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Research Report

Immunohistochemical characterization and quantitativeanalysis of neurons in the myenteric plexus of theequine intestine

Christiane Freytaga,b,c, Johannes Seegerb, Thomas Siegemunda,d, Jens Groschea,e,Astrid Groschef, David E. Freemanf, Gerald F. Schusserc, Wolfgang Härtiga,⁎aPaul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, GermanybDepartment of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43,D-04103 Leipzig, GermanycLarge Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 11,D-04103 Leipzig, GermanydClinical Haemostaseology and Adult Haemophilia Care Center, Center of Internal Medicine, Faculty of Medicine,University of Leipzig, GermanyeInterdisciplinary Center for Clinical Research (IZKF), Faculty of Medicine, University of Leipzig, GermanyfDepartment of Large Animal Clinical Science, College of Veterinary Medicine, University of Florida, Gainesville, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +49 341 97 25749E-mail address: hartig@medizin.uni-leipz

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.09.070

A B S T R A C T

Article history:Accepted 19 September 2008Available online 7 October 2008

The present study was performed on whole-mount preparations to investigate the chemicalneuroanatomy of the equinemyenteric plexus throughout its distribution in the intestinal wall.The objective was to quantify neurons of the myenteric plexus, especially the predominantcholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution ofvasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples fromdifferent defined areas of the small intestine and the flexura pelvina were taken from 15 adulthorses.After fixationandpreparationof the tissue, immunofluorescence labelingwasperformedon free floating whole-mounts. Additionally, samples used for neuropeptide staining wereincubatedwith colchicine to reveal theneuropeptide distributionwithin theneuronal soma. Theevaluation was routinely accomplished using confocal laser-scanning microscopy. Forquantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied incombination with the detection of the marker enzymes for cholinergic neurons and nitrergicnerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than thenitrergic one in several different locations of the small intestine. On the contrary, the nitrergicneurons outnumber the cholinergic neurons in the flexura pelvina of the large colon.Furthermore, ganglia are more numerous in the small intestine compared with the largecolon, but ganglion sizes are bigger in the large colon. However, comparison of the entirepopulation of neurons in the different locations of the gut showed no difference. The presentstudyadds further data on the chemoarchitectureof themyenteric plexuswhichmight facilitatethe understanding of several gastrointestinal disorders in the horse.

© 2008 Elsevier B.V. All rights reserved.

Keywords:HorseMyenteric plexusWhole-mountImmunofluorescenceCholine acetyltransferaseNitric oxide synthase

.ig.de (W. Härtig).

er B.V. All rights reserved.

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1995). A smooth muscle enzymatic digestion techniqueproducing whole-mounts was only occasionally applied

1. Introduction

The enteric nervous system (ENS) is an intrinsic neuronalnetwork within the gut wall, extending over the entirelength of the gastrointestinal tract (Furness, 2006). The ENSis responsible for the intrinsic control and coordination ofmotility, blood flow and secretion to support normaldigestion (Grundy and Schemann, 2005). It consists of twoganglionated plexuses: the submucosal plexus is locatednear the luminal side between the mucosa and circularmuscle layer, and the myenteric plexus is embeddedbetween the outer longitudinal and inner circular musclelayer of the intestine (Hansen, 2003).

The ENS is the largest accumulation of neurons outsidethe central nervous system containing millions of neurons,e.g., about 100 million in man (Furness et al., 2003). Itsintrinsic functional components can be distinguished assensory, inter- and motoneurons (Furness, 2000). Sensoryneurons which are often referred to as intrinsic primaryafferent neurons (IPAN) are excited by chemical, mechanicaland thermal stimuli. Motoneurons innervate effectors likemuscle cells and blood vessels, whereas interneuronsintegrate information into the enteric network (Furness,2000).

Different neurophysiological and morphological propertiesare used to characterize this large assemblage of neurons(Bornstein et al., 1994; Brehmer et al., 1999; Lomax andFurness, 2000). One unique feature for identification andclassification of neuronal subpopulations is their neurochem-ical coding: the expression of a specific combination ofneurotransmitters, neuromodulators and neurochemicals(Furness et al., 1995). More than 25 substances have beensuggested to be involved in neurotransmission within thegastrointestinal tract (McConalogue and Furness, 1994). Forexample, acetylcholine, one of themajor neurotransmitters inthe ENS can be found in excitatory motoneurons andinterneurons, whereas nitric oxide is mainly located ininhibitory motoneurons (Brookes, 2001). Other neuropeptides,like tachykinines, vasoactive intestinal polypeptide (VIP) andneuropeptide Y act as important secondary and co-transmit-ters (McConalogue and Furness, 1994).

