1Department of Physiology and Biophysics, 2Enteric Neuroscience Program, and 3Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905
Submitted 10 July 2003 ; accepted in final form 9 August 2003
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ABSTRACT |
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prevertebral ganglia; colonic mechanoreceptors; intestinointestinal reflex
Previous studies in vitro demonstrated that colonic distension activates a cholinergic mechanosensory afferent pathway from the colon to the inferior mesenteric ganglion (IMG) in the guinea pig and superior mesenteric ganglion (SMG) in the guinea pig and mouse (3, 5, 20, 24, 26). The colonic mechanosensory afferent nerves that project to IMG neurons in guinea pig and to SMG neurons in mouse are believed to function as volume detectors, because excitatory cholinergic synaptic input to ganglion neurons increases when colonic intraluminal volume is increased during filling and decreases when colonic intraluminal volume decreases during emptying (1, 21, 25). However, it is not known whether colonic mechanosensory synaptic input to IMG and SMG neurons is a result of increased volume (stretch) per se or due to increased colonic wall tension, as would be expected as a consequence of increased intraluminal volume, according to the Law of LaPlace. On the other hand, colonic mechanosensory synaptic input to the guinea pig IMG was previously shown to increase when colonic contractions increased and to decrease when colonic contractions were reduced (26), supporting the concept of in-series tension receptors in colonic afferent nerves projecting to the IMG.
The muscle coat of the colon is made up of an outer longitudinal muscle layer and an inner circular layer (8), the latter constricting the gut lumen during contraction, the former shortening it. It is possible that there are mechanoreceptor populations in both muscle layers that respond to changes in muscle stretch or muscle tension and relay that information to prevertebral ganglion neurons. We investigated this possibility by examining in vitro the relationship between colonic wall tension and colonic mechanosensory synaptic input to the mouse SMG. Changes in colonic wall tension and intracellular electrical activity from SMG neurons were simultaneously monitored during spontaneous colonic contractions, during colonic distension with fluid, and during manual stretch of the colon wall. The results showed that colonic mechanosensory fast excitatory synaptic input to mouse SMG neurons increased when muscle tension decreased before contraction and during circumferential but not longitudinal colonic stretch. In contrast, fast excitatory synaptic input to SMG neurons decreased and was often absent at peak tension of contraction. These results suggest that cholinergic mechanosensory colonofugal neurons that project to the SMG function as stretch receptors arranged in parallel to the circular muscle layer.
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MATERIALS AND METHODS |
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Dissection Procedures to Make SMG-Colon Preparations
Further dissection was made to obtain a preparation consisting of a 2.5- to 3.0-cm-long segment of colon connected to the SMG via the inferior mesenteric artery, colonic nerves, and intermesenteric nerve, as previously described (20). In some experiments, the colon segment connected to the SMG was cut open longitudinally off-center to the antimesenteric border to make a flat sheet of the colon. Colon-SMG preparations were transferred to a two-compartment organ bath, the colon (intact tube or flat sheet) pinned out in one compartment, the SMG in the other compartment, and the colonic nerves, inferior mesenteric artery, and vein were draped over the partition separating the chambers and covered with moistened strips of tissue paper. Each compartment was continuously perfused at 6-7 ml/min with oxygenated Krebs solution warmed to 35-37°C. Colonic contractions, intraluminal pressure, or both were monitored as follows. Intact colon segments were cannulated with a small glass tube connected to a pressure-monitoring system at the anal end and a small glass tube connected to a plastic syringe at the oral end (Fig. 1, top). The pressure-monitoring system consisted of a Statham pressure transducer connected via a three-way valve to a fluid-filled reservoir as previously described (21). When the valve to the reservoir was opened, fluid could flow from the colon to the reservoir during contraction and flow back into the colon during relaxation. When the valve was closed, fluid could not leave the colon during contraction (i.e., isovolumetric condition). The glass cannula at the anal end was attached to a Statham force transducer, which measured tension changes in the long axis of the colon segment. The transducer was attached to a manipulator that could be moved backward to stretch the colon in its long axis (longitudinal stretch). After securing the ends of the colon to the glass cannulas, the colon was stretched to approximate the in vivo resting length. To measure tension in the circular axis of the colon segment, a thin steel pin (1.5 cm long) was inserted through the wall of the colon and was attached via a silk suture to a glass hook, which was attached to a Statham force transducer, which was attached to a micromanipulator. In some experiments, a latex balloon inserted into the colon lumen from the orad end and connected to a syringe filled with air was used to distend the colon (Fig. 1, middle). In flat sheet preparations (Fig. 1, bottom), the colon was pinned down, mucosal side up, along the mesenteric edge, maintaining the approximate in vivo longitudinal length. A 1.5-cm-long steel pin was inserted through the opposite edge and was connected by silk thread to a Statham force transducer attached to a micromanipulator. The oral edge of the sheet was also secured with a row of pins, maintaining the approximate in vivo circumferential length. A 1.5-cm-long steel pin was placed through the opposite (anal) edge and was connected via silk thread to a Statham force transducer attached to a micromanipulator. With this arrangement, contractions along the longitudinal and circular muscle axes could be recorded and stretch of the colon could be applied in both axes.
