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Journal of Physiology (2002), 543.1, pp. 211-220
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.019042
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ABSTRACT |
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Cold-sensitive C afferents of the urinary bladder were studied in adult cats anaesthetised with-chloralose. The bladder was catheterised for fluid instillations and bladder pressure recordings. Pelvic nerve branches were stimulated electrically close to the bladder. Evoked afferent activity was recorded from dissected filaments of the ipsilateral S1-S2 dorsal roots. Responsive afferents were identified using the 'marking technique', based on activity-dependent decrease in C fibre conduction velocity. Of 108 examined bladder C afferents, 14 were activated by innocuous cooling of the bladder wall. Their conduction velocities ranged from 0.6 to 1.7 ms-1 and their activity dependent decrease in conduction velocity was <10 %. All nine cold-sensitive afferents tested responded to menthol exposure. Cold-sensitive C afferents failed to respond to bladder filling with body-warm saline and to active bladder contractions. These characteristics indicate that the cold-sensitive C afferents of the bladder resemble cutaneous cold receptors rather than cold-sensitive mechanoreceptors or nociceptors. It is concluded that the bladder wall is endowed with cold receptors with unmyelinated C afferents in the pelvic nerves and that these afferents are responsible for the bladder cooling reflex.
(Received 17 February 2002; accepted after revision 27 May 2002)
Corresponding author C. Jiang: Department of Biomedicine and Surgery, Faculty of Health Sciences, S-581 85 Linköping, Sweden. Email: chonghe.jiang{at}mcb.liu.se
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INTRODUCTION |
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The ordinary micturition reflex is activated by bladder mechanoreceptors, which in the adult cat mainly belong to the A
afferent group (Iggo, 1955; De Groat, 1975; Häbler et al. 1993). Clinical observations suggest, however, that other types of bladder receptors may also induce reflex activation of the detrusor, i.e. during inflammation or bladder cooling (Bors & Blinn, 1957). Experimental studies in cat and man have revealed a bladder cooling reflex that does not originate from bladder mechanoreceptors (Fall et al. 1990; Lindström & Maziéres, 1991; Geirsson et al. 1993, 1999). The reflex can be evoked by infusion of cold fluid in the bladder at volumes and pressures well below threshold for activation of the mechanoreceptors, the sensitivity of which is in fact decreased by cooling.
From its temperature threshold and menthol sensitivity, the conclusion was reached that the bladder cooling reflex originates from bladder receptors with properties similar to cutaneous cold receptors (Lindström & Maziéres, 1991; Geirsson, 1993). The threshold of the reflex is above 30 °C in the cat, i.e. well above noxious cold temperatures (Leem et al. 1993; Campero et al. 1996). Furthermore, its temperature threshold is shifted towards higher temperatures by a brief exposure of the bladder to menthol. The afferents of the responsible receptors, located in the pelvic nerve, were proposed to be unmyelinated C fibres. In keeping with this idea, the electrically evoked C fibre reflex of the bladder is facilitated by bladder cooling or menthol exposure (Maziéres et al. 1998).
In the present study, we tried to identify bladder afferents responsive to innocuous cold and menthol. Bladder afferents were recorded in sacral dorsal roots, with focus on those belonging to the C fibre category. Activated C fibres were identified by a spike delay procedure ('marking technique'; Torebjörk & Hallin, 1974; Thalhammer et al. 1994; Schmelz et al. 1995). Findings are compared with recent results for cutaneous cold C afferents (Gee et al. 1996; Campero et al. 2001).
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METHODS |
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Ten adult cats of either sex were used for the experiments. Anaesthesia was induced with an alphaxalone-alphadolone mixture (Saffan, Glaxovet, 2 ml kg-1 I.M.) followed by -chloralose (55 mg kg-1 I.V., supplemented as necessary). An adequate depth of anaesthesia was ascertained by the lack of blood pressure and heart rate changes after strong paw-pinches or electrical stimulation of bladder C afferents. Central body temperature was monitored with a thermode in the lower oesophagus and maintained between 37.5 and 38.5 °C by a feed-back controlled heating device. Mean arterial pressure was maintained above 110 mmHg by a slow I.V. infusion of a bicarbonate-buffered Ringer-glucose solution. Animals were killed at the end of the experiments by an overdose of the anaesthetics, followed by severance of the heart. Experimental procedures were approved by the animal research ethical committee of Linköping in accordance with Swedish law.
