Scratching Behavior

METHODS
Animals
The present experiments were approved by the Animal Care and Use Committee of our university (No. IZ 27 - 117, 139). The studies were conducted in male C57/ BL6ｊ mice (21–27 g). The mice were housed in a controlled light (lighted from 8:00 to 20:00) and temperature (23–25°C) environment. Food and water were freely available.

Drugs
Morphine hydrochloride (Takeda Pharmaceutical, Tokyo, Japan) was dissolved in physiological saline. The dosage of morphine was determined based on previous study(１：学論文) TRPV1 antagonist SB366791 (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in vehicle (ethanol, saline 1: 9). The dosage of SB366791 was determined as previously described(２：Diana Spicarova Neuropharmacology.2014:81

Scratching behavior was counted as previously described(4:倉石モデルの論文). The mice were habituated each day under the same conditions of observation 2 days before starting this study. The mice were individually put back into the observation cage (12 cm × 9 cm × 14 cm) to permit acclimation for 30 min.
After acclimation, the mice were administered one of the following drugs intrathecally: morphine (0.1, 0.3, or 1.0 nmol), SB366791 (0.01, 0.03, or 0.1 nmol), combination dose of morphine 0.3 nmol + SB366791 (0.01, 0.03, or 0.1 nmol), saline and vehicle. Scratching behavior was videotaped for 60 min after intrathecal administration under unmanned conditions. The temporal and total numbers of scratches by the hind paws during the first 60 min after intrathecal injection were

Intrathecal morphine at 0.3 nmol group was significantly higher numbers of scratches compared with that in the saline group (127.5 ± 23.2 : P ＝ 0.001 ; F3, 20 = 9.2) (Fig 1A).
The peak of scratching behavior was at 10–20 min in the morphine 0.3 nmol groups and at 0–10 min in the morphine 1.0 nmol group, and the number of scratches decreased after these times in all the groups (Fig 1B). Scratching behavior in intrathecal SB366791 0.1 nmol group was not significantly different from that in vehicle group (P > 0.99). Intrathecal morphine at 0.3 nmol group significantly increased scratching compared with that in the saline group (P < 0.001). On the contrary, the morphine 0.3 nmol + SB366791 0.01, 0.03, or 0.1 nmol groups did not increase scratching compared with vehicle group (P = 0.17, P = 0.83, P = 0.99, respectively; F6, 35 = 11.1). Intrathecal SB366791 dose-dependently reduced the scratching behavior induced by morphine 0.3 nmol (Fig 2). In addition, the total number of scratches in the combination of intrathecal morphine 0.3 nmol + intrathecal SB366791 0.03, or 0.1 nmol groups were significantly inhibited scratching behavior compared with that in the intrathecal morphine 0.3 nmol group (P = 0.0063, P = 0.001, respectively