Heterogeneity in Primary Nociceptive Neurons: From...

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Arch Pharm Res Vol 33, No 10, 1489-1507, 2010 DOI 10.1007/s12272-010-1003-x 1489 Heterogeneity in Primary Nociceptive Neurons: From Molecules to Pathology Eduardo Gascon and Aziz Moqrich Institut de Biologie du Développement de Marseille Luminy, UMR 6216 CNRS-Université de la Méditerranée, Campus de Luminy case 907, 13288 Marseille Cedex 09, France (Received August 9, 2010/Revised August 19, 2010/Accepted August 20, 2010) Pain sensation (nociception) is an alarm system aiming to signal the presence of potentially or actually harmful stimuli. In our hazard-rich environment, pain initiates the necessary reac- tions to prevent or limit tissue damage in response to noxious inputs playing therefore a cru- cial survival role. Specialized noxious stimuli detectors, called primary nociceptive neurons or nociceptors transduce and convey pain information to the central nervous system. Unlike other sensory systems, pain sensation could be evoked by a vast range of external or internal stimuli. Nearly any of the environmental stimuli could be potentially noxious depending on their nature and/or intensity and/or duration. Early studies at the beginning of the 20 th cen- tury identified a discrete number of nociceptive neuronal types according to their electrophys- iological responses or their degree of myelination. However, the advent of molecular biology techniques revealed an extraordinary diversity among nociceptors. Such heterogeneity likely reflects the evolutionary adaptation required to respond to an extremely variety of circum- stances. Key words: Dorsal root ganglia, Nociceptors, Ion channels, Pain INTRODUCTION Due to its unpleasant nature, it is generally assum- ed that pain is something we had better live without. However, survival of an individual critically depends on its ability to rapidly react to potentially harmful stimuli. Pain perception alerts us to damage and re- cruits protective responses (i.e. rapid withdrawal) aiming to prevent us from serious injury. In addition, nociceptive system has evolved to endow injured tissue with extra protection. Following tissue damage, pain thresholds could be dramatically decreased so that innocuous stimuli become painful (allodynia) and noxious insults evoke an exacerbated response (hyper- algesia) (Basbaum et al., 2009). This nociceptive plas- ticity is thought to promote healing by preventing dam- aged tissue from further insults. From a Darwinian perspective, the most endowed individuals protect them- selves from pain-evoking situations and therefore will survive more consistently. In human, mutations that lead to a pain-free phenotype clearly illustrates that abolition of pain is a ‘negative factor’ for survival and these individuals are permanently affected by burns, wounds and fractures and their life expectancy is drastically shortened (Indo, 2001; Indo et al., 2001; Mardy et al., 2001). Unfortunately, under some pathological conditions, nociceptive signaling might start to misrepresent the sensory inputs and, at times, might be completely in- dependent of sensory stimulation causing pain which is no longer beneficial for the organism. It is estimated that over one-third of the world’s population suffers from persistent or recurrent pain. Moreover, persist- ent pain, even at moderate levels, is often highly in- validating compromising working ability and/or every- day life activities. Finally, clinicians have few, if any, consistently effective means of relief chronic pain devoid of severe side effects. Development of novel agents against pain requires a more profound knowledge of how pain signals are initially interpreted and subse- Correspondence to: Aziz Moqrich, Institut de Biologie du Dével- oppement de Marseille Luminy, UMR 6216 CNRS-Université de la Méditerranée, Campus de Luminy case 907, 13288 Marseille Cedex 09, France Tel: 0491269765, Fax: 0491269748 E-mail: [email protected] REVIEW

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Arch Pharm Res Vol 33, No 10, 1489-1507, 2010DOI 10.1007/s12272-010-1003-x

1489

Heterogeneity in Primary Nociceptive Neurons: From Molecules to Pathology

Eduardo Gascon and Aziz MoqrichInstitut de Biologie du Développement de Marseille Luminy, UMR 6216 CNRS-Université de la Méditerranée, Campusde Luminy case 907, 13288 Marseille Cedex 09, France

(Received August 9, 2010/Revised August 19, 2010/Accepted August 20, 2010)

Pain sensation (nociception) is an alarm system aiming to signal the presence of potentially oractually harmful stimuli. In our hazard-rich environment, pain initiates the necessary reac-tions to prevent or limit tissue damage in response to noxious inputs playing therefore a cru-cial survival role. Specialized noxious stimuli detectors, called primary nociceptive neurons ornociceptors transduce and convey pain information to the central nervous system. Unlikeother sensory systems, pain sensation could be evoked by a vast range of external or internalstimuli. Nearly any of the environmental stimuli could be potentially noxious depending ontheir nature and/or intensity and/or duration. Early studies at the beginning of the 20th cen-tury identified a discrete number of nociceptive neuronal types according to their electrophys-iological responses or their degree of myelination. However, the advent of molecular biologytechniques revealed an extraordinary diversity among nociceptors. Such heterogeneity likelyreflects the evolutionary adaptation required to respond to an extremely variety of circum-stances.Key words: Dorsal root ganglia, Nociceptors, Ion channels, Pain

INTRODUCTION

Due to its unpleasant nature, it is generally assum-ed that pain is something we had better live without.However, survival of an individual critically dependson its ability to rapidly react to potentially harmfulstimuli. Pain perception alerts us to damage and re-cruits protective responses (i.e. rapid withdrawal)aiming to prevent us from serious injury. In addition,nociceptive system has evolved to endow injuredtissue with extra protection. Following tissue damage,pain thresholds could be dramatically decreased sothat innocuous stimuli become painful (allodynia) andnoxious insults evoke an exacerbated response (hyper-algesia) (Basbaum et al., 2009). This nociceptive plas-ticity is thought to promote healing by preventing dam-aged tissue from further insults. From a Darwinian

perspective, the most endowed individuals protect them-selves from pain-evoking situations and therefore willsurvive more consistently. In human, mutations thatlead to a pain-free phenotype clearly illustrates thatabolition of pain is a ‘negative factor’ for survival andthese individuals are permanently affected by burns,wounds and fractures and their life expectancy isdrastically shortened (Indo, 2001; Indo et al., 2001;Mardy et al., 2001).

Unfortunately, under some pathological conditions,nociceptive signaling might start to misrepresent thesensory inputs and, at times, might be completely in-dependent of sensory stimulation causing pain whichis no longer beneficial for the organism. It is estimatedthat over one-third of the world’s population suffersfrom persistent or recurrent pain. Moreover, persist-ent pain, even at moderate levels, is often highly in-validating compromising working ability and/or every-day life activities. Finally, clinicians have few, if any,consistently effective means of relief chronic pain devoidof severe side effects. Development of novel agentsagainst pain requires a more profound knowledge ofhow pain signals are initially interpreted and subse-

Correspondence to: Aziz Moqrich, Institut de Biologie du Dével-oppement de Marseille Luminy, UMR 6216 CNRS-Université dela Méditerranée, Campus de Luminy case 907, 13288 MarseilleCedex 09, FranceTel: 0491269765, Fax: 0491269748E-mail: [email protected]

REVIEW

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1490 E. Gascon and A. Moqrich

quently transmitted and perpetuated.Nociceptors have emerged as attractive targets for

novel pharmacological strategies for a number of rea-sons. First, nociceptive neurons act as the first ele-ment in the pain sensory machinery transducing noxi-ous stimuli and transmitting nociceptive informationto the central nervous system. Thus, effective nocicep-tor inhibition (i.e. local anesthetics) is known to resultin robust analgesia. Second, long-term modificationsin primary nociceptive neurons under pathologicalconditions (i.e. inflammation or nerve injury) largelycontribute to clinical pain. Targeting these neuronswould therefore prevent the development of chronicpain syndromes. Third, nociceptors are peripheralneurons, drugs designed to interfere with nociceptorfunction might therefore circumvent most (central)side effects of existing painkillers such as opioids.

