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The postganglionic parasympathetic fibers originating in the otic ganglion are distributed in several branches of the trigeminal mandibular nerve: an HRP study in the guinea pig
386
Brain Research, 411 (1987) 386-390 Elsevier
BRE 22266
The postgangtionic parasympathetic fibers otic gang.on are distributed in several tri inal mandibular the guinea
in of the udy in
Luis A.G. Segade, David Suarez Quintanilla and Jose Maria Suarez Nufiez Department of Anatomy, Faculty of Medicine. Santiago de Compostela (Spain) IAccepted 10 February 1987) Key words: Horseradish peroxidase- Otic ganglion; Trigeminal nerve: Salivary gland; Parasympathetic system: Guinea pig
Application of HRP to the proximal stumps of the ramifications of the trigeminal nerve shows that all those belonging to the mandibular branch contain parasympathetic fibers originating in the otic ganglion. The nerve with the largest proportion of these fibers is the auriculotemporal nerve (50-60% of all labeled neurons), while the smallest percentages are found in the lingual nerve and motor root (about 5% each). The presence of otic fibers in the inferior alveolar, mylohyoid, buccal and motor branches of the trigeminal nerve has not hitherto been reported. In the guinea-pig the otic ganglion (Arnold's ganglion) is located (Figs. 1 and 2c-f) in a position very similar to that of the human otic ganglion, lateroventral to the mandibular branch of the tngeminal nerve (whereas in rats it is separated from the fifth nerve by the basisphenoid bone3). It is generally accepted that this ganglion receives preganglionic parasympathetic fibers from the inferior salivatory nucleus, and that its postganglionic fibers innervate the parotid gland via the auriculotemporal nerve 5'12. Neurons belonging to the inferior salivatory nucleus have recently been located in the rhombencephalon by electrophysiological methods 9 or by retrograde transport of horseradish peroxidase 2"3"7"17-19"22"24.The axons projecting from this nucleus course peripherally in the glossopharyngeal and superior laryngeal nerves and in the cervical branch of the vagus nerve 19'24. The H R P studies of Contreras et al. 3 and Hamilton and Norgren 7 have confirmed that some terminate at the otic ganglion, which they presumably reach via the tympanic and lesser petrosal nerves. None of the above studies attempted detailed description of the course of the postganglionic fibers
originating in the otic ganglion, though Contreras et al. 3 confirmed the presence of otic ganglion fibers in the auriculotemporal nerve of the rat. and also found a few in the lingual nerve. Those studies in which H R P has been systematically applied to all the various branches of the mammalian trigeminal nerve have been concerned with the semilunar ganglion 1' 10.13 the trigeminal mesencephalic nucleus 6'15 or the description of trigeminal primary afferents ~ 10.14.z0.2~ As a step towards establishing the peripheral areas innervated by postganglionic otic ganglion fibers, we have undertaken to determine all those branches of the trigeminal nerve containing such fibers. Thirty-three female guinea pigs weighing 200-350 g were anesthetized with 15-25 mg/kg of Nembutal and supplementary doses of ethyl ether. The nerve branch to be studied was exposed, transected and carefully isolated from surrounding tissue with parafilm and cottonwool pads before crystals of H R P (Serva) were applied for 45-275 min to the proximal stump. The sites of H R P application in the ophthalmic, maxillary and mandibular nerve branches o f the fifth cranial nerve are shown in Fig. 1. The first few
Correspondence: L.A.G. Segade, Department of Anatomy, Faculty of Medicine. Santiago de Compostela. Spare. 0006-8993/87/$03.50 © 1987Elsevier Science Publishers B.V. (Biomedical Division)
387 mg of o-tolidine to 100 ml of a 0.225% solution of pyrocatechol in 0.05 M citric acid/ammonium acetate buffer of pH 4.85), and then treating them at room temperature for 15-20 min with a fresh batch of the same mixture supplemented with 35 ml/liter of 0.3%
","
,',:
.
.
My.
