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. 2001 Sep 1;535(Pt 2):565–578. doi: 10.1111/j.1469-7793.2001.00565.x

Spatial and temporal coordination of junction potentials in circular muscle of guinea-pig distal colon

Nick J Spencer 1, Grant W Hennig 1, Terence K Smith 1
PMCID: PMC2278784  PMID: 11533145

Abstract

  1. In isolated, stretched, flat-sheet preparations of guinea-pig distal colon, simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells to map the region of smooth muscle at which spontaneous junction potentials (sJPs) were coordinated in both space and time.

  2. Spontaneous inhibitory junction potentials (sIJPs) and excitatory junction potentials (sEJPs) were recorded from all animals with varying frequencies and amplitudes (up to 25 mV).

  3. Large amplitude (≥ 9 mV) sIJPs or sEJPs with near-identical amplitudes and time courses were recorded synchronously from two CM cells, even when the two electrodes were separated by up to 11 mm in the circumferential axis and ≤ 4 mm in the longitudinal axis. However, smaller (< 9 mV) sIJPs or sEJPs were less coordinated and exhibited greater variability in their times to peak.

  4. The standard deviation (s.d.) for the time difference between the peaks of sJPs was related to the amplitude of the events and the distance between the electrodes. The s.d. for large amplitude JPs (∼30 ms), which was less than that for small JPs (∼150 ms), remained constant across the circumferential axis (at least up to 6 mm), but declined rapidly for distances ≥ 2 mm in the longitudinal axis.

  5. Current injection (up to 8 nA) into a single CM cell elicited electrotonic potentials in neighbouring CM cells, only when the two electrodes were separated by less than 100 μm circumferentially. Beyond 50 μm electronic potentials were rarely detected.

  6. Tetrodotoxin (TTX; 1 μm) abolished all sJPs, whereas hexamethonium (300 μm) either abolished, or substantially reduced all sJPs.

  7. Nitro-l-arginine (l-NA; 100 μm) abolished the slow repolarisation phase of sIJPs without any apparent effect on the amplitude of sIJPs. Apamin abolished the fast, initial component of sIJPs, suggesting synchronous release of two inhibitory neurotransmitters during the sIJP. Atropine (1 μm) abolished sEJPs.

  8. No sJPs were recorded from the CM layer when it was separated from the myenteric plexus.

  9. In conclusion, sIJPs and sEJPs in colonic CM occur synchronously over large regions of the smooth muscle syncitium. The results are discussed in relation to the idea that spontaneous junction potentials in colonic CM are not monoquantal events, but are generated by the simultaneous release of transmitter from many release sites, due to the synchronous activation of many enteric motor neurons.

In many regions of the gastrointestinal tract, smooth muscle cells exhibit brief, nerve-mediated spontaneous hyperpolarisations and depolarisations referred to as spontaneous inhibitory junction potentials (sIJPs) and excitatory junction potentials (sEJPs), respectively. In other autonomic neuromuscular junctions, sEJPs have been reported in the smooth muscle of the vas deferens (guinea-pig: Burnstock & Holman 1962; Tomita, 1967; mouse: Holman et al. 1977; Brock & Cunnane, 1988), arteriolar smooth muscle (Hirst & Nield, 1980; Kuriyama & Suzuki, 1981), seminal vesicles (Kajimoto et al. 1972) and urinary bladder (Bramich & Brading, 1996). These events are tetrodotoxin resistant and have been assumed to represent the spontaneous release of sympathetic neurotransmitter (Burnstock & Holman, 1962; Bennett, 1972; Stjarne & Astrand, 1984; Cunnane & Stjarne, 1984; Stjarne, 2000) from single vesicles (Del Castillo & Katz, 1954). Since only single quanta underlie the sEJP in these preparations, it has been shown that during simultaneous intracellular recordings from pairs of CM cells, the sEJP occurs synchronously only over a small area of muscle cells (see Tomita, 1967; Bramich & Brading, 1996). In fact, Tomita (1967) concluded that in the guinea-pig vas deferens the spatial decay of the spontaneous junction potential was very sharp and similar to the spatial decay of electrotonic potentials evoked by intracellular polarisation.

In contrast to other autonomic neuromuscular junctions (Burnstock & Holman, 1961; Tomita, 1967; Hirst, 1977; Bramich & Brading, 1996), spontaneous junction potentials in intestinal smooth muscle require action potential conduction along motor neurons, since they are abolished by tetrodotoxin (Smith, 1989; Spencer et al. 1998a). Although it is clear that sEJPs in intestinal smooth muscle are predominantly due to transient release of acetylcholine from cholinergic motor neurons (since they are abolished by atropine; Smith et al. 1992), the neurotransmitter(s) underlying sIJPs in the colon are unknown. In the circular muscle layer of the mouse colon, sIJPs are resistant to inhibition of nitric oxide (NO) synthesis, but are abolished by apamin, suggesting that small conductance ‘SK’-type K+ channels are involved (Spencer et al. 1998b). In the guinea-pig ileum, sIJPs were also abolished by apamin (Smith, 1989), but the identity of the underlying transmitter also remains unclear.

Many studies have investigated the nature of the evoked IJP and EJP following electrical stimulation of isolated segments of mammalian bowel; however, little attention has focused on the mechanisms underlying spontaneous junction potentials. It is known that following increasing intensities of electrical stimulation, it is possible to grade the amplitude of the evoked IJP (Tomita, 1972; Bywater et al. 1981), which suggests that many inhibitory motor neurons innervate each CM cell and that they can be recruited to generate a larger amplitude evoked junction potential. This is particularly noteworthy, since in the mouse colon and other intestinal smooth muscles, the waveform of evoked IJPs (following electrical stimulation of many inhibitory motor neurons) is similar to the waveform of spontaneous IJPs of similar amplitude (Furness, 1969; Smith et al. 1992; Spencer et al. 1998b). This raises the question as to what mechanisms generate the spontaneous junction potential in the bowel? Is it due to monoquantal release of transmitter, as is the case in the vas deferens or arterioles where only a small population of muscle cells is polarised simultaneously, or is it due to synchronous firing of many motor neurons which may polarise the smooth muscle over a large area of the syncitium?

In this study we have made simultaneous intracellular recordings from pairs of CM cells to investigate the area of smooth muscle over which the spontaneous potentials show coordinated activity.

METHODS

Preparation of tissues

Guinea-pigs weighing 200-350 g were killed by inhalation of a rising concentration of CO2, 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, flushed clean with modified Krebs solution (∼25-28 °C: see composition below), and placed immediately into a Petri dish containing Krebs’ solution.

Dissection procedure

The preparation was incised along the mesenteric border and pinned flat with the mucosa uppermost in a Sylgard-lined Petri dish. Using sharp dissection, the mucosa and submucosa were peeled off to expose the underlying circular muscle layer. Preparations were pinned taught in the organ bath, such that the distance between either circumferential edge was ∼11-14 mm. The position of the microelectrodes could be readily adjusted to record from pairs of CM cells. The preparations were transferred and re-pinned serosal side down in a recording chamber (∼8 ml capacity) with a base consisting of a microscope coverslip that was laminated with a fine layer (∼2-3 mm deep) of Sylgard silicon (Dow Corning Corp., Midland, MI, USA). When preparations were pinned to the Sylgard, a distance of 6 mm in the circumferential axis represented approximately half of the circumference of the colon.

Electrical recordings from the circular muscle layer

Simultaneous intracellular recordings were made using two independently mounted micromanipulators (model M3301R; WPI Inc., Sarasota, FL, USA). 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 and digitised at 660 Hz to 1.5 kHz on a PC using Axoscope software (version 8.0; Axon Instruments, Foster City, CA, USA). Experiments were performed at 36-37 °C in the presence of nifedipine (1-2 μm).

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. Apamin, atropine sulphate, hexamethonium bromide (Hex), nitro-l-arginine (l-NA), nifedipine and tetrodotoxin (TTX) were all obtained from Sigma Chemical Co. (St Louis, MO, USA).

Analysis of data

Analysis of spontaneous junction potentials (sJPs) was performed using custom-written routines on short samples of recordings of electrical activity (∼2-3 min in duration) that were re-sampled to 100 Hz. A smoothing algorithm (100 ms average, 5 iterations) and an average baseline algorithm (10 s, 3 iterations) were applied to reduce high frequency noise, and for calculation of amplitudes of spontaneous junction potentials respectively.

The first recording was used to identify the peaks of sEJPs and sIJPs by locating inflexion points. The amplitude, calculated as the difference between the peak of the junction potential and the average baseline was calculated for each sJP along with the maximum slope (dV/dt), and time to half-maximum amplitude.

To determine if sJPs in the second recording were coordinated in time and had similar amplitudes to sJPs in the first recording, two different search routines were used.

The first routine used the time at the peak of a sJP in the first recording and looked for any inflexion (sIJP or sEJP) in the second recording that was closest in time to that point. Once the peak of a sJP was located in the second trace, the amplitude, maximum slope and time to half-maximum amplitude of the second peak was calculated. Results from this analysis were used to compare the similarity of the amplitudes of sJPs between the two recordings. Regression analysis was used to summarise the variability in the amplitude of sJPs between the two recordings and was expressed as the correlation coefficient r (see example in Fig. 6).

Figure 6. Coordination of spontaneous junction potentials from two circular muscle cells when the recording electrodes were separated in the longitudinal axis.

Figure 6

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Aa, simultaneous recordings from two CM cells separated by 1 mm in the longitudinal axis show synchronised sIJPs and sEJPs. Ab, the similarity of the two recordings is shown when the two recordings are superimposed. Ba, in the same animal, the two electrodes separated by 4 mm, sIJPs and sEJPs in CM1 and CM2 are now uncoordinated. Note, sIJPs are indicated (see *) in CM1 that were not correlated with sIJPs in CM2. Bb, the recordings shown in Ba are superimposed.

The second routine, instead of searching for any inflexion in the second recording, looked only for sJPs that were similar to those in the first trace (e.g. if there was an IJP in the first recording, the routine would only return the closest IJP in the second recording). Results from this analysis were used to compare differences in time between similar sJPs, and were expressed as standard deviations calculated at three different amplitude levels (1-5, 5-9 and > 9 mV).

All results are expressed as means ±s.e.m. One-way and two-way factorial ANOVA statistical tests were used to compare differences between groups. A probability of less than 0.05 was considered significant. Scheffé's post hoc tests were used. n refers to the number of animals on which observations were made.

RESULTS

Simultaneous intracellular recordings from two circular muscle cells

Simultaneous intracellular recordings were made from 156 pairs of CM cells (n = 31 animals) in the presence of nifedipine (1-2 μm). The membrane potential from all cells in the CM layer was highly unstable and revealed sIJPs and sEJPs of varying amplitudes. All preparations were dominated by sIJPs, but a proportion of cells also showed sEJPs within the same cells, an example of which is shown in Fig. 1A. The mean resting membrane potential, taken during the quiescent periods between ongoing sIJPs and sEJPs, was -35 ± 1 mV (range: -24 to -48 mV; 214 cells; n = 31).

Figure 1. Spontaneous electrical activity in circular muscle when connected and separated from the myenteric plexus.

Figure 1

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A, simultaneous intracellular recordings from two circular muscle cells (CM1 and CM2) separated by 200 μm in the longitudinal axis. Spontaneous IJPs and EJPs occur synchronously at both electrodes. B, effects of tetrodotoxin (TTX) on sJPs. TTX (1 μm), applied at arrow, immediately abolished all sIJPs and sEJPs. No miniature events could be resolved in TTX. C, when the CM was separated from the myenteric plexus, no spontaneous potentials were observed in the presence or absence of TTX. The recording shown was in the presence of TTX (1 μm).

Effects of blocking neural activity and cholinergic transmission on spontaneous junction potentials

To test the involvement of the enteric nervous system in the generation of sIJPs and sEJPs, tetrodotoxin (TTX) was added to the organ bath. In eight out of eight animals tested, TTX (1 μm) immediately abolished all junction potentials in both muscle layers (Fig. 1B). In the presence of TTX, no minature events could be resolved.

To examine the possibility that cholinergic neuromuscular transmission was involved in the generation of the sEJPs, atropine was applied to the colon, and in all of six animals tested, atropine (1 μm) consistently abolished all sEJPs.

The effect of blockade of fast nicotinic synaptic potentials on enteric neurons was then tested by application of hexamethonium. In three out of five animals tested, Hexamethonium (300 μm) abolished all spontaneous potentials, while in the remaining two animals, sIJPs were less frequent, but still persisted, suggesting that in addition to nicotinic transmission, some non-nicotinic neurotransmission may be involved in excitation of the enteric motoneurons (see Spencer et al. 2000).

Electrical activity in circular muscle when separated from the myenteric plexus

To test whether spontaneous potentials could be recorded from the CM layer when it was isolated from the myenteric plexus (i.e. when only the CM motor nerve terminals are present in the muscle) the CM layer was carefully dissected away from the LM and myenteric plexus and impalements were performed in CM strips. In six preparations (n = 4), we could not resolve any spontaneous potentials over the recording noise when the CM was disconnected from the myenteric plexus. In three preparations (n = 2), TTX (1 μm) was applied to the perfusing solution and no detectable change in the recording noise was noted (Fig. 1C).

Characteristics of spontaneous IJPs and EJPs

The majority of sIJPs and sEJPs were less than 6 mV in amplitude; however, large sIJPs and sEJPs were also recorded that often reached 20 mV in amplitude (Fig. 2A). An amplitude histogram of sIJPs and sEJPs was constructed and the data followed a Gaussian distribution (Fig. 2A). It was found that there were approximately three times as many sIJPs that were > 6 mV in amplitude as sEJPs (1034 sIJPs compared with 314 sEJPs) (154 pairs of CM cells; n = 31 animals).

Figure 2. Plots showing the characteristics of spontaneous IJPs and EJPs.

Figure 2

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A, relationship between the amplitudes (mV) of sJPs and occurrence (frequency histogram). The majority of sJPs were less than 6 mV in amplitude and followed a Gaussian distribution. The prevalence of sIJPs > 6 mV was three times greater than sEJPs > 6 mV (1034 vs. 314: n = 152). B, relationship between amplitude and maximum slope (dV/dt). Larger amplitude sIJPs (-ve) or sEJPs (+ve) were linearly related to the maximum slope, such that for every 1 mV increase in amplitude, the maximum slope increased 5 mV s−1 (y = 4.96x+ 0.14; r2= 0.92; n = 30). This relationship was reflected in the time to half-maximum (t1/2 max, C), where larger amplitude sJPs reached half-maximum amplitude at approximately the same time (150-200 ms, n = 30) as moderately sized junction potentials. Junction potentials less than 5 mV showed varying times to half-maximum, which suggests uncoordinated summation of individual motoneuron outputs to the circular muscle.

When the maximum slopes (dV/dt) of sEJPs and sIJPs were compared with the relative amplitudes of spontaneous potentials, a linear relationship was found, such that increasing amplitudes of sEJPs and sIJPs showed a corresponding increase in their maximum dV/dt (Fig. 2B). The increasing rate-of-rise with amplitude suggested that larger sJPs were depolarising/hyperpolarising faster than smaller potentials (Fig. 2B). This indicated that the peaks of sJPs would occur at approximately the same time, regardless of amplitude. Indeed, this was the case as the time to half-maximum amplitude of sJPs from 5 mV to 25 mV was constant around 180 ms (Fig. 2C).

Correlation of junction potential amplitudes with distance

Circumferential axis

To examine whether sJPs in the CM layer were local events that occurred in a small population of muscle cells, or whether they could be recorded synchronously over large regions of smooth muscle, simultaneous recordings were made from two CM cells at increasing electrode separation distances. To do this, we first recorded from two CM cells when the two electrodes were separated by 100 μm (n = 8) in the circumferential axis. The distance between the two electrodes was then increased in successive steps to 200 μm (n = 8), 500 μm (n = 8), 1 mm (n = 13), 2 mm (n = 8), 3 mm (n = 7), 4 mm (n = 9) and 6 mm (n = 7).

In all animals tested, when the two electrodes were separated by 100-200 μm circumferentially, both small and large amplitude sIJPs and sEJPs always had similar amplitudes (Fig. 3A). This similarity was reflected in a linear relationship between the amplitudes of sJPs from both recording sites (see Fig. 4A). As the distance between the electrodes was increased, even up to 6 mm circumferentially, the amplitudes of sIJPs and sEJPs still remained remarkably similar at both recording electrodes (Fig. 3C and Fig. 4B). In fact, in three pairs of CM cells (n = 2), when we separated the two recording electrodes by 11 mm in the circumferential axis (i.e. the distance from one circumferential edge of the colon to another) sIJPs still remained phase locked, with near-identical amplitudes at both electrodes, suggesting that the entire circumference of the colon had been polarised simultaneously (Fig. 5).

Figure 3. Simultaneous recordings from the circular muscle layer at varying distances in the circumferential axis.

Figure 3

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A, simultaneous recordings from two circular muscle cells (CM1 and CM2), when the two electrodes were separated by 200 μm in the circumferential axis. Large sIJPs can be seen to occur synchronously at both recording electrodes. B, the similarity in the recordings is shown when the traces are superimposed. C, in the same animal, the electrodes were separated by 6 mm in the circumferential axis and large sIJPs are still shown to occur synchronously. Small sIJPs were not as well correlated at the two electrodes (see *). D, the traces in CM1 and CM2 are superimposed.

Figure 4. Individual examples of the coordination of the amplitude of spontaneous junction potentials when compared at the two recording electrodes at 100 μm and 6 mm in the circumferential and longitudinal axis.

Figure 4

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In the circumferential axis, spontaneous junction potentials closely resembled each other regardless of the separation between the electrodes (A and B). In the longitudinal axis, spontaneous junction potentials were well coordinated at small separations between the electrodes (C); however, at greater separations (> 3 mm), the amplitude of the spontaneous junction potentials became uncoordinated (D). For example, at 6 mm longitudinal separation, a large sIJP recorded in the first electrode occurred with a small sEJP at the second electrode (see arrow). Note coordinated sEJPs occur in the upper right quadrant and sIJPs in the lower left quadrant.

Figure 5. Simultaneous recordings from pairs of circular muscle cells separated by various distances in the circumferential axis.

Figure 5

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A, when the simultaneous recordings were made from two CM cells, separated by 11 mm in the circumferential axis (i.e. the distance from one circumferential edge to the other), sIJPs can still be seen to occur synchronously. B, in another pair of CM cell recordings separated by 4 mm in the circumferential axis, two sEJPs can be seen to have occurred synchronously at both recording sites. To confirm that the events were not due to a passive flow of membrane current between the two electrodes, brief current pulses (300 pA) were passed out of each recording electrode (i.e. into CM1 and CM2). It can be seen that electrotonic potentials generated at each current-passing electrode were not recorded at the second recording site.

