An understanding of the events initiating vago-vagal reflexes requires knowledge of mechanisms of transduction by vagal afferents. Such information presumes an understanding of receptor morphology and location. Anatomic studies have recently characterized two types of vagal afferents, both putative mechanoreceptors distributed in gastrointestinal (GI) smooth muscle. These two receptors are highly specialized in that they 1) are morphologically distinct, 2) have different smooth muscle targets, 3) form complexes with dissimilar accessory cells, and 4) vary in their regional distributions throughout the GI tract. By comparison, information on the architecture and regional distributions of other classes of vagal afferents, notably chemoreceptors, has only begun to accumulate. Progress on the study of the two mechanoreceptors, however, illustrates general principles and delineates experimental issues that may apply to other submodalities of vagal afferents. By extension from morphological and physiological observations on the two species of smooth muscle endings, it is reasonable to hypothesize that additional classes of vagal receptors are also differentiated morphologically and that they vary in structure, accessory cells, regional distributions, and other features. A full appreciation of vago-vagal reflexes will require thorough structural and regional analyses of each of the types of vagal receptors within the GI tract.
- visceral afferents
gastrointestinal (gi) vago-vagal reflexes are initiated when vagal afferents transduce energy associated with events in the gut. Transduction is the process in which a receptor, that is a first-order afferent or an accessory cell that interacts with such an afferent, translates energy from a stimulus into electrical signals that can be propagated by the nervous system. As the definition implies, studies of transduction in visceral afferents need to specify the receptor, the stimulus energy, and the resulting cellular signaling events. Even though the structural complexities of the gut and the resulting difficulties in achieving spatial and temporal stimulus control within the GI tract make refined study of its interoceptors difficult, a consideration of the formal elements of the definition of transduction serves to focus some key issues.
Besides the definition of transduction, other lessons from recent progress in elucidating other afferent systems also offer useful perspective on the analysis of vago-vagal reflexes. In particular, the breakthroughs of the last two decades in understanding transduction in other afferent systems (e.g., vision, audition, olfaction, taste) have involved isolating and characterizing the receptor cell and the site of stimulation, specification of the energetic event that is translated to a neural signal, cloning of the membrane-bound receptors, and identification of the second-messenger pathways (cf. Ref.30). For each of the well-characterized senses, advances have occurred by an iterative series of complementary and parallel observations specifying more precisely the receptor and the stimulus. Then, after the receptor has been thoroughly delineated and the stimulus energy accurately characterized, the membrane events and intracellular signaling pathways have been identified.
In the case of vago-vagal reflexes and the afferents that initiate them, research has commonly focused on the stimulus, with less attention given to the receptor side of the equation. Vago-vagal reflexes have been conventionally studied by delivering mechanical or chemical stimuli to the luminal surface of the gut. When integrative reflexes have been the focus of the work, the circuitry and its functions have been typically inferred from physiological and/or behavioral responses. When transduction events have been the focus, the operations of vagal afferents have been inferred from extracellular or intracellular recordings of nodose ganglion neurons. In both strategies, emphasis has been most frequently on the stimulus and responses in the circuit but not on the receptor. By applying incrementally smaller stimuli to progressively more restricted sites, by minimizing the latencies between stimulation and a detectable response, by employing variations in the molecular structure or the configuration of the stimuli, by employing blockers and nerve cuts to delimit the mechanism, and by using other reductionistic strategies, this approach works to specify the effective stimulus, where and how it works, and how many steps may be involved in converting a GI stimulus into a response. This strategy has been used in a number of highly innovative experiments and has made significant progress in identifying some of the effective (and ineffective) stimuli that directly, or indirectly, initiate vago-vagal reflexes. These experiments have been reviewed in recent papers in this journal (e.g., Refs. 5and 24; also see, for example, Refs. 10,12, 17, 23, 25, and27).
