THE JOURNAL OF COMPARATIVE NEUROLOGY 349~129-147 (1994)

Innervation of the Larynx, Pharynx, and Upper Esophageal Sphincter of the Rat

JAMES B. KOBLER, SUBIMAL DATTA, RAJ K. GOYAL,

Harris Peyton Mosher Laryngological Research Laboratory, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114 (J.B.K., E.J.B.),

Department of Psychiatry, Harvard Medical School, Boston, Massachusetts 021 15 (S.D.), and Department of Gastroenterology, Beth Israel Hospital, Boston, Massachusetts 02 115 (R.K.G.)

AND ELIZABETH J. BENECCHI

ABSTRACT We identified a ‘semicircular’ compartment of the rat thyropharyngeus muscle at the

pharyngoesophageal junction and used the glycogen depletion method to determine how the fibers of this muscle (as well as all others of the pharynx and larynx) are innervated by different cranial nerve branches. The semicircular compartment appears anatomically homologous to the human cricopharyngeus muscle, an important component of the upper esophageal sphincter. While we found very little overlap in the muscle targets of the pharyngeal, superior laryngeal and recurrent laryngeal nerves within the pharynx and larynx, the semicircular muscle receives a dual, interdigitating innervation from two vagal branches: the pharyngeal nerve and a branch of the superior laryngeal nerve we call the dorsal accessory branch. After applying horseradish peroxidase to either of these two nerves, we compared the distribution and number of cells labeled in the brainstem. The dorsal accessory branch conveys a more heterogeneous set of efferent fibers than does the pharyngeal nerve, including the s o n s of pharyngeal and esophageal motor neurons and parasympathetic preganglionic neurons. The observed distribution of labeled motor neurons in nucleus ambiguus also leads us to suggest that the semicircular compartment is innervated by two subsets of motor neurons, one of which is displaced ventrolateral to the main pharyngeal motor column. This arrangement raises the possibility of functional differences among semicircular compartment motor neurons correlated with the observed differences in brainstem location of cell bodies. D 1994 Wiley-Liss, Inc.

Key words: motor neuron, vagus nerve, deglutition, brainstem, dysphagia

The mammalian upper esophageal sphincter (UES) nor- mally maintains closure of the upper end of the esophagus, preventing tracheobronchial aspiration and entry of air into the esophagus during inspiration. Disorders of UES motor control may affect either resting tone or swallow-induced opening, leading to pulmonary aspiration of gastrointesti- nal contents and dysphagia, respectively. The main muscu- lar component of the UES is the cricopharyngeus muscle, a caudal subdivision of the inferior pharyngeal constrictor muscle (see review by Shapiro and Goyal, 1988). Unlike other pharyngeal constrictor muscle fibers, which insert obliquely into a midline raphe posteriorly, cricopharyngeus muscle fibers form a continuous semicircular loop around the junction of the pharynx and esophagus. Anteriorly, the cricopharyngeus muscle fibers attach to the cricoid carti- lage. The broad posterior lamina of the cricoid cartilage is a rigid structure against which the cricopharyngeus can compress the hypopharyngeal lumen. The more rostra1 pharyngeal constrictors show a phasic burst of activity during swallowing while the cricopharyngeus is tonically

active, relaxing briefly as the bolus passes through (An- drew, 1956b; Shipp et al., 1970; Murakami et al., 1972; Asoh and Goyal, 1978).

The motor innervation of the UES is difficult to deter- mine in a precise manner because the lower pharynx and adjacent esophagus receive contributions from several branches of the vagus nerve, namely, the recurrent laryn- geal nerve (RLN), the superior laryngeal nerve (SLN) and the pharyngeal plexus (Lemere, 1932; Sprague, 1944), and because these branches anastomose with each other. It is of some importance to establish the nerve supply of the UES, for neurectomy of the lower parts of the pharyngeal plexus has shown promise as a method for improving both swallow- ing and the ability to use tracheoesophageal speech in some laryngectomy patients, presumably by reducing residual

Accepted April 27,1994. Address reprint requests to Dr. James B. Kobler, Mosher Laryngological

Research Laboratory, Massachussetts Eye and Ear Infirmary, 243 Charles St., Boston, MAO2114.

O 1994 WILEY-LISS. INC.

130 J.B. KOBLER ET AL.

stained lightly with 0.5% osmium tetroxide to enhance their visibility. In two cases the branching patterns of the pharyngeal and laryngeal nerves were drawn in detail. After the pharynx, larynx and associated nerves had been completely dissected, the specimens were fixed in 10% formalin. For the specimen illustrated in Figure 1, seg- ments of each nerve branch were removed after fixation, dehydrated and embedded in Epon. Sections 0.5 km in thickness were then cut on an ultramicrotome and were stained with toluidine blue. Each section was photo- graphed, the myelinated fibers were counted and the peri- meters of the external diameters of each myelinated fiber were measured with a computer-based planimeter. A value for the fiber’s diameter was computed from the perimeter data as if the fiber had a circular cross-section.

In addition, three animals were perfused with aldehyde fixatives, and the whole head and neck region of each was removed, decalcified in EDTA, embedded in celloidin and sectioned at 20 km. A complete set of serial sections was obtained in the transverse, sagittal and coronal planes. Sections were stained with hematoxylin and eosin, eosin- methylene blue or cresylecht violet.

Glycogen depletion experiments Rats (n = 21) were anesthetized and suspended in a

supine position within a cylindrical coil of plastic tubing that was perfused with water warmed to 37°C. Their heads were secured in a stereotaxic frame using ear bars and a snout clamp. Following a midline incision of the neck, the salivary glands were dissected bluntly aside and the sterno- hyoid, sternothyroid, and omohyoid muscles were cut and retracted on the side to be prepared for stimulation. Prior to pharyngeal nerve (PN) stimulation, the posterior belly of the digastric was detached from the temporal bone and retracted anteriorly. The nerve to be stimulated was dis- sected gently, freed from surrounding connective tissue and was placed on bipolar stainless steel hook electrodes. The isolated nerve was then covered with warm mineral oil to help isolate it electrically from surrounding tissues and to minimize dehydration. The shock train parameters and total stimulus time were varied somewhat between animals in an effort to get the most complete depletion possible. The paradigm found to be most effective consisted of 300 ms trains of shocks delivered at 50 Hz, separated by 100 ms intervals and continued for a total duration of 25 minutes. The stimulus current level was adjusted to provide maximal contraction as observed visually and then the level was doubled. In addition, we found that two procedures greatly enhanced our ability to achieve thorough depletion: (1) at the onset of simulation a dose of 0.1 cc of 1:10,000 units of epinephrine was given subcutaneously to facilitate glycogen utilization and help inhibit its uptake; and (2) after the 20-30 minutes of stimulation, an overdose of Nembutal (0.5 to 1.0 cc) was given intraperitoneally, resulting in cardiac and respiratory arrest within several minutes. Stimulation was then continued for 30 seconds following respiratory arrest. Following stimulation, the each animal was quickly exsanguinated and the larynx and pharynx were removed en bloc. The block of tissue was mounted on a piece of cork with Tissue-tek and was rapidly frozen in melting isopentane cooled to -159°C in liquid nitrogen. Serial 10-16 km sections were cut in a horizontal plane (parallel to the long axis of the pharyngeal lumen) using a cryostat. Every other section was stained by the periodic- acid Schiff method (Bancroft and Cook, 1984). The control

UES tone (Singer et al., 1986). A better understanding of UES innervation could also provide some clues to the developmental and evolutionary origins of the UES muscle, since the different vagal branches are associated with the derivatives of different branchial arches (Williams and Warwick, 1980).

The central control mechanisms of the UES are of interest from both practical and basic viewpoints. Failure of the UES to relax leads to severe dysphagia that might be amenable to drug therapy if more was known about the physiology and pharmacology of UES motor neurons and their inputs. The current treatment is a surgical myotomy of the cricopharyngeus. In a more general context, the pharyngeal constrictors appear to be a further example of a muscle group with compartments specialized for specific tasks, a form of organization that has been the subject of recent studies correlating muscle architecture with moto- neuron pool organization (see review by Windhorst et al., 1989). Since the pattern of activity of the UES muscle is different from that of the other pharyngeal constrictors, the inputs and/or intrinsic properties of UES motor neurons must differ from those of other pharyngeal motor neurons.

The rat is now a widely used species for studying the anatomy, physiology and pharmacology of brainstem motor neurons and interneurons involved in swallowing (Andrew, 1956a,b; Bieger et al., 1977; Kessler and Jean, 1985; Bieger and Hopkins, 1987; Cunningham and Sawchenko, 1989; Altschuler et al., 1989, 1991; Wang and Bieger, 1991). However, the architecture, innervation and function of the rat UES have received little attention. The classic work of Andrew (1956a,b) showed that there are physiological similarities between rat and other mammals in the patterns of activity in the UES region, suggesting that common principles of UES control can be studied in this species. Toward this end we have now identified the muscle fiber targets of the different laryngeal and pharyngeal nerve branches in the rat using the method of glycogen depletion (Kugelberg and Edstrom, 1968). In this method, a periph- eral nerve branch is electrically stimulated until the muscle fibers that it innervates are depleted of glycogen. The number and distribution of depleted fibers are determined histochemically, revealing a complete innervation territory with great resolution. This method is thus ideally suited for determining the targets of nerves that become intermixed and that are impossible to sort out by dissection or routine histology. Once the peripheral targets of these nerves have been defined in this way we are also in a better position to interpret the results of application of retrograde tracers to the same nerves. We thus also used retrograde transport of horseradish peroxidase to define the distribution of cells in the brainstem that participate in the innervation of the UES.

