EXPERIMENTAL
NEUROLOGY
65, 99- 106 (1979)
Trigeminal Nerve and Temporomandibular of the Cat: A Horseradish Peroxidase JOHN H. ROMFH, Departments
of Anatomy
Received
NORMAN
F. CAPRA, AND GLENN
and Pharmacology, University of Mississippi Jackson, Mississippi 39216
September
18, 1978; revision
received
February
Joint Study B. GATIPON’ Medical
Center,
2, 1979
The mesencephalic nucleus of the trigeminal nerve is considered to contain the cell bodies of the first-order neurons that have peripheral connections in the temporomandibular joint capsule. Through use of retrograde transport of horseradish peroxidase, this concept is challenged. The results indicate that a relatively specific region of the mandibular division of the trigeminal ganglion of the cat contains the first-order neurons innervating the temporomandibular joint capsule.
INTRODUCTION The sensory nerves which innervate joint capsules mediate pain and proprioceptive impulses. These afferent fibers have their cell bodies within spinal ganglia. However, the temporomandibular joint is thought to be innervated by sensory neurons in the mesencephalic nucleus of the trigeminal nerve (2, 4, 20). The temporomandibular joint capsule contains both free nerve endings that may transmit pain impulses and modified Golgi-Mazzoni end-organs which serve as proprioceptive mechanoreceptors (1 l- 13). These endorgans are supplied by the deep temporal nerve, the nerve to the masseter muscle, and the auriculotemporal nerve (3, 5, 8, 9). The deep temporal nerve and nerve to the masseter, but not the auriculotemporal nerve, contain fibers from the mesencephalic nucleus (5). Abbreviations: HRP-horseradish peroxidase. ’ This project was supported in part by National Institutes of Health grant NS 14147, National Institute of Dental Research grant DE 03746, and BRSG grant 5 507 RR05386 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health. 99 0014-4886/79/070099-08$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
100
ROMFH,
CAPRA,
AND
GATIPON
In electrophysiological studies (12, 13) mesencephalic nucleus neurons did not respond to movement of a surgically isolated temporomandibular joint preparation. The present investigation attempted to locate the primary neurons innervating the temporomandibular joint, whether within the ganglion or mesencephalic nucleus of the trigeminal nerve, or both. To accomplish this, the horseradish peroxidase (HRP) technique was used. METHODS Eight young adult cats were used. Horseradish peroxidase (Sigma Type VI) dissolved in saline (2 mg HRP/SO ~1 saline) was injected into the temporomandibularjoint of each animal. Also four of eight animals received injections of HRP into the contralateral masseter and temporalis muscles. Ketamine (Ketaset, W. A. Butler, 50 mg/kg, i.m.) was used as an anesthetic agent in all experiments. Injection of HRP into the joint capsule was accomplished by one of two methods. The first involved mounting a Hamilton microliter syringe fitted with a 30-gauge needle into a David Kopf electrode carrier. Prior to each experiment, measurements for stereotaxic placement of the injection needle were made using a dried cat skull (Carolina Biological Supply) placed in the stereotaxic instrument. The electrode carrier holding the syringe was adjusted so that the needle could be advanced into the mandibular notch, and stereotaxic coordinates were recorded for injection of the medial, intermediate, and lateral aspects of the joint capsule. Then the skull was removed and an anesthetized animal was mounted in the stereotaxic device. To facilitate penetration of the injection needle, a small skin incision was made at the point of entry. By advancing the electrode carrier slowly, a successful insertion into the capsule was determined by a slight movement of the mandible. A total of 50 ~1 HRP solution was injected into the capsule. In the second method the temporomandibular joint and intact joint capsule were exposed surgically, thus allowing injection of HRP solution under direct visualization. Finally, to inject the contralateral masseter and temporalis muscles, a small skin incision was made parallel to the zygomatic bone to expose the muscles. They were injected at multiple loci with small amounts of HRP solution. The total amount of solution injected was 50 ~1. In six animals, injections of HRP were repeated 24 to 72 h after the initial one. Twenty-four hours later, these animals were anesthetized and killed by transcardiac perfusion with a fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (PH 7.3 to 7.4). The other two animals received only a single injection, One was killed
