Substance P- and tyrosine hydroxylase- containing neurom in the human dorsal motor nucleus of the...

THE JOURNAL OF COMPARATIVE NEUROLOGY 335~109-122 (1993)

Substance P- and Tyrosine Hydroxylase-Containing Neurons

in the Human Dorsal Motor Nucleus of the Vagus Nerve

XU-FENG HUANG, GEORGE PAXINOS, PAUL HALASZ, DEBORAH MCRITCHIE, AND ISTVAN TORK*

Schools of Anatomy (X.-F.H., I.T., D.M., P.H.) and Psychology (G.P.), University of New South Wales, Kensington, Sydney 2033, NSW, Australia

ABSTRACT The aim of this study was to provide a comprehensive account of the topography,

morphology, and frequencies of the substance P- and tyrosine hydroxylase-containing neurons in the human dorsal motor nucleus of the vagus nerve. The morphology of immunoreactive neurons was studied and the variations of the cell distributions were presented by three- dimensional computer reconstructions. Three types of substance P-like immunoreactive neurons were identified. They were predominantly located in the dorsointermediate, centrointermediate, caudointermediate, and caudal division of the dorsal motor nucleus of the vagus nerve. The morphology of substance P-like immunoreactive neurons varied according to the subnuclei in which they were found, Three types of tyrosine hydroxylase-like immunoreactive neurons were identified, mainly in the periphery of the dorsal motor nucleus of the vagus nerve, including the medial fringe, ventrointermediate, and dorsointermediate subnuclei of the 10. Many cells throughout the ventrointermediate subnucleus of the dorsal motor nucleus of the vagus nerve are seen ventrally to intermingle with the tyrosine hydroxylase neurons of the intermediate reticular zone.

Computer reconstructions provided a three-dimensional view of the positions of substance P- and tyrosine hydroxylase-like immunoreactive neurons within the subdivisions of the dorsal motor nucleus of the vagus nerve. The uneven distribution of substance P- and tyrosine hydroxylase-like immunoreactive neurons within the subdivisions suggests an involvement of these substances in some, but not all, autonomic functions of the dorsal motor nucleus of the vagus nerve. 0 1993 Wiley-Liss, Inc.

Key words: autonomic, chemoarchitecture, medulla, A2

The dorsal motor nucleus of the vagus nerve (10) is the major source of parasympathetic preganglionic efferent fibers to the autonomic organs. The location of the human 10 commences 2 mm caudal to the pontomedullary junction and extends about 14 mm further caudally, until the level of pyramidal decussation. The neurons of the human 10 display heterogeneous clustering and have been classified into six types. Rostrocaudally, the human 10 features nine subnuclei, the dorsorostral (DoR), ventrorostral (VeR), rostrointermediate (RoI), dorsointermediate (DoI), centrointermediate (CeI), ventrointermediate (VeI), caudointermediate (CaI), and caudal (Ca) (Huang et al., '93). These subnuclei have been grouped in three major divisions (rostral, intermediate, and caudal).

Substance P (SP) is an undecapeptide (Von Euler and Gaddum, '31) which remains fairly stable for postmortem

periods of up to 72 hours in human (Gale et al., '78; Emson et al., '80; Cooper et al., '81). The substance P-like immunoreactivity is widely but unevenly distributed in the central nervous system of experimental animals (Kanazawa and Jessell, '76; Cue110 and Kanazawa, '78; Sasek et al., '84 in rats; Krukoff et al., '85 in cat) and humans (Hokfelt et al., '76; Kanazawa et al., '77; Gale et al., '78; Emson, '79; Emson et al., '80; Snyder, '80; Langevin and Emson, '82; Charnay et al., '83; Beach et al., '84, '87a; Del Fiacco et al., '84; Sakamoto et al., '85; Bouras et al., '86; Mai et al., '86;

Accepted April 16, 1993. *Professor Istvan Tork, our long-time colleague and friend, died on

November 21,1992 at the age of 53. Address reprint requests to Dr. Xu-Feng Huang, School of Psychology,

University of New South Wales, Kensington, Sydney 2033, NSW, Australia.

O 1993 WILEY-LISS, INC.

110 X.-F. HUANG ET AL.

bodies or black melanin-containing cell bodies (Bogerts, '81; Saper and Petito, '82). A number of investigators have demonstrated the existence of catecholamine immunoreactivity in the human medulla oblongata including the 10 (Nobin and Bjorklund, '73; Lew et. al., '76; Pearson et al., '83, '90; Kitahama et al., '85; Halliday et al., '88).

The present study is concerned primarily with the distribution pattern of substance P- and tyrosine hydroxylase- containing neurons in the human 10, especially as this pattern relates to the recently identified subnuclei of the 10 (Huang et al., '93). Although the existence of substance P-like immunoreactive (SP-LI) and tyrosine hydroxylase immunoreactive (TH-LI) neurons in the human 10 has been reported by several researchers, it was of particular interest in this study to determine the precise rostrocaudal extent of these neuronal populations and to investigate their morphology in depth. Computer-assisted techniques were extensively applied in this study in order to obtain quantitative data relating to number, size, and shape of the SP- and TH-containing neurons in the human 10. Follow- ing completion of chemoarchitectonic and topographical analyses, the precise localization of the SP- and TH- containing neurons were defined by three-dimensional computer reconstructions. Neurochemical differences among the nine subnuclei of the human 10 were established.

Huang et al., '92). Comparative studies of the central nervous system reveal that the distribution of substance P-like immunoreactivity displays a remarkable homology, though there is also evidence for some species differences (Ljungdahl et al., '78; Nomura et al., '82, '87; Del Fiacco et al., '83, '84).