To determine the distribution and complexity of therelevant structures of the ENS, previous studies were mainlyperformed on whole-mount preparations, e.g., the isolatedmyenteric plexus without surrounding tissue. Rodents,especially guinea pigs, are the preferred animal model toinvestigate the ENS (Furness et al., 1988; Lomax and Furness,2000; Sang and Young, 1998). Nevertheless, data are availableon the ENS of humans (Ganns et al., 2006) and of severallarge animals such as sheep (Pfannkuche et al., 2003), pigs(Brehmer et al., 2002) and cattle (Balemba et al., 1999). Ingeneral, studies on the ENS of horses were performed ontransverse and horizontal paraffin or cryostat sections,neglecting its areal distribution (Domeneghini et al., 2004;Doxey et al., 1995; Schusser and White, 1994; Burns andCummings, 1993). However, equine whole-mounts obtainedby micro-dissection technique were exclusively achievedusing tissue from young foals (Pearson, 1994; Doxey et al.,

(Burns and Cummings, 1991). Because of strong connective-tissue bonds, Doxey et al. (1995) stated that intestinal layersof adult horses cannot be separated cleanly in order toobtain whole-mounts.

To our knowledge, we present here the first study on themyenteric plexus of the adult horse using whole-mountsobtained by a micro-dissection technique. The previouslymentioned technical problems have prevented a moredetailed (immuno)histochemical analysis of the equine ENSwhich is of interest considering the high incidence ofgastrointestinal tract disturbances. Motility dysfunctionscaused by disorders of control or smooth muscle functionoccur frequently in the horse (Burns et al., 1989; Dabareinerand White, 1995; Schusser et al., 2000). Furthermore, diseasesdirectly linked to the ENS, e.g., the Lethal White FoalSyndrome (Metallinos et al., 1998) and alterations in the ENSof horses suffering from equine dysautonomia (Cottrell et al.,1999) are often reported. Therefore, our aimwas to obtain newdata on the chemoarchitecture of the equine ENS underphysiological conditions which might allow a better under-standing of pathological changes.

2. Results

2.1. HuC/D as marker revealing neurons andganglia properties

The pan-neuronal marker HuC/D belonging to the family ofELAV (embryonic lethal abnormal visual) RNA binding-pro-teins was used to reveal the entire neuronal population. Thisproduced a consistent intense staining of neuronal somataand nuclei. In general, the majority of neuronal processes aswell as nucleoli remained unstained. Ganglia of the smallintestine appeared in a round, oval or triangular formation.Other ganglia displayed a crescent- or butterfly shape. In thesmall intestine, gangliaweremainly orientatedwith their longaxis parallel to the circular muscle layer. Ganglia withtriangular and square appearances predominated in theflexura pelvina, with edges sending long offshoots in theperipheral area. Occasionally, other shapes were observed inthe large intestine.

In order to classify the ganglionic organization, the entirepopulation of myenteric neurons was considered, not onlythose neurons organized in ganglia, but also ectopicneurons, defined as solitary neurons or as small groupsconsisting of 2 to 4 cells. The occurrence of ectopic neuronsand neurons organized in ganglia is demonstrated in Fig. 1.Data on the different sizes of ganglia and ectopic neuronsare summarized in Fig. 2. Taken together, in the smallintestine more neurons were organized in smaller gangliacompared to the pelvic flexure. Ganglia were subdivided intofour groups based on the number of neurons: 5 to 10 (mini-ganglia), 11 to 50, 51 to 150 and >150 neurons. On the onehand, all locations of the small intestine studied showedgreater numbers of ectopic neurons and ganglia sized 11 to50 neurons compared to the flexura pelvina (p<0.05). On theother hand, the flexura pelvina was characterized by a larger

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

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number of ganglia sized 51 to 150 and >150 compared to thesmall intestine (p<0.05). The largest ganglion in the flexurapelvina contained over 900 neurons while in the smallintestine no ganglia with more than 300 neurons could bediscovered. However, mini-ganglia appeared to be present inthe 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-duodenumdescendens, 55 300-flexura duodeni caudalis, 52 000-duodenumascendens, 58 400-jejunum, and 57 500-flexura pelvina. Ingeneral, no difference could be verified between the locationsof the small and large intestine studied, except the duodenumascendens showinga lowerneuronal packingdensity comparedto the jejunum.

