Mechanosensitive Pelvic Nerve Afferent Fibers Innervating the Colon of the Rat are Polymodal in Character

X. Su and G. F. Gebhart

The University of Iowa, College of Medicine, Department of Pharmacology, Bowen Science Building, Iowa City, Iowa 52242

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Su, X. and G. F. Gebhart. Mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat are polymodal in character. J. Neurophysiol. 80: 00-00, 1998. This report describes the chemical and thermal sensitivity of mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat. A total of 51 fibers in the S1 dorsal root, identified by electrical stimulation of the pelvic nerve, were studied. An approximately 7 cm length of descending colon was isolated in situ to permit intracolonic perfusion and distension with Krebs solution. Reproducibility of responses to repetitive colorectal distension (CRD, 40 mmHg, 30 s, every 4 min) was documented. All fibers gave monotonic, incrementing responses to graded CRD (5 to 60 mmHg). Increases (n = 6) or decreases (n = 6) in pH of the perfusate failed to produce any change in resting activity or responses to CRD. Infusion of bile salts increased the resting activity of 6/6 fibers in a concentration-dependent manner, but did not affect the magnitude of responses to CRD. After intracolonic instillation of an inflammatory soup (bradykinin 10-5 M, PGE2 10-5 M, serotonin 10-5 M, histamine 10-5 M and KCl 10-3 M), 13/22 fibers exhibited sensitization of responses to CRD. Seventy-three percent of 45 fibers tested responded to intracolonic perfusion of heated Krebs solution. The estimated threshold for response was 45°C and response magnitude increased with the temperature. A smaller proportion (30%) of 37 fibers tested responded to intracolonic perfusion of cold Krebs solution. The estimated threshold for response was 28°C. Of 36 fibers tested, 8 were activated by both heat and cold; typically, fibers activated by heat did not respond to cold. In a sample of 26 fibers tested for response to all three modalities of stimulation, 11 responded to mechanical, chemical and thermal stimuli; the remaining 15 responded to mechanical and either chemical or thermal stimulation. Changes in intracolonic pressure in response to chemical and thermal stimuli were also evaluated. Inflammatory soup and bile salts did not change intracolonic pressure; heat and cold produced a modest decrease and increase in muscle tension, respectively. These results document that mechanosensitive pelvic nerve afferent fibers are also chemosensitive and/or thermosensitive, supporting the notion that visceral mechanoreceptors in general are likely polymodal in character.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Sherrington (1906) was the first to note that the cutaneous nociceptors he proposed as detectors of stimuli that threaten or damage tissue were polymodal ("anelectic"), although the term polymodal was not used in this context until much later. Bessou and Perl (1969) termed polymodal those C-fiber afferents in cutaneous nerves that responded to mechanical, thermal and particularly noxious chemical (e.g., acid) stimuli. In cutaneous nerves, several groups of nociceptors with diverse receptive properties exist (for recent overview, see Campbell and Meyer 1996). Many cutaneous C-fiber afferents belong to the class of polymodal nociceptive neurons that respond to mechanical, thermal and chemical stimuli (for recent overview, see Perl 1996). Polymodal receptors are also widely distributed in other tissues, including joints (Schmidt 1996), skeletal muscle (Kumazawa and Mizumura 1977; Mense 1996), dura (Bove and Moskowitz 1997), cornea (Gallar et al. 1993), splanchnic nerve afferents innervating the mesentery (Adelson et al. 1996 1997) and superior spermatic nerve innervation of the testis and/or epididymis (Kumazawa and Mizumura 1980a, b; Kumazawa et al. 1987).

In the gastrointestinal tract, afferent nerve terminals are present in the mucosa, muscularis and serosa. Visceral afferent units typically have been classified according to whether their principal activity is associated with mechanical, thermal or chemical stimuli, although many respond to more than one stimulus (for review, see Sengupta and Gebhart 1994b). For example, it has long been known that mechanosensitive mucosal afferent fibers are often also sensitive to chemical stimuli (Iggo 1957). Others have since documented that mechanosensitive afferent fibers innervating colon or urinary bladder muscle also respond to chemical stimuli (e.g., bradykinin, acetic acid, mustard oil, xylenes; Floyd et al. 1977; Haupt et al. 1983; Häbler et al. 1990; Jänig and Koltzenberg 1990; Sengupta and Gebhart 1994a; Sengupta et al. 1996; Su et al. 1997a, b). Longhurst (1995) has extensively studied the chemosensitivity of afferent fibers innervating the stomach, proximal small intestine, and mesentery, many of which are also mechanosensitive.

Thermosensitive afferent fibers have been reported in the vagus and splanchnic nerves (Von Euler 1947; Riedel 1976) and it has been documented that thermoreceptors exist in the esophagus (EL Ouazzani and Mei 1982), stomach and duodenum (Gupta et al. 1979) and intestine (Rawson et al. 1969; Rawson and Quick 1972). In humans, warm and cold receptors are distributed along the gastrointestinal tract (Villanova et al. 1997) and anal canal and rectum (Cervero et al. 1987; Miller et al. 1987). El Ouazzanni and Mei (1982) considered warm, cold, and mixed receptors in the digestive tract to be specific thermoreceptors sensitive only to warm or cold stimuli and not to mechanical and chemical stimuli. Receptors that respond to both mechanical and thermal stimuli have been described in duodenum (Cottrell 1984) and colon and rectum (Clifton et al. 1976).

Much of the recent literature has focused on the mechanosensitivity of gastrointestinal receptors (e.g., Blumberg et al. 1983; Haupt et al. 1983; Jänig and Koltzenburg 1991; Sengupta and Gebhart 1994a, b) and their potential role in visceral nociception. Virtually all such studies have used balloons to distend hollow organs, making it experimentally difficult to also test the application of chemical or thermal stimuli. We have argued that the adequate stimuli for such receptors are unknown, and that they are likely polymodal (Gebhart and Sengupta 1994; Sengupta and Gebhart 1994b). Accordingly, the objective of the present study was to examine the chemical and thermal sensitivity of mechanosensitive pelvic nerve afferent fibers innervating the rat colon. We adapted the control device used previously for distension of the colon (Gebhart and Sengupta 1995) to provide constant pressure colonic distension with Krebs solution, to which we could add chemicals or change temperature of the solution. Preliminary reports of some of these data have been presented previously (Gebhart 1996; Su et al. 1996).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

General procedures

Male Sprague-Dawley rats weighting 410-530 gm (Harlan, Indianapolis, IN) were used. Food, but not water, was withheld for 24 h before an experiment. Rats were anesthetized initially with 40-45 mg/kg i.p. sodium pentobarbital (Nembutal,® Abbott Laboratories, North Chicago, IL); anesthesia was maintained by infusion of pentobarbital (5-10 mg/kg/h i.v.). A femoral artery and vein were catheterized for measurement of arterial pressure and administration of drugs, respectively; the trachea was also cannulated. Rats were paralyzed with pancuronium bromide (initial 0.3 mg/kg i.v.; supplemental 0.2-0.3 mg/kg/h i.v.) and subsequently ventilated with room air (55-60 strokes/min, 3-4 ml stroke volume). Mean arterial blood pressure was monitored continuously and was maintained at >80 mmHg by i.v. injection of 5% dextrose in saline given in a bolus of 1-1.5 ml as required. Core body temperature was maintained at 36°C by a hot-water-circulating heating pad placed under the rat and an overhead feedback-controlled heat lamp (thermoprobe inserted into the thoracic esophagus). At the end of experiments, rats were killed by an overdose of i.v. pentobarbital. The experimental protocol was approved by the Institutional Animal Care and Use Committee of The University of Iowa.

Surgical procedures

The lower abdomen was exposed by a 4-5 cm long incision laterally at the left flank. The urinary bladder was emptied and catheterized (PE-100) through the fundus and urine was constantly evacuated via the fundic catheter. An approximately 7 cm length of descending colon was exposed and isolated in situ. The blood supply and nerves innervating the colon remained intact. A catheter (diameter 5 mm) was inserted into and ligated in the proximal end of the descending colon. Another catheter (diameter 5 mm) was inserted via the anus and similarly ligated, thus permitting intracolonic perfusion or distension of the colon with Krebs-Henseleit (Krebs) solution.

The left testis, vas deferens, and seminal vesicle were tied and removed. The prostate was reflected laterally to access the major pelvic ganglion and pelvic nerve. The pelvic nerve was isolated from the surrounding fatty tissues and a pair of Teflon® insulated stainless steel wires stripped at the tips were wrapped around the pelvic nerve and sealed with nonreactive Wacker® gel (Wacker Silicone Corp., Adrian, MI). The hypogastric, pudendal, and femoral nerves were isolated and transected. The sciatic nerve was approached through the ischiatic notch and transected. The lateral tail nerve was approached at the root of the tail and transected and the abdomen was closed with silk sutures.

The lumbosacral spinal cord was exposed by laminectomy (T13-S2), and the rat was suspended from thoracic vertebra and ischia spinal clamps. The dorsal skin was reflected laterally and tied to make a pool for mineral oil. The dura was carefully removed and the spinal cord was covered with warm (37°C) mineral oil.

Recording of afferent nerve action potentials

The S1 dorsal root was identified and decentralized close to its entry to the spinal cord. Recordings were made from the distal cut end of the central processes of primary afferent fibers. A length of nerve fiber was draped over a black micro-base plate immersed in warm (37°C) mineral oil. The dorsal rootlet was split into thin bundles and a fine filament was isolated from the bundle to obtain a single unit. Electrical activity of the single unit was recorded by placing the teased fiber over one arm of a bipolar silver-silver chloride electrode; a fine strand of connective tissue was placed across the other pole of the electrode. Action potentials were monitored continuously by analog delay, displayed on a storage oscilloscope after initial amplification through a low-noise AC differential amplifier, processed through a window discriminator and counted using the spike2/CED 1401 data acquisition program. Peri-stimulus time histograms (1 s or 10 s binwidth), intracolonic pressure, intracolonic temperature, colonic distending pressures and blood pressure were displayed on-line continuously. Data were also recorded on magnetic tape for off-line analysis.

Experimental protocol

Pelvic nerve input to the S1 dorsal root was identified first by electrical stimulation of the pelvic nerve (single pulse 0.5 ms square-wave at 3-8mA). Single fibers were classified on the basis of their conduction velocities (CV), determined by estimating with a piece of thread the distance between stimulation and recording sites and dividing the conduction distance by conduction time. Fibers with a CV <2.5 m/s were considered unmyelinated C-fibers and those with a CV >2.5 m/s were considered thinly myelinated Adelta -fibers. The isolated colon was connected to a pressurized fluid reservoir through the proximal catheter and intracolonic pressure was measured through a fine catheter (polyethylene tubing, PE-60) placed in the colon from the proximal end. The pressure reservoir was connected to a distension control device via a low-volume pressure transducer (see Gebhart and Sengupta 1995). At rest, 37°C Krebs solution (0 mmHg) remained in the colon. For phasic, constant pressure distension (5-60 mmHg, 30 s), 37°C Krebs solution was introduced via the proximal catheter and the distal catheter was clamped. The experimental arrangement is illustrated in Fig. 1.


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 1. Experimental arrangement.

If a fiber responded to brief phasic colorectal distension (CRD; 40 mmHg, 2-3 s), a stimulus-response function (SRF) to phasic distending pressures of 5, 10, 20, 30, 40 and 60 mmHg, 30 s at 4-min intervals was determined. Responses to repeated CRD (10 trials of 40 mmHg, 30 s at 4 min intervals) were examined in six fibers. Thermal and chemical stimulation of the colon was produced by changing the temperature or composition of the Krebs solution with which the isolated colon was perfused. To monitor the temperature of the perfusate, a thermoprobe (Physitemp, type IT-1E) was introduced into the colon via the anal catheter. A total of 51 pelvic nerve afferent fibers were studied, nine of which were studied following partial characterization of another fiber in the same experiment. After characterizing responses to CRD, responses to thermal (heat and/or cold) stimulation generally were tested before examining responses to chemical stimulation (pH, inflammatory soup, bile salts; see below); thermosensitivity was not tested in five fibers.

Thermal stimulation of the colon was produced by ramp increases or decreases in temperature (37°C to 50-60°C or 37°C to 20°C, approximately 480 s) without changing intracolonic pressure while outflow was open. Responses to CRD (40 mmHg, 30 s) during the peak temperature were tested in some experiments by clamping the distal catheter. The responses of some fibers to repeated heat or cold stimuli (10 min intertrial interval) were also tested.

Chemical stimulation of the colon was produced by changing the pH of the perfusate, by perfusion with an inflammatory soup (IS; bradykinin 10-5 M, PGE2 10-5 M, serotonin 10-5 M, histamine 10-5 M and KCl 10-3 M, pH 7.35 or pH 5.50; Handwerker and Reeh 1991), or by adding bile salts (BS) to the perfusate. The pH of the perfusate was adjusted by adding HCl or NaOH to the Krebs solution. The effects of these chemical stimuli on spontaneous activity and responses of fibers to CRD were determined with the chemical-containing fluid in the colon; the colon was flushed with 37°C Krebs solution after chemical stimulation. Testing of other stimuli followed a recovery interval of 40-60 min (at which time responses to CRD returned to control).

Data analysis

The resting activity of a fiber was counted for 60 s before CRD and response was determined as the increase in discharge during distension above its resting activity. In some cases where chemical stimulation of the colon variably increased resting activity, the overall mean change in resting activity was taken into account to determine the response to CRD. SRFs to graded CRD were plotted for each individual fiber and a least-squares regression line was obtained from the linear part of the SRF. The regression line was then extrapolated to the ordinate (representing distension pressure) to estimate response threshold.

To estimate the response threshold to thermal stimulation, the mean and standard deviation (SD) of the resting activity was determined. Threshold was defined as the temperature at which unit activity increased >2 sd above resting activity. For fibers with no or low background activity, the response threshold was considered that temperature at which the fiber began and continued to discharge. Unit activity during thermal stimulation was counted in 10 s bins and the maximum response during thermal stimulation was defined as that bin with the greatest number of counts.

All data are expressed as mean ± SE. Results were analyzed using Student's t-test or an ANOVA (ANOVA) for repeated measures; P < 0.05 was considered statistically significant.

Chemicals and Drugs

Krebs solution of the following composition (mM): NaCl 118.0, KCl 0.7, NaHCO3 24.0, MgSO3 1.2, CaCl2 2.5, KH2PO4 1.1, and glucose 10.0, pH 7.3-7.4, was prepared from chemicals purchased from Sigma Chemical Co. (St. Louis, MO). BS (a mixture of sodium cholate and sodium deoxycholate), histamine hydrochloride (MW: 184.1), serotonin hydrochloride (MW: 212.7), PGE2 (MW: 352.5) and bradykinin (MW: 1060.2) were purchased from Sigma and dissolved in distilled water.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Fiber Sample

Thirty-one of the 51 distension-sensitive pelvic nerve afferent fibers studied (61%) were unmyelinated C-fibers with a mean CV of 2.0 ± 0.1 m/s (range, 1-2.5 m/s) and 20 (39%) were thinly myelinated Adelta -fibers with a mean CV of 5.2 ± 0.9 m/s (range, 2.8-14 m/s). Forty-nine fibers were spontaneously active (mean, 1.3 ± 0.3 imp/s; range, 0.01-7.1 imp/s); two C-fibers had no resting activity. The characteristics of this sample of S1 pelvic nerve afferent fibers are similar to what we found in earlier studies in which the colon was distended with air using a 7 cm long latex balloon (Sengupta and Gebhart 1994a; Su et al. 1997a; Su and Gebhart 1998; Table 1). All fibers gave sustained, phasic responses to fluid distension that were temporally linked to the distending stimulus (e.g., see Fig. 3). None gave on-off type, rapidly adapting responses suggestive of sensitivity to movement of fluid across the mucosa, but the tissue location of these mechanosensitive fibers cannot be determined from these experiments.

