Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 µm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 µM) or tetrodotoxin (1 µM) either reduced the frequency of SDs or temporily abolished activity, whereas nifedipine (1 µM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca2+ channels.
interstitial cells of Cajal; slow wave; pacemaker cell
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INTRODUCTION |
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MOST INTRACELLULAR MICROELECTRODE recordings of electrical activity in the smooth muscle of the gastrointestinal tract have been made from the relatively thick circular muscle (CM) layer. These studies (5, 7, 10-12, 17, 25, 27-30, 41) have consistently reported spontaneous depolarizations (SDs) in membrane potential (slow waves) in a variety of species. In contrast to the CM, very few intracellular recordings have been made from the longitudinal muscle (LM) layer that largely exhibits spontaneous action potentials superimposed on slow waves (5-7, 12, 21, 22, 29, 34, 35). As a result, little is known about the electrical events underlying the movements of the LM. During propulsive movements, such as peristalsis, it is now clear that in both the small and large bowels, the LM and CM contract and relax together (31-32). Moreover, in stretched preparations during nonpropulsive movements, the LM layer is characterized by a high degree of spontaneous activity (5, 6, 12, 14, 18, 21, 22, 34, 35, 37, 38) yet, little is known about the ionic mechanisms underlying rhythmicity in the LM.
Although slow waves are common in intestinal smooth muscle of many mammalian species, they are rarely reported from the guinea pig intestine with microelectrodes (21, 22, 27). In the guinea pig ileum, Kuriyama and Tomita (22) reported that the LM fired irregular bursts of action potentials, or alternatively, a tonic discharge of action potentials. There have been very few intracellular recordings from LM in guinea pig colon (34, 35). A recent study from this region found that the LM fired spontaneous burst action potentials in unparalyzed preparations (34, 35). Slow waves have been reported from the guinea pig distal colon using extracellular suction electrodes (28).
Little is known of the morphological characteristics of LM cells in guinea pig distal colon. When neurobiotin is injected into one of these LM cells (dimensions 300 × 10 µm), it often spreads into neighboring cells (35). Also, calcium waves, which appear to depend on the conduction of action potentials, spread across many (>50) LM cells (37). These observations suggest that LM cells in the guinea pig distal colon are coupled, but by what mechanism remains unclear.
Interestingly, electron microscopy studies of the colon have found little evidence for functional gap junctional coupling between LM cells and between interstitial cells of Cajal at the level of the myenteric plexus and LM (2, 7, 8, 40). Further support for this comes from a recent study (40) using electron microscopy to show that immunoreactivity to connexin 43 was not observed in regions lacking gap junctions. Therefore, the mechanisms of propagation of slow waves and action potentials in LM remains unclear. It has been suggested that both low-resistance pathways through gap junctions and electric field coupling between adjacent smooth muscle cells (39) may coordinate the spread of excitability in gastrointestinal smooth muscles. It is clear, however, that electrotonic potentials, which decrease exponentially with distance, can be generated in LM cells of guinea pig intestine (4) and taenia coli (22), suggesting that current flow between neighboring cells is possible within this muscle layer. It was recently found with simultaneous calcium imaging of LM and CM (38) and during simultaneous electrical recordings from LM and CM (35) that neither spontaneous calcium waves nor action potentials in the LM ever propagated into the CM and vice versa. This suggests poor electrotonic coupling between the two muscle layers, supporting previous findings with electron microscopy studies. In contrast to myenteric interstitial cells of Cajal (ICC-MY), intramuscular ICC (ICC-IM) make close contacts with CM (9), usually <20 nm, and have been shown to have morphological connection via gap junctions to neighboring smooth muscle (2, 9).
One of the major obstacles in characterizing the propagation of action potentials in smooth muscle is that they usually generate contraction and muscle movement therefore, making microelectrode impalements difficult. As a result, Ca2+-channel blockers are commonly used to paralyze the muscle (prevent action potentials) and aid microelectrode impalements. However, it has recently become clear that muscular paralysis has profound effects on the spontaneous neuronal activity in the bowel (19, 20, 36), which may have pronounced effects on slow-wave activity. For example, it has long been known that ongoing enteric neuronal inputs can modify colonic slow waves (25). To avoid these limitations, we have recorded from the LM layer in the absence of an antagonist for L-type Ca2+ channels. Therefore, the major aims of this study were to 1) characterize the spontaneous electrical activity in the LM layer of a contracting (unparalyzed) tissue, 2) determine the ionic basis of this activity, and 3) investigate the extent of propagation of spontaneous action potentials around the circumference of the colon using simultaneous intracellular recordings from pairs of LM cells. A preliminary form of these data has been presented in abstract form (34).
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METHODS |
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Guinea pigs weighing 200-350 g were euthanized by CO2 inhalation overdose, 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 were removed, the mesenteric attachment was trimmed away, and the lumen was flushed clean with modified Krebs solution composition.
Dissection Procedure
The distal colon was opened along the mesenteric attachment, and the terminal distal region was pinned to the base of a Sylgard (Dow Corning)-lined Petri dish, so that the mucosal surface faced uppermost. The mucosa and submucosa were then sharp dissected from this open region to expose the underlying CM. Strips of CM were then dissected from the preparation to expose the underlying myenteric plexus and LM layer (as shown previously in Ref. 35). Therefore, these preparations included an island of LM and associated myenteric plexus that remained in neural continuity with the CM. This dissection procedure enabled us to clearly visualize the myenteric ganglia and underlying LM in one region while also identifying the thicker CM in the same field of view as we have previously described (35). Therefore, microelectrodes could be positioned so as to record unequivocally from LM cells. In vitro preparations studied were ~25 mm in length, and when pinned out in a recording chamber, the circumferential axis measured 11-13 mm (approximately twice their resting width). In all experiments, preparations were pinned serosal side down, in a recording chamber whose base consisted of a microscope coverslip lightly coated with a fine layer of Sylgard silicon (Dow Corning, Midland, MI). Unambiguous identification of the LM layer was aided by the use of an inverted microscope (model CK2; Olympus, Napa, CA). In some experiments, we recorded from LM strips devoid of any myenteric ganglia. In these preparations, LM strips were peeled off the myenteric plexus. These were between 2-5 × 10-15 mm in length when stretched to approximately twice their resting width. Confirmation that these preparations were devoid of myenteric ganglia was readily made with the use of an inverted microscope and by immunohistochemical studies that revealed these tissues only contained ICC-IM (see Fig. 10B).Electrical Recording Technique
Simultaneous intracellular microelectrode recordings were made from LM cells with the use of two independently mounted microelectrodes whose fine positioning could be adjusted using two micromanipulators (model M3301L; World Precision Instruments, Sarasota, FL). Electrodes were filled with 1.5 M KCl solution and had resistances of about 120 MAnalysis of the Coordination of Electrical Activity Between Two Recording Sites
Recordings of membrane potential changes from two separate recording electrodes were imported into a modified version of NIH Image 1.62 software. The two traces were resampled (1,000 Hz for 0.1 and 1.0 mm; 500 Hz for 13 mm electrode separation) and smoothed (25-ms average, 2 iterations). The two traces were then differentiated (sampled in time steps of 1 ms for 0.1 and 1.0 mm and 2 ms for 13 mm electrode separation) and plotted against each other such that the rates of change in membrane potential (dV/dt) from electrode 1 were plotted on the x-axis, and the rates of change in membrane potential from electrode 2 were plotted on the y-axis (see Fig. 2). Ideally, if electrical activity (i.e., depolarization and repolarization phases) was identical and coincident at both electrodes, this would be reflected as a line extending at 45° through both positive and negative quadrants, as the changes in membrane potential in one axis were matched by the changes in membrane potential in the other axis. In practice, however, even the waveforms of well coordinated electrical activities differ considerably, because their electrical waveforms have varying rates of rise and decay. In addition, shifting pacing sites and differing conduction pathways between the two electrodes produce irregular conduction velocities that also contribute to meandering trajectories that tend to the 45° line (see Fig. 2A). In contrast, any activity occurring solely in one electrode and not at the other would appear as a single line along its respective axis; unrelated activities at both electrodes generating a cruciform appearance (see Fig. 2, D and E, 1 mm circumferential separation).To quantify the degree of coordination of electrical activity between the two electrodes, the following method was used. The proportion of the total trace, in which positive (depolarizing) membrane potential changes occurred at both electrodes, was calculated by adding all the time points located in the upper right hand quadrant (trace 1 positive/trace 2 positive quadrant; see Fig. 2A) and dividing it by the total number of time points spent in all quadrants (excluding any time points less than the electrode noise level of 25-50 µV/ms). Thus this proportion expressed the percentage of the total trace in which coordinated depolarizations (CDs) were present.
It was expected that at greater electrode separation distances the time
taken for the action potential to spread through the LM syncitium would
increase. To account for this delay, a sequence of plots was
constructed where the two recordings of membrane potential were time
shifted with respect to one another (4.0 to +4.0 s, 5-ms increments).
If the two traces still had coordinated electrical activity, this would
appear as a distinct peak of %CD at some time shift (see Fig.
2C). Examples can be seen in Fig. 3, A and
D. An average plot of %CD versus time shift was constructed for each electrode separation (n = 9 for 0.1 and 1 mm;
n = 5 for 13 mm). Using the time at which peak
coordination occurred, traces were then phase shifted by this amount,
and revised trajectory plots were constructed that showed the most
synchronized orbits that could be obtained for this level of
coordination. For example, the more coordinated trajectory in Fig.
2B is the result of phase shifting the trajectories shown in
Fig. 2A by
15 ms, which represents the average conduction
delay between the two sites.
Immunohistochemistry
Freshly dissected segments of distal colon were incised along the mesenteric border and pinned out in a small Sylgard-lined Petri dish in room temperature Krebs solution. The mucosa and submucosa were sharp dissected off the tissue so that the CM layer faced uppermost. The tissues were then fixed in acetone at 4°C for 10 min. Tissues were then washed several times with PBS buffer (0.01 M) for ~2 h to remove the acetone. Preparations were then blocked for 1 h using 1% BSA at room temperature, and then the primary antibody (ACK2; GIBCO-BRL, Gaithersburg, MD) was applied (5 µg/ml) overnight. In the morning, preparations were then washed for 3-4 h with PBS at room temperature and the secondary antibody was applied (IGG raised against rat) and tagged with FITC (1:100 dilution) for 1 h. Tissues were then rinsed in PBS for ~3-4 h (changing solutions every 20 min), and a confocal microscope (Bio-Rad MRC 600) was used to visualize ICC using excitation wavelength appropriate for FITC fluorescence (488 nM). Z-series of up to 11 images through depths of 1 µm were collected, and final images were projected using Bio-Rad COMOS software.Drugs and Solutions
The following drugs were used throughout the current study: atropine sulfate, nifedipine, and TTX, (all from Sigma, St. Louis, MO). Nifedipine was prepared at a stock concentration of 10Measurements and Statistics
Student's paired t-tests were used where appropriate. A minimum significance level of P < 0.05 was used throughout. The use of n in the results section refers to the number of animals on which observations were made, and data are presented as means ± SE. Measurements of amplitude and half-width and time-to-peak response were made using Axoscope 8.0 (Axon Instruments, Foster City, CA). The propagation velocity of action potentials was established by dividing the distance an action potential traveled over the time taken to travel this distance (measured from the half-amplitude point on the rising phase of the spike). Because SDs in LM usually generated smooth muscle action potentials and it was not possible to clearly discern the amplitude of the spike from the amplitude of the SD, in RESULTS, the amplitudes of the action potentials include the amplitude of the underlying SD. These were measured from 10% peak amplitude point (on the rising phase) to the peak of the action potential. ![]() |
RESULTS |
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Spontaneous Electrical Activity in Unparalyzed LM
In a spontaneously contracting, stretched preparation of distal colon, simultaneous intracellular recordings were made from 61 pairs of LM cells (n = 20). In all LM cells, the resting membrane potential was highly unstable and showed SDs that often reached threshold for the generation of smooth muscle action potentials (Fig. 1). When SDs did not reach threshold for the generation of action potentials, the mean amplitude of the SD was 12 ± 1 mV (range 4-24 mV, 66 LM cells, n = 20; taken from the 10% peak amplitude point of the rising phase of the SD) and had a mean half duration of 198 ± 22 ms (range 15-802 ms, 66 LM cells, n = 20). There was some variability in the mean interval of SDs between animals (mean interval 3.4 ± 0.4 s, range 0.3-12.8 s, n = 20). The mean resting membrane potential of the LM, measured after dislodgement of the electrode was
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Extent of Propagation of Action Potentials in LM
Circumferential axis.
Using simultaneous recordings from pairs of LM cells, investigations
were made as to how far spontaneous action potentials and SDs would
propagate around the circumference of the distal colon. In nine pairs
of LM cell recordings (n = 8), when the two electrodes
were separated by 100 µm circumferentially, SDs and associated action
potentials were phase locked between the two recording electrodes (Fig.
1). The mean propagation time between the two electrodes at 100 µm
separation was 32 ± 9 ms (range 6-81 ms, 9 LM pairs,
n = 8). This gave a mean propagation velocity of 3.1 mm/s circumferentially (range 1.2 to 18 mm/s, 9 LM pairs, n = 8). The coordinated electrical activity was also
apparent in plots of dV/dt (Fig.
2, A and B), where
the majority of activity occurred in the trace 1 + ve,
trace 2 + ve, trace 1 ve, and trace
2
ve quadrants. In some instances, the location where the
action potential was detected first was not stable, at first leading
for a period of time in one electrode before moving closer to the other
electrode. This can be seen in the example in Fig. 2A, where
a bifurcation in the shape of the orbit occurred producing two distinct
depolarizing trajectories that gave rise to a "wishbone shape"
(c.f., arrows 1 and 2 in Figs. 2A and
6D). This suggests that the location of pacemaker activity
is not fixed for long periods of time.
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Longitudinal axis.
SDs and action potentials were found to propagate considerably more
rapidly in the longitudinal axis than in the circumferential axis. In
the longitudinal axis, the mean conduction time for slow waves to
propagate over 1 mm was 17.3 ± 4.1 ms (range 9-25 ms, 4 LM
pairs, n = 3). This gave a mean propagation velocity of
57.8 mm/s (range 40-111 mm/s, 4 LM pairs, n = 3).
In five of five pairs of LM cells (n = 3), the activity
of SDs and action potentials was compared between the two electrodes
when separated by 1 mm in the longitudinal axis. At this separation
distance, spontaneous action potentials were found to consistently
propagate between the two electrodes (Fig.
7, A and B).
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Is There a Role for TTX-Sensitive Na+ Current in Rhythmicity and Action Potential Generation in LM?
TTX was applied to the colon to test whether blockade of enteric nervous activity may modify spontaneous electrical activity in LM. The effects of TTX on rhythmic activity were somewhat variable between animals. In four of the five animals, TTX caused an initial block or marked reduction of all spontaneous spiking without any significant effect on the resting membrane potential (control 37.9 ± 3.2 mV, TTX 38.9 ± 3.3 mV, P = 0.38, n = 5) (Fig. 8A). In three of these animals, interestingly, rhythmic spiking resumed spontaneously in the LM layer after 3-10 min. In one animal, however, TTX abolished spontaneous activity and was not restored, even 20 min after the application of the drug. In another animal, TTX did not abolish spontaneous spiking, but slowed the frequency of rhythmical spiking. In the four animals where spontaneous action potential firing was restored while in the maintained presence of TTX, it was found that there was no significant effect of TTX on the spike amplitude (control 29.3 ± 4.6 mV, TTX 25.3 ± 5.1 mV, n = 5, P = 0.38) or half duration (11.3 ± 2.5 ms, TTX 26.2 ± 16.3 ms, P = 0.39, n = 5). In these four animals where spontaneous spiking was finally restored, there was no overall significant effect of TTX on the interval between action potentials (control 1.33 ± 0.46 s, TTX 7.82 ± 5.95 s, P = 0.32, n = 4).
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Effects of Nifedipine and Atropine on SDs and Action Potentials
Slow waves reported in other intestinal smooth muscles such as the mouse ileum (41) and guinea pig stomach (11) are resistant to L-type Ca2+ channel blockers such as nifedipine. We tested whether nifedipine would affect the SDs and action potentials in the LM. In all of five animals tested, nifedipine (1 µM) abolished action potentials and also, surprisingly, the underlying SD (Fig. 8C). Resting membrane potential was not significantly modified by blockade of L-type Ca2+ currents (control 43 ± 1 mV, nifedipine 42 ± 2 mV, P = 0.88, n = 4) (Fig. 8C). To test whether activity in cholinergic motor neurons may be involved in rhythmic activity in the LM, we applied atropine to the colon. Atropine (1 µM) was applied to four spontaneously contracting tissues and was found to hyperpolarize the LM layer by 8 ± 2 mV (n = 4). In three of these four animals, atropine caused an increase in the interval between SDs and spikes (Fig. 8B) or a brief (<5 min) abolition of all activity after which SDs returned and normal spiking resumed. In one animal, SDs and spikes were abolished and not restored after 5 min after atropine infusion.Does Electrical Hyperpolarization Inhibit Action Potential Propagation?
We tested whether the invasion of action potentials from distant sites could be inhibited by local membrane hyperpolarization of LM cells, via local current injection into single LM cells. To do this, we recorded from two LM cells simultaneously, when the two electrodes were separated by 1 mm in the longitudinal axis. In four pairs of LM cells (n = 3), spontaneous action potentials were found to be phase locked between the two electrodes, and action potentials clearly propagated between the two electrodes. In these preparations, when brief hyperpolarizing currents (range 1-8 nA) were passed of one microelectrode, the membrane was locally hyperpolarized by up to 50 mV at the current passing electrode (after neutralization of the voltage drop across the tip resistance). Also, interestingly, there was no inhibitory effect on the propagation of the action potentials from one electrode to the other (Fig. 9). That is, local hyperpolarization, even up to 80 mV at the current passing electrode did not modify the characteristics of spontaneous action potentials. The control spike amplitude and half durations before hyperpolarization were 28.3 ± 2 mV and 26.1 ± 9.1 ms, respectively, and during hyperpolarization were 31.8 ± 2.5 mV and 25.8 ± 7.2 ms (n = 4). These values were not significantly different from one another (amplitude P = 0.30; half duration P = 0.91).
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Electrical Activity in Isolated LM Strips
We tested whether the SDs we recorded from the LM of intact segments of distal colon would still occur if we separated the LM from the myenteric plexus and CM layer. In seven of eight animals tested, SDs and rhythmically firing action potentials were still recorded from isolated LM strips that were not different from the activity recorded from LM in intact segments of colon (Fig. 10A). The mean interval between action potentials was 1.2 ± 0.2 s (n = 7), where the mean action potential half duration and amplitude were 25 ± 4 ms and 31 ± 2 mV (n = 7), respectively. There was no significant difference in the interval between action potentials from isolated and intact segments of distal colon (P > 0.05; Student's unpaired t-test). In one of these eight animals, the LM was electrically silent. When the preparations were observed visually, it could be seen that each action potential was associated with brief contractions of the LM strip. Nifedipine (1 µM) abolished SDs and action potentials (Fig. 10B) and did not reveal any underlying unitary events (n = 5), nor display membrane noise, as has been reported in the gastric antrum of the guinea pig (16). When these LM strips were stained for c-Kit, ICC-IM were present (Fig. 10C), as was found in the intact preparations.
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ICC in the Distal Colon
Recent evidence has shown that the ICC, which are mostly located near the myenteric plexus (ICC-MY), are pacemaker cells underlying the initiation of slow waves in CM (11). Although the presence of ICCs has been reported in the proximal colon of the guinea pig (3, 26), their existence in the distal colon is not known. c-Kit immunohistochemistry has been routinely used as a selective technique for labeling ICC in the guinea pig gastrointestinal tract.We consistently observed at least two classes of ICC in the distal
colon, similar to the different classes of ICC that have been
previously reported in the proximal colon of the guinea pig (3,
26). Dense c-Kit-like immunoreactivity (c-Kit-LI) was found at
the myenteric plexus (ICC-MY), where Kit-positive cells surrounded
myenteric ganglia and densely innervated internodal strands (Fig.
11A). In addition to
ICC-MY, branching ICC-IM were also present in both the LM and CM
layers. ICC-IM showed polarized projections and were orientated in the
long axis of both the LM and CM fibers. An example of ICC-IM in CM and
LM is shown in Fig. 11, B and C, respectively.
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DISCUSSION |
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There have been remarkably few studies regarding the intracellular electrical activity of the LM muscle of the small bowel and even less is known about the colon. In this study, we have characterized the electrical activity of the LM layer in stretched preparations of distal colon that are free to contract spontaneously. There are a number of major findings of the current study. First, rhythmic SDs and action potentials occur not only in intact preparations, but also in isolated strips of LM, devoid of myenteric ganglia and CM. Second, the ionic mechanisms underlying SDs and action potentials in LM are critically dependent on the opening of L-type Ca2+ channels. Third, the circumferential propagation and coordination of SDs and action potentials are restricted to localized domains; activity rarely propagating at distances >1 mm around the circumference.
Ionic Dependence of Electrical Activity
In previous studies (7, 10, 11, 41), slow waves have been reported in the CM of isolated bowel from many species and such activity has been shown to persist in the presence of dihydropyridines, such as nifedipine. Therefore, the upstroke phase of these slow waves is clearly generated by an inward current not carried by Ca2+ through L-type Ca2+ channels. However, in this study, we report the existence of rhythmic SDs and action potentials abolished by nifedipine, suggesting that L-type Ca2+ channels are critically important for the cyclical ionic conductances in this muscle layer. It is also noteworthy that the resting membrane potential of LM cells in our study was aboutThe waveform of SDs showed some variation between animals; from the classic slow-wave configuration, where a clear plateau phase was observed, to small prepotentials that did not have a marked plateau phase but were associated with spontaneous rhythmical action potential firing. In the latter case, spontaneous action potentials appeared to resemble the slow diastolic potential observed in cardiac action potential (see Fig. 7B) and the spontaneous spike discharges reported in the T. coli (22). Similar to our findings, Kuriyama and Tomita (22) also reported that the amplitude of the slow potentials in the Taenia "... varies not only from preparation to preparation, but also from cell to cell and it is sometimes difficult to distinguish clearly between the slow potential and the spike."
Effects of Intracellular Current Injection on Propagated Activity
Interestingly, when currents were passed of the recording electrode, either hyperpolarizing or depolarizing, there was no detectable difference in the amplitude of the action potentials (Fig. 9). It is not clear why even large currents (Extent of Action Potential Propagation
The extent of propagation of spontaneous action potentials around the circumference of the colon was investigated using simultaneous recordings from pairs of LM cells. When recordings were made 100 µm apart in the circumferential axis, spikes were always phase locked and clearly propagated between the two electrodes. However, when recordings were made 1 mm apart circumferentially, only two of nine pairs of recordings showed unambiguous propagation from one electrode to the other, suggesting that spikes die out rapidly within 1 mm around the circumference of the large bowel. In support of this, when recordings were made at 13 mm apart circumferentially, activities were always independent at the two recording sites. In contrast, action potentials were well correlated in the longitudinal direction, at least up to 1 mm (the maximum distance investigated). Ca2+ waves, which appear to depend on action potential propagation, in the LM of guinea pig ileum and distal colon have also been shown to propagate over localized zones often <1 mm in the circumferential direction and up to 10 mm in the longitudinal direction, which also shift over time. Also, recent multielectrode array studies of the surface of the feline duodenum (muscle layer unspecified) demonstrate that spikes, unlike slow waves, are confined to small "patches" of activity, which are usually <40 µm (23). Thus, in the absence of stimulation, the LM is unlikely to ever contract synchronously around the entire circumference but rather generate localized constrictions that would periodically change direction as the dominant pacing site shifted. This hypothesis is supported by recent video imaging of LM contractions observed to originate in small centers of activity (pacing sites) around the circumference of the LM that periodically merge and change location to possibly produce mixing movements (14).Chaotic Electrical Activity
Meandering trajectories produced by coordinated action potentials recorded from closely associated sites (see Figs. 2 and 6) suggest that the electrical activity in LM has the characteristics of a chaotic system (see Ref. 42). The bifurcating, fairly stable orbits [see Figs. 2A (arrowheads) and 6D], which deviate from the 45° line, suggest that recording sites that have coordinated muscle activity are subject to the periodic dominance by at least two major pacing regions (24, 37). Presumably, a pacing region is formed by spontaneous action potential firing in one dominant area of muscle, which entrains neighboring regions of muscle. Two pacing regions, combined with local changes in conductance, produce changes in interval and waveform and periodic reversal in direction of action potential conduction.Role of the Enteric Nerves in Spontaneous Electrical Activity in LM
TTX and atropine either reduced the frequency of SDs and action potentials or, alternatively, caused a temporary abolition of all spontaneous activity. The observation that TTX did not affect the action potential configuration or underlying SD, suggests that Na+ influx is probably not a major player in the inward current underlying the upstroke of SD or the action potential. The reason for the reduction in frequency or temporary abolition of SD activity after TTX is unclear. We suspect that atropine and TTX may have acted to remove cholinergic tone in the muscle (33). In support of this, we have found that in TTX, the further addition of ACh or TEA depolarized the LM and restored spontaneous rhythmicity (unpublished observations). It is not known, however, how TTX or atropine modified SDs in tissues where the resting membrane potential was not altered or how recovery after these blockers occurs. Perhaps, the removal of the effects of ongoing neurotransmission by these drugs leads to adaptation of stretch sensitive elements in the tissue.Propagation Velocities of Action Potentials in Longitudinal and Circumferential Axis
A wide range of variation was noted in the propagation velocity of spikes around the circumference of the colon, suggesting that spike propagation velocity is not constant. Similar observations have been reported in this tissue using Ca2+ imaging techniques (37, 38). Action potentials in LM were found to propagate ~19 times faster in the longitudinal axis of the colon compared with the circumferential axis. These propagation velocities measured with intracellular recording techniques are similar to the values reported in recent Ca2+ imaging experiments of the distal colon, which calculated that Ca2+ waves propagate about ten times faster in the longitudinal axis than the circumferential axis. In the urinary bladder, spontaneous action potentials were recently reported to propagate ~40 times faster in the axial direction compared with the transverse direction (13). Interestingly, Brookes et al. (2a) recently reported that extracellular recordings from the CM of guinea pig ileum revealed that action potentials do not propagate further than 1 mm in the longitudinal axis.In conclusion, SDs and action potentials occur rhythmically in the LM layer of the distal colon, and such activity propagates over discrete localized zones in the circumferential axis of the bowel (often <1 mm). Spikes in LM are unlikely to propagate around the entire circumference of the colon, and this may be responsible for generating local constrictions within the LM, possibly underlying the mixing movements associated with this muscle layer. Also, in direct contrast to the slow waves reported in the CM layer of other mammals, SDs in LM are critically dependent on the opening of L-type Ca2+ channels. Such rhythmicity appears to be under ongoing enteric neuronal control.
