Number and Distribution of Intraganglionic Laminar Endings in the Mouse Esophagus as Demonstrated with Two Different Immunohistochemical Markers
Department of Anatomy I, University of ErlangenNuremberg, Erlangen, Germany
Correspondence to: M. Raab, Institut für Anatomie, Lehrstuhl I, Krankenhausstr. 9, D-91054 Erlangen, Germany. E-mail: marion.raab{at}anatomie1.med.uni-erlangen.de
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Summary |
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Key Words: P2x2 VGLUT2 vagal afferents anterograde tracing cuprolinic blue myenteric ganglia C57Bl/6 mice
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Introduction |
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Investigation of structurefunction relationships of the various types of visceral afferent nerve endings in the digestive tract is still in its infancy (Costa et al. 2004). In particular, further studies on the functional role of IGLEs in various parts of the gastrointestinal tract would be greatly facilitated by the availability of specific immunohistochemical markers that label the entire population of IGLEs in a given organ. This would make the detection of numerical alterations of IGLEs, e.g., in mutant mice, more feasible. Such studies, which were aimed at elucidating the significance of IGLEs by loss of function in certain knockout mice, had to utilize the more laborious and time-consuming anterograde tracing technique from nodose ganglion for labeling of IGLEs (Fox et al. 2001
; Raab et al. 2003
). Unfortunately, calretinin, which labels specifically IGLEs in the rat esophagus (Dütsch et al. 1998
; Kressel and Radespiel-Tröger 1999
), cannot be used for this purpose in the mouse (Castelucci et al. 2003
; Raab and Neuhuber 2003
) or in other parts of the gastrointestinal tract in rat (Raab M and Neuhuber WL, unpublished data). Thus, the aim of this study was to compare two recently described candidate markers for IGLEs, i.e., P2x2 receptor (Castelucci et al. 2003
) and VGLUT2 (Raab and Neuhuber 2003
,2004
), as to their suitability as both qualitatively and quantitatively specific markers for IGLEs in the esophagus. C57Bl/6 mice were chosen because they are widely used as background for various mutant mouse strains.
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Materials and Methods |
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For all procedures performed on animals, the federal animal welfare legislation implemented by the local government was followed.
Anterograde Tracing and Tissue Preparation in Mice
For structural identification of IGLEs, anterograde tracing from the nodose ganglion was performed as described before (Raab and Neuhuber 2003; Raab et al. 2003
). In brief, mice were anesthetized by using a combination of ketamine hydrochloride (100 mg/kg, Ketavet; Pharmacia Upjohn, Erlangen, Germany) and xylazine (5 mg/kg, Rompun; Bayer Vital, Leverkusen, Germany) injected intramuscularly, and the left cervical vagus and the nodose ganglion were carefully exposed by a midline incision. WGA-HRP (0.3 µl, 4% in NaCl; Sigma, Deisenhofen, Germany) was pressure-injected through a glass micropipette into the left nodose ganglion (n=3). After suturing the skin incision, animals were warmed, closely monitored, and allowed to recover in a single cage. For tracing controls and quality checks, see Raab et al. (2003)
.
After survival times of 3840 hr, animals were deeply anesthetized with a lethal dose of Thiopental [250 mg/kg intraperitoneally (IP), Trapanal; Byk Gulden, Konstanz, Germany]. When mice were unresponsive to nociceptive stimuli, abdomen and thorax were opened and 10 ml of Ringer solution containing 2500 IU/500 ml heparin was injected through the left ventricle. Mice were then perfused transcardially with 80 ml of 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). This was followed by 20 ml of 15% sucrose phosphate buffer (pH 7.4).
Esophagi from the level of the cricoid cartilage to the gastroesophageal junction were removed and transferred to 15% sucrose phosphate buffer (pH 7.4) at 4C overnight.
Upper, middle, and lower thirds of the esophagus were mounted on Tissue-Tek (GSV1; Slee Technik, Mainz, Germany), rapidly frozen in methylbutane at 70C, and stored at 20C. Twelve-µm-thick longitudinal-directed sections were cut in a cryostat and mounted on poly-L-lysine-coated slides.
