CHAPTER 3 Peripheral Pain Mechanisms and Nociceptor Sensitization

MICHAEL S. GOLD

Pain has been categorized by duration (acute vs. chronic), denoted as mechanically insensitive nociceptors) but develop location (superficial or deep; cutaneous, bone/joint, muscle, or spontaneous activity and mechanosensitivity after exposure to viscera), and cause or type (inflammatory, neuropathic, can- inflammatory and other endogenous mediators. These types cer). Generally, activation of and/or ongoing activity in specific of nociceptors—low threshold and sleeping—as well as other subpopulations of primary afferent neurons underlies the expe- subpopulations of sensory neurons that may contribute to the rience of pain regardless of how it is categorized. Accordingly, sensation of pain following tissue injury are considered further primary afferents are key players in understanding mechanisms in the discussion of sensitization in the following section. of, and managing, pain. Sir Charles Sherrington anticipated by many decades the ex- Functional Characterization of Nociceptors istence sensory receptors that respond to noxious stimuli, that he called nociceptors and thereby provided for us the opera- As suggested earlier, there are many types of nociceptors, our tional definition of stimuli that are noxious (i.e., stimuli that knowledge of which has been advanced by human psychophys- damage or threaten damage of tissue). Two considerations are ical studies while recording from afferent fibers (Box 3.1). In important to this discussion. First, Sherrington functionally de- human skin, for example, there exist nociceptors that respond fines the nociceptor by its response to a noxious stimulus (e.g., only to mechanical, only to cold thermal, or only to hot thermal a nociceptive withdrawal reflex, pain). Second, the definition of stimuli as well as those that are insensitive to both mechani- an applied stimulus as noxious is based on the response to the cal and heat stimuli (mechanically insensitive or sleeping no- stimulus applied to skin and subcutaneous structures. ciceptors).1,2 The most abundant is the polymodal nociceptor, Sherrington’s definition of a nociceptor continues to the which responds to mechanical, thermal, and chemical stimuli. present day. However, the term has undergone change and In general, nociceptors that innervate skin have the broadest challenge over the past 100 years and may be nearing the end range of modality selectivity, whereas nociceptors innervating of its utility in the face of the growing understanding of the deeper structures tend to be less modality-selective and more heterogeneity in the neurons that may not fit so comfortably polymodal in character.3 For example, mechanical sensitivity is under this umbrella term. It is therefore important to consider, a prominent feature of visceral and joint nociceptors because within the context of our current knowledge, how a nociceptor stimuli adequate for their activation include hollow organ dis- is defined, identified, and studied, as the interpretation of this tension and overrotation, respectively. Many of these nocicep- information will directly affect the management of pain. tors also respond to chemical and/or thermal stimuli as well, All would agree that a nociceptor is a sensory receptor although the functional significance of thermal sensitivity in which, when activated or active, can contribute to the expe- deep tissues is uncertain. An important characteristic of poly- rience of pain. Nociceptors are present in skin, muscle, joints, modal nociceptors, whether the modalities of stimulation to and viscera, although the density of innervation (i.e., the num- which they respond are two or all three, is that when sensitized ber and distribution of sensory endings) varies between and (e.g., by an inflammatory insult), responses to the other mo- within tissues. As originally described by Sherrington, a noci- dality or modalities of stimuli to which it responds are all in- ceptor is the peripheral sensory terminal (i.e., the site of energy creased.4 That is, it is not only the mechanosensitive modality, transduction—see following section), although commonly the for example, that becomes sensitized, but other modalities to term is used to also include the cell body (in a dorsal root, which it responds are sensitized as well. trigeminal, or nodose ganglion) and its central termination in With respect to mechanosensitivity, nociceptors at the op- the spinal cord or brainstem. Beyond this, agreement about im- posite extremes of sensitivity are most illustrative of the limita- portant features of nociceptors is less uniform. One of the best tions of even a functional definition of a nociceptor. Nociceptors examples of why this definition is becoming problematic is the with low mechanical thresholds for response and those with observation that injury-induced changes in the central nervous very high mechanical thresholds for response (i.e., sleeping system underlie the emergence of allodynia, pain in response to nociceptors) are both clinically important. Mechanosensitive normally innocuous stimuli. Allodynia is problematic for this sensory neurons with low thresholds for response have long definition of nociceptor because pain is mediated by activity been classified as non-nociceptors (because it was considered in low-threshold afferents that in the absence of tissue injury that nociceptors had to have response thresholds in the nox- would, if anything, contribute to the suppression of pain. That ious range). Some mechanosensitive skin, joint, and many vis- is, these afferents would not be considered nociceptors despite ceral sensory neurons have low thresholds for response (i.e., in the fact that they contribute to the experience of pain. the nonnoxious range) but possess characteristics that suggest Furthermore, because stimuli adequate for activation of an important role in pain. First, they encode stimulus inten- nociceptors differ between tissues (e.g., tissue damage is not sity well into the noxious range and, moreover, typically give always required), defining a noxious stimulus has become a greater responses to all intensities of stimulation than do noci- challenge. For example, some nociceptors in skin and joints ceptors with high mechanical thresholds for response. Second, and most nociceptors in the viscera have low thresholds for me- they sensitize after tissue insult. Unlike nociceptors with low chanical activation that do not conform to the condition that mechanical thresholds, mechanically insensitive or sleeping stimulus intensity must be either damaging or threaten dam- nociceptors normally provide no information to the central age. Further, so-called “silent” or “sleeping” nociceptors are nervous system but after tissue insult become spontaneously unresponsive to intense mechanical stimulation (and are better active and mechanosensitive.5

1

Rathmell5e_Ch003.indd 1 3/23/18 12:12 AM 2 PART ONE BASIC CONSIDERATIONS

BOX 3.1 Microneurography The development of a method to record from human nerve fibers in Electrical search strategies have revealed a wider range of no- situ,191,192 termed microneurography, provided an unparalleled opportu- ciceptors, including190 A-mechanoheat (AMH), which have similar nity to expand our knowledge about peripheral sensory receptors, includ- heat thresholds as CMH (C-polymodal) fibers and also typically re- ing nociceptors. The method involves percutaneous insertion of the tip spond to chemical stimuli; C-mechanonociceptors (CM), C-heat (CH);

of a sharp, insulated metal microelectrode into a nerve (e.g., peroneal C-mechano- and heat-insensitive (CMiHi, or sleeping nociceptors);

or radial nerve) and the application of search stimuli to sites distal to and C-mechanoinsensitive-histamine responsive (CMiHis1, or itch the electrode. In earlier work, mechanical search stimuli (e.g., von Frey fibers193). Microneurography has also been extended to psycho- filaments) were used, and accordingly, only mechanosensitive afferents physical study of pathologic pain states in humans. In a study of were studied. An electrical search stimulus (surface electrode), however, patients suffering from erythromelalgia, a condition characterized

has become favored because the electrical stimulus identifies afferent by painful, red, and hot extremities, a proportion of CMiHi fibers fibers independent of sensitivity to natural stimulation. After an afferent were found to be spontaneously active or sensitized to mechanical

fiber is isolated, the innervation territory can be drawn on the skin and stimuli. Because CMiHi fibers also mediate the axon flare reflex, the adequate, natural stimulus/stimuli determined. their hyperexcitability was considered to contribute to the patients’ Because microneurography can be easily coupled with a psychophys- ongoing pain and tenderness as well as the redness and warming in ical approach, human subjects are able to describe stimulus-produced this pain syndrome. In patients with painful peripheral neuropathy, 194 experiences (e.g., pain) while recording from single afferent fibers. Ochoa et al. reported hyperexcitability in CMH and CMiHi fibers. Microneurography also has been expanded to include intraneural elec- Signs of hyperexcitability included reduced thresholds to mechanical trical stimulation of the fiber through the recording electrode, providing and heat stimuli, spontaneous activity, and increased responses to additional insight into the qualities of sensation produced, for example, stimulation. In diabetic neuropathic pain patients, Ørstavik et al.195

by low- and high-frequency stimulation in addition to qualities associated found that the ratio of CMH to CMiHi fibers was reduced by about with natural stimulation. Microneurography has confirmed in psychophysi- 50%, apparently due to loss of mechanical and heat responsiveness cal experiments sensations associated with activation of rapidly adapting in CMH fibers. (flutter, vibration) and slowly adapting (pressure) cutaneous mechanore- These and future studies will help to understand which nociceptors

ceptors, A-mechanonociceptors (AM[mechano]; sharp pain), C-polymodal no- (and nonnociceptors) in which conditions contribute to spontaneous, on-

ciceptors (CM[mechano]H[heat]; dull, burning [heat] pain), and group IV muscle going pain as well as stimulus-evoked pain and what therapeutic strate- nociceptors (cramping pain). gies are most effective.

Identification of Putative Nociceptors functional heterogeneity is manifest both within the context of nociceptive signaling (i.e., subpopulations of nociceptors may As indicated earlier, nociceptors are defined classically in a func- underlie distinct “types” of pain such as cold allodynia or ther- tional context. However, in experimental situations where func- mal hyperalgesia) and in the context of nonnociceptive func- tion cannot be assessed, other criteria to classify a neuron as a tion (i.e., such as the maintenance of tissue integrity). nociceptor have been advanced. These include the presence or Even the two functional properties that have been viewed as absence of axon myelination, cell size, and/or cell content (e.g., common to all nociceptors, that they encode stimulus intensity peptide or ion channel) as well as central termination pattern. into the noxious range and they sensitize, are, at best, generalities. Sensory neurons commonly identified as nociceptors are those As noted earlier, the so-called mechanically insensitive afferents with unmyelinated (C-fiber) axons, small cell body diameters shown to play such a prominent role in visceral pain11 and the (,20 or 25 m), that terminate in the superficial layers of the burning pain associated with capsaicin12 do not appear to code spinal cord dorsal horn. The presence or absence of certain stimulus intensity in the noxious range. Similarly, a variety of markers (e.g., the tetrodotoxin (TTX)-resistant sodium chan- nociceptive afferents may be desensitized under the appropriate nel NaV1.8, the transient receptor potential vanilloid receptor conditions.13–16 The link between the stimulus–response prop- TRPV1) have been used to identify subsets of nociceptors. More erties of a given subpopulation of afferents and the functional recently, with the ability to analyze many genes expressed in a role of these neurons in the context of nociceptive signaling single cell, patterns of gene expression have been used to fur- has been further complicated by results of selective ablation/ ther define subpopulations of nociceptors.6,7 With respect to cell silencing studies in mice, where modality specific deficits in no- body size and myelination, it should be appreciated that there ciceptive processing are associated with the silencing of specific exist some large diameter cells with heavily myelinated and rap- subpopulations of afferents despite electrophysiologic data sug- idly conducting axons that have been documented functionally gesting that the majority of the afferents silenced/ablated are to be nociceptors.8 Conversely, many nonnociceptors have un- polymodal. For example, the majority of cutaneous neurons myelinated axons, and thus, axon myelination or cell diameter in the mouse that express the Mas1-related G protein-coupled cannot be applied as reliable criteria to define a nociceptor. Sim- receptor D (MrgprD) are polymodal nociceptors,17 yet mice in ilarly, identifying nociceptors by content or by what genes they which this subpopulation of neurons has been ablated respond express has limitations. Two examples of this are that (1) cells normally to changes in temperature, demonstrating, instead, a other than nociceptors express TRPV1 and (2) the features of relatively selective mechanosensitivity deficit.18 Conversely, the the subset of nociceptors in the mouse skin that stain positive voltage-gated sodium channel (VGSC) NaV1.8 is enriched in for the isolectin B4 do not apply to the visceral innervation.9 nociceptive afferents that are responsive to both noxious ther- And although patterns of gene expression may enable identi- mal (TRPV1‑expressing) and mechanical (MrgprD-expressing) fication of additional subpopulations of nociceptive sensory stimuli, yet mice in which NaV1.8‑expressing neurons have neurons,10 the extent to which any particular pattern reliably been ablated respond to noxious heat but not noxious mechan- reveals a neuron’s function remains to be established. ical pressure or cold stimuli.19 Minimally, these results suggest The fact that no single criterion can be used to identify all that the behavioral response to noxious stimulation involves a nociceptors combined with the growing number of examples to number of factors in addition to the types of afferents that are the ways in which these neurons are heterogeneous is at the core activated. of a growing concern over the utility of the term. In addition All of this heterogeneity does not bode well for the devel- to the anatomical, biochemical, and physiologic heterogeneity opment of broadly acting novel analgesics. That is, given our in the afferent population, generally referred to as nociceptors, current understanding of the complexity of peripheral pain there is functional heterogeneity. As will be discussed later, this mechanisms, it seems unlikely that the range of intensities

