Fekete, Éva

Mária, Csoknya

English

Fekete Éva

Mária Csoknya

University of Szeged

A < IA B I O L . S/l lil l). 33. pp. 97 K M (19X7) F L U O R E S C E N C E C H A R A C T E R I Z A T I O N O F T H E N E R V E P L E X U S E S IN T H E S M A L L I N T E S T I N E O F T H E C H I C K E N E . F E K E T E 1 A N D M . C S O K N Y . V ' Depurimcnt n! Zoology. Anita Jtizscl L'niicrsin H—6701 Sieged. /'.O B. 6W. Hungary ' Department <4 Zoology, Janus Punnonius Lniwrsity Pics. Hungary (Received March 15. 19X6) Abstract The enteric nerve plexuses in ihe small intestine of the chicken has been investigated in sections and stretch preparations by means of the silver staining and the glyoxylic acid histoOuorcsccncc methods. Both techniques revealed the presence of two submucosal plexuses in the small intestine of ihe chicken. Using the histoOuorcsccncc technique some of the nerve cell bodies located in ihe perivascular plexuses showed monoaminc-specific fluorescence. The appearance and distribution of nerve elements in the chicken small intestine has been studied also with electronmicroscope. Profiles containing dense-core vesicles in different sizes have been claimed to contain catecholamines. Key words: enteric nerve plexuses, catecholamine, chicken, small intestine Introduction Most of the s t ructural and funct ional studies concerning the organizat ion of the enteric nervous system were performed on mammal ian intestine (GUNN. 1968; G A B E L L A , 1979 ; F U R N E S S a n d C O S T A , 1980 ; S C H E U E R M A N a n d S T A C H . 1984) . Data on the innervation of the al imentary tract of the birds were mostly based on staining techniques as meth>lenc blue and silver me thods (ABRAHAM, 1936; C S O K N Y A a n d B E N E D E C Z K Y in p r i n t ; KOLOSSOW et a l . 1 9 3 2 ; M l C H A I L a n d KARAMANDLIDIS, 1967). Using fluorescence histochemical me thods (BF.NNETT and MALMKORS, 1970) it was shown that the basic pat tern of the adrenergic inner-vation of the al imentary tract in the birds were comparab le to that in mammals (COSTA and GABELLA, 1971; STACH. 1984). All these techniques revealed several cont rary concerning the organiza t ion of the enteric nervous system. Because of the very small number or even the lack of fluorescent nerve cell bodies observed in the al imentary tract of the chicken (READ and BURNSTOCK, 1968; BENNETT and MALMFORS, 1970; ALI and MCLELLAND, 1978) it has been suggested that all the catecholamine (CA) conta ining axons in the intestine of the domestic fowl arise f rom extrinsic ganglions. Some au thors report on a secondary meshwork of submucosal plexus (ALI and MCLELLAND. 1978). On the bases of ul trastructural studies (BENNETT and COBBS. 1969) the major i ty of the terminals contained a mixed F. Fi kl il \M> M Csoksa \ populat ion of small and large granular vesicles which suggest thai at least some of the nerves handle with catecholamines. During present work using silver impregnation and glyoxylic acid induced fluorescence methods we tried to clear up some of these contraversaries. Materials and methods The small intestine of ihe 6 days old chicken «as investigated. The basic histological structure of the small intestine « a s studied by hematein-eosin stained paraffin sections Detection of ihe enteric nerve plexuses «a s carried out on sections ( J O jim) stained by Bit I st H O W S K V - G R O S - C M N \ silver method For ihe histochenucal detection of monoamines ihe sucrose-phosphalc-glyoxylic acid (SPG) method (Di L v T O R K I and Si R C . I O V W M «as applied lo «holemount stretch preparates of chicken small intestine. After dissection, ihe mucosal and muscle lasers «ere separated and incubated in reaction mivlure containing 6.X g sucrose, J.2 g KH.PO, and I g glyoxylic acid (GA) in UK) ml of distilled water ai 4"C for 25 min The lavers «ere then stretched separately on microscope slides, blotted « i ih blotting paper and dried under cool air for about half an hours. Finally the specimens were placed in an oven at for 4 nun. and mounted «i ih liquid paraffin. The preparations were viewed through a Leit/ Orthoplan microscope equipped with IIBO 5« W super pressure mercury lamp and an E-J tiller block. Tissue blocks for electron microscopy were taken after \ascular perfusion »i ih ice-cold K V R N O W S K V fixative. Small pieces of the small intestine were kept in the same fixative lor : hours. Alter postlixation in : • „ osmium teroxid the specimens were dehydrated in ascending ethanol series and embedded in Durcupan. Tissue blocks «ere contrasted «iih saturated uranyl acetate in 75"n ethanol and sections were reeonlrasied «i ih lead citrate, then studied under TESLA BS 5tH> electronmieroscope Results and discussion The basic histological structure of the chicken small intestine is similar to that of mammal ian pat tern (Table I.a.). The thick epithelium is characterist ic to the absorptive tissues. The muscularis mucosae consist of one longitudinal layer of smooth muscle cells. The submucosa is reduced to a few connective tissue fibres. The muscularis externa has a highly developed inner circular layer and a relatively poorly developed outer longitudinal layer covered by the tunica serosa in the whole length of the small intstine. Both the histochemical and histological me thods we Table I. ,i. Detail of a hematein-eosin stained cross section from the small intestine of the chicken. ep—epithelium, su—submucosa. me musculariscxterna.se serosa x 4M) b. One ganglion in the plexus mycnlcricus after silver impregnation. Arrows indicate the large, mainly multipolar nerve cells. xMM) <•. Ganglion in the plexus myentericus after GA induced fluorescence IXnsc holes (arrows) represent the non-fluorescence cell bodies in the ganglion, s smooth libres. V \aricosc libres xMXl (/. Silver impregnated libres of the plexus submucosus externus. characterized by ganglions « i th a few cell bodies (arrows) and a small number of libres, x NX) e. Fine network of fibers from plexus submucosus externus. with a very intense G A induced fluorescence, x 600 F U O R K S O NCI C H A R A C T E R I Z A T I O N O F I HK N I R M PLKXIJSIS 4 4 100 É . F H K K T K A N D M . C S O K N Y A used dur ing the present studies were suitable to visualize the enteric nerve plexuses in the small intestine of the chicken (Table l.b-e.. 2.a-e ). Af te r silver impregna t ion the nerve fibers of the plexus myentericus appeared as a compac t aggregates between the longitudinal and circular muscle layer (Table l .b .) . All the nerve cell bodies were located in ganglions (Table l.b.c.). Both in the silver impregnated sections and on the G A treated wholemounts two networks of plexus submucosus were identified (Table l .d.e. . 2 . a . b ) . In the plexus submucosus in ternus (Table 2.b.) which lies close to the tunica muscularis mucosae the individual nerves could much better be recog-nized than in plexus myentericus. This plexus fo rms a regular pat tern with fibers running to the mucosa. The plexus submucosus externus can be recognized as a separate entity lying close against the inner side of the circular muscle layer. Mos t of the nerve cell bodies are also aggregated into gangl ions a l though the sizes of the cells and the ganglions are smaller than in Auerbach"s plexus. It was demons t r a t ed with glyoxylic acid that a large part of these nerves were catecholaminergic. T h e myen-teric plexus conta ins the greatest density of the ca techolaminergic fibers (Table I.e.). At many places in the gangl ions large dense holes su r rounded by basket- l ike fluorescent varicose and non-varicose axons have been observed (Table I.e.). It is highly probable that dense holes related to nonfluorescent enteric nerve cell bodies . The smooth appea rance of some nerves and varicosity of o thers might be the indica-tion of the different role of these nerves. We suppose that the varicose segments represent the active segments in chemical neurotransmission while the smoo th have mainly conduct ing role. The submucosal plexus in stretch p repa ra t ions were distr ibuted in two discrete plane (Table 2.b.). In plexus submucosus in te rnus a lmost all the fibers were varicose in appearance (Table 2.b.) The fibers of the plexus submucosus externus have a less intense fluorescence (Table I.e.. 2.b.) which sug-gests a lower concentra t ion of catecholamine. T h e fibers of the plexus submucosus internus were always in close connect ion to the perivascular plexuses (Table 2.d.). T h e topographical difference between the two submucosa l plexuses might suggest funct ional difference, too. O u r suggestion is that the fibers of the p r imary o r external meshwork may influence mucosal funct ion directly however the fibers of Table 2. a. Plexus submucosus internus alter silver impregnation. Single nerve cell body is characteristic to this plexus, x 600 b. G A induced picture of plexus submucosus internus (I) superlayered on the plexus submucosus externus (E). x 600 GA induced nerve cell body (arrow) in the perivascular plexus of the chicken small intestine. x 600 </. Close relation of the plexus submucosus internus (I) and blood vessel (BV) in the chicken small intestine. Both the submucosus and the perivascular plexuses show and intensive G A induced fluorescence, x 600 e. Small soliter multipolar cell (arrow) in the intestinal wall, with a well defined GA induced lluorcscencc. x X00 F L U O R E S C E N C E C H A R A C T E R I Z A T I O N OK THE NERVE PLEXUSES 1 0 1 the inner plexus act by influencing the blood flow. Even though there are a lot of contrary about the ul t ras t ructural appea rance of aminergic nerves in the gut, profiles conta ining dense-core vesicles in different size have been claimed to conta in c a t e c h o l a m i n e s ( B E N N E T T a n d M A L M F O R S . 