THE ANATOMICAL RECORD 210:73-85 (1984) Morphology of Presumptive Slowly Adapting Receptors in Dog Trachea JANE M. KRAUHS Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, T X 77550 ABSTRACT The three-dimensional branching pattern and ultrastructure of afferent myelinated fibers and their terminals located in the trachealis muscle of the dog are described. The afferent endings are believed to be those of the slowly adapting stretch receptors of the trachea. They have structural features typical of mechanoreceptors: distal to the loss of myelin, their shape becomes more irregular and the cytoplasm is filled with mitochondria, glycogen, and osmiophilic bodies. In some places the cell membrane is attached directly to basal lamina without interposition of a Schwann cell. A bundle of unmyelinated fibers accompanies each myelinated fiber and continues for an undetermined distance beyond (luminal to) terminations of the myelinated fiber. The unmyelinated fibers contain many round, clear vesicles and a few dense-cored vesicles and are also attached directly to basal lamina in places. Three-dimensionalreconstruction of three receptors revealed three quite different branching patterns, but all included apparent rings as part of more or less contorted terminal regions (some neurons apparently having more than one terminal region). No obvious structural basis for the activation of receptors by transverse but not longitudinal stretch was found. Slowly adapting stretch receptors of the trachea are believed (Fillenz and Widdicombe, 1972) to contribute to the HeringBreuer inflation reflex, which inhibits further inspiration of air when the lungs are inflated. The axons of these receptors in the dog are known to travel in the vagus nerve and are believed, because of their conduction velocities, to be myelinated (Coleridge et al., 1965; Sampson and Vidruk, 1975).Their terminals have been localized by electrophysiological methods (Bartlett et al., 1976) to the trachealis muscle in the posterior wall of the trachea (Fig. 1).The receptors are activated by transverse but not longitudinal stretching of the trachea (Bartlett et al., 1976; Sant’Ambrogio et al., 1980) and are believed to be arranged (at least functionally)in series with the transversely oriented trachealis muscle (Bartlett et al., 1976). The structure of the slowly adapting receptors, especially as it may relate to the muscle, is therefore of interest. A few studies of afferent terminals in human bronchial smooth muscle stained with methylene blue (Larsell and Dow, 1933), in 0 1984 ALAN R. LISS, INC. human airways stained with silver or osmium tetroxide-zinciodide (Jabonero and Sabadell, 1972)or with gold chloride (Sampaolo and Sampaolo, 19581, and in silver-stained dog tracheal and bronchial smooth muscle (Elftman, 1943)have revealed complex structures but not as much detail as might be desired (see also the literature review by Fillenz and Widdicombe, 1972). von Diiring et al. (1974) have recently published an ultrastructural study and three-dimensional drawing of afferent nerve terminals in the lamina propria and smooth muscle of rat bronchi. However, no ultrastructural studies of probable slowly adapting afferents in the trachea have been published. Hoyes and Barber (1980) found mitochondria-filled nerve profiles in the trachealis muscle of guinea pig and suggested that they were mechanosensitive, but they were not Received June 13, 1983; accepted March 2, 1984. Jane M. Krauhs’s present address is Northrop Services, Inc., Life Sciences Laboratory, P.O. Box 34416, Houston, TX 77234. Address reprint requests to Dr. Jane M. Krauhs, P.O. Box 1128, League City, TX 77573. 74 J.M. KRAUHS Cross Section of Posterior Wall Lamina Propria (Incl. Elastic Fibers) Elastic Fiber &rent i\ Nerve Fibers (From Vagus) Fig. 1. Schematic drawing of the posterior surface of the trachea, drawn in a longitudinally stretched state to show annular ligaments between cartilaginous rings. The posterior wall is composed of trachealis muscle (a smooth muscle), connective tissue (collagen, elastic f i bers, and fibroblasts), and nerves and blood vessels (not shown). studied in detail. A discussion of the literature and brief summary of some of the findings reported here have appeared in a recent review article (Sant’Ambrogio, 1982). MATERIALS AND METHODS Eight dogs provided by the Animal Care Center a t the University of Texas Medical Branch were anesthetized with a n intravenous injection of sodium pentobarbitone (30 mgkg) and continued administration of anesthetic by a catheter in the right femoral vein. Dissection and electrophysiological location of areas containing the terminals of slowly adapting stretch receptors were done as described by Mortola and Sant’Ambrogio (1979). After stretch receptors had been located, the dog was sacrificed by clamping the heart, and part of the extrathoracic trachea was removed immediately and placed in cold 3% glutaraldehyde and 0.05 M piperazineN,N’bis(2-ethane sulfonic acid) (PIPES buffer). The layer containing trachealis muscle was separated from the lamina propria and epi- thelium and was cut with scissors into pieces a few millimeters on a side. After fixation overnight in fresh fixative at 5”C, specimens were rinsed in 0.05 M PIPES, postfixed in 1%OsO4 in 0.05 M PIPES, dehydrated in ethanol series, and flat-embedded in a medium-soft Epon mixture. They were then cut into two or three pieces smaller in area, sectioned for light microscopy (3-pm sections), and stained with p-phenylenediamine and toluidine blue. Selected sections were remounted and sectioned for electron microscopy (silver sections) as described by Krauhs and Salinas (1980). Thick and thin sections were cut tangential to the trachea wall instead of being cut as cross sections, and all pieces were thick-sectioned through the entire thickness of trachealis muscle. Thin sections were stained with uranyl acetate and lead citrate. Three fibers (from two dogs) and their terminal regions were chosen for three-dimensional reconstruction, and a photomicrograph of each thick section of these was taken. The profiles of each axon and its terminals were outlined on each photomicrograph, and edges of the surrounding smooth muscle cells were outlined. Some of the thick sections were resectioned for electron microscopy so that such features as myelination could be checked. The profiles of nerve and muscles were traced onto tracing paper, and muscle cell outlines were traced onto acetate sheets 0.03 in thick to prepare a scale model of each specimen. The paper and plastic sheets were aligned by using muscle cells and other relatively constant features of the sections as fiducials. The plastic was cut out between muscle cells so that pipe cleaners representing the nerve could be placed between them. After a rough pipe-cleaner model had been made by using the paper tracings, the plastic sheets were stacked from the luminal side outward, and the nerve model was fit into them to correlate with the paper tracings. Tape was used to hold the pipe cleaners and plastic sheets in place. A medical artist, Lee Rose, used the plastic and pipe cleaner model and the paper tracings to portray the three-dimensional structure of the nerve. RESULTS At the light microscope level, sections were examined for myelinated fibers approaching or infiltrating the trachealis muscle. Such fibers were indeed found (Fig. 2), often in association with dark-staining, irregular profiles which proved, in the electron micro- MORPHOLOGY OF SLOWLY ADAPTING TRACHEAL RECEPTORS 75 Fig. 2. Photograph taken through a light microscope with a x 10 objective lens. At least one myelinated nerve fiber (open arrow) accompanies a capillary (C) into the connective tissue between strands of smooth muscle (MI. Nerve endings can be seen clearly (closed arrows) even at this low magnification. The arrowhead indicates the nerve ending profile shown at higher magnification in Figure 6. Bar = 20 bm. x700. scope, to be afferent nerve terminals. The myelinated fibers, which were about 4 to 5 pm in diameter, accompanied capillaries into the connective tissue between smooth muscle cells. Upon examination in the electron microscope (Fig. 3), the myelinated fibers were found to be accompanied by one or more bundles of unmyelinated fibers of various diameters (about 0.25 to 0.65 pm). The whole complex of fibers and capillary pursued a tortuous course through the connective tissue. At the point illustrated in Figure 3, the myelinated fiber contained numerous microtubules but few mitochondria. More distally, however (Fig. 4),mitochondria constituted a much larger proportion of the cytoplasmic volume. The myelinated fiber became more irregular in shape, finally lost its myelin (Fig. 5), and sometimes began to branch. Distal to the loss of myelin it is called a “premyelinated” axon, according to the terminology of Rees (1967). The cytoplasm was filled with mitochondria, microtubules, vesicles of var- ious shapes and sizes (generally larger than synaptic vesicles), membranous whorls, apparently solid osmiophilic bodies, and deposits of glycogen granules (Fig. 6). Some very large profiles (up to 10 pm in diameter) containing mitochondria, osmiophilic bodies, and glycogen were seen as well as smaller profiles containing mostly mitochondria. The terminal profile illustrated in Figure 6 had two processes which contained no large organelles. Such processes were not very frequently observed, however. Although most of the surface of the sensory ending was covered by Schwann cell, in some places, including parts of the processes, the membrane of sensory terminal was exposed directly to the extracellular space. There was usually a narrow gap, then a layer of basal lamina which might be attached to other components (particularly collagen) of the connective tissue. In one section (Fig. 7) a n apparent afferent terminal was in direct contact with a process of smooth muscle cell. Although there were some vesicles in the terminal, the contact point did not appear to be Fig. 3. Electron micrograph of a myelinated fiber found to give rise to afferent terminals near trachealis muscle. The fiber is accompanied by a bundle of unmy- elinated fibers W). There are few organelles other than microtubules in the cytoplasm of the myelinated fiber. Bar = 2 pm. x 10,400. MORPHOLOGY OF SLOWLY ADAPTING TRACHEAL RECEPTORS 77 Fig. 4. Electron micrograph of a different receptor, at a point closer to a terminal region. The cytoplasm is becoming filled with mitochondria, and some osmiophilic granules (arrowheads) can be seen. Bar = 1pm. ~13,500. a classical synapse. Except for this one case, terminals were always separated from muscle by connective tissue. The unmyelinated fibers, which did not seem to branch, also appeared to have a type of terminal region which may be more like a “varicosity” than a terminal or synapse. Such regions contained many clear, round vesicles, a few dark-cored vesicles, mitochondria, and small glycogen deposits, but no whorls or osmiophilic bodies (Fig. 8a,b). Patches of their membranes were bare of Schwann cells also; such areas were not necessarily adjacent to concentrations of vesicles. The unmyelinated fibers appeared to travel more distally (toward the trachea lu- 78 J.M. KRAUHS Fig. 5. Loss of myelin in a receptor from a different dog. The slender nerve profile has myelin at only one end. It appears to give off a small recurrent branch (asterisk). Bar = 2 pm. x 10,400. men) than the terminals of the myelinated fibers, but they have not yet been followed far enough to determine whether or not they go to the ventral border of the trachealis muscle. Reconstruction of three myelinated fibers (some of which may have actually been pairs of myelinated fibers) indicates that the terminals have a highly complex structure and that this structure may vary considerably (Fig. 9). The first drawing (Fig. 9a) depicts two nerve ending regions (at left and bottom of drawing) derived from one or more myelinated fibers. There were several small myelinated branches for which terminal regions were not found. The exact number of myelinated fibers could not be determined; the tangled configuration indicates that more than one fiber is present, but both terminal regions may have originated from the same fiber. The terminal regions contained loops, and their surfaces were actually more irregular than depicted in the drawing. The second receptor (Fig. 9b) appeared to consist of a single myelinated fiber for some distance. It eventually branched, with one branch turning backward before forming a n extensive terminal region with its main axis parallel to the muscle and the other branch forming a small