Antibodies and Reagents
The primary and secondary antibodies used in this study are listed in Table 1 and Table 2. Secondary antibodies were fluorochrome-conjugated F(ab')2 fragments or Alexa Fluor-conjugated affinity-purified antibodies that react with IgG heavy chains and all classes of immunoglobulin light chains from rabbit. Tyramide amplification reagents were purchased from Life Science Products (PerkinElmer Wallace; Freiburg, Germany).
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Because of the known unspecific staining of myenteric cell bodies in the mouse esophagus with the guinea pig VGLUT2 antibody (Raab and Neuhuber 2004), we used in all wholemount preparations the rabbit VGLUT2 antibody. In our previous study we have shown in detail that IGLEs were identified by both VGLUT antibodies with comparable specificity (Raab and Neuhuber 2004
). Due to the species origin of the P2x2 antibody (rabbit), we applied the guinea pig VGLUT2 antibody for the triple staining.
Other controls included omission of the primary antibodies or replacement by normal serum.
Tyramide Amplification Combined with VGLUT2 and P2x2 Immunohistochemistry
For tracer detection with the tyramide amplification method (Kressel 1998), we used the upper and middle thirds of esophagi from WGA-HRP-injected mice (n=3). Briefly, slides were dried for 60 min and rehydrated in a 0.1 M TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) for 5 min. Then they were pretreated in freshly prepared NaBH4 (0.5 mg/ml; Fluka, Sigma) in TN buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl) for 15 min. After rinsing in TNT buffer, the slides were permeabilized in 0.1% Triton X-100 in TN buffer for 15 min and again washed in TNT buffer. The preincubation solution (containing TNT buffer and 2% BSA) was left for 1 hr. The slides were rinsed in TNT buffer for another 5 min and then incubated in biotinylated tyramide diluted 1:50 in amplification diluent (NEN; Life Science Products) for 15 min. Following a washing step in TNT buffer, the primary VGLUT2 antibody (guinea pig VGLUT2, 1:1500 in TNT buffer) and the P2x2 antibody (rabbit P2x2, 1:250 in TNT buffer) were applied overnight at 4C. The primary antibodies were detected by incubation with donkey anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes Inc., Eugene, OR) and donkey anti-guinea pig Cy5 (1:100; Dianova, Hamburg, Germany) in TNT buffer. Incorporated biotintyramide was visualized by adding streptavidin-Cy3 (1:1000; Dianova) to the last incubation solution. After a final washing step in TN buffer, the slides were mounted in TBSglycerol at pH 8.6. Twenty-five anterogradely WGA-HRP-labeled IGLEs from all esophageal sections examined were randomly selected for triple-channel confocal analysis.
Esophageal Wholemount Preparation
Mice were deeply anesthetized with a lethal dose of Thiopental (250 mg/kg IP, Trapanal; Byk Gulden). When they were unresponsive to nociceptive stimuli, the abdomen and the thorax were opened and mice were perfused transcardially with 10 ml of Ringer solution containing 500 IU/100 ml heparin. After this prewash, a small polyethylene tube was inserted through the esophagus to the stomach to distend the esophagus before fixation started. Mice were then perfused transcardially with 80 ml of 3% paraformaldehyde (for VGLUT2 immunohistochemistry; n=8) or Zamboni fixative (for P2x2 immunohistochemistry; 2% paraformaldehyde and 0.2% picric acid; n=7), respectively, in 0.1 M phosphate buffer (pH 7.4) followed by 20 ml of 15% sucrose phosphate buffer (pH 7.4). Paraformaldehyde-fixed tissue is optimal for VGLUT2 immunohistochemistry in central and peripheral nervous system (Fremeau et al. 2001; Tong et al. 2001
; Raab and Neuhuber 2003
; Alvarez et al. 2004
). In our opinion, Zamboni's fixative was superior over paraformaldehyde for P2x2 immunohistochemistry because it reduced unspecific background staining as already described by Castelucci et al. (2003)
.
The esophagus from the level of the cricoid cartilage to the gastroesophageal junction was removed and transferred to phosphate buffer (pH 7.4) at 4C. Esophagi were freed from adhering connective tissue including main vagal nerve trunks under a dissecting microscope and opened approximately along the ventral midline. Wholemounts of muscularis and mucosa were prepared by separating both layers along the submucosal plane using jeweler's forceps and stored in 0.1 M phosphate buffer (pH 7.4) overnight at 4C.
VGLUT2 and P2x2 Immunohistochemistry in Esophageal Wholemount Preparations
To locate VGLUT2 and P2x2, respectively, in esophageal wholemount preparations by immunohistochemistry, wholemounts were incubated with 5% normal donkey serum containing 0.5% Triton X-100, 0.05% Thimerosal (T-5125; Sigma), and 1% BSA in Tris-buffered saline (TBS) for 2 hr at room temperature. After rinsing in TBS buffer, wholemounts were then exposed for 3 days at 4C to antibodies raised against VGLUT2 (rabbit VGLUT2 1:1000; n=8) and P2x2 (1:250; n=7), respectively. Both primary antibodies contained 0.5% Triton X-100, 0.05% Thimerosal, and 1% BSA in TBS. After washing with TBS, wholemounts were incubated with donkey anti-rabbit secondary antibody coupled to Alexa Fluor 488 (1:1000; Molecular Probes, Inc.) for 4 hr at room temperature. Secondary antibody contained 0.5% Triton X-100, 0.05% Thimerosal, and 1% BSA in TBS. Wholemounts were rinsed again with TBS and coverslipped in TBSglycerol (pH 8.6).
Quantification, Distribution, and Mapping of IGLEs
IGLEs were quantified and their distribution was investigated with both marker substances VGLUT2 (n=8) and P2x2 (n=7). All counts were made by the same individual. IGLEs were identified by their typical appearance of laminar aggregates with fine terminal puncta interspersed as shown before (Castelucci et al. 2003; Raab and Neuhuber 2003
). All identified IGLEs were counted in each wholemount from the cricoid level until the beginning of the smooth muscle cells of the lower esophageal sphincter (LES). For microscopic analysis and documentation, a Leica Aristoplan fluorescence microscope (Leica; Bensheim, Germany) was used.
For mapping the distribution of IGLEs labeled with the two different immunohistochemical markers, three representative wholemounts from each series were chosen. The contours of the wholemounts and location of immunostained IGLEs were transferred to scale paper using the ocular's reticule and x/y coordinates of the microscope's stage. Contours were recorded at steps of 1 mm and IGLEs at steps of 0.2 mm, respectively.
Distribution of WGA-HRP-labeled IGLEs in Esophageal Wholemounts
For analyzing the distribution of IGLEs labeled by WGA-HRP tracing from the nodose ganglion, we used esophageal wholemounts prepared in a previous study only for quantification of IGLEs (n=6) (Raab et al. 2003).
Distribution of Myenteric Ganglia in Esophageal Wholemounts
The distribution of myenteric ganglia in the mouse esophagus was determined in cuprolinic blue-stained wholemounts (n=4). Cuprolinic-blue histochemistry provides selective and complete staining of enteric neurons (Heinicke et al. 1987; Holst and Powley 1995
; Karaosmanoglu et al. 1996
; Phillips et al. 2004
). We used wholemounts of the muscularis propria that were prepared previously for quantification only (Raab et al. 2003
).
Single neurons or clusters of enteric neurons were considered enteric ganglia. The distribution of enteric ganglia was determined using the same protocol as for IGLEs.
To compare different methods of demonstrating IGLEs and enteric ganglia, we normalized the length of the esophageal wholemounts to control for different degrees of shrinkage during dehydration in cuprolinic blue and TMB (3,3',5,5'-tetramethylbenzidine) procedures. Also, minor individual variations of esophageal length were adjusted by this normalization procedure. We set the length of every esophageal wholemount as 100% and divided it into 20 transverse segments. Numbers of IGLEs and enteric ganglia, respectively, were counted and mapped in these segments from the level of the cricoid to the LES in each wholemount and the means of data pooled from all wholemounts (WGA-HRP n=6; VGLUT2 n=3; P2x2 n=3; cuprolinic blue n=4) were transferred into a line diagram using Microsoft Excel (2002) software (Microsoft Corporation; Microsoft Deutschland, Munich, Germany) to demonstrate the distribution along the esophagus.
