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. 2006 Dec;209(6):733–743. doi: 10.1111/j.1469-7580.2006.00653.x

Chemical coding of myenteric neurons with different axonal projection patterns in the porcine ileum

Carsten Jungbauer 1, Tobias M Lindig 1, Falk Schrödl 1, Winfried Neuhuber 1, Axel Brehmer 1
PMCID: PMC2049006  PMID: 17118061

Abstract

The aim of this study was to perform an immunohistochemical characterization of two different myenteric neuron types of the pig displaying opposite axonal projections. These were type I neurons equipped with lamellar dendrites that projected mainly orally, and type VI neurons that displayed typical axonal dendrites and projected anally. Double immunostainings of longitudinal muscle/myenteric plexus wholemounts from ileal segments of four pigs were performed to visualize neurofilaments (NF) in combination with calcitonin gene-related peptide (CGRP), leu-enkephalin (ENK) and substance P (SP), respectively. Triple immunostainings of wholemounts, using antibodies against neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) as well as against VIP and galanin (GAL), were performed. We found that 78% of type I neurons immunoreacted to ENK, 21% to CGRP and 24% to SP. The NF-positive type I neurons co-reactive for one of the three above markers displayed mostly frayed outlines of both their somal contours and their broadened dendritic endings. By contrast, most of the non-coreactive type I neurons displayed rather sharply outlined somata and dendrites. No type I neuron immunoreacted to nNOS, VIP or GAL and none of the type VI NF-reactive neurons reacted to CGRP, ENK or SP. All type VI neurons investigated displayed immunoreactivity for nNOS, 92% of which were co-reactive for VIP. Co-reactivity for VIP and GAL was found in 69% of type VI neurons, 21% were positive for VIP but negative for GAL, 9% were negative for both GAL and VIP, and 1% were positive for GAL but negative for VIP. We conclude that there are two subpopulations of morphological type I neurons. One of these displays mainly oral projections and could not be further characterized in this study. The other, which may correspond to neurons innervating the longitudinal and circular muscle layers, were partly immunoreactive for ENK, CGRP and/or SP. Type VI neurons are immunoreactive for nNOS frequently co-localized with VIP and, partly, also GAL. These may be inhibitory motor neurons and are different from VIP/GAL-coreactive minineurons described earlier.

Keywords: chemical coding, enteric nervous system, gut, innervation, neuron type

Introduction

A central feature of the neuronal organization of the enteric nervous system (ENS) is that neurons which form synaptically coupled chains transmit action potentials towards more orally or anally lying regions. These form the basis for ascending and descending reflex pathways within the gut wall, an important prerequisite for peristalsis, for example. The cellular components of these pathways, including their morphological, chemical, physiological and pharmacological aspects, have been most thoroughly studied in the guinea-pig (Costa et al. 1996; Brookes, 2001; Furness, 2006). Although our knowledge about enteric circuits in other species is far more fragmentary, there are a number of experimental results indicating that general principles of enteric neuronal organization are preserved. On the other hand, there are numerous differences in detail between different mammalian species. These differences concerning, for example, the chemical coding of enteric neurons, are known from the rat (Sundler et al. 1993; Mann et al. 1997, 1999), the mouse (Sang et al. 1997; Furness et al. 2004; Nurgali et al. 2004), the pig (Brehmer et al. 1999; Timmermans et al. 2001; Brown & Timmermans, 2004) and humans (Wattchow et al. 1997; Porter et al. 2002; Brehmer et al. 2004a).

In the pig, in contrast to other species, the identification of a number of enteric neuron types (beyond the original basic classification of Dogiel, 1899), was performed morphologically, including observations on their axonal projection patterns, based on silver-impregnated specimens (Stach, 1989). Of the six myenteric neuron types described by Stach, five are typical for the ileum of pigs. Of these six, type II, IV and V neurons were also characterized immunohistochemically; that is, over and above the classification of their principal cholinergic phenotype, there exists at least one known additional marker or marker combination which is typical, if not specific, for each of the three populations (Scheuermann et al. 1987; Hens et al. 2000; Brehmer et al. 2002b). In contrast, chemical coding of type I and VI neurons is at present only known with regard to their immunoreactivity for cholinergic and nitrergic markers (Timmermans et al. 1994; Brehmer & Stach, 1997; Brehmer et al. 1998, 2004b). Type I neurons, displaying mainly short, frequently lamellar-shaped dendrites, are the only known neuron population in the pig with mainly oral axonal projections. They are immunoreactive for cholinergic markers (Brehmer et al. 2004b). Although substance P (SP) has been shown to be present in some type I neurons, this has not been investigated more thoroughly (Scheuermann et al. 1991b,c). Type VI neurons display specific axonal dendrites emerging from the axon hillock and the initial axonal segment. They project mainly anally in the myenteric plexus, are immunoreactive for nitrergic markers (Timmermans et al. 1994; Brehmer & Stach, 1997) and display additional reactivity for the peripheral form of choline acetyltransferase (pChAT; Tooyama & Kimura, 2000; Brehmer et al. 2004b).

In this study, we investigated the presence or absence of immunoreactivity for several markers within the perikarya of type I and type VI neurons. For type I neurons, these were calcitonin gene-related peptide (CGRP), leucine-enkephalin (ENK) and SP because these peptides were found previously in some neurons displaying type I morphology (Scheuermann et al. 1991a,b; Brehmer et al. 2002b). Of these, ENK and tachykinins (one of them being SP) have also been detected in ascending interneurons and excitatory motor neurons of the guinea-pig ENS (Furness, 2006). For type VI neurons, the markers chosen were vasoactive intestinal peptide (VIP) and galanin (GAL) because VIP is partly co-localized with neuronal nitric oxide synthase (nNOS) in the pig (Brown & Timmermans, 2004) as well as in other species (e.g. the guinea-pig; Furness, 2006) and GAL has been shown to be co-localized with VIP in the so-called ‘minineurons’ of the pig (Stach, 1989). Additionally, immunoreactivity for the markers mentioned above in the secondary and tertiary branches of the myenteric plexus was recorded. These components of the myenteric plexus are related to the innervation of the circular and longitudinal muscle, respectively (Furness, 2006).

Materials and methods

Four pigs (both sexes) aged between 12 and 15 weeks were killed in a slaughterhouse. The European Communities Council Directive and animal welfare protocols approved by the local government were followed. Ileal segments about 20 cm in length were taken from the region lying approximately 1 m orally of the ileocecal orifice and transferred to the laboratory within iced Krebs solution. Thereafter, segments were rinsed in Krebs solution at room temperature and transferred into Dulbecco's modified Eagle's medium (DME/F12-Ham, Sigma Chemical Co., St Louis, MO, USA) containing 10 mg mL−1 antibiotic-antimycotic (Sigma), 50 µg mL−1 gentamycin (Sigma), 2.5 µg mL−1 amphotericin B (Sigma), 10% fetal bovine serum (Sigma), 4 µm nicardipine and 2.1 mg mL−1 NaHCO3, bubbled with 95% O2 and 5% CO2 at 38.5 °C for approximately 2 h. Subsequently, they were incubated for 5–6 h in the same medium with 100 µm colchicine added to enhance peptide immunoreactivities within the neuronal somata.

Segments were then immersion fixed under distension in a solution of 4% formalin in 0.1 m phosphate buffer (pH 7.4) at room temperature for 2–3 h. Wholemounts (about 2 cm in length and 1 cm in width) containing the longitudinal muscle with adhering myenteric plexus were prepared.

They were then pre-incubated for 30 min in 0.05 m Tris-buffered saline (TBS; pH 7.4) containing 1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.05% thimerosal and 5% normal donkey serum. After a rinse in TBS for 10 min, they were incubated in a solution containing BSA, Triton X-100, thimerosal (concentrations see above) and the primary antibodies (Table 1) for 48 h (4 °C). After an overnight rinse in TBS at 4 °C, the secondary antibodies (Table 1) were added within the same solution as the primary antibodies (4 h; room temperature) followed by a rinse in TBS (overnight; 4 °C) and wholemounts were mounted with TBS/glycerol (1 : 1, pH 8.6). Incubations of wholemounts in solutions lacking primary antisera served as negative controls.

Table 1.

List of antibodies

Antigen Host Dilution Source
Primary antisera
  Calcitonin gene-related peptide rabbit 1 : 400 T-4032; Bachem
  Galanin rabbit 1 : 1000 ICH 7100; Peninsula
  Leu-enkephalin rabbit 1 : 200 4140-0204; Biotrend
  Neurofilament mouse 1 : 100 0168; Beckman Coulter
  Neuronal nitric oxide synthase rabbit 1 : 400 Dr Mayer; University of Graz
  Substance P rabbit 1 : 200 4107; Bachem
  Vasoactive intestinal peptide guinea-pig 1 : 200 16071; Progen
Secondary antisera
  Cy2, donkey anti-mouse 1 : 200 715-225-150; Dianova
  Cy3, donkey anti-guinea-pig 1 : 800 706-165-148; Dianova
  Cy3, donkey anti-rabbit 1 : 1000 711-165-152; Dianova
  Cy5, donkey anti-rabbit 1 : 100 711-175-152; Dianova
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Examination of type I neurons was performed in 12 wholemounts, four double stained for ENK/neurofilaments (NF), four double stained for CGRP/NF and four double stained for SP/NF (in all cases one wholemount from each pig). In each wholemount, 25 NF-reactive type I neurons were selected randomly, in a meander-like fashion, and their reactivity or non-reactivity for each one of the three above markers was recorded. Investigation of type VI neurons was performed in eight wholemounts, four triple stained for nNOS/VIP/NF and four triple stained for GAL/VIP/NF (in both cases one wholemount from each pig). The further procedure corresponded to that for type I neurons except that co-reactivity or non-coreactivity of NF-stained type VI neurons for two markers (nNOS and VIP or GAL and VIP) was evaluated.

Neuron portraits (see Figs 1–4) were prepared by creating extended-focus images with a confocal laser scanning microscope (Bio-Rad MRC 1000 attached to a Nikon diaphot 300, equipped with a krypton–argon laser, American Laser Corp., Salt Lake City, UT, USA). The filter settings were 568-nm excitation/filter 605 DF322 (for Cy3), 488-nm excitation/filter 522 DF32 (Cy2) and 647-nm/680 DF322 (Cy5). A 40× oil-immersion objective lens (numerical aperture 1.3) was used, with zoom factors varying between 1.5 and 2.0. Figures shown were completed utilizing Confocal Assistant 4.02, Adobe Photoshop CS and CorelDraw 11.

Fig. 1.

Fig. 1

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Co-immunoreactivity for leu-enkephalin (ENK) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were positive for ENK (a′,b′; filled arrowheads) whereas a minority were negative (c′,d′; empty arrowheads). NF-stained neurons in both (a) and (b) displayed frayed outlines of their somata and most of their short dendrites had similarly frayed endings. The NF-stained type I neurons in (c) and (d) display sharply contoured somal and dendritic outlines. Arrowed scale bars (25 µm) point orally.

Fig. 4.

Fig. 4

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Co-immunoreactivity of neurofilament (NF)-stained type VI neurons for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP; a), as well as for galanin (GAL) and VIP (b). In (a), two type VI neurons (filled arrowheads; arrows = axons; axonal dendrites are seen in between both markings) are co-reactive for both nNOS (a′) and VIP (a″; filled arrowheads). In (b), three type VI neurons (filled arrowheads; arrows = axons, one axon is beneath another type VI neuronal soma) are co-reactive for both GAL and VIP (b′,b″; filled arrowheads). A fourth neuron (left side) without distinct axonal dendrites is also co-reactive for both GAL and VIP. Arrowed scale bars (50 µm) point orally.

Documentation of the secondary and tertiary components of the myenteric plexus was carried out using a digital camera system (Spot-RT-realtime, Visitron Systems, Munich, Germany) attached to a Leica Aristoplan microscope (16× dry objective) and SPOT advanced software (Version 3.5.6 for Windows, Diagnostic Instruments, Laredo, TX, USA).

Results

We observed no type I neurons that immunoreacted to GAL or VIP and no type VI neurons that immunoreacted to ENK, CGRP or SP.

Co-immunoreactivity of NF-reactive type I neurons for ENK, CGRP or SP

Neurons identified as type I displayed, in general, short, partly branched dendrites, frequently with broadened, lamellar endings. Two subtypes could be observed. In one, both the soma and the dendrites were strongly stained and showed distinct outlines (e.g. Figs 1c,d, 2a,b and 3a,b), whereas in the other subtype their outlines were not sharply contoured, the dendrites were very short, not branched and displayed frayed endings (Figs 1a, 2d and 3d). However, both types did not always occur separately (e.g. Figs 1b and 3c). Tracking axonal courses over longer distances, i.e. beyond the next or even the next-but-one neighbouring ganglion, was only possible in the case of the sharply outlined type I neurons. Their axons ran mostly orally. A few type I neurons displayed short, lamellar dendrites emerged from the initial axonal segment (e.g. see Fig. 3b). Despite this occasional feature, they were unequivocally type I neurons, based on their dendritic shapes and axonal courses as described earlier by Stach (1980, 1989).