Fig. 1. The sites of HRP application in the branches of the trigeminal nerve, with the number of animals involved in each case. Au., auriculotemporal; Bu., buccal; C.T., chorda tympani; D.Ma., deep masseters; D.Te., deep temporals; Et., ethmoidal; Fr., frontal; G.g., semilunar ganglion (Gasser's ganglion); I.AI., inferior alveolar; In., infraorbital; Li., lingual; man. b., mandibular branch; max. b., maxillary branch; Me., mental; My., mylohyoid; Na., nasal; O.g., otic ganglion; oph.b., ophthalmic branch; Pt., pterygopalatine; S.Ma., superficial masseter; T.n., trigeminal nerve.
animals examined were allowed 48-72 h of postoperative survival, but subsequently this time was shortened to 24-40 h, which gave better labeling of otic ganglion neurons. Once the survival time had elapsed, the animals were reanesthetized and perfused through the ascending aorta, first with a physiological saline solution warmed to 37 °C and containing 10,000 IU of heparin and 0.125 % of amyl nitrite; and then, over 30 rain, with 400 ml of a 2.5% solution of glutaraldehyde in 0.1 M phosphate buffer of pH 7.2 followed by 400 ml of the same solution with 5% saccharose added. Gasser's ganglion and the otic ganglion were immediately removed in a single block, transferred to a 2.5% solution of glutaraldehyde in phosphate buffer with 30% of saccharose, and left for 6-8 h, at 2 - 4 °C. Horizontal 40 Bm sections were then cut serially in a cryostat, mounted on slides moistened with 0.75% gelatine and left to dry for 30-75 rain. In sections from the first few animals studied the presence of H R P was revealed by tetramethylbenzidine 16, but for most of the material the otolidine/pyrocatechol method was used 23. Essentially, this procedure consists of first dipping the sections for 5-10 rain in a preincubation medium (prepared by adding 1.75 ml of dimethylsulfoxide containing 50
H202. No labeled neurons were detected in the otic ganglion after application of H R P to ramifications of the ophthalmic and maxillary branches of the trigeminal nerve (specifically, the nasal, frontal and infraorbital-superior alveolar nerves), but when the mandibular branch was investigated labeled neurons were observed in the ganglion, no matter on which nerve the H R P had been deposited. When application had taken place close to the otic and semilunar ganglia (for example, on investigating the auriculotemporal or buccal nerves (Fig. 2a, d) or the anterior mandibular root (Fig. 2f)), artifactual perivascular, glial and pial cell uptake in the otic ganglion and surrounding nerves was evident, and clusters of erythrocytes appeared. However, HRP-bearing neurons were easily distinguished on the basis of cellular morphology, location and fine diffuse-granular pattern of reaction product deposition. The greatest number of nerve cells were labeled when H R P was applied to the auriculotemporal nerve (Table I). This population made up some 50-60% of the total number of cells labeled after treatment of the various trigeminal mandibular branches. Next in order of HRP-bearing neuron population were the inferior alveolar and buccal nerves, followed by the mylohyoid nerve. The ramifications for which application of H R P produced the smallest numbers of labeled ganglion cells were the lingual nerve and the trigeminal motor root (the deep temporal and masseter nerves). The present findings are the first anatomical evidence that postganglionic fibers from the otic ganglion enter all the ramifications of the trigeminal mandibular nerve. It is well known that at least some of the postganglionic fibers originating in the otic ganglion stimulate secretion in the parotid gland, which they are assumed to reach via the auriculotemporal nerve -~'~2. Our results show that in the guinea pig 5 0 - 6 0 % of the otic parasympathetic fibers entering the mandibular division course with this nerve. A large number of these fibers probably have secretomotor function in the parotid. However. besides the parotid,
388
H Fig. 2. HRP-labeled cells in the otic ganglion after application of the enzyme to the proximal stumps of the following tngeminal mandibular nerves: the auriculotemporal (a), the anterior mandibular branch on exit from the foramen ovale (b), the inferior alveolar (c), the buccal (d), the lingual (e) and the common root of the deep temporals and the masseters (f-g). Scales: 200~m in a and b, and 500 /tin in c-f (g is an enlargement of the otic ganglion shown in f). All sections were processed by the o-tolidine/pyrocatechol method: In a and b, elongate arrows show HRP-bearing parasympathetic neurons and short arrows non-neuronallabeledcetls. The labeled fibers in d belong to the medial temporal nerve, which in some guinea pigs (not those used to investigate the temporals!) is joined at the root to the buccal nerve, from which it cannot be separated, bu., buccal fibers; G.g., Gasser's ganglion; O.g., otic ganglion; p.mand~b., posterior mandibular branch. s u b m a n d i b u l a r and sublingual glands, there are also numerous small salivary glands in the buccal mucosa, whose lower region is i n n e r v a t e d chiefly by the buccal and inferior alveolar nerves and to a lesser extent by the mylohyoid. It therefore seems likely that the otic ganglion n e u r o n s labeled by administration of
H R P to these nerves stimulate salivation in these smaller glands. It should be borne in mind that the territories of the 3 nerves overlap; the tooth pu!p, for example, being innervated by both the inferior alveolar and mylohyoid nerves251 The fact that of the 3 it is the mylohyoid which innervates the smallest area
389 TABLE I Mean number of neurons labeled in the otic ganglion after HRP administration to the branches of the trigeminal mandibular nerve (counts were made on sections prepared by the o-tolidine/ pyrocatechol method) Nerve
No. of valid animals
No. of labeled neurons per animal (mean +_S.D.)
