The Journal of Physiology Neuroscience

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1 J Physiol (2012) pp The Journal of Physiology Neuroscience Inhibition of micturition reflex by activation of somatic afferents in posterior femoral cutaneous nerve Changfeng Tai 1,BingShen 1, Abhijith D. Mally 1, Fan Zhang 1,2, Shouguo Zhao 1,3, Jicheng Wang 1, James R. Roppolo 4 and William C. de Groat 4 1 Department of Urology, University of Pittsburgh, Pittsburgh, PA, USA 2 Department of Urology, China Rehabilitation Research Center, School of Rehabilitation Medicine, Capital Medical University, Beijing, China 3 Department of Biomedical Engineering, Beijing Jiaotong University, Beijing, China 4 Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA Key points Activation of afferents in the posterior femoral cutaneous nerve (PFCN) can reflexively induce efferent firing in the pudendal nerve and pudendal afferent firing via a motor sensory coupling. Activation of pudendal afferent nerves can inhibit the micturition reflex, suggesting PFCN stimulation might also inhibit the micturition reflex. This study discovered a somato-bladder inhibitory reflex elicited by electrical or tactile stimulation of cutaneous afferents in the PFCN, but excluded the involvement of pudendal nerves. This PFCN-bladder inhibitory reflex could be utilized to develop new neuromodulation therapies for lower urinary tract disorders. Abstract This study determined if activation of somatic afferents in posterior femoral cutaneous nerve (PFCN) could modulate the micturition reflex recorded under isovolumetric conditions in α-chloralose anaesthetized cats. PFCN stimulation inhibited reflex bladder activity and significantly (P < 0.05) increased bladder capacity during slow infusion of saline or 0.25% acetic acid (AA). The optimal frequency for PFCN stimulation-induced bladder inhibition was between 3 and 10 Hz, and a minimal stimulation intensity of half of the threshold for inducing anal twitching was required. Bilateral pudendal nerve transection eliminated PFCN stimulation-induced anal twitching but did not change the stimulation-induced bladder inhibition, excluding the involvement of pudendal afferent or efferent axons in PFCN afferent inhibition. Mechanical or electrical stimulation on the skin surface in the PFCN dermatome also inhibited bladder activity. Prolonged (2 30 min)pfcn stimulation induced a post-stimulation inhibition that persists for at least 2 h. This study revealed a new cutaneous-bladder reflex activated by PFCN afferents. Although the mechanisms and physiological functions of this cutaneous-bladder reflex need to be further studied, our data raise the possibility that stimulation of PFCN afferents might be useful clinically for the treatment of overactive bladder symptoms. (Received 20 March 2012; accepted after revision 2 August 2012; first published online 6 August 2012) Corresponding authors C. Tai: Department of Urology, University of Pittsburgh, 700 Kaufmann Building, Pittsburgh, PA 15213, USA. cftai@pitt.edu Abbreviations AA, acetic acid; CMG, cystometrogram; PFCN, posterior femoral cutaneous nerve; T, threshold. DOI: /jphysiol

2 4946 C. Tai and others J Physiol Introduction It is known that electrical stimulation of pudendal nerve afferent axons can inhibit the micturition reflex (Tai et al. 2006, 2011a). It is also known that activation of afferents in the posterior femoral cutaneous nerve (PFCN) can reflexively induce efferent firing in the pudendal nerve (McMahon et al. 1982) to elicit sphincter muscle contractions that in turn can induce pudendal afferent firing via a motor sensory coupling (Lagunes-Cordoba et al. 2010). Therefore, it seems logical to hypothesize that stimulation of somatic afferents in PFCN will inhibit the micturition reflex indirectly by activation of the pudendal efferent and afferent pathways. However, to our knowledge this putative somato-bladder inhibitory reflex mechanism has never been examined. Sphincter and pelvic floor reflexes evoked by PFCN afferents innervating the thigh and perineum have been shown to increase vaginal wall tone that may play a role in mating behaviour (Lagunes-Cordoba et al. 2010). A PFCN afferent suppression of bladder activity may be another reflex mechanism that occurs during mating. This study was undertaken in anaesthetized cats to determine: (1) if electrical stimulation of PFCN can inhibit the micturition reflex, (2) if mechanical or electrical stimulation of cutaneous afferents in the PFCN dermatome on the surface of the thigh inhibits reflex micturition, and (3) if axonal pathways in the pudendal nerve are involved in the PFCN