MATERIALS AND METHODS The experimental animals were male albino Wistar rats

obtained from Charles River Labs, weighing between 200 g and 300 g. They were anesthetized with Nembutal(35-50 mgikg, i.p.) for surgical procedures and prior to perfusion with fixative.

Anatomy of the rat pharynx The anatomy of the rat pharyngeal muscles and nerves

was studied in many fresh specimens by microdissection under a Zeiss operating microscope. The nerves were

INNERVATION OF RAT PHARYNX AND LARYNX 131

TABLE 1. Summary of the Locations of Retrograde Tracer Applications and Injections

Injection site Tracer used Tracer amount n

Superior laryngeal nerve HRP crystal 4 Thyropharyngeus muscle CT-HRP 0.1-0.5 pl 10

Pharyngeal nerve HRP Crystal 3 DAB-SLN HRP crystal 4 Esophagus CT-HRP 0.1-10 p1 5 Cricothyroid m. CT-HRP 0.1-0.2 pl 3 Glossopharyngeal nerve HRP crystal 2 Cervical vagus HRP crystal 1 Posterior belly of digastric m. CT-HRP 0.1-0.2 pl 1 Recurrent laryngeal nerve HRP crystal 1 Fascia of surgical site HRP crystal 2 Total 36

animals were maintained anesthetized for two hours, which was approximately the time it took to perform the nerve stimulation experiments, and were then sacrificed and processed like the experimental cases. In a separate series of animals, horizontal sections through the pharynx were stained for succinic dehydrogenase (Nachlas et al., 1957).

HRP histochemistry Anesthesia and surgical exposures were the same as

those used for nerve stimulation. Horseradish peroxidase (HRP; Sigma type VIA) or HRP conjugated to cholera toxin B-subfragment (CT-HRP) were used as tracers. CT-HRP was used to produced a more extensive filling of dendrites. For nerve applications, a tiny pellet of the tracer was applied to the severed nerve branch and the branch was isolated from surrounding tissues by plastic film. The tracer was left in contact with the nerve for about 20 minutes with occasional mechanical teasing of the nerve stump, after which the plastic film was removed and the entire operative field was rinsed thoroughly with saline. To control for possible HRP spread to thoracic or abdominal viscera, in two experiments involving HRP application to a branch of the SLN, the cervical vagus was tightly ligated with 5-0 silk sutures and the vagal trunk distal to the sutures was cauterized with a bipolar cautery. For muscle injections, 0.1-0.2 p1 CT-HRP was injected into the pharyn- geal or esophageal muscles through a beveled micropipet epoxied to a Hamilton syringe. In some experiments the pharyngeal muscle was approached by a midline transec- tion of the larynx to visualize the pharyngeal wall directly. Table 1 summarizes the number of animals used for each type of injection. After a survival period of 24 hours, the animals were perfused intracardially with a fixative contain- ing 1.25% glutaraldehyde and 1% paraformaldehyde. The brains were then immersed in 0.1 M phosphate buffer, pH 7.6, containing 30% sucrose for 24 hours and then 40-60 pm transverse frozen sections were cut on a sliding micro- tome. The sections were processed for HRP histochemistry with tetramethylbenzidine as a chromogen and were coun- terstained with neutral red (Mesulam, 1982). The distribu- tion of HRP-labeled cells in the brainstem was plotted with the aid of a camera lucida and a computer-based 3-D reconstruction system (Joseph et al., 1985) and was also documented photographically.

Analysis methods: Glycogen depletion The number and distribution of glycogen-depleted muscle

fibers was documented by observation, photographically and with a computer-interfaced video microscope (Nikon Optiphot microscope with 10 x PlanApochromat lens, Xy- bion CCD-50 camera, Neotech frame-grabber, Macintosh

Quadra 800 computer, NIH Image software). The percent- age of depleted fibers was determined for each muscle by counting the number of depleted and nondepleted muscle fibers. We counted and measured the staining intensity of each muscle fiber from montages of digitized images of muscle cross-sections using NIH-Image software. A small circle was placed over the center of each fiber to mark and count it and the integrated gray level value was determined for the area of the spot. Optical densities (OD) were computed by calibrating the video system with blanks of known optical density. Histograms of optical density showed clear bimodal distributions with little overlap between peaks (as in Fig. 12). For counting purposes we categorized all fibers in the low OD peak as depleted and all fibers in the high OD peak as nondepleted. Because of the vagaries of sectioning it was not always possible to count every fiber in a muscle; however, in all but a few cases it was possible to count enough fibers to categorize the muscles according to the relatively broad percentile ranges used in Table 1.

RESULTS General anatomy

In two animals the fine details of the innervation of the larynx and pharynx were studied by microdissection and osmium tetroxide staining. In both cases the patterns of nerve branching were very similar. One of these cases is illustrated in Figure 1. The conformation of the larger branches was consistent with our observations made in the large series of animals prepared surgically for nerve stimu- lation or injection.

We found that four divisions of the pharyngeal constrictor muscles could be readily identified in the rat. The thyropharyngeal and hypopharyngeal divisions are most clearly observed from the posterior view; they form the bulk of the posterior pharyngeal wall (Fig. 1). The origins of the thyropharyn- geus from the thyroid cartilage and of hyopharyngeus from the hyoid bone are easily observed. These two divisions insert obliquely into the midline pharyngeal raphe, which in turn is suspended from the skull base. The stylopharyn- geus muscle enters the body of the pharynx between these two muscles. The fibers of the stylopharyngeus interweave with those of the palatopharyngeus and appear to insert into the submucosal connective tissue.

According to House (19531, there are pterygo-, palato-, glosso-, and salpingo-pharyngeal components to the rat pharyngeal musculature, of which the glossopharyngeal muscle is the only one with a constrictive orientation, the others being directed longitudinally. In most of our experi- ments the pharynx was detached from the skull base for frozen sectioning and it was therefore not possible to trace all pharyngeal muscle fascicles to their origins in order to discriminate between these different components. There is a separate inner muscle compartment deep to the thyropha- ryngeal and hyopharyngeal muscles, referred to here as the palatopharyngeus muscle, but that probably also contains small contingents of salpingopharyngeal, glossopharyngeal and pterygopharyngeal fibers. Some fibers of the palatopha- ryngeus arch around the posterior pharyngeal wall insert- ing near, but not crossing, the midline.

Near the junction of the pharynx and esophagus are muscle fibers that arch across the posterior pharyngeal wall to the opposite side without inserting into the midline raphe. These fibers are overlapped by the more oblique

Myology of the pharyngeal muscles.

132 J.B. KOBLER ET AL.

.. TRACHEA

Fig. 1. Dorsal surface of the rat pharynx, upper esophagus and associated nerve branches as visualized in a single case by rnicrodissec- tion and osmium tetroxide staining. The semicircular muscle (SM) is normally somewhat more obscured by the overlying thyropharyngeus muscle (TP). The stylopharyngeus muscles (SP) have been rotated anteriorly from their normal positions. A comparison of right and left sides shows typical variations in the branching and distribution of the pharyngeal nerve (PN). IX, glossopharyngeal nerve; X, vagus nerve; SLN, superior laryngeal nerve; RLN, recurrent laryngeal nerve; HP, hyopharyngeus muscle (appears foreshortened in this view); int, inter- nal branch of SLN; ext, external branch of SLN; d.a.b., dorsal accessory branch of SLN; r.a., ramus anastomoticus; nerve-branch labels “a-k” are for reference to other figures and to text. Scale bar = 1 mm.

fibers of the thyropharyngeus, some of which have been removed for illustrative purposes in Figure 1. We will refer to this division as the semicircular muscle (SM). In careful examination of serial sections through this region, no insertion of pharyngeal muscle fibers on the cricoid carti- lage could be found. Thus, it appears that the rat does not have a strictly defined cricopharyngeus muscle although the SM resembles its general location and semicircular fiber arrangement.

Because we were particularly interested in the innerva- tion of the SM, we sought to establish whether this region was a continuation of the upper esophagus or part of the

Fig. 2. A. Horizontal section through the pharyngeal and esopha- geal muscles stained histochemically for succinic dehydrogenase. Note difference in staining of fibers in semicircular muscle (SM) and esophagus (ESO). TP, thyropharyngeus muscle; PP, palatopharyngeus muscle. B Enlargement of area enclosed by box in A. Scale bars = 200 p n .

pharyngeal muscle proper. Figure 2 shows a horizontal section through this region that has been stained for succinic dehydrogenase. Note that the staining of the SM compartment is similar to that of the rest of the thyropha- ryngeus muscle and considerably darker than the adjacent esophagus. A similar difference in oxidative activity was noted between the esophagus and cricopharyngeus (CP) in the guinea pig by Bonington et al. (1987). The SM and thyropharyngeus muscle fibers are also larger in diameter than the adjacent esophageal muscle fibers. These observations suggest that the SM is of pharyngeal, not esophageal origin.

