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Ultrastructural characterization of pharyngeal and esophageal motoneurons in the nucleus ambiguus of the rat

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Ultrastructural Characterization of
Pharyngeal and Esophageal Motoneurons
in the Nucleus Ambiguus of the Rat
Department of Anatomy (T.H., K.Z.) and Physiology (Y.Y.), Hyogo College of Medicine,
Nishinomiya, Hyogo 663, Japan
The viscerotopic organization of the upper alimentary tract has been established in the
nucleus ambiguus, but there is little information about the morphology of the individual
neurons innervating the pharynx and esophagus. We studied the ultrastructure of pharyngeal
(PHI, cervical esophageal (CE),and subdiaphragmatic esophageal (SDE) motoneurons labeled
by retrogradely transported wheat germ agglutinin conjugated horseradish peroxidase (WGAHRP) in the compact formation of the nucleus ambiguus. WGA-HRP was injected into the
lower pharynx, or the cervical and subdiaphragmatic esophagus of male rats. The retrogradely
labeled PH neurons in the rostral portion of the compact formation were large (26.1 x 50.1 km,
906.7 pm2), polygonal, and contained well-developed cell organelles with a round nucleus.
Subsurface cisterns connected with rough endoplastic reticulum were often present near the
postsynaptic membrane. Both CE and SDE neurons in the compact formation were mediumsized, round or oval, and contained well-developed cell organelles, although the SDE neuron
was significantly larger than the CE neuron (24.9 x 33.6 pm, 593.0 pm2 in the SDE neuron,
and 19.5 x 30.2 km, 440.3 pm2 in the CE neuron).
The average number of axosomatic terminals in a sectional plane was largest in PH
neurons (29.0),smaller in CE neurons (7.91, and smallest in SDE neurons (4.2).The number of
axosomatic terminals containing round vesicles (Gray’s type I) was almost equal to that of
terminals containing pleomorphic vesicles (Gray’s type 11) in PH and CE neurons, but there
were few Gray’s type I1 axosomatic terminals in SDE neurons. Desmosome-like junctions at
somato-somatic or somato-dendritic apposition were often present in the area surrounding
SDE neurons. There were also small unlabeled neurons (9.5 x 18.1 km, 131.8 pm2) in the
compact formation of the nucleus ambiguus. The small neurons contained poorly developed cell
organelles and an irregular shaped nucleus with invaginated nuclear membrane, and had no
Nissl bodies.
These results indicate that PH neurons have the characteristics of somatic motoneurons,
and that CE and SDE neurons are similar to visceral motoneurons.
1996 Wiley-Liss, Inc.
Indexing terms: retrograde tracing study, WGA-HRP, swallowing, deglutition, electron microscopy
The nucleus ambiguus, as one of the original nuclei of the
vagus nerve, innervates the soft palate, pharynx, esophagus, larynx, and heart (Lawn, 1966a; Yoshida et al., 1981;
Holstege et al., 1983; Pasaro et al., 1983; Fryscak et al.,
1984). Thus, this nucleus plays an important role in
swallowing, peristalsis, vocalization, and cardiovascular
function. Lawn (1966b) reported that the nucleus ambiguus of the rabbit is composed of a rostral compact
formation which is divided into principal and medial columns, and has a diffuse formation. In recent years, retrograde tracing studies with horseradish peroxidase (HRP)
have revealed the topographic organization between the
subnuclei and the innervated organs (Bieger and Hopkins,
1987).It has been shown that the compact formation of the
nucleus ambiguus (AmC) contains esophageal motoneurons, the semicompact formation contains palatopharyngeal motoneurons, the loose formation contains laryngeal
motoneurons, and the external formation contains cardiopulmonary efferent neurons.
The pharynx has striated muscles concerned with swallowing and breathing. The upper esophagus has a thin
muscle layer concerned with swallowing and peristalsis,
Accepted January 16,1996
Tetsu Hayakawa, Department of Anatomy, Hyogo College of Medicine.
Mukogawa, Nishinomiya, Hyogo 663, Japan.
