Brain Research, 586 (1992) 162-165 ~'~ 1992 Elsevier Science Publishers B.V. All rights reserved 0006.8993/92/$05,00
162
BRES L~i2S5
Trigeminal nerve endings of lingual mucosa and musculature of the rat S e t s u k o S u e m u n e ~', T o s h i k a z u N i s h i m o r i a, M i t s u t e r u H o s o i a, Y o s h i t a k a S u z u k i b, H i r o m i c h i T s u r u b, T o s h i t s u g u K a w a t a ~, K a z u o Y a m a u c h i ~ a n d N o r i h i k o M a e d a ~ /st Dc,partmenrt of ~ Oral Anatomy and h Prosthodontics and ' Department of Orthodontics, tliroshima Unirersity School of Dentistry, lliroshuna (Japan)
(Accepted 28 April 1992)
Key words': llorseradish i)eroxidase; Trigeminal sensory receptor; Anterior rat tongue
Ih)rseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the trigeminal ganglion of adult rats to label the peripheral sensory receptors of the tongue. The conjugate was transported anterogradely to all the ipsilateral fungiform papillae and filiform papillae. Some labeled fibers crossed over to the contralateral papillae. In the intrinsic tongue muscle undulating nerve fibers along or across muscle fibers were often observed, and fl~rmedsimple spiral endings.
The sensory innervation of the anterior part of the tongue is derived from the lingual branch of the trigeminal nerve and the chorda tympani branch of the facial nerve. The general somatosensation (e,g. tactile, thermal and noxiou,,;) and gustatory sensation of the anterior tongue are mediated by the lingual nerve and the chorda tympani, respectively, in t'act it has been estimated that the trigeminal nerve contributes about
75% of the total number of papillary axons found in each fungiform papilla =',". This nerve serves not only to transmit peripheral sensation but also has a trophic effect. For example, Mizuno et al. =~ reported that the cuticular spines of filiform papillae dropped off, exfoliation increased, and ulcerous changes appeared, within 48 h in the lingual mucosa of the cat after transection of the lingual nerve, in contrast it was demonstrated by a study involving sensory nerve denervation that no nerve endings entered the epithelium of the filiform papillae in the tongue of the hamster =v, and no end organs were detectable by the silver method in the filifnrm papillae of the cat tongue I~. Additionally immunohistochemical studies showed that a few neurofilamcnt-protein (NFP)-positive nerve fibers entered the connective tissue core in the filiform papillae of cattle and rats ~', and substance P (SP)-positive fibers never
supplied the filiform papillae of the rat tongue ='~,Thus, concerning the nerve supply to the lingual mucosa species differences have been observed by morphologi.. cal studies in mammals. On the other hand afferent fiber.~ in the hypoglossal nerve have been physiologically shown to respond to the mechanical stimulation produced by stretching of the tongue "~''t''~'l'~, but not to tactile ,~timuli applied to the surface of the tongue ='='='~. Moreover, by the trans-ganglionic transport technique we found that most of the cell bodies of the primary afferent fibers in the hypoglossal nerve of the cat were located in the trigeminal ganglion =2, These facts led us to study the nerve-endings in the rat tongue that selectively originate from the trigeminal nerve, Experiments were performed on 6-week-old Sprague-Dawley rats weighing 2(X)-300 g, Thirteen animals were anesthetized via i,p, injection of sodium pentobarbital (0,06 mg/g body weight), The unilateral trigeminal ganglion was surgically exposed, and 2-5/~1 of 5% HRP-WGA (Toyobo Ltd,, Japan) entrapped in a I% hypo-allergenic polyacrylamide gel was injected by means of a glass micropipette (80-100 #m in diameter) into several sites to label all the mandibular ganglion cells,
('orre.v,ondenr(,: N. Macda, Ist Department of Oral Anatomy, Hiroshima University, School of Dentistry, Kasumi I chome, Minami-ku, }liroshima 734,Japan.
163
'
.o
R
I
C q
.,
fli,
i
~~
~
-
~r
. . . .
