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Localization of motoneurons innervating the posterior belly of the digastric muscle: A comparative anatomical study by the HRP method
Neuroscience Letters, 12 (1979) 47--52
47
© Elsevier/North-Holland Scientific Publishers Ltd.
LOCALIZATION OF M O T O N E U R O N S I N N E R V A T I N G THE P O S T E R I O R BELLY O F THE DIGASTRIC MUSCLE: A COMPARATIVE ANATOMICAL STUDY BY THE H R P METHOD
K O J Y U R O M A T S U D A , M A S A N O R I U E M U R A , YOSHIKI TAKEUCHI, M A M O R U K U M E , R Y O T A R O M A T S U S H I M A and N O B O R U M I Z U N O *
Department of OralAnatomy, School of Dentistry,Hiroshima University,Hiroshima 734 and (N.M.) Department of Anatomy, Faculty of Medicine, Kyoto University,Kyoto 606 (Japan) (Received November 9th, 1978) (Revised versionreceived December 8th, 1978) (Accepted December 9th, 1978)
SUMMARY
Motoneurons innervating the posterior belly of the digastric muscle were identified in the m o n k e y , cat, dog, guinea pig and rat by the HRP method. After injections of horseradish peroxidase (HRP) into the posterior belly of the digastric muscle, t w o groups of HRP-labeled motoneurons were observed; the rostral group was seen as a small cluster of neurons in the lateral reticular area along the medial border of the descending r o o t of the facial nerve, and the neurons of the caudal group were distributed among the ascending r o o t fibers of the facial nerve. The distribution pattern of these neurons corresponded to that of the accessory facial nucleus neurons. The accessory facial nucleus was lacking in the rabbit in which the posterior digastric (PD) muscle is nonexistent.
It is well known that the anterior and posterior bellies of the digastric muscle receive m o t o r innervation respectively from the trigeminal and facial nerves, and that the anterior digastric (AD) motoneurons innervating the anterior belly of the digastric muscle are located within the m o t o r nucleus of the trigeminal nerve [6,8,9]. As regards the site of origin of m o t o r nerve supplying the posterior belly of the digastric muscle, however, there has n o t been unanimity of opinions of previous investigators. According to Szent~gothai [ 14] the posterior digastric (PD) motoneurons innervating the posterior belly of the digastric muscle in the cat are located in the accessory facial nucleus.
*To whom all correspondence should be sent
48 This opinion was supported by Mizuno et al. [9] in an horseradish peroxidase (HRP) study in the cat and rat. On the other hand, the facial nucleus was suggested to contain PD motoneurons in the rabbit [ 16], guinea pig [10], opossum [3], rat [7] and possum [11]. Morphology of the digastric muscle is variable among mammals [ 2,12]. In the m o n k e y the digastric muscle is divided into anterior and posterior bellies by an intermediate tendon with the form of a round cable. In the cat, dog, guinea pig and rat, however, the intermediate tendon of the muscle is cast into a thin plate and the muscle appears as a single-bellied one. On the other hand, the posterior belly of the muscle is non-existent in the rabbit. In the present study attempts were made to identify PD motoneurons and to examine topographical relationship between PD and AD motoneurons in the monkey, cat, dog, guinea pig, rat and rabbit. Injections of HRP into the digastric muscle were carried out under general anesthesia (Nembutal 30--35 mg/kg i.v.) in 6 monkeys (1 rhesus, 4 pig-tailed and 1 Japanese monkeys), 12 cats, 5 dogs, 10 rabbits, 15 guinea pigs and 12 rats. Prior to HRP injections in cats, dogs, guinea pigs and rats, the anterior and posterior bellies of the muscle were separated from each other by severing the muscle at the tendinous insertion; the cut ends of the muscle bellies were closed tightly with thread. Injections of 2--5% HRP ( T o y o b o Grade-I-C, RZ: 3.4), dissolved in sterile 0.9% saline containing 2% dimethylsulfoxide, were performed into the anterior and/or posterior belly of the digastric muscle as described elsewhere [9]. After survival time of 15--48 h the animals were perfused intravitally under deep anesthesia through the ascending aorta with 7--10% formalin in 0.9% saline adjusted to pH 7.3 with sodium bicarbonate. The brain stems were removed immediately and placed in 0.1 M phosphate buffer (pH 7.6) containing 30% sucrose at 4°C for 2--4 days. Subsequently transverse or parasagittal frozen sections through the lower brain stem were made serially at 60-pm thickness, and treated for the histochemical demonstration of HRP [4,13]. These sections were then m o u n t e d onto gelatincoated slides and counterstained with 1% cresylecht violet. After HRP injections into the anterior belly of the digastric muscle in the animals, neurons labeled with HRP were seen in the ventromedial division of the trigeminal m o t o r nucleus [8,9]. On the other hand, when HRP was injected into the posterior belly of the digastric muscle, two groups of HRP-
Fig. 1. Distribution pattern of PD (black stars) and AD (black circles) motoneurons is shown diagrammatically in series of projection drawings of cross sections of the lower brain s~ems of a monkey, cat and rabbit. The drawings are arranged from rostral (top) to caudal (bottom), and the numbers indicate the position of the serial sections of 60-~m thickness. The section Nr. i corresponds to the most rostral level of AD mononeuron group. Double and single arrow-heads point to the rostral and caudal groups of PD motoneurons, respectively, a, Root of abducens nerve; Ab, abducens nucleus; F, facial nucleus; f, root of facial nerve; Os, superior olive; Vm, motor nucleus of trigeminal nerve; Vp, main sensory nucleus of trigeminal nerve; Vs, spinal tract of trigeminai nerve.
