Propagation of individual spikes as "patches" of activation in isolated feline duodenum

Wim J. E. P. Lammers

Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Asynchrony of spikes has made it difficult to study the spatial and temporal behavior of spikes in the gastrointestinal system. By simultaneously recording from a large number of closely spaced electrodes, we investigated the propagation of individual spikes. Recordings were performed from the serosal surface of the isolated feline duodenum at 240 sites simultaneously. Analysis of the tracings made it possible to reconstruct the propagation of individual spikes. Spikes propagate in the longitudinal and circumferential directions in self-limiting areas or "patches." Conduction within patches may occur in the orad or aborad direction irrespective of the direction of the slow wave. Most of the patches are smaller (<40 mm2), although inhomogeneous activation by the preceding slow wave may increase their size. Stimulation by ACh, TTX, or tetraethylammonium does not affect the average patch size but does increase significantly their number and distribution in the duodenum [from 26% (control) to 56%, 61%, and 72%, respectively]. In conclusion, individual spikes activate limited areas or patches in the small intestine, and pharmacological stimulation increases the number and distribution of these patches. In the small intestine, this pattern of activation would induce localized contractions. Contraction could be modulated by the size, number, and distribution of spike patches.

slow waves; contraction; myometrium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SMALL INTESTINE CAN PRODUCE a variety of contraction patterns, different from that caused by the peristaltic reflex, variously described as pendular, segmenting, mixing, stationary, propagating, or otherwise (1, 2, 6). These movements are caused by the emergence of spikes or "action potentials" on top of the depolarization induced by the propagating slow wave (3, 12). The temporal relationship among the slow wave, the spike burst, and the ensuing contraction have been described (1, 10). Spikes can occur immediately after the upstroke of the slow wave and within a limited period of 1-2 s after that moment (26, 30), and the resulting contractions are also limited to this time frame (10, 28).

There is, however, not much information available describing the spatial behavior of spikes, especially related to their pattern of propagation. It has been suggested that spikes are able to propagate (3), and some reports mention asynchrony of conduction beyond a few millimeters (9, 14, 37). Reentry has been postulated as a mechanism for this "asynchrony" (27), but data confirming or negating this concept have not been presented. Sancholuz et al. (29) warned that "records of spike bursts from a single electrode site do not accurately reflect activity beyond that one point site" and that regions of preferential spike activity could be present and "might be revealed by experiments with large numbers of closely spaced electrodes." In this study, high-density electrical recordings revealed that the propagation of individual spikes is self-limited and excites relatively small areas of the small intestine. Parts of this study have been presented previously in abstracts (21, 22).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mongrel cats of either sex (average body wt 3.1 kg) were, after an overnight fast, anesthetized with xylazine (10 mg/kg) and ketamine (10 mg/kg) administered intramuscularly. After a midabdominal incision, a segment of ~20 cm of the upper part of the small intestine was removed and placed in a dissecting dish filled with warm, oxygenated Tyrode solution. The duodenum was further dissected to a length of ~10 cm, flushed clean with Tyrode, and opened along the mesenteric border. The final preparation was positioned in a Perspex tissue bath with the serosal side facing upward.

The tissue bath (content 200 ml) was perfused at a rate of 100 ml/min with a modified Tyrode solution of the following composition (mM): 130 NaCl, 4.5 KCl, 2.2 CaCl2, 0.6 MgCl2, 24.2 NaHCO3, 1.2 NaH2PO4, and 11 glucose. The solution was saturated with a mixture of 95% O2-5% CO2; pH was 7.35 ± 0.05, and temperature was kept at 37 ± 0.5°C. A custom-made electrode assembly used to map the spread of electrical activity consisted of 240 Teflon-coated silver wires (diameter 0.3 mm) arranged in a 24 × 10 rectangular array with an interelectrode distance of 2 mm. The tip of the electrodes, glued together in a block of dental acrylic, extended 3 mm below the acrylic to permit flow of the solution between the extracellular electrodes and along the serosal surface of the tissue. Unipolar electrograms were recorded from all 240 electrodes using a large silver plate at one side of the tissue bath as the indifferent electrode. Each electrode was connected through shielded wires to individual alternating current preamplifiers (gain 4,000), and the recorded signals were subsequently filtered (bandwidth 2-400 Hz), digitized (8 bits, 1 kHz sampling rate per channel), multiplexed, and stored on a modified video recorder. Details of the experimental setup and the recording system have been presented in previous communications (16, 20).

Continuous recordings were made from the isolated duodenum during a control period of 30 min, after which ACh (2 × 10-8 M, Merck), tetraethylammonium (TEA, 1 mM, Sigma), or TTX (3 × 10-6 M, Sigma) was added to the circulating Tyrode for an additional recording period of 30 min. After the experiment, the tape was replayed at 10-min intervals for periods of 1 min and the slow waves and spikes occurring during these periods along a longitudinal row of 24 electrodes were counted. Thereafter, a representative 4- to 12-s window was identified in each period and all 240 recorded electrograms were transferred from tape to a personal computer for the reconstruction of the activation sequences.

The method of reconstructing the propagation pattern of slow waves was presented in a previous communication (20). Briefly, all electrograms, after digital filtering to remove 50-Hz interference, were displayed on a screen in groups of 24 (Fig. 1A) and the time at which each slow wave occurred in each trace was visually determined by the time of the maximum negative slope and marked with a cursor. After this analysis was performed for all recordings, the local activation times of the slow waves were plotted in a grid representing the location of the recording electrodes. To visualize the pattern of propagation of the slow wave, isochrones were drawn manually around areas activated in steps of 0.5 s. Figure 1B displays the activation map of a slow wave, which in this example had two sites of origin, one in the middle left of the segment (t = 0.2 s) and a second in the caudal part of the tissue (t = 0.0 s). Propagation of the slow wave occurred from both foci with orad conduction in the upper part and collision of the two slow waves occurring in the lower segment of the preparation.


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Fig. 1.   Method of reconstructing propagation of individual slow waves and spikes in isolated feline duodenum. Inset, location of 24 × 10 mapping electrode assembly in relation to an isolated strip of duodenum. In this case, only central 7 columns of electrodes were in contact with tissue. Remaining 3 columns are left out in following maps. A: electrograms recorded from 1 electrode column depicting slow wave activity followed, in most electrograms, by a single spike. Short vertical bars overlaid on each spike indicate moment of activation at each site. Times are related (in ms) to occurrence of first spike (t = 0 ms at site indicated by open arrow in C). B: activation pattern of slow wave propagation with isochrones drawn every 0.5 s. C: all measured spike activation times (in ms) on grid representing electrode matrix. Spike isochrones were drawn manually around areas activated in steps of 100 ms. D: final spike map. Actual activation times have been left out and replaced with filled dots to indicate degree of resolution. Isochrones, star to indicate point of origin, and arrow included to help in visualizing pattern of spike propagation. From a single point of origin, spike propagated rapidly along the longitudinal (orad) direction and more slowly in circumferential direction. After 100-400 ms, spike propagation stopped spontaneously in all directions, describing an isolated "patch" of activity. Size of patch and conduction velocity of spike as measured in longitudinal direction are indicated.

In this study, a first attempt was made to reconstruct the pattern of activation of the spikes, and the procedure is illustrated in Fig. 1, C and D. As is visible in some leads in Fig. 1A, a single spike occurred after the slow wave. The moment at which every spike occurred, as determined by the maximum negative slope, was marked and indicated with a short vertical bar. Occasionally, the positive slope was more rapid then the negative slope and it was then chosen as the moment of activation (electrode 12). These local activation times were related, in milliseconds, to the time at which the first spike occurred (t = 0 ms), which in this case was at the electrode indicated by the open arrow in Fig. 1C. After all spike deflections had been individually marked, the activation times of the spikes were plotted in a grid representing the locations of the recording electrodes (Fig. 1C). If the recording was too poor or if the spike was absent from a particular trace, the electrode site was left empty. Isochrones were then manually drawn around areas activated by spikes in steps of 100 ms. Figure 1D shows the final format of the spike map, in which the actual recording times have been left out for the sake of clarity. Instead, filled circles provide an impression of the spatial resolution of the map, whereas the isochrones help to visualize the pattern of propagation of the spike. In this case, the spike originated from an area located in the lower middle part (indicated by star in Fig. 1D) and propagated towards the orad part of the duodenum for a distance of ~36 mm. It can also be seen that the spike did not activate the whole area of available tissue but propagation was limited to an area in the upper center of the preparation. We have found that propagation of spikes is always limited to such circumscribed areas and are using the term "patch" to describe such localized events.

