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Dendritic ramifications of trigeminal motor neurons innervating jaw-closing muscles of rats
Journal of the Neurological Sciences, 1988, 86:251-264
251
Elsevier
JNS 03033
Dendritic ramifications of trigeminal motor neurons innervating jaw-closing muscles of rats F. S. F. Mong 1, Y.C. Chen 2 and C . H . L u 3 ~Department of Anatomical Sciences, The University of Texas Dental Branch, Houston, TX (U.S.A.), 2Department of Anatomy, Sun Yat-Sen University of Medical Sciences, Guangzhou (People's Republic oJ China), and 3Department of Anatomy, Peking Union Medical University, Beijing (People's Republic of China) (Received 11 December, 1987) (Revised, received 12 April, 1988) (Accepted 12 April, 1988)
SUMMARY
A cholera toxin subunit conjugated horseradish peroxidase (CTHRP) was found to be very useful in labelling the dendrites of motoneurons. CTHRP was injected individually to jaw-closing muscles (temporalis, masseter, and medial pterygoid) of rats, and their motoneurons including the dendrites were labelled and studied. The results show that the motoneuron cell bodies innervating temporalis, masseter, and medial pterygoid muscles are located in the trigeminal motor nucleus in dorsal, ventromedial and ventrolateral position. The dendrites of these motoneurons extend radially into mesencephalic nucleus, supratrigeminal nucleus, pontine reticular formation, trigeminal sensory nucleus and even into the bundles of the ascending root of the facial nerve. These dendrites may serve as an extended surface for various synaptic contacts to the jaw closing motoneurons. The possibility that they may also have presynaptic influence on the input to the trigeminal motoneurons is also discussed.
Key words: Trigeminal motor nucleus; Jaw-closing motoneurons; Dendrites; Choleratoxin-conjugated HRP
Correspondence to: Dr. Franz S.F. Mong, Dept. of Anatomical Sciences, The University of Texas Dental Branch, P.O. Box 20068, Houston, TX 77225, U.S.A. Abbreviations: Ascending root of facial nerve, ARF; central canal, CC; cochlear nucleus, CN; facial nucleus, FN; genu of facial nerve, GF; inferior olive, IO; longitudinal fasciculus ofpons, LFP; mesencephalic nucleus of trigeminal, ME5; motor root of trigeminal, MR5; motor nucleus of trigeminal, MN5; pontine nuclei, PN; pontine reticular formation, PRF; periventricular gray, PVG; raphe nucleus, RN; superior olive, SO; superior salivary nucleus, S SN; supratrigeminal nucleus, STN; spinal tract of trigeminal, ST5; sensory nucleus of trigeminal, SN5; ventricle, V. 0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
252 INTRODUCTION There have been many studies on the mgeminal motor nucleus concerning the location of the cell bodies ofmotoneurons innervating the muscles of mastication. Early investigations (Szentagothai 1949; Vedral and Matzke 1967) using degeneration methods, showed that neurons innervating jaw-closing muscles were ventrally located in the trigeminal motor nucleus. Later, however, with accurate horseradish peroxidase (HRP) tracing technique, it was shown that these neurons were more dorsally located (Limwongse and DeSantis 1977). In fact, in the dorsal portion of the nucleus, the temporalis neurons are dorsal while the masseter and medial pterygoid neurons are ventromedially and ventrolaterally located (Mizuno et al. 1975; Sasamoto I979). Despite the accurate localization of these motor neurons in the trigeminal motor nucleus, their dendritic distribution to the nearby nuclear groups has not been thoroughly investigated. Dendrites provide an elaboration of the surface area of neurons. The traditional approach for studying dendrites is Golgi's silver impregnation technique (Scheibel and Scheibel 1970). Silver impregnation is usually non-specific. however, and since background dendrites stain simultaneously, interpretation of results is often difficult. In a recent communication (Szekely and Matesz 1987), the dendritic geometry of trigeminal motoneurons were studied by applying cobalt lysin solution to the central nerve stumps of various jaw muscles in the frog. However, the specific projections of dendrites into various nuclear groups were not mentioned, lntracellular microinjections of free HRP into various anterior horn cells of spinal cord were carried and their dendritic ramifications were studied by camera lucida drawings (Ruigrok et al. 1985; Cameron et al. 1983). Such laborous work has not been carried out for trigeminal motoneurons. Recently, it was found that if HRP was conjugated with subunit B of cholera toxin (CTHRP), it was 30-50 times more sensitivethan free HRP, particularly in labelling the dendrites ofmotoneurons (Trojanowski et al. 1981a,b, 1982; Wan et al. 1982a, b; Furicchia and Goshgarian 1987). The increased sensitivity would allow a minute quantity of CTHRP be injected to a specific muscle and prevent the problem of leakage to the surrounding muscles. Thus, labelling of undesired neurons and dendrites could be avoided. The specific mechanism of the increased sensitivity of CTHRP is unknown but was suggested due to the differences in the transport of CTHRP and free HRP across the neuronal cell membrane. The ligand cholera toxin bound to GM1 ganglioside receptor on neuronal cell membrane. This specific interaction between ligands and its receptor was followed by an active adsorptive endocytosis ofligand-conjugated HRP. In contrast, there were no receptors for free HRP and the transport was due to passive fluid phase endocytosis. The active process could transport probes in greater quantity and faster than the passive process (Trojanowski et al. 1981a, b, 1982: Wan et al. 1982a,b). Ledeen (1978) has observed a high concentration of GM 1 ganglioside receptors at the neuromuscular junctions. The purpose of the present investigation, therefore, is to inject CTHRP into jaw-closing muscles of the rat and to study the dendritic distribution of their motoneurons.
