Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurones of the vagal motor nucleus in the adult rat

Neuroscience Letters, 80 (1987) 141 146 141 Elsevier Scientific Publishers Ireland Ltd.

NSL 04816

Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurones of the vagal motor nucleus in the adult rat

Monique Manier, Patrick Mouchet and Claude Feuerstein Laboratoire de Physiologie section Neurophysiologie, C.N.R.S. Jeune Equipe 3808. Pavillon de Neurologie,

CHU de Grenoble, Grenoble (France)

(Received 4 February 1987; Revised version received 11 May 1987; Accepted 21 May 1987)

Key wor&v Immunohistochemistry; Tyrosine hydroxylase; Choline acetyltransferase; Vagal motor nucleus; Co-localization; Spinal cord; Rat

Catecholaminergic nerve cell bodies have been recently identified in the rat spinal cord. They lie in the rostral cervical segments and at the lumbosacral junction. Among them, many are located in parasympathetic areas. This finding led us to investigate the interactions between these catecholaminergic neurones and the cholinergic ones. To address this question, we performed sequential immunocytochemical detection of choline acetyltransferase (CHAT) and tyrosine hydroxylase (TH) in the same sections. We could then identify the co-expression of both TH and ChAT-like immunoreactivities (LI) in some perikarya of the cervical spinal cord and medulla oblongata. Such cells are located in the caudal extension of the dorsal motor nucleus of the vagus nerve (DMNX) as well as in the caudal part of the medullary DMNX itself. Such a co-expression of TH-LI and ChAT-LI could not be found in the lumbosacral region, another parasympathetic territory where cell bodies displaying TH-LI were intermingled with those containing CHAT- LI. This is one of the first demonstrations of the co-existence of catecholaminergic and cholinergic phenotypes in some neurones of the adult mammalian nervous system. These observations also support the presence of catecholaminergic efferents within the vagus nerve.

T h e p re sence o f c a t e c h o l a m i n e r g i c cell bod ie s in the ra t sp inal c o r d has been

d e m o n s t r a t e d qu i t e r ecen t ly [5, 12, 15]. A m o n g such cells, s o m e c o n t a i n b o t h ty ros ine

h y d r o x y l a s e - ( T H - L I ) and d o p a m i n e - f l - h y d r o x y l a s e - l i k e i m m u n o r e a c t i v i t i e s , the o th -

ers d i sp l ay ing on ly T H - L I [12]; no p e r i k a r y a c o n t a i n i n g p h e n y l e t h a n o l a m i n e - N -

m e t h y l t r a n s f e r a s e - l i k e i m m u n o r e a c t i v i t y c o u l d be de t ec t ed in the spinal cord . Ca t e -

c h o l a m i n e r g i c cell bod ie s h a v e been f o u n d to be m o r e n u m e r o u s in the ros t ra l cervi -

cal s e g m e n t s [5, 12, 15] a n d at the l u m b o s a c r a l j u n c t i o n [5, 12]. In these regions , m a n y

c a t e c h o l a m i n e r g i c cells a re l oca t ed in p a r a s y m p a t h e t i c a reas [12], p r o m p t i n g then an

i nves t i ga t i on o f the r e l a t ions b e t w e e n these n e u r o n e s and the cho l ine rg i c p a r a s y m -

pa the t i c ones . T h e r e f o r e , cho l i ne ace ty l t r an s f e r a se ( C H A T ) a n d ty ros ine h y d r o x y l a s e

Correspondence: C. Feuerstein, Laboratoire de Physiologie section Neurophysiologie, C.N.R.S. Jeune Equipe 3808, Pavillon de Neurologie, CHU de Grenoble, BP 217 X, F-38043 Grenoble cedex, France.

0304-3940/87/$ 03.50 @ 1987 Elsevier Scientific Publishers Ireland Ltd.