stimulation-induced inhibition of bladder activity. The experiments revealed a new cutaneous-bladder reflex that increases bladder capacity. This reflex, which may suppress voiding during mating, might be activatible by devices used for neuromodulation and therefore be useful clinically for treatment of lower urinary tract dysfunctions such as overactive bladder (Andersson & Wein, 2004). Methods All protocols involving the use of animals in this study were approved by the Animal Care and Use Committee at the University of Pittsburgh. Experimental set-up The experiments were conducted in 26 cats (2.2 kg to 3.9 kg, 10 male and 16 female) under α-chloralose anaesthesia (65 mg kg 1, I.V. supplemented as necessary) after initial isoflurane (2 3% in O 2 ) anaesthesia for surgery. Heart rate and blood oxygen level were measured by a pulse oximeter (9847V, Nonin Medical, Inc., Plymouth, MN, USA) with the sensor attached to the tongue. Systemic blood pressure was monitored throughout the experiment via a catheter inserted in the right carotid artery. A tracheotomy was performed and a tube was inserted to keep the airway patent. A catheter for I.V. infusion was introduced into the right cephalic vein. The ureters were cut and drained externally. A double lumen catheter was inserted through the urethra into the bladder and secured by a ligature around the urethra. One lumen of the catheter was connected to a pump to infuse the bladder with saline or 0.25% acetic acid (AA) at a rate of ml min 1 and the other lumen was connected to a pressure transducer to measure the pressure change in the bladder. The PFCN and the common pudendal nerve (including both motor and sensory branches) were exposed from the right side via a 3 4 cm incision between the tail and the sciatic notch. A tripolar cuff electrode (NC223pt, MicroProbe Inc., Gaithersburg, MD, USA) was implanted on the PFCN and then connected to a stimulator (S88, Grass Medical Instruments, Quincy, MA, USA). A suture was looped around the right common pudendal nerve for transecting the nerve by pulling the suture out at the end of the experiment. A suture was also placed around the left common pudendal nerve for the same purpose. Then the skin incisions were closed by sutures. Stimulation protocol Uniphasic rectangular pulses (0.2 ms pulse width) of 1 40 Hz frequency were delivered to the PFCN via the cuff electrode. The intensity threshold (T) for inducing anal twitching was determined at 5 Hz by gradually increasing the stimulation intensity. Then, multiples of the threshold intensity were used for PFCN stimulation. In the first group of experiments (n = 10 cats), the effect of PFCN stimulation was tested during repeated cystometrogram (CMG) that consisted of a slow infusion of saline or AA starting with an empty bladder until the threshold volume (i.e. bladder capacity) that triggers the first large amplitude (>30 cmh 2 O) long duration (>20 s) micturition contraction (see Fig. 1A). Initially, two or three control CMGs were performed during saline infusion without stimulation to obtain the control bladder capacity and evaluate reproducibility. Then, PFCN stimulation of 5 Hz at different intensities (0.25, 0.5, 1, and 2T) was applied during repeated saline CMGs (see Fig. 1A). This was followed by two control saline CMGs without stimulation and a series of repeated saline CMGs with PFCN stimulation at 1T intensity but different frequencies (1 40 Hz) (see Fig. 2A). Then, the infusion was changed from saline to 0.25% AA in order to irritate the bladder, activate bladder afferent C-fibres, and induce bladder overactivity. During repeated AA CMGs, PFCN stimulation of 5 Hz at 1T or 2T intensity was applied to inhibit the AA-induced bladder overactivity and increase

3 J Physiol Cutaneous inhibition of micturition reflex 4947 the bladder capacity (see Fig. 3A). Finally, the pudendal nerves were transected bilaterally and PFCN stimulation of 5Hzat1T intensitywasappliedagainduringaacmgto determine if the inhibition was maintained (see Fig. 4C). The inhibitory effect was quantified by measuring the increase in bladder capacity during PFCN stimulation. Stimulation was applied at the start of each CMG, and both the stimulation and the infusion were stopped at the onset of the first reflex micturition contraction. The bladder was emptied after each CMG and a 5 10 min rest period was inserted between CMGs to allow the distended detrusor to recover. In the second group of experiments (n = 16 cats), the post-stimulation effect of prolonged (30 min) PFCN stimulation was examined by performing repeated saline CMGs. Initially two or three CMGs were performed without stimulation