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Brainstem origin of the efferent components of the cervical vagus nerve in the house musk shrew, Suncus murinus
Journal of the Autonomic Nervous System 71 Ž1998. 55–63
Brainstem origin of the efferent components of the cervical vagus nerve in the house musk shrew, Suncus murinus a,)
Moo Ho Won
, Koji Matsuo d , Seung Mook Jo a , Tae-Cheon Kang b, Yang Seok Oh c , Chang Do Choi a , Junjoh Kitoh d
a
Department of Anatomy, College of Medicine, Hallym UniÕersity, Chunchon 200-702, South Korea Department of Anatomy, College of Medicine, Seoul National UniÕersity, Seoul 110-799, South Korea c Experimental Animal Center, College of Medicine, Hallym UniÕersity, Chunchon 200-702, South Korea d Institute for Laboratory Animal Research, Nagoya UniÕersity School of Medicine, Nagoya 466, Japan b
Received 24 February 1998; revised 30 March 1998; accepted 31 March 1998
Abstract The brainstem origin of the efferent neurons of the vagus nerve in the house musk shrew, an animal species which has been recently used in researches on emesis, was studied using the retrograde tracing method. The vagus nerve was exposed and cut at the mid-cervical level below the nodose ganglion. Horseradish peroxidase was applied to the proximal end of the cut nerve. The brainstem was sectioned and processed histochemically with the tetramethylbenzidine method. The horseradish peroxidase injection into the vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve and ambigual nuclear complex. Labelled neurons in the dorsal motor nucleus of the vagus nerve, constituting approximately 80% of the total labelled neurons, formed a longitudinal column whose length varied from 3.4 to 3.8 mm. Half of labelled neurons in this nucleus were found at the level between the area postrema and 0.6 mm rostral to it. The ambigual nuclear complex was made up of two major longitudinal divisions; the dorsal division corresponded to the ambiguus nucleus and the ventral division was identified as the external formation of the ambiguus nucleus. Our results suggest that in the Suncus murinus the neuroanatomical feature of the dorsal motor nucleus of the vagus nerve is similar to those of other mammals, but ambigual nuclear complex must be somewhat different between mammals. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dorsal motor nucleus of the vagus nerve; Ambigual nuclear complex; Retrograde tracing; Horseradish peroxidase; Insectivore
1. Introduction The vagus nerve is involved in many autonomic and various regulatory functions and has heterogeneous components, i.e. preganglionic parasympathetics; visceral afferents that follow their peripheral distribution; motoneuAbbreviations: AN, Ambiguus nucleus; ANC, Ambigual nuclear complex; ANc, Compact formation of ambiguus nucleus; ANe, External formation of ambiguus nucleus; ANl, Loose formation of ambiguus nucleus; AP, Area postrema; DMX, Dorsal motor nucleus of the vagus nerve; HRP, Horseradish peroxidase; NST, Nucleus of the solitary tract; RF, Reticular formation; ST, Solitary tract; STNV, Spinal tract and nucleus of the trigeminal nerve; V, The fourth ventricle; X, Vagus nerve; Xa, Afferent fibers of the vagus nerve; Xe, Efferent fibers of the vagus nerve; XII, Hypoglossal nucleus ) Corresponding author. Tel.: q82 361 2401614; fax: q82 361 561614; e-mail: [email protected] 0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 6 2 - 9
rons to the pharynx, larynx and upper esophagus; gustatory fibers from near the epiglottis; and somatic afferents from the external ear ŽChen et al., 1996; Haxhiu and Loewy, 1996; Kinami et al., 1997; Ladic and Buchan, 1996; Myers et al., 1996.. Brainstem neurons associated with autonomic and regulatory functions are difficult to identify neuroanatomically because they often do not aggregate in histologically welldefined nuclei. The exact locations of some of the brainstem nuclei may be related to the migration of the cells during development. The primitive dorsal visceral efferent column of the medulla oblongata separates into dorsal and ventral portions ŽBystrzycka and Nail, 1985.. The general visceral efferent neurons remain in the dorsal motor nucleus of the vagus nerve ŽDMX., while the special visceral efferent neurons supplying branchial musculature migrate to form the ambiguus nucleus ŽAN. ŽAltschuler et al.,
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1991; Bieger and Hopkins, 1987; Bystrzycka and Nail, 1985; Hopkins et al., 1984; Lawn, 1996.. The central efferent organization of the vagus nerve and vagal efferent neurons projecting to some viscera has been documented by techniques based on retrograde tract-tracing methods in many vertebrates, including virtually all common laboratory species. Vagal efferent axons originate from either the DMX or vicinity of the AN ŽAltschuler et al., 1991; Gwyn et al., 1985; Hamilton et al., 1987; Kalia and Mesulam, 1980; Kressel et al., 1994; Okumura and Namiki, 1990; Withington-Wray and Spyer, 1988.. Recently a small animal, the insectivore Suncus murinus, has been described as a new animal model for researches on emesis ŽMiyachi, 1996; Selve et al., 1994;
Ueno et al., 1987.. This new model is not commonly used in pharmacological laboratories, but some basic experiments have already been performed ŽMiyachi, 1996; Okada et al., 1995; Selve et al., 1994; Torii et al., 1993; Ueno et al., 1987.. In addition, the S. murinus is one of the most primitive and phylogenetic earliest eutherians, and therefore the relationship of primates to this order is closer than that to rodents usually used in pharmacological research ŽSelve et al., 1994.. In this regard different anatomical and functional organization may account for different susceptibility to or even ability to vomit and may be an explanation for inability of rat, mouse, guinea pig or rabbit to vomit in contrast to ferret, dog, cat and man ŽSelve et al., 1994.. With the necessity to identify the vagal innervation
Fig. 1. Three-dimensional computer reconstruction of the labelled DMX and AN within the left brainstem of the house musk shrew after HRP injection into the cervical vagus nerve. A part of the lateral and ventral sides of the brainstem is cut. Rostral ŽR., caudal ŽC., dorsal ŽD. and V Žventral. represent the directions of position. A: Medial view of the left brainstem. Note the location of the AP and relation to the DMX and AN. B: Anteromedial view of the left brainstem. C–F: Caudo-rostral four parts of B. Note the shape of the cut surface on each nucleus. Scale bar s 1 mm.
