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The Journal of Physiology logoLink to The Journal of Physiology
. 2005 Feb 24;564(Pt 3):829–847. doi: 10.1113/jphysiol.2005.083600

Synchronization of enteric neuronal firing during the murine colonic MMC

Nick J Spencer 1, Grant W Hennig 1, Eamonn Dickson 1, Terence K Smith 1
PMCID: PMC1464464  PMID: 15731189

Abstract

DiI (1,1′didodecyl-3,3,3′,3′-tetramethylindocarbecyanine perchlorate) retrograde labelling and intracellular electrophysiological techniques were used to investigate the mechanisms underlying the generation of spontaneously occurring colonic migrating myoelectric complexes (colonic MMCs) in mice. In isolated, intact, whole colonic preparations, simultaneous intracellular electrical recordings were made from pairs of circular muscle (CM) cells during colonic MMC activity in the presence of nifedipine (1–2 μm). During the intervals between colonic MMCs, spontaneous inhibitory junction potentials (IJPs) were always present. The amplitudes of spontaneous IJPs were highly variable (range 1–20 mV) and occurred asynchronously in the two CM cells, when separated by 1 mm in the longitudinal axis. Colonic MMCs occurred every 151 ± 7 s in the CM and consisted of a repetitive discharge of cholinergic rapid oscillations in membrane potential (range: 1–20 mV) that were superimposed on a slow membrane depolarization (mean amplitude: 9.6 ± 0.5 mV; half-duration: 25.9 ± 0.7 s). During the rising (depolarizing) phase of each colonic MMC, cholinergic rapid oscillations occurred simultaneously in both CM cells, even when the two electrodes were separated by up to 15 mm along the longitudinal axis of the colon. Smaller amplitude oscillations (< 5 mV) showed poor temporal correlation between two CM cells, even at short electrode separation distances (i.e. < 1 mm in the longitudinal axis). When the two electrodes were separated by 20 mm, all cholinergic rapid oscillations and IJPs in the CM (regardless of amplitude) were rarely, if ever, coordinated in time during the colonic MMC. Cholinergic rapid oscillations were blocked by atropine (1 μm) or tetrodotoxin (1 μm). Slow waves were never recorded from any CM cells. DiI labelling showed that the maximum projection length of CM motor neurones and interneurones along the bowel was 2.8 mm and 13 mm, respectively. When recordings were made adjacent to either oral or anal cut ends of the colon, the inhibitory or excitatory phases of the colonic MMC were absent, respectively. In summary, during the colonic MMC, cholinergic rapid oscillations of similar amplitudes occur simultaneously in two CM cells separated by large distances (up to 15 mm). As this distance was found to be far greater than the projection length of any single CM motor neurone, we suggest that the generation of each discrete cholinergic rapid oscillation represents a discreet cholinergic excitatory junction potential (EJP) that involves the synaptic activation of many cholinergic motor neurones simultaneously, by synchronous firing in many myenteric interneurones. Our data also suggest that ascending excitatory and descending inhibitory nerve pathways interact and reinforce each other.

Colonic migrating myoelectric complexes (colonic MMCs) are periodic electrical or contractile complexes that propagate over large lengths of colon and which probably aid in the movement of colonic content. The colonic MMC was first identified more than 20 years ago in conscious dogs and had been studied solely using in vivo extracellular recording techniques (Schuurkes & Tukker, 1980; Fioramonti & Bueno, 1983; Sarna, 1985). It is now clear that there are some major differences between the colonic MMC compared with the classic MMC recorded from the small bowel and stomach of non-ruminents. For example, the colonic MMC occurs during both the fasted and fed state, whereas the MMC in the small bowel only occurs in the fasted state of non-raminants (Sarna, 1985). Early studies of the colonic MMC had been restricted to larger mammals, such as the dog or pig (Fioramonti & Bueno, 1983; Sarna, 1985). However, more recently, it has become clear that owing to their small size, the whole colon can be removed from mice and the colonic MMC preserved and recorded from in vitro preparations (Wood, 1973; Brann & Wood, 1976; Fida et al. 1997; Mule et al. 1999; Bush et al. 2000; Brierley et al. 2001; Powell & Bywater, 2001; Spencer & Bywater, 2002; reviewed in Spencer, 2001). In these studies, it has also been found that colonic MMCs are abolished by tetrodotoxin or hexamethonium, confirming the original suggestions of Wood (1973), that the enteric nervous system is critical for colonic MMC generation.

The electrical correlates of colonic MMCs in mice have been determined using intracellular microelectrode recordings from circular muscle (CM) cells in isolated full length preparations of colon (Bywater et al. 1989; Spencer et al. 1998a, b, c). The colonic MMC consists of a brief increased burst of inhibitory junction potentials (IJPs) followed by a slow depolarization. Superimposed on the rising and plateau phase of the slow depolarization is a discharge of cholinergic rapid oscillations in membrane potential that result from the release of acetylcholine from intrinsic motor neurones, since they are blocked by muscarinic antagonists (Spencer et al. 1998b; Spencer, 2001). Calcium entry during action potential firing upon these fast oscillations is responsible for the strong phasic contractions of the CM that provides for the bulk of the contraction associated with the colonic MMC (Bywater et al. 1989; Spencer et al. 1998b, c).

In contrast to the cholinergic rapid oscillations, the underlying slow depolarization during the MMC results from non-cholinergic mechanisms. The slow depolarization underlying each colonic MMC results largely from a ‘turning off’ of an ongoing neurally mediated tonic inhibition of the CM. This disinhibition in the mouse colon involves presynaptic inhibition of the release of all inhibitory neurotransmitter(s) simultaneously that normally maintains the CM in a hyperpolarized state between MMCs. That is, the apamin-sensitive and -resistant fast IJP and the slow nitrergic IJP are all inhibited simultaneously (Spencer et al. 1998a; Spencer, 2001). These more recent observations in mouse colon support the earlier proposal by Christensen et al. (1978) that migrating spike bursts in the isolated cat colon are generated by a process of disinhibition of the muscle.

The mechanisms that generate colonic MMCs are not fully understood (see Spencer, 2001 for review). It remains unclear what mechanisms generate the repetitive discharge of cholinergic rapid oscillations that occur in the CM during the rising and plateau phase of the colonic MMC (see Bywater et al. 1989; Lyster et al. 1995; Spencer, 2001). It is unknown whether these rapid cholinergic oscillations are due to postsynaptic myogenic slow wave mechanisms similar to myenteric potential oscillations (MPOs) observed in the dog colon (see Smith et al. 1987). In this case they would have to be induced by acetylcholine released from continuous activity in motor nerves and have mechanisms similar to acetylcholine-induced slow waves in the small intestine of guinea-pigs (Bolton, 1971). Alternatively, these rapid cholinergic oscillations in membrane potential could be discreet excitatory junction potentials (EJPs) due to repetitive firing of cholinergic motor neurones.

The major aim of this study was to determine the mechanisms that generate the repetitive cholinergic rapid oscillations in CM membrane potential that occur during each murine colonic MMC (see Bywater et al. 1989; Spencer, 2001). A major finding of this study suggests that these repetitive cholinergic oscillations in the CM are in fact a repetitive discharge of discrete cholinergic EJPs, generated by the repetitive activation of many CM motor neurones, rather than due to postsynaptic mechanisms in the CM itself in response to a continuous neural release of ACh. Furthermore, during the colonic MMC the rapid cholinergic oscillations (EJPs) are coordinated over relatively large lengths (∼15 mm) of bowel suggesting that the firing of interneurones are synchronized during the MMC. This synchrony of interneurone firing is important in generating the colonic MMC and help explain why in earlier studies it had been demonstrated that a finite length of bowel is necessary for generating the MMC (i.e. ∼20 mm in cat colon, see Christensen et al. 1978).

Methods

Preparation of tissues

C57BL/6 mice (20–90 days old) of either sex were humanely killed by inhalation of anaesthetic (Nembutal) and cervical dislocation, in accordance with the animal ethics committee of the University of Nevada School of Medicine. The whole colon (without the caecum) was removed and placed immediately into a Sylgard (Dow Corning)-lined Petri dish containing oxygenated Krebs' solution (see composition below) at room temperature. A midline incision (up to 25 mm in length) was made from the anus towards the proximal colon along the mesenteric attachment. The proximal colon was left as an intact tube (i.e. no dissections were performed). The opened end of the colonic segment was pinned with the serosal side uppermost, mucosal side down, to a Sylgard-lined organ bath (∼10 ml capacity). Microelectrode impalements were made into CM cells from the serosal side of the opened portion of the distal segment.

