CB1 receptor radioligand binding was performed using a Multiscreen Filtration System with Durapore 1.2-μM filters (Millipore, Bedford, MA) as described previously (Hillard et al., 1995a). Incubations (total volume = 0.2 mL) were carried out using TME buffer containing 1 mg/mL bovine serum albumin (TME/BSA). Membranes (10 μg protein per incubate) were added to the wells containing 0.25, 0.5, 1.0 or 2.5 nM [3H]CP 55,940, a cannabinoid CB1 receptor agonist. Ten μM Δ9-tetrahydrocannabinol was used to determine non-specific binding. KD and Bmax values were determined by nonlinear curve fitting to the single site binding equation using GraphPad Prism (San Diego, CA, USA).
