Localization of Masticatory Motoneurons in the Cat and Rat by Means of Retrograde Axonal Transport of Horseradish Peroxidase NOBORU MIZUNO, AKIRA KONISHI AND MANABU S A T 0 Depurtment of Anutomy, Faculty of Medicine, Kyoto University, Kyoto, J a p a n
ABSTRACT Topographical localization of motoneurons supplying the masticatory muscles was investigated in the cat and rat, utilizing retrograde axonal transport of horseradish peroxidase. Following iqjection of horseradish peroddase i n each masticatory muscle, motoneurons labelled with peroxidase were seen to be aggregated into a cluster within the motor nucleus of the trigeminal nerve. Such clusters of peroxidase-labelled motoneurons innervating each masticatory muscle were demarcated more sharply in kittens than in adult animals. The pattern of the nuclear representation of the masticatory muscles was found to be essentially the same in the cat and rat; it could be summarized as follows: The motor nucleus of the trigeminal nerve could be divided cytoarchitectonically into the dorsolateral and ventromedial divisions; the former was seen in almost the whole rostrocaudal extent of the nucleus, while the latter was localized at the levels of caudal two thirds of the nucleus. In the dorsolateral division, the temporal muscle was represented dorsally and dorsomedially, the masseter muscle ventrolaterally, and the pterygoid muscles ventromedially. In the ventromedial division, the anterior digastric muscle was represented dorsomedially, and the mylohyoid muscle ventrolaterally. I t was also confirmed that the motoneurons supplying the posterior digastric muscle were localized in the accessory facial nucleus.
The applicability of retrograde axonal transport of horseradish peroxidase for cartography of motoneurons has recently been demonstrated in the oculomotor, hypoglossal and spinal nerves (Kristensson and Olsson, '71; Kristensson et al., '71; Gacek, '74). The present study was designated to demonstrate topographical localization of the masticatory motoneurons in the motor nucleus of the trigeminal nerve in the cat and rat by utilizing the method of retrograde axonal transport of horseradish peroxidase. Topographical localization of the masticatory motoneurons in the cat has been investigated systematically only by Szentagothai ('49) and Vedral and Matzke ('67). The data presented by both of these investigators have shown that masticatory rnuscles situated in more ventral parts of the head are represented in more dorsal portions of the motor nucleus of the trigeminal nerve than more cranially situated masticatory muscles. The present study, J. COMP. NEUR., 164: 105116.
however, will describe a different principle of the nuclear representation of masticatory muscles within the motor nucleus of the trigeminal nerve in the cat and rat, as determined by the new method. MATERIALS A N D METHODS
Eight newborn kittens of age 1-18 days, 31 cats (500-3500 g body weight) and 28 albino rats (180-400 g body weight) were used in the present study. The animals were anesthetized with intraperitoneal sodium pentobarbital (2.535 mglkg). After exposing the temporal, masseter, mylohyoid, anterior digastric, posterior digastric or pterygoid muscles on the left side of the animals, 2% solution of horseradish peroxidase (Sigma Type VI) dissolved in sterile 0.9% saline was injected within the muscle belly with a fine caliber intradermic needle attached to a syringe calibrated in 0.02 milliliter. The injections were performed very carefully until the injected muscle became discolored with peroxidase; 105
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if escape of peroxidase from injected muscle was noted, and if spread of the peroxidase to neighboring muscles was indicated by discoloration of the muscles, the animal was excluded from the experiment. After administration of horseradish peroxidase the incisions in the skin were sutured closed, and the animals were allowed to survive for 20-48 hours. After survival periods the animals were again anesthetized deeply with intraperitoneal sodium pentobarbital (40-50 mglkg) and perfused through the ascending aorta or the left cardiac ventricle with 10% formalin in 0.9% saline adjusted to pH 7.6 with sodium