Histochem Cell Biol (2014) 141:393–405
DOI 10.1007/s00418-013-1163-0
Original Paper
What neurons hide behind calretinin immunoreactivity
in the human gut?
Nicholas Beuscher · Samir Jabari · Johanna Strehl ·
Winfried Neuhuber · Axel Brehmer 
Accepted: 22 October 2013 / Published online: 8 November 2013
© Springer-Verlag Berlin Heidelberg 2013
Abstract  Calretinin (CALR) is often used as an immu-
nohistochemical marker for the histopathological diag-
nosis of human intestinal neuropathies. However, little is
known about its distribution pattern with respect to specific
human enteric neuron types. Prior studies revealed CALR
in both myenteric and submucosal neurons, most of which
colabel with choline acetyl transferase (ChAT). Here, we
specified the chemical code of CALR-positive neurons in
small and large intestinal wholemounts in a series of 28
patients. Besides other markers, we evaluated the labeling
pattern of CALR in combination with vasoactive intesti-
nal peptide (VIP). In colonic submucosa, CALR and VIP
were almost completely colocalized in about three-quarters
of all submucosal neurons. In the small intestinal submu-
cosa, both the colocalization rate of CALR and VIP as
well as the proportion of these neurons were lower (about
one-third). In the myenteric plexus of both small intestine
and colon, CALR amounted to 11 and 10 %, respectively,
whereas VIP to 5 and 4 % of the whole neuron population,
respectively. Colocalization of both markers was found
in only 2 and 3 % of myenteric neurons, respectively. In
section specimens, nerve fibers coreactive for CALR and
VIP were found in the mucosa but not in the muscle coat.
Summarizing the present and earlier results, CALR was
found in at least one submucosal and two myenteric neu-
ron populations. Submucosal CALR+/VIP+/ChAT± neu-
rons innervate mucosal structures. Furthermore, CALR
N. Beuscher · S. Jabari · W. Neuhuber · A. Brehmer (*) 
Institute of Anatomy I, University of Erlangen-Nuremberg,
Krankenhausstraße 9, 91054 Erlangen, Germany
e-mail: axel.brehmer@fau.de
J. Strehl 
Institute of Pathology, University of Erlangen-Nuremberg,
Krankenhausstraße 8‑10, 91054 Erlangen, Germany
immunoreactivity in the myenteric plexus was observed
in morphological type II (supposed primary afferent) and
spiny type I (supposed inter- or motor-) neurons.
Keywords  Calcium-binding protein · Enteric nervous
system · Neuron type · nNOS · Somatostatin
Introduction
After an early description of the distribution pattern of cal-
retinin (CALR) in human gastrointestinal tissues (Walters
et al. 1993), immunohistochemistry for this calcium-bind-
ing protein has been increasingly applied in pathohistologi-
cal diagnostics for intestinal neuropathies (Barshack et al.
2004; Guinard-Samuel et al. 2009; Holland et al. 2011;
Kapur et al. 2009; Knowles et al. 2009; Morris et al. 2013).
However, in contrast to the guinea pig, the most commonly
used animal model in enteric neuroscience (Costa et al.
1996; Furness 2006), the distribution pattern of CALR in
different human enteric neuron types is scarcely known
(Brehmer 2006).
Kustermann et al. (2011) distinguished two differ-
ent human submucosal neuron types, one of them being
immunoreactive for CALR while the other for somatostatin
(SOM). Subsequently, Beyer et al. (2013) found that most
human CALR-reactive, but also other submucosal neurons
are cholinergic. Like in the guinea pig and other laboratory
animals, cholinergic markers label quite different human
enteric neuron types and are, beyond identification of cho-
linergic neurons, hardly suited for further immunohisto-
chemical discrimination.
Recently, investigating the submucosal and mucosal
layers of chagasic megacolon, Jabari et al. (2012a) found
a wide colocalization of CALR with vasoactive intestinal
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peptide (VIP). This peptide has a variety of actions in the
gastrointestinal tract (Grider 1989; Neunlist et al. 2003;
Toumi et al. 2003; Ekblad and Bauer 2004; Brenneman
2007). The expression of VIP in human submucosal neu-
rons was shown earlier (Domoto et al. 1990; Crowe et al.
1992; Dhatt and Buchan 1994; Accili et al. 1995; Anlauf
et al. 2003). Its mapping with respect to defined human
enteric neuron types has been initiated only in the myen-
teric plexus (Brehmer 2006; Lindig et al. 2009; Schuy et al.
2011).
Our present investigation aimed at answering the ques-
