Download Abstract Introduction The structural organization of the myenteric

A C T A B I O L . S Z E G E D . 3 3 . PP. 8 5 — 130 ( 1 9 8 7 )
C Y T O C H E M I C A L DETECTION OF N A D H - D I A P H O R A S E
POSITIVE NERVE C E L L S
IN T H E ALIMENTARY CANAL OK F R O G
R. G A B R I E L 1 . K . HALASY1 a n d M . C S O K N Y A 3
'Department of Zoology. Attila József University
H—6701 Szeged. P.O.B. 659. Hungary
Department of Zoology. Janus Pannonius University
Pécs. Hungary
(Received: May 15. 1986)
Abstract
Authors studied the nerve cells of the myenteric plexus of the frog alimentary canal using the
N B T / N A D H - d i a p h o r a s e technique, as well as silver impregnation and transmission elcclron microscope. It was concluded lhat the nerve cells form groups o i ganglia in the stomach: on the contrary they
show dispersed localization in the small intestine, while both ganglionic and solitary neuronal arrangements arc delectable in the transitional duodenal section. The size of the nerve cells varies through the
length of the intestinal canal (50—200 ^m"). The myenteric plexus is situated in the connective tissue of
the subserosa. in this way differing from the higher Vertebrates plexuses. The neuronal density ranges
from 600 to 1100 cells/cm : throughout the lenght of the alimentary canal. The organization of the cellular
elements shows a transition between the innervation by dispersed nerve cells and the enteric nervous
system of ganglionic arrangement.
Key words: frog intestine, myenteric plexus. N B T / N A D H diaphorase. "whole mourn", cell count
Introduction
T h e structural o r g a n i z a t i o n of the myenteric plexus has been studied by several
a u t h o r s at various sections of the alimentary canal (FLLOGAMO a n d ViGULIANl.
1954; GABELLA, 1971; IRWIN, 1931; LEAMING a n d C A U N A . 1961; O K H U B O ,
1936a
a n d b). T h e studies firstly deal with the innervation of the m a m m a l intestine, a n d in
this relation also give a c c o u n t on cell count d a t a referring to area unit. Studies of this
kind have been launched by the N A D H - t e t r a s o l i u m reductase technique, introduced by GABELLA, (1969). This technique electively stains the nerve cells with good
reproducibility, a n d c o n t r a r y t o the classical staining p r o c e d u r e s , d e m o n s t r a t e s
practically every nerve cell. In the possession of this specific a n d highly effective
technique, it seemed o b v i o u s to study the myenteric plexus of the frog (Runu
esculent a L.) enteric n e r v o u s system, since literary d a t a are rather scant in respect to
its o r g a n i z a t i o n — particularly in regard to the upper sections of the intestinal canal
(GUNN, 1951). T h e a i m of o u r studies was t o examine the myenteric plexus of the
frog intestine f r o m morphological aspect, a n d within this, by m e a n s of the detection
86
R
GABRIIL. K
HAIASY
AND M
CSOKNYA
a n d c o u n t i n g of the N A D H - d i a p h o r a s e positive nerve cells, we wished to o b t a i n
new (comparative) d a t a on the innervation of the studied intestine sections.
Materials and methods
Mature male and female adult frogs I Rana e.u ulenia L.; were used in o u r studies The method of
(1969) was used for the histocheraical reaction. The animals were decapitated and the distal end
of the intestinal canal was tied down, then ihe intestine » a s filled u p with Krcbs-solution After tilling up.
the other end of the intestine was also lied down, then the whole intestinal tract was placed into 0 . 3 %
Triton-solution, followed by washing for 2x5 minutes. Then the tilled u p intestine was placed into
incubating solution, the composition of which was the following: 2.5 mg of N B T dissolved in 5 ml of
tridistilled water. 5 ml of 0.1 M phosphate buffer (pH 7.3). 10 mg ol N A D U dissolved in 10 ml of
iridistilled water. The intestine was kept in the incubating solution till the a p p e a r a n c e of the d a r k blue
colour reaction ( 4 0 - 6 0 min). T h e fixation of the samples in 10". neutral formaline lasted lor at least 12
hours Using safety razor blade, about I cm slices were cut f r o m the intestine, perpendicularly to the
longitudinal axis, and "whole m o u n t " specimens were prepared f r o m the slices. During the preparation
of the specimens it turned out that the complete muscle layer (tunica muscuians) could be removed
GABELLA
without d a m a g e to the nerve plexus.
The method of B I E L S C H O W S K Y - G R O S - C A I
NA
was used for the silver impregnation (Li
CALNA. 1961).
AMINO
and
„
F o r the purpose of electronmicroscopic studies, transcardial perfusion was performed with fixative
containing 1% glutaraldehyde and 2 % paraformaldehyde. 0.2 M Na-cacodylate buffer was used for the
preparation of the fixative. Following 2 hours of immersion post-fixation, the material was washed in 0.2
M phosphate buffer containing. 7.5% saccharose. 2 % buffered osmium was used for osmification. then
the material was dehydrated in ascending alcohol series. During dehydration "block contrasting was
performed in the solution of 7 5 % alcohol saturated with uranylacctatc. Using propylcnc-oxidc
intermedium, the samples were embedded in D u r c u p a n . T h e ultrathin sections were also contrasted with
lead citrate according to REYNOLDS. The electronmicroscopic figure » a s prepared by Tesla BS 540
electronmicroscope.
Results
The light microscopic studies of the specimens prepared by the N A D H d i a p h o r a s e cytochemical reaction showed the m o r p h o l o g y of the myenteric plexus
t o be strongly variable t h r o u g h o u t the length of the intestinal canal. Using the
present m e t h o d to study the esophagus could not be accomplished since the
proximal end of the intestine could only be tied d o w n on the a c c o u n t of this section
of the intestine.
T h e nerve cells form g r o u p s of ganglia at the area of the s t o m a c h (Fig. I), with
3 - 1 0 n e u r o n s occupied in each ganglion. T h e average distance between the ganglia
is a b o u t 100 n m . but distances of only 3 0 - 4 0 nm were also f o u n d between some
neighbouring ganglia. T h e ganglia of the s t o m a c h arc generally polygonal. T h e
section of the d u o d e n u m close to the s t o m a c h a s s u m e s an interesting a p p e a r a n c e
f r o m the viewpoint of n e u r o n a l a r r a n g e m e n t . Solitary n e u r o n s are frequent here,
with the s i m u l t a n e o u s occurrence of ganglional a r r a n g e m e n t (Fig. 2). T h e size of the
cells s h o w s great variation. T h e cell profile ranges f r o m 50 to 200 n m : in case of both
C Y T O C H E M I C A L D E T E C T I O N OK N A D H - D I A P H O R A S I POSITIVE NI RVE
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the s t o m a c h a n d the p r o x i m a l end of the d u o d e n u m . In the s t o m a c h , a b o u t 5 0 % of
the cells are large: m o r e than 150 n m \ o n e third are small (50—75 nm 2 ), while the
r e m a i n i n g p a r t s show m o d e r a t e profile. F r o m the processes of the nerve cells,
generally only oné c a n be followed till a greater distance f r o m the cell b o d y (Fig. 3a).
T h e histochemical reaction causes well detectable g r a n u l a t i o n in the p l a s m a of the
nerve cells, a n d it can be observed well close t o the cell b o d y , in the initials of the
processes (Fig. 3b).
T h e ganglionic a r r a n g e m e n t of the nerve cells d i s a p p e a r s completely at the
distal part of the d u o d e n u m as well as in the small intestine: the cells are a r r a n g e d
sporadically w i t h o u t showing even distribution (Fig. 4). Here the localization of
n e u r o n s f r o m each o t h e r is at distances of 25—75 n m .
In the e n v i r o n s of the blood vessels entering f r o m the mesenterium, the
n e u r o n a l density of the gut is higher then on the antimesenterical side. T h e profile of
the n e u r o n s is small (50—75 nm 2 ). A few larger n e u r o n s are also occassionally
observable at the distal end of the d u o d e n u m a n d in the small intestine, but these are
consequently f o u n d a m o n g the entering of mesenterial vessels (Fig. 5a). T h e size of
these n e u r o n s surpasses t h a t of the large n e u r o n s of the 2 0 0 - 2 5 0 n m 2 profile. T h e s e
large n e u r o n s are m u l t i p o l a r (Fig. 5b). T h e n e u r o n s were f o u n d in the sections of
d u o d e n u m a n d small intestine, resp., had o n e or two processes (Fig. 6).
T h e n e u r o n s were c o u n t e d per intestinal section, a n d the results obtained are
given in T a b l e I. T h e n u m b e r of n e u r o n s per area unit differs a c c o r d i n g to various
intestinal sections. T h e lowest a m o u n t was found at the initial sections of the
s t o m a c h (619 cells/cm : ), while an increase was manifested at the p y l o r u s section (948.
cells/cm").
T h e d u o d e n u m cell c o u n t s h o w e d slight decrease c o m p a r e d to the pylorus (772
cells/cm"), but the rest of the small intestine p a r t s was f o u n d t o be m o r e a b u n d a n t in
nerve cells: the n u m b e r of cells was f o u n d to be the highest here f r o m the studied
sections ( a b o u t 1000 cells/cm : ). At the s a m e time, n o significant difference was
manifested in respect to cell n u m b e r between the proximal a n d distal e n d s of the
small intestine, as observable in the c o l u m n d i a g r a m (Fig. 7).
T h e results of the N A D H - d i a p h o r a s e p r o c e d u r e serving f o r the detection of the
n e u r o n s evidence the m a j o r i t y of the n e u r o n s are localized on the o u t e r surface of
the tunica muscularis. This a r r a n g e m e n t has also been verified by the results of the
silver impregnation a n d electron microscopic studies. T h e silver i m p r e g n a t i o n light
microscope figure (Fig. 8b) s h o w s that the myenteric plexus is situated on the o u t e r
surface of the muscle layer, a n d this is s u p p o r t e d by the figure p r e p a r e d f r o m the
specimen labelled by the d i a p h o r a s e technique (Fig. 8a). O n the electron microscopic picture (Fig. 9) this a r r a n g e m e n t is even clearer: the edge of the s m o o t h
muscle layer; the centrally located n e u r o n a n d the n e u r o p i l e in the direct
n e i g h b o u r h o o d : f u r t h e r m o r e , the wide connective tissue layer of the consecutive
subserosa, mainly c o m p o s e d of collagen fibers are all well recognizable in the left
u p p e r corner of the Figure.
R
GABRII-L. K
HALASV AND M
CSOKNYA
Discussion
It is evident f r o m b o t h literary d a t a (GABELLA. 1969; 1971; 1979) a n d o u r
study results that the histochemical detection of N A D H - d i a p h o r a s e activity is
suitable for the selective d e m o n s t r a t i o n of the nerve cells, f o r the d e t e r m i n a t i o n of
neuron n u m b e r , a s well as for studies o n the cellular a r r a n g e m e n t in the f r o g
intestinal canal.