Rathmell5e_Ch003.indd 2 3/23/18 12:12 AM CHAPTER 3 Peripheral Pain Mechanisms and Nociceptor Sensitization 3

within any one modality (thermal, mechanical) that is encoded Four distinct events are necessary for a nociceptor to con- by a nociceptor and initiates nociceptive transmission involves vey information to the central nervous system about noxious only a single voltage- or ligand-gated channel. Similarly, even stimuli impinging on peripheral tissues (Fig. 3.1—the events if the ability to sensitize is one means of functionally defining are discussed fully in the following paragraphs). First, “energy” a peripheral neuron as a nociceptor, the endogenous mediators from the stimulus (mechanical, thermal, or chemical) must be and factors that contribute to an increase in the excitability converted into an electrical signal. This process, referred to as of nociceptors (i.e., sensitization) are numerous and synergetic signal transduction, results in a generator potential or depolar- and differ in different pain conditions (e.g., inflammation, ization of the peripheral terminal. Second, the generator poten- nerve injury). A sampling of the complexities and contributors tial must initiate an action potential, the rapid “all or nothing” to sensitization are discussed in the following section. change in membrane potential that constitutes the basic unit of Why include in a clinical textbook of pain management a electrical activity in the nervous system. This process is some- chapter on peripheral pain mechanisms and nociceptors? There times referred to as transformation. Third, the action potential are many reasons. First, in most cases, blockage of peripheral must be successfully propagated from the peripheral terminal nociceptor activity removes the “drive” for the experience of to the central terminal. And fourth, the propagated action po- pain. Further, if a primary goal is to develop mechanism-based tential invading the central terminal must drive a sufficient in- strategies for pain management, it is critical that character- crease in intracellular calcium ions to enable release of enough istics of a key player—the nociceptor—are fully understood. transmitter to initiate the whole process once again in the Finally, nociceptor characteristics change as the local environ- second-order neuron. Distinct sets of proteins underlie each of ment in which they reside changes (inflammation, nerve injury, these processes and are, therefore, the targets of a wide variety etc.). We discuss in the following section how nociceptors are of therapeutic interventions. activated, differ in different tissues, and contribute to the ex- perience of pain and how their behavior changes when they STIMULUS TRANSDUCTION become sensitized. Where possible, we have added relevant An important implication of the fact that nociceptive afferents clinical examples and have inserted text boxes to elaborate on terminate in “free nerve endings” is that they are not dependent key issues. on other cell types for the transduction of a noxious stimulus. That is, proteins responsible for transduction should be intrin- Nociceptor Characteristics sic to the nociceptor. Consistent with this suggestion, isolated sensory neurons are responsive to thermal (both heating27,28 ANATOMY OF THE NOCICEPTOR and cooling29,30), mechanical,31,32 and a wide variety of chemical As stated earlier, nociceptors are sensory neurons with a stimuli, including both endogenous33,34 and exogenous35,36 com- cell body located in dorsal root, trigeminal, or nodose gan- pounds that activate nociceptors in vivo. Proteins involved in glia. All sensory neurons arising from these ganglia are the transduction of each stimulus modality have been identified pseudo-unipolar neurons with a central process terminating (see Gold37 for review). in the central nervous system (e.g., spinal dorsal horn) and a With the exception of transient receptor potential (TRP) chan- peripheral process terminating in a peripheral target such as nels (see the following discussion), two-pore potassium channels the skin, muscle, or viscera. Both central and peripheral pro- (K2P), and possibly the acid-sensing ion channels (ASICs), chemo- cesses terminate in a branching pattern referred to as a termi- transducers are, in general, only activated by chemical stimuli nal arbor. The extent of the peripheral arbor depends on the (and not also mechanical or thermal stimuli) and encompass afferent type and site of innervation with the general rule that various families of proteins that respond to specific molecules the higher the spatial resolution for sensory discrimination, such as adenosine triphosphate (ATP)38 and protons.39 There is the smaller the terminal arbor. In contrast to low-threshold also compelling evidence to support the suggestion that different afferents that are responsive to nonnoxious stimuli such as members of the TRP superfamily underlie thermal transduction brush or vibration and which terminate in specialized struc- of temperatures ranging from the very cold (TRPA140) to the very tures, such as Ruffini endings or Merkel disks,20,21 nociceptors hot (TRPV241), with receptors for cool,42 warm,43,44 and hot45 in are said to have “free” (unencapsulated) nerve endings be- between. Subsequent research, however, suggests that in contrast cause peripheral terminals of these afferents do not appear to to traditional chemoreceptors, TRP family members are not be associated with any specific cell type.22 modality-specific as all are activated by specific chemicals46 and Both light and electron micrographic analyses of peripheral several contribute to mechanical transduction.32,47,48 nociceptor terminals reveal complex anatomical structures. Because the only sensation associated with TRPV1 activation As suggested earlier, the structure of the terminal arbor varies is pain, this receptor has received considerable attention from with target of innervation. There is also evidence that subpop- pain researchers. Data from an array of studies paint a picture ulations of nociceptive afferents have distinct terminal arbor of TRPV1 as an excellent example of a polymodal receptor; it patterns within the same structure. For example, the terminal is activated by exogenous compounds such as capsaicin and arbor morphology of cutaneous C fibers varies according to resiniferatoxin, endogenous compounds ranging from protons whether the C fiber is peptidergic (i.e., expresses substance P to lipids, as well as noxious heat.49 TRPV1 is also present on the or calcitonin gene-relative peptide) or expresses the MrgprD.23 central terminals of nociceptive afferents where it also facilitates On the other hand, the terminal morphology of a subpopula- transmission of noxious mechanical stimuli.50–52 Furthermore, tion of high-threshold A fibers that appear to mediate the pain excessive activation of the receptor with compounds such as cap- associated with hair pull24 is similar to that of a subpopulation saicin results in desensitization of the nociceptive terminal to all of low-threshold mechanosensitive A fibers that appear to un- modes of stimuli, a process that underlies the therapeutic efficacy derlie the sensation of gentle stroking of the skin.25 Evidence of topical capsaicin application.53 More recently, intrathecal ap- also suggests that distinct but overlapping subpopulations of plication of resiniferatoxin has been used to selectively ablate the nociceptors may signal distinct aspects of the painful experi- central terminals of TRPV1 containing nerve terminals, resulting ence (i.e., sensory/discriminative vs. emotional/motivational),26 in a sustained block of nociceptive transmission.54,55 There is also suggesting that it may someday be possible to selectively treat evidence that TRPV1 receptor antagonists may have some anal- the suffering associated with chronic pain while still enabling gesic efficacy, although their therapeutic potential may be limited patients to appropriately respond to noxious stimuli in their by a small but significant hyperthermia associated with systemic environment. administration of blood–brain barrier permeable analogs.53

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4. Transmitter release

Spike Initiation Therapeutics Local anesthetics Tissue TCAs 5. Naive Insult COX inhibitors K ch. openers

P .Ectopic activity K؉ Ch Kv P KIR BK ؉ SK Na Ch. NaV1.7 2؉ NaV1.8 Ca Ch. NaV1.9 CaV3.2 P 3. Spike propagation

HCN Ch. C HCN1 C 2. Spike initiation

1. Transduction A C

Transduction Naive Tissue Insult Mediator Therapeutics ATP P2X3 Antagonist PGE2 COX inhibitor NGF Anti-NGF GPCRs TRK TNFα Anti-TNFα EP NGF B1, 2 Artemin Cytokine 5HT1A, 2, 7 TNFα IL1β TRPV1 TRPV1 TRPV4 TRPV4 TRPA1 ASIC-3 TRPA1 ASIC-3 Cav3.2 TRPM8 P2×3 P P P

B 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.

Rathmell5e_Ch003.indd 4 3/23/18 12:12 AM CHAPTER 3 Peripheral Pain Mechanisms and Nociceptor Sensitization 5

Spike Propagation Therapeutics Tissue Local anesthetics TCAs Naive Insult COX inhibitors

.Na؉ Ch. Na؉ Ch NaV1.6 NaV1.6 NaV1.7 NaV1.8

D

Ectopic Activity

Naive Tissue Insult Therapeutics Local anesthetics TCAs COX inhibitors Anti-TNF-␣

E FIGURE 3.1 (continued) These changes are brought about by actions of inflammatory mediators such as ATP, prostaglandin E2, NGF, and TNF-a 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. (continued)

Rathmell5e_Ch003.indd 5 3/23/18 12:12 AM 6 PART ONE BASIC CONSIDERATIONS

Transmitter Release

Naive Tissue Insult Inhibitory Ionotropic R. GABA-A NaϩCh. P NaV1.8

Excitatory Inhibitory P GPCRs GPCRs EP OR B1,2 ␣-AR

Excitatory ϩ lonotropic R. Ca2ϩCh. K Ch. Kv P2X CaV2.2 Therapeutics BK TRPV1 CaV2.1 COX inhibitors CaV1.3 OR agonists AR agonists Benzodiazepine receptor agonist Large dense core vesicle—substance P, CGRP Kϩ channel openers Small clear vesicle—Glutamate Ca2ϩ channel blockers F 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 (a-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 a2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.

Of the three modalities of noxious stimuli, molecular mech- within hours of transection.61 Despite the slow progress in this anisms of mechanotransduction remain the most elusive. Many area, identification of a nociceptor specific mechanotransducer mechanically sensitive proteins have been identified,56 but none blocker remains an active area of investigation because of its appears to be both necessary and sufficient for mechanotrans- therapeutic potential in light of the fact that mechanical hy- duction in nociceptive afferents. Data from null mutant mice, persensitivity is the primary complaint associated with the vast where the deletion of a single putative mechanotransducer re- majority of chronic pain syndromes.62–64 sults in an increase in mechanosensitivity in one population Whereas mechanotransduction is an intrinsic property of of afferents and a decrease in others,57–59 suggests that several many nociceptors, there is evidence that epithelial cells may different proteins are likely to work together in specific sub- also be mechanosensitive.65–67 Because these cells may store and populations of afferents to enable responses to specific forms of release transmitters such as ATP, it has been suggested that af- mechanical stimuli (e.g., stretch or compression). This picture ferent activity evoked with mechanical stimulation of periph- is complicated further by the observation that some mechano- eral structures such as the bladder,65 gastrointestinal tract,68 transducers such as piezo2 contribute to mechanotransduction and skin66,67,69 may be secondary to the transduction event that in specific subsets of sensory neurons.60 That even these more has occurred in the epithelial cell that subsequently releases a specialized forms of mechanosensitivity reflect intrinsic prop- chemical mediator capable of activating nearby nociceptor ter- erties of afferents are suggested by the emergence of mecha- minals. The implication of this mechanism from a therapeutic nosensitivity at the severed ends of a subpopulation of axons perspective is that it may be possible to attenuate mechanical

