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Motor neurons of the laryngeal nerves.

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THE ANATOMICAL RECORD 230551-556 (1991)
Motor Neurons of the Laryngeal Nerves
J.W. PATRICKSON, T.E. SMITH, AND S.-S.ZHOU
Department of Anatomy, Loma Linda University, Loma Linda, California (J.W.P., T.E.S.);
Department of Anatomy, Suzhou Medical College, Suzhou, Jiangsu,
People’s Republic of China (S.-S.Z.)
ABSTRACT
The present study was undertaken to determine the relationship
between the motor neurons of the superior and recurrent laryngeal nerves within
the nucleus ambiguus. The retrograde transport of horseradish peroxidase was
utilized to identify the motor neurons subsequent to its application to the proximal
transected end of the superior and recurrent laryngeal nerves. Labeled superior
laryngeal motor neurons were distributed ventrolaterally in the rostral portion of
the nucleus. The recurrent laryngeal motor neurons were distributed throughout
the nucleus with two distinct populations: a rostral group and a caudal group. The
rostral group overlaps the motor neurons of the superior laryngeal nerve. The
caudal group occupies that portion of the nucleus that is classically described for
the recurrent laryngeal nerve. Additional superior laryngeal nerve labeled
perikarya were found in the dorsal motor nucleus of the vagus. This study defines
the rostral distribution of the recurrent laryngeal nerve motor neurons and suggests that this rostral group is a component of the neuroanatomical substrate that
is involved in the co-activation of the laryngeal abductors controlling the laryngeal
aperture.
The articulation of the larynx by the intrinsic muscles of the larynx plays a n important role in the modulation of respiration. This is achieved by changing the
aperture of the airway and thus the resistance to air
flow. Of the laryngeal muscles, the posterior cricoarytenoid and the cricothyroid muscles are the most
dominant in this function. The contraction of the posterior cricoarytenoid muscles results in the abduction
of the vocal cords, increasing the glottic chink. The
cricothyroid muscles when contracted tense the vocal
cords and increase the anteroposterior diameter of the
laryngeal aperture (for review, see Sasaki and Isaacson, 1988). During the inspiratory phase of respiration,
there is first a n increase of the laryngeal aperture primarily by the co-activation of the posterior cricoarytenoid and the cricothyroid muscles followed by
the activation of the diaphragm.
The cricothyroid muscle is innervated by the superior laryngeal nerve (SLN) while the posterior cricoarytenoid and the remaining intrinsic laryngeal
muscles (thyroarytenoid, lateral cricoarytenoid, and
the interarytenoid) are innervated by the recurrent laryngeal nerve (RLN). The motor neurons innervating
these muscles are somatotopically distributed in the
nucleus ambiguus (Gacek, 1975; Kalia and Mesulam,
1980a,b; Hinrichsen and Ryan, 1981; Yoshida et al.,
1982; Pasaro e t al., 1983; Bieger and Hopkins, 1987).
Of the two nerves, SLN and RLN, the motor neurons of
the SLN (cricothyroid) are the most rostrally placed.
The motor neuron pool of the RLN, a s one would expect
based on the number of muscles supplied, is larger and
more complex than that of the SLN. The motor neurons
of the posterior cricoarytenoid are the most rostrally
placed. The remaining motor neurons of the intrinsic
laryngeal muscles are distributed progressively caudal
0 1991 WILEY-LISS. INC
within the nucleus with extensive overlapping (Hinrichsen and Ryan, 1981; Hisa et al., 1984; Bieger and
Hopkins, 1987; Okubo et al., 1987). However, there are
conflicting reports regarding the distribution of the
SLN versus the RLN motor neuron pools. The cricothyroid motor neurons are consistently reported as being
localized in the rostral ambiguus. The posterior cricoarytenoid motor neurons, on the other hand, are described by some investigators as extending caudally
from the middle of the nucleus (Bieger and Hopkins,
1987; Hisa et al., 1984; Yoshida e t al., 1982) while others localize these neurons a s extending caudally from
the most rostral limits of the nucleus (Hinrichsen and
Ryan, 1981; Gacek, 1975). Recently, Yajima and Hayashi (1989) recorded evoked antidromic potentials
from motor neurons within the nucleus ambiguus in
response to both superior and recurrent laryngeal
nerve stimulation. This observation would suggest a
unique relationship must exist between the motor neurons of the recurrent and superior laryngeal nerves.