TEMPOROMANDIBULAR
JOINT
INNERVATION
101
24 h and the other 48 h after the injection by the same procedure as the preceding animals. Serial frozen sections (30 pm thick) were made of the trigeminal ganglia and that part of the brain stem extending from the level of the abducens nucleus caudally, to the posterior commissure rostrally. Both were processed for visualization of neurons containing HRP using procedure 8 of the blue reaction method as described by Mesulam (15). Briefly described, this technique utilizes benzidine as a chromagen instead of 3,3’-diaminobenzidine. Benzidine produces a blue granular reaction product which is easily visualized with conventional bright-field microscopy. For easy visualization of the entire section, each was counterstained with neutral red. RESULTS Under bright-field illumination, the distinctive blue granules in the perikaryon were readily identified. Neurons containing few or lightly stained granules were verified by dark field examination. The number of labeled cells varied from one experiment to the next and labeled neurons, although distinctive, could not be used for quantitative analysis. Many variables must be considered in evaluating the results of HRP studies. Of particular importance are variables encountered in tissue fixation which occasionally result in failure of labeled neurons to show enzyme activity (16). Age of the animal and method of injection of HRP must also be considered (7). CATS TMJ 1,2, and 3. Each of these cats received two unilateral injections into either the right or left temporomandibular joint according to the methods described. The results were qualitatively identical and will be considered together. Neurons containing prominent blue granules were observed in the caudal and lateral regions of the mandibular portion of the ipsilateral trigeminal ganglion. These cells constituted a heterogenous population 20 to 60 pm in diameter (Figs. 1, 2). There was no evidence of retrograde transport of HRP to the contralateral trigeminal ganglion or to any brain stem nuclei; including those known to contribute fibers to the trigeminal nerve. CATS TMJ 4, 5, and 8. These animals received two injections of HRP solution in the left temporomandibular joint and in the right masseter and temporalis muscles. After injections of the left temporomandibular joint, labeled neurons in the ipsilateral trigeminal ganglion were identified in the same approximate region as in the prior group of animals, i.e., the most caudal and lateral area (Figs. 1,2). Peroxidase reaction product was not found in cells of any brain stem nuclei on the left side. Injections of the temporalis and masseter
102
ROMFH, CAPRA, AND GATIPON
FIG. 1. A composite reconstruction of the left trigeminal ganglion of the cat. The solid squares (U) designate the general area of the ganglion in which the cell bodies were labeled by the injection of horseradish peroxidase into the ipsilateral masse& and temporalis muscles. The open circles (0) indicate that portion of the ganglion in which the cell bodies were labeled by injection of horseradish peroxidase into the ipsilateral temporomandibular joint. A-ophthalmic division, B-maxillary division, and C-mandibular division.
FIG. 2. Photomicrograph taken from a section of the trigeminal ganglion of the cat. This cluster of five labeled cell bodies resulted from injection of horseradish peroxidase into the temporomandibular joint. This group of neurons demonstrates the range of size and the intensity of the labeling by the horseradish peroxidase. Bar represents 25 pm.
