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Most of what is written and believed about pain and nociceptors originates from studies of the “somatic” (non-visceral) sensory system. As a result, the unique features of visceral pain are often overlooked. In the clinic, the management of visceral pain is typically poor, and drugs that are used with some efficacy to treat somatic pain often present unwanted effects on the viscera. For these reasons, a better understanding of visceral sensory neurons—particularly visceral nociceptors—is required. This review provides evidence of functional, morphological, and biochemical differences between visceral and non-visceral afferents, with a focus on potential nociceptive roles, and also considers some of the potential mechanisms of visceral mechanosensation.
The majority of what is known about pain and nociceptors originates from studies of “somatic” structures (i.e., non-visceral components of the body, principally skin). Nevertheless, the most common pain produced by disease (and the most difficult to manage) is that originating from the internal organs (i.e., visceral pain), and the characteristics of visceral innervation differ significantly from other tissues. Visceral pain may result from direct inflammation of a visceral organ (e.g., inflammatory bowel disease, pancreatitis, appendicitis), occlusion of bile or urine flow (e.g., kidney stones), or from functional visceral disorders [e.g., irritable bowel syndrome (IBS)]. Add to this list angina, painful bladder syndrome (interstitial cystitis), gastroesophageal reflux disease, endometriosis, and dyspepsia, and the widespread impact of visceral disease becomes clear. Most basic and clinical pain research has focused on somatic (principally cutaneous) tissue, which has significantly influenced strategies for pain management. As a result, the unique features of visceral pain and innervation have remained underappreciated, and thus visceral pain management is typically poor. Moreover, visceral nociceptors are intrinsically different from cutaneous and most other non-visceral nociceptors. We provide here a review of the visceral sensory system and highlight some of the features that distinguish it from non-visceral systems. The visceral system encompasses a large number of organs, from the eyes (technically, the brain is also a visceral organ) down to the genitourinary organs, and so this review will focus on our laboratory’s current primary area of experimental expertise: the lower gastrointestinal tract (principally colon) and bladder.
The principal extrinsic afferent nerves innervating visceral organs are anatomically associated with sympathetic and parasympathetic nerves and are accordingly named (e.g., pelvic nerve afferent), although they are not part of these efferent, autonomic, pathways. Most extrinsic visceral afferent neurons have cell bodies in dorsal root ganglia (DRG) and terminate in the spinal cord (spinal afferents); visceral afferent fibers in the vagus nerve, with cell bodies in the nodose and adjacent jugular ganglia, terminate in the brainstem nucleus tractus solitarius. There are two features that are unique to the visceral sensory innervation: 1) most organs also have an intrinsic innervation (e.g., the enteric nervous system of the gastrointestinal tract) and 2) each organ is innervated by two different extrinsic nerves (e.g., the distal colon and urinary bladder are innervated by the pelvic and splanchnic nerves). Although there are likely important functional interactions between the intrinsic and extrinsic visceral innervations, their anatomical relationship and means of intercommunication are largely unknown. Figure 1 summarizes the visceral sensory innervation, using the gut as an example.
A key difference between visceral and non-visceral sensory neurons is the degree to which their peripheral terminals are specialized. For example, cutaneous afferents can have one of many different sensory endings to transduce stimuli into electrical energy (e.g., Merkel cells, Ruffini endings, Pacinian corpuscles), whereas only two types of specialized ending have been reported in visceral afferents: intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs). Both types have limited distributions (e.g., near sphincters), are specific to muscular vagal or pelvic innervation, and are less intricate than their non-visceral counterparts [for review, see (1, 2)]. IGLEs and IMAs appear to be low-threshold mechanoreceptors and are therefore less likely to be involved in detecting noxious events. Most spinal visceral afferents are believed to have primitive, unencapsulated endings (like non-visceral nociceptors), with no specialized structure and one or a few punctate receptive fields.
Visceral and non-visceral afferents encode different types of information: the conscious experiences generated by the visceral sensory system are not initiated by non-visceral afferents. For example, the sensation of nausea does not arise from the skin, and vice versa, one cannot detect cutting of the gut [for review, see (3, 4)]. Conscious sensations arising from the viscera, in addition to pain, include organ filling, bloating and distension, dyspnea, and nausea, whereas non-visceral afferent activity gives rise to sensations such as touch, pinch, heat, cutting, crush, and vibration. Both sensory systems can detect chemical stimuli.
