Progress in molecular biology and translational science, volume 131

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ISBN: 978-0-12-801389-2
ISSN: 1877-1173
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CONTRIBUTORS
Seena K. Ajit
Department of Pharmacology & Physiology, Drexel University College of Medicine,
Philadelphia, Pennsylvania, USA
E. Alfonso Romero-Sandoval
Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of
Pharmacy, Clinton, South Carolina, USA
Carolina Burgos-Vega
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Julie A. Christianson
Department of Anatomy and Cell Biology, School of Medicine, University of Kansas
Medical Center, Kansas City, Kansas, USA
Vaskar Das
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Gregory Dussor
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Jill C. Fehrenbacher
Department of Pharmacology and Toxicology; Stark Neuroscience Research Institute, and
Department of Anesthesiology, Indiana University School of Medicine, Indianapolis,
Indiana, USA
Sarah J.L. Flatters
Wolfson Centre for Age-Related Diseases, King’s College London, London, United
Kingdom
Sandrine M. Ge´ranton
Department of Cell and Developmental Biology, University College London, London,
United Kingdom
Mohab Ibrahim
Department of Anesthesiology, University of Arizona, Tucson, Arizona, USA

Kufreobong E. Inyang
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Nathaniel A. Jeske
Department of Oral and Maxillofacial Surgery, UT Health Science Center, San Antonio,
Texas, USA
Jungo Kato
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

xiii


xiv

Contributors

Arkady Khoutorsky
Department of Biochemistry, Rosalind and Morris Goodman Cancer Research Centre,
McGill University, Montre´al, Quebec, Canada
Benedict J. Kolber
Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania, USA
Marguerite K. McDonald
Department of Pharmacology & Physiology, Drexel University College of Medicine,
Philadelphia, Pennsylvania, USA
Ohannes K. Melemedjian
Department of Neural and Pain Sciences, School of Dentistry, University of Maryland,
Baltimore, Maryland, USA
Aaron D. Mickle
Department of Pharmacology, The University of Iowa Roy J. and Lucile A. Carver College
of Medicine, Iowa City, Iowa, and Department of Anesthesiology, Washington University
School of Medicine, St. Louis, Missouri, USA

Durga P. Mohapatra
Department of Pharmacology; Department of Anesthesia, The University of Iowa Roy J. and
Lucile A. Carver College of Medicine, Iowa City, Iowa, and Department of Anesthesiology,
Washington University School of Medicine, St. Louis, Missouri, USA
Jamie Moy
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Amol Patwardhan
Department of Anesthesiology, University of Arizona, Tucson, Arizona, USA
Angela N. Pierce
Department of Anatomy and Cell Biology, School of Medicine, University of Kansas
Medical Center, Kansas City, Kansas, USA
Steven A. Prescott
Neurosciences and Mental Health, The Hospital for Sick Children, and Department of
Physiology, University of Toronto, Toronto, Ontario, Canada
Theodore J. Price
Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
Reza Sharif-Naeini
Department of Physiology and Cell Information Systems Group, McGill University,
Montreal, Quebec, Canada
Andrew J. Shepherd
Department of Pharmacology, The University of Iowa Roy J. and Lucile A. Carver College
of Medicine, Iowa City, Iowa, and Department of Anesthesiology, Washington University
School of Medicine, St. Louis, Missouri, USA
Justin Sirianni
Department of Anesthesiology, University of Arizona, Tucson, Arizona, USA


Contributors

xv

Robert E. Sorge
Department of Psychology, University of Alabama at Birmingham, Birmingham,
Alabama, USA
Camilla I. Svensson
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
Sarah Sweitzer
Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of
Pharmacy, Clinton, South Carolina, USA
Andrew Michael Tan
Department of Neurology and Center for Neuroscience and Regeneration Research,
Yale University School of Medicine, New Haven; Rehabilitation Research Center, Veterans
Affairs Connecticut Healthcare System, West Haven, and Hopkins School, New Haven,
Connecticut, USA
Keri K. Tochiki
Department of Cell and Developmental Biology, University College London, London,
United Kingdom
Stacie K. Totsch
Department of Psychology, University of Alabama at Birmingham, Birmingham,
Alabama, USA
Megan E. Waite
Department of Psychology, University of Alabama at Birmingham, Birmingham,
Alabama, USA


PREFACE
When we were approached to assemble and edit this volume, we were
immediately faced with a dilemma: there are many excellent pain textbooks
that already exist, why should we set out to create a new one? After some
time, and some perusing of the venerable Textbook of Pain, which has just

received a fresh update, it occurred to us that there was indeed room for
a new textbook on pain. While the existing texts are unquestionably excellent, they largely fail to touch on exciting new areas of research emerging
from young investigators in the field. There are many young investigators
who were on the frontlines of pain research in the laboratories of wellknown figures in the field and now have their own independent laboratories
continuing their exciting lines of investigation. Therefore, we decided to set
out to create a textbook with a slightly different agenda. Rather than focusing on specific topics, we decided to assemble a group of the leading young
investigators in the field of pain research, give them some guidance on our
overall goals, and set them loose to create the chapters they would like to see
based on their most exciting new areas of research.
The title of this book is: “Molecular and Cell Biology of Pain.” A book
with such a title could have 100 chapters and take up an entire bookshelf. This
volume is not meant to be comprehensive, not by any stretch of the imagination, but it is meant to be exciting and new. We hope that these are topics
that are largely not covered in existing texts in the field but we also hope that
these topics will have staying power in the field. To that end, the group of
investigators assembled for this volume have already agreed, in principle,
to update this volume periodically as our research areas continue to progress.
We hope that the evolution of this title over the coming years will give a sort
of history of the research endeavors engaged by the authors of these chapters.
We are indebted to our mentors, who are many and whom we assume
know who they are by this point. We are also indebted to the institutions,
University of Arizona and University of Texas at Dallas that have given us
the opportunity to do this work. We would like to thank the production team
at Elsevier and especially Helene Kabes for her work and collaboration on this
project. Most importantly, we want to thank our colleagues who agreed to
take on this project and without whom this volume most certainly would
not exist. We value your academic collaboration, your scientific accomplishments at such an early career stage, and your continued friendship.
xvii