Nonetheless, recent work in the last decade hasrevealed that drugs acting at nociceptors are still achallenge. It is now clear that, from a molecular pointof view, nociceptors encompass a remarkably hetero-geneous populations of neurons (Chen et al., 2006;Woolf and Ma, 2007). Indeed, transduction and trans-mission of noxious stimuli entail a myriad of ion chan-nels, membrane receptors, signaling molecules andneuropeptides/neurotransmitters. A single nociceptoris thus equipped with a precise set of transducing/signaling molecules allowing it to react to a definednumber of stimuli. This molecular diversity (nociceptormolecular signature) might simply reflect the extraor-dinary versatility required to respond to a wide rangeof potential noxious insults. How this molecular di-versity is generated? what is its functional relevance?and how molecularly distinct nociceptors behave inresponse to damage are fundamental questions thatremain poorly understood.

In this review, we will briefly recapitulate the mech-anisms of nociceptors generation and diversification.We will next highlight recent progress on the under-standing of the palette of ion channels involved inpain perception and how differences at the molecularlevel generate a remarkable functional heterogeneityamong nociceptive neurons. Finally, a speculativechapter will be devoted to how different nociceptorssubsets might contribute to chronic pain states.

MECHANISMS OF NOCICEPTIVE DIVER-SIFICATION

Major types of adult nociceptive neuronsNociceptors are the specialized types of peripeheral

sensory neurons responding to noxious stimuli andtissue damage (Sherrington, 1903). Cell bodies of noci-

ceptive neurons are located in dorsal root ganglia (DRGs)and trigeminal ganglia (TG). The axons bifurcateshortly after leaving the cell body and splits to onecentral (directed to the dorsal horn of the spinal cordor the brain stem) and one peripheral branch (inner-vating peripheral tissues such as the skin, muscle orviscera).

Studies conducted in the early 90s uncovered speci-fic trophic dependencies of distinct DRG neuronalsubsets (Barbacid, 1995). Thus, it was observed thatNGF-TrkA is essential for the survival of nociceptorsduring embryonic development (Smeyne et al., 1994).Later studies revealed that trophic requirements ofnociceptive neurons shifted in the first post-natalweek(s). A subset of nociceptive neurons (comprisingtwo thirds of the total population) is seen to downre-gulate TrkA and expresses Ret, the receptor of theGDNF-family ligands. The remaining neurons main-tain NGF-TrkA activity (Molliver et al., 1997; Woolfand Ma, 2007). Interestingly, these two major subsetsof molecularly defined nociceptive neurons display anumber of common molecular, histological and anato-mical features. Adult TrkA+ neurons contain neuro-peptides (i.e. calcitonin gene-related peptide, CGRP)and are also known as peptidergic neurons. They arealso low-myelinated (A-delta) fibers, projecting to thestratum spinosum of the epidermis and to the mostsuperficial layers of the dorsal horn of the spinal cord(lamina I and outer lamina II). Conversely, Ret+ neuronsare non-peptidergic and were firstly identified by theirability to bind to isolectin B4 (IB4). These neurons arenon-myelinated (C fibers), terminate in the stratumgranulosum in the skin and project centrally to deeperlayers of the dorsal horn (inner lamina II). Moreover,it has been postulated that inflammatory and neuro-pathic pain relate to TrkA+ and Ret+ nociceptors, re-spectively. Although electrophysiological studies haverevealed an intrinsic heterogeneity within these twopopulations (Bessou and Perl, 1969; Burgard et al., 1999;Petruska et al., 2000; Cain et al., 2001; Vydyanathanet al., 2005), this classification of nociceptors is stillwidely accepted.

Generation of nociceptive neuronsDRG neurons, including nociceptors, arise from

neural crest stem cells (NCCs) that delaminate fromthe dorsal part of the neural tube (Anderson, 2000;Marmigere and Ernfors, 2007). During NCC migration,sensory neurogenesis occurs in three successive waves(Ma et al., 1999; Maro et al., 2004; Marmigere andErnfors, 2007). The first one is dependent on the pro-neural transcription factor Neurogenin2 (Ngn2) where-as progenitors of the second one express another mem-

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ber of the family, Neurogenin1 (Ngn1). The cells inthe third wave come from the boundary cap, a cellularstructure located at the boundary between the centraland peripheral nervous system. The mechanisms un-derlying nociceptor cell fate determination are stillunder thorough investigation.

In a pioneering work, Moqrich et al. (2004) demon-strated that expression of TrkC from trkA locus re-sulted in a fate switch in prospective nociceptors thatbecome proprioceptive neurons. This piece of datastrongly suggests that neurotrophin signaling is ne-cessary for survival and for neuronal subtype specifi-cation. It has been shown that most TrkA+ neuronsdepend on Ngn1 (Ma et al., 1999). Other transcriptionfactors such as Brn3a and Klf7 are required for themaintenance, but not the initiation of TrkA expression(Marmigere and Ernfors, 2007). Both Brn3a and Klf7bind to a defined enhancer sequence upstream of exon1 of TrkA gene (Lei et al., 2001; Ma et al., 2000, 2003)and loss of Brn3a results in decreased TrkA expres-sion (Huang et al., 1999). A recent study also reporteda loss of TrkA+ neurons in the absence of the LIM-homeodomain transcription factor Islet1 (Sun et al.,2008). However none of these transcription factors arespecifically expressed in developing nociceptors sug-gesting that they might play a more general role inDRG neurogenesis. In agreement with this hypothe-sis, TrkC expression seems also to be dependent onBrn3a activity (Huang et al., 1999) and a loss of TrkBneurons is also detected in Islet1 conditional knockoutmice (Sun et al., 2008).

Much attention has been focused on Runx1, a runtdomain transcription factor. Unlike Ngn1, Brn3a orKlf7, runx1 is expressed exclusively in TrkA+ neuronsat early embryonic stages (Levanon et al., 2002; Chenet al., 2006). In addition, Runx1 overexpression in thechick neural tube leads to ectopic TrkA expression sug-gesting that Runx1 might regulate TrkA expressiononset (Marmigere et al., 2006). However, other studiesargue against this possibility. On one hand, endogen-ous Runx1 expression is initiated some time after TrkAexpression. On the other, targeted deletion of Runx1in DRG neurons has no effect on TrkA expression (Chenet al., 2006; Yoshikawa et al., 2007). Moreover, Runx1overexpression does not perturb TrkA in mouse em-bryonic DRGs (Kramer et al., 2006). These results in-dicate that Runx1 is unlikely to be involved in noci-ceptors’ early cell fate determination.

The role of extracellular signals such as Wnt, SonicHedgehog (Shh) or Notch in fate determination of NCCshas been widely documented (Raible and Ungos, 2006;Marmigere and Ernfors, 2007). Whether these (orothers) signaling cascades are also involved in specifi-

cation of different neuronal subtypes remains yet tobe determined.

