Brain Research, 137 (1977) 145-149 (C) Elsevier/North-Holland Biomedical Press
145
Short Communications
Salivatory neurons innervate the submandibular and sublingual glands in the rat: horseradish peroxidase study
TOHRU HIURA Hiura Dental Hospital, 18-3 Akitamachi, Tokushimashi 770 (Japan)
(Accepted July 21st, 1977)
There are many anatomical 4,5,7,s and physiologicaP, 2,6 studies done on the bulbar salivary center by many workers. Among these studies, using the method of retrograde degeneration after lesions involving the chorda tympani and the tympanic branch of the glossopharyngeal nerve in the dog 7,8 and kitten 5, it has been reported that the salivary center was situated in the lateral reticular formation of the ipsilateral side of the lesion. On the contrary, using the histochemical technique for cholinesterase, Shute and Lewis 4 reported that the rat's salivary center was a collection of nerve cells flanking the lateral and medial aspect of the facial genu at its rostral end and there were no salivary cells in the lateral reticular formation. Therefore, the present study was taken in an attempt to clarify these discrepancies between the results obtained by the retrograde degeneration method and those of the histochemical technique for cholinesterase. In the present study, the method based on the retrograde axonal transport of horseradish peroxidase (HRP) was adopted in the rat. This study was carried out in 10 Sprague-Dawley rats weighing about 80 g. The H R P was applied to the preganglionic fibers existed along the duct of the submandibular gland. Namely, under pentobarbital anesthesia, the preganglionic fibers with the duct of the submandibular gland were crushed with a forceps or cut with a pair of scissors. The H R P was applied in dry substance around the crushed or cut area of the nerves time and again over a period of 2-3 h. After 2 days' survival the animals were reanesthetized with pentobarbital and fixed by transcardiac perfusion with physiologic saline followed by a mixture of 2 ~ paraformaldehyde and 1.25~ glutaraldehyde in 0.1 M phosphate buffer. The brain was removed from the skull and fixed some hours in the same fixative. Then, the brain was placed in 0.1 M phosphate buffer containing 5 ~ sucrose and kept at 4 °C for 24-48 h and frozen sections were cut at 50/zm in transverse plane. The sections were transferred from 5 700phosphate buffered sucrose to 0.2 M Tris buffer solution for 20 min. They were then incubated for 10 min at room temperature in a bath containing 0.05 ~o 3,3'-diaminobenzidine tetrahydrochloride and 0.01 ~ H202 in 0.2 M Tris buffer. Following this, they were washed three
146
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Fig. 1. Diagrams of the locations of HRP-labeled neurons after the application of HRP to the submandibular branch of chorda tympani. Abbreviations: C.r., corpus restiforme; D.v.n., descending vestibular nucleus; F.s., solitary tract; G.VII, genu of facial nerve; M.v.n., medial vestibular nucleus; N.c.tr., nucleus corporis trapezoidei; N.Deit., Deiters', nucleus; N.f.s., nucleus of the solitary tract; N.VIII, nucleus cochlearis ventralis; Ol.s., nucleus olivaris superior; r.f. VII aft., afferent fibers in 7th cranial nerve; Tr.sp.V, spinal trigeminal tract; VII, nucleus n. facialis.
147
Fig. 2. Dark-field photomicrographs of HRP-labeled neurons at the level of Fig. 12. These cells are located in the lateral aspect of the reticular formation. × 200. times in distilled water and mounted on slides with alcohol gelatine. They were counterstained with cresylecht violet. As the results of the observation, the H R P labeled cells could be seen in all cases. The fact that most labeled cells could be seen is shown in Fig. 1. Fig. 11 6 is illustrated at 150 #m intervals and each figure is a composite of the location of the H R P labeled cells appearing in 3 serial sections. The H R P labeled cells were seen in the reticular formation at the level between the most caudal end of the root of the facial nerve and the rostral part of the facial nucleus. At the most rostral level (Fig. 11), 4 labeled cells were seen in the reticular formation medial to the most caudal end of the root of the facial nerve. Twenty-six and 14 labeled cells could be seen at the level of Fig. 12 and Fig. 13 respectively. The afferent fibers of the facial nerve that transverse the dorsal part of the spinal trigeminal tract and the most rostral part of the facial m o t o r nucleus appears in Fig. 12, and the most rostral part of the solitary tract is seen in Fig. Is. These cells illustrated in Fig. 12,3 were seen in the reticular formation ventral to Deiters' nucleus, medial to the spinal trigeminal nucleus and dorsal to the facial motor nucleus. The distribution of these cells extended in the dorsoventral direction of the lateral aspect of the reticular formation. At the level of Fig. 14,5.6, the labeled cells were seen in the area of caudal prolongation of the same region of rostral ones, but decreased in number (a total of 5). Almost all of the labeled cells were the medium-sized
148 multipolar type (15-20/~m diameter), but only 2 cells (of a total of 49) were small-size d (7-10 # m diameter, Fig. 2). The results obtained in the rest of the cases were identical to those shown in Fig. 1. Yagita and Hayama 7 observed the chromatolytic cells after the incision of the chorda tympanic nerve in the ipsilateral lateral reticular formation ventromedial to Deiters' nucleus and dorsal to the facial motor nucleus in dog. In addition, Yagita 8 reported that retrograde cellular degeneration occurred in the lateral reticular formation at the level of the nucleus ambiguus and the facial motor nucleus after cutting the tympanic branch of the glossopharyngeal nerve in dog. Torvik 5 obtained the same results of Yagita and Hayama 7, and Yagita s in kittens. Namely, he reported that retrograde cellular degeneration occurred in the medium- or small-sized cells in the lateral reticular formation which was in accordance with the nucleus parvicellularis named by Meessen and Olszewski 3. On the contrary, Shute and Lewis 4 have described that, by the histochemical technique for cholinesterase, the salivary center was a collection of nerve cells flanking the lateral and medial aspect of the facial genu at its rostral end and there were no salivatory cells in the lateral reticular formation. From the results of the present study, the salivary cells that give rise to preganglionic fibers to the submandibular and sublingual glands were localized in the ipsilateral reticular formation. The results support the findings of Yagita and Hayama 7 and Torvik 5. However, no labeled cells could be seen in the area demonstrated as the salivary nucleus by Shute and Lewis 4. Therefore, it is doubtful that a collection of nerve cells demonstrated by the histochemical technique for cholinesterase are the real salivary ceils. Salivary secretion can be obtained by Wang 6 in cat, and Magoun and Beaton 2 in monkey, by electrophysiological methods. The points where salivary secretion can be obtained by electrical stimulus were more widely distributed than the area confirmed by the retrograde cellular degeneration and HRP methods. Therefore, it seems that these points contain some neural elements other than the salivary cells, such as the solitary fasciculus and its nucleus and the trigeminal nuclear complex. From the results of the present study in the rat, it can be said that the salivary cells that give rise to preganglionic fibers to the submandibular and sublingual glands are localized in the ipsilateral reticular formation, which is in accordance with the rostral part of the nucleus parvicellularis named by Meessen and Olszewski 3. There remains, however, the possibility that the salivary nucleus by Shute and Lewis a has some relation to salivary secretion, as the absence of HRP labeling in neuron cell bodies should not be considered conclusive evidence that those cells do not supply a projection to the injected site.
1 Kawamura, Y. and Funakoshi, M., Studies on the neurophysiologic mechanisms of salivary secretion, Med. J. Osaka Univ., 8 (1958) 433443. 2 Magoun, H. W. and Beaton, L. E., The salivatory motor nuclei in the monkey, "4mer. J. Physiol., 136 (1942) 720-725. 3 Meessen, H. and Olszweski, J., ,4 Cytoarchitectonic Atlas o f the Rhombencephalon o f the Rabbit, S. Karger, Basel, 1949. 4 Shute, C. C. D. and Lewis, P. R., The salivatory centre in the rat, J. Anat. (Lond.), 94 (1960) 59-73.
149 5 Torvik, A., Die Lokalisation des Speichelzentrum bei der Katze, Z. mikr.-anat. Forsch., 63 (1957) 317-326. 6 Wang, S. C., Localization of the salivatory center in the medulla of the cat, J. Neurophysiol., 6 (1943) 195-202. 7 Yagita, K. and Hayama, S., Uber das Speichelsekretionscentrum, Neurol. Zbl., 28 (1909) 738-753. 8 Yagita, K., Weitere Untersuchungen fiber das Speichelzentrum, Anat. Ariz., 35 (1910) 70--75.
