peripheral pain mechanisms and nociceptor sensitization

1

C H A P T E R 3

Peripheral Pain Mechanisms and Nociceptor Sensitization

MICHAEL S. GOLD

Pain has been categorized by duration (acute vs. chronic), location (superficial or deep; cutaneous, bone/joint, muscle, or viscera), and cause or type (inflammatory, neuropathic, can-cer). Generally, activation of and/or ongoing activity in specific subpopulations of primary afferent neurons underlies the expe-rience of pain regardless of how it is categorized. Accordingly, primary afferents are key players in understanding mechanisms of, and managing, pain.

Sir Charles Sherrington anticipated by many decades the ex-istence sensory receptors that respond to noxious stimuli, that he called nociceptors and thereby provided for us the opera-tional definition of stimuli that are noxious (i.e., stimuli that damage or threaten damage of tissue). Two considerations are important to this discussion. First, Sherrington functionally de-fines the nociceptor by its response to a noxious stimulus (e.g., a nociceptive withdrawal reflex, pain). Second, the definition of an applied stimulus as noxious is based on the response to the stimulus applied to skin and subcutaneous structures.

Sherrington’s definition of a nociceptor continues to the present day. However, the term has undergone change and challenge over the past 100 years and may be nearing the end of its utility in the face of the growing understanding of the heterogeneity in the neurons that may not fit so comfortably under this umbrella term. It is therefore important to consider, within the context of our current knowledge, how a nociceptor is defined, identified, and studied, as the interpretation of this information will directly affect the management of pain.

All would agree that a nociceptor is a sensory receptor which, when activated or active, can contribute to the expe-rience of pain. Nociceptors are present in skin, muscle, joints, and viscera, although the density of innervation (i.e., the num-ber and distribution of sensory endings) varies between and within tissues. As originally described by Sherrington, a noci-ceptor is the peripheral sensory terminal (i.e., the site of energy transduction—see following section), although commonly the term is used to also include the cell body (in a dorsal root, trigeminal, or nodose ganglion) and its central termination in the spinal cord or brainstem. Beyond this, agreement about im-portant features of nociceptors is less uniform. One of the best examples of why this definition is becoming problematic is the observation that injury-induced changes in the central nervous system underlie the emergence of allodynia, pain in response to normally innocuous stimuli. Allodynia is problematic for this definition of nociceptor because pain is mediated by activity in low-threshold afferents that in the absence of tissue injury would, if anything, contribute to the suppression of pain. That is, these afferents would not be considered nociceptors despite the fact that they contribute to the experience of pain.

Furthermore, because stimuli adequate for activation of nociceptors differ between tissues (e.g., tissue damage is not always required), defining a noxious stimulus has become a challenge. For example, some nociceptors in skin and joints and most nociceptors in the viscera have low thresholds for me-chanical activation that do not conform to the condition that stimulus intensity must be either damaging or threaten dam-age. Further, so-called “silent” or “sleeping” nociceptors are unresponsive to intense mechanical stimulation (and are better

denoted as mechanically insensitive nociceptors) but develop spontaneous activity and mechanosensitivity after exposure to inflammatory and other endogenous mediators. These types of nociceptors—low threshold and sleeping—as well as other subpopulations of sensory neurons that may contribute to the sensation of pain following tissue injury are considered further in the discussion of sensitization in the following section.

Functional Characterization of NociceptorsAs suggested earlier, there are many types of nociceptors, our knowledge of which has been advanced by human psychophys-ical studies while recording from afferent fibers (Box 3.1). In human skin, for example, there exist nociceptors that respond only to mechanical, only to cold thermal, or only to hot thermal stimuli as well as those that are insensitive to both mechani-cal and heat stimuli (mechanically insensitive or sleeping no-ciceptors).1,2 The most abundant is the polymodal nociceptor, which responds to mechanical, thermal, and chemical stimuli. In general, nociceptors that innervate skin have the broadest range of modality selectivity, whereas nociceptors innervating deeper structures tend to be less modality-selective and more polymodal in character.3 For example, mechanical sensitivity is a prominent feature of visceral and joint nociceptors because stimuli adequate for their activation include hollow organ dis-tension and overrotation, respectively. Many of these nocicep-tors also respond to chemical and/or thermal stimuli as well, although the functional significance of thermal sensitivity in deep tissues is uncertain. An important characteristic of poly-modal nociceptors, whether the modalities of stimulation to which they respond are two or all three, is that when sensitized (e.g., by an inflammatory insult), responses to the other mo-dality or modalities of stimuli to which it responds are all in-creased.4 That is, it is not only the mechanosensitive modality, for example, that becomes sensitized, but other modalities to which it responds are sensitized as well.

With respect to mechanosensitivity, nociceptors at the op-posite extremes of sensitivity are most illustrative of the limita-tions of even a functional definition of a nociceptor. Nociceptors with low mechanical thresholds for response and those with very high mechanical thresholds for response (i.e., sleeping nociceptors) are both clinically important. Mechanosensitive sensory neurons with low thresholds for response have long been classified as non-nociceptors (because it was considered that nociceptors had to have response thresholds in the nox-ious range). Some mechanosensitive skin, joint, and many vis-ceral sensory neurons have low thresholds for response (i.e., in the nonnoxious range) but possess characteristics that suggest an important role in pain. First, they encode stimulus inten-sity well into the noxious range and, moreover, typically give greater responses to all intensities of stimulation than do noci-ceptors with high mechanical thresholds for response. Second, they sensitize after tissue insult. Unlike nociceptors with low mechanical thresholds, mechanically insensitive or sleeping nociceptors normally provide no information to the central nervous system but after tissue insult become spontaneously active and mechanosensitive.5

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2 PART ONE BASIC CONSIDERATIONS

functional heterogeneity is manifest both within the context of nociceptive signaling (i.e., subpopulations of nociceptors may underlie distinct “types” of pain such as cold allodynia or ther-mal hyperalgesia) and in the context of nonnociceptive func-tion (i.e., such as the maintenance of tissue integrity).

Even the two functional properties that have been viewed as common to all nociceptors, that they encode stimulus intensity into the noxious range and they sensitize, are, at best, generalities. As noted earlier, the so-called mechanically insensitive afferents shown to play such a prominent role in visceral pain11 and the burning pain associated with capsaicin12 do not appear to code stimulus intensity in the noxious range. Similarly, a variety of nociceptive afferents may be desensitized under the appropriate conditions.13–16 The link between the stimulus–response prop-erties of a given subpopulation of afferents and the functional role of these neurons in the context of nociceptive signaling has been further complicated by results of selective ablation/silencing studies in mice, where modality specific deficits in no-ciceptive processing are associated with the silencing of specific subpopulations of afferents despite electrophysiologic data sug-gesting that the majority of the afferents silenced/ablated are polymodal. For example, the majority of cutaneous neurons in the mouse that express the Mas1-related G protein-coupled receptor D (MrgprD) are polymodal nociceptors,17 yet mice in which this subpopulation of neurons has been ablated respond normally to changes in temperature, demonstrating, instead, a relatively selective mechanosensitivity deficit.18 Conversely, the voltage-gated sodium channel (VGSC) NaV1.8 is enriched in nociceptive afferents that are responsive to both noxious ther-mal (TRPV1-expressing) and mechanical (MrgprD-expressing) stimuli, yet mice in which NaV1.8-expressing neurons have been ablated respond to noxious heat but not noxious mechan-ical pressure or cold stimuli.19 Minimally, these results suggest that the behavioral response to noxious stimulation involves a number of factors in addition to the types of afferents that are activated.