253 MATERIALS AND METHODS Thirty male Sprague-Dawley rats (350-500 g) were anesthetized with chloral hydrate (40 mg/100 g wt., i.p.). A small incision over the temporalis, masseter and medial pteryoid muscles was made and 5 #1 of 0.4~"o CTHRP (provided by Professor X.S.T. Wan, Peking Union Medical University, Beijing, China; for preparation of CTHRP, see Gonatas et al. 1979) was injected into each individual muscle. Only one muscle was injected in each rat. For control, a similar amount of saline was injected into corresponding muscles of control rats. All injections were made by means of a glass micropipette inserted into a polyethylene tube filled with glycerine and connected to a 50-/~1 Hamilton syringe. Jaw-closing muscles were chosen because they were large and easily accessible with minor surgery. Rarely was there any leakage. If any leakage was suspected, it was quickly absorbed with gauze followed by a saline rinse. Finally, the skin was closed. Survival time was 24 h as were the cases of previous studies (Trojanowski et al. 1981a,b, 1982; Wan et al. 1982a,b). The rats were anesthetized again with chloral hydrate (45 mg/100 g wt., i.p.) and perfused via the ascending aorta with 0.9~o saline followed by a fixative containing 1 Y/oparaformaldehyde, 1.25 ~/oglutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and finally with 10~ phosphate-buffered sucrose. The brain was removed after perfusion and immersed in 10 ~o phosphate-buffered sucrose solution overnight. The brain stem was then embedded in OCT compound (Lab. Tek product, Miles Laboratories, Inc.) and frozen sections (40 #m) were cut in a cryostat horizontally, sagittally, and coronally. Serial sections were collected in buffered sucrose solution and processed for HRP histochemistry according to Mesulam's TMB method (Mesulam 1978; 0.3 ~o H202). They were then mounted sequentially on chrome-alum coated slides, air dried, and counter-stained in 1~o neutral red solution. CTHRP injection sites have been thoroughly studied previously (Wan et al. 1982b) and therefore, was not repeated.
RESULTS Figures 1,2, and 3 are camera lucida drawings from the representative cross, coronal, and sagittal sections of brain stem respectively. Major nuclear groups are indicated for the purpose of orientation. These nuclear groups are identified using Paxinos and Watson's atlas (1986) as a reference. The dimensions of the trigeminal motor nucleus are 900 #m dorsoventrally, 780 #m mediolaterally, and 930 #m rostrocaudally. Saline-injected control rats showed no HRP reaction. Cross sections
In cross sections, the somata of temporalis muscle (Fig. 4) were located dorsally while that ofmasseter (Fig. 5) and medial pterygoid (Fig. 6) were located ventromedially and ventrolaterally, respectively. Their axons, forming the motor root of the trigeminal nerve (MR 5), exit ventrolaterally and rostrally.
254
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:~ xx
Figs. 1, 2 and 3. Camera lucida drawings of representative section of cross (Fig. t ), coronal (Fig. 2), and sagittat (Fig. 3) planes of brainstem. Major nuclear groups are labelled {see Abbreviations) for the orientation of the remaining photographs. Within the motor nucleus, open circles represent the location of motoneurons for temporalis muscle, triangles that for medial pterygoid muscle, and solid squares that for masseter muscle. The number of the symbols does not ihdicate the number of neurons. Shaded a r e a indicates the field where dendrites of j a w - d o s i n g motoneurons extend to. R = rostral. D = dorsal. L = lateral, C = caudal.
255
C~
Y
R
V
SN5
®
PN
The dendrites of the temporalis motoneurons can be seen extending dorsally into trigeminal mesencephalic nucleus (ME 5) and supratrigeminal nucleus (STN, Fig. 7) and medially into pontine reticular formation just medial to the motor nucleus (PRF, Fig. 8). The dendrites of masseter motoneurons extend dorsally into supratrigeminal nucleus (STN), mesencephalic nucleus (ME 5), and medially into pontine reticular formation just medial to motor nucleus (PRF). Thus, the direction is similar to that of the temporalis motoneurons. The dendrites of medial pterygoid neurons expand across the field to the dorsomedial part of the nucleus (Fig. 9) and project into the areas as those of temporalis and masseter motoneurons. Coronal sections
In coronal sections, the dendrites of temporalis neuron extend rostrally and caudally into the pontine reticular formation (Fig. 10). They also extend laterally into the trigeminal sensory nucleus (Fig. 11). In Figs. 10 and 11, the axons of trigeminal ganglionic neurons travelling in the spinal trigeminal tract are also labelled. Termination of these axons in the spinal sensory nucleus is evident. Some caudally extending
/
Figs. 4, 5 and 6. Cross-sections o f p o n s showing locations ofternporalis neurons (Fig, 4), masseter neurons (Fig. 5), and medial pterygoid (Fig. 6). Note the somata of temporalis are dorsal while that o f m a s s e t e r and medial pterygoid are ventromedially and ventrotaterally located in the trigeminal motor nucleus. D = dorsal, L = lateral; bar = 200 t~m-
257
Figs. 7 and 8. Close-up view (10 x ) of cross-section of pons showing dendrites of temporalis neurons projecting close to mesencephalic nucleus (ME5) and to supratrigeminal nucleus (STN, Fig. 7) and into pontine reticular formation (PRF, Fig. 8). Arrows indicate dendrites. D = dorsal, L = lateral; bar = 90 um.
258
Fig. 9. Close-up view (10 x ) on cross-section of pons showing medial pterygoid neurons located on the ventrolateral portion of the trigeminal motor nucleus (MN5). Their axons (a) exit ventrolateraUy,while their dendrites (d) extend dorsomedially. Those dendrites projecting into mesenecphali¢ nucleus cannot be seen in this section, but in the adjacent sections. D = dorsal. L = lateral; bar = 90 era.
dendrites can be seen traversing into the bundles of the ascending root o f the facial nerve (Figs. 10 and 11). The dendrites o f the medial pterygoid motoneurons reach caudomedially into the pontine reticular formation and rostrolateraUy into the trigeminal sensory nucleus (Fig. 12). The distribution of dendrites ofmasseter motoneuron is similar to that of medial pterygoid neurons.
