The effect of pituitary adenylate cyclase-activating polypeptide on elevated plus maze behavior and hypothermia induced by morphine withdrawal

Assessment of naloxone-precipitated withdrawal jumping in mice treated with graded doses of morphine. Graded doses of morphine (mg/kg, s.c. per injection) or saline were given twice daily for 5days (day 1, 20; day 2, 40; day 3, 60; day 4, 80). Mice were also treated once a day with either PACAP (500ng/2μl) or aCSF i.c.v. 30min after morphine injection. On day 5, 30min prior to test either PACAP or aCSF was injected. Naloxone (1mg/kg, s.c.) or saline was administered 2h after the final injection of morphine at a dose of 100mg/kg, and the jump latency was immediately measured. Therefore, we injected morphine or saline 2h; PACAP or aCSF 30min; naloxone or saline 0min prior to jumping behavior. Number of mice: control: 9, morphine withdrawal mice: 10, morphine withdrawal mice+PACAP: 9, mice treated with PACAP: 9. Bars represent the latency of jump, vertical lines on the top of the bars denote S.E.M., ^∗p<0.05 vs. mice given morphine and naloxone.

The effect of PACAP on hypothermia induced by naloxone in mice treated with morphine. Treatment protocol and the number of mice were the same as naloxone-precipitated withdrawal jumping experiment. The body temperatures of all animals were measured 15, 30, 60min after naloxone injection. Bars represent the decreasing of body temperature, vertical lines on the top of the bars denote S.E.M., ^∗p<0.05 vs. mice given morphine and naloxone.

The effect of naloxone on EPM behavior in mice treated with morphine in absence of PACAP. The graded doses of morphine (mg/kg, s.c. per injection) or saline were given twice daily for 3days (day 1, 10; day 2, 20; day 3, 40). On day 4, naloxone (0.1 and 0.2mg/kg, respectively) or saline was administered 2h after the final injection of morphine at a dose of 20mg/kg, and the EPM behaviors were measured 5min after naloxone injection. Therefore, we injected morphine or saline 2h; naloxone or saline 5min prior to EPM assessment. Number of mice: control: 12, mice treated with morphine: 10, morphine withdrawal mice (0.1mg/kg): 8, morphine withdrawal mice (0.2mg/kg): 13. Bars represent the open-arm time/total time rate and the number of open arm entries/total entries rate, vertical lines on the top of the bars denote S.E.M., ^∗p<0.05 vs. control mice and mice treated with morphine.

The effect of naloxone on EPM behavior in mice treated with morphine in the presence of PACAP. Morphine and naloxone treatments were the same as outlined with Fig. 3. Mice were also treated once a day with either PACAP (500ng/2μl) or aCSF i.c.v. 30min after morphine injection for 3days. On day 4, PACAP or aCSF administrated 30min prior to test. Accordingly, we injected morphine or saline 2h; PACAP or aCSF 30min; naloxone or saline 5min prior to EPM test. Number of mice: control: 9, mice treated with morphine: 10, morphine withdrawal mice: 10, morphine withdrawal mice+PACAP: 8. Bars represent the open-arm time/total time rate, vertical lines on the top of the bars denote S.E.M., ^∗p<0.05 vs. control mice and mice treated with morphine.

The effects of PACAP on motor activity in controls and mice treated with morphine challenged with naloxone. Treatment protocol and the number of mice were the same as outlined with Fig. 4. Bars represent the total activity, vertical lines on the top of the bars denote S.E.M., ^∗p<0.05 compared with mice treated with morphine. PACAP significantly increased the total activity vs. mice treated with morphine. ^∗p<0.05.