Distribution of P2X3 receptor immunoreactivity in myenteric ganglia of ...

Histochem Cell Biol (2009) 131:13–27 DOI 10.1007/s00418-008-0498-4

ORIGINAL PAPER

Distribution of P2X3 receptor immunoreactivity in myenteric ganglia of the mouse esophagus Christine Kestler · Winfried L. Neuhuber · Marion Raab

Accepted: 14 August 2008 / Published online: 20 September 2008 © Springer-Verlag 2008

Abstract Intraganglionic laminar endings (IGLEs) represent the major vagal aVerent terminals throughout the gut. Electrophysiological experiments revealed a modulatory role of ATP in the IGLE-mechanotransduction process and the P2X2-receptor has been described in IGLEs of mouse, rat and guinea pig. Another purinoceptor, the P2X3-receptor, was found in IGLEs of the rat esophagus. These Wndings prompted us to investigate occurrence and distribution of the P2X3-receptor in the mouse esophagus. Using multichannel immunoXuorescence and confocal microscopy, P2X3immunoreactivity (-iry) was found colocalized with the vesicular glutamate transporter 2 (VGLUT2), a speciWc marker for IGLEs, on average in three-fourths of esophageal IGLEs. The distribution of P2X3 immunoreactive (-ir) IGLEs was similar to that of P2X2-iry and showed increasing numbers towards the abdominal esophagus. P2X3/P2X2colocalization within IGLEs suggested the occurrence of heteromeric P2X2/3 receptors. In contrast to the rat, where only a few P2X3-ir perikarya were described, P2X3 stained perikarya in »80% of myenteric ganglia in the mouse. Detailed analysis revealed P2X3-iry in subpopulations of nitrergic (nNOS) and cholinergic (ChAT) myenteric neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a (P2X2)-P2X3 receptor-mediated pathway especially in the abdominal portion of the mouse esophagus. Keywords Intraganglionic laminar endings · Enteric nervous system · Vagus · Purinergic transmission · Mouse (C57Bl/6)

C. Kestler · W. L. Neuhuber · M. Raab (&) Institut für Anatomie, Lehrstuhl I, Universität Erlangen-Nürnberg, Krankenhausstr. 9, 91054 Erlangen, Germany e-mail: [email protected]

Introduction In the last three decades, adenosine triphosphate (ATP) has become widely recognized as a fast synaptic transmitter in both central and peripheral nervous systems (Bardoni et al. 1997; Burnstock 1978; Dunn et al. 2001; Edwards et al. 1992; Evans et al. 1992). In the gastrointestinal tract there is abundant evidence that ATP acts as a neurotransmitter, being released from either extrinsic sympathetic eVerent nerves or from intrinsic enteric neurons (Burnstock 1990; Galligan and Bertrand 1994; McConalogue et al. 1996). ATP is also released from tissues during stretch and physical damage (Bodin and Burnstock 2001; Knight et al. 2002; Nakamura and Strittmatter 1996; Vlaskovska et al. 2001), leading to the suggestion that purinergic transmission may play an important role in nociception in damaged or inXamed tissue (Burnstock 2001). Two major families of ATP receptors (purinergic receptors, purinoceptors) have been identiWed as P1 and P2, showing diVerent aYnities to adenosine and ATP/adenosine diphosphate (ADP), respectively (Burnstock 1978; 1996). The P2 receptors fall into two families, P2X and P2Y (Abbracchio and Burnstock 1994; Burnstock and Kennedy 1985). The P2X receptors are multimeric proteins that combine to form a ligand-gated cation channel (Khakh et al. 2001; North 2002) with almost equal permeability to Na+, K+, and signiWcant permeability to Ca2+ (Evans et al. 1996). There are seven P2X receptor subunit proteins (P2X1–P2X7) with each subunit having two membrane spanning domains (Burnstock and King 1996; Khakh et al. 2001; North 2002). The P2X subunits are able to assemble into functional homo- and hetero-oligomeric channels (Khakh et al. 2001). The subunit composition of P2X receptors determines their pharmacological and functional properties (North and Surprenant 2000). Most of these

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receptors are widely distributed not only throughout the central and peripheral nervous systems, but also in nonneuronal tissues (Burnstock et al. 2004; Dunn et al. 2001). Both immunohistochemistry and molecular biology indicated that the P2X3 receptor subunit is the main type to be expressed by sensory neurons. While the non-peptidergic subpopulation of small nociceptive neurons in dorsal root ganglia express predominantly P2X3 receptors, nodose ganglion neurons express mixed populations of P2X2 and heteromeric P2X2/3 receptors (Bradbury et al. 1998; Chen et al. 1995; Dunn et al. 2001; Lewis et al. 1995). In the enteric plexus ATP functions as a neurotransmitter mediating fast-excitatory synaptic transmission (ESPSs; Galligan et al. 2000), and is involved in intrinsic enteric reXex pathways associated with motor complex formation (Brierley et al. 2001). The ATP may also be involved in gastrointestinal aVerent functions exciting mechanosensors in esophagus and intestine (Kirkup et al. 1999; Page et al. 2002). Immunocytochemical studies have identiWed several P2X purinergic receptors widely distributed in the enteric neurons of the intestine. However, the subunit composition of functional P2X receptors has not been established. It has been suggested that the enteric neurons of the small intestine of diVerent species including human express predominantly P2X2 receptors (Castelucci et al. 2002; LePard et al. 1997; Zhou and Galligan 1996), while a minority heterologously expresses P2X1 and P2X3 receptors (Facer et al. 2001; Poole et al. 2002; Van Nassauw et al. 2002; Yiangou et al. 2000; Zhou and Galligan 1996). In the rat esophagus only a few myenteric perikarya were found to express P2X2 or P2X3-receptors (Wang and Neuhuber 2003), probably indicating signiWcant species diVerences or/and diVerences between diVerent segments of the gastrointestinal tract. Distribution and further characterization of immunopositive myenteric neurons within the esophagus or cell counts were not determined in that study. Intraganglionic laminar endings (IGLEs; Rodrigo et al. 1975) are derived from nodose ganglion neurons (Berthoud and Powley 1992; Neuhuber 1987; Rodrigo et al. 1982) and represent the most prominent terminal structures of vagal aVerent Wbers throughout the gastrointestinal tract (Berthoud et al. 1997a; Phillips and Powley 2000; Wang and Powley 2000). They originate from relatively coarse myelinated branching nerve Wbers and more or less extensively enwrap the enteric ganglia sandwiched between the outer and inner layers of the tunica muscularis (Rodrigo et al. 1975). For better diVerentiation of laminae and puncta within one IGLE displayed by immunoXuorescence the term “single IGLE-bouton” (SIB) was introduced by Ewald et al. 2006. The intimate relationship of IGLEs to connective tissue layers enclosing myenteric ganglia renders them ideally suited to participate in deformations during peristal-