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ACKNOWLEDGEMENTS |
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We thank Yulia Bayguinov and Sean Ward for expert assistance with confocal microscopy and immunohistochemistry.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-45713 (to T. K. Smith) and PO1-DK-41315 (to core facilities of the program project).
Address for reprint requests and other correspondence: T. K. Smith, Dept. of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 (E-mail: tks{at}physio.unr.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00345.2001
Received 3 August 2001; accepted in final form 15 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barr, L,
Dewey M,
and
Berger W.
Action potentials can propagate along small strands of smooth muscle.
Pflügers Arch
380:
165-170,
1970.
2.
Berezin, I,
Huizinga JD,
and
Daniel EE.
Structural characterization and interconnections of interstitial cells of Cajal in myenteric plexus and circular muscle of canine colon.
Can J Physiol Pharmacol
68:
1419-1431,
1990[ISI][Medline].
2a.
Brookes, SJH,
D'Antona G,
Zagorodnyuk VP,
Humphreys CMS,
and
Costa M.
Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea-pig ileum.
Neurogastroenterol Mot
13:
519-531,
2001[ISI][Medline].
3.
Burns, AJ,
Herbert TM,
Ward SM,
and
Sanders KM.
Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry.
Cell Tissue Res
290:
11-20,
1998[ISI].
4.
Bywater RA and Taylor GS. Non-cholinergic excitatory and
inhibitory junction potentials in the circular smooth muscle of
guinea-pig ileum. J Physiol (Lond) 374: 153-164.
5.
Cheung, DW,
and
Daniel EE.
Comparative study of the smooth muscle layers of the rabbit duodenum.
J Physiol (Lond)
309:
13-27,
1980[Abstract].
6.
Chow, E,
and
Huizinga JD.
Myogenic electrical control activity in longitudinal muscle of human and dog colon.
J Physiol (Lond)
392:
21-34,
1987[Abstract].
7.
Daniel, EE,
and
Berezin I.
Interstitial cells of Cajal: are they major players in control of gastrointestinal motility?
J Gastrointest Motil
4:
1-24,
1992.
8.
Daniel, EE,
and
Wang YF.
Gap junctions in intestinal smooth muscle and interstitial cells of Cajal.
Microsc Res Tech
47:
309-320,
1999[ISI][Medline].
9.
Daniel, EE,
and
Posey-Daniel V.
Neuromuscular structures in opposum esophagus: role of interstitial cells of Cajal.
Am J Physiol Gastrointest Liver Physiol
246:
G305-G315,
1984
10.
Dickens, EJ,
Edwards FR,
and
Hirst GDS
Selective knockout of intramuscular interstitial cells reveals their role in the generation of slow waves in mouse stomach.
J Physiol (Lond)
531:
827-833,
2001
11.
Dickens, EM,
Hirst GDS,
and
Tomita T.
Identification of rhythmically active cells in guinea-pig stomach.
J Physiol (Lond)
514:
515-531,
1999
12.
Hara, Y,
Kubota M,
and
Szurszewski JH.
Electrophysiology of smooth muscle of the small intestine of some mammals.
J Physiol (Lond)
372:
501-520,
1986[Abstract].
13.
Hashitani, H,
Fukuta H,
Takano H,
Klemm MF,
and
Suzuki H.
Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.
J Physiol (Lond)
530:
273-286,
2001
14.
Hennig, GW,
Gallager S,
Grandi P,
and
Smith TK.
Role of longitudinal muscle in mixing movements (Abstract).
Gastroenterology
120:
A169,
2001.
15.
Hirst, GD,
Holman ME,
and
McKirdy HC.
Two descending nerve pathways activated by distension of guinea-pig small intestine.
J Physiol (Lond)
244:
113-127,
1975[Abstract].
16.
Edwards, FR,
Hirst GDS,
and
Suzuki H.
Unitary nature of regenerative potentials recorded from circular smooth muscle of guinea-pig antrum.
J Physiol (Lond)
519:
235-250,
1999
17.
Koh, SD,
Sanders KM,
and
Ward SM.
Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine.
J Physiol (Lond)
513:
203-213,
1998
18.
Kosterlitz, JH,
and
Lees GM.
Pharmacological analysis of intrinsic intestinal reflexes.
Pharmacol Rev
30:
301-339,
1963.
19.
Kunze, WAA,
Clerc N,
Bertrand PP,
and
Furness JB.
Contractile activity in intestinal muscle evokes action potential discharge in guinea-pig myenteric neurons.
J Physiol (Lond)
517:
547-561,
1999
20.
Kunze, WAA,
Furness JB,
Bertrand PP,
and
Bornstein JC.
Intracellular recording from myenteric neurons that respond to stretch.