xviii

Preface

Finally, it is our sincere hope that this book will find its way into lecture
halls throughout the world. We have aimed the material at graduate and
medical students and we think it can give these students an interesting snapshot of the forefront of the field. If you are a student reading this foreword,
we hope you find something in this book that inspires discovery. All of us
started in your shoes aspiring to learn and make a contribution to mankind
through scientific discovery. We wish you the best, and we look forward to
reading the volume that will be written by the coming generations.
THEODORE J. PRICE
GREGORY DUSSOR


CHAPTER ONE

An Introduction to Pain Pathways
and Pain “Targets”
Vaskar Das1
Behavioral
and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, USA
1
Corresponding author: e-mail address:

Contents
1. An Introduction to Pain and Pain Pathways
1.1 Neuropathic pain
1.2 Inflammatory Pain
2. Ion Channels, Receptors, and Other “Targets” for Persistent Inflammatory or
Neuropathic Pain

2.1 Ion channels
2.2 Sodium channels
2.3 Calcium channels
2.4 K+ channels
2.5 Receptors
2.6 Purinergic receptors
2.7 Toll-like receptors
2.8 PAR receptors
2.9 Glutamate receptors
2.10 AMPA receptors
2.11 NMDA receptors
2.12 Metabotropic glutamate receptors
2.13 Opioid receptors
2.14 TRPV receptors
2.15 Prostaglandin (prostanoid) E2
2.16 Pronociceptive neurotransmitters
3. Summary
Acknowledgments
References

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Abstract
The purpose of this chapter is to provide a brief introduction to the anatomy and physiology of pain pathways from peripheral nociceptors to central nervous system areas
involved in the perception and modulation of pain. This chapter also provides a short
introduction to major types of persistent pain: neuropathic and inflammatory persistent
pain, and gives an overview of some important molecular targets that are thought to
mediate these types of pain. These targets, which include ion channels, receptors, and
Progress in Molecular Biology and Translational Science, Volume 131
ISSN 1877-1173
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2015 Elsevier Inc.
All rights reserved.

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Vaskar Das

some neurotransmitters, are further discussed in the context of their relevance as potential drug targets for the better treatment of pain in patients with persistent pain. Finally,
this chapter introduces several important concepts in pain research that will be primary
topics for chapters that come later in the book.

1. AN INTRODUCTION TO PAIN AND PAIN PATHWAYS
The International Association for the Study of Pain (IASP) has defined
pain as “an unpleasant sensory and emotional experience associated with
actual or potential tissue damage, or described in terms of such damage.”1
When asked to describe their pain, individuals variously described it in terms
of severity (mild, moderate, severe), duration (acute or chronic), and type
(nociceptive, inflammatory, neuropathic).2
Nociceptive pain is the normal acute pain sensation produced by activation of nociceptors in skin, viscera, and other internal organs in the absence
of sensitization.3–7 It may occur as a result of mechanical, thermal, or chemical noxious stimulation and is variously described as an aching or throbbing
kind of pain.5,6,8,9 Nociceptive pain comprises four main stages: transduction (i.e., action at receptors in the periphery), transmission (i.e., action
potentials along axons), perception (i.e., cortical processing of nociceptive
input), and modulation (i.e., engagement of descending circuits).4,10–12
Noxious stimuli are first detected by mechanical, thermal, and chemical
nociceptors found on specialized nerve endings present in skin (cutaneous),
viscera, and other internal or external organs.8,9,13,14 Nociceptive impulses
are transmitted from the periphery to the spinal cord via primary afferent
nerve fibers which may be unmyelinated or myelinated.3,15–20 The central
nervous system (CNS) components of this pathway constitute particular
anatomical connections in the spinal cord, brain stem, thalamus, and cortex
(the “pain pathway”), linking the sensory inflow generated in high threshold
primary afferents with those parts of the CNS responsible for conscious

awareness of painful sensations21 (Fig. 1). Unmyelinated nerve fibers are
small diameter C-fibers with diameters in the range 0.4–1.2 μm.22,23
Myelinated primary afferent nerve fibers are the Að-fibers (2–6 μm diameter), whereas the thinly myelinated nerve fibers are the Aβ-fibers (>10 μm
diameter).23,24 Primary afferent C-fibers and Að-fibers are responsible for
transmission of noxious stimuli whereas Aβ-fibers transmit innocuous,
mechanical stimuli such as touch.21–24 Put simply, nociceptors collect information from noxious stimuli which are transmitted by C-fibers and


An Introduction to Pain Pathways and Pain “Targets”

3

Figure 1 Simplified schematic diagram of the pain pathway. Pain begins with detection
of damage or potentially damaging stimuli by nociceptive neurons in the periphery that
can transduce this signal into transmission toward the CNS. The first synapse in this
pathway is in the dorsal horn, where these projection neurons can send pain-related
information onto multiple brain areas. Pain perception occurs in the brain and can
be modulated by different centers in the brain. The brain also sends modulatory inputs
back down to the spinal cord to induce pain modulation.