2.2. Cholinergic and nitrergic neurons

The cholinergic and nitrergic subpopulations of the myen-teric neurons were studied based on triple fluorescence

Fig. 2 – Prevalence of different sized ganglia in diverse locationsstudied, larger numbers of ectopic neurons and ganglia containingIn the large intestine, higher numbers of ganglia sized 51–150 necompared to the small intestine. However, no differences were fare presented in %±standard error of mean.

labeling of HuC/D, ChAT and NOS. Homogeneous immuno-reactivity (IR) of the soma without nucleus labeling was acharacteristic feature of NOS-staining. Nitrergic neuronalprocesses were only stained at their origin and could not befollowed over a long distance. One subpopulation ofcholinergic neurons was intensely stained, while othercells exhibited only weak ChAT-IR (Figs. 3A and B). ChATand VAChT were shown to be co-expressed in neuronalprocesses (Fig. 3C). As presented in Fig. 4, a higher rate ofcholinergic neurons was detected in the duodenal and in thejejunal specimens than in the flexura pelvina (p<0.05). Incontrast, more nitrergic neurons could be seen in the flexurapelvina compared to all locations of the small intestinestudied (p<0.05) as presented in Fig. 5. Furthermore, a smallportion of cells co-expressing ChAT and NOS was observed.The number of these cells appeared to be higher in the smallintestine than in the flexura pelvina. On the contrary, thenumber of neurons labeled only by HuC/D (HuC/D-only)without additional ChAT- or NOS-immunoreactivity wasnearly identical in the flexura pelvina and all the locationsof the small intestine studied (Table 1).

In general, neuron sizes displayed a greater somal areain the duodenal specimens compared to the jejunum andflexura pelvina, regardless to their neurochemical charac-teristics (p<0.05). Furthermore, cholinergic neurons hadlarger soma sizes than the nitrergic population regardlessto the location of the gut examined (p<0.05) as shown inTable 2.

2.3. Calretinin-expressing neurons

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

of the equine intestine. In all locations of the small intestine11 to 50 neuronswere found compared to the large intestine.urons and ganglia comprising >151 neurons were identifiedound regarding the occurrence of mini-ganglia. Mean values

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

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long, polar process and several small, stubby and to someextent arborized processes around the somata. Furthermore,multipolar and roundish cells, sending a number of longprocesses, could be detected in the equine myenteric plexus.However, a few of the CR-expressing cells did not showstaining of any processes.

About 6.5% of the entire neuronal population stained byHuC/D was CR-positive (Table 3). The majority of theseneurons co-expressed ChAT and only a few cells were CR-monolabeled. The appearance of the CR-population in the

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

2.4. Neurons containing vasoactive intestinal polypeptide

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

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

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and to some extent arborized processes could be detectedaround the soma. Moreover, a frequent co-localization of VIPand NOS was detectable (Fig. 6C). On the contrary, neurons co-expressing VIP and CHAT were only occasionally found (datanot shown).

3. Discussion

3.1. Pan-neuronal labeling

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

Fig. 5 – Proportion of nitrergic population in different locations odisplayed similar sizes of the nitrergic subpopulation. In contrastneurons compared to the small intestine (p<0.05). Mean values a

proteins according to cell activity (Ganns et al., 2006). Thedistribution of HuC/D-IR also in processes indicates high cellactivity, whereas HuC/D-IR restricted to the nucleus repre-sents low cell activity. Furthermore, a high variability ofHuC/D-expression in older individuals is well-known (Phil-lips et al., 2004b). In addition, a neuronal drop-out in theENS of older horses was not detectable (Burns and Cum-mings, 1991). Comparisons of different pan-neuronal mar-kers in the ENS such as HuC/D, PGP 9.5, Cuprolinic Blue andthe tract-tracer Fluoro-gold, have found HuC/D and Cupro-linic Blue to be superior (Phillips et al., 2004a). In thepresent study, there were no neurons labeled with otherneuronal markers that were missing HuC/D-IR, demonstrat-ing the reliability of HuC/D as a pan-neuronal marker in theequine ENS.

