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 M(2), M(3), and M(4) 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 Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) 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 M(3) 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 M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.