169
J. Anat. (1990), 173, 169-176 With 3 figures Printed in Great Britain
A comparison of age-related changes in neuron number in the dorsal motor nucleus of the vagus and the nucleus ambiguus of the mouse R. R. STURROCK
Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland
(Accepted 19 June 1990) INTRODUCTION
The vagus nerve has two motor nuclei: the dorsal motor nucleus, which is a general visceral efferent nucleus, and the nucleus ambiguus, which is a special visceral efferent, or branchiomotor nucleus. The dorsal motor nucleus contains medium-sized neurons and small neurons (Aldskogius, 1978; McLean & Hopkins, 1981; Laiwand, Werman & Yarom, 1987). The medium-sized neurons are preganglionic parasympathetic motor neurons and the small neurons are interneurons. If the vagus nerve is severed the medium-sized neurons degenerate but the small neurons are unaffected, which confirms that they do not contribute to the vagus nerve (Aldskogius, 1978; Laiwand et al. 1987). The preganglionic neurons of the dorsal motor nucleus supply the thoracic and abdominal viscera but not the extrathoracic trachea (Kalia & Mesulam, 1980b), with a large proportion (90-95 %) of fibres supplying the stomach (Leslie, Gwyn & Hopkins, 1982; Shapiro & Miselis, 1985) with collaterals to other viscera. There is some controversy as to whether the dorsal motor nucleus does or does not supply cardio-inhibitory fibres to the heart but there is evidence that, in the cat, some preganglionic neurons of the dorsal motor nucleus project to the cardiac plexus (Kalia & Mesulam, 1980 b). The nucleus ambiguus contains branchiomotor neurons which supply the pharynx, larynx and oesophagus (Lawn, 1966a; Davis & Nail, 1984; Bieger & Hopkins, 1987; Portillo & Pasaro, 1988a) and preganglionic neurons which supply thoracic viscera (Bieger & Hopkins, 1987) and the forestomach (Shapiro & Miselis, 1985). Two problems which occur in studies of the nucleus ambiguus are that authors disagree about which structures should be included within the nucleus (Davis & Nail, 1984; Bieger & Hopkins, 1987) and the actual structure of the nucleus varies between species, with descriptions in the rabbit (Lawn, 1966b; Davis & Nail, 1984), cat (Kalia & Mesulam, 1980a; Davis & Nail, 1984) and rat (Bieger & Hopkins, 1987) all differing. When an earlier investigation of age-related changes in the retrofacial nucleus was carried out (Sturrock, 1988 a), the available evidence (Szentagothai, 1952; Kerr, 1969) indicated that the retrofacial nucleus was a general visceral efferent nucleus, but since then it has been shown to be a branchiomotor nucleus which should probably be considered to be part of the nucleus ambiguus (Bieger & Hopkins, 1987; Portillo & Pasaro, 1988b). It innervates oesophageal muscles and perhaps contributes to the ventral respiratory group of muscles (Sasaki et al. 1989). Bieger & Hopkins (1987) proposed that the nucleus ambiguus should be divided into four parts - a compact formation (the retrofacial nucleus), a semicompact formation, a loose formation and
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an external division. According to Bieger & Hopkins (1987) the compact formation projects to the oesophagus, the semicompact formation supplies the pharyngeal constrictors and cricothyroid, and the loose formation supplies the remaining intrinsic muscles of the larynx. The fourth part, the external division, gives rise to the general visceral efferent component of the nucleus ambiguus, mainly supplying the heart. The present study set out to investigate changes in the number of preganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus, and in the number of branchiomotor neurons in the nucleus ambiguus. In view of the work of Bieger & Hopkins (1987) it was initially decided to include the retrofacial nucleus as part of the nucleus ambiguus, but as the results showed that neuron loss with ageing was not uniform throughout the nucleus ambiguus the results for the retrofacial nucleus (compact formation) and the remainder of the nucleus will be considered separately. MATERIALS AND METHODS
The material consisted of 6,tm serial sections of brains of mice aged 6, 15, 25, 28. and 31 months. The right halves were sectioned in the sagittal plane and stained with Lapham's stain (Lapham, Johnstone & Brundjar, 1964) and the left halves were sectioned in the coronal plane and stained with haematoxylin and eosin. Three sets of sections were used at each age. Full details of fixation and subsequent processing have already been given (Sturrock, 1987). The brains had been bisected in the midsagittal plane, but in the majority of brains the plane of section was not precisely in the midline. The most caudal part of the dorsal motor nucleus of the vagus lies very close to the midline and in most sets of sections the nucleus was completely intact in only the sagittal or coronal set but not both. The coronal sets containing the intact dorsal motor nucleus at 15 and 31 months did not include sections of the medulla caudal to the cerebellum, and therefore could not be used for neuron number estimations since the dorsal motor nucleus extends caudally beyond the cerebellum. For these reasons the sets of sections used for dorsal motor nucleus neuron counts were three sagittal sets at 6 months, two sagittal sets at 15 months, two sagittal and one coronal set at 25 and 28 months and one sagittal set at 31 months. The