Afferent excitability changes during micturition
The excitability of 67 afferents was examined during micturition evoked by either bladder distension or brainstem (PMC) stimulation in decerebrated adult cats. Within the first sacral segment (S1), 19 urethral afferents (mean conduction velocity, 38.9 ± 11.4 m s
−1) and 34 afferents (mean conduction velocity, 41.6 ± 16.9 m s
−1) associated with a hindlimb cutaneous (CCF, CCS, LCS, SP) or perineal nerves were sampled. The remaining 14 afferents studied were hindlimb cutaneous (CCF, CCS, LCS, SP) afferents (mean conduction velocity, 41.7 ± 11.3 m s
−1) terminating within the fifth to seventh lumbar segments. The depth of the intra-spinal stimulating microelectrode used for excitability testing of afferents in the S1 segment ranged from 210 to 1730 μ
m from the dorsal surface of the spinal cord and from 502 to 1730 μ
m in the lumbar segments. Results were obtained during voiding characterized by a simultaneous bladder pressure increase and decreased EUS activity during which time fluid was expelled from the animal.
Urethral afferent excitability during micturition
Seven of the 19 urethral afferents displayed a biphasic excitability change during either brainstem- (
Fig. 1A) or distension-evoked (
Fig. 1B) micturition. This biphasic response was characterized by a decrease in the intra-spinal current threshold when the EUS activity ceased and the bladder contracted followed by an increase in the required intra-spinal current when the EUS activity resumed at the end of the void. The mean peak decrease in intra-spinal current was 11 ± 7 % and the increase was 21 ± 26 %, as measured from the prevoid baseline.
Along with the abrupt biphasic response at the time of the micturition reflex, Fig. 1B shows one of two urethral afferents that displayed a progressive decrease in excitability over several minutes during bladder filling prior to micturition. This same pattern was seen in both afferents during repeated filling and voiding cycles. In both of these afferents, the step-like decreases in the intra-spinal stimulus current seen during the filling period were coincident with progressive reductions in EUS electroneurogram activity. While this pattern of change in excitability and EUS activity was seen in only two animals in the present study, similar progressive decreases in EUS activity (or motoneurone membrane potential) during the later stages of slow bladder filling have been reported before (Fedirchuk & Shefchyk, 1993; see Fig. 1 of Shimoda et al. 1992). Superimposed on the gradual intra-spinal current threshold decrease was sometimes an additional small decrease in threshold at the time of the bladder contraction. In both afferents, the intra-spinal current threshold increased to above control levels immediately after the void as was characteristic of the biphasic response described earlier. In these cases, control levels were measured minutes earlier before the gradual decrease in current threshold manifested itself.
In addition to the biphasic excitability changes observed, four of 19 urethral afferent fibres displayed a monophasic increase in excitability when the EUS activity was suppressed and the bladder contracted (not shown). Three of the 19 afferents displayed a decrease in excitability or PAH during the reflex void and the remaining five afferents showed no measurable change in excitability. The excitability changes in the urethral afferents during micturition are summarized in Table 1.
| Table 1 Summary of excitability changes in afferents during micturition |
Excitability changes in urethral afferents evoked by segmental afferent stimulation
Although some urethral afferents did not display an excitability change during micturition, all the afferents examined underwent PAD in response to stimulation of at least one source of segmental afferents.
Figure 2A shows the typical pattern of intra-spinal current threshold changes produced in a urethral afferent with stimulation of either perineal or hindlimb cutaneous afferents at 5
T. Stimulation at 5
T was selected since it was the lowest stimulus strength that produced a reliable excitability increase and had been used previously to examine sacral cutaneous afferent pathways (
Fedirchuk et al. 1992a,
b). Similar to the results reported for the common sensory pudendal nerve by
Angel et al. (1994), PAD could be evoked by conditioning stimulation of nerves carrying afferents of either proximal perineal (cutSPud, SFP) or more distal hindlimb receptive fields (CCF, Sural).
| Figure 2PAD evoked in two urethral afferents (conduction velocity: A, 45 m s−1; B, 42 m s−1) terminating in S1 produced by trains of stimuli at varying strengths (indicated by filled bars above the traces) to cutaneous (A) and hindlimb muscle (more ...) |
Urethral afferents showed PAD evoked by hindlimb muscle nerve stimulation at 5T, but not at 2T, presumably reflecting a predominant group II muscle afferent-evoked PAD (Jack, 1978; Edgley & Jankowska, 1987; Jankowska & Riddell, 1994). In a total of 68 tests in 17 urethral afferents in which both 2T and 5T stimulus strengths were tested, 5T but not 2T strengths evoked an excitability increase 97 % of the time. Figure 2B shows the change in intra-spinal stimulation current evoked by electrical stimulation of gastrocnemius muscle afferents in one of the two afferents in which PAD was evoked with < 2T stimulation. It is noteworthy that, in both these cases, it was stimulation of the gastrocnemius afferents that evoked the PAD and that a considerable proportion of group II muscle afferents in the gastrocnemius nerve were activated at strengths < 2T (Jack, 1978). A summary of the incidence of PAD measured in urethral afferents evoked by stimulation of various segmental nerves at 5T is shown in Fig. 3. Many of the nerves produced PAD in at least 50 % of the afferents tested and the mean decrease in intra-spinal stimulation current ranged from 7 to 19 %.
| Figure 3 Summary of the incidence of excitability changes evoked in urethral afferents by stimulation of various segmental afferents at 5T |
Modulation of urethral afferent-evoked EPSPs during micturition
Intracellular recordings were made from eight sacral sphincter motoneurones that received polysynaptic (central latency > 2.5 ms) excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of urethral pudendal afferents (1.6-5
T). The motoneurone resting membrane potential ranged from -45 to -71 mV. Due to the absence of any urethral afferent-evoked EPSPs in hindlimb motoneurones, no evaluation of urethral-evoked EPSPs in hindlimb motoneurones not undergoing membrane potential or conductance changes during micturition could be undertaken (see previous work by
Fedirchuk et al. 1994).
The most commonly observed pattern of modulation of urethral-evoked EPSPs during micturition is shown in Fig. 4. In this EUS motoneurone, during the void and initial period of membrane hyperpolarization (Fig. 4Ab and Bb), the peak amplitude of the urethral-evoked EPSP was diminished to 10 % of its prevoid control amplitude. In the remaining part of the void when the membrane was still hyperpolarized, the EPSP recovered to 25 % of the control value (Fig. 4Ac and Bc). This pattern of modulation of urethral-evoked EPSPs was observed in six sphincter motoneurones that hyperpolarized by 4-10 mV during micturition. On average, in these motoneurones the peak amplitude of the urethral-evoked EPSPs was diminished to 36 ± 19 % of the prevoid value. In one EAS motoneurone which depolarized by several millivolts during the void, the EPSP was reduced to 67 % of the control amplitude. In another EAS motoneurone which did not show a change in membrane potential during voiding, the amplitude of the urethral-evoked EPSP was unchanged. While not measured directly in the current protocol, it is quite likely that there was a conductance change in the motoneurones that hyperpolarized or depolarized during voiding (see Fedirchuk & Shefchyk, 1993; Fedirchuk et al. 1994).
| Figure 4 Modulation of urethral pudendal-evoked excitatory postsynaptic potentials recorded in an EUS motoneurone during distension-evoked micturition |
Excitability of hindlimb cutaneous and perineal afferents during micturition
PAD was observed during micturition in 25 out of 34 afferents including 16 hindlimb cutaneous (6 CCF, 9 Sural and 1 SP) and nine perineal (6 cutSPud and 3 SFP) afferents terminating in S1 (see
Table 1). There was no excitability change observed during either distension- or PMC-evoked micturition in nine afferents, although stimulation of segmental afferents in the absence of micturition did produce increases in the excitability in each afferent. The mean maximum change in the intra-spinal current threshold measured during voiding ranged from 6 ± 1 % (Sural), 10 ± 3 % (SFP), 12 ± 7 % (cutSPud) and 14 ± 8 % (CCF).
Figure 5A illustrates typical data from one of the 17 afferents in which the onset of the decrease in intra-spinal current threshold coincided with the onset of increased bladder pressure and decreased EUS activity (indicated with a vertical dashed line). In another four afferents, the excitability change did not appear immediately at the onset of the void but was delayed to the time at which the peak bladder pressure occurred (not shown). As shown in
Fig. 5B, the lack of a tight coupling between the timing and changes in the intra-spinal current threshold and the micturition reflex was also evident in afferents in which the current threshold remained elevated for seconds to several minutes past the void and the point at which the EUS activity resumed and the void was over. In summary, 80 % of the hindlimb afferents and just over 50 % of the perineal afferents examined in S1 showed evidence of PAD at some time during distension-evoked micturition, although the onset and offset of the excitability change with respect to both bladder pressure and EUS activity was not always tightly linked.
While the direction of the excitability changes (e.g. increase) observed during distension-evoked voids (Fig. 5) and PMC-evoked voiding (Fig. 6) were similar, the decrease in the intra-spinal current threshold during PMC-evoked voids was on average 8 % larger than that observed during distension-evoked voiding. In eight afferents it was possible to compare the evoked excitability changes during both distension- and PMC-evoked voiding. In three afferents, the decrease in the intra-spinal stimulus current was greatest during PMC-evoked voiding (mean = 12 ± 5 %) compared with the mean change of 8 ± 2 % measured during distension-evoked voiding. During PMC stimulation there was evidence of PMC stimulus-locked inhibition of the EUS activity but, as evident in Fig. 6A and B, the largest excitability changes occurred during the evoked void and were not sustained throughout the time of brainstem stimulation. Thus the PAD seen during PMC stimulation was not probably due solely to a stimulus-locked activation of descending systems but also reflected the activation of the micturition circuitry for a period of time during the stimulation. The other five afferents did not display an excitability change during either distension- or PMC-evoked voiding.
In sharp contrast to the afferents examined in S1, 13 afferents (10 Sural, 2 CCF and 1 SFP) sampled between mid L5 and rostral L7 failed to display a change in excitability (not shown) during either distension- or PMC-evoked voiding (see Table 1). Only one Sural afferent recorded from L7 displayed a 5 % increase in the intra-spinal current beyond control values (i.e.PAH). All the fibres tested in the lumbar segments received PAD in response to conditioning stimulation of other segmental cutaneous or muscle afferents.
Segmental afferent-evoked excitability changes in hindlimb and perineal sacral afferents
Twenty-eight afferents (10 CCF, 3 LCS, 12 CCS, and 3 SP) were tested for both cutaneous- and muscle afferent-evoked excitability changes. All of the fibres showed an increase in excitability in response to conditioning stimulation of either lumbar or sacral urethral, cutaneous or muscle afferents. The sites of intra-spinal stimulation and the afferent terminals sampled were located throughout the dorsal and intermediate spinal grey. Several previous studies (
Eccles et al. 1963;
Jankowska et al. 1993;
Angel et al. 1994) have reported similar or slightly greater excitability changes in cutaneous or joint afferents by stimulation of either cutaneous or group II muscle afferents although only the study by
Angel et al. (1994) was done in a comparable non-anaesthetized preparation.
The cutaneous afferents associated with the different hindlimb nerves examined appeared similar to one another in terms of their sources of PAD although some differences may not have been evident due to the small sample size for some afferents. Figure 7 shows typical changes in the intra-spinal current threshold in three sural afferents and a CCF afferent evoked by stimulation of either cutaneous and urethral (Fig. 7A) or muscle nerves (Fig. 7B-D) at various stimulus strengths. The incidence and magnitude of the excitability increases in the two branches of the sural nerve (lateral and caudal sural) and the CCF afferents with conditioning stimulation of various segmental afferents are summarized in Fig. 8A. The data represented in this figure include not only the 28 afferents in which both cutaneous and muscle afferents were tested but also additional afferents in which only cutaneous afferents were tested. The mean decrease in intra-spinal current threshold with conditioning stimulation (5T) of hindlimb cutaneous afferents was 16 ± 8 % (range, 5-32 %), 13 ± 8 % (range, 5-38 %) for perineal afferents (cutSPud and SFP) and 10 ± 7 % (range, 5-26 %) with stimulation of the urethral nerve.
| Figure 7 Cutaneous and muscle afferent-evoked PAD in four hindlimb cutaneous afferents terminating in S1 |
| Figure 8 Incidence of PAD and the magnitude of the intra-spinal current threshold changes evoked in various hindlimb cutaneous afferents with stimulation of cutaneous (A) or muscle (B) afferents |
With regard to the muscle afferent-evoked excitability changes, in 30 of 31 tests when muscle nerve stimulation was tested at both 5T and 2T, only 5T strengths evoked excitability changes. Conditioning stimulation at 2T (near maximum group I strength) was examined 84 times in 28 afferents with an average of three different muscle nerves tested on each afferent. A decrease in the intra-spinal current threshold of ≥ 5 % was found in only one of 28 fibres (a CCF afferent). However, while this single CCF afferent showed an increase in excitability with 2T PBSt stimulation, strengths < 2T produced no changes.
Figure 7B shows that stimulation of either a flexor (PBSt) or an extensor muscle nerve (Q, GS) at > 2T evoked a decrease in intra-spinal current threshold and that in almost every case more than one muscle nerve was effective in a given afferent. Although only tested in a few fibres, combined simultaneous conditioning stimulation of two effective muscle nerves at 5T did not increase the magnitude of the evoked PAD beyond that observed with conditioning of a single muscle nerve. There was also no evidence of a reduction of the excitability increase with simultaneous stimulation of two nerves using a 20 ms condition-test interval and both threshold and subthreshold conditioning stimulation strengths. In 13 of the 27 afferents that received PAD with 5T (but not with 2T) stimulation strengths, additional increases in the strength of the conditioning stimuli above 5T (e.g. 10T) resulted in a further 4 % decrease in the intra-spinal current threshold (as evident in Fig. 7B with Q stimulation and Fig. 7D with PBSt stimulation).
The PAD evoked with 5T conditioning nerve stimulation in the pooled sample of hindlimb afferents was examined to reveal the muscle nerves most effective in evoking PAD, presumably by group II muscle afferent activation. The histogram in Fig. 8B shows that stimulation of afferents of either flexor (PBSt) or extensor (GS, Q) muscle nerves was effective; about 75 % of all fibres received PAD from PBSt nerve stimulation, about 25 % from GS stimulation, and just over 80 % of the fibres received PAD from 5T Q stimulation (note, however, the small sample size). In addition, the mean decrease in intra-spinal current threshold evoked by muscle afferents at 5T was 9 ± 4 % as compared with that evoked by conditioning stimulation of cutaneous afferents (16 ± 8 %).