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Plexus muscularis profundus and associated interstitial cells. I. Light microscopical studies of mouse small intestine

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THE ANATOMICAL RECORD 203:115-127 (1982)
Plexus Muscularis Profundus and Associated Interstitial Cells.
I. Light Microscopical Studies of Mouse Small Intestine
Anatomy Department C, University of Copenhagen, 1 Universitetsparken,
DK-2100 Copenhagen 0,Denmark
The zinc iodidelosmic acid (ZIO)method was used in a modification that selectively stained nerves and associated interstitial cells of Cajal (ICC )
of muscularis externa. Due to its selectivity the method allowed a detailed
stereoscopical analysis of whole mounts with respect to the topography and morphology of these elements. The method thus assisted and expanded our ultrastructural studies. The ZIO staining allowed a distinction of four morphologically
different interstitial cell types (ICC-I-IV)confined to four compartments. The
stained components were: (1)A rich plexus of highly ramified intestitial cells (ICC11) in the subserous layer. (2) Auerbachs plexus with an associated extensive
plexus of interstitial cells (ICC-I)in close contact with tertiary fasciculi. (3)Nerve
fasciculi of the outer division of the circular muscle layer. These formed a nerve
plexus in a well-definedplane in the outermost cell layers (plexusmuscularis superficialis),with few fasciculi located internal to this plexus. A few bipolar interstitial
cells (ICC-IV)were associated with nerve fasciculi of this region. (4)A nerve plexus
located in the region between the two subdivisions of the circular muscle, plexus
muscularis profundus (PMP).PMP was revealed throughout the small intestine as
a continuous network of elongated, circularly oriented meshes. The pattern of connections between PMP and the other enteric plexuses was studied stereoscopically. Ganglion cells intrinsic to PMP occurred widely scattered. Interstitial cells
associated with PMP (ICC-111)were arranged in a plexifonn manner; their morphology and relations to nerves were investigated in great detail. A selective innervation of ICC-I11via axons of PMP was strongly supported.
Plexus muscularis profundus (PMP)is a welldefined nerve plexus, sandwiched between two
subdivisions of the circular muscle layer of the
small-intestinal muscularis externa: an outer,
thick layer and an inner, very thin layer (Cajal,
1893,1911; Li, 1937,1940; Taxi, 1965; Gabella,
1972, 1974; Duchon et al., 1974; Rumessen et
al., 1982; Thuneberg, 1982). A nerve plexus at
this specific location has been identified in all
mammals examined. Its function is unknown,
but the potential importance of PMP as a
significant component of the enteric nervous
system was clearly demonstrated by Gabella
(1972): In guinea-pig ileum PMP contains
almost two-thirds of all vesiculated axon terminals counted within the circular layer.
In recent reviews it has been stressed that
the inner division of the circular muscle is
richly innervated by PMP (Gabella, 1979). the
0003-276W82/2031-0115$04.00 0 1982 Alan R. Liss. Inc.
branches of which are “running between the innermost layers of muscle cells” (Furness and
Costa, 1980). In contrast, our ultrastructural
studies (Rumessen et al. 1982; Thuneberg,
1982) on mouse intestine show conclusively
that PMP specifically innervates the outer,
main layer of circular muscle. Furthermore, the
innervation is mediated by interstitial cells of
Cajal (type 111:ICC-111; Rumessen et al., 1982;
Thuneberg, 19821, identical with the “hybrid
cells” of dog intestine (Duchon et al., 1974;
Daniel, 1977). In accordance with the latter
authors we have found that PMP preferentially
innervates ICC-111, which are coupled by
numerous gap junctions with cells of the outer,
main division only. This specific arrangement
stresses the potential importance of ICC-I11as
Received July 2,1981; accepted December 2, 1981.