For a number of years it has been appreciated that substance P is not only a sensory neurotransmitter for general afferent fibers (Emson, '79; Snyder, '80; Hokfelt et al., '80; Del Fiacco et al., '84; Cuello et al., '851, but is also involved in extensive autonomic functions, such as cardiac control (in the rat, Helke et al., '80a,b; or see Talman and Reis, '811, stomach and oesophageal functions (in the pig and toad, Lindh et al., '83; Gibbins et al., '87), and respiratory functions (in the toad, Gibbins et al., '87).

Since Pernow ('53) first recognised that substance P is a constituent of the vagus nerve, many immunocytochemical studies have demonstrated its presence in somata and fibers within the solitary vagal complex, including the 10, nucleus of the solitary tract, and area postrema (Cuello and Kanazawa, '78; Ljungdahl et al., '78; Helke et al., '80a,b; Armstrong et al., '81, '82; Voorn and Buijs, '83). Peripher- ally, substance P plays an important role in autonomic sensory transmission with some autonomic sensory neurons releasing substance P both at the central and periph- eral end (Gilbert et al., '80). This has been demonstrated in the aortic nerve afferents of the rat (Helke et al., '80a; Gillis et al., 'SO), splanchnic nerve of the rat (Lundberget al., '781, oesophagus of the toad (Gibbins et al., '87), and pylorus of the stomach of the guinea pig (Lindh et al., '83). Application of substance P in the rat dorsal motor nucleus of the vagus nerve has been reported to increase blood pressure and heart rate (Granata and Woodruff, '80; Petty and Reid, '81; but see Talman and Reis, '81). Consistent with these reports, numerous and often conspicuous cell groups and fiber bundles have been observed in the vagal system.

Tyrosine hydroxylase is a stable chemical compound in postmortem brain tissue and reveals the location of catecholamine cells (Beach et al., '87a,b, in 36 hours). As first described in the rat brain by Dahlstrom and Fuxe ('64), catecholamine neurons are unevenly distributed in the human central nervous system (Olson et al., '73; Nobin and Bjorklund, '73; Gaspar et al., '85). The demonstration of the human catecholamine neuron has basically relied on the distribution of catecholaminergic immunoreactive cell

MATERIALS AND METHODS Three human brains from adult individuals (2 males and

1 female, mean age 67 years) were obtained with an average postmortem delay of 13.5 hours. This is a continuation of a previous study which examined the cytoarchitecture of the human dorsal motor nucleus of the vagus nerve (Huang et al., '93). This study was approved by the Human Ethics Committees at the University of the New South Wales under the New South Wales Transplantation and Anatomy Act. No signs of cerebral disease were evident from clinical histories or postmortem examination.

Histological procedures Three brains were fixed by perfusion with 10 litres of 4%

paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 through the vertebral and internal carotid arteries after flushing the blood vessels with 4 litres phosphate-buffered saline (PBS) (perfusing speed was 120 mliminute). The

10 12 Am Ca CaI

CeI c u DGi Do1 DRt EO Gi Gr icp In I0 IRt LPGi LRt

cc

dorsal motor nucleus of the vagus nerve hypogiossai nucleus ambiguus nucleus caudal subnucleus of the 10 caudointermediate subnucleus of the 10 central canal centrointermediate subnucleus of the 10 cuneate nucleus dorsal gigantocellular nucleus dorsointermediate subnucleus of the 10 dorsal reticular nucleus epiolivary lateral reticular nucleus gigantocellular reticular nucleus gracile nucleus inferior cerebellar peduncle intercalated nucleus of the medulla inferior olive intermediate reticular zone lateral paragigantocellular nucleus lateral reticular nucleus

Abbreviations

MeF MRt MVe PCRt PCom PrH PY Ro Rob RoI Sol sol SP-LI s p 5 c Sp5I SpVe TH-LJ VeI VRt

medial fringe subnucleus of the 10 medial reticular nucleus of the medulla medial vestibular nucleus parvicellular reticular nucleus paracommissural solitary nucleus prepositus hypoglossal nucleus pyramidal tract nucleus of Roller raphe obscums nucleus rostrointermediate subnucleus of the 10 nucleus of the solitary tract solitary tract substance P-like immunoreactive spinal trigeminal nucleus, caudal part spinal trigeminal nucleus, interpolar part spinal vestibular nucleus tyrosine hydroxylase-like immunoreactive ventrointermediate subnucleus of the 10 ventral reticular nucleus of the medulla

HUMAN DORSAL MOTOR NUCLEUS OF VAGUS

brainstems were postfixed for a maximum of 24 hours, then cut into small blocks, and cryoprotected in 30% sucrose buffer containing 0.2% sodium azide for two weeks before serial 50 pm sections were cut in the transverse plane on a freezing microtome. Every fourth section was collected for each particular stain. Different series of sections were treated immunohistochemically with either antibodies for the peptide substance P, tyrosine hydroxylase, or stained with cresyl violet for Nissl substance to identify the cytoarchitecture and exclude any pathological states.

Immunohistochemical procedures For immunohistochemistry, sections were washed in

0.8% hydrochloric acid (HC1) in 50% alcohol for 30 minutes for the abolition of endogenous peroxidase activity. The primary antibodies, rat anti-substance P from Sera-Lab, U.K. or rabbit anti-TH from Oxford, diluted at 1:1,000 or 1: 1,500 in PBS containing 0.1% Triton X-100 (PBS-Triton), was then applied for one week at 4°C. After three washings in PBS-Triton for 10 minutes each, sections were incubated in the biotinylated secondary antibody (Vector Laborato- ries, diluted at 1 : l O O in Tris HC1 for one hour and then incubated in the avidin-biotin-peroxidase complex (Vector Laboratories) diluted at 1 : l O O in PBS-Triton for one hour. Sections were washed in Tris HCl for 30 minutes before treating them with 0.05% diaminobenzidine tetrahydrochlo- ride (Sigma) in Tris HCl (pH 7.4) and 0.007% hydrogen peroxide in the presence of 0.04% nickel ammonium sulphate. Nickel ammonium sulphate allows enhancement of the peroxidase reaction (black) to be further distinguished from melanin pigment (brown). The specificity of the antiserum has been tested previously (Halliday and Tork, '89) and control reactions were performed by omission of the primary antisera. No peroxidase reaction occurred in these experiments. For photography, Kodak Technical Pan black and white film was used and the film was developed with Kodak Technidol LC developer.