sections containing the most medial and most lateral neurons of the dorsal motor nucleus were identified in the sets of sagittal sections and similarly the sections containing the most rostral and most caudal neurons were identified in the sets of coronal sections. It was decided to count every 10th sagittal and every 20th coronal section (since the nuclei are much longer than they are wide). In order to ensure random sampling of the nucleus a number, R, between one and ten (sagittal sections) or between one and twenty (coronal sections) was obtained from a random number table and the number of neurons in the Rth section and in every 10th or 20th section thereafter was counted. Only preganglionic parasympathetic neurons, which could be recognised by a characteristic peripheral rim of Nissl substance (Fig. 2; Olszewski & Baxter, 1954), were recorded. The mean neuronal nuclear diameter was calculated as described previously (Sturrock, 1987). The total number of neurons was estimated by multiplying the number counted by the total number of sections containing the nucleus, dividing by the number of sections used for counts and applying the correction formula devised by Abercrombie (1946). In order to investigate possible changes in nuclear diameter with age, the mean nuclear diameter of preganglionic neurons was measured in three sets of coronal sections at each age. This was possible even when the nucleus was incomplete. The medial and lateral boundaries of the nucleus ambiguus, excluding the
171 Ageing vagal efferent nuclei retrofacial nucleus, were identified in three sets of sagittal sections at each age, and every 10th section was used for counts, with the position of the first section counted being determined at random as described above. Only branchiomotor neurons (see Olszewski & Baxter, 1954) were recorded. The estimates of total neuron number were obtained in a similar manner to that described above. In the earlier investigation of the retrofacial nucleus (Sturrock, 1988a) no counts were carried out at 15 months. The number of neurons in the retrofacial nucleus at 15 months of age was therefore estimated in three sets of sagittal sections as described previously (Sturrock, 1988 a). One set of sagittal sections from a 6 months old mouse which had been used previously for counts of the retrofacial nucleus did not contain a complete dorsal motor nucleus. In order to use the same set of sections for neuron counts in the dorsal motor nucleus and in the nucleus ambiguus wherever possible, this set was replaced by another 6 months set in which the dorsal motor nucleus was intact. The number of neurons in the retrofacial nucleus in this new set of sections was calculated, and replaced the result from the earlier study.
RESULTS
The dorsal motor nucleus of the vagus is easy to identify in both sagittal (Fig. 1) and coronal sections. The preganglionic parasympathetic neurons have a peripheral rim of Nissl substance around the perikaryon (Fig. 2) and from 25 months of age the perikaryon is packed with lipofuscin granules which are easily identified in Laphamstained material. The retrofacial nucleus, or compact formation of the nucleus ambiguus, is also very easily recognised by its small size and the tight packing of its neurons (Sturrock, 1988a). The remainder of the nucleus ambiguus is much more difficult to delineate. A semicompact part can be identified caudal to the compact formation but its boundaries are indistinct, especially where it tapers off rostrally into the loose formation which, as its name implies, consists of widely scattered branchiomotor neurons (Fig. 3) extending to the caudal end of the lateral reticular nucleus. It was not possible to identify clearly the boundary between the semicompact and loose formations. The difficulty of accurately identifying the semicompact and loose parts of the nucleus ambiguus is reflected in the standard errors, which are proportionally much greater than those in the well-defined retrofacial nucleus and dorsal motor nucleus (Table 1). It was easier to identify the nucleus in sagittal sections than in coronal sections, which is why sagittal sections were used for counts, but care had to be taken to exclude the occasional large reticular neuron and to clearly differentiate the cells of the nucleus ambiguus from those of the adjacent lateral reticular nucleus. This was less of a problem in older brains because the neurons of the lateral reticular nucleus contained large quantities of lipofuscin from 25 months of age whereas branchiomotor neurons of the nucleus ambiguus accumulated little or no lipofuscin even at 31 months. The dorsal motor nucleus loses neurons from 25 months of age (Table 1) and the decrease in number is statistically significant (F (4,7) = 36-06: P > 0-001). There is a statistically significant loss of neurons from the nucleus ambiguus from 15 months of age (Table 1) (F (4, 10) = 14-08; P > 0-001), but examination of the numerical changes in the retrofacial nucleus and the remainder of the nucleus ambiguus shows that neuron number in the retrofacial nucleus does not begin to decline until after 25 months of age whereas the loss of neurons in the remainder of the nucleus starts after 15 months (Table 1). Neuronal nuclear diameter (Table 2) does not vary significantly with age in either
172
R. R. STURROCK
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Aa s,' ;nt@
*!h.
