Propagation and Neural Regulation of Calcium Waves inLongitudinal and Circular Muscle Layers of Guinea PigSmall Intestine

RANDEL J. STEVENS, NELSON G. PUBLICOVER, and TERENCE K. SMITHBiomedical Engineering Program, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada

Background & Aims: The relative movements of longitu-dinal muscle (LM) and circular muscle (CM) and therole that nerves play in coordinating their activities hasbeen a subject of controversy. We used fluorescentvideo imaging techniques to study the origin andpropagation of excitability simultaneously in LM andCM of the small intestine. Methods: Opened segmentsof guinea pig ileum were loaded with the Ca21 indicatorfluo-3. Mucosal reflexes were elicited by lightly depress-ing the mucosa with a sponge. Results: SpontaneousCa21 waves occurred frequently in LM (1.2 s21) andless frequently in CM (3.2 min21). They originated fromdiscrete pacing sites and propagated at rates 8–9times faster parallel (LM, 87 mm/s; CM, 77 mm/s)compared with transverse to the long axis of musclefibers. The presence of Ca21 waves in one muscle layerdid not affect the origin, rate of conduction, or range ofpropagation in the other layer. The extent of propaga-tion was limited by collisions with neighboring waves orrecently excited regions. Simultaneous excitation ofboth muscle layers could be elicited by mucosal stimu-lation of either ascending or descending reflex path-ways. Neural excitation resulted in an increase in thefrequency of Ca21 waves and induction of new pacingsites without eliciting direct coupling between layers.Conclusions: Localized, spontaneous Ca21 waves oc-cur independently in both muscle layers, promotingmixing (pendular or segmental) movements, whereasactivation of neural reflexes stimulates Ca21 wavessynchronously in both layers, resulting in strong peristal-tic or propulsive movements.

To understand motility patterns in the gastrointesti-nal tract, it is important to discern how longitudinal

(LM) and circular muscles (CM) act together to produceboth mixing and peristaltic movements. The 2 musclelayers are innervated by different populations of motorneurons with different distributions,1–3 suggesting thatthey can be regulated independently by nerves. Motorneurons to the LM of the small intestine are mainly(,95%) excitatory, whereas those to the CM are bothexcitatory and inhibitory (with roughly equal contribu-

tions).3 Movements of either muscle alone should producemixing of chyme4; however, relative movements of themuscle layers and the role that nerves play in coordinat-ing their activities have been a subject of controversy formany years. Recently, Wood5 argued that the LM and CMlayers cannot contract or relax together because ‘‘. . . theanatomical arrangement of the circular and longitudinalmuscle coats and the laws of geometry dictate that theyare antagonistic muscles (i.e., shortening of one opposesshortening of the other).’’ Bayliss and Starling6 in 1899were aware of the possibility of such passive mechanicalinteractions between the muscles, but considered them tobe ‘‘negligible’’ with their recording system, whichrevealed synchronous contractions and relaxations of the2 layers during peristalsis in the canine small intestine.

Other studies involving the small intestine have alsosuggested contrasting views of conduction between musclelayers, including: (1) movements of the 2 layers aresynchronous; (2) muscle layers are electrically coupled;(3) movements are 180° out of phase with each other; and(4) there is no correlation between movements of the 2muscle layers.7 In canine small intestine, studies haveshown that muscles appear to move 180° out of phasewith respect to each other, whereas in the stomach theyappear to contract together.8 Melville et al.9 used videomethods to monitor markers along the length of thefeline duodenum and concluded that contractions in LMare independent of those in CM. Similar conclusions werereached by Yokoyama and North10 who found no correla-tion between spontaneous action potentials in LM andCM of the guinea pig small intestine. At the threshold forperistalsis, however, both muscle layers exhibited burstsof action potentials at the same time, suggesting thatactivation of neural reflex pathways could coordinateactivities.

Abbreviations used in this paper: ACh, acetylcholine; CM, circularmuscle; ICC, interstitial cells of Cajal; KRB, Krebs–Ringer’s bicarbon-ate; LM, longitudinal muscle; ROI, region of interest.

r 2000 by the American Gastroenterological Association0016-5085/00/$10.00

doi:10.1053/gg.2000.7042

GASTROENTEROLOGY 2000;118:892–904

Studies of peristalsis using the Trendelenburg prepara-tion also emphasized differences in the movements of the2 muscle layers of the guinea pig small intestine duringpropulsion.11 During peristalsis, there was an initialcontraction of LM (type I contraction) that was laterfollowed by a propagating contraction of the CM (type IIcontraction). During the type II contractions, LM ap-peared to relax. These studies, as well as others showingthat the 2 muscles respond differently to drugs andelectrical field stimulation, led Kottegoda12 to put forththe now widely held notion of reciprocal innervation:when excitatory nerves to one layer are activated to causecontraction, inhibitory nerves to the other layer areactivated at the same time to cause relaxation.