,
Fig. 1. A: frontal section of the rat tongue, 0.3 mm posterior t'rom the tongue tip showing labeled endings in the ipsilateral (arrows) and contralateral (arrowheads) papillae. The open arrow indicates the central sulcus. B: labeled nerve endings in fungiform and filiform (arrowheads) papillae. A taste bud (arrow) is free of reaction products. C: inferior surface of the tongue. D: cross-section of fungiform and filiform papillae. Labeled fibers entered into the epithelium. E: undulating labeled fibers across the muscle fibers. F: simple spiral nerve endings in the muscle fibers, sc, stratum corneum; e, oral epithelium; Ip, lamina propria; Bar = 50 p,m.
164 After 24 h, the anesthetized animals were perfused through the ascending aorta with 300 ml of heparinized saline, followed immediately by 500 ml of fixative (1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). The fixative was then flushed out with 300 ml of 10% sucrose in 0.1 M phosphate buffer, pH 7.4. Thereafter the tongue was removed and stored overnight in 30% sucrose phosphate buffer solution at 4°C. Transverse frozen sections of 50-/zm thickness were serially cut, and the sections were processed according to the tetramethylbentizine (TMB) protocol of Mesulum m. These sections were dried overnight on chrome-alum-gelatin-coated slides, and then counterstained with 1% Neutral red. All treated sections were observed under an Olympus BH-2 light microscope. HRP injected into the trigeminal ganglion was transported anterogradely to the ipsilateral anterior tongue. Bundles of labeled fibers coursed through the deep musculature of the tongue. Some groups of axons diverged from the nerve plexus and bifurcated in the upper submucosa or lamina propria. The trigeminai nerve provides innervation to all the fungiform and filiform papillae of the ipsilateral tongue. A few fascicles of fibers filled with reaction product crossed contralaterally and innervated fungiform and filiform papillae. They were localized near the central sulcus of the anterior tongue (Fig. IA). in a single fungiform papilht a few thick-caliber fibers innervated the center of its core and bifurcated several times. They formed multiple branches flanking the taste bud and ended in the epithelial layer. The terminals were most densely located in the surface of the ¢pith¢li,I layer around the taste pore under the stratum corneum. The taste buds were free from labeled fibers. Fine-caliber fibers invaded the fungiform papilla surrounding the connective tissue core, and terminated in the regions between the epithelial layer and lamina propria (Fig. I B). A single or a few fibers innervated each fUiform papilla without ramifying, and terminated as simple nerve endings. Thick fibers appeared to end in the lamina propria, and many of the fine-caliber ones coursed through the lamina propria, and terminated in the epithelial layer (Fig. IB and D), Simple axons innervated the papillae of the ventral surface of the rat tongue and ended within the lamina propria or intraepithelial layer (Fig, IC), Free endings were observed in the tongue muscle, Also a single undulating fiber derived from the nerve bundle traveling to the lingual mucosa branched extensively in an area of 300 × 1000/zm, and often terminated as a simple spiral ending (Fig, I E and F).
in the present study, we examined the distribution and appearance of the trigeminal nerve endings in the lingual mucosa of the anterior tongue and those of intramuscular nerve endings in the tongue. We found that each fungiform papilla contained a single taste bud, which was distinguished by its lightly stained cells oriented parallel to the long axis of the papilla, ,rod that the non-taste epithelium around the taste bud was abundantly innervated. The arrangement and superficial location of t h e s e trigeminal terminal endings in the fungiform papillae suggest that the papillae may well be adapted for relatively sensitive somatosensation, possibly mechanosensation. Moreover, the fact that not only the fungiform but also the filiform papillae w e r e innervated by the trigeminal n e r v e strongly suggests that the filiform papillae may also receive peripheral stimuli and be involved in the conduction of mechanosensory information. However, we can not completely deny that the fibers innervating the filiform papillae may be involved only in the trophic effect, because it was earlier reported that the fibers lack Sp t3. Interestingly, we detected simple spiral nerve endings that originated from the trigeminal ganglion, although no muscle spindles have been observed in the rat tongue, These endings around non-encapsulated muscle fibers resembled the extrafusal endings in the mammalian extraocular muscle t's°x'ts. As it has been suggested that these special endings in the eye muscle of various animals may be involved in receiving the stretch sensation t''s our finding suggests that similar types of sensory receptor receiving the stretch sensation may also exist in the rat tongue, In conclusion, our present results well demonstrate that these afferents, whose cell bodies are located in the trigeminal ganglion, possibly course with the hypoglossal nerve bundle and project to the trigeminal sensory nuclear complex to modulate the movement of the tongue.