intercalated, regulatory cells in circular muscle
function (Daniel,1977).The “interstitialcells of
Cajal” (ICC) were described by Cajal (1911)in
two locations related to the intestinal muscularis externa: associated with (1)PMP and (2)
Auerbach’s plexus. In a previous light and electron microscopical study (Thuneberg, 1982)we
have accumulated evidence in favor of a role as
intestinal pacemaker cells of ICC (type I: ICCI) associated with Auerbach’s plexus. The
study of the organization of a network of highly
ramified ICC-I was carried out by selective
staining of ICC-I with supravital methylene
blue. In order to expand our ultrastructural
observations on ICC-111 (which do not take up
methylene blue), we have developed a modification of the zinc iodide/osmic acid (ZIO)method,
which selectively stains the intestinal nerves
and associated ICC, including ICC-111. The
study of whole mounts of the muscularis,
stained with ZIO, and observed with stereoscopical methods, has allowed a precise and
thorough description of the general organization of ICC and nerves throughout the small intestine. The present investigation provides a
basis for our accompanying ultrastructural
study (Rumessen et al., 1982) and expands the
observations made by Cajal (1911), Li (1940).
and Taxi (1965).
Adult albino mice of either sex were used.
After cervical dislocation, segments of
duodenum, jejunum, and ileum were excised
and processed for light microscopy.
The excised segments were immediately
transferred to a modified Tyrode solution
(Caprilli e t al., 1970). Then papaverine (1 mM)
was added. The segments were immersed for
5-10 minutes, at an initial temperature of
37 “C, declining to room temperature. This procedure caused an apparent complete relaxation
of smooth muscle cells, providing comparable
material from different parts of the intestine.
The segments were flattened and slightly distended by strips of dental wax (TruwaxDentsply) introduced into the lumen. Subse
quently, 1-cm pieces were cut and immersed in
a freshly prepared ZIO mixture comprising
0.4%OsO,, 2.4% ZnIz (Merck)(Maillet, 1959).
The segments were immersed for 4, 10, 24,48,
72 hours, and some for 7 and 14 days at room
temperature. Some preparations were not subjected to either papaverine or slight distension,
and others were subjected to only one of these
procedures. Following ZIO-staining/fixation
whole mounts of the muscularis externa mea-
suring about 15-20 mm2 were cautiously
dissected under a stereomicroscope by means
of small-caliber hypodermic needles.
The isolated muscle layers were mounted
with Aquamount (Gurr biol. reagent, Searle).
The preparations were left to harden for 1-2
days at room temperature before examination.
For optimal microscopical examination of the
inner circular muscle layer most preparations
were mounted with this layer upward. Sphincter regions were not included in the material.
Further examination was carried out, and all
photos taken, on a Zeiss photomicroscope
equipped with differential interference contrast (Nomarski) optics. Nerves and ICC were
studied by stereoscopical methods. A polarizing filter system (Michel, 1967)which converts
the ordinary binocular microscope to a
stereomicroscope allowed true stereoscopical
view a t all magnifications. Limitations due to
interpretation of sectioned material were thus
overcome. Stereo-pairs(Figs.4-7) should be examined by means of an ordinary binocular
stereo-viewer or naked-eye stereopsis. Nakedeye stereopsis with inversion (convergent
visual axes) will produce a reversal of desired
Our previous investigations (Thuneberg,
1982) have revealed four main types of interstitial cells of Cajal (ICC) related to the
muscularis externa of mouse small intestine.
ICC-I are situated between the main muscle
layers, associated with Auerbach’s plexus (AP)
in a plexiform manner, ICC-I1 form a plexus in
the subserous layer, ICC-I11 are associated
with PMP, and ICC-IV are disposed along
intra-muscular nerve fasciculi in the outer,
main layer of circular muscle.
In initial experiments, it was observed that
preincubation of the intestinal segments in
papaverine (see Materials and Methods)
prevented a heavy, unspecific staining of
smooth muscle cells, which otherwise occurred
in contracted segments, so that nerves and ICC
were totally obscured. Subsequent to this
preincubation the ZIO mixture selectively
stained nervous elements in all regions of the
muscularis externa and of the submucous
layer, with a distinct outline of single axons
and varicosites. ZIO staining of the different
elements followed a characteristic time scale.
