Male NIH Swiss mice were used for activity evaluation. On experimental days, mice were weighed, marked, and returned to the home cage. Doses were then calculated and prepared for intraperitoneal (ip) injection. To generate dose effect functions, individual animals were removed from the home cage, injected with saline or various doses of 25CN-NBOH (0.3, 3 and 10mk/kg) and placed into an observation cage containing fresh bedding. Ten minutes following this injection, an overhead camera was activated and behavior was recorded for 10 min.

The day after transfection, tsA201 cells were split into poly-D-lysine-coated 96-well tissue culture plates in inositol-free DMEM supplemented with 10% dialyzed fetal bovine serum, penicillin (100 U mL−1), streptomycin (100 mg mL−1), and 4 μCi mL−1 myo-[2-3H]inositol (GE Healthcare, Buckinghamshire, U.K.). Two days after transfection, cells were washed with assay buffer 1 (Hanks’ balanced saline solution (HBSS) containing 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and 1 mg mL−1 BSA, pH 7.4) and preincubated in 100 μL assay buffer 1 for 4 h at 37 °C, where the buffer was replaced after 2 h. The cells were then washed and subsequently incubated in 50 μL of assay buffer 2 (HBSS containing 1 mM CaCl2, 1 mM MgCl2, and 20 mM LiCl) for 30 min at 37 °C. Following this incubation, the cells were stimulated with 50 μL of the NBOH-2C-CN in assay buffer 2 for 30 min at 37 °C. The reactions were stopped by exchanging the buffer with 50 μL ice-cold 10 mM formic acid and incubating the cells at 4 °C for at least 30 min. Yttrium silicate scintillation proximity assay beads (GE Healthcare, Buckinghamshire, U.K.) were used for measuring radioactivity from generated [3H]-IP