• chemical coding;
  • enteric nervous system;
  • gut;
  • innervation;
  • neuron type;
  • submucosal plexus


  1. Top of page
  2. Abstract
  6. Acknowledgements

In human myenteric plexus, calretinin (CALR) and somatostatin (SOM) coexist in Dogiel Type II neurons, which were considered as intrinsic primary afferent neurons in the guinea pig. The aims of this study were to test if also human submucosal neurons costain immunohistochemically for CALR and SOM and whether these or other neurons display Type II morphology. Two sets of submucosal wholemounts of small and large intestine from 29 patients (median age 65 years) were triple stained for CALR, SOM, and human neuronal protein Hu C/D (HU, a pan-neuronal marker) as well as for CALR, SOM, and peripherin (PER), respectively. Only exceptionally, neurons coreactive for both CALR and SOM were found. The three major groups of neurons were CALR-/HU-coreactive (CALR-neurons), SOM-/HU-coreactive (SOM-neurons), and HU-alone-positive neurons. We observed significantly more CALR-neurons in the external submucosal plexus (ESP) of all regions and more SOM-neurons in the internal submucosal plexus (ISP), although with substantial interindividual variations. Comparisons of small vs. large intestine revealed more SOM-neurons (ESP: 29% vs. 4%, ISP: 40% vs. 13%) but fewer CALR-neurons (ESP: 37% vs. 77%, ISP: 21% vs. 67%) in small intestine. Morphologically, CALR-neurons had multiple processes; in some cases, we identified multidendritic/uniaxonal neurons. In contrast, SOM-neurons had mostly only one process. The functions of both populations as possible primary afferent neurons, interneurons, secretomotor neurons, or vasomotor neurons are discussed. Future morphochemical distinction of these groups may reveal different subgroups. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

During the last 4 decades, studies of the enteric neuronal circuitry were primarily done in the guinea pig. In both the myenteric and submucosal nerve plexus of this laboratory animal, a number of studies revealed different types of neurons, which could be distinguished, for example, by morphology, neurochemistry, their connections, and, last but not least, by their distribution onto these two ganglionated plexus (Furness,2006). So far, at least four submucosal neuron populations have been identified in the guinea pig, one of them was termed as submucosal intrinsic primary afferent neurons (IPANs). These display Dogiel Type II morphology, that is, they have several long, axonal processes. They account for about one-tenth of the submucosal neuron population and display a quite similar chemical code to their myenteric counterparts in this species; for example, they are cholinergic and immunoreactive, among others, for calbindin and a neuronal nuclei antibody (Evans et al.,1994; Furness et al.,2003; Van Nassauw et al.,2005; Furness,2006). In another view, Dogiel Type II neurons have been considered as interneurons because of synaptic contacts among themselves (Wood,1994). More recently, it has been shown in the guinea pig that also other populations function as sensory, for example, mechanosensory, neurons (Spencer and Smith,2004; Smith et al.,2007; Schemann and Mazzuoli,2010). Thus, a given neuron type may fulfill not only one but several functions.

It was early recognized that interspecies differences hamper direct transfer of findings in animals to human (Gershon et al.,1994). Since about 2 decades, increasing efforts were undertaken to distinguish myenteric neurons also in human intestines and to correlate these findings with results from animal studies (Wattchow et al.,1995,1997; Porter et al.,1996,1997,2002; Pimont et al.,2003; Brehmer,2006; Murphy et al.,2007). These and other articles revealed mainly candidates for human myenteric interneurons and motorneurons, whereas other studies addressed the chemical diversity of human submucosal neurons (Hoyle and Burnstock,1989; Domoto et al.,1990; Crowe et al.,1992; Timmermans et al.,1992; Dhatt and Buchan,1994; Accili et al.,1995; Porter et al.,1999; Hens et al.,2001; Michel et al.,2005).

On the basis of the immunohistochemical demonstration of their cytoskeleton by a neurofilament antibody, we have described pseudouniaxonal or multiaxonal, nondendritic Type II neurons in the myenteric plexus of the human small intestine, suggested to be IPANs (Brehmer et al.,2004b). Triple labeling revealed, besides neurofilament, calretinin (CALR), somatostatin (SOM), and substance P immunoreactivity in most of these neurons. Weidmann et al. (2007) showed that the colocalization of CALR and SOM in human myenteric neurons of the small intestine is highly indicative for Type II neurons, putative IPANs. This chemical code is different from that of the guinea pig and also of other laboratory animals (Brehmer,2007).

The architecture of the human submucosal plexus differs from that of small laboratory animals. It consists of at least two different networks (Schabadasch,1930; Brehmer et al.,2010). The external submucosal plexus (ESP), lying near the outer border of the submucosa, is mostly monolayered and displays a wide-meshed, angular network. The internal submucosal plexus (ISP) is frequently multilayered, occupies about the inner half of the submucosa and displays more narrow, irregular meshes. The ganglia of the ISP frequently resemble grapes, whereas the neurons of ESP ganglia are rather embedded in the contours of joining nerve strands. Interconnecting strands between the two plexus appear regularly coiled and sometimes contain intercalated ganglia. We have discussed diverging views onto the architecture of the human submucosal plexus (Brehmer et al.,2010). Nevertheless, the basis for this study on submucosal neurons is the concept of two submucosal plexus in human.

This work was undertaken to ascertain whether coimmunostaining for CALR and SOM may also be indicative for Type II neurons in human submucosal neurons. To this end, we first evaluated the proportions of CALR/SOM-positive neurons in submucosal wholemounts triple stained for CALR, SOM, and the human neuronal protein Hu C/D (HU), a pan-neuronal marker (Phillips et al.,2004; Ganns et al.,2006). Second, we evaluated shapes of submucosal neurons by coapplication of an antibody against peripherin (PER). We found that PER antibodies, because of their stronger and more reliable staining in our submucosal samples, were more useful for structural demonstration of submucosal neurons and nerve fibers than neurofilament antibodies, which were used in the myenteric plexus (Brehmer,2006; Brehmer et al.,2010).