First to appear (incubation times 2-3 hours)
was AP, followed by intramuscular axons,
PMP, and the submucous plexus (PS) (4-10
hours). ICC generally appeared later (10-24
hours), and no further ZIO staining occurred
after incubation times of more than 72 hours.
Nervous elements
AP, confined to the space between the main
muscle layers, exhibited a plexiform appearance of ganglia and of primary, secondary,
and tertiary fasciculi. The tertiary plexus was
generally irregularly arranged, and appeared
little or variably developed.
In a well-defined plane in the outer part of the
outer circular muscle layer was revealed a
plexus of parallel, regularly spaced, and largely
unbranching nerve fasciculi (plexusmuscularis
(strati circularis) superficialis (PMS), Thuneberg, 1982). These nerve fasciculi extended
from primary or secondary fasciculi of A P to
the interstices of the circular muscle layer, thus
invariably located at the inner aspect of AP.
The fasciculi were very seldom obliquely or
transversely interconnected, but showed bifurcations of single fasciculi continuing as two
parallel fasciculi, separated 12-18 pm from
each other. The width of the fasciculi was 3-4
pm (Figs. 1, 5, 6) .
A prominent PMP was confined to a welldefined plane, separated from the submucosa
by the layer of one to two muscle cells of the inner subdivision of the circular muscle layer
(Fig. 2). The plexus was encircling the intestinal wall in its whole circumference. No difference in overall arrangement was observed in
different parts of the small intestine. The main
constituents of PMP were larger (primary),circularly oriented fasciculi separated 30-40 pm
and measuring 4-7 pm in width. These fasciculi
were thus more widely separated and of a larger
width than fasciculi of PMS and of approximately the same width as true secondary
fasciculi of A P (Figs. 1, 2). Via oblique or
transverse interconnections these fasciculi
formed the largest meshes, generally elongated
in the direction of the main axis of the circularmuscle cells and with sizes varying between 40
X 200 pm and 10 X 15 pm. The large (primary)
meshes were often subdivided by finer fasciculi
or single axons. Higher magnifications of
fasciculi revealed preferentially stained
varicose axons. Varicosities (1-4 pm in
diameter) were separated 1 pm to several pm
from one another within a single axon.
Schwann cells related to PMP could be
distinguished with certainty from ICC-111.
Schwann cells were stained only very occasionally and usually only empty swellings
within the fasciculi indicated the location of
Schwann cell nuclei (Fig.2). When present, the
staining of Schwann cells was very characteristic, as the nucleus was invariably the most
colored part, the cytoplasm being only faintly
In a small number of preparations ganglionlike formations were observed as a part of
PMP, clearly separated from submucosa by the
muscle cells comprising the inner subdivision
of the circular muscle layer. As neurons were
most often unstained by the ZIO mixture, their
presence in PMP was indicated by the occurrence of the typical basketlike networks of
stained axons surrounding groups of two to
five or single (unstained) ganglion cells. The
neurons inside the ganglionlike formations
were identified at higher magnification, using
differential interference contrast (DIC)optics,
which revealed their rounded nuclei and large
spherical nucleoli (Fig. 3). very similar to
ganglion cells of AP. Stereoscopy a t high magnifications established the location of ganglion
cells and pericellular networks with certainty
to the level of PMP, as neurons intrinsic to this
The main part of connections to PMP appeared to be derived from AP, and f a r fewer
connections were observed between PMP and
PS. Perivascular connections originating in the
zone of mesenteric attachment were not investigated. The stereoscopic investigations revealed connections between AP and PMP, with
a perpendicular course in relation to the circular muscle layer (Figs. 4,5,7).These connections originated from primary or secondary
elements of AP and often gave off branches to
supply PMS (Fig. 5 ) . The only connections between PMP and PS were revealed by a few of
the large perpendicular connections between
AP and PMP continuing to connect with PS
(Fig. 4). Infrequent, perpendicular connections between PMP and PMS were revealed
(Fig. 6). Occasionally fasciculi left the plane of
PMS to traverse the circular muscle coat for a
long distance nearly parallel to the muscle
fibers, and ultimately merge with PMP. Thus,
PMS, PMP, and the connections mentioned
constituted all intramuscular nerves.