to obtain the control bladder capacity and evaluate reproducibility. Then, two groups of experiments were conducted: (1) control group without stimulation (n = 8 cats) and (2) treatment group with PFCN stimulation (n = 8 cats) (see Fig. 5). In the treatment group the bladder volume was maintained at a volume slightly above the bladder capacity to induce isovolumetric rhythmic bladder contractions. Then, 5 Hz PFCN stimulation at 1T intensity was applied for 30 min to inhibit the isovolumetric contractions. After the 30 min stimulation, five CMGs were performed within a h period to examine the change of bladder capacity. At the end of the fifth CMG, the bladder volume was again maintained at a volume slightly above the bladder capacity to induce isovolumetric rhythmic contractions, during which a second 30 min PFCN stimulation was applied to inhibit the contractions. The post-stimulation effect on bladder capacity induced by the second 30 min stimulation was evaluated by another five CMGs repeated within h after the termination of the second stimulation treatment. In the control group the procedures similar to those used in the treatment group were performed, but the PFCN stimulation was not applied during either the first or the second 30 min treatment period. Instead, the isovolumetric rhythmic bladder contractions were allowed to continue during each 30 min period. The bladder was emptied after each CMG and a 5 10 min rest period was inserted between CMGs to allow the distended detrusor to recover. The 30 min stimulation duration was chosen based on our previous study of tibial nerve stimulation (Tai et al. 2011b). After testing for a post-stimulation effect, the bladder was infused with saline to a volume slightly above the bladder capacity to induce isovolumetric rhythmic bladder contractions. Mechanical stimulation of a broad skin area of the posterior thigh including the PFCN dermatome (Burgess et al. 1974) was applied by lightly stroking the skin area repeatedly (2 3 times s 1 for s, stroke length 2 3 cm) using a cotton swab to inhibit the isovolumetric rhythmic bladder contractions (see Fig. 7). Then, a self-adhesive pad electrode (FE10ND, Grass Technologies, West Warwick, RI, USA) 1 cm in diameter was attached to the most effective skin area identified during mechanical stimulation. Another pad electrode was inserted underneath the skin via a small incision facing up to the skin surface electrode to electrically stimulate the PFCN branches in the skin area between the two pad electrodes during saline CMGs (see Fig. 8A). Finally, the pudendal nerves were transected bilaterally and PFCN stimulation (5 Hz, 1T) via the cuff electrode was applied again during saline CMGs to determine if the inhibition was maintained (see Fig. 4A). Data analysis For the repeated CMG recordings, the bladder capacities were measured and normalized to the measurement of the first control CMG in each experimental group. Normalization of bladder capacities avoided the large variation among the control bladder capacities caused by individual animal differences. PFCN inhibition is also better measured as percentage change relative to the control bladder capacity rather than as a change in absolute values (i.e. ml). Repeated measurements in the same animal during the same experiment were averaged. The normalized data from different animals are presented as means ± SEM. One-way ANOVA followed by Dunnett s multiple comparison, two-way ANOVA followed by Bonferroni multiple comparison, or Student s t test was used to detect statistical significance (P < 0.05). Results PFCN stimulation inhibits the micturition reflex during saline infusion The inhibitory effect of PFCN stimulation on the micturition reflex was dependent on both stimulation intensity and frequency (Figs 1 2). When applied during saline CMG at 5 Hz, PFCN stimulation significantly (P < 0.05) increased bladder capacity to 194 ± 34%, 199 ± 26%, and 226 ± 36% of control (6.2 ± 1.2 ml, n = 10) at0.5t, 1T, and2t intensity, respectively (Fig. 1). Increasing intensity from 0.5T to 2T did not significantly increase bladder capacity. When different frequencies were comparedat 1T intensity, PFCN stimulation significantly (P < 0.05) increased bladder capacity to 162 ± 17%, 168 ± 19% and 159 ± 15% of control (8.3 ± 1.4 ml, n = 10) at 3 Hz, 5 Hz and 10 Hz, respectively, but was ineffective at 1 Hz and Hz (Fig. 2). This inhibitory effect was not completely reversible after stimulation

