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. 2002 Oct 18;545(Pt 2):629–648. doi: 10.1113/jphysiol.2002.028647

A rhythmic motor pattern activated by circumferential stretch in guinea-pig distal colon

Nick J Spencer 1, Grant W Hennig 1, Terence K Smith 1
PMCID: PMC2290691  PMID: 12456839

Abstract

Simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells, at the oral and anal ends of a segment of guinea-pig distal colon, to investigate the neuronal mechanisms underlying faecal pellet propulsion. When a minimum degree of circumferential stretch was applied to sheet preparations of colon, recordings from CM cells revealed either no ongoing junction potentials, or alternatively, small potentials usually < 5 mV in amplitude. Maintained circumferential stretch applied to these preparations evoked an ongoing discharge of excitatory junction potentials (EJPs) at the oral recording site (range: 1-25 mV), which lasted for up to 6 h. The onset of each large oral EJP was time-locked with the onset of an inhibitory junction potential (IJP) at an anal recording electrode, located 2 cm from the oral recording. Similar results were obtained in isolated intact tube preparations of colon, when recordings were made immediately oral and anal of an artificial faecal pellet. The amplitudes of many large (> 5 mV) oral EJPs were linearly related to the amplitudes of anal IJPs occurring 20 mm apart. In the absence of an L-type Ca2+ channel blocker, action potentials occurred on each large oral EJP. Synchronized discharges of stretch-activated EJPs and IJPs were preserved following pretreatment with capsaicin (10 μm), were unaffected by nifedipine (1 μm) and did not require the mucosa or submucous plexus. EJPs and IJPs were abolished by hexamethonium (300 μm) or tetrodotoxin (1 μm), but persisted in the presence of pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 μm) or an NK3 tachykinin receptor antagonist (Neurokinin A 4-10; 100 nm to 5 μm). In summary, maintained circumferential stretch of the distal colon activates a population of intrinsic mechanosensory neurons that generate repetitive firing of ascending excitatory and descending inhibitory pathways to CM. These mechanosensory neurons, which may be interneurons, are stretch sensitive, rather than muscle tension sensitive, since they are resistant to muscular paralysis. We suggest the synchrony in onset of oral EJPs and anal IJPs over large regions of colon is due to synchronous synaptic activation of ascending and descending interneurons.

It has been known for more than one hundred years that local stimulation of the intestine elicits a polarized neuronal reflex in the neighbouring smooth muscles, consisting of a contraction oral to and relaxation anal to the stimulus (Bayliss & Starling, 1899, 1900). These polarized responses are commonly referred to as the peristaltic reflex and have been demonstrated in the large intestine of many mammalian species (Bayliss & Starling, 1900; Crema et al. 1970; Mackenna & McKirdy, 1972; Costa & Furness, 1976; Grider & Makhlouf, 1990; Smith et al. 1992b; Grider, 1994; Smith & McCarron, 1998; Spencer et al. 1999a; Spencer & Smith, 2001a). However, in the small intestine, a number of studies have found difficulty in demonstrating a pronounced relaxation anal to a stimulus. Most commonly, in the small bowel, local distension or mucosal stimuli evoke a contraction oral and anal to a stimulus, rather than relaxation of the smooth muscle (Alvarez & Starkweather, 1919; Hukuhara et al. 1936; Röden, 1937; Alvarez, 1940; Brookes et al. 1999; Spencer et al. 1999b).

The mechanisms underlying the peristaltic reflex are likely to be different between the small and large intestine. This is probably due to differences in neural projections (Messenger & Furness, 1990; Costa et al. 1996; Lomax & Furness, 2000) and the electrophysiological properties (Wade & Wood, 1988a,b; Bornstein et al. 1994; Messenger et al. 1994; Lomax et al. 1999; Tamura et al. 2001) of myenteric neurons between the small and large intestine. A major difference between the small and large intestine is that the longitudinal muscle in the small intestine has a predominantly excitatory innervation (Spencer et al. 1999b, 2000), whereas in the colon it receives both a strong inhibitory as well as excitatory innervation (Lomax & Furness, 2000; Neunlist et al. 2001; Spencer & Smith 2001a). Also, the differences in the velocity of peristaltic waves in these different regions of the bowel may be related to differences in their neural circuitry. For example, peristalsis in the guinea-pig small intestine, which has a mostly fluid content, is rapid (≈30 mm s−1; Kosterlitz & Robinson, 1957; Tsuji et al. 1992; Hennig et al. 1999; Spencer et al. 1999b, 2001a). In contrast, peristalsis in the guinea-pig proximal colon, which has a viscous fluid content, is approximately 4 mm s−1 (D'Antona et al. 2001), whereas the propagation velocity of faecal pellets in the distal colon is about 1 mm s−1 (Costa & Furness, 1976; Foxx-Orenstein & Grider, 1996; Kadowaki et al. 1996; Smith et al. 2002). However, it is clear that peristalsis in the small and large intestine is critically dependent upon the enteric nervous system, as these coordinated motor patterns are abolished by tetrodotoxin.

Colonic propulsion is complex and several mechanisms may contribute to the propulsion of faecal pellets. Crema et al. (1970) first demonstrated that the peristaltic reflex could be preserved in isolated preparations of guinea-pig and feline distal colon, devoid of extrinsic neural inputs. Costa & Furness (1976) then showed that propagation of a faecal pellet down the guinea-pig distal colon is associated with activation of local reflexes involving ascending excitatory and descending inhibitory neuronal pathways. In addition, spontaneous, neurally mediated, contractile waves (and electrical complexes) sweep down the large bowel in many different species, including the guinea-pig (Wood, 1973; Christensen et al. 1974; Costa & Furness, 1976; Sarna, 1985; Bywater et al. 1989; Smith & McCarron, 1998; Bush et al. 2000; D'Antona et al. 2001; Spencer, 2001). However, it has been demonstrated in the guinea-pig distal colon that these waves, which travel at 0.3 mm s−1, are of insufficient strength to occlude the lumen (D'Antona et al. 2001). More recently, we have shown that the neural activity generated by a faecal pellet is more complex than previously supposed since artificial pellets held at a fixed location within the colon can themselves generate rhythmic contractile complexes (duration ≈50 s; frequency ≈0.3 c min−1) that originate just oral to the pellet and sweep anally at an apparent conduction velocity of ≈1 mm s−1 (Smith et al. 2001, 2002). The generation of these evoked migrating complexes, which exert considerable propulsive force, are dependent upon muscle tone, similar to peristaltic waves in the guinea-pig small intestine (Spencer et al. 2001a).

In this study, we have further investigated the patterns of neural activity that are generated by stretch, similar to that produced by a faecal pellet. We demonstrate that an additional neural mechanism may also contribute to pellet propulsion, since circumferential stretch of the distal colon evokes another repetitively discharging colonic motor pattern. A major finding is that mechanosensory neurons underlying ascending excitation and descending inhibition in the distal colon are resistant to L-type Ca2+ channel blockers, or smooth muscle paralysis. The results are discussed in relation to the idea of convergence of ascending and descending interneurons. A preliminary account of these findings has been published in abstract form (Spencer & Smith, 2001b).

Methods

Guinea-pigs of either sex, weighing 200-350 g were killed by exposure to CO2 in a rising concentration, in accordance with the Animal Ethics Committee of the University of Nevada School of Medicine. The abdominal cavity was opened and the terminal 10 cm of distal colon was removed. After the mesenteric attachment was trimmed away, the lumen was flushed clean with modified Krebs’ solution (composition below).

Protocol for recording from two circular muscle cells simultaneously in an intact tube preparation of colon

An artificial faecal pellet was created by covering a naturally expelled pellet with Silastic (Dow Corning Corp., Midland, MI, USA) silicon elastic and allowing this to dry. This allowed micropins to partially penetrate the pellet and stabilize the smooth muscle of the colon, so that microelectrodes could then be used to record from the CM cells. In an unparalysed tube preparation of distal colon, the pellet was inserted into the oral end of the colon and allowed to propagate anally (see Costa & Furness, 1976; D'Antona et al. 2001; Smith et al. 2001). Once the pellet reached the middle of the segment it was held in position by anchoring a cotton thread that was attached to the back of the pellet. Nifedipine (1-2 μm) was then applied to the colon to paralyse the musculature so that simultaneous recordings could be made from two CM cells immediately oral and anal of the pellet (See Fig. 1A).

Figure 1. Simultaneous recordings at either end of a faecal pellet.

Figure 1

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A, an artificial faecal pellet was held in position in the middle of an isolated segment (≈6 cm in length) of distal colon by a thread, which prevented its expulsion. Simultaneous intracellular recordings were then made from two circular muscle (CM) cells immediately oral and anal of the pellet. Nifedipine (1 μm) was present to paralyse smooth muscle. EJPs (Ba) and IJPs (Bb) were simultaneously recorded from CM cells at the oral and anal ends of the faecal pellet, respectively. Note the similar onset of oral EJPs and anal IJPs (see vertical dashed lines).

Protocol for recording from two circular muscle cells simultaneously in isolated sheet preparations of colon

The distal colon was opened along the mesenteric attachment and the terminal distal region was pinned to the base of a Sylgard (Dow Corning)-lined Petri dish, so that the mucosal surface faced uppermost. The mucosa and submucosa were then dissected from this opened region to expose the underlying CM. The preparations studied were 20-40 mm in length, and when pinned out in a recording chamber the circumferential axis measured 10-12 mm.

In all experiments, these dissected preparations were pinned serosal side down on the base of a recording chamber the bottom of which consisted of a microscope coverslip that was lightly coated with a fine layer of Sylgard silicon. Unambiguous identification of the CM layer was aided by the use of an inverted microscope (Olympus, CK2; Napa, CA, USA). To test the effects of circumferential stretch, the segment of colon (20 mm in length) was pinned under a minimum degree of circumferential stretch, visualized as corrugations between either circumferential edge. Simultaneous recordings were made from two CM cells in unstretched preparations, so that the two recording electrodes were separated by 20 mm in the longitudinal axis, as shown diagrammatically in Fig. 2A. In these experiments, the circumferential cut edges of the colon measured 6-8 mm in width. Once recordings had been made from completely flaccid, unstretched preparations, these same preparations were repinned under maintained maximal circumferential stretch. In these experiments, the circumferential cut edges of the colon measured 10-12 mm in width.

Figure 2. Effect of circumferential stretch localized to the opened segment in the middle of an intact tube preparation.

Figure 2

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A, simultaneous intracellular recordings were made from the circular muscle (CM) at either end of the stretched opened portion of the segment. Ba and Bb, an ongoing discharge of oral EJPs that were often synchronized with anal IJPs was observed. C, an expanded time period represented by the filled bar shown in Ba. D, relationship between oral EJP amplitudes and anal IJP amplitudes. Small junction potentials (< 5 mV) are largely unrelated in amplitude between the two recording sites. Events larger than 5 mV follow a near linear relationship (R2 + 0.33, where y + −0.59x + 0.273). E, relationship between the time difference of oral EJPs and anal IJPs plotted with respect to the amplitudes of events (mainly IJPs) recorded at the anal electrode. Events smaller than 5 mV show large degrees of time difference between the peaks of EJPs and IJPs, whereas events > 5 mV show a greater degree of synchronization in time. Negative Δt (-Δt) values represent the anal IJP occurring slightly prior to the oral EJP, whereas positive Δt (+Δt) values represent the IJPs occurring slightly after the oral EJPs. The positive values on the ordinate represent small depolarizing events occurring at the anal electrode.

In an additional series of experiments, a region of colon was incised in the longitudinal axis (2 cm in length) as described above, but the oral and anal extremities of these preparations were preserved, as shown in Fig. 2A. The opened region, alone, was pinned under maintained circumferential stretch to the base of the Sylgard-lined organ bath (Fig.2A) and in these preparations the mucosa and submucous plexus were dissected off the opened region only, to facilitate microelectrode impalements into the circular muscle cells.

Electrical recording technique

Intracellular microelectrode recordings were made from two CM cells simultaneously, using two independently mounted microelectrodes, the fine positioning of which could be adjusted using two micromanipulators (model M3301L; World Precision Instruments, Sarasota, FL, USA). Electrodes were filled with 1.5 m KCl solution and had resistances of about 120 MΩ. Electrical signals were amplified using a dual input high impedance amplifier (Axoprobe 1A; Axon Instruments Inc., Foster City, CA, USA), using two Axon HS-2 headstages (Gain 0.1L). Output signals from the amplifier were digitized on an A-D converter, and filtering frequencies ranging from 0.66-1.5 KHz were used. Recordings were simultaneously visualized and recorded onto a PC running Axoscope (version 8.0; Axon Instruments) and also onto a digital 4-channel oscilloscope (Gould 1604, Ilford, UK).

Comparison of stretch-activated junction potentials with junction potentials evoked by electrical stimulation

We sought to compare the latencies and waveform of EJPs and IJPs evoked by circumferential stretch with those evoked by electrical nerve stimulation. To do this, we mounted transmural stimulating wires in the middle of a 2-cm-long segment of colon, so that the recording electrodes were equidistant from the oral and anal recording electrodes (1 cm from the oral and anal recording sites). This way a controlled instantaneous stimulus could be delivered to the colon, so that we could be sure that ascending excitatory and descending inhibitory nerve pathways had been activated simultaneously. Transmural electrical stimuli were delivered using single shocks (pulse width: 0.4 ms, 20-50 V), via a Grass S44 stimulator (Grass Instruments, Quincy, MA, USA).

Drugs and solutions

The following drugs were used throughout the current study: capsaicin, hexamethonium, nifedipine, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), tetrodotoxin (TTX) and the NK3 receptor antagonist [Trp7,β-Ala8]-Neurokinin A 4-10. All drugs were obtained from the Sigma Chemical Co. (St Louis, MO, USA) except for [Trp7,β-Ala8]-Neurokinin A 4-10, which was obtained from Peninsula Laboratories (Belmont, CA, USA). Nifedipine was prepared at a stock concentration of 10−2m in ethanol and diluted to a final concentration of 10−6m in Krebs’ solution. Hexamethonium was made up in distilled water at a stock concentration of 10−2m. [Trp7,β-Ala8]-Neurokinin A 4-10 was diluted in distilled water, made up at a stock concentration of 10−2m and used at a final bath concentration of 500 nm to 5 μm. 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 Krebs’ solution was gassed continuously with a mixture containing 3 % CO2-97 % O2 (v/v), pH 7.3-7.4.

Measurements and statistics

Students’ paired t tests or analysis of variance (ANOVA; with Newman-Keuls post hoc tests) were used where appropriate. A minimum significance level of P < 0.05 was used throughout. The use of ‘n’ in the results section refers to the number of animals on which observations were made and data are presented as means ± standard error of the mean (s.e.m.). Measurements of amplitude, half-width and time to peak of junction potentials were made using Axoscope 8.0 (Axon Instruments, Foster City, CA, USA).

Analysis of data

The methods used to analyse and compare recordings have been described previously (Spencer et al. 2001b). Briefly, voltage data were exported as a text file and imported into a custom written program (OpenGL-based) in which the traces were resampled to 250-300 Hz and smoothed (36 ms moving average; 5 iterations). An average baseline (5-10 s) was calculated to follow slow undulations in voltage while ignoring faster events (e.g. junction potentials). Inflexions in the voltage traces were detected and the peaks were subtracted from the average baseline to calculate junction potential amplitudes. Once peaks had been located in both traces, the peak in the second trace which was closest in time to a reference peak in the first trace was identified. Plots were then constructed of the amplitude of the closest peaks (see Fig. 4C) and an R2 value was calculated. Also, the standard deviation for the time difference between the peaks of oral EJPs and anal IJPs was determined and referred to throughout the Results as s.d. (see Table 1). Histograms of the amplitudes of junction potentials (JPs) were constructed with a bin size amplitude of 1 mV. To visualize the relative change in voltage of events (EJPs and IJPs) in both traces, the traces were differentiated (4 ms timesteps) and plotted against each other, such that the rate-of-rise of JPs in the anal electrode was plotted on the x-axis, whilst the rate of rise of JPs in the oral electrode was plotted on the y-axis. If the plot skewed to one axis only, this indicated that changes in that electrode were occuring disproportionately faster than in the other electrode (e.g. Fig. 5Ab). If both events were undergoing the same changes in voltage at the same time, these traces would fall along a linear 45 deg line between the two axes.

Figure 4. The distribution of excitatory and inhibitory junction potentials evoked by stretch at the oral and anal recording electrodes.

Figure 4

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A shows the number of IJPs and EJPs recorded from unstretched preparations. Note, the majority of events are small, typically < 5 mV and no clear polarity is apparent. In contrast, when circumferential stretch was applied to the same preparations shown in A, a clear polarity was apparent in the distribution of junction potentials at the oral and anal recording electrodes (B). EJPs preferentially occurred at the oral electrode and IJPs at the anal electrode. Note, the amplitude and frequency of IJPs and EJPs is also substantially increased following circumferential stretch. The black columns show the potentials recorded from the oral electrode and the open columns show the potentials recorded from the anal electrode. C shows the relationship between the amplitudes of oral EJPs and anal IJPs in stretched preparations. There is a near linear relationship for the junction potentials larger than 5 mV, but events < 5 mV are essentially unsynchronized and do not show a relationship in amplitude (data from 5 animals). D, plot showing the time difference (in milliseconds) between the peaks of spontaneous events occurring at the oral (mainly EJPs) and anal (mainly IJPs) electrodes plotted against the amplitudes of events recorded at the anal recording electrode, in stretched preparations. It can be seen that small amplitude EJPs and IJPs show greater variability in the time difference between peaks than larger amplitude events. Negative Δt (-Δt) values represent the peaks of anal IJPs occurring slightly prior to the peaks of oral EJPs, whereas positive Δt (+Δt) values represent the peaks of anal IJPs occurring slightly after the peaks of oral EJPs. The positive values on the ordinate represent depolarizing events occurring at the anal electrode (data from 9 animals).

Table 1.

Statistical comparison of the effects of PPADS, an NK3 receptor antagonist and nifedipine on stretch-activated EJPs and IJPs

Control PPADS (10μm) P value Control Nifedipine (1 μm) P value Control NK3-blocker (1 μm) P value
Correlation coefficient (R2) 0.47 ± 0.1 0.44 ± 0.09 0.69 0.24 ± 0.83 0.36 ± 0.08 0.41 0.38 ± 0.09 0.41 ± 0.05 0.55
Maximum correlation coefficient (R2max) 0.61 ± 0.1 0.46 ± 0.13 0.12 0.46 ± 0.1 0.66 ± 0.06 0.07 0.64 ± 0.09 0.64 ± 0.06 0.94
s.d. (ms) 82.67 ± 23.07 109.6 + 17.84 0.51 95.0 ± 9.923 92.66 ± 10.24 0.73 96.40 ± 14.64 87.58 ± 6.25 0.41
Δt(ms) −21.75 ± 17.77 −95 ± 42.36 0.07 −53 ± 80.33 −23.75 ± 25.26 0.66 −39.6 ± 24.48 −72.0 ± 39.87 0.15
n 4 4 – 4 4 – 5 5 –
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No significant difference was detected with any of the drugs on the correlation coefficient (R2) of the amplitudes of oral EJPs and anal IJPs, the maximum R2 (R2max) the standard deviation (s.d.) for the time difference between peaks of synchronized EJPs and IJPs, or the Δt (ms) for EJPs and IJPs. The R2max value represents the maximum correlation coefficient of the amplitudes of oral EJPs and anal IJPs obtained throughout any recording, while the Δt values refer to the degree of time shift required (in ms) to provide the greatest synchronization between the rate of rise of EJPs and IJPs. This means that the IJP recordings required a time shift (in the order of 20–95 ms to the left, i.e. –ve values), to be best aligned with the corresponding rate of rise of the EJP recording.

Figure 5. Comparison of the latencies of onset and time courses of EJPs and IJPs evoked by single-shot electrical stimuli versus EJPs and IJPs evoked by circumferential stretch.

Figure 5

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Aa shows a simultaneous recording from two CM cell recordings that have been superimposed following stretch. Note, the peak of the EJP occurred approximately 100 ms prior to the peak of the IJP (see dotted line). Ab, both the oral EJP and anal IJP recordings shown in Aa have been differentiated and plotted against each other. Y-axis values are from the oral recording electrode and represent differentiated EJP recordings, while the x-axis values are from the anal electrode and represent differentiated IJP recordings. During the onset of both oral EJPs and anal IJPs, the rate-of-rise of the EJP was greater than that of the IJP. Ba, graded oral EJPs and anal IJPs were evoked by graded single-pulse stimuli (0.3 ms, 20-40 V). Note, electrically evoked EJPs reached their peak prior to the peak of the IJP. Therefore, stretch-evoked EJPs and IJPs showed the same properties as electrically evoked EJPs and IJPs. Bb, differentiated traces of electrically evoked junction potentials shown in Ba. During the onset of electrically evoked EJPs and IJPs, the rate-of-rise of oral EJPs was also greater than that of anal IJPs. Ac and Bc show the degree of time shift required to observe the greatest degree of synchronization between the rates-of-rise of EJPs and IJPs. In Ac the IJP trace was shifted 199 ms to the left, while in Bc the IJP trace was shifted 191 ms to the left.

Results

Simultaneous recordings from two circular muscle cells in an intact tubular preparation

In an intact tube preparation, simultaneous intracellular recordings were made from two circular muscle cells immediately oral and anal to an artificial faecal pellet, in the presence of nifedipine (Fig. 1A). At the oral recording electrode, we consistently recorded an ongoing discharge of EJPs of irregular amplitude (up to 10 mV; Fig. 1Ba). When recordings were made from the anal recording electrode (Fig. 1A), an ongoing discharge of IJPs was recorded, which also showed a wide variation in amplitude (up to 10 mV; Fig. 1Bb). During simultaneous recordings from two CM cells (8 pairs, n = 4), it was noted that there were periods in which many EJPs orally occurred at the same time as IJPs anally, despite the two recording electrodes being separated by ≈20 mm in the longitudinal axis. An example of this is shown in Fig. 1B.

Effects of localized circumferential stretch in intact preparations

We tested whether circumferential stretch applied to an opened segment of colon (as shown in Fig. 2A) would also evoke oral EJPs and anal IJPs similar to those recorded from intact tube preparations (as shown in Fig. 1A). Unlike the intact tube preparations described above using an artificial faecal pellet (Fig. 1A), in these latter preparations the middle segment of the tube preparation was opened and pinned flat under maintained circumferential stretch (see Fig. 2A). The mucosa and submucous plexus were removed from this opened region to facilitate microelectrode impalements (Fig. 2A) and nifedipine (1 μm) was present in the perfusion solution. In total, 13 pairs of simultaneous recordings were made from two CM cells, in 9 guinea-pigs, using the recording configuration shown in Fig. 2A. In 11 of these 13 pairs of CM cells (n = 9) a prolonged discharge of synchronized oral EJPs and anal IJPs was recorded. A typical example of this is shown in Fig. 2Ba and Bb. Oral EJPs and anal IJPs greater than 5 mV in amplitude were considered to occur synchronously if their peaks occurred within 80 ms of each other (see Fig. 2E). In 2 out of the 13 pairs of CM cells (from 2 out of the 9 animals) oral EJPs and anal IJPs discharged in an unsynchronized fashion. The summary data from these nine animals is presented graphically in figures 2D and E. It is shown that a near-linear relationship is maintained between the amplitudes of large oral EJPs and anal IJPs in these preparations. Also, the time difference between the peaks of oral EJPs and anal IJPs was found to be closely related to the amplitude of the junction potentials, such that larger amplitude IJPs anally were associated with a reduction in time difference between the peaks of anal IJPs and oral EJPs (Fig. 2E).