TEMPOROMANDIBULAR
JOINT INNERVATION
103
FIG. 3. Dark-field photomicrograph taken from a section at the level of the trigeminal motor nucleus. This cluster of four labeled multipolar neurons resulted from injection of horseradish peroxidase into the ipsilateral temporalis or masseter muscles. Bar represents 25 pm.
muscles on the right resulted in peroxidase uptake by neurons in the trigeminal ganglion, the motor nucleus, and the mesencephalic nucleus. The right trigeminal ganglion contained labeled perikarya rostrally disposed in the mandibular portion of the ganglion (Fig. 1). Labeled cells were not found in the more caudal regions. Prominent labeling of multipolar neurons in the right motor nucleus of the trigeminal nerve (Fig. 3) and in the right mesencephalic nucleus of the trigeminal nerve was observed (Fig. 4). Labeled cells in the mesencephalic nucleus were found from isthmus to midinferior colliculus levels of the brain stem. TM./ 6. This animal was killed 24 h after unilateral injections of the right temporomandibular joint. The injections were not repeated. A few scattered perikarya were labeled in the ipsilateral trigeminal ganglion and none were detected in the brain stem. TMJ 7. Single injections of HRP solution in the right temporomandibular
104
ROMFH, CAPRA, AND GATIPON
FIG. 4. Photomicrograph of a cluster of neurons of the mesencephalic nucleus of the trigeminal nerve. The labeled perikaryon resulted from the injection of horseradish peroxidase into the temporalis or masseter muscles. Bar represents 25 pm.
joint and in the contralateral muscles of mastication were performed. After 48 h, the animal was anesthetized and killed by perfusion fixation and tissues were processed according to the methods outlined previously. Labeled neurons, indicating retrograde transport of HRP, were not found in any tissues examined. DISCUSSION
AND SUMMARY
In seven animals, injections of HRP solution into the temporomandibular joint labeled neurons in the ipsilateral trigeminal ganglion. These neurons are restricted to the caudolateral portion of the ganglion. In the cat this region of the ganglion is overlaid by a piece of the bony tentorium. Perhaps this bony shelf has hampered electrophysiological studies of this portion of the ganglion which appears related to innervation of the temporomandibular joint. Mesencephalic nucleus neurons are known to innervate muscles of mastication (1, 5, 6, 10, 14, 17-19). In the present study, injection of two
TEMPOROMANDIBULAR
105
JOINT INNERVATION
muscles of mastication labeled neurons of both the ipsilateral mesencephahc nucleus and ipsilateral motor nucleus of the trigeminal. In addition, the muscular injections labeled a few neurons of the ipsilateral trigeminal ganglion; these cells are more rostrally situated in the ganglion than those labeled by injection of the joint. Joint injections did not label any neurons of the mesencephalic nucleus of the trigeminal nerve. This finding is supported by the electrophysiological work of Kawamura and associates (11-13). They surgically isolated the temporomandibular joint in cats by partially dissecting the temporalis, masseter, and external (lateral) pterygoid muscles. These procedures were carefully carried out so as not to damage the surrounding nerves and blood vessels (13). Next they recorded from cells in the lower brain stem which responded to mechanical stimulation of the joint capsule or to hinge-like movement of the condyle (13). None were found in the region of the mesencephalic nucleus, rather they were located in the dorsal parts of the bulbar and spinal trigeminal sensory nuclei (13). The results obtained by Kawamura et al. are in agreement with the present study because HRP labels the first neuron in the circuit (7), in this case the trigeminal ganglion, whereas electrophysiological studies located the second-order neuron within the bulbar and spinal trigeminal sensory nuclei. These latter nuclei are known to have secondary fiber connections with the ganglion. Of interest are observations concerning the use of double injections of HRP and increased survival time. The present results indicate greater transport of HRP by loading selected areas of the adult peripheral nervous system. The animals that received a single injection demonstrated few, if any, labeled cells in either the ganglion or the brain stem. However one must not overlook the necessity of carefully limiting the injection area from surrounding tissues when using this type of modification. This study shows that the afferent fibers of the temporomandibular joint have their cell bodies within a relatively restricted and identifiable area of the trigeminal ganglion. This area is situated in the posteriolateral portion of the ganglion and is overlaid by a shelf of bone. The results of the present study offer no evidence that the temporomandibular joint received fibers from the mesencephalic nucleus. REFERENCES 1. ALVARADO-MALLART,
2. 3.
