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NEUROREGULATION AND MOTILITY

Activation of neural circuitry and Ca²⁺ waves in longitudinal and circular muscle during CMMCs and the consequences of rectal aganglionosis in mice

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada

Submitted 31 July 2006 ; accepted in final form 26 September 2006

	ABSTRACT

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In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contribute to dysmotility and pseudoobstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions or the effects of aganglionosis on the generation of Ca²⁺ waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease [endothelin type B receptor-deficient (Ednrb^s–l/Ednrb^s–l) mice], the Ca²⁺ indicator fluo-4 was used to simultaneously monitor the temporal activation and spread of intercellular Ca²⁺ waves in the LM and CM during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, Ca²⁺ waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discreet Ca²⁺ waves fired simultaneously in both muscle layers, where the propagation velocity of Ca²⁺ waves significantly increased, as did the rate of initiation and number of collisions between Ca²⁺ waves. Hexamethonium (300 µM) or atropine (1 µM) prevented synchronized firing of Ca²⁺ waves. In the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice, not only were CMMCs absent, but Ca²⁺ waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discreet Ca²⁺ waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in Ca²⁺ wave firing between the LM and CM and an absence of CMMCs.

colon; myenteric; peristalsis; colonic migrating motor complexes

A major step forward in the understanding of human Hirschsprung's disease came about from the discovery of spontaneously mutated strains of mice, such as the piebald lethal mouse (17, 31–34). Piebald homozygote lethal offspring [endothelin type B receptor (EDNRB)-deficient (Ednrb^s–l/Ednrb^s–l) mice] develop congenital rectal aganglionosis that leads to megacolon and death shortly after birth. Ednrb^s–l/Ednrb^s–l mice are now known to have a mutation in the Ednrb gene, and, at a similar time as to when this mutation was first identified in Ednrb^s–l/Ednrb^s–l mice, other investigators realized that a subset of human patients with Hirschsprung's disease also carry mutations in EDNRB (19). It is for this reason that the piebald mouse strain has proved an invaluable model for studies on human Hirschsprung's disease and is increasingly becoming the focus of much attention.

Normal motor activity recorded from the isolated whole colon in wild-type mice has been shown in a variety of laboratories to consist of regularly occurring colonic migrating motor complexes (CMMCs; for a review, see Ref.23). Wood et al. (34) first described these events as "...migrating contractile complexes" that are clearly neural in origin and occur about every 1–2 min. It was suggested that these were the motor correlates of extracellularly recorded spike bursts and that they "...may be the electromechanical basis for organized propulsion of intraluminal fecal pellets." More recently, these events have been described simply as CMMCs (1, 5–7, 9, 23–25). The mechanisms that generate CMMCs involve complex intrinsic neural circuitry that is only recently being unraveled (23, 25). To date, however, all of the electrophysiological knowledge gathered on CMMCs in mice has been from the circular muscle (CM) layer (7, 25). Unfortunately, there have been no electrophysiological recordings made from the longitudinal muscle (LM) layer of the mouse colon, and this has led to a major weakness in our understanding of CMMC generation in wild-type mice, let alone mutant strains of mice that develop rectal aganglionosis.

In this study, we used Ca²⁺-imaging techniques to determine 1) the mechanisms by which the two muscle layers of the mouse colon are activated during CMMCs and 2) how the properties of Ca²⁺ waves underlying phasic contractions are modified in the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mutant mice, where CMMCs are absent. In brief, we showed that during the intervals between CMMCs in wild-type mice, Ca²⁺ waves discharge largely asynchronously between the LM and CM. However, during CMMCs, discreet Ca²⁺ waves switch to a highly synchronized firing state between both muscle layers that involves synaptic activation of myenteric interneurons and excitatory motor neurons to both muscle layers. In the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice, Ca²⁺ waves propagate faster in the hypertrophic musculature but discharge sporadically and show consistently poor temporal synchronization between both muscle layers.