Fig. 2.

Fig. 2

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Co-immunoreactivity for calcitonin gene-related peptide (CGRP) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were negative for CGRP (a′,b′; empty arrowheads), whereas a minority were positive (c′,d′; filled arrowheads). Most type I neurons negative for CGRP had sharply outlined somata and dendrites (a), whereas most CGRP-positive neurons had frayed contours (d). Arrowed scale bars (25 µm) point orally.

Fig. 3.

Fig. 3

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Co-immunoreactivity for substance P (SP) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were negative for SP (a′,b′; empty arrowheads), whereas a minority were positive (c′,d′; filled arrowheads). Most type I neurons negative for SP had sharply outlined somata and dendrites (a), whereas most SP-positive neurons had frayed contours (d). Arrowed scale bars (25 µm) point orally.

Counts of NF-reactive type I neurons are given in Table 2. The majority of type I neurons investigated (78/100) were immunoreactive for ENK. Neurons immunoreactive for CGRP (21/100) and SP (24/100) each represented a minority of the total type I. It was notable that for all three peptides, the immunopositive neurons were mostly of the frayed outlined subtype. This is emphasized in Figs 1–3, where the most contrasting examples are shown in images (a) and (d).

Table 2.

Counts of 4 × 25 NF-reactive type I neurons in four wholemounts derived from different pigs and double stained for NF and the three markers listed

ENK CGRP SP
Positive Negative Positive Negative Positive Negative
pig 1 20  5  6 19  6 19
pig 2 19  6  6 19  4 21
pig 3 21  4  5 20  7 18
pig 4 18  7  4 21  7 18
total 78 22 21 79 24 76
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The accumulation of immunostained boutons on type I neurons did not correlate strongly with somal reactivity. Some neurons immunoreactive for ENK, CGRP or SP were surrounded extensively by boutons reactive for the same marker (Figs 1a, 2c and 3c). In other cases, immunopositive neurons were scarcely apposed by reactive boutons (Figs 2d and 3d). In turn, ENK-, CGRP- or SP-negative neurons were partly surrounded by intensely reactive boutons (Fig. 1c,d) and partly not (Figs 2b and 3b).

Co-immunoreactivity of NF-reactive type VI neurons for VIP and GAL

These neurons had, in addition to somal dendrites, slender, tapering dendrites characteristically emerging from the initial axonal segment (Fig. 4a,b).

Counts are given in Table 3. In our NF/VIP/nNOS specimens, the majority of investigated type VI neurons displayed co-reactivity for VIP and nNOS (92/100). The eight remaining neurons were positive for nNOS but negative for VIP.

Table 3.

Results of counts of 4 × 25 NF-reactive type VI neurons each in four wholemounts, respectively, derived from different pigs and triple stained for NF, VIP and nNOS or for NF, GAL and VIP

VIP+/nNOS+ VIP+/nNOS– VIP–/nNOS+ VIP–/nNOS–
pig 1 22 0 3 0
pig 2 24 0 1 0
pig 3 23 0 2 0
pig 4 23 0 2 0
total 92 0 8 0
VIP+/GAL+ VIP+/GAL– VIP–/GAL+ VIP–/GAL–
pig 1 16  6 0 3
pig 2 16  6 0 3
pig 3 19  5 0 1
pig 4 18  4 1 2
total 69 21 1 9
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Similarly, the majority of type VI neurons were co-immunoreactive for GAL and VIP (69/100). Reactivity for VIP without co-reactivity for GAL was displayed by 21 of 100 neurons. Thus, 90 of 100 neurons in these specimens were reactive for VIP. In nine of 100 type VI neurons, we found neither GAL nor VIP immunoreactivity, whereas one neuron was postive for GAL but negative for VIP. Thus, 70 of 100 type VI neurons were immunoreactive for GAL.

We were unable to observe close proximity between VIP- and GAL-immunoreactive perikarya and accumulations of surrounding VIP- and GAL-immunoreactive boutons.

Immunoreactivity of nerve fibres in the secondary and tertiary components of the myenteric plexus

Secondary strands of the myenteric plexus were found on the inner side of the main plexus consisting of the primary strands, which run parallel to the circular muscle fibres. Numerous fibres of this part of the myenteric plexus were immunoreactive for VIP, GAL, ENK and SP. CGRP-reactive fibres could be found but were less frequent (Fig. 5a–e).

Fig. 5.

Fig. 5

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Immunoreactivity for vasoactive intestinal peptide (VIP), galanin (GAL), leucine-enkephalin (ENK), substance P (SP) and calcitonin gene-related peptide (CGRP) in nerve fibres of the secondary (a–e) and tertiary (a′–e′) components of the myenteric plexus. The longitudinal axis of the wholemount is orientated horizontally (according to the scale bar = 100 µm). Secondary strands are situated on the inner (circular muscle) side of the main plexus (‘Pr’ = primary strands of the myenteric plexus) and run perpendicularly, and the tertiary strands are located on the outer (longitudinal muscle) side and run horizontally. VIP- and GAL-immunoreactive fibres are abundant in both the secondary (a,b; arrows) and the tertiary component (a′,b′; arrows) of the myenteric plexus. By contrast, ENK and SP immunoreactivity were found in fibres of the secondary (c,d; arrows) but only scarcely in the tertiary component (c′,d′; arrows). In (e), CGRP is seen in some fibres of the secondary strands (arrows) but only exceptionally in tertiary fibres (e′, arrow).

Tertiary strands of the myenteric plexus were observed on the outer side of the main plexus running parallel to the longitudinal muscle fibres. Numerous VIP- and GAL-immunoreactive fibres were found (Fig. 5a′,b′). By contrast, tertiary fibres positive for ENK, SP and CGRP were rare (Fig. 5c′–e′).

In general, only a few fibres of the secondary and no fibres of the tertiary component of the myenteric plexus were immunoreactive for NF (not shown).

Discussion

This study has presented further evidence that immunohistochemical specificity of neurons of the pig myenteric plexus mirrors morphological specificity. Neurons displaying different dendritic architectures and axonal projection patterns (Stach, 1989) display different chemical codes. Type I neurons, which project mainly orally, did not express immunoreactivity for VIP or GAL, whereas type VI neurons, with predominant anal projections, did not show immunoreactivity for the markers shown in type I neurons (CGRP, ENK, SP).

Type I neurons

The original definition of enteric type I neurons (Dogiel, 1899) in guinea-pig, human and other mammals has been specified and extended by Stach (1980, 1989) for enteric neurons of the pig. According to Stach's definition, only neurons with lamellar dendrites and (mostly) oral axonal projections were classified as type I neurons.

In this study, we placed all neurons displaying short, broadened dendrites in a common category, namely type I neurons. We suggest that the actual Stach type I neurons in the pig, which were originally characterized in silver-impregnated wholemounts, are equivalent to those NF-reactive neurons that showed strongly stained and distinctly outlined somata and dendrites (Brehmer et al. 2002a, 2004b). Their axons could be followed easily, mostly running in the oral direction as described earlier (Stach, 1980). This is consistent with our results obtained by using silver impregnation (reviewed in Brehmer et al. 1999). NF immunohistochemistry appears to visualize additional neurons displaying the (formal) criteria of type I neurons given by Dogiel (1899), i.e. short dendrites with flat, broadened endings. As described above, we were not able to distinguish clearly between these two subgroups in the present study. However, neurons immunoreactive for CGRP, ENK or SP mostly displayed frayed outlines of both their NF-stained somata and dendritic endings. These neurons resembled, to a degree, the stubby (type I) neurons recently observed in human intestine (Brehmer et al. 2005). As the axons of these porcine neurons were not as strongly stained as those of the former subgroup, they could not be followed over longer distances. Although the co-expression pattern of ENK and SP was not investigated in this study, it is possible that some of the ENK- and SP-immunoreactive neurons described here correspond, on the basis of their shape, to the ascending (ENK+/SP+) and/or descending (ENK+/SP–) longitudinal muscle motor neurons demonstrated by DiI-tracing and immunohistochemistry (Hens et al. 2002). However, we found only few ENK- and SP-reactive fibres in the tertiary component of the myenteric plexus, which is considered to be the gateway of innervation of the longitudinal muscle layer (Furness, 2006). Our results regarding ENK are in line with those of Porcher et al. (2000), who demonstrated numerous reactive nerve fibres in the circular muscle of pig small intestine but not in the longitudinal muscle layer.

