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NEUROSCIENCE |
1 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Abstract |
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40% of the thickness of CM layer from the entire preparation did not significantly disrupt, nor reduce the degree of correlation between oral EJPs and anal IJPs, suggesting that critical sensory elements did not lie adjacent to the submucosal plexus. It is concluded that mechanosensory transmission that underlies repetitive firing of ascending excitatory and descending inhibitory neuronal pathways is critically dependent upon sensory elements within the CM layer. These elements are likely to activate stretch-sensitive interneurons in the myenteric plexus. No evidence was found to suggest that the connectivity between the LM and the myenteric plexus was required for mechanotransduction.
(Received 13 March 2006;
accepted after revision 3 August 2006;
first published online 3 August 2006)
Corresponding author T. K. Smith: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. Email: tks{at}unr.edu
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Introduction |
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Reflex responses activated by stretch or mucosal stimulation appear to be mediated by two different intrinsic sensory neurons that converge upon common interneurons and motor neurons (Smith et al. 1991, 1992a; Furness et al. 1995; Spencer & Smith, 2004). Myenteric after-hyperpolarizing (AH) neurons project into the intestinal villi (Furness et al. 1990; Neunlist et al. 1999; reviewed in Kunze & Furness, 1999 and Sanders & Smith, 2003) and are directly activated by chemicals applied to the mucosa (Smith, 1994, 1996; Kunze et al. 1995). AH neurons are therefore likely to be the sensory neurons mediating mucosal reflexes. The enteric sensory neurons mediating stretch reflexes, on the other hand, are more controversial and appear to be mediated by both AH neurons (Kunze et al. 1998) and mechanosensitive S (fast synaptic input)-interneurons (Spencer & Smith, 2004; reviewed in Smith et al. 2005).
Myenteric AH neurons, in addition to responding to chemical stimulation of the mucosa, also respond to both stretch and contraction with a discharge of action potentials (Kunze et al. 1998; Kunze & Furness, 1999). The response of AH neurons to stretch, like their response to contraction, results from an increase in active muscle tension (tone), since their activity is abolished by drugs that abolish muscle tension (isoproterenol or nicardipine) despite maintained stretch (Kunze et al. 1998). Thus the stretch-activated firing in AH neurons is generated by the muscle's resistance to stretch, rather than by passive lengthening of the muscle fibres. In both the small and large intestine of the guinea-pig, anally propagating peristaltic waves activated by balloon distension are blocked by drugs that reduce muscle tension when applied around the site of stimulation, suggesting that AH neurons are likely to be important in the initiation of these waves (Spencer et al. 2001a; Smith et al. 2003).
In addition to the low-frequency ongoing peristaltic waves (see Smith et al. 2003), we have recently shown that maintained stretch applied to the distal colon also activates another motor pattern that is independent of muscle tone (Spencer et al. 2002a, 2003). This more rapid motor pattern activated by circumferential stretch consists of a repetitive firing of peristaltic reflex pathways, i.e. an ongoing discharge of oral excitatory junction potentials (EJPs) that are temporally synchronized with an ongoing discharge of anal inhibitory junction potentials (IJPs). This ongoing discharge of oral EJPs and anal IJPs, which occurs at the same time in both the longitudinal muscle (LM) and circular muscle (CM), is unaffected by muscle paralysis with nifedipine and by removal of the mucosa and submucous plexus (Spencer et al. 2002a, 2003). When the oral EJPs reach threshold to elicit action potentials they elicit a vigorous contraction of the muscle that is likely to also contribute to moving a pellet anally (Spencer et al. 2002a; Smith et al. 2003). AH neurons were found to be electrically silent in these stretched colonic preparations, which is perhaps not surprising since both smooth muscle layers lacked tone because most of these experiments were carried out in the presence of nifedipine (Spencer & Smith, 2004). This activity appears to be mediated by a population of myenteric, stretch-sensitive mechano-sensory ascending and descending S-interneurons that exhibit an ongoing discharge of action potentials and proximal process potentials that is unaffected by synaptic blockade or muscle paralysis (Spencer & Smith, 2004). Surprisingly some of the fine dendrites of these filamentous mechano-sensitive interneurons projected down through the ganglia and ran within and parallel to the CM fibres (Spencer & Smith, 2004). By analogy with stretch-sensitive muscle spindles in skeletal muscle, this, in parallel arrangement with the CM, may be ideal for transducing muscle stretch rather than muscle tension (see Smith et al. 2005).
Given the dendritic projections of these sensory interneurons, we were therefore particularly interested in this study in determining whether either the LM or CM was necessary for mechanically transducing the stretch-sensitive input required for maintaining the ongoing discharge of reflex pathways in the guinea-pig distal colon, that were independent of muscle tone.
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Methods |
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Guinea-pigs weighing 200–350 g were killed 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 was removed, flushed clean with modified Krebs solution (25°C: see composition below), and placed immediately into a Petri dish containing Krebs solution.
Dissection procedure
The preparation was incised along the mesenteric border and pinned flat with the mucosa uppermost in a Sylgard-lined Petri dish. Using sharp dissection, the mucosa and submucosa were peeled off to expose the underlying circular muscle layer.
Longitudinal muscle myenteric plexus preparations (LMMP).
In these preparations the CM was removed by peeling off strips of CM from the myenteric plexus, using standard sharp dissection. At both the oral and anal cut ends of the preparation, full-width strips of CM (<1 mm wide) were left intact to allow simultaneous microelectrode recordings into two CM cells situated at either end of the tissue. In other studies, simultaneous recordings were made from a CM cell in the strip and an adjacent LM cell situated 100 µm apart at the same end of the LMMP preparation (see Spencer & Smith, 2001).
Circular muscle myenteric plexus preparations (CMMP). CMMP preparations were prepared by removing the LM. The LM was removed by teasing up muscle fibres at the oral end, pinching these fibres with a pair of curved forceps and peeling the LM off the preparation, in an oral to anal direction. This easily removed the LM, which often came off as wide strips of muscle, without damaging the myenteric plexus that remained intact upon the CM.
After dissection, both the LMMP and the CMMP preparations were cut to 20 mm in length and transferred to and pinned serosa side down to the base of a recording chamber (8 ml capacity). The base of the recording chamber, which was mounted on the stage of an inverted microscope (Olympus, CK2; Napa, CA, USA), consisted of a microscope coverslip that was laminated with a fine layer (2–3 mm deep) of Sylgard (Dow Corning Corp. Midland, MI, USA). The preparations were stretched circumferentially, the distance between either circumferential edge was 11–14 mm, which represents approximately twice the circumference of the unstretched colon (6 mm). In both CMMP and LMMP preparations, the entire preparation was stretched uniformly in the circumferential axis. with no longitudinal stretch imposed.
Simultaneous intracellular recordings from pairs of circular muscle cells
In both types of preparations (i.e. LMMP or CMMP), simultaneous intracellular recordings were routinely made from the CM at either end of the colon, using two independently mounted micromanipulators (model M3301R; WPI Inc., Sarasota, FL, USA; see Spencer et al. 2002a). In other studies, simultaneous microelectrode recordings were made from both the CM strip and the adjacent LM at either the oral or anal end of the LMMP preparation (see Spencer et al. 2003). Microelectrodes (i.d. 0.5 mm) were filled with 1.5 M KCl solution and had tip resistances of about 100 M
. Electrical signals were amplified using a dual input Axoprobe 1A amplifier, and digitized at between 660 Hz and 1.5 kHz on a PC using Axoscope software (version 8.0; Axon Instruments, Foster City, CA, USA).
To ensure that the myenteric plexus was undamaged in these preparations, transmural stimulating electrodes were mounted in the middle of the preparation. If the myenteric plexus was undamaged, then single-shot stimuli 20–40 V, duration of 0.5 ms applied with a Grass stimulator (S44) evoked an oral EJP and an anal IJP in both muscles (see Spencer et al. 2003).
NADPH-diaphorase histochemistry
As a further test for the integrity of the myenteric plexus in preparations from which the LM or CM had been removed, histochemical staining for NADPH diaphorase was routinely performed following each experiment. Whole-mount colonic preparations were fixed for 4 h in a 4% paraformaldehyde solution, then washed four times (30 min each wash) using a phosphate-buffered saline solution. Then reduced NADP (NADPH)-diaphorase staining was applied, following the same protocol as that used by Song et al. (1993).
Drugs and solutions
Preparations were perfused with Krebs solution at 35–36°C. Also, nifedipine (1–2 µM) was present in the Krebs perfusion solution in order to facilitate intracellular recording from the CM or LM, since it reduces spontaneous and evoked contractions by abolishing muscle action potentials (Spencer et al. 2002b).
The composition of the modified Krebs solution was (mM): NaCl, 120.35; KCl, 5.9; NaHCO3, 15.5; NaH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; and glucose, 11.5. Nifedipine and tetrodotoxin (TTX) were obtained from Sigma Chemical Co. (St Louis, MO, USA).
Measurements and statistics
Student's paired t tests were used where appropriate. A minimum significance level of P < 0.05 was used throughout. The use of n in the Results section refers to the number of animals on which observations were made, and data are presented as means ± S.E.M. Measurements of amplitude and half width, and time to peak response were made using Axoscope 8.0 (Axon Instruments, Foster City, CA, USA).
Analysis of data
The methods used to analyse and compare electrical recordings have been previously described (Spencer et al. 2001b). Briefly, voltage data were exported as a text file and imported into a custom-written program (OpenGL-based) in which the traces were resampled to 250–300 Hz and smoothed (36 ms moving average; 5 iterations). An average baseline (5–10 s) was calculated to follow slow undulations in voltage while ignoring faster events (e.g. junction potentials). Inflexions in the voltage traces were detected, and the peaks were subtracted from the average baseline to calculate junction potential amplitudes. Once peaks had been located in both traces, the peak in the second trace which was closest in time to a reference peak in the first trace was identified. Plots were then constructed of the changes in junction potential amplitudes versus the time difference between the closest oral EJP and anal IJP peaks that occurred at opposite ends of the tissue. To determine the degree of temporal correlation between synchronized oral EJPs and anal IJPs activated by stretch, plots of correlation coefficient (R) were calculated. These plots show how the degree of temporal correlation (synchronization) between oral EJPs and anal IJPs changes when an imposed time shift is applied only to the oral recording of up to 5 s positive or negative of real time. We refer to ‘real time’ as the time at which both intracellular recordings were made simultaneously from the oral and anal regions of CM, without imposing any time shifting of oral or anal recordings. At the anal recording electrode, many depolarizing events were detected. These events could represent small EJPs or myogenic ‘rebound’ depolarizations that immediately follow some IJPs. Rebound depolarizations can be identified over EJPs by their resistance to atropine, and were not temporally synchronized with any oral EJPs at the oral recording electrode.
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Results |
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We have previously reported that in the guinea-pig distal colon, if circumferential stretch is applied to the intact colon, or circumferential stretch is applied to sheet preparations of colon with both the LM and CM intact, an ongoing discharge of coordinated oral EJPs and anal IJPs occurs synchronously in both the LM and CM (Spencer et al. 2002a, 2003). This ongoing reflex activity is activated by stretch alone; and is independent of muscle tone, since it occurs even after the muscle has been paralysed with nifedipine (see Fig. 1A). In the experiments described in this study, we applied maintained circumferential stretch to both intact preparations, CMMP preparations and LMMP preparations, to the same degree. This varied from a slack diameter of 6 mm, to a diameter of 11–14 mm under stretch, which represents an increase in circumferential slack diameter of between 85% and 130%. In these preparations, like those described below, the mucosa and submucous plexus were removed and the preparation pinned serosal surface down, so as to facilitate simultaneous microelectrode impalements into the underlying CM at both ends of the preparation. The fact that this ongoing motor pattern occurred following removal of the mucosa and submucous plexus implies that the sensory elements underlying this activity must be in either the myenteric plexus, the LM or the CM.