4 4948 C. Tai and others J Physiol (Figs1A and 2A). This post-stimulation effect was further studied in the following result section. PFCN stimulation inhibits the micturition reflex during AA infusion Bladder irritation by 0.25% AA induced bladder overactivity and significantly (P < 0.05) reduced bladder capacity to 21 ± 5% of saline control capacity (10.8 ± 1.7 ml, n = 8) (Fig. 3). PFCN stimulation at 5 Hz inhibited AA-induced bladder overactivity and significantly (P < 0.05) increased bladder capacity to 43 ± 10% and 62± 12% of saline control capacity at intensity of 1T and 2T, respectively (Fig. 3A and B). Effect of pudendal nerve transection on PFCN inhibition of micturition reflex Bilateral transection of pudendal nerves did not change the inhibitory effect of PFCN stimulation on the micturition reflex during either saline infusion (Fig. 4A and B)orAA infusion (Fig. 4C and D). During saline infusion, PFCN stimulation (5 Hz, 1T) significantly (P < 0.05) increased bladder capacity to 147 ± 9% and 154 ± 20% of control (8.1 ± 1.4ml, n = 5) before and after pudendal nerve transection, respectively (Fig. 4B). During AA infusion, PFCN stimulation significantly (P < 0.05) increased the small capacity (15 20% of saline control) of the irritated bladder to 43 ± 10% and 46± 8% of saline control (11.4 ± 1.8 ml, n = 7), respectively (Fig. 4D). Bilateral pudendal nerve transection that eliminated the stimulation-induced anal twitching did not change control bladder capacity during saline infusion, but significantly (P < 0.05) decreased control bladder capacity during AA infusion. Prolonged PFCN stimulation elicits a persistent post-stimulation increase in bladder capacity Post-stimulation increase in bladder capacity was first observed during repeated CMG when we examined different stimulation intensities or frequencies (Figs 1A and 2A). In order to confirm and quantify this post-stimulation inhibition, five CMGs were performed in a h period after a prolonged (30 min) PFCN stimulation (5 Hz, 1T) was applied to eight cats that had not received any prior stimulation. Figure 1. Inhibition of micturition reflex by stimulation of posterior femoral cutaneous nerve is dependent on stimulation intensity A, stimulation of different intensities was applied during repeated CMGs during intravesical saline infusion. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = 4 V. T is the threshold intensity for inducing anal twitching. The black bars under the CMG traces indicate the stimulation duration. B, bladder capacity was significantly increased by stimulation of intensity 0.5Tand above. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity T = V. n = 10 cats. Significantly (P < 0.05) different from control.

5 J Physiol Cutaneous inhibition of micturition reflex 4949 As shown in Fig. 5A the bladder capacity in control experiments remained relatively constant during repeated CMGs performed over 3 4 h which included two 30 min periods of constant bladder distension to induce rhythmic bladder contractions but without PFCN stimulation. However when 5 Hz PFCN stimulation was applied continuously during the first 30 min period at 1T intensity to inhibit the rhythmic bladder contractions (Fig. 5B), the bladder capacity was significantly increased following the stimulation but then slightly decreased with time (Fig. 5B). However, a second 30 min PFCN stimulation again increased bladder capacity and maintained a large bladder capacity during the next h period after the stimulation (Fig. 5B). In a series of eight cats, complete inhibition occurred in eight cats during the first 30 min stimulation, while it occurred in seven cats during the second 30 min stimulation. The post-stimulation inhibitory effect of 30 min PFCN stimulation is summarized in Fig. 6. In the control un-stimulated animals, bladder capacity did not significantly (P > 0.05) change during 10 repeated CMGs performed over a 3 4h period. On the other hand bladder capacity was significantly (P < 0.05) increased during the initial CMG after the first 30 min PFCN stimulation, and the second 30 min stimulation further increased the bladder capacity and maintained a significantly large bladder capacity for at least 2 h (Fig. 6). Figure 2. Inhibition of micturition reflex by stimulation of posterior femoral cutaneous nerve is dependent on stimulation frequency A, stimulation of different frequencies was applied during repeated CMGs during intravesical saline infusion. Stimulation: pulse width 0.2 ms, intensity 1T = 6 V. T is the threshold intensity for inducing anal twitching. The bars under the CMG traces indicate the stimulation duration. B, bladder capacity was significantly increased by stimulation of frequency 3 10 Hz. Stimulation: pulse width 0.2 ms, intensity T = V. n = 10 cats. Significantly (P < 0.05) different from control.