Að-fibers through the dorsal root ganglia to the superficial laminae I/II of
the dorsal horn of the spinal cord.20,23 Að-fibers transmit impulses from
the dorsal horn to deeper laminae (III–IV) of the spinal cord and onto higher
centers in the brain via the spinothalamic tracts.20 Dorsal horn neurons comprise (i) projection neurons, (ii) local interneurons, and (iii) propriospinal
neurons.20,25 Although projection neurons are the primary means for transferring sensory information from the spinal cord to the brain, they are only a
small fraction of the total number of cells in the dorsal horn.23,26 Many


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Vaskar Das

projection neurons have axons that cross the midline and ascend to multiple
areas of the brain including the thalamus, periaqueductal gray matter, lateral
parabrachial area of the pons, and various parts of the medullary reticular
formation.27 These neurons are also involved in activation of endogenous
descending inhibitory pathways that modulate dorsal horn neurons.26
Activity-dependent synaptic plasticity in the spinal cord that generates
postinjury pain hypersensitivity together with the cellular and molecular
mechanisms responsible for this form of neuronal plasticity are termed
“central sensitization.”21 Neuroplastic changes relating to the function,
chemical profile, or structure of the peripheral nervous system are
encompassed by the term “peripheral sensitization” and encompass changes
in receptor, ion-channel, and neurotransmitter expression levels.28,29
Central sensitization in the spinal cord includes sensitization and disinhibition mechanisms, and supraspinally there are functional changes such as
enlargement of receptive fields.30,31 In the CNS, there are also changes in
the dynamic interplay between neuronal structures and activated glial
cells,30,32,33 a topic covered in depth in Chapter “Nonneuronal Central
Mechanisms of Pain: Glia and Immune Response” by E. Alfonso
Romero-Sandoval and Sarah Sweitzer.
Following tissue injury and inflammation, vasoactive mediators such as
histamine, substance P (SP), serotonin (5-HT), nitric oxide (NO), prostaglandins (PGs), and bradykinin are released which activate nociceptors
resulting in nociception.13 This in turn can induce release of pronociceptive
neurotransmitters such as SP, calcitonin gene-related peptide (CGRP),
dynorphin (Dyn), neurokinin A (NKA), glutamate, adenosine triphosphate
(ATP), NO, PGs, and neurotrophins such as brain-derived neurotropic factor (BDNF), from primary afferents either in the periphery or at the first synapse in the dorsal horn of the spinal cord.13,20,22,34,35 More recently, the
important role of proinflammatory cytokines (e.g., tumor necrosis factoralpha (TNF-α), interleukin-1β, interleukin-18, etc.) in peripheral and central sensitization mechanisms associated with persistent pain states has begun
to be appreciated.36
Many C-fibers express transient receptor potential vanilloid 1 (TRPV1)
receptors and hence are sensitive to the vanilloid, capsaicin, which is a highaffinity ligand for TRPV1 receptors.37 TRPV1-expressing C-fibers may be

further subdivided into two major classes:
(i) those that contain the neuropeptides, SP, and CGRP, express the highaffinity nerve growth factor (NGF) receptor, TrkA, and are developmentally dependent on NGF,34,38,39 and


An Introduction to Pain Pathways and Pain “Targets”

5

(ii) those that express isolectin B4, (IB-4), the P2X3 purinergic receptor,40
fluoride-resistant acid phosphatase, do not contain SP or CGRP,41 and
are dependent on glial cell line-derived neurotrophic factor (GDNF).34

1.1 Neuropathic pain
The IASP has defined neuropathic pain as “Pain initiated or caused by a primary lesion or dysfunction in the nervous system.”1,42 Neuropathic pain is
variously described by patients as having one or more of the following qualities: burning, tingling, electric shock like, and stabbing or pins and
needles.5,42,43 The appearance of abnormal sensory signs such as allodynia
in response to innocuous (nonnoxious) stimulation and/or hyperalgesia in
response to noxious stimulation is common.43 When neuropathic pain is
evoked, it may be classified as having dysesthetic, hyperalgesic, or allodynic
properties depending upon the dynamic or static characteristics of the
stimulus.44
In recent years, it has begun to be appreciated that the pathobiology of
various neuropathic pain subtypes may differ.45,46 Hence, multiple research
groups have focussed on developing and validating rodent models of each of
these neuropathic pain conditions.47,48 These more relevant rodent models
of neuropathic pain have considerable potential not only in terms of
unraveling the neurobiology of each of these neuropathic pain subtypes
but also in terms of identifying novel targets for discovery of new efficacious
and well-tolerated analgesics to improve relief of these persistent pain
conditions.43,49

Inflammatory and neuroimmune mechanisms contribute to both
peripheral and central sensitization that underpin the pathobiology of neuropathic pain.50 Following peripheral nerve injury, inflammatory cells
including mast cells, neutrophils, macrophages, and T-lymphocytes contribute to peripheral sensitization and hyperexcitability of injured and adjacent
noninjured primary afferent nerve fibers.50 In the CNS, activation of glial
cells including microglia and astrocytes leads to the production and secretion
of various proinflammatory mediators that promote neuroimmune activation and can sensitize the central terminals of primary afferent and
second-order neurons to increase the intensity and duration of pain.50–53
However, rodent models of neuropathic pain allow behavioral pain
responses such as mechanical allodynia in response to application of a nonnoxious stimulus (light pressure) to the hindpaws and/or hyperalgesia in
response to application of noxious stimuli (pressure, heat, cold) to the