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Histochem Cell Biol (2009) 131:13–27

sis subserving a mechanosensory function as low-threshold tension sensors (Neuhuber 1987; Neuhuber and Clerc 1990; Phillips and Powley 2000) which has been proven in vitro by electrophysiological experiments (Zagorodnyuk and Brookes 2000; Zagorodnyuk et al. 2001). Ultrastructural investigations have revealed small clear vesicles clustered at membrane specializations suggesting synaptic contacts of IGLEs onto myenteric neurons (Neuhuber 1987; Neuhuber and Clerc 1990; Neuhuber et al. 2006; Phillips and Powley 2007). VGLUT2- (Raab and Neuhuber 2003; 2004; Zagorodnyuk et al. 2003) and VGLUT1-immunoreactivity (-iry) (Ewald et al. 2006; Kraus et al. 2007; Zagorodnyuk et al. 2003) has been detected in esophageal IGLEs of rat, mouse, and guinea pig, indicating vesicular storage and possibly also release of glutamate from primary vagal aVerent endings. The VGLUT2-iry has been proven to serve as a speciWc marker for qualitative and quantitative investigations of IGLEs of the mouse esophagus (Raab and Neuhuber 2003; 2004; 2005). In addition, substance P (SP) was found in IGLEs of mouse and rat esophagus (Kressel and Radespiel-Tröger 1999; Raab and Neuhuber 2004). Electrophysiological experiments demonstrated that ATP may play a modulatory role in the IGLE-mechanotransduction process, but the nature of the P2X receptor involved is yet unclear (Zagorodnyuk et al. 2003). Both P2X2 and P2X3 were detected in 80 and 82%, respectively, of IGLEs of the rat esophagus (Wang and Neuhuber 2003). Recently, P2X2 purinergic receptor-iry was described in 50% of IGLEs of the mouse (Raab and Neuhuber 2005). We have therefore immunohistochemically investigated the occurrence and distribution of the P2X3 receptor-iry in the mouse esophageal myenteric ganglia, with special emphasis on both IGLEs and myenteric neurons and focused on the following questions: (1) how is the distribution of the P2X3 receptor subunit bearing structures along the esophagus? Are these structures also shifted to lower esophageal parts comparable to the P2X2 receptor bearing IGLEs (Raab and Neuhuber 2005)? (2) Is there a colocalization of P2X2 and P2X3 receptor subunits, probably indicating occurrence of heteromultimeric P2X2/3 receptors within IGLEs or myenteric neurons?

Materials and methods Animals Thirteen adult mice (C57Bl/6, The Jackson Laboratory, Bar Habor, USA; stock number 000664, inbred) were used. The federal animal welfare legislation implemented by the local government was followed for all procedures performed on the animals.


Tissue preparation The mice were deeply anesthetized with an overdose of carbon dioxide, followed by a lethal dose of Thiopental (Trapanal; Byk Gulden, Konstanz, Germany; 250 mg/kg i.p.). As soon as they were unresponsive to nociceptive stimuli, the abdomen and the thorax were opened, and the mice were perfused transcardially with 20 ml Ringer solution containing 1,000 IE/100 ml heparin followed by 80 ml ZamboniWxative (2% paraformaldehyde in 0.1 M phosphate buVer and 0.2% picric acid) and Wnally 10 ml 15% sucrose phosphate buVer (pH 7.4). The esophagus from the level of the cricoid cartilage to the gastroesophageal junction was removed, transferred to 15% sucrose phosphate buVer (pH 7.4) at 4°C, and freed from adhering connective tissue, including the main vagal nerve trunks, under a dissecting microscope. The following day the esophagi were divided into three segments of similar length, which deWned the cervical (= upper third), thoracic (= middle third) and abdominal (= lower third) parts. These segments were mounted in Tissue-Tek (GSV 1, Slee Technik, Mainz, Germany), rapidly frozen in methylbutan at ¡70°C and stored at ¡20°C. Antibodies and reagents For P2X3 immunostaining we used P2X3 antibodies raised in rabbit and guinea pig against a 15 amino acid peptide corresponding to amino acids 383–397 from the carboxyterminus of the rat P2X3 receptor protein (rP2X3; Chemicon, Temecula, California, USA; Code AB5895; gpP2X3; Chemicon; AB5896). Table 1 List of primary antibodies and antisera used for immunohistochemistry

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To test the speciWcity of both the rP2X3 and gpP2X3 antibodies in the mouse esophagus, we blocked the antibody binding sites by preincubation with the corresponding peptide (P2X3 control protein, Chemicon, USA; Code AG356). As suggested by the supplier, preadsorption of the respective antibody with the one, two and tenfold antigen excess was performed overnight at 4°C. The next day the antigen-antibody mixture was spun at 14.000 g for 10 min to sediment the precipitating antigen-antibody complexes and thus avoid high background staining. The supernatant was then used instead of the primary antibody. For counterstaining of IGLEs the rVGLUT2 antibody was used in the preadsorption control of the gpP2X3-receptor antibody. All primary and secondary antibodies used in this study are listed in Tables 1 and 2. Double-label immunohistochemistry Double-label immunohistochemistry was used for the detection of rP2X3 and VGLUT2, an established speciWc marker for IGLEs (Raab and Neuhuber 2003; 2004; 2005). We also combined rP2X3 with markers for primary spinal aVerents (CGRP) and nitrergic (nNOS), cholinergic (ChAT) and monaminergic (TH) myenteric neuronal cell bodies. The gpP2X3 was used for P2X2-receptor and S100 Protein double-immunostainings (Table 1). Twelve-m thick cryostat sections were mounted on poly-L-lysine-coated slides and dried for 1 h at room temperature. Sections were preincubated with 5% normal donkey serum (DAKO, Hamburg, Germany) containing 0.5% Triton X-100 (Merck, Darmstadt, Germany), 1% bovine

Antigen (abbreviation)

Host

Working dilution

Source and code

P2X3

rabbit

1:800

Chemicon, Temecula, California, USA; Code AB5895

P2X3

guinea pig

1:250

Chemicon, Temecula Code AB5896

P2X2

rabbit

1:250

Chemicon, Temecula, Code AB5244

Vesicular glutamate transporter 2 (VGLUT2)

rabbit

1:1000

Synaptic Systems, Göttingen, Germany, Code 135 102

Vesicular glutamate transporter 2 (VGLUT2)

guinea pig

1:1,500

Chemicon, Temecula, Code AB5907

Choline acetyltransferase (ChAT)

goat

1:100

Chemicon, Temecula, Code AB144P-200UL

Neuronal nitric oxide synthase (nNOS)

guinea pig

1:1,500

Progen, Heidelberg, Germany; Code 16059

Tyrosine hydroxylase (TH)

sheep

1:2,000

Novus Biologicals, Littleton, USA; Code NB300-110

Calcitonin gene-related peptide (CGRP)

goat

1:1,000

Bio Trend, Cologne, Germany; Code 1720-9007

S100 Protein (S100)

rabbit

1:200

DakoCytomation, Glostrup, Denmark; Code N1573

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Table 2 List of secondary antibodies and detection systems used for immunohistochemistry Secondary antibodies and detection systems

Working dilution

Source and code

ALEXA Fluor® 488-conjugated donkey anti-rabbit

1:1,000

Molecular Probes, Eugene, Oregon, USA, distributed by: MoBiTec, Göttingen, Germany; Code A21206

Cy3-conjugated donkey anti-guinea pig

1:1,000

Jackson Immunoresearch, USA, distributed by: Dianova, Hamburg, Germany; Code 706-165-148

ALEXA Fluor® 555-conjugated donkey anti-goat

1:1,000

Molecular Probes, USA; Code A-21432

ALEXA Fluor® 555-conjugated donkey anti-sheep

1:1,000

Molecular Probes, USA; Code A-21436

serum albumin (BSA; Roth, Karlsruhe, Germany) in TrisbuVered saline (TBS, pH 7.4; Roth) for 1 h at room temperature. After being rinsed in TBS, the sections were incubated with primary antibodies against rP2X3 and gpP2X3, respectively, and either VGLUT2, calcitonin gene-related peptide (CGRP), P2X2 receptor, S100 Protein (S100), or neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Working dilutions and suppliers are listed in Table 1. All primary antibodies were diluted in TBS containing 1% BSA and 0.5% Triton X-100, and incubated overnight at room temperature. After being washed with TBS, the sections were incubated with donkey anti-rabbit secondary antibodies coupled to ALEXA Fluor® 488, donkey anti-guinea pig secondary antibodies coupled to indocarbocyanine (Cy3), donkey anti-goat secondary antibodies coupled to ALEXA Fluor® 555 or donkey anti-sheep secondary antibodies coupled to ALEXA Fluor® 555 for 1 h at room temperature. For working dilutions used and suppliers see Table 2. All secondary antisera contained 0.5% Triton X-100 and 1% BSA in TBS buVer. The sections were rinsed again with TBS, coverslipped with TBS-glycerol (pH 8.6) and viewed under a Leica Xuorescence microscope (Aristoplan, Leica, Bensheim, Germany). For controls, primary antibodies were replaced by TBS containing 0.5% Triton X-100 and 1% BSA (all antibodies) or preadsorbed (rP2X3 and gpP2X3 this study; for P2X2 see Castelucci et al. 2002; for gpVGLUT2 and rVGLUT2 see Raab and Neuhuber 2004). All primary antibodies used in double-label experiments were Wrst tested in single incubations and the results were checked with those of doubleimmunohistochemistry. The dilution of primary antibodies used has been determined by performing dilution series. All double-label experiments were cross-checked with diVerent secondary antibodies. Analysis of P2X3/VGLUT2 immunostaining In order to get more detailed information about the location and co-location of the P2X3 receptor within IGLEs doubleimmunohistochemistry for P2X3 and VGLUT2 was

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performed. Altogether we evaluated 174 VGLUT2-ir IGLEs: 62 in the upper, 57 in the middle and 55 in the lower third of the esophagus. The pattern of P2X3 labeling allowed qualitative diVerentiation of VGLUT2 immunopositive IGLEs into three groups: a “high colocalization” and “partial/low colocalization” subgroup, respectively, in contrast to a “no colocalization” group. Colocalization of the “highly colocalized” type was morphologically deWned as a colocalization over the whole ganglionic area, resulting in a huge amount of yellow spots. By contrast colocalization of the “partial/low colocalization” type was assumed as a colocalization only over some parts of the ganglionic area and only in a few spots, resulting in a small amount of yellow spots. In the “no colocalization” group no spot of colocalization of VGLUT2- and P2X3-iry was found within the same proWle. Chemical coding of P2X3 immunopositive myenteric neuronal cell bodies The myenteric ganglia were identiWed by VGLUT2 immunostaining. As anterograde tracing studies in both rat (Neuhuber et al. 1998) and mouse (Raab et al. 2003) indicated that virtually all myenteric ganglia in the esophagus were innervated by IGLEs, we also compared the distribution of myenteric ganglia stained with cuprolinic blue histochemistry with that of IGLEs immunostained for VGLUT2. We found that the number of cuprolinic blue-stained myenteric ganglia was similar to that of VGLUT2 immunostained IGLEs in the esophagus (Raab and Neuhuber 2005). For determining the chemical code of P2X3 immunopositive perikarya, ganglia from all three parts of the esophagus were randomly selected and analyzed by confocal laser scanning microscopy. For quantiWcation of P2X3/ChAT costained myenteric neuronal cell bodies a sample of 149 myenteric ganglia was analyzed (48 upper-third, 54 middlethird, 47 lower-third). For quantiWcation of P2X3/nNOS immunopositive myenteric cell bodies a sample of 120 myenteric ganglia (39 upper-third, 40 middle-third, 41 lower-third), and for P2X3/TH co-stained myenteric perikarya a sample of 50 myenteric ganglia (15 upper-third, 15 middle-third, 20 lower-third) was evaluated.


For counting of co-stained myenteric cell bodies all single optical sections of the confocal z-series each as well as the projections were studied to avoid counting errors. Confocal microscopy and image analysis Confocal images were obtained by using a BioRad MRC 1000 confocal laser scanning system (BioRad, Hemel Hamstead, UK) equipped with a three-line krypton-argon laser (American Laser, Salt Lake City,Utah, USA) and attached to a Nikon Diaphot 300 microscope (Nikon, Düsseldorf, Germany). The Wlter settings of the BioRad confocal scanner for double-channel analysis were: 488-nm excitation for ALEXA Fluor® 488 (Wlter DF32), 568-nm excitation for Cy3 and ALEXA Fluor® 555 (Wlter 605 DF322). A 60x oil immersion objective lens (numerical aperture 1.4) was used. Zoom factors varied between 1.0 and 3.6. Extended-focus images or z-series of up to 13 optical sections at z-increments of 0.6–1.0 m were scanned. Images of 768 £ 512 pixels were obtained, and the two channels of each z-series were merged into an 8-bit RGB tif-Wle by using confocal assistant software 4.02. In order to test for colocalization, single sections at the same focus plane were taken out of these z-series, and the two channels were merged. After recording by confocal laser scanning microscopy, no alternations of image Wles by additional image processing were performed. In order to document details of close appositions of P2X3 to CGRP immunostained varicose Wbers, selected areas of images were electronically enlarged (“zoom-ins”). For the Wgure arrangement, color images were imported from confocal assistant software into Photoshop software

Fig. 1 a–f SpeciWcity of rP2X3 and gpP2X3 antibodies. a rP2X3 shows coarse lamellar and Wne granular immunostaining (arrows in a) typical for intraganglionic laminar endings (IGLEs; single optical section). In b additional perikaryal labeling of myenteric neurons is seen (asterisks indicate neuronal nuclei; single optical section). The red bordered asterisk is situated in the centre of the nucleus of a myenteric perikaryon and is surrounded by three nucleoli. Distinguishing the P2X3-ir myenteric neuronal cell body from P2X3-ir IGLEs or varicosities is not possible in this single immunostaining. c Extended-focus projection of a myenteric ganglion after a twofold block with the

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(Adobe Photoshop CS, version 8.0.1, Adobe Systems, Unterschleißheim, Germany). Photoshop and Coreldraw software (Coreldraw, version 11, Corel, Dublin, Ireland) were used in order to apply text and scale bars, to adapt brightness and contrast, and to organize the Wnal layout.