Nociceptors were originally defined as receptors that respond to noxious stimuli, particularly those that damage or threaten to damage skin. Thus, they were defined in a functional context. As our understanding of nociceptors has increased, however, attempts to redefine the nociceptor have generated much debate and little agreement. Furthermore, a “noxious stimulus” is semantically distinct from a “painful stimulus,” a concept that has evolved from the descriptions of “nocicipient” cutaneous receptors by Sherrington at the beginning of the twentieth century (5).
“Pain” is a psychological state, defined by the International Association for the Study of Pain (IASP) as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (6). This concept, and the distinction between nociception and pain, has been appreciated for some time, as the following quotation from 1900 shows (7):
The stimuli which evoke pain may be characterised as ‘excessive.’ It might almost be asserted that ‘excess’ is that quality of a stimulus in virtue of which it becomes ‘adequate’ for the sense of pain. ‘Excessive’ in this application connotes ‘harmful,’ or ‘to be avoided,’ e.g. by muscular action for resistance or escape. The ‘excess’ of the stimulus may lie in its intensity, or in its extensity (spatial or temporal).
Potential confusion arises from the IASP’s definition (6) of a noxious stimulus as “one which is damaging to normal tissues,” thereby excluding the potential for damage that is considered under the definition of “pain”. Thus, a nociceptor is a peripheral sensory receptor (considered colloquially to be the entire neuron, including its peripheral and central terminals and soma) that signals actual tissue damage.
One problem with these definitions is demonstrated by the existence in visceral organs of low-threshold mechanosensory afferents (sometimes also called “wide dynamic range”) that proportionally encode organ distension from low, physiological (non-noxious), distending pressures through to pressures that are noxious (Figure 2A and B). Similarly, joint afferents and some cutaneous afferents also have low mechanical response thresholds and encode well into the noxious range; however, neither slowly nor rapidly adapting mechanosensitive afferents in skin encode into the noxious range, or sensitize (see below). The current definition thus appears to omit low-threshold visceral afferents from the classification of nociceptor, as no matter how prolonged a non-noxious stimulus may be, low-threshold mechanoreceptors will not signal a noxious event until the stimulus intensity increases. Silent (or sleeping) nociceptors offer another complication. These neurons are unusual in that they are insensitive to all but the highest intensity of mechanical stimulation. However, inflammatory chemicals “awaken” these nociceptors and induce spontaneous activity and mechanosensitivity in the noxious range.
Accordingly, we propose that a visceral nociceptor (or indeed, a nociceptor in any tissue) is a sensory receptor that, when activated, can produce a reflex or response that is protective or adaptive (e.g., withdrawal, guarding, vocalization); can encode stimulus intensity in the noxious range; and can sensitize (i.e., give increased responses to noxious intensities of stimulation after insult or exposure to chemical mediators such as those produced during inflammation). Requirement for the latter two capabilities reveals that most of the visceral sensory innervation is nociceptive in character, particularly during organ insult. Most (70-80%) mechanosensitive visceral afferents have low thresholds for activation in the physiological range; the remainder have high thresholds and are commonly considered to represent the population of visceral nociceptors. Nevertheless, most low-threshold mechanosensitive visceral afferents encode into the noxious range and generally give greater responses than their high-threshold counterparts (Figure 2A). They also sensitize after organ insult, giving increased responses to both innocuous and noxious intensities of stimulation. These findings argue for potential roles of both low- and high-threshold mechanoreceptive visceral afferents in visceral pain conditions.
With an ever-increasing number of in vitro methods available to the pain researcher, the identification of nociceptors often relies on cellular characterization, such as size or biochemical markers, rather than functional definitions. For example, all small-diameter, capsaicin-sensitive DRG neurons (that is, those that either express the capsaicin receptor TRPV1 or respond to application of capsaicin) are sometimes considered as nociceptors. The reliability of biochemical targets, such as TRPV1, to act as nociceptor markers is discussed below.