1970; G O R D O N - W E E K S , 1981) . Accepting this agreement o u r present ul t rastructural observa t ions indicate the presence of catecholaminergic nerves in both the myenteric and submucosus 102 T F E K E T E A N D M . C S O K N Y A plexuses of the chicken small intestine. In a good agreement with o u r light microscopic investigations the number of nerve bundles was much higher in myenteric plexus than in submucous plexus. Most of the axons were g rouped into bundles (Table 3.a.) lying mainly parallel to the smooth muscle cells. Profiles in nerve bundles were sometimes surrounded by Schwann-cell processes while o the r s were in close association with the muscle cells (Table 3.a.) The axon profiles in bo th plexuses contained heterogeneous vesicle popula t ion even though the dominancy of dense-core vesicles was characteristic. The diameter of these vesicles was a r o u n d 50—100 A (Table 3.a.b ). This indicates that most of the nerves in the enter ic plexuses handle different kind of catecholamines. Besides t ransmit ter vesicles the axon profiles con ta in mitochondria and multivesicular bodies. These detailed results allow us to make the conclusion that large part of the nerves in the enter ic plexuses in the chicken small intestine are catecholaminergic . and except the perivascular plexuses, where intensively fluorescent cell bodies were noticed all the aminergic nerves are extrinsic in origin. Table 3. a. Nerve bundle in the chickcn small intestine (nb). surrounded by glial processes (sch). Arrow shows a neuromuscular junction, sm—smooth muscle cell, x 13 000 h. Nerve bundles with different axon profiles in the chicken small intestine Arrow shows a characteristic profile with heterogeneous vesicle populat ion: dense-core (50—IOOA in diameter), small agranular and flattened vesicles can be seen, x 22 000 1 0 4 £ F E K E T E A N D M C S O K N Y A References A L I . H.A. and M C L E L L A N D . J . ( 1 9 7 8 ) : Avian enteric nerve plexuses. A histochemical study. Cell Tissue Res. IH9. 5 3 7 - 5 4 8 . A B R A H A M . A . (1936): Beitrage zur Kenntnis der Innervation des Vogeldarmes. Z. Zcllforsch. 23. 737-745. B E N N E T T . T. and C O B B . J . L . S . ( 1 9 6 9 ) : Studies on the avian gizzard: Auerbach s plexus. - Z Zellforsch. 99. 1 0 9 - 1 2 0 . B E N N E T T . T . and M A L M E O R S . T . ( 1 9 7 1 ) : Fluorescence histochemical observation on Auerbach ' s plexus and the problem on the inhibitory of the gut. — J. Physiol. 218. 77-78. C O S T A . M. and G A B E L L A , G . (1971): Adrenergic innervation of the alimentary canal. — Z. Zcllforsch. 122. 357-377. C S O K N Y A , M . and B E N E D E C Z K Y , I . (1986): Cell types of the enteric nerve plexuses of the chicken (Gallus domeslicus L.). — Acta Biol. Szeged. 32. 93-102. FURNESS. J.B. and COSTA, M. (1980): Types of nerves in the enteric nervous system. — Neurosci. 5. 1-20. G A B F . L L A . G . (1979): Innervation of the gastrointestinal tract. — Int. rev. Cytol. 59. 129-193. G O R D O N - W E E K S . P R . ( 1 9 8 1 ) : Neuroscience properties of nerve endings with small granular vesicles in the distal colon and rectum of the guinea-pig. — Neurosci. 6. 1-20. GUNN. M. (1968): Histological and histochemical observations on the myenteric and submucous plexuses of mammals . — J. Anat . (Lond.) 102. 223-239. K O L O S S O W . N . G . . S A B U S S O W . G . H . and I W A N O W , J .F. ( 1 9 3 2 ) : Zur Innervation des Verdauungskanales der Vögel. — Z. mikr. anat . Forsch. 30. 2 5 7 - 2 9 4 . M I C H A I L . S . and K A R A M A N D L I D I S . A. ( 1 9 6 7 ) : Morphologie du plexus myenterique d 'Aucrbach de I'intestine. — Acta ana t . 67. 4 2 4 - 4 3 6 . R E A D , J . B . and B U R N S T O C K . G . ( 1 9 6 8 ) : Comparat ive histochemical studies of adrenergic nerves in the enteric plexuses of vertebrate large intestine. — Comp. Biochcm. Physiol. 27. 5 0 5 - 5 1 7 . S C H E U E R M A N N . D . W . and S T A C H . W . (1984): Fluorescence microscopic study of the architecture and structure of an adrenergic network in the plexus myentcricus (AUERBACH), plexus submucous cxtcrnus (SCHABADASCH) and plexus submucous internus (MEISSNER) of the porcin small intestine. — Acta anal. 119. 49-59. DE LA T O R R E , J . C . and S U R G E O N . J . W . ( 1 9 7 6 ) : Histochemical fluorescence of tissue and brain monoamines: results in using the sucrose-phosphate-glyoxylic acid (SPG) method. Neuroscience / . 4 5 1 - 4 5 3 . 

Fluorescence characterization of the nerve plexuses in the small intestine of the chicken

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Fluorescence characterization of the nerve plexuses in the small intestine of the chicken

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