Differential amplifier

After induction of anesthesia with urethane, the left ureter was catheterized, and the EUS‐EMG electrodes implanted (per above). The bladder catheter was then connected via a three‐way connector to an infusion pump for saline infusion (Braintree Scientific, Braintree, MA, USA) and a pressure transducer (World Precision Instruments [WPI, LLC]; Sarasota, FL, USA) for bladder pressure recording. The three EUS‐EMG electrodes were connected to a differential amplifier (A‐M Systems, Sequim, WA, USA). The animal was placed in a supine position on a surgical table in a reverse Trendelenburg position and a container coupled to a weight transducer (WPI, LLC) was placed under the rat for urine collection and measurement. Ten urination cycles were elicited with saline (rate: 0.12 ml/min). After the recording of 10 fill/void cycles, the abdominal wall was re‐opened and a flared PE‐60 catheter inserted into the bladder dome as described above, and the opening re‐closed. The ureter catheter was sealed and replaced with the dome catheter by connection to the pressure transducer and pump for the collection of 10 more fill‐void cycles. CMG parameters analyzed included baseline basal pressure, inter‐contraction interval, bursting period duration, void pressure, and maximal bladder pressure.

Mice were lightly anesthetized with 15% urethane dissolved in water (1.25 g/kg). Once tail pinch reflexes were lost, mice were secured into a surgical plate with body temperature maintained at 37°C. A hole was drilled through the skull above the right forelimb somatosensory cortex (FLS1) so a 1–2 MΩ glass micropipette filled with HEPES-buffered Artificial Cerebral-Spinal Fluid (ACSF) could be inserted into the brain 200–300 μm below the cortical surface. Evoked potentials were amplified (1000×) and filtered between 1 and 1000 Hz with a differential amplifier (A-M Systems). A single 5 ms deflection of the forelimb with a piezoelectric wafer (∼300 μm deflection) was used to evoke cortical field potentials every 10 s and averaged over 45 trials. Cortical responses were collected for up to 60 min after topical application of 50 mM GABA_A agonist muscimol or 100 μM of the inverse agonist L-655,708.