tion whether CALR immunoreactivity in the human gut
labels a specific neuron population or is rather unspecifi-
cally distributed onto various enteric neurons types. There-
fore, we quantified the colocalization pattern of CALR
and VIP in both the two submucosal and the myenteric
plexus of the main small intestinal and colonic regions. As
the majority of human myenteric VIP+ neurons costain
for the neuronal nitric oxide synthase (NOS; Schuy et al.
2011), we included also this marker in our investigation.
To estimate the proportions of neuronal subpopulations, the
human neuronal protein Hu C/D (HU) served as general
neuronal marker (Ganns et al. 2006), whereas peripherin
(PER) was used for morphological analysis (Kustermann
et al. 2011). In order to enable conclusions about axonal
projections, the distribution pattern of immunohistochemi-
cally stained nerve fibers within intestinal layers and gan-
glia was studied.
Materials and methods
Tissue handling
The Ethics Committee of the University of Erlangen-
Nuremberg approved the use of human tissues. We used 28
samples derived from 26 tumor patients (only tissue gained
from the non-tumor infiltrated borders of the resected gut
segments were used) and from 2 body donors of the Insti-
tute of Anatomy (postmortem delay less than 6 h). The
median age of these 28 persons (14 females and 14 males)
was 70 years (range between 53 and 83 years).
Intestinal segments were transported in physiological
saline (pH 7.3) on ice to the laboratory. Upon arrival (in
case of tumor patients up to 6 h after surgical resection),
specimens were rinsed in Krebs solution at room tem-
perature and transferred to Dulbecco’s modified Eagle’s
medium (DME/F12-Ham, Sigma Chemical Company, St.
Louis, MO, USA) containing 10 mg/ml antibiotic–antimy-
cotic (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 h.
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Histochem Cell Biol (2014) 141:393–405
For fixation, samples were divided. The larger pieces
(dedicated for wholemount preparation) 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 h. The smaller pieces (dedicated for sections) were
frozen at −70 °C in methylbutan after cryoprotection with
15 % sucrose in 0.1 M PB.
For the following immunohistochemical incubations,
longitudinal muscle–myenteric plexus, submucosal (each
about 1 × 1.5 cm), and pieces of mucosal wholemounts as
well as cryostat sections parallel to the gut longitudinal axis
were prepared.
Immunohistochemistry
Antibodies used for the following incubations are listed in
Table  1. One set of wholemounts (myenteric plexus and
submucosal) was triple-stained for VIP, CALR, and HU. A
set of sections was double-stained for VIP and CALR. A
second set of wholemounts (submucosal and mucosal) was
quadruple stained for SOM, NOS, VIP, and HU. A third
set of wholemounts (submucosal) was dedicated for mor-
phological analysis and quadruple stained for VIP, PER,
CALR, and SOM.
Incubations included the following steps: preincubation
of wholemounts for 2 h (sections 1 h) 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, Tri-
ton X-100, thimerosal (see above), and the primary anti-
bodies for 72 h (4 °C; sections overnight). After an over-
night rinse in TBS at 4 °C, specimens were incubated with
secondary antibodies in the same solution as for the pri-
mary antibodies (4 h; room temperature; sections 1 h) fol-
lowed by a rinse in TBS (overnight; 4 °C).
In all specimens, we applied a lipofuscin reduction pro-
tocol: incubation in ammonium acetate buffer (pH 5.0) con-
taining 1 mM CuSO4 for 120 min followed by a short rinse
in distilled water (Schnell et al. 1999; Brehmer et al. 2004a).
This protocol was more effective in myenteric than in other
wholemounts. As mentioned earlier, only material that did
not display neuronal autofluorescence different from lipo-
fuscin pattern and present without any kind of staining was
included in this study (Kustermann et al. 2011).
Thereafter, specimens were mounted with TBS–glycerol
(1:1; pH 8.6). Submucosal wholemounts were first mounted
with their mucosal side up. After evaluation of the internal
submucosal plexus (ISP), wholemounts were reversed and
again mounted with the outer side up for the analysis of the
external submucosal plexus (ESP). Mucosal wholemounts
were mounted with their abluminal side up, for the analysis
of the basal part of the mucosal plexus.
Histochem Cell Biol (2014) 141:393–405 395

