Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon

Nick J. Spencer, Grant W. Hennig, and Terence K. Smith

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 µm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. Nomega -nitro-L-arginine (L-NA; 100 µM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.

circular muscle; inhibitory junction potential; excitatory junction potential; myenteric neuron; peristaltic reflex; peristalsis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THERE HAS BEEN MUCH CONTROVERSY regarding the relative movements of the longitudinal (LM) and circular muscle (CM) layers throughout the gastrointestinal (GI) tract. Still today, the relative movements of the LM and CM layers in the GI-tract are the subject of much investigation. Bayliss and Starling (2, 3) first reported that the two muscle layers of the bowel contracted together during ascending excitation or relaxed together during the descending inhibitory phase of the peristaltic reflex. Since their work, many other investigators have also shown that the two muscle layers of the bowel contract simultaneously or relax simultaneously during reflex stimulation or peristalsis (6, 9, 12, 19, 26, 33, 36-38, 41, 44, 45).

However, other investigators (see Ref. 1 for reviews) have criticized the original observations of Bayliss and Starling (2, 3), because it has been observed that the two muscle layers appear to contract out of phase with respect to each other during peristalsis or reflex stimulation (1, 15, 16, 18, 20, 29, 48) or during the migrating motor complex (30). Wood (49) has argued that the two muscle layers cannot contract or relax together because "... the laws of geometry dictate that they are antagonistic muscles." Perhaps most noteably, Kottegoda (21, 22) proposed the idea of reciprocal innervation, whereby he stated that the LM and CM "..do not contract simultaneously but are activated reciprocally so that when one muscle layer contracts the other relaxes..." His studies were performed on the guinea pig ileum. However, recent studies (38, 44, 45) in the same tissue have found no evidence to support the work of Kottegoda. The problem with unrestrained mechanical recordings is that movements of one muscle layer can passively influence the movements of the other muscle layer (see Ref. 25). This can lead to erroneous assumptions regarding the intrinsic innervation of the two muscles during reflex activity (2, 3, 6, 9, 12, 19, 25). In both the small and large intestine, more sophisticated dissection techniques designed to eliminate such mechanical interactions (33, 36-38, 44) and calcium imaging of the two muscle layers (45) have consistently shown that the enteric neural circuits are wired so that the LM and CM contract together and relax together during reflex activity. Similarly, ultrasonographic techniques applied to the human esophagus have demonstrated a similar relationship between the two muscle layers (28a, 50). Furthermore, when simultaneous intracellular recordings have been made from the two muscle layers of the guinea pig distal colon, it was found that both the LM and CM always received synchronous inhibitory junction potentials during the descending inhibitory reflex and synchronous excitatory junction potentials during the ascending excitatory reflex (41). However, the later studies used phasic and likely supramaximal stimuli that recruit large populations of common interneurons. Other forms of motility that do not rely on such intense stimulation may recruit different neural pathways leading to relatively independent activities of the two muscles such as occur during spontaneous movements (27, 39, 45). A number of studies has suggested that the two muscle layers can have different thresholds for activation (6, 14, 19, 20).

The existence of polarized neuronal reflexes in the gastrointestinal tract has been known since the turn of the last century (2, 3). How the enteric nervous system is wired to cause polarized neuronal reflexes has not been resolved. Recently, we showed that maintained circumferential stretch of the guinea pig distal colon activates a colonic motor pattern that consists of an ongoing discharge of excitatory junction potentials (EJPs) in the CM layer at the oral end of colon, which occur at the same time as inhibitory junction potentials (IJPs) in the CM located 20 mm anally (40a). One of the major findings of our previous study was that stretch activated a population of mechanosensory neurons that was resistant to nifedipine and smooth muscle paralysis (40a). This is vastly different from what is known about mechanosensory neurons in the small intestine. Activity in these neurons has been shown to be dependent on muscle tone or contraction rather than stretch per se, because their excitability is abolished by muscle paralysis (isoprenaline and L-type Ca2+ channel blockers) despite maintained stretch (23, 24). Therefore, in the distal colon, a population of mechanosensory neurons underlying repetitively firing oral EJPs and anal IJPs appear to be resistant to muscle paralysis (40a). In this study, we have used stretched preparations of colon to correlate the electrical activities in both the LM and CM layers. We show that under maintained circumferential stretch, EJPs discharge synchronously to the LM and CM orally at the same time as IJPs discharge to the LM and CM anally. Furthermore, synchronized junction potentials in both muscle layers are linearly related in amplitude, suggesting that different populations of motor neurons to both the LM and CM are synaptically activated at the same time by common enteric interneurons, some of which may be mechanosensory.


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Guinea pigs of either sex, weighing 200-350 g, were euthanized by CO2 inhalation overdose in accordance with the Animal Ethics Committee of the University of Nevada School of Medicine. The abdominal cavity was opened, and the terminal 10 cm of distal colon, ~2 cm oral to the rectum, were removed. The mesenteric attachment was trimmed away, and the lumen was flushed clean with modified Krebs solution.