Kottegoda’s conclusions are, however, based on me-chanical studies that sometimes fail to eliminate possiblepassive mechanical interactions between the 2 musclelayers. Contraction of the CM may lead to passivelengthening of the LM and vice versa.6,13,14 Recentstudies, which have focused on designs to avoid theseinteractions, have shown that in response to peristalticwaves, point activation of enteric reflexes, and extrinsicnerve stimulation, both muscle layers in the small andlarge intestine contract and relax together,14–20 as origi-nally suggested by Bayliss and Starling.6 In addition,recent less invasive techniques such as video imaging(guinea pig small intestine)21 and ultrasonography (hu-man esophagus)22 have also shown synchronous contrac-tion and relaxation of the 2 muscle layers after activationof enteric reflexes, suggesting that excitatory and inhibi-tory motor neurons to both layers can be activated at thesame time. Although Hennig et al.21 found that the 2muscle layers contracted together during the initial phaseof peristaltic contractions, they also observed a lengthen-ing of the intestine when CM contraction becameluminally occlusive.

Electrophysiological studies have generally suggestedthat slow waves and their associated contractions occursynchronously in both muscle layers of the intestine.23–25

This was thought to occur because slow waves generatedin the LM spread electrotonically into the CM via bridgesof muscle cells spanning the 2 layers.23 Although musclefibers have been observed to occasionally bridge the 2muscle layers,26 there seems to be little or no electrotoniccoupling27or spread of action potentials10 between theLM and CM in the guinea pig ileum. Recent reports alsosuggest that indirect coupling may exist because slowwaves appear to arise from common networks of pace-maker cells (interstitial cells of Cajal [ICC]) in thevicinity of the myenteric plexus located between the 2layers.28

Calcium is the intracellular mediator linking electricalexcitation with muscle contraction. Recently, we intro-duced low-light video imaging techniques to directlyvisualize the origin and spread of Ca21 waves in theguinea pig large intestine29 and canine gastric antrum.30

In this study, we extend these techniques to visualize thepropagation of Ca21 waves simultaneously in the LM andCM layers of the guinea pig small intestine. We showthat Ca21 waves occur in both layers, but a wave in 1layer has no effect on the origin, rate of propagation, orrange of spread of waves in the other layer. Neuralinduction of new pacing sites at the same time in bothmuscles provides a mechanism for synchronized contrac-tion of the 2 layers during peristalsis.

Materials and Methods

Muscle PreparationGuinea pigs weighing between 250 and 300 g were

killed by asphyxiation in a CO2 chamber, followed by severingof the carotid arteries and exsanguination, in accordance withthe requirements of the Animal Ethics Committee at theUniversity of Nevada, Reno, which follows the NationalInstitutes of Health guidelines (NIH Publication no. 80-23;revised 1978). All efforts were made to minimize suffering ofanimals and the number of animals used in these experiments.Segments (5–6 cm) of small intestine were removed from theregion 10–15 cm above the ileocecal junction. Segments wereopened along the mesenteric border and pinned flat across anoptical window with the mucosa uppermost to the bottom of acustom-designed organ chamber (Figure 1), as describedpreviously.1,29,31 A rectangular region (1 3 1 cm) of mucosaabove the optical window was removed to avoid excessivebackground light from entering the field of view. Tissues werecontinuously perfused with oxygenated Krebs–Ringer’s bicar-bonate (KRB) solution for 2–3 hours at 37°C before dyeloading.

Loading Muscles With FluorescentIndicator

After equilibration, the area of the tissue with exposedmuscle was treated with 5 3 1026 mol/L of the Ca21-sensitiveindicator fluo-3 acetoxymethyl ester, 0.01% dimethyl sulfox-ide, and 0.025% of the noncytotoxic detergent cremophor ELfor 15–25 minutes. LM and CM in the dissected island lyingover the optical window could be loaded independently (or atthe same time) with fluo-3 by pipetting the Ca21 indicatorunder the serosal surface to load the LM or onto the submucosalsurface to load the CM.29–32 When dye loading was complete,tissues were again allowed to equilibrate in KRB solution foran additional 15–20 minutes at 37°C before the experiments.

Evoked Neural Responses

Ascending or descending neural reflexes were evokedby mechanically stimulating the mucosa 10 mm anal or oral to

May 2000 NEURAL REGULATION OF CALCIUM WAVES 893

the recording site, respectively.1,33,34 Mucosal stimulation wasperformed using sponges (diameter, 4 mm) that lightlydepressed the villi. Sponges were mounted to custom-built,computer-controlled solenoids (Figure 1).29,34

Data Acquisition and Analysis

Muscles were illuminated at 470 6 20 nm, andfluorescent emissions . 510 nm (e.g., Figure 2A) wererecorded as a relative measure of intracellular Ca21 concentra-tion ([Ca21]i) using an intensified charge-coupled devicecamera (Dage-MTI model SIT-66X, Michigan City, IN) at-tached to the videoport of an inverted epifluorescence micro-scope (Nikon Diaphot, Tokyo, Japan).31 Usually, a 103 fluorobjective (Nikon) was used, yielding a field of view of 1.2 31.0 mm. To compute conduction velocities in the long axis ofmuscle fibers, a 23 objective and low-power (e.g., 1.63) relaylens were sometimes used (6.0 3 5.0–mm field of view).Images were stored on a computer-controlled videocassetterecorder (Sanyo model GVR-S950, Itasca, IL) at a rate of 30frames per second.

