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Brain stem afferents of hypoglossal neurons in the rat
Brain Research, 269 (1983) 4 ~ 5 5
47
Elsevier
Brain Stem Afferents of Hypoglossal Neurons in the Rat*
ROSEMARY C. BORKE, MARTIN E. NAU and ROBERT L. RINGLER, Jr.
Department of A natomy, UniformedServices University, 4301 Jones Bridge Road, Bethesda, MD 20814 (U.S.A. (Accepted November 16th, 1982)
Key words: hypoglossal neurons - afferents to hypoglossal neurons - horseradish peroxidase - retrograde tracing
The origin of afferent connections of the hypoglossal nucleus in rats was investigated using horseradish peroxidase (HRP) as a retrograde tracer. Pressure injections (0.15-0.17/~l) of 15% HRP were introduced into the rostral, middle and caudal portions of the nucleus. Projections to the hypoglossal nucleus originated from 3 regions of the brainstem: the reticular formation, the spinal V complex and the nucleus of the solitary tract. Bilateral projections with ipsilateral predominance came from the lateral reticular formation: the dorsal aspect of the nucleus reticularis parvocellularis and its caudal continuation, the nucleus reticularis dorsalis. Fewer projections emerged from two nuclei of the medial reticular formation. The dorsal part of the nucleus reticularis ventralis at the spinomedullary junction contributed bilateral with mainly contralateral input to hypoglossal neurons. A few labeled neurons were situated bilaterally in the nucleus reticularis gigantocellularis of the rostral medulla. The input from the spinal V complex originated from the dorsal aspect along most of its length but particularly from the pars interpolaris and oralis subdivisions. Labeled neurons were located primarily in the posterior portion of the nucleus of the solitary tract. Projections from the spinal V complex and the solitary nucleus exhibited ipsilateral predominance. These results suggest that somatic and visceral centers of the rat brainstem play an important role in the control of the activity ofhypoglossal motoneurons. INTRODUCTION
The tongue is a muscular organ that assists in mastication, deglutition, and vocalization. The arrangement of the lingual muscles and the reflex mechanisms of the tongue account for the highly organized and controlled movements of the tongue during these actions. While numerous cranial nerves play an important part in the sensory feedback regulation of tongue movements, the hypoglossal nerve alone provides motor innervation to the muscle mass. Topographical representation of the main branches of the hypoglossal nerve and the lingual muscles within this cranial nerve motor nucleus has been determined in rats by recent experiments using retrograde tracers 15 and cytoarchitectonic methods 24. Although sources of input to the hypoglossal neurons in rats have only been summarized in preliminary reports 2.5~9, afferent mechanisms involved in hypoglossal re-
flexes have been the subject of numerous physiological studies 12,17,22,28,29~34,41.Since physiological data have implicated certain regions of the brain stem as providing input to the hypoglossal nucleus, anatomical localization of their precise origin is necessary for a complete understanding. The object of this study was to identify brainstem afferents to the hypoglossal nucleus and to interpret their significance. MATERIALS AND METHODS
Eighteen adult rats of the Osborn-Mendel strain were used for this investigation. The rats were anesthetized with sodium pentobarbital (30 mg/kg) and small amounts (0.15 0.17 ~1) of 15% H R P were injected into the hypoglossal nucleus stereotaxically using micropipettes with a tip diameter of 20-30/~m. The majority of the injections were introduced into the rostral portion of the hypoglossal nucleus to avoid involvement
* A preliminary report of this work was presented at the American Association of Anatomists, 95th Annual Session at Indianapolis, IN, April 4-8, 1982. 0006-8993/83/$03.00 ~,'~1983 Elsevier Science Publishers B.V.
