J Physiol Volume 513, Number 2, 531-541, December 1, 1998
The Journal of Physiology (1998), 513.2, pp. 531-541
© Copyright 1998 The Physiological Society
The C fibre reflex of the cat urinary bladder
L. Mazières, C.-H. Jiang and S. Lindström
Department of Biomedicine and Surgery, Faculty of Health Sciences, University of Linköping, S-581 85 Linköping, Sweden
Received 11 March 1998; accepted after revision 19 August 1998
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ABSTRACT |
- Reflexes evoked in bladder parasympathetic neurones by electrical stimulation of bladder C afferent fibres were studied in cats anaesthetized with
-chloralose. The responses were compared with the ordinary micturition reflex evoked by low-threshold A
afferents from bladder mechanoreceptors and mediated by a spino-ponto-spinal reflex pathway.
- The bladder was catheterized for fluid instillations and pressure recordings. Efferent reflex discharges were recorded from the cut central end of a small distal bladder branch of the pelvic nerve. The remaining bladder pelvic nerve branches were stimulated electrically close to the bladder.
- Stimulation at C afferent intensity evoked a late reflex discharge in bladder pelvic efferents in all animals. The response was centrally mediated, had a latency of 150-250 ms, and was much weaker after stimulation on the contralateral nerve.
- The bladder C fibre reflex differed in several functional aspects from the ordinary A micturition reflex. It could be evoked at a low rate of stimulation, with an empty bladder and no background activity from bladder mechanoreceptors. In this situation, the normal A micturition reflex is not elicited. The C fibre reflex also survived an acute spinalization at a low thoracic level.
- The C fibre reflex was strongly inhibited by dorsal clitoris or dorsal penis nerve stimulation, an effect that was maintained after spinalization. It was facilitated by bladder or urethra exposure to cold and menthol, stimuli that activate specific cold-sensitive receptors associated with unmyelinated C afferents.
- It is concluded that the central pathway of the C fibre reflex is spinal and partly separate from that of the ordinary micturition reflex. These observations are in keeping with the clinical finding that a bladder cooling reflex can be elicited in patients with disturbed descending control of the bladder.
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INTRODUCTION |
The ordinary micturition reflex is activated by low-threshold mechanoreceptors in the bladder wall (Iggo, 1955; De Groat & Ryall, 1969). In the cat, these receptors have myelinated afferents, mainly of the A type, and they reach the sacral spinal cord via the pelvic nerves. The central course of the micturition reflex pathway is complex and not fully clarified. It involves, however, an excitatory spino-ponto-spinal reflex arc with bladder-specific preganglionic parasympathetic neurones as the final target in the sacral cord (Barrington, 1921; De Groat, 1975, 1990).
Recently, unmyelinated afferents from the bladder have attracted much interest. Such afferents have been visualized in several species, including man, using antibodies against various neuropeptides (Kawatani et al. 1986; Smet et al. 1996). Clinical and experimental studies with capsaicin, a drug that inactivates certain C afferents, suggest that these afferents may play a role in a number of pathological conditions of the urinary bladder (Bahns et al. 1987; Maggi et al. 1989; Fowler et al. 1994). It has also been proposed that they may be responsible for the recovery of reflex micturition after complete spinal lesion (De Groat, 1990; De Groat et al. 1990).
A few bladder C afferents have been identified as originating from nociceptors on the basis of their high mechanical threshold and/or response to noxious chemicals (Häbler et al. 1990). Others appear to originate from cold-sensitive receptors, sensitized by menthol and with dynamic temperature thresholds in the innocuous range (Lindström et al. 1990). In the cat, the latter afferents are responsible for a bladder-to-bladder cooling reflex (Fall et al. 1990; Lindström & Mazières, 1991). A bladder-to-bladder cooling reflex with similar innocuous threshold temperature can also be demonstrated in certain conditions in humans (Bors & Blinn, 1957; Geirsson, 1993; Geirsson et al. 1993b, 1994). Thus C afferents from the bladder appear to be endowed with specific receptors and to induce or facilitate bladder reflex activity.
Little is known about how bladder parasympathetic neurones respond to the C afferent input. In the present study we have tried to identify and characterize a bladder reflex evoked by electrical stimulation of such afferents. A bladder C afferent reflex has previously been identified by De Groat and collaborators (De Groat et al. 1981; De Groat, 1990), although never described in detail.
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METHODS |
Fifteen 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 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 feedback-controlled heating device. Heart rate and blood pressure were also monitored. Mean arterial pressure was maintained above 120 mmHg by a slow I.V. infusion of a bicarbonate-buffered Ringer-glucose solution. 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 intravesical pressure recordings. If not otherwise specified, the bladder was filled with body-warm saline to a volume below threshold for a spontaneous micturition reflex. In some experiments, a second catheter was inserted in the distal direction to allow separate perfusion of the urethra. The animals were killed at the end of the experiment by an overdose of the anaesthetics, followed by severance of the heart. The experimental procedures were approved by the Animal Research Ethical Committee of Linköping in accordance with Swedish law.
Bladder efferent activity was recorded from the proximal stump of a small pelvic nerve branch transected close to the bladder (Fig. 1). The filament was always distal to some parasympathetic ganglia and contained a mixture of pre- and postganglionic axons. The remaining pelvic nerve branches to the bladder were left in continuity. For stimulation, they were dissected free for about 10 mm close to the bladder, excluding nerve branches directed towards urethra (or other visceral organs), and lifted from the surrounding tissue onto pairs of silver hook electrodes. Stimulation electrodes were also placed on the ipsi- and contralateral dorsal clitoris (or penis) branches of the pudendal nerve. To isolate the stimulation sites from surrounding structures the exposed nerves were covered by paraffin oil in a pool formed by sewn-up skin flaps. The temperature of the pool was maintained at 36-38°C by a heating lamp. In three experiments, the hypogastric nerves and the sympathetic chains (below the L6 ganglia) were transected bilaterally. Laminectomies were also performed to allow for spinalization at the level of the T10-T11 segments (seven cats) or for unilateral transection of the S1-S3 dorsal roots (two cats) in the course of the experiment.
Nerves were stimulated with short rectangular pulses (0·2 ms) in order to increase the threshold separation between myelinated and unmyelinated afferents. For comparison between experiments, the strength of stimulation was expressed in multiples of the threshold intensity, i.e. the lowest stimulus strength that would elicit reflex activity in bladder pelvic efferents, determined with the bladder filled. In some animals, the pelvic nerve was cut about 20 mm central to the stimulation site at the end of the experiment and mounted for recording so that the threshold intensity for different nerve fibre components could be determined. Such recordings confirmed that the reflex threshold was close to threshold for bladder A afferent fibres. If not otherwise stated, the reflexes were evoked by a train of three pulses at 10 ms intervals with a repetition rate of 0·5-1 Hz. Latencies were measured from the first pulse in the train to onset of a particular reflex discharge.
Nerve signals were amplified with differential amplifiers equipped with appropriate filters, displayed on an oscilloscope and photographed for off-line analysis. Efferent reflex responses were also full-wave rectified, digitized and averaged (20 or 32 responses). For quantification, the areas under the averaged reflex responses were measured by computer. In addition, the nerve signals were displayed on a multichannel chart recorder and stored on a DC tape recorder, together with a recording of the intravesical pressure, heart rate and blood pressure. The peripheral conduction distance between the stimulation and recording sites of the pelvic nerve and the spinal cord was measured by dissection at the end of the experiments. It ranged from 95 to 117 mm in different experiments.
In four experiments, the effect of bladder and/or urethra cooling on the C fibre reflex was studied. To avoid concomitant activation of bladder A mechanoreceptor afferents during testing, two procedures were used for cold stimulation while keeping the bladder flaccid and empty. Cold (8°C) or body-warm fluid was either circulated through the bladder via a pair of catheters or perfused through the distal urethra. In both cases the bladder outlet was kept open to prevent bladder inflation, and hence mechanoreceptor activation. In some trials in each experiment the sensitivity of bladder or urethra cold receptors was enhanced by menthol exposure (0·6 mM in saline, 2 min) prior to the cold stimulation (Lindström & Mazières, 1991). Before testing, the menthol solution was washed out by body-warm saline.
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Figure 1. Schematic diagram of experimental arrangements
The spinal cord and sympathetic nerves to the bladder were transected during the course of some experiments. Further details are given in the text.
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RESULTS |
Identification of the bladder C fibre reflex
Electrical stimulation of the bladder pelvic nerve consistently evoked prolonged reflex discharges in pelvic efferents to the bladder. The response was complex, with several components activated at different stimulation intensities (Fig. 2).