Image Analysis

Analysis of videofluorescent signals was performedusing both custom-designed and commercially available soft-ware (Adobe PhotoShop, San Jose, CA). Images were digitized(640 3 480 pixels) using a frame grabber (Data Translationmodel 3152, Marlboro, MA).31 Because each frame could beanalyzed individually using the videocassette recorder, a tempo-ral sampling rate of 30 frames per second could be achieved.

To view propagation, series of contrast-enhanced images

were computed by storing an initial frame (with no apparentactivity) as a ‘‘background image’’ (first frame in Figures 3–8).This image was then subtracted from each subsequent frame.To further aid visualization, all contrast-enhanced frames aredisplayed as ‘‘negative images,’’ in which dark areas correspondto regions of increased fluorescent intensity. Conduction veloci-ties were measured by determining the distance traveled by awave front over a single digitized frame (i.e., over a 33.3-millisecond interval).

To monitor Ca21 transients over prolonged periods, regionsof interest (ROIs) in the shape of a parallelogram (usuallyrectangular) were selected (Figure 2A) with elongated sidesaligned to the long axis of muscle fibers in each muscle layer (orlayers). In some cases, muscle fibers were not preciselyorthogonal to each other and sampling parallelograms wereconstructed at appropriate angles31 (Figures 7 and 8). Pixelintensities for each ROI were summed and normalized for area(Figure 2B). To determine temporal relationship betweenactivities in different ROIs, event time plots were constructedby thresholding summed intensities and representing eachCa21 transient as a vertical line segment (Figure 2C). All eventtime plots were verified by manually viewing video data on aframe-by-frame basis.

Statistics are reported as mean 6 SD. All image series inFigures 3–8 are incremented in time from left to right and topto bottom. In addition, all frames are orientated so that thevertical direction is aligned (approximately) with the long axisof LM fibers (oral to anal) and the horizontal directionrepresents the long axis of CM fibers (as illustrated in Figure2A).

Solutions

The KRB used in this study contained (in mmol/L)NaCl, 120.4; KCl, 5.9; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 15.5;NaH2PO4, 1.2; and dextrose, 11.5 (Fisher Scientific, Pitts-burgh, PA). This solution had a pH of 7.3–7.4 at 37.5°C whenbubbled to equilibrium with 97% O2–3% CO2. Fluo-3 waspurchased from Molecular Probes (Eugene, OR). Dimethylsulfoxide, atropine, nicardipine, tetrodotoxin, and cremophorEL were obtained from Sigma Chemical Co. (St. Louis, MO).

Results

By monitoring fluorescent intensities of the indi-cator fluo-3, Ca21 waves could be observed in LM only(serosal loading), in CM only (submucosal loading), or atthe same time in both muscle layers (see Materials andMethods and Figure 2). Ca21 waves in either muscle layerwere completely eliminated by the L-type Ca21 channelantagonist nicardipine (1 mmol/L; n 5 6).

Origin and Spread of Excitability in LM

Spontaneous Ca21 waves (frequency, 1.2 6 0.5/s;n 5 14 tissues) emerged from discrete sites and propa-gated with anisotropic conduction velocities through the

Figure 1. Fluorescent imaging and neural stimulation apparatus. Themucosa was removed to create an island of exposed LM and CMsituated atop a quartz window positioned over a microscope objective.Ascending or descending neural pathways were evoked by loweringsponges attached to computer-controlled solenoids placed eitheranally or orally to the recording region, respectively. Tissues within thefield of view were irradiated by 470 6 20 nm light, and fluorescence .

510 nm was collected using an intensified camera.

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LM layer of the ileum (Figure 3A). These events weresimilar to those we observed in LM of the distal colon.29

Propagation velocities were approximately 8 times fasterparallel (87.3 6 22 mm/s; vertical direction in the field

of view of Figure 3A; n 5 50 events from 4 tissues)compared with orthogonal (10.8 6 4.0 mm/s; n 5 50events from 14 tissues) to the long axis of the LM fibers(Figure 3A). Spontaneous Ca21 waves within the LMlayer emerged from discrete sites within fields of view(Figure 3A–D) and typically spread outside the field ofview. Ca21 waves entered the field of view with nopreference from the oral, anal, or circumferential direc-tions (Figure 3E). Five percent of Ca21 waves (3 of 60events in 5 tissues) emerged from discrete sites and couldbe seen to initially spread rapidly along the length offibers, but failed to propagate for any significant distanceorthogonal to the long axis of LM fibers (Figure 3B).These ‘‘minimum conducting bundles’’ were 250 6 85µm (n 5 30 events from 5 tissues; range, 110–400 µm) inwidth.

The extent of propagation of Ca21 waves in thecircumferential axis was limited by encountering refrac-tory or suppressed regions, occasionally for no apparentreason (Figure 3C and E, wave IV), or by colliding withother Ca21 waves from neighboring regions (Figure 3Dand E, wave V). When opposing Ca21 waves collided,they did not propagate past each other (Figure 3D, frames5 and 6; 3E, wave V; and 3F, dashed lines), suggestingthat each wave is followed by a wake of refractoriness.Each collision resulted in the annihilation of a pair ofpropagating wave fronts parallel to the long axis ofmuscle fibers (without affecting the wave fronts associ-ated with each event propagating in opposite directions).

