Volume 115, Issue 2, Pages 314-329 (August 1998) Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters  Sean M. Ward, Gerard Morris, Lee Reese, Xuan-Yu Wang, Kenton M. Sanders  Gastroenterology  Volume 115, Issue 2, Pages 314-329 (August 1998) DOI: 10.1016/S0016-5085(98)70198-2 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Isolation of muscles of the pyloric sphincter and LES. (A) A stomach with segments of the esophagus (E) and duodenum (D) attached. (B) A stomach opened along the lesser curvature from the gastric antrum (GA) through the duodenum segment. (C) The same preparation after removal of the mucosa. The pyloric sphincter (PS) is clearly visible as a thickened band of circular muscle at the junction between the stomach and duodenum. (D) A stomach opened along the greater curvature to reveal the junction between the esophagus and the stomach. (E) The same preparation after opening the gastroesophageal junction through the esophagus. (F) The same preparation after removal of the mucosa. The LES is visible as a band of thickened musculature. F, fundus. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Distribution of ICC in the murine LES. (A) A cryostat cross section through the circular (cm) and longitudinal (lm) muscle layers (section cut along the long axis of the longitudinal layer) of a wild-type (+/+) mouse. IC-IM are observed in both layers (arrows). Circular cells appear as points and longitudinal IC-IM as elongated cells with ovoid nuclear region and long, unbranching processes. No immunoreactivity was observed in the submucosal region(s). (B) A confocal reconstruction of the entire tunica muscularis from a flattened whole mount. This shows the relative density of the IC-IM in the circular and longitudinal muscle layers. IC-IM were completely absent in W/Wv animals. (C) A cryostat section and (D) a flattened whole-mount preparation from W/Wv animals. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Distribution of ICC in the murine pyloric sphincter. (A) A cryostat cross section featuring the various classes of ICC within the pyloric sphincter. ICC in the myenteric region (IC-MY) are brightly labeled in the plane between the circular (cm) and longitudinal (lm) muscle layers. As in the LES, IC-IM are found in both muscle layers, running parallel with the muscle fibers. We also observed cells with c-Kit–LI along the surface separating the submucosa (s) from the circular muscle, running perpendicular to the circular muscle layer (*). (B) A confocal image featuring the IC-IM of the circular muscle layer. The cells are long and spindle-shaped and run parallel to the circular muscle fibers. (C) A whole mount showing IC-IM (arrows) and IC-MY (arrowheads). (D) A cryostat section revealing the absence of IC-IM and cells with c-Kit–LI at the submucosal surface in W/Wv animals. IC-MY were apparently unaffected by the W/Wv mutations. (E and F) Confocal images of whole mounts featuring the circular muscle layer and the region of the myenteric plexus. Note the abundance of IC-MY (arrowheads). Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Distribution of NOS-containing neurons in the LES and pyloric sphincter as determined by NADPH diaphorase histochemistry. (A, B, D, and E) Whole-mount images of the myenteric plexus from +/+ and W/Wv animals at different levels of resolution. We could detect no differences between the number, size, or distribution of cell bodies within the myenteric plexus. (C and F) Varicose fibers within the circular muscle layer. No difference in the density of innervation was detected. (G, H, J, and K) Whole mounts of the pyloric sphincters of +/+ and W/Wv animals. (I–L) Varicose fibers within the circular muscle layer. No differences in the density of innervation of this region were detected in +/+ and W/Wv animals. Summaries of neuron and varicose fiber densities can be found in Table 1. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Confocal micrographs of NOS-like immunoreactivity in the (A) LES and (B) pyloric sphincters. Whole mounts of the LES and pyloric sphincter fixed with formaldehyde showed NOS-like immunoreactivity in varicose nerve fibers (arrowheads) and cells with profiles suggestive of IC-IM (arrows). The varicose fibers were in close proximity to the IC-IM. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Double-labeling of cells with c-Kit–LI (IC-IM) and NOS-like immunoreactivity. Whole mounts of the (A–C) LES and (D–F) pyloric sphincter fixed with acetone showed clear labeling of IC-IM with both antibodies. A and D show immunoreactivity to NOS (FITC), and B and E show c-Kit–LI (Texas red). C and F show colocalization of both fluorophores (yellow) in IC-IM (arrows). The NOS immunoreactivity was reduced in varicose nerve fibers in acetone-fixed preparations. Scale bars apply to all figures. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Mechanical responses to EFS of the LES. (A) Responses to 5- and 10-Hz stimulation (1-minute trains; 0.5-millisecond pulses; black bars). Responses under NANC conditions (see Materials and Methods) were characterized by a relaxation of the muscle during the period of stimulation and a rebound contraction upon cessation of the stimulation. L-NAME (200 μmol/L) blocked the relaxation phase and rebound contractions and converted the response during the stimulation at 10 Hz to a contraction. (C) Responses to EFS of a strip from a W/Wv animal. In all cases, EFS elicited a contractile response during the stimulation and the poststimulus rebound response did not occur. (D) L-NAME had little or no effect in W/Wv animals. (E) A graph showing a summary of experiments in wild-type (n = 5) and W/Wv animals (n = 5). Frequency-dependent inhibitory responses during stimulation of wild-type muscles (●) were decreased by L-NAME (○). The response during stimulation of muscles from W/Wv animals was a frequency-dependent contraction during the period of stimulation (■), which was not significantly affected by L-NAME (2). Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Mechanical responses of the pyloric sphincter to EFS. (A) Responses to 5- and 10-Hz stimulation (1-minute trains; 0.5-millisecond pulses; black bars). (B) Responses under NANC conditions (see Materials and Methods) were characterized by a relaxation of the muscle during the period of stimulation and a rebound contraction on cessation of the stimulation. L-NAME (200 μmol/L) blocked the relaxation phase and rebound contractions. (C) Responses to EFS of a strip from a W/Wv animal. In all cases, EFS elicited a contractile response during the stimulation and the poststimulus rebound response failed to occur. (D) L-NAME slightly increased the contractions of muscles of W/Wv animals during stimulation. (E) A graph showing a summary of experiments in wild-type (n = 8) and W/Wv animals (n = 5). Frequency-dependent inhibitory responses during stimulation of wild-type muscles (●) were decreased by L-NAME (○). The response during stimulation of muscles from W/Wv animals was a frequency-dependent contraction during the period of stimulation (■), which was slightly increased by L-NAME (2). Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Changes in membrane potential elicited by EFS of LES muscles. (A) Responses to 1–20 Hz stimulation (1-second trains at arrows). The inhibitory junction potentials were characterized by a rapid hyperpolarization followed by a slow hyperpolarization phase. (B) L-NAME (200 μmol/L) greatly reduced both phases of the inhibitory junction potential. Recordings in A and B were made from the same cell. (C) In W/Wv animals, inhibitory junction potentials were smaller in amplitude and consisted only of the rapid phase of hyperpolarization. (D) These events were not significantly affected by L-NAME. Recordings in C and D were made from the same cell. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 10 Summaries of electrical responses to EFS of wild-type and W/Wv LES muscles. (A) Amplitudes (fast component) of frequency-dependent inhibitory junction potentials (IJPs) in wild-type animals before (●) and after (○) L-NAME. (B) IJP duration in wild-type animals before (●) and after (○) L-NAME. The amplitudes and durations of IJPs significantly decreased at all frequencies (96 impalements from 24 animals). In W/Wv animals (54 impalements from 18 animals), (C) amplitudes and (D) durations of IJPs were reduced (●) and unaffected (○) by L-NAME. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 11 Changes in membrane potential elicited by EFS of pyloric sphincter muscles. (A) Responses to 1–20 Hz stimulation (1-second trains at arrows). The inhibitory junction potentials (IJPs) were characterized by a rapid hyperpolarization followed by a rebound depolarization. (B) L-NAME (200 μmol/L) reduced both phases of the IJP. Recordings in A and B were made from the same cell. (C) In W/Wv animals, IJPs were smaller in amplitude. (D) These events were not significantly affected by L-NAME. Recordings in C and D were made from the same cell. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 12 Summaries of electrical responses to EFS in pyloric sphincters of wild-type and W/Wv animals. (A) Amplitudes of frequency-dependent inhibitory junction potentials (IJPs) in wild-type animals before (●) and after (○) L-NAME. (B) IJP duration in wild-type animals before (●) and after (○) L-NAME. The amplitudes and durations of IJPs were significantly decreased at all frequencies (P < 0.05). In W/Wv animals, the (C) amplitude and (D) duration of IJPs were significantly smaller than observed in wild-type animals (●; P < 0.05) and were unaffected by L-NAME (○; P > 0.05). The IJPs in W/Wv animals were approximately the same amplitudes and durations as wild-type animals in the presence of L-NAME (P > 0.4; analysis of variance). Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 13 Postjunctional responses to SNP. Traces in each panel are superimposed recordings from single cells showing responses to several concentrations of SNP (10−8 to 10−4 mol/L). The traces in each panel are lined up on the time of SNP application (arrow). SNP was applied at the concentrations noted throughout the period of each recording, and the wash periods between SNP doses were eliminated. (A and B) Responses in LES muscles of wild-type (+/+) and W/Wv animals, respectively. (C and D) Responses in pyloric muscles wild-type (+/+) and W/Wv animals, respectively. Gastroenterology 1998 115, 314-329DOI: (10.1016/S0016-5085(98)70198-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions