Harvester

[³H]-8-OH-DPAT was used as a selective 5-HT_1AR agonist. The affinity (K_D) and maximal number of binding sites (B_max) were measured by saturation binding experiments over a radioligand concentration range of 0.1–14 nM. The affinity shift was determined by measuring the K_D obtained in saturation binding assays performed in the absence and presence of six concentrations of Zn (0.01–5 mM). Competition studies were performed with 2.5 nM of [³H]-8-OH-DPAT in the presence of various concentrations of orthosteric agonist serotonin in the absence and presence of Zn at two concentrations (10 and 500 μM). Non-specific binding was estimated in the presence of 10 μM 5-HT. The incubation buffer consisted of 50 mM Tris–HCl (pH 7.7), 10 mM MgCl₂, 10 mM pargyline and 0.1 % ascorbic acid. Radioligand binding assays were performed by incubating 30 μg of protein of the membrane suspension in 96-well microtitre plates for 60 min at room temperature with shaking, in a total volume of 200 μl. The binding reactions were stopped by filtration through GF/C Unifilter plates using a harvester (PerkinElmer). The plate filters were dried, and 20 μl of Ultima Gold MV (PerkinElmer) was added. Radioactivity was measured using a MicroBeta TriLux counter (PerkinElmer).