The Journal of Pain, Vol 10, No 9 (September), 2009: pp 895-926Abstract: Central sensitization represents an enhancement in the function of neurons and circuits innociceptive pathways caused by increases in membrane excitability and synaptic efficacy as well as to

reduced inhibition and is a manifestation of the remarkable plasticity of the somatosensory nervous

system in response to activity, inflammation, and neural injury. The net effect of central sensitization

is to recruit previously subthreshold synaptic inputs to nociceptive neurons, generating an increased

or augmented action potential output: a state of facilitation, potentiation, augmentation, or ampli-

fication. Central sensitization is responsible for many of the temporal, spatial, and threshold changes

in pain sensibility in acute and chronic clinical pain settings and exemplifies the fundamental contri-

bution of the central nervous system to the generation of pain hypersensitivity. Because central sen-

sitization results from changes in the properties of neurons in the central nervous system, the pain is

no longer coupled, as acute nociceptive pain is, to the presence, intensity, or duration of noxious

peripheral stimuli. Instead, central sensitization produces pain hypersensitivity by changing the sen-

sory response elicited by normal inputs, including those that usually evoke innocuous sensations.

Perspective: In this article, we review the major triggers that initiate and maintain central sensiti-zation in healthy individuals in response to nociceptor input and in patients with inflammatory and

neuropathic pain, emphasizing the fundamental contribution and multiple mechanisms of synaptic

plasticity caused by changes in the density, nature, and properties of ionotropic and metabotropic

glutamate receptors.

2009 by the American Pain SocietyKey words: Central sensitization, inflammatory pain, neuropathic pain, scaffolding protein, hetero-

synaptic facilitation.

Editors Note: This article is 1 in a series of invited CriticalReview articles designed to celebrate The Journal ofPains 10th year anniversary of publication.

Acute nociceptive pain is that physiological sensa-tion of hurt that results from the activation ofnociceptive pathways by peripheral stimuli of suf-

ficient intensity to lead to or to threaten tissue damage(noxious stimuli).374 Nociception, the detection of nox-ious stimuli,282 is a protective process that helps prevent

injury by generating both a reflex withdrawal from thestimulus and as a sensation so unpleasant that it resultsin complex behavioral strategies to avoid further contactwith such stimuli. An additional important phenomenonthat further enhances this protective function is the sen-sitization of the nociceptive system that occurs afterrepeated or particularly intense noxious stimuli, so thatthe threshold for its activation falls and responses to sub-sequent inputs are amplified.132,376,380 In the absence ofongoing tissue injury, this state of heightened sensitivityreturns over time to the normal baseline, wherehigh-intensity stimuli are again required to initiate noci-ceptive pain; the phenomenon is long lasting but notpermanent. The nociceptor-induced sensitization of thesomatosensory system is adaptive in that it makes the sys-tem hyperalert in conditions in which a risk of furtherdamage is high, for example, immediately after exposureto an intense or damaging stimulus. This sensitization is

Supported by the National Institutes of Health and the Fondation pour laRecherche Medicale.Address reprint requests to Dr Clifford J.Woolf, Neural Plasticity ResearchGroup, Department of Anesthesia and Critical Care, Massachusetts Gen-eral Hospital and Harvard Medical School, Charlestown, MA 02129.E-mail: cwoolf@partners.org

1526-5900/$36.00

2009 by the American Pain Societydoi:10.1016/j.jpain.2009.06.012

895Critical Review

Central Sensitization: A Generato

by Central Neural Plasticity

Alban Latremoliere and Clifford J. WoolfNeural Plasticity Research Group, Department of AnesthesHarvard Medical School, Charlestown, Massachusetts.r of Pain Hypersensitivity

ia and Critical Care, Massachusetts General Hospital and

Available online at www.sciencedirect.com

the expression of use-dependent synaptic plasticity trig-gered in the central nervous system (CNS) by thenociceptor input and was the first example of centralsensitization, discovered 26 years ago.369 Since then,we have learned that a number of different forms offunctional, chemical, and structural plasticity can sensi-tize the central nociceptive system to produce painhypersensitivity under both normal and pathological cir-cumstances, some of which are persistent.In many clinical syndromes, pain is no longer protec-

tive. The pain in these situations arises spontaneously,can be elicited by normally innocuous stimuli (allodynia),is exaggerated and prolonged in response to noxiousstimuli (hyperalgesia), and spreads beyond the site ofinjury (secondary hyperalgesia). Central sensitizationhas provided a mechanistic explanation for many ofthe temporal, spatial, and threshold changes in pain sen-sibility in acute and chronic clinical pain settings and hashighlighted the fundamental contribution of changes inthe CNS to the generation of abnormal pain sensitivity.Although phenomenologically central sensitizationmay appear to be comparable to peripheral sensitiza-tion, it differs substantially, both in terms of the mole-cular mechanisms responsible and its manifestation.Peripheral sensitization represents a reduction in thresh-old and an amplification in the responsiveness of noci-ceptors that occurs when the peripheral terminals ofthese high-threshold primary sensory neurons areexposed to inflammatory mediators and damaged tis-sue46,105,124,242 and, in consequence, is restricted to thesite of tissue injury.124 Although peripheral sensitizationcertainly contributes to the sensitization of the nocicep-tive system and thereby to inflammatory pain hypersen-sitivity at inflamed sites (primary hyperalgesia), itnevertheless represents a form of pain elicited by activa-tion of nociceptors, albeit one with a lower thresholddue to the increased peripheral transduction sensitivity,and generally requires ongoing peripheral pathologyfor its maintenance. Peripheral sensitization appears toplay a major role in altered heat but not mechanical sen-sitivity, which is a major feature of central sensitization.Central sensitization, in contrast to peripheral sensiti-

zation, co-opts novel inputs to nociceptive pathwaysincluding those that do not normally drive them, suchas large low-threshold mechanoreceptor myelinatedfibers to produce Ab fibermediated pain.376 It also pro-duces pain hypersensitivity in noninflamed tissue bychanging the sensory response elicited by normal inputsand increases pain sensitivity long after the initiatingcause may have disappeared and when no peripheralpathology may be present. Because central sensitizationresults from changes in the properties of neurons in theCNS, the pain is no longer coupled, as acute nociceptivepain is, to the presence, intensity, or duration of particu-lar peripheral stimuli. Instead, central sensitizationrepresents an abnormal state of responsiveness or in-creased gain of the nociceptive system. The pain iseffectively generated as a consequence of changeswithin the CNS that then alter how it responds to sensory

896 Central Sensitizationinputs, rather than reflecting the presence of peripheralnoxious stimuli. In this respect, central sensitization rep-resents a major functional shift in the somatosensory sys-tem from high-threshold nociception to low-thresholdpain hypersensitivity. We all experience pain as arisingfrom out there, and, in consequence, imagine that itis actually triggered by noxious stimuli where we feelthe pain. Central sensitization reveals, however, thatthis in many cases is a sensory illusion; specific alterationsin the CNS can result in painful sensations occurring inthe absence of either peripheral pathology or noxiousstimuli, and the target for treatment in these situationsmust be the CNS not the periphery.Central sensitization corresponds to an enhancement

in the functional status of neurons and circuits in noci-ceptive pathways throughout the neuraxis caused by in-creases in membrane excitability, synaptic efficacy, ora reduced inhibition. The net effect is that previouslysubthreshold synaptic inputs are recruited to generatean increased or augmented action potential output,a state of facilitation, potentiation, or amplification.The reason that these cellular changes alter the systemso profoundly is that normally only a small fraction ofthe synaptic inputs to dorsal horn neurons contributeto their action potential output.373 Nociceptive-specificneurons, for example, although dominated by largemonosynaptic and polysynaptic synaptic potentialsfrom nociceptors in their receptive field, typically alsohave small-amplitude synaptic inputs from low-thresh-old afferents and from nociceptor inputs outside theirreceptive fields, which constitute a subliminal fringethat normally does not drive the output of the cells(Fig 1). Recruiting these subthreshold inputs to the out-put of a neuron markedly alters its receptive field prop-erties, with profound changes in receptive fieldthreshold, spatial, and temporal properties (Fig 2). Thisprovides an opportunity for rapid functional plasticitythat can be revealed experimentally by increasing theexcitability of the neuron or by blocking inhibitory trans-mitters. After administration of GABA or glycine recep-tor antagonists, for example, Ab inputs are recruited toneurons in the superficial dorsal horn,17 and pain-likebehavior can be elicited by movement of just a fewhairs.289 The receptive field of somatosensory neuronsare, therefore, not fixed or hard wired, but are insteadhighly malleable. This malleability or plasticity is the sub-strate for the functional effects of central sensitization,and the means is a change in synaptic efficacy.When neurons in the dorsal horn spinal cord are sub-

ject to central sensitization, they exhibit some or all thefollowing: development of or increases in spontaneousactivity, a reduction in the threshold for activation byperipheral stimuli, increased responses to suprathresholdstimulation, and an enlargement of their receptive fields(Fig 2). Several features appear particular to central sen-sitization: conversion of nociceptive-specific neurons towide-dynamic neurons that now respond to both innoc-uous and noxious stimuli, progressive increases in theresponses elicited by a standard series of repeated innoc-uous stimuli (temporal windup), an expansion of thespatial extent of their input, and changes that outlast

: Generator of Pain Hypersensitivity by Central Neural Plasticityan initiating trigger.132,368,369,372,376 These electro-physiological changes correlate remarkably with the

lesion sites. Central sensitization may also play a funda-mental role in the abnormal and widespread pain sensi-tivity in patients with fibromyalgia.6,68,301-303 Given themajor role of central sensitization in the generation ofclinical pain hypersensitivity, it is essential that we under-stand the triggers and mechanisms responsible for theinduction and maintenance of the switch in the somato-sensory system from the physiological state, in which thesensory experiences evoked by low-intensity stimuli(innocuous sensations) and noxious stimuli (pain) arequite distinct and separate, to a dysfunctional hypersen-sitive system in which this discrimination is lost.

The Discovery of Central SensitizationThe first evidence for a central component to acute

pain hypersensitivity was provided in 1983.369 Electro-physiological recordings from single biceps femorisa-motoneuron axons were used to measure the outputof the nociceptive system, in this case the flexor reflexwithdrawal response elicited by noxious stimuli (Fig 3).These recordings revealed, as expected, that under nor-

First, electric stimulation of Ab sensory fibers began toelicit responses in themotor neurons after the condition-ing noxious heat stimuli, whereas these inputs elicited noresponse before. Second, a local anesthetic block of thesite of the peripheral injury did not result in collapse ofthe expanded receptive fields: The change was autono-mous once it was triggered by the peripheral input.Finally, the hypersensitivity produced by the noxiousheat could bemimicked in extent and duration by a brief20-second low-frequency electrical stimulation of thesural nerve only at C-fiber strength, which producedchanges lasting for tens of minutes. The interpretationof all these data was that noxious heat stimulation, byactivating C-fiber nociceptors, had induced a centralplasticity of the nociceptive system,whichwas thereaftercapable of responding to stimuli outside of the injuryarea and to low-threshold afferents that previously didnot activate the nociceptive system. This led to the artic-ulation of a more general hypothesis that brief trains ofnociceptor C-fiber input could trigger or conditiona long-lasting sensitization of the nociceptive system(an effect termed central sensitization) by producingdevelopment in human experimental subjects aftera noxious conditioning input of allodynia (particularlydynamic tactile or brush-evoked allodynia), the temporalsummation of repeated low-intensity stimuli from an in-nocuous sensation to pain, with afterpain on cessationof the stimulus, and widespread secondary hyperalgesia.These changes can be elicited in human volunteers bynoxious stimulation of the skin (as with topical or intra-dermal capsaicin or repeated heat stimuli340) and in thegastrointestinal tract by exposure to low pH solutions.272

Central sensitization contributes to neuropathic37 andinflammatory pain,26,274,395 migraine,35 and irritablebowel syndrome.253 In these patients, it is involved inproducing abnormal responsiveness to noxious andinnocuous stimuli and a spread of tenderness beyond

Figure 1. Subthreshold synaptic inputs. The substrate for receptivspecific rat dorsal horn neuron revealing subthreshold synaptic inperipheral sensory inputs that produce sufficiently large-amplitupotential discharge (A and B). This constitutes the receptive field ofield can evoke synaptic inputs that are too small normally to profringe or low-probability firing fringe, which can be recruited iffield. In this particular neuron, a standard pinch stimulus applieand B but clear subthreshold synaptic inputs at C. Modified from

Latremoliere and Woolfmal conditions there was no spontaneous activity inthe motor neurons and that their activation requireda noxious mechanical or thermal stimulus to the skin.These neurons had high-threshold nociceptive-specificreceptive fields restricted to the toes or hind paw, inkeeping with their activation only as part of the flexionwithdrawal reflex. After repeated peripheral noxiousheat stimuli sufficient to generate mild inflammationof the hind paw, however, an increased excitability ofthe motor neurons was detected that lasted for severalhours and included a reduction in threshold and enlarge-ment of the cutaneous receptive fields. The flexor motorneurons were now no longer nociceptive-specific butcould be activated by low-intensity (innocuous) periph-eral inputs such as light touch.369 Three experimentsshowed that this change in receptive field propertieswas due to alterations in the CNS and not the periphery.

e field plasticity. Intracellular in vivo recordings from a nociceptive-puts. The output of somatosensory neurons is determined by thosede monosynaptic and polysynaptic potentials to evoke an actionr firing zone of the neuron. However, stimuli outside the receptiveduce action potential outputs (C), and this constitutes a subliminalsynaptic efficacy is increased, to expand and change the receptived to points A, B, and C evoked only action potentials at points AReference 365.

897activity-dependent changes in the functional propertiesof neurons in the dorsal horn of the spinal cord and

that this contributed both to postinjury flexor reflex andpain hypersensitivity.Before the discovery of central sensitization, the recep-

tive field properties of dorsal horn neurons was thoughtto be fixed by the geometry of their dendrites relative tothe central terminals of sensory axons.33 Although plas-ticity of the receptive fields of dorsal horn neurons hadbeen shown to occur after peripheral nerve injury, this

was thought to be due to a loss of presynaptic inhibitionincreasing synaptic input from silent or ineffectivesynapses and not to plasticity in dorsal horn neurons.71

After the first demonstration of central sensitization inflexor motor neurons, essentially identical changeswere soon described in many studies in lamina I andV neurons in the dorsal horn of the spinalcord55,79,173,176,192,287,365,372 (Fig 3) as well as in spinal

Figure 2. Expansion of receptor fields during central sensitization. Recruiting subthreshold synaptic inputs to the output of a noci-ceptive-specific neuron can markedly alter its receptive field properties, producing changes in receptive field threshold and spatialextent. When neurons in the dorsal horn spinal cord are subject to activity-dependent central sensitization, they exhibit some orall the following: development of or increases in spontaneous activity, a reduction in threshold for activation by peripheral stimuli,increased responses to suprathreshold stimulation, and enlargement of their receptive fields. The examples in this figure of intracel-lular recordings of rat dorsal horn neurons show the cutaneous receptive fields before central sensitization (premustard oil) and afterthe induction of central sensitization (post mustard oil) and indicate how subthreshold nociceptive (pinch, top) and low-threshold(brush, bottom) inputs in the low-probability firing fringe (LPFF) are recruited by central sensitization. Central sensitization was pro-duced by topical application of mustard oil, which generates a brief burst of activity in TRPA1-expressing nociceptors and resulted inthe neural equivalent of secondary hyperalgesia (top) and tactile allodynia (bottom). Note that the mustard oil (conditioning input)was applied to a different area (in red) from the test pinch or brush inputs (in blue), so that the changes observed are due to hetero-synaptic facilitation. Modified from Reference 364.898 Central Sensitization: Generator of Pain Hypersensitivity by Central Neural Plasticity

nucleus pars caudalis (Sp5c),36 thalamus78 (Fig 3), amyg-dala,219,220 and anterior cingulate cortex.364 Morerecently, functional magnetic resonance imaging, posi-tron emission tomography, and magnetoencephalogra-phy have revealed in human subjects that several otherbrain structures implicated in pain (parabrachial nucleus,periaqueductal gray [PAG], superior colliculus, prefron-tal cortex) also exhibit changes compatible withincreases in excitability corresponding to central sensiti-zation187,209,212,244,283 (Fig 3).

Activity-Dependent Central SensitizationThe original description of central sensitization

referred to an activity- or use-dependent form of func-tional synaptic plasticity that resulted in pain hypersensi-tivity after an intense noxious stimulus. This plasticitywas triggered by the activity evoked in dorsal horn neu-rons by input from C-nociceptors, as after repeated heatstimuli above 49C,369 electrical stimulation of C-fibers(1 Hz for 10 to 20 seconds),358 and chemical activationof nociceptors by irritant compounds such as allyl isothio-cyanate (mustard oil)372 and formalin, which both actthrough the TRPA1 channel135,199 as well as capsaicin,which activates TRPV1 channels.158 To induce central sen-sitization, the noxious stimulus must be intense,repeated, and sustained. Input from many fibers isrequired over tens of seconds; a single stimulus, such asa pinch, is insufficient. Peripheral tissue injury is not nec-essary, although the degree of noxious stimulation that

Figure 3. Schematic representation of the structures exhibitingcentral sensitization. The first evidence for central sensitizationwas generated in 1983 by revealing injury-induced changes inthe cutaneous receptive field properties of flexormotor neuronsas an integrated measure of the functional plasticity in the spi-nal cord. A conditioning noxious stimulus resulted in long-last-ing reductions in the threshold and an expansion of thereceptive field of the motor neurons that was shown to be cen-trally generated. Essentially identical changes were thendescribed in lamina I and V neurons in the dorsal horn of the spi-nal cord (b) as well as in spinal nucleus pars caudalis (Sp5c), thal-amus (c), amygdala, and anterior cingulate cortex. Imagingtechniques have revealed several brain structures in human sub-jects that exhibit changes compatible with central sensitization(blue dots).

Latremoliere and Woolfproduces frank tissue injury almost always induces cen-tral sensitization, so that the phenomenon is veryprominent after post-traumatic or surgical injury. Inter-estingly, nociceptor afferents innervating muscles orjoints produce a longer-lasting central sensitizationthan those that innervate skin.358

Once the phenomenon had been shown to be robust,easily activated, and detected in both preclinical andhuman subjects, the issue then was what molecularmechanisms were responsible. The first major mechanis-tic insight was that the induction and maintenance ofacute activity-dependent central sensitization wasdependent on NMDA receptors,379 revealing a key in-volvement of glutamate and its receptors. We nowappreciate from 2 decades of investigation by manylabs that central sensitization comprises 2 temporalphases, each with specific mechanisms. The early phos-phorylation-dependent and transcription-independentphase results mainly from rapid changes in glutamate re-ceptor and ion channel properties.376 The later, longer-lasting, transcription-dependent phase drives synthesisof the new proteins responsible for the longer-lastingform of central sensitization observed in several patho-logical conditions.376 We will review the current under-standing of these mechanisms.

Triggers of Activity-Dependent CentralSensitizationGlutamate, the fast transmitter of primary afferent

neurons, binds to several receptors on postsynapticneurons in the dorsal horn of spinal cord, including ion-otropic amino-3-hydroxy-5-methyl-4-isoxazole propio-nate (AMPA), N-methyl-D-Aspartate (NMDA), andKainate (KA) receptors and several metabotropic (G-pro-tein coupled) glutamate receptor subtypes (mGluR). Inthe superficial laminae of the dorsal horn, AMPAR andNMDAR are present in virtually every synapse and are ar-ranged in a mosaic-like manner, whereas mGluRs sit atthe extremities of the postsynaptic density zone(PSD).10,11,15,247At the subunit level, NMDAR is a tetramerthat contains 2 low-affinity glycine-binding NR1 subunitsand 2 subunits from the 6 different NR2A-D or NR3A/B sub-units.305 The most common NMDA complexes in the dor-sal horn are composed of NR1-NR2A/B subunits.