Diversification of nociceptorsRecent studies have started to unravel the different

mechanisms that drive nociceptors diversification.Several reports have unambiguously established thatRunx1 plays a central role in this process (Chen et al.,2006; Kramer et al., 2006; Marmigere et al., 2006;Yoshikawa et al., 2007). Using two independent gen-etic strategies to knockout Runx1 during DRG devel-opment, it has been shown that loss of Runx1 selec-tively impairs non-peptidergic maturation. Indeed, inthe absence of Runx1, most adult nociceptive neuronsare devoid of Ret but retain TrkA and CGRP expres-sion. Ret expression in Runx1 conditional knockoutmice remain confined to a subset of proprioceptiveTrkC+ neurons (Chen et al., 2006; Yoshikawa et al.,2007). In this line, embryonic Runx1 overexpressionresults in the complete inhibition of peptidergic markerssuch as CGRP (Kramer et al., 2006). These observa-tions together provide strong evidence that Runx1 isthe master regulator of peptidergic vs. non-peptidergicdifferentiation.

Runx1 begins to be expressed shortly after TrkA ex-pression and the vast majority of prospective nocicep-tors (~90% at E14.5) are Runx1+ during embryonicdevelopment (Levanon et al., 2002; Chen et al., 2006).The mechanism by which Runx1 expression is turnedon still remains unknown but it is reported that onlynociceptors arising from Ngn1+ precursors are able toexpress Runx1 (Ma et al., 1999; Kramer et al., 2006).Peptidergic neurons most likely are derived from Ngn2lineage and from boundary cap cells as a default cellfate in the absence of Runx1 whereas those generatedfrom the second wave (Ngn1) might express Runx1and be committed to a non-peptidergic phenotype. How-ever, several lines of experimental evidence argueagainst this contention. Quantitatively, those nocicep-tors devoid of Runx1 (about 1/10 of total DRG neurons)could not account for the final population of peptider-gic neurons (roughly 1/3 of total DRG neurons). In add-ition, Runx1 has been shown to be necessary for theexpression of some peptidergic markers (Chen et al.,2006) such as some Trp channels indicating that, atleast, a subset of peptidergic neurons might derive fromRunx1 lineage. In line with this hypothesis, a recentstudy reported by us, provided genetic evidence thathalf of peptidergic neurons do derive from prospectivenociceptors that expressed Runx1 at a precise embryo-nic developmental stage (Gascon et al., 2010).

How is Runx1 expression maintained/extinguishedin this population of prospective nociceptors? Luo et

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al. (2007) reported that NGF-TrkA signaling is requir-ed to maintain Runx1 expression and is thereforeessential for non-peptidergic maturation. Since allnociceptors, both peptidergic and nonpeptidergic, re-quire NGF for proper development (Smeyne et al., 1994),these observations strongly suggest that additionalfactor(s), in concert with NGF, ensure the extinction ofRunx1 in a subset of prospective peptidergic neurons.Our recent work identified Hepatocyte Growth Factor(HGF) and its high affinity receptor Met are one suchfactor (Gascon et al., 2010). It was demonstrated thatMet loss results in an excessive number of Runx1+

neurons in the adult DRG. More importantly, TrkAand Runx1, which label non-overlapping neuronal po-pulation in the adult wild type DRG, could be found inthe same nociceptive neurons of Met conditional knock-out mice. This observation suggests that TrkA andRunx1 do not properly segregate in the absence of Metand argue that it critically participates in the processof Runx1 extinction. In agreement with these data, Metconditional knockout mice exhibit a marked decreasein the number of CGRP+ neurons. In summary, thesefindings suggest that the nociceptors activating factorsMet and TrkA would switch off Runx1 expression andbecome peptidergic neurons whereas those respondingexclusively to NGF would maintain Runx1 and differ-entiate into non-peptidergic nociceptors.

ION CHANNELS INVOLVED IN NOCICE-PTION

One of the most likely reasons of nociceptors heter-ogeneity is the extraordinary variety of potential nox-ious insults. Indeed, all stimuli, if intense enough, couldbe harmful. Likewise, early electrophysiological datarevealed that nociceptors display a high threshold forstimulation compared to other sensory neurons (i.e.touch receptors in the skin). This basic feature ensuresthat most activities could be carried out pain-freewhereas intense stimuli could elicit pain sensation.

Recent advances in the sensory neurons researchreveals molecular mechanisms underlying varioustypes of stimuli transduced to neural signals gettingtransmitted to the central nervous system for painperception. These studies identified a number of ionchannels which are activated in response to definedsensory inputs. In addition, many other ion channels(and some ionotropic receptors) are involved in thegeneration and transmission of action potentials innociceptors. Each nociceptor is thought to express aparticular set of these channels (nociceptor molecularsignature) and therefore to respond to an exquisiterange of stimuli and environmental conditions. Differ-

ent combinations of molecular transduction/transmis-sion machinery in nociceptors might ensure the func-tional diversity required for optimal pain perceptionin response to most (if not all) of the potentially harm-ful stimuli.

Ion channels involved in pain detection: TRPchannels

Pain detection entails an initial phase in which thenociceptor transduces the impinging noxious stimulusinto a neural (electrical) signal. During evolution, mostsensory systems have acquired specialized types of ionchannels for signal transduction. These channels sharea common mechanism of action: upon specific stimu-lation, their opening generates inward currents thatlead to depolarization and increased excitability of thesensory neuron.

Ion channels responsible for the detection of painlargely belong to the family of Transient PotentialChannels (TRP).These channels have been involved inolfaction, taste vision, osmoregulation, mechanosensa-tion and temperature perception (Clapham, 2003;Venkatachalam and Montell, 2007; Song and Yuan,2010). The involvement of TRP channels in pain is underintensive scrutiny (Patapoutian et al., 2009; Stucky etal., 2009). Here, we will focus on those properties ofTRP channels that make them particularly suitablefor nociception.

TRP channels comprise proteins with six transmem-brane domains and cytoplasmic N- and C-termini. TRPproteins preferentially assemble as homotetramers toform cation-permeable channels (Voets et al., 2005).They are highly unusual among other ion channelsfamilies as they display a remarkable diversity of spe-cific activation mechanisms being able to respond toall major classes of external and internal inputs, in-cluding light, sound, chemicals, temperature, osmolarityand mechanical stimuli (Clapham, 2003; Venkatachalamand Montell, 2007).

Currently, 28 TRP channels have been identified inmammals and based on their sequence homology, areclassified into six subfamilies: TRPC, TRPV, TRPM,TRPA, TRPP and TRPML (Montell, 2005; Venkata-chalam and Montell, 2007). A role of TRP channels inpain was first suggested by the discovery that TRPV1could be activated by noxious heat (Caterina et al.,1997, 1999). We will briefly describe the most relevantaspects of TRP channels whose role in nociception hasbeen firmly established, TRPV1, TRPV2, TRPV3,TRPV4, TRPA1 and TRPM8.

TRPV1As stated above, TRPV1 is the first member of TRP

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family to be linked to pain perception. TRPV1 is acti-vated by a wide range of stimuli including chemicalssuch as capsaicin or allicin (Caterina et al., 1997;Salazar et al., 2008), acid pH (Caterina et al., 1999;Caterina and Julius, 2001), endogenous lipids (Zygmuntet al., 1999; Gunthorpe et al., 2000; Hwang et al., 2000;Huang et al., 2002; Patwardhan et al., 2010) andnoxious heat (>42oC) (Caterina et al., 1997, 1999). It istherefore considered as a polymodal transducer.