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Vaskar Das

hindpaws, to be quantified.43,49 The three most commonly used rodent
models of neuropathic pain involve induction of a unilateral chronic constriction injury of the sciatic nerve,54 partial sciatic nerve ligation,55 and
L5 spinal nerve ligation.56 Over the past two decades, these models have
been used to identify numerous neuropathic pain “targets” encompassing
various receptors, ion channels, and enzymes, as well as to assess novel pain
therapeutics in development.43,49
More recently, rodent models of varicella zoster virus-induced neuropathic pain, antiretroviral drug-induced neuropathic pain, cancer
chemotherapy-induced neuropathic pain, bone cancer pain, and multiple
sclerosis-induced neuropathic pain have been developed.43,57,58 It is hoped
that through use of these more sophisticated rodent pain models, it will be
possible to gain an enhanced understanding of the pathobiology of these
conditions. Additionally, it may be possible to identify novel “druggable”
targets for drug discovery aimed at producing novel analgesics with
improved efficacy and reduced adverse-event profiles for improved relief

of these chronic pain conditions in the clinical setting.49,59
Persistent ongoing pain secondary to nerve injury is underpinned by
considerable complexity and plasticity at multiple levels of the neuraxis.49,58,60–62 Following peripheral nerve injury, ectopic firing of injured
and uninjured afferents induces neuroplastic changes and “central
sensitization” in the spinal cord and the brain, underpinned by both neuronal and nonneuronal mechanisms49 (Fig. 2). Intensive research over the past
two decades has revealed a large number of receptors, enzymes, and ion
channels as potential novel targets for drug discovery programs aimed at

Figure 2 Simplified diagram of mechanisms of neuropathic pain. Peripheral nerve
injury causes many changes in the function and phenotype of injured fibers, but an
important property for neuropathic pain is the generation of ectopic activity (action
potentials without any stimulus, signified by lightning). This ectopic activity drives spontaneous pain and plasticity in the dorsal horn and brain that may underlie clinical features like allodynia.


An Introduction to Pain Pathways and Pain “Targets”

7

producing new drugs for the relief of neuropathic pain.63–65 Although several molecules have entered preclinical and clinical development, very few
have been approved by regulatory agencies for clinical use.66 Hence, this
large unmet medical need is driving research in this field.

1.2 Inflammatory Pain
Inflammatory pain is precipitated by an insult to the integrity of tissues at the
cellular level. One of the characteristic features of inflammatory states is that
normally innocuous stimuli can produce pain.67 Inflammation is classically
associated with pain (dolor), heat (calor), redness (rubor), swelling (tumor),
and loss-of-function (function laesa).67,68 Examples of inflammatory pain
include pain secondary to tissue injury and infection as well as rheumatoid
arthritis.69,70 Following tissue injury, nociceptors in the affected tissue become

sensitized due to the release of proinflammatory mediators from damaged cells
and blood vessels at the site of injury as well as from immune cells that invade
the injured site.71 This topic is covered in detail in Chapter “Peripheral
Scaffolding and Signaling Pathways in Inflammatory Pain by Jeske Nathan.”
Inflammatory mediators including protons, 5HT, histamine, adenosine,
bradykinin, prostaglandin E2 (PGE2), NO, IL-1, TNF-α, interleukin-6
(IL-6), leukemia inhibitory factor, and NGF5,72,73 contribute to nociceptor
sensitization so that innocuous stimuli are detected as painful (allodynia) or
there is an exaggerated response to noxious stimuli (hyperalgesia).34,74 The
central terminals of primary afferent nerve fibers (first-order neurons) are
located in the superficial layers (laminae I/II) of the dorsal horn of the spinal
cord.13 Synaptic input from these terminals to second-order neurons in the
spinal cord transfers information created by action potentials in primary
afferents secondary to peripheral noxious stimuli (depending on intensity
and duration), to the thalamus, and then onto the cerebral cortex in the
brain.13 Synaptic function at the central terminals of first-order neurons is
regulated by neurotransmitter release, primarily involving glutamate, and
neuroactive peptides like substance P and CGRP.60–62,75–78
In inflammatory pain, peripheral inflammation induces a phenotypic
switch in primary sensory neurons to induce a change in their neurochemical character and properties.62 This topic is covered in some depth in Chapters “Translation Control of Chronic Pain by Ohannes K. Melemedjian and
Arkady Khoutorsky,” “Regulation of Gene Expression and Pain States by
Epigenetic Mechanisms by S.M. Ge´ranton and K.K. Tochiki,” and
“Commonalities between Pain and Memory Mechanisms and their Meaning for Understanding Chronic Pain by Theodore J Price and Kufreobong E
Inyang.” In brief, this is underpinned by alterations in transcription and


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Vaskar Das

translation of various receptors and ion channels to induce central sensitization by virtue of a change in the level of synaptic input produced by the sensitized afferent nerve fibers62 (Fig. 3). Put simply, continuous inputs from
sensitized nociceptive afferents can activate or trigger central sensitization
that is characterized by a reduced threshold of dorsal horn neurons to noxious stimulation16,79–84 (Fig. 3). There is expansion of the receptive fields of
dorsal horn neurons,85,86 and temporal summation of slow postsynaptic
potentials resulting in a cumulative depolarization and a prolonged after discharge or “wind up” of dorsal horn neurons.15 There is also increased excitability of the flexion reflex in response to peripheral stimulation.82,87 The
neural mechanisms underlying central sensitization involve excitatory amino
acids (EAAs, e.g., mainly L-glutamate) acting at AMPA and NMDA receptors with the net result being persistent activation of the NMDA receptor84
to allow Ca2+ entry into neurons and activation of numerous intracellular
signaling pathways.62
SP-expressing C-fibers and BDNF-expressing dorsal root ganglion
(DRG) neurons both have a significant role in inflammation-induced central sensitization after exposure of their peripheral terminals to inflammatory
mediators and NGF released from immune cells.62,88,89 Ectopic firing of
sensitized terminals increase Aβ-mediated synaptic input to superficial dorsal
horn neurons62,90 and induction of cyclooxygenase-2 (COX-2) expression
levels to drive production of PGE2.62,91,92 Retrograde transport of NGF
from the peripheral terminals of C-fibers to the dorsal root ganglia induces
upregulation of TRPV1 expression and activation (phosphorylation) of p38
MAPK.39,93 This in turn leads to upregulated synthesis and release of

