Bladder afferent sensitivity in wild-type and TRPV1 knockout mice

Abstract

Understanding bladder afferent pathways may reveal novel targets for therapy of lower urinary tract disorders such as overactive bladder syndrome and cystitis. Several potential candidate molecules have been postulated as playing a significant role in bladder function. One such candidate is the transient receptor potential vanilloid 1 (TRPV1) ion channel. Mice lacking the TRPV1 channel have altered micturition thresholds suggesting that TRPV1 channels may play a role in the detection of bladder filling. The aim of this study was therefore to investigate the role of TRPV1 receptors in controlling bladder afferent sensitivity in the mouse using pharmacological receptor blockade and genetic deletion of the channel. Multiunit afferent activity was recorded in vitro from bladder afferents taken from wild-type (TRPV+/+) mice and knockout (TRPV1-/-) mice. In wild-type preparations, ramp distension of the bladder to a maximal pressure of 40 mmHg produced a graded increase in afferent activity. Bath application of the TRPV1 antagonist capsazepine (10 mum) caused a significant attenuation of afferent discharge in TRPV1+/+ mice. Afferent responses to distension were significantly attenuated in TRPV1-/- mice in which sensitivity to intravesical hydrochloric acid (50 mm) and capsaicin (10 microm) were also blunted. Altered mechanosensitivity occurred in the absence of any changes in the pressure-volume relationship during filling indicating that this was not secondary to a change in bladder compliance. Single-unit analysis was used to classify individual afferents into low-threshold and high-threshold fibres. Low threshold afferent responses were attenuated in TRPV1-/- mice compared to the TRPV1+/+ littermates while surprisingly high threshold afferent sensitivity was unchanged. While TRPV1 channels are not considered to be mechanically gated, the present study demonstrates a clear role for TRPV1 in the excitability of particularly low threshold bladder afferents. This suggests that TRPV1 may play an important role in normal bladder function.

Figure 1. Afferent response to bladder distension in the mouse

A, representative sequential rate histogram of the afferent response to a control distension (with isotonic saline at a rate of 100 μl min⁻¹). B, raw multiunit afferent nerve activity in response to ramp distension. C, increase in intravesical pressure due to infusion of saline. Note that two phases can clearly be identified, a first phase in which the pressure increases slowly and the afferent response increases rapidly and a second phase where there is a more rapid increase in intravesical pressure and a slower increase in afferent activity.

Figure 2. The effect of capsazepine on afferent response to ramp distension

A, whole nerve afferent response to distension before (control) and after application of 10 μm capsazepine in wild-type mice (P < 0.001 2-way ANOVA). B, the whole nerve response has been normalized to the number of single units in the afferent bundles (see Methods). The variability in the whole nerve responses arising because of the different number of single units that contribute to each nerve bundle is eliminated by this procedure making the statistical difference after treatment more robust (P < 0.001, 2-way ANOVA).

Figure 3. Effect of capsaicin and hydrochloric acid on bladder afferent activity

A, representative trace showing a marked increase in baseline afferent discharge in the presence of 100 μm capsaicin in TRPV1+/+. B, representative trace showing response during intravesical application of 50 mm HCl in a wild-type preparation. C, bar graph showing the magnitude of the increase in afferent discharge in response to HCl and capsaicin in TRPV1+/+ and TRPV1−/− mice (P < 0.05, t test).

Figure 4. The response to ramp distension in TRPV1+/+ and TRPV1−/− mice

A, afferent response to increasing intravesical pressure. Whole nerve discharge is normalized to the number of single units in the afferent bundles (P < 0.001, two-way ANOVA and Bonferroni post test). B, pressure–volume curve during distension in the TRPV1+/+ and TRPV1−/− mice. Note the curves are identical indicating that bladder compliance is not different in the TRPV1−/− mouse.

Figure 5. Single unit analysis of bladder afferent responses to distension

A, representative traces of increased intravesical pressure, concurrent increase in afferent discharge and whole nerve spike rate histogram. Waveform analysis (see Methods) revealed 5 distinct single units that could be clearly separated using the Principle Component analysis function in Spike2 software (C). Individual spike shapes are coloured differently in the wavemark channel and presented in separate histograms in panel B. Overlaid action potentials are shown in the insets which illustrates the distinct amplitude and duration of these single units. Each inset represents a period of 2 ms. Note that each unit has a distinct threshold and response profile which is used to classify individual units into LT and HT afferents. In this example the lower 3 afferents were classified as LT and the upper 2 as HT.

Figure 6. Stimulus–response profile of low threshold (A) and high threshold (B) bladder afferents

Note that the LT afferents have a blunted response profile in the TRPV1−/− mice compared to wildtype (P < 0.001, 2-way ANOVA and Bonferroni post test). The response profile of HT afferents is unchanged.