Confocal Microscopy and Image Analysis
Confocal images were obtained by using a Bio-Rad MRC 1000 confocal laser scanning system (Bio-Rad; Hemel Hempstead, UK) equipped with a three-line kryptonargon laser (American Laser Corporation; Salt Lake City, UT) and attached to a Nikon Diaphot 300 microscope (Nikon; Düsseldorf, Germany). The filter settings of the Bio-Rad confocal scanner for triple label were 488-nm excitation for Alexa Fluor 488 (filter DF32), 568-nm excitation for Cy3 (filter 605 DF322), and 647-nm excitation for Cy5 (filter 680 DF322). A x60 oil-immersion objective lens (numerical aperture 1.4) was used. Zoom factors varied between 1.2 and 2.3. Extended-focus images or z-series of up to eight optical sections at z-increments of 1.0 µm were created. In some cases, z-stacks were compressed into one focus plane ("all-in-focus"). Images of 768 x 512 pixels were obtained, and the three channels of each z-series were merged into an 8-bit RGB tif-file by using Confocal Assistant software 4.02 (2). To test for colocalization, single sections at the same focus plane were taken out of these z-staples, and the two or three channels, respectively, were merged.
After recording by confocal laser scanning microscopy, no alterations of image files by additional image processing was performed. For the printing process, color images were imported into Photoshop software (Adobe Photoshop CS, version 8.0.1; Adobe Systems, Unterschleißheim, Germany). To apply text and scale bars, adapt brightness and contrast of the pictures, and organize the final layouts for printing both Photoshop (Adobe Systems) and Coreldraw software (Coreldraw, version 11; Corel Corporation, Dublin, Ireland) were used. Images were printed on Epson Premium Semigloss Photo Paper (S041332, A4; Epson, Düsseldorf, Germany) using an Epson Stylus Photo 2000P inkjet printer.
Statistical Analysis
Hypotheses about differences between numbers of IGLEs stained with the different markers were tested with MannWhitney U-test; p0.05 was required for statistical significance.
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Results |
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The pattern of P2x2 labeling allowed differentiation of WGA-HRP/VGLUT2 immunopositive IGLEs into three groups:
P2x2 immunostaining without concomitant tracer labeling was never seen.
No specific staining was found in control sections.
VGLUT2 and P2x2 in Esophageal Wholemounts
Immunolabeling for VGLUT2 showed fine contiguous immunopositive dots in profusely arborizing laminar structures enveloping myenteric ganglia as typically described previously for IGLEs (Raab and Neuhuber 2003). In wholemount preparations, VGLUT2 immunoreactive fibers that connected with the clusters of lamellae and fine dots were additionally displayed (Figure 2A, arrows). Again, myenteric neuronal cell bodies were immunonegative (Raab and Neuhuber 2003
).
As described before (Castelucci et al. 2003), P2x2 receptor-ir occurred in clusters of punctiform and lamellar structures covering parts of, or entire, esophageal myenteric ganglia as well as in myenteric neuronal cell bodies. In wholemount preparations, P2x2 immunostaining appeared coarser, making the contours of ganglionic areas more pronounced than in VGLUT2 immunostaining (Figure 2B). P2x2 immunopositive fibers leading into interconnecting strands were also shown (Figure 2B, arrows).
In both groups of wholemounts, all immunostained IGLEs were counted from the level of the cricoid to the beginning of the smooth muscle cells of the LES. We found a significant difference (p<0.001) between the numbers of IGLEs per esophageal wholemount counted with VGLUT2 and P2x2 immunohistochemistry: the mean number of VGLUT2 immunopositive IGLEs was 661 ± 39, whereas P2x2-immunopositive IGLEs accounted for only 285 ± 72 (Table 3).
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In P2x2-labeled esophageal wholemount preparations, the distribution of immunopositive IGLEs was significantly different. In the upper and middle parts (between the cricoid level and the 65% mark) we found only between 3 and 13 IGLEs (on average: 9 IGLEs) per segment. In the lower middle and lower parts (70% to 95% mark), the number of immunopositive IGLEs ranged between 17 and 29 IGLEs per segment (on average: 23 IGLEs) and increased to a maximum of 32 IGLEs per segment at the 95% mark before dropping again in the area of the LES (at the 100% mark).
