sis for Pain Perception

Volume 7, Number 4, 2001

s REVIEW

Molecular Basis for the Perception of Pain

It is perhaps presumptuous to talk about the molecular basis of a subjective sensation such as pain, but defined conformational changes in membrane proteins, controlled by a family of extra- and intracellular messenger molecules, are known to underlie the activation of sensory nerve terminals and the process of synaptic neurotransmission, which are necessary for pain perception. Furthermore, a subset of neurotransmission processes has a permissive, and possibly exclusive, role in pain perception. Clearly, the experience of pain in the clinical sense with all its affective components of unpleasantness and suffering cannot yet be fully understood in molecular terms, but the process of nociception, whereby the signal generated as a result of tissue damaging or potentially damaging peripheral stimuli reaches and evokes neuronal activity in the central nervous system, is becoming better characterized. Recent advances in neurobiology have given us insights that are already helping improve understanding of the events that lead to a patient experiencing pain and, it is hoped, will also lead to more successful treatment strategies. NEUROSCIENTIST 7(4):282292, 2001
KEY WORDS Pain, Neurotransmission, Chemical messengers

The Process of Nociception The peripheral nerve fibers are the first structures to become activated following tissue damage or inflammation. They carry a number of voltage and ligand-gated ion channels, which, together with associated G-proteincoupled receptors, are responsible for the generation of the nerve impulses that travel into the CNS and carry sensation to the level of consciousness. The small, unmyelinated sensory C-fibers, which are thought to perform the major role in signaling pain, in addition to containing the transduction elements common to all sensory fibers, are now known to have some specific channels and receptors that are not found on larger myelinated sensory fibers nor on neurons within the central nervous system. These transducers are under the control of cytokine and neurotrophin systems, which, in turn, are activated by tissue damage resulting in transducer up-regulation or in some cases de novo activation (induction). The nature of the neurotransmission process changes with maintained tissue damage or inflammation and leads to activation of sensory fibers that are normally silent (silent nociceptors) and, in some cases, the recruitment of large sensory fibers, which normally do not signal pain, into the pain-signaling process. Once impulses have reached the level of the spinal cord, excitatory neurotransmitters carry the sensory message onto relay neurons, which, via additional synaptic pathAddress correspondence to: Dr. R. G. Hill, Neuroscience Research Centre, Merck, Sharp and Dohme Research Laboratories, Terlings Park, Harlow, Essex, CM20 2QR UK; phone: 44 (0) 1279 440168; fax: 44 (0) 1279 440713 (e-mail: [email protected]).

ways, connect to the higher brain centers. Again, large and small primary afferent fibers share some of the same signaling systems within the spinal dorsal horn, but small fibers have a repertoire of neurotransmitter substances, many of which are neuropeptides, that are not found in larger non-nociceptive fibers (at least under normal circumstances). Part of the change in response seen in reaction to persistent nociceptive signaling caused by maintained inflammation or tissue damage and leading to intractable chronic pain (Fig. 1; Cervero and Laird 1991) is due to a reprogramming of the way in which the dorsal horn of the spinal cord and other central circuits respond to sensory inputs. This can be due to an up-regulation of presynaptic transmitter synthesis and release, to induction or up-regulation of postsynaptic receptors or channels, or to a physical rewiring of circuitry caused by the expression of factors leading to sprouting of afferent fiber terminals or the production of additional postsynaptic dendritic spines (Hatada and others 2000). The processes involved in the mechanisms of chronic pain are similar to those involved in the phenomenon of long-term potentiation, believed by many to underlie the laying down of memory. The higher centers of the brain, where the most subtle processing of impulse traffic takes place, are the areas about which, at present, least is known. One reason for this is that there is no clearly identifiable pain center in the brain (but see Perl 1998). Areas known to be involved in somatosensory perception are activated after noxious stimuli but so are areas in the limbic system, perhaps related to the distress and suffering aspects of pain and other areas important in autonomic function and defense reactions

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Fig. 1. This diagram illustrates the progression from baseline nociception to neuropathic pain. In phase 1, nociceptors respond to brief, nondamaging high-intensity stimuli, especially those that are chemical and thermal in nature. The trains of action potentials that are generated cease immediately when the stimulus is removed. In phase 2, either tissue damage or inflammation is produced and the silent nociceptors are activated. Nociceptors respond to thermal, mechanical, and chemical stimuli with lowered thresholds and larger responses. When a noxious stimulus is applied, the responses generated may outlast the stimulus and there can be changes in receptor, channel, and enzyme production driven by cytokines and neurotrophins. Changes can also occur in the CNS as a result of maintained input producing wind up of responses, neurotrophin production, immediate early gene induction, and transmitter and receptor synthesis induction. In phase 3, either nerve or CNS damage has led to reprogramming of central circuits so that pain may be self-sustaining even in the absence of peripheral inputs. The involvement of large A sensory fibers may lead to allodynia where gentle touch such as stroking with a feather may be perceived as painful. Prolonged sensory input to the dorsal horn of the spinal cord may have caused excitotoxic death of inhibitory interneurons, thus further increasing the excitability of nociceptive transmission. (Diagram from Cervero and Laird 1991 with permission.)