TRPV1−/− mice have provided unexpected resultsconcerning the physiological role of TRPV1 in nocicep-tion (Caterina et al., 2000; Davis et al., 2000). Giventhe similar activation threshold of TRPV1 and heat-gated currents observed in cultured DRG (Cesare andMcNaughton, 1996), in vitro studies have predictedthat TRPV1 might be important for the perception ofnoxious heat. Several lines of evidences suggest thatthe role of TRPV1 as a noxious heat sensor is likely tobe negligible. First, Caterina et al. found that with-drawal latencies in a variety of behavioral tests areunchanged until 50oC in mutant animals (a tempera-ture much higher than TRPV1 activation threshold)(Caterina et al., 2000); moreover, an independentlygenerated strain of TRPV1−/− animals show no behav-ioral defects even at 52.5oC (Davis et al., 2000). Second,heat-sensitive C-fibers in TRPV1−/− mice had an acti-vation threshold indistinguishable from wild typeanimals (Woodbury et al., 2004). Third, heat-sensitivefibers from the skin are predominately IB4+ (Lu et al.,2001) and co-expression between IB4 and TRPV1 hasbeen shown to be rather low in mice (Zwick et al.,2002; Woodbury et al., 2004). These results togetherstrongly suggest that receptors other than TRPV1might account for nouxious heat perception underacute conditions.

There is, however, no doubt about the involvementof TRPV1 in thermal hyperalgesia produced by in-flammation. Thermal hyperalgesia induced by Com-plete Freund’s Adjuvant (CFA) could be alleviated byinjection of TRPV1 antagonist in the wild type animals(Yu et al., 2008). More importantly, several groupshave reported a severe impairment of thermal but notmechanical hyperalgesia in TRPV1−/− mice (Caterinaet al., 2000; Davis et al., 2000; Jin and Gereau, 2006;Kawamata et al., 2008). Withdrawal latency afterheat stimulation is dramatically reduced in wild typemice following inflammation. Importantly, these la-tencies remain unchanged in mutant mice before andafter inflammation suggesting that most (if not all)heat-induced inflammatory response is dependent onTRPV1.

Subsequent research has clearly demonstrated an

enhancement of TRPV1 currents after inflammation.Two independent (and probably overlapping) mecha-nisms account for this effect. The first one (sensitiza-tion) endows nerve terminals with the possibility torapidly react to tissue damage/inflammation. Sensiti-zation results from a shift in the temperature depend-ency towards lower temperatures, thus reducing thethreshold for channel activation and increasing theamplitude of channel responses to above-thresholdinputs. Extensive work highlighted the importance ofTRPV1 phosphorylation for inflammatory sensitization(Roberts and Connor, 2006; Cheng and Ji, 2008; Uceyleret al., 2009). Inflammatory mediators (i.e. bradykinin,ATP, histamine or prostaglandins) acting through theirG-protein coupled receptors could activate proteinkinase Cε (Bhave et al., 2003; Amadesi et al., 2006)and/or protein kinase A (Zhang et al., 2008) and there-fore phosphorylate TRPV1. In addition, TRPV1 is alsodirectly sensitized by some growth factor releasedduring tissue damage such as NGF (Bonnington andMcNaughton, 2003; Zhuang et al., 2004). NGF-depend-ent sensitization is largely linked to protein kinase Cphosphorylation in the downstream of ERK/PI3kinase pathway (Bonnington and McNaughton, 2003;Zhuang et al., 2004).

The second mechanism involves an upregulation ofTRPV1 expression and/or insertion into the plasmamembrane of nociceptive terminals. Compared to chan-nel sensitization, increasing the proteins levels couldbe a possible method to maintain hypersensitivity overlonger time periods. In this regard, NGF and TumorNecrosis Factor alpha (TNFα) are the best character-ized factors inducing TRPV1 upregulation. NGF elicitsboth an augmentation of TRPV1 levels (Ji et al., 2002)and the membrane insertion of pre-existing TRPV1protein (Zhang et al., 2005) via p38 MAPK and Srckinase, respectively. TNFα seems to use the same down-stream cascade (p38 MAPK) to trigger rapid (less than1h) TRPV1 upregulation (Jin and Gereau, 2006;Hensellek et al., 2007). Recent data indicate that manypro-inflammatory agents could mimic such effects(Hucho and Levine, 2007).

These observations suggest that TRPV1’s contribu-tion in the perception of noxious heat is rather limitedunder acute conditions in contrast to the many studiesthat have highlighted its pivotal role in thermalhyperalgesia secondary to tissue injury/inflammation.More importantly, considering that most of inflamma-tory mediators converge on TRPV1 activity; these re-sults indicate that the TRPV1 channel is a key effectorin such processes.

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Other TRPV channelsOther TRPV members have been shown to be

activated at different range of temperatures. TRPV2responds to increasing temperatures with a thresholdof activation of 52oC indicating a role in detecting acutenoxious heat (Caterina et al., 1999). Intriguingly,TRPV2 exhibits little overlap with TRPV1 being foundin myelinated sensory fibers that are mechanicallysensitive (Caterina et al., 1999; Ma, 2002).

TRPV3 is considered as a warm sensor because ofits activation threshold (around 31oC) (Peier et al.,2002b; Smith et al., 2002; Xu et al., 2002). A remark-able feature of TRPV3 is its expression; it is highlyabundant in keratinocytes (Peier et al., 2002b; Smithet al., 2002; Xu et al., 2002; Chung et al., 2004) butalmost undetectable in rodent DRG neurons (Peier etal., 2002b; Xu et al., 2002). Interestingly, Trpv3−/− micedisplay a deficit not only in sensing warm tempera-tures but also noxious ones (Moqrich et al., 2005). Con-verse to the TRPV1−/− mice, no impairment of thermalor mechanical hyperalgesia could be observed. Due tothis phenotype and the absence of TRPV3 in DRGterminals, it has been suggested that keratinocytesprimarily detect warm and, at least partially, noxioustemperatures (Moqrich et al., 2005). In agreementwith this finding, a recent report showed that ATP-release from activated keratinocytes could transmittemperature information to sensory neurons (Mandadiet al., 2009). Further confirmation of this finding byusing conditional deletion of TRPV3 in kertainocyteswill lend further supports to the proposed cooperationbetween skin cells and sensory nerve endings.

Although initially described as an osmolarity detect-or (Liedtke et al., 2000), TRPV4 is also gated by warmtemperatures (24-34oC) (Watanabe et al., 2002; Chunget al., 2003) and various chemicals (Gao et al., 2003;Vriens et al., 2004). Data obtained from Trpv4−/− micesupports the contention that this channel might beinvolved in thermal and mechanical nociception, mainlyin pathological conditions. Similar to TRPV3, the siteof TRPV4 action (keratinocytes or primary sensoryneurons) remains debatable (Alessandri-Haber et al.,2003; Chung et al., 2003, 2004; Mizuno et al., 2003).