  1. Top of page
  2. Abstract
  6. Acknowledgements

Tissue Processing

The use of human tissues for these experiments was approved by the Ethics Committee of the University of Erlangen-Nuremberg. In Table 1, donor characteristics were listed (segment, age, and gender; median age 65 years). Most of the samples were derived from patients suffering from tumors. Here, only tissue gained from the nontumor infiltrated borders of the resected gut segments was used. Some of the segments were derived from body donors to the Institute of Anatomy in those rare cases in which the postmortem delay was 6 hr or less.

Table 1. Counts and percentages of neurons in submucosal wholemounts stained for SOM, CALR, and HU
Segment dataESPISP
SOM (%)CALR (%)SOM + CALR (%)HU (%)TotalSOM (%)CALR (%)SOM + CALR (%)HU (%)Total
  1. Percentages were rounded, mostly to whole numbers except the third line (Du 45 m: 0.4%) to indicate a value different from zero. “Segment data” include: intestinal segmenta, age, and gender of patient.

  2. aDu, duodenum; Je, jejunum; Il, ileum; Ca, colon ascendens; Ct, colon transversum; Cd, colon descendens; Cs, colon sigmoideum.

Du 45 m5180771113430.463477
Du 84 m8370551612120158330
Du 86 f042058641924057214
Je 32 m5836062245922019290
Je 42 f36200441604713040286
Je 80 f4626721765113135358
Je 82 m36310331065219029231
Je 82 f59110301974421035365
Je 84 m41320261764819033320
Il 7 m12550332122846026304
Il 35 f4580381133826036325
Il 37 m26500241725013037404
Il 57 m242056187718075346
Il 61 m12760121842364013291
Il 82 m47370161974914038346
Ca 52 f392051181575010442
Ca 61 f1780201711068022286
Ca 66 m7780151371275013401
Ct 21 f2680304442458018393
Ct 68 f787061261959022271
Ct 72 f4860101111551034383
Ct 72 f08701314448808298
Ct 76 f10700202592047033287
Cd 44 f18301689766026193
Cd 45 m275022379178120332
Cd 71 m1680312321264023371
Cs 27 m281017293375021348
Cs 65 f885071071377010181
Cs 68 f5780171552354023345

Intestinal segments were transported in physiological saline (pH 7.3) on ice to the laboratory. Upon arrival (in case of patients tissue probes up to 6 hr after resection), specimens were rinsed in Krebs solution at room temperature and transferred to Dulbecco's modified Eagle's medium (DME/F12-Ham, Sigma Chemical Company, St. Louis, MO) containing 10 mg/mL antibiotic-antimycotic (Sigma), 50 μg/mL gentamycin (Sigma), 2.5 μg/mL amphotericin B (Sigma), 10% fetal bovine serum (Sigma), 4 μM nicardipine, and 2.1 mg/mL NaHCO3, bubbled with 95% O2 and 5% CO2 at 37°C for 1–2 hr.

For fixation, samples were pinned on a Sylgard-lined Petri dish and transferred to 4% formalin in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2–4 hr. Some segments were frozen at −70°C in methylbutan after cryoprotection with 15% saccharose in 0.1 M PB. For the following incubations, three submucosal wholemounts (about 1 cm × 1.5 cm) from each segment were prepared.

Additionally, cryostat sections parallel to the gut longitudinal axis of undissected, frozen wholemounts were triple stained for PER, SOM, and CALR (see below).


Each two (in total 58) of these three wholemounts were triple stained for SOM, CALR, and HU as well as for SOM, CALR, and PER. Incubations included the following steps: preincubation of wholemounts for 2 hr (sections 1 hr) in 0.05 M TBS (pH 7.4) containing 1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.05% thimerosal, and 5% normal donkey serum. After rinsing in TBS for 10 min, the wholemounts/sections were incubated in a solution containing BSA, Triton X-100, thimerosal (see above), and the primary antibodies (Table 2) for 72 hr (4°C; sections overnight). After an overnight rinse in TBS at 4°C, specimens were incubated in an equivalent solution as for the primary antibodies but with secondary antibodies added (Table 2; 4 hr; room temperature; sections 1 hr) followed by a rinse in TBS (overnight; 4°C).

Table 2. Antisera
Primary antisera
 CalretininRabbit1:5007699/3H; Swant (Switzerland)
 Human neuronal protein HuC/HuD (anti-HuC/D)Mouse1:50A21271; Mobitec (Germany)
 Peripherin (C-19)Goat1:200sc-7604; Santa Cruz (Germany)
 Somatostatin (YC7)Rat1:200sc-47706; Santa Cruz (Germany)
Secondary antisera
 ALEXA Fluor 488, donkey anti-rabbit 1:1,000A-21206; Mobitec (Germany)
 ALEXA Fluor 488, donkey anti-goat 1:1,000A-11055; Mobitec (Germany)
 ALEXA Fluor 647, donkey anti-mouse 1:1,000A-31571; Mobitec (Germany)
 ALEXA Fluor 647, donkey anti-rabbit 1:1,000A-31573; Mobitec (Germany)
 Cy™3, donkey anti-rat IgG 1:500712-165-153; Dianova (Germany)

Thereafter, specimens were mounted with TBS-glycerol (1:1; pH 8.6). Wholemounts were first mounted with their external side up. After evaluation of the ESP, wholemounts were reversed and again mounted with the inner, mucosal side up.