Dissection procedure: direct visualization of LM and CM cells. The distal colon was opened along the mesenteric attachment, and the terminal distal region was pinned to the base of a Sylgard (Dow Corning, Midland, MI)-lined Petri dish so that the mucosal surface faced uppermost. The mucosa and submucosa were then sharp dissected from this opened region to expose the underlying CM. Small strips of CM (1-2 mm wide) were then dissected off the far oral and anal extremities of the preparation to expose the underlying myenteric plexus and LM layer only at the extremities, as we have previously described (40-42). This dissection procedure enabled us to clearly visualize the myenteric ganglia and underlying LM in one region while also identifying the thicker CM in the same field of view (see Fig. 1 in Ref. 41). Microelectrodes could then be positioned so as to record unequivocally from LM or CM cells or both simultaneously. The electrical activities and characteristics of junction potentials in LM and CM cells were vastly different and readily identified (see below). The in vitro preparations studied were a maximum of 20 mm in length and, when pinned out under maximal circumferential stretch in a recording chamber, the circumferential axis measured between 10 and 12 mm. Naturally expelled pellets from guinea pigs used in this study measured ~5-6 mm in diameter. In all experiments, preparations were pinned serosal-side down in a recording chamber whose base consisted of a microscope coverslip that was lightly coated with a fine layer of Sylgard silicon (Dow Corning). Identification of the LM layer was aided by the use of an inverted microscope (Olympus, CK2; Napa, CA).


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Fig. 1.   A simultaneous recording from 2 circular muscle (CM) cells in stretched segments of guinea pig distal colon. A: schematic of the preparations used. A segment of colon was incised in the longitudinal axis and pinned under maintained circumferential stretch. The mucosa and submucous plexus were removed. Two recording electrodes were used to impale CM cells at the oral and anal cut ends of colon. Bi and Bii are a simultaneous recording from 2 CM cells in the absence of nifedipine. Excitatory junction potentials (EJPs) and action potentials occur at the oral recording site. These occur at the same time as Inhibitory junction potentials (IJPs) at the anal recording located 20 mm anally. Di and Dii are in another animal in the presence of nifedipine, the same motor pattern was recorded, but the action potentials were abolished. Ci and Cii are an expanded time scale of the single EJP/IJP complex highlighted by the black bar in Bi. Ei and Eii are a single EJP/IJP complex as an expanded recording represented by the black bar in Di and Dii. Note that the action potential truncates the repolarization phase of the EJP (c.f. Ci and Ei).

Preparations used to record electrical activity from LM and CM cells. Two different preparations were used for recordings in the study. The majority of recordings was made from LM and CM cells at the terminal oral and anal cut ends of colon, (<100 µm from each cut end) as shown schematically in Fig. 1A. These preparations were 20 mm in length and, when pinned under maximal circumferential stretch, measured between 11 and 13 mm wide. These preparations were devoid of mucosa and submucous plexus but contained all the CM layer. The other preparation used was identical to the one described, except that intact regions of colon (10 mm in length) were preserved oral and anal of the stretched region, as shown schematically in Fig. 3A.

Simultaneous video imaging and electrophysiological recordings. During stretch-activated EJPs and IJPs, we correlated in time the spontaneous longitudinal movements of the colon, with the electrical activity occurring simultaneously in the smooth muscle. To do this, two independent digital video cameras (model WV-BP330; Panasonic CCTV) were used. This enabled us to correlate in time intracellular electrical recordings with spontaneous movements of the colon. One was mounted on an inverted microscope (Olympus CK2); the other was mounted so as to videotape the oscilloscope displaying the intracellular electrical activities. The outputs of these two cameras were connected to a digital video mixer (model MX-1 NTSC; Videonics) to combine both colonic movements and simultaneously occurring electrical events. The combined outputs from the mixer were visualized directly onto a black and white monitor (model SSM-930; Sony, Tokyo, Japan), and a hardcopy of these images was recorded and stored on a VHS tape recorder (model VWM-390; Sanyo). A ×10 objective was used to visualize a 1-mm2 field of view at the anal cut end of colon. Microelectrode impalements were made within 100-200 µm of this area of muscle that was monitored for muscle movement. Spatiotemporal maps of the longitudinal colonic movements were generated using the method previously described (19). These maps could then be correlated in time with electrophysiological recordings (Fig. 2).