3.2. Morphology and sizes of ganglia

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

f the equine intestine. All regions of the small intestine, the flexura pelvina contained a larger proportion of nitrergicre presented in %±standard error of mean.

Table 1 – OccurrenceofneuronsexpressingHuC/D-onlyandneuronsco-expressing cholineacetyltransferase (ChAT)andnitric oxide synthase (NOS)

Location NeuronsexpressingHuC/D-only

(%)

Neuronsco-expressingChAT andNOS (%)

Duodenum descendens 42.9±3.1 0.81±0.29Flexura duodeni caudalis 42.7±2.7 0.91±0.32Duodenum ascendens 43.1±2.3 1.64±0.38Jejunum 42.8±2.2 1.20±0.39Flexura pelvina 42.4±1.6 0.12±0.06

Neurons without additional ChAT- and NOS-immunoreactivitywere determined as HuC/D-only nerve cells. No differences couldbe detected comparing the different locations of the equineintestine examined.Meanvalues are presented in%±standard errorof mean.

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geometrical features (Gabella, 1971 and 1987). Moreover, wefound that the long axis of the myenteric ganglia wasorientated parallel on the circular muscle layer, confirmingearlier results of Pearson (1994). This orientation documentedin various small and large animals seems to be a distinctivefeature of the myenteric plexus (Gabella, 1987, Santer andBaker 1988). Close apposition of the neuronal structures to thegut wall allows reorganization of the ganglia in response toconstant mechanical activity from muscle contraction andrelaxation (Gabella, 1990). The parallel orientation of the longaxis on the thicker circular muscle layer may facilitate thereorganization of the ganglia embedded between the musclelayers.

For analysis of ganglion sizes, a precise definition of gangliaboundaries is mandatory. According to Phillips et al., (2004b)theminimal demarcation line between ganglia was defined asdouble the-length of an average sized neuron. In the smallintestine, about 50 to 54% of the ganglia contained 11 to 50neurons, while in the flexura pelvina only 24% of the gangliawere in this range. In contrast, in the flexura pelvina about21% of the ganglia contained 51–150 neurons and about 27%contained more than 150 neurons, whereas in the smallintestine 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 inthe colon between 60 to 100 neurons which might beexplained by the difficulties of defining ganglia boundaries.Other studies on the horse small intestine using paraffinsections documented 1 to 10 neurons or 3.5 neurons per

Table 2 – Summary of somata sizes of cholinergic (choline acetysynthase-expressing) neurons in different locations of the equ

Duodenumdescendens

Flexura dcaud

Cell sizes (μm2) of cholinergicneurons

1055±44 1063±

Cell sizes (μm2) of nitrergicneurons

916±45 938±

In general, cholinergic neurons displayed larger perikarya compared to theerror of mean.

ganglia (Pogson et al., 1992; Scholes et al., 1993). Thechemoarchitecture of the myenteric plexus in its naturalareal expanse cannot be analyzed on transversal paraffinsections, which could explain the different results. Moreover,we recorded ectopic neurons, defined as 1 to 4 neurons notorganized in ganglionic structures. In the small intestine,higher numbers of ectopic neurons were documented withinthe duodenum ascendens (9.6% of all neurons) than in theflexura pelvina (3.8%). In the guinea pig ileum, 10% of all nervecells were defined as ectopic applying the same pan-neuronalmarker Hu (Abalo et al., 2005). On the contrary, Costa et al.(1996) revealed only 1.1% as ectopic in the guinea pig ileumusing PGP 9.5 as a “pan-neuronal” marker. Comparisons ofdifferent studies appear problematic because of differingdefinitions of ectopic neurons and the use of different pan-neuronal markers. In conclusion, the ganglionic pattern in theflexura pelvina is more tightly organized compared to thesmall intestine of the horse; the flexura pelvina displays largeganglia and low numbers of ectopic neurons compared to thesmall intestine of the horse.

