Okajimaslia Anat. Jpn., 61(4): 287-310. October 1984

L ocalization of the of Origin of Efferent Fibers in the Glossopharyngeal, Vagus and Accessory in the Rat by Means of Retrograde Degeneration and Horseradish Peroxidase Methods

By

Yong Li LU and Hisashi SAKAI

Department of Anatomy, School of Medicine, Nagoya University, Nagoya 466, Japan

-Received for Publication, July 4,1984-

Key Words: Dorsal motor nucleus of vagus, Ambiguus nucleus, Localization, Retrograde degeneration, HRP.

Summary: In order to pursue further the possible localization of the functional centres which belong to the group of the glossopharyngeal, vagus and accessory nerves, the motoneurons of these nerves and their major branches in the rat were examined by the retrograde degeneration method and the horseradish peroxidase (HRP) method. The results obtained are based on the examination of 62 rats which were divided into five groups. The results obtained are as follows: (A) The vagus arises from 80-90% of neurons of the dorsal motor nucleus of the vagus (DMV), the total neurons of the ambiguus nucleus (AM) except for a few cells occupying the ventral part of its rostral region, the neurons of the reticular formation between the DMV and the AM, and the neurons of the lateral reticular formation ventrolateral to the AM. (B) The motoneurons of the innervating the laryngeal muscles comprise 20% of the total cells in the DMV and 50% of the neurons in the rostral one-third of the AM, but the motoneurons of the recurrent nerve are present only in the caudal two-thirds of the AM. (C) Fifty to 60% of the neurons of the DMV supply the abdominal organs. The cell mass forms a series of columns, which is located in the lateral part of the rostral region, the central and medial parts of the middle region and the medial part of the caudal region of the DMV. (D) The glossopharyngeal nerve does not arise from the DMV but from the neurons which are located at a more rostral level than the DMV and from the most rostral and the ventro-rostral parts of the AM. (E) The results also indicate that the consists of elements not only from the vagus nerve but also from the internal branch of the accessory nerve. The neurons of the internal branch of the accessory nerve are scattered in the caudal region of the DMV and the AM. The external branch of the accessory nerve does not arise from the DMV and the AM, but from neurons located in the lateral area of the anterior horns extending from the first to the fourth cervical segments of the . These findings were consistently ipsilateral to the side operated on.

It is well known that the efferent fibers (AM), which contain many neurons involv- in the vagus nerve originate from two ing respiratory, cardiovascular, and gastro- distinct nuclei, the dorsal motor nucleus of intestinal functions (Whitteridge 1948. Getz vagus (DMV) and the ambiguus nucleus and Sirnes 1949, Paintal 1953, Mohiuddin

*Present address: Department of Anatomy , China Medical College, Shenyong, China

287 288 Yong Li LU and H. Sakai

1953, Mitchell and Warwick 1955, Calaresu the previous studies. In order to pursue the and Cottle 1965, Sugimoto et al. 1979, possible topographical location of the Kalia and Mesulam 1980a, 1980b). Further- functional centres which belong to the more, determination of a possible specific group of the glossopharyngeal, vagus and topographical arrangement within these accessory nerves, the motoneurons of nuclei has been attempted. Earlier, Molhant efferent fibers in these nerves and their (1910) and Getz and Sirnes (1949) provided major branches in the rat have been ex- anatomical reports with regard to the DMV amined by means of the methods of the in rabbits and described the definite topo- retrograde degeneration and the retrograde graphical localization in relation to various transport of HRP. visceral organs in this nucleus by the retro- In the present study, in order to deter- grade degeneration method. Since then, mine the presence or absence of localiza- many other studies in several animal species, tion of the motoneuron in the DMV and cat (Mohiuddin 1953, Smolen and Truex the AM, the vagus trunk and its branches 1978), monkey (Mitchell and Warwick innervating the larynx via the superior 1955), sheep (Bell and Lawn 1955, Welento laryngeal and the recurrent nerves and and Flieger 1974), buffalo (Rao and Sahu branches innervating the abdominal organs 1974), and fowl (Watanabe 1968), have via the anterior and posterior abdominal indicated the definite topographical localiza- trunks of the vagus were cut and examined. tion within this nucleus by the same meth- In addition, in order to determine whether od. In addition to these studies similar the DMV anastomoses with other nerves, attempts have been made at topographical the glossopharyngeal nerve and the internal location in the AM of the rabbit (Lawn and external branches of the accessory 1966a, 1966b). nerve were also cut and examined. Recently, the retrograde transport of horseradish peroxidase (HRP) has been Materials and Methods used to identify more exactly the cells of origin of efferent fibers supplying the A total of 60 male and female Wistar visceral organs (Todo et al. 1977, Yama- rats weighing 200-500 g were used in the moto et al. 1977, Satomi et al. 1978, Nosaka present experiment. The animals were et al. 1979, Kalia and Mesulam 1980a, anesthetized with 5% sodium pentobarbital 1980b, Wild 1981, Contreras et al. 1980, (i.p. 50 mg/kg). All animals were divided Nomura and Mizuno 1981, 1982). into five experimental groups and operated However, since certain differences are on. The materials were prepared by the recognizable among the species, the loca- methods of retrograde degeneration and tions of the motoneurons in the species retrograde transport of HRP. The numbers do not seem to be fully described as yet. of animals used, plane transected and Few studies have been carried out on the procedures employed for each group are DMV and the AM of the rat. In addition, illustrated in Figs. 1A, I B and Table 1. there are some questions: do the efferent a) Retrograde degeneration method fibers of the vagus nerve originate from The animals under anesthesia were nuclei other than these two, and do these operation on and the nerves were transected two nuclei give the fibers to nerves other at five sites in the cranial and cervical than the vagus nerve? With regard to these regions and the region of the glandular questions, there are discrepancies among stomach (Figs. 1A and 1B). Under an Neurons of Origin in Rat Gloopharyngeal. Vagu and AcceN%or Nerves 289