The ability to identify the source (spatial location) of cutaneous pain is excellent, and the ability to identify that of joint and muscle pain is generally good; in contrast, visceral pain is diffuse in character and poorly localized. Two factors contribute to this difference. First, relative to non-visceral structures, the viscera are sparsely innervated. It is estimated that fewer than seven percent of spinal afferents in the DRG project to the viscera [see (1,8,9,10)], and only a fraction of these convey input to the central nervous system that will be perceived. This sparse innervation is compensated for in the spinal cord, where visceral terminations arborize widely over several spinal segments and even to the contralateral spinal cord (11). Second, spinal neurons that receive visceral input also receive convergent input from skin or deeper structures (including other viscera), producing referred pain. For example, cardiac pain (angina) is typically referred to the left arm and shoulder (but skin, joint, or muscle pain is not referred from shoulder to heart). In addition, whereas pain can be evoked from virtually all non-visceral structures, parenchymous viscera (e.g., liver and pancreas) do not give rise to pain unless the organ is inflamed or the organ capsule is distorted, for example by a tumor. Finally, visceral pain is commonly associated with greater emotional valence and exaggerated autonomic reflexes, although the former is a central phenomenon not to be confused with nociception.
We discuss here some of the differences between visceral and non-visceral nociceptors—with the caveat that the majority of studies are done in the absence of a physiologically (functionally) defined nociceptor population.
The most obvious parameter for distinguishing cell types is size. It is generally accepted that DRG neurons are bimodally distributed in terms of soma size, resulting in the designation of “(large) light” or “small dark” neurons. The former have myelinated A-fibers and somata with dense neurofilament (typically detected using antibodies raised against neurofilament protein, such as RT97 (12)); the latter have unmyelinated C-fibers and are significantly less dense. Generally, the somata of visceral afferents in DRG are larger than those considered to be non-visceral nociceptors (13, 14).
Generally, smaller-diameter neurons have myelinated Aδ- or unmyelinated C-fiber processes, whereas myelinated Aα/β-fibers can be found on cells of most sizes. Up to eighty percent of visceral DRG somata can have C-fibers, whereas fewer than forty percent generally have Aδ-fibers (15,16). An exception, however, can be found in the perianal mucosa, where the distribution is reversed: 23% C-fibers, 77% Aδ-fibers (16). Visceral Aβ-fibers are rarely encountered. In contrast, L4 DRG neurons with projections in the sciatic nerve (a non-visceral nerve that innervates skin and muscle) show a bias towards Aα/β-fibers (≥ 69%), with few Aδ- (approximately 15%) or C- (7-17%) fibers (17, 18). A similar pattern has been shown in guinea pig neurons that innervate the left hind limb and flank; the bias shifts to C-fibers in an identified nociceptive population of these afferents (19). In addition to nociceptive Aδ- and C-fibers, non-visceral nociceptors can also have Aβ-fibers [for review, see (20)]. It is important to appreciate that studies such as these do not necessarily reflect the exact proportions of each type of fiber present; the data are subject to varying selection dynamics and other experimental parameters chosen by the investigator.
Visceral and non-visceral afferents also differ in their spinal cord terminations. Spinal visceral afferent fibers terminate in the superficial dorsal horn, lamina V, and around the central canal, an area also referred to as lamina X (21, 22). In contrast, the terminal fields of cutaneous nociceptive afferent neurons are much smaller than those of visceral afferents and terminate throughout the spinal dorsal horn (11, 23). Non-visceral nociceptors also terminate in the superficial dorsal horn and lamina V, and thus converge on some of the same second order spinal neurons as visceral afferents; this convergence may account for the perception of pain from referred visceral sensations. Furthermore, visceral C-fibers have significantly more extensive spinal terminations (more terminal regions in different laminae and at multiple levels of the spinal cord) than the “nest-like” terminations of non-visceral C-fibers that are found principally in laminae I and II (11).
The vast majority (typically, 70–90%) of visceral afferent cell bodies in the DRG stain positive with antibody for the calcitonin gene-related peptide (CGRP) (Table 1) (24-35). In contrast, DRG cell bodies of the non-visceral sensory system are far less likely to manifest positive immunostaining for CGRP. For example, approximately four-fifths of mouse colonic DRG somata are positive for CGRP, compared to about one-fourth of cells in the whole DRG population (24), one-third to one-half of cutaneous afferents (25, 36, 37), and one-fifth to one-third of muscle afferents (37). It has also been noted that visceral neurons that are positive for CGRP show a more intense signal than do positive non-visceral neurons (38).