Fig. 1. Light micrographs of retrogradely wheat germ agglutininhorseradish peroxidase (WGA-HRP)labeled cells in the nucleus ambiguus. A: Retrogradely labeled cells in the rostra1 portion of the
compact formation (AmC)and the semicompact formation (AmS) of the
nucleus ambiguus after WGA-HRP injection into the lower pharynx,
frontal section. B: Retrogradely labeled cells in the AmC after WGAHRP injection into the cervical esophagus, frontal section. C: Retrogradely labeled cells in the AmC after WGA-HRP injection into the
subdiaphragmatic esophagus, sagittal section. Scales = 100 km.
whereas the lower esophagus has a well-developed muscle especially about neurons identified as pharyngeal and
layer participating in autonomic peristalsis (Fryscak et al., esophageal motoneurons.
1984). Thus, the AmC may contain not only somatic
We investigated the cytology and ultrastructural features
motoneurons, but also general visceral motoneurons. A of the pharyngeal and esophageal motoneurons labeled by
dendritic architectonic study using cholera toxin conju- retrogradely transported wheat germ agglutinin conjugated
gated horseradish peroxidase (Altschuler et al., 1991) horseradish peroxidase (WGA-HRP) in the AmC. Comparshowed that the pharyngeal motoneurons have extensive ing the pharyngeal, cervical esophageal, and subdiaphragdendrites that extend into the adjacent reticular formation. matic esophageal motoneurons, we also attempted to clarify
Dendrites of the subdiaphragmatic esophageal motoneu- what kind of axon terminals each neuronal somata receive
rons were confined to the AmC, and made bundles running in the AmC.
rostrocaudally. This study suggests that the character of
pharyngeal motoneurons is different from that of subdiaMATERIALS AND METHODS
phragmatic esophageal motoneurons. In addition, although
it has been noted that the AmC neurons receive direct
Twelve male Sprague-Dawley rats weighing 250-300 g
projections from the nucleus of the solitary tract (Loewy were used in this study. All surgical procedures were
and Burton, 1978; Stuesse and Fish, 1984; Cunningham carried out under sodium pentobarbital (30 mgikg, i.p.)
and Sawchenko, 1989; Barrette et al., 19941, the presence of anesthesia. The injection of WGA-HRP was made with a
interneurons in the AmC has been suggested in physiologi- glass micropipette (tip diameter 50-80 pm) affixed to a
cal studies of swallowing activity (Bieger, 1984; Kessler and 10 pl Hamilton syringe. To expose the lower pharynx, the
Jean, 1985). These reports suggest that several kinds of sternohyoid muscle and the posterior digastric muscle were
neurons are present in the AmC. There is, however, little removed. The pipette was inserted obliquely into the left
information about the ultrastructural features of the AmC, inferior constrictor muscle. Two microliters of 4% WGA-
Fig. 2. Low power electron micrograph of retrogradely WGA-HRP labeled pharyngeal motoneuron in
the AmC. Arrows indicate axosomatic terminals, open arrows indicate retrogradely transported WGA-HRP
reaction products. E indicates well-developed rough endoplasmic reticulum (rER) forming a Nissl body.
Scale = 5 km.
HRP were injected three times into the pharynx between
the hyoid bone and thyroid cartilage. The injection of the
cervical esophagus was in the esophageal mucous membrane between the thyroid gland and the sternum. Three
injections of 2 ~1 each were made. The injection of the
subdiaphragmatic esophagus was in the area between the
ventral surface of diaphragm and the cardia of the stomach.
Four injections of 2 ~1 each were made. Cotton swabs were
used during the injections to avoid spread of the tracer to
adjacent structures.
Three days after injection, the animals were reanesthetized and perfused first with 100 ml saline and then 500 ml
of 1%glutaraldehyde-1% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were immediately
removed and placed in the same fixative for 1 hour. Serial
frontal or sagittal sections were cut at 70 Fm with a
Vibratome. Every second section through the nucleus
ambiguus was processed for light microscopic study with
tetramethyl benzidine (TMB) at pH 3.3 according to the
method ofMesulam (1978).
In order to carry out electron microscopic studies, alternate sections were collected and processed with TMB at pH
5.8 by the method of Hayakawa and Zyo (1992). Briefly, the
sections were presoaked with 40 ml of 0.025% ammonium
heptamolybdate containing 2 mg TMB dissolved in 1 ml
ethanol in 0.1 M phosphate buffer at pH 5.8. Then 40 ~1 of
3% H202per 40 ml solution was added every 5 minutes up
to 30 minutes. After a brief rinse, the section was postfixed
with 2% Os04 in 0.1 M phosphate buffer at pH 6.0 for 2
hours at 45°C. The sections were then dehydrated and
embedded with Epon 812 (Olucha et al., 1985). Ultrathin
sections were collected and stained with uranyl acetate and
Reynold’s solution, and examined with a JEOL 1200 EX
transmission electron microscope.
Retrogradely labeled neurons cut through the plane of
the nucleolus were sampled randomly from each case, and
photographed at a final magnification at x 13,000 to carry
out quantitative electron microscopic analyses. Prints were
used to make montages of somatic profiles. Then a planimeter and a curvimeter were used to calculate the width,
length and area of the neuronal somata, the number of
axosomatic terminals, percentages of the two different
morphological classes of terminals, such as terminals containing round vesicles (Gray’stype I) and terminals containing pleomorphic vesicles (Gray’s type 11; Gray, 1959), and
the ratio of the surface length of neuronal somata and the
surface length covered with axosomatic terminals (the
bouton covering ratio; BCR; Nakamura 1975). Student’s
t-test ( P < 0.05) was used to compare the mean values for
the cytoarchitectonic data of each neuron.
Fig. 3. Electron micrographs of axosomatic terminals in the pharyngeal motoneuron. A Terminal containing round synaptic vesicles and
forming asymmetric synaptic contact (Gray’s type I). Arrow indicates
asymmetric synaptic contact associated with subsynaptic densities
(Taxi body). Open arrow indicates retrogradely transported WGA-HRP
reaction product. B: Terminal containing pleomorphic synaptic vesicles
and forming symmetric synaptic contact (Gray’s type 11). Arrow
indicates symmetric synaptic contact, and arrowhead indicates a densecored vesicle. C: Many Gray’s type I (R) and type I1 (P) axosomatic
terminals presenting in a line. Note strands of rER coming close to the
postsynaptic membrane and forming subsurface cisterns (curved arrow). Open arrow indicates retrogradely WGA-HRP transported reaction product. Scales = 0.5 pm.
Labeled pharyngeal (PH) neurons were multipolar, and
their dendrites expanded to the adjacent reticular formaThe compact formation of the nucleus ambiguus (AmC)
is easily distinguishable from other subnuclei owing to the tion (Fig. 1A).
Electron microscopy of P H neurons revealed many typiconcentration of cell somata and the prominent clear area
created by sparse myelinated fibers. Since it is easy to trim cal electron-dense, needle-shaped or crystalline WGA-HRP
the AmC in osmium fixed Vibratome sections, we studied reaction products in the neuronal somata or dendrites
labeled cells in the AmC. When WGA-HRP was injected into (Figs. 2 and 3C). No axon terminals contained WGA-HRP
the lower pharynx, many retrogradely labeled neurons were reaction products. PH neurons were large (26.1 & 1.2 x
observed in the rostral portion of the AmC including the 50.1 2 2.7 pm, mean & S.E.),polygonal, triangular or spindlerostral extension of the Amc (Bieger and Hopkins, 1987). shaped, with a mean area of 906.7 ? 62.0 pm2 (Table 1).A
There were also many other labeled neurons located in the light prominent round nucleus was present at the center of
semicompact formation ventral and caudal to the AmC. the soma and had one nucleolus. The neurons had well-
TABLE 1. Mean Cytoarchitectonic Data, Axosomatic Terminals, Gray's
Type I and I1 Terminals, and Bouton Covering Ratio (BCR) in the
Pharyngeal (PHI,Cervical Esophageal (CE),Subdiaphragmatic Esophageal
(SDE),and Small Unlabeled Neurons in the Compact Formation of the
Nucleus Ambiguus (Mean % S.E.)
