Signaling mechanisms mediating muscarinic enhancement of GABAergic synaptic transmission in the spinal cord

Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M₂, M₃, and M₄ subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca²⁺-free solution. Inhibition of voltage-gated Ca²⁺ channels with Cd²⁺ and Ni²⁺ largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M₃ subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M₂/M₄ mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca²⁺ influx and voltage-gated Ca²⁺ channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M₂/M₄ mAChRs in the spinal dorsal horn.