Sagittal sections In sagittal sections, all motor neurons have dendrites extending rostrally and caudally into the reticular formation. In addition, the temporalis neurons have dendrites extending dorsally into mesencephalic neurons (Fig. 13) while those of masseter
259
Fig. 10. Coronal section ofbrainstem showing neurons and their dendrites oftemporalis muscle (see Fig. 2 for orientation). Major nuclear groups are outlined (see Abbreviation). Note, the incoming axons (short arrows) from trigeminal ganglionic neurons are labelled and can be seen in spinal trigeminal tract (ST5). Some of them (long arrows) can be seen in the sensory trigeminal nucleus (SN5). Note that dendrites also extend rostrally and caudally into pontine reticular formation (PRF) and into the ascending root of the facial nerve (ARF). R = rostral, L = lateral; bar = 200 ktm.
ARF J
Fig. 11. Close-up view (10 × ) of an adjacent section of Fig. 10. Note dendrites (short arrows) extend into sensory trigeminal nucleus (SN5) and ascending root of facial nerve (ARF). Long arrows indicate the incoming axons oftrigeminal ganglionic neurons terminating in the sensory trigeminal nuclei (SN5), as that of Fig. 10. Bar = 90 #m.
260
It
-
Fig. 12. Coronal section of brain stem showing medial pterygoid motoneurons and dendrites (see Fig.2 for orientation). Dendrites extend rostrolaterally into sensory trigeminal nucleus (SN5) and caudomedially into pontine reticular formation (PRF). R = rostral, L = lateral; bar = 200 #m. (Fig. 14) and medial pterygoid also have dendrites in the vicinity of mesencephalic neurons although not as clearly shown as those of the temporalis neurons.
DISCUSSION A few aspects of the methodology employed need to be addressed. The advantage of using C T H R P in labelling motoneurons and, particularly, its dendrites is the superior sensitivity of C T H R P over free H R P . This superiority was well documented (Trojanowski et al. 198 la,b, 1982; W a n et al. 1982a, b; Furicchia and Goshgarian 1987) and was not due to an artifact of the volume of the injected solution, o f the injection site area, or o f the survival time. The fact that C T H R P is 3 0 - 5 0 t i m e s more sensitive than free H R P could allow the investigator to reduce substantially the quantity o f C T H R P injected into a specific muscle and, thus, reduce the possibility o f leaking to the surrounding muscles resulting in labelling unrelated motoneurons. In our study, the unrelated neurons such as those o f facial nucleus or of nucleus ambiguus have never been observed. It is likely that a minute volume injected into a muscle may not label
261
Fig. 13. A close-up view (10 x ) of a sagittal section of brainstem showing temporalis motoneurons and dendrites (see Fig. 3 for orientation). Dendrites (arrow) extend into mesencephalic nucleus (ME5) which is just ventral to superior cerebellar peduncle (SCP). Note that mesencephalic neurons are labelled by CTHRP in this case. D = dorsal, R = rostral; bar = 90/~m. all motoneurons in the trigeminal motor nucleus. The purpose of the present investigation, however, was to delineate the dendritic ramification and not to quantify the number of motoneurons which was investigated previously (Limwongse and DeSantis 1977). With this superior sensitivity of C T H R P , further studies need to be carried out to establish the optimal maximal volume injected so that all motoneurons innervating a specific muscle could be labelled in the trigeminal motor nucleus. Lastly, gamma motoneurons were not studied in the present investigation as was the case in previous studies (Limwongse and DeSantis 1977; Mizuno et al. 1975). Muscle spindles are enclosed by a connective tissue capsule which might serve as a barrier for C T H R P to enter and to label either the gamma motoneurons or. mesencephalic neurons. This is probably the reason why, in our study, mesencephalic neurons were labelled only occasionally. In addition to the confirmation of previous studies in regard to the specific location of jaw-closing motor neurons in the trigeminal motor nucleus (Mizuno et al. 1975; Sasamoto 1979), the present investigation also reveals that the dendritic distributions of jaw-closing motor neurons are quite extensive. They extend radially from the
262
Fig. 14. A sagittal section of brain stem showing masseter motoneurons and dendrites t see Fig. 3 for orientation). A few dendrites (arrows) extending dorsally toward the mesencephalicnucleus (ME 5) which is not labelled by CTHRP. Other dendrites extend rostrally into pontine reticular formation(PRF). Axons (a) exit ventrorostrally. SCP = superior cerebellar peduncle, D = dorsal. R = rostral; bar = 200 ~tm. motor nucleus into mesencephalic nucleus, supratrigeminal nucleus, pontine reticular formation, and spinal trigeminal sensory nucleus. Some dendrites also extend caudally into the bundles of the ascending root of the facial nerve. On the other hand, previous studies showed that, in rats, the cortical input to trigeminal motor neurons was largely indirect and relayed via pontine reticular formation (Travers and Norgren 1983; Vornov and Satin 1983). Other inputs-into the trigeminal motor nucleus include mesencephalic nucleus, red nucleus (Travers and Norgren 1983), bilateral supratrigeminal nuclei (Mizuno et al. 1978), pontine raphe nucleus (Vornov and Sutin 1983) trigeminal sensory nucleus, and reticular formations of medulla (Travers and Norgren 1983; Vornov and Sutin 1983). Thus, the trigeminal motor nucleus can receive axonal inputs from various nuclear groups and projects, by way of its dendrites, into various nuclear groups in the vicinity. The nuclear groups further away from trigeminal motor nucleus (e.g,, red nucleus, medullary reticular formation) have no dendritic extensions from jaw-closing motoneurons. I t is possible that an extensive dendritic distribution would be necessary to collect the synaptic input necessary for the functions of the trigeminal motor nucleus, because the-nucleus is not