! Barker, D,, Hunt C,(', and Mclntyre A,K,, llandhook of Sensory Phy,~'iolo~, 1/ol. Iii, Springer-Verlag, Berlin 1974, 2 Beidler, L,M,, Innervation of rat fungiform papilla. In C, Pfaffmann (Ed,), Olfa('tion and Taste. I/ol. i11, New York, Rockfeller University Press, 1969, pp. 352-369. 3 Biota, S,, Afferent influences on tongue muscle activity: a morphological and physiolo#cal study in the cat, Actu Physiol. Scand., 19, Suppl, 170 (1960) 1-97, 4 C[mpcr, S,, Afferent impulses in the hypoglossal nerve on stretching the cat's tongue, J, Physiol., 126 (1954) 32. 50mper, S, and Fillenz, M,, Afferent discharges in response to stretch from the extraocular muscles of the cat and monkey and the innervation of these muscles. J. Physiol., 127 (1955) 400-.413. 6 Daniel, P,M,, Spiral nerve endings in the extrinsic eye muscles of man, J, AnaL, 80 (1946) 189-192,
165 7 Dogiel, A.S., Die Endigungen der sensiblen nerven in den augenmuskeln und deren sehnen beim menschen und den saugetieren, Arch. Mikr, Anat., 68 (1906) 501-526. 8 Farbman, A.I. and HeUekant, G., Quantitative analyses of the fiber population in rat cborda tympani nerves and fungiform papillae, Am. J. Anat., 153 (1978) 509-522, 9 Hanson, J. and Widen, L,, Afferent fibres in the hypoglossal nerve of cat, Acta Physiol. Scand., 79 (1963) 24-36. 10 Mesulum, M,-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents, J. Histochem. Cytochem., 26 (1978) 106-117. ! ! Mizuno, N., Akimoto, C., Mochizuki, K. and Matsushima, R., Experimental studies of afferent fibers in the hypoglossal nerve in the cat: a scanning electron microscopic observation on the lingual mucosa following transection of the nerve, and a degeneration study with silver impregnation methods, Arch. Histol. Jpn., 35 (1973) 99-113, 12 Nazruddin, Suemune, S., Shirana, Y., Yamauchi, K. and Shigenaga, Y., The cells of origin of the hypoglossal afferent nerves and central projections in the cat, Brain Res., 49{)(1989) 219-235. 13 Nishimoto, T., Akai, M., inagaki, S., Shiosaka, S., Shimizu, Y.,
14 15 16
17
18 19
Yamamoto, K., Senba, E., Sakanaka, M., Takatsuki, K., Hara, Y., Takagi, H., Matsuzaki,T., Kawai, Y., and Tohyama, M., On the distribution and origins of substance P in the papillae of the rat tongue: an experimental and immunohistochemical study, J. Comp. Nenrol., 207 (1982) 85-92. Porter, R., Lingual mechanoreceptors activated by muscle twitch, J. Physiol., 183 (1966) 101-111. Sas, J., Appeitauer, C., Atypical muscle spindles in the extrinsic eye muscles of man, Acta Anat., 55 (1963) 311-322. Sato, O., Maeda, T., Kobayashi, S., lwanaga, T. and Fujita, T., Filiform papillae as a sensory apparatus in the tongue: an immunohistochemical study of nervous elements by use of neurofilament protein (NFP) and S-100 protein antibodies, Cell Tissue Res., 252 (1988) 231-238. Whitehead, M.C., Beeman, C.S. and Kinsella, B.A., Distribution of taste and general sensory nerve endings in fungiform papillae of the hamster, Am. J. Anat., 173 (1985) 185-201. Winkelmann, R.K., The mammalian end-organ in oral tissue of the cat, J. D. Res,, 41 (1962) 207-212. Zapata, P. and Torrealba, G., Mechanosensory units in the hypoglossal nerve of the cat, Brain Res., 32 (1971) 349-367.
