EXPERIMENTAL
NEUROLOGY
254-262 (1978)
59,
Commissural Interneurons for Masticatory Motoneurons: A Light and Electron Microscope Study Using the Horseradish Peroxidase Tracer Technique NOBORU MIZUNO, SAKASHI NOMURA, KAZUO ITOH, YASUHISA NAKAMURA, AND AKIRA KONISHI l Department
of Anatomy,
Faculty of Medicine, and College Kyoto University, Kyoto 606, Japan Received
November
of
Medical
Technology,
1,1977
Commissural interneurons for masticatory motoneurons of the cat were investigated using the horseradish peroxidase tracing technique. After injection of horseradish peroxidase into the motor trigeminal nucleus, neurons labeled with the enzyme were seen contralaterally around the motor trigeminal nucleus, mainly in the supra- and intertrigeminal regions and the lateral tegmental regions close to the medial border of the motor trigeminal nucleus, and additionally within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei. After placing lesions in the supra- and intertrigeminal regions, degenerated axon terminals were found electron microscopically in the motor trigeminal nucleus contralateral to the lesion. In accordance with these findings, axon terminals in the motor trigeminal nucleus were labeled orthodromically with horseradish peroxidase injected contralaterally into the supra- and intertrigeminal regions ; both terminals filled with round synaptic vesicles and those containing flattened vesicles were labeled with the enzyme. The existence of many commissural neurons around the motor trigeminal nucleus would be consistent with the intricacy of bilateral mechanisms of jaw movements. INTRODUCTION
Interneurons for masticatory motoneurons have been suggested to distribute in the supratrigeminal and intertrigeminal regions; the supratrigeminal region is a reticular area capping the motor trigeminal nucleus dorsally and rostrodorsally, and the intertrigeminal region is a narrow reticular area interposed between the motor and the main sensory trigeminal 1 Dr. Konishi is at the College of Medical Technology. photographic work of Mr. Akira Uesugi. 254 0014~4886/78/0592-0254$02.00/O Copyright All rights
Q 1978 by Academic Press, of reproduction in any form
Inc. reserved.
The authors acknowledge
the
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
255
nuclei lfor review, see (3, 11, 19, 20) 1. Tl 1e existence of commissural interneurons projecting contralaterally to masticatory motoneurons of the cat was indicated electrophysiologically in the supratrigeminal region (1, 7, 14). After placing a lesion in the supra- and/or intertrigeminal region, fiber degeneration was shown within the motor trigeminal nucleus and the supra- and intertrigeminal regions contralateral to the lesion in the cat (3, ll), monkey (11, 20), and rat (19). In the present study attempts were made to examine the distribution pattern of commissural interneurons for masticatory motoneurons and to investigate electron microscopically the axon terminals of commissural interneurons by utilizing the method of retrograde and orthograde axonal transport of horseradish peroxidase. An abstract of part of the present findings was published elsewhere (12). MATERIALS
AND
METHODS
The experiments were carried out on young adult cats (1.5 to 3.0 kg) anesthetized with intraperitoneal Nembutal (35 mg/kg). In 19 cats a single injection of 0.05 ~1 50% horseradish peroxidase (Toyobo GradeI-C, RZ: 3.4) d issolved in 20/o dimethylsulfoxide (6) or in sterile 0.9% saline was attempted stereotaxically by means of a 1-J Hamilton syringe into the motor trigeminal nucleus and/or its adjacent regions. After survival periods of 24 to 48 h, 12 of the 19 cats were deeply anesthetized and perfused through the ascending aorta with 1 to 1.5 liters 7% formalin in 0.9% saline or 0.1 M phosphate buffer (pH 7.3). Brain stems were cut serially at 60-pm thicknesses in the frontal plane on a freezing microtome after 1 to 2 days of soaking in Millonig’s buffer containing 30% sucrose. Sections were incubated in a medium containing hydrogen peroxide, 3,3’-diaminobenzidine tetrahydrochloride, and p-cresol, as described by Streit and Reubi (22), washed, mounted, and counterstained with cresyl violet. For electron microscopic work, brains from 7 of 19 cats subjected to injection of peroxidase were perfused with 2 liters of a mixture composed of 4% paraformaldehyde and 0.5% glutaraldehyde in Millonig’s buffer (PH 7.4). Brain stems were removed immediately and sectioned transversely at loo-pm thicknesses on a Vibratome, floated on a bath of perfusion fixative or Millonig’s buffer, and incubated 20 min at 20 to 25°C in a slightly modified Graham-Karnovsky’s medium (2). Subsequently, small tissue slices containing the motor trigeminal nucleus were cut, using razor blades under a dissecting microscope, and postfixed 40 min in a chilled 2% solution of osmium tetroxide in the same Millonig’s buffer used for preparing the perfusion fixative. Embedding was done in an epoxy resin after dehydration in a graded series of ethanol. The location
256
MIZUNO
ET
AL.