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.t.: 4 I:.. _
]
W7:..::
.1
W
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Fig. 1. The dorsal motor nucleus of the vagus appears as a tightly packed group of cells lying between the nucleus of the tractus solitarius (NTS) and the hypoglossal nucleus (XII). Sagittal section of a 25 months old mouse stained with Lapham's stain. x 200. Fig. 2. Preganglionic parasympathetic neurons of the dorsal motor nucleus (arrows) can be identified by a dark peripheral rim of Nissl substance. Lapham's stain. x 500. Fig. 3. In the caudal part of the nucleus ambiguus motor neurons (arrows) are widely scattered. Lapham's stain. x 500.
the retrofacial nucleus (F (4, 10) = 0-92; NS) or the rest of the nucleus (F (4, 10) = 0 95; NS) but the nuclear diameter of preganglionic parasympathetic neurons increases significantly after 25 months of age (F (4, 10) = 6-63; P < 0 01). DISCUSSION
As, in most of the sets of sections, the dorsal motor nucleus of the vagus was intact in only one half of the brain, estimates of neuron number at 25 and 28 months of age were carried out in two right halves and one left half of the brains and the possibility
Ageing vagal efferent nuclei
173 Table 1. Estimated number of preganglionic parasympathetic neurons (+ S.E.M.) in the dorsal motor nucleus of the vagus and estimated total number of branchiomotor neurons (± S.E.M.) in the nucleus ambiguus and the number ofneurons (± S.E.M.) in the retrofacial nucleus and in the remainder of the nucleus ambiguus Age (months)
Dorsal motor nucleus of X
6 15 25 28 31
1318+21 1330+70' 1271 +42 927+27
Total nucleus ambiguus
Retrofacial nucleus
587+4 537+35 477+29 395+22 8202 351 +29 All estimations from three brains except 1 two * Results from Sturrock (1988 a).
Remainder of nucleus ambiguus
203+4 204+5 209 +4*
384+7 333+36 268+29 181+11* 214+31 157+4* 194+26 brains and 2 one brain.
Table 2. Estimated mean nuclear diameter (± S.E.M.) in the dorsal motor nucleus of the vagus, the retrofacial nucleus and the remainder of the nucleus ambiguus Age (months)
Dorsal motor nucleus IX (m)
Retrofacial nucleus (m)
Remainder of nucleus ambiguus (m)
6 15 25 28 31
12-2+04 11 8+0-2 11-8+0-6 13-4+0-1 13-8 +0-3
10-9+0-3* 11 0+0 1 11-2±0-2*
11-4+0-1 11-7+0-2 11-6+0-1 11-3+0-1 11-4±0-3
*
10.8+01*
10.9+0.1* Results from Sturrock (1988 a).
of right-left asymmetry affecting the results has to be considered. In both cases, however, the estimated number of neurons in the coronal sections (left half) lay somewhere between the two estimations for the sets of sagittal sections (right half). Although only one intact dorsal motor nucleus was available at 31 months the estimated number of neurons in the nucleus at this age was consistent with the loss of neurons found between 25 and 28 months. This loss of dorsal motor nucleus neurons after 25 months of age may be secondary to loss of ganglionic neurons, possibly as a consequence of age-related changes in the viscera supplied. In his quantitative study on the rat myenteric plexus Gabella (1971) refers to unpublished observations of a dramatic reduction in the number of nerve cells in the myenteric plexus of the ageing guinea-pig. Unfortunately no further information regarding the proportion of neurons lost nor the age of the guinea-pigs was given. In a recently published abstract Jew & Wang (1990) described a substantial loss of neurons from the myenteric plexus of the colon of guinea-pigs, rats and mice with increasing age, but no ages were given. They reported a decrease in neuron number from 9290 to 3762 in the guinea-pig, but no figures were given for the other species. The myenteric plexus is a complex structure and only a small proportion of its neurons receive direct input from the vagus (Gershon, 1981); nevertheless, if there is a large decrease in neuron number in the myenteric plexus with age this could be responsible for the late loss of the dorsal motor nucleus neurons which supply it since a high proportion of general visceral efferent vagal fibres supply the gastro-intestinal tract (Leslie et al. 1982; Shapiro & Miselis, 1985). In contrast to the dorsal motor nucleus, the nucleus ambiguus begins to lose neurons after 15 months of age but this neuron loss is not uniform throughout the
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R. R. STURROCK
nucleus. The retrofacial nucleus, or compact formation, shows no evidence of neuron loss until after 25 months. Bieger & Hopkins (1987) have shown that the retrofacial nucleus gives rise only to oesophageal motor neurons in the rat. Other authors have claimed that the retrofacial nucleus also supplies the cricothyroid muscle (Yoshida et al. 1982; Davis & Nail, 1984). These disparate results may reflect differences between the species investigated and if this is the case one might expect the mouse nucleus ambiguus to conform to the rodent pattern described by Bieger & Hopkins (1987) in their carefully controlled study of the rat rather than to that of the rabbit or cat. It would therefore appear that oesophageal motor neurons do not decrease in number until after 25 months of age, whereas there is a reduction in the number of laryngeal and pharyngeal motor neurons after 15 months of age. Thus oesophageal motor neurons begin to decline in number at the same time as dorsal motor nucleus neurons which are visceromotor to a large part of the