Quantitative analysis The principal guide to the identification of anatomical

structures of SP-LI neurons was based on the neighbouring Nissl-stained sections following two descriptions: Cytoarchi- tecture o f the Human Brain Stem by Olszewski and Baxter ('541, and The Human Nervous System, edited by Paxinos ('90). An advantage of the more recent description is that clear boundaries of nuclei are produced as a result of aceytylcholinesterase histochemical reactions. Evidence for the existence of subnuclei in human 10 is presented else- where (Huang et al., '93).

A complete cell count could only be done on one whole brain for substance P and tyrosine hydroxylase. In order to allow for some statistical comparison to be conducted, cells from the left hemisphere of another brain were counted. Unfortunately, the regions of interest (the dorsal motor nucleus of the vagus nerve) in the right hemisphere were torn during sectioning and therefore cell counting was rendered impossible. Difficulties in serially sectioning such an extensive nucleus (14 mm in its rostral-caudal exten- sion) were encountered because sections are easily lost when dealing with a nucleus of that size. However, the particular emphasis in this study was not on the provision of a detailed statistical analysis but rather on chemoarchitecture in terms of distribution of substance P- and tyrosine hydroxylase-containing neurons in the dorsal motor nucleus of the vagus nerve.

111

A microscope driven by an IBM compatible computer with the application of the Magellan Computer Program (Halasz and Tork, '89) was used to obtain data concerning cell plotting, cell counting, measuring of cell size and calculation of cell eccentricity. These data were also used for the three-dimensional computer reconstruction of the chemoarchitecture (SP-LI and TH-LI) of the 10.

Determination of cell position The cells on both sides of each section were plotted. The

data for substance P or tyrosine hydroxylase were entered into separate computer files. Finally, the plotted cellular distributions were reconstructed in three dimensions by the Magellan Computer Program (Halasz and Tork, '89).

Assessment of cell number Sections were serially cut in groups of four out of which

one was selected for substance P, tyrosine hydroxylase and Nissl. For each substance P and/or tyrosine hydroxylase section taken, the number of cells counted were multiplied by four to give an estimation of total cell number. Only substance P- and tyrosine hydroxylase-containing neurons were counted in this experiment (Nissl reported previously; Huang et al., '93). No correction factors were used to adjust the cellular counts because: (I) thick sections (50 pm) were used in this study to diminish the counting error, and (2) determination of whether or not the soma was wholly within the section was obvious when a clear profile of the immunoreacted neurons was present. Only the neurons showing a clear profile with at least one stained prominent process were counted in present study (Mai et al., '86). These procedures minimized the double counting of bi- sected cells. Unlike the use of colchicine to facilitate labelling in experimental animals, this procedure cannot be done in humans and therefore not all cells can be assumed to be labelled.

Assessment of cell size and shape In order to determine cell size and shape, the Magellan

Computer Program transforms a tracing of the cell's perimeter into an ellipsoid. This procedure facilitates the calculation of the average diameter and eccentricity (EC) of the cell. For more details please refer to a previous study (Huang et al., '93). The amount of possible tissue shrinkage caused by staining procedures was not calculated in present study.

Proportion of the substance P- and tyrosine hydroxylase-like immunoreactive neurons

In order to obtain the percentages of SP-LI and TH-LI neurons in the 10, the results from a previous article were used, which included an estimation of total Nissl count (Huanget al., '93). In addition, the sets of sections immuno- stained for substance P and tyrosine hydroxylase were obtained from the same brain, thus affording direct comparison.

RESULTS Substance P- and tyrosine hydroxylase-like immunoreac-

tive neurons show predilection for certain subnuclei of the human dorsal motor nucleus of the vagus nerve (10). The neurons are mainly medium-sized and round and fusiform. The small neurons as well as the presumed interneurons,


Type IV and Type VI, respectively (Huang et al., '931, reveal no evidence of SP- or TH-like immunoreactivity. The total number of SP-LI and TH-LI neurons accounts for about 37% of the presumed vagal motoneurons of the 10 (compared with cresyl violet-stained neurons).

Morphology of substance P-like immunoreactive neurons in the dorsal

motor nucleus of the vagus nerve The majority of SP-LI neurons in the 10 are medium to

large with a mean cellular diameter of 26 2 5 pm (range: 14 to 50 km). SP-LI neurons of the 10 differ from SP-LI neurons in the subadjacent intermediate reticular zone in that the cells of the 10 are medium to large, and ovoid, triangular, and fusiform (Fig. 2), while those of the intermediate reticular zone are small and fusiform. The mean ratio of longest to shortest diameters of the SP-LI somata in the 10 is 1.8 f 0.5. Contacts of immunoreactive proximal dendrites and cell bodies with immunoreactive varicose fibers are frequently seen. The human 10 contains an estimated total of 2,040 f 28 SP-LI neurons, accounting for 16% of the total number of the neurons stained for Nissl substance. The left side of Figure 5 presents a three- dimensional computer reconstruction of the topographical localization of SP-LI neurons in the 10.

Substance P-like immunoreactive neuronal types and their distributions

On the basis of the cellular size and shape, three types of SP-LI neurons were identified in the 10, corresponding to Types I, I1 and I11 observed in Nissl preparation (Huang et al., '93).