Table 1  Antisera Antigen Host Dilution Source

Primary antisera
Calretinin (CALR) Rabbit 1:500 7699; Swant, Switzerland
Human neuronal protein HuC/D (HU) Mouse 1:50 A21271; Mobitec, Germany
Neuronal nitric oxide synthase (NOS) Rabbit 1:400 Mayer et al. (1990, 1991)
Peripherin (PER) Goat 1:200 sc-7604; Santa Cruz, Germany
Somatostatin (SOM) Rat 1:200 sc-47706; Santa Cruz, Germany
Vasoactive intestinal peptide (VIP) Guinea pig 1:500 T-5030; Bachem, CA/USA
Secondary antisera Donkey-
Alexa Fluor 488 -anti-goat 1:1,000 A11055; Mobitec, Germany
Alexa Fluor 488 -anti-rabbit 1:1,000 A21206; Mobitec, Germany
Alexa Fluor 647 -anti-rabbit 1:1,000 A31573; Mobitec, Germany
Cy 3 -anti-guinea pig 1:1,000 706-165-148; Dianova, Germany
Cy 3 -anti-rat 1:500 712-165-153; Dianova, Germany
DyLight 405 -anti-mouse 1:100 715-475-150; Dianova, Germany
DyLight 405 -anti-rat 1:100 712-475-153; Dianova, Germany
DyLight 649 -anti-guinea pig 1:500 706-495-148; Dianova, Germany

Controls

With the exception of the VIP antibody (see below), negative

controls for antibodies used here (omission of primary anti-
bodies from incubation protocol as described above) were car-
ried out earlier (Brehmer et al. 2005; Kustermann et al. 2011).
Preabsorption tests for antibodies against CALR, PER, and
SOM were described previously (Kustermann et al. 2011).
In this study, we tested the specificity of the VIP anti-
body (antigen: H-3775; Bachem, CA/USA). Preabsorptions
with 20- and 50-fold excess of VIP antigen (Fig. 1) were
performed overnight at 4 °C. The antigen–antibody mix-
tures were spun at 20,000g for 20 min to sediment precipi-
tating antigen–antibody complexes and avoid high back-
ground staining. The supernatants were then used in place
of the primary antibodies.

Image acquisition, quantification

Wholemounts were evaluated using a confocal laser-scan-

ning microscope system (Nikon Eclipse E1000-M; Nikon
Digital Eclipse C1; Tokyo, Japan) equipped with a quadru-
ple laser configuration: 405-nm diode laser, 488-nm argon
laser, 543-nm helium–neon laser, 647-nm diode laser (from
Coherent, Santa Clara, CA/USA and Melles Griot Inc.,
Carlsbad, CA/USA). The four laser lines were assigned
to varying colors in the figures. To allow comparisons
with neuron populations represented in two earlier papers
(Kustermann et al. 2011; Beyer et al. 2013), SOM reactivi-
ties were coded red and VIP blue, respectively.
For the reduction of unspecific background fluores-
cence, a BIO1-Filterset (DAPI/Cy5 for C1-Detector; AHF
Analysentechnik, Tübingen, Germany) was added. Two dry
Fig. 1  Preabsorption control for the antibody against vasoactive
intestinal peptide (VIP blue), counterstained for (PER green) and
depicted as single optical sections through submucosal ganglia (left
side merged images; right side VIP images, respectively). Whereas
incubations using the supernatants after preabsorption with 20-fold
antigen excess resulted in weak neuronal labeling for VIP (a′ filled
asterisk), incubation after 50-fold antigen excess resulted in no neu-
ronal staining (b′ empty asterisk)
objective lenses (20× and 40×, numerical apertures 0.75 and
0.95, respectively) were used. Z-series dedicated to quantita-
tive analysis used a zoom factor of 2.0 in all sessions, and
z-steps were 2 μm. The figures were prepared using Volocity
Demo 6.1.1, Adobe Photoshop CS4 and CorelDRAW X6.

