Activation of group I mGlu receptors contributes to facilitation of NMDA receptor membrane current in spinal dorsal horn neurons after hind paw inflammation in rats

Abstract

The interaction between the group I metabotropic glutamate (mGlu) receptors and N-methyl-D-aspartate (NMDA) receptors plays a critical role in spinal hyperexcitability and hyperalgesia. The cellular mechanisms underlying this interaction remain unknown. Utilizing an ex vivo spinal slice preparation from young adult rats, we investigated the group I mGlu receptor modulation of NMDA receptor-mediated current in superficial dorsal horn neurons by patch clamp recording after complete Freund's adjuvant (CFA)-induced hind paw inflammation. We show that NMDA receptor-mediated dorsal root stimulation-evoked EPSC (eEPSC) and NMDA-induced current was enhanced in the inflamed rats, compared to naïve rats and this effect was attenuated by AIDA (1 mM), a group I mGlu receptor antagonist. There were also increases in the frequency and amplitude of miniature excitatory postsynaptic currents in the presence of tetrodotoxin, suggesting enhanced presynaptic glutamate release probability and postsynaptic membrane responsiveness in inflamed rats. DHPG (10 μM), a selective group I mGlu receptor agonist, further facilitated NMDA receptor-mediated eEPSC and NMDA-induced current in inflamed rats. The DHPG-produced facilitation of NMDA-induced current was blocked by intracellular dialysis of GDP-beta-S (1 mM), a G protein antagonist, and BAPTA (15 mM), an intracellular calcium chelating agent; and by pretreatment with U73,122 (10 μM), a PLC inhibitor, or 2-APB (100 μM), an IP₃-receptor antagonist. These findings support the hypothesis that signal transduction coupling between group I mGlu receptors and NMDA receptors underlies the activation of NMDA receptors in spinal hyperexcitability and hyperalgesia.

Introduction

Metabotropic glutamate (mGlu) receptors, a family of G protein-linked receptors, are classified into groups I (mGlu1,5 receptors), II (mGlu2,3 receptors), and III (mGlu4,6,7,8 receptors) based on their signal transduction pathways, pharmacology profiles, and sequence homology (Conn and Pin, 1997). While groups II and III mGlu receptors are dominantly located presynaptically, group I mGlu receptors are expressed primarily on postsynaptic membranes, although a presynaptic localization has also been observed (Jia et al., 1999, Neugebauer, 2002, Shigemoto et al., 1997). Group I mGlu receptors are distributed in the superficial spinal dorsal horn (Berthele et al., 1999, Guo et al., 2004), and implicated in a variety of pain conditions (Adwanikar et al., 2004, Guo et al., 2004, Neugebauer, 2002, Scotland and Coderre, 2007). It has been well established that the N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in synaptic plasticity, dorsal horn hyperexcitability and hyperalgesia (Dubner and Ruda, 1992, Guo and Huang, 2001, Ren et al., 1992, Woolf and Thompson, 1991).

Studies suggest that interactions between the mGlu and NMDA receptors underlie the mechanism of modulation of neuronal plasticity (Alagarsamy et al., 2001, Guo et al., 2004, Millan, 1999). In CNS neurons, NMDA receptors are linked to mGlu receptors in the postsynaptic membrane (Guo et al., 2004, Naisbitt et al., 1999). Activation of mGlu receptors enhances the effects of ionotropic glutamate receptor function (Budai and Larson, 1998, Neugebauer, 2002, Stanfa and Dickenson, 1998). Previous studies have shown that mGlu receptor agonists enhance NMDA-mediated increase in intracellular calcium concentration and NMDA-induced currents in dorsal horn neurons (Bleakman et al., 1992, Bond and Lodge, 1995, Cerne and Randić, 1992). The response to mGlu receptor agonists is potentiated in the spinal cord in hyperalgesic but not naive animals, and this effect is reversed by an NMDA receptor antagonist (Boxall et al., 1998). Intrathecal administration of the mGlu1,5 receptor agonist (S)-3,5-DHPG in mice produced hyperalgesia that was attenuated by NMDA receptor antagonists and silencing of the NR2B subunit of the NMDA receptor by RNA interference (Gabra et al., 2007). We have shown that a group I mGlu receptor-NMDA receptor coupling cascade leads to phosphorylation of the NMDA receptor and initiation of spinal dorsal horn hyperexcitability and behavioral hyperalgesia after inflammation (Guo et al., 2004).

Despite converging evidence, the intracellular signaling mechanisms underlying the interaction between mGlu and NMDA receptors in inflammatory pain are still largely unknown. In the present study we performed ex vivo patch clamp recordings from spinal dorsal horn neurons in young adult rats to directly assess the intracellular mechanisms involved in the modulation of NMDA receptor-mediated current by inflammation and group I mGlu receptor activation. The results show that NMDA receptor-mediated membrane current was enhanced in complete Freund's adjuvant (CFA)-inflamed rats and this effect was attenuated by antagonism of group I mGlu receptors. DHPG, a selective agonist of group I mGlu receptors, further facilitated NMDA receptor current in inflamed rats and the mechanisms underlying this facilitation involved postsynaptic intracellular G protein, the PLC pathway and IP₃-sensitive Ca²⁺ release.