	METHODS

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Preparation of tissues. Ednrb^s–l/Ednrb^s–l homozygote lethal offspring of the piebald mouse strain and their experimental controls (C57BL/6 mice) of either sex (20–90 days old) were euthanized humanely by inhalation anesthetic (Nembutal) followed by cervical dislocation, in accordance with University of Nevada Animal Ethics Committee guidelines. All Ednrb^s–l/Ednrb^s–l homozygote offspring were bred in the animal facility at the University of Nevada by intercrossing Ednrb^s/Ednrb^s–l and Ednrb^s/Ednrb^s–l heterozygote siblings (19). The original Ednrb^s/Ednrb^s–l heterozygote breeder pair was obtained from Jackson Laboratories. The use and treatment of all animals was approved by the Institutional Animal Use and Care Committee of the University of Nevada. Following euthanasia, the entire colon was removed, and a midline incision was made along the mesenteric border along the distal half (3 cm) of the (7–8 cm long) full-length preparation. This opened region was pinned mucosal side down in a Sylgard-lined petri dish containing oxygenated Krebs-Ringer buffer (KRB; see composition in Drugs and solutions) at room temperature. Strips of the LM were dissected off the colon to reveal the myenteric plexus and underlying CM (Fig. 1A). This approach was essential to facilitate penetration of the Ca²⁺ indicator into both muscle layers simultaneously to be able to simultaneously image activity in both muscle layers. The preparation was transferred to a Sylgard-lined organ bath (10-ml capacity). Ca²⁺ waves were only visualized in the distal 15 mm of the colon that was ganglionic in C57BL/6 mice and aganglionic in Ednrb^s–l/Ednrb^s–l mutant mice.

View larger version (101K): [in this window] [in a new window]   Fig. 1. Intercellular Ca2+ waves in the circular muscle (CM) and longitudinal muscle (LM) during intervals between colonic migrating motor complexes (CMMCs). A: images using a x10 objective of Ca2+ waves in the LM and CM. In the top portion of the field of view, the LM was dissected away (above the dotted line), leaving the myenteric plexus intact on the serosal surface of the CM. In the bottom portion of the field of view, the LM was left intact (see the dotted line). The direction of LM fibers was from left to right, and that of CM fibers was from top to bottom. B: the top three images show the propagation of a Ca2+ wave in the CM [0 ms (top left), 67 ms (top middle), and 133 ms (top right)]. *The site of initiation of a Ca2+ wave (in the top middle image). Note that the Ca2+ wave traveled more rapidly along CM fibers and more slowly orthogonal (left to right) to CM fibers (see top middle and top right images). In the bottom left image (200 ms), a second wave emerged on the left while the original wave was still propagating to the left side of the field of view. This original wave in the CM collided with the second wave and was annihilated [bottom middle image (267 ms)]. The second wave also did not propagate beyond the collision site. Also, in the bottom three images, the emergence of a wave in the LM could be observed. This Ca2+ wave propagated quickly along muscle fibers and more slowly orthogonal to LM fibers. C: images of Ca2+ waves in the CM (top portion of the field of view) and LM (below the dotted line) using a lower-power x4 objective. In this series of frames, the images were differentiated to enhance contrast and revealed the most rapid changes in Ca2+, which were at the crests of the waves. Following the first frame, Ca2+ waves were initiated in both the CM (* above the dotted line, frame at 35 ms) and LM (* below the dotted line, frame at 35 ms). These waves spread slowly orthogonal to CM fibers and orthogonal to LM fibers (105, 140, and 175 ms). The Ca2+ wave in the LM faded as it appeared to hit the edge of the LM (175 ms). Note that in this low-power image, the Ca2+ wave in the LM (70–140 ms) and CM (105 ms) appeared to split into two waves traveling in opposite directions from its site of initiation.

Ca²⁺ imaging. To better portray Ca²⁺ activity over long recording periods, spatiotemporal (ST) maps were constructed. Ca²⁺ fluorescence was averaged along the length of smooth muscle cells across the field of view for each row in the image. This resulted in a single column of averaged pixels. This procedure was repeated for each frame, and the resulting columns of averaged pixels were placed along side each other (time starting on the left and continuing to the right). The white vertical lines visible in ST maps (e.g., Figs. 2 and 5) indicate that a particular row of smooth muscle cells were active. If activity spread across the field of view, this would appear as an angled white line in ST maps.

View larger version (106K): [in this window] [in a new window]   Fig. 2. Changes in properties of Ca2+ waves in LM and CM layers before and during a CMMC. Spaciotemporal (ST) maps were constructed for both the CM (A and D) and LM (B and E). In this example, Ca2+ waves in the CM (A) were sporadic, often being initiated and terminating in similar zones along the bowel, whereas in the LM (B) Ca2+ waves discharged in a regular tonic fashion. Closest-peak analysis comparing the time at which Ca2+ waves occurred in the LM layer to the closest Ca2+ wave in the CM layer showed little coordination of firing between the two muscle layers (maximum change in time: 3 s; C). During the CMMC, the frequency of the ongoing discharge of Ca2+ waves in the CM increased (D) and the LM became active for an extended period (E). During the burst of Ca2+ waves associated with the CMMC, closest-peak analysis showed that the LM and CM fired near simultaneously (change in time: 0 s; F). An example of LM and CM firing during the CMMC is shown in G. Two Ca2+ waves were initiated in the CM (*), each propagating in both directions. At 133 ms, a Ca2+ wave appeared in the LM at the bottom of the field of view and propagated upward. After 200 ms, the two CM Ca2+ waves collided in the middle of the field of view and annihilated each other. H: examples of the pattern of propagation of Ca2+ waves in the CM layer during the CMMC (taken between dotted lines in D). Note that there were often numerous sites of initiation (*) and collisions () and that the propagation velocity varied considerably from wave to wave, giving the appearance of the "zigzag"-type pattern seen.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Hypertrophy induced in the LM and CM of Ednrbs/Ednrbs–l and Ednrbs–l/Ednrbs–l mutant mice. A–C: photomicrographs of sections of the far distal colon (3 mm from the rectum) stained for hematoxylin and eosin. A: normal LM and CM layers in C57BL/6 mice. B and C: sections of the colon from Ednrbs/Ednrbs–l heterozygote and Ednrbs–l/Ednrbs–l homozygote lethal mice, respectively. It can be seen that both piebald mice exhibited a similar degree of hypertrophy in both the LM and CM. D: cumulative graphical representation of the relative changes in muscle wall thickness in both the CM and LM. The most prominent increase in hypertrophy was detected in the aganglionic rather than ganglionic region of the colon (from n = 4 animals).

Protocol for hematoxylin-eosin staining. Isolated whole preparations of the mouse colon were cut into several segments (labeled as the ileum, cecum, proximal, midcolon, or distal colon) for cross sections and placed in labeled cassettes. Cassettes were mounted on a tissue processor (Sakura VIP 5, Torrance, CA) where the tissues are then infiltrated with paraffin. Upon the completion of processing, tissues were embedded in paraffin and sectioned at 4 µm thickness. Slides were air dried overnight and placed in staining racks, where they were stained with hematoxylin and eosin on a staining machine (Sakura DRS-601). Once stained, slides were placed on a coverslipper (Sakura VIP 5) and permanently mounted. Slides were then given a microscopic quality check to make sure proper trimming, embedding, sectioning, and staining had been achieved. The same procedure was used to stain both C57BL/6 wild-type and piebald mutant mouse colon preparations. To measure the thickness of the LM and CM walls, a bright-field Leitz Diaplan microscope and a Leica LEI-750 camera, running metamorph software (version 3.0. Metamorph imaging software), were used with lens objectives of x10 and x20 magnification.

Drugs and solutions. The composition of KRB was (in mM) 120.35 NaCl, 5.9 KCl, 15.5 NaHCO₃, 1.2 NaH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, and 11.5 glucose. Atropine, hexamethonium bromide, and nicardipine were all obtained from Sigma Chemical (St. Louis, MO). For Ca²⁺ imaging, a solution consisting of 25 µg fluo-4 (FluoroPureTM-AM, Molecular Probes, Eugene, OR), 0.02% DMSO, and 0.01% nontoxic detergent Cremophor EL was used.

