BrainResearchBulletin,Vol. 24. pp. 81-87. o Pergamon Press plc, 1990. Printed in the U.S.A.
0361-9230/90 $3.00 + .00
Afferent Fibers in the Hypoglossal Nerve: A Horseradish Peroxidase Study in the Cat Y O S H I K I T A K E U C H I , * T O S H I O H A Y A K A W A , ' ~ H I R O K I S. O Z A K I , * J U N Z O KITO,:~ T A K A H I R O S A T O D A § A N D R Y O T A R O M A T S U S H I M A §
*Department of Anatomy, Kagawa Medical School, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-07, Japan ~Department of Anatomy and $1nstitute for Laboratory Animal Research Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan and §Department of Oral Anatomy, School of Dentistry Hiroshima University, 1-2-3 Kasumi, Hiroshima 734, Japan R e c e i v e d 19 July 1989
TAKEUCHI, Y., T. HAYAKAWA, H. S. OZAKI, J. KITO, T. SATODA AND R. MATSUSHIMA. Afferentfibers in the hypoglossal nerve: A horseradishperoxidase study in the cat. BRAIN RES BULL 24(1) 81-87, 1990.--The existence of afferent fibers in the cat hypoglossal nerve was studied by transganglionic transport of horseradish peroxidase (HRP). Injections of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the hypoglossal nerve resulted in some retrograde labeling of cell bodies within the superior ganglia of the ipsilateral glossopharyngeal and vagal nerves. A few labeled cell bodies were also present ipsilaterally within the inferior ganglion of the vagal nerve and the spinal ganglion of the C1 segment. Some of the labeled glossopharyngeal and vagal fibers reached the nucleus of the solitary tract by crossing the dorsal portion of the spinal trigeminal tract. Others distributed to the spinal trigeminal nucleus pars interpolaris and to the ventrolateral part of the medial cuneate nucleus by descending through the dorsal portion of the spinal trigeminal tract. In the spinal cord these descending fibers, intermingling with labeled dorsal root fibers, distributed to laminae I, IV-V and VII-VIII of the C 1 and C2 segments. Additional HRP experiments revealed that the fibers in laminae VII-VIII originate mainly from dorsal root of the C1 segment. Afferent fibers
Hypoglossal nerve
Ganglion cells
Central distribution
HRP
Cat
METHOD
THE existence of afferent fibers in the hypoglossal nerve has been proposed by recording of afferent impulses in the isolated filaments of the hypoglossal nerve (4,8). Furthermore, some other physiological studies, in which stimulation of the hypoglossal nerve evoked vascular reactions (10,32) and reflex activation of the facial (19, 30, 35) and laryngeal muscles (29,35), provided evidence for the existence of such afferent fibers. The interest in these studies was focused on the mediation of the hypoglossofacial and hypoglossolaryngeal reflexes by afferent fibers which join the vagal nerve at the level of the inferior (nodose) ganglion. Anatomical studies have also demonstrated the existence of afferent fibers in the hypoglossal nerve, but led to varied conclusions concerning the sites of the parent cell bodies and the course of the afferent fibers. Sensory cells have been reported to be present along the course of the hypoglossal nerve (11, 24-26, 31-33, 36) and within the cervical spinal ganglion (6, 15, 34). Other investigations have revealed that afferent fibers join the vagal nerve and cell bodies of the fibers are located within the inferior (nodose) ganglion (32,36). However, recent studies have shown that the cell bodies are present within the superior (jugular) ganglion (6,22). This variety of observations implies the existence of various pathways of the afferent fibers in the hypoglossal nerve. The aim of the present study is, therefore, to investigate the ganglion cells and central distribution of the afferent fibers in the cat hypoglossal nerve utilizing transganglionic transport of WGA-HRP.
Twenty-one cats weighing 0.7 to 3.4 kg were used for the present study. All surgical procedures were done under general anesthesia with pentobarbital soda (40 mg/kg IP). In the series of the present experiments, the hypoglossal nerve was exposed widely around the common carotid artery. After identification of the superior radix of the ansa cervicalis, 2-5 I~1 of a solution of 0.2% WGA-HRP (Toyobo) containing 10% HRP (Toyobo, Grade-I-C) was injected into the proximal end of the hypoglossal nerve, cut at the portion distal to the superior radix, using a 10-1xl Hamilton microsyringe (11 cats). In the other experiments, 2-4 I~1 of the HRP was injected into the same portion of the hypoglossal nerve after additional transections of the superior radix of the ansa cervicalis (3 cats), the dorsal root of the CI segment (4 cats) and both the superior radix and dorsal root (3 cats). In all of the above experiments, to prevent leakage of injected HRP, the proximal end of the hypoglossal nerve was tied immediately with a thread after the injections. After a survival time of 2-3 days, the animals were anesthetized again and sacrificed by perfusion through the ascending aorta with 0.1 M phosphate buffer (pH 7.4) followed by a fixative containing 8% formalin in 0.1 M phosphate buffer. The brain stem and spinal cord were removed together with the superior and inferior ganglia of the glossopharyngeal and vagal nerves, geniculate ganglion, trigeminal ganglion and C1-C3 spinal ganglia on the side ipsilat-
81
J
82
TAKEUCHI ET AL. X l nerve
C, ~C"c,,,
GH
TH
OsHHS4~C THi FIG. 1. Schematic representation of relationship between the hypoglossal nerve and the ansa cervicalis. Arrows x, y and z indicate the site of transection of the hypoglossal nerve, superior radix of the ansa cervicalis and dorsal root of the C 1 segment, respectively. CI, CII, CIII, first, second and third cervical dorsal root; GH, geniohyoid muscle; OHi, inferior belly of omohyoid muscle; OHs, superior belly of omohyoid muscle; SH, sternohyoid muscle; ST, sternothyreoid muscle; TH, thyreohyoid muscle; XII nerve, hypoglossal nerve.