 
View this table:
[in this window] [in a new window]
 
TABLE 1. Summary of characteristics of pelvic nerve mechanosensitive afferent fibers innervating the colon of the rat


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 3. Response patterns of pelvic nerve afferent fibers to colorectal distension (CRD, 30 s). A: Examples illustrated as peri-stimulus time histograms (1 s binwidth) of responses of an adapting and nonadapting fiber to 40 mmHg CRD. B: comparison of responses of adapting (n = 34) and nonadapting (n = 17) fibers, plotted as mean impulses/s during the first 10 s of CRD or during the last 10 s of CRD (5-60 mmHg).

Mechanosensitivity

All 51 fibers gave monotonically increasing responses to graded CRD. As in previous studies in which the colon was distended with air and a balloon, the majority of pelvic nerve fibers had low thresholds (LT) for response to fluid distension of the colon (<10 mmHg; n = 46). Five fibers had high-thresholds (HT) for response (>25 mmHg). Individual SRFs of LT and HT afferent fibers are shown in Fig. 2 and the corresponding insets show the mean SRFs for each group of fibers. The mean extrapolated thresholds for response of LT and HT pelvic nerve afferent fibers to graded CRD was 2.4 ± 0.4 and 28.5 ± 0.8 mmHg, respectively.


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 2. Responses of pelvic nerve afferent fibers in the S1 dorsal root to graded colorectal distension (CRD, 30 s). A: individual stimulus-response functions (SRFs) of 46 fibers that responded at low threshold to CRD. The inset illustrates the mean SRF; the mean extrapolated response threshold was 2.4 ± 0.4 mmHg. B: individual SRFs of 5 fibers that responded at high-threshold to CRD. The inset illustrates the mean SRF; the mean extrapolated response threshold was 28.5 ± 0.8 mmHg.

RESPONSE PATTERN. Similar to balloon CRD (Sengupta and Gebhart 1994a), two different response patterns to fluid CRD were noted. One group of fibers (n = 34) gave an initial, dynamic response to CRD that adapted slowly during maintained distension. A second group of fibers (n = 17) gave incrementing responses to CRD during fluid distension. Examples are given in Fig. 3A; Fig. 3B summarizes responses over the pressure range studied. In a posthoc analysis, we examined whether the pattern of response to CRD was associated with sensitivity to chemical and/or thermal stimuli. The 17 nonadapting fibers studied are identified in Table 2. Aside from the observation that four of the five HT fibers were nonadapting, there are no obvious correlations.

 
View this table:
[in this window] [in a new window]
 
TABLE 2. Summary of responses of pelvic nerve mechanosensitive afferent fibers to chemical and thermal stimuli

REPRODUCIBILITY OF RESPONSES. Six fibers were tested for responses to repetitive CRD at 40 mmHg (30 s). None of the fibers exhibited any change in response magnitude or pattern to repeated distension at a 4 min interval between distensions. Fig. 4 shows the response a C-fiber to 10 successive colonic distensions and the responses of each of 6 fibers to repeated CRD.


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 4. Reproducibility of responses to repeated colorectal distension (CRD, 40 mmHg for 30 s). A: example of responses of an unmyelinated fiber (1.71 m/s) to 10 repeated distensions applied every 4 min. Responses of the fiber are illustrated as peristimulus time histograms (1 s binwidth); phasic distending pressure is presented below. B: summary responses of 6 fibers to repeated CRD plotted as the mean increase in impulses/s over the resting activity against the number of trials. `A' indicates the example illustrated in panel A.

Chemosensitivity

Responses to pH. The effect of transiently changing the pH of the perfusate on spontaneous activity and responses to 40 mmHg CRD was tested on 8 LT fibers (3 Adelta - and 5 C-fibers). Four fibers were exposed to both low (3.0) and high (11.0) pH (e.g., Fig. 5A); two fibers each were exposed for about 8 min to only low pH or only high pH. Neither the spontaneous activity nor mechanosensitivity of any of the fibers tested were affected; an example is given in Fig. 5A and the data are summarized in Fig. 5B and C; see also Table 2.


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 5. Effects of pH. A: Responses of a pelvic nerve afferent fiber, illustrated as peristimulus time histograms (1 s binwidth), to colorectal distension (CRD, 40 mmHg, 30 s) before (control) and during intracolonic instillation of pH3 or pH11 Krebs solution. Neither resting nerve activity of the 6 fibers tested (B) nor responses of the same fibers to 40 mmHg CRD (C) were affected by changing intracolonic pH.

RESPONSE TO BILE SALTS (BS). The mechanosensitive properties of 6 fibers (5 LT [3 Adelta - and 2 C-fibers] and 1 HT C-fiber) were tested before and after sequential instillation of 0.1%, 0.5% and 1% BS into the colon. The mean resting activity of the 6 fibers increased from a mean 0.2 ± 0.1 imp/s to 0.5 ± 0.3, 3.2 ± 1.2 (P < 0.05) and 4.2 ± 1.6 (P < 0.05) imp/s, respectively (Fig. 6B). No fibers exhibited sensitization of responses to CRD after instillation of BS in the colon (Fig. 6C). Fig. 6A illustrates an example of responses to CRD of an unmyelinated LT fiber in the presence of BS.