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Fig. 2.   Simultaneous video imaging of longitudinal muscle (LM) movement and intracellular recording of electrical activity in 2 CM cells. Ai and Aii show a simultaneous recording from 2 CM cells in the absence of nifedipine, where recordings were made within 200 µm of the anal cut end of colon. The electrodes were separated by 100 µm in the circumferential axis. Both the oral and anal ends of colonic muscle were free to move spontaneously. B is a spatiotemporal map of spontaneous longitudinal movement generated at the same time as the ongoing discharge of IJPs were recorded in Ai and Aii. Contraction of LM is represented by distortion lines that deviate toward the oral end of colon. On 3 occasions (see arrows), an IJP occurs simultaneously in 2 CM cells at the same time as LM displacement occurs, suggesting that LM was excited (contracting) orally at the same time as the CM was inhibited anally. The vertical white lines represented by the arrows show the similarity in latency between the onset of the CM IJPs and the LM movement. During some IJPs, however, no clear correlation was detected with respect to LM movement.

Electrical recording technique. Intracellular microelectrode recordings were made from LM and CM cells simultaneously using two independently mounted microelectrodes, whose fine positioning could be adjusted using two micromanipulators (model M3301L; World Precision Instruments, Sarasota, FL). Microelectrode impalements into LM and CM cells were made at distances of <300 µm from each other at either the far oral or anal cut ends of the isolated colon. Electrodes were filled with 1.5 M KCl solution and had tip resistances of ~120 MOmega . Electrical signals were amplified using an Axoprobe 1A (Axon Instruments, Foster City, CA), whose outputs were connected to a digidata 1200 Interface (Axon Instruments) and then into a four-channel digital oscilloscope (Gould 1604; Ilford, Essex, UK). All electrical signals were also recorded onto a personal computer running Axoscope version 8.0 (Axon Instruments). For simultaneous recordings from four smooth muscle cells, three independent amplifiers were used: the Axoprobe 1A described above and two independent WPI electrometers (model FD 223). Filtering frequencies of 0.66-1.5 kHz were used.

Drugs and solutions. The following drugs were used throughout this current study: apamin, atropine, L-NA, and nifedipine. All drugs were obtained from Sigma (St. Louis, MO). Nifedipine was prepared at a stock concentration of 10-2 in ethanol and diluted to a final concentration of 10-6 M in Krebs solution. The composition of the modified Krebs solution was (in mM): 120.35 NaCl, 5.9 KCl, 15.5 NaHCO3, 1.2 NaH2PO4, 1.2 1.2 MgSO4, 2.5 CaCl2, and 11.5 glucose. The Krebs solution was gassed continuously with a mixture containing 3% CO2-97% O2 (vol/vol), pH 7.3-7.4.

Measurements and statistics. For statistical comparison of membrane potential changes before and after drug additions, Student's paired t-tests were used. A minimum significance level of P < 0.05 was used throughout. In the majority of experiments, multiple recordings were made from the same animals. However, in the RESULTS, the use of n always refers to the number of different animals on which observations were made. Data are presented as means ± SE. Measurements of amplitude and half width and time to peak response were made using Axoscope 8.0 (Axon Instruments).

Analysis of data. The data in this study were analyzed using the same procedures as described in our previous study (39). In brief, membrane voltage recordings were exported as text files and imported into a custom-written program (OpenGL-based). The traces were resampled to 250-300 Hz and smoothed (36-ms moving average; 5 iterations), and an average baseline (5-10 s) was calculated to follow slow undulations in voltage and disregard faster events (such as junction potentials). To characterize the relative changes in EJPs and IJPs that were recorded during simultaneous impalements, each membrane potential trace was differentiated (4-ms time step) and plotted against each other, such that the rate of rise of junction potentials in the anal electrode was plotted on the x-axis, whereas the rate of rise of junction potentials in the oral electrode was plotted on the y-axis. If the plot skewed to any particular axis, this indicated changes in that electrode were occurring disproportionally faster than in the other electrode. If both events were undergoing the same changes in voltage at the same time, these traces would fall along a linear 45° line between the two axes.


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General observations. In total, 207 CM cells were recorded from 46 animals and 94 LM cells were recorded from 24 animals. As we have previously described (40a), in preparations in which maintained circumferential stretch was applied, all recordings from CM cells showed that resting membrane potentials were highly unstable and revealed an ongoing discharge of IJPs or EJPs. Taken between the peaks of EJPs and troughs of IJPs, the mean resting membrane potential of CM cells was -35.3 ± 0.54 mV (122 cells). Impalements into LM cells also showed ongoing bursts of IJPs and EJPs, but these were readily distinguishable from CM cells. The major difference between junction potentials in LM and CM cells was that EJPs and IJPs in LM cells were considerably smaller in amplitude and showed slower rates of rise compared with CM cells. The mean resting membrane potential of LM cells was -30.2 ± 0.7 mV (29 cells); this value was significantly depolarized compared with CM cells (P < 0.01; Student's unpaired t-test).