Using techniques that label whole nerve bundles (Table 1 and Box 1), rather than just cell bodies in the DRG, ninety percent or more of visceral afferents are reactive to anti-CGRP immunostaining, compared to only about 20–50 percent of skin and 30–70 percent of muscle afferents (38-41). Approximately one-third of functionally identified non-visceral nociceptors in the guinea pig are CGRP-positive (42), although proportions vary among tissues (e.g., hairy versus glabrous skin). If only C- and Aδ-fibers are considered, this value is just under half of nociceptive units. We are unaware of any visceral correlate to these experiments for comparison.
One method to identify visceral or non-visceral spinal afferents is to use retrograde labeling of whole nerve bundles and study their cell bodies in the DRG. Some of these nerves are described here, including their afferent innervation and the DRG levels at which labeled neurons have been found in the rat. Data are collated from a number of studies [72-76].
|Population||Nerve||Aferent innervation||Principal DRG levels|
|Visceral||splanchnic (SPL)||visceral organs||T8–T12|
|pelvic (PN)||visceral organs||L6 and S1|
|Non-visceral||genitofemoral (GF)||muscle||L1 and L2|
|sciatic (SCI)||muscle and skin||L4–L5|
Subpopulations of afferent neurons that bear the CGRP marker are known as “peptidergic. ” Classically, peptidergic neurons are reported to express the nerve growth factor receptor trkA; in contrast, non-peptidergic neurons express Ret, the receptor for glial cell line–derived neurotrophic factor. It has been suggested that nonpeptidergic neurons also bind the Griffonia simplicifolia-dervied isolectin IB4 and express the purinergic P2X3 receptor. Immunoreactivity patterns for trkA correlate well with those for CGRP; for example, 75% of bladder and 43% of skin DRG somata express trkA (25), and in situ hybridization studies show trkA mRNA in ninety percent of visceral (pelvic nerve), 20% of muscle (gastrocnemius nerve), and 48% of cutaneous (saphenous nerve) afferents (43). Thus, a greater proportion of visceral DRG somata, relative to non-visceral DRG somata, contain CGRP and are, therefore, potential nociceptors. Any line of argument that maintains that visceral afferents are predominantly peptidergic, however, should be tempered by the caveat that a strict distinction between “peptidergic” and “nonpeptidergic” may not be completely suitable for this group of neurons.
Among DRG somata, cutaneous afferents are over ten times more likely to bind IB4 than are visceral afferents [40 percent vs 3 percent; 72 hours after retrogradely labeling whole nerve trunks (44)]. Few muscle DRG somata (approximately 3%) bind this lectin (44). There are currently few studies detailing the binding of IB4 in afferents retrogradely labeled from specific organs, but in the case of colon and bladder, the proportion of afferents that bind IB4 has been estimated at six to thirty percent (24, 25, 34, 35), whereas retrograde labeling from skin indicates that twenty to forty percent of cutaneous afferents bind IB4 (25,36). These data suggest that visceral afferents are biased towards the peptidergic classification; however, it is worth noting that over ninety percent of colonic DRG somata that bind IB4 (i.e., presumed to be “non-peptidergic”) stain positive for CGRP (whereas only about twenty percent of all IB4-binding DRG somata test positive for CGRP; David R. Robinson, PhD thesis, University of Cambridge, 2004). Also, although the functional significance is unknown, these IB4- and CGRP-positive neurons display lower-intensity IB4 staining than afferents that bind IB4 but are not CGRP-positive (24). In any event, these observations suggest that the biochemical phenotype visceral afferents can be distinguished from that of non-visceral sensory neurons. This difference is reinforced by the finding that colonic afferents are far less likely to test positive for both the P2X3 receptor and IB4 binding, relative to the rate that both markers appear together in the general DRG population (24).