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hypersensitivity in several peripheral tissues with the appropri- levels in sensory neurons,82,83 and intracellular calcium influence ate antagonist of the responsible chemoreceptors on nocicep- activity of calcium,84 potassium,85 and chloride86 channels in tive afferents.70 Consistent with this idea, there is evidence that sensory neurons. Conversely, the sodium/potassium/chloride mechanical hypersensitivity observed in several visceral struc- cotransporter and the bicarbonate/chloride exchangers in sen- tures can be attenuated with ATP receptor antagonists.71 That sory neurons influence intracellular chloride concentrations, a similar mechanism may contribute to chemotransduction, at which, in turn, influence the impact of type A -aminobutyric least in the intestine, was recently suggested by the observation acid (GABAA) on sensory neurons.87 that enterochromaffin cells are not only activated by a vari- The results of several recent studies suggest that influencing ety of chemicals but are electrically excitable and form synap- the magnitude and/or spread of the generator potential may tic connections with primary afferents.72 However, serotonin underlie the next generation of therapeutics. Because of the appears to be the primary transmitter underlying the signaling limitations of the nonspecific nerve block produced by local an- from these cells to primary afferents, possibly accounting for esthetics, strategies that enable a more selective afferent block •AQ1• the therapeutic efficacy of 5-HT3 receptor antagonists for the are an active area of investigation. In one such strategy, a viral treatment of visceral pain.73 vector was used to drive expression of an ionotropic glycine Aberrant expression of transducers may play a significant receptor, an inhibitory ligand-gated ion channel not normally role in chronic pain associated with tissue injury. The presence present in sensory neurons. Sensory neurons innervating spe- of a functional transducer at a site other than the peripheral cific targets such as the skin or the bladder could be selectively terminal may underlie the emergence of ectopic activity and infected by injecting the viral vector into the intended target contribute to ongoing or spontaneous pain. Such changes have tissue. This strategy enabled the reversal of hypersensitivity been most extensively detailed following nerve injury, where, with the local administration of glycine.88 A similar approach as mentioned earlier, mechanical sensitivity is detectable in involves the use of optogenetics, or light-activated proteins that cut axons within hours of injury61 and may persist in neuro- can be selectively expressed in targeted cell types. Inhibitory mas indefinitely.74 Similarly, chemosensitivity, particularly to light-activated ion pumps, such as halorhodopsin or archaer- adrenergic agonists, develops at both the cut ends of nocicep- hodopsin,89 have been used to study the role of specific subpop- tive afferents75 as well as within the ganglia itself,76 contrib- ulations of afferents in pain, where both viral vector-based90–92 uting to ectopic activity arising from both the site of injury and site-specific recombinase technology93 have been used. Ex- and from the ganglia. The emergence of ectopic activity arising pression of these light-activated proteins can be restricted to from sites distant to the site of injury may explain why inter- specific subpopulations of afferents via a variety of approaches ventions targeting the site of injury are unsuccessful. including the site of infection, the virus serotype used,90 as well as cell-specific promoters.92 Given the spatial and temporal PASSIVE ELECTROPHYSIOLOGIC PROPERTIES AND control of the afferent inhibition afforded by these strategies, THE SPREAD OF THE GENERATOR POTENTIAL combined with the ability to selectively target subpopulations A generator potential at a primary afferent terminal ending is of afferents, the therapeutic potential is tremendous. not equivalent to an action potential propagated along that afferent’s axon. The generator potential decays over distance ACTION POTENTIAL GENERATION from the point of origin as a function of membrane resistance, VGSCs are responsible for the upstroke of the action poten- membrane capacitance, and internal resistance of the nerve ter- tial in virtually all excitable tissue. As their name implies, these minal and may or may not be propagated beyond the terminal channels are gated (opened and closed) by changes in mem- ending. Generally considered stable properties, there is evi- brane potential. VGSCs are generally composed of an  subunit dence for dynamic remodeling of the terminal arbor of central and up to two  subunits. The  subunit is a large molecule nervous system neurons77 which may influence both membrane (200 kD) that contains all features necessary for a functional capacitance and internal resistance; it remains to be determined channel including voltage sensor, ion selectivity filter, channel whether such changes may also impact passive conduction pore, and inactivation gate.94 Ten a subunits have been identi- of generator potentials in peripheral terminals. In contrast, a fied, nine of which form functional channels in heterologous number of ion channels have been identified that may establish expression systems. The channels encoded by each of these sub- resting membrane resistance and therefore the spread of the units can be distinguished by a combination of pharmacologic generator potential within the terminal arbor.78 This issue is im- and biophysical properties. Eight of these nine  subunits are portant to nociceptive signaling because action potential initia- present in the nervous system, all eight of which are present in tion does not always occur at the site of stimulus transduction.79 nociceptive afferents95 at one point during development, with Consequently, the magnitude of the generator potential at the six of these detectable in the adult.96  Subunits influence chan- site of action potential initiation must be greater than or equal nel gating properties as well as trafficking and localization in to action potential threshold. Thus, at least in some fiber types, the plasma membrane.97 Four  subunits have been identified, it may be possible to block nociceptor signaling and therefore at least three of which are present in nociceptive afferents.95 pain, with manipulations such as the local administration of The VGSC  subunit primarily responsible for action potassium (K1) channels openers80,81 that decrease membrane potential initiation in the majority of nociceptors is NaV1.8. resistance and therefore spread the generator potential. This subunit is unique in several ways. First, it is normally Although ion channels have been the focus of research into only expressed in primary afferents98,99 where it is primarily ex- mechanisms controlling neuronal excitability of peripheral neu- pressed in nociceptors.100 This unique pattern of distribution, rons, recent evidence suggests that ion pumps and exchangers in combination with its primary function in spike initiation, may contribute as well. Some pumps and exchangers, such makes it an ideal target for novel therapeutics.101 Although as the sodium/potassium ATPase and the sodium/calcium ex- in no way mitigating the therapeutic potential of this chan- changer, are electrogenic; that is, they generate net flux of ions nel, recent evidence suggests that it may contribute to action when active and therefore may contribute directly to resting potential propagation in the distal peripheral axon in addition membrane potential and consequently neuronal excitability. to its role in action potential initiation.102 Second, NaV1.8 has They may also contribute indirectly, via an influence on intra- a relatively high threshold for activation. Whereas many other cellular ion concentrations and consequently the activity and/or VGSCs begin to activate at membrane potentials between 250 actions of other ion channels. For example, the sodium/calcium and 240 mV, a depolarization to 230 mV or greater is neces- exchanger, and the calcium ATPase control intracellular calcium sary to activate NaV1.8.103 This feature may explain, at least

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in part, why greater intensity stimuli are generally needed for bursting activity, as the channels underlie a sustained mem- nociceptor activation. Third, NaV1.8 is relatively resistant to brane depolarization after a single action potential that pro- steady-state inactivation, a voltage-dependent process whereby vides the driving force for the initiation of subsequent action channels residing in a closed or resting state transition to an potentials.117 This feature has led some to speculate that selec- inactive state before they ever get a chance to open. Recov- tive T-type channel blockers may be particularly effective for ery from the inactivated state requires membrane hyperpolar- treating paroxysmal pain116 such as that associated with tri- ization; thus, inactivated channels cannot contribute to the geminal neuralgia. upstroke of the action potential. Even a small sustained depo- The focus on NaV1.8 has been on its role in action poten- larization to 250 mV can inactivate virtually all other VGSCs. tial initiation in the periphery. Nevertheless, there is also evi- However, NaV1.8 is still fully available for activation at this dence that the channels are present and functional at central membrane potential.103 Fourth, NaV1.8 recovers from inacti- nociceptor terminals.118,119 At the central terminal, the channel vation rapidly.104 These last two features enable the channel to appears to facilitate the spread of the invading action potential underlie sustained activity in the face of a persistent depolar- throughout the terminal arbor and consequently the release of ization that might be observed in the presence of inflammatory transmitter from the primary afferent. There is also a grow- mediators. Fifth, NaV1.8 is resistant to cooling-induced inacti- ing body of evidence suggesting that action potentials may also vation.105 Other VGSCs are completely inactivated at tempera- be initiated at the central terminals of nociceptors, where they tures at or below 18 C. However, NaV1.8 is still functional at are conducted antidromically to the periphery.120 This activity, temperatures down to 4 C, enabling the burning pain associ- referred to as the dorsal root reflex, appears to play a signifi- ated with noxious cold stimuli. Sixth, in contrast to all but one cant role in the neurogenic inflammation that develops follow- other VGSC  subunit (NaV1.9), NaV1.8 is resistant to TTX ing tissue injury. and is therefore referred to as a TTX-resistant channel. Whereas NaV1.8 is critical for action potential initiation ACTION POTENTIAL PROPAGATION in nociceptors, data from the study of rodent sensory neurons NaV1.8 and NaV1.7 underlie action potential generation and suggests that in many of these afferents, NaV1.8 works in con- even propagation over the first 5 to 10 mm of peripheral axon. cert with another VGSC  subunit, NaV1.7.106 The NaV1.7 However, a different set of VGSCs underlies action potential subunit has unique features enabling it to play a significant role propagation into the central nervous system in the absence in spike initiation.107 That this channel may play a critical role of tissue injury. NaV1.6 appears to be the subunit primarily in nociceptor activity is suggested by the recent discovery of in- responsible for propagation in both myelinated and unmyelin- dividuals possessing both gain-of-function and loss-of-function ated axons of both nociceptive and nonnociceptive afferents,121 point mutations in this subunit. Strikingly, two distinct pain although data from a small molecule inhibitor of NaV1.7 sug- syndromes—primary erythermalgia (PE) and paroxysmal ex- gests this channel may contribute to action potential conduc- treme pain disorder (PEPD)—reveal the specific impact of gain- tion as well122 (but see Zhang et al.113 for data suggesting this of-function mutations.108 PE is associated with burning pain in channel may not be as selective as originally thought). Unfor- the hands and feet, and PEPD is initially associated with pain tunately, the distribution of NaV1.6 in the peripheral nervous in the rectum that ultimately progresses to include trigeminal system in combination with its widespread expression in the structures including the eye and jaw. The unique distribution of central nervous system precludes selective block of propagation these pain syndromes, in light of the widespread distribution of in nociceptors via a NaV1.6 specific mechanism. Nevertheless, NaV1.7 in the peripheral nervous system as well as neuroen- block of these channels with local anesthetics and/or TTX, or docrine tissues, highlights the importance of other channels in more recently small interfering RNA knockdown,123 remains sculpting the response properties of sensory neurons. Further- an effective means of blocking input into the central nervous more, in contrast to the impact of the gain-of-function muta- system. tions, loss-of-function mutations that result in nonfunctional channels are associated with a complete insensitivity to pain.109 TRANSMITTER RELEASE Although the data from patients with these rare genetic muta- Voltage-gated calcium channels (VGCC) are primarily respon- tions is intriguing, recent evidence suggests the role of NaV1.7 sible for the initial influx of calcium necessary for initiation in nociceptive processing may be more complicated than origi- of machinery underlying the release of neurotransmitters. Like nally anticipated. That is, evidence from null mutant mice sug- VGSCs, these multisubunit channels consist of a large  sub- gests that the loss of function phenotype may not only involve unit that contains all of the features necessary for a functional sympathetic postganglionic neurons110 but a compensatory channel as well as a  subunit. Ten  subunits have been iden- upregulation of the endogenous opioid enkephalin in primary tified, encoding channels that are commonly defined by their afferents.111 Furthermore, electrophysiologic analysis of noci- threshold for activation or pharmacologic properties.124 T-type ceptive afferents in patients with gain-of-function mutations in channels (CaV3.1 to CaV3.3), as mentioned earlier, have a these channels suggests that at least at rest, the afferents are hy- low threshold for activation, whereas all others have a high poexcitable.112 These observations raise the possibility that the threshold for activation. The high-threshold channels are fur- pain phenotypes associated with mutations in NaV1.7 reflect ther subdivided based on their sensitivity to specific channel developmental processes independent of any ongoing influence blockers: L-type channels (CaV1.1 to CaV1.4) are blocked of the channel in controlling afferent excitability. Furthermore, by dihydropyridines such as nimodipine, N-type channels in contrast to isolated sensory neurons from the rat, where (CaV2.2) are blocked by the snail toxin -conotoxin GVIA, NaV1.7 appears to contribute to action potential initiation, re- P/Q-type channels (CaV2.1) are blocked by the spider toxin cent evidence suggests that this may not be the case in human -agatoxin IVA, and R-type channels (CaV2.3) are blocked by sensory neurons.113 the spider toxin SNX-482.125 In contrast to VGSCs, VGCCs are Low voltage–activated, or T-type, calcium channels may not effectively targeted to the plasma membrane in the absence 114 126 also contribute to spike initiation in the periphery. Whereas of the 2 subunit complex. A single  subunit also appears there is compelling evidence that these channels are present in to be important for efficient gating.127 All VGCC subtypes are high density in low-threshold D-hair afferents,115 there is also present in nociceptors. And whereas there is evidence that all evidence that they may be present in a subpopulation of noci- high-threshold calcium channels may contribute to transmitter ceptors as well.116 The biophysical properties of these channels release, N-type channels appear to play a dominant role in the enable them to play a particularly important role in mediating release of transmitter from nociceptors.128