The urethra and bladder neck were exposed extraperitoneally by a ventral approach. A thin catheter, inserted into the bladder through a slit in the proximal urethra, was used for fluid infusions and pressure recordings (Fig. 1A). Pelvic nerve branches to the bladder were identified on the left side and prepared for stimulation by gently wrapping a pair of wire electrodes around them. The electrodes were 4-5 mm apart, with the cathode proximal and placed about 10-15 mm from the bladder. They were shielded from the surrounding tissue by a soft isolating sheath and firmly fixed in place by tying the cable to the bladder catheter. Care was taken not to touch or manipulate the bladder during the surgical procedures in order not to sensitise bladder C afferents. For the same reason, we did not attempt to attach a thermoprobe to the bladder wall. The abdominal incision was closed with a few sutures to maintain the bladder at central core temperature.
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Figure 1. Effect of cold stimulation on bladder wall temperature A, schematic diagram of experimental arrangement. B, bladder wall temperature change induced by bladder infusions of saline (5 ml) at different temperatures, plotted against the fluid take-out temperature. The fluid was left in the bladder for 60 s and then rapidly withdrawn. The temperature of this fluid corresponds to the mean temperature of the fluid in the bladder. C, time course of change in bladder wall temperature after bladder infusions (5 and 10 ml) of fluid at 4 °C; measurements were obtained with the thermoprobe in contact with the inner ( | ||
A laminectomy of the L6-L7 vertebrae was performed to expose the appropriate dorsal roots. All ipsilateral nerves from the S1-S3 segments, except the pelvic, were transected to eliminate disturbing background afferent activity. It was especially important to sever all branches to tail muscles. In four cats the S1-S3 dorsal or ventral roots were cut bilaterally in the course of the experiment to abolish reflex contractions of the detrusor. For the recordings, the animals were rigidly fixed to a frame by clamps attached to the sacrum and a rostral vertebra. The exposed tissue was covered with paraffin oil in a pool formed by sewn-up skin flaps. The temperature of the pool was kept between 36 and 38 °C with a heating lamp.
Bladder C afferents, identified as such by electrical stimulation of pelvic branches to the bladder, were recorded in thin filaments split from the left S1 or S2 dorsal roots. The filaments typically contained a few A and C afferents from the bladder. An isolated constant current stimulator giving unipolar square-wave pulses of variable amplitude was used for stimulation. With 0.5 ms pulse duration, thresholds were 20-40 µA for A afferents and 1-3 mA for C afferents. Conduction velocity of afferents was estimated from their response latency and the distance between stimulating and recording sites (range 72-123 mm), was measured at the end of each experiment. Afferents were classified as C fibres if they had conduction velocities below 2.0 ms-1. The nerve signals were amplified with differential amplifiers equipped with appropriate filters and displayed on an oscilloscope and a digital multi-channel chart recorder together with the intravesical pressure.
A spike delay procedure (the 'marking technique') was used to identify cold sensitive C afferents (Torebjörk & Hallin, 1974; Thalhammer et al. 1994; Schmelz et al. 1995; see Results). This technique allowed several C afferents to be evaluated simultaneously, thereby increasing the efficacy in the sampling of responsive units (cf. Häbler et al. 1990). This was important in order to minimise the number of animals required for the study. Another advantage was that a bladder origin of studied cold-sensitive C afferents could be ascertained without extensive denervation of the pelvic region. A consequence was that ongoing cold-evoked activity could not be ascribed with certainty to specific C afferents, and therefore such recordings were not stored.
The marking technique involved continuous electrical stimulation of the bladder pelvic nerve at low frequency (0.5 Hz) to evoke reference spikes in the studied afferents while the bladder was filled with small volumes of saline at different temperatures. The fluid was left in the bladder for 30-60 s and then rapidly withdrawn. To ensure stable conditions for electrical stimulation, spike latencies were only measured in periods when the bladder was empty and relaxed. After fluid evacuation the first measurements were typically obtained within 5 s. Latency changes were estimated from means of 5-10 consecutive electrically evoked responses obtained before and after, cold stimulation. Results are given as means ± 95 % confidence interval (c.i.). The occurrence of cold-unresponsive C afferents in recorded filaments served as a control for stability of stimulation and recording and also excluded a general cooling effect on afferents central to the stimulation site.
To activate cold receptors, a similar procedure was used as in previous studies of the bladder cooling reflex in cats and man (Fall et al. 1990; Geirsson, 1993; Geirsson et al. 1993). The bladder was rapidly filled with 5-10 ml saline, depending on bladder capacity of the individual animal, at a temperature of 4-8 °C. Such infusions resulted in an estimated mean fluid temperature in the bladder of 20-25 °C as judged from the fluid withdrawn after 30-60 s (Fall et al. 1990; Lindström & Maziéres, 1991). The stimulus temperature at the receptors in the bladder wall was considerably higher (see below). After each cold infusion, the bladder was re-warmed by brief instillations of body-warm saline. Repeated trials with cold stimulation of the bladder were used to ascertain reproducibility of the response of individual cold-sensitive C fibres.
The actual change in bladder wall temperature imposed by the used cold stimuli was estimated in two cats by performing a number of bladder infusions with simultaneous recording of the bladder wall temperature. A small thermocouple probe (CIE 305, Taiwan; probe type K) was applied manually so that it gently touched the outside of the bladder dome. In other trials, the probe was inserted into the bladder (along the catheter), so that it stayed in contact with the mucosa during the filling of small volumes (
10 ml). Both procedures gave very similar changes in wall temperature (Fig. 1C). Infusions of 5 ml at 4 °C reduced the wall temperature within 20 s to a plateau of 7.2 ± 1.1 (mean ± c.i.; n = 9; inside) and 6.7 ± 0.8 °C (n = 6; outside) below initial level. With a double volume of cooling fluid at 4 °C the wall temperature decreased by 9.8 ± 1.1 °C (n = 6, outside). After bladder evacuation the wall temperature returned very gradually over several minutes towards the original level.
To relate these measurement to trials with cold stimulation of C afferents (and previous studies of the bladder cooling reflex), the change in wall temperature was plotted against the mean temperature of the fluid withdrawn from the bladder (Fig. 1B). Points represent individual trials with infusates of different temperature. Confirming earlier assumptions (Fall et al. 1990; Lindström & Maziéres, 1991), some cooling of the bladder wall was detected at the fluid take-out temperatures in the range 30-33 °C. These values correspond to the threshold temperature of the bladder cooling reflex as previously determined (Fall et al. 1990).
C afferents were also tested after bladder exposure to menthol (0.6 mM in saline, 1 min), known to sensitise cold receptors reversibly (Hensel & Zotterman, 1951; Schäfer et al. 1986). In two experiments, response specificity of cold-sensitive C afferents was evaluated by infusing hot (52-55 °C; estimated bladder wall temperature 40-44 °C) or acid (pH 4.8) fluid into the bladder. Pressure sensitivity was tested by filling the bladder with body-warm saline to passive pressures above 2-3 kPa or by allowing the innervated bladder to contract.
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RESULTS |
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Identification of cold-sensitive C afferents
Bladder C afferents were searched for in S1 or S2 dorsal root filaments during continuous electrical stimulation of the bladder pelvic nerve at low rate and high intensity (0.5 Hz, >50 
threshold for A fibres). The C afferents were recognised by their high threshold (> 20 threshold) and long latency (>40 ms), corresponding to a conduction velocity below 2 ms-1 (conduction distance 72-123 mm). A total of 108 bladder C afferents with conduction velocities ranging from 0.4 to 1.9 ms-1 were thus identified and tested for their response to cold stimulation.
Cold sensitivity of C afferents was demonstrated using the 'marking technique' (Torebjörk & Hallin, 1974; Thalhammer et al. 1994; Schmelz et al. 1995). The procedure rests on the principle that, when activated by adequate or electrical stimuli above a certain frequency, unmyelinated afferents undergo a decrease in their conduction velocity (Gasser, 1935). Hence, an increase in spike latency of a bladder C afferent would occur during and after, any imposed activity (Fig. 2A). In this case, a cold-unresponsive C afferent was stimulated electrically for 60 s at the indicated frequency. The sample records show the last response of each stimulation period. The spike latency of this particular C afferent was increased from 67 ms at control stimulation (0.5 Hz) to 89 ms at 8 Hz (133 %). This change corresponded to a decrease in spike conduction velocity from 1.3 to 1.0 ms-1.
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Figure 2. Application of the marking technique for bladder C afferents A, a cold unresponsive C afferent stimulated electrically for 1 min at the indicated frequency. Sample records were obtained at the end of the stimulation period. Note progressive increase in spike latency with increasing stimulation frequency. Resting conduction velocity 1.1 ms-1. B, plot of relative spike latency increase at different stimulation frequencies. The latency change is expressed as a percentage of control conduction time at 0.5 Hz. Unit in A, | ||
The frequency-dependent delay of the same and two other C afferents are plotted in Fig. 2B. Filled symbols indicate a cold-sensitive C afferent. All units were delayed more than 10 ms after one minute at their highest tested frequency. However, when the latency increase was expressed as a percentage of the control latency, the cold-sensitive C afferent was less delayed than the two cold-unresponsive units. At 4 Hz the delay was 5.4 % for the cold-sensitive C afferent compared with 10.0 and 11.3 % for the unresponsive C afferents (Campero et al. 2001).
A typical result with the spike delay procedure applied during bladder cooling is shown in Fig. 3A-C. Five bladder C afferents could be resolved in the recordings from this filament (A, upper trace). Three afferents with spike latencies 115, 127 and 139 ms, corresponding to conduction velocities of 1.1, 1.0 and 0.9 ms-1, are shown in the time-expanded records below. On bladder cooling to an estimated bladder wall temperature of 31 °C (cf. Fig. 1), the spike of the last unit in this triad was delayed by 11 ms (8 %), while the spike latency of the other two afferents was unchanged. Recordings obtained a few minutes later, after bladder re-warming, showed complete recovery of the spike latency of the last afferent to the control value (C).
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Figure 3. Identification of cold-sensitive C afferents from the bladder Recordings in A-C show afferent responses in an S2 dorsal root filament evoked by electrical stimulation of bladder branches of the ipsilateral pelvic nerve at 0.5 Hz. Spikes in upper, slow sweep speed recordings, are from C afferents, three of which are detailed in three consecutive time expanded records below. At normal bladder temperature (A), the control conduction velocity of illustrated units was 1.1, 1.0 and 0.9 ms-1, respectively. Records in B were obtained after bladder cooling by the infusion of 5 ml saline at 4 °C (estimated bladder wall temperature 31 °C). Following this cold stimulus, the spike latency of the last unit in the assembly (*) was delayed by 11 ms (8 %), while the spikes of the other two afferents remained unchanged. The spike latency change of the last unit was fully reversible as shown by records in C, obtained after bladder re-warming to 38 °C. Time calibration uppermost in C (80 ms) is for upper traces, the one below (20 ms) is for time expanded traces. Histograms to the right show the distribution of conduction velocities for all cold-sensitive and cold-unresponsive C afferents examined. Shaded area in upper histogram marks cold-sensitive C afferents tested for menthol sensitivity, evoked by the natural cold stimulation of the bladder. The constant latency of the two other units in trials with bladder cooling demonstrates that the stimulation and recording conditions were stable during the testing procedure and that the cooling effect on the nerve centrally to the stimulation electrode was negligible. | ||
Three additional trials with bladder cooling showed a similar picture. The spikes of the first two C afferents had unchanged latencies, while the spike of the last C afferent was delayed 7-11 ms (mean 9.3 ms; 6.7 %). The latency increase was highly significant in each cooling trial, with confidence intervals below ± 0.5 ms (based on the latency of the first 10 traces after bladder evacuation). The delay of the same cold-sensitive C afferent, when tested with electrical stimulation (plot, Fig. 2) suggested an impulse frequency of 4-5 Hz.
Fourteen C afferents were identified with significant spike delays after bladder cooling. Their spike latency increase ranged from 2.0 to 11.5 ms, corresponding to changes in conduction time of 2.9-9.7 % (mean 5.7 %). The cold-sensitive C afferents were found in seven filaments split from caudal S1 and rostral S2 rootlets. Four filaments contained more than one cold-sensitive C afferent. In all but one filament, there were also cold-unresponsive C afferents that showed no significant change in spike latency on bladder cooling (20 afferents). Another 31 filaments from the S1-S2 dorsal roots contained only cold-unresponsive bladder C afferents (74 units). This distribution suggests that cold sensitive afferents tend to be grouped in a small proportion of S1-S2 dorsal rootlets. Conduction velocities of cold-sensitive (0.6-1.7 ms-1) and cold-unresponsive (0.4-1.9 ms-1) bladder C afferents were similar (Fig. 3, histograms).
Effect of menthol
Menthol is known to shift the temperature-response curve of cutaneous cold receptors towards higher temperatures (Hensel & Zotterman, 1951; Schäfer et al. 1986) and to exaggerate the bladder cooling reflex (Lindström & Maziéres, 1991; Geirsson, 1993). The substance also affected tested cold-sensitive C afferents of the bladder (Fig. 4). The illustrated filament contained several resolvable spikes from bladder C afferents, two of which are displayed in the time expanded records (latencies 113 and 142 ms, conduction velocities 1.1 and 0.9 ms-1). Only the first spike in this pair was delayed by bladder cooling (9 ms; middle group of traces, Fig. 4 A). This value is close to the mean latency increase in five bladder cooling trials with the same afferent (8.8 ms; 7.8 %; range 6.2 - 8.8 %).
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Figure 4. Effect of menthol on bladder C afferent. Records in A and B show afferent activity in an S2 dorsal root filament evoked by electrical stimulation of the bladder pelvic nerve, as in Fig. 3. Time expanded records below display two C afferents with conduction velocities 1.1 and 0.9 ms-1. The first of these units (*) was cold sensitive as shown by its increased spike latency (9 ms, 8 %; A, middle pair of traces) after bladder cooling (4 ml at 4 °C, estimated bladder wall temperature 32 °C). The spike latency returned to normal after bladder re-warming (A, lower pair of traces). After exposure to menthol (0.6 mM, 1 min), the spike of the same afferent was delayed 2 ms already at body temperature (B, menthol + warm). The spike became even further delayed (12 ms) upon cold exposure (B, menthol + cold). The menthol effect was fully reversible as shown by the lowermost records in B, obtained 40 min after the menthol exposure. Note that the second unit in the time expanded records was completely unaffected by all these manipulations. The time calibration lowermost in B is 80 ms for the upper trace in A. The diagram in C shows spike latency of the first C afferent in A-B during bladder cooling before and after menthol exposure. All latencies were taken immediately after the thermal stimulation with the bladder empty (mean ± c.i. of 10 first responses). | ||
Bladder exposure to menthol (0.6 mM, 1 min, followed by a rinse with body-warm saline) delayed the spike of this cold-sensitive unit by 2 ms (Fig. 4B). This change suggests that the afferent became active at body temperature. Bladder cooling within 5 min after menthol exposure caused a larger delay of the spike (12 ms) than cooling alone (Fig. 4B, middle group of traces). The spike latency of the responsive afferent returned to control values after a recovery period of about 40 min (Fig. 4B, lower trace). The described latency changes were highly significant as illustrated graphically in Fig. 4C. The relative increase in spike conduction time was 1.8 % at body temperature and 10.6 % after cold stimulation. The second cold-unresponsive C afferent was unaffected by menthol or menthol plus bladder cooling.
A total of 9/14 cold-sensitive C afferents were tested with menthol exposure (histogram, Fig. 3). All were clearly menthol sensitive. Their resting warm spike latency increased on average by 2.4 ms (range 0.5-4 ms; P < 0.01), corresponding to a mean increase of 3.0 % (range 1.0-6.8 %). Their cold response was shifted by a similar amount, giving an overall mean increase in latency of 5.8 ms (range 2.5-14 ms) above control values, corresponding to 7.9 % (range 5.1-12.4 %). None of the tested cold-unresponsive C afferents had their spike latency changed by menthol (n = 15). Nine of these 15 unresponsive C afferents were recorded together with cold- and menthol-sensitive units in the same filaments.
Recovery of spike latency
After a cooling stimulus, the latency of cold-sensitive C afferents recovered slowly over several minutes after bladder evacuation (Fig. 5A). In the illustrated case, the spike was still delayed by about 2 ms, 3.5 min after bladder emptying. Bladder re-warming at this time immediately restored the spike latency to its control value. This behaviour of the cold-sensitive C afferents is explained by the slow recovery of the bladder wall temperature after a cold stimulus (Fig. 1C). Thus, the C afferent remained active at a low rate during the same period, an activity that was stopped by the bladder re-warming. Bladder infusion of body-warm fluid was regularly used to speed up the return to the control spike latency of cold-sensitive C afferents.
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Figure 5. Recovery time course and lack of pressure response of a bladder cold-sensitive C afferent A, recovery time course after bladder cold stimulation, same cold-sensitive C afferent as in Fig. 4. Each point is the mean of 5-10 consecutive responses (c.i. | ||
Response specificity
In experiments with preserved bladder innervation, cold fluid infusions typically produced higher intravesical pressures than equal volumes of body-warm fluid. The difference was mainly due to evoked bladder cooling reflexes (Fall et al. 1990). To exclude the possibility that the observed cold response of C afferents was due to mechanical stimulation, reflex contractions were eliminated in two experiments by transecting the S1-S3 ventral roots bilaterally. Two cold-sensitive C afferents were studied in these experiments. The spike latency of one such unit after warm and cold stimulation is plotted in Fig. 5B against the induced intravesical pressure. As before, the latency was measured with an empty bladder while the plotted pressure represents the mean value in the preceding period with fluid in the bladder. A range of pressures, 3-4 times higher than typical thresholds for A mechanoreceptor activation, was produced by varying the infusion speed. Similar intravesical pressures were obtained with the warm and cold fluids but only the latter increased the spike latency of the afferent. Obviously, this C afferent was sensitive to the cold stimulus per se and not to changes in bladder pressure. Also, the other cold-sensitive C afferent was insensitive to changes in bladder pressure.
None of the other cold-sensitive C afferents showed any spike delay after infusion of warm fluid, not even when these infusions were large enough to induce bladder contractions. To exclude the eventuality that this lack of change was due to rapid recovery during the bladder evacuation, four cold-sensitive C afferents were tested for latency changes also during ongoing contractions with body warm fluid in the bladder. This departure from our usual procedure required cats with intact ventral roots. The spike latency of the studied cold-sensitive C afferents was completely unchanged by contractions that evoked vigorous activation of A mechanoreceptors in the same filaments.
Most cold-unresponsive C afferents did not display any low threshold mechanosensitivity. However, in one experiment with transected ventral roots, three mechanosensitive C afferents were encountered after an intentional overdistension of the bladder (filled to a pressure of 10 kPa). The spikes of these units were delayed by passive filling of the bladder with body-warm fluid up to pressures in the 3-10 kPa range. All three were cold unresponsive. No cold-sensitive unit was encountered after the overdistension of the bladder.
Hot (estimated bladder wall temperature 40-44 °C) and acid (pH 4.8) fluids were tried with nine C afferents, two of which were cold sensitive. One of the latter had its spike delayed by the warm fluid, none responded to the low pH. The response of the cold-sensitive unit to the high temperature might correspond to the 'paradoxical cold' response of cutaneous cold receptors (Dodt & Zotterman, 1952; Leem et al. 1993). The hot fluid had no effect on the cold-unresponsive C afferents but the acid fluid clearly delayed the spike for one of them.
Bladder A afferents
A afferents were regularly recorded in the same filaments as the C afferents. Their spike latency was unchanged by bladder cooling and they did not have any lasting activity after the bladder was emptied. Thus, in keeping with previous studies we found no evidence for cold-sensitive bladder A afferents (Fall et al. 1990; Lindström & Maziéres, 1991).
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DISCUSSION |
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A group of C afferents was identified that responded to innocuous cooling of the bladder and to menthol exposure. Their axons travelled in the pelvic nerve and entered the sacral spinal cord via the S1-S2 dorsal roots. This finding confirms our previous inference, based on indirect observations of the bladder cooling reflex (Fall et al. 1990, Lindström & Maziéres, 1991; Maziéres et al. 1998), that the bladder is endowed with cold-sensitive receptors similar to cutaneous-cold receptors.
Identification of responsive afferents
The procedure used to identify these cold-activated C afferents deserves some comment. It relied on the recording of changes in their conduction velocity when exposed to their natural stimulus, i.e. cooling of the bladder wall. This 'marking technique' is now well established for studies of C afferents both in animals and man (Torebjörk & Hallin, 1974; Thalhammer et al. 1994; Schmidt et al. 1995; Gee et al. 1996; Campero et al. 2001).
The present results could, in principle, be affected by two methodological problems. Temperature is known to influence the conduction velocity of peripheral nerves, thus cooling could result in a 'non-specific' decrease in afferent conduction velocity (Franz & Iggo, 1968). Displacement of the stimulation electrodes in the distal direction during bladder filling could also result in a spike latency increase. We tried to avoid such errors by applying the electrical stimulation to the bladder nerves at some distance from the cooled organ. In addition, latencies were only estimated in a standard situation with the bladder empty and relaxed. That these precautions were sufficient is confirmed by the finding that unresponsive C afferents with unchanged spike latency could be recorded in the same filaments as the cold-sensitive C afferents and that the behaviour of the different C afferents was consistent over several cooling trials. It follows that the spike delays of the studied C afferents were due to natural stimulation of their receptors in the bladder wall.
Receptor properties
The cold-sensitive C afferents of the bladder resemble lingual (Hensel & Zotterman, 1951; Schäfer et al. 1986), episcleral (Gallar et al. 1993) and cutaneous-cold receptors (Iriuchijima & Zotterman, 1960; Hensel et al. 1960; Gee et al. 1996) rather than cold-responsive mechanoreceptors (Iggo, 1969) or nociceptors (Leem et al. 1993, Campero et al. 1996). Typical cold receptors are lacking in low threshold mechanosensitivity. The same was true for the present cold-sensitive C afferents. They did not react to bladder contractions or passive filling, up to pressures around 3 kPa - stimuli which give a vigorous activation of bladder mechanoreceptors of A type (Iggo, 1955; Häbler et al. 1993). Like cutaneous-cold receptors, the bladder equivalents responded to small cooling steps above 30 °C, i.e. well above the highest temperature observed for cold activation of cutaneous nociceptors (Leem et al. 1993; Campero et al. 1996). They were also silenced by warming, manifested as a rapid return of their spike latency to control values on filling the bladder with warm fluid.
The cold-sensitive afferents of the bladder all had conduction velocities in the C fibre range. They responded with a less than 10 % increase in spike latency to either bladder cooling or electrical stimulation at 4-5 Hz. Such a moderate activity-dependant decrease in conduction velocity appears to be a common feature of cutaneous cold sensitive C afferents both in rat (Gee et al. 1996) and man (Campero et al. 2001). While cutaneous-cold receptors have long been associated with the A afferent group in humans and other primates, they belong to the C fibres in lower mammals, like cats and rats (Iriuchijima & Zotterman, 1960; Hensel et al. 1960; Gee et al. 1996). Recent studies suggest that cutaneous-cold sensitive C afferents might also be quite numerous in humans (Campero et al. 2001).
Menthol effects
It was not feasible to determine the dynamic threshold temperature of the bladder cold-sensitive C afferents with any accuracy. They were instead tested for menthol sensitivity, another distinguishing feature of cold receptors (Hensel & Zotterman, 1951; Schäfer et al. 1986). All nine of the nine cold-sensitive C afferents tested were sensitive to menthol, while none of the 15 tested cold-unresponsive C afferents were affected.
Menthol at a low concentration is known to selectively sensitise cold receptors by shifting their temperature response curve toward higher temperatures (Hensel & Zotterman, 1951; Schäfer et al. 1986). It has a similar effect on the bladder cooling reflex both in cats (Lindström & Maziéres, 1991) and man (Geirsson, 1993). This finding together with the high dynamic threshold temperature of the bladder cooling reflex formed the basis for the proposition that the reflex originated from bladder cold receptors (Fall et al. 1990).
Recently, menthol has been found to enhance a cooling-induced inward current in a small proportion of dorsal root ganglion cells (Reid & Flonta, 2001). An appropriate cold- and menthol-sensitive ion channel, named CMR1 or TRPM8 and belonging to the TRP family of excitatory channels, has also been characterised (McKemy et al. 2002; Peier et al. 2002). Analysis with in situ hybridisation indicates that the channel is expressed in a selective group of small dorsal root ganglion cells with unmyelinated axons, in adults apparently without co-expression with VR1, CGRP or IB4 markers of nociceptive neurones (Peier et al. 2002). Thus, molecular biology gives additional credence to the idea that menthol sensitivity can be used to identify a particular type of cold receptor.
Perceptually, menthol enhances the sensation of cold without affecting thresholds of cold pain or other sensations (although menthol has anaesthetic or desensitising effects in high concentration; Green, 1992; Cliff & Green, 1994; Yosipovitch et al. 1996). When infused in the human bladder, subjects with preserved sensibility report an increased sensation of cold referred to the bladder region (Geirsson, 1993). The cooling effect of menthol is presumably related to the fact that the substance, at an appropriate concentration, stimulates cold receptors to fire impulses at a low rate at body temperature (Hensel & Zotterman, 1951). The same was true for the cold-sensitive C afferents of the bladder. After menthol infusion, they all had their spikes somewhat delayed even without bladder cooling, an effect that was completely reversible within an hour.
Other bladder C afferents
None of the cold-unresponsive units, constituting 87 % of our total sample of bladder C afferents, had low threshold mechanosensitivity. In this respect, bladder C afferents in cats (Häbler et al. 1993) differ from those in rats, which seem to have many C afferents that respond to low intravesical pressures (Sengupta & Gebhart, 1994; Shea et al. 2000). One problem with the interpretation of the rat studies is that the published cystometrograms showed abnormally low bladder compliances, implying that C afferent sensitisation cannot be ruled out. In the present study, three cold-unresponsive C afferents with moderate mechanosensitivity were encountered after an intentional overdistension of the bladder. One further C afferent responded to a low pH. Such observations suggest that the cold-unresponsive C afferents may originate from chemoreceptors or nociceptors in the bladder wall. The latter afferents could respond either to bladder overdistension or to inflammation (Häbler et al. 1990). In order not to compromise the specificity of the cold-sensitive units, such stimuli were actively avoided.
The bladder cooling reflex
The congruence between the bladder cooling reflex and the studied C afferents goes beyond their menthol sensitivity. The bladder-cooling reflex involves afferents in the pelvic nerve. It is abolished by the transection of pelvic nerve branches to the bladder while lesions of the hypogastric nerves and caudal sympathetic chain are without effect (Fall et al. 1990). The bladder-cooling reflex displays a long lasting after-discharge upon withdrawal of the cold fluid from the bladder, as do the C afferents. In both cases, this after-effect is abolished by bladder re-warming. In agreement with the poor mechanosensitivity of the cold-sensitive C afferents, the bladder cooling reflex is largely insensitive to bladder contractions. Relevant in this context is the observation that bladder mechanoreceptors are insensitive to menthol and become less pressure responsive on bladder cooling (Lindström & Maziéres, 1991).
In the cat, a segmental bladder C-fibre reflex has been identified that survives acute spinalisation (Maziéres et al. 1998). This reflex has been postulated to play a crucial role in the recovery of bladder reflexes after spinal injury (De Groat, 1997). Although several types of C afferents may contribute to the bladder C-fibre reflex, it is easily facilitated by bladder and urethral cooling or menthol exposure (Maziéres et al. 1998). Thus the C-fibre reflex is, at least, partly related to the bladder-cooling refIex. Both the A micturition reflex and the C-fibre reflex have afferents and efferents in the pelvic nerves but their pathways are partly separate at the spinal level. Bladder C afferents from both nociceptors and cold receptors seem to terminate in lamina I-II of the dorsal horn (Jiang & Hermanson, 1996; Birder et al. 1999), while A afferents terminate close to the pre-ganglionic neurones (De Groat et al. 1997). For the C-fibre reflex one or more spinal interneurones are apparently intercalated between the terminal region in the dorsal horn and the target pre-ganglionic neurones.
Functional considerations
The finding of cold receptors in the bladder wall may seem rather puzzling. However, thermoreceptors occur at other sites within the abdominal cavity (Rawson & Quick, 1972; Riedel, 1976; El Quazzani & Mei, 1979; Gupta et al. 1979) and may be important components of the thermoregulatory system of the body (Hensel, 1981). The cold receptors of the bladder wall may have a similar role in the regulation of central body temperature. The bladder cooling reflex may have evolved as an extra benefit to facilitate the release of urine formed by cold diuresis (Fall et al. 1990). Such a mechanism would be particularly useful in small individuals with a large surface-to-volume ratio.
In humans, the bladder cooling reflex is a neonatal reflex (Geirsson et al. 1993) that becomes suppressed by descending signals after the age of four. In adults, the reflex reappears after lesions that affect the controlling descending system (Geirsson et al. 1999). The responsible pathway remains to be identified. Even so, the bladder cooling reflex can be used to demonstrate a preserved segmental spinal reflex arch for the bladder in patients with spinal cord injury (Bors & Blinn, 1957). With this approach, functional connections between the bladder and the spinal cord were unexpectedly found for a majority of infants born with myelomeningocele (Gladh & Lindström, 1999).
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Acknowledgements
This investigation was supported by the Swedish Medical Research Council (project no 04767) L. M was supported by a grant from INSERM (project 921106) and by IRME.
Author's present address
L. Maziéres: Service de Rééducation Neurologique, Hôpital de la Salpêtrière, 47-83 boulevard de l' Hôpital, 75651 Paris Cedex 13, France.
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, 
) or outer (
) surface of the bladder wall. Note change in scale of the time axis and the slow recovery of the bladder wall temperature.
(1.1 ms-1), another cold-unresponsive C afferent,