Auriculotemporal Inferior alveolar Buccal Mylohyoid Lingual Motor branches (masseters + deep temporals) Anterior mandibular nerve (buccal + masseters + deep temporals)
3 2 3 2 2
2296 _+279 451 _+73 415 _+60 314 ± 53 231 _+41
2
170 + 41
2
518 + 84
would explain why relatively few neurons were labeled after deposition of tracer on this nerve. The presence of a small n u m b e r of otic fibers in the lingual nerve, which has already been r e p o r t e d in the rat 3, suggests that the secretory activity of the submandibular and sublingual glands may be controlled by both the superior and inferior salivatory nuclei. Such dual control is already suspected in the case of the parotid gland, which appears to be related to both the inferior nucleus (via fibers synapsing in the otic ganglion after coursing in the glossopharyngeal nerve) and the superior nucleus (via the facial nerve s and, presumably, the chorda tympani4). One of our future objectives is to resolve these questions by examining the otic ganglion and brainstem labeling following application of H R P to the parotid, submandibular and sublingual glands.
1 Arvidsson, J. and Gobel, S., An HRP study of the central projections of primary trigeminal neurons which innervate tooth pulps in the cat, Brain Research, 210 (1981) 1-16. 2 Bradley, R.M., Mistretta, Ch.M., Bates, C.A. and Killackey, H.P., Transganglionic transport of HRP from the circumvallate papilla of the rat, Brain Research, 361 (1985) 154-161. 3 Contreras. R.J., Gomez, M.M. and Norgren, R., Central origins of cranial nerve parasympathetic neurons in the rat, J. Comp. Neurol., 190 (1980) 373-394. 4 Diamant, H. and Wilberg, A., Does the chorda tympani in man contain secretory fibres for the parotid gland? Acta Oto-Larvngol., 6(I (1965) 255-264. 5 Gabella, G., Structure o f the Autonomic Nervous System, Chapman and Hall, London, 1976, 214 pp.
W h e n tracer was applied to the m o t o r root (masseter and deep temporal nerves), extreme care was taken to isolate them from the buccal nerve and so prevent its taking up any stray H R P . Fig. 2f shows that only fibers entering the m o t o r branches were in fact labeled. F u r t h e r m o r e , labeled neurons continued to a p p e a r in the otic ganglion when H R P was applied further along the deep masseter, superficial masseter and deep temporal nerves; and, in another set of experimental animals, 3 0 - 4 0 labeled cells were also observed when the tracer was injected directly into individual masticatory muscles. It is therefore evident that not all the fibers originating in the otic ganglion stimulate salivation. Indeed, this might have been expected, since histochemical studies of other cranial parasympathetic ganglia tl have shown them to contain more than one kind of neuron. In the present case, it seems likely that the few postganglionic otic fibers running to the masticatory muscles may have vasodilatory function, thus complementing the action of the s e c r e t o m o t o r fibers on the salivary glands by facilitating the supply of energy to the masticatory muscles. In conclusion, the results of this study clearly show that the otic ganglion innervates not only the parotid gland, but also other areas of the orofacial region. These probably comprise the s u b m a n d i b u l a r and sublingual glands, other salivary glands in the buccal mucosa, and the blood vessels of the masticatory muscles.
This work was carried out with the aid of a research grant from the 'Instituto de Ciencias Neurol6gicas Barri6 de la Maza'.
6 Gottlieb, S., Taylor, A. and Bosley, M.A., The distribution of afferent neurones in the mesencephalic nucleus of the fifth nerve in the cat, J. Comp. Neurol., 228 (1984) 273-283. 7 Hamilton, R.B. and Norgren, R., Central projections of gustatory nerves in the rat, J. Comp. NeuroL, 222 (1984) 560-577. 8 Holmberg, J., On the nerves of the parotid gland. In N. Emmelin and Y. Zonerman (Eds.), Oral Physiology, Pergamon, Oxford, 1972, pp. 17-19. 9 Ishizuka, K.I. and Murukami, T., Responses of inferior salivatory neurons to stimulation of trigeminal sensory branches, Exp. Neurol., 91 (1986) 269-276. 10 Jacquin, M.F., Semba, K., Egger, M.D. and Rhoades, R.W., Organization of HRP-labeled trigeminal mandibular
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14
15
16
17
18
primary afferent neurons in the rat, J. Comp. Neurol., 215 (1983) 397-420. Koelle, W.A. and Koelle G.B., The localization of external or functional acetylcholinesterase at the synapses of autonomic ganglia, J. Pharmacol. Exp. Ther., 126 (1959) 1-8. Lazorthes, G., Le SystOme Nerveux P~riphOrique. Masson, Paris, 1971. Marfurt, C.F., The somatotopic organization of the cat trigeminal ganglion as determined by the horseradish peroxidase technique, Anat. Rec.. 201 (1981) 105-118. Marfurt, C.F., The central projections of trigeminal primary afferent neurons in the cat as determined by the transganglionic transport of horseradish peroxidase, J. Comp. Neurol., 203 (1981) 785-798. Matesz, C., Peripheral and central distribution of fibres of the mesencephalic trigeminal root in the rat, Neurosci. Lett., 27(1981) 13-17. Mesulam, M.-M., Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neuronal pathways - - axonal transport, enzyme histochemistry and light microscopic analysis. In M.-M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley, Chichester, 1982, pp. 1-152. Nicholson, J.E. and Severin, C.M., The superior and inferior salivatory nuclei in the rat, Neurosci. Lett., 21 (198t) 149-154. Nomura, S. and Mizuno, N., Central distribution of afferent and efferent components of the glossopharyngeal
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nerve: an H R P study in the cat. Brain Research. 236 (1982~ 1-t3 Nomura. S and Mizuno. N.. Central distribution of efferent and afferent components of the cervical branches of the vagus nerve: an H R P study in the cat. Anat. Ernbrvol.. 166 11983) 1-18. Nomura. S.. Yasui Y.. Takada. M. ,rod Mizuno, N.. lrigeminal primary afferent neurons projecting directly to the solitary nucleus in the cat: a transganglionic and retrograde horseradish study, Neurosci. Lett.. 50 (19841 257 262 Panneton. W.M and Burton. It_. Corneal and periocular representation within the trigeminal sensory complex in the cat studied with transganglionic transport of horseradish peroxidase. J. Comp Neurol.. 199 ( 19811327 -344 Satomi H.. Yamamoto. T.. Ise. 11 and ]~akahashi. K.. Identification of the inferior salivatorv nucleus in the cat as studied by H R P bathings of the transected glossopharyngem nerve root. Neurosci. Lett. 11 I [0791 259-263. Segade, L A G . . Pyrocatechot a s , stabilizing agent for otolidine and o-dianisidine: a sensitive new method for H R P neurohistochemistry, J. Hirnforseh.. m press. Sweazy, R D . and Bradley, R.M., ( e n t r a l connectmns ot the lingualtonsillar branch of the gloss~pharyngeal nerve and the superior laryngeal nerve in lamb. J Comp. Neurol.. 245 ~1986~ 471-482. Wilson. S.. Johns. P and Fuller. P.M., Accessory mnervanon of mandibular anterior teeth in cats: a horseradish peroxidase study. Brain Research. 29811984~ 392-396.
