Opposing functions of spinal M₂, M₃, and M ₄ receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 μM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M₂ and M ₄ subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M₃ KO and M ₁/M₃ double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M₂/M₄ double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N- methylpiperidine methiodide, an M₃ subtypepreferring antagonist. In M₂ or M₄ single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M₃ subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M₂ and M₄ subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M₂ and M₄ subtypes in the spinal cord of rats.