For the most part, physiological experiments have proceeded with little information about the afferent receptor morphology or location. In lieu of structural information, the experiments have employed a set of assumptions that, as the observations reviewed below indicate, are false. One of these propositions is a widely believed assumption that the vagus supplies the GI tract with simple, undifferentiated free nerve endings. Furthermore, much of the classic work on vago-vagal reflexes assumed, either implicitly or by default, that it is practical to understand vagal afferent transduction without knowing the morphology of the endings, the structure of their accessory tissues, the receptive fields of the endings, and, finally, the densities or the regional distributions of the endings.
In contrast, in the present survey, we approach vago-vagal reflexes from the perspective of the receptor morphology, and we take issue with these two conventional assumptions. The “simple ending” idea is sharply contradicted by present evidence. The “transduction can be understood without knowing the receptor” idea is highly problematic. To put the issue in perspective, it is hard to conceive of the dramatic progress in the visual system having occurred only from the stimulus side and if rods and cones were not well characterized, if the pigmented epithelium were not described, if ganglion cell receptive fields were not understood, or if receptor distributions in the fovea and the peripheral retina were not appreciated. Similar arguments are equally compelling for the successes in auditory, olfactory, and gustatory physiology.
As noted in an earlier review in this journal (8), the lining of the GI tract is the largest vulnerable body surface exposed to the external environment, and sensory mechanisms of this system participate in coordinating absorption of food, motility, secretion, tissue defense, and vascular perfusion as well as behaviors such as food intake and toxin avoidance. The extent and the irony of the problem that structural studies of receptors in this extensive body surface have been neglected in GI physiology are both underscored by the observation that more structural work is available on the electroreceptors found in electric fish (and, for that matter, on several other exotic classes of receptors from the annals of zoology) than on the vagal receptors in the GI tract.
Recently, however, some progress has been made in characterizing vagal afferents in terms of structural/functional considerations, and examples of this progress are summarized below. Some of the implications of a fuller characterization of the receptor are also discussed here.
VAGAL AFFERENT SUBMODALITIES
For many species, anatomic surveys have not yet established the size of the afferent pool supplied to the gut by the vagus. As a general index, however, the abdominal vagus of the rat has ∼16,250 afferents and, by comparison, ∼6,000 efferents (22). Since most of the vagal projections in the abdomen are to the GI tract, these figures are likely to be good estimates of the total pool of vagal fibers found in the gut. Given that the nodose ganglion, the source of the afferent fibers, does not display exceptional changes in numbers of perikarya across species that have been evaluated, the number of vagal afferents to the GI tract in the rat may be a reasonable approximation for other species as well. Regionally, vagal afferent endings are most densely distributed in the proximal GI tract (esophagus, stomach, and small intestine), although they are located throughout the length of the gut (31). A comparison of the relatively modest number of vagal afferents innervating the gut and the extensive mass and surface area of the GI tract (cf. Ref.8) suggests that individual fibers would be expected to branch extensively and to produce multiple endings within their target sites. This prediction holds for the two vagal afferents to the GI tract for which we have the most structural information (seevagal mechanoreceptors).
Electrophysiological as well as physiological experiments have established that most of these afferents appear to be either mechano- or chemoreceptors, and most of the work on vago-vagal reflexes has focused on these two submodalities. Although other submodalities (e.g., thermoreceptors) have also been described (e.g., Ref. 13), little if anything is known about the mechanisms of transduction in these additional types of endings, and they will not be covered in the present survey. A preponderance of vagal fibers relay low-threshold mechano- or chemoreceptor signals associated with the movement and digestion of nutrients. In the distal bowel, innervation from dorsal root visceral afferents predominates and a preponderance of these fibers are involved in detecting high-threshold mechanical or nociceptive events (28). These nonvagal, high-threshold afferents are also not covered in the present review.
Structure and distribution of the receptors.