3.3. Neuronal packing densities

Many studies have been carried out to determine neuronaldensity related to the serosal area (Furness, 2006). Karaos-manoglu et al. (1996) have shown a relation between theneuronal density and the grade of tissue stretching prior tofixation: increasing the tissue stretching by 32% resulted in a31% reduction of the neuronal packing density in thejejunum–ileum segment of the guinea pig. Nevertheless,neuronal packing density, defined as neurons/cm2 ganglionicarea, was found to be stretch-independent which allowsreproducible and comparable measurements (Karaosmano-glu et al., 1996). In contrast to the stretch-dependent non-neuronal tissue, the ganglionic area consists of neurons,enteric glial cells and neuropil, but is devoid of connectivetissue, blood vessels or smooth muscle cells (Gabella, 1990;Natali et al., 2000). The neuron packing density of themyenteric plexus of the small and large intestine in theguinea pig accounted for 240 000 to 250 000 neurons/cm2

ganglionic area, whereas in the rat 200 000 to 250 000 neuronscould be detected in the small intestine and 130 000 neuronsin the large intestine (Karaosmanoglu et al., 1996, Phillips etal., 2004b). In the equine myenteric plexus, we calculated aneuron packing density of 52 000 to 58 000 neurons/cm2

ganglionic area. Although Gabella (1990) specified the neuro-nal densities as neurons/cm2 serosal area, mice had the

ltransferase-containing) neurons and nitrergic (nitric oxideine intestine

uodenialis

Duodenumascendens

Jejunum Flexurapelvina

39 1050±44 985±33 905±28

48 911±38 799±43 775±26

nitrergic neurons (p<0.05). Mean values are presented in μm2±standard

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

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highest and sheep the lowest numbers. Furthermore, theneuropil of the mice accounted for 1/2 and that of the sheep3/4 of the ganglionic area in the small intestine (Gabella,1990). On the same subject, increasing divergence (numbersof neurons innervated by one axon) and convergence(numbers of axons innervating one neuron) are detectablewhen homologous neuronal structures of large animals arecompared with those of smaller species (Purves and Licht-man, 1985; Purves et al., 1986). Discrepancies between our

data on the horse and those on small laboratory animals canbe related to the substantial differences between the speciescompared.

3.4. Cholinergic neurons

Acetylcholine is one of themost important transmitters in theENS which can be found in excitatory motoneurons, inter-neurons and IPAN (Costa et al., 2000). For the detection of

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

Duodenumdescendens

Flexura duodenicaudalis

Duodenumascendens

Jejunum Flexurapelvina

CR-expressing neurons (%) 6.6±0.8 7.4±1.2 5.7±0.7 6.8±0.8 5.5±0.7CR-only (%) 0.23±0.2 0.49±0.2 0.08±0.1 0.18±0.1 0.44±0.2ChAT neurons co-expressing CR (%) 14.4±1.6 15.6±2.2 13.3±1.7 17.1±2.4 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 wereCR-monolabeled (CR-only). There were no significant differences in the distribution of CR-positive neurons in the different gut locationsexamined. Mean values are presented in %±standard error of mean.

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cholinergic neurons different cholinergic markers such as theenzyme catalyzing the transmitter synthesis (ChAT), thetransmitter degrading enzyme (acetylcholinesterase), thevesicular acetylcholine transporter (VAChT) and the cholinetransporter are commonly used (Leaming and Cauna, 1961;Usdin et al., 1995; Costa et al., 2000; Harrington et al., 2007). Inthe present study, ChAT simultaneously revealed strongly andweakly immunoreactive neurons with homogenous stainingof the cytoplasm. Similar staining features were revealed inthe ENS of the guinea pig (Li and Furness, 1998; Chiocchettiet al., 2003). Verifying the cholinergic phenotype, simulta-neous labeling of ChAT and VAChT showed extensive co-expression in neuronal processes, but lack of VAChT-IR inperikarya. Sang and Young (1998) demonstrated VAChT-IR inenteric neuronal processes of mice, but not in somata. Incontrast, VAChT-ir somata were detected in the equine ENS(Domeneghini et al., 2004). The use of different primaryantibodies might partially explain the differing results. Inthe equine small intestine about 36% of all neurons wereshown to be cholinergic, while the flexura pelvina contained24% ChAT-ir nerve cells. In contrast, 60% of the neurons in thesmall intestine of mice and 65% in the human ileum displayedChAT-IR (Porter et al., 1996; Sang and Young, 1998). Addition-ally, a cholinergic phenotype was reported for 57% (of allneurons) in the large intestine of the guinea pig and for 63% ofthe human enteric neurons (Porter et al., 1996; Lomax andFurness, 2000). Because different “pan-neuronal” markersused in order to quantify cholinergic subpopulations, e.g.,PGP 9.5, neuron-specific enolase or anti-nerve cell body, acomparison between different studies can be difficult.