263 large and the neurons in it are closely packed. It is worthwhile to mention that physiological studies in cats showed that monosynaptic jaw reflexes are strong and yet, the contacts between mesencephalic axons and trigeminal motor neurons were very limited (Appenteng et al. 1978). Luschei (1987) speculated that the majority of these contacts might be on the dendrites extending out of the nucleus into the region above it (Luschei 1987). The present study, using rats, provides support for such an interpretation. The dendrites from trigeminal motor neurons in the rat project dorsally out of the nucleus into close vicinity of mesencephalic neurons. Alternatively, it is also possible that the dendritic extensions are responsible for the reciprocal modulations of axonal inputs. Trigeminal motor neurons not only receive axonal input from the nuclei mentioned above, but also project back to most of those nuclei, as revealed by the current study. Although the present study could not suggest that these dendrites might serve as presynaptic terminals, such a possibility was proposed by previous investigators, e.g., in the olfactory bulb (Hirata 1964; Andres 1965; Rail et al. 1966), lateral geniculate body (Guillery 1969; Lieberman 1973), medial geniculate body (Morest 1971), and thalamus (Harding 1971; Ralston 1971). It must be emphasized, however, that the cited examples of dendrites serving as presynaptic terminals are all sensory system receiving input from periphery. The current observation deals only with the motor output. Whether dendrites of motor system can serve as presynaptic terminals remains to be determined. The observation that the dendrites of trigeminal motor neurons extend to trigeminal sensory nucleus is interesting. In the present study, the central processes of trigeminal ganglionic neurons traveling in the spinal trigeminal tract and their termination in the trigeminal sensory nucleus could be visualized. Previous studies (Nomura and Mizuno 1985; Luschei 1987) showed that mesencephalic axons could also terminate in the trigeminal sensory nucleus. Thus, a synaptic arrangement involving mesencephalic terminals, incoming semilunar ganglionic terminals, the dendrites of trigeminal motoneurons and the neurons of sensory nucleus may take place.This possibility and the possibility that dendrites of trigeminal motoneurons might serve as presynaptic terminals are being studied by electron microscopy. REFERENCES Andres, K.H. (1965) Der Feinbau des Bulbus Olfactorius der Ratte, Z. Zellforsch. Mikrosk. Anat., 65: 530-561. Appenteng, K., M.J. O'Donovan, G. Somjen, J.A. Stephens and A. Taylor (1978) The projection of jaw elevator muscle spindle afferents to fifth nerve motoneurons in the cat, J. Physiol. (London), 279: 409-423. Cameron, W. E., D. B. Averill and A.J. Berger(1983) Morphologyof cat phrenic motor neurons as revealed by intracellular injection of horseradish peroxidase, J. Comp. Neurol., 219: 70-80. Furicchia, J.V. and H.G. Goshgarian (1987) Dendritic organization of phrenic motoneurons in the adult rat, Exp. Neurol., 96: 621-634. Gonatas, N.K., C. Harper, T. Mizutani and J.O. Gonatas (1979) Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem., 27: 728-734. Guillery, R.W. (1969) The organization of synaptic interconnections in the laminae of the dorsal lateral geniculate nucleus of the cat, Z. Zellforsch. Mikrosk. Anat., 96: 1-38. Harding, B.N. ( 1971) Dendro-dendritic synapses,including reciprocalsynapses,in the ventrolateralnucleus of the monkey thalamus, Brain Res., 34: 181-185.
264 Hirata, Y. (1964) Some observations on the fine structure of the synapses in the olfactory bulb with particular reference to the atypical synaptic configurations, Arch. Histol. ,lap., 24:293-302 Ledeen, R.W. (1978) Ganglioside structures and distribution: are they localized at the nerve ending? J. Supramol. Struct., 8: 1-17. Lieberman, A.R. (1973) Neurons with presynaptlc perikaria and presynaptic dendrites in the lateral geniculate nucleus, Brain Res.. 59: 35-59. Limwongse. V. and M. DeSantis C1977) Cell body locations and axonal pathways of neurons innervating muscles of mastication in the rat, Am. d. Anat., 149: 477-488. Luschei, E. S. (1987) Central projections of the meseneephalic nucleus of the fifth nerve: an autoradiographic study, J. Comp. Neurol., 263: 137-145. Mesulam, M.M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26: 106-117. Miznno, N.. A. Konishi and M. Sato (t975) Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol.. 164:105-116. Miznno, N., S. Nomura, K. Itah, Y. Nakamura and A. Konishi (1978) Commissural interneurons for masticatory motoneurons: a light and electron microscope study using the horseradish peroxidase tracer technique, Exp. NeuroL, 59: 254-262. Morest, D.K. (1971) Dendrodendritic synapses of cells that have axons: the fine structure of the Golgi type II cell in the medial geniculate body of the cat, Z. Anat. Entwieldungsgesch., 133: 216-246. Numura, S. and N. Mizuno (1985)Differential distribution of cell bodies and central axons ofmesencephalic trigeminal nucleus neurons supplying the jaw-closing muscles and periodontal tissue: a transganglionic tracer study in the cat, Brain Res., 359: 311-319. Paxinos, G. and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press. New York. Rail. W., G. Shepherd, T.S. Reese and M.W. Brightman (1966) Dendro-dendritic synaptic pathway for inhibition in the olfactory bulb, Exp. Neurol., 14: 44-56. Ralston, H.J. (1971) Evidence for presynaptic dendrites and a proposal for their mechanism of action. Nature, 230: 585-587. Ruigrok, T.J.H., A. Crowe and H.J.T. Donkelaar (19851 Dendrite distribution of identified motoneurons in the lumbar spinal cord of the turtle Pseudemys scripta elegans, J. Comp. NeuroL, 238: 275-285. Sasamoto, K. (1979) Motor nucleus representation of masticatory muscles in the rat, Jap. J. Physiol.. 29: 739-747. Scheibel, M.E. and A.B. Scheibel (1970) Organization of spinal motoneuron dendrites in bundles. Exp. Neurol., 28:106-112. Szekely, G. and C. Matesz (1987) Trigemmal motoneurons with disparate dendritic geometry innervate different muscle groups in the frog, Neurosci. Lett., 77: 161-165. Szentagothai, J. (1949) Functional representation in the motor trigeminal nucleus. J. Comp. Neurol.. 90: 111-120.