202,215,243

AMPAR is also a tetramer, and its most abundant sub-units in the dorsal horn of the spinal cord are the calcium(Ca21)-permeable subunits GluR1 and GluR3 and thenonCa21-permeable GluR2 subunit.252 In basal condi-tions, inhibitory interneurons appear to preferentiallyexpress GluR1, whereas the excitatory neurons appearto express mostly GluR2.145,338 The AMPAR complex canalso be a GluR1/GluR2 heteromer,11 in which case the re-ceptor displays mainly GluR2 properties.233 A subpopula-tion of lamina I neurons lacking the NK1 receptor andexpressing the GluR4 (Ca21-permeable) subunit hasbeen identified.248 ThemGluR family is composed of 8 re-ceptors that form 3 groups, based on their sequence sim-ilarities and their coupling with specific Ga-proteins.54

Group I mGluRs (mGluR1 and 5) are coupled with Gaq-proteins (whose activation causes an increase of

899

[Ca21]i), whereas group II (mGluR 2 and 3) and group III(mGluR4, 6, 7 and 8) are coupled with Gai/o-proteins.All mGluRs except for mGluR6 and 8 are expressed inthe spinal cord,346 whereas only mGluR6 appears not tobe expressed by primary afferent neurons.39 In addition,a lamina-specific pattern of expression has been charac-terized for mGluR1a (lamina V), mGluR5 (laminasI-II),10,247 and mGluR2/3 (lamina II inner),12 suggestingprecise and distinct physiological roles for the differentsubtypes.Activation of NMDAR is an essential step in both ini-

tiating and maintaining activity-dependent central sen-sitization as its blockade by noncompetitive (MK801) orcompetitive (D-CPP) NMDAR antagonists prevent andreverse the hyperexcitability of nociceptive neuronsinduced by nociceptor conditioning inputs184,379 andconditional deletion of NR1 abolishes NMDA synapticinputs and acute activity-dependent central sensitiza-tion.300 NMDAR is both a trigger and effector of centralsensitization. Under normal conditions, the NMDARchannel is blocked in a voltage-dependent manner bya magnesium (Mg21) ion sitting in the receptorpore.198 Sustained release by nociceptors of glutamateand the neuropeptides substance P and CGRP leads tosufficient membrane depolarization to force Mg21 toleave the NMDAR pore, whereupon glutamate bindingto the receptor generates an inward current.198 Re-moval of this voltage-dependent block is a major mech-anism for rapidly boosting synaptic efficacy and allowsentry of Ca21 into the neuron, which then activatesnumerous intracellular pathways that then contributeto the maintenance of central sensitization. In additionto the critical role of NMDAR in increasing the excitabil-ity nociceptive neurons, activation of group I mGluRs byglutamate also appear important for the developmentof central sensitization. Although these receptors donot participate to basal nociception,221,392 their act-ivation is necessary for activity-dependent central sen-sitization mediated by C-fibers.14,67,165,296,392,393 Incontrast, activation of group II-III mGluRs isassociated with a reduction of capsaicin-induced centralsensitization.296

Substance P (SP), which is co-released with glutamateby unmyelinated peptidergic nociceptors, is alsoinvolved in the generation of central sensitiza-tion.8,146,183,192,365 Substance P binds to the neurokinin-1 (NK1) G-proteincoupled receptor, which is expressedby spinothalamic, spinoparabrachial, and spino-PAGneurons101 and causes a long-lastingmembrane depolar-ization,112 and contributes to the temporal summationof C-fiberevoked synaptic potentials80,388,389 as well asto intracellular signaling. Ablation of NK1-positive neu-rons in the spinal cord leads to a reduction in capsaicin-evoked central sensitization, confirming the importanceof projecting neurons expressing the substance P recep-tor in this phenomenon.146,192 Calcitonin gene-relatedpeptide (CGRP), also synthesized by small diameter sen-sory neurons, potentiates the effects of SP367 and partic-ipates in central sensitization through postsynaptic

900 Central SensitizatioCGRP1 receptors, which activate PKA and PKC.307,308

CGRP also enhances release of brain-derived neurotro-phic factor (BDNF) from trigeminal nociceptors,34 whichmay contribute to its involvement in migraine and otherprimary headaches.82,103

BDNF is a neurotrophic factor and synaptic modulatorthat is synthesized by nociceptor neurons and releasedinto the spinal cord404 in an activity-dependentmanner,19

where it also has a role in the production of central sensi-tization.113,144,330 On binding to its high-affinity trkBreceptor, BDNF enhances NMDAR-mediated C-fiberevoked responses144 and causes activation of severalsignaling pathways in spinothalamic track neurons, in-cluding ERK141,245,292 and PKC.293

The inflammatory kinin bradykinin is produced in thespinal cord in response to intense peripheral noxiousstimuli and acts through its Gq-coupled B2 receptor,which is expressed by dorsal horn neurons41,359 andboosts synaptic strength by activating PKC, PKA, andERK.152 ERK is also activated by a serotoninergic (5-HT)descending input involving the ionotropic 5-HT3 recep-tor143,310,313,396 and possibly the 5-HT7 GS-coupled recep-tor.29 Fig 4 summarizes the key known synaptic triggersof central sensitization.

Signaling Pathways and Activity-Dependent Central SensitizationAn increase in intracellular Ca21 beyond a certain

threshold level appears to be the key trigger for initiat-ing activity-dependent central sensitization. Calcium in-flux through NMDAR appears to be particularlyprominent in the induction phase but can also occurthrough calcium-permeable AMPARs,159,355 voltage-gated calcium channels,52,376 as well as from releasefrom intracellular microsomal stores in response to acti-vation of several metabotropic receptors106,182 (Fig 5, Athrough C). Why is the calcium-induced activation of in-tracellular kinases so important? The reason is that iono-tropic NMDA and AMPA glutamate receptors can bephosphorylated on several key residues located on theirC-terminus,40,42 and this post-translational modificationchanges their activity as well as their trafficking to orfrom the membrane,40,161 which with similar post-trans-lational changes in other ion channels, produces thefunctional changes that manifest as central sensitization(Fig 6, A through C). AMPAR subunit GluR1 residueSer831 is phosphorylated by protein kinase C (PKC) andcalcium-calmodulin-dependent protein kinase II (CaM-KII); Ser845 is the phosphorylation target of PKA, andGluR2 has 1 main site for phosphorylation by PKC, onSer880.40 For the NR1 subunit of NMDAR, PKC phosphor-ylates Ser896, whereas PKA has 2 potential phospho-rylation sites on Ser890 and Ser897.168,333 NMDARconductance properties are modified by phosphoryla-tion of tyrosine residues located on the NR2A andNR2B subunits at 3 potential sites (Tyr1292, 1325, or1387 for NR2A and Tyr1252, 1336, or 1472 for NR2B),through activation of nonreceptor tyrosine kinasessuch as Src or Fyn.42,106,107,268

n: Generator of Pain Hypersensitivity by Central Neural PlasticityStimulation of AMPAR and group I mGluRs89,106,118,281

participate with NMDAR in the activation of the

intracellular pathways that sustain central sensitization.These include the PLC/PKC pathway, through openingof Ca21 channels on the endoplasmic reticulum,85,391

the phosphatidylinositol-3-kinase (PI3 K) pathway,246

and the mitogen-activated protein kinase (MAPK) path-way that involves the extracellular signal-regulated ki-nases (ERK1 and ERK2), which are 44- and 42-kDa Ser/Thr kinases, respectively, with 90% sequence identity,and the cAMP response element binding protein(CREB).130,138,141,357,363 One way that ERK and CREB are

activated is throughanelevation in intracellular Ca21 suf-ficient to drive a calmodulin-induced stimulation of ad-enylyl cyclases 1 and 8, whose cAMP production in turnactivates PKA and subsequent cascade(s).363

The activation of ERK by phosphorylation is regulatedby a core signaling module that consists of an apicalMAPK kinase kinase (MAP3 K), a MAPK kinase (MEK or

Figure 4. Central sensitization triggers: Schematic representa-tion of key synaptic triggers of central sensitization. (A), Modelof the synapse between the central terminal of a nociceptor anda lamina I neuron under control, basal conditions. mGluR recep-tors sit at the extremities of the synapse and are linked to theendoplasmic reticulum (ER). Note that NMDAR channels areblocked by Mg21 in the pore (black dot). After a barrage ofactivity in the nociceptor (B), the primary afferent presynapticterminal releases glutamate that binds to AMPAR, NMDAR(now without Mg21), and mGluR, as well as substance P, CGRP,and BDNF, which bind to NK1, CGRP1, and TrkB receptors,respectively. B2 receptors are also activated by spinally producedbradykinin. NO is produced by several cell types in the spinalcord and can act presynaptically and postsynaptically.

Figure 5. Sources of Ca21 in the synapse of nociceptive neuronsfor inducing central sensitization. (A), Model of a nociceptordorsal horn neuron synapse under control, nonactivated condi-tions. After nociceptor input (B), activation of NMDAR andmGluR result in a rapid increase of [Ca21]i that activates PKCand CaMKII, 2 major effectors of central sensitization. (C), Rep-resentation of a synapse during peripheral inflammation-in-duced central sensitization, where there is a shift from GluR2/3to GluR1-containing AMPARs that enables, along with volt-age-dependent calcium channels andNMDAR, entry of Ca21andwhich, together with activation of the G-coupled MGluR, NK1,B2, and CGRP1 receptors, which release intracellular Ca21stores,recruits PKC and CaMKII, strengthening the excitatory synapse.