Protocol for simultaneous recordings from circular muscle cells

Simultaneous intracellular recordings were made from pairs of CM cells using two independently mounted micromanipulators (model M3301R; WPI Inc., Sarasota, FL, USA). The position of the microelectrodes could be readily adjusted to record from CM cells located up 20 mm from each other. Microelectrodes (i.d. 0.5 mm) were filled with 1.5 m KCl solution and had tip resistances of about 100 MΩ. Electrical signals were amplified using a dual input Axoprobe 1A amplifier (Axon Instruments) and digitized at 1 kHz on a PC running Axoscope software (version 8.0; Axon Instruments). Experiments were performed at 35–36°C and always in the presence of nifedipine (1–2 μm). Microelectrode impalements were made into CM cells located at the mid and distal regions of colon, between 1 and 2 cm from the anal sphincter.

DiI-retrograde labelling of circular muscle motor neurones

Whole preparations of mouse colon were pinned flat (mucosa uppermost) in a Sylgard-lined Petri dish. The mucosa and submucosa were dissected away. A glass bead (diameter: 212–300 μm; Sigma, G-1277), coated in DiI (1,1′didodecyl-3,3,3′,3′-tetramethylindocarbecyanine perchlorate; Molecular Probes, D-838) dissolved in methanol was then applied to the CM muscle at the antimesenteric border. For the retrograde tracing of motor neurones innervating the circular muscle the bead was applied to the circular muscle after removal of the submucosa (see Vogalis et al. 2000; Brookes, 2001). For the retrograde tracing of interneurones the bead was applied to an exposed myenteric ganglia following removal of strips of longitudinal muscle (see Brookes, 2001). The tissue was washed several times with sterile Krebs' solution and was incubated (37.0°C, 5.0% CO2) for 72 h in culture medium (F-12 HAM nutrient mixture with l-glutamine (Gibco, 12396-016); 5.0% fetal bovine serum, 2.0% antibiotic/antimycotic solution and 5 × 10−3% nifedipine). The culture medium was changed every 24 h. After incubation the tissue was fixed for 1.5 h at room temperature in 0.1 m phosphate buffered saline (17.418 g K2HPO4; 13.6 KH2PO4; 9.0 g, NaCl per litre of 18.0 Ω H2O; pH 7.2) containing 4.0% paraformaldehyde. Following several washes in the 0.1 m phosphate buffered saline, the longitudinal muscle was removed to expose the myenteric plexus. Preparations were viewed under an Olympus BX50W1 upright microscope fitted with epifluoresence using a water immersion lens. The position of the DiI-labelled neurones with respect to the application site was measured with a vernier gauge (Peacock) with precision movements of 2.5 μm. The x-axis was parallel to the longitudinal muscle. The y-axis was parallel to the circular muscle. The application site was arbitrarily assigned the coordinates (0,0). DiI-labelled neurones were defined as descending or ascending if their cell body was located orally or anally to the DiI application site, respectively.

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. Atropine sulphate, tetrodotoxin, hexamethonium sulphate and nifedipine were obtained from Sigma Chemical Co.

Analysis of data

The methods to analyse and compare membrane potential recordings from smooth muscle have recently been described (Spencer et al. 2002). Briefly, membrane potential recordings from two CM cells were exported as text files and imported into a custom-written program (OpenGL-based) whereby the recordings 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 and an R2 value was calculated. To visualize the relative change in voltage of events (EJPs and IJPs) in both traces, the traces were differentiated (4 ms time step)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 changes in that electrode were occurring disproportionately faster than in the other electrode. If both events were undergoing the same changes in voltages at the same time, these traces would fall along a linear 45 deg line between the two axes. Measurements of amplitude, duration and interval between colonic MMCs were made using Axoscope (Version 8.0, Axon Instruments). The n value in the Results section refers to the number of animals on which experiments were performed. In some animals, more than one impalement (recording) was made. In these cases, the number of cells recorded from is also stated, in addition to the n number.

Results

General observations

When microelectrode impalements were made into all CM cells from the mid to distal region of mouse colon, the resting membrane potentials were found to be highly unstable and showed an ongoing discharge of spontaneous junction potentials (44 cells, n = 15). Spontaneous IJPs were most commonly recorded and were observed in every CM cell from every preparation from each mouse (n = 15). Spontaneous IJPs ranged in amplitude from < 1 mV in amplitude (i.e. within the recording noise) up to 20 mV. The mean resting membrane potential of CM cells, taken between the troughs of spontaneous IJPs was −45.7 ± 1.2 mV (44 cells, n = 15).

In addition to spontaneous IJPs, colonic MMCs were also recorded every 151 ± 6.8 s (range: 95.9–230.6 s; n = 15). Colonic MMCs consisted of a slow membrane depolarization with a superimposed discharge of rapid positive-going oscillations in membrane potential (Fig. 1), which were abolished by atropine (1 μm; n = 3) or tetrodotoxin (1 μm; n = 3). Because they are dependent upon release of acetylcholine from cholinergic motor nerves they are referred to as cholinergic rapid oscillations (see Bywater et al. 1989). The mean interval between each cholinergic rapid oscillation was 0.52 ± 0.07 s (range: 0.36–0.77 s; n = 15): mean frequency, 1.9 Hz (range: 1.29 to 2.7 Hz; n = 15), where the mean amplitude of each rapid oscillation (recorded during the rising and plateau phase of each colonic MMC) was 10.6 ± 0.42 mV (range: 0.4 to 21.5 mV; n = 15). The mean amplitude of the slow depolarization underlying each colonic MMC was 9.6 ± 0.5 mV (range: 2.0 to 22.1 mV; n = 15), where the mean half-duration of each slow depolarization was 25.9 ± 0.7 s (measured at half-maximal amplitude between the rising and recovery phases). It was noted that immediately preceding the depolarizing phase of many colonic MMCs, a period of membrane hyperpolarization occurred, as previously described (see Bywater et al. 1989; Lyster et al. 1995; Spencer et al. 1998a). This period of inhibition has been suggested to represent descending inhibition prior to the colonic MMC contraction (see Spencer et al. 1998a), and measured 8.5 ± 0.66 mV (range: 2.0 to 25.6 mV; n = 15) in amplitude.

Figure 1. Synchronization of cholinergic rapid oscillations during the colonic MMC.

Figure 1

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A, schematic of colon preparation. Simultaneous intracellular electrical recordings were made from pairs of circular muscle (CM) cells during the colonic MMC. The entire colon was left intact, and a single longitudinal incision was made into the distal colon along the mesenteric attachment (∼25 mm in length). The longitudinal muscle was pinned uppermost (i.e mucosa down). Simultaneous recordings were made from two CM cells (through the LM layer). B, a simultaneous recording from two CM cells separated by 1 mm in the longitudinal axis. During the intervals between colonic MMCs, spontaneous IJPs occurred asynchronously between the two electrodes. During the rising phase of each colonic MMC, cholinergic rapid oscillations of similar amplitudes occurred at the same time at both electrodes. C, the bar a, shown in panel B is shown on expanded time scale. Individual cholinergic rapid oscillations of similar amplitudes can be seen to occur simultaneously over 1 mm of CM. The bar labelled b in panel B, is shown on expanded time scale in D. Note, during the intervals between colonic MMCs, spontaneous IJPs occur asynchronously. No EJPs are seen during this period.

Simultaneous recordings from two circular muscle cells in the longitudinal axis during the colonic MMC

It has been shown that in the guinea-pig colon, circular muscle motor neurones have short projections to the CM layer, that are typically < 4 mm in the longitudinal axis (Neunlist & Schemann, 1997). Similar results have been found in the guinea-pig small intestine (Brookes, 2001) and mouse colon (see DiI projections below). Acetylcholine is a major excitatory neurotransmitter released from circular muscle motor neurones (Furness & Costa, 1987; Steele et al. 1991; Brookes, 2001). Therefore, in light of this knowledge, we were particularly interested in determining how far along the colon would cholinergic rapid oscillations in the CM remain temporally synchronized during the colonic MMC. To answer this, two independent recording microelectrodes were used to record from two CM cells that were initially separated by 1 mm in the longitudinal axis, then progressively the electrode separation distance was increased until cholinergic rapid oscillations in the two CM cells became temporally uncoordinated. The degree of temporal synchrony between these cholinergic rapid oscillations was tested during colonic MMCs at electrode separations of 1, 7, 15 and 20 mm.

When the two electrodes were separated by 1 mm in the longitudinal axis, it was found that during the intervals between colonic MMCs, spontaneous IJPs showed poor temporal coordination, regardless of their amplitudes (Fig. 1D and Supplemental material). However, when colonic MMCs spontaneously propagated along the colon, it was found that a discharge of cholinergic rapid oscillations occurred simultaneously in both CM cells (Fig. 1B and C and Supplemental material). This discharge of rapid oscillations occurred during the rising and plateau phase of the MMC and ceased during the repolarizing phase. Supplemental material shows a real time recording of a single colonic MMC complex recorded from two CM cells at an electrode separation distance of 1 mm. The cholinergic rapid oscillations and slow depolarization component of a single colonic MMC can be seen. The correlation coefficient of junction potentials recorded during the intervals between colonic MMCs was compared with the correlation coefficient of cholinergic rapid oscillations during the rising phase of the colonic MMC and is shown in Fig. 2E. It can be seen that during the intervals between colonic MMCs, the correlation coefficient is low when the two CM cells were separated by 1 mm (where R values lie between 0.013 and 0.081). However, during each colonic MMC, R values range between 0.83 and 0.91. Figure 2 also shows summary data of the absolute changes in amplitude of individual cholinergic rapid oscillations occurring during five colonic MMC cycles (Fig. 2C). It can be seen that the largest amplitude rapid oscillations were consistently recorded during the rising phase of each colonic MMC (Fig. 2C). Moreover, it was found that the rate of rise of individual cholinergic rapid oscillations occurring simultaneously in both CM cells were highly correlated, since when their rates of rise were plotted against each other, the plots lay along a 45 deg line (Fig. 3Ab). The degree of temporal synchrony between discrete cholinergic rapid oscillations in two CM cells was related to the amplitude of individual events. For example, it was consistently found that the larger amplitude cholinergic rapid oscillations (i.e. > 5 mV) showed the greatest degree of temporal synchrony between the two CM cells. The smaller amplitude events (< 5 mV) were always poorly correlated in time, even over an electrode separation of 1 mm. This is shown in Fig. 3Aa, where most of the cholinergic rapid oscillations recorded were < 5 mV, and showed poor temporal correlation between the two CM cells. However, consistently, the larger amplitude events (5–20 mV events) showed the greatest temporal synchrony between the two CM cells, where the peaks of discrete cholinergic rapid oscillations were separated by at most up to 80 ms. This is shown in the cumulative summary data in Fig. 3Aa (n = 8). The slow membrane depolarization phase underlying each colonic MMC was also well correlated in time between the two CM cells (at 1 mm separation), where each depolarization occurred simultaneously Fig. 1B.

Figure 2. Characteristics of cholinergic rapid oscillations that occur in the CM layer during the colonic MMC.

Figure 2

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A shows a schematic representation of the preparation used for simultaneous recording from the two CM cells, when the recording electrodes were separated by 1 mm in the longitudinal axis. B shows a simultaneous recording from two CM cells during the colonic MMC. C shows a histogram of the changes in absolute amplitude of individual cholinergic rapid oscillations occurring during the different phases of the colonic MMC cycle shown in panel B. It can be seen that individual cholinergic rapid oscillations greater than 5 mV are rare or absent during the intervals between colonic MMCs. The largest cholinergic rapid oscillations occurred during the rising phase of each colonic MMC and steadily decrease in amplitude during the plateau and repolarization phase. The histogram is plotted in real time with respect to the recordings in A. D shows the time differences between the peaks of individual cholinergic rapid oscillations and IJPs occurring during the different phases of the colonic MMC cycle. During the intervals between colonic MMCs, the time difference between asynchronously occurring IJPs is usually > 200 ms. However, during the rising phase of colonic MMCs, where cholinergic rapid oscillations occur synchronously, the time difference between their peaks is < 50 ms (see each *). E shows the relative changes in correlation coefficient of all cholinergic rapid oscillations and IJPs occurring during the 5 colonic MMCs shown in panel A. During the intervals between MMCs, the correlation coefficient is low (i.e junction potentials poorly correlated). However, during the rising phase of each colonic MMC individual cholinergic rapid oscillations are highly correlated (R2 range from 0.85 to 1.00).

Figure 3. Histogram plots showing the changes in temporal synchronization of cholinergic rapid oscillations with respect to their amplitudes.

Figure 3

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Histograms show the temporal characteristics of cholinergic rapid oscillations recorded from two CM cells at various electrode separation distances during the colonic MMC. Simultaneous recordings were made from two CM cells at electrode separations of: A, 1 mm; B, 7 mm; C, 15 mm and D, 20 mm, in the longitudinal axis. Aa, plot shows the time difference between the peaks of cholinergic rapid oscillations recorded from two CM cells separated by 1 mm. The y-axis indicates the absolute amplitude of EJPs in the CM. The x-axis shows the time difference between the peaks of cholinergic rapid oscillations at the two sites in milliseconds. Note, as the amplitude of the cholinergic rapid oscillations increases, the time difference (in ms) between the peaks of individual events decreases. As the amplitude of cholinergic rapid oscillation decreases in the two CM cells, the time difference between the peaks of each event increases. Ab shows a plot of the differentiated traces of CM membrane potential in both CM cells plotted against each other. It can be seen that the rate of rise of most cholinergic rapid oscillations in one CM cell are similar to those recorded from the other CM cell located 1 mm away, because as the rate of rise of these events in one CM cell closely follows the rate of rise of similar events occurring in the other CM cell. However, as the electrode separation distance increases (to 7, 15 and 20 mm; cf. Bb, Cb and Db), there is a gradual decrease in the correlation of the two differentiated recordings. At 20 mm separation, Da, all larger amplitude cholinergic rapid oscillations showed poor temporal correlation with each other, suggesting they do not occur at the same time. Similarly, the time difference between cholinergic rapid oscillations in each CM cell show poor temporal correlation the greater the electrode separation distance (i.e. cf. Bb, Cb and Db).

We were particularly interested in whether discrete cholinergic rapid oscillations occurring during each colonic MMC would still remain temporally synchronized when the distance between the two recording electrodes was increased. It was found that at 7 mm separation, similar to the results at 1 mm separation, the larger amplitude events (i.e. > 5 mV) were still well correlated during the rising and plateau phases of each colonic MMC (Fig. 3Ba), whereas the smaller events were less temporally synchronized (n = 8). Again, the larger amplitude events were separated by typically < 80 ms (Fig. 3Ba; n = 8). The slow membrane depolarization underlying each colonic MMC was also well correlated in time at electrode separations of 7 mm. When the electrode separation was then increased to 15 mm, remarkably the larger amplitude discrete cholinergic rapid oscillations were still exceptionally well synchronized in time and of similar amplitudes over this entire spatial distance, during the colonic MMC (see Fig. 4C and Supplemental material). The smaller amplitude EJPs recorded at this same distance selectively ‘dropped out’ of synchrony with each other and were never correlated in both time and amplitude, see * in Fig. 4C. (Supplemental material) shows a real time recording of a single colonic MMC complex recorded from two CM cells, at an electrode separation distance of 15 mm.) The summary data for electrode recordings made at 15 mm apart, from five different animals is shown (Fig. 3Ca and Cb). The major difference between recordings at 15 mm compared with 1 and 7 mm was that the smaller amplitude cholinergic rapid oscillations (< 5 mV) were, as mentioned, never coordinated in time between the two CM cells. This is shown in the differentiated plot (Fig. 3Cb), where the rates of rise of discrete events, rarely, if ever, followed a 45 deg line, as in Fig. 3Ab, where the smaller amplitude events were well correlated at 1 mm separation during the colonic MMC. At 15 mm electrode separation, it was often possible to identify the direction of propagation of colonic MMCs, as the slow membrane depolarization underlying each colonic MMC showed a phase lag in onset between the two CM cells (see Fig. 4B). Figure 5 shows a summary of changes in the amplitude of cholinergic rapid oscillations recorded during each colonic MMC cycle. It can be seen that, as with recordings from 1 mm, the largest amplitude events occurred during the rising phase of each colonic MMC. When the correlation coefficient, R, was compared during each colonic MMC cycle at 15 mm, R values lay between 0.02 and 0.34 during the intervals between colonic MMCs, whereas during the colonic MMC, R values increased between a range of 0.55–0.64. These values were clearly not as well correlated as the R values found at 1 mm electrode separation (see Fig. 2E).

Figure 4. Simultaneous recordings from two circular muscle cells during the colonic MMC.

Figure 4

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A, schematic of the preparation and electrodes separated by 15 mm in the longitudinal axis. B, recording shows three colonic MMCs, where the slow depolarization component of the colonic MMC does not occur simultaneously at this distance, that is, there is a propagation latency in the onset of the slow depolarization between CM1 and CM2. C, however, the discharge of cholinergic rapid oscillations that occurs during the rising and plateau phases still shows that many larger amplitude cholinergic rapid oscillations are temporally synchronized, with the same amplitudes. The smaller amplitude cholinergic rapid oscillations (see *) are less temporally correlated.

Figure 5. Changes in correlation coefficient of IJPs and cholinergic rapid oscillations recorded between two CM cells at increasing electrode separation distances of 1, 7, 15 and 20 mm.

Figure 5

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a shows the changes in R during the rising phase of colonic MMCs. Note, R2 is high at 1 mm and stays at ∼0.5 even at 15 mm electrode separation. Only at 20 mm separation is the R value low, showing poor correlation of cholinergic rapid oscillations and IJPs. b shows similar changes during the plateau phase as in a. c shows the R values during the intervals between colonic MMCs. Note that the correlation of cholinergic rapid oscillations and IJPs is poor at all electrode separation distances during this phase of the MMC.

When the two recording electrodes were separated by 20 mm, colonic MMCs never occurred simultaneously between the two CM cells (n = 5). It was also found that both the small and large amplitude cholinergic rapid oscillations and IJPs occurred independently in the two CM cells (Figs 3D and 6).

Figure 6. Characteristics of spontaneous IJPs (in the presence of atropine) occurring in the CM prior to the onset of colonic MMCs.

Figure 6

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A, schematic of the preparation used for recording simultaneously from two CM cells (CM1 and CM2) separated by 7 mm. B, shows a typical recording of three colonic MMCs in the presence of atropine (1 μm). In the presence of atropine, to block the cholinergic rapid oscillations, a repetitive discharge of IJPs can be recorded prior to the onset of the slow depolarization component of the colonic MMC. Note, the frequency of IJPs in the CM (∼2 Hz) was similar to the frequency of cholinergic rapid oscillations that are normally recorded in the absence of atropine (∼2 Hz), suggesting that myenteric interneurons were responsible for synaptically activating inhibitory and excitatory motor neurons at the same frequency. C, shows an expanded portion of the trace represented by the black bar in panel B. D, shows the traces in panel C superimposed showing their temporal synchronicity. E, shows an expanded portion of the trace represented by the black bar in panel B. Between colonic MMCs, spontaneous IJPs become smaller in amplitude and asynchronous again. F, shows CM1 and CM2 in panel E now superimposed.

Changes in the correlation coefficient of junction potentials during the different phases of the colonic MMC cycle

The changes in correlation coefficient of junction potentials were determined during the different phases of the colonic MMC cycle. The greatest degree of correlation was found during the rising phase of each colonic MMC at 1 mm separation (see a in Fig. 5A), then decreased at 7 mm electrode separation and remained consistent to a electrode separation of 15 mm. Only when the two electrodes were separated by 20 mm were EJPs and IJPs poorly correlated, as shown by R values decreasing to ∼0.3. When the plateau and repolarizing phases of individual cholinergic rapid oscillations and IJPs were compared at increasing electrode separations (see graph b in Fig. 5A), it was found that R values were ∼0.65 at 1 mm separation and remained similar to 15 mm electrode separation. Only when the electrodes were separated to 20 mm did the R values again decrease to ∼0.2 and were poorly correlated. When the correlation coefficient of junction potentials was compared during the intervals between colonic MMCs (see graph c in Fig. 5A), it was found that all potentials were poorly correlated (R values < 0.3), regardless of the electrode separation (see graph c in Fig. 5A).

Synchronized IJPs in two circular muscle cells during the descending inhibitory phase of the colonic MMC in the presence of atropine

Consistent with previous studies (Bywater et al. 1989; Spencer et al. 1998c), we found that a period of membrane hyperpolarization (descending inhibition) occurred immediately prior to the depolarizing phase of many colonic MMCs. In 6 out of 15 animals, recordings from the distal region of colon revealed a repetitive discharge of IJPs that could be recorded simultaneously in the two CM cells immediately prior to the onset of the cholinergic rapid oscillations. To confirm these events were in fact IJPs, we applied atropine (1 μm) to the perfusing solution, which has been consistently shown to block cholinergic rapid oscillations, and EJPs in the mouse colon (Bywater et al. 1989; Lyster et al. 1993; Spencer et al. 1998a). In animals where the cholinergic rapid oscillations were prominent, atropine (1 μm) was applied to the perfusing solution. Despite the two CM cells being separated by 7 mm in the longitudinal axis (see Fig. 6), IJPs occurred at the same time and with similar amplitudes, suggesting that a large population of inhibitory motor neurones were synaptically activated at the same time over a large spatial region during the descending inhibitory phase. Note, most interesting was the observation that the interval between fast IJPs recorded in the presence of atropine was always remarkably similar to the interval between individual cholinergic rapid oscillations when recorded in the absence of atropine. In fact, no significant difference was found when the mean interval between cholinergic rapid oscillations (during the plateau phase of the colonic MMC) was compared with the mean interval between IJPs (recorded in atropine) during the descending inhibitory phase of the colonic MMC (Fig. 6C). In the presence of atropine, the mean interval between IJPs that were temporally synchronized in two CM cells over 7 mm separation, during the colonic MMC was 0.58 ± 0.01 s (range: 0.5–0.63 s; n = 6). The mean amplitude and half-duration of fast IJPs recorded during the descending inhibitory phase of the colonic MMC was 7.8 ± 0.3 mV (range: 3–15 mV; n = 4) and 328 ± 8.2 ms (range: 225–421 ms; n = 4).

Simultaneous recordings from two circular muscle cells in the circumferential axis during the colonic MMC

Since cholinergic rapid oscillations occurring during the colonic MMC were found to occur simultaneously over long lengths of colon, we wished to also determine whether they remain temporally synchronized around the circumference of the colon. To determine this, the two recording microelectrodes were used to impale to CM cells simultaneously at both circumferential edges of the cut preparation (approximately 7–8 mm apart), as shown in Fig. 7. We found that in all preparations (n = 3) the slow depolarization and cholinergic rapid oscillations underlying the colonic MMC occurred at the same time across the circumference. In particular, cholinergic rapid oscillations occurred synchronously at the two circumferential recording sites, an example of which is shown in Fig. 7. Spontaneous IJPs occurring between complexes showed little, or no temporal synchrony between colonic MMCs (see Fig. 7).

Figure 7. Simultaneous recording from two CM cells at both circumferential edges of the distal colon.

Figure 7

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A, schematic of the preparation used. The two recording electrodes were impaled into two CM cells within 100 μm of either circumferential cut edge, as shown. B shows a typical recording of normal colonic MMC activity recorded at each circumferential edge. The colonic MMC characteristics were normal and cholinergic rapid oscillations were synchronized in both time and amplitude at each circumferential edge. The bar shown in C is expanded in panel D to show the synchronized cholinergic rapid oscillations. Note, the smaller amplitude cholinergic rapid oscillations were less correlated in time and amplitude. Also, the spontaneous IJPs occurred asynchronously between complexes; this can be seen by comparing the rapid downward deflections or hyperpolarizations in lower trace with those in the upper trace of Fig. 9B.

Projections of circular muscle motor neurones and ascending and descending interneurones

The retrograde neuronal tracer DiI was used to map the projection lengths of individual CM motor neurones along the length of mouse colon (see Vogalis et al. 2000; Brookes, 2001). This was performed to determine whether a single CM motor neurone could project (and hence possibly release) acetylcholine over 15 mm of colon simultaneously, to account for the temporally synchronized EJPs we have recorded above. When DiI was applied to the CM layer, a distinct projection pattern was identified. A similar distribution of DiI-labelled circular muscle motor neurones (CMMNs) were found oral and anal to the DiI application site (51% oral to the DiI site: 49% anal, n = 13) (see Fig. 8A). CMMNs were found to have a projection length of 0.62 ± 0.2 mm (mean ± s.e.m.) around the circumferential axis of the colon and 1.37 ± 0.1 mm along the longitudinal (oral to anal) axis (560 motor neurones, n = 13). The maximum circumferential projection of all labelled CMMNs was 3.9 mm, whereas the maximum longitudinal projection of any labelled CMMN was 2.8 mm. No further attempts were made in this study to distinguish inhibitory from excitatory CM motor neurones based on morphology or chemical coding. When DiI was applied to myenteric ganglia, a distinctly different population of neurones was labelled (Fig. 8B). It is assumed that DiI applied to the ganglia will label not only motor neurones, but also ascending and descending interneurones (see Brookes, 2001). Since the maximum projection length of any given CM motor neurone was < 3 mm along the colon, we suggest that any neurones labelled longer than 3 mm must be interneurones (see Porter et al. 2002). DiI applied to myenteric ganglia labelled orally projecting (ascending) and anally projecting (descending) interneurones with projections up to 13 mm oral and anal to the DiI site (mean ascending projection: 5.9 ± 0.1 mm; mean descending interneurone length: 5.2 ± 0.3 mm; n = 11; see Fig. 8B).