q r
Ij-
,$
C
3
50
Fig. 2. Dark-field photomicrography of a parasagittal section through AD motoneuron group of a rabbit. The cluster of HRP-labeled AD motoneurons extends caudally to the level of the descending root of the facial nerve (f). Bar = 300 um. labeled n e u r o n s were observed; the rostral group was seen as a small cluster o f n e u r o n s along t h e medial b o r d e r o f the descending r o o t o f the facial nerve, and the n e u r o n s o f the caudal g r o u p were s o m e w h a t s c a t t e r e d a m o n g the asc e n d i n g r o o t fibers o f t h e facial nerve. T h e m o r p h o l o g y o f these PD m o t o n e u r o n s was similar t o t h a t o f the facial m o t o n e u r o n s [ 5 ] . In the m o n k e y and rat t h e PD m o t o n e u r o n s o f the caudal group were m o r e n u m e r o u s t h a n t h o s e o f t h e rostral group. In o t h e r animals studied, h o w e v e r , the n u m b e r o f PD m o t o n e u r o n s in t h e caudal g r o u p was a l m o s t equal t o t h a t in the rostral group (Fig.l). When H R P was injected i n t o t h e digastric muscle in the rabbit, in which the p o s t e r i o r belly o f the muscle is lacking, the cluster o f HRP-labeled n e u r o n s in the v e n t r o m e d i a l division o f the trigeminal m o t o r nucleus was observed t o e x t e n d caudally t o the area medial t o t h e d e s c e n d i n g r o o t o f the facial nerve, w h e r e t h e rostral g r o u p o f PD m o t o n e u r o n s was identified in each o f o t h e r animals studied (Figs. 1 and 2). T h e d i s t r i b u t i o n p a t t e r n o f PD m o t o n e u r o n s i d e n t i f i e d on t h e p r e s e n t s t u d y c o r r e s p o n d e d t o t h a t o f n e u r o n s o f the nucleus nervi facialis accessorius o f T a b e r [15] or the r e t r o t r i g e m i n a l nucleus of B e r m a n [1] described in the cat. As p o i n t e d o u t b y SzentAgothai [14] in the cat and c o r r o b o r a t e d b y M i z u n o e t al. [ 9] in t h e cat and rat, the rostral pole o f the cluster o f PD m o t o n e u r o n s c o n s t i t u t i n g the accessory facial nucleus was c o n t i n u o u s ros:
51
Fig. 3. Photomicrograph of a parasagittal section through the brain stem of a guinea pig, showing distribution of AD and PD motoneurons labeled with HRP injected simultaneously into the AD and PD muscles. The rostral and caudal groups of PD motoneurons are indicated by a double and single arrows, respectively. Abbreviations are as in Fig. 1. Bar = 300 um
trally, with a gap of short distance, to the caudal pole of the cluster of AD motoneurons in the ventromedial division of the trigeminal motor nucleus in the monkey, dog and guinea pig (Figs. 1 and 3). ACKNOWLEDGEMENTS
The technical assistance of Mr. Kiwamu Tanaka and the photographic help of Mr. Akira Uesugi are gratefully acknowledged. REFERENCES 1 Berman, A.L., The Brain Stem of the Cat, A Cytoarchitectonic Atlas with Stereotaxic Coordinates, The University of Wisconsin Press, Madison, 1968, 175 pp. 2 Bijvoet, W.F., Zur vergleichended Morphologie des Musculus digastricus mandibulae bei den S~iugetieren, Z. Morphol. Anthropol., 11 (1908) 249--316. 3 Don, R., Falls, W. and Martin, G.G., The motor nucleus of the facial nerve in the opossum (Didelphis marsupialis virginiana), its organization and connections, J. comp. Neurol., 152 (1973) 373--402. 4 Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302.