Type I SP-LI neurons are the largest in the 10 (mean diameter 32 f 4 pm) and intensely immunoreactive (Fig. 2A). Type I SP-LI neurons are mainly ovoid and triangular and the short dendrites arising from their somata appear in the 50 pm section (Ec = 1.6 i 0.3). Type I SP-LI neurons are mainly found in the intermediate division, especially in the dorsointermediate subnucleus, with moderate and sparse distribution in the caudal and rostral divisions, respectively (Fig. 4). The total number of Type I SP-LI neurons in the 10 was approximately 812 f 101, accounting for 40% SP-LI neurons of the 10.

Type I1 SP-LI neurons are medium (mean diameter 24 f 2 km) with varying degrees of immunoreactivity (Fig. 2B). In the lightly reactive cells, the nucleus of the cells is distinguishable (Fig. 2B, 1 and 2). Type I1 SP-LI neurons are round and oval (Ec = 1.7 t 0.3) and are the second common type of SP-positive neurons in the 10 (38%). Type I1 SP-LI neurons are seen throughout the intermediate and

caudal divisions (Fig. 4), especially from 2 mm caudal to and 4 mm rostral to the obex, with sparse distribution in the rostral division. The total number of Type I1 SP-LI neurons was estimated to be 779 t 87.

Type I11 SP-LI neurons are fusiform or bipolar in shape and vary in size (mean diameter = 27 2 5 Fm; Ec = 2.5 f 0.4) in transverse sections (Fig. 2C). Large Type 111 SP-LI neurons reacted intensively while the small Type I11 SP-LI neurons immunoreacted lightly (Fig. 2C2). Type I11 SP-LI neurons account for 22% of SP-LI neurons of the 10, and amounted to 449 2 34. Although these neurons are found throughout the intermediate and caudal divisions of the 10, they are distributed mainly in the area 3 mm rostral to the obex (Fig. 4).

Distribution of substance P-like immunoreactive neurons in the subnuclei of the dorsal motor nucleus of the vagus nerve SP-LI neurons, fibers and terminals are unevenly distrib-

uted in the human 10 (Fig. 1). The intermediate division of the 10 contains the majority of SP-LI neurons, accounting for 63% of the total. The caudal division contains 37% of the SP-LI neurons in the 10, whilst the rostral division rarely displays SP-LI neurons.

Rostra1 division of the dorsal motor nucleus of the vagus nerve

Although only few SP-LI neurons can be detected in the rostral division, the entirety of the rostral division of the 10 is densely punctuated by a diffuse pattern of punctiform and beaded SP-LI varicosities (Fig. lA,A).

Intermediate division of the dorsal motor nucleus of the vagus nerve

The majority of SP-LI neurons of the 10 are contained in the intermediate division. The mean cellular diameter of the SP-LI neurons within the intermediate division is 26 ? 5 pm. The mean ratio of the longest to shortest diameters of the SP-LI somata is 1.8 2 0.5. The estimated total number of SP-LI neurons was 1,291 t 26, accounting for 15% the number of presumed motoneurons established by cresyl violet staining. The intermediate division displays an uneven distribution of SP-LI fibers and terminals. Beaded and discrete varicosities gathering around SP-LI soma and proximal dendrites are always seen in the intermediate division.

The RoI is a small subnucleus of the 10 lying in the most rostral part of the intermediate division of the 10. The predomi-

The rostrointermediate subnucleus of the 10 (Rol).

Fig. 1. Photomacrographs of the substance P-like immunoreactive (SP-LI) and tyrosine hydroxylase-like immunoreactive (TH-LI) neurons and fibers (or terminals) in 50 pm transverse sections through the human medulla, displaying the human dorsal motor nucleus of the vagus nerve (10) at four levels. The left column (A-D) shows SP-like immunoreactivity; the middle column (A'-D') shows TH-like immunoreactivity. Sections A' to D' are directly adjacent to the sections A to D, affording a close comparison of SP-LI and TH-LI neurons and fibers in the 10. The line drawings in the right column (A"-D) display the medullary region from which the sections on the left were taken. A, A', and A are approximately 6.4 mm rostral to obex; B, B', and B are approximately 5.2 mm rostral to obex; C, C', and C are approximately 0.2 mm rostral to obex; and D, D', and D are approximately 2.4 mm caudal to obex. Photomacrographs A and A' display the rostral division

of the 10. In A there is evidence of moderate punctiform SP-LI varicosities but no evidence of positive neurons. In A' there is evidence of many TH-LI neurons on the medial and dorsal aspects of the 10. Photomacrographs B, B', C, and C' display the intermediate division of the 10. In B and C there is evidence of many SP-LI neurons and of an uneven distribution of SP-LI fibers. B' and C' display many TH-LI neurons on the medial and ventral sides of the 10. Further, many TH-LI neurons can be seen outside the lateral border of the 10, an area that belongs to the solitary nucleus (B' and C'). Note that the area of the ventrointermediate subnucleus of the 10 (VeI; C) is sparsely populated with SP-LI neurons but densely populated with TH-LI neurons (VeI; C'). Photomacrographs D and D' display the caudal division of the 10: This division contains few widely distributed SP-LI fibers or terminals (D) and some TH-LI neurons (D').

HUMAN DORSAL MOTOR NUCLEUS OF VAGUS 113

I 1 Rostra1

D”

Figure 1


nant type of SP-LI neurons in the RoI is Type I1 (medium- sized) neurons. The mean cell diameter of the SP-LI neurons within the RoI is 26 i 5 pm while cells range in size from 19 to 38 pm. The mean ratio of the longest to shortest diameters of the SP-LI somata is 2.0 ? 0.5. Only a light background of fibers is present in the RoI which makes the individual somata and their dendrites easily visible. The SP-LI neurons invested in turn by numerous positive granules were always in evidence. The estimated total number of SP-LI neurons was 44 i 3, accounting for only 2 8 of SP-LI neurons in the 10.