(see Hutchison and others 1999). The complexity of the human forebrain makes observations in lower animals at this level of limited value, whereas at the spinal level, where more is understood about structure and function, there are fewer differences between higher and lower mammalian species. Knowledge of the function of supraspinal circuits in pain perception in man is now improving as a result of data from imaging techniques such as PET and fMRI. Although they do not have molecular levels of resolution, they can visualize evoked activity in areas of the brain, such as the anterior cingulate cortex, which are activated in response to pain but not to lower intensity peripheral sensory inputs (Hutchison and others 1999). Molecular neurobiology thus has new target brain areas on which it can focus in

the future to identify specific molecules that may have a role in determining what distinguishes pain from other sensations. Sometimes pain can arise centrally (e.g., thalamic pain), and this may be a result of ischemic damage following a stroke. The mechanism probably involves a loss of inhibitory control as inhibitory interneurons are generally the most sensitive to ischemia. Impulse traffic into and within the CNS is controlled by descending inhibitory pathways coming down to the spinal cord from the brain, as well as by local inhibitory circuits. The effectiveness of this inhibition may be an important factor in determining the intensity of pain. This review will explore how molecular neurobiology is helping our understanding of the nociceptive process. A number of recent books have

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Box 1: Human Genetic Evidence that Specific Genetic Defects Lead to Changes in Pain Perception
Patients lacking functional Trk-A receptors have defects in pain perception. It has been observed by neurologists for many years that occasional patients present with an inability to feel pain, usually because they have suffered injury especially to joints as a result of not knowing when safe limits had been exceeded. It has now been shown that at least some of these patients have mutations in their Trk-A gene, which is likely to be the cause of the congenital insensitivity to pain with anhydrosis (CIPA) from which they suffer (Indo and others 1996). Trk-A is a tyrosine kinase receptor through which nerve growth factor (NGF) exerts its effects. This neurotrophin is important for the development of many sensory and autonomic neurons and is also an important part of the mechanism in the adult by which sensory nerve fibers respond to damage and regenerate. The stimulation of Trk-A by NGF released following tissue damage can also cause hyperalgesia, and this was a common side effect in clinical trials of the administration of NGF to treat diabetic neuropathy. Deletion, splice, and missense mutations in the tyrosine-kinase domain were seen in three unrelated patients with CIPA. Mice with the Trk-A gene deleted have similar but not identical deficits to the human patients. Other defects in the NGF signaling system such as in the production of NGF itself or in the low-affinity receptor p75 were not detected in the patients studied, although the corresponding knockout mice do show defective nociception. It should be stressed that changes in the NGF/trkA system are not the only way in which pain sensitivity can be altered by genetic factors, and recently it has been shown that there is angiotensin-converting enzyme polymorphism in patients with neuropathic pain and that the ACE/DD genotype is a significant risk factor (Kimura and others 2000). Patients with mutations on chromosome 19 leading to changes in P/Q type calcium channel function suffer from familial hemiplegic migraine (FHM). Familial hemiplegic migraine is a rare autosomal dominant subtype of migraine with aura. It is characterized by attacks in which transient hemiparesis accompanies the usual migraine symptoms. Migraine attacks typically last 1 to 3 days and present with unilateral pulsating headache. Four different mutations have now been identified in families with FHM on chromosome 19p13.1 in a single gene that encodes the (1A Ca channel subunit that is the functional unit of P/Q type voltage-gated Ca channels. This gene, CACNA1A, codes a protein of about 2400 amino acids. Each of the described mutations produces a missense mutant that codes for substitution of a highly conserved residue. The mutations are sometimes associated with a specific phenotype, and, for example, T666M produces FHM with progressive cerebellar ataxia, whereas V714A produces FHM symptoms only (Ophoff and others 1998). When whole cell and isolated patch recordings were made from HEK293 cells that had been transfected with cDNA carrying the FHM mutations, it was shown that both gain and loss of function resulted. Surprisingly, the mutated channels sometimes seemed to switch on and off, perhaps as a result of an unidentified factor (Hans and others 1999). This may be a necessary correlate of the episodic nature of the disease.

dealt with aspects of this topic and may help the interested reader get deeper into the subject matter (Max 1999; Wall and Melzack 1999; Wood 2000). Where Our New Knowledge Comes From The explosion in molecular biology has allowed us to detect changes in gene expression, both peripherally and centrally, in response to activity in sensory neurons; to locate areas of induced gene expression and translation in peripheral and central nervous systems; and to study the localization and function of specific protein products of gene translation (see Wood 2000). The proteins of interest can now readily be expressed transiently or stably in immortalized cell lines, which can be used as living test tubes to examine their function (e.g., see Hans and others 1999; Jordt and others 2000). Moreover, site-directed mutagenesis (Jordt and others 2000) can be used to alter the composition of a protein to establish areas of critical functionality, and the assembly of protein sub284 THE NEUROSCIENTIST

units into functional heteromers (Hans and others 1999) can be examined. The study of human genetics can provide evidence for the importance of some molecular systems in pain perception, for example, in the case of those rare individuals with a chronic insensitivity to pain (Indo and others 1996) or with painful disorders with a strong familial component, such as migraine headache (see Ophoff and others 1998; Hans and others 1999; Box 1). The information that is rapidly coming into the public domain from the human genome project will facilitate such studies. It is tempting to suggest that there must be an important genetic component in many chronic pain states as not every patient that experiences a zoster infection suffers the distress of postherpetic neuralgia and injury to peripheral nerves does not automatically lead to neuropathic pain. If we could understand at the genetic level why some patients were more likely to be victims of intractable pain, then improved treatments might result (see Box 1). A more practical and immediately addressable use of genetics for the pain scientist is

Molecular Basis for Pain Perception

the use of gene deletion or gene-overexpressing mutant animals to ask direct questions about the importance of particular systems coded by these genes in pain. This has become very important during the last 5 years with many different systems being studied (see Box 2). The cloning of novel genes expressing proteins with relevance to nociception has allowed the manufacture of very selective antibodies raised to synthetic peptides, which represent a unique part of the protein sequence. This has allowed high-resolution histology of the location of the target proteins in the nervous systems of a number of species including man. In the case of G-proteincoupled receptors, it has also allowed the study of function as activation of the receptor is signaled by internalization of the antibody-labeled receptor, and this internalization also allows neurotransmitter-coupled toxins to be used to specifically lesion populations of neurons bearing the receptor (see Nichols and others 1999). Studies on Peripheral Nociceptors The sensory nerve fibers, which are activated when damaging or potentially damaging stimuli are applied to the tissues of the body, are termed nociceptors. These are primarily the smallest unmyelinated slow-conducting C-fibers, but the grouping also includes some of the smallest myelinated fibers, the A fibers. There is some uncertainty about precisely how these fibers are induced to fire action potentials, but it is likely that mechanical, chemical, and temperature inputs all play a part. Those nociceptors that respond to mechanical stimuli are normally silent and become mechanosensitive after damaging or inflammatory events (Schmidt and others 2000), unlike larger sensory fibers, which have specific stretch receptors. It is thus possible that the mechanosensitivity of C-nociceptors is indirect, by way of chemical mediators, although it has recently been suggested that up to 35% of small DRG neurons do have gadolinium-sensitive stretch receptors (McCarter and others 1999). The C-nociceptors respond to heat and to chemical stimuli in their baseline state but show enhanced responses after tissue damage or inflammation (Schmidt and others 2000). The larger, A low-threshold mechanoreceptors become important in the allodynia seen in some neuropathic pain states where light touch becomes extremely painful (see Woolf and Salter 2000). Cell surface proteins have been used to separate C-nociceptors into major classes, for example, according to whether they bind the plant lectin IB-4 or express the trk-A receptors at which nerve growth factor (NGF) acts. In a detailed study of the properties of IB-4expressing DRG cells (Stucky and Lewin 1999), it was found that they were less responsive to noxious heat, had larger tetrodotoxin (TTX)-resistant currents, and had longer action potential durations. Sensitivity to capsaicin has also been used as a criterion, and those nociceptors that do not bear VR-1 receptors (i.e., are capsaicin-insensitive) (Caterina and Julius 1999) but have heteromeric P2X 2/3 purine receptors have been