Fig. 1. Diagrams showing the five levels (1 to 5) of transected sites in the glossopharyngeal, vagus and accessory nerves. A, in the cranial and cervical regions. B, in the glandular stomach region.

Table 1. Schematic presentation of the sites of neurectomy 290 Yong Li LU and H. Sakai operation microscope, the nerves were hours postoperatively, and then deeply re- exposed and dissected free from the sur- anesthetized and perfused through the left rounding tissue. A small piece of the nerve, ventricle with 300-500 ml of 0.9% saline about 0.3-1.0 cm in length, was excised solution at room temperature. This was to avoid contact of the terminals. After followed by 500 ml of 1.0% paraformal- the operation, the animals were allowed dehyde and 1.25% glutaraldehyde in 0.1 M to survive for 8-14 days. After anesthetiza- phosphate buffer (pH 7.2-7.4) and then tion by the same method the animals were by 500 ml of 10% sucrose in the same perfused through the left ventricle with buffer. The brain with the cervical segment 10% formalin, then the brains with the of the spinal cord was removed and stored cervical segments of the spinal cord were in 10% sucrose in phosphate buffer, pH removed and postfixed in 10% formalin 7.2-7.4, for 2-24 hours at 4°C, and then for three to four weeks. Then the brains cut transversely into serial 50 pm thick were washed, dehydrated and embedded sections on the freezing microtome. The in paraffin. Serial 10 pm thick sections sections were treated by the tetramethyl- were made transversely in all materials. benzidine (TMB) method of Mesulam The first of every four sections was stained (1981) for the histochemical demonstration with cresyl violet and examined. The judge- of HRP, then mounted on gelatinized ment as a degenerative cell was based on slides and counterstained with 1% neutral three standards: (1) neuronal swelling of red solution. They were examined micro- the cell body, (2) eccentricity of the nuclei, scopically under bright-field and dark- (3) fragmentation of the Nissl substance. field illumination. The counting of degenerative neurons The present results are almost all based was based on those counted in the first on observations obtained by the two meth- of every four sections. Then multiplying ods at the same sites except for the internal by four gave the total number of degen- branch of the accessory nerve which was erative neurons. The proportion of degen- treated only by the retrograde degenera- erative neurons in normal cells was deter- tion method because this branch is too mined as previously described and found to short for HRP bathing. be about 5,000 cells in each DMV in the rat (Li' and Sakai 1984). Results b) HRP method As in the method described above, the Group 1. The motoneurons of the vagus nerves were transected and dissected free nerve from the surrounding tissues which were In the first group of 12 rats, the right or covered with a piece of parafilm in order left cervical vagus trunk was transected at to avoid any undetected uptake of HRP. the level just distal from the nodose gan- The proximal cut end of the nerve was glion. In these animals, degenerative or inserted into a piece of polyethylene tubing HRP-labeled cells were found in the follow- which was filled with 10-20 pl of 30% ing four regions. HRP (Sigma. type VI) dissolved in 0.9% a) The DMV saline solution. The open end of the tube In the cases of investigations by the was sealed with vaselineand aronalpha. degenerative method, in the DMV on the After the animals recovered from anes- side operated on, the typical retrograde thesia they were undisturbed for 36-48 degenerative cells were found throughout Neurons of Origin in Rat Glossonharvngeal, Valais and Accessory Nerves 29 the region of the nucleus. The cells showed of the AM were found to be degenerated a swollen appearance, tigrolysis and an or labeled when either of the two methods eccentric position of the nuclei. Cell count- was used (Figs. 6 and 7). Undegenerated ing in five animals showed that 80-90% or unlabeled cells were located in the rostra- of the neurons of the DMV were degen- ventral part (Fig. 6). erated (Fig. 2). c) The reticular formation between the In six animals examined by the HRP DMV and the AM method, HRP-labeled cells were found in After application of the HPR method. every region of the ipsilateral DMV. These scattered HRP-labeled cells were found in cells formed a spindle-shaped longitudinal an intermediate zone between the DMV column which was elongated rostro-caudal- and the AM. These cells were located along ward about 3-4 mm. The majority of the the tracts of the vagus nerve and were labeled cells were located at the level just mainly rostra! to the DMV. They were rostral to the obex. Almost all of the cells different in size, about 10-25 jim in the more than 20 pm in the long axis were long axis and 8-12 pm in the short axis labeled. No labeled cells were found in a (Fig. 4). small portion of round or oval small cells, d) The lateral reticular formation and constituted less than 20% of the total After the application of HRP to the cells in the DMV (Figs. 3,4 and 5). cervical trunk of the vagus nerve, HRP- b) The AM labeled cells were also seen in the lateral Investigation by the two methods re- reticular formation located ventrolateral vealed that the AM of the rat is located in to the AM and were more numerous than the lateral area of the reticular formation those of the reticular formation between in the lower brain stem, and forms a con- the DMV and the AM. However, compared tinuous elongated cell mass about 3 mm in to the AM neurons, these seemed to be length rostro-caudalward. It was found to quite similar in shape but a little smaller. be composed of large and medium-sized A few labeled cells were located in a more neurons, and is divided into the rostra! and ventral position, and in some sections the caudal regions according to the distribu- cells lay as close as 200 pm from the sur- tion of the cells. The rostra! region com- face of the ventral medulla (Fig. 7). The prised one-third of the total length of the affected cells mentioned above were nucleus and contained most of the cells found ipsilaterally. in this nucleus. The rostral region is sub- In addition, in this group the cells of the divided into the dorsal and ventral parts and the afferent fibers of by a region