Brain Research, 411 (1987) 386-390 Elsevier
BRE 22266
The postgangtionic parasympathetic fibers otic gang.on are distributed in several tri inal mandibular the guinea
in of the udy in
Luis A.G. Segade, David Suarez Quintanilla and Jose Maria Suarez Nufiez Department of Anatomy, Faculty of Medicine. Santiago de Compostela (Spain) IAccepted 10 February 1987) Key words: Horseradish peroxidase- Otic ganglion; Trigeminal nerve: Salivary gland; Parasympathetic system: Guinea pig
Application of HRP to the proximal stumps of the ramifications of the trigeminal nerve shows that all those belonging to the mandibular branch contain parasympathetic fibers originating in the otic ganglion. The nerve with the largest proportion of these fibers is the auriculotemporal nerve (50-60% of all labeled neurons), while the smallest percentages are found in the lingual nerve and motor root (about 5% each). The presence of otic fibers in the inferior alveolar, mylohyoid, buccal and motor branches of the trigeminal nerve has not hitherto been reported. In the guinea-pig the otic ganglion (Arnold's ganglion) is located (Figs. 1 and 2c-f) in a position very similar to that of the human otic ganglion, lateroventral to the mandibular branch of the tngeminal nerve (whereas in rats it is separated from the fifth nerve by the basisphenoid bone3). It is generally accepted that this ganglion receives preganglionic parasympathetic fibers from the inferior salivatory nucleus, and that its postganglionic fibers innervate the parotid gland via the auriculotemporal nerve 5'12. Neurons belonging to the inferior salivatory nucleus have recently been located in the rhombencephalon by electrophysiological methods 9 or by retrograde transport of horseradish peroxidase 2"3"7"17-19"22"24.The axons projecting from this nucleus course peripherally in the glossopharyngeal and superior laryngeal nerves and in the cervical branch of the vagus nerve 19'24. The H R P studies of Contreras et al. 3 and Hamilton and Norgren 7 have confirmed that some terminate at the otic ganglion, which they presumably reach via the tympanic and lesser petrosal nerves. None of the above studies attempted detailed description of the course of the postganglionic fibers
originating in the otic ganglion, though Contreras et al. 3 confirmed the presence of otic ganglion fibers in the auriculotemporal nerve of the rat. and also found a few in the lingual nerve. Those studies in which H R P has been systematically applied to all the various branches of the mammalian trigeminal nerve have been concerned with the semilunar ganglion 1' 10.13 the trigeminal mesencephalic nucleus 6'15 or the description of trigeminal primary afferents ~ 10.14.z0.2~ As a step towards establishing the peripheral areas innervated by postganglionic otic ganglion fibers, we have undertaken to determine all those branches of the trigeminal nerve containing such fibers. Thirty-three female guinea pigs weighing 200-350 g were anesthetized with 15-25 mg/kg of Nembutal and supplementary doses of ethyl ether. The nerve branch to be studied was exposed, transected and carefully isolated from surrounding tissue with parafilm and cottonwool pads before crystals of H R P (Serva) were applied for 45-275 min to the proximal stump. The sites of H R P application in the ophthalmic, maxillary and mandibular nerve branches o f the fifth cranial nerve are shown in Fig. 1. The first few
Correspondence: L.A.G. Segade, Department of Anatomy, Faculty of Medicine. Santiago de Compostela. Spare. 0006-8993/87/$03.50 © 1987Elsevier Science Publishers B.V. (Biomedical Division)
387 mg of o-tolidine to 100 ml of a 0.225% solution of pyrocatechol in 0.05 M citric acid/ammonium acetate buffer of pH 4.85), and then treating them at room temperature for 15-20 min with a fresh batch of the same mixture supplemented with 35 ml/liter of 0.3%
","
,',:
.
.
My.
Fig. 1. The sites of HRP application in the branches of the trigeminal nerve, with the number of animals involved in each case. Au., auriculotemporal; Bu., buccal; C.T., chorda tympani; D.Ma., deep masseters; D.Te., deep temporals; Et., ethmoidal; Fr., frontal; G.g., semilunar ganglion (Gasser's ganglion); I.AI., inferior alveolar; In., infraorbital; Li., lingual; man. b., mandibular branch; max. b., maxillary branch; Me., mental; My., mylohyoid; Na., nasal; O.g., otic ganglion; oph.b., ophthalmic branch; Pt., pterygopalatine; S.Ma., superficial masseter; T.n., trigeminal nerve.