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of the upper alimentary tract in this species, we had reported about the brainstem topology of the vagal motoneurons projecting to the esophagus and stomach in the S. murinus ŽWon et al., 1998.. However, the organization of the central origin of the vagus nerve in this species remains to be identified. Therefore, we have used the retrograde neuronal transport of horseradish peroxidase ŽHRP. to determine the central organization of the efferent neurons of the cervical vagus nerve and compared our results with the findings of previous investigations in other mammals.
2. Materials and methods 2.1. Animals and retrograde tracing We used the house musk shrew as experimental animal, S. murinus, belonging to the Insectivora. A total of 13 male or female adults weighing between 35 and 60 g ŽInstitute for Laboratory Animal Research, Nagoya Uni-
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versity School of Medicine, Japan. were anesthetized by injecting sodium pentobarbital Ž0.05 mlr10 g b.wt., i.p.. prior to operation and subsequent experimental procedures. After anesthesia, the left Ž n s 5., the right Ž n s 5. or both sides Ž n s 3. of the vagus nerve were exposed at the mid-cervical level. The nerve was then placed over a small sheet of Parafilm and cut at the mid-cervical level below the nodose ganglion with microdissection scissors. Two milligrams of crystalline HRP ŽToyobo, Japan. was applied to the proximal end of the cut nerve with fine forceps and dissolved in 10 m l saline and allowed to dry to a tacky consistency for 30–40 min, after which the area around the nerve was sealed with paraffin wax. The animal was then sutured and returned to its home cage. 2.2. HRP histochemistry After 24 h, the animals were reanesthetized and perfused transcardially with 150 ml of physiological saline, followed by 300 ml of fixative solution containing 1%
Fig. 2. Histograms showing mean numbers of the labelled neurons in the DMX ŽA., AN ŽB. and external formation of the ambiguus nucleus ŽANe. ŽC. in the house musk shrew following injection of HRP into the cervical vagus nerve. The horizontal axis of each histogram indicates the rostrocaudal location Žmm., where 0 was set as the AP. The vertical axis shows the number of HRP-labelled neurons.
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paraformaldehyde and 1.5% glutaraldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4. for 40 min at room temperature. And then the animals were followed by a 30 min perfusion with 100 ml of 10% sucrose solution in the same buffer. After perfusion, the brain and upper cervical spinal cord was removed and stored in 20% sucrose solution overnight at 48C. Serial transverse sections of 50 m m thickness were
cut with a freezing microtome ŽKamato Koki, Japan.. The sections were then processed, using a modified technique of Mesulam Ž1982., with tetramethylbenzidine ŽTMB. as chromogen for the demonstration of HRP reaction product, then placed on gelatin-coated slides and counterstained with Richardson’s solution. Series of sections were dehydrated in alcohol, cleared with xylene, and coverslipped.
Fig. 3. Photomicrographs showing retrogradely labelled neurons and fibers in the transverse sections of the brainstem after bilateral HRP injection into the cervical nerves. Neurons are labelled in the DMX and ANC. Note the labelled afferent fibers ŽXa. and efferent fibers ŽXe.. The levels of A and B represent 1.2 and 0.4 mm rostral to the area postrema, respectively. ANc, compact formation of ambiguus nucleus; ANe, external formation of ambiguus nucleus; ANl, loose formation of ambiguus nucleus; Lt, left side; NST, nucleus of the solitary tract; Rt, right side; XII, hypoglossal nucleus. Scale bar s 500 m m.
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2.3. Three-dimensional reconstruction and tissue analysis For the shape and location of the DMX and AN within the brainstem, views of a three-dimensional reconstruction of the DMX and AN based on the experiments were made. All transverse sections through the left brainstem Ž n s 4. were used, and the outline of the DMX and AN area containing HRP labelled cells were drawn with a microscope driven by a personal computer. The images of the specimens in the microscope and on the computer screen were optically mixed with a conventional drawing tube. The data was reconstructed as a three-dimensional image by a computer program ŽOZ, Olympus.. All the retrogradely labelled neurons found in all transverse sections
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through the brainstem Ž n s 7. were counted and represented the histogram. To document the distribution of labelled neurons, photographs were also taken using bright-field illumination. The level of the area postrema ŽAP. was used as the reference along the rostrocaudal axis; in the following description, the levels rostral to the AP were indicated by a plus sign and the levels caudal to the AP by a minus sign. 3. Results Following application of HRP to the cervical vagus nerve in the house musk shrew, retrogradely labelled neurons formed ipsilaterally two completely separate groups
Fig. 4. A: Labelled neurons in the right DMX at the level of 0.3 mm rostral to the area postrema after HRP injection into the right vagus nerve. B and C: High magnification of ambigual nuclear complex. ANc and ANl of the ambiguus nucleus could be distinguished morphologically. Note that the ANe is located ventromedially to the ambiguus nucleus. Scale bar s 250 m m.
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in the brainstem, a dorsomedial and a ventral cell column. These corresponded to the DMX and ambigual nuclear complex ŽANC.. A few labelled neurons were also identified in the intermediate zone between the DMX and ANC. The greatest concentration of labelled neurons was identified in the DMX, constituting approximately 80% of the total labelled neurons. The labelled neurons were observed throughout the entire length of the DMX. However, some neurons in the small ventrolateral part of the DMX were not labelled. Labelled neurons in the DMX formed a longitudinally oriented column that extended rostrally from the C1 level of the spinal cord Žapproximately 2.0 mm caudal to the AP. to the level caudal to the facial motor nucleus Žapproximately 1.6 mm rostral to the AP. ŽFigs. 1 and 2.. The rostrocaudal length of the DMX varied from 3.4 to 3.8 mm. The caudal tapering end of the DMX was closed dorsal to the hypoglossal nucleus. The middle portion of the DMX, however, expanded laterally in a cell group lying dorsolaterally to the hypoglossal nucleus. The rostral portion of the DMX, which was relatively broader than the caudal part, was situated immediately adjacent to the ependyma of the floor of the fourth ventricle ŽFigs. 1, 3 and 4A.. Half Žabout 47%. of the labelled neurons in the DMX were found at the level between the AP and q0.6 mm ŽFig. 2A.. In addition to the labelled neurons in the DMX, there were a few scattered labelled neurons around the DMX, in the nucleus of the solitary tract ŽNST.. A detailed morphological description of individual neurons after HRP injection into cervical vagus nerve was not possible because almost all the neurons were filled with HRP reaction products. DMX neurons were mainly medium-sized and polymorphic ŽFig. 4A.. The second major population of labelled neurons was confirmed within the AN and comprised about 12% of the total labelled neurons. Labelled AN neurons were relatively larger than labelled DMX neurons. Labelled neurons in the AN were distributed over a total rostrocaudal extent of 1.8 mm Žapproximately 1r2 of the entire DMX length. from 1.5 mm rostrally to the AP and 0.2 mm caudally to the AP ŽFigs. 1 and 2B.. Topographic organization of labelled neurons within the AN was obvious. The numerous labelled neurons packed in the rostral part Žbetween q1.1 and q1.5 mm. comprised a compact formation ŽANc., and the labelled neurons in the caudal part Žbetween y0.2 and q0.5 mm. comprised a loose formation ŽANl. ŽFig. 4B and C.. In the present study, we could not find a semicompact formation that seemed to be located between the ANc and ANl, where few labelled cells were observed ŽFig. 2B.. Ventral and ventromedial to the AN, a diffuse cell population Žapproximately 7% of the total labelled neurons. forming the external formation of the ambiguus nucleus ŽANe. was identified. Neurons in this region were significantly smaller in size than those identified in the AN, and comprised the cell column of approximately 1.2 mm in length. The ANe was located between q0.2 and
q1.4 mm, and coursed separately from the AN virtually over its entire length ŽFig. 2C, Fig. 4B and C.. Although the majority of labelled neurons were located in the DMX and AN, a few labelled neurons were found in the intermediate zone between the DMX and AN. Following the injection of HRP into the cervical vagus nerve, the axons of the labelled DMX neurons were arranged in one bundle traversing the medullary reticular formation. Labelled fiber fascicles of the DMX neurons were seen throughout a rostrocaudal extent of about 2.9 mm, beginning at the level of q1.4 mm and extending caudally to the level of y1.5 mm. There was no overlap in the rostrocaudal extent between the sites in which vagal afferent fibers entered the dorsolateral medulla oblongata and vagal efferent fibers were seen to exit from the ventral medulla oblongata. The labelled fascicles of DMX efferents coursed directly to their exit on the ventral medulla oblongata, without traversing the spinal tract and nucleus of the trigeminal nerve ŽSTNV.. The labelled axons from the AN joined directly the efferent fibers from the DMX ŽFigs. 3 and 4B and C..
4. Discussion In the present study, the retrogradely labelled vagal efferent neurons were located primarily in the DMX and ANC following application of HRP to the cervical vagus nerve of the house musk shrew. Nearly all of the DMX neurons were retrogradely labelled and they were distributed over a considerable longitudinal extent, from the upper cervical spinal cord to the level caudal to the facial motor nucleus. The majority of the labelled neurons in the brainstem were observed in the DMX. The greatest concentration of labelled neurons in the DMX was located around the AP. In general, these findings are in agreement with previous studies in various species: rat ŽFox and Powley, 1985; Kalia and Sullivan, 1982; Karim and Leong, 1980; Scharoun et al., 1984., guinea pig ŽJou et al., 1993., hamster ŽMiceli and Malsbury, 1985., ferret ŽRanson et al., 1993; Withington-Wray and Spyer, 1988., mink ŽRanson et al., 1993., cat ŽKalia, 1981; Kalia and Mesulam, 1980; Nomura and Mizuno, 1983., dog ŽChernicky et al., 1983. and monkey ŽGwyn et al., 1985; Hamilton et al., 1987.. Although most of the neurons in the DMX of the house musk shrew were retrogradely labelled, a few neurons in the lateral and ventrolateral parts of the nucleus were not labelled. Some authors suggested that these unlabelled neurons may project to certain visceral sites or other regions of the brain in the rat ŽKalia and Sullivan, 1982., ferret ŽKnox et al., 1994. and monkey ŽHamilton et al., 1987.. In support of this assumption, some neurons in the DMX of the cat have been shown to project rostrally to or through the pons ŽKing, 1980; McLean and Hopkins, 1982. and to the cerebellum ŽZheng et al., 1982..