Area lpm'i
Width ipml
Length < hm 1
No. ansometic
No. Gray's type I
terminals 1% 1
No. Gray's type I1
terminals i % l
PH neuron
( n = 25)
CE neuron
(n = 27)
SDE neuron
(n = 23)
Small neuron
( n = 25)
906.7 i 62.0
26.1 r 1.2
50.1 2 2 . 7
29.0 Z 1.7
440.3 Z 32.2
19.5 t 0.9
30.2 z 1.2
7.9 2 1.4
593.0 r 27.1
24.9 2 0.8
33.6 ? 1.3
4.2 i 0.4
1 3 1 . 8 6.6
9.5 2 0.3
18.1 2 0.8
4.1 2 0.4
15.1 ? 1.2
13.8 t 0.9
37.6 t 1.8
4.5 t 0.9
3.4 2 0.6
11.3 z 2.2
3.3 2 0.3
0.8 2 0.2
4.9 2 0.5
2.0 2 0.4
2.1 z 0.3
11.9 Z 1.4
developed cell organelles, that is, many mitochondria, lysosomes, Golgi apparatus, numerous ribosomes, and many
prominent rough endoplasmic reticulum (rER) that were
accumulated into multiple parallel arrays forming many
Nissl bodies (Fig. 2).
Axosomatic terminals in the AmC were classified by the
morphological differentiation of their synaptic vesicles as
round (diameter, about 40 nm) and pleomorphic (about
20 x 50 nm). Terminals containing round vesicles generally formed asymmetric synaptic contacts (Fig. 3A), whereas
those containing pleomorphic vesicles formed symmetric
synaptic contacts (Fig. 3B). We considered the terminals
containing round vesicles to be Gray's type I terminals, and
those containing pleomorphic vesicles to be Gray's type I1
terminals (Gray, 1959). Small dense-cored vesicles (60-70
Fig. 4. Low power electron micrograph of two retrogradely labeled cervical esophageal motoneurons
(C1, C2). Arrows indicate axosomatic terminals, open arrows indicate retrogradely transported WGA-HRP
reaction products, and curved arrow indicates dendritic bundles. E, well-developed rER. S, small unlabeled
neuron. Scale = 5 pm.
Fig. 5. Electron micrographs of Gray’s type I (A) and type I1 (B)
axosomatic terminals of the cervical esophageal neuron (CE). A:
Terminal containing round vesicles contacting a dendrite and the soma
of CE neuron with asymmetric synaptic contact (arrow). B: Terminal
containing pleomorphic vesicles contacting two dendrites and the soma
of a CE neuron with symmetric synaptic contact (arrow). Arrowhead
indicates a dense-cored vesicle. D, dendrite; S, soma. Scales = 0.5 pm.
nm) were present in both types of terminals (Figs. 3B, 5B,
and 7A). Subsynaptic densities (Taxi bodies, Taxi, 1961)
were sometimes associated with the postsynaptic membrane of Gray’s type I terminals (Fig. 3A). PH neurons
received many axosomatic terminals which often contacted
each other and formed a row (Fig. 3C). The rER was often
close to the postsynaptic membranes of Gray’s type I or
type I1 terminals, and formed subsurface cisterns. These
cisterns extended to the postsynaptic membranes of two
terminals (Fig. 3C). There were neither synapse en passant,
nor axoaxonic terminals. The number of axosomatic terminals in a sectional plane was 29.0 t 1.7 (mean 2 S.E.),and
the size was large (1.71 ? 0.03 pm, n = 723). The number
of Gray’s type I terminals was almost equal to that of type I1
terminals in the axosomatic terminals (Table 1). The
bouton covering ratio (BCR)was 37.6 t 1.8%of the surface
of the neuronal somata.