of the motor trigeminal nucleus was verified in semithin sections which were cut from each block and stained with 0.576 toluidine blue in 1% borax. Ultrathin sections were stained with lead acetate or lead citrate and viewed with a Hitachi HU-12 electron microscope. Unstained sections were also examined. In 12 cats, electrolytic lesions were placed stereotaxically in the supratrigeminal regions as described elsewhere (11). After survival periods of 2 to 4 days, the cats were perfused transcardially with the aldehyde mixture as described above. Tissue slices containing the motor trigeminal nucleus contralateral to the lesion were postosmicated and embedded in the epoxy resin. Ultrathin sections were stained and examined electron microscopically as described above. The locus and extent of the lesions were checked by routine histological methods. RESULTS In 5 of 12 cats which were injected with horseradish peroxidase and perfused for light microscopic work, injection sites were confined within the motor trigeminal nucleus and its vicinities, including more or less the supratrigeminal and intertrigeminal regions (Fig. la). The pattern of
FIG. 1. Photomicrographs of cross sections through a site of injection of horseradish peroxidase into the motor trigeminal nucleus (a), and through an electrolytic lesion (arrow) placed in the supratrigeminal area rostrodorsal to the motor trigeminal nucleus (b). r-Motor root of trigeminal nerve. a, X 63 ; b, X 9.
INTERNEVRONS
a
FOR
MASTICATORY
I
b
MOTONEURONS
257
‘I
FIG. 2. Projection drawings made from sections through rostra1 (a) and middle (b) levels of the motor trigeminal nucleus, showing the distribution pattern of neurons labeled with horseradish peroxidase injected contralaterally into and around the motor trigeminal nucleus (M). BC-brachium conjunctivum; PBparabrachial nucleus; r-motor root of trigeminal nerve; T-main sensory trigeminal nucleus. distribution of neurons labeled with peroxidase was quite similar among these cats (Fig. 2). enzyme were small or medium size, and were
transported retrogradely Neurons labeled with the distributed mainly in the
FIG. 3. Photomicrographs taken from a cross section through the middle level of the motor trigeminal nucleus. Neurons labeled with horseradish peroxidase injected contralaterally into the motor trigeminal nucleus are seen in the lateral tegmental areas (a) immediately adjacent to the medial border of the motor trigeminal nucleus (M), and in the supratrigeminal (S) and intertrigeminal (I) regions (b). Survival time, 36 h. T-Main sensory trigeminal nucleus. X 78.
258
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ET
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degenerated axodendritic synaptic terminals within the motor FIG. 4. Electron-dense trigeminal nucleus of a cat with a lesion in the contralateral supratrigeminal region. The degenerated terminal in (a) is filled with round synaptic vesicles, and that in (b) contains flattened vesicles. Arrows indicate synaptic sites of degenerated terminals. Survival time, 3 days. X 32,400.
supra- and intertrigeminal regions (Fig. 3b) and the lateral tegmental regions immediately adjacent to the medial border of the motor trigeminal nucleus (Fig. 3a). Additionally, neurons labeled with peroxidase were often seen within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei (Fig. 2). Even in ordinary Nisslstained preparations, these commissural neurons would be distinguishable from large motoneurons, but it might be rather difficult to discriminate them from small motoneurons or neurons of the main sensory trigeminal nucleus and the parabrachial nuclei. In 7 of 12 cats subjected to stereotaxic operation, electrolytic lesions were produced successfully in the supratrigeminal region (Fig. lb). The survival periods after the operation were 60 to 84 h. In all cats, electrondense degenerated axon terminals were found within the confines of the motor trigeminal nucleus contralateral to the lesion (Fig. 4). Other features of possible degeneration of axon terminals, such as the increase of neuro-
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
259
5. Axon terminals within the motor trigeminal nucleus which were labeled FIG. with horseradish peroxidase injected contralaterally into the supratrigeminal region. Electron-dense peroxidase granules (arrows) are seen both in axon terminals filled with round synaptic vesicles (R) and in those containing flattened vesicles (F). Survival time, 40 h. a; X 27,000; b, X 103,500.