Simultaneous recordings were made from two CM cells at either end of a stretched-sheet preparation that was pinned under maintained circumferential stretch, as shown in Fig. 1A. In this preparation, it was found that a repetitive discharge of EJPs (and action potentials) occurred at the oral end of colon. These oral EJPs were found to occur at the same time as IJPs in the CM located 20 mm anally (Fig. 1, Bi and Bii). This coordinated motor pattern was routinely recorded for up to 5 h following the application of maintained stretch. On some occasions, EJPs were recorded at the anal cut end of colon and IJPs at the oral cut end (see below).

Simultaneous video imaging of LM movement with intracellular electrical activity in the CM. Because stretch activated ongoing EJPs and action potentials in CM orally, at the same time as IJPs in CM anally (Fig. 1, Bi), we then sought to investigate whether any LM contractions would correlate in time with IJPs recorded from the CM. To do this, we simultaneously recorded from CM cells in the absence of nifedipine, while also using video recordings to monitor muscle movements. In four animals, we recorded electrical activity in CM cells anally at the same time as recording the movements of the LM on video. An example of these recordings is shown Fig. 2. It was noted that some of the IJPs recorded from the CM at the anal end of colon (Fig. 2A) could be correlated in time with the onset of LM shortenings recorded orally (Fig. 2B). This suggested that action potentials were occurring in LM cells orally at the same time as IJPs in CM anally. In the spatiotemporal map shown (Fig. 2B), the distortion of the white lines represents movement (either contraction or relaxation) of the preparation. In this case, on three occasions at the same time as the IJP occurred in the CM (see arrows), the colon contracted from the oral region, and this is represented by distortion toward the oral axis (Fig. 2B). If preparations were not spontaneously active, the distortion line would be represented as a straight line from the left to right side of the figure. Therefore, these observations prompted us to directly record the electrical activity in the LM layer at the same time as recordings were made from the CM layer.

Simultaneous recordings from LM and CM cells at the oral and anal regions of colon. We investigated whether LM cells would show similar electrical activities as CM cells under maintained circumferential stretch. To do this, four independent microelectrodes were used in the recording configuration shown in Fig. 3A. This allowed us to directly record and compare the stretch-activated junction potentials in LM and CM orally with those occurring in the LM and CM anally. In total, 14 quadruple recordings were made simultaneously from LM and CM orally at the same time as LM and CM cells located 20 mm anally (n = 11). In all animals studied (n = 11), it was clear that once stable impalements were made, EJPs discharged simultaneously in both the LM and CM orally at the same time as IJPs in the LM and CM anally. A typical recording using this technique is shown in Fig. 3B. As described above, the characteristics of junction potentials in LM cells were readily distinguished from CM cells. In LM cells, EJPs and IJPs had consistently smaller amplitudes than IJPs recorded from CM cells. A major distinguishing feature of CM cells was the presence of ongoing "fast" apamin-sensitive membrane fluctuations (IJPs). Apamin-sensitive IJPs were not routinely detected from LM cells.


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Fig. 3.   Simultaneous recordings from 4 smooth muscle cells: 2 LM and 2 CM cells. A: schematic of the intact tube preparation used. Four independent microelectrodes were impaled simultaneously: 1 electrode into an LM cell and 1 into a CM cell at anal end of colon, while at the same time as 1 electrode into a LM cell and 1 into a CM cell at the oral end of the stretched preparation. B: a typical simultaneous recording from 4 muscle cells using the protocol shown in A. It can be seen that EJPs occur synchronously in both the LM and CM at the oral end of the colon at the same time as IJPs occur synchronously in both the LM and CM 20 mm anally. C: 4 recordings are superimposed. It can be seen that oral EJPs and anal IJPs occur simultaneously at either end of the stretched region. Note, on occasion, an EJP discharged synchronously in both the LM and CM orally, but no accompanying IJPs occurred anally (see arrow).

Simultaneous recordings from LM and CM cells at the anal end of colon. Because it was noted that the characteristics of IJPs and EJPs appeared to be different between LM and CM cells, we recorded simultaneously from a single LM cell and CM cell (separated by <200 µm) to compare characteristics of junction potentials between muscle layers. In total, we recorded from 25 pairs of LM and CM cells (n = 20). In 23 of the 25 pairs of cells, IJPs occurred simultaneously in both muscle layers. A typical example of synchronized IJPs in the LM and CM at the anal end of the colon is shown in Fig. 4A. In two pairs of LM and CM cell recordings, IJPs and EJPs occurred essentially asynchronously between the two muscle layers. On an expanded time scale, it was noted that there were major differences between IJPs in the LM and CM. In the LM, the amplitudes of IJPs were always smaller than IJPs in CM (Fig. 4, A and C), and the rate of rise of IJPs in LM was considerably less than those of CM IJPs (Fig. 4D). The cumulative summary data of simultaneous recordings from LM and CM cells made from seven preparations (n = 5) is shown in Fig. 4C, where we plotted the amplitudes of IJPs in LM with those in CM. In general, a 20-mV IJP in CM usually corresponds with an IJP of ~7 mV in LM when the two events are synchronized.


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Fig. 4.   Simultaneous recordings from LM and CM at the anal region of colon during maintained circumferential stretch. A: simultaneous recording from a LM cell and a CM cell when separated by <100 µm. IJPs occur synchronously in both the LM and CM layers. Note, the amplitudes of IJPs in the 2 muscle layers are linearly related, such that a large IJP in the LM corresponds with a large IJP in the CM. Small-amplitude IJPs in the CM usually do not correlate with an IJP in the LM (see * in A). Also, note that the amplitudes of the IJPs in CM were always larger than those in the LM. B: superimposed recordings of A. C: summary data from 8 preparations (n = 6) are plotted in which IJP amplitudes in CM are consistently larger than IJPs in LM. D: membrane potential recordings of IJPs in both muscle layers were differentiated and plotted against each other. In these plots, it is evident that during the onset of synchronized IJPs in both muscle layers, the rate of rise of IJPs in CM was greater than the corresponding IJP in the LM. This is represented by the trajectories consistently tending toward the x-axis during the onset of IJPs before deviating toward the y-axis.

Simultaneous recordings from LM and CM cells at the oral end of colon. We also compared the differences between synchronized EJPs in LM and CM cells. Simultaneous recordings were made from LM and CM cells <300 µm from each other and within 100 µm of the oral cut end of colon. In total, 12 pairs of recordings were made from LM and CM cells (n = 9). In all simultaneous recordings, it was consistently found that EJPs occurred synchronously in the two different muscle layers (Fig. 5A). Although, there were major differences in the waveform of EJPs in LM compared with CM. EJPs in CM were always larger in amplitude than those of the CM (Fig. 5, A and C) and the rate of rise of EJPs in CM was also considerably greater than that of EJPs in LM (Fig. 6D). The cumulative summary data for the amplitudes of the EJPs is pooled and plotted in Fig. 5, C and D. It can be seen that the amplitudes of EJPs that occurred synchronously in the LM and CM were linearly related, such that a larger amplitude EJP in the CM correlated well with a larger EJP in the LM. Interestingly, although EJPs occurred synchronously in the LM and CM, when current pulses (400 pA-2.5 nA) were passed out of one electrode into either an LM or CM cell, electrotonic potentials were never detected in the other muscle layer at distances of 100 µm from the current-passing electrode (6 pairs of LM and CM cells; n = 3).


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Fig. 5.   Simultaneous recordings from LM and CM at the oral region of colon maintained under circumferential stretch. A: a simultaneous recording from a single LM cell and a single CM cell (separated by <100 µM). The dual recordings were made within 100 µm of the oral cut end of colon. EJPs discharge synchronously in both the LM and CM. Note, EJPs in the 2 muscle layers were linearly related, such that a large EJP in the LM was associated with a large EJP in the CM. B: superimposed recordings from the recordings shown in A. C: graph showing the relationship between EJPs in the LM and CM. The data are a summary of 7 preparations (n = 5). EJPs in the 2 muscle layers are linearly related in amplitude. Note, EJPs in CM were consistently larger in amplitude than EJPs in LM. D: membrane potential recordings of EJPs in LM and CM were differentiated and plotted against each other. The graph of differentiated recordings shows that during the onset of synchronized IJPs, the rate of rise of EJPs in CM was greater than the rate of rise of EJPs in LM cells. This is represented by the trajectories consistently tending toward the x-axis during the onset of IJPs before deviating toward the y-axis.



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Fig. 6.   Effects of inhibition of nitric oxide synthesis and the K+-channel blocker apamin on stretch-evoked IJPs in LM and CM. A: a control simultaneous recordings from LM and CM cells at the anal end of colon. IJPs occur synchronously in the 2 muscle layers. B: Nomega -nitro-L-arginine (100 µM) abolished the "slow" IJP in the LM layer and left L-NA-resistant "fast" IJPs in CM cells, confirming that impalements were made from different muscle layers. The time course of the repolarization phase of IJPs in the CM layer was reduced in L-NA. C: in the presence of L-NA, further addition of apamin (500 nM) abolished fast IJPs in CM and induced small EJPs that were observed only in the CM layer. All recordings are from the same animal.

Effects of L-NA and apamin on stretch-activated IJPs in LM and CM. Because major differences were noted in the characteristics of stretch-activated IJPs in LM and CM cells, we sought to compare the sensitivity of these IJPs to an inhibitor of nitric oxide (NO) synthesis; and the bee venom apamin. In three of three animals tested, L-NA (100 µM) consistently abolished the slow IJPs in LM cells (Fig. 6B) but did not affect the amplitude of the fast IJP of the CM (n = 5; Fig. 6B). L-NA significantly depolarized the CM cells from -36.6 ± 2.6 to -24.3 ± 0.6 mV (P = 0.025; 5 cells; n = 5) but caused no significant depolarization of the LM cells (30.5 ± 4.5 to 29.6 ± 3.8 mV; P = 0.77; n = 4). In the presence of L-NA, the further addition of apamin (500 nM) always abolished the L-NA-resistant fast IJPs in CM cells (Fig. 6C) from 29.8 ± 1.7 to 26.8 ± 1.6 mV (P = 0.013; 5 cells; n = 5).

