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. 2012 Jul 18;221(4):303–310. doi: 10.1111/j.1469-7580.2012.01546.x

The distribution of HCN2-positive cells in the gastrointestinal tract of mice

Shu Yang 1,*, Cheng-jie Xiong 2,*, Hai-mei Sun 1, Xiao-shuang Li 1, Guo-quan Zhang 1, Bo Wu 1, De-shan Zhou 1
PMCID: PMC3458249  PMID: 22803609

Abstract

HCN2 channels are involved in the spontaneous rhythmic activities of some CNS neurons and act by generating If current. The gastrointestinal (GI) tract is known to be capable of spontaneous rhythmic activity; however, the possible role of HCN2 channels in this organ has not yet been elucidated. This study investigated the distribution of HCN2-positive cells in the mouse GI tract using immunohistochemistry. To identify the nature of these HCN2 cells, anti-ChAT and anti-Kit antibodies were used to co-label neurons and the interstitial cells of Cajal (ICCs), respectively. Additionally, differences in the distribution of HCN2-positive cells within the GI tract were also analyzed. Our results showed that HCN2 channels were mainly located within the myenteric neurons of the enteric nervous system in the GI tract. Double-staining revealed that HCN2-positive neurons were labeled by ChAT, indicating that these HCN2-positive cells are also cholinergic neurons. Although the HCN2-positive cells were not stained by the anti-Kit antibody, their processes were in close proximity to ICCs around the myenteric plexus region. Moreover, several differences in the distribution of HCN2 in the stomach, small intestine and colon were partly consistent with the regional differences in the spontaneous rhythmic activities of these organs. Basing on the role HCN2, we suggested that HCN2 channels facilitate the release of Ach from cholinergic neurons to affect the GI peristalsis by acting on M receptors on the ICCs. However, the HCN2 channels are not directly involved in spontaneous slow-wave initiation by ICCs.

Keywords: ChAT, gastrointestinal tract, HCN2, interstitial cells of Cajal, myenteric plexus

Introduction

Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) was first identified in the rabbit sino-atrial node (DiFrancesco et al. 1986). It was later demonstrated that HCN channels are extensively expressed in the heart and central nervous system, including the neocortex, hippocampus, basal ganglia, and dorsal root ganglion (Notomi & Shigemoto, 2004; Postea & Biel, 2011). The HCN channel family consists of four homologous members (HCN1–HCN4), which differ from each other in their activation kinetics and their responses to cAMP (Mistrik et al. 2005). Of these, HCN2 had a ubiquitous distribution throughout most brain regions and the cardiac conduction system of the heart (Shi et al. 1999; Monteggia et al. 2000; Notomi & Shigemoto, 2004). It has been reported that HCN controls the generation of If (funny current) or Ih (hyperpolarizing-activated current), a slowly developing inward current, which plays a major role in cardiac pace-making and rate modulation as well as neuronal excitability (Beck & Yaari, 2008). HCN channels are unique in that they are activated upon hyperpolarization of the membrane potential and their activities are dependent on the intracellular cAMP concentration (DiFrancesco & Tortora, 1991). Evidence has shown that HCN2 functions as a genuine pacemaker and plays a key role in supporting the generation of a stable and regular heart beat (Ludwig et al. 2003).

Gastrointestinal (GI) peristalsis has a spontaneous rhythm, which results from the interstitial cells of Cajal (ICCs) and is mediated by the enteric nervous system (ENS). ICCs are thought to be the GI pacemaker and act by generating and propagating spontaneous slow-waves. ICCs are also believed to mediate both inputs from the ENS to the GI smooth muscles and excitatory and inhibitory neurotransmission (Rumessen & Thuneberg, 1996; Sanders et al. 2006; Ward & Sanders, 2006). ICCs are divided into four subgroups based on their morphological features and location within the gut: ICC-SM (of the submucosal surface of the circular muscle layer in the colon), ICC-IM (of the intramuscular layer), ICC-DMP (of the deep muscular plexus of the small intestine), and ICC-MY (of the myenteric plexus). Of these, ICC-MY and ICC-SM have several processes connected to one another, which respectively form an independent cellular network and play a major role in pacemaking for GI peristalsis (Sanders et al. 2006). It remains unknown whether GI peristalsis is similar to the spontaneous firing of cardiac tissue and whether HCN2 channels have a role in the spontaneous rhythmic activity of the GI tract.