eral to the injections. These brain stem, spinal cord and ganglia were stored 1-3 days in 0.1 M phosphate buffer containing 30% sucrose. Serial 60 ixm-thick transverse sections were cut on a freezing microtome. Sections were processed for the demonstration of HRP reaction product according to the tetramethyl benzidine (TMB) protocol of Mesulam (20) and were counterstained with neutral red. RESULTS HRP injections are made into the hypoglossal nerve, leaving the superior radix of the ansa cervicalis and the dorsal root of the C1 segment intact (Fig. 1, arrow x). In this experiment the injected HRP is clearly restricted along the course of the hypoglossal nerve as a small brown mass. Heavy HRP labeling was found in the hypoglossal motor fibers and their cell bodies ipsilaterally (Fig. 2). Some labeled cells are detected within the ipsilateral superior ganglia of the glossopharyngeal and vagal nerves. Within the superior ganglion of the glossopharyngeal nerve the labeled cells are observed to be relatively compact (Fig. 3A) while within the ganglion of the vagal nerve these are confined to the region close to the vagal nerve (Fig. 3B). A few labeled cells are present within the inferior ganglion of the vagal nerve (Fig. 3C) and the spinal ganglion of the C1 segment (Fig. 3D). Very few labeled cells occur within the inferior ganglion of the glossopharyngeal nerve, mandibular branch of the trigeminal ganglion and spinal ganglia of the C2 and C3 segments. No cell labeling is observed within the geniculate ganglion. The transganglionic labeling of the central processes of these
FIG. 2. Photomicrograph of retrogradely labeled neurons of the hypoglossal nucleus after HRP injections into the cut proximal end of the hypoglossal nerve. Calibration bar = 200 p,m.
HYPOGLOSSAL AFFERENT FIBERS
83
B
¢ \ t
FIG. 3. Photomicrographs of retrogradely labeled cell bodies within the superior ganglion of the glossopharyngeal nerve (A), superior (B) and inferior ganglion (C) of the vagal nerve, and spinal ganglion of the CI segment (D) after HRP injections into the cut proximal end of the hypoglossal nerve without transection of the superior radix of the ansa cervicalis and the dorsal root of the C1 segment. Note that labeled cell bodies are relatively compact within the superior ganglion of the glossopharyngeal nerve (arrow in A). Asterisk indicates the superior ganglion of the vagal nerve. IXn, glossopharyngeal nerve; Xn, vagal nerve. Calibration bars = 200 Ixm in A-D.
ganglia can be followed into ipsilateral brain stem areas. Labeled glossopharyngeal and vagal fibers enter the brain stem from the lateral surface at the level of the facial nucleus and the rostral end of the inferior olive, respectively (Figs. 4A, B, and 5A, B). After crossing the dorsal portion of the spinal trigeminal tract, these labeled glossopharyngeal and vagal fibers join the solitary tract and extend caudally. These fibers distribute mainly to the interstitial and medial subdivisions of the nucleus of the solitary tract (Figs. 4C and 5C). A few fibers also distribute to the parvocellular and commissural subdivisions (Fig. 4D-F). On occasion, commissural fibers are found in the region just dorsal to the central canal (Figs. 4F and 5F).
A few labeled fibers of the glossopharyngeal nerve and some labeled fibers of the vagal nerve form a descending bundle in the dorsal portion of the spinal trigeminal tract. From this bundle, labeled fibers distribute to the caudal portion of the spinal trigeminal nucleus pars interpolaris immediately adjacent to the spinal trigeminal tract (Figs. 4D, E and 5D). At the level of the rostral portion of the spinal trigeminal nucleus pars caudalis, the descending fibers are located along the medial border of the spinal trigeminal tract. At this level, termination areas appear to be present in the ventrolateral part of the medial cuneate nucleus (Figs. 4E, F and 5E). At the C1 level, the descending fibers intermingle with labeled dorsal root fibers located in the dorsolat-
84
TAKEUCHI E T AL.
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i
/
FIG. 4. Distribution of labeled fibers in the lower brain stem and upper cervical spinal cord after HRP injections into the cut proximal end of the hypoglossal nerve without transection of the superior radix of the ansa cervicalis and dorsal root of the C 1 segment. Amb, ambiguus nucleus; AP, area postrema; Corn, commissural nucleus; Cum, medial cuneate nucleus; FN, facial nucleus; Gr, gracil nucleus; IO, inferior olivary nucleus; NST, nucleus of solitary tract; Spc, parvocellular solitary nucleus; Vsp, spinal trigeminal nucleus; Vt, spinal trigeminal tract; IXn, glossopharyngeal nerve; Xn, vagal nerve; XII, hypoglossal nucleus.
eral funiculus. In the gray matter, these labeled fibers distribute to laminae I, IV-V and VII-VIII of Rexed (27) from the medial aspect of the dorsal horn (Figs. 4G and 6). The labeled descending fibers are traced as far as the C2 segment, but there are no differences in the pattern of distribution of the labeled fibers as compared to that of the C1 segment (Fig. 4H). In order to determine the course of the afferent fibers in the hypoglossal nerve, transection of the superior radix of the ansa cervicalis and/or the dorsal root of the C 1 segment is made prior to HRP injections into the hypoglossal nerve. In the case of transection of the superior radix (Fig. 1, arrow y), the results of labeled cells within the ganglia and pattern of central distribution of labeled fibers are similar to those in the experiment described above. On the other hand, after transection of the dorsal root of the C 1 segment (Fig. 1, arrow z) or both the dorsal root and superior radix, the only difference is a substantial reduction of labeled fibers in laminae VII-VIII. DISCUSSION
The present study confirms the existence of afferent fibers in the cat hypoglossal nerve and demonstrates the distribution of the afferent fibers in the lower brain stem and spinal cord. Sensory fibers in the hypoglossal nerve are indicated to be conveyed
centrally through the glossopharyngeal and vagal nerves and the upper cervical dorsal roots. Many electrophysiological investigations on the hypoglossal afferents in the cat referred to a probable location of the corresponding cell bodies within the inferior ganglion of the vagal nerve (10, 12, 29, 30, 35). In anatomical studies, the afferent fibers were often reported to join the vagal nerve via the hypoglossonodose branch. Tarkhan and Abou-E1-Naga (32) showed that extirpation of the inferior ganglion of the vagal nerve in the dog resulted in degeneration of approximately 5% of the fibers in the hypoglossal nerve. Zimny et al. (36) also found chromatolytic changes in cells of the inferior ganglion in the cat and dog after transection of the hypoglossal nerve. However, in recent HRP studies, cells of the afferent fibers were reported to be located predominantly within the superior ganglion in the rat (22) and dog (6). The present study indicates that in the cat the hypoglossal nerve connects with the vagal nerve and that the afferent fibers are more likely to originate from cells within the superior ganglion rather than from neurons within the inferior ganglion of the vagal nerve. With respect to connections of the hypoglossal nerve with the glossopharyngeal nerve, Chibuzo and Cummings (6) suggested the existence of a course along the lingual branch of the glossopharyngeal nerve because of the presence of HRP-labeled cells within the ganglia of the glossopharyngeal nerve following injection of HRP into the intrinsic lingual muscle. However, the specific ganglion and the degree of labeling were not described in detail. With regard to this point, the present study has shown connections of the hypoglossal nerve with the glossopharyngeal nerve and that the major origin of such afferent fibers is from cells within the superior ganglion. The present results show that the afferent fibers in the hypoglossal nerve end in the spinal trigeminal nucleus, cuneate nucleus, nucleus of the solitary tract and the C 1 and C2 segments of the spinal cord. A similar distribution of the afferent fibers was reflected in central projections of primary afferent fibers originating from cells within the superior ganglion of the glossopharyngeal nerve in the monkey (2), superior ganglion of the vagal nerve in the cat (1,23) and inferior ganglion of the vagal nerve in the rat and cat (7, 9, 16). The projections to the upper cervical spinal cord may originate from both the dorsal root of the C1 segment as well as the glossopharyngeal and vagal nerves. Previous authors noted in the rabbit (34), dog (6) and cat (17) that cell bodies of afferent fibers in the hypoglossal nerve are present within the spinal ganglion of the C1 segment. This finding is well in accordance with that obtained from the present HRP experiments. The present study also reveals that the afferent fibers do not come from the ansa cervicalis, but from the dorsal root at the C1 segment. Interestingly, the terminal fields of the fibers are laminae VII-VIII. This distribution of labeled fibers is well reflected by the central projection of the dorsal roots of the C1 segment (14). The remaining labeled fibers in laminae I and I V - V represent mainly the termination of primary afferent fibers of the glossopharyngeal and vagal nerves. In fact, Kerr (16) and Rhoton et al. (28) using the degeneration method demonstrated an exclusive termination of the glossopharyngeal and vagal fibers in lamina IV at the C1-C3 segments in the cat and monkey while Beckstead and Norgren (2) using the autoradiographic method revealed termination of the vagal fibers in laminae I-IV at the C1 segment in the monkey. Furthermore, of particular interest in the present study is the distribution of labeled fibers to lamina I because this layer is reported to be a major relay for nociceptive information (3, 5, 13, 18, 21). Therefore, the present findings indicate that afferent fibers in the hypoglossal nerve play an important role in conveying information of muscular pain mainly through the glossopharyngeal and vagal nerves.