Longitudinal axis

Simultaneous recordings were made from two CM cells at increasing electrode separation distances in the longitudinal axis, in successive steps from 100 μm (n = 10), 200 μm (n = 10), 500 μm (n = 9), 1 mm (n = 9), 2 mm (n = 8), 3 mm (n = 8), 4 mm (n = 8) and 6 mm (n = 6). When the two microelectrodes were separated by 100 μm, the amplitudes of sJPs at both recording sites were found to be linearly related (Fig. 4C), as was the case for equivalent distances of separation in the circumferential axis (cf. Fig. 4A and C). That is, as the amplitude of the sJP increased at one electrode, it also increased to a similar degree at the second electrode (Fig. 6Aa). However, as the mean distance between the two electrodes increased past 2 mm (range: 1-4 mm), the amplitudes of the events became more uncorrelated (Fig. 6Bb), such that sEJPs and sIJPs were never coordinated when the two electrodes were separated by 6 mm (n = 6) in the longitudinal axis. In fact, in only one of eight animals were sIJPs correlated at 4 mm.

Correlation coefficients of the amplitudes of spontaneous junction potentials

Over the range of electrode separation distances tested, it was found that the correlation between the amplitudes of sJPs at the two recording sites remained remarkably stable in the circumferential axis at r∼0.9, even up to 6 mm separation (Fig. 3C and Fig. 7). A slight, but statistically significant decline in the correlation coefficient was noted beyond 3 mm separation compared with 100 and 200 μm separation (Fig. 7).

Figure 7. Correlation coefficients of the amplitudes of spontaneous junction potentials from the two recording sites at different distances of separation in the circumferential (□) and longitudinal (•) axes.

Figure 7

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In the circumferential axis, correlation coefficients remained stable across all distances of separation. In the longitudinal axis, correlation coefficients were high at less than 500 μm separation, but dramatically declined at greater distances of separation, particularly beyond 2 mm, indicating poor correlation between the recordings. *P < 0.05 compared with same axis (subscript number denotes the other distance of separation in which a statistical difference was found). †P < 0.05 compared with the same distance of separation in the other axis.

Interestingly, in the longitudinal axis, the level of correlation between the amplitude of sJPs from both recording sites was maintained, and even had a tendency to increase (not significant) up to an electrode separation of 2 mm (Fig. 7). At electrode separations greater than 2 mm, the correlation coefficients declined rapidly, indicating that there were few sJPs that were of similar amplitudes at the two recording sites (Fig. 6). Correlation coefficients less than ∼0.7 are indicative of poor correlation (Fig. 7). An example of the poor correlation between the amplitudes of sJPs at the two recording sites, at 4 mm longitudinal separation is shown in Fig. 6Ba.

Correlation of the timing of spontaneous junction potentials

Circumferential axis

We correlated the time difference between sJPs reaching their peaks, with respect to the relative amplitudes of sJPs at the two recording sites, and the distance between the recording electrodes. In the circumferential axis, increasing electrode separation distances had little effect on the time difference between sJPs at the two recording sites, both traces being superimposable (cf. Fig. 8A and B). However, there were consistent differences in the time difference between sJPs with regard to the different amplitudes of sJPs. The peaks of large amplitude sJPs were well synchronised (Fig. 8A and B), evidenced by low (∼30 ms; Fig. 9A) standard deviations in the time between the peaks of sJPs. In contrast, the time at which smaller amplitude sJPs reached their peaks was much more variable between the two recordings sites (Fig. 9A).

Figure 8. Relationship between the time difference (Δt) between the peaks of spontaneous junction potentials and their amplitudes when recordings were made at 100 μm and 6 mm electrode separation in the circumferential and longitudinal axis.

Figure 8

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A and B, in the circumferential axis, small amplitude sIJPs and sEJPs were not well correlated in time between the two electrodes. However, the larger amplitude sIJPs were particularly well coordinated in time at the two recording sites (see A and B). C and D, in the longitudinal axis at close electrode separations (see C), it can be seen that the larger amplitude sIJPs showed greater synchronisation than smaller potentials, as is shown in A and B. However, at large electrode separation distances longitudinally (D), the time between the peaks of the sJPs varied widely, indicating poor coordination. Note increased scatter of points in D.

Figure 9. Time variation between spontaneous junction potentials from both recording sites at different distances of electrode separation in the circumferential and longitudinal axis.