filaments or the enlargement of synaptic vesicles, were also seen. These degenerated axon terminals were observed most frequently on the dendritic profiles of medium size; no degenerated synaptic terminals were found on the somatic profiles. Of the total 46 electron-dense synaptic terminals so far found, 20 were filled with round synaptic vesicles and 16 contained flattened ones; in the remaining 10 the shapes of synaptic vesicles could not be discerned clearly. In three of seven cats injected with peroxidase and perfused for electron microscopic work, injection sites were almost confined within the motor trigeminal nucleus and the supra- and intertrigeminal regions. In neuropils within the motor trigeminal nucleus contralateral to the injection site, axon terminals labeled with peroxidase transported orthodromically could be observed; the enzyme in axon terminals was seen as electron-dense granules in metal-stained specimensas well as in unstained ones (Fig. 5). In accordance with the findings obtained from the degeneration study
260
MIZUNO
ET
AL.
described above, the peroxidase granules were found in axon terminals filled with round synaptic vesicles as well as in those containing flattened vesicles. DISCUSSION Commissural interneurons projecting contralaterally to masticatory motoneurons were suggested to exist in the supra- and intertrigeminal regions (1, 7, 11, 14, 19, 20). In the present study, after injection of horseradish peroxidase in the motor trigeminal nucleus, neurons labeled with peroxidase were found contralaterally not only in the supra- and intertrigeminal regions, but also in the lateral tegmental areas close to the medial border of the motor trigeminal nucleus; these regions appear to be included in the area R described in the rabbit (IO) and cat (3), where various propriobulbar fibers were reported to terminate (3, 4, 11, 13, 15, 16, 21). In addition, small- or medium-size cells labeled with peroxidase were often seen within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei. However, even in the supra- and intertrigeminal regions, where neurons labeled with the enzyme were found most numerously, many neurons remained unlabeled. This may indicate that these regions contain many neurons other than the commissural interneurons for the motor trigeminal nucleus ’ [cf. ( 16, 17) 1. Interneurons for masticatory motoneurons in the supratrigeminal region were assumed electrophysiologically to be located within the supratrigeminal nucleus (1, 5, 7, 14), which was first described in the mouse (9) and later also in the cat (8, 11, 23) and monkey (11). However, the extent of the supratrigeminal region, where neurons labeled with peroxidase were observed in the present study, was much wider than that of the supratrigeminal nucleus. Kidokoro et al. (7) recorded inhibitory postsynaptic potentials from masseteric motoneurons of the cat monosynaptically after stimulation of the supratrigeminal region of the ipsilateral as well as the contralateral side. The inhibitory nature of commissural interneurons in the supratrigeminal region was also indicated by Goldberg and Nakamura ( 1) and Nakamura et al. (14). After placing the lesion in the supratrigeminal region, however, both axon terminals filled with round synaptic vesicles and those containing flattened vesicles were degenerated within the contralateral motor trigeminal nucleus. Because some axons of neurons in the intertrigeminal region were described as running through the supratrigeminal region (9, 19, ZO), lesions produced in the supratrigeminal region would damage not only neurons of the supratrigeminal area, but also those of the intertrigeminal region. Therefore, degenerated axon terminals found in the motor trigeminal nucleus after making the lesion
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
261