Three types of mechanoreceptors have been inferred from functional and morphological observations, and two of these types of vagal afferent endings terminate within the smooth muscle wall of the gut. Of all of the vagal afferent endings in the GI tract, the two mechanoreceptors in the smooth muscle are the ones about which we have the most structural information. The first of the endings to be characterized structurally is the intraganglionic laminar ending (IGLE; Refs. 15 and26). An IGLE consists of a plate of terminal puncta that lies parallel to muscle layers and that is situated at the surface of myenteric ganglion, effectively at the boundary between a ganglion and one of the smooth muscle layers (see Fig. 1). The IGLE appears to be anchored to both the ganglion and the over- or underlying muscle layer by glial processes (14), and it has been hypothesized that this biophysical arrangement allows the IGLE to transduce shearing forces produced in association with changes of tension (both active and passive) and stretch in the muscle wall. If these terminal plates formed at the distal tips of the vagal afferents utilize accessory tissues as part of the receptor unit, then presumably the accessory elements must be either the glial cells that tie the plates of puncta to the ganglion and adjacent muscle sheet or the cells of the myenteric ganglia.
An individual vagal afferent fiber that forms an IGLE will typically ramify into several short terminal telodendria, each of which ends in an individual IGLE. These terminal branches issue separate IGLEs onto several neighboring myenteric ganglia or poles of these ganglia. Presumably, the receptive field of such an afferent corresponds to the area of the muscle wall that all of the clusters of IGLE plates on its telodendria contact (cf. Ref. 35). In some cases, individual fibers give off collaterals that travel considerable distances after diverging to terminate in separate assemblages of IGLEs, perhaps providing the multiple distinct receptive fields reported for some fibers (2). Although the number of vagal afferent fibers supplied to the GI tract might seem relatively modest, in light of the extensive areas covered by the system of IGLE plates at the end of an individual fiber, the vagus appears to supply an extensive network of overlapping receptive fields in the wall of the gut. Vagal afferents supply IGLEs to essentially the entire length of the GI tract, but they are concentrated most densely in the rostral GI tract and become progressively less dense along the length of the GI tract (e.g., Refs. 3 and 31).
The second type of vagal afferent found in GI smooth muscle differs dramatically from the IGLE in both location and morphology. This ending has only recently been described (4) and still more recently named the intramuscular array (IMA; cf. Ref. 31). An IMA consists of a series of long terminal telodendria located within either the circular or longitudinal muscle sheets and that run parallel to smooth muscle fibers of the sheet and to each other. These arrays of telodendria are interconnected by short branches or cross bridges (Fig.2). The separate terminal telodendria forming one of the arrays lie on a scaffolding formed by the interstitial cells of Cajal, which also lie within the smooth muscle sheets and which run parallel to muscle fibers (4, 7). On the basis of both their morphology and their distributions and concentrations within the muscle wall, it has been hypothesized that these IMAs appear to be specialized to transduce stretch of the organ wall (20). If the IMAs have accessory cells complexed with them to form receptor units, then the accessory tissue is presumably the interstitial cell of Cajal network. Mutant mice with disturbances of this network have losses of vagal IMAs (7), suggesting that the interstitial scaffolding provides either trophic or structural support for the IMAs. As it enters the muscle wall, an individual vagal afferent often branches to terminate in two or more separate IMAs in neighboring areas of the smooth muscle (see Ref.20). Presumably the receptive fields of such afferents correspond to the area encompassed by the IMA or IMA complex at their peripheral terminals.
The two classes of endings in smooth muscle differ as strikingly in their regional distributions as in their morphology, tissue targets, and accessory cells. With the use of a neural tracer that comprehensively labels afferent terminals in the gut (i.e., wheat germ agglutinin-horseradish peroxidase) and a whole mount sampling technique, the regional distributions of the different types of afferents can be inventoried (Fig. 3; see also Refs. 19 and 31). IGLEs are widely distributed throughout the gut and occur in highest densities in the antrum and corpus of the stomach and the initial segment of the duodenum; their densities are reduced in the forestomach, in the region of GI sphincters, and in the distal small and large intestines. IMAs have much more limited distributions, with concentrations in the forestomach (in both longitudinal and circular muscle layers) and in sphincters (in the circular muscle of the sphincters). IMAs are rare in the intestines and appear to occur almost exclusively at strategic sites such as flexures and perhaps functional valves.