gut, and this may indicate a relationship between oesophageal motility and that of the remainder of the gut. Motor neuron loss from the nucleus ambiguus begins much earlier than neuron loss from any other cranial nerve motor nucleus. There is no loss of motor neurons from the abducens (Sturrock, 1989), trochlear, oculomotor (Sturrock, 1991 b) or hypoglossal nuclei (Sturrock, 1991 a) up to 31 months of age. Motor neuron number does not begin to fall until after 28 months in the trigeminal motor nucleus (Sturrock, 1987) nor until after 25 months in the facial nucleus (Sturrock, 1988 b). It is surprising that there is an early decline in motor neuron supply to such physiologically important muscles as the laryngeal and pharyngeal muscles. The earlier suggestion that a high level of motor activity might prevent motor neuron loss (Sturrock, 1989) does not seem to apply in the case of the nucleus ambiguus since laryngeal and pharyngeal muscles are constantly in use during respiration and deglutition. The nucleus ambiguus also differs from other ageing cranial nerve motor nuclei that lose neurons in that neuronal nuclear diameter does not increase with age in either the retrofacial nucleus or other parts of the nucleus ambiguus. Dorsal motor nucleus neuronal diameter does increase after 25 months of age at the time when neuron number is falling. The significance of the increase in neuronal nuclear diameter is unknown. In the sets of sections used in the present study an increase in neuronal nuclear diameter has been found in most, but not all, brainstem nuclei which lose neurons with age. An increase in neuronal diameter has never been observed in nuclei in which neuronal number remains stable, with the solitary exception of the supraoptic nucleus (Sturrock, 1990), but neurons in this nucleus are neurosecretory neurons and are probably not comparable with neurons in the cranial nerve nuclei since neurosecretory neuronal nuclei vary in size with neurosecretory activity (Paterson & Leblond, 1977). SUMMARY
The numbers of preganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus and of branchiomotor neurons in the nucleus ambiguus were estimated in mice aged 6, 15, 25, 28 and 31 months. The number of preganglionic neurons fell from 1318 at 6 months to 820 at 31 months with neuron number remaining stable until after 25 months. The number of branchiomotor neurons in the nucleus ambiguus fell from 587 at 6 months to 351 at 31 months, the significant decline in number beginning after 15 months. The retrofacial nucleus, or compact formation of the nucleus ambiguus, did not lose neurons until after 25 months whereas significant neuron loss from the remainder of the nucleus began after 15 months.
Ageing vagal efferent nuclei
175
REFEREN CES
ABERCROMBIE, M. (1946). Estimation of nuclear population from microtome sections. Anatomical Record 94, 239-247. ALDSKOGIUS, H. (1978). Fine structural changes in nerve cell bodies of the adult rabbit dorsal motor vagal nucleus during axon reaction. Neuropathology and Applied Neurobiology 4, 323-341. BIEGER, D. & HOPKINS, D. A. (1987). Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus. Journal of Comparative Neurology 262, 546-562. DAVIS, P. J. & NAIL, B. S. (1984). On the location and size of laryngeal motoneurons in the cat and rabbit. Journal of Comparative Neurology 230, 13-32. GABELLA, G. (1971). Neuron size and number in the myenteric plexus of the newborn and adult rat. Journal of Anatomy 109, 81-95. GERSHON, M. D. (1981). The enteric nervous system. Annual Review of Neuroscience 4, 227-272. JEW, J. W. & WANG, Y. (1990). Age-related changes in myenteric plexus of colon of three mammalian species. Anatomical Record 226, 47A. KALIA, M. & MESULAM, M.-M. (1980a). Brain stem projections of sensory and motor components of the vagus complex in the cat. 1. The cervical vagus and nodose ganglion. Journal of Comparative Neurology 193, 435-465. KALIA, M. & MESULAM, M.-M. (1980b). Brain stem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, cardiac and gastrointestinal branches. Journal of Comparative Neurology 193, 467-508. KERR, F. W. L. (1969). Preserved vagal visceromotor function following destruction of the dorsal motor nucleus. Journal of Physiology 202, 755-769. LAIWAND, R., WERMAN, R. & YAROM, Y. (1987). Time course and distribution of motoneuronal loss in the dorsal motor vagal nucleus of guinea pig after cervical vagotomy. Journal of Comparative Neurology 256,
527-537. LAPHAM, L. W., JOHNSTONE, M. A. & BRUNDJAR, K. H. (1964). A new paraffin method for the combined staining of myelin and glial fibers. Journal of Neuropathology and Experimental Neurology 23, 156-160. LAWN, A. M. (1966a). The localization in the nucleus ambiguus of the rabbit of the cells of origin of motor nerve fibres in the glossopharyngeal nerve and various branches of the vagus nerve by means of retrograde degeneration. Journal of Comparative Neurology 127, 293-306. LAWN, A. M. (1966b). The nucleus ambiguus of the rabbit. Journal of Comparative Neurology 127, 307-320. LESLIE, R. A., GWYN, D. G. & HOPKINS, D. A. (1982). The central distribution of the cervical vagus nerve and gastric afferent and efferent projection in the rat. Brain Research Bulletin 8, 37-43. McLEAN, J. H. & HOPKINS, D. A. (1981). A light and electron microscopic study of the dorsal motor nucleus of the vagus nerve in the cat. Journal of