GASTROENTEROLOGY 2010;138:1091–1101
Sensory and Motor Innervation of the Crural Diaphragm by the
Vagus Nerves
RICHARD L. YOUNG,*,‡ AMANDA J. PAGE,*,‡,§ NICOLE J. COOPER,* CLAUDINE L. FRISBY,* and
L. ASHLEY BLACKSHAW*,‡,§
*Nerve-Gut Research Laboratory, Hanson Institute, Department of Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, South Australia, Australia;
‡
Discipline of Medicine, Faculty of Health Sciences and §Discipline of Physiology, School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide,
South Australia, Australia
BACKGROUND & AIMS: During gastroesophageal re-
flux, transient lower esophageal sphincter relaxation and
crural diaphragm (CD) inhibition occur concomitantly.
Modifying vagus nerve control of transient lower esoph-
ageal sphincter relaxation is a major focus of develop-
ment of therapeutics for gastroesophageal reflux disease,
but neural mechanisms that coordinate the CD are
poorly understood. METHODS: Nerve tracing and im-
munolabeling were used to assess innervation of the
diaphragm and lower esophageal sphincter in ferrets.
Mechanosensory responses of vagal afferents in the CD
and electromyography responses of the CD were recorded
in novel in vitro preparations and in vivo. RESULTS:
Retrograde tracing revealed a unique population of vagal
CD sensory neurons in nodose ganglia and CD motor
neurons in brainstem vagal nuclei. Anterograde tracing
revealed specialized vagal endings in the CD and phre-
noesophageal ligament—sites of vagal afferent mechano-
sensitivity recorded in vitro. Spontaneous electromyogra-
phy activity persisted in the CD following bilateral
phrenicotomy in vivo, while vagus nerve stimulation
evoked electromyography responses in the CD in vitro
and in vivo. CONCLUSIONS: We conclude that vagal
sensory and motor neurons functionally innervate
the CD and phrenoesophageal ligament. CD vagal
afferents show mechanosensitivity to distortion of
the gastroesophageal junction, while vagal motor
neurons innervate both CD and distal esophagus and
may represent a common substrate for motor control
of the reflux barrier.
Keywords: Crural Diaphragm; Vagus Nerve; Retrograde
Tracing; Anterograde Tracing; Electrophysiology.
W ith a prevalence of 10%–20% in the adult popula-
tion and up to 50% in infants, gastroesophageal
reflux disease is one of the most common diseases of the
upper gastrointestinal tract.4,5 The major barrier against
reflux is formed by the lower esophageal sphincter to-
gether with the partly superimposed crural diaphragm
(CD). Combined pH-metric and manometric studies have
documented that transient lower esophageal sphincter
relaxations (TLESR) account for most reflux episodes in
healthy subjects and gastroesophageal reflux disease pa-
tients (for review, see Mittal et al6). During TLESR, there
is simultaneous relaxation of the lower esophageal
sphincter (LES), profound inhibition of activity in the
CD, and esophageal shortening, permitting reflux of gas-
tric content into the esophagus. Reduction of TLESRs
may consequently be an effective means to prevent reflux
from occurring.7,8
TLESR is triggered via a vagal pathway involving acti-
vation of gastric vagal mechanoreceptors, which provide
input to nuclei in the medulla oblongata of the central
nervous system, which in turn coordinate episodic acti-
vation of vagal pathways to inhibitory enteric neurons
ALIMENTARY TRACT
supplying smooth muscle of the LES (for review, see
Blackshaw9). Inhibition of the CD that accompanies
BASIC–
TLESR has been thought, like diaphragmatic motor con-
trol in general, to be mediated via the phrenic nerves,
such that medullary nuclei provide inhibitory signals to
the phrenic motor nucleus in the spinal cord, which shut
down rhythmic output via ␣-motor neurons to the
CD—an independent descending pathway to that con-
trolling the LES. However, a number of studies have
questioned the sole involvement of the phrenic nerve in
reflex control of CD inhibition.10 –12 Although gastric
afferent fibers in the vagus nerves are important in trig-
gering TLESR, and LES vagal efferents are important in
smooth muscle relaxation, the role of the vagus in the
sensory and motor innervation of the CD and adjacent
structures is unknown. We hypothesized that, in order to
demonstrate such tight coordination with the LES, the
CD must receive direct motor control via the vagus nerve.
It was important to test this hypothesis in a species
that displays TLESR and gastroesophageal reflux, so we
chose the ferret.13,14 We took a multifaceted approach,
Abbreviations used in this paper: CD, crural diaphragm; ChAT, cho-
line acetyltransferase; CTB, cholera toxin subunit B; DMN, dorsal motor
nucleus; EMG, electromyography; HRP, horseradish peroxidase; LES,
lower esophageal sphincter; NG, nodose ganglia; PFA, paraformalde-
hyde; TLESR, transient lower esophageal sphincter relaxation; VAChT,
vesicular acetylcholine transporter; WGA, wheat germ agglutinin.
© 2010 by the AGA Institute
0016-5085/10/$36.00
doi:10.1053/j.gastro.2009.08.053
 1092 YOUNG ET AL
mapping the connections and neurochemistry of vagal
innervation of the reflux barrier, and investigating the
functional role of the vagus nerve in both sensory and
motor control in isolated preparations of the CD.
Methods
Animal Preparation
Experiments were performed on adult ferrets
(0.6 –1.4 kg) of either sex. All studies were performed in
accordance with the Australian code of practice for the
care and use of animals for scientific purposes and with
the approval of the Animal Ethics Committees of the
Institute of Medical and Veterinary Science (Adelaide,
Australia) and the University of Adelaide. Animals had
free access to water and a standard carnivore diet and
were fasted overnight prior to experimentation.
Restricted Volume Retrograde Tracing
Ferrets were anesthetized with isoflurane (2%–3%
in oxygen) and a midline laparotomy performed to access
the dorsal surface of the left and right diaphragm. In 26
animals, the viscera were retracted, the diaphragm gently
tensioned laterally, and 2 ␮L fluorescein isothiocyanate
conjugate of cholera toxin-B subunit (CTB-fluorescein
isothiocyanate; Sigma-Aldrich, Castle Hill, Australia) or
ALIMENTARY TRACT
Alexa Fluor 555 conjugate (CTB-AF555; Invitrogen, Glen
Waverley, Australia) injected beneath the peritoneal lin-
BASIC–
ing of the CD using a Hamilton microsyringe equipped
with a 34-gauge needle (total volume ⫽ 4 ␮L). The
injection sites were 5 mm bilateral and adjacent to the
esophageal hiatus and parallel to the orientation of the
left and right crus muscle fibers. In 14 of these ferrets,
dual-site tracing was performed by injecting 2 ␮L alter-
nate tracer into the peritoneal lining of the dorsolateral
costal diaphragm 10 –15 mm bilateral of midline (n ⫽ 6),
or into the dorsal and ventral subserosa of the distal
esophagus (n ⫽ 8). After injection, injection sites were
dried, the laparotomy incision closed with individual
sutures, and antibiotic (Terramycin, Pfizer, West Ryde,
Australia, 10 mg/kg) and analgesic (Metacam, Boehringer
Ingelheim, North Ryde, Australia, 2.5 mg/kg) adminis-
tered subcutaneously. All ferrets recovered well from sur-
gery and were routinely monitored. After 4 days, ferrets
were given a lethal dose of sodium pentobarbitone (180
mg kg⫺1 intraperitoneally), the stomach filled with 60 mL
4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered
saline, pH 7.4 at 4°C via an oral polyethylene tube, and
the ferret perfused transcardially with 500 mL heparin-
ized saline (1000 IU/L) followed by 1000 mL 4% PFA at
4°C. The nodose ganglia (NG), brainstem, and cervical
spinal cord with attached dorsal root ganglia were then
removed, fixed overnight at 4°C in PFA, then cryopro-
tected in 30% sucrose at 4°C for 24 – 48 hours. Frozen
transverse sections were then cut serially at 20 ␮m
through the rostrocaudal axis of the NG, brainstem, and
GASTROENTEROLOGY Vol. 138, No. 3
C5 and C6 spinal cord (and dorsal root ganglia). Control
experiments for tracer injection are detailed in Supple-
mentary Material.
Anterograde Tracing
Ferrets were anesthetized as noted, a midline in-
cision made, and the left NG exposed by blunt dissection.
A 30-gauge needle was used to pierce the ganglion sheath
and a glass capillary, drawn to a 25-␮m tip, was used to
deliver 5 ␮L wheat germ agglutinin conjugated to horse-
radish peroxidase (WGA-HRP; Sigma Aldrich) via pneu-
matic injection. After injection, injection sites were dried,
the incision closed, and antibiotic (Terramycin, 10 mg/
kg) and analgesic (Metacam, 2.5 mg/kg) administered
subcutaneously. All ferrets recovered well from surgery
and were routinely monitored. After 4 days, ferrets were
given a lethal dose of pentobarbitone and perfuse-fixed as
mentioned previously. The stomach and reflux barrier
were removed, the stomach was divided into ventral and
dorsal halves along the lesser and greater curvatures and
pinned flat. The submucosa and mucosa were removed
from the stomach, leaving the muscle layers and myen-
teric plexus intact, which were fixed in 4% PFA overnight
at 4°C. WGA-HRP was then subject to tyramide signal
amplification with a tyramide signal amplification-Biotin
Tyramide kit (Perkin-Elmer Life Sciences; Boston, MA)
followed by detection with streptavidin Alexa Fluor 546
conjugate (Invitrogen). Dorsal and ventral stomach spec-
imens were then whole-mounted on gelatin-coated slides,
dried overnight, and vagal afferent endings visualized by
fluorescence microscopy as positive control for antero-
grade tracing. The reflux barrier was prepared for section-
ing by reducing the CD surrounding the esophageal
hiatus to a 2-cm square and fixing the specimen over-
night at 4°C in 4% PFA with an angled support inserted
through the residual esophagus to preserve barrier anat-
omy. The specimen was then cryoprotected in 30% su-
crose at 4°C for 24 to 48 hours, then cryosectioned in
transverse (20 ␮m) through the dorsal-ventral axis. WGA-
HRP in vagal endings was visualized by epifluorescence
and confocal microscopy in alternate serial sections of
the reflux barrier— one series using the tyramide signal
amplification protocol described here, and the alternate
series following direct immunohistochemistry for WGA.
Immunohistochemistry, Visualization, and
Quantification
Immunoreactivity for choline acetyltransferase
(ChAT) and neuronal nitric oxide synthase were detected
in traced brainstem sections, while vesicular acetylcholine
transporter (VAChT) and WGA were detected in traced
sections of the reflux barrier. Details of assay conditions,
visualization, and quantification are detailed in Supple-
mentary Material.
March 2010
Isolated Ferret CD Vagal Afferent
Preparation
This preparation was based on our isolated gas-
troesophageal preparation, which has been described pre-
viously.15,16 Ferrets were given a lethal dose of sodium
pentobarbitone (180 mg kg⫺1, intraperitoneally), exsan-
guinated, and thoracic organs and diaphragm removed
together and placed in a modified Krebs solution com-
prising (in mM): 118.1 NaCl, 4.7 KCl, 25.1 NaHCO3, 1.3
NaH2PO4, 1.2 MgSO4.7H2O, 1.5 CaCl2, 1.0 citric acid, and
11.1 glucose, bubbled with 95% O2⫺5% CO2, pH 7.4 at
4°C. Thoracic organs were removed and the vagus nerve
cleared of connective tissue. The diaphragm with residual
esophagus was pinned out flat in one chamber of a
2-chamber organ bath, perfused with modified Krebs
solution at 34°C. The L-type calcium channel antagonist
nifedipine (1 ␮M) was then added to the superfusing
solution to suppress smooth muscle activity. The vagus
nerves were led into an adjacent chamber through a small
hole, bathed in mineral oil, and dissected into strands for
single-fiber electrophysiological recordings. The location
of receptive fields of vagal afferent endings within the
CD, phrenoesophageal ligament, and associated connec-
tive tissue was determined by mechanical stimulation
with a brush, then high-threshold mechanical responses
were classified using a blunt glass rod.17 Accurate quan-
tification of mechanical responses of CD vagal afferents
was measured by the focal movement of calibrated von
Frey hairs across the receptive field without applying
stretch; stretch was applied across receptive fields by the
use of calibrated weights and a pulley-cantilever system.17
Single neurons were discriminated offline using Spike2
(Cambridge Electronic Design, Cambridge, UK) based on
action potential shape, duration, and amplitude. Peri-
stimulus time histograms and instantaneous frequency
plots were generated from these neurons using Spike2
analysis. In some experiments, receptive fields were
marked with carbon particles and the surrounding tissue
removed, fixed overnight at 4°C in PFA, then immuno-
histochemistry performed by incubating with rabbit
antiserum against the general neural marker protein gene
product (PGP9.5), followed by detection with streptavi-
din Alexa Fluor 546 conjugate (Invitrogen). The specimen
was then whole-mounted on gelatin-coated slides, dried
overnight, and individual vagal afferent endings visual-
ized by fluorescence microscopy.
Isolated Ferret CD Vagal Efferent
Preparation
Tissue collection and preparation was identical
to that for vagal afferent preparation, except that the
phrenic nerves were preserved and also cleared of connec-
tive tissue. The diaphragm with residual esophagus was
pinned out in a single-chamber organ bath and perfused
with modified Krebs solution at 34°C. Vagus or phrenic
nerve trunks were stimulated individually via hook elec-
A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1093
trodes using a pulse generator and a constant-current
stimulus unit (JRAK, Melbourne, Australia); a 5-V elec-
trical stimulus of 0.5 Hz and 0.5-ms pulse width was used
to evoke electromyograms (EMG) in esophagus, CD, or
costal diaphragm. Stimulation of the phrenic nerve trunk
was used, as the CD branch was difficult to distinguish in
ferrets. EMG responses were recorded via fine bipolar
electrodes inserted into the CD parallel with the muscle
fibers, 1–2 mm adjacent the esophageal hiatus. Bipolar
intramuscular electrodes were also placed in the ventral
wall of the distal esophagus or in the lateral portion of
the costal diaphragm for validation of EMG responses. In
4 separate preparations, after EMG responses had been
determined, a ganglioplegic drug, hexamethonium (100
␮M), was added to the superfusing Krebs solution and
allowed to equilibrate for 20 minutes, after which EMG
responses in the CD were retested. Subsequently, a neu-
romuscular junction-blocking drug, pancuronium (5
␮M), was added to the superfusing solution, and EMG
responses retested after 20 minutes. This was performed
to establish whether EMG responses in the CD were
mediated through vagal postganglionic collateral inner-
vation, the neuromuscular junction, or by direct muscle
excitation. Time control experiments were also per-
formed in which there was no change in EMG responses
over a comparable duration. Details of sequential dissec-
ALIMENTARY TRACT
tion experiments are presented in Supplementary Mate-
rial.
BASIC–
In Vivo Ferret CD Experiments
The function of vagal innervation of the CD was
also assessed in anesthetized ferrets subject to bilateral
thoracic phrenicotomy and vagus nerve stimulation. Full
details of these experiments are presented in Supplemen-
tary Material.
Results
Vagal Innervation of the CD
To investigate the possibility of a vagal innerva-
tion of the CD, we observed labeling in vagal neurons
after tracer injections into the CD (Figure 1A). We found
unique labeling of 45 ⫾ 13 vagal afferent neurons per
NG that were distributed throughout the ganglion (Fig-
ure 1B). This contrasted with earlier studies tracing from
gastric muscle with larger volumes, in which 11.2%
(⬃2600) of vagal afferents were labeled per NG.18,19 In
brainstem, a total of 133 ⫾ 14 vagal motor neurons were
labeled bilaterally from the CD in each animal; these were
predominantly located rostral from the level of the obex
within lateral regions of the dorsal motor nucleus (DMN;
Figure 1E–1F). In contrast, tracer injected bilaterally into
the dorsolateral costal diaphragm did not label any vagal
neurons. As expected, tracer injection in the distal esoph-
agus labeled a population of 123 ⫾ 9 vagal afferent
neurons per NG with a general distribution, and 69 ⫾ 11
 1094 YOUNG ET AL GASTROENTEROLOGY Vol. 138, No. 3
ALIMENTARY TRACT
BASIC–

Figure 1. Retrograde tracing of vagal nerve pathways from the crural diaphragm (CD) and distal esophagus in ferrets. (A) Abdominal surface view
of the diaphragm showing injection sites of retrograde tracers cholera toxin subunit B (CTB)-AF555 (CD, red) and CTB-fluorescein isothiocyanate
(distal esophagus, green). (B) Transverse view of left nodose ganglia (NG) showing a vagal afferent traced from the CD with CTB-AF555 (white). (C)
Low magnification view of left NG showing afferents traced from the CD (CTB-AF555, red) and distal esophagus (CTB-fluorescein isothiocyanate,
green). (D) High-magnification view of left NG showing vagal afferents traced separately, and a rare afferent dual-traced from the CD (CTB-AF555,
red) and distal esophagus (CTB-fluorescein isothiocyanate, green). (E) Diagram of the ferret rostral brainstem adapted from Boissonade et al.20 (F)
Transverse view of the rostral brainstem depicted in (E, boxed) showing a vagal motor neuron in the dorsal motor nucleus (DMN) traced from the CD
by CTB-AF555 (white). (G) High-magnification view of the DMN showing vagal motor neurons traced separately and dual traced (yellow) from the CD
(CTB-AF555, red) and distal esophagus (CTB-fluorescein isothiocyanate, green); weakly filled dendrites are evident within the nucleus centralis
following distal esophagus tracing. Note: (B–D, and G) are intentionally imaged at lower contrast to identify nontraced neuron populations. Key for
image (I): sg, subnucleus gelatinosus; mn, medial subnucleus of the nucleus tractus solitarius (NTS); ncom, commissural subnucleus of NTS; DMN,
dorsal motor nucleus of the vagus; XII, hypoglossal nucleus; IC, nucleus intercalatus; cc, central canal; TS, solitary tract; d/dln, dorsal/dorsolateral
subnucleus; v/vln, ventral/ventrolateral subnucleus; nI, intermediate subnucleus; ni, interstitial subnucleus. Scale bar (B, D, G) ⫽ 50 ␮m, (C) ⫽ 500
␮m, (E, F) ⫽ 200 ␮m.

motor neurons in the DMN, predominantly rostral from
the level of the obex. Esophageal motor neurons in the
nucleus ambiguus were occasionally traced, as expected.
Vagal motor neurons traced from the distal esophagus
also showed weakly filled dendrites concentrated within
the nucleus centralis of the nucleus tractus solitarius, the
presumed terminal area of esophageal vagal afferents
(Figure 1G).20,21 This was not apparent in vagal motor
neurons traced from the CD. Vagal afferents were gener-
ally traced separately from CD or distal esophagus, al-
though 8 ⫾ 6 double-labeled neurons were identified per
NG (Figure 1C and 1D). In contrast, a large population of
vagal motor neurons were double-labeled from the distal
esophagus and CD (46 ⫾ 9 neurons, Figure 1G). Data on
spinal innervation of the CD and on control experiments
for tracer injection are presented in Supplementary Figure 1
and in Supplementary Material.
Because there was strong evidence for a vagal sensory
innervation of the CD, we sought to visualize the struc-
ture of their peripheral endings. This was achieved by
anterograde tracing from the left NG with WGA-HRP.
Strikingly, vagal fibers and endings were labeled within
the CD and phrenoesophageal ligament. In contrast, no
endings were labeled within the costal diaphragm. Vagal
endings within the CD were evident as discrete regions of
fine punctate labeling at high-power magnification, often
with clear evidence of the entry of a single parent axon
(Figure 2A). Vagal punctate endings were identified pre-
dominantly within connective tissue at the abdominal
surface of the CD, ipsilateral to the injected ganglion.
March 2010 A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1095

Figure 2. Anterograde tracing of vagal nerve pathways to the crural diaphragm (CD) and phrenoesophageal ligament in ferrets. (A) High magnifi-
cation extended focal image (EFI) of a vagal punctate ending filled by wheat germ agglutinin– horseradish peroxidase (WGA-HRP) (with parent axon)
located within connective tissue of the abdominal CD surface. (B) EFI image of vagal punctate endings in connective tissue of the abdominal CD
surface, close to muscle fascia. (C) High magnification view of a punctate ending associated with CD muscle [M]. (D) Vagal fibers immunopositive for
WGA in connective tissue of the abdominal CD surface. (E) Vagal laminar endings labeled by WGA-HRP in the phrenoesophageal ligament; these
were located adjacent the anchor point with the thoracic distal esophagus and bilateral to the esophageal hiatus. ESO, esophagus; PEL, phreno-
esophageal ligament (inset box depicts vagal ending shown in F). (F) 3-dimensional confocal projection view (Z ⫽ 40 ␮m, 1 ␮m slice) of a labeled vagal
laminar ending showing detailed tertiary structure and a parent axon entering at the apex. Scale bar (A, B, D) ⫽ 10 ␮m, (C) ⫽ 50 ␮m, (E) ⫽ 100 ␮m,
(F) ⫽ 25 ␮m.

ALIMENTARY TRACT
They were also found in lower numbers within the phre- (Figure 2D) and punctate endings in the CD; these data

noesophageal ligament at the esophageal hiatus and in
connective tissue at the thoracic surface of the CD. These
endings were also found clustered in fascia of the CD
muscle (Figure 2B), occasionally in direct contact with
individual muscle fibers (Figure 2C). Vagal punctate end-
ings showed a limited distribution in the CD, restricted
laterally to within 400 ␮m of the esophageal hiatus and
within the 5 mm dorsoventral span of the esophagus.
Labeling by WGA-HRP tracer was subsequently con-
firmed by the WGA antiserum, which labeled vagal fibers
BASIC–
confirm the specificity of the tracing method.
Following anterograde tracing, a second type of vagal
ending was identified only within the phrenoesophageal
ligament. Vagal laminar endings were labeled within the
phrenoesophageal ligament, oriented in the oral-aboral
axis adjacent to the tether point of the ligament with the
esophageal serosa (Figure 2E). These laminar endings
were frequently located in-series with each other and
bilateral to the esophageal hiatus and were absent from
lateral regions of the ligament near the insertion with CD

Figure 3. Vagal motor neurons that innervate the crural diaphragm (CD) express markers of excitatory neurotransmission. (A) High magnification
view of the dorsal motor nucleus showing vagal motor neurons traced from the CD (cholera toxin subunit B [CTB]-AF555, red) immunopositive for
choline acetyltransferase (ChAT) (green). (B) A CD vagal motor neuron (CTB-AF555, red) immunonegative for neuronal nitric oxide synthase (green).
Scale bar (A, B) ⫽ 50 ␮m.
 1096 YOUNG ET AL GASTROENTEROLOGY Vol. 138, No. 3
ALIMENTARY TRACT
BASIC–

Figure 4. Functional innervation of the crural diaphragm (CD) by vagal afferents. (A) Thoracic surface view of the diaphragm showing major
structures and mechanosensory sites recorded within the CD in the single vagal fibers. Individual vagal afferent fiber responses recorded to
high-threshold mechanical stimuli (blunt probing with rod, B) and low-threshold stimuli (10 mg von Frey hair, C) in the right crus muscle of the CD.
(D) A “multimodal” vagal afferent fiber located in the left crus-phrenoesophageal ligament that responded to von Frey hair stimuli (70 –1000 mg, D)
and to stretch applied across the receptive field. (E) High-magnification view of PGP9.5 immunoreactive tissue whole-mount corresponding to the
marked receptive field recorded in (D); a putative vagal laminar ending in connective tissue possessing distinct arborizing laminar structures and a
clear parent axon is evident. Scale bar (E) ⫽ 25 ␮m.

muscle. Confocal imaging of these endings revealed a
distinct tertiary structure, comprised of profusely ar-
borizing laminar structures similar to that described for
intraganglionic laminar endings;22 a distinct entry point
of a parent axon was visible at the apex of a number of
these endings (Figure 2F). Additional images of vagal
fibers and endings labeled within the CD and phrenoe-
sophageal ligament are provided in Supplementary Fig-
ure 2.
Neurochemistry of Vagal Innervation of
the CD
To identify the neurochemical phenotype of vagal
motor neurons that innervate the CD, we performed
immunolabeling in retrograde-labeled brainstem. We ob-
served that vagal motor neurons in the DMN were fre-
quently immunopositive for ChAT as expected, with the
majority showing heavy immunolabeling for ChAT in
cytoplasm and processes. In contrast, immunolabeling for
neuronal nitric oxide synthase was sparse in the DMN, with
only a few motor neurons labeled in rostrolateral regions.
Vagal motor neurons retrogradely traced from the CD
were exclusively ChAT immunopositive (Figure 3A) and
neuronal nitric oxide synthase immunonegative (Figure
3B). Immunoreactivity for VAChT was detected at CD
motor end-plates in anterograde-labeled sections of the
reflux barrier, as expected (Supplementary Figure 2E);
however, none of the identified vagal structures were
immunopositive for VAChT. These findings indicate
that vagal punctate endings in the CD and laminar
March 2010
Figure 5. Functional innervation of the crural diaphragm (CD) by vagal
motor neurons. (A) Electromyography (EMG) responses recorded in the
right crus muscle of the CD in response to stimulation of individual
phrenic and vagus nerve trunks. (B) Single EMG waveform correspond-
ing to responses in (A). (C) Frequency-dependent fatigue of EMG re-
sponses of right crus muscle to right vagus nerve trunk stimulation at
frequencies of 2.5–50 Hz.
endings in the phrenoesophageal ligament were of
vagal afferent origin, and that vagal motor endplates
were not filled from efferent fibers of passage during
NG injections.
Functional Innervation of the CD by Vagal
Afferent Neurons
To determine the function of vagal sensory inner-
vation of the CD, we tested the mechanosensitivity of
vagal afferent fibers in an in vitro diaphragm preparation.
We observed receptive fields of vagal mechanoreceptors
within the CD muscle (in association with muscle bun-
dles of the right crus), within the phrenoesophageal lig-
ament, and in connective tissue adjacent to the distal
esophagus and CD (Figure 4A). Receptive fields were
small (1–3 mm2) and distinct. Two types of vagal mech-
anoreceptor were identified in the CD; those that re-
sponded only to high-intensity blunt probing with a glass
rod (Figure 4B), corresponding to high-threshold mech-
anoreceptors, and those that responded to focal compres-
sion with von Frey hairs with forces as low as 10 mg,
corresponding to low-threshold mechanoreceptors (Fig-
ure 4C). These receptors did not respond to stretch up to
10 g, and were generally silent at rest.
Two types of vagal mechanoreceptors were identified
within the phrenoesophageal ligaments that were dis-
tinct from types in the CD. These responded either to
calibrated von Frey hairs, as “dynamic mechanorecep-
tors,” or responded both to calibrated von Frey hairs and
to stretch applied across the receptive field, as “multimo-
dal afferents” (Figure 4D). In comparison with our pre-
A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1097
vious in vitro recordings of gastroesophageal mech-
anoreceptors in ferrets, vagal afferents with receptive
fields in the CD or phrenoesophageal ligament were
rare and represented ⬍5% of all vagal afferents encoun-
tered in this preparation. To determine if mechanore-
ceptive sites in the phrenoesophageal ligament con-
tained anatomically distinct endings, we performed
immunolabeling for PGP9.5 in tissue specimens fol-
lowing electrophysiology. At marked mechanorecep-
tive sites in the phrenoesophageal ligament we ob-
served endings in connective tissue that possessed
distinct tertiary and arborizing laminar structures
(Figure 4E) that were identical to anterograde-identi-
fied vagal laminar endings.
Functional Innervation of the CD by Vagal
Motor Neurons
To determine the function of vagal motor inner-
vation of the CD, we tested the responses of CD muscle
to vagal stimulation in an in vitro diaphragm preparation
and in vivo. Electrical stimulation of either phrenic nerve
in vitro produced EMG responses within the dorsolateral
costal diaphragm and in crus muscles of the CD, as
expected (Figure 5A and 5B). EMG responses evoked by
phrenic nerve stimulation comprised compound action
potentials of amplitude in direct proportion to that of
ALIMENTARY TRACT
the stimulus, with ipsilateral predominance. Electrical
stimulation of either vagus nerve produced EMG re-
BASIC–
sponses within the distal esophageal striated muscle, as
expected, but also evoked EMG responses within crus
muscles of the CD (Figure 5A and 5B). EMG responses
evoked in the CD by stimulation of the dedicated phrenic
nerve input were 2.9-fold larger than following vagal
stimulation, as would be expected from the increased
density of motor neurons traced from the CD in the
phrenic motor nucleus than in the DMN (see Figure 1,
Supplementary Figure 1). Vagal evoked EMG in the CD
was free of artefact, showed frequency-dependent fatigue
(⬎10 Hz) and was therefore characteristic of a muscle
Figure 6. Direct innervation of crural diaphragm (CD) neuromuscular
junctions by vagal motor neurons. Electromyography (EMG) responses
recorded in right crus muscle in response to stimulation of the right
phrenic and vagus nerve trunks in the absence (left), or presence of the
autonomic ganglion blocker, hexamethonium (100 ␮M, middle) or neu-
romuscular junction blocker, pancuronium (5 ␮M, right). Drugs were
administered to superfusing Krebs; responses recorded were after a
30-minute equilibration period.
 1098 YOUNG ET AL GASTROENTEROLOGY Vol. 138, No. 3

Figure 7. Effects of bilateral phrenicotomy on electromyography (EMG) responses of the of crural diaphragm (CD) in vivo. (A) EMG responses
recorded in left crus muscle in anesthetized, ventilated ferrets prior to (left), and following bilateral phrenicotomy (middle, right). (A, right) Expanded
view of tonic EMG spikes present in CD (arrows) but absent from costal diaphragm (COSD) following phrenicotomy. Note: Cardiac electric
interference is evident in both COSD and CD recordings. (B) EMG responses of COSD and CD following bilateral phrenicotomy and 20-V 0.5-ms
pulse width vagal stimulation, showing short latency evoked EMG responses in the CD (filled arrowhead) and longer variable latency reflex EMG
responses in CD (open arrowhead).
ALIMENTARY TRACT

response (Figure 5C). These responses were not due to of the CD that is excitatory and tonically active in vivo.
current spread from the esophagus, as phrenic stimula- Our data suggest that vagal afferents may sense stimuli
BASIC–

tion did not produce EMG responses in the distal esoph-
agus, nor did vagus stimulation produce EMG responses
in dorsolateral costal diaphragm. Blockade of ganglionic
transmission with hexamethonium (100 ␮M) failed to
significantly alter vagal evoked EMG responses in the CD
in this preparation, whereas pancuronium (5 ␮M) inhib-
ited all evoked EMG responses in the CD by blockade at
phrenic and vagal motor endplates (Figure 6). In in vivo
experiments, bilateral phrenicotomy in anesthetized and
ventilated ferrets led to a complete loss of EMG activity
in the costal diaphragm, while spontaneous EMG activity
continued in the CD (Figure 7A). Subsequent electrical
stimulation of the intact vagus trunks evoked short-latency
EMG spikes in the CD, and increased EMG frequency after
a longer and more variable latency (Figure 7B). Full details
of these experiments are provided in Supplementary Mate-
rial.
Discussion
Our major findings of this study are that the
vagus nerve provides a functional sensory and motor
innervation of the CD. We have characterized high- and
low-threshold vagal mechanoreceptors in CD muscle and
dynamic and multimodal mechanoreceptors within the
phrenoesophageal ligament. Our anatomical studies con-
firm that specialized vagal afferent endings exist in these
locations. Furthermore, we have characterized the anat-
omy and function of a direct vagal motor innervation
specific to the phrenoesophageal ligament and CD. We
have also established that vagal motor neurons provide
innervation of the LES and CD, and may represent
common targets of a central pattern generator for
TLESR and gastroesophageal reflux. A summary dia-
gram depicting this functional neurocircuitry associ-
ated with motor control of the LES and CD is shown
in Figure 8.
Vagal Afferent Innervation of the CD
Results of this study provide the first direct ana-
tomical and functional evidence that a small population
of vagal afferents directly and uniquely innervates the CD
in ferrets. Vagal afferent cell bodies were not labeled after
retrograde tracing from the costal diaphragm, nor were
vagal endings anterogradely traced from the NG to the
costal diaphragm, indicating that the CD is unique in
receiving a dual sensory innervation from the phrenic
and vagus nerves. Vagal afferents in the NG were occa-
sionally double-labeled following tracing from the LES
and CD, raising the intriguing possibility that single
vagal afferents may receive convergent sensory input
from the LES and CD, as is the case with those innervat-
ing other regions of the stomach.23
Vagal afferent laminar endings identified within the
phrenoesophageal ligament in this study possessed a
tertiary structure comparable with gastroesophageal mech-
anoreceptive intraganglionic laminar endings. Afferents
March 2010
Figure 8. Neurocircuitry of motor control of the lower esophageal
sphincter (LES) and crural diaphragm (CD). A central pattern generator
for transient lower esophageal sphincter relaxation (TLESR) coordinates
premotor input to LES- and CD-projecting vagal motor neurons in dor-
sal motor nucleus (DMN), and to CD-projecting phrenic motor neurons
in spinal cord. During a TLESR, this pattern generator provides premo-
tor excitation to vagal motor neurons that excite postganglionic nonad-
renergic, noncholinergic enteric neurons within the LES. Simultaneously,
premotor inhibition is provided to three tonically active circuits: (i) to vagal
motor neurons that excite postganglionic cholinergic enteric neurons
within the LES, (ii) to vagal motor neurons that directly excite motor
endplates within the CD, and (iii) to phrenic motor neurons via a yet to be
defined pathway, possibly involving the DMN.37 In this manner, dual and
synchronous output from this central pattern generator will relax the
LES and inhibit the CD, creating a common cavity that favors reflux.
Note: this simplified diagram does not show bilateral innervation of the
LES or CD, or innervation of the costal diaphragm.
recorded from this location in vitro also responded to
calibrated von Frey hairs and to stretch, similar to esoph-
ageal tension-mucosal receptors reported in this spe-
cies.15 These laminar endings may therefore sense axial
distension of the LES or stretch in the phrenoesophageal
ligament. In addition, the extraluminal location of vagal
laminar and punctate endings within the reflux barrier
and their relevant mechanical sensitivity suggest they
respond to perturbed barrier anatomy or to changing
posture— conditions known to strongly influence the fre-
quency of TLESR.24 –26 Future experiments involving se-
lective dennervation of these CD vagal afferents will re-
veal whether they serve as additional triggers proposed
for TLESR.27
Vagal Motor Innervation of the CD
Results of this study show that vagal motor neu-
rons provide a direct innervation to the CD, but not the
A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1099
costal diaphragm in ferrets, a finding recently corrobo-
rated by another group.28 Therefore, the CD is equipped
with innervation for both respiratory and anti-reflux bar-
rier control, consistent with evidence of a dual functional
role.29 –31 A population of nitrergic vagal motor neurons
that directly innervate the LES in ferrets has been de-
scribed previously within the rostrocaudal DMN.21 We
confirmed this population of nitrergic motor neurons,
but showed that CD vagal motor neurons were exclu-
sively cholinergic and therefore excitatory, expressing
ChAT centrally and VAChT at peripheral endings. Stud-
ies in the isolated diaphragm preparation confirmed that
vagal motor innervation to the CD was functionally ex-
citatory, and that EMG responses in crus muscles oc-
curred, as expected, at a classical neuromuscular junction
because responses to vagal stimulation were abolished by
pancuronium. A similar innervation of striated muscle by
vagal parasympathetic nerves has been described previ-
ously, in esophageal striated muscle of cats32 and pigs.33
Our finding that vagal motor innervation of the CD was
tonically active and independent of cardiac or respiratory
modulation in vivo indicates separate pathways of central
control of the CD, as proposed by earlier work.10 In
addition, vagal reflex activation of EMG responses in the
CD in vivo provides strong evidence of a bilateral and
multisynaptic central pathway that converges on DMN
ALIMENTARY TRACT
neurons that innervate the CD.
Because of the significant contribution of cholinergic
BASIC–
tone to basal LES pressure34 and tonic activity of CD
vagal motor neurons revealed in the current study, a
TLESR event is likely to involve dual central inhibition of
excitatory vagal inputs to the LES and CD. This inhibi-
tion must occur simultaneously with inhibition of
phrenic motor drive to the CD and with activation of
inhibitory vagal inputs to the LES. Indeed, LES and CD
projecting vagal motor neurons may represent an impor-
tant point of convergence of a central pattern generator
for gastroesophageal reflux. Although our data clearly
show different mechanisms of excitatory input to the
CD, it is important to note that our experiments do not
address the central mechanism resulting in inhibition of
this input during TLESR. This is an important subject
for further study, which must now include both central
vagal and phrenic pathways as candidates.
In addition to the pathways we have described in this
study, there is evidence for a peripheral mechanism
of reflex inhibition of the CD. Liu and colleagues12,35
showed that distension of the distal esophagus in cats
triggered relaxation of the esophagogastric junction via a
pathway that was unaffected by bilateral phrenicotomy
or vagotomy. It is important to note, however, that pat-
terns of CD inhibition differ in duration and degree of
inhibition during TLESR compared to those following
esophageal distension.36 Our evidence of a functional
sensory and motor vagal innervation of the CD provides
strong support for a central vagal pathway that may exert
 1100 YOUNG ET AL
dual control of the LES and CD during TLESR. The
current data, however, does not refute the existence of a
peripheral mechanism for CD inhibition in response to
distension of the distal esophagus; indeed both mecha-
nisms may operate at the CD under different conditions.
In conclusion, these data provide important new clues
to the role of the vagus and CD in the control of the
antireflux barrier. Results here show that the CD and
phrenoesophageal ligament are uniquely innervated by
vagal afferent and motor neurons and that specialized
vagal endings within these tissues correlate with sensory
and motor responses recorded within the CD. It is likely,
therefore, that a central pattern generator for TLESR acts
within the DMN to differentially control vagal motor
neurons innervating the LES and to inhibit vagal motor
neurons innervating the CD, together with central inhi-
bition of phrenic motor neurons. The identity of molec-
ular targets specific to vagal innervation of the CD may
provide new possibilities for the pharmacotherapy of
gastroesophageal reflux disease. In addition, these data
provide important new information on the consequences
of surgical manipulation of the gastroesophageal junc-
tion and should help to refine and direct surgical strat-
egies.
ALIMENTARY TRACT
Supplementary Material
Note: To access the supplementary material
BASIC–
accompanying this article, visit the online version of
Gastroenterology at www.gastrojournal.org, and at doi:
10.1053/j.gastro.2009.08.053.
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A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1101
Received December 16, 2008. Accepted August 19, 2009.
Reprint requests
Address requests for reprints to: Richard L. Young, PhD,
Nerve-Gut Research Laboratory, Hanson Institute, Frome Road,
Adelaide, South Australia 5000, Australia. e-mail:
richard.young@adelaide.edu.au
Acknowledgments
Early accounts of this work were presented at the Digestive
Disease Week meetings of the American Gastroenterological
Association in Los Angeles, CA (2006),1 Washington, DC (2007),2
and San Diego, CA (2008).3
Conflicts of interest
The authors disclose no conflicts.
Funding
This work was supported by a grant from AstraZeneca. L. Ashley
Blackshaw was supported by a National Health and Medical
Research Council (Australia) Senior Research Fellowship.
AstraZeneca was not involved in the study design or collection,
analysis, or interpretation of data.
ALIMENTARY TRACT
BASIC–
1101.e1 YOUNG ET AL
Supplementary Materials and Methods
Control Experiments for Tracer Injection
Control experiments were also performed to test
for tracer spread in dual-injected animals and to assess
tracer uptake within the intraperitoneal space. Two sep-
arate ferrets were injected with CTB-AF555 (CD) and
CTB-fluorescein isothiocyanate (distal esophagus) and
perfused at 2 hours following tracing. Transverse sections
of the reflux barrier were then prepared to directly assess
tracer distribution. In an additional 2 ferrets, 4 ␮L CTB-
AF555 was injected into the intraperitoneal space adja-
cent to the diaphragm to test for tracer leakage. Survival
times and tissue processing were the same as those fol-
lowing diaphragm tracing.
Immunohistochemistry
Immunoreactivity for ChAT and neuronal nitric
oxide synthase (nNOS) were detected in traced brainstem
sections, while VAChT and WGA were detected in traced
sections of the reflux barrier. ChAT, nNOS, and VAChT
were detected using rabbit antisera from a number of
suppliers; WGA was detected using a goat antiserum
from Vector Laboratories (Burlingame, CA) (Supplemen-
tary Table 1). ChAT, nNOS, and VAChT primary anti-
bodies were visualized using a goat secondary antibody
conjugated to Alexa Fluor 488 (AF488 green; Invitrogen),
while WGA was visualized using Alexa Fluor 546 (AF546
red; Invitrogen). Sections were air dried at room temper-
ature for 10 minutes and rinsed twice in phosphate-
buffered saline ⫹ 0.2% Triton X-100 (PBS-T, pH 7.4;
Sigma-Aldrich) to facilitate antibody penetration. Fol-
lowing 2-hour incubation at 37°C with primary antisera
(1:200 in 10% PBS-T) unbound antibody was removed
with PBS-T washes and slides incubated for 1 hour at
room temperature with secondary antisera (1:200 in PBS-
T). Slides were given final PBS-T washes, drained, and
mounted with ProLong Antifade (Invitrogen). The spec-
ificities of these primary antisera for ChAT,1 nNOS,2 and
VAChT3 have been well-characterized in previous studies,
including homologous and heterologous preabsorption.
In addition, we confirmed positive control labeling in
target tissues for ChAT, VAChT (rat cerebellum), and
nNOS (mouse intestinal myenteric neurons) prior to use
in ferret tissues. Slides in which the primary antiserum
was omitted showed no labeling and served as negative
controls.
Visualization and Quantification
High-magnification epifluorescent images were
obtained on a conventional epifluorescence microscope
(BX-51; Olympus, Australia) equipped with specific exci-
tation filters and images acquired on a Photometrics
CoolSnapfx monochrome digital camera (Roper Scien-
tific, Tucson, AZ). A differential interference contrast
(Nomarski) stage was also used for imaging brainstem
sections which were overlaid to align cytoarchitectural
GASTROENTEROLOGY Vol. 138, No. 3
features of nucleus tractus solitarius subnuclei described
in ferret with target immunoreactivity and retrograde
tracer. All fluorescence images were imported unmodified
into V⫹⫹ imaging software v 4.0 (Digital Optics, Auck-
land, New Zealand), pseudocolored, and merged for com-
posite images; luminance intensity was not adjusted. An-
terogradely traced vagal endings in the reflux barrier were
further imaged by confocal microscopy (Leica SP5) and
3-dimensional projection images generated using Leica
Confocal Software (Leica Microsystems, Australia).
Sequential Dissection Experiments in the
Isolated Ferret CD Vagal Efferent
Preparation
In 2 preparations, a sequential dissection was per-
formed on vagal nerve trunks to determine the route of
innervation of the CD. In this experiment, a cut was
made 1 cm above the gastroesophageal junction in the
left vagus trunk and the caudal trunk dissected free of
preparation. Vagal stimulation was performed as detailed
above, and EMG responses were recorded in the left CD
muscle. The vagal trunk was then cut sequentially at 1.5,
2, 2.5, and 3 cm above the gastroesophageal junction and
EMG responses recorded at each intervention; the same
protocol was performed on the right vagus nerve trunk
with EMG responses recorded in the right CD muscle.
In Vivo EMG Experiments
Vagal innervation of the CD was also assessed in a
whole-animal preparation. Ferrets were anesthetized with
pentobarbitone sodium (50 mg/kg intraperitoneally),
and the right carotid artery and jugular vein were cannu-
lated for blood pressure recordings and administration of
intravenous anesthetic, respectively. Supplemental doses
of pentobarbitone were administered as required to abol-
ish the hindlimb pinch-withdrawal reflex. The right and
left vagus nerves were isolated and a tracheotomy per-
formed; body temperature was maintained at 37–39°C
via a heating pad. A midline laparotomy was performed
to access the abdominal surface of the left diaphragm. In
6 animals, the viscera were retracted, the diaphragm
gently tensioned laterally, and fine bipolar electrodes
secured in the left CD 1–2 mm adjacent the esophageal
hiatus; these were isolated using Parafilm (Pechiney Plas-
tic Packaging, Chicago, IL). The viscera were then re-
leased and the laparotomy closed. A thoracotomy and
median sternotomy were then performed in the 7– 8 rib
space and ventilation commenced (15 mL, 30 breaths per
minute). The left and right phrenic nerves were isolated
and a snare attached for quick access. Fine bipolar elec-
trodes were then placed in the thoracic surface of the left
ventrolateral costal diaphragm. Blood pressure and EMG
activity in the left CD and costal diaphragm were re-
corded simultaneously and analysed offline using Spike2
(Cambridge Electronic Design).
After 10 minutes of stable baseline recording from CD,
costal diaphragm, and blood pressure the thoracic phrenic
March 2010
nerves were sectioned and the ensuing EMG activity re-
corded. Intact vagus nerve trunks were then individually
stimulated via hook electrodes, using a pulse generator
and a constant-current stimulus unit delivering a single
20-V electrical stimulus of 0.5-ms pulse width.
Drugs and Solutions
Hexamethonium bromide was obtained from Sig-
ma-Aldrich. Pancuronium bromide was obtained from
AstraZeneca (North Ryde, Australia). All drug dilutions
were prepared and diluted in isotonic saline on the day of
experimentation.
Supplementary Results
Spinal Innervation of the CD
To determine the location of afferent and efferent
cell bodies innervating the diaphragm we performed ret-
rograde tracing by bilateral injection of CTB-fluorescein
isothiocyanate or CTB-AF555 into the CD. As expected,
this resulted in retrograde labeling of a small population
of spinal afferent neurons in cervical dorsal root ganglion
(DRG) and motor neurons in the ventromedial C5 and
C6 ventral horn of the spinal cord (Supplementary Figure
1). Similar findings were made when tracer was injected
into the costal diaphragm, but not when injected into the
distal esophagus, as expected. When different tracers
were injected in the CD and costal diaphragm in the
same experiments, separate afferent neurons were labeled
in DRG (Supplementary Figure 1A), and separate motor
neurons were labeled in the ventral horn (Supplementary
Figure 1B). Labeled motor neurons were intermingled
within a compact cell group representing the phrenic
motor nucleus. All of these exhibited large multipolar
perikarya that gave rise to several dendrites that extended
radially from the cell body. Double-labeled motorneu-
rons were occasionally identified in these animals (Sup-
plementary Figure 1C).
Control Experiments for Tracer Injection
In order to determine if tracer spread into adja-
cent regions could explain our findings, we observed
peripheral tracer spread 2 hours after injection into re-
spective target tissues. This showed CTB-AF555 injected
into the CD was restricted to skeletal muscle of the CD,
while CTB-fluorescein isothiocyanate injected into the
esophagus was evident only in the serosa of the distal
esophagus and in the inner portion of the phrenoesopha-
geal ligament (data not illustrated). Control intraperito-
neal injection of the same volume of either tracer weakly
labeled a small number of NG neurons (⬍1%) and DMN
neurons, presumably due to uptake by vagal motorneu-
rons and afferents innervating abdominal viscera (data
not illustrated). This weak tracing contrasted with the
bright labeling seen with tracing from CD, costal dia-
phragm, or distal esophagus, and was at a level below
A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1101.e2
that used to define positive tracing in other experiments.
Thus, we were confident that the labeling we observed in
tracing experiment, from specific targets could not have
arisen from nonselective uptake.
Anterograde Tracing Positive Control
Anterograde tracing labeled specialized vagal af-
ferent endings within the myenteric plexus and muscular
layers of the ferret stomach, corresponding to intragan-
glionic laminar endings (Supplementary Figure 2A) and
intramuscular arrays, as observed in many species. Intra-
ganglionic laminar endings were concentrated within the
gastric fundus and body, with fewer labeled toward the
greater curvature and were more abundant in the ventral
specimen of the stomach. Tracing of gastric intragangli-
onic laminar endings thus provided a confirmation of
effective anterograde tracing of afferent endings in indi-
vidual animals.
Sequential Dissection Experiments
Sequential dissection experiments revealed that
motor innervation of the CD by the vagus arose bilater-
ally via nerve branches that diverged from each trunk of
the thoracic vagus 1–2 cm above the gastroesophageal
junction and ran within muscle layers of the distal esoph-
agus prior to innervating the CD (data not illustrated).
In Vivo EMG Experiments
Spontaneous EMG activity in the left CD and ven-
trolateral costal diaphragm were reliably recorded in these
in vivo experiments (Figure 7A). Bilateral phrenicotomy led
to a complete loss of EMG activity in the costal diaphragm,
after which animals were artificially ventilated. Spontaneous
EMG activity continued in the CD at a rate of 0.3 ⫾ 0.1 per
second. This nonphrenic EMG activity was not phase-
locked to cardiac electrical activity or ventilation, suggesting
a separate central coordination.
Stimulation of intact vagus nerve trunks in ventilated
and phrenicotomized ferrets evoked short-latency EMG
spikes in the left CD (but not costal diaphragm) with a
latency consistent with input from motorneurons with
conduction velocities of A␦ fiber (8.3 ⫾ 0.3 m/s). This
stimulus also produced reflex increase in EMG activity
after a longer and more variable latency. Stimulation of
the right vagus increased the EMG frequency in the left
CD immediately following the stimulus by 14-fold (0.3 ⫾
0.1 vs 3.8 ⫾ 0.7 per second in 1-second poststimulation;
Figure 7B), while stimulation of the left vagus increased
EMG frequency in the left CD by 4-fold (0.3 ⫾ 0.1 vs
1.1 ⫾ 0.5 per second). These data provide strong evidence
of a bilateral, multisynaptic central pathway converging
on DMN neurons that innervate the CD.
We also performed additional experiments to assess ef-
fects of ipsilateral and bilateral vagotomy on diaphragm
EMG in phrenic-intact, spontaneous breathing ferrets.
These data were inconclusive as the loss of the Hering-
1101.e3 YOUNG ET AL
Breuer reflex upon vagotomy led to increased EMG activity
(amplitude) in both compartments of the diaphragm.
Supplementary References
1. Consonni S, Leone S, Becchetti A, et al. Developmental and
neurochemical features of cholinergic neurons in the murine cere-
bral cortex. BMC Neurosci 2009;10:18.
GASTROENTEROLOGY Vol. 138, No. 3
2. Christie AE, Edwards JM, Cherny E, et al. Immunocytochemical
evidence for nitric oxide- and carbon monoxide-producing
neurons in the stomatogastric nervous system of the crayfish
Cherax quadricarinatus. J Comp Neurol 2003;467:293–306.
3. Weihe E, Schutz B, Hartschuh W, et al. Coexpression of cholin-
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March 2010 A VAGAL INNERVATION OF THE CRURAL DIAPHRAGM 1101.e4