Results P2X3-immunoreactivity in the mouse esophagus The P2X3-iry was detected in sections from all esophageal levels. The distribution of P2X3-iry was mainly restricted to Wbers within and areas between inner and outer striated muscle layers. The P2X3-iry showed a Wne granular labeling pattern in the myenteric ganglionic neuropil (arrows in Fig. 1a) as well as in myenteric neuronal cell bodies (Fig. 1b). In this consistent staining, created by many Wne Xuorescent dots, neuronal cell nuclei appeared as circumscribed areas of less Xuorescence intensity (stars in Fig. 1b). The borders of P2X3 immunopositive myenteric neuronal perikarya were often hard to distinguish from surrounding P2X3-iry belonging to surrounding IGLEs or varicosities of the myenteric neuropil (Fig. 1b). The P2X3 immunopositive myenteric cell bodies were found on average in 79% (137 out of 174) myenteric ganglia sampled from all esophageal levels. The distribution of P2X3-ir myenteric neuronal perikarya showed a shift towards the lower parts: in the upper third we found a signiWcantly lower rate (58%; 36 out of 62), while thoracic and abdominal parts showed similar high proportions of P2X3-ir neuronal cell bodies: 89% (51 out of 57) and 91%

control peptide of rP2X3. SpeciWc staining is no longer detectable. Nevertheless some unspeciWc background staining of striated muscles and myenteric ganglia are still remaining. The border of the myenteric ganglion is outlined. d–f Co-incubation with unblocked rVGLUT2 (red; e) and tenfold blocked gpP2X3 (green; d; single optical section out of a z-series of six pictures). There is no colocalization of red and green label indicating that IGLE labeling by gpP2X3 is eYciently blocked by preadsorption. The border of the myenteric ganglion is outlined in d. Scale bars 20 m for a–f

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(50 out of 55), respectively. The number of P2X3-ir neuronal perikarya per ganglion varied from one to four immunopositive neurons depending on the esophageal parts and the size of the ganglion. In order to test the speciWcity of both the rabbit and guinea pig P2X3-receptor staining, we performed preadsorption controls with the corresponding P2X3 peptide. After blocking with a twofold (rP2X3-antibody) and tenfold (gpP2X3-antibody), respectively, amount of antigen, we no longer found immunostaining in myenteric neuropil and myenteric neuronal cell bodies (Fig. 1c, d–f), although a Wne granular background staining in myenteric ganglia and surrounding striated muscle layers remained in the rP2X3 preadsorption (Fig. 1c). To ascertain that preadsorbed P2X3 failed to label IGLEs, we additionally performed double incubation with tenfold blocked pgP2X3 and unblocked rVGLUT2 (Fig. 1d–f). No spots of colocalization were found. Immunostainings of both P2X3 antibodies were carefully compared in single incubations and showed identical staining patterns. No labeling was found in control sections that were processed without primary antibodies. P2X3 and IGLE-marker VGLUT2 Immunolabeling using gpVGLUT2 appeared as described before: Wne contiguous immunopositive dots resulting in

Fig. 2 a–i Double immunostaining for P2X3 and IGLE-marker VGLUT2 in the mouse esophagus. For better distinguishing P2X3 immunostained IGLEs from myenteric neuronal cell bodies P2X3-labeled ganglia were chosen which contain no P2X3 immunopositive myenteric cell bodies. a–c Single optical sections of a highly colocalized IGLE immunolabeled for rP2X3 (a; green) and VGLUT2 (b; red). The whole ganglionic area contains co-stained SIBs displayed as numerous yellow spots in the merge (c). Arrows point to areas co-staining for P2X3-iry and VGLUT2-iry. d–f Single optical sections of a

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profusely arborizing laminar aggregates that enveloped myenteric ganglia in a manner typical for IGLEs (Fig. 2b, e, h; Raab and Neuhuber 2004). We also observed the previously described unspeciWc immunostaining in several myenteric neuronal cell bodies (for preadsorption of the gpVGLUT2 see Raab and Neuhuber 2004). Altogether we evaluated a sample of 174 VGLUT2-ir IGLEs: 62 in the upper, 57 in the middle and 55 in the lower third of the esophagus. The overall rate of P2X3 colocalization was 65% (40 out of 62), 77% (44 out of 57) and 92% (50 out of 55), respectively, for upper, middle and lower thirds of esophagus. The pattern of P2X3 labeling allowed diVerentiation of VGLUT2 immunopositive IGLEs into three groups: 1. High colocalization: VGLUT2 immunopositive IGLEs with concomitant P2X3 immunostaining distributed over the whole ganglionic area resulting in a huge amount of yellow spots of colocalization (21% of IGLEs; 37 out of 174; arrows in Fig. 2a–c). 2. Partial/low colocalization: VGLUT2 immunopositive IGLEs with only partial P2X3 co-staining (56% of IGLEs; 97 out of 174; Fig. 2d–f) resulting in some yellow spots, indicating colocalization in several SIBs. 3. No colocalization: VGLUT2 immunopositive IGLEs without any P2X3 labeling within the integrity of the laminae and puncta of the IGLE (23% of IGLEs; 40 out

partly/lowly colocalized IGLE immunostained for rP2X3 (d; green) and VGLUT2 (e; red), respectivly. The merge (f) reveals only a few yellow SIBs, resulting of an overlap of red and green within the same proWle, which are pointed out by white arrows. g–i Not colocalized IGLE. g, h Single optical sections through a myenteric ganglion immunostained for P2X3-ir (g; green) and VGLUT2-ir (h; red), respectively. Arrowheads point to spots of intimate proximity of both varicosities, partly appearing yellow in the merge (i). We found no co-localization within the same proWle. Scale bars 20 m for a–i


of 174; Fig. 2 g–i). Additional P2X3 immunostaining within myenteric neurons and varicose Wbers of the ganglionic neuropil, respectively, were seen. P2X3 labeled IGLE-like structures were never found without VGLUT2-ir within the mouse esophagus. We detected increasing percentages of colocalization towards the lower esophagus. In the upper third 35% (22 out of 62) evaluated IGLEs were not colocalized, 48% (30 out of 62) were partly colocalized in a few spots and 16% (10 out of 62) IGLEs were highly colocalized over the whole ganglionic area. In sections from thoracic parts we found 23% (13 out of 57) IGLEs with no colocalization, 61% (35 out of 57) IGLEs with a low degree of colocalization and 16% (9 of 62) IGLEs with a high degree of colocalization. In the lower third IGLEs 9% (5 out of 55) were not, 58% (32 out of 55) were lowly and 33% (18 out of 55) IGLEs were highly colocalized. Thus, we could demonstrate that P2X3 is highly expressed by nearly one-fourth of mouse esophageal IGLEs. Another 56% of esophageal IGLEs contain smaller amounts of P2X3-iry. However, P2X3 cannot serve as a speciWc marker for IGLEs in the mouse esophagus, as it was also found in a considerable number of myenteric neurons (Fig. 1b). P2X3/P2X2 colocalization In order to test for possible hetero-oligomeric P2X2/3 channels in esophageal myenteric ganglia we performed doubleimmunostaining for both P2X3- and P2X2 receptors. As described before, P2X2 receptor-iry occurred in clusters of

