Visualization of origins and propagation of excitation in
canine gastric smooth muscle
Randel J.
Stevens,
Jeffery S.
Weinert, and
Nelson G.
Publicover
Biomedical Engineering Program, Department of Physiology and Cell
Biology, University of Nevada School of Medicine, Reno, Nevada
89557
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ABSTRACT |
The origin and
spread of excitation were visualized with fluo 3 fluorescence in
tissues isolated from canine gastric antrum. Sheets of circular muscle
(5 × 6 mm) had at least 1 (30%) and up to 3 discrete slow-wave
pacing sites located near the longitudinal-circular muscle boundary,
whereas similarly sized longitudinal sheets had an average of 5 sites
(range 3-12 sites) that initiated
Ca2+ waves. Superimposed
fluorescent oscillations (circular muscle) and spikes (longitudinal
muscle) were seen to initiate and propagate as distinct events,
separate from their underlying activities. Average propagation
velocities transverse (6-7 mm/s) and parallel (39-45 mm/s) to
the long axis of muscle fibers were similar for each type of event in
circular and longitudinal tissues; however, distinct regions where
velocities of some (but not all) events decreased by up to an order of
magnitude were present. The distance propagated by individual events
was limited by collisions with concurrent excitable events or recently
activated regions. Complex patterns of excitation in gastrointestinal
smooth muscle arise as a result of interactions between multiple pacing
sites, heterogeneous conduction velocities, and the interplay of
adjacent pacemaker domains.
electrophysiology; calcium; fluorescence
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INTRODUCTION |
GASTRIC PERISTALSIS occurs when slow waves originating
near the greater curvature of the corpus propagate aborally toward the
pylorus (4, 12, 32). At the cellular level, the strength of peristaltic
contractions is influenced by the degree of slow-wave depolarization
(5) and the coexistence of other excitable events such as spikelike
action potentials (12) and superimposed oscillations in membrane
potential (5, 12, 37). However, the overall efficacy of peristalsis can
be affected by a number of additional factors including the locations
of pacemaker activity (1, 32), the spontaneous frequencies at each site
(13), the synchronization of sites, the excitation conduction
velocities in each axis, and the distances traveled in each
direction (25). Although considerable progress toward understanding the
membrane and intracellular pathways that regulate cellular excitability
has been made (3, 10, 17), less is known about intercellular
interactions that affect the coordination of peristalsis. In this
study, the origin and propagation of excitation in gastric tissue
segments were directly observed by low-light, fluorescent video techniques.
Most studies of the origin and propagation of excitation in smooth
muscle have employed multiple electrodes to detect arrival times of
electrical activity in isolated tissue segments. Early studies using
extracellular electrodes suggest that complex synchronized spikes of
electrical activity arise from the summation of functional muscle units
and not individual muscle fibers (2). These studies also demonstrate
that conduction velocities in the long axis of muscle fibers are ~10
times greater than those found in transverse directions (21). By
measuring arrival times at two intracellular micropipettes in
cross-sectional preparations, Bauer and his colleagues (1) were the
first to suggest on the basis of electrophysiological evidence that
gastric slow waves originate in the circular layer region near the
myenteric border, although Thuneberg (36) had earlier reached this
conclusion on the basis of morphological data. The existence of
so-called "hot-spots" or preferred locations in tissues where the
majority of slow waves originate was demonstrated by using
triangulation algorithms and event arrival times at three intracellular
microelectrodes (28). In each of these studies, it has been assumed
that unitary events emerge and propagate via direct pathways with
uniform conduction velocities throughout tissue segments (26). Although
these techniques provide estimates of pacing locations and their
variability, the presence of muscle bundles (6), septal structures
(38), different cell types, and other inhomogeneities (7, 23) suggests
that propagation velocities and pathways may not be uniform.
More recently, Lammers and his colleagues (13, 14) employed an array of
240 extracellular surface electrodes covering an area of 15 × 16 mm to investigate the propagation of slow waves in the rat myometrium
(14) and rabbit duodenum (13). They developed "isochrones"
(contour maps indicating wave fronts at specific times) in order to
show the progression of electrical events. These maps provided some of
the first evidence of the complexity of conduction patterns and
pathways in smooth muscle, although the ability to accurately identify
the origin and exact pathways of individual events was somewhat limited
by the spatial resolution of the recording system (1 mm).
In the present study, the origin, propagation velocities, conduction
pathways, and extent that Ca2+
waves propagate in circular and longitudinal muscle layers of the
stomach were visualized. Video sequences demonstrate that complex
interactions among tissue regions arise as a result of Ca2+ waves emerging from multiple
pacing sites. Ca2+ transients
associated with slow waves in circular and longitudinal muscles,
oscillations in fluorescence superimposed during slow waves in circular
muscle, and rapid spikelike changes in fluorescence superimposed during
slow waves in longitudinal muscle each originate as unitary events from
discrete locations. Excitation patterns are also influenced by
temporally and spatially inhomogeneous conduction velocities,
collisions of events, morphological boundaries, and tissue injury.
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METHODS |
Muscle preparation.
Mongrel dogs of either sex were killed with pentobarbital sodium (30 mg/kg body wt). After the abdomen was opened, the entire stomach was
removed and placed in a bath containing Krebs-Ringer bicarbonate (KRB)
solution. Sheets of muscularis from the ventral surface, 7-9 cm
oral to the pyloric sphincter, were removed from the underlying mucosa.
In some experiments, the muscularis was dissected to expose a
cross-sectional preparation (12 × 4 mm) containing both muscle
layers as previously described (1). In other experiments, strips (16 × 10 mm) were carefully dissected to form sheets of tissue
consisting of either longitudinal or circular muscle.