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Two populations of labelled neurons were distinguished in the ANC in the present study. The large neurons constituted the AN, and the medium-sized neurons lying ventral to the AN constituted the ANe. In some species, including the rat ŽKalia and Sullivan, 1982., ferret and mink ŽRanson et al., 1993., cat Kalia and Mesulam, 1980. and monkey ŽHamilton et al., 1987., it was reported that the AN comprises three distinct subnuclei: the ‘retrofacial nucleus’ rostrally, the ‘ambiguus nucleus proper’ intermediately, and the ‘retroambigual nucleus’ caudally. HRP injection into the supranodosal vagus nerve of the rat ŽAltschuler et al., 1991; Bieger and Hopkins, 1987. resulted in AN labelling that comprised a rostral esophagomotor ‘compact formation’, an intermediate pharyngolaryngomotor ‘semicompact formation’ and a caudal laryngomotor ‘loose formation’, whereas, HRP injection into the infranodosal vagus nerve resulted in the semicompact formation was not defined. Injection of HRP into the pharyngeal branch of the vagus nerve, which exits at the level of the nodose ganglion, resulted in retrograde labelling localized selectively in the semicompact formation ŽAltschuler et al., 1989; Bieger and Hopkins, 1987.. In the present study, the AN of the house musk shrew could be subdivided into two parts corresponding to the compact formation and to the loose formation. We injected HRP at mid-cervical level below the nodose ganglion in the vagus nerve. Therefore, we could not observe a labelled neuronal population corresponding to the semicompact formation. It has been considered in the rat ŽAltschuler et al., 1989; Bieger and Hopkins, 1987. that the neurons of the semicompact formation of the AN project to the pharyngeal musculature by branches exiting the vagus nerve at the level of the nodose ganglion ŽAltschuler et al., 1989; Bieger and Hopkins, 1987.. Therefore, the central origin of the pharyngeal efferent innervation from the AN remains to be identified in the house musk shrew. In the ferret and mink ŽRanson et al., 1993; Withington-Wray and Spyer, 1988., a discrete subdivision was identified ventral to the AN, which was named the ‘ ventral ambiguus nucleus’. In the present study, a similar subdivision was identified and the corresponding region was termed ANe. It was also reported in the rat that the ventral and ventrolateral divisions of the ANC correspond to the ‘external formation’, which has been referred to as the periambigual area ŽBieger and Hopkins, 1987; Bystrzycka and Nail, 1985; Kalia and Sullivan, 1982; Nosaka et al., 1979; Stuesse, 1982.. In the house musk shrew, neurons in the ANe were found to be smaller in size than those observed in the AN. This latter finding is in agreement with ferret and mink studies ŽRanson et al., 1993; Withington-Wray and Spyer, 1988.. The brainstem origin of preganglionic cardiac motoneurons has been studied in some species using retrograde transport techniques. However, individual reports have differed as to the location of the preponderance of labelled neurons. Some implicated the DMX or the ANe as the
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principal nucleus of origin ŽNosaka et al., 1979, 1982., while others showed both nuclei and the intermediate zone as important loci ŽCiriello and Calaresu, 1982; Standish et al., 1995.. The discrepancies among these results may be due to technical parameters, since tracers has been injected either directly into the heart ŽKalia, 1981; Stuesse, 1982., or into cardiac nerves ŽCiriello and Calaresu, 1982; Hopkins et al., 1984; Karim and Leong, 1980; Nosaka et al., 1979.. Furthermore, the problem of spread of the injected tracer is endemic with all studies of this nature, and may be a possible source of the confusion ŽFox and Powley, 1989.. Recently it has been reported in the muskrat that preganglionic cardiac motoneurons were principally located in the ANe ŽPannenton et al., 1996., which, thus, contained general visceral efferent motoneurons. Therefore, the ANe of the house musk shrew may not innervate the special visceral structures, i.e. the striated musculature of the upper digestive tract and larynx, but may represent cardiac preganglionics by certain branches of the vagus nerve. On the other hand, it was reported that, at rostral levels of the ANC in the ferret and mink ŽRanson et al., 1993., the ANC gives rise to a third subdivision of labelled neurons named the ‘medial ambiguus nucleus’. Similar subdivisions of the ANC have also been observed in the rat ŽHinrichsen and Ryan, 1981. and rabbit ŽLawn, 1996.. In the present study, however, we could not observe the corresponding neuronal population. The presence of labelled neurons in the intermediate zone between the DMX and AN was reported in some mammals ŽHamilton et al., 1987; Kalia, 1981; Ranson et al., 1993.. In the house musk shrew these neurons were also identified, and were similar in size to those of the DMX. This finding differs from the monkey ŽHamilton et al., 1987. whose intermediate zone neurons are similar to AN neurons. It is unknown whether this species difference is physiologically significant. In the house musk shrew, the labelled efferent and afferent fibers of the vagus nerve were observed to course separately in the brainstem. The efferent fascicle of the DMX neurons coursed directly in a ventral direction, not through the STNV. This finding differs from the rat ŽKalia and Sullivan, 1982., in which efferent fibers from DMX are divided into a number of small fiber bundles that do not traverse the STNV and are so widely separated from the afferent fibers. In the cat ŽKalia and Mesulam, 1980; Nomura and Mizuno, 1983., these fibers course through the STNV as one group. On the other hand, in the ferret and mink ŽRanson et al., 1993. these fibers from DMX are separated from the afferent fibers that course through the STNV. In conclusion, our results in the house musk shrew showed that the efferent components of the vagus originated from the DMX, AN and ANe. The morphological feature of the DMX was similar to other mammals, but AN and ANe were different from other mammals.
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Acknowledgements The authors would like to thank Mr Hiroki Kawashima for his assistance of programming for three-dimensional reconstruction and histogram on computer, and Mr Akihito Takei for his support of experimental animals management. This work was supported by Korea Science and Engineering Foundation grants KOSEF-2312-4069 ŽWon, M.H.. and Nagoya University School of Medicine grants ŽKitoh, Z...
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M.H. Won et al.r Journal of the Autonomic NerÕous System 71 (1998) 55–63 and gastric connections to the central nervous system determined by the transport of horseradish peroxidase. Brain Res. Bull. 13, 573–583. Selve, N., Friderichs, E., Reimann, W., Reinartz, S., 1994. Absence of emetic effects of morphine and loperamide in Suncus murinus. Eur. J. Pharmacol. 256, 287–293. Standish, A., Enquist, L.W., Escardo, J.A., Schwaber, J.S., 1995. Central neuronal circuit innervating the rat heart defined by transneuronal transport of pseudorabies virus. J. Neurosci. 15, 1998–2012. Stuesse, S.L., 1982. Origins of cardiac vagal preganglionic fibers: a retrograde transport study. Brain Res. 236, 15–25. Torii, Y., Saito, H., Mastuki, N., 1993. Induction of emesis in Suncus murinus by pyrogallol, a generator of free radicals. Br. J. Pharmacol. 111, 431–434.
