Abstract

Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M₂ and M₄ receptor subtypes, the two G_i/G_o-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M₂ and M₄ mAChRs [M₂/M₄ double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M₂/M₄ double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M₂ receptor single-KO mice, but retained full analgesic activity in M₄ receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M₂ and M₄ receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M₄ than for M₂ receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M₂ and M₄ mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.