THE ANATOMICAL RECORD 256:412–424 (1999) Neuromuscular Organization of the Canine Tongue LIANCAI MU AND IRA SANDERS* Grabscheid Voice Center, Department of Otolaryngology, The Mount Sinai Medical Center, New York, New York 10029-6574 ABSTRACT The tongue manipulates food while chewing and swallowing, dilates the airway during inspiration, and shapes the sounds of speech in humans. While performing these functions the tongue morphs through many complex shapes. At present it is not known how the muscles of the tongue perform these complex shape changes. The difficulty in understanding tongue biomechanics is partly due to gaps in our knowledge regarding the complex neuromuscular anatomy of the tongue. In this study the motor and sensory nerve anatomy of four canine tongues was studied with Sihler’s stain, a technique that renders most of the tongue tissue translucent while counterstaining nerves. An additional tongue specimen was serially sectioned to provide a reference for the muscle structure of the tongue. The hypoglossal nerve (XII) has approximately 50 primary nerve branches that innervate all intrinsic and extrinsic tongue muscles. Two extrinsic muscles, the styloglossus and hyoglossus, are innervated by about three to four branches from the lateral division of the XII. The third extrinsic muscle, the genioglossus, is composed of oblique and horizontal compartments, which receive about ten nerve branches from the medial division of the XII. The intrinsic muscles are composed of many neuromuscular compartments. On each side, the superior longitudinal muscle had an average of 40 distinct muscle fascicles that spanned the length of the tongue. Each of the fascicles is supplied by a nerve branch. The inferior longitudinal muscle had a similar organization. Each of the transverse and vertical muscles is composed of over 140 separate muscle sheets, and every sheet is innervated by a separate terminal nerve. The muscle sheets from the vertical and transverse alternate their orientation 90° throughout the length of the tongue. It is concluded that the intrinsic canine tongue muscles are actually composed of groups of neuromuscular compartments that are arranged in parallel (longitudinal muscles) or in a precise alternating sequence (transverse and vertical muscles). This arrangement suggests that the compartments from the different tongue muscles could cooperate to control the three-dimensional contractile state of their local area. This hypothesis could explain how many different tongue shapes are formed, and is supported by physiologic evidence. Anat Rec 256:412–424, 1999. r 1999 Wiley-Liss, Inc. Key words: tongue; innervation; tongue muscles; neuromuscular compartment; Sihler’s stain The tongue is a complex muscular organ constituted of several intrinsic and extrinsic muscles, innervated by several cranial nerves (Weddell et al., 1940; Miller et al., 1964) and involved in several important physiological tasks such as mastication, swallowing, taste, respiration, and human speech (Abd-El-Malek, 1955; MacNeilage and Sholes, 1964; Sauerland and Mitchell, 1970). Although the tongue is capable of performing complex movements, our r 1999 WILEY-LISS, INC. current understanding of the contributions of specific tongue muscles to precise movement patterns is limited. *Correspondence to: Ira Sanders, MD, Department of Otolaryngology, Box 1189, The Mount Sinai Medical Center, New York, NY 10029-6574. Received 22 October 1998; Accepted 23 July 1999 NEUROMUSCULAR ORGANIZATION OF THE TONGUE Knowing the neuromuscular organization of the tongue is crucial for understanding how the tongue functions. However, details concerning the nerve supply and muscular architecture have never been demonstrated in wholemount tongue specimens from any mammal. This is largely due to the fact that the muscles of the tongue interweave in complex ways, making it technically impossible to trace the muscle fascicles and nerve branches by traditional microscopic dissection. It is known that four nerves supply innervation to the tongue. The hypoglossal (XII) nerve supplies motor innervation to all tongue muscles (Miller et al., 1964). The lingual nerve, a branch of the mandibular division of the trigeminal nerve, provides both gustatory and general sensory fibers to the tongue. The gustatory component is contributed by the chorda tympani branch of the facial nerve which connects with the lingual nerve. Finally, the glossopharyngeal (IX) nerve terminates in the mucosa and the vallate papillae at the posterior third of the tongue to supply general sensation and taste (Lawn, 1966; Yamamoto, 1975). These classical descriptions on the innervation of the tongue are mainly based on the findings obtained by gross dissection, nerve degeneration, neural tracing techniques, and electrophysiological methods (AbdEl-Malek, 1938; Lawn, 1966; Yamamoto, 1975; Lowe, 1980; Hellstrand, 1981; Chibuzo and Cummings, 1982). Although much information about the innervation of the tongue has been directly and indirectly provided by the methods