All of this heterogeneity does not bode well for the devel-opment of broadly acting novel analgesics. That is, given our current understanding of the complexity of peripheral pain mechanisms, it seems unlikely that the range of intensities

Identification of Putative NociceptorsAs indicated earlier, nociceptors are defined classically in a func-tional context. However, in experimental situations where func-tion cannot be assessed, other criteria to classify a neuron as a nociceptor have been advanced. These include the presence or absence of axon myelination, cell size, and/or cell content (e.g., peptide or ion channel) as well as central termination pattern. Sensory neurons commonly identified as nociceptors are those with unmyelinated (C-fiber) axons, small cell body diameters (,20 or 25 m), that terminate in the superficial layers of the spinal cord dorsal horn. The presence or absence of certain markers (e.g., the tetrodotoxin (TTX)-resistant sodium chan-nel NaV1.8, the transient receptor potential vanilloid receptor TRPV1) have been used to identify subsets of nociceptors. More recently, with the ability to analyze many genes expressed in a single cell, patterns of gene expression have been used to fur-ther define subpopulations of nociceptors.6,7 With respect to cell body size and myelination, it should be appreciated that there exist some large diameter cells with heavily myelinated and rap-idly conducting axons that have been documented functionally to be nociceptors.8 Conversely, many nonnociceptors have un-myelinated axons, and thus, axon myelination or cell diameter cannot be applied as reliable criteria to define a nociceptor. Sim-ilarly, identifying nociceptors by content or by what genes they express has limitations. Two examples of this are that (1) cells other than nociceptors express TRPV1 and (2) the features of the subset of nociceptors in the mouse skin that stain positive for the isolectin B4 do not apply to the visceral innervation.9 And although patterns of gene expression may enable identi-fication of additional subpopulations of nociceptive sensory neurons,10 the extent to which any particular pattern reliably reveals a neuron’s function remains to be established.

The fact that no single criterion can be used to identify all nociceptors combined with the growing number of examples to the ways in which these neurons are heterogeneous is at the core of a growing concern over the utility of the term. In addition to the anatomical, biochemical, and physiologic heterogeneity in the afferent population, generally referred to as nociceptors, there is functional heterogeneity. As will be discussed later, this

BOX 3.1 Microneurography

The development of a method to record from human nerve fibers in situ,191,192 termed microneurography, provided an unparalleled opportu-nity to expand our knowledge about peripheral sensory receptors, includ-ing nociceptors. The method involves percutaneous insertion of the tip of a sharp, insulated metal microelectrode into a nerve (e.g., peroneal or radial nerve) and the application of search stimuli to sites distal to the electrode. In earlier work, mechanical search stimuli (e.g., von Frey filaments) were used, and accordingly, only mechanosensitive afferents were studied. An electrical search stimulus (surface electrode), however, has become favored because the electrical stimulus identifies afferent fibers independent of sensitivity to natural stimulation. After an afferent fiber is isolated, the innervation territory can be drawn on the skin and the adequate, natural stimulus/stimuli determined.

Because microneurography can be easily coupled with a psychophys-ical approach, human subjects are able to describe stimulus-produced experiences (e.g., pain) while recording from single afferent fibers. Microneurography also has been expanded to include intraneural elec-trical stimulation of the fiber through the recording electrode, providing additional insight into the qualities of sensation produced, for example, by low- and high-frequency stimulation in addition to qualities associated with natural stimulation. Microneurography has confirmed in psychophysi-cal experiments sensations associated with activation of rapidly adapting (flutter, vibration) and slowly adapting (pressure) cutaneous mechanore-ceptors, A-mechanonociceptors (AM

[mechano]; sharp pain), C-polymodal no-

ciceptors (CM[mechano]

H[heat]

; dull, burning [heat] pain), and group IV muscle nociceptors (cramping pain).

Electrical search strategies have revealed a wider range of no-ciceptors, including190 A-mechanoheat (AMH), which have similar heat thresholds as CMH (C-polymodal) fibers and also typically re-spond to chemical stimuli; C-mechanonociceptors (CM), C-heat (CH); C-mechano- and heat-insensitive (CM

iH

i, or sleeping nociceptors);

and C-mechanoinsensitive-histamine responsive (CMiHis1, or itch

fibers193). Microneurography has also been extended to psycho-physical study of pathologic pain states in humans. In a study of patients suffering from erythromelalgia, a condition characterized by painful, red, and hot extremities, a propor tion of CM

iH

i fibers

were found to be spontaneously active or sensitized to mechanical stimuli. Because CM

iH

i fibers also mediate the axon flare reflex,

their hyperexcitability was considered to contribute to the patients’ ongoing pain and tenderness as well as the redness and warming in this pain syndrome. In patients with painful peripheral neuropathy, Ochoa et al.194 repor ted hyperexcitability in CMH and CM

iH

i fibers.

Signs of hyperexcitability included reduced thresholds to mechanical and heat stimuli, spontaneous activity, and increased responses to stimulation. In diabetic neuropathic pain patients, Ørstavik et al.195 found that the ratio of CMH to CM

iH

i fibers was reduced by about

50%, apparently due to loss of mechanical and heat responsiveness in CMH fibers.

These and future studies will help to understand which nociceptors (and nonnociceptors) in which conditions contribute to spontaneous, on-going pain as well as stimulus-evoked pain and what therapeutic strate-gies are most effective.


CHAPTER 3 PERIPHERAL PAIN MECHANISMS AND NOCICEPTOR SENSITIzATION 3

Four distinct events are necessary for a nociceptor to con-vey information to the central nervous system about noxious stimuli impinging on peripheral tissues (Fig. 3.1—the events are discussed fully in the following paragraphs). First, “energy” from the stimulus (mechanical, thermal, or chemical) must be converted into an electrical signal. This process, referred to as signal transduction, results in a generator potential or depolar-ization of the peripheral terminal. Second, the generator poten-tial must initiate an action potential, the rapid “all or nothing” change in membrane potential that constitutes the basic unit of electrical activity in the nervous system. This process is some-times referred to as transformation. Third, the action potential must be successfully propagated from the peripheral terminal to the central terminal. And fourth, the propagated action po-tential invading the central terminal must drive a sufficient in-crease in intracellular calcium ions to enable release of enough transmitter to initiate the whole process once again in the second-order neuron. Distinct sets of proteins underlie each of these processes and are, therefore, the targets of a wide variety of therapeutic interventions.

STIMULUS TRANSDUCTIONAn important implication of the fact that nociceptive afferents terminate in “free nerve endings” is that they are not dependent on other cell types for the transduction of a noxious stimulus. That is, proteins responsible for transduction should be intrin-sic to the nociceptor. Consistent with this suggestion, isolated sensory neurons are responsive to thermal (both heating27,28 and cooling29,30), mechanical,31,32 and a wide variety of chemical stimuli, including both endogenous33,34 and exogenous35,36 com-pounds that activate nociceptors in vivo. Proteins involved in the transduction of each stimulus modality have been identified (see Gold37 for review).

With the exception of transient receptor potential (TRP) chan-nels (see the following discussion), two-pore potassium channels (K2P), and possibly the acid-sensing ion channels (ASICs), chemo-transducers are, in general, only activated by chemical stimuli (and not also mechanical or thermal stimuli) and encompass various families of proteins that respond to specific molecules such as adenosine triphosphate (ATP)38 and protons.39 There is also compelling evidence to support the suggestion that different members of the TRP superfamily underlie thermal transduction of temperatures ranging from the very cold (TRPA140) to the very hot (TRPV241), with receptors for cool,42 warm,43,44 and hot45 in between. Subsequent research, however, suggests that in contrast to traditional chemoreceptors, TRP family members are not modality-specific as all are activated by specific chemicals46 and several contribute to mechanical transduction.32,47,48

Because the only sensation associated with TRPV1 activation is pain, this receptor has received considerable attention from pain researchers. Data from an array of studies paint a picture of TRPV1 as an excellent example of a polymodal receptor; it is activated by exogenous compounds such as capsaicin and resiniferatoxin, endogenous compounds ranging from protons to lipids, as well as noxious heat.49 TRPV1 is also present on the central terminals of nociceptive afferents where it also facilitates transmission of noxious mechanical stimuli.50–52 Furthermore, excessive activation of the receptor with compounds such as cap-saicin results in desensitization of the nociceptive terminal to all modes of stimuli, a process that underlies the therapeutic efficacy of topical capsaicin application.53 More recently, intrathecal ap-plication of resiniferatoxin has been used to selectively ablate the central terminals of TRPV1 containing nerve terminals, resulting in a sustained block of nociceptive transmission.54,55 There is also evidence that TRPV1 receptor antagonists may have some anal-gesic efficacy, although their therapeutic potential may be limited by a small but significant hyperthermia associated with systemic administration of blood–brain barrier permeable analogs.53

within any one modality (thermal, mechanical) that is encoded by a nociceptor and initiates nociceptive transmission involves only a single voltage- or ligand-gated channel. Similarly, even if the ability to sensitize is one means of functionally defining a peripheral neuron as a nociceptor, the endogenous mediators and factors that contribute to an increase in the excitability of nociceptors (i.e., sensitization) are numerous and synergetic and differ in different pain conditions (e.g., inflammation, nerve injury). A sampling of the complexities and contributors to sensitization are discussed in the following section.