Ca21 waves typically propagated 11.6 6 4.5 mm (n 550 events from 4 tissues) in the long axis of LM fibers andterminated by encountering refractory regions or bycolliding with other Ca21 waves. Termination in this axisresulted in excited regions with thin, finger-like projec-tions (Figure 4A). The locations of these finger-likeprojections were dynamic, generally changing from eventto event.

Ca21 waves propagating in the long axis of LM fiberswere also frequently observed to propagate collaterallyaround recently excited (i.e., refractory) regions. Figure4B shows an example of how pathways of Ca21 waves canbe influenced by the presence of a refractory region. ACa21 wave propagated anally through the center of thefield of view (frames 1–6), leaving this area transitorilyrefractory. A second Ca21 wave emerged just to the left ofthe field of view and propagated adjacent to, but not into,the refractory region (frames 8–10). This wave sweptaround the refractory region, reentering the lower portionof the field of view (frames 10–15) and propagatingalongside the region. After a delay, reexcitation of theregion initially left refractory by the first wave eventuallyoccurred by circumferential spread inward from adjacent

A

Figure 2. Imaging processing steps used to generate event time plotsover extended periods. (A) In the background fluorescent image,rectangular ROIs (white rectangles) within the field of view (1.2 3 1.0mm) were selected to favor the detection of Ca21 transients within LM(long axis of muscle fibers oriented vertically) or CM (long axis ofmuscle fibers oriented horizontally). (B) Summed pixel intensities fromeach ROI were normalized for area. Large peaks indicate the spread ofCa21 waves in LM (upper trace) and CM (lower trace), whereas smallerpeaks in the upper (*) and lower (**) traces result from Ca21

transients sweeping through portions of ROIs in the opposing layer.(C) An amplitude threshold to detect the larger peaks was used toconstruct event time plots for each muscle layer.

May 2000 NEURAL REGULATION OF CALCIUM WAVES 895

Figure 3. Propagation of Ca21

waves in LM. (A) The leftmostframe shows background fluores-cence of LM. The subsequentsequence (frames 2–6, 0–133milliseconds) shows the emer-gence and anisotropic propaga-tion of a Ca21 wave. Note thatthe wave propagates rapidly(leaving the field of view by frame3) in the direction of the LMfibers (vertical) and less rapidlyorthogonal to the long axis ofthe muscle fibers (horizontal orcircumferential direction). Bar 5

300 µm. (B) Propagation wasrapid in the direction of LM fi-bers (vertical) but was limited to,200 µm in the circumferentialaxis. Occasionally propagationorthogonal to LM fibers stoppedfor no apparent reason (C and E,wave IV; arrow indicating lack ofconduction). In most cases,propagation orthogonal to LMfibers stopped on collision withCa21 waves propagating fromthe opposite direction (D and E,wave V). Event time plots (E,using 3 ROIs of background im-age in A) show propagation fromboth circumferential directions(left-to-right waves I and VI; right-to-left waves II and III). (F )Summed fluorescent intensitiesduring a collision of circumferen-tially propagating Ca21 waves inwhich the superimposed dashedwave forms indicate the positionof Ca21 waves if they had propa-gated through each other with aconstant conduction velocity.

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excited regions (frames 15–18). Extremely complex pat-terns of excitation can arise rapidly as a result of spreadaround refractory regions.

Origin and Spread of Excitability in CM

Spontaneous Ca21 waves (3.2 6 0.2 events/min;n 5 14 tissues) were observed much less frequently inCM compared with longitudinal tissue. The relativeabsence of Ca21 waves in CM was not caused byinsufficient loading of the muscle with fluo-3, becauseafter a period of mucosal stimulation, there was a markedincrease in the density and frequency of observed Ca21

waves (see below). In some preparations in which bothlayers were loaded with fluo-3 (3 of 50 events in 4tissues), it was possible to study the activity in CMduring lulls in the almost continual activity of thelongitudinal layer. Ca21 waves within the CM layerexhibited many of the same characteristics as those seenin LM. They were observed to emerge repeatedly fromdiscrete sites and propagate anisotropically (Figure 5A).Propagation velocities were approximately 9 times fasterparallel (77.7 6 13.5 mm/s; 50 events in 4 tissues) thanorthogonal (8.4 6 1.8 mm/s; 100 events from 14 tissues)to the long axis of CM fibers. Infrequently (2 events of 50from 4 tissues), Ca21 waves emerged from discrete sitesand propagated rapidly in the long axis but failed to

spread very far in the short axis of the CM fibers. Thisresulted in narrow bands (i.e., ‘‘minimum conducting bundles’’)of muscle excitation (200 6 20 µm; n 5 30 events from 5tissues; range, 150–300 µm) that extended across the width ofthe field of view (Figure 5B). Ca21 waves entered the field ofview with no directional preference (e.g., Figure 5E, waves I,II, III, and V), but sometimes ceased to propagate throughthe field of view (Figure 5C and E, wave VI) for noapparent reason.