General observations in stretched isolated sheet preparations

In total, 173 CM cells from 33 guinea-pigs were recorded from in the presence of nifedipine (1-2 μm). In stretched preparations, all CM cell recordings revealed an ongoing discharge of EJPs or IJPs that occurred as frequently as every 0.5 s and ranged in amplitude from 1-30 mV. The mean interval between junction potentials was 5.2 ± 0.4 s, n = 13, when a 4 mV cut-off threshold filter was used. The overall median was 3.8 s and the mode was 2.3 s (n = 13). The range of intervals between preparations was 27.7 ± 7.0 s (max.: 28.6 s; min.: 0.9 s). The mean resting membrane potential was −36.0 ± 0.6 mV (84 cells; n = 30), similar to values recently reported (Spencer et al. 2001b). This represented the number of CM cells in which dislodgement of the electrode was recorded, and an accurate measurement of membrane potential could be ascertained. All junction potentials recorded from CM cells were abolished by tetrodotoxin (1 μm; n = 3), as we have previously reported for this tissue (Smith et al. 1992b; Spencer et al. 2001b).

Effects of circumferential stretch in isolated sheet preparations of colon

We tested whether junction potentials would occur in unstretched isolated sheet preparations and, if so, would these potentials occur when recordings were made adjacent to the oral and anal ends of a uniformly stretched segment of colon (20 mm in length), as shown diagrammatically in Fig. 3Ba. In these preparations, influences from descending and ascending nerve pathways were minimized at the oral and anal ends of the tissue, respectively. When simultaneous recordings were made from two CM cells in unstretched preparations, either no activity was recorded (Fig. 3Ab and Ac), or small junction potentials (< 5 mV) occurred in CM cells. These events occurred asynchronously between the oral and anal recording sites. The mean resting membrane potential of CM cells in unstretched preparations was −39.2 ± 1.1 mV (13 cells; n = 4). However, when circumferential stretch was applied to these same preparations (Fig. 3Ba), a consistent finding was that an ongoing discharge of EJPs was recorded at the oral electrode, occurring at the same time as IJPs at the anal electrode. A typical example of this is shown in Fig.3Bb and Bc. In 55 out of 72 pairs of simultaneous recordings from two CM cells (n = 33), prolonged discharges of synchronized oral EJPs and anal IJPs were recorded. These bursts of EJPs and IJPs were recorded for at least 3-4 h following the application of circumferential stretch. Interestingly, large EJPs and IJPs were consistently recorded within 100 μm of the oral and anal ends of colon, as can be seen in Fig. 3Bb and Bc. In the remaining 17 of 72 pairs of CM cells, EJPs and IJPs occurred largely asynchronously between the oral and anal recording sites, or showed only occasional synchronized events. The mean resting membrane potential of CM cells in stretched preparations was −35.6 ± 1.4 mV (15 cells; n = 4). This value was significantly depolarized compared to unstretched preparations (P + 0.042; Student's unpaired t test). Histograms were constructed showing the summary data from five animals, and the ranges in amplitude of EJPs and IJPs recorded at oral and anal electrodes were plotted (Fig. 4A and B). It can be seen in Fig. 4A, that most events were small (< 6 mV) and did not preferentially occur at either the oral or anal electrode. However, in stretched preparations, a clear polarity was observed, whereby large EJPs were predominantly recorded from the oral recording site, and large IJPs from the anal recording site (Fig. 4B). Many large EJPs recorded at the oral end of the colon were linearly related to the amplitudes of IJPs recorded at the anal end (Fig. 4C). That is, when a large EJP occurred orally, it was usually associated with a large IJP located 20 mm anally. The summary data from five animals are shown in Fig. 4C. It is apparent that the amplitudes of most small events (< 5 mV) recorded at the oral and anal electrodes were largely unrelated. We also compared the variation in time difference between the peaks of small and large oral EJPs and anal IJPs. This is presented in Fig. 4D, in which the cumulative data from nine animals are shown. The amplitudes of IJPs recorded at the anal electrode were plotted with respect to the time difference between the peaks of synchronized oral EJPs and anal IJPs. It can be seen that the larger the amplitude of an anally occurring IJP, the less variation in time difference there was between the peaks of large oral EJPs and anal IJPs (Fig. 4D). Smaller amplitude oral EJPs and anal IJPs, however, showed much greater variation in difference between their peaks (Fig. 4D). Stretch-activated oral EJPs were consistently observed to reach their peak amplitude more rapidly than anal IJPs; this was further investigated, see below. In general, the findings in sheet preparations of colon from which the mucosa and submucous plexus were removed were no different to those in an intact tube preparation. This suggests that the mechanosensory neuron(s) underlying the initiation of EJPs and IJPs is located in the myenteric plexus and does not require the submucous plexuses or mucosa, as shown for the guinea-pig ileum (Smith et al. 1991).

Figure 3. Effects of maintained circumferential stretch in an isolated opened ‘sheet’ preparation of distal colon.

Figure 3

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Schematic illustration of simultaneous recordings made from the CM at either end (within 100 μm of the cut ends) of an unstretched (Aa) and a circumferentially stretched (Ba) preparation. In unstretched preparations, no pronounced junction potentials were observed at either the oral (Ab) or anal (Ac) ends of the tissue. However, when the same preparation was stretched and repinned to twice its resting circumferential slack diameter, an ongoing discharge of oral EJPs (Bb) synchronized with anal IJPs (Bc) was observed. Recordings in Bb and Bc were made 20 min after those shown in Ab and Ac. C, an expanded portion of the recording represented by the filled bar in Bb and Bc. Note, the simultaneous onset of oral EJPs and anal IJPs, and the similarity in amplitudes of oral EJPs and anal IJPs.

Comparison of stretch-activated junction potentials with electrical stimulus-evoked junction potentials

It was noted that stretch-activated EJPs showed consistently different characteristics when compared to IJPs. We sought to investigate whether these differences may be due to differences in neuro-neuronal transmission or neuromuscular transmission. To do this, we tested whether electrical nerve stimuli would also evoke oral EJPs and anal IJPs, with similar differences to those observed in stretch-evoked junction potentials. In four animals, recordings were made from two CM cells simultaneously, using the preparation described in Fig. 3Ba. Transmural wires were positioned in the middle of the preparation and single pulses (0.4 ms, 20-50 V) were delivered to the colon while impalements were made in two CM cells equidistantly (1 cm) oral and anal of the stimulating wires. It was found that electrical stimulus-evoked EJPs also had briefer durations than IJPs, and that the rate-of-rise of EJPs was greater than IJPs (n = 4; Fig. 5Ba). There were no obvious differences between the characteristics and latencies of stretch-evoked EJPs and IJPs compared with electrical stimulus-evoked EJPs and IJPs (cf. Fig. 5Aa with 5Ba). To compare the relative rates-of-rise of EJPs and IJPs evoked by both types of stimuli, we differentiated both the EJPs and IJPs in stretched preparations and in electrically stimulated preparations and plotted these differentiated traces against each other (Fig. 5Ab and Bb). In these differentiated plots it is apparent that under either stimulation condition, during the onset of both the oral EJP and anal IJP, the rate-of-rise of the EJP is greater than the rate-of-rise of the IJP. This is represented by Fig. 5Ab, the rate-of-rise of the EJP is plotted against the rate-of-rise of the IJP. It can be seen that the trajectories follow the y-axis (+CM dV/dt) before deviating toward the x-axis (-CM dV/dt).

Electrophysiological mapping of the excitatory and inhibitory fields in circular muscle

To investigate whether stretch-activated junction potentials would occur simultaneously across the whole length of colon, or whether these events were restricted to the oral and anal regions of colon, three independent microelectrodes were used. This enabled us to impale three CM cells simultaneously across the full length of colon. Each electrode was initially separated by 6 mm, so that one muscle cell was impaled at the far oral end, one at the far anal end, and one in the middle of the preparation, as shown schematically in Fig. 6A. In all animals tested (n = 10), it was found that junction potentials occurred in the middle of the colon at the same time as EJPs orally and IJPs anally (Fig. 6Ca-c). These recordings showed that while EJPs discharged orally and IJPs anally, in the middle of the preparation there was greater degree of variation in terms of the polarity of the junction potentials observed, ranging from discrete EJPs or IJPs, to biphasic potentials consisting of an EJP-IJP complex (see no.3 in Fig.6B). In Fig. 6A, the relative electrode recording positions along the colon are shown. The ranges in amplitude of junction potentials recorded at each site along the colon have been plotted in Fig. 6B. It can be seen that while some large IJPs discharged in the middle of the preparation, many EJPs were also recorded from this same recording site. When impalements were made 3 mm in from the oral and anal cut ends (see nos 2 and 4 in Fig. 6B), it was clear that EJPs and IJPs similar in amplitude to those recorded from the oral and anal cut ends of colon were recorded.

Figure 6. Simultaneous recordings from three circular muscle cells.

Figure 6

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A, diagrammatic representation of the circumfentially stretched preparation (12 mm in length) used for multiple (3 electrode) recordings. Three CM cell recordings were made simultaneously from the oral (CM 1), middle (CM 3) and anal regions (CM 5), as shown. Each electrode (CM1, CM3 and CM5) was separated by 6 mm. Single electrode recordings were then also made at distances of 3 mm in from the oral and anal extremities. B, graphical representation of the amplitudes of EJPs and IJPs recorded at each site. Each vertical line represents the range of junction potential amplitudes obtained from a different animal (total of 5 animals) at each site along the colon. B shows that EJPs were predominantly recorded from CM1 (see group 1). In the middle of the preparation (CM3), there was greater variability in the polarity of junction potentials recorded (see group 3). At the anal end (CM5), predominantly IJPs were recorded (see group 5). Note that some small IJPs occurred at the oral recording site and some EJPs at the anal recording site. Also, at distances of 3 mm from the oral cut end, predominantly large EJPs were still recorded from CM2 (see group 2). Similarly, large IJPs were also recorded at CM4 (see group 4), showing that at least 3 mm of CM is polarized simultaneously at the oral and anal extremities. Ca, Cb and Cc, typical recordings from three CM cells. It can be seen that when EJPs discharge orally and IJPs anally, IJPs occur in the middle of the preparation, suggesting that excitatory and inhibitory motor neurons are activated simultaneously across the entire length of the colon. D shows the single EJP-IJP complex highlighted by the filled bar in Ca. E shows the same three CM cells superimposed.

Effects of sectioning the colon on synchronized ascending excitatory and descending inhibitory nerve pathways

We sought to investigate whether a minimum length of colon was required to preserve the discharge of oral EJPs and anal IJPs evoked by stretch. Therefore, we tested whether sectioning the colon into shorter segments would disrupt synchronized ascending excitatory and descending inhibitory pathways. In control experiments, a 20 mm segment of colon was used (Fig. 7Aa). In all animals tested (n = 5), simultaneous recordings were initially made from two CM cells at the terminal oral and anal ends of the colon (see Fig. 7Aa). Electrodes were impaled within 100 μm of each cut end to minimize synaptic outputs from descending interneurons at the oral electrode, and ascending interneurons at the anal electrode. In all segments of colon 20 mm in length (n = 5), we observed synchronized oral EJPs and anal IJPs (Fig. 7Ba). Once stable discharges of robust EJPs and IJPs were detected in a given preparation, we sectioned the terminal 5 mm of colon from the anal end, so that the colon was reduced to 15 mm in length. Simultaneous recordings were then, again, made from two CM cells at the far oral and anal extremities of these colonic preparations of reduced length. In all animals (n = 5), it was found that when the colon was sectioned to 15 mm and even to 10 mm in length large EJPs still occurred orally that were synchronized in time with large IJPs anally (Fig. 7Ab and Bb). The correlation coefficient of the amplitude of oral EJPs and anal IJPs was reduced in 10 mm segments, but was not statistically significantly different from that for 20 mm segments (Fig. 7C; P > 0.05; one-way ANOVA). As the colon was sectioned to 7 mm or less (Fig. 7Ac and Bc), there was a marked reduction in the correlation coefficient (R2) between junction potentials that occurred synchronously at the oral and anal ends (Fig. 7C). In no preparations were synchronized oral EJPs and anal IJPs ever recorded when the colon was reduced to 3 mm in length (Fig. 7Ad and Bd), and these were rarely recorded at 7 mm length. Also, it is most noteworthy that the amplitudes of junction potentials were dramatically reduced when the colon was progressively reduced in length. The changes in amplitude and coordination are represented by the changes in correlation coefficient (R2) versus the length of the colon (Fig. 7C). The summary data for five animals is presented in Fig. 7C. There was no significant difference in the mean resting membrane potentials of CM cells in 20-mm-long preparations and those in 3-mm-long sectioned preparations (20-mm: 36.7 ± 1.2; 3-mm: 35.8 ± 1.7; 11 cells, n = 4; P + 0.37; Student's unpaired t test).

Figure 7. Effects of segment length on synchronized junction potentials.

Figure 7

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Aa-d, diagrammatic representations of the different sizes of circumferentially stretched preparations used for simultaneous recordings from CM at either end of the tissue. Ba, synchronized oral EJPs and anal IJPs in a 20-mm-long preparation. Bb, coordinated firing of oral EJPs and anal IJPs when the colon was cut in half to 10 mm. Note that the junction potentials were coordinated but of reduced amplitude. Bc, in 7-mm-long segments, coordinated oral EJPs and anal IJPs were rarely observed. Bd, no synchronized activity was detected in 3-mm-long segments, in which junction potentials were usually < 5 mV. All recordings were made 1 h after sectioning the same tissue. C, changes in correlation coefficient of oral EJPs and anal IJPs following sectioning of the distal colon. On the y-axis, the correlation coefficient (R2) is plotted against the length of the colon (in mm). R2 was not significantly (P > 0.05, #) different between 20-, 15- and 10-mm-long preparations. In 7- and 3-mm-long preparations, R2 was significantly (P < 0.05, *) smaller than for 10-mm or longer preparations.

Effects of hexamethonium and PPADS on stretch-activated EJPs and IJPs

Hexamethonium was used to test for involvement of fast nicotinic transmission in stretch-activated junction potentials. Immediately upon infusion into the organ bath, hexamethonium (300 μm) abolished the ongoing IJPs and EJPs, without causing a change in resting membrane potential (Fig. 8A). Prior to the application of hexamethonium, the resting membrane potentials of the CM cells were 35.8 ± 1.5 mV, while in the presence of hexamethonium they were 34.4 ± 1.5 mV (P + 0.43; 13 pairs of CM cells; n = 8). Since purinergic transmission has been shown to play a major role in synaptic transmission of the intestine (Galligan & Bertrand, 1994; Galligan et al. 2000; Bian et al. 2000; Spencer et al. 2000) and colon (LePard et al. 1997), we tested for a role of purinergic transmission underlying stretch-activated EJPs or IJPs. The effect of PPADS was to significantly reduce the amplitude of the fast component of the IJP to 45 % of control amplitudes (control: 16.6 ± 3.2 mV; in PPADS: 7.5 ± 1.5 mV; n = 5; P + 0.03) and this was reversible upon washout (Fig. 8B). However, even after prolonged infusion for periods greater than 1 h, no change in the frequency of stretch-activated EJPs or IJPs was detected (Fig. 8B). The amplitudes of oral EJPs were not significantly modified by PPADS (control: 12.6 ± 1.3 mV; in PPADS: 12.8 ± 1.9 mV; n = 5; P + 0.98). Neither the standard deviation for the time difference between peaks of EJPs and IJPs, nor the correlation coefficient of the amplitudes of synchronized EJPs and IJPs were statistically modified by PPADS (see Table 1). There was also no change in the resting membrane potentials following addition of PPADS (control: −38.5 ± 1.9 mV to PPADS: −38.0 ± 1.8 mV; P + 0.48; 7 pairs of CM cells; n = 5).

Figure 8. Effects of hexamethonium and PPADS on stretch-activated junction potentials in circular muscle.

Figure 8

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Aa and b show a simultaneous recording from two CM cells at either end of an opened sheet preparation (see Fig. 3Ba). Hexamethonium (300 μm; see grey bar) immediately abolished all synchronized activity (oral EJPs with anal IJPs). Ba and b, application of PPADS (10 μm; see grey bar) reduced the amplitude of the fast IJP, but had no detectable effect on the latency of onset or firing frequency of oral EJPs or anal IJPs. PPADS had no detectable effect on EJP amplitudes, as shown in panel Ba. Upon washout of PPADS, the fast IJP recovered. The figure shows a 3 h simultaneous recording. Arrow 1, an EJP-IJP complex recorded in control solution and on an expanded time scale (see Ca and b). Arrow 2, an EJP-IJP complex in the presence of PPADS (expanded time scale in Da and b) where the fast IJP is blocked. Arrow 3, recovery of the fast IJP during washout (expanded time scale in Ea and b).

Simultaneous recordings from unparalysed stretched preparations: effects of nifedipine

In guinea-pig small intestine, muscle paralysis with an L-type Ca2+ channel blocker abolishes stretch-evoked firing in mechanosensory neurons of the myenteric plexus (Kunze et al. 1998, 1999). Since the ascending EJPs and descending IJPs we recorded in the CM were activated by stretch, we were particularly interested in whether these nerve pathways would be affected by nifedipine. In the absence of nifedipine, seven pairs of simultaneous recordings were made from two CM cells (n = 4) in a sheet preparation that was pinned under maintained circumferential stretch (as shown in Fig. 3Ba). These tissues were free to contract spontaneously at 36 °C, in the absence of any pharmacological agents. In all seven pairs of recordings, EJPs (with or without superimposed action potentials) were recorded at the oral recording site; these were synchronized in time with the onset of IJPs at the anal recording electrode, as described above. A typical example of a stretch-activated discharge of oral EJPs (with action potentials) and anal IJPs is shown in Fig. 9Aa and Ba. No difference was found between the junction potentials recorded in the presence or absence of nifedipine (1-2 μm) (Fig. 9A; Table 1). When the preparations were observed visually, it was found that each EJP that was accompanied by an action potential generated a robust contraction of the CM at the oral end of the colon. The mean amplitude and half-duration of action potentials in CM cells were 30.9 ± 2.3 mV and 36.7 ± 8.7 ms (7 cells; n = 3), respectively. The resting membrane potential of unparalysed CM cells was −37 ± 2.6 mV (5 cells, n = 2). In all seven pairs of CM cell recordings, action potential firing was essentially restricted to the oral recording electrode. Occasional spikes were recorded at the anal electrode in four of the seven pairs of CM cell recordings. We applied nifedipine (1 μm) to these four spontaneously contracting preparations. It was found that the action potentials were always abolished, but none of the other characteristics of EJPs or IJPs were affected by muscle paralysis. It is noteworthy that nifedipine did not affect the characteristics of the EJPs. This suggests that the inward current generating the EJP does not involve L-type Ca2+ channels. We compared the correlation coefficients (R2) of the peak amplitudes of synchronized oral EJPs and anal IJPs, and the variations in time difference between peaks of synchronized EJPs and IJPs (see Table 1). None of these parameters were significantly modified by nifedipine. This strongly suggests that a population of mechanosensory neurons in the distal colon is insensitive to muscle paralysis.

Figure 9. Effects of muscle paralysis on stretch-activated junction potentials.

Figure 9

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Simultaneous recording of synchronized oral EJPs (Aa) and action potentials, with anal IJPs (Ab) recorded 20 mm away. Ba and b, simultaneous recording from two CM cells after the application of nifedipine to the same animal. Nifedipine abolished action potentials on oral EJPs, but did not affect the synchronized activity.

Effects of an NK3 antagonist on stretch-activated ascending excitatory and descending inhibitory neuronal pathways

It has recently been shown that intrinsic mechanosensory (afterhyperpolarizing, AH) neurons in the guinea-pig small intestine receive and transmit predominantly via slow excitatory postsynaptic potentials (slow EPSPs) (Kunze et al. 1993). These potentials are reduced or abolished by NK3 receptor antagonists (Alex et al. 2001; Neunlist et al. 1999). In light of this, we were particularly interested in whether NK3-mediated transmission was involved in the generation of stretch-activated EJPs and IJPs in the distal colon. During simultaneous recordings from two CM cells, we found no significant effect of the selective NK3 receptor antagonist, neurokinin A 4-10 (100 nm to 5 μm), on the amplitude of oral EJPs (control: 8.1 ± 1.0 mV; in NK3 antagonist: 7.5 ± 1.7 mV; n = 4; P + 0.62), or anal IJPs (control: 9.6 ± 3.0 mV; in NK3 antagonist: 8.2 ± 2.1 mV; n = 4; P + 0.28). In control solution, oral EJPs had a median amplitude and mode of 7 mV, respectively, while in an NK3 antagonist, EJPs had a median amplitude of 7 mV and mode of 5 mV. In control solution, IJPs had a median amplitude of 9 mV and mode of 7 mV, while in an NK3 antagonist these values were (mode: 4 mV; median: 4 mV). Also, there was no overall significant effect of the antagonist on either the correlation coefficient of the amplitudes of EJPs and IJPs, or the standard deviation of synchronized junction potentials, even after prolonged application of the antagonist for up to 2 h (Fig. 10 and Table 1). Also, there was no change in the resting membrane potentials of the CM cells after prolonged exposure to Neurokinin A 4-10 (control: 32 ± 1.6 mV; Neurokinin A: 32 ± 1.5 mV; P + 1.0; n = 4).

Figure 10. Effects of an NK3 receptor antagonist on stretch-activated EJPs and IJPs recorded in the presence of nifedipine.

Figure 10

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Aa and Ba, simultaneous recording from two CM cells reveals synchronized EJPs orally, with IJPs anally (recording electrodes 20 mm apart). Expanded control traces represented by the black bar are shown in Ab and Bb. Ca and Da show that in the presence of an NK3 receptor antagonist (1 μm) there was no change in the characteristics of the synchronized oral EJPs and anal IJPs, as shown in the expanded traces Cb and Db, as shown in the expanded traces Cb and Db, which are represented by the black bar.