bicarbonate or with a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde in Millonig buffer (pH 7.4). The brains were removed immediately and kept overnight in the Millonig buffer or 0.05 M Tris-HC1 buffer (pH 7.6) containing 30% sucrose, and then transverse frozen sections through the lower brainstem were made serially at 60 Fm thickness. The sections were treated with 3, 3’-diamino-benzidine tetrahydrochloride and hydrogen peroxide as described by Graham and Karnovsky (‘66). These sections were mounted on glass slides coated with a chrome alumgelatin (Pappas, ’71), and counterstained lightly with 1% cresyl violet. Complete sets of serial sections were thus obtained through the brainstem from rostral levels of the facial nucleus to the most rostral levels of the pons. The cells labelled with the rusty-red peroxidase-granules were plotted on cytoarchitectonic projection drawings of representative cross sections of the motor trigeminal nucleus (fig. 1). Most of the masticatory muscles investigated in the present study were easily exposed for peroxidase-injection, but the pterygoid muscles were rather difficult to access for injection. Although in six cats and four rats the pterygoid muscles exposed through submandibular approach were injected with horseradish peroxidase, peroxidase tended to spread both in the lateral and medial pterygoid muscles and it was also difficult to see if the injected peroxidase was spread to the whole extent of the injected muscle. Hence, in additional five cats, horseradish peroxidase was injected within all of the masticatory muscles except for pterygoid muscles; in these animals non-labelled motoneurons
in the motor nucleus of the trigeminal nerve were observed to complement the results obtained from observation of the peroxidase-labelled pterygoid motoneurons. Separate peroxidase-labelling of the lateral pterygoid muscle from the medial pterygoid muscle, however, could not be achieved in the present study. RESULTS
In both the cat and rat the motor nucleus of the trigeminal nerve could be divided cytoarchitectonically into two divisions; the larger dorsolateral and the smaller ventromedial divisions. The dorsolateral division was seen in almost the whole rostrocaudal extent of the motor trigeminal nucleus, while the ventromedial division was localized at the levels of caudal two thirds of the nucleus. The motoneurons of the ventromedial division were further grouped into the dorsomedial and the ventrolateral subdivisions; the vast majority of motoneurons in the ventrolateral subdivision were smaller in size than those in the dorsomedial subdivision (fig. 1). Following injection of horseradish peroxidase in each of the masticatory muscle, motoneurons labelled with peroxidase were seen to be aggregated into a cluster within the motor nucleus of the trigeminal nerve. Although these clusters of motoneurons innervating each masticatory muscle appeared to be overlapped with each other to a certain extent in adult animals, they were fairly well demarcated in kittens. The pattern of nuclear representation of the masticatory muscles revealed in the present study was essentially the same in the cat and rat. After injection of horseradish peroxidase in the temporal (7 cats and 5 rats), pterygoid (6 cats and 4 rats) or masseter (4 cats and 4 rats) muscle, nerve cells labelled with peroxidase were found in the dorsolateral division of the motor trigeminal nucleus; the temporal motoneurons were localized dorsally and dorsomedially, pterygoid motoneurons ventromedially, and masseter motoneurons ventrolaterally (figs. 1, 2). The nuclear areas corresponding to the representation field of the pterygoid muscles were free of peroxidase-labelled cells in the five cats subjected to injection of horseradish peroxidase in all masticatory muscles except for the pterygoid muscles (fig. 2c). A few
LOCALIZATION OF MASTICATORY MOTONEURONS