145
Short Communications
Salivatory neurons innervate the submandibular and sublingual glands in the rat: horseradish peroxidase study
TOHRU HIURA Hiura Dental Hospital, 18-3 Akitamachi, Tokushimashi 770 (Japan)
(Accepted July 21st, 1977)
There are many anatomical 4,5,7,s and physiologicaP, 2,6 studies done on the bulbar salivary center by many workers. Among these studies, using the method of retrograde degeneration after lesions involving the chorda tympani and the tympanic branch of the glossopharyngeal nerve in the dog 7,8 and kitten 5, it has been reported that the salivary center was situated in the lateral reticular formation of the ipsilateral side of the lesion. On the contrary, using the histochemical technique for cholinesterase, Shute and Lewis 4 reported that the rat's salivary center was a collection of nerve cells flanking the lateral and medial aspect of the facial genu at its rostral end and there were no salivary cells in the lateral reticular formation. Therefore, the present study was taken in an attempt to clarify these discrepancies between the results obtained by the retrograde degeneration method and those of the histochemical technique for cholinesterase. In the present study, the method based on the retrograde axonal transport of horseradish peroxidase (HRP) was adopted in the rat. This study was carried out in 10 Sprague-Dawley rats weighing about 80 g. The H R P was applied to the preganglionic fibers existed along the duct of the submandibular gland. Namely, under pentobarbital anesthesia, the preganglionic fibers with the duct of the submandibular gland were crushed with a forceps or cut with a pair of scissors. The H R P was applied in dry substance around the crushed or cut area of the nerves time and again over a period of 2-3 h. After 2 days' survival the animals were reanesthetized with pentobarbital and fixed by transcardiac perfusion with physiologic saline followed by a mixture of 2 ~ paraformaldehyde and 1.25~ glutaraldehyde in 0.1 M phosphate buffer. The brain was removed from the skull and fixed some hours in the same fixative. Then, the brain was placed in 0.1 M phosphate buffer containing 5 ~ sucrose and kept at 4 °C for 24-48 h and frozen sections were cut at 50/zm in transverse plane. The sections were transferred from 5 700phosphate buffered sucrose to 0.2 M Tris buffer solution for 20 min. They were then incubated for 10 min at room temperature in a bath containing 0.05 ~o 3,3'-diaminobenzidine tetrahydrochloride and 0.01 ~ H202 in 0.2 M Tris buffer. Following this, they were washed three
146
G.VII
•
•
~
~l~mm•
/
-
: •"~
~
N~VI
~
VII kaff.
\~"" ~
\
\
\ \".v~ •
Y~.~
•
"
II
ZY. ,,~\~ • s~ ~III
I
Fig. 1. Diagrams of the locations of HRP-labeled neurons after the application of HRP to the submandibular branch of chorda tympani. Abbreviations: C.r., corpus restiforme; D.v.n., descending vestibular nucleus; F.s., solitary tract; G.VII, genu of facial nerve; M.v.n., medial vestibular nucleus; N.c.tr., nucleus corporis trapezoidei; N.Deit., Deiters', nucleus; N.f.s., nucleus of the solitary tract; N.VIII, nucleus cochlearis ventralis; Ol.s., nucleus olivaris superior; r.f. VII aft., afferent fibers in 7th cranial nerve; Tr.sp.V, spinal trigeminal tract; VII, nucleus n. facialis.