	RESULTS

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Spontaneous intercellular Ca²⁺ waves in the LM and CM during intervals between CMMCs. During the intervals between CMMCs in C57BL/6 control mice, spontaneous intercellular Ca²⁺ waves were commonly observed in both the LM and CM layers (n = 8), an example of which is shown in Fig. 1, B and C. Ca²⁺ waves occurred more frequently in the LM (1.3 ± 0.3 waves/s) than in the CM (0.5 ± 0.3 waves/s, P < 0.05, n = 8 preparations). Ca²⁺ waves fired asynchronously between each muscle layer, i.e., discreet intercellular Ca²⁺waves occurred independently in both muscle layers 66% of the time, where each wave propagated anisotropically through each muscle layer. We found no evidence to suggest that the generation of any Ca²⁺ waves in either the LM or CM electrotonically spread into the neighboring muscle layer to activate a Ca²⁺ wave in both layers simultaneously, as has been reported previously. Ca²⁺ waves propagated comparatively slower orthogonal to CM (3.8 ± 0.2 mm/s, n = 8) or LM (6.6 ± 0.5 mm/s, n = 8) fibers (Table 1) and considerably more rapidly along or parallel to the muscle fibers (see Fig. 1, B and C). Thus, Ca²⁺ waves in each muscle layer were readily distinguishable from one another since their fast and slow conducting axes through the LM were orthogonal to those Ca²⁺ waves in the CM and vice versa (Fig. 1, B and C). Spontaneous Ca²⁺ waves in either muscle layer originated and terminated in discreet zones of muscle (100- to 400-µm width; see Fig. 1, B and C), suggesting that these zones may represent morphological distinct muscle bundles or regions of muscle activated by terminals of motor neurons innervating a particular region of muscle.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Changes in properties of Ca2+ waves in the CM during CMMCs and in rectal aganglionosis. A: during CMMCs, the distance traveled by Ca2+ waves orthogonal to CM fibers significantly increased compared with spontaneous waves between CMMCs. However, in the aganglionic segment of endothelin receptor type B-deficient (Ednrbs–l/Ednrbs–l) mice, the mean distance of propagation was not significantly different from control. B: during CMMCs, the velocity of Ca2+ wave propagation in the CM also significantly increased in C57BL/6 control mice. Interestingly, the propagation velocity in aganglionic muscles was significantly higher than even during CMMCs in wild-type mice. C: there was a dramatic increase in numbers of collisions between Ca2+ waves during CMMCs. Very few collisions occurred between CMMCs or in the aganglionic segment. D: the rate at which Ca2+ waves were initiated in the CM dramatically increased during CMMCs. Note that we only measured waves that originated and terminated within the field of view. A wave could propagate away from its site of initiation in opposite directions; therefore, one-half of the wave could terminate outside the field of view. To avoid this, the distance a wave traveled was measured from its site of initiation to its point of termination; therefore, the measured propagation distances are likely to represent the minimum distance a wave could travel. Some waves may have traveled twice this distance (*P < 0.01). Data were extracted from control (n = 5), CMMC (n = 4) and Ednrbs–l/Ednrbs–l (n = 8) mice.

Pharmacological sensitivity of Ca²⁺ waves in the LM and CM. The generation of CMMCs in the mouse colon has been shown to involve a discharge of cholinergic excitatory junction potentials (EJPs) in the CM layer (7, 23, 25). In light of this, we tested whether atropine would prevent the generation of Ca²⁺ waves in both the LM and CM during CMMCs. Atropine (1 µM) or hexamethonium (300 µM) consistently abolished Ca²⁺ waves in both muscle layers (n = 4). Ca²⁺ waves were also blocked by nicardipine (1–2 µM), an L-type Ca²⁺ channel antagonist, suggesting that they are caused by an influx of Ca²⁺ as an action potential propagates through the smooth muscle syncitium.

Properties of Ca²⁺ waves in the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice. It was of particular interest to determine whether the properties of Ca²⁺ waves that underlie the phasic contractions in the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice would be different from C57BL/6 controls. In both LM and CM layers, spontaneous Ca²⁺ waves were routinely recorded from the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice (n = 8). The mean propagation velocity of Ca²⁺ waves in the CM was found to be significantly higher in the aganglionic distal colon compared with C57BL/6 mice (P < 0.05; n = 8; Fig. 3B). The mean frequency of Ca²⁺ wave discharges in the LM and CM was found to be 0.57 ± 0.05 and 1.72 ± 0.11 Hz (211 and 301 waves, respectively, n = 8). Interestingly, the number of collisions between Ca²⁺ waves was low in the aganglionic distal colon (Fig. 3C). In the CM but not LM, it was possible to ascertain the number of collisions between Ca²⁺ waves. Only 11 detectable collisions occurred from 301 waves (n = 8 animals). It was not possible to reliably determine how many collisions occurred in the LM layer due to the removal of part of the LM (see METHODS).

The most noteable difference in the characteristics of Ca²⁺ waves in the aganglionic distal colon was the reduced temporal synchrony between the two muscle layers (Fig. 4, A and B). In 78% of the discreet Ca²⁺ waves that fired in the CM, it was not possible to temporally correlate the onset of any discreet Ca²⁺ wave with another Ca²⁺ wave in the LM (within a 100-ms time window, n = 8; Fig. 4, A and B). In 22% of the Ca²⁺ waves identified in the CM, a Ca²⁺ wave could be also identified in the LM (within a 100-ms window of the onset of the Ca²⁺ wave in the CM).

View larger version (76K): [in this window] [in a new window]   Fig. 4. Asynchronous firing of Ca2+ waves between the LM and CM in the aganglionic rectum of Ednrbs–l/Ednrbs–l mutant mice. A and B: ST maps of Ca2+ wave activity imaged simultaneously in LM and CM layers. Time is represented from the top to bottom, whereas the distance of Ca2+ wave propagation is represented from the left to right. It can be seen that Ca2+ waves in the two muscle layers occurred independently and rarely discharged simultaneously. C: sequence of Ca2+ waves in the LM. In the top left image (0 ms), a single Ca2+ wave was initiated, which then spread in the direction of the arrow to the top middle image (67 ms). At 133 ms, the top right image showed a second Ca2+ wave in the LM that was propagating toward the first wave initiated at 0 ms. At 267 ms (bottom middle image), the two waves collided. During these Ca2+ waves in the LM, there was no accompanying Ca2+ wave in the underlying CM layer. D: a single propagating Ca2+ wave in the CM. At 67 ms (top middle image), the CM Ca2+ wave was initiated, which propagated as one continuous wave from the right side of the image to the left (see the direction of the arrow). Eventually, at 333 ms (bottom right image), the wave propagated outside the field of view. No evidence of any accompanying LM Ca2+ wave was shown when the Ca2+ wave in the CM was active.

	DISCUSSION

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In this study, we used Ca²⁺ imaging of intact whole preparations of the murine colon to determine how the temporal activation and spread of Ca²⁺ waves in LM and CM fibers changed during CMMCs in wild-type mice and, second, to determine how the properties of Ca²⁺ waves were modified in the aganglionic rectum of Ednrb^s–l/Ednrb^s–l mutant mice that do not exhibit CMMCs. A number of findings arose from this study. Namely, during CMMCs in wild-type mice, a discharge of discreet Ca²⁺ waves occurs simultaneously in both LM and CM layers. This temporal synchronization of Ca²⁺ waves between the two muscle layers involves the synaptic activation of many myenteric interneurons and cholinergic excitatory motor neurons. Second, during CMMCs, the orthogonal propagation velocity and distance traveled by discreet Ca²⁺ waves increases, as do the numbers of initiation sites and collisions between Ca²⁺ waves. However, in the aganglionic rectum of Ednrb^s–l/Ednrb^s–l mice, although the propagation velocity of Ca²⁺ waves was significantly increased, Ca²⁺ waves between the two muscle layers showed consistently poor temporal synchronization.