contralaterally in the supratrigeminal area were considered to arise from commissural interneurons in the supra- and intertrigeminal regions. Within the motor trigeminal nucleus, axon terminals of both types, those filled with round synaptic vesicles as well as those containing flattened vesicles, were also found to be labeled orthodromically with horseradish peroxidase injected contralaterally into the supra- and intertrigeminal regions. Thus, if the assumption that synaptic vesicle shape is significantly related to the function of the synapse is valid [for review, see (18) 1, the present data would certainly suggest that both excitatory ancl inhibitory commissural interneurons may exist around the motor trigeminal nucleus. The presence of a well-organized interneuronal system around the motor trigeminal nucleus would be consistent with the intricate bilateral coordination existing in the movements of the jaw. REFERENCES J., AND Y. NAICAXYRA. 1968. Lingually induced inhibition of masseteric motoneurones. Expcvictltia 24 : 371-373. GRAHAI~, R. C., AND M. J. KARNOVSICY. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastruckural cytochemistry by new technique. J. Histocl?cnt. Cytochcm. 14 : 291-302. HOLSTEGE, G., AICD H. G. J. M. KUYPERS. 1977. Propriobulbar fibre connections to the trigeminal, facial and hypoglossal motor nuclei. I. An anterograde degeneration study. Brai 100 : 239-264. HOLSTEGE, G., H. G. J. M. KUYPERS, AND J. J. DEKICER. 1977. The organization of the bulbar fibre connections to the trigeminal, facial and hypoglossal motor nuclei. II. An autoradiographic tracing study in cat. Bvai~ 100: 265-286. JERZE, C. R. 1963. The function of the nucleus supratrigeminalis. J. Ncwophysiol. 26 : 393-402. KEEFER, D. A., W. B. SPATZ, AND U. MISGELD. 1976. Golgi-like staining of neocortical neurons using retrogradely transported horseradish peroxidase. Nczlrosci. Lctt. 3 : 233-237. KIDOKORO, Y., K. KUBOTA, S. SHCTO, ASD R. SUMINO. 1968. Possible interneurons responsible for reflex inhibition of motoneurons of jaw-closing muscles from the inferior dental nerve. J. NcuropfzysioE. 31 : 709-716. KUYPERS, H. G. J. M., AND J. D. TUERK. 1964. The distribution of the cortical fibres within the nuclei cunettus and gracilis in the cat. J. &at. 98: 143-162. LOREXTE, DE N6, R. 1922. Contribucibn al conocimiento de1 nervio trigemino. Libvo e7t Ho7zor de D7z. S. Ramd>t y Ccljal, Mdyn, Madrid 2: 13-39. MEESSEN, H., AND J. OLSZE~~SKI. 1949. C~toc1vc~lfitrfitor7~~r~7~~ a4tlas dcr Rautod~im des Ka72iclrc77s. Larger, Basel. MIZUNO, N. 1970. Projection fibers from the main sensory trigeminal nucleus and the supratrigeminal region. J. Camp. Nczirol. 139 : 457472. MIZUNO, N., AND A. KONISHI. 1975. An electron microscope study of supratrigeminal fibers to the motor nucleus of the trigeminal nerve. ,4nat. Rec. 181 : 538. MIZUNO, N., Y. NAICAMURA, AXD N. IWAHORI. 1974. Central afferent fibers to trigeminal motor system. Bzllt. ?‘okyo h/fed. Dmt. Uuiv. (SttfibZ.) 21 : 19-21.
1. GOLDBERG,
2.
3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13.
L.
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ET
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14. NAKAMURA, Y., S. MORI, AND H. NAGASHIMA. 1973. Origin and central pathways of crossed inhibitory effects of afferents from the masseteric muscle on the masseteric motoneuron of the cat. Brnin Res. 57 : 29-42. 15. NAUTA, W. J. H., AND H. G. J. M. KUYPERS. 1958. Some ascending pathways in the brain-stem reticular formation. Pages 3-30 in H. H. JASPER, L. D. PROCTOR, R. S. KNIGHTON, W. C. NOSHAY, AND R. T. COSTELLO, Eds., Reticular Forwzatio~t of the Brain. Little, Brown, Boston. 16. NORGREN, R., AND C. M. LEONARD. 1973. Ascending central gustatory pathways. J. Con@. N~ur01. 150 : 217-238. 17. NORGREN, R., AND C. PFAFFMANN. 1975. The pontine taste area in the rat. Brain Res. 91: 99-117. 18. PETERS, A., S. L. PALAY, AND H. DEF. WEBSTER. 1976. The Fine Structure of the Nervous System: The Nezlrons and Supporting Cells. Saunders, Philadelphia. 19. SMITH, R. L. 1973. The ascending fiber projections from the principal sensory trigeminal nucleus in the rat. J. Camp. Neacrol. 148 : 423-446. 20. SMITH, R. L. 1975. Axonal projections and connections of the principal sensory trigeminal nucleus in the monkey. J. Camp. Neural. 163: 347-376. 21. STEWART, W. A., AND R. B. KIXG. 1963. Fiber projections from the nucleus caudalis of the spinal trigeminal nucleus. J. Cow@. Neural. 121: 271-286. 22. STREIT, P., AND J. C. REUBI. 1977. A new and sensitive method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system. Brain Res. 126: 530-537. 23. TORVIK, A. 1957. The ascending fibers from the main trigeminal sensory nucleus. An experimental study in the cat. Am. J. And. 100 : 1-15.