Effects of atropine on ascending excitatory and descending inhibitory nerve pathways to LM and CM. We tested whether release of acetylcholine was involved in ascending excitatory and descending inhibitory nerve pathways to the two different muscle layers. Immediately on infusion of atropine into the bath, atropine caused a significant and sustained hyperpolarization (Fig. 7A) of all LM cells by 14 ± 2 mV (7 cells; n = 5) from a mean value (control: 31.0 ± 1.3 to 45.7 ± 2.3 mV; P < 0.01; 7 cells; n = 5) and in CM cells by 13 ± 1 mV (14 cells; n = 8) from a mean value of (control: 32.5 ± 1.2 to 45.3 ± 1.5 mV; P < 0.01; 14 cells; n = 8). Also, atropine (1 µM) consistently abolished EJPs in both the LM and CM. No evidence of noncholinergic excitation was observed. Interestingly, at the oral recording electrode, in addition to abolishing the EJP, small IJPs were recorded in three of eight animals. An example of this ascending inhibition is shown in Fig. 7C. In these three animals, it was possible to record IJPs that occurred synchronously in both the LM and CM at both the oral and anal recording electrodes (see Fig. 7C).


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Fig. 7.   Effects of atropine on stretch-activated ascending excitatory and descending inhibitory pathways. Simultaneous recordings were made from 2 LM and 2 CM cells, as shown schematically in Fig. 3A. A: simultaneous impalements into a single LM and CM cell at the oral region of colon at the same time as an LM and CM cell at the anal region. Note that most EJPs discharge synchronously to the LM and CM orally at the same time as IJPs to the LM and CM located 20 mm anally. Addition of atropine (1 µM; shaded bar) caused a hyperpolarization of all 4 cells and increased the amplitude of IJPs in both muscle layers anally while abolishing EJPs orally. Note that in the presence of atropine, small IJPs were observed at the oral end once the oral EJPs were abolished. This is highlighted by the expanded trace represented by the filled bars marked b (control) and c (in atropine) in A. The periods represented by b and c are shown on expanded time scale in B and C. B: the control period highlighted by the bar b. C: the changes that occur in atropine and the recording highlighted by c in A.

Repetitive activation of evoked EJPs and IJPs. In light of the observation that stretch-activated EJPs and IJPs occurred with short intervals between their onset, we tested whether EJPs and IJPs could be evoked repetitively without showing rundown or fatigue. In six animals, ganglionic stimuli were applied to the myenteric plexus using a fine artists paint brush on the region where the mucosa and submucous plexus had been dissected away, as in Fig. 8A. Simultaneous recordings were then made from two CM cells at the oral and anal regions, and ganglionic compression stimuli were applied to the colon as rapidly as could be applied (Fig. 8). In all animals, EJPs were evoked in the CM orally (Fig. 8B) and IJPs anally (Fig. 8B), and we did not observe accommodation or rundown of these pathways, at least over the time with which impalements could be maintained during stimulation. These evoked polarized reflexes could also be evoked when compression stimuli were applied to myenteric ganglia devoid of CM.


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Fig. 8.   Effects of repetitive compression stimuli applied to local myenteric ganglia and CM. In preparations devoid of mucosa and submucous plexus, simultaneous recordings were made from 2 CM cells at the oral and anal ends of colon, as shown schematically in A. A fine artist's paint brush was used to press on local myenteric ganglia located between the oral and anal recording electrodes. B: simultaneous recording from 2 CM cells made from the recording configuration shown in A. B, top shows that EJPs are evoked repetitively at the oral recording electrode following repetitive ganglionic stimulation applied every 1 s. At the same time, IJPs are evoked at the anal recording electrode. Note, that the evoked EJPs and IJPs do not show rundown or fatigue following repeated stimulation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In preparations of distal colon where maintained circumferential stretch was applied, ongoing discharges of oral EJPs and anal IJPs were found to occur synchronously in both the LM and CM layers. These results show that excitatory motor neurons to both the LM and CM must be simultaneously activated orally at the same time as inhibitory motor neurons to both the LM and CM anally. These stretch-activated neural pathways were abolished by hexamethonium, but are unaffected by NK3 receptor antagonists (40a). This suggests that fast nicotinic rather than slow NK3-mediated synaptic transmission between enteric neurons is essential for the generation of this motor pattern. From studies in the guinea pig small (5, 7, 8, 13, 31) and large intestine (28), it is clear that there is no known morphological connection between motor neurons to the LM or CM. This is particularly noteworthy because we found that EJPs or IJPs occurred synchronously in the two different muscle layers. Therefore, the only way in which the separate populations of excitatory and inhibitory motor neurons can be activated simultaneously to both the LM and CM is by common inputs from ascending and descending interneurons.

