Visceromotor response (VMR) recordings

Mice were anesthetized with urethane (IP,1.2g/Kg). The depth of anesthesia was monitored with respiration rate, heart rate, and the response to noxious pinch of the hindpaw. A balloon, made from polyethylene (length, 1.5 cm; diameter, 0.9 cm) affixed to the end of PE-60 tubing, was used to distend the colon. The balloon was inserted transanally until the proximal end of the balloon was 0.5 cm from the anal verge (total balloon insertion = 2 cm) and secured to the mouse tail with tape. Balloon distention of the colon to 60 mmHg, referred to as colorectal distention (CRD), was applied for 10 s every four min. The response to three stimuli were collected before and after infusion of test agents. The CRD balloon was designed to enable infusion of the test agents rostral to the balloon using a catheter (PE-10 tubing) running parallel of PE-60 tubing through the balloon and finishing 2 mm outside rostral of the balloon. The volume of each intracolonic infusