FUNCTIONAL ROLES OF INTERSTITIAL CELLS OF CAJAL IN THE GI TRACT OF RATS

Abstract

Interstitial cells of Cajal (ICC) are distributed through the gastrointestinal (GI) tract, but the functional role of these cells comes primarily from studies of mice. Whether functions of ICC are similar in larger animals is largely speculative. We investigated whether the Kit mutation in Ws/Ws rats had consequences on ICC populations in stomach, small intestine, and colon and whether loss of ICC resulted in functional defects similar to Kit mutations in mice. Immunohistochemical labeling with c-KIT or ANO1 antibodies revealed loss of intramuscular ICC (ICC-IM) and reduced myenteric ICC (ICC-MY) in stomachs of Ws/Ws mutants. Disruption of ICC-MY networks but not ICC within the deep muscular plexus (ICC-DMP) was observed in the small intestine. ICC in the proximal colon were reduced, but no population was absent. ICC loss in the stomach caused loss of spontaneous transient depolarizations, reduced pacemaker activity and reduced responses to cholinergic and nitrergic nerve stimulation. Loss of ICC-MY in the small intestine resulted in abnormal pacemaker activity, but neural responses appeared to be normal. In the proximal colon tonic inhibition due to ongoing nitrergic neural inputs was reduced, spontaneous spike complexes were less rhythmic and nitrergic neural responses were reduced. Apamin-sensitive inhibitory neural responses were retained throughout the GI tract. In summary Ws/Ws rats have lesions in ICC and functional deficits similar, but not identical to Kit mutant mice. These larger animals with more robust GI muscles may be useful for investigations into the role of ICC in normal and abnormal GI motility.

NEW AND NOTEWORTHY

The physiological roles of interstitial cells of Cajal throughout the gastrointestinal tract have been derived predominantly from studies of mice. We sought to determine if reduction in ICC in the rat, a commonly used animal for studies of GI motor functions, lead to functional deficits. Ws/Ws rats display reduced ICC leading to a disruption in pacemaker activity and neuroeffector responses. Our results provide additional evidence for the functions of ICC in the GI tract.

Graphical Abstract

Two basic classes of ICC were identified morphologically in rat GI muscles: i) ICC in the plane of the myenteric plexus (ICC-MY) and cells within muscle bundles in the circular and longitudinal muscle layers, intramuscular ICC or ICC-IM. Because of the specific location of ICC-IM within the region of the deep muscular plexus in the small intestine, these cells are also referred to as ICC-DMP. Studies on mice have suggested different roles for these ICC, and therefore ICC functions are classified (2^nd column) as pacemaker activity (ICC-MY) and neurotransmission (ICC-IM/ICC-DMP) in stomach, small intestine and colon. In the gastric fundus, ICC-IM also generated spontaneous transient depolarizations, events known to regulate membrane potential and contribute to neurotransduction. Third column shows diverse defects noted in the electrical and mechanical behaviors of Ws/Ws rats. Some of these defects are equivalent to reports on mouse mutants with defective ICC populations, but there are also important differences, such as the disordered networks of ICC-MY in the gastric antrum and aberrant slow wave activity and the partial loss of ICC-MY in the small intestine. This summary should provide a road map for investigators wanting to use rat GI tissues and organs to investigate the functional roles of ICC.

INTRODUCTION

ICC located along the intermuscular plane between the circular and longitudinal muscle layers of the gastrointestinal tract are pacemaker cells that generate electrical slow waves and phasic contractions in many regions of the GI tract.¹ Intramuscular ICC (ICC-IM) dispersed within the muscle layers of the stomach and colon and at the level of the deep muscular plexus in the small intestine (AKA ICC-DMP) mediate cholinergic excitatory and nitrergic inhibitory neurotransmission. ^2–6 ICC-IM are also mechanosensitive cells that mediate stretch dependent regulation of pacemaker frequency in the gastric antrum, ^7,8 and critical for the formation of vagal afferent intramuscular arrays in the stomach. ^9,10

Most studies investigating the functions of ICC in the GI tract have utilized mouse models. Intraperitoneal injection of the c-KIT neutralizing antibody, ACK2, into newborn mice, or organotypic culture of embryonic or newborn intestines blocks the development and/or maintenance of ICC networks and demonstrated the importance of ICC in pacemaker activity.^11–13 These experiments were performed concurrently with studies of W/W^V and Sl/Sl^d mutant mice that have mutations in Kit and stem cell factor (or steel), causing functional loss of the receptor tyrosine kinase activity of c-KIT.^14–16 W/W^V and Sl/Sl^d mice also have disrupted ICC networks in the small intestine and loss of pacemaker activity.^17–20 Gastric lesions were also observed in these mice with loss of ICC-IM and reduced or abolished cholinergic or nitrergic neurotransmission. ^{2,3, 21–23} Additional support for these conclusions came from experiments using mice with inducible expression of diphtheria toxin (DTA) in ICC. Activation of DTA expression caused defective pacemaker activity in the small intestine and defects in neurotransmission in the colon. ²⁴

Mice, particularly strains harboring cell-specific genetic modifications, have been extremely useful for understanding the role of ICC in GI muscles, however experimental limitations are imposed by the small size of GI organs in these animals. Currently it is only assumed that the functions of ICC are common throughout mammalian species. Therefore, we sought to expand understanding of the consequences of ICC loss with studies of another and larger animal species. Ws/Ws rats have deletion of 12 bases, encoding a Val-Lys-Gly-Asn sequence downstream from the tyrosine autophosphorylation site in the c-KIT. This sequence is important for function and conserved in mouse and human c-KIT and in mouse and human c-fms kinases (colony-stimulating factor-1 receptor). Disruption in these four amino acids leads to reduced receptor tyrosine kinase activity, ²⁵ and loss of specific classes of ICC in the GI tract. ^26,27 In this study we tested the hypothesis that loss-of-function mutations in Kit would result in changes in basal electrophysiology and defects in responses to enteric neurotransmission in rat GI muscles. To test this hypothesis, we compared the distribution of ICC throughout the GI tracts of wildtype and Ws/Ws rats and measured functional characteristics of muscles from these animals.

MATERIALS AND METHODS

Animals and tissue preparation.

Ws/Ws rats and their age matched wildtype (+/+) siblings were obtained from WsRC-Ws/⁺ breeder pairs purchased from Japan SLC, Inc. (Shizuoka, Japan). Animals were housed in a 12/12-hour light-dark cycle and supplied with food and water ad libitum before being euthanized. Animals were anesthetized by isoflurane (Baxter, Deerfield, IL, USA) inhalation and exsanguinated following cervical dislocation. The GI tracts from 1 cm above the lower esophageal sphincter to 1 cm above the internal anal sphincter was removed and placed in Krebs’-Ringer bicarbonate buffer (KRB). Stomachs were opened along the lesser curvature, small intestines and proximal colons were opened along the mesenteric border, and contents washed with KRB. Tissues were subsequently pinned to the Sylgard elastomer (Dow Corning, Inc) base of a dissecting dish and the mucosa removed by sharp dissection revealing the underlying circular muscle layer.

All animals were maintained, and the experiments performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures used were approved by the Institutional Animal Use and Care Committee at the University of Nevada.

Morphological studies.

Immunohistochemical studies were carried out on whole mounts as previously described.²⁸ Briefly whole mount tissues were stretched to 110% of their resting length and width before being fixed in either Zamboni’s fixative (2% formaldehyde plus 0.2% picric acid in 0.1 M phosphate buffer for 15 min at 4°C) or paraformaldehyde (4% w/v in 0.01M phosphate buffer for 15 min at 4°C). Tissues that were fixed in Zamboni’s were washed three times (15 min) with dimethyl sulfoxide (DMSO) and three times (10 min) with phosphate buffered saline (PBS; 0.01 M, pH 7.4). Following fixation, preparations were washed overnight in PBS. Incubation of tissues in BSA (3%) for 1 h at room temperature containing Triton X-100 (0.5%) was used to reduce non-specific antibody binding. Tissues were incubated in primary antibodies at 4°C overnight. Following incubation in primary antibodies, tissues were washed and incubated separately in appropriate secondary antibodies (Alexa Fluor 488 and 594; Thermo Fisher Scientific Inc., Waltham, MA, USA, diluted to 1:1000 in PBS for 1 hr. at room temperature). Control tissues were prepared by either omitting primary or secondary antibodies from the incubation solutions. All the antisera were diluted with 0.5% Triton X 100 in 0.01M PBS (pH 7.2). The antibodies used in this study are listed in Table I.

Table I.

Details of antibodies used for immunohistochemistry.

Primary Antibody Target	Resource	Cat No. RRID No.	Clone	Host	Dilution	Fixative
c-KIT	R&D Systems Inc., Minneapolis, MN, USA	AF1356	Poly-	Goat	1:100	Zamboni’s
ANO1	Abcam, Cambridge, MA, USA	AB323181	Poly-	Rabbit	1:800	Zamboni’s
vAChT	Millipore Corp. (Temecula, CA, USA)	SAB4200559	Poly-	Rabbit	1:500	Zamboni’s
	UC Davis/NIH NeuroMab Facility. Department of Physiology and Membrane Biology, UC Davis, Davis CA, USA.	AB_2188148 (RRID No.)	Mono	Mouse	1:500	Paraform
nNOS	R&D Systems Inc., Minneapolis, MN, USA	AF2416	Poly-	Goat	1:100	Zamboni’s
	Piers Emson, Mol. Sci. Group, Cambridge, UK	N/A	Poly-	Sheep	1:500	Paraform
PDGFRα	R&D Systems Inc., Minneapolis, MN, USA	AF1062	Poly-	Goat	1:100	Zamboni’s

N.B. To avoid cross reactivity of antibodies in double labeling immunohistochemical experiments, the host species of the primary antibodies was chosen to avoid labeling or cross-reactivity issues.

Tissues were examined with a Zeiss LSM 510 Meta (Jena, Germany), Nikon A1R (Melville, NY, USA) or Leica Stellaris (Wetzlar, Germany) confocal microscopes with appropriate excitation wavelengths. Confocal micrographs were digital composites of Z-series scans of 10–20 optical sections through a depth of 10–20 μm. Final images were constructed, and montages were assembled using Zeiss LSM 5 Image Examiner, Nikon NIS Elements or Leica LAS X and converted to Tiff files for final processing in Adobe Photoshop CS5 software (Adobe Co., Mountain View, CA, USA) and Photoshop 7.0 and Corel Draw X8 (Corel Corp. Ontario, Canada).

Physiological experiments.

After removing the mucosa and submucosa, gastric fundus, antrum, small intestine and colon muscle strips (approximately 10 mm wide × 10 mm in length for most regions, 5 mm in length for the gastric antrum) were cut and pinned to the Sylgard (Dow Corning Corp., Midland, MI, USA) elastomer-lined floor of a recording chamber. Stomachs was cut along the lesser curvature. Gastric fundus and antral tissues (5×10 mm) were taken from along the greater curvature, while intestinal and colonic muscles were taken from the anti-mesenteric border. In some experiments, gastric fundus and antral muscles were re‐pinned serosal surface uppermost and the longitudinal muscle layer dissected away. Single bundles of circular muscle (diameter 50–150 μm, length 400–800 μm) were dissected free and pinned in a recording chamber.²⁹ Electrical activities of circular muscle cells were made, and signals recorded onto a PC running AxoScope 9.0 (Axon Instruments, Union City, CA, USA). ²¹ Small intestinal experiments were performed in the presence of nifedipine (1 μM) to reduce contractions and facilitate impalements of cells for extended periods of time where stated. Proximal colon experiments were also performed in the presence of nifedipine to reveal post-junctional neural responses. Neural responses were also elicited as previously described.²¹ Electric field stimulation (EFS, 0.1–0.5 ms duration pulses were delivered for 1 second at a frequency of 1–20 Hz). Specific agonists or antagonists were added to the bathing solution at the stated concentrations to evaluate cholinergic, nitrergic and purinergic motor responses.

Solutions and drugs.

The bath chamber was constantly perfused with oxygenated KRB of the following composition (mM): NaCl 118.5; KCl 4.5; MgCl₂ 1.2; NaHCO₃ 23.8; KH₂PO₄ 1.2; dextrose 11.0; CaCl₂ 2.4. The pH of the KRB was 7.3 – 7.4 when bubbled with 97% O₂ - 3% CO₂ at 37 ± 0.5°C. Muscles were left to equilibrate for at least 1 hour before experiments were initiated. Atropine, N^ω-nitro-L-arginine (L-NNA), apamin (Sigma, St Louis, MO, USA) and MRS2500 (Tocris; Minneapolis, MN, USA) were dissolved in de-ionized H₂O before being diluted in KRB to the final concentration stated in the results section. In some electrophysiological experiments nifedipine (Sigma) was dissolved in ethanol at a stock concentration of 10 mM before being added to the perfusion solution at a final concentration of 1 μM.

Analysis of data.

Data are expressed as means ± standard errors of the mean. The student’s t test was used where appropriate to evaluate differences in the data. P values less than 0.05 were taken as a statistically significant difference. The “n” values reported in the text refer to the number of muscle strips used for each experimental protocol. Each muscle strip used in “n” values were taken from a separate animal. The electrical parameters that were analyzed were i) resting membrane potential (RMP), ii) slow wave amplitude, iii) frequency and iv) ½ maximal duration for antral and small intestine slow waves. The amplitudes and durations of post junctional excitatory and inhibitory neural responses were also measured. Figures displayed were made from digitized data using Adobe Photoshop 7.0 (Adobe Co., Mountain View, CA, USA) and Corel Draw X7 (Corel Corp. Ontario, Canada).

RESULTS

Gastric Fundus

ICC in the fundus of wildtype and Ws/Ws rats.

Immunohistochemical analysis of whole mount preparations revealed the presence of spindle shaped c-KIT immunopositive intramuscular ICC (ICC-IM) within the circular (CM) and longitudinal (LM) muscle layers of the gastric fundus of wildtype animals. ICC-IM were typically 225 ± 15 μm in length and ran parallel to the smooth muscle cells of the muscle layer in which they were located (Fig. 1A). Cells with similar morphology and distribution as ICC-IM were labeled with antibodies against the Ca²⁺-activated Cl^- channel, ANO1 (Fig. 1B) and double labeling with c-KIT antibodies confirmed ANO1⁺ cells were ICC (Fig. 1C). Double labeling of ANO1⁺ cells with antibodies against vesicular acetylcholine transporter (vAChT) showed that varicose vAChT⁺ nerve fibers were closely associated with ANO1⁺ ICC-IM (Fig. 1D-F & J). Cell processes labeled with antibodies against neuronal nitric oxide synthase (nNOS) were also closely associated with ANO1⁺ ICC-IM (Fig. 1G-I & 1K). Similar immunohistochemical analysis of gastric fundus muscles from Ws/Ws rats showed that c-KIT⁺ and ANO1⁺ cells were greatly reduced in numbers (Fig. 2A-C,D & G). However, no defects were noted in the distribution of vAChT⁺ and nNOS⁺ nerve fibers in the Ws/Ws rat fundus (Fig. 2E,F,H,I,J & K).

Figure 1. c-KIT and ANO1-immunopositive ICC-IM in the gastric fundus of wildtype animals and their close anatomical relationship with enteric motor nerves.

Panel A, shows the distribution of c-KIT⁺-ICC-IM (red) within the circular and longitudinal muscle layers (arrows and arrowheads, respectively). Panel B, shows a similar distribution of ANO1⁺-ICC-IM within the circular and longitudinal muscle layers (green; arrows and arrowheads, respectively). Panel C, is a merged image of Panels A and B showing cellular co-localization of c-KIT and ANO1⁺- ICC-IM in the gastric fundus (yellow; arrows and arrowheads respectively). Panel D, ANO1⁺-ICC-IM within the circular layer (red; arrows). Panel E, vesicular acetylcholine transporter (vAChT; green; arrowheads)-immunopositive nerve fibers within the circular layer. Panel F, merged image of Panels D and E showing the close morphological relationship between vAChT⁺ nerve fibers and ICC-IM in the gastric fundus. Panel G, ANO1⁺-ICC-IM within the circular layer (red; arrows). Panel H, neuronal nitric oxide synthase (nNOS; green; arrowheads)-immunopositive nerve fibers within the circular layer. Panel I, merged image of Panels G and H revealing the close morphological relationship of nNOS⁺ nerve fibers with ICC-IM. Panels J and K, higher power images of the regions outlined by the dashed white rectangles in Panels F and I, showing the relationship between vAChT⁺ cholinergic excitatory and nNOS⁺ inhibitory nerves (arrowheads) and ICC-IM (arrows), respectively. Scale bars are as indicated in Panels C, F, I, J & K are representative of the appropriate series of panels, where applicable.

Figure 2. Absence of c-KIT and ANO1-immunopositive ICC-IM in the gastric fundus of Ws/Ws mutant animals.

Panel A, reveals the absence of c-KIT⁺-ICC-IM within the circular and longitudinal muscle layers. Panel B, ANO1⁺-ICC-IM are also absent in both muscle layers. Panel C, merged image of Panels A and B. Panel D, absence of ANO1⁺ ICC-IM, however, vAChT⁺ nerve fibers (Panel E, green; arrowheads) are distributed within the circular muscle layer similar to wildtype controls. Panel F, merged image of Panels D and E show the absence of ANO1⁺ ICC-IM but presence of vAChT nerve fibers (arrowheads). Panel G, similar to Panel D, ANO1⁺ ICC-IM are absent in the gastric fundus of Ws/Ws mutants however, Panel H, nNOS⁺ nerve fibers (arrowheads) were present in a distribution and density similar to wildtype animals. Panel I, merged image of Panels G and H, displaying the absence of ANO1⁺ ICC-IM but the presence of nNOS⁺ nerve fibers (arrowheads). Panels J and K, higher power images of the regions outlined by the dashed white rectangles in Panels F and I, showing the presence of vAChT⁺ cholinergic excitatory (arrowheads) and nNOS⁺ inhibitory nerves (arrowhead) within the circular layer but the absence of ICC-IM. Scale bars are as indicated in Panels C, F, I, J & K are representative of the appropriate series of panels, where applicable.

Electrical activity and responses to enteric nerve stimulation were abnormal in the Ws/Ws gastric fundus.

Resting membrane potentials (RMPs), recorded via intracellular impalements of SMCs in sheets of fundus muscle (10 mm × 10 mm), averaged −43 ± 0.3 mV. Slow waves are absent in the gastric fundus. Membrane potentials were unstable, consisting of irregular oscillations, referred to as spontaneous transient depolarizations (STDs)^2,29,30 (n = 8; Fig. 3A). RMPs were also recorded from tiny bundles of CM (diameter 100–200 μm, length 400–800 μm). These tissues, likely to have higher input resistances than sheets of muscle, had average RMPs of −49±1.5 mV (n=4) and displayed larger amplitude fluctuations (Fig. 3C). Ongoing STDs have also been recorded in fundus muscles of mice.^2,29,30 In contrast RMPs of fundus muscles from Ws/Ws rats were quiescent electrically and averaged −42±1 mV (n=8; Fig. 3B).). Recordings from isolated CM bundles from fundus Ws/Ws rats also showed quiescent RMPs that averaged −50±3 mV (n =3; Fig. 3D).

Figure 3. Electrical activities and post-junctional neural responses from the gastric fundus of wildtype and Ws/Ws mutant animals.

Panels A and B, shows the electrical activities recorded from sheets of gastric fundus muscles from wildtype and Ws/Ws mutant rats, respectively. The spontaneous membrane noise recorded from wild type muscles (Panel A) was absent from Ws/Ws mutants (Panel B). Panels C and D, show the electrical activity recorded from isolated fundus circular muscle bundles from a wildtype control and a Ws/Ws mutant respectively. The spontaneous discharge of STDs recorded from wildtype tissues (Panel C) was absent from Ws/Ws mutants (Panel D). Panel E, shows post-junctional neural responses recorded from a wildtype animal in response to electrical field stimulation (EFS) under control conditions and in the presence of L-NNA (100 μM; Panel F) and L-NNA and atropine (1 μM; Panel G). EFS (0.3 ms; 5 and 20 Hz for 1 sec) was delivered at the arrow heads under each trace. Panel E, Under control conditions EFS evoked a fast and slow IJP. In some preparations a transient EJP was observed. Panel F, After L-NNA the slow component of the IJP was abolished (dashed lines in E) and unmasked a larger EJP (arrows) that was abolished by atropine (Panel G). Panels H-J, shows neural responses from the gastric fundus of a Ws/Ws mutant rat under similar conditions. Under control conditions (Panel H) and in L-NNA (Panel I), there was an absence of cholinergic EJPs and slow nitrergic IJP responses in the Ws/Ws mutant tissue to EFS. Panel J, atropine had little or no effect on post-junctional neural responses. Panels K and L, Neural responses to EFS (20 Hz) in L-NNA, displayed at a faster sweep speed from a wildtype control (Panel K) and Ws/Ws mutant (Panel L). Note the EJP present in wildtype fundus (arrow) was absent in the Ws/Ws mutant tissue.

Electrical field stimulation of fundus CM caused frequency-dependent, bi-phasic inhibitory junction potentials (IJPs), consisting of an initial rapid hyperpolarization phase followed by more sustained hyperpolarization. At 5 Hz for 1 sec, EFS evoked IJPs that averaged 10.7±2 mV in amplitude, persisted for 3.2±0.1 seconds and were followed by a smaller, secondary phase, 3.0±0.6 mV in amplitude, that persisted for 14.1±0.9 seconds (Fig. 3E; n=8). In some preparations small-amplitude, transient depolarizations were observed before onset of the IJPs. Addition of L-NNA (100 μM) reduced the initial component of the IJPs to 6.0±1.4 mV and abolished the secondary, sustained component (P=0.073 and P=0.0006, respectively. In the presence of L-NNA, a rapid depolarization termed excitatory junction potential (EJP) was revealed (Fig. 3F & K). Atropine (1 μM, added in the presence of L-NNA), blocked EJPs and slightly increased the duration of the remaining IJP to 10±1.6 mV at 5 Hz (Fig 3G).

In Ws/Ws fundus muscles, EFS evoked monophasic IJPs that were smaller in amplitude and shorter in duration than in wildtype muscles. At 5 Hz, the IJP amplitude averaged 3.5±0.3 mV and 2.4±0.2 seconds in duration (Fig. 3H). L-NNA (100 μM) had little effect on IJPs in Ws/Ws fundus muscles (IJP amplitude averaged 3.0 ± 0.4 mV in amplitude and 2.6 ± 0.14 seconds in duration (P=0.243 and P=0.313, for amplitude and duration respectively compared to control; n = 6; Fig. 3I). Addition of L-NNA did not unmask EJPs in Ws/Ws fundus muscles (Fig. 3I & 3L). In LNNA and atropine IJPs were also not affected (4.2±0.4 mV in amplitude and 3.0±0.2 seconds in duration at 5 Hz; (P=0.09 and P=0.232, for amplitude and duration respectively compared to control (n = 6; Fig. 3J).

After addition of L-NNA and atropine, IJPs were prevalent at higher frequencies of EFS (5–20 Hz), and these events were observed in both wildtype and Ws/Ws tissues. Apamin (0.3 μM) reduced the amplitude and duration of IJPs resistant to L-NNA in wildtype (Figs. 4A & B) and W/Ws (Figs. 4C & D) fundus muscles. A summary of the effects of apamin on IJPs in wildtype and Ws/Ws rat fundus is shown in Fig. 4E & F, respectively.

Figure 4. Inhibitory responses in both wildtype and Ws/Ws mutant fundus were attenuated by apamin.

Panel A, In the presence of L-NNA (100 μM) and atropine (1 μM), EFS (5&10 Hz) evoked robust IJPs that were attenuated by apamin (0.3 μM; Panel B). Panels C and D, In L-NNA and atropine EFS (5&10 Hz) also evoked IJPs in Ws/Ws fundus muscles that were also attenuated by apamin (0.3 μM). Panels E and F, Summary of the amplitude of EFS evoked inhibitory neural responses (IJPs) in the presence of L-NNA and atropine (black bars) and after the addition apamin (white bars), in the of both wildtype (Panel E) and Ws/Ws mutant fundus tissues (Panel F). * P < 0.05 and ** P < 0.01.

Gastric Antrum

ICC in the antrum of wildtype and Ws/Ws rats.

c-KIT and ANO1 immunohistochemistry was performed on whole mount preparations of gastric antrum, 5 to 1 mm from the pyloric sphincter, from wildtype and Ws/Ws rats to characterize the distribution and density of ICC in this region of the stomach. A dense anastomosing network of ICC was observed in the plane of the myenteric plexus (ICC-MY; Fig. 5A) in wildtype antrums. Double labeling with antibodies against ANO1, revealed that c-KIT⁺ ICC-MY were ANO1⁺ (Fig. 5B & C). Spindle-shaped, c-KIT⁺ ICC-IM were found within the circular muscle layer (Fig. 5D), and these cells were also ANO1⁺ (Fig. 5E & F). Double labeling with antibodies against vAChT and nNOS and c-KIT or ANO1 showed that processes of enteric motor neurons were closely associated with ICC-IM (Fig. 5G-N). A similar close morphological relationship between vAChT⁺ or nNOS⁺ neurons and ICC-MY was not apparent.

Figure 5. c-KIT and ANO1-immunopositive ICC-MY and ICC-IM in the gastric antrum of wildtype animals and their close anatomical relationship with enteric motor nerves.

Panel A, c-KIT⁺ ICC-MY (red, arrows) and Panel B, ANO1⁺ ICC-MY (green, arrowheads) in the gastric antrum. Panel C, merged image of Panels A and B, showing cellular co-colocalization of c-KIT and ANO1 in ICC-MY (yellow, arrows). Panel D, c-KIT⁺ ICC-IM (red, arrows) and Panel E, ANO1⁺ ICC-IM (green, arrowheads) within the circular muscle layer. Panel F, merged image of Panels D and E showing cellular co-localization of c-KIT and ANO1 (yellow, arrows) in ICC-IM within the circular muscle layer. Panels G and H, ANO1⁺ ICC-IM (red, arrows) and vAChT⁺ nerve fibers (green, arrowheads) within the circular muscle layer, respectively. Panel I, merged image of Panels G and H showing the close morphological relationship between ANO1⁺ ICC-IM and vAChT⁺ excitatory nerve fibers (arrows) within the antral circular muscle layer. Panels J and K, c-KIT⁺ ICC-IM (red, arrows) and nNOS⁺ inhibitory nerve fibers (green, arrowheads) within the circular muscle layer, respectively. Panel L, merged image of Panels J and K showing the close anatomical relationship between nNOS⁺ inhibitory nerve fibers and c-KIT⁺ ICC-IM (arrows) within the antral circular muscle layer. Panels M and N, higher power images of the regions outlined in Panels I and L by the dashed white rectangles, showing the close anatomical relationship between vAChT⁺ cholinergic excitatory (M; arrowhead) and nNOS⁺ inhibitory nerves (N, arrowhead) and ANO1⁺ and c-KIT⁺ ICC-IM (arrows) within the circular layer of the antrum at higher magnification. Scale bars are as indicated in Panels C, F, I, L, M & N are representative of the appropriate series of panels, where applicable.

c-KIT⁺ and ANO1⁺ ICC-MY were reduced in gastric antrums of Ws/Ws rats and often appeared patchy in whole mount preparations (Fig. 6A-C). Similar to fundus, ICC-IM were greatly reduced or absent in the antrum of Ws/Ws rats (Fig. 6 D-I). Labeling with vAChT or nNOS antibodies showed apparently normal distributions of both classes of nerve fibers within the circular muscle layer (Fig. 6E,F & H,I). These findings are consistent with observations made from Ws/Ws rats using cryostat sections or with electron microscopy,^31,32 and with the loss of ICC-IM in W/W^v and Sl/Sld mice.^3,21

Figure 6. c-KIT and ANO1-immunopositive ICC-MY and ICC-IM in the gastric antrum of Ws/Ws mutants and their anatomical relationship with enteric motor nerves.

Panels A-C, c-KIT⁺ (A; red arrows) and ANO1⁺ (B; green, arrowheads) ICC-MY between the circular and longitudinal muscle layers at the level of the myenteric plexus. Panel C, merged image showing cellular co-localization of c-KIT and ANO1 in ICC-MY (yellow; arrows) that were disrupted and patchy in nature. Panels D-F, bundles of ANO1⁺ ICC-IM (D; red; arrows) did not form discrete morphological relationships with vAChT⁺ nerve fibers (E; green; arrowheads). Panel F, merged image of panels D and E showing a lack of close anatomical relationship between vAChT⁺ nerve fibers and ICC-IM (arrows and arrowheads, respectively). Panels G-I, ANO1⁺ ICC IM and ICC-MY (G; red; arrows) at the level of the myenteric plexus and lack of morphological association with nNOS⁺ enteric ganglia (H; green; arrowheads). Panel I; merged image of panels G and H, showing lack of relationship between ICC-IM and ICC-MY with enteric neurons. Panels J-L, ANO1⁺ ICC-IM (J; red; arrows) and nNOS⁺ nerve fibers (K; green; arrowheads) showing no apparent morphological relationship. Panel L, merged imaged of panels J and K. Scale bars in panels C, F, I & L = 50 μm are representative of the appropriate series of panels.

Spontaneous electrical activity in gastric antrums of wildtype and Ws/Ws rats.

The RMP of the gastric antrum 3 mm from the pyloric sphincter averaged −62±0.5 mV in gastric antrum of wildtype rats, and electrical slow waves, 19.2±2 mV in amplitude and 6.3±0.3 sec in ½ maximal duration, occurred at 4.2±0.2 cycles min⁻¹ (n=14; Fig. 7A). RMP was more depolarized in antral muscles of Ws/Ws rats, averaging −57±1 mV (n=25; P=0.0001), and slow wave activity was highly variable. Muscles of 12 animals displayed slow waves averaging 17.7±2.0 mV and 6.9±0.5 sec in ½ maximal duration at a frequency of 3.3±0.4 cycles min⁻¹ (Fig. 7B). However, slow waves were small in amplitude, 3.2±1.4 mV and occurred at 2.8±0.4 cycles min⁻¹ in another 6 animals (Fig. 7C) and not resolved in an additional group of 7 animals (Fig. 7D). No significant difference was noted in RMPs of muscles with small amplitude slow waves and those without resolvable slow waves (P=0.151). RMPs from isolated antral circular muscle bundles averaged −59±2 mV (n=3) and displayed ongoing discharge of STDs (Fig. 7E), while RMPs of circular muscle bundles from Ws/Ws rats averaged −56±1 mV (n=3) and did not display STDs (Fig. 7F).

Figure 7. Spontaneous electrical activity and post-junctional neural responses recorded from the gastric antrum of wildtype and Ws/Ws mutant rats.

Electrical activity recorded from the gastric antrum of a wildtype control (Panel A) and a Ws/Ws mutant (Panels B-D). Slow wave activity was similar (Panel B), reduced in amplitude (Panel C) or absent (Panel D) in Ws/Ws mutants compared to wildtype controls. Panels E and F show the electrical activity recorded from isolated antral circular muscle bundles from a wildtype control and a Ws/Ws mutant, respectively. The discharge of spontaneous transient depolarizations (STDs) recorded from wildtype tissues (Panel E) was absent from Ws/Ws mutants (Panel F). Panels G-I, shows post-junctional neural responses to EFS (arrowheads) from a wildtype animal under control conditions (Panel G), in the presence of L-NNA (100 μM; Panel H) and L-NNA and atropine (1 μM; Panel I). EFS produced frequency dependent IJPs and phase advancement of slow waves. After the addition of L-NNA an excitatory junction potential was unmasked at both frequencies (Panel H; arrows) that was inhibited by atropine. Panels J-L, shows neural responses from the antrum of a Ws/Ws mutant rat under similar conditions. There was an absence of slow waves and post-junctional nitrergic and cholinergic responses in Ws/Ws mutant tissues in response to EFS delivered at the arrowheads.

Post-junctional neural responses in gastric antrums of wildtype and Ws/Ws rats

EFS evoked differential neural responses in antral muscles of wildtype and Ws/Ws rats. In the circular muscle wildtype rats EFS caused small EJPs followed immediately by much larger amplitude, frequency-dependent IJPs (Fig. 7G). L-NNA (100 μM) potentiated EJPs following EFS (Fig. 7H) and IJP amplitude and duration was reduced by L-NNA. In the presence of L-NNA, atropine (1 μM) abolished EJPs and increased the amplitude of the IJP (Fig. 7I). These findings are similar to what has been previously described except for the presence of EJPs in L-NNA.³³ EFS evoked frequency-dependent IJPs in antral muscles of Ws/Ws (Fig. 7J) rats, and L-NNA (100 μM) had little or no effect on the IJPs (Fig. 7K). Atropine (1 μM) also had little or no effect on the amplitude or duration of IJPs (Fig. 7L).

We also tested whether antral IJPs resistant to L-NNA were affected by apamin. EFS evoked frequency-dependent IJPs in wildtype and Ws/Ws rats in the presence of L-NNA and atropine (Fig. 8A & C). Apamin (0.3 μM) reduced the amplitude of IJPs at all frequencies in muscles of both animals (Fig. 8B & D, respectively). The effects on apamin on IJPs (1–20 Hz) in the presence of L-NNA and atropine are summarized in Fig. 8E & F. No statistical difference was noted between the effects of apamin on IJPs in muscles of wildtype or Ws/Ws rats. We previously reported that IJPs resistant to L-NNA in mouse antrum are mediated via P2Y1 receptors (P2Y1R)³⁴. MRS2500 (1 μM) reduced IJPs at all frequencies of EFS in muscles of wildtype (Fig. 9A & B) and Ws/Ws rats (Fig. 9C & D). Fig 9E & F shows a summary of the effects of MRS2500 on IJPs recorded from wildtype and Ws/Ws antrums evoked in the presence of L-NNA and atropine, respectively. No differences were noted between the effects of MRS2500 on IJPs in muscles of wildtype or Ws/Ws rats.

Figure 8. Effect of SK3 channel blocker apamin on inhibitory neural responses of wildtype and Ws/Ws mutant antrum tissues.

Panel A, shows neuroeffector responses to EFS (5&10 Hz, arrowheads) in the presence of L-NNA (100 μM) and atropine (1 μM) in a wildtype antrum. Note the phase advancement of slow waves. Panel B, shows a reduction in inhibitory neural responses after the addition of apamin (0.3 μM), the phase advancement of slow waves was also reduced. Panel C, shows similar inhibitory neuroeffector responses to EFS (5&10 Hz; arrowheads) of a Ws/Ws antral muscle, in the in the presence of L-NNA (100 μM) and atropine (1 μM). Panel D, apamin (0.3 μM) attenuated these neural responses at all frequencies. Panels E and F, summary of the effects of apamin on IJP amplitude (frequencies 1–20 Hz) in wildtype controls (E) and Ws/Ws mutants (F) in the presence of L-NNA and atropine (black bars). Apamin reduced IJPs in both wildtype and Ws/Ws mutants (white bars) at all frequencies tested compared to IJPs in L-NNA and atropine. * P < 0.05 and ** P < 0.01.

Figure 9. Effect of P2Y1R antagonist, MRS2500 on inhibitory neural responses of wildtype and Ws/Ws mutant antrum tissues.

Panel A, neuroeffector responses of a wildtype antrum following EFS (5&10 Hz, arrowheads) in the presence of L-NNA (100 μM) and atropine (1 μM). Note the slow wave advancement at 10 Hz. Panel B, shows a reduction in inhibitory neuroeffector responses after the addition of MRS2500 (1 μM). Panel C, EFS evoked neural inhibitory responses of a Ws/Ws antral muscle in the presence of L-NNA (100 μM) and atropine (1 μM). Panel D, in L-NNA and atropine, MRS2500 (1 μM) attenuated these responses at both frequencies shown (arrowheads). Panels E and F, A summary of the effects of MRS2500 on IJP amplitude in wildtype controls (Panel E) and Ws/Ws mutants (Panel F). IJPs in the presence of L-NNA and atropine (black bars) were significantly attenuated by MRS2500 at all frequencies in both tissues (white bars). ** P < 0.01 and *** P < 0.001.

In mice GI muscles P2Y1 receptors are highly expressed in PDGFRα⁺ cells, a distinct, secondary population of interstitial cells in the SIP syncytium.^35–36 Wildtype and Ws/Ws rats displayed no apparent differences in the density or distribution of PDGFRα⁺ cells (Fig. 18).

Figure 18. Expression of PDGFRα⁺ cells throughout the GI tracts of wildtype and Ws/Ws mutant rats.

Panels A and B, PDGFRα⁺ cells in the fundus of a wildtype (Panel A; arrows) and a Ws/Ws mutant animal (Panel B; arrows). Panels C and D, PDGFRα⁺ cells in the antrum of a wildtype (Panel C; arrows) and a Ws/Ws mutant animal (Panel D; arrows). Panels E and F, PDGFRα⁺ cells in the small intestine of a wildtype (Panel E; arrows) and a Ws/Ws mutant animal (Panel F; arrows). Panels G and H, PDGFRα⁺ cells in the colon of a wildtype (Panel G; arrows) and a Ws/Ws mutant animal (Panel H; arrows). There was no apparent difference in the density and distribution of PDGFRα⁺ cells throughout the GI tracts of wildtype and Ws/Ws mutant animals. Scale bars are as indicated in panels B, D, F and H and are representative of both wildtype and Ws/Ws mutant tissues.

Small Intestine

ICC in the small intestines of wildtype and Ws/Ws rats

The distribution and density of ICC were also examined in the jejunums of wildtype and Ws/Ws mutant rats. Two distinct populations of ICC were observed in whole mount preparations of wildtype muscles. C-KIT⁺ ICC-MY were observed within the intermuscular plane between the circular and longitudinal muscle layers, forming a dense anastomosing network of cells. Fine processes extending from oval cell bodies formed interconnections with processes of adjacent ICC-MY (Fig. 10A, D &G). These cells were also ANO1⁺ (Fig. 10B &C). A second population of ICC was found at the level of the deep muscular plexus (ICC-DMP). These cells ran parallel with circular smooth muscle cells. Fine processes extended from the main axis of ICC-DMP and contacted processes of neighboring ICC-DMP, forming a network of these cells (Fig. 10A &J).

Figure 10. Distribution of ICC in the small intestine of wildtype controls and their close anatomical relationship with enteric motor nerves.

Panels A and B show c-Kit⁺ ICC-MY (Panel A; red; arrows) and ANO1⁺ ICC-MY (Panel B; green; arrowheads) in the jejunum of a wildtype animal. Panel C is a merged image of Panels A & B showing cellular colocalization of c-KIT and ANO1 (yellow; arrows). Panels D-F show cellular localization of ANO1⁺ICC (D; red; arrows) and vAChT⁺ nerves (E; green; arrowheads) at the level of the myenteric plexus. Little cellular apposition of both cell types occurred at this level in the jejunum. Panels G-I show cellular localization of ANO1⁺ICC (G; red; arrows) and nNOS⁺ nerves (E; green; arrowheads) through the wall of the small intestine. Panel I is a merged image of Panels G and H. Panels J-L, images at the level of the deep muscular plexus (DMP). Panel J ANO1⁺ ICC-DMP (red; arrows) and Panel K, nNOS⁺ nerve fibers (green; arrowheads). Panel L is a merged image of Panels J and K showing close cellular apposition between nNOS⁺ nerve fibers (arrowheads) and ANO1⁺ ICC-DMP (arrows). Panel M, higher power image of the region outlined by the dashed white rectangle in Panel L, showing the close anatomical relationship between nNOS ⁺ inhibitory nerves (arrowheads) and ICC-DMP (arrows) within the circular layer of the small intestine. Scale bars are as indicated in Panels C, F, I, L & M are representative of the appropriate series of panels, where applicable.

Double labeling with c-KIT or ANO1 antibodies and vAChT or nNOS antibodies showed that although ICC-MY are distributed on both sides of myenteric ganglia, close associations with nerve fibers were not apparent (Fig. 10D-F and 10G-I). However, ICC-DMP formed very close associations with vAChT⁺ and nNOS⁺ nerve fibers (Fig. 10J-L &M).

The distribution of ICC-MY and structure of the ICC-MY network was abnormal in the jejunums of Ws/Ws rats. c-KIT and ANO1⁺ ICC-MY were absent in some areas of the intestine and occurred only in small clusters in other regions (Fig.11A-C). ICC-DMP appeared to be distributed normally in Ws/Ws small intestines, as others also described (Fig. 11A-C,D,F,G,I,J,K). ³¹ The distribution of vAChT⁺ and nNOS⁺ nerve processes and close associations with ICC-DMP also appeared normal in Ws/Ws rats (Fig. 11D-F, G-I, J,K).

Figure 11. Distribution of c-KIT⁺ ICC in the small intestine of Ws/Ws mutants and their close anatomical relationship with enteric motor nerves.

Panels A and B show c-Kit⁺ ICC (Panel A; red) and ANO1⁺ ICC (Panel B; green) in the jejunum of a Ws/Ws mutant. A dense population of ICC-DMP are identified by arrows. A few patchy ICC-MY are identified with an arrowhead, in other regions ICC-MY were absent. Panel C is a merged image of Panels A & B showing cellular colocalization of c-KIT and ANO1 in ICC-MY and ICC-DMP (yellow; arrows and arrowhead). Panels D-F show localization of c-KIT⁺ ICC-DMP (D; red; arrows) and vAChT⁺ nerves (E; green; arrowheads). Panel F, Close apposition of ICC-DMP and vAChT⁺ nerves occurred at the level of the deep muscular plexus (arrows). Panels G-I, c-KIT⁺ ICC-DMP (G; red; arrows) and nNOS⁺ nerves (H; green; arrowheads). Panel I, is a merged image of Panels G and H showing close cellular apposition at the level of the deep muscular plexus (arrows). Panels J and K, higher power images of the regions outlined by the dashed white rectangles in Panels F and I, showing the close anatomical relationship between v-AChT⁺ excitatory and nNOS ⁺ inhibitory nerves and c-KIT⁺ ICC-DMP within the circular layer (arrows and arrowheads). Scale bars are as indicated in Panels C, F, I, J & K are representative of the appropriate series of panels, where applicable.

Spontaneous electrical activity in small intestines of wildtype and Ws/Ws rats.

Intracellular electrical recordings were made from jejunal circular muscles of wildtype and Ws/Ws rats. RMPs of wildtype animals averaged −67±1.0 mV and rhythmic slow wave activity averaging 30±1 mV in amplitude and 2.9±0.3 secs in duration occurred at frequency of 14±2 cycles min⁻¹ (Fig. 12A). Slow waves displayed two general waveforms across all preparations. In some muscles, the upstroke depolarization was followed by a partial repolarization to a plateau phase that was sustained for approximately 1 sec before repolarization to RMP. In other muscles, the upstroke was not resolved as a distinct component, and the slow waves consisted of a monophasic sinusoidal-like waveform. In some cases, a small, initial primary depolarization in membrane potential, 5±0.5 mV in amplitude was recorded. This primary component of the upstroke phase of the slow wave had a rate-of-rise of 6±1 mV s^-1. The secondary component was larger in amplitude and had a faster rate of rise, averaging 33±2 mV s⁻¹ (n=6).

Figure 12. Electrical activities and neural responses from the small intestine of wildtype and Ws/Ws mutants.