The extent that a Ca21 wave traveled along the bowel(slow axis) was generally limited by collisions (Figure 5Dand E, wave IV). Colliding Ca21 waves in the CM did notpropagate past each other (Figure 5E, wave IV; andFigure 5F) and (as in LM) terminated in a single event ofsimilar magnitude to the waves before the collision(Figure 5F). Ca21 wave propagation in the long axis ofthe CM fibers resulted in an entire excited band of musclethat invariably encompassed the entire width (5.3 6 1.2mm) of the preparation, spreading as a planar wave frontanally, orally, or (most frequently) in both directions.

Activities in Both LM and CM Layers

By adjusting the focal plane of the microscope, wewere able to visualize muscle fibers and Ca21 waves inboth muscle layers at the same time, thereby monitoringthe excitability in LM and CM simultaneously. A typical

Figure 4. Termination of Ca21

waves in LM. (A) Frames 2–12(0–333 milliseconds) illustratetermination of an LM Ca21 wavethat results in numerous ‘‘finger-like’’ projections in the directionof the long axis of LM fibers.Bar 5 300 µm. (B) Initial frames1–6 (0–166 milliseconds) showan LM Ca21 wave that stoppedpropagating in circumferential di-rections near the center of thefield of view (region labeled 1). Asecond Ca21 wave (initiated inregion labeled 2 in frame 8)propagated collaterally aroundthis recently excited region.Frame 14 (with dotted lines atthe locations of wave frontsadded for clarity) shows that re-gions 2 and 3 are excited,whereas region 1 is not. After aprolonged delay, the secondwave finally enveloped region 1(frames 17 and 18).

May 2000 NEURAL REGULATION OF CALCIUM WAVES 897

Figure 5. Propagation of Ca21

waves in CM. (A) Frames 2–6(0–133 milliseconds) show theemergence and anisotropic prop-agation of a Ca21 wave in CMwith rapid conduction in the longaxis of CM fibers (horizontal)and slower conduction along thelength of the intestine (vertical).Bar 5 300 µm. (B) A Ca21 waveemerged (frame 1) in CM andpropagated rapidly in frames 1–3(0–66 milliseconds) across thefield of view but failed to propa-gate in frames 4–6 (100–166milliseconds) along the length ofthe intestine (limited to ,250µm). (C) As illustrated in frames1–6 (0–166 milliseconds), occa-sionally propagation orthogonalto CM fibers stopped for no ap-parent reason (E, wave VI; arrowindicating lack of conduction).(D) As shown in frames 1–6(0–166 milliseconds), in mostcases propagation in CM alongthe intestine stopped upon colli-sion with another Ca21 wave(see frames 3–6; E, wave IV, andF ). (E) Event time plots (using 3ROIs shown in the backgroundimage in A) show propagationfrom either direction along thegut (oral to anal, waves I, II, V,and VI; anal to oral, wave III). (F )Summed fluorescent intensitiesduring a collision of anally andorally propagating Ca21 waves inCM in which superimposeddashed wave forms indicate theposition of Ca21 waves if theyhad propagated through eachother with a constant velocity.

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Figure 6. Ca21 waves recordedsimultaneously in both layers.Ca21 waves were viewed in bothLM (long axis of LM fibers ori-ented vertically) and overlyingCM (long axis of CM fibers ori-ented horizontally). Arrows indi-cate the direction of propagationof Ca21 waves in the slow axesof LM and CM. (A) An estab-lished Ca21 wave within the LM(first frame) did not affect theability of Ca21 waves to propa-gate in CM (frames 2–12; 33–333 milliseconds). Bar 5 300µm. (B) Conversely, a Ca21 waveestablished within the CM(frames 1–6; 0–166 millisec-onds) did not obstruct the propa-gation of a Ca21 wave in theunderlying LM (frames 7–12;199–366 milliseconds).

A

B

Figure 7. Ascending excitatoryreflex. (A) Frames 2–12 (0–333milliseconds) show the induc-tion of multiple pacing sites inLM (labeled A and B) and CM(labeled I–III) in response to de-pression of the mucosa 10 mmanal to the recording site. Bar 5

300 µm. (B) The upper traceshows a time line of the applica-tion of the stimulus, and the barabove the stimulus trace showsthe time over which frames in Awere recorded. Event time plotswere constructed based on in-tensities in CM (CM 1 and CM 2)and LM (LM 1) from the 3 ROIsin the background image in A.Stimulation elicited new pacingsites in both muscle layers andincreased overall Ca21 wave fre-quency (ascending excitation).An expanded time scale duringstimulation shows that someevents in CM did not propagatethroughout the field of view andindividual events in LM do notcoincide with activity in the CM.

May 2000 NEURAL REGULATION OF CALCIUM WAVES 899

example of the simultaneous propagation of Ca21 wavesin both muscle layers is shown in Figure 6. In Figure 6A,a Ca21 wave in the LM entered the field of view from theleft and propagated circumferentially (frames 2–12)across the field of view. Then a Ca21 wave in the CMspread with a similar conduction velocity in an oral-to-anal direction, passing over without disturbing the wavein the LM (frames 3–12). As the waves overlapped, lightintensity increased above levels generated by a Ca21 wavewithin a single muscle layer. This arose as a result of thecamera summing fluorescence from the 2 muscle layersand not because of increased intracellular [Ca21] in theseregions.