H Y P O G L O S S A L A F F E R E N T FIBERS
85
D
ST
iNST
Vsp
FIG. 5. Photomicrographs of labeled glossopharyngeal (A) and vagal nerves (B), and distribution of labeled fibers in the nucleus of the solitary tract (C), spinal trigeminal nucleus pars interpolaris (D), medial cuneate nucleus (E) and commissural fibers (F). Note that descending fibers are located in the dorsal portion of the spinal trigeminal tract (asterisk in D). Arrowheads indicate commissural fibers just dorsal to the central canal. CC, central canal; Cure, medial cuneate nucleus; iNST, interstitial subdivision of nucleus of solitary tract; mNST, medial subdivision of nucleus of solitary tract, ST, solitary tract; Vsp, spinal trigeminal nucleus; Vt, spinal trigeminal tract. Calibration bars = 100 izm in A-F.
T A K E U C H I ET AL.
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FIG. 6. Photomicrograph of labeled fibers in the spinal cord of the C1 segment. Note that these fibers distribute to laminae I and IV-V. I-V, Rexed's laminae I V . Calibration bar= 200 p.m.
ACKNOWLEDGEMENTS The authors would like to thank Professor Kazuo Yamashita for his advice and encouragement and Mr. Shinkichi Terada for technical assistance.
REFERENCES 1. Arvidson, J.; Thomander, L. An HRP study of the central course of sensory intermediate and vagal fibers in peripheral facial nerve branches in the cat. J. Comp. Neuroh 223:35-45; 1984. 2. Beckstead, R. M.; Norgren, R. An autoradiographic examination of the central distribution of the trigeminal, facial, glossopharyngeal, and vagal nerves in the monkey. J. Comp. Neurol. 184:455-472; 1979. 3. Bennett, G. J.; Abdelmoumene, M.; Hayashi, H.; Hoffert, M. J.; Dubner, R. Spinal cord layer I neurons with axon collaterals that generate local arbors. Brain Res. 209:421-426; 1981. 4. Blom, S. Afferent influences on tongue muscle activity. Acta Physiol. Scand. 49(Supph 170):1-97; 1960. 5. Burstein, R.; Cliffer, K. D.; Giesler, G. J., Jr. Direct somatosensory projections from the spinal cord to the hypothalamus and telencephalon. J. Neurosci. 7:4159-4164; 1987. 6. Chibuzo, G. A.; Cummings, J. F. The origins of the afferent fibers to the lingual muscles of the dog, a retrograde labeling study with horseradish peroxidase. Anat. Rec. 200:95-101; 1981. 7. Contreras, R. J.; Beckstead, R. M.; Norgren, R. The central projections of the trigeminal, facial, glossopharyngeal and vagal nerves: an autoradiographic study in the rat. J. Auton. Nerv. Syst. 6:303-322; 1982. 8. Cooper, S. Afferent impulses in the hypoglossal nerve on stretching the cat's tongue. J. Physiol. (Lond.) 126:32P; 1954. 9. Cottle, M. K. Degeneration studies of primary afferents of IXth and Xth cranial nerves in the cat. J. Comp. Neurol. 122:329-345; 1964.
10. Downman, C. B. B. Afferent fibers of the hypoglossal nerve. J. Anat. 73:387-395; 1939. 11. Froriep, A.; Beck, W. Uber das Vorkommen dorsaler hypoglossuswurzeln mit Ganglion, in der Reihe der Saugetiere. Anat. Anz. 10:688--696; 1895. 12. Hanson, J.; Widen, L. Afferent fibers in the hypoglossal nerve of cat. Acta Physiol. Scand. 79:24-36; 1970. 13. Hylden, J. K.; Hayashi, H.; Dubner, K.; Bennett, G. J. Physiology and morphology of the lamina I spinomesencephalic projection. J. Comp. Neurol. 247:505-515; 1986. 14. Imai, Y.; Kusama, T. Distribution of the dorsal root fibers in the cat. An experimental study with the Nauta method. Brain Res. 13: 338-359; 1969. 15. Kendall, B.; Crobin, K. B.; Harrison, F. The sensory innervation of the spinal accessory and tongue musculature in the rhesus monkey. Brain 62:191-197; 1939. 16. Kerr, F. W. L. Facial, vagal and glossopharyngeal nerves in the cat. Arch. Neurol. 6:264-281; 1962. 17. Langworthy, O. R. A study of the innervation of the tongue musculature with particular reference to the proprioceptive mechanism. J. Comp. Neurol. 36:273-293; 1924. 18. Light, A. R.; Trevino, D. L.; Perl, E. R. Morphological features of functionally defined neurons in the marginal zone and substantia geratinosa of the spinal dorsal horn. J. Comp. Neurol. 186:151-172; 1979.