mentioned above, the complete nerve branching patterns of these nerves within the tongue have never been demonstrated. The motor innervation of the tongue is by the XII nerve, which bifurcates into lateral and medial divisions that supply the retrusor and protrusor muscle groups, respectively (Abd-El-Malek, 1938; Hellstrand, 1981). However, the specific muscles supplied by the two divisions of the XII nerve appear not to be the same in different species. It has been reported that in the human a smaller lateral division supplies hyoglossus, styloglossus, and inferior longitudinal muscles while the medial division supplies several branches to the genioglossus and intrinsic tongue muscles (Abd-El-Malek, 1938). In contrast, the medial division of the XII in the cat also supplies branches to the hyoglossal and styloglossal muscles (Hellstrand, 1981). There is little information of the terminal branching pattern inside these muscles. Recently, an old technique, the Sihler’s stain, has been modified to study the neuromuscular organization of complex structures such as the larynx (Wu and Sanders, 1992; Sanders et al., 1993a,b; Mu et al., 1994; Sanders et al., 1994; Wu et al., 1994; Sanders and Mu, 1998) and the human pharynx (Mu and Sanders, 1996, 1998a). Sihler’s stain has the unique capability of rendering a large postmortem specimen translucent while counterstaining the entire nerve supply. In the specimens processed by Sihler’s stain, the peripheral nerve supply and arrangement of individual muscles can be seen in their normal three-dimensional positions. By careful dissection of the specimen every motor nerve can be followed to its terminal branches within each muscle, and every sensory nerve branch can be traced to the termination in the mucosa. In addition, small connections between the motor and sensory nerves can be visualized. In this study the whole-mount Sihler’s stain was used to investigate: 1) the entire branching and distribution of all 413 motor and sensory nerves to the tongue; and 2) the arrangement of individual muscles and their intramuscular nerve supply. MATERIALS AND METHODS Five tongues were obtained from adult mongrel dogs which were sacrificed for another, unrelated experiment. Four tongues were stained with Sihler’s stain to study the nerve supply and muscular organization and one was sectioned and stained with hematoxylin and eosin (H&E) to demonstrate the internal organization of individual tongue muscles. All procedures for animal care and treatment were approved by the Mount Sinai Medical Center’s CLAS. The Sihler’s stained specimens were initially dissected, and then the gross nerve branches and arrangement of the tongue muscles were examined. Finally, each tongue muscle with its supplying nerve was excised off from the tongue body to study its intramuscular nerve supply pattern. Procedures for Modified Sihler’s Stain Solutions needed for Sihler’s stain. 1) 10% formalin. 2) 3% KOH (potassium hydroxide) (3 drops of 3% hydrogen peroxide are added into 100 ml 3% KOH). 3) Sihler’s solution I (1 part glacial acetic acid, 1 part glycerin and 6 parts 1% aqueous chloral hydrate). 4) Sihler’s solution II (1 part stock Ehrlich’s hematoxylin, 1 part glycerin and 6 parts 1% aqueous chloral hydrate). 5) 0.05% lithium carbonate solution. 6) 50% and 100% glycerin. Staining steps. 1) Fixation of the tongues studied in 10% formalin for 4–8 weeks. 2) Maceration in 3% KOH solution for 3 weeks with several changes. 3) Decalcification in Sihler’s solution I for 2 weeks with several changes. 4) Staining in Sihler’s solution II for 4 weeks. 5) Destaining in Sihler’s solution I for 3–4 hr. 6) Darkening the nerves in 0.05% lithium carbonate solution for 1 hr. 7) Clearing in 50% glycerin for 2–3 days. 8) Preserving the stained specimens in 100% glycerin with a few thymol crystals. Microdissection and Photography The Sihler’s stained tongues were preserved in 100% glycerin for 1–2 months and then dissected with the aid of a dissecting microscope (TYP 3555110; Wild, Heerbrugg, Switzerland) at a magnification of ⫻10–30 by using microsurgical instruments after transillumination by a Xenon light source (model 610; Karl Storz, Endoscopy-America, Culver City, California). The relationship between the individual tongue muscles and orientation of the muscle fascicles of a given muscle were carefully examined and recorded. The nerves supplying each of the muscles were traced from their main trunks to the terminations. The completed specimens were photographed with an OM-4 414 MU AND SANDERS Olympus camera (Tokyo, Japan), using a 50, 38, or 20 mm lens to show the muscle organization and nerve branching patterns of the tongue. The numbers and branching points of the major nerve branches derived from the XII, IX and lingual nerves were recorded. RESULTS The neuromuscular organization of the canine tongue was demonstrated by means of Sihler’s stain (n ⫽ 4) and H&E staining (n ⫽ 1). Histological examination of the cross sections stained with H&E allowed us to localize the individual muscles