The third wholemount was divided into two parts, which were used as negative controls for CALR and SOM, respectively. Negative controls for antibodies against HU and PER were carried out earlier (Ganns et al.,2006; Brehmer et al.,2010); the PER antigen and related preabsorption protocol is given by the provider (Santa Cruz: “Blocking peptide available for competition studies, sc-7604 P [100-μg peptide in 0.5-mL PBS containing <0.1% sodium azide and 0.2% BSA]”). The halves were incubated in solutions lacking alternately CALR or SOM antisera. In a number of submucosal wholemounts, we observed that there was neuronal autofluorescence partly different from lipofuscin pattern, which could not be reduced by the protocol used in earlier studies of the myenteric plexus (Schnell et al.,1999; Brehmer et al.,2004a). Subsequently, on additional wholemount pieces of the corresponding segments, we observed this autofluorescence also without any kind of staining. These segments were excluded from this study, and only those 29 segments that displayed no staining in negative controls were used (Table 1).

Furthermore, using another submucosal wholemount per region, we tested the specificity of the antibodies by preabsorption tests for the antigens SOM (as Santa Cruz was unable to provide us with the antigen for their SOM antibody listed in Table 2, we were forced to acquire the suitable antigen by Mobitec/Germany: Somatostatin14, code number 24277) and CALR (6-His human Calretinin by Swant/Switzerland). Preabsorptions of the two antibodies (Table 2) with twofold and fivefold excess of SOM antigen (Fig. 1A,B) and with fivefold excess of CALR antigen (Fig. 1C; according to supplier's instructions), respectively, were performed overnight at 4°C. The antigen–antibody mixtures were spun at 20,000g for 20 min to sediment precipitating antigen–antibody complexes and avoid high background staining. The supernatants were then used in place of the primary antibodies, respectively.

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Figure 1. Preabsorption controls for antibodies against somatostatin (SOM; A, B) and calretinin (CALR; C), counterstained for peripherin (PER) and depicted as single optical sections through submucosal ganglia (left side: merged images; right side: SOM-red and CALR-blue images, respectively). Incubations using the supernatants after preabsorption with twofold antigen excess resulted in weak neuronal labeling for SOM (A′), incubation after fivefold antigen excess resulted in no staining (B′). Similarly, incubations using the supernatants after preabsorption with fivefold antigen excess resulted in no staining for CALR (C′). Bars = 50 μm.

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Image Acquisition and Quantitative Evaluation

For quantitative evaluation (wholemounts counterstained with HU), each 15 ganglia or single neurons lying outside of ganglia in interganglionic nerve strands were selected randomly in a meander-like fashion, first from the outer surface of the wholemount preparation (ESP), thereafter from the inner, mucosal side of the wholemount (ISP). Ganglia or neurons located within coiled interconnecting strands (between the two plexus) were not considered. Three-line z-series through myenteric ganglia (z-steps 2 μm) were created using a Nikon Eclipse E1000-M microscope (Tokyo, Japan) equipped with a confocal laser scanning system (Nikon Digital Eclipse C1), laser configuration: 488-nm Argon laser, 543-nm Helium-Neon laser [both from Melles Griot, Carlsbad, CA], and 638-nm Diode laser [Coherent, Santa Clara, CA]). A 20× dry objective lens (numerical aperture 0.75) was applied, and the zoom factor was set to 2.0 in scanning sessions dedicated for quantitative evaluation (HU-counterstained specimens). All counts were carried out on these z-series using the FreeViewer software (EZ-C1 3.30) of Nikon. We tried to carefully discriminate neurons lying at the same x-y- but at different z-positions to avoid false-positive recordings of neurons (e.g., one double-stained instead of two single-stained, overlapping neurons).

For morphological evaluation (wholemounts counterstained with PER), a 60× oil immersion objective lens (numerical aperture 1.4) was additionally applied, and the zoom factors varied between 1 and 3.

The figures are all-in-focus projections of z-series and were prepared using Adobe Photoshop CS (8.0.1) and CorelDRAW X4.

Statistical Analysis

After pooling the corresponding single values within each segment, we tested whether CALR-, SOM-, and HU-immunoreactive neurons were evenly distributed between the two submucosal plexus or not. The dependence of the two traits (relative neuron number and location: that is, the significance of the differences in percentages of immunoreactive neurons between the ESP and ISP) was tested using the chi-squared statistic on a 2 × 2 contingency.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Neuronal Staining Pattern

In Fig. 2, examples of ganglia of the ESP and the ISP of the small intestine (Fig. 2A,B) and the large intestine (Fig. 2C,D) are depicted. CALR and SOM generally displayed cytoplasmic staining of neuronal somata and of nerve fibers within interganglionic strands. Perikaryal CALR staining appeared rather homogeneous, that of SOM-positive somata was more granular. In some cases, initial parts of processes were seen in CALR staining, whereas some SOM-positive neurons displayed one longer (axonal?) process. HU labeled strongly the neuronal nuclei, with sparing the nucleoli. Frequently, there was also moderate staining of the cytoplasm. In few cases, there was additional HU labeling of some surrounding varicose nerve fibers.

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Figure 2. No coimmunoreactivity of somatostatin (SOM; middle column) and calretinin (CALR; right column) in neurons of the human submucosal plexus counterstained for human neuronal protein Hu C/D (HU, merged with SOM and CALR; left column). Projected z-series images. A, B: Ganglia of the external and the internal submucosal plexus, respectively (42 years, jejunum, female); C, D: ganglia of the external and internal submucosal plexus, respectively (colon ascendens, 52 years, female). In each row, some neurons were representatively marked: arrows indicate neurons immunoreactive for SOM (filled) but negative for CALR (empty), and arrowheads point at neurons immunoreactive for CALR (filled) but negative for SOM (empty). All SOM- and CALR-positive neurons are coreactive for HU. In C, the marked HU-positive neuron (filled triangle) is negative for both SOM and CALR (empty triangles). Bar = 50 μm for A–D.

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Quantitative Estimations in HU-Counterstained Wholemounts

Table 1 gives the individual counts and percentages. In Fig. 3, percentages per segment are summarized and represented graphically. Three major groups of neurons could be identified, neurons costaining for CALR and HU (CALR-neurons), for SOM and HU (SOM-neurons), as well as neurons reactive only for HU (HU-alone neurons).

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Figure 3. Mean proportions of neurons per segment stained for human neuronal protein Hu C/D (HU; upper percentage values in the columns, white letters), calretinin (CALR; middle percentage values within the columns), and somatostatin (SOM; lower percentage values within the columns) of the external (A) and the internal (B) submucosal plexus (original counts in Table 1). In all segments, there were significantly more SOM-positive neurons in the internal than in the external plexus. With one exception (colon descendens), there were significantly more CALR-positive neurons in the external than in the internal plexus. Proportions of HU-positive neurons differed significantly only in the ileum, the colon sigmoideum, and the whole small intestine. Neurons coimmunoreactive for both CALR and SOM did not account for more than 1% and, consequently, were not visualized as separate portions of columns. Du, duodenum; Je, jejunum; Il, ileum; Ca, colon ascendens; Ct, colon transversum; Cd, colon descendens; Cs, colon sigmoideum; SI, small intestine; LI, large intestine. Numbers below the segments = sums of counts per segment.

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We found substantial individual variations of proportions, for example, the minimal vs. maximal values for SOM-neurons in the ESP were 0% vs. 58% and in the ISP 0% vs. 59%. CALR-neurons in the ESP amounted between 11% and 92% in the ISP between 3% and 88%. HU-alone neurons ranged between 5% and 77% (both minimal and maximal values in the ESP).

Nevertheless, CALR- and SOM-neurons were not evenly distributed onto both plexus. Summarizing the values of each segment, there were significantly more SOM-neurons in the ISP than in the ESP (P < 0.01 in all segments). Two individual regions showed the reversed relation (jejunum, 82, female; colon descendens, 45, male).

Vice versa, we counted more CALR-neurons in the ESP than in the ISP of all segments (P < 0.01 in all segments except the descending colon, where the difference was not significant). Three individual regions displayed the reversed relationship (jejunum, 82 years, female; colon transversum, 7, female; colon descendens, 45, male).

Although the numbers of HU-alone neurons ranged widely (between 14% and 63%, Fig. 3), there were only small proportional differences between ESP and ISP in each segment: only the ileum (P < 0.01), the whole small intestine (P < 0.01), and the sigmoid colon (P < 0.05) revealed significant differences.

From these results, it is obvious that neurons costaining for CALR and SOM were rare, in only 4 of 29 segments (Table 1). With one exception (7% in the ESP of “jejunum 80 female”), these neurons did not account for more than 1% and, consequently, were not visualized as separate portions of columns in Fig. 3.

Morphology of PER-Counterstained Neurons

As it is obvious from Fig. 2, neurons in larger ganglia of both submucosal plexus are tightly packed. The same was seen in triple-stained wholemounts with PER instead of HU. The great majority of submucosal neurons displayed PER immunoreactivity; however, the more neurons were inside a ganglion the less we were able to identify details beyond sizes and shapes of perikarya. This was due to a combination of various factors: small sizes of neurons, thin processes which were—mainly in centers of ganglia—weekly stained or not visible at all. Thus, the following observations refer to neurons lying singly or in small ganglia or at the periphery of larger ganglia.

CALR/PER- and SOM/PER-immunoreactive neurons differed markedly in their morphology as revealed by their PER staining.

SOM/PER-neurons appeared unipolar, that is, they had one prominent, single process emerging from a smoothly contoured cell body which could be followed for some distance, for example, to the next or next but one ganglion (Fig. 4A). In rare cases, we were able to trace the process up to a branching point (Fig. 4B), that is, characteristic of a pseudouniaxonal phenotype. Only exceptionally, we observed two processes emerging from one cell body.

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Figure 4. Morphology of submucosal neurons as demonstrated by their immunostaining for peripherin (PER) and their coimmunoreactivity for somatostatin (SOM) or calretinin (CALR). Projected z-series images. A: The arrow points at a unipolar neuron coimmunoreactive for both PER and SOM (filled) but negative for CALR (empty). The triangles point at a group of three neurons coimmunoreactive for PER and CALR (filled) but negative for SOM (empty). These neurons display a multipolar appearance (colon transversum, 21 years, female, ISP). B: A pseudounipolar neuron coreactive for PER and SOM (filled arrows) but negative for CALR (empty arrowhead). The course of the axon is marked with arrowheads, and its branching point with an asterisk (colon descendens, 81 years, male, ISP). C: A neuron coreactive for PER and CALR (filled triangles) but negative for SOM (empty triangle) displays a multipolar, more precisely a multidendritic, uniaxonal phenotype (colon transversum, 21 years, female, ESP). D: A neuron coreactive for PER and CALR (filled triangles) but negative for SOM (empty triangle) displays a multipolar appearance, a clear distinction between axon and dendrites is not possible (colon transversum, 21 years, female, ISP). All bars = 50 μm.

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CALR/PER-neurons appeared multipolar, that is, several processes emerged from the cell body but only occasionally we were able to differentiate morphologically between one axon and several dendrites (Fig. 4C). In other cases, processes could not be differentiated (Fig. 4D).

Distribution Patterns of SOM- and CALR-Reactive Nerve Fibers Within Layers of the Gut Wall

In Fig. 5, a section through a small intestinal segment is depicted. Both SOM- and CALR-nerve fibers were found to be largely absent from the external muscle layers (Fig. 5A) but abundant, though frequently not colocalized, in mucosal layers (Fig. 5B). This is in contrast to the distribution pattern of PER that was abundantly found within muscle layers and in the basal parts of the mucosa but only scarce or even lacking in the more superficial part of the mucosa. Additionally, SOM-positive nerve fibers were located around submucosal blood vessels (not shown).