Following WGA-HRP injection into the cervical esophagus, many retrogradely labeled neurons were found in the
rostra1 two-thirds of AmC (Fig. 1B). There were no anterogradely labeled terminals. The dendrites of the cervical
esophageal (CE) neurons expanded dorsoventrally or rostrocaudally. Electron microscopic study revealed that CE
neurons were medium-sized (19.5 & 0.9 x 30.2 2 1.2 pm,
mean 2 S.E.), round or oval, and had a mean area of 440.3
? 32.2 pm2 (Table 1).A light prominent round nucleus was
present with a nucleolus. The neurons contained welldeveloped cell organelles of many mitochondria, lysosomes,
ribosomes, Golgi apparatus, and rER forming Nissl bodies
(Fig. 4). The amount of rER was smaller than that of PH
neuron. Many dendritic bundles ran rostrocaudally in the
center of the AmC (Fig. 4). The number of axosomatic
terminals in a sectional plane was 7.9 1.4 (mean S.E.),
and the size was 1.27 0.03 pm (n = 214). CE neurons
received both Gray’s type I and type I1 axosomatic terminals (Fig. 5 ) . The number of Gray’s type I axosomatic
terminals was almost equal to that of type I1 terminals
(Table 1).The BCR was small (11.3 t 2.2%). The surface
not in contact with axosomatic terminals was covered with
processes of glial cells, dendrites, and myelinated or unmyelinated axons. Sometimes two neuronal somata of CE
neurons apposed each other. There was, however, no
characteristic structure at that site.
When WGA-HRP was injected into the subdiaphragmatic
esophagus, many retrogradely labeled neurons were found
within the AmC (Fig. 1C). The dendrites of subdiaphragmatic esophageal (SDE) neurons formed bundles running
rostrocaudally within the AmC. No anterogradely labeled
terminals were present. The ultrastructure of SDE neurons
was similar to that of CE neurons (Fig. 6). SDE neurons
were medium-sized (24.9 ? 0.8 x 33.6 ? 1.3 pm,
mean ? S.E.); round, oval, or spindle-shaped; and had a
mean area of 593.0 2 27.1 pm2. However, they were
significantly larger than CE neurons (t = 3.63, P < 0.001).
The neurons contained a light prominent round nucleus
and well-developed cell organelles. They had a smaller
amount of rER than did PH neurons. The number of
axosomatic terminals in a sectional plane was small
(4.2 2 0.4, mean ? S.E.), and their size was small
(1.16 0.06 pm, n = 96). Most of the axosomatic terminals
were Gray’s type I (Fig. 7A). The BCR was also small
(4.9 0.5%).The surface of neuronal somata was generally
smooth, and covered with glial cell processes, myelinated or
unmyelinated axons, dendrites, and other somatic membranes.
SDE neurons were often apposed to dendrites (Fig. 7B) or
other SDE neuronal somata (Fig. 7C) with an adherent
junction which was similar to a desmosome. The length of
the adherent junction was about 300 nm, and its intercellular space was 20 nm. Both somatic and dendritic membranes on this site were thick and electron dense (Fig. 7D).
There was, however, neither a dense mat of filamentous
material, nor a slender intermediate line in the midline of
the intercellular space. Thus it was different from the gap
junction. There were 1.74 0.21 (mean 2 S.E.) adherent
junctions in a sectional plane. Adherent junctions were also
present at the dendro-dendritic apposition of neuropil
adjacent to SDE neurons.
PH neurons were significantly larger in area than SDE
neurons (t = 4.64, P < 0.001), and SDE neurons were
significantly larger than CE neurons (t = 3.63, P < 0.001),
making the latter the smallest (Fig. 9A). SDE neurons had
significantly fewer axosomatic terminals than did CE neurons (t = 2.65, P < 0.0121,whereas PH neurons had significantly more than did CE neurons (t = 9.78, P < 0.001)
(Fig. 9B). The minimum number of axosomatic terminals in
Fig. 6. Low power electron micrograph of retrogradely WGA-HRP labeled subdiaphragmatic esophageal
neuron. Arrows indicate axosomatic terminals, open arrows indicate retrogradely transported WGA-HRP
reaction products, curved arrow indicates somato-dendritic junction which is shown in Fig. 7B. E,
well-developed rER. Scale = 5 Km
CE neuron was 2, the maximum was 39, the mean was 7.9,
and the standard deviation was 7.01. Although the number
of Gray’s type I and I1 terminals were almost equal in P H
and CE neurons, SDE neurons had fewer type I1 terminals
(Table 1).