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Ueno, S., Matsuki, N., Saito, H., 1987. Suncus murinus: a new experimental model in emesis research. Life Sci. 41, 513–518. Withington-Wray, D.J., Spyer, K.X., 1988. The brainstem localization of vagal preganglionic neurones in the ferret Mustela putorius furo. Q. J. Exp. Physiol. 73, 439–441. Won, M.H., Matsuo, K., Oh, Y.S., Kitoh, J., 1998. Brainstem topology of the vagal motoneurons projecting to the esophagus and stomach in the house musk shrew, Suncus murinus. J. Auton. Nerv. Syst. 68, 171– 181. Zheng, Z.H., Dietrichs, E., Walberg, F., 1982. Cerebellar afferent fibers from the dorsal motor vagal nucleus in the cat. Neurosci. Lett. 32, 113–118.
Brainstem origin of the efferent components of the cervical vagus nerve in the house musk shrew, Suncus murinus a,)
Moo Ho Won
, Koji Matsuo d , Seung Mook Jo a , Tae-Cheon Kang b, Yang Seok Oh c , Chang Do Choi a , Junjoh Kitoh d
a
Department of Anatomy, College of Medicine, Hallym UniÕersity, Chunchon 200-702, South Korea Department of Anatomy, College of Medicine, Seoul National UniÕersity, Seoul 110-799, South Korea c Experimental Animal Center, College of Medicine, Hallym UniÕersity, Chunchon 200-702, South Korea d Institute for Laboratory Animal Research, Nagoya UniÕersity School of Medicine, Nagoya 466, Japan b
Received 24 February 1998; revised 30 March 1998; accepted 31 March 1998
Abstract The brainstem origin of the efferent neurons of the vagus nerve in the house musk shrew, an animal species which has been recently used in researches on emesis, was studied using the retrograde tracing method. The vagus nerve was exposed and cut at the mid-cervical level below the nodose ganglion. Horseradish peroxidase was applied to the proximal end of the cut nerve. The brainstem was sectioned and processed histochemically with the tetramethylbenzidine method. The horseradish peroxidase injection into the vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve and ambigual nuclear complex. Labelled neurons in the dorsal motor nucleus of the vagus nerve, constituting approximately 80% of the total labelled neurons, formed a longitudinal column whose length varied from 3.4 to 3.8 mm. Half of labelled neurons in this nucleus were found at the level between the area postrema and 0.6 mm rostral to it. The ambigual nuclear complex was made up of two major longitudinal divisions; the dorsal division corresponded to the ambiguus nucleus and the ventral division was identified as the external formation of the ambiguus nucleus. Our results suggest that in the Suncus murinus the neuroanatomical feature of the dorsal motor nucleus of the vagus nerve is similar to those of other mammals, but ambigual nuclear complex must be somewhat different between mammals. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dorsal motor nucleus of the vagus nerve; Ambigual nuclear complex; Retrograde tracing; Horseradish peroxidase; Insectivore
1. Introduction The vagus nerve is involved in many autonomic and various regulatory functions and has heterogeneous components, i.e. preganglionic parasympathetics; visceral afferents that follow their peripheral distribution; motoneuAbbreviations: AN, Ambiguus nucleus; ANC, Ambigual nuclear complex; ANc, Compact formation of ambiguus nucleus; ANe, External formation of ambiguus nucleus; ANl, Loose formation of ambiguus nucleus; AP, Area postrema; DMX, Dorsal motor nucleus of the vagus nerve; HRP, Horseradish peroxidase; NST, Nucleus of the solitary tract; RF, Reticular formation; ST, Solitary tract; STNV, Spinal tract and nucleus of the trigeminal nerve; V, The fourth ventricle; X, Vagus nerve; Xa, Afferent fibers of the vagus nerve; Xe, Efferent fibers of the vagus nerve; XII, Hypoglossal nucleus ) Corresponding author. Tel.: q82 361 2401614; fax: q82 361 561614; e-mail: [email protected] 0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 6 2 - 9
rons to the pharynx, larynx and upper esophagus; gustatory fibers from near the epiglottis; and somatic afferents from the external ear ŽChen et al., 1996; Haxhiu and Loewy, 1996; Kinami et al., 1997; Ladic and Buchan, 1996; Myers et al., 1996.. Brainstem neurons associated with autonomic and regulatory functions are difficult to identify neuroanatomically because they often do not aggregate in histologically welldefined nuclei. The exact locations of some of the brainstem nuclei may be related to the migration of the cells during development. The primitive dorsal visceral efferent column of the medulla oblongata separates into dorsal and ventral portions ŽBystrzycka and Nail, 1985.. The general visceral efferent neurons remain in the dorsal motor nucleus of the vagus nerve ŽDMX., while the special visceral efferent neurons supplying branchial musculature migrate to form the ambiguus nucleus ŽAN. ŽAltschuler et al.,
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1991; Bieger and Hopkins, 1987; Bystrzycka and Nail, 1985; Hopkins et al., 1984; Lawn, 1996.. The central efferent organization of the vagus nerve and vagal efferent neurons projecting to some viscera has been documented by techniques based on retrograde tract-tracing methods in many vertebrates, including virtually all common laboratory species. Vagal efferent axons originate from either the DMX or vicinity of the AN ŽAltschuler et al., 1991; Gwyn et al., 1985; Hamilton et al., 1987; Kalia and Mesulam, 1980; Kressel et al., 1994; Okumura and Namiki, 1990; Withington-Wray and Spyer, 1988.. Recently a small animal, the insectivore Suncus murinus, has been described as a new animal model for researches on emesis ŽMiyachi, 1996; Selve et al., 1994;
Ueno et al., 1987.. This new model is not commonly used in pharmacological laboratories, but some basic experiments have already been performed ŽMiyachi, 1996; Okada et al., 1995; Selve et al., 1994; Torii et al., 1993; Ueno et al., 1987.. In addition, the S. murinus is one of the most primitive and phylogenetic earliest eutherians, and therefore the relationship of primates to this order is closer than that to rodents usually used in pharmacological research ŽSelve et al., 1994.. In this regard different anatomical and functional organization may account for different susceptibility to or even ability to vomit and may be an explanation for inability of rat, mouse, guinea pig or rabbit to vomit in contrast to ferret, dog, cat and man ŽSelve et al., 1994.. With the necessity to identify the vagal innervation
Fig. 1. Three-dimensional computer reconstruction of the labelled DMX and AN within the left brainstem of the house musk shrew after HRP injection into the cervical vagus nerve. A part of the lateral and ventral sides of the brainstem is cut. Rostral ŽR., caudal ŽC., dorsal ŽD. and V Žventral. represent the directions of position. A: Medial view of the left brainstem. Note the location of the AP and relation to the DMX and AN. B: Anteromedial view of the left brainstem. C–F: Caudo-rostral four parts of B. Note the shape of the cut surface on each nucleus. Scale bar s 1 mm.