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(TH), the specific synthesizing enzymes of acetylcholine and catecholamines respectively, have been detected in the rat spinal cord and caudal medulla oblongata by sequential immunocytochemistry performed on the same sections.

Male Wistar rats (250-300 g) were used. They were perfused through the ascending aorta with an isotonic saline solution followed by 4% paraformaldehyde in phosphate buffer at pH 7.4, according to a procedure described previously [12]. Selected parts of the brain and the spinal cord were removed: the caudal medulla oblongata, the rostral cervical segments and the lumbosacral junction (L6-SI). They were postfixed (3 h in the same fixative) and immersed for 16 h in a 15% sucrose solution in phosphate buffer. Sections (20/tm thick) were then cut with a cryotome at - 18°C. The whole subsequent immunohistochemical procedure was carried out on floating sections. After 30 min preincubation in 1% hydrogen peroxide and 1 h in phosphate- buffered saline (PBS, potassium phosphate buffer 0.02 M, pH 7.5 with 0.14 M NaCI) containing 1% normal goat serum (Miles), the cryotome sections were washed in PBS pH 7.5 containing 0.3% Triton X-100 (PBST) and incubated for 36 h at 4°C with monoclonal anti-ChAT antibodies (2 Ftg per ml in PBST containing 1% normal goat serum) raised in rat (Boehringer-Mannheim) [6]. After washing in PBST, the sections were incubated for 1.5 h in a goat serum raised against rat IgG (Jackson Immunore- search Labs., 1:10). After further washing in PBST and incubation in rat peroxidase-- antiperoxidase (PAP) complex (Seralab 1:50 in PBS), according to Sternberger's PAP technique [16], the sections were rinsed in PBS, then in Tris-HC1 buffer 0.05 M pH 7.5 and reacted with 0.05% diaminobenzidine and 0.01% hydrogen peroxide in Tris- HCI buffer for ChAT-LI visualization. The second step included rinsing in PBST and incubation for 60 h at 4°C in a rabbit anti-TH antiserum [17] (1:2000 in PBST containing normal goat serum 1:100). Following a rinse in PBST, the sections were treated for 2.5 h in fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit serum (Cappel, 1:2000) and normal goat serum (1:100 in PBST) according to the indirect immunofluorescence technique of Coons [4]. After washing in PBS, the sections were mounted on gelatin-coated slides, coverslipped in glycerol-potassium hydrogen car- bonate and examined with a Zeiss microscope either by light transillumination for PAP detection, or under fluorescence epi-iUumination for FITC.

The distribution of ChAT-LI perikarya and TH-LI ones was examined on the same sections. The precise location of these neurones was delineated in reference to adjacent sections stained with Cresyl violet. At rostral cervical levels, ChAT-LI cell bodies were found in the ventral horn where they correspond to the motoneurone pool, in a region dorsal to the central canal and in a more lateral situation within the intermediary grey matter. Few ChAT-LI perikarya were observed in the dorsal horn. The TH-LI cell bodies were always less numerous than the ChAT-LI ones. They were localized in the two restricted sites previously described, namely the lateral white matter and a zone situated dorsally to the central canal [12]. The latter localization was then the only restricted zone where the distribution of ChAT-LI and TH-LI perikarya overlap. Moreover, in this region, ChAT-like and TH-like immunoreactivities could be often seen in the same perikarya (Fig. 1). These cell bodies had the shape and the size that we had previously described for the TH-LI neurones located in this territory [12]. They were round or fusiform, of medium size (15-20/~m) and often with long processes tranversely oriented. They were all located in the cervical part

143

B

i i~%1 ~ ~ ~ii~i~ :

i t

~t C D

Fig. 1. Typical double-labeled cells visualized in the tirst cervical segment dorsally to the central canal. A ( x 158) and C ( x 396): perikarya displaying ChAT-LI observed under light illumination as brown images. B ( × 158) and D ( x 396): the same areas as those seen in A and C respectively but under fluorescence epiillumination and revealing perikarya containing TH-LI. Asterisks show the location of the central canal. In each set of paired microscope images (A, B and C, D) the arrows point to some of the cells which display both immunoreactivities (ChAT-LI and TH-LI): these cells are detectable both under light microscopic visualization and fluorescence illumination.

o f the dor sa l m o t o r nuc l eus o f the v a g u s n e r v e ( D M N X ) . A t this level, d o u b l e - l a b e l e d

cells a d j o i n n u m e r o u s s i n g l e q a b e l e d C h A T - L I neu rons . A t the m o s t c a u d a l levels,

nea r ly all T H - L I p e r i k a r y a ( a r o u n d 2 pe r 2 0 - ~ m - t h i c k sec t ion) s e e m e d to be a lso

C h A T c o n t a i n i n g . Th i s p r o p o r t i o n d e c r e a s e d p rog re s s ive ly r o s t r a l w a r d , whi le the

n u m b e r o f T H - L I cells pe r sec t ion inc reased g radua l ly . A t the b u l b o s p i n a l j u n c t i o n ,

the a b s o l u t e a m o u n t o f T H - L I cell b o d i e s pe r sec t ion was n e a r 15, whi le on ly 40%

seemed to d i sp lay a lso C h A T - l i k e i m m u n o r e a c t i v i t y .

In the c a u d a l m e d u l l a o b l o n g a t a , d o u b l e - l a b e l e d cells were still p r e sen t in the

D M N X . T h u s , all the T H - L I p e r i k a r y a o f the D M N X were C h A T c o n t a i n i n g , wh ich

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Fig. 2. Location of TH-LI (triangles), ChAT-LI (circles) and double-labeled (asterisks) perikarya at the Ca cervical level (upper drawing) and at a medullary level 1.5 mm caudal to the obex (inferior drawing). DH, dorsal horn; VH, ventralhorn; Arab, ambiguus nucleus; Py, pyramidal tract; Sol, nucleus of the solitary tract; Sp5C, spinatis nucleus of the trigeminal nerve, pars caudalis; XII, nucleus of the hypoglossal nerve.

corresponded to about 10% the amount of the ChAT-LI cell bodies present at this level in the DMNX. Such cells lay between many single-labeled ChAT-LI perikarya. The vagal group was dearly distinct from the more dorsal TH-LI cell bodies of the nucleus of the solitary tract and from the ventral ChAT-LI motoneurones of the hypoglossal nucleus. Laterally, there was also a clearcut separation between the ChAT-LI cells of the ambiguus nucleus and the TH-LI ones of the Al noradrenergic cell group. Within the medulla oblongata, a transversal plane situated 0.5 mm caudal to the area postrema could define the rostral limit beyond which no double-labeled neurones could be detected.

In the sacral spinal cord, besides the motoneurone pool of the ventral horn, CHAT- like immunoreactivity was also present in cells belonging to the intermediolateral parasympathetic nucleus and in perikarya scattered throughout the central grey matter. The two TH-LI groups that we described previously in the marginal part of the dorsal horn and in the intermediary grey matter [12] were also visible. In the parasympathetic area, ChAT-LI and TH-LI perikarya were tightly mingled, but double- labeled cells were never observed.

The specificity of ChAT-LI and TH-LI labeling was checked by the complete dis- appearance of the respective staining when only ChAT or TH antiserum was omitted from the procedure.

