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The rectum receives specialized extrinsic afferent innervation by stretch-sensitive, low threshold, slowly adapting mechanoreceptors, with transduction sites shown to correspond to rectal intraganglionic laminar endings (IGLEs). Rectal IGLEs are located in myenteric ganglia, surrounded by the longitudinal and circular smooth muscle layers; in this study we investigated the mechanical stimuli to which they respond. Mechanoreceptors had graded responses to highly focal transmural compression with von Frey hairs. They were activated when stretched circumferentially by imposed increases of both length and load. Under both conditions, firing typically occurred in bursts associated with phasic muscle contractions. However, many contractions did not evoke firing. Longitudinal stretch also evoked firing, again associated with contractile activity. Thus, mechanoreceptors did not show directional sensitivity. Two agonists that excited smooth muscle directly (0.1 μm[β-Ala8]-neurokinin A (4–10) and 1 μm carbachol) activated rectal mechanoreceptors, but not in the presence of Ca2+-free solution or when preparations were kept entirely slack. We measured the dimensions, in both longitudinal and circumferential axes, of receptive fields during smooth muscle contractile activity, using video micrography. Contractile activity within the receptive field often differed significantly from the behaviour of the preparation as a whole, providing an explanation for many of the discrepancies between gross contractility and firing. Simultaneous contraction of both muscle layers within the receptive field was the strongest predictor of mechanoreceptor activation. Our results suggest that rectal mechanoreceptors do not act simply as tension receptors: rather they appear to detect mechanical deformation of myenteric ganglia – especially flattening – associated with stretch of the receptive field, or contractions of smooth muscle within the receptive field.
Distension activates a number of motor reflexes within the gastrointestinal tract. Over relatively short distances, these are mediated via the enteric nervous system, which contains intrinsic primary afferent neurones, interneurones and motor neurones arranged in functional circuits (Furness & Costa, 1987; Wood, 1994; Gershon, 1999). However, many distension-evoked reflexes involve neural pathways outside the gut. Thus, gastric accommodation, receptive relaxation, entero-gastric and entero-enteric reflexes all involve sympathetic and parasympathetic efferent pathways to the gut (Szurszewski & King, 1989; Mulak & Bonaz, 2004). These are activated either by enteric neurones that project to prevertebral ganglia (‘viscerofugal neurones’), or by extrinsic afferent neurones projecting to the central nervous system. Rectal distension activates extrinsic excitatory defaecatory reflexes mediated via pelvic nerve pathways (Yamanouchi et al. 2002), and in humans, damage to pelvic pathways plays a key role in many cases of obstructed defaecation (Denny-Brown & Robertson, 1935). Reduced sensitivity to rectal distension is associated with disordered defaecation in children and adults with spinal damage (Greving et al. 1998). Extrinsic afferent neurones also give rise to conscious sensations from the gut, such as feelings of fullness, bloating and pain, and in the rectum, urgency. Vagal afferent mechanoreceptors, with cell bodies in the nodose ganglion, are believed to mediate predominantly non-nociceptive sensations from the gut wall, especially the upper gut. Spinal afferent neurones are responsible for mediating predominantly mechano-nociceptive sensations: detecting noxious levels of distension, and activating central pain pathways (Sengupta & Gebhart, 1994).
Studies in patients with spinal lesions have indicated that although thoraco-lumbar spinal pathways are involved in mechano-nociception, sacral spinal pathways may mediate non-noxious sensations (Lembo et al. 1994). Recent studies have provided a cellular basis for these observations. Lynn et al. (2003) demonstrated the presence of low threshold, slowly adapting mechanoreceptors in the guinea pig rectum, which are very rare – or absent – more proximally in the colon. Dye filling studies have shown that the transduction sites of these low threshold mechanoreceptors correspond to specialized flattened endings within myenteric ganglia in the rectum called rectal IGLEs, or rIGLEs (Lynn et al. 2003; Olsson et al. 2004). These anatomically defined endings are rare or absent more proximally in the colon. Thus, in the rectum, but not the colon, a specialized class of low threshold afferent fibres exists which signals a wide range of distension.
Vagal mechanoreceptors to the upper gut bear many similarities to the specialized rectal mechanoreceptors described above. Both types of afferents have low mechanical thresholds, respond over a wide range of distension, and have a slowly adapting response to maintained stretch. Vagal mechanoreceptors have been shown to transduce mechanical deformation at specialized, flattened, branching structures (IGLEs) located within the myenteric ganglia that are structurally similar to rIGLEs (Zagorodnyuk & Brookes, 2000; Zagorodnyuk et al. 2001; Lynn et al. 2003). Both IGLEs and rIGLEs have been described to act like in-series tension receptors, their firing determined primarily by wall tension rather than length (Iggo, 1955; Blackshaw & Grundy, 1990; Page & Blackshaw, 1998; Zagorodnyuk et al. 2001; Lynn et al. 2003). This is despite the fact that anatomically these endings lie parallel to the muscle whose tension they reflect. In this study, we aimed to determine the adequate mechanical stimulus for specialized rectal mechanoreceptors, in terms of longitudinal and circular smooth muscle length and tension, within identified receptive fields.
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Adult male and female guinea pigs (250–350 g) were killed by a blow to the occipital region and exsanguinated as approved by the Animal Welfare Committee of Flinders University. Specimens of distal bowel with attached rectal nerve trunks were excised, flushed of contents, and opened up into a flat sheet. The preparation was superfused at 3 ml min−1 with Krebs solution (mm; NaCl, 118; KCl, 4.75; NaH2PO4, 1.0; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2.5; glucose, 11), chilled to approximately 10°C, and bubbled with 95% O2–5% CO2. The mucosa was removed by sharp dissection and three to seven small extrinsic nerve trunks (20–40 μm in diameter) were dissected free of connective tissue for a length of 2–6 mm. We defined the rectum as the region innervated by rectal nerve trunks emerging from the pelvic ganglia. The preparation was then cut down to size and pinned – serosal side up, with 50 μm tungsten pins – along one side.
Afferent dissection and recordings
The extrinsic nerve trunks and a strand of connective tissue were led under a coverslip partition into a recording chamber and sealed with silicon grease (Ajax, Australia). The recording chamber was then filled with paraffin oil; platinum wire electrodes were touched on a nerve trunk and on the connective tissue strand. Recorded signals were amplified and filtered through an isolated bio-amplifier (ISO-80, World Precision Instruments), then digitized with either a micro1401 (Cambridge Electronic Design) run with an IBM-compatible personal computer using Spike2 software (v.4.12, Cambridge Electronic Design), or a Powerlab/16SP (ADInstruments) and recorded on a Macintosh personal computer using Chart software (ADInstruments). The main chamber containing the preparation was superfused at 3 ml min−1 with oxygenated Krebs solution, at 34°C.
Activation of stretch-sensitive units
The edge of the preparation was attached to an array of hooks, resembling a miniature garden rake. This was attached to a ‘tissue stretcher’ consisting of a microprocessor-controlled stepper motor with an in-series isometric force transducer (DSC 46-1001-01, Kistler Morse). The preparation was maintained at approximately 1 mN basal force for the duration of experiments. Using this apparatus, the tissue could be stretched at rates of 10–5000 μm s−1 over distances of 1–5 mm, while recording the tension developed by the tissue (stretching by ‘imposed length’– see Fig. 1A). In other experiments, stretch was applied by applying an ‘imposed load’ to the tissue. In these cases, the rake was attached to the arm of an isotonic transducer via a pulley, and the counterweight was increased as either a step change by hanging weights of 10–50 mN or by infusing fluid into a reservoir to increase the counterweight in a graded fashion (Fig. 1B). Using this set-up, stretch was applied by ‘imposed load’, while recording the contractions of the tissue with the isotonic transducer. In all cases, a resting load of 5 mN was applied. Using these methods, stretch could be applied in either the longitudinal and/or circumferential axis of the preparation. It should be pointed out that fully isometric recordings from smooth muscle preparations are not achievable because serial elastic elements in the tissue allow some shortening of smooth muscle cells during contraction. Likewise, true isotonic recording conditions cannot be achieved because finite loads applied to the preparation stretch passive elastic components of the tissue, and force per cross-sectional area necessarily changes as the muscle shortens. The terms ‘near-isotonic’ and ‘near-isometric’ are used throughout this study for clarity to highlight the practical limitations of our recording techniques.