H Y P O G L O S S A L A F F E R E N T FIBERS
19. Lindquist, C.; Martensson, A. Reflex responses induced by stimulation of hypoglossal afferents. Acta Physiol. Scand. 77:234--240; 1969. 20. Mesulam, M.-M. Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26:106-117; 1978. 21. Miletic, V.; Hoffert, M. J.; Ruda, M. A.; Dubner, R.; Shigenaga, Y. Serotonergic axonal contacts on identified cat spinal dorsal horn neurons and their correlation with nucleus raphe magnus stimulation. J. Comp. Neurol. 228:129-141; 1984. 22. Neuhuber, W.; Mysicka, A. Afferent neurons of the hypoglossal nerve of the rat as demonstrated by horseradish peroxidase tracing. Anat. Embryol. (Berl.) 158:349-360; 1980. 23. Nomura, S.; Mizuno, N. Central distribution of primary afferent fibers in the Arnold's nerve (the auricular branch of the vagus nerve): a transganglionic HRP study in the cat. Brain Res. 292:199-205; 1984. 24. Pearson, A. A. Sensory type neurons in the hypoglossal nerve. Anat. Rec. 85:365-375; 1943. 25. Pearson, A. A. Further observations on the intramedullary sensory type neurons along the hypoglossal nerve. J. Comp. Neurol. 82: 93-100; 1945. 26. Pearson, A. A. Hypoglossal nerve ganglion in man. Anat. Rec. 139:316; 1961.
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27. Rexed, B. The cytoarchtectonic organization of the spinal cord in the cat. J. Comp. Neurol. 96:415-495; 1952. 28. Rhoton, A. L.; O'Leary, J. L.; Furguson, J. P. The trigeminal, facial, vagal and glossopharyngeal nerves in the monkey. Arch. Neurol. 14:530-541; 1966. 29. Sauerland, E. K.; Mizuno, N. Hypoglossal nerve afferents: elicitation of a polysynaptic hypoglossolaryngeal reflex. Brain Res. 10:256-258; 1968. 30. Tanaka, T. Afferent projections in the hypoglossal nerve to the facial neurons of the cat. Brain Res. 99:140-144; 1975. 31. Tarkhan, A. A. Uber das Vorhandensein afferenter Fasem im Nervus hypoglossus. Arch. Psychiat. Nervenkr. 105:475--483; 1936. 32. Tarkhan, A. A.; Abou-E1-Naga, I. Sensory fibers in the hypoglossal nerve. J. Anat. 81:23-32; 1947. 33. Tarkhan, A. A.; Abd EI-Malek, S. On the presence of sensory nerve cells on the hypoglossal nerve. J. Comp. Neurol. 93:219-228; 1950. 34. Yee, J.; Harrison, F.; Corbin, K. B. The sensory innervation of the spinal accessory and tongue musculature in the rabbit. J. Comp. Neurol. 70:305-314; 1939. 35. Zapata, P. G.; Torrealba, G. Reflex effects evoked by stimulation of hypoglossal afferent fibers. Brain Res. 445:19-29; 1988. 36. Zimny, R.; Sobusiak, T.; Matlosz, Z. The afferent components of the hypoglossal nerve. J. Himforsch. 12:83-100; 1970.
0361-9230/90 $3.00 + .00
Afferent Fibers in the Hypoglossal Nerve: A Horseradish Peroxidase Study in the Cat Y O S H I K I T A K E U C H I , * T O S H I O H A Y A K A W A , ' ~ H I R O K I S. O Z A K I , * J U N Z O KITO,:~ T A K A H I R O S A T O D A § A N D R Y O T A R O M A T S U S H I M A §
*Department of Anatomy, Kagawa Medical School, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-07, Japan ~Department of Anatomy and $1nstitute for Laboratory Animal Research Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan and §Department of Oral Anatomy, School of Dentistry Hiroshima University, 1-2-3 Kasumi, Hiroshima 734, Japan R e c e i v e d 19 July 1989
TAKEUCHI, Y., T. HAYAKAWA, H. S. OZAKI, J. KITO, T. SATODA AND R. MATSUSHIMA. Afferentfibers in the hypoglossal nerve: A horseradishperoxidase study in the cat. BRAIN RES BULL 24(1) 81-87, 1990.--The existence of afferent fibers in the cat hypoglossal nerve was studied by transganglionic transport of horseradish peroxidase (HRP). Injections of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the hypoglossal nerve resulted in some retrograde labeling of cell bodies within the superior ganglia of the ipsilateral glossopharyngeal and vagal nerves. A few labeled cell bodies were also present ipsilaterally within the inferior ganglion of the vagal nerve and the spinal ganglion of the C1 segment. Some of the labeled glossopharyngeal and vagal fibers reached the nucleus of the solitary tract by crossing the dorsal portion of the spinal trigeminal tract. Others distributed to the spinal trigeminal nucleus pars interpolaris and to the ventrolateral part of the medial cuneate nucleus by descending through the dorsal portion of the spinal trigeminal tract. In the spinal cord these descending fibers, intermingling with labeled dorsal root fibers, distributed to laminae I, IV-V and VII-VIII of the C 1 and C2 segments. Additional HRP experiments revealed that the fibers in laminae VII-VIII originate mainly from dorsal root of the C1 segment. Afferent fibers