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punctiform and lamellar structures covering parts of, or entire esophageal myenteric ganglia, i.e., IGLEs in mouse and rat (Castelucci et al. 2003; Raab and Neuhuber 2005; Wang and Neuhuber 2003) (Fig. 3b). Analysis of 13 representative esophageal myenteric ganglia in merged twochannel single optical sections revealed a high degree of colocalization of P2X3- and P2X2-iry in IGLEs, indicating co-expression of both receptors within SIBs, suggesting the presence of a heteromeric P2X2/3-receptor (Fig. 3a–c). In all P2X2-ir IGLEs examined additional P2X3-iry was found, in 92% (12 out of 13) highly colocalized but in 8% (1 out of 13) not colocalized indicating P2X2-iry within the IGLE without corresponding P2X3-iry. The P2X3-iry occurring within these myenteric ganglia most likely belonged to varicose Wbers traversing the ganglion or dendrites of P2X3-ir myenteric neurons. No P2X2-ir myenteric neuronal cell bodies were found in the ganglia examined, while P2X3 stained various myenteric perikarya, as described above. P2X3 and nNOS In order to Wnd out whether P2X3 is contained in nitrergic myenteric neurons, we performed double-immunostaining for rP2X3 and nNOS in esophagi of three mice. Altogether we examined 120 myenteric ganglia, 40 in the cervical, 39 in the thoracic and 41 in the abdominal esophagus. Immunolabeling for nNOS resulted in homogeneous cytoplasmic staining of numerous neuronal cell bodies and proximal dendrites and in varicose Wbers traversing the ganglionic neuropil (Fig. 4b).

Fig. 3 a–c Double immunostaining for P2X3 and P2X2 in the mouse esophagus. a, b Single optical sections through a myenteric ganglion immunolabeled for P2X3 (a; green) and P2X2 (b; red). The white arrows (a–c) point to spots of colocalization of P2X3- and P2X2-iry with-

in SIBs, appearing yellow in the merge (c). The asterisk indicates the nucleus of a P2X3 immunopositive, P2X2 immunonegative myenteric perikaryon (a–c). Scale bar 20 m

Fig. 4 a–c Double immunostaining for P2X3 and nNOS in the mouse esophagus. a, b Single optical sections through a myenteric ganglion immunolabeled for P2X3 (a; green) and nNOS (b; red). The white asterisks (a–c) indicate the nucleus of a P2X3 and nNOS co-stained and colocalized myenteric neuron. The white arrow points to spots of

colocalization of P2X3 and nNOS immunopositive varicosities within the neuropil, appearing yellow in the merge (c). The arrowhead indicates spots of close proximity of P2X3-iry and a dendrite of a nNOSimmunopositive myenteric neuronal perikaryon, appearing yellow in the merge (c). Scale bar 20 m

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In double-immunohistochemistry for nNOS and P2X3 we found co-stained myenteric neuronal cell bodies at all esophageal levels with an increasing tendency in lower parts (Fig. 4a–c). While in the upper third only 19% (36 out of 188) of P2X3-ir neuronal perikarya also stained for nNOS, the corresponding numbers increased from 28% (64 out of 230) in the middle third to 31% (79 out of 256) in the lower third. Additionally, we detected colocalization within the same proWle, as well as close contacts between P2X3 and nNOS positive varicosities (arrow in Fig. 4a–c) within the myenteric neuropil. The number of myenteric ganglia with colocalized varicosities also increased towards lower esophageal levels. In contrast, the rate of no (38% in upper, 5% in middle and 2% in lower esophagus) or low P2X3/ nNOS colocalization (30% of cervical, 15% of thoracic and 7% of abdominal sections), respectively, continuously decreased towards the stomach. Furthermore, close contacts between nNOS-positive varicosities and P2X3-ir neuronal perikarya and vice versa (arrowhead in Fig. 4a–c) were found. No staining was observed in control sections. P2X3 and ChAT Altogether 149 myenteric ganglia positive for P2X3 were examined (48 cervical, 54 thoracic and 47 from the abdominal esophagus, respectively). ChAT immunostaining of


neuronal cell bodies, axons and proximal dendrites was found as dense or sometimes faint granular staining (Fig. 5b). Regarding all ganglia examined, we found P2X3/ ChAT double-stained myenteric perikarya at all levels of the esophagus with a slightly increasing tendency in the middle and lower esophageal parts (22% (51 out of 233) in upper, 28% (77 out of 277) in middle and 27% (48 out of 179) in lower esophagus, respectively; Fig. 5a–c). Nevertheless, we noticed marked inter-individual diVerences in the distribution of P2X3/ChAT-ir neurons in the respective animals, probably indicating an inconsistent distribution along the esophagus. Additionally, ChAT-ir varicose Wbers passing through the ganglionic neuropil were observed in almost all ganglia (Fig. 5b). Colocalization of P2X3- and ChAT-ir within these varicose Wbers as well as close contacts of Wbers stained for either marker was seen (Fig. 5a–c). No staining was observed in controls. P2X3 and TH TH immunohistochemistry primarily stained varicose Wbers traversing the myenteric neuropil as previously described (Raab and Neuhuber 2004). In addition, a small number of TH positive neuronal perikarya were found at all levels of the esophagus (Fig. 6b). In some of these neurons TH immunolabeling was faint. Analysis of 50 representative

Fig. 5 a–c Double immunostaining for P2X3 and ChAT in the mouse esophagus. a, b Single optical sections through a myenteric ganglion immunolabeled for P2X3 (a; green) and ChAT (b; red). The asterisks in (a–c) signify the nucleus of a P2X3/ChAT co-stained myenteric neuronal cell body. The arrows indicate spots of colocalization of P2X3

and ChAT immunopositive varicosities within the neuropil, appearing yellow in the merge (c). Site of intimate proximities of P2X3 and ChAT immunopositive varicosities within the myenteric neuropil are displayed in yellow in the merge (c; arrowheads). Scale bar 20 m

Fig. 6 a–f Double immunostaining for P2X3 and TH in the mouse esophagus. a, b, d, e Single optical sections through a myenteric ganglion immunolabeled for P2X3 (a, d; green) and TH (b, e; red). The white asterisks in (a–c) indicate the nucleus of a P2X3 and TH costained myenteric neuronal perikaryon. Sites of close contacts between

P2X3-iry and TH immunopositive varicosities within the myenteric neuropil are shown in yellow in the merge (c, f; arrowheads a–f). No spots of colocalization were seen within the same proWle. Scale bar 20 m

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myenteric ganglia (15 in cervical, 15 in thoracic and 20 in abdominal esophagus) revealed TH/P2X3 co-staining in 2% (2 out of 114) in the upper, in 5% (4 out of 79) in the middle and in 3% (5 out of 196) P2X3-ir neuronal cell bodies in the lower esophagus (Fig. 6a–c). Sites of close proximity of TH-ir varicose Wbers and P2X3-iry, resulting in yellow mixed color at the site of contact were seen (Fig. 6d–f), but no colocalization within the same proWle could be detected. Controls were free of labeling.