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of the upper alimentary tract in this species, we had reported about the brainstem topology of the vagal motoneurons projecting to the esophagus and stomach in the S. murinus ŽWon et al., 1998.. However, the organization of the central origin of the vagus nerve in this species remains to be identified. Therefore, we have used the retrograde neuronal transport of horseradish peroxidase ŽHRP. to determine the central organization of the efferent neurons of the cervical vagus nerve and compared our results with the findings of previous investigations in other mammals.
2. Materials and methods 2.1. Animals and retrograde tracing We used the house musk shrew as experimental animal, S. murinus, belonging to the Insectivora. A total of 13 male or female adults weighing between 35 and 60 g ŽInstitute for Laboratory Animal Research, Nagoya Uni-
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versity School of Medicine, Japan. were anesthetized by injecting sodium pentobarbital Ž0.05 mlr10 g b.wt., i.p.. prior to operation and subsequent experimental procedures. After anesthesia, the left Ž n s 5., the right Ž n s 5. or both sides Ž n s 3. of the vagus nerve were exposed at the mid-cervical level. The nerve was then placed over a small sheet of Parafilm and cut at the mid-cervical level below the nodose ganglion with microdissection scissors. Two milligrams of crystalline HRP ŽToyobo, Japan. was applied to the proximal end of the cut nerve with fine forceps and dissolved in 10 m l saline and allowed to dry to a tacky consistency for 30–40 min, after which the area around the nerve was sealed with paraffin wax. The animal was then sutured and returned to its home cage. 2.2. HRP histochemistry After 24 h, the animals were reanesthetized and perfused transcardially with 150 ml of physiological saline, followed by 300 ml of fixative solution containing 1%
Fig. 2. Histograms showing mean numbers of the labelled neurons in the DMX ŽA., AN ŽB. and external formation of the ambiguus nucleus ŽANe. ŽC. in the house musk shrew following injection of HRP into the cervical vagus nerve. The horizontal axis of each histogram indicates the rostrocaudal location Žmm., where 0 was set as the AP. The vertical axis shows the number of HRP-labelled neurons.
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paraformaldehyde and 1.5% glutaraldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4. for 40 min at room temperature. And then the animals were followed by a 30 min perfusion with 100 ml of 10% sucrose solution in the same buffer. After perfusion, the brain and upper cervical spinal cord was removed and stored in 20% sucrose solution overnight at 48C. Serial transverse sections of 50 m m thickness were
cut with a freezing microtome ŽKamato Koki, Japan.. The sections were then processed, using a modified technique of Mesulam Ž1982., with tetramethylbenzidine ŽTMB. as chromogen for the demonstration of HRP reaction product, then placed on gelatin-coated slides and counterstained with Richardson’s solution. Series of sections were dehydrated in alcohol, cleared with xylene, and coverslipped.
Fig. 3. Photomicrographs showing retrogradely labelled neurons and fibers in the transverse sections of the brainstem after bilateral HRP injection into the cervical nerves. Neurons are labelled in the DMX and ANC. Note the labelled afferent fibers ŽXa. and efferent fibers ŽXe.. The levels of A and B represent 1.2 and 0.4 mm rostral to the area postrema, respectively. ANc, compact formation of ambiguus nucleus; ANe, external formation of ambiguus nucleus; ANl, loose formation of ambiguus nucleus; Lt, left side; NST, nucleus of the solitary tract; Rt, right side; XII, hypoglossal nucleus. Scale bar s 500 m m.
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2.3. Three-dimensional reconstruction and tissue analysis For the shape and location of the DMX and AN within the brainstem, views of a three-dimensional reconstruction of the DMX and AN based on the experiments were made. All transverse sections through the left brainstem Ž n s 4. were used, and the outline of the DMX and AN area containing HRP labelled cells were drawn with a microscope driven by a personal computer. The images of the specimens in the microscope and on the computer screen were optically mixed with a conventional drawing tube. The data was reconstructed as a three-dimensional image by a computer program ŽOZ, Olympus.. All the retrogradely labelled neurons found in all transverse sections
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through the brainstem Ž n s 7. were counted and represented the histogram. To document the distribution of labelled neurons, photographs were also taken using bright-field illumination. The level of the area postrema ŽAP. was used as the reference along the rostrocaudal axis; in the following description, the levels rostral to the AP were indicated by a plus sign and the levels caudal to the AP by a minus sign. 3. Results Following application of HRP to the cervical vagus nerve in the house musk shrew, retrogradely labelled neurons formed ipsilaterally two completely separate groups
Fig. 4. A: Labelled neurons in the right DMX at the level of 0.3 mm rostral to the area postrema after HRP injection into the right vagus nerve. B and C: High magnification of ambigual nuclear complex. ANc and ANl of the ambiguus nucleus could be distinguished morphologically. Note that the ANe is located ventromedially to the ambiguus nucleus. Scale bar s 250 m m.