6 4950 C. Tai and others J Physiol Stimulation of PFCN dermatome inhibits the micturition reflex After conducting the post-stimulation test, mechanical or electrical stimulation of PFCN dermatome was further investigated. The bladder was filled with saline to a volume exceeding the threshold for eliciting a large amplitude micturition reflex contraction and then maintained under constant volume conditions to induce large amplitude contractions ( cmh 2 O) that occurred at regular intervals (1 2 per min) over the course of many hours (Fig. 7). During the isovolumetric rhythmic bladder contractions, mechanical stimulation of the PFCN dermatome was tested in four cats by lightly stroking the skin repeatedly (2 3 times s 1 for s, stroke length 2 3 cm) using a cotton swab. The effective skin areas for inducing bladder inhibition are marked on the left side in Fig. 7 with the ineffective areas marked on the right side. The numbers in the circled skin areas indicated the number of cats exhibiting inhibitory or non-inhibitory responses indicated by arrows to representative recordings in Fig. 7. In seven cats at the most effective skin area (i.e. the shadowed area marked with the number 4 in Fig. 7), a pad electrode was attached on the skin surface with another pad electrode inserted underneath the skin via a small incision caudal to the electrode site (see Fig. 8A). Then the skin between the two pad electrodes (1 cm in diameter) was electrically stimulated during saline infusion CMG (Fig. 8A). Electrical stimulation of 5 Hz at 1T intensity significantly (P < 0.05) increased the bladder capacity to 120 ± 8% of control (12.7 ± 2ml,n = 7) (Fig. 8B). Discussion This study discovered a new somato-bladder reflex elicited by electrical or tactile stimulation of cutaneous afferents in the PFCN. Activating this reflex by direct stimulation of PFCN significantly inhibits the normal reflex bladder activity during saline distension (Figs 1 and2)aswellastheoveractivereflexbladderactivity during AA irritation (Fig. 3). The optimal frequency for PFCN stimulation to induce bladder inhibition is between 3 and 10 Hz (Fig. 2) and the minimal effective intensity is half of the threshold for inducing reflex anal twitching (Fig. 1). Bilateral pudendal nerve transection that eliminates PFCN stimulation-induced anal twitching does not change the stimulation-induced bladder inhibition (Fig. 4). Prolonged PFCN stimulation induces a post-stimulation increase in bladder capacity that persists for at least 2 h (Figs 5 and 6). Mechanical or electrical stimulation of PFCN dermatome also inhibits the reflex bladder activity (Figs 7 and 8). These results indicate that activation of PFCN afferents can trigger an inhibitory reflex to the bladder in addition to an excitatory Figure 3. Stimulation of posterior femoral cutaneous nerve inhibits bladder overactivity induced by 0.25% acetic acid (AA) irritation A, stimulation of different intensities was applied during repeated CMGs during intravesical AA infusion. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = 4 V. T is the threshold intensity for inducing anal twitching. The black bars under the CMG traces indicate the stimulation duration. B, bladder capacity was significantly increased by stimulation of intensity 1 2T. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity T = 4 8 V. n = 8cats. Significantly (P < 0.05) different.

7 J Physiol Cutaneous inhibition of micturition reflex 4951 reflex to the peri-vaginal muscles that may facilitate mating function (Lagunes-Cordoba et al. 2010). Although our study identified a prominent inhibitory effect of PFCN afferent activity on the micturition reflex, it did not confirm our original hypothesis that the inhibition would be mediated by reflex activation of the pudendal efferent pathways to the anal and urethral sphincters (McMahon et al. 1982) that in turn activates pudendal afferent pathways via a motor sensory coupling (Lagunes-Cordoba et al. 2010). Bilateral pudendal nerve transection eliminated the anal twitching induced by PFCN but did not change the bladder inhibition induced by PFCN stimulation (Fig. 4). Thus other mechanisms for PFCN inhibition must be important including (1) reflex activation of sympathetic pathways in the hypogastric nerve by PFCN stimulation that in turn can inhibit bladder smooth muscle and/or suppress synaptic transmission in bladder parasympathetic ganglia (de Groat & Lalley, 1972; de Groat & Saum, 1972; de Groat & Theobald, 1976), and (2) suppression of synaptic transmission in the micturition reflex pathway in the spinal cord or brain by afferent input from the PFCN. Further studies are needed to examine these mechanisms. Figure 4. Inhibition of micturition reflex by stimulation of posterior femoral cutaneous nerve is maintained after bilateral transection of the pudendal nerves during intravesical saline (A and B) or 0.25% acetic acid infusion (C and D) Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T. T is the threshold intensity for inducing anal twitching; T = 3 8 V. n = 5catsinB, n = 7catsinD. Significantly (P < 0.05) different.