Hypoglossal nerve
Ganglion cells
Central distribution
HRP
Cat
METHOD
THE existence of afferent fibers in the hypoglossal nerve has been proposed by recording of afferent impulses in the isolated filaments of the hypoglossal nerve (4,8). Furthermore, some other physiological studies, in which stimulation of the hypoglossal nerve evoked vascular reactions (10,32) and reflex activation of the facial (19, 30, 35) and laryngeal muscles (29,35), provided evidence for the existence of such afferent fibers. The interest in these studies was focused on the mediation of the hypoglossofacial and hypoglossolaryngeal reflexes by afferent fibers which join the vagal nerve at the level of the inferior (nodose) ganglion. Anatomical studies have also demonstrated the existence of afferent fibers in the hypoglossal nerve, but led to varied conclusions concerning the sites of the parent cell bodies and the course of the afferent fibers. Sensory cells have been reported to be present along the course of the hypoglossal nerve (11, 24-26, 31-33, 36) and within the cervical spinal ganglion (6, 15, 34). Other investigations have revealed that afferent fibers join the vagal nerve and cell bodies of the fibers are located within the inferior (nodose) ganglion (32,36). However, recent studies have shown that the cell bodies are present within the superior (jugular) ganglion (6,22). This variety of observations implies the existence of various pathways of the afferent fibers in the hypoglossal nerve. The aim of the present study is, therefore, to investigate the ganglion cells and central distribution of the afferent fibers in the cat hypoglossal nerve utilizing transganglionic transport of WGA-HRP.
Twenty-one cats weighing 0.7 to 3.4 kg were used for the present study. All surgical procedures were done under general anesthesia with pentobarbital soda (40 mg/kg IP). In the series of the present experiments, the hypoglossal nerve was exposed widely around the common carotid artery. After identification of the superior radix of the ansa cervicalis, 2-5 I~1 of a solution of 0.2% WGA-HRP (Toyobo) containing 10% HRP (Toyobo, Grade-I-C) was injected into the proximal end of the hypoglossal nerve, cut at the portion distal to the superior radix, using a 10-1xl Hamilton microsyringe (11 cats). In the other experiments, 2-4 I~1 of the HRP was injected into the same portion of the hypoglossal nerve after additional transections of the superior radix of the ansa cervicalis (3 cats), the dorsal root of the CI segment (4 cats) and both the superior radix and dorsal root (3 cats). In all of the above experiments, to prevent leakage of injected HRP, the proximal end of the hypoglossal nerve was tied immediately with a thread after the injections. After a survival time of 2-3 days, the animals were anesthetized again and sacrificed by perfusion through the ascending aorta with 0.1 M phosphate buffer (pH 7.4) followed by a fixative containing 8% formalin in 0.1 M phosphate buffer. The brain stem and spinal cord were removed together with the superior and inferior ganglia of the glossopharyngeal and vagal nerves, geniculate ganglion, trigeminal ganglion and C1-C3 spinal ganglia on the side ipsilat-
81
J
82
TAKEUCHI ET AL. X l nerve
C, ~C"c,,,
GH
TH
OsHHS4~C THi FIG. 1. Schematic representation of relationship between the hypoglossal nerve and the ansa cervicalis. Arrows x, y and z indicate the site of transection of the hypoglossal nerve, superior radix of the ansa cervicalis and dorsal root of the C 1 segment, respectively. CI, CII, CIII, first, second and third cervical dorsal root; GH, geniohyoid muscle; OHi, inferior belly of omohyoid muscle; OHs, superior belly of omohyoid muscle; SH, sternohyoid muscle; ST, sternothyreoid muscle; TH, thyreohyoid muscle; XII nerve, hypoglossal nerve.