M.
R.,
C. BATINI,
C. BUISSERET-DELMAS,
AND
J. CORVISIER.
1975. Trigeminal representation of the masticatory and extraocular proprioceptors as revealed by horseradish peroxidase retrograde transport. Exp. Brain Res. 23: 167179. BARR, M. L. 1974. The Human Nervous System, 2nd ed. Harper & Row, New York. BERNICK, S. 1962. The vascular and nerve supply to the temporomandibular joint of the rat. Oral Surg. 15: 488-498.
ROMFH, CAPRA, AND GATIPON
106
M. B. 1976. Human Neuroanatomy ,7th ed. Williams & Wilkins, Baltimore. K. B. 1949. Observations on the peripheral distribution of fibers arising in the mesencephalic nucleus of the fifth cranial nerve. J. Comp. Neural. 73: 153-177. CORBIN, K. B., AND F. HARRISON. 1940. Function of mesencephalic root of fifth cranial nerve. J. Neurophysiol. 3: 423-435.
4. CARPENTER,
5. 6.
CORBIN,
7. COWAN, W. M., AND M. CUBNOD. Neuronal Connectivity. Elsevier,
1975.
The Use of Axonal
Transport
for
Studies
of
Amsterdam/Oxford/New York. 8. FRANKS, A. T. 1964. Studies on the innervations of the temporomandibular joint and lateral pterygoid muscle in animals. J. Dem. Res. 43: 947-948. 9. GREENFIELD, B. E., AND B. WYKE. 1966. Reflex innervation of the temporomandibular joint. Nature (London) 211: 940-941. 10. JERGE. C. R. 1963. Organization and function of the trigeminal mesencephalic nucleus. J. Neurophysiol.
11. 12. 13. 14.
15. 16.
17.
18. 19.
20.
26: 379-392.
Y. 1970. A role of atferents for mandibular and lingual movements. Pages 170-194 in J. F. BOSMA, Ed., Second Symposium on Oral Sensation and Perception. Thomas, Springfield. KAWAMURA, Y., AND T. MAJIMA. 1964. Temporomandibularjoint’s sensory mechanisms controlling activities of the jaw muscles. J. Dent. Res. 43: 150. KAWAMURA, Y., T. MAJIMA, AND I. KATO. 1967. Physiologic role of deep mechanoreceptor in temporomandibular joint capsule. J. Osaka Univ. Dent. Sch. 7: 63-76. MANNI, E., R. BORTOLAMI, V. E. PETTOROSSI, E. CALLEGARI, AND M. L. LUCCHI. 1977. Reflex and reticular modulation of first-order proprioceptive neurons of the mesencephalic trigeminal nucleus. Arch. &al. Biol. 115: 20-37. MESULAM, M.-M. 1976. The blue reaction product in horseradish peroxidase neurohistochemistry: Incubation parameters and visibility. J. Histochem. Cytochem. 24: 12731280. ROSEN, D. L., AND M.-M. MESULAM. 1978. Fixation variables in horseradish peroxidase neurohistochemistry. I. The effects of fixation time and perfusion procedures upon enzyme activity. J. Histochem. Cytochem. 26: 28-39. SMITH, R. D., H. Q. MARCARIAN, AND W. T. NIEMER. 1967. Bilateral relationships of the trigeminal mesencephalic nuclei and mastication. J. Comp. Neural. 131: 79-92. SMITH, R. D., H. Q. MARCARIAN, AND W. T. NIEMER. 1968. Direct projections from the masseteric nerve to the mesencephalic nucleus. J. Comp. Neurol. 133: 495-502. SZENTAGOTHAI, J. 1948. Anatomical considerations of monosynaptic reflex arc. J. Neurophysiol. 11: 445-454. WILLIAMS, P. L., AND R. WARWICK. 1975. Functional Neuroanatomy of Man. Saunders, Philadelphia. KAWAMURA,
NEUROLOGY
65, 99- 106 (1979)
Trigeminal Nerve and Temporomandibular of the Cat: A Horseradish Peroxidase JOHN H. ROMFH, Departments
of Anatomy
Received
NORMAN
F. CAPRA, AND GLENN
and Pharmacology, University of Mississippi Jackson, Mississippi 39216
September
18, 1978; revision
received
February
Joint Study B. GATIPON’ Medical
Center,
2, 1979
The mesencephalic nucleus of the trigeminal nerve is considered to contain the cell bodies of the first-order neurons that have peripheral connections in the temporomandibular joint capsule. Through use of retrograde transport of horseradish peroxidase, this concept is challenged. The results indicate that a relatively specific region of the mandibular division of the trigeminal ganglion of the cat contains the first-order neurons innervating the temporomandibular joint capsule.