Effects of capsaicin

We attempted to determine whether capsaicin-sensitive extrinsic nerves mediated this colonic motor pattern, as has been suggested by Grider & Jin (1994) for the stretch activated peristaltic reflex in rat colon. We therefore exposed the preparation to capsaicin for 10 min, which is twice as long as necessary for capsaicin to desensitize extrinsic primary afferent nerves in the distal colon (Weber et al. 2001). Immediately upon infusion of capsaicin (10 μm; n = 4), the coordination between oral EJPs with anal IJPs was disrupted (Fig. 11). However, following washout (≈15 mins) of the drug the motor pattern was restored to its control level (Fig. 11).

Figure 11. Effects of capsaicin on stretch-activated EJPs and IJPs.

Figure 11

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Aa and b, synchronized oral EJPs and anal IJPs were recorded from CM cells at either end of a stretched preparation (20-mm-long). Capsaicin (10 μm) immediately disrupted this coordinated motor pattern (not shown). Ba and b, following washout of capsaicin (10 μm), which was applied for 10 min, the synchronized activity was restored.

Discussion

We have identified a colonic motor pattern that is activated by maintained circumferential stretch of the distal colon. This motor pattern consists of an ongoing discharge of polarized neuronal pathways that generate oral EJPs and anal IJPs in the CM. These nerve pathways discharge for many hours in response to stretch and occur regardless of whether the smooth muscle layers are paralysed by an L-type Ca2+ channel blocker. This suggests that the underlying mechanosensory neurons are activated independently of smooth muscle tone or contraction and do not undergo desensitization during prolonged stimulation.

Effects of muscular paralysis on stretch-activated ascending excitatory and descending inhibitory nerve pathways

In previous studies on the guinea-pig small intestine, it has been shown that IJPs can be evoked in the CM anal to (Hirst & McKirdy, 1974; Hirst et al. 1975; Smith et al. 1988, 1991) and EJPs oral to (Smith et al. 1988, 1991) a local stimulus. That is, polarized neuronal reflexes can be evoked by stimulation, in the presence or absence of L-type Ca2+ channel blockers. However, ongoing discharges of polarized nerve pathways have not been reported previously in either the small or large intestine. Our results suggest that the population of mechanosensory neurons in the distal colon, which underlie repetitively firing ascending EJPs and descending IJPs, are stretch-dependent rather than muscle tone-dependent.

Synchronous firing of ascending and descending interneurons

When recordings were made from CM cells within 100 μm of the cut ends of stretched sheets of colon (Fig. 2Ba), synchronized large oral EJPs and anal IJPs (> 20 mV) were consistently recorded. At the oral and anal cut ends of the colon, excitatory and inhibitory motor neurons can only be excited preferentially by ascending interneurons or descending interneurons, respectively, or by circumferentially projecting neurons. Our findings show that this colonic motor pattern was not due to selective removal of descending and ascending nerve pathways, at the cut ends of the preparation, as proposed by Waterman et al. (1994) for peristalsis in the small intestine. This is because in our experiments, the colonic motor pattern we have identified is still preserved in the middle of a long preparation (see Fig. 2A and B, and Fig. 6C). Therefore, the polarity of this motor pattern is caused by the projections of ascending and descending neurons activated within the stretched region. In light of these results then, it is difficult to envisage that only one class of interneuron would stimulate inhibitory motor neurons located anally at the same time as excitatory motor neurons located orally. Our findings suggest that at least one class of ascending interneuron and one class of descending interneuron were being synaptically activated at the same time to cause these synchronized oral EJPs and anal IJPs. The synaptic outputs of ascending interneurons would stimulate many excitatory motor neurons located orally (to cause an EJP) while at the same time descending interneurons would stimulate many inhibitory motor neurons located anally (to cause an IJP).

Relationship between oral EJPs and anal IJPs

The frequency histogram of the junction potentials revealed that the majority of events in both stretched and unstretched preparations were small (< 5 mV), and largely uncoordinated in time between the oral and anal regions. In contrast, the amplitudes of the larger oral EJPs and anal IJPs at either cut end of the colon were linearly related (Fig. 4C). That is, the larger EJPs and IJPs not only occurred synchronously at either end of the colon, but also showed similar changes in amplitude, despite the recording electrodes being separated by 10-20 mm. This coordination suggests that activity in many ascending and descending interneurons becomes synchronized, to cause the simultaneous recruitment of many excitatory motor neurons orally and inhibitory motor neurons anally. Presumably the smaller junction potentials are produced by motor neurons activated by many unsynchronized interneurons, or by a smaller population of synchronized interneurons. This results in local junction potentials that may occur independently between the oral and anal recording sites.

In addition, when electrical stimuli were delivered to the centre of the colon, the time courses of the evoked oral EJPs and anal IJPs were similar to those activated by stretch. The electrically and stretch-evoked EJPs were briefer than IJPs, and reached their peak amplitude approximately 100 ms prior to the IJPs. This means that, in both instances, ascending excitatory and descending inhibitory pathways were activated simultaneously. Therefore, the time difference between the peaks of the oral EJPs and anal IJPs probably reflects the more rapid activation of the inward membrane current underlying the cholinergic EJP than the outward K+ current underlying the IJP, rather than differences in timing between activation of ascending and descending nerve pathways.

What is the minimum length of preparation required for synchronized firing of ascending excitatory and descending inhibitory pathways ?

A major observation from this study was that a minimum length of colon (between 7-10 mm) was required to preserve and record the colonic motor pattern consisting of synchronized discharges of oral EJPs and anal IJPs. Oral EJPs and anal IJPs, which were coordinated in time in stretched preparations of 10-20 mm in length, were markedly attenuated when the colon was sectioned into segments of 7 mm or less. The fact that preparations < 7 mm long showed poor synchronization between oral EJPs and anal IJPs is consistent with a role for interneuron integrity being essential for the coordination of these neural pathways. In support of this, morphological studies have shown that the majority of ascending interneurons at least in the guinea-pig intestine are 5-8 mm in length (Brookes et al. 1997). Interestingly, most AH neurons, at least in the guinea-pig ileum and distal colon, have been shown to preferentially project circumferentially (Bornstein et al. 1991; Brookes et al. 1995; Lomax et al. 1999). If AH neurons were involved in the generation of stretch-activated EJPs and IJPs, then it is unclear why they could not still generate such activity in relatively short segments of distal colon (7 mm in length), in which circumferential integrity had been preserved. Longitudinal sections would not disrupt monosynaptic circumferential pathways, from AH cells to motor neurons, in preparations 7 mm in length. It seems possible that enteric interneurons themselves can coordinate the rapid synchronization of oral EJPs and anal IJPs without involvement from AH cells.

Electrophysiological mapping of the extent of the excitatory and inhibitory fields in circular muscle

In stretched preparations, we were able to map the extent of the excitatory and inhibitory fields in the circular muscle that were active during ascending excitation and descending inhibition. Using three recording electrodes, it was revealed that mixed junction potentials (i.e pure IJPs or EJPs, or biphasic EJPs and IJPs) occurred in the middle region of a stretched segment of distal colon (Fig. 6B3), whilst at the same time large EJPs discharged orally and IJPs anally (Fig. 6Ca-c). These electrical recordings showed that inhibitory and excitatory motor neurons must be activated simultaneously over the entire 20 mm segment of colon. It was found that the EJP occurred with similar amplitudes in two CM cells, for at least the first 3 mm from the oral end (cf. nos 1 and 2 in Fig. 6B). Similarly, at the anal end of the colon, each IJP was found to simultaneously polarize at least the first 3 mm of CM (cf. nos 4 and 5 in Fig. 6B). These electrophysiological recordings are consistent with the projection lengths of excitatory and inhibitory motor neurons to the circular muscle in the proximal colon (Neulinst et al. 2001) and distal colon (P. Dickenson, P. Vanden Berghe and T. K. Smith, unpublished observations) of guinea-pig. These morphological studies show that motor neurons to the CM project at a maximal distance of ≈3 mm along the length of colon.