The Do1 is the most dorsal part of the intermediate division of the 10. The majority of SP-LI neurons in the Do1 are Type I, which are large, triangular, or multipolar (Fig. 2A). These SP-LI neurons display multiple primary dendrites radiat- ing in all directions. The mean cell body diameter of the SP-LI neurons within the Do1 is 29 * 3 pm, while cells range in size from 18 to 50 pm. The mean ratio of the longest to shortest diameters of the SP-like immunoreactive somata is 1.7 2 0.5. The estimated total number of SP-LI neurons was 410 i 15, accounting for 20% of SP-LI neurons in the 10.

The CeI lies ventral to the dorsointermediate subnucleus. The predominant type of SP-LI neurons in the CeI is Type 11, which is medium and oval (Fig. 2B). The mean cellular diameter of the SP-LI neurons within the CeI is 27 ? 0.1 pm while cells range in size from 17 to 40 pm. The mean ratio of the longest to shortest diameters of the SP-LI somata is 1.8 i 0.5. The estimated total number of SP-LI neurons was 432 t 51, accounting for 21% of SP-LI neurons in the 10.

The CaI is the largest subnucleus of the 10 lying in the most caudal part of the intermediate division. The SP-LI neurons in the CaI consist of Type I11 (fusiform) and Type I1 (ovoid) neurons (Fig. 2C,B). The mean cellular diameter of the SP-LI neurons within the CaI is 27.5 2 0.8 pm, while cells range in size from 19 to 39 pm. The mean ratio of the longest to shortest diameters of the SP-LI somata is 1.8 i 0.4. The estimated total number of SP-LI neurons was 321 i 28, accounting for 16% of SP-LI neurons in the 10. It should be noted that bipolar SP-LI neurons were much more numerous than multipolar SP-LI neurons in the CaI.

The ventrointermediate subnucleus of the 10 (Vel). The VeI lies in the rostroventral part of the intermediate division, ventral to the centrointermediate subnucleus of the 10 (Fig. 1C). Type I1 (medium) ovoid neurons are the predominant SP-LI neurons in the CeI (Fig. 2B). The mean cellular diameter of SP-LI neurons within the VeI is 23 2 3 pm while cells range in size from 14 to 33 pm. The mean ratio of the longest to shortest diameters of the SP-LI somata is 2.0 t 0.5. The estimated total number of SP-LI neurons was 84 + 26, accounting for only 4% of SP-LI neurons in the 10. The VeI is densely populated by a patchy pattern of punctiform and beaded SP-LI varicosities.

The dorsointermediate subnucleus of the 10 (Dol).

The centrointermediate nucleus of the 10 (Cel).

The caudointermedinte subnucleus of the 10 (CaI).

Caudal division of the dorsal motor nucleus of the vagus nerve (Ca)

SP-LI neurons in the Ca are predominantly Type I1 (medium) ovoid neurons (Fig. 2B). The mean cellular diameter of the SP-LI neurons of the Ca is 2.4 i 0.5 pm, the smallest in the 10 (Fig. 41, while cells range in size from 13 to 37 pm. The mean ratio of the longest to shortest

diameters of the SP-LI somata is 1.8 i 0.5. The estimated total number of SP-LI neurons was 749 ? 19, accounting for 37% of SP-LI neurons in the 10.

Morphology of tyrosine hydroxylase-like immunoreactive neurons in the dorsal

motor nucleus of the vagus nerve The majority of TH-LI neurons are medium to large with

a mean cellular diameter of 25 i 5 km (while cells range: 16 to 43 pm). The TH-LI somata are oval, round, and fusiform (Fig. 3). The mean ratio of the longest to shortest TH-LI somata is 1.9 ? 0.5. The human 10 contains an estimated total of 2,601 i 26 TH-LI neurons, accounting for 21% of total number of Nissl-stained presumed motoneurons. Fig- ure 5 is a three-dimensional computer reconstruction of the topographical localization of TH-LI neurons in the 10.

On the basis of the cellular size and shape, three types of TH-LI neurons were identified in the 10, corresponding to Types I, I1 and I11 observed in cresyl violet-stained preparation (Huanget al., ’93).

Type I TH-LI neurons are the largest TH-LI neurons in the 10 (mean diameter 33 3 pm) and are mainly oval and triangular (Ec = 1.5 ? 0.3). Usually, each large TH-LI neuron has two, but occasionally three, primary cylindrical dendrites (nearly straight) which become thinner in caliber gradually and can be followed for one or two millimetres in the plane of a 50 pm transverse section (Fig. 3A). Occasion- ally, the branches are a shorter distance from the soma. No preferential orientation could be detected for these TH-LI dendrites. The total number of large TH-LI neurons was approximately 627 2 111, accounting for 24% of TH-LI neurons in the 10. Type I TH-LI neurons are predominantly found in the area 6 mm rostral to the obex in the medial fringe and the ventrointermediate subnuclei of the 10.

Type I1 TH-LI neurons are medium (mean diameter 23 ? 5 pm). Their somata are ovoid (Ec = 1.7 ? 0.3) and their dendrites are much thinner than those of Type I TH-LI neurons (Fig. 3B). They are the most common type of TH-LI neurons in the 10 (47%). Some of these cells are present in every 50 pm section through the 10. The total number of Type I1 TH-LI neurons was estimated to be 1,199 i 112.

Type I11 TH-LI neurons vary in size in transverse section (mean diameter 24 i 5 km; Fig. 3C). They are fusiform (Ec = 2.7 ? 0.4). Usually, Type I11 TH-LI neurons have two long cylindrical dendrites extending more than 2 mm. These neurons are found predominantly in the area 6 mm rostral from the obex, but are also found in the rostral and caudal extremes of the 10. The estimated total number of TH-LI neurons was 747 2 211, accounting for 29% of TH-LI neurons in the 10.