Latremoliere and Woolf 901

MKK), and the downstream ERK. Many different signal-ing pathways can induce ERK activation in addition toits canonical ras/raf pathway, and this can be readily de-tected immunohistochemically in the dorsal horn withinminutes of peripheral noxious stimuli130 (Fig 7). Thepresence of phosphorylated ERK reveals the anatomicaldistribution of those neurons whose intracellular signal-ing has been activated by the nociceptor input and arepresumably undergoing the synaptic changes that con-stitute central sensitization120,131,141,152 (Fig 7). In thespinal cord, ERK is only activated in neurons in responseto intense peripheral noxious stimulation, effectivelyidentical to those stimuli that induce central sensitiza-tion,130,363 suggesting that ERK phosphorylation is a bet-ter marker of the neural plasticity that mediates centralsensitization than c-Fos, which can be activated by lowthreshold stimuli that do not induce central sensitiza-tion.126 Because the ERK pathway is composed of suc-cessive protein kinases, each of which can be activatedby several different signaling pathways,131 most of thetriggers of central sensitization such as NMDAR, groupI mGluR, TrkB, NK1, or CGRP1 converge to activateERK120,130,131,171 (Fig 8). Once activated, ERK producestranslational and post-translational effects that partici-pate in the maintenance of central sensitization in spi-nal cord neurons.131,387 The post-translational effectsinclude an increase of NMDAR function through phos-phorylation of its NR1 subunit152,293 (Fig 6, A), recruit-ment of AMPAR to the membrane93,254 (Fig 6, B)leading to an increase in AMPAR, and NMDAR currentsboosting synaptic efficacy.152 Furthermore, ERK pro-duces a decrease in K1 currents through phosphoryla-tion of the residue Ser616 of Kv4.2 channels leadingto an increase in membrane excitability118,119 (Fig 6,A). Transcriptional changes are mediated by activationof CREB as well as other transcription factors, whichdrives expression of several genes including c-Fos,NK1, TrkB, and Cox-2131 (Fig 6, C). Inhibition of ERKactivation using inhibitors of MEK reducesbehavioral measures of activity-dependent centralsensitization.137,142

Nitric oxide (NO) synthesized by either neuronal or in-ducible NO synthases in the dorsal horn381 also has a rolein central sensitization.381-383 Potential mechanisms forNO actions include the cGMP synthesis cascade, nitrosyla-tion of membrane channels, ADP-ribosylation, and pro-duction of reactive species.64,278 The NO-cGMP pathwayinvolves soluble guanylate cyclase, which is expressedby NK1-positive spinothalamic neurons, as well as inhib-itory interneurons.73 Fig 8 summarizes key intracellularpathways whose activation contributes to the genera-tion of central sensitization.

Effectors of Activity-Dependent CentralSensitizationAMPAR and NMDAR phosphorylation during central

sensitization increases the activity/density of thesereceptors, leading to postsynaptic hyperexcitabi-lity.32,86-88,134,178,345,394,407,408 The first phase of central

Figure 6. Contribution of PKC, CaMKII, PKA, and ERK activa-tion to central sensitization. (A), Phosphorylation by PKC, CaM-KII, PKA, and ERK cause changes in the threshold and activationkinetics of NMDA and AMPA receptors, boosting synapticefficacy. ERK also produces a decrease in K1 currents throughphosphorylation of Kv4.2 channels, increasing membrane excit-ability. (B), PKA, CaMKII, and ERK promote recruitment ofGluR1-containing AMPAR to themembrane from vesicles storedunder the synapse. (C), Transcriptional changes mediated byactivation of CREB and other transcription factors drivingexpression of genes including c-Fos, NK1, TrkB, and Cox-2, toproduce a long-lasting strengthening of the synapse.

902 Central Sensitization: Generator of Pain Hypersensitivity by Central Neural Plasticity

sensitization is a fast augmentation of excitatory gluta-matergic synapses in the superficial dorsal horn thatstrengthens nociceptive transmission and recruits non-nociceptive input to the pathway. This is achieved byphosphorylation of numerous receptor and ion channeltargets that lead to changes in threshold, channel kinet-ics, and voltage dependence, as well as amodification inthe trafficking of the receptors to the synapse (Fig 6, Aand B). On noxious stimulation, PKA phosphorylatesGluR1 subunits,87,88,214 leading to an insertion of thesereceptors into the synapse84,93 and thereby an increasein synaptic strength.20 Phosphorylation of GluR1-con-taining AMPAR by PKC and CaMKII also increases theexcitability of nociceptive neurons.40,88

NR1 phosphorylation by PKA408 or PKC32,407 partici-pates in the development of hypersensitivity97,262,345 byincreasing the response of NMDARs to glutamate.44,259

Phosphorylation of the NR2B subunit of NMDAR, medi-ated through Src activation, increases the opening ofthe receptor channel268 and prevents endocytosis of acti-vated receptors by disrupting the binding site of AP-2,a protein involved in clathrin-coated endocytosic vesicleformation.42 Decoupling Src interaction with NMDAR

Figure 7. Key effectors of central sensitization in the dorsalhorn. Subcutaneous injection of capsaicin, a potent inducer ofcentral sensitization, causes rapid activation of ERK and CREB(upper panels) as well as a PKC-induced phosphorylation ofthe NR1 subunit of NMDAR in c-Fospositive neurons of the su-perficial laminas of the spinal cord (middle panels). ERK activa-tion participates in transcriptional and post-translationalchanges, CREB activation promotes transcription of severalgenes involved in central sensitization. NR1 phosphorylationby PKC increases NMDAR activity. ERK and PKC are activatedthrough the increase of intracellular calcium that occurs duringstimulation of nociceptive fibers (lower panels). Signals areshown in pseudocolor from blue (weak intensity) to red (strongintensity). ERK and CREB staining are from Reference 141; NR1phosphorylation staining from Reference 32, and calcium imag-ing from Reference 179.

Latremoliere and Woolfblocks NR2B phosphorylation and reduces formalin-induced and inflammatory pain without altering basalnociceptive pain.178

Activation of PKC contributes to hyperexcitability innociceptive neurons by several different pathways.First, PKC reduces the Mg21 block of NMDAR andincreases the probability of channel opening, facilitat-ing the activated state of NMDAR.44 Second, activa-tion of PKC decreases inhibitory transmission at thesegmental level by reducing GABA and glycine tonicinhibition174 and the descending inhibition drivenfrom the PAG.175 Disinhibition, mediated by whatevermeans, leaves dorsal horn neurons more susceptibleto activation by excitatory inputs including non-noci-ceptive A-fibers, and is 1 of the major mechanismstriggering and maintaining central sensitiza-tion.17,289,341,390 Finally, PKC contributes with PKA tothe activation of ERK in a manner that requires theircoactivation and is triggered by the central releaseof bradykinin.152

Activation of guanylate cyclase seems to be the majorway that NO contributes to the induction of sen-sitization275,322,403 through increases in neuronalexcitability and a reduction in inhibition,173,176,275 al-though an NO-mediated activation of ADP-ribosyltrans-ferase may participate in the maintenance of centralsensitization.403

Global Features of Activity-DependentCentral SensitizationThe key features of acute activity-dependent central

sensitization are that it is induced with a short latency(seconds) by intense, repeated, or sustained nociceptorinputs and typically lasts for tens of minutes to several

Figure 8. Key intracellular pathways contributing to the gen-erationof central sensitization. NMDARactivation causes activa-tion of PKC, CaMKII, and ERK (black arrows); GluR1-containingAMPAR activate PKC (red arrow); NK1 and CGRP1 receptors acti-vate PKC, PKA, and ERK (orange and brown arrows, respec-tively); TrkB s activates of PKC and ERK (purple arrows); andmGluR, via release of Ca21 from microsomal stores, activatesPKC and ERK (gray arrows). Note that most of the triggers ofcentral sensitization: Activation of NMDAR, mGluR, TrkB, NK1,CGRP1, or B2 converge to activate ERK.

903hours in the absence of further nociceptor input. It

generally requires activation of NMDA receptors for itsinduction, and these receptors contribute to its mainte-nance. Nevertheless, as reviewed above, multiple differ-ent triggers can contribute to the establishment of thisform of central sensitization: glutamate acting onNMDAR, but also on AMPAR and mGluR, the neuropep-tides substance P and CGRP, the kinin bradykinin, as wellas BDNF and NO (Fig 4). The reason so many differenttransmitters, modulators, and their receptors are in-volved is that it is not their specific action that is impor-tant but rather that they are released directly from orinduced in response to nociceptor afferent activity, andeach can separately or together initiate the activationof those multiple intracellular signaling pathways thatlead to the establishment of hyperexcitability in dorsalhorn neurons (Figs 6 and 8). In other words, there aremany parallel inputs to dorsal horn neurons that canindependently or cooperatively initiate central sensitiza-tion. Elevation in intracellular calcium, by whatevermeans, is 1 major trigger, activating multiple calcium-de-pendent kinases that act on receptors and ion channelsto increase synaptic efficacy (Figs 5 and 6). Many centralsensitization-inducing inputs also activate ERK, and thisMAPK appears to have a pivotal role, contributing to in-creases in AMPA and NMDA currents as well as reducingpotassium currents (Fig 6, A). However, even this kinasemay not be essential. Other kinases such as PKC, PKA,and Src can, independent of ERK, also modulate iono-tropic receptors to lead to an increase in synaptic efficacy(Fig 6, A).What has become clear is that there is no single defin-

ing molecular mechanism of central sensitization butrather that it is a general phenomenon, one that pro-duces distinct changes in somatosensory processing butnevertheless can be mediated by several different pro-cesses that, in response to nociceptor input, can (1) in-crease membrane excitability, (2) facilitate synapticstrength, or (3) decrease inhibitory influences in dorsalhorn neurons. Similarly, the effectors of this plasticityare multiple: changes in the threshold and activationkinetics of NMDA and AMPA receptors and in their traf-ficking to the membrane, alterations in ion channels toincrease inward currents and reduce outward currents,and reductions in the release or activity of GABA and gly-cine (Fig 9). These changes produce dramatic alterationsin function. However, they are usually relatively short-lasting and reversible. Phosphatases will dephosphory-late receptors and ion channels resetting their activityto baseline levels, trafficking to the membrane willreverse by endocytosis, and, with time, the increasedgain of the nociceptive neurons will fade, at least inthe absence of any further triggering inputs.186,400-402

Different, transcription-dependent changes are requiredfor longer-lasting effects, and these generally do not oc-cur in response only to nociceptor activity but are theconsequence of peripheral inflammation and nerve in-jury (see below). Activity-dependent central sensitiza-tion, even though it increases pain sensitivity, is in mostsituations an adaptive mechanism. Unlike nociceptive

904 Central Sensitizatiopain, which warns of potential damage in response tonoxious stimuli, central sensitization creates a situationinwhich pain is elicited by innocuous stimuli. This changeis protective, because it helps healing by limiting use ofan injured body part until the injury is fully repaired.Central sensitization becomes pathological, however, ifinflammation persists, as with rheumatoid arthritis, inwhich no healing occurs, and in situations in which cen-tral sensitization becomes autonomous and is main-tained in the absence of active peripheral pathology.Central sensitization represents not only a state in whichpain can be triggered by less intense inputs but in whichthe central sensitization itself can be maintained bya lower level or different kind of input. Ongoing activityin C-fibers, even at levels that do not elicit central sensi-tization in basal conditions, is sufficient to maintain cen-tral sensitization once it has been induced for prolongedperiods (days).153 Furthermore, although nociceptorinput is required to trigger central sensitization, pheno-typic changes in myelinated fibers after inflammationand nerve injury can enable these afferents to acquirethe capacity to generate central sensitization (see later).

