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. 2010 Feb 8;588(Pt 7):1153–1169. doi: 10.1113/jphysiol.2009.185421

The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal

Rachael R Roberts 1, Melina Ellis 1, Rachel M Gwynne 1, Annette J Bergner 2, Martin D Lewis 3, Elizabeth A Beckett 4, Joel C Bornstein 1, Heather M Young 2
PMCID: PMC2853002  PMID: 20142273

Abstract

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC-MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC-MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/Wv embryos, which lack KIT+ ICC-MP and slow waves. Third, KIT-immunoreactive cells with the morphology of ICC-MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC-MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L-type Ca2+ channel antagonist nicardipine (2.5 μm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC-MP, but entry of extracellular Ca2+ through L-type Ca2+ channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

Introduction

In mature animals, intestinal motility patterns result from interactions between enteric neurons, interstitial cells of Cajal (ICC) and intrinsic smooth muscle mechanisms (Hasler, 1999; Bornstein et al. 2004; Farrugia, 2008; Sanders, 2008; Huizinga & Lammers, 2009). Pharmacological studies have shown that the main intestinal motility patterns in mature animals – segmentation, peristalsis and migrating motor complexes (MMCs) – all depend on neuronal activity (Hasler, 1999; Gwynne et al. 2004). Furthermore, in infants with Hirschsprung's disease, in which enteric neurons are absent from the distal regions of the bowel, and in animal models of Hirschsprung's disease, functional motor patterns cannot be detected in the aganglionic region, which is consequently unable to propel gut contents anally (Huizinga et al. 2001; De Giorgio et al. 2004; Ro et al. 2006; Roberts et al. 2008). In vivo studies of W/Wv mice, which have an impaired network of myenteric ICC (ICC-MP) in the small intestine (Ward et al. 1994), have shown that the propagation of contents along the small intestine is delayed and peristaltic movements are abnormal (Der-Silaphet et al. 1998). Thus, enteric neurons and ICC-MP, as well as normal smooth muscle contractile activity (Angstenberger et al. 2007), are required for normal intestinal motility patterns in mature animals.

Although fetuses receive their nutrient supply via the placenta, fetuses swallow amniotic fluid and gastrointestinal motility commences well prior to birth (Ross & Nijland, 1998; Burns et al. 2009). In humans (McLain, 1963) and mice (Anderson et al. 2004), gut contents (amniotic fluid and meconium) are propelled along the gastrointestinal tract during fetal stages. However, very little is known about the mechanisms controlling intestinal motility prior to birth.

In the colon of late embryonic and newborn mice, spontaneous contractions are present, but the contractions do not require neurons for their generation as they persist in the presence of tetrodotoxin, and are present in late embryonic mutant mice that lack enteric neurons (Roberts et al. 2007; Lindley et al. 2008). Surprisingly, spontaneous neurally mediated motility patterns are not present in the mouse colon until at least 1 week after birth (Roberts et al. 2007).

The types of motility patterns present in the small intestine during fetal and early postnatal development, and the mechanisms controlling motility, have not previously been examined. In the duodenum, it is essential that motility patterns that mix and propel gut contents are present immediately after birth, when the placental nutrient supply is severed. The period immediately after birth has therefore been termed the ‘neonatal starvation period’ (Kuma et al. 2004). In the present study, we investigated the types of motility patterns, and the roles of neurons and ICC-MP, in the duodenum and colon of pre- and neonatal mice. Propagating contractions, which we termed ‘ripples’, were first detected in the duodenum of embryonic day (E)13.5 mice and the colon of E14.5 mice. Notably, we showed that ripples were not mediated by neurons or ICC-MP, and are thus myogenic in origin. Neurally mediated contractions were first detected in the duodenum prior to birth, at E18.5, which is at least 1 week before neurally mediated contractions are present in the colon.

Methods

All experiments were approved by the Anatomy & Cell Biology, Neuroscience, Pathology, Pharmacology, and Physiology Animal Ethics Committee of the University of Melbourne. The experiments comply with The Journal of Physiology policy on animal experimentation (Drummond, 2009).

Mice

Wild-type and RetTGM/+ mice (Enomoto et al. 2001) were on a C57BL/6 background. Female WB/ReJ-W/+ (W/+) and male C57BL/6J-WV/+ (WV/+) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). To generate RetTGM/TGM embryos, RetTGM/+ mice were mated. To identify RetTGM/TGM embryos, samples of ileum from each embryo were processed for NADPH diaphorase histochemistry (Young et al. 1992); RetTGM/TGM embryos lack NADPH diaphorase stained neurons in the intestine (Ward et al. 1999). To obtain W/Wv embryos, female W/+ and male WV/+ mice were mated. Total RNA was extracted from the liver and colon of each of the progeny, and genotyping was performed as described previously (Beckett et al. 2007). The day on which a vaginal plug was detected was designated E0.5. Pregnant mice were killed by cervical dislocation, and postnatal day (P)0–P10 mice by decapitation. The stomach and small and large intestine were dissected and placed in oxygenated (95% O2 and 5% CO2) physiological saline as described previously (Roberts et al. 2007).

Spatiotemporal mapping

Motility patterns were revealed using spatiotemporal mapping (Hennig et al. 1999). The entire length of colon, the entire length of small intestine (E12.5–E16.5) or the proximal 50% of the small intestine (E18.5–P10) were placed in an organ bath and superfused with physiological saline at 6 ml min−1. After 45 min equilibration, video images were captured using a digital video camera mounted on a dissecting microscope (Roberts et al. 2007). Spatiotemporal maps of intestinal movement were generated using software developed in-house (Gwynne et al. 2004). Vertical slice analysis was used to quantify diameter changes (Gwynne et al. 2004), and Fast Fourier Transform analysis was applied to obtain data at single points (MatLab subroutine) to reveal contraction frequencies. All quantitative data are given as mean ± standard error of the mean, and t tests were used to compare data statistically.

Drugs

Tetrodotoxin (TTX, 1 μm, Alomone Laboratories, Jerusalem, Israel), NG-nitro-l-arginine (NOLA, 100 μm, Sigma-Aldrich, St Louis, MO, USA), cobalt chloride (1 mm, Ajax, NSW, Australia), nicardipine (2.5 μm, Sigma-Aldrich) and nifedipine (2.5 μm, Sigma-Aldrich) were initially made up in distilled water or dimethyl sulphoxide (DMSO) (nicardipine and nifedipine) as stock solutions of 1 mm TTX, 100 mm NOLA, 1 m cobalt chloride, 2.5 mm nicardipine and 2.5 mm nifedipine. Final drug concentrations were achieved by adding the drug to the physiological saline used to superfuse the tissue.

Immunohistochemistry

Intact preparations of E14.5, E16.5 and E18.5 duodenum and colon and opened preparations of P0 duodenum and colon were fixed in 4% formaldehyde and then processed for immunohistochemistry using antibodies to the pan-neuronal marker Hu (human anti-Hu, 1:2000) (Fairman et al. 1995), and KIT (rabbit anti-KIT, 1:100, Calbiochem). The primary antisera were localized using donkey anti human-Texas Red (1:100, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and biotinylated donkey anti-rabbit (1:100, Jackson) followed by streptavidin-Cy5 (1:100, GE Healthcare, Little Chalfont, UK). Preparations were imaged on Zeiss Pascal or META confocal microscopes.

Electrophysiology

The first 3–5 mm of the duodenum of E16.5, E18.5, P0, P1 and P4 mice was opened along the mesenteric border and pinned out with the mucosa facing upwards. The anal end of each segment was folded back on itself and pinned to allow access to the circular muscle via the serosa. Circular muscle cells were impaled with glass microelectrodes (100–200 MΩ) filled with 1 m KCl as described previously (Gwynne & Bornstein, 2007).

Results

Motility in the duodenum and colon of E12.5–P10 mice and the role of neurons

Spontaneous motility patterns in the duodenum and colon of fetal and neonatal mice were examined using spatiotemporal mapping, in which video recordings of segments of duodenum and colon in vitro were used to construct maps of gut diameter as functions of intestinal length and time (Hennig et al. 1999; Gwynne et al. 2004).

E12.5

Contractile activity was not detected in the duodenum or colon of E12.5 wild-type mice in spatiotemporal maps (Fig. 1A), by Fourier analysis (Fig. 1C) or by vertical slice analysis (Fig. 1D) (n= 4).

Figure 1. Contractile activity in the E12.5 and E14.5 duodenum.

Figure 1

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A and B, spatiotemporal maps of contractile activity in the E12.5 (A) and E14.5 (B) duodenum. A, propagating contractions were not observed in the E12.5 duodenum. B, in the E14.5 duodenum, contractions that propagated orally (arrow pointing to the left), anally (arrow pointing to the right) and bidirectionally (double-headed arrow) were present. C and D, vertical slice analysis to reveal frequency (C) and diameter change (D) was performed on the preparations of E12.5 and E14.5 duodenum shown in A and B at the locations indicated by the white dotted lines. C, in the E12.5 duodenum, a contraction frequency could not be detected (green line), but in the E14.5 duodenum (blue line), contractions at a variety of frequencies were present. D, the diameter of the E12.5 duodenum was relatively constant (green line), whereas there were regular contractions (open arrows) and relaxations (arrows) in the E14.5 duodenum (blue line). E–G, effects of tetrodotoxin (TTX, 1 μm) on propagating contractions in the E14.5 duodenum. E, the frequency of contractions in the presence of TTX was not significantly different from control conditions (n= 6). F and G, spatiotemporal maps of a preparation of E14.5 duodenum under control conditions (F) and following the addition of TTX (G). Propagating contractions persisted in the presence of TTX.

E13.5

In E13.5 wild-type mice, regular, shallow contractions that propagated for short distances bidirectionally, orally or anally were detected in some preparations of duodenum (5/10), but not in the colon (0/10) (data not shown). We have previously reported similar contractions in the colon of neonatal mice, which we called ‘ripples’ (Roberts et al. 2007).

E14.5

Ripples that propagated both orally and anally were observed in all preparations of duodenum (n= 18) (Fig. 1B) and in 11/19 preparations of colon from E14.5 mice. In the duodenum, Fourier analysis revealed a single dominant contraction frequency (Fig. 1C), and vertical slice analysis of diameter change revealed clear and regular changes in gut diameter (Fig. 1D) that were not present in E12.5 mice (Fig. 1D).

In the presence of tetrodotoxin (TTX, 1 μm), which blocks action potentials dependent on inward Na+ currents, contractions were present in spatiotemporal maps of E14.5 duodenum (n= 6; Fig. 1F and G) that did not differ in frequency (Fig. 1E) or diameter change from controls. Contractions were also present in 5/7 preparations of E14.5 colon following the addition of TTX; in the 2/7 preparations in which contractions did not persist in the presence of TTX, there were also no contractions following wash-out.

E16.5

Contractions (ripples) that propagated both orally and anally were observed in all preparations of duodenum (n= 15) and colon (n= 15). The frequency of contractions in the E16.5 duodenum (2.6 ± 0.3 contractions min−1) was significantly greater than in the E14.5 duodenum (0.9 ± 0.2, P < 0.008). However, there was no significant difference in the proportional diameter change between E14.5 and E16.5 duodenum (29.1 ± 3.6% in E14.5 versus 21.0 ± 5.4% in E16.5, P > 0.2). The frequency and speed of contractions in the E16.5 duodenum were significantly higher than in the E16.5 colon, but there was no significant difference in the proportional diameter change between the E16.5 duodenum and colon (Fig. 2A).

Figure 2. Quantitative data of the properties of ripples (propagating contractions) in the E16.5 duodenum and colon (A), the effects of TTX on ripples in the E16.5 duodenum (B) and the effects of an absence of neurons on ripples in the E16.5 duodenum (C).

Figure 2

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A, propagating contractions occurred at significantly lower frequencies and speeds in the E16.5 colon (n= 15) compared to the E16.5 duodenum (n= 15), but there was no significant difference in the proportional change in diameter that occurred during contractions (middle graph). B, TTX (1 μm) had no effect on the frequency, proportional change in diameter during contractions or resting diameter in the E16.5 duodenum (n= 6). C, the frequency of contractions and the proportional change in diameter during contractions in the duodenum of E16.5 RetTGM/TGM mice (n= 6) was not significantly different from Ret+/TGM or Ret+/+ littermates (n= 6).

The role of neurons in ripples was investigated using TTX and by examining the intestine of Ret null mutant mice, which lack enteric neurons along the entire intestine (Schuchardt et al. 1994). TTX had no significant effect on contraction frequency, diameter change or resting diameter in the E16.5 duodenum (n= 6; Fig. 2B). RetTGM/TGM embryos (n= 6) were identified by screening segments of caudal small intestine using NADPH diaphorase histochemistry; the small intestine of RetTGM/TGM embryos lack NADPH diaphorase-stained neurons (Roberts et al. 2007). Ret+/+ and Ret+/TGM embryos were not distinguished from each other as there is no difference in enteric neuron densities in adult Ret+/− mice from Ret+/+ mice (Gianino et al. 2003). Propagating contractions were observed in the duodenum and colon of E16.5 RetTGM/TGM mice (Movie 2, supplemental material) and their littermates (Movie 1, supplemental material); contraction frequencies and diameter changes in E16.5 RetTGM/TGM mice were not significantly different from those in E16.5 Ret+/+ or Ret+/TGM mice (Fig. 2C).

Thus, ripples appear to be the only form of motility present in the duodenum and colon of E16.5 mice, and, as at E14.5, they are independent of neurons.

E18.5 duodenum

In 11/19 preparations of E18.5 duodenum, there were prominent clusters of contractions (Fig. 3A and D), which we termed ‘contraction complexes’. Contraction complexes occurred at a frequency of 0.19 ± 0.03 contractions min−1 (n= 11), and were clearly distinguished from higher frequency, individual, shallow contractions by Fast Fourier Transform analysis and vertical slice analysis (Fig. 3C and D). The majority of complexes (9/11; 81%) propagated orally. Contraction complexes in the E18.5 duodenum were abolished by TTX and so were neurally mediated (Fig. 3G). The higher frequency, TTX-resistant, shallow contractions, which occurred at a frequency of around 14 min−1 (Fig. 3C), were present in all preparations, including those without contraction complexes (Fig. 3B). The nitric oxide synthase inhibitor, NOLA, increased the frequency of contraction complexes (Fig. 3G) and induced contraction complexes in preparations that did not possess contraction complexes under control conditions (Fig. 3E and F).

Figure 3. Contractile activity in the E18.5 duodenum.

Figure 3

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A and B, spatiotemporal maps from two different preparations of E18.5 duodenum. Both preparations showed high frequency, shallow contractions (white arrows). One of the preparations (A) also exhibited prominent clusters of contractions, which we termed ‘contraction complexes’ (black arrows). C and D, vertical slice analysis to reveal frequency (C) and diameter change (D) was performed on the preparations shown in A and B at the locations indicated by the blue and green dotted lines. The contraction complexes occur at low frequency (around 0.2 contractions min−1; red arrow in C) and result in large changes in gut diameter (red arrows in D). Higher frequency contractions (around 15 contractions min−1; black arrow in C) are also present. E and F, spatiotemporal maps of a preparation of E18.5 duodenum, which under control conditions did not exhibit contraction complexes (E), but following the addition of NOLA (100 μm), contractions complexes (arrows) were induced (F). G, quantification of the effects of NOLA (100 μm) and TTX (1 μm) on the frequency of contraction complexes in the E18.5 duodenum. The frequency of contraction complexes was significantly higher in the presence of NOLA than in control conditions (n= 11), and TTX abolished contraction complexes (n= 11). The mean frequency of the contraction complexes in control preparations shown in panel G includes preparations that did possess contraction complexes under control conditions.

E18.5 colon

Contraction complexes were not observed in the E18.5 colon, but shallow contractions (ripples) similar to those observed in the E16.5 colon were present.

Meconium is present in the colon of E18.5 mice (Anderson et al. 2004). This allowed us to test whether ripples could produce a net propulsion of colonic content and to determine whether meconium affected the properties of ripples. The relationship between the presence of meconium, neurons and contractions was examined in the colon in E18.5 Ret+/+/Ret+/TGM and RetTGM/TGM mice. Meconium was observed in the colon of all RetTGM/TGM mice (n= 7) (Fig. 4B) and Ret+/+ or Ret+/TGM mice (Ret+/+ and Ret+/TGM embryos were not distinguished) (n= 7). The movement of small clumps of meconium was examined. Within a 15 min recording period, each clump of meconium moved anally 2.6 ± 2.4% of the colonic length in Ret+/+ or Ret+/TGM mice (n= 2) and 2.0 ± 0.8% of the segment in RetTGM/TGM mice (n= 7). In the colon of Ret+/+ or Ret+/TGM mice and RetTGM/TGM mice, contractions were seen in spatiotemporal maps in both meconium-containing and meconium-free regions of the colon (Fig. 4A). There was no significant difference between the frequency of contractions within meconium-containing and meconium-free regions of Ret+/+ or Ret+/TGM mice and RetTGM/TGM mice (Fig. 4C). There was, however, a significantly larger change in the proportional diameter during contractions in meconium-free regions (Fig. 4D), which was probably due to the lack of luminal contents.

Figure 4. Spatiotemporal map of contractile activity and photograph of the colon of an E18.5 Ret−/− mouse, and quantification of the frequency of contractions and proportional change in diameter during contractions.

Figure 4

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A and B, spatiotemporal map of contractile activity (A) and photograph (B) of the colon of an E18.5 Ret−/− mouse. The dark blue horizontal lines in the spatiotemporal map are artifacts. Meconium is present in the anal end of the segment (B), and contractile activity was present in both meconium-containing and meconium-free regions (A). C and D, quantification of the frequency of contractions (C) and proportional change in diameter during contractions (D) in meconium-containing and meconium-free regions. There was no difference in the frequency of contractions (C), but there was a significantly larger change in diameter during a contraction in meconium-free regions (D) (n= 7).

Postnatal mice

Motility in the neonatal colon has been examined previously (Roberts et al. 2007). Hence in the current study, motility was only examined in the duodenum in postnatal mice.

P0 duodenum

Contraction complexes and high frequency motility patterns were seen in spatiotemporal maps from 4/4 pinned preparations and 6/12 cannulated preparations of P0 duodenum (Fig. 5A and B). In cannulated preparations at 0 cmH2O, the frequency of complexes was 0.08 ± 0.05 cycles min−1 (n= 6), which did not differ significantly from pinned preparations (0.21 ± 0.13 cycles min−1; n= 4). However, the contractions comprising the complexes were more tightly clustered in cannulated preparations than in pinned preparations (Fig. 5A and B). NOLA induced contractions complexes in 2/6 cannulated preparations that did not possess complexes under control conditions.

Figure 5. Spatiotemporal maps of contractile activity of P0 and P10 duodenum.

Figure 5

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A and B, spatiotemporal maps of contractile activity of pinned (A) and cannulated (B) preparations of P0 duodenum. Propagating complexes are present in both preparations (arrows), but are more tightly clustered in the cannulated preparation (B). C, quantification of contraction frequency from vertical slices in control (cannulated) preparations (black squares) and following TTX (red diamonds). Low frequency contractions (dotted oval) are present only in control preparations. The higher frequency contractions represent slow waves and probably ripples. D–F, spatiotemporal maps from preparations of P10 duodenum. Contraction complexes are not present at resting intraluminal pressures (D), but are induced by intraluminal pressures of 2 cmH2O (E). Contraction complexes in the P10 duodenum are abolished by NOLA (100 μm) (F).

To determine whether different motility patterns could be identified by contraction frequency, software based on the Fast Fourier Transform was used. The power spectra calculated were examined for peaks, based on the assumption that the different motility patterns would exhibit discrete frequencies. A low frequency pattern was present in control maps, but was absent in maps from preparations exposed to TTX (1 μm) (Fig. 5C); this pattern represents neurally mediated contraction complexes as they were absent from spatiotemporal maps from TTX-treated preparations. A range of higher frequency contractions was detected that persisted in the presence of TTX (Fig. 5C).

P10 duodenum

At 0 cmH2O, high frequency contractile activity was observed in all preparations (Fig. 5D). Vertical slice analysis of spatiotemporal maps revealed a frequency of 30.1 ± 0.9 cycles min−1 (n= 10) for non-complexed background contractions. Contraction complexes were observed in only 2/10 preparations at 0 cmH2O. These cycled at 0.19 ± 0.22 complexes min−1 and propagated orally.

Contraction complexes were induced in 7/8 duodenal preparations that lacked contraction complexes under resting conditions by increasing the intra-luminal pressure to 2 cmH2O (Fig. 5E). These contraction complexes cycled at 0.37 ± 0.07 contractions min−1. Eighty-eight per cent of distension-induced complexes were overall orally directed and 12% travelled anally. The orally directed complexes propagated at −42.6 ± 5.9 mm min−1, and anally directed complexes propagated at 46.1 ± 13.4 mm min−1. In the presence of NOLA (n= 4), neither orally nor anally directed complexes could be distinguished (Fig. 5F). In addition to contraction complexes, higher frequency activity was detected in spatiotemporal maps at 2 cmH2O. The frequency of this activity was 29.8 ± 1.5 cycles min−1, which did not differ significantly from the frequency at resting intraluminal pressure.

In summary, these data show that ripples (propagating contractions that are not neurally mediated), are present in the duodenum from E13.5 and the colon from E14.5. Neurally mediated contraction complexes were present in the duodenum from E18.5, but both the luminal pressure required to induce these complexes and the role of nitric oxide in modulating the complexes change during the first postnatal week.

Involvement of ICC-MP in motility patterns

To examine a possible role for ICC-MP in ripples, three approaches were taken: (a) intracellular electrical recordings were made from the circular muscle of the duodenum, (b) motility patterns were examined in W/Wv embryos, and (c) the morphological development of ICC-MP was examined using antibodies to KIT.

Development of slow waves in duodenal circular muscle of E16.5–P4 mice

To determine when pacemaker activity can first be detected, intracellular recordings were made from the circular muscle of opened duodenal preparations from E16.5, E18.5, P0, P1 and P4 mice. Recordings were performed in the presence of the L-type voltage-dependent Ca2+ channel (VDCC) antagonist, nicardipine (2.5 μm), to minimise smooth muscle contractions. However, preparations from all age groups still showed some spontaneous contractile activity in the presence of nicardipine, which made it difficult to maintain impalements. The majority of impalements from which data were analysed lasted between 2 and 20 min (maximum duration 68 min).

E16.5

A total of eight preparations from five litters at E16.5 were studied. The mean resting membrane potential (RMP) of circular muscle cells was −54 ± 1 mV (mean ±s.e.m.). The first recordings after the equilibration period revealed very little electrical activity; however, in 6/8 preparations small amplitude depolarizations (mean amp 5.0 ± 0.5 mV, mean duration 1.1 ± 0.1 s) were observed (Fig. 6A), at least 2 h after the recordings were commenced (3–4 h after the initial dissection). The depolarisations were irregular in frequency and amplitude and were usually infrequent, although in one preparation they occurred at an average frequency of 14.2 ± 2.5 peaks min−1 (Fig. 6A). Regular slow wave activity was not present in any of the E16.5 preparations.

Figure 6. Electrophysiological recordings from duodenal circular muscle at different ages.

Figure 6

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The boxed regions in A, C, D, F and G are shown on an expanded time scale on the right. A shows the small irregular depolarizations seen in some E16.5 preparations. These were also at recorded at the other ages (see also arrows in D, P0). B is an example from an E 18.5 preparation showing the slow oscillations in membrane potential seen in preparations from each age group. These are seen occurring regularly in E, alternating with short periods of slow wave activity. C, D, F and G are recordings of slow waves at E18.5, P0, P1 and P4, respectively. The expanded traces on the right show the development of a more distinct plateau on the downward phase of the slow wave cycle with age (see arrows).

E18.5

Recordings were performed in 11 preparations of duodenum from eight litters. The mean RMP of circular muscle cells was −51 ± 1 mV, which was significantly more depolarised than at E16.5 (P < 0.001). Regular slow waves were observed in 4/11 preparations (Fig. 6C) but they were not observed until approximately 3–4 h after recordings had commenced. They ranged in frequency from 12 to 17 cycles min−1 (median 14.3, s.d. 2.1) and had amplitudes ranging from 6 to 22 mV (mean 11.8 ± 1.8). Slow waves in the E18.5 duodenum usually did not have a plateau on the downward phase (Fig. 6C). In 9/11 preparations, small irregular depolarisations similar to those seen at E16.5 were observed and the mean amplitude (4.7 ± 0.4 mV) and duration (duration 1.2 ± 0.2 s) of individual peaks was not significantly different from those seen at the younger age (both P= 0.6; data not shown). These depolarisations were often observed earlier in the recording period in the absence of any other membrane potential activity, but would also persist after regular slow waves developed. In addition, in 4/11 preparations (only 1 of which developed slow waves), a much slower membrane potential oscillation was present ranging between 4.1 and 4.7 cycles min−1 (median 4.35, s.d. 0.3; mean amplitude 5.5 ± 0.7 mV; Fig. 6B). This activity was often seen early in the recording period.

P0

Recordings from the duodenal muscle were performed in eight preparations from four litters of P0 mice. The mean RMP of P0 circular muscle cells was −54 ± 0.8 mV. Slow waves were recorded in 8/8 P0 preparations (Fig. 6D and E) and ranged in frequency from 11 to 22 cycles min−1 (median 15.3, s.d. 3.8) and amplitude from 7 to 15 mV (mean 10.1 ± 1.3). Similar to E16.5 and E18.5 preparations, regular slow wave activity was not seen until several hours after recording had commenced. The mean frequencies and amplitudes of slow waves were not different between P0 and E18.5 preparations (both P= 0.6); however P0 slow waves often contained a small plateau on the downward phase (Fig. 6D) which was rarely seen at E18.5 (Fig. 6C). The small irregular depolarisations in membrane potential seen at both E16.5 and E18.5 were observed in every P0 preparation, although not in every cell impaled, and did not differ in mean amplitude (4.8 ± 0.4 mV) or duration (0.9 ± 0.1 s) from those at younger ages (P= 0.9 and P= 0.2 respectively, Fig. 6D). Slow membrane potential oscillations similar to those seen at E18.5 were also observed at P0 (6/8 preparations) and these did not differ in frequency (range 3–7 cycles min−1, median 4.2, s.d. 2.0) or amplitude (6.7 ± 0.3 mV) from E18.5 preparations (P= 0.6 and P= 0.9 respectively). In one preparation, slow membrane oscillations occurred regularly after three to four slow waves (Fig. 6E).

P1

Slow waves were recorded in 3/3 preparations of duodenum from P1 mice (2 litters). The mean RMP of P1 circular muscle cells was −57 ± 3 mV. The slow waves ranged in frequency from 18 to 25 cycles min−1 (median 24.1, s.d. 3.3) and in amplitude from 9 to 22 mV (mean 16.0 ± 1.5, Fig. 6F). A distinct plateau was seen on the downward phase of almost every slow wave cycle at P1 (Fig. 6F). Slow membrane potential oscillations were seen in 2 of 3 preparations and had similar properties to those seen at the younger ages. Small irregular depolarisations were recorded amidst slow wave activity in one P1 preparation.

P4

Slow waves were recorded in all four duodenal preparations at P4 (2 litters). The mean RMP of P4 circular muscle cells was −60 ± 1 mV. The frequency ranged from 28 to 31 cycles min−1 (median 30.2, s.d. 1.0) and the amplitude ranged from 11 to 23 mV (mean 16.2 ± 1.0). The slow waves showed a more exaggerated plateau on the downward phase compared with P1 slow waves (Fig. 6G). Slow membrane potential oscillations were not seen in any P4 preparations. Small depolarisations were seen in 2 of the 4 preparations but were not recorded at every location impaled. The frequency of P4 slow waves was significantly faster than those seen at E18.5 (P < 0.0001) indicating that slow wave frequency increases during development.

These recordings demonstrate that slow waves are first detected at E18.5 in the duodenum, and hence are unlikely to underlie ripples, which are present from E13.5.

Motility in the duodenum of E13.5 and E16.5 W/Wv mice

E13.5 W/Wv mice

Duodenal motility was examined in one litter (n= 7) of E13.5 mice from the mating of a W/+ female with a Wv/+ male. All preparations showed regular, propagating contractions (ripples), including those from W/Wv embryos (n= 2) (Fig. 7A and B).

Figure 7. Spatiotemporal maps of contractile activity in the duodenum of the progeny of matings of W/+ females with Wv/+ males.

Figure 7

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A and B, E13.5 duodenum. Contractions that propagated anally (arrow pointing to the right), orally (arrow pointing to the left) or bidirectionally (double arrow) were observed in all progeny, including those that were later genotyped as being +/+ (A) or W/Wv (B) embryos. C and D, E16.5 duodenum. Contractions that propagated anally, orally or bidirectionally were observed in all progeny, including +/+ (C) and W/Wv (D) embryos.

E16.5 W/Wv mice

The duodenum from two litters of E16.5 mice from matings of W/+ females with Wv/+ males were examined. All preparations showed ripples (propagating contractions), including those from W/Wv embryos (n= 4) (Fig. 7A and B). In all genotypes, the ripples were similar to those observed in the duodenum of E16.5 control (C57/Bl6) mice. The frequency of ripples in the duodenum of W/Wv embryos (1.4 ± 0.6 contractions min−1, n= 4) was not significantly different from that in wild-type (+/+) embryos (1.3 ± 0.4 contractions min−1, n= 5; t test, P= 0.84).

Previous studies have shown that in late embryonic W/Wv mice, there is an absence of KIT+ ICC-MP and slow waves (Beckett et al. 2007). As we have demonstrated that ripples are present in W/Wv mice, our data suggest that ripples are not mediated by ICC-MP.

Morphological development of ICC-MP

The morphological development of ICC-MP was examined using KIT immunostaining in wholemount preparations of duodenum and colon from E14.5, E16.5, E18.5 and P0 wild-type mice. The location of myenteric neurons was determined using antibodies to the pan-neuronal marker, Hu.

In the duodenum and colon of E14.5 mice, KIT staining was observed on the surface of most mesenchymal (Hu-negative) cells at the same focal plane as the myenteric Hu+ neurons, as well as mesenchymal cells on the serosal side of the myenteric plexus (Fig. 8A–C); these cells have been identified previously as precursors of both ICC-MP and longitudinal muscle cells (Torihashi et al. 1997; Kluppel et al. 1998).

Figure 8. Confocal microscope images of KIT and Hu (to localize neurons) immunoreactivity.

Figure 8

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A and B, single optical sections of the E14.5 colon. Many mesenchymal cells surrounding myenteric Hu+ neurons show KIT staining on their cell surface (A), but they do not exhibit the morphological features of ICC-MP. Almost all mesenchymal cells between myenteric ganglia and the serosa show KIT staining on their cell surface (B). The vertical slice at the bottom shows the location of the slices shown in A (level of myenteric plexus) and B (between the myenteric plexus and the serosa). C–F, projected images through the myenteric region of E14.5 (C), E16.5 (D), E18.5 (E) and P0 (F) duodenum. There are some process-bearing KIT+ cells (arrows in D) in the E16.5 duodenum, but KIT+ cells with the morphological characteristics of ICC-MP are not present until E18.5 (arrows in E). ICC-MP in the P0 duodenum (F) form a network and are similar in appearance to ICC-MP in the adult duodenum reported by other studies.

In the duodenum and colon of E16.5 mice, there was a population of cells in a similar focal plane to the myenteric Hu+ neurons that showed strong KIT immunostaining (Fig. 8D). Some of the KIT+ myenteric cells possessed one to two processes, but the cells did not exhibit the characteristic morphology of mature ICC-MP and they did not form a network (Fig. 8D). Process-bearing myenteric KIT+ cells were closely associated with myenteric neurons and were more common in the E16.5 duodenum than colon. Mesenchymal cells of the serosal side of the Hu+ myenteric neurons (presumed longitudinal muscle precursors) now showed only weak KIT immunostaining.

By E18.5, KIT+ cells at the level of the myenteric plexus in the duodenum were similar in morphology to ICC-MP in postnatal mice (Ward et al. 1997) and formed a network characteristic of ICC-MP (Fig. 8E). Myenteric KIT+ cells in the colon were more immature in appearance than in the duodenum. The appearance of the KIT+ ICC-MP in the P0 duodenum was very similar to that in mature mice (Fig. 8F).

These data demonstrate that KIT+ cells with the morphological characteristics of ICC-MP are not observed in the duodenum or colon until E18.5. As ripples are present from E13.5 in the duodenum, the observations are consistent with ripples not being mediated by ICC-MP.

Source of calcium for ‘ripple’ muscle contraction

To identify some of the mechanisms underlying the myogenic contractions (ripples) that are present in mid- and late-embryonic mice, the effects of cobalt chloride and nicardipine on contractile activity in the duodenum and colon of E16.5 and E18.5 mice were examined.

E16.5

Cobalt chloride (1 mm), which blocks extracellular Ca2+ entry through VDCCs and receptor-operated channels, abolished contractions in both the E16.5 duodenum (n= 8) and colon (n= 8) (Fig. 9A, B, E and F). Contractions in the E16.5 duodenum were also abolished by the L-type VDCC antagonist, nicardipine (2.5 μm) (n= 8; Fig. 9C and D). Unlike the duodenum, the effects of nicardipine varied between preparations of E16.5 colon. In 3/7 colon preparations, contractions were abolished by nicardipine. In 4/7 preparations, however, ripples persisted in the presence of nicardipine (Fig. 9G and H). In these preparations, contractions were still observed 45 min after the addition of nicardipine to the organ bath.

Figure 9. Source of calcium for contractile activity in the E16.5 duodenum and E16.5 colon.

Figure 9

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A–D, spatiotemporal maps of E16.5 duodenum. Propagating contractions, which are present under control conditions (A and C), are abolished by cobalt chloride (1 mm) (B) or nicardipine (2.5 μm) (D). E–H, spatiotemporal maps of E16.5 colon. The propagating contractions that are seen under control conditions (E and G), are abolished by cobalt chloride (1 mm) (F), but in some preparations (H) they persist in the presence of nicardipine (2.5 μm).

E18.5

All motility in the E18.5 duodenum, including complexes and the shallow contractions, was abolished by both cobalt chloride and nicardipine. Contractions in the E18.5 colon were also abolished by cobalt chloride (n= 5). Contractions persisted in 8/13 preparations of colon following the addition of nicardipine, but the frequency and percentage diameter change were significantly reduced compared to control conditions (Fig. 10). The effect of another L-type Ca2+ channel blocker, nifedipine (2.5 μm), was also examined in the E18.5 colon. Contractions also persisted following the addition of nifedipine (n= 4/4).

Figure 10. Quantitative data on frequency (A) and proportional change in diameter (B) that occurred in the E18.5 colon following nicardipine (2.5 μm).

Figure 10

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These data come only from preparations (8/11) where ripples persisted following addition of nicardipine. Although nicardipine did not abolish contractions, it significantly reduced both the frequency (A) and change in diameter (B).

These data show that ripples in the duodenum require Ca2+ entry through L-type VDCCs, whereas in the colon, additional channels are also involved.

Discussion

The main conclusion of this study is that prominent propagating contractions are present in the duodenum and colon of fetal mice. We called these propagating contractions ‘ripples’, as this was the term given to TTX-resistant contractions in the proximal colon of adult guinea-pigs (D’Antona et al. 2001). Ripples are not mediated by neurons or ICC-MP, and must therefore be myogenic in origin. In the duodenum, the myogenic contractions appear to become entrained by slow waves after ICC-MP develop around E18.5.

Role of neurons in duodenal and colonic motility during fetal development

Enteric neurons are essential for segmentation, peristalsis and migrating motor complexes in the intestine of mature animals (Furness et al. 1995; Hasler, 1999; Bornstein et al. 2004; Gwynne et al. 2004). Enteric neurons arise from the neural crest. In mice, neural crest-derived cells have colonized the entire small intestine by E11.5 and the large intestine by E14.5 (Kapur et al. 1992; Young et al. 1998). A subpopulation of crest-derived cells starts to express pan-neuronal markers including PGP9.5, Hu and neurofilament-M while they are colonizing the gut (Baetge & Gershon, 1989; Young et al. 1999; Young et al. 2002; Barlow et al. 2008; Hao et al. 2009). Thus, enteric neurons, as defined by the expression of pan neuronal markers, are present in the small intestine from E11.5, the proximal colon from E12.5 and the distal colon from E14.5 (Hao & Young, 2009). Our data showed, however, that ripples were not mediated by neurons as ripples were present in the duodenum and colon of Ret null mutant mice that lack enteric neurons, and were unaffected by TTX. Neurally mediated contraction complexes were not present in the duodenum until E18.5, which is approximately 1 day prior to birth. We have previously shown that neurally mediated colonic migrating motor complexes (CMMCs), a propulsive motility pattern in the mouse colon (Spencer et al. 1998; Spencer et al. 2003; Heredia et al. 2009), are not present until approximately 1 week after birth (Roberts et al. 2007). Thus, there is a considerable delay between the first appearance of neurons and the development of neurally mediated motility patterns in both the duodenum and colon. This delay is presumably due to the time involved for different types of neurons involved in motility circuits to develop and form the appropriate connections with each other and target tissues. Furthermore, like ripples, neurally mediated motility patterns commence in the small intestine before the colon. Interestingly, although neurally mediated motility patterns develop in the duodenum just prior to birth, and are presumably important for the absorption of milk-derived nutrients immediately after birth, neurally mediated motility patterns are not required in the colon until at least 1 week postnatally (Roberts et al. 2007).

In the small intestine of adult mice, neurally mediated propagating contraction complexes do not occur at resting intraluminal pressures, but can be induced by increasing the intraluminal pressure (Huizinga et al. 1998; Abdu et al. 2002; Seerden et al. 2005; Neal et al. 2009). The current study showed that contractile complexes also did not occur in most preparations of duodenum from P10 mice at resting intraluminal pressures, but were induced by an increase in intraluminal pressure. In contrast, contractile complexes were recorded at resting intraluminal pressures in most duodenal preparations from E18.5 and P0 mice. It therefore appears that the threshold of intraluminal pressure required to induce propagating complexes increases during the first postnatal week.

Electrical recordings from the muscle and measurements of contractile activity have shown that distension of the adult mouse small intestine induces bursts of action potentials and contractions separated by periods of quiescence (Huizinga et al. 1998; Abdu et al. 2002). Blockade of nitric oxide synthesis abolishes the periods of quiescence so there is continuous generation of actions potentials (Huizinga et al. 1998). In the duodenum of P10 mice (current study) and adult mice (J. C. Bornstein & M. Ellis, unpublished observation), we have found that contraction complexes cannot be distinguished in the presence of NOLA using spatiotemporal mapping, probably because of increased contractile activity in the quiescent periods. Spatiotemporal mapping experiments in the current study showed that NOLA had different effects on contraction complexes in the duodenum at different ages – NOLA increased the frequency of contraction complexes and induced complexes in preparations without contractile complexes in E18.5 and P0 mice, but contraction complexes could not be distinguished in the presence of NOLA in the duodenum of P10 mice. Thus, the sensitivity of the neuronal circuitry underlying contraction complexes to the effects of blockade of nitric oxide synthesis in the duodenum changes during development. A recent study has also reported changes in the relative contribution of nitric oxide signalling in neurotransmission to the longitudinal muscle of the guinea-pig ileum during postnatal development (Bian et al. 2009).

During zebrafish development, the first motility patterns observed are also unaffected by TTX, but at later stages, motility patterns are dependent on neural activity (Holmberg et al. 2007; Kuhlman & Eisen, 2007). Although infants with Hirschsprung's disease illustrate that neurons are essential for the propulsion of gut contents after birth in humans, it is possible there are also neuron-independent motility patterns in fetal humans that precede the development of neurally mediated motor patterns.

Ripples are not mediated by ICC-MP

Contractions were previously reported in the colon of newborn mice that were blocked by culturing the colon in the presence of KIT function blocking antibodies, suggesting strongly that the contractions are mediated by ICC-MP (Lindley et al. 2008). However, multiple lines of evidence in the current study suggest that ICC-MP do not generate ripples – although ripples were present from E13.5 in the duodenum, slow waves and KIT+ cells with the morphological features of ICC-MP were not detected in the duodenum prior to E18.5. Although cultured ICC progenitors have been reported to show slow wave activity (Lorincz et al. 2008), ripples were present in the duodenum of E16.5 W/Wv embryos, and occurred at a similar frequency to E16.5 wild-type embryos; the small intestine of W/Wv embryos has been previously shown to lack ICC-MP and slow waves (Beckett et al. 2007). The KIT-dependent contractions in the distal colon of newborn mice reported by Lindley et al. (2008) also differ from the ripples reported in the current study in the role of neurons: TTX increases the frequency of contractions in the distal colon, showing that neurons normally inhibit the frequency of contractions (Lindley et al. 2008), whereas in our studies of the duodenum and colon of late embryonic and neonatal mice, ripple properties were unaffected by TTX and were not significantly different in Ret null mutant mice that lack enteric neurons. Thus the contractile activity in the distal colon of newborn mice reported by Lindley et al. (2008) appears to be a different motor activity from the ripples reported in the current study.

Intracellular recordings from duodenal circular muscle revealed slow waves in some preparations of E18.5 mice, which coincided with the development of cells with the morphology of mature ICC-MP. KIT+ ICC-MP and slow waves are also present in the jejunum of E19 mice (Torihashi et al. 1997; Ward et al. 1997). As electrical activity was found to be sensitive to L-type Ca2+ channel blockers in the small intestine of newborn, but not P2 and older, mice, it was suggested that the electrical activity in newborn mice is generated primarily by smooth muscle cells (Liu et al. 1998). However, we recorded slow waves in the duodenum of E18.5 and neonatal mice in the presence of L-type Ca2+ channel blockers, and they are therefore likely to originate from the activity of ICC-MP.

In the duodenum of E18.5, P0 and P10 mice, vertical slice and Fast Fourier Transform analysis of the spatiotemporal maps revealed two motility patterns based on contraction frequency: low frequency contraction complexes that were neurally mediated as they were blocked by TTX, and higher frequency contractions, which had a similar frequency to that of slow waves. For example, in the E18.5 duodenum, the frequency of slow waves determined from intracellular recordings was 15 cycles min−1, and the high frequency contractions in the spatiotemporal maps occurred at a frequency of about 14 cycles min−1. The ripples that were present in the duodenum from E13.5 could no longer be identified in the late embryonic and postnatal duodenum, and slow waves appear to underlie the higher frequency contractions from E18.5 onwards. It is likely that myogenically mediated ripples become entrained by slow waves following the development of ICC-MP in the E18.5 duodenum.

Are ripples propulsive?

During fetal development, gut contents (containing amniotic fluid, epithelial debris, bile) are propelled along the gut (McLain, 1963; Anderson et al. 2004). Ripples are the only motility pattern we detected prior to E18.5 in the duodenum (this study) and prior to P10 in the colon (Roberts et al. 2007), suggesting that ripples are responsible for the propulsion of meconium. The frequency of ripples was similar in meconium-free and meconium-containing regions of colon, which shows that the presence of meconium does not influence ripple frequency. Although we did not detect an anal bias in the direction of propagation of ripples, clumps of meconium in both E18.5 normal and E18.5 aganglionic (from RetTGM/TGM mice) colon moved anally at a rate of around 8–10% of the length of the colon per hour. Further studies are required to determine whether ripples alone, or ripples in combination with other phenomena (for example, pressure from additional contents entering the oral end), are responsible for the propulsion of meconium.

Ripples require entry of extracellular Ca2+ through L-type VDCCs

Ripples in the E16.5 and E18.5 duodenum and colon were abolished by blockade of extracellular Ca2+ entry using cobalt chloride, and were also sensitive to blockade of L-type VDCCs, although neither nicardipine nor nifedipine completely blocked ripples in the colon. Thus, some non-L-type(s) of Ca2+ channels also appear to contribute to ripples in the fetal colon. The role of intracellular Ca2+ stores in ripples was not examined. Further studies are required to identify the non-L-type VDCCs involved in ripples in the colon and the mechanisms underlying the generation of ripples.

Conclusions

Ripples, the first propagating intestinal motility patterns in fetal mice, are not mediated by neurons or ICC-MP. Neurally mediated motility patterns are first observed in the duodenum just prior to birth, but the luminal pressure required to induce these contractile complexes and the role of nitric oxide in regulating the complexes, change during the first postnatal week. Neurally mediated motility patterns do not develop in the colon until around P10, and so the propulsion of gut contents along the colon after birth does not initially require neurons. Hence, different mechanisms controlling intestinal motility develop at different times – myogenic motor patterns develop first, and subsequently neurally and ICC-MP-mediated mechanisms develop and become the dominant motor patterns.

Acknowledgments

This work was funded by ARC Discovery Grant DP0878755 to H.M.Y. and J.C.B., NHMRC Project Grant 454351 to J.C.B., and NHMRC Project Grant 565319 to E.A.B. We thank Miles Epstein for the Hu antiserum, Hideki Enomoto and Jeff Milbrandt for the RetTGM mice, and Aaron Citti for excellent technical assistance.

Glossary

Abbreviations

ICC

interstitial cells of Cajal

ICC-MP

interstitial cells of Cajal at the level of the myenteric plexus

NOLA

nitro-l-arginine

RMP

resting membrane potential

TTX

tetrodotoxin

VDCC

voltage-dependent Ca2+ channel

Author contributions

Conception and design: R.R., E.B., J.B., H.Y.; collection and analysis of data: R.R., M.E., R.G., A.B., M.L., H.Y.; data interpretation: R.R., M.E., R.G., A.B., M.L., E.B., J.B., H.Y.; manuscript writing: R.R., M.E., R.G., E.B., J.B., H.Y.; manuscript revision: M.E., R.G., A.B., M.L. All authors approved the final version of the manuscript. All experiments were performed at the University of Melbourne except the experiments on the Kit (W) mutant mice, which were performed at the University of Adelaide.

Supplemental material

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