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Review

Pain and purinergic signaling

Makoto Tsuda, Hidetoshi Tozaki-Saitoh, Kazuhide Inoue⁎

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,Higashi, Fukuoka 812-8582, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +81 92 642 4729.E-mail address: inoue@phar.kyushu-u.ac.

0165-0173/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainresrev.2009.11.003

A B S T R A C T

Article history:Accepted 11 November 2009Available online 18 November 2009

A growing body of evidence indicates that extracellular nucleotides play important roles in theregulation of neuronal and glial functions in the nervous system through P2 purinoceptors. P2purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropicreceptors (P2Y). P2X receptors (seven types; P2X1–P2X7) contain intrinsic pores that open bybinding with ATP, and P2Y receptors (eight types; P2Y1, 2, 4, 6, 11, 12, 13 and 14) are activatedby nucleotides and couple to intracellular second-messenger systems through heterotrimericG-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons inphysiological and pathophysiological conditions. Studies have shown thatmicroglia, a type ofglial cells known as residentmacrophages in the CNS, express several subtypes of P2X and P2Yreceptors, and these receptors play a key role in pain signaling in the spinal cord underpathological conditions such as by peripheral nerve injury (called neuropathic pain). Withinthe spinal dorsal horn, peripheral nerve injury leads to a progressive series of changes inmicroglia including morphological hypertrophy of the cell body and proliferation, which areconsidered indicative of activation. These activated microglia upregulate expression of P2X/Yreceptors (e.g., P2X4 and P2Y12). Importantly, pharmacological, molecular and geneticmanipulations of the function or expression of these microglial molecules stronglysuppress neuropathic pain. We expect that further investigation to determine how ATPsignaling via P2X receptors participates in the pathogenesis of chronic painwill lead to a betterunderstanding of the molecular mechanisms of pathological pain and provide clues for thedevelopment of new therapeutic drugs.

© 2009 Elsevier B.V. All rights reserved.

Keywords:PurinoceptorNeuropathic painMicrogliaSpinal cord

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2232. Molecular structure and pharmacological profiles of purinergic receptors . . . . . . . . . . . . . . . . . . . . . . . . 224

2.1. P2X receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2242.2. P2Y receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

3. Purinergic signaling and neuropathic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2253.1. P2X4 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2253.2. P2X7 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

jp (K. Inoue).

er B.V. All rights reserved.

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3.3. P2Y6 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2283.4. P2Y12 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

1. Introduction

In 1972, Burnstock proposed new roles of nucleotides asneurotransmitters (Burnstock, 1972) even though it wasprimarily recognized that intracellular ATP is the source offree energy to maintain life and nucleotides are keymoleculeswithin cells. In 1993, the first receptors for nucleotides, calledP2 purinoceptors, were cloned (Lustig et al., 1993; Webb et al.,1993). Afterwards, numerous subtypes of these receptors werealso cloned, and subsequently scientists began to graduallyaccept the “purinergic nervous system”. Now P2 purinoceptorsare divided into two families, ionotropic receptors (P2X) andmetabotropic receptors (P2Y) (Abbracchio et al., 2006; Khakh etal., 2001; Ralevic and Burnstock, 1998). Accumulating evidenceindicates that nucleotides are released and leaked from non-excitable cells as well as neurons and are involved in cell-to-cell communication in physiological and pathophysiologicalconditions (Burnstock, 2008). We finally have learned that theplayground of nucleotides may expand throughout the entirebody. One of the most exciting findings is that glia cells makeup over 70% of the total cell population in the central nervoussystem (CNS). Glial cells are classified into astrocytes,oligodendrocytes and microglia. Astrocytes express manytypes of P2X/Y receptors and release ATP in response tovarious stimuli or even spontaneously, and communicatewith neurons at synapses, microglial cells and vascular wallsat capillaries (Abbracchio et al., 2006; Inoue et al., 2007).Microglia, known as resident macrophages of the CNS, alsoexpress many types of P2 receptors (Farber and Kettenmann,2006; Inoue et al., 2007; Inoue, 2008). Although it was longbelieved that glial cells in the nervous system were onlyphysical and nutrient supports for neurons, a growing body ofevidence has dramatically changed this classical view andindicates that neuron–glia interactions are a key componentto understand CNS functions. In addition to crucial roles innormal physiological conditions, glial cells also play impor-tant roles in pathophysiological conditions of the CNSincluding psychiatric disorders, physical trauma, and chronicpain (Miller, 2005).

Pain in response to noxious stimuli can act as an earlywarning device that alerts us to the presence of damagingstimuli. This normal ‘good’ pain usually goes away soon afterthe nociceptive stimulus is removed. In contrast, sometimespain persists for a long time. In general, chronic pain may becategorized into inflammatory pain and neuropathic pain.Inflammatory pain is the result from inflammatory responsesto trauma in peripheral tissues. This type of pain, however,still has a physiological significance in that it may assistwound repair since contact with the damaged area isminimized. Also, inflammatory pain usually goes away afterthe tissue damage is repaired and can be generally managedby treatment with known analgesics. However, neuropathic

pain does not go away even though the initial damage hashealed. It typically occurs after nerve damage that can beinduced by bone compression in cancer, diabetes, infection,autoimmune disease or physical injury (Baron, 2006). Inaddition to spontaneous pain and hyperalgesia (the increasedpain perception of noxious stimuli), a troublesome symptomof neuropathic pain is pain hypersensitivity to normallyinnocuous stimuli, a phenomenon known as tactile allodynia.This type of pain is refractory to currently available treat-ments, such as non-steroidal anti-inflammatory drugs andopioids (Scholz and Woolf, 2002; Woolf and Mannion, 1999).We are now beginning to understand that neuropathic pain isnot just a symptom of disease but is a consequence ofdisordered functioning of the nervous system (Scholz andWoolf, 2002; Woolf and Mannion, 1999; Woolf and Salter,2000). Unraveling the mechanisms of pain hypersensitivitycaused by nerve damage is therefore essential for thedevelopment of new therapeutic drugs for neuropathic pain.

Accumulating evidence from diverse animal models ofneuropathic pain suggests that neuropathic pain mightinvolve aberrant excitability of the nervous system. Notably,in primary sensory ganglia and in the dorsal horn of the spinalcord multiple functional and anatomical alterations followperipheral nerve injury (Costigan et al., 2009; Scholz andWoolf, 2002; Woolf and Salter, 2000). While it has long beenconsidered that there are relevant changes in neurons,emerging lines of evidence have revealed that they alsooccur in glial cells, especially microglia (Marchand et al.,2005; Scholz and Woolf, 2007; Suter et al., 2007; Tsuda et al.,2005; Watkins et al., 2001). After peripheral nerve injury,microglia in the normal state (traditionally called ‘resting’microglia) in the spinal dorsal horn are converted to anactivated state through a series of cellular and molecularchanges (Echeverry et al., 2008; Suter et al., 2007; Tsuda et al.,2005). Microglia change their phenotype to the activated formfollowing altered expression of multiple molecules, includingcell surface receptors, intracellular signaling molecules, anddiffusible factors. By responding to extracellular ligands,activated microglia can evoke various cellular responses,such as migration toward afflicted sites and secretion ofproinflammatory factors. Importantly, pharmacological, mo-lecular and genetic manipulations of the function or expres-sion of these microglial molecules strongly modulateneuropathic pain (Clark et al., 2007; Griffin et al., 2007; Jin etal., 2003; Obata et al., 2007; Tanga et al., 2005; Tsuda et al., 2003,2004; Zhuang et al., 2005) and hyperexcitability of dorsal hornneurons (Coull et al., 2005; Keller et al., 2007). A growing bodyof evidence indicates that purinergic receptors are expressedin activated microglia under such pain conditions and thatP2X and P2Y receptors-mediated signaling critically contri-butes to the development and maintenance of neuropathicpain. In this article, we focus on recent developments that

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further the understanding of mechanisms by which P2X andP2Y receptors onmicroglia in the spinal cord participate in thepathogenesis of neuropathic pain. In addition, we summarizethe accumulated evidence from immunohistochemical, bio-chemical, molecular and behavioral studies, and we alsoprovide a framework to address the major questions thatcurrently remain unanswered.

2. Molecular structure and pharmacologicalprofiles of purinergic receptors

2.1. P2X receptors

P2X receptors belong to a family of ligand-gated ion-channelsand are cation-selective channels with almost equal perme-ability to Na+ and K+, and significant permeability to Ca2+

(Khakh et al., 2001; North, 2002; Ralevic and Burnstock, 1998).Molecular cloning has so far identified seven genes encodingP2X receptor subunits (P2X1–P2X7). All P2X subunits possesstwo transmembrane regions and have intracellular N- and C-terminals and a long extracellular loop between transmem-brane domains (Khakh et al., 2001; North, 2002; Vial et al.,2004). One third of the amino acids in the extracellular loop areconserved in at least six P2X subunits, suggesting they areinvolved in ATP binding (Vial et al., 2004). While theintracellular N-terminal regions have relatively similarlengths of amino acids, the length of the C-terminals divergesconsiderably from 30 residues in P2X6 to 240 in P2X7 (North,2002; Vial et al., 2004). In addition, there is a putative motif forphosphorylation by protein kinase C and A in the N- and C-terminals, respectively (North, 2002), and by ecto-proteinkinase C at the ectodomain (Wirkner et al., 2005).