and make some measures. Sihler’s stained specimens allowed us to observe the nerve supply and muscle organization in three-dimensional positions. Organization of the Tongue Muscles The canine tongues were about 16 cm in length (n ⫽ 3). The width of the dorsal surface on each side was measured to be ⬃19 mm for the anterior, ⬃25 mm for the middle, and ⬃20 mm for the posterior tongue (n ⫽ 3). The thickness of the tongue close to the midline was ⬃6 mm, 16 mm, and 22 mm for the anterior, middle, and posterior tongue, respectively (n ⫽ 1). As demonstrated by H&E (Fig. 1A) and Sihler’s stain (Figs. 1B–E, 2A), the tongue muscles except for genioglossus (a midline muscle) were found to be organized into three layers: superior, middle, and inferior. The thickness of the three layers is different. The superior layer accounts for ⬃18%, the middle layer for ⬃59%, and the inferior layer for ⬃23% of the whole thickness of the tongue (Fig. 1A). The superior layer which lies just beneath the dorsum of the tongue consists of only the superior longitudinal (SL) muscle (Figs. 1A–C). The thickness of this layer in the middle portion of a semi-tongue was measured ⬃1 mm, 2 mm, and 2.2 mm for the anterior, middle, and posterior tongue, respectively (n ⫽ 1). This muscle courses from the tongue base and hyoid bone to the tip of the tongue. On each side, the SL was found to be composed of ⬃26, 35, and 58 fascicles on average for the anterior, middle, and posterior tongue, respectively (n ⫽ 3) (Figs. 1A–C). Cross section showed that the SL fascicles tend to be oval in shape and are arranged into a row under the dorsum of the tongue (Fig. 1A). The sizes of the fascicles are different. The fascicles in the medial half are larger than those in the lateral half of the tongue. Two large fascicles close to the median fibrous septum were recognized (Figs. 1B,C). The diameter for each of the two largest fascicles was measured to be ⬃1 mm for the anterior, 2 mm for the middle, and 2.5 mm for the posterior tongue. The most lateral fascicles are the smallest in size (Fig. 1A). The diameter of these smaller fascicles was measured to be ⬃0.6 mm. The middle layer consists of both the transverse and vertical muscles (Figs. 1A, D). The thickness of this layer was measured to be ⬃3 mm for the anterior, 7 mm for the middle, and 13 mm for the posterior tongue (n ⫽ 1). Sihler’s stain showed that on each side either transverse or vertical muscle comprises ⬃143 muscular sheets 126⬃157) on average (n ⫽ 3). The muscular sheets of the two muscles alternate from the tip to the root of the tongue. The transverse muscular sheets course from the median fibrous septum to the lateral margin of the tongue, whereas the vertical muscular sheets travel between the dorsum and ventral mucosa of the tongue. Therefore, the muscle fibers of either the transverse or vertical muscles are relatively shorter than those of other muscles. The length of the transverse muscle fibers was measured to be ⬃22 mm. The vertical muscle fibers were ⬃4 mm for the anterior, 14 mm for the middle, and 20 mm for the posterior tongue (n ⫽ 1). The inferior layer that is situated just above the mucosa of the ventral aspect of the tongue consists of the styloglossus, hyoglossus, and inferior longitudinal muscles (Figs. 1A, E). The thickness of this layer was ⬃1.5 mm, 2.8 mm, and 3.5 mm for the anterior, middle, and posterior tongue, respectively (n ⫽ 1). The styloglossus which enters the tongue caudolaterally and mixes up with the hyoglossus about 2 cm posterior to the hyoid bone (Fig. 1E). The inferior longitudinal muscle was noted to consist of numerous fascicles that are overlapped. These fascicles are much more in number and smaller in size than those of the superior longitudinal fascicles (Fig. 1A). Branching, Distribution, and Connections of the Nerves to the Canine Tongue After processed with Sihler’s stain and trimmed by microscopic dissection, all the nerves innervating the tongue were visualized. Figure 1E demonstrates the distribution of the XII, IX, and lingual nerves within a Sihler’s stained canine tongue. The branching patterns of the individual nerves to the canine tongue are described as follows: The hypoglossal nerve (XII). The details regarding the branching of the XII are illustrated in Figures 2A and B. During its course within the tongue, the XII nerve gives off about 48 (45⬃52) (n ⫽ 3) first-order nerve branches to supply all the tongue muscles at different levels. On approaching the lateral aspect of the tongue base, the XII nerve gives off three to four early branches that correspond to the classically described lateral division. These branches leave the main trunk of the XII within a short distance (about 2 cm) and innervate the styloglossus, hyoglossus, and inferior longitudinal muscles. The medial division runs anteriorly to the tip of the tongue (about 14 cm long). During its course the medial division gives off several groups of first-order nerve branches to supply other tongue muscles. First, it gives off a single branch about 4–5 mm distal to the lateral division to supply the geniohyoid muscle. It then sends three to four nerve branches to the superior longitudinal muscle, three nerve branches to the horizontal part, and seven to eight nerve branches to the oblique part of the genioglossus muscle. At the level of the genioglossus muscle, the medial division gives off four to five branches to the posterior transverse and vertical muscles. In the anterior tongue, the medial division gives off about 26 (24⬃29) (n ⫽ 3) first-order branches to innervate the anterior transverse and vertical muscles. The glossopharyngeal nerve (IX). The IX nerve was found to reach the tongue on the lateral margin and travel beneath the dorsum of the tongue. It usually gives off three to four first-order branches, which were measured to be about 5–6 cm in length (n ⫽ 4), to supply the mucosa and vallate papillae in the posterior one-third of the tongue (Figs. 1E, 3A,B). NEUROMUSCULAR ORGANIZATION OF THE TONGUE 415 Fig. 1. Three layers of the canine tongue muscles. A: A cross section of the right middle tongue stained with hematoxylin and eosin, showing the three layers of the tongue muscles. Three bars indicate the superior (S), middle (M), and inferior (I) layers of the tongue muscles, respectively. L, lateral margin of the tongue; D, dorsal surface of the tongue; V, ventral surface of the tongue. 7.5⫻. B: A Sihler’s stained canine tongue, showing the superior layer of the tongue musculature (ventral view). The middle and inferior layers have been removed. Note that the superior layer (e.g., superior longitudinal muscle) is composed of two large fascicles (large solid arrow) close to the midline (double arrowheads) and numerous small fascicles (small arrow) lateral to the midline. The open arrow indicates a vallate papilla. T, tip of the tongue; B, base of the tongue. C: Higher power view of the superior layer of the anterior tongue in B, showing the organization pattern of the superior longitudinal muscle fascicles. 3⫻. D: Higher power view of the middle layer of the Sihler’s stained anterior tongue. Note that this layer is composed of numerous alternating sheets of the transverse (T) and vertical (V) muscles. This layer is also seen on the cross section of the tongue in A. 9⫻. E: The inferior layer of the tongue musculature demonstrated by Sihler’s stain (ventral view). Note that this layer consists of the styloglossus (SG), hyoglossus (HG) and inferior longitudinal (IL) muscles. This layer is also demonstrated by the cross section of the tongue stained with hematoxylin and eosin stain in A. The Sihler’s stained canine tongue shows that the hypoglossal (XII) and lingual (LN) nerves travel on the ventral aspect of the tongue to supply the muscles and mucosa, respectively. The glossopharyngeal (IX) nerve supplies the mucosa and vallate papillae in the posterior tongue. In this specimen the genioglossus and inferior longitudinal muscles have been removed. a, anterior division of the lingual nerve; p, posterior division of the lingual nerve. In the canine posterior tongue, there are two to three vallate papillae on each side (Figs. 3A,B). It was found that the first or medial branch of the IX is the major contributor to the nerve supply of the vallate papillae. In general, the posterior vallate papilla receives its innervation from the first branch of the IX, while the anterior vallate papilla from both the first and second branches of the IX nerve (Fig. 3A). Each of the vallate papillae is usually supplied by two to three terminal branches which connect each other, pass through the center of the papilla, and extend to the free end of the papilla on the dorsal surface of the tongue. The lingual nerve (LN). The LN reaches the tongue at the middle third and comes into the tongue ventrally (Fig. 1E). It divides into a posterior (p) and an anterior (a) division. The posterior division, which has a relatively shorter length and a smaller innervation area than the anterior one, sends four to five branches which pass through the tongue muscles to supply the posterior mucosa of the middle third of the tongue. In addition, the posterior division of the LN also sends nerve branches to innervate the vallate papillae (Fig. 3B). The anterior division of the LN runs forward along with the medial division of the XII to the tip of the tongue. During its course, this division gives off about 10 (n ⫽ 3) first-order branches and numerous twigs (Fig. 4A). These nerve branches pass through the tongue musculature to supply the mucosa of the anterior third and anterior part of the middle third of the tongue. Some terminal twigs of the LN were found to terminate taste buds in the mucosa of the anterior tongue (Fig. 4B). Neural connections between the XII, IX, and lingual nerves. Numerous neural connections between the XII, IX, and lingual nerves were found in all specimens studied (Figs. 5A–D). In Figure 5A, three squares indicate the sites and patterns of the connections commonly seen in the canine tongue. In this specimen most muscles have been removed and the nerve plexus formed by XII and lingual nerves was placed beside the tongue in order to show the neural connections. The connections between the lingual and XII nerves were observed in the plexus and in the tongue muscles. In addition, the connections between the IX and lingual nerves were also recognized. Crossing innervation of the XII and lingual nerves. In one specimen, the XII was found to send a branch at the conjunction between the anterior and middle thirds of the tongue to cross the midline to supply the muscle on the contralateral side (Fig. 6A). The lingual nerve also sends nerve branches in the anterior tongue to cross the median septum of the tongue to supply the mucosa on the contralateral side (Fig. 6B). 416 MU AND SANDERS Figure 1 (Continued). NEUROMUSCULAR ORGANIZATION OF THE TONGUE 417 Fig. 2. Branching and distribution of the XII nerve within the canine tongue. A: A schematic photograph of a canine tongue (ventral view) that illustrates the branching pattern of the XII nerve within the three layers of the tongue muscles on one side. The genioglossus (GG) muscle is retracted laterally. 1, the inferior layer which is separated and removed to the lateral side; 2, the anterior part of the middle layer; 3, the posterior part of the superior layer which is seen after the middle layer is removed. SG, styloglossus; HG, hyoglossus; IL, inferior longitudinal muscle; T, transverse muscle; V, vertical muscle; SL, superior longitudinal muscle; GG, genioglossus muscle; O, oblique compartment of the GG muscle; H, horizontal compartment of the GG muscle; L, lateral division of the XII nerve; M, medial division of the XII nerve. The asterisks indicate the nerve branches to the posterior transverse and vertical muscles which have been removed. The arrow indicates the nerve branch to the geniohyoid muscle. B: Higher power view of the XII nerve branching pattern in the posterior tongue, demonstrating the numbers and branching points of the muscle nerves derived from the XII. M, medial division of the XII nerve; L, lateral division of the XII nerve; PTV, nerve branches to the posterior transverse and vertical muscles; SL, nerve branches to the superior longitudinal muscle; GH, the nerve branch to the geniohyoid muscle; GG, genioglossus muscle; HG, hyoglossus muscle; SG, styloglossus muscle; LN, lingual nerve. 3⫻. The Intramuscular Innervation Patterns of the Individual Tongue Muscles Styloglossus, hyoglossus, and inferior longitudinal muscles. The styloglossus, hyoglossus and inferior Figure 2A shows the three layers of the tongue muscles. From the ventral to the dorsal aspect of the tongue, the muscle layers are the inferior (1), middle (2), and superior (3), respectively. The XII nerve provides motor nerve supply to all the three layers of the tongue muscles. The inferior layer of the muscles is supplied by the lateral division, whereas the middle and superior layers by the medial division of the XII nerve. The intrinsic musculature studied includes the transverse, vertical, and superior longitudinal muscles. The extrinsic musculature studied includes the genioglossus, hyoglossus, and styloglossus muscles. Each of these muscles was found to have its own specific intramuscular innervation pattern as described below. longitudinal muscles consist of the inferior muscle layer of the tongue. They receive their nerve supply from the lateral division of the XII nerve. The lateral division actually consists of three to four separate nerve branches which are derived from the XII nerve at different levels within a short distance (Figs. 1E, 2A,B). The first two branches which are smaller and shorter than the third one supply the posterior part of the styloglossus and hyoglossus muscles. The third branch gives off three to four second-order branches forming a nerve bundle. These nerve branches run toward the tip of the tongue and give off many terminal branches along their courses to supply the inferior longitudinal, styloglossus and hyoglossus muscles. 418 MU AND SANDERS Fig. 3. Branching and distribution of the IX nerve within the canine tongue (ventral view). A: After entering the tongue base the IX nerve gives off three to four nerve branches to supply the vallate papillae (arrows) and the mucosa of the posterior tongue. The first or medial branch mainly contributes innervation to the vallate papillae. 3⫻. B: Higher power view of the nerve supply of the vallate papillae in A. Note that in addition to the IX the posterior division of the lingual nerve (LN) also gives off small nerve branches (solid arrows) to innervate the vallate papillae (open arrows). 6⫻. Fig. 4. Branching and distribution of the lingual nerve (LN) within the canine tongue. A: Higher power view of Sihler’s stained right anterior canine tongue. The arrowheads indicate that the dorsal mucosa (D) (left side) has been separated from the muscles (M) (right side). Note that the anterior division of the LN (aLN) and the medial division of the XII (mXII) form a nerve bundle, running forward to the tip of the tongue (top). Nerve branches derived from the aLN supply the mucosa and taste buds. The square indicates the area of magnification for B. 3⫻. B: Higher power view of the area boxed in A. Note that the terminal branches of the aLN innervate taste buds (arrows). 