The following muscles were identified in the rat larynx: posterior cricoarytenoid, crico- thyroid, thyroarytenoid, and lateral cricoarytenoid. We did not observe an interarytenoid muscle. Two additional muscles were identified that have not previously been described: one, which we call the rostral cricoarytenoid, originates from the rostral and medial part of the posterior lamina of the cricoid cartilage and inserts on the lateral face of the arytenoid cartilage; the other, which we call the cricovocal muscle, originates just lateral to the rostral cricoarytenoid muscle and inserts on a small bar of cartilage anterior to the vocal folds near the base of the epiglottis.

MHology of the larynx.

INNERVATION OF RAT PHARYNX AND LARYNX

Innervation ofpharyngeal muscles. Grossly, the phar- ynx appears to be innervated from two sources: the pharyn- geal nerve (PN; Fig. l), which originates near the nodose ganglion of the vagus nerve and divides into several branches on the posterior aspect of the pharyngeal musculature, and the SLN. The SLN has three main branches: an internal sensory branch, a motor branch to the cricothyroid muscle and a branch that innervates the upper esophagus, the region of the junction between the pharynx and esophagus and that gives off a communicating twig, the ramus anasto- moticus, to the RLN. We will refer to this third branch as the ‘dorsal accessory branch of the SLN’ (DAB-SLN). A similar branching pattern of the SLN has been noted in earlier studies on the rat (Andrew, 1956a; Bieger and Hopkins, 1987). In histological material, clusters of gan- glion cells were frequently observed along the course of the DAB-SLN, particularly near its origin (Fig. 3A,B). Innerva- tion of the stylopharyngeus muscle by the glossopharyngeal nerve was clearly observed; however, innervation of in- trapharyngeal parts of this muscle by the PN could not be

133

ruled out. Branches of both the glossopharyngeal and PN coursed anteriorly and entered the hyopharyngeal muscle but only the PN appeared to terminate there.

For the specimen illustrated in Figure 1, the nerve branches labeled with lower case letters were excised, embedded in plastic and sectioned to count and measure myelinated axons. Counts of myelinated axons are shown in Figure 4 and histograms comparing fiber diameters are presented in Figure 5. Statistical comparisons of the fiber diameter distributions (t-test) for this one specimen showed that the fibers in the PN branch (branch g) were signifi- cantly larger (mean diameter = 6.7 p,m) than fibers in branches d, e, or k ( P < .0001). Fibers in the branch to the SM were significantly larger (mean diameter = 4.3 p,m; P < .0001) than those in the branches to the esophagus. Finally, there was no significant difference ( P = 2 2 ) in diameter measurements between the two branches to the esophagus, whether they originated from the SLN (branch e: mean diameter = 3.4 pm) or from the RLN (branch k: mean diameter = 3.5 pm).

Fig. 3. A Horizontal section through the caudal part of the thyropharyngeus muscle (TP) and its semicircular compartment (SM). The dorsal accessory branch of the superior laryngeal nerve (DAB-SLN) can be seen in the connective tissue near the muscle. B: Higher power

view showing ganglion cell bodies (arrowheads) in the DAB-SLN. In this case the SLN was electrically stimulated and the tissue was stained for glycogen with the periodic acid-Schiff (PAS) method. Depleted fibers are unstained. Scale bars = 100 wm.

134

SUPERIOR LARYNGEAL PHARYNGEAL

J.B. KOBLER ET AL.

NERVE (a) NERVE (I)

aXOM (b) INTERNAL

(h) ROSTRAL BRANCH

138

(c) BRANCH TO CRICOTHYROID MUSCLE

105

DORSAL ACCESSORY BRANCH OF THE SLN

(d) BRANCH TO PHARYNGO- ESOPHAGEAL JUNCTloN ZONE

(e) BRANCH TO UPPER ESOPHAGUS

(t) BRANCHTO RECURRENT LARYNGEAL NERVE

38

Fig. 4. Schematic representation of the superior laryngeal and pharyngeal nerves and their myelinated fiber populations. Small letters refer to Figure 1, where the actual nerve branches are depicted. The

thicknesses of the lines are proportional to number of myelinated fibers in each branch. Note that the dorsal accessory branch has more fibers than the external branch to the cricothyroid muscle.

Glycogen depletion results Densitometric measurements. Examples of densitomet-

ric data are presented first to show the clear-cut difference in staining intensity between stimulated and unstimulated fibers. Densitometric measurements of PAS staining of the pharyngeal muscles, comparing stimulated and unstimu- lated sides and control animals where neither side was stimulated, are shown in Figure 6. Except for the SM, where depleted fibers were found bilaterally, there was little overlap between histograms of fiber staining ipsilat- era1 or contralateral to the stimulation or between histo- grams of depleted and control muscles. The small amount of overlap is consistent with the occasional lightly staining fibers that were seen in control animals. The variations in the control histograms reflect both natural differences between muscles in their glycogen contents and slight differences in histological processing between individual experiments.

A summary of the results for animals in each stimulation category is presented in Table 2 and the results are described below for individual nerves.

Following stimulation of the main trunk of the SLN, large numbers of fibers within the cricothyroid muscle were depleted (Table 2, Fig. 7). The percentage of depleted fibers in the cricothyroid muscle ranged from about 50% to >99%. Possible reasons for obtaining less than complete depletion in some cases are explored in the Discussion.

In addition, a few depleted fibers were found in the thyropharyngeus muscle ipsilateral to the stimulation in two cases. More striking was the large number of depleted fibers seen in both the ipsilateral and contralateral SM after SLN stimulation (Fig. 8B). Given the midline-spanning structure of the SM, it was not surprising to find depleted fibers on both sides following unilateral nerve stimulation. A quantitative analysis of innervation fractions is pre- sented below. Depleted fibers were also numerous in the upper esophagus after SLN stimulation (Fig. 91, with a

Superior laryngeal nerue.

;uln(g) 20 15

10 5 0 101

0 2 4 8 8 1 0 1 2 1 4

branch to '4 I bl semicirdarm.(d)

branch from DAB-SLN 15 I to esophagus (e) tL

Axon Diameter (pm)

Fig. 5. Histograms of myelinated fiber diameters for vagus nerve branches innervating above, at, and below the semicircular muscle. Small letters in parentheses refer to Figures 1 and 4. A Branch to hyopharyngeus. B: Branch to SM. C: Branch from DAB-SLN to esophagus. D: Branch from RLN to esophagus. Dashed lines indicate range of fiber diameters in the branch to the SM.

INNERVATION OF RAT PHARYNX AND LARYNX 135

HYOPHARYNGEUS PALATOPHARY NGEUS STYLOPHARY NGEUS LOCATION OF FIBERS

TO STIMULUS RELATIVE 1 0 0

IPSILATERAL

CONTROL CASE (no stirnulation)

IPSILATERAL

12 40 20

30 15 8

20 10

10 5 4

0 0 0 0 0.25 0.5 0 0.25 0.5 0 0.25 0.5

optical density of muscle fibers

THYROPHARYNGEUS SM-PHARYNGEAL N. STlM SM-SLN STlM

25 20

z

L l 5 CONTRALATERAL 9

- u 5 0

40

30

CONTROLCASE 20 (no stimulation)

10

0 0 0.25 0.5 0 0.25 0.5 0 0.25 0.5

optical density of muscle fibers

Fig. 6. Histograms showing the optical density of PAS-stained muscle fibers in typical glycogen depletion experiments. Shaded bars are to aid in visual alignment of histograms. For each of the pharyngeal muscles, measurements were made of comparable regions on the stimulated and unstimulated sides following pharyngeal nerve stimula-

tion and for one control animal. In the SM-SLN STIM case the superior laryngeal nerve was stimulated. Fibers with more intense staining for glycogen have a greater optical density. Histograms for the semicircular muscle show bimodal distributions because of partial innervation by the pharyngeal and superior laryngeal nerves.