Figure 3 Simplified diagram of mechanisms of inflammatory pain. Activation of
immune cells during inflammation leads to the release of inflammatory mediators that
act on nociceptors. Many of these inflammatory mediators from immune cells directly
activate or modulate the activity of nociceptors. This can drive spontaneous pain and
plasticity in the dorsal horn and brain that underlies clinical features including allodynia
and hyperalgesia.


An Introduction to Pain Pathways and Pain “Targets”

9

proinflammatory cytokines among which IL-1β and TNF-α contribute to
the development of central sensitization by enhancing excitatory and reducing inhibitory currents, and by activating induction of COX-2.62,91,94 As a
result, GluR2-containing AMPA receptor activation allows entry of Ca2+
into neurons,62,95 and this mechanism generates as much Ca2+ influx as
occurs with NMDA receptor activation during inflammatory pain.62,95

2. ION CHANNELS, RECEPTORS, AND OTHER “TARGETS”
FOR PERSISTENT INFLAMMATORY OR
NEUROPATHIC PAIN
Intensive research over the past two decades has revealed a vast array
of ion channels, receptors, transporters, and enzymes that are potential
“druggable” targets for use in discovery programs aimed at developing
the next generation of analgesic drugs.66 Examples of “pain targets” for
potential modulation by novel analgesic agents include voltage-gated
sodium channels (Nav1.3, Nav1.7, and Nav1.8),12 voltage-gated potassium
channels (Kv1.4) is the sole Kv1 subunit expressed in smaller diameter sensory neurons96–98 suggesting that homomeric Kv1.4 channels predominate
in Aδ and C-fibers arising from these cells.97 By contrast, larger diameter
neurons associated with mechanoreception and proprioception express
high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 subunits,
suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.97,99–102 Additional “pain
targets” include voltage-gated calcium channels (VGCC) (α2δ subunits;
Cav2.2, Cav3.1, Cav3.2, Cav3.3),103 acid-sensing ion channels
(ASICs)104 covered to some extent in Chapter “Meningeal Afferent Signaling and the Pathophysiology of Migraine by Carolina Burgos-Vega, Jamie
Moy and Greg Dussor,” NMDA receptors, TRPV1 receptors, covered in
Chapter “Sensory TRP Channels: The Key Transducers of Nociception
and Pain by Aaron D. Mickle, Andrew J. Shepherd and Durga P.
Mohapatra,” NKA, purinergic receptors, toll-like receptors (TLRs),
protease-activated receptors (PAR) receptors, opioid receptors, the norepinephrine transporter, and cyclooxygenases (COX-1/2).105 Many of these
“pain targets” are described briefly in the following sections and in more

detail throughout this volume, as noted and summarized in Fig. 4.

2.1 Ion channels
Ion channels play a key role in nociception and are involved in sensory
transduction (TRPV1), regulation of neuronal excitability (potassium


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Vaskar Das

Figure 4 Mechanisms of inflammation-induced pain in the periphery. Tissue damage
causes an immune response and the release of inflammatory mediators that act on
nociceptors. As described in the text, peripheral nociceptors are finely tuned to detect
mediators released by immune cells via the expression of receptors that bind these
ligands and the presence of ion channels that are altered by the signaling pathways
downstream of activation of these receptors.

channels), action potential propagation (sodium channels, ATP-gated channels, ASICs), and presynaptic release of various neurotransmitters (calcium
channels).106

2.2 Sodium channels
Voltage-gated sodium channels are considered a major target for the development of novel therapies for improving pain management, as the ectopic
firing of primary afferents is associated with abnormal sodium channel
regulation.107–109
Sodium channels comprise an α-subunit containing a voltage-gated
sodium-selective aqueous pore and one or two smaller ancillary β-subunits.110
Sodium channel subtypes differ in their sensitivity to block by tetrodotoxin
(TTX) with six isoforms being sensitive (TTXs: Nav1.1, Nav1.2, Nav1.3,
Nav1.4, Nav1.6, Nav1.7) to block by nanomolar concentrations of TTX

An Introduction to Pain Pathways and Pain “Targets”