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In myenteric and submucosal wholemounts, each 15
ganglia or single neurons lying outside of ganglia in inter-
ganglionic nerve strands was selected randomly in a mean-
der-like fashion, first from the inner, mucosal side of the
wholemount preparation (for evaluation of the ISP), there-
after from the outer side of the wholemount (for the ESP).
All counts were carried out on z-series of the ganglia, using
the Nikon FreeViewer software (EZ-C1 3.30) and Voloc-
ity Demo 6.1.1. In mucosal wholemounts, each 10 z-series
of consecutive viewfields (meander-like selection) of the
mucosal plexus was recorded. These 10 viewfields cor-
responded to an area of about 1 mm2. Only neurons lying
within the honeycomb-like meshes (see below) of the
mucosal plexus were considered (Kramer et al. 2011).
We tried to carefully discriminate neurons lying at the
same x–y- but at different z-positions to avoid double
counting of neurons.
Results
Colocalization of CALR and VIP
Examples of ganglia are depicted in Fig. 2. Results of
counts per small and large intestinal regions are listed in
Table 2 and are summarized in Fig. 3.
Neuron counts in submucosal wholemounts. In the small
intestine, most submucosal neurons reactive for CALR
or VIP were coreactive for the other marker, respectively.
These CALR/VIP-coreactive neurons amounted to 31 %
of all HU-stained neurons in both submucosal plexus.
Between 1 % (ISP) and 7 % (ESP) of neurons were reac-
tive only for CALR, whereas 16 % (ISP) and 14 % (ESP)
were reactive for VIP only (Fig. 2a). In the large intestine,
almost complete colocalization of CALR and VIP was
found (Fig. 2b). Only between 1 and 3 % of all submucosal
neurons immunoreactive for either CALR or VIP were not
coreactive for the other marker, respectively. In addition,
CALR/VIP-coreactive neurons were the largest colonic
submucosal population (79 and 78 % of HU-stained neu-
rons, respectively). Both CALR and VIP immunoreactivi-
ties were restricted to the somata of submucosal neurons,
and morphological evaluation of the process architecture
was not possible.
Neuron counts in myenteric plexus wholemounts. In con-
trast to the submucosal plexus, neurons reactive for CALR
or VIP or both represented a minority of all HU-stained
myenteric neurons (14 % in the small and 11 % in the large
intestine). As a further difference to the submucosal plexus,
CALR/VIP-coreactive neurons represented only a small
proportion of HU-stained, myenteric neurons, 2 % (small
intestine) and 3 % (large intestine), respectively. Gener-
ally, most neurons were visible exclusively with their cell
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Histochem Cell Biol (2014) 141:393–405
bodies. However, some larger myenteric CALR neurons
displayed processes that were distinctly stained. Of these
neurons, some had smooth somal surfaces and one single
or two long processes; these neurons were never VIP-core-
active (Fig. 2c). Others were obviously dendritic with one
additional long process leaving the ganglion of origin (pre-
sumably multidendritic, uniaxonal morphology). Of the lat-
ter neurons, some were coreactive for VIP (Fig. 2d).
Nerve fiber analysis in sections. In the mucosal layers of
both small and large intestinal regions, extensive colocali-
zation of CALR and VIP was found (Fig. 4a). In contrast,
intramuscular nerve fibers were reactive only for VIP, not
for CALR (Fig. 4b).
Neuron counts in submucosal wholemounts stained
for VIP, SOM, HU, and NOS
A representative ganglion of this staining combination is
depicted in Fig. 5. Results of counts along small and large
intestinal regions are listed in Table 3 and are summarized
in Fig. 6.
We found three large populations present throughout.
VIP-positive neurons amounted between 32 % (ISP) and
39 % (ESP) in the small and between 72 % (ISP) and
74 % (ESP) in the large intestine. SOM-positive neurons
accounted for 36 % (ISP) and 24 % (ESP) in the small
and for 14 % (ISP) and 10 % (ESP) in the large intes-
tine. Neurons positive only for HU ranged between 11 %
(colonic ESP) and 34 % (small intestinal ESP). Although
frequently few NOS neurons were found in most speci-
mens with the exception of two jejunal and one ascending
colonic specimen. Additionally, in four specimens (one
duodenum, one ileum, one ascending, and one transverse
colon), few NOS neurons were found only in the ESP but
not in the ISP.
Besides, colocalizations of two of the three non-general
markers were observed only occasionally. SOM/VIP-core-
active neurons were observed in up to 3 % in jejunal seg-
ments, NOS/VIP neurons in up to 1 % in some colonic seg-
ments (Table 3). SOM/NOS-coreactive neurons were found
not at all.
Observations in wholemounts stained for PER, VIP,
and SOM
If observable (Kustermann et al. 2011), VIP neurons were
generally dendritic (Fig. 7), whereas SOM neurons dis-
played one single long process (not depicted). VIP- and
SOM-reactive varicosities, respectively, were abundant
within the ganglia. Both were found in close vicinity of
VIP-reactive perikarya and dendrites. Besides, some SOM-
reactive perikarya were surrounded by SOM-reactive bou-
tons (not depicted).
Histochem Cell Biol (2014) 141:393–405 397
Fig. 2  Different degrees of neuronal colocalization of vasoactive
intestinal peptide (VIP blue) and calretinin (CALR yellow) in sub-
mucosal (a, b) and myenteric (c, d) specimens counterstained with
the human neuronal protein Hu C/D (HU green). a Small intestinal
submucosal plexus: some neurons costain for VIP and CALR, their
colors turn from blue or yellow, respectively, into different shades
of blue-gray/white (merged picture). Other neurons stain only for
VIP (one example: arrow) or CALR (one example: arrowhead), they
remain blue or yellow in the merged picture. HU-only-positive neu-
rons (negative for both VIP and CALR) remain green in the merged
picture (asterisk). b Colonic submucosal plexus: most neurons cos-
tain for VIP and CALR (color in the merged picture: blue-gray to
white), one neuron (asterisk) is negative for both VIP and CALR
(asterisk). c Myenteric plexus: most HU-positive neurons are nega-
tive for both VIP and CALR. One CALR-positive neuron (arrow) has
smooth outlines and a single, branched process. d Myenteric plexus:
one VIP/CALR-coreactive neuron (arrow) displays dendritic outlines
and a single long process. (All-in-focus-projections; sample data: a
external submucosal plexus (ESP) of duodenum, 70 years, female; b
ESP of ascending colon, 75 years, male; c myenteric plexus of ileum,
71 years, male; d myenteric plexus of ascending colon, 77 years,
male.)
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Table 2  Proportions of neurons Segment CALR only* CALR VIP* VIP only* HU only Neuron
in specimens stained for CALR, n = patients (%) (%) (%) (%) number
VIP, and HU
Duodenum n = 3 ISP 1 17 15 67 699
ESP 2 22 15 61 560
MP 6 1 4 89 1,185
Jejunum n = 3 ISP 1 19 30 50 803
ESP 1 21 26 52 473
MP 10 2 4 83 747
Ileum n = 3 ISP 3 57 2 38 781
ESP 18 50 1 31 496
MP 10 3 1 86 954
Σ small intestine ISP 1 31 16 52 2,283
n = 9 ESP 7 31 14 48 1,529
MP 9 2 3 86 2,886
Colon ascendens ISP 0 82 2 16 745
n = 3 ESP 1 77 4 18 537
MP 7 4 1 88 880
Colon transversum ISP 1 76 5 18 608
n = 3 ESP 3 75 4 18 373
MP 7 2 1 90 873
Colon descendens ISP 2 77 1 20 817
n = 3 ESP 2 79 1 18 637
MP 6 4 1 89 1,165
Colon sigmoideum ISP 0 81 1 18 778
n = 3 ESP 1 80 2 16 500
ISP internal submucosal plexus,
ESP external submucosal MP 7 3 1 89 1,354
plexus, MP myenteric plexus Σ large intestine ISP 1 79 2 18 2,948
* All neurons immunoreactive n = 12 ESP 2 78 3 17 2,047
for CALR and/or VIP were also MP 7 3 1 89 4,272
coreactive for HU

Neuron counts in mucosal wholemounts stained for VIP,
SOM, HU, and NOS
In the mucosal plexus, only few neurons were found.
However, mucosal neurons displaying colocalizations of
the three non-general markers occur disproportionately
high as compared with the submucosa (Table 4): SOM/
VIP-positive neurons (11 % in small intestine; 6 % in the
colon), NOS/VIP neurons (6 %; 5 %), and NOS/VIP/SOM
neurons (12 %; 9 %), which were even not observed in the
submucosa.
Discussion
Here, we characterized the human enteric neuronal sub-
types labeled by CALR. We focused on submucosal neu-
rons, although comparison with myenteric and mucosal
neurons should allow integration into the overall immu-
nohistochemical map of the human enteric nervous
system.
We found almost complete colocalization of CALR and
VIP in a majority of human colonic submucosal neurons. In
small intestinal submucosal neurons, both the proportions
of CALR and/or VIP neurons and the amount of colocali-
zation of both markers were somewhat lower; nonetheless,
the majority of CALR- or VIP-reactive neurons was core-
active also for the other marker, respectively. Furthermore,
VIP, SOM, and NOS immunoreactivities generally labeled
separate submucosal neuron populations, whereas colocal-
izations of these three markers were rare. In Table 5, we
have compared the proportions of the two main neuronal
populations (CALR/VIP vs. SOM) derived from the studies
of Kustermann et al. (2011), Beyer et al. (2013), and our
present results. We have confined this overview to the colon
as CALR and VIP were almost completely colocalized here
(in slight contrast to the small intestine).
Submucosal CALR+/VIP+/ChAT± neurons
Kustermann et al. (2011) showed that, in human colon,
CALR-positive, multidendritic neurons account for the

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Histochem Cell Biol (2014) 141:393–405 399

Fig. 3  Amount of colocaliza-
tion of calretinin (CALR) with
vasoactive intestinal peptide
(VIP) in neurons of the internal
submucosal, the external
submucosal, and the myenteric
plexus. Percentages are related
to the total number of neurons
stained for the human neuronal
protein Hu C/D (HU). All
neurons represented were HU-
reactive including the “CALR
only*” and “VIP only*”
neurons

majority of submucosal neurons. Beyer et al. (2013) found
the great majority of submucosal CALR neurons costain for
the cholinergic marker choline acetyltransferase (ChAT).
Here, we observed almost complete colocalization of CALR
and VIP in colonic submucosal neurons. Thus, the major-
ity (about three-quarters, Table 5) of submucosal neurons
in the human colon are characterized by the chemical code
CALR+/VIP+/ChAT± and a multidendritic morphology.
In the small intestine, submucosal CALR neurons
account for less than 50 % and the colocalization rate of
CALR and VIP is somewhat lower, although the majority
of neurons reactive for either CALR or VIP is coreactive
also for the other marker, respectively. However, it is not yet
clear whether the lower colocalization rate of CALR and
VIP indicates variability or different neuron populations.
Nerve fibers coreactive for both CALR and VIP were
found in the mucosa and submucosal ganglia but not in
the muscular coat. Here, we did not focus the perivascular
nerves that will be dealt with separately. Since colocalization
of both markers is not common in the myenteric plexus (see
below), we conclude that the target of submucosal CALR/
VIP-coreactive neurons is the mucosa. It must be noted that,
by combining retrograde tracing with subsequent immuno-
histochemistry, some VIP-reactive, submucosal neurons
projecting to the circular muscle layer were observed (Por-
ter et al. 1999). Furthermore, with regard to the close apposi-
tions of VIP-reactive nerve boutons onto VIP-reactive sub-
mucosal neurons, an interneuronal role cannot be excluded.
In the mucosa, VIP has been shown to exert stimulatory
effects onto the maintenance of the mucosal barrier (Neun-
list et al. 2003; Toumi et al. 2003; Ben-Horin and Chow-
ers 2008; Neunlist et al. 2013). In addition, VIP may have
also neuroprotective effects (Brenneman 2007; Ekblad and
Bauer 2004). During extensive neuronal destruction in cha-
gasic megacolon, neurons containing VIP were shown to
resist selectively in both submucosal and myenteric plexus
(Jabari et al. 2012a, b).
Submucosal SOM+ neurons
These neurons were shown to be cholinergic through-
out and, by their majority, to contain substance P (Beyer

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400
Fig. 4  Distribution pattern of vasoactive intestinal peptide(VIP)-
and calretinin(CALR)-reactive as well as coreactive nerve fibers in
sections through the gut wall. In mucosal nerve fibers (a), VIP and
CALR coexist widely (in a′ indicated by the merged color white). In
intramuscular nerve fibers (b), VIP without CALR is abundant (in b′,
Fig. 5  Populations of submucosal neurons differentiated by immuno-
histochemical quadruple staining for the human neuronal protein Hu
C/D (HU green), vasoactive intestinal peptide (VIP blue), calretinin
(CALR red), and neuronal nitric oxide synthase (NOS yellow). One
example of each population is marked: vertical arrow a HU-/SOM-
et al. 2013). They express the chemical code SOM+/
ChAT+/SP± and are characterized morphologically by
their unipolar (supposed pseudouniaxonal) appearance
(Kustermann et al. 2011; Beyer et al. 2013). In the small
intestine, they account for about one-third of submucosal
neurons, whereas in the colon, for about one-tenth. In the
submucosa of chagasic megacolon, they were, in contrast
to CALR+ neurons, almost completely missing (Jabari
et al. 2012a). Similarly to submucosal CALR+/VIP+
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Histochem Cell Biol (2014) 141:393–405
the blue color is unchanged despite the merged picture). Asterisk gan-
glion of the inner submucosal plexus; arrowhead nerve strand of the
external submucosal plexus; arrow ganglion of the myenteric plexus.
(Single optical sections; sample data: descending colon, 53 years,
male.)
coreactive neuron; horizontal arrow a HU/NOS-coreactive neuron;
vertical arrowhead a HU/VIP-coreactive neuron; horizontal arrow-
head a HU-only-positive neuron. Further combinations of coreactivi-
ties were observed very infrequently. (All-in-focus-projection; sample
data: external submucosal plexus of ileum, 71 years, male.)
neurons and as discussed earlier (Kustermann et al. 2011;
Beyer et al. 2013), the SOM+/ChAT+/SP± neurons have
their main innervation target(s) in the mucosa as primary
afferent, antisecretory, vasoactive, and/or interneurons.
Submucosal NOS+ neurons
In the human myenteric plexus, neurons costaining for
NOS, VIP, and neurofilaments were morphologically
Histochem Cell Biol (2014) 141:393–405 401