The general procedure to load the
Ca2+-sensitive dye fluo 3 and
record fluorescence in these tissues has been reported previously (22).
Briefly, muscles were placed in 35-mm petri dishes and allowed to
equilibrate for 2-3 h in KRB at 37°C. The bottoms of dishes
were replaced with thin (no. 1) glass coverslips and coated with a
clear resin (Sylgard; Dow Corning). Tissues were pinned rigidly to
reduce any movement artifact. Preparations were treated with 5 × 10
6 M fluo 3-AM, 0.01%
DMSO, and 0.025% noncytotoxic detergent cremophor EL for 35-45
min. When dye loading was complete, muscle strips were allowed to
equilibrate for an additional 15 min at 37°C in KRB before use.
Data acquisition.
Muscle strips were illuminated at 470 ± 20 nm, and fluorescent
signals >510 nm were recorded as a measure of excitation. Unloaded tissues produced no significant changes in fluorescence during rhythmic
contractile activity. Although fluo 3 (when used alone) does not
provide an opportunity to perform ratiometric calibration, increases in
fluorescence likely result from increases in cytosolic Ca2+ concentration
([Ca2+]i).
We have previously reported increases in
[Ca2+]i
associated with slow waves in these tissues by using the ratiometric dye indo 1 (22). Fluorescent signals were collected with an intensified
video camera (Hammamatsu C1966 type 20 or Dage-MTI model SIT-66X)
attached to the video port of an inverted epifluorescence microscope
(Nikon Diaphot). Images were stored on a computer-controlled videocassette recorder (Sanyo model GVR-S950) at a rate of 30 frames/s.
The microscope was modified to include a platform (custom built) to
stabilize a micromanipulator (Narishige model MM-3) suitable for
intracellular recordings. Cells were impaled with glass microelectrodes filled with 3 M KCl having resistances in the range of 30-50 M
. Transmembrane potentials were measured by a standard, high-impedance electrometer (World Precision Instruments model 7000). The
same micromanipulator was used to lower "broken" unfilled glass
microelectrodes (tip diameter up to 0.2 mm) onto tissues to explore the
effects of localized injuries on pacing sites.
To archive image data while preserving the temporal relationship
between transmembrane potentials and fluorescent video signals, membrane potentials were converted to a range of audio frequencies (1-8 kHz) with a custom-designed circuit. A voltage-to-frequency converter (Burr-Brown) allowed membrane potential to be recorded on one
audio channel of the videocassette recorder while fluorescent video
images were simultaneously recorded. When data were played back through
the videocassette recorder, membrane potential was retrieved with a
frequency-to-voltage converter (Burr-Brown). The temporal phase shift
introduced by converting a voltage to a frequency-modulated
signal and back to a voltage was less than one video frame
(33 ms).
Data analysis.
Analysis of the video fluorescence signals was performed with both
custom-designed and commercially available (Adobe Photoshop) software.
Images were digitized (640 × 480 pixels) with a frame grabber
(Data Translation model 3152) that has the ability to separately
control the gain and offset of the video signal, allowing background
light levels to be subtracted before amplification. Membrane potentials
were acquired with an analog-to-digital converter (Precision
Electronics model PCL-711B). Because each frame on the videocassette
recorder could be accessed individually, temporal resolution of video
data was 33.3 ms (i.e., 30 frames/s).
To view propagation, series of contract-enhanced images were computed
by storing an initial frame (with no apparent activity) as a
"background image." This image was subtracted from each
subsequent frame. An intensity threshold was then applied to each pixel
within a frame to enhance contrast. To further aid visualization, all contrast-enhanced frames are displayed as "negative images" in which dark areas indicate regions of increased fluorescence intensity. The net effect of applying a threshold is to make the decision whether
the region represented by each pixel is in the excited state (dark) or
not (light). No other determinations from the amplitude of fluorescent
signals were made [except to display raw summed fluorescence
traces (see Figs. 1 and 2) and to note that there was no substantial
increase in fluorescence when 2 events "collide"].
Quantitative measurements were restricted to those involving space
(e.g., pacing location) or time (e.g., spontaneous frequency at each
location) during the initiation, spread, and termination of activity.
Conduction velocities were measured by determining the distance
traveled by a wave front over each digitized image (i.e., over a 33-ms
interval). Statistics are reported as means ± SDs.
Solutions.
The KRB used in this study contained (in mM) 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 134 Cl
, 15.5 HCO
3, 1.2 H2PO
4, and 11.5 dextrose (all
Fisher Scientific). This solution had a pH of 7.3-7.4 at
37.5°C when bubbled to equilibrium with 97%
O2-3% CO2. Fluo 3 was purchased from
Molecular Probes (Eugene, OR). DMSO, TTX, and cremophor EL were from
Sigma (St. Louis, MO).
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RESULTS |
Types of excitation in smooth muscle.
The Ca2+-sensitive dye fluo 3 was
selected to monitor the origin and propagation of excitation because of
its high absorbance (molar extinction coefficient) and low background
fluorescence when not bound to
Ca2+ (8). Excitation of fluo 3 at
long wavelengths avoids the intrinsic tissue fluorescence inherent in
smooth muscles (22). In addition, because of its dissociation constant
(Kd) and high
quantum efficiency on Ca2+
binding, fluo 3 undergoes a substantial increase in fluorescence intensity over the range of Ca2+
levels found in gastrointestinal tissues (22). Because fluo 3 does not
directly monitor membrane potential, experiments to establish the
relationship between propagated events visualized by fluo 3 fluorescence and those recorded by traditional electrophysiological techniques were performed.
Figure
1A shows
an example of the initiation and spread of a typical
Ca2+ wave recorded from a
cross-sectional preparation isolated from the antrum. The
frame at time 0 shows a
Ca2+ wave emerging within the
field of view in the region near the longitudinal-circular muscle
boundary. Subsequent frames show rapid spread of the wave front in the
direction parallel to the long axis of circular muscle fibers (oriented
approximately vertically in the series of images) and slower
propagation transverse to this direction. Circular muscle within the
entire field of view remained in the excited state throughout the full
duration of the slow wave (4 s; individual frames not shown). In
cross-sectional preparations, Ca2+
waves associated with excitable events in the circular layer were never
observed to spread (above threshold levels) into the longitudinal or
mucosal layers.

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Fig. 1.
Types of excitation in circular muscle.
A, top
left: background fluorescent image of cross-sectional
preparation showing longitudinal (L), circular (C), and mucosal (M)
regions. Contrast-enhanced images (A,
0-133 ms) show regions of elevated cytosolic
Ca2+ concentration
([Ca2+]i)
associated with a slow wave as it emerged near the
longitudinal-circular border and propagated throughout entire circular
muscle layer. B and
C: intracellular membrane potentials
and fluorescence intensities in region of electrode (summed intensities
from 9 × 9 pixels centered on electrode; location indicated by
arrow in A).
Ca2+ transients superimposed on
some slow waves (C) appear as
synchronous (dotted lines) oscillations in fluorescence intensity and
membrane potential. Scale bar = 2 mm.
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A myocyte in the circular layer near the longitudinal boundary was
impaled with an intracellular microelectrode at the location indicated
by the arrow in the background image (see
METHODS) in Fig.
1A, top
left. The upper traces in Fig. 1,
B and
C, show summed fluorescence in the
immediate region of the impaled cell. The lower traces show electrical
slow waves recorded simultaneously from this cell over the same period
of time. Circular muscle cells near the longitudinal boundary in
dye-loaded and illuminated tissues had resting membrane potentials of
72 ± 5 mV and depolarized to plateau potentials of 31 ± 3 mV with a spontaneous slow-wave frequency of 1.5 ± 0.5 min
1
(n = 11). On the basis of
observations of hundreds of slow waves simultaneously recorded with
fluorescent images, all slow waves were associated with
Ca2+ waves that propagated
throughout the circular layer, and no
Ca2+ waves were observed in
circular muscle without a concurrent electrical slow wave.
In some cases, fluctuations in fluorescence intensity were superimposed
during the plateau phase of a slow wave. These superimposed "oscillations" are discrete events that emerge from multiple
sites (not generally the site of origin of the ongoing slow wave) and can be seen in video records to propagate over substantial distances (from <1 mm up to the entire field of view). In Fig.
1C, fluctuations appear as small
oscillations in summed fluorescence records (upper trace) correlated
temporally with oscillations in membrane potential (lower trace). These
oscillations are more prominent in tissues removed from distal regions
of the stomach (data not shown).
In cross-sectional preparations, the longitudinal muscle layer was
generally too thin to accurately track the propagation of excitation.
Therefore imaging was performed on sheets of longitudinal muscle (with
the circular layer removed) viewed from the serosal side. Figure
2A shows
an example of the emergence of a
Ca2+ wave that spreads rapidly
parallel to the long axis of the longitudinal fibers (oriented
approximately vertically in Fig. 2A)
and more slowly in the transverse direction to envelop the entire field of view within 133 ms. A myocyte was impaled at the location indicated by the arrow in the background image of Fig.
2A (top
left). In Fig. 2, B
and C, membrane potentials from this
cell are plotted on the same time scale as summed fluorescence
intensities from a small region of pixels that included the impaled
cell. Preparations had transmembrane potentials of
52 ± 5 mV
at the most negative point of voltage excursions and depolarized to 34 ± 5 mV (excluding depolarizations associated with
fluorescence spikes; n = 20 regions within 8 tissues). Electrical slow waves in longitudinal muscles (as
shown in Fig. 2, B and
C) were consistently associated with propagated Ca2+ waves in video
sequences. When observations of
Ca2+ transients were isolated to
specific locations within a field of view, events were observed at a
mean frequency of 15 ± 3 min
1
(n = 20 tissues).

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Fig. 2.
Types of excitation in longitudinal muscle. A sheet of longitudinal
muscle (background frame; A,
top left) with no circular muscle
attached was viewed from the serosal side. Contrast-enhanced images
(A, 0-133 ms) show propagation of
Ca2+ transient associated with a
longitudinal slow wave. B and
C: comparison of intracellular
membrane potentials (electrode location indicated by arrow in
A) with fluorescent intensities
recorded simultaneously in region of electrode (summed intensities from
81 pixels). During some Ca2+ waves
(C), superimposed fluorescent spikes
were observed in some cells within region of elevated fluorescence.
Scale bar = 2 mm.
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The electrical record in Fig. 2C is an
example in which a spikelike action potential occurred during the slow
wave. The action potential was associated with a superimposed sharp
rise in fluorescence (upper trace) that could be seen in video images
to propagate only over limited distances (<1 mm) within the region of
elevated [Ca2+]i
associated with the slow wave. In stomach, not all preparations produced action potentials superimposed on longitudinal slow waves. In
addition, in preparations where action potentials were observed, they
were not seen in all sweeps of elevated fluorescence (observed in 10 of
60 Ca2+ sweeps in cells from 10 tissues). In other words, rapid fluctuations in fluorescence
superimposed on the elevated Ca2+
transient associated with a slow wave were often observed to sweep
through the region of a microelectrode impalement without electrical
spikelike action potentials from an individual cell being recorded.
Origin of excitation.
The nature of the origin of slow waves and whether slow waves are
initiated from a single location have been the subject of debate for a
number of years (24). Figure 3 shows an
example in which the emerging phase of a
Ca2+ transient associated with a
slow wave has been captured in a narrow region along the
circular-longitudinal muscle boundary. These frames demonstrate the
emergence of a Ca2+ wave as a
unitary event. If left undisturbed (i.e., without neural blockade or
other tissue manipulations), Ca2+
waves (associated with electrical slow waves; see Fig.
1B) originated from one to three
distinct pacemaker locations in circular sheets (6 × 5 mm) over
30-min observation periods (n = 12).
In 33% of preparations, a single pacing site was detected. The
remaining tissues had two to three distinct sites where at least some
events from each site could be observed throughout the observation
period.

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Fig. 3.
Ca2+ waves in circular muscle
arise near circular-longitudinal boundary. Top left:
background fluorescence image of a cross-sectional preparation showing
L, C, and M regions. Contrast-enhanced images show emergence (0 ms) of a Ca2+ wave near
longitudinal-circular boundary. Propagation of
Ca2+ wave (up to 233 ms) resulted
in a region of elevated
[Ca2+]i
that encompassed entire circular muscle. Scale bar = 2 mm.
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Similarly sized sheets of longitudinal muscle contained many more
spontaneous pacing sites. In 20 tissues viewed over 30 min, there was
an average of 5 (range 3-12) distinct pacing locations. Longitudinal sites appeared to be more transient than those in the
circular muscle layer. When viewed over prolonged periods (30-60
min), new sites where activity had not previously been observed
occasionally appeared while other active sites disappeared. In tissues
that were not manipulated throughout the observation period, no causes
for the appearance or disappearance of these sites were apparent.
Figure 4 demonstrates the
reproducibility of these discrete pacing locations in a
longitudinal muscle preparation. The emergence of four contiguous
Ca2+ waves is shown. The
first two Ca2+ waves (Fig. 4,
A and
B) originated from the same
location to within the temporal and spatial resolution of the imaging
system (pixel resolution ~16 µm). The third event (Fig.
4C) emerged from a clearly distinct
site and was followed by an event (Fig.
4D) from the same location as
that for the first two events. Individual pacing sites, once
established in circular or longitudinal muscle preparations, were not
observed to vary (to within the spatial resolution of the imaging
system) throughout prolonged observation periods (>30 min). The
invariant locations of individual pacing sites were not affected by
interspersed events from other sites.

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Fig. 4.
Repeated activity from longitudinal pacing sites.
A-D:
contrast-enhanced images of 4 contiguous events at 0 (initial event
detection), 67, and 133 ms. A and
B: event originating from same site
(to within temporal and spatial resolution of imaging system).
C: event originating from a different
site before pacing activity returned to original site
(D). Slight variations in size of
excited region appear at 0 ms because spontaneous generation of events
is not synchronized with frame rate (30 frames/s) of camera. Scale bar = 2 mm.
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Synchronization of pacemakers.
As noted previously, one-third of circular muscle preparations revealed
a single pacing site when viewed over an observation period of 30 min.
Circular tissues with multiple sites demonstrated periods in which one
site might dominate (initiating 50-100% of Ca2+ waves) for periods of 5 min
or more (up to the 30-min observation period). Then dominance might
shift to another site before returning to the initial site. In other
words, if an event initiated at one site, the next event was most
likely to begin at the same site. Except for this observation, there
were no clear patterns of initiation among multiple sites in circular
muscle strips.
On the other hand, repeatable patterns of activity from multiple pacing
sites in longitudinal muscle preparations were frequently observed.
These patterns varied in complexity from two sites initiating alternating events for brief periods to intricate patterns involving numerous sites. An example of complex synchronized activity in the
longitudinal layer is illustrated in Table
1. Patterns often showed some variability
(as in Table 1); however, the initiation of a new series was often
recognized by activity at a pacing site that appeared to initiate the
sequence and/or a prolonged delay (lasting up to 2 min) after the
previous sequence. There was no correlation between pacing sites with
the highest intrinsic frequency and those that initiated repeat
patterns.
Conduction velocities.
In circular muscle, maximum propagation velocities of
Ca2+ waves transverse to the long
axis of muscle fibers averaged 6.2 ± 3.0 mm/s
(n = 12 tissues), whereas in
longitudinal muscle the average was 7.0 ± 3.0 mm/s
(n = 12 tissues). At standard 30 frame/s video rates, variations in conduction velocities in the
direction of the long axes of muscle fibers could only be assessed over one to three frames. In longitudinal muscle, the average propagation velocity of Ca2+ waves in this
direction was estimated as 45.0 ± 5.0 mm/s
(n = 6 tissues). In circular muscle,
where propagation distances are generally longer, the mean conduction
velocity was 39.0 ± 9.0 mm/s (n = 12 tissues). It was difficult to accurately measure the propagation
velocities of superimposed activities (fluorescence oscillations and
spikes) because of short propagation distances and the need to
discriminate wave fronts within fluorescence levels that were already
elevated; however, conduction velocities were in the same range as
those for the underlying Ca2+ waves.
It has previously been shown that slow-wave conduction velocity is
dependent on the interval since depolarization during electrically paced activity in circular muscle of the canine antrum (27). Video
sequences illustrate that this relation is also apparent during
spontaneous activity in longitudinal tissues. There is generally
insufficient spontaneous variation in slow-wave frequency to readily
demonstrate the relation in circular muscle segments; however,
longitudinal muscle segments have multiple pacing sites, and most
events do not propagate throughout the entire field of view. This
results in isolated regions or "islands" within tissues where the
interval since depolarization varies substantially. Figure
5 shows an example of conduction velocities
measured transverse to the long axis of muscle fibers in regions where
spontaneous variations in the interval since the previous
depolarization were generated. Although a linear regression has been
drawn through these data, there was an upper limit to conduction
velocities (i.e., after prolonged intervals).

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Fig. 5.
Conduction velocities increase with increased interevent interval.
Conduction velocities transverse to long axis of muscle fibers as a
function of time since previous excitation (measured at end of
Ca2+ wave) and initiation of a new
event were measured. All events ( ) were generated spontaneously in
different regions of a longitudinal tissue segment. The least-squares
linear regression
(A t B) superimposed on data has
a slope (A) of 16.7 mm/s2.
B,
y-intercept;
t, time.
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In addition to temporal variations in conduction velocities, video
sequences clearly show discrete spatial regions where propagation varies significantly. These bands were found in both circular and
longitudinal muscle strips. Figure 6 shows
an example of one region in the longitudinal layer. Of five
Ca2+ waves that originated from
the same pacing site, the first three events (Fig. 6,
A-C)
had an average conduction velocity of 7 mm/s before entering the region
of slowed conduction. Conduction slowed by as much as an order of
magnitude within the region (frames 12-17). The original conduction velocities were
then restored once events emerged from the region. The fourth event
(Fig. 6D) slowed and then stopped in
the region where conduction was delayed. The termination of an event
within a region of slowed conduction was a common finding (30% of 30 sequential events during the experiment associated with Fig. 6). On the
other hand, ~10% of excitable events (e.g., Fig.
6E) propagated through the same
region without an observable delay.

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Fig. 6.
Region of slowed conduction. Conduction velocities of
Ca2+ waves were measured on a
frame-by-frame basis transverse to long axis of longitudinal muscle
fibers in region of slowed conduction. Wave fronts repeatedly
approached region at velocity of 5-7 mm/s
(A-C).
Velocity decreased by up to 1 order of magnitude within region
(frames 12-17) before initial
conduction velocity was restored on other side of region.
D: excitation stopped within region.
E: illustration of an event that
propagated without delay through same region.
A-D show sequential events, and
E shows an event that occurred 3 events (as in A-C) after event shown
in D.
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Wide ranges in conduction velocities in regions where propagation
slowed were observed. Propagation velocities varied from a 10-fold
decrease (as in Fig. 6) to only a slight delay. Slowed conduction was
independent of the direction of travel through the region of delayed
conduction (data not shown).
Extent of propagation.
In all preparations of isolated circular muscle
(n = 12),
Ca2+ waves consistently spread
throughout the entire circular layer, even when viewed at low optical
magnification (up to 2 cm on a side). This range of spread included any
adjacent pacing locations (up to 2) as well as regions excited by these
adjacent sites.
In longitudinal muscles, most (but not all) of the
Ca2+ waves from one pacing site
were impeded from propagating through the "domain" (the total
region or area covered by an event originating from a single pacing
site) of an adjacent pacing site. Propagation generally stopped when
events encountered tissue regions recently excited; however, domains
were not rigid. Figure 7 shows an example in which two pacemakers (sites A and B in the background image of Fig.
7A) initiated events that swept into
each other's domains. The frames in Fig.
7A show an event starting at site A
and sweeping through an active pacing site at site B (frame at 133 ms).
Figure 7B shows the next event
emerging from site B to sweep through and beyond the pacing site at
site A (frame at 167 ms). All longitudinal tissues
(n = 14) demonstrated at least some
events that swept into adjacent domains. In some cases in longitudinal
muscle, a single Ca2+ wave could
be seen to sweep through at least three distinct pacing sites.

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Fig. 7.
Propagated Ca2+ waves can overlap
pacemaker domains. Background image of a sheet of longitudinal muscle
with circular muscle removed (A,
top left) shows location of 2 pacing
sites (A and B). A:
Ca2+ wave that originated at site
A and propagated across field of view, encompassing site B (frame at
133 ms). Subsequent event originated at site B and propagated in
opposite direction through syncytium to encompass site A
(B). Scale bar = 2 mm
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The frequencies of activation and extent of
Ca2+ wave propagation produced
complex sequences of activity in both longitudinal and circular muscle
sheets (see, for example, Fig. 9B).
It was difficult to predict overall patterns of excitation; however, propagation consistently stopped, and pacing was not observed in
recently excited regions. In fact, it was possible to observe "collisions" of actively propagating events. Figure
8 shows an example of two
Ca2+ waves that were spontaneously
initiated at approximately the same time, separated by 3 mm in the
direction transverse to the long axis of longitudinal muscle fibers.
The two waves of excitation approached each other and then collided
(frame at 167 ms). Regions excited as a result of two or more wave
fronts that encountered each other generated fluorescence levels that
were the same as those generated by propagated events from a single
pacemaker site. In other words, there was no summation of
fluorescence despite multiple excitatory pathways.

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Fig. 8.
Collision of 2 Ca2+ waves.
Contrast-enhanced images show excitation in a sheet of longitudinal
muscle viewed from serosal side. Two spatially separate
Ca2+ waves spontaneously arose
nearly simultaneously (0-33 ms). Activity spread rapidly in
direction parallel to long axis of muscle fibers and more slowly in
transverse direction, resulting in a collision wave front at
133-167 ms. Scale bar = 2 mm.