Activity-Dependent Central Sensitizationand Synaptic PlasticityThat the activity-dependent synaptic plasticity in the

dorsal horn responsible for central sensitization is revers-ible differs from the permanent activity-dependent syn-aptic change in the cortex that leads to long-termmemory, long-term potentiation (LTP), in which the effi-cacy only of activated synapses is changed. Synapticchanges with some resemblance to cortical LTP do occurin the spinal cord, that is, a form of homosynaptic poten-tiation. However, the major synaptic alteration under-lying activity-dependent central sensitization isheterosynaptic potentiation, in which activity in 1 setof synapses enhances activity in nonactivated synapses,typically by sensitizing the entire neuron, somethingthat never occurs with cortical LTP.Homosynaptic potentiation is a type of use-dependent

facilitation of a synapse evoked by activation of thatsame synapse (Fig 10, A). Classic LTP in the CA1 regionof the hippocampus is formally defined as input-specifichomosynaptic facilitation27,164,384 and is dependent onNMDAR activation, Ca21 influx, and activation of Ca21-dependent intracellular signaling pathways, notablythe CaMKII pathway.164 Although the increased Ca21 isrelatively widespread in neurons after tetanic condition-ing stimulation of afferents,128,182,260 only the stimulatedsynapse is potentiated.164,260 The development of LTP by2 independent synapses using asynchronous pairingstimulation has been described in the hippocampus,but, once again, only conditioned synapses are poten-tiated.123

One form of homosynaptic facilitation in spinal cordneurons is windup, in which the action potential dis-charge elicited by a low-frequency (0.5 to 5 Hz) train ofidentical C-fiber strength stimuli gets larger on each suc-cessive stimulus200 (Fig 11). Windup is the result of the

n: Generator of Pain Hypersensitivity by Central Neural Plasticityactivation of NK1 and CGRP1 receptors after release ofsubstance P and CGRP from peptidergic nociceptors to

produce a cumulative membrane depolarization fromthe temporal summation of slow synaptic potentials.290

This then enables activation of NMDAR by removal oftheMg21 block, further boosting the responses in a non-linear fashion63,72,331,379 (Fig 11). The stimuli that inducewindup (repeated C-fiber stimulation) can lead to centralsensitization,379 and, although windup is often consid-ered to be an aspect of central sensitization, it is insteadthe reflection of activity-dependent excitability increasesin neurons during a nociceptor conditioning paradigmrather than changes that follow such inputs, which iswhen central sensitizationmanifests.Windup disappearswithin tens of seconds of the end of the stimulus train asthe membrane potential returns to its normal restinglevel (Fig 11).Another form of homosynaptic facilitation occurs in

NK1-positive lamina I neurons in the dorsal horn neuron.This has been termed LTP, although, unlike classic hippo-

effects on pain sensitivity are not permanent instead last-ing, like central sensitization for a few hours, with noevident change equivalent to long-term memory. Per-haps, therefore, to avoid confusion with cortical plastic-ity, the term LTP should be avoided for homosynapticpotentiation in the spinal cord because the changes inthe dorsal horn are long-lasting (hours) rather thanlong-term (persistent). The original description of thislong-lasting homosynaptic potentiation in the dorsalhorn referred to an activity-dependent facilitation ofexcitatory postsynaptic currents in spinoparabrachialneurons in response to high-frequency (tetanic burst;100 Hz) stimulation of C-fibers.127,177,271 The physiologi-cal relevance of this phenomenon was questionable be-cause C-fibers do not fire at such high frequencies.Conditioning C-fiber stimulation at a low frequency(2 Hz) was subsequently shown also to elicit a long-last-ing homosynaptic potentiation in lamina I spino-PAG

Figure 9. Multiple cellular processes lead to central sensitization. Central sensitization is not defined by activation of a single mo-lecular pathwaybut rather represents the altered functional status of nociceptive neurons. During central sensitization, these neuronsdisplay 1 or all of the following: i, development of or an increase in spontaneous activity; ii, reduction in threshold for activation; andiii, enlargement of nociceptive neuron receptive fields. These characteristics can be produced by several different cellular processesincluding increases in membrane excitability, a facilitation of synaptic strength, and decreases in inhibitory transmission (disinhibi-tion). Similarly, these mechanisms can be driven by different molecular effectors including PKA, PKC, CaMKII, and ERK1/2. Thesekinases participate in changes in the threshold and activation kinetics of NMDA and AMPA receptors and in their trafficking to themembrane, cause alterations in ion channels that increase inward currents and reduce outward currents, and reduce the release oractivity of GABA and glycine.Latremoliere and Woolfcampal LTP, this form of homosynaptic facilitationappears not to be persistent, or at least the functional905neurons but not in spinoparabrachial neurons.128 Thislow-frequency potentiation is dependent on elevations

contribute to either secondary hyperalgesia or tactileallodynia. Repeated nociceptor input, such as thatgenerated by capsaicin, will simultaneously generateboth a potentiation of the activated C-fiber synapses(homosynaptic), and, unlike LTP in the hippocampus,also a potentiation of neighboring nonactivated syn-apses (heterosynaptic). It seems likely, therefore, thatlong-lasting potentiation in projecting dorsal hornneurons is simply a restricted aspect of the generalwidespread changes induced in these neurons by noci-ceptor activity.Heterosynaptic potentiation appears to dominate the

functional sensorymanifestations of use-dependent cen-tral sensitization. After injection of capsaicin, for exam-ple, the thresholds of sensory fibers innervating thearea surrounding the injection site are not modi-fied,23,157,340,365 but pain hypersensitivity in these areasis prominent and depends on centrally mediated hetero-synaptic facilitation. The same argument holds for theactivation of pain in response to tactile stimulation or

Figure 11. Action potential windup. Windup is the conse-quence of a cumulative membrane depolarization resultingfrom the temporal summation of slow synaptic potentials. Un-der normal conditions, low-frequency stimulations of C-fibers(0.2 Hz) cause steady neuronal discharges (A) as the membranepotential has sufficient time to return to resting potential be-tween stimuli. At a frequency of 0.5 Hz or higher, activation ofNK1 and CGRP1 receptors by release of substance P and CGRPfrom peptidergic nociceptors produces a cumulative increasein membrane depolarization. This then enables activation ofNMDAR by removal of the voltage-dependent Mg21 block, fur-ther boosting the responses in a non-linear fashion (B). Windupdisappears within tens of seconds of the end of the stimulusin Ca21, which activates PLC, PKC, CaMKII, and NOS.128

Capsaicin and formalin injection also evoke a homosy-naptic long-lasting potentiation, as manifested by an en-hancement of C-fiberevoked synaptic potentials afterthe capsaicin/formalin evoked conditioning input.128

Capsaicin is, of course, also a potent inducer of activity-dependent central sensitization,294,340,383 characterizedby the production of secondary hyperalgesia and tactileallodynia. However, both of these particular forms ofpain hypersensitivity reflect heterosynaptic and not ho-mosynaptic facilitation. Indeed, heterosynaptic facilita-tion characterizes most major changes in the receptivefield properties of neurons and in pain sensitivity, in pre-clinical models, and human subjects368,369,376 (Fig 10, B).Interestingly, healthy human subjects receiving high-

frequency stimulation of C-fibers exhibit increased pain

Figure 10. Homo synaptic and heterosynaptic facilitation. (A),Hoa synapse evoked by activation of that same synapse (in red). Apotentiation is commonly called long-term potentiation (LTP). LTPalgesia. (B), Heterosynaptic facilitation represents a form of activitditioning input, red) augments subsequent activity in other, nonpotentiation is responsible for the major sensory manifestationsthreshold afferents (allodynia) and spread of pain sensitivity to no

906 Central Sensitizationtrain as the membrane potential returns to its normal restinglevel (B). Modified from Reference 323.in the stimulated region, quite possibly caused by homo-synaptic facilitation, but also show evidence of heterosy-naptic facilitation, as manifested by dynamic mechanicalallodynia in adjacent nonstimulated areas.149 Thecombination of the homosynaptic potentiation of condi-tioning nociceptor inputs and the heterosynaptic facili-tation of nonconditioned fibers in the nociceptivepathway constitutes central sensitization.Heterosynaptic facilitation represents a form of

activity-dependent facilitation where activity in 1 setof synapses (the conditioning input) augments subse-quent activity in another nonactivated group of syn-apses (the test input) (Fig 10, B). For homosynapticpotentiation, the test and conditioning inputs arethe same; for heterosynaptic facilitation they are dif-ferent. LTP-like phenomena in spinobrachial neuronscan only account for the augmentation of the sameC-fiber inputs that evoked the facilitation and cannot

mosynaptic potentiation is a form of use-dependent facilitation ofnonconditioned synapse (green) is not potentiated. This type of-like homosynaptic potentiation can contribute to primary hyper-y-dependent facilitation in which activity in 1 set of synapses (con-activated groups of synapses (test input, green). Heterosynapticof use-dependent central sensitization: pain in response to low-ninjured areas (secondary hyperalgesia).

