0.5 mg/kg (two injections/week)

Adult, male, Sprague-Dawley rats (200-250g; Harlan Labs, Indianapolis, IN) were sacrificed by decapitation, the brains rapidly removed and dissected on ice. Striatal tissue was homogenized in 30 vol. ice-cold 0.32 M sucrose using a glass homogenizer and Teflon pestle. The crude nuclear fraction (P1), the pellet formed from centrifugation of the pellet at 1,000 x g for 10 mitt; the crude synaptosomal fraction (P2), the pellet formed by the centrifugation of the resulting supernatant at 20,000 x g for 30 min; Subfractionation of the crude synaptosomal fraction (P2) was performed by centrifugation of freshly prepared P2 fraction suspended in 0.32 M sucrose over a discontinuous gradient of 0.8 M and 1.2 M sucrose at 110,000 x g for 70 min. Three subfractions were collected. The myelin fraction (P2A) was collected at the 0.32 M/O.8 M sucrose interface; the synaptosomal fraction (P2B) at the 0.8 /1.2 M sucrose interface; and the mitochondrial fraction (P2C), the final pellet. All resulting fractions were resuspended in assay buffer and centrifuged at 100,000 x g for 30 min. The resulting pellets were used for receptor binding assays. The final fraction pellets were resuspended in assay buffer (50 mM Tris-HCl, 5 mM KCI, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4 at 23°C) to yield a final protein concentration of 400 mkg/tube. Binding assays were performed in duplicate in disposable polystyrene tubes. Saturation analyses were performed in the major subcellular fractions to determine radioligand affinity. For saturation analyses, eight concentrations of [3H]quinpirole (43 Ci/mmol; NEN DuPont Research Products, Boston, MA) were used (0.1 to 30 nM). Because [3H]quinpirole exhibited similar affinity in the major subcellular fractions, single point analyses were used for subsequent experiments. For single point studies, the final concentration of [3H]quinpirole was ~2 nM Binding was initiated by the addition of resuspended subcellular fractionate. The total assay volume was 0.5 ml. Tubes were incubated at 23°C for 4 h