phosphate-buffered saline (3×10 min) between steps: 1.5% hydrogen peroxide for 30 min, 4% horse serum and 0.1% Triton X-100 for 2 h, monoclonal primary antibody (1 : 10,000; Immunostar, Hudson, Wisconsin, USA) overnight at 4°C, horse antimouse antibody for 2 h (1 : 200; Vector Laboratories, Burlingame, California, USA), and avidin–biotin complex (Vector Laboratories) for 1 h. Tyrosine hydroxylase was visualized in 50 mM Tris buffer with diaminobenzidine (Vector Laboratories) using a Leica DM LFS microscope (Leica Microsystems, Wetzlar, Germany). Optical density was quantified using a Leica DFC 480 camera (Leica camera, Solms, Germany) and Image Pro-Plus 4.1 software (MediaCybernetics, Bethesda, Maryland, USA). Lesion size was calculated by comparing 1.4×1.0 mm areas of 6-OHDA-treated and untreated dorsolateral neostriatum with adjacent cerebral cortex as background.
For Westerns, rats were sacrificed by decapitation under anesthesia. Brains were removed and whole neostriatum dissected and frozen. Protein was extracted with lysis buffer (50 mM Tris, 10 mM ethylenediaminetetra-acetic acid, 1% Triton-X 100, 10 nM phenylmethylsulfonyl fluoride) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, Indiana, USA), then quantified with a Pierce protein assay kit (Rockford, Illinois, USA). Samples (15 mg) were diluted 1 : 2 in Laemmli sample buffer (Sigma-Aldrich), heated at 100°C for 5 min, then separated on a 10% Criterion Tris–HCl polyacrylamide gel (Bio-Rad; 200 V, 1 h). Proteins were transferred to polyvinylidene diflouride membranes (30 V, 13 h), then washed in Tris-buffered saline with 1% Tween-20 (pH 7.4; 3×10 min) between steps: 5% nonfat dry milk for 1 h, monoclonal primary antibody (1 : 10 000; Immunostar) for 2 h, and goat antimouse antibody (1 : 3000; Bio-Rad) for 1 h. Proteins were visualized using ECF (Amersham, Piscataway, New Jersey, USA) according to supplier's instructions, then scanned and quantified using the Typhoon fluorescence detection system (Typhoon 9410 Variable Mode Imager, GE Healthcare, Piscataway, New Jersey, USA; Emission filter: 520 BP40, Laser: 488 Blue2).
Data were analyzed with SPSS (Chicago, Illinois, USA) with P≤0.05. A mixed design Time×Treatment analysis of variance was used to analyze dyskinesias over the 3-h sessions, and t-tests were used to analyze total dyskinesia scores in the 3-h session and the first hour after l-DOPA. When data violated homogeneity of variance assumptions, Greenhouse–Geisser corrections or t-tests in which equal variance was not assumed were used for repeated or between-subjects data, respectively, yielding fractional degrees of freedom.