Is slow synaptic transmission via AH neurons involved in the generation of ascending excitatory and descending inhibitory pathways ?

Myenteric AH neurons in the guinea-pig small intestine have been shown to transmit to other neurons predominantly via slow EPSPs (Kunze et al. 1993), which typically last for about 20 s and are usually abolished by NK3 receptor antagonists (Alex et al. 2001). Interestingly, however, in the same preparation, slow EPSPs in some classes of myenteric S neurons are resistant to NK3 antagonism (Thornton & Bornstein, 2002). In the guinea-pig colon, however, slow EPSPs in myenteric AH neurons are significantly reduced by NK3 receptor antagonists (Neunlist et al. 1999). Neural network models of peristalsis in the small intestine depend upon slow EPSPs being evoked in first order interneurons by intrinsic sensory neurons (Thomas et al. 1999). In these models, higher order interneurons transmit to other interneurons and motor neurons via fast excitatory postsynaptic potentials (fEPSPs) that lead to a wave of excitation in the muscle. In our study, however, oral EJPs and anal IJPs were ongoing and occurred as rapidly as every 0.5 s, which is obviously much briefer than the time course of a slow EPSP. In light of the short intervals between the onset of junction potentials, it is difficult to envisage how periodic activation of slow EPSPs by intrinsic sensory neurons (which often rise to threshold over seconds), could activate CM motor neurons as frequently as every 0.5 s. Although hexamethonium abolished junction potentials in the guinea-pig distal colon, an NK3 blocker was without effect on any of the characteristics of the ongoing EJPs or IJPs. This suggests that although fast nicotinic transmission is critical for the generation of the ongoing motor activity, slow synaptic transmission via NK3 receptors is unlikely to be of great importance in these stretch-activated nerve pathways. In addition, the ongoing polarized neural activity was unaffected by muscle paralysis. If the findings of Kunze et al. (1998, 1999) in the small intestine apply to the distal colon, then this suggests that AH sensory neurons, the activity of which is dependent upon muscle tone and contraction, rather than stretch per se, are unlikely to be mediating this activity. These conclusions are substantiated by simultaneous recordings from myenteric neurons and circular muscle cells in guinea-pig distal colon (Spencer & Smith, 2001b). We found that although many myenteric S neurons exhibit brief (≈50 ms) discharges of fast EPSPs that occur immediately prior to the onset of EJPs and IJPs, myenteric AH neurons were found to be electrically silent (Spencer & Smith, 2001b).

Other mechanosensory neurons

In our studies this stretch-activated motor pattern persisted after capsaicin pretreatment, suggesting that this ongoing activity is mediated by intrinsic sensory neurons rather than by activation of extrinsic primary afferent terminals (axon reflexes). Capsaicin, which desensitizes small diameter C-fibres, has no direct effects on myenteric neurons (Takaki & Nakayama, 1989). In addition, studies using capsaicin have suggested that extrinsic afferents do not participate directly in peristalsis in guinea-pig ileum (Bartho & Holzer, 1995).

Previous studies have suggested that different sensory neurons may mediate stretch reflexes and mucosal reflexes in the guinea-pig small intestine (Smith et al. 1991, 1992a) and rat large intestine (Grider & Jin, 1994). From their experiments, Grider & Jin (1994) proposed that the mucosal reflex and stretch reflex were mediated by intrinsic and extrinsic sensory neurons respectively. Our conclusion that intrinsic neurons mediate the stretch-activated motor pattern in the guinea-pig distal colon is supported by the work of Furness et al. (1995) in the guinea-pig small intestine. They demonstrated that both stretch and mucosal reflexes were preserved following extrinsic denervation, suggesting that both reflexes were mediated by intrinsic sensory neurons.

Our studies clearly demonstrate that the myenteric neurons involved in the colonic motor pattern we have identified here have mechanosensory properties that are vastly different from AH neurons in the small intestine, which have been proposed to be the intrinsic primary afferent neuron (IPAN; see Kunze et al. 1999, 2000). We suggest that the intestine must exhibit an additional population of intrinsic sensory neurons, which have mechanosensory transduction mechanisms that are dependent upon stretch, rather than muscle shortening, since their activity is resistant to nifedipine. If myenteric AH neurons in the distal colon are also inactivated by muscle paralysis, as they are in the small intestine (Kunze et al. 1998), this raises the question as to what is the mechanosensory neuron generating large EJPs and IJPs in CM? Our most likely explanation for these findings is that myenteric interneurons are mechanosensory and are activated by stretch. In support of this, Kunze et al. (1998) found that in two out of nine orally-projecting uniaxonal neurons in the guinea-pig ileum, spontaneous action potentials persisted when membrane hyperpolarization was imposed on these neuronal cell bodies, suggesting that these action potentials were generated by proximal process potentials away from the cell body, perhaps in sensory endings. It has been shown that ascending interneurons in the small intestine (Brookes et al. 1997; Smith et al. 1999) are highly excitable; and indeed, Brookes et al. (1999) have proposed that bursting activity in ascending interneurons may underlie the initiation of peristalsis, at least in the small intestine. However, ascending interneurons in the distal colon have somewhat different properties than those in the small intestine. They have filamentous processes, receive prominent fast synaptic inputs, are rapidly adapting with AH neuron-like characteristics (intermediate afterhyperpolarization) (Lomax et al. 1999) and fire spontaneously in stretched preparations (N. J. Spencer & T. K. Smith, unpublished observations). It is possible that these particular ascending neurons may be stretch sensitive, rather than tone sensitive. This could explain why junction potentials in the distal colon occur in paralysed stretched preparations.

Functional role of synchronized EJPs and IJPs

How repetitively firing EJPs and IJPs in stretched colonic segments are involved in motility is unclear. We speculate that the role of this ongoing motor activity may underlie a slow anally directed propulsion of faecal pellets. The synchronous onset of the fast IJP anally could limit the spread of action potential conduction initiated by the oral EJP, in the same way that inhibition appears to limit the spread of calcium waves (Stevens et al. 1999). This may lead to localized excitation above and inhibition below a single pellet or several pellets, which would produce a net aboral movement of contents. Although not commented on in their study it appears from their spatio-temporal maps of faecal pellet propulsion in the same preparation, that a slow conduction (velocity ≈0.04-0.10 mm s−1) of faecal pellets can occur (see Fig. 5 in D'Antona et al. 2001).

The results of our study suggest that the stretch-dependent ongoing motor activity is a ‘hard wired’ neural circuit, which does not require inputs from either the mucosa or submucous plexus. Also, this particular motor activity is independent of muscle tone. In contrast, we have recently described another motor pattern activated by a stationary pellet in the distal colon, which consists of rhythmic peristaltic waves that resemble in wave form spontaneous migrating complexes (see Introduction); these are critically dependent upon muscle tone (Smith et al. 2001, 2002) and an intact mucosa (our unpublished observations). This latter motor pattern, which lasts ≈60 s and occurs about every 3 min, appears to underlie rapid propulsion (peristalsis) of faecal pellets.

Conclusions

In summary, maintained circumferential stretch of the distal colon activates a colonic motor pattern that consists of an ongoing discharge of synchronized ascending excitatory and descending inhibitory neuronal pathways to the CM. In contrast to what is known about AH neurons in the small bowel, the mechanosensory neurons underlying these neural pathways are resistant to L-type Ca2+ channel blockers and muscle paralysis. Therefore the intestine appears to have two intrinsic sensory systems that can detect changes in smooth muscle length as well as tone. This is analogous to the somatic nervous system, in which muscle spindles and Golgi tendon organs within the same muscle provide complementary information about the mechanical state of the muscle, its length and its degree of tension, respectively.

Acknowledgments

Financial support for this project was provided by the National Institutes of Health of the USA (grant no. NIDDK RO1 DK45713).

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