Figure 8. Projections of DiL-labelled CM motorneurons and interneurons.

Figure 8

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A, projections of DiI-labelled CM motor neurones. DiI applied to the CM was found to label CM motor neurones that showed greater circumferential projection than longitudinal (oral to anal) projection. All CM motor neurones were found to project < 3 mm along the longitudinal axis of the colon, but up to ∼4 mm around the circumference of the colon. All CM motor neurones had projections that lay circumferential to the DiI site of application. B, projections of DiI-labelled interneurones. DiI applied to a ganglion was found to label ascending and descending interneurones as well as CM motor neurones. Myenteric neurones were considered to be interneurones if they projected for distances > 3 mm along the longitudinal axis of the colon.

Simultaneous recordings from circular muscle cells at the oral cut end and middle region of colon

To test the functional role of ascending interneurones in the generation of the colonic MMC, recordings were made adjacent to the far oral cut end of colon (< 50 μm from the cut). At this site, we could not visualize any myenteric ganglia oral of the recording electrode. Therefore, we suggest that no descending interneurones could provide functional synaptic outputs to any myenteric ganglia adjacent to the oral cut end, as they have been cut off during sectioning of the colon. As a control recording, we also always recorded simultaneously from a CM cell in the middle region of colon (i.e. away from either the oral or anal cut ends), so that myenteric neurones in this region could receive functional synaptic outputs from both ascending and descending interneurones. The two recording microelectrodes that were used to impale the two CM cells simultaneously had an electrode separation distance of 15 mm (see Fig. 9).

Figure 9. Effects of oral lesions through the myenteric plexus on colonic MMC activity.

Figure 9

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A, a schematic of the preparation used for simultaneous recording from two circular muscle cells, separated by 15 mm. One electrode adjacent to the oral cut end (< 50 μm from the cut), while the other in the middle region of colon. B shows a simultaneous recording from two CM cells. Note, in CM1, only the slow depolarizing phase of the colonic MMC is observed. That is, the descending inhibitory phase (slow hyperpolarization) is absent. In contrast, at CM2 (in the middle region of colon), the descending inhibitory phase and the slow depolarization phase is preserved. C shows an expanded region of panel B. Note that the slow depolarization in CM1 occurs at the same time as the descending inhibitory phase (slow hyperpolarization) at CM2, suggesting that ascending and descending interneurones are activated at the same time. D, expanded portion of trace in B, represented by the bar. Note, some EJPs in CM1 occur at the same time as IJPs in CM2, suggesting that ascending excitatory and descending inhibitory neuronal pathways fire simultaneously.

The major finding from these simultaneous recordings was that at the oral cut end, the slow depolarization component of the colonic MMC was present and also the repetitive cholinergic oscillations, but no descending inhibition (slow hyperpolarization) was ever recorded (see Fig. 9B). The amplitude of the slow depolarization underlying each colonic MMC, when recorded at the oral cut end was 8.3 ± 0.7 mV (range: 2–16 mV, n = 14), while the same colonic MMC slow depolarization recorded (at the same time) from the middle region of colon had a mean peak amplitude of 11.4 ± 1.1 mV (range: 2–28 mV; n = 14). These amplitudes were significantly different (P < 0.05; Student's paired t test; n = 14). It is particularly noteworthy that the onset of slow MMC depolarization recorded at the oral cut end could be temporally correlated with the onset of the descending inhibitory phase of the colonic MMC recorded 15 mm in the middle region of colon (see Fig. 9C). At the oral cut end, cholinergic rapid oscillations (EJPs) were recorded during the rising phase and plateau phase of colonic MMCs, but spontaneous IJPs were considerably less common, or usually absent (Figs 9 and 10). The repetitive discharge of cholinergic rapid oscillations recorded during each colonic MMC at the oral cut end occurred with a mean interval of 0.43 ± 0.01 s (range: 0.31–0.62 s; n = 14), while recordings 15 mm anally showed that the EJP interval was not significantly different (0.59 ± 0.02 s; n = 14; range, 0.42–1.1 s; P= 0.052). The mean amplitude of the cholinergic rapid oscillations that occurred at the oral cut end was 5.7 ± 0.64 mV (range: 3.2–11.0 mV) and this value was significantly less than their mean amplitude during the colonic MMC in the middle region of colon (mean: 7.79 ± 0.54 mV, P= 0.029; n = 14).

Figure 10. Effects of recording from CM cells adjacent to oral and anal lesions through the myenteric plexus.

Figure 10

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A shows the position of the recording electrode < 50 μm from the oral cut end. B shows representative recordings from 4 different animals of electrical activity between colonic MMCs at this site. Note, predominantly EJPs, and not IJPs, are recorded. C and D, recordings from the anal cut end. Note, predominantly IJPs are recorded. One small EJP (see *) occurs in 1 animal.

The resting membrane potential of CM cells recorded adjacent to the oral cut end of colon were −43.4 ± 2.3 μV (14 cells, n = 14). These values were not significantly different from resting membrane potentials of CM cells recorded at the middle region of colon (P > 0.05, n = 8; Student's paired t test).

Simultaneous recordings from circular muscles at the anal cut end and middle region of colon

To test the functional role of descending interneurones in the generation of the colonic MMC, recordings were made adjacent to the far anal cut end of colon (< 50 μm from the cut). At this site, we suggest that ascending interneurones were cut off during the sectioning of the colon and therefore no functional synaptic outputs of ascending pathways exists at this site. Recordings were made simultaneously from two CM cells, one cell (< 50 μm) from the anal cut end while the other 15 mm from the anal cut end, in the middle region of colon. The major finding when recordings were made from these two sites was that at the anal cut end, only the descending inhibitory (slow hyperpolarization) phase of the colonic MMC was recorded (Fig. 11B). The slow depolarization component of the colonic MMC and the repetitive discharge of cholinergic rapid oscillations were never observed at this anal cut end (Fig. 11B). Moreover, spontaneous EJPs were essentially absent at the anal cut end (Fig. 10D). In a small proportion of cells, some small amplitude EJPs (< 5 mV) could be resolved (Fig. 10D). On the other hand, spontaneous IJPs were commonly recorded from the anal cut end (Fig. 10D). At the same time that recordings were made from the anal cut end, control recordings from the middle region of colon again showed both normal descending inhibitory and excitatory components of the colonic MMC were present (Fig. 11B). That is, the repetitive discharge of cholinergic rapid oscillations and the slow depolarization phase of colonic MMC were still recorded and robust in amplitude (up to 20 mV). It is again noteworthy that the onset of descending inhibitory phase of the colonic MMC recorded at the anal cut end occurred at the same time as the descending inhibitory phase recorded in the middle region of colon (see Fig. 11B). The mean resting membrane potential of CM cells recorded adjacent to the anal cut end of colon was −45.3 ± 2.0 μV (range: 8 cells, n = 8). These values were not significantly different from resting membrane potentials of CM cells recorded at either the oral cut end, or middle region of colon (P > 0.05, n = 8, Student's paired t test).

Figure 11. Effects of anal lesions through the myenteric plexus on colonic MMC activity.

Figure 11

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A, schematic of the preparation used for simultaneous recording from two CM cells during the colonic MMC. The two recording electrodes were separated by 15 mm; one electrode impaled in the middle region of the preparation, the other < 50 μm from the anal cut end. B, a simultaneous recording from two CM cells located at the position shown in A. At CM1 normal colonic MMC activity occurs with a descending inhibitory phase and slow depolarizing (excitatory) phase. At the same time, however, in CM2 only the descending inhibitory phase was recorded. No excitation was recorded at this site. C shows an expanded portion of the trace in B, represented by the bar. D, note that the amplitude of the descending inhibitory phase (slow hyperpolarization) in CM2 is significantly less than that shown in CM1 from the middle region of colon.

Discussion

When simultaneous intracellular electrical recordings were made from two CM cells located as little as 1 mm apart along the length of the colon, it was found that, during the intervals between colonic MMCs, spontaneous IJPs occurred asynchronously at the two sites and were variable in amplitude. This result suggests that there was little spread of electrical activity between neighbouring rings of circular muscle cells and that the inhibitory motor neurones in myenteric ganglia supplying these areas of muscle were firing in an unsynchronized manner.

In contrast, during the colonic MMC, the cholinergic rapid oscillations in membrane potential and IJPs at the two sites were found to be synchronized and to have similar amplitudes. The larger amplitude rapid oscillations exhibited synchronized activity over distances of up to 15 mm along the length of colon. This distance long the gut was far greater than the length that any single CM motor neurone could innervate, since CM motor neurones had projections less than 3 mm along the length of colon, consistent with previous findings by others in the guinea-pig proximal colon (Neunlist et al. 1997) and ileum (Smith et al. 1988; Brookes & Costa, 1990; Brookes, 2001). In fact, much of this projection length is through the myenteric plexus rather than through the muscle, since dye fills into single motor neurones in mouse colon show that they innervate a narrow band of CM, less than 200 μm wide (see Fig. 5 in Nurgali et al. 2004; N. J. Spencer & T. K. Smith, unpublished results).

It is known that in a smooth muscle syncytium any local current injected into a single point source will rapidly dissipate through a three dimensional syncytium (see Spencer et al. 2001, 2002). Therefore, we suggest that the only means by which cholinergic rapid oscillations in membrane potential or IJPs could be synchronized in both time course and amplitude over such a large spatial distance (i.e. ∼15 mm) along the bowel, is if the underlying membrane current is: (1) synchronized in time in all the CM cells over this large distance, and (2) if the underlying membrane current is of similar intensity over this 15 mm region of CM. Therefore, a large population of excitatory and inhibitory motor neurones along the colon must be synaptically activated at the same time in order to give a synchronized release of transmitter onto the muscle in order to produce simultaneous electrical events of similar amplitude along the bowel. The activity in these motor neurones is coordinated by the synchronous firing of interneurones. Presumably, the synapses from many interneurones converge onto common motor neurones (see Spencer & Smith, 2004). The amplitude of any discreet IJP or EJP at a particular site in the muscle reflects the number of motor neurones synaptically activated at the same time and the degree to which their activation was temporally synchronized with other motor neurones.

During the colonic MMC are the rapid cholinergic oscillations composed of discreet EJPs?

In previous studies of mouse colon, cholinergic rapid oscillations were reported to occur in the CM layer during the colonic MMC, but the nature of these oscillations was unknown (Bywater et al. 1989). What was clear was that they occurred at a consistent frequency of ∼2 Hz and were abolished by atropine. Therefore, the suggestion was raised that they involved the release of acetylcholine from cholinergic motor neurones (Bywater et al. 1989). In the current study, we have recorded the same cholinergic oscillations in the CM layer and also at a consistent frequency of ∼2 Hz during the colonic MMC. However, a major unanswered question was what mechanism generates this repetitive discharge of oscillations at ∼2 Hz? One possibility is that the cholinergic rapid oscillations in membrane potential are due to postsynaptic myogenic slow wave mechanisms in the CM. Although, it has been shown that in the presence of neuronal antagonists which block the colonic MMC, further bath application of carbachol to stimulate muscarinic receptors on the smooth muscle was found to induce a slow membrane depolarization of the CM, but failed to induce the cholinergic rapid oscillations, suggesting that prolonged exposure or activation of muscarinic receptors on CM cells was not a prerequisite for the generation of the rapid oscillations (Lyster et al. 1995). We suggest, as a result of the current study, that the cholinergic rapid oscillations reported previously (see Bywater et al. 1989; Spencer, 2001), represent a repetitive discharge of discrete cholinergic EJPs, generated by repetitive firing of many intrinsic cholinergic motor neurones, rather than by a postsynaptic mechanism in the CM cells themselves in response to prolonged exposure of acetylcholine to muscarinic receptors. Since cholinergic EJPs occur in the CM cells at a frequency of ∼2 Hz during the colonic MMC, we suggest that the CM motor neurones must also be firing at a similar frequency. Further support for our notion that the repetitive EJPs occurring during the colonic MMC were generated by interneurones and motor neurones firing at ∼2 Hz comes from experiments when the cholinergic rapid oscillations were abolished by atropine. It was noted that a repetitive discharge of fast IJPs could also be recorded at the same frequency (∼2 Hz) (as the EJPs in the absence of atropine) during the onset of the colonic MMC. During the onset of the colonic MMC, IJPs occurring at the two recording electrodes also became temporally locked and occurred with the same amplitudes over distances that far exceed the projection length of a single CM motor neurone. We have recently shown that the ongoing discharge of EJPs and IJPs in the CM of the guinea-pig colon is produced by the discreet discharge of fEPSPs in motor neurones (Spencer & Smith, 2004).

How do myenteric interneurones synchronize firing of many motor neurones during colonic MMCs?

The mechanism by which myenteric interneurones periodically synchronize their firing to generate colonic MMCs is not clear. What is clear is that when brief trains of electrical stimuli are applied to local sites along the mouse colon, they can readily evoke a premature colonic MMC, which, once initiated, will propagate along the whole length of colon without decrement in amplitude (Spencer & Bywater, 2002). This suggests that the neuronal circuitry underlying the colonic MMC involves a regenerative process to sustain a propulsive contraction along the full length of large bowel.

Crosstalk between ascending and descending interneurones

When recordings were made adjacent to the oral cut end of the colon, we found that the slow hyperpolarizing (inhibitory) phase of the colonic MMC was consistently absent from this oral site, suggesting that descending inhibitory nerve pathways had been severed. In addition, at the oral site, the repetitive discharge of cholinergic rapid oscillations (EJPs) and the slow depolarizing component of the colonic MMC were readily recorded but the amplitude of both were significantly reduced suggesting that descending nerve pathways synapse with and reinforce ascending excitatory nerve pathways. In contrast, when recordings were made adjacent to the anal cut end of the colon, only the slow hyperpolarizing (inhibitory) component of the colonic MMC was recorded, and this too was of reduced amplitude, suggesting also that ascending nerve pathways synapse with and reinforce descending inhibitory nerve pathways. Our data suggest a proposed interaction between interneurones in ascending excitatory and descending inhibitory nerve pathways as shown schematically in Fig. 12. It is particularly noteworthy that Brookes et al. (2001) observed that stretch-activated peristaltic waves in isolated flat sheet preparations of guinea-pig ileum consistently failed to invade the terminal anal end of intestine. These investigators found that peristaltic contraction amplitudes significantly decreased in a progressive fashion, over the most aboral 10–15 mm of intestine. Brookes et al. (2001) suggested that a possibility was that ‘a reduction in ascending pathways as the most likely explanation for the decline in amplitude of the contraction further orally’.

Figure 12. Diagrammatic representation of the enteric neuronal circuit underlying colonic MMC generation.

Figure 12

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During the intervals between colonic MMCs, there is asynchronous firing in many myenteric interneurones leading to asynchronous firing of CM motor neurones and hence uncoordinated spontaneous IJPs and cholinergic EJPs (rapid oscillations) in the CM. During the onset of the colonic MMC, the firing of many ascending and descending myenteric interneurones becomes temporally synchronized. Therefore, many ascending interneurones (cell bodies in pink) and many descending interneurones (cell bodies in light blue) synaptically activate each other. The synaptic outputs of ascending interneurones activate a large population of cholinergic CM motor neurones (in red) simultaneously, where excitatory CM motor neurones project locally or orally to the CM, while inhibitory CM motor neurones (darker blue) project largely anally to the CM, consistent with other mammals. Since individual CM motor neurones project < 3 mm along the colon, then during the colonic MMC, many cholinergic motor neurones must become synaptically activated at the same time to cause discrete EJPs to have the same amplitudes and temporal synchronization over 15 mm of CM. A, at the oral cut end of colon, ascending excitation is preserved via ascending interneurones, but descending inhibition is absent. B, as the colonic MMC migrates distally and approaches the anal cut end, the descending inhibitory phase of the colonic MMC is preserved, via desceding interneurones, but ascending excitation is absent.

What is the sensory neurone underlying the colonic MMC?