One functional P2X receptor channel is presumably formedby a number of P2X subunits, as is considered to be the case inother ligand-gated ion channels. The number of subunits of aP2X receptor has been proposed to be three (Jiang et al., 2003;Nicke et al., 1998, 2003; Stoop et al., 1999) or four (Ding andSachs,2000; Kim et al., 1997). Recently, we analyzed the architectureand ATP-induced structural changes in P2X4 receptors usingfast-scanning atomic force microscopy (AFM) and revealed atrimer structure (Shinozaki et al., 2009). The trimer structure ofP2X receptors is also demonstrated in P2X2 receptors (Barrera etal., 2005). The trimer structure of P2X channels is stronglysupported by a recent study revealing the three-dimensionalcrystal structure of P2X4 receptor at a high resolution (Kawate etal., 2009). It appears that all three subunits are either identical,making P2X4 receptors homomeric, rather than identical, orheteromeric receptors. Biochemical studies have shown the co-assembly of P2X1/5 (Torres et al., 1998), P2X2/3 (Lewis et al.,1995), P2X2/6 (King et al., 2000), P2X4/6heteromeric receptors (Leet al., 1998). In addition, there is a report that P2X2/3 receptorsare likely to contain oneP2X2and twoP2X3subunits (Jianget al.,2003; Wilkinson et al., 2006).

Each of the seven homomeric P2X receptors and at least fourheteromeric receptors show different (but partly overlapping)electrophysiological and pharmacological properties in terms ofcurrent kinetics, desensitization rates and sensitivities toagonists and antagonists (Jarvis and Khakh, 2009; Khakh et al.,2001; North, 2002; Ralevic and Burnstock, 1998). When each one

or twoof themareexpressedheterologously in cells, ATPevokesa rapid- or slow-inactivating inward current. The former is seenincells expressingeither P2X1or P2X3 receptors, and the latter isseen in those expressing all other receptors including the fourheteromeric receptors (Khakh et al., 2001; North, 2002). Further-more, repetitive activation of P2X1 and P2X3 receptors by ATPcausesamarkeddesensitizationofATP-induced responses.α,β-Methylene ATP (αβmeATP), an analogue of ATP, is a usefulagonist for basic identification of P2X receptors containing P2X1or P2X3 (i.e., P2X1, P2X3, P2X1/5 and P2X2/3), although αβmeATPalso activatesP2X4/6 (Khakhetal., 2001;North, 2002; Ralevic andBurnstock, 1998).As for antagonists, suraminandpyridoxalpho-sphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) are antago-nistic to almost all P2X receptors except rat P2X4 receptors (Buellet al., 1996). However, it should be noted that suramin alsoblocks several neurotransmitter receptors (e.g., NMDA andGABAA receptors) (Nakazawa et al., 1995; Peoples and Li, 1998).2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) at a nanomolarrange selectively blocks P2X1, P2X3 and P2X2/3 receptors(Virginio et al., 1998) and at high concentration also blocksP2X4 receptors (Virginio et al., 1998). Furthermore, diinosinepentaphosphate (Ip5I) is a very potent antagonist to P2X1 (Kinget al., 1999) and P2X3 receptors but, interestingly, not to P2X2and P2X2/3 receptors (Dunn et al., 2000). In 2002, the firstselective antagonist for P2X3 (and P2X2/3), A-317491, wasdeveloped (Jarvis et al., 2002). A-317491 blocks the responsesmediated by P2X3 or P2X2/3 receptors in a competitive fashionwithout any effect on other receptors, enzymes or ion-channels(Jarvis et al., 2002). Furthermore, several selective antagonistsfor P2X7 receptors, A-740003 (Honore et al., 2006), A-438079(McGaraughty et al., 2007), A-804598 (Donnelly-Roberts et al.,2009) and AZ11645373 (Stokes et al., 2006), have been developedrecently. A-317491 and A-438079 are commercially available.

2.2. P2Y receptors

The first G protein-coupled P2Y receptors were cloned in 1993(Lustig et al., 1993; Webb et al., 1993), and there are currentlyeight accepted P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,P2Y12, P2Y13, and P2Y14. The missing numbers represent eithernonmammalian orthologs or receptors that have some sequencehomology to P2Y receptors but for which there is no functionalevidence of responsiveness to nucleotides (Abbracchio et al.,2006). P2Y receptors are 308–377 amino acid proteins with amass of 41–53 kDa after glycosylation. The seven transmem-brane (TM) domain tertiary structure of P2Y receptors iscommon to other G protein-coupled receptors (Fig. 1). P2Y areactivated by purine or pyrimidine nucleotides or sugar-nucleo-tides with subtype-dependent heterotrimeric G-proteins(Abbracchio et al., 2006). Site-directed mutagenesis studieshave shown that some positively charged residues in TM3, 6and 7 are crucial for receptor activation by nucleotides (Jiang etal., 1997). Each P2Y receptor binds to a single heterotrimeric Gprotein (Gαq/11 for P2Y1, 2, 4, 6), although P2Y11 can couple toboth Gαq/11, and Gαs, whereas P2Y12 and P2Y13 couple to Gαi