Why include in a clinical textbook of pain management a chapter on peripheral pain mechanisms and nociceptors? There are many reasons. First, in most cases, blockage of peripheral nociceptor activity removes the “drive” for the experience of pain. Further, if a primary goal is to develop mechanism-based strategies for pain management, it is critical that character-istics of a key player—the nociceptor—are fully understood. Finally, nociceptor characteristics change as the local environ-ment in which they reside changes (inflammation, nerve injury, etc.). We discuss in the following section how nociceptors are activated, differ in different tissues, and contribute to the ex-perience of pain and how their behavior changes when they become sensitized. Where possible, we have added relevant clinical examples and have inserted text boxes to elaborate on key issues.

Nociceptor CharacteristicsANATOMY OF THE NOCICEPTORAs stated earlier, nociceptors are sensory neurons with a cell body located in dorsal root, trigeminal, or nodose gan-glia. All sensory neurons arising from these ganglia are pseudo-unipolar neurons with a central process terminating in the central nervous system (e.g., spinal dorsal horn) and a peripheral process terminating in a peripheral target such as the skin, muscle, or viscera. Both central and peripheral pro-cesses terminate in a branching pattern referred to as a termi-nal arbor. The extent of the peripheral arbor depends on the afferent type and site of innervation with the general rule that the higher the spatial resolution for sensory discrimination, the smaller the terminal arbor. In contrast to low-threshold afferents that are responsive to nonnoxious stimuli such as brush or vibration and which terminate in specialized struc-tures, such as Ruffini endings or Merkel disks,20,21 nociceptors are said to have “free” (unencapsulated) nerve endings be-cause peripheral terminals of these afferents do not appear to be associated with any specific cell type.22

Both light and electron micrographic analyses of peripheral nociceptor terminals reveal complex anatomical structures. As suggested earlier, the structure of the terminal arbor varies with target of innervation. There is also evidence that subpop-ulations of nociceptive afferents have distinct terminal arbor patterns within the same structure. For example, the terminal arbor morphology of cutaneous C fibers varies according to whether the C fiber is peptidergic (i.e., expresses substance P or calcitonin gene-relative peptide) or expresses the MrgprD.23 On the other hand, the terminal morphology of a subpopula-tion of high-threshold A fibers that appear to mediate the pain associated with hair pull24 is similar to that of a subpopulation of low-threshold mechanosensitive A fibers that appear to un-derlie the sensation of gentle stroking of the skin.25 Evidence also suggests that distinct but overlapping subpopulations of nociceptors may signal distinct aspects of the painful experi-ence (i.e., sensory/discriminative vs. emotional/motivational),26 suggesting that it may someday be possible to selectively treat the suffering associated with chronic pain while still enabling patients to appropriately respond to noxious stimuli in their environment.



P P P

P2X3 AntagonistCOX inhibitorAnti-NGF

ATPPGE2NGFTNFα Anti-TNFα

TherapeuticsMediatorTissue Insult

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B1, 25HT1A, 2, 7

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FIGURE 3.1 Nociceptive afferents terminate as free nerve endings in skin and other tissues. A: Their principal sensory functions consist of (1) transduction of external or internal chemical or physical stimuli into generator potentials, (2) transformation of a generator potential into an action potential, (3) propagation of the action potential toward the central nervous system, and (4) release of neurotransmitters and neuromodulators into the superficial dorsal horn of the spinal cord or brainstem. Nociceptive afferents also release transmitters in the periphery, a process that contributes a neurogenic component to inflammation (not shown). The sensory neuron cell body (soma, 5) appears to be a site critical for the integration of neural activity. Proteins and signaling molecules are delivered to the soma via axonal transport mechanisms (not shown) under normal conditions, which may contribute to aberrant or ectopic activity under pathologic conditions. Many of the proteins responsible for each of these processes, both under normal and pathologic conditions, have been identified. Although not a complete list, several lines of data implicate each of the proteins and mediators illustrated in each subpanel. B: Transduction: In naive tissue, proteins thought to play a role in mechanotransduction include transient receptor po-tential vanilloid type 4 (TRPV4), acid-sensing ion channel type 3 (ASIC-3), and the low-threshold voltage-gated calcium channel (VGCC) CaV3.2. Several different classes of TRP channels are involved in transduction of changes in temperature from noxious cold (TRPA1, ankyrin type 1), cool (TRPM8, melastatin type 8), warm (TRPV4), and hot (TRPV1, vanilloid type 1). Many chemoreceptors are present in nociceptive afferents including those involved in the response to tissue acidosis (TRPV1 and ASIC-3), noxious organic compounds (e.g., aldehydes at TRPA1), and endogenous chemicals (e.g., ATP at P2X3, the ionotropic purine receptor type 3). A wide variety of other receptors for both pro- and anti-inflammatory (not shown) mediators are also pres-ent on the terminals of nociceptive afferents. These include G protein-coupled receptors (GPCRs) responsive to E-type prostaglandins (EP), bradykinin (B) types 1 and 2, and serotonin (5-HT) types 1A, 2, and 7. Tyrosine receptor kinases (TRK), responsive to trophic factors such as nerve growth factor (NGF) and artemin, are present as are receptors for cytokines such as TNF- and interleukin 1-. Also depicted are transmitters such as ATP stored in epithelial cells. Following tissue insult, there are changes in nociceptive terminals that result in both an increase in sensitivity to noxious stimuli as well as the emergence of membrane depolarization. There are increases in the density of several transducers as well as posttranslational modifi-cations (depicted as phosphorylation, P) that increase channel activity or sensitivity such that the transducers are activated by lower intensity stimuli.