suggested to have a special role in mechanical allodynia (Tsuda and others 2000). The response of fibers to heat received additional interest following the cloning of VR-1 and its congener VRL-1 (see Reichling and Levine 2000). A useful review of the factors recently identified as being expressed by sensory neurons can be found in Caterina and Julius (1999). Opening of ion channels, principally Na channels, is the means by which nociceptor fibers are induced to fire. We now know, because of recent cloning efforts, that there are at least six different proteins present in sensory neurons that may function as Na channels. Two of these are found elsewhere in the nervous system also and are sensitive to blockade by the Na channel toxin TTX, but four appear to be unique to sensory neurons. These are PN-1, which has a rapid activation, is present in small and large cells, and is TTX-sensitive; SNS/PN-3 and NaN/SNS2, which are restricted to small neurons and are TTX-resistant; and NaG, which is also found in glial cells but so far has not been shown to be functional (Waxman and others 1999). The recent report that, in pathological circumstances at least, there is an endogenous pentapeptide that can act as an Na channel blocker (Brinkmeier and others 2000) may mean we need to change our way of thinking about how such channels are activated and controlled. Perhaps the most common cause of clinical pain is inflammation. This may be due to a chronic disease process such as arthritis, or it can follow trauma. There are well-defined chemical changes that take place in the tissues as a result of an active disease process. The pH of the tissue falls, and a variety of messengers (generically called algogens as they cause pain either directly or in concert with other factors) including cytokines, prostanoids, histamine, 5-HT, peptides, and acetylcholine are released into the extracellular space close to nociceptor terminals and also interact with immune-competent cells releasing other messengers (Fig. 2). The interaction of the peptide bradykinin and the cytokine interleukin systems seems to be especially important (Phagoo and others 1999). Interleukin-1- increases the expression of both the B1 receptor and the production of metabolites of bradykinin that are the natural ligands for the B1 recep10 9 tor. These ligands, des-Arg -kallidin and des-Arg bradykinin in turn increase the levels of IL-1 mRNA. Release of bradykinin following tissue damage both activates B2 receptors directly and increases the synthesis of the B1 receptors on which the bradykinin metabolites act. This adds to the rich chemical mixture, which directly activates sensory nerve terminals and sensitizes them to thermal and mechanical stimuli, thereby causing them to fire, sending trains of action potentials toward the dorsal horn of the spinal cord where their central terminals release peptides and other messengers to activate spinal relay neurons (Fig. 3). The activation of these fibers also sends action potentials toward the periphery, and additional chemical messengers are released from the peripheral terminals to further complicate the recipe of the inflammatory soup (Fig. 2).

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Fig. 2. Diagram to illustrate the factors impinging on peripheral nociceptor fibers that may be responsible for their discharge and sensitization following noxious stimuli. This should be compared with the scheme outlined in Figure 1. Clinically, only those situations in which tissue damage or inflammation actually occurs will be important. As a result, the nociceptor fibers will be exposed to a mixture of potential algogens, some of which act directly to cause action potential generation and others that act as sensitizing agents. Further details are given in the text. Redrawn from Abrahams and Munglani (2000) with permission from Ashley Publications, Ltd.

The most abundant peptide and most potent vasodilator in primary afferent fibers is calcitonin generelated peptide, (CGRP) (Williamson and others 1997), and inflammatory mediators will release this peptide from DRG neurons (Averbeck and others 2000). Lowered pH is sufficient to produce release in the presence of extracellular Ca, as is the presence of bradykinin, but other mediators such as 5-HT and prostaglandin E2 are ineffective when applied individually but will add to the effects of bradykinin. Activation of VR-1 may be an important component of the mechanism as application of the VR-1 antagonist capsazepine reduced the CGRP release produced by co-application of bradykinin, 5-HT, and PGE2. Increases in blood flow, which are partly neurogenic (Williamson and others 1997), carry some of the chemical milieu away from the site of inflammation but can also lead to further nociceptor activation as a result of increase in tissue temperature. The pH of damaged or inflamed tissue falls to be significantly more acid than under baseline conditions. The application of low pH solutions intradermally or to a blister base in humans is intensely painful. We now know that there are specific receptors on sensory

neurons that are activated by low pH and that other receptive processes are sensitized by low pH (Caterina and Julius 1999). Interestingly, the capsaicin receptor (VR-1) has a receptor site on its extracellular part that detects the presence of protons (Jordt and others 2000), and it may be a combination of low pH and an endogenous ligand (possibly a lipoxygenase product) (Hwang and others 2000) that activates this particular chemical nociceptor. Other sensory neuron receptor channels that respond to pH, ASIC and DRASIC, produce potential changes that are transient and not likely to produce sustained firing; thus, their role in signaling pain is as yet uncertain (Caterina and Julius 1999). What Happens after Peripheral Nerve Damage? Neuropathic pain (produced as a result of nerve damage) can be very difficult to treat and may be extremely persistent. Some of the ideas about why this is so are outlined in Figure 1, and there is an increasing opinion that genetic factors may also play a part (Box 1). Injury to a nerve causes an inflammatory reaction (Bennett
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Fig. 3. Diagram to illustrate some of the factors controlling the excitation of neurons within the dorsal horn of the spinal cord as a result of impulses arriving in nociceptor fibers. This simplified schematic shows the multiple channels of transmission that lead to neuronal activation using the example of a fiber expressing both glutamate and peptide transmitter systems. The arrival of an Na channel operated action potential in the sensory nerve terminal triggers Ca entry via N- and possibly P/Q-type Ca channels. Glutamate and peptides are released and act on receptors on the proximate spinal dorsal horn neuron. Glutamate acts through AMPA receptors to produce a rapid depolarization, which relieves the Mg block of the NMDA receptor channel. Activation of the NMDA receptor leads to a longer lasting depolarization, and activation of mGLUR5 metabotropic receptors and peptide receptors further increases the excitability of the relay neuron. Postreceptor events lead to activation of protein kinases, mobilization of intracellular Ca, and induction of immediate early genes. Production of nitric oxide (NO) and prostaglandins (PG) feeds back on the afferent terminal to produce additional transmitter release. See text for further details. Redrawn from Abrahams and Munglani (2000) with permission from Ashley Publications, Ltd.