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Factors that influence pacing site frequency and location.
In the absence of tissue manipulations (see next paragraph), pacing
locations appeared to be relatively stable over a time scale of 30 min.
Occasionally a site in longitudinal muscle preparations was lost or
gained within this period. To test the possibility that neural activity
might influence pacemaker activity, sheets of longitudinal muscle
(n = 8) were treated with TTX (1 µM;
preincubated for 10 min). In 60% of tissues, one or more pacemaker
sites that were previously active became quiescent and/or new sites
emerged. This turnover in pacemaker sites was somewhat greater than the rate in untreated tissues; however, the overall number of pacemaker sites (average of 5) and spontaneous frequency (16 ± 3 min
1) remained
approximately the same. In addition, there were no differences in
average spontaneous frequencies or patterns of excitation in tissues
cold-stored for extended periods (up to 24 h).
It has long been suspected that tissue injury might stimulate or
disrupt pacemaker sites in gastrointestinal muscles (18, 19).
Experiments were designed to test whether, well-defined, localized
injury might alter the frequency or location of pacing activity. Figure
9 shows an example of the effects of local
injury created by penetration with a fine microelectrode in a
longitudinal muscle preparation. Four pacing sites (locations shown in
Fig. 9A) were active before injury
(Fig. 9B). Penetration of the tissue by an unfilled glass micropipette (0.2 mm in diameter) caused increased
fluorescence in the region around the pipette. Away from the site of
injury (>1 mm) there was no influence on membrane potential (on the
basis of intracellular recordings), and video images show that the
region of elevated
[Ca2+]i
was confined to a radius of <2 mm. Immediately after penetration (and
withdrawal of the micropipette) a burst of activities from all sites,
including a new site within the region of elevated fluorescence (site 5 in Fig. 9), ensued. The new site generated the highest frequency of
spontaneous activity (although this was not a consistent finding), and
activity from the new site persisted throughout the observation period
(30 min). Within 2 min after injury, a previously active site (site 1 in Fig. 9) stopped producing events. No further events were initiated
from site 1 throughout the rest of the observation period (>30 min).

View larger version (38K):
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|
Fig. 9.
Patterns of excitation are altered by tissue injury.
A: locations of 5 pacing sites and
site of injury resulting from tissue penetration and immediate
withdrawal of a 0.2-mm-diameter unfilled glass micropipette (arrow;
image taken immediately after injury).
B: sequence of spontaneous events from
4 active pacing sites just before injury. Immediately after injury
(C), there was a burst of activity
from all pacemaker sites. Site 5, which was previously quiescent,
became active and site 1 became quiescent 2 min after injury. Scale bar = 2 mm.
|
|
It was difficult to predict the specific effects of an injury within a
preparation. In 70% of tissues injured by a 0.2-mm glass micropipette,
new sites were formed (n = 14 injuries
in 6 preparations). In 28% of preparations, activity in one or more sites ceased immediately after penetration and additional sites were
lost within minutes after injury. An injury created by a much larger
(0.4-mm diameter) tungsten electrode on penetration resulted in four of
eight sites ceasing initiating events and in the emergence of six new
pacing sites to generate activity. New sites appeared up to at least 7 mm away from the site of penetration; however, effects even further
away (outside of the field of view) could not be ruled out. Data
suggest that syncytial excitation is not governed by single pacemakers
but rather results from complex interactions among numerous potential sites.
 |
DISCUSSION |
Fluorescence imaging by using
Ca2+-sensitive dyes to detect
regions of excitation provides an effective technique to examine the
dynamics and interactions of activities at the tissue level. Imaging in
the gastrointestinal tract has a particular advantage over similar
techniques employed, for example, in the heart (20) or brain (11),
because propagation velocities are relatively slow and wave fronts can
generally be resolved at standard video rates (25-30 frames/s).
The present work demonstrates that fluorescence video imaging can be
used to assess 1) the origin of
tissue activation, 2) the spread of
excitation including conduction velocities and the range of propagation
of individual events, and 3)
alterations in patterns of excitation by factors such as neural
blockade and localized injury.
With a single-stage intensified camera, there was insufficient
fluorescence (without signal averaging that would result in a loss of
temporal resolution) to employ narrow-bandwidth filters to collect
paired images for dual-wavelength, calibrated measures of changes in
[Ca2+]i.
In canine gastric antral tissues, we have previously demonstrated (22)
using the ratiometric fluorescent indicator indo 1 that slow waves and
contractions are associated with concurrent elevations in
[Ca2+]i.
In the present study, we have not conclusively shown that increases in
fluo 3 fluorescence associated with oscillations during the plateau
phase of slow waves in circular muscle and spikes in longitudinal
muscle result solely from an increase in [Ca2+]i. These activities might,
for example, be influenced by changes in cell volume. Nonetheless,
fluorescence video sequences can be used to assess the initiation,
spread, and termination of each of these forms of activity at the
tissue level.
Resting membrane potentials in circular muscle preparations loaded with
fluo 3 in cells near the myenteric border were within the same range of
values (
70 ± 4 mV) previously reported by numerous laboratories for preparations not illuminated or loaded with a fluorescent indicator (1, 5, 12, 37). Similarly, plateau potentials
recorded in cells loaded with fluo 3 are in the same range (
31 ± 3 mV) as that for untreated tissues (1, 5). Spontaneous slow-wave
frequencies are also consistent with previously reported results (1.6 ± 0.6 min
1) for
nonloaded tissues (31). The characteristic waveform of slow waves
appears to be unaffected by dye loading, including the presence of
oscillations during the plateau phase (Fig.