136

TABLE 2. Summary of All Glycogen Depletion Experiments'

J.B. KOBLER ET AL.

~~~

Nerve stimulated

Muscle None Superior laryngeal Recurrent laryngeal Pharyngeal Glossopharyngeal

5 6 4 Stylopharyngeus - _ - - - - - - - _ _ - _ _ _ - - _ Hypopharyngeus _ _ - - - - - - _ _ _ _ _ 5 5 5 3 5 - - -

5 5 5 5 5 a Palatopharyngeus a a Thyropharyngeus I 1 - 1 1 1 1 1 - - - - - - - 5 6 5 5 1 - Ipsilateral semicircular 1 1 - b 2 2 3 3 - - 1 - - b 3 3 3 3 - - - Contralateral semicircular - - - b 1 2 3 2 - - - - - b 2 3 3 3 - - - Esophagus _ _ - + t + + + + + + + + - + - + - - - - Cricothyroid _ _ _ 5 4 4 6 5 - - - - I - - - - - - - - Posterior cricoarytenoid - - - - - - - - 4 4 3 6 6 - - - - - - - - Lateral cricoarytenoid 1 1 - - - - - - 5 3 3 6 6 - - - - - - - - Thyroarytenoid - - _ 1 - - 1 - 5 3 4 6 6 - - - - - - - - Rostralcricnarytenoid - - - - - - - - 2 2 2 6 - - - Cricovocd - - - - - - - - 2 2 2 6 6 - - - - - - - -

18 19 Case number 10 11 17 1 5 6 7 16 2 8 9 14 15 4 12 13 20 21 3

'Code B fibers depleted 6 95100% 5 75-95x 4 5&75% 3 25-50? 2 5-25% 1 0% < n < 5%

_ _ - - - - - - - _ _ - -

0% -

a: Because stylopharyngeus fibers interdigitate with palatopharyngeus fibers, we Cannot be sure about identity of depleted fibers seen in palatopharyngeus in these cases b: Histology inadequate for counting. t : For esophageal muscle only, the presence 1 + 1 of depleted fibers is scored since It was not possible to count all esophageal fibers.

preponderance of depleted fibers located in the deeper layer of the esophageal muscle. Roughly half of the fibers in the upper 3-4 mm of the esophagus were depleted in the SLN stimulation cases. The findings of depleted fibers in esopha- gus, pharynx and SM following SLN stimulation were replicated in five animals. On the basis of the gross dissec- tions, it seems likely that the innervation of these struc- tures is conveyed by the DAB-SLN.

Muscle fibers in the hyopharyngeus, thyropharyngeus, SM, and palatopharyngeus were depleted by stimulation of the PN. This nerve appears to provide complete innervation of all of these muscles except for the SM, whose innervation we have shown comes in part from the SLN. In two cases there were small numbers of depleted fibers in the cervical esophagus following PN stimulation. Depletion in the SM following I" stimulation is shown in Figures 8A and 10. Figure 8A shows that the pattern of depletion after PN stimulation is biased towards the rostral part of the SM. Figure 10 shows a horizontal section where depletion of the SM following PN stimulation can be seen bilaterally. Palatopharyngeus muscle fibers, some of which form the palatopharyngeal sphincter described by Nakano and Muto (1985), were depleted only ipsilateral to stimula- tion, indicating that these fibers do not cross the midline.

Pharyngeal nerve.

TABLE 3. Results of Counts to Determine the Percentage of Fibers in the SM Depleted by Stimulation of Either the PN or the SLN

Percent fibers Percent fibers depleted depleted Difference

ipsilateral contralateral (%)

Pharyngeal n . stimulation cnse 12 case 13 case 20 case 21

mean (S.D.)

case 5 ease 6 case 7 case 16

mean (S.D.1

Superior laryngeal n stirnulation

49.6 44.5 44.5 45.7 46.1 (2.4)

6.2 23.3 28.3 28.0 21.4 110.4)

24 7 33 4 30 9 33 6 30 7 (4 21

4 0 22 9 28 1 23 5 19 6 (10 7)

24.9 11.1 13.6 12.1 15.4 (6.4)

2.2 0.4 0.2 4.5 1 8 (2.0)

Quantifying the innervation o f the SM. From the per- centage of depleted fibers found in the semicircular muscle after PN and SLN stimulation experiments, we can attempt to reconstruct the relative contributions of each nerve to the overall innervation of this muscle. These data are summarized in Table 3. Whichever nerve was stimulated, more fibers were depleted ipsilateral to the stimulation, suggesting that some SM fibers do not cross the midline (a mean of 15.4% of PN-innervated fibers and a mean of 1.8% of SLN-innervated fibers). Totaling up the innervation as seen in a cross-section of the SM on one side, one would then sum 46.1% from the ipsilateral PN, 30.7% from the contralateral PN, 21.4% from the ipsilateral SLN and 19.6% from the contralateral SLN, yielding a sum of 117.8%. The difference between this value and the expected sum of 100% could be due to: (1) miscategorizing light- staining nonstimulated fibers as depleted, (2) polyneuronal innervation of some SM fibers by more than one motor nerve, or (3) variability due to a small sample size.

Examples of glycogen deple- tion resulting from RLN stimulation are shown in Figure 11. In this case, complete depletion of the intrinsic laryn- geal muscles (apart from the cricothyroid), including the newly described cricovocal and rostral cricoarytenoid muscles, was obtained. The esophagus was partially de- pleted in a manner that appeared complimentary to the SLN stimulation cases. A small number of pale staining muscle fibers were found in the SM in 215 cases of RLN stimulation.

Glossopharyngeal nerve. Glossopharyngeal nerve stimu- lation depleted muscle fibers in the stylopharyngeus muscle, as expected. This nerve does not appear to supply efferent fibers to any other pharyngeal muscles. In one case a small number of fibers in the palatopharyngeus muscle also appeared to be depleted, but these fibers might have been part of the stylopharyngeus.

Recurrent laryngeal nerve.

Results of retrograde tracer experiments Nerve injections. Since the general topography of pha-

ryngeal motor neurons in the nucleus ambiguus has been

INNERVATION OF RAT PHARYNX AND LARYNX 137

Fig. 7. Results of stimulating the SLN on PAS staining of cricothy- roid muscle. A Unstimulated side. B: Stimulated side. This is a typical result of the glycogen depletion method, with a clear distinction between depleted and nondepleted fibers. CC, cricoid cartilage; LCA, lateral cricoarytenoid muscle; CT, cricothyroid muscle. Scale bar = 200 km.

described in detail previously for the rat (Bieger and Hopkins, 1987) we will concentrate on new results pertain- ing to the efferent components of the DAB-SLN, particu- larly in comparison to the PN.

Examples of retrogradely labeled cells are shown in Figures 12 and 13 and the distributions of labeled cells resulting from DAB-SLN and PN injections in two cases are compared in Figure 14. Labeled cells were counted in each case and these values together with summary statistics are found in Table 4. Inspection of this table shows that the pattern of labeling differed significantly depending on which nerve was injected (P = .0001, chi square test).

Injection of either the PN or the DAB-SLN labeled cells of the nucleus ambiguus in a cytoarchitectural division called the semicompact formation (SCF; Lawn, 1966; Bieger and Hopkins, 1987; Fig. 12A). Within this division we distin- guished between cells that were restricted to a well- delineated, or main, column and cells that were more scattered ventral and lateral to this column near its rostral end (Fig. 12C). Application of HRP to the PN labeled a mean of 173 cells distributed over the whole length of the main column and only a few cells in the ventrolateral

region. Application of HRP to the DAB-SLN labeled a mean of 42 cells in the rostral half of the main column and a mean of 34 cells in the ventrolateral region. Figure 13 illustrates the difference in distribution of labeled of cells after injec- tion of these two nerves.

The compact formation (CF), a cell column extending rostral and slightly medial to the SCF, contained 39 labeled cells on the average after injection of the DAB-SLN (Fig. 13B) but only a mean of two cells following injection of the PN. Similarly, more small fusiform cells (mean = 16) were labeled in the external formation of the nucleus ambiguus in DAB-SLN cases than in PN cases (mean = 4). These cells typically showed moderate to light staining. The external formation is a poorly delineated region peripheral to the main cell clusters of the nucleus, which contains small preganglionic neurons that are characterized by a lack of CGRP immunoreactivity (Lee et al., 1992). Lacking the more definitive immunostaining evidence, we categorized these neurons by their location well outside the semicom- pact formation and by their small fusiform somata.

After DAB-SLN injections, a sizeable number (mean = 110) of heavily labeled cells was consistently found in the rostral part of the dorsal motor nucleus of the vagus nerve (DMV). From three PN injection cases a total of only four cells were found labeled in the DMV. Following one PN injection, labeled cells were also found rostrally, dorsal to the facial motor nucleus (Fig. 14).