1999), and this may also occur as a result of infection. Immune-competent cells are found in the endoneurial compartment, and at the central end of the nerve there is glial activation. The postinfection pain associated with the nerve damage caused by viral infection with herpes zoster is one of the commonest neuropathic pains, and neuropathic pain is also seen as a result of HIV infection. Recently, it was demonstrated that intrathecal administration of the HIV envelope glycoprotein, gp120, produces hyperalgesia and allodynia in the rat but that heat-denatured gp120 was without effect (Milligan and others 2000). In mice with a chronic constriction injury to one sciatic nerve, it has been observed that the resulting thermal hyperalgesia and mechanical allodynia can be prevented by applying antibodies to the cytokine and tumour necrosis factor- (TNF ) to the nerve (Lindenlaub and others 2000). Increased sensitivity to noxious and non-noxious stimuli may also be linked to increased expression of receptors for algogens. Chronic constriction injury to rat sciatic nerve has been shown to produce an up-regulation of bradykinin B2 receptor message in the DRG ipsilateral to the injury within
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2 days, followed by an increase in the message for B1 receptors at 14 days postinjury (Levy and Zochodne 2000). One feature of sensory nerve damage is that although the axons may degenerate, the cell bodies in the DRG survive, and thus regrowth of axons is possible (and may in some cases be linked to the production of pain). The survival of sensory neurons has been linked to expression of HSP27, a molecular chaperone that can also stabilize microfilaments (Lewis and others 1999). There has been much discussion over which is the most appropriate nerve lesion to model human neuropathic pain (see Bennett 1999), but it is probable that none of the current experimental lesions adequately mimics the changes that give rise to clinical neuropathic pain. One common factor is that, qualitatively, the increase in channel and receptor expression that can be detected after experimental nerve lesions in animals is driven by trophic factors and inflammatory cytokines (vide infra). The sympathetic nervous system is also implicated in changes in nociception after nerve damage (see Munglani and Hill 1999).

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Box 2: The Use of Transgenic Animals and Gene Knock Down in the Study of Mechanisms of Nociception
Knockout animals A large number of publications have appeared in the last 5 years in which gene-deleted mice have been used to study nociceptive processes, and the reader is referred to Zimmer and Usdin (2000) for a general description of the approach and a detailed account of those studies related to opioid receptors. Targeted disruption of the gene of interest is performed in embryonic stem (ES) cells derived from J129 sv mice. Typically, a PGK-neo cassette is substituted for a critical region of the gene in a targeting vector made using 129sv genomic DNA and then introduced into the ES cells. ES cell clones are then selected for correct expression of the targeting vector, injected into C57Bl/6J blastocysts, which are then implanted into pseudopregnant foster mothers. Progeny are screened (depending on maternal strain used, this may be initially by coat color) and classified according to genotype established from tail biopsy specimens. Mice in a pure J129 genetic background are not suitable for behavioral experiments, and it is common to back-cross the offspring with C57Bl/6 mice to introduce as much of this strain as possible into the mix. It is sometimes possible to breed from a colony of homozygotic mice, but in a number of cases the homozygotes do not survive or breed as well as wild-type mice, so homozygotic animals for experimental use are derived from a colony of heterozygotes. It is also useful to have heterozygotes for study as they will shed light on the possible functionality of partial deletion of the gene product. Good examples of the approach described above can be found in the studies of disruption of the B2 bradykinin receptor by Borkowski and others (1995) and on the TTX resistant Na channel SNS/PN-3 by Akopian and others (1999). It is interesting to note that none of the transgenic animals studied so far out of a list including substance P, opioid, nicotinic, muscarinic, histamine cannabinoid or growth factor receptors, Ca channels, Na channels, VR-1, CGRP, or amylin show changes that are profound enough to suggest that any of the gene products deleted has a principle role in the control of nociceptive thresholds. The picture that is emerging supports the view that multiple substances and systems acting in parallel are responsible for generating and transmitting the nociceptive message into the CNS. There also appear to be multiple inhibitory and trophic systems that modulate the excitatory inputs. An important caveat with respect to all studies on gene-disrupted or knockout animals is that part of the phenotype may be due to adaptive, compensatory changes triggered by the absence of a gene product from the earliest stages of development (e.g., such as the increase in expression of PN-1 seen in the SNS null mutant mice by Akopian and others 1999). The technology for inducible knockouts that allow gene deletion once an animal has reached adulthood is currently unreliable. Antisense oligonucleotides The best approach currently available for gene knockdown in adult animals and in nonmurine species is by administration of oligonucleotides (OGN), which are antisense to a critical coding region of the gene that is to be blocked. This is not always successful, and the use of the technique has to be empirical. To affect genes in sensory neurons, the best results have been obtained by administering the OGN intrathecally so that they are taken up by afferent fibers and either retrogradely transported or diffused into the DRG cell bodies. Mismatch or nonsense sequence OGN can be used as controls for unspecific effects. In the case of SNS/PN-3, for example, Porreca and others (1999) designed an antisense 23mer to a unique sequence of the gene and controlled the experiment with a similar sized missense sequence. The OGNs were injected in a dose of 45 g intrathecally twice daily to rats that had previously been subjected to ligation of L5/6 spinal nerves. After 48 h of this treatment with the antisense OGN, expression of SNS/PN-3 in DRG was reduced whereas that of PN-1 was unaffected. There was also a reduction in the thermal hyperalgesia and tactile allodynia produced as a result of the nerve ligation. The missense OGN was without effect on Na channel expression or on the nociceptive responses of the animals. Cessation of antisense OGN administration was followed by recovery of the hyperalgesia and allodynia within 48 h, consistent with the 26-h half-life for Na channel turnover. If experiments are adequately controlled, this approach can be valuable. It is noteworthy that Porreca and others (1999) also administered an antisense OGN to NaN/SNS2 in a similar experiment, but in this case although protein production was knocked down they did not observe a change in the nociceptive behavior of spinal nerveligated rats.