terminal region near the branching point. A myelinated portion of the second branch appeared to cross over a myocyte along with the first branch (top of drawing), but whether or not it had any terminals in that region could not be determined. It appears possible that two myelinated fibers had a close association over part of their length and could not always be distinguished from each other; if only one fiber was involved, its structure must be quite complex and apparently unlike that of the other two receptors. The third receptor (Fig. 9c) was located at the edge of a muscular region, perhaps at a point at which trachealis muscle was attached to the connective tissue attached to the edge of a ring of cartilage. This receptor MORPHOLOGY OF SLOWLY ADAPTING TRACHEAL RECEPTORS Fig. 6. Nerve profile in a thin section cut from the thick section in Figure 2. Features typical of sensory receptors in general and mechanoreceptors in particular are seen: a large number of mitochondria (MI, dense or osmiophilic bodies (0) of various sizes, and accumulations of glycogen (G). In addition there are two processes was relatively simple in structure, but, as in the others, sensory terminals were highly contorted and included loops of various sizes. Several small terminal branches were derived from a major loop, proximal to which there was at least one small myelinated branch for which no terminal region was found. The terminal regions extended to the very edge of the muscle and even slightly beyond. Branches forming the terminal region were roughly perpendicular to the path of the myelinated fiber and direction of the muscle myofibrils. Sometimes profiles of the myelinated fiber terminals occurred in “rows” parallel to the smooth muscle cells (Fig. 10). In Figure 10, the two processes of one profile are roughly parallel to the muscle cell. Sometimes colla- 79 (PI which are at least partially uncovered by the thin Schwann cell sheath that invests most of the profile. At least one layer of basal lamina (BL) covers cell membranes that would otherwise be exposed directly to the extracellular matrix. Bar = 1 pm. X21,400. gen bundles could be seen to form a wavy pattern with terminal profiles in between. Such regularity was observed only rarely, however. DISCUSSION The nerve terminals described here are believed to be those of the slowly adapting receptors because they arise from myelinated fibers (Fillem and Widdicombe, 19721, their ultrastructure is typical of that of mechanoreceptors, and they are located entirely within the trachealis muscle layer (Bartlett et al., 1976).The fibers of the slowly adapting receptors are the only afferent fibers known to be distributed to the trachealis muscle. However, I have not succeeded in labeling terminals, as with horseradish peroxidase, 80 J.M. KRAUHS Fig. 7. A mitrochondria-filled profile in contact with a process of a smooth muscle cell (M). Bar = 1pm. ~ 2 3 , 3 0 0 . which were identified by electrophysiology as slowly adapting receptors. The terminals were not abundant; some pieces of muscle contained no recognizable afferent terminals. Ultrastructure of the axons and terminals was typical of mechanoreceptors (discussed by Krauhs, 1979). A few small, round vesicles were observed in these terminals, but not as many as in presumptive aortic baroreceptor terminals (Krauhs, 1979). Perhaps the greater amount of experimental manipulation of the tracheal receptors resulted in release of vesicles. A number of larger vesicles of various electron lucencies and shapes were found in the terminals, but their function, as well as that of the osmiophilic bodies, is unknown. Structure of these afferent terminals was quite different from that of the pulmonary stretch receptors in rat bronchi investigated by von Diiring et al. (1974), who did not describe large, mitochondria-filled profiles. In many places the Schwann cell covering of the sensory endings was incomplete. Although this phenomenon may be due in part to tissue shrinkage, it has been noted in other mechanoreceptors (Tranum-Jensen, 1975; Krauhs, 1979), and it is the more exposed portions of sensory terminals that are generally thought to be sites of transduction. The cell membrane was attached to basal lamina