21

varicose Wbers was studied (e.g. zoom-ins 1–3 of Fig. 7a– c). If spots of yellow mixed color changed position relative to the green and red proWles when followed through the confocal series, this was interpreted as close apposition rather than colocalization (zoom-ins 1–3 of Fig.7a–c). Nevertheless, in some cases the question of colocalization versus close proximity is beyond the resolution limit of confocal microscopy and can ultimately only be proven by electron microscopy. P2X3 and S100

P2X3 and CGRP CGRP immunostaining showed Wne varicose Wbers with rare ramiWcations in the neuropil of myenteric ganglia (Fig. 7b). In all myenteric ganglia examined (n = 12) no CGRP-ir could be detected within neuronal perikarya resembling the pattern previously described (Dütsch et al. 1998; Raab and Neuhuber 2003; Sang and Young 1998). Analysis in merged two-channel single optical sections revealed no spots of colocalization within the same proWle but close apposition of P2X3-iry and CGRP-ir varicosities (yellow) at the site of contact, indicating proximity beyond the resolution of the confocal technique (arrows in Fig. 7c, zoom-ins 1–3). For distinguishing true colocalization within the same proWle from close apposition of P2X3-ir and CGRP-ir varicosities the whole myenteric ganglion was analyzed by confocal microscopy. Z-series with up to 13 single optical sections were taken and the course of the

Fig. 7 a–c Double immunostaining for P2X3 and CGRP in the mouse esophagus. a, b Single optical sections through a myenteric ganglion immunolabeled for P2X3 (a; green) and CGRP (b; red). In the P2X3labeled green channel (a, c) several myenteric neuronal cell bodies are displayed (asterisks indicate neuronal nuclei). CGRP immunostains varicose Wbers within the myenteric neuropil (b). No spots of colocalization were seen within the same proWle. In the merge (c) areas of close proximity between CGRP-ir varicose Wbers and P2X3-ir are pointed at by arrows (c; yellow at the site of contact). Bars 20 m

S100-ir showed homogenous staining of cell bodies of enteric glial cells and their processes while sparing myenteric perikarya. Analysis of 9 myenteric ganglia in merged two-channel single optical sections revealed no P2X3-iry within the cell bodies of enteric glial cells, while for their delicate processes intermingling with IGLEs, varicose Wbers and dendrites of myenteric neurons a reliable statement is hard to make from confocal images. In several areas, we found close contacts and interdigitations between P2X3-ir and S100-ir glial processes, resulting in yellow mixed color at the site of contacts (arrows in Fig. 8a–c). Single consecutive optical section analysis of areas suspicious for colocalization, revealed interdigitations of both immunoreactivities. Again, in some cases deWnite statements on colocalization may require electron microscopy.

Zoom-ins 1–3 higher magniWcations of the white-framed boxes are arranged below every image of the respective channels and the merged image. Zoom-ins 1, 3 are consecutive single optical sections 1.0 m above (Zoom-in 1) and 1.0 m below (Zoom-in 3) the level of Zoomin 2. In all three planes no spots of colocalization within the same proWle could be found but the migration of both immunoreactivities. Arrows point to close contacts between P2X3- and CGRP-labeled varicosities (Zoom-ins 1–3)

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22


Fig. 8 a–c Double immunostaining for P2X3 and S100 in the mouse esophagus. a, b Single optical sections through a myenteric ganglia immunolabeled for P2X3 (a; green) and S100 (b; red). The white asterisk in a and c indicate the nucleus of a P2X3 immunostained myenteric neuronal perikaryon. The white bordered red asterisks in (b and c) indicate the nuclei of enteric glial perikarya. White arrows point to

areas with close contacts and interdigitations between P2X3-ir and S100-ir glial processes, resulting in yellow mixed color at the site of contacts (arrows in a–c). In some parts exclusion from colocalization is not possibly and might only be reliably done using electron microscopy. Scale bar 20 m

Discussion

without intervening glial processes, between varicosities, and between varicosities and neuronal cell bodies, electron microscopy is indispensable.

The aim of this study was to elucidate the relationships of the purinergic P2X3 receptor subunit to vagal IGLEs, intrinsic cholinergic, nitrergic and monaminergic myenteric neurons, spinal primary aVerents, postganglionic sympathetic Wbers and enteric glial cells in the mouse esophagus. As in rat (Wang and Neuhuber 2003), immunoreactivity for the P2X3 receptor subunit is found on average in about three-fourth of IGLEs as identiWed by VGLUT2 immunostaining. Also comparable to the rat (Wang and Neuhuber 2003), CGRP positive spinal aVerent Wbers were consistently immunonegative for P2X3. Colocalization of P2X3and P2X2 receptor within one SIB led to the suggestion that subpopulations of IGLEs express besides the P2X2 receptor also the hetero-oligomeric P2X2/3 receptor. In contrast to the rat esophagus, where only a small number of P2X3-ir myenteric neurons were described (Wang and Neuhuber 2003), we detected P2X3-ir myenteric neuronal cell bodies in the mouse in nearly 80% of myenteric ganglia. More detailed investigations revealed P2X3 receptor-iry in subpopulations of nitrergic (nNOS), cholinergic (ChAT) and monaminergic (TH) myenteric neurons. Additionally, P2X3 receptor-iry was colocalized in ChAT-ir and nNOS-ir varicosities. Enteric glial cells were found immunonegative for P2X3-iry, although a deWnite statement may require electron microscopy.

Methodological considerations In some of the P2X3-double immunostainings (e.g. P2X3/ CGRP, P2X3/S100) we found close contacts of both immunoreactivities. Although analysis of single optical confocal sections extracted from z-series could solve the issue of colocalization or close proximity, in some cases this was beyond the resolution limit. Two factors that may contribute to an overestimation of possible contacts in confocal analysis are the limited resolution of the technique, and the eVects of oblique sections through the surface of nerve cells (Mann et al. 1997). For analysis of direct contacts, i.e.,