TRPA1TRPA1 was initially described as a cold sensing

receptor (<17oC) (Story et al., 2003). From this originaldescription, a significant controversy has been gener-ated around the physiological stimuli activatingTRPA1. Few reports suggest that TRPA1 is gated bycold temperatures (Story et al., 2003; Bandell et al.,2004; Karashima et al., 2009) and mechanical stimuli(Kwan et al., 2009). These results, however, were not

replicated by other researchers (Jordt et al., 2004;Bautista et al., 2006). The generation of two independ-ent TRPA1−/− mice did not resolve this issue (Bautistaet al., 2006; Kwan et al., 2006). Most probably, thepresence of other cold activated channels (i.e. TRPM8)might mask an overt cold phenotype in the TRPA1−/−

animals.It is well known that TRPA1 acts as a chemical

sensor. TRPA1 binds to a large number of pungent orirritating chemicals containing reactive electrophilessuch as allyl isothiocyanate (mustard oil), cinnamal-gehyde (cinnamon), acolein (gas exhaust), allicin (gar-lic) and formaldehyde (formalin) (Patapoutian et al.,2009; Stucky et al., 2009). It is also generally acceptedthat several inflammatory mediators such as brady-kinin activate TRPA1 (Bandell et al., 2004; Wang et al.,2008). Moreover, TRPA1−/− animals exhibit a markeddecrease in thermal and mechanical hyperalgesiafollowing bradykinin injection (Bautista et al., 2006;Kwan et al., 2006). Thus, much like TRPV1, TRPA1seems to be a multi-functional nocitransducer mostlyinvolved in inflammatory pain. In agreement with this,TRPV1 and TRPA1 are expressed in largely overlap-ping subsets of nociceptors (Bautista et al., 2005;Kobayashi et al., 2005).

TRPA1 is the only member of the TRP family withan extended ankryin repeat domain in the N-terminus(Clapham, 2003). This is not the only remarkable fea-ture of this channel. Interestingly, activation of TRPA1by these reactive groups is particularly unusual. In-stead of transiently binding to a precise domain of thechannels, electrophiles covalently react with free cys-teine residues of the TRPA1 (Macpherson et al., 2007).It has been reported that covalent binding is physiolo-gically relevant for TRPA1 activation.

TRPM8The last identified TRP channel, TRPM8 is reported

to be involved in perception of cold sensation. TRPM8detects cooling temperatures over a broad range. Whenexpressed on a heterologous cell system, temperatures<24-28oC start to evoke membrane currents, and thecurrents reach maximum near 10oC (McKemy et al.,2002; Peier et al., 2002a). In addition, TRPM8 is acti-vated by cooling compounds such as menthol (McKemyet al., 2002; Peier et al., 2002a). The analysis ofTRPM8−/− mice has clearly established its role in sens-ing innocuous cool temperatures (Bautista et al., 2007;Colburn et al., 2007; Dhaka et al., 2007). However,noxious cold responses are only partially affected inmutant animals suggesting that other channels mightcompensate for lack of TRPM8. To entertain the con-troversy about TRPA1 cold temperature sensitivity, a

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recent work just showed that mice lacking both TRPA1and TRPM8 responded to noxious cold stimuli simi-larly to mice lacking TRPM8 alone (Knowlton et al.,2010). Absence of additive effect due to loss of bothTRPA1 and TRPM8 indicates that noxious coldtransducers other than TRPA1 might exist.

Distribution of TRPM8 in primary sensory neuronshas long remained unknown. Recently, a TRPM8-GFPreporter line has been generated and it is now clearthat TRPM8 is mostly present in non-nociceptiveneurons and only a minority would be associated tonociceptive markers (Takashima et al., 2007; Dhakaet al., 2008). These findings nicely correlate to theclassical electrophysiological classification of low- (po-tentially TRPM8+) and high-threshold (mostly TRPM8−)cold sensitive fibers (Takashima et al., 2007; Dhaka etal., 2008) as well as to the phenotype observed inTRPM8−/− mice. A recent study in which most nocicep-tive DRG neurons were ablated in vivo clearly demon-strated that, in DRG deprived of nociceptive neurons,TRPM8 levels remained almost identical to wild typeDRG (Abrahamsen et al., 2008) confirming that mostTRPM8 is expressed outside of nociceptive neurons.Interestingly, a near complete loss of cold nocifensivebehavior was observed in these mice whereas the cool-ing responses to acetone was preserved (Abrahamsenet al., 2008).

Ion channels involved in pain transmissionAs in most neuronal types, voltage-gated sodium

channels (VGSCs) subserve the generation and pro-pagation of action potentials. VGSCs are closed atresting membrane potential. In response to depolari-zation, they cycle through activated, inactive and re-priming states (Dib-Hajj et al., 2009). Transient open-ing at the activated state allows a sodium flow gener-ating an inward current crucial for the upstroke of theaction potential. Most VGSCs rapidly inactivates andthen undergo conformational changes to restore re-sponsiveness.

VGSCs are heteromultimers of large α-subunits andsmall (auxiliary) β-subunits (Catterall, 2000). α-sub-units are sufficient to form a functional channel. β-subunits and other cytosolic partners of VGSCs modu-late trafficking of the channel and/or its biophysicalproperties (Catterall, 2000). At present, nine isoformsof α-subunits (Nav1.1-Nav1.9) are known. Althoughthey exhibit a similar overall structure, each subunithas different kinetics (cycling properties) and voltagethresholds. According to their responses to the tetro-dotoxin (TTX), VGSCs are divided into two categories;TTX-sensitive VGSCs are blocked by nanomolar con-centrations of TTX whereas TTX-resistant subunits re-

quire micromolar concentrations to be inhibited.At least five different VSGCs (TTX-sensitive Nav1.1,

Nav1.6 and Nav1.7 and TTX-resistant Nav1.8 and Nav1.9)are expressed in DRGs neurons. Nav1.1 is mainly foundin large neurons, Nav1.6 in medium to large neuronand Nav1.7, Nav1.8 and Nav1.9 are preferentially ex-pressed in small nociceptive neurons (Akopian et al.,1996; Black et al., 1996; Cummins et al., 1999; Dib-Hajjet al., 1999). Interestingly, individual DRG neuronscould express more than one subunit. More than du-plicating function, controlled expression of specificVGSC subtypes is necessary for a proper function ofperipheral pain processing (Rush et al., 2007).

It is now well established that transcriptional andpost-transcriptional control of VGSCs expression de-termines neuronal excitability. A regulatory sequence(referred as to RE-1) upstream of VGSCs could be re-cognized by a number of transcription factors (REST,NSRF etc.) (Kraner et al., 1992). These transcriptionfactors have an intrinsic repressive activity and blockVGSCs transcription, for example, in the glial cells.Interestingly, double stranded RNA molecules thathave this RE-1 sequence switch the activity of trans-cription factors from a repressor to an activator role.These regulatory RNA molecules are found in neuronalprecursors (Kuwabara et al., 2004). In addition, VGSCare subjected to alternative splicing. Two developmen-tal splice variants (embryonic and adult) presentingspecific biophysical properties have been described forNav1.3 and Nav1.7 (Chatelier et al., 2008). DRG neuronsexpress an unique repertoire of splice variants (Dietrichet al., 1998; Raymond et al., 2004). More importantly,splicing has been shown to be modified in response toNGF (Akopian et al., 1999) opening the intriguingpossibility that mediators released after tissue injurycould also affect nociceptive excitability.