of low density. The dorsal the vagus nerve were also labeled (Figs. 3, part formed a rather compact mass of 4 and 5). medium-sized neurons, while the ventral Group 2. The motoneurons of the laryn- part consisted of loosely arranged large geal nerves neurons and was continuous with the caudal In this group, 15 rats were used, and in region (Fig. 6). The caudal region comprised 9 of them the superior laryngeal nerve was two-thirds of the total length of the nucleus, transected at the level near its derivation but contained only a small part of the total from the vagus and the recurrent nerve was number of cells of this nucleus (Fig. 8). also transected near the larynx. In these In this group, except for a few cells in experiments both methods of treatment the rostro-ventral part, almost all neurons were used in order to observe the distribu- 292 Yong Li LU and H. Sakai tion of motoneurons innervating the laryn- AM (Fig. 12); no labeled cells were found geal muscles. The results indicated that in the DMV. The distribution of these about 20% of the cells in the rostra! and labeled cells extended rostro-caudally about middle regions and scattered cells in the 2,000 pm in length. The cells were multi- caudal region of the DMV were degenerated polar in shape and larger than those of the or labeled. The majority of affected cells rostral region of the AM. In three animals were located in the dorsal part of the nu- the average number of labeled cells per cleus (Figs. 9 and 10). Furthermore, many rat was about 200-250. degenerated or labeled cells were found in Group 3. The motoneurons of the ab- the AM ipsilaterally. In the rostral one- dominal vagus trunk third of the AM, about half of the total In 13 rats of this group, the anterior or cells in the dorsal part and a few cells in posterior abdominal trunk of the vagus nerve the ventral part were affected (Fig. 11), was transected and treated by bcth the de- while in the caudal two-thirds of the nucleus, generation and HRP methods at the abdomi- almost all cells were affected, although this nal level of the oesophagus. regions contained only a small portion of When the anterior trunk was transected the total cells of this nucleus. and treated, the affected cells were seen in In order to localize the cells of origin in the left DMV, but in the posterior trunk, the laryngeal nerve, the other 6 rats were the result was vice versa. The results indi- used for the HRP method. The superior cated that about 50-60% of the total cells laryngeal and the recurrent nerves were cut in the DMV sent axons to the abdominal separately. When HRP was applied to only organs. These cells formed a continuous the superior laryngeal nerve, HRP-labeled slender column which extended obliquely cells were found in the rostra] and middle from the rostral to the caudal region of the regions of the DMV and the rostral one- nucleus. In the rostral region of the DMV, third of the AM, but no labeled cells were the cell column was located in the lateral found in the caudal region of the AM. The part of the nucleus, and 20-30% of the labeled cells comprised about 20% of the cells located in this region were degenerated total cells in the DMV and were located or labeled (Fig. 13). In the middle region of mainly in the dorsal part of the rostral the DMV, the cell column was located in and middle regions of this nucleus. In the the central and medial parts of the nucleus rostra] one-third of the AM, 400-500 (Fig. 14). The cells located in this region labeled cells were found per rat. The dis- formed the densest and widest extension tribution of the labeled cells extended in the column and made up about 70% of rostro-caudally over a length of about 1,000 the cells of the region. In the caudal region ptm and its rostra! end reached to the caudal of the DMV, the cell column was located pole of the motor nucleus of the facial in the most medial part of the nucleus nerve. The number of affected cells in the (Fig. 15). From the rostral end of the caudal DMV and the rostra! region of the AM was region, the cell column became slender. In nearly equal to those mentioned above, the most caudal part the average number of in which HPR was applied to the superior affected cells was 3-5 per section. The laryngeal and recurrent nerves at the same affected cells comprised approximately 20% time. of the total cells in this region. When HRP was applied only to the re- In addition, a few labeled cells were also current nerve, labeled cells were found only seen in the dorsal part of the rostra! one- in the caudal two-thirds of the ipsilateral third of the ipsilateral AM. The average Neurons of Origin in Rat Glossopharyngeal, \'agus and AL.cessory Nerves 293 number of labeled cells seen was 3-5 per The accessory nerve consists of the section (Fig. 16). The results obtained in internal and external branches. After leaving the anterior trunk were similar to those in the , the internal branch of the posterior trunk. the accessory nerve was close to the vagus Group 4. The motoneurons of the glos- nerve and joined the nodose ganglion of the sopharyngeal nerve vagus nerve immediately. Since the branch In this group of 10 rats, the glossopharyn- is too short to bathe with HRP, in 2 of 8 geal nerve was transected at the level after it rats it was transected at its juncture with passed through the jugular foramen. No de- the vagus nerve and treated by the degen- generation or labeled cells were found in the eration method. In these 2 animals, a few DMV, but affected cells forming a distinct degenerative cells were scattered in the compact column were found in the ipsi- caudal region of the DMV and the AM. The lateral reticular formation rostral to the results indicated that the vagus nerve con- DMV. This column began at the level just tains elements not only from the vagus rostral to the DMV and extended rostrally nerve but also from the internal branch of about 800 ,um. In the transverse section, the accessory nerve. The efferent fibers these cells were located ventrolateral to the arising from these cells emerged through medial division of the solitary nucleus (Fig. the jugular foramen, and formed a part of 17) and were approximately 150 pm and the accessory nerve. The branch joined the 120 pm in maximum mediolateral and vagus nerve immediately after leaving the ventro-dorsal lengths