Murphy et al. (2007) quantified ChAT-expressing neuronsin the human large intestine applying the pan-neuronalmarker Hu and determined that this subpopulation was 48%of the neurons. Discrepancies with our results may beexplained by the fact that the ChAT-antibody we used did

Table 4 – Primary antibodies used for immunohistochemistry

Primary antibody Host Dilution Fluorochrome

HuC/D Mouse 1:100 Cy3 InvitNeuronal-NOS Rabbit 1:200 Cy2 TranNeuronal-NOS Mouse 1:50 Cy3 BD BChAT Goat 1:25 Cy5 MillipVAChT Rabbit 1:200 Cy2 SynaCR Rabbit 1:300 Cy2 SwanVIP Rabbit 1:200 Cy2 Diaso

not recognize all cholinergic neurons in the equine ENS. In theperipheral tissue, two types of ChAT mRNA arising fromalternative splicingwere detected: a peripheral (p)-ChAT and acommon (c)-ChAT (Tooyama and Kimura, 2000). In the centralnervous system, cChAT represents the exclusive ChAT-form(Tooyama and Kimura, 2000). Moreover, in the porcine ileum22.4% of the neurons were labeled by both ChAT forms and31% expressed cChAT alone, while 27.8% of the neuronsdisplayed pChAT exclusively (Brehmer et al., 2004). In addi-tion, investigations in the guinea pig ENS revealed a sub-population of cholinergic neurons expressing pChAT alone(Chiocchetti et al., 2003). Further elucidation of the equinecholinergic population in themyenteric plexus is required alsousing antibodies directed against pChAT.

3.5. Nitrergic neurons

Nitrergic neurons revealed by immunolabeling of their markerenzyme NOS are known to be inhibitory motoneurons anddescending interneurons in the guinea pig small intestine(Furness 2000). Furthermore, it is known that nitrergic neuronsinnervate and support interstitial cells of Cajal (ICC), whichhave a putative role in control of intestinal motility (Hudson etal., 1999; Choi et al., 2007; Iino et al., 2008). In the equinemyenteric plexus, nitrergic activity was successfully demon-strated (Rakestraw et al., 1996). We identified 20 to 22% of theHuC/D-labeled neurons in the small intestine as nitrergicneurons, while in the flexura pelvina 33% displayed NOS-IR.Using different pan-neuronal markers, the nitrergic compo-nent of all neurons in the myenteric plexus comprises 26% inmice, 20% in human small intestine and 19% in guinea pigileum (Furness et al., 1994; Sang and Young, 1996; Belai andBurnstock, 1999). Moreover, in the large intestine NOS-IR wasexpressed in 35% of mouse enteric neurons and in 39% ofguinea pig enteric neurons (Sang and Young, 1996; Lomax and

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rogen, Karlsruhe, Germany Phillips et al., 2004asduction Labs, Heidelberg, Germany Lüth et al., 2000ioscience, Heidelberg, Germany Sasaki et al., 2000ore, Billerica, MA, USA Li and Furness, 1998ptic Systems, Göttingen, Germany Härtig et al., 2007t, Bellinzona, Switzerland Schwaller et al., 1993rin, Dietzenbach, Germany Kawaguchi and Kubota, 1996

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Furness, 2000). In the human colon, quantification based onHuC/D revealed 43% nitrergic neurons (Murphy et al., 2007).Differences between the data on human nitrergic populationsand our results may be related to an age-dependent increaseof nitrergic neurons in humans, as the patients examined hadan average age of 68 years (Belai and Burnstock, 1999; Murphyet al., 2007). In addition, six different splicing variants of theenzyme have been identified in the human ENS and thedevelopment of new antibodies could support a more detailedanalysis of nitrergic subpopulations (Saur et al., 2000; Breh-mer, 2006). Moreover, there is evidence for a pacemakeractivity in the flexura pelvina of the horse (Sellers, 1982;Burns and Cummings, 1991). Further investigation especiallyof different sampling sites of the colon ascendens is needed toincrease information about these subjects.