The ENS, independent of the sympathetic and parasympathetic nervous systems, is composed of the submucosal plexus and the myenteric plexus. The ENS regulates GI peristalsis by releasing neurotransmitters, such as acetylcholine (Ach), norepinephrine, 5-HT and variety of neuropeptides. Xiao et al. (2004) demonstrated that HCN2 is highly expressed in Dogiel II/AH neurons of the myenteric plexus of the mouse distal colon. It is believed that the Dogiel II/AH neuron is a type of excitatory neuron that can exhibit If current (Galligan et al. 1990; Rugiero et al. 2002). Thus, HCN2 might be involved in the regulation process of ENS on GI peristalsis. However, the localization and distribution of HCN2 channels and their possible role in the GI tract remain unclear. Therefore, the goal of the present study is to investigate the localization and distribution of HCN2 channels and their relationship with ICCs and myenteric neurons in the mouse GI tract using double immunohistochemistry. Moreover, we also analyze their potential role in the spontaneous rhythmic activities of the GI tract. This work may provide a new target for the treatment of GI motility disorders in the clinical setting.

Materials and methods

Animals and whole-mount preparations

Twenty-seven BALB/c mice (aged 2–3 months, weighed 18–26 g) of either sex were used (purchased from the animal center of Third Military Medical University). The mice were sacrificed by cervical dislocation and performed in accordance with the University Health Guide for the Care and Use of Laboratory Animals. The entire GI tract was removed under sterile conditions and fully washed with pre-cooled 0.01 m (pH 7.4) phosphate-buffered saline (PBS). The stomach, small intestine and colon were inflated with 100% acetone for Kit staining or Zamboni's fixative (2% paraformaldehyde, 1.5% saturated picric acid solution, 0.1 m phosphate buffer, pH 7.3) for HCN2 and ChAT staining and then immersed in the same fixative for 6 h (4 °C). Three segments (each 0.5–1 cm in size) were randomly cut from the gastric fundus, corpus, antrum, duodenum, jejunum, ileum, ascending colon, transverse colon and descending colon, respectively. For whole-mount preparations, the mucosa was removed, and the longitudinal muscle layer containing the myenteric plexus was dissected with the aid of an anatomical microscope. The prepared muscle layers were trimmed into 0.5 × 0.5 cm pieces and placed in 0.01 m PBS at 4 °C for (double) immunostaining.

Immunohistochemistry

For immunohistochemistry, the specimens from nine mice were rinsed three times in 0.01 m PBS containing 0.5% Triton for 5 min per rinse at 25 °C. Endogenous peroxidase was inhibited with 3% H2O2-methanol for 20 min, and non-specific binding sites were blocked with 2% bovine serum albumin (BSA) for 30 min at 25 °C. The specimens were incubated with rabbit anti-HCN2 polyclonal antibody (1 : 100, APC-030; Alomone, Israel) in a humid chamber for 2 h at 25 °C and then for 24 h at 4 °C. The HCN1-4 channels are closely related to each other and share homology of about 60%. However, they differ from each other in their cytoplasmic N- and the C-terminus. Rabbit anti-HCN2 polyclonal antibody specifically recognized the intracellular epitope, (amino acid residues 147–161) of the HCN2 channel from human, rat and mouse samples. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1 : 100; Dako, Denmark) was used as the secondary antibody. For visualization, the DAB staining kit (Sigma, USA) was used according to manufacturer's guidelines. After washing with PBS, the specimens were examined with Olympus BX51 microscope (Olympus, Japan). The control specimens were processed in the same manner, but the primary antibody was omitted. Additional specificity of anti-HCN2 polyclonal antibody was demonstrated by the pre-adsorption experiment in which the primary anti-HCN2 polyclonal antibody (1 : 100) was adsorbed with corresponding antigen peptide (APC-030; Alomone, Israel).