respectively. The col- brain stem. Actually, the internal branch umn may correspond to the inferior sali- was regarded as a part of the vagus nerve vatory nucleus giving rise to the secreto- but not as a part of the accessory nerve. motor and vasodilator components of the In the other 6 rats, the external branch glossopharyngeal nerve. of the accessory nerve was transected and Degenerative or HRP-labeled cells were treated by the two methods. In these ani- also seen in the AM. The most rostral region mals, no affected cells were found in the of the AM extended to the level of the DMV and the AM. However, affected cells caudal pole of the facial nucleus (Fig. 20). were present in the lateral area of the Caudally, it was continuous with the ventral anterior horn at the level of the first to the part of the rostral one-third of the AM (Figs. fourth cervical segments of the spinal cord 18 and 19). The affected cells in the most (Fig. 21). These cells were typical multi- rostral and ventro-rostral parts of the AM polar motor type cells, the same as motor were larger than those in its dorso-rostral neurons in the anterior horn of the spinal part. These cells were mostly pyramidal in cord. Such cell group is usually designated shape and more than 30 pm in diameter. as spinal accessory nucleus and gives off In a group of 2 rats, the glossopharyngeal the fibers to the and sternomastoid nerve and the pharyngeal branch of the muscles. vagus nerve were transected simultaneously and treated by the HRP method. The labeled Discussion cells were found not only in all regions mentioned above but also in the rostral 1) Localization of the motoneurons of the half of the DMV. vagus nerve Group 5. The motoneurons of the accessory From group 1 of the present experiments, nerve it may be concluded as follows: The efferent 294 Yong Li Lii and H. Sakai fibers of the vagus nerve arise from 80-90% neurons give off the fibers to the other of the neurons of the ipsilateral DMV, most nerves. On this point, most of the previous neurons of the ipsilateral AM, the neurons studies based on the retrograde degeneration of the reticular formation between the DMV method gave unsatisfactory resolution be- and the AM and the neurons of the lateral cause of the difficulties in judging the degen- reticular formation ventrolateral to the AM. erative small cell. However, compared to In this group, because the transection was that method, the HRP method used mainly done at the level just distal to the nodose in the present study was more useful al- ganglion, almost all of these affected cells though there were no significant differences may be considered neurons sending efferent in the results obtained by the two methods. fibers to the vagus nerve. The present results showed that all medium- The results described above showed that sized and some small cells in the DMV give the neurons of the DMV and the AM give off off fibers to the vagus nerve. Such cells were the efferent fibers to the vagus nerve, and 80-90% of the total number. The other 10— this fact is in agreement with the previous 20% of the DMV cells were smaller than the studies (Getz and Sirnes 1949, Smolen and affected cells. These are probably inter- Truex 1978, Kalia and Mesulam 1980a). neurons. Recently, an electron microscope However, the question of whether the study by McLean and Hopkins (1982) also neurons of the reticular formation also send demonstrated that two populations may fibers to the vagus nerve has not been exist in 20-25% of the small cells of the resolved. In the present study, a substantial DMV. One population appears to have number of HRP-labeled cells was seen in the projections ascending to higher levels of the reticular formation between the DMV and , while the other, more numerous, the AM and the lateral reticular formation population may be interneurons. Further- ventrolateral to the AM. A recent study of more, a few affected cells were seen in the Nosaka et al. (1979) revealed the presence DMV when the branch of the accessory of labeled cells in the reticular formation nerve was examined. As this branch was following HRP injection into the cardiac regarded as a part of the vagus nerve, it may branch of the vagus nerve. Sugimoto et al. be concluded that the DMV neurons never (1979) and Stuesse (1982) have also ob- give off the fibers to any nerves other than served HRP-labeled cells in these reticular the vagus nerve. formation after injection of HRP into the With respect to the vagal fibers arising heart, and they assumed that these labeled from the contralateral DMV, doubt still cells give rise to the cardiac fibers to the remains. Earlier, Mitchell and Warwick heart. In group 2 and 3 of the present ex- (1955) reported that a small proportion of periments no HRP-labeled cells were found the vagal fibers decussate within the medulla in these reticular formations after HRP and there is evidence of an interchange of application to the vagal fibers supplying the fibers between the contralateral nerves in larynx and abdominal organs. On the basis the peripheral plexuses. Some recent studies of this result, it may be considered that the (Smolen and Truex 1978, Karim and Seng cells in these regions give off the vagal fibers 1980) again support this opinion and indi- to the heart and the other thoracic organs. cate that a small number of the DMV In addition, there are two questions: (1) neurons send axons to the contralateral How many neurons of the DMVgive off the vagus nerve. On the other hand, De Vito et fibers to the vagus nerve? (2) Do such al. (1974) and Kalia and Mesulam (1980a) Neurons of Origin in Rat G/oopharyngeal. Vagus and Accessor) Ncrve,, 295 demonstrated that the DMV neurons supply the DMV neurons give off the fibers to the axons only ipsilaterally. This result is wel superior laryngeal nerve. but not to the in accordance with those of group 1, 2 an recurrent laryngeal nerve. 