Figure 6A shows that the spread of a Ca21 wave in LMdoes not affect propagation in the circular layer. Theseries of frames in Figure 6B show the opposite sequenceof activities in the same tissue. The trail of refractorinessleft by a Ca21 wave in the CM layer propagating anallyacross the field of view (frames 1–7) did not affect theemergence or circumferential spread of a Ca21 wave in theunderlying LM layer (frames 7–12). Spontaneous Ca21

waves in both muscles were blocked by atropine (1mmol/L; 5 tissues), suggesting they are induced bycholinergic activity.

Neurally Induced Excitation of CM and LMLayers

As we have shown recently,20 mucosal stimulationof the ileum produces synchronous contractions of bothLM and CM layers both above (ascending excitation) andbelow (descending excitation) the site of stimulation.Experiments were performed to determine if activation ofexcitatory reflex pathways affects the spread of Ca21

waves within and between each muscle layer.Mechanical stimulation of the mucosa increased the

frequency of Ca21 waves both oral (ascending excitation:LM from 1.7 6 0.8 to 11.9 6 3.1 s21 and CM from0.07 6 0.01 to 12.8 6 2.8 s21; 5 responses from 5tissues) and anal (descending excitation: LM from 1.4 60.6 to 13.4 6 2.6 s21 and CM from 0.06 6 0.01 to13.9 6 3.0 s21; 5 responses from 5 tissues) to thestimulus. Events were initiated from multiple pacing

A

B

Figure 8. Descending excita-tory reflex. (A) Frames 2–12 (0–333 milliseconds) show the in-duction of multiple pacing sitesin LM (labeled A–C) and CM(labeled I–V) in response to de-pressing the mucosa 10 mmoral to the recording site. Bar 5

300 µm. (B) The upper traceshows a time line of the applica-tion of the stimulus, and the barabove the stimulus trace showsthe time over which frames in Awere recorded. Event time plotswere constructed based on in-tensities in CM (CM 1 and CM 2)and LM (LM 1) from the 3 ROIsin the background image in A.New pacing sites in both musclelayers and increases in overallpacing frequency (descending ex-citation) were elicited by muco-sal stimulation. The expandedtime scale during stimulationshows that some events in CMdid not propagate throughout thefield of view, and activity in LM isnot coupled with events in CM.

900 STEVENS ET AL. GASTROENTEROLOGY Vol. 118, No. 5

sites, at the same time in both muscle layers (Figures 7and 8). The neurally evoked increase in the number ofpacing sites seems to be the basis for both ascending anddescending excitation.1,16,19,20,34,35 Exploded views ofevent traces illustrate that during stimulation of neuralreflexes, there was a reduced temporal correlation of Ca21

waves induced across the CM (Figure 7B, CM 1 vs. CM 2;Figure 8B, CM 1 vs. CM 2) and no correlation betweenwaves induced in CM with those induced in LM (Figure7B, CM 1 and CM 2 vs. LM 3; Figure 8B, CM 1 and CM2 vs. LM 3).

Reflexly induced increases in the frequency of Ca21

waves in both the LM and CM layers were abolished byatropine (1 mmol/L) and tetrodotoxin (1 mmol/L).

Discussion

Motility patterns in the gastrointestinal tract areprimarily coordinated by slow waves initiated in ICC thatappear to spread electrotonically into underlying smoothmuscle cells where they can generate Ca21-dependentaction potentials,28,36,37 and by the activation of intrinsicand extrinsic nerves that regulate these activities.15,19

Action potential firing in smooth muscles triggers Ca21

influx, which is the primary activator of muscularcontraction.28

Using fluorescent imaging techniques, we have beenable to observe elevations in intracellular [Ca21] thatpropagate in both LM and CM layers of the guinea pigsmall intestine. These Ca21 waves depend on spontaneousrelease of acetylcholine (ACh) from cholinergic motorneurons because they are blocked by atropine (which alsosuggests some degree of cholinergic tone).20

Spontaneous Ca21 waves are more frequent in LM thanin CM. They are eliminated by nicardipine (L-typechannel blocker) and are therefore likely to be initiated bysmooth muscle action potentials,28,38 which, like localcontractions,9,21,39 are more frequent in LM than inCM.10,25 The action potential dependence of Ca21 wavesis also supported by the observation that they propagatealong the long axis of muscle fibers with conductionvelocities that are similar to velocities (67–88 mm/s) ofaction potentials along strips of guinea pig taenia coli.40

It is generally considered that in most smooth muscletissues, action potentials are initiated by slow waves.However, classical slow waves are rare in open-sheetpreparations of guinea pig ileum, presumably becausecutting the preparation may lead to a lack of entrainmentbetween pacemaking cells (presumably ICC).41 Actionpotentials in these opened-sheet preparations are likelyinitiated by fluctuations in membrane potential causedby the random coupling of slow wave generators. This

coupling may be enhanced by ACh, which increases thefrequency and amplitude of slow waves.32,42 Alterna-tively, action potentials could be produced by ACh actingdirectly on muscles, which can also generate oscillationsin membrane potential similar to slow waves.43 Themaximal extent of propagation of action potentials isdetermined by the area of muscle depolarized by the slowwave and limited temporally by the repolarization phaseof the slow wave.44

Spontaneous Ca21 waves in LM exhibit characteristicssimilar to those we recently reported in LM of the distalcolon.29 The constant shifting in pacing sites, which isespecially characteristic of LM, probably prevents expo-sure of cells to sustained high levels of intracellular Ca21,which may be toxic to individual muscle cells.45,46

Overall, the shifting pacing sites can maintain a basallevel of contractile activity at the tissue level.

Spread of Excitability

Propagation velocities of Ca21 waves are similarand show a similar anisotropy with respect to orientationof muscle fibers within LM and CM. In both layers, wavesare 8–9 times faster parallel compared with perpendicularto the long axis of muscle fibers (Figures 3 and 5). Thisanisotropy in the conduction velocities of Ca21 waveslikely reflects the difference in electrotonic couplingparallel and transverse to each muscle fiber38 which, inturn, is a determinant of action potential velocity in the 2axes.

Conduction in each axis of LM should produce com-plex shearing forces that stir chyme by generating localregions of rapid wall shortening. These regions spreadmore slowly in the circumferential direction until theycollide with adjacent Ca21 waves (Figure 3). Spontaneouslocal contraction of LM on one side of the intestinal tuberesults in a decrease in the radius of curvature on that sideof the lumen. During contraction, chyme is bidirection-ally displaced relative to the inner wall of the lumen,exposing the contents to new regions of mucosa, which inturn promotes mixing and absorption. These asymmetriccontractions of LM produced by local, anisotropicallypropagating Ca21 waves may contribute to the ‘‘squirm-ing’’ motion (Figure 9A) of the small intestine commonlyobserved in vivo and in situ.

In contrast, anisotropic propagation velocities of Ca21

waves in CM allow for rapid conduction around thecircumference of the intestine. This should organizeexcitability of this layer into rings of contraction (Figure4) that propagate slowly in the short axis of CM fibers(along the gut) until they encounter another recentlyexcited region. These slowly conducting rings of contrac-

May 2000 NEURAL REGULATION OF CALCIUM WAVES 901

tion produce a ‘‘squeezing’’ of the lumen (Figure 9B) topromote additional mixing and absorption.

Minimum Motor Units

In both layers there seems to be a minimum widthof muscle that can support the spread of Ca21 transientsparallel to muscle fibers, but not for a significant distanceorthogonal to the long axis of muscle fibers. This width,which is ,200 µm in LM and ,250 µm in CM, isconsistent with the concept of a minimum width (motorunit) of parallel smooth muscle fibers necessary to sustainaction potential propagation.48,49 The width of LM motorunits is similar to the extent of spread of terminal axons ofLM motor neurons,2 suggesting that these bands ofmuscle may be directly activated by release of ACh fromindividual motor neurons. Similarly, the motor unit inCM may be the width of the ring of muscle innervated bythe circumferential spread of axonal collaterals from CMmotor neurons.

Extent of Propagation

The extent of propagation varies from the mini-mum width of muscle that can sustain propagation(‘‘minimum conducting bundle’’) to complete sweepsacross the field of view. Ca21 waves originating from anyone pacing site in a particular muscle layer are notrepeatedly restricted by identifiable structures. Instead,waves can propagate through any neighboring region andaround regions of the same layer recently excited and/orin a refractory state. The exact dynamics of Ca21 wavetermination varied somewhat in each muscle layer. In thelong axis of CM fibers, the extent of propagation seems tobe limited by the circumference of the lumen. Ca21 wavepropagation in this axis is rapid and always extended tothe edges of a segment. This suggests that in a tube, awave is only limited by colliding with itself (producing aring-like constriction). In the short axis of both muscle

layers, the extent of propagation is limited by (1)collisions with neighboring Ca21 waves, (2) encounteringregions that have recently been excited (i.e., that arerefractory), and less often (3) stopping for no apparentreason. In all cases, Ca21 wave fronts stop as a plane (astraight line when viewed in 2 dimensions) of excitabil-ity.

Parallel to the long axis of longitudinal fibers, eventsterminated with an uneven profile, generating finger-likeprojections (Figure 4) resulting from either intermittentbranch-point failure of action potentials to cross anasto-moses joining muscle bundles26 or encountering regionsthat had recently been excited. Wave fronts spreadaround refractory regions, forming multiple finger-likeprojections (collateral pathways). Some refractory regionsare as small as minimum motor units. When a waveencounters such a region there is often an apparent delayas the Ca21 wave takes collateral pathways around theregion in the slower axis. Collisions and branch-pointfailure of action potentials restrict contractions in thelong axis of the LM from sweeping the entire length ofthe intestine, thus maintaining regional regulation ofcontraction. In addition, these finger-like terminationsresult in a more gradual transition in excitability betweensegments of small intestine in the excited state vs. thosein a nonexcited state, in turn avoiding cell damage byreducing large strain gradients across the transition zone.