Double-immunohistochemistry

To determine the nature of the HCN2-positive cells, the preparations from nine mice were double-labeled as the following: (i) anti-HCN2 rabbit polyclonal antibody and anti-ChAT (a marker for cholinergic nerves) goat polyclonal antibody (1 : 100, AB144; Chemicon, USA) as primary antibodies (Itoh et al. 2011), and Cy3-conjugated donkey anti rabbit IgG antibody (1 : 100; Chemicon, USA) and FITC-conjugated donkey anti goat IgG antibody (1 : 100; Chemicon, USA) as secondary antibodies, respectively. The anti-ChAT goat polyclonal antibody was raised against ChAT, which mainly distributed in the cholinergic neurons of ENS in mouse GI tract (Schemann et al. 1993; Mongardi et al. 2009; Chandrasekharan et al. 2011); (ii) anti-HCN2 rabbit polyclonal antibody and anti-Kit (a marker for ICCs) mouse monoclonal antibody (1 : 100, ACK2; Dako, Denmark) as the primary antibodies (Mei et al. 2009b), and FITC-conjugated donkey anti rabbit IgG antibody (1 : 100; Chemicon, USA) and Cy3-conjugated rabbit anti mouse IgG antibody (1 : 100; Dako, Denmark) as secondary antibodies, respectively. The anti-Kit rat monoclonal antibody (ACK2) was raised against Kit, which specifically stained ICCs in the GI tract (Ordög et al. 1999; Rich et al. 2002). After incubation and washing, the specimens were mounted with Fluorescent Mounting Medium (Thermo Electron Co.) and examined with a BX51 fluorescence microscope (Olympus, Japan) or TCS SP5 confocal laser scanning microscope (Leica, Germany) with an excitation wave-length appropriate for FITC (488 nm) or Cy3 (550 nm). The control specimens were prepared as described above.

Image analysis and statistics

Photographs were taken in nine random fields per whole-mount preparation (three preparations from each segment in the GI tract of five mice) based on stereological rules at a magnification of 10× under the BX51 microscope. The numbers of HCN2-labeled cells, which were defined as positive cells automatically by image-pro plus 5.0 software (Media Cybernetics) according to the IR intensity, were counted; the areas of ganglia, which were outlined artificially, were also automatically measured and recorded. The number density of HCN2-positive cells was determined by dividing the total number of HCN2-positive cells by the total area of the fields. The area density of the ganglia was derived by dividing the total area of the ganglia by the total area of field. Statistical analyses were performed with the spss 13.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± SD and compared using a one-way analysis of variance (anova). A 2P value of 0.05 was adopted.

Results

Distribution of HCN2-positive cells

The specificity of anti-HCN2 was established by the pre-adsorptive control in which no positive staining was seen in the myenteric neurons of GI tract. HCN2 were mainly distributed in the myenteric plexus of the mouse GI tract from the gastric fundus to the descending colon. Immunoreactivity (IR) for HCN2 was found within the cell membrane and/or cytoplasm but not in the nuclei. HCN2-positive cells were round or ovoid with indistinct dendrites; in these cells, the IR in the soma was much more prominent compared with the IR in the processes (Fig. 1). These HCN2-positive cells appeared to be neurons based on their location and morphology. Dozens of HCN2-positive cells were located in each ganglion, and the primary nerve bundles connecting ganglia were also immunostained.

Fig. 1.

Fig. 1

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A set of photographs of HRP-DAB immunohistochemistry showing the distribution of the HCN2-positive neurons in the mouse GI tract. IR for HCN2 was identified in the cell membrane and/or cytoplasm. HCN2-positive cells were round or ovoid with indistinct processes.

To minimize the effects of traction and folding during preparation on the number density estimations, we prepared the specimens in their natural flat state. Moreover, to normalize the number density analysis, the area density of ganglia was also estimated on different GI segments; the area density was less affected by preparation traction and more constant during preparations (Karaosmanoglu et al. 1996). Results indicated that the distribution of the number density of HCN2-positive cells was in line with that of the area density of ganglia (data not shown), thereby, the number density analysis was omitted in this study.

In the stomach, the number density of HCN2-positive cells was highest in the antrum and lowest in the fundus (P < 0.05) (Fig. 2A). In the small intestine, the number density of HCN2-positive neurons in the ileum was significantly greater than in either duodenum or jejunum (P < 0.05); there was no difference between duodenum and jejunum (Fig. 2B). In the colon, the number density of HCN2-positive neurons in the transverse colon was the greatest when compared with the ascending and descending colon (P < 0.05) (Fig. 2C). It was noted that the number density of HCN2-positive neurons was the greatest in the colon, in which the density was higher than that in either the stomach or the small intestine (P < 0.05) (Fig. 2D).

Fig. 2.

Fig. 2

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Graphs showing the number density of HCN2-positive neurons in different segments of the mouse GI tract. (A) Comparison of the density of HCN2-positive neurons between the gastric fundus, corpus and antrum. (B) Comparison of density of HCN2-positive neurons between the duodenum, jejunum and ileum. (C) Comparison of the density of HCN2-positive neurons between the ascending colon, transverse colon and descending colon. (D) Comparison of the density of HCN2-positive neurons between the stomach, small intestine and colon. *P < 0.05.

Double-labeling with ChAT and HCN2

Ach is synthesized in the neuronal soma and then transported to axonal terminals. In this process, the rate-limiting enzyme, ChAT, catalyses Ach synthesis; therefore, ChAT is a marker for cholinergic neurons. Double-labeling with HCN2 and ChAT was used to explore the nature of HCN2-positive cells. Immunofluorescence staining showed that ChAT-positive ganglia, connecting nerve bundles and nerve fibers, generally existed in the myenteric plexus of the GI tract. Dozens of ChAT-positive neurons were located within a ganglion, and a few ChAT-positive neurons were also located in the connections of primary nerve bundles. ChAT-positive neurons were distinct, round or oval, and had slim axons and indistinct dendrites. IR for ChAT stained both the membrane and cytoplasm but not nuclei (Fig. 3 was taken from the transverse colon and the same IR for ChAT was also identified in other segments of the GI but not shown). Double-labeling revealed that almost HCN2-positive cells completely overlapped with ChAT staining, indicating that these HCN2-IR cells were cholinergic neurons (Fig. 4 was taken from the gastric antrum, jejunum and transverse colon, respectively, and the same IR for HCN2 and ChAT was also identified in other segments of the GI but is not shown). There were a few voids which were labeled neither with ChAT nor HCN2 in the myenteric ganglion; these voids were more frequent in stomach and small intestine than in colon (Fig. 4).

Fig. 3.

Fig. 3

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(A–C) Photographs of immunofluorescence for ChAT were taken with a fluorescence microscope to show the myenteric plexus in the transverse colon. IR for ChAT was identified within neurons, nerve bundles connecting ganglia and nerve fibers. ChAT-positive neurons were located in ganglia and connecting primary nerve bundles. Neurons were distinct, round or oval, with slim axons and indistinct dendrites. The same IR for ChAT was also identified in other segments of the GI.

Fig. 4.

Fig. 4

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Double-immunofluorescence for HCN2 and ChAT with confocal laser scanning microscopy. Row A showed ChAT staining (green), Row B, HCN2 staining (red) and row C, the merged double-labeling for HCN2 and ChAT in the gastric antrum, jejunum and transverse colon, respectively. Double-labeling results suggested that HCN2-positive neurons were ChAT-positive cholinergic neurons in the GI tract. Arrows indicated voids which were labeled with neither ChAT or HCN2 in the myenteric ganglion. The same IR for HCN2 and ChAT was also identified in other segments of the GI.

Double-labeling with HCN2 and Kit

To determine whether HCN2 was expressed on ICCs, which initiate spontaneous GI rhythms, double-labeling with Kit and HCN2 was performed. As seen above, HCN2-positive neurons in the myenteric plexus were densely arranged in ganglia (Fig. 5A). It was noted that the Kit-positive ICCs were oval or spindle-shaped, with two or three long, thin processes around the myenteric plexus region. They formed an independent cellular network with one another via gap junctions between their cytoplasmic processes (Fig. 5B). Double-labeling demonstrated that ICCs were not co-immunostained for HCN2. However, the HCN2-positive neurons were in close proximity to the processes of ICCs (Fig. 5C).

Fig. 5.