2C), often seen in tissues taken
from more distal regions of the stomach (5, 12, 37). In video
sequences, average Ca2+ wave
propagation velocities were in the same range as that previously reported for slow waves (6.5 ± 4 mm/s transverse and 39 ± 8 mm/s parallel to the long axis of muscle fibers) on the basis of
techniques using multiple intracellular microelectrodes (26).
Propagation velocities of Ca2+
waves in the longitudinal muscle layer are similar to those in circular
muscle, suggesting that mechanisms of conduction in the two layers may
be similar.
Taken together, these data suggest that dye loading and tissue
illumination do not significantly alter the characteristic electrical
activities, spontaneous frequencies, or ability of events to propagate
through gastric syncytia. On the other hand, tissue penetration by
microelectrodes can alter sites of pacemaker activity (Fig. 9). Thus
commonly employed intracellular and extracellular techniques using
microelectrodes might alter excitation patterns in some preparations.
Fluorescence imaging as a measure of excitation.
At the tissue level, previous work has shown that there is a tight
correlation between slow-wave depolarization, increases in
[Ca2+]i,
and the subsequent production of force (22). The present study confirms
and extends the correlations between electrical depolarizations and
increases in fluorescence to include oscillations in membrane potential
during the plateau phase of the slow wave and spikelike depolarizations
superimposed atop slow waves in the longitudinal layer. In circular
muscle, fluorescent oscillations during the slow wave are spatially
confined to the region of elevated [Ca2+]i
associated with the slow wave. Except for this constraint, superimposed
oscillations originate and propagate as independent events, distinct
from the underlying rise in
[Ca2+]i.
The similarity in range of conduction velocities compared with
slow-wave propagation and the presence of collisions between oscillations suggest that the excitability mechanisms associated with
oscillations are similar to those of other excitable events in smooth
muscle. Their role may be to enhance the strength of contractions
during slow waves by stimulating additional transient Ca2+ entry without elevating
[Ca2+]i
in a sustained fashion, which might be harmful to the cell.
In longitudinal muscle preparations, the underlying rhythmicity is
similar to so-called myenteric potential oscillations that have been
observed in canine distal colon (34). Slow waves recorded by
electrophysiological techniques were consistently associated with
concurrent Ca2+ waves. On the
other hand, superimposed "spikes" or brief fluctuations (<0.5
s) in fluorescence observed in video sequences were not always
associated with spikelike depolarizations or action
potentials recorded intracellularly. It has previously been suggested,
on the basis of microelectrode recordings, that these action potentials do not propagate in gastric and duodenal tissues (33). Video sequences
indicate that fluorescence transients associated with spikes do have
discrete sites of origin and propagate over short distances. Similar to
fluorescence oscillations in the circular layer, they are confined to a
region of elevated
[Ca2+]i
dictated by underlying activity. However, unlike oscillations, spikes
in the longitudinal layer were not observed in all cells within a
region of elevated fluorescence (superimposed atop the Ca2+ transient) by
electrophysiological techniques. These observations are consistent with
the notion that the spread of high-frequency components of electrical
activity might be strongly attenuated by the syncytial nature of the
tissue (24).
Origin of activation.
It has previously been suggested that gastric (28) and other smooth
muscles (13, 15, 16) contain hot spots or preferred locations where excitable events are initiated. Fluorescence video images verify that this is the case for both the longitudinal and
circular muscle layers in the stomach. It appears that both layers
contain numerous "potential" pacing sites, because new sites can
readily be induced after a localized injury. However, during
spontaneous activity, a small number of sites dominate pacemaker
activity during all forms of activation within the two muscle layers.
To within the spatial and temporal resolution of video images
(revealing small muscle bundles), events repeatedly initiate from these
same sites, even when events from other sites are interspersed (Fig.
4).
In cross-sectional muscle preparations,
Ca2+ transients associated with
electrical slow waves consistently originate from the circular-longitudinal muscle boundary, initiating waves of excitation that simultaneously propagate parallel and transverse to the long axis
of circular fibers (Fig. 3). The locations of the sites of origin are
consistent with the notion promoted by a number of laboratories that
interstitial cells of Cajal associated with the myenteric plexus region
may serve the role of the pacemaker in the stomach and other intestinal
muscles (9, 29, 30).
Longitudinal tissues contain more active pacing sites than similarly
sized circular tissues. It is not clear whether the reason for this is
the higher intrinsic rate of activity in the longitudinal layer or that
the extent of propagation from each site is more confined. There is
likely an interplay involving the number of pacemakers, intrinsic
frequencies, and extent of propagation around each site. In some cases,
there is clearly a coordination or synchronization among longitudinal
pacemakers that can produce complex patterns of activity lasting up to
at least 1 h (Table 1). TTX (1 µM) can partially disrupt these
patterns, although the overall frequency of excitation in gastric
tissues remains unchanged. This suggests some form of neural
modulation; however, the continued presence of patterned activity
suggests that at least some aspects of the coordination of activation
are governed by non-TTX-dependent mechanisms.
Propagation of excitation.
Historically, it has been suggested that pacemakers control a region or
domain within a tissue segment (32). In circular muscles, all
Ca2+ waves spread throughout the
entire field of view including any nearby pacing sites. Thus it is
likely that the size of any domain in circular muscle includes the
entire circumference of the organ along the long axis of circular
muscle fibers and segments greater than the field of view
(2 cm under low magnification) along the length of the stomach. In
contrast, in longitudinal muscles (e.g., Fig. 2), most (but not all)
events, from a single pacing site, did not propagate through adjacent
domains. Propagation usually stopped when events encountered regions
recently excited by adjacent pacemakers. Regions excited by the
collision of two events (e.g., Fig. 8) were not distinguishable from
unitary events in the same region. However, rigid pacing domains were
not observed consistently (e.g., Fig. 7). All longitudinal tissues
demonstrated at least some events that swept into adjacent pacemaker
regions including pacing sites. This loose regional influence of
domains may be a part of the segmental level of control in
gastrointestinal muscles.