9⫻. NEUROMUSCULAR ORGANIZATION OF THE TONGUE Figure 5. (Legend, overleaf.) 419 420 MU AND SANDERS Fig. 6. Contralateral innervation of the XII and lingual (LN) nerves within a canine tongue. A: Higher power view of the median fibrous septum in the middle tongue. Note that a nerve branch from the XII nerve (arrow) on left side crosses the median septum (M) to innervate the superior longitudinal muscle on the contralateral side. 12⫻. B: Higher power view of the median fibrous septum in the anterior tongue. Note that the LN on the right side sends nerve branches (arrow) crossing the median septum (M) to supply the mucosa on the contralateral side. 12⫻. Transverse and vertical muscles. When the inferior muscle layer (styloglossus, hyoglossus, and inferior longitudinal muscles) of the tongue was removed, the middle muscle layer (transverse and vertical muscles) was identified (Fig. 2A). Of the intrinsic tongue muscles, the transverse and vertical muscles have relatively shorter muscle fibers than others. Both muscles receive their nerve supply from the medial division of the XII nerve (Figs. 2A, 7A,B). The medial division of the XII nerve on each side travels along the midline and runs to the tip of the tongue. It gives off more than 20 first-order branches and numerous twigs to the transverse and vertical muscu- lar sheets. Each of the muscular sheets is generally innervated by a small twig even though numerous connections exist between them (Fig. 7B). Superior longitudinal muscle. When the inferior and middle muscle layers of the tongue were removed, the superior layer (superior longitudinal muscle) was seen (Figs. 1A–C). The nerve branches to the superior longitudinal muscle is derived from the medial division of the XII nerve (Fig. 2A). Shortly after the lateral division of the XII is given off, four to five first-order branches are derived from the medial division to supply the superior longitudinal muscle. These nerve branches subdivides into many Fig. 5. Neural connections between different nerves within the canine tongue. A: A Sihler’s stained canine tongue from which most tongue muscles have been removed to show the neural connections between the hypoglossal (XII), lingual (LN), and glossopharyngeal (IX) nerves. The plexus formed by the XII and LN was dissected and placed beside the tongue. The square a indicates the connections between the lateral division of the XII nerve and the anterior (a) division of the LN. The square b indicates the connections between the medial division of the XII and the posterior (p) division of the LN. The square c indicates the connections between the XII and the LN and between the LN and IX nerves. B: Higher power view of square a in A, showing the neural connections (arrows) in the plexus formed by the LN and XII nerves. 6⫻. C: Higher power view of square b in A, demonstrating the neural connections (arrows) between the LN and XII nerves in the posterior tongue. 6⫻. D: Higher power view of square c in A, illustrating the neural connections between the LN and XII nerves (small arrow) and between the LN and IX nerves (large arrow). 6⫻. NEUROMUSCULAR ORGANIZATION OF THE TONGUE 421 Fig. 7. Innervation of the transverse (T) and vertical (V) muscles in the canine tongue. A: A Sihler’s stained right semi-tongue, showing that the T and V muscles receive their nerve supply from the medial division (M) of the XII. L, lateral division of the XII nerve; LN, lingual nerve. The box indicates the area of magnification for B. B: Higher power view of the nerve supply of the anterior transverse (T) and vertical (V) muscles. Note that almost every sheet of the T and V muscles has a terminal branch that innervates both. 6⫻. second-order branches, each of which generally supplying a fascicle. The nerve branch supplying a muscle fascicle always gives off finer twigs during its course (Fig. 8). Genioglossus muscle. The genioglossus is a midline muscle of the tongue. It originates from the mandible and inserts into the tongue body and hyoid bone. In the dog, this muscle was found to comprise at least two distinct neuromuscular compartments, horizontal and oblique. The muscle fibers in the horizontal compartment insert into the tongue base and the hyoid bone, whereas those in the oblique compartment insert into the tongue body. Multiple nerve branches that are derived from the medial division of the XII nerve supply the genioglossus muscle. The medial division of the XII gives off more than 10 nerve branches at different levels to supply this muscle. These nerve branches travel from the insertional end to the original end of the muscle and supply individual muscular compartments (Figs. 2A,B, 9A). The intramuscular innervation patterns are different between the two compartments (Fig. 9B). Three to four first-order nerve branches travel along the muscle fibers and span the whole length of the horizontal compartment. During their courses they Fig. 8. Higher power view of the nerve supply of the superior longitudinal muscle. Note that each of the muscle fascicles is supplied by a nerve branch which runs along the muscle fascicle, distributing terminal branches at regular intervals, whereas each of the two larger fascicles is supplied by several nerve branches. 