Linear relationship between the amplitudes of IJPs and EJPs in LM and CM. The amplitudes of synchronized oral EJPs and anal IJPs were linearly related in the two different muscle layers. That is, when a large EJP occurred in the CM orally, a large EJP also consistently occurred in the LM (Fig. 4C). Similarly, at the anal end of the colon, when a large IJP occurred in the CM, a large IJP also occurred in the LM (Fig. 4B). This means that under stretch, inhibitory motor neurons to the LM were synaptically activated at the same time as inhibitory motor neurons to the CM. Furthermore, we suggest that the amplitudes of synchronized junction potentials in both muscle layers is directly related to the number of interneurons that had been synaptically recruited. Our results also suggest that when a larger amplitude oral EJP or anal IJP occurs synchronously in both muscle layers, it is either due to 1) recruitment of more interneurons, which activates a larger population of motor neurons to cause a larger amplitude event, or, alternatively 2) a similar number of interneurons had been recruited, but their neural activity is more coordinated. This means that the motor neurons to the two different muscle layers would be activated in a more coordinated fashion.

Pharmacological differences between IJPs and EJPs in LM and CM cells. In CM cells, stretch-activated IJPs had a pronounced fast and "slow" component, consistent with previous studies in this preparation (32, 41, 47). The fast component was abolished by apamin and markely reduced by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, whereas, the slow component was dependent on NO, because it was abolished by L-NA. In contrast, in LM cells, only a slow component was consistently observed during the IJP, suggesting that NO is the predominant transmitter underlying the IJP in this muscle layer. Similar results were obtained when distension-evoked IJPs were compared in the LM and CM layers (41). The presence of a fast- and slow-component IJP has been described in the CM of guinea pig small intestines (10, 11, 17), although in contrast to the large intestine, IJPs are rarely recorded from the LM layer in the ileum (10).

In the present study, the addition of atropine consistently abolished EJPs in both the LM and CM and in three of eight animals unmasked IJPs in both muscle layers at the oral end of colon, as shown previously (41). Our laboratory has previously reported (32, 41) the presence of ascending inhibition in the guinea pig distal colon. Also, atropine always caused a large membrane hyperpolarization, suggesting that both muscle layers in these stretched preparations are under a large degree of ongoing cholinergic tone. Similar findings have been reported during mechanical recordings from the same preparation (36, 37). It is interesting to note that IJPs were rare in LM cells when the CM had been completely removed (40), suggesting, perhaps, that compression by both muscle layers is required to activate the stretch-sensitive neurons underlying repetitively firing oral EJPs and anal IJPs.

Characteristics of stretch-activated EJPs and IJPs in longitudinal and circular muscle. Stretch-activated junction potentials had different characteristics between LM and CM cells. Although discharging synchronously in both muscle layers, the amplitudes and rates of rise of IJPs in CM were always considerably larger than those in LM. This is most probably due to the fast apamin-sensitive IJP, which preferentially occurs in CM cells. However, the rate of rise and amplitude of EJPs were also consistently less in LM compared with CM. A possible explanation for the differences in rate of rise and amplitudes of EJPs and IJPs in the LM and CM is that in the distal colon, there is a larger population of CM motor neurons compared with LM motor neurons, as there appears to be in the guinea pig proximal colon (28). Presumably, this would result in a lower density of motor innervation of LM cells compared with CM cells. If the amount of transmitter release at LM cells is less than that at CM cells, then the amplitude and rate of rise of the junction potentials in LM would also be comparatively less than those in CM (see Ref. 4). We have recently shown (40) that the CM and LM layers of the guinea pig distal colon contain intramuscular interstitial cells of Cajal (ICC-IM). These ICC-IM have been shown to be transducers of neuromuscular transmission in gastric smooth muscle (46). In the distal colon, there may also be differences between ICC-IM in LM and CM cells and this could account for the differences in the characteristics of EJPs and IJPs between the two muscle layers. Alternatively, it is possible that fewer LM motor neurons are synaptically activated than CM motor neurons during this ongoing polarized motor pattern. In addition, the differences in amplitudes and rate of rise between the junction potentials may represent differences in the cable properties of the two muscles. Although, both electrical measurements (39, 41) and calcium imaging (45) suggest that action potentials and their underlying calcium waves exhibit similar conduction velocities in both muscles. However, despite these differences between the LM and CM, during peristalsis or reflex stimulation, the LM is still able to contract and relax as forcefully as the CM (33, 36, 37-39). In light of the fact that L-NA abolished IJPs in LM cells, it is unlikely that junction potentials in LM result from electrotonic spread of IJPs generated in the CM. This is because in the presence of L-NA, large-amplitude, fast IJPs in CM did not show any corresponding IJP in the LM. Furthermore, the dissection technique excluded this possibility, because the CM layer was removed from the site where recordings were made in the LM.