Previously, it was assumed that propagation in gastrointestinal smooth
muscles proceeds at uniform rates parallel and transverse to the long
axis of muscle fibers (27). Video sequences of the spread of
Ca2+ waves demonstrate that this
is not the case. There appears to be a basal, more rapid rate of
conduction (6-10 mm/s transverse to muscle fibers) in most regions
of a tissue, with spatially well-defined areas of slowed conduction.
Slowed conduction generally occurs repeatedly at the same anatomical
sites (see Fig. 6). The degree of slowing varies from event to event at
a given site, and in some cases events were observed to proceed without
slowing through these sites (e.g., Fig.
6E). These data suggest that there may be a mechanism to regulate the overall rate of conduction in a
syncytium by controlling conduction through specific regions within a
tissue. The electrophysiological mechanism or mechanisms that cause
delayed conduction (e.g., reduced membrane resistance, elevated
cell-to-cell junctional resistance) have not been determined in this
study. It is possible that regions of delayed conduction may be
associated with septal structures or other morphological features (6,
38).
Regions of slowed conduction may also play an important role in
regulating the extent of propagation. When viewed in terms of elapsed
time (as opposed to conduction velocities), an event can spend
25-50% or more of its overall lifetime in regions of delayed
conduction (see Fig. 6). Thus not only might these regions influence
rates of conduction, they can also be the most likely locations for
event termination. This may provide an opportunity for influencing the
extent and rate of propagation (neural or otherwise) by directly
modulating these specific locations without controlling the entire syncytium.
It has previously been shown (26) that conduction velocities in
circular muscle depend on the "interevent" interval (the time
since a region was last excited). The present study (e.g., Fig. 5)
furthers this concept to spontaneously initiated events in
two-dimensional sheets of longitudinal muscle. Conduction velocity (transverse to muscle fibers) increases with longer interevent intervals. This property of the syncytium may contribute to the regulation of activation within a syncytium by limiting rates of
conduction into regions recently excited. This might be particularly important in the longitudinal muscle layer where there is a greater density of distinct regions influenced by different pacing sites.
Although fluorescence imaging reveals a spatially heterogeneous range
of conduction velocities, when viewed macroscopically, average
conduction velocities are similar to those measured with multiple
electrodes (28). Video data can be used to augment electrophysiological
results, although some care must be exercised when comparing
fluorescence imaging results with those obtained by traditional
microelectrode techniques. For example, in longitudinal muscles, Ca2+ waves and spikes
generally do not sweep throughout an entire tissue (at the scale of a
few millimeters), so individual electrodes do not record all events in
a given field of view viewed within video sequences. Thus measurements
of frequencies must be confined to a specific location within the
tissue to produce results comparable to those obtained by
microelectrode-based techniques. In addition, in longitudinal muscles
the most rapid fluorescence transients (see Fig. 2) do not always
result in a "spike" potential in individual cells recorded
electrophysiologically even though transients can be seen to sweep
through the region of a microelectrode recording.
Alterations in tissue excitability.
In the antrum, neural blockade generated only minor shifts in pacing
locations, with no effect on frequency. These data are in sharp
contrast to recently reported results (35) from guinea pig colon where
the enteric nervous system appears to play a major role in regulating
both the number of pacing sites and the frequency of activity at each
site. Differences may be due to different functions within these
regions of the gastrointestinal tract. Locally initiated peristaltic
contractions in the colon appear to be under strong neural control,
whereas the initiation of contractions that generally sweep through the
antral region of the stomach appears to be largely independent of
neural input.
For some time, there has been speculation that injury might generate an
effect on pacemaker activity in the stomach (18, 19). Fluorescence
imaging clearly shows that injury can induce new pacemaker activity in
the vicinity of the injury site. However, the effects of injury were
confined neither to the induction of new sites nor to the region of
injury. Pacemaker sites can be lost or gained after localized injury.
In most tissues, both processes were observed: some pacemaker sites
were lost while others were gained within a short period after injury
(Fig. 9). Pacemaker sites (lost or gained) can be near the injury
(i.e., near the site of elevated fluorescence) or at distances up to at
least several millimeters away from the injury.
Because of collateral propagation around a localized injury site, there
is a minimal effect on the time of arrival of activation, even a
short distance away (<1 mm) from the site of a localized injury. The
physiological consequences of a confined injury may be less associated
with disrupting conduction pathways. More significantly, injury appears
to alter the initiation and sequence of excitation throughout the
entire syncytium.
In summary, the origin, propagation, and extinction of activity can
generate complex patterns of excitation in three-dimensional gastrointestinal syncytia. Ca2+
transients associated with slow waves, oscillations, and spikes are
confined both morphologically and functionally to tissue regions; however, each contributes uniquely to overall tissue excitation. Even
small tissue segments with few pacing sites can generate a rich
repertoire of activity.
 |
ACKNOWLEDGEMENTS |
This grant was supported by National Institute of Diabetes and
Digestive and Kidney Diseases grant DK-32176.
 |
FOOTNOTES |
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: N. G. Publicover, Biomedical Engineering Program, Dept. of Physiology and
Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: nelson{at}unr.edu).
Received 3 November 1998; accepted in final form 14 May 1999.
 |
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