Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia

Abstract

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5-/- mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5-/- mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5-/- mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.

Fig 1.

Morphine-induced CPP in M₅^+/+ and M₅^−/− mice. (A) CPP studies were carried out as described under Materials and Methods. Morphine-associated place conditioning is substantially reduced in M₅^−/− mice (129SvEv/CF1 hybrids resulting from homozygous matings, age-matched mice). *, P < 0.05, **, P < 0.01. Significantly different from the corresponding M₅^+/+ morphine-treated groups (ANOVA, Bonferroni's test, n = 10–13). a, significantly different from the corresponding saline groups (P < 0.05). (B) The reduction in preference for the morphine (5 mg/kg i.p.)-paired chamber is also observed in M₅^−/− mice generated by intermating heterozygous M₅^+/− mice (129SvEv/CF1 hybrids, littermates) or in isogenic M₅^−/− mice (129SvEv). The CPP responses to 5 mg/kg morphine shown in A (129SvEv/CF1 hybrids, age-matched) are included for comparison. Data obtained using the two parental strains (CF1 and 129SvEv) are presented as controls. **, P < 0.01. Significantly different from the two parental strains and the corresponding M₅^+/+ morphine-treated groups (ANOVA, Bonferroni's test, n = 8, 12).

Fig 2.

Morphine-induced increases in dopamine levels in the Acb of M₅^+/+ and M₅^−/− mice. Dialysate dopamine concentrations in the Acb of M₅^−/− mice (129SvEv/CF1 hybrids, age-matched) are greatly reduced following acute morphine administration (arrow, 25 mg/kg i.p.). Each point represents the mean ± SEM of dopamine concentrations in individual samples accumulated over 15 min of perfusion (0.6 μl/min). *, P < 0.05, **, P < 0.01. Significantly different from corresponding time point in M₅^−/− mice (repeated measures ANOVA, Tukey's test, n = 13, 16).

Fig 3.

Morphine-induced analgesia and development of acute morphine tolerance in M₅^+/+ and M₅^−/− mice. (A) The lack of M₅ receptors has no significant effect on morphine (i.p.) analgesia. HP, hot-plate; TF, tail-flick (n = 9–12). (B) M₅^+/+ and M₅^−/− mice do not differ in the development of acute tolerance to morphine analgesia. Tolerance was assessed by determining the analgesic potency of morphine (10 mg/kg i.p.) 24 hr after administration of a single dose of morphine (100 mg/kg i.p.). a, Significantly different from the corresponding naive groups, P < 0.01, n = 9–14. All experiments were carried out with 129SvEv/CF1 hybrid mice.

Fig 4.

Development of tolerance to the analgesic actions of morphine chronically administered to M₅^+/+ and M₅^−/− mice. Disruption of the M₅ receptor gene has no significant effect on the development of tolerance to the analgesic effects of chronically administered morphine (75 mg s.c. pellet). Analgesia measurements were carried out at the indicated time points (M, morphine pellet; P, placebo pellet) (n = 7–16, 129SvEv/CF1 hybrids).

Fig 5.

Immunoblot analysis of morphine-induced Fos-B expression in the Acb of M₅^+/+ and M₅^−/− mice. (A) The magnitude of Fos-B expression in the Acb of M₅^−/− mice chronically treated with morphine pellets (75 mg s.c., 7 days) is reduced relative to M₅^+/+ mice. (Inset) Immunoblot of Fos-B expression in M₅^+/+ (+/+) and M₅^−/− (−/−) mice implanted with placebo (P) or morphine (M) pellets. a, significantly different from all other groups, P < 0.01, ANOVA, Bonferroni's test, n = 7 or 8. (B) Concurrent treatment of M₅^+/+ mice with morphine (75 mg s.c. pellet, 7 days) and atropine (15 mg/kg per day, s.c., 7 days) mimics the effect of the M₅ receptor mutation on Fos-B expression in the Acb. (Inset) Immunoblot of Fos-B expression in M₅^+/+ mice implanted with placebo (P) or morphine (M) pellets alone or combined with atropine administered via osmotic minipumps. a, significantly different from all other groups, P < 0.01, ANOVA, Bonferroni's test, n = 4 or 5. All experiments were carried out with 129SvEv/CF1 hybrid mice.

Fig 6.

Naloxone-induced morphine withdrawal symptoms in M₅^+/+ and M₅^−/− mice. The number of occurrences of the indicated withdrawal behaviors (A, jumps; B, wet-dog shakes; C, paw tremors; D, teeth chattering) were recorded over a 30-min period following administration of naloxone (1 mg/kg i.p.) to mice chronically treated with morphine (75 mg s.c. pellet, 7 days). The severity of morphine withdrawal symptoms was less pronounced in M₅^−/− mice, independent of whether M₅^−/− mice were generated by homozygous (129SvEv/CF1 hybrids, age-matched mice) or heterozygous matings (129SvEv/CF1 hybrids, littermates). Similar results were obtained with isogenic M₅^+/+ and M₅^−/− mice (129SvEv). Data obtained using the two parental strains (CF1 and 129SvEv) are also included for control purposes. *, P < 0.05; **, P < 0.01. Significantly different from the corresponding M₅^+/+ groups, ANOVA, Bonferroni's post hoc test, n = 8–26.

Fig 7.

Naloxone-induced conditioned place aversion in M₅^+/+ and M₅^−/− mice. M₅^−/− mice (129SvEv mice) chronically treated with morphine (75 mg s.c. pellet, 7 days) show a reduction in the magnitude of naloxone (1 mg/kg i.p.)-induced conditioned place aversion. a, significantly different from M₅^+/+ pretest and naloxone/morphine-treated M₅^−/− test groups, P < 0.01; b, significantly different from M₅^−/− pretest group, P < 0.05; ANOVA, Bonferroni's test, n = 10–12.