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Figure 5. Section through the small intestinal wall labeled for peripherin (PER, left side: merged image), somatostatin (SOM), and calretinin (CALR). In AA″, the external muscle layers with a myenteric ganglion (asterisk in A) lying between them are represented, whereas the muscle layers (mainly the circular, below) contain numerous PER-positive nerve fiber profiles (A: green); in A′ and A″, SOM- and CALR-nerve profiles are only sporadically present, respectively. In BB″, mucosal layers are represented. The muscularis mucosae is marked by the triangle (B), and villi are directed downward. Both SOM- and CALR-positive nerve fibers are abundant within the villi. Frequently, they are not colocalized (B′, B″: filled vs. empty arrows mark SOM- and CALR-fibers not reactive for the other marker, respectively) but, also costaining for SOM and CALR was observed (arrowheads). Bar = 100 μm.

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Close Appositions of SOM-Positive Nerve Fibers Around SOM/PER-Positive Neurons

Some of the SOM/PER-coreactive perikarya were observed to be closely surrounded by SOM-reactive nerve fibers (Fig. 6). However, this was not found in all cases (Fig. 4A,B). Such close appositions were not observed for CAR-positive fibers.

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Figure 6. Close appositions of somatostatin (SOM)-positive nerve fibers on SOM/peripherin(PER)-coreactive perikarya (three examples were marked with arrows in AC). In B, one of the few neurons coreactive for both calretinin (CALR; blue) and SOM (red) is marked by a triangle (the pink neuron in B″). Bars = 50 μm.

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  1. Top of page
  2. Abstract
  6. Acknowledgements

Our previous studies in the human myenteric plexus revealed that the coexistence of CALR and SOM in the small intestine labels morphological Type II neurons, that is, putative IPANs (Brehmer et al.,2004b; Weidmann et al.,2007). However, this study failed to demonstrate this coexistence in human submucosal Type II neurons. Unexpectedly, we found that CALR and SOM labeled two separate neuron populations, which could also be distinguished by their PER-stained morphology.

A general structural dichotomy of submucosal neurons into multipolar and unipolar (occasionally bipolar) neurons was already depicted and/or described by early authors in different species, for example, in dog (Koelliker,1896), guinea pig (Ramón y Cajal,1911), pig (Rossi,1929), cattle (Sokolova,1931), and in human (Stöhr,1949). Although Dogiel (1899), the founder of the classification of enteric neurons, reported that he investigated also submucosal ganglia, his depictions and descriptions refer mainly to myenteric neurons (Brehmer,2006).

In the pig small intestine, Stach (1977,1980,1981,1982,1989) not only described a number of morphological neuron types beyond the three types of Dogiel but also introduced a more exact nomenclature for describing the structure of neuronal processes. For example, neurons with several processes (“multipolar”) were categorized into uniaxonal, multidendritic neuron Types I and III–VII, nondendritic, multiaxonal Type II, and multidendritic, multiaxonal Type II neurons. Thereafter, in the guinea pig, the axonal character of the multiple processes of Type II neurons has been corroborated also electrophysiologically (Hendriks et al.,1990). As a special variant of the nondendritic, multiaxonal Type II neurons, there exist pseudouniaxonal Type II neurons displaying a single primary axon, which divides, sometimes multiply, into secondary and tertiary axonal branches running in different directions (Stach,1977,1981). Although mostly written in German, it is worthwhile and rewarding to study the illustrations of Stach's publications, even today.

Quantitative Observations

In this study, we have used HU as a pan-neuronal marker. This antibody labels neuronal nuclei (without nucleoli) and the surrounding cytoplasm but, usually, not processes. However, in some specimens of this and earlier studies, we also found Hu-labeled varicose nerve fibers. The possible upregulation of HU antigen under certain circumstances (age, diseases, and medications of patients) and its subsequent transport into axons was discussed earlier (Ganns et al.,2006).

Although we observed substantial variations in numbers and proportions (more pronounced in the small intestine), the general distribution pattern of CALR- and SOM-neurons onto the two submucosal plexus was consistent throughout all regions investigated, that is, there were more SOM-neurons in the ISP and more CALR-neurons in the ESP within each region. Only in the descending colon, this difference was not significant. This distribution pattern points to a functional diversity between the two plexus. Furthermore, marked quantitative differences between small intestinal and colonic regions were obvious. Retrospectively, because of the large proportion of CALR-nerve elements especially in colonic submucosa (77% in ESP and 67% in ISP), CALR immunostaining seems eligible as a quite “general” neuronal marker in pathohistology of submucosal and mucosal layers (Barshack et al.,2004; Guinard-Samuel et al.,2009; Kapur et al.,2009; Knowles et al.,2009). However, the proportion of myenteric CALR-positive neurons in corresponding large intestinal segments is not yet determined. In the small intestine, it ranged between 7% and 20% (Weidmann et al.,2007). From this rather low proportion in the myenteric plexus and the wide absence of CALR fibers from the external muscle layers (Fig. 5) it may be concluded that PER may be a good marker for the external muscle and CALR for the mucosal layers.

Remarkably, proportions of neurons negative for both CALR and SOM, that is, HU-alone neurons in this study, did not differ between ESP and ISP. In other words, although the proportions of CALR- and SOM-neurons differed between ESP and ISP, their summed proportion did (largely) not differ. The significance of this observation is presently unclear.

As to the substantial quantitative variability observed here and earlier (Beck et al.,2009), the fact that human samples of gut available for basic research are generally much more heterogeneous than those from laboratory animals has to be taken into account. This is due to, for example, the different ages of patients and body donors, their different nutritional habits, life and disease histories, medications, etc.