52 5 Kume, M., Uemura, M., Matsuda, K., Matsushima, R. and Mizuno, N., Topographical representation of peripheral branches of the facial nerve within the facial nucleus: a HRP study in the cat, Neurosci. Lett., 8 (1978) 5--8. 6 Landgren, S. and Olsson, K.A., Localization of evoked potentials in the digastric, mas seteric, supra- and intertrigeminal subnuclei of the cat, Exp. Brain Res., 26 (1976) 299--318. 7 Martin, M.R. and Lodge, D., Morphology of the facial nucleus of the rat, Brain Res., 123 (1977) 1--12. 8 Matsuda, K., Uemura, M., Kume, M., Matsuda, R. and Mizuno, N., Topographical representation of masticatory muscles in the motor trigeminal nucleus in the rabbit: a HRP study, Neurosci. Lett., 8 (1978) 1--4. 9 Mizuno, N., Konishi, A. and Sato, M., Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. comp. Neurol., 164 (1975) 105--116. 10 Papez, J.W., Subdivisions of the facial nucleus, J. comp. Neurol., 43 (1927) 159--191. 11 Provis, J., The organization of the facial nucleus of the brush-tailed possum (Trichosurus vulpecula), J. comp. Neurol., 172 (1977) 177--188. 12 Scapino, R.P., Function of the digastric muscle in carnivores, J. Morphol., 150 (1976) 843--860. 13 Streit, P. and Reubi, J.C., A new and sensitive method for axonalty transported horseradish peroxidase (HRP) in the pigeon visual system, Brain Res., 126 (1977) 530-537. 14 Szent~igothai, J., Functional representation in the motor trigeminal nucleus, J. comp. Neurol., 90 (1949) 111--120. 15 Taber, E., The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of the cat, J. comp. Neurol., 116 (1961) 27--70. 16 Van Gehuchten, A., Recherches sur l'origine rdele des nerfs craniens. II. Nerf facial (suite). J. Neurol. (Bruxelles), 3 (1898) 293--303. 17 Vraa-Jensen, G.F., The Motor Nucleus of the Facial Nerve, With a Survey of the Efferent Innervation of the Facial Muscles, Munksgraard. Copenhagen, 1942, 157 pp.
47
© Elsevier/North-Holland Scientific Publishers Ltd.
LOCALIZATION OF M O T O N E U R O N S I N N E R V A T I N G THE P O S T E R I O R BELLY O F THE DIGASTRIC MUSCLE: A COMPARATIVE ANATOMICAL STUDY BY THE H R P METHOD
K O J Y U R O M A T S U D A , M A S A N O R I U E M U R A , YOSHIKI TAKEUCHI, M A M O R U K U M E , R Y O T A R O M A T S U S H I M A and N O B O R U M I Z U N O *
Department of OralAnatomy, School of Dentistry,Hiroshima University,Hiroshima 734 and (N.M.) Department of Anatomy, Faculty of Medicine, Kyoto University,Kyoto 606 (Japan) (Received November 9th, 1978) (Revised versionreceived December 8th, 1978) (Accepted December 9th, 1978)
SUMMARY
Motoneurons innervating the posterior belly of the digastric muscle were identified in the m o n k e y , cat, dog, guinea pig and rat by the HRP method. After injections of horseradish peroxidase (HRP) into the posterior belly of the digastric muscle, t w o groups of HRP-labeled motoneurons were observed; the rostral group was seen as a small cluster of neurons in the lateral reticular area along the medial border of the descending r o o t of the facial nerve, and the neurons of the caudal group were distributed among the ascending r o o t fibers of the facial nerve. The distribution pattern of these neurons corresponded to that of the accessory facial nucleus neurons. The accessory facial nucleus was lacking in the rabbit in which the posterior digastric (PD) muscle is nonexistent.
It is well known that the anterior and posterior bellies of the digastric muscle receive m o t o r innervation respectively from the trigeminal and facial nerves, and that the anterior digastric (AD) motoneurons innervating the anterior belly of the digastric muscle are located within the m o t o r nucleus of the trigeminal nerve [6,8,9]. As regards the site of origin of m o t o r nerve supplying the posterior belly of the digastric muscle, however, there has n o t been unanimity of opinions of previous investigators. According to Szent~gothai [ 14] the posterior digastric (PD) motoneurons innervating the posterior belly of the digastric muscle in the cat are located in the accessory facial nucleus.