Dependence of intercellular Ca²⁺ waves on action potentials. Ca²⁺ waves appear to arise from Ca²⁺ influx during a smooth muscle action potential since, like muscle action potentials, they are blocked by L-type Ca²⁺ channel antagonists (13, 15, 26, 29, 30). Therefore, a Ca²⁺ wave is likely due to the sequential rise in Ca²⁺ within each muscle cell produced by an action potential as it propagates through the smooth muscle syncitium rather than a spread of Ca²⁺ from muscle cell to muscle cell. In the present study, we found that the velocity of Ca²⁺ waves orthogonal to the muscle fibers was similar for both the LM and CM. These values were similar to those we reported previously for the LM of the guinea pig ileum (30), the LM and CM of the guinea pig colon (29), and the LM of the murine colon (13). They were also consistent with the velocities of propagating action potentials in the LM of the guinea pig colon measured orthogonal to the muscle fibers with two intracellular microelectrodes (15). Previously, we had estimated the velocity of Ca²⁺ waves parallel to muscle fibers to be 8–10 times faster than perpendicular to the direction of the fibers (29, 30).

Multiple initiation sites and multiple collisions during CMMCs. It is particularly noteworthy that when Ca²⁺ waves discharged during CMMCs, they appeared to make a zigzag appearance across the CM. This was because they were found to be not a single propagating wave but rather composed of multiple discreet Ca²⁺ waves. This zigzag appearance is caused by an increase in both the numbers of sites at which Ca²⁺ waves were initiated and due to annihilating collisions between Ca²⁺ waves (13, 29, 30). This suggests that the synchronized EJPs that occur in the muscle during the CMMC (25) are initiating action potentials at many discreet sites across the muscle at about the same time. The resulting action potentials (Ca²⁺ waves) can only propagate orthogonally a short distance before colliding with another Ca²⁺ wave. Our recent electrophysiological study (26) using two microelectrodes support these conclusions since we have shown that action potentials could readily propagate over 100 µm across the LM, but they were rarely coordinated over a distance of 1 mm across the LM of the guinea pig colon. As we have previously suggested (13, 30), collisions prevent any one Ca²⁺ wave initiation site from controlling large regions of muscle. It may seem contradictory that individual Ca²⁺ waves travel further during the CMMC yet are subject to more collisions. This maybe due to the fact that they also travel faster; therefore, each wave can propagate over a larger area of muscle before it collides with another wave.

Ca²⁺ waves and EJPs during CMMCs. One of the major findings of the present study was that during CMMCs, Ca²⁺ waves propagated over larger distances orthogonal to the muscle fibers and at a higher velocity across the smooth muscle syncitium. It is known that during CMMCs, there is a repetitive discharge of cholinergic EJPs in the CM layer that become temporally synchronized over large regions of the CM layer (up to 15 mm in the longitudinal axis and across the entire width of the colon) (25). It is likely that if action potential threshold is reached during the EJP, then a considerably larger area of smooth muscle will have its resting membrane potential at or near action potential threshold that may result in Ca²⁺ waves spreading over larger distances at a higher velocity. Also, the release of ACh during the CMMC is likely to change the conductance properties of the smooth muscle syncitium, which is likely to promote the faster propagation of action potentials. It is particularly noteworthy that intracellular electrophysiological recordings from the CM during the CMMC demonstrates that the frequency of cholinergic EJPs is 1.9 Hz (7, 23, 25), which is remarkably similar to the frequency of Ca²⁺ waves in both muscle layers during the CMMC. This similarity in EJP and Ca²⁺ wave frequency , together with the observation that Ca²⁺ waves were blocked by atropine, supports the idea that the discharge of suprathreshold cholinergic EJPs in both muscles may be the underlying electrical event responsible for the initiation of Ca²⁺ waves. Indeed, in the small intestine of other laboratory animals such as the guinea pig, the MMC in the small intestine has also been recorded (10, 11) and shown to be abolished by atropine or hexamethonium (11), suggesting that in other regions of the intestine, MMC generation is also critically dependent on the release of ACh from cholinergic motor neurons. Since single cholinergic motor neurons project for relatively short distances along the intestine (2, 4, 8), it seems highly likely that many myenteric interneurons and motor neurons are activated simultaneously to generate a single EJP in gastrointestinal smooth muscle (25).