Table 3  Proportions of Segment/n =  VIP only* SOM SOM HU only NOS NOS Neuron
submucosal neurons in patients (%) VIP* (%) only* (%) (%) only* (%) VIP* (%) number
specimens stained for VIP,
SOM, HU, and NOS Duodenum n = 3 ISP 23 0 44 33 0 0 802
ESP 32 0 32 34 2 0 432
Jejunum n = 3 ISP 26 2 43 28 0 0 735
ESP 35 3 26 35 1 0 394
Ileum n = 4 ISP 45 0 26 28 1 0 997
ESP 49 0 15 34 2 0 828
Σ small intestine ISP 32 1 36 30 1 0 2,534
n = 10 SP 39 1 24 34 2 0 1,654
Colon ascendens ISP 73 1 18 8 0 0 878
n = 3 ESP 75 0 13 9 2 1 759
Colon transver- ISP 74 1 17 7 1 0 1,193
sum n = 4 ESP 73 0 17 8 2 0 733
Colon descend- ISP 68 0 11 19 2 0 1,268
ens n = 5 ESP 72 0 6 14 7 1 1,203
ISP internal submucosal plexus, Colon sigmoi- ISP 72 0 12 13 3 1 1,042
ESP external submucosal plexus deum n = 3 ESP 78 0 6 12 4 1 583
* All neurons immunoreactive Σ large intestine ISP 72 0 14 13 1 0 4,381
for VIP, SOM, and/or NOS n = 15 ESP 74 0 10 11 4 1 3,278
were also coreactive for HU

Fig. 6  Proportions of sub-
mucosal neurons stained for
vasoactive intestinal peptide
(VIP), somatostatin (SOM) and
neuronal nitric oxide synthase
(NOS) and their combinations
observed. Percentages are
related to the total number of
neurons counterstained for the
human neuronal protein Hu C/D
(HU). All neurons represented
were HU-reactive including the
VIP-only*, SOM-only*, and
NOS-only* neurons

characterized as spiny type I neurons (Brehmer et al.
2006; Lindig et al. 2009). They amounted up to 7 % of
the whole myenteric neuron population (Schuy et al.
2011). In addition, these authors found a small population
of myenteric VIP+ (NOS-negative) neurons (2 %) and a
substantial population of NOS+ (VIP-negative) neurons
(up to 40 %). In our present material, submucosal NOS+
neurons represented a small population (between 1 and
4 % of HU-stained neurons) and were mostly VIP-nega-
tive. Moreover, they were not found in every submucosal
wholemount. We suggest, in accordance with the interpre-
tation in guinea pig gut (Furness 2006), that these submu-
cosal NOS+ neurons may correspond to displaced myen-
teric neurons and may innervate the external (especially

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402
Fig. 7  Morphology of vasoactive intestinal peptide (VIP)-reactive
submucosal neurons and appositions made of VIP- and somatosta-
tin (SOM)-reactive varicosities, respectively. a A single VIP-positive
neuron and b, c small ganglia consisting of two and three VIP-posi-
tive neurons, respectively, are dendritic as revealed by their peripherin
(PER) counterstaining. The neurons are surrounded by (blue) VIP-
circular) muscular layer (Krammer et al. 1994; Timmer-
mans et al. 1994).
Myenteric CALR+ and VIP+ neurons
In contrast to the submucosal plexus, myenteric neurons
staining for CALR and/or VIP represent a minority of the
HU-stained whole neuron population. Furthermore, only
a small proportion colocalized both CALR and VIP. This
was not surprising since both peptides label two distinct
small intestinal neuron populations. CALR-reactivity was
found in morphological type II, supposed primary afferent
neurons (Brehmer et al. 2004b; Weidmann et al. 2007). In
the colonic myenteric plexus, a similar analysis still has
to be done. VIP was observed in spiny type I, supposed
13
Histochem Cell Biol (2014) 141:393–405
and (red) SOM-positive varicosities (arrowheads). Note that merged,
VIP/SOM-coreactive appositions are virtually absent (a–c all-in-
focus-projections; a′/b′/c′–a′″/b′″/c′″: single optical sections; sam-
ple data: a external submucosal plexus (ESP) of descending colon,
70 years, female; b ESP of transverse colon, 68 years, female; c ESP
of ileum, 70 years, female)
descending inter- or motorneurons (Brehmer et al. 2006;
Lindig et al. 2009; Schuy et al. 2011). In some of our pre-
sent specimens, strong CALR staining in neurons coreac-
tive for VIP enabled the identification of their multiden-
dritic, uniaxonal morphology. These neurons resembled
spiny type I neurons, beneath neurofilaments, NOS, VIP
(see above), and ChAT (Beck et al. 2009), some of the
spiny type I neurons seem to costain also for CALR.
Mucosal neurons
The occasional presence of mucosal neurons has been
discussed recently (Kramer et al. 2011). Both the results
of Beyer et al. (2013) and the present study suggest
that the chemical coding of mucosal neurons does not
Histochem Cell Biol (2014) 141:393–405 403