8 4952 C. Tai and others J Physiol The PFCN afferent inhibition of bladder activity was very effective, producing a complete inhibition of reflex bladder contractions under isovolumetric conditions and eliciting a marked increase in bladder capacity that is equivalent to the increases in bladder capacity induced by stimulation of other somatic afferent nerves (e.g. pudendal, tibial, or afferent innervation of the foot) (Tai et al. 2010, 2011a,b). There are also other similarities but also some differences between the modulatory effects elicited by the various nerves. For example the optimal frequency (3 10 Hz, Fig. 2) for PFCN inhibition is similar to that for pudendal nerve inhibition in cats (Tai et al. 2011a), but is different from tibial or foot stimulation that is effective in inhibiting the bladder at both low (5 Hz) and high (20 30 Hz) frequencies (Tai et al. 2010, 2011b). On the other hand, PFCN stimulation can induce persistent post-stimulation inhibition (Figs 5 and 6) similar to that induced by tibial nerve stimulation (Tai et al. 2011b), whereas pudendal nerve stimulation does not induce post-stimulation inhibition (Tai et al. 2012b). These comparisons indicate that the neural mechanisms underlying PFCN inhibition of the micturition reflex might not be the same as either pudendal or tibial nerve inhibition. Our previous study in cats (Tai et al. 2012a) revealed a role of opioid receptors in tibial inhibition of the bladder overactivity, whereas our recent unpublished data indicate that opioid receptors are not involved in pudendal inhibition of bladder overactivity. Thus each somato-bladder reflex mechanism may exhibit a unique set of properties. Identifying the neurotransmitters/receptors involved in somatic inhibition (pudendal, tibial, or PFCN) of the micturition reflex will ultimately reveal the underlying synaptic mechanisms and may provide new pharmacological targets to treat overactive bladder symptoms (Andersson & Wein, 2004). Reflex bladder activity is initiated by bladder Aδ- and C-fibre afferent axons travelling in the pelvic nerve to the sacral spinal cord. Under normal physiological conditions bladder distension generates afferent firing in bladder Aδ-fibres that triggers a spinobulbospinal micturition Figure 5. Post-stimulation inhibitory effect by stimulation of posterior femoral cutaneous nerve is evident during repeated CMG testing A, bladder capacity was not significantly changed during repeated CMGs in the absence of nerve stimulation. Note: after the initial and 5th CMG, the bladder was maintained for 30 min in a distended condition. B,bladder capacity was significantly increased during the first CMG following a 30 min stimulation. It was further increased after the second 30 min stimulation. Five repeated CMGs (1st 5th and 6th 10th) were performed within h after each 30 min period of stimulation. The vertical dashed line indicates the control bladder capacity. The horizontal bar indicates the 30 min stimulation. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = 7 V. T is the threshold intensity for inducing anal twitching.

9 J Physiol Cutaneous inhibition of micturition reflex 4953 Figure 6. Prolonged post-stimulation inhibition induced by 30 min stimulation of posterior femoral cutaneous nerve After the second 30 min stimulation, the post-stimulation inhibitory effect lasted more than h. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = 3 8 V. T is the intensity threshold for inducing anal twitching. Statistically significant difference between the control and treatment groups. Vertical dashed line indicates the time of the second 30 min stimulation. Control: n = 8 cats. Stimulation: n = 8cats. reflex (Fowler et al. 2008), while bladder afferent C-fibres are silent(habler et al. 1990). However in pathological conditions, bladder afferent C-fibres are activated that trigger a spinal micturition reflex causing bladder overactivity (Cheng et al. 1999; Szallasi & Fowler, 2002; Fowler et al. 2008). In this study, saline distension of the bladder was used to activate the afferent Aδ-fibres and mimic the normal physiological conditions, while AA was used to irritate the bladder, activate the afferent C-fibres, and mimic pathological conditions. Reflex bladder activity was inhibited during either saline distension (Figs 1 and 2) or AA irritation (Fig. 3) indicating that PFCN afferent input suppressed micturition reflexes mediated by either bladder afferent Aδ-fibres or C-fibres. PFCN inhibition of C-fibre afferent mediated bladder overactivity suggests a potential clinical application of PFCN stimulation for treating overactive bladder symptoms. This study also raises the possibility that new neuromodulation therapies might be developed to treat overactive bladder. Currently sacral, pudendal, or tibial neuromodulation has been applied clinically to treat overactive bladder patients (van Kerrebroeck et al. 2007; MacDiarmid et al. 2010; Peters et al. 2010). However, Figure 7. Isovolumetric bladder contractions are inhibited by mechanical stimulation of skin areas (shaded) innervated by posterior femoral cutaneous nerve Number in the circled area indicates the number of animals in which the effects (inhibition or no inhibition) were observed. A total of 4 cats were tested. The bar under the bladder pressure trace indicates the stimulation duration. Mechanical stimulation was applied by repeatedly stroking the skin area using a cotton swab. Bladder was distended by saline. The relative locations of main nerve trunks are illustrated on the right side for posterior femoral cutaneous nerve (PFCN), pudendal nerve (Pud), tibial nerve (Tibial), and peroneal nerve (Peroneal).