Travers. J. B. and R. Norgren (1983) Afferent projections to the normal and noradrenergically hyper-innervated trigeminal motor nucleus, J. Comp. Neurol., 220: 280-298. Trojanowski, J.O. Gonatas and N.K. Gonatas (1981a) A light and electronmicroscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system, J. Neurocytol., 10: 441-456. Trojanowski, J.Q., J.O. Gonatas and N.K. Gonatas (t981b) Conjugates of horseradish peroxidase with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers. Brain Res., 223: 381-385. Trojanowski, J. Q., J.O. Gonatas and N.K. Gonatas (1982) Horseradish peroxidase conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP, Brain Res., 231 : 33-50. Vedral, D. F. and H. A. Matzke (1967) Topographical localization of the muscles of mastication in the motor nucleus of the trigeminal nerve in the cat, J. Hirnforsch., 9: 565-569. Vornov, J.J. and J. Sutin (1983) Brain stem projections to the normal and noradrenergically hyperinnervated tngeminal motor nucleus, J. Comp. NeuroL, 214: 198-208. Wan, X. S.T., J.Q. Trojanowski, J.O. Gonatas and C. N. Liu (1982a) Dendritic distribution ofhypoglossal neurons, Exp. Neurol., 78: 167-175. Wan, X.S.T.. J.Q. Trojanowski and J.O. Gonatas (1982b) Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: their uptake and clearance, transganglionic and retrograde transport and sensitivity, Brain Res., 243: 215-224.
251
Elsevier
JNS 03033
Dendritic ramifications of trigeminal motor neurons innervating jaw-closing muscles of rats F. S. F. Mong 1, Y.C. Chen 2 and C . H . L u 3 ~Department of Anatomical Sciences, The University of Texas Dental Branch, Houston, TX (U.S.A.), 2Department of Anatomy, Sun Yat-Sen University of Medical Sciences, Guangzhou (People's Republic oJ China), and 3Department of Anatomy, Peking Union Medical University, Beijing (People's Republic of China) (Received 11 December, 1987) (Revised, received 12 April, 1988) (Accepted 12 April, 1988)
SUMMARY
A cholera toxin subunit conjugated horseradish peroxidase (CTHRP) was found to be very useful in labelling the dendrites of motoneurons. CTHRP was injected individually to jaw-closing muscles (temporalis, masseter, and medial pterygoid) of rats, and their motoneurons including the dendrites were labelled and studied. The results show that the motoneuron cell bodies innervating temporalis, masseter, and medial pterygoid muscles are located in the trigeminal motor nucleus in dorsal, ventromedial and ventrolateral position. The dendrites of these motoneurons extend radially into mesencephalic nucleus, supratrigeminal nucleus, pontine reticular formation, trigeminal sensory nucleus and even into the bundles of the ascending root of the facial nerve. These dendrites may serve as an extended surface for various synaptic contacts to the jaw closing motoneurons. The possibility that they may also have presynaptic influence on the input to the trigeminal motoneurons is also discussed.
Key words: Trigeminal motor nucleus; Jaw-closing motoneurons; Dendrites; Choleratoxin-conjugated HRP
Correspondence to: Dr. Franz S.F. Mong, Dept. of Anatomical Sciences, The University of Texas Dental Branch, P.O. Box 20068, Houston, TX 77225, U.S.A. Abbreviations: Ascending root of facial nerve, ARF; central canal, CC; cochlear nucleus, CN; facial nucleus, FN; genu of facial nerve, GF; inferior olive, IO; longitudinal fasciculus ofpons, LFP; mesencephalic nucleus of trigeminal, ME5; motor root of trigeminal, MR5; motor nucleus of trigeminal, MN5; pontine nuclei, PN; pontine reticular formation, PRF; periventricular gray, PVG; raphe nucleus, RN; superior olive, SO; superior salivary nucleus, S SN; supratrigeminal nucleus, STN; spinal tract of trigeminal, ST5; sensory nucleus of trigeminal, SN5; ventricle, V. 0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
252 INTRODUCTION There have been many studies on the mgeminal motor nucleus concerning the location of the cell bodies ofmotoneurons innervating the muscles of mastication. Early investigations (Szentagothai 1949; Vedral and Matzke 1967) using degeneration methods, showed that neurons innervating jaw-closing muscles were ventrally located in the trigeminal motor nucleus. Later, however, with accurate horseradish peroxidase (HRP) tracing technique, it was shown that these neurons were more dorsally located (Limwongse and DeSantis 1977). In fact, in the dorsal portion of the nucleus, the temporalis neurons are dorsal while the masseter and medial pterygoid neurons are ventromedially and ventrolaterally located (Mizuno et al. 1975; Sasamoto I979). Despite the accurate localization of these motor neurons in the trigeminal motor nucleus, their dendritic distribution to the nearby nuclear groups has not been thoroughly investigated. Dendrites provide an elaboration of the surface area of neurons. The traditional approach for studying dendrites is Golgi's silver impregnation technique (Scheibel and Scheibel 1970). Silver impregnation is usually non-specific. however, and since background dendrites stain simultaneously, interpretation of results is often difficult. In a recent communication (Szekely and Matesz 1987), the dendritic geometry of trigeminal motoneurons were studied by applying cobalt lysin solution to the central nerve stumps of various jaw muscles in the frog. However, the specific projections of dendrites into various nuclear groups were not mentioned, lntracellular microinjections of free HRP into various anterior horn cells of spinal cord were carried and their dendritic ramifications were studied by camera lucida drawings (Ruigrok et al. 1985; Cameron et al. 1983). Such laborous work has not been carried out for trigeminal motoneurons. Recently, it was found that if HRP was conjugated with subunit B of cholera toxin (CTHRP), it was 30-50 times more sensitivethan free HRP, particularly in labelling the dendrites ofmotoneurons (Trojanowski et al. 1981a,b, 1982; Wan et al. 1982a, b; Furicchia and Goshgarian 1987). The increased sensitivity would allow a minute quantity of CTHRP be injected to a specific muscle and prevent the problem of leakage to the surrounding muscles. Thus, labelling of undesired neurons and dendrites could be avoided. The specific mechanism of the increased sensitivity of CTHRP is unknown but was suggested due to the differences in the transport of CTHRP and free HRP across the neuronal cell membrane. The ligand cholera toxin bound to GM1 ganglioside receptor on neuronal cell membrane. This specific interaction between ligands and its receptor was followed by an active adsorptive endocytosis ofligand-conjugated HRP. In contrast, there were no receptors for free HRP and the transport was due to passive fluid phase endocytosis. The active process could transport probes in greater quantity and faster than the passive process (Trojanowski et al. 1981a, b, 1982: Wan et al. 1982a,b). Ledeen (1978) has observed a high concentration of GM 1 ganglioside receptors at the neuromuscular junctions. The purpose of the present investigation, therefore, is to inject CTHRP into jaw-closing muscles of the rat and to study the dendritic distribution of their motoneurons.