48
of the dorsal motor nucleus of the vagus (DMX). Injections were limited to the hypoglossal nucleus in 10 animals. In 5 other animals, the injection spread included the hypoglossal nucleus, DMX and varying portions of the adjacent reticular formation. After survival times of 2472 h, rats were anesthetized with sodium pentobarbital and killed by intracardiac perfusion with Ringer’s solution followed by dilute and concentrated aldehydego. The brains were removed, postfixed in the dilute aldehyde mixture for 2 h, cut into blocks and immersed overnight in a 30% sucrose-buffer solution. Frozen serial sections of the entire brain and the cervical spinal cord were cut at 30 pm in a transverse plane. Alternate tissue sections were processed according to the method of LaVaill or Mesulam19 using 3,3-diaminobenzidine (DAB) and tetramethylbenzidine (TMB) as chromagens. The reacted sections were mounted on chrome-alum gelatinized slides and lightly counterstained with cresyl violet or Neutral red. The location of the injection site was confirmed and serial sections through the brain and cervical spinal cord were examined using brightand dark-field illumination. The distribution of labeled neurons was mapped on sequential tracings through the brain and spinal cord. RESULTS
Separate injections of HRP into the rostral, middle, and caudal portions of the hypoglossal nucleus, in general furnished the same information about the sources of afferent connections of this cranial nerve nucleus. However, those injections into the rostra1 portion of the hypoglossal nucleus were accomplished without involvement of DMX. The locations of labeled neurons in the brain stem after these injections were remarkably consistent and varied for individual animals only in the number of retrogradely labeled neurons. In all cases, the XIIth nerve contained anterograde label. The sources of input to hypoglossal neurons can be conveniently presented in a single synthetic description of the results of a case in which HRP was introduced into
the rostra1 portion of the hypoglossal nucleus 1 mm above the level of the obex. The micropipette traversed the dorsal half of the nucleus and spread of the exogenous protein was limited to the boundaries of the ipsilateral hypoglossal nucleus(Fig. lC,). The greatest number of labeled neurons was identified in the lateral reticular formation of the medulla and pons, specifically the nucleus reticularis parvocellularis (RPc) (Fig. IA-D). This projection was bilateral with ipsilateral predominance. Along most of the length of the hypoglossal nucleus, labeled neurons with fusiform and triangular-shaped somata (Fig. 2A) occupied the dorsal aspect of RPc. Fewer labeled cells were located in the intermediolateral portion of RPc. The labeled neurons decreased in number and became continuous with those cells labeled in nucleus reticularis dorsalis (RD) at caudal medullary levels (Fig. 1E). Rostra1 to the hypoglossal nucleus, labeled neurons were situated bilaterally in the oral pole of RPc (Figs. lA, B and 2B). Labeled neurons also were identified in 2 cell groups of the medial reticular formation: (1) the nucleus reticularis gigantocellularis (RGc); and (2) its caudal continuation, the nucleus reticularis ventralis (RV) (Fig. 1BF). Medium- and large-sized neurons with long dendritic processes were labeled in RGc (Fig. 2C). Cell labeling in RGc and RV at levels adjacent to the hypoglossal nucleus was bilateral (Fig. IC-E). At the spinomedullary junction, these connections originated primarily from the contralateral RV (Fig. 1F). Axons of these labeled neurons coursed medially rostra1 to the pyramidal decussation, and entered the ventral aspect of the hypoglossal nucleus in caudal regions of the medulla. Another source of input to hypoglossal neurons was from the spinal V complex. A longitudinal column of labeled neurons extended from the pars interpolaris subdivision at medullary levels to the principal sensory nucleus of V(PrSV) at pontine levels. Labeling in the spinal V nucleus was confined to neurons in the dorsal part of the nucleus (Fig. IA-E). The majority of input from the spinal trigeminal nucleus originated from medium sized neurons in the pars
49
HRP-40
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(~)
RGc
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I
"
°
B RV
E
o
/I
i'/\"~o
C
C1 Fig. !. Serial reconstruction of labeled neurons in the brainstem after injection of HRP into the rostral hypoglossal nucleus, q is a dark-field micrograph of the injection site and spread of the enzyme.
50
Fig. 2. A: labeled s0mata and processes of neurons in the ipsilateral nucleus reticularis parvocellularis adjacent to the rostral portion of the hypoglossal nucleus, x 160. B: HRP-positive neuronal cell bodies in the ipsilateral nucleus reticularis parvocellularis above the level of the hypoglossal nucleus, x 320. C: HRP reaction product filling a neuron located in the ipsilateral nucleus reticularis gigantocellularis, x 320. D: 3 labeled neurons situated in the dorsal aspect of the ipsilateral spinal V complex, pars oralis. × 320. E: HRP-positive neuron in the ipsilateral spinal V complex, pars interpolaris. The axon (arrow) projects medially through the dorsolateral reticular formation toward the hypoglossal nucleus, x 320. F: labeled neurons (arrows) in the posterior portion of the ipsilateral solitary nucleus adjacent to the hypoglossal nucleus. X 320.
51 oralis (SpVO) (Fig. 2D) and interpolaris (SpVI) (Fig. 2E) subdivisions (Fig. 1B-D). The projections from the trigeminal complex were predominantly ipsilateral. Neurons labeled in the nucleus of the solitary tract (SN) were limited to medullary levels that coincided with cells in the XIIth nucleus (Fig. 1C-E). Somata of these neurons were spindleshaped and their axons entered the dorsolateral angle of the hypoglossal nucleus (Fig. 2F). Only occasional labeled cells were identified in the ipsilateral D M X at the same medullary levels (Fig. 1C, D). No labeled neurons were identified in more rostral brainstem regions or in any cortical area. H R P injections into the middle and caudal regions of the hypoglossal nucleus invariably produced spread of the tracer into DMX. Retrograde neuronal labeling included the same areas identified by rostral hypoglossal injections, but with additional labeling of cells in PrSV, SN and the nuclei of the medullary reticular formation. Cells also were labeled in certain rostral areas of the brainstem, namely, the nucleus reticularis pontis oralis, the central gray region and the paraventricular nucleus of the hypothalamus. Injections of HRP into middle levels of the XIIth nucleus, DMX, the adjacent reticular formation and portions of the medial longitudinal fasciculus (Fig. 3D~) produced the same results as those experiments previously described with 3 additional loci of labeled neurons: neurons in the intermediate and ventral portions of the reticular formation of the pons and medulla (Fig. 3A-F), neurons in the intermediate and ventral parts of SpVO and PrSV (Fig. 3A, B), and the vestibular nuclei, particularly the medial and inferior cell groups (Fig. 3B). DISCUSSION