Figure 1. Preparations used to study effects of stretch on mechanoreceptor activity A, the preparation was pinned along one edge, serosa uppermost, and connected via a metal rake on the other edge to a microprocessor-controlled stepper motor. Changes in length of the preparation were imposed and the tension developed by the tissue was recorded via the isometric force transducer. B, the preparation was similarly pinned down one edge, but the rake on the other edge was attached to a thread that ran, via a pulley, to an isotonic length transducer. Stretch was applied by increasing the counterweight on the transducer while recording the resulting changes in length of the preparation, which was free to shorten. By aligning the preparation appropriately, it was possible to stimulate and record from longitudinal and/or circular muscle with either preparation.
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Local mechanical distortion of the tissue was applied with von Frey hairs with tip diameters < 50 μm (0.1–1.10 mN). ‘Hotspots’ were then identified, and their positions marked on the tissue using fine carbon particles attached to the tip of the hair. These hotspots have previously been shown to correspond to rectal IGLEs in myenteric ganglia (Lynn et al. 2003). For this reason we refer to these sites in the text as ‘putative rIGLEs’. Stretch-sensitive units (n= 8, N= 4) were investigated for their sensitivity to von Frey hairs (0.1, 0.2, 0.3, 0.5, 0.8, 1.1 mN) under imposed length conditions.
Video imaging and spatiotemporal mapping
The location of one unit's receptive field was marked with four small opaque markers (area less than 1 mm2) placed on the preparation, after identifying hotspots with von Frey hairs. The preparation was then cut down to an L-shape, and a 1 mm wide hook was used to record longitudinal muscle tension aligned with the identified receptive field. A 5–6 mm wide hook was attached to record circular muscle force. Movements of the opaque markers were recorded at high magnification on a digital video camera recorder (DCR-TRV11E, Sony) fitted to the dissecting microscope. Video images were synchronized with electrophysiological and mechanical recordings by placing a small flashing LED in the corner of the video image, and recording the driving pulses in Chart (ADInstruments) together with electrical and mechanical activity. The area defined by the four markers, and the length of the receptive field in longitudinal and circular axes, was calculated from each frame of digital video using purpose-designed in-house software.
Rhythmic muscle contractions were induced by application of [β-Ala8]-neurokinin A fragment 4–10 ([β-Ala8]-NKA(4–10); 0.1 μm, Sigma) or carbachol (0.06 μm, Sigma) to the bath. Tetrodotoxin was from Alomone Laboratories.
Single units were identified and discriminated using Spike2 software or Chart Spike Histogram software; when single units could not be discriminated, recordings were discarded. Single unit activity was presented and analysed using impulses per second except where instantaneous frequency was used. Data are presented as mean ±s.e.m. unless otherwise stated; n refers to the number of single units and N to the number of animals. Statistical comparison was carried out using Student's t test and one- or two-way analysis of variance as appropriate; P < 0.05 was considered significant.