NEUROLOGY
254-262 (1978)
59,
Commissural Interneurons for Masticatory Motoneurons: A Light and Electron Microscope Study Using the Horseradish Peroxidase Tracer Technique NOBORU MIZUNO, SAKASHI NOMURA, KAZUO ITOH, YASUHISA NAKAMURA, AND AKIRA KONISHI l Department
of Anatomy,
Faculty of Medicine, and College Kyoto University, Kyoto 606, Japan Received
November
of
Medical
Technology,
1,1977
Commissural interneurons for masticatory motoneurons of the cat were investigated using the horseradish peroxidase tracing technique. After injection of horseradish peroxidase into the motor trigeminal nucleus, neurons labeled with the enzyme were seen contralaterally around the motor trigeminal nucleus, mainly in the supra- and intertrigeminal regions and the lateral tegmental regions close to the medial border of the motor trigeminal nucleus, and additionally within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei. After placing lesions in the supra- and intertrigeminal regions, degenerated axon terminals were found electron microscopically in the motor trigeminal nucleus contralateral to the lesion. In accordance with these findings, axon terminals in the motor trigeminal nucleus were labeled orthodromically with horseradish peroxidase injected contralaterally into the supra- and intertrigeminal regions ; both terminals filled with round synaptic vesicles and those containing flattened vesicles were labeled with the enzyme. The existence of many commissural neurons around the motor trigeminal nucleus would be consistent with the intricacy of bilateral mechanisms of jaw movements. INTRODUCTION
Interneurons for masticatory motoneurons have been suggested to distribute in the supratrigeminal and intertrigeminal regions; the supratrigeminal region is a reticular area capping the motor trigeminal nucleus dorsally and rostrodorsally, and the intertrigeminal region is a narrow reticular area interposed between the motor and the main sensory trigeminal 1 Dr. Konishi is at the College of Medical Technology. photographic work of Mr. Akira Uesugi. 254 0014~4886/78/0592-0254$02.00/O Copyright All rights
Q 1978 by Academic Press, of reproduction in any form
Inc. reserved.
The authors acknowledge
the
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
255
nuclei lfor review, see (3, 11, 19, 20) 1. Tl 1e existence of commissural interneurons projecting contralaterally to masticatory motoneurons of the cat was indicated electrophysiologically in the supratrigeminal region (1, 7, 14). After placing a lesion in the supra- and/or intertrigeminal region, fiber degeneration was shown within the motor trigeminal nucleus and the supra- and intertrigeminal regions contralateral to the lesion in the cat (3, ll), monkey (11, 20), and rat (19). In the present study attempts were made to examine the distribution pattern of commissural interneurons for masticatory motoneurons and to investigate electron microscopically the axon terminals of commissural interneurons by utilizing the method of retrograde and orthograde axonal transport of horseradish peroxidase. An abstract of part of the present findings was published elsewhere (12). MATERIALS
AND
METHODS
The experiments were carried out on young adult cats (1.5 to 3.0 kg) anesthetized with intraperitoneal Nembutal (35 mg/kg). In 19 cats a single injection of 0.05 ~1 50% horseradish peroxidase (Toyobo GradeI-C, RZ: 3.4) d issolved in 20/o dimethylsulfoxide (6) or in sterile 0.9% saline was attempted stereotaxically by means of a 1-J Hamilton syringe into the motor trigeminal nucleus and/or its adjacent regions. After survival periods of 24 to 48 h, 12 of the 19 cats were deeply anesthetized and perfused through the ascending aorta with 1 to 1.5 liters 7% formalin in 0.9% saline or 0.1 M phosphate buffer (pH 7.3). Brain stems were cut serially at 60-pm thicknesses in the frontal plane on a freezing microtome after 1 to 2 days of soaking in Millonig’s buffer containing 30% sucrose. Sections were incubated in a medium containing hydrogen peroxide, 3,3’-diaminobenzidine tetrahydrochloride, and p-cresol, as described by Streit and Reubi (22), washed, mounted, and counterstained with cresyl violet. For electron microscopic work, brains from 7 of 19 cats subjected to injection of peroxidase were perfused with 2 liters of a mixture composed of 4% paraformaldehyde and 0.5% glutaraldehyde in Millonig’s buffer (PH 7.4). Brain stems were removed immediately and sectioned transversely at loo-pm thicknesses on a Vibratome, floated on a bath of perfusion fixative or Millonig’s buffer, and incubated 20 min at 20 to 25°C in a slightly modified Graham-Karnovsky’s medium (2). Subsequently, small tissue slices containing the motor trigeminal nucleus were cut, using razor blades under a dissecting microscope, and postfixed 40 min in a chilled 2% solution of osmium tetroxide in the same Millonig’s buffer used for preparing the perfusion fixative. Embedding was done in an epoxy resin after dehydration in a graded series of ethanol. The location
256
MIZUNO
ET
AL.
of the motor trigeminal nucleus was verified in semithin sections which were cut from each block and stained with 0.576 toluidine blue in 1% borax. Ultrathin sections were stained with lead acetate or lead citrate and viewed with a Hitachi HU-12 electron microscope. Unstained sections were also examined. In 12 cats, electrolytic lesions were placed stereotaxically in the supratrigeminal regions as described elsewhere (11). After survival periods of 2 to 4 days, the cats were perfused transcardially with the aldehyde mixture as described above. Tissue slices containing the motor trigeminal nucleus contralateral to the lesion were postosmicated and embedded in the epoxy resin. Ultrathin sections were stained and examined electron microscopically as described above. The locus and extent of the lesions were checked by routine histological methods. RESULTS In 5 of 12 cats which were injected with horseradish peroxidase and perfused for light microscopic work, injection sites were confined within the motor trigeminal nucleus and its vicinities, including more or less the supratrigeminal and intertrigeminal regions (Fig. la). The pattern of
FIG. 1. Photomicrographs of cross sections through a site of injection of horseradish peroxidase into the motor trigeminal nucleus (a), and through an electrolytic lesion (arrow) placed in the supratrigeminal area rostrodorsal to the motor trigeminal nucleus (b). r-Motor root of trigeminal nerve. a, X 63 ; b, X 9.
INTERNEVRONS
a
FOR
MASTICATORY
I
b
MOTONEURONS
257
‘I
FIG. 2. Projection drawings made from sections through rostra1 (a) and middle (b) levels of the motor trigeminal nucleus, showing the distribution pattern of neurons labeled with horseradish peroxidase injected contralaterally into and around the motor trigeminal nucleus (M). BC-brachium conjunctivum; PBparabrachial nucleus; r-motor root of trigeminal nerve; T-main sensory trigeminal nucleus. distribution of neurons labeled with peroxidase was quite similar among these cats (Fig. 2). enzyme were small or medium size, and were
transported retrogradely Neurons labeled with the distributed mainly in the
FIG. 3. Photomicrographs taken from a cross section through the middle level of the motor trigeminal nucleus. Neurons labeled with horseradish peroxidase injected contralaterally into the motor trigeminal nucleus are seen in the lateral tegmental areas (a) immediately adjacent to the medial border of the motor trigeminal nucleus (M), and in the supratrigeminal (S) and intertrigeminal (I) regions (b). Survival time, 36 h. T-Main sensory trigeminal nucleus. X 78.
258
MIZUNO
ET
AL.
degenerated axodendritic synaptic terminals within the motor FIG. 4. Electron-dense trigeminal nucleus of a cat with a lesion in the contralateral supratrigeminal region. The degenerated terminal in (a) is filled with round synaptic vesicles, and that in (b) contains flattened vesicles. Arrows indicate synaptic sites of degenerated terminals. Survival time, 3 days. X 32,400.