Four sources of hypoglossal input have been identified by these retrograde tracer experi•ments (Fig. 4): portions of the medial and lateral reticular formation, the spinal V nucleus and the nucleus of the solitary tract. Previous results from anterograde studies in cats established the existence of a projection from the lateral reticu-
lar formation (LRF) and the adjoining spinal V nucleus to the ipsilateral hypoglossal nucleus m,ll, while bilateral terminations from these same sources were identified in rats 27. Isotope diffusion at the injection site prevented either of these studies from determining whether the LRF, the spinal V nucleus or both nuclei projected to hypoglossal neurons. The present retrograde tracer work suggests that both nuclei provide separate input to hypoglossal motoneurons, but LRF provides the primary source of XIIth nucleus afferents. The origin of the L R F projections was localized primarily to the dorsal and intermediate parts of RPc along its length but particularly from neurons adjacent to the rostro-caudal extent of the XIIth nucleus. Prior Golgi studies supported the likelihood that axons from the dorsolateral reticular formation terminate on hypoglossal dendrites outside the borders of this cranial nerve nucleus 7,8. Limitation of the injection site to the hypoglossal nucleus in many animals in the present experiments provides evidence that a substantial population of neurons in RPc end on postsynaptic sites within the hypoglossal nucleus. Neurophysiologica117,21,33 and anatomical data 6,38,39 indicate that RPc receives afferents from secondary sources of hypoglossal input identified in the present work: the spinal V nucleus and the solitary nucleus. RPc therefore functions as a relay station to the hypoglossal nucleus from dual origins: cortical 43 and peripheral 36 sources which lack direct connections with the XIIth nucleus and somatic and visceral centers of the brainstem that also project directly to hypoglossal neurons. RPc may act as the site for the convergence and integration of hypoglossal reflexes from spatially separated sources for complex movements of the tongue associated with somatic and visceral afferents. In the present work, the small n u m b e r of labeled cells in the medial reticular formation (MRF) coincides with the failure of anterograde studies in cats to disclose significant projections from M R F to motoneurons of cranial nerves m,ll. A short report failed to specify the M R F nuclei giving rise to hypoglossal afferents in rats 9. The present results of labeled cells in the dorsolateral
52
HRP-25
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\-o F
Fig. 3. Serial reconstruction of sites of labeled neurons in the brainstem after injection of H RP into the middle level of the hypoglossal nucleus DMX, adjacent reticular formation and the medial longitudinal fasciculus. D] is a dark-field micrograph of the injection site and spread of enzyme.
53 aspect of RGc and RV agree with and further specify findings in cats that the dorsal reticular formation projected to the XIIth nucleus 3,26 but the ventral part did not 1,3. Since Golgi studies have shown axon collaterals emerging from the main axons of neurons in the reticular formation 32'37, sparse labeling of some neurons in RGc suggests that collaterals from the main axons may be involved in the hypoglossal projection from RGc. The present study is the first to demonstrate that neuronal connections to the hypoglossal nucleus originate only from the dorsal aspect of the spinal V nucleus and therefore appear topographically related to the mandibular division of the trigeminal nerve 14. These findings provide an anatomical basis for physiological reports suggesting a disynaptic pathway to the hypoglossal nucleus from various branches of the mandibular nerve 13,22,28.35,42.These observations also provide a more precise localization of the source of spinal V connections to the XIIth nucleus. The principal sensory nucleus of V has been suggested as the site of secondary neurons in the disynaptic pathway from the mandibular nerve to the hypoglossal nucleus 28. According to the present data, cells in the dorsal parts of inter-
polaris and oralis, which receive innervation from perioral and intraoral structures4°, provide the major projections from the spinal V nucleus to hypoglossal neurons. Results also indicate that axons of these neurons terminate within the hypoglossal nucleus, rather than on hypoglossal dendrites in the reticular formation, as has been previously suggested 28. Previous anterograde studies on whether the solitary nucleus sends direct projections to the hypoglossal nucleus have been hampered by contamination problems involving isotope spread to RPc 4,23. The present finding of labeled cells in the solitary nucleus, limited to medullary levels of the XIIth nucleus, coincides primarily with the posterior solitary nucleus. Therefore, direct connections from the solitary nucleus may be associated primarily with central mechanisms involved in general visceral afferents rather than the gustatory-hypoglossal reflex. Anatomical 23 and physiological41 data indicate that multisynaptic connections between the anterior solitary nucleus and the hypoglossal nucleus are involved in the gustatory-hypoglossal reflex. Our results failed to disclose significant retrograde transport of H R P to cells in the brainstem region rostral to the pons after H R P injections
S,in., V Dorsal Portion Intorpoiarls Oralis
Hypoglossal Nucleus
I Lateral Reticular Formation
(RPc& RD) Medial Reticular Formation
(RGc & RV)
Fig. 4. Summary diagram of brainstem afferents of hypoglossal neurons in rats. Broad arrow indicates primary source of hypoglossal input.