Enteric glial cells are identified as a reservoir of L-argininewhich is a precursor of nitric oxide (NO) (Nagahama et al.,2001). NOS catalyzes the conversion of L-arginine into citrul-line and NO (Prince and Gunson, 1993). In general, all nitrergicneurons displayed smaller perikarya compared to the choli-nergic neurons regardless to their location. Smaller cell sizesof nitrergic neurons may be explained by the extra-neuronalreservoir of the precursor substances L-arginine in glial cellsand the fact that this gaseous neurotransmitter is not stored invesicles.

3.6. Co-expression of ChAT and NOS

The triple staining HuC/D/ChAT/NOS revealed a small propor-tion of neurons co-expressing ChAT and NOS (0.1 to 2%). Thesame marker combination was reported for 4% of double-stained ChAT/NOS-neurons in the human colon (Murphy etal., 2007). In the duodenumof the guinea pig, co-localization ofpChAT and NOS was detected in 3.6% of all neurons, whereasin 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 asdescending interneurons (Costa et al., 2000; Furness, 2000).

3.7. Calretinin-expressing neurons

Excitatory motoneurons innervating the outer longitudinalmuscle and ascending interneurons in themyenteric plexus ofthe guinea pig small intestine are CR-ir (Furness, 2000). In theequine myenteric plexus, we have shown CR-IR in neuronsand neuronal processes almost entirely co-localized withChAT. CR-containing moto- and interneurons of the guineapig are cholinergic as well (Brookes, 2001). We identified mostof the CR-expressing neurons in the investigated parts of thehorse ENS as Dogiel type I neurons, while cells displayingDogiel type II morphologywere rarely detectable. Additionally,CR-ir neuronal processes did not form a tertiary plexus,confirming earlier results that only small animals with athin stratum longitudinale have a tertiary plexus in order toinnervate this muscle layer (Llewellyn-Smith et al., 1993;Furness et al., 2000). In contrast, neuronal processes of largeanimals form a network within the longitudinal muscle layer(Furness et al., 2000). Furthermore, excitatorymotoneurons forthe longitudinal muscle layer were distinguishable fromascending interneurons displaying smaller soma areas (Pom-

polo and Furness 1993). In this study, all CR-labeled neuronswere characterized by a largely identical cell body size givingno possibility for differentiation. Retrograde labeling studiescould further elucidate functional aspects of the equine, CR-expressing neurons.

3.8. Neurons containing vasoactive intestinalpolypeptide (VIP)

In the ganglionic area, we detected strong VIP-IR in neurons aswell as in neuronal processes. Some neurons displayed anaccumulation of VIP-IR in parts of their soma, apparentlyresulting from colchicine utilization. Colchicine inhibitsaxonal transport of neuropeptides and is useful for theirvisualization also in the perikarya (Ekblad and Bauer, 2004).VIP-expressing cells were identified as Dogiel type I neuronsshowing a single long process and several short, lamellardendrites. Furthermore, distinctive co-localization of VIP andNOS was detected in the myenteric neurons of the horse. VIPandNOS co-expression iswell known inmyenteric neurons of,e.g., guinea pig, dog and man (Wang et al., 1998; Lomax andFurness, 2000; Brehmer et al., 2006). Inhibitorymotoneurons inguinea pig and man contain VIP and NOS among othertransmitters (Porter et al., 1997; Brookes, 2001). VIP-ir neuronalprocesseswere identified in the circularmuscle layer of horses(Burns and Cummings 1993) suggesting that VIP acts as atransmitter in motoneurons. Moreover, we showed rare co-localization of VIP and ChAT in equine myenteric neurons inline with data on the co-expression of VIP and ChAT indescending interneurons of guinea pig and man (Wattchow etal., 1997; Furness, 2000).