The capsaicin receptor, TRPV1, is often regarded as a marker for nociceptors. The majority of visceral DRG somata test positive for TRPV1 (Table 1) (24, 39, 45, 46). In contrast, cutaneous (36, 39,45) and muscle afferents (39) are far less likely to contain the receptor. The few studies that have functionally identified nociceptors report that only a very small number of cutaneous nociceptors contain TRPV1. For example, one study showed only thirteen percent of tested nociceptors (both C- and A-fiber afferents) innervating mouse skin were TRPV1-positive (47). In the colon, animals lacking the TRPV1 receptor are significantly less sensitive to colorectal distension (Figure 2C) than wild-type littermates (48). Although not specifically identified as nociceptors, approximately half of colonic serosal mechanoreceptors respond to capsaicin (49). In innervation of cat viscera, all C-fibers and 38% of A-fibers are capsaicin-sensitive (50).
An illustration of both the complexity of the definition of the nociceptor and the role of TRPV1 can be found in a less commonly studied animal species. The African naked mole-rat, Heterocephalus glaber, expresses functional TRPV1 receptors, but does not exhibit pain behavior in response to capsaicin applied to the hind paw, unless substance P, which is absent in cutaneous C-fibers (51), is first administered as an intrathecal injection (52). This observation raises an interesting question: Are the afferent neurons that are obviously capable of encoding a noxious stimulus to be considered nociceptors, even if such “nociception” depends on a pharmacological intervention?
Although visceral afferents can be distinguished from their non-visceral counterparts, they do not appear to form a homogeneous population (Table 1). Indeed, the colon and bladder are innervated by afferents associated with two different nerves: afferents that follow the splanchnic nerves and have cell bodies in thoracolumbar DRG, and afferents that follow the pelvic nerve and have cell bodies in lumbrosacral DRG. Studying the mechanosensitivity of single afferent fibers that innervate the colon reveals five different classes of mechanosensory primary afferent (53) (Figure 3). Two of these are expressed in specific afferent populations, with the remaining three (i.e., serosal, muscular, and mucosal) found in both the splanchnic and pelvic pathways. Mesenteric afferents, an afferent class that is not observed in the pelvic nerve, constitute half the splanchnic innervation of the colon. Similarly, muscular/mucosal afferents have been reported in the pelvic, but not splanchnic, innervation and have been likened to a population of vagal afferents. This prompts the question of whether the pelvic nerve is fulfilling a “vagal-like” role in those more distal portions of the gastrointestinal tract that the vagus nerve does not innervate. (Pelvic afferent terminals in the colon are predominantly found distal to those from the lumbar splanchnic nerve). Generally, pelvic afferents have lower mechanical thresholds for activation but respond more intensely to a given stimulus than do splanchnic afferents. Chemical differences are also apparent between these two visceral afferent subpopulations. For example, significantly more splanchnic than pelvic afferents (66% versus 11%) respond to the direct application of bradykinin (BK), a chemical mediator released following tissue injury, to the receptive ending (54). BK-responsive pelvic afferents (two of nineteen fibers tested) were mechanosensitive, whereas the BK-responsive splanchnic population included mechanically insensitive as well as mechanically sensitive afferents. If a purely serosal afferent population is studied (chosen because they are equally represented in both the pelvic and splanchnic nerves), more splanchnic than pelvic afferents respond to activation of either P2X or TRPV1 receptors (49), findings that are confirmed immunohistochemically (35, 49).
Similar findings have been reported in the rat bladder, an organ that also receives dual innervation through the pelvic and splanchnic nerves. Whole-cell patch clamp electrophysiology of cultured retrograde labeled DRG neurons has revealed that almost all pelvic afferent cell bodies respond to the P2X agonist α,β-methyleneATP, whereas only half of the splanchnic afferents responded (55). Although practically all the neurons studied responded to capsaicin, those from pelvic DRG evoked a significantly greater current.
The peptide content of splanchnic and pelvic afferent cell bodies in the DRG, based on immunostaining for CGRP, has been reported to be similar. On the other hand, there appears to be a difference in the non-peptidergic population, as more pelvic than splanchnic afferent cell bodies bind IB4, although variations in the use of retrograde tracers and fluorophphres have produced some inconsistencies in results (35, 56). Of the two IB4-binding populations, splanchnic afferent cell bodies tend to show lower intensity in staining than does the pelvic population (DR Robinson and GA Hicks, unpublished). A higher proportion of IB4-binding afferents has also been reported in splanchnic, as compared to pelvic, afferents that innervate the rat bladder (55).