Based on these divergent opinions, the present study
was undertaken to examine the distribution of, and the
relationship between, the SLN and RLN motor neuron
pools.
MATERIALS AND METHODS
Experiments were performed on 29 Sprague Dawley
rats weighing 200-310 g. The animals were anesthe-
Received July 16, 1990; accepted January 10, 1991.
Address reprint requests to J.W. Patrickson, Ph.D., Dept. of Anatomy, Lorna Linda University, Lorna Linda, CA 92350.
552
J.W. PATRICKSON ET AL.
tized with a 12% aqueous solution of chloral hydrate
(0.3 mlilOO g body weight i.p.).
Surgical Procedure
A ventral cervical midline incision was made, and
the larynx and trachea were exposed by blunt dissection. With the aid of a dissecting microscope, the laryngeal nerves were dissected free from the surrounding
tissue using hand-pulled glass micro-probes. For optimum tracer uptake, it is imperative that the transected
nerve be free of blood. The vascular tissue, where possible, was therefore dissected away from the nerves
prior to transection. Care was taken to prevent trauma
to the nerves during these manipulations. At a point
immediately proximal to the larynx, yet before any
branching may have occurred, the nerve was obliquely
transected.
HRP Application
Wheat germ agglutin-horseradish peroxidase (WGAHRP) was applied to the transected nerve by one of two
methods, each yielding similar results. In ten animals,
the nerve was isolated from the surrounding tissue by
packing the area with gel-foam. Dry crystals of WGAHRP were applied directly to the transected nerve for
30 minutes. At the end of the allotted time, the excess
tracer was sponged from the nerve and the area
flushed with saline. In 13 animals, the isolated nerve
was inserted into a small-diameter plastic tubing and
the proximal end sealed with Gi-Mask (Coltene Inc.), a
non-toxic dental impression material. The tube was
then filled with a 20% WGA-HRP saline solution and
sealed. This technique allows for the continuous bathing of the transected nerve stump for the allotted transport time of 48 hours. In most cases, the procedure was
applied to one nerve per animal. In six animals, the
tracer was applied to both nerves using the tube
method, the SLN on one side and the RLN on the other.
The wound was closed with stainless-steel wound clips.
Tissue Preparation and Processing
Forty-eight hours post HRP application, the animal
was deeply anesthetized and perfused through the
aorta with 250 ml of saline at room temperature (25°C).
This was followed with 500 ml of fixative consisting of
2% paraformaldehyde and 0.75% glutaraldehyde in 0.1
M phosphate buffer (pH 7.4) at 25°C. For cryoprotection, the fixative was followed with 250 ml 10% sucrose
in 0.1 M phosphate buffer (pH 7.4) a t 4°C. The brain
was removed and placed in sucrose-buffer solution a t
4°C for approximately 24 hours. Frozen 30 pm serial
sections in the coronal, sagittal, or horizontal planes
were made. The tissues were incubated using tetramethylbenzidine (TMB) a s the chromagen following
the protocol of Mesulam (1978) with minor modifications. The sections were mounted on gelatin-coated
slides, air dried, dehydrated, and cover slipped. The
specimens were examined under light- and darkfield
illumination for the presence of HRP positive neurons.
RESULTS
Both methods of exposing the central end of the
transected laryngeal nerves to WGA-HRP resulted in
a n intense labeling of the motor neurons and presented
a near Golgi-type staining, thus allowing for the visu-
alization of the cell processes. There were two cell types
based on their size and processes: First, there were
small cells of approximately 21 pm in diameter with
short and thin dendritic processes that were observed
consistently in the rostral area of nucleus ambiguus.
The other were larger (approximately 35 pm in diameter) multipolar cells with rather large dendritic processes. These cells were distributed along the entire
length of the nucleus ambiguus.
