2.3. Evaluation of Scratching Behavior

Scratching behavior was analyzed as an indicator of pruritus. Mice were acclimatized to their environment for 2 weeks before the experiments. Mice were randomly allocated into different treatment groups (n = 6 (H4R-induced scratching behavior in four different mouse strains) and n = 9 (effects of TRP channels on histamine induces itch) for each group). Group sizes were determined from a power analysis with the software G*Power 3.1.9.2. A co-worker blinded to the experimental protocol randomized animals into these groups. One day before each experiment, the rostral part of the neck was clipped with electric clippers. To measure strain dependent differences in the response to H4R-induced itch, the H4R agonist 4-MH (500 nmol/L NaCl) was injected intradermally (i.d.) into the neck. Application of 50 µL sterile saline (0.9% NaCl) was used as vehicle control. The strain with the most pronounced scratching response was used for further experiments.

HC-030031 (60 mg/kg), Capsazepine (6 mg/kg) or SB366791 (0.5 mg/kg) were given intraperitoneal (i.p.; 200 µL) 45 min before injection of histamine (25 nmol/L), HTMT (100 nmol/L), 4-MH (50 nmol/L) or ST-1006 (50 nmol/L). For the experiments in knockout mice, histamine (800 nmol/L), 4-MH (500 nmol/L) or ST-1006 (100 nmol/L) were injected intradermally. After injection of the histamine receptor agonists, mice were placed in observation chambers and recorded on video for 30 min. Afterwards, scratching bouts were analyzed in a blinded manner. According to Kuraishi et al. (1995), a scratching bout was defined as a series of scratching movements by a hind paw in the area around the injection site until the paw was licked by the mouse or placed on the ground [15].