54 confined to the hypoglossal nucleus. Although direct cortical connections to hypoglossal nuclei have not been demonstrated in rats39, midbrain regions including the interstitial nucleus of Caja125, the periaqueductal gray25, and the mesencephalic nucleus of V ~8,2° have been considered to supply diffuse input to the hypoglossal nucleus. Our failure to obtain appreciable label of neurons in any of these regions after injections confined to the hypoglossal nucleus suggested that axonal endings from midbrain regions, if present, may terminate mainly on distal hypoglossal dendrites situated outside the nuclear boundaries. The spread of HRP injection into the dorsal and lateral medullary reticular formation in the experiments of Panneton and Martin25 and the absence of synaptic potentials in hypoglossal nucleus after midbrain stimulation35 support this possibility. Certain rostral brainstem regions did contain retrogradely labeled neurons if the spread of the injection included the overlying cells of DMX.
These findings concur with those of others in which connections to DMX originated from the paraventricular nucleus of the hypothalamus, PrSV, reticular formation nuclei and the solitary nucleus31. Large injections involving the reticular formation and portions of the medial longitudinal fasciculus as as well as the hypoglossal and vagal motor nuclei served as a cumulative control in that inputs to the hypoglossal nucleus, DMX, and connections of the reticular formation and the medial longitudinal fasciculus were demonstrated.
REFERENCES
11 Holstege, G., Kuypers, H. G. J. M. and Decker, J. J., The organization of the bulbar fibre connections to the trigeminal facial and hypoglossal motor nuclei. II. An autoradiographic tracing study in the cat, Brain, 100 (1977) 265-286. 12 Hunter, I. W. and Porter, R., Glossopharyngeal influences on hypoglossal motoneurons in the cat, Brain Research, 74 (1974) 161-166. 13 Kaiga, Y., Linguo-hypoglossalreflex elicited by mechanical stimulation in rabbits, J. Osaka Odont. Soc., 43 (1980) 449-460. 14 Kerr, F. W. L., The divisional organization of afferent fibers of the trigeminal nerve, Brain, 86 (1963) 721-732. 15 Krammer, E. B., Rath, T. and Lischka, M. F., Somatopic organization of the hypoglossal nucleus: a HRP study in the rat, Brain Research, 170 (1979) 533-537. 16 LaVail, J. H., The retrograde transport method, Fed. Proc., 34(1975) 1618-1624. 17 Lowe, A. A., Excitatory and inhibitory inputs to hypoglossal motoneurons and adjacent reticular formation neurons in cats, Exp. Neurol., 62 (1978) 3(~ 47. 18 Matesz, C., Peripheral and central distribution of fibres of the mesencephalic trigeminal root in the cat, Neurosci. Lett., 27 (1981) 13-17. 19 Mesulam, M.-M., Tetramethylbenzidine horseradish peroxidase neurohistochemistry. A non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 10~ 117. 20 Mizuno, N. and Sauerland, E. K., Trigeminal proprioceptive projections to the hypoglossal nucleus and the cervical ventral gray column, J. comp. Neurol., 139 (1970) 215-226.
1 Abols, I. A. and Basbaum, A. I., Afferent connections of the rostral medulla of the cat: a neural substrate for midbrain medullary interactions in the modulation of pain, J. comp. Neurol., 297 (1981) 285-295. 2 Aides, L. D., Afferent projections to the hypoglossal nuclei in the rat and cat, A nat. Rec., 196 (1980) 7A. 3 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. comp. Neurol., 178 (1978) 209-224. 4 Beckstead, R. M., Morse, J. R. and Norgren, R., The nucleus of the solitary tract in the monkey: projections to the thalamus and brain stem nuclei, J. comp. NeuroL, 190 (1980) 259-282. 5 Borke, R. and Nau, M., Afferent connections of the rat hypoglossal nucleus, A nat. Rec., 202 (1982) 18A- 19A. 6 Brodal, A., NeurologicalAnatomy in Relation to Clinical Medicine, 3rd Ed., Oxford University Press, New York, 1981, 1053 pp. 7 Ramon y Cajal, S., Histologie du Systbme Nerveux de l'Homme et des Vertebras, Vol. 1, Azoulay, L. (Trans), Paris, Maloine, 1909, pp. 70~712. 8 Cooper, M. H., Neurons of the hypoglossal nucleus of the rat, Otolaryng. HeadNeck Surg., 89 (1981) 10-15. 9 Cooper, M. H. and Fritz, H. P., Input of rat hypoglossal nucleus from the reticular formation, Anat. Rec., 199 (1981) 58A. 10 Holstege, G. and Kuypers, H. G. J. M., Propriobulbar fibre connections to the trigeminal, facial and hypoglossal motor nuclei. I. An anterograde degeneration study in the cat, Brain, 100 (1977) 239-264.
ACKNOWLEDGEMENTS
The author is grateful to Dr. Malcolm Carpenter for his thoughtful suggestions and comments. Thanks are given to Lillian Magruder for careful typing of the manuscript. This work was supported by the Department of Defense, USUHS, Department of Defense Grant C07019.