Nerves were neither observed within the
longitudinal muscle, nor in the subserous layer.
Interstitial cells associated with
PMP (ICC-111)
ICC-I11 exhibited a characteristic staining
pattern, as their nuclei were only faintly
stained, although sometimes with nucleoli
standing out in negative contrast. Cytoplasm
of ICC-I11 was well delimited and invariably of
a darker shade than that of the nuclei. Precise
discrimination of axon bundles and cell processes of ICC-I11 was possible, since ICC-I11
cytoplasm was of different shades of gray in
contrast to the deep black, varicose axons.
ICC-I11were demonstrated in all parts of the
small intestine, along its whole circumference,
constituting a plexus of somewhat irregular
outlines, with nuclei oriented in the same plane,
and spaced with regular intervals. Thus ICCI11 formed a flat, two-dimensional cellular
plexus in the same plane as PMP, intermingling with this (Fig. 8).
The nuclei of ICC-I11showed some variation
in size and shape. ICC-I11 had oval or rounded
nuclei, flattened in the radial direction, while
measuring about 13-25 pm X 6 pm in the tangential plane. The longest axis of the nucleus
was oriented in the direction of the muscle cells
of the circular layer. Nucleoli (three to five)
often were arranged in a longitudinal row (Fig.
11). Cells with larger nuclei most often were
bipolar with more abundant perinuclear cyto-
A, Single axon of PMP
AP, Auerbach’s plexus
DIC. Differential interference contrast (Nomarski)
F, Large (primary) fasciculus of PMP
N, Nucleus of ICC-111 (unless specified)
PMP, Plexus muscularis profundus; deep plexus of circular
PMS. Plexus muscularis superficialis; superficial plexus of
circular muscle
Fig. 1. PMS (arrows)located in theouter part of theouter
subdivision of the circular muscle layer. Fasciculi are
regularly spaced in a well-defined plane at the luminal aspect
of AP. of which a primary fasciculus is labeled. Doubleheaded arrow: main axis of smooth muscle cells in the circular layer. X 610. (All figures show nerve plexuses and interstitial cells as they appear in the tangential plane of ZIOstained whole mounts.)
Fig. 2. PMP (arrows) confined to the plane between the
thick outer and thin inner subdivision of the circular muscle
layer. Fasciculi are interconnected, forming elongated
meshes. Fasciculi of PMP are thicker and more widely
spaced as compared with fasciculi of PMS (Fig. 1).S : location
of nucleus of Schwann cell (unstained).Double-headed arrow:
a s in Figure 1. X 610.
Fig. 3. Group of four ganglion cells constituting a part of
PMP. Nuclei and large rounded nucleoli (arrows) are seen.
Ganglion cells are surrounded by a network of axons. DIC. X
plasm, whereas cells with smaller nuclei often
exhibited two to four and occasionally five
primary processes in combination with a
marked scarcity of perinuclear cytoplasm. In
comparison, nuclei of smooth muscle cells
averaged 25 X 6 pm.