9⫻. 422 MU AND SANDERS give off several terminal branches. Seven to eight firstorder nerve branches supply the oblique compartment. The nerve branches in the oblique compartment are arranged in a grid-form (Figs. 2A, 9B). DISCUSSION Fig. 9. Innervation pattern of the canine genioglossus (GG) muscles. A: The medial division (M) of the XII gives off nerve branches to the GG muscle. GH, geniohyoid muscle; LN, lingual nerve; L, lateral division of the XII nerve; O, oblique compartment of the GG muscle; H, horizontal compartment of the GG muscle. B: Higher power view of the right GG muscle in A. Note that the horizontal (H) and oblique (O) compartments of the GG muscle have their own specially organized innervation patterns. The nerve branches to the H compartment travel the whole length of the muscle, whereas those to the O compartment are arranged in a grid-form. XII, hypoglossal nerve; LN, lingual nerve; PTV, nerve branches to the posterior transverse and vertical muscles. 3⫻. In this study the entire nerve supply of a mammalian tongue was demonstrated for the first time. The branching and distribution patterns of the motor (XII) and sensory (IX and LN) nerves to the canine tongue were studied from the main nerves to the terminal branches. Largely this was due to the use of the Sihler’s stain, which has the unique ability to render a whole specimen such as the tongue translucent, while still allowing the nerves and muscles to be studied in their original three-dimensional relationships. However, to fully demonstrate these relationships each specimen must be meticulously microdissected. With this combination of techniques several important observations could be made regarding the canine tongue. Each of the intrinsic tongue muscles appears to actually be a group of separate neuromuscular compartments. The motor nerve supply to these muscles, although highly complex, has a regular and predictable pattern. Other observations made were that there were numerous neural connections between the LN and XII or between the LN and the IX, and that the innervation of the XII and LN nerves crosses the midline. As to the motor innervation of the tongue, Abd-El-Malek (1938) and Hellstrand (1981) reported that the XII nerve in man and cats is divided into a lateral and a medial division and our findings agree with this basic description. However, we did not find the medial division of the XII nerve in the dog to supply the hyoglossus and styloglossus muscles as reported in the cat (Hellstrand, 1981). In the dog the medial division of the XII sends branches to supply the superior longitudinal muscle immediately after the lateral division is given off. This is not in agreement with the finding obtained from the cat reported by Hellstrand (1981), who observed that the nerve branches to the superior longitudinal muscle branch off the medial division more distally. These observations indicate that the distribution of the XII nerve is different between species. In this study, the branching order of the XII nerve from the root to the tip of the tongue was found to be: 1) the styloglossal branch; 2) the hyoglossal branch; 3) the geniohyoid branch; 4) the inferior longitudinal branch; 5) the superior longitudinal branch; 6) the branches to the horizontal part of the genioglossus muscle; 7) the posterior transverse and vertical muscular branches; 8) the branches to the oblique part of the genioglossus muscle; and 9) the anterior transverse and vertical muscular branches. The numbers of the first-order nerve branches to each muscle are different, ranging from one to two (styloglossus muscle) to 10 (genioglossus muscle). In addition, the entrance of the first-order nerve branches to each muscle is also different. For example, the nerve branches to the styloglossus, hyoglossus, and superior longitudinal muscles enter these muscles at their original ends (posterior part of the tongue), whereas the nerve branches to the genioglossus enter the muscle at its insertional end. Since the transverse and vertical muscles are arranged in alternating sheets from the base to the tip of the tongue, they receive their nerve supply from different levels along the course of the medial division of the XII nerve. NEUROMUSCULAR ORGANIZATION OF THE TONGUE The sensory innervation of the canine tongue was also studied. The results obtained from Sihler’s stain are in agreement with those reported by previous authors (Miller et al., 1964; Lawn, 1966; Yamamoto, 1975) who reported that the lingual nerve provides sensory fibers to the anterior two-thirds of the tongue mucosa, while