Neurotrophins, Other Trophic Factors, and Sensory Neurons Different populations of sensory neurons seem to have differing neurotrophin dependence. Proprioceptive and cutaneous mechanoreceptive neurons are dependent upon NT-3 acting at trk-C as demonstrated by the loss

of such neurons in NT-3 or trk-C knockout mice (Box 2), whereas thermoreceptive and nociceptive neurons are dependent on NGF acting at trk-A (Silos-Santiago 2000). Rat pups exposed to an anti-NGF antibody in utero were found to be unresponsive to noxious stimuli and had lost many of their small sensory neurons when examined histologically. Both the C-unmyelinated and

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the small myelinated A neurons appear to be NGF dependent, especially those neurons that are neuropeptide releasing. A separate population of non-neurotrophindependent small sensory neurons is now known to exist. These are not peptide containing and may use purines as their major transmitters. They are characterized by the binding of IB-4 and appear to be dependent on glial cell line-derived neurotrophic factor (GDNF) acting at a receptor called c-ret, although during development many of these neurons go through a phase of NGF dependence (McMahon and Bennett 2000). It is noteworthy that both neurotrophin and GDNF-dependent nociceptors are capsaicin sensitive, via expression of VR-1 receptors, whereas expression of other signaling receptors and channels (e.g., B1, PN-3/SNS) seems to be primarily under the control of NGF. It is possible to show that neurotrophins are involved in the nociceptive changes produced by inflammation because the resulting hyperalgesia is reversed by antibodies to NGF or by a weak trk-A receptor antagonist, ALE-0540 (Owolabi and others 1999). Some of these effects are produced directly via neurons by increase in expression of nociceptive peptides or their receptors, whereas others are produced indirectly by immunecompetent cells such as the mast cells that carry trk-A receptors. Exogenous administration of NGF to the peripheral tissues can cause hyperalgesia within minutes (Shu and Mendell 1999). BDNF, a ligand for trk-B receptors, is up-regulated in trk-A-bearing cells after inflammation and retrogradely transported. As activation of trk-B also sensitizes nociceptors and produces hyperalgesia, BDNF may also be an important factor in neuropathic changes (Shu and Mendell 1999). However, BDNF has been shown to have some antinociceptive effects when given intrathecally and has been postulated to facilitate descending inhibition. Implantation of immortalized cells, derived from embryonic rat neurons and transfected to express BDNF, to the lumbar subarachnoid space in rats, reduced the cold and tactile allodynia and thermal hyperalgesia following chronic constriction injury of one sciatic nerve (Cejas and others 2000). Other less obvious substances such as the peptide galanin also appear to have a trophic role after nerve damage (Kerr and others 2000; Sten Shi and others 1999). Chronic intrathecal galanin administration to rats produces hyperalgesia, galanin production is elevated following peripheral nerve injuries that cause hyperalgesia, and galanin knockout mice fail to show the expected increases in nociceptive sensitivity after peripheral nerve injury (Kerr and others 2000). Recently, it was shown that cAMP response elementbinding protein (CREB) contributes to the increased neurite outgrowth of sensory neurons following the release of vasoactive intestinal polypeptide (VIP) and activity-dependent neurotrophic factor (ADNF) (White and others 2000). Such molecules can help repair the damage and restore normal function or can exacerbate the pathological changes leading to a chronic pain state.