Comparative Neurology 195, 157-175. OLSZEWSKI, J. & BAXTER, D. (1954). Cytoarchitecture of the Human Brain Stem. Basel & New York: Karger. PATERSON, J. A. & LEBLOND, C. P. (1977). Increased proliferation of neuroglia and endothelial cells in the supraoptic nucleus and hypophysial neural lobe of young rats drinking hypertonic sodium chloride solution. Journal of Comparative Neurology 175, 373-390. PORTILLO, F. & PASARO, R. (1988a). Location of bulbospinal neurons and of laryngeal motoneurons within the nucleus ambiguus of rat and cat by means of retrograde fluorescent labelling. Journal of Anatomy 159, 11-18. PORTILLO, F. & PASARO, R. (1988 b). Location of motoneurons supplying the intrinsic laryngeal muscles of rats. Horseradish peroxidase and fluorescent double-labelling study. Brain, Behaviour and Evolution 32, 220-225. SASAKI, H., OTAKE, K., MANNEN, H., EzURE, K. & MANABE, M. (1989). Morphology of augmenting inspiratory neurons of the ventral respiratory group in the cat. Journal of Comparative Neurology 282, 157-168. SHAPIRO, R. E. & MISELIS, R. R. (1985). The central organization of the vagus nerve innervating the stomach of the rat. Journal of Comparative Neurology 238, 473-488. STURROCK, R. R. (1987). Changes in the number of neurons in the mesencephalic and motor nuclei of the trigeminal nerve in the ageing mouse. Journal of Anatomy 151, 15-25. STURROCK, R. R. (1988a). Loss of neurons from the retrofacial nucleus of the mouse in extreme old age. Journal of Anatomy 160, 195-199. STURROCK, R. R. (1988b). Loss of neurons from the motor nucleus of the facial nerve in the ageing mouse brain. Journal of Anatomy 160, 189-194. STURROCK, R. R. (1989). Stability of neuron and glial number in the abducens nerve nucleus of the ageing mouse brain. Journal of Anatomy 166, 97-101. STURROCK, R. R. (1990). Stability of neuronal and glial number in the ageing mouse supraoptic nucleus. Anatomischer Anzeiger (In the Press.) STURROCK, R. R. (1991 a). Stability of motor neuron and interneuron number in the hypoglossal nucleus of the ageing mouse brain. Anatomischer Anzeiger (In the Press.) STURROCK, R. R. (1991 b). Stability of motor neuron number in the oculomotor and trochlear nuclei of the ageing mouse brain. Journal of Anatomy (In the Press.)
R. R. STURROCK SZENTAGOTHAI, J. (1952). The general visceral efferent column of the brain stem. Acta morphologica Academiae scientiarum hungaricae 2, 313-328. YOSHIDA, Y., MIYAZAKI, T., HIRANO, M., SMN, T. & KANASEKI, T. (1982). Arrangement of motoneurons innervating the intrinsic laryngeal muscles of cats as demonstrated by horseradish peroxidase. Acta otolaryngologica 94, 329-334.
176
J. Anat. (1990), 173, 169-176 With 3 figures Printed in Great Britain
A comparison of age-related changes in neuron number in the dorsal motor nucleus of the vagus and the nucleus ambiguus of the mouse R. R. STURROCK
Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland
(Accepted 19 June 1990) INTRODUCTION
The vagus nerve has two motor nuclei: the dorsal motor nucleus, which is a general visceral efferent nucleus, and the nucleus ambiguus, which is a special visceral efferent, or branchiomotor nucleus. The dorsal motor nucleus contains medium-sized neurons and small neurons (Aldskogius, 1978; McLean & Hopkins, 1981; Laiwand, Werman & Yarom, 1987). The medium-sized neurons are preganglionic parasympathetic motor neurons and the small neurons are interneurons. If the vagus nerve is severed the medium-sized neurons degenerate but the small neurons are unaffected, which confirms that they do not contribute to the vagus nerve (Aldskogius, 1978; Laiwand et al. 1987). The preganglionic neurons of the dorsal motor nucleus supply the thoracic and abdominal viscera but not the extrathoracic trachea (Kalia & Mesulam, 1980b), with a large proportion (90-95 %) of fibres supplying the stomach (Leslie, Gwyn & Hopkins, 1982; Shapiro & Miselis, 1985) with collaterals to other viscera. There is some controversy as to whether the dorsal motor nucleus does or does not supply cardio-inhibitory fibres to the heart but there is evidence that, in the cat, some preganglionic neurons of the dorsal motor nucleus project to the cardiac plexus (Kalia & Mesulam, 1980 b). The nucleus ambiguus contains branchiomotor neurons which supply the pharynx, larynx and oesophagus (Lawn, 1966a; Davis & Nail, 1984; Bieger & Hopkins, 1987; Portillo & Pasaro, 1988a) and preganglionic neurons which supply thoracic viscera (Bieger & Hopkins, 1987) and the forestomach (Shapiro & Miselis, 1985). Two problems which occur in studies of the nucleus ambiguus are that authors disagree about which structures should be included within the nucleus (Davis & Nail, 1984; Bieger & Hopkins, 1987) and the actual structure of the nucleus varies between species, with descriptions in the rabbit (Lawn, 1966b; Davis & Nail, 1984), cat (Kalia & Mesulam, 1980a; Davis & Nail, 1984) and rat (Bieger & Hopkins, 1987) all differing. When an earlier investigation of age-related changes in the retrofacial nucleus was carried out (Sturrock, 1988 a), the available evidence (Szentagothai, 1952; Kerr, 1969) indicated that the retrofacial nucleus was a general visceral efferent nucleus, but since then it has been shown to be a branchiomotor nucleus which should probably be considered to be part of the nucleus ambiguus (Bieger & Hopkins, 1987; Portillo & Pasaro, 1988b). It innervates oesophageal muscles and perhaps contributes to the ventral respiratory group of muscles (Sasaki et al. 1989). Bieger & Hopkins (1987) proposed that the nucleus ambiguus should be divided into four parts - a compact formation (the retrofacial nucleus), a semicompact formation, a loose formation and
170
R. R. STURROCK
an external division. According to Bieger & Hopkins (1987) the compact formation projects to the oesophagus, the semicompact formation supplies the pharyngeal constrictors and cricothyroid, and the loose formation supplies the remaining intrinsic muscles of the larynx. The fourth part, the external division, gives rise to the general visceral efferent component of the nucleus ambiguus, mainly supplying the heart. The present study set out to investigate changes in the number of preganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus, and in the number of branchiomotor neurons in the nucleus ambiguus. In view of the work of Bieger & Hopkins (1987) it was initially decided to include the retrofacial nucleus as part of the nucleus ambiguus, but as the results showed that neuron loss with ageing was not uniform throughout the nucleus ambiguus the results for the retrofacial nucleus (compact formation) and the remainder of the nucleus will be considered separately. MATERIALS AND METHODS
The material consisted of 6,tm serial sections of brains of mice aged 6, 15, 25, 28. and 31 months. The right halves were sectioned in the sagittal plane and stained with Lapham's stain (Lapham, Johnstone & Brundjar, 1964) and the left halves were sectioned in the coronal plane and stained with haematoxylin and eosin. Three sets of sections were used at each age. Full details of fixation and subsequent processing have already been given (Sturrock, 1987). The brains had been bisected in the midsagittal plane, but in the majority of brains the plane of section was not precisely in the midline. The most caudal part of the dorsal motor nucleus of the vagus lies very close to the midline and in most sets of sections the nucleus was completely intact in only the sagittal or coronal set but not both. The coronal sets containing the intact dorsal motor nucleus at 15 and 31 months did not include sections of the medulla caudal to the cerebellum, and therefore could not be used for neuron number estimations since the dorsal motor nucleus extends caudally beyond the cerebellum. For these reasons the sets of sections used for dorsal motor nucleus neuron counts were three sagittal sets at 6 months, two sagittal sets at 15 months, two sagittal and one coronal set at 25 and 28 months and one sagittal set at 31 months. The sections containing the most medial and most lateral neurons of the dorsal motor nucleus were identified in the sets of sagittal sections and similarly the sections containing the most rostral and most caudal neurons were identified in the sets of coronal sections. It was decided to count every 10th sagittal and every 20th coronal section (since the nuclei are much longer than they are wide). In order to ensure random sampling of the nucleus a number, R, between one and ten (sagittal sections) or between one and twenty (coronal sections) was obtained from a random number table and the number of neurons in the Rth section and in every 10th or 20th section thereafter was counted. Only preganglionic parasympathetic neurons, which could be recognised by a characteristic peripheral rim of Nissl substance (Fig. 2; Olszewski & Baxter, 1954), were recorded. The mean neuronal nuclear diameter was calculated as described previously (Sturrock, 1987). The total number of neurons was estimated by multiplying the number counted by the total number of sections containing the nucleus, dividing by the number of sections used for counts and applying the correction formula devised by Abercrombie (1946). In order to investigate possible changes in nuclear diameter with age, the mean nuclear diameter of preganglionic neurons was measured in three sets of coronal sections at each age. This was possible even when the nucleus was incomplete. The medial and lateral boundaries of the nucleus ambiguus, excluding the
171 Ageing vagal efferent nuclei retrofacial nucleus, were identified in three sets of sagittal sections at each age, and every 10th section was used for counts, with the position of the first section counted being determined at random as described above. Only branchiomotor neurons (see Olszewski & Baxter, 1954) were recorded. The estimates of total neuron number were obtained in a similar manner to that described above. In the earlier investigation of the retrofacial nucleus (Sturrock, 1988a) no counts were carried out at 15 months. The number of neurons in the retrofacial nucleus at 15 months of age was therefore estimated in three sets of sagittal sections as described previously (Sturrock, 1988 a). One set of sagittal sections from a 6 months old mouse which had been used previously for counts of the retrofacial nucleus did not contain a complete dorsal motor nucleus. In order to use the same set of sections for neuron counts in the dorsal motor nucleus and in the nucleus ambiguus wherever possible, this set was replaced by another 6 months set in which the dorsal motor nucleus was intact. The number of neurons in the retrofacial nucleus in this new set of sections was calculated, and replaced the result from the earlier study.