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The dominant role N-type channels play in mediating trans- to sensitization include products of arachidonic acid metabo-

mitter release from nociceptive afferent terminals makes them lism (e.g., prostaglandin E2 [PGE2]), histamine, serotonin, pro- an ideal target for both endogenous and exogenous analgesics. tons and ATP, but the list has grown quite extensively and now Opioid and adrenergic receptor agonists act through inhibitory also includes cytokines, chemokines, growth factors, peptides, G protein-coupled receptors which enable inhibition of VGCCs etc., some of which are released from immune competent cells via two major intracellular pathways. The first is a rapid, mem- attracted to the site of insult (e.g., macrophages), from nearby brane delimited pathway involving G protein -subunit dis- cells (e.g., mast cells), or from nociceptor (and other) nerve termi- placement of the VGCC  subunit, resulting in a “sleepy” or nals (e.g., peptides). Interestingly, despite the variety of mediators “unwilling” channel that requires a larger membrane depolar- capable of producing nociceptor sensitization, several appear to ization for channel opening.127 The second pathway involves play particularly important roles. This list includes prostanoids, more traditional second messenger kinase dependent signaling as evidenced by the antihyperalgesic efficacy of nonsteroidal an- with a slower onset and offset.129 Interestingly, neither pathway ti-inflammatory drugs (NSAIDs) that act via inhibition of cyclo- results in complete VGCC block, yet both result in a dramatic oxygenase and thus prostanoid synthesis.136,137 More recently, the inhibition of transmitter release. This amplification effect re- importance of tumor necrosis factor alpha (TNF-a) in chronic flects the fact that there is considerable cooperativity of cal- inflammatory conditions has been highlighted by the antino- cium in mediating vesicle fusion to the cell membrane that is ciceptive efficacy of compounds such as etanercept which are necessary for transmitter release; four to five calcium ions are designed to bind and inactivate TNF-a released at sites of inflam- needed to trigger vesicle fusion.130 This amplification effect is mation.138,139 Finally, nerve growth factor (NGF) appears to play also likely to facilitate the use of relatively low concentrations a major role in orchestrating a variety of signaling cascades nec- of the N-type channel blocker SNX-111 (Prialt), enabling the essary for an inflammatory response and therefore has also been block of transmitter release from nociceptive afferent terminals targeted with antibody-based strategies.140–144 in the superficial dorsal horn while minimizing side effects as- The mechanisms that trigger changes in nociceptor excit- sociated with block of channels at more distant sites. Finally, ability are not fully known. A growing body of evidence sug- although additional mechanisms likely contribute to the ther- gests that despite what appears to be a bewildering assortment apeutic efficacy of drugs like gabapentin and pregabalin, that of mediators and membrane receptors, there are a relatively

have been shown to bind to the 2 subunit, the most com- small number of intracellular pathways that underlie their pelling model involves the inhibition of membrane traffick- actions. For example, both prostaglandins and bradykinin, ing of the α subunit. That is, following nerve injury, there is which are among the most extensively studied inflammatory

a dramatic increase in 21 subunit expression in nociceptive mediators, act at G protein-coupled receptors. Two major afferents.131 This increase in expression appears to facilitate G protein-dependent­ pathways have been implicated. One the trafficking of VGCC α subunits to nociceptive afferent cen- involves a stimulatory G protein, Gs, which drives activation tral terminals, which in turn, appears to facilitate transmitter of adenylate cyclase, resulting in an increase in cyclic adenosine release and consequently the increase in pain associated with monophosphate (cAMP) and the activation of protein kinase nerve injury. These compounds block the increase in α subunit A (PKA).145,146 The other involves a Gq-dependent pathway, re- 132 trafficking via an 21-dependent mechanism. sulting in the activation of phospholipase C, the liberation of Although VGCCs play a dominant, if not essential, role in diacylglycerol (DAG) and IP3, and the subsequent activation the increase in intracellular calcium needed for transmitter re- of protein kinase C (PKC).147,148 The PKC- isoform appears to lease, recent evidence suggests other calcium regulatory pro- play a particularly important role in nociceptor sensitization. teins contribute to the regulation of the amplitude and duration Other mediators that appear to play important roles in noci- of the increase in intracellular calcium and, consequently, trans- ceptor sensitization, such as TNF- and interleukin (IL) 1, mitter release. These include the sarco-endoplasmic reticulum utilize a mitogen-activated protein kinase (MAPK)-dependent ATPase/ryanodine receptor system,133,134 the plasma membrane pathway ultimately resulting in the activation of p38.149 Still calcium ATPase,135 mitochondria,135 and the sodium/calcium other mediators directly activate ion channels. For example, exchanger.83 Consistent with the suggestion, these calcium reg- protons (which increase in concentration during inflammation) ulatory proteins influence nociception, selective genetic knock- act at TRPV1 and ASICs. ASIC-3 is important to pain associ- down of the sodium/calcium exchanger, a regulatory protein ated with ischemia, such as that which occurs during angina, that attenuates the calcium transient, in a subpopulation of no- and deep muscle pain where protons and lactic acid accumu- ciceptive afferents decreases nociceptive threshold.83 late. In contrast, ATP and its metabolites act at ionotropic P2X and metabotropic P2Y receptors to modulate nociceptor Nociceptor Sensitization excitability. The relatively novel, if not paradoxical, mechanism of sensiti- Sensitization is a characterizing feature of nociceptors; nonno- zation involving ionotropic GABAA receptors that we described ciceptors do not sensitize following tissue insult. Sensitization in the action potential generation section serves as an example represents an increase in nociceptor excitability, which is ex- of the dynamic interplay between second messenger signaling pressed and defined as an increase in response to a noxious cascades and the regulation of ion channels. Activation of these stimulus. Sensitization is also typically accompanied by a re- receptors are normally inhibitory, even in nociceptive afferents duction in the threshold for activation and occasionally by the where they drive membrane depolarization, because of the rel- development of ongoing, spontaneous activity. Nociceptor sen- atively high intracellular chloride concentration maintained sitization is the cause of primary hyperalgesia (i.e., increased in these neurons.150 The GABAA-mediated depolarization in pain produced by stimulation at the site of tissue insult) and these neurons is inhibitory, as suggested earlier, because of the is important because nociceptor sensitization is the trigger for decrease in membrane resistance, referred to as shunting, as initiation of an increase in excitability of central neurons in the well as the depolarization-induced activation of low-threshold nociceptive pathway, an event termed central sensitization. voltage-gated potassium channels.151 In the presence of persistent An increase in nociceptor excitability is a reflection of changes inflammation, there is a constitutive shift in the balance of ty- in the behavior of nociceptor voltage- and/or ligand-gated ion rosine kinase to tyrosine phosphatase activity, such that there is channels produced by actions of endogenous substances either a net increase in protein phosphorylation. This increase results released or synthesized at the site of tissue insult or attracted in a net increase in functional GABAA receptors in the plasma there. Endogenous substances considered classically to contribute membrane because of a decrease in receptor internalization.152

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The result is an increase in GABA-evoked current and an in- carrageenan, or inflammatory mediators such as IL-6.148 These crease in the magnitude of the GABA-evoked depolarization. changes were manifest up to weeks after the complete resolution These changes are further augmented by a decrease in the den- of hypersensitivity associated with the initiating stimulus and sity of low-threshold potassium current.151 The result is a shift were most prominently manifest as a dramatic increase in the in GABA signaling, from inhibition to excitation. duration of the hypersensitivity associated with a subsequent This picture gets a little more complicated when one consid- challenge, although an increase in the magnitude of the hyper- ers the ceramide/sphingosine 1-phosphate (S1P) system because sensitivity has also been described. That is, although the intra-

ceramide, a potent proinflammatory sphingolipid-generated de dermal injection of PGE2 into naive skin results in a relatively novo from hydrolysis of sphingomyelin or synthesis from ser- transient hypersensitivity lasting ,1 hour, the same injection ine and palmityl CoA, can directly activate protein kinases and into primed skin results in hypersensitivity detectable for more phosphatases as well as serve as a precursor to S1P, which acts via than 24 hours. This phenomenon has been proposed to contrib- G protein-coupled receptors to sensitize nociceptive neurons.153 ute to the transition from acute to chronic pain.161 And although a common set of second messengers, including In the context of a discussion of mechanisms underlying phosphatidylinositol 3-kinase, phospholipase C, and extracel- nociceptor sensitization, the mechanisms implicated in the lular signal-regulated kinase (ERK), underlie the acute sensitizing manifestation of hyperalgesic priming are notable for several actions of trophic factors such as NGF, these mediators signal via reasons. First, although there are data suggesting changes within a distinct class of receptors that contain intrinsic tyrosine kinase the central nervous system, notably involving a descending activity, referred to as tropomyosin receptor kinase, or Trk recep- dopaminergic input to the superficial dorsal horn, contribute tors.154 Channels underlying transduction and spike initiation, in to the manifestation of hyperalgesic priming,162 the bulk of data particular TRPV1 and NaV1.8, respectively, appear to be final generated so far suggest that changes in nociceptive afferents common targets for this diverse array of mediators and second may play a critical role in the emergence of chronic pain. Sec- messenger pathways.155 Phosphorylation of specific residues on ond, as further evidence of the differential role of distinct sub- the channel or associated proteins results in increases in channel populations of nociceptive afferents in nociceptive signaling, density and/or increases in channel function. Importantly, many two distinct forms of hyperalgesic priming have been described, inflammatory mediators including ceramide/SP1 and NGF drive where type I depends on a subpopulation of neurons that do long-term increases in nociceptor excitability via changes in gene not express the neuropeptides calcitonin gene-related peptide expression, and mechanisms contributing to long-term changes (CGRP) or substance P and bind the plant lectin IB4,163 whereas in gene expression, such as alterations in DNA acetylation and type II depends on a subpopulation of neurons that do express methylation, have emerged as potential therapeutic targets.156,157 these neuropeptides and do not bind IB4.164 Third, there appear Although mechanisms underlying changes in gene expres- to be sex differences in the mechanisms underlying both the sion associated with the emergence of persistent pain have initiation (type I)165 and maintenance (type II)164 of the primed received considerable attention, there is a growing body of state. And fourth, although a cAMP response element binding evidence suggesting that changes in protein translation are protein (CREB)-dependent change in gene expression appears also important. One of the first studies to implicate a change to be necessary for the establishment of the primed state, local in the regulation of protein translation per se in manifestation translation appears to be critical for the manifestation of the of persistent pain was focused on the role of TRPV1 in per- more robust response observed in primed state, at least for sistent inflammatory pain. The authors observed that inflam- type I priming.166 Given the length of some nociceptive affer- mation is associated with an increase in TRPV1 protein in the ents, in particular those innervating the distal appendages, local absence of a detectable increase in messenger RNA.158 It was control of translation may prove to be an important therapeutic subsequently demonstrated that inflammatory mediators such target for the treatment of pain. 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- Clinical Implications of tion of protein synthesis.159 The protein initiation step involves Nociceptor Function the assembly of a complex of eukaryotic initiation factors (eIF) eIF4E and G among others, as well as the dissociation of the As researchers have begun to explore the basis for chronic pain eIF4E binding protein (4EBP), from eIF4E. At least two distinct syndromes generally associated with specific body regions and/ signaling pathways have been identified underlying the actions or organs (e.g., temporomandibular joint disorder [TMJD], of NGF and IL-6. In addition to the activation of PI3K, NGF inflammatory bowel disease [IBD], irritable bowel syndrome activation of TrkA drives the activation of PI3K which subse- [IBS], or painful bladder syndrome [formerly interstitial cys- quently activates Akt (also known as protein kinase B). Akt titis or IC]), a number of common themes have emerged that then activates the mammalian target of rapamycin (mTOR), are likely to impact future treatment approaches. First, spe- which facilitates the initiation of translation via facilitating the cific mechanisms underlying injury-induced sensitization of dissociation of 4EBP from eIEF4E, as well as the activation of nociceptors vary as a function of target of innervation. For eIF4G, via the phosphorylation of both proteins. In contrast, example, inflammation-induced sensitization of masseter IL-6 drives the activation of ERK, which subsequently acti- muscle afferents appears to reflect a decrease in a specific sub- vates MAP-kinase interacting kinase (MNK), which facilitates population of voltage-gated potassium channels.167 The same the initiation of translation via the phosphorylation of eIF4E channels do not appear to contribute to the sensitization of (which facilitates the eIF4E/G complex formation).159 Interest- TMJ afferents.168 Similarly, inflammation-induced increases in ingly, this rapid regulation of translation contributes not only the excitability of bladder sensory neurons appear to reflect one to the magnitude and duration of both the mechanical and pattern of changes in voltage-gated169 ion channels, whereas thermal hypersensitivity associated with acute inflammation the inflammation-induced increase in the excitability of sensory and the actions of inflammatory mediators like NGF and IL-6 neurons innervating the stomach,170–172 ileum,173,174 or colon175 but also the manifestation of more persistent changes in noci- reflect other patterns. Although tissue-specific patterns of -in ceptive processing, referred to as hyperalgesic priming.160 flammation may contribute to these differences between sub- Hyperalgesic priming is the term used to describe the changes populations of afferents, differences persist when the response in nociceptive processing associated with an initial inflammatory to inflammatory mediators is studied in vitro.176,177 These obser- insult, such as that associated with the subcutaneous injection of vations imply that it may be possible, if not necessary, to treat