Exposure of the superior laryngeal nerve to the neuronal tracer resulted in HRP positive neurons in the
ipsilateral rostral nucleus ambiguus (Fig. 1A). These
neurons were distributed in the ventrolateral region of
the nucleus. The labeled area was found to extend for
a n average of 430 pm rostrally from a point 420 pm
rostral to obex. No labeled cells were found caudal to
this area. The cells (average 71 in number) consisted
primarily of small spindle-shaped and small (but more
typical looking) multipolar motor neurons. The spindle
cells were always dorsomedial to the latter, the majority of which were found to occupy the most rostral portion of the nucleus. In addition to the nucleus ambiguus, labeled neurons were identified within the dorsal
motor nucleus of the vagus (Fig. 1B). These vagal neurons (average 69 in number) were found a t a level 338
pm rostral to obex and to extend rostrally for approximately 454 pm.
Labeled neurons of the recurrent laryngeal nerve
were identified over a much wider area of the ipsilatera1 nucleus ambiguus. Two distinct populations of
neurons were identified within the nucleus, a rostral
and caudal group. The rostral labeling was found ventrolaterally within the nucleus and extended for a n
average of 260 pm rostrally from a point 525 pm rostral to obex and consisted primarily of the small spindle-shaped cells (Fig. 2C). The caudal group of cells
extended for a n average of 740 pm and were distributed rostrally from a point 409 pm caudal to obex or a t
the level of pyramidal decussation. The neurons of this
group consisted primarily of the larger multipolar-type
motor neurons (Figs. 2A,B). Labeling of the dorsal motor nucleus of the vagus via the recurrent laryngeal
nerve was found to be virtually nonexistent, with a n
average of three labeled cells per animal.
The dimensions of the motor neuron pool of the recurrent laryngeal nerve within nucleus ambiguus reveal two interesting phenomena: First, there is a gap in
the labeling of recurrent laryngeal motor neurons
within the nucleus ambiguus, thereby creating a rostral and caudal group. This gap (void in labeling) was
found to extend rostrally for approximately 200 pm
from a level 330 pm rostral to obex. Second, the dimensions further reveal a n area of overlap in the ventrolateral pole of nucleus ambiguus between the two laryngeal nerves. To recapitulate, the motor neurons
contributing to the superior laryngeal nerves began
420 pm rostral to obex and continued rostrally for 430
pm. The neurons contributing to the recurrent laryngeal nerve were located 525 pm rostral to obex and
continued rostrally for 260 pm. These data suggest a n
area of overlap between the neuron pools of the superior and recurrent laryngeal nerves spanning 260 pm.
To further demonstrate this gap and overlap, horizontal sections were made following the bilateral exposure
of laryngeal nerves alternatively and contralateral to
MOTORNEURONSOFTHELARYNGEALNERVES
553
Fig. 1. Brightfield photomicrographs taken in the coronal plane demonstrating HRP labeled motor
neurons of the superior laryngeal nerve. A: Ventrolateral region of rostral nucleus ambiguus. Bar = 40
pm. B: Dorsal motor nucleus of vagus. Dorsal = top; medial = left; Bar = 30 pm.
each other (i.e., the recurrent on the right and the superior on the left). This arrangement provided for a
more precise method of comparison (Fig. 31, which confirmed our previous observations.
DISCUSSION
In the Results we described a n area within the nucleus ambiguus that is common to both the superior
and recurrent laryngeal nerves. The methodology of
whole nerve exposure to the tracer material utilized in
this study maximizes the possibility of identifying the
total motor neuron population and the distribution for
each laryngeal nerve. The superior laryngeal nerve in
addition to its efferent projection to the cricothyroid
muscle contains a small component to the inferior constrictor muscles of the pharynx and to the proximal
muscularis externa of the esophagus (Bieger and Hopkins, 1987), the motor neurons of which are distributed
with those of the cricothyroid. The location of the superior laryngeal nerve motor neurons described in this
study are similar to that described for the cricothyroid
in the rabbit (Okubo et al., 1987); cat (Gacek, 1975;
Davis and Nail, 1984; Pasaro e t al., 1983); dog (Hisa et
al., 1984); and rat (Hinrichsen and Ryan, 1981; Bieger
and Hopkins, 1987), with minor species differences.