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in the brainstem, a dorsomedial and a ventral cell column. These corresponded to the DMX and ambigual nuclear complex ŽANC.. A few labelled neurons were also identified in the intermediate zone between the DMX and ANC. The greatest concentration of labelled neurons was identified in the DMX, constituting approximately 80% of the total labelled neurons. The labelled neurons were observed throughout the entire length of the DMX. However, some neurons in the small ventrolateral part of the DMX were not labelled. Labelled neurons in the DMX formed a longitudinally oriented column that extended rostrally from the C1 level of the spinal cord Žapproximately 2.0 mm caudal to the AP. to the level caudal to the facial motor nucleus Žapproximately 1.6 mm rostral to the AP. ŽFigs. 1 and 2.. The rostrocaudal length of the DMX varied from 3.4 to 3.8 mm. The caudal tapering end of the DMX was closed dorsal to the hypoglossal nucleus. The middle portion of the DMX, however, expanded laterally in a cell group lying dorsolaterally to the hypoglossal nucleus. The rostral portion of the DMX, which was relatively broader than the caudal part, was situated immediately adjacent to the ependyma of the floor of the fourth ventricle ŽFigs. 1, 3 and 4A.. Half Žabout 47%. of the labelled neurons in the DMX were found at the level between the AP and q0.6 mm ŽFig. 2A.. In addition to the labelled neurons in the DMX, there were a few scattered labelled neurons around the DMX, in the nucleus of the solitary tract ŽNST.. A detailed morphological description of individual neurons after HRP injection into cervical vagus nerve was not possible because almost all the neurons were filled with HRP reaction products. DMX neurons were mainly medium-sized and polymorphic ŽFig. 4A.. The second major population of labelled neurons was confirmed within the AN and comprised about 12% of the total labelled neurons. Labelled AN neurons were relatively larger than labelled DMX neurons. Labelled neurons in the AN were distributed over a total rostrocaudal extent of 1.8 mm Žapproximately 1r2 of the entire DMX length. from 1.5 mm rostrally to the AP and 0.2 mm caudally to the AP ŽFigs. 1 and 2B.. Topographic organization of labelled neurons within the AN was obvious. The numerous labelled neurons packed in the rostral part Žbetween q1.1 and q1.5 mm. comprised a compact formation ŽANc., and the labelled neurons in the caudal part Žbetween y0.2 and q0.5 mm. comprised a loose formation ŽANl. ŽFig. 4B and C.. In the present study, we could not find a semicompact formation that seemed to be located between the ANc and ANl, where few labelled cells were observed ŽFig. 2B.. Ventral and ventromedial to the AN, a diffuse cell population Žapproximately 7% of the total labelled neurons. forming the external formation of the ambiguus nucleus ŽANe. was identified. Neurons in this region were significantly smaller in size than those identified in the AN, and comprised the cell column of approximately 1.2 mm in length. The ANe was located between q0.2 and
q1.4 mm, and coursed separately from the AN virtually over its entire length ŽFig. 2C, Fig. 4B and C.. Although the majority of labelled neurons were located in the DMX and AN, a few labelled neurons were found in the intermediate zone between the DMX and AN. Following the injection of HRP into the cervical vagus nerve, the axons of the labelled DMX neurons were arranged in one bundle traversing the medullary reticular formation. Labelled fiber fascicles of the DMX neurons were seen throughout a rostrocaudal extent of about 2.9 mm, beginning at the level of q1.4 mm and extending caudally to the level of y1.5 mm. There was no overlap in the rostrocaudal extent between the sites in which vagal afferent fibers entered the dorsolateral medulla oblongata and vagal efferent fibers were seen to exit from the ventral medulla oblongata. The labelled fascicles of DMX efferents coursed directly to their exit on the ventral medulla oblongata, without traversing the spinal tract and nucleus of the trigeminal nerve ŽSTNV.. The labelled axons from the AN joined directly the efferent fibers from the DMX ŽFigs. 3 and 4B and C..
4. Discussion In the present study, the retrogradely labelled vagal efferent neurons were located primarily in the DMX and ANC following application of HRP to the cervical vagus nerve of the house musk shrew. Nearly all of the DMX neurons were retrogradely labelled and they were distributed over a considerable longitudinal extent, from the upper cervical spinal cord to the level caudal to the facial motor nucleus. The majority of the labelled neurons in the brainstem were observed in the DMX. The greatest concentration of labelled neurons in the DMX was located around the AP. In general, these findings are in agreement with previous studies in various species: rat ŽFox and Powley, 1985; Kalia and Sullivan, 1982; Karim and Leong, 1980; Scharoun et al., 1984., guinea pig ŽJou et al., 1993., hamster ŽMiceli and Malsbury, 1985., ferret ŽRanson et al., 1993; Withington-Wray and Spyer, 1988., mink ŽRanson et al., 1993., cat ŽKalia, 1981; Kalia and Mesulam, 1980; Nomura and Mizuno, 1983., dog ŽChernicky et al., 1983. and monkey ŽGwyn et al., 1985; Hamilton et al., 1987.. Although most of the neurons in the DMX of the house musk shrew were retrogradely labelled, a few neurons in the lateral and ventrolateral parts of the nucleus were not labelled. Some authors suggested that these unlabelled neurons may project to certain visceral sites or other regions of the brain in the rat ŽKalia and Sullivan, 1982., ferret ŽKnox et al., 1994. and monkey ŽHamilton et al., 1987.. In support of this assumption, some neurons in the DMX of the cat have been shown to project rostrally to or through the pons ŽKing, 1980; McLean and Hopkins, 1982. and to the cerebellum ŽZheng et al., 1982..