Consistent with its role in pain transmission, a num-ber of human mutations affecting VGSCs expressed inDRG nociceptors have been associated to pain syn-dromes. Mutations in the Nav1.7 gene resulting in anon-functional channel lead to the complete absence ofpain confirming the importance of Nav1.7 for humanpain perception (Cox et al., 2006; Goldberg et al.,2007). Moreover, a number of other mutations leadingto an abnormally active protein (shift of the voltage-dependency to more negative potentials, impairedinactivation etc) have been linked to erythermalgia(Drenth et al., 2005) and paroxysmal extreme paindisorder (Cummins et al., 2004), two clinical conditionscharacterized by violent pain attacks. A recent report(Choi et al., 2010) described a new mutation affectingthe function of the adult but not the embryonic spliceisoform Nav1.7. Patients carrying such a mutation

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exhibit a much later onset of erythermalgia con-firming that relative expression of splice isoforms cancontribute to time-dependent manifestation of thefunctional phenotype. Mice deficient for Nav1.7 alsoexhibited a severe disruption (or abolishment) inacute and inflammatory pain confirming a major roleof this subunit in pain perception (Nassar et al., 2004).

Recent work uncovered important clues in Nav1.8functions. Expression of this channel seems to berestricted to nociceptive neurons in the DRG andtrigeminal ganglia (Zimmermann et al., 2007). Nav1.8exhibit a number of remarkable electrical properties(Renganathan et al., 2001): i) rapid recovery from in-activation; ii) more depolarized potentials for activa-tion; iii) unlike other VGSCs, it maintains its activityat cold temperatures (Zimmermann et al., 2007). Inagreement with these data, responses to noxious coldstimuli (cold plate 0oC) were severely reduced inNav1.8−/− mice (Zimmermann et al., 2007). Mutantanimals also exhibit a decreased pain-related behaviorunder inflammatory conditions (Akopian et al., 1996;Kerr et al., 2001). Upregulation of expression (Tanakaet al., 1998) and sensitization (Gold et al., 1996) mightaccount for the observed effects of Nav1.8 in inflam-mation.

The role of other VGSC in pain is poorly understood.Nav1.9 is expressed in nociceptive neurons. Inflam-matory mediators seem to potentiate Nav1.9-mediatedcurrents and therefore increase nociceptor excitability(Rush and Waxman, 2004; Maingret et al., 2008).However, Nav1.9−/− mice display only minor phenoty-pic defects (Leo et al., 2010). Finally, Nav1.1 is widelyexpressed in the peripheral and central nervous sys-tem. Most human mutations are associated with epi-lepsy and only some link to an autosomal dominantform of migraine (Dichgans et al., 2005). Its functionin sensory neurons remains to be identified.

OTHER PROTEINS INVOLVED IN NOCI-CEPTION

We will briefly mention here a number of otherproteins (receptors and ion channels) that have beeninvolved in the nociception

Voltage-gated calcium channels (VGCCs)Pharmacological experiments have provided evi-

dence that VGCCs participate in pain perception. Block-ers of L-type VGCCs (i.e. nifedipine or verapamil) areable to reduce mechanical and thermal pain (Todorovicet al., 2004) as well as the inflammatory-dependentrelease of substance P (Vedder and Otten, 1991). Simi-larly, intravenous injection of mibefradil, an antagonist

of T-type VGCCs, induces analgesia (Todorovic et al.,2001). In this line, knockout mice lacking T-type chan-nels showed an exaggerated response to visceral pain.Finally, the role of N-type VGCCs remains controver-sial as contradictory data have been obtained withtwo different knockout strains (Hatakeyama et al.,2001; Kim et al., 2001).

Acid sensing ion channels (ASICs) Acid Sensing Ion Channels (ASICs) are a family of

protons activated channels principally expressed inneurons. Four genes encoding seven subunits (ASIC1a,ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3 and ASIC4)have been identified so far in mammals (Gitterman etal., 2005). Expression of ASIC1b and ASIC3 is restri-cted to peripheral sensory neurons (Chen et al., 1998;Bassler et al., 2001). Because they are activated by ex-tracellular acid (one of the major noxious stimuli),ASICs has been proposed to participate in pain per-ception. In this line, Deval et al. provided compellingevidence that ASIC3 mediates in vivo detection ofacidic pain and contributes to inflammatory hyperal-gesia in the rat (Deval et al., 2008). However, data ob-tained from ASIC knockout mice have failed to dem-onstrate a clear function of these channels in acidic orprimary inflammatory pain (Chen et al., 2002).

Potassium channelsK+ channels display tremendous diversity in terms

of structure and function. It is thought that this diver-sity accounts for a variety of functions in differentneuronal types (Trimmer and Rhodes, 2004). Severaltypes of K+ channels are present in sensory neuronsbut their role in pain perception is largely unknown.Nonetheless, recent work suggested that K+ channelsplay important roles in nociceptors. Thus, it has beenshown that potassium channels of the TREK/TRAAKfamily control pain produced by mechanical stimula-tion and both heat and cold pain perception in mice(Noel et al., 2009). In this line, it has been reportedthat K+ channels critically modulate activation thresh-old in trigeminal cold sensitive neurons (Madrid et al.,2009). Finally, Liu et al. reported that Kv7.2/Kv7.3channels are also involved in bradykinin-evoked pain(Liu et al., 2010). Clearly, further work in the comingyears is required to precisely define the functions andisoforms of K+ channels in pain detection.

Other channelsResearch is in progress aimed at identifying novel

ion channels involved in nociception potentially usefulas targets for drug design. Chloride channels (Liu etal., 2010), serotoninergic ionotropic receptors 5-HT3R

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(McCleane et al., 2003) or aquoporin1 (Zhang andVerkman, 2010) are only some examples of potentialattractive candidates.

BUILDING UP NOCICEPTORS FUNCTIO-NAL HETEROGENEITY

As stated in the introduction, it is believed that noci-ceptors functional diversity reflects the wide spectrumof existing noxious stimuli. Studies conducted in thelast decade have shed much light into the molecularmechanisms involved in the transduction/transmis-sion of different modalities of noxious stimuli. Thus,TRPV1 is present in most heat-sensitive nociceptorsand Nav1.8 is required for transmission of pain in-formation at cold temperatures. One salient feature ofthose ion channels participating in pain transductionis their versatility; most of them are activated by anumber of different stimuli (i.e. TRPV1 is opened inresponse to heat or capsaicin) and are therefore consi-dered as polymodal. In this context, TRPA1 is theparadigm of flexibility; a single mechanism (covalentmodification of cysteine residues) allows the channelto detect the presence of a varied palette of unrelatedchemicals. Multimodality of ion channels might beimportant in terms of economy by limiting the numberof transducing designs necessary for the detection of apotentially unlimited number of different insults.

Identification of such molecules is just the first stepin understanding the existing functional diversity ofnociceptive neurons. Nociceptor function is believed tobe more complex than the simple summation of ionchannels. The challenge will be illustrated usingnoxious cold as a model.

