M2, M3, and M4 receptor subtypes contribute to muscarinic potentiation of GABAergic inputs to spinal dorsal horn neurons

The spinal cholinergic system and muscarinic receptors are important for regulation of nociception. Activation of spinal muscarinic receptors produces analgesia and inhibits dorsal horn neurons through potentiation of GABAergic inputs. To determine the role of receptor subtypes in the muscarinic agonist-induced synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons using whole-cell voltage-clamp recordings in rat spinal cord slices. The muscarinic receptor agonist oxotremorine-M dose-dependently (1-10 mu M) increased GABAergic sIPSCs but not miniature IPSCs. The potentiating effect of oxotremorine-M on sIPSCs was completely blocked by atropine. In rats pretreated with intrathecal pertussis toxin to inactive inhibitory G(i/o) proteins, 3 mu M oxotremorine-M had no significant effect on sIPSCs in 31 of 55 (56%) neurons tested. In the remaining 24 44%) neurons in pertussis toxin-treated rats, oxotremorine-M caused a small increase in sIPSCs, and this effect was completely abolished by subsequent application of 25 nM 4-diphenylacetoxy-N- methylpiperidine methiodide (4-DAMP), a relatively selective M-3 subtype antagonist. Furthermore, himbacine ( 1 mu M), a relatively specific antagonist for M-2 and M-4 subtypes, produced a large reduction in the stimulatory effect of oxotremorine-M on sIPSCs, and the remaining effect was abolished by 4-DAMP. Additionally, the M-4 receptor antagonist MT-3 toxin ( 100 nM) significantly attenuated the effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that M-2 and M-4 receptor subtypes play a predominant role in muscarinic potentiation of synaptic GABA release in the spinal cord. The M-3 subtype also contributes to increased GABAergic tone in spinal dorsal horn by muscarinic agonists.