supra- and intertrigeminal regions (Fig. 3b) and the lateral tegmental regions immediately adjacent to the medial border of the motor trigeminal nucleus (Fig. 3a). Additionally, neurons labeled with peroxidase were often seen within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei (Fig. 2). Even in ordinary Nisslstained preparations, these commissural neurons would be distinguishable from large motoneurons, but it might be rather difficult to discriminate them from small motoneurons or neurons of the main sensory trigeminal nucleus and the parabrachial nuclei. In 7 of 12 cats subjected to stereotaxic operation, electrolytic lesions were produced successfully in the supratrigeminal region (Fig. lb). The survival periods after the operation were 60 to 84 h. In all cats, electrondense degenerated axon terminals were found within the confines of the motor trigeminal nucleus contralateral to the lesion (Fig. 4). Other features of possible degeneration of axon terminals, such as the increase of neuro-
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
259
5. Axon terminals within the motor trigeminal nucleus which were labeled FIG. with horseradish peroxidase injected contralaterally into the supratrigeminal region. Electron-dense peroxidase granules (arrows) are seen both in axon terminals filled with round synaptic vesicles (R) and in those containing flattened vesicles (F). Survival time, 40 h. a; X 27,000; b, X 103,500.
filaments or the enlargement of synaptic vesicles, were also seen. These degenerated axon terminals were observed most frequently on the dendritic profiles of medium size; no degenerated synaptic terminals were found on the somatic profiles. Of the total 46 electron-dense synaptic terminals so far found, 20 were filled with round synaptic vesicles and 16 contained flattened ones; in the remaining 10 the shapes of synaptic vesicles could not be discerned clearly. In three of seven cats injected with peroxidase and perfused for electron microscopic work, injection sites were almost confined within the motor trigeminal nucleus and the supra- and intertrigeminal regions. In neuropils within the motor trigeminal nucleus contralateral to the injection site, axon terminals labeled with peroxidase transported orthodromically could be observed; the enzyme in axon terminals was seen as electron-dense granules in metal-stained specimensas well as in unstained ones (Fig. 5). In accordance with the findings obtained from the degeneration study
260
MIZUNO
ET
AL.
described above, the peroxidase granules were found in axon terminals filled with round synaptic vesicles as well as in those containing flattened vesicles. DISCUSSION Commissural interneurons projecting contralaterally to masticatory motoneurons were suggested to exist in the supra- and intertrigeminal regions (1, 7, 11, 14, 19, 20). In the present study, after injection of horseradish peroxidase in the motor trigeminal nucleus, neurons labeled with peroxidase were found contralaterally not only in the supra- and intertrigeminal regions, but also in the lateral tegmental areas close to the medial border of the motor trigeminal nucleus; these regions appear to be included in the area R described in the rabbit (IO) and cat (3), where various propriobulbar fibers were reported to terminate (3, 4, 11, 13, 15, 16, 21). In addition, small- or medium-size cells labeled with peroxidase were often seen within the confines of the motor and main sensory trigeminal nuclei and the parabrachial nuclei. However, even in the supra- and intertrigeminal regions, where neurons labeled with the enzyme were found most numerously, many neurons remained unlabeled. This may indicate that these regions contain many neurons other than the commissural interneurons for the motor trigeminal nucleus ’ [cf. ( 16, 17) 1. Interneurons for masticatory motoneurons in the supratrigeminal region were assumed electrophysiologically to be located within the supratrigeminal nucleus (1, 5, 7, 14), which was first described in the mouse (9) and later also in the cat (8, 11, 23) and monkey (11). However, the extent of the supratrigeminal region, where neurons labeled with peroxidase were observed in the present study, was much wider than that of the supratrigeminal nucleus. Kidokoro et al. (7) recorded inhibitory postsynaptic potentials from masseteric motoneurons of the cat monosynaptically after stimulation of the supratrigeminal region of the ipsilateral as well as the contralateral side. The inhibitory nature of commissural interneurons in the supratrigeminal region was also indicated by Goldberg and Nakamura ( 1) and Nakamura et al. (14). After placing the lesion in the supratrigeminal region, however, both axon terminals filled with round synaptic vesicles and those containing flattened vesicles were degenerated within the contralateral motor trigeminal nucleus. Because some axons of neurons in the intertrigeminal region were described as running through the supratrigeminal region (9, 19, ZO), lesions produced in the supratrigeminal region would damage not only neurons of the supratrigeminal area, but also those of the intertrigeminal region. Therefore, degenerated axon terminals found in the motor trigeminal nucleus after making the lesion
INTERNEURONS
FOR
MASTICATORY
MOTONEURONS
261
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