of small neurons localized in the ventral aspects of the dorsolateral division at the middle levels of the motor trigeminal nucleus were also free from peroxidase-labelling in all animals observed in the present study (fig. 1). These small neurons were tentatively assumed as the motoneurons supplying the tensor tympani and the tensor veli platini muscles; in these small trigeminal muscles horseradish peroxidase was not injected in the present study. In the ventromedial division of the motor trigeminal nucleus, neurons labelled with horseradish peroxidase were seen following peroxidase-injection in the anterior digastric (3 cats and 4 rats) or the mylohyoid (3 cats and 3 rats) muscle; cell clusters of the anterior digastric and the mylohyoid motoneurons were found to correspond roughly to the dorsomedial and the ventrolateral subdivisions in the ventromedial division of the nucleus, respectively (figs. 1, 3a-3c). In 2 cats and 2 rats horseradish peroxidase was injected in the posterior digastric muscle. In these animals small clusters of peroxidase-labelled motoneurons were found in the rostrodorsal aspects of the facial nucleus, and in the areas lateral to the ascending root of the facial nerve, and medial to the descending root of the facial nerve (fig. 3d). The rostra1 pole of the cluster of the posterior digastric motoneurons was situated at the levels 60-120 pm (one or two sections) more caudally to the caudal pole of the ventromedial division of the motor trigeminal nucleus. A few of medium-sized or small cells in the mesencephalic nucleus of the trigeminal nerve were also found to be labelled with horseradish peroxidase injected into the temporal or masseter muscle of the rat (fig. 3e). Such labelling of neurons in the mesencephalic trigeminal nucleus was not recognized in the cat. DISCUSSION
The motor nucleus of the trigeminal nerve in both the cat and rat could be divided cytoarchitectonically into the dorsolateral and ventromedial divisions, as described in the cat by Taber ('61). These two divisions of the motor trigeminal nucleus correspond to the dorsal and ventral divisions designated in the cat by Berman ('68),or to the gamma and beta nuclei
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reported in the rabbit by Meessen and Olszewski ('49). The present study has demonstrated that motoneurons supplying each masticatory muscle were aggregated into a cluster within the motor nucleus of the trigeminal nerve, and that the pattern of nuclear representation of the masticatory muscles was essentially the same in the cat and rat. Within the dorsolateral division of the motor trigeminal nucleus, the temporal muscle was represented dorsally and dorsomedially, the pterygoid muscles ventromedially, and the masseter muscle ventrolaterally. Within the ventromedial division of the motor trigeminal nucleus, the anterior digastric muscle was represented in the dorsomedial subdivision, and the mylohyoid muscle in the ventrolateral subdivision. Such pattern of nuclear representation of the masticatory muscles, however, is not in accordance with that reported by earlier investigators, who have studied the nuclear representation in the cat by examining degenerated motor end-plates and motor fibers after placing small lesions stereotaxically in the motor nucleus of the trigeminal nerve (Szentagothai, '49) or by detecting the retrograde cell reaction following removal of the motor nerves to the masticatory muscles (Vedral and Matzke, '67). The most marked discrepancies between the present findings and those of the earlier investigators are concerned with localization of the temporal and mylohyoid motoneurons. According to Szentagothai ('49) and Vedral and Matzke ('67), the temporal muscle was represented ventromedially in the motor trigeminal nucleus, and the data of Szentagothai ('49) showed that the mylohyoid motoneurons were localized in the dorsal aspects at the most cranial levels of the motor trigeminal nucleus. Jacobs ('70) also postulated on the basis of the embryologic study of the human fetus that the mylohyoid and the anterior digastric muscles were represented dorsally in the motor trigeminal nucleus, and that the motoneurons innervating the temporal, masseter and pterygoid muscles were localized in the intermediate and ventral parts of the motor trigeminal nucleus. In the present study, however, the temporal motoneurons were always found in the most dorsal portions of the motor trigeminal nucleus, and no motoneurons