eral to the injections. These brain stem, spinal cord and ganglia were stored 1-3 days in 0.1 M phosphate buffer containing 30% sucrose. Serial 60 ixm-thick transverse sections were cut on a freezing microtome. Sections were processed for the demonstration of HRP reaction product according to the tetramethyl benzidine (TMB) protocol of Mesulam (20) and were counterstained with neutral red. RESULTS HRP injections are made into the hypoglossal nerve, leaving the superior radix of the ansa cervicalis and the dorsal root of the C1 segment intact (Fig. 1, arrow x). In this experiment the injected HRP is clearly restricted along the course of the hypoglossal nerve as a small brown mass. Heavy HRP labeling was found in the hypoglossal motor fibers and their cell bodies ipsilaterally (Fig. 2). Some labeled cells are detected within the ipsilateral superior ganglia of the glossopharyngeal and vagal nerves. Within the superior ganglion of the glossopharyngeal nerve the labeled cells are observed to be relatively compact (Fig. 3A) while within the ganglion of the vagal nerve these are confined to the region close to the vagal nerve (Fig. 3B). A few labeled cells are present within the inferior ganglion of the vagal nerve (Fig. 3C) and the spinal ganglion of the C1 segment (Fig. 3D). Very few labeled cells occur within the inferior ganglion of the glossopharyngeal nerve, mandibular branch of the trigeminal ganglion and spinal ganglia of the C2 and C3 segments. No cell labeling is observed within the geniculate ganglion. The transganglionic labeling of the central processes of these
FIG. 2. Photomicrograph of retrogradely labeled neurons of the hypoglossal nucleus after HRP injections into the cut proximal end of the hypoglossal nerve. Calibration bar = 200 p,m.
HYPOGLOSSAL AFFERENT FIBERS
83
B
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FIG. 3. Photomicrographs of retrogradely labeled cell bodies within the superior ganglion of the glossopharyngeal nerve (A), superior (B) and inferior ganglion (C) of the vagal nerve, and spinal ganglion of the CI segment (D) after HRP injections into the cut proximal end of the hypoglossal nerve without transection of the superior radix of the ansa cervicalis and the dorsal root of the C1 segment. Note that labeled cell bodies are relatively compact within the superior ganglion of the glossopharyngeal nerve (arrow in A). Asterisk indicates the superior ganglion of the vagal nerve. IXn, glossopharyngeal nerve; Xn, vagal nerve. Calibration bars = 200 Ixm in A-D.
ganglia can be followed into ipsilateral brain stem areas. Labeled glossopharyngeal and vagal fibers enter the brain stem from the lateral surface at the level of the facial nucleus and the rostral end of the inferior olive, respectively (Figs. 4A, B, and 5A, B). After crossing the dorsal portion of the spinal trigeminal tract, these labeled glossopharyngeal and vagal fibers join the solitary tract and extend caudally. These fibers distribute mainly to the interstitial and medial subdivisions of the nucleus of the solitary tract (Figs. 4C and 5C). A few fibers also distribute to the parvocellular and commissural subdivisions (Fig. 4D-F). On occasion, commissural fibers are found in the region just dorsal to the central canal (Figs. 4F and 5F).
A few labeled fibers of the glossopharyngeal nerve and some labeled fibers of the vagal nerve form a descending bundle in the dorsal portion of the spinal trigeminal tract. From this bundle, labeled fibers distribute to the caudal portion of the spinal trigeminal nucleus pars interpolaris immediately adjacent to the spinal trigeminal tract (Figs. 4D, E and 5D). At the level of the rostral portion of the spinal trigeminal nucleus pars caudalis, the descending fibers are located along the medial border of the spinal trigeminal tract. At this level, termination areas appear to be present in the ventrolateral part of the medial cuneate nucleus (Figs. 4E, F and 5E). At the C1 level, the descending fibers intermingle with labeled dorsal root fibers located in the dorsolat-
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TAKEUCHI E T AL.
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i
/
FIG. 4. Distribution of labeled fibers in the lower brain stem and upper cervical spinal cord after HRP injections into the cut proximal end of the hypoglossal nerve without transection of the superior radix of the ansa cervicalis and dorsal root of the C 1 segment. Amb, ambiguus nucleus; AP, area postrema; Corn, commissural nucleus; Cum, medial cuneate nucleus; FN, facial nucleus; Gr, gracil nucleus; IO, inferior olivary nucleus; NST, nucleus of solitary tract; Spc, parvocellular solitary nucleus; Vsp, spinal trigeminal nucleus; Vt, spinal trigeminal tract; IXn, glossopharyngeal nerve; Xn, vagal nerve; XII, hypoglossal nucleus.
