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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine Christiane Freytag a,b,c , Johannes Seeger b , Thomas Siegemund a,d , Jens Grosche a,e , Astrid Grosche f , David E. Freeman f , Gerald F. Schusser c , Wolfgang Härtig a,⁎ a

Paul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43, D-04103 Leipzig, Germany c Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 11, D-04103 Leipzig, Germany d Clinical Haemostaseology and Adult Haemophilia Care Center, Center of Internal Medicine, Faculty of Medicine, University of Leipzig, Germany e Interdisciplinary Center for Clinical Research (IZKF), Faculty of Medicine, University of Leipzig, Germany f Department of Large Animal Clinical Science, College of Veterinary Medicine, University of Florida, Gainesville, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

The present study was performed on whole-mount preparations to investigate the chemical

Accepted 19 September 2008

neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall.

Available online 7 October 2008

The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of

Keywords:

vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from

Horse

different defined areas of the small intestine and the flexura pelvina were taken from 15 adult

Myenteric plexus

horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed

Whole-mount

on free floating whole-mounts. Additionally, samples used for neuropeptide staining were

Immunofluorescence

incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The

Choline acetyltransferase

evaluation was routinely accomplished using confocal laser-scanning microscopy. For

Nitric oxide synthase

quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse. © 2008 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +49 341 97 25749. E-mail address: hartig@medizin.uni-leipzig.de (W. Härtig). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.070


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1.

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Introduction

The enteric nervous system (ENS) is an intrinsic neuronal network within the gut wall, extending over the entire length of the gastrointestinal tract (Furness, 2006). The ENS is responsible for the intrinsic control and coordination of motility, blood flow and secretion to support normal digestion (Grundy and Schemann, 2005). It consists of two ganglionated plexuses: the submucosal plexus is located near the luminal side between the mucosa and circular muscle layer, and the myenteric plexus is embedded between the outer longitudinal and inner circular muscle layer of the intestine (Hansen, 2003). The ENS is the largest accumulation of neurons outside the central nervous system containing millions of neurons, e.g., about 100 million in man (Furness et al., 2003). Its intrinsic functional components can be distinguished as sensory, inter- and motoneurons (Furness, 2000). Sensory neurons which are often referred to as intrinsic primary afferent neurons (IPAN) are excited by chemical, mechanical and thermal stimuli. Motoneurons innervate effectors like muscle cells and blood vessels, whereas interneurons integrate information into the enteric network (Furness, 2000). Different neurophysiological and morphological properties are used to characterize this large assemblage of neurons (Bornstein et al., 1994; Brehmer et al., 1999; Lomax and Furness, 2000). One unique feature for identification and classification of neuronal subpopulations is their neurochemical coding: the expression of a specific combination of neurotransmitters, neuromodulators and neurochemicals (Furness et al., 1995). More than 25 substances have been suggested to be involved in neurotransmission within the gastrointestinal tract (McConalogue and Furness, 1994). For example, acetylcholine, one of the major neurotransmitters in the ENS can be found in excitatory motoneurons and interneurons, whereas nitric oxide is mainly located in inhibitory motoneurons (Brookes, 2001). Other neuropeptides, like tachykinines, vasoactive intestinal polypeptide (VIP) and neuropeptide Y act as important secondary and co-transmitters (McConalogue and Furness, 1994). To determine the distribution and complexity of the relevant structures of the ENS, previous studies were mainly performed on whole-mount preparations, e.g., the isolated myenteric plexus without surrounding tissue. Rodents, especially guinea pigs, are the preferred animal model to investigate the ENS (Furness et al., 1988; Lomax and Furness, 2000; Sang and Young, 1998). Nevertheless, data are available on the ENS of humans (Ganns et al., 2006) and of several large animals such as sheep (Pfannkuche et al., 2003), pigs (Brehmer et al., 2002) and cattle (Balemba et al., 1999). In general, studies on the ENS of horses were performed on transverse and horizontal paraffin or cryostat sections, neglecting its areal distribution (Domeneghini et al., 2004; Doxey et al., 1995; Schusser and White, 1994; Burns and Cummings, 1993). However, equine whole-mounts obtained by micro-dissection technique were exclusively achieved using tissue from young foals (Pearson, 1994; Doxey et al.,

1995). A smooth muscle enzymatic digestion technique producing whole-mounts was only occasionally applied (Burns and Cummings, 1991). Because of strong connectivetissue bonds, Doxey et al. (1995) stated that intestinal layers of adult horses cannot be separated cleanly in order to obtain whole-mounts. To our knowledge, we present here the first study on the myenteric plexus of the adult horse using whole-mounts obtained by a micro-dissection technique. The previously mentioned technical problems have prevented a more detailed (immuno)histochemical analysis of the equine ENS which is of interest considering the high incidence of gastrointestinal tract disturbances. Motility dysfunctions caused by disorders of control or smooth muscle function occur frequently in the horse (Burns et al., 1989; Dabareiner and White, 1995; Schusser et al., 2000). Furthermore, diseases directly linked to the ENS, e.g., the Lethal White Foal Syndrome (Metallinos et al., 1998) and alterations in the ENS of horses suffering from equine dysautonomia (Cottrell et al., 1999) are often reported. Therefore, our aim was to obtain new data on the chemoarchitecture of the equine ENS under physiological conditions which might allow a better understanding of pathological changes.