Regional distribution of the tyrosine hydroxylase immunoreactive neurons in the

dorsal motor nucleus of the vagus nerve The TH-LI neurons are distributed in specific regions of

the 10 (Fig. 1). The intermediate division of the 10 contains the majority of the TH-LI neurons. TH-LI cells are distributed throughout the medial fringe, the rostrointermediate, the ventrointermediate, and the dorsointermediate subnuclei (Fig. lB‘,C’). In the rostral division of the 10, TH-LI neurons are found only in the medial side (Fig. lA’), whilst in the caudal division of the 10, TH-LI neurons are found


Dorsoi n termed i ate

n =410_+ 15

10 20 30 40

Centrointermediate

,,-n=432k51

Dorsointermediate

10 20 30 40

Ventrointermediate

clu x = 27.0 + 0.1 6

m 4

U 2 20 S

m E l o 2

m c ; 10 20 30 40 f Q) S

L

Caudointermediate

n = 321k 28 40[ X =27.5+0.8

10 20 30 40

Caudal Subnucleus

10 20 30 40

Medial Fringe

n =I370 & 21

10 20 30 40

Caudal Subnucleus

n = 383 +22

10 20 30 40 Cell Diameter (Lm)

Fig. 4. Histograms depict the size and number of somata in those subnuclei of the human dorsal motor nucleus of the vagus nerve (10) that show substance P- and tyrosine hydroxylase-like immunoreactive (SP-LI and TH-LI) neurons. Left column: The dorsointermediate subnucleus contains SP-LI neurons with larger average cell diameter than the centrointermediate and caudointermediate subnuclei, while the caudal division displays the smallest average cell diameter. The

10 20 30 40 Cell Diameter (pm)

centrointermediate and caudointermediate subnuclei contain primarily medium SP-LI neurons. Right column: The medial fringe subnucleus contains the majority of TH-LI neurons and these cells show the largest average cell diameter. The dorsointermediate subnucleus contains only a small number of TH-LI neurons and these neurons have the smallest average cell diameter. The ventrointermediate and caudal subnuclei contain TH-LI neurons with medium average cell diameter.


only in the ventral side (Fig. 1D'). There are no TH-LI neurons in the ventrorostral, dorsorostral, centrointermediate, and caudointermediate subnuclei of the 10. Lateral to the 10, the intermediate subnucleus of the solitary tract contains many TH-LI neurons.

This subnucleus lies along the medial aspect of other components of the 10, including the rostral, intermediate, and caudal divisions (Fig. 1A' to D'). The MeF contains the majority of TH-LI neurons (Fig. 4). The TH-LI neurons in the MeF are predominantly Type I1 (54%), but Type I (18%) and Type I11 (28%) are also seen. The mean diameter of TH-LI neurons in the MeF is 26.3 I ~ I 1.3 pm (ranging in size from 17 to 40 pm). The mean ratio of the longest to shortest diameters of the TH-LI somata is 1.9 t 0.6. The TH-LI dendrites of the MeF can often be followed laterally into the dorsointermediate, centrointermediate, and ventrointermediate subnuclei of the 10, ventrally into the intermediate reticular zone, and medially into the intercalatus nucleus (Fig. 1A' to C'). The estimated total number of TH-LI neurons in the medial fringe was 1,370 t 21, accounting for 53% of TH-LI neurons of the entire 10.

This subnucleus is the smallest subnucleus of the 10, and is located in the most rostral part of the intermediate division of the 10. Type I TH-LI neurons (large) can often be seen in RoI (mean d iameter of s o m a t a = 26 f 5 p m ; Ec = 2.3 ? 0.8). The estimated total number of TH-LI neurons in RoI was only 67 -+ 19, accounting for 3% of TH-LI neurons in the 10.

The dorsointermediate subnucleus of the 10 (Dol). Al- though the Do1 is composed of the largest neurons of the 10, it is only small neurons which display TH-like immunoreactivity (mean diameter 24.0 -+ 1.3 pm, Fig. 4). TH-LI neurons in the Do1 are fusiform and oval (Ec = 2.1 ? 0.7) and their estimated total was 283 -+ 46, accounting only for 11% of TH-LI neurons of the entire 10.

The ventrointermediate subnucleus of the 10 (Vel). The ventral rim of VeI contains the second largest popula- tion of TH-LI neurons in the 10 (Fig. 4). The VeI contains predominantly Type I1 TH-LI neurons (48961, but also contains many Type I (33%) and some Type I11 (19%) TH-LI neurons. The mean diameter of TH-LI neurons in VeI is 25.0 -t 1.9 pm (ranging in size from 17 to 41 pm). The mean ratio of the longest to shortest diameters of the TH-LI somata is 1.8 t 0.4. The TH-LI dendrites are seen to extend from the VeI into the intermediate reticular nucleus (Fig. 1B'). The estimated total number of TH-LI neurons in VeI was 498 + 22, accounting for 19% TH-LI neurons of the 10.

The ventral rim of Ca displays many TH-LI neurons (Fig. 1D'). The Ca contains predominantly Type I1 TH-LI neurons, but also contains many Type 111, and a few Type I TH-LI neurons (Fig. 3). The mean diameter of TH-LI neurons in Ca is 26.2 t 1.1 pm (rangingin size from 16 to 31 pm). The mean ratio of the longest to shortest diameters of the TH-LI somata is 2.1 ? 0.6. The estimated total number of TH-LI neurons in Ca was 383 f 22, accounting for 15% of TH-LI neurons in the 10.

The medial fringe subnucleus o f the 10 (MeF).

The rostrointermediate subnucleus of the 10 (Rol).

The caudal subnucleus of the 10 (Ca).

DISCUSSION The present study provides an account of the distribution

and preliminary quantitation (size, shape and number) of

substance P- and tyrosine hydroxylase-containing neurons in the human dorsal motor nucleus of the vagus nerve (10). It confirms the existence of SP-LI and TH-LI neurons in the 10 and describes their morphological features and location. Thirty-seven percent of the presumed vagal motoneurons of the 10 stained for Nissl substance displayed either SP-LI or TH-LI immunoreactivity (16% and 21%, respectively). All SP-LI and TH-LI neurons belonged to the category of presumed motoneurons (Type I to 111, Huang et al., '93). There was a predilection for the different morphological categories of SP-LI and TH-LI for various subnuclei of the 10, confirming the organizational scheme (Huang et al., '93).

The existence of SP-LI neurons in the 10 has been reported in the human adult and newborn (Del Fiacco et al., '83, '84; Halliday et al., '88, '90; Pioro et al., '90). However, the precise localization and types of SP-LI neurons of the 10 have not hitherto been reported. This study found the distribution of SP-LI neurons in the subnuclei of the 10 to be uneven and the SP-LI cell types in the subnuclei of the 10 to be heterogeneous. The majority of SP-LI neurons are found in the intermediate division, 37% in the caudal division and very few, if any, in the rostral division.

In the sections stained for Nissl substance, the human 10 displays six distinctive neuronal types (Huang et al., '93); only half of them (Types I to 111) were found to display SP-like immunoreactivity in the present study. It is of interest that different types of SP-LI neurons are predominantly concentrated in specific subnuclei of the nucleus. Thus, the majority of large, triangular or polygonal SP-LI neurons (Type I) are distributed in the dorsointermediate subnucleus of the 10; the majority of medium ovoid SP-LI neurons (Type 11) occupy the rostrointermediate, centrointermediate, caudointermediate subnuclei, and caudal division, and the majority of medium, fusiform SP-LI neurons (Type 111) are located in the caudointermediate subnucleus. In contrast, a minimal number of SP-LI neurons is displayed in the ventral portion (VeI) which is comprised primarily of small neurons (equivalent to Type IV neurons stained for Nissl substance), and the medial fringe, which contains a large proportion of pigmented neurons (equivalent to Type V neurons stained for Nissl substance, Huang et al., '93).

Analysis of substance P-immunoreactive fibers and their possible origin

SP-LI fibers in the human 10 display predominantly the beaded appearance as per the descriptions of Pioro et al. ('90). A similar pattern of distribution of SP-LI fibers has also been seen in the human solitary nucleus (Pioro et al., 'go), anterior and medial hypothalamic region, globus pallidus, and granular and supragranular layers of the hippocampus (Mai et al., '86; Pioro et al., '90). From the present observations, areas displaying a high density of SP-LI varicosities in the human 10 include the ventrointermediate and medial fringe subnuclei (punctiform); this extends the observations of Pioro et al. ('90) who com- mented that SP-LI varicosities are found predominantly in the caudal extent of the human 10. There is evidence to suggest that certain SP-LI fibers form direct connections with the SP-LI neurons of the 10; although the precise origin of the SP-LI fibers is unknown (Pioro et al., '901, it is possible that the distribution of the punctiform pattern of the SP-LI fibers within the human 10 reflects terminal arborization of intrinsic or afferent neurons, which estab-


SP-immunoreactive neurons TH -i m mu no reactive neuron s

Posterior L,( views

Lateral views

Fig. 5. Computer-generated three-dimensional reconstructions of substance P-like immunoreactive (SP-LI; left) and tyrosine hydroxylase immunoreactive (TH-LI; right) neurons in the human dorsal motor nucleus of the vagus nerve based on cell plots of serial transverse sections (one in every eight sections were plotted). Each dot represents eight immunoreactive neurons. In order to make it seem as though the cells were within the sections, it was necessary to scatter the dots randomly along their z (rostrocaudal) coordinate through a distance equivalent with the spacing of the sections (400 pm). In the posterior views, the sections were at a tilt so that the longitudinal axis of the

brainstem was at 55" from the horizontal plane. In the lateral views, the sections were tilted 3" laterally and the longitudinal axes of the brainstem were kept at 55" from the horizontal plane. In the inferior views, all selected sections were overlapped. Consideration of all three-dimensional reconstructions shows that in a rostrocaudal direc- tion, the TH-LI neurons extend much more rostrally than do the SP-LI neurons. The SP-LI neurons display a more concentrated arrangement, while the TH-LI neurons are more diffuse. Scale bar applies to all six reconstructions.


lish direct axonal reflexes with the motoneurons. Further, in the rat, there have been reports of high substance P receptor concentrations in the 10 (Manaker and Rizio, '89).

Comparison of the pigmented and tyrosine hydroxylase-like immunoreactive neurons

in the human dorsal motor nucleus of the vagus nerve

It is well known that the 10 is a parasympathetic preganglionic nucleus, the neurons of which are generally considered to be cholinergic in nature and to send preganglionic fibers to supply the majority of autonomic organs (Satoh et al., '83). Areas at the periphery of the human 10 (medial fringe subnucleus, ventral side of the ventrointermediate subnucleus, and ventral side of the caudal division), unlike the central core, display many pigmented neurons which are black when observed in cresyl violet- stained preparation (Olszewski and Baxter, '54; Bazelon et al., '67). The black pigment (or melanin) is considered to be the waste product of catecholamine metabolism (Das et al., '78; Saper and Petito, '82) and the black pigmented cells display catecholaminergic immunoreactivity (Baker et al., '89). The present study shows that the medial fringe, ventrointermediate subnuclei, and caudal division of the human 10 contain the majority of TH-LI neurons of the 10 (89%); this is in agreement with previous reports of the location of pigmented neurons in the human 10 (Olszewski and Baxter, '54; Bazelon et al., '67).

In the present study, it was found that approximately 50% of TH-LI neurons of the 10 are pigmented. This finding is in agreement with the observation of Halliday et al. ('88) that pigment in neurons is not a reliable marker for medullary catecholamine-synthesizing neurons (in their study, 65% of TH-LI neurons contained melanin pigment). Although the catecholamine A2 cell group is primarily located in the 10 and the solitary nucleus (Dahlstrom and Fuxe, '64), there are species differences in the precise localization of TH-LI neurons related to the 10. For example, in pygmy marmosets, the majority of catecholaminergic neurons are found lateral to the 10 proper, specifically in the intermediate subnucleus of the solitary tract (Jacobo- witz and MacLean, '781, while in the human, 56% of TH-LI cells within the periphery of the 10 are located in the medial fringe, as in the rat (Armstrong et al., '90). In contrast, the area corresponding to the medial fringe of the 10 in the pygmy marmosets displays only a few catecholamine neurons (Jacobowitz and MacLean, '78).

