Labeling of vagal motoneurons and central afferents after
injection of cholera toxin B into the airway
lumen
J. Julio
Pérez Fontán and
Christine R.
Velloff
The Edward Mallinckrodt Department of Pediatrics, Washington
University School of Medicine, St. Louis, Missouri 63110
 |
ABSTRACT |
We tested the hypothesis
that application of the subunit B of cholera toxin (CTB) to the airway
mucosa would produce labeling of neuronal somata and sensory fibers in
the medulla oblongata. Using 125I-CTB as a tracer, we
demonstrated first that CTB is transported across the tracheal
epithelium, but once in the airway wall, it remains confined to the
subepithelial space and lamina propria. Despite the rarity of
intrinsic neurons in these areas, intraluminal CTB labeled
10-60 neurons/rat in the nucleus ambiguus and a smaller number
of neurons in the dorsal motor nucleus of the vagus. Well-defined sensory fiber terminals were also labeled in the commissural, medial,
and ventrolateral subnuclei of the nucleus of the tractus solitarius.
Approximately 50 and 90% of the neurons labeled by intraluminal CTB
were also labeled by injections of FluoroGold into the tracheal
adventitia and lung parenchyma, respectively. These findings
demonstrate that a substantial number of medullary vagal motoneurons
innervate targets in the vicinity of the airway epithelium. These
neurons do not appear to be segregated anatomically from vagal
motoneurons that project to deeper layers of the airway wall or lung parenchyma.
parasympathetic system; airway ganglia; airway epithelium; retrograde neuronal markers
 |
INTRODUCTION |
THE AIRWAYS receive
the majority of their excitatory innervation through parasympathetic
nerve fibers, the bodies of which are located in the nucleus ambiguus
and dorsal motor nucleus of the vagus (12, 14, 15, 17,
28). These fibers are carried by the vagus nerves to the airway
walls where they are thought to synapse obligatorily with an intrinsic
network of airway ganglia (1). The function of the ganglia
is to modify and relay central parasympathetic inputs to airway smooth
muscle, glands, and blood vessels (8, 24).
The diversity of the functions carried out by these various tissues
would suggest that airway parasympathetic preganglionic neurons must be
segregated anatomically to reflect the location and physiological role
of their ultimate effectors. Early studies that used retrograde
neuronal tracers indicated that similar to the upper alimentary tract
(2, 5) (with which they share a common embryological
origin), the airways have a segmental representation in the
organization of the vagal preganglionic neurons (17). Unfortunately, tracer methodologies cannot be applied easily to elucidate whether different tissue structures within an airway segment
have a specific representation centrally. The main difficulty resides
not only with localizing the tracer injections accurately in the
desired tissue but also with preventing the injected material from
diffusing to adjacent areas (11). As a result, the only viable experimental approach to the question of tissue-specific innervation has been to measure physiological airway responses to
exogenous stimulation of specific groups of medullary neurons. Using
such a technique, Haselton et al. (13) demonstrated that unlike the nucleus ambiguus, activation of neurons in the dorsal motor
nucleus of the vagus with homocysteic acid does not increase total lung
resistance in dogs. This observation has led to the speculation
(15) that neurons projecting to the airways from the
dorsal medulla innervate only airway mucus glands and blood vessels.
In the present study, we tested the hypothesis that transepithelial
transport of the subunit B of cholera toxin (CTB) deposited into the
tracheal lumen would label a distinctive population of preganglionic
parasympathetic neurons and sensory fibers in the rat medulla. This
hypothesis was based on the recent demonstration that CTB, a widely
used retrograde and anterograde neuronal tracer (10, 22),
undergoes transcytotic transport from the apical to the basolateral
membrane of polarized intestinal epithelial cells (21). We
performed three sequential experiments. First, we used radiolabeled CTB
to confirm that a transport mechanism similar to that described for the
intestine exists in the airway epithelium where CTB is translocated
from the airway lumen to the subepithelial space and lamina propria.
With such information in hand, we then analyzed the topographic
distribution of the medullary neurons and sensory fibers labeled by
intraluminal CTB. Finally, we examined the question of whether the
neuronal population labeled with intraluminal CTB has innervation
targets outside the airway mucosa. This was accomplished by counting
the number of neurons double labeled by injections of CTB into the
tracheal lumen and FluoroGold into the dorsal wall of the trachea or
the lung parenchyma.
 |
METHODS |
All experiments were performed in male Sprague-Dawley rats
(weight, 350-500 g; age, 10-16 wk; Charles River
Laboratories, Wilmington, MA) following protocols approved by the
Washington University (St. Louis, MO) Animal Studies Committee. The
rats were kept at 23°C with access to standard rat feed and water.
Transepithelial transport of radiolabeled CTB in the airways.
We performed two series of experiments with 125I-CTB to
determine whether, as we hypothesized, CTB is transported across the airway epithelium and if so, to define its subsequent distribution in
the airway wall. For both series, 125I-CTB was prepared as
described previously (19) with 2 mCi of Na125I
(New England Nuclear, Boston, MA) and 2 IODO-BEADS (Pierce Chemical, Rockford, IL) in a final volume of 200 µl with a concentration of
0.08% and a specific activity of 256 counts · min
1 · fM
1.
The first series of experiments included six rats and was designed to
characterize the uptake of 125I-CTB by the tracheal
epithelium. A tracheostomy was performed near the thoracic inlet under
pentobarbital sodium anesthesia (50 mg/kg ip). The distal trachea was
cannulated, and positive-pressure ventilation was begun. A 10- to
30-µl volume of 125I-CTB suspension was instilled in the
proximal tracheal segment. After periods of 3, 5, or 7 h (2 rats/period), the rats were killed with an overdose of pentobarbital
sodium. The tracheae were then removed, placed in buffered 4%
paraformaldehyde fixative for 24 h, and embedded in paraffin. The
paraffin blocks were cut transverse to the tracheal axis into 10-µm
sections that were deparaffinized and mounted on glass slides. Finally,
the slides were coated with photographic emulsion (NTB2, Eastman Kodak,
Rochester, NY) and processed for autoradiographic analysis. The time
elapsed from the experiment to the performance of the autoradiographs
was 7 days.
The second series of experiments included eight rats and was aimed at
examining the intrapulmonary distribution of 125I-CTB
injected into the tracheal lumen. The rats were anesthetized with
halothane (1-4%), and the trachea was cannulated through the
mouth with a 14-gauge intravenous catheter that was advanced to the
midtrachea. A 30-µl volume of 125I-CTB suspension was
then injected into the tracheal lumen. The catheter was finally
removed, allowing the rat to breathe spontaneously while it recovered
from anesthesia. The injected rats were kept in a radioactive
containment hood for 3 h (2 rats), 24 h (3 rats), 4 days (1 rat), or 7 days (2 rats). At the end of this period, each rat was
killed with an overdose of pentobarbital sodium. The thoracic organs
were exposed through an extended median sternotomy. The pulmonary
vasculature was then perfused through a cannula inserted into the
pulmonary artery with 0.1 M sodium phosphate-buffered saline followed
by 4% buffered paraformaldehyde. The trachea and lungs, esophagus, and
duodenum were removed and stored in 4% paraformaldehyde overnight. Two
0.2- to 0.4-cm-thick coronal sections were cut, one from the apex and
the other from the base of each lung, and mounted on glass slides.
Coronal 0.1-mm sections of the extrathoracic and intrathoracic trachea
and esophagus and intact portions of the duodenum and ileum were
mounted similarly. The slides were covered with plastic wrap and
covered with photographic film to obtain contact autoradiographs.
Samples from the same tissues were embedded in paraffin and processed
for microscopic autoradiographic analysis as described above.
Intratracheal instillation of CTB.
These experiments sought to characterize the topography of neurons and
fibers labeled in the medulla by intraluminal CTB. We devised three
different methods for the introduction of CTB into the tracheal lumen.
Each method contained specific modifications to minimize neuronal
labeling via pathways other than transepithelial transport.
In a first group of rats (n = 6), a 200-µl volume of
0.1% CTB suspension (List Biological Laboratories, Campbell, CA) was injected through an endotracheal cannula made from a 6-cm section of
PE-190 tubing. The cannula was inserted through the mouth under halothane (0.5-4%) anesthesia. After the CTB injection, the lungs were ventilated with a rodent ventilator (Harvard Apparatus, South Natick, MA) for 5-10 min through the same endotracheal cannula used for the injections. The cannula was removed on initiation of
spontaneous breathing after discontinuation of mechanical ventilation.
In a second group (n = 10 rats), the anterior surface
of the trachea was exposed through a midline incision under
pentobarbital sodium anesthesia. A 33-gauge needle (Hamilton, Reno, NV)
was then inserted through one of the intercartilaginous spaces. After a
small volume of air was aspirated to ascertain that the needle tip was
not in contact with the tracheal wall, a 10-µl volume of 0.1% CTB
suspension was injected into the tracheal lumen. The objective of this
transmural technique was to avoid any potential disruption of the
tracheal mucosa by the endotracheal cannula. Although the needle
puncture interrupted continuity of the epithelial lining, we judged
that this interruption was too small to result in substantial contact
of deep tracheal tissues with the injectate.
Finally, in a third group of rats (n = 10), a 50-µl
volume of 0.1% CTB was instilled directly into the tracheal lumen
through a tracheostomy. The tracheostomy made it possible to occlude
the larynx for a period of 24 h after the instillation, thereby
preventing entry of CTB into the gastrointestinal tract. The rats were
anesthetized with pentobarbital sodium. After the neck was shaved, a
rhomboidal section of skin was removed to prevent obstruction of the
tracheostomy stoma by redundant neck tissue. A midline incision was
made through the subjacent muscle and fascia to expose the trachea. The
first intercartilaginous space was incised transversely, leaving the dorsal wall of the trachea intact. The adventitia was then sewn at the
edges of the tracheal stoma to the subcuticular layer of the skin with
interrupted suture on a noncutting needle, carefully avoiding
perforation of the tracheal mucosa. The stitches were positioned to
create a small ridge of free skin, which, on tightening the suture
line, became apposed to the edge of the tracheal stoma, protecting it
from contact with tracheal secretions. The larynx was packed with
cotton through the stoma before the CTB suspension was deposited inside
the tracheal lumen with a small Silastic tube. After a period of
24 h, which we deemed sufficient for the disappearance of CTB from
the lumen based on autoradiographic determinations with
125I-CTB, the suture was cut and the laryngeal packaging
was removed. The tracheostomy was sutured, this time placing the
stitches through the tracheal cartilage to prevent dehiscence.
Double-labeling experiments.
The objective of these experiments was to determine whether the
parasympathetic preganglionic neurons labeled by intraluminal CTB have
concurrent innervation targets outside the airway mucosa. We used two
labeling strategies.
In the first (n = 11 rats), we combined instillation of
CTB into the tracheal lumen with injections of FluoroGold
(Fluorochrome, Denver, CO) into the adventitial membrane that covers
the dorsal wall of the trachea. Both the longitudinal trunk and
superficial muscular neuronal plexi, which comprise the majority of the
intrinsic neurons of the trachea, are embedded in this membrane
(3, 9, 33). The bodies and, presumably, the short
dendrites of the neurons from these plexi, however, are separated from
the tracheal mucosa by several tissue barriers including the trachealis
muscle. The injections were performed under a dissecting microscope
while the rats were anesthetized with halothane piped into the
inspiratory limb of the ventilator. CTB (100 µl of a 0.1%
suspension) was injected through an endotracheal cannula as described
in Intratracheal instillation of CTB. FluoroGold
(0.5 µl of a 4% solution) was injected with a glass micropipette
mounted on a micromanipulator (Stoelting, Wood Dale, IL). For this
purpose, we first lifted the tracheal adventitia immediately anterior
to the tracheoesophageal angle with the tip of the pipette. We then
advanced the pipette for a short distance in an oblique dorsal
direction that kept the tip of the pipette oriented away from the
tracheal lumen. The injectate was divided into two to five injections
distributed over the length of the extrathoracic trachea. After each
injection, the site was blotted with a cotton-tipped probe; at the
completion of the injections, the bed of the incision was rinsed with
warm saline before the muscle and skin layers were closed with
continuous sutures.
In the second strategy (n = 6 rats), we combined
intratracheal injections of CTB with injections of FluoroGold into the
lung parenchyma. In previous studies (12, 17, 27, 28), we
and others have shown that retrograde neuronal markers injected into the lung tissue label numerous vagal motoneurons in the nucleus ambiguus and dorsal motor nucleus of the vagus. This finding is surprising because the lung periphery of mature mammals is considered to be devoid of parasympathetic ganglia (31) and therefore
offers no apparent innervation targets for preganglionic neurons. The experiment described here sought to establish whether the same neurons
can be labeled by parenchymal injections of FluoroGold and by
intraluminal CTB (which we also found confined to an area lacking
intrinsic neurons). CTB (100 µl of a 0.1% suspension) was injected
through an endotracheal cannula as described in Intratracheal instillation of CTB. FluoroGold (2 µl of a 4% solution) was
injected with a micropipette into the apical lobe of the right lung,
which was exposed through a thoracotomy of the fourth intercostal space performed with the animal under halothane anesthesia. Five to ten injections were performed to a 1- to 2-mm depth below the visceral
pleura, and lung volume was maintained constant to avoid tearing the
lung tissue. After each injection, the pleural surface was blotted with
a cotton-tipped probe; at the completion of all injections, the lung
surface was washed thoroughly with warm normal saline and allowed to
dry for 3-5 min before the thoracotomy was closed by layers. A
small Silastic tube was left in the pleural space to evacuate residual
air until the rat resumed spontaneous breathing. To rule out the
possibility that FluoroGold entering the lung air spaces during the
injections could undergo subsequent absorption, we examined the brain
stems of six additional rats injected with 10 µl of 15% FluoroGold
solution into the tracheal lumen. These injections were carried out
through an endotracheal cannula with the rats under halothane
anesthesia in a fashion similar to that described for CTB.
Identification of labeled neurons and sensory fibers in the
brain.
On recovery from anesthesia, the rats were returned to the holding
facility where they remained for a period of 11-13 days. At the
end of this period, each rat was anesthetized with pentobarbital sodium. The heart and large vessels were exposed, and heparin (1,000 U/kg) was injected directly into the right ventricle. A cannula was
then inserted into the aortic root, and the systemic circulation was
perfused with 0.1 M sodium phosphate-buffered saline followed by 4%
buffered paraformaldehyde (pH 7.40), allowing the effluent to exit
through a nick in the right atrium. Finally, the brain and spinal cord
were removed, placed in 4% paraformaldehyde for 2 days, and stored in
buffered 30% sucrose until sectioned.
The brain stem and cervical spinal cord were cut into transverse
50-µm sections on a freezing microtome. A 1-in-5 series of these
sections (amounting to 16 sections/rat medulla) was placed for 30 min
at room temperature in a blocking solution of 5% donkey serum (Sigma)
in 0.3% Triton X-100 buffered with 0.02 M potassium phosphate. The
section series was then incubated overnight at room temperature with
primary antisera against CTB (1:10,000 dilution, goat-raised; List
Biological Laboratories), FluoroGold (1:500 dilution, rabbit-raised;
Chemicon International, Temecula, CA), or both (double-labeling
studies). A few representative medullary sections were incubated with
CTB antiserum and a guinea pig-raised antiserum that recognizes
residues 24-130 of the rat,
-preprotachykinin [1:500;
courtesy of J. E. Krause Neurogen, Branford, CT
(23)] to identify CTB-labeled sensory fibers that
contained tachykinins (C fibers). After they were rinsed with buffered
saline solution, the sections were placed for 3-4 h in either a
1:100 dilution of biotinylated anti-IgG antiserum (single-labeling
studies) or 1:50 dilutions of fluorescent-labeled [FITC and
tetramethylrhodamine isothiocyanate (TRITC)] anti-IgG antisera
(double-labeling studies) at room temperature (all raised in donkey;
Jackson ImmunoResearch Laboratories, West Grove, PA). After thorough
washing with buffered saline, the sections exposed to biotinylated
antisera were incubated for 1 h with an avidin-horseradish
peroxidase conjugate (VECTASTAIN ABC kit, Vector Laboratories,
Burlingame, CA) and stained with 0.05% diaminobenzidine
tetrahydrochloride in potassium phosphate-buffered saline containing
0.003% hydrogen peroxide. Staining was intensified by sequential
treatment with silver nitrate for 1 h at 56°C, 0.2% gold
chloride for 15 min at room temperature, and 5% sodium thiosulfate for
5 min, also at room temperature. The tissue was then counterstained with 0.6% thionin in 0.2 M acetic acid buffer. The sections exposed to
fluorescent antisera were simply washed with buffered saline. All the
processed sections were mounted on gelatinized glass slides, covered
with a buffered glycerol solution (with 0.1%
p-phenylenediamine if appropriate to reduce fading during
fluorescent viewing), and protected with glass coverslips.
Localization of intrinsic neurons in the rat trachea.
The topographic distribution of the intrinsic neurons of the trachea
has been analyzed qualitatively and quantitatively in a variety of
species including rats (3, 4, 6, 7, 9, 33). The
descriptions resulting from these analyses, however, have varied widely
depending on the methodology used to identify the neurons and whether
the investigators concentrated their attention only on cholinergic
parasympathetic ganglia associated with the nerve trunks. Accordingly,
to facilitate the interpretation of our findings, we obtained
transverse paraffin-embedded sections of the trachea and esophagus and
the lungs of six rats of age and weight analogous to those of the rats
used in the preceding experiments. After deparaffinization, the
sections were incubated with a rabbit-raised antiserum against rat
neurofilament M (Chemicon International) followed by a FITC-labeled
donkey anti-rabbit IgG antibody to stain neurons and nerve fibers.
Data analysis.
Brain and cervical spinal cord sections treated with biotinylated and
fluorescence-labeled secondary antisera were examined with bright-field
and fluorescence microscopy, respectively. Labeled neuronal somata and
nerve fibers were annotated on a computerized chart of the rat brain
(25), which, after some modification, was also used to
illustrate the position of the neurons in Figs. 4, 7, 9, and
10. For analysis purposes, labeled neurons were counted in
medullary sections corresponding to ~0.5-mm intervals, starting 15 mm
below the bregma. Some of the figures, however, contain images
selected from intermediate sections for their clarity. The total
neuronal counts in the nucleus ambiguus and dorsal motor nucleus of the
vagus were analyzed for differences between methods of CTB injection
with a Kruskal-Wallis single-factor analysis of variance by ranks.
Numerical variables are presented as means ± SD or as medians and
10th and 90th percentiles, depending on whether the individual values
followed a normal distribution. All the microphotographs shown in the
illustrations were scanned digitally into Adobe Photoshop (Adobe
Systems, San Jose, CA) for display and labeling. Whole image color and
contrast were adjusted during the scanning process to reproduce the
conditions viewed under the microscope.
 |
RESULTS |
Transepithelial transport of radiolabeled CTB in the
trachea.
The first series of experiments that used radiolabeled CTB examined the
distribution of 125I-CTB in the wall of a tracheal segment
bound proximally by the larynx and distally by a tracheostomy
immediately proximal to the thoracic inlet. Autoradiographic analysis
of tracheal sections confirmed that CTB is indeed transported across
the tracheal epithelium (Fig. 1).
The radioactive tracer was detected both in the epithelial cells lining the tracheal lumen and in the subepithelial space and
lamina propria at all the time points selected for tracheal fixation
(3-7 h after the instillation). In the lamina propria, the tracer
was concentrated in cells with the morphological characteristics of
macrophages. Unless bound to these cells, radioactivity was absent from
the tracheal lumen, even in the rats killed 3 h after instillation
of CTB. We found no radioactive tracer in the deeper layers of the
submucosa, trachealis muscle, or tracheal adventitia. Small amounts of
radioactivity were present in the lumen of the esophagus, especially in
its cervical section, where it probably accumulated as a result of
diminished swallowing activity during the prolonged anesthesia.
125I-CTB was not incorporated into the esophageal mucosa.

View larger version (133K):
[in this window]
[in a new window]
|
Fig. 1.
Autoradiographic demonstration of transepithelial
transport of cholera toxin B (CTB) in rat tracheae. A:
bright-field examination demonstrates accumulation of exposed
photographic granules, denoting the presence of radioactive tracer in
the epithelium and subepithelial space 3 h after instillation of
125I-CTB into the tracheal lumen. B and
C: dark-field (top) and bright-field
(bottom) images obtained 3 (B) and 7 (C) h after instillation of 125I-CTB into the
tracheal lumen show that CTB remained confined to the subepithelial
space and lamina propria. There was no radioactive signal in deeper
layers of the tracheal wall or in the lumen. In the lamina propria, the
tracer was concentrated in cells with morphological characteristics of
macrophages (inset). C, tracheal cartilage; TM, trachealis
muscle. D: dark-field (left) and bright-field
(right) autoradiographs illustrate the presence of
radiolabeled CTB in the lumen (EL) but not in the walls of the
midthoracic esophagus 7 h after instillation of
125I-CTB into the tracheal lumen. Arrowheads, luminal
surface of the esophageal mucosa. Bars, 25 µm.
|
|
Distribution of radiolabeled CTB in the airways and
lungs.
The second series of experiments studied the localization of
radioactivity in the lungs after injection of a bolus of
125I-CTB suspension into the trachea. In this instance, the
radiolabeled protein was allowed to progress distally into the airways
not only to obtain information about its dispersion in the lungs but also to determine whether the transepithelial absorption observed in
the trachea occurred in smaller airways. Contact autoradiographs obtained at various times (3 h to 7 days) after the injection demonstrated accumulations of the radiolabeled tracer in the tracheal and bronchial epithelia and in the lumen of both conducting airways and
distal air spaces. Radioactivity was distributed in a patchy pattern
throughout the lung parenchyma (Fig. 2),
each patch corresponding to the area subtended by an individual
bronchus. At the times studied, the esophagus, duodenum, and ileum had
no detectable radioactive residue.

View larger version (79K):
[in this window]
[in a new window]
|
Fig. 2.
Contact autoradiographic image of the lungs,
trachea, esophagus, and small intestine obtained from 8 rats at the
indicated times after injection of 125I-CTB into the
tracheal lumen. d, Day. For each rat, the tissue samples include a 1-cm
longitudinal segment of the midthoracic esophagus (e), coronal sections
from the cervical (top) and thoracic (bottom)
portions of the trachea (arrows) and esophagus, coronal sections of the
2 lung apexes (top) and bases (bottom), and
longitudinal sections of the duodenum and terminal ileum. Radioactive
signal was detected only in the trachea and lungs in this 3-h exposure.
The signal was strongest in the lungs where it adopted a patchy pattern
caused by accumulation of the tracer in distal air spaces subtended by
a bronchus (see Fig. 3). Some of the lung sections contained no
radioactivity, probably as a result of the nonhomogeneous distribution
of the 125I-CTB in the lungs. Even after longer exposures,
we found no signs of radioactive tracer in the small intestine. The
intense radioactive signal seen in the section of the cervical trachea
at 4 days of survival corresponds to a small ring of thyroid tissue. No
signal was detected in other sections of the trachea in this rat at the
same exposure.
|
|
Microscopic analysis demonstrated the concentration of
125I-CTB in the epithelium and subepithelial space of the
intrapulmonary airways from main stem bronchi to small bronchioles
(Fig. 3). Radioactive tracer was
present in these locations at all the time points included in the
experiment, from 3 h to 7 days after the injections. The signal
intensity, however, decreased noticeably with time. We found no signs
of radiolabeled CTB in the bronchial adventitia or in the peribronchial
or perivascular tissue. Unlike the epithelium of the conducting
airways, the epithelial cells lining the peripheral air spaces had no
obvious uptake of radioactivity. The radiolabeled protein appeared to
form deposits on the alveolar surfaces of the epithelium. Although we
cannot exclude that CTB was transported into the interstitial space of
the acinus, the persistence of CTB alveolar deposits 7 days after the
injections contrasted sharply with the absence of radioactivity in the
airway lumina, suggesting that the radiolabeled marker was not absorbed by alveolar cells and thus could not be removed from the air spaces by
blood or lymphatic vessels.

View larger version (165K):
[in this window]
[in a new window]
|
Fig. 3.
Autoradiographic microscopic images of the lung
parenchyma and intrapulmonary bronchi after injection of
125I-CTB into the tracheal lumen. A and
B: dark-field demonstration of patches of radioactivity in
the distal air spaces 3 h (A) and 7 days (B)
after the injections. The patches correspond to the areas subtended by
subsegmental bronchi. Some of these can be identified by the presence
of radioactive signal in the bronchial epithelium. C and
D: dark-field microphotographs revealing accumulation of
radioactive tracer in epithelial cells and in the subepithelial spaces
of both large (3 h postinjection; C) and small (1 day
postinjection; D) bronchi. E: bright-field image
of the wall of a lobar bronchus 3 h after the injection showing
that CTB was confined to the epithelium. F: bright-field
image of the distal air spaces 1 day after the injection, demonstrating
that the radiolabeled protein remained attached to the alveolar surface
of the epithelium but did not appear to undergo substantial transport
into the interstitial space. Bars, 50 µm.
|
|
Neuronal soma and sensory fiber labeling by intratracheal
CTB.
Injection of CTB into the tracheal lumen produced extensive labeling of
both neuronal somata and sensory fibers in the medulla. Labeled neurons
were found in all 6 rats injected through the endotracheal cannula, in
5 of 10 rats injected through the tracheal wall, and in all 6 rats that
survived after being injected through a tracheostomy.
Neuronal somata were labeled bilaterally in the areas of both the
nucleus ambiguus and dorsal motor nucleus of the vagus. The neurons
labeled in the nucleus ambiguus were typically multipolar and were
either concentrated in the compact formation or distributed more
loosely in the external formation, sometimes straying in close
proximity to the anterior medullary surface (Fig.
4). Overall, CTB-labeled neurons formed a
3- to 4-mm column in the ventral medulla, extending from the area often
referred to as the nucleus retroambigualis to the caudal end of the
facial nucleus. The density of the column increased notably
between 13 and 11.5 mm below the bregma (Fig.
5). The neurons labeled in the dorsal
nucleus of the vagus were more sparse (Fig.
6) and more homogeneously distributed along the caudal-cranial axis than those labeled in the nucleus ambiguus. The method used to inject CTB into the tracheal lumen had no
effect on the distribution (Fig. 5) or number (Table
1) of labeled neurons, even though the
doses of CTB varied substantially with each method.

View larger version (113K):
[in this window]
[in a new window]
|
Fig. 4.
Bilateral labeling of nucleus ambiguus neurons
(bregma 12.3 mm) by CTB (0.1%, 200 µl) injected into the tracheal
lumen. Labeled neurons were present both in the compact and external
formations of the nucleus. CTB labeling is demonstrated with
diaminobenzidine tetrachloride staining. NA, nucleus ambiguus; LPGi,
lateral paragigantocellular nucleus. [Modified from Paxinos and Watson
(25).]
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Distribution of parasympathetic preganglionic neurons
labeled by injecting CTB into the tracheal lumen via three different
approaches: through an endotracheal cannula (A), with a
needle placed through the tracheal wall (B), and directly
into the distal trachea through a sublaryngeal tracheostomy after
occluding the laryngeal lumen to prevent CTB entry into the
gastrointestinal tract (C). Labeled neuronal soma counts in
the left (LNA) and right (RNA) nucleus ambiguus and left (LDMV) and
right (RDMV) dorsal motor nucleus of the vagus are plotted at 0.5-mm
intervals from bregma (B) 15 mm to bregma 11.5 mm. Each symbol
represents an individual rat. There were no significant differences
(Kruskal-Wallis test) in the distribution or total number of labeled
neurons regardless of disparities in CTB dose or mode of administration
(see text).
|
|

View larger version (111K):
[in this window]
[in a new window]
|
Fig. 6.
Distribution of sensory fibers labeled by intraluminal
CTB in the area of the left nucleus of the tractus solitarius of a rat.
CTB labeling is demonstrated with diaminobenzidine tetrachloride
staining. Left, boxed areas on right at higher
magnification. Densest fiber labeling was in the commissural subnucleus
(com) in the caudal medulla and in the medial (med) and ventrolateral
(vl) subnuclei rostral to the obex. The 3 caudalmost sections also show
isolated neuronal somata labeled in the DMV. Cu, cuneate nucleus; Gr,
nucleus gracilis; XII, hypoglossal nucleus.
|
|
Sensory fibers were also labeled bilaterally by CTB.
Labeled fibers and fiber terminals formed distinctive fields in the
area of the nucleus of the tractus solitarius (Fig. 6), converging in
the commissural subnucleus caudally and in the medial and ventrolateral subnuclei more rostrally. Labeled sensory fibers became rare rostral to
13 mm below the bregma. Only a small proportion of the fibers labeled
by CTB were immunoreactive for
-preprotachykinin (Fig. 7), possibly denoting that CTB has a
greater affinity for thick than for thin fibers as proposed for its
horseradish peroxidase conjugate (30).

View larger version (108K):
[in this window]
[in a new window]
|
Fig. 7.
Relationship between the locations of sensory fibers labeled by
intraluminal CTB and a tachykinin-immunoreactive fiber field in the
dorsal medulla of a rat (bregma 13.75 mm). Top:
-preprotachykinin (a precursor of substance P)
immunoreactivity in the nucleus of the tractus solitarius and adjacent
areas of the dorsal medulla (solid rectangle on line drawing).
Immunoreactive fibers are stained red with a tetramethylrhodamine
isothiocyanate-conjugated (TRITC) antiserum.
Bottom: double exposure of area enclosed by open square in
top showing CTB-labeled (stained green by
FITC-conjugated antiserum) and
-preprotachykinin-immunoreactive nerve fibers (red, as in
top). Few double-stained fibers are seen, even in areas
where the 2 fiber fields overlap, suggesting that intraluminal CTB
predominantly stained thick myelinated fibers. AP, area postrema; CC,
central canal; med, medial subnucleus of the nucleus of the tractus
solitarius; NTS, nucleus of the tractus solitarius; vl, ventrolateral
subnucleus of the nucleus of the tractus solitarius. CC and dorsal
surface of medulla have been outlined for clarity. [Modified from
Paxinos and Watson (25).]
|
|
Double-labeling experiments.
Labeling of neuronal somata by CTB injected intraluminally and
FluoroGold injected into the tracheal adventitia coexisted in all of
the 11 rats that underwent the double injections. The topographic
distribution of the neurons labeled by the two markers was similar
(Fig. 8). The number of neurons labeled
by FluoroGold, however, was considerably larger (Table
2), especially in the area of the dorsal
motor nucleus of the vagus where labeled neuronal somata usually
exceeded 50. Double labeling, indicating uptake of the two markers by
the same neuron, was present at all levels of the medulla (Fig.
9). Approximately half the neurons
labeled by CTB in the nucleus ambiguus were also labeled by FluoroGold (median proportion in all rats = 50%, 10th percentile = 15%, 90th percentile = 73%).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
Distribution of parasympathetic preganglionic neurons labeled in
the RNA and LNA by CTB injected into the tracheal lumen and FluoroGold
(FG) injected into either the dorsal aspect of the tracheal adventitia
(A) or the apical lobe of the right lung (B).
Counts of single (CTB or FG) and double-labeled (CTB + FG) neurons
are plotted at 0.5-mm intervals from 14.5 to 11.5 mm caudal to the
bregma. Each symbol represents an individual rat. The large proportion
of double-labeled neurons suggests that the parasympathetic neurons
labeled by intraluminal CTB also have innervation targets in the
trachea and lung tissue.
|
|

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 9.
Double exposure demonstrating simultaneous labeling of NA
neurons (bregma 12.5 mm) by CTB instilled through a tracheostomy into
the tracheal lumen and FluoroGold injected into the dorsal aspect of
the tracheal adventitia. Neuronal somata labeled by CTB and FluoroGold
are colored in red and green, respectively, by TRITC- and
FITC-conjugated antisera. Double-labeled neurons appear yellow by the
superposition of the 2 colors (arrowheads). [Modified from Paxinos and
Watson (25).]
|
|
Neuronal soma labeling by both markers also coexisted in all six rats
injected with CTB into the tracheal lumen and FluoroGold into the
right apical lobe. In this case, however, double labeling was present
in the majority of the CTB-labeled neurons (90 ± 18% for nucleus
ambiguus neurons), indicating a surprising identity between the two
neuronal populations (Figs. 8 and 10).

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 10.
Double photographic exposures demonstrating
labeling of neuronal somata (bregma 13 mm) in the DMV (top)
and NA (bottom) after injections of CTB into the tracheal
lumen and FluoroGold into the apical lobe of the right lung. Neurons
labeled by CTB and FluoroGold are colored in red and green,
respectively, by TRITC- and FITC-conjugated antisera. Three neurons in
the compact formation of the NA are labeled by both retrograde markers
and thus appear yellow by the superposition of the 2 colors
(arrowhead). [Modified from Paxinos and Watson (25).]
|
|
FluoroGold produced no detectable labeling of sensory fibers. We also
found no neuronal somata labeled by FluoroGold in any of the six rats
injected with this marker in the tracheal lumen.
Localization of parasympathetic ganglia in the rat trachea.
In the trachea, the majority of the neurofilament M-immunoreactive
cells were located in the dorsal adventitia either in association with
the longitudinal nerve trunks that run between the trachea and the
esophagus or immediately superficial to the trachealis muscle (Fig.
11). A smaller number of neuronal
somata were found in the submucosa, also in the vicinity of the
trachealis muscle and, more rarely, in the lamina propria, coming in
close proximity to the tracheal epithelium. In the lung tissue,
neuronal somata were present only in the adventitial surfaces of large
bronchi and pulmonary vessels (Fig. 11).

View larger version (112K):
[in this window]
[in a new window]
|
Fig. 11.
Distribution of neurofilament M-immunoreactive cells
(shown by FITC-conjugated antisera) in the dorsal tracheal wall of the
trachea (top) and intrapulmonary bronchi. In the trachea,
the majority of these cells are located on the adventitial side of the
trachealis muscle (TM) in association with the longitudinal nerve
trunks (open arrow) or the trachealis muscle itself (solid arrow). A
few neurons and fibers are present in the submucosa (solid arrowhead).
Two neurons are located in the lamina propria (open arrowheads). In the
bronchi, neuronal somata are distributed throughout the adventitia of
large bronchi, separated from the bronchial lumen by the epithelium,
submucosa, and muscularis layers.
|
|
 |
DISCUSSION |
Our results demonstrate that CTB is transported from the luminal
to the basal surface of airway epithelial cells where it is taken up by
a substantial number of vagal sensory and motor fibers.
Immunohistochemical identification of the labeled motoneurons revealed
the existence of an unexpectedly dense innervation of the tracheal and
bronchial subepithelial space by medullary vagal motoneurons. (Our
observations suggest that it is less likely that CTB was absorbed by
alveolar cells, but we cannot exclude that some labeled neurons
projected to interstitial targets). Contrary to our stated hypothesis,
however, these motoneurons lack a distinctive topographic distribution
compared with motoneurons labeled by direct injection of retrograde
tracers into the tracheal wall or the lung parenchyma (12, 14,
15, 17, 27, 28). Moreover, many of the neurons labeled by CTB
via the transepithelial route appear to have concurrent innervation
targets in the tracheal adventitia and in the lung tissue. These
findings contradict the idea that vagal motoneurons are segregated
anatomically by the functions of their effector tissues in the airways.
They also raise the possibility that some medullary neurons innervate
airway targets without interposition of intrinsic neurons.
Transepithelial transport of CTB and labeling of
medullary neurons.
The pathway of absorption and subsequent disposition of CTB in the
trachea recapitulates much of what is known about the transport of
cholera toxin in the intestine. When placed in contact with the luminal
pole of intestinal cells, the two subunits of the toxin, A and B (CTB),
undergo vesicular transport initiated by the binding of CTB to a
ganglioside GM1 receptor on the cell surface (20, 21). The
process, which interestingly is three times more efficient for CTB
alone than for the two subunits combined (21), directs the
toxin to the basolateral surface of the cell where subunit A exerts its
catalytic action. In the intestinal wall, CTB binds to macrophage-like
cells analogous to the ones that we observed in the trachea, a property
that may facilitate the strong immunogenicity of the toxin
(29).
The idea that once it is transported across the epithelium, CTB remains
confined to the subepithelial space and lamina propria is crucial to
our interpretation of the neuron- and fiber-labeling patterns reported
here. This idea is substantiated by the absence of radioactivity in the
tracheal and bronchial submucosa, muscle, and adventitia after
intraluminal injections of 125I-CTB. Even when present in
the tracheal lamina propria, the radioactive tracer was associated with
macrophage-like cells, which may have carried it there from the
subepithelial space.
Selectivity of retrograde and anterograde neuron labeling.
In the classic conception of the airway parasympathetic system, vagal
motoneurons innervate tissue effectors only through synapses with
airway parasympathetic ganglia (1). However, ganglia are
rarely found in the lamina propria and are absent altogether from the
bronchial subepithelial space and the alveolar interstitium (3,
9, 33). How then could a retrograde marker such as CTB, which is
not transferred transsynaptically or taken up by intact fibers of
passage (22), have labeled such a large number of
medullary neurons from these locations?
To answer this question, we must first consider the possibility
that medullary vagal motoneurons were labeled by CTB at sites other
than the subepithelial space or lamina propria (nonselective labeling)
of the airway. CTB was injected into the airways at doses greater than
those used by other investigators in direct tissue injections. These
large doses, however, do not appear to have resulted in nonspecific
labeling of neurons as shown best by the absence of labeled neurons on
the side of the medulla ipsilateral to the cervical vagotomies. Deep
injury to the tracheal or laryngeal mucosa during endotracheal
cannulation may have placed luminal CTB in potential contact with
preganglionic fibers innervating the deeper neuronal plexi. We believe
that such an event is unlikely, however, because the intensity of
neuron and sensory fiber labeling was unchanged when CTB was injected
through the tracheal wall with a needle or was placed directly into the
tracheal lumen through a tracheostomy. Furthermore, when we injected
FluoroGold intraluminally after cannulating the trachea with a similar
technique, we found no labeled neurons in the medulla. Swallowing of
CTB transported to the pharynx by the retrograde clearance mechanisms
of the trachea may have also placed the neuronal marker within the
reach of gastrointestinal parasympathetic neurons. Radiolabeled CTB was
certainly not transported into the walls of the pharynx or esophagus,
which are the only segments of the upper alimentary tract in which
motor innervation originates primarily from the nucleus ambiguus
(5). Although gastrointestinal absorption of CTB could
account for labeling of some neurons in the dorsal motor nucleus of the
vagus, extensive neuron labeling via the stomach or intestine seems
unlikely. Laryngeal occlusion did not alter the topographic location or
quantity of the neurons labeled by CTB injected through a tracheostomy.
Moreover, the pattern of sensory fiber labeling did not coincide with
that reported after injections of anterograde neuronal markers into upper gastrointestinal organs. Consistent with the findings of other
studies of the sensory innervation of the trachea and lungs (15,
18), the highest density of labeled sensory terminals was in the
commissural subnucleus of the nucleus of the solitary tract below the
obex and in the medial and ventrolateral subnuclei above the obex. In
contrast, Altschuler et al. (2) reported sensory fiber
labeling of the central and gelatinous subnuclei after injections of
anterograde tracers into the esophagus and stomach of the rat.
Innervation targets of neurons labeled retrogradely by
CTB.
If we exclude nonselective labeling, we are left with only two
plausible explanations for our findings. The first is that CTB labeled
a population of medullary vagal motoneurons that provide innervation to
the few intrinsic neurons found in the tracheal lamina propria (see
Fig. 11). The function of these neurons can only be presumed from their
location. In animals that, like the dog, have a well-developed system
of tracheal glands, intrinsic neurons located in this layer or in the
neighboring submucosa can be seen sending nerve fibers to the glandular
epithelium (33). It is therefore reasonable to speculate
that the lamina propria and submucosal neurons play a role in the
regulation of tracheal secretions. Even if these neurons are indeed the
target of the medullary neurons labeled by intraluminal CTB, it is
difficult to reconcile their rarity in the trachea (they are absent
altogether from the bronchial mucosa) with the abundance of CTB-labeled
neuronal somata in the medulla.
This disparity prompts a second explanation, this one based on the idea
that some medullary vagal motoneurons may provide direct innervation to
epithelial or vascular effector organs in the airway mucosa. Such a
proposal is not without precedent in other divisions of the
parasympathetic system. Specifically, both degeneration studies after
eyeball enucleation and retrograde tracer experiments indicate that the
ciliary muscle receives a substantial proportion of its
parasympathetic nerves directly from midbrain neurons, bypassing the
ciliary ganglion (16, 32). Although further experimental
evidence is needed, a similar arrangement for the vagal innervation of
the airways and lungs could also explain the extensive labeling of
medullary neurons by CTB, FluoroGold, and even pseudorabies virus
injected into the lung parenchyma (12, 27, 28) where
ganglion neurons are also rare.
Specificity of vagal innervation: double-labeling experiments.
Regardless of the ultimate identity of their innervation targets, many
of the individual medullary neurons labeled by intraluminal CTB were
also labeled by FluoroGold injections into the tracheal adventitia or
the lung parenchyma. This finding, which was highly consistent between
rats, denotes a surprising lack of specificity in the distribution of
parasympathetic inputs in different tissues within the airway wall.
The FluoroGold injections into the tracheal adventitia were designed to
reach the vicinity of the longitudinal nerve and superficial muscular
plexi, which contain the majority of the intrinsic neurons of the
mammalian trachea (3, 9, 26, 33). Because of the existence
of a natural cleavage plane between the trachea and the esophagus, the
small volume of injectate, and the precautions taken to orient the
pipette away from the tracheal lumen, we believe that the injected
FluoroGold remained separated from the tracheal submucosa or mucosa.
The extensive FluoroGold labeling of neuronal somata in the dorsal
motor nucleus of the vagus suggests that the spread of these injections
included endings from motor fibers innervating the esophagus, an
unavoidable consequence of the proximity of this organ to the trachea
(and also an additional proof that the injections were located on the
dorsal side of the tracheal adventitia).
The FluoroGold injections into the right lung were intended to label
what we believed to be a population of vagal motoneurons committed to
the innervation of intrapulmonary ganglia. This interpretation needs to
be revised, however, in light of the current results. Specifically, the
fact that most of the neurons labeled by intraluminal injections of CTB
were also labeled by FluoroGold has two potential implications to the
contrary. First, there is clearly a subset of neurons labeled by the
intraparenchymal injections, the endings of which are within the reach
of CTB molecules translocated across the airway epithelium. This is,
once again, unlikely to be the case in large bronchi where ganglia
reside (24, 31, 33) because in those locations, the bodies
of the ganglion neurons are separated from the mucosa and submucosa by
a considerable diffusion distance. In addition, the finding that
FluoroGold injections into one single lobe reached most of the
medullary neurons labeled transepithelially by CTB implies that many of
these neurons have multilobar projections and may therefore be involved
in the innervation of multiple airway segments.
Summary.
Our results demonstrate a novel use of CTB as a
transepithelial neuronal marker in the airways. The medullary
parasympathetic neurons and fibers identified through this route,
however, lack both distinctive topographic characteristics and target
specificity. These findings support the notion that it is the role of
the diverse populations of airway intrinsic neurons (9,
24) to impart functional selectivity to the parasympathetic
outflow carried by the vagus nerves to the airways.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Arthur D. Loewy for advice and technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-57998.
Address for reprint requests and other correspondence: J. J. Pérez Fontán, Dept. of Pediatrics, Washington Univ.
School of Medicine, St. Louis Children's Hospital, One Children's
Place, St. Louis, MO 63110 (E-mail:
fontan{at}kids.wustl.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 January 2000; accepted in final form 27 July 2000.
 |
REFERENCES |
1.
Akasu, T,
and
Nishimura T.
Synaptic transmission and function of parasympathetic ganglia.
Prog Neurobiol
45:
459-522,
1995[ISI][Medline].
2.
Altschuler, SM,
Bao X,
Bieger D,
Hopkins DA,
and
Miselis RR.
Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and the spinal trigeminal tracts.
J Comp Neurol
283:
248-268,
1989[ISI][Medline].
3.
Baker, DG,
McDonald DM,
Basbaum CB,
and
Mitchell RA.
The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry.
J Comp Neurol
246:
513-526,
1986[ISI][Medline].
4.
Baluk, P,
and
Gabella G.
Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves.
J Comp Neurol
285:
117-132,
1991.
5.
Bieger, D,
and
Hopkins DA.
Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus.
J Comp Neurol
262:
546-562,
1987[ISI][Medline].
6.
Chiang, CH,
and
Gabella G.
Quantitative study of the ganglion neurons in the mouse trachea.
Cell Tissue Res
246:
243-252,
1986[ISI][Medline].
7.
Chiang, CK.
Distribution of ganglion neurons in the trachea of the rat.
Acta Anat Nippon
68:
607-616,
1993.
8.
Coburn, RF.
Neural coordination of excitation of ferret trachealis muscle.
Am J Physiol Cell Physiol
246:
C459-C466,
1984[Abstract].
9.
Dey, RD,
Altemus JB,
Mayer RB,
Said SI,
and
Coburn RF.
Neurochemical characterization of intrinsic neurons in ferret tracheal plexus.
Am J Respir Cell Mol Biol
14:
207-216,
1996[Abstract].
10.
Ericson, H,
and
Blomqvist A.
Tracing of neuronal connections with cholera toxin subunit B: light and electron microscopic immunohistochemistry using monoclonal antibodies.
J Neurosci Methods
24:
225-235,
1988[ISI][Medline].
11.
Fox, EA,
and
Powley TL.
False-positive artifacts of tracer strategies distort autonomic connectivity maps.
Brain Res Rev
14:
53-77,
1989[ISI][Medline].
12.
Hadziefendic, S,
and
Haxhiu MA.
CNS innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus.
J Auton Nerv Syst
76:
135-145,
1999[ISI][Medline].
13.
Haselton, JR,
Solomon IC,
Motekaitis AM,
and
Kaufman MP.
Bronchomotor vagal preganglionic cell bodies in the dog: an anatomic and functional study.
J Appl Physiol
73:
1122-1129,
1992[Abstract/Free Full Text].
14.
Haxhiu, MA,
Jansen ASP,
Cherniack NS,
and
Loewy AD.
CNS innervation of airway-related parasympathetic preganglionic neurons: a transneuronal labeling study using pseudorabies virus.
Brain Res
618:
115-134,
1993[ISI][Medline].
15.
Haxhiu, MA,
and
Loewy AD.
Central connections of the motor and sensory vagal systems innervating the trachea.
J Auton Nerv Syst
57:
49-56,
1996[ISI][Medline].
16.
Jaeger, RJ,
and
Benevento LA.
A horseradish peroxidase study of the innervation of the internal structures of the eye.
Invest Ophtalmol Vis Sci
19:
575-583,
1980[Abstract].
17.
Kalia, M,
and
Mesulam MM.
Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches.
J Comp Neurol
193:
467-508,
1980[ISI][Medline].
18.
Kubin, L,
Kimura H,
and
Davies RO.
The medullary projections of afferent bronchopulmonary C fibers in the cat as shown by antidromic mapping.
J Physiol (Lond)
435:
207-228,
1991[Abstract].
19.
Lencer, WI,
Chu SHW,
and
Walker WA.
Differential binding kinetics of cholera toxin to intestinal microvillus membrane during development.
Infect Immun
1987:
3126-3130,
1987.
20.
Lencer, WI,
de Almeida JB,
Moe S,
Stow JL,
Ausiello DA,
and
Madara JL.
Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation.
J Clin Invest
92:
2941-2951,
1993[ISI][Medline].
21.
Lencer, WI,
Moe S,
Rufo PA,
and
Madara JL.
Transcytosis of cholera toxin subunits across model human intestinal epithelia.
Proc Natl Acad Sci USA
92:
10094-10098,
1995[Abstract].
22.
Luppi, PH,
Fort P,
and
Jouvet M.
Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons.
Brain Res
534:
209-224,
1990[ISI][Medline].
23.
MacDonald, MR,
Takeda J,
Rice CM,
and
Krause JE.
Multiple tachykinins are produced and secreted upon posttranslational processing of the three substance P precursor proteins,
-,
-, and
-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants.
J Biol Chem
264:
15578-15592,
1989[Abstract/Free Full Text].
24.
Myers, AC,
Undem BJ,
and
Weinreich D.
Electrophysiological properties of neurons in guinea pig bronchial parasympathetic ganglia.
Am J Physiol Lung Cell Mol Physiol
259:
L403-L409,
1990[Abstract/Free Full Text].
25.
Paxinos, G,
and
Watson C.
The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1997.
26.
Pérez Fontán, JJ,
Cortright DN,
Krause JE,
Velloff CR,
Karpitskyi VV,
Carver TW, Jr,
Shapiro SD,
and
Mora BN.
Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat.
Am J Physiol Lung Cell Mol Physiol
278:
L344-L355,
2000[Abstract/Free Full Text].
27.
Pérez Fontán, JJ,
Diec CT,
and
Velloff CR.
Bilateral distribution of vagal motor and sensory nerve fibers in the rat's lungs and airways.
Am J Physiol Regulatory Integrative Comp Physiol
279:
R713-R728,
2000[Abstract/Free Full Text].
28.
Pérez Fontán, JJ,
and
Velloff CR.
Neuroanatomical organization of the parasympathetic bronchomotor system in developing sheep.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R121-R133,
1997[Abstract/Free Full Text].
29.
Shimodori, S,
Nada O,
Sakamoto N,
Hirose R,
and
Kawana T.
The occurrence of macrophage-like cholera toxin uptake cells in the intestinal villi of suckling rats.
Pathology
28:
58-64,
1996[ISI][Medline].
30.
Valtschanoff, JG,
Phend KD,
Bernardi PS,
Weinberg RJ,
and
Rustioni A.
Amino acid immunocytochemistry of primary afferent terminals in the rat dorsal horn.
J Comp Neurol
346:
237-252,
1994[ISI][Medline].
31.
Weichselbaum, M,
Everett AW,
and
Sparrow MP.
Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2.
Am J Respir Cell Mol Biol
15:
703-710,
1996[Abstract].
32.
Westheimer, G,
and
Blair S.
The parasympathetic pathways to internal eye muscles.
Invest Ophtalmol
12:
193-197,
1973.
33.
Yamamoto, Y,
Ootsuka T,
Atoji Y,
and
Suzuki Y.
Morphological and quantitative study of the intrinsic nerve plexuses of the canine trachea as revealed by immunohistochemical staining of protein gene product 9.5.
Anat Rec
250:
438-447,
1998[ISI][Medline].
Am J Physiol Lung Cell Mol Physiol 280(1):L152-L164
1040-0605/01 $5.00
Copyright © 2001 the American Physiological Society