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innervating the anterior digastric and the mylohyoid muscles were encountered at the levels of rostral one third of the motor trigeminal nucleus. In these respects, the present findings are in agreement with those of Parhon and Nadejde ('06) and Willems ('11); according to these investigators, the mylohyoid motoneurons were localized in the medial and ventral parts of the motor trigeminal nucleus in the dog and rabbit. The vast majority of the mylohyoid motoneurons were found to be smaller in size than the anterior digastric motoneurons. Smaller motoneurons are generally considered to be the gamma motoneurons or the motoneurons supplying the slow or tonic muscle fibers. Since no muscle spindles have been demonstrated clearly in the mylohyoid muscle of the cat and rat (Cooper, '60; Hosokawa, 'Sl), the mylohyoid motoneurons of small size can not be regarded as the gamma motoneurons. Therefore, if the assumption that motoneurons innervating slow or tonic muscle fibers tend to be smaller in size than those supplying fast or phasic muscle fibers is correct (Granit et al., '56; Eccles et al., '58; Henneman et al., '65; Henneman and 01son, '65; Burke, 'SS), the mylohyoid muscle can be considered to be composed chiefly of slow or tonic muscle fibers. The present study confirmed the findings of Szentagothai ('49) that the posterior digastric muscle was innervated by a small group of motoneurons lying dorsocranially from the facial nucleus on the lateral side of the ascending root fibers of the facial nerve. This small group of motoneurons corresponds to the nucleus nervi facialis accessorius of Taber ('61) or the retrotrigeminal nucleus of Berman ('68). As pointed out by Szentagothai ('49), the accessory facial nucleus appeared to be continuous rostrally to the trigeminal motoneurons innervating the anterior digastric muscle, although a gap of short distance was always noted between these two groups of motoneurons. It is well known that the motor nucleus of the trigeminal nerve innervates the tensor tympani and the tensor veli palatini muscles in addition to the masticatory muscles. According to Szentagothai ('49), the motoneurons supplying these small
trigeminal muscles were localized in the ventral portions of the motor trigeminal nucleus. The nuclear representation of these muscles was not investigated in the present study. It was observed, however, that at the middle levels of the motor trigeminal nucleus small clusters of small motoneurons existed in the ventral aspects of the dorsolateral division of the nucleus, and that none of these small motoneurons were labelled with horseradish peroxidase in the present study. Thus, these small motoneurons were tentatively assumed to be motoneurons innervating the tensor tympani and the tensor veli palatini muscles. In addition to motoneurons of the masticatory muscles, nerve cells of the mesencephalic nucleus of the trigeminal nerve in the rat have been reported to be labelled with horseradish peroxidase injected into the temporal or masseter muscle (Mizuno et al., '74). Recently, Furstman et al. ('75) have also shown that nerve cells in the spinal and trigeminal ganglia were labelled by retrograde axonal transport of horseradish peroxidase applied to the tooth pulps or sciatic nerve in the rat. In the present study, however, no cells in the mesencephalic trigeminal nucleus were labelled with peroxidase injected into the masticatory muscles of the cat. Since the number of layers of sheet cells of the muscle spindle capsules has been known to be generally more numerous in the cat than in the rat (Barker, '74; Corvaja et al., '69; Landon, 'SS), the capsule of the muscle spindle in the cat was considered to be more resistant to penetration of horseradish peroxidase than that in the rat (Shantha and Bourne, '68). Thus, it was postulated that horseradish peroxidase injected in the muscle might gain access to nerve terminals within the muscle spindles more easily in the rat than in the cat. If this postulation is correct, gamma motoneurons in the rat should be labelled with peroxidase more easily than those in the cat. No confirmatory evidences supporting this assumption, however, could be obtained in the present study. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mr. Akira Uesugi for his ex-
LOCALIZATION OF MASTICATORY MOTONEURONS
cellent photographic work and to Mrs. Tomoko Uchida for her helpful assistance in the preparation of this manuscript. LITERATURE CITED Barker, D. 1974 The morphology of muscle receptors. In: Handbook of Sensory Physiology, Vol. 3, Pt. 2, Muscle Receptors. C. C. Hunt, ed. Springer, Berlin, pp. 1-190. Berman, A. L. 1968 The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates. The University of Wisconsin Press, Madison. Burke, R. E. 1968 Firing patterns of gastrocnemius motor units. J. Physiol. (London), 196:
631-654. Cooper, S. 1960 Muscle spindle and other muscle receptors. In: The Structure and Function of Muscles, Vol. 1. G. H. Bourne, ed. Academic Press, New York, pp. 381420. Corvaja, N., V. Marinozzi and 0. Pompeiano 1969 Muscle spindles in the lumbrical muscle of the adult cat. Electron microscopic observations and functional considerations. Arch. ital. Biol., 107: 365-543. Eccles, J. C., R. M. Eccles and A. Lundberg 1958 The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (London), 142: 275-291. Furstman, L., S. Saporta and L. Kruger 1975 Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat. Brain Res., 84: 320-324. Gacek, R. R. 1974 Localization of neurons supplying the extraocular muscles in the kitten using horseradish peroxidase. Exp. Neurol., 44;
381403. Graham, R. C.,and M. J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase i n the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-
302. Granit, R., H. D. Henatsch and G. Steg 1956 Tonic and phasic ventral horn cells differentiated by post-tetanic potentiation in cat extensors. Acta Physiol. Scand, 37: 114-126. Henneman, E.,and C. B. Olson 1965 Relations between structure and function in the design of skeletal muscles. J. Neurophysiol., 28: 581-
598. Henneman, E., G. Somjen and D. 0. Carpenter 1965 Functional significance of cell size i n spinal motoneurons. J . Neurophysiol., 28: 560-
580.
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Hosokawa, H. 1961 Proprioceptive innervation of striated muscles in the territory of cranial nerves. Texas Rept. Biol. Med., 19: 405-464. Jacobs, M. J. 1970 The development of the human motor trigeminal complex and accessory facial nucleus and their topographic relations with the facial and abducens nuclei. J. Comp. Neur., 138: 161-194. Kristensson, K., and Y. Olsson 1971 Retrograde axonal transport of protein. Brain Res., 29:
363-365. Kristensson, K., Y. Olsson and J. Sjostrand 1971 Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve. Brain Res., 32: 399-406. Landon, D. N. 1966 Electron microscopy of muscle spindles. In: Control and Innervation of Skeletal Muscle. B. L. Andrew, ed. Livingston, Edinburgh, pp. 9 6 111, Meessen, H.,and J. Olszewski 1949 Cytoarchitektonischer Atlas des Rautenhirn des Kaninchens. Karger, Basel. Mizuno, N., N. Iwahori, Y. Nakamura, A. Konishi and M. Sat0 1974 Fiber connections of trigeminal motor system. Advance neurol. Sci. (Tokyo), 18: 971-984.(in Japanese). Pappas, P. W. 1971 The use of a chrome alumgelatin (subbing) solution as a general adhesive for paraffin sections. StainTechnol., 46: 121-124. Parhon, C., and G. Nadejde 1906 Nouvelle contribution A l'btude des localisations dans les n o y a w des nerfs craniens et rachidiens chez l'homme et chez le chien. J. Neurol. (Bruxelles), 10: 129-140.
Shantha, T. R., and G. H. Bourne 1968 The perineural epithelium - a new concept. In: The Structure and Function of Nervous Tissue, Vol. 1, Structure I. G.H. Bourne, ed. Academic Press, New York, pp. 379460. Szentagothai, J. 1948 The representation of facial and scalp muscles in the facial nucleus. J. Comp. Neur., 88: 207-220. 1949 Functional representation in the motor trigeminal nucleus. J. Comp. Neur., 90:
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111-120. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat. J. Comp. Neur., 1 1 6: 27-70. Vedral, D. F., and H. A. Matzke 1967 Topographical localization of the muscles of mastication in the motor nucleus of the trigeminal nerve i n the cat. J. Hirnforsch., 9: 565569. Willems, E. 1911 Localisation motrice et kinesthesique. Les noyaux masticateur et mesencephalique du trijumeau chez le lapin. Le N&vraxe, 12: 1-225.
