Mutations in genes encoding members of the GDNF and endothelin-3 (Et-3) signaling pathways can cause Hirschsprung's disease, a congenital condition associated with an absence of enteric neurons in the distal gut. GDNF signals through Ret, a receptor tyrosine kinase, and Et-3 signals through endothelin receptor B (Ednrb). The effects of Gdnf, Ret, and ET-3 haploinsufficiency and a null mutation in ET-3 on spontaneous motility patterns in adult and developing mice were investigated. Video recordings were used to construct spatiotemporal maps of spontaneous contractile patterns in colon from postnatal and adult mice in vitro. In Ret+/− and ET-3+/− mice, which have normal numbers of enteric neurons, colonic migrating motor complexes (CMMCs) displayed similar properties under control conditions and following inhibition of nitric oxide synthase (NOS) activity to wild-type mice. In the colon of Gdnf+/− mice and in the ganglionic region of ET-3−/− mice, there was a 50–60% reduction in myenteric neuron number. In Gdnf+/− mice, CMMCs were present, but abnormal, and the proportion of myenteric neurons containing NOS was not different from that of wild-type mice. In the ganglionic region of postnatal ET-3−/− mice, CMMCs were absent, and the proportion of myenteric neurons containing NOS was over 100% higher than in wild-type mice. Thus impairments in spontaneous motility patterns in the colon of Gdnf+/− mice and in the ganglionic region of ET-3−/− mice are correlated with a reduction in myenteric neuron density.
- enteric nervous system
- glial cell line-derived neurotrophic factor
hirschsprung's disease is a congenital condition that affects 1 of 5,000 human births and is characterized by colonic stasis due to the absence of enteric neurons in the distal gut. Mutations in genes encoding members of the glial cell line-derived neurotrophic factor (GDNF) and endothelin-3 (Et-3) signaling pathways account for ∼50% of the cases of Hirschsprung's disease in humans (22).
The effects of mutations in genes encoding members of the GDNF and Et-3 signaling pathways on the development of the enteric nervous system (ENS) in mice have been extensively investigated at the cell and molecular levels (22, 29). However, little is known about the physiological consequences of mutations in genes encoding members of these signaling pathways during development or in mature animals other than the absence of transit through the region lacking enteric neurons.
GDNF signals through the receptor tyrosine kinase, Ret (1). Ret−/− mice and Gdnf−/− mice lack neurons from the small and large intestines and die at birth (14, 28, 33, 38, 42), but Ret+/− and Gdnf+/− mice are viable and fertile. Ret+/− mice have approximately normal numbers of enteric neurons, but there is about a 50% reduction in the number of enteric neurons in Gdnf+/− mice (20, 43). The only physiological study performed to date showed that circular muscle and longitudinal muscle contractility in response to electrical field stimulation was reduced in both Ret+/− mice and Gdnf+/− mice (20). However, the effects of Gdnf or Ret haploinsufficiency on complex motility patterns have yet to be examined.
Et-3 signals through endothelin receptor B (Ednrb). ET-3−/− and Ednrb−/− mice lack enteric neurons in the distal colon and are a long-established mouse model of Hirschsprung's disease (12, 34, 37). Although the ENS of ET-3+/− and Ednrb+/− mice was thought to be normal, a recent study has shown that Ednrb+/− mice have a small aganglionic region in the most distal regions of the large intestine and impaired colonic motor activity (35). The presence of a “megacolon,” an abnormal dilatation of the colon, proximal to the aganglionic region in ET-3−/− and Ednrb−/− mice indicates that there is little, or no, propulsion of intestinal contents through the aganglionic region, but it is unknown whether there are nonpropulsive motility patterns in the aganglionic region or whether there are defects in motility patterns in the colon of ET-3+/− mice.
In this study we used spatiotemporal mapping, a high-resolution method for the analysis of gut diameter as a function of length along the intestine and of time (4, 23), to examine spontaneous motility patterns in the colon of postnatal and mature wild-type, Ret+/−, and Gdnf+/− mice and postnatal ET-3−/− and ET-3+/− mice in vitro. This method is extremely powerful because 1) many minutes of activity can be compressed into a single small map, 2) motility patterns can be quantified, and 3) subtle motor patterns that would be difficult to detect with other methods can be detected (5, 13, 21, 23, 36, 46). Furthermore, the technique is less invasive than most other in vitro techniques, which is an advantage for studies of motility in gut from postnatal mice, which is small and fragile.
Colonic migrating motor complexes (CMMCs) are the most prominent spontaneous motility pattern seen in the mouse colon in vitro and are neurally mediated, rhythmic, anally propagating contractions that occur every 2–5 min (6, 8–10, 45, 46, 50). The frequency of CMMCs is enhanced by inhibition of nitric oxide (NO) synthase (NOS) (18, 36, 47). During development, CMMCs are not present in postnatal day 6 (P6) mice, but can be revealed by NOS inhibition (36). A role for NO in the modulation of motility in developing zebrafish has also been reported (25). Accordingly, we also examined the density of myenteric neurons and the proportion of the myenteric neurons that express NOS to determine whether changes in the density of neurons or the proportion of NOS neurons are correlated with altered motility. Our results show that CMMCs are impaired in mice in which there is a significant reduction in myenteric neuron density. Furthermore, in the ganglionic region of the colon of ET-3−/− mice where there was a decrease in the density of myenteric neurons plus a 100% increase in the proportion of neurons expressing NOS, CMMCs could not be detected.
Wild-type, Gdnf+/−, Ret+/−, ET-3+/−, and ET-3−/− mice, all raised on a C57BL/6 background strain, were used. Male mice, 8–12 wk old, were used for adult motility experiments. Male and female P4–P14 mice were used to examine motility in postnatal mice. The Gdnf+/− mice were originally generated by Pichel et al. (33), the Ret+/− mice were generated by Enomoto et al. (15), and the ET-3+/− mice were purchased from Jackson Laboratories (Bar Harbor, ME). The ET-3+/− mice were originally generated by Baynash et al. (3) by targeted inactivation of the ET-3 gene. The ET-3+/− mice were raised on a 129 background strain when purchased, but they were subsequently backcrossed for >10 generations onto a C57Bl6 background. Mice were killed by cervical dislocation (adults), CO2 gas (P10–P14), or decapitation (P4–P8) as approved by the Animal Experimentation Ethics Committee of The University of Melbourne. The cecum to most distal colon was removed and placed in an oxygenated (95% O2 and 5% CO2) physiological saline (room temperature) of the following composition (in mM): 118 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 25 NaHCO3, 11 d-glucose.
The colon was placed in an organ bath containing two adjacent chambers of dimensions 19 mm × 110 mm × 16 mm. This enabled the examination of two adult cannulated colonic preparations simultaneously, or up to four preparations from animals aged P4–P12. Saline was continuously superfused through each chamber at a flow rate of 6 ml/min. Adult tissues were emptied of intestinal contents and cannulated at the oral and anal ends. P4–P14 colon was attached via pins at the oral and anal ends to a dish lined with Silicone elastomer (Sylgard 184, Dow Corning).
Video Imaging of Colonic Motility In Vitro
Spontaneous motility patterns were examined by spatiotemporal mapping of the entire colon from adult and postnatal mice in vitro as described previously (36). Briefly, video images were captured with a Logitech QuickCam for adult tissues or by a Canon DM-MVX150i digital video camera mounted on a dissecting microscope for developing gut. The video image captured the entire length of the colonic segment. Images were captured at a rate of 15 frames/s with a resolution ranging between 400 × 320 pixels (Cannon DM under dissecting microscope, P4) and 128 × 96 pixels (Logitech QuickCam, adult) and acquired to computer in AVI format. For each individual frame of the video, software developed using the MATLAB 7.0.4 system (version 1.2.7) converted an image of the intestine into a silhouette (21, 36). A spatiotemporal map of the movements of the intestine was generated by counting the number of vertical pixels for each horizontal pixel in the silhouette (21). The diameter of the intestine at each point was plotted as a color function of space (distance along the segment) and time (each video frame corresponds to one time point giving a resolution of 67 ms) on a two-dimensional image.
Following a 1-h equilibration, video images were recorded for 45 min in control, drug, and washout conditions. A nonrecording period of 15 min during the time of addition or washout of drugs into the bath separated each recording period.
From the spatiotemporal maps, the interval between CMMCs was calculated as the time difference between the initiation of consecutive contractions at the proximal end of the colonic segment, duration was measured as the time for which contraction was maintained at the midpoint of the colonic segment for each CMMC, and resting diameter and percentage diameter change were measured at the midpoint of the colonic segment. The percentage propagated was calculated by the placement of a vertical line at the points of initiation and termination of each individual CMMC in a spatiotemporal map. The difference between these two points, as calibrated to the intestinal length, was determined by analysis software developed in house. A similar method was used to determine the initiation site of CMMCs. The mapping technique, which allows simultaneous examination of activity at all points along the intestinal length, allowed the direction of CMMCs to be detected largely by eye. When necessary, this was confirmed by expanding the time scale of the map and comparing the CMMC contraction with a horizontal line drawn from its point of initiation.
Drugs used were tetrodotoxin (TTX, 1 μM; Alomone Laboratories, Jerusalem, Israel), nitro-l-arginine (NOLA, 100 μM; RBI, Natick, MA), and granisetron hydrocholoride (1 μM; SKB GlaxoSmithKline; Middlesex, UK). All drugs were initially made up in distilled water to form stock solutions. Final concentrations of drug were achieved by adding appropriate aliquots of the stock to the saline used to superfuse the tissue.
The colon was dissected from adult and P10 wild-type and Gdnf+/− mice, and P10 wild-type, ET-3+/−, and ET-3−/− mice, opened along the mesenteric border, stretched, and pinned flat on balsa wood. The tissue was fixed overnight in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2 at 4°C. Preparations were washed with phosphate buffer, and the mucosa and some of the circular muscle was removed by dissection. Whole mount preparations of myenteric plexus plus longitudinal muscle were stained with primary antibodies [anti-NOS raised in sheep, 1:2,000 (51); anti-Hu (pan-neuronal marker) raised in human, 1:2,000 (16)]. The preparations were then washed in phosphate buffer and exposed to secondary antisera (donkey anti-goat FITC 1:100, Jackson Immunoresearch; donkey anti-human Texas red, 1:100, Jackson Immunoresearch). The preparations were viewed on a confocal microscope. To quantify the number of Hu+ cells and the proportion Hu+ cells that were NOS+ in myenteric ganglia in adult Gdnf+/− and wild-type mice, a Z series was taken with a ×20 lens in adjacent fields of a 3 × 3 tile scan (total area = 1.59 mm2) at one-third (proximal) and two-thirds (distal) along the length of the colon between the cecum and the anal end. The Z series were projected and the total numbers of Hu+ and NOS+ cells were counted. In P10 ET-3+/−, ET-3−/−, and their wild-type littermates, Hu+ cells in myenteric ganglia of the colon were present in multiple layers and it was not possible to count accurately the total number of Hu+ cells from projected Z series. Hence, single optical sections were taken through the middle of ganglia in two adjacent fields of the proximal colon and in the distal colon using a ×40 lens (total area = 0.09 mm2), and the relative densities of Hu+ and NOS+ cells were compared between ET-3+/−, ET-3−/−, and wild-type littermates. The density of Hu+ and NOS+ cells were measured in 1) the proximal colon, immediately adjacent to the cecum, 2) one-third the distance between the cecum and the anal end, and 3) two-thirds the distance between the cecum and the anal end.
At the conclusion of some motility experiments, ET-3−/− and littermate mouse colon was stained using NADPH-diaphorase histochemistry to determine the presence and length of the aganglionic region. Although NADPH diaphorase only stains NOS neurons, it is an accurate method for localizing the aganglionic zone (35). The colon was removed with care to include the anus and opened along the mesenteric border and attached to balsa wood with pins fixed overnight in 4% formaldehyde. The preparations were washed with phosphate buffer stained with β-NADPH 1 mg/ml dissolved in 3.0 mM NBT in 0.1 m Tris·HCl pH 8.0 and Triton-X. Solutions were left for 30–45 min at 37°C as described previously (52).
Data are reported as means ± SE and were analyzed by paired t-tests, ANOVAs, or χ2 as appropriate.
Spontaneous Colonic Motility in Adult Wild-Type, Ret+/−, and Gdnf+/− Mice
Spontaneous motility patterns were examined by spatiotemporal mapping of the entire colon of adult and postnatal wild-type Ret+/− and Gdnf+/− mice. Ret−/− and Gdnf−/− mice die at birth (14, 28, 33, 38, 42) and consequently were not used in this study. Ret+/− and Gdnf+/− mice are viable and fertile. Ret+/− mice have normal numbers of enteric neurons, but Gdnf+/− mice have been reported to have ∼50% less enteric neurons than wild-type mice in the small and large intestines (20, 43).
Comparison of CMMCs in adult wild-type, Ret+/−, and Gdnf+/− mice.
CMMCs are spontaneous, regularly occurring, migrating contractions that are seen in the mouse colon in vitro and have been well characterized by a variety of techniques (6, 36, 45, 50). CMMCs were observed in adult wild-type, Ret+/−, and Gdnf+/− mice. They were initiated in the proximal colon (within 10% of the colonic length from the oral end of the segment) and propagated anally (Fig. 1I, A, D, and G). The properties of CMMCs in adult Ret+/− mice did not differ significantly from wild-type mice in frequency, velocity, duration, percentage of the length of the colon along which CMMCs propagated, diameter change, or site of initiation (P > 0.05; n = 18; Fig. 2). In contrast, in adult Gdnf+/− mice (n = 19) there was a significant increase in the interval between CMMCs (Fig. 2A) and a significant decrease in the percentage of the length of the colon along which CMMCs propagated (Fig. 2D), percentage diameter change (Fig. 2E), and resting diameter of the tissue (Fig. 2F) compared with both wild-type or Ret+/− mice. There was no significant difference in the velocity (Fig. 2B), duration (Fig. 2C), or initiation site (Fig. 2G) of CMMCs between adult Gdnf+/−, Ret+/−, and wild-type mice.
Effect of NOS inhibition on CMMCs in adult mice.
Previous studies have shown that NOS inhibition increases the frequency and velocity of CMMCs in wild-type mice (6, 36, 47). In the present study, NOLA (100 μM) significantly decreased the interval between CMMCs and increased the velocity of CMMCs in adult Ret+/− and wild-type mice (P < 0.05; n = 8 wild-type, n = 7 Ret+/−; Fig. 1I, B and E; Fig. 3, A and B), although the duration, percentage of the length of the colon along which CMMCs propagated, percentage diameter change, and resting diameter were not significantly changed (Fig. 3, C–F). In contrast, NOLA had no significant effect on any of the properties of CMMCs examined in Gdnf+/− mice (Fig. 1I H; Fig. 3, A–F).
Effect of the 5-HT3 receptor antagonist granisetron on CMMCs in adult mice.
5-HT3 receptor antagonists inhibit CMMC frequency (7). To determine whether only NO-mediated modulation of CMMCs is affected by Gdnf haploinsufficiency, we examined the effects of the 5-HT3 receptor antagonist granisetron (1 μM) on CMMCs in adult wild-type, Gdnf+/−, and Ret+/− mice. In wild-type mice, the interval between CMMCs was significantly increased by granisetron and the velocity of CMMCs was reduced (P < 0.05; n = 8; Fig. 4, A and B); other parameters were unchanged (Fig. 4, C and D). Ret+/− mice displayed a significant increase in interval between CMMCs (P < 0.05; n = 7; Fig. 4A) but no significant change in any of the other properties (Fig. 4, B–D). None of the properties of CMMCs in Gdnf+/− mice was altered significantly by granisetron (P > 0.05; n = 8; Fig. 4, A–D). Thus both 5-HT3- and NO-mediated modulation of CMMCs are perturbed in Gdnf+/− mice.
Density of myenteric neurons and the proportion of myenteric neurons that is NOS+ in adult Gdnf+/− mice compared with wild-type mice.
Previous studies have shown that the density of enteric neurons is lower in Gdnf+/− mice than wild-type mice (20, 43), but it has not been established whether this is due to a selective loss of a particular class or classes of neurons or whether most or all classes of enteric neurons are reduced in density. Since NOS inhibition is less effective in regulating CMMCs in Gdnf+/− mice (see Effect of NOS inhibition on CMMCs in adult mice), we compared the density of myenteric neurons and the proportion of myenteric neurons that are NOS+ in the proximal and distal colon of adult wild-type and Gdnf+/− mice using antibodies to Hu (to label all neurons) and NOS (Fig. 5I). There was a 62% reduction in the density of myenteric neurons (Hu+ cells) in the proximal colon and a 55% reduction in the distal colon of adult Gdnf+/− mice compared with wild-type mice (P < 0.001; n = 6; Fig. 5IIA). However, in both the proximal and distal colon, the percentage of myenteric neurons that was NOS+ was similar in Gdnf+/− and wild-type mice (Fig. 5IIB). These data suggest that Gdnf haploinsufficiency does not selectively affect NOS neurons.
Spontaneous Colonic Motility in Postnatal Wild-Type Ret+/− and Gdnf+/− Mice
Motility in p10-p14 mice.
We have previously reported that CMMCs are observed in wild-type mice at P10 (36). In the present experiments, pinned uncannulated colonic preparations from P10–P14 wild-type or Ret+/− mice displayed CMMCs that were initiated proximally (Fig. 6F) and migrated anally (data not shown). These contractions did not display significant differences in interval or duration from cannulated preparations from adult wild-type or Ret+/− mice (Table 1, Fig. 6, A and C). However, in both wild-type and Ret+/− P10–P14 mice, there was a significant reduction in percentage of the length of the colon along which CMMCs propagated anally and percentage diameter change compared with adults (Table 1). In P10–P14 Ret+/− mice, the velocity of CMMCs was lower than that observed in adults (Table 1) and in P10–P14 wild-type mice (Fig. 6B). In P10–P14 Gdnf+/− mice, there was a significantly reduced incidence of CMMCs (P < 0.05). Contractile activity propagating from the oral end was only seen in 4 of 10 preparations and persisted for a maximum of 45 min. These contractions were disorganized and thus their properties could not be quantified. Three of these preparations were treated with 1 μM TTX, which abolished all contractions.
Effect of NOLA.
Bath applied NOLA (100 μM) reduced the interval between CMMCs in P10–P14 wild-type and Ret+/− mice (Fig. 6A). The velocity (Fig. 6B) and percentage of the colonic segment propagated (Fig. 6D) by CMMCs was also significantly increased by NOLA in both wild-type and Ret+/− mice, but CMMC duration (Fig. 6C) was not significantly changed. In P10–P14 Gdnf+/− mice, motility remained disorganized in the presence of NOLA.
Motility in P6 mice.
As previously described (36), CMMCs were not detected in P6 wild-type mice under control conditions, and the only motility patterns observed were shallow contractions, termed “ripples,” which propagated in both directions (n = 6). These high-frequency contractions were revealed by zooming in on regions of spatiotemporal maps. However, CMMCs were induced in the colon of P6 wild-type mice after the addition of NOLA (100 μM) to the organ bath (Fig. 1II B). Like wild-type mice, in P6 Ret+/− mice, only ripples were observed under control conditions; however, CMMCs were induced after the addition of NOLA (n = 6; Fig. 1II, D and E). CMMCs in P6 wild-type mice and Ret+/− mice were abolished by TTX but ripples were unaffected (data not shown). In P6 Gdnf+/− mice, CMMCs could not be detected under control conditions or in the presence of NOLA (n = 5) (Fig. 1II, G–I). Ripples, however, were present.
Motility in P4 mice.
In P4 wild-type mice, we have previously shown that CMMCs are not detected, even after addition of NOLA; the only motility observed is TTX-insensitive ripples (36). In the present study, ripples were the only motility pattern observed in P4 wild-type, Ret+/−, and Gdnf+/− mice. Ripples were observed in the colon from mice of all ages, including adults.
Spontaneous Colonic Motility in Et-3+/− and Et-3−/− Mice
Mice with mutations in the Et-3 signaling pathway are the best established model of Hirschsprung's disease (19). ET-3−/− mice exhibit skin pigmentation defects in addition to colonic aganglionosis and can be distinguished from ET-3+/− and wild-type mice by their spotted phenotype. Motility in ET-3−/− mice was examined in experiments performed in parallel with a littermate control. Littermates were later genotyped by PCR to identify them as either ET-3+/− or wild-type mice.
Motility is severely impaired in postnatal Et-3−/− mice, but not in postnatal Et-3+/− mice.
MOTILITY IN P8–P12 ET-3−/− MICE.
The ET-3−/− mice used were on a C57Bl6 background (see methods); in 18 litters examined, the maximal viable age was P12. ET-3−/− mice possessed a bloated abdomen and were also smaller in size than their littermates. Colonic motility was examined in P8 (n = 3), P10 (n = 9), P11 (n = 1), and P12 (n = 7) ET-3−/− mice. In 60% of preparations (12 of 20), no contractile activity was observed in any region of the colon in control conditions (Fig. 7I, b2 and c2). In 8/20 preparations, some contractile activity was observed in various locations along the colon, but the contractions were disorganized and did not propagate significant distances. NOLA did not induce CMMCs in any region, including the proximal colon, in any preparation from P8–P12 ET-3−/− mice.
Although NADPH diaphorase activity is a marker of NOS neurons only, NADPH diaphorase histochemistry can be used to identify the extent of aganglionic regions in mouse models of Hirschsprung's disease (35). Eight preparations in which video images were recorded were stained with NADPH diaphorase histochemistry at the end of the experiment (and prior to the processing of video images) so the location of the aganglionic region could be identified. Three of these preparations were inactive, and five demonstrated some disorganized contractions. The aganglionic region comprised 82.5 ± 10.3% of the entire colonic segment. CMMCs were not observed in either the ganglionic or aganglionic regions (Fig. 7I, a2, b2, and c2). In preparations with a long aganglionic region, spatiotemporal maps were inactive.
MOTILITY IN P8–P12 ET-3+/− MICE.
ET-3+/− mice showed no significant differences in CMMC interval, duration, velocity, percentage of the length of the colon along which CMMCs propagated, or initiation site from wild-type mice (Fig. 8). Furthermore, NOLA significantly reduced the interval and increased the velocity of CMMCs in both wild-type and ET-3+/− mice (Fig. 8, A and B).
MOTILITY IN P6 MICE.
In P6 mice, CMMCs were induced by the application of NOLA in both ET-3+/− mice and wild-type mice (n = 4); however, no organized activity was observed in ET-3−/− mice under control conditions or in the presence of NOLA (n = 4) (data not shown).
Ripples in postnatal Et-3−/− and Et-3+/− mice.
Ripples were seen in spatiotemporal maps in both ganglionic and aganglionic regions in postnatal ET-3−/−, ET-3+/−, and wild-type mice at all ages examined. These appeared as short, shallow constrictions that propagated in the oral and/or anal direction (Fig. 9).
Immunohistochemical and histochemical studies of P10 Et-3+/−, wild-type, and Et-3−/− mice.
Longitudinal muscle/myenteric plexus preparations from P10 wild-type and ET-3+/− and ET-3−/− mice were stained with antibodies to NOS and Hu. There was no significant difference between wild-type and ET-3+/− mice in the density of Hu+ cells or the proportion of cells that was NOS+ in the proximal third or distal third of the colon (Fig. 7III). Examination of preparations from P10 ET-3−/− mice confirmed that at least 70% of the colon was aganglionic, and the transition zone between the ganglionic and aganglionic zone was in the proximal colon, adjacent to the cecum. In the proximal (ganglionic) region, the density of neurons was lower than in equivalent regions of the colon of wild-type or ET-3+/− mice (Fig. 7IIIa). However, the proportion of neurons that expressed NOS in the ganglionic region of ET-3−/− mice was significantly higher (over twofold) than in wild-type mice (Fig. 7IIIb).
NOS+ nerve terminals were present in the circular muscle up to 100 μm caudal to the most caudal NOS cell body (Fig. 7IIe), confirming that at least some NOS neurons project anally to the muscle in the mouse colon (40). A small number of Hu+ and NOS+ cells were observed in the distal colon of one preparation from an ET-3−/− mouse (Fig. 7IIf). Some of these were NOS+ and most were associated with thick extrinsic fiber bundles. These were likely to be derived from the sacral neural crest.
A recent study has reported the presence of an aganglionic region in the most caudal segment of the large intestine of Ednrb+/− mice (35). However, in the present study, NADPH diaphorase staining did not reveal an aganglionic region in the distal colon or rectum of ET-3+/− mice.
Mice with mutations in genes encoding members of the Gdnf or Et-3 signaling pathways have provided fundamental information about the roles of these pathways in the cell and molecular biology of ENS development and the etiology of Hirschsprung's disease. However, little is known about the functional consequences of mutations in these genes on gut motility. Our results show that the integrity of spontaneous motility patterns is correlated with myenteric neuron density. Although the experiments were performed in vitro, migrating motor complexes have been described in many animals in vivo (17, 41, 44), and it is very likely that defects in neuronal function in mutant mice detected in vitro will also have consequences in vivo. Current methods for recording motility patterns in vivo are less amenable to pharmacological manipulation and are less sensitive than in vitro methods.
Motility in Mice With Mutations in Genes Encoding Members of the Gdnf Signaling Pathway
The present data show that the properties of CMMCs in adult Ret+/− mice do not differ significantly from those of wild-type mice. This is consistent with the earlier report that the density of myenteric neurons in the colon of adult Ret+/− mice does not differ significantly from that in adult wild-type mice (20).
Gianino et al. (20) reported that electrically evoked longitudinal and circular muscle contractions and release of the neurotransmitters substance P and vasoactive intestinal peptide were severely impaired in adult Ret+/− mice compared with wild-type mice. However, none of the properties of CMMCs examined in adult Ret+/− mice were significantly different from those of adult wild-type mice. The reasons for the discrepancy between the two studies are unclear but are very unlikely to include to lack of sensitivity in the spatiotemporal mapping method. This can detect very small changes in diameter; in the present study constrictions of 100 μm were easily resolved in adult tissues with higher resolutions being obtained with the neonatal preparations imaged via a dissecting microscope. Most measured properties of CMMCs in postnatal Ret+/− mice did not differ significantly from those of postnatal wild-type mice. However, CMMC velocity was significantly slower in P10–P14 Ret+/− mice than in wild-type mice, which suggests there are subtle differences in enteric circuitry between developing Ret+/− and wild-type mice.
Adult Gdnf+/− mice exhibited abnormal CMMCs (Fig. 1, Fig. 2). Furthermore, unlike wild-type and Ret+/− mice, CMMCs in Gdnf+/− mice were unaffected by NOS inhibition or 5-HT3 receptor antagonism. Thus there must be defects in the neural circuitry mediating CMMCs involving NO and 5-HT3-mediated transmission in Gdnf+/− mice. P6–P14 Gdnf+/− mice also show delayed development of the neural circuitry mediating CMMCs, since organized CMMCs were not present, even in most P14 Gdnf+/− mice.
Impaired motility was suggested by previous studies of Gdnf+/− mice. Shen et al. (43) reported a high incidence (83%) of fecal retention and relative inability to form pellets in Gdnf+/− mice. Gianino et al. (20) reported that electrically evoked contractions of the colonic muscle and transmitter release were substantially smaller in Gdnf+/− than in wild-type colon. The differences between CMMCs in Gdnf+/− and wild-type colon, however, are too small to correspond to the roughly 80% reduction in responses to nerve stimulation reported by Gianino et al., who also reported similar reductions in Ret+/− mice, which contrasts with the present study.
Phenotype of neurons in Gdnf+/− mice.
Previous studies have not established whether the decrease in myenteric neuron density in Gdnf+/− mice is due to losses of specific subclasses of myenteric neurons or a decrease in the density of all classes of myenteric neurons. Our data show that the proportion of myenteric neurons that express NOS is similar in wild-type and Gdnf+/− mice. This suggests that there may be a decrease in the density of all classes of myenteric neurons in Gdnf+/− mice. Gianino et al. (20) found that proliferation of enteric neuron precursors is impaired in embryonic day 12 (E12) Gdnf+/− mice, which is before most classes of enteric neurons have developed (29). Defects in proliferation from early in development would probably lead to a decrease in density of all types of enteric neurons.
Motility in Mice With Mutations in Genes Encoding Members of the Et-3 Signaling Pathway
ET-3−/− mice have been used as a mouse model of Hirschsprung's disease for many years because they exhibit terminal aganglionosis. We used ET-3−/− mice on a C57Bl6 background and the aganglionic zone comprised >70% of the colon, considerably longer than the aganglionic zone in ET-3−/− mice on a 129 background (3). The penetrance and severity of the aganglionosis in mice with mutations in other Hirschsprung's susceptibility genes, such as Sox10, vary with strain (30). ET-3−/− mice on a C57Bl6 background rarely lived beyond P12, so adult mice were not studied.
The properties of CMMCs in P8–P12 ET-3+/− mice did not differ significantly from those of wild-type mice. In contrast, CMMCs in Ednrb+/− mice are abnormal and the density of myenteric neurons in the proximal, middle, and distal colon of Ednrb+/− mice is significantly lower than in wild-type mice. Furthermore, Ednrb+/− mice possess a small aganglionic zone at the distal end of the colon (35). However, we did not observe a terminal aganglionic zone in ET-3+/− mice, and the density of myenteric neurons in the colon of ET-3+/− mice did not differ from that of wild-type. Furthermore, the percentage of myenteric neurons expressing NOS was the same in ET-3+/− mice as in wild-type. Thus, unlike Ednrb haploinsufficiency (35), ET-3 haploinsufficiency does not appear to affect colonic myenteric neuron density or spontaneous motility patterns. Combined, these data imply that in Ednrb haploinsufficient mice, insufficient Ednrb is produced for normal development of the ENS whereas sufficient Et-3 is produced for normal enteric neuron development in ET-3 haploinsufficient mice.
CMMCs were not observed in ET-3−/− mice, even in the ganglionic region of the colon. Similarly, CMMCs are absent from the ganglionic region of Ednrb−/− mice (35). The absence of CMMCs in the ganglionic region of the colon of ET-3−/− mice may be due to insufficient density of neurons to generate functional CMMCs (see below), to abnormal proportions of different neuronal sub-types in the enteric circuitry (see below), or to absence of ascending pathways arising from the aganglionic region. Ascending pathways may be critical for activation of cholinergic motor neurons mediating the contractions during CMMCs (48).
Phenotype of myenteric neurons in colon of Et-3+/− and Et-3−/− mice.
The density of neurons in the postcecal colon of ET-3−/− mice was reduced by ∼50% compared with wild-type mice. Interestingly, the proportion of neurons that expressed NOS was significantly (over twofold) higher in the ganglionic region of ET-3−/− mice than in equivalent regions of wild-type mice; ∼75% of neurons in the postcecal colon of ET-3−/− mice were NOS+, compared with ∼30% in wild-type mice. Thus other classes of myenteric neurons must be reduced in number, are absent from this region, or have changed their phenotype. This is consistent with a previous quantitative study of myenteric neurons in adult ET-3−/− mice on a different background strain (39). Although both the entire colon of Gdnf+/− mice and the region proximal to the aganglionic zone in ET-3−/− mice exhibited an approximate 50% reduction in neuron density, the reduced neuron density was accompanied by an increased percentage of NOS neurons in ET-3−/− mice, but not Gdnf+/− mice. Recently Clarke et al. (11) reported a significant loss of NOS neurons, but no change in cholinergic neurons, in the ileum of adult PrP-SCA7-92Q transgenic mice, which phenocopy many aspects of human spinocerebellar ataxia type 7, a neurodegenerative disorder. These mice also demonstrate abnormal colonic transit. Thus gut dysmotility can occur when there is no change (Gdnf+/− mice), an increase (ganglionic region of ET-3−/− mice), or a decrease (PrP-SCA7-92Q) in the proportion of myenteric NOS neurons.
Ripples in Et-3−/− mice.
Although CMMCs were absent from the aganglionic region of the ET-3−/− colon, another motor pattern was seen. These were the ripples that are also prominent in developing mouse colon from E18.5 to P6, prior to the normal onset of CMMCs (36). Ripples were also the major motor pattern in the neonatal colon of Gdnf+/− mice, in which the development of CMMCs is delayed. We have previously reported that ripples are seen between the CMMCs in adult mice (36). Furthermore, they occur independently of the presence of enteric neurons and do not appear to result from the activity of interstitial cells of Cajal. TTX-insensitive motility patterns have also recently been reported in developing zebrafish (24).
Spencer et al. (46) have demonstrated waves of increased Ca2+ concentration that propagate through the circular and longitudinal smooth muscle both between and during CMMCs. These are seen, albeit in less organized form, in the aganglionic region of the colon of Ednrb−/− mice, suggesting that they may correspond to the ripples seen in this and our previous study. Further experimentation is required to confirm that these Ca2+ waves and ripples share a common mechanism.
In humans with Hirschsprung's disease, motility problems commonly persist after surgical removal of the aganglionic segment (2, 53). Our data using mouse models of Hirschsprung's disease show that 1) neuron density is reduced proximal to the aganglionic region, 2) spontaneous neurally mediated motility patterns are abnormal (Gdnf+/− mice) or absent (ET-3−/− mice) in regions of the gut where there is a reduction in myenteric neuron density, and 3) there are changes in the proportion of NOS neurons in the region proximal to the ganglionic zone in ET-3−/− mice. CMMCs are also absent from the ganglionic region of the colon of Ednrb+/− mice, which have reduced numbers of myenteric neurons (35). Some of these mechanisms are likely to contribute to the motility problems suffered by patients with Hirschsprung's disease following surgery.
Role of Nitric Oxide in CMMCs
Much remains unknown about the neural circuit underlying the initiation and modulation of CMMCs. The present study provides information regarding the role of NO in CMMCs. Tonic release of NO is thought to hold the muscle hyperpolarized between CMMCs, and during a CMMC nitrergic transmission is suppressed (18, 27, 47). However, the present study indicates an entirely different role for NO in CMMCs. NOS inhibition reduced the interval between CMMCs and their rate of propagation, but not their duration, the distance over which they propagate, or the relative diameter change during contractions. Furthermore, inhibition of NOS had no effect on the basal diameter between CMMCs, indicating that NO does not set the basal tone between contractions despite the marked depolarizations seen with the same NOS inhibitor in intracellular recording studies. This suggests that NO does not modulate the properties of CMMCs via feedback from the muscle.
The enhanced frequency of CMMCs in wild-type and Ret+/− mice seen with inhibition of NOS and insensitivity of CMMCs in Gdnf+/− mice to inhibition of NOS indicates that NO is unnecessary for activation or maintenance of activity within the neural circuit that generates this motor pattern. It appears that NO modulates the mechanism that activates this neural circuit, which may account for the enhanced propagation velocity seen with NOS inhibition.
The present study shows a strong relationship between neuron density and the integrity of spontaneous motility patterns, CMMCs, in a number of mouse models of Hirschsprung's disease. In regions lacking enteric neurons (distal two-thirds of colon of ET-3−/− mice), CMMCs could not be detected; in regions in which there is a reduction in enteric neuron density from the normal state (entire colon of Gdnf+/− mice and proximal colon of ET-3−/− mice), CMMCs are absent or abnormal; and in mice where enteric neuron density is similar to wild-type mice (Ret+/− mice and ET-3+/− mice), CMMCs are essentially normal. A correlation between neuron number and integrity of motility patterns has recently been reported in zebrafish larvae (26). The correlation between neuron number and motility may be useful not only in understanding how the ENS develops in the normal and impaired state but also in making predictions about aging, where myenteric neuron number has been shown to decrease (see Refs. 31 and 49).
Some funding for this study was provided by the National Health and Medical Research Council of Australia (Grant 454351).
We thank Hideki Enomoto and Jeff Milbrandt for providing the Ret mice, Heiner Westphal for the Gdnf mice, Miles Epstein and Piers Emson for antibodies, and Marlene Hao for assistance with PCR genotyping.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 the American Physiological Society