11

and three isoforms being resistant to micromolar concentrations of TTX
(Nav1.5, Nav1.8, Nav1.9).111
Acute inflammatory and neuropathic pain can be attenuated or abolished
by local treatment with sodium channel blockers,112 showing that peripheral
nociceptive input is dependent on the presence of functional voltage-gated
sodium channels. Four voltage-gated sodium channel subtypes (Nav1.3,
Nav1.7, Nav1.8, and Nav1.9) are of greatest interest in pain due to their
selective expression in peripheral nerves.107,113–128 For first-order sensory
neurons, Nav1.8 is expressed by the cell body, peripheral terminals, and central terminals within the dorsal horn of the spinal cord.110 Anatomical and
electrophysiological evidence indicates that expression of Nav1.9 is largely
restricted to nociceptive Aδ- and C-fibers.110
Highlighting the importance of Nav1.7 in inflammatory pain (Fig. 4),
levels of expression of Nav1.7 are increased in sensory nerve terminals by
inflammation12,128 and following ablation of Nav1.7 in nociceptive neurons,
inflammatory pain responses are greatly reduced.129 Additionally, dominant
gain-of-function mutations in SCN9A, the gene encoding Nav1.7 to
increase DRG neuron excitability are thought to be causal in two inherited
chronic pain disorders in humans, viz, erythromelalgia, characterized by
burning pain and skin redness in the extremities, and paroxysmal extreme
pain disorder, characterized by skin flushing, rectal, periocular, and perimandibular pain evoked principally by mechanical stimuli.109,130 Additionally, congenital indifference to pain due to rare recessive loss-of-function
mutations in SCN9A mean that although individuals so-affected are of normal intelligence, they often fail to recognize and report pain in response to
injury or infection which can lead to early mortality.131
Conditional knockout of SCN9A in mice abolished mechanical pain,
inflammatory pain, and reflex withdrawal responses to noxious heat recapitulating the pain-free phenotype in humans with SCN9A loss-of-function

mutations.128 Interestingly, as conditional knockout of SCN9A in both sensory and sympathetic neurons in mice with spinal nerve transection, markedly reduced neuropathic pain behavior in these animals, neuropathic pain
therefore appears to involve interaction between sensory and sympathetic
neurons.128
In neuropathic pain, levels of expression of Nav1.3 are increased in damaged peripheral nerves and this is highly correlated with the appearance of a
rapidly repriming sodium current in small DRG neurons consistent with the
notion that Nav1.3 channels make a key contribution to neuronal hyperexcitability in neuropathic pain.12,105,110,132–135


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2.3 Calcium channels
VGCCs play an important role in synaptic transmission and nociceptive
signaling.103,136–138 N-type (Cav2.2) VGCCs are expressed in high density
in the cell bodies of primary afferents in the dorsal root ganglia and on
presynaptic terminals that form synapses with second-order neurons in the dorsal horn.139–144 Cav2.2 channels regulate calcium entry, modulate neurotransmitter release, and lead to changes in sensory nerve excitability for the
modulation of pain.67,145–147 Mice lacking Cav2.2 channels have reduced pain
responses secondary to inflammation and peripheral nerve injury.95,103,148,149
T-type VGCCs (Cav3.1, Cav3.2, and Cav3.3) are expressed on the cell
bodies and peripheral terminals of primary afferents, where they contribute
to initiation of the action potential to regulate neuronal excitability.103,150–152
Multiple studies in rodent pain models have implicated the α2δ1 subunit
of presynaptic calcium channels as having an important role in persistent pain
states.153 This is emphasized by clinical studies showing that the antineuropathic drugs, gabapentin, and pregabalin that are ligands at the α2δ1
subunit, have efficacy for the relief of neuropathic pain.154–157 The mechanisms of action of these drugs are still controversial, but their widespread
use for neuropathic pain disorders is highlighted in Chapter “Chronic Pain
Syndromes, Mechanisms, and Current Treatments by Justin Sirianni,
Mohab Ibrahim and Amol Patwardhan.”

2.4 K+ channels
Voltage-gated K+ (Kv) channel subunits are expressed in DRG neurons and
have an important physiological role in the regulation of membrane potentials in excitable tissues including nociceptive neurons.101,102,158–160 The Kv
channel subunit Kv1.4 is the sole Kv1 α subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in Aδ
and C-fibers arising from these cells.97,99,100 Additionally, these neurons are
presumably nociceptors, because they also express the TRPV1 capsaicin
receptor, CGRP, and/or Na+ channel SNS/PN3/Nav1.8.97,99,161–164
However, larger diameter neurons associated with mechanoception and
proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or
other Kv1 α subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these
cells.97,99,100,164 As the opening of K+ channels leads to hyperpolarization
of the cell membrane and so decreased nerve cell excitability, several Kv
channels are implicated as possible targets for novel pain therapeutics. For


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13

example, A-type potassium currents contribute significantly to neuronal
excitability and central sensitization in the dorsal horn of the spinal cord
in inflammatory pain.100,165–169
However, abnormal hyperexcitability of primary sensory neurons plays
an important role in neuropathic pain.164,166 Kv channels regulate neuronal
excitability by affecting the resting membrane potential and influencing the
repolarization and frequency of the action potential and may therefore play a
key role in ectopic activity that develops in peripheral nerves driving neuropathic pain.163,164 Additionally, diabetes primarily reduces Kv channel
activity in medium and large DRG neurons.164 Increased BDNF activity
in these neurons likely contributes to the reduction in Kv channel function
through TrkB receptor stimulation in painful diabetic neuropathy.164

2.5 Receptors
In inflammatory pain, multiple receptor classes located on nociceptors are
modulated by vasoactive mediators released from damaged tissues and
immune cells that invade the inflamed tissues.170

2.6 Purinergic receptors
ATP activates P2X purinergic receptors, especially P2X1, P2X3, or P2X7
receptors, to produce pain.171–173 Currently, a range of preclinical studies
are investigating a role for P2X receptors in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction.173 Some
of these have been progressed into clinical trials for rheumatoid arthritis,
pain, and cough.173
P2X3 receptors, located exclusively on small diameter nociceptivefibers, are implicated in inflammatory pain and P2X4 receptors on microglia
in the dorsal horn of the spinal cord are implicated in the pathogenesis of
neuropathic pain.174 Antagonists at the P2X7 receptor also reduce pain
behaviors in rodent models of inflammatory and neuropathic pain, again
highlighting that purinergic glial–neuronal interactions are important modulators of noxious nociceptive neurotransmission.175 Hence, P2X3, P2X4,
and P2X7 receptors are potential targets for novel therapeutics for the treatment of inflammatory and neuropathic pain conditions.176,177