INTRODUCTION The sensory nerves which innervate joint capsules mediate pain and proprioceptive impulses. These afferent fibers have their cell bodies within spinal ganglia. However, the temporomandibular joint is thought to be innervated by sensory neurons in the mesencephalic nucleus of the trigeminal nerve (2, 4, 20). The temporomandibular joint capsule contains both free nerve endings that may transmit pain impulses and modified Golgi-Mazzoni end-organs which serve as proprioceptive mechanoreceptors (1 l- 13). These endorgans are supplied by the deep temporal nerve, the nerve to the masseter muscle, and the auriculotemporal nerve (3, 5, 8, 9). The deep temporal nerve and nerve to the masseter, but not the auriculotemporal nerve, contain fibers from the mesencephalic nucleus (5). Abbreviations: HRP-horseradish peroxidase. ’ This project was supported in part by National Institutes of Health grant NS 14147, National Institute of Dental Research grant DE 03746, and BRSG grant 5 507 RR05386 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health. 99 0014-4886/79/070099-08$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
100
ROMFH,
CAPRA,
AND
GATIPON
In electrophysiological studies (12, 13) mesencephalic nucleus neurons did not respond to movement of a surgically isolated temporomandibular joint preparation. The present investigation attempted to locate the primary neurons innervating the temporomandibular joint, whether within the ganglion or mesencephalic nucleus of the trigeminal nerve, or both. To accomplish this, the horseradish peroxidase (HRP) technique was used. METHODS Eight young adult cats were used. Horseradish peroxidase (Sigma Type VI) dissolved in saline (2 mg HRP/SO ~1 saline) was injected into the temporomandibularjoint of each animal. Also four of eight animals received injections of HRP into the contralateral masseter and temporalis muscles. Ketamine (Ketaset, W. A. Butler, 50 mg/kg, i.m.) was used as an anesthetic agent in all experiments. Injection of HRP into the joint capsule was accomplished by one of two methods. The first involved mounting a Hamilton microliter syringe fitted with a 30-gauge needle into a David Kopf electrode carrier. Prior to each experiment, measurements for stereotaxic placement of the injection needle were made using a dried cat skull (Carolina Biological Supply) placed in the stereotaxic instrument. The electrode carrier holding the syringe was adjusted so that the needle could be advanced into the mandibular notch, and stereotaxic coordinates were recorded for injection of the medial, intermediate, and lateral aspects of the joint capsule. Then the skull was removed and an anesthetized animal was mounted in the stereotaxic device. To facilitate penetration of the injection needle, a small skin incision was made at the point of entry. By advancing the electrode carrier slowly, a successful insertion into the capsule was determined by a slight movement of the mandible. A total of 50 ~1 HRP solution was injected into the capsule. In the second method the temporomandibular joint and intact joint capsule were exposed surgically, thus allowing injection of HRP solution under direct visualization. Finally, to inject the contralateral masseter and temporalis muscles, a small skin incision was made parallel to the zygomatic bone to expose the muscles. They were injected at multiple loci with small amounts of HRP solution. The total amount of solution injected was 50 ~1. In six animals, injections of HRP were repeated 24 to 72 h after the initial one. Twenty-four hours later, these animals were anesthetized and killed by transcardiac perfusion with a fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (PH 7.3 to 7.4). The other two animals received only a single injection, One was killed