T h e localization of the myenteric plexus is s o m e w h a t different f r o m that is
usual in the case of m a m m a l species. T h e cellular elements a r e situated on the o u t e r
side of the tunica muscularis. while in the case of higher V e r t e b r a t e s the usual localization is between the circular a n d longitudinal layers of the muscle sheat. T h e cause
of the deviation might be the weak development of the longitudinal muscle layer,
since this layer s t a n d s of only 1—4 cell r o w s in f r o g s , thus the a x o n s necessary f o r
innervation can easily penetrate through it. O u r o b s e r v a t i o n s regarding the s h a p e of
the diaphorase-positive nerve cells is in c o r r e l a t i o n with the d a t a of GUNN, (1951)
obtained by the silver impregnation m e t h o d , a c c o r d i n g to which b o t h small a n d
large cells a r e f o u n d in the small intestine; f u r t h e r m o r e , the localization of the large
cells rarely detectable at this site is also t h o u g h t by this a u t h o r to be in the
s u r r o u n d i n g s of the large veins entering f r o m the m e s e n l c r i u m . T h e r e have been n o
d a t a so far c o n c e r n i n g to the a r r a n g e m e n t of the nerve cells in the f r o g s t o m a c h . O u r
studies have proved on the o n e hand the ganglional a r r a n g e m e n t , on the o t h e r h a n d
that the small a n d large n e u r o n s in the s t o m a c h ganglia are f o u n d simultaneously
beside each other.
DOGIEL (1896. 1899) classified the vegetative n e u r o n s on the basis of their
processes. N o w a d a y s , however, it seems more expedient to take the cell size into
consideration l o o . a c c o r d i n g to the suggestion of LEAMING a n d CAUNA, (1961) as
well a s GUNN, (1959. 1968). Based on newer studies, a third basic cell type can also
be f o u n d in the myenteric plexus of the chicken intestinal tract (CSOKNYA a n d
BENEDECZKY, under publication).
G u n n also had m a d e an a t t e m p t t o differentiate the small a n d large n e u r o n s
f r o m the viewpoint of transmitter c o n t c n t . h o w e v e r , she could not achieve una m b i g u o u s results (GUNN. 1968). Newer studies (VINCENT el al. 1983a a n d b. 1986)
have shown that the c o m p a r i s o n of the results of the d i a p h o r a s e reaction with those
of the acetylcholine-esterase reaction c a n successfully be applied in the central
n e r v o u s system. P e r f o r m i n g the reactions separately, the n e u r o n s giving b o t h histochemical reactions could be identified on consecutive sections. S u p p o r t i n g the result
of the acetylcholine-esterase reaction with choline-acetyltransferase reaction.
VINCENT et al. (1983a) came t o the conclusion t h a t the N A D P H - d i a p h o r a s e posilivity of certain central n e r v o u s system structures a s s u m e s cholinergic t r a n s m i s s i o n .
In their o t h e r studies, however, these a u t h o r s could not u n a m b i g u o u s l y s u p p o r t this
statement (VINCENT et al. 1983b). In a recent p u b l i c a t i o n . VINCENT et al. (1986)
have been successful in proving the coexistence of the cholinergic-peptidergic system
in respect t o the ascending reticular cholinergic systems. In o u r o p i n i o n , b o t h the
cholinergic a n d adrenergic n e u r o n s of the intestinal myenteric plexus b e c a m e
C Y T O C H E M I C A L DETECTION OK N A D H - D I A P H O R A S I
POSITIVE N I RVE C U L L S
X9
stained d u r i n g the course of o u r experiments. This statement is s u p p o r t e d by the
fact, that in case of consecutive diaphorase- a n d acetylcholinesterase reaction
p e r f o r m e d on the same " w h o l e m o u n t " p r e p a r a t i o n , such cells arc found that show
only diaphorase-activity (GABRIEL, unpublished d a t a ) .
We could n o t find a n y literary d a t a on nerve cell c o u n t concerning the f r o g
intestine. O u r d a t a show 2—7 times less cells per intestinal section than the s u m m a r izing d a t a of GABELLA. (1979) related t o m a m m a l species. T h e r e are n o d a t a ,
however, on the tissue v o l u m e innervated by certain ganglia or cells. Calculations in
this respect a r e rather difficult since the muscle layer is not of the same thickness
even t h r o u g h o u t the same intestinal section. O n the o t h e r h a n d , distending with
Krebs-solution p r o d u c e s the elongation of the intestinal muscle cells. T h e r e f o r e ,
these d o not show their original volume d u r i n g the c o u r s e of m e a s u r e m e n t , a n d preliminary m e a s u r e m e n t s are not possible o w i n g to the filling up the whole intestine.