Fig. 5

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One set of photographs of the confocal laser scanning microscope showing the results of the double-labeling with HCN2 and Kit immunofluorescence in the jejunum. (A) IR for HCN2 (green). Arrow indicated a neuron. (B) IR for Kit (red). Arrows indicated the soma (white) and the process (blue). (C) Double-labeling for HCN2 and Kit, suggesting that ICCs were not co-immunostained for HCN2. Arrow indicated that the HCN2-positive neurons were in close proximity to the processes of ICCs. The similar results were also seen in other segments of the GI.

Discussion

The present study demonstrated that HCN2 channels were generally expressed in the myenteric neuronal soma and their neurites of the mouse GI tract. Double-labeling revealed that HCN2-positive neurons are cholinergic, meaning that they synthesize and release the ENS neurotransmitter Ach. In the myenteric plexus, Ach has multiple functions in the inter-neurons, sensory neurons and motor neurons and functions to control the contractions and relaxations of smooth muscle cells of the GI tract through the nerve terminals derived from cholinergic neuronal somas (Vasilyev & Barish, 2002; Robinson & Siegelbaum, 2003). Ach released from cholinergic neurons excites M receptors on the smooth muscle cells and thereby moderates GI peristalsis (Uchiyama & Chess-Williams, 2004). Although there was no direct evidence on the precise physiological effects of HCN2 channels in the GI tract, Linden et al. (2003) reported that HCN2 channels participate in the abnormal firing observed in GI inflammatory diseases. There are two major electrophysiologically defined neuron types of the ENS: the AH neurons and the S neurons. AH neurons, which are immunoreactive for HCN2, exhibit If current in the mouse distal colon (Xiao et al. 2004). It has been shown that AH neurons regulate neuronal excitability in the inflamed guinea-pig distal colon (Linden et al. 2003), indicating that HCN2 channels were likely critical in the regulation of neuronal excitability in the GI tract. If would change the resting membrane potential of AH neurons, increasing their sensitivity to chemical and/or mechanical stimulations, and thereby increasing the neuronal electric activities for the modulation of GI peristalsis (Galligan et al. 1990). Previous studies have shown that HCN2 channels were distributed in the axonal terminals, where they modulated the release of Ach in the CNS (Phillips & Powley, 2007). Therefore, we propose that HCN2 channel-associated If current might be involved in Ach release from the cholinergic neurons of the myenteric plexus and thereby regulate peristalsis of the GI tract. Moreover, our results showed that a few voids which were labeled neither with ChAT nor HCN2 were also encountered in the myenteric ganglion; and these voids were more frequent in stomach and small intestine than in colon (see Fig. 4). Since the neuronal population in the myenteric plexus almost entirely comprises cholinergic neurons and nitrergic neurons, two mutually exclusive sub-populations, such voids might correspond to the nitrergic neurons. Nitrergic neurons in ENS act as inhibitory motor neuron, which would co-regulate the GI motility with excitatory cholinergic neurons. However, whether HCN2 is expressed by the nitrergic as well as the cholinergic neurons needs further direct investigation by double-labeling with HCN2 and neuronal nitric oxide synthase (nNOS), a marker of nitregic neurons in ENS.

The origin of slow-waves, which was believed to be the basic pacemaker for the GI tract, has been debated for many years. Studies on isolated smooth muscle cells failed to demonstrate a capability to generate slow-wave activity (Farrugia, 1999); however, isolated ICCs were invariably found to have a rhythmic Cl-current (Tokutomi et al. 1995). Studies of mutant mice lacking subpopulations of ICCs provided further support for the role of ICCs in the generation of slow-waves (Huizinga et al. 1995). ICCs are now considered to be a pacemaker that generates and propagates spontaneous slow-waves within the GI tract. ICCs are classified into four types according to their location in GI tract and they are spindle-shaped or satellite-like, with two to five long processes that repeatedly branch to form a cellular network via gap junctions. ICCs also form gap junctions with smooth muscle cells. Of these, ICC-MY and ICC-SM are considered to be the pacemakers that generate slow-waves that are then propagated along the network of ICCs to reach the smooth muscle cells via gap junctions (Sanders et al. 2006; Mei et al. 2009a). The slow-wave is the base of the rhythmic contractions of the GI tract. However, in our study, no double-labeled ICC-MY and ICC-SM with HCN2 were observed in the GI tract, and HCN2 channels correlated with spontaneous pacing were only found in neurons forming independent networks from the ICCs, indicating that HCN2 channels are not involved in the initiation of slow-waves generated by ICCs. However, it was noted that HCN2-positive neurons and their terminal endings exist in physical proximity with the cellular network of the ICCs, suggesting that the ICCs, which do express Ach receptors, may be involved in the mediation of Ach transmission of the HCN2-positive neurons (Kim et al. 2006). This finding further suggests that such ICCs respond to Ach, based on monitoring the membrane potentials (Faussone-Pellegrini et al. 2007). Therefore, HCN2-positive neurons regulated GI peristalsis not only through Ach, directly exciting M receptors on smooth muscle cells, but also indirectly via ICC-based mediation (Uchiyama & Chess-Williams, 2004).