2.7 Toll-like receptors
Proinflammatory central immune signaling contributes significantly to the
initiation and maintenance of heightened pain states because recent


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discoveries have implicated the innate immune system, in particular, pattern
recognition TLRs in triggering these proinflammatory central immune signaling events.178 There is considerable interest in the targeting of TLRs on

immune cells for the prevention and treatment of cancer, infection, inflammation, and autoimmune diseases.179 In neuropathic pain, “TLR4 receptors
on peripheral immune cells (e.g., monocytes/macrophages, dendritic cells,
and immune-related cells such as keratinocytes)”170 as well as “activated
microglia in the CNS” appear to have a key role in the establishment of this
condition.180–183 Acute TLR4 antagonism attenuates neuropathic pain
behavior and potentiates opioid antinociception.184 Hence, TLR4 appears
to be a possible target for therapeutic intervention for relief of neuropathic
pain and for augmenting opioid analgesia. As already noted in an earlier section of this literature review, dysregulation of chemokines (Section 1) and
their receptors (Section 2.1; Fig 4), particularly fractalkine and its CX3CR1
receptor, appear to play a key role in neuroimmune signaling that contributes
significantly to the pathobiology of neuropathic pain.185 This target is discussed in more detail in Chapter “Role of Extracellular Damage-Associated
Molecular Pattern Molecules (DAMPS) as Mediators of Persistent Pain by
Jungo Kato and Camilla I Svensson.”

2.8 PAR receptors
PARs are G-protein coupled receptors (GPCRs) that have a unique activation mechanism involving specific proteolytic cleavage of the amino-terminal
sequence by serine proteases.186–188 PAR1 is expressed by primary afferent
neurons and can modulate nociception.189 PAR2 is expressed by SP- and
CGRP-containing primary afferents.189 Activation of PAR2 induces the
release of the pronociceptive neurotransmitters, SP, and CGRP from both
peripheral and central terminals of primary DRG neurons.189 PAR4 modulates nociceptive responses in normal and inflammatory conditions such that a
PAR4 agonist alleviated inflammatory pain in rats.159,188,190–196

2.9 Glutamate receptors
Glutamate is the major EAA neurotransmitter in the central nervous system
and is found in at least 70% of sensory neurons in the DRGs.197 It is released
from the central terminals of primary afferents and has an important role in
nociceptive neurotransmission.197 Glutamate acts via two main receptor
classes, iGluRs (ionotropic), and mGluRs (metabotropic) with iGluRs further subdivided into AMPA, NMDA, and kainate receptors.72,198 AMPA

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and NMDA receptors are directly coupled to cation-permeable ion channels
whereas metabotropic glutamate receptors (mGluRs) are coupled via
G-proteins to soluble second messengers.53,72,198–200 Brief nociceptive stimuli primarily activate AMPA receptors whereas stimuli of more prolonged
duration activate NMDA receptors.35,201 Astroglial cells remove excess glutamate from the extracellular space and express the glutamate reuptake transporters, GLAST/EEAT-1 (excitatory amino acid transporter) and glutamate
transporters EEAT-2/GLT-1.202–207

2.10 AMPA receptors
AMPA receptors have an important role in acute spinal processing of nociceptive and nonnociceptive inputs.208 Activation of AMPA receptors by
glutamate results in potent depolarization of dorsal horn neurons to remove
the Mg2+ block, and hence activate NMDA receptors resulting in calcium
influx and initiation of a cascade of downstream signaling events.209 Because
AMPA receptors also have roles in many other CNS functions,208 they are
generally regarded as being unsuitable targets for development of novel pain
therapeutics.208

2.11 NMDA receptors
NMDA receptors (Fig. 1) are located in the superficial and deeper laminae of
the spinal dorsal horn on the central terminals of primary afferents as well as
on membranes that are postsynaptic to the primary afferent.210 All NMDA
receptors display a certain degree of voltage-dependent Mg2+ block and
marked permeability to Ca2+ after removal of the Mg2+ block
(Fig. 1).198,211–214 Spinal NMDA receptors have an important role in
“central sensitization” in the spinal cord in persistent pain states.215 Although
molecules targeting NMDA receptors have potential for the relief of persistent pains such as neuropathic pain, NMDA receptors are involved in normal physiological functions and so the first generation of these agents were
hampered by CNS side effects in the analgesic dose range.216

2.12 Metabotropic glutamate receptors
Group I mGluRs in laminae I/II of the spinal dorsal horn play an important
role in the transduction of nociceptive input from C-fibers.217 There are
three mGluR classes containing eight cloned mGluRs and in vivo studies
show that these are not involved in acute nociceptive signaling.218–220
Group I mGluRs (mGlu1 and 5) are implicated in central sensitization


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and persistent nociception.221 By contrast, activation of group II mGluRs
(mGlu2/3) alleviates neuropathic and inflammatory pain.221 This topic is
discussed in more detail in Chapter “mGluRs Head to Toe in Pain by
Benedict J. Kolber.”