3 of the present experiments in which the In addition, in relation to localization affected cells were found only in the ipsi of the AM neurons supplying the two lateral DMV and the AM. As a conclusion laryngeal nerves, the results indicated that therefore, it may be assumed that the decus the motoneurons of the superior laryngeal sation is absent in the vagus nerve. nerve are located in the rostral one-third 2) Localization of the motoneurons inner of the AM, especially in the dorsal part. vating the organs while the motoneurons of the recurrent Group 2 and 3 of the present experiment! laryngeal nerve are located in the caudal revealed certain differences in the topo two-thirds of the AM. This result disagrees graphical arrangement within the DMV an with that of Kalia and Mesulam (1980b) in the AM. the cat. They stated that the caudal part According to the results of group 2, the of the AM makes no contribution to the motoneurons of the superior and recurren. larynx muscles. laryngeal nerves are located in the rostra The results obtained from group 3 of the half of the DMV and the greater part of the present experiment further showed the AM except for its rostro-ventral part. Fur localization of the motoneurons in the thermore, the present results indicate tha. DMV. In this group, the anterior and poste-. the motoneurons of the superior laryngea rior abdominal trunks of the vagus supplying nerve are clearly separated from those o: the abdominal organs were examined. The the recurrent nerve. The motoneurons results showed that the affected cells form- the superior laryngeal nerve were found t( ing a continuous spindle-shaped column be mainly located in the dorsal part of th( were located in the lateral at the rostral rostral half of the DMV and the rostral one level, in the central and medial at the middle third of the AM, but the motoneurons level, and the medial at the caudal level of the recurrent laryngeal nerve were locate( the DMV. only in the caudal two-thirds of the AM In the earlier studies using the degenera- In previous studies, the question 01 tion method, the rostral half or the rostral whether the laryngeal nerve receives fiber two-thirds of the DMV were shown to be from the neurons of the DMV or not re associated with the fibers supplying the mained unsolved. The present results sup abdominal organs (Molhant 1910, Getz port the opinion of Hinrichsen and Ryar and Sirnes 1949, Mohiuddin 1953, Bell (1981), i.e. these cells are located withir 1960, Mitchell and Warwick 1955, Rao and both the DMV and the AM. Some report: Sahu 1974). More recently, Coil and Norgren have indicated that such cells are locate( (1979) used the HRP method in this field, only in the AM, not in the DMV (Schweizei and reported that the caudal region of the et al. 1981, Lawn 1966a). According to th( nucleus also gives off subdiaphragma tic results of the present experiment, abou. fibers to the vagus nerve. In the present 20% of the cells in the DMV were labelec experiment, it was also shown clearly that after HRP bathing in the superior laryngea the neurons sending axons to the abdominal nerve but no labeled cells were seen in th( trunk of the vagus nerve are located in the DMV after HRP bathing in the recurren. caudal region of the DMV. laryngeal nerve. This finding shows thai As to the question of whether the ab- 296 Yong Li LU and H. Sakai dominal trunk of the vagus nerve arises from present study, neurons in these regions are the AM neurons or not, most of the previous considered to be motoneurons of the glosso- studies did not reveal any AM neurons send- pharyngeal nerve innervating the pharyngeal ing fibers to the abdominal trunk (Lewis et muscles. al. 1970, Dennison et al. 1981, Bell 1960). 4) Localization of the motoneurons of the However, Kalia and Mesulam (1980b) found accessory nerve labeled cells in the AM following injection Mitchell and Warwick (1955) reported of HRP into the lung, heart and stomach. that the rostral and caudal ends of the DMV In group 3 of the present experiment, using give rise to part of the components of the the HRP method, a few labeled cells were fibers running in the glossopharyngeal nerve also found in the dorso-rostral part of the and the cranial portion of the accessory AM. This result is noteworthy because it nerve. Therefore, this nucleus was re-named indicates that the AM neurons innervate not the dorsal glossopharyngeo-accessory nu- only the striated muscles, such as the laryn- cleus. In the present study, a few degenera- geal muscles, but also the smooth muscles tive cells were found to be scattered in the of the lung and stomach. caudal region of the DMV and the AM when 3) Localization of the motoneurons of the the internal branch of the accessory nerve glossopharyngeal nerve was transected. Since the fibers belonged to With respect to the question of whether the internal branch joined to the nodose the neurons in the DMV also give off the ganglion immediately after branching, these fibers to the glossopharyngeal nerve, some were regarded as a part of the vagus nerve authors reported that the rostral region of but not the accessory nerve. It is obvious the nucleus does give off fibers to the glos- that the external branch forms the main sopharyngeal nerve (Addens 1933, Mitchell trunk of the accessory nerve. When it was and Warwick 1955, Welento and Flieger transected and examined by the two meth- 1974). In group 4 of the present experiment, ods, no affected cells were found in these there were no labeled cells in the DMV, but two nuclei. Therefore, it was concluded that labeled cells were present in the area rostral the vagus nerve contains elements not only to the DMV, which seems to correspond to from the vagus nerve, but also from the the inferior salivatory nucleus (Satomi et internal branch of the accessory nerve, and al. 1979, Contreras et al. 1980) or the that the DMV and the AM are not associated dorsal glossopharyngeal motor nucleus (Wild with the external branch of the accessory 1981). nerve. As to the localization of the AM neurons giving fibers to the glossopharyngeal nerve, Acknowledgements the present study showed that these are located in the most rostral and the ventro- The authors would like to thank Profes- rostra] one-third of the nucleus. In these sor M. Yasuda and Drs. Y. Takeuchi and J. regions, the labeled cells were not seen when Kitoh for their critical comments on this the cervical vagus trunk was transected, as manuscript, and Mr. F. Sanda for his excel- shown in group 1. According to the data lent technical assistance. obtained from the retrograde degeneration study in rabbits (Lawn, 1966b), the rostral region of the AM contains the motoneurons supplying the pharyngeal muscles. In the Neurons of Origin in Rat Giossopharyngeal, Vagus and Accessory Nerves 297