These neurons are located in the ventrolateral region
of the nucleus.
The motor neurons of the recurrent laryngeal nerve
in its most rostral distribution are within the ventrolateral region of nucleus ambiguus overlapping those
of the superior laryngeal nerve. These neurons are seen
as a discrete population of the RLN that is distinct from
those classically described. The distribution of these
more rostrally placed recurrent laryngeal nerve motor
neurons using neuronal tracers was first described by
Gacek (1975) in the kitten and later by Hinrichsen and
Ryan (1981) in the rat. Subsequent to these studies,
other reports on the laryngeal motor neuron failed to
describe this rostral population of cells (Hisa e t al.,
1984; Bieger and Hopkins, 1987; Yoshida et al., 1982).
The uniqueness of this study is the exposure of each
laryngeal nerve to the tracer contralateral to one another and visualizing the labeled neurons in the horizontal plane. In so doing, the distribution of the neurons for each nerve could be compared in the same
animal.
No attempts were made in this study to identify the
somatotopic organization of nucleus ambiguus in reference to each laryngeal muscle since numerous studies have been conducted on this subject. In brief, the
cricothyroid motor neurons (via the superior laryngeal
nerve) are most rostral, followed by the posterior cricoarytenoid and the remaining intrinsic laryngeal motor neurons, the thyroarytenoid, lateral cricoarytenoid,
and interarytenoid more caudally and overlapping
each other (Hinrichsen and Ryan, 1981; Bieger and
Hopkins, 1987). Somatotopically, the rostral group of
recurrent laryngeal motor neurons is a component of
the posterior cricoarytenoid motor neuron pool, the
larger population of which is within the more rostral
portion of the caudal group (Hinrichsen and Ryan,
1981). Because of the somatotopic organization of the
cricothyroid and the posterior cricoarytenoid motor
neurons relative to superior and recurrent laryngeal
nerves, the whole nerve preparation was utilized in
this study in lieu of the individual muscle injections
which are always at risk to the undesirable spread of
tracer material to neighboring structures.
The recent electrophysiological study on the motor
neurons of the recurrent and superior laryngeal nerve
by Yajima and Hayashi (1989) reported on neurons
whose axons traverse both laryngeal nerves to innervate the intrinsic muscles of the larynx. They have
suggested that these axons branch midway between
554
J.W. PATRICKSON E T AL.
Fig. 2. A series of brightfield photomicrographs taken in the coronal
plane demonstrating recurrent laryngeal motor neurons and their
arrangement a t various levels within the nucleus ambiguus. A. Caudalmost region of the nucleus. B: At the level of obex. C: Ventrolateral
area of the rostral area of the nucleus. Motor neurons in panels A and
B are from the caudal group, while those of panel C are from the
rostral group. Dorsal = top; medial = left; bar = 40 pm.
cated ventrolaterally within the nucleus ambiguus.
The remaining intrinsic muscles of the larynx are adductors. The motor neuron pools of the adductors are
located dorsally and are more caudally placed with extensive overlapping of the motor neuron pool for each
muscle (Hisa et al., 1984; Okubo et al., 1987; Hinrichsen and Ryan, 1981, Gacek, 1975).
Motor neuron size may be of functional significance
relative to its effector organ. The intrinsic laryngeal
muscles are fast contracting (Syrovy and Gutmann,
1971) and contain no muscle spindles (Raman and Devanandan, 1989). Similar to most skeletal muscles,
there are two types of muscle fibers found in the laryngeal muscles, one with low actomyosin ATPase activity
(type I) and the other with high actomyosin ATPase
activity (type 11) (Rosenfield et al., 1982; Sahgal and
Hast, 1974). Type I muscle fibers are slow contracting
and fatigue resistant. In contrast, the type I1 fibers are
the soma and the effector. The functional significance fast contracting and fatigue rapidly. Type I fibers are
of these laryngeal motor neurons are yet to be deter- innervated by small motor neurons. These fibers are
mined; however, this neuronal arrangement along easily recruited (low threshold), control fine movewith those of the presently described overlapping ros- ments, and are capable of prolonged, slow rhythmic
tral group may represent a component of the efferent discharge. The fiber ratio per neuron is small. The type
system that is involved in the coordination and co-ac- I1 muscle fibers discharge a t higher thresholds, have
tivation of the respective laryngeal muscles, the abduc- high discharge rates, and develop more tension but fatigue faster. These fibers are innervated by large motor
tors, during the respiratory cycle.