FIGURE 3.1 (continued) These changes are brought about by actions of inflammatory mediators such as ATP, prostaglandin E2, NGF, and TNF- that can directly depolarize nociceptive terminals, drive posttranslational changes via the activation of second messenger cascades, and/or alter the expression of transducers via influencing transcription and/or translation. All of these processes may be facilitated as a result of an increase in the release of mediators from epithelial cells, resident (mast) cells, and recruited (macrophages) immune cells. Several therapeutics currently in use or in development act via suppressing the actions of proinflammatory mediators. The specific pattern of changes and mediators depends on many factors, including the type and site of injury, time after injury, and previous history of the injured tissue as well as age and sex, which also influence the relative efficacy of the therapeutic intervention. C: Spike initiation: The appropriate anatomical distribution of ion channels is critical for normal function. A number of ion channels play an important role in determining the threshold for spike initiation and upstroke of the spike. Action potential threshold appears to be critically regulated by potassium (K1) channels, which include voltage-gated K1 channels (KV), inward rectifying K1 channels (KIR), two-pore K1 channels (K2P), large-conductance calcium-modulated K1 channels (BK), and small conductance calcium-dependent K1 channels (SK). The nonselective inward rectifying cation channel (HCN) also contributes to action potential threshold. In some cases, the low-threshold calcium channel (CaV3.2) may contribute to action potential threshold, but when present, CaV3.2 appears to play a more prominent role in mediating burst activity. The voltage-gated sodium channel (VGSC) NaV1.9 may also contribute to establishing action potential threshold. Finally, the channels responsible for the upstroke of the action potential include the VGSCs NaV1.7 and NaV1.8. As with transduction, there are a number of changes in ion channels that affect action potential threshold and spike initiation to increase in the excitability of nociceptive afferents in the presence of insult. These changes include a decrease in K1 channel density and/or current and an increase in CaV3.2, HCN, and NaV channel density and/or activity. These changes are the result of both posttranslational modifications and/or changes in transcription driven by the same inflammatory mediators that influence transduction. Thus, several of the therapeutics listed under transduction may also act by inhibiting changes in channels underlying spike initiation. Other drugs may act via direct inhibition of the ion channels underlying spike initiation (which include local anesthetics, tricyclic antidepressants [TCAs], and several cycloox-ygenase inhibitors). K1 channel openers such as retigabine may also have efficacy in increasing the threshold for spike initiation. D: Action potential propagation: The ion channels underlying action potential propagation are distinct from those underlying spike initiation. The channel most prominently implicated in action potential propagation is the VGSC NaV1.6. In myelinated axons, NaV1.6 is clustered at nodes of Ranvier, whereas in unmyelinated axons, the channel is distributed throughout the axon. Following insult, however, the pattern of channel expression can change, including redistribution of VGSCs NaV1.7 and/or NaV1.8 to the cell membrane. The dependence of action potential propagation on VGSCs confers the therapeutic efficacy of sodium channel blocking compounds such as local anesthetics, TCAs, and some cyclooxygenase (COX) inhibitors. E: Ectopic activity: As is true for all neurons in the absence of tissue insult, the soma serves as the supply depot for the rest of the neuron, synthesizing and packaging the proteins, transmitters, and lipids that will be used throughout the cell. Although the soma is capable of generating action potentials and is likely depolarized in response to neural activity in the axons, it is not necessary for action potentials to invade the soma for information to propagate from the periphery to the central nervous system. In nociceptive afferents, the VGSC NaV1.8 can be the primary, if not the only sodium channel in the cell underlying action potential generation. In the presence of nerve injury, however, changes in the soma and/or proximal axon can include an increase in transducer pro-teins that may make the soma responsive to mechanical, thermal, and chemical stimuli, an increase in sodium channels that may lead to membrane instability (manifesting as oscillatory behavior), and an increase in inflammatory mediators and their receptors. The result of such changes is that the soma and/or proximal axon may become a source of aberrant or ectopic activity. An important implication of this activity is that local administration of therapeutic agents to block activity arising from peripheral terminals may not provide pain relief. Given the source of much of this activity, therapeutic interventions designed to block sodium channels and/or inhibit the actions of inflammatory mediators are predicted to have the greatest efficacy.

Na� Ch.NaV1.6 NaV1.6

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(continued)



Of the three modalities of noxious stimuli, molecular mech-anisms of mechanotransduction remain the most elusive. Many mechanically sensitive proteins have been identified,56 but none appears to be both necessary and sufficient for mechanotrans-duction in nociceptive afferents. Data from null mutant mice, where the deletion of a single putative mechanotransducer re-sults in an increase in mechanosensitivity in one population of afferents and a decrease in others,57–59 suggests that several different proteins are likely to work together in specific sub-populations of afferents to enable responses to specific forms of mechanical stimuli (e.g., stretch or compression). This picture is complicated further by the observation that some mechano-transducers such as piezo2 contribute to mechanotransduction in specific subsets of sensory neurons.60 That even these more specialized forms of mechanosensitivity reflect intrinsic prop-erties of afferents are suggested by the emergence of mecha-nosensitivity at the severed ends of a subpopulation of axons

within hours of transection.61 Despite the slow progress in this area, identification of a nociceptor specific mechanotransducer blocker remains an active area of investigation because of its therapeutic potential in light of the fact that mechanical hy-persensitivity is the primary complaint associated with the vast majority of chronic pain syndromes.62–64

Whereas mechanotransduction is an intrinsic property of many nociceptors, there is evidence that epithelial cells may also be mechanosensitive.65–67 Because these cells may store and release transmitters such as ATP, it has been suggested that af-ferent activity evoked with mechanical stimulation of periph-eral structures such as the bladder,65 gastrointestinal tract,68 and skin66,67,69 may be secondary to the transduction event that has occurred in the epithelial cell that subsequently releases a chemical mediator capable of activating nearby nociceptor ter-minals. The implication of this mechanism from a therapeutic perspective is that it may be possible to attenuate mechanical

P

Tissue Insult

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InhibitoryIonotropic R.

GABA-A

Na�Ch.NaV1.8

K�Ch.KvBK

Ca2�Ch.CaV2.2CaV2.1CaV1.3

TherapeuticsCOX inhibitorsOR agonistsAR agonistsBenzodiazepine receptor agonistK� channel openersCa2� channel blockers

Large dense core vesicle—substance P, CGRP

Small clear vesicle—Glutamate

Excitatorylonotropic R.

P2XTRPV1

InhibitoryGPCRs

OR�-AR

ExcitatoryGPCRs

EPB1,2

P

FIGURE 3.1 (continued) F: Transmitter release: The release of transmitter at the central terminals of nociceptive afferents is essential for transmis-sion of nociceptive information to the central nervous system. This process is calcium-dependent—extracellular calcium enters the central terminal generally via high-threshold VGCCs that are activated following invasion of the action potential into the central terminals. N-type channels (CaV2.2) are the most abundant, but P/Q-type (CaV2.1) and L-type (CaV1.3) are also present. CaV2.2 is most readily modulated following activation of inhibitory GPCRs, serving as the primary mechanism for the therapeutic efficacy of intrathecal opioid receptor and alpha adrenergic receptor (-AR) agonists. Transmitters present in nociceptive afferents are generally packaged in vesicles referred to as small clear vesicles, which generally contain the ex-citatory amino acid glutamate, and large dense-core vesicles, which contain, among other things, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP). There are a number of excitatory ionotropic receptors, including P2X3 and TRPV1, that appear to facilitate transmitter release from the central terminals. Inhibition of the central terminal may involve activation of voltage-gated (KV) and calcium-modulated (BK) K1 chan-nels. Under normal conditions, presynaptic ionotropic g-aminobutyric acid (GABA) receptors (GABAA) play a major role in mediating presynaptic inhibi-tion of the central terminals of nociceptive afferents. The VGSC that appears to play a major role in enabling spike invasion of the central terminals of nociceptive afferents is NaV1.8. Finally, excitatory GPCRs (e.g., EP and 1, 2 receptors) are also present. As with other steps in the process, a number of changes occur in the central terminals of nociceptive afferents after tissue insult that contribute to the transmission of nociceptive informa-tion. These include an increase in the

2

1 subunit in VGSCs. This subunit is important for trafficking channels to the membrane and, importantly, is

a binding site for gabapentin and pregabalin. There is also an increase in neuropeptide expression, the emergence of additional excitatory receptors, modulation of ion channels such as NaV1.8, and a decrease in K1 currents that facilitate nociceptive signaling. Interestingly, there is a growing body of evidence suggesting that there may be changes in GABAA receptor signaling as well such that activation of these receptors may become excitatory secondary to changes in the regulation of intracellular chloride. This issue is complicated by the fact that benzodiazepine receptor agonists may have therapeutic efficacy in the presence of tissue insult which appears to involve, at least in part, activation of presynaptic receptors. From the therapeu-tic perspective, it is important to note that following tissue insult, in particular that associated with inflammation, there may be an increase in the expression of inhibitory receptors, ultimately facilitating the therapeutic efficacy of opioid and adrenergic receptor agonists. Additional therapeutic interventions may also involve inhibitors of VGCCs, K1 channel openers, and inhibitors to inflammatory mediators.

