Behavioral assessment of itch (neck model)

Mice were handled at least 5 days before performing the behavioral experiments. All animals were acclimatized to the observation chamber for 3 days before the behavioral experiments. The pruritogen-induced neck-scratching test was performed as described previously [34]. The nape of the neck of mice was shaved the day before the experiment. The animals were placed individually in the observation chamber and allowed to habituate to it for 30 min. Chloroquine (200 μg/50 μL) was injected intradermally into the nape of the neck, and the mice were immediately placed into the observation chamber. Subsequently, the scratching behaviors were video recorded at 60 frames/s for 30 min in an unmanned environment, and the video was then played back to assess the scratching behavior. The scratching behavior was quantified by counting the number of scratching bouts, which consisted of one or more rapid back-and-forth hind-paw motions on the intradermal injection site. Counting of scratching behavior was performed in a blinded manner. The duration of scratch bouts and scratch frequency in each bout was not analyzed in this study.