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in the vagus nerve. J. Comp. Neurol., 121: S. and Konishi, A.: The site of origin of 341-359, 1953. cardiac preganglionic fibers of the vagus 27) Rao, G. S. and Sahu, S.: The localization nerve: An HRP study in the cat. Neurosci. within the dorsal motor nucleus of the Lett., 12: 53-58, 1979. vagus in the buffalo (Bubalus bubalis). Acta 34) Todo, K., Yamamoto, T., Satomi, H., Ise, H., Anat., 90: 388-393, 1974. Takatama, H. and Takahashi, K.: Origins of 28) Satomi, H., Yamamoto, T., Ise, H. and vagal preganglionic fibers to the sino-atrial Takahashi, K.: Identification of the inferior ani atrio-ventricular node regions in the cat salivatory nucleus in the cat as studied by heart as studied by the horseradish peroxidase HRP bathings of the transected glossopharyn- method. Brain Res., 130: 545-550, 1977. geal nerve root. Neurosci. Lett., 11: 259- 35) Watanabe, T.: A study of retrograde degenera- 263, 1979. tion in the vagal nuclei of the fowl. Jap. J. 29) Satomi, H., Yamamoto, T., Ise, H. and Vet. Sci., 30: 331-340, 1968. Takatama, H.: Origins of the parasympathetic 36) Welento, J. and Flieger, S.: Distribution of fibers to the cat intestine as demonstrated the nerve centres in the nucleus dorsalis nervi by the horseradish peroxidase method. Brain vagi and nucleus ambiguus in the sheep. Pol. Res., 151: 571-578, 1978. Arch. Wet., 17: 7-24, 1974. 30) Schweizer, H., Ruebsamen, R. and Ruehle, 37) Whitteridge, D.: Afferent nerve fibers from C.: Localization of brain stem motoneurons the heart and lungs in the cervical vagus. J. innervating the laryngeal muscles in the Physiol., 107: 496-512, 1948. rufous horseshoe bat, Rhinolophus rouxi. 38) Wild, J. M.: identification and localization Brain Res., 230: 41-50, 1981. of the motor nuclei and sensory projections 31) Smolen, A. J. and Truex, R. C.: The dorsal of the glossopharyngeal, vagus, and hypo- motor nucleus of the vagus nerve of the cat: glossal nerves of the cockatoo (Cacatua Localization of preganglionic neurons by roseicapilla), Cacatuidae. J. Comp. Neurol., quantitative histological methods. Anat. Rec., 203: 351-377, 1981. 189: 555-566, 1978. 39) Yamamoto, T., Satomi, H., Ise, H. and Taka- 32) Stuesse, S. L.: Origins of cardiac vagal pre- hashi, K.: Evidence of the dual innervation ganglionic fibers: A retrograde transport of the cat stomach by the vagal dorsal motor study. Brain Res., 236: 15-25, 1982. and medial solitary nuclei as demonstrated 33) Sugimoto, T., Itoh, K., Mizuno, N., Nomura, by the horseradish peroxidase method. Brain Res., 122: 125-131, 1977.