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pain arising from a specific structure with a specific interven- of stimulation also are now able to activate nociceptors. In tion. Second, specific mechanisms underlying insult-induced addition, spontaneous activity may develop. In the aggregate, sensitization of nociceptors also vary as a function of the type central nervous system input from sensitized nociceptors, of insult. For example, acute phosphorylation-dependent mod- awakened sleeping nociceptors, and spontaneously active noci- ulation of the voltage-gated sodium channel NaV1.8 results in ceptors is significantly increased. For example, approximately an increase in current which contributes to an inflammation- 24% of human cutaneous C fibers are sleeping nociceptors,190 induced increase in nociceptor excitability.178,179 In contrast, comprising significant new input to the central nervous system following traumatic nerve injury, redistribution of NaV1.8 to if awakened. Consequently, the amount of neurotransmitters the axons of uninjured afferents appears to be necessary for (as well as perhaps their relative proportions) released onto the expression of mechanical hypersensitivity associated with central neurons is increased, which in turn alters the excitabil- nerve injury.180 The dynamic allodynia that often develops after ity of central neurons. The increase in excitability of central nerve injury, however, likely represents more than only a redis- neurons is manifest as an increase in the size of the cutaneous tribution of NaV1.8 (Box 3.2). receptive field (i.e., secondary hyperalgesia) or area of tender- With respect to the viscera, because each organ receives in- ness referred from deep structures, particularly the viscera. nervation from two nerves, the effect of organ insult can be Although the principal focus of study of mechanisms of central different in the two groups of sensory neurons that innervate sensitization has been the spinal cord, it should be appreciated the organ (e.g., Wang et al.,181 Traub182). These observations that nociceptor-driven changes in central excitability extend underscore the importance of developing diagnostic criteria throughout the central nervous system. that enable identification of the factors primarily responsible for ongoing pain. Third, as clearly indicated by the discussion References of hyperalgesic priming, the history of the nociceptor influences the response to subsequent challenge. Furthermore, there is ev- 1. Torebjork E. Nociceptor activation and pain. Philos Trans R Soc Lond B Biol Sci 1985;308(1136):227–234. idence of a developmental window within which injury may 2. Torebjork E. Human microneurography and intraneural microstimulation 183,184 produce permanent changes in nociceptors. With the de- in the study of neuropathic pain. Muscle Nerve 1993;16(10):1063–1065. velopment of more specific therapeutic tools, patient history 3. McGuire C, Boundouki G, Hockley JRF, et al. Ex vivo study of human vis- may become a critical factor in the identification of the most ceral nociceptors. Gut 2018;67:86–96. 4. Hockley JR, Tranter MM, McGuire C, et al. P2Y receptors sensitize mouse appropriate intervention. Fourth, there is evidence for sex and human colonic nociceptors. J Neurosci 2016;36(8):2364–2376. differences in both the excitability of different groups of no- 5. Weidner C, Schmelz M, Schmidt R, et al. Functional attributes discriminat- ciceptors185 as well as the response to tissue injury.186,187 These ing mechano-insensitive and mechano-responsive C nociceptors in human differences appear to be mediated, at least in part, through the skin. J Neurosci 1999;19(22):10184–10190. actions of gonadal hormones and may contribute to sex dif- 6. Goswami SC, Thierry-Mieg D, Thierry-Mieg J, et al. Itch-associated pep- tides: RNA-Seq and bioinformatic analysis of natriuretic precursor peptide ferences in the manifestation of a number of chronic pain syn- B and gastrin releasing peptide in dorsal root and trigeminal ganglia, and the dromes. There is also evidence for age-dependent changes in spinal cord. Mol Pain 2014;10:44. nociceptor function.188 Given the ever-growing proportion of 7. Hu G, Huang K, Hu Y, et al. Single-cell RNA-seq reveals distinct injury aging adults, this particular issue in is need of further investiga- responses in different types of DRG sensory neurons. Sci Rep 2016;6:31851. 8. Djouhri L, Lawson SN. Abeta-fiber nociceptive primary afferent neurons: tion. Finally, all of these factors interact with what are clearly a review of incidence and properties in relation to other afferent A-fiber 189 genetic differences in pain and analgesic mechanisms. neurons in mammals. Brain Res Brain Res Rev 2004;46(2):131–145. As indicated earlier, the consequences of tissue insult are not 9. La JH, Feng B, Kaji K, et al. Roles of isolectin B4-binding afferents in col- limited only to changes in the excitability of nociceptors and orectal mechanical nociception. Pain 2016;157(2):348–354. 10. Usoskin D, Furlan A, Islam S, et al. Unbiased classification of sensory the awakening of sleeping nociceptors. Because sensitization neuron types by large-scale single-cell RNA sequencing. Nat Neurosci leads to an increased response to noxious stimuli and a de- 2015;18(1):145–153. crease in response threshold, previously nonnoxious intensities 11. Feng B, La JH, Schwartz ES, et al. Long-term sensitization of mechanosen- sitive and -insensitive afferents in mice with persistent colorectal hypersensi- tivity. Am J Physiol Gastrointest Liver Physiol 2012;302(7):G676–G683. 12. Wooten M, Weng HJ, Hartke TV, et al. Three functionally distinct classes of BOX 3.2 Afferent Contributions to Neuropathic Pain C-fibre nociceptors in primates.Nat Commun 2014;5:4122. and Allodynia 13. Weng Y, Batista-Schepman PA, Barabas ME, et al. Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception. Mol Pain One of the most striking positive symptoms of neuropathic pain is dy- 2012;8:75. namic mechanical allodynia, a term used to describe pain resulting 14. Giniatullin R, Nistri A. Desensitization properties of P2X3 receptors shap- from light tactile stimuli that would never be considered noxious in the ing pain signaling. Front Cell Neurosci 2013;7:245. absence of nervous system injury. This phenomena can be recapitu- 15. Lukacs V, Yudin Y, Hammond GR, et al. Distinctive changes in plasma membrane phosphoinositides underlie differential regulation of TRPV1 in lated with a subcutaneous injection of capsaicin.196 Data from detailed 197 nociceptive neurons. J Neurosci 2013;33(28):11451–11463. psychophysical analysis in combination with microneurography and 16. Smith TP, Smith SN, Sweitzer SM. Endothelin-1 induced desensitization in 198 dorsal horn recording in animal models all suggest that dynamic me- primary afferent neurons. Neurosci Lett 2014;582:59–64. chanical allodynia reflects activity in low-threshold afferents that signal 17. Rau KK, McIlwrath SL, Wang H, et al. Mrgprd enhances excitability in pain as a result of changes in the central nervous system. The exact specific populations of cutaneous murine polymodal nociceptors.J Neurosci nature of these changes, generally referred to as central sensitization, 2009;29(26):8612–8619. is still debated (e.g., see Polgar and Todd199 and Schoffnegger et al.200), 18. Cavanaugh DJ, Lee H, Lo L, et al. Distinct subsets of unmyelinated primary but it is clear that they depend on activity in nociceptive afferents. sensory fibers mediate behavioral responses to noxious thermal and mechan- Another possibility, however, is that low-threshold afferents undergo a ical stimuli. Proc Natl Acad Sci USA 2009;106(22):9075–9080. phenotypic switch such that they begin to transmit information to the 19. Abrahamsen B, Zhao J, Asante CO, et al. The cell and molecular basis of central nervous system as if they were nociceptive afferents. Consis- mechanical, cold, and inflammatory pain.Science 2008;321(5889):702–705. 20. Johnson KO. The roles and functions of cutaneous mechanoreceptors. Curr tent with this possibility, there is evidence following peripheral nerve in- Opin Neurobiol 2001;11(4):455–461. jury that a subpopulation of putative nonnociceptive afferents begins to 21. Mearow KM, Diamond J. Merkel cells and the mechanosensitivity of normal 201 express the neuropeptide, substance P, although this change does and regenerating nerves in Xenopus skin. Neuroscience 1988;26(2):695–708. 202 not appear to be necessary for the expression of allodynia. Evidence 22. KruMger L, Kavookjian A, Kumazawa T, et al. Nociceptor structural spe- of a third possibility, which would involve sprouting of nonnociceptive cialization in canine and rodent testicular “free” nerve endings. J Comp afferents into more superficial layers of the dorsal horn to enable low Neurol 2003;463(2):197–211. threshold drive of nociceptive dorsal horn neurons,203 remains largely 23. Zylka MJ, Rice FL, Anderson DJ. Topographically distinct epidermal no- unsubstantiated.204–206 ciceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 2005;45(1):17–25.