ICC-I11 showed considerable variation in
their pattern of ramification. The primary processes extended from the sparse perinuclear
cytoplasm and were slender (2-3 pm) or had a
broader, laminar appearance. They gave off
secondary and tertiary processes, not infrequently terminating in a complex arborizing
formation (Figs. 9, 10).Almost all ICC-111 had
very fine, short “twigs,” either at the most
distal parts of the processes, or at several sites
along the larger processes, as well as extending
from the perinuclear cytoplasm. Thus, the pattern of branching could be described as a mixture of dichotomic (main processes) and monopodic (“twigs”).ICC-I11 most often had their
main axis oriented in the direction of the muscle cells of the circular layer (Fig. 8), but
multipolar ICC with primary and secondary
processes extending for a considerable distance in all directions in the plane of PMP
were often observed (Figs. 9, 13). Some main
processes of ICC-111, crossing the outer
circular-muscle cells at an angle, showed small
expansions closely following the contours of
the smooth muscle cells (Fig. 13). Occasionally
processes of ICC-111 were followed deep into
the outer circular layer. ICC-I11 could be seen
to be in apparent contact with each other in
perinuclear areas (Fig. 1 l),or by means of distal
processes overlapping each other for a distance
(Fig. 8).Some ICC-I11 seemed to be enveloped
by secondary processes of neighboring ICC111.
Relations to nerves. In preparations with
well-developed staining of nerves and ICC, all
ICC-I11encountered were in close contact with
axonal varicosities, at some point or another.
ICC-111 with nuclei apposed to larger fasciculi
of PMP, often seemed to supply the latter with
an incomplete sheath of primary processes,
which often followed the fasciculi for several
hundred microns. ICC-I11 with no apparent
perinuclear association to axons, always contacted larger fasciculi via primary or secondary
processes (Fig.9).Single axons were often seen
to follow closely the contours of ICC-I11 processes and perinuclear cytoplasm (Figs. 13,14).
This close relationship of ICC-111 to varicose
axons could not be explained as a passive close
apposition of the involved structures in a
Fig. 4 . Stereo-pair showing connection (arrowhead) between a submucous ganglion (G)
and PMP (F).
This connection can be followed (arrows)toward AP (out of focus).X 440.
Fig. 6. Stereo-pair showing connection (small arrows) be.
tween afasciculusof PMS(broad arrow) and PMP(F).X 370.
Fig. 5. Stereo-pair of the same area as Figure 4. Focus is
a t AP. The fasciculus connecting PMP and AP contacts a
fasciculus of PMS (small arrow) and merges with a
ganglionated strand of AP (broad arrow). X 440.
Fig. 7. This stereo-pair shows a perpendicularly oriented
connection (arrow head) between PMP (focus) and AP. G:
location of a ganglion cell in AP. X 740.
Fig.8. Survey of PMPand ICC-111. ICC-111show anoval
nucleus and two to five primary processes. The finer cell processes form a complex pattern. Double-headed arrow: main
axis of smooth muscle cells of the circular layer. X 550.
Fig. 9. A multipolar ICC-111 spanning between two
larger fasciculi of PMP. This cell exhibits a very complex pat-
tern of ramification, and an extensive development of
cytoplasmic "twigs" (arrows). DIC. X 1,200.
Fig. 10. Abipolar ICC-111.showing anextensivedevelopment of cytoplasmic "twigs" (arrows). Preterminal
varicosities are in close contact with the cell (arrowheads).
DIC. X 1.530.
restricted space, because it very often seemed
that larger fasciculi of P M P gave off a single or
a few axons to pursue, and selectively supply,
ICC-I11 with terminal (Figs. 11, 12, 16)and/or
preterminal (Figs. 13, 14, 15)varicosities, in a
manner very suggestive of innervation. It was
observed that varicosities of larger size (approximately 3-4 pm) more often were in apparent selective contact with ICC-I11 than
could be expected from their overall distribution (Figs. 11,12, 15).Nomarski microscopy indicated that contacts to ICC-I11 revealed as
single axons actually were single and not accompanied by other unstained axons (Figs. 14,
16). Other single axons leaving fasciculi and
terminating among smooth muscle cells but
not associated with ICC-I11 were very rare,
although they have been observed. Smooth
muscle cells of the inner subdivisions of the circular muscle layer (Fig. 17)sometimes showed
a forklike branching not observed in the outer
circular muscle cells, but finer branching was
never observed.