The reflex component with lowest threshold appeared at an intensity when only A afferents were activated. In the illustrated case, this component was maximal at 3 times threshold (Fig. 2A and D). It had a latency of 115 ms, as measured from the first stimulus pulse to the onset of the response. This A component corresponds to the ordinary micturition reflex and could be evoked only when the bladder was partially filled. Its long latency is attributed to a central polysynaptic pathway with a relay in the pontine micturition centre (Barrington, 1921; De Groat, 1975).
With stronger stimuli (4-15 times threshold), an earlier response appeared with a latency of about 20 ms (Fig. 2B). This discharge was mainly due to a peripheral ganglionic reflex evoked by antidromic activation of branching preganglionic efferents (De Groat & Ryall, 1969). It was never seen on stimulation of the contralateral pelvic nerve (cf. Fig. 8B) and it survived decentralization of the ipsilateral pelvic ganglia by proximal transection of the pelvic and hypogastric nerves (cf. Fig. 3B).
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Figure 2. Reflex discharges in a bladder pelvic nerve branch evoked by electrical stimulation of ipsilateral bladder nerves
Pairs of records in A-C show the same response at two different sweep speeds. A, response evoked by a train of three stimuli (10 ms apart) at an intensity of 3 × threshold. This response originates from A bladder mechanoreceptor afferents and corresponds to the normal micturition reflex. B, responses evoked by a similar brief train of stimuli at higher intensity, 15 × threshold. The early discharge is due to a peripheral ganglionic reflex (see text). C, responses evoked at 60 × threshold. Note that the late reflex component in the upper trace is lacking in the corresponding records of A and B. This late reflex was evoked by stimulation of unmyelinated bladder C afferents. D, averaged rectified recording of 32 reflex responses at 3 × threshold (as in A); E, similar averaged recording of reflexes at 60 × threshold (as in C). The ganglionic (G), C fibre (C) and A fibre components of the reflex are indicated. The arrows in D and E indicate the first stimulus in the train; stimulus repetition rate, 0·5 Hz; bladder partially filled with saline. The time calibration in C corresponds to 250 ms for upper traces, and 100 ms for lower traces in the pairs A-C. The time calibration in E is also for D.
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A third reflex component with a latency of about 230 ms was evoked at still higher stimulation intensities (above 30 times threshold; Fig. 2C and E). From afferent recordings it was clear that C afferents were being recruited at these intensities. The strength-duration curve for this late reflex differed from that of the A component and was typical for unmyelinated afferents (Fig. 3A). With long stimulation pulses (5 ms, rheobase), the late component had a threshold of 250 µA compared with 19 µA for the A component, a 13-fold difference. With shorter pulses the difference became even larger, as indicated by the diverging curves in Fig. 3A. Similar strength-duration curves for the two reflex components were obtained in two other experiments. These properties, the high threshold and the characteristic strength-duration curve, demonstrate that the described late reflex component originates from unmyelinated bladder C afferents.
A similar bladder-to-bladder C fibre reflex was observed in all animals. It was easily evoked in three cats with the hypogastric nerves transected bilaterally and the sympathetic chains cut below the L6 ganglia (Fig. 3B, upper trace). In the same animals, the reflex was abolished by a subsequent transection of the pelvic nerve central to the most proximal pelvic ganglia (Fig. 3B, lower trace). Thus the C fibre reflex had both its afferents and efferents in the pelvic nerve. In two other experiments with preserved sympathetic innervation of the bladder, the C fibre reflex was abolished by selective transection of the S1-S3 dorsal roots at the side of stimulation and recording. A crossed A reflex could still be evoked by stimulating the opposite pelvic nerve, indicating that the efferent pathway was functional. Normal A and C fibre reflexes were evoked on the non-lesioned side. These experiments demonstrate that the C fibre reflex was centrally mediated with its afferents in the sacral dorsal roots. Thus they exclude the remote possibility that the reflex was mediated by a peripheral pathway through lumbo-sacral sympathetic ganglia (Kuo et al. 1984).
Characteristics of the C fibre reflex
The latency of the C fibre reflex varied considerably between animals (range, 150-250 ms). In situations with a concomitant A reflex, the onset of the C fibre reflex often overlapped in time with the A response. The latter had a latency of 90-130 ms and a duration of about 100 ms. The shorter latency values for the C fibre reflex were frequently obtained in situations when the A response was small or absent (see below). Even when A and C fibre reflexes merged in time, the two reflexes could be differentiated by careful grading of the stimulus strength as illustrated in Fig. 4. The threshold intensity for the A reflex was 50 µA in this experiment, and the response was maximal at 4 times this intensity (Fig. 4A (upper row of traces) and B). A large ganglionic response developed at higher intensities and, in parallel, the A reflex was suppressed (Fig. 4A (10-30 times threshold) and B). This suppression was presumably due to a combination of impulse collision, refractoriness and recurrent inhibition (De Groat & Ryall 1968b) evoked by antidromic activation of preganglionic fibres. A first trace of a C fibre reflex appeared at 30 times threshold and the reflex was maximal at 100 times (Fig. 4A (last trace) and B). The latency of this high-threshold component was only slightly longer than that of the A component (150 ms compared with 130 ms). In all other respects, it was a typical C fibre reflex.
The C fibre reflex was much increased by temporal summation and most easily elicited by a brief train of stimuli (three shocks at 10 ms intervals; Fig. 5A).
It was more frequency sensitive than the A reflex and virtually abolished at frequencies above 2 Hz (Fig. 5B). Note in the illustrated recordings that the A components were unaffected (Fig. 5A and B, bottom trace). The C fibre reflex also differed from the A component in that it was largely indifferent to changes in intravesical pressure. Thus the C fibre reflex was regularly evoked at low rates of stimulation (0·5-1·5 Hz), with the bladder empty or non-contracting. The A reflex, on the other hand, required some background facilitation and was not evoked by our pattern of stimulation when the bladder was empty. To obtain an A reflex it was necessary either to fill the bladder above threshold for bladder mechanoreceptors, or to stimulate the nerve repetitively at rates above 3-5 Hz.
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Figure 5. Temporal facilitation and depression of the bladder C fibre reflex
A, bladder C fibre reflexes (C) evoked by 1-3 stimuli at 10 ms intervals, 0·5 Hz, 80 × threshold; the bottom trace shows an A reflex evoked at 2 × threshold. B, C fibre reflexes evoked at different stimulus repetition rates (0·5-3·5 Hz, 3 shocks at 80 × threshold). Note the pronounced depression of the C fibre reflex at the highest frequency and the lack of effect on the early A component. All recordings are from the same animal, bladder partly filled. Each trace is the average of 20 responses. The time calibration in B refers to all records.
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The recordings in Fig. 6 show this difference in more detail. They were obtained with a small volume of fluid in the bladder, well below threshold for a spontaneous micturition reflex. With a non-contracting bladder, stimulation at low repetition rate (1 Hz) and at A intensity (three shocks at 2·5 times threshold) evoked no reflex response, as exemplified by the records from stimulus 18 and 25 in a long sequence of stimuli (Fig. 6A). After the twenty-fifth stimulus, a micturition contraction was triggered by the insertion of two extra stimuli in rapid succession (arrows). These two stimuli produced enough temporal facilitation to allow an A reflex to be evoked by the following stimulus (28). The reflex, marked by an asterisk, had a latency of about 100 ms (as best seen from the lower time-expanded trace). With the development of a detrusor contraction, the subsequent stimuli evoked similar A reflexes (stimulus 36) until the contraction spontaneously died away (stimulus 40).
The situation was entirely different with stimulation at C fibre intensity (50 times threshold; Fig. 6B). Already the first stimuli evoked C fibre reflexes, as exemplified by the response to the fifth stimulus. The latency of this reflex discharge was about 210 ms, i.e. twice as long as that of the A reflex, which explains why it cannot be seen in the time-expanded trace. The C fibre reflex induced small detrusor contractions that eventually resulted in facilitation of the A reflex (stimulus 6; cf. lower traces of stimuli 5 and 6), which in turn initiated a micturition contraction. As before, the A response subsided with the contraction (cf. stimuli 18 and 24) while the late C fibre reflex remained, albeit somewhat smaller (stimulus 24, upper trace). The late reflex suppression is presumably due to a central inhibitory process that terminates the micturition contraction (Lindström et al. 1984; Kruse et al. 1992). The described procedure was repeated several times in eight experiments with similar results. Bladder mechanoreceptors are strongly activated by detrusor contractions (Iggo, 1955; Bahns et al. 1987) and may then provide background facilitation of the central reflex pathway (s) of the bladder. Apparently, the A reflex is very much dependent on such background facilitation, while the C fibre reflex is not.
Spinalization
The effect of an acute spinal cord transection (at a low thoracic level) on the C fibre reflex was studied in seven experiments. The A micturition reflex is claimed to be abolished by such a lesion (De Groat & Ryall, 1969; De Groat et al. 1981). The C fibre reflex survived in all cases, although was somewhat decreased in amplitude. Recordings from one spin
alization experiment are shown in Fig. 7. Reflex discharges were evoked at the intensity maximal for the recruitment of A (10 times threshold; Fig. 7A) and C fibres (100 times threshold; Fig. 7B). The high stimulation intensity added a late C fibre component to the A reflex as revealed by the superimposed traces (Fig. 7C). A corresponding late, high-threshold component could be recorded within 1 min after the spinal cord transection (Fig. 7E). Stimulation at maximal A intensity revealed only a trace of a response at shorter latency (Fig. 7D).
The remaining A component was only 9 % of the control response as estimated from the areas of the rectified responses. The corresponding value for the C fibre reflex was 88 % (measured as the ratio (E - D)/(B - A) × 100; see Fig. 7). The relative proportion of the A and C fibre components remained the same for the entire observation period after the spinalization (about 2 h).
Another example of a surviving C fibre reflex after acute spinalization is shown in Fig. 8C. There was also in this case a minuscule short-latency discharge at A stimulation intensity while the C fibre reflex was about 70 % of the control. Obviously, the C fibre reflex is mainly mediated by a spinal reflex pathway and does not require an intact loop via the pontine micturition centre.
Another difference between the A and C fibre reflexes is emphasized in Fig. 8. A sizeable A reflex could regularly be evoked by stimulation of either the ipsilateral or contralateral bladder pelvic nerves. In contrast, the C fibre reflex was primarily unilateral. When the contralateral nerve was stimulated, the response was dominated by an early A component with only a trace of a late C fibre reflex (Fig. 8B). Acute spinalization completely eliminated all crossed reflex responses (Fig. 8D). Similar observations were obtained in two other animals. Thus the segmental C fibre reflex is mainly mediated by an ipsilateral pathway.
Inhibition
Both the A and C fibre reflexes were effectively suppressed by conditioning stimulation of afferents in the dorsal clitoris or penis nerves (seven experiments). For the C fibre reflex the inhibitory effect was readily demonstrated also after an acute spinalization (Fig. 9A and B), indicating that the inhibitory pathway is at least partly segmental in nature. The inhibitory effects originated from medium to small myelinated afferents activated at stimulation intensities below 20 times threshold (Fig. 9C).
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Figure 9. Suppression of the C fibre reflex by conditioning stimulation of the ipsilateral dorsal penis nerve (DP)
The averaged records in A represent the test C fibre response evoked by bladder pelvic nerve stimulation at 100 × threshold and 0·5 Hz. This test response was conditioned by a continuous stimulation of the DP nerve at 10 × threshold and 10 Hz (B). Both responses were obtained after an acute spinalization at T11 and are averages of 32 responses. The diagram in C shows the suppression of the C fibre reflex at different intensities of stimulation of the DP nerve.
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Effect of bladder cooling and menthol exposure
Since some C afferents from the bladder originate from cold-sensitive receptors (Lindström et al. 1990), it was of interest to test whether the C fibre reflex could be influenced by activation of such receptors. The experiments were performed so that concomitant stimulation of bladder mechanoreceptors was avoided to exclude interference with any evoked A reflex component. To that end, the bladder was maintained empty with an open outlet during the testing (see Methods), a situation in which the A afferents are normally silent. Accordingly, electrical stimulation of the bladder pelvic nerve elicited only a late, high-threshold C fibre reflex.
Bladder cooling regularly enhanced the C fibre reflex (eleven trials in two experiments, P < 0·01, sign test). The facilitation was more pronounced after bladder exposure to menthol (0·6 mM in saline, 2 min), which is known to sensitize cold receptors and to potentiate the bladder cooling reflex (Lindström & Mazières, 1991). In fact menthol exposure alone, without cold stimulation, greatly enhanced the C fibre reflex (three trials in two experiments). The effect of menthol and bladder cooling was fully reversible within 1 h.
A similar facilitation was consistently found after activation of cold-sensitive receptors in the distal urethra, receptors that are known to contribute to the bladder cooling reflex (Fall et al. 1990). These experiments are more illustrative since the cold-stimulated afferents differed from those that were electrically stimulated. The bladder C fibre reflex was evoked, as before, by stimulation of bladder pelvic nerve branches (Fig. 10A). The C fibre reflex was unaffected by a slow perfusion of the distal urethra with body-warm saline (Fig. 10B), but was greatly enhanced by a similar perfusion with cold (8°C) fluid (Fig. 10C). As with bladder cooling, the effect was fully reversible (not illustrated). The illustrated records were obtained in a spinalized cat, about 15 min after the cord transection. A similar facilitation was also easily shown in intact animals (fifteen trials in two experiments; P < 0·01, sign test).
The bladder C fibre reflex was also facilitated by urethral exposure to menthol. The records in Fig. 10D and E are from another experiment, before spinalization. They were obtained without any flow through the urethra. Shortly after menthol exposure, the bladder C fibre reflex had increased to more than twice its control size (Fig. 10D and E), an effect which was reversed within 50 min (Fig. 10F). Similar effects of urethral menthol exposure were observed in nine trials in four experiments.
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Figure 10. Effect of urethral cooling and menthol exposure on the bladder C fibre reflex
Averaged recordings of C fibre reflexes evoked by ipsilateral bladder pelvic nerve stimulation at maximal intensity (100 × threshold). The recordings were obtained with the bladder empty and open to avoid any A reflex component (see text). A, control response; B and C, responses evoked during urethral perfusion with body-warm (B) and cold (8 °C) saline (C). The animal was spinalized. Note that the C fibre reflex was much enhanced by the cold perfusion. D and E, effect of urethral exposure to menthol. The recordings are from another experiment in a cat with an intact spinal cord and were obtained without any flow through the urethra. D, control C fibre reflex; E, response evoked 4 min after a brief urethral exposure to menthol (0·6 mM in saline); F, recovery 50 min later. The bladder C fibre reflex was more than doubled by the menthol exposure. Each trace is the average of 20 rectified responses. The time calibration in F is for all records.
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DISCUSSION |
Two centrally mediated bladder-to-bladder reflexes could be distinguished by graded electrical stimulation of afferents in the bladder pelvic nerve. Both reflexes had their afferent and efferent limbs in the pelvic nerve. One was the well-known micturition reflex originating from bladder mechanoreceptor (A) afferents (De Groat, 1975, 1990), and the other an excitatory reflex elicited by stimulation of unmyelinated C afferents. The properties of these reflexes indicate that they differ not only in afferent supply but also in the organization of their central pathways.
The bladder C fibre reflex was identified by its high threshold to electrical stimulation, its typical strength- duration relation and its long latency. In different animals the estimated latency varied between 150 and 250 ms, compared with 90-130 ms for the A reflex. In the latter case, the long latency is mainly attributed to the conduction time in a spino-ponto-spinal reflex loop (De Groat & Ryall, 1969; De Groat, 1975). For the C fibre reflex, a considerable part of the latency could be consumed in the peripheral afferent path. Depending on the conduction velocity of the involved C afferents, ranging from 0·5 to 1·9 m s-1 (Lindström et al. 1990), the afferent conduction time could be anything from 50-60 to 190-230 ms (with a conduction distance of 95-117 mm). The efferent conduction time would be much less, about 10 ms, since the efferent activity was partly recorded from preganglionic (myelinated) axons (De Groat & Ryall 1968a; De Groat et al. 1982). This leaves the possibility for a quite long central delay, possibly in the same range as for the A reflex. Since the C fibre reflex survives an acute spinalization, this time is entirely consumed in a segmental pathway.
The organization of the spinal relay of the C fibre reflex is difficult to determine due to its long and variable central delay. The pronounced temporal facilitation of the reflex suggests that the pathway is polysynaptic. That the reflex is suppressed by repetitive stimulation at frequencies above 2 Hz seems to be related to the properties of the central pathway. Bladder C afferents follow stimulation frequencies of 5-10 Hz (S. Lindström, C.-H. Jiang & L. Mazières, unpublished observations) and the efferent preganglionic neurones even higher frequencies (De Groat & Saum, 1976). As for the A system, reflex bladder contractions are optimal at an afferent stimulation frequency as high as 20 Hz (Ebner et al. 1992).
The fact that the C fibre reflex, in contrast to the A reflex, can be evoked without background facilitation from bladder mechanoreceptors, is probably related to its partly separate segmental pathway. The C fibre reflex was little facilitated by A afferents (see Figs 7-9), whether activated by electrical stimulation or by bladder contractions. This lack of facilitation could imply that the two reflexes were mediated by separate preganglionic neurones. However, it is also possible that they had the same preganglionic neurones as a final common path. In this case, the explanation could be that bladder A afferents have dual central pathways, one excitatory and one inhibitory (Mazières et al. 1993). If both pathways were active in the present experimental situation, any facilitation could be counterbalanced by inhibition. To differentiate between these possibilities it would be necessary to record from individual bladder preganglionic neurones.
The existence of a bladder C fibre reflex in cats was first reported in a review by De Groat and collaborators (De Groat et al. 1981). They emphasized the prominence of the C fibre reflex in chronic spinal animals and proposed that this reflex would be responsible for the recovered micturition in this state. Our finding that the reflex consistently occurs in normal animals might be ascribed to our regular use of temporal facilitation with brief trains of stimuli. We also found that the reflex can be evoked within minutes after spinalization. In contrast, De Groat and collaborators stated that several days were necessary for the development of a C fibre reflex after spinalization (De Groat et al. 1981; De Groat, 1990).
There is agreement that the reflex is primarily ipsilateral in spinal animals. As found here, the reflex was not changed by transection of the sympathetic supply to the bladder while it was strongly inhibited by activation of genital afferents. The latter effect remained after spinalization which emphasizes the importance of the segmental control of the C fibre reflex pathway(s). A small A component was observed in several experiments after the acute spinalization. A segmental excitatory pathway from A bladder afferents has not been reported in cats, but a low-threshold, short-latency spinal reflex is regularly found in spinal rats (Steers & De Groat, 1988; Mallory et al. 1989). Whether this spinal reflex pathway plays a role in micturition of chronic spinal cats remains to be elucidated.
Our interest in the bladder C fibre reflex stemmed from the identification of a bladder cooling reflex that depended on stimulation of specific cold-sensitive receptors in the bladder and urethral wall (Fall et al. 1990; Lindström & Mazières, 1991). These receptors resemble cutaneous cold receptors in being sensitized by menthol, but they differ in having unmyelinated C afferents (Iggo, 1955; Lindström et al. 1990). As expected, the electrically evoked C fibre reflex was consistently facilitated by bladder as well as by urethral cooling and/or menthol exposure, thus confirming convergence in the pathway. It should be kept in mind that only a fraction of the identified C afferents from the bladder are cold sensitive; others seem to originate from nociceptors (Häbler et al. 1990) and possibly from chemoreceptors. Such afferents may also contribute to the bladder-to-bladder C fibre reflex, although this possibility has not yet been explored.
The bladder cooling reflex was first described by Bors & Blinn (1957) in humans with spinal cord lesions. The finding that in the cat the C fibre reflex survives an acute spinalization and is facilitated by bladder or urethral cooling, points to a similar organization in the two species. The bladder cooling reflex cannot be demonstrated in normal awake adults, but it occurs in patients with supraspinal lesions affecting the central control systems of the bladder (Geirsson et al. 1993a, b). It is also expressed in neurologically normal infants and children up to the age of four (Geirsson et al. 1994). This age dependence suggests that the bladder cooling reflex has its main functional role early in life. We have already proposed that the reflex may help the individual to rid itself of an unnecessary thermal ballast in the bladder when under cooling stress, and that such a mechanism may be particularly valuable in small individuals with unfavourable body surface-to-volume ratio (Fall et al. 1990).
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Acknowledgements
This investigation was supported by the Swedish Medical Research Council (project 04767) and by Torsten and Ragnar Söderberg's Stiftelser. L. M. was supported by a grant from INSERM (project 921106).
Corresponding author
S. Lindström: Department of Biomedicine and Surgery, Faculty of Health Sciences, University of Linköping, S-581 85 Linköping, Sweden.
Email: sivert.lindstrom{at}ibk.liu.se
Author's present address
L. Mazières: Service de Rééducation Neurologique, Hôpital de la Salpêtrière, 47-83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France.
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