TEMPOROMANDIBULAR
JOINT
INNERVATION
101
24 h and the other 48 h after the injection by the same procedure as the preceding animals. Serial frozen sections (30 pm thick) were made of the trigeminal ganglia and that part of the brain stem extending from the level of the abducens nucleus caudally, to the posterior commissure rostrally. Both were processed for visualization of neurons containing HRP using procedure 8 of the blue reaction method as described by Mesulam (15). Briefly described, this technique utilizes benzidine as a chromagen instead of 3,3’-diaminobenzidine. Benzidine produces a blue granular reaction product which is easily visualized with conventional bright-field microscopy. For easy visualization of the entire section, each was counterstained with neutral red. RESULTS Under bright-field illumination, the distinctive blue granules in the perikaryon were readily identified. Neurons containing few or lightly stained granules were verified by dark field examination. The number of labeled cells varied from one experiment to the next and labeled neurons, although distinctive, could not be used for quantitative analysis. Many variables must be considered in evaluating the results of HRP studies. Of particular importance are variables encountered in tissue fixation which occasionally result in failure of labeled neurons to show enzyme activity (16). Age of the animal and method of injection of HRP must also be considered (7). CATS TMJ 1,2, and 3. Each of these cats received two unilateral injections into either the right or left temporomandibular joint according to the methods described. The results were qualitatively identical and will be considered together. Neurons containing prominent blue granules were observed in the caudal and lateral regions of the mandibular portion of the ipsilateral trigeminal ganglion. These cells constituted a heterogenous population 20 to 60 pm in diameter (Figs. 1, 2). There was no evidence of retrograde transport of HRP to the contralateral trigeminal ganglion or to any brain stem nuclei; including those known to contribute fibers to the trigeminal nerve. CATS TMJ 4, 5, and 8. These animals received two injections of HRP solution in the left temporomandibular joint and in the right masseter and temporalis muscles. After injections of the left temporomandibular joint, labeled neurons in the ipsilateral trigeminal ganglion were identified in the same approximate region as in the prior group of animals, i.e., the most caudal and lateral area (Figs. 1,2). Peroxidase reaction product was not found in cells of any brain stem nuclei on the left side. Injections of the temporalis and masseter
102
ROMFH, CAPRA, AND GATIPON
FIG. 1. A composite reconstruction of the left trigeminal ganglion of the cat. The solid squares (U) designate the general area of the ganglion in which the cell bodies were labeled by the injection of horseradish peroxidase into the ipsilateral masse& and temporalis muscles. The open circles (0) indicate that portion of the ganglion in which the cell bodies were labeled by injection of horseradish peroxidase into the ipsilateral temporomandibular joint. A-ophthalmic division, B-maxillary division, and C-mandibular division.
FIG. 2. Photomicrograph taken from a section of the trigeminal ganglion of the cat. This cluster of five labeled cell bodies resulted from injection of horseradish peroxidase into the temporomandibular joint. This group of neurons demonstrates the range of size and the intensity of the labeling by the horseradish peroxidase. Bar represents 25 pm.
TEMPOROMANDIBULAR
JOINT INNERVATION
103
FIG. 3. Dark-field photomicrograph taken from a section at the level of the trigeminal motor nucleus. This cluster of four labeled multipolar neurons resulted from injection of horseradish peroxidase into the ipsilateral temporalis or masseter muscles. Bar represents 25 pm.
muscles on the right resulted in peroxidase uptake by neurons in the trigeminal ganglion, the motor nucleus, and the mesencephalic nucleus. The right trigeminal ganglion contained labeled perikarya rostrally disposed in the mandibular portion of the ganglion (Fig. 1). Labeled cells were not found in the more caudal regions. Prominent labeling of multipolar neurons in the right motor nucleus of the trigeminal nerve (Fig. 3) and in the right mesencephalic nucleus of the trigeminal nerve was observed (Fig. 4). Labeled cells in the mesencephalic nucleus were found from isthmus to midinferior colliculus levels of the brain stem. TM./ 6. This animal was killed 24 h after unilateral injections of the right temporomandibular joint. The injections were not repeated. A few scattered perikarya were labeled in the ipsilateral trigeminal ganglion and none were detected in the brain stem. TMJ 7. Single injections of HRP solution in the right temporomandibular
104
ROMFH, CAPRA, AND GATIPON
FIG. 4. Photomicrograph of a cluster of neurons of the mesencephalic nucleus of the trigeminal nerve. The labeled perikaryon resulted from the injection of horseradish peroxidase into the temporalis or masseter muscles. Bar represents 25 pm.
joint and in the contralateral muscles of mastication were performed. After 48 h, the animal was anesthetized and killed by perfusion fixation and tissues were processed according to the methods outlined previously. Labeled neurons, indicating retrograde transport of HRP, were not found in any tissues examined. DISCUSSION
AND SUMMARY
In seven animals, injections of HRP solution into the temporomandibular joint labeled neurons in the ipsilateral trigeminal ganglion. These neurons are restricted to the caudolateral portion of the ganglion. In the cat this region of the ganglion is overlaid by a piece of the bony tentorium. Perhaps this bony shelf has hampered electrophysiological studies of this portion of the ganglion which appears related to innervation of the temporomandibular joint. Mesencephalic nucleus neurons are known to innervate muscles of mastication (1, 5, 6, 10, 14, 17-19). In the present study, injection of two
TEMPOROMANDIBULAR
105
JOINT INNERVATION
muscles of mastication labeled neurons of both the ipsilateral mesencephahc nucleus and ipsilateral motor nucleus of the trigeminal. In addition, the muscular injections labeled a few neurons of the ipsilateral trigeminal ganglion; these cells are more rostrally situated in the ganglion than those labeled by injection of the joint. Joint injections did not label any neurons of the mesencephalic nucleus of the trigeminal nerve. This finding is supported by the electrophysiological work of Kawamura and associates (11-13). They surgically isolated the temporomandibular joint in cats by partially dissecting the temporalis, masseter, and external (lateral) pterygoid muscles. These procedures were carefully carried out so as not to damage the surrounding nerves and blood vessels (13). Next they recorded from cells in the lower brain stem which responded to mechanical stimulation of the joint capsule or to hinge-like movement of the condyle (13). None were found in the region of the mesencephalic nucleus, rather they were located in the dorsal parts of the bulbar and spinal trigeminal sensory nuclei (13). The results obtained by Kawamura et al. are in agreement with the present study because HRP labels the first neuron in the circuit (7), in this case the trigeminal ganglion, whereas electrophysiological studies located the second-order neuron within the bulbar and spinal trigeminal sensory nuclei. These latter nuclei are known to have secondary fiber connections with the ganglion. Of interest are observations concerning the use of double injections of HRP and increased survival time. The present results indicate greater transport of HRP by loading selected areas of the adult peripheral nervous system. The animals that received a single injection demonstrated few, if any, labeled cells in either the ganglion or the brain stem. However one must not overlook the necessity of carefully limiting the injection area from surrounding tissues when using this type of modification. This study shows that the afferent fibers of the temporomandibular joint have their cell bodies within a relatively restricted and identifiable area of the trigeminal ganglion. This area is situated in the posteriolateral portion of the ganglion and is overlaid by a shelf of bone. The results of the present study offer no evidence that the temporomandibular joint received fibers from the mesencephalic nucleus. REFERENCES 1. ALVARADO-MALLART,
2. 3.
