Sex chromosome complement affects nociception and analgesia in newborn mice

Abstract

In animal studies of nociception, females are often more sensitive to painful stimuli, whereas males are often more sensitive to analgesia induced by mu-agonists. Sex differences are found even at birth, and in adulthood are likely caused, at least in part, by differences in levels of gonadal hormones. In this report, we investigate nociception and analgesia in neonatal mice and assess the contribution of the direct action of sex chromosome genes in hotplate and tail withdrawal tests. We used the 4 core genotypes mouse model, in which gonadal sex is independent of the complement of sex chromosomes (XX vs XY). Mice were tested at baseline and then injected with mu-opioid agonist morphine (10 mg/kg) or with the kappa-opioid agonist U50,488H (U50, 12.5 mg/kg) with or without the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (0.1 mg/kg). On the day of birth, XX mice showed faster baseline latencies than XY in tail withdrawal, irrespective of their gonadal type. Gonadal males showed greater effects of morphine than gonadal females in the hotplate test, irrespective of their sex chromosome complement. U50 and morphine were effective analgesics in both tests, but MK-801 did not block the U50 effect. The results suggest that sex chromosome complement and gonadal secretions both contribute to sex differences in nociception and analgesia by the day of birth.

Perspective: Sex differences in pain may stem not only from the action of gonadal hormones on pain circuits but from the sex-specific action of X and Y genes. Identification of sex chromosome genes causing sex differences could contribute to better pain therapy in females and males.

Figure 1

In the baseline tail withdrawal tests for all experiments, XX mice showed faster latencies than XY mice, irrespective of their gonadal sex (*p=0.02). Mean latencies were XY male 0.92, XY female 0.86, XX male 0.80, XX female 0.70 seconds.

Figure 2

Results of the hotplate and tail withdrawal assays in the morphine study. A. Averaging over all drug (including saline) and time conditions in the hotplate test, males had a higher %Maximum Possible Effect (%MPE) than females (*p=0.009). B. The graphs shows %MPE averaged across time. In the hotplate test, morphine at 10mg/kg had significant analgesic effect (*p=0.000001) relative to the 0–1 mg/kg morphine group. The response to morphine tended to be higher in males than females irrespective of their sex chromosome complement (p=0.06 sex by drug interaction; +, p=0.02 comparison of males vs. females at 10mg/kg). C. Graphs show results averaged across all drug doses, including saline, so that the main effects of sex and sex chromosome complement can be seen. In the tail-withdrawal test, the %MPE over time differed as a function of both sex and sex chromosome complement because XX males had a more variable response than the other three groups (sex by sex chromosome by time interaction, p=0.035). D and E. In the tail withdrawal test, there was a statistically significant interaction of time, sex chromosome, sex, and drug (p=0.015), meaning that the effects of sex chromosome, sex, and drug were different at different times. To illustrate those differences, the %MPE are graphed separately for T=15 and T=45 minutes. At T=15 minutes after morphine injection, there was a differential effect of morphine on XX vs. XY females (+, p=0.07 in XY females and not significant for XX females) but not between XX and XY males (* both p<0.05). At 45 minutes after injection (T=45), the effect of morphine was significant only in XY males (**p =0.0002).

Figure 3

Hotplate and tail withdrawal assays in the U50 study. A, Averaging across all groups before and after injection in the hotplate test, the U50 group had longer latencies than controls (U50 vs. sal, ***p<0.0001), but MK did not block the effect of U50 (U50-MK vs. sal, ***p<0.0001). B, Hotplate latencies as a function of time and sex and sex chromosome complement. There was a significant interaction of time and sex chromosome complement -- XX and XY mice had longer latencies than XX mice only at T=90 (*p<0.05). To show this interaction, which did not depend on drug treatment, data from all drug treatment groups, including saline, are combined for each of the FCG groups. C, Averaging across time and sex and sex chromosome complement, U50 with or without MK significantly increased latencies in the tail withdrawal test (U50 vs. sal, and U50-MK vs. sal, both ***p<0.00001).