Independence of the Two Muscle Layers

For sometime there has been controversy overwhether the 2 muscle layers can act independently (seeintroduction). Studies of spontaneous contractions sug-gest that they can because events occur much more oftenin LM than in CM.9,21,39 Action potentials are common inLM to the degree that it is difficult to find a region voidof activity for any extended period of time. Localcontractions in LM sum to produce the overall pendularor rocking movements that are characteristic of thislayer.6,21,39 In circular tissues of the small intestine, Ca21

waves seem to be generated only when slow wavespropagating through bulk CM are of sufficient amplitudeto depolarize spiking cells in the innermost dense circularregion. The combination of slow waves and actionpotentials may be required for ring-like contraction ofCM.49

The current study clearly shows that Ca21 transients inthe LM and CM layers in the guinea pig small intestineare not coupled because Ca21 waves occurring in 1 musclelayer do not induce new pacing sites, affect propagation,or stop propagation from occurring in the opposing layer.In addition, although refractoriness limits the extent ofpropagation in 1 layer, there seems to be no influence ofthe refractory period on Ca21 waves in the other layer.

Figure 9. Patterns of force development in the ileum. (A) Spontane-ous Ca21 waves in LM generate a radially asymmetric force about thelumen resulting in a ‘‘squirming’’ motion. (B) Spontaneous Ca21

waves in CM produce ring-like contractions of the lumen resulting in a‘‘squeezing’’ motion. In both cases, propagation of Ca21 waves isgoverned primarily by the properties of the syncytium. (C) Upon neuralstimulation, new pacing sites are induced in both muscle layers duringperistalsis. The overall spread of this region of increased Ca21 wavedensity is governed by the spread of excitability in the enteric nervoussystem.

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If Ca21 waves could spread from 1 layer to the other,overall wave fronts would not spread in a planar fashiontransverse to the long axis of muscle fibers, but wouldpropagate diagonal to the long axis in each layer. Theexact direction would depend on the relative magnitudeof the fastest velocities in each layer. Because the fastestconduction velocities parallel to the long axis of eachmuscle layer are similar, the observed direction ofpropagation would be approximately 45° relative to thelong axis in each muscle layer. By maintaining separateactivities, tissues are able to maintain greater regionalcontrol over excitability. If there were communicationbetween muscle layers, the slower conducting axis wouldbe circumvented by rapid propagation velocities in theother muscle and would not play a role in movement ofchyme. In contrast, independent activities in each musclelayer allow the slow-conduction velocities, which aremore consistent with viscoelastic properties of chyme, topromote mixing.

Effects of Neural Reflexes

Mucosal reflex stimulation increases the numberof pacing sites and the frequency of Ca21 waves at thesame time in LM and CM layers both above (ascendingexcitation) and below (descending excitation) the site ofstimulation. These increases, which mirror the rapidaction potential firing of muscles during peristalsis,10,50

suggest they are the basis for synchronous reflex contrac-tions of LM and CM layers observed both oral (ascending)and anal (descending excitation) to mucosal stimuli.20

In the guinea pig distal colon, a similar induction ofnew pacing sites occurs in LM oral to the stimulus,although anal to the stimulus Ca21 waves were com-pletely blocked while the mucosa was depressed.29 Thisdescending inhibition is followed by excitation or induc-tion of new pacing sites (‘‘off excitation’’) after thetermination of the stimulus.29 The suppression of Ca21

waves seems to underlie the nitric oxide–mediated analrelaxation observed in the colon.17 In contrast, the role ofdescending inhibitory pathways in the ileum remainsunclear; reflex-evoked fast inhibitory junction poten-tials51 might regulate propulsion by reducing the rateand strength of the anal contraction as it propagates downthe bowel.20

During stimulation, new pacing sites are inducedrandomly (both spatially and temporally) in both musclelayers; consistent with the notion that although thesensory neurons and interneurons in the reflex pathwaysare likely to be common to both muscle layers, theexcitatory motor neurons innervating each layer aredifferent.1–3 Neural stimulation causes frequent collisionsbetween waves propagating in the short axis that origi-nate from adjacent pacing sites, producing a more

synchronous excitation. This confinement of excitation tothin slivers of LM ensures that contractions occur at thesame time around the gut to sum and produce uniformshortening of a segment of intestine.29 In CM, collisionsbetween induced neighboring bands of excitation pro-mote stationary ring-like contractions that are confinedto neurally excited regions along the short axis of the CM.Therefore, collisions between Ca21 waves stop the spreadof excitation through the muscle, ensuring that thenerves directly control the sequential activation of theCM along the intestine during peristalsis (Figure 9C).The rate of spread of the synchronous induction of pacingsites in both layers during a peristaltic wave, whichtravels at approximately 25–30 mm/s down the ileum,20

is controlled by the speed of propagating neural activitywithin the reflex pathways of the enteric nervous system.

Conclusions

Ca21 waves occur independently in each musclelayer of the guinea pig ileum. A key factor for thisindependence is spike generation from multiple, discretepacing sites (regardless of electrotonic spread or commonslow wave generators between the muscles). This lack ofcoupling between muscles allows for independent move-ments during mixing. Neural induction of new pacingsites at the same time in each muscle allows for synchro-nized contraction of the 2 layers during peristalsis.

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Received August 9, 1999. Accepted January 13, 2000.Address requests for reprints to: Terence K. Smith, Ph.D.,

Department of Physiology and Cell Biology, MS 352, University ofNevada School of Medicine, Reno, Nevada 89557. e-mail:tks@physio.unr.edu; fax: (775) 784-6903.

Supported by a grant from the National Institutes of Health,NIDDK 45713 (to T.K.S.).

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