Figure 9

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Larger amplitude junction potentials consistently had smaller time variations between the peaks of sJPs from both electrodes (▵). A, the time variation remained stable at all distances of separation in the circumferential axis. However, in the longitudinal axis (B), the time variation of large spontaneous potentials increased noticeably at separations greater than 3 mm, indicating that these potentials were not well correlated. *P < 0.05 compared with 1-5 mV and **P < 0.05 compared with 5-9 mV amplitudes in the same axis. †P < 0.05 compared with same amplitudes in the other axis. s.d., standard deviation.

Longitudinal axis

The difference in time between the peaks of sJPs at small electrode separations in the longitudinal axis (< 200 μm) was very similar to the standard deviations measured at the same distances of electrode separation in the circumferential axis (cf. Fig. 8A and C). Larger amplitude sJPs were also well synchronised (s.d.∼30 ms; Fig. 9B), whereas smaller sJPs were less synchronised, with standard deviations approaching 150 ms. Between 200 μm and 2 mm electrode separation there was an increase in the standard deviation of smaller sJPs (< 5 mV) compared with circumferential separation, but not with the larger amplitude sJPs which remained well synchronised in time (cf. squares and triangles in Fig. 9B). However, beyond 2 mm separation distance there was a dramatic increase in the overall time difference between peaks, particularly with the large amplitude sJPs (Fig. 8D, and see ▵ in Fig. 9B), that was significantly different compared with circumferential separation (Fig. 9, cf. ▵ in A and B). The standard deviation of medium sized JPs (5-9 mV) also tended to increase with increasing separation distance in the longitudinal axis (see • in Fig. 9B). However, because of the variation between animals, a statistical difference was not obtained. These results suggest that small sJPs are less synchronised in the longitudinal axis compared with the circumferential axis, and that large amplitude sJPs become unsynchronised in both time and amplitude at distances that are usually greater than ∼2 mm in the longitudinal axis.

Effects of local current injection into circular muscle

To confirm that the similarity in sIJP and sEJP amplitudes over large areas of CM was not due to local current dissipation from a single point source (i.e. transmitter release from distant varicosities), current was passed from one microelectrode, and the effect on membrane potential at the second electrode was observed. When the two electrodes were separated by < 50 μm, large electrotonic potentials were capable of being elicited at the second electrode (Fig. 9A). However, when the electrodes were separated by 100 μm or greater, even large current injections (1-8 nA) via one electrode was only capable of generating small electrotonic potentials (< 2-3 mV) at the second electrode, or alternatively no electrotonic potentials at all. The current-voltage relationship of the CM layer was found to be linear over a range of potentials, with no evidence of rectification at either positive (1-8 nA, n = 4) or negative potentials (-1 to -8 nA, n = 8) (Fig. 10B). The input resistance of the CM cells ranged from 1.2 to 2.2 MΩ (n = 3-8). It was not possible to identify whether action potentials could be evoked in response to outward (depolarising) current injection, since experiments were performed in nifedipine.

Figure 10. Effects of current injection into a single circular muscle cell.

Figure 10

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A, when the two electrodes were separated by 25 μm in the circumferential axis, inward and outward current clamp (+ve 1-8 nA to -ve 1-8 nA) through one electrode generated electrotonic potentials in the neighbouring CM. B, voltage-current (V-I) relationship of the CM layer. A near-linear V-I can be seen, with no pronounced rectification at positive or negative potentials. C, effects of l-NA and apamin on sIJPs in CM layer: a, sIJPs in CM in control solution; b, following addition of l-NA (100 μm), the slow repolarisation phase of sIJPs was abolished, without any effect on the amplitude of the ‘fast’ sIJPs; c, in the presence of l-NA, the further addition of apamin (500 nm) abolished l-NA-resistant sIJPs and induced small sEJPs.

Effects of l-NA and apamin on spontaneous IJPs in the CM layer

To test the possibility that release of nitric oxide may underlie the generation of sIJPs in the CM layer, l-NA (100 μm) was added to the bath. Addition of l-NA (100 μm) depolarised the resting membrane potential CM cells in three of six animals, while in the three other animals l-NA had no effect (control: 43.2 ± 3.4 to 36.8 ± 2.2 mV; n = 6, P = 0.12). The waveform of sIJPs was altered by l-NA by substantially decreasing the slow repolarisation phase of sIJPs in the CM layer, and revealed a ‘fast’ sIJP in all six animals tested (Fig. 10Cb).

The existence of l-NA-resistant sIJPs suggests that at least two inhibitory neurotransmitters were being released synchronously from the CM motor neurons. To determine whether small conductance SK-type K+ channels were involved, apamin (500 nm) was added to preparations already exposed to l-NA and was found to abolish l-NA-resistant sIJPs (n = 6) (Fig. 10Cc). To confirm that apamin (500 nm) alone did not block the sIJPs in both muscle layers, it was applied first, which substantially reduced the amplitude of the ‘fast’ component of sIJPs in the CM layer. The further addition of l-NA to these animals abolished the residual slower component of sIJPs. In 4 of the 9 animals that were exposed to apamin (with or without l-NA present), sEJPs were induced in the CM layer (Fig. 10Cc) and these were abolished by atropine.