In addition to these pharyngeal and esophageal motoneurons, we found other small neurons in the AmC (Figs. 4 and
8). These small neurons were not always labeled by the
WGA-HRP, and were generally situated at the marginal
area in the AmC. They were small (9.5 ? 0.3 x 18.1 +- 0.8
km, mean ? S.E.), oval or spindle-shaped, and had a mean
area of 131.8 ? 6.6 pm2 (Table 1).They had a characteristic
irregular shape, and a dark nucleus with one nucleolus. The
nuclear membrane was vigorously invaginated (Figs. 4 and
8A). The neuronal somata had a small rim of dark cytoplasm containing many ribosomes, several mitochondria, a
few lysosomes, Golgi apparatus, and a few strands of rER.
There was no Nissl body (Fig. 4). Sometimes the outer
nuclear membrane extended into the cytoplasm, and connected with rER or formed subsurface cisterns (Fig. 8).
There were a few axosomatic terminals (4.1 2 0.4,
mean 2 S.E.),their size was 1.34 ? 0.05 pm, and the BCR
was 11.9 t 1.4%.The number of Gray’s type I axosomatic
terminals was almost equal to that of the type I1 terminals
(Table 1).The small neurons were often apposed to the
other motoneurons (Fig. 8B), glial cells and dendrites. The
intracellular space was less than 5 nm, but there were no
adherent junctions at these appositions.
This paper is the first to describe the ultrastructural
character of ambiguous neurons projecting to the pharynx
and the esophagus. PH neurons are large, and have many
well-developed cell organelles, including many subsurface
cisterns, and many axosomatic terminals in a sectional
plane (29.0, mean). SDE neurons are medium, have welldeveloped cell organelles, a few axosomatic terminals (4.2,
mean), and appose other somata or dendrites across adherent junctions. CE neurons are similar to SDE neurons in
shape. However, they may represent a mixed population of
P H and SDE neurons in a number of the axosomatic
terminals (Fig. 9B). There are also small neurons that do
not project to the alimentary tract. The small neuron has an
irregular-shaped nucleus, and a small cytoplasm containing
less-developed cell organelles.
When TMB was used as a chromogen, retrogradely
transported WGA-HRP reaction products were present as a
crystalline, needle-shaped, electron-dense materials in the
somata and dendrites. Membranes and structures of la-
Fig. 7. A Terminal containing round vesicles contacting a dendrite
(D) and the retrogradely labeled subdiaphragmatic esophageal (SDE)
neuron with asymmetric synaptic contact (arrow). Open arrow indicates retrogradely transported WGA-HRP reaction product, arrowhead
indicates a dense-cored vesicle. B: Somatokbdendritic (D) junction
(arrow) that is indicated by the curved arrow in Fig. 6. Arrowhead
indicates a somatic spine. C:Somato-somaticjunction (arrow) between
two labeled SDE neurons. Open arrows indicate retrogradely transported WGA-HRP reaction products. N, nucleus. D: Adherent junction
(arrow) connecting the soma of SDE neuron (S) and a dendrite (D).
Scale = 0.5 p m in A, D and 1 pm in B, C.
beled neurons were well preserved (Hayakawa and Zyo,
1992). Since the reaction products did not label all dendrite
areas, we did not study dendrites and axodendritic terminals. It has been reported that transsynaptic and transneuronal transport of WGA-HRP requires a long survival time
after injection of a large amount of tracer (Henry et al.,
1985; Porter et al., 1985; Itaya, 1987). We used a small
amount of WGA-HRP and short survival times. Light
microscopic observation from the Vibratome sections
showed no anterogradely or retrogradely transsynaptic
transport in the medullary nuclei.
Light microscopic studies using retrograde tracing methods have revealed that there are topographic projections
between the subnuclei of the nucleus ambiguus and the
innervated organs (Kalia and Mesulam, 1980a,b; Fryscak et
al., 1984; Shapiro and Miselis, 1985; Bieger and Hopkins,
1987; Portillo and Pasaro, 1988). That is, the AmC contains
neurons projecting to the cervical, thoracic, and subdiaphragmatic esophagus, the semicompact formation to the
pharyngeal and cricothyroid muscles, and the loose formation to the intrinsic laryngeal muscle except for the cricothyroid muscle. There are also pharyngeal motoneurons lo-
cated in the rostral portion and the rostral extension of the
AmC (Bieger and Hopkins, 1987; Altschuler et al., 1991).