253 MATERIALS AND METHODS Thirty male Sprague-Dawley rats (350-500 g) were anesthetized with chloral hydrate (40 mg/100 g wt., i.p.). A small incision over the temporalis, masseter and medial pteryoid muscles was made and 5 #1 of 0.4~"o CTHRP (provided by Professor X.S.T. Wan, Peking Union Medical University, Beijing, China; for preparation of CTHRP, see Gonatas et al. 1979) was injected into each individual muscle. Only one muscle was injected in each rat. For control, a similar amount of saline was injected into corresponding muscles of control rats. All injections were made by means of a glass micropipette inserted into a polyethylene tube filled with glycerine and connected to a 50-/~1 Hamilton syringe. Jaw-closing muscles were chosen because they were large and easily accessible with minor surgery. Rarely was there any leakage. If any leakage was suspected, it was quickly absorbed with gauze followed by a saline rinse. Finally, the skin was closed. Survival time was 24 h as were the cases of previous studies (Trojanowski et al. 1981a,b, 1982; Wan et al. 1982a,b). The rats were anesthetized again with chloral hydrate (45 mg/100 g wt., i.p.) and perfused via the ascending aorta with 0.9~o saline followed by a fixative containing 1 Y/oparaformaldehyde, 1.25 ~/oglutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and finally with 10~ phosphate-buffered sucrose. The brain was removed after perfusion and immersed in 10 ~o phosphate-buffered sucrose solution overnight. The brain stem was then embedded in OCT compound (Lab. Tek product, Miles Laboratories, Inc.) and frozen sections (40 #m) were cut in a cryostat horizontally, sagittally, and coronally. Serial sections were collected in buffered sucrose solution and processed for HRP histochemistry according to Mesulam's TMB method (Mesulam 1978; 0.3 ~o H202). They were then mounted sequentially on chrome-alum coated slides, air dried, and counter-stained in 1~o neutral red solution. CTHRP injection sites have been thoroughly studied previously (Wan et al. 1982b) and therefore, was not repeated.
RESULTS Figures 1,2, and 3 are camera lucida drawings from the representative cross, coronal, and sagittal sections of brain stem respectively. Major nuclear groups are indicated for the purpose of orientation. These nuclear groups are identified using Paxinos and Watson's atlas (1986) as a reference. The dimensions of the trigeminal motor nucleus are 900 #m dorsoventrally, 780 #m mediolaterally, and 930 #m rostrocaudally. Saline-injected control rats showed no HRP reaction. Cross sections
In cross sections, the somata of temporalis muscle (Fig. 4) were located dorsally while that ofmasseter (Fig. 5) and medial pterygoid (Fig. 6) were located ventromedially and ventrolaterally, respectively. Their axons, forming the motor root of the trigeminal nerve (MR 5), exit ventrolaterally and rostrally.
254
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Figs. 1, 2 and 3. Camera lucida drawings of representative section of cross (Fig. t ), coronal (Fig. 2), and sagittat (Fig. 3) planes of brainstem. Major nuclear groups are labelled {see Abbreviations) for the orientation of the remaining photographs. Within the motor nucleus, open circles represent the location of motoneurons for temporalis muscle, triangles that for medial pterygoid muscle, and solid squares that for masseter muscle. The number of the symbols does not ihdicate the number of neurons. Shaded a r e a indicates the field where dendrites of j a w - d o s i n g motoneurons extend to. R = rostral. D = dorsal. L = lateral, C = caudal.
255
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PN
The dendrites of the temporalis motoneurons can be seen extending dorsally into trigeminal mesencephalic nucleus (ME 5) and supratrigeminal nucleus (STN, Fig. 7) and medially into pontine reticular formation just medial to the motor nucleus (PRF, Fig. 8). The dendrites of masseter motoneurons extend dorsally into supratrigeminal nucleus (STN), mesencephalic nucleus (ME 5), and medially into pontine reticular formation just medial to motor nucleus (PRF). Thus, the direction is similar to that of the temporalis motoneurons. The dendrites of medial pterygoid neurons expand across the field to the dorsomedial part of the nucleus (Fig. 9) and project into the areas as those of temporalis and masseter motoneurons. Coronal sections
In coronal sections, the dendrites of temporalis neuron extend rostrally and caudally into the pontine reticular formation (Fig. 10). They also extend laterally into the trigeminal sensory nucleus (Fig. 11). In Figs. 10 and 11, the axons of trigeminal ganglionic neurons travelling in the spinal trigeminal tract are also labelled. Termination of these axons in the spinal sensory nucleus is evident. Some caudally extending
/
Figs. 4, 5 and 6. Cross-sections o f p o n s showing locations ofternporalis neurons (Fig, 4), masseter neurons (Fig. 5), and medial pterygoid (Fig. 6). Note the somata of temporalis are dorsal while that o f m a s s e t e r and medial pterygoid are ventromedially and ventrotaterally located in the trigeminal motor nucleus. D = dorsal, L = lateral; bar = 200 t~m-
257
Figs. 7 and 8. Close-up view (10 x ) of cross-section of pons showing dendrites of temporalis neurons projecting close to mesencephalic nucleus (ME5) and to supratrigeminal nucleus (STN, Fig. 7) and into pontine reticular formation (PRF, Fig. 8). Arrows indicate dendrites. D = dorsal, L = lateral; bar = 90 um.