4. Experimental procedures

4.1. Tissue preparation

A first set of tissue samples was taken from 8 horses thatshowed no sign of gastrointestinal disturbances at localabattoirs near Leipzig, Germany. The samples (5×5 cm) weretaken from several locations of the small intestine (duodenumdescendens, flexura duodeni caudalis, duodenum ascendens,mid jejunum) and of the large colon (flexura pelvina).Furthermore, the College of Veterinary Medicine (Universityof Florida, Gainesville, USA) provided additional samples ofthe flexura pelvina from 7 horses. The age of the animalsranged from 4 to 22 years.

All specimens were rinsed clean and transported in cooledcarbogen bubbled Krebs solution, pH 7.4 (in mM: 117 NaCl, 25NaHCO3, 1.2 NaH2PO4, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 11.5 mMglucose, 1 μM nifedipine; all chemicals from Sigma, Tauf-kirchen, Germany). Nifedipine was added to relax the smoothmuscle by blocking calcium-channels and this facilitatedfurther handling of the tissues. For fixation in 4% paraformal-dehyde saturated with picric acid, the samples were dissectedfree from the mucosal layer, stretched out and pinned flat inSylgard®-covered Petri dishes (Dow Corning, Auburn, MI, USA;24 h, 5°C). The fixed specimens were rinsed in 0.1 Mphosphate-buffered saline (PBS, pH 7.4) and stored in 0.1 MPBS containing 0.1% NaN3.

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Tissue used for neuropeptide staining was incubated withcolchicine in order to enhance immunosignals in the myen-teric neuronal somata. Specimens dissected free from themucosal layer were placed in a sterile culture medium(Dulbeccos modified eagles medium) for 24 h and kept in anincubator environment of 37°C, 95%O2 and 5%CO2. The culturemedium was enriched with 100 IU/ml penicillin, 100 μg/mlstreptomycin, 20 μg/ml gentamicin, 1.24 μg/ml amphotericin B,1 μM nifedipine, 80 μM colchicine and 10% heat-inactivatedfetal calf serum (all chemicals from Sigma, Taufkirchen,Germany). During the culture period, the samples wereconstantly moved on a rocking tray. All devices, instrumentsand solutions used in order to accomplish colchicine incuba-tion weremaintained under sterile conditions. The specimenswere fixed and stored as described above.

Further tissue assessment was achieved by using astereomicroscope with an integrated light source (StemiSV11, Zeiss Jena, Germany). The myenteric plexus was care-fully dissected free from the serosal layer, the circular andlongitudinal muscle layer and, if necessary, from the rem-nants of mucosal layer. This preparation was successfullycarried out on maximally stretched tissue samples. Untilfurther investigation, the whole-mounts were stored in 0.1 MPBS containing 0.1% NaN3.

4.2. Immunofluorescence labeling

Multiple immunofluorescence labeling was performed onfree floating preparations which had been extensivelyrinsed with 0.1 M Tris-buffered saline, pH 7.4 (TBS). Blockingof unspecific binding sites and permeabilization wasachieved by incubating the tissues in 5% donkey serumand 0.3% Triton-X100 diluted in TBS. Next, whole-mountswere transferred into a cocktail of primary antibodiesdiluted in the same blocking solution for 18 h. Primaryantibodies used, their hosts, dilutions, suppliers and refer-ences are listed in Table 4. All markers were demonstratedsimultaneously by fluorescent, highly purified secondarydonkey antibodies (from Dianova, Hamburg, Germany)conjugated to green fluorescent Cy2, red fluorescent Cy3or infrared light-emitting Cy5. All secondary antibodiesdirected against rabbit, mouse or goat IgGs were used at20 μg/ml in TBS containing 2% bovine serum albumin for1 h. Controls were performed by omission of primaryantibodies resulting in the expected absence of cellularlabeling by fluorochromated secondary antibodies. Allwhole-mounts were finally washed extensively with TBS,rinsed briefly in distilled water, mounted onto fluorescence-free 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 differentsubpopulations were routinely carried out with a confocallaser scanning microscope (cLSM 510 Meta, Zeiss Oberkochen,Germany). The cLSM also allowed the detection of Cy5-signalswhich were color-coded in blue. Furthermore, the neuronalpacking densities, defined as neurons/cm2 ganglionic areawere determined with cLSM.

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

Acknowledgments

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

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