The identity of the intrinsic sensory neurone, or pacemaker neurone, that generates colonic MMC rhythmicity in mouse is unclear. It is unlikely to have properties that are similar to the intrinsic sensory neurones described so far (see below). What is clear is that the intrinsic pacemaker frequency can be readily increased or decreased using pharmacological agents such as 5-HT, l-NA or atropine (Fida et al. 1997; Bush et al. 2000; Spencer, 2001). Recordings from myenteric neurones in the mouse colon (Furukawa et al. 1986; Nurgali et al. 2004) have found that both AH and S-type neurones exist with similar properties to those found in the guinea-pig colon (Wade & Wood, 1988; Wood, 1994; Lomax et al. 1999; Spencer & Smith, 2004). One major feature of the enteric neural circuitry which generates the murine colonic MMC is that it is resistant to smooth muscle tension or tone, since colonic MMCs can still propagate along the colon even in the presence of nifedipine (Bywater et al. 1989). This suggests that myenteric AH neurones in the mouse colon are probably unlikely to be involved in the generation of the colonic MMC, since the excitability and activation of these neurones, at least in guinea-pig small intestine, has been shown to be highly dependent upon smooth muscle tone or tension and become inactivated by L-type Ca2+ blockers (Kunze et al. 1998). Interestingly, in the guinea-pig distal colon, we have found that a population of mechanosensory neurones exists which generate rhythmic peristaltic waves and this class of neurone is dependent upon smooth muscle tension, since nifedipine abolishes peristaltic wave generation when applied selectively to the site of distension (Smith et al. 2003). Importantly, the colonic MMC in mouse also occurs in a flaccid colon; therefore any intrinsically active pacemaker neurone is unlikely to have properties that are similar to the mechanosensory S-interneurones that have recently been shown to exist in guinea-pig colon since, although they are resistant to smooth muscle tension or tone, they appear to be dependent upon stretch for their activation (Spencer & Smith, 2004).

Do slow waves play any role in the generation of the colonic MMC?

In this study, slow waves were never recorded from any CM cells. These observations are highly consistent with other intracellular electrical recordings made from the mouse colon (Bywater et al. 1989; Lyster et al. 1995; Ward et al. 1997; Spencer et al. 1998a, 1998b, 1999c). Since slow waves are rarely recorded from the CM of the mouse colon, and the colonic MMC is abolished by neuronal antagonists, we find no evidence to suggest that slow waves play a role in colonic MMC generation. Interestingly, in the isolated mouse small intestine, slow waves are very commonly recorded, as are MMCs in this region in vitro (Bush et al. 2000; Spencer et al. 2003). However, interestingly, MMC generation in the mouse small intestine also still occurs in W/Wv mutant mice in which slow waves and pacemaker-type interstitial cells of Cajal at the level of the myenteric plexus (ICC-MY) are absent (Spencer et al. 2003).

Conclusions

In summary, we suggest that the generation of each colonic MMC involves a stochastic process, whereby the firing of many myenteric ascending and descending interneurones becomes temporally synchronized (Fig. 12). The synaptic outputs of these interneurones are suggested to cause the simultaneous activation of many orally projecting cholinergic excitatory motor neurones and anally projecting inhibitory motor neurones. This explains why cholinergic EJPs or IJPs of the same amplitudes and time courses can be recorded simultaneously over large lengths of colon. We suggest that, similar to other mammals studied, EJPs are generated by largely ascending neural pathways, and IJPs by descending neural pathways (Smith et al. 1990, 1991; Spencer et al. 2002; Spencer & Smith, 2001). These ascending and descending interneurones provide the synaptic outputs to temporally synchronize the firing of many cholinergic motor neurones, causing each discrete EJP, and inhibitory motor neurones to cause each IJP (Fig. 12). Our working hypothesis is that during the colonic MMC ascending and descending interneurones synaptically converge upon each other in a similar way that accounts for the coordinated firing of EJPs and IJPs in the guinea-pig distal colon (Spencer et al. 2002; Smith et al. 2005). However, during the ascending excitatory phase of the murine colonic MMC, few, if any IJPs are recorded in the muscle, since inhibitory transmitter release is compromised by presynaptic mechanisms (see Spencer et al. 1998a).

Supplementary Material

Supplemental Data
jphysiol_2005.083600_index.html (920B, html)

Acknowledgments

This study was supported by a grant from the National Institute of Health (USA) (RO1 NIDDK 45713) awarded to T.K.S. and N.J.S. We wish to acknowledge the helpful comments of Dr David Hirst, John Curtin School of Medicine, Australian National University.

Supplemental material

The online version of this paper can be accessed at:

10.1113/jphysiol.2005.083600

http://jp.phy soc.org/cgi/content/full/jphysiol.2005.083600/DC1 and contains two video files (10 and 10 MB).

This material can also be found at:

