Sensory Fibers of the Pelvic Nerve Innervating the Rat's Urinary Bladder

V. K. Shea,1 R. Cai,1 B. Crepps,1 J. L. Mason,2 and E. R. Perl1

 1Department of Cell and Molecular Physiology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7545; and  2Department of Pathology, Physicians and Surgeons, Columbia University, New York, New York 10027


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Shea, V. K., R. Cai, B. Crepps, J. L. Mason, and E. R. Perl. Sensory Fibers of the Pelvic Nerve Innervating the Rat's Urinary Bladder. J. Neurophysiol. 84: 1924-1933, 2000. Much attention has been given to the pelvic nerve afferent innervation of the urinary bladder; however, reports differ considerably in descriptions of afferent receptor types, their conduction velocities, and their potential roles in bladder reflexes and sensation. The present study was undertaken to do a relatively unbiased sampling of bladder afferent fibers of the pelvic nerve in adult female rats. The search stimulus for units to be studied was electrical stimulation of both the bladder nerves and the pelvic nerve. Single-unit activity of 100 L6 dorsal root fibers, activated by both pelvic and bladder nerve stimulation, was analyzed. Sixty-five units had C-fiber and 35 units had Adelta -fiber conduction velocities. Receptive characteristics were established by direct mechanical stimulation, filling of the bladder with 0.9% NaCl at a physiological speed and by filling the bladder with solutions containing capsaicin, potassium, or turpentine oil. The majority (61) of these fibers were unambiguously excited by bladder filling with 0.9% NaCl and were classified as mechanoreceptors. All mechanoreceptors with receptive fields on the body of the bladder had low pressure thresholds (<= 10 mmHg). Receptive fields of units with higher thresholds were near the ureterovesical junction, on the base of the bladder or could not be found. Neither thresholds nor suprathreshold responses could be related to conduction velocity. Bladder compliance and mechanoreceptor thresholds were influenced by the stage of the estrous cycle: both were lowest in proestrous rats and highest in metaestrous rats. Mechanoreceptors innervating the body of the bladder and the region near the ureterovesical junction showed two patterns of responsiveness to slow bladder filling. One group of units exhibited increasing activity with increasing pressure up to 40 mmHg, while the other group showed a peak in activity at pressures below 40 mmHg followed by a plateau or decrease in activity with increasing pressure. It is proposed that differences in stimulus transduction relate to the different response patterns. Thirty-nine units failed to respond to bladder filling. Eight of these were excited by intravesical potassium or capsaicin and were classified as chemoreceptors. The remaining 31 units were not excited by any stimulus tested. Chemoreceptors and unexcited units had both Adelta and C afferent fibers. We conclude that the pelvic nerve sensory innervation of the rat bladder is complex, may be sensitive to hormonal status, and that the properties of individual sensory receptors are not related in an obvious manner to the conduction velocity of their fibers.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The pelvic nerves are the principal pathway for afferent input related to urinary continence, voiding, and the sensations of bladder fullness, discomfort, and pain (de Groat 1993, 1997; Jänig and Koltzenburg 1993). Surprisingly, despite considerable work, a consistent picture of the sensory receptor types in this pathway, their roles in bladder reflexes and in sensations has yet to emerge.

Several studies suggest that the receptive properties of the bladder's afferent neurons in the pelvic nerve are correlated with the conduction velocities of their fibers. In the cat, it is reported that Adelta units form a homogeneous group of sensitive "tension" mechanoreceptors, while the few C fiber units excited by bladder stimulation respond only to very elevated pressure (30-50 mmHg) or irritant chemicals (Bahns et al. 1987; Häbler et al. 1990, 1993a,b). In the rat, both C and Adelta fibers are excited by bladder distension but on the average those with C fibers are reported to have higher pressure thresholds and respond less vigorously than those with Adelta fibers (Dmitrieva and McMahon 1996; Morrison 1997). Pelvic nerve afferent fibers described as initially unresponsive to bladder distension with 0.9% NaCl but excited by intravesical potassium or nerve growth factor ("silent" afferents) generally are reported to conduct at C velocities (Dmitrieva and McMahon 1996; Jiang and Morrison 1995).

A different picture emerges from the studies in the rat by Sengupta and Gebhart (1994b) and Su et al. (1997). They found no demonstrable relationship between conduction velocity and functional properties of pelvic nerve bladder mechanoreceptors. Units unresponsive to mechanical stimulation of pelvic structures conducted at both C and Adelta velocities.

