Acetic acid–induced writhing test

The acetic acid–induced writhing test was performed as described (55). Mice were preadministered with H4 (20, 30 μg/kg i.p.), HNTX-III (100 μg/kg i.p.), or morphine (100 μg/kg i.p.), whereas the control group received an equal volume of saline 15 min before intraperitoneal injection of 200 μl 1% (v/v) acetic acid. Mice were individually placed into open polyvinyl cages (30 cm × 40 cm × 30 cm). Abdominal writhes were defined as a wave of contraction of the abdominal musculature followed by extension of the hind limbs. The intensity of nociceptive responses was quantified by counting the total number of writhings between 0 and 30 min after the acetic acid administration. The percentage of inhibition was calculated by the equation as follows: Inhibition% = (Writhing Number_vehicle–Writhing Number_drug)/Writhing Number_vehicle × 100%.