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In order to investigate the possible role of the LM in transducing this ongoing peristaltic reflex activity, we removed the LM, without affecting the myenteric plexus, which normally adheres to the CM in the colon (Furukawa et al. 1986; Spencer & Smith, 2004). When circumferential stretch was applied to these sheet preparations of colon (from which the LM had been removed), interestingly, an ongoing discharge of synchronized oral EJPs and anal IJPs still occurred in the CM layer, whose characteristics were indistinguishable from those when both the muscle layers remained intact with the myenteric plexus (Spencer et al. 2002a, 2003). In total, 34 pairs of simultaneous recordings were made from two CM cells (from n = 15 animals), at the oral and anal ends of the stretched tissue, that was devoid of LM. Figure 1B shows the recording configuration and a typical example of this ongoing reflex activity. Oral EJPs and anal IJPs greater than 5 mV in amplitude were considered to occur synchronously if their peaks occurred within 80 ms of each other (Fig. 2A and B). When the peaks of oral EJPs were cross-correlated with the peaks of IJPs recorded from the anal end of the colon, it was found that there was a high degree of temporal correlation, where R2 values reached 0.26 (n = 15) (Fig. 2B). When the two electrical traces occurring at either end of the preparation were phase shifted up to +5 or –5 s from t = 0, it was found the R2 values decreased rapidly and became uncoordinated when traces were shifted by >±1 s from t = 0 (Fig. 2B). The distribution of the amplitudes of oral EJPs and anal IJPs that occur in these CMMP preparations is shown in Fig. 3A and B. The most frequently occurring EJPs in the CM were between 0 and 2 mV in amplitude, and these had mean interval of 3.0 ± 0.5 s (n = 15). In general, it was found that the larger the amplitude of synchronized oral EJPs or anal IJPs, the less frequently occurred (Fig. 3). The largest-amplitude oral EJPs reached 22 mV, where the mean interval between successive synchronized oral EJPs and anal IJPs in the range of 20–22 mV was 46.4 ± 36.5 s (n = 15) (Fig. 3A). At the oral and anal electrodes, a clear polarity in the nature of junction potentials was identified. That is, although both IJPs and EJPs could be recorded at both the oral and anal recording sites in CMMP preparations, the largest-amplitude and most frequently occurring events were always EJPs at the oral electrode and IJPs at the anal electrode (see distribution graph in Fig. 3A and B).
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Recording from LMMP preparations
To test the role of neural elements in the CM as a site for mechanotransduction of ongoing stretch-activated junction potentials, we now left the LM intact, but sharp dissected off as much CM as possible from the entire preparation, except for two full-width strips of CM (<1 mm wide) at either end of the preparation (see Fig. 1C). These two small strips of CM allowed us to make simultaneous recordings from two CM cells, while the majority of the CM was removed. In these classic LMMP-type preparations, surprisingly, no coordinated oral EJPs or anal IJPs were ever observed (Fig. 1C). In these preparations, an irregular baseline activity <5 mV in amplitude was recorded (Figs 1C and 2C and D). The activities recorded by the two electrodes at either end of the tissue were temporally uncoordinated (see Fig. 2C and D). A frequency distribution of the amplitudes of residual junction potentials recorded from these LMMP preparations (from n = 10 animals) is shown in Fig. 3C and D. In contrast to the CMMP, cross-correlation of the small uncoordinated junction potentials in the CM cells revealed a poor correlation coefficient of <0.01 (see Fig. 2D), and furthermore, phase shifting the two traces by up to +5 s or –5 s from t = 0, failed to modify the poorly correlated events.
This lack of ongoing coordinated reflex activity was not due to damage of the myenteric plexus during dissection, since when intracellular recordings were made simultaneously from adjacent cells in both the LM and CM, transmural stimulation applied to the centre of the preparation consistently evoked EJPs that occurred at the same time in both the LM and CM at the oral end of the tissue, and IJPs that occurred at the same time in both muscles at the anal end of the tissue (Fig. 4).
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Since nifedipine does not completely block all smooth muscle contraction, it might be argued that some residual level of muscle tension still exists in the musculature of the guinea-pig colon that could possibly activate tension-sensitive, rather than purely stretch-sensitive myenteric neurons. Therefore, to test this, we applied the smooth muscle relaxant papavarine (10 µM) to the colon after stretch-activated junction potentials had been activated in the presence of nifedipine. In tissue samples from three animals, we found no detectable, nor significant effect of papavarine on the degree of correlation between synchronized oral EJPs and anal IJPs in the CM layer (nifedipine: R2 = 0.4 ± 0.06; papavarine: R2 = 0.37 ± 0.05; n = 3; P > 0.05) (see Fig. 5B and C). Interestingly, the RMP of CM cells did not significantly change from the presence of nifedipine, to a solution containing nifedipine and papavarine (10 µM) (control: 48.1 ± 0.96 mV; papavarine: 46.2 ± 0.6 mV; n = 3; P = 0.17). Under these conditions in the combined presence of nifedpine and papavarine, it is reasonable to assume there is negligible active muscle tension in the musculature, yet stretch-activated junction potentials persisted.
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Effects of removing increasing length segments of circular muscle from the myenteric plexus
Since removal of the majority of the CM from the myenteric plexus abolished stretch-activated junction potentials in both the LM and CM, it was of particular interest to determine whether continuity of the CM along the entire length of the preparation was important for sustained activation of the underlying mechanosensory neural elements. To test this, we first sharp dissected a 7-mm-wide full-thickness strip of CM from the middle region of the 20-mm-long preparation of colon (see Fig. 6C). Then in the same preparation, we increased the width of the dissected strip of CM to 14 mm (Fig. 6E). In these preparations, under these conditions, a significant and graded decrease in the correlation coefficient was detected (Fig. 7E). In preparations with 7 mm of width of CM removed from the myenteric plexus, synchronized oral EJPs and anal IJPs were still recorded, but the degree of correlation was significantly smaller than in the same preparations when all the CM remained intact with the plexus (n = 4; Fig. 6 and Fig. 7E). When a 14-mm-wide segment of CM was sharp dissected from the myenteric plexus, no synchronized oral EJPs and anal IJPs were ever recorded, and R2 values approached zero (i.e. no coordination in either amplitude or temporal onset) (Figs 6F and 7E).
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In three animals, we investigated whether the mechanosensory neural elements lay close to the submucous plexus. To test this we sharp dissected off approximately 40% of the thickness of the CM layer from the entire 20-mm-long preparation (see Fig. 7D). In these preparations, we found no detectable, or significant difference in the R2 value (degree of correlation between oral EJPs and anal IJPs) in the remaining CM layer (Fig. 7C–E; n = 3). When these preparations were fixed and visualized under bright field microscope it was found that the thickness of the CM was 52.5 ± 2.5 µm once sharp dissected, and was 82.5 ± 2.5 µm in undissected preparations CM (n = 3). This means that removal of approximately 37% of the depth of the CM failed to affect mechanotransduction or neuromuscular output to the CM.
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Discussion |
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The major finding of the current study is that removal of the CM layer from the myenteric plexus abolishes the stretch-induced ongoing discharge of ascending excitatory and descending inhibitory neuronal pathways to both the LM and CM layers. The loss of this neural activity was not due to damage of the myenteric plexus, since NADPH diaphorase staining after each experiment revealed that the myenteric plexus remained intact. Furthermore, transmural electrical nerve stimuli consistently evoked robust oral EJPs and anal IJPs in these preparations, where their amplitudes could be graded in amplitude according to stimulus intensity. In direct contrast, in preparations devoid of most of the CM, junction potentials were usually <5 mV, and no synchronized oral EJPs or anal IJPs ever occurred. Taken together, these observations suggest that mechanosensory transduction underlying stretch-induced firing of ascending excitatory and descending inhibitory pathways is critically dependent upon connectivity between the myenteric plexus and the CM layer. Importantly, removal of approximately 37% of the depth of the CM failed to affect the ongoing EJPs and IJPs, suggesting that the sensory elements transducing this ongoing peristaltic reflex activity lie within the CM close to the myenteric plexus.
In contrast, removal of the LM from the myenteric plexus did not affect stretch-activated ascending excitatory and descending inhibitory neuronal pathways. These preparations that were devoid of LM exhibited ongoing oral EJPs and anal IJPs that occurred at a similar frequency and exhibited the same degree of temporal coordination as observed in preparations in which both muscle layers were intact (see Spencer et al. 2002a, 2003).
We tested whether continuity of the CM layer along the length of the preparation was a critical element underlying the mechanosensory activation of repetitive oral EJPs and anal IJPs. Interestingly, it was found that stretch-activated junction potentials were not abolished when a 7-mm-wide strip of CM was removed from the myenteric plexus (Fig. 7D and E), although there was a significant reduction in the degree of correlation between oral EJPs and anal IJPs (Fig. 7E). When 14 mm of CM was sharp dissected off the myenteric plexus from either the oral or anal cut ends, a further graded significant decrease in the correlation of junction potentials occurred, such that synchronized oral EJPs and anal IJPs were no longer recorded (Fig. 6F). These observations suggest that although continuity of the CM layer along the length of colon is not critical for the activation of synchronized oral EJPs and anal IJPs, it is clear that a graded increase in the removal of CM from the myenteric plexus results in a graded decrease in the level of synchronized oral EJPs and anal IJPs.
Spontaneous neuronal activity is weak in classic LMMP preparations of colon
Both Wade & Wood (1988a,b) and Lomax et al. (1999) studied the electrical characteristics of myenteric neurons in the guinea-pig distal colon, using the classic longitudinal muscle, myenteric plexus (LMMP) preparation, i.e. where the CM was removed from the myenteric plexus, but the LM layer was preserved. Despite the fact that the LM in these preparations was presumably stretched both longitudinally and circumferentially, both groups reported a relative lack of spontaneous activity in myenteric neurons. Lomax et al. (1999) found that only 3 out of 145 myenteric S-neurons showed ongoing spontaneous fast EPSPs. In contrast, when we recorded from the same length preparations, but preserved the CM intact with the myenteric plexus, essentially all S-neurons exhibited ongoing spontaneous fast EPSPs and action potentials (Spencer & Smith, 2004). The only difference between the two studies was that we preserved much of the CM intact with the myenteric plexus. These findings absolutely support our conclusions in this study, in that stretch-activated neuronal activities that underlie ascending excitatory and descending inhibitory neuronal pathways are critically dependent upon the CM, but not the LM. Since the only difference between the studies of Lomax et al. (1999) and our study was the presence of the CM in our preparations, this led us to believe that the CM is critical for mechanosensory transmission. In preparations with the CM removed from the myenteric plexus where there was an absence of any stretch-activated junction potentials in either muscle layer, our observation that in these same preparations transmural stimulation evoked robust oral EJPs and anal IJPs in both muscle layers showed that interneuronal communication along the myenteric plexus was intact.
Previous studies strongly suggest that there are at least two distinct classes of intrinsic sensory neuron in the intestine (Smith et al. 1991, 1992a; Spencer & Smith, 2004): AH neurons (Holman et al. 1972; Hirst et al. 1974, 1975; Bornstein et al. 1991; Smith et al. 1992a; Kunze et al. 1998, 1999, 2000) and myenteric S-interneurons (Spencer & Smith, 2004).