animals examined were allowed 48-72 h of postoperative survival, but subsequently this time was shortened to 24-40 h, which gave better labeling of otic ganglion neurons. Once the survival time had elapsed, the animals were reanesthetized and perfused through the ascending aorta, first with a physiological saline solution warmed to 37 °C and containing 10,000 IU of heparin and 0.125 % of amyl nitrite; and then, over 30 rain, with 400 ml of a 2.5% solution of glutaraldehyde in 0.1 M phosphate buffer of pH 7.2 followed by 400 ml of the same solution with 5% saccharose added. Gasser's ganglion and the otic ganglion were immediately removed in a single block, transferred to a 2.5% solution of glutaraldehyde in phosphate buffer with 30% of saccharose, and left for 6-8 h, at 2 - 4 °C. Horizontal 40 Bm sections were then cut serially in a cryostat, mounted on slides moistened with 0.75% gelatine and left to dry for 30-75 rain. In sections from the first few animals studied the presence of H R P was revealed by tetramethylbenzidine 16, but for most of the material the otolidine/pyrocatechol method was used 23. Essentially, this procedure consists of first dipping the sections for 5-10 rain in a preincubation medium (prepared by adding 1.75 ml of dimethylsulfoxide containing 50
H202. No labeled neurons were detected in the otic ganglion after application of H R P to ramifications of the ophthalmic and maxillary branches of the trigeminal nerve (specifically, the nasal, frontal and infraorbital-superior alveolar nerves), but when the mandibular branch was investigated labeled neurons were observed in the ganglion, no matter on which nerve the H R P had been deposited. When application had taken place close to the otic and semilunar ganglia (for example, on investigating the auriculotemporal or buccal nerves (Fig. 2a, d) or the anterior mandibular root (Fig. 2f)), artifactual perivascular, glial and pial cell uptake in the otic ganglion and surrounding nerves was evident, and clusters of erythrocytes appeared. However, HRP-bearing neurons were easily distinguished on the basis of cellular morphology, location and fine diffuse-granular pattern of reaction product deposition. The greatest number of nerve cells were labeled when H R P was applied to the auriculotemporal nerve (Table I). This population made up some 50-60% of the total number of cells labeled after treatment of the various trigeminal mandibular branches. Next in order of HRP-bearing neuron population were the inferior alveolar and buccal nerves, followed by the mylohyoid nerve. The ramifications for which application of H R P produced the smallest numbers of labeled ganglion cells were the lingual nerve and the trigeminal motor root (the deep temporal and masseter nerves). The present findings are the first anatomical evidence that postganglionic fibers from the otic ganglion enter all the ramifications of the trigeminal mandibular nerve. It is well known that at least some of the postganglionic fibers originating in the otic ganglion stimulate secretion in the parotid gland, which they are assumed to reach via the auriculotemporal nerve -~'~2. Our results show that in the guinea pig 5 0 - 6 0 % of the otic parasympathetic fibers entering the mandibular division course with this nerve. A large number of these fibers probably have secretomotor function in the parotid. However. besides the parotid,
388
H Fig. 2. HRP-labeled cells in the otic ganglion after application of the enzyme to the proximal stumps of the following tngeminal mandibular nerves: the auriculotemporal (a), the anterior mandibular branch on exit from the foramen ovale (b), the inferior alveolar (c), the buccal (d), the lingual (e) and the common root of the deep temporals and the masseters (f-g). Scales: 200~m in a and b, and 500 /tin in c-f (g is an enlargement of the otic ganglion shown in f). All sections were processed by the o-tolidine/pyrocatechol method: In a and b, elongate arrows show HRP-bearing parasympathetic neurons and short arrows non-neuronallabeledcetls. The labeled fibers in d belong to the medial temporal nerve, which in some guinea pigs (not those used to investigate the temporals!) is joined at the root to the buccal nerve, from which it cannot be separated, bu., buccal fibers; G.g., Gasser's ganglion; O.g., otic ganglion; p.mand~b., posterior mandibular branch. s u b m a n d i b u l a r and sublingual glands, there are also numerous small salivary glands in the buccal mucosa, whose lower region is i n n e r v a t e d chiefly by the buccal and inferior alveolar nerves and to a lesser extent by the mylohyoid. It therefore seems likely that the otic ganglion n e u r o n s labeled by administration of
H R P to these nerves stimulate salivation in these smaller glands. It should be borne in mind that the territories of the 3 nerves overlap; the tooth pu!p, for example, being innervated by both the inferior alveolar and mylohyoid nerves251 The fact that of the 3 it is the mylohyoid which innervates the smallest area
389 TABLE I Mean number of neurons labeled in the otic ganglion after HRP administration to the branches of the trigeminal mandibular nerve (counts were made on sections prepared by the o-tolidine/ pyrocatechol method) Nerve
No. of valid animals
No. of labeled neurons per animal (mean +_S.D.)