RESULTS
The dorsal motor nucleus of the vagus is easy to identify in both sagittal (Fig. 1) and coronal sections. The preganglionic parasympathetic neurons have a peripheral rim of Nissl substance around the perikaryon (Fig. 2) and from 25 months of age the perikaryon is packed with lipofuscin granules which are easily identified in Laphamstained material. The retrofacial nucleus, or compact formation of the nucleus ambiguus, is also very easily recognised by its small size and the tight packing of its neurons (Sturrock, 1988a). The remainder of the nucleus ambiguus is much more difficult to delineate. A semicompact part can be identified caudal to the compact formation but its boundaries are indistinct, especially where it tapers off rostrally into the loose formation which, as its name implies, consists of widely scattered branchiomotor neurons (Fig. 3) extending to the caudal end of the lateral reticular nucleus. It was not possible to identify clearly the boundary between the semicompact and loose formations. The difficulty of accurately identifying the semicompact and loose parts of the nucleus ambiguus is reflected in the standard errors, which are proportionally much greater than those in the well-defined retrofacial nucleus and dorsal motor nucleus (Table 1). It was easier to identify the nucleus in sagittal sections than in coronal sections, which is why sagittal sections were used for counts, but care had to be taken to exclude the occasional large reticular neuron and to clearly differentiate the cells of the nucleus ambiguus from those of the adjacent lateral reticular nucleus. This was less of a problem in older brains because the neurons of the lateral reticular nucleus contained large quantities of lipofuscin from 25 months of age whereas branchiomotor neurons of the nucleus ambiguus accumulated little or no lipofuscin even at 31 months. The dorsal motor nucleus loses neurons from 25 months of age (Table 1) and the decrease in number is statistically significant (F (4,7) = 36-06: P > 0-001). There is a statistically significant loss of neurons from the nucleus ambiguus from 15 months of age (Table 1) (F (4, 10) = 14-08; P > 0-001), but examination of the numerical changes in the retrofacial nucleus and the remainder of the nucleus ambiguus shows that neuron number in the retrofacial nucleus does not begin to decline until after 25 months of age whereas the loss of neurons in the remainder of the nucleus starts after 15 months (Table 1). Neuronal nuclear diameter (Table 2) does not vary significantly with age in either
172
R. R. STURROCK
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Fig. 1. The dorsal motor nucleus of the vagus appears as a tightly packed group of cells lying between the nucleus of the tractus solitarius (NTS) and the hypoglossal nucleus (XII). Sagittal section of a 25 months old mouse stained with Lapham's stain. x 200. Fig. 2. Preganglionic parasympathetic neurons of the dorsal motor nucleus (arrows) can be identified by a dark peripheral rim of Nissl substance. Lapham's stain. x 500. Fig. 3. In the caudal part of the nucleus ambiguus motor neurons (arrows) are widely scattered. Lapham's stain. x 500.
the retrofacial nucleus (F (4, 10) = 0-92; NS) or the rest of the nucleus (F (4, 10) = 0 95; NS) but the nuclear diameter of preganglionic parasympathetic neurons increases significantly after 25 months of age (F (4, 10) = 6-63; P < 0 01). DISCUSSION
As, in most of the sets of sections, the dorsal motor nucleus of the vagus was intact in only one half of the brain, estimates of neuron number at 25 and 28 months of age were carried out in two right halves and one left half of the brains and the possibility
Ageing vagal efferent nuclei
173 Table 1. Estimated number of preganglionic parasympathetic neurons (+ S.E.M.) in the dorsal motor nucleus of the vagus and estimated total number of branchiomotor neurons (± S.E.M.) in the nucleus ambiguus and the number ofneurons (± S.E.M.) in the retrofacial nucleus and in the remainder of the nucleus ambiguus Age (months)
Dorsal motor nucleus of X
6 15 25 28 31
1318+21 1330+70' 1271 +42 927+27
Total nucleus ambiguus
Retrofacial nucleus
587+4 537+35 477+29 395+22 8202 351 +29 All estimations from three brains except 1 two * Results from Sturrock (1988 a).
Remainder of nucleus ambiguus
203+4 204+5 209 +4*
384+7 333+36 268+29 181+11* 214+31 157+4* 194+26 brains and 2 one brain.
Table 2. Estimated mean nuclear diameter (± S.E.M.) in the dorsal motor nucleus of the vagus, the retrofacial nucleus and the remainder of the nucleus ambiguus Age (months)
Dorsal motor nucleus IX (m)
Retrofacial nucleus (m)
Remainder of nucleus ambiguus (m)
6 15 25 28 31
12-2+04 11 8+0-2 11-8+0-6 13-4+0-1 13-8 +0-3
10-9+0-3* 11 0+0 1 11-2±0-2*
11-4+0-1 11-7+0-2 11-6+0-1 11-3+0-1 11-4±0-3
*
10.8+01*
10.9+0.1* Results from Sturrock (1988 a).
of right-left asymmetry affecting the results has to be considered. In both cases, however, the estimated number of neurons in the coronal sections (left half) lay somewhere between the two estimations for the sets of sagittal sections (right half). Although only one intact dorsal motor nucleus was available at 31 months the estimated number of neurons in the nucleus at this age was consistent with the loss of neurons found between 25 and 28 months. This loss of dorsal motor nucleus neurons after 25 months of age may be secondary to loss of ganglionic neurons, possibly as a consequence of age-related changes in the viscera supplied. In his quantitative study on the rat myenteric plexus Gabella (1971) refers to unpublished observations of a dramatic reduction in the number of nerve cells in the myenteric plexus of the ageing guinea-pig. Unfortunately no further information regarding the proportion of neurons lost nor the age of the guinea-pigs was given. In a recently published abstract Jew & Wang (1990) described a substantial loss of neurons from the myenteric plexus of the colon of guinea-pigs, rats and mice with increasing age, but no ages were given. They reported a decrease in neuron number from 9290 to 3762 in the guinea-pig, but no figures were given for the other species. The myenteric plexus is a complex structure and only a small proportion of its neurons receive direct input from the vagus (Gershon, 1981); nevertheless, if there is a large decrease in neuron number in the myenteric plexus with age this could be responsible for the late loss of the dorsal motor nucleus neurons which supply it since a high proportion of general visceral efferent vagal fibres supply the gastro-intestinal tract (Leslie et al. 1982; Shapiro & Miselis, 1985). In contrast to the dorsal motor nucleus, the nucleus ambiguus begins to lose neurons after 15 months of age but this neuron loss is not uniform throughout the
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R. R. STURROCK
nucleus. The retrofacial nucleus, or compact formation, shows no evidence of neuron loss until after 25 months. Bieger & Hopkins (1987) have shown that the retrofacial nucleus gives rise only to oesophageal motor neurons in the rat. Other authors have claimed that the retrofacial nucleus also supplies the cricothyroid muscle (Yoshida et al. 1982; Davis & Nail, 1984). These disparate results may reflect differences between the species investigated and if this is the case one might expect the mouse nucleus ambiguus to conform to the rodent pattern described by Bieger & Hopkins (1987) in their carefully controlled study of the rat rather than to that of the rabbit or cat. It would therefore appear that oesophageal motor neurons do not decrease in number until after 25 months of age, whereas there is a reduction in the number of laryngeal and pharyngeal motor neurons after 15 months of age. Thus oesophageal motor neurons begin to decline in number at the same time as dorsal motor nucleus neurons which are visceromotor to a large part of the gut, and this may indicate a relationship between oesophageal motility and that of the remainder of the gut. Motor neuron loss from the nucleus ambiguus begins much earlier than neuron loss from any other cranial nerve motor nucleus. There is no loss of motor neurons from the abducens (Sturrock, 1989), trochlear, oculomotor (Sturrock, 1991 b) or hypoglossal nuclei (Sturrock, 1991 a) up to 31 months of age. Motor neuron number does not begin to fall until after 28 months in the trigeminal motor nucleus (Sturrock, 1987) nor until after 25 months in the facial nucleus (Sturrock, 1988 b). It is surprising that there is an early decline in motor neuron supply to such physiologically important muscles as the laryngeal and pharyngeal muscles. The earlier suggestion that a high level of motor activity might prevent motor neuron loss (Sturrock, 1989) does not seem to apply in the case of the nucleus ambiguus since laryngeal and pharyngeal muscles are constantly in use during respiration and deglutition. The nucleus ambiguus also differs from other ageing cranial nerve motor nuclei that lose neurons in that neuronal nuclear diameter does not increase with age in either the retrofacial nucleus or other parts of the nucleus ambiguus. Dorsal motor nucleus neuronal diameter does increase after 25 months of age at the time when neuron number is falling. The significance of the increase in neuronal nuclear diameter is unknown. In the sets of sections used in the present study an increase in neuronal nuclear diameter has been found in most, but not all, brainstem nuclei which lose neurons with age. An increase in neuronal diameter has never been observed in nuclei in which neuronal number remains stable, with the solitary exception of the supraoptic nucleus (Sturrock, 1990), but neurons in this nucleus are neurosecretory neurons and are probably not comparable with neurons in the cranial nerve nuclei since neurosecretory neuronal nuclei vary in size with neurosecretory activity (Paterson & Leblond, 1977). SUMMARY
The numbers of preganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus and of branchiomotor neurons in the nucleus ambiguus were estimated in mice aged 6, 15, 25, 28 and 31 months. The number of preganglionic neurons fell from 1318 at 6 months to 820 at 31 months with neuron number remaining stable until after 25 months. The number of branchiomotor neurons in the nucleus ambiguus fell from 587 at 6 months to 351 at 31 months, the significant decline in number beginning after 15 months. The retrofacial nucleus, or compact formation of the nucleus ambiguus, did not lose neurons until after 25 months whereas significant neuron loss from the remainder of the nucleus began after 15 months.
Ageing vagal efferent nuclei
175
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