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Electrical Recordings
Intracellular recordings from SMG neurons were made with glass microelectrodes filled with 3 M KCl (electrode resistances were 40-80 M) and a WP M707 electrometer (World Precision Instruments) as previously described (20). Electrical and mechanical signals were simultaneously displayed on a Tektronix 5113 oscilloscope and a Gould 2400 chart recorder and were stored on FM tape (Hewlett-Packard 3964A recorder). To assess the frequency of fast synaptic potentials, tape recordings were played back to a strip chart recorder by using a chart speed of 100 mm/s. Fast excitatory postsynaptic potentials (fEPSPs) and action potentials (APs) in the traces were counted manually. "N" refers to the number of SMG-colon preparations, hence the number of ganglia studied, whereas "n" refers to the number of ganglion neurons studied.
Drugs
In some experiments, nicardipine or nifedipine (both from Sigma) were added to the solution superfusing the colon to inhibit muscle contraction. Stock solutions of 10-2 M were made in ethanol, and then they were diluted to a final concentration of 2-3 x 10-6 M in Krebs solution.
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RESULTS |
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Tension was simultaneously measured in the longitudinal and circular muscle axes of colon segments that were connected to a fluid-filled reservoir that received fluid from the colon during contraction and returned fluid into the segment during relaxation. Intracellular recordings were made from 13 SMG neurons (N = 8). All of the neurons received ongoing fEPSPs or a combination of fEPSPs and APs. Fluid (0.05-0.10 ml) added to empty colon segments initiated large emptying contractions that occurred at a frequency of 1/min, with peak amplitude of longitudinal muscle contraction preceding that of the circular muscle contraction. Fluid distension also immediately increased the frequency of fEPSPs and APs in all SMG neurons tested. In the example shown in Fig. 2, synaptic input was also increased just before the onset of circular and longitudinal muscle contraction when tension either remained at baseline level or decreased before the onset of contraction. Synaptic input then decreased when muscle tension increased with contraction and was almost abolished at peak tension of contraction. The frequency of synaptic input returned to baseline levels when tension decreased after contraction.
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The relationship between colonic wall tension and synaptic input to SMG neurons was further investigated in another four preparations under conditions in which the colon was prevented from emptying during contraction (isovolumetric contraction) or prevented from refilling during relaxation (Fig. 3). Similar to the previous results, the frequency of synaptic input to SMG neurons (5 out of 5 neurons tested) increased before the onset of emptying contractions, was markedly decreased at peak tension during contraction, and returned to basal levels during relaxation. However, when refilling of the colon was prevented, synaptic input to the neurons remained diminished until the colon was allowed to refill. On refilling, synaptic input to the neurons increased to basal levels. In contrast, when the colon was prevented from emptying during contraction, synaptic input to SMG neurons did not diminish until the colon was allowed to empty. These results support our previous findings that SMG neurons receive volume-sensitive colonic afferent excitatory synaptic input (21, 25) and further suggest that colonic afferent synaptic input to SMG neurons was sensitive to stretch of the colon wall but not to increases in active tension of the muscle layers accompanying colonic contraction.