258
Fig. 9. Close-up view (10 x ) on cross-section of pons showing medial pterygoid neurons located on the ventrolateral portion of the trigeminal motor nucleus (MN5). Their axons (a) exit ventrolateraUy,while their dendrites (d) extend dorsomedially. Those dendrites projecting into mesenecphali¢ nucleus cannot be seen in this section, but in the adjacent sections. D = dorsal. L = lateral; bar = 90 era.
dendrites can be seen traversing into the bundles of the ascending root o f the facial nerve (Figs. 10 and 11). The dendrites o f the medial pterygoid motoneurons reach caudomedially into the pontine reticular formation and rostrolateraUy into the trigeminal sensory nucleus (Fig. 12). The distribution of dendrites ofmasseter motoneuron is similar to that of medial pterygoid neurons.
Sagittal sections In sagittal sections, all motor neurons have dendrites extending rostrally and caudally into the reticular formation. In addition, the temporalis neurons have dendrites extending dorsally into mesencephalic neurons (Fig. 13) while those of masseter
259
Fig. 10. Coronal section ofbrainstem showing neurons and their dendrites oftemporalis muscle (see Fig. 2 for orientation). Major nuclear groups are outlined (see Abbreviation). Note, the incoming axons (short arrows) from trigeminal ganglionic neurons are labelled and can be seen in spinal trigeminal tract (ST5). Some of them (long arrows) can be seen in the sensory trigeminal nucleus (SN5). Note that dendrites also extend rostrally and caudally into pontine reticular formation (PRF) and into the ascending root of the facial nerve (ARF). R = rostral, L = lateral; bar = 200 ktm.
ARF J
Fig. 11. Close-up view (10 × ) of an adjacent section of Fig. 10. Note dendrites (short arrows) extend into sensory trigeminal nucleus (SN5) and ascending root of facial nerve (ARF). Long arrows indicate the incoming axons oftrigeminal ganglionic neurons terminating in the sensory trigeminal nuclei (SN5), as that of Fig. 10. Bar = 90 #m.
260
It
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Fig. 12. Coronal section of brain stem showing medial pterygoid motoneurons and dendrites (see Fig.2 for orientation). Dendrites extend rostrolaterally into sensory trigeminal nucleus (SN5) and caudomedially into pontine reticular formation (PRF). R = rostral, L = lateral; bar = 200 #m. (Fig. 14) and medial pterygoid also have dendrites in the vicinity of mesencephalic neurons although not as clearly shown as those of the temporalis neurons.
DISCUSSION A few aspects of the methodology employed need to be addressed. The advantage of using C T H R P in labelling motoneurons and, particularly, its dendrites is the superior sensitivity of C T H R P over free H R P . This superiority was well documented (Trojanowski et al. 198 la,b, 1982; W a n et al. 1982a, b; Furicchia and Goshgarian 1987) and was not due to an artifact of the volume of the injected solution, o f the injection site area, or o f the survival time. The fact that C T H R P is 3 0 - 5 0 t i m e s more sensitive than free H R P could allow the investigator to reduce substantially the quantity o f C T H R P injected into a specific muscle and, thus, reduce the possibility o f leaking to the surrounding muscles resulting in labelling unrelated motoneurons. In our study, the unrelated neurons such as those o f facial nucleus or of nucleus ambiguus have never been observed. It is likely that a minute volume injected into a muscle may not label
261
Fig. 13. A close-up view (10 x ) of a sagittal section of brainstem showing temporalis motoneurons and dendrites (see Fig. 3 for orientation). Dendrites (arrow) extend into mesencephalic nucleus (ME5) which is just ventral to superior cerebellar peduncle (SCP). Note that mesencephalic neurons are labelled by CTHRP in this case. D = dorsal, R = rostral; bar = 90/~m. all motoneurons in the trigeminal motor nucleus. The purpose of the present investigation, however, was to delineate the dendritic ramification and not to quantify the number of motoneurons which was investigated previously (Limwongse and DeSantis 1977). With this superior sensitivity of C T H R P , further studies need to be carried out to establish the optimal maximal volume injected so that all motoneurons innervating a specific muscle could be labelled in the trigeminal motor nucleus. Lastly, gamma motoneurons were not studied in the present investigation as was the case in previous studies (Limwongse and DeSantis 1977; Mizuno et al. 1975). Muscle spindles are enclosed by a connective tissue capsule which might serve as a barrier for C T H R P to enter and to label either the gamma motoneurons or. mesencephalic neurons. This is probably the reason why, in our study, mesencephalic neurons were labelled only occasionally. In addition to the confirmation of previous studies in regard to the specific location of jaw-closing motor neurons in the trigeminal motor nucleus (Mizuno et al. 1975; Sasamoto 1979), the present investigation also reveals that the dendritic distributions of jaw-closing motor neurons are quite extensive. They extend radially from the
262
Fig. 14. A sagittal section of brain stem showing masseter motoneurons and dendrites t see Fig. 3 for orientation). A few dendrites (arrows) extending dorsally toward the mesencephalicnucleus (ME 5) which is not labelled by CTHRP. Other dendrites extend rostrally into pontine reticular formation(PRF). Axons (a) exit ventrorostrally. SCP = superior cerebellar peduncle, D = dorsal. R = rostral; bar = 200 ~tm. motor nucleus into mesencephalic nucleus, supratrigeminal nucleus, pontine reticular formation, and spinal trigeminal sensory nucleus. Some dendrites also extend caudally into the bundles of the ascending root of the facial nerve. On the other hand, previous studies showed that, in rats, the cortical input to trigeminal motor neurons was largely indirect and relayed via pontine reticular formation (Travers and Norgren 1983; Vornov and Satin 1983). Other inputs-into the trigeminal motor nucleus include mesencephalic nucleus, red nucleus (Travers and Norgren 1983), bilateral supratrigeminal nuclei (Mizuno et al. 1978), pontine raphe nucleus (Vornov and Sutin 1983) trigeminal sensory nucleus, and reticular formations of medulla (Travers and Norgren 1983; Vornov and Sutin 1983). Thus, the trigeminal motor nucleus can receive axonal inputs from various nuclear groups and projects, by way of its dendrites, into various nuclear groups in the vicinity. The nuclear groups further away from trigeminal motor nucleus (e.g,, red nucleus, medullary reticular formation) have no dendritic extensions from jaw-closing motoneurons. I t is possible that an extensive dendritic distribution would be necessary to collect the synaptic input necessary for the functions of the trigeminal motor nucleus, because the-nucleus is not
263 large and the neurons in it are closely packed. It is worthwhile to mention that physiological studies in cats showed that monosynaptic jaw reflexes are strong and yet, the contacts between mesencephalic axons and trigeminal motor neurons were very limited (Appenteng et al. 1978). Luschei (1987) speculated that the majority of these contacts might be on the dendrites extending out of the nucleus into the region above it (Luschei 1987). The present study, using rats, provides support for such an interpretation. The dendrites from trigeminal motor neurons in the rat project dorsally out of the nucleus into close vicinity of mesencephalic neurons. Alternatively, it is also possible that the dendritic extensions are responsible for the reciprocal modulations of axonal inputs. Trigeminal motor neurons not only receive axonal input from the nuclei mentioned above, but also project back to most of those nuclei, as revealed by the current study. Although the present study could not suggest that these dendrites might serve as presynaptic terminals, such a possibility was proposed by previous investigators, e.g., in the olfactory bulb (Hirata 1964; Andres 1965; Rail et al. 1966), lateral geniculate body (Guillery 1969; Lieberman 1973), medial geniculate body (Morest 1971), and thalamus (Harding 1971; Ralston 1971). It must be emphasized, however, that the cited examples of dendrites serving as presynaptic terminals are all sensory system receiving input from periphery. The current observation deals only with the motor output. Whether dendrites of motor system can serve as presynaptic terminals remains to be determined. The observation that the dendrites of trigeminal motor neurons extend to trigeminal sensory nucleus is interesting. In the present study, the central processes of trigeminal ganglionic neurons traveling in the spinal trigeminal tract and their termination in the trigeminal sensory nucleus could be visualized. Previous studies (Nomura and Mizuno 1985; Luschei 1987) showed that mesencephalic axons could also terminate in the trigeminal sensory nucleus. Thus, a synaptic arrangement involving mesencephalic terminals, incoming semilunar ganglionic terminals, the dendrites of trigeminal motoneurons and the neurons of sensory nucleus may take place.This possibility and the possibility that dendrites of trigeminal motoneurons might serve as presynaptic terminals are being studied by electron microscopy. REFERENCES Andres, K.H. (1965) Der Feinbau des Bulbus Olfactorius der Ratte, Z. Zellforsch. Mikrosk. Anat., 65: 530-561. Appenteng, K., M.J. O'Donovan, G. Somjen, J.A. Stephens and A. Taylor (1978) The projection of jaw elevator muscle spindle afferents to fifth nerve motoneurons in the cat, J. Physiol. (London), 279: 409-423. Cameron, W. E., D. B. Averill and A.J. Berger(1983) Morphologyof cat phrenic motor neurons as revealed by intracellular injection of horseradish peroxidase, J. Comp. Neurol., 219: 70-80. Furicchia, J.V. and H.G. Goshgarian (1987) Dendritic organization of phrenic motoneurons in the adult rat, Exp. Neurol., 96: 621-634. Gonatas, N.K., C. Harper, T. Mizutani and J.O. Gonatas (1979) Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem., 27: 728-734. Guillery, R.W. (1969) The organization of synaptic interconnections in the laminae of the dorsal lateral geniculate nucleus of the cat, Z. Zellforsch. Mikrosk. Anat., 96: 1-38. Harding, B.N. ( 1971) Dendro-dendritic synapses,including reciprocalsynapses,in the ventrolateralnucleus of the monkey thalamus, Brain Res., 34: 181-185.
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Travers. J. B. and R. Norgren (1983) Afferent projections to the normal and noradrenergically hyper-innervated trigeminal motor nucleus, J. Comp. Neurol., 220: 280-298. Trojanowski, J.O. Gonatas and N.K. Gonatas (1981a) A light and electronmicroscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system, J. Neurocytol., 10: 441-456. Trojanowski, J.Q., J.O. Gonatas and N.K. Gonatas (t981b) Conjugates of horseradish peroxidase with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers. Brain Res., 223: 381-385. Trojanowski, J. Q., J.O. Gonatas and N.K. Gonatas (1982) Horseradish peroxidase conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP, Brain Res., 231 : 33-50. Vedral, D. F. and H. A. Matzke (1967) Topographical localization of the muscles of mastication in the motor nucleus of the trigeminal nerve in the cat, J. Hirnforsch., 9: 565-569. Vornov, J.J. and J. Sutin (1983) Brain stem projections to the normal and noradrenergically hyperinnervated tngeminal motor nucleus, J. Comp. NeuroL, 214: 198-208. Wan, X. S.T., J.Q. Trojanowski, J.O. Gonatas and C. N. Liu (1982a) Dendritic distribution ofhypoglossal neurons, Exp. Neurol., 78: 167-175. Wan, X.S.T.. J.Q. Trojanowski and J.O. Gonatas (1982b) Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: their uptake and clearance, transganglionic and retrograde transport and sensitivity, Brain Res., 243: 215-224.