As anyone who has experienced “gas” or bloating can attest, the distension of the gut can be an unpleasant and sometimes intensely painful experience. As genetically modified mice have become more widely available, there has been increased interest in the study of molecules that mediate visceral mechanosensation and hypersensitivity (Table 2). These studies have implicated a number of different molecules, including two members of the TRP family of receptors, TRPV1 and TRPV4, and the acid-sensing ion channels (ASICs).
As indicated above, five different types of afferent fiber have been reported in the colon, each of which is responsive to different forms of mechanical stimulation and has its own putative functional role. An overview of these, along with the differences seen between the splanchnic and pelvic innervation of the colon, can be found in Figure 3.
TRPV1 knockout mice show a significant reduction in their behavioral (visceromotor) response to colorectal distension as well as in afferent fiber responses to stretch (48); similarly, urinary bladder and jejunal afferent subpopulations exhibit reduced mechanosensitivity (57, 58). It also appears that TRPV1 plays a role in hypersensitivity to mechanical stimulation in models of colon (48) and bladder hypersensitivity (interstitial cystitis) (59). This evidence of a role for TRPV1 in visceral hypersensitivity—and thus, presumably, in visceral pain—extends to non-visceral tissues, because mice that lack the TRPV1 receptor do not exhibit the enhanced referred mechanical hypersensitivity to the hind paw following cystitis that is seen in wild type controls (59).
A relative of TRPV1, the TRPV4 receptor, has also been implicated in visceral mechanosensation and may be most important in the colon; TRPV4 receptor mRNA content is significantly greater in colonic DRG cell bodies (with more in splanchnic than pelvic nerve DRG) compared to the cell bodies of gastric or non-visceral afferents (56). Furthermore, mechanical responses of colon afferents are reduced, and response thresholds are greater, in TRPV4 receptor knockout mice, consistent with reduced behavioral responses to colorectal distension (56). Conversely, the intracolonic administration of a TRPV4 receptor–selective agonist results in a dose-dependent increase in the responses of mice to colorectal distension (60).
The proton-sensing ion channels of the ASIC family have been investigated as potential visceral mechanotransducers using knockout mice that lack ASIC1a, ASIC2, or ASIC3 channels. The loss of ASIC1a appears to result in an increase in mechanosensitivity throughout the gastrointestinal tract, including the colon, whereas deficiency in ASIC2 results in different mechanical responses, depending on the target (in the colon it also results in increased mechanosensitivity) (61, 62). CGRP release from the colon is unchanged by knockout of ASIC2 (63). In contrast, knocking out ASIC3 leads to reduced colonic mechanosensitivity, both with respect to stretch in single-fiber experiments and behavioral assessment of the visceromotor response to colorectal distension (48, 62).
We have reviewed evidence here that visceral nociceptors—or, more accurately, visceral afferents with the potential to transmit nociceptive information—differ from non-visceral (somatic) afferents in a number of ways, including their morphology and the channels and receptors they contain. They also differ in the consequences of their activation. In humans, visceral pain has a number of characteristics that distinguish it from pain originating from non-visceral structures, and these differences are most likely responsible for the symptoms experienced by patients with a visceral disease such as IBS. Somatic pain relief strategies typically work poorly for the management of visceral pain, and a better understanding of the visceral nociceptor (along with central mechanisms not discussed here) is vital to the development of new therapies for visceral pain management. The way forward would be easier if we were able to identify visceral nociceptors by characteristics other than response to noxious stimuli. Although CGRP- and TRPV1-containing DRG somata are more common in visceral sensory neurons, these and other potential surrogates do not reliably distinguish visceral from non-visceral nociceptors. Until such a marker (or constellation of markers) is found, identification of nociceptors requires functional assessment.
David R. Robinson, PhD, received his doctoral degree, with a PhD studentship from GlaxoSmithKline, from the University of Cambridge, where he studied the characterization of the distal colon with particular emphasis on colonic hypersensitivity. He is now a Postdoctoral Associate at the Pittsburgh Center for Pain Research, University of Pittsburgh, where his research interests remain focused around colonic hypersensitivity.
Gerald F. Gebhart, PhD, is Professor and Director of the Center for Pain Research at the University of Pittsburgh School of Medicine. He has published more than 350 articles in journals and books. His interests in the pain field include descending modulation of pain (mechanisms of descending facilitation, secondary hyperalgesia, and chronic pain conditions) and visceral pain.