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To further examine the relationship between colonic muscle tension and excitatory synaptic input to SMG neurons, an additional three SMG-colon preparations were studied. In these experiments, 0.03-0.05 ml of fluid was added to distend the colon and it was not allowed to empty its contents during contraction. Examples of results from two experiments are shown in Fig. 4. Distension of the colon with fluid resulted in a transient decrease in longitudinal muscle tension, a prolonged decrease in circular muscle tension, and an increase in frequency of synaptic input to SMG neurons (6 out of 6 neurons tested). Distension also initiated phasic contractions and increases in circular and longitudinal muscle tension. In the recording shown in Fig. 4A, excitatory synaptic input to the SMG neuron decreased when muscle tension increased, whereas synaptic input increased when the muscle layers relaxed and tension decreased. On removal of distension, there was a transient contraction of the colon and the frequency of excitatory synaptic input to the neuron decreased to the basal level seen before distension. In the other experiment, illustrated in Fig. 4B, the colon segment was distended twice with fluid. Accompanying the first distension (+0.03 ml) was a brief transient decrease in longitudinal muscle tension, a sustained decrease in circular muscle tension, and an increase in excitatory synaptic input to the neuron. The second distension (+0.07 ml) produced a transient increase in longitudinal but not circular muscle tension and a marked increase in frequency of synaptic input to the neuron that persisted for the duration of the distension. On removal of the distension, there was an increase in longitudinal and circular muscle tension and an immediate decrease in synaptic input. These results also suggest that the increased synaptic input to SMG neurons accompanying the increase in intracolonic volume was due to increased stretch (length) but not active tension (contraction) of the colon wall.
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Effect of Colonic Stretch on Excitatory Synaptic Input to SMG Neurons
Longitudinal stretch. The effect of longitudinally applied colonic stretch on excitatory synaptic input to SMG neurons (n = 19) was tested in 15 SMG-colon preparations. Tubular colon segments were manually stretched in the long axis by increasing their length (by 10-20%) with a micromanipulator. Before, during, or after longitudinal stretch, the segments were either distended with 0.05 ml of fluid (N = 12 preparations) or with an intraluminal balloon (N = 3 preparations) to test the response of the SMG neurons to colonic distension. In all 19 neurons tested, distension with fluid (n = 15) or with an intraluminal balloon (n = 4) resulted in an increase in synaptic input to SMG neurons, whereas manual stretch of the colon in the long axis did not (Fig. 5). In the recording shown in Fig. 5A, note that the frequency of synaptic input to the neuron did not increase when the colon was stretched by increasing its length 10% and then by a further 5% more than its resting length. Similarly, in the recording shown in Fig. 5B, the frequency of synaptic input to the SMG neuron did not increase when the colon segment was stretched to 10% of its resting length, but synaptic input to the neuron did increase on balloon distension and input remained elevated when longitudinal stretch was terminated during distension. When distension was released, synaptic input immediately decreased. Since in most of the experiments on longitudinal stretch of the colon only one or two neurons in each ganglion were tested, it is possible that neurons in a distinct location in the ganglion might receive input from longitudinal muscle afferents and were not sampled. To rule out this possibility, two additional preparations were studied in which an attempt was made to sample neurons throughout the SMG. Of 16 neurons tested, 14 showed an increase in synaptic input when the colon was distended with fluid but none showed an increase when the colon was stretched to 15-20% beyond its resting length.
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Circumferential stretch. Colon tube preparations were resistant to manual stretching in the circular axis. When segments (N = 4) were stretched in the direction of the circular axis, the circular muscle immediately contracted, making it difficult to further stretch the colon without causing a marked distortion in circumferential shape of the colon. Accompanying these events, there were no increases in frequency of synaptic inputs to any of five SMG neurons tested.