162
BRES L~i2S5
Trigeminal nerve endings of lingual mucosa and musculature of the rat S e t s u k o S u e m u n e ~', T o s h i k a z u N i s h i m o r i a, M i t s u t e r u H o s o i a, Y o s h i t a k a S u z u k i b, H i r o m i c h i T s u r u b, T o s h i t s u g u K a w a t a ~, K a z u o Y a m a u c h i ~ a n d N o r i h i k o M a e d a ~ /st Dc,partmenrt of ~ Oral Anatomy and h Prosthodontics and ' Department of Orthodontics, tliroshima Unirersity School of Dentistry, lliroshuna (Japan)
(Accepted 28 April 1992)
Key words': llorseradish i)eroxidase; Trigeminal sensory receptor; Anterior rat tongue
Ih)rseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the trigeminal ganglion of adult rats to label the peripheral sensory receptors of the tongue. The conjugate was transported anterogradely to all the ipsilateral fungiform papillae and filiform papillae. Some labeled fibers crossed over to the contralateral papillae. In the intrinsic tongue muscle undulating nerve fibers along or across muscle fibers were often observed, and fl~rmedsimple spiral endings.
The sensory innervation of the anterior part of the tongue is derived from the lingual branch of the trigeminal nerve and the chorda tympani branch of the facial nerve. The general somatosensation (e,g. tactile, thermal and noxiou,,;) and gustatory sensation of the anterior tongue are mediated by the lingual nerve and the chorda tympani, respectively, in t'act it has been estimated that the trigeminal nerve contributes about
75% of the total number of papillary axons found in each fungiform papilla =',". This nerve serves not only to transmit peripheral sensation but also has a trophic effect. For example, Mizuno et al. =~ reported that the cuticular spines of filiform papillae dropped off, exfoliation increased, and ulcerous changes appeared, within 48 h in the lingual mucosa of the cat after transection of the lingual nerve, in contrast it was demonstrated by a study involving sensory nerve denervation that no nerve endings entered the epithelium of the filiform papillae in the tongue of the hamster =v, and no end organs were detectable by the silver method in the filifnrm papillae of the cat tongue I~. Additionally immunohistochemical studies showed that a few neurofilamcnt-protein (NFP)-positive nerve fibers entered the connective tissue core in the filiform papillae of cattle and rats ~', and substance P (SP)-positive fibers never
supplied the filiform papillae of the rat tongue ='~,Thus, concerning the nerve supply to the lingual mucosa species differences have been observed by morphologi.. cal studies in mammals. On the other hand afferent fiber.~ in the hypoglossal nerve have been physiologically shown to respond to the mechanical stimulation produced by stretching of the tongue "~''t''~'l'~, but not to tactile ,~timuli applied to the surface of the tongue ='='='~. Moreover, by the trans-ganglionic transport technique we found that most of the cell bodies of the primary afferent fibers in the hypoglossal nerve of the cat were located in the trigeminal ganglion =2, These facts led us to study the nerve-endings in the rat tongue that selectively originate from the trigeminal nerve, Experiments were performed on 6-week-old Sprague-Dawley rats weighing 2(X)-300 g, Thirteen animals were anesthetized via i,p, injection of sodium pentobarbital (0,06 mg/g body weight), The unilateral trigeminal ganglion was surgically exposed, and 2-5/~1 of 5% HRP-WGA (Toyobo Ltd,, Japan) entrapped in a I% hypo-allergenic polyacrylamide gel was injected by means of a glass micropipette (80-100 #m in diameter) into several sites to label all the mandibular ganglion cells,
('orre.v,ondenr(,: N. Macda, Ist Department of Oral Anatomy, Hiroshima University, School of Dentistry, Kasumi I chome, Minami-ku, }liroshima 734,Japan.
163
'
.o
R
I
C q
.,
fli,
i
~~
~
-
~r
. . . .