These results are the first evidence for the presence of both TH-like and ChAT-like immunoreactivities in the same neuronal cells of the upper cervical cord and the adjacent medulla oblongata. The specificity of the two primary antisera used here has been evaluated extensively in previous studies [6, 17]. In addition, the distribution

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of the spinal and medullary TH-LI neurones shown in this study has been already reported [5, 12]. In the same way, the distribution of ChAT-LI cells that we observed in the spinal cord was similar to that recently described by Borges and Iversen [2], with the same antiserum. In fact, the main problem of specificity which might arise in our study results from an eventual binding of the antibodies used for the second step of the experiment (TH immunostaining) to the reagents used for the detection of CHAT. This appears unlikely since: (1) no fluorescence was visible on sections where only the TH antiserum was omitted; (2) in the same section, double-immunola- beled cells were found intermingled with many other perikarya displaying only one immunolabeling. Furthermore, in the same cervical sections, 3 groups of cell bodies were often visible: those containing ChAT-like immunoreactivity exclusively (motoneurones), those located in the lateral white matter which contained TH-like-immunoreactivity exclusively and the double-labeled perikarya of the DMNX (Fig. 2). An erroneous interpretation due to the superposition of different neuronal profiles seems also improbable since the double-labeled perikarya were always of identical shape and they were clearly seen for the same adjustment of the focus, whether they were examined through light microscopy (DAB precipitate visualization) or under fluorescence illumination. Moreover, double labeling was never observed in the sacral parasympathetic area in spite of the frequent apparent overlapping of ChAT-LI and TH- LI cell bodies in this territory.

Even though our observations do not provide direct identification of the transmit- ters themselves, the localization of such neurones displaying both TH and ChAT immunolabeling strongly suggests that they contain catecholamines and acetylcholine. Indeed catecholamine histofluorescence [15] allowed the visualization of perikarya in this region, where vagal cholinergic neurones also exist. However, the double-labeled cells that we described here have not yet been proved able to synthetize catecholamines and acetylcholine.

The study of the relationship between the expression of the catecholaminergic phe- notype and the cholinergic one has promoted extensive investigations devoted to the peripheral sympathetic nervous system, both in vitro [13] and in vivo [10]. In vivo, both phenotypes co-exist transiently in the postganglionic neurones which innervate eccrine sweat glands [10]. To our knowledge, such a co-existence has not yet been found in adult peripheral neurones. In the central nervous system, the presence of TH immunoreactivity has been reported to be also expressed in some ~-motoneurones [3], but not in other known cholinergic cells. However, in our present work as in a previous one [12], no TH-like immunoreactivity could be identified in the rat motoneurones. Our results are therefore the first evidence showing that some neu- tones in the central nervous system are able to co-express both phenotypes and that this phenomenon is not restricted to transient periods of the development, since it persists into adulthood. The neuronal population involved may give new insight into the mechanisms regulating the phenotypic expression of neurotransmitters.

The localization of the double-labeled neurones in the DMNX strongly suggests their belonging to the vagal system. The presence of catecholaminergic efferent fibres within the vagus nerve has already been postulated by some authors [7, 9, 14] but refuted by others [1]. Our results give support to the former contention. In addition we show that the putative catecholaminergic component of the vagal nerve is not dis-

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t inc t f r o m the c h o l i n e r g i c one , at least fo r the ef ferents o f the c a u d a l p a r t o f the vaga l

nuc leus . T h e use o f r e t r o g r a d e t r a c ing m e t h o d s m i g h t he lp to a sce r t a in such a

hypo thes i s . In this case, the phys io log i ca l i m p l i c a t i o n s s h o u l d d e p e n d on the ident i ty

o f the i n n e r v a t e d s t ruc tu res , w h i c h p r o b a b l y i nc lude the u p p e r ga s t ro - in t e s t i na l t ract .

Th i s w o r k was s u p p o r t e d by g ran t s f r o m C . N . R . S . ( A T P 3266, J e u n e E q u i p e

3808), F o n d a t i o n p o u r la R e c h e r c h e M6d ica l e , U n i v e r s i t 6 de G r e n o b l e 1 and H o s p i -

tal C e n t r e o f G r e n o b l e . W e t h a n k M. A r l u i s o n ( C . R . N . S . U A 884, U n i v e r s i t 6 Par is

6) fo r his he lp fu l adv ice , J. T h i b a u l t fo r p r o v i d i n g us wi th T H a n t i s e r u m , Dr . A . M .