Rathmell5e_Ch003.indd 11 3/23/18 12:12 AM 12 PART ONE BASIC CONSIDERATIONS

24. Ghitani N, Barik A, Szczot M, et al. Specialized mechanosensory nociceptors 57. Price MP, Lewin GR, McIlwrath SL, et al. The mammalian sodium chan- mediating rapid responses to hair pull. Neuron 2017;95(4):944.e4–954.e4. nel BNC1 is required for normal touch sensation. Nature 2000;407(6807): 25. Bai L, Lehnert BP, Liu J, et al. Genetic identification of an expansive mechano- 1007–1011. receptor sensitive to skin stroking. Cell 2015;163(7):1783–1795. 58. Shin JB, Martinez-Salgado C, Heppenstall PA, et al. A T-type calcium chan- 26. Braz JM, Nassar MA, Wood JN, et al. Parallel “pain” pathways arise from nel required for normal function of a mammalian mechanoreceptor. Nat subpopulations of primary afferent nociceptor. Neuron 2005;47(6):787–793. Neurosci 2003;6(7):724–730. 27. Cesare P, McNaughton P. A novel heat-activated current in nociceptive 59. Kwan KY, Glazer JM, Corey DP, et al. TRPA1 modulates mechanotransduc- neurons and its sensitization by bradykinin. Proc Natl Acad Sci U S A tion in cutaneous sensory neurons. J Neurosci 2009;29(15):4808–4819. 1996;93:15435–15439. 60. Ranade SS, Woo SH, Dubin AE, et al. Piezo2 is the major transducer of me- 28. Reichling DB, Levine JD. Heat transduction in rat sensory neurons by cal- chanical forces for touch sensation in mice. Nature 2014;516(7529):121–125. cium-dependent activation of a cation channel. Proc Natl Acad Sci U S A 61. Michaelis M, Blenk KH, Vogel C, et al. Distribution of sensory proper- 1997;94(13):7006–7011. ties among axotomized cutaneous C-fibres in adult rats. Neuroscience 29. Reid G, Flonta ML. Physiology. Cold current in thermoreceptive neurons. 1999;94(1):7–10. Nature 2001;413(6855):480. 62. Drew LJ, Rugiero F, Cesare P, et al. High-threshold mechanosensitive ion 30. Thut PD, Wrigley D, Gold MS. Cold transduction in rat trigeminal ganglia channels blocked by a novel conopeptide mediate pressure-evoked pain. neurons in vitro. Neuroscience 2003;119(4):1071–1083. PLoS One 2007;2:e515. 31. McCarter GC, Reichling DB, Levine JD. Mechanical transduction by rat 63. Park SP, Kim BM, Koo JY, et al. A tarantula spider toxin, GsMTx4, reduces dorsal root ganglion neurons in vitro. Neurosci Lett 1999;273(3):179–182. mechanical and neuropathic pain. Pain 2008;137(1):208–217. 32. Vilceanu D, Stucky CL. TRPA1 mediates mechanical currents in the plasma 64. St. Sauver JL, Warner DO, Yawn BP, et al. Why patients visit their doctors: membrane of mouse sensory neurons. PLoS One 2010;5(8):e12177. assessing the most prevalent conditions in a defined American population. 33. Bevan S, Yeats J. Protons activate a cation conductance in a sub-population Mayo Clin Proc 2013;88(1):56–67. of rat dorsal root ganglion neurones. J Physiol 1991;433:145–161. 65. Birder LA. More than just a barrier: urothelium as a drug target for urinary 34. Krishtal OA, Marchenko SM, Obukhov AG. Cationic channels activated by bladder pain. Am J Physiol Renal Physiol 2005;289(3):F489–F495. extracellular ATP in rat sensory neurons. Neuroscience 1988;27(3):995–1000. 66. Baumbauer KM, DeBerry JJ, Adelman PC, et al. Keratinocytes can modu- 35. Heyman I, Rang HP. Depolarizing responses to capsaicin in a subpopulation late and directly initiate nociceptive responses. Elife 2015;4. doi:10.7554 of rat dorsal root ganglion cells. Neurosci Lett 1985;56:69–75. /eLife.09674 36. Bautista DM, Jordt SE, Nikai T, et al. TRPA1 mediates the inflam- 67. Zappia KJ, Garrison SR, Palygin O, et al. Mechanosensory and ATP release matory actions of environmental irritants and proalgesic agents. Cell deficits following keratin14-Cre-Mediated TRPA1 deletion despite absence 2006;124(6):1269–1282. of TRPA1 in murine keratinocytes. PLoS One 2016;11(3):e0151602. 37. Gold MS. Molecular biology of sensory transduction. In: McMahon SB, 68. Wynn G, Rong W, Xiang Z, et al. Purinergic mechanisms contribute to Koltzenburg M, Tracey I, et al, eds. Wall and Melzack’s Textbook of Pain. mechanosensory transduction in the rat colorectum. Gastroenterology 6th ed. London: Elsevier; 2013:31–47. 2003;125(5):1398–1409. 38. Burnstock G. Physiology and pathophysiology of purinergic neurotransmis- 69. Mizumoto N, Mummert ME, Shalhevet D, et al. Keratinocyte ATP release sion. Physiol Rev 2007;87(2):659–797. assay for testing skin-irritating potentials of structurally diverse chemicals. 39. Wemmie JA, Price MP, Welsh MJ. Acid-sensing ion channels: advances, ques- J Invest Dermatol 2003;121(5):1066–1072. tions and therapeutic opportunities. Trends Neurosci 2006;29(10):578–586. 70. Burnstock G. Purinergic P2 receptors as targets for novel analgesics. Phar- 40. Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-like channel ex- macol Ther 2006;110(3):433–454. pressed in nociceptive neurons, is activated by cold temperatures. Cell 71. Xu GY, Shenoy M, Winston JH, et al. P2X receptor-mediated visceral 2003;112(6):819–829. hyperalgesia in a rat model of chronic visceral hypersensitivity. Gut 41. Caterina MJ, Rosen TA, Tominaga M, et al. A capsaicin-receptor­ homologue 2008;57(9):1230–1237. with a high threshold for noxious heat. Nature 1999;398(6726):436–441. 72. Bellono NW, Bayrer JR, Leitch DB, et al. Enterochromaffin cells are gut che- 42. McKemy DD, Neuhausser WM, Julius D. Identification of a cold recep- mosensors that couple to sensory neural pathways. Cell 2017;170(1):185. tor reveals a general role for TRP channels in thermosensation. Nature e16–198.e16. 2002;416(6876):52–58. 73. Mawe GM, Hoffman JM. Serotonin signalling in the gut—functions, dysfunc- 43. Xu H, Ramsey IS, Kotecha SA, et al. TRPV3 is a calcium-permeable tions and therapeutic targets. Nat Rev Gastroenterol Hepatol 2013;10(8): temperature-sensitive cation channel. Nature 2002;418(6894):181–186. 473–486. 44. Güler AD, Lee H, Iida T, et al. Heat-evoked activation of the ion channel, 74. Janig W, Grossmann L, Gorodetskaya N. Mechano- and thermosensitivity of TRPV4. J Neurosci 2002;22(15):6408–6414. regenerating cutaneous afferent nerve fibers. Exp Brain Res 2009;196:101–114. 45. Caterina MJ, Schumacher MA, Tominaga M, et al. The capsaicin receptor: 75. Korenman EM, Devor M. Ectopic adrenergic sensitivity in damaged periph- a heat-activated ion channel in the pain pathway. Nature 1997;389(6653): eral nerve axons in the rat. Exp Neurol 1981;72:63–81. 816–824. 76. Devor M, Janig W, Michaelis M. Modulation of activity in dorsal root 46. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76: ganglion neurons by sympathetic activation in nerve-injured rats. J Neuro- 387–417. physiol 1994;71(1):38–47. 47. Kwan KY, Allchorne AJ, Vollrath MA, et al. TRPA1 contributes to cold, 77. Mantyh PW, DeMaster E, Malhorta A, et al. Receptor endocytosis and den- mechanical, and chemical nociception but is not essential for hair-cell trans- drite reshaping in spinal neurons after somatosensory stimulation. Science duction. Neuron 2006;50(2):277–289. 1995;268(5217):1629–1632. 48. Alessandri-Haber N, Joseph E, Olayinka D, et al. TRPV4 mediates pain-related 78. Gold MS, Gebhart GF. Nociceptor sensitization in pain pathogenesis. Nat behavior induced by mild hypertonic stimuli in the presence of inflammatory Med 2010;16(11):1248–1257. mediator. Pain 2005;118(1–2):70–79. 79. Carr RW, Pianova S, Brock JA. The effects of polarizing current on nerve ter- 49. Mickle AD, Shepherd AJ, Mohapatra DP. Sensory TRP channels: the key trans- minal impulses recorded from polymodal and cold receptors in the guinea-pig ducers of nociception and pain. Prog Mol Biol Transl Sci 2015;131:73–118. cornea. J Gen Physiol 2002;120(3):395–405. 50. Honore P, Wismer CT, Mikusa J, et al. A-425619 [1-isoquinolin-5-yl-3-(4-­ 80. Zhang XF, Gopalakrishnan M, Shieh CC. Modulation of action potential trifluoromethyl-benzyl)-urea], a novel transient receptor potential type V1 firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons. Neu- receptor antagonist, relieves pathophysiological pain associated with in- roscience 2003;122(4):1003–1011. flammation and tissue injury in rats.J Pharmacol Exp Ther 2005;314(1): 81. Passmore GM, Selyanko AA, Mistry M, et al. KCNQ/M currents in sensory 410–421. neurons: significance for pain therapy.J Neurosci 2003;23(18):7227–7236. 51. Mrozkova P, Spicarova D, Palecek J. Hypersensitivity induced by activation 82. Gemes G, Oyster K, Pan B, et al. Painful nerve injury increases plasma of spinal cord par2 receptors is partially mediated by trpv1 receptors. PLoS membrane Ca21-ATPase activity in axotomized sensory neurons. Mol Pain One 2016;11(10):e0163991. 2012;8:46. 52. Nishio N, Taniguchi W, Sugimura YK, et al. Reactive oxygen species en- 83. Scheff NN, Yilmaz E, Gold MS. The properties, distribution and function hance excitatory synaptic transmission in rat spinal dorsal horn neurons by of Na(1)-Ca(21) exchanger isoforms in rat cutaneous sensory neurons. activating TRPA1 and TRPV1 channels. Neuroscience 2013;247:201–212. J Physiol 2014;592(pt 22):4969–4993. 53. Moran MM, Szallasi A. Targeting nociceptive transient receptor potential 84. Tang Q, Bangaru ML, Kostic S, et al. Ca21-dependent regulation of Ca21 channels to treat chronic pain: current state of the field [published online currents in rat primary afferent neurons: role of CaMKII and the effect of ahead of print September 19, 2017]. Br J Pharmacol. doi:10.1111/bph.14044. injury. J Neurosci 2012;32(34):11737–11749.

54. Karai L, Brown DC, Mannes AJ, et al. Deletion of vanilloid receptor 85. Zhang X-L, Mok L-P, Katz EJ, et al. BKCa currents are enriched in a subpop- 1-­expressing primary afferent neurons for pain control. J Clin Invest 2004; ulation of adult rat cutaneous nociceptive dorsal root ganglion neurons. Eur 113(9):1344–1352. J Neurosci 2010;31:450–462. 55. Brown DC, Agnello K, Iadarola MJ. Intrathecal resiniferatoxin in a dog 86. Vaughn AH, Gold MS. Ionic mechanisms underlying inflammatory mediator-­ model: efficacy in bone cancer pain.Pain 2015;156(6):1018–1024. induced sensitization of dural afferents. J Neurosci 2010;30(23):7878–7888. 56. Delmas P, Hao J, Rodat-Despoix L. Molecular mechanisms of mechano- 87. Zhu Y, Zhang XL, Gold MS. Activity-dependent hyperpolarization of transduction in mammalian sensory neurons. Nat Rev Neurosci 2011;12(3): EGABA is absent in cutaneous DRG neurons from inflamed rats.Neurosci - 139–153. ence 2014;256:1–9.