PLATE 1 EXPLANATION OF FIGURES
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The pattern of nuclear representation of the masticatory muscles is shown semi-diagrammatically in a series of projection drawings of the motor trigeminal nucleus of a newborn kitten. The serial sections of 60 pm thickness are numbered from caudal to rostral level; the thirteenth section corresponds to the most rostral level of the nucleus. The temporal (T), pterygoid (P) and masseter (M) motoneurons are localized in the dorsolateral division of the motor trigeminal nucleus, while the anterior digastric (D) and mylohyoid (H) motoneurons in the ventromedial division. Arrows indicate clusters of small motoneurons supplying assumedly the tensor veli palatini and tensor tympani muscles.
LOCALIZATION OF MASTICATORY MOTONEURONS Noboru Mizuno, Akira Konishi a n d Manabu Sato
PLATE 1
2.
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PLATE 2 EXPLANATION O F F I G U R E S
Fig. 2 Clusters of motoneurons supplying the temporal, pterygoid and masseter muscles within the dorsolateral division of the motor trigeminal nucleus. Arrows point to motoneurons of the ventromedial division of the motor trigeminal nucleus. X 70. Photomicrograph of a cross section through a middle level of the motor trigeminal nucleus of a rat, showing a cluster of peroxidase-labelled temporal motoneurons (T). Temporal motoneurons (T) labelled with peroxidase at a middle level of the motor trigeminal nucleus of a newborn kitten. Compare with figure 2a, and note the same pattern of distribution of peroxidaselabelled temporal motoneurons in the cat and rat. Photomicrograph of a cross section through a caudal level of the motor trigeminal nucleus of a newborn kitten, in which horseradish peroxidase was injected in all masticatory muscles except for the pterygoid muscles. The temporal (T), masseter (M) and anterior digastric (D) motoneurons are labelled with peroxidase, whereas the pterygoid motoneurons (P) are free from labelling. The mylohyoid motoneurons are not seen in this section because of obliquity of the cut plane. Masseter motoneurons labelled with peroxidase at a middle level of the motor trigeminal nucleus of a newborn kitten.
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LOCALIZATION OF MASTICATORY M O T O N E U R O N S Noboru Mizuno, Akira K o n i s h i a n d M a n a b u Sato
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
Fig. 3 Clusters of motoneurons innervating the suprahyoid muscles, and peroxidase-labelled cells in the mesencephalic trigeminal nucleus. Larger arrows indicate the dorsomedial margin of the dorsolateral division of the motor trigeminal nucleus. Anterior digastric motoneurons (D)labelled with horseradish peroxidase in the ventromedial division of the motor trigeminal nucleus of an adult cat. Motoneurons (H) localized ventrolaterally in the ventromedial division of the nucleus are not labelled. X 70. Peroxidase-labelled mylohyoid motoneurons (H) in the ventromedial division of the motor trigeminal nucleus of a young cat. Motoneurons (D) localized dorsomedially in the ventromedial division of the nucleus are not labelled. x 70. The anterior digastric (D, labelled) and the mylohyoid (H, unlabelled) motoneurons i n the ventromedial division of the motor trigeminal nucleus of a rat. As i n the cat, the anterior digastric motoneurons are localized dorsomedially, and the mylohyoid motoneurons ventrolaterally. X 70. Peroxidase-labelled posterior digastric motoneurons in the accessory facial nucleus, localized medial to the descending root of the facial nerve (FN) of a rat. X 180. Peroxidase-labelled, medium-sized neurons (arrows) i n the mesencephalic trigeminal nucleus of a rat, in which horseradish peroxidase was injected in the temporal muscle. X 270.
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LOCALIZATION OF MASTICATORY MOTONEURONS Noboru Mizuno, Akira Konishi and Manabu Sato
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