CALR-/PER-Coimmunoreactive Neurons

Most of these neurons were coreactive for PER. Unfortunately, because of the tight packing of cells mainly in central parts of larger ganglia, the architecture of their processes was difficult to analyze. Wherever possible to observe, CALR immunoreactivity coincided with multiple processes of neurons. At present, we cannot decide whether CALR-/PER-neurons represent a homogeneous population with common functions. Only in some cases, we were able to differentiate their processes into axon and dendrites, that is, these neurons were uniaxonal and multidendritic. Others may be multiaxonal, either dendritic or nondendritic (Stach,1989; Stach et al.,2000; Brehmer,2006). Future studies will address the question whether there are subpopulations of CALR-reactive neurons that might fulfill different roles. Based on their distribution pattern within our sections, they may act as motor neurons to the muscularis mucosae, secretomotor neurons, or short projecting interneurons (Domoto et al.,1990; Porter et al.,1999).

Although CALR immunostaining is often used in pathohistological diagnostics as potent marker for enteric nerves (see above), little is known about its distribution onto different human enteric neuron types. As mentioned above, in the myenteric plexus, CALR together with SOM labels putative IPANs (Brehmer et al.,2004b; Weidmann et al.,2007). In both studies, we additionally observed a number of small, dendritic, obviously non-Type II CALR-positive myenteric neurons, which remain to be further characterized, like their submucosal counterparts of this study.

SOM-/PER-Coimmunoreactive Neurons

To these neurons, the same applies as to CALR-/PER-neurons, that is, the architecture of processes of a number of SOM-/PER-neurons could not be analyzed by the reason mentioned above. Wherever visible, SOM-immunoreactive neurons had only one process. We suggest that this process is an axon that may divide regularly in some distance from the soma as observed in some fortunate cases. Thus, some of these neurons resemble nondendritic, pseudouniaxonal Type II neurons and might act as primary afferent neurons. Another suggestion was raised by Hens et al. (2001). These authors, by combined retrograde tracing in mucosa from infants gut and subsequent immunohistochemistry, demonstrated submucosal and myenteric SOM-reactive neurons (without specific morphological analysis) and discussed their putative antisecretory role. De Fontgalland et al. (2008) found SOM-reactive, perivascular nerve fibers in human submucosa and suggested an intrinsic origin. It may be either that these fibers arise from myenteric SOM-positive neurons or that processes of submucosal SOM-neurons run within these networks indicating that at least some SOM-containing neurons are involved in regulation of blood flow. A potential extrinsic source of SOM-positive nerve fibers in the gut may be prevertebral ganglia (Quartu et al.,1993). The close appositions of SOM-reactive nerve fibers to some SOM-reactive perikarya found in our study may point to an interneuronal role of SOM-reactive neurons. As there seem to exist different intrinsic and extrinsic SOM-positive populations, this question has to be addressed by further morphochemically distinguishing studies.

In the guinea-pig submucosal plexus, Type II neurons represent one of four large populations are suggested to be IPANs and amounted to about 11% of all submucosal neurons (Furness et al.,2003; Furness,2006). Among other markers, they display immunoreactivities for calbindin and tachykinins. In the rat, submucosal Type II neurons were identified and stained for CALR, neurofilaments, and substance P but not for calcitonin gene-related peptide (CGRP), whereas myenteric Type II neurons of this species displayed reactivity for CGRP (Mitsui,2009,2010). In the mouse submucosa, three different neuron types were identified but, among them, no putative IPANs (Wong et al.,2008; Mongardi Fantaguzzi et al.,2009) although such neurons could be identified in the mouse myenteric plexus (Furness et al.,2004). In larger laboratory animals, pig and lamb, Type II neurons were present both in the myenteric and the two submucosal plexus and displayed CGRP reactivity throughout (Scheuermann et al.,1987; Timmermans et al.,2001; Chiocchetti et al.,2006; Wolf et al.,2007), whereas in the horse, only submucosal CGRP-reactive neurons occurred (Chiocchetti et al.,2009). This peptide has been found only in a minority of human myenteric Type II neurons (Brehmer,2007). As far as known, Type II neurons throughout different species are all cholinergic. In detail, both the locations and further chemical codes of these putative IPANs differ. These interspecies differences highlight the need of basic morphochemical analyses even in human. These will further characterize and, likely, subdifferentiate these two populations and address their single (Furness,2006) or even multiple (Smith et al.,2007; Schemann and Mazzuoli,2010) function(s).


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  2. Abstract
  6. Acknowledgements