10 4954 C. Tai and others J Physiol sacral or pudendal neuromodulation requires surgery to permanently implant a stimulator (InterStim stimulator, Medtronic Inc., Minneapolis, MN, USA) and insert a tined electrode in S3 sacral formen or close to the pudendal nerve. Although tibial neuromodulation is minimally invasive, only involving a needle insertion at the ankle to stimulate the tibial nerve, it requires frequent clinical visits for a 30 min stimulation once per week for initial 12 weeks followed by maintenance therapy once every 2 3 weeks (Peters et al. 2009; MacDiarmid et al. 2010). Compared to sacral, pudendal, or tibial neuromodulation, PFCN neuromodulation therapy could be fully non-invasive by attaching skin surface electrodes to the posterior thigh Figure 8. Inhibition of the micturition reflex by stimulation of the skin area innervated by posterior femoral cutaneous nerve A, CMG recordings and stimulation location. The stimulated skin area was sandwiched by two pad electrodes. The bar under bladder pressure trace indicates the stimulation duration. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = 40 V. T is the threshold intensity for inducing anal twitching. B, bladder capacity was significantly increased by the skin stimulation. n = 7 cats. Stimulation: frequency 5 Hz, pulse width 0.2 ms, intensity 1T = V. Significantly (P < 0.05) different. area to directly stimulate the PFCN branches innervating the skin (Figs 7 8) or to stimulate transcutaneously the PFCN beneath the skin. Since prolonged (2 30 min) PFCN stimulation like the tibial nerve stimulation in cats (Tai et al. 2011b) induces a persistent post-stimulation inhibition lasting for hours (Figs 5 and 6), a stimulation protocol similar to tibial neuromodulation might also be used in PFCN neuromodulation to apply a 60 min or longer stimulation once per week. A more frequent stimulation protocol might also be employed if necessary because PFCN stimulation could be fully non-invasive and be self-administered by the patient without frequent clinical visits. In addition to the non-invasive approach, PFCN neuromodulation therapy could also utilize an implantable stimulator and insert an electrode close to the PFCN for stimulation. This type of PFCN neuromodulation approach would have a significant advantage over sacral or pudendal neuromodulation because the PFCN is more superficial than sacral root or pudendal nerve and thereby easily accessible. It may also have a higher efficacy than non-invasive approach because more afferent fibres of PFCN could be activated by the electrode close to the main branch of PFCN. A clinical trial of PFCN neuromodulation to treat overactive bladder symptoms is feasible due to its non-invasive feature, or its easy accessibility for implantation of a commercially available stimulator (InterStim stimulator, Medtronic Inc.). It is worth noting that the bladder contraction is prolonged when PFCN stimulation inhibits the micturition reflex and significantly increases the bladder capacity (Figs 1 4). This is probably due to the fact that the larger bladder volume during PFCN stimulation generates more tension on the bladder wall and thereby more afferent firing in the bladder afferent nerves. The increased bladder afferent activity enhances the micturition reflex resulting in prolonged bladder contraction. This result also indicates that PFCN stimulation inhibits the micturition reflex by increasing the threshold level for bladder afferent activity to trigger a micturition reflex. However, once the micturition reflex is triggered the magnitude of the reflex is not suppressed. This would be beneficial for a clinical application to increase the bladder storage without interrupting voiding function. Despite the potential clinical application of PFCN stimulation, the physiological function of PFCN afferent inhibition of micturition reflex is unclear. A recent study in cats (Lagunes-Cordoba et al. 2010) has speculated that the reflex from PFCN afferents to pudendal efferent might help to maintain the peri-vaginal muscle contractions during mating. In addition PFCN afferent inhibition of micturition reflex would be desirable during mating. Further investigation of the PFCN afferent evoked visceral reflexes is definitely warranted.