There continues to be some controversy as to whether TH-LI or pigmented neurons do, in fact, belong to the 10 or to neutral territory between the 10 and adjacent nuclei. Several investigators have argued in favour of the tradi- tional point of view that the 10 is a parasympathetic preganglionic nucleus and thus, wholly cholinergic in nature (Brodal, '81) and that the pigmented (or catecholaminergic) cells are either found outside or are ectopically located in the 10 (Garver and Sladek, '75; Jacobowitz and MacLean, '78; Bogerts, '81). However, by using double labelling techniques (horseradish peroxidase retrograde tracing and immunohistochemical reactivity), it has recently been shown in experimental animals that those catecholamine-containing neurons constitute one of the components of the vagus nerve (Ritchie et al., '82; Saw- chenko, '83; Kalia et al., '84; Gwyn et al., '85; Manier et al., '87; Armstrong et al., '90, but see Blessing et al., '85, '86). In concordance with the above studies in experimental

animals, the results of the present study indicate that some TH-LI neurons lie within the proper boundaries of the human 10, specifically that the rostrointermediate, and dorsointermediate subnuclei and ventral part of the ventrointermediate display such cells (Fig. 1A' to D').

The question of whether the medial fringe is a part or a subnucleus of the 10, a part or a subnucleus of the intercalated nucleus of the medulla or a totally separate nucleus on its own needs to be considered. According to some criteria, the medial fringe area is unlike the other subnuclei of the 10; for example, the medial fringe subnucleus displays very low acetylcholinesterase histochemical reactivity compared to high activity in the 10 (Huang et al., '931, and possesses no SP-LI neurons but many TH-LI neurons. I t can also be contrasted with the intercalated nucleus of the medulla which displays medium acetylcholinesterase reactivity. Although it is hard to compare experimental animals and humans in terms of the precise localization of the TH-LI labelled neurons, there have been some suggestions that the area in the rat which corresponds to the medial fringe in the human 10 sends TH-LI fibers to join the vagus nerve (Ritchie et al., '82; Kalia et al., '84; Armstrong et a]., '90). In addition, the evidence presented by Manaker and Rizio ('89) and Allen et al. ('88), that the highest thyrotropin-releasing hormone (TRH) and angiotensin I1 receptors concentrations are in the medial fringe of the rat 10, adds further weight to the concept that the medial fringe is a subdivision of the 10 (Tork et al., '90). In recent observations on the demonstration of calcitonin receptor in the human medulla, the medial fringe displays high levels of binding like most of the subnuclei of the 10 and unlike the intercalated nucleus of the medulla (Sexton, Paxinos, Mendelsohn, and Huang, unpublished observations).

Comparison of the location of the substance P- and tyrosine hydroxylase-like

immunoreactivity in the dorsal motor nucleus of the vagus nerve

The present study shows different regional distributions of SP- and TH-containing neurons; however, there is complementarity between the two types of neurons. While the SP-LI neurons are found in the main body of the 10, the TH-LI neurons tend to be concentrated within the periphery, such as the medial fringe subnuclei, and the ventral side of the ventrointermediate subnucleus and the ventral side of the caudal division.

Nickel ammonium sulphate was used to enhance the peroxidase reaction (black) so as to differentiate it from the melanin pigment (brown). This allowed the examination of the interrelations between the SP-LI neurons and the pigmented neurons. The present investigation showed that SP-LI neurons send fibers to the pigmented neurons located in the medial fringe subnucleus. Unfortunately, this result cannot be rendered in figures because only black and white film was used in this study. This observation is consistent with the reports by a number of other investigators who concluded that catecholaminergic cell groups such as Al, A2, A6, A9, and A12 (as identified in the human by their content of melanin pigment) are surrounded by SP-LI fibers and terminals (Iversen, '82; Mai et al., '86). Consis- tent with the anatomical proximity revealed in these studies, catecholamine cell groups are excited by local application of substance P (Magnusson et al., '76). I t is likely that


the two systems, substance P and the catecholamines, interact within the human 10.

On postmortem examination, the brains of patients with Huntington's disease reveal a decreased level of substance P in the substantia nigra and basal ganglia (Kanazawa et al., '77; Gale et al., '78; Emson et al., '80). Although there is still no clear explanation why Parkinsonian patients have autonomic symptoms (Wooten, 'go), postmortem examination of Parkinsonian brains reveal decreased levels of substance P in the dorsal motor nucleus of the vagus nerve (Halliday et al., '90). The possible involvement of substance P in Huntington's and Parkinson's diseases stresses the importance of a detailed knowledge of its distribution in the normal human central nervous system.

The present study describes the distribution patterns of SP- and TH-containing neurons in the human 10 in order to provide the skeleton for determining the functional organization of this parasympathetic preganglionic nucleus. It demonstrates a cytochemical heterogeneity in the human 10 as reported in experimental animals (Dahlstrom and Fuxe, '64; Ljungdahl et al., '78; Del Fiacco et al., '84; Bouras et al., '86; Mai et al., '86). It suggests the possibility that both SP- and TH-containing neurons may be involved in a variety of vagal functions.

ACKNOWLEDGMENTS This study was supported by grants from the National

Health and Medical Research Council of Australia, Na- tional Heart Foundation, Australian Research Council, and Sandoz Foundation for Gerontological Research. We thank Dr. Annette Dorfman for editorial assistance, Alicia Fritchle for graphic illustration, and Sharlane Hamblet for skillful technical assistance.

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