Structure of nociceptor ‘endings’
Abstract and Keywords
Determining the association of specific sense organs with a specific sensory ‘quality’ or ‘modality’ has been a long and controversial quest, largely successful in relating the variety of elaborate sensitive mechanoreceptors to the complex events constituting tactile and kinaesthetic sensations. The evidence inferred for assigning the large corpuscular elements supplied by large, myelinated, fast-conducting afferent axons has been secured by combined electrophysiological and morphological observations. Thus about one half of afferent axons, derived from approximately one million large sensory ganglion cells in humans, can be accounted for in terms of both sensory function and an associated complex end-organ. The other half, constituting the thin, unmyelinated, slowly conducting axons originating principally in small sensory ganglion cells, remains elusive. No other sensation has eluded the designation of a specific structure as successfully as the nociceptor.
Introduction: The identification of nociceptors
Determining the association of specific sense organs with a specific sensory ‘quality’ or ‘modality’ has been a long and controversial quest, largely successful in relating the variety of elaborate sensitive mechanoreceptors to the complex events constituting tactile and kinaesthetic sensations. The evidence inferred for assigning the large corpuscular elements supplied by large, myelinated, fast-conducting afferent axons has been secured by combined electrophysiological and morphological observations and thus about one-half of afferent axons, derived from approximately one million large sensory ganglion cells in humans, can be accounted for in terms of both sensory function and an associated complex end-organ. The other half, constituting the thin, unmyelinated, slowly conduct-ing axons originating principally in small sensory ganglion cells, remains elusive, although it is generally conceded that the vast majority probably constitute the nociceptive sense organs underlying the variety of sensory qualities usually described as ‘pain’. No other sensation has eluded the designation of a specific structure as successfully as the nociceptor, and indeed it should be noted that some modern, seminal reviews, including the widely studied ‘gate control theory’ of pain (Melzack and Wall 1965), denied the very existence of nociceptors until the electrophysiological studies of Iggo and of Perl and their collaborators confirmed the widely held nineteenth century belief that cutaneous sensa-tion was punctate and that each quality was associated with a discrete morphological arrangement serving as a sense organ, a view championed by Max von Frey (see Peri, Chapter 1, this volume). By the end of the nineteenth century, it was generally believed that ‘free nerve endings’ associated with thin, largely unmyelinated axons provided the morphological substrate for pain, and Sherrington (1900) noted that those sructures that he believed to be innervated solely by unmyelinated axons were zones from which only a sensation of pain could be elicited; these were the cornea, the dental pulp, and the internal surface of the tympanic membrane. Although the sensory quality attributable to stimulating these regions may be argued and the fibre spectrum innervating them extends into the thinly myelinated range in some species, the intuitive correlation between free endings, thin axons, and pain seemed basically sound.
An exclusionary principle proved more elusive, and the association of thin, largely unmyelinated axons with thermal sensations and the poorly understood qualities of tickle and itch at the other end of the hedonic spectrum presents a problem that remains unresolved. Aside from the uncertainty concerning the several sensory qualities asso-ciated with thin, unmyelinated, slowly conducting axons possessing a relatively simple terminal structure, it has been argued that many afferent axons are rarely, if ever, likely to be associated with any ‘sensation’ and that these axons primarily subserve an efferent or effector role, playing a role in the body's defence system as ‘noceffectors’ (Kruger 1987; Kruger et al. 1989). The electrophysiological observation of thin axons conducting in the C-fibre range that are apparently unexcitable by natural stimuli (Meyer et al. 1991) (p. 38 ) also renders fallible any exclusionary principle for attributing a specific morphology to nociception. Indeed, there are remarkably few examples of electrophysiologically identified receptive field ‘spots’ that have been studied in detail and, even in those studied in serial sections by electron microscopy, there can be no guarantee that there is only a single axon terminal in the marked zone. The only direct evidence that a single C fibre may merit a ‘pain’ designation derives from microstimulation of a presumed single fibre where the character and locus of the receptive field can be matched with the human subect's sensory report (Ochoa and Torebjörk 1989). The anatomical isolation of solitary axons in man (Fig. 2.1) provides one of the notable criteria in establishing a specific ‘labelled line’ serving pain. In the following account, the description of the characteristics of axon terminals probably associated with nociception and perhaps pain, is tempered by the caveats mentioned above and the certainty that there are other alternatives (including at least thermoreceptors and sensitive C mechanoreceptors) for the sensory modalities associated with ‘free nerve endings’. Fortunately, popular beliefs cannot substitute for objective scientific evidence and the prevailing curent opinion that ‘free’ nerve endings constitute nociceptors should be considered an inference quite susceptible to other interpretations.
(p. 39 ) ‘Silent nociceptors’
The number and proportion of thin afferent fibres that might be difficult or unlikely to be detected by means of conventional electrophysiological exploration could logically con-stitute a very substantial population. Although some experienced investigators indicate that they are rather rare in cutaneous nerves (Kress et al. 1992), if a sufficient range of mechanical and thermal stimuli is applied, the proportion of Inexcitable units ranges from 10 per cent (Bessou and Perl 1969), up to 28 per cent or about half of the C fibres (Handwerker et al. 1991b) in rat skin nerves (Lynn and Carpenter 1982; Pini et al. 1990) and 48 per cent of AS fibres and 30 per cent of C fibres in monkey skin (Meyer et al. 1991b). The proportions can be considerably higher in deep joint and muscle nerves (Kniffki et al. 1976; Schaible and Schmidt 1983, 1985, 1988a; Grigg et al. 1986), but many of them can be sensitized by inflammation and can be excited by hypertonic KC1, leading Schaible and Schmidt (1988b) to call them ‘sleeping’ rather than ‘silent’ units. It can be argued that it is dangerous to infer inexcitability from negative observations (Lynn 1991), but the large proportion of apparently unresponsive fibres in visceral nerves (Janig and Koltzenburg 1990) that can be rendered active by inflammation has led to the proposal that there is a large class of visceral receptor fibres that are mainly concerned with signalling tissue injury; they become active only after inflammation of a peripheral organ (McMahon and Koltzenburg 1990a, 1990b),for example, as demonstrated in urinary bladder thin fibres (Habler 1990, 1993).
Chemical stimuli, especially those that might be released consequent to inflammation (such as bradykinin, histamine, serotonin, prostaglandins, and leukotrienes), can sensitize high-threshold mechanoreceptors (for example, Perl et al. 1976; Martin et al. 1987, 1988; Cohen and Perl 1990; Dray et al. 1992), but it is likely that there is a novel class of thin afferent fibres that are chemoreceptive mediating chemogenic pain (Lang et al. 1990; LaMotte et al. 1991, 1992) and there are suggestive data for C-fibre chemoreceptors signalling itch (for example, Torebj8rk and Ochoa 1981; Tuckett and Wei 1987; Wei and Tuckett 1991) as well as evidence of a ‘sensitive’ C mechanoreceptor class (Kumazawa and Perl 1977; Shea and Perl 1985a and b) that discharge when excited by gentle, moving tactile stimuli that might conceivably elicit a human sensory report of ‘tickle’. It also should be noted that a substantial portion of the unmyelinated population includes sympathetic postganglionic efferents (Baron and Janig 1988), but the ‘insensitive’ (silent or sleeping) class of C-fibres is amply demon-strable after surgical sympathectomy (Meyer et al. 1991a; Davis et al. 1993).
Sensitive C mechanoreceptors
A substantial number of unmyelinated afferents can be excited by innocuous mechanical stimuli (Douglas and Ritchie 1957; Iggo 1960; Bessou et al. 1971), and, as noted, it has been suggested that their discharge may be related to itch (Handwerker et al. 1991a; McMahon and Koltzenburg 1992). In human neuronography, C units appear to be almost exclusively activated by noxious or by thermal stimuli (Torebjörk and Hallin 1974) and perhaps gentle tactile stimuli in trigeminal neuralgia (Nordin 1990), but this may be a consequence of conspicuous regional differences, wherein sensitive C mechan-oreceptors are evident in forelimb hairy skin but absent in distal glabrous skin in monkeys (Kumazawa and Perl 1977), a finding recently corroborated in humans (Vallbo et al. 1993).
(p. 40 ) Thermoreceptive endings
Extensive electrophysiological evidence for both ‘cold’ and ‘warm’ fibres conducting at velocities consistent with unmyelinated axons (reviewed by Spray 1986) supports the view championed by Weddell et al. (1955) that both types of thermoreceptors are subserved by ‘free’ nerve endings. The early opinions purporting to associate Krause end bulbs with cold receptors and Ruffini endings with warm receptors is still represented in some textbooks, although the mechanoreceptive properties of these elaborate ‘corpus-cular’ endings are firmly established. The sequential dissociation of cold and warm sensations with local anaesthetic block (Fruhstorfer et al. 1974) is consistent with electrophysiological evidence of a smaller proportion of ‘cold’ than ‘warm’ C fibres. There is only limited evidence of a distinctive terminal morphology in a cold receptor (Hensel 1974) and there is virtually nothing known about warm receptors, but findings obtained in the free nerve endings in the pit organs of crotaline snakes (Bleichmar and De Robertis 1962; Terashima et al. 1970), known to be infrared thermoreceptors (Bullock and Fox 1957), may provide some insights into the characteristics of the vesicles found in afferent axon terminals (see below).
Finally, it has been argued that there may be a class of thin afferent axons that lack a sensory role, largely subserving efferent influences (Kenins 1981), possibly of a ‘trophic’ nature (Kruger 1988; McMahon and Koltzenburg 1990a,1990b). There is an extensive literature dating from the beginning of the century (Bayliss 1901) for an ‘axon reflex’ in which vasodilatation and plasma extravasation can be elicited in sympathectomized, cutaneous nerves, specifically in the slowly conducting fibres (Hinsey and Gasser 1930, Kenins 1981), and largely comprising a neuropeptide-dependent ‘effector’ action of ‘sensory’ nerves (Gamse et al. 1980; Lembeck and Gamse 1982; Maggi and Meli 1988).
The classification of small sensory ganglion cells
The main thrust of this review deals with the fine structural characterization of the terminals derived from thin axons, but it is becoming increasingly evident that the smaller-diameter dorsal root and trigeminal ganglion cells express extraordinary biochemical diversity compared with their larger counterparts that give rise to the thick, myelinated axons associated with a variety of specific, sensitive mechanorecep-tors. Chapter 3 in this volume, by Lawson, expands upon this subject in detail, but a few broad features may prove relevant for solving the problem of modality assign-ment—a crucial issue for inferring nociceptive function in unmyelinated afferent axons.
A convenient and widely used means of identifying thin sensory axons, is based on the presumption that sensory ganglion neurones manufacture molecules unique to that population. The discovery of substance Pand other neuropeptides in a restricted population of sensory neurones and their axons proved of special interest and value, because peptides are found predominantly in numerous small cells and their thin axons, which, by inference, probably include a substantial proportion of nociceptors. The tissues innervated by peptidergic axons are largely sites from which pain can be elicited and, since a vast number of unmyelinated and thinly myelinated axons possess (p. 41 ) nociceptive properties that have enabled them to be identified electrophysiologically, it has been inferred that ‘sensory’ neuropeptides are selectively limited to nociceptive neurones. Selectivity is a key issue, but it also must be established that a ‘sensory’ neuropeptide is not expressed by other neurones. Fortunately, the most prevalent peptides found in small sensory ganglion cells, calcitonin gene-related peptide (CGRP) and galanin (Ju et al. 1987; Klein et al. 1990) are apparently absent from sympathetic postganglionic neurones. Although many sympathetic neurones are sur-ounded by peptidergic axon terminals, especially those containing tachykinins and CGRP, these are presynaptic (Silverman and Kruger 1989), and evidence for perikaryal labelling in sympathetics is weak. On the other hand, there are numerous parasympa-thetic postganglionic neurones in the several ganglia examined in detail (Silverman and Kruger 1989) that have been shown to express substantial quantities of the prevalent ‘sensory’ neuropeptides. Thus, some proportion of peptide-labelled unmyelinated axons is probably autonomic, although numerically they constitute only a minor population.
A relatively unexplored feature of the ganglion cell bodies giving rise to the thin axons purported to be nociceptors is the variety of axon morphology. Figure 2.2 illustrates some peptidergic (CGRP-immunoreactive) cells with a variety of axon diameters and, inter-estingly, some axons display a variety of beaded appearances near the soma. Peripheral (distal) axons with varicosities have been recognized since the nineteenth century, but it remains to be determined whether axonal form constitutes a specific class, a functional variant of all classes or an artefact (Ochs and Jersild 1990), although the latter would not readily explain the axonal varieties exhibited within the ganglion itself (Fig. 2.2).
(p. 42 ) The literature on peptidergic sensory neurones is beyond the scope of this survey (see Lawson, Chapter 3, this volume), but several features relevant to the classification of nociceptors derive from the selective distribution of neuropeptides and their receptors. A prominent feature of the ‘sensory’ peptidergic endings is their relation to autonomic targets containing smooth muscle and glands, especially blood vessels and sphincters (Kruger et al. 1989; Silverman and Kruger 1989). This contrasts with a largely non-peptidergic unmyelinated fibre population, characterized by specific glycoconjugates and traceable with specific lectins (Silverman and Kruger 1990a). The latter population constitutes the fluoride-resistant acid phosphatase (FRAP)-positive small sensory ganglion cells, once thought to prevail only in rodents (Silverman and Kruger 1988a). This subset of small, ‘type B‘, sensory ganglion cells has been shown to express α 1,3-galactosyl-extended lactoseries carbohydrate epitopes that can be labelled by specific monoclonal antibodies (Dodd and Jessell 1985; Lawson et al. 1985). The cell-surface glycoconjugates expressed by these cells render them readily labelled by galactose and N-acetylgalactosamine-binding lectins. The lectin-reactive cell surface carbohydrate expression enables the labelling of axonal processes of a substantial proportion of the unmyelinated fibre population (Streit et al. 1986) which is largely distinct from the peptidergic population of dorsal root ganglia (DRG) neurones (Nagy and Hunt 1982; Silverman and Kruger 1990) and most prominently in their peripheral terminal distribution, for example in whole mounts where lectin-reactive axons appear to be unrelated to the small blood vessel distribution, which is contrastingly heavily decorated with peptidergic endings (see below and Fig. 2.11). The implication of the differential distribution is that those peptidergic axons containing the potent vasodi-lator, CGRP, are predominantly involved in vascular regulation and may more readily serve an effector role than the lectin-reactive axon terminals that appear to distribute in a ‘free’ fashion lacking any obvious target cells and corresponding receptor binding sites. The possibility that the non-peptidergic, lectin-positive neurones might be strictly nociceptive and may constitute a distinctive sensory class has not been supported by electrophysiological studies (see Lawson, Chapter 3, this volume). It should also be noted that peptidergic sensory axons are abundantly distributed in the avascular cornea (Silverman and Kruger 1989) and that CGRP-immunoreactive axons include sensitive mechanoreceptors (for example, Hoheisel et al. 1994) and thus also are unlikely to constitute a single functional class in either afferent or efferent terms.
The ‘noceffector’ concept
This concept arose from the need to account for the distribution of peptidergic thin ‘sensory’ axons whose efferent roles are not likely to be in the form of vascular effector actions or ‘axon reflexes’ and are more probably of a trophic nature (Kruger 1988). The argument derived originally from observation of the extraordinarily rich innervation of the dentinal tubules of molar teeth by CGRP-immunoreactive afferent fibres (Silverman and Kruger 1987) where only a small number of fibres might be required for ‘nociceptive’ function in the exceedingly rare instance of injury and pain from this location. This and other examples of innervation that presumably are not nociceptive are discussed below. The putative ‘effector’ roles in terms of some ‘trophic’ influences have been widespread for peptidergic afferents (Micevych and Kruger 1992), but there is no direct evidence that these constitute the ‘silent’ or ‘sleeping’ nociceptor afferent class.
(p. 43 ) Selective degeneration of nociceptive axons has been deduced from experiments based on systemic administration of capsaicin to rodents (Jancsó et al. 1977). In newborn animals, use of this agent results in extensive elimination of unmyelinated axons and small sensory ganglion cells accompanied by a behavioural deficit consisting of reduced responsiveness to thermal and noxious mechanical stimuli (reviewed by Buck and Burks 1986; Fitzgerald 1983). Fine structural examination reveals severe depletion of peri-vascular fibres (Papka et al. 1984) and of intraepithelial endings in the ear drum (Yeh and Kruger 1984) and epidermis (Kruger et al. 1985) following neonatal capsaicin treatment; there is no evident loss following chemical sympathectomy (Kruger et al. 1985), although the capsaicin-induced loss of peptidergic fibres has been questioned by other workers (Kashiba et al. 1990). In adult rats, following capsaicin treatment the sensory defect is evident without obvious loss of sensory ganglion cells or in dorsal root fibre counts (Chung et al. 1985) despite obvious degeneration of distal axons (Hoyes and Barber 1981), but quantitative analysis of subepidermal axon bundles suggests severe loss of the distal portions of unmyelinated nerves without detectable loss in their supplying cutaneous (sural) nerve (Chung et al. 1990). These authors also suggest that these intact axons do not regenerate (at least within 112 days) consistent with the prolonged selective action of this toxin (Lynn et al. 1987). doo et al. (1969) claim that there is long-term mitochondrial damage in the small sensory ganglion cells associated with the sensory disturbance induced by capsaicin.
The distribution of thin afferent axons does not appear to obey any simple rule based on sensory requirements, probable exposure to noxious stimuli, or putative effector roles, but nineteenth century histologists had already established that the innervation of the skin differed considerably in different locations and surmised that these variants should relate to features of sensory discrimination. It is beyond the scope of this brief survey to review systematically the broad range of regional variation for which some general articles are available; we select here observations relevant to a few specialized regions of the body whose examination provides some insight into the problem of identifying nociceptors.
The principal emphasis has generally been placed on the structure of sense organs in glabrous digital skin because of its prominent role in exploratory behaviour. The descrip-tion by Cauna (1980) of thin axonal endings in human digital skin opened the modern period in which a range of variants were sought, following his description of three types of ‘free’ nerve endings open; beaded; and plain. Their thin axons distribute vertically in a ‘punctate pattern’, some apparently terminating in the dermis and others penetrating the epidermal basal lamina, invaginating the basal layer. Cauna (1973, 1980) also noted a branched ‘penicillate’ distribution, more common in hairy human skin. An example of the branching pattern from a thinly myelinated axon from the lip of a marsupial is illustrated in Fig. 2.6. The meaning of such distinctions in functional terms (that is, modality) is largely unknown, and the few morphological studies of electrophysiologically characterized endings generally exploited the idiosyncratic features of specific tissues.
(p. 44 ) Intraepidermal axons
The existence of thin axons penetrating far up into the epidermis was illustrated in the mid-nineteenth century in a drawing by Langerhans (1868) and confirmed by numerous histologists using silver impregnation methods, but tracing their vertical course proved more elusive by electron microscopy (Breathnach 1971; Cauna 1959, 1980; Kadaroff 1971). The sensory nature of intraepithelial axons was demonstrated by electron microscopic autoradiography in the palatal epithelium employing a radioactive axonal transport label (Yeh and Byers 1983). Numerous studies have revealed peptidergic intraepidermal axons distributed in a manner resembling that of the more general axonal marker, protein gene product 9.5 (PGP9.5), suggesting that a large proportion of intraepidermal fibres are peptidergic (see, for example, Gibbins et al. 1985; Kruger et al. 1985, 1989; Dalsgaard et al. 1989; Karanth et al. 1991; Ribeiro da Silva et al. 1991; Hosoi et al. 1993; Kennedy and Wendelschafer-Crabb 1993; Rice et al. 1993). It should be emphasized that these axons do not penetrate above the stratum spinosum and that their sensory function has not been established. The peptidergic axons have been implicated in effector functions in regulating Langerhans (‘dendritic’) cell phenotypic expression (Hosoi et al. 1993; Hsieh et al. in press) and keratinocyte proliferation (Kjartansson and Dalsgaard 1987).
Among the puzzles in interpreting the functional significance of intraepidermal unmyelinated axons are findings concerning calcium-binding proteins Immunoreactiv-ity for calbindin (CB) and calretinin (CR) appears to be associated with rapidly adapting sensitive mechanoreceptors in both cutaneous and deep avian tissues and these proteins are apparently absent in slowly adapting axons (Duc et al. 1993). The identification of CR-immunoreactive intraepidermal axons in rat digital skin by Duc et al. (1994) presents a special problem as these workers have now established the general principle of calcium-binding proteins in mammalian rapidly adapting mechanoreceptive axons. The high affinity of these proteins for calcium could signify a role in controlling intracellular Ca2+ levels and thereby modulate impulse generation properties, but comparison of CB and CR amino acid sequences in diverse species indicates that calcium-binding sites are not the most conserved regions of these proteins, and it has been suggested that the interspersed segments actually may be crucial in interacting with intracellular macromolecules, independently of the role of calcium ions in transduction (Parmentier 1990). Whether these proteins are singularly associated with rapidly adapting mechanoreceptors remains uncertain because it has often been inferred that intraepidermal axons extending deep into the stratum spinosum of glabrous skin derive from unmyelinated parent axons and thus probably constitute ‘nociceptors’, unlike the thinly myelinated ‘delta’ mechano-nociceptors terminating in or near the basal stratum (Kruger et al. 1981) in cat hairy skin. The nature of deep epidermal ‘free’ nerve endings enveloped by keratinocytes and terminating below the stratum corneum merits inquiry concerning a possible role as sensitive mechanoreceptors with axons conducting at C-fibre velocities. In this context, it should be noted that the intrapithelial axons that penetrate almost to the surface of the cornea (see Fig. 2.7) identified by Maclver and Tanelian (1993) include C ‘nociceptors’ that respond to delicate mechanical stimuli, although in this location their excitation usually results in sensory reports of discomfort, if not pain, in human subjects (see Belmonte and Gallar, Chapter 6, this volume).
(p. 45 ) Thinly myelinated (Aδ) endings
The thinly myelinated axons that lose their myelin sheath and penetrate to the basal layer of the epidermis described by Cauna (1980) have been noted in various cutaneous sites, including several electron microscopic studies thereof (Fig. 2.3). In one early study (Hensel or al. 1974), a terminal of this type, identified electrophysiologically as a ‘cold’ spot receptive field, was illustrated by a drawing, but there apparently is no evidence to date that enables morphological distinction between ‘cold spots’ or the mechanorecep-tive ‘spots’ of nociceptors. In a study of cat hairy skin, Kruger et al. (1981) examined a series of units identified as conducting in the myelinated Aδ conduction velocity range, and were able to identify axon terminals in the basal epidermis at identified nociceptive ‘spots’ that were not found in the intervening insensitive regions of the receptive field (Fig. 2.4). The several electron microscopic studies of epidermal basal layer endings reveal aggregations of vesicles and mitochondria, but little else that might distinguish these ‘terminals’ as specialized sense organs and, in the absence of serial sections before and after encountering the putative ending, there can be no assurance as to whether or not a branch might continue further into the stratum spinosum since such axons have been observed by many workers and in diferent species. Examples traced from thinly myelinated cutaneous axon to their presumptive terminal containing an accumulation of mitochondria and a few vesicles (Fig. 2.5) and the terminal branching pattern (Fig. 2.6) are illustrated here in marsupial skin.
A recent study by Maclver and Tanelian (1993) in which thin polymodal fibres were studied electrophysiologically, while observed in the living cornea (in vitro) using epifluorescence, offers much promise. The mechanical-and heat-sensitiveδfibres were associated with horizontally directed processes described as basal epithelial branched ‘leashes’ that could be distinguished from the ‘delicate stranded endings’ traced into superficial epithelial layers and which are associated with ‘polymodal’ C fibres. Although MacIver and Tanelian (1993) present no fine structural data, their findings clearly distinguish separate classes of ‘free nerve endings’ that can be functionally interpreted in the context of the several accounts of corneal innervation (Zander and Weddell 1951; Matsuda 1968; Hoyes and Barber 1976; Tervo et al. 1979; Rozsa and Beuerman 1982; Tanelian and Beuerman 1984; Silverman and Kruger 1988b, 1989; Jones and Marfurt 1991; Ogilvy et al. 1991). The leash-like’ arrangement of the basal fibres and endings (p. 48 ) associated with Aδ fibres (Fig. 2.7) may account for the directional selectivity of these mechano-sensitive units (MacIver and Tanelian 1993), but this is probably not a functionally homogeneous population as it comprises units with high and low mechan-ical thresholds as well as some with heat sensitivity (Belmonte et al. 1991; Belmonte and Gallar, Chapter 6, this volume) and, although some of these fibres are peptidergic (Silverman and Kruger 1989; Jones and Marfurt 1991), there is a lectin-reactive popula-tion (Silverman and Kruger 1988b) that is predominantly associated with thinner C fibres.
The corneal C fibres are of particular interest, in that they not only clearly penetrate vertically into the depths of the epithelium close to the surface, but also are the only sensory endings that undergo continuous growth and remodelling, spectacularly observed continuously in the living cornea by Harris and Purves (1989). While it is tempting to assume that these axons are nociceptive and polymodal, often possessing low mechanical threshold, thermal sensitivity and chemosensitivity, this is a complex problem for which the morphological correlates require further detailed examination.
The innervation of the interior of teeth is numerically rich and of special interest because the only sensory quality reported on its stimulation in humans is invariably intense pain. The extensive innervation of dental pulp blood vessels is not surprising, although much of it is presumably of sympathetic origin. Peptidergic ‘sensory’ fibres are also quite numerous, but the unexpected finding is the richness of CGRP-immunoreactive axons within the coronal dentinal tubules (Silverman and Kruger 1987), which in terms of density (that is, number of axons/unit area) is richer than the exposed, superficial cornea and any other tissue we know of (Fig. 2.8). It was largely this finding that led to the proposal of the ‘noceffector’ concept (Kruger 1988), because afferent activation of these axons would seem unlikely in normal circumstances. It should also be noted that pulpal afferents apparently derive from numerous large ganglion cells with myelinated axons, many of which contain calcium-binding proteins (Ichikawa et al. 1994; Sugimoto et al. (p. 49 ) 1988), reducing the likelihood that these proteins might be exclusive mechanoreceptor markers unless the retrograde fluorescent label used in these experiments leaked to periodontal mechanoreceptors—an unlikely occurrence.
Pulpal innervation appears to be especially disconcerting for sustaining some general principles concerning nociceptors. The sensory report by pulpal excitation, even using stimuli that would be innocuous when applied to other tissues, is invariably intense pain in humans, yet it clearly contains some myelinated axons derived from large, light ganglion cells. However, the fibre spectrum of the trigeminal nerve differs from other somatic afferent nerves in possessing a higher ratio of myelinated to unmyelinated fibres. If these fibres serve a nociceptive function, especially encased in a crystalline protective casing of enamel, it is not obvious why numerous axons would be useful as required for signalling ‘pain’.
This structure is particularly interesting in possessing an outer surface constituting the thinnest epidermis of the entire body and innervated by thick and thin axons but lacking in specialized corpuscular endings, although it is clearly suitable for tactile function. The (p. 50 ) inner mucosal surface facing the middle ear cavity is a sparsely innervated thin epithelium innervated solely by unmyelinated axons, and it is generally believed that their excitation can only elicit the sensory quality of slow, prolonged pain. These fibres can be visulized conveniently in whole-mount preparations (Cohn and Kruger 1986) and traced to their single ending (Fig. 2.9). Fine structural analysis (Yeh and Kruger 1984) reveals superficial axon terminals lying immediately beneath the mucosal basal lamina, and many of these endings contain granular core vesicles and mitochondria (Fig. 2.10) typical of peptidergic endings (see (Fig. 2.15) and also seen in the intraepidermal terminals in the outer surface (Yeh and Kruger 1984), These peptidergic axons are extensively, but not totally, eliminated by neonatal capsaicin treatment.
In the context of the previous discussion of tooth pulp innervation, it is worth noting that inflammation of the tooth and of the mucosa of the tympanic membrane both result in intense, ‘slow’ pain but with vast differences in the density of ‘free’ nerve endings representing the extremes of highest and lowest numerically.
This organ has been exploited as an optimum region for electrophysiological study of visceral nociceptors largely through the use of an in vitro preparation developed by Kumazawa et al. (1987). The testes are rarely associated with pain except when elicited by a mechanical insult or an inflammatory process. The sense organs are distributed principally in the tunica vasculosa protected by the collagenous coat of the tunica albuginea, an arrangement that is convenient for identifying the locus of receptive fields in a confined sheet suitable for electron microscopy as well as for whole-mount staining. The whole-mount preparation revealed a propitious vehicle for distinguishing between peptidergic sensory fibres and the large population of generally thinner lectin-reactive (p. 51 )
The distribution of axons in the relatively flat sheet of the tunica vasculosa also facilitated electron microscopic analysis of serial sections through electrophysiologically delimited receptive field ‘spots’ of characterized ‘polymodal’ fibres (Fig. 2.12). Kuma-zawa and his colleagues conservatively avoid commitment to designating these (p. 52 )
Nervi vasorum and nervi nervorura
The vascular tree throughout the body was shown to be richly innervated by substance P-and CGRP-immunoreactive sensory axons (Furness et al. 1982; Kruger et al. 1989), and numerous studies have implicated these fibres with the plasma extravasation and vasodilatation accompanying inflammation (see Holzer 1988; Maggi and Meli 1988 for reviews). There is direct evidence that individual nociceptive axons, when excited electrically, can elicit these peripheral efferent actions (Kenins 1981). These vasoactive fibres are capsaicin-sensitive and their perivascular distribution has been demonstrated using antibodies to several peptides in virtually every tissue examined thus far. Most papers illustrate the rich innervation of large arterial branches, but there are numerouss endings surrounding small vessels entering capillary beds that are distinct, though less numerous, surrounding veins (Kruger et al. 1989). In frozen sections, some fibres appear to traverse the mural smooth muscle, but this is probably an artefact and we have never seen a labelled terminal at the electron microscopic level that actually penetrates the adventitia. Thus, it is highly unlikely that there is any direct synaptic action on smooth muscle, but numerous investigators have noted peptidergic axons traceable to the vicinity of mast cells (Fig. 2.14) whose release of histamine when excited to degranulate probably also contributes to ‘neurogenic’ vascular effects (Coderre et al. 1989; Dimi-triadou et al. 1991). (p. 53 )
(p. 55 ) The innervation of nerve bundles resembles that of the vasa nervorum and thus many of the sympathetic as well as of the sensory peptidergic axons surrounding nerves (nervi nervorum) probably exert their effect directly on blood vessels (Hromada 1963; Furness et al. 1982; Appenzeller et al. 1984; Dhital and Appenzeller 1988). There is suggestive evidence that peptidergic fibres to vessels and nerves might be implicated in pathological conditions associated with chronic pain (Lincoln et al. 1993; Zochodne 1993), but the mode of action is poorly understood. Bove and Light (1994) have identified nociceptive fields associated with neurovascular bundles electrophysiologically and suggest that these are related to epineurial CGRP immunoreactivity (Bove and Light 1993), some of which may be non-vascular. A direct effect of nociceptive terminals on or associated with intraneurial axonal bundles is difficult to establish, but putative terminals can be detected with electron microscopy, a striking example of which is illustrated in Fig. 2.15. However, unless the parent axon is traced in its course through the epineural sheath, serial sections showing the disappearance of these rare presumptive ‘terminals’ should be interpreted with caution, although large clusters of peptidergic vesicles are not generally evident in sagittal sections of axonal varicosities. Unmyelinated axons terminating within small peripheral nerves have been shown in serially reconstructed sections in joint capsules and tendons (Andres et al. 1985; Heppelmann et al. 1990). The localization of peptides to granular ‘dense core’ vesicles using colloidal gold immuno-cytochemistry was established earlier by Gulbenkian et al. (1986). It should also be noted that small vessels penetrate the nerve sheaths and these presumably might be accom-panied by small penetrating axons.
The peptidergic innervation of glands, especially by CGRP because it is lacking in sympathetic postganglionics, provides some clues to the complex pattern of secretory regulation (see Silverman and Kruger 1989). In general, the acinar portions of exocrine glands receive rather sparse sensory (that is, CGRP-immunoreactive) supply, although there is an abundance of perivascular intraglandular fibres (for example, dermal sweat glands; Kruger et al. 1989), and the densest pattern of glandular innervation is associated with excretory ducts and especially their orifices. It should be noted, however, that there are exceptions to such generalizations among the various nasal glands, for example, the Bowman's glands associated with the olfactory epithelium, and the acini of several nasal serous glands are amply supplied with CGRP-immunoreactive fibres, in contrast to the scant innervation of the mucous vomeronasal gland acini. The duct orifices strikingly supplied with sensory peptidergic fibres (for example, Bowman, von Ebner, and vomeronasal glands) imply an efferent role in regulation of secretions, especially in conjunction with chemosensory epithelia, but several exceptions remain unexplained (Silverman and Kruger 1989). In the absence of data suggesting that the efferent axons to glandular acini, ducts, and orifices serve a sensory role, it is tempting to relegate this rich thin-fibre innervation to an effector role, some of which is unrelated to regulation of smooth muscle. Nociceptive protective reflexes may be mediated by some of these fibres, but it would be difficult to account for their number and pattern of distribution strictly on the basis of nociceptive function. (p. 56 )
Specialized secretory epithelia
The watery secretions of low protein content, and differing in ionic content substantially from plasma and extracellular fluid, do not appear to be regulated by peptidergic axons; they are conspicuous in their absence from the ocular ciliary processes, the stria vascularis of the inner ear, and the choroid plexuses (Silverman and Kruger 1989). This condition contrasts with the peptidergic innervation of all types of sensory epithelia.
Specialized sensory epithelia
The innervation of the renewable chemosensory epithelia—the neural olfactory and vomeronasal mucosae, as well as perigemmal axons surrounding taste buds and their orifices (Silverman and Kruger 1989, 1990b)—may be related to the turnover of cells in these renewable epithelia and the putative efferent regulation of mitogenesis by peptide release (Kruger 1987), rather than to joining central somatic nociceptive pathways. Neither of these functional inferences are likely to account for the sensory peptidergic (p. 57 ) innervation of the non-mitotic vestibular and cochlear hair cells (see Silverman and Kruger 1989). This constitutes another region in which a sensory ‘nociceptive’ role for these fibres is not evident, and their possible efferent function in regulating the sensitivity of these sense organs has not been elucidated.
The vibrissae of those species using their ‘whiskers’ as exploratory organs (for example, rodents) are among the regions most richly supplied with unmyelinated axons, most prominently to the vibrissal follicle—sinus complex constituting the inner conical body (ICB). Andres (1966) illustrated circumferential ‘lanceolate’ endings derived from thin myelinated axons and numerous branching unmyelinated axons in the rat—the latter notably sparse in the rabbit and cat. Many of the unmyelinated endings are associated with parallel collagen bundles and are partially encapsulaed by septal cell processes; these are interpreted as Ruffin endings (Munger and Halata 1983; Renehan and Munger 1986). There are three sets of sensitive mechanoreceptors supplied by myelinated axons associated with the ICB including those ending on Merkel cells, small lamellated corpuscles (in monkey), and the lanceolate endings associated with the basal lamina (Halata and Munger (1980), These all obviously possess an unmyelinated terminal portion, but it is unlikely that they serve a nociceptive role.
The ICB of sinus hairs has been studied using high-voltage electron microscopy and serial reconstrucion of the extraordinarily dense array of small bundles of principally unmyelinated axons that terminate in a succession of cytoplasmic expansions resembling a cluster of grapes (Fig. 2.16). The endings are packed with organelles, principally mitochondria, and vesicles of both clear round and dense-core varieties, and they remain surrounded by a thin sheath of Schwann cell and basal lamina such that they are never observed as single ‘free’ endings (Mosconi et al. 1993). There are two distinct sets of unmyelinated axons: one derived from the deep nervous plexus to the lower third of the blood sinus where they branch and run upwards and the other from the superficial plexus, which are topographically more likely to discharge on pulling or swelling of the blood sinus. The latter are more likely to serve as nociceptors. Both plexuses also contain some thinly myelinated axons.
Other sets of unmyelinated axons surround the mouth of the sinus hair follicle and are deep in the outer root sheath of the hair shaft (Rice 1993; Rice et al. 1993). The location and large number of these branched endings surrounding the ICB would suggest that they are unlikely to serve a purely nociceptive function. Although many of the axons display peptide immunoreactivity (CGRP, galanin, and substance P), there is also a prominent lectin-reactive axonal population, indicating that there is probably a functionally heterogeneous innervation pattern (Mosconi et al. 1991).
Common fur or down hairs are also innervated by circumferential arrays of highly branched unmyelinated axons in their terminal distribution, some of which derive from myelinated parent axons and are arranged in a Ruffini-like pattern (Halata 1988; Halata and Munger 1981). The larger guard hairs are prominently innervated by large myelinated axons that serve as velocity-responsive sensitive mechanoreceptors (see Burgess and Perl 1973). The sensory function of hairs is generally to provide a lever for enhancing mechanosensitivity and it is doubtful that they serve an important role in nociception, although pain can be elicited by pulling on all types of hair For those hairs (p. 58 ) most heavily innervated by unmyelinated axons, that is, sinus hairs, it is unlikely that they serve principally as nociceptors. Perhaps these fibres possess an efferent role with respect to altering the dynamic properties of the blood sinus, but the mechanism is unknown.
Fine structure of axon terminals
Recognition of the terminal receptive portion of a distal axon derived from a sensory ganglion cell may be ambiguous for unmyelinated axons unless fully reconstructed in serial sections that extend beyond the terminal in all directions. Most mechanoreceptor afferent fibres end in an encapsulated form, at least in the sense that the perineurial epithelium can be traced to a surrounding corpuscle or sensory apparatus with which it is continuous (Halata 1975), whereas unmyelinated axons often lose their perineurial layer (except perhaps some of its collagen component) before terminal expansion is reached (see Fig. 2.6). It is conceivable that the entire segment of such axons distal to its perineurial epithelial ensheathment constitutes the ‘receptive’ portion of the axon and there are examples of unensheathed segments along the course of nerves (for example, Yokota 1984). The ‘terminal’ portion, as defined by the absence of perineurial epithelium, may have multiple sectors of organelle aggregations (cf. vesicles and mitochondria) and there is no direct evidence to preclude the possibility that the entire distal process serves as a ‘transducer’, especially for C nociceptors that respond to chemical stimuli and possess what appear to be multiple ‘endings’ for a single axon (Heppelmann et al. 1990). The issue (p. 59 ) is not trivially semantic, for there is a distinction between multiple branches of a single axon each with its own terminus and multiple sensor zones along the path of one axon (or branch) each constituting a ‘terminal’ zone.
Ultimately, most authors have relied on defining the cytoplasmic features of putative endings that distinguish them from other portions of the distal unmyelinated parent axon. There is general agreement, in numerous papers, that mitochondria accumulate in the region of impulse generation in all sensory terminals, although in very thin terminals, a common feature of C fibres, there may be only a few, and similar clusters of mitochondria might be found in the parent nerve trunk The pattern of cristae and intramitochondrial matrix density differs in individual terminals, often in the same field. Although this may be an irrelevant feature for determining a functional role and is generally ignored in the literature, mitochondrial matrix density is related to functional activity in sensory ganglion cells (Wong-Riley and Kageyama 1986). A number of reports illustrate accumulation of glycogen granules in terminals (see Andres and von During 1973; Halata 1975), and it is likely that their apparent absence in many studies is related to the method of tissue preservation and avoidance of anoxia prior to fixation. However, glycogen also is found along the entire axonal length. A feature that has not been noted in any studies is the ‘receptor matrix’ (Andres 1969; Andres and von During 1973), an amorphous, faintly filamentous region located in a focal sector of the terminal often associated with microtubules. Failure to identify this in most reports on un-myelinated axon terminals with nociceptor candidacy also may be related to fixation methodology even when electron micrographs are of obvious high quality, but for the present judgement should be postponed.
The presence of dense bodies (for example, Figs 2.12 and 2.15) is evident in many micrographs from numerous reports and a few also reveal small vacuolar inclusions or organelles, but these are unlikely to serve as diagnostic markers for identifying a ‘free’ nerve terminal. The all-important issue of vesicle accumulation is generally accepted as a particularly reliable criterion for identifying a receptive terminal zone, but there are many micrographs in the literature of putative endings with few or no vesicles. This may be, in part, a sampling problem and we shall deal with the vesicles in some detail below. Finally, there is the issue of identifying a region of bare axolemma lacking envelopment by Schwann cell cytoplasm associated with one or more of the above-mentioned features. A sampling of the growing literature and our own experience indicate that a thin layer of Schwann cell processes and its basal lamina constitute an interface that attenuates the usefulness and accuracy of the term ‘free’ nerve ending, unless one finds comfort and mitigation in the examples of those small sectors of the axolemma that protrude from one surface of the Schwann cell (Fig. 2.12 (b)). The site of the receptive or transductive surface, the nature of endocytotic and exocytotic processes and especially of peptide release, and the role of the Schwann cell in the interaction between sensory terminals and their surround must be better understood before the concept of a ‘free’ ending acquires meaning.
The nature of the vesicles found in the peripheral axon terminals of sensory ganglion cells is of special interest because, unlike the central (dorsal root) branch of the same axon, the distal ‘sensory’ terminal lacks synaptic contact and function and there is clearly no known (p. 60 ) requirement for neurotransmitter release. The latter statement may not be strictly correct, at least for peptidergic terminals serving an efferent role, especially when the peptide target can be identified by determining the cellular localization of peptide receptor binding sites. A functional role of ‘sensory neuropeptides’ in impulse generation or even as potent modulators of membrane events in these afferent terminals has not been demonstrated persuasively. It seems most likely that peptides serve a quasi-hormonal role in the various target tissues where receptor-binding sites have been identified. In this context, the role of vesicles containing peptides may be simply to fuse with the axolemma at a locus suitable for release; their effector action is known for smooth muscle but it is probably important in a variety of other cell types expressing sensory neuropeptide receptors. A dramatic example of a receptor-binding site in a lymph node, in addition to those in smooth muscle, where an effector target site might be expected, is illustrated in Fig. 2.17. There also has been some speculation that peptides, for example, CGRP, may play a role in impulse initiation (Beckers et al. 1992), but this has not been demonstrated.
We shall not attempt to survey the growing body of literature indicating that various peptides can be localized within granular or ‘dense-core’ vesicles, using colloidal gold immunocytochemistry as illustrated in Fig. 2.15 (see Gulbenkian et al. 1986; Ribeiro da Silva et al. 1991). The form of these peptide (or protein-precursor)-containing electron (p. 61 ) dense vesicles is apparently susceptible to perturbation by various fixation methods such that preservation of the vesicle membrane and the distinct visualization of a halo around a dense interior is not evident in all published micrographs. It should be emphasized that the exclusivity of sequestration of peptides in dense vesicles has not been firmly established in all clases. Furthermore, it is likely that some of the dense-core vesicles are of the catecholaminergic variety (Price and Mudge 1983) and possess distinctly greater electron density. Clear, spherical vesicles are mostly commonly found in sensory axon terminals (Whitear 1974), and it can be inferred reasonably that the same range of excitatory and inhibitory neurotransmitters identified in the synaptic terminals of the spinal cord and brainstem are manufactured and apparently indiscriminately trans-ported to both central and peripheral terminals. The nature and variety of these ‘transmitters’ is reviewed by Lawson (Chapter 3, this volume), but their functional, clearly non-synaptic role in peripheral endings is unknown. Some of the most intensively studied synaptic transmitters, popularly known as ‘classical’, for example acetylcholine and glutamate, are known to act upon non-neural elements. There is also a panoply of substances associated with pain and capable of eliciting impulse discharge in nocicep-tors, including bradykinins and free radicals. Nitric oxide, a recent ‘molecule of the year’, implicated in pain (Dawson et al. 1992; Meller and Gebhart 1993) is found in a variety of putative nociceptive neurones and their endings (Verge et al. 1993; Bscheidl et al. 1994). Its actions on a variety of cell types involved in inflammatory reactions obfuscate its neurogenic role and its specificity.
On strictly morphological grounds, it is unlikely that there is a homogeneous population of clear vesicles, for, in addition to some pleomorphic variants, there is a range of sizes, and, in some studies employing methods optimal for preservation of membrane ultrastructure, distinct vesicle coats or cages can be discerned. Recent studies of the ‘free’ nerve endings of the infra-red sensitive pit organ of crotaline snakes (Terashima and Jiang 1993; Terashima et al. 1995) suggest that temperature can alter the size, number, and coats of vesicles in this receptor terminal and Fig. 2.18, taken from Terashima's studies, illustrates the typically larger size of the large spherical coated vesicles. We are unaware of attempts to determine whether the latter are clathrin-coated and exert a distinctive role in endocytosis and exocytosis. The role of the clear, uncoated, vesicles in transmitter cycling, discussed in the extensive literature on synaptic endings (see Heuser and Reese 1973; Basbaum and Heuser 1979; Zimmer-man 1979; Tauc 1982; Heuser 1989; Valtorta et al. 1990), apparently has not attracted systematic investigation in nociceptive endings, nor has the putative role of neuro-transmitters or neuropeptides in impulse generation been examined in a morphological context. Techniques for examining the mechanism of uptake of axonal transport ‘markers’ can be applied readily to this perplexing question. The absence of terminal transporters for neuropeptides and the lack of information concerning the presence and distribution of vesicle transporters of transmitters have impeded our understand-ing of vesicle function in non-synaptic sensory terminals. Stimulated release of neuropeptides has been established convincingly (Olgart et al. 1977; Fujimori et al. 1990), and many of their cellular targets have been identified by receptor-binding autoradiography, but the peripheral target cells or hypothetical axon terminal auto-receptors for transmitters involved in rapid depolarization and impulse generation remain remarkably unexplored. (p. 62 )
Any survey of morphological studies of putative nociceptor endings reveals that these sense organs have been characterized in very bare detail. A vast literature abounds with non-descriptive constructs implying that nociceptors are ‘simple’ or ‘primitive’, an unlikely condition for which evidence is grossly deficient, and, despite a consensus for physiological diversity, we lack a satisfactory taxonomy based on structure. Reluctance to accept the very existence of ‘nociceptors’ until late in this century may account in part for dilatory progress, but their study has also been hampered by the inherent difficulty of characterizing a structure that requires some degree of damage in order to be identified.
A vast proportion of the thin fibres whose endings might be involved in pain or any sensation related to mechanical, thermal, or chemical stimuli, are arranged in the periphery in a pattern and in numbers suggestive of efferent actions that are indepen-dent of nociceptive protective reflexes and instead may be involved in physiological trophic roles in the absence of actual or threatened tissue damage. Peptide-mediated efferent mechanisms, particularly of vascular regulation, may be controlled by impulse generation due to noxious stimuli, although dependence upon afferent activity and peptide release has not been established. The ‘noceffector’ concept implies that many afferent fibres are effectors and that they are rarely, if ever, activated by noxious stimuli (for example, those innervating glands, ducts, sphincters, taste buds, endosteum, (p. 63 ) dentinal tubules, etc.), and others (such as those innervating cochlear and vestibular hair cells, olfactory epithelium, and sympathetic ganglion cells) may lack any afferent or sensory role whatever.
Another population of apparently non-peptidergic sensory ganglion cells emitting thin, principally unmyelinated axons characterized by specific membrane glycoconju-gates and co-localized markers (monoclonal antibodies, lectins, fluoride-resistant acid phosphatase) are not distributed in any identified relation to peripheral target effectors, nor have their electrophysiological correlates been secured. The ostensible ‘transmitters’ at sensory terminals have been inferred largely from identification of these substances in central (dorsal horn) terminals (acetylcholine, y-aminobutyric acid (GABA), excitatory amino acids, purines, etc.) and their influence upon events in putative nociceptive endings is obscure. The molecular specificity of neuromodulators found within, or exerting an influence upon, ‘nociceptive’ endings offers promise in classifying ‘noci-ceptors’ as do the many substances that clearly influence or vigorously excite discharge (bradykinins, nitric oxide, and reactive oxygen species). A practical taxonomy of ‘nociceptors’ based on molecular specificity or on morphological specialization remains elusive, but the popularly held concept of ‘free’ nerve endings and their identity with nociceptors has probably outlived its usefulness.
We are indebted to our colleagues who are too numerous to mention for discussions of the content of this chapter, to Ms Deborah Anderson for preparing the immunogold electron micrograph as well as for assistance in asembling the figures, and to Ramon Colinayo and Lisa Chen for manuscript preparation. The electron micrographs from marsupial skin were obtained from studies in Hamburg supported by the Deutsche Forschungsgemeinschaft Ha 1194/3–1 and the previously unpublished rat electron micrographs were obtained at UCLA supported by a National Institute of Health, grant NS-5685 (a Javits Investigator award).
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