2.

Results

2.1. HuC/D as marker revealing neurons and ganglia properties The pan-neuronal marker HuC/D belonging to the family of ELAV (embryonic lethal abnormal visual) RNA binding-proteins was used to reveal the entire neuronal population. This produced a consistent intense staining of neuronal somata and nuclei. In general, the majority of neuronal processes as well as nucleoli remained unstained. Ganglia of the small intestine appeared in a round, oval or triangular formation. Other ganglia displayed a crescent- or butterfly shape. In the small intestine, ganglia were mainly orientated with their long axis parallel to the circular muscle layer. Ganglia with triangular and square appearances predominated in the flexura pelvina, with edges sending long offshoots in the peripheral area. Occasionally, other shapes were observed in the large intestine. In order to classify the ganglionic organization, the entire population of myenteric neurons was considered, not only those neurons organized in ganglia, but also ectopic neurons, defined as solitary neurons or as small groups consisting of 2 to 4 cells. The occurrence of ectopic neurons and neurons organized in ganglia is demonstrated in Fig. 1. Data on the different sizes of ganglia and ectopic neurons are summarized in Fig. 2. Taken together, in the small intestine more neurons were organized in smaller ganglia compared to the pelvic flexure. Ganglia were subdivided into four groups based on the number of neurons: 5 to 10 (miniganglia), 11 to 50, 51 to 150 and >150 neurons. On the one hand, all locations of the small intestine studied showed greater numbers of ectopic neurons and ganglia sized 11 to 50 neurons compared to the flexura pelvina (p < 0.05). On the other hand, the flexura pelvina was characterized by a larger


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Fig. 1 – Organization of the myenteric neuronal population. The myenteric neuronal population was divided in ectopic neurons occurring solitary or in small groups up to four neurons and in neurons organized in ganglia. Mean values are presented in % ± standard error of mean.

number of ganglia sized 51 to 150 and >150 compared to the small intestine (p < 0.05). The largest ganglion in the flexura pelvina contained over 900 neurons while in the small intestine no ganglia with more than 300 neurons could be discovered. However, mini-ganglia appeared to be present in the same frequency in all locations. Furthermore, the neuronal packing density was calculated. The following neuronal packing densities, defined as neurons/ cm2 ganglionic area, were determined: 55 100-duodenum descendens, 55 300-flexura duodeni caudalis, 52 000-duodenum ascendens, 58 400-jejunum, and 57 500-flexura pelvina. In general, no difference could be verified between the locations of the small and large intestine studied, except the duodenum ascendens showing a lower neuronal packing density compared to the jejunum.

2.2.

Cholinergic and nitrergic neurons

The cholinergic and nitrergic subpopulations of the myenteric neurons were studied based on triple fluorescence

55

labeling of HuC/D, ChAT and NOS. Homogeneous immunoreactivity (IR) of the soma without nucleus labeling was a characteristic feature of NOS-staining. Nitrergic neuronal processes were only stained at their origin and could not be followed over a long distance. One subpopulation of cholinergic neurons was intensely stained, while other cells exhibited only weak ChAT-IR (Figs. 3A and B). ChAT and VAChT were shown to be co-expressed in neuronal processes (Fig. 3C). As presented in Fig. 4, a higher rate of cholinergic neurons was detected in the duodenal and in the jejunal specimens than in the flexura pelvina (p < 0.05). In contrast, more nitrergic neurons could be seen in the flexura pelvina compared to all locations of the small intestine studied (p < 0.05) as presented in Fig. 5. Furthermore, a small portion of cells co-expressing ChAT and NOS was observed. The number of these cells appeared to be higher in the small intestine than in the flexura pelvina. On the contrary, the number of neurons labeled only by HuC/D (HuC/D-only) without additional ChAT- or NOS-immunoreactivity was nearly identical in the flexura pelvina and all the locations of the small intestine studied (Table 1). In general, neuron sizes displayed a greater somal area in the duodenal specimens compared to the jejunum and flexura pelvina, regardless to their neurochemical characteristics (p < 0.05). Furthermore, cholinergic neurons had larger soma sizes than the nitrergic population regardless to the location of the gut examined (p < 0.05) as shown in Table 2.

2.3.

Calretinin-expressing neurons

Expression of calretinin (CR) was analyzed in tissues triplestained for HuC/D, ChAT and CR (Figs. 6A and B). The majority of CR-expressing cells displayed a weaker immunoreactivity in the soma than in the nerve fibers. Only a few somata showed a strong and intense staining similar to the fibers. CRpositive cells were either observed alone or in small groups of up to 4 cells organized in clusters or in line next to each other. Elongated or slightly oval-shaped cells were seen, having one

Fig. 2 – Prevalence of different sized ganglia in diverse locations of the equine intestine. In all locations of the small intestine studied, larger numbers of ectopic neurons and ganglia containing 11 to 50 neurons were found compared to the large intestine. In the large intestine, higher numbers of ganglia sized 51–150 neurons and ganglia comprising >151 neurons were identified compared to the small intestine. However, no differences were found regarding the occurrence of mini-ganglia. Mean values are presented in % ± standard error of mean.


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Fig. 3 – Immunohistochemical labeling of cholinergic and nitrergic neurons in myenteric plexus of the jejunum (A) and in the flexura pelvina (B). The pan-neuronal marker HuC/D (Cy3, red) stained all cholinergic and nitrergic neurons, while a portion of cells was HuC/D-monolabeled (A, A′″). Nitrergic neurons and processes (Cy2, green) were revealed by immunolabeling of nitric oxide synthase (NOS) (A′). Cholinergic neurons (Cy5, blue) labeled by choline acetyltransferase (ChAT) displayed strong or weak ChAT-immunoreactivity (A″). Cholinergic processes co-expressed vesicular acetylcholine transporter (VAChT) and ChAT, while cholinergic neurons were stained by ChAT only (C). Scale bars: in (B), also valid for (A) = 100 μm, and in (C) = 20 μm.

long, polar process and several small, stubby and to some extent arborized processes around the somata. Furthermore, multipolar and roundish cells, sending a number of long processes, could be detected in the equine myenteric plexus. However, a few of the CR-expressing cells did not show staining of any processes. About 6.5% of the entire neuronal population stained by HuC/D was CR-positive (Table 3). The majority of these neurons co-expressed ChAT and only a few cells were CRmonolabeled. The appearance of the CR-population in the

flexura pelvina was indistinguishable from those in all locations of the small intestine studied.

2.4.

Neurons containing vasoactive intestinal polypeptide

After colchicine incubation, an accumulation of VIP-IR could be seen in the ganglionic area, while interganglionic fiber tracts were not prominent. VIP-containing cells displayed an elongated or slightly oval-shaped cell body with one long process. Apart from the long process, several short, lamellar


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57

Fig. 4 – Proportion of cholinergic population in different locations of the equine intestine. All regions of the small intestine displayed similar sizes of the cholinergic subpopulation. In contrast, the flexura pelvina contained a smaller proportion of cholinergic neurons compared to the small intestine (p < 0.05). Mean values are presented in % ± standard error of mean.

and to some extent arborized processes could be detected around the soma. Moreover, a frequent co-localization of VIP and NOS was detectable (Fig. 6C). On the contrary, neurons coexpressing VIP and CHAT were only occasionally found (data not shown).

3.

Discussion

3.1.

Pan-neuronal labeling

The pan-neuronal marker HuC/D was applied to analyze the ENS in different species, e.g., rat and human (Phillips et al., 2004a; Ganns et al., 2006). Hu-proteins belonging to the family of ELAV RNA-binding proteins have been shown to be highly conserved over evolution (Nabors et al., 1998) which allows the use of antibodies directed against HuC/D in various animal species. HuC/D staining in the horse ENS was intense in neuronal nuclei and cell bodies, whereas the majority of neuronal processes and nucleoli remained unstained. Only occasionally, stained nuclei surrounded by unstained somata were observed. Comparable staining results were documented in the myenteric plexus of adult rats (Phillips et al., 2004a). Heterogenic immunoreactivity of HuC/D can be attributed to different expression of Hu-

proteins according to cell activity (Ganns et al., 2006). The distribution of HuC/D-IR also in processes indicates high cell activity, whereas HuC/D-IR restricted to the nucleus represents low cell activity. Furthermore, a high variability of HuC/D-expression in older individuals is well-known (Phillips et al., 2004b). In addition, a neuronal drop-out in the ENS of older horses was not detectable (Burns and Cummings, 1991). Comparisons of different pan-neuronal markers in the ENS such as HuC/D, PGP 9.5, Cuprolinic Blue and the tract-tracer Fluoro-gold, have found HuC/D and Cuprolinic Blue to be superior (Phillips et al., 2004a). In the present study, there were no neurons labeled with other neuronal markers that were missing HuC/D-IR, demonstrating the reliability of HuC/D as a pan-neuronal marker in the equine ENS.