147
Fig. 2. Dark-field photomicrographs of HRP-labeled neurons at the level of Fig. 12. These cells are located in the lateral aspect of the reticular formation. × 200. times in distilled water and mounted on slides with alcohol gelatine. They were counterstained with cresylecht violet. As the results of the observation, the H R P labeled cells could be seen in all cases. The fact that most labeled cells could be seen is shown in Fig. 1. Fig. 11 6 is illustrated at 150 #m intervals and each figure is a composite of the location of the H R P labeled cells appearing in 3 serial sections. The H R P labeled cells were seen in the reticular formation at the level between the most caudal end of the root of the facial nerve and the rostral part of the facial nucleus. At the most rostral level (Fig. 11), 4 labeled cells were seen in the reticular formation medial to the most caudal end of the root of the facial nerve. Twenty-six and 14 labeled cells could be seen at the level of Fig. 12 and Fig. 13 respectively. The afferent fibers of the facial nerve that transverse the dorsal part of the spinal trigeminal tract and the most rostral part of the facial m o t o r nucleus appears in Fig. 12, and the most rostral part of the solitary tract is seen in Fig. Is. These cells illustrated in Fig. 12,3 were seen in the reticular formation ventral to Deiters' nucleus, medial to the spinal trigeminal nucleus and dorsal to the facial motor nucleus. The distribution of these cells extended in the dorsoventral direction of the lateral aspect of the reticular formation. At the level of Fig. 14,5.6, the labeled cells were seen in the area of caudal prolongation of the same region of rostral ones, but decreased in number (a total of 5). Almost all of the labeled cells were the medium-sized
148 multipolar type (15-20/~m diameter), but only 2 cells (of a total of 49) were small-size d (7-10 # m diameter, Fig. 2). The results obtained in the rest of the cases were identical to those shown in Fig. 1. Yagita and Hayama 7 observed the chromatolytic cells after the incision of the chorda tympanic nerve in the ipsilateral lateral reticular formation ventromedial to Deiters' nucleus and dorsal to the facial motor nucleus in dog. In addition, Yagita 8 reported that retrograde cellular degeneration occurred in the lateral reticular formation at the level of the nucleus ambiguus and the facial motor nucleus after cutting the tympanic branch of the glossopharyngeal nerve in dog. Torvik 5 obtained the same results of Yagita and Hayama 7, and Yagita s in kittens. Namely, he reported that retrograde cellular degeneration occurred in the medium- or small-sized cells in the lateral reticular formation which was in accordance with the nucleus parvicellularis named by Meessen and Olszewski 3. On the contrary, Shute and Lewis 4 have described that, by the histochemical technique for cholinesterase, the salivary center was a collection of nerve cells flanking the lateral and medial aspect of the facial genu at its rostral end and there were no salivatory cells in the lateral reticular formation. From the results of the present study, the salivary cells that give rise to preganglionic fibers to the submandibular and sublingual glands were localized in the ipsilateral reticular formation. The results support the findings of Yagita and Hayama 7 and Torvik 5. However, no labeled cells could be seen in the area demonstrated as the salivary nucleus by Shute and Lewis 4. Therefore, it is doubtful that a collection of nerve cells demonstrated by the histochemical technique for cholinesterase are the real salivary ceils. Salivary secretion can be obtained by Wang 6 in cat, and Magoun and Beaton 2 in monkey, by electrophysiological methods. The points where salivary secretion can be obtained by electrical stimulus were more widely distributed than the area confirmed by the retrograde cellular degeneration and HRP methods. Therefore, it seems that these points contain some neural elements other than the salivary cells, such as the solitary fasciculus and its nucleus and the trigeminal nuclear complex. From the results of the present study in the rat, it can be said that the salivary cells that give rise to preganglionic fibers to the submandibular and sublingual glands are localized in the ipsilateral reticular formation, which is in accordance with the rostral part of the nucleus parvicellularis named by Meessen and Olszewski 3. There remains, however, the possibility that the salivary nucleus by Shute and Lewis a has some relation to salivary secretion, as the absence of HRP labeling in neuron cell bodies should not be considered conclusive evidence that those cells do not supply a projection to the injected site.
1 Kawamura, Y. and Funakoshi, M., Studies on the neurophysiologic mechanisms of salivary secretion, Med. J. Osaka Univ., 8 (1958) 433443. 2 Magoun, H. W. and Beaton, L. E., The salivatory motor nuclei in the monkey, "4mer. J. Physiol., 136 (1942) 720-725. 3 Meessen, H. and Olszweski, J., ,4 Cytoarchitectonic Atlas o f the Rhombencephalon o f the Rabbit, S. Karger, Basel, 1949. 4 Shute, C. C. D. and Lewis, P. R., The salivatory centre in the rat, J. Anat. (Lond.), 94 (1960) 59-73.
149 5 Torvik, A., Die Lokalisation des Speichelzentrum bei der Katze, Z. mikr.-anat. Forsch., 63 (1957) 317-326. 6 Wang, S. C., Localization of the salivatory center in the medulla of the cat, J. Neurophysiol., 6 (1943) 195-202. 7 Yagita, K. and Hayama, S., Uber das Speichelsekretionscentrum, Neurol. Zbl., 28 (1909) 738-753. 8 Yagita, K., Weitere Untersuchungen fiber das Speichelzentrum, Anat. Ariz., 35 (1910) 70--75.