J Physiol (Lond)
506:
827-842,
1998
21.
Kuriyama, H,
Osa T,
and
Toida N.
Electrophysiological study of the intestinal smooth muscle of the guinea-pig.
J Physiol (Lond)
191:
239-255,
1967[ISI][Medline].
22.
Kuriyama, H,
and
Tomita T.
The responses of single smooth muscle cells of guinea-pig taenia coli to intracellularly applied currents and their effect on the spontaneous electrical activity.
J Physiol (Lond)
178:
270-289,
1965[ISI].
23.
Lammers, WJ.
Propagation of individual spikes as "patches" of activation in isolated feline duodenum.
Am J Physiol Gastrointest Liver Physiol
278:
G297-G307,
2000
24.
Nagai, T,
and
Prosser C.
Patterns of conductions in smooth muscle.
Am J Physiol
204:
910-914,
1963[ISI].
25.
Sanders, KM,
and
Smith TK.
Enteric neural regulation of slow waves in circular muscle of the canine proximal colon.
J Physiol (Lond)
377:
297-313,
1986[Abstract].
26.
Seki K, Zhou DS, and Komuro T. Immunohistochemical study of the
c-Kit expressing cells and connexin 43 in the guinea-pig digestive
tract. J Auton Nerv Syst 68: 182-187.
27.
Smith, TK.
Spontaneous junction potentials and slow waves in the circular muscle layer of guinea-pig ileum.
J Auton Nerv Syst
27:
147-152,
1989[ISI][Medline].
28.
Smith, TK,
Bywater RAR,
Taylor GS,
and
Holman ME.
Electrical responses of the muscularis externa to distension of the isolated guinea-pig distal colon.
J Gastrointest Motil
4:
145-156,
1992.
29.
Smith, TK,
Reed BJ,
and
Sanders KM.
Interaction of two electrical pacemakers in the circular muscle of the canine proximal colon.
Am J Physiol Cell Physiol
252:
C290-C299,
1987[Abstract].
30.
Smith, TK,
Reed BJ,
and
Sanders KM.
Electrical pacemakers of the canine proximal colon are functionally innervated by inhibitory motor neurons.
Am J Physiol Cell Physiol
256:
C466-C477,
1989
31.
Smith, TK,
and
Robertson WJ.
Synchronous movements of the longitudinal and circular muscle during peristalsis in the isolated guinea-pig distal colon.
J Physiol (Lond)
506:
563-577,
1998
32.
Spencer, N,
McCarron S,
and
Smith TK.
Sympathetic inhibition of ascending and descending interneurons during the peristaltic reflex in the guinea-pig distal colon.
J Physiol (Lond)
519:
539-550,
1999
33.
Spencer, N,
Walsh M,
and
Smith TK.
Does the guinea-pig ileum obey the "law of the intestine"?
J Physiol (Lond)
517:
889-898,
1999
34.
Spencer, NJ,
and
Smith TK.
Propagation of action potentials in longitudinal muscle of guinea-pig distal colon (Abstract).
Gastroenterology, Suppl
120:
1702,
2001.
35.
Spencer, NJ,
and
Smith TK.
Simultaneous intracellular recordings from longitudinal and circular muscle during the peristaltic reflex in guinea-pig distal colon.
J Physiol (Lond)
533:
787-799,
2001
36.
Spencer, NJ,
Smith CB,
and
Smith TK.
Role of muscle tone in peristalsis in guinea-pig small intestine.
J Physiol (Lond)
530:
295-306,
2001
37.
Stevens, RJ,
Publicover NG,
and
Smith TK.
Induction and organization of Ca2+ waves by enteric nervous reflexes.
Nature
399:
62-66,
1999[ISI][Medline].
38.
Stevens, RJ,
Publicover NG,
and
Smith TK.
Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea-pig small intestine.
Gastroenterology
118:
892-904,
2000[ISI][Medline].
39.
Vigmond, EJ,
and
Bardakjian BJ.
Role of cellular orientation in electrical coupling between gastrointestinal smooth muscle.
Ann Biomed Eng
26:
703-711,
1998[ISI][Medline].
40.
Wang, YF,
and
Daniel EE.
Gap junctions in gastrointestinal muscle contain multiple connexins.
Am J Physiol Gastrointest Liver Physiol
281:
G533-G543,
2001
41.
Ward, SM,
Burns AJ,
Torihashi S,
and
Sanders KM.
Mutation of the proto-oncogene c-Kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
J Physiol (Lond)
480:
91-97,
1994[Abstract].
42.
Williams, GP.
Chaos Theory Tamed. Characteristics of Chaos. Part IV. Washington, DC: Joseph Henry, 1997, p. 209-237.