,
Fig. 1. A: frontal section of the rat tongue, 0.3 mm posterior t'rom the tongue tip showing labeled endings in the ipsilateral (arrows) and contralateral (arrowheads) papillae. The open arrow indicates the central sulcus. B: labeled nerve endings in fungiform and filiform (arrowheads) papillae. A taste bud (arrow) is free of reaction products. C: inferior surface of the tongue. D: cross-section of fungiform and filiform papillae. Labeled fibers entered into the epithelium. E: undulating labeled fibers across the muscle fibers. F: simple spiral nerve endings in the muscle fibers, sc, stratum corneum; e, oral epithelium; Ip, lamina propria; Bar = 50 p,m.
164 After 24 h, the anesthetized animals were perfused through the ascending aorta with 300 ml of heparinized saline, followed immediately by 500 ml of fixative (1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). The fixative was then flushed out with 300 ml of 10% sucrose in 0.1 M phosphate buffer, pH 7.4. Thereafter the tongue was removed and stored overnight in 30% sucrose phosphate buffer solution at 4°C. Transverse frozen sections of 50-/zm thickness were serially cut, and the sections were processed according to the tetramethylbentizine (TMB) protocol of Mesulum m. These sections were dried overnight on chrome-alum-gelatin-coated slides, and then counterstained with 1% Neutral red. All treated sections were observed under an Olympus BH-2 light microscope. HRP injected into the trigeminal ganglion was transported anterogradely to the ipsilateral anterior tongue. Bundles of labeled fibers coursed through the deep musculature of the tongue. Some groups of axons diverged from the nerve plexus and bifurcated in the upper submucosa or lamina propria. The trigeminai nerve provides innervation to all the fungiform and filiform papillae of the ipsilateral tongue. A few fascicles of fibers filled with reaction product crossed contralaterally and innervated fungiform and filiform papillae. They were localized near the central sulcus of the anterior tongue (Fig. IA). in a single fungiform papilht a few thick-caliber fibers innervated the center of its core and bifurcated several times. They formed multiple branches flanking the taste bud and ended in the epithelial layer. The terminals were most densely located in the surface of the ¢pith¢li,I layer around the taste pore under the stratum corneum. The taste buds were free from labeled fibers. Fine-caliber fibers invaded the fungiform papilla surrounding the connective tissue core, and terminated in the regions between the epithelial layer and lamina propria (Fig. I B). A single or a few fibers innervated each fUiform papilla without ramifying, and terminated as simple nerve endings. Thick fibers appeared to end in the lamina propria, and many of the fine-caliber ones coursed through the lamina propria, and terminated in the epithelial layer (Fig. IB and D), Simple axons innervated the papillae of the ventral surface of the rat tongue and ended within the lamina propria or intraepithelial layer (Fig, IC), Free endings were observed in the tongue muscle, Also a single undulating fiber derived from the nerve bundle traveling to the lingual mucosa branched extensively in an area of 300 × 1000/zm, and often terminated as a simple spiral ending (Fig, I E and F).
in the present study, we examined the distribution and appearance of the trigeminal nerve endings in the lingual mucosa of the anterior tongue and those of intramuscular nerve endings in the tongue. We found that each fungiform papilla contained a single taste bud, which was distinguished by its lightly stained cells oriented parallel to the long axis of the papilla, ,rod that the non-taste epithelium around the taste bud was abundantly innervated. The arrangement and superficial location of t h e s e trigeminal terminal endings in the fungiform papillae suggest that the papillae may well be adapted for relatively sensitive somatosensation, possibly mechanosensation. Moreover, the fact that not only the fungiform but also the filiform papillae w e r e innervated by the trigeminal n e r v e strongly suggests that the filiform papillae may also receive peripheral stimuli and be involved in the conduction of mechanosensory information. However, we can not completely deny that the fibers innervating the filiform papillae may be involved only in the trophic effect, because it was earlier reported that the fibers lack Sp t3. Interestingly, we detected simple spiral nerve endings that originated from the trigeminal ganglion, although no muscle spindles have been observed in the rat tongue, These endings around non-encapsulated muscle fibers resembled the extrafusal endings in the mammalian extraocular muscle t's°x'ts. As it has been suggested that these special endings in the eye muscle of various animals may be involved in receiving the stretch sensation t''s our finding suggests that similar types of sensory receptor receiving the stretch sensation may also exist in the rat tongue, In conclusion, our present results well demonstrate that these afferents, whose cell bodies are located in the trigeminal ganglion, possibly course with the hypoglossal nerve bundle and project to the trigeminal sensory nuclear complex to modulate the movement of the tongue.