From the spike maps, the maximum extent of the spike patches in the longitudinal and circumferential directions and the area activated by the spike were measured. In Fig. 1, the longitudinal and circumferential dimensions were ~36 and 8 mm, respectively, and the area excited was 208 mm2 (area covered by entire mapping electrode in this experiment was 672 mm2). An attempt was also made to measure the conduction velocity of the spikes. For this purpose, two sites were selected that were located in an area of uniform conduction and at a distance of at least 10 mm apart. As shown in Fig. 1D, the two chosen sites (at t = 39 ms and t = 216 ms) were located 14 mm distant from each other. From this electrode distance and the time difference (216 - 39 = 177 ms), the conduction velocity was measured (7.9 cm/s). No attempts were made to measure the conduction velocities in the circumferential direction because the distance covered was usually too small to provide a reliable measurement. The same procedure was also used to measure the longitudinal conduction velocity of the slow wave (Fig. 1B; 1.3 cm/s).

In most cases, instead of a single spike (as shown in Fig. 1), several spikes were found after each slow wave. An automated sorting routine was used to distinguish one spike from the next (15). The software essentially sorts clusters of spikes from each other based on spatial and temporal parameters. In this study, best sorting results were achieved with a time difference between spikes of 150 ms and a spatial distance of 4 mm. After the sorting routine, spike patches were individually displayed and their shape and size were confirmed by visual analysis of the signals.

Statistical analysis was performed where appropriate with the paired Student's t-test or the chi 2 test. The study conformed to the locally approved guidelines for care and use of laboratory animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The pattern of propagation of the spike presented in Fig. 1D describes several major features of spike patches: 1) the origin of the spike is located at a different site from that of the slow wave; 2) spike conduction may occur in a different direction from that of the slow wave; and 3) conduction of the spike is limited to a restricted area. Action potentials, however, are usually not restricted to a single discharge but can occur repeatedly after a single slow wave. Figure 2A displays a column of 24 electrograms, recorded in the longitudinal direction, as indicated in the maps in Fig. 2, B-D. As shown by the electrograms and visible in Fig. 2B, a slow wave propagated uniformly in the aborad direction except for the most caudal part of the segment, which was activated from another pacemaker. This slow wave activity was followed by several spike discharges visible in most electrograms. The number of spikes recorded in each electrogram, however, was not constant and ranged from 0 to 3. Further analysis from all 240 electrograms made it possible to group these spikes into seven different clusters chronologically labeled 1-7. Fig. 2C presents the propagation pattern of three such individual spike patches, labeled 2, 4, and 6. The spike in patch 2 was initiated at a site indicated by the star close to electrode 7, and conduction occurred in the longitudinal and circumferential directions for a distance of 4-8 mm before it stopped. The spike in patch 6 occurred a few seconds later, from a locus located close to electrode 18. The propagation of this spike was once more spontaneously limited in the orad and circumferential directions, whereas its propagation in the caudal direction went beyond the border of the mapping electrode.


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Fig. 2.   Typical spike activities after a single slow wave during control. A: 24 electrograms recorded from electrodes positioned in a longitudinal direction as shown by corresponding circled numbers in B-D. Most traces showed several spikes that were clustered in groups 1-7 as indicated by ellipses in A. B: propagation of slow wave, which conducted predominantly from orad to caudad. C: activation sequence of spike propagation in spike patches 2, 4, and 6. Isochrones in spike patches were drawn every 100 ms. Spikes originated at circumscribed locations, indicated by stars, and propagated in several directions for 100-400 ms before terminating abruptly. D: composite spike map displays boundaries of all 7 spike patches, showing considerable variations in size, location and overlap between patches. Because of this overlap, some electrode sites were activated by >1 spike patch (i.e., electrode 10 was activated by patches 2, 3, and 4). Area activated by a spike varied from 20 (patch 1) to 156 (patch 4) mm2. Most of mapped area, however, was not excited by any spike at all, and only 93 of the 216 (= 43%) recording electrodes registered >= 1 spikes.

Patch 4 shows a more complex activation pattern with two origins of spike activity as indicated by the two stars, located ~10 mm distant from each other. Spike conduction occurred from both sites, propagating toward each other until they collided after ~300 ms. This analysis is based on the assumption that two separate patches were initiated more or less at the same time and could not be separated from each other in a reliable manner. Such difficulties in delineating single patches of activity occurred infrequently (n = 22, 8%) and most spike patches showed a single origin of activity (n = 241; 92%).