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Presence of the homomeric P2X2, (P2X3) and the heteromeric P2X2/3 receptors, respectively, in subpopulations of vagal IGLEs VGLUT2-ir has been found to be a reliable marker for both qualitative (Raab and Neuhuber 2003; 2004) and quantitative (Raab and Neuhuber 2005) analysis of IGLEs in the mouse esophagus, suggesting that virtually all IGLEs in the mouse esophagus are VGLUT2-immunopositive. This enabled us to elucidate the relationship of P2X3 receptor-iry and IGLEs. Double-immunostaining for VGLUT2 and P2X3 receptor subunit showed an overall colocalization of 77% within mouse esophageal IGLEs. Vice versa, P2X3 IGLE-like labeling without corresponding VGLUT2 has not been observed, emphasizing the value of VGLUT2 as a reliable qualitative and quantitative marker for IGLEs in the mouse esophagus. Exact colocalization rates of VGLUT2/P2X3 and P2X2/P2X3, respectively, were not determined in this study because of the Wne granular staining pattern of P2X3iry and the simultaneous location on IGLEs, traversing cholinergic and nitrergic varicose Wbers and myenteric neurons. Counting of P2X3 immunopositive dots therefore would cause an overestimation of P2X3-ir spots. Analysis of the distribution of P2X3/VGLUT2 immunopositive IGLEs along the esophagus revealed an increase towards the abdominal part. This data concur well with results obtained in the rat esophagus where 82% of IGLEs examined were described to be P2X3 immunopositive (Wang and Neuhuber 2003). The distribution of P2X3-ir IGLEs along the esophagus was not determined in that study, not least because of visualization of IGLEs with their speciWc marker calretinin which is known to be diVerently distributed along the rat esophagus with highest levels in cervical and decreasing numbers towards the lower parts (Kressel and Radespiel-Tröger 1999). The P2X2 receptor subunit was found within 50% of mouse esophageal IGLEs


and serves as a reliable qualitative but not quantitative marker for IGLEs (Raab and Neuhuber 2005). In our previous study P2X2-immunopositive IGLEs were rarely found in upper and middle esophagus but increasingly in its lower parts, comparable to our Wndings of the distribution of the P2X3 receptor subunit. However, Castelucci et al. found P2X2 receptor-iry in the majority of esophageal IGLEs, particularly in the thoracic esophagus (Castelucci et al. 2003). Nevertheless, in that study no exact quantiWcation of esophageal IGLEs along the esophagus was performed. In an electrophysiological study fewer than 50% of mouse vagal mechanoreceptors were excited by ,-meATP (Page et al. 2002). This argues against P2X2 receptor expression by the majority of IGLEs. P2X2 receptors have been demonstrated in both nodose and dorsal root ganglion neurons as well as on their central and peripheral extensions to be highly co-localized with P2X3 receptors in rat and monkey (Bradbury et al. 1998; Dunn et al. 2001; Hübscher et al. 2001; Petruska et al. 2000; Vulchanova et al. 1997, 1996; Wang and Neuhuber 2003; Xiang et al. 1998). Furthermore, electrophysiological studies have identiWed both, homomeric P2X2 and P2X3 as well as functional P2X2/3 heteromultimeric channels in cultured nodose and petrosal ganglion cells (Chen et al. 1995; Lewis et al. 1995; Prasad et al. 2001; Thomas et al. 1998; Virginio et al. 1998), indicating that the P2X2 receptor subunit may form heteromers with the P2X3 receptor subunit in vagal aVerent terminals. Colocalization of P2X3- and P2X2 receptor-ir within one SIB may indicate the occurrence of heteromeric P2X2/3 receptors within mouse esophageal IGLEs. In our sample 92% of P2X2-ir IGLEs showed a high degree of colocalization with the P2X3-receptor-iry, while 8% of P2X2-ir IGLEs were P2X3-immunonegative, indicating the occurrence of heteromeric P2X2/3 but also homomeric P2X2receptors within a subpopulation of IGLEs. Although the presence of both P2X2 and P2X3 within one SIB was not directly tested in rat, our results are in line with the suggestion of Wang and Neuhuber that heteromeric P2X2/3 receptors are also expressed in IGLEs in the rat esophagus (Wang and Neuhuber 2003). Electrophysiological results from IGLEs of the guinea pig esophagus allow to assign the response to ,-meATP to P2X2, P2X3 and P2X2/3 receptors, respectively (Zagorodnyuk et al. 2003). While homomeric P2X2 receptor subunits are not activated by ,-meATP, homomeric P2X3 receptor subunits desensitize within a few tens of milliseconds. In contrast, heteromeric P2X2/3 receptors do not rapidly desensitize. The IGLEs of the guinea pig esophagus did not rapidly desensitize which provides further support for the occurrence of heteromeric P2X2/3 receptors (Zagorodnyuk et al. 2003). Thus, the P2X receptor sought after, probably involved in modulation of IGLEs is likely to be the heteromeric P2X2/3 receptor.

23

P2X3 receptor subunit-ir in subpopulations of cholinergic and nitrergic enteric neurons in the esophagus Altogether, P2X3-iry was found in about 80% of myenteric perikarya throughout all esophageal levels with increasing tendency towards lower parts. In this study myenteric ganglia were identiWed by VGLUT2-immunostaining. As anterograde tracing studies in both rat (Neuhuber et al. 1998) and mouse (Raab et al. 2003) indicated that virtually all myenteric ganglia in the esophagus were innervated by IGLEs, we also compared the distribution of myenteric ganglia stained with cuprolinic blue histochemistry with that of IGLEs immunostained for VGLUT2. We found that the number of cuprolinic blue-stained myenteric ganglia was similar to that of VGLUT2 immunostained IGLEs in the esophagus (Raab and Neuhuber 2005). Additionally, there was no IGLE found in the esophagus without myenteric neurons. The P2X2-iry in mouse myenteric neuronal cell bodies were described in the ileum and distal colon (Castelucci et al. 2003) but not in the esophagus, except for the rare P2X2 immunopositive perikarya in its lower parts (M. Raab, own unpublished observations). These results contradict the possible presence of signiWcant numbers of P2X2/3 heteromultimers within myenteric neurons of the mouse esophagus which were suggested in guinea pig ileum and distal colon (Poole et al. 2002). In the rat esophagus a small number of myenteric neurons were described to immunostain either for P2X2 or P2X3 (Wang and Neuhuber 2003). Most data concern the presence of P2X2 or P2X3 receptoriry on myenteric neurons in more aboral regions of the gut, mostly ileum and distal colon in diVerent species including human (Castelucci et al. 2003; 2002; Facer et al. 2001; Poole et al. 2002; Van Nassauw et al. 2002; Xiang and Burnstock 2004; Yiangou et al. 2001). There, the P2X2 receptor has been found to be widely expressed, whereas the P2X3 receptor subunit is only found on a small subpopulation of about 10–15% (Bian et al. 2003; LePard et al. 1997; North 2002; Xiang and Burnstock 2004; Zhou and Galligan 1996). Thus, ratios of P2X2 versus P2X3 immunopositive myenteric neurons in ileum and distal colon are almost reversed in the esophagus. Further specifying of these P2X3 receptor-ir esophageal myenteric neurons revealed colocalization with the nitrergic marker nNOS in 27%, again with increasing tendency towards lower esophageal parts. The P2X3-iry was frequently colocalized with nNOS-iry also in varicosities of the ganglionic neuropil, indicating extensive coexistence of P2X3 and nNOS in nitrergic enteric interneurons, typically representing inhibitory neurons (Aimi et al. 1993; Boeckxstaens et al. 1991; Furness 2006). The colocalization of P2X3-iry and nNOS-iry has been described in inhibitory motor neurons and ascending interneurons of the guinea pig intestine (Poole et al. 2002; Van Nassauw et al. 2002).

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24

Another 26% (176 of 689) of P2X3-ir esophageal myenteric neurons co-stained for the cholinergic marker ChAT, although a consistent distribution could not be found because of a high inter-individual variability. Examination of the ganglionic neuropil revealed colocalization of P2X3- and ChAT-iry within varicosities, suggesting that P2X3/ChAT-ir neurons in the esophagus represent cholinergic interneurons (Kuramoto and Brookes 2000). As in our previous studies (Ewald et al. 2006; Kraus et al. 2007; Raab and Neuhuber 2004), we found close relationships of P2X3-iry and TH-immunopositive varicose Wbers, which may largely correspond to postganglionic sympathetics originating in para- and prevertebral ganglia. Additionally, we found a small percentage (3%) of P2X3/ TH-ir myenteric neuronal cell bodies along the mouse esophagus, probably representing intrinsic catecholaminergic enteric neurons, which were described in some regions of the gut, including the esophagus of diVerent species (Anlauf et al. 2003; Furness and Costa 1974; Raab and Neuhuber 2004). Functional considerations ATP is known to be released from enteric glial cells (Burnstock 2007) and from myenteric perikarya, most likely from inhibitory intrinsic neurons (nNOS/VIP-ir) due to a stimulus, e.g., distension (Bian et al. 2003; Bodin and Burnstock 2001; Knight et al. 2002; McConalogue et al. 1996; Van Nassauw et al. 2002; Vlaskovska et al. 2001). Therefore, involvement of ATP in local tension perception and its modulation has been considered (McConalogue et al. 1996; Zagorodnyuk et al. 2003). The ATP is also released from tissues during physical damage, suggesting that purinergic transmission may play an essential role in nociception in damaged or inXamed tissue (Bodin and Burnstock 2001; Burnstock 2001). At central synapses, ATP is co-released with glutamate or originates from glial cells, (Pankratov et al. 2006). If ATP is also co-released from glutamatergic (VGLUT1-ir) myenteric neurons (Ewald et al. 2006; Kraus et al. 2007) has further to be determined. Within myenteric neurons, P2X3 mediated fast ESPSs have been described to be often followed by hyperpolarization, which resulted in reduction of neuronal excitability in the sense of inhibitory co-transmission (Bertrand 2007; Thomas and Bornstein 2003). Equipped with both P2X2 and P2X3 receptors (this study; Castelucci et al. 2003; Wang and Neuhuber 2003; Xiang and Burnstock 2004), IGLEs might serve as a major target for ATP released from myenteric neurons. Almost all vagal mechanosensors in the guinea pig esophagus, i.e., IGLEs, and about 50% of mouse esophageal IGLEs were excited by ATP (Page et al. 2002; Zagorodnyuk et al. 2003). Thus, it appears that IGLEs are more than “simple”

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tension detectors and may subserve additional functions that are poorly understood. Activation of presynaptic P2X receptors was found to facilitate quantal release of glutamate and elicited terminal action potentials and therefore resulted in synchronized glutamate release due to direct Ca2+ entry into presynaptic terminals in neurons of the spinal cord dorsal horn (Gu and MacDermott 1997; Nakatsuka and Gu 2006). In addition to the enhancement of presynaptic glutamate release, the activation of P2X receptors in the dorsal horn was also found to elicit SP release (Nakatsuka and Gu 2006). Both, glutamate and SP appear to be contained in synaptic vesicles of IGLEs (Kressel and Radespiel-Tröger 1999; Raab and Neuhuber 2003; 2004) and possibly exert inXuences onto myenteric neurons. A modulation of excitability of IGLEs via ATP, which has already been described (Zagorodnyuk et al. 2003) might be due to P2X2 or P2X3 or P2X2/3 receptor activity and may tone both glutamate and SP release. Another potential role which might be assigned to the P2X3 and P2X2/3 receptors, respectively, was the regulation of dysmotility and pain (Galligan and North 2004). Administration of a selective P2X3 antagonist, A-317491, has been shown to eVectively block both hyperalgesia and allodynia in diVerent animal models of pathological pain (Donnelly-Roberts et al. 2008). Activation of heteromeric P2X2/3 receptors appears to modulate longer-lasting nociceptive sensitivity associated with nerve injury or chronic inXammation (Jarvis 2003; Nakagawa et al. 2007). Although P2X3 receptors are not sensitive to pH, recombinant P2X2 receptors have been shown to be strongly pH sensitive (Wildman et al. 1998) and P2X2/3 receptors are also pH sensitive but to a lesser extent (Stoop et al. 1997). This suggests that the sensitivity of P2X2/3 receptors might be enhanced in inXammatory conditions with acidosis. Low pH has also been shown to augment the excitatory actions of ATP in the sense of a local inXammatory mediator (Li et al. 1996; Page et al. 2002). It is tempting to speculate that the prevalence of purinergic receptors on IGLEs in the lower esophagus represents the anatomical basis for protective clearing reXexes during acidic reXux and disturbed motility in esophagitis (Bremner et al. 1993; Helm et al. 1982; ShaWk et al. 2005). Moreover, under conditions of persistent nociceptive input, activation of other P2 receptors (e.g., P2X4 and P2X7) may also serve to maintain nociceptive sensitivity through neuronal-glial cell interactions or via sensitization (via P2Y) of other nociceptive receptors such as TRPV1 channels (Donnelly-Roberts et al. 2008). Most IGLEs in the esophagus and stomach are resistant to capsaicin (Berthoud et al. 1997b), which corresponds to a lack of immunohistochemically detectable vanilloid receptor VR1/TRPV1 (Patterson et al. 2003). However, chemical or inXammatory challenge may enhance the expression of TRPV1 (Mat-


thews et al. 2004) or acid-sensitive ion channels (Holzer 2003) that could be relevant for the pathogenesis of mechanical hypersensitivity or non-cardiac chest pain arising in the esophagus.

Conclusion P2X3-iry was found colocalized with VGLUT2 in 77% of esophageal IGLEs. The distribution of P2X3-ir IGLEs was similar to that of P2X2-ir and showed increasing numbers towards the abdominal esophagus. P2X3/ P2X2-colocalization within the majority of IGLEs suggested besides homomeric P2X2-receptors, the occurrence of heteromeric P2X2/ 3 receptors. P2X3 additionally stained perikarya in »80% of myenteric ganglia and detailed analysis revealed P2X3-iry in subpopulations of both nitrergic (nNOS) and cholinergic (ChAT) neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a P2X2, (P2X3) and P2X2/3 receptor-mediated pathway especially in the abdominal portion of the mouse esophagus probably involved in pathogenesis of mechanical hypersensitivity or non-cardiac chest pain arising in the esophagus. Acknowledgments The skilful technical assistance of Karin Löschner, Hedwig Symowski and Andrea Hilpert is gratefully acknowledged. This study was supported by Johannes und Frieda Marohn-Stiftung, Erlangen.

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