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Discussion |
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Specific Markers for IGLEs
Investigations on structurefunction relationships in both physiological and pathophysiological conditions often benefit from availability of specific immunohistochemical markers for the neuronal structure in question. Although a mechanosensor function of IGLEs was established in electrophysiological experiments in vitro (Zagorodnyuk and Brookes 2000), their functional significance for various aspects of gastrointestinal physiology and pathophysiology in the intact organism remains to be elucidated (Phillips and Powley 2000
). A popular approach for revealing the functional significance of a given neuronal structure is to search for correlation of its absence with disturbed functions in knockout mice. This was first achieved in NT-4/ mutant mice that almost completely lacked IGLEs in the duodenum and showed disturbed satiety mechanisms (Fox et al. 2001
). In that study, the numerical deficiency of IGLEs was verified using anterograde tracing. Circumventing this laborious and time-consuming approach by labeling of IGLEs with a specific marker would greatly facilitate screening of various neurotrophin or other mutants for numerical alterations of IGLEs along the gastrointestinal tract. The first marker that could be used specifically for IGLEs in the rat esophagus was calretinin (Dütsch et al. 1998
). It was found highly colocalized with tracer transported anterogradely from the nodose ganglion (Kressel 1998
; Kressel and Radespiel-Tröger 1999
). In contrast to rats, IGLEs in the mouse esophagus showed no, or only low, calretinin content (Castelucci et al. 2003
; Raab and Neuhuber 2003
). Recently, two immunohistochemical markers were identified that specifically labeled IGLEs also in the mouse: the purinergic P2x2 receptor (Castelucci et al. 2003
) and the vesicular glutamate transporter 2 (VGLUT2) (Raab and Neuhuber 2003
,2004
). In contrast to stomach and intestines where subdiaphragmatic truncal vagotomy could be used to test for vagal origin of IGLEs stained for P2x2 (Castelucci et al. 2003
), this approach is not feasible in the esophagus. In the present study, the vagal sensory origin of P2x2-immunostained structures was determined by anterograde WGA-HRP tracing from the nodose ganglion followed by tyramide amplification. We found triple colocalization in about one half of IGLEs indicating that P2x2 is contained only in a subset of IGLEs in the esophagus of C57Bl/6 mice.
Quantification of IGLEs in Esophageal Wholemounts
The first quantification of IGLEs in the mouse esophagus was performed by anterograde WGA-HRP tracing from the left nodose ganglion to study the dependency of IGLEs on neurotrophins (Raab et al. 2003). Especially in NT-3 knockout mice with reduced neuron numbers, thus reduced size of nodose ganglion (Raab et al. 2003
), tracer injections into this ganglion are difficult, possibly leading to quantitatively suboptimal results. This could be circumvented by using an immunohistochemical marker that specifically and quantitatively labels IGLEs along the whole esophagus. Multilabel immunohistochemistry for identification of a variety of functionally relevant molecules in IGLEs would be more readily performed.
The mean number of IGLEs in the esophagus of C57Bl/6 mice obtained in the tracing study was 349 ± 27 (Raab et al. 2003). Immunostaining of esophageal wholemounts demonstrated, in contrast to the unilateral anterograde tracing, IGLEs derived from both nodose ganglia. The numbers of IGLEs obtained with VGLUT2 immunohistochemistry (661 ± 39; present study) matched approximately the 2-fold number of IGLEs found in our previous tracing study (349 x 2 = 698). Recent data in the mouse (Castelucci et al. 2003
), as well as earlier experiments in rat esophagus (Neuhuber et al. 1998
), indicated that the IGLE per ganglion ratio in the esophagus was close to 1.0. The mean number of myenteric ganglia counted in cuprolinic blue-stained esophageal wholemounts was
685 ± 42. Thus, the IGLE per ganglion ratio calculated from both tracing and VGLUT2 immunohistochemical results is
1.0, confirming earlier data. Therefore, VGLUT2 may serve as a quantitative marker for IGLEs in the mouse esophagus.