The external formation contains cardiopulmonary efferent
neurons. No double-labeled cells were observed in the AmC
when two different fluorescent tracers were injected at the
cervical and subdiaphragmatic esophagus, respectively
(Bieger and Hopkins, 1987). Our results agreed with theirs,
and showed four kinds of neurons in the AmC, that is, PH,
CE, SDE, and small neurons.
The motor trigeminal nucleus of the rat is composed of
large-, medium-, and small-sized neurons. The large neurons are characterized ultrastructurally by a light round
nucleus, organelle-rich cytoplasm containing numerous
cistern arrays of rER, and many axosomatic terminals. The
percentage of terminals containing round vesicles is 42.14%,
and BCR is 77.5% (Card et al., 1986). They considered this
neuron to be an a-motoneuron. Its morphology is similar to
that of PH neuron, implying that PH neurons may be
a-motoneurons. Similar large motoneurons have been reported in the hypoglossal nucleus of the rat (Boone and
Aldes, 19841, the facial nucleus of the opossum (Falls and
King, 19761, the abducens nucleus of the cat (Spencer and
Fig. 8. A Electron micrograph of a small unlabeled neuron in the
AmC. Note invaginated nuclear membrane connecting a strand of rER
in the small rim of cytoplasm (arrow). B: Small neuron directly
apposing a pharyngeal neuron (PH)without an adherent junction (open
arrow). Note outer nuclear membrane (large arrow) connectingsubsurfacecistern (small arrows). N, nucleus; R,axosomatic terminal containinground synapticvesicles. Scales = 1 pm.
Sterling, 19771, and the trochlear nucleus of the cat (Bak excitatory, whereas Gray’s type I1 terminals are inhibitory
and Choi, 1974). There are many subsurface cisterns (Uchizono, 1965; Peters et al., 19911, P H and CE neurons
beneath postsynaptic membranes in P H neurons. These receive many excitatory and inhibitory inputs, whereas
subsurface cisterns are often detected by immunohistochem- SDE neurons receive almost exclusively excitatory inputs to
istry with antibodies against subsurface cisterns (con- the somata.
An interesting finding of our study was that SDE neunexin32) in the facial nucleus and the hypoglossal nucleus
(Yamamoto et al., 1991). These subsurface cisterns may be rons were apposed to other somata and dendrites without
active in controlling cellular calcium mobilization (McBur- astrocytic intervention. SDE neurons were apposed to the
ney and Neering, 1987).
other neurons by adherent junctions. Recently, Hopkins
The dorsal motor nucleus of the vagus is composed of ( 1995) reported somato-somatic or somatodendritic apposimedium-sized and small neurons. The medium-sized neu- tions by puncta adherensis in the AmC. They are the same
rons are characterized by a light round or oval nucleus, a structures of our cell apposition at SDE neurons. Milner et
well-developed cell cytoplasm, and a few (five to eight) al. (1995) reported that labeled vagal motoneurons in the
axosomatic terminals in neuronal profile. The small neuron AmC were sometimes apposed to the other neurons, and
is round or elongate, and contains scanty cytoplasm and a n that the appositions were not characterized by adherent
invaginated nucleus (McLean and Hopkins, 1981; Baude et junctions. Because the neurons in their study were labeled
al., 1992; Milner et al., 1995). Since SDE and CE neurons by WGA-HRP injection at the cut end of the cervical vagus
are similar to the medium-sized neurons of the dorsal nerve, they may be CE motoneurons. A similar apposition
motor nucleus of the vagus in their morphology, they of neurons was present in the inferior olivary nucleus
correspond to the parasympathetic efferent neurons of the (Sotelo et al., 1974). This may be associated with electronic
viscera. The rat, however, has striated muscle throughout coupling between apposing neurons (Llinas et al., 1974).
the length of the esophagus, then, these neurons may not Since esophageal peristalsis is produced by synchronous
exert autonomic parasympathetic activity but voluntary esophageal muscle contraction induced by excitation or
movement on the esophagus. SDE neurons are significantly inhibition of SDE motoneurons (Bieger, 1984, 1991), our
larger than CE neurons. This may be because the muscle findings provide some anatomical bases for this synchrolayer of the subdiaphragmatic esophagus is far more devel- nized discharge in the AmC.