55 21 Morimoto, T., Kato, I. and Kawamura, L., Studies on functional organization of the hypoglossal nucleus, J. Osaka Univ. Dent. Sch., 6 (1966)75-87. 22 Morimoto, T., Takata, M. and Kawamura, Y., Effect of lingual nerve stimulation on hypoglossal motoneurons, Exp. Neurol., 22 (1968) 174-190. 23 Norgren, R., Projections from the nucleus of the solitary tract in the rat, Neuroscience, 3 (1978) 207-218. 24 Odutola, A. B., Cell grouping and Golgi architecture of the hypoglossal nucleus of the rat, Exp. Neurol., 52 (1976) 356-371. 25 Panneton, W. M. and Martin, G. F., Midbrain projections to the trigeminal facial and hypoglossal nuclei in the opossum. A study using axonal transport techniques, Brain Research, 168 (1979) 493-512. 26 Petras, J. M., Cortical, tectal and tegmental fiber connections in the spinal cord of the cat, Brain Research, 6 (1967) 275-324. 27 Petrovicky, P., The reticulo-raphe connection and experimental study using the method of anterograde degeneration and the method of retrograde horseradish peroxidase transport tracing in the rat, J. Hirnforsch., 22 (1981) 32-45. 28 Porter, R., The synaptic basis of a bilateral lingual-hypoglossal reflex in cat, J. PhysioL (Lond.), 190 (1967) 611-627. 29 Porter, R., Cortical actions on hypoglossal motoneurones in cats: a proposed role for a common internuncial cell, J. Physiol. (Lond.), 193 (1967) 295-308. 30 Reese, T. S. and Karnovsky, M. J., Fine structural localization of a blood brain barrier to exogenous peroxidase, J. Cell Biol., 34 (1967) 20%217. 31 Rogers, R. C., Kita, H., Butcher, L. and Dovin, D., Afferent projections to the dorsal motor nucleus of the vagus, Brain Res. Bull., 15 (1980) 365-373. 32 Scheibel, M. E. and Scheibel, A. B., Structural substrates for integrative patterns in the brain stem reticular core. In H. H. Jasper and L. D. Proctor (Eds.), Reticular Formation of the Brain, Little Brown, Boston, 1958, pp. 3155.
33 Sessle, B. J., Excitatory and inhibitory inputs to single neurons in solitary tract nucleus and adjacent reticular formation, Brain Research, 53(1973) 319- 331. 34 Sumi, T., Synaptic potentials of hypoglossal motoneurons and their relation to reflex deglutition, Jap. J. Physiol., 19 (1969) 68-79. 35 Sumino, R. and Nakamura, Y., Synaptic potentials of hypoglossal motoneurons and a common inhibitory interneuron in the trigemino-hypoglossal reflex, Brain Research, 73 (1974) 439-454. 36 Torvik, A., Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures. An experimental study in rat, J. comp. Neurol., 106(1956)51 141. 37 Valverde, F., Reticular formation of the pons and medulla oblongata. A Golgi study, J. comp. NeuroL, 116 (1961) 71-100. 38 Valverde, F., A new type of cell in the lateral reticular formation of the brain stem, J. comp. NeuroL, 117 (1961) 189- 195. 39 Valverde, F., Reticular formation of the albino rat's brain stem: cytoarchitecture and corticofugal connections, J. cornp. Neurol., 119 (1962) 25-53. 40 Wall, P. D. and Taub, A., Four aspects of the trigeminal nucleus and a paradox, J. Neurophysiol., 25 (1962) 11(~ 126. 41 Yamamoto, T., Fujiwara, T., Matsuo, R. and Kawamura, Y., Hypoglossal motor nerve activity elicited by taste and thermal stimuli applied to the tongue in rats, Brain Research, 238 (1982) 89-104. 42 Yokota, T., Nishikawa, Y. and Ohno, S., A hypoglossal reflex elicited by mechanical stimulation of the mandibular mucosa in the cat, Jap. J. Physiol,, 28 (1978) 659667. 43 Zimmerman, E. A., Chambers, W. W. and Liu, C. N., An experimental study of the anatomical organization of the cortico-bulbar system in the albino rat, J. comp NeuroL, 123 (1964) 301-324.
47
Elsevier
Brain Stem Afferents of Hypoglossal Neurons in the Rat*
ROSEMARY C. BORKE, MARTIN E. NAU and ROBERT L. RINGLER, Jr.
Department of A natomy, UniformedServices University, 4301 Jones Bridge Road, Bethesda, MD 20814 (U.S.A. (Accepted November 16th, 1982)
Key words: hypoglossal neurons - afferents to hypoglossal neurons - horseradish peroxidase - retrograde tracing
The origin of afferent connections of the hypoglossal nucleus in rats was investigated using horseradish peroxidase (HRP) as a retrograde tracer. Pressure injections (0.15-0.17/~l) of 15% HRP were introduced into the rostral, middle and caudal portions of the nucleus. Projections to the hypoglossal nucleus originated from 3 regions of the brainstem: the reticular formation, the spinal V complex and the nucleus of the solitary tract. Bilateral projections with ipsilateral predominance came from the lateral reticular formation: the dorsal aspect of the nucleus reticularis parvocellularis and its caudal continuation, the nucleus reticularis dorsalis. Fewer projections emerged from two nuclei of the medial reticular formation. The dorsal part of the nucleus reticularis ventralis at the spinomedullary junction contributed bilateral with mainly contralateral input to hypoglossal neurons. A few labeled neurons were situated bilaterally in the nucleus reticularis gigantocellularis of the rostral medulla. The input from the spinal V complex originated from the dorsal aspect along most of its length but particularly from the pars interpolaris and oralis subdivisions. Labeled neurons were located primarily in the posterior portion of the nucleus of the solitary tract. Projections from the spinal V complex and the solitary nucleus exhibited ipsilateral predominance. These results suggest that somatic and visceral centers of the rat brainstem play an important role in the control of the activity ofhypoglossal motoneurons. INTRODUCTION
The tongue is a muscular organ that assists in mastication, deglutition, and vocalization. The arrangement of the lingual muscles and the reflex mechanisms of the tongue account for the highly organized and controlled movements of the tongue during these actions. While numerous cranial nerves play an important part in the sensory feedback regulation of tongue movements, the hypoglossal nerve alone provides motor innervation to the muscle mass. Topographical representation of the main branches of the hypoglossal nerve and the lingual muscles within this cranial nerve motor nucleus has been determined in rats by recent experiments using retrograde tracers 15 and cytoarchitectonic methods 24. Although sources of input to the hypoglossal neurons in rats have only been summarized in preliminary reports 2.5~9, afferent mechanisms involved in hypoglossal re-
flexes have been the subject of numerous physiological studies 12,17,22,28,29~34,41.Since physiological data have implicated certain regions of the brain stem as providing input to the hypoglossal nucleus, anatomical localization of their precise origin is necessary for a complete understanding. The object of this study was to identify brainstem afferents to the hypoglossal nucleus and to interpret their significance. MATERIALS AND METHODS
Eighteen adult rats of the Osborn-Mendel strain were used for this investigation. The rats were anesthetized with sodium pentobarbital (30 mg/kg) and small amounts (0.15 0.17 ~1) of 15% H R P were injected into the hypoglossal nucleus stereotaxically using micropipettes with a tip diameter of 20-30/~m. The majority of the injections were introduced into the rostral portion of the hypoglossal nucleus to avoid involvement
* A preliminary report of this work was presented at the American Association of Anatomists, 95th Annual Session at Indianapolis, IN, April 4-8, 1982. 0006-8993/83/$03.00 ~,'~1983 Elsevier Science Publishers B.V.
48
of the dorsal motor nucleus of the vagus (DMX). Injections were limited to the hypoglossal nucleus in 10 animals. In 5 other animals, the injection spread included the hypoglossal nucleus, DMX and varying portions of the adjacent reticular formation. After survival times of 2472 h, rats were anesthetized with sodium pentobarbital and killed by intracardiac perfusion with Ringer’s solution followed by dilute and concentrated aldehydego. The brains were removed, postfixed in the dilute aldehyde mixture for 2 h, cut into blocks and immersed overnight in a 30% sucrose-buffer solution. Frozen serial sections of the entire brain and the cervical spinal cord were cut at 30 pm in a transverse plane. Alternate tissue sections were processed according to the method of LaVaill or Mesulam19 using 3,3-diaminobenzidine (DAB) and tetramethylbenzidine (TMB) as chromagens. The reacted sections were mounted on chrome-alum gelatinized slides and lightly counterstained with cresyl violet or Neutral red. The location of the injection site was confirmed and serial sections through the brain and cervical spinal cord were examined using brightand dark-field illumination. The distribution of labeled neurons was mapped on sequential tracings through the brain and spinal cord. RESULTS
Separate injections of HRP into the rostral, middle, and caudal portions of the hypoglossal nucleus, in general furnished the same information about the sources of afferent connections of this cranial nerve nucleus. However, those injections into the rostra1 portion of the hypoglossal nucleus were accomplished without involvement of DMX. The locations of labeled neurons in the brain stem after these injections were remarkably consistent and varied for individual animals only in the number of retrogradely labeled neurons. In all cases, the XIIth nerve contained anterograde label. The sources of input to hypoglossal neurons can be conveniently presented in a single synthetic description of the results of a case in which HRP was introduced into
the rostra1 portion of the hypoglossal nucleus 1 mm above the level of the obex. The micropipette traversed the dorsal half of the nucleus and spread of the exogenous protein was limited to the boundaries of the ipsilateral hypoglossal nucleus(Fig. lC,). The greatest number of labeled neurons was identified in the lateral reticular formation of the medulla and pons, specifically the nucleus reticularis parvocellularis (RPc) (Fig. IA-D). This projection was bilateral with ipsilateral predominance. Along most of the length of the hypoglossal nucleus, labeled neurons with fusiform and triangular-shaped somata (Fig. 2A) occupied the dorsal aspect of RPc. Fewer labeled cells were located in the intermediolateral portion of RPc. The labeled neurons decreased in number and became continuous with those cells labeled in nucleus reticularis dorsalis (RD) at caudal medullary levels (Fig. 1E). Rostra1 to the hypoglossal nucleus, labeled neurons were situated bilaterally in the oral pole of RPc (Figs. lA, B and 2B). Labeled neurons also were identified in 2 cell groups of the medial reticular formation: (1) the nucleus reticularis gigantocellularis (RGc); and (2) its caudal continuation, the nucleus reticularis ventralis (RV) (Fig. 1BF). Medium- and large-sized neurons with long dendritic processes were labeled in RGc (Fig. 2C). Cell labeling in RGc and RV at levels adjacent to the hypoglossal nucleus was bilateral (Fig. IC-E). At the spinomedullary junction, these connections originated primarily from the contralateral RV (Fig. 1F). Axons of these labeled neurons coursed medially rostra1 to the pyramidal decussation, and entered the ventral aspect of the hypoglossal nucleus in caudal regions of the medulla. Another source of input to hypoglossal neurons was from the spinal V complex. A longitudinal column of labeled neurons extended from the pars interpolaris subdivision at medullary levels to the principal sensory nucleus of V(PrSV) at pontine levels. Labeling in the spinal V nucleus was confined to neurons in the dorsal part of the nucleus (Fig. IA-E). The majority of input from the spinal trigeminal nucleus originated from medium sized neurons in the pars
49
HRP-40
/ A RV
D
y/.', \ I
(~)
RGc
I
I
"
°
B RV
E
o
/I
i'/\"~o
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C1 Fig. !. Serial reconstruction of labeled neurons in the brainstem after injection of HRP into the rostral hypoglossal nucleus, q is a dark-field micrograph of the injection site and spread of the enzyme.