The excellent technical assistance of Karin Löschner, Stefanie Link, Anita Hecht, Andrea Hilpert, Hedwig Symowski, and Inge Zimmermann is gratefully acknowledged.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  • Accili EA, Dhatt N, Buchan AM. 1995. Neural somatostatin, vasoactive intestinal polypeptide and substance P in canine and human jejunum. Neurosci Lett 185: 3740.
  • Barshack I, Fridman E, Goldberg I, Chowers Y, Kopolovic J. 2004. The loss of calretinin expression indicates aganglionosis in Hirschsprung's disease. J Clin Pathol 57: 712716.
  • Beck M, Schlabrakowski A, Schrödl F, Neuhuber W, Brehmer A. 2009. ChAT and NOS in human myenteric neurons: co-existence and co-absence. Cell Tissue Res 338: 3751.
  • Brehmer A. 2006. Structure of enteric neurons. Adv Anat Embryol Cell Biol 186: 194.
  • Brehmer A. 2007. The value of neurofilament-immunohistochemistry for identifying enteric neuron types—special reference to intrinsic primary afferent (sensory) neurons. In: ArlenRK, editor. New research on neurofilament proteins. New York: Nova Science Publishers. p 99114.
  • Brehmer A, Blaser B, Seitz G, Schrödl F, Neuhuber W. 2004a. Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic, neurofilament immunoreactive myenteric neuron types of human small intestine. Histochem Cell Biol 121: 1320.
  • Brehmer A, Croner R, Dimmler A, Papadopoulos T, Schrödl F, Neuhuber W. 2004b. Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine. Auton Neurosci 112: 4959.
  • Brehmer A, Rupprecht H, Neuhuber W. 2010. Two submucosal nerve plexus in human intestines. Histochem Cell Biol 133: 149161.
  • Chiocchetti R, Bombardi C, Mongardi-Fantaguzzi C, Venturelli E, Russo D, Spadari A, Montoneri C, Romagnoli N, Grandis A. 2009. Intrinsic innervation of the horse ileum. Res Vet Sci 87: 177185.
  • Chiocchetti R, Grandis A, Bombardi C, Lucchi ML, Dal Lago DT, Bortolami R, Furness JB. 2006. Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation. Cell Tissue Res 323: 183196.
  • Crowe R, Kamm MA, Burnstock G, Lennard-Jones JE. 1992. Peptide-containing neurons in different regions of the submucous plexus of human sigmoid colon. Gastroenterology 102: 461467.
  • De Fontgalland D, Wattchow DA, Costa M, Brookes SJ. 2008. Immunohistochemical characterization of the innervation of human colonic mesenteric and submucosal blood vessels. Neurogastroenterol Motil 20: 12121226.
  • Dhatt N, Buchan AM. 1994. Colocalization of neuropeptides with calbindin D28k and NADPH diaphorase in the enteric nerve plexuses of normal human ileum. Gastroenterology 107: 680690.
  • Dogiel AS. 1899. Ueber den Bau der Ganglien in den Geflechten des Darmes und der Gallenblase des Menschen und der Säugethiere. Arch Anat Physiol Anat Abt (Leipzig) 130158.
  • Domoto T, Bishop AE, Oki M, Polak JM. 1990. An in vitro study of the projections of enteric vasoactive intestinal polypeptide-immunoreactive neurons in the human colon. Gastroenterology 98: 819827.
  • Evans RJ, Jiang MM, Surprenant A. 1994. Morphological properties and projections of electrophysiologically characterized neurons in the guinea-pig submucosal plexus. Neuroscience 59: 10931110.
  • Furness JB. 2006. The enteric nervous system. Oxford: Blackwell.
  • Furness JB, Alex G, Clark MJ, Lal VV. 2003. Morphologies and projections of defined classes of neurons in the submucosa of the guinea-pig small intestine. Anat Rec A Discov Mol Cell Evol Biol 272: 475483.
  • Furness JB, Robbins HL, Xiao J, Stebbing MJ, Nurgali K. 2004. Projections and chemistry of Dogiel type II neurons in the mouse colon. Cell Tissue Res 317: 112.
  • Ganns D, Schrödl F, Neuhuber W, Brehmer A. 2006. Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine. Histol Histopathol 21: 4151.
  • Gershon MD, Kirchgessner AL, Wade PR. 1994. Functional anatomy of the enteric nervous system. In: JohnsonLR, editor. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven Press. p 381422.
  • Guinard-Samuel V, Bonnard A, De Lagausie P, Philippe-Chomette P, Alberti C, El Ghoneimi A, Peuchmaur M, Berrebi-Binczak D. 2009. Calretinin immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung disease. Mod Pathol 22: 13791384.
  • Hendriks R, Bornstein JC, Furness JB. 1990. An electrophysiological study of the projections of putative sensory neurons within the myenteric plexus of the guinea-pig ileum. Neurosci Lett 110: 286290.
  • Hens J, Vanderwinden JM, De Laet MH, Scheuermann DW, Timmermans JP. 2001. Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine. J Neurochem 76: 464471.
  • Hoyle CH, Burnstock G. 1989. Galanin-like immunoreactivity in enteric neurons of the human colon. J Anat 166: 2333.
  • Kapur RP, Reed RC, Finn LS, Patterson K, Johanson J, Rutledge JC. 2009. Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung Disease. Pediatr Dev Pathol 12: 615.
  • Knowles CH, De Giorgio R, Kapur RP, Bruder E, Farrugia G, Geboes K, Gershon MD, Hutson J, Lindberg G, Martin JE, Meier-Ruge WA, Milla PJ, Smith VV, Vandervinden JM, Veress B, Wedel T. 2009. Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group. Acta Neuropathol 118: 271301.
  • Koelliker A. 1896. Handbuch der Gewebelehre des Menschen. II. Bd.: Nervensystem des Menschen und der Thiere. Leipzig: Engelmann.
  • Michel K, Zeller F, Langer R, Nekarda H, Kruger D, Dover TJ, Brady CA, Barnes NM, Schemann M. 2005. Serotonin excites neurons in the human submucous plexus via 5-HT3 receptors. Gastroenterology 128: 13171326.
  • Mitsui R. 2009. Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon. Cell Tissue Res 337: 3743.
  • Mitsui R. 2010. Immunohistochemical characteristics of submucosal Dogiel type II neurons in rat colon. Cell Tissue Res 340: 257265.
