2.1. σ1R radioligand binding in guinea pig cortex

Male Hartley guinea pig cortices were dissected from freshly harvested brains (shipped cold in phosphate-buffered saline (PBS) buffer from BioReclamation IVT) and frozen at -80 °C for future use. On test day, thawed guinea pig cortices were suspended and homogenized in 20 volumes (w/v) (10 mM Tris.HCl, 0.32M Sucrose, pH 7.4 at 25 °C) with a glass-teflon apparatus and centrifuged (∼1,200 rpm) for 10 min at 4 °C. The supernatant was collected in a clean tube and the pellet re-suspended in 10 ml of cold buffer and centrifuged again (∼1,200 rpm) for 10 min at 4 °C. The supernatants were pooled together and centrifuged (20,000 rpm) for 15 min at 4 °C. The final pellet was suspended in ice-cold binding buffer at 50 mg/ml concentration (original wet weight). A Bradford protein assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration present in the tissue preparation (1.25 mg/ml). All test compounds were freshly dissolved in 30% DMSO and 70% H₂O to a stock concentration of 1 mM or 100 μM. To assist the solubilization of free-base compounds, 10 μl of glacial acetic acid was added along with the DMSO (in place of 10 μl final H₂O volume). Each test compound was then diluted into 10 half-log serial dilutions using 30% DMSO as the vehicle. Radioligand competition experiments were conducted in 96-well plates containing 300 μl fresh binding buffer, 50 μl of diluted test compound, 100 μl of tissue preparation (125 μg/well total protein amount), and 50 μl of radioligand diluted in binding buffer ([³H]-(+)-pentazocine: 3 nM final concentration, ARC, Saint Louis, MO). Nonspecific binding was determined using 10 μM PRE-084 and total binding was determined with 30% DMSO vehicle (3% DMSO final concentration). All compound dilutions were tested in triplicate and the competition reactions started with the addition of the tissue preparation and incubated for 120 min at room temperature. The reaction was terminated by filtration through Perkin Elmer Uni-Filter-96 GF/B, presoaked for 120 min in 0.05% polyethylenimine, using a Brandel 96-Well Plates Harvester Manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed 3 times with 3 ml (3 × 1 ml/well) of ice cold binding buffer. 65 μL Perkin Elmer MicroScint 20 Scintillation Cocktail was added to each well and filters were counted using a Perkin Elmer MicroBeta Microplate Counter (calculated efficiency: 31%). IC50 values for each compound were determined from inhibition curves and K_i values were calculated using the Cheng-Prusoff equation; K_d values for [³H]-(+)-pentazocine (σ₁R: 5.18 nM) and B_max (1091 fmol/mg) were determined via separate homologous competitive binding experiments. K_i values were determined from at least 3 independent experiments and are reported as mean ± SEM.