F



levels in sensory neurons,82,83 and intracellular calcium influence activity of calcium,84 potassium,85 and chloride86 channels in sensory neurons. Conversely, the sodium/potassium/chloride cotransporter and the bicarbonate/chloride exchangers in sen-sory neurons influence intracellular chloride concentrations, which, in turn, influence the impact of type A g-aminobutyric acid (GABAA) on sensory neurons.87

The results of several recent studies suggest that influencing the magnitude and/or spread of the generator potential may underlie the next generation of therapeutics. Because of the limitations of the nonspecific nerve block produced by local an-esthetics, strategies that enable a more selective afferent block are an active area of investigation. In one such strategy, a viral vector was used to drive expression of an ionotropic glycine receptor, an inhibitory ligand-gated ion channel not normally present in sensory neurons. Sensory neurons innervating spe-cific targets such as the skin or the bladder could be selectively infected by injecting the viral vector into the intended target tissue. This strategy enabled the reversal of hypersensitivity with the local administration of glycine.88 A similar approach involves the use of optogenetics, or light-activated proteins that can be selectively expressed in targeted cell types. Inhibitory light-activated ion pumps, such as halorhodopsin or archaer-hodopsin,89 have been used to study the role of specific subpop-ulations of afferents in pain, where both viral vector-based90–92 and site-specific recombinase technology93 have been used. Ex-pression of these light-activated proteins can be restricted to specific subpopulations of afferents via a variety of approaches including the site of infection, the virus serotype used,90 as well as cell-specific promoters.92 Given the spatial and temporal control of the afferent inhibition afforded by these strategies, combined with the ability to selectively target subpopulations of afferents, the therapeutic potential is tremendous.

ACTION POTENTIAL GENERATIONVGSCs are responsible for the upstroke of the action poten-tial in virtually all excitable tissue. As their name implies, these channels are gated (opened and closed) by changes in mem-brane potential. VGSCs are generally composed of an subunit and up to two subunits. The subunit is a large molecule (200 kD) that contains all features necessary for a functional channel including voltage sensor, ion selectivity filter, channel pore, and inactivation gate.94 Ten subunits have been identi-fied, nine of which form functional channels in heterologous expression systems. The channels encoded by each of these sub-units can be distinguished by a combination of pharmacologic and biophysical properties. Eight of these nine subunits are present in the nervous system, all eight of which are present in nociceptive afferents95 at one point during development, with six of these detectable in the adult.96 Subunits influence chan-nel gating properties as well as trafficking and localization in the plasma membrane.97 Four subunits have been identified, at least three of which are present in nociceptive afferents.95

The VGSC subunit primarily responsible for action potential initiation in the majority of nociceptors is NaV1.8. This subunit is unique in several ways. First, it is normally only expressed in primary afferents98,99 where it is primarily ex-pressed in nociceptors.100 This unique pattern of distribution, in combination with its primary function in spike initiation, makes it an ideal target for novel therapeutics.101 Although in no way mitigating the therapeutic potential of this chan-nel, recent evidence suggests that it may contribute to action potential propagation in the distal peripheral axon in addition to its role in action potential initiation.102 Second, NaV1.8 has a relatively high threshold for activation. Whereas many other VGSCs begin to activate at membrane potentials between 250 and 240 mV, a depolarization to 230 mV or greater is neces-sary to activate NaV1.8.103 This feature may explain, at least

hypersensitivity in several peripheral tissues with the appropri-ate antagonist of the responsible chemoreceptors on nocicep-tive afferents.70 Consistent with this idea, there is evidence that mechanical hypersensitivity observed in several visceral struc-tures can be attenuated with ATP receptor antagonists.71 That a similar mechanism may contribute to chemotransduction, at least in the intestine, was recently suggested by the observation that enterochromaffin cells are not only activated by a vari-ety of chemicals but are electrically excitable and form synap-tic connections with primary afferents.72 However, serotonin appears to be the primary transmitter underlying the signaling from these cells to primary afferents, possibly accounting for the therapeutic efficacy of 5-HT3 receptor antagonists for the treatment of visceral pain.73

Aberrant expression of transducers may play a significant role in chronic pain associated with tissue injury. The presence of a functional transducer at a site other than the peripheral terminal may underlie the emergence of ectopic activity and contribute to ongoing or spontaneous pain. Such changes have been most extensively detailed following nerve injury, where, as mentioned earlier, mechanical sensitivity is detectable in cut axons within hours of injury61 and may persist in neuro-mas indefinitely.74 Similarly, chemosensitivity, particularly to adrenergic agonists, develops at both the cut ends of nocicep-tive afferents75 as well as within the ganglia itself,76 contrib-uting to ectopic activity arising from both the site of injury and from the ganglia. The emergence of ectopic activity arising from sites distant to the site of injury may explain why inter-ventions targeting the site of injury are unsuccessful.

PASSIVE ELECTROPHYSIOLOGIC PROPERTIES AND THE SPREAD OF THE GENERATOR POTENTIALA generator potential at a primary afferent terminal ending is not equivalent to an action potential propagated along that afferent’s axon. The generator potential decays over distance from the point of origin as a function of membrane resistance, membrane capacitance, and internal resistance of the nerve ter-minal and may or may not be propagated beyond the terminal ending. Generally considered stable properties, there is evi-dence for dynamic remodeling of the terminal arbor of central nervous system neurons77 which may influence both membrane capacitance and internal resistance; it remains to be determined whether such changes may also impact passive conduction of generator potentials in peripheral terminals. In contrast, a number of ion channels have been identified that may establish resting membrane resistance and therefore the spread of the generator potential within the terminal arbor.78 This issue is im-portant to nociceptive signaling because action potential initia-tion does not always occur at the site of stimulus transduction.79 Consequently, the magnitude of the generator potential at the site of action potential initiation must be greater than or equal to action potential threshold. Thus, at least in some fiber types, it may be possible to block nociceptor signaling and therefore pain, with manipulations such as the local administration of potassium (K1) channels openers80,81 that decrease membrane resistance and therefore spread the generator potential.

Although ion channels have been the focus of research into mechanisms controlling neuronal excitability of peripheral neu-rons, recent evidence suggests that ion pumps and exchangers may contribute as well. Some pumps and exchangers, such as the sodium/potassium ATPase and the sodium/calcium ex-changer, are electrogenic; that is, they generate net flux of ions when active and therefore may contribute directly to resting membrane potential and consequently neuronal excitability. They may also contribute indirectly, via an influence on intra-cellular ion concentrations and consequently the activity and/or actions of other ion channels. For example, the sodium/calcium exchanger, and the calcium ATPase control intracellular calcium

•AQ1•



bursting activity, as the channels underlie a sustained mem-brane depolarization after a single action potential that pro-vides the driving force for the initiation of subsequent action potentials.117 This feature has led some to speculate that selec-tive T-type channel blockers may be particularly effective for treating paroxysmal pain116 such as that associated with tri-geminal neuralgia.

The focus on NaV1.8 has been on its role in action poten-tial initiation in the periphery. Nevertheless, there is also evi-dence that the channels are present and functional at central nociceptor terminals.118,119 At the central terminal, the channel appears to facilitate the spread of the invading action potential throughout the terminal arbor and consequently the release of transmitter from the primary afferent. There is also a grow-ing body of evidence suggesting that action potentials may also be initiated at the central terminals of nociceptors, where they are conducted antidromically to the periphery.120 This activity, referred to as the dorsal root reflex, appears to play a signifi-cant role in the neurogenic inflammation that develops follow-ing tissue injury.