! Barker, D,, Hunt C,(', and Mclntyre A,K,, llandhook of Sensory Phy,~'iolo~, 1/ol. Iii, Springer-Verlag, Berlin 1974, 2 Beidler, L,M,, Innervation of rat fungiform papilla. In C, Pfaffmann (Ed,), Olfa('tion and Taste. I/ol. i11, New York, Rockfeller University Press, 1969, pp. 352-369. 3 Biota, S,, Afferent influences on tongue muscle activity: a morphological and physiolo#cal study in the cat, Actu Physiol. Scand., 19, Suppl, 170 (1960) 1-97, 4 C[mpcr, S,, Afferent impulses in the hypoglossal nerve on stretching the cat's tongue, J, Physiol., 126 (1954) 32. 50mper, S, and Fillenz, M,, Afferent discharges in response to stretch from the extraocular muscles of the cat and monkey and the innervation of these muscles. J. Physiol., 127 (1955) 400-.413. 6 Daniel, P,M,, Spiral nerve endings in the extrinsic eye muscles of man, J, AnaL, 80 (1946) 189-192,
165 7 Dogiel, A.S., Die Endigungen der sensiblen nerven in den augenmuskeln und deren sehnen beim menschen und den saugetieren, Arch. Mikr, Anat., 68 (1906) 501-526. 8 Farbman, A.I. and HeUekant, G., Quantitative analyses of the fiber population in rat cborda tympani nerves and fungiform papillae, Am. J. Anat., 153 (1978) 509-522, 9 Hanson, J. and Widen, L,, Afferent fibres in the hypoglossal nerve of cat, Acta Physiol. Scand., 79 (1963) 24-36. 10 Mesulum, M,-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents, J. Histochem. Cytochem., 26 (1978) 106-117. ! ! Mizuno, N., Akimoto, C., Mochizuki, K. and Matsushima, R., Experimental studies of afferent fibers in the hypoglossal nerve in the cat: a scanning electron microscopic observation on the lingual mucosa following transection of the nerve, and a degeneration study with silver impregnation methods, Arch. Histol. Jpn., 35 (1973) 99-113, 12 Nazruddin, Suemune, S., Shirana, Y., Yamauchi, K. and Shigenaga, Y., The cells of origin of the hypoglossal afferent nerves and central projections in the cat, Brain Res., 49{)(1989) 219-235. 13 Nishimoto, T., Akai, M., inagaki, S., Shiosaka, S., Shimizu, Y.,
14 15 16
17
18 19
Yamamoto, K., Senba, E., Sakanaka, M., Takatsuki, K., Hara, Y., Takagi, H., Matsuzaki,T., Kawai, Y., and Tohyama, M., On the distribution and origins of substance P in the papillae of the rat tongue: an experimental and immunohistochemical study, J. Comp. Nenrol., 207 (1982) 85-92. Porter, R., Lingual mechanoreceptors activated by muscle twitch, J. Physiol., 183 (1966) 101-111. Sas, J., Appeitauer, C., Atypical muscle spindles in the extrinsic eye muscles of man, Acta Anat., 55 (1963) 311-322. Sato, O., Maeda, T., Kobayashi, S., lwanaga, T. and Fujita, T., Filiform papillae as a sensory apparatus in the tongue: an immunohistochemical study of nervous elements by use of neurofilament protein (NFP) and S-100 protein antibodies, Cell Tissue Res., 252 (1988) 231-238. Whitehead, M.C., Beeman, C.S. and Kinsella, B.A., Distribution of taste and general sensory nerve endings in fungiform papillae of the hamster, Am. J. Anat., 173 (1985) 185-201. Winkelmann, R.K., The mammalian end-organ in oral tissue of the cat, J. D. Res,, 41 (1962) 207-212. Zapata, P. and Torrealba, G., Mechanosensory units in the hypoglossal nerve of the cat, Brain Res., 32 (1971) 349-367.
