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 μm) 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 μm) 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.

Key points: The prevalence of bladder conditions such as overactive bladder syndrome and urinary incontinence significantly increases with age, but how bladder function is altered by ageing is unclear. Sensory nerves together with the epithelial lining of the bladder known as the urothelium play a key role in mediating bladder function. In aged male mice we find a significant increase in natural bladder voiding, augmented afferent nerve firing during bladder filling and a significant increase in urothelial responses to purinergic receptor stimulation. This suggests that with ageing there is increased purinergic transmission in the mouse bladder which may lead to increased sensation and result in bladder hypersensitivity. These findings help us better understand how the function of the bladder may be affected by advancing age. The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age‐related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial‐afferent signalling in the mouse bladder. Three‐month‐old control and 24‐month‐old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X₃ receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X₃). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.

Objective: To investigate the direct effect of onabotulinumtoxin A ( O na B ot A ) on bladder afferent nerve activity and release of ATP and acetylcholine ( ACh ) from the urothelium. Materials and Methods: Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension. Intraluminal and extraluminal ATP , ACh , and nitric oxide ( NO ) release were measured using the luciferin–luciferase and A mplex ^® R ed assays ( M olecular P robes, C arlsbad, CA , USA ), and fluorometric assay kit, respectively. O na B ot A ( 2U ), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application. Whole‐nerve activity was analysed to classify the single afferent units responding to physiological (low‐threshold [ LT ] afferent <15 mmHg) and supra‐physiological (high‐threshold [ HT ] afferent >15 mmHg) distension pressures. Results: Bladder distension evoked reproducible pressure‐dependent increases in afferent nerve firing. After exposure to O na B ot A , both LT and HT afferent units were significantly attenuated. O na B ot A also significantly inhibited ATP release from the urothelium and increased NO release. Conclusion: These data indicate that O na B ot A attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that O na B ot A may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves.

Summary: The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five‐month‐old(young) and 24‐month‐old (aged) mice were used. H₂O₂, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H₂O₂‐induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N‐acetyl‐cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8‐mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.

Background: Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro‐immune association in the human bowel.Methods: Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age‐related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell‐nerve micro‐anatomical association were investigated by histological and immune staining.Key Results: Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo‐sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P‐immunoreactive (SP‐IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP‐IR nerves.Conclusions & Inferences: Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell‐nerve association suggest a compensatory mechanism for sensory neurodegeneration.