As described in the previous section, a single TRPchannel (TRPM8) seems to largely mediate cold sensa-tion. Nonetheless, psychophysical studies conductedin humans indicate that cold temperature elicits abroad array of sensations ranging from freshness toovert pain (Chen et al., 1996; Belmonte et al., 2009).Cooling of the skin by just a few degrees can be sensedas a cold sensation. Cooling skin to 5-10oC can pro-duce a mixture of cold and painful prickle sensations.Finally, cold stimuli below 5oC typically produce painthat can have a prickly, burning or aching quality. Ina pioneering study, Madrid et al. proposed that theexcitatory effects of TRPM8 activation could be tunedby excitability brakes, namely outward K+ currents(Madrid et al., 2009). Indeed, it was demonstrated thatthe temperature threshold of activation in menthol-responding (TRPM8+) neurons depends on the presenceof thermosensitive leak or background K+ channels(Kv1.1 and Kv1.2 containing channels) that are closed

by cooling. These channels allow K+ outflow at mem-brane potentials that are subthreshold for action po-tential generation therefore decreasing neuronal ex-citability. Consequently, a reverse correlation betweenthese K+ currents and temperature threshold wasobserved in cold-sensitive neurons. In that context, ithas been proposed that innocuous cold temperaturesare transduced via cold-sensitive TRPM8+ neuronshaving low or no such K+ currents. Colder tempera-tures might allow the progressive recruitment of cold-sensitive units with increasing density of K+ currents.Noxious cold might activate specific high-thresholdcold neurons. However, electrophysiological data indi-cate that the picture is still too simplistic. These speci-fic high-threshold cold neurons probably underlie thecommon sensation of cold pain accompanying suddentemperature changes (Mauderli et al., 2003). This po-pulation seems to be functionally distinct from conven-tional C-type polymodal nociceptive neurons that ap-pear to require more prolonged and/or intense coolingfor activation (Wahren et al., 1989). In addition tomenthol, polymodal nociceptors also express TRPV1and therefore respond to capsaicin. It has been postu-lated that burning perception associated to noxiouscold might derive from indirect TRPV1 activation inthese nociceptive units (Belmonte et al., 2009). A recentreport compared the electrophysiological properties oftwo populations of TRPM8 cold-sensitive neurons:those insensitive to capsaicin (TRPV1−) and those re-sponding to it (TRPV1+) (Xing et al., 2006). Interestingly,only one major difference was found between thoseneuronal subsets. A large majority of TRPM8+ TRPV1-

neurons (>90%) were inhibited by TTX whereas morethan 60% of TRPM8+ TRPV1+ neurons were insensi-tive to TTX blocking. Since the only Na channel knownto remain functional at low temperatures (Nav1.8) isTTX-resistant, these observations together suggest anincreasingly complex scenario for cold pain transduc-tion. Noxious cooling might be initially perceived ascold discomfort through specific high-threshold coldneurons (TRPM8+ TRPV1− Nav1.8−). These insults donot decrease skin temperature enough to inactivateNa channels. More intense cold stimuli (but under thethreshold of Na channel inactivation) might recruitpolymodal nociceptors (TRPM8+ TRPV1+ Nav1.8−) andmight be perceived as a different quality (i.e. prickl-ing). Finally, those inputs whose intensity and/or dur-ation results in Na channel inhibition are exclusivelytransmitted via Nav1.8 containing nociceptors. Thefinal perception (burning, prickling, aching etc.) mightbe dependent on the number and type of nociceptorsactivated (TRPM8+ TRPV1− Nav1.8+ or TRPM8+ TRPV1+

Nav1.8+). In conclusion, the mixed sensation triggered

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by cold stimuli is likely due to contributions from func-tionally different cold-sensitive primary afferents allexpressing a common cold transducer (TRPM8).

Intensity and the exposure time of the insult areinversely-related prime determinants of injury. Thus,the higher the intensity shorter the exposure timerequired for a given amount of damage. In order to pre-vent damage, nociceptive system needs to be capableof detecting transient and very intense insults as wellas sustained and, somehow, less severe inputs. A veryrecent published report provided new insights into themechanisms used by nociceptors to integrate temporalinformation about the stimulus (Plaghki et al., 2010).It has long been appreciated that painful informationis transmitted through unmyelinated C-fibers andthinly myelinated Ad-fibers. Given the different con-duction velocities of these fibers, Plaghki et al. exam-ined the involvement of distinct subset of nociceptorsfor the detection of the same stimulus presented underdifferent timescales (transient versus sustained). Todemonstrate this, they studied noxious heat responsesin humans using the benefits of Laser technology. Byincreasing laser power, the investigators were able toreach a defined skin temperature (monitored using aninfrared camera) in a shorter time. The working hypo-thesis was rather simple. If all nociceptive neurons(independently of the myelination level) have the sametemperature threshold, the latency of nocifensive re-sponses will be independent of the application timeand will reflect activation of the Ad fibers (those trans-mitting information at a faster speed). Conversely, ifthe threshold is different in those two populations, thelatency will vary according to the exposure time. Re-cording a number of healthy individuals clearly dem-onstrated that different latencies exist and that C-fibers threshold is about 3-4oC lower than that of Adfibers. These results demonstrate that if the physicalaggression is rapid, the system is much faster to reactbut require stronger stimuli. In contrast, slowly-actinginsults leading to a gradual warming-up triggered muchslower reactions but at relatively lower temperatures.Whether these different thresholds for noxious heatrely, as for cold perception, on other channels limitingneuronal excitability remains to be explored.

To sum up, these findings are compatible with a scen-ario in which the great heterogeneity of nociceptiveneurons is built up by a combinatorial approach. Inthe first level of this hierarchical organization, combi-nations of a limited number of molecular transducers(most belonging to TRP channels family) determinethe basic modalities to which these neurons mightrespond. In the next level, excitability modulators (i.e.K+ channels) subtly sculpts the responsiveness of sens-

ory units capable of reacting to the same stimulusfurther expanding their functional diversity. Finally,other molecular and anatomical features (i.e. thedegree of myelination) might provide another level offunctional heterogeneity. Combination at all levels islikely to generate a myriad of functionally distinctnociceptive neurons. It is noteworthy to highlight thattiny differences in the molecular composition of noci-ceptors might account for the quantitative and quali-tative differences in pain perception observed amongindividuals. This combinatorial model also illustratesthat translation from molecules to function (in termsof electrical activity) and from function to perceptionis a challenging task.

NOCICEPTORS DIVERSITY IN PATHOL-OGY

In pathological conditions, nociceptive system un-dergoes a number of plastic changes leading to decre-ased pain thresholds (allodynia and hyperalgesia) orspontaneous pain sensation (pain in the absence ofsensory inputs). These changes mostly operate on pri-mary nociceptors though central mechanisms are alsoimplicated (Latremoliere and Woolf, 2009). Pathologi-cal conditions have been grouped into two large cat-egories depending on the mechanism of action. Inflam-matory pain encompass all those conditions resultingin tissue damage and inflammation (i.e. burning, mech-anical traumatism, exposure to toxic chemicals etc.)whereas neuropathic pain refers to those pathologicalsituations primarily affecting nociceptive neurons (i.e.diabetic neuropathy, axotomy etc.).

Inflammatory painNociceptive terminals are equipped with a broad

palette of ‘inflammatory receptors’ aiming to detectmolecular mediators released after tissue injury andthe secondary inflammatory reaction. Upon activa-tion, these receptors elicit an increase of nociceptorexcitability and, consequently, lower pain thresholds.Enhanced nociceptive responses after tissue damage/inflammation play a physiological role in limiting fur-ther injury of the affected area as well as in promotinghealing.

Many inflammatory mediators such as histamine,bradykinin, ATP, NGF or prostaglandins have beenknown to alter pain threshold resulting in allodyniaand hyperalgesia. Three basic mechanisms have beenproposed to rapidly increase nociceptor excitability: i)sensitization of nociceptive ion channels transducers(i.e. TRPV1); ii) switch of resting membrane potentialto more depolarized potentials (i.e. closing K+ channels);

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and iii) modification of the properties of action poten-tial generators (i.e. inhibition of fast inactivation ofNa+ channels). Using bradykinin as an example, wewill illustrate the molecular complexity of that pro-cess. Bradykinin is one of the most potent algogenicsubstances found in nature. Injection of bradykininhas been shown to elicit nocifensive responses in vivo(Mizumura et al., 2009). About 40-60% of cutaneousreceptors respond to bradykinin in vivo (Khan et al.,1992) and these responses are largely mediated throughG-protein coupled receptor BK2 (Boyce et al., 1996;Pesquero et al., 2000). Bradykinin is known to act atdifferent levels. It induces sensitization of TRPV1(Chuang et al., 2001) and TRPA1 (Wang et al., 2008)increasing their probability of activation. It could alsopotentiate Nav1.9 currents generating subthresholdamplification and increased excitability (Maingret etal., 2008). Finally, it could alter the ‘voltage clamp’ con-trolling resting membrane potential and threshold foraction potential generation by the simultaneous inhi-bition of K+ channels (Kv7) and activation of calcium-dependent Cl− channels (Liu et al., 2010).

Although the final cellular effectors are largelyshared, it has been shown that different inflammatoryagents might act preferentially on precise targets(Linley et al., 2010). Thus, NGF drives TRPV1 upre-gulation (Ji et al., 2002) and sensitization (Zhu andOxford, 2007) whereas bradykinin mainly alters the‘voltage clamp’ (Linley et al., 2008; Liu et al., 2010).Operating at different levels might allow inflamma-tory mediators to synergize and ensure that subse-quent sensitization of the system even in cases oflimited damage. Indeed, it has been shown that someresponses could only be evoked when several factorsare applied conjointly (inflammatory soup) (Maingretet al., 2008). In addition, one could imagine that thispartial target specificity might be important quantita-tively (more the tissue damage, more the inflammatorymolecules released, more the nociceptors recruited)and qualitatively (different kind of lesion, differentcomposition of the inflammatory soup, impact on dif-ferent subsets of nociceptors). Finally, it should beemphasized that molecular heterogeneity among noci-ceptors and subtle differences between individualsmight probably explain the different clinical outcomeafter similar inflammatory insults.

In most cases, after tissue repairing, inflammatoryreaction ceases and nociceptive threshold returns tobasal levels. However, under some circumstances, painpersists in the absence of injury/inflammation. Mecha-nisms underlying this spontaneous inflammatory painare not well understood. Most experimental evidencepoint to K+ channels and their control of resting mem-

brane potential (Linley et al., 2008). Finally, if tissuecould not be repaired ad integrum, inflammation failsto stop or noxious agent remains active, inflammatorypain is perpetuated and becomes chronic. It is believ-ed that chronic insults alter gene expression profile ofprimary nociceptive neurons. Thus, Nav1.3, Nav1.7,Nav1.8 and TRPV1 are upregulated for extendedperiods of time following persistent inflammation(Gould et al., 1998, 1999; Strickland et al., 2008) andmight account for gauging the duration of pain underthese conditions.

Neuropathic painNeuropathic pain mostly arises from injury or dise-

ase of peripheral neurons. Unlike inflammatory pain,underlying molecular mechanisms of neuropathic painremains poorly understood (Harriott and Gold, 2009).One of the main reasons might be the heterogeneity ofetiological origins. Thus, neuropathic pain could de-velop following traumatic insults (i.e. nerve transec-tion), metabolic disorders (i.e. diabetes), infections (i.e.HIV neuropathy), and exposure to toxic chemicals (i.e.chemotherapy) or immune diseases (i.e. multiple scler-osis). In addition, other distortion factors such as theexposure time or the site of injury might enhance theheterogeneity in the clinical presentation and physio-pathology of neuropathic pain.

Evidence from clinical and animal experiments sug-gests that aberrant afferent activity is at the origin ofthe initiation and maintenance of neuropathic pain.Central changes may influence the intensity and qual-ity but seem insufficient for the overt manifestation ofneuropathic pain (Dubner, 2004; Harriott and Gold,2009). It is currently assumed that spontaneous acti-vity in neuropathic pain reflects the ongoing activa-tion of a subset of nociceptive afferents and/or non-nociceptive neuron that switch their phenotype as aconsequence of the lesion (Harriott and Gold, 2009).The driving force underlying this activity is derivedfrom a combination of terminal sensitization (Campbelland Meyer, 2006), the upregulation of ion channelssuch as Nav1.3 (Waxman et al., 1994) or HCN channels(Jiang et al., 2008) and the increase of action potentialgeneration in the central projections of nociceptors(dorsal root reflex) (Willis, 1999). Due to injury-induc-ed changes in VGCCs (Li et al., 2006), this latter effectseem to facilitate neurotransmitter release at thespinal cord and greatly contribute to pain generation.Indeed, gabapentin-mediated analgesia in neuropathicpain models depends on its ability to modulate cal-cium channels at central terminals (Taylor, 2009).

A paucity in the amount of information about theorigin of neuropathic pain, the role of different subsets

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of nociceptors has not been explored. Interestingly,phenotypic switch of certain non-nociceptive neuronsresults in ectopic discharges and contributes to neuro-pathic pain (Devor, 2009). These findings highlightthe importance of a more universal analysis (includingall DRG neurons and even vegetative neurons) in orderto further understand the generation and perpetua-tion of neuropathic pain.

PERSPECTIVES AND CONCLUDING RE-MARKS

Although intensive work in the last years hasenabled a significant advance in the molecular under-standing of pain perception, there are still fundamentalquestions that remain unanswered. From a molecularpoint of view, it is necessary to identify ion channelsinvolved in other noxious systems (especially thosetransducing mechanical pain), to get a panoramicimage of the existing combinations of ion channelspresent in nociceptors and to define how interactionsbetween all these channels determine neuronal excit-ability. In addition, there is a need to expand our viewabout how restricted populations of nociceptors aregenerated during development and early post-natalweeks. Runx1 is the master regulator of this processbut it is likely that a refined network of transcriptionand environmental factors fine tune the diversifica-tion of downstream of Runx1. It is critical to under-stand the mechanism by which different populationsof neurons react to pathological conditions, how theyare modified and how these changes lead to patholo-gical pain. Indeed, a recent study suggests that, inDrosophila, hyperalgesia and allodynia might arisethrough independent genetic pathways opening theintriguing possibility that they might reflect the re-sponse of specific nociceptors subsets (Babcock andGalko, 2009). Finally, focused efforts are needed in thepharmacological field. Nociceptors have the potentialto become the pharmacological target of future pain-killers as they are the first contact elements of thepain pathway, they are located peripherally, and theyexhibit a broad palette of molecular targets for phar-macological blocking. These future therapies are likelyto be a la carte. Molecular understanding of pain per-ception would foster the generation of a wide range ofpharmacological inhibitors operating at different mo-lecular targets. One could expect that, in the near fu-ture, development of specific methodologies might alsoallow to easily test variations in nociceptive responsesand therefore to infer the specific nociceptive molecu-lar signature of each individual. This review (togetherwith genetic variations) integrates the current medical

data and would allow selecting the most appropriatedrug combination according to the nature of thepathological insult.

ACKNOWLEDGEMENTS

We thank S. Alonso and S. Gaillard for their valu-able comments on the manuscript. This work wassupported by grants from CNRS: the ATIP-programfor young investigators, the National Research Agency(ANR-Neuro2006) to Aziz Moqrich and the MedicalResearch Foundation (FRM) to Eduardo Gascon.

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