Explanation of Figures

Abbreviations in Figures a : common carotid artery NIX : glossopharyngeal nerve AM : ambiguus nucleus NRP : nucleus reticularis parvocellularis CC : central canal NTS : nucleus tractus solitarii DMV : dorsal motor nucleus of vagus NX : vagus nerve DP : dorsal part of rostral region of AM NXI : accessory nerve FV : fourth ventricle TX : tractus solitarius NH : nucleus nervi hypoglossi VP : ventral part of rostral region of AM Neurons of Origin in Rat Glossopharyngeal. Vagus and Accessory Nerves 299

PLATES 300 Yong Li I,u and H. Sakai

Explanation of Figures

Plate I

Photomicrographs of the DMV from the first group in which the cervical vagus nerve was examined at the level just distal to the nodose ganglion of the vagus nerve.

Fig. 2. Topographical photomicrograph of a transverse section through the middle region of the DMV. 12 days after the operation. Note that almost all cells in this nucleus show retrograde degeneration with a swollen appearance, an eccentric nucleus and fragmented Nissl granules. Cresyl violet stain. Bar = 125 pm.

Figs. 3, 4 and 5. Dark-field photomicrographs of the DMV in an animal in which the cervical vagus nerve was bathed in HRP. Three transverse sections: middle (Fig. 3), rostral (Fig. 4) and caudal (Fig. 5) regions. Retrogradely labeled neurons are seen in the rostral to caudal region of the nucleus and in the reticular formation ventral to the DMV (slender arrows in Fig. 4). In addition, labeled cells are found in the contralateral nucleus of the tractus solitarius. Bar = 125 pm. Neurons of Origin in Rat Glossopharyngeal, Vagus and Accessory Nerves 301 Plate I 302 Yong Li Li' and H. Sakai

Plate II

Photomicrographs of the AM from the first group in which the cervical vagus nerve was examined at the level just distal to the nodose ganglion of the vagus nerve.