Relationship Between Muscle Tension, Intracolonic Volume, and Excitatory Synaptic Input to SMG Neurons
Intraluminal pressure, volume of intraluminal content, longitudinal or circular muscle contractions, and synaptic input to SMG neurons (n = 44) were recorded simultaneously in 26 preparations in which the colon segment was connected to a fluid-filled reservoir, allowing it to empty on contraction and refill with fluid on relaxation. Figure 6A shows results from 1 of 17 preparations in which tension was measured in the longitudinal axis, and Fig. 6B shows tension measured in the circular muscle axis in 1 of 9 preparations. In all of the preparations, longitudinal or circular muscle tension and colonic intraluminal pressure increased during contraction, whereas colonic intraluminal volume decreased as fluid was expelled from the colon into the reservoir. Peak longitudinal muscle tension preceded peak colonic intraluminal pressure (Fig. 6A), whereas peak circular muscle tension coincided with peak intraluminal pressure (Fig. 6B). In all 44 neurons tested, synaptic input increased just before contraction, and input then decreased and was sometimes completely abolished as colonic muscle tension and intraluminal pressure increased and intracolonic volume decreased during contraction. In the traces illustrated in Fig. 6B, two successive spontaneous colonic contractions are shown with tension recorded from the circular axis. Note that an increase in synaptic input to the neuron coincided with a decrease in colonic intraluminal pressure and an increase in intraluminal volume before the onset of circular muscle contraction. Note also that the peak tension of the second contraction was larger than the first and that synaptic input to the neuron was less at the peak of the higher-amplitude second contraction compared with the first. These results suggest that increased synaptic input to SMG neurons was associated with stretch of the colon wall that occurred during increases in intracolonic volume. This hypothesis was tested in 9 of the 26 preparations in which the colon segments were manually stretched in the longitudinal direction while synaptic input to SMG neurons was simultaneously recorded. In 11 of 11 SMG neurons tested, synaptic input did not increase when colon segments were stretched in the longitudinal direction (up to 20% increase of resting length), similar to the previous results that longitudinal stretch of the colon did not increase synaptic input to SMG neurons. On the other hand, attempts to manually stretch three colon preparations in the circular muscle axis were unsuccessful, because the colon contracted when stretch was applied and further stretch unevenly distended the shape of the colon. These manipulations were not accompanied by an increase in synaptic input to any of six SMG neurons tested, similar to the previous results.
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Colonic Muscle Tension and Synaptic Input to SMG Neurons in Flat Sheet Preparations
Flat sheet preparations of the colon were made to directly test whether synaptic input to SMG neurons increased during stretch of the longitudinal muscle, circular muscle, or both. The flat colon sheets, unlike the tubular segments of colon, could be readily stretched in both the circular and longitudinal axes. For these experiments, the sheet was pinned out to its approximate resting in vivo length in both axes and the colon was stretched 1-2 mm (10-15% beyond resting dimensions) in the circular axis and 3-4 mm (15% beyond resting length) in the longitudinal axis. The effect of longitudinal and circular stretch of the colon on synaptic input to SMG neurons was examined in three flat sheet preparations. All five neurons tested exhibited ongoing fEPSPs and APs before the colon was stretched, and synaptic activity increased when the colon sheet was stretched in the circular axis but not when it was stretched in the longitudinal axis (Fig. 7A). During maintained stretch in the longitudinal axis, there was no increase in fEPSP frequency until manual stretch in the circular axis was applied (Fig. 7B). In these experiments, circumferential stretch of the colon sheet produced a small increase in tension in the longitudinal muscle layer in addition to the large increase in circular muscle tension (Fig. 7). However, the increase in longitudinal tension produced by circular muscle stretch was much smaller than the increase in longitudinal tension produced when the colon was stretched in the longitudinal axis, the latter being insufficient to cause an increase in synaptic input to SMG neurons. These results suggest that circumferential but not longitudinal colonic stretch activated mechanosensory input to SMG neurons.
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Effect of Muscle Relaxants on Stretch-Induced Increase in Synaptic Input to SMG Neurons
Contractile activity in flat sheet preparations of the colon was inhibited by adding the L-type Ca2+ channel antagonists nifedipine (2 µM) or nicardipine (3 µM) to the superfusate. In the presence of nifedipine (6 neurons tested in 3 preparations) or nicardipine (1 neuron tested in 1 preparation), circumferential stretch of the colon sheet to 15% beyond resting length resulted in increased synaptic input to the neurons, similar to the response of cells to the same circumferential stretch before relaxing the colon (Fig. 8).
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DISCUSSION |
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Muscle tension receptors in abdominal viscera are defined as slowly adapting mechanoreceptors whose discharge rate is related to the amount of tension in the muscle layers of the viscus (15). Through an analogy to muscle spindles and Golgi tendon organs in skeletal muscle, muscle tension receptors in abdominal viscera are similarly classified into two types, depending on their response to distension and active contraction of the viscus (10, 14). "In-series" receptors (e.g., Golgi tendon organs) increase their firing frequency during active contraction and passive distension of the viscus. "In-parallel" receptors (e.g., muscle spindles) increase their discharge rate when the viscus is passively distended but decrease their discharge rate when they are "unloaded" during contraction. In the present study, it was found that afferent excitatory synaptic input to SMG neurons increased during colonic relaxation, decreased during colonic (isotonic) contraction, and increased during circumferential stretch of the colon. This behavior is what would be predicted from an in-parallel tension receptor. Thus we interpret our results to indicate that cholinergic mechanosensory afferent nerves from the colon to the SMG function as in-parallel receptors.