eral funiculus. In the gray matter, these labeled fibers distribute to laminae I, IV-V and VII-VIII of Rexed (27) from the medial aspect of the dorsal horn (Figs. 4G and 6). The labeled descending fibers are traced as far as the C2 segment, but there are no differences in the pattern of distribution of the labeled fibers as compared to that of the C1 segment (Fig. 4H). In order to determine the course of the afferent fibers in the hypoglossal nerve, transection of the superior radix of the ansa cervicalis and/or the dorsal root of the C 1 segment is made prior to HRP injections into the hypoglossal nerve. In the case of transection of the superior radix (Fig. 1, arrow y), the results of labeled cells within the ganglia and pattern of central distribution of labeled fibers are similar to those in the experiment described above. On the other hand, after transection of the dorsal root of the C 1 segment (Fig. 1, arrow z) or both the dorsal root and superior radix, the only difference is a substantial reduction of labeled fibers in laminae VII-VIII. DISCUSSION
The present study confirms the existence of afferent fibers in the cat hypoglossal nerve and demonstrates the distribution of the afferent fibers in the lower brain stem and spinal cord. Sensory fibers in the hypoglossal nerve are indicated to be conveyed
centrally through the glossopharyngeal and vagal nerves and the upper cervical dorsal roots. Many electrophysiological investigations on the hypoglossal afferents in the cat referred to a probable location of the corresponding cell bodies within the inferior ganglion of the vagal nerve (10, 12, 29, 30, 35). In anatomical studies, the afferent fibers were often reported to join the vagal nerve via the hypoglossonodose branch. Tarkhan and Abou-E1-Naga (32) showed that extirpation of the inferior ganglion of the vagal nerve in the dog resulted in degeneration of approximately 5% of the fibers in the hypoglossal nerve. Zimny et al. (36) also found chromatolytic changes in cells of the inferior ganglion in the cat and dog after transection of the hypoglossal nerve. However, in recent HRP studies, cells of the afferent fibers were reported to be located predominantly within the superior ganglion in the rat (22) and dog (6). The present study indicates that in the cat the hypoglossal nerve connects with the vagal nerve and that the afferent fibers are more likely to originate from cells within the superior ganglion rather than from neurons within the inferior ganglion of the vagal nerve. With respect to connections of the hypoglossal nerve with the glossopharyngeal nerve, Chibuzo and Cummings (6) suggested the existence of a course along the lingual branch of the glossopharyngeal nerve because of the presence of HRP-labeled cells within the ganglia of the glossopharyngeal nerve following injection of HRP into the intrinsic lingual muscle. However, the specific ganglion and the degree of labeling were not described in detail. With regard to this point, the present study has shown connections of the hypoglossal nerve with the glossopharyngeal nerve and that the major origin of such afferent fibers is from cells within the superior ganglion. The present results show that the afferent fibers in the hypoglossal nerve end in the spinal trigeminal nucleus, cuneate nucleus, nucleus of the solitary tract and the C 1 and C2 segments of the spinal cord. A similar distribution of the afferent fibers was reflected in central projections of primary afferent fibers originating from cells within the superior ganglion of the glossopharyngeal nerve in the monkey (2), superior ganglion of the vagal nerve in the cat (1,23) and inferior ganglion of the vagal nerve in the rat and cat (7, 9, 16). The projections to the upper cervical spinal cord may originate from both the dorsal root of the C1 segment as well as the glossopharyngeal and vagal nerves. Previous authors noted in the rabbit (34), dog (6) and cat (17) that cell bodies of afferent fibers in the hypoglossal nerve are present within the spinal ganglion of the C1 segment. This finding is well in accordance with that obtained from the present HRP experiments. The present study also reveals that the afferent fibers do not come from the ansa cervicalis, but from the dorsal root at the C1 segment. Interestingly, the terminal fields of the fibers are laminae VII-VIII. This distribution of labeled fibers is well reflected by the central projection of the dorsal roots of the C1 segment (14). The remaining labeled fibers in laminae I and I V - V represent mainly the termination of primary afferent fibers of the glossopharyngeal and vagal nerves. In fact, Kerr (16) and Rhoton et al. (28) using the degeneration method demonstrated an exclusive termination of the glossopharyngeal and vagal fibers in lamina IV at the C1-C3 segments in the cat and monkey while Beckstead and Norgren (2) using the autoradiographic method revealed termination of the vagal fibers in laminae I-IV at the C1 segment in the monkey. Furthermore, of particular interest in the present study is the distribution of labeled fibers to lamina I because this layer is reported to be a major relay for nociceptive information (3, 5, 13, 18, 21). Therefore, the present findings indicate that afferent fibers in the hypoglossal nerve play an important role in conveying information of muscular pain mainly through the glossopharyngeal and vagal nerves.
H Y P O G L O S S A L A F F E R E N T FIBERS
85
D
ST
iNST
Vsp
FIG. 5. Photomicrographs of labeled glossopharyngeal (A) and vagal nerves (B), and distribution of labeled fibers in the nucleus of the solitary tract (C), spinal trigeminal nucleus pars interpolaris (D), medial cuneate nucleus (E) and commissural fibers (F). Note that descending fibers are located in the dorsal portion of the spinal trigeminal tract (asterisk in D). Arrowheads indicate commissural fibers just dorsal to the central canal. CC, central canal; Cure, medial cuneate nucleus; iNST, interstitial subdivision of nucleus of solitary tract; mNST, medial subdivision of nucleus of solitary tract, ST, solitary tract; Vsp, spinal trigeminal nucleus; Vt, spinal trigeminal tract. Calibration bars = 100 izm in A-F.
T A K E U C H I ET AL.
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FIG. 6. Photomicrograph of labeled fibers in the spinal cord of the C1 segment. Note that these fibers distribute to laminae I and IV-V. I-V, Rexed's laminae I V . Calibration bar= 200 p.m.
ACKNOWLEDGEMENTS The authors would like to thank Professor Kazuo Yamashita for his advice and encouragement and Mr. Shinkichi Terada for technical assistance.
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