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Two populations of labelled neurons were distinguished in the ANC in the present study. The large neurons constituted the AN, and the medium-sized neurons lying ventral to the AN constituted the ANe. In some species, including the rat ŽKalia and Sullivan, 1982., ferret and mink ŽRanson et al., 1993., cat Kalia and Mesulam, 1980. and monkey ŽHamilton et al., 1987., it was reported that the AN comprises three distinct subnuclei: the ‘retrofacial nucleus’ rostrally, the ‘ambiguus nucleus proper’ intermediately, and the ‘retroambigual nucleus’ caudally. HRP injection into the supranodosal vagus nerve of the rat ŽAltschuler et al., 1991; Bieger and Hopkins, 1987. resulted in AN labelling that comprised a rostral esophagomotor ‘compact formation’, an intermediate pharyngolaryngomotor ‘semicompact formation’ and a caudal laryngomotor ‘loose formation’, whereas, HRP injection into the infranodosal vagus nerve resulted in the semicompact formation was not defined. Injection of HRP into the pharyngeal branch of the vagus nerve, which exits at the level of the nodose ganglion, resulted in retrograde labelling localized selectively in the semicompact formation ŽAltschuler et al., 1989; Bieger and Hopkins, 1987.. In the present study, the AN of the house musk shrew could be subdivided into two parts corresponding to the compact formation and to the loose formation. We injected HRP at mid-cervical level below the nodose ganglion in the vagus nerve. Therefore, we could not observe a labelled neuronal population corresponding to the semicompact formation. It has been considered in the rat ŽAltschuler et al., 1989; Bieger and Hopkins, 1987. that the neurons of the semicompact formation of the AN project to the pharyngeal musculature by branches exiting the vagus nerve at the level of the nodose ganglion ŽAltschuler et al., 1989; Bieger and Hopkins, 1987.. Therefore, the central origin of the pharyngeal efferent innervation from the AN remains to be identified in the house musk shrew. In the ferret and mink ŽRanson et al., 1993; Withington-Wray and Spyer, 1988., a discrete subdivision was identified ventral to the AN, which was named the ‘ ventral ambiguus nucleus’. In the present study, a similar subdivision was identified and the corresponding region was termed ANe. It was also reported in the rat that the ventral and ventrolateral divisions of the ANC correspond to the ‘external formation’, which has been referred to as the periambigual area ŽBieger and Hopkins, 1987; Bystrzycka and Nail, 1985; Kalia and Sullivan, 1982; Nosaka et al., 1979; Stuesse, 1982.. In the house musk shrew, neurons in the ANe were found to be smaller in size than those observed in the AN. This latter finding is in agreement with ferret and mink studies ŽRanson et al., 1993; Withington-Wray and Spyer, 1988.. The brainstem origin of preganglionic cardiac motoneurons has been studied in some species using retrograde transport techniques. However, individual reports have differed as to the location of the preponderance of labelled neurons. Some implicated the DMX or the ANe as the
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principal nucleus of origin ŽNosaka et al., 1979, 1982., while others showed both nuclei and the intermediate zone as important loci ŽCiriello and Calaresu, 1982; Standish et al., 1995.. The discrepancies among these results may be due to technical parameters, since tracers has been injected either directly into the heart ŽKalia, 1981; Stuesse, 1982., or into cardiac nerves ŽCiriello and Calaresu, 1982; Hopkins et al., 1984; Karim and Leong, 1980; Nosaka et al., 1979.. Furthermore, the problem of spread of the injected tracer is endemic with all studies of this nature, and may be a possible source of the confusion ŽFox and Powley, 1989.. Recently it has been reported in the muskrat that preganglionic cardiac motoneurons were principally located in the ANe ŽPannenton et al., 1996., which, thus, contained general visceral efferent motoneurons. Therefore, the ANe of the house musk shrew may not innervate the special visceral structures, i.e. the striated musculature of the upper digestive tract and larynx, but may represent cardiac preganglionics by certain branches of the vagus nerve. On the other hand, it was reported that, at rostral levels of the ANC in the ferret and mink ŽRanson et al., 1993., the ANC gives rise to a third subdivision of labelled neurons named the ‘medial ambiguus nucleus’. Similar subdivisions of the ANC have also been observed in the rat ŽHinrichsen and Ryan, 1981. and rabbit ŽLawn, 1996.. In the present study, however, we could not observe the corresponding neuronal population. The presence of labelled neurons in the intermediate zone between the DMX and AN was reported in some mammals ŽHamilton et al., 1987; Kalia, 1981; Ranson et al., 1993.. In the house musk shrew these neurons were also identified, and were similar in size to those of the DMX. This finding differs from the monkey ŽHamilton et al., 1987. whose intermediate zone neurons are similar to AN neurons. It is unknown whether this species difference is physiologically significant. In the house musk shrew, the labelled efferent and afferent fibers of the vagus nerve were observed to course separately in the brainstem. The efferent fascicle of the DMX neurons coursed directly in a ventral direction, not through the STNV. This finding differs from the rat ŽKalia and Sullivan, 1982., in which efferent fibers from DMX are divided into a number of small fiber bundles that do not traverse the STNV and are so widely separated from the afferent fibers. In the cat ŽKalia and Mesulam, 1980; Nomura and Mizuno, 1983., these fibers course through the STNV as one group. On the other hand, in the ferret and mink ŽRanson et al., 1993. these fibers from DMX are separated from the afferent fibers that course through the STNV. In conclusion, our results in the house musk shrew showed that the efferent components of the vagus originated from the DMX, AN and ANe. The morphological feature of the DMX was similar to other mammals, but AN and ANe were different from other mammals.
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Acknowledgements The authors would like to thank Mr Hiroki Kawashima for his assistance of programming for three-dimensional reconstruction and histogram on computer, and Mr Akihito Takei for his support of experimental animals management. This work was supported by Korea Science and Engineering Foundation grants KOSEF-2312-4069 ŽWon, M.H.. and Nagoya University School of Medicine grants ŽKitoh, Z...
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