50
Fig. 2. A: labeled s0mata and processes of neurons in the ipsilateral nucleus reticularis parvocellularis adjacent to the rostral portion of the hypoglossal nucleus, x 160. B: HRP-positive neuronal cell bodies in the ipsilateral nucleus reticularis parvocellularis above the level of the hypoglossal nucleus, x 320. C: HRP reaction product filling a neuron located in the ipsilateral nucleus reticularis gigantocellularis, x 320. D: 3 labeled neurons situated in the dorsal aspect of the ipsilateral spinal V complex, pars oralis. × 320. E: HRP-positive neuron in the ipsilateral spinal V complex, pars interpolaris. The axon (arrow) projects medially through the dorsolateral reticular formation toward the hypoglossal nucleus, x 320. F: labeled neurons (arrows) in the posterior portion of the ipsilateral solitary nucleus adjacent to the hypoglossal nucleus. X 320.
51 oralis (SpVO) (Fig. 2D) and interpolaris (SpVI) (Fig. 2E) subdivisions (Fig. 1B-D). The projections from the trigeminal complex were predominantly ipsilateral. Neurons labeled in the nucleus of the solitary tract (SN) were limited to medullary levels that coincided with cells in the XIIth nucleus (Fig. 1C-E). Somata of these neurons were spindleshaped and their axons entered the dorsolateral angle of the hypoglossal nucleus (Fig. 2F). Only occasional labeled cells were identified in the ipsilateral D M X at the same medullary levels (Fig. 1C, D). No labeled neurons were identified in more rostral brainstem regions or in any cortical area. H R P injections into the middle and caudal regions of the hypoglossal nucleus invariably produced spread of the tracer into DMX. Retrograde neuronal labeling included the same areas identified by rostral hypoglossal injections, but with additional labeling of cells in PrSV, SN and the nuclei of the medullary reticular formation. Cells also were labeled in certain rostral areas of the brainstem, namely, the nucleus reticularis pontis oralis, the central gray region and the paraventricular nucleus of the hypothalamus. Injections of HRP into middle levels of the XIIth nucleus, DMX, the adjacent reticular formation and portions of the medial longitudinal fasciculus (Fig. 3D~) produced the same results as those experiments previously described with 3 additional loci of labeled neurons: neurons in the intermediate and ventral portions of the reticular formation of the pons and medulla (Fig. 3A-F), neurons in the intermediate and ventral parts of SpVO and PrSV (Fig. 3A, B), and the vestibular nuclei, particularly the medial and inferior cell groups (Fig. 3B). DISCUSSION
Four sources of hypoglossal input have been identified by these retrograde tracer experi•ments (Fig. 4): portions of the medial and lateral reticular formation, the spinal V nucleus and the nucleus of the solitary tract. Previous results from anterograde studies in cats established the existence of a projection from the lateral reticu-
lar formation (LRF) and the adjoining spinal V nucleus to the ipsilateral hypoglossal nucleus m,ll, while bilateral terminations from these same sources were identified in rats 27. Isotope diffusion at the injection site prevented either of these studies from determining whether the LRF, the spinal V nucleus or both nuclei projected to hypoglossal neurons. The present retrograde tracer work suggests that both nuclei provide separate input to hypoglossal motoneurons, but LRF provides the primary source of XIIth nucleus afferents. The origin of the L R F projections was localized primarily to the dorsal and intermediate parts of RPc along its length but particularly from neurons adjacent to the rostro-caudal extent of the XIIth nucleus. Prior Golgi studies supported the likelihood that axons from the dorsolateral reticular formation terminate on hypoglossal dendrites outside the borders of this cranial nerve nucleus 7,8. Limitation of the injection site to the hypoglossal nucleus in many animals in the present experiments provides evidence that a substantial population of neurons in RPc end on postsynaptic sites within the hypoglossal nucleus. Neurophysiologica117,21,33 and anatomical data 6,38,39 indicate that RPc receives afferents from secondary sources of hypoglossal input identified in the present work: the spinal V nucleus and the solitary nucleus. RPc therefore functions as a relay station to the hypoglossal nucleus from dual origins: cortical 43 and peripheral 36 sources which lack direct connections with the XIIth nucleus and somatic and visceral centers of the brainstem that also project directly to hypoglossal neurons. RPc may act as the site for the convergence and integration of hypoglossal reflexes from spatially separated sources for complex movements of the tongue associated with somatic and visceral afferents. In the present work, the small n u m b e r of labeled cells in the medial reticular formation (MRF) coincides with the failure of anterograde studies in cats to disclose significant projections from M R F to motoneurons of cranial nerves m,ll. A short report failed to specify the M R F nuclei giving rise to hypoglossal afferents in rats 9. The present results of labeled cells in the dorsolateral
52
HRP-25
LV
iI \
D
• I RPoC
A
D1
\~
o';//." ..