To assess the regional difference in the distribution of HCN2-positive neurons, we estimated the density of such neurons in different GI segments. To avoid the effects of preparation traction on this analysis, the area density of ganglia was also estimated on different GI segments, as this measure was more constant during preparations. It was noted that similar results were obtained using both the number density of neurons and the area density of ganglia (data not shown), indicating that the number density analysis of neurons was not obviously influenced by preparations. Our results demonstrate that the number density of HCN2-positive neurons was the highest in the colon and the lowest in the small intestine, with intermediate numbers measured for the stomach. In the stomach, the number density of HCN2-positive neurons increased gradually but significantly from the proximal (fundus) to the distal (antrum). In the small intestine, there were no significant differences in the number density of HCN2-positive neurons between the duodenum and jejunum; however, the number density was significantly greater in the ileum than in either the duodenum or jejunum. The number density of HCN2-positive neurons in the transverse colon was significantly greater than that of the ascending and descending colon. These results are consistent with previous studies on neuron counts in the myenteric plexus of guinea pigs (Karaosmanoglu et al. 1996). The observed regional differences in the HCN2 neuronal distribution are probably related to the distinct functions of the HCN2 neurons in different GI segments. It has been reported that the frequency of slow-waves in the stomach increases from the proximal to the distal. The frequency of slow-waves is faster in the transverse colon than in either the ascending or descending colon. The observed regional differences in slow-wave frequency are consistent with the regional distribution of the number density of HCN2-positive neurons in the stomach and colon. Slow-waves, which are initiated by ICCs and propagate to smooth muscle, are not action potentials; therefore, they cannot directly induce the contraction of GI smooth muscle. However, slow-waves decrease the threshold for excitability to trigger the action potential and corresponding GI peristalsis. HCN2-positive neurons and their terminal endings are close to the ICCs. Thus, we assumed that HCN2 channels may enhance Ach release to generate action potentials on the basis of slow-waves for GI peristalsis. Nevertheless, the number density of HCN2-positive neurons does not seem to relate to the frequency of the slow-waves in the small intestine, where they are more frequent in the duodenum than in the jejunum or ileum. This may be compensated by the overlap of high-frequency slow-waves in the antrum and low-frequency in the duodenum. Diamant & Bortoff (1969) interpreted this gradual decrease in intestinal slow-waves according to the vibration theory. However, the precise relationship between slow-wave frequency and HCN2-positive neuron number in the GI tract will require additional investigation.

In summary, HCN2 channels are mainly expressed in myenteric neurons with some regional differences in the mouse GI tract. These regional differences are partly adapted to the corresponding electric activities of the different segments of the GI tract. HCN2-positive cells are also immunoreactive for ChAT, indicating that HCN2 channels might be able to facilitate Ach release from cholinergic neurons. Moreover, although HCN2-positive neurons are not immunoreactive for Kit, the neuronal somas and neurites were in close proximity to the process of ICCs, indicating that HCN2-positive neurons are likely involved in the regulation of GI peristalsis in both ways, i.e. by directly exciting M receptors on smooth muscle cells and indirectly mediating ICCs with Ach signaling. Our results may provide a new clue for future studies of the mechanisms underlying the spontaneous rhythmic motility and associated motility disorders of the GI tract.

Acknowledgments

This work was supported in part by grants Nos 30971349 and 81101769 from the National Science Foundation of China (NSFC) and by grant No. PHR201007113 from the Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (IHLB).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Pre-adsorption control by immunofluorescence staining was taken from the transverse colon.

joa0221-0303-SD1.docx (546.4KB, docx)

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