M.
R.,
C. BATINI,
C. BUISSERET-DELMAS,
AND
J. CORVISIER.
1975. Trigeminal representation of the masticatory and extraocular proprioceptors as revealed by horseradish peroxidase retrograde transport. Exp. Brain Res. 23: 167179. BARR, M. L. 1974. The Human Nervous System, 2nd ed. Harper & Row, New York. BERNICK, S. 1962. The vascular and nerve supply to the temporomandibular joint of the rat. Oral Surg. 15: 488-498.
ROMFH, CAPRA, AND GATIPON
106
M. B. 1976. Human Neuroanatomy ,7th ed. Williams & Wilkins, Baltimore. K. B. 1949. Observations on the peripheral distribution of fibers arising in the mesencephalic nucleus of the fifth cranial nerve. J. Comp. Neural. 73: 153-177. CORBIN, K. B., AND F. HARRISON. 1940. Function of mesencephalic root of fifth cranial nerve. J. Neurophysiol. 3: 423-435.
4. CARPENTER,
5. 6.
CORBIN,
7. COWAN, W. M., AND M. CUBNOD. Neuronal Connectivity. Elsevier,
1975.
The Use of Axonal
Transport
for
Studies
of
Amsterdam/Oxford/New York. 8. FRANKS, A. T. 1964. Studies on the innervations of the temporomandibular joint and lateral pterygoid muscle in animals. J. Dem. Res. 43: 947-948. 9. GREENFIELD, B. E., AND B. WYKE. 1966. Reflex innervation of the temporomandibular joint. Nature (London) 211: 940-941. 10. JERGE. C. R. 1963. Organization and function of the trigeminal mesencephalic nucleus. J. Neurophysiol.
11. 12. 13. 14.
15. 16.
17.
18. 19.
20.
26: 379-392.
Y. 1970. A role of atferents for mandibular and lingual movements. Pages 170-194 in J. F. BOSMA, Ed., Second Symposium on Oral Sensation and Perception. Thomas, Springfield. KAWAMURA, Y., AND T. MAJIMA. 1964. Temporomandibularjoint’s sensory mechanisms controlling activities of the jaw muscles. J. Dent. Res. 43: 150. KAWAMURA, Y., T. MAJIMA, AND I. KATO. 1967. Physiologic role of deep mechanoreceptor in temporomandibular joint capsule. J. Osaka Univ. Dent. Sch. 7: 63-76. MANNI, E., R. BORTOLAMI, V. E. PETTOROSSI, E. CALLEGARI, AND M. L. LUCCHI. 1977. Reflex and reticular modulation of first-order proprioceptive neurons of the mesencephalic trigeminal nucleus. Arch. &al. Biol. 115: 20-37. MESULAM, M.-M. 1976. The blue reaction product in horseradish peroxidase neurohistochemistry: Incubation parameters and visibility. J. Histochem. Cytochem. 24: 12731280. ROSEN, D. L., AND M.-M. MESULAM. 1978. Fixation variables in horseradish peroxidase neurohistochemistry. I. The effects of fixation time and perfusion procedures upon enzyme activity. J. Histochem. Cytochem. 26: 28-39. SMITH, R. D., H. Q. MARCARIAN, AND W. T. NIEMER. 1967. Bilateral relationships of the trigeminal mesencephalic nuclei and mastication. J. Comp. Neural. 131: 79-92. SMITH, R. D., H. Q. MARCARIAN, AND W. T. NIEMER. 1968. Direct projections from the masseteric nerve to the mesencephalic nucleus. J. Comp. Neurol. 133: 495-502. SZENTAGOTHAI, J. 1948. Anatomical considerations of monosynaptic reflex arc. J. Neurophysiol. 11: 445-454. WILLIAMS, P. L., AND R. WARWICK. 1975. Functional Neuroanatomy of Man. Saunders, Philadelphia. KAWAMURA,