ACTION POTENTIAL PROPAGATIONNaV1.8 and NaV1.7 underlie action potential generation and even propagation over the first 5 to 10 mm of peripheral axon. However, a different set of VGSCs underlies action potential propagation into the central nervous system in the absence of tissue injury. NaV1.6 appears to be the subunit primarily responsible for propagation in both myelinated and unmyelin-ated axons of both nociceptive and nonnociceptive afferents,121 although data from a small molecule inhibitor of NaV1.7 sug-gests this channel may contribute to action potential conduc-tion as well122 (but see Zhang et al.113 for data suggesting this channel may not be as selective as originally thought). Unfor-tunately, the distribution of NaV1.6 in the peripheral nervous system in combination with its widespread expression in the central nervous system precludes selective block of propagation in nociceptors via a NaV1.6 specific mechanism. Nevertheless, block of these channels with local anesthetics and/or TTX, or more recently small interfering RNA knockdown,123 remains an effective means of blocking input into the central nervous system.

TRANSMITTER RELEASEVoltage-gated calcium channels (VGCC) are primarily respon-sible for the initial influx of calcium necessary for initiation of machinery underlying the release of neurotransmitters. Like VGSCs, these multisubunit channels consist of a large sub-unit that contains all of the features necessary for a functional channel as well as a subunit. Ten subunits have been iden-tified, encoding channels that are commonly defined by their threshold for activation or pharmacologic properties.124 T-type channels (CaV3.1 to CaV3.3), as mentioned earlier, have a low threshold for activation, whereas all others have a high threshold for activation. The high-threshold channels are fur-ther subdivided based on their sensitivity to specific channel blockers: L-type channels (CaV1.1 to CaV1.4) are blocked by dihydropyridines such as nimodipine, N-type channels (CaV2.2) are blocked by the snail toxin -conotoxin GVIA, P/Q-type channels (CaV2.1) are blocked by the spider toxin -agatoxin IVA, and R-type channels (CaV2.3) are blocked by the spider toxin SNX-482.125 In contrast to VGSCs, VGCCs are not effectively targeted to the plasma membrane in the absence of the 2 subunit complex.126 A single subunit also appears to be important for efficient gating.127 All VGCC subtypes are present in nociceptors. And whereas there is evidence that all high-threshold calcium channels may contribute to transmitter release, N-type channels appear to play a dominant role in the release of transmitter from nociceptors.128

in part, why greater intensity stimuli are generally needed for nociceptor activation. Third, NaV1.8 is relatively resistant to steady-state inactivation, a voltage-dependent process whereby channels residing in a closed or resting state transition to an inactive state before they ever get a chance to open. Recov-ery from the inactivated state requires membrane hyperpolar-ization; thus, inactivated channels cannot contribute to the upstroke of the action potential. Even a small sustained depo-larization to 250 mV can inactivate virtually all other VGSCs. However, NaV1.8 is still fully available for activation at this membrane potential.103 Fourth, NaV1.8 recovers from inacti-vation rapidly.104 These last two features enable the channel to underlie sustained activity in the face of a persistent depolar-ization that might be observed in the presence of inflammatory mediators. Fifth, NaV1.8 is resistant to cooling-induced inacti-vation.105 Other VGSCs are completely inactivated at tempera-tures at or below 18 C. However, NaV1.8 is still functional at temperatures down to 4 C, enabling the burning pain associ-ated with noxious cold stimuli. Sixth, in contrast to all but one other VGSC subunit (NaV1.9), NaV1.8 is resistant to TTX and is therefore referred to as a TTX-resistant channel.

Whereas NaV1.8 is critical for action potential initiation in nociceptors, data from the study of rodent sensory neurons suggests that in many of these afferents, NaV1.8 works in con-cert with another VGSC subunit, NaV1.7.106 The NaV1.7 subunit has unique features enabling it to play a significant role in spike initiation.107 That this channel may play a critical role in nociceptor activity is suggested by the recent discovery of in-dividuals possessing both gain-of-function and loss-of-function point mutations in this subunit. Strikingly, two distinct pain syndromes—primary erythermalgia (PE) and paroxysmal ex-treme pain disorder (PEPD)—reveal the specific impact of gain-of-function mutations.108 PE is associated with burning pain in the hands and feet, and PEPD is initially associated with pain in the rectum that ultimately progresses to include trigeminal structures including the eye and jaw. The unique distribution of these pain syndromes, in light of the widespread distribution of NaV1.7 in the peripheral nervous system as well as neuroen-docrine tissues, highlights the importance of other channels in sculpting the response properties of sensory neurons. Further-more, in contrast to the impact of the gain-of-function muta-tions, loss-of-function mutations that result in nonfunctional channels are associated with a complete insensitivity to pain.109 Although the data from patients with these rare genetic muta-tions is intriguing, recent evidence suggests the role of NaV1.7 in nociceptive processing may be more complicated than origi-nally anticipated. That is, evidence from null mutant mice sug-gests that the loss of function phenotype may not only involve sympathetic postganglionic neurons110 but a compensatory upregulation of the endogenous opioid enkephalin in primary afferents.111 Furthermore, electrophysiologic analysis of noci-ceptive afferents in patients with gain-of-function mutations in these channels suggests that at least at rest, the afferents are hy-poexcitable.112 These observations raise the possibility that the pain phenotypes associated with mutations in NaV1.7 reflect developmental processes independent of any ongoing influence of the channel in controlling afferent excitability. Furthermore, in contrast to isolated sensory neurons from the rat, where NaV1.7 appears to contribute to action potential initiation, re-cent evidence suggests that this may not be the case in human sensory neurons.113

Low voltage–activated, or T-type, calcium channels may also contribute to spike initiation in the periphery.114 Whereas there is compelling evidence that these channels are present in high density in low-threshold D-hair afferents,115 there is also evidence that they may be present in a subpopulation of noci-ceptors as well.116 The biophysical properties of these channels enable them to play a particularly important role in mediating



to sensitization include products of arachidonic acid metabo-lism (e.g., prostaglandin E2 [PGE2]), histamine, serotonin, pro-tons and ATP, but the list has grown quite extensively and now also includes cytokines, chemokines, growth factors, peptides, etc., some of which are released from immune competent cells attracted to the site of insult (e.g., macrophages), from nearby cells (e.g., mast cells), or from nociceptor (and other) nerve termi-nals (e.g., peptides). Interestingly, despite the variety of mediators capable of producing nociceptor sensitization, several appear to play particularly important roles. This list includes prostanoids, as evidenced by the antihyperalgesic efficacy of nonsteroidal an-ti-inflammatory drugs (NSAIDs) that act via inhibition of cyclo-oxygenase and thus prostanoid synthesis.136,137 More recently, the importance of tumor necrosis factor alpha (TNF-) in chronic inflammatory conditions has been highlighted by the antino-ciceptive efficacy of compounds such as etanercept which are designed to bind and inactivate TNF- released at sites of inflam-mation.138,139 Finally, nerve growth factor (NGF) appears to play a major role in orchestrating a variety of signaling cascades nec-essary for an inflammatory response and therefore has also been targeted with antibody-based strategies.140–144

The mechanisms that trigger changes in nociceptor excit-ability are not fully known. A growing body of evidence sug-gests that despite what appears to be a bewildering assortment of mediators and membrane receptors, there are a relatively small number of intracellular pathways that underlie their actions. For example, both prostaglandins and bradykinin, which are among the most extensively studied inflammatory mediators, act at G protein-coupled receptors. Two major G protein- dependent pathways have been implicated. One involves a stimulatory G protein, Gs, which drives activation of adenylate cyclase, resulting in an increase in cyclic adenosine monophosphate (cAMP) and the activation of protein kinase A (PKA).145,146 The other involves a Gq-dependent pathway, re-sulting in the activation of phospholipase C, the liberation of diacylglycerol (DAG) and IP3, and the subsequent activation of protein kinase C (PKC).147,148 The PKC- isoform appears to play a particularly important role in nociceptor sensitization. Other mediators that appear to play important roles in noci-ceptor sensitization, such as TNF- and interleukin (IL) 1, utilize a mitogen-activated protein kinase (MAPK)-dependent pathway ultimately resulting in the activation of p38.149 Still other mediators directly activate ion channels. For example, protons (which increase in concentration during inflammation) act at TRPV1 and ASICs. ASIC-3 is important to pain associ-ated with ischemia, such as that which occurs during angina, and deep muscle pain where protons and lactic acid accumu-late. In contrast, ATP and its metabolites act at ionotropic P2X and metabotropic P2Y receptors to modulate nociceptor excitability.

The relatively novel, if not paradoxical, mechanism of sensiti-zation involving ionotropic GABAA receptors that we described in the action potential generation section serves as an example of the dynamic interplay between second messenger signaling cascades and the regulation of ion channels. Activation of these receptors are normally inhibitory, even in nociceptive afferents where they drive membrane depolarization, because of the rel-atively high intracellular chloride concentration maintained in these neurons.150 The GABAA-mediated depolarization in these neurons is inhibitory, as suggested earlier, because of the decrease in membrane resistance, referred to as shunting, as well as the depolarization-induced activation of low-threshold voltage-gated potassium channels.151 In the presence of persistent inflammation, there is a constitutive shift in the balance of ty-rosine kinase to tyrosine phosphatase activity, such that there is a net increase in protein phosphorylation. This increase results in a net increase in functional GABAA receptors in the plasma membrane because of a decrease in receptor internalization.152

The dominant role N-type channels play in mediating trans-mitter release from nociceptive afferent terminals makes them an ideal target for both endogenous and exogenous analgesics. Opioid and adrenergic receptor agonists act through inhibitory G protein-coupled receptors which enable inhibition of VGCCs via two major intracellular pathways. The first is a rapid, mem-brane delimited pathway involving G protein g-subunit dis-placement of the VGCC subunit, resulting in a “sleepy” or “unwilling” channel that requires a larger membrane depolar-ization for channel opening.127 The second pathway involves more traditional second messenger kinase dependent signaling with a slower onset and offset.129 Interestingly, neither pathway results in complete VGCC block, yet both result in a dramatic inhibition of transmitter release. This amplification effect re-flects the fact that there is considerable cooperativity of cal-cium in mediating vesicle fusion to the cell membrane that is necessary for transmitter release; four to five calcium ions are needed to trigger vesicle fusion.130 This amplification effect is also likely to facilitate the use of relatively low concentrations of the N-type channel blocker SNX-111 (Prialt), enabling the block of transmitter release from nociceptive afferent terminals in the superficial dorsal horn while minimizing side effects as-sociated with block of channels at more distant sites. Finally, although additional mechanisms likely contribute to the ther-apeutic efficacy of drugs like gabapentin and pregabalin, that have been shown to bind to the 2 subunit, the most com-pelling model involves the inhibition of membrane traffick-ing of the α subunit. That is, following nerve injury, there is a dramatic increase in 21 subunit expression in nociceptive afferents.131 This increase in expression appears to facilitate the trafficking of VGCC α subunits to nociceptive afferent cen-tral terminals, which in turn, appears to facilitate transmitter release and consequently the increase in pain associated with nerve injury. These compounds block the increase in α subunit trafficking via an 21-dependent mechanism.132

Although VGCCs play a dominant, if not essential, role in the increase in intracellular calcium needed for transmitter re-lease, recent evidence suggests other calcium regulatory pro-teins contribute to the regulation of the amplitude and duration of the increase in intracellular calcium and, consequently, trans-mitter release. These include the sarco-endoplasmic reticulum ATPase/ryanodine receptor system,133,134 the plasma membrane calcium ATPase,135 mitochondria,135 and the sodium/calcium exchanger.83 Consistent with the suggestion, these calcium reg-ulatory proteins influence nociception, selective genetic knock-down of the sodium/calcium exchanger, a regulatory protein that attenuates the calcium transient, in a subpopulation of no-ciceptive afferents decreases nociceptive threshold.83

Nociceptor SensitizationSensitization is a characterizing feature of nociceptors; nonno-ciceptors do not sensitize following tissue insult. Sensitization represents an increase in nociceptor excitability, which is ex-pressed and defined as an increase in response to a noxious stimulus. Sensitization is also typically accompanied by a re-duction in the threshold for activation and occasionally by the development of ongoing, spontaneous activity. Nociceptor sen-sitization is the cause of primary hyperalgesia (i.e., increased pain produced by stimulation at the site of tissue insult) and is important because nociceptor sensitization is the trigger for initiation of an increase in excitability of central neurons in the nociceptive pathway, an event termed central sensitization.

An increase in nociceptor excitability is a reflection of changes in the behavior of nociceptor voltage- and/or ligand-gated ion channels produced by actions of endogenous substances either released or synthesized at the site of tissue insult or attracted there. Endogenous substances considered classically to contribute



carrageenan, or inflammatory mediators such as IL-6.148 These changes were manifest up to weeks after the complete resolution of hypersensitivity associated with the initiating stimulus and were most prominently manifest as a dramatic increase in the duration of the hypersensitivity associated with a subsequent challenge, although an increase in the magnitude of the hyper-sensitivity has also been described. That is, although the intra-dermal injection of PGE2 into naive skin results in a relatively transient hypersensitivity lasting 1 hour, the same injection into primed skin results in hypersensitivity detectable for more than 24 hours. This phenomenon has been proposed to contrib-ute to the transition from acute to chronic pain.161

In the context of a discussion of mechanisms underlying nociceptor sensitization, the mechanisms implicated in the manifestation of hyperalgesic priming are notable for several reasons. First, although there are data suggesting changes within the central nervous system, notably involving a descending dopaminergic input to the superficial dorsal horn, contribute to the manifestation of hyperalgesic priming,162 the bulk of data generated so far suggest that changes in nociceptive afferents may play a critical role in the emergence of chronic pain. Sec-ond, as further evidence of the differential role of distinct sub-populations of nociceptive afferents in nociceptive signaling, two distinct forms of hyperalgesic priming have been described, where type I depends on a subpopulation of neurons that do not express the neuropeptides calcitonin gene-related peptide (CGRP) or substance P and bind the plant lectin IB4,163 whereas type II depends on a subpopulation of neurons that do express these neuropeptides and do not bind IB4.164 Third, there appear to be sex differences in the mechanisms underlying both the initiation (type I)165 and maintenance (type II)164 of the primed state. And fourth, although a cAMP response element binding protein (CREB)-dependent change in gene expression appears to be necessary for the establishment of the primed state, local translation appears to be critical for the manifestation of the more robust response observed in primed state, at least for type I priming.166 Given the length of some nociceptive affer-ents, in particular those innervating the distal appendages, local control of translation may prove to be an important therapeutic target for the treatment of pain.

Clinical Implications of Nociceptor FunctionAs researchers have begun to explore the basis for chronic pain syndromes generally associated with specific body regions and/or organs (e.g., temporomandibular joint disorder [TMJD], inflammatory bowel disease [IBD], irritable bowel syndrome [IBS], or painful bladder syndrome [formerly interstitial cys-titis or IC]), a number of common themes have emerged that are likely to impact future treatment approaches. First, spe-cific mechanisms underlying injury-induced sensitization of nociceptors vary as a function of target of innervation. For example, inflammation-induced sensitization of masseter muscle afferents appears to reflect a decrease in a specific sub-population of voltage-gated potassium channels.167 The same channels do not appear to contribute to the sensitization of TMJ afferents.168 Similarly, inflammation-induced increases in the excitability of bladder sensory neurons appear to reflect one pattern of changes in voltage-gated169 ion channels, whereas the inflammation-induced increase in the excitability of sensory neurons innervating the stomach,170–172 ileum,173,174 or colon175 reflect other patterns. Although tissue-specific patterns of in-flammation may contribute to these differences between sub-populations of afferents, differences persist when the response to inflammatory mediators is studied in vitro.176,177 These obser-vations imply that it may be possible, if not necessary, to treat

The result is an increase in GABA-evoked current and an in-crease in the magnitude of the GABA-evoked depolarization. These changes are further augmented by a decrease in the den-sity of low-threshold potassium current.151 The result is a shift in GABA signaling, from inhibition to excitation.