Fig. 11. Two ICC-I11 apparently contacting each other
(arrowheads). The cell to the right appears to be innervated
by the terminal part of a varicose single axon (arrows).A row
of nucleoli is seen in negative contrast in the samecell. X 960.
Fig. 12. ICC-I11 with terminal varicosities abutting on
its cytoplasm (arrow).The varicosities are larger than most
others seen. This cell has three primary processes, which are
delicately branched. DIC. X 960.
Fig. 13. An ICC-I11 with four main processes. Two processes are closely followed by a varicose single axon (arrows).
The other processes, which traverse the muscle cells, are
slightly expanded and appear to follow the contours of the
faintly stained smooth muscle cells (arrowheads). X 960.
Fig. 14. ICC-I11 in close contact with single axons and
preterminal varicosities. These are disposed along the
perinuclear cytoplasm (arrowheads) and along a primary
process (arrow). X 1,530.
Fig. 15. Multipolar ICC-111 apparently innervated by
preterminal varicosities (small arrows). The varicosities impress ICC-I11 cytoplasm. Broad arrows: ICC-I11 processes.
X 1,380.
Fig. 16. Bipolar ICC-I11 apparently innervated by terminal varicosities on a single axon leaving a larger fasciculus
of PMP. The selective nature of the contact is evident (small
arrows). Broad arrows: ICC-I11 processes. DIC. X 1,370.
Fig. 17. A smooth muscle cell of the inner division of the
circular layer. The cell branches in a forklike fashion
(arrows).DIC. X 550.
Relations to other types of ICC (I, 11, IV). In
most preparations a rich plexus of ICC-I confined to the space between the main muscle
layers was revealed. ICC-I were stained with
fine detail of processes including the finest and
most distal ramifications. An intimate association of the tertiary elements of AP with ICC-I
was observed (Fig. 18). Nuclear features of
ICC-I were much like nuclei of ICC-111.
Although no contacts between ICC-I and ICCI11 were seen, processes of both cell types were
traced in the bulk of the circular muscle.
ICC-IV were only occasionally stained. They
were bipolar cells with very long, largely unbranching processes, closely apposed fasciculi
of PMS,and other intramuscular fasciculi (Fig.
20). No contacts between ICC-IV and ICC-I11
were seen.
The subserous plexus of ICC-I1 was also
demonstrated, showing a lower cellular density
than the plexus of ICC-I (Fig. 19).The cells had
three to five long primary processes, branching
in a largely dichotomic pattern, apparently interconnecting with processes of other ICC-11.
Contact points between individual ICC-I1were
difficult to distinguish. The main axis of ICC-I1
was oriented roughly in the direction of the
muscle cells of the longitudinal layer. Nuclear
features were similar to other types of ICC. No
contacts between ICC-I1 and other ICC or
nerves were observed.
Osmic acidliodide staining of peripheral
autonomic nerves was introduced by Champy
(1913) and modified and improved by Maillet
(1959) (osmic acidlzinc iodide). Initially it was
claimed (Coujard, 1943) that the osmic
acidiodide methods rendered adrenergic
nerves selectively visible, but this was later
questioned and discredited (e.g., Hillarp, 1959).
The problems concerning the specificity of the
ZIO methods are still not solved (see Pellegrino
de Iraldi, 1977).
At the light microscopical level, ZIO staining
is highly selective for unmyelinated nerves, using incubation times of 3-4 hours (Maillet,
1963, 1968/69;present report). With prolonged
incubation time a wider spectrum of cell types
is stained, most notable in this context the
ICC. The preincubation of smooth muscle
tissue in papaverine as in the present study,
seems to further increase the specificity of ZIO
methods, preventing heavy unspecific staining
of contracted smooth muscle cells.