DISCUSSION

The major finding of this study was that TTX-sensitive sIJPs and sEJPs in the CM layer of the colon can be recorded synchronously over large regions of the smooth muscle syncitium. This suggests that spontaneous potentials in the colon are not monoquantal events as described in other autonomic neuromuscular junctions (see Introduction), but rather are generated by the simultaneous release of transmitter from multiple release sites, due to the synchronous activation of many enteric motor neurons.

Correlation of spontaneous IJPs and EJPs in the circumferential and longitudinal axis

Large sJPs (i.e. > 9 mV) in the CM layer were generally well correlated in both time course and amplitude over at least 6 mm around the circumferential axis, since independent recordings from two distant sites were superimposable. The fact that large amplitude sJPs were recorded synchronously at opposite ends of the circumference of the colon (i.e. 11 mm in the circumferential axis) suggests that many inhibitory motor neurons around the circumference of the colon fired synchronously (see below). However, the distance over which large sJPs were synchronised in time and amplitude was markedly shorter in the longitudinal axis (mean ∼2 mm; maximum 4 mm) than in the circumferential axis (Fig. 9B). This is consistent with the view that the nerve endings of circular muscle motor neurons preferentially exhibit circumferential orientation (Bornstein et al. 1986; Smith et al. 1988; Brookes & Costa, 1990; Brookes et al. 1991). Interestingly, since the nerve terminals of enteric motoneurons in the muscle only project up to 30-50 % around the circumference of the gut wall (Brookes & Costa, 1990; Brookes et al. 1991), then in the light of our data that sIJPs can occur synchronously at either circumferential extremity (i.e. 11 mm apart), this means that the generation of these sIJPs must involve synchronous firing of many interneurons (and/or sensory neurons) that synapse with many inhibitory motoneurons. This is further supported by the observation that sJPs were hexamethonium sensitive. Also, sectioning the preparation into small segments either abolishes or dramatically reduces the amplitude and frequency of spontaneous potentials (N. J. Spencer & T. K. Smith, unpublished observations).

On the other hand, the smaller amplitude sJPs were much less correlated in time and distance in both axes. The larger standard deviations in timing between smaller amplitude sJPs (Fig. 9) is likely to result from less synchronised activation of a smaller population of motor neurons. The summation of many unsynchronised synaptic potentials tends to produce a waveform that has a more variable slope, amplitude and time to peak (Fig. 2C). This was particularly visible in the large variance in the time to half-maximum of small sJPs (< 5 mV; Fig. 2C).