http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp826/tjp826sm.htm

References

  1. Bolton TB. On the nature of the oscillations of the membrane potential (slow waves) produced by acetylcholine or carbachol in intestinal smooth muscle. J Physiol. 1971;216(2):403–18. doi: 10.1113/jphysiol.1971.sp009532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brann L, Wood JD. Motility of the large intestine of piebald-lethal mice. Am J Dig Dis. 1976;21:633–640. doi: 10.1007/BF01071956. [DOI] [PubMed] [Google Scholar]
  3. Brierley SM, Nichols K, Grasby DJ, Waterman SA. Neural mechanisms underlying migrating motor complex formation in mouse isolated colon. Br J Pharmacol. 2001;132(2):507–517. doi: 10.1038/sj.bjp.0703814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brookes SJ. Retrograde tracing of enteric neuronal pathways. Neurogastroenterol Motility. 2001;13:1–18. doi: 10.1046/j.1365-2982.2001.00235.x. [DOI] [PubMed] [Google Scholar]
  5. Brookes SJ, Costa M. Identification of enteric motor neurons which innervate the circular muscle of the guinea-pig small intestine. Neuroscience Lett. 1990;118:227–230. doi: 10.1016/0304-3940(90)90633-k. [DOI] [PubMed] [Google Scholar]
  6. Brookes SJ, O'Antona G, Zagorodnyuk UP, Humphreys CM, Costa M. Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea-pig ileum. Neurogastroenterol Motil. 2001;13:519–531. doi: 10.1046/j.1365-2982.2001.00290.x. [DOI] [PubMed] [Google Scholar]
  7. Bush TG, Spencer NJ, Watters N, Sanders KM, Smith TK. Spontaneous migrating motor complexes occur in both the terminal ileum and colon of the C57BL/6 mouse in vitro. Auton Neurosci. 2000;84(3):162–168. doi: 10.1016/S1566-0702(00)00201-0. [DOI] [PubMed] [Google Scholar]
  8. Bywater RAR, Small RC, Taylor GS. Neurogenic slow depolarisations and rapid oscillations in circular muscle of mouse colon. J Physiol. 1989;413:505–519. doi: 10.1113/jphysiol.1989.sp017666. 10.1007/BF00594181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christensen J, Anuras S, Arthur C. Influence of intrinsic nerves on electromyogram of cat colon in vitro. Am J Physiol. 1978;234:E641–E647. doi: 10.1152/ajpendo.1978.234.6.E641. [DOI] [PubMed] [Google Scholar]
  10. Fida R, Lyster DJ, Bywater RA, Taylor GS. Colonic migrating motor complexes (CMMCs) in the isolated mouse colon. Neurogastroenterol Motil. 1997;9(2):99–107. doi: 10.1046/j.1365-2982.1997.d01-25.x. 10.1046/j.1365-2982.1997.d01-25.x. [DOI] [PubMed] [Google Scholar]
  11. Fioramonti J, Bueno L. Diurnal changes in colonic motor profile in conscious dogs. Dig Dis Sci. 1983;28:257–264. doi: 10.1007/BF01295121. 10.1007/BF01295121. [DOI] [PubMed] [Google Scholar]
  12. Furness JB, Costa M. The Enteric Nervous System. New York: Churchill Livingstone; 1987. [Google Scholar]
  13. Furukawa K, Taylor GS, Bywater RAR. An intracellular study of myenteric neurons in the mouse colon. J Neurophysiol. 1986;55:1395–1406. doi: 10.1152/jn.1986.55.6.1395. [DOI] [PubMed] [Google Scholar]
  14. Kunze WA, Furness JB, Bertrand PP, Bornstein JC. Intracellular recording from myenteric neurons of the guinea-pig ileum that respond to stretch. J Physiol. 1998;506:827–842. doi: 10.1111/j.1469-7793.1998.827bv.x. 10.1111/j.1469-7793.1998.827bv.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lomax AE, Sharkey KA, Bertrand PP, Low AM, Bornstein JC, Furness JB. Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the guinea-pig distal colon. J Auton Nerv Syst. 1999;76:45–61. doi: 10.1016/s0165-1838(99)00008-9. 10.1016/S0165-1838(99)00008-9. [DOI] [PubMed] [Google Scholar]
  16. Lyster DJK, Bywater RAR, Taylor GS. Regulation of the frequency of myoelectric complexes in the mouse colon. J Gastrointest Motil. 1993;5:143–151. [Google Scholar]
  17. Lyster DJ, Bywater RA, Taylor GS. Neurogenic control of myoelectric complexes in the mouse isolated colon. Gastroenterol. 1995;108:1371–1378. doi: 10.1016/0016-5085(95)90684-3. [DOI] [PubMed] [Google Scholar]
  18. Mule F, D'Angelo S, Tabacchi G, Amato A, Serio R. Mechanical activity of small and large intestine in normal and mdx mice: a comparative analysis. Neurogastroenterol Motil. 1999;11(2):133–139. doi: 10.1046/j.1365-2982.1999.00142.x. 10.1046/j.1365-2982.1999.00142.x. [DOI] [PubMed] [Google Scholar]
  19. Neunlist M, Schemann M. Projections and neurochemical coding of myenteric neurons innervating the mucosa of the guinea-pig proximal colon. Cell Tissue Res. 1997;287:119–125. doi: 10.1007/s004410050737. 10.1007/s004410050737. [DOI] [PubMed] [Google Scholar]
  20. Nurgali K, Stebbing MJ, Furness JB. Correlation of electrophysiological and morphological characteristics of enteric neurons in the mouse colon. J Comp Neurol. 2004;468:112–124. doi: 10.1002/cne.10948. 10.1002/cne.10948. [DOI] [PubMed] [Google Scholar]
  21. Porter AJ, Wattchow DA, Brookes SJ, Costa M. Cholinergic and nitrergic interneurones in the myenteric plexus of the human colon. Gut. 2002;51:70–75. doi: 10.1136/gut.51.1.70. 10.1136/gut.51.1.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Powell AK, Bywater RA. Endogenous nitric oxide release modulates the direction and frequency of colonic migrating motor complexes in the isolated mouse colon. Neurogastroenterol Motil. 2001;13(3):221–228. doi: 10.1046/j.1365-2982.2001.00260.x. 10.1046/j.1365-2982.2001.00260.x. [DOI] [PubMed] [Google Scholar]
  23. Sarna SK. Cyclic motor activity; migrating motor complex: 1985. Gastroenterol. 1985;89:894–913. doi: 10.1016/0016-5085(85)90589-x. [DOI] [PubMed] [Google Scholar]
  24. Schuurkes JA, Tukker JJ. The interdigestive colonic motor complex of the dog. Arch Int Pharmacodyn Ther. 1980;247:329–334. [PubMed] [Google Scholar]
  25. Smith TK, Bornstein JC, Furness JB. Distension-evoked ascending and descending reflexes in the circular muscle of guinea-pig ileum: an intracellular study. J Auton Nerv Syst. 1990;29:203–217. doi: 10.1016/0165-1838(90)90146-a. 10.1016/0165-1838(90)90146-A. [DOI] [PubMed] [Google Scholar]
  26. Smith TK, Bornstein JC, Furness JB. Interactions between reflexes evoked by distension and mucosal stimulation: electrophysiological studies of guinea-pig ileum. J Auton Nerv Syst. 1991;34:69–75. doi: 10.1016/0165-1838(91)90009-r. 10.1016/0165-1838(91)90009-R. [DOI] [PubMed] [Google Scholar]
  27. Smith TK, Furness JB, Costa M, Bornstein JC. An electrophysiological study of the projections of motor neurones that mediate non-cholinergic excitation in the circular muscle of the guinea-pig small intestine. J Auton Nerv Syst. 1988;22(2):115–128. doi: 10.1016/0165-1838(88)90085-9. 10.1016/0165-1838(88)90085-9. [DOI] [PubMed] [Google Scholar]
  28. Smith TK, Oliver GR, Hennig GW, Vanden Berghe P, Kang SK, Spencer NJ. A muscle tone-dependent migrating motor pattern in guinea-pig distal Colon. J Physiol. 2003;551:955–969. doi: 10.1113/jphysiol.2003.049163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith TK, Reed JB, Sanders KM. Interaction of two electrical pacemakers in muscularis of canine proximal colon. Am J Physiol. 1987;252(3 Part 1):C290–C299. doi: 10.1152/ajpcell.1987.252.3.C290. [DOI] [PubMed] [Google Scholar]
  30. Smith TK, Spencer NJ, Hennig GW. Sensory transduction in the enteric nervous system. Physiol News. 2005;58:23–25. http://physoc.org/publications/pn/issuepdf/58/23--25pdf. [Google Scholar]
  31. Spencer NJ. Control of migrating motor activity in the colon. Curr Opin Pharmacol. 2001;1(6):604–610. doi: 10.1016/s1471-4892(01)00103-5. 10.1016/S1471-4892(01)00103-5. [DOI] [PubMed] [Google Scholar]
  32. Spencer NJ, Bywater RAR. Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse. Neurogastroenterol Mot. 2002;14:657–665. doi: 10.1046/j.1365-2982.2002.00367.x. 10.1046/j.1365-2982.2002.00367.x. [DOI] [PubMed] [Google Scholar]
  33. Spencer NJ, Bywater RAR, Holman ME, Taylor GS. Inhibitory neurotransmission in the circular muscle layer of mouse colon. J Auton Nerv Syst. 1998c;70:10–14. doi: 10.1016/s0165-1838(98)00045-9. 10.1016/S0165-1838(98)00045-9. [DOI] [PubMed] [Google Scholar]
  34. Spencer NJ, Bywater RAR, Taylor GS. Disinhibition during myoelectric complexes in mouse colon. J Autonomic Nervous System. 1998a;71:37–47. doi: 10.1016/s0165-1838(98)00063-0. 10.1016/S0165-1838(98)00063-0. [DOI] [PubMed] [Google Scholar]
  35. Spencer NJ, Bywater RA, Taylor GS. Evidence that myoelectric complexes in the isolated mouse colon may not be of myogenic origin. Neurosci Lett. 1998b;250(3):153–156. doi: 10.1016/s0304-3940(98)00461-3. 10.1016/S0304-3940(98)00461-3. [DOI] [PubMed] [Google Scholar]
  36. Spencer NJ, Hennig GW, Smith TK. Spatial and temporal coordination of junction potentials in circular muscle of guinea-pig distal colon. J Physiol. 2001;535:565–578. doi: 10.1111/j.1469-7793.2001.00565.x. 10.1111/j.1469-7793.2001.00565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Spencer NJ, Hennig GW, Smith TK. A rhythmic motor pattern activated by circumferential stretch in guinea-pig distal colon. J Physiol. 2002;545:629–648. doi: 10.1113/jphysiol.2002.028647. 10.1113/jphysiol.2002.028647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spencer NJ, Sanders KM, Smith TK. Migrating motor complexes do not require electrical slow waves in the mouse small intestine. J Physiol. 2003;553:881–893. doi: 10.1113/jphysiol.2003.049700. 10.1113/jphysiol.2003.049700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spencer NJ, Smith TK. Simultaneous intracellular recordings from longitudinal and circular muscle during the peristaltic reflex in guinea-pig distal colon. J Physiol. 2001;533:787–800. doi: 10.1111/j.1469-7793.2001.00787.x. 10.1111/j.1469-7793.2001.00787.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Spencer NJ, Smith TK. Mechanosensory S-neurons rather than AH neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon. J Physiol. 2004;558:577–596. doi: 10.1113/jphysiol.2004.063586. 10.1113/jphysiol.2004.063586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Steele PA, Brookes SJH, Costa M. Immunohistochemical localization of cholinergic neurons in the myenteric plexus of guinea-pig small intestine. Neuroscience. 1991;45:227–239. doi: 10.1016/0306-4522(91)90119-9. 10.1016/0306-4522(91)90119-9. [DOI] [PubMed] [Google Scholar]
  42. Vogalis F, Hillsley K, Smith T. Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon. J Neurosci Meth. 2000;96(1):25–34. doi: 10.1016/s0165-0270(99)00180-6. 10.1016/S0165-0270(99)00180-6. [DOI] [PubMed] [Google Scholar]
  43. Wade PR, Wood JD. Electrical behavior of myenteric neurons in guinea pig distal colon. Am J Physiol. 1988;254:G522–G530. doi: 10.1152/ajpgi.1988.254.4.G522. [DOI] [PubMed] [Google Scholar]
  44. Ward SM, Harney SC, Bayguinov JR, McLaren GJ, Sanders KM. Development of electrical rhythmicity in the murine gastrointestinal tract is specifically encoded in the tunica muscularis. J Physiol. 1997;505:241–258. doi: 10.1111/j.1469-7793.1997.241bc.x. 10.1111/j.1469-7793.1997.241bc.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wood JD. Electrical activity of the intestine of mice with hereditary megacolon and absence of enteric ganglion cells. Am J Dig Dis. 1973;18:477–488. doi: 10.1007/BF01076598. 10.1007/BF01076598. [DOI] [PubMed] [Google Scholar]
  46. Wood JD. Physiology of the enteric nervous system. In: Johnson LR, editor. Physiology of the Gastrointestinal Tract. ed. New York: Raven Press; 1994. pp. 423–482. In. [Google Scholar]

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