and P2Y14 couples to Gαi/o (Abbracchio et al., 2006). In responseto nucleotide activation, recombinant P2Y receptors eitheractivate the phospholipase C (PLC)-IP3 pathway through Gαq/11,which in turn releases intracellular calcium and activatesprotein kinase C, or affect adenylyl cyclase and alter cAMP

Fig. 1 – Schematic illustration of potential mechanisms by which P2X and P2Y receptors in activated microglia modulate painsignaling in the dorsal horn. Activated microglia after nerve injury increase the expression of P2X4, P2Y6 and P2Y12. The P2X4and P2X7 receptors are activated by ATP which is presumably released from primary sensory neurons (or astrocytes), and inturn cause a rise in the intracellular calcium and activation of p38MAPK. P2X receptors activation leads to the release ofbioactive diffusible factors, such as BDNF and other proinflammatory factors (cytokines and chemokines). BDNF causes acollapse of the transmembrane anion gradient in dorsal horn lamina I neurons presumably through the downregulation ofKCC2, which in turn renders GABA and glycine affects depolarizing, rather than hyperpolarizing, in these neurons. Microglialfactors may also interact with excitatory synapses of neighboring dorsal horn neurons and enhance the excitability in dorsalhorn neurons. The net hyperexcitability in the dorsal horn pain network by these factors from activated microglia may beresponsible for neuropathic pain.

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levels through Gαi/o (Abbracchio et al., 2006). Furthermore,recent studies have shown that several P2Y receptors (P2Y2,P2Y6 and P2Y12) also coupled to Gα12/13, and nucleotidestimulation also activates Rho (Liao et al., 2007; Nishida et al.,2008).

In contrast to P2X receptors, P2Y receptors are activated notonly by ATP but also by its metabolite ADP and othernucleotides such as UTP and UDP (Abbracchio et al., 2006).For P2Y1 receptors, ADP is a more potent agonist than ATP(their 2-methylthio derivatives are even more potent). Themost effective antagonists to display selectivity for the P2Y1receptor areMRS2179 (Boyer et al., 1998), MRS2279 (Boyer et al.,2002) and MRS2500 (Kim et al., 2003). P2Y2 receptors are fullyactivated by equivalent concentrations of ATP and UTP,whereas ADP and UDP are much less effective agonists. P2Y6receptors are selective for UDP (Abbracchio et al., 2006). A 1,4-di-(phenylthioureido) butane derivative (MRS2578) (Mame-dova et al., 2004) has been shown to selectively inhibit UDP-

induced phospholipase C activity through P2Y6 receptors.P2Y12 receptors are also activated selectively by ADP andblocked by the 5′-triphosphate derivative AR-C69931MX (Ingallet al., 1999) (although this antagonist also blocks P2Y11 andP2Y13 receptors (Abbracchio et al., 2006; Communi et al., 1999)).Recent studies have demonstrated that the active metaboliteof the antiplatelet drug clopidogrel is a potent and selectiveantagonist of P2Y12 receptors (Savi and Herbert, 2005).

3. Purinergic signaling and neuropathic pain

3.1. P2X4 receptor

A clue to identifying P2X4Rs in the spinal cord as required forneuropathic pain first came frompharmacological investigationof pain behavior after nerve injury using the P2X receptorantagonists TNP-ATP and PPADS (Tsuda et al., 2003) that are

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known to be sensitive and insensitive to P2X4, respectively(Jarvis and Khakh, 2009; Khakh et al., 2001). Because nerveinjury-induced allodynia was effectively suppressed by TNP-ATP but not by PPADS, it was inferred that tactile allodyniadependsuponP2X4Rs in the spinal cord (Tsuda et al., 2003). Thishypothesis is substantially supported by recent findings show-ing that both mice treated spinally with a P2X4 antisenseoligonucleotide and mice lacking P2X4 show attenuated tactileallodynia after nerve injury (Tsuda et al., 2003, 2009; Ulmann etal., 2008). Expression of P2X4 receptors in the spinal cord, afternerve injury, markedly increased specifically in microglia,indicating that tonic activation of P2X4 receptors in microgliais necessary for sustaining allodynia (Tsuda et al., 2003).Moreover, it was found that spinal administration of P2X4-stimulated microglia caused otherwise normal rats to developallodynia. Therefore, P2X4 receptor activation inmicroglia is notonly necessary but is also sufficient to cause tactile allodynia(Tsuda et al., 2003).

The mechanisms by which microglia are crucial forproducing neuropathic pain must involve signaling fromactivated microglia to dorsal horn neurons, but how? Coullet al. (2005) demonstrated that P2X4-stimulated microgliainduced the hyperexcitability of dorsal horn neurons. In spinalcord slices of rats that had displayed allodynia after intrathe-cal administration of ATP-stimulated microglia, a positiveshift of the anion reversal potential (Eanion) in spinal lamina Ineurons was found, which caused GABA-evoked depolariza-tion, rather than hyperpolarization, in these neurons (Coull etal., 2005). Moreover, TNP-ATP acutely reversed the depolariz-ing shift in Eanion in lamina I neurons after nerve injury (Coullet al., 2005). These results imply that spinal microgliastimulated by P2X4 receptors cause neuropathic pain througha rise in intracellular Cl− in spinal lamina I neurons.Furthermore, Keller et al. (2007) have shown that local spinaladministration of ATP-stimulated microglia changed thephenotype of in vivo spinal lamina I output neurons, suchthat they relay innocuous mechanical input (Keller et al.,2007), which may account for allodynia. Recently, Coull et al.(2005) determined the role of brain-derived neurotrophicfactor (BDNF) as a signaling factor between microglia anddorsal horn lamina I neurons. It was found that intrathecalapplication of BDNF mimicked tactile allodynia and thedepolarizing shift in Eanion in lamina I neurons by peripheralnerve injury or by intrathecal administration of P2X4-stimu-lated microglia. Interference of signaling between BDNF andits receptor (TrkB) prevented tactile allodynia caused byperipheral nerve injury or by intrathecal administration ofP2X4-stimulated microglia. Activating P2X4 onmicroglial cellscaused the release of BDNF(Coull et al., 2005; Trang et al., 2009),an effect that is dependent on activation of p38 (Trang et al.,2009), a member of mitogen-activated protein kinase that hasbeen reported to be implicated in neuropathic pain (Jin et al.,2003; Tsuda et al., 2004). Interestingly, P2X4-mediated BDNFrelease was abolished by inhibiting SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated exocytosis. Thus, these results indicate that P2X4-stimulated microglia release BDNF as a crucial factor to signalto lamina I neurons, causing a collapse of their transmem-brane anion gradient and subsequent neuronal hyperexcit-ability (Fig. 1). The γ-aminobutyric acidA (GABAA) receptor-

mediated depolarization might also produce excitationthrough voltage-sensitive Ca2+ channels and N-methyl-D-aspartate (NMDA) receptors. There is evidence that severalproinflammatory cytokines that are known to be released frommicroglia (Inoue, 2002; Inoue, 2006) modulate excitatorysynaptic transmission. Spinal injection of interleukin-1β (IL-1β) enhanced C-fiber-evoked responses and windup in wide-dynamic-range dorsal horn neurons (Reeve et al., 2000).Exogenous application of IL-1β enhanced NMDA receptor-mediated Ca2+ responses via activation of the tyrosine proteinkinase Src (Viviani et al., 2003). IL-1β was also reported todecrease GABAA receptor-mediated currents (Wang et al.,2000). A recent study has clearly demonstrated a powerfulrole for proinflammatory cytokines in excitatory or inhibitorysynaptic transmission and on neuronal activity in thesuperficial dorsal horn neurons (Ikeda et al., 2007; Kawasakiet al., 2008b). Also, inhibition of microglial ERK decreasedhyper-responsiveness of dorsal horn neurons in spinal cordinjured rats through a reduction of prostaglandin E2 produc-tion (Zhao et al., 2007). Thus, the net enhanced transmission inthe dorsal horn pain network by these factors might beresponsible for nerve injury-induced neuropathic pain.

From the above evidence, upregulation of P2X4 receptorexpression in microglia is therefore a key process in neuro-pathic pain. How peripheral nerve injury increases expressionof P2X4 receptors in microglia is not clearly understood.Activating both toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2, another pattern-recognition receptor) in cultured microglia increased theexpression of P2X4 at the mRNA level (Guo et al., 2006), butthe functional relevance of these receptors in vivo is stillunknown. Studies in our laboratory have recently shown thatthe extracellular matrix protein, fibronectin, increased ex-pression of mRNA and protein of P2X4 receptors in primarycultured microglial cells (Nasu-Tada et al., 2006; Tsuda et al.,2010). Cultured microglia stimulated by fibronectin alsoshowed an enhanced ATP-induced Ca2+ influx (Nasu-Tada etal., 2006). The level of fibronectin protein was elevated in thedorsal horn 3–7 days after nerve injury (Nasu-Tada et al., 2006),the time when P2X4 protein levels start to increase (Tsuda etal., 2003). Blockade of fibronectin receptors attenuated nerveinjury-induced P2X4 upregulation and allodynia (Tsuda et al.,2008a). It was also found that the upregulation of P2X4receptors in the spinal cord and allodynia after spinal nerveinjury was significantly suppressed by intrathecal adminis-tration of echistatin (Tsuda et al., 2008a). Furthermore,intrathecal delivery of fibronectin increased P2X4 expressionand produced allodynia, a behavior that was not evoked inP2X4-deficient mice. These results suggest that the fibronec-tin-integrin signaling system participates in the upregulationof P2X4 expression after nerve injury and subsequent neuro-pathic pain (Tsuda et al., 2008a).