The nucleus ambiguus, especially P H and CE neurons,
oped than that of the cervical esophagus (Fryscak et al.,
are closely related to swallowing (Miller, 1982; Jean, 1984;
Altschuler et al. (1991) reported that the dendritic arbori- Kessler and Jean, 1985). An electrophysiological study
zation of both P H and CE neurons extended into the indicated that swallowing represents an integrated activity
adjacent reticular formation, whereas the dendrites of the of motoneurons and interneurons (Kessler and Jean, 1985).
thoracic esophageal and SDE neurons were confined to the We found that there were small neurons in the AmC that do
AmC. Thus, they suggested that P H and CE neurons not project to the alimentary t,ract. Their morphology is
received divergent multiple afferents which may control the similar to that of the small interneurons in the dorsal motor
motor function of swallowing, and also that SDE neurons nucleus of the vagus nerve (McLean and Hopkins, 1982).
received convergent afferents which control peristalsis. If Afferent inputs of the swallowing reflex originating from
the notion is correct that Gray’s type I terminals are the surface of the tongue, the soft palate, and the pharynx
O S D E n.
MCE n.
14 r
B P H n.
f3Small n.
4 l
l - l l - l l - l l - l
Cell size (area pm2)
MCE n.
B P H n.
10 r
6 c
2 ,
1 2 1 5 1 8 2 1 2 4 27 3 0 3 3 3 6 3 9 4 2 45
N o . of axosomatic terminals
Fig. 9. A Histograms showing the number of cells per unit area in a sectional plane of the
subdiaphragmatic esophageal (SDE), cervical esophageal (CE), pharyngeal (PHI, and small unlabeled
neurons (Small).B: The number of cells per number of axosomatic terminals in a sectional plane of SDE,
CE, and PH neurons.
enter the nucleus of the solitary tract and the sensory
trigeminal nucleus (Miller, 1982; Altschuler et al., 1989).
Although the presence of interneurons in the AmC as well
as interneurons in the adjacent reticular formation has
been suggested in physiological studies of swallowing activity (Andrew, 1956; Bieger, 1984; Kessler and Jean, 19851,
the transneuronal tracing study of pseudorabies virus
showed that direct projections from the nucleus of the
solitary tract to the AmC (Barrette et al., 1994). And there
are a few small neurons in the marginal area of the AmC or
the AmC itself projecting to the nucleus of the solitary tract
(Ross et al., 1981; Mtui et al., 1995). Thus they may
correspond the small neurons not projecting to the alimentary tract, and regulate swallowing and peristaltic activity
of the AmC via the nucleus of the solitary tract.
The AmC receives descending projections from the
nucleus of the solitary tract, the parabrachial nucleus, the
midbrain periaqueductal gray matter, and the paraventricular hypothalamic nucleus (Loewy and Burton, 1978; Saper
and Loewy, 1980; Stuesse and Fish, 1984; Fulwiler and
Saper, 1984; Ter Horst et al., 1984; Ross et al., 1985; Van
Bockstaele et al., 1989; Herbert et al., 1990; Zheng et al.,
1995).A neuropharmacological study suggests that excitatory as well as inhibitory peptidergic neurotransmitters
may contribute to swallowing and peristaltic rhythmic
generation at the motoneuronal level (Bieger, 1991). Neurons containing vasopressin and oxytocin project to the
AmC from the paraventricular hypothalamic nucleus (Buijs,
1978; Luiten et al., 19851, and neurons containing somatostatin and enkephalin project to the AmC from the nucleus
of the solitary tract (Cunningham and Sawchenko, 1989;
Cunningham et al., 1991). Enkephalin containing terminals form symmetric synaptic contacts, and may send
inhibitory inputs to CE neurons (Milner et al., 1995). The
existence of calcitonin gene-related peptide (Takami et al.,
1985;Lee et al., 19921,galanin (Moore, 19891,and acetylcholine (Kimura et al., 1984; Ruggiero et al., 1990; Houser et
al., 1983) has recently been reported in the neurons of the
AmC. The AmC neurons may exert influence on the
pharynx and alimentary tract with these neurotransmitters.
The authors thank Ms. M. Hatta and Mr. N. Okamura for
their technical assistance.
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