While the main objective of the retrograde labeling experiments was to compare PN and DAB-SLN efferent components, the results of direct muscle injections have some relevance to assessing the validity of those results. Because the semicircular muscle is very small, difficult to visualize in the live animal and not isolated from the contiguous esophageal and oblique thyro- pharyngeal muscle, it is not possible to confine an injection to this compartment. In ten cases, we made small pressure injections of the lower thyropharyngeus with the intent of centering these injections on the semicircular muscle. These injections could label efferents to the thyropharyn- geus, including the semicircular muscle, esophageal effer- ents to the pharyngoesophageal junction, and visceromotor efferents to autonomic targets in the wall and mucosa of the pharynx. Following these injections, labeled cells were found in the dorsal motor nucleus of the vagus nerve and the nucleus ambiguus. The set of labeled cells in the nucleus ambiguus in those five cases where the injections and processing were of good quality was a superset of the cells labeled by the more restricted PN and DAB-SLN nerve injections. That is, we found cells in the semicompact formation both in the main column and in a more scattered distribution at the rostral pole of the nucleus. These observations are consistent with labeling of semicompact formation efferents projecting to the pharynx by both the pharyngeal and the superior laryngeal nerves. In some cases there were more neurons labeled in the semicompact formation than were seen on the average by injections of either the PN or the DAB-SLN alone. In those regions where the labeled cells are likely to be autonomic efferents, the DMV and the external formation, there were fewer retrogradely labeled cells after direct muscle injection than were seen following DAB-SLN injections. This observation would be consistent with DAB-SLN visceromotor efferents having targets predominantly within the ganglia observed within this nerve.

Muscle injections.

138 J.B. KOBLER ET AL.

Fig. 8. This figure compares the pattern of depletion in the semicir- cular muscle (sm) following either PN stimulation (A) or superior laryngeal nerve stimulation (B). Note that the patterns are complimen-

tary with PN stimulation-induced depletion biased towards the rostral end of the SM and SLN-induced depletion biased towards the caudal end. Scale bar = 200 pm.

DISCUSSION The pattern of peripheral innervation

The glycogen depletion method can help identify the specific muscle fibers innervated by a particular motor nerve. Particularly convincing are cases where a whole muscle is completely depleted while adjacent muscles are stained normally. While a number of our experiments were this clear-cut, others seemed to achieve only partial depletion. There are several factors that could influence the efficacy of depletion: (1) the shock level may not have been above threshold for all nerve fibers; (2) some nerve fibers might have been damaged during the preparation or stimu- lation procedure; (3) fatigue at some neuromuscular junc- tions may have occurred before depletion was complete; (4) some nondepleted fibers may have been metabolically resis- tant to glycogen depletion (Rosser et al., 1992); and (5) the nerve to be stimulated may consist of more than one trunk. Negative as well as positive evidence for innervation can aid in interpretation of the glycogen depletion results. For example, the cricothyroid muscle was not always com- pletely depleted following SLN stimulation; however, stimu- lation of the PN and glossopharyngeal nerves never de- pleted fibers in the cricothyroid muscle and stimulation of

Overview.

the RLN depleted only a few cricothyroid muscle fibers in 1 out of 5 cases. Therefore, the partial depletion in the cricothyroid in 3 out of 5 cases was probably due to less than optimal efficacy in stimulation-induced depletion. Such observations, coupled with the evidence from cases in which complete depletion was obtained, leads to the following conclusions: (1) The RLN provides the innervation of the intrinsic laryngeal muscles, except for the cricothyroid muscle. It also shares innervation of the cervical esophagus with the SLN and it on occasion provides some minor innervation of the semicircular muscle. (2) The PN supplies all the muscle fibers of the pharyngeal constrictors and about 3/5 of the semicircular muscle. It innervates the deeper pharyngeal muscles that we have grouped under the palatopharyngeus, but which may also include glosso-, pterygo- and salpingopharyngeal components. On occasion some of its axons reach a few muscle fibers of the upper esophagus. (3) The glossopharyngeal nerve provides the sole motor innervation of the stylopharyngeus. (4) The SLN innervates the cricothyroid muscle, about 2/5 of the semicir- cular muscle, a few fibers in the more rostral thyropharyn- geus and about half of the upper 5 mm of the esophagus. (5) On occasion, fibers of the SLN reach intrinsic laryngeal

INNERVATION OF RAT PHARYNX AND LARYNX 139

pharyngeal constrictor fibers insert into a midline raphe. The semicircular compartment is called the cricopharyn- geus muscle in human (see review by Shapiro and Goyal, 1988) and other species because of its origin from the caudolateral cricoid cartilage. The region of the junction between the pharynx and the esophagus in the rat has also been referred to as the cricopharyngeus (House, 1953). In the present study of the rat, however, we have not been able to trace any pharyngeal muscle fibers to the cricoid carti- lage by microdissection or by examination of over 20 serially sectioned specimens. We have therefore referred to it simply as the “semicircular muscle” (SM) to distinguish it from the more rostra1 and structurally different thyropha- ryngeus muscle. While there are clear differences between species in the anterior attachments of the inferior constric- tor muscle, the semicircular part that encircles the pharyn- geal-esophageal junction appears to be a constant feature. Therefore, we propose that the rat SM is functionally, evolutionarily, and possibly homologous with the human CP.

In man the innervation of the pharyngeal constrictor muscles derives from a plexus made up largely of vagal branches, including contributions from the RLN, the SLN, pharyngeal nerves that arise from the vagus more proximally than the SLN, the glossopharyngeal nerve (thought to supply sensory fibers to the pharyngeal mucosa), and laryngopharyngeal branches from the sympathetic trunk (Sprague, 1944; Williams and Wanvick, 1980). Recent microdissection work by Mebis et al. (1993) indicates that the most significant innervation of the human cricopharyngeus is via the pharyn- geal branches of the vagus nerve with a minor contribution from the recurrent laryngeal nerve. Figure 15 summarizes pharyngeal innervation in a number of species. Branches of the SLN innervate the inferior constrictor and communi- cate with branches with other pharyngeal nerves in all species where pharyngeal innervation has been described. It is clear from this diagram that the anastomoses between different branches makes it impossible to determine by dissection how the fibers are ultimately sorted within the pharyngeal muscles.

In the rat, most of the pharyngeal constrictor muscula- ture is innervated by a pharyngeal nerve that originates near the nodose ganglion and divides into 4-5 main branches near the pharynx (Bieger and Hopkins, 1987; this study). The SLN sends branches to the pharyngoesophageal junc- tion and upper esophagus and communicates with the RLN. The glossopharyngeal nerve sends a twig to the stylopharyngeus and has several more robust branches that penetrate the pharyngeal constrictor muscles, presumably en route to the mucosa of tongue and pharynx. There are no major anastomoses between the SLN and the PN external to the pharynx although there may be some between intramuscular fascicles. The most detailed study of the SLN and its branches in the rat was made by Andrew (1956a). His measures of axon number and diameter were very similar to those obtained in our study. Andrew de- scribed a branch to the cricothyroid muscle and a branch running more posteriorly, which we have called the dorsal accessory branch of the SLN (DAB-SLN). Andrew assumed that all the efferent fibers in the DAB-SLN were destined to innervate the cervical esophagus.

Peripheral inneruation-evidence from physiology. An- drew (195613) recorded from muscle fibers and from SLN branches to characterize the activity patterns of individual

SM innervation-evidence from dissection.

Fig. 9. Results of SLN stimulation on PAS staining of the esopha- gus. A: Low power view. B: Higher power view. Note higher proportion of depleted fibers in the inner layer of the esophageal muscle (boundary between layers is marked with line). OL, outer layer; IL, inner layer; M, mucosal epithelium. Scale bars = 100 Fm in A, 50 )*m in B.

muscles that lie next to the cricothyroid muscle, including the thyroarytenoid muscle and the lateral cricoarytenoid. Similarly, the RLN may occasionally innervate a few fibers in the cricothyroid muscle that are next to other intrinsic laryngeal muscles. In such cases of apparently spurious innervation, the amount of crossover is extremely small and probably of no functional significance. Because of the functional and clinical importance of the UES, we next consider in some detail the implications of the dual innerva- tion of the SM.

The presence of a semicir- cular, sphincter-like compartment at the junction of the esophagus and the pharynx appears to be a common mammalian feature. By semicircular, we refer to muscle fibers that for the most part span the midline, inserting at each end on laryngeal cartilage. In contrast, all other

The rat semicircular muscle.

140 J.B. KOBLER ET AL.

Fig. 10. Results of stimulating the PN on the PAS staining of the pharyngeal constrictor muscles. Note the nearly complete depletion of the hyopharyngeus (HP) and the thyropharyngeus (TP) components. The semicircular (SM) is partially depleted bilaterally. No depleted

fibers were seen in the esophagus (ESO) in this case, although a few were present in two other cases. THY, thyroid cartilage. Scale bar = 400 km.

motor units. Units with tonic, respiratory-modulated activ- ity were found in three places: the branch to the cricothy- roid muscle, the dorsal accessory branch (branch #3 of Andrew) and in muscle fibers of the pharyngoesophageal junction at the level of the cricoid cartilage. These units were typically most active during inspiration and were briefly inhibited during swallowing. This type of activity was seen regardless of whether or not there was a propul- sive wave generated in the esophagus. Electromyographic recordings from the region of the SM showed that after the inhibitory period, activity was increased during a brief period of “postinhibitory excitation.” When propulsive waves did occur in the esophagus, a second type of nerve fiber was activated in the DAB-SLN that fired a brief burst

coincident with the peristaltic wave and was otherwise inactive.

Asoh and Goyal (1978) found that a branch of the pharyngeal plexus provided innervation of both the inferior pharyngeal constrictor and the CP in the opossum. Stimula- tion of this branch increased luminal pressure at the level of the CP and transection caused a cessation in the tonic electromyographic activity in the CP and inferior pharyn- geal constrictor. SLN section had no effect on tonic activity. The SLN of the opossum is a slender nerve running anterior to the pharyngeal muscles without giving off branches. It should be noted that marsupials such as the opossum have fused cricoid and thyroid cartilages and hence somewhat different anatomy in this region.

INNERVATION OF RAT PHARYNX AND LARYNX 141

Fig. 11. Effect of stimulation of the right recurrent laryngeal nerve on glycogen content in some of the intrinsic laryngeal muscles. A: Low power view of horizontal section of larynx. The small letters in this panel show the locations of the enlargements in B-D. B: Enlargement showing thyroarytenoid fibers on control side. C: Enlargement of thyroarytenoid fibers on the stimulated side. Dark staining features are

nerve bundles. D: Enlargement showing lateral cricoarytenoid (LCA) fibers on left and cricothyroid (CT) fibers on right separated by connective tissue and nerve fascicles. TA, thyroarytenoid muscle; LCA, lateral thyroarytenoid muscle; CT, cricothyroid muscle; TP, thyropha- ryngeus muscle; CRI, cricoid cartilage; THY, thyroid cartilage; ARY, arytenoid cartilage. Scale bars = 100 km.

Evidence from this study. The results of our study show that the SM of the rat receives a dual innervation from both the PN and the SLN. Counts of depleted fibers ipsilateral and contralateral to the stimulation showed that the pharyngeal nerves supply about 3/5 of those fibers that cross the midline while the dorsal accessory branches supply about K of that fiber population. About 17% of the

SM muscle fibers stop at the midline and 85% of these fibers are innervated by the pharyngeal nerves. The SLN also innervates a small number of more rostrally located muscle fibers within the thyropharyngeus muscle and numerous muscle fibers in the upper esophagus. Our measurements of DAB-SLN nerve fiber diameters in one case showed that these fibers are significantly smaller on the average than

142 J.B. KOBLER ET AL.

Fig. 12. Photomicrographs of retrogradely labeled cells in the nucleus ambiguus (NA). A Cells labeled in the semicompact formation (SCF) of NA after HRP application to the PN. Similar results were obtained with DAB-SLN labeling but fewer cells per section were labeled. B: Cells labeled in the compact formation (CF) of the NA after

the fibers in the PN. This observation suggests that SM motor units might differ functionally from other pharyn- geal constrictor motor units and would be consistent with the presence of small and/or slow twitch, tonically active motor units in the SM (Burke, 1981). A larger series of animals would be useful in future studies to more fully characterize the fiber populations in these nerves.

Based on the glycogen depletion data, we know that three types of efferent fibers are conveyed by the dorsal accessory branch of the SLN: those to the nonsemi- circular part of the thyropharyngeus, those to the semicir- cular part of the thyropharyngeus, and those to the esopha- gus. Andrew’s physiological results showed there are several functional unit types in the DAB-SLN. If we assume that the part of the pharyngoesophageal junction from which Andrew recorded tonic activity was the SM, we can make

Synthesis.

HRP application to the DAB-SLN. C: Cells ventral to the caudal end of the CF labeled after HRP application to the DAB-SLN. D: Cells in the rostra1 semicompact formation labeled after cholera-toxin-HRP was injected into the cricothyroid muscle. Scale bar = 100 km.

some plausible hypotheses regarding the correlation of unit types and muscle targets. The assumption that the SM has tonic activity is reasonable given the tonic muscle fiber activity Andrew recorded from this general region (the exact location was poorly defined) and the large body of evidence from other species. We thus hypothesize that type 1 units (tonic respiratory-modulated activity) correspond to the fibers that innervate the SM, and that type 2 fibers (which fire only during esophageal propulsive wave) inner- vate the upper esophagus. Units that fire like type 2 units but that are recruited in the buccopharyngeal stage of swallowing could innervate the nonsemicircular part of the thyropharyngeus. Alternatively, there might be tonic and nontonic motor units within the SM. These hypotheses can be tested in future experiments that directly correlate the response properties motor units with their locations.

INNERVATION OF RAT PHARYNX AND LARYNX 143

Fig. 13. Comparison of labeling in nucleus ambiguus after HRP application to DAB-SLN (A) or PN (B). These are transverse sections through the rostral pole of nucleus ambiguus. The DAB-SLN injection (A) labeled two clusters of cells at this level. The dorsomedial cluster corresponds to the main semi-compact column labeled after PN injec- tion (B). Cells ventrolateral to the main column were infrequently labeled by PN injections. Scale bar = 100 pm.

Correlations between brainstem labeling and peripheral targets

Dorsal motor nucleus of the vagus nerve (DMV). The extent of labeling in the DMV following SLN injections has not been described in detail previously. A large population of labeled cells was located in the DMV after DAB-SLN injections but only an occasional labeled cell was seen there after PN injections. The labeled cells were in the anterior

half of the DMV, extending from 100-200 pm anterior to the obex to a level slightly anterior to the posterior border of the facial motor nucleus. This cell column is clearly part of the DMV posteriorly, but in its rostral half these cells would be hard to distinguish from the nucleus of the solitary tract if they were not labeled. Since the DMV is largely devoted to innervation of stomach and other abdominal viscera (Leslie et al., 1982; Shapiro and Miselis, 1985; Altschuler et al., 1989; Okumura and Namiki, 1990), we performed control experiments to rule out spread of tracer to other areas. Ligating and cauterizing the vagus nerve just distal to its anastomosis with the SLN had no effect on the number or pattern of cells labeled by DAB-SLN injection. Another observation that argues against tracer spread is the absence of any contralateral labeling in the DMV or in other cranial nerve motor nuclei, which would be expected if there was generalized spread of tracer in the neck or by way of the vascular system. We also note that the staining of these cells was dark and comparable to those labeled in compact and semicompact formations of the nucleus ambiguus. The existence of a sizable preganglionic parasympathetic compo- nent in the DAB-SLN is consistent with the large numbers of ganglion cells associated with this nerve (see Fig. 3), although connections between DMV cells and these ganglia have yet to be demonstrated.

Labeling in the DMV after SLN and laryngeal injections has been observed previously. Basterra et al. (1987) found labeling in the DMV in guinea pig after intralaryngeal HRP injection and attributed it to labeling of the visceromotor component of the larynx. Altschuler et al. (1989) dealt mainly with the sensory components of the vagus nerve and their projections to NTS subdivisions; however, they do illustrate a few retrogradely labeled cells in the DMV after injection of the SLN and after injection of the esophagus. The peripheral targets of the autonomic fibers in the DAB-SLN are unknown, but may include postganglionic neurons innervating glands and smooth muscle associated with the larynx and cervical esophagus.