\-o F
Fig. 3. Serial reconstruction of sites of labeled neurons in the brainstem after injection of H RP into the middle level of the hypoglossal nucleus DMX, adjacent reticular formation and the medial longitudinal fasciculus. D] is a dark-field micrograph of the injection site and spread of enzyme.
53 aspect of RGc and RV agree with and further specify findings in cats that the dorsal reticular formation projected to the XIIth nucleus 3,26 but the ventral part did not 1,3. Since Golgi studies have shown axon collaterals emerging from the main axons of neurons in the reticular formation 32'37, sparse labeling of some neurons in RGc suggests that collaterals from the main axons may be involved in the hypoglossal projection from RGc. The present study is the first to demonstrate that neuronal connections to the hypoglossal nucleus originate only from the dorsal aspect of the spinal V nucleus and therefore appear topographically related to the mandibular division of the trigeminal nerve 14. These findings provide an anatomical basis for physiological reports suggesting a disynaptic pathway to the hypoglossal nucleus from various branches of the mandibular nerve 13,22,28.35,42.These observations also provide a more precise localization of the source of spinal V connections to the XIIth nucleus. The principal sensory nucleus of V has been suggested as the site of secondary neurons in the disynaptic pathway from the mandibular nerve to the hypoglossal nucleus 28. According to the present data, cells in the dorsal parts of inter-
polaris and oralis, which receive innervation from perioral and intraoral structures4°, provide the major projections from the spinal V nucleus to hypoglossal neurons. Results also indicate that axons of these neurons terminate within the hypoglossal nucleus, rather than on hypoglossal dendrites in the reticular formation, as has been previously suggested 28. Previous anterograde studies on whether the solitary nucleus sends direct projections to the hypoglossal nucleus have been hampered by contamination problems involving isotope spread to RPc 4,23. The present finding of labeled cells in the solitary nucleus, limited to medullary levels of the XIIth nucleus, coincides primarily with the posterior solitary nucleus. Therefore, direct connections from the solitary nucleus may be associated primarily with central mechanisms involved in general visceral afferents rather than the gustatory-hypoglossal reflex. Anatomical 23 and physiological41 data indicate that multisynaptic connections between the anterior solitary nucleus and the hypoglossal nucleus are involved in the gustatory-hypoglossal reflex. Our results failed to disclose significant retrograde transport of H R P to cells in the brainstem region rostral to the pons after H R P injections
S,in., V Dorsal Portion Intorpoiarls Oralis
Hypoglossal Nucleus
I Lateral Reticular Formation
(RPc& RD) Medial Reticular Formation
(RGc & RV)
Fig. 4. Summary diagram of brainstem afferents of hypoglossal neurons in rats. Broad arrow indicates primary source of hypoglossal input.
54 confined to the hypoglossal nucleus. Although direct cortical connections to hypoglossal nuclei have not been demonstrated in rats39, midbrain regions including the interstitial nucleus of Caja125, the periaqueductal gray25, and the mesencephalic nucleus of V ~8,2° have been considered to supply diffuse input to the hypoglossal nucleus. Our failure to obtain appreciable label of neurons in any of these regions after injections confined to the hypoglossal nucleus suggested that axonal endings from midbrain regions, if present, may terminate mainly on distal hypoglossal dendrites situated outside the nuclear boundaries. The spread of HRP injection into the dorsal and lateral medullary reticular formation in the experiments of Panneton and Martin25 and the absence of synaptic potentials in hypoglossal nucleus after midbrain stimulation35 support this possibility. Certain rostral brainstem regions did contain retrogradely labeled neurons if the spread of the injection included the overlying cells of DMX.
These findings concur with those of others in which connections to DMX originated from the paraventricular nucleus of the hypothalamus, PrSV, reticular formation nuclei and the solitary nucleus31. Large injections involving the reticular formation and portions of the medial longitudinal fasciculus as as well as the hypoglossal and vagal motor nuclei served as a cumulative control in that inputs to the hypoglossal nucleus, DMX, and connections of the reticular formation and the medial longitudinal fasciculus were demonstrated.
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ACKNOWLEDGEMENTS
The author is grateful to Dr. Malcolm Carpenter for his thoughtful suggestions and comments. Thanks are given to Lillian Magruder for careful typing of the manuscript. This work was supported by the Department of Defense, USUHS, Department of Defense Grant C07019.
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