Table 4  Proportions of mucosal neurons in specimens stained for VIP, SOM, HU, and NOS
Segment n = patients VIP only SOM VIP SOM only HU only NOS only NOS VIP NOS VIP Neuron
(%) (%) (%) (%) (%) (%) SOM (%) number

Duodenum n = 3 12 0 43 9 0 0 36 17
Jejunum n = 3 0 50 50 0 0 0 0 2
Ileum n = 4 62 6 13 8 0 11 0 27
Σ small intestine 35 11 29 7 0 6 12 46
n = 11
Colon ascendens 0 0 0 0 0 0 0 0
n = 3
Colon transversum 10 5 29 5 6 6 39 18
n = 4
Colon descendens 55 0 34 11 0 1 0 51
n = 5
Colon sigmoideum 46 14 23 6 0 11 0 32
n = 3
Σ large intestine 42 6 29 8 1 5 9 101
n = 17

Table 5  Comparison of percentage values of SOM-, CALR-/VIP-reactive, and other neurons in human colonic submucosa
Colon SOM CALR/VIP Other
KUS (%) BEY (%) Pres (%) KUS (%) BEY (%) Pres KUS BEY (%) Pres

ISP 13 <25 14 67 <69 72 20 >6 14

ESP 4 <12 10 77 <82 75 19 >6 15

Values were taken from Kustermann et al. (2011; KUS), Beyer et al. (2013; BEY) and the present (pres) investigation. For the latter, results of
quadruple staining including VIP were considered. Since Beyer et al. (2011) have not used a pan-neuronal marker, their values were marked with
“<” or “>”, respectively

mirror that of the adjacent (inner) submucosal neuron pop-
ulation. Neurons containing ChAT/CALR/substance P or
ChAT/CALR/SOM/substance P (Beyer et al. 2013) as well
as SOM/VIP or NOS/SOM/VIP (present study) were rare
or even absent from the submucosal but surprisingly pre-
sent in the mucosal plexus. Thus, both quantitatively and
immunohistochemically, mucosal neurons seem to be no
useful indicators for evaluating the state of the enteric nerv-
ous system, e.g., in biopsies.
and mostly not colocalized with VIP. Importantly, and
also in contrast to the submucosal neurons, the myenteric
CALR-positive neurons belong to at least two different neu-
ron types, morphological type II, supposed primary afferent,
and spiny type I, supposed motor, and/or interneurons.
Given that CALR is often used as an immunohistochem-
ical marker for the histopathological diagnosis of human
intestinal neuropathies, it is important to consider its dif-
ferential distribution onto at least one submucosal and two
myenteric neuron types.

Conclusions Acknowledgments  The excellent technical assistance of Karin

CALR immunoreactivity in the submucosal plexus cannot
be equated to that observed in the myenteric plexus.
In the submucosal nerve plexus, CALR labels a distinct
neuron population with the chemical code CALR+/VIP+/
ChAT± and a multidendritic appearance. These neurons,
innervating mainly the mucosa, amount for about three-
quarters of all colonic and about one-third of all small
intestinal submucosal neurons.
In contrast, in the myenteric plexus, CALR is expressed
in only about one-tenth of the myenteric neuron population
Löschner, Stefanie Link, Anita Hecht, Andrea Hilpert, and Hedwig
Symowski is gratefully acknowledged. Furthermore, we thank Jochen
Lennerz (Ulm) for fruitful discussion.
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