: Generator of Pain Hypersensitivity by Central Neural PlasticityAb fiber inputs during central sensitization. It is no sur-prise, then, that spinal LTP shares major mechanisms

with central sensitization (NMDAR, Ca21, kinases, andNO) because it is very likely that the phenomenon of cen-tral sensitization includes both homosynaptic and heter-osynaptic facilitations triggered by the same process; themajor difference is that heterosynaptic potentiation re-sults from the spread of signaling from the conditioningsynapse to other synapses in the neuron182,270,371 (Fig 10,B). Homosynaptic changes will contribute with periph-eral sensitization to primary hyperalgesia,128,270 whereasheterosynaptic facilitation alone is responsible for sec-ondary hyperalgesia and allodynia.Although several different forms of LTP have been

characterized in the hippocampus,188,224,384 spreadingor heterosynaptic LTP has not been reported, eventhough release of Ca21 from intracellular stores can causea spread of long-term depression (LTD) to neighboring,unstimulated synapses.227 What, then, is responsible forheterosynaptic facilitation in dorsal horn neurons? Twomajor candidates are the activation of mGluRs and NO.mGluRs are coupled to the Ca21 channels of the endo-plasmic reticulum85 and play an important role in centralsensitization.7 Consequently, the release of intracellularCa21 in spinal cordneurons onmGluR activationmaypar-ticipate in spreading facilitation fromconditioned synap-ses to neighboring test synapses. NO is also a majoreffector of spinal cord neuronal plasticity211,309 and dif-fuses rapidly from the site of its production to producemultiple effects at a distance via its downstream signal-ing pathways, and in this way may contribute to the het-erosynaptic facilitation characteristic of centralsensitization. It is certainly likely that these and otherspreading signals cooperate to produce the wide-spread synaptic facilitation so characteristic to centralsensitization. Scaffolding proteins play a major role inthe addressing of specific kinases to the synapse and rep-resent another potential mechanism for widespreadsynaptic facilitation. A recent study has shown that inthe hippocampus, CaMKII activation is restricted to thesynaptic bouton of a conditioned synapse, thus onlyallowing homosynaptic facilitation at that specificsite.164 It is likely in thedorsal horn that CaMKII activationwill be much more widespread and indeed the dendritesof dorsal horn neurons lack synaptic boutons.

Central Sensitization in PathologicalSettingsIn addition to its role in rapidly and reversibly sensitiz-

ing the nociceptive system by activity-dependentchanges in synaptic strength and excitability, central sen-sitization also contributes to the longer-lasting andsometimes persistent pain hypersensitivity present inpathological situations involving inflammation anddamage to the nervous system. The molecular and cellu-lar mechanisms involved include some that are also re-sponsible for activity-dependent central sensitizationand others that are unique to either inflammation ornerve injury. NMDAR,47,193,255,280,315 AMPAR,180,239

Latremoliere and Woolfgroup I mGluR,7,65,75,92,102,118,221,288,392,405 group II-IIImGluR,45,104,194,207,286,398 BDNF,144,179,190,230 SP andCGRP,2,4,163 NO,50,316 and bradykinin241 have all beenshown to contribute both to the development of centralsensitization and to pain hypersensitivity in inflamma-tory and neuropathic pain models.

Inflammatory PainPeripheral inflammation induces a phenotypic switch

in primary sensory neurons that comprises a change intheir neurochemical character and properties due to al-terations in transcription and translation. We will onlydiscuss here those changes that relate specifically to cen-tral sensitization by virtue of changes in the synapticinput produced by the afferents and will not reviewthemajor changes that also alter peripheral transductionsensitivity and membrane excitability (peripheral sensiti-zation), although of course, anything that increases noci-ceptor afferent inputwill also indirectly lead to increasedcentral sensitization. Large DRG neurons begin, unlike intheir naive condition, to express SP and BDNF when theirperipheral terminals are exposed to inflammatory signalsand nerve growth factor (NGF).191,223 Consequently, acti-vation of the myelinated fibers by low-intensity innocu-ous stimuli now releases these neuropeptides in thespinal cord, and conditioning stimulation of the affer-ents acquires the capacity to generate central sens-itization, something they normally cannot do185,190,223

(Fig 12). After peripheral inflammation, Ab-mediatedsynaptic input to superficial dorsal horn neurons is sub-stantially increased from the very low levels found innoninflamed animals.16 TrkA-expressing nociceptors, in-stead of a phenotypic switch, begin to express higherlevels of neuropeptides and other NGF-dependentproteins as a result of exposure to the increased NGF pro-duced by inflammation.370,375

A critical central pathway for the generation of inflam-matory pain hypersensitivity involves induction of cyclo-oxygenase-2 (Cox-2) in dorsal horn neurons, to drive pro-duction of prostaglandin E2 (PGE2).269,349 PGE2 binds toits EP2 GPCR on dorsal horn neurons to potentiate AM-PAR and NMDAR currents,152 activate nonselective cat-ion channels,18 and reduce ininhibitory glycinergicneurotransmission by blocking glycinergic receptorswith a3 subunits9,111,152,213 (Fig 12). PGE2 also acts onEP4 receptors on presynaptic terminals to increase trans-mitter release.350 The importance of the central neuronalinduction of COX-2 to inflammatory hyperalgesia is re-vealed by conditional deletion of COX-2 only in neurons,which results in the retention of peripheral inflamma-tion and heat hyperalgesia but an almost complete lossof mechanical pain hypersensitivity.350

Under normal conditions, microglia are the onlyimmunocompetent cells of the nervous system66,362 andconstantly probe or survey the CNS parenchyma tomain-tain homeostasis.62,225 After peripheral inflammation,some spinal cord microglial cells change their shape,function, and chemical expression.115,258,311,312 In partic-ular, p38MAPK is activated311,312 and leads to the synthe-sis and release of pro-inflammatory cytokines,115,258

907amongwhich, IL-1b and TNF-a contribute to the develop-ment of central sensitization by enhancing excitatory

and reducing inhibitory currents and by activating induc-tion of COX-2142,269 (Fig 12).Neurons in the superficial lamina of the dorsal horn

usually display a GluR2 AMPAR phenotype (ie, areCa21-impermeable)252; however, peripheral inflamma-tion triggers a shift from GluR2/3 to GluR1-containingAMPARs at the membrane159,238,355 (see PSD Proteinsand AMPAR Recycling and Subunit Switch, below). Un-der these conditions, activation of AMPAR elicits entryof Ca21, which can then participate in the activation ofthe signaling pathways that drive central sensitization.Ca21-permeable AMPARs appear to be a major sourceof the [Ca21]i increase in inflammatory pain, generatingas much Ca21 influx as with NMDAR activation.182 Thefunctional state of NMDAR is also modified in responseto peripheral inflammation, with phosphorylation ofNR2B subunits by Src resulting in increased activity ofthe receptors106,107,178 and in their maintenance at thesynapse.42 Finally, peripheral inflammation also pro-motes group I mGluR insertion into the membrane(mGluR5) and closer to the synapse (mGluR1), therebyfurther clustering these receptors at the synapse.247

Neuropathic PainAfter peripheral nerve injury, damaged and nondam-

aged A- and C-fibers begin to generate spontaneousaction potentials. Because these do not arise from the pe-ripheral terminal, it is a form of ectopic input.70,74 Suchinput in C-fibers can initiate and then maintain activity-dependent central sensitization in the dorsal horn.154

However, because of chemical and structural changes in

Figure 12. Central sensitization in pathological settings. A,Representation of the superficial lamina of the dorsal horn ofthe spinal cord. Nociceptive peptidergic fibers contact lamina Iand II outer (I and IIo) neurons that express GluR2-containingAMPAR (in blue). Some of these neurons project to the thala-mus, parabrachial nucleus, and PAG. Nociceptive nonpeptider-gic fibers contact neurons in dorsal lamina II inner (IIid), whichalso express GluR2-containing AMPAR. Non-nociceptive largefibers contact deeper laminae but also send collaterals to inhib-itory interneurons in the ventral part of lamina II (IIiv).222 B,Alterations in the superficial lamina in inflammatory pain. Be-cause large DRG neurons begin to express SP and BDNF, stimula-tion of these afferents acquires the capacity to generate centralsensitization. Neurons now express GluR1-containing AMPAR attheir synapse (in red), resulting in an increase of Ca21 influx ontheir activation. Some spinal cord microglial cells are activatedand release factors that contribute to the development of cen-tral sensitization by enhancing excitatory and reducing inhibi-tory currents. C, Representation of superficial lamina inneuropathic pain states. After peripheral nerve injury, there isa loss of C-fiber central terminals and the sprouting of myelin-ated A-b fibers from deep to superficial lamina. Injured sensoryneurons in the dorsal root ganglion exhibit a change in tran-scription that alters their membrane properties, growth, andtransmission. Large fibers begin to express substance P, BDNF,and the synthetic enzymes for tetrahydrobiopterin, an essentialcofactor for NOS and can drive central sensitization. Recruit-ment and activation of microglial cells is an essential step inthe development of pain after nerve injury and to triggercentral sensitization by releasing proinflammatory cytokinesthat increase neuronal excitability and BDNF that inducesa switch in Cl- gradients, resulting in a loss of inhibition. Lossof inhibition is also caused by excitotoxic apoptosis of inhibitoryinterneurons.

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908 Central Sensitization: Generator of Pain Hypersensitivity by Central Neural Plasticity

A fibers,56,229,386 input in these afferents can also beginto drive central sensitization.69 Injured, and to a muchlesser extent, noninjured sensory neurons in the dorsalroot ganglion exhibit a massive change in transcriptionthat alter their membrane properties, growth, and trans-mitter function.56,231,232,386 These changes are muchgreater than those that occur in response to peripheralinflammation,where only a few tens of transcripts are al-tered in the DRG195 and involve altered expression ofabout 1000 transcripts, including ion channels, receptors,transmitters, and the molecular machinery necessary foraxon regeneration.56,261 Among the many changes,large fibers begin to express new transmitters and neuro-modulators including substance P and BDNF and the syn-thetic enzymes for tetrahydrobiopterin, an essentialcofactor for NOS. Stimulation of non-nociceptive fibersnow triggers release of factors that can drive central sen-sitization.24,56,91,229,327,386

Structural changes also contribute to altered synapticfunction. Peripheral nerve injury leads to a transgan-glionic degeneration of C-fiber terminals in laminaII.13,136 This loss of presynaptic input, together with thetriggering of increases in the intrinsic axonal growth ca-pacity as part of the regenerative response of the injuredneurons, provides an opportunity and the molecularmeans for myelinated A-b fibers to sprout from laminaeIII-IV into laminae I-II andmake contact with nociceptive-specific neurons.166,191,285,377,378 The original experi-ments describing the sprouting phenomenon were con-ducted using cholera toxin B subunit as a selectivetracer for A-fibers as well as single axonal label withHRP. The selectivity of this toxin after peripheral nerveinjury is somewhat controversial.125,339 Nevertheless,immunostaining for c-Fos activation and electrophysio-logical recordings have clearly established that periph-eral nerve injury causes large myelinated fibers tobegin to drive nociceptive neurons in superficiallamina.24,151,235,366