View larger version (42K):
[in this window]
[in a new window]
 
FIG. 6. Effects of bile salts (BS). A: Responses of a pelvic nerve afferent fiber, illustrated as peristimulus time histograms (1 s binwidth), to colorectal distension (CRD, 40 mmHg, 30 s) before (control), during intracolonic instillation of 0.05%, 0.1%, 0.5% and 1% BS and 60 min after flushing BS from the colon (recovery). B: Mean resting activity of 6 fibers tested before (control) and during intracolonic instillation of BS. C: Summary of responses (mean impulses/s) of the same 6 fibers to CRD (40 mmHg, 30 s) before (control) and during intracolonic instillation of BS.

RESPONSES TO INFLAMMATORY SOUP (IS). The mechanosensitive properties of 22 fibers (20 LT [9 Adelta - and 11 C-fibers] and 2 HT C-fibers) were tested before and after instillation of IS into the colon (13 fibers at pH 7.35; 9 fibers at pH 5.5). IS remained in the colon for the duration of these experiments (60 min). The resting activity of 10/22 fibers was unaffected (pH 7.35, 5 LT [3 Adelta - and 2 C-fibers]; pH 5.5, 5 LT [1 Adelta - and 4 C-fibers]); the resting activity of 12 fibers (pH 7.35, 6 LT [4 Adelta - and 2 C-fibers] and 2 HT C-fibers; pH 5.5, 4 LT [1 Adelta - and 3 C-fibers]) increased significantly from a mean 0.8 ± 0.5 imp/s to 1.43 ± 0.5 and 2.59 ± 0.8 imp/s 30 and 60 min, respectively, after intracolonic instillation of IS (F = 4.11, P <0.05; Fig. 7B). These 12 fibers also exhibited sensitization of responses to graded CRD 30 and 60 min after intracolonic instillation of IS (an example is shown in Fig. 7A). An additional LT C-fiber did not show an increase in resting activity, but its response to CRD after pH 5.5 IS was increased. Fig. 7C illustrates responses of these 13 sensitized fibers 60 min after intracolonic instillation of either pH 7.35 (F = 11.56, P < 0.001) or pH 5.5 (F = 6.97, P < 0.01) IS. Although pH 5.5 IS produced an apparent greater sensitization than did pH 7.35 IS, the difference is not statistically significant (F = 1.09. P = 0.30).


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 7. Effects of inflammatory soup (IS). A: Responses of a pelvic nerve afferent fiber, illustrated as peristimulus time histograms (1 s binwidth), to graded colorectal distension (CRD, 5 to 60 mmHg, 30 s) before (control) and 30 and 60 min after intracolonic instillation of IS. IS remained in the colon for the duration of these experiments (60 min). B: Resting activity before (control) and 30 and 60 min after intracolonic instillation of IS (pH 7.35 or pH 5.5). C: Summary of responses of pelvic nerve afferent fibers to CRD before (control, con) and 60 min after intracolonic instillation of IS (pH 7.35, n = 8; pH 5.5, n = 5).

The mean threshold for response, another indication of sensitization, decreased in 7/13 fibers with response thresholds > 3 mmHg before IS treatment (range: 3-30 mmHg). Response threshold decreased from a mean 11.8 ± 4.1 to 3.8 ± 1.5 (P < 0.05) and 2.6 ± 1.6 (P < 0.05) mmHg before and 30 and 60 min, respectively, after pH 7.35 or 5.5 IS treatment. Pretreatment response thresholds of the remaining 6 sensitized fibers (increased responses to CRD) were near 0 mmHg and could not have decreased.

Thermosensitivity

The responses of mechanosensitive afferent fibers to intracolonic perfusion of hot and/or cold Krebs solution were also tested. The method of colonic perfusion did not allow us to determine precisely the temperature at the location of the endings, but three types of receptors were distinguished according to the apparent temperature at which they were activated. Heat receptors responded to temperatures >= 42°C, cold receptors responded to temperatures <= 30°C, and mixed receptors responded to both heat and cold thermal stimuli.

RESTING ACTIVITY. Forty-five fibers were tested for response to heat stimulation and 33 (73%) responded (an example is given in Fig. 8A). Mean resting activity increased from 1.5 ± 0.4 to a mean maximum 17.3 ± 2.3 imp/s (n = 33) during intracolonic perfusion with 50°C Krebs solution; the mean estimated response threshold was 44.9 ± 1.2°C. Three of 45 fibers failed to respond during the increase in colonic temperature, but exhibited an afterdischarge when heat stimulation was terminated. Eighteen fibers were also tested for possible sensitization of response to a second heat stimulus 10 min after the first stimulus (Fig. 8B). One fiber responded only to the 1st trial and two fibers responded only to the 2nd trial. Although some fibers exhibited variability in response threshold and/or magnitude between the two trials, the mean response thresholds of the first and the second trials were not different (45.0 ± 1.4°C and 45.4 ± 1.3°C, respectively) and the mean maximum magnitude of response to heat on the first and the second trials also did not differ (17.5 ± 2.9 imp/s and 14.5 ± 3.0 imp/s, respectively).


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 8. Effects of thermal stimulation. A and C: Responses of different pelvic nerve afferent fibers, illustrated as peristimulus time histograms (1 s binwidth), to intracolonic instillation of hot or cold Krebs solution, respectively; intracolonic temperatures are shown below. B and D: response thresholds (left axes) and response magnitudes to 40 mmHg colorectal distension (40 mmHg, 30 s; right axes) of pelvic nerve afferent fibers on different trials of heat (B) or cold (D) stimuli applied intracolonically.

Thirty-seven fibers were tested for response to cold stimulation and 11 (30%) responded (Fig. 8C). Resting activity increased from a mean 0.3 ± 0.1 to 14.2 ± 4.5 imp/s during intracolonic perfusion with 20°C Krebs solution. The mean estimated response threshold was 27.7 ± 1.4°C. No fiber exhibited an afterdischarge when cold stimulation was terminated. Six fibers were also tested for responses to a second cold stimulus 10 min after the first stimulus. All responded to both trials (Fig. 8D); the response threshold for one fiber decreased about 11°C, but the overall mean response threshold did not change significantly between trials (28.2 ± 2.0°C versus 26.3 ± 1.7°C). The mean maximum response to cold stimulation decreased from 18.5 ± 5.8 to 14.3 ± 4.6 imp/s (P > 0.05).

Of 36 fibers tested, 8 were activated by both heat and cold; typically, fibers activated by heat did not respond to cold.

RESPONSE TO CRD. The responses of 30 fibers (25 heat-sensitive and 5 heat-insensitive; 18 adapting and 12 nonadapting) to CRD were also tested during the peak temperature (50°C) by clamping colonic outflow. Twenty-two fibers (17 heat-sensitive and 5 heat-insensitive; 14 adapting and 8 nonadapting) exhibited a significant increase in the initial 10 s dynamic response to CRD (from 26.8 ± 2.2 to 41.4 ± 2.8 imp/s, P < 0.01) during heat stimulation and adapted quickly during maintained 40 mmHg distension. In eight heat-sensitive fibers (4 adapting and 4 nonadapting), the magnitude of responses to CRD during the second heat stimulation was significantly reduced (P < 0.05). The data are summarized in Fig. 9A.


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 9. Summary of effects of intracolonic instillation of hot or cold Krebs solution on response patterns of pelvic nerve afferent fibers to colorectal distension (CRD, 40 mmHg, 30 s). Responses at peak temperature of heat (A) or cold (B) are illustrated as mean peristimulus time histograms (1 s binwidth).

The responses of 29 fibers (8 cold-sensitive and 21 cold-insensitive) to CRD were also tested at 20°C. All fibers gave a decreased response to 40 mmHg CRD (from 28.8 ± 2.4 to 12.2 ± 1.8 imp/s; P < 0.01); data are summarized in Fig. 9B.

Conduction velocity (CV)

There were no differences in the distribution of CVs of heat or cold sensitive and insensitive fibers (Fig. 10). CV was also measured in some fibers after heat (n = 15), cold (n = 22), BS (n = 3) and IS (n = 7) stimuli; no changes in CV were noted.


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 10. Frequency histograms of conduction velocity of temperature-sensitive and temperature-insensitive pelvic nerve afferent fibers. n, number of fibers.

Responses of colonic muscles

To determine whether chemical or thermal stimuli affected muscle tone of the colon, intracolonic pressure was recorded during some trials. Heat produced a mean decrease in intracolonic pressure from 7.3 ± 2.4 to 3.9 ± 1.9 mmHg (n = 8; P > 0.05) and cold produced a slight increase in intracolonic pressure (from 6.4 ± 2.8 to 7.4 ± 2.2 mmHg, n = 7; P > 0.05). BS and IS did not change intracolonic pressure.

Polymodal responses

Twenty-six mechanosensitive fibers were exposed to chemical (BS or IS) and thermal stimuli. Eleven fibers responded to all three modalities of stimulation: mechanical, chemical and thermal stimuli (7 sensitive only to heat, 3 sensitive to both heat and cold, and 1 sensitive only to cold); 15 fibers responded to either chemical or thermal stimulation (in addition to distension). There were no differences in the thermo- and/or chemosensitivity between Adelta - and C-fibers (Fig. 11).


View larger version (67K):
[in this window]
[in a new window]
 
FIG. 11. Summary of response properties of 25 mechanosensitive pelvic nerve afferent fibers that were tested for response to chemical (bile salts or inflammatory soup) and thermal stimuli. There were no differences in the distribution of response properties between Adelta - and C-fibers.

All but 4 of the remaining 25/51 mechanosensitive fibers in which only either chemical or thermal stimuli were tested responded to the second stimulus. Thus, among the 51 fibers studied, 47 (92%) responded to another modality of stimulation (either chemical and/or thermal) in addition to the distending mechanical stimulus used to initially characterize the fiber. Table 2 summarizes the response of all fibers tested.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In previous studies, using constant pressure air distension of a balloon inserted in the colon, we documented the presence of low- and high-threshold pelvic nerve afferent fiber populations innervating the colon and also that responses to repetitive colonic distension were stable (Sengupta and Gebhart 1994a; Su et al. 1997a; Su and Gebhart 1998). Similarly, in the present study, using Krebs solution to distend the colon in situ, we found populations of low- and high-threshold pelvic nerve afferent fibers in the S1 dorsal root. We also documented that responses to repeated fluid distension of the colon are stable, thus permitting quantitative evaluation of alterations in mechanosensitivity produced by chemical stimuli.

Chemosensitivity of mechanosensitive afferents

A number of studies have reported that mechanosensitive visceral afferent fibers are also sensitive to chemicals, principally algesic or irritant chemical stimuli (see Introduction). We previously reported that intracolonic instillation of acetic acid or mustard oil can sensitize mechanosensitive afferent fibers innervating the colon (Sengupta et al. 1996; Su et al. 1997a). In complementary studies, urinary bladder instillation of irritant xylenes or mustard oil was shown to sensitize mechanosensitive pelvic nerve afferent fibers innervating the bladder (Su et al. 1997b). In the present study, intracolonic instillation of IS increased resting activity and the magnitude of response of 13/22 fibers to CRD throughout the range of distending pressures tested. IS also reduced the threshold for response of fibers which had pre-IS response thresholds >3 mmHg. Numerous potential mediators are released and synthesized during tissue injury and inflammation and one chemical does not likely represent the adequate stimulus. That multiple putative mediators constitute an adequate stimulus was suggested by Handwerker and Reeh (1991). In an in vitro skin-nerve preparation, applying an IS induced greater excitation (80%) of nociceptive C-fibers (Kessler et al. 1992) than did application of serotonin (20%), bradykinin (50%) (Lang et al. 1990) or histamine (14%) (Koppert et al. 1993). Bove and Moskowitz (1997) reported that 79% of nasociliary nerve fibers innervating the dura are sensitive to a combination of inflammatory mediators similar to the composition of IS used here. Handwerker and Reeh (1991) further documented that 40% of cutaneous polymodal receptors were excited by increasing the H+ concentration to a pH of 5.2. Similarly, we noted that the effect of IS on responses to CRD was enhanced by increasing the H+ concentration to pH 5.5.

We did not find that transient exposure of the colon to low or high pH Krebs perfusate changed either resting activity or responses to CRD of afferent fibers. In support, colonic mucosal afferent fibers in the ventral root of the cat colon were also not sensitive to 0.1 N HCl (Clifton et al. 1976). We previously reported that pH 3.0 acetic acid (2 ml, 2.5%) retained in the rat colon for 15 min sensitized about one-half of a sample of 32 mechanosensitive pelvic nerve fibers to 60-100 mmHg balloon CRD when tested 30-60 min after acetic acid instillation (Sengupta et al. 1996). Acetic acid can cause mucosal barrier dysfunction (Gardiner et al. 1995) and can lead to neutrophil infiltration, hemorrhage and necrosis (Lowe and Noronha-Blob 1993). Exposure of the colonic mucosa to low pH in the present experiments was of relatively short-duration and fluid CRD was limited to 40 mmHg; changes in resting activity or responses to CRD were not seen in the sample tested. BS did not increase the magnitude of response to noxious CRD, but BS did significantly increase the resting activity of afferent fibers. BS are normally present in the colon (Variyam 1992; Rodrigues et al. 1995) where they reportedly stimulate synthesis of LTB4, an inflammatory mediator in inflammatory bowel disease (Dias et al. 1994). Rather than sensitizing visceral afferent fibers, LTB4 has been reported to decrease responses of abdominal visceral afferent fibers (Pan et al. 1995). In the present study, all 4 HT fibers tested with BS or IS were affected whereas 9/25 LT fibers were unresponsive. This is supported by Pan and Longhurst's (1996) report that ischemia-sensitive abdominal afferent fibers had higher thresholds to mechanical distension whereas ischemia-insensitive receptors had lower thresholds to mechanical distension.

Thermosensitivity of mechanosensitive afferents

Thermal stimuli are adequate for some visceral sensory fibers, producing both reflex responses (e.g., bladder cooling reflex, Fall et al. 1990) and conscious sensation (e.g., drinking a cold beverage on a empty stomach; Webber et al. 1980). We found that 33/45 fibers tested were heat sensitive, 11/37 fibers were cold sensitive and 8/36 fibers responded to both hot and cold Krebs solution. Thermosensitive fibers were estimated to have response thresholds of 28°C for cold sensitive fibers and 45°C for heat sensitive fibers. Apparent sensitization or desensitization was observed in a few fibers by repeat stimulation with heat or cold, but generally response thresholds and response magnitudes to thermal stimulation were similar on the second presentation of the thermal stimulus 10 min after the first stimulus. Sensitization, particularly to repeated heat stimulation, might have been expected to develop, but the number of trials (two) and interval between trials (10 min) was limited in the present experiments. These thermosensitive pelvic nerve fibers resemble thermoreceptors in skin and the testis and epididymis (Iggo 1959; Kumazawa and Mizumura 1980a). Compared with other visceral thermoreceptors, pelvic nerve thermoreceptors in the colon have similar response thresholds to those in the vagus nerve in the gastroesophageal region (EL Ouazzani and Mei 1982) and in the gastrointestinal vagal territory (EL Ouazzani and Mei 1979). Splanchnic cold receptors have also been described in the gastrointestinal tract (Gupta et al. 1979) and receptors that respond to both mechanical and thermal stimuli have been described in the duodenum (Cottrell 1984). Mechanosensitive mucosal afferent fibers in sacral ventral roots innervating the colon and rectum of the cat also responded to hot and cold temperatures (Clifton et al. 1976).

Kumazawa et al. (1987) reported that chemical responses of polymodal receptors can be modulated by temperature. Similarly, we noted that responses to mechanical stimulation decreased during cold stimulation. It could be that the decrease in temperature altered stimulus transduction or CV (most likely the former). CV of the fibers, tested by electrical stimulation at a site distant from the receptor, did not change. Heat stimulation produced mixed effects. In 22 fibers, there was an increase in the dynamic component of the response to CRD; in 8 fibers, attenuated responses to CRD were observed at the peak temperature. As in earlier work (Sengupta and Gebhart 1994a), we noted two patterns of response to CRD. Adapting and nonadapting responses of colonic afferents have also been noted by others (Blumberg et al. 1983; Jänig and Koltzenburg 1991). As suggested previously (Sengupta and Gebhart 1994a), the initial dynamic response likely represents an increase in muscle tension that develops during active resistance to distension offered by the muscle and of intrinsic excitatory reflexes produced by the myenteric plexus. The firing rate then slowly declines as muscle tension decreases during the "receptive" reflex; with termination of distension, the firing often ceases because of the sudden loss of muscle tension. Thermal stimulation appeared to have its greatest effect on the dynamic component of the response. The mechanisms and possible functional role of altered mechanosensitivity by thermal stimuli remains to be studied. It is conceivable that changes in adaptation by heat can be caused by changes in the excitability of the spike generator membrane during prolonged distension (e.g., heat may increase the activity of an electrogenic Na-K+ pump or membrane inactivation of Na+; cold may have an opposite effect).

Polymodal receptors

The effects of chemical and thermal stimuli reported here could be due to a change in conduction of nerve action potentials or compliance of the colon and not on neuron sensory endings (receptors). In the present study, CVs of the fibers remained unaffected after intracolonic application of IS, BS and hot or cold perfusate. It seems unlikely, however, that changes in CV of the axon, measured distant from the colon, would be produced by intracolonic treatments. Recording of intracolonic pressure revealed that cold and heat stimulation can produce a slight increase or decrease in intracolonic pressure, respectively; IS and BS did not change the tension of the smooth muscle. In experiments in rats pretreated with loperamide to paralyze the smooth muscle, we observed similar effects of IS, BS, heat and cold (Su and Gebhart, unpublished data). Accordingly, the chemical and thermal effects reported here likely occur at receptors associated with afferent nerves innervating the colon; that is, visceral afferent fibers innervating colon are polymodal in character. Like the high proportion (90%) of polymodal receptors in testis (Kumazawa and Mizumura 1980a,b; Kumazawa et al. 1987), we found that 92% of mechanosensitive receptors in the colon are polymodal (i.e., respond to at least two stimulus modalities).

We found no differences between polymodal Adelta - and C-fibers in the pelvic nerve in response to thermal and/or chemical stimuli. It has been noted in cutaneous nerve that units with faster CVs generally have lower mechanical thresholds and also are less responsive to heat stimulation and algesic substances. It was proposed by Kumazawa (1996), however, that the criterion most important in classifying a sensory receptor as transmitting nociceptive information is not CV, but whether it can encode changes in the tissue surrounding the receptor terminals that are produced by noxious events.

Detailed information about the response-properties of polymodal receptors is still lacking. Szolcsányi (1993) reported that polymodal nociceptive axons terminate in a "chain of beads," which were considered to be multiple sensor sites. Some of these sensor beads are able to be activated by mechanical stimuli directly or indirectly by releasing chemical mediators from the tissue (Hamill et al. 1992). Other sites are preferentially depolarized by noxious heat or chemical stimuli (Szolcsányi 1993). Most studies of polymodal receptors, including this one, have focused on responses to applied stimuli and do not address either the precise location of the receptors or transduction mechanisms. We acknowledge that some responses reported here could be indirect (e.g., through release of an endogenous chemical from specialized cells in the tissue which activates the nerve terminal) and cannot document that the afferent fibers recorded here and characterized as polymodal contain on their peripheral terminal receptors for all three stimulus energies. Functionally, however, these fibers are polymodal in character and transmit information from the tissue about mechanical, thermal and chemical stimuli.

Functional Significance

Visceral afferent fibers, in the presence of tissue inflammation, can become sensitized, contribute to central hyperexcitability and lead to visceral hyperalgesia. Most mechanosensitive colon afferent fibers have a wide dynamic range of response and can be sensitized to nonnoxious intensities of distension. There are a number of clinical conditions, categorized as functional bowel disorders, including nonulcer dyspepsia, noncardiac chest pain and irritable bowel syndrome, that are characterized by discomfort and pain in the absence of tissue inflammation or apparent pathology (Mayer and Gebhart 1994). These disorders are complex and involve both peripheral and central contributions. It seems apparent now that a significant component of the discomfort and pain associated with the functional bowel disorders is associated with altered sensory input or altered integration in the CNS. The knowledge of adequate stimuli for receptors in the gastrointestinal tract and improved knowledge of their basic physiology will help understand the extent to which peripheral contributions from the organ itself or visceral receptors contribute to the altered sensations and pain that characterize functional bowel disorders.

    ACKNOWLEDGEMENTS

  The authors thank Mike Burcham for preparation of the graphics. This study was supported by NIH award NS-19912; Xin Su was supported in part by an award from Pfizer Central Research, Sandwich, UK.

    FOOTNOTES

  Address for reprint requests: X. Su, Dept. of Pharmacology, The University of Iowa, Bowen Science Bldg., Iowa City, IA 52242.

  Received 13 April 1998; accepted in final form 29 July 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society