DAB-SLN injections also la- beled more cells in the EF of nucleus ambiguus than did PN injections. Cells in the EF differ from other efferent cell bodies in nucleus ambiguus in that they do not stain immunohistochemically for CGRP (Lee et al., 1992); this test should be applied in the future to better characterize these DAB-SLN efferents. While many EF cells apparently innervate the heart (Lee et al., 19921, our results support those of Bieger and Hopkins (1987) who also labeled small fusiform cells ventrolateral and dorsolateral to the CF by injections of the SLN or esophagus. The targets of these neurons is unknown; possibly they are destined for either laryngeal or esophageal visceral structures. I t is even possible that some could reach the thoracic vagus and heart by way of the ramus anastomoticus joining the DAB-SLN and RLN. The source of preganglionic innervation of one smooth muscle target, the trachealis muscle, has not been identified, although electrophysiological studies indicate these efferents may be conveyed by the SLN in the dog (Brown et al., 1980; Coleridge et al., 1982). The pregangli- onic efferents to the muscularis mucosa of the esophagus, a very thin layer of smooth muscle that lies between the striated muscle layers and the epithelium, are also uniden- tified and could be either in the DMV or in the nucleus ambiguus.

Numerous previous studies (Yoshida et al., 1981; Fryscak et al., 1984; Hudson and

External formation (EF).

Compact formation (CF).

- - 0.5

J.B. KOBLER ET AL.

i a i

Semicompact / Formation

Millimeters to Left of Midline

\ 2.0 \ \1.5 1 .o

'-1 I I Millimeters to Left of Midline

Fig. 14. Plot of cells labeled from pharyngeal or DAB-SLN nerve injections as viewed in a horizontal projection. Left: Distribution of labeled cells after HRP application to the PN. Cells are in SCF and dorsal t o facial motor nucleus. Right: Distribution of labeled cells after

Cummings, 1985; Bieger and Hopkins, 1987) and our own unpublished data show that the esophageal (striated muscle) motor neurons are located in the CF. On average, 39 cells were labeled in the CF following DAB-SLN injection. They were found throughout the rostral-caudal extent of the CF with a preponderance in the more dorsal and peripheral parts of this column. These findings are consistent with the glycogen depletion results demonstrating that the SLN innervates cervical esophagus and with the observations that the DAB-SLN sends branches into the cervical esopha- gus. Occasional cells were also seen in the CF after PN injection, again consistent with observations of occasional esophageal muscle fibers depleted after PN stimulation.

The SCF contains pala- tal, pharyngeal and cricothyroid motor neurons (Szen- tagothai, 1943; Lawn, 1966; Gacek, 1975; Wetzel et al., 1980; Yoshida et al., 1980, 1981, 1982; Hinrichsen and

Semicompact formation (SCF).

HRP application to the DAB-SLN. Cells were located in SCF, CF, dorsal motor-nucleus of the vagus nerve (DMV) and ventrolateral area of nucleus ambiguus.

Ryan, 1981; Kalia and Sullivan, 1982; Nomura and Mizuno, 1983; Holstege et al., 1983; Hisa et al., 1984; Davis and Nail, 1984; Hudson, 1986; Bieger and Hopkins, 1987; Grelot et al., 1989; Altschuler et al., 1991). Our finding that DAB-SLN efferent cell bodies are located rostral to PN efferent cell bodies in the SCF is consistent with earlier studies in rat which showed that thyropharyngeus motor neurons are located more rostral than hyopharyngeus motor neurons (Bieger and Hopkins, 1987). The pharyngeal constrictor motoneuron cell bodies are largely confined to a discrete column about 100 pm in diameter in the posterior three-fourths of the subdivision. In the anterior quarter of the SCF, where the SCF overlaps with the CF, there is a slightly broader distribution of pharyngeal motor neurons ventral and lateral to the CF. Bieger and Hopkins (1987) noted that the lower constrictor (thyropharyngeus) motor neurons tend to be more ventral, anterior and contiguous

INNERVATION OF RAT PHARYNX AND LARYNX 145

TABLE 4. Counts of Retrogradely Labeled Cells Following HRP Injections into Either the PN or the DAB-SLN

Number of retrogradely labeled cells

Nucleus ambiguus subdivision

Semicompact Dorsal to Semicompact formation Compact External

formation (ventrolateral) formation formation DMV VII Total

Pharyngeal nerve Injections

Case 2 1 Case 35 Case 37

Mean number iS.D.i Mean vercent of cells ver nuc.

ambiguus subdivision DAB-SLN injections

Case 29 Case 34 Case 36 Case 37

Mean number Mean percent of cells per nuc.

ambiguus subdivision

176 146 197

173 (25.6)

95.1 (2.71

47 58 17 45 41.8 (17.51

31.2 (10.9)

2 2 6 3.3 (2.31

1.7 (-9)

29 28 27 53 34 3 (12 5)

26.0 (5.11

1 3 1 1.7 (1.2)

1.0 (0.8)

28 35 37 56 39.0 (12.0)

30.3 (8.31

with cricothyroid motor neurons, while the upper constric- tor (hyopharyngeus) motor neurons are more dorsal and are roughly in line with the motor neurons for other intrinsic laryngeal muscles. This topography, together with physiological considerations, led them to hypothesize that the motor neurons supplying the pharyngo-esophageal junction share similar inputs as cricothyroid motor neurons while the more oral parts of the pharyngeal constrictors share common inputs with other laryngeal motor neurons. Our results are consistent with this idea. In particular, the injections of the DAB-SLN resulted in labeling of cells in the main semicompact formation column and a cluster located more ventrolateral at the rostra1 pole of the nucleus near the cricothyroid motor neuron pool. (Fig. 14). If anatomical location and functional properties are related, as Bieger and Hopkins suggest, there may be two functional types of motor units in the SM. This would be consistent with the observation by Andrew (1956b) that some units in the DAB-SLN have tonic activity while others behave like upper pharyngeal motor neurons. We also have unpub- lished observations that type 1 (slow twitch) muscle fibers make up a relatively small fraction of the SM. Further experiments are necessary to define the physiological prop- erties of different pharyngeal motor neuron populations within nucleus ambiguus and to correlate them with muscle architecture.

Clinical relevance. A clear understanding of the pat- tern of innervation of the cricopharyngeus muscle has obvious clinical value. It has been observed that some patients have dysphagia and difficulties in acquisition of functional tracheoesophageal speech following laryngec- tomy, apparently due to a high residual tone in the cricopha- ryngeus or to spasm of the cricopharyngeus in response to injection of air into the esophagus (Singer et al., 1986; Horowitz and Sasaki, 1993). Myotomy of the cricopharyn- geus is commonly performed in conjunction with laryngec- tomy, but this may weaken the pharyngeal wall. In one series of patients, Singer et al. (1986) were able to improve tracheoesophageal speech by severing 3-5 branches of the pharyngeal plexus in the vicinity of the hypopharynx. The study by Mebis et al. (19931, which shows a heavy innerva- tion of the human cricopharyngeus by pharyngeal nerve branches, provides the clearest anatomical basis for this surgical approach to date. As shown in our study, greater

2 1 0 182 1 0 17 169

10 3 0 217 4.3 (4.91 1.3 (1.51 5.7 (9.8) 189.3 124 81

2.2 (2.21

25 69 0 198 13 121 0 255 10 136 0 227 17 114 0 285 16.3 (16.51 110 “28.8) 0 241.3 (37.31

12.5 (4.6)

resolution of muscle fiber targets and their relationship to the overall architecture of the muscle can be achieved using the glycogen depletion method, particularly in a region where the convergence and anastomoses between nerve branches is complex. If similar data could be obtained for the human pharynx, it would be particularly relevant in cases where ablation of one set of inputs is considered as a therapeutic approach.

The role of the CP in oropharyngeal dysphagia is also not fully understood. A differential distribution of CP motor neurons in the central nervous system (CNS) could explain the remaining tone and CP prominence in some patients with pharyngeal paralysis due to stroke if the lesion hap- pened to spare an anatomically separate CP motor neuron group.

ACKNOWLEDGMENTS The authors thank Drs. Joe C. Adams, Robert E. Hillman

and Nelson Y.-S Kiang for helpful comments on the manu- script. This work was supported by NIDCD grant DC0129 and NIDDK grant DK-31092.

LITERATURE CITED Altschuler, S.M., X. Bao, D. Bieger, D.A. Hopkins, and R.R. Miselis (1989)

Viscerotopic representation of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol. 283:248-268.

Altschuler, S.M., X. Bao, D. Bieger, and R.R. Miselis (1991) Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat. J. Comp. Neurol. 309:402-414.

Andrew, B.L. (1956a) A functional analysis of the myelinated fibers of the superior laryngeal nerve of the rat. J. Physiol. (Land.) 133:420-432.

Andrew, B.L. (1956b) The nervous control of the cervical esophagus of the rat during swallowing. J. Physiol. (Land.) 134:729-740.

Asoh, R., and R.K. Goyal (1978) Manometry and electromyography of the upper esophageal sphincter in the opossum. Gastroenterology 74:514- 520.

Bancroft, J.D., and H.C. Cook (1984) Manual of Histological Techniques. Edinburgh: Churchill Livingstone, pp. 102-103.

Basterra, J., C. Chumbley, and P.N. Dilly (1987) Topographic distribution of laryngeal motor neurons in the nucleus ambiguus of the guinea pig studied by horseradish peroxidase (HRP) technique. Acta Otolaryngol. lO3:105-110.

146 J.B. KOBLER ET AL.

RAT - THIS STUDY