in the same manner as in the rat aortic baroreceptors (Krauhs, 19791, but in the trachea there was generally one layer of basal lamina around receptor profiles, a fact that increases the possibility that the large amount Fig. 8. Groups of unmyelinated fibers which were near the terminals of myelinated fibers. Many clear vesicles can be seen, as well as a few cored vesicles (arrowhead, b). In some places (arrowheads, a), the nerve membrane is directly exposed to extracellular connective tissue, and hemidesmosomes may be observed on Schwann cell membranes (arrows, a). A glycogen deposit (G) can be seen in b. Bars = 0.5 pm. a, X25,lOO; b, X30,i'OO. 82 J.M. KRAUHS b C Fig. 9. Drawings by Lee Rose of three different receptors (a,b,c) reconstructed from serial thick sections. Receptors are drawn as seen from the luminal side. The myelinated part of each fiber is darker than the unmyelinated part, and the proximal end of each myelinated fiber is on the right in each drawing. The outline of muscle cells at the cut surface on the luminal side is shown as a dark solid line. The lighter solid and dashed lines represent the cut surface on the side away from the lumen, and shading is used to show muscle tissue protruding into intracellular spaces between the surfaces. The double-headed arrows indicate the direction of muscle fibers and probable direction of stretch for the receptors. Fig. 10. Electron micrograph of a thin section cut from the thick section shown in Figure 2. Note the almost row-like arrangement of Schwann cell nuclei (N) and afferent terminal profiles (TI, one of which is seen a t higher magnification in Figure 6. Its processes are marked with arrows. C, collagen; M, muscle. Bar = 2 pm. X10,400. 84 J.M. KRAUHS of basal lamina around the aortic baroreceptors is a result of the large amount of stress on the aorta. However, hemidesmosomes were often seen on tracheal Schwann cells where they were exposed to extracellular connective tissue (Fig. 8a), basal lamina being the structure to which they were attached. Hemidesmosomes are thought to aid in attaching cells to the substrate, particularly in tissues, such as epithelium, which are exposed to unusually great mechanical stress (Fawcett, 1981). Only one receptor profile was seen in contact with a smooth muscle cell (Fig. 7). Hoyes and Barber (1980) observed some mitochondria-filled nerve profiles in intimate contact with muscle in guinea pig trachea; the frequency of this type of contact may be different for different species. The importance of such contact, especially when it seems infrequent, cannot now be determined. Myelinated fibers and their terminals were accompanied by a t least one bundle of unmyelinated fibers of varying diameter. These were quite similar to unmyelinated fibers associated with aortic baroreceptors in the rat (Krauhs, 1979). The unmyelinated fibers in rat aorta did not appear to continue even for the whole length of the terminals of the myelinated fiber, but those in the dog trachea continued distally beyond the myelinated fiber terminations. They were not followed to their ends, but it appears possible that they continue into the lamina propria as do the presumed pulmonary stretch receptors described by von Diiring et al. (1974). The membranes of unmyelinated fibers were directly attached to basal lamina in many places, and, although no synapses were observed, it is tempting to speculate that their vesicles may be released after a proper stimulus. The three-dimensional structure of the receptors (Fig. 9) was generally similar to those published for afferent terminals in canine (Elftman, 1943)and human (Larsell and Dow, 1933; Sampaolo and Sampaolo, 1958; Jabonero and Sabadell, 1972) airway smooth muscle. Three different receptors had considerably different branching patterns, but all contained apparent rings. Although no section that contained a complete circular structure was found, some Y and U shapes were observed. A single fiber could give rise to more than one terminal region, and some of the major branches were roughly perpendicular to the direction of muscle contraction. Some terminal regions extended over a rela- tively long distance parallel to the long axis of the myocytes. In general only