  • Mongardi Fantaguzzi C, Thacker M, Chiocchetti R, Furness JB. 2009. Identification of neuron types in the submucosal ganglia of the mouse ileum. Cell Tissue Res 336: 179189.
  • Murphy EM, Defontgalland D, Costa M, Brookes SJ, Wattchow DA. 2007. Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase. Neurogastroenterol Motil 19: 126134.
  • Phillips RJ, Hargrave SL, Rhodes BS, Zopf DA, Powley TL. 2004. Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers. J Neurosci Methods 133: 99107.
  • Pimont S, Bruley Des Varannes S, Le Neel JC, Aubert P, Galmiche JP, Neunlist M. 2003. Neurochemical coding of myenteric neurones in the human gastric fundus. Neurogastroenterol Motil 15: 655662.
  • Porter AJ, Wattchow DA, Brookes SJ, Costa M. 1997. The neurochemical coding and projections of circular muscle motor neurons in the human colon. Gastroenterology 113: 19161923.
  • Porter AJ, Wattchow DA, Brookes SJ, Costa M. 1999. Projections of nitric oxide synthase and vasoactive intestinal polypeptide-reactive submucosal neurons in the human colon. J Gastroenterol Hepatol 14: 11801187.
  • Porter AJ, Wattchow DA, Brookes SJ, Costa M. 2002. Cholinergic and nitrergic interneurones in the myenteric plexus of the human colon. Gut 51: 7075.
  • Porter AJ, Wattchow DA, Brookes SJ, Schemann M, Costa M. 1996. Choline acetyltransferase immunoreactivity in the human small and large intestine. Gastroenterology 111: 401408.
  • Quartu M, Polak JM, Del Fiacco M. 1993. Neuropeptides in the human celiac/superior mesenteric ganglionic complex: an immunohistochemical study. J Chem Neuroanat 6: 7999.
  • Ramón y Cajal S. 1911. Histologie du système nerveux de l'homme et des vertébrés. Paris: Maloine.
  • Rossi O. 1929. Contributo alla conoscenza degli apparati nervosi intramurali dell' intestino tenue. Arch Ital Anat 26: 632644.
  • Schabadasch A. 1930. Intramurale Nervengeflechte des Darmrohrs. Z Zellforsch Mikrosk Anat 10: 320385.
  • Schemann M, Mazzuoli G. 2010. Multifunctional mechanosensitive neurons in the enteric nervous system. Auton Neurosci 153: 2125.
  • Scheuermann DW, Stach W, De Groodt-Lasseel MH, Timmermans JP. 1987. Calcitonin gene-related peptide in morphologically well-defined type II neurons of the enteric nervous system in the porcine small intestine. Acta Anat 129: 325328.
  • Schnell SA, Staines WA, Wessendorf MW. 1999. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47: 719730.
  • Smith TK, Spencer NJ, Hennig GW, Dickson EJ. 2007. Recent advances in enteric neurobiology: mechanosensitive interneurons. Neurogastroenterol Motil 19: 869878.
  • Sokolova ML. 1931. Zur Lehre von der Cytoarchitektonik des peripherischen autonomen Nervensystems. II. Die Architektur der intramuralen Ganglien des Verdauungstrakts des Rindes. Z Mikrosk Anat Forsch 23: 552570.
  • Spencer NJ, Smith TK. 2004. Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon. J Physiol 558.2: 577596.
  • Stach W. 1977. Der Plexus submucosus externus (Schabadasch) im Dünndarm des Schweins. I. Form, Struktur und Verbindungen der Ganglien und Nervenzellen. Z Mikrosk Anat Forsch 91: 737755.
  • Stach W. 1980. Zur neuronalen Organisation des Plexus myentericus (Auerbach) im Schweinedünndarm. I. Typ I-Neurone. Z Mikrosk Anat Forsch 94: 833849.
  • Stach W. 1981. Zur neuronalen Organisation des Plexus myentericus (Auerbach) im Schweinedünndarm. II. Typ II-Neurone. Z Mikrosk Anat Forsch 95: 161182.
  • Stach W. 1982. Zur neuronalen Organisation des Plexus myentericus (Auerbach) im Schweinedünndarm. III. Typ III-Neurone. Z Mikrosk Anat Forsch 96: 497516.
  • Stach W. 1989. A revised morphological classification of neurons in the enteric nervous system. In: SingerMV, GoebellH, editors. Nerves and the gastrointestinal tract. Lancaster: Kluwer. p 2945.
  • Stach W, Krammer HJ, Brehmer A. 2000. Structural organization of enteric nerve cells in large mammals including man. In: KrammerHJ, SingerMV, editors. Neurogastroenterology from the basics to the clinics. Dordrecht: Kluwer. p 320.
  • Stöhr P. 1949. Mikroskopische Studien zur Innervation des Magen-Darmkanales V. Z Zellforsch Mikrosk Anat 34: 154.
  • Timmermans JP, Hens J, Adriaensen D. 2001. Outer submucous plexus: an intrinsic nerve network involved in both secretory and motility processes in the intestine of large mammals and humans. Anat Rec 262: 7178.
  • Timmermans JP, Scheuermann DW, Barbiers M, Adriaensen D, Stach W, Van Hee R, De Groodt-Lasseel MH. 1992. Calcitonin gene-related peptide-like immunoreactivity in the human small intestine. Acta Anat (Basel) 143: 4853.
  • Van Nassauw L, Wu M, De Jonge F, Adriaensen D, Timmermans JP. 2005. Cytoplasmic, but not nuclear, expression of the neuronal nuclei (NeuN) antibody is an exclusive feature of Dogiel type II neurons in the guinea-pig gastrointestinal tract. Histochem Cell Biol 124: 369377.
  • Wattchow DA, Brookes SJ, Costa M. 1995. The morphology and projections of retrogradely labeled myenteric neurons in the human intestine. Gastroenterology 109: 866875.
  • Wattchow DA, Porter AJ, Brookes SJ, Costa M. 1997. The polarity of neurochemically defined myenteric neurons in the human colon. Gastroenterology 113: 497506.
  • Weidmann S, Schrödl F, Neuhuber W, Brehmer A. 2007. Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine. Histochem Cell Biol 128: 399407.
  • Wolf M, Schrödl F, Neuhuber W, Brehmer A. 2007. Calcitonin gene-related peptide: a marker for putative primary afferent neurons in the pig small intestinal plexus? Anat Rec 290: 12731279.
  • Wong V, Blennerhassett M, Vanner S. 2008. Electrophysiological and morphological properties of submucosal neurons in the mouse distal colon. Neurogastroenterol Motil 20: 725734.
  • Wood JD. 1994. Physiology of the enteric nervous system. In: JohnsonLR, editor. Physiology of the gastrointestinal tract. New York: Paven Press. p 423482.