Results following 7 days of morphine treatment Table 1 and Fig.

B . Kest et al. Brain Research 879 2000 17 –22 19 and all mice received a single 10 mg kg morphine 4.160.63; females: 3.960.37, Day 4 males 3.860.41; injection. Withdrawal latencies were re-assessed at 30-min females: 4.060.29 or Day 8 males: 4.160.37; females: intervals for 2 h. Mice were then subject to 3 days of 4.260.40. In addition, male and female morphine ED 50 morphine injections according to the dosing schedule values obtained on Day 1, prior to the start of the 3- or above. On Day 4, baseline latencies and morphine analge- 7-day tolerance induction protocol, were practically identi- sia were again determined at 30-min intervals for 2 h. cal and were thus pooled. As illustrated in Fig. 1, 3 days of Withdrawal latencies at peak analgesia 30 min for each morphine administration produced a rightward shift in the sex on Day 1 and 4 were converted to MPE scores morphine dose–response curve in both sexes on Day 4 [post-drug latency–baseline latency cutoff latency– relative to Day 1. Table 1 shows that the resultant baseline latency3100], and compared. The area under the morphine ED estimates was significantly increased on 50 time3latency curve AUC; min3s for males and females Day 4 males: 12.2 mg kg; females: 28.6 mg kg relative was used to calculate total analgesia AUC maximum to Day 1 males: 4.1 mg kg; females: 6.2 mg kg in both possible AUC and compared. males and females, indicative of tolerance. However, the magnitude of tolerance was different between sexes. 2.6. Data analysis Whereas morphine potency was similar in males and females on Day 1, equivalent dose–response shifts and Morphine dose–response data were analyzed using the ED estimate increases were not obtained on Day 4 Fig. 50 BLISS -21 computer program. This program maximizes the 1 and Table 1, respectively. In females, there was an log-likelihood function to fit a parallel set of Gaussian approximately 4.6-fold rightward shift in the morphine sigmoid curves to the dose–response data, and provides dose–response curve relative to an only three-fold shift in ED values, 95 confidence intervals CI, and estimates males Fig. 1, resulting in changes in morphine potencies 50 of relative potency [42]. Baseline tail-withdrawal latencies, on Day 4 relative to Day 1 of 0.34 in males but 0.22 in and peak and total morphine analgesia between sexes on females Table 1. ED estimates from morphine naive 50 Day 1 was compared using an independent t-test. A two- mice Day 1 in morphine-treated mice and Day 4 in saline way one within, one between repeated measures ANOVA control mice did not differ within or between sex, was used to compare peak and total morphine analgesia confirming that there were no significant initial differences on Days 1 and 4. An a level of 0.05 was used for all in morphine sensitivity between sexes, and indicating no comparisons. effect of the repeated injection protocol. Although morphine tolerance is increased in both sexes when morphine treatment and or cumulative are increased

3. Results following 7 days of morphine treatment Table 1 and Fig.

1, sex differences in tolerance were still observed. 3.1. Dose–response data Females displayed a significantly larger seven-fold right- ward shift in the dose–response curve on Day 8 ED : 50 Mean baseline tail-withdrawal latencies did not differ 43.1 compared to only a 4.8-fold shift for males ED : 50 between males and females on either Day 1 males: 19.5 Fig. 1 and Table 1, resulting in relative potencies Fig. 1. Cumulative dose–response curves for morphine analgesia on the tail-withdrawal test in male and female mice. Curves were obtained before Day 1 and after 3 Day 4; n: males513, females514 or 7 days Day 8; n: males512, females512 of repeated tid and escalating morphine injections in separate experiments. For clarity, Day 1 data was pooled from the two experiments n: males525, females526. ED estimates derived from curves 50 indicate significant P,0.05 loss of analgesic potency in both sexes, but which was greater in females than males, on Day 4 and 8 relative to Day 1. 20 B Table 1 a Morphine analgesic tolerance in male and female CD-1 mice Treatment Day 1 Day 4 Day 8 [ED 95 CI potency ratio] [ED 95 CI potency ratio] 50 50 Male Morphine 4.1 2.9–5.8 12.2 9.3–15.8 0.34 19.5 13.9–27.0 0.21 Saline – 5.0 3.1–8.1 6.5 4.5–9.1 Female b b Morphine 6.2 4.8–8.1 28.6 22.5–36.2 0.22 43.1 29.9–62.1 0.14 Saline 6.0 3.7–9.4 6.5 4.6–9.1 a Separate groups of mice received subcutaneous saline or morphine injections for 3 and 7 days using an escalating dosing schedule. Morphine ED and 50 95 confidence intervals CI estimates mg kg were derived from cumulative dose–response curves obtained on Day 1, 4, and 8, respectively see Fig. 1. Day 1 data from the two experiments were pooled for clarity. Potency ratio indicates proportion of analgesia retained after chronic treatment determined by ED Day 1 ED Day 4 or 8. 50 50 b Denotes significantly greater reduction in relative potency i.e., tolerance than males. relative to Day 1 of 0.21 in males and 0.14 in females. morphine analgesia for Day 1 and 4 revealed decreased Again, a comparison of morphine naive mice from Day 1 peak morphine analgesia on Day 4 relative to Day 1 which and Day 8 indicate no effect from repeated injection did not differ between sex. Similar results were obtained following this longer 7-day injection paradigm in either for total analgesia. Although there was a significant sex. repeated measures effect F 542.41; P,0.001, in- 1,14 dicating a decrease in morphine analgesia, between Days 1 3.2. Time–response data and 4, there was no effect of sex F 50.84; n.s. or 1,14 their interaction F 50.14; n.s.. Similar to our ob- 1,14 The analgesic time course for a single 10 mg kg servations for dose–response studies, mean baseline tail- injection of morphine is shown in raw data form in Fig. 2. withdrawal latencies did not differ between males Peak morphine analgesia occurred by 30 min post-injection 4.360.66 and females 4.260.49. for both sexes, and there was no difference in corre- sponding MPE scores at that time. A two-way one between, one within ANOVA for sex F 50.44; n.s.,

4. Discussion