Rathmell5e_Ch003.indd 12 3/23/18 12:12 AM CHAPTER 3 Peripheral Pain Mechanisms and Nociceptor Sensitization 13

88. Goss JR, Cascio M, Goins W, et al. HSV delivery of a ligand-regulated 117. White G, Lovinger DM, Weight FF. Transient low-threshold Ca21 current endogenous ion channel gene to sensory neurons results in pain control triggers burst firing through an after depolarizing potential in an adult following channel activation. Mol Ther 2011;19(3):500–506. mammalian neuron. Proc Natl Acad Sci USA 1989;86:6802–6806. 89. Rivnay J, Wang H, Fenno L, et al. Next-generation probes, particles, and 118. Gu JG, MacDermott AB. Activation of ATP P2X receptors elicits glutamate proteins for neural interfacing. Sci Adv 2017;3(6):e1601649. release from sensory neuron synapses. Nature 1997;389(6652):749–753. 90. Boada MD, Martin TJ, Peters CM, et al. Fast-conducting mechanorecep- 119. Jeftinija S. The role of tetrodotoxin-resistant sodium channels of small pri- tors contribute to withdrawal behavior in normal and nerve injured rats. mary afferent fibers.Brain Res 1994;639(1):125–134. Pain 2014;155(12):2646–2655. 120. Willis WD Jr. Dorsal root potentials and dorsal root reflexes: a double-edged 91. Iyer SM, Montgomery KL, Towne C, et al. Virally mediated optogenetic sword. Exp Brain Res 1999;124(4):395–421. excitation and inhibition of pain in freely moving nontransgenic mice. Nat 121. Wittmack EK, Rush AM, Craner MJ, et al. Fibroblast growth factor ho- Biotechnol 2014;32(3):274–278. mologous factor 2B: association with Nav1.6 and selective colocalization at 92. Li B, Yang XY, Qian FP, et al. A novel analgesic approach to optogenet- nodes of Ranvier of dorsal root axons. J Neurosci 2004;24(30):6765–6775. ically and specifically inhibit pain transmission using TRPV1 promoter. 122. Alexandrou AJ, Brown AR, Chapman ML, et al. Subtype-selective small Brain Res 2015;1609:12–20. molecule inhibitors reveal a fundamental role for Nav1.7 in nociceptor 93. Daou I, Beaudry H, Ase AR, et al. Optogenetic silencing of Nav1.8-positive electrogenesis, axonal conduction and presynaptic release. PLoS One afferents alleviates inflammatory and neuropathic pain.eNeuro 2016;3(1). 2016;11(4):e0152405. doi:10.1523/ENEURO.0140-15.2016. 123. Xie W, Strong JA, Zhang JM. Local knockdown of the NaV1.6 sodium 94. Catterall WA, Hulme JT, Jiang X, et al. Regulation of sodium and cal- channel reduces pain behaviors, sensory neuron excitability, and sympathetic cium channels by signaling complexes. J Recept Signal Transduct Res sprouting in rat models of neuropathic pain. Neuroscience 2015;291:317–330. 2006;26(5–6):577–598. 124. Catterall WA, Perez-Reyes E, Snutch TP, et al. International Union of Phar- 95. Gold MS. Ion channels: recent advances and clinical applications. In: Flor macology. XLVIII. Nomenclature and structure-function relationships of H, Kaslo E, Dostrovsky JO, eds. Proceedings of the 11th World Congress voltage-gated calcium channels. Pharmacol Rev 2005;57(4):411–425. on Pain. Seattle, WA: IASP Press; 2006:73–92. 125. Dolphin AC. Voltage-gated calcium channels and their auxiliary sub- 96. Ho C, O’Leary ME. Single-cell analysis of sodium channel expression in dor- units: physiology and pathophysiology and pharmacology. J Physiol sal root ganglion neurons. Mol Cell Neurosci 2011;46(1):159–166. 2016;594(19):5369–5390. 97. Isom LL. Sodium channel beta subunits: anything but auxiliary. Neurosci- 126. Davies A, Hendrich J, Van Minh AT, et al. Functional biology of the entist 2001;7(1):42–54. alpha(2)delta subunits of voltage-gated calcium channels. Trends Pharma- 98. Sangameswaran L, Delgado SG, Fish LM, et al. Structure and function col Sci 2007;28(5):220–228. of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to 127. Ikeda SR. Voltage-dependent modulation of N-type calcium channels by sensory neurons. J Biol Chem 1996;271(11):5953–5956. G-protein beta gamma subunits. Nature 1996;380(6571):255–258. 99. Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated 128. Rycroft BK, Vikman KS, Christie MJ. Inflammation reduces the contri- sodium channel expressed by sensory neurons. Nature 1996;379(6562): bution of N-type calcium channels to primary afferent synaptic transmis- 257–262. sion onto NK1 receptor-positive lamina I neurons in the rat dorsal horn. J 100. Djouhri L, Fang X, Okuse K, et al. The TTX-resistant sodium channel Physiol 2007;580(pt 3):883–894. Nav1.8 (SNS/PN3): expression and correlation with membrane properties in 129. Ewald DA, Matthies HJ, Perney TM, et al. The effect of down regulation of rat nociceptive primary afferent neurons. J Physiol 2003;550(pt 3):739–752. protein kinase C on the inhibitory modulation of dorsal root ganglion neu- 101. Jarvis MF, Honore P, Shieh CC, et al. A-803467, a potent and selective ron Ca21 currents by neuropeptide Y. J Neurosci 1988;8(7):2447–2451. Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory 130. Schneggenburger R, Neher E. Presynaptic calcium and control of vesicle pain in the rat. Proc Natl Acad Sci USA 2007;104(20):8520–8525. fusion. Curr Opin Neurobiol 2005;15(3):266–274. 102. Klein AH, Vyshnevska A, Hartke TV, et al. Sodium channel Nav1.8 un- 131. Luo ZD, Chaplan SR, Higuera ES, et al. Upregulation of dorsal root gan-

derlies TTX-resistant axonal action potential conduction in somatosensory glion 2 calcium channel subunit and its correlation with allodynia in spi- C-fibers of distal cutaneous nerves.J Neurosci 2017;37(20):5204–5214. nal nerve-injured rats. J Neurosci 2001;21(6):1868–1875. 103. Elliott AA, Elliott JR. Characterization of TTX-sensitive and TTX-resistant 132. Bauer CS, Nieto-Rostro M, Rahman W, et al. The increased trafficking of sodium currents in small cells from adult rat dorsal root ganglia. J Physiol the calcium channel subunit alpha2delta-1 to presynaptic terminals in neu- (Lond) 1993;463(39):39–56. ropathic pain is inhibited by the alpha2delta ligand pregabalin. J Neurosci 104. Gold MS, Zhang L, Wrigley DL, et al. Prostaglandin E(2) Modulates 2009;29(13):4076–4088. TTX-R I(Na) in Rat Colonic Sensory Neurons. J Neurophysiol 2002;88(3): 133. Cheng LZ, Lü N, Zhang YQ, et al. Ryanodine receptors contribute to the 1512–1522. induction of nociceptive input-evoked long-term potentiation in the rat spinal 105. Zimmermann K, Leffler A, Babes A, et al. Sensory neuron sodium channel cord slice. Mol Pain 2010;6:1. doi:10.1186/1744-8069-6-1. Nav1.8 is essential for pain at low temperatures. Nature 2007;447(7146): 134. Duncan C, Mueller S, Simon E, et al. Painful nerve injury decreases sarco-­ 855–858. endoplasmic reticulum Ca21-ATPase activity in axotomized sensory neurons. 106. Rush AM, Dib-Hajj SD, Liu S, et al. A single sodium channel mutation Neuroscience 2013;231:247–257. produces hyper- or hypoexcitability in different types of neurons. Proc Natl 135. Shutov LP, Kim MS, Houlihan PR, et al. Mitochondria and plasma mem- Acad Sci U S A 2006;103(21):8245–8250. brane Ca21-ATPase control presynaptic Ca21 clearance in capsaicin-­ 107. Cummins TR, Dib-Hajj SD, Waxman SG. Electrophysiological properties sensitive rat sensory neurons. J Physiol 2013;591(pt 10):2443–2462. of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J 136. Gupta A, Bah M. NSAIDs in the treatment of postoperative pain. Curr Neurosci 2004;24(38):8232–8236. Pain Headache Rep 2016;20(11):62. 108. Dib-Hajj SD, Geha P, Waxman SG. Sodium channels in pain disorders: 137. Eccleston C, Cooper TE, Fisher E, et al. Non-steroidal anti-­inflammatory pathophysiology and prospects for treatment. Pain 2017;158(suppl 1): drugs (NSAIDs) for chronic non-cancer pain in children and adolescents. Co- S97–S107. chrane Database Syst Rev 2017;(8):CD012537. 109. Cox JJ, Reimann F, Nicholas AK, et al. An SCN9A channelopathy 138. Willrich MA, Murray DL, Snyder MR. Tumor necrosis factor inhibitors: causes congenital inability to experience pain. Nature 2006;444(7121): clinical utility in autoimmune diseases. Transl Res 2015;165(2):270–282. 894–898. 139. Jing S, Yang C, Zhang X, et al. Efficacy and safety of etanercept in the 110. Minett MS, Nassar MA, Clark AK, et al. Distinct Nav1.7-dependent pain treatment of sciatica: a systematic review and meta-analysis. J Clin Neuro- sensations require different sets of sensory and sympathetic neurons. Nat sci 2017;44:69–74. Commun 2012;3:791. 140. Jimenez-Andrade JM, Martin CD, Koewler NJ, et al. Nerve growth factor 111. Minett MS, Pereira V, Sikandar S, et al. Endogenous opioids contribute to sequestering therapy attenuates non-malignant skeletal pain following frac- insensitivity to pain in humans and mice lacking sodium channel Nav1.7. ture. Pain 2007;133(1–3):183–196. Nat Commun 2015;6:8967. 141. Koewler NJ, Freeman KT, Buus RJ, et al. Effects of a monoclonal an- 112. Kist AM, Sagafos D, Rush AM, et al. SCN10A mutation in a patient with tibody raised against nerve growth factor on skeletal pain and bone erythromelalgia enhances C-fiber activity dependent slowing.PLoS One healing after fracture of the C57BL/6J mouse femur. J Bone Miner Res 2016;11(9):e0161789. 2007;22(11):1732–1742. 113. Zhang X, Priest BT, Belfer I, et al. Voltage-gated Na1 currents in human 142. Sabsovich I, Wei T, Guo TZ, et al. Effect of anti-NGF antibodies in a dorsal root ganglion neurons. Elife 2017;6. doi:10.7554/eLife.23235. rat tibia fracture model of complex regional pain syndrome type I. Pain 114. Snutch TP, Zamponi GW. Recent advances in the development of T-type 2008;138(1):47–60. calcium channel blockers for pain intervention [published online ahead of 143. Wild KD, Bian D, Zhu D, et al. Antibodies to nerve growth factor reverse print July 12, 2017]. Br J Pharmacol. doi:10.1111/bph.13906. established tactile allodynia in rodent models of neuropathic pain without 115. Bernal Sierra YA, Haseleu J, Kozlenkov A, et al. Genetic tracing of Cav3.2 tolerance. J Pharmacol Exp Ther 2007;322(1):282–287. T-type calcium channel expression in the peripheral nervous system. Front 144. Denk F, Bennett DL, McMahon SB. Nerve growth factor and pain mecha- Mol Neurosci 2017;10:70. nisms. Annu Rev Neurosci 2017;40:307–325. 116. Todorovic SM, Jevtovic-Todorovic V. The role of T-type calcium channels 145. Gold MS, Levine JD, Correa AM. Modulation of TTX-R INa by PKC and in peripheral and central pain processing. CNS Neurol Disord Drug Tar- PKA and their role in PGE2-induced sensitization of rat sensory neurons in gets 2006;5(6):639–653. vitro. J Neurosci 1998;18(24):10345–10355.