We recently identified Lyn as a critical signalingmolecule forP2X4 upregulation in microglia (Tsuda et al., 2008b). Lyn-deficient microglial cells showed a deficit in increased P2X4expression caused by fibronectin. The level of Lyn expressionwas increased exclusively in microglia after nerve injury, andLyn-knockout mice exhibited a striking reduction in upregula-tion of P2X4 and tactile allodynia after nerve injury. Lyn tyrosinekinase distinctly activated the phosphatidylinositol 3-kinase

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(PI3K)-Akt and mitogen-activated protein kinase kinase (MAPKkinase, MEK)-extracellular signal-regulated kinase (ERK) signal-ing cascades (Tsuda et al., 2010). Signaling through the PI3K-Aktpathway induceddegradationof p53 viamouse doubleminute 2(MDM2) in a proteasome-dependentmanner. The consequenceof an attenuated repressive effect of p53may be associatedwithenhanced P2X4 gene expression. On the other hand, activatedMEK-ERK signaling in microglia exposed to fibronectin en-hanced eukaryotic translation initiation factor 4E (eIF4E)phosphorylation status via activated MAPK-interacting proteinkinase-1 (MNK1), which may play a role in regulating P2X4expression at translational levels. Themolecular machinery forthe upregulation of P2X4 expression in microglia is nowconsidered to be as shown in Fig. 2.

There is evidence that P2X4 receptors inmicroglial cells arelocated predominantly within lysosomal compartments andare targeted there by their N- and C-terminal motifs (Qureshiet al., 2007). Notably, P2X4 receptors remain stable withinlysosomes and resist degradation by virtue of their N-linkedglycans. It is of particular interest that stimulating lysosome

Fig. 2 – Schematic representation of the hypotheticalmechanisms involved in fibronectin-induced P2X4upregulation in microglia. Extracellular fibronectin binds toα5β1 integrins on microglial cells, which leads to activationof the PI3K-Akt andMEK-ERK signaling cascades through Lyntyrosine kinase. Signaling through the PI3K-Akt pathwayinduces degradation of p53 via MDM2 in aproteasome-dependent manner. The reduction of therepressive effect of p53 may enhance P2X4 gene expression.Activated MEK-ERK signaling in microglia exposed tofibronectin enhances eIF4E phosphorylation status viaactivated MNK1, which may play a role in regulating P2X4expression at translational levels.

exocytosis, for example, using a Ca2+ ionophore, resulted inthe trafficking of P2X4 to the plasmamembrane (Qureshi et al.,2007). Given that P2X4 activity on the plasma membrane ofmicroglia is critical for microglial function, it is conceivablethat mechanisms underlying P2X4 recycling between thelysosome and the plasma membrane are important forneuropathic pain.

The above evidence indicates that P2X4 might be apotential therapeutic target to treat neuropathic pain. How-ever, there is no antagonist to strongly inhibit P2X4 receptors.We have recently demonstrated that some antidepressantsand anticonvulsants clinically used in patients with neuro-pathic pain have inhibitory effects on ATP-evoked Ca2+

response in cells expressing recombinant P2X4 receptors andprimary cultured microglial cells (Nagata et al., 2009). Amongthe drugs used, paroxetine has the most potent inhibitoryeffect. Intrathecal administration of paroxetine and fluvox-amine, but not citalopram, produced an anti-allodynic effectin an animal model of neuropathic pain, which correlatedwith the potency of inhibition of rat P2X4 receptors. Interest-ingly, it was found that the anti-allodynic effect of paroxetineis insensitive to 5-HT receptor antagonists and was alsoobserved in neuropathic pain animals with depleted 5-HT inthe spinal cord. These results suggest that the anti-allodyniceffect of antidepressants may be independent of the spinal 5-HT system and may be mediated by inhibiting P2X4 receptors(Nagata et al., 2009).