The long-standing idea that nearly all vesical mechanoreceptors are in series tension receptors, responsive to both distension and to contraction (Iggo 1955), has recently been challenged. Morrison (1997) describes vesical mechanoreceptors in the rat that respond to distension but not to contraction and defines them as "volume" receptors.

Many clinical disorders of the bladder such as interstitial cystitis present primarily with sensory symptoms. Knowledge of the normal afferent innervation is a necessary prerequisite to understanding these disorders. Experience with cutaneous afferent neurons has shown the use of "natural" stimulation as a search can bias the findings and requires repeated and potentially damaging stimuli to uncover units with high thresholds (Burgess and Perl 1967; Perl 1996). Previous studies of bladder afferent units are open to this criticism because they employed repeated bladder distensions with 0.9% NaCl or intravesical instillation of other chemicals to identify bladder receptors from the mixed population of pelvic sensory fibers. Additionally, only units responsive to the stimulus employed can be identified as forming part of the bladder's innervation. The major aim of our study was to provide a representative survey of the properties of pelvic nerve afferent fibers innervating the bladder of the rat. The goal was to include units regardless of their mechanical or chemical sensitivity while also avoiding the complications of unnatural stimulation as much as possible. Preliminary reports of some of these observations have been presented (Mason et al. 1995; Shea et al. 1995).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adult, virgin female Sprague-Dawley rats (200-400 g) were housed one to three animals per cage under institutional care on a 12:12 h light-dark cycle. Vaginal smears were obtained by lavage on the day of terminal experiments at approximately 9:00 am, 2 h after lights were turned on. The animals were anesthetized with urethan (1.5 g/kg ip); supplemental doses were administered through a cannula in the external jugular vein as needed to maintain deep anesthesia (judged by the absence of corneal and limb withdrawal reflexes). The trachea was cannulated and the animals allowed to respire spontaneously. Body temperature was maintained by a heated support. At the end of experiments, the animals were killed by an overdose of urethan. This protocol was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Surgical preparation

Abdominal and pelvic structures were exposed by an incision along the flank on the left side. The bladder was emptied and a two-barrel cannula (PE50) with a heat-flared end was inserted into the dome of the bladder and secured by silk suture. One barrel was attached to a pressure transducer for continuous measurement of intravesical pressure. Solutions were introduced into and removed from the bladder through the second barrel (Fig. 1A). The ureters were cannulated (PE10) to allow urine to drain externally and were ligated close to the bladder. The urethra was ligated externally, completing a closed system.



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Fig. 1. A: schematic diagram of the procedures used to identify and record from pelvic nerve afferent units innervating the urinary bladder. Unitary discharge was recorded from fine filaments dissected from the L6 dorsal root and evoked by electrical stimulation of both the bladder nerves distal to the major pelvic ganglion (MPG) and the pelvic nerve. The ureters and the external urethra were ligated forming a closed system. Fluids were instilled through the infusion cannula while intravesical pressure was continuously monitored. B: for receptive field exploration, the bladder was divided into 3 regions: the body, the vicinity of the ureterovesical junction (uvj), and the base. The receptive fields of units responsive to direct mechanical probing of the bladder were mapped to one of these regions.

The left bladder nerves were identified and carefully dissected distal to the major pelvic ganglion (MPG). The left pelvic nerve was identified and cleared from surrounding tissue proximal to the MPG. The sciatic nerve and accessible nerves at the base of the tail were cut to reduce somatic afferent activity and thereby facilitate isolation of bladder afferent units. The lumbosacral region of the spinal cord was exposed by laminectomy and the dura was carefully opened. The left L6 dorsal root was identified and cut close to its entrance to the spinal cord.

Electrophysiological recordings

Pairs of Teflon-coated silver wire (0.005 in.) electrodes, stripped at the tips, were placed around the bladder nerves and the pelvic nerve. Both electrodes were encased in Wacker SilGel (Sengupta and Gebhart 1994a). A pool was formed by tying the dorsal skin margins to a metal ring and filled with warm (37°C) mineral oil with the animal positioned on its side so that the bladder was accessible for direct mechanical stimulation.

Fine filaments were dissected from the L6 dorsal root and placed across shielded bipolar platinum electrodes. Afferent activity was amplified, made audible through a loudspeaker, and viewed on an oscilloscope. Filaments were teased until a unique unitary discharge was evoked by electrical stimulation (0.1-0.5 ms square wave pulse, 0.33 Hz) from both the bladder nerves and from the pelvic nerve which could be clearly distinguished from all other neural activity. Under the conditions of this study, nerve stimulation rarely evoked bladder contractions and no assessment of responses to natural stimulation was carried out within 30 s of electrical stimulation. Conduction velocities were estimated from the latency of response to electrical stimulation of the pelvic nerve and the conduction distance; no account was made for stimulus utilization time. In separate experiments to estimate the ranges of conduction velocities expected of afferent units of the pelvic nerve, compound action potentials were evoked by electrical stimulation of L6 dorsal root and recorded in the pelvic nerve or were evoked by stimulation of the pelvic nerve and recorded in the L6 dorsal root.

Characterization of bladder afferent neurons

Mechanically responsive receptive fields of single bladder units were sought by gently probing the serosal surface of the bladder with the smoothed, rounded tip of a glass rod. The approximate position of a receptive field, if found, was located on a schematic representation of the accessible regions of the bladder divided into three segments: the body, the vicinity of the ureterovesical junction (uvj), and the base (Fig. 1B).

Pelvic nerve bladder afferent units included in this survey were assessed for their responses during slow filling of the bladder with room temperature 0.9% NaCl at 100 µl/min, a rate achieved in the conscious rat during diuresis (Pollock et al. 1986). Slow filling of the empty bladder was initiated after a minimum of 1 min of control observations of ongoing activity and starting intravesical pressure (-2 to 4 mmHg). Filling was continued until intravesical pressure was 40 mmHg or greater at which point the infusion cannula was disconnected and the bladder was allowed to empty.

Some units were subsequently tested for responses to intravesical instillation of a volume (~75% of that resulting in 40 mmHg during slow filling with 0.9% NaCl) of solution containing capsaicin, turpentine oil, or KCl. The chemical test solutions remained in the bladder for about 1 min. To minimize potential damage to the bladder secondary to repeated filling against a closed outlet, one unit (64 cases) or two to three units in one filament (5 cases) were studied in a given experiment. In 11 experiments 2-3 units were studied consecutively. No unit was studied after chemicals other than 0.9% NaCl had been instilled into the bladder. Stock solutions of 100 mM capsaicin(Sigma) in ethanol were diluted with 0.9% NaCl. KCl was dissolved in deionized water at a concentration of 300 mM, and lower concentrations were made by dilution with 0.9% NaCl. Turpentine oil was diluted 50:50 with mineral oil.

Data acquisition and analyses

Analog signals of unitary activity and intravesical pressure were simultaneously displayed on a chart recorder and stored on magnetic tape. The neural activity was analyzed after digital conversion using an impulse shape recognition program adapted for MS-DOS computers (from Schmittroth in Bessou and Perl 1969); it was used to confirm the unitary nature of the discharges evoked by bladder stimuli with those evoked from electrical stimulation of the bladder and the pelvic nerves. Graphs and statistical analyses were made using Prism 2 (Graphpad Software) or Microsoft Excel. Averaged data are presented as means ± SE.

The volume of 0.9% NaCl at which intravesical pressure (between bladder contractions) increased 10 mmHg (V10) above resting pressure was determined to provide an estimate of bladder compliance. Ongoing (resting) activity of a unit was defined as the mean activity (in 4-s bins) occurring during the 60-75 s before initiation of slow filling of the bladder with 0.9% NaCl and expressed as impulses/s. The thresholds of mechanoreceptive units responding during slow filling of the bladder with 0.9% NaCl were estimated by establishing the intravesical pressure (above resting) and volume from the time during filling at which activity increased. For units with no ongoing activity, intravesical pressure and volume at the time of occurrence of the first impulse were defined as the threshold values. For units exhibiting ongoing activity, thresholds were defined as the intravesical pressure and volume at the time when activity (in 4-s bins) exceeded the mean +3 SD of ongoing activity.

Relationships between mechanoreceptor activity and intravesical pressure were established by comparing at 1-s intervals the neural activity and the corresponding intravesical pressure (from initiation of filling to the time when the system was opened to air). Intravesical pressures and the corresponding unitary activity were averaged at 5-mmHg intervals, and average unitary activity was graphed as a function of intravesical pressure. The short-term effects of solutions containing capsaicin or KCl were evaluated by comparing activity during and following instillation of capsaicin or KCl to that evoked by the same volume of 0.9% NaCl.

Vaginal smears were air-dried and stained with Wrights-Giemsa. Based on smears obtained from 69 animals, 8 rats were in proestrus, 24 in estrus, 11 in metaestrus, and 26 in diestrus on the morning of the experiments.

Results were statistically analyzed using the following methods: Student's t-test for comparing two samples, ANOVA followed by Tukeys' post test for comparison of more than two groups, and chi 2 tests for contingency tables.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cystometrograms

Under the experimental conditions used in this study (Fig. 1A), the intravesical pressure changes during filling (cystometrograms) consisted of a slowly varying component with transient changes often superimposed. The compliance of the bladder determined during the first filling in 68 experiments varied significantly (P < 0.05) in relationship to the stage of the rat's estrous cycle (Fig. 2).



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Fig. 2. Effect of the stage of the estrous cycle on bladder compliance. The average V10 values (volume of 0.9% NaCl required to increase intravesical pressure 10 mmHg above resting pressure) determined during the 1st slow filling of the bladder in 68 experiments are plotted as a function of the stage of the estrus cycle. Eight rats were in proestrus (p) on the morning of the experiment, 24 in estrus (e), 10 in metaestrus (m), and 26 in diestrus (d). * Significantly different from all other groups (P < 0.05).

Bladder sensory units

In 80 experiments the responses of 100 single units, excited by electrical stimulation of both the bladder and the pelvic nerves, were recorded in left L6 dorsal roots. The selection of units studied was based solely on a response to electrical stimulation of the pelvic and bladder nerves. The conduction velocity of the individual fibers ranged from 0.5 to 10.6 m/s (Fig. 3A). Compound action potentials, such as that illustrated in Fig. 3B, were characterized by two deflections. The faster deflection in this example was biphasic and reflected activity in fibers conducting between about 2 and 10 m/s. Conduction velocities calculated from the leading edge of the slower (C) deflections ranged from 0.8 to 1.4 m/s. The C conduction velocity values are in agreement with those reported for the initial deflection of the slowest component of compound action potentials evoked by stimulation of bladder nerves and recorded in the pelvic nerve (Purinton et al. 1976). This evidence, together with the unimodal distribution of units conducting <= 1.7 m/s (Fig. 3A), led to the classification of units conducting <= 1.7 m/s as C fibers and those conducting more rapidly as Adelta fibers. On these criteria, 65 units of our sample were C fibers and 35 units were Adelta fibers, a relative proportion of C and Adelta units that agrees closely with a previous systematic analysis of bladder nerve afferent fibers in rat in which units conducting under 2.5 m/s were considered to be C fibers (Vera and Nadelhaft 1990).



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Fig. 3. Conduction velocities. A: distribution of conduction velocities (m/s) of pelvic nerve bladder units: all units sampled, mechanoreceptive units excited by slow filling of the bladder with 0.9% NaCl, chemoreceptive units unresponsive to slow filling of the bladder with 0.9% NaCl but excited by intravesical capsaicin or KCl (KCl-responsive units indicated with *) and units unexcited by bladder filling with 0.9% NaCl and all other stimuli tested. B: an example of a compound action potential evoked by stimulation of L6 dorsal root (cut centrally) and recorded from the pelvic nerve (cut distally). Arrow indicates the onset of the 0.5-ms electrical stimulus. Approximate conduction velocities (calculated by dividing the conduction distance of 50 mm by latency) are shown below.

Sixty-one units were unambiguously excited by slow filling of the bladder with 0.9% NaCl and were classified as mechanoreceptors. Ipsilateral mechanically excitable receptive fields were located for 52 of these. Eight of the 39 units that failed to respond to filling of the bladder with 0.9% NaCl were excited when the intravesical solutions contained capsaicin or KCl and were categorized as chemoreceptors. The remaining 31 units unresponsive to bladder filling with 0.9% NaCl failed to respond to the tested chemicals and were classified as "unexcited," although two of these were minimally responsive to direct probing of the bladder. The likelihood of a unit exhibiting mechanical sensitivity did not vary with the estrous cycle (P > 0.05). Properties of the units forming these groups are summarized in Table 1.


                              
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Table 1. Summary of the characteristics of bladder afferent units of the rat's pelvic nerve

The conduction velocities (Fig. 3) of the units forming the various groups were similar; differences in mean values (Table 1) could be attributed to chance (P > 0.05). Ongoing activity appeared in 36/100 bladder units and was more common (P < 0.0001) for distension-responsive mechanoreceptors than for chemoreceptors or unexcited units (Table 1). The occurrence and magnitude of ongoing activity in all bladder units and in bladder mechanoreceptors were unrelated to the stage of the estrous cycle (P > 0.05)

Mechanoreceptors

Twenty-five mechanoreceptors had receptive fields on the body of the bladder. Receptive fields were either punctate (n = 18), ~5-mm-long ovals (n = 5) or so sensitive that they could not be properly mapped (n = 2). Eighteen units were excited by probing near the uvj. Twelve uvj units had punctate receptive fields, while six were excited by probing a larger continuous area. Two of these larger receptive fields were semicircles around the uvj, similar to the receptive field of a bladder unit described by Berkley et al. (1990). The receptive fields of all nine units on the base of the bladder were punctate.

Six units, excited by bladder filling with 0.9% NaCl, could not be excited by direct probing the accessible regions of the bladder. These units could have been responsive to intense mechanical stimuli or could have innervated the urethra (Bahns et al. 1987). Receptive fields were not sought for three units responsive to filling.

The mean conduction velocities (Table 2) of units innervating the body, uvj, and base of the bladder were similar; however, units for which no receptive field could be located conducted more rapidly than those with defined fields (P < 0.05).


                              
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Table 2. Characteristics of 58 bladder mechanoreceptors by their receptive field locations

Ongoing activity was observed in one-half of the mechanoreceptors (Table 1). It ranged from 0.01 to 1.9 imp/s, and was not related to the receptive field location (P > 0.05). Ongoing activity in rat pelvic nerve bladder mechanoreceptors has been reported previously (Dmitrieva and McMahon 1996; Sengupta and Gebhart 1994b; Su et al. 1997) but not in the cat (Bahns et al. 1987; Häbler et al. 1990, 1993a,b); this feature may be species specific.

Examples of the responses of mechanoreceptors to slow bladder filling are illustrated in Fig. 4. Units illustrated in Fig. 4, A-C, were C fibers, and that illustrated in Fig. 4D was an Adelta fiber.



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Fig. 4. Responses of pelvic nerve bladder mechanoreceptors to bladder filling with 0.9% NaCl. Top portions graph unitary discharge as impulses/s (binwidth, 1 s), and the bottom portions show intravesical pressure as a function of time. Filling (100 µl/min) began at the time of the upward arrow. The system was opened to atmospheric pressure at the time indicated by the downward arrow. Recordings ended at the asterisk. Abscissa scale division marks are at 1-min intervals. A: unit (1.3 m/s) with a receptive field on the body of the bladder. B: unit (1.3 m/s) with a receptive field near the ureterovesical junction (uvj). C: unit (1.0 m/s) with a receptive field on the base of the bladder. D: unit (10 m/s) minimally responsive to filling but not excited by probing of the bladder. Note the different ordinate scale for the graph of pressure in D. RF, receptive field.

Volume and pressure thresholds are illustrated in Fig. 5A. Forty-eight units (36 C and 12 Adelta fibers) had pressure thresholds <10 mmHg. Three C-fiber units and one Adelta unit had thresholds between 10 and 20 mmHg, while nine units (5 C and 4 Adelta fibers) only responded after intravesical pressure exceeded 25 mmHg.



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Fig. 5. Thresholds of mechanoreceptors estimated from responses to slow filling of the bladder with 0.9% NaCl. A: pressure (mmHg) thresholds () and volume (µl) thresholds () of 61 mechanoreceptors. B: pressure (left) and volume (right) thresholds of 61 mechanoreceptors as a function of conduction velocity. C: mean + SE pressure (left) and volume (right) thresholds of mechanoreceptors studied in rats shown by vaginal smears to be in proestrus (p, n = 8), estrus (e, n = 21), metaestrus (m, n = 11), and diestrus (d, n = 21). * Mean significantly different from all other groups (P < 0.05).

All units with receptive fields on the bladder body had low thresholds (<= 10 mmHg and 250 µl). Units with receptive fields near the uvj or on the bladder base and those for which receptive fields could not be located, included elements with both low and higher distension thresholds. The mean pressure and volume thresholds reflected this distribution (Table 2). Maximal excitation of the higher threshold units could occur during isovolumic detrussor contractions against an obstructed outlet; under such conditions conceivably their activity could contribute to experiences of discomfort and pain.

No relationship appeared between the conduction velocities of individual mechanoreceptors and their pressure or volume thresholds. The mean pressure and volume thresholds of C (6.7 ± 1.3 mmHg; 143 ± 33 µl, mean ± SE) and Adelta units (12.0 ± 2.6 mmHg; 223 ± 51 µl) were similar and differences in means could be attributed to chance (P > 0.05). The substantial number of low-threshold mechanoreceptors with C fibers is difficult to rationalize with the suggestion that the micturition reflex in rats is initiated by Adelta mechanoreceptive fibers (Mallory et al. 1989).

Mean mechanical thresholds varied systematically with the stage of the rat's estrus cycle. Units studied in proestrus had the lowest while those tested in metaestrus the highest thresholds (Fig. 5C).

There were two response patterns to slow filling of the bladder with 0.9% NaCl for units with receptive fields on the bladder body and near the uvj. One type was characterized by continuously increasing firing during filling (e.g., Fig. 4B). The second pattern exhibited peak frequencies at lower than the maximum pressures reached during filling followed by a plateau or a decrease in activity at higher pressures (e.g., Fig. 4A). Of 29 units in which the intravesical pressure exceeded 40 mmHg, 12 displayed continuously rising activity as a function of intravesical pressure (Fig. 6A), while 17 reached peak discharge at pressures <40 mmHg (Fig. 6B). The two response sequences we noted are nearly identical to two of three patterns of responses by pelvic nerve mechanoreceptors to bladder filling under pentobarbital sodium anesthesia (Moss et al. 1997). These patterns are probably not secondary to the contractile state of the bladder because similar response patterns occur under experimental conditions in which cystometrograms show little evidence of bladder contractions (Häbler et al. 1993a,b; Moss et al. 1997). Both C and Adelta fibers displayed plateaus or decreases in activity to increases in pressure.



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Fig. 6. A and B: pressure-response relationships of individual mechanoreceptors with receptive fields on the body or near the uvj. A: units (10 C and 2 Adelta fibers) exhibiting increasing activity with increasing pressure up to 40 mmHg. B: units (11 C and 6 Adelta fibers) exhibiting maximum activity at pressures below 40 mmHg (*, unit illustrated in Fig. 4A). C: averaged responses to filling the bladder with 0.9% NaCl of C (n = 43) and Adelta (n = 17) mechanoreceptors as a function of intravesical pressure. D: averaged responses of mechanoreceptors with receptive fields on the body (n = 25), uvj (n = 18), and base (n = 9) of the bladder and those not excited by direct mechanical stimulation (none, n = 6).

Distension-evoked activity of most mechanoreceptors ceased when the bladder was allowed to empty. However, four C-fiber units displayed elevated activity that continued during and up to 1 min after bladder emptying. These resemble a group of Adelta units in rat described by Morrison (1997) proposed to signal that the bladder is empty.

Responses averaged as a function of intravesical pressure did not differ for mechanoreceptors with Adelta and with C fibers (Fig. 6C); however, location of receptive field did correlate with average responsiveness (Fig. 6D). No relationships were noted between the stage of the rat's estrous cycle and the suprathreshold response patterns or averaged responsiveness of bladder mechanoreceptors.

One of five mechanoreceptors tested increased activity in response to 150 and 300 mM KCl for about 3 min, the increase ceasing when the bladder was rinsed with 0.9% NaCl. Capsaicin excited 8 of the 10 mechanoreceptors on which it was tested. Figure 7 compares the responses of a mechanoreceptor to slow and to rapid filling with 0.9% NaCl and to the same volume of solutions containing capsaicin. Activity during instillation of 30 and 100 µM capsaicin was greater than that evoked by 0.9% NaCl or by 1 µM capsaicin. Increased ongoing activity was observed after removal of capsaicin in seven units. This increase disappeared in 2-8 min for six units, although the bladder was not rinsed; one unit continued to show activity for at least 10 min. The responses of mechanoreceptors to KCl and capsaicin are summarized in Table 3.



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Fig. 7. Responses (impulses/s; binwidth, 1 s) of a mechanoreceptor (1.0 m/s, receptive field on the body of the bladder) to slow filling of the bladder with 0.9% NaCl (left, starting at the upward arrow) and to rapid instillation (0.3 ml total in 0.1 ml boluses, upward arrows) of 0.9% NaCl and solutions containing varying concentrations of capsaicin (CAP). Each solution was removed at the downward arrows. Abscissa scale marks are at 1-min intervals.


                              
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Table 3. Responses of bladder mechanoreceptors to intravesical KCl and capsaicin

Chemoreceptors

Thirty-nine units did not respond to slow filling with 0.9% NaCl to intravesical pressures of at least 40 mmHg. Two of this group were excited by intravesical KCl, and six, unresponsive to KCl, were excited by intravesical capsaicin.

KCL-RESPONSIVE UNITS. One C unit responded weakly to probing the bladder base. When the bladder was filled with a solution of 75 mM KCl, activity increased but displayed no relationship to intravesical pressure, continued after the KCl was drained, and was not affected by repeated filling. These observations suggest a selective excitation by KCl and not by mechanical events. An Adelta unit with no mechanically demonstrable receptive field failed to respond to either 150 or 300 mM KCl. However, subsequent distension and slow filling with 0.9% NaCl resulted in excitation with an estimated threshold of 8 mmHg, suggesting that exposure to KCl had induced mechanical sensitivity.

CAPSAICIN-RESPONSIVE UNITS. Six units, unresponsive to filling with 0.9% NaCl or KCl solutions (75-300 mM), were excited by intravesically applied capsaicin. The sensitivity and responses of capsaicin-responsive units varied. Three Adelta units were excited by the only concentration of capsaicin tested. One of these gave a burst of activity 8 min after 50 µM capsaicin, while the other two responded promptly at concentrations of 0.5 and 10 µM. A fourth Adelta unit was excited by 10 mM but not by 100 µM capsaicin, and the excitation continued for at least 10 min. One C unit responded promptly to 10 mM capsaicin (lowest concentration tested) and activity continued for over 30 min. Responses of the other C unit are illustrated in Fig. 8. Excitation occurred at 1 mM capsaicin. As shown, subsequent distension with the 10% ethanol in 0.9% NaCl vehicle and 0.9% NaCl evoked minimal responses that had not been seen prior to 1 mM capsaicin. However, the activity evoked without capsaicin was far less than that initiated when it was present. Development of mechanoresponsiveness by the other five capsaicin-responsive units was not systematically tested: however, two of the five units were electrically inexcitable after exposure of the bladder to capsaicin.



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Fig. 8. Responses (impulses/s; binwidth, 1 s) of a capsaicin-responsive unit (conduction velocity, 0.6 m/s; no receptive field, not excited by slow filling) to rapid instillation (upward arrows) and removal (downward arrows) of 0.6 ml of 0.9% NaCl, KCl solution (150 mM), varying concentrations of capsaicin (CAP), and 10% ethanol in 0.9% NaCl (ETOH, vehicle for 10 mM capsaicin). Abscissa scale marks are at 1-min intervals.

Unexcited units

Table 4 summarizes the properties of units excited neither by filling nor by the tested chemical stimuli. Two unexcited units with ongoing activity and receptive fields may have been mechanoreceptors with thresholds >40 mmHg (Sengupta and Gebhart 1994b). It is unlikely that the remaining 29 units innervated structures other than the bladder because all units were excited by electrical stimulation of both the pelvic and bladder nerves. Some unexcited units might have been mechanoreceptors or chemoreceptors injured during the surgical preparations. Others may have been thermoreceptive (Fall et al. 1990). An intriguing possibility is that these units represent a subset of chemoreceptors only responsive to conditions not present in our experiments (e.g., inflammation).


                              
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Table 4. Observations on units not excited by any stimulus tested


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Conduction velocities and afferent characteristics

In the present study the samples of both C and of Adelta fibers included mechanoreceptors, chemoreceptors, and unexcited units. Additionally, the properties of mechanoreceptors with C fibers were indistinguishable from those with Adelta fibers, consistent with earlier reports by Sengupta and Gebhart (1994b) and Su et al. (1997). The lack of relationship between conduction velocity and peripheral receptive properties of bladder afferent fibers in the rat is difficult to reconcile with recent descriptions of the properties of their soma. Results from whole cell patch-clamp recordings from dissociated bladder neuronal soma in the rat suggest that the bladder is innervated by two principal populations of L6 and S1 dorsal root ganglion neurons. About three-fourths are small and presumed to have peripheral C fibers. The electrical properties of these small soma are suggested to correlate with tonically active high-threshold C-fiber bladder nociceptors. The larger soma are suggested to be associated with rapidly adapting low-threshold mechanoreceptors with Adelta fibers (Yoshimura and de Groat 1997; Yoshimura et al. 1996, 1998). The proportions of small and larger soma and the proportions of our C and Adelta fibers are similar and consistent with the likelihood that the small soma are associated with C fibers and the larger soma with Adelta fibers. However, our observations showed that more than one-half of pelvic nerve bladder C fibers are mechanoreceptors with low thresholds (<10 mmHg). Moreover, we uncovered no relationship between conduction velocity and receptor type. One possible explanation is that the units sampled here in vivo are different from those encountered after dissociation and short-term culture. Another possibility is that the electrical properties of bladder neuronal soma do not correlate in an obvious manner with their peripheral receptive properties.

While the proportions and general properties of pelvic nerve bladder mechanoreceptors described here were similar to those described by Sengupta and Gebhart (1994b) and Su et al. (1997), some quantitative differences are notable. Nearly all mechanoreceptors in those previous studies had ongoing activity, while only one-half of the sample had activity prior to stimulation in our study. The frequency of ongoing activity, when present in the previous studies, was nearly four times that observed here. Likewise, the average activity in response to intravesical pressures of 40 mmHg (estimated from stimulus-response graphs in Sengupta and Gebhart 1994b and Su et al. 1997) was roughly three times greater than that observed in the current experiments. The reasons for these differences may relate to alterations in the bladder or its afferent innervation as a consequence of sudden, possibly noxious pressure steps used for identification and characterization of bladder units in the previous studies.

Arrangement of bladder mechanoreceptors relative to contractile elements

Two patterns of activity in response to intravesical suprathreshold pressure could reflect differences in transduction mechanisms. It is generally accepted that most bladder mechanoreceptors encode intravesical pressure and respond in a manner consistent with their receptive terminals being in series with bladder smooth muscle (Iggo 1955). Morrison (1997, 1999) has argued that regulation of bladder function additionally requires receptors dedicated the encoding bladder volume, reviving a possibility suggested previously (Arlhac 1972; Coolsaet et al. 1993; Morrison 1997; Uemura et al. 1973). A distinction between in series and in parallel receptors may be hard to establish. Intravesical pressure, the variable used most commonly to infer the contractile state of the bladder wall, does not relate in a simple manner to receptive field length, tension, or mechanoreceptor activity (Downie and Armour 1992). In addition, the bladder wall is known to exhibit micromotion with local contractions stretching immediately surrounding bladder tissues (Coolsaet et al. 1993). Temporal coincidence of increased mechanoreceptor activity with increased intravesical pressure or tension is not proof that receptive terminals are in series with smooth muscle. Likewise, absence of responses during contractions cannot be taken as prima facie evidence that the terminals are in parallel with smooth muscle; the smooth muscle cells associated with a set of receptive terminals may not be those producing the measured tension. Only when intravesical pressure happens to reflect the contractile state of elements directly associated with receptive terminals could a relationship to tension be unambiguous.

If receptors specific for bladder volume exist, they would be among bladder units excited by mechanical stimulation. Bladder mechanoreceptors with discharge rates that peak or plateau and do not increase with further elevations of pressure, similar to those illustrated in Fig. 6B, are commonly encountered (Downie and Armour 1992; Häbler et al. 1993a; Winter 1971). Such units may represent a population with terminals in parallel with smooth muscle elements and thereby may encode volume especially at low intravesical pressures.

These considerations have important consequences. If experimental manipulations increase the compliance of the bladder, units with in parallel receptive terminals will appear to be sensitized if their responses are considered only in relationship with intravesical pressure. These same units would appear unchanged if only volume were measured. If the mammalian bladder is served by a significant population of mechanoreceptors operating in parallel with bladder smooth muscle and most responsive to increases in length (volume), interpretation of results from experiments in which solely pressure is determined, without consideration of volume, could be misleading.

Chemosensitivity of pelvic nerve bladder receptors

Most mechanoreceptors and a few mechanically unresponsive units were excited by capsaicin. Intravesical capsaicin evokes decreases in the bladder volume for the first desire to void, bladder capacity, and pressure thresholds for micturition as well as sensations ranging from warmth to severe pain in human patients (Cruz et al. 1997; Geirsson et al. 1995; Lazzeri et al. 1996; Maggi et al. 1989) and bladder hyperactivity and behaviors suggestive of pain in awake rats (Pandita et al. 1997). It is possible that excitation of mechanoreceptors by capsaicin leads to bladder hyperactivity in rats and to decreased bladder volume to first urge, decreased bladder capacity, and lowered pressure thresholds for micturition in humans. The human burning and pain and behaviors indicative of pain in rats after intravesical capsaicin could be mediated by excitation of mechanically unresponsive units, urethral units of the pudendal nerve (Lecci et al. 1994), or sensory units of the hypogastric nerve.

Estrus cycle effects

Changes in hormonal status affect the sensitivity of sensory units innervating female reproductive organs in rats. Such changes could be important for mating and conception (Berkley et al. 1990; Robbins et al. 1990). Effects of ovarian cycles have also been demonstrated on bladder inflammation (Bon et al. 1997) and somatic motor responses to colorectal distension (Sapsed-Byrne et al. 1996). The present results indicate that the average bladder compliance and average thresholds of mechanoreceptors are lowest in proestrus and greatest in metaestrus. It is probable that the changes in compliance and in sensory receptor threshold are related; however, the loci of the effects (bladder muscle and its innervation or central processing) remain to be determined.


    ACKNOWLEDGMENTS

The authors thank S. Derr and R. Hoyle-Thacker for excellent editorial assistance.

This study was supported by National Institutes of Health Grants DK-47590 and NS-14899.


    FOOTNOTES

Address for reprint requests: V. K. Shea, Dept. of Cell and Molecular Physiology, CB 7545, 187 Med. Sci. Res. Bldg., University of North Carolina-CH, Chapel Hill, NC 27599-7545.

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.

Received 18 June 1999; accepted in final form 22 June 2000.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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