Fig. 2D displays a composite spike map in which the boundaries of all seven patches after this single slow wave are superimposed on each other. It is evident from this composite map that 1) the order in which spike patches occurred, from orad to caudad, was related to the spatial sequence of propagation of the preceding slow wave; 2) there was a considerable variation in shape and size of individual patches; 3) there was considerable overlap between some of the spike patches, indicating that several sites were activated by more than one patch; and 4) a large area of the tissue was not excited at all. To quantify this last point, the number of sites at which at least one spike was found (93 electrodes) was related to the total number of recording sites (216 electrodes) and expressed as a percentage (= total patch area; 43%).

The number, dimensions, area, frequency and total patch area of 195 patches, accumulated during a 30-min control period in 12 experiments, are shown in Table 1. The length of the average spike patch (12.1 mm) is significantly longer than its width (5.8 mm; P < 0.001). Each patch, on average, covers an area of 57 mm2, whereas the total area covered by all spike patches after a single slow wave was relatively low (26%), indicating that, during control, most of the preparation was not excited at all. Furthermore, the distribution of the area of spike patches (Fig. 3) shows that small patches occurred much more frequently than larger spike patches. In Table 2, the conduction velocity of spikes was compared with the conduction velocity of the slow waves and demonstrates that spikes, in the longitudinal direction, propagate approximately six times faster than slow waves (P < 0.001).

                              
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Table 1.   Size, frequency, and spatial distribution of individual spike patches



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Fig. 3.   Frequency histogram of area of spike patches during control (n = 195) and during application of ACh (n = 119), TTX (n = 113), or tetraethylammonium (TEA; n = 154). In all these situations large majority of patches were <100 mm2, with patches <20 mm2 occurring in ~40% of cases. Addition of ACh, TTX, or TEA had no effect on normalized distribution of spike patches (chi 2 test; P > 0.01).


                              
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Table 2.   Longitudinal conduction velocities

The direction of propagation of the spike was also compared with that of the preceding slow wave. As in the case of the conduction velocity measurements, this comparison was only made in the longitudinal direction. In 34.3% of the cases (n = 58), the longitudinal direction of propagation of the spike was in the same direction as that of the preceding slow wave, whereas in 32.5% of the patches (n = 55), propagation in the patch occurred in the opposite direction. In 56 patches (33.1%), spike conduction in a patch occurred both in the orad and in the aborad direction and therefore showed a mixture of dromic and antidromic conduction compared with the conduction direction of the preceding slow wave. Conduction of the spike within the patch did not therefore seem to depend on the direction of propagation of the preceding slow wave.

Effects of ACh, TTX, and TEA on spike patches. In an attempt to perturb the behavior of spike patches, ACh, TTX, or TEA was added to the superfusing fluid (4 experiments each), and the results are also presented in Table 1. All three drugs had no effect on the average dimensions or size of spike patches, although the frequency of spikes after the slow waves was significantly increased. As shown in Fig. 3, the distribution of the size of spike patches remained essentially unchanged (P > 0.01) because again smaller spike patches occurred much more frequently than larger patches. Finally, as presented in Table 2, the three drugs had no significant effect on the conduction velocities of the slow wave or of the spikes.

The most important effect of these three drugs was the significant increase in the total area covered by spike patches. Table 1 presents the increase in total patch area induced by ACh, TTX, and TEA, which increased from 26% during control to 56%, 61%, and 72%, respectively. This indicates that more spike patches activated together a larger size of the preparation by the application of these drugs than was the case during control. An example of the effect of these drugs is displayed in Fig. 4, in which composite spike maps from controls and during ACh, TTX, and TEA administration are compared. It is evident from these results that the major excitatory action of these three drugs is to induce a larger number of spike patches to occur. These spike patches were distributed throughout the tissue, thereby exciting a larger area than during control.


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Fig. 4.   Composite spike maps from a control experiment (A) and during application of ACh, TTX, or TEA (B-D, respectively). Top panels display a typical electrogram recorded during each condition registering a slow wave followed by 1 or more spikes. Locations of these recording electrodes are indicated with a circled symbol in corresponding maps, and spike numbers indicate individual spike patch in map. In each map, boundaries of all spike patches recorded after these single slow waves are superimposed on each other in chronological sequence and show variability in size, locations, and overlap between patches. With application of ACh, TTX, or TEA, considerable increase in number and distribution of spike patches is evident. Some patches have been slightly shifted to reveal underlying patches. In D, approximate locations of underlying patches 3, 10, 16, and 18 are indicated by horizontal lines.