Rathmell5e_Ch003.indd 13 3/23/18 12:12 AM 14 PART ONE BASIC CONSIDERATIONS

146. Taiwo YO, Bjerknes LK, Goetzl EJ, et al. Mediation of primary afferent pe- and active electrophysiological properties. J Neurophysiol 2004;91(6): ripheral hyperalgesia by the cAMP second messenger system. Neuroscience 2524–2531. 1989;32(3):577–580. 177. Harriott AM, Gold MS. Electrophysiological properties of dural afferents 147. Ahlgren SC, Levine JD. Protein kinase C inhibitors decrease hyperalgesia in the absence and presence of inflammatory mediators. J Neurophysiol and C-fiber hyperexcitability in the streptozotocin-diabetic rat.J Neuro- 2009;101(6):3126–3134. physiol 1994;72(2):684–692. 178. Fitzgerald EM, Okuse K, Wood JN, et al. cAmp-dependent phosphoryla- 148. Aley KO, Messing RO, Mochly-Rosen D, et al. Chronic hypersensitivity tion of the tetrodotoxin-resistant voltage- dependent sodium channel Sns. J for inflammatory nociceptor sensitization mediated by the epsilon isozyme Physiol (Lond) 1999;516(pt 2):433–446. of protein kinase C. J Neurosci 2000;20(12):4680–4685. 179. Gold MS, Reichling DB, Shuster MJ, et al. Hyperalgesic agents increase 149. Jin X, Gereau RW. Acute p38-mediated modulation of tetrodotoxin-­ a tetrodotoxin-resistant Na1 current in nociceptors. Proc Natl Acad Sci resistant sodium channels in mouse sensory neurons by tumor necrosis USA 1996;93(3):1108–1112. factor-alpha. J Neurosci 2006;26(1):246–255. 180. Gold MS, Weinreich D, Kim CS, et al. Redistribution of Na(V)1.8 in unin- 150. Price TJ, Cervero F, Gold MS, et al. Chloride regulation in the pain path- jured axons enables neuropathic pain. J Neurosci 2003;23(1):158–166. way. Brain Res Rev 2009;60(1):149–170. 181. Wang G, Tang B, Traub RJ. Differential processing of noxious colonic 151. Zhu Y, Lu SG, Gold MS. Persistent inflammation increases GABA-induced input by thoracolumbar and lumbosacral dorsal horn neurons in the rat. J depolarization of rat cutaneous dorsal root ganglion neurons in vitro. Neu- Neurophysiol 2005;94(6):3788–3794. roscience 2012;220:330–340. 182. Traub RJ. Evidence for thoracolumbar spinal cord processing of inflamma- 152. Zhu Y, Dua S, Gold MS. Inflammation-induced shift in spinal GABA(A) sig- tory, but not acute colonic pain. Neuroreport 2000;11(10):2113–2116. naling is associated with a tyrosine kinase-dependent increase in GABA(A) cur- 183. Ruda MA, Ling QD, Hohmann AG, et al. Altered nociceptive neuronal circuits rent density in nociceptive afferents. J Neurophysiol 2012;108(9):2581–2593. after neonatal peripheral inflammation.Science 2000;289(5479):628–631. 153. Salvemini D, Doyle T, Kress M, et al. Therapeutic targeting of the cera- 184. Ren K, Anseloni V, Zou SP, et al. Characterization of basal and re-inflammation-­ mide-to-sphingosine 1-phosphate pathway in pain. Trends Pharmacol Sci associated long-term alteration in pain responsivity following short-lasting neo- 2013;34(2):110–118. natal local inflammatory insult.Pain 2004;110(3):588–596. 154. Kelleher JH, Tewari D, McMahon SB. Neurotrophic factors and their in- 185. Cairns BE, Hu JW, Arendt-Nielsen L, et al. Sex-related differences in hibitors in chronic pain treatment. Neurobiol Dis 2017;97(pt B):127–138. human pain and rat afferent discharge evoked by injection of glutamate 155. Gold MS, Caterina MJ. Molecular biology of nociceptor transduction. In: into the masseter muscle. J Neurophysiol 2001;86(2):782–791. Basbaum AI, Bushnell MC, eds. The Senses: A Comprehensive Reference. 186. Bartley EJ, Fillingim RB. Sex differences in pain: a brief review of clinical San Diego, CA: Academic Press; 2008:43–74. and experimental findings.Br J Anaesth 2013;111(1):52–58. 156. Denk F, McMahon SB. Chronic pain: emerging evidence for the involvement 187. Mogil JS, Bailey AL. Sex and gender differences in pain and analgesia. Prog of epigenetics. Neuron 2012;73(3):435–444. Brain Res 2010;186:141–157. 157. Niederberger E, Resch E, Parnham MJ, et al. Drugging the pain epigenome. 188. Wang S, Davis BM, Zwick M, et al. Reduced thermal sensitivity and Nat Rev Neurol 2017;13(7):434–447. Nav1.8 and TRPV1 channel expression in sensory neurons of aged mice. 158. Ji RR, Samad TA, Jin SX, et al. p38 MAPK activation by NGF in primary Neurobiol Aging 2006;27(6):895–903. sensory neurons after inflammation increases TRPV1 levels and maintains 189. Foulkes T, Wood JN. Pain genes. PLoS Genet 2008;4(7):e1000086. heat hyperalgesia. Neuron 2002;36(1):57–68. 190. Schmidt R, Schmelz M, Forster C, et al. Novel classes of responsive and unre- 159. Melemedjian OK, Asiedu MN, Tillu DV, et al. IL-6- and NGF-induced sponsive C nociceptors in human skin. J Neurosci 1995;15(1, pt 1):333–341. rapid control of protein synthesis and nociceptive plasticity via convergent 191. Hagbarth KE, Vallbo AB. Mechanoreceptor activity recorded percutane- signaling to the eIF4F complex. J Neurosci 2010;30(45):15113–15123. ously with semi-microelectrodes in human peripheral nerves. Acta Physiol 160. Moy JK, Khoutorsky A, Asiedu MN, et al. The MNK-eIF4E signaling axis Scand 1967;69(1):121–122. contributes to injury-induced nociceptive plasticity and the development of 192. Hagbarth KE, Vallbo AB. Afferent response to mechanical stimulation of chronic pain. J Neurosci 2017;37(31):7481–7499. muscle receptors in man. Acta Soc Med Ups 1967;72(1):102–104. 161. Reichling DB, Levine JD. Critical role of nociceptor plasticity in chronic 193. Schmelz M, Schmidt R, Weidner C, et al. Chemical response pattern of dif- pain. Trends Neurosci 2009;32(12):611–618. ferent classes of C-nociceptors to pruritogens and algogens. J Neurophysiol 162. Kim JY, Tillu DV, Quinn TL, et al. Spinal dopaminergic projections control 2003;89(5):2441–2448. the transition to pathological pain plasticity via a D1/D5-mediated mecha- 194. Ochoa JL, Campero M, Serra J, et al. Hyperexcitable polymodal and nism. J Neurosci 2015;35(16):6307–6317. insensitive nociceptors in painful human neuropathy. Muscle Nerve 163. Joseph EK, Levine JD. Hyperalgesic priming is restricted to isolectin 2005;32(4):459–72. B4-positive nociceptors. Neuroscience 2010;169(1):431–435. 195. Ørstavik K, Namer B, Schmidt R, et al. Abnormal function of C-fibers in 164. Araldi D, Ferrari LF, Levine JD. Hyperalgesic priming (type II) induced patients with diabetic neuropathy. J Neurosci 2006;26(44):11287–11294. by repeated opioid exposure: maintenance mechanisms. Pain 2017;158(7): 196. Baumann TK, Simone DA, Shain CN, et al. Neurogenic hyperalgesia: the 1204–1216. search for primary cutaneous afferent fibers that contribute to capsaicin-­ 165. Joseph EK, Parada CA, Levine JD. Hyperalgesic priming in the rat demon- induced pain and hyperalgesia. J Neurophysiol 1991;66(1):212–227. strates marked sexual dimorphism. Pain 2003;105(1–2):143–150. 197. Torebjörk HE, Lundberg LE, LaMotte RH. Central changes in processing 166. Ferrari LF, Bogen O, Reichling DB, et al. Accounting for the delay in the of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in transition from acute to chronic pain: axonal and nuclear mechanisms. humans. J Physiol (Lond) 1992;448:765–780. J Neurosci 2015;35(2):495–507. 198. Weng HR, Dougherty PM. Response properties of dorsal root reflexes in 167. Harriott AM, Dessem D, Gold MS. Inflammation increases the excitability of cutaneous C fibers before and after intradermal capsaicin injection in rats. masseter muscle afferents. Neuroscience 2006;141(1):433–442. Neuroscience 2005;132(3):823–831. 168. Flake NM, Gold MS. Inflammation alters sodium currents and excitability of 199. Polgar E, Todd AJ. Tactile allodynia can occur in the spared nerve injury temporomandibular joint afferents. Neurosci Lett 2005;384(3):294–299. model in the rat without selective loss of GABA or GABA(A) receptors 169. Yoshimura N, de Groat WC. Increased excitability of afferent neurons in- from synapses in laminae I-II of the ipsilateral spinal dorsal horn. Neuro- nervating rat urinary bladder after chronic bladder inflammation.J Neuro- science 2008;156(1):193–202. sci 1999;19(11):4644–4653. 200. Schoffnegger D, Ruscheweyh R, Sandkuhler J. Spread of excitation 170. Bielefeldt K, Ozaki N, Gebhart GF. Mild gastritis alters voltage-sensitive across modality borders in spinal dorsal horn of neuropathic rats. Pain sodium currents in gastric sensory neurons in rats. Gastroenterology 2002; 2008;135(3):300–310. 122(3):752–761. 201. Noguchi K, Dubner R, De Leon M, et al. Axotomy induces preprotachy- 171. Dang K, Bielefeldt K, Gebhart GF. Gastric ulcers reduce A-type potassium kinin gene expression in a subpopulation of dorsal root ganglion neurons. currents in rat gastric sensory ganglion neurons. Am J Physiol Gastrointest J Neurosci Res 1994;37(5):596–603. Liver Physiol 2004;286(4):G573–G579. 202. Hughes DI, Scott DT, Riddell JS, et al. Upregulation of substance P in 172. Sugiura T, Dang K, Lamb K, et al. Acid-sensing properties in rat gastric sen- low-threshold myelinated afferents is not required for tactile allodynia in sory neurons from normal and ulcerated stomach. J Neurosci 2005;25(10): the chronic constriction injury and spinal nerve ligation models. J Neurosci 2617–2627. 2007;27(8):2035–2044. 173. Moore BA, Stewart TM, Hill C, et al. TNBS ileitis evokes hyperexcitability 203. Woolf CJ, Shortland P, Coggeshall RE. Peripheral nerve injury triggers cen- and changes in ionic membrane properties of nociceptive DRG neurons. tral sprouting of myelinated afferents. Nature 1992;355(6355):75–78. Am J Physiol Gastrointest Liver Physiol 2002;282(6):G1045–G1051. 204. Hughes DI, Scott DT, Todd AJ, et al. Lack of evidence for sprouting of 174. Stewart T, Beyak MJ, Vanner S. Ileitis modulates potassium and sodium Abeta afferents into the superficial laminas of the spinal cord dorsal horn currents in guinea pig dorsal root ganglia sensory neurons. J Physiol after nerve section. J Neurosci 2003;23(29):9491–9499. 2003;552(pt 3):797–807. 205. Shehab SA, Spike RC, Todd AJ. Do central terminals of intact myelin- 175. Beyak MJ, Ramji N, Krol KM, et al. Two TTX-resistant Na1 currents in ated primary afferents sprout into the superficial dorsal horn of rat spi- mouse colonic dorsal root ganglia neurons and their role in colitis-induced nal cord after injury to a neighboring peripheral nerve? J Comp Neurol hyperexcitability. Am J Physiol Gastrointest Liver Physiol 2004;287(4): 2004;474(3):427–437. G845–G855. 206. Woodbury CJ, Kullmann FA, McIlwrath SL, et al. Identity of myelinated 176. Gold MS, Traub RJ. Cutaneous and colonic rat DRG neurons differ cutaneous sensory neurons projecting to nocireceptive laminae following with respect to both baseline and PGE2-induced changes in passive nerve injury in adult mice. J Comp Neurol 2008;508(3):500–509.

Rathmell5e_Ch003.indd 14 3/23/18 12:12 AM QUERY: AQ1: Please confirm if 5-HT3 should be defined here. Please reconcile.

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