Distension-sensitive mechanosensory nerves in visceral muscle that are found in the vagus, splanchnic, and pelvic nerve trunks are mostly of the in-series type (9, 14). They appear to be arranged in series with the longitudinal muscle, because their rate of firing increases during longitudinal stretch of the viscus or when longitudinal muscle tension increases during contraction. These afferent nerves, whose cell bodies are located in vagal ganglia and dorsal root ganglia of the spinal cord, connect the abdominal viscera with the central nervous system. In-series longitudinal muscle tension receptors are believed to act as sensors of the degree of distension and level of contractile activity in the viscus (15). They are often referred to as tension receptors because they encode information about the degree of tension in the gut wall. In contrast, the present study suggests that the mechanosensory colonofugal nerves that project to the SMG monitor changes in intracolonic volume during filling and emptying of the colon by sensing the degree of stretch or length of the circular muscle, not the longitudinal muscle layer. They appear to encode information about the diameter of the colon wall. Thus colonofugal nerves projecting to SMG neurons appear to carry a qualitatively different message about the mechanical state of the colon from that conveyed from the colon to the central nervous system by extrinsic primary mechanosensory afferent nerves.
Gastrointestinal distension-responsive afferent nerves are generally considered to be muscle afferent nerves (14, 22) because their endings are in the muscularis, as demonstrated functionally by showing that removal of the mucosa does not impair responses to distension (4) and anatomically by anterograde tracing studies (2). To determine the functional location (longitudinal muscle layer, circular muscle layer, or both) of mechanosensory colonofugal afferent nerves, we used a flat sheet preparation of the colon wall and measured tension in the direction of the long axis of the circular muscle layer and in the direction of the long axis of the longitudinal muscle while recording mechanosensory afferent synaptic input to SMG neurons. When the colon wall was stretched in the circular muscle direction, afferent input to SMG neurons increased. In contrast, stretch of the colon wall in the direction of the long axis of the longitudinal muscle did not increase mechanosensory afferent synaptic input to SMG neurons. We infer from these results that cholinergic mechanosensory colonofugal neurons that project from the colon to the SMG are mechanically connected in parallel with the circular muscle layer. Mechanosensory nerves in parallel with the circular muscle would be excited by stretch of the circular muscle but would be offloaded when that muscle layer contracts. Accordingly, we speculate that the colonic mechanosensory afferent pathway to the SMG might operate as follows during filling and emptying of the colon: an increase in colonic intraluminal volume during filling increases the circumference of the colon, causing a tangential lengthening of the circular muscle layer, which in turn activates mechanosensory afferent nerves that are mechanically connected to it, resulting in an increased frequency of afferent synaptic input to SMG neurons. Contraction and emptying of the colon decreases the circumference of the colon and unloads the mechanoreceptor endings of the colonofugal nerves, resulting in a diminished synaptic input to the SMG. This conclusion is tentative because the anatomic location and arrangement of the mechanosensory endings within the muscle layers have not been determined and because SMG neurons receive mechanosensory synaptic input indirectly from secondary or higher-order neurons in the mechanosensory pathway (20, 21). Additional studies are also needed to determine the mechanism(s) by which stretch of the colon wall is transduced to excite the mechanosensory endings.
A major function of the distal colon is to store and evacuate feces. As the colon wall is stretched during filing of the colon with feces, stretch-induced depolarization and AP generation in smooth muscle cells as well as stretch-induced peristaltic reflex activity result in contractions to empty the colon. Noradrenaline release from sympathetic nerves can inhibit colonic contractions by inhibiting excitatory motor neurons and perhaps also by a direct effect on the smooth muscle cells. An increase in excitatory synaptic input to SMG neurons during stretch of the colon would be expected to increase the probability of AP firing in the neurons and to increase noradrenergic output to the colon. In addition, inhibitory sympathetic reflexes, in which distension of a portion of the colon results in inhibition of contraction in adjacent or more proximal parts, are important in tonic inhibition of colonic motility. Thus the physiological function of stretch-sensitive mechanosensory information relayed from the colon to SMG neurons might be to dampen the tendency of the colon to contract as it fills and thus to allow it to fill more completely.
In summary, the present results suggest that mechanosensory afferent nerves projecting from the colon to the sympathetic prevertebral ganglion neurons provide information about changes in diameter of the colon wall, not colonic wall tension.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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