the IX supplies the posterior third mucosa of the tongue and the vallate papillae. In addition, we found that the lingual nerve also innervates the vallate papillae. The relationship among the lingual, XII and IX nerves in the tongue, which is an important aspect of the neuroanatomy of the organ, was also examined by using Sihler’s stain. Earlier studies have demonstrated the peripheral connections between the lingual and XII nerves in the man, dog, pig, rabbit (Fitzgerald and Law, 1958) and cat (Fitzgerald and Law, 1958; Blom, 1960; Hellstrand, 1981). Sihler’s stain revealed numerous neural connections not only between the lingual and XII, but also between the lingual and IX nerves. The neural connections between the lingual and XII were mainly found at the junction of the posterior and middle third of the tongue, in the plexus formed by the lingual and XII nerves, and within the intrinsic tongue muscles. The connections between the lingual and IX were mainly located at the junction of the posterior and middle thirds of the tongue. Fitzgerald and Law (1958) observed two connection patterns, a lateral and medial lingual-hypoglossal connections. The lateral lingual-hypoglossal connection is formed by the connection of one or two branches of the lingual nerve with the lateral division of the XII nerve. This connection pattern was found in 90% (36/40) of dissections in man. The medial lingual-hypoglossal connection is formed by the connection of one or more branches of the lingual nerve with the branches of the medial division of the XII nerve. This connection pattern was revealed in 40% (16/40) of human dissections. Although the nature and physiological significance of the connecting nerves have never been evaluated because of technical difficulties, the data concerning the location and patterns of the neural connections will be helpful for further studies. Sihler’s stain also revealed contralateral innervation of the XII and lingual nerves in one canine tongue specimen. This finding is not consistent with the traditional concept that the XII, lingual and IX nerves innervate the ipsilateral musculature and mucosa, respectively (Weddell et al., 1940; Miller et al., 1964; Lowe, 1980). Although a conclusive statement cannot be made at this point, it is reasonable to assume that with complete unilateral paralysis of the XII nerve some reinnervation of the paralyzed muscles by the contralateral side could occur. Further studies will be worthwhile to study the extent of contralateral tongue innervation in mammals by means of Sihler’s stain and to estimate its functional and clinical significance by using electrophysiological techniques. In this study, the neuromuscular organization pattern of the tongue was demonstrated in much greater detail than previously shown. Recent studies have confirmed that some muscles traditionally described to be single muscles are actually composed of smaller neuromuscular compartments that may have different functions (English and Letbetter, 1982a,b; Windhorst et al., 1989). By using anatomical, histological, and histochemical criteria such as orientation of the muscle fascicles, distribution of muscle fiber types, and intramuscular nerve supply pattern, the compartmentalized neuromuscular organization 423 of many mammalian muscles has been demonstrated in both the large (English and Letbetter, 1982a,b) and smaller muscles (Richmond et al., 1985; Richmond and Armstrong, 1988; Zaretsky and Sanders, 1992; Sanders et al., 1994; Mu and Sanders, 1998b). This concept of neuromuscular compartments within a single muscle (English and Letbetter, 1982a,b; Windhorst et al., 1989) appears to characterize the tongue muscles. In fact the evidence for separate neuromuscular compartments in the tongue muscles is so strong it may be more accurate to refer to them as tongue muscle groups. Recent studies have indicated that the regional activation of the tongue muscles appears to shape and regulate the various motions of the tongue (Napadow et al., 1999). It is possible that the individual tongue muscle compartments have some degree of independent motor function, and this would help explain how a small number of tongue muscles can cause a large number of different tongue shapes and movements. Confirming this functional organization requires much more experimentation. However, the details of the nerve branching will be helpful for determining functions of individual tongue muscles or their compartments by electrical stimulation of specific nerve branches. In addition, it will be useful for localizing the motor neurons in the hypoglossal nucleus which innervate a compartment within a given tongue muscle by injecting horseradish peroxidase (HRP) into the compartment or muscle layer as revealed by Sihler’s stain. Clinically, determining the exact branching patterns of the nerves to the tongue will be helpful for designing neurosurgical procedures to restore sensory or motor function to the paralyzed or non sensate tongue. 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