It is now clear that trophic factors have multiple roles and ways of signaling; for example, NGF uses cell surface trk receptors to maintain cell survival, but the internalized receptor following ligand binding is necessary to trigger cell differentiation (Zhang and others 2000). What Happens in the CNS? All central secondary sensory neurons receive inputs from afferent fibers releasing glutamate and carry glutamate receptors (NMDA and non-NMDA ionotropic subtypes and metabotropic receptors, especially mGLUR5; Bordi and Ugolini 2000). In addition, those neurons that receive input from C-fibers are also activated by peptides (such as substance P and CGRP) and purines (Fig. 3). Once signals have entered the dorsal horn of the spinal cord, they are modulated by inhibitory processes operated both by local inhibitory interneurons acting both presynaptically on transmitter release and postsynaptically on relay neuron excitability. The main local inhibitory transmitter is GABA, which acts on all types of transmission, although there is a strong rationale for believing that it has a major role in controlling nociceptive sensitivity (Hammond 1997) and there are inhibitory neuropeptides, such as the endogenous opioids, which have a more restricted distribution. Recently, an endogenous cannabinoid system was identified that may have an important role in reducing sensitivity to pain (see Walker and others 1999). Molecular pharmacology, largely by expression cloning, has now revealed that there are subtypes of most of the target receptors for these messengers. Cloning and expression of voltage-gated ion channels is taking longer, as the level of complexity is higher, the proteins involved are larger, and analysis of structure is less advanced than for G-protein-coupled receptors. Excitatory processes involve mainly Ca and Na channels, with N-type and P/Q-type Ca channels being the main regulators of transmitter release from primary afferents (Hans and others 1999). Inhibitory processes are regulated by Cl channels and by K channels, and, although a full treatment is outside the scope of this article, the diversity of ion channels is now realized to be just as great as that of receptors. The functions of receptors and channels overlap and, for example, many voltage-gated Ca channels are negatively coupled to G-protein receptors. Some receptors and most ion channels are multimeric, that is, made up of multiple subunits, and there are both homomers and heteromers to consider, adding yet another level of complexity. The cloning and expression of receptors and channels, the recognition of species differences in sensitivity, and the development of high throughput functional screening techniques have led to the production of potent and specific antagonists and in many cases agonists of most of the receptors and some of the channels of interest. These are available as tools to study function and as potential therapeutic agents, so that it is becoming possible to selectively block just some components of sensory transmission. Although

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this holds the potential for design of analgesic drugs with fewer side effects, initial experience with blockers of the action of substance P at NK1 receptors, for example, in clinical pain trials showed a lack of analgesic activity (Boyce and Hill 2000). It may be necessary to acknowledge that, as with the inflammatory soup that activates peripheral nociceptors, the sensory input signaling pain in the dorsal horn of the spinal cord may be carried by a melange of substances released in concert, such that blocking the effects of any one substance may not adequately reduce the signal enough to give clinical pain relief. We are gaining a better understanding of the postreceptor systems within the neuron, and it is possible that these may provide novel sites of intervention at the level of, say, the protein kinases. The idea of a postsynaptic receptor complex is under revision, and the use of proteomics allows a level of complexity never before imagined. For example, a recent study on the downstream signaling associated with the NMDA receptor complex identified 77 proteins with identified or potential roles (Husi and others 2000). We are now learning that some systems that were assumed to be specifically associated with peripheral inflammation may also be important within the CNS. It has been established using immunocytochemical techniques, selective blocking drugs, or transgenic animals that, for example, bradykinin B1 receptors and cox-2 are both involved in regulating the nociceptive threshold of the spinal cord. Brain-derived TNF has been postulated to mediate some of the changes associated with neuropathic pain (Ignatowski and others 1999). It may be especially important in nociception to acknowledge that there are systems downstream of the neurotransmitter receptors that play a vital role. Noxious stimuli will lead to activation of the extracellular signal-regulated protein kinases (ERK) (Ji and others 1999) and the transcription factor CREB in neurons within the DHSC and also the DRG (White and others 2000). ERK is downstream of both PKC and PKA, and ERK activation seems to be necessary for the second phase of the behavioral response to intraplantar formalin and shows activation and phosphorylation after a range of intense noxious stimuli, probably due to NMDA receptor activation (Ji and others 1999). CREB phosphorylation (possibly as a result of ERK activation) may contribute to CRE-operated transcription and the establishment of chronic nociceptive changes (Fig. 1; White and others 2000; but also see Woolf and Salter 2000). Recent work on rat pups (far more immature than human infants) shows that the plastic changes associated with inflammatory hyperalgesia occur in neonates (Ruda and others 2000). Transgenic Animals and Antisense Techniques in the Identification of Systems Important in Nociception This approach of gene deletion has already been of real benefit. It has helped establish that systems previously