3.2. P2X7 receptor

The first evidence indicating the involvement of P2X7 receptorsin pain was provided by a study using P2X7-deficient mice.These mice exhibited reduced thermal and mechanical hyper-sensitivities after partial sciatic nerve ligation (Chessell et al.,2005). Recently, several groups have developed novel, selectiveinhibitors of P2X7 receptors and have examined their effects onneuropathic pain. Systemic (i.p.) administration of A-740003and A-438079, structurally unrelated P2X7 antagonists, reducedtactile allodynia in three different models of neuropathic painin rats (Honore et al., 2006). Furthermore, A-438079 (i.v.)reduced innocuous stimuli-evoked activity of in vivo dorsalhorn neurons in neuropathic rats, but not in sham-operatedrats (McGaraughty et al., 2007). The role of microglial P2X7receptors in neuropathic pain remains unclear because of alack of data showing the effect of intrathecal administration ofthese antagonists. Interestingly, recent evidence has indicateda structural and functional interaction between P2X7 and P2X4receptors (Casas-Pruneda et al., 2009; Guo et al., 2007). Studieshave shown co-immunoprecipitation of both receptor subtypes(Boumechache et al., 2009; Guo et al., 2007), but it might not bedue to the formation of heterotrimeric complex but to the closeassociation of neighboring P2X4 and P2X7 homotrimers(Boumechache et al., 2009; Nicke, 2008). Furthermore, incontrast to the predominant localization of P2X4 in intracellu-lar lysosome (as mentioned above), P2X7 is predominantlydistributed in the cell surface. If the trafficking of P2X4 to theplasma membrane is enhanced in activated microglia, afunctional interaction between these two receptors mightoccur. Therefore, it is of particular interest to investigate theinteraction between P2X4 and P2X7 in neuropathic pain.

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3.3. P2Y6 receptor

Microglial cells also express several G-protein-coupled recep-tors. In our recent study, it was found that microglial cells inculture express P2Y6 receptors (Koizumi et al., 2007) and thatafter peripheral nerve injury P2Y6 mRNA expression ismarkedly increased in the spinal cord, the time course ofwhich is parallel with that of tactile allodynia (unpublishedobservation). The function of P2Y6 in microglia remainsunknown, but we recently revealed that this receptor medi-ated microglial phagocytosis. It was found that applying UDP,an agonist for P2Y6, to primary cultured microglial cellsfacilitated phagocytosis (Koizumi et al., 2007). Neuronal injuryinduced by administration of kainic acid (KA) caused upregu-lation of P2Y6 receptors in microglia in the hippocampus. KA-evoked neuronal injury resulted in an increase in extracellularUTP, which was immediatelymetabolized into UDP in vivo andin vitro. UDP, leaked from injured neurons, caused P2Y6receptor-dependent phagocytosis. The P2Y6 receptor is upre-gulated when neurons are damaged, and could function as asensor for phagocytosis by sensing diffusible UDP signals.However, the role of upregulated P2Y6 in the spinal cord underneuropathic pain conditions remains unknown, and furtherstudies are required to determine its role.

3.4. P2Y12 receptor

Two recent studies have revealed that P2Y12 receptors are oneof the key regulators for neuropathic pain (Kobayashi et al.,2008; Tozaki-Saitoh et al., 2008). It was demonstrated thatintrathecally administered P2Y12 receptor antagonists, suchas MRS2395 and AR-C69931MX, or antisense oligonucleotidefor P2Y12 receptors significantly suppressed development ofneuropathic pain after spinal nerve injury (Tozaki-Saitoh etal., 2008) and partial sciatic nerve injury (Kobayashi et al.,2008). A crucial finding was established using P2Y12-knockoutmice. Genetic ablation of P2Y12 receptors failed to producetactile allodynia (Tozaki-Saitoh et al., 2008). Strategic advancesin targeting P2Y12 receptors were considered because of itsrestricted expression in the CNS. Several lines of evidencerevealed that P2Y12 receptor expression is specifically ob-served in brain and spinal cord resident microglia but is notobserved in fms- or CD11b-positive peripheralmacrophages inspleen (Haynes et al., 2006; Kobayashi et al., 2006; Pausch et al.,2004; Sasaki et al., 2003). In line with these facts, we couldconsider that P2Y12 receptor inhibition results from P2Y12receptor-gated signal modulation in the CNS. p38MAPK is asignalingmoleculewhose phosphorylation after partial sciaticnerve injury is almost completely suppressed by an antagonistor antisense oligonucleotide for P2Y12 receptors (Kobayashi etal., 2008). We have also shown that a single administration ofthe P2Y12 receptor antagonists AR-C69931MX (intrathecally)or clopidogrel (orally) to nerve-injured rats produced a strikingalleviation of established tactile allodynia (Tozaki-Saitoh etal., 2008). These effects by P2Y12 receptor antagonists are incontrast to the effects of another P2Y12 receptor antagonist,MRS2395, and a P2Y12 receptor antisense oligonucleotide,which did not reverse pain behaviors (Kobayashi et al., 2008).The exact reason for this difference remains open to question.In addition, it must be noted that P2Y12 receptors are

predominantly expressed in platelets in the periphery andinhibition of this receptor is one of the adopted mechanismsfor anti-platelet agents (Gachet, 2005).

