Head Twitch Response and Intracerebroventricular Injections

Intracerebroventricular injections were performed as described previously (26). Head twitch responses were assessed as described previously (10). ICR mice were anesthetized and placed in a stereotaxic instrument (Narishige, Tokyo, Japan). A G-4 cannula (Eicom, Kyoto, Japan) was implanted, −0.4 mm posterior, 1.0 mm lateral, and 2.3 mm ventral from the bregma. After cannula implantation, each mouse was given 1 mg/kg buprenorphine (Sigma-Aldrich) to relieve pain and housed individually for at least 10 days before performing head-twitch experiments. Thirty minutes before DOI (Sigma-Aldrich) treatment, PACAP (10 pmol) was diluted in Ringer’s solution (1:100, Fuso Pharmaceutical Industries, Osaka, Japan) and a 3 μl volume was injected at an infusion rate of 1 μl/min using a microinjection pump (KD Scientific, MA, USA). For the pretreatment of the PAC1 antagonist, PACAP_6-38 (100 pmol) were diluted and injected in the same way 30 min before PACAP treatment. The mice were individually placed in observation cages (19 × 10 × 11 cm) for a 30 min habituation period. They were then intraperitoneally injected with either saline or DOI, which were prepared just before use, and recordings were made for a duration of 60 min. Scoring began immediately after injection by trained observers who were blind to the treatment. The head twitch response is a distinctive paroxysmal head-twitching behavior that is easily distinguished from head-bobbing, lateral movements of the head and grooming. The intracerebroventricular injection was judged successful if the third ventricle was stained by Evans blue.