Panel A, spontaneous slow waves recorded from a wildtype animal. Panels B and C show the lack of slow waves (B) or only small oscillations in membrane potential (C)recorded from intestinal tissues of Ws/Ws mutants, respectively. Slow waves were absent in 4 animals, but small oscillations in membrane potential were observed in 2 animals. Panel D, post-junctional neural responses to EFS (5 and 10 Hz; arrowheads) under control conditions and Panel E, in the presence of L-NNA (100 μM) and Panel F, L-NNA and atropine (1 μM) in a wildtype animal. Under control conditions and in the presence of L-NNA, EFS evoked a phase advancement in slow waves and depolarization in membrane potential. In L-NNA (100 μM), phase advancement persisted, and the depolarization was potentiated. Panel F, both phase advancement and membrane depolarization were blocked by atropine, revealing a fast IJP. Panels G-I, shows neural responses from a Ws/Ws mutant under similar control conditions. Panel G, Under control conditions EFS evoked IJPs. Panel H, L-NNA (100 μM) reduced the amplitude and duration of IJPs. Panel I, atropine increased the amplitude of the IJP that persisted in the presence of L-NNA in Ws/Ws mutant intestines. Microelectrode impalements in the small intestine were performed in the presence of nifedipine (1μM) to maintain extended impalements.

RMPs of circular muscle cells from the jejunums of Ws/Ws rats were more depolarized, averaging−55±1 mV (n=6; P =0.0001 compared to wildtype controls). Spontaneous slow waves were not recorded in 4 of the 6 muscle preparations (Fig. 12B), however small oscillations averaging 4±0.2 mV in amplitude with a frequency of 16±4 cycles min⁻¹ were recorded in 2 of the 6 Ws/Ws rat muscles studied (Fig. 12C).

Post-junctional neural responses in small intestine of wildtype and Ws/Ws rats.

ICC-DMP appeared normal and formed close associations with enteric motor neurons in Ws/Ws rats (Fig. 11), ³¹ so we tested whether normal post-junctional neural responses occurred in these rats. EFS (1–20 Hz) of intestinal muscles from wildtype animals resulted in tri-phasic responses, consisting of a short duration IJP, followed by phase advancement of slow waves that was followed by a more sustained depolarization (EJP; Fig. 12D). L-NNA (100 μM) potentiated the amplitude and duration of the EJP for several slow wave cycles following EFS (Fig. 12E). In the continued presence of L-NNA, atropine (1 μM) blocked the phase advancement and sustained EJP and unmasked a transient IJP (Fig 12F). These data suggest that at least 3 neurotransmitters mediate response to EFS in the rat small intestine.

It was somewhat difficult to compare responses to EFS in Ws/Ws rats with wildtype muscles due to the lack of slow waves in Ws/Ws muscles. EFS evoked frequency dependent IJPs (Fig. 12G) that were slightly reduced in amplitude and duration by L-NNA (100 μM) at all frequencies tested (Fig. 12H). In the continued presence of L-NNA, atropine (1 μM) slightly increased the amplitude of the IJPs but did not affect duration (Fig. 12I).

The effects of apamin were tested on the L-NNA and atropine resistant post-junctional responses in wildtype and Ws/Ws muscles. In the presence of L-NNA and atropine, apamin (0.3 μM) abolished IJPs at 1 & 3 Hz and significantly reduced the amplitude of IJPs at 5–20 Hz in wildtype controls (Fig. 13A,B & E). Apamin also reduced the amplitude of IJPs in Ws/Ws jejunums but to a lesser extent than in wildtype tissues (Fig. 13C,D & F). A summary of the effects of apamin on IJPs in the presence of L-NNA and atropine in wildtype and WsWs mutant intestines is shown in Figs. 13E & F, respectively.

Figure 13. Apamin reduced the amplitude of IJPs in the small intestine of wildtype controls and Ws/Ws mutants.

Panel A, in the presence of L-NNA (100 μM) and atropine (1 μM), EFS evoked a fast IJP at 5 and 10 Hz (arrowheads). Panel B, IJPs recorded in L-NNA and atropine were inhibited by apamin (0.3 μM). Panel C, shows EFS evoked IJPs in a Ws/Ws intestine in the presence of L-NNA (100 μM) and atropine (1.0 μM). Panel D, apamin significantly reduced the amplitude of EFS evoked IJPs recorded in the presence of L-NNA (100 μM) and atropine (1 μM). Panels E & F, summary of the effects of apamin on IJP amplitude from wildtype controls (E) and Ws/Ws mutants (F). Apamin significantly reduced IJPs at all frequencies in both wildtype and Ws/Ws antral tissues (white bars) compared to L-NNA and atropine (black bars). * P < 0.05 and ** P < 0.01. Microelectrode impalements in the small intestine were performed in the presence of nifedipine (1μM) to maintain impalements.

Proximal colon

ICC in the proximal colons of wildtype and Ws/Ws rats and their relationship to enteric nerves.

In the proximal colon c-KIT and ANO1⁺ ICC were widely distributed throughout the plane of the myenteric plexus (ICC-MY) and within the circular and longitudinal muscle layers (ICC-IM). ICC-MY were distributed on both the circular and longitudinal muscle sides of myenteric ganglia (Fig. 14A-F) but did not appear to form close associations with enteric nerve fibers. ICC-IM were abundantly distributed within the circular muscle layer and formed close contacts with vAChT⁺ and nNOS⁺ nerve fibers (Fig. 14G-I & Fig. J-L), respectively. There were areas where ICC-IM appeared disrupted. Higher power images of the relationship between vAChT⁺ and nNOS⁺ nerves and ICC-IM are shown in Figure 14M & N, respectively.

Figure 14. c-KIT and ANO1-immunopositive ICC-MY and ICC-IM in the proximal colon of wildtype animals and their close anatomical relationship with enteric motor nerves.

Panel A, c-KIT⁺ ICC-MY (red, arrows) and ICC-IM (red; arrowheads). Panel B, ANO1⁺ ICC-MY (green, arrows) and ICC-IM (green; arrowheads). Panel C, merged image of Panels A and B, showing the cellular co-localization of c-KIT⁺ and ANO1⁺ ICC (yellow, arrows and arrowheads). Panel D, ANO1⁺ ICC-MY (red, arrows) and Panel E, vAChT⁺ ganglia and nerve fibers (green, arrows showing ganglia and arrowheads showing nerve fibers) within the circular muscle layer. Panel F, merged image of Panels D and E showing ICC-MY encasing nerve ganglia (arrows) and vAChT⁺ nerve fibers closely apposed to ICC-IM between ganglia. Panel G, ANO1⁺ ICC-IM (red, arrows) and Panel H, vAChT⁺ nerve fibers (green, arrowheads) within the circular muscle layer. Panel I, merged image of Panels G and H showing vAChT⁺ nerve fibers in close apposition with ICC-IM (arrows). Panel J, ANO1⁺ ICC-IM (red, arrows) and Panel K, nNOS⁺ nerve fibers (green, arrowheads) within the circular muscle layer. Panel L, merged image of Panels J and K showing nNOS⁺ nerve fibers in close morphological apposition with ICC-IM. Panels M and N, higher power images of the regions outlined by the dashed white rectangles in Panels I and L, showing the close anatomical relationship between vAChT⁺ and nNOS ⁺ inhibitory nerves (arrowheads) and ICC-DMP (arrows) within the circular layer of the proximal colon. Scale bars are as indicated in Panels C, F, I, L, M and N are representative of the appropriate series of panels, where applicable.

c-KIT and ANO1⁺ ICC-MY networks were less dense in the proximal colons of Ws/Ws rats in comparison to wildtype animals and there were areas between patches devoid of ICC and processes (Fig. 15A-C). The distribution of ICC-IM in circular muscle was also not homogenous and areas of reduced density or devoid of ICC-IM were noted (Fig. 15D-F). In areas where ICC-IM were observed vAChT⁺ and nNOS⁺ fibers were equally abundant as wildtype animals and closely aligned with ICC-IM (Fig. 15G-I & M-O). In the region of the myenteric plexus where ICC-MY were observed they did not appear to form a close apposition with enteric nerve fibers (Fig. 15J-L).

Figure 15. c-KIT and ANO1-immunopositive ICC-MY and ICC-IM in the proximal colon of Ws/Ws mutants and their relationship with enteric motor nerves.

Panel A, c-KIT⁺ ICC-MY (red, arrows) and Panel B, ANO1⁺ ICC-MY (green, arrows). Panel C, merged image of Panels A and B, showing the cellular co-localization of c-KIT⁺ and ANO1⁺ ICC (yellow, arrows). ICC-MY networks were patchy in nature. Panel D, c-KIT⁺ ICC-IM (red, arrows) and Panel E, ANO1⁺ ICC-IM within the circular muscle layer (green; arrowheads). Panel F, merged image of panels D and E showing cellular co-localization of c-KIT and ANO1 (yellow; arrows). There were areas that ICC-IM appeared less dense or disrupted. Panels G-I, ANO1⁺ ICC-IM (G; red; arrows) and vAChT⁺ nerve fibers (H; green arrowheads) within the circular layer. Merged image (panel I) showing close apposition between vAChT⁺ nerve fibers and ICC-IM (arrows). Panel J, c-KIT⁺ ICC-MY (red; arrows) and ICC-IM (red, arrowheads) at the level of the myenteric plexus. Panel K, nNOS⁺ neurons and nerve fibers (arrowheads). Panel L, merged image of panels J and K, nNOS⁺ nerve fibers form close apposition with ICC-IM (arrowheads) but no apparent relationship between nNOS⁺ nerves and ICC-MY (arrows). Panels M-O, ANO1⁺ ICC-IM (M; red; arrows), Panel N, nNOS⁺ nerve fibers (green; arrowheads) and Panel O, merged image of panels M and N showing nNOS⁺ nerve fibers forming close morphological apposition with ICC-IM within the circular muscle layer (arrows). Panels P and Q, higher power images of the regions outlined by the dashed white rectangles in Panels I and O, showing the close anatomical relationship between vAChT⁺ and nNOS ⁺ inhibitory nerves (arrowheads) and ICC-IM (arrows) within the circular layer of the proximal colon. Scale bars are as indicated in Panels C, F, I, L, O, P and Q are representative of the appropriate series of panels, where applicable.

Spontaneous electrical activity of the proximal colons of wildtype and Ws/Ws mutant animals.

RMPs of circular muscle cells averaged −48 ± 1.7 mV and complex waveforms occurred spontaneously that consisted of slow membrane depolarization and a discharge of action potentials (n=6; Fig. 16A). RMPs were similar in Ws/Ws rat proximal colon and averaged −48±2.7 mV (P=0.94 compared to wildtype animals; n=6). The slow, rhythmic depolarizations were absent but bursts of action potentials occurred and occasionally appeared less organized (Fig. 16B). In wildtype and Ws/Ws proximal colon membrane potentials between spike complexes were very noisy (Fig. 16 A&B). These experiments were performed in the absence of nifedipine (1μM) to reveal spike complexes.

Figure 16. Spontaneous electrical activity and EFS evoked post-junctional neural responses in wildtype and Ws/Ws proximal colons.

Panel A, Spontaneous activity of the proximal colon from a wildtype animal under control conditions consisting of an irregular membrane potential and slow membrane depolarizations with superimposed spontaneous action potential complexes. Panel B, Spontaneous activity of Ws/Ws mutants was similar to that of wildtype controls, except the slow depolarization in membrane potential was less pronounced. Panels C-E, in wildtype tissues under control conditions, EFS (single pulse 0.3 & 0.5 ms at arrowheads) evoked a bi-phasic inhibitory post-junctional response consisting fast (fIJP) and slow inhibitory junction potential (sIJP) components (dashed lines). Panel D, L-NNA (100 μM) depolarized membrane potential and inhibited the sIJP. Panel E, In the continued presence of L-NNA, atropine (1.0 μM) had little effect on the fIJP. Panel F, EFS of Ws/Ws mutant colons evoked a fIJP followed by a frequency-dependent rebound excitation. Panel G, L-NNA (100 μM) produced a slight depolarization in membrane potential, potentiated the fIJP and reduced the rebound excitation. Panel H, In the continued presence of L-NNA, atropine had little effect on the EFS evoked post-junctional response. EFS evoked neural responses were performed in the presence of nifedipine (1μM) to unmask nerve evoked post-junctional responses.

Neural responses in proximal colons of wildtype and Ws/Ws rats.

EFS of proximal colon muscles was performed in the presence of nifedipine (1 μM) to facilitate long-term intracellular impalements and reveal post-junctional responses. Single pulses of EFS (0.1, 0.3 & 0.5 ms in duration) evoked bi-phasic IJPs consisting of a fast-transient component (fIJP) followed by a slower, sustained hyperpolarization phase (sIJP). The fIJP (0.5 ms pulse duration) was 18.5±2.0 mV, and the sIJP 11.2±1.8 mV in amplitude (Fig. 16C; n=6). L-NNA (100 μM) depolarized colonic tissues to −36±1.2 mV. L-NNA slightly but not significantly increased the amplitude of the fIJP (20.2.0±2.0 mV; P=0.88). L-NNA abolished sIJPs (Fig. 16D). In the continued presence of L-NNA, atropine (1 μM) had no effect on RMP and did not change the amplitude of the fIJP (20.3± 2 mV; Fig. 16E; P=0.77).

Post-junctional responses to EFS were significantly different in proximal colons of Ws/Ws rats. Single pulses of EFS (0.5 ms duration) evoked fIJPs averaging 12.8±4.1 mV in amplitude (Fig. 16F; P=0.71 compared to wildtype controls). sIJPs were recorded in 5 of 7 muscles, and these events were smaller than the equivalent response in wildtype muscles (i.e., 4.8±1.5 mV in amplitude; P= 0.004 compared to wildtype muscles). IJPs in Ws/Ws colons were followed by a post-stimulus depolarization response consisting of one or more action potentials (Fig. 16F). L-NNA (100 μM) depolarized Ws/Ws colons to a lesser but insignificant extent than that in wildtype muscles, i.e. to −41±2.6 mV (P=0.093 compared to wildtype controls). L-NNA did not significantly affect the amplitude or duration of the fIJP (13.3±2.7 mV; P=0.089) but abolished sIJPs in muscles when present (Fig. 16G). In L-NNA, atropine (1 μM) had no effect on RMP but slightly but not significantly increased the amplitude of the fIJP to 15.8±3.2 mV (Fig. 16H; P= 0.285).

The fIJP in proximal colons of mice, rats and humans is mediated by P2Y1R.³⁶ We therefore tested MRS2500 to confirm this conclusion in wildtype and Ws/Ws rat colons. fIJPs were inhibited in both wildtype and Ws/Ws colons at all pulse durations tested (0.1, 0.3 and 0.5 ms). In L-NNA and atropine, the fIJP evoked by EFS (0.5 ms pulse duration) was reduced by MRS2500 (1 μM) from 20.3±2 mV to 1.6±0.5 mV in wildtype colons (P=0.0007; n=6) and from 15.8±3.2 mV to 0.7±0.3 in Ws/Ws colons (P=0.0009; n=6). There was no statistical significance between wildtype and Ws/Ws muscles (P=0.22; Fig. 17). A summary of the effects of MRS2500 on fIJPs in colonic muscles of wildtype and Ws/Ws rats is shown in Fig. 17E & F, respectively.

Figure 17. The P2Y1R antagonist, MRS2500 inhibited the fIJP in wildtype and Ws/Ws proximal colons tissues.

Panel A, In the presence of L-NNA (100 μM) and atropine (1.0 μM), EFS (single pulse 0.3 & 0.5 ms delivered at arrowheads) evoked a fIJP in wildtype tissues. Panel B, the fIJP was inhibited by MRS2500 (1.0 μM). Panel C, in the presence of L-NNA (100 μM) and atropine (1.0 μM), EFS also evoked a fIJP in Ws/Ws colonic tissues that was inhibited by MRS2500 (1.0 μM, Panel D). Panels E and F, summary of the effects of EFS (0.1, 0.3 and 0.5 ms pulse duration) on fIJPs in the presence of L-NNA (100 μM) and atropine (1.0 μM; black bars). Increasing the duration of EFS augmented the amplitude of the fIJP and this was significantly inhibited by MRS2500 (1.0 μM) in both wildtype (Panel E) and Ws/Ws colonic tissues (Panel F; white bars). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Expression of PDGFRα⁺ interstitial cells in GI tracts or wildtype and Ws/Ws rats

It has been demonstrated that enteric inhibitory responses caused by release of purinergic neurotransmitters are mediated by P2Y1 receptors expressed not by SMCs or ICC, but by a second class of interstitial cells known as PDGFRα⁺ cells.³⁷ Purinergic responses, indicated by the inhibitory effects of MRS2500, persisted in GI muscles of Ws/Ws rats. Therefore, we sought to determine whether the distribution of PDGFRα⁺ cells was affected in GI muscles of Ws/Ws rats. Immunohistochemical analysis revealed little or no differences in the distribution or density of PDGFRα⁺ cells in the fundus, antrum, small intestine, or proximal colons of Ws/Ws rats compared to wildtype controls (Fig. 18).

DISCUSSION

Interstitial cells of Cajal (ICC) and PDGFRα⁺ cells are specialized populations of cells that have important and distinct functional roles in the gastrointestinal motility. Much of the evidence defining the role of these cells has been obtained from studies of mice.^1–6 A few studies have previously investigated the role of ICC in rat GI muscles, but morphological studies conducted in concert with functional studies are rare. ^38–40 The tunica muscularis of the rat stomach has many similarities with human, consisting of an external longitudinal and internal circular muscle. In the region of the gastro–esophageal junction there is a further layer, internal to the circular muscle, the oblique or sling muscle, similar again to human.

Genetically modified mice have been a very powerful approach to learning about cell signaling and the molecular behaviors of ICC within intact muscles, but genetically modified data have been less available due to constraints on techniques to generate these animals. Now with gene editing the availability of rat genetic models is increasing, and the similarities in behaviors and systemic functions often more closely resemble those in humans.^41–43 It often is difficult to perform assessments of GI motility functions in mice due to their small size and relative fragility of tissues. Thus, animals of moderate size, such as rats may in many circumstances facilitate more representative disease models and more robust experimental models to investigate GI motility functions and dysfunction. Since c-KIT and ANO1 are now well recognized markers for ICC throughout the GI tracts of all species studied to date, including rat in the present study, we used antibodies raised against these proteins interchangeably.^44–46 In the present study we found that ICC function in analogous ways in rats as deduced previously from mouse studies. Thus, future studies of the involvement of ICC in motility disorders may be facilitated by the findings of the current study.