Expression levels of P2Y12 receptors are dramaticallyincreased in microglial cells ipsilateral to peripheral nerveinjury in the spinal cord after nerve injury (Kobayashi et al.,2008; Tozaki-Saitoh et al., 2008) but down-modulated in brainmicroglia activated by slice preparation (Haynes et al., 2006). Amajor difference in each experiment was the proximal ordistal neuronal damage, which might induce different signalcascades to invoke microglial responses. Indeed, upregulationof P2Y12 receptor mRNA is also observed in a facial nerveinjury model (Sasaki et al., 2003). Detailed mechanisms ofexpressional regulation of P2Y12 receptors remain to beelucidated.

Many studies that have investigated in depth the mecha-nismof ATP-mediated chemotaxis inmicroglia have identifiedP2Y12 and P2X4 receptors as primary sensors for extracellularATP (Honda et al., 2001; Irino et al., 2008; Kanazawa et al., 2002;Ohsawa et al., 2007). Further studies have added evidenceindicating that P2Y12 receptors are implicated in the motilityof microglial cell bodies and processes in vivo or ex vivo(Davalos et al., 2005; Haynes et al., 2006; Kurpius et al., 2007).Thus, low ATP concentrations in the extracellular spaceactivate chemotaxis of microglia at the injured or inflamedsite. We already know that P2Y12 receptors are unlikely to benecessary to convert spinalmicroglia to the activated state andto increase their cell numbers because morphological andnumerical alterations of microglia were not influenced by thelack of P2Y12 receptors or by the administration of the P2Y12receptor antagonist AR-C69931MX in the spinal dorsal hornafter spinal nerve injury (Tozaki-Saitoh et al., 2008). One ofremaining possibilities regarding ATP-induced microglialchemotactic activity is that P2Y12 receptor activity inmicrogliamay influence the abilities of microglia to extend the tips oftheir branched processes toward neighboring pain transmis-sion neurons, which may in turn affect microglia–neuroncommunications.

4. Concluding remarks

We have primarily focused on the role of purinoceptorsexpressed in spinal microglia in neuropathic pain caused byperipheral nerve injury. Pharmacological, molecular andgenetic manipulations of the function or expression of thesepurinoceptors have influenced nerve injury-induced painbehaviors and hyperexcitability of the dorsal horn painpathway. Therefore, purinoceptor-mediated spinal microgliaactivity make a critical contribution to pathologically en-hanced pain processing in the dorsal horn, and microglialpurinoceptors might be promising targets for treating neuro-pathic pain. Recently, novel and established drugs that targetP2X and P2Y receptors have been reported to be effective inmodels of pain. Of note, a more recent study has revealed thethree-dimensional crystal structure of P2X4 receptor. Al-though the structure was solved in the absence of ATP,cavities between subunits thought to be the ATP binding sitewere also found. Thus, the P2X4 structure will be highlyinformative for designing new drugs targeting P2X4 receptor.

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A predicted therapeutic benefit of interfering with microglialP2X and P2Y receptors may be that normal pain sensitivitywould be unaffected because expression or activity of most ofthese molecules are upregulated or enhanced predominantlyin activated microglia in the spinal cord where damagedsensory fibers project. In addition to microglia, recent studieshave also identified astrocyte-specific molecules and demon-strated a critical role of spinal astrocytes in neuropathic pain,particularly in the maintenance phase (Ji et al., 2006; Katsuraet al., 2008; Kawasaki et al., 2008a; Zhuang et al., 2006).Extracellular nucleotides are also a strong modulator ofastrocytes in the brain including the dorsal horn of the spinalcord (Fam et al., 2000; Salter and Hicks, 1994). How astrocyticpurinoceptors participate in neuropathic pain and interactwith microglial purinoceptors is now open for investigation. Itis expected that an increased understanding of the functionsof purinoceptors will provide us with exciting insights intopain mechanisms and clues to develop new therapeuticagents for the management of neuropathic pain.

Acknowledgments

M.T., H.T.-S. and K.I. are supported by a grant from theMinistry of Education, Culture, Sports, Science and Technol-ogy of Japan. M.T. acknowledges support from the MochidaMemorial Foundation for Medical and Pharmaceutical Re-search and the Astellas Foundation for Research on MetabolicDisorders.

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