*To whom all correspondence should be sent
48 This opinion was supported by Mizuno et al. [9] in an horseradish peroxidase (HRP) study in the cat and rat. On the other hand, the facial nucleus was suggested to contain PD motoneurons in the rabbit [ 16], guinea pig [10], opossum [3], rat [7] and possum [11]. Morphology of the digastric muscle is variable among mammals [ 2,12]. In the m o n k e y the digastric muscle is divided into anterior and posterior bellies by an intermediate tendon with the form of a round cable. In the cat, dog, guinea pig and rat, however, the intermediate tendon of the muscle is cast into a thin plate and the muscle appears as a single-bellied one. On the other hand, the posterior belly of the muscle is non-existent in the rabbit. In the present study attempts were made to identify PD motoneurons and to examine topographical relationship between PD and AD motoneurons in the monkey, cat, dog, guinea pig, rat and rabbit. Injections of HRP into the digastric muscle were carried out under general anesthesia (Nembutal 30--35 mg/kg i.v.) in 6 monkeys (1 rhesus, 4 pig-tailed and 1 Japanese monkeys), 12 cats, 5 dogs, 10 rabbits, 15 guinea pigs and 12 rats. Prior to HRP injections in cats, dogs, guinea pigs and rats, the anterior and posterior bellies of the muscle were separated from each other by severing the muscle at the tendinous insertion; the cut ends of the muscle bellies were closed tightly with thread. Injections of 2--5% HRP ( T o y o b o Grade-I-C, RZ: 3.4), dissolved in sterile 0.9% saline containing 2% dimethylsulfoxide, were performed into the anterior and/or posterior belly of the digastric muscle as described elsewhere [9]. After survival time of 15--48 h the animals were perfused intravitally under deep anesthesia through the ascending aorta with 7--10% formalin in 0.9% saline adjusted to pH 7.3 with sodium bicarbonate. The brain stems were removed immediately and placed in 0.1 M phosphate buffer (pH 7.6) containing 30% sucrose at 4°C for 2--4 days. Subsequently transverse or parasagittal frozen sections through the lower brain stem were made serially at 60-pm thickness, and treated for the histochemical demonstration of HRP [4,13]. These sections were then m o u n t e d onto gelatincoated slides and counterstained with 1% cresylecht violet. After HRP injections into the anterior belly of the digastric muscle in the animals, neurons labeled with HRP were seen in the ventromedial division of the trigeminal m o t o r nucleus [8,9]. On the other hand, when HRP was injected into the posterior belly of the digastric muscle, two groups of HRP-
Fig. 1. Distribution pattern of PD (black stars) and AD (black circles) motoneurons is shown diagrammatically in series of projection drawings of cross sections of the lower brain s~ems of a monkey, cat and rabbit. The drawings are arranged from rostral (top) to caudal (bottom), and the numbers indicate the position of the serial sections of 60-~m thickness. The section Nr. i corresponds to the most rostral level of AD mononeuron group. Double and single arrow-heads point to the rostral and caudal groups of PD motoneurons, respectively, a, Root of abducens nerve; Ab, abducens nucleus; F, facial nucleus; f, root of facial nerve; Os, superior olive; Vm, motor nucleus of trigeminal nerve; Vp, main sensory nucleus of trigeminal nerve; Vs, spinal tract of trigeminai nerve.