Quantification of P2x2-immunostained IGLEs in esophageal wholemounts yielded results significantly different from those obtained with VGLUT2 immunohistochemistry. Only 285 ± 72 (43% of the VGLUT2 counts) IGLEs were found to be P2x2 immunopositive. These data obtained in optimal fixed tissue (Zamboni) are in line with results from combined anterograde tracing and immunohistochemistry, where only 50% of tracer-labeled IGLEs also stained for P2x2. These immunohistochemical results are also consistent with electrophysiological findings in C57Bl/6 mice, showing that <50% of esophageal vagal mechanoreceptors, i.e., IGLEs, were excited by
,ß-methylene ATP, the ligand of the P2x2 receptor (Page et al. 2002
). Nevertheless, our findings contrast with a recent report that P2x2-immunopositive IGLEs supplied all myenteric ganglia, in particular in the thoracic parts of the mouse esophagus (Castelucci et al. 2003
). One possible explanation for this numerical discrepancy might be strain differences. Our experiments were performed on esophagi of C57Bl/6 mice, whereas Castellucci et al. used BalbC mice. In the rat esophagus
80% of IGLEs immunopositive for calretinin co-stained for the P2x2 receptor (Wang and Neuhuber 2003
). On the other hand, nearly all tracer-labeled IGLEs in the guinea pig esophagus were shown to express P2x2 and, consistent with this,
,ß-methylene ATP has been described to cause powerful excitation of almost all esophageal vagal mechanoreceptors in that species (Zagorodnyuk et al. 2003
). These data suggest both strain and species differences of receptor expression in vagal afferents.
In summary, our results indicate that in C57Bl/6 mice only a subpopulation of IGLEs in the esophagus expressed the purinergic P2x2 receptor, whereas VGLUT2 apparently labeled almost all IGLEs. Thus, VGLUT2 immunostaining may be superior over P2x2 immunohistochemistry for quantitative analysis of esophageal IGLEs in this mouse strain.
Distribution of IGLEs as Demonstrated with Different Markers in Esophageal Wholemounts
IGLEs immunopositive for VGLUT2 were distributed along the esophagus similar to those labeled after WGA-HRP injections into the nodose ganglion (Raab et al. 2003). Likewise, VGLUT2-immunostained IGLEs matched the distribution of cuprolinic blue-stained myenteric ganglia. In the area of the esophago-gastric junction, the number of IGLEs decreased slightly ahead of that of enteric ganglia. Thus, about half of enteric ganglia at the LES level were probably not associated with IGLEs. The double-peak distribution and the significant decrease of IGLEs in the area of the LES found in the mouse is in line with the distribution of IGLEs described in rat esophagus (Wang and Powley 2000
). Because wholemount preparation is difficult in the thick wall of the esophago-gastric junction, another reason for that earlier decrease might be insufficient penetration of the antibody through the thick muscularis of this region. Also, individual variability of the distribution might be a further reason for this effect. Nevertheless, VGLUT2 yielded similar numbers and comparable distribution of esophageal IGLEs in the mouse as anterograde WGA-HRP tracing. Thus, VGLUT2 may serve as a selective marker for IGLEs in the mouse esophagus also suitable for quantitative analysis.
The distribution of IGLEs as found with P2x2 immunolabeling differed markedly from that of VGLUT2-labeled IGLEs. In the upper and middle esophagus only a few IGLEs contained P2x2, whereas in the lower parts the number of IGLEs staining for the purinergic P2x2 receptor significantly increased before the drop at the level of the LES. In the area of the esophago-gastric junction (90100% mark), P2x2 immunostaining showed as many IGLEs as found with anterograde WGA-HRP tracing. In this particular area, P2x2 may be as useful as VGLUT2 as a quantitative marker. Whether this remarkable distribution of P2x2 containing IGLEs indicates specific functional properties especially required in the lower parts of the esophagus should be further investigated.
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Acknowledgments |
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The skillful technical assistance of Anita Hecht, Andrea Hilpert, Karin Löschner, Stephanie Link, Hedwig Symowski, and Inge Zimmermann is gratefully acknowledged. Special thanks to Falk Schrödl for stimulating discussion and to Jürgen Wörl for providing the mice.
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Footnotes |
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