3.2.

Morphology and sizes of ganglia

The present findings display distinctive form variations of equine ganglia in the small and large intestines. Our results confirm data from previous studies on features of the myenteric plexus of large mammals like cattle and sheep such as heterogenic forms (Gabella, 1987; Balemba et al., 1999). Myenteric ganglia of small laboratory animals, e.g., mice, rats and guinea pigs, are elongated and show homologous

Fig. 5 – Proportion of nitrergic population in different locations of the equine intestine. All regions of the small intestine displayed similar sizes of the nitrergic subpopulation. In contrast, the flexura pelvina contained a larger proportion of nitrergic neurons compared to the small intestine (p < 0.05). Mean values are presented in % ± standard error of mean.


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Table 1 – Occurrence of neurons expressing HuC/D-only and neurons co-expressing choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) Location

Duodenum descendens Flexura duodeni caudalis Duodenum ascendens Jejunum Flexura pelvina

Neurons expressing HuC/D-only (%)

Neurons co-expressing ChAT and NOS (%)

42.9 ± 3.1 42.7 ± 2.7 43.1 ± 2.3 42.8 ± 2.2 42.4 ± 1.6

0.81 ± 0.29 0.91 ± 0.32 1.64 ± 0.38 1.20 ± 0.39 0.12 ± 0.06

Neurons without additional ChAT- and NOS-immunoreactivity were determined as HuC/D-only nerve cells. No differences could be detected comparing the different locations of the equine intestine examined. Mean values are presented in % ± standard error of mean.

geometrical features (Gabella, 1971 and 1987). Moreover, we found that the long axis of the myenteric ganglia was orientated parallel on the circular muscle layer, confirming earlier results of Pearson (1994). This orientation documented in various small and large animals seems to be a distinctive feature of the myenteric plexus (Gabella, 1987, Santer and Baker 1988). Close apposition of the neuronal structures to the gut wall allows reorganization of the ganglia in response to constant mechanical activity from muscle contraction and relaxation (Gabella, 1990). The parallel orientation of the long axis on the thicker circular muscle layer may facilitate the reorganization of the ganglia embedded between the muscle layers. For analysis of ganglion sizes, a precise definition of ganglia boundaries is mandatory. According to Phillips et al., (2004b) the minimal demarcation line between ganglia was defined as double the-length of an average sized neuron. In the small intestine, about 50 to 54% of the ganglia contained 11 to 50 neurons, while in the flexura pelvina only 24% of the ganglia were in this range. In contrast, in the flexura pelvina about 21% of the ganglia contained 51–150 neurons and about 27% contained more than 150 neurons, whereas in the small intestine only 13 and 1.5% of the ganglia displayed these sizes. Differing from our results, Burns and Cummings (1991) documented in the equine jejunum ganglia up to 20 and in the colon between 60 to 100 neurons which might be explained by the difficulties of defining ganglia boundaries. Other studies on the horse small intestine using paraffin sections documented 1 to 10 neurons or 3.5 neurons per

ganglia (Pogson et al., 1992; Scholes et al., 1993). The chemoarchitecture of the myenteric plexus in its natural areal expanse cannot be analyzed on transversal paraffin sections, which could explain the different results. Moreover, we recorded ectopic neurons, defined as 1 to 4 neurons not organized in ganglionic structures. In the small intestine, higher numbers of ectopic neurons were documented within the duodenum ascendens (9.6% of all neurons) than in the flexura pelvina (3.8%). In the guinea pig ileum, 10% of all nerve cells were defined as ectopic applying the same pan-neuronal marker Hu (Abalo et al., 2005). On the contrary, Costa et al. (1996) revealed only 1.1% as ectopic in the guinea pig ileum using PGP 9.5 as a “pan-neuronal” marker. Comparisons of different studies appear problematic because of differing definitions of ectopic neurons and the use of different panneuronal markers. In conclusion, the ganglionic pattern in the flexura pelvina is more tightly organized compared to the small intestine of the horse; the flexura pelvina displays large ganglia and low numbers of ectopic neurons compared to the small intestine of the horse.

3.3.

Neuronal packing densities

Many studies have been carried out to determine neuronal density related to the serosal area (Furness, 2006). Karaosmanoglu et al. (1996) have shown a relation between the neuronal density and the grade of tissue stretching prior to fixation: increasing the tissue stretching by 32% resulted in a 31% reduction of the neuronal packing density in the jejunum–ileum segment of the guinea pig. Nevertheless, neuronal packing density, defined as neurons/cm2 ganglionic area, was found to be stretch-independent which allows reproducible and comparable measurements (Karaosmanoglu et al., 1996). In contrast to the stretch-dependent nonneuronal tissue, the ganglionic area consists of neurons, enteric glial cells and neuropil, but is devoid of connective tissue, blood vessels or smooth muscle cells (Gabella, 1990; Natali et al., 2000). The neuron packing density of the myenteric plexus of the small and large intestine in the guinea pig accounted for 240 000 to 250 000 neurons/cm2 ganglionic area, whereas in the rat 200 000 to 250 000 neurons could be detected in the small intestine and 130 000 neurons in the large intestine (Karaosmanoglu et al., 1996, Phillips et al., 2004b). In the equine myenteric plexus, we calculated a neuron packing density of 52 000 to 58 000 neurons/cm2 ganglionic area. Although Gabella (1990) specified the neuronal densities as neurons/cm2 serosal area, mice had the

Table 2 – Summary of somata sizes of cholinergic (choline acetyltransferase-containing) neurons and nitrergic (nitric oxide synthase-expressing) neurons in different locations of the equine intestine

Cell sizes (μm2) of cholinergic neurons Cell sizes (μm2) of nitrergic neurons

Duodenum descendens

Flexura duodeni caudalis

Duodenum ascendens

Jejunum

Flexura pelvina

1055 ± 44

1063 ± 39

1050 ± 44

985 ± 33

905 ± 28

916 ± 45

938 ± 48

911 ± 38

799 ± 43

775 ± 26

In general, cholinergic neurons displayed larger perikarya compared to the nitrergic neurons (p < 0.05). Mean values are presented in μm2 ± standard error of mean.


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Fig. 6 – Cholinergic and calretinin (CR)-expressing neurons in the myenteric plexus of the duodenum ascendens (A) and the flexura pelvina (B); nitrergic neurons showing VIP-co-localization in the flexura duodeni caudalis (C). The pan-neuronal marker HuC/D (Cy3, red) stained all cholinergic and calretinin-expressing neurons, while a portion of neurons was HuC/D-monolabeled (A, A′″). CR-containing neurons and processes (Cy2, green) co-expressed choline acetyltransferase (ChAT) (A′, A′″). Cholinergic neurons (Cy5, blue) showed different intensities of ChAT-immunolabeling (A″). Neurons containing vasoactive intestinal polypeptide (VIP) were frequently immunoreactive for nitric oxide synthase (NOS) (C). Scale bars: in (B), also valid for (A) = 100 μm, and in (C) = 10 μm.

highest and sheep the lowest numbers. Furthermore, the neuropil of the mice accounted for 1/2 and that of the sheep 3/4 of the ganglionic area in the small intestine (Gabella, 1990). On the same subject, increasing divergence (numbers of neurons innervated by one axon) and convergence (numbers of axons innervating one neuron) are detectable when homologous neuronal structures of large animals are compared with those of smaller species (Purves and Lichtman, 1985; Purves et al., 1986). Discrepancies between our

data on the horse and those on small laboratory animals can be related to the substantial differences between the species compared.

3.4.

Cholinergic neurons

Acetylcholine is one of the most important transmitters in the ENS which can be found in excitatory motoneurons, interneurons and IPAN (Costa et al., 2000). For the detection of


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Table 3 – Summary of the distribution of calretinin (CR)-expressing cells in the different locations of the equine intestine

CR-expressing neurons (%) CR-only (%) ChAT neurons co-expressing CR (%)

Duodenum descendens

Flexura duodeni caudalis

Duodenum ascendens

Jejunum

Flexura pelvina

6.6 ± 0.8 0.23 ± 0.2 14.4 ± 1.6

7.4 ± 1.2 0.49 ± 0.2 15.6 ± 2.2

5.7 ± 0.7 0.08 ± 0.1 13.3 ± 1.7

6.8 ± 0.8 0.18 ± 0.1 17.1 ± 2.4

5.5 ± 0.7 0.44 ± 0.2 14.6 ± 1.4

The CR subpopulation was referred to the entire neuronal myenteric population ascertained by the pan-neuronal marker HuC/D. Furthermore, the portion of cholinergic, choline acetyltransferase (ChAT)-expressing neurons co-stained with CR was identified. Moreover, a few cells were CR-monolabeled (CR-only). There were no significant differences in the distribution of CR-positive neurons in the different gut locations examined. Mean values are presented in % ± standard error of mean.

cholinergic neurons different cholinergic markers such as the enzyme catalyzing the transmitter synthesis (ChAT), the transmitter degrading enzyme (acetylcholinesterase), the vesicular acetylcholine transporter (VAChT) and the choline transporter are commonly used (Leaming and Cauna, 1961; Usdin et al., 1995; Costa et al., 2000; Harrington et al., 2007). In the present study, ChAT simultaneously revealed strongly and weakly immunoreactive neurons with homogenous staining of the cytoplasm. Similar staining features were revealed in the ENS of the guinea pig (Li and Furness, 1998; Chiocchetti et al., 2003). Verifying the cholinergic phenotype, simultaneous labeling of ChAT and VAChT showed extensive coexpression in neuronal processes, but lack of VAChT-IR in perikarya. Sang and Young (1998) demonstrated VAChT-IR in enteric neuronal processes of mice, but not in somata. In contrast, VAChT-ir somata were detected in the equine ENS (Domeneghini et al., 2004). The use of different primary antibodies might partially explain the differing results. In the equine small intestine about 36% of all neurons were shown to be cholinergic, while the flexura pelvina contained 24% ChAT-ir nerve cells. In contrast, 60% of the neurons in the small intestine of mice and 65% in the human ileum displayed ChAT-IR (Porter et al., 1996; Sang and Young, 1998). Additionally, a cholinergic phenotype was reported for 57% (of all neurons) in the large intestine of the guinea pig and for 63% of the human enteric neurons (Porter et al., 1996; Lomax and Furness, 2000). Because different “pan-neuronal” markers used in order to quantify cholinergic subpopulations, e.g., PGP 9.5, neuron-specific enolase or anti-nerve cell body, a comparison between different studies can be difficult. Murphy et al. (2007) quantified ChAT-expressing neurons in the human large intestine applying the pan-neuronal marker Hu and determined that this subpopulation was 48% of the neurons. Discrepancies with our results may be explained by the fact that the ChAT-antibody we used did

not recognize all cholinergic neurons in the equine ENS. In the peripheral tissue, two types of ChAT mRNA arising from alternative splicing were detected: a peripheral (p)-ChAT and a common (c)-ChAT (Tooyama and Kimura, 2000). In the central nervous system, cChAT represents the exclusive ChAT-form (Tooyama and Kimura, 2000). Moreover, in the porcine ileum 22.4% of the neurons were labeled by both ChAT forms and 31% expressed cChAT alone, while 27.8% of the neurons displayed pChAT exclusively (Brehmer et al., 2004). In addition, investigations in the guinea pig ENS revealed a subpopulation of cholinergic neurons expressing pChAT alone (Chiocchetti et al., 2003). Further elucidation of the equine cholinergic population in the myenteric plexus is required also using antibodies directed against pChAT.

3.5.

Nitrergic neurons

Nitrergic neurons revealed by immunolabeling of their marker enzyme NOS are known to be inhibitory motoneurons and descending interneurons in the guinea pig small intestine (Furness 2000). Furthermore, it is known that nitrergic neurons innervate and support interstitial cells of Cajal (ICC), which have a putative role in control of intestinal motility (Hudson et al., 1999; Choi et al., 2007; Iino et al., 2008). In the equine myenteric plexus, nitrergic activity was successfully demonstrated (Rakestraw et al., 1996). We identified 20 to 22% of the HuC/D-labeled neurons in the small intestine as nitrergic neurons, while in the flexura pelvina 33% displayed NOS-IR. Using different pan-neuronal markers, the nitrergic component of all neurons in the myenteric plexus comprises 26% in mice, 20% in human small intestine and 19% in guinea pig ileum (Furness et al., 1994; Sang and Young, 1996; Belai and Burnstock, 1999). Moreover, in the large intestine NOS-IR was expressed in 35% of mouse enteric neurons and in 39% of guinea pig enteric neurons (Sang and Young, 1996; Lomax and

Table 4 – Primary antibodies used for immunohistochemistry Primary antibody HuC/D Neuronal-NOS Neuronal-NOS ChAT VAChT CR VIP

Host

Dilution

Fluorochrome

Supplier

Reference

Mouse Rabbit Mouse Goat Rabbit Rabbit Rabbit

1:100 1:200 1:50 1:25 1:200 1:300 1:200

Cy3 Cy2 Cy3 Cy5 Cy2 Cy2 Cy2

Invitrogen, Karlsruhe, Germany Transduction Labs, Heidelberg, Germany BD Bioscience, Heidelberg, Germany Millipore, Billerica, MA, USA Synaptic Systems, Göttingen, Germany Swant, Bellinzona, Switzerland Diasorin, Dietzenbach, Germany

Phillips et al., 2004a Lüth et al., 2000 Sasaki et al., 2000 Li and Furness, 1998 Härtig et al., 2007 Schwaller et al., 1993 Kawaguchi and Kubota, 1996