q r
Ij-
,$
C
3
50
Fig. 2. Dark-field photomicrography of a parasagittal section through AD motoneuron group of a rabbit. The cluster of HRP-labeled AD motoneurons extends caudally to the level of the descending root of the facial nerve (f). Bar = 300 um. labeled n e u r o n s were observed; the rostral group was seen as a small cluster o f n e u r o n s along t h e medial b o r d e r o f the descending r o o t o f the facial nerve, and the n e u r o n s o f the caudal g r o u p were s o m e w h a t s c a t t e r e d a m o n g the asc e n d i n g r o o t fibers o f t h e facial nerve. T h e m o r p h o l o g y o f these PD m o t o n e u r o n s was similar t o t h a t o f the facial m o t o n e u r o n s [ 5 ] . In the m o n k e y and rat t h e PD m o t o n e u r o n s o f the caudal group were m o r e n u m e r o u s t h a n t h o s e o f t h e rostral group. In o t h e r animals studied, h o w e v e r , the n u m b e r o f PD m o t o n e u r o n s in t h e caudal g r o u p was a l m o s t equal t o t h a t in the rostral group (Fig.l). When H R P was injected i n t o t h e digastric muscle in the rabbit, in which the p o s t e r i o r belly o f the muscle is lacking, the cluster o f HRP-labeled n e u r o n s in the v e n t r o m e d i a l division o f the trigeminal m o t o r nucleus was observed t o e x t e n d caudally t o the area medial t o t h e d e s c e n d i n g r o o t o f the facial nerve, w h e r e t h e rostral g r o u p o f PD m o t o n e u r o n s was identified in each o f o t h e r animals studied (Figs. 1 and 2). T h e d i s t r i b u t i o n p a t t e r n o f PD m o t o n e u r o n s i d e n t i f i e d on t h e p r e s e n t s t u d y c o r r e s p o n d e d t o t h a t o f n e u r o n s o f the nucleus nervi facialis accessorius o f T a b e r [15] or the r e t r o t r i g e m i n a l nucleus of B e r m a n [1] described in the cat. As p o i n t e d o u t b y SzentAgothai [14] in the cat and c o r r o b o r a t e d b y M i z u n o e t al. [ 9] in t h e cat and rat, the rostral pole o f the cluster o f PD m o t o n e u r o n s c o n s t i t u t i n g the accessory facial nucleus was c o n t i n u o u s ros:
51
Fig. 3. Photomicrograph of a parasagittal section through the brain stem of a guinea pig, showing distribution of AD and PD motoneurons labeled with HRP injected simultaneously into the AD and PD muscles. The rostral and caudal groups of PD motoneurons are indicated by a double and single arrows, respectively. Abbreviations are as in Fig. 1. Bar = 300 um
trally, with a gap of short distance, to the caudal pole of the cluster of AD motoneurons in the ventromedial division of the trigeminal motor nucleus in the monkey, dog and guinea pig (Figs. 1 and 3). ACKNOWLEDGEMENTS
The technical assistance of Mr. Kiwamu Tanaka and the photographic help of Mr. Akira Uesugi are gratefully acknowledged. REFERENCES 1 Berman, A.L., The Brain Stem of the Cat, A Cytoarchitectonic Atlas with Stereotaxic Coordinates, The University of Wisconsin Press, Madison, 1968, 175 pp. 2 Bijvoet, W.F., Zur vergleichended Morphologie des Musculus digastricus mandibulae bei den S~iugetieren, Z. Morphol. Anthropol., 11 (1908) 249--316. 3 Don, R., Falls, W. and Martin, G.G., The motor nucleus of the facial nerve in the opossum (Didelphis marsupialis virginiana), its organization and connections, J. comp. Neurol., 152 (1973) 373--402. 4 Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302.
52 5 Kume, M., Uemura, M., Matsuda, K., Matsushima, R. and Mizuno, N., Topographical representation of peripheral branches of the facial nerve within the facial nucleus: a HRP study in the cat, Neurosci. Lett., 8 (1978) 5--8. 6 Landgren, S. and Olsson, K.A., Localization of evoked potentials in the digastric, mas seteric, supra- and intertrigeminal subnuclei of the cat, Exp. Brain Res., 26 (1976) 299--318. 7 Martin, M.R. and Lodge, D., Morphology of the facial nucleus of the rat, Brain Res., 123 (1977) 1--12. 8 Matsuda, K., Uemura, M., Kume, M., Matsuda, R. and Mizuno, N., Topographical representation of masticatory muscles in the motor trigeminal nucleus in the rabbit: a HRP study, Neurosci. Lett., 8 (1978) 1--4. 9 Mizuno, N., Konishi, A. and Sato, M., Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. comp. Neurol., 164 (1975) 105--116. 10 Papez, J.W., Subdivisions of the facial nucleus, J. comp. Neurol., 43 (1927) 159--191. 11 Provis, J., The organization of the facial nucleus of the brush-tailed possum (Trichosurus vulpecula), J. comp. Neurol., 172 (1977) 177--188. 12 Scapino, R.P., Function of the digastric muscle in carnivores, J. Morphol., 150 (1976) 843--860. 13 Streit, P. and Reubi, J.C., A new and sensitive method for axonalty transported horseradish peroxidase (HRP) in the pigeon visual system, Brain Res., 126 (1977) 530-537. 14 Szent~igothai, J., Functional representation in the motor trigeminal nucleus, J. comp. Neurol., 90 (1949) 111--120. 15 Taber, E., The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of the cat, J. comp. Neurol., 116 (1961) 27--70. 16 Van Gehuchten, A., Recherches sur l'origine rdele des nerfs craniens. II. Nerf facial (suite). J. Neurol. (Bruxelles), 3 (1898) 293--303. 17 Vraa-Jensen, G.F., The Motor Nucleus of the Facial Nerve, With a Survey of the Efferent Innervation of the Facial Muscles, Munksgraard. Copenhagen, 1942, 157 pp.