Mechanosensory neurons in the distal colon. We have recently described a complex migrating motor pattern in the guinea pig distal colon that is sensitive to muscle tone (34, 35). In this study, however, we report the presence of another stretch-activated motor pattern that consists of repetitively firing oral EJPs and anal IJPs. This ongoing motor pattern is resistant to nifedipine. This shows that a population of mechanosensory neurons in the distal colon are stretch sensitive rather than muscle tone or contraction sensitive (40a). Our findings in the current study are vastly different from what is known about mechanosensory neurons in the guinea pig small intestine (23, 24). In this latter preparation, myenteric AH neurons, which have been shown to be mechanosensory, are inactivated by antagonists that cause muscle paralysis, such as nifedipine or isoprenaline. In our studies, we have shown that circumferential stretch activates a population of mechanosensory neurons in the presence of nifedipine (40a). Consistent with this hypothesis, we have shown that after-hyperpolarizing (AH) neurons in the distal colon are quiescent during this ongoing motor activity in stretched but paralyzed segments of distal colon (42). A possible candidate underlying this stretch-sensitive but tone-independent motor activity may be ascending interneurons (see discussion in Ref. 40a). Therefore, the intestine appears to have different sensory modalities similar to the somatic nervous system (SNS) in that it can detect both muscle tone and contraction (similar to Golgi tendon organs in SNS) and muscle stretch (similar to muscle spindles in SNS).

Synchronous activation of ascending and descending interneurons. A major observation of the current study was that large oral EJPs and anal IJPs were consistently recorded in both the LM and CM cells, when recordings were made within 100 µm of either the oral or anal cut end of colon. At the oral end of the short, stretched segment of the distal colon (see Fig. 1), synaptic outputs of ascending interneurons are preferentially preserved, while at this same site, the outputs of descending interneurons had been removed as a result of sectioning the colon. Conversely, at the anal end of colon, synaptic outputs of descending interneurons had been selectively preserved, whereas the outputs of ascending interneurons had been removed. The fact that EJPs orally discharged at the same time as IJPs anally strongly suggests that a large population of ascending and descending interneurons are likely to fire synchronously. Ascending interneurons would synaptically activate excitatory motor neurons to both the LM and CM orally, at the same time many descending interneurons activate many inhibitory motor neurons to the LM and CM. However, this motor activity still occurs when the ends of the unstretched segment are preserved (see Fig. 3). Therefore, these polarized reflex pathways are activated by stretch, rather than being an artifact of a short preparation, where influences from descending and ascending nerve pathways have been removed at the oral and anal ends, respectively.

Synchronous neuromuscular inputs to the LM and CM. Our findings show that under maintained circumferential stretch, the enteric nervous system is "hard wired" to cause synchronized excitation of both muscle layers orally at the same time as synchronized inhibition of both muscle layers anally. Reciprocal neuromuscular inputs were never recorded. Our findings in the current study add further support to our previous electrophysiological studies (41), in which we showed that transient distension or mucosal stimuli of the same preparation evoked IJPs synchronously in both the LM and CM during descending inhibition and EJPs synchronously in the LM and CM during ascending excitation.

In conclusion, the local stretch-activated intrinsic motor program described here, which is independent of muscle tone, is likely to contribute to fecal pellet propulsion along the distal colon. This motor pattern involves the coordinated activation of common ascending and descending interneurons that synapse with separate populations of excitatory and inhibitory motor neurons, respectively, to both the LM and CM. The coordination of excitation (EJP + action potential) above with inhibition (IJP) below of both muscles would ensure that each firing of these synchronized neural reflex pathways would tend to displace the bolus anally. The shortening of the LM may be expected to pull the muscle bundles of the contracting CM together (28a). This combined thickening of both muscles would increase the contracting muscle mass and, thereby, net force behind the bolus (28a, 50). Also, the simultaneous oral contraction of both muscles would insure that the LM just oral to the pellet would contribute to propulsion by providing a force vector directed down the bowel (36). At the same time, the inhibition of both muscles would facilitate accommodation below the bolus.

In contrast to propulsion, storage of fecal pellets in the distal colon is promoted by activation of extrinsic sympathetic reflexes that inhibit transmitter (ACh) release from common interneurons driving both muscles and by directly relaxing both the LM and CM (37).


    ACKNOWLEDGEMENTS

This study was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-45713) awarded to T. K. Smith. G. W. Henning was supported by a C. J. Martin Fellowship (Australia).


    FOOTNOTES

Address for reprint requests and other correspondence: T. K. Smith, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: tks{at}physio.unr.edu).

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

First published October 16, 2002;10.1152/ajpgi.00291.2002

Received 19 July 2002; accepted in final form 3 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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