In support of this concept, the overlapping of the mo- neurons with a high fiber ratio per neuron (Warmolts,
tor neuron populations to the cricothyroid and the pos- 1981; Burke et al., 1982; Burke et al., 1973; Burke and
terior cricoarytenoid muscles appears to be a functional Tsairis, 1974).
grouping of the motor neurons relative to that of its
Although the laryngeal muscles contain both fiber
effector. These muscles are abductors; when co- types, there are more type I fibers in the abductors and
activated, they enlarge the aperture of the laryngeal a preponderance of type I1 in the adductors (Teig et al.,
airway. The motor neurons of these abductors are lo- 1978).Functionally, the fast action of the adductors are
MOTOR NEURONS OF TH ELARYNGEALNERVES
OBEX
555
to the laryngeal area. This visceral component is not
visualized when the muscles of the larynx are exposed
to neuronal tracers (Hinrichsen and Ryan, 19811, but
is, rather, with injections into the laryngeopharyngeal
areas including the mucosa or, a s in this study, with
the exposure of the proximal end of the transected
nerves. Contrary to the findings of Kalia and Mesulam
(1980a,b), the present results show a substantial contribution of the dorsal motor nucleus of the vagus to the
larynx, primarily via the superior laryngeal nerve.
This visceromotor component is unilateral. This observation is supported by similar observations in the monkey (Yoshida et al., 1982) and guinea pig (Basterra e t
al., 1988). Kalia and Mesulam (1980a,b) reported bilateral innervation to the larynx of the cat. This apparent
contradiction, if not due to species differences, is resolved in that relatively large volumes of neuronal
tracer was injected into the laryngeal mucosa; thus, the
possibility of diffusion across the midline. If this is the
case, then the results would be mistaken as a bilateral
projection.
To conclude, the present study has affirmed the rostral placement of the superior laryngeal nerve motor
neurons within the ventrolateral subdivision of nucleus ambiguus. There are two distinct populations of
recurrent laryngeal nerve motor neurons within nucleus ambiguus: a rostral and a caudal. The most rostral component overlaps those of the superior laryngeal
nerve and may represent a component of the neuroanatomical substrate for the co-activation of the laryngeal abductors.
LITERATURE CITED
Fig. 3. A projected tracing of the lower brain stem and spinal cord in
the horizontal plane illustrating labeled laryngeal motor neurons
(stippled area) within nucleus ambiguus following the exposure of the
laryngeal nerves (superior, left; recurrent, right) to HRP in the same
animal. Arrow indicates gap between the rostral and caudal groups of
the recurrent laryngeal nerve motor neurons. lfp: longitudinal fasciculus pons; ml: medial lemniscus; mlf: medial longitudinal fasciculus;
m5: motor trigeminal nucleus; s5: sensory root trigeminal nerve. Bar
= 1 mm.
essential for the protection of the airway. The cricothyroid muscle tenses the vocal cord and requires prolonged contraction. Both the posterior cricoarytenoid
and the cricothyroid muscles are tonically active during the inspiratory phase of respiration (Woodson et
al., 1989; Horiuchi and Sasaki, 1978; Konrad and Rattenborg, 1969). This rhythmic co-activation of these
muscles is best suited to a larger ratio of type I fibers.
The neurons of overlap are the small type which also
innervate the type I fibers of the abductors. This arrangement of the overlapping neurons may represent
the neuroanatomical substrate for the co-activation of
the abductors during eupnea.
The presence of labeled neurons in the dorsal motor
nucleus of the vagus via the laryngeal nerves demonstrates that these nerves provide visceral innervation
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