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Furness, 2000). In the human colon, quantification based on HuC/D revealed 43% nitrergic neurons (Murphy et al., 2007). Differences between the data on human nitrergic populations and our results may be related to an age-dependent increase of nitrergic neurons in humans, as the patients examined had an average age of 68 years (Belai and Burnstock, 1999; Murphy et al., 2007). In addition, six different splicing variants of the enzyme have been identified in the human ENS and the development of new antibodies could support a more detailed analysis of nitrergic subpopulations (Saur et al., 2000; Brehmer, 2006). Moreover, there is evidence for a pacemaker activity in the flexura pelvina of the horse (Sellers, 1982; Burns and Cummings, 1991). Further investigation especially of different sampling sites of the colon ascendens is needed to increase information about these subjects. Enteric glial cells are identified as a reservoir of L-arginine which is a precursor of nitric oxide (NO) (Nagahama et al., 2001). NOS catalyzes the conversion of L-arginine into citrulline and NO (Prince and Gunson, 1993). In general, all nitrergic neurons displayed smaller perikarya compared to the cholinergic neurons regardless to their location. Smaller cell sizes of nitrergic neurons may be explained by the extra-neuronal reservoir of the precursor substances L-arginine in glial cells and the fact that this gaseous neurotransmitter is not stored in vesicles.

3.6.

Co-expression of ChAT and NOS

The triple staining HuC/D/ChAT/NOS revealed a small proportion of neurons co-expressing ChAT and NOS (0.1 to 2%). The same marker combination was reported for 4% of doublestained ChAT/NOS-neurons in the human colon (Murphy et al., 2007). In the duodenum of the guinea pig, co-localization of pChAT and NOS was detected in 3.6% of all neurons, whereas in the colon 21.3% of all neurons co-expressed both markers (Nakajima et al., 2000). In the guinea pig small intestine, neurons containing ChAT and NOS were identified as descending interneurons (Costa et al., 2000; Furness, 2000).

3.7.

Calretinin-expressing neurons

Excitatory motoneurons innervating the outer longitudinal muscle and ascending interneurons in the myenteric plexus of the guinea pig small intestine are CR-ir (Furness, 2000). In the equine myenteric plexus, we have shown CR-IR in neurons and neuronal processes almost entirely co-localized with ChAT. CR-containing moto- and interneurons of the guinea pig are cholinergic as well (Brookes, 2001). We identified most of the CR-expressing neurons in the investigated parts of the horse ENS as Dogiel type I neurons, while cells displaying Dogiel type II morphology were rarely detectable. Additionally, CR-ir neuronal processes did not form a tertiary plexus, confirming earlier results that only small animals with a thin stratum longitudinale have a tertiary plexus in order to innervate this muscle layer (Llewellyn-Smith et al., 1993; Furness et al., 2000). In contrast, neuronal processes of large animals form a network within the longitudinal muscle layer (Furness et al., 2000). Furthermore, excitatory motoneurons for the longitudinal muscle layer were distinguishable from ascending interneurons displaying smaller soma areas (Pom-

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polo and Furness 1993). In this study, all CR-labeled neurons were characterized by a largely identical cell body size giving no possibility for differentiation. Retrograde labeling studies could further elucidate functional aspects of the equine, CRexpressing neurons.

3.8. Neurons containing vasoactive intestinal polypeptide (VIP) In the ganglionic area, we detected strong VIP-IR in neurons as well as in neuronal processes. Some neurons displayed an accumulation of VIP-IR in parts of their soma, apparently resulting from colchicine utilization. Colchicine inhibits axonal transport of neuropeptides and is useful for their visualization also in the perikarya (Ekblad and Bauer, 2004). VIP-expressing cells were identified as Dogiel type I neurons showing a single long process and several short, lamellar dendrites. Furthermore, distinctive co-localization of VIP and NOS was detected in the myenteric neurons of the horse. VIP and NOS co-expression is well known in myenteric neurons of, e.g., guinea pig, dog and man (Wang et al., 1998; Lomax and Furness, 2000; Brehmer et al., 2006). Inhibitory motoneurons in guinea pig and man contain VIP and NOS among other transmitters (Porter et al., 1997; Brookes, 2001). VIP-ir neuronal processes were identified in the circular muscle layer of horses (Burns and Cummings 1993) suggesting that VIP acts as a transmitter in motoneurons. Moreover, we showed rare colocalization of VIP and ChAT in equine myenteric neurons in line with data on the co-expression of VIP and ChAT in descending interneurons of guinea pig and man (Wattchow et al., 1997; Furness, 2000).

4.

Experimental procedures

4.1.

Tissue preparation

A first set of tissue samples was taken from 8 horses that showed no sign of gastrointestinal disturbances at local abattoirs near Leipzig, Germany. The samples (5 × 5 cm) were taken from several locations of the small intestine (duodenum descendens, flexura duodeni caudalis, duodenum ascendens, mid jejunum) and of the large colon (flexura pelvina). Furthermore, the College of Veterinary Medicine (University of Florida, Gainesville, USA) provided additional samples of the flexura pelvina from 7 horses. The age of the animals ranged from 4 to 22 years. All specimens were rinsed clean and transported in cooled carbogen bubbled Krebs solution, pH 7.4 (in mM: 117 NaCl, 25 NaHCO3, 1.2 NaH2PO4, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 11.5 mM glucose, 1 μM nifedipine; all chemicals from Sigma, Taufkirchen, Germany). Nifedipine was added to relax the smooth muscle by blocking calcium-channels and this facilitated further handling of the tissues. For fixation in 4% paraformaldehyde saturated with picric acid, the samples were dissected free from the mucosal layer, stretched out and pinned flat in Sylgard®-covered Petri dishes (Dow Corning, Auburn, MI, USA; 24 h, 5°C). The fixed specimens were rinsed in 0.1 M phosphate-buffered saline (PBS, pH 7.4) and stored in 0.1 M PBS containing 0.1% NaN3.


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Tissue used for neuropeptide staining was incubated with colchicine in order to enhance immunosignals in the myenteric neuronal somata. Specimens dissected free from the mucosal layer were placed in a sterile culture medium (Dulbeccos modified eagles medium) for 24 h and kept in an incubator environment of 37°C, 95% O2 and 5% CO2. The culture medium was enriched with 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 μg/ml gentamicin, 1.24 μg/ml amphotericin B, 1 μM nifedipine, 80 μM colchicine and 10% heat-inactivated fetal calf serum (all chemicals from Sigma, Taufkirchen, Germany). During the culture period, the samples were constantly moved on a rocking tray. All devices, instruments and solutions used in order to accomplish colchicine incubation were maintained under sterile conditions. The specimens were fixed and stored as described above. Further tissue assessment was achieved by using a stereomicroscope with an integrated light source (Stemi SV11, Zeiss Jena, Germany). The myenteric plexus was carefully dissected free from the serosal layer, the circular and longitudinal muscle layer and, if necessary, from the remnants of mucosal layer. This preparation was successfully carried out on maximally stretched tissue samples. Until further investigation, the whole-mounts were stored in 0.1 M PBS containing 0.1% NaN3.

4.2.

Immunofluorescence labeling

Multiple immunofluorescence labeling was performed on free floating preparations which had been extensively rinsed with 0.1 M Tris-buffered saline, pH 7.4 (TBS). Blocking of unspecific binding sites and permeabilization was achieved by incubating the tissues in 5% donkey serum and 0.3% Triton-X100 diluted in TBS. Next, whole-mounts were transferred into a cocktail of primary antibodies diluted in the same blocking solution for 18 h. Primary antibodies used, their hosts, dilutions, suppliers and references are listed in Table 4. All markers were demonstrated simultaneously by fluorescent, highly purified secondary donkey antibodies (from Dianova, Hamburg, Germany) conjugated to green fluorescent Cy2, red fluorescent Cy3 or infrared light-emitting Cy5. All secondary antibodies directed against rabbit, mouse or goat IgGs were used at 20 μg/ml in TBS containing 2% bovine serum albumin for 1 h. Controls were performed by omission of primary antibodies resulting in the expected absence of cellular labeling by fluorochromated secondary antibodies. All whole-mounts were finally washed extensively with TBS, rinsed briefly in distilled water, mounted onto fluorescencefree glass slides, air dried and coverslipped using Entellan (Merck, Darmstadt, Germany).

4.3.

Analysis of data

Evaluation of the tissues, cell sizes and occurrence of different subpopulations were routinely carried out with a confocal laser scanning microscope (cLSM 510 Meta, Zeiss Oberkochen, Germany). The cLSM also allowed the detection of Cy5-signals which were color-coded in blue. Furthermore, the neuronal packing densities, defined as neurons/cm2 ganglionic area were determined with cLSM.

For the quantitative evaluation of ganglion sizes, we used a fluorescence microscope Axioplan (Zeiss) and the related software (Axiovision 3.1). All data are expressed as mean ± standard error of mean. The Student's t-Test was used to compare data that was normally distributed (Chiocchetti et al., 2006) according to the Kolmogorow–Smirnow test. The level of significance was set at p < 0.05.

Acknowledgments The technical support of Dr. Helga Pfannkuche, Mrs. Petra Philipp and Mrs. Ute Bauer is gratefully acknowledged. The authors thank Dr. Douglas D. Rasmusson (Halifax, NS, Canada) for critical reading of an earlier version of the manuscript.

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Analysis of neurons in the myenteric plexus  

Neurons in the myenteric plexus

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