Fig. 6. Topographical photomicrograph of a transverse section through the rostral region of the AM. This photograph is of the same section as shown in Fig. 2. The left part of the figure shows the medial side. The rostral region of the AM is subdivided into the dorsal part (DP) and the ventral part (VP). Note that all cells in the DP are degenerated typically, while a few undegenerated cells are seen in the VP. These undegenerated cells are larger than those in the DP. Cresyl violet stain. Bar = 125 Am.

Figs. 7 and 8. Dark-field photomicrographs of the rostra], (Fig. 7) and caudal regions (Fig. 8) of the AM obtained from an animal in which the cervical vagus nerve was bathed in HRP. Fig. 7 shows the same result as in Fig. 6. Note that neurons of the lateral reticular formation (arrows) are present in the area ventro-lateral to the AM. These neurons are smaller than those of the AM. Fig. 8 shows that the neurons in the caudal region of the AM are also labeled. These cells are larger than those of the rostral region of the AM, but are fewer in number. Bar = 125 ;Am. Neurons of Origin in Rat Glossopharyngeal, Vagus and A( ( e,sory ticrves 303 Pidte II 304 Yong Li LU and H. Sakai

Plate III

Photomicrographs of the DMV and the AM from the second group in which the superior laryngeal nerve and recurrent laryngeal nerve were examined.

Figs. 9 and 10. Topographical photomicrograph (Fig. 9) and dark-field photomicrograph (Fig. 10) of sections cut through the middle region of the DMV after the superior and recurrent laryngeal nerves were transected or bathed in HRP. Fig. 9 is from an animal 13 days after transection. The affected cells are the larger of the two types of cells in the DMV, and make up about 20% of the total number. Bar = 125 pm.

Fig. 11. Dark-field photomicrograph of the rostral region of the AM in the same animal as that shown in Fig. 9. Note that the degenerated cells are located mainly in the dorsal part of the rostral region of the AM and make up about 50% of the total number. Bar = 125 Mm.

Fig. 12. Dark-field photomicrograph of the caudal region of the AM after the recurrent laryngeal nerve was bathed with HRP. Nearly all cells in this area are labeled. Bar = 125 Mm. Neurons of Origin in Rat Glossopharyngeal. Vagus and Accessorl Nerves 305 Plate III 306 Yong Li Lii and H. Sakai

Plate IV

Photomicrographs of the DMV and the AM in the third group in which the abdominal trunk of the vagus was treated by the HRP method.

Figs. 13, 14 and 15. Topographical photomicrographs of the DMV. Fig. 13 shows the rostral region of the nucleus. The HRP-labeled cells are located mainly in the lateral part in the medio-lateral direction of the nucleus (deep staining cells). Bar = 100 Am. Fig. 14 shows the middle region of the nucleus. The HRP-labeled cells are located mainly in the central and the medial parts in the medio-lateral direction of the nucleus (deep staining cells). Bar = 100 Am. Fig. 15 shows the caudal region of the nucleus. The HRP-labeled cells are located mainly in the medial part in the medio-lateral direction of the nucleus (deep staining cells). Bar = 100 ,um.

Fig. 16. Dark-field photomicrograph of the rostral region of the AM. A few cells in the dorsal part of the rostral region of the nucleus are labeled. Bar = 125 um. Neurons of Origin in Rat Glossopharyngeal, Vagus and Accessory Ne n es 307 Plate IV 308 Yong Li Lii and H. Sakai

Plate V

Figs. 17-20 show the photomicrographs in the fourth group in which the glossopharyngeal nerve was treated by the HRP method. Fig. 17. Dark-field photomicrograph of the medulla at a level more rostral than the most rostral pole of the DMV. A mass of labeled cells (arrow) is seen in the area ventrolateral to the NTS, and is nearly round in shape. It corresponds to the inferior salivary nucleus. Bar = 200 Am. Figs. 18 and 19. Dark-field photomicrograph (Fig. 18) and topographical photomicrograph (Fig. 19) in the rostral region of the AM from the same animal as in Fig. 17. The labeled cells are seen only in the ventral part of the nucleus. Bar = 125 Arn. Fig. 20. Dark-field photomicrograph of a level rostral to that in Figs. 18 and 19, showing that the neurons in the most rostral part of the AM also give off the glossopharyngeal nerve. Bar = 125 Am.

Fig. 21. Topographical photomicrograph of the first segment of the cervical spinal cord in the fifth group, in which the external branch of the accessory nerve was treated by the FIRP method, showing that the labeled cells are located in the lateral area of the anterior horn (arrow). Bar = 250 Mm. Neurons of Origin in Rat Glossopharyngeal, Vagus and Accessory \ erves 309 Plate V