Effect of pattern of activation of preceding slow wave. Occasionally, it was possible to detect larger than normal spike patches, and this seemed to be related to abnormal patterns of slow wave propagation. An example of such a phenomenon is shown in Fig. 5. Figure 5A displays a column of 24 electrograms recorded in the longitudinal direction as indicated in the maps, Fig. 5B presents the propagation pattern of the slow waves, and Fig. 5C depicts the propagation pattern of the spike encircled in the electrograms in Fig. 5A. Two slow waves propagated in this region. The first arose from an area located outside the recording area, which appeared in the mapped area close to electrode 24 (t = 0.0 s). The second slow wave originated close to the center adjacent to electrode 14 (t = 0.2 s). Both slow waves propagated towards each other in the caudal part of the map and collided against each other in the area indicated by the dashed line at the level of electrodes 18 and 19.


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Fig. 5.   Occurrence of a large spike patch after a nonuniform pattern of propagation of preceding slow wave. A: set of 24 electrograms recorded from longitudinal array of numbered electrodes indicated in maps. B: pattern of slow wave propagation (isochrones every 0.5 s) identifying 2 sites of origin; 1 in middle of map and 1 distal to map. Consequently, the 2 slow waves propagated toward each other at bottom of map, and dashed line indicates general area where these 2 slow waves collided. C: propagation pattern of spike encircled in A. Isochrones in patch were drawn every 100 ms. Spike originated from a site distant from origin of slow waves and conducted predominantly in aborad direction, covering a distance of 32 mm in ~350 ms. Comparison between spike map and slow wave map reveals that spike conducted over origin of central slow wave, across area of collision between the 2 slow waves, and into area depolarized by lower slow wave.

After the slow wave, a spike was initiated in the upper part of the preparation close to electrode 5. From this site, the spike propagated in the orad direction for a short distance (~6 mm) and in the aborad direction for a much longer distance (32 mm). The total area activated by this patch was 232 mm2, which is much larger than most spikes (Fig. 3). Comparison of the spike map (Fig. 5C) with the pattern of propagation of the preceding slow wave (Fig. 5B) reveals that the caudad propagating spike was conducting initially retrogradely over the area activated by the orally propagating slow wave. The spike then propagated over the area of origin of that slow wave, across the site of collision between the two slow waves, and into the area excited by the lower slow wave.

The relationship between the pattern of propagation of the preceding slow wave and the size of the spike patches was analyzed for all control experiments, and the results are presented in Table 3. The patterns of propagation of the slow wave preceding the spikes were classified as either uniform or nonuniform. Uniform conduction occurred when the slow wave originated from a single pacemaker and propagated homogeneously in either the orad or the aborad direction. Nonuniform conduction took place when one or more pacemakers were located in the mapped area with conduction occurring in both the orad and the aborad direction or when a collision occurred between two propagating slow waves. As presented in Table 3, in the case of uniform slow wave propagation, 82 of the analyzed spike patches were smaller than 160 mm2 and only one (1%) was larger, whereas in the case of nonuniform conduction, larger spike patches occurred much more frequently (16%; P < 0.001). These results show that patches of much larger than average area can occur. Moreover, such large patches may follow inhomogeneously propagating slow waves. Even in this situation, however, the large majority of patches are still small (84%; Table 3).

                              
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Table 3.   Pattern of slow wave propagation and size of spike patches


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that individual spikes propagate in the longitudinal and the circumferential direction. All spike propagation is limited to circumscribed areas that we term patches. In addition, we show that spikes usually originate from single sites. Sites of spike initiation are located at different sites from those at which slow waves are initiated. Spike conduction may occur in both the orad and the aborad direction and is independent of the direction of propagation of the preceding slow wave. In the isolated feline duodenum, the area activated by individual spike patches is on average quite small (57 mm2), although larger than normal spike patches may occur, especially after inhomogeneous propagation of the slow waves. Stimulation by ACh, TTX, or TEA does not influence the size of the patches but significantly initiates more patches covering a larger area of the duodenum.

That spikes propagate was actually demonstrated quite some time ago. Bülbring et al. (3) showed that spikes conducted along the isolated taenia coli of the guinea pig. Using microelectrode impalements, they measured a conduction velocity of 6.7-8.8 cm/s. This is comparable to our values, measured using a different technology, and in the feline duodenum (8.5 ± 3.3 cm/s; Table 2). Tomita (36, 37), also in the guinea pig taenia coli, and Nagai and Prosser (27), in the cat small intestine, reported similar results. Kuriyama et al. (14), in the jejunum of the guinea pig, measured a much smaller value (2.1 cm/s). This low value may have been caused by the complex architecture of bundle bifurcations, which would decrease overall conduction velocities. Other reports suggest that "spike potentials either are not conducted, or pass over very short distances" (1). Most reviews do not mention the matter of spike conduction.

Careful reading of the literature also indicates the possibility of limitations in spike conduction. Bülbring et al. (3) reported that asynchrony of spikes could occur between different sites just a few millimeters away. Daniel and Chapman (6) stated that spikes are transmitted along "very short distances, usually less than 0.5 cm." Nagai and Prosser (27) mentioned that spikes were frequently asynchronous at sites separated by >5 mm in the longitudinal direction and 2 mm in the transverse direction. They postulated that spikes could reenter previously excited areas, thereby inducing asynchrony and multiple spikes. Although it is possible for reentry to occur in smooth muscles (18), we have never seen reentering spikes in the isolated duodenum. All these observations, however, can be explained by our observation that spike conduction is confined within the area of a patch and that multiple patches may be superimposed on each other in the wake of a single slow wave (Fig. 2).

There is hardly any information as to the natural direction of propagation of individual spikes within these patches. Specht (32), presenting a good recording from the cat small intestine in vitro, concluded that "action potentials seemed to arise from the middle of the slow wave and travel in both directions over the area depolarized by the slow wave." Our results confirm and extend his observations in that spikes could conduct from their sites of origin in all directions, longitudinally and circumferentially, orally and aborally. Furthermore, spike conduction is highly anisotropic, with a dominant direction and a longer pathway in the longitudinal direction. Therefore, it could be argued that these spike propagations occur mainly in the longitudinal muscle layer, because the electrotonic space constant, measured in ileal longitudinal muscle, was 10 times higher in the axial than in the transversal direction (5). In addition, the spike conduction recorded in this study is an active propagation, because the longitudinal distance of spike propagation in an average patch (12 mm) is much longer than the passive axial space constant (0.6 mm) measured in longitudinal guinea pig ileum (5), even if the difference in species is considered. In other words, we do not believe that a patch is the spatial representation of a locally induced spike with an anisotropic region of exponentially decaying electronic potential. Such exponentially decaying potentials are not visible in the longitudinally recorded spike potentials in Figs. 1, 2, and 5.

Clearly, there are marked differences between the propagation of spikes and that of slow waves. There are differences in conduction velocity, in direction of propagation, and in the fact that spikes are limited to patches whereas slow waves continue to propagate until they are blocked by the edges of the tissue, by the end of the organ such as the gastroduodenal junction (19), or by collision with another slow wave (Fig. 5). Such differences imply that slow waves and spikes are two distinctly different electrical phenomena that may occur in different tissues; slow waves would originate from the interstitial cells of Cajal (13, 31), whereas spike patches occur in the longitudinal muscle layer. Consequently, the basic mechanisms for the propagation of these two distinct electrical phenomena would be quite different.

It is critical to differentiate between the propagation of individual spikes, as studied in this paper, and the propagation of groups or bursts of spikes (7, 29, 30, 34). Several reports have shown that "the sequential appearance of spike bursts at adjacent aboral electrodes within a particular slow-wave cycle may be interpreted as propagative spike burst spread" (7). Our study offers an extension and clarification of these previous reports in the sense that we have analyzed the propagation of individual spikes that occur within these clusters or bursts of spikes. The patches described in this study would then form the elements of the "bursts" investigated in previous reports. An indication of the relationship between patches and bursts can be seen in Fig. 4, in which the composite spike maps display the overlap of 9-22 spike patches after a particular slow wave. Such a spatial "cluster" of spike patches together form a burst in the time domain pictured in the electrograms above the maps.

Spikes occur during the depolarization of the cell membrane induced by the slow wave (12, 35), most probably in the longitudinal muscle, but also possible in the inner circular muscle layer (10). This depolarization starts immediately after the upstroke of the slow wave and may last for 1-2 s before gradual repolarization as the resting potential is restored (8). Spikes are therefore more likely to occur in the initial one-third of the slow-wave cycle (26).

One of the crucial findings in this study is that propagation of spikes is limited in both the longitudinal and the circumferential direction. To appreciate the spatial relationship between the slow wave depolarization and the limited spike propagation, two factors must be taken into account. In the first place, evidence from microelectrode studies demonstrates that spikes only occur while the tissue is depolarized by the slow wave (3, 8). The second point relates to the fact that the longitudinal conduction of the spike is much faster than that of the slow wave (8.5 vs. 1.4 cm/s). From these points, three different scenarios describing the relationship between the spike and the slow wave can be visualized (Fig. 6).


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Fig. 6.   Schematic diagram of spatial and temporal relationship between depolarization of slow wave and spike patches. In all diagrams, depolarization of slow wave is represented by shaded segment, which propagates aborally as indicated by small arrows at edges of depolarization and repolarization. In A, a spike is initiated in middle of depolarized area and is shown propagating rapidly in all directions and beyond borders of depolarization and repolarization. In B, spike propagation is initiated at same site but its propagation is limited to depolarized area, thereby inducing a ring contraction. In C, propagation of a spike is restricted to a limited domain or "patch," which would induce a weak contraction. In D, several patches of different sizes and degree of overlap occur in area of slow wave depolarization. Diagram illustrates proposed mechanism of generating stronger contractions by recruiting more spike patches.

In the first scenario (Fig. 6A), the depolarization by the aborad propagating slow wave may allow for the initiation of a spike but does not limit its propagation. Because of the higher conduction velocity of the spike compared with that of the slow wave, the spike would propagate rapidly through the depolarized area and beyond, into areas yet to be depolarized by the advancing slow wave. This obviously would lead to constant spasm of the intestine and shows how important it is that conduction of spikes is limited to the depolarized area.

In Fig. 6B, this dependence of the spike conduction on the slow wave depolarization has been incorporated. Once a spike is initiated, it will conduct rapidly throughout the depolarized area until it reaches the orad and aborad borders of the slowly propagating slow wave. Consequently, the area of the spike patch would be similar to the depolarized area, and, because the slow wave usually propagates aborally, the spike patch would have the shape of a ring around the intestinal tube. The expected contraction in this case would be that of a segment or a ring and, most importantly, would always be of that shape and magnitude.

The reality is better described with the addition of another factor in the scenario (Fig. 6C), the fact that individual spike conduction is limited to a restricted area or patch. In this situation, the size of the patch and therefore the ensuing contraction could be much smaller than the size of the depolarized area. This would, by itself, generate much weaker contractions than in previous scenarios. Moreover, by adding the possibility of activating more patches within the same depolarized area (Fig. 6D), stronger contractions up to ring contractions could also be generated using the same mechanism. This situation is then very similar to what we measured when the preparation was excited by the addition of drugs as shown in Fig. 4. In other words, varying the number of patches creates the possibility of grading the magnitude of the contraction.

It is useful at this point to compare differences and similarities between the conduction pattern of spikes in the feline duodenum and those of spikes in the pregnant uterus of the rat, as measured with the same technology (17). Before spikes are considered, however, it should be pointed out that the slow wave depolarizations that trigger the spikes differ in the two organs. In the myometrium, the upstroke velocity of the depolarization is so low (24) that a detectable extracellular signal is not produced and therefore cannot be recorded using our technology.

In both tissues, a burst of spikes may occur after the slow depolarization of smooth muscle. The major difference between spike conduction in the myometrium and that in the duodenum is that myometrial spike conduction is not self-limited as it is in the duodenum; in other words, spike patches do not occur in the uterus (17). In this sense, the myometrium more closely resembles the scenario described in Fig. 6B, in which spike conduction continues throughout the area of slow wave depolarization. Therefore, myometrial spike conduction, once initiated, is only limited by the area of depolarization (Fig. 6B) or by collision against other spikes. This, in turn, may lead to more complex and erratic conduction patterns that can easily degenerate into reentry of the impulse, the appearance of circus movements, and fibrillation (18). In the small intestine, however, the situation is very different, and the fact that spike propagation is limited to patches could be a mechanism to prevent such conduction arrhythmias.

The mechanisms that limit the size of individual spike patches are not clear. The fact that the patches are larger in the longitudinal than in the circumferential direction may point to an anatomic contribution to spike propagation, assuming that conduction occurs predominantly in the longitudinal muscle layer (5). In the taenia coli, it has been suggested that spikes propagate within "functional units" of muscle bundles (36, 37). In that concept, muscle cells are clustered together into bundles with good intercellular connections within the bundle and poor or absent gap junctions between separate bundles. Similarly, in the canine colon, Ba2+-induced spikelike action potentials were coordinated within single circular muscle lamellae but not necessarily across the septa that separated these lamellae (23). However, in the feline duodenum, if spike patches were limited by anatomically located barriers, one would expect spike patches to be located at distinct and fixed location in the tissue, akin to motor units in skeletal muscle. Instead, the spike patches we measured could occur anywhere in the tissue and showed a large amount of overlap with each other (Figs. 2 and 4). In addition, we were able to show that in the case of inhomogeneous conduction of the slow wave, it is possible to provoke larger than normal spike patches, suggesting that other factors play a role in determining the size of spike patches.

Recently, Stevens et al. (33), by monitoring the conduction of Ca2+ waves in an isolated longitudinal layer of guinea pig colon, showed that these waves also spread much faster in the longitudinal than in the axial direction, and that they "occasionally stopped abruptly at locations that had previously supported propagation" (33). In addition, Ca2+ waves could be blocked by collision against each other or with adjacent neurally suppressed regions. It is tempting to speculate that these local propagating Ca2+ waves are the intracellular sequel of the spike patches described in this study. However, some caution must also be expressed, because we are dealing with differences in species, in organs, and in behavior to TTX.

The spike patches that occur in the small intestine are probably responsible for weak movements of the muscular wall. Melville et al. (25) described longitudinal oscillations in rhythm with the slow wave and, in a mechanical model, showed that such longitudinal movements could induce intestinal fluid movements. Bass et al. (1) described the relationship between the number and amplitude of spikes that occurred in phase with the slow waves and type I contractions. Oigaard and Dorph (28) described the correlation in time and magnitude between the occurrence of spikes and pressure waves in the human small intestine, with small spikes inducing weak movements and "larger" spikes provoking stronger contractions. Christensen et al. (4) showed that pressure peaks, in the human duodenum, occurred at multiples of the period of the slow wave cycle and that the "physical length of the contractions producing these pressure peaks can be less than 2 cm." Bühner and Ehrlein (2) showed the difference between stationary and propagating contractions and showed that both the anatomic location in the intestine and the ingested nutrients may affect this relationship. Recently, Hennig et al. (11) also reported rhythmic longitudinal muscle oscillations in the guinea pig ileum that could be in phase with the slow wave frequency. In our experiments, during control, there were probably too few spikes to generate strong segmental or ring contractions. Because the area activated by spikes was on average only 26% of the total tissue area (Table 1), we would expect in that situation only isolated local contractions to occur, which in turn would stretch neighboring parts of the duodenum, thereby leading to small and weak movements of the intestinal wall. When the level of excitation in the tissue is increased, however, after the addition of ACh, TTX, or TEA, the number and distribution of spike patches can be significantly increased (Table 1 and Fig. 4) and their localized contractions are likely to merge together into segmental or ring types of contraction patterns.

In conclusion, this study has shown that the dynamics of spikes are more complex than hitherto conceived. The spatial and temporal characteristics of contraction in the small intestine seem to be determined not only by the direction of propagation of the slow wave but also by whether or not spikes are generated after these slow waves. In addition, the fact that spikes show propagation in different directions and in restricted areas suggests another level of dynamics between the original depolarization by the slow wave and the resulting contraction pattern of the gut. This level of complexity seems to suggest the possibility of modulating the level of excitation and therefore of contraction between the weakest of movements and strong ring contractions. Future work therefore must concentrate on the factors that determine the spatial and temporal modulation of spike patches.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical expertise provided by B. Stephen and Dr. O. Pozzan and the editorial assistance of J. R. Slack.


    FOOTNOTES

This work was supported by the Research Committee of the Faculty of Medicine & Health Sciences, United Arab Emirates University (Grants P/92/05 and P/93/14).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. J. E. P. Lammers, Dept. of Physiology, Faculty of Medicine and Health Sciences, PO Box 17666, Al Ain, United Arab Emirates (E-mail: wlammers{at}uaeu.ac.ae).

Received 5 May 1999; accepted in final form 1 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 278(2):G297-G307
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