the complex terminal regions were premyelinated; major branches remained myelinated until they branched further. In some cases, small myelinated branches did not appear to form terminal regions. No evidence was found for a simple morphological arrangement of receptors in series with smooth muscle. In addition to the gross directionality described in the preceding paragraph, in a few receptors small processes devoid of mitochondria (Fig. 6 ) were observed to emanate from large profiles in a direction parallel to the muscle fibers, but these were rare. The processes were reminiscent of those described by von Diiring et al. (1974) on lanceolate terminals and seemed to be in contact with collagen, basal lamina, and microfibrils. Collagen, which was plentiful around the receptors (Fig. lo), is likely to be a determinant of the viscoelastic properties of the trachealis muscle, upon which the adapting properties of tracheal receptors depend (Davenport et al., 1981). ACKNOWLEDGMENTS Expert technical assistance was provided by Norman Salinas and Rodney Nunley. I thank Karen Rex and Dr. Franca Sant’Ambrogio for dissection of the dogs and physiological location of receptor areas and Dr. Giuseppe Sant’Ambrogio for critical reading of the manuscript. Supported by grant HL29697 from the National Institutes of Health. LITERATURE CITED Bartlett, D., Jr., P. Jeffery, G. Sant’Ambrogio, and J.C.M. Wise (1976) Location of stretch receptors in the trachea and bronchi of the dog. J . Physiol. (Lond.), 258 409-420. Coleridge, H.M., J.C.G. Coleridge, and J.C. Luck (1965) Pulmonary afferent fibres of small diameter stimulated by capsaicin and by hyperinflation of the lungs. J. Physiol. (Lond.), I79:248-262. Davenport, P.W., F.B. Sant’Ambrogio, and G. Sant’Ambrogio (1981) Adaptation of tracheal stretch receptors. Respir. Physiol., 44:339-349. Elftman, A.G. (1943) The afferent and parasympathetic innervation of the lungs and trachea of the dog. Am. J. Anat., 721-21. Fawcett, D.W. (1981) The Cell, 2nd ed. W.B. Saunders Co., Philadelphia, pp. 156-158. Fillenz, M., and J.C. Widdicombe (1972)Receptors of the lungs and airways. In: Handbook of Sensory Physiology, Vol. IIII1. Enteroceptors. E. Neil, ed. SpringerVerlag, Berlin, pp. 81-112. Hoyes, A.D., and P. Barber (1980) Innervation of the trachealis muscle in the guinea-pig: A quantitative ultrastructural study. J. Anat., 130:789-800. Jabonero, V., and J. Sabadell (1972) Die sensible Innervation der glatten Muskulatur der Luftwege. Z. Mikrosk. Anat. Forsch., 86:213-243. MORPHOLOGY OF SLOWLY ADAPTING TRACHEAL RECEPTORS Krauhs, J.M. (1979)Structure of rat aortic baroreceptors and their relationship to connective tissue. J. Neurocytol., 8401-414. Krauhs, J.M., and N.L. Salinas (1980) Ultrastructural study of unencapsulated vertebrate mechanoreceptor terminals facilitated by double staining and resectioning of thick plastic sections. J. Neurosci. Methods, 3:175-182. Larsell, O., and R.S. Dow (1933) The innervation of the human lung. Am. J. Anat., 52:125-146. Mortola, J.P., and G. Sant’Ambrogio (1979)Mechanics of the trachea and behavior of its slowly adapting stretch receptors. J. Physiol. Gond.), 286577490, Rees, P.M. (1967)Observations on the fine structure and distribution of presumptive baroreceptor nerves at the carotid sinus. J. Comp. Neurol., 131:517-548. Sampaolo, G., and C.L. Sampaolo (1958) Aspetti morfologici delle espansioni sensitive in rapport0 con i fas- 85 cetti rnuscolari della trachea dell’uomo. Boll. SOC.Ital. Biol. Sper., 34:1219-1222. Sampson, S.R., and E.H. Vidruk (1975) Properties of “irritant” receptors in canine lung. Respir. Physiol., 25:9-22. Sant’Ambrogio, G. (1982) Information arising from the tracheobronchial tree of mammals. Physiol. Rev., 62531-567. Sant’Ambrogio, G., J.P. Moi-tola, and F.B. Sant’Ambrogio (1980) Response of tracheal slowly adapting stretch receptors t o longitudinal forces. Respir. Physiol., 41:323-332. Tranum-Jensen, J. (1975)The ultrastructure of the sensory end-organs (baroreceptors) in the atrial endocardium of young mini-pigs. J. Anat., 119255-275. von During, M., K.H. Andres, and J. Iravani (1974) The fine structure of the pulmonary stretch receptor in the rat. Z. Anat. Entwicklungsgesch., 143:215-222.