The third type of vagal mechanoreceptor has been identified electrophysiologically and is postulated to be mucosal, but the morphology of this type of ending has not been clearly determined. Unlike the smooth muscle endings, which appear to be slowly adapting, the mucosal receptors seem to be fast adapting.
Stimuli transduced by mechanoreceptors.
A consideration of the disparate geometries of the IGLE and the IMA illustrates the thesis that structural knowledge of the receptor is needed. As we have reviewed elsewhere (20), the architectures of the two endings as well as their accessory tissues and their regional distributions suggest that they must transduce different mechanical forces (or, if they transduce the same forces, then they must translate these forces into dissimilar neural codes). We have proposed that their structural features are consistent with the IGLE transducing tension and the IMA detecting stretch. In particular, IGLEs have the distribution throughout the gut and the architecture to make them sensitive to detecting complex rhythmic motor activity, whereas IMAs have the regional concentrations and the morphological features to register sustained nonrhythmic adjustments in length or stretch. Details of the morphologies of the two endings also make it more practical to consider the design of focal and selective stimuli that should probe the transduction processes of these afferents. As we have discussed, the mechanical stimuli that have been traditionally used to stimulate vagal afferents are too indiscriminate and too nonspecific, confounding tension and stretch, to yield precise characterization of afferent processing.
Of the three species of vagal mechanoreceptors, only the effective stimuli for the IGLEs have been determined with specificity. In a powerful paradigm that minimizes some of the hurdles that have stalled analysis of vagal afferent transduction and that promises to refine such analyses, Zagorodnyuk et al. (35) performed acute experiments recording from a vagal axon near an isolated square of gut wall, in an in vitro situation that facilitates mapping the unit's receptive field, subjecting it to specific mechanical forces (e.g., circumferential extension), and then filling its axon with an anterograde tracer that will identify its receptor ending(s). With this preparation, the investigators were able to work with identified IGLEs from the guinea pig cardia (as well as esophageal wall; see Ref.34) and provided characterization of their receptive fields, adaptation profiles, thresholds, and additional electrophysiological characteristics.
No comparable analyses characterizing the response properties of identified IMAs have been performed. In an attempt to assess retrospectively whether earlier recording work done on vagal afferents that were not identified as to terminal structure might suggest that a particular pattern of electrophysiological responses was correlated with the IMA on the basis of regional pattern of responses or some other feature, we recently reviewed the literature on GI mechanoreceptors that had accumulated before the recent characterizations of the receptor architecture (20). For the most part, earlier electrophysiological analyses proved to have sample sizes that were too small and/or to lack enough stimulus control and other information to make any assignments, but the review did underscore several of the controls required and a number of the issues that need to be addressed with additional work. In addition, the survey did suggest that, as outlined above, the available information is consistent with the IGLEs serving as tension receptors and the IMAs comprising length or stretch detectors.
In stressing how little is known about transduction, we do not mean to imply that nothing is known about the sensitivity of vagal afferents innervating the gut. Considerable strides have been made in the last decade in identifying hormones, neuromodulators, and other agents that modify ongoing and elicited activity in afferents. In the case of vagal mechanoreceptors, for example, CCK (e.g., Ref. 27) GABA (e.g., Ref. 17), leptin (e.g., Ref. 32), purinergic compounds (18), and opioid receptor agonists (e.g., Ref. 16), among other neuroactive agents, tune their sensitivity. This work, however, can only achieve a certain level of precision if the classes of mechanoreceptors are not distinguished, if direct effects on first-order afferents cannot be discriminated from indirect effects associated with actions on smooth muscle or enteric neurons, and if agents that effect transduction cannot be distinguished from other compounds that affect posttransduction events in the afferents. A fuller understanding of the receptor apparatus and its cascade of transduction events is the type of information that will make such discriminations practical and will make complete assessment of the earlier observations feasible.