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Rectal intraganglionic laminar endings (rIGLEs) have previously been shown to correspond to the transduction sites of low threshold, slowly adapting mechanoreceptors that are present in the guinea pig rectum, but not distal colon (Lynn et al. 2003). In the present study, punctate mechanoreceptor sites were shown to give highly sensitive, graded responses to focal transmural compression with von Frey hairs. In one way, rectal mechanoreceptors responded as if they were true tension receptors: they increased firing when muscle spontaneously contracted against a fixed length (i.e. under near-isometric conditions). However, under near-isotonic conditions – where the muscle could shorten against a fixed load – rectal mechanoreceptors did not behave as tension receptors. Typically, they showed variable bursts of firing, which usually coincided with the onset of contractions that subsequently led to shortening of the preparation. By definition, true tension receptors should fire at a nearly constant rate under constant load conditions (subject only to changes in cross-sectional area, according to the law of Laplace). Muscle contraction evoked by an NK2 receptor agonist increased mechanoreceptor excitability under both near-isotonic, and near-isometric conditions. Rectal mechanoreceptors were also activated by stretch in both circumferential and longitudinal axes of the gut, irrespective of whether this was evoked by imposed load or by imposed length. It should be noted that such stretches often triggered reflex activity in the orthogonal muscle layers, complicating the interpretation of their effects on mechanoreceptors. However, neither length changes nor tension changes measured from the longitudinal or circular muscle layers of the whole preparation fully predicted firing of mechanoreceptors. High-resolution video microscopy indicated that much of the variability in firing was due to highly localized contractions around the receptive field which were not necessarily detected by transducers attached to the preparation as a whole.
Directional sensitivity of rectal mechanoreceptors
Stretch in the longitudinal and circumferential axes of the gut, together with vertical compression of transduction sites, all evoked firing. In addition, contractions of the preparation under both near-isometric and near-isotonic conditions also evoked firing. The complex nature of mechanoreceptor responses is perhaps not surprising given that their transduction sites correspond to branching flattened nerve endings (rIGLEs) that occupy a substantial part of myenteric ganglia (Lynn et al. 2003). These endings are, by location, both parallel to smooth muscle and also in-series to it. It seems reasonable to assume that rIGLEs detect distortion of myenteric ganglia. The fact that putative rIGLEs investigated here respond to both longitudinal and circular muscle stretch, and contraction, suggests that they have little directional sensitivity.
Mechanoreceptor firing and contractility
In previous studies of extrinsic afferents to the large bowel, muscle activity of the gut has typically been recorded either in vivo, using a large balloon to measure intraluminal pressure (Janig & Koltzenburg, 1990, 1991; Sengupta & Gebhart, 1994; Thewissen et al. 2000), or in flat sheet preparations, ex vivo, where stretch has been applied without recording mechanical responses (Page & Blackshaw, 1998; Lynn & Blackshaw, 1999). In the present study, we applied mechanical stimuli and recorded muscle responses in the longitudinal, and circular axes, with transducers coupled to the tissue with stiff metal ‘rakes’ which were from 2 to 10 mm in length. This provided higher resolution mechanical recordings than previous studies. Nevertheless, the mechanical state of the muscle was poorly predictive of afferent firing. The results of the spatiotemporal mapping studies provided a simple explanation for this finding. By monitoring both longitudinal and circular dimensions within the receptive field of mechanoreceptors, it was found that contractile activity within the receptive field could not be predicted accurately from transducer recordings from the preparation as a whole, even when the receptive field was in a preparation only 2–5 mm wide. This is readily explained by the observation that electrical activity in segments of gut propagates rapidly and over long distances along bundles of smooth muscle fibres, but over shorter distances propagates perpendicularly to bundles. Thus, in our study, a receptive field that was less than 1 mm wide could have quite different mechanical activity (‘micro-contractions’) than that recorded from the full width of the preparation. There also seemed to be some variability between individual mechanoreceptors.
The firing of most units was more closely related to longitudinal rather than circular muscle microcontractions. This seems to be at variance with our observation that rectal mechanoreceptors responded equally to circular and longitudinal stretch. We can speculate on possible causes of this apparent discrepancy. In most cases, preparations were pinned along an edge close to the receptive field, which may have restricted circular muscle contractions more than longitudinal muscle contractions measured with the video mapping system. It is also possible that reflex interactions between the muscle layers may have contributed differently to stretch-evoked responses and responses to agonist-induced contractions.