thought to be important in nociception on the basis of circumstantial evidence actually are involved, and in other cases it has indicated that the systems coded by the deleted gene were perhaps not as important as previously thought. This approach has also allowed the study of the putative effects of a pharmacological antagonist where adequately potent and selective receptor antagonists and channel blockers are not currently available. A few examples will be described in some detail to illustrate the scope of this area of work, but a broader view of the topic is given in Box 2. In the study of the role of bradykinin receptors in nociception, the situation has been complicated in the past by the fact that there are two receptors to be considered, the constitutive B2 receptor where bradykinin itself is the preferred agonist and the inducible B1 receptor where the des-Arg metabolites of bradykinin or its precursor kallidin are the preferred agonists. As there are no suitable small molecule antagonists available that are adequately selective for either of these receptors, the only way to study them in isolation was to make knockout mice with one or the other gene deleted. This has now been done, and the phenotype of these animals demonstrates convincingly that both the B2 and B1 receptors have a role in pain perception but that the B1 receptor may be the most important if the phenotype of the knockout animals is a reliable guide. In mice with a targeted disruption of the B2 receptor gene (Box 1; Borkowski and others 1995), tissue responses to exogenous bradykinin were absent. In nociception assays in these mice (Rupniak and others 1997), mechanical hyperalgesia was not produced by intraplantar bradykinin nor by carageenan inflammation in contrast to the situation in wild-type animals. However, nociception was not completely absent in the knockout mice as normal responses were evoked by intraplantar formalin, and thermal hyperalgesia was produced following injection of complete Freunds adjuvant. It therefore appeared that the role of the remaining B1 receptor was potentially more important than that of the B2 site. Recently, mice with a disruption of the B 1 receptor gene have become available (Pesquero and others 2000) and show both reduced peripheral inflammatory responses and a significant deficit in thermal and chemical nociception. Unexpectedly, these mice have also shown us that the major site at which B1 receptor function is likely to be important is within the dorsal horn of the spinal cord. A channel of particular interest to nociception is the TTX-resistant Na channel (SNS or PN-3) preferentially expressed in C-nociceptors (Akopian and others 1999; Waxman and others 1999). It has been difficult to decide just how important this channel may be in nociception in vivo because (as mentioned earlier) there are at least six different Na channel proteins expressed in dorsal root ganglion neurons and at present there are no blocking drugs that adequately discriminate between them. A SNS null mutant mouse has now been engineered and has been found to have deficits in mechanical and thermal nociception with delayed development

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of inflammatory hyperalgesia (Akopian and others 1999). Interestingly, this mouse shows an up-regulation of expression of TTX-sensitive Na currents that may make the changes in the nociceptive phenotype less marked than would be the effects of a drug selectively blocking SNS in a wild-type animal. One additional benefit of the SNS null mutant has been that it has allowed the discovery of the physiological signature of a second TTX-resistant channel, named NaN, by studying the electrophysiological properties of DRG neurons taken from these mice (Cummins and others 1999). Where knockout animals are not yet available or not feasible (at present, the technology only appears to be facile in the mouse) or where there is an adaptive change produced by removal of a gene product early in development (as in the SNS mutant described above), injection of antisense oligonucleotides has been used, with mixed success, to temporarily prevent translation of a functional protein. This approach has allowed confirmation of the role of the TTX-resistant Na current PN-3/SNS in nociception without the complication of simultaneous up-regulation of PN-1 and has been interpreted as suggesting that NaN/SNS2 may be less important in nociception, and it has extended the experimental observations to the rat (Porreca and others 1999). A similar approach has allowed the discovery that the peripheral action of opioid agonists on visceral nociception is not operated through the known opioid receptor (Joshi and others 2000). The Importance of Empiricism Most of the drugs that are available for treating intractable pain are not classical analgesics but are anticonvulsants (gabapentin, valproate, lamotrigine) or antidepressants (amitriptyline). Some anticonvulsants and antidepressants are not useful for treating pain, so there is clearly some additional property in those agents that is useful and which transcends their conventional pharmacology. As the existing agents are nonideal because of side effects and suboptimal efficacy, it is important to establish how these drugs relieve pain so as to design analogues that act only at those sites that are pivotal for pain relief. The availability of cloned receptors channels and enzymes allows the screening of existing agents to establish key activities, but even then a multiplicity of targets remains such that it is necessary to combine such studies with transgenic animal work (e.g., is amitriptyline still analgesic in a SNS -/- mouse?). The way in which anaesthetic agents act has received intense study recently, and actions at specific sites on the GABA-A receptor complex (Mascia and others 2000) and on two pore potassium channels (Patel and others 1999) have been demonstrated. Conclusions As illustrated in this review, which has of necessity been very selective and has omitted large areas of important research, the field is moving ever more quickly.
Volume 7, Number 4, 2001

This is the most exciting time to be working in the basic science of pain perception. The number of possible targets for novel analgesic drugs is now legion, but a real challenge lies in making the correct choice of target from the plethora available. There is a risk that by making drugs extremely selective for a single molecular target we will lose clinical efficacy at the same time we lose unwanted side effects. However, our ability to decide which of the many gene products is the most relevant to pain perception should increase our chances of making the correct choice. Perhaps the most immediate clinical benefit is likely to come from a molecular understanding of how some of the empirical treatments for intractable pain actually work and the consequential design of targeted drugs to exploit this knowledge. References
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