A reduction in the synthesis, release, or activity of in-hibitory transmitters leads to a state of disinhibition,whose net functional effects are very similar to that pro-duced by increases in synaptic strength of excitatory syn-apses and in membrane excitability.289,341,390 Inneuropathic pain states, there is substantial disinhibitionin the superficial dorsal horn with loss of GABAergic anda reduction in glycinergic inhibitory currents210 that canbe attributed, at least in part, to apoptosis of inhibitoryinterneurons.277 This neuronal death appears to be theresult of an NMDAR-induced excitotoxicity that developsover time rather than to the large amount of glutamatereleased centrally at the time of nerve injury.277 One lab-oratory failed to find significant loss of neurons or of GA-BAergic content in the dorsal horn of neuropathic painanimal models.249-251 The reasons for this discrepancyare not clear but may reflect technical differences inhow the studies were performed. Interestingly, the re-duction in glycinergic neurotransmission caused by theactivation of EP2 receptors after peripheral inflamma-tion does not appear to operate after nerve injury, fur-

Latremoliere and Woolfther indicating that some inflammatory andneuropathic pain mechanisms differ.117Another mechanism contributing to the reduction insegmental inhibition in a subpopulation of lamina I neu-rons in the spinal cord after nerve injury is dependent onBDNF effects on an anion transporter, changing aniongradients across neuronal membrane to alter the inhibi-tory efficacy of GABA. Under normal conditions, theintracellular concentrations of Cl- are maintained bythe opposed effects of Cl--cotransporter K1-Cl- exporter2 channels (KCC2) and Na1-K1-Cl- exporter 1 channels(NKCC1). KCC2 drives Cl- ions out of the cells (alongwith K1) and NKCC1 is responsible for an influx of K1,Na1, and Cl- into the cells. The net effect of these 2 co-transporters is a steady-state Cl- concentration gradientin which opening of Cl- channels (such as GABAA recep-tors) causes entry of Cl- into the neuron and hyperpolar-izes the neurons. After peripheral nerve injury, BDNFreleased by activated microglial cells results in a reduc-tion of KCC2 expression in a subset of neurons in the su-perficial lamina of the dorsal horn.57,58,203 Consequently,activation of GABAA receptors by GABA result in a dimi-nution or absence of Cl- entry into the cell and thus a dis-inhibition of these nociceptive neurons57,58,179,203

(Fig 12). As after peripheral inflammation, there is alsoan increase in descending excitatory controls from theRVM in the brainstem after peripheral nerve injury, aswell as a reduction of descending inhibitory con-trols.60,95,351,356

Peripheral nerve injury causes a massive activation of,and change in, glial cells in the spinal cord as well as in-filtration of peripheral immune-competent cells, notablymacrophages and T-cells.38,317,360 The extent and dura-tion of the changes in microglia and astrocytes is muchgreater than in response to peripheral inflammation. Ac-tivated microglia produce and release trophic factors,neurotransmitters, cytokines, and reactive oxygen spe-cies263,361 and appear to play an essential step in the de-velopment of pain after nerve injury by triggeringcentral sensitization through their interaction with neu-rons.133,160,162,201,206,256,257,352 Numerous signals triggermicroglial activation and recruitment, including ATPand NO,62,81,225 cytokines, and chemokines, some ofwhich are released by injured sensory neurons and othersby microglial cells themselves or by astrocytes andT-cells.1,3,66,76,204,348,361,362 Release of cytokines by micro-glia increases neuronal excitability through activation ofERK and CREB.131,142 Activated microglia also releaseBDNF and NO,58,116 promoting segmental disinhibi-tion.57 Finally, microglia can also provoke neuronaldeath by producing ROS, pro-apoptotic cytokinessuch as TNF,121 and by a diminished glutamateuptake.48,326,332 T-cells produce specific cytokines suchas IFN-g, which reduce GABAergic currents in the dorsalhorn354 through activation of IFN-g receptors353 andalso activate and recruit microglia. Astrocytes also be-come activated after peripheral nerve injury,98,131,205

with a slower onset and more prolonged time coursethan microglia, and may play more of a role in the main-tenance of neuropathic pain hypersensitivity thanmicro-glia.94,399,406 What seems clear is that multiple different

909mechanisms operate after nerve injury to increase excit-ability and reduce inhibition.

Scaffolding Proteins, Synaptic Plasticity,and Central Sensitization DuringInflammation and After Nerve InjuryThe proteins that make up the PSD can drive a major

functional reorganization of synapses, modifying post-synaptic efficacy by altering receptor density at themem-brane and producing switches from Ca21-impermeableto Ca21-permeable AMPARs (Fig 13). The PSD is not sim-ply a structural landmark of the synapse but contains el-ements essential both for the formation of the synapseand for changes in its properties. Absence of scaffoldingproteins or specific disruption of their binding sites re-sults in a dramatic reduction in synaptic plasticity be-cause the proteins contribute both to transcriptionaland post-translational events. They initiate signaling cas-cades that lead to the activation of transcription factors,traffic newly synthesized receptors to the PSD, andaddress kinases and phosphatases to specific receptorsin a stimulus-dependent manner. Although the involve-ment of the PSD in synaptic plasticity in the cortex ismuch better established than in the spinal cord, thereis increasing evidence for a major role for the PSD inchanging synaptic efficacy in response to peripheralinflammation and nerve injury.The PSD consists of cytoskeletal proteins, signaling

molecules, membrane receptors, and scaffolding pro-teins.234 Scaffolding proteins are families of proteinscharacterized by their ability to interact with numerouspartners, and these proteins form the dense molecularstructure of the postsynaptic component of the synapse.A particularly abundant component of the PSD are pro-teins containing a specific peptidergic domain calledPDZ, which is named after the protein in which the se-quence was first identified (postsynaptic density protein95 [PSD-95]/discs large/zonula occludens 1). This family ofproteins includes, among hundreds of members, the 4membrane-associated guanylate kinases (MAGUK):PSD-95, PSD-93, synapse associated protein (SAP)-97,SAP-102. The MAGUKs represent the most abundantscaffolding protein family in the PSD234 and are charac-terized by 3 PDZ domains, an Src homology region(SH3) domain, and a guanylate kinase-like (GK) do-main,148 making them central elements of the synapsescaffold. The prime binding protein for theMAGUK fam-ily is the NMDAR subunit NR2,155 but it also binds to thetransmembrane AMPAR regulatory proteins (TARPs),43

nonreceptor tyrosine kinases,329 nNOS,30,31 GKAP,217

and AKAP79/150.53 MAGUKs can be seen as the func-tional scaffold of the PSD and are essential for the struc-tural integrity of synapses but also modulate theinsertion of glutamate receptors into the synapse andphysically bring together key enzymes to thePSD.59,148,167,197,265 Knock-down of PSD-95 and PSD-93,as well as targeted mutagenesis of the residues requiredfor their protein:protein interaction, both prevent andreduce central sensitization in normal conditions321 aswell as in inflammatory319,323,397 and neuropathic painmodels99,320,323,397 but do not alter nociception or loco-

910 Central Sensitizatiomotor functions.319-321,323Another member of the PDZ family, stargazin, isa 4-transmembrane domain protein whose putative sec-ondary structure is close to the Ca21-channel g subunit,and was named g2.170 Stargazin however, does notplay an important role in neuronal Ca21 channels170

but is instead highly concentrated in the PSD andco-immunoprecipitates with GluR1, 2, and 4.43,335 Theprotein is amajor AMPAR partner, alongwith the 4 otherisoforms, g3, g4, g7, and g8,140,335 which form the TARPsubgroup.335 Stargazin traffics AMPAR from the endo-plasmic reticulum to the extrasynaptic membrane.43,334

Once stargazin and AMPAR are addressed to the extrasy-naptic membrane, their recruitment to the synapserequires interaction of the C-terminus segment of starga-zin with PSD-95.21,276 Activity-dependent phosphoryla-tion of stargazin by PKC and CaMKII342 producesa massive insertion of AMPAR into the membrane,337

whereas stargazins dephosphorylation by PP1 or PP2Breduces the number of AMPAR at the synapse.337 In addi-tion, via the interaction between stargazins ectodomainand the glutamate binding region of AMPAR, stargazinmodulates the activity of AMPAR by slowing channel de-activation and desensitization and increasing the affinityof the receptors for glutamate,49,334,336 thereby potenti-ating synaptic strength. Disruption of stargazin in thespinal cord inhibits the second phase of formalin-in-duced pain and reduces the heat hyperalgesia causedby intraplantar CFA injection.318 Recently, cornichon ho-molog 2 (CNIH-2) and cornichon homolog 3 (CNIH-3)have been found to be novel partner proteins for AMPARin the CNS.279 Cornichon proteins bind to GluR1-4 AM-PAR and, as for stargazin, they promote AMPAR surfaceexpression and slow their deactivation kinetics.279 Be-cause their expression in the CNS is estimated to be in70% of neurons, and because cornichon and stargazinappear to be mutually exclusive,279 the determinationof their presence in spinal cord neurons and their rolein central sensitization is something that needs to be in-vestigated.EphrinB-ephBR receptor interactions participate in

NMDAR clustering at the PSD.90 EphBRs are receptor ty-rosine kinases expressed by postsynaptic neurons,whereas EphrinB is anchored to the presynaptic mem-brane.156 The kinase activity of EphBR is not requiredfor the initial clustering of NMDAR at the synapse butis essential for their maintenance.61 Stimulation ofEphBR potentiates NMDAR-induced Ca21 influx andthe phosphorylation of CREB through the activation ofthe nonreceptor tyrosine kinase src314 and recruits CaM-KII to the synapse236 to increase NMDAR activity.314 Acti-vation of EphBR in the spinal cord induces thermalhyperalgesia (but not allodynia),22,299 without modify-ing nociception.22 Inhibition of EphRB prevents or re-verses inflammatory22,291 and neuropathic150,299 painand prevents establishment of NMDAR-induced spinalcord LTP.299 More specifically, targeted disruption ofthe coupling between EphRB-activated Src and NR2Balso prevents the development of central sensitizationwithout altering basal nociceptive transmission.178 In ad-

n: Generator of Pain Hypersensitivity by Central Neural Plasticitydition, sustained nociceptive activity leads to an

upregulation and reorganization of presynaptic EphB in-creasing EphB-EphRB interaction.22,298,299