O u r present results suggest that the d e v e l o p m e n t of the myenteric plexus of the
f r o g intestine s h o w s an intermediary state phylogenically at the various sections of
the intestine: the myenteric plexus of the s t o m a c h is ganglionic, the small intestine
c o n t a i n s sporadically a r r a n g e d nerve cells. In case of chicken as well as m a m m a l s , it
is known (DOG1EL, 1896, 1899) that the whole myenteric plexus s h o w s ganglional
a r r a n g e m e n t . T h e alimentary canal of the developing chicken a n d m a m m a l ,
however, d o e s not show clearly this kind of a r r a n g e m e n t , since the cells still migrate
a n d there is also an increase in their a b s o l u t e n u m b e r with f u r t h e r development
(GABELLA.
1971).
O u r results o b t a i n e d with silver i m p r e g n a t i o n a n d transmission electron
microscope have u n a m b i g u o u s l y s u p p o r t e d o u r a s s u m p t i o n s regarding the localization of the plexus, a c c o r d i n g to which the nerve plexus is not localized at the " u s u a l "
place between the two muscle layers, but r a t h e r outside this, in the subserosa.
T h e m o r p h o l o g i c a l a n d functional significance of this characteristic
localization is t o be clarified by further studies.
References
and B E N E D E C Z K Y . I . ( 1 9 8 6 ) : Cell types of the enteric nerve plexuses in the chicken. — Acta
Biol. Szeged. 32. 9 3 - 1 0 2 .
DOGIEL, A.S. (1896): Zwei Arten sympatischer Nervenzellen. — Anat. Anz. I I . 679-687.
DOGIEL. A.S. (1899): Über den Bau der Ganglien in den Geflechten des Darmes und der Gallenblase des
Menschen und der Saugetiere. — Arch. Anat. Physiol. 5. 130-159.
F I L O G A M O , G . and V I G U L I A N I . F . ( 1 9 5 4 ) : Richerche sperimentali sulla correlazione tra estenzione del
territorio di inncrvazione e grandezza e numero dellc cellule gangliari del plesso mienterico (di
Auerbach) nel cane. — Riv. patol. nerv. ment. 75. 1-32.
G A B E L L A , G . (1967): Neuron number in the myenteric plexus in newborn and adult rats. — Experientia
CSOKNYA, M .
23.
52-54.
(1969): Detection of nerve cells by a histochemical technique. — Experienlia 25. 218-219.
(1971): Neuron size and number in the myenteric plexus of the newborn and adult rat. — J.
Anat. 108. 81-95.
G A B E L L A . G . ( 1 9 7 9 ) : Innervation of the gastrointestinal tract. — Int. Rev. Cyt. 5 0 . 1 2 9 - 1 9 3 .
GABELLA. G .
GABELLA. G .
R
90
GABRIEL, K
HALASY
AND M
CSOKNYA
GUNN. M (1951): A sludy of the enteric plexuses in some amphibians. — Q. Jl. Microsc. Sei. 92. 55-77.
GUNN. M. (1959): Cell types in the myenteric plexus of the cat.
J. C o m p . Neurol / / / , 83-100.
G I N N . M. (1968): Histological a n d histochcmical observations on the myenteric a n d s u b m u c o u s
plexuses of m a m m a l s .
J Anat. 102. 223-229
IRWIN. D A (1931): T h e a n a t o m y of Auerbach s plexus. — Am J Anat. 49. 141-166.
LEAMING. D.B and CAUNA. N. (1961): A qualitative and quantitative s t u d y of the myenteric plexus in the
small intestine of the cat. — J. A n a l 95. 160-169
O K I I U B O . K (1936a): Studien über d a s intramurale Nervensystem des Vcrdauungskanals I I . Die plexus
mycntericus und plexus subserosus des Meerschweines.