The results from spatiotemporal mapping of microcontractions also explain why, under near-isotonic conditions, some contractions of the circular muscle were associated with decreased firing of rectal mechanoreceptors (Figs 3 and 4). This may have occurred when circular muscle shortening outside the receptive field effectively ‘unloaded’ mechanoreceptor endings in an area that did not contract. In these ways, microcontractile activity around the receptive field may decrease the correlation between length and force recorded with transducers, and afferent firing.
Ganglionic distortion as an adequate stimulus
In the light of our findings, it is worth considering the true nature of the adequate stimulus for rectal mechanoreceptors. Recent studies have demonstrated that similar endings of vagal afferents in the upper gut probably detect distortion via the opening of stretch-activated ion channels (Zagorodnyuk et al. 2003). Preliminary data suggest that this is also likely to be the case for rectal mechanoreceptors (authors' unpublished observations). For this to be the case, there must be physical movement of part, but not all, of the mechanotransducing molecular complex located in the membrane of rIGLEs. Rectal IGLEs are branching flattened structures that are not aligned with either longitudinal or circular muscle, but rather appear to course in all directions through myenteric ganglia, often close to the ensheathing connective tissue (Lynn et al. 2003; Olsson et al. 2004). Mechanosensitive ion channel complexes are unlikely to show preferential alignment with either the longitudinal or circumferential axis of the gut. Thus, distortion of ganglia in any direction would necessarily activate a subset of channels and excite endings. Any movement of tissue that either compresses or stretches ganglia could activate rIGLEs. We suggest that contraction of smooth muscle fibres above or below a ganglion would increase their rigidity and thus compress the interposed ganglion. Simultaneous contraction of both muscle layers should thus be particularly effective in activating mechanosensitive endings, as was observed. Several studies have shown that simultaneous contractions of longitudinal and circular muscle commonly occur during neurogenic propulsive activity (Brookes et al. 1999; Hennig et al. 1999; Spencer et al. 1999; Spencer & Smith, 2001) and during myogenic activity evoked by pacemaker cells, which are common to both muscle layers (Dickens et al. 1999). In addition, contractions of distant smooth muscle that stretch the receptive field should cause flattening of the ganglion (to preserve volume), and thus also activate mechanoreceptors.
Significance of multiple transduction sites
Individual rectal mechanoreceptors have previously been shown to have multiple hotspots spread over a few square millimetres of tissue, each capable of responding to localized transmural compression with a von Frey hair (Lynn et al. 2003). We have previously shown, using simple arithmetic modelling, that the multiple transduction sites of similar mechanoreceptors in the oesophagus do not function independently (Zagorodnyuk & Brookes, 2000). Rather, action potentials from the most strongly activated site ‘reset’ other transduction sites, and thus determine the overall firing frequency of the unit. A similar mechanism has recently been proposed for rabbit pulmonary receptors (Yu & Zhang, 2004). If, as seems likely, the same mechanism operates for rectal mechanoreceptors, the presence of multiple mechanosensitive sites spread over several square millimetres of the gut wall would tend to smooth out variations in firing frequency caused by the localized nature of contractile activity. Thus, rectal mechanoreceptors should encode the state of contractile activity of the most active region of muscle anywhere within the receptive field, at any particular moment.
Physiological significance of transduction
The present study has shown that specialized rectal mechanoreceptors are typically activated during muscle contraction, whether this occurs under near-isotonic conditions or under near-isometric conditions. It is known that faecal matter moving in the gut of the guinea pig causes deformation of myenteric ganglia in which these endings reside (Gabella, 1990). It also triggers neurogenic motor activity involving both longitudinal and circular muscle (Costa & Furness, 1976). In the present study, stretch of the gut wall not only evoked activity of rectal mechanoreceptors directly, but also triggered neurogenic contractions in the stretched muscle layer and in the orthogonal layer, via a combination of neurogenic and myogenic mechanisms (see Fig. 7). There is extensive evidence that activation of rectal mechanoreceptors, presumably by both distension and by contractile activity of the rectal wall, activate extrinsic and intrinsic reflexes that play a key role in defaecatory motor behaviour (Yamanouchi et al. 2002).