The original Stach type I neurons, which were mainly negative for the markers applied here, will need to be further characterized immunohistochemically (Brehmer et al. 2004b). They may be ascending interneurons which have not yet been identified in the pig ENS (Timmermans et al. 2001; Brown & Timmermans, 2004), but this is still highly speculative.

CGRP was one of the first neuroactive substances to be immunohistochemically identified within the pig small intestine in a morphologically defined neuron type, namely the putative primary afferent type II neuron (Scheuermann et al. 1987). CGRP has thus long been considered as a marker peptide for putative primary afferent neurons in the pig ENS (Scheuermann et al. 1991a; Timmermans et al. 1997). However, it is possible that the neurons displaying type I-like morphology already represent the second non-type-II neuron population within the pig ENS, which is immunoreactive for CGRP (the others are the type V neurons; Brehmer et al. 2002a,b). Thus, CGRP can no longer be considered as a specific marker peptide for putative primary afferent neurons in the pig ENS.

Type VI neurons

The coexistence of nNOS and VIP is not uncommon in enteric neurons (Furness, 2006) and both co-operate in the relaxation of intestinal smooth muscle (Grider, 1993). In the guinea-pig, subgroups of neurons displaying this phenotype were identified as descending interneurons and inhibitory motor neurons. In the pig, it has been suggested that myenteric neurons displaying co-reactivity for both markers are inhibitory motor neurons (Brown & Timmermans, 2004). Also in the pig, however, neurons displaying co-reactivity for VIP and GAL (so-called ‘minineurons’, which were found in all three ganglionated plexuses; Stach, 1989) were suggested to be non-nitrergic secretomotor and vasomotor neurons (Timmermans et al. 2001; Brown & Timmermans, 2004). GAL may play multiple roles in the ENS of pig and other mammals where it may serve as an interneuronal, secretomotor or inhibitory smooth muscle motor peptide (Bishop et al. 1986; Brown et al. 1990; Schmidt et al. 1993; Anselmi et al. 2005). In this study, we demonstrated the presence of both VIP and GAL in morphologically defined type VI neurons (Stach, 1989), which have previously been shown to be nitrergic (Timmermans et al. 1994; Brehmer & Stach, 1997). There are several arguments suggesting that the VIP/GAL minineurons and the VIP/GAL-type VI neurons are different populations. First, myenteric type VI neurons project anally within the myenteric plexus (Stach, 1989). Because, on the one hand, we found numerous VIP- and GAL-reactive nerve fibres in both the secondary and the tertiary strands of the myenteric plexus, but, on the other hand, we did not find marked accumulations of VIP-/GAL-immunoreactive boutons around VIP-/GAL-reactive type VI neurons (this would be indicative for an interneuronal role), we suggest that these type VI neurons may be inhibitory muscle motor neurons. Secondly, VIP/GAL minineurons have been found in all three ganglionated plexuses of the pig small intestine whereas type VI neurons are absent from the inner submucosal plexus. Thirdly, it is very likely that VIP/GAL minineurons are non-nitrergic, as there are only very few nitrergic neurons in the inner submucosal plexus (Brehmer et al. 1998; Van Ginneken et al. 1998). Future studies will address the exact morphological and chemical distinction of these neuronal populations.

Concluding remarks

There are two subpopulations of morphological type I neurons. The first, which corresponds to the type I neurons described by Stach based on silver impregnation, displays mainly oral projections. They may be ascending interneurons, but further chemical characterization is necessary to confirm this. The other may correspond to motor neurons innervating the longitudinal and circular muscle layers, characterized so far by tracing combined with immunohistochemistry. The latter neurons are partly immunoreactive for ENK, CGRP and/or SP. Thus, CGRP can no longer be considered as a specific marker for type II neurons in the pig ENS as this peptide is also present in some type I neurons (this study) and in type V neurons (Brehmer et al. 2002a,b).

Type VI neurons are immunoreactive for nNOS, frequently co-localized with VIP and GAL. They differ from VIP/GAL-coreactive minineurons described earlier and may be inhibitory motor neurons. Minineurons may represent vasomotor and/or secretomotor neurons.

Acknowledgments

The excellent technical assistance of Karin Löschner and Stephanie Link is gratefully acknowledged. We are also grateful to Patricia Heron (Erlangen) for checking the manuscript.

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