A smooth muscle tone-dependent stretch-activated migrating motor pattern in isolated guinea-pig distal colon

doi:10.1113/jphysiol.2003.049163

A smooth muscle tone-dependent stretch-activated migrating motor pattern in isolated guinea-pig distal colon

Terence K. Smith, Gavin R. Oliver, Grant W. Hennig, Deirdre M. O'Shea, Pieter Vanden Berghe, Sok Han Kang and Nick J. Spencer

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

We have investigated the tone dependence of the intrinsic nervous activity generated by localized wall distension in isolated segments of guinea-pig distal colon using mechanical recordings and video imaging of wall movements. A segment of colon was threaded through two partitions, which divided the colon for pharmacological purposes into oral, stimulation and anal regions. An intraluminal balloon was located in the stimulation region between the two partitions (12 mm apart). Maintained colonic distension by an intraluminal balloon or an artificial faecal pellet held at a fixed location generated rhythmic (frequency 0.3 contractions min^-1; duration ~60 s) peristaltic waves of contraction. Video imaging of colonic wall movements or the selective application of pharmacological agents suggested that peristaltic waves originated just oral ( 4 mm) to the pellet and propagated both orally (~11 mm s^-1) and anally (~1 mm s^-1). Also, during a peristaltic wave the colon appears to passively shorten in front of a pellet, as a result of an active contraction of the longitudinal muscle oral to the pellet. Faecal pellet movement only occurred when a rhythmic peristaltic wave was generated. Rhythmic peristaltic waves were abolished in all regions by the smooth muscle relaxants isoproterenol (1 µM), nicardipine (1 µM) or papavarine (10 µM), and by the neural antagonists tetrodotoxin (TTX; 0.6 µM), hexamethonium (100 µM) or atropine (1 µM), when added selectively to the stimulation region. Nicardipine, atropine, TTX, or hexamethonium (100 µM) also blocked the evoked peristaltic waves when selectively added to the oral region. N-nitro-L-arginine (L-NA; 100 µM) added to the anal region reduced the anal relaxation but increased the anal contraction, leading to an increase in the apparent conduction velocity of each peristaltic wave. In conclusion, maintained distension by a fixed artificial pellet generates propulsive, rhythmic peristaltic waves, whose enteric neural activity is critically dependent upon smooth muscle tone. These peristaltic waves usually originate just oral to the pellet, and their apparent conduction velocity is generated by activation of descending inhibitory nerve pathways.

(Resubmitted 16 June 2003; accepted after revision 7 July 2003; first published online 7 July 2003)
Corresponding author T. K. Smith: Department of Physiology and Cell Biology, Anderson Medical Building/352, University of Nevada School of Medicine, Reno, NV 89503, USA. Email: tks{at}physio.unr.edu

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

The mechanisms underlying propulsion of faecal contents along the large intestine are not well understood (Sanders & Smith, 2003). What is clear is that the rate of faecal pellet propulsion along the murine colon can vary and be a staggered rather than a smooth continuous event (Brann & Wood, 1976). When several faecal pellets are present in the isolated guinea-pig distal colon, usually the most distal pellet will break away from the proximal pellets and propagate anally (D'Antona et al. 2001). Under these circumstances, activation of descending inhibitory nerve pathways by proximal faecal pellets may reduce the effectiveness of ascending excitatory reflexes generated by more distal pellets (Costa & Furness, 1976; D'Antona et al. 2001).

If propulsion along the isolated distal colon relies on the continuous activation of ascending excitatory and descending inhibitory nervous pathways by a moving pellet (see Costa & Furness, 1976), it is unclear why a pellet should ever stop and what factors might reinitiate propulsion. In this situation, pellet propulsion may be facilitated or inhibited by any spontaneous contractile or relaxing activity, including propagating spontaneous colonic migrating motor complexes (CMMCs) that can occur in an empty intestine and change their direction of propagation from cycle to cycle (Wood, 1973; Brann & Wood, 1976; Costa & Furness, 1976; Sarna et al. 1984; Bywater et al. 1989; Bush et al. 2000; D'Antona et al. 2001).

Intrinsic and extrinsic inhibitory neural reflexes reduce smooth muscle tone and probably limit pellet propulsion (see Spencer et al. 1999a, 2001a). Smooth muscle tone has been shown to be important for both the initiation and conduction of a peristaltic wave in the small intestine (Spencer et al. 2001b). However, the relationship between colonic smooth muscle tone and motility is unclear, and they are usually treated as unrelated phenomena. McKirdy (1978) found that the smooth muscle relaxant papaverine blocked low-frequency, high-amplitude pressure 'spikes' evoked by distension of short segments of rabbit distal colon. In contrast, CMMCs in the murine colon appear to be tone independent, since their propagation and frequency are unaffected by muscle paralysis with nifedipine (Bywater et al. 1989; Powel et al. 2002). In the human large bowel, both smooth muscle tone and motility increase following feeding, the increase in tone being dependent upon cholinergic nerve activity (see reviews by Camilleri & Ford, 1998; Bassotti et al. 1999). Abnormal changes in smooth muscle tone and motility are indicative of gut pathology. Both muscle tone and motility decrease in some delayed colonic transit disorders, e.g. chronic constipation and megacolon. In contrast, in carcinoid diarrhoea both muscle tone and propagated contractions are increased (see Camilleri & Ford, 1998).

An increase in muscle tone is likely to directly increase the activity of myenteric AH (after-hyperpolarizing/Dogiel type II neurons) sensory neurons (see Kunze & Furness, 1999). Stretch-induced or contraction-induced action potential firing in AH sensory neurons is dependent upon smooth muscle tone and contraction rather than stretch per se, since it is abolished by nicardipine and isoprenaline, which directly relax smooth muscle, despite maintained stretch (Kunze et al. 1998, 1999). Increases in the excitability of AH neurons will probably excite most interneurons and motor neurons of the intestine (see Smith et al. 1992a; Sanders & Smith, 2003). A possible sensory role for tone-dependent AH neurons is underscored by the fact that peristalsis in the small intestine is dependent upon muscle tone (Spencer et al. 2001b). Many myenteric AH neurons in the guinea-pig colon have electrophysiological and morphological characteristics that are similar to those in the small intestine (Wade & Wood, 1988; Smith, 1994; Lomax et al. 1999; Neunlist et al. 1999; Lomax & Furness, 2000; Tamura et al. 2001; Sanders & Smith, 2003). Colonic AH neurons also largely project circumferentially and send processes down into the mucosa (Smith, 1994; Neunlist et al. 1999; Lomax et al. 1999). Therefore, it is possible that activity in colonic AH neurons may also be dependent upon smooth muscle tone, but this has not been tested.

In the somatic nervous system, both stretch-sensitive muscle spindles and tone-sensitive Golgi tendon organs within the same skeletal muscle bundle give complementary information about muscle length and force, respectively. Recent investigations suggest the possibility that two analogous sensory systems may also participate in the regulation of intestinal movements. Recently, we have described a stretch-activated motor pattern in guinea-pig distal colon that is independent of smooth muscle tone and requires only an intact myenteric plexus (Spencer et al. 2002, 2003). This motor pattern consists of an ongoing coordinated discharge of ascending excitatory and descending inhibitory reflex pathways that are resistant to muscle paralysis. Therefore this stretch-activated motor pattern is more likely to be coordinated by mechanosensory interneurons than AH sensory neurons (see Spencer et al. 2002, 2003). So the question we addressed in this study is whether a different neurally mediated motor pattern exists in the isolated guinea-pig distal colon that requires smooth muscle tone for its activation.

We demonstrate that maintained distension by a stationary faecal pellet in isolated guinea-pig distal colon can generate neurally mediated rhythmic peristaltic waves that depend upon muscle tone.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Guinea-pigs of either sex weighing approximately 250-350 g were killed by inhalation of a rising concentration of CO₂; the protocol was approved by the animal ethics committee of the University of Nevada School of Medicine. The abdominal cavity was incised in the longitudinal axis and 16 cm of distal colon taken from 4 cm above the anus was removed. Faecal pellets were expelled from the colon by placing the segment of colon in a beaker of oxygenated Krebs Ringer solution (composition below) at 36 °C for approximately 30 min (see Costa & Furness, 1976).

A 6 cm long segment of distal colon was pinned via the mesentery to the base of a non-partitioned organ bath (volume 100 ml). An artificial Perspex pellet (length 11 mm; maximum diameter 4 mm) with a thread attached was inserted into the oral end of the colon and stopped in the middle of the segment. It was then anchored by the attached thread to the organ bath, or to an isometric transducer via a pulley wheel to measure any propulsive force on the fixed pellet (Fig. 1C).

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Figure 1. Partitioned bath and periodic neural activity generated by a fixed pellet
A, partitioned bath: a segment of distal colon was threaded through two partitions (12 mm apart) which divided the colon into oral, stimulation (where balloon was inflated) and anal segments. Tension transducers were attached to the colonic segment: oral (T_O1 and T_O2 were 10 mm apart; T_O1 was 4 mm above the partition or 10 mm from T_S), over the balloon (T_S, 6 mm from partitions) and below the balloon/pellet (T_A, 10 mm anal of T_S or 4 mm anal of partition). B, rhythmic peristaltic waves were observed when a fixed balloon was inflated to provide maintained distension. C, an artificial pellet was allowed to propel some distance down the bowel; then it was held in position by being connected via a thread to an isometric tension transducer that measured the force on the pellet (T_P). The pellet also generated rhythmic peristaltic waves that appeared to propagate distally. The peristaltic waves exerted considerable propulsive force on the pellet (see top trace, T_P) that lasted for the duration of the wave. The waves were neural in origin since they were blocked by hexamethonium (100 µM) applied to the whole colon.

Partitioned bath

In other experiments a segment of colon (6 cm long) was threaded through greased small holes in a rubber diaphragm located in each of two Perspex partitions. The partitions effectively divided the colon for pharmacological purposes into an oral segment, a middle or stimulation segment, and an anal segment (Fig. 1A; and Smith & McCarron, 1998; Spencer et al. 1999a, 2000, 2001b). The colonic segment was anchored in each chamber by pinning the mesentery to Sylgard (Dow Corning, Midland, MI, USA)-coated blocks in each chamber. Each of the three chambers contained modified Krebs solution (see below) at 37.0 ± 0.5 °C, which was replaced every 20-30 min. The Krebs solution in each chamber was separately gassed (3 % CO₂-97 % O₂) in order to maintain the solution at a pH at 7.3-7.4. A balloon (No. 6 Fogarty Embolectomy Catheter, Baxter Heathcare Corp., CA, USA) was inserted into the oral end of the lumen of the segment and carefully positioned within the colon in the middle (or stimulation) chamber (Fig. 1A). The width (11 mm) of the stimulation (middle) chamber was chosen so that it was the approximate length of a faecal pellet. The No. 6 catheter was chosen because this balloon could be inflated to mimic the oval dimensions (maximum diameter 4-5 mm, length 11 mm) of a faecal pellet. The balloon was inflated with distilled water via a syringe connected to the catheter. After mounting the preparation, it was not perturbed for a 2 h equilibration period prior to either inflating the balloon or inserting an artificial faecal pellet into the oral end of the segment.

Recording mechanical activity of the circular muscle

Three or four stainless steel clips (micro-serrefines; Fine Science Tools, Foster City, CA, USA) were attached to the wall of the colon in a direction that was orthogonal to the segment so as to record the activity of the circular muscle layer. The clips were placed oral ( 4 mm and 14 mm) and anal ( 4 mm) to the partitions, and to the wall in the middle of the colon lying over the balloon or an artificial pellet (Fig. 1A). Suture silk was used to connect each clip to a force transducer (model TST125C; Biopac Systems Inc., Santa Barbara, CA, USA). The tension of the oral clips was set to 5 mN, so as to minimize local reflex stimulation of the bowel. The tension over the balloon, however, was initially adjusted so that it would give a resting tension of 5 mN when inflated. Tension was monitored continuously using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (BIOPAC Systems, Inc., Santa Barbara, CA, USA) (see Spencer et al. 1999a; Bush et al. 2000).

Avoidance of leaks between chambers

Leaks between chambers were avoided by: (1) making only small holes (~1 mm) in each rubber diaphragm of a partition; (2) by inserting grease carefully in and around the holes in the partitions (Dow Corning high vacuum grease); (3) by ensuring that the tubing and end of the embolectomy catheter lay firmly within the holes of the partitions. (4) The fluid in each chamber was kept at different levels throughout the experiment so that leaks between chambers could be readily detected (Smith & McCarron, 1998; Spencer et al. 1999a). (5) Also, following the determination of the effects of a drug added to a specific chamber, the other chambers were emptied in turn so that possible leaks between chambers could be detected.

Video recording of colonic movements and spatio-temporal map analysis

High resolution video imaging of colonic wall movements were monitored in an isolated segment of distal colon containing an anchored or moving artificial faecal pellet (see Fig. 2B and Fig. 4; Hennig et al. 1999; D'Antona et al. 2001). After the mesentery had been dissected away, remaining fragments of mesentery could be tracked to provide a measure of longitudinal displacement (Fig. 4). Changes in diameter of the intact segment of colon were followed over time by converting the colonic images into a silhouette and then plotting the diameter along the colon at each time point (33 ms) as a spatio-temporal diameter or D map (Hennig et al. 1999, 2002; D'Antona et al. 2001).

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Figure 2. Origin of rhythmic peristaltic waves.
A, the distension-evoked rhythmic peristaltic waves usually appeared to start at nearly the same time at the 4 recording sites. The contraction over the balloon (T_S) was often the most prolonged, whereas the contraction at the anal recording site (T_A) was sometimes preceded by a relaxation. The peristaltic wave appeared to propagate rapidly in an oral direction (T_O1 to T_O2; velocity = V_O1-O2); more slowly over the proximal half of the pellet (T_O1 to TS; velocity = V_O1-S), and even slower over the latter half of the pellet (T_O1 to T_A; velocity = V_O1-A). The apparent conduction velocity of the wave was measured at the half-amplitude point of a contraction and usually with reference to the contraction T_O1 just behind the pellet or balloon, since this was the first point to register a contraction and this contraction usually had the maximum rate of rise. B, spatio-temporal diameter or D map showing the development of two rhythmic peristaltic waves (RPWs) originating behind an artificial pellet. Distance is horizontal, time is downwards. The black vertical streak down the middle of the map represents the dilated colon caused by the stationary pellet. Approximately 4 waves of contraction (white diagonal streaks indicated by arrows) were associated with each peristaltic wave. In this example, these contractions originated at different distances oral to the pellet and propagated towards the pellet. Each peristaltic wave produced some anal displacement of the fixed pellet as seen by the horizontal displacement of the black vertical streak caused by the fixed pellet. Note that the minimum (contraction) and maximum (dilatation) diameters of the colon on the spatio-temporal D map were 2.3 mm (white) and 5.5 mm (black), respectively.

Analysis of data and statistical methods

Frequency, duration and amplitude of contractile complexes were measured using Acqknowledge 3.2.6 (BIOPAC Systems, Inc.) and tests for statistical significance were made using Sigma Plot 5.0 (Jandel Scientific, San Rafael, CA, USA). The duration of each contraction was determined from the 50 % or 10 % amplitude points. The interval between contractions was measured between the respective half-amplitude points on the rising phase (see Bush et al. 2000). Propagation velocity was determined by calculating the delay between the 50 % amplitude points of coordinated peaks in both the proximal and distal traces and relating it to the distance between the clips.

Statistical comparisons of data were performed using Student's (paired or unpaired) t tests or ANOVA, and a minimum level of significance was reached at P < 0.05. In the Results section, n refers to the number of animals from which tissue was taken. All data are presented as means ± S.E.M. (standard error of the mean).

Drugs and solutions

The composition of the modified Krebs solution was (mM): NaCl, 120.35; KCl, 5.9; NaHCO₃, 15.5; NaH₂PO₄, 1.2; MgSO₄, 1.2; CaCl₂, 2.5; and glucose, 11.5. The solution was gassed continuously with a mixture containing 3 % CO₂-97 % O₂ (v/v), pH 7.3-7.4.

The following drugs were used throughout the course of these experiments: atropine sulphate, hexamethonium bromide, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), isoproterenol, nicardipine, N-nitro-L-arginine (L-NA), papavarine, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), substance P and tetrodotoxin (TTX) (all from Sigma Chemical Co., St Louis, MO, USA).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Peristaltic waves evoked by a fixed pellet

When an artificial faecal pellet or an intraluminal balloon was held at a fixed location in the lumen of the isolated distal colon, rhythmic peristaltic waves of contraction were observed (Fig. 1B and C). These had a frequency of 0.30 ± 0.05 contractions min^-1 (range 0.06-0.9 contractions min^-1; n = 18) and continued throughout the period of distension (2.2 ± 0.3 h; up to 4 h). Between peristaltic waves, smaller amplitude (29.3 ± 0.3 % of peristaltic waves) but higher frequency contractions (10.7 ± 0.05 contractions min^-1) were recorded. These events had a duration of 3.9 ± 0.6 s (Fig. 1B). Rhythmic peristaltic waves could be graded according to the level of distension. However, these results are not presented in this study since throughout the experiments reported here we used a constant distension level; this was chosen to mimic the average distension (maximum diameter 4-5 mm wide) provided by a natural faecal pellet.

Rhythmic peristaltic waves were neural in origin and dependent upon cholinergic neuro-neuronal transmission since they were blocked by tetrodotoxin (TTX; 0.6 µM; n = 8) or hexamethonium (100 µM; n = 8) respectively, added to the whole segment (Fig. 1B). These waves appeared to be initiated on the oral side of the pellet, since the rate of rise and time to peak of their associated contractions decreased distally (T_O1 to T_A; Fig. 1C and Fig. 2A; Table 1). The rate-of-rise of the contractions on the oral side of the pellet (T_O1 and T_O2) were similar (P > 0.01) (Fig. 2; Table 1). However, the rate of rise of the oral contractions were ~2-3 times more rapid (P < 0.05) than the contraction over the pellet (T_S), and ~3-5 times faster (P < 0.01) than the contraction just below the pellet (T_A) (Table 1). Also, the amplitude of a peristaltic contraction was significantly (P < 0.5) greater over the pellet (T_S), compared to the contraction oral (T_O1 and T_O2) or anal (T_A) to the pellet (Table 1). The amplitude of the anal contraction (T_A) was significantly (P < 0.05) smaller than the contraction over the pellet (T_S) (Fig. 2A; Table 1).

The duration (at 10 % amplitude) of the evoked peristaltic waves increased distally to ~60 s, such that the duration of T_S and T_A was longer than that of the oral contraction (T_O1 or T_O2; P < 0.01). Similarly, the half-duration of the contraction associated with a rhythmic peristaltic wave increased anally (Table 1). The delay ( $Delta$ t; measured at half-amplitude from T_O1; Fig. 2A) in the onset of peristaltic contractions increased distally (i.e. T_O1 to T_S and T_O1 to T_A; Table 1). This delay between contractions appeared to be due to the activation of descending inhibitory nerve pathways, since the contractions over (T_S) and below (T_A) the balloon became increasingly delayed in onset. This apparent delay in the anal contractions was more pronounced at T_A than at T_S; the contraction at T_A sometimes being preceded by an anal relaxation (Fig. 2A). Tension recordings of circular muscle activity at more distal sites suggested that these contractile waves could spread at least 3 cm distal to the pellet.

Conduction velocity of peristaltic waves

Conduction velocities were calculated with reference to the half-amplitude point on the rising phase of the oral contraction (T_O1, 4 mm above oral partition) since usually this had the fastest rising phase and was the first point to register a contraction. Oral to the pellet, peristaltic waves spread rapidly in an oral direction over the distance from T_O1 to T_O2 at an apparent conduction velocity (V_O1-O2) of 11.3 ± 4.9 mm s^-1 (Fig. 2A; Table 1). However, the velocity of the wave on the oral side of the pellet was variable, since in some preparations (20 % from n = 15) the peristaltic wave appeared to spread distally from T_O2 to T_O1 (=10 mm) towards the rear of the pellet at a velocity (V_O2-O1) of 2.1 ± 1.5 mm s^-1 (see Fig. 2B). The wave propagated more slowly over the proximal half of the pellet from T_O1 to T_S, at a velocity (V_O1-S) of 1.2 ± 0.3 mm s^-1. However, the apparent rate of propagation of the peristaltic wave appeared to decrease further over the distal half of the pellet, since the apparent conduction velocity (V_O1-A) over T_O1 to T_A was 0.67 ± 0.03 mm s^-1 (P < 0.05, n = 15).

Tension recordings and video imaging of a fixed pellet suggested that rhythmic peristaltic waves consistently originated just oral of a pellet ( 4 mm), since this site was first to register a contraction. However, the oral cluster of contractions associated with a peristaltic wave could arise at different distances proximal to the pellet (Fig. 2B).

Propulsive force on a pellet

We developed a technique to monitor the net propulsive force on a stationary pellet (Fig. 1C). Using this technique, each rhythmic peristaltic wave was found to exert considerable propulsive force (154.5 ± 6.6 mN; n = 5) on the pellet that lasted for the duration of the contraction (Fig. 1C and Fig. 2C). The peak tension on the pellet was much greater than the level of the maximum circular muscle contraction, suggesting that contraction of the longitudinal muscle was contributing to the force on the pellet (Fig. 1C) (see Smith & Robertson, 1998; Spencer et al. 1999a, 2003). This is also suggested by the fact that if the mesentery was not pinned tightly then the anal clip tended to move orally for 3-4 mm during a peristaltic wave, suggesting a shortening of the longitudinal muscle (see below and Brann & Wood, 1976).

If the string attached to the pellet was severed then the pellet propagated anally as soon as a rhythmic peristaltic contraction occurred, a phenomenon that was most noticeable in preparations where rhythmic peristaltic wave frequency was low.

Video imaging of peristalsis

We examined propulsion along the isolated distal colon using video imaging to track the movements of an artificial pellet down the bowel. In a number of colonic preparations (n = 5 out of 26), it was noted that faecal pellet did not propagate continuously from the oral to the anal end of the colon, but rather showed an intermittent or staggered propagation. A pellet, once introduced into the lumen at the oral end of the colon often took a considerable time to start spontaneously moving down the bowel (15 ± 3 min). Sometimes the pellet needed to be pushed down the lumen of the colon some ~5-10 mm from the oral end before it started spontaneously moving. Figure 3 shows an example of intermittent or staggered propagation. After being introduced into the oral end of the colon the pellet remained stationary for some time before being propelled a short distance (~10 mm) down the colon. It then stopped and remained in position for up to ~2.5 min before again moving a short distance down the colon. Movement of the pellet was associated with a rapid increase in the frequency of spontaneous contractions behind the pellet that propagated in an oral direction (see expanded panel, Fig. 3B).

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Figure 3. Staggered pellet propulsion
A, spatio-temporal diameter (D) map of pellet propulsion down the distal colon. Distance along colon is horizontal; time is downwards. Dark area represents pellet. White dashed lines represent frames of pellet position. B, expanded region of spatio-temporal map, showing orally propagating contractions originating from behind the pellet (white arrows) associated with displacement of the pellet. Note that the minimum and maximum diameters of the colon on the spatio-temporal D map were 2.26 mm (white) and 5.73 mm (black), respectively.

Even when a pellet appeared to move continuously down the colon its trajectory was not smooth but rather discontinuous (see Fig. 4). By video imaging remnants of mesentery along the mesenteric border, the longitudinal movements of the colon could also be followed as a pellet propagated (Fig. 4). It was clear that the mesenteric markers were displaced towards the approaching pellet, suggesting shortening of the longitudinal muscle in front of the pellet (see second trace 5 s, Fig. 4A). However, when these two markers lie over the anal side of the pellet they remain approximately the same distance apart (see white arrow) relative to the initial frame (white brackets), suggesting that the longitudinal muscle below the pellet is not actively shortening. However, the two markers move closer together when the pellet displacement is such that they lie over the oral portion of the pellet. As the pellet passes in front of the markers, which now lie just oral to the pellet, they are closest together (dark arrow), implying active contraction of the longitudinal muscle.

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Figure 4. Continuous pellet propulsion
A, sequence of frames showing the displacement of a pellet along the colon. Dashed white lines track two remnants of closely spaced mesentery. B, spatio-temporal diameter (D) map of pellet propulsion down the distal colon. Distance along colon is horizontal; time is downwards. Dark area represents pellet. Note that the velocity of pellet propulsion varies along the colon (see dark thick line which shows the propagation of the pellet from the oral to the anal end of the colon). The dark, downward wavy lines are due to displacement over time of fragments of mesentery. By following the two closely spaced mesenteric markers in particular the longitudinal movements of the colon can be tracked during the movement of the pellet (see white dashed lines in A and closely spaced wavy lines in B indicated by the white brackets). As the pellet approaches these fragments of mesentery they are displaced orally, suggesting shortening of the segment in front of the pellet. However, when these two markers lie over the anal side of the pellet, the distance between them slightly increases (see white arrow in B) relative to the initial frame (white brackets) suggesting the possibility that the longitudinal muscle is relaxing. The two markers do, however, get closer together when the pellet displacement is such that the two mesenteric markers lie over the oral half of the pellet, suggesting active longitudinal muscle shortening. As the pellet passes in front of the markers, which now lie just oral to the pellet, they are closest together (see dark arrow in B), implying that active contraction of the longitudinal muscle also occurs oral to the pellet. Note that the minimum and maximum diameters of the colon were 1.97 mm (white) and 5.13 mm (black), respectively.

Role of muscle tone

We were interested in whether smooth muscle tone was necessary for generation of neurally mediated rhythmic peristaltic waves in the distal colon. To determine this, several smooth muscle relaxants were applied selectively to the stimulation chamber. Isoproterenol (1 µM; n = 5), a $beta$ -adrenergic agonist, or papaverine (10 µM; n = 5), a cAMP phospodiesterase inhibitor, relaxed the muscle when applied to the stimulation chamber only, but also blocked the contractions associated with rhythmic peristaltic waves in all three chambers (Fig. 5). Lower concentrations of isoprenaline (0.1 µM) decreased the frequency of rhythmic peristaltic waves from 0.40 ± 0.01 to 0.29 ± 0.02 contractions min^-1 (P < 0.05; n = 5) and their amplitude by 34.5 ± 1.5 % of control, but did not significantly relax the muscle. Lower concentrations of papavarine (1 µM; n = 4), however, did not significantly affect the frequency of rhythmic peristaltic waves or reduce muscle tone.

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Figure 5. Effects of smooth muscle relaxants on rhythmic peristaltic waves
A, isoproterenol (1 µM) added to the stimulation chamber (Stim Ch) abolished the evoked rhythmic peristaltic waves in all chambers, and reduced muscle tone in the stimulation chamber. B, similarly, papaverine (10 µM) in the stimulation chamber (Stim Ch) also abolished the rhythmic peristaltic waves and lowered smooth muscle tone in this chamber only.

The muscarinic antagonist atropine has been shown to reduce colonic smooth muscle tone (Spencer et al. 1999a) and hyperpolarize both the longitudinal and circular muscle (Spencer et al. 2003). We tested the effects of atropine when applied to the stimulation chamber. Atropine (1 µM; n = 6) relaxed the muscle in this chamber only and also blocked the rhythmic peristaltic waves occurring in all three chambers (Fig. 6A). Furthermore, nicardipine (1 µM; n = 6), an L-type channel blocker and smooth muscle relaxant that blocks action potential firing and calcium waves in smooth muscle (Hennig et al. 2002; Stevens et al. 1999, 2000; Spencer et al. 2002a), also relaxed the muscle when added to the stimulation chamber and blocked the rhythmic peristaltic waves (Fig. 6B). When the site of distension was paralysed with any of these drugs, slowly increasing the intensity of the distension stimulus to twice the diameter of a pellet did not reinitiate the rhythmic peristaltic waves.

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Figure 6 Effects of muscarinic antagonist and L-type calcium channel blocker on rhythmic peristaltic waves
A, atropine (1 µM) in the stimulation chamber (Stim Ch) abolished the evoked rhythmic peristaltic waves and reduced muscle tone. B, nicardipine (1 µM) in the stimulation chamber (Stim Ch) also abolished the rhythmic peristaltic waves. C, atropine (1 µM) in the oral chamber (Oral Ch) abolished rhythmic peristaltic waves and reduced muscle tone. D, nicardipine (1 µM) in the oral chamber (Oral Ch) also abolished rhythmic peristaltic waves and reduced muscle tone. Note that these drugs only lowered muscle tone in the chambers to which they were applied.

When atropine (1 µM; n = 5) was selectively added to the oral chamber, it relaxed the smooth muscle in this chamber only, but again also blocked the rhythmic peristaltic waves in all three chambers (Fig. 6C). These effects of atropine in the stimulation and oral chambers suggested that cholinergic tone/contraction may also be important for the generation and propagation of the distension-evoked rhythmic peristaltic waves. To further examine this effect, nicardipine (1 µM) or isoprenaline (1 µM) was also added to the oral chamber. It was found that isoprenaline in the oral chamber also blocked the peristaltic waves in all three chambers (n = 3). In five out of six experiments, nicardipine applied to the oral chamber also abolished the rhythmic peristaltic waves in all three chambers; an example is shown in Fig. 6D. However, in one other preparation, nicardipine added to the oral chamber abolished the contractions associated with the rhythmic peristaltic waves in this chamber only, the contractions in the other chambers being only slightly reduced. Addition of hexamethonium (100 µM) to the oral chamber, with the nicardipine still present, blocked the rhythmic peristaltic waves.

Atropine (1 µM) in the anal chamber reduced the peristaltic contractions by ~80 % (control area 204.3 ± 4.3 mN s vs. atropine 42.0 ± 2.2 mN s; n = 3; P < 0.001) in this chamber only. Nicardipine (1 µM; n = 3) or isoprenaline (1 µM; n = 3) in the anal chamber blocked peristaltic contractions in this chamber only, but had no effect on contractions in the other chambers.

Effects of substance P

In preparations where distension-evoked peristaltic waves were either absent or of low frequency, substance P was applied to the stimulation chamber to determine whether increases in smooth muscle tone around the distended balloon could induce peristaltic waves. Substance P (0.1 µM), which can have direct excitatory effects on smooth muscle (see Keef et al. 1992) as well as on myenteric AH neurons (Katayama & North, 1978), transiently increased smooth muscle tone by 18.3 ± 1.2 mN (n = 5). Superimposed on this increase in tone were 8.6 ± 0.4 rhythmic contractions that occurred at a frequency of 0.8 ± 0.04 contractions min^-1 (10 % duration in stimulation chamber = 37.6 ± 0.2 s, n = 5). These contractions, which occurred in all chambers, exhibited similar characteristics to rhythmic peristaltic waves. Both the muscle tone and the amplitude of the rhythmic contractions gradually waned; however, the contractions, which lasted 6.2 ± 0.9 min, endured beyond the return of tension to baseline values.

Origin of peristaltic waves: partitioned chamber experiments

To further investigate the origin of the rhythmic peristaltic waves evoked by localized maintained distension, we selectively blocked nervous transmission in each of the three recording chambers.

Neural antagonists in stimulation chamber. Tetrodotoxin (0.6 µM; TTX; n = 7) or hexamethonium (100 µM; n = 7) applied to the stimulation chamber consistently abolished the contractions associated with the waves in all three chambers (Fig. 7A and B).

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Figure 7. Effect of neural blockade on rhythmic peristaltic waves
A, tetrodotoxin (TTX; 0.6 µM) in the stimulation chamber (Stim Ch) abolished rhythmic peristaltic waves evoked by maintained distension of an intraluminal balloon in all chambers. B, hexamethonium (100 µM) in the stimulation chamber (Stim Ch) also abolished the rhythmic peristaltic waves. C, TTX (0.6 µM) in the oral chamber (Oral Ch) also abolished the rhythmic peristaltic waves in all chambers. D, similarly, hexamethonium (100 µM) in the oral chamber (Oral Ch) usually abolished the rhythmic peristaltic waves in all chambers. Note that although TTX lowered muscle tone in the chamber to which it was applied hexamethonium did not.

Neural antagonists in oral chamber. Most interestingly, TTX (0.6 µM; n = 7) in the oral chamber also consistently abolished the contractions associated with the rhythmic peristaltic waves in all three chambers (Fig. 7C). This suggests that the contractile waves originate oral to the distension site. The effects of hexamethonium, which blocks neuro-neuronal transmission, in the oral chamber were slightly more variable than TTX. Hexamethonium (100 µM) in the oral chamber also abolished the rhythmic peristaltic waves in all three chambers in 7 out of 10 experiments (Fig. 7D). In the other three experiments, however, hexamethonium added to the oral chamber (100 µM) only reduced the size of the contractions associated with the rhythmic peristaltic waves by ~20-30 %. Further addition to the oral chamber (on top of the hexamathonium) of PPADS (100 µM), which blocks the P2X receptors that mediate fast synaptic transmission on enteric neurons (Galligan & Bertrand, 1994; Spencer et al. 2000) abolished the rhythmic peristaltic waves (not shown).

Neural antagonists in anal chamber. The effects of blockade of neurotransmission in the anal chamber were considerably more variable than in the other chambers. TTX in the anal chamber either increased (n = 4) the frequency (as in Fig. 8), decreased (n = 2) the frequency, or had no effect (n = 4) on the frequency of rhythmic peristaltic waves in the other chambers. Overall, in these experiments, there was no consistent effect of TTX in the anal chamber on the amplitude, duration and frequency of rhythmic peristaltic waves (control = 0.28 ± 0.03 contractions min^-1; TTX = 0.33 ± 0.06 contractions min^-1; P > 0.05; n = 6). However, in another three experiments, TTX when added to the anal chamber abolished the rhythmic peristaltic waves in all three chambers. Hexamethonium (100 µM; n = 4) added to the anal chamber reduced the amplitude of rhythmic contractions in this chamber by 33.0 ± 0.6 % but had no effect on the rhythmic contractions in the other chambers.

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Figure 8. Effect of TTX in the anal chamber on rhythmic peristaltic waves
TTX (0.6 µM) in the anal chamber (Anal Ch) did not abolish the evoked rhythmic peristaltic waves, although in this example TTX reduced their frequency. However, the further addition of TTX (0.6 µM) to the oral chamber (Oral Ch) abolished the peristaltic waves.

Origin of peristaltic waves

To test how far oral of the pellet the peristaltic waves originated, the inflated balloon was placed 8 mm anal to the oral partition (the anal partition having been removed). In this situation, when TTX was applied to the oral chamber the rhythmic peristaltic waves continued. However, if the balloon was slowly and carefully pulled orally the waves were blocked when the oral end of the balloon was within 4 mm of the oral partition (n = 5), suggesting that the peristaltic waves were generated within this distance behind the pellet.

Responses to nicotinic agonist

Since the peristaltic waves were abolished by hexamethonium in the oral chamber, we sought to determine whether DMPP (20 µM, n = 5; nicotinic agonist) could activate both descending excitatory and inhibitory nerve pathways. Immediately upon application of DMMP to the oral chamber, a robust contractile response occurred in all three chambers that was similar in waveform to a peristaltic wave. This response consisted of a prolonged contraction (42.2 ± 2.2 s) in the oral chamber and a brief relaxation (5.2 ± 0.6 s) followed by a prolonged contraction (53.0 ± 1.2 s) in the middle and anal chambers.

Role of inhibitory nerves in regulating conduction of the rhythmic peristaltic wave

Nitric oxide-dependent descending inhibitory pathways are important for coordinating colonic peristalsis not only by relaxing the muscle ahead of a bolus but also by delaying the wave of descending excitation (see Smith & Robertson, 1998; Smith & McCarron, 1998). Therefore, we attempted to determine whether the apparent conduction velocity of these rhythmic peristaltic waves is regulated by activation of nitric oxide-dependent descending inhibitory nerve pathways.

L-NA (10 µM; n = 5) added to the anal chamber significantly reduced the anal relaxation during a peristaltic wave and exposed an early contractile response, an example of which is shown in Fig. 9. The effect of L-NA was to effectively increase the apparent conduction velocity (V_01-A) of a peristaltic wave over the distal half of the pellet from 0.56 ± 0.04 mm s^-1 to 0.93 ± 0.08 mm s^-1 (P < 0.05; n = 5). Interestingly, L-NA applied to the anal chamber had no significant effect on the frequency and amplitude of rhythmic peristaltic waves in the other chambers.

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Figure 9. Inhibitory nerves and apparent conduction velocity of peristaltic waves
A, two evoked rhythmic peristaltic waves showing a prominent anal relaxation response (T_A). B, L-NA (10 µM) in the anal chamber (Anal Ch) reduced the relaxation response and increased the contractile response (T_A) in this chamber associated with the peristaltic wave. The apparent conduction velocity V_O1-A was increased following addition of L-NA to the anal chamber (see dotted lines). Note that sometimes the oral contraction T_O1 was preceded by a relaxation (# ascending inhibition).

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

We have investigated whether smooth muscle tone is necessary for the activation of rhythmic peristaltic waves in the guinea-pig distal colon. A major finding of the current study is that distension by a fixed artificial faecal pellet or an intraluminal balloon evokes rhythmically occurring peristaltic waves that are propulsive. The intrinsic neural activity underlying these peristaltic waves is critically dependent upon smooth muscle tone. A major observation was that once initiated, peristaltic waves in the colon did not require the constant movement of a pellet down the colon for their continued propagation to occur. Similarly, we have found that localized stimulation of the empty small intestine can give rise to a propagating peristaltic wave that occurs without the continued distension provided by a moving column of fluid (Spencer et al. 1999b).

Video imaging and pharmacological experiments suggest that the rhythmic peristaltic waves in the colon usually originate just oral ( 4 mm) to a pellet/balloon and propagate both orally and anally. The orally propagating waves appear to be made up of a cluster of phasic contractions.

Rhythmic peristaltic waves

Our evidence that rhythmic peristaltic waves generated by a fixed pellet exhibit similar characteristics to normal peristalsis in the distal colon is supported by the following observations. (1) They are neurally mediated and dependent upon cholinergic neuro-neuronal transmission, as is faecal pellet propulsion along the isolated guinea-pig distal colon (Frigo & Lecchini, 1970; Costa & Furness, 1976; Tonini et al. 2001). (2) These waves appear to migrate anally with an apparent conduction velocity that is similar to faecal pellet propulsion along the guinea-pig distal colon (0.6-1.4 mm s^-1: Crema et al. 1970; Frigo & Lecchini, 1970; Costa & Furness, 1976; Foxx-Orenstein & Grider, 1996; Kadowaki et al. 1996; Smith & Robertson, 1998; D'Antona et al. 2001). (3) On releasing the thread holding a pellet at a fixed location, the pellet was only propelled anally when a rhythmic peristaltic wave occurred. (4) In colonic segments where propulsion was staggered, the pellets only moved when a rhythmic peristaltic wave was generated.

The observation that a fixed, maintained stimulus can evoke these rhythmic peristaltic waves may seem unusual, since enteric neural reflex stimulation of the colon usually evokes a phasic response (ascending contraction and descending relaxation) of the muscle (Crema et al. 1970; Costa & Furness, 1976; Grider & Makhlouf, 1990; Smith & McCarron, 1998; Spencer et al. 1999a; Spencer & Smith, 2001). However, McKirdy (1978) found that maintained fluid distension of small segments (10-20 mm) of rabbit distal colon generated 'high-amplitude, low-frequency' (frequency 0.3-3 min^-1) periodic rises in intraluminal pressure.

Rhythmic peristaltic waves are dependent upon muscle tone

Gregersen & Christensen (2000) succinctly stated that 'tone especially designates the resistance to stretch that characterizes tissue containing muscle'. Although it is well established that activation of enteric excitatory and inhibitory motor neurons can increase and decrease colonic smooth muscle tone, respectively, it is generally unappreciated that muscle tone itself can upregulate activity in the enteric nervous system (see Kunze & Furness, 1999 and Introduction).

The neurally mediated rhythmic peristaltic waves observed in this study were abolished by the smooth muscle relaxants isoproterenol, nicardipine or papaverine, when applied to the stimulation chamber. Therefore, activity in the intrinsic neurons underlying these waves is dependent upon muscle tone. Stretch was not as important as muscle tone in initiating these rhythmic peristaltic waves, since further increases in distension of the balloon in the presence of a muscle relaxant did not reinitiate rhythmic peristaltic waves. Our finding that atropine in the stimulation chamber reduced muscle tone and also blocked peristaltic waves suggests that smooth muscle tone around the pellet was largely being generated by local cholinergic reflexes.

Presumably, these antagonists reduced or abolished spontaneous action potential firing and associated calcium waves in longitudinal and circular muscle layers that are enhanced by stretch-activated cholinergic motor nerve activity (Stevens et al. 1999, 2000; Hennig et al. 2002; Spencer et al. 2002a). However, stretch-activated myogenic mechanisms, which are independent of nerves, may also contribute to smooth muscle tone around the pellet (Bülbring, 1955; Spencer et al. 2002a).

Complex neural circuitry underlying rhythmic peristaltic waves

Anally propagating peristaltic contractions appear to be dependent upon activation of descending excitatory as well as inhibitory nerve pathways that are an important component of peristalsis in both the small intestine (Hirst et al. 1975; Brookes et al. 1999; Spencer et al. 1999b, 2000, 2001b) and colon (Crema et al. 1970; Frigo & Lecchini 1970; Costa & Furness, 1976; Smith & McCarron, 1998; Smith & Robertson, 1998; Spencer et al. 1999a; Stevens et al. 1999). Both the descending excitatory and descending inhibitory nerve pathways in the distal colon were found to be activated in most cases from just behind the pellet, since TTX or hexamethonium (or hexamethonium plus PPADS) consistently blocked the peristaltic waves when applied to the oral chamber. The descending excitatory and descending inhibitory nerve pathways appear to be excited at the same time since the onset of the contraction in the stimulation chamber occurred at the same time as the onset of the anal relaxation. Also, the fact that DMPP could activate a similar wave suggests that both descending excitatory and descending inhibitory nerve pathways are probably activated via nicotinic receptors.

Presumably, muscle tone-dependent AH sensory neurons activated around the pellet can also excite ascending interneurons (see Lomax & Furness, 2000). Ascending interneurons can be 8 mm long in the guinea-pig small intestine (see Brookes et al. 1997) and maybe of a similar length in the large intestine (Spencer et al. 2002b), i.e. half the length of a pellet. Ascending interneurons may in turn activate descending inhibitory nerve pathways (see Spencer et al. 2002b) and perhaps descending excitatory nerve pathways from behind the pellet. Ascending interneurons also activate excitatory motor neurons to produce oral muscle tone/contraction (ascending excitatory reflex; see Grider & Makhlouf, 1990). Therefore, neurons whose activity is muscle tone and contraction dependent (presumably AH sensory neurons; see Introduction) are also likely to be activated oral to the pellet. The observation that on some occasions TTX blocked peristaltic waves when added to the anal chamber suggests the possibility that ascending interneurons can be activated by descending nervous pathways as proposed by Brookes et al. (2001) for the small intestine. Thus this complex motor pattern probably involves the activation of an intricate neural network (see Thomas et al. 1999), which becomes rhythmic when stretched, and has a superimposed neural polarity, leading to net oral excitation and anal inhibition, where descending inhibitory nerve pathways regulate conduction of the wave.

Apparent conduction velocity

A peristaltic wave appears to originate from behind a pellet where it propagates rapidly (11 mm s^-1) in an oral direction but more slowly (1 mm s^-1) in the anal direction. The question is, how can velocities of propagation in these two directions be so different, when it has been shown that nervous conduction through the enteric nervous system is very rapid compared to either of these velocities? Conduction velocities along myenteric axons that give rise to FEPSPs are fast (~400-600 mm s^-1; see Stebbing & Bornstein, 1996) compared to the speed of peristaltic waves. Our results suggest that the different oral and anal propagation velocities of peristaltic waves result from the differences in the extent of activation of underlying inhibitory nerve pathways. We show that descending inhibitory nerve pathways are more strongly activated over the distal half of the pellet, where they produce a slowing of the peristaltic wave. This conclusion is supported by the observation that following antagonism of nitric oxide-mediated relaxation in the anal chamber, a faster apparent conduction velocity for the peristaltic wave was observed. It is possible that ascending inhibitory pathways (Hukuhara & Miyake, 1959; Smith et al. 1992b; Spencer & Smith, 2001), which are much weaker than the descending inhibitory pathways, also direct the fast oral propagation of the peristaltic wave. Therefore, the apparent conduction velocity results largely from a competition between the effects of excitatory and inhibitory transmitter responses on the muscle (see Smith & McCarron, 1998) rather than a slow conduction of neural activity down the bowel.

The control of the spread of a descending peristaltic wave in the colon appears to be somewhat analogous to that in the oesophagus (Yamoto et al. 1992). Blockade of nitric oxide-mediated relaxation in the oesophageal body produces an almost synchronous contraction down the oesophagus.

Relative movements of the longitudinal muscle and circular muscle

It is usually assumed that the longitudinal muscle actively shortens in front of a moving pellet (Brann & Wood, 1976; Wood 1986), and contracts anal to the point of enteric reflex stimulation (Hukhuhara & Miyake, 1959; Grider, 2003). In the current study where we have videoed colonic wall movements, our data suggest that during pellet propulsion the longitudinal muscle actively contracts above and over the oral side of a pellet (Fig. 4). This raises the possibility that much of the observed initial shortening of the segment in front of a moving pellet reported previously by others could be in large part be due to a passive mechanical artifact; active oral contraction of the longitudinal muscle pulling the relaxed segment below over the pellet. We suggest that both muscles are contracting together and relaxing together during a rhythmic peristaltic wave. This conclusion is supported by our previous studies where we have shown that motor neurons to the longitudinal and circular muscle of the distal colon are simultaneously activated by common interneurons, so that during peristalsis (Smith & Robertson, 1998), a migrating motor complex evoked by mucosal stimulation (Smith & McCarron, 1998; Spencer et al. 1999b) and peristaltic reflex activity (Smith & McCarron, 1998; Spencer & Smith, 2001; Spencer et al. 1999b, 2003), both muscles contract together at the same time and relax together at the same time. Our studies support the earlier observations by Bayliss & Starling (1899, 1900), who found that during intestinal propulsion, both muscles contract together on the oral side and relax together on the anal side of a moving bolus.

Physiological significance rhythmic peristaltic waves

As well as the low-frequency (~0.3 contractions min^-1), muscle tone-dependent, rhythmic peristaltic waves reported here, we have also reported a higher frequency (~5-11 contractions min^-1) stretch-dependent, but tone-independent, ongoing reflex activity generated by a stationary pellet (see Spencer et al. 2002b, 2003). This ongoing reflex activity consisted of excitatory junction potentials that were phase locked with inhibitory junction potentials in the circular muscle at the oral and anal ends, respectively, of a stretched region of colon. These two different colonic motor patterns both found in the guinea-pig distal colon may be driven by two different intrinsic sensory neurons (see Smith et al. 1991, 1992a). The rhythmic peristaltic waves may be generated by a sensory neuron that registers muscle tone and contraction, whereas the ongoing reflex activity is driven by another sensory neuron that is tone independent but sensitive to muscle stretch. This is analogous to the two sensory systems in the somatic nervous system where Golgi tendon organs and muscle spindles within the same muscle bundle give complementary information about the muscles force development and length, respectively.

In the guinea-pig distal colon, these two motor patterns may allow a stationary distal pellet to break away from the inhibitory influences generated by more proximal pellets (see Introduction). Also, the staggered nature of pellet propulsion along the distal colon could be due to a pellet requiring sufficient time in a new location to generate the neural activity to trigger a rhythmic peristaltic wave. Any inhibitory nerve activity that reduces smooth muscle tone may then decrease the likelihood of generating a tone-dependent peristaltic wave and thus slow pellet propulsion.

Conclusions

Maintained local distension by a fixed faecal pellet generates long duration low-frequency rhythmic peristaltic waves in the guinea-pig distal colon that are strongly propulsive and are critically dependent upon muscle tone. The apparent distal propagation of these waves appears in large part to be due to a competition between inhibitory and excitatory responses on the muscle, organized by activation of descending inhibitory and excitatory nerve pathways from behind the pellet. This fixed pellet model of peristalsis should provide a useful model for studying the pharmacology of colonic propulsion.

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Acknowledgements

This study was supported by a grant from the National Institute of Health (USA) RO1 DK 45713 to T.K.S. G.W.H is a CJ Martin Research Fellow (007160 NHMRC, Australia). P.V.B is a postdoctoral Fellow of the Fund for Scientific Research, Flanders (Belgium).

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				N. J. Spencer, E. J. Dickson, G. W. Hennig, and T. K. Smith Sensory elements within the circular muscle are essential for mechanotransduction of ongoing peristaltic reflex activity in guinea-pig distal colon J. Physiol., October 15, 2006; 576(2): 519 - 531. [Abstract] [Full Text] [PDF]

				N. J. Spencer, G. W. Hennig, E. Dickson, and T. K. Smith Synchronization of enteric neuronal firing during the murine colonic MMC J. Physiol., May 1, 2005; 564(3): 829 - 847. [Abstract] [Full Text] [PDF]

				N. J. Spencer and T. K. Smith Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon J. Physiol., July 15, 2004; 558(2): 577 - 596. [Abstract] [Full Text] [PDF]