The nonreceptor tyrosine kinase family also includesFyn, which phosphorylates NR2 subunits268 and bindsto PSD-93 to phosphorylate NR2A/B

273 and could playa role in the maintenance of neuropathic pain.5

NMDA receptors are structurally connected withgroup I metabotropic glutamate receptors througha complex composed of PSD-95, GKAP, Shank1, andHom-er1b/c. GKAP binds to the GK domain of PSD-95147,217

and recruits Shank1 to the PSD via their PDZ do-

main.216,264 Shank1 then binds to the EVH1 domain ofhomer1b/c.343 Homer1b/c proteins have a coiled-coilstructure in their C-terminus region that enables themto form homotetramers or heterotetramers.284 This as-sembly of Homers leaves 3 available EVH1 domains thatcan bind several other targets such as group I mGluRs(but not group 2 or 3mGluRs),28 inositol triphosphate re-ceptors (IP3R), or the actin cytoskeleton.284,344 The inter-action between Homer1b and IP3R and between Shank1and Homer1b/c controls local Ca21 release from theendoplasmic reticulum upon mGluRs activation.266,267

Figure 13. Role of scaffolding proteins in central sensitization. Representation of the post synaptic density (PSD) region of a synapseof a nociceptive neuron in the superficial lamina of the spinal cord under basal conditions (A) and during inflammatory pain (B). Theproteins that make up the PSD can drive a major functional reorganization of synapses, modifying postsynaptic efficacy by alteringreceptor density at themembrane and producing switches fromCa21-impermeable to Ca21-permeableAMPARs. In addition, scaffold-ing proteins mediate the addressing of kinases to the receptors, thereby increasing their activity. Under normal conditions (A), neu-rons mostly express GluR2-containing AMPAR that are anchored to the PSD via GRIP-1. NMDAR are recycled in an activity-dependentmanner via PICK-1 and NSF. During inflammation (B), there is an endocytosis of GluR2-containing AMPAR (initiated by PKC phosphor-ylation of GluR2) along with a loss of NSF that prevents their reinsertion into the synapse. GluR1-containing AMPAR are expressed atthemembrane, where their activity is increased by phosphorylation with PKA as well as by the ectodomain of stargazin, whose C-ter-minus segment is phosphorylated by PKC and CaMKII. MAGUK can participate to increase glutamate receptors density at the synapse,and phosphorylated stargazin promotes AMPAR and NMDAR clustering. Scaffolding proteins such as AKAP79/150 and the MAGUKsalso address kinases to specific synaptic position at the right time to phosphorylate AMPAR and NMDAR subunits, thereby increas-ing their activity. The Homer-Shank1-GKAP-MAGUK complex couples mGluR and NMDAR to intrasomal Ca21 stores, so that activationof glutamate receptors leads to high levels of Ca21 in the PSD that activate PKC and CaMKII, also recruited to the PSD by scaffoldingproteins. PKC, CaMKII, and other kinases converge to activate ERK, which reduce K1 channels activity and promote transcriptionalactivity.

Latremoliere and Woolf 911

Homer1a, a short isoform of Homer1b/c that lacks thecoiled-coil structure, is an immediate early gene acti-vated on neuronal activity and participates in remodel-ing synapses in an activity-dependent manner.129

Homer1a is upregulated in dorsal horn neurons afterthe subcutaneous injection of formalin or CFA324 aswell as transiently after peripheral nerve injury.208 Fac-tors responsible for Homer1a activation include NMDAR,ERK1/2 and Src.208,324 Knock-down of Homer1a increasespain-like behaviors specific to central sensitization andnot those associated with peripheral sensitization,324

whereas overexpression reduced inflammatory pain-like behavior without altering basal nociception.324

Homer1a probably causes a disruption of the clusteringproperties of Homer1b/c protein and of Ca21 release onNMDAR or mGluR activation.324 In contrast, homer1b ac-tivation leads to an increase of AMPAR activity after thestimulation ofmGluR, whereas activation of Homer1a in-hibits this.347 In addition, Homer1b/c proteins could playan important role in clustering group 1 mGluRs at thesynapse after CFA injection,247 an effect that is reducedby Homer1a overexpression.385 Homer proteins are,therefore, convergent factors that potentially link majorglutamate receptors with Ca21 stores in neurons, and thebalance between Homer1b/c and Homer1a may play animportant role in the development and maintenance ofcentral sensitization.

PSD Proteins and AMPAR Recycling andSubunit SwitchStargazinmaybe important in creating the switch from

GluR2- to GluR1-containing AMPAR in response to pe-ripheral inflammation159,355 by specifically addressingGluR1-containing AMPARs to the synapse and thenstrengthening their activity via its ectodomain.334 Periph-eral inflammation leads via PKA to a phosphorylation ofSer831 and Ser845 on GluR1 in the spinal cord,180 which,inassociationwithCaMKII activation,promotesa transfer

Figure 14. Synaptic scaffolding proteins and the switch toGluR1-containing AMPAR after peripheral inflammation. Underbasal conditions, GluR2-containing AMPARs are associated inthe synapsewith stargazin andGRIP-1. The C-terminus of starga-zin binds to PSD-95 (1). Peripheral inflammation leads to gluta-mate release from nociceptors and AMPA and NMDA receptoractivation. NMDAR activation leads to a Ca21 influx that acti-vates PKC (2). Activated PKC phosphorylates GluR2-containingAMPARs at ser880, which leads to a loss of affinity for stargazinand GRIP-1, allowing PICK-1 to bind to the receptor (3). TheGluR2-containing AMPAR and PICK-1 complex undergo endocy-tosis, whereas NSF is downregulated, preventing reinsertion ofGluR2-containing AMPAR into the synapse. Meanwhile, periph-eral inflammation also leads to activation of PKA that promotesGluR1-containing AMPAR to be inserted into themembrane (4).Because GluR2-containing AMPARs are internalized and theCa21-permeable GluR1-containing AMPAR are inserted intothe membrane, there is a switch from GluR2- to GluR1-contain-ing AMPAR (5 and 6). Increased [Ca21]i causes an activation ofPKC and CaMKII, which phosphorylate the C-terminus of starga-zin, increasing its affinity for PSD-95 (CaMKII) and modifying itsectodomain (PKC),which causes increasedGluR1 affinity for glu-tamate and single-channel conductance and a higher channelopening probability, further increasing Ca21 influx (7).

:

912 Central Sensitization: Generator of Pain Hypersensitivity by Central Neural Plasticity

of GluR1-containing AMPARs to the membrane,84 wherethey are maintained by synaptic activity.189

GluR2-containing AMPARs are associatedwith high af-finity to GRIP-1, which clusters the receptors at the syn-apse,77,114 whereas PICK-1 is a critical element foractivity-dependent endocytosis of the receptor.51,96,226

NMDAR activation leads to a PKC-mediated phosphory-lation of GluR2 at Ser880,172,238 which decreases GluR2saffinity for GRIP-1, whereas the increase in intracellularCa21 recruits PICK-1 to the synapse,51,108,238 where it re-duces the clustering of GluR2-containing AM-PAR51,172,196,238,304,328 (Fig 14). On endocytosis, vesiclescan either be inserted back into the synapse under theaction of NSF,122,226,228,297 which disrupts the [GluR2-PICK-1] complex,109 or be maintained out of the synapsethrough PICK-1.172 During inflammation, NSF expressionis reduced in the spinal cord,139 thereby preventingGluR2-containing AMPAR reinsertion into the synapse.The net effect of these complex changes is an increase

in GluR1 and a decrease in GluR2 containing AMPAR atthe synapse (Fig 14). Once inserted into the synapse,GluR1-containing AMPAR remain there for as long asthere is glutamate release,83,189 and their activation ispotentially increased by stargazins ectodomain, therebyfurther promoting the Ca21-dependent pathways re-quired for maintenance of central sensitization. In addi-tion, the phosphorylation of stargazins C-terminusdomain by PKC and CaMKII342 increases its affinity forPSD-95, thereby re-enforcing the clustering of AMPARwith NMDAR at the synapse.276

After peripheral nerve injury, GluR2 and GRIP-1 are up-regulated in dorsal horn neurons,100,110 whereas PICK-1expression is not modified and NSF is downregulated.100

Peptides that disrupt the binding of GRIP-1 or NSF withGluR2 partially decrease neuropathic pain-like behav-iors.100 PICK-1 is also required for Gi/o-coupled mGluR7trafficking to the membrane.306 Activation of mGluR7can block P/Q-type Ca21 channels240 and reduces thepain caused by injection of capsaicin218 or peripheralnerve injury.237 The upregulation of GRIP-1 but not ofPICK-1 in neuropathic pain models would promote AM-PARmaintenance at the synapse but not that of mGluR7,resulting in increased excitability in these cells.Finally, the A kinase-anchoring protein 79/150

(AKAP79/150) binds to PSD-9553 and is a scaffold for pro-tein kinases and phosphatases,53,169 specifically traffick-ing enzymes within the PSD to increase (kinases) orreduce (phosphatases) synaptic transmission. WhenAKAP79/150 recruits PKA181,295 or PKC325 in the PSD, itpromotes insertion of new AMPAR to the membrane

(via PKA) as well as increasing their activity (via PKC). Incontrast, recruitment of PP2B25,169 triggers AMPARendocytosis.25 AKAP79/150 may function therefore asa master switch of central sensitization by promotingphosphorylation or dephosphorylation of stargazin andAMPAR.

ConclusionBefore central sensitization was discovered, there

were 2 major models of pain. The first was that it wasa labeled-line system, in which specific pain pathwayswere activated only by particular peripheral pain stim-uli and that the amplitude and duration of pain wasdetermined solely by the intensity and timing of theseinputs. The second model evoked gate controls inthe CNS, which, by opening or closing, enabled or pre-vented pain. Neither model envisaged, however, thatpain may arise as a result of changes in the propertiesof neurons in the CNS: central sensitization. We nowappreciate that there are indeed specific nociceptivepathways and that these are subject to complex facili-tating and inhibitory controls; both models were inpart correct. We also know though, that changes inthe functional properties of the