Involvement of microglial P2X7 receptors and downstream signaling pathways in long-term potentiation of spinal nociceptive responses

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Abstract

Tetanic stimulation of the sciatic nerve (TSS) produces long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal cord. This potentiation is considered to be a substrate for long-lasting sensitization in the spinal pain pathway. Because microglia have previously been shown to regulate the induction of spinal LTP, we hypothesize that P2X7 receptors (P2X7R), which are predominantly expressed in microglia and participate in the communication between microglia and neurons, may play a role in this induction. This study investigated the potential roles of P2X7Rs in spinal LTP and persistent pain induced by TSS in rats. OxATP or BBG, a P2X7R antagonist, prevented the induction of spinal LTP both in vivo and in spinal cord slices in vitro and alleviated mechanical allodynia. Down-regulation of P2X7Rs with P2X7-siRNA blocked the induction of spinal LTP and inhibited mechanical allodynia. Double immunofluorescence showed colocalization of P2X7Rs with the microglial marker OX-42, but not with the astrocytic marker GFAP or the neuronal marker NeuN. Intrathecal injection of BBG suppressed the up-regulation of microglial P2X7Rs and increased expression of Fos in the spinal superficial dorsal horn. Further, pre-administration of BBG inhibited increased expression of the microglial marker Iba-1, phosphorylated p38 (p-p38), interleukin 1β (IL-1β) and GluR1 following TSS. Pre-administration of the IL-1 receptor antagonist (IL-1ra) blocked both the induction of spinal LTP and the up-regulation of GluR1. These results suggest that microglial P2X7Rs and its downstream signaling pathways play a pivotal role in the induction of spinal LTP and persistent pain induced by TSS.

Introduction

Tetanic stimulation of the sciatic nerve (TSS) has previously been shown to produce long-lasting hyperalgesia and allodynia in rats (Ying et al., 2006, Zhang et al., 2005). Electrophysiological studies have also shown that long-term potentiation (LTP) of C-fiber/lamina I neuron synapses in the spinal cord is induced by TSS (Sandkuhler, 2007). Induction of this LTP is blocked by antagonists of spinal N-methyl-D-aspartic acid (NMDA) and NK-1 receptors, both of which are know to function in the spinal pain pathways (Liu and Sandkuhler, 1995, Liu and Sandkuhler, 1997, Sandkuhler and Liu, 1998, Rygh et al., 1999). It is therefore plausible that spinal LTP is the cellular mechanism underlying central sensitization of nociceptive signals (Ji et al., 2003, Sandkuhler, 2007).

Increasing evidence suggests that microglia play an important role in the modulation of neuronal plasticity and the induction of neuropathic pain (Watkins et al., 2007, Watkins et al., 2009, Watkins and Maier, 2002). In our previous studies, we showed that disruption of microglial and astrocytic function blocked both tetanic stimulation-induced spinal LTP and pain behaviors (Ma and Zhao, 2002, Ying et al., 2006). More recently, other investigators showed that activation of Src-family kinases (SFKs) in microglia in spinal dorsal horn controls the direction of TSS-induced LTP and that the cytokine tumor necrosis factor α (TNF-α) is involved in LTP induction (Zhong et al., 2010). Cytokines that are primarily released from microglia, including interleukin 1β (IL-1β) and TNF-α, have also been shown to modulate the induction of LTP in the hippocampus by increasing the surface expression and phosphorylation of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Cunningham et al., 1996, Beattie et al., 2002, Ikeda et al., 2007, Pedersen et al., 2009). Although the contribution of microglia to the spinal LTP has been confirmed, the exact mechanisms are uncertain.

P2X7 receptors (P2X7R) are abundantly expressed in microglia and have been proposed to play a pivotal role in the cross-talk between microglia and neurons (Sperlagh et al., 2006, Yu et al., 2008). Activation of P2X7Rs by ATP or BzATP significantly blocked the uptake of glutamate into microglia (Morioka et al., 2008). Also, disruption of P2X7Rs by oxidized ATP (oxATP), a selective P2X7R antagonist, prevents the release of proinflammatory cytokines IL-1β and TNF-α (Clark et al., 2010, Hide et al., 2000, Hughes et al., 2007, Labrousse et al., 2009). Activation of P2X7Rs increases phosphorylation of p38 mitogen activated protein kinase (MAPK), which functions upstream in the IL-1β signaling pathway (Solle et al., 2001, Donnelly-Roberts et al., 2004, Ferrari et al., 2006, Sperlagh et al., 2007). Moreover, deletion of the P2X7R gene abolishes chronic inflammatory and neuropathic pain in P2X7−/− mice (Chessell et al., 2005). Furthermore, blockade of P2X7Rs by a P2X7R antagonist reduces nerve injury-induced spontaneous firing of spinal neurons and pain behaviors (McGaraughty et al., 2007). Taken together, these results suggest that microglial P2X7Rs may regulate tetanic stimulation-induced spinal LTP through a similar signaling pathway.

In this study, we demonstrate that microglial P2X7Rs and the downstream IL-1β signaling pathway play pivotal roles in the induction of spinal LTP and behavioral persistent pain induced by TSS.

Section snippets

Animals and reagents

Adult male Sprague–Dawley rats (180–350 g) (Animal Center, Chinese Academy of Sciences, Shanghai) were housed in single plastic cages in a light (12:12 light/dark cycle) and temperature (23 ± 2 °C) controlled room with standard rodent chow and water available ad libitum. The treatment of the animals conformed to the guidelines of the protocols of the International Association for the Study of Pain concerning the use of laboratory animals.

OxATP and Brilliant Blue G (BBG), P2X7R antagonists, were

Blockade of the induction of spinal LTP by P2X7R antagonists and P2X7-siRNA

In consistent with our previous studies (Ma and Zhao, 2001, Ying et al., 2006, Zhang et al., 2005), the C-fiber-evoked field potentials by electrical stimulation (0.5 ms, 20–30 V) were recorded at the depth of 300–500 μm from the surface of the spinal cord. Four consecutive C-fiber-evoked field potentials were averaged as control. The baseline of C-fiber-evoked field potentials was stable during the recording periods. Tetanic stimulation of the sciatic nerve (0.5 ms, 100 Hz, 40 V, 10 trains of 2-s

Discussion

The main findings of this study are: (1) Activation of spinal P2X7Rs by tetanic stimulation of the sciatic nerve is intimately linked with the induction of spinal LTP of C-fiber-evoked field potentials and long-lasting behavioral pain hypersensitization; (2) P2X7Rs are expressed in spinal cord microglia; (3) Disruption of P2X7R signaling by antagonists or introduction of siRNA into spinal microglia blocked the spinal LTP and alleviated pain hypersensitization; and (4) IL-1β signaling pathway

Acknowledgments

This work was supported by grants from National Basic Research Program of China (Nos. 2006CB500800, 2007CB512502 and 2007CB512303) and grants from National Nature Science Found of China (No. 3083004). We are grateful to Dr. David W. Saffen for the improvement of the language for the whole manuscript.

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