This picture gets a little more complicated when one consid-ers the ceramide/sphingosine 1-phosphate (S1P) system because ceramide, a potent proinflammatory sphingolipid-generated de novo from hydrolysis of sphingomyelin or synthesis from ser-ine and palmityl CoA, can directly activate protein kinases and phosphatases as well as serve as a precursor to S1P, which acts via G protein-coupled receptors to sensitize nociceptive neurons.153 And although a common set of second messengers, including phosphatidylinositol 3-kinase, phospholipase Cg, and extracel-lular signal-regulated kinase (ERK), underlie the acute sensitizing actions of trophic factors such as NGF, these mediators signal via a distinct class of receptors that contain intrinsic tyrosine kinase activity, referred to as tropomyosin receptor kinase, or Trk recep-tors.154 Channels underlying transduction and spike initiation, in particular TRPV1 and NaV1.8, respectively, appear to be final common targets for this diverse array of mediators and second messenger pathways.155 Phosphorylation of specific residues on the channel or associated proteins results in increases in channel density and/or increases in channel function. Importantly, many inflammatory mediators including ceramide/SP1 and NGF drive long-term increases in nociceptor excitability via changes in gene expression, and mechanisms contributing to long-term changes in gene expression, such as alterations in DNA acetylation and methylation, have emerged as potential therapeutic targets.156,157

Although mechanisms underlying changes in gene expres-sion associated with the emergence of persistent pain have received considerable attention, there is a growing body of evidence suggesting that changes in protein translation are also important. One of the first studies to implicate a change in the regulation of protein translation per se in manifestation of persistent pain was focused on the role of TRPV1 in per-sistent inflammatory pain. The authors observed that inflam-mation is associated with an increase in TRPV1 protein in the absence of a detectable increase in messenger RNA.158 It was subsequently demonstrated that inflammatory mediators such as NGF and IL-6 could rapidly increase protein translation via the phosphorylation of proteins responsible for control over the rate-limiting step of protein translation, that of the initia-tion of protein synthesis.159 The protein initiation step involves the assembly of a complex of eukaryotic initiation factors (eIF) eIF4E and G among others, as well as the dissociation of the eIF4E binding protein (4EBP), from eIF4E. At least two distinct signaling pathways have been identified underlying the actions of NGF and IL-6. In addition to the activation of PI3K, NGF activation of TrkA drives the activation of PI3K which subse-quently activates Akt (also known as protein kinase B). Akt then activates the mammalian target of rapamycin (mTOR), which facilitates the initiation of translation via facilitating the dissociation of 4EBP from eIEF4E, as well as the activation of eIF4G, via the phosphorylation of both proteins. In contrast, IL-6 drives the activation of ERK, which subsequently acti-vates MAP-kinase interacting kinase (MNK), which facilitates the initiation of translation via the phosphorylation of eIF4E (which facilitates the eIF4E/G complex formation).159 Interest-ingly, this rapid regulation of translation contributes not only to the magnitude and duration of both the mechanical and thermal hypersensitivity associated with acute inflammation and the actions of inflammatory mediators like NGF and IL-6 but also the manifestation of more persistent changes in noci-ceptive processing, referred to as hyperalgesic priming.160

Hyperalgesic priming is the term used to describe the changes in nociceptive processing associated with an initial inflammatory insult, such as that associated with the subcutaneous injection of



of stimulation also are now able to activate nociceptors. In addition, spontaneous activity may develop. In the aggregate, central nervous system input from sensitized nociceptors, awakened sleeping nociceptors, and spontaneously active noci-ceptors is significantly increased. For example, approximately 24% of human cutaneous C fibers are sleeping nociceptors,190 comprising significant new input to the central nervous system if awakened. Consequently, the amount of neurotransmitters (as well as perhaps their relative proportions) released onto central neurons is increased, which in turn alters the excitabil-ity of central neurons. The increase in excitability of central neurons is manifest as an increase in the size of the cutaneous receptive field (i.e., secondary hyperalgesia) or area of tender-ness referred from deep structures, particularly the viscera. Although the principal focus of study of mechanisms of central sensitization has been the spinal cord, it should be appreciated that nociceptor-driven changes in central excitability extend throughout the central nervous system.

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pain arising from a specific structure with a specific interven-tion. Second, specific mechanisms underlying insult-induced sensitization of nociceptors also vary as a function of the type of insult. For example, acute phosphorylation-dependent mod-ulation of the voltage-gated sodium channel NaV1.8 results in an increase in current which contributes to an inflammation- induced increase in nociceptor excitability.178,179 In contrast, following traumatic nerve injury, redistribution of NaV1.8 to the axons of uninjured afferents appears to be necessary for the expression of mechanical hypersensitivity associated with nerve injury.180 The dynamic allodynia that often develops after nerve injury, however, likely represents more than only a redis-tribution of NaV1.8 (Box 3.2).

With respect to the viscera, because each organ receives in-nervation from two nerves, the effect of organ insult can be different in the two groups of sensory neurons that innervate the organ (e.g., Wang et al.,181 Traub182). These observations underscore the importance of developing diagnostic criteria that enable identification of the factors primarily responsible for ongoing pain. Third, as clearly indicated by the discussion of hyperalgesic priming, the history of the nociceptor influences the response to subsequent challenge. Furthermore, there is ev-idence of a developmental window within which injury may produce permanent changes in nociceptors.183,184 With the de-velopment of more specific therapeutic tools, patient history may become a critical factor in the identification of the most appropriate intervention. Fourth, there is evidence for sex differences in both the excitability of different groups of no-ciceptors185 as well as the response to tissue injury.186,187 These differences appear to be mediated, at least in part, through the actions of gonadal hormones and may contribute to sex dif-ferences in the manifestation of a number of chronic pain syn-dromes. There is also evidence for age-dependent changes in nociceptor function.188 Given the ever-growing proportion of aging adults, this particular issue in is need of further investiga-tion. Finally, all of these factors interact with what are clearly genetic differences in pain and analgesic mechanisms.189

As indicated earlier, the consequences of tissue insult are not limited only to changes in the excitability of nociceptors and the awakening of sleeping nociceptors. Because sensitization leads to an increased response to noxious stimuli and a de-crease in response threshold, previously nonnoxious intensities

One of the most striking positive symptoms of neuropathic pain is dy-namic mechanical allodynia, a term used to describe pain resulting from light tactile stimuli that would never be considered noxious in the absence of nervous system injury. This phenomena can be recapitu-lated with a subcutaneous injection of capsaicin.196 Data from detailed psychophysical analysis in combination with microneurography197 and dorsal horn recording in animal models198 all suggest that dynamic me-chanical allodynia reflects activity in low-threshold afferents that signal pain as a result of changes in the central nervous system. The exact nature of these changes, generally referred to as central sensitization, is still debated (e.g., see Polgar and Todd199 and Schoffnegger et al.200), but it is clear that they depend on activity in nociceptive afferents. Another possibility, however, is that low-threshold afferents undergo a phenotypic switch such that they begin to transmit information to the central nervous system as if they were nociceptive afferents. Consis-tent with this possibility, there is evidence following peripheral nerve in-jury that a subpopulation of putative nonnociceptive afferents begins to express the neuropeptide, substance P,201 although this change does not appear to be necessary for the expression of allodynia.202 Evidence of a third possibility, which would involve sprouting of nonnociceptive afferents into more superficial layers of the dorsal horn to enable low threshold drive of nociceptive dorsal horn neurons,203 remains largely unsubstantiated.204–206

BOX 3.2 Afferent Contributions to Neuropathic Pain and Allodynia



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peripheral pain mechanisms and nociceptor sensitization

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