Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M 2 /M 4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA B receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M 2 -KO and M 4 -KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M 3 subtype and the M 2 and M 4 subtypes. This study demonstrates that stimulation of the M 2 and M 4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M 3 subtype potentiates synaptic glycine release. Furthermore, GABA B receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.The cholinergic system and muscarinic acetylcholine receptors (mAChRs) are important for the regulation of nociceptive transmission in the spinal cord. In this regard, blocking of mAChRs in the spinal cord causes a large decrease in the nociceptive threshold (Zhuo and Gebhart, 1991). Intrathecal administration of mAChR agonists or acetylcholinesterase inhibitors produces a potent analgesic effect in many species, including rats, mice, and humans
