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Morphology of presumptive slowly adapting receptors in dog trachea.

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THE ANATOMICAL RECORD 210:73-85 (1984)
Morphology of Presumptive Slowly Adapting Receptors
in Dog Trachea
Department of Physiology and Biophysics, University of Texas Medical
Branch, Galveston, T X 77550
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
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.
Cross Section
of Posterior Wall
Lamina Propria
(Incl. Elastic Fibers)
Elastic Fiber
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
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).
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
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.
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-
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
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
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.
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-
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
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
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-
(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,
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,
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
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.
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
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.
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
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).
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.
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