There have not been any electrical recordings made from the LM layer of the mouse colon. We have shown previously in the guinea pig distal colon that the LM and CM both received a synchronized discharge of cholinergic EJPs in response to maintained circumferential stretch that was due to the synchronous activation of excitatory motor neurons to both the LM and CM (27). It is likely that a similar arrangement of enteric circuitry may be responsible for the simultaneous discharge of activity in both muscle layers in the mouse colon (Fig. 6) (for a review, see Ref. 22).

View larger version (34K): [in this window] [in a new window]   Fig. 6. Neural circuitry underlying the activation of Ca2+ waves in the LM and CM during the CMMC. The schematic diagram shows the possible activation of enteric excitatory nerve pathways during the CMMC. Descending interneurons (Desc Ints) activate common ascending interneurons (Asc Ints), which, in turn, activate excitatory motor neurons (EMNs) to the CM and LM. During the CMMC, ACh is released from excitatory motor neurons to activate muscarinic receptors on the LM and CM at the same time. This induces rhythmic depolarizing potentials in both muscles, which initiate action potential firing. The action potentials in both muscles are independent of each other. Ca2+ influx during the action potential induces a Ca2+ wave in the muscle, which propagates rapidly along muscle fibers and more slowly across muscle fibers. The rapid propagation of a Ca2+ wave parallel to CM fibers ensures that the gut contracts as a ring, whereas the slower orthogonal spread of Ca2+ waves dictates how wide these narrow contracting rings or bands of muscle are organized along the gut.

Properties of Ca²⁺ waves in the aganglionic distal colon of Ednrb^s–l/Ednrb^s–l mice. We found that despite the absence of enteric nerves, the aganglionic smooth muscle was readily able to generate myogenic contractions (19) and Ca²⁺ waves in both the LM and CM. The mechanisms underlying these contractions have been elusive. In this study, the major finding in the aganglionic segment was that the two muscle layers fired almost exclusively out of phase with one another, likely due to the absence of interneurons and motor neurons to synchronize their firing. Surprisingly, the propagation velocity of Ca²⁺ waves was significantly faster in the hypertrophic region. It is known that the conduction of action potentials is increased in thicker nerve axons (28). A similar phenomenon may also account for these results in the hypertrophic aganglionic segment.

Conclusions. We have shown that during CMMCs in wild-type mice, a rapid discharge of Ca²⁺ waves occurs simultaneously in both smooth muscle layers. Since no evidence of electrotonic conduction was found between the two muscle layers, we suggest that the underlying mechanism must involve synchronized firing of myenteric interneurons and excitatory motor neurons to both muscle layers that results in simultaneous EJPs and action potentials. The net response is likely to lead to simultaneous shortening of both muscle layers during the propulsion of a fecal pellet. The absence of enteric ganglia in the distal colon of Ednrb^s–l/Ednrb^s–l mice leads to sporadic and largely asynchronous firing of Ca²⁺ waves in both muscle layers.

	GRANTS

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This study was funded by National Institutes of Health (NIH) Grants P20 RR-018751 (to N. J. Spencer), R01 DK-45713 (to T. K. Smith), and DK-41315 (to K. M. Sanders). Laboratory imaging was supported by NIH Grant P20 RR-018751.(14)

	FOOTNOTES

 

Address for reprint requests and other correspondence: N. Spencer, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (e-mail: nick{at}unr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* N. J Spencer and B. Bayguinov contributed equally to this work.

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