Jap. J Med. Sei. Anat. 6. 31-37.
OKHUBO. K (1936b): Studien über d a s intramurale Nervensystem des V c r d a u u n g s k a n a l s III. Affe und
Mensch. - J a p J. Med. Sei. A n a l . 6. 219-247.
V I N C E N T . S R.. S A T O H . K . . A R M S I R O N G . M D. and F I B I G E R . H C . (1983a): N A D H - d i a p h o r a s c : a
selective histochcmical marker for the cholinergic n e u r o n s of the pontine reticular f o r m a t i o n . —
Ncurosci. Lett. 43. 31-36.
V I N C E N T . S R . . S T A I N E S , W.A. and F I B I G E R . H . C . (1983b): Histochcmical d e m o n s t r a t i o n of separate
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VINCENT,
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K..
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PANULA.
P..
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(1986):
Neuropeptides and N A D P H - d i a p h o r a s e activity in the ascending cholinergic reticular system in
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WALKER, D.G. (1963): A survey of dehydrogenases in the various epithelial cells in the rat. — J. Cell.
Biol. 17. 255-277.
Tabic I. Results of the cell count
cclls/unit
(nu./cm-)
average
Section of the
intestine
cell count
(total)
area
(mm!)
stomach cardia
524"
523 b
634'
72
104
101
728
503
627
619
stomach pylorus
241445"
393'
21
48
48
1100
927
818
948
duodenum
363*
462 t
43
66
X44
700
772
small intestine
proximal
367'
379"
210'
45
35
21
814
1083
1000
966
small intestine
distal
351 1
I24 6
36
12
975
1033
1004
The labcllings a. b. c mean the cell count data of the total a m o u n t of specimens prepared f r o m three different
experimental animals.
C Y T O C H E M I C A L D E T E C T I O N OK N A D H - D I A P H O R A S I POSITIVE NI R V E
CULLS
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Legend to figures
Fig. I.
T h e majority of the nerve cells in the stomach form g r o u p s of ganglia (arrow.) 200 x
Fig. 2.
Both ganglional and solitary n e u r o n s are observable in the transitional section of the stomachd u o d e n u m . 200 x
G : ganglion. S: solitary cell
Fig. 3a.
In general, the cells possess well detectable processes. 400 x
Fig. 3b.
Strong granulation can mainly be observed in the neuronal processes (arrow.) 800 x
Fig. 4.
The neurons of the small intestine d o not form g r o u p s of ganglia and do not show even distribution (arrows: cells). 200 x
Fig. 5a.
Large n e u r o n s can be seen (arrows) in the surroundings of the vessels entering from the
mcscntcrium. 200 x
Fig. 5b
Several processes are recognizable (arrows) projecting f r o m the large cells. 800 x
Fig. 6.
The small neurons mostly possess I — 2 processes at the area of the small intestine.
The processes are indicated by arrows. 200 x
Fig. 7.
On the column diagram the neuron density of the intestinal canal can be seen according to
the various sections of the intestine.
Fig. 8a.
The plexus is localized outside the muscle layer: smc: smooth muscle cells, n: neuron. 200 x
Fig. 8b.
The plexus (indicated by arrows) is situated at the outer edge of the muscle layer, beneath the
connective tissue limiting the intestine from outside. 200 x
Fig. 9.
Detail of the myenteric plexus. In the upper left corner of the Figure the detail of a smooth muscle
cell (smc) is observable, with a neuron (n) in the neighbourhood, close t o which a neuropil (np)
can be seen. The thick layer of the subserosa. packed with the collagen fibres (co). is situated near
the outer surface of the intestine. 25 000 x
92
R
GABRIEL. К
HALAS'»
AND M .
CSOKNVA
C V T C X H H M K ' A L 1)1 I K I I O M I H
N A D H - D I A P M O R A S I POSIIIVI NI KVI
(ILLS
R
GABKIEL. К
HALASY
ANO M
CSOKNYA