Control of Glycinergic Input to Spinal Dorsal Horn Neurons by Distinct Muscarinic Receptor Subtypes Revealed Using Knockout Mice

Abstract

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

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

“…Recording electrodes with a resistance of 5-10 megaohms were pulled from borosilicate glass capillaries and filled with internal solution containing 135.0 mM potassium gluconate, 5.0 mM KCl, 2.0 mM MgCl 2 , 0.5 mM CaCl 2 , 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM ATP-Mg, 0.5 mM Na-GTP, 1.0 mM GDP␤S, and 10.0 mM lidocaine N-ethyl bromide, adjusted to pH 7.2-7.4 with 1 M KOH (290 -300 mosM). GDP␤S, a general G protein inhibitor, was added to the internal solution to block the postsynaptic effect mediated by mAChR agonists through G proteins (27,30).…”

“…EPSCs were elicited by electrical stimulation (0.2 ms, 0.3-0.6 mA, 0.2 Hz) through a bipolar tungsten electrode placed on the dorsal root, which stimulates both A␦-and C-fibers (27,30).…”

Modulation of pain transmission by G-protein-coupled receptors

“…Further investigations using mAChR subtype knockout mice have revealed that activation of the M 2 and M 4 subtypes inhibits spinal GABA and glycine release. In contrast, stimulation of the M 3 subtype potentiates both GABAergic and glycinergic input to dorsal horn neurons in mice (Zhang et al, 2006a;Zhang et al, 2006b). It is not yet clear how reduction in GABA and glycine synaptic release by spinal M 2 and M 4 subtypes contributes to the analgesic effect of mAChR agonists in mice.…”

“…However, the mAChR agonists primarily inhibit GABAergic and glycinergic synaptic transmission in the spinal dorsal horn of wildtype mice (Zhang et al, 2006a;Zhang et al, 2006b). Further investigations using mAChR subtype knockout mice have revealed that activation of the M 2 and M 4 subtypes inhibits spinal GABA and glycine release.…”

Current and Future Issues in the development of spinal agents for the management of pain

“…M1/3/5 receptors induce membrane depolarization by enhancing cation currents and inhibiting potassium channels [285,286], while M2 and M4 channels activate potassium channels and inhibit some voltage-gated Ca channels (especially Ca v2.2 ), leading to hyperpolarization and block of transmitter release [287]. M 2 and M 4 knockout animals show a robust attenuation of the analgesic effects produced by muscarinic agonists [165,288,289] Interestingly, stimulation of M 2 and M 4 receptors inhibit spinal glycinergic function which might lead to a paradoxical hyperpathia, whereas stimulation of the M3 activation potentiates synaptic glycine release [290]. The events underlying the effects upon spinal nociceptive processing have been proposed to reflect several mechanisms.1) effect of muscarinic receptor subtypes in reducing afferent terminal excitability (reducing glutamate release) [289]; and, 2) activation of muscarinic receptors on glycinergic and GABAergic interneurons enhancing dorsal horn inhibitory amino acid release through muscarinic subtypes, such as the M2 and M4 receptors [290,291].…”