CRCOTHYROID MUSCLE

ESOPHAGUS

SEMICIRCULAR M.

u

THY ROPHARYNGEU S

HYOPHARYNGEUS

PALATOPHARYNGEUS

STYLOPHARYNCEUS

TREE SHREW - SPRAGUE ('44)

I' ESOPHAGUS

CRICOTHYROID MUSCLE

INFERIOR PHARYNGEAL CWSTRMOR

MIDDLE PHARYNGEAL CONSTRICTOR

SUPERIOR PHARYNGEAL CONSTRICTOR

SPlLOPHARYNQUS

CERATOHYOIDEUS

HUMAN - SPRAGUE ('44)

CRICOTHYROID MUSCLE

INF ERlOR PHARYNGEAL

PHARYNGEAL 3 MIDDLE PHARYNGEAL CONSTRICTOR

SUPERIOR PHARYNGEAL CONSTRICTOR

I I b STYLOPHARYNCfUS I X

DOG - LEMERE ('32)

ESOPHAGUS 7 3 PHARYNGEAL

CRICOTHYROID MUSCLE

INF ERlOR PHARYNGEAL CONSTRlCTOR

MIDDLE PHARYNGEAL CONSTRICTOR

SUPERIOR PHARYNGEAL CONSTRCTOR

MACAQUE - SPRAGUE ('44)

ESOPHAGUS

INFERIOR PHARYNGEAL CONSTRICTOR

PHARYNGEAL

CONSTWCTOR

SUPERIOR PHARYNGEAL CONSTRICTOR

STYLOPHARYMUS E CERATOHYOIDEUS

TARSIER - SPRAGUE ('44)

CRCOTHYROID MUSCLE

UNLABELED BR.1 lNFERIDR b PHARYNGEAL CONSTRMOR

PHARYNGEAL MIDDLE PHARYNGEAL CONSTRICTOR

SUPERIOR PHARWGEAL CCNSTRlCTOR

STYLDFliARYNQUS -E CERATOHYOIDEUS

Fig. 15. Comparison of the distribution of the superior laryngeal, pharyngeal and glossopharyngeal nerves in six species. Interconnections between branches a re represented by single vertical lines but may represent multiple anastomoses.

Bieger, D., and D.A. Hopkins 11987) Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: The nucleus ambiguus. J. Comp. Neurol. 262:54&562.

Bieger, D., S.A. Giles, and C.H. Hockman (1977) Dopaminergc influences on swallowing. Neuropharmacology 16243-252.

Bonington, A,, I. Whitmore, and M. Mahon (1987) A histological and histochemical study of the cricopharyngeus muscle in the guinea pig. J. Anat. 153:151-161.

Brown, J.K., A.R. Leff, M.J. Frey, B.R. Reed, and W.M. Gold (1980) Physiological and pharmacological properties of the canine trachealis muscle in vivo. J. Appl. Physiol. 49.84-94.

Burke, R.E. (1981) Motor units: Anatomy, physiology and functional organization. In V.B. Brooks (ed): Handbook of Physiology, Sec. 1, The Nervous System, Vol. 2, Motor Control, Part 1. Washington, D.C.: American Physiological Society pp. 345-442.

Coleridge, J.C.G., H.M. Coleridge, A.M. Roberts, M.P. Kaufman. and D.G. Baker (1982) Tracheal contraction and relaxation initiated by lung and somatic afferents in dogs. J. Appl. Physiol. 52984-990.

Cunningham, E.T., and P.E. Sawchenko (1989) A circumscribed projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: Anatomical evidence for somatostatin-28-iinmunoreactive interneurons subserving reflex control of esophageal motility. J. Neurosci. 9:1668- 1682.

Davis, P.J., and B.S. Nail (1984) On the location and size of laryngeal motoneurons in the cat and rabbit. J. Comp. Neurol. 230: 13-32.

Fryscak, T., W. Zenker, and D. Kantner (1984) Afferent and efferent innervation of the rat esophagus. A tracing study with horseradish peroxidase and nuclear yellow. Anat. Embryol. 170:63-70.

Gacek, R.R. (1975) Localization of laryngeal motor neurons in the kitten. Laryngoscope 85: 1841-1860.

Grelot, L., J.C. Barillot, and A.L. Bianchi 11989) Central distributions of the efferent and afferent components of the pharyngeal branches of the vagus and glossopharyngeal nerves: An HRP study in the cat. Exp. Brain Res. 78:327-335.

Hinrichsen, C.F.L., andA.T. Ryan (19811 Localization of laryngeal motoneu- rons in the rat: Morphological evidence for dual innervation? Exp. Neurol. 74:341-355.

Hisa, Y., F. Sato, Y. Suzuki, K. Yanohara, M. Hyuga, and 0. Mizukoshi (1984) The localization of motoneurons innervating the canine pharyn- geal constrictor muscles in the posterior pharynx by the fluorescent double-labeling technique. Arch. Otorhinolaryngol. 241t83-87.

Holstege, G., G. Graveland, C. Bijker-Biemond, and I. Schuddeboom (1983) Location of motoneurons innervating soft palate, pharynx and upper esophagus. Anatomic evidence for a possible swallowing center in the pontine reticular formation. Brain Behav. Evol. 23:47-62.

INNERVATION OF RAT PHARYNX AND LARYNX 147

House, E.L. (1953) A myology of the pharyngeal region of the albino rat. Anat. Rec. 126t363-381.

Hudson, L.C. (1986) The origins of innervation of the canine caudal pharyngeal muscles: An HRP study. Brain Res. 374t413-8.

Hudson, L.C., and J.F. Cummings (1985) The origins of innervation of the esophagus of the dog. Brain Res. 326:125-136.

Joseph, M.P., J.J. Guinan, Jr., B.C. Fullerton, B.E. Norris, and N.Y.S. Kiang (1985) Number and distribution of stapedius motoneurons in cats. J. Comp. Neurol. 232.43-54.

Kalia, M., and J.M. Sullivan (1982) Brainstem projections of sensory and motor components of the vagus nerve in the rat. J. Comp. Neurol. 211248-264.

Kessler, J.P., and A. Jean (1985) Identification of the medullary swallowing regions in the rat. Exp. Brain Res. 57256-263.

Kugelherg, E., and L. Edstrom (1968) Differential histochemical effects of muscle contractions on phosphorylase and glycogen in various types of fibers: Relation to fatigue. J. Neurol. Neurosurg. Psychiat. 311415-423.

Lawn, A.M. (1966) The localization, in the nucleus ambiguus of the rabbit, of the cells of origin of motor nerve fibers in the glossapharyngeal nerve and various branches of the vagus nerve by means of retrograde degenera- tion. J. Comp. Neurol. 127.293-306.

Lee, B.H., R.B. Lynn, H.3 . Lee, R.R. Miselis, and S.M. Altschuler (1992) Calcitonin gene-related peptide in nucleus ambiguus motoneurons in rat: viscerotopic organization. J. Comp. Neurol. 320t531-543.

Lemere, F. (1932) Innervation of the larynx. I. Innervation of laryngeal muscles. Am. J. Anat. 51t417-437.

Mebis, J., K. Geboes, and V. Desmet (1993) Innervation of the human esophagus. In J. Janssens (ed): Gastrointestinal Motility Disorders. Belgium: K.U. Leuven, pp. 29-46.

Mesulam, M.-M. (1982) Principles of horseradish peroxidase neurochemis- try and their applications for tracing neural pathways-axonal trans- port, enzyme histochemistry and light microscopic analysis. In M.-M. Mesulam (ed): Tracing Neural Connections with Horseradish Peroxi- dase. NewYork: John Wiley & Sons, Ltd., pp. 1-151.

Murakami, Y., H. Fukuda, and J.A. Kirchner (1972) The cricopharyngeus muscle. An electrophysiological and neuropharmacological study. Acta Otol. suppl. 311:l-19.

Nachlas, M.M., K.C. Tsou, E. DeSouza, S.C. Cheng, and A.M. Seligman (1957) Cytochemical demonstration of succinic dehydrogenase by the use of a new P-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5.420.

Nakano, T., and H. Muto (1985) Anatomical observations in the pharynx of the mouse with special reference to the nasopharpgeal hiatus (Wood Jones). Acta Anat. 121t147-152.

Nomura, S., and N. Mizuno (1983) Central distribution of efferent and afferent components of the cervical branches of the vagus nerve. A HRP study in the eat. Anat. Embryol. 166:l-18.

Okumura, T., and M. Namiki (1990) Vagal motor neurons innervating the stomach are sitespecifically organized in the dorsal motor nucleus of the vagus nerve in rats. J. Auton. Nerv. Syst. 29t157-162.

Rosser, B.W.C., B.J. Norris, and P.M. Nemeth (1992) Metabolic capacity of individual muscle fibers from different anatomic locations. J. Histochem. Cytochem. 40:815-825.

Shapiro, J., and R.K. Goyal (1988) Disorders of the upper esophageal sphincter. In M.P. Fried (ed): The Larynx: A Multidisciplinary Approach. Boston: Little Brown and Co., pp. 293-317.

Shapiro, R.E., and R.R. Miselis (1985) The central organization of the vagus nerve innervating the stomach of the rat. 3. Comp. Neurol. 238:473-488.

Shipp, T., W.W. Deatsch, and K. Robertson (1970) Pharyngoesophageal muscle activity during swallowing in man. Laryngoscope 80:l-16.

Singer, M.I., E.D. Blom, and R.C. Hamaker 11986) Pharyngeal plexus neuroectomy for alaryngeal speech rehabilitation. Laryngoscope 96.50- 54.

Sprague, J.M. (1944) The innervation of the pharynx in the rhesus monkey and the formation of the pharyngeal plexus in primates. Anat. Rec. 90:197-208.

Szentagothai, J. (1943) Die lokalisation der Kehlkopfmuskulatur in den Vaguskerne. J. Anat. EntwickL-Gesch. 112t704-710.

Wang, Y.T., and D. Bieger (1991) Role of solitarial GABAergic mechanisms in control of swallowing. Am. J. Physiol. 261:R639-R646.

Wetzel, D.M., D.B. Kelly, and B.A. Campbell (1980) Central control of ultrasonic vocalizations in neonatal rats: I. Brain stem motor nuclei. J. Comp. Physiol. Psychol. 94t596-605.

Williams, P.L., and R. Warwick (1980) Gray's Anatomy, 36th edition. Philadelphia: W.B. Saunders Co., pp. 1311-1312.

Windhorst, U., T.M. Hamm, and D.G. Stuart (1989) On the function of muscle and reflex partitioning. Behav. Brain Sciences 12t62.9-681.

Yoshida, Y., T. Miyazaki, M. Hirano, T. Shin, T. Totoki, and T. Kanaseki 11980) Localization of motoneurons supplying the cricopharyngeal muscle in the cat studied by means of the horseradish peroxidase method. Neurosci. Lett. 18:1-4.

Yoshida, Y., T. Miyazaki, M. Hirano, T. Shin, T. Totoki, and T. Kanaseki (1981) Localization of efferent neurons innervating the pharyngeal constrictor muscles and the cervical esophagus muscle in the cat by means of the horseradish peroxidase method. Neurosci. Lett. 22t91-95.

Yoshida, Y., T. Miyazaki, M. Hirano, T. Shin, and T. Kanaseki (1982) Arrangement of motoneurons innervating the intrinsic laryngeal muscles of cats as demonstrated by horseradish peroxidase. Acta Otol. 94:329- 334.