The location of the mechanosensory transduction sites that generate stretch-activated oral EJPs and anal IJPs in the colon was previously unclear. In the canine jejunum it was suggested that the stretch receptors mediating the inhibitory reflex were in the LM, because removal of this layer, but not the CM, abolished this reflex activity (Hukuhara et al. 1960). However, others have suggested that in guinea-pig colon ‘... the longitudinal muscle seems not essential for propulsion’ (Crema et al. 1970). Interestingly, Ohkawa & Prosser (1972) reported that extracellularly recorded spike discharges in myenteric neurons could be evoked by pressing on ganglia with a fine glass probe, whereas pressing on the connective tissues or the neighbouring muscle was ineffective. Importantly, probing around a ganglion initiates generator potentials or action potentials in AH neurons, suggesting that some processes of AH neurons within the myenteric plexus are mechanosensitive (Kunze et al. 2000). These ‘receptive sites’ were located in the ganglion from which recordings were made, and ‘. . . did not extend over the interganglionic connectives or adjacent longitudinal muscle’ (Kunze et al. 2000). Presumably the tension-sensitive processes of AH neurons should be in series with the muscle, as tension-sensitive Golgi tendon organs are associated with skeletal muscle. To date, the evidence suggests that the mechanosensory endings of AH neurons, at least in the small intestine, have no specialized ending in either the LM or CM, but probably lie within the myenteric plexus.
In contrast, our data are strongly consistent with the idea that the dendrites of filamentous stretch-sensitive ascending and descending interneurons that project through myenteric ganglia and run within and parallel to the CM fibres are indeed mechanosensory. We speculate that when circumferential stretch is applied, the mechanosensitive dendrites of these interneurons become activated, which initiates the ongoing coordinated reflex activity, providing stretch is maintained (Fig. 8). Therefore, these mechanosensory S-interneurons are critical for transducing stretch-sensitive, but muscle tone-independent ongoing peristaltic reflex activity (Spencer & Smith, 2004). When we recorded from interneurons in preparations where the CM was intact but the LM removed, they exhibited spontaneous action potentials, proximal process potentials that were insensitive to synaptic blockade with low-Ca2+ solution, and spontaneous fast excitatory post synaptic potentials (FEPSPs) that were blocked by low-Ca2+ solution (Spencer et al. 2004). In contrast, in preparations where the CM was removed but the LM was intact, interneurons were electrically quiescent until current was injected into their soma, or FEPSPs were evoked by focal electrical stimulation of fibre tracts (Lomax et al. 1999). The difference in these studies can be attributed to the fact that spontaneous action potentials in the soma of interneurons are probably initiated by proximal process potentials, which result from action potentials in the stretch-sensitive sensory processes of these cells that project to the underlying CM (see Spencer et al. 2004). Presumably, the excited interneurons in our preparations synapse with each other, giving rise to FEPSPs in other interneurons (see Fig. 8; Smith et al. 2005; Spencer et al. 2005). Removal of the CM probably destroys these mechanosensitive dendrites and prevents the activation of mechanosensitive interneurons. It is, at present, unknown whether the dendrites of interneurons within the CM are themselves stretch sensitive or part of a more complex sensory apparatus analogous to muscle spindles in skeletal muscle. Such sensory elements in the CM that lie in parallel with CM could be the intramuscular interstitial cells of Cajal (ICC-IM) (see Smith et al. 2005). In the stomach, ICC-IM run parallel to and within the CM, and are stretch sensitive and also mediate neurotransmission to the CM (see Beckett et al. 2004; Won et al. 2005).
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t
= 0). Any imposed time shift of up to ±5 s did not improve the degree of correlation. C, in LMMP preparations, the amplitudes of EJPs and IJPs in CM at the oral and anal ends was usually small (<5 mV) and uncoordinated. The peaks of small junction potentials at the oral and anal ends of colon showed no temporal correlation, giving rise to noise. D, the correlation coefficient between events in CM at the oral end and those at the anal end of the colonic preparation was zero in LMMP preparations.
), as in LMMP-preparations. At 36°C and in the presence of nifedipine, there was no significant change in the length of the preparation evoked by graded increases in stretch when the CM was removed from the myenteric plexus. B and C, the further addition of the smooth muscle relaxant, papavarine (10 µM) after nifedipine to the colon did not affect stretch-activated firing of oral EJPs and anal IJPs, suggesting that muscle tension is not required in mechanotransduction. Error bars represent S.E.M.