11 J Physiol Cutaneous inhibition of micturition reflex 4955 References Andersson KE & Wein AJ (2004). Pharmacology of the lower urinary tract: Basis for current and future treatments of urinary incontinence. Pharmacol Rev 56, Burgess PR, English KB, Horch KW & Stensaas LJ (1974). Patterning in the regeneration of type I cutaneous receptors. JPhysiol236, Cheng CL, Liu JC, Chang SY, Ma CP & de Groat WC (1999). Effect of capsaicin on the micturition reflex in normal and chronic spinal cord-injured cats. Am J Physiol Regul Integr Comp Physiol 277, R786 R794. de Groat WC & Lalley PM (1972). Reflex firing in the lumbar sympathetic outflow to activation of vesical afferent fibres. J Physiol 226, de Groat WC & Saum WR (1972). Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat. JPhysiol220, de Groat WC & Theobald RJ (1976). Reflex activation of sympathetic pathways to vesical smooth muscle and parasympathetic ganglia by electrical stimulation of vesical afferents. JPhysiol259, Fowler CJ, Griffiths D & de Groat WC (2008). The neural control of micturition. Nat Rev Neurosci 9, Habler HJ, Janig W & Koltzenburg M (1990). Activation of unmyelinated afferent fibers by mechanical stimuli and inflammation of the urinary bladder in the cat. JPhysiol425, Lagunes-Cordoba R, Hernandez PR, Raya JG & Munoz-Martinez EJ (2010). Functional coupling between motor and sensory nerves through contraction of sphincters in the pudendal area of the female cat. J Neurophysiol 103, MacDiarmid SA, Peters KM, Shobeiri SA, Wooldridge LS, Rovner ES, Leong FC, Siegel SW, Tate SB & Feagins BA (2010). Long-term durability of percutaneous tibial nerve stimulation for the treatment of overactive bladder. JUrol 183, McMahon SB, Morrison JFB & Spillane K (1982). Plasticity of urinary bladder reflexes evoked by stimulation of pudendal afferent nerves after chronic spinal cord injury in cats. J Physiol 328, Peters KM, Killinger KA, Boguslawski BM & Boura JA (2010). Chronic pudendal neuromodulation: expanding available treatment options for refractory urologic symptoms. Neurourol Urodyn 29, Peters KM, MacDiarmid SA, Wooldridge LS, Leong FC, Shobeiri SA, Rovner ES, Siegel SW, Tate SB, Jarnagin BK, Rosenblatt PL & Feagins BA (2009). Randomized trial of percutaneous tibial nerve stimulation versus extended-release tolterodine: results from the overactive bladder innovative therapy trial. JUrol182, Szallasi A & Fowler CJ (2002). After a decade of intravesical vanilloid therapy: still more questions than answers. Lancet Neurol 1, TaiC,ChenM,ShenB,WangJ,LiuH,RoppoloJR&deGroat WC (2011a). Plasticity of urinary bladder reflexes evoked by stimulation of pudendal afferent nerves after chronic spinal cord injury in cats. Exp Neurol 228, Tai C, Larson JA, Ogagan PD, Chen G, Shen B, Wang J, Roppolo JR & de Groat WC (2012a). Differential role of opioid receptors in tibial nerve inhibition of nociceptive and non-nociceptive bladder reflexes in cats. Am J Physiol Renal Physiol 302, F Tai C, Shen B, Chen M, Wang J, Roppolo JR & de Groat WC (2011b). Prolonged poststimulation inhibition of bladder activity by tibial nerve stimulation in cats. Am J Physiol Renal Physiol 300, F385 F392. TaiC,ShenB,ChenM,WangJ,LiuH,RoppoloJR&deGroat WC (2010). Suppression of bladder overactivity by activation of somatic afferent nerves in the foot. BJU Int 107, Tai C, Shen B, Wang J, Liu H, Subbaroyan J, Roppolo JR & de Groat WC (2012b). Bladder inhibition by intermittent pudendal nerve stimulation in cat using transdermal amplitude-modulated signal (TAMS). Neurourol Urodyn 31, Tai C, Smerin SE, de Groat WC & Roppolo JR (2006). Pudendal-to-bladder reflex in chronic spinal-cord-injured cats. Exp Neurol 197, van Kerrebroeck PE, van Voskuilen AC, Heesakkers JP, Nijholt AABL, Siegel S, Jonas U, Fowler CJ, Fall M, Gajewski JB, Hassouna MM, Cappellano F, Elhilali MM, Milam DF, Das AK, Dijkema HE & van den Hombergh U (2007). Results of sacral neuromodulation therapy for urinary voiding dysfunction: outcomes of a prospective, worldwide clinical study. JUrol178, Author contributions Experiments were performed at the University of Pittsburgh. All authors contributed to the conception and design of the experiments, the collection, analysis and interpretation of data, and drafting the article or revising it critically for important intellectual content. All authors have approved the final version of the manuscript. Acknowledgements This study is supported by NIH under grants DK , DK and DK