Relationship to other neuromuscular junctions

The findings of this study suggest that sJPs in the colon are different from sEJPs reported in other neuromuscular regions (e.g. vas deferens, bladder, seminal vesicles or arterioles). The recording of sJPs in these other tissues has led to the idea that the sEJP represents the release of single quanta of sympathetic transmitter from a varicosity in close apposition to the smooth muscle (Burnstock & Holman, 1962; Cunnane & Stjarne, 1984). Single quanta only polarise a small population of muscle cells close to the release site, owing to the rapid dissipation of current into the smooth muscle syncitium (Tomita, 1967; Hirst & Nield, 1980; Hirst & Edwards, 1989). Also, sEJPs are not dependent upon action potential conduction along a nerve because they are insensitive to TTX (Cunnane & Stjarne, 1984). Tomita (1967) reported that sEJPs in the vas deferens occurred asynchronously at two electrodes, even when separated by only 100 μm. Similarly, Bramich & Brading (1996) reported that sEJPs in the urinary bladder were asynchronous at two intracellular microelectrodes when the electrodes were separated by only 40 μm. However, Bramich & Brading (1996) observed that when extrinsic nerves were stimulated, evoked EJPs of similar amplitude and time course were recorded at the two sites. A major difference between evoked and spontaneous EJPs in the arterioles (Hirst & Nield, 1980), or vas deferens (see Burnstock & Holman, 1962; Cunnane & Stjarne, 1984) is that the time course of the sEJP is very brief compared with the evoked EJP from electrical stimulation of extrinsic nerves. The evoked EJP is due to the simultaneous depolarisation of the whole muscle bundle because of the synchronous release of transmitter from many release sites throughout the muscle, rather than release of transmitter from a point source, as is the case with the sEJP (see Hirst & Nield, 1978). In contrast, the larger sJPs we report in the colon are clearly not local events since they can be recorded synchronously, with near-identical amplitudes and time courses, over large regions of smooth muscle (up to 11 mm × 4 mm). Therefore the large amplitude sJPs cause synchronous polarisation of the entire circumference of the colon. Importantly, sIJPs and sEJPs in colonic muscle have been shown to exhibit the same time course and amplitude as an evoked JP from electrical stimulation or reflex distension (Furness, 1969; Smith, 1989; Smith et al. 1992; Spencer et al. 1998a). Therefore, the spontaneous polarisation of large areas of circular muscle in the colon are due to coordinated synchronous firing of many enteric motor neurons. It seems highly unlikely that even the smaller sJPs in the colon could be due to passive flow of current originating from transmitter release at distant varicosities, as local injection of current through one microelectrode rapidly dissipated into the CM layer and electrotonic potentials were rarely, if ever, detected at distances greater than 100 μm from the current-passing electrode. The short length constants suggest that the correlation and coordination of junction potentials is due to the synchronous release of a similar output of transmitter from the motor nerve terminals that project over these distances. Also, we have not been able to resolve any minature quantal events, even in isolated CM devoid of enteric ganglia (Fig. 1C). This suggests that either: (1) there is no spontaneous transmitter release from isolated varicosities in the intestine, or (2) the membrane current underlying ‘quantal’ events is dissipated so rapidly into the smooth muscle syncitium that the events are within the recording noise. Alternatively, (3) spontaneous quantal events may not be detectable by recording from the smooth muscle because most nervous activity in the intestine appears to be relayed or transduced to the muscle through interstitial cells of Cajal (ICC) (Ward et al. 2000). Also, (4) since the activation of sJPs in intestinal smooth muscle involves second messenger systems, it is possible that synchronous release of inhibitory (NO) or excitatory (ACh) transmitter from many release sites is necessary to raise intracellular second messengers in the muscle sufficiently to open channels underlying sIJP or sEJP formation. Spontaneous JPs in the colon also show a long latency of channel activation compared with the sEJPs recorded at other autonomic neuromuscular junctions which are mediated via ligand-gated ion channels (Brock & Cunnane, 1992) in which events due to discrete ‘quanta’ can be clearly resolved from the recording noise. Clearly the sIJP and sEJP in colonic smooth muscle represent the summed activity of release sites from multiple axon terminal varicosities, due to the synchronous firing of many motor neurons.

Spontaneous junction potentials and synchronous activity in motor neurons

It has been demonstrated that graded short duration electrical stimuli can evoke IJPs or EJPs that show graded increases in amplitude with increasing stimulus intensity (Tomita, 1972). This suggests that more than one motor neuron innervates any given muscle cell and that it is possible to recruit more inhibitory or excitatory CM motor neurons to cause a large evoked junction potential. In light of this, it seems that since evoked and spontaneous JPs in the colon can show identical amplitudes (see above) it is likely that the great majority of the enteric inhibitory and excitatory motoneurons that innervate a particular region of muscle must fire simultaneously to generate the large spontaneous sIJPs and sEJPs, respectively. It has been estimated that circular smooth muscle cells are innervated by approximately 400 inhibitory motor neurons per millimetre length of bowel (Furness & Kunze, 1999). ‘If a single axon contributes 0.5 mV to the IJP …’ (Bornstein et al. 1986), then assuming this is so, given the caveats (see 3 and 4) above, then a 25 mV sIJP in the colon could be due to the synchronous activation of at least 50 inhibitory motor neurons converging onto the muscle around the recording site. Interestingly, in the same tissue, Furness (1969) described spontaneous junction potentials in the CM layer that only occurred in < 1 % of cells. He stated that ‘… experiments were carried out with the tissue pinned out without stretching’. In his study, the maximum amplitude of sIJPs was 3 mV and that of sEJPs, 8 mV (Furness, 1969). In our study, all impalements showed both sIJPs and sEJPs, and these often reached 20 mV in amplitude. It is quite likely that the spontaneous potentials we describe here are a result of the greater degree of circumferential stretch applied to the colon.

Conclusions

Spontaneous junction potentials in colonic smooth muscle are clearly not generated by single quanta, but involve the simultaneous release of transmitter from multiple release sites. The variability in amplitude of spontaneous junction potentials is likely to be due to a variable number of enteric motor neurons recruited synchronously, combined with slight differences in the timing of transmitter release, rather than one motor neuron releasing a variable output of transmitter. Large amplitude sEJPs and sIJPs (i.e. ∼20-30 mV) can be recorded synchronously over large regions: up to 11 mm in the circumferential axis and between 1 and 4 mm in the longitudinal axis of circular muscle.

Acknowledgments

We wish to acknowledge the helpful comments of Professor D. G. S. Hirst, Department of Zoology, University of Melbourne. Financial support for this project was provided by the National Institute of Health (USA) (Grant No. RO1 DK45713 to T.K.S).

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