Like the spontaneous, loss-of-function mutations in Kit in W/W^V mice, c-KIT is also compromised in Ws/Ws rats. This study thus confirms that c-KIT is an important factor for the normal development of ICC in rats. The Ws mutation is due to deletion of 12 bases encoding 4 amino acids in Kit (ie, Val-Lys-Gly-Asn) that are conserved in mouse and human c-KIT kinases.²⁵ The mutation results in loss-of-function in the receptor tyrosine kinase activity but does not totally disable c-KIT. Ws/Ws homozygotes were previously shown to have lesions in specific types of ICC, ^27,31 as in W/W^V mice. Also similar to mouse mutants is the non-total loss of ICC in Ws/Ws rats, and this is why the model is useful to understand the role of specific types of ICC but cannot be used to characterize the functions of ICC universally. Thus, as provided in the current study, region by region characterization of functions in stomach, small bowel and colon is likely to provide a useful catalogue of behaviors linked to ICC in rats (see Table II).

Table II:

Distribution and functions of ICC deduced from studies of mice and rats.

Species	Organ	ICC type	Relative lesion	Excitatory neurotransmission	Inhibitory neurotransmission	Pacemaker activity	STDs
Mouse +/+	Stomach (fundus)	ICC-IM	0	+	+	-	+
Mouse +/+	Stomach (antrum)	ICC-IM	0	+	+	+	+
		ICC-MY	0	-	-	+	+
Mouse +/+	Small Intestine	ICC-DMP	0	+	+	-	+
		ICC-MY	0	-	-	+	?
Mouse +/+	Colon	ICC-IM	0	+	+	-	+
		ICC-MY	0	-	?	+	+
Mouse W/WV	Stomach (fundus)	ICC-IM	****	***	***	-	****
Mouse W/WV	Stomach (antrum)	ICC-IM	***	***	*	-	-
		ICC-MY	*	-	-	****	?
Mouse W/WV	Small Intestine	ICC-DMP	0	+	+	-	-
		ICC-MY	***	-	-	**	?
Mouse W/WV	Colon	ICC-IM	*	***	**	?	*
		ICC-MY	*	?	?	**	+
Rat +/+	Stomach (fundus)	ICC-IM	0	+	+	-	+
Rat +/+	Stomach (antrum)	ICC-IM	0	+	+	?	+
		ICC-MY	0	?	?		+
Rat +/+	Small Intestine	ICC-DMP	0	+	+	-+	-+
		ICC-MY	0	?	?	+	+
Rat +/+	Colon	ICC-IM	0	+	+	?	+
		ICC-MY	0			+	?
Rat Ws/Ws	Stomach (fundus)	ICC-IM	****	****	***	-	****
Rat Ws/Ws	Stomach (antrum)	ICC-IM	***	***	*	?	****
		ICC-MY	*			**	?
Rat Ws/Ws	Small Intestine	ICC-DMP	0	**	+	-	?
		ICC-MY	***	?	?	***	?
Rat Ws/Ws	Colon	ICC-IM	**	**	**	?	*
		ICC-MY	*	?	?	*	+

+. = Activity present

0. = Unresolved loss

- = Not present

* = Minor loss

** = Moderate loss

*** = Severe loss

**** = Ablation

? = Not known

In the stomachs of Ws/Ws rats intramuscular ICC (ICC-IM) were greatly reduced or absent in the both the fundus and antrum. Although ICC-IM were lost, no morphological changes in SMCs were noted in previous studies of these mutants. ^32,47 Disruption of ICC-IM resulted in loss of STDs which have been shown to be an important element in setting RMP and excitability of the SIP syncytium in gastric muscles and likely to be regulated by inputs from enteric motor neurons.^48,49 The close associations between the processes of enteric motor neurons and ICC-IM have been examined in several animal species including mouse, guinea-pig, rat, dog monkey and human, ^{3,32, 50–53} and ultrastructural studies have shown there are specialized, synaptic-like contacts between nerve varicosities and ICC-IM and gap junctions between ICC-IM and neighboring smooth muscle cells. ^28,32,51 Proteins identified in pre- and post-synaptic membranes suggest synaptic-like connectivity as PSD93 and PSD95 are expressed at these junctions. ^28,51 The functional role of ICC in enteric motor neurotransmission was examined in mouse models with Kit mutations (i.e. W/W^V and Sl/Sl^d mutants), and loss of ICC-IM was found to reduce cholinergic and nitrergic neural responses in stomach, lower esophageal and pyloric sphincters and proximal colon. ^{2–4,21–23, 54} Similar consequences of reduced ICC-IM were observed in Ws/Ws rats in the current study in which nitrergic and cholinergic neural responses were greatly diminished, leaving only purinergic responses intact. A difference between the present study and a previous published study was the existence of EJPs in the antrum of wildtype rats when the tissues were in L-NNA and MRS2500. In the previous study EJPs were not evoked under similar conditions.³³ This difference may have been due to the location where the stimulating electrodes were placed. An interesting difference from W/W^V mice was noted in antral muscles of Ws/Ws rats where partial defects in the ICC-MY network were observed, and these lesions were associated with inconsistent recordings of slow wave activity. Some cells impaled displayed nearly normal slow waves, but these events were small in amplitude or absent in other cells. Lack of continuity of the ICC-MY network may compromise proximal to distal propagation of slow waves, and therefore Ws/Ws rats might be a good model of the electrical disturbances occurring in diabetes, in which similar patchy distribution of ICC-MY has been reported to occur.⁵⁵ The lack of continuity of antral ICC-MY networks or impaired contractile activity of the small intestine may contribute to the significantly increased bile acids that have been reported in the stomachs of Ws/Ws mutants.⁵⁸

Ws/Ws rats displayed disrupted ICC-MY networks, but ICC-DMP were relatively intact in jejunal tissues. Associated with loss of ICC-MY was loss of pacemaker activity, however in 2 animals small amplitude slow waves were recorded from small intestinal muscles. This activity might have originated from small clusters of ICC-MY that persisted in some animals. Cholinergic excitatory and nitrergic inhibitory responses appeared to be normal in the small intestine, however direct comparison of these events was difficult due to the absence of slow wave activity in the mutant rats. These characteristics were similar to what has been reported previously in W/W^V and Sl/Sl^d mice that lack ICC-MY but not ICC-DMP in the small intestine. ^5,17,19

ICC populations were retained in proximal colons of Ws/Ws rats, but patchy reductions in ICC networks were evident, as previously reported.⁵⁷ Spontaneous spike complexes persisted in these mutants, however these events were not as well organized as in wildtype controls. Post‐junctional neural responses were abnormal in proximal colons of Ws/Ws rats. There was a significant reduction in the nitrergic component of tonic inhibition in Ws/Ws colonic muscles, as L-NNA did not depolarize these muscles to the same extent as wildtype controls. Nitrergic inhibitory neural responses evoked by EFS were also reduced in Ws/Ws colonic muscles. W/W^V mice also display patchy distributions of ICC in the colon and reduced tonic inhibition and reduced post-junctional neural responses.⁵⁷

Similar also to neural responses in W/W^V mice, the fast component of IJPs (fIJPs) was conserved throughout the GI tracts of Ws/Ws rats, suggesting that ICC are not involved in these responses. In mice, rats and non-human primate antrum and colons, the fIJP are known to be mediated through binding of purinergic neurotransmitters to P2Y1 receptors and activation of apamin sensitive small conductance Ca²⁺-activated K⁺ channels, which are expressed dominantly in another type of specialized interstitial cell, PDGFRα ⁺ cells. ^{34–37, 58,59} Immunohistochemical analysis revealed no apparent differences in the density and distribution of PDGFRα ⁺ cells throughout the GI tracts of Ws/Ws and wildtype muscles. The presence of PDGFRα ⁺ cells and purinergic inhibitory responses suggest that the well-described morphological unit, known as the SIP syncytium, is also a functional component of rat GI muscles.^1,46,60,61

In summary, c-Kit is an essential signaling element required for development of ICC in the mouse GI tract, and mutations, such as the Ws/Ws mutation in Kit, has similar consequences on the development, distribution and functions of ICC in rats. We catalogued ICC morphological and functional lesions in stomach, small intestine and proximal colon of Ws/Ws rats and showed that loss of ICC results in similar defects in electrical pacemaker activity and responses to inputs from enteric motor neurons. The larger size of these animals, robust nature of GI muscles and similarities of functions to the human GI tract may make Ws/Ws rats useful models for future investigations into the significance of ICC in normal and abnormal GI motility.