Skf81297

The functional [³⁵S]GTPγS binding experiments were performed as previously described^{59 (link),60 (link)}, with modifications for optimizing the binding assay stimulated with a D₁R agonist^{61 (link)}. Briefly the membrane homogenates were incubated at 30 °C for 60 min in buffer (pH 7.4) composed of 25 mM HEPES, 120 mM NaCl, 20 mM MgCl₂, 1.8 mM KCl, and 1 mM sodium deoxycholate containing 20 MBq/0.05 cm^{3 (link)} [³⁵S]GTPγS (0.05 nM) and increasing concentrations (10^{—10 (link)} to 10^{—5 (link)}M) of the selective D₁R full agonist, SKF81297 (Tocris Bioscience, Bristol, United Kingdom)^{62 (link)}. The experiments were performed in the presence of excess GDP (10 µM) in a final volume of 1 mL. Total binding was measured in the absence of test compounds, whereas non-specific binding was determined in the presence of 10 µM unlabeled GTPγS and subtracted from the total binding. The difference represented basal activity. The reaction was terminated by rapid filtration under vacuum (Brandel M24R Cell Harvester), and washed three times with 5 mL of ice-cold 0.1 M phosphate (pH 7.4) buffer through Whatman GF/B glass fibers. The radioactivity of the dried filters was detected in an UltimaGold MV aqueous scintillation cocktail on a Packard Tricarb 2300TR liquid scintillation counter. [³⁵S]GTPγS binding experiments were performed in triplicate and were repeated at least three times.

Four different drug solutions were prepared in this experiment: L-dopa, D1-like receptor agonist SKF81297 (Andersen and Jansen, 1990 (link)), D2-like receptor agonist Ropinirole (Millan et al., 2002 (link), 2004 (link)) and a mixture of SKF81297 and Ropinirole. All the drugs were dissolved in sterile saline (0.9%), aliquoted, and stored in -20°C. The dose was 1 μg for L-dopa, 0.2 μg for SKF81297 and Ropinirole, all in 0.2 μL saline. For mixed SKF81297 and Ropinirole injection, 0.2 μg SKF81297 and 0.2 μg Ropinirole were dissolved in 0.2 μL saline. Ropinirole and SKF81297 were purchased from Tocris; L-dopa (L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride) was purchased from Sigma.

The DR1 agonist SKF-81297 (1 μM), the D2-like receptor agonist quinpirole (10 μM), the selective DR3 agonist pramipexole (10 μM), the DR4 agonist PD-168077 (200 nM), the selective DR4 antagonist L-745870 (500 nM), the dopamine (200 μM), the n-methy-d-aspartate (NMDA) receptor antagonist D-AP5 (50 μM) and CCH (5 μM) were purchased from Tocris Bioscience. Stock solutions, at thousand times the final concentration, were made up in water or in DMSO, and stored in individual aliquots at 20°C. Final solutions were prepared freshly on the day of the experiment. Drugs were applied to the recording ACSF 15 min after steady-state γ power was reached. The dead volume and bath volume was kept such that final concentration was reached in the bath in about 5 min.

Synthetic agonists for RARα (BMS753), RARβ (BMS641) and RARγ (CD666) (abbreviated as agoRARα, agoRARβ, agoRARγ in the figures), were synthesized in our laboratories. BMS753 was prepared as described in the original patent⁶² . BMS641 was synthesized as reported^{63 (link)}. CD666 was prepared following the procedure described in the original publication^{64 (link)}. Haloperidol (Sigma, ref. H-1512) was dissolved in water containing 5% acetic acid and used at 10 µM final concentration. SKF81297 (Tocris, ref. 1447) was prepared in sterile water and used at 1 µM final concentration. Dopamine (Sigma, ref. 8502) was dissolved in darkness in the presence of 500 µM ascorbic acid (Sigma ref., A4403), and used at 1 µM final concentration. Control cells were treated in sterile water.