2.13 Opioid receptors
Opioid receptors are members of the superfamily of 7-transmembrane spanning region GPCRs that are coupled to intracellular effectors via
G-proteins, mainly of the inhibitory type, i.e., Gi,o.25,222,223 There are high
densities of opioid receptors in various brain regions, the dorsal horn of the
spinal cord, on peripheral nerve terminals, in peripheral tissues including the
gastrointestinal tract and on immune cells (Fig. 2).198,224,225
In the 1990s, three opioid receptor types, viz, μ (MOP), δ (DOP), and κ
(KOP) were cloned, and more recently multiple splice variants of these
receptors, particularly the MOP receptor, have been identified.222,226–228
The endogenous ligands for opioid receptors include the endomorphins
(highly MOP selective), β-endorphin (equal MOP and DOP selectivity),
met- and leu-enkephalin (more selective at DOP than MOP), and Dyn

(KOP selective).229–231 Opioid agonists activate opioid receptors to produce
potent analgesia by activation of the descending inhibitory system to inhibit
ascending excitatory nociceptive transmission (Fig. 2).224,232,233 Studies
using MOP receptor knockout mice show that the antinociceptive and
other effects of morphine and most other clinically available opioid analgesics, are produced secondary to activation of the MOP receptor.222,223,234–239 Studies using rodents show that peripheral opioid
actions are increased in inflammation, suggesting that peripherally selective
opioid analgesics may have benefit as future analgesic agents that are devoid
of CNS side effects.25,222

2.14 TRPV receptors
TRPV1 receptor (Fig. 4) is a ligand-gated nonselective cation channel
expressed on primary afferent sensory neurones that can be activated by
exogenous agents (e.g., capsaicin), endogenous substances (e.g., bradykinin,
ethanol, nicotine, anandamide, and insulin) as well as by heat (>43 °C) and
low pH.240–243 Following activation of TRPV1, there is a rapid increase in
intracellular Ca2+ concentrations resulting in nociceptive signal transduction
via C-fibers to produce pain in humans and pain behaviors in animals.243,244
TRPV1 knockout mice exhibit reduced thermal nociception and a loss of
inflammatory thermal hyperalgesia.242,245 However, both NGF and GDNF


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elicit thermal hyperalgesia during peripheral inflammation via an increase in
TRPV1 expression.246 Expression of these two growth factors follows different time courses and they act on distinctive subpopulations of DRG neurons.246 Intradermal injection of capsaicin and NGF produce heat
hyperalgesia via activation of their respective receptors, viz, TRPV1 and
TrkA on sensory nerve terminals. Moreover, PI3K induces heat
hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent

manner, and the PI3K pathway also appears to play a role that is distinct from
ERK (Fig. 4) by regulating the early onset of inflammatory pain.39,93,247,248
TRP channels are discussed in more detail in Chapter “Sensory TRP Channels: The Key Transducers of Nociception and Pain by Aaron D. Mickle,
Andrew J. Shepherd and Durga P. Mohapatra.”

2.15 Prostaglandin (prostanoid) E2
Inflammatory pain hypersensitivity is regulated by prostaglandin receptors
(EP1, EP2, EP3, EP4 receptors; Fig. 4).249 At the site of inflammation,
PGE2 sensitizes peripheral nociceptors via activation of EP2 receptors that
are present on the peripheral terminals of high threshold sensory nerve fibers
by reducing the nerve firing threshold and increasing responsiveness, which
is the key phenomenon of peripheral sensitization.249,250
Following tissue injury, the synthesis of PGE2 in the spinal cord91 contributes to central sensitization251 and increased excitability of spinal dorsal
horn neurons.249 NSAIDs inhibit prostaglandin synthesis through nonselective inhibition of constitutively expressed cyclooxygenase COX-1 as
well as the inducible isoform COX-2.252–254

2.16 Pronociceptive neurotransmitters
2.16.1 Nitric oxide
In the CNS, NO is synthesized primarily from the precursor, L-arginine, by
the enzyme nitric oxide synthase (NOS).255 There are three NOS isoforms,
viz, neuronal (nNOS), endothelial (eNOS), and inducible nitric oxide
synthase (iNOS), with nNOS having a role in the modulation of nociceptive
transmission in the spinal cord.255,256 Following glial cell activation in the
CNS, NO is produced by iNOS, to further sensitize nociceptive neurones
and contribute to the maintenance of central sensitization in persistent pain
conditions.256 NO produced in excess by iNOS and nNOS is implicated in
inflammatory and neuropathic pain, and so iNOS and nNOS inhibitors have
been investigated as potential novel agents for alleviation of these chronic
pain conditions.257

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2.16.2 Nerve growth factor
NGF levels increase during inflammation258–260 resulting in sensitization of
primary afferents to noxious thermal, mechanical, and chemical stimuli via
upregulated synthesis of TRPV1 receptors as well as SP, CGRP, and bradykinin receptors261–265 (Fig. 4). Anti-NGF therapeutics have advanced
to clinical trials in humans and have shown broad efficacy but also severe,
albeit rare, side effects. NGF signaling is discussed in more detail in Chapters
“Translation Control of Chronic Pain by Ohannes K. Melemedjian and
Arkady Khoutorsky” and “Commonalities between Pain and Memory
Mechanisms and their Meaning for Understanding Chronic Pain by
Theodore J Price and Kufreobong E Inyang.”

3. SUMMARY
Chronic pain is notoriously difficult to treat and so there is a large
unmet clinical need for new treatments to alleviate pain. Rodent models
of neuropathic and inflammatory pain have had a huge impact on our basic
understanding of pain and pain plasticity, a feature that will be discussed
throughout this volume. These basic science investigations have led to
the development of a broad variety of pain targets, some of which were discussed briefly here and some of which will be discussed in great detail
throughout the rest of this book. The goal of this line of research is to
develop a new generation of pain therapies with broad efficacy and limited
side effects.

ACKNOWLEDGMENTS
Dr. Das is grateful to Professors Maree Smith at University of Queensland and Theodore
Price at University of Texas at Dallas for editing of the chapter.

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