Auriculotemporal Inferior alveolar Buccal Mylohyoid Lingual Motor branches (masseters + deep temporals) Anterior mandibular nerve (buccal + masseters + deep temporals)
3 2 3 2 2
2296 _+279 451 _+73 415 _+60 314 ± 53 231 _+41
2
170 + 41
2
518 + 84
would explain why relatively few neurons were labeled after deposition of tracer on this nerve. The presence of a small n u m b e r of otic fibers in the lingual nerve, which has already been r e p o r t e d in the rat 3, suggests that the secretory activity of the submandibular and sublingual glands may be controlled by both the superior and inferior salivatory nuclei. Such dual control is already suspected in the case of the parotid gland, which appears to be related to both the inferior nucleus (via fibers synapsing in the otic ganglion after coursing in the glossopharyngeal nerve) and the superior nucleus (via the facial nerve s and, presumably, the chorda tympani4). One of our future objectives is to resolve these questions by examining the otic ganglion and brainstem labeling following application of H R P to the parotid, submandibular and sublingual glands.
1 Arvidsson, J. and Gobel, S., An HRP study of the central projections of primary trigeminal neurons which innervate tooth pulps in the cat, Brain Research, 210 (1981) 1-16. 2 Bradley, R.M., Mistretta, Ch.M., Bates, C.A. and Killackey, H.P., Transganglionic transport of HRP from the circumvallate papilla of the rat, Brain Research, 361 (1985) 154-161. 3 Contreras. R.J., Gomez, M.M. and Norgren, R., Central origins of cranial nerve parasympathetic neurons in the rat, J. Comp. Neurol., 190 (1980) 373-394. 4 Diamant, H. and Wilberg, A., Does the chorda tympani in man contain secretory fibres for the parotid gland? Acta Oto-Larvngol., 6(I (1965) 255-264. 5 Gabella, G., Structure o f the Autonomic Nervous System, Chapman and Hall, London, 1976, 214 pp.
W h e n tracer was applied to the m o t o r root (masseter and deep temporal nerves), extreme care was taken to isolate them from the buccal nerve and so prevent its taking up any stray H R P . Fig. 2f shows that only fibers entering the m o t o r branches were in fact labeled. F u r t h e r m o r e , labeled neurons continued to a p p e a r in the otic ganglion when H R P was applied further along the deep masseter, superficial masseter and deep temporal nerves; and, in another set of experimental animals, 3 0 - 4 0 labeled cells were also observed when the tracer was injected directly into individual masticatory muscles. It is therefore evident that not all the fibers originating in the otic ganglion stimulate salivation. Indeed, this might have been expected, since histochemical studies of other cranial parasympathetic ganglia tl have shown them to contain more than one kind of neuron. In the present case, it seems likely that the few postganglionic otic fibers running to the masticatory muscles may have vasodilatory function, thus complementing the action of the s e c r e t o m o t o r fibers on the salivary glands by facilitating the supply of energy to the masticatory muscles. In conclusion, the results of this study clearly show that the otic ganglion innervates not only the parotid gland, but also other areas of the orofacial region. These probably comprise the s u b m a n d i b u l a r and sublingual glands, other salivary glands in the buccal mucosa, and the blood vessels of the masticatory muscles.
This work was carried out with the aid of a research grant from the 'Instituto de Ciencias Neurol6gicas Barri6 de la Maza'.
6 Gottlieb, S., Taylor, A. and Bosley, M.A., The distribution of afferent neurones in the mesencephalic nucleus of the fifth nerve in the cat, J. Comp. Neurol., 228 (1984) 273-283. 7 Hamilton, R.B. and Norgren, R., Central projections of gustatory nerves in the rat, J. Comp. NeuroL, 222 (1984) 560-577. 8 Holmberg, J., On the nerves of the parotid gland. In N. Emmelin and Y. Zonerman (Eds.), Oral Physiology, Pergamon, Oxford, 1972, pp. 17-19. 9 Ishizuka, K.I. and Murukami, T., Responses of inferior salivatory neurons to stimulation of trigeminal sensory branches, Exp. Neurol., 91 (1986) 269-276. 10 Jacquin, M.F., Semba, K., Egger, M.D. and Rhoades, R.W., Organization of HRP-labeled trigeminal mandibular
390
ll
12 13
14
15
16
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