In the present report, all interstitial elements
stained with ZIO methods are referred to as
ICC, but other cell types may occasionally be
stained. Indeed, cells interpreted as fibroblasts
have been demonstrated with ZIO methods
(Stach, 1963; Maillet, 1968/69; Stockinger and
Graf, 1965; Garret, 1965)as have macrophages
(Stach, 1963, 1969; Clara et al., 1968; Maillet,
1968/69).Both macrophagelike cells and fibroblastlike cells have been identified electron
microscopically in muscularis externa of
mouse small intestine (Rumessen et al., 1982;
Thuneberg, 1982). With respect to the identification if ICC-I and ICC-I1 the results obtained in the present study with ZIO methods
are in accordance with supravital methylene
blue studies (Thuneberg, 1982).With respect to
the level of PMP it is reasonable to assume that
those ZIO-positive cells which are preferentially supplied with varicose axons are identical
with the innervated ICC-I11 identified ultrastructurally (Rumessen et al., 1982; Thuneberg, 1982).
In the following our results will be discussed,
as they relate to four distinct regions of the
Longitudinal muscle layer and serosa
The absence of nerves within the subserous
compartment and the longitudinal muscle
layer confirms results obtained with methods
other than ZIO (Thuneberg, 1982).
Apart from the present results, light microscopical illustrations of ICC-I1 have been provided only by Richardson (1960, silver impregnation), and Thuneberg (1982, methylene
blue). The latter method seems to be unsatisfactory as applied to ICC-11, since only occasional single or small groups of ICC-I1 are
stained. Electron microscopy of ICC-I1
(Thuneberg, 1982) reveals a fibroblastlike
Fig. 18. The plexus of ICC-I at the level of AP. The cells
are extensively branched and exhibit close relations t o tertiary elements of A P (arrows).N: nuclei of ICC-I. X 290 and
X 720.
Fig. 19.a. The subserous plexus of ICC-11. This plexus
has a lower cellular concentration than the plexus of ICC-I
(Fig. 18a). Note that nerves are absent. X 290. b. A larger
magnification of a single ICC-11, branching in a dichotomic
pattern. The main axis of the cell is roughly in the
longitudinal direction. N: nucleus. X 610.
Fig. 20. An ICC-IV with nucleus (N) and processes (arrows) associated with a fasciculus of PMS. ICC-IV are
generally bipolar with long unbranching processes. X 720.
ultrastructure. The nature of these cells is
obscure at the moment.
The region between the main muscle layers
As a consequence of the simultaneous ZIO
staining of nerves and ICC, we have observed
that the finest strands of Auerbachs plexus
(tertiary fasciculi) exhibit very close contacts
with the extensive plexus of ICC-I;the nature of
these contacts deserves further investigation,
as they may be of importance to the concept of a
role of ICC-I in the origin or modulation of electrical impulses in mouse small intestine, as suggested by Thuneberg (1982).
Outer part of the circular muscle layer
Regularly spaced axon bundles, oriented
parallel to the muscle cells of the circular layer
and situated invariably at the inner aspect of
the main plexus of Auerbach (Schabadasch,
1930; Stohr, 1957) have generally been regarded as a component of the secondary plexus
of Auerbach. In mouse small intestine evidence
exists (Thuneberg, 1982) that these fasciculi
are actually located within the interstices of
the outermost cell layer of the circular muscle
coat (hence the term plexus muscularis (strati
circularis) superficialis) (PMS).The present investigation provides further evidence of a very
regular and well-developednerve plexus at this
Apart from the present findings, interstitial
cells (ICC-IV)apposed to PMS and to oblique
or perpendicular fasciculi within the outer division of the circular muscle layer have been investigated only electron microscopically
(Thuneberg, 1982). ICC-IV exhibit a fibroblastlike ultrastructure. Nothing is known of
their function.
Region between outer and inner subdivision
of the circular muscle coat
Variable definitions of the term “plexus
muscularis profundus” occur in the literature.