F o o t e fo r Eng l i sh c o r r e c t i o n s a n d M r s . M . J . G a l l e t fo r t y p i n g the m a n u s c r i p t .

1 Blessing, W.W., Willoughby, J.C. and Joh, T.H., Evidence that catecholamine synthesizing perikarya in rat medulla oblongata do not contribute axons to the vagus nerve, Brain Res., 348 (1985) 397~,00.

2 Borges, L.F. and Iversen, S.D., Topography of choline acetyltransferase immunoreactive neurons and fibers in the rat spinal cord, Brain Res., 362 (1986) 140-148.

3 Chan-Palay, V., Engel, A.G., Palay, S.L. and Wu, J.Y., Synthesizing enzymes for four neuroactive substances in motor neurons and neuromuscular junctions: light and electron microscopic immunocytochemistry, Proc. Natl. Acad. Sci. USA, 79 (1982) 6717~721.

4 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic, New York, 1958, pp. 399-422.

5 Dietl, M., Arluison, M., Mouchet, P., Feuerstein, C., Manier, M. and Thibault, J., Immunohistochem- ical demonstration of catecholaminergic cell bodies in the spinal cord of the rat, Histochemistry, 82 (1985) 385-389.

6 Eckenstein, F. and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterisation and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363 368.

7 Gwyn, D.G., Ritchie, T.C. and Coulter, J.D., The central distribution of vagal catecholaminergic neurons which project into the abdomen in the rat, Brain Res., 328 (1985) 139-144.

8 Houser, C.R., Crawford, G.D., Barber, R.P., Salvaterra, P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Res., 266 (1983) 97 119.

9 Kalia, M., Fuxe, K., Goldstein, M., Harfstrand, A., Agnati, L.F. and Coyle, J.T., Evidence for the existence of putative dopamine, adrenaline and noradrenaline containing vagal motor neurons in the brainstem of the rat, Neurosci. Lett., 50 (1984) 57--62.

10 Landis, S.C., Development of cholinergic sympathetic neurons: evidence for neurotransmitter plasticity in vivo, Fed. Proc. Fed. Am. Soc. Exp. Biol., 42 (1983) 1633-1638.

11 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on alternative nomenclature (Ch l-Ch6), Neuroscience, 10 (1983) 1185-1201.

12 Mouchet, P., Manier, M., Dietl, M., Feuerstein, C., B6rod, A., Arluison, M., Denoroy, L. and Thi- bault, J., Immunohistochemical study of catecholaminergic cell bodies in the rat spinal cord, Brain Res. Bull., 16 (1986) 341 - 353.

13 Potter, D.D., Furshpan, E.J. and Landis, S.C., Transmitter status in cultured rat sympathetic neurons: plasticity and multiple functions, Fed. Proc. Fed. Am. Soc. Exp. Biol., 42 (1983) 1626--1632.

14 Ritchie, T.C., Westlund, K.N., Bowker, R.M., Coulter, J.D. and Leonard, R.B., The relationship of the medullary catecholamine containing neurones to the vagal motor nuclei, Neuroscience, 7 (1982) 1471-1482.

15 Singhaniyom, W., Wreford, N.G.M. and Giildner, F.H., Asymmetric distribution of catecholamine containing neuronal perikarya in the upper cervical spinal cord of rat, Neurosci. Lett., 41 (1983) 91 97.

16 Sternberger, L.A., The unlabeled antibody peroxidase-antiperoxidase (PAP) method. In L.A. Stern- berger (Ed.), Immunocytochemistry, Wiley, New York, 1979, pp. 104-169.

17 Thibault, J., Vidal, O. and Gros, F., In vitro translation of mRNA from rat pheochromocytoma tu- mors, characterization of tyrosine hydroxylase, Biochem. Biophys. Res. Commun., 99 ( 1981) 960-968.

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Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurones of the vagal motor nucleus in the adult rat