Supplementary Figure 1. Retrograde tracing of spinal nerve pathways from the crural diaphragm (CD) in ferrets. (A) Transverse view of dorsal root
ganglion (DRG) at C6 level showing spinal afferent neurons traced separately from CD (cholera toxin subunit B [CTB]-AF555, red) and costal
diaphragm (CTB-fluorescein isothiocyanate, green). (B) Transverse high-magnification view of C6 spinal cord showing CD motorneurons and
processes in the ventral horn traced with CTB-AF555 (white). (C) High-magnification view of C6 ventral horn showing motor neurons traced
separately, and jointly from the CD (CTB-AF555, red) and costal diaphragm (CTB-fluorescein isothiocyanate, green). Scale bar (A, C) ⫽ 100 ␮m, (B) ⫽
200 ␮m.

Supplementary Figure 2. Anterograde tracing of vagal nerve pathways to the crural diaphragm (CD) and phrenoesophageal ligament in ferrets.
(A) Vagal intraganglionic laminar ending;(IGLE) in whole mount of stomach filled by anterograde tracing from the left nodose ganglia (NG) with wheat
germ agglutinin– horseradish peroxidase (WGA-HRP) (white). (B) High-magnification extended focal image (EFI) of vagal punctate endings filled by
WGA-HRP within connective tissue of the abdominal CD surface. (C) Low-magnification view of a vagal punctate ending at the base of a CD muscle
fascia near the esophageal hiatus. AB CT, connective tissue of the abdominal CD surface. (D) Vagal punctate endings immunopositive for WGA in
the abdominal CD surface. (E) High-magnification view of a motor endplate in the CD immunopositive for vesicular acetylcholine transporter (VAChT);
none of the identified vagal structures were immunopositive for VAChT. (F) EFI view of a vagal laminar ending in the phrenoesophageal ligament. Scale
bar (A) ⫽ 500 ␮m, (B, D, E) ⫽ 10 ␮m, (C) ⫽ 100 ␮m, (F) ⫽ 25 ␮m.
1101.e5 YOUNG ET AL GASTROENTEROLOGY Vol. 138, No. 3

Supplementary Table 1. Antisera Used for Localization and

Classification of Vagal Innervation
of the Crural Diaphragm
Antisera
Target Dilution Supplier Category no.
ChAT 1:100 Millipore AB144P
VAChT 1:500 Phoenix Biotech H-V007
nNOS 1:200 Zymed Laboratories 61-7000
WGA 1:1000, goat Vector Laboratories AS-2024
antibody
PGP 1:500, anti-human Ultraclone RA95101

NOTE. All primary antibodies were directed to C-termini and were

rabbit anti-rat antibodies unless indicated.
ChAT, choline acetyltransferase; nNOS, neuronal nitric oxide syn-
thase; PGP, protein gene product 9.5; VAChT, vesicular acetylcholine
transporter; WGA, wheat germ agglutinin.