Cajal (1893, 1911) described a rich plexus of
nerves and ICC situated at the internal surface
of muscularis externa of the mammalian gastrointestinal tract (“plexussous-musculeux ou
musculaire profound). That Cajal referred to
this location only is evident from his drawings
of Golgi-impregnated guinea pig intestine (Cajal, 1911).SinceCajaldidnotdividethecircular
layer of muscularis externa of mammalian
small intestine in two separate layers (aslater
reported by Li, 1937, 1940), he did not
distinguish clearly between small and large in-
testine with respect to PMP. No local plexiform
concentration of nerve fibers has been reported
within the circular muscle layer of mammalian
large intestine, but a plexus of nerves and ICC
perhaps corresponding to “plexus sous-musculeux” of Cajal (1911) has been described just
luminal to the circular muscle of colon in guinea
pig and rat (Stach, 1969, 1972).Stach refers to
this plexus as “plexus entericus (submucosus)
extremus” and regards it as the outermost extension of plexus submucosus. This nerve
plexus may be homologous to PMP of small intestine, with which it seems to share some
similarities, regarding morphology and relations to ICC. It would be of interest to investigate the relation between PMP and its
possible homologue in colon with respect to
presumptive transitions and ultrastructural
Consequently, the term plexus muscularis
(strati circularis) profundus should be
restricted to the nerve plexus situated between
the two divisions of the circular muscle layer of
small intestine (Cajal, 1893, 1911; Li, 1937,
1940; Taxi, 1965; Gabella, 1972,1974; Duchon
et al., 1974; Rumessen et al., 1982; Thuneberg,
1982), and the designation of PMP to all intramuscular nerve bundles of the circular muscle coat in all regions of the digestive tract
(Schabadasch, 1930; Ohkubo, 1937a,b; Stohr,
1957; Stach, 1969, 1972; Schardt and van der
Zypen, 1974) should be abandoned. Similarly,
our findings disagree with a concept of PMP as
situated in the interstices of the innermost
layer of muscle cells (Furnessand Costa, 1980).
Our investigations have revealed PMP as an
extensive nerve plexus, confined to the space
between the outer and inner subdivisions of the
circular muscle layer of mouse small intestine.
PMP is equally developed along the entire
length of the small intestine. Most nerve fibers
present in PMP seem to be derived from AP,
and connections to PS are sparse. Ganglion
cells are occasionally seen, but are very widely
scattered, which explains why they up to now
have escaped both light and electron microscopic characterization.
The arrangement of nerves in the circular
muscle layer in two well-defined planes (PMS
and PMP respectively) should be considered in
studies of the nervous regulation of the muscle
cells of the circular layer. The unequal distribution of fasciculi suggests that PMS and PMP
maintain different functions. Our investigations indicate that PMS and other intramuscular fasciculi are engaged with innervation of the outermost layers of the circular
muscle (Thuneberg, 1982),whereas PMP preferentially innervates ICC-I11(Rumessen et al.,
1982; Thuneberg, 1982).
I t has been suggested that PMPprimarily innervates the inner subdivision of the circular
muscle layer (Gabella, 1974,1979),but other ultrastructural reports have emphasized the role
of associated interstitial cells (ICC-III),as anatomically intercalated between nerve terminals
and the outer division of the circular muscle
layer. ICC-I11 establish gap junctions to muscle cells of the outer division of the circular
layer as well as to each other (Duchon et al.,
1974; Rumessen et al., 1982; Thuneberg, 1982).
Their close relations to vesicle-containing
nerve terminals are well documented (Taxi,
1965;Ono, 1967; Duchon et al., 1974; Yamamoto, 1977; Rumessen et al., 1982; Thuneberg,
1982), and we have found synapselike formations (Rumessen et al., 1982;Thuneberg, 1982).
The present study shows that the relation of
nerves to ICC-I11 is not a passive or random
phenomenon, but that ICC-I11 very often are
selectively contacted by axons of PMP and,
furthermore, that this arrangement is a general
feature throughout the small intestine. A concept of ICC-I11 as primary regulatory cells is
further developed and strongly supported by
our accompanying ultrastructural study
(Rumessen et al., 1982).
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