Intracellular basis of vagal mechanoreceptor.
Although three types of mechanoreceptors have been provisionally identified on the basis of their structures and/or response patterns and the differing architectures of the two in smooth muscle have been described, the analyses of these endings have not reached the precision of being able to stipulate the biophysical events that translate shearing, tension, stretch, or displacement into electrical signals in the first-order afferents. Parsimony would suggest that it may be possible to extrapolate from other mechanoreceptor systems, and considerable information is available for some of these prototypes. From such extrapolations, it would seem probable that stretch-activated channels (which have been shown to exist in some nodose neurons; see Ref. 29) might participate in the transduction of mechanical stimuli and that the specific morphology, accessory tissue matrix, and distribution of the different afferents would tune the afferents to particular forces. As an example of the utility of having the receptors specified, it has been possible to begin such analyses and to associate calcium-binding proteins such as calretinin with IGLEs, particularly those in the esophagus (e.g., Refs. 6and 11).
Structure and distribution of the receptors.
Compared with the characterizations of the structure of smooth muscle mechanoreceptors, even less is known about the morphology of vagal chemoreceptors. Mucosal endings were described in the anatomic work of Hill (9), although at the time vagal origin of the fibers was not established. More recently, they have been identified as vagal by injecting anterograde tracers into the nodose ganglion (1,21). These vagal endings are putative chemoreceptors (or at least some of them are putative chemoreceptors; others may be the mucosal mechanoreceptors that have been defined electrophysiologically). Labeled vagal terminals are widely distributed in lamina propria and situated among the intestinal crypts. Vagal afferents also enter and ramify in individual villi of the mucosa. In general, these vagal afferents appear to divide into terminal processes within the villi, to run in conjunction with the network of fibroblasts or fibroblast-like cells with the villi, and to end near, or even apposed to, the basal side of epithelial cells. These endings are found in close proximity to enterochromaffin cells as well. The concept that the fibroblast network serves as accessory tissue and provides a structural and/or trophic support for the vagal afferents in the mucosa, one akin to the interstitial cell of Cajal scaffolding associated with IMAs, is a reasonable hypothesis. Vagal mucosal afferents also have been observed to make putative contacts with mast cells in jejunal villi (33).
Most of the information available is based on a spatially very limited sample and comes from the duodenum (cf. Refs. 1,21, and 31), although vagal afferents innervate the jejunal mucosa (33) and occur as far distal as the crypts of the colon (31). Whether these mucosal endings have specializations in terms of their densities, their locations, and their finer architectures that distinguish them into subclasses responsible for different submodalities is unclear. In general, structural analyses have not yet begun to assess the divergence of individual fibers, to differentiate types or subclasses (presumably modalities), or to map the distributions of such endings. Whereas single mechanoreceptor fibers have been found to end in a multiplicity of IGLEs or IMAs, it is unclear how extensive individual vagal afferents to the mucosa may be. To classify and analyze individual fibers, it will be necessary to use protocols that label a few individual fibers in their entirety, without staining so many neurites or other elements that it is impractical to distinguish the components of a single ending (e.g., the protocols used to label the single processes in Figs. 1 and 2). Conversely, to quantify the topographic distributions of such endings, protocols that label all fibers are needed (for example the wheat germ agglutinin-horseradish peroxidase protocol used for the inventories in Fig. 3 and illustrated in other papers, e.g., Refs. 19 and 31).
Stimuli transduced by vagal chemoreceptors.
Although studies have not yet examined transduction mechanisms of any class of morphologically identified mucosal ending, a variety of electrophysiological experiments have been performed (e.g., Ref.13) and many physiological experiments have made progress in identifying stimuli affecting vagal afferent activity (e.g., Refs.10 and 25) or engaging vagal reflexes (e.g., Refs.5 and 24). Two complementary approaches have converged on the issue of the sensitivities of first-order vagal afferents, but they have not in a strict sense dealt with transduction by identified vagal receptors.
One of the approaches involves demonstrating that vagal mucosal afferents (typically monitored by recording from nodose ganglion perikarya or from teased vagal fibers), much like vagal smooth muscle mechanoreceptors, are sensitive to several neurotransmitters, neuromodulators, hormones, cytokines, and other endogenous neuroactive signals (e.g., Ref. 10). Although some of these signals could be involved specifically in the transduction cascade, most of these signals probably operate on vagal afferent sensitivity once the primary stimulus energy has been transduced (as suggested, for example, by the careful analyses of CCK; see Ref. 25).
The other approach that has implications for the sensitivities of vagal afferents complements the first by demonstrating that first-order vagal afferents (typically assessed at the level of the perikarya in the nodose ganglion or, in some cases, in cross-sections of the entire vagus) contain binding sites for many of the transmitters, neuromodulators, hormones, neurotrophic factors, and cytokines that have been shown to modulate vagal afferent activity (e.g., Ref.36). For the most part, however, such experiments cannot yet link a perikaryon that has been characterized electrophysiologically or by receptor immunochemistry with a particular ending morphology.
Until the morphological specializations, including the structural associations with any accessory cells, of the different vagal afferent receptors are described and mapped, it will be difficult to distinguish those substances that play an obligatory role in the transduction pathway from those chemicals that may modulate the (posttransduction) sensitivity of the different afferents or that modify the local (pretransduction) milieu so as to alter either the subsequent sensitivity of vagal afferents or the probabilities of transduction events occurring.
In other, more thoroughly characterized sensory systems, analysis of the afferent traffic that initiates reflexes has been approached by identification of each of the basic elements that participate in signal transduction. This strategy has involved the use of complementary functional and structural approaches that progressively specify both the stimulus and the receptor apparatus. In no instance has afferent transduction been adequately characterized without knowledge of the receptor. Delineation of the stimulus helps focus morphological analyses of the receptors, and, in turn, structural characterization of the receptor helps clarify the features of the stimulus that are transduced. With more precise understanding of both the stimulus and the receptor, it then becomes practical to elucidate which channels and membrane-bound or intracellular receptors are found in the endings and which intracellular signaling events are downstream of the transduction process. Recent developments in the analysis of GI afferent mechanisms suggest that the same formula will yield success in developing a full explanation of vago-vagal reflexes.
At least three specific applications of fuller characterizations of the structural features of vagal receptors initiating the vago-vagal reflexes can be envisioned. First, understanding the architecture, distribution, and accessory tissue of a particular type of receptor should make it practical to design more selective stimuli that do not confound a variety of energy dimensions (e.g., active and passive tension) and complicate analyses of stimulus transduction. Second, fuller characterizations of the morphology and organization of vagal afferent terminals should make it more practical to make informed hypotheses and judgments about a number of classic sensory issues applied to the GI tract (e.g., poly- or unimodal, narrow or broad tuning, labeled lines or cross-fiber codes, center-surround organization or other sharpening mechanisms). Third, identification of the particular receptor apparatuses for the individual submodalities should make it much more practical to evaluate which receptors (or channel proteins, or second messengers, etc.) are localized in the different submodalities of afferents, not just more generally in an undefined subpopulation of afferents in the vagus nerve or the nodose ganglion.
Such progress should help bring the afferent systems of the gut up to the refined level of analysis that has been reached for many of the senses, including vision, audition, olfaction, taste, and even the electric sense. At that plane, our understanding of vagal afferents will augment needed work on vago-vagal reflexes and will make it feasible to use more successfully the tools of autonomic pharmacology and molecular biology to address disorders of GI physiology.
We were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-27627 and DK-61317.
Address for reprint requests and other correspondence: T. L. Powley, 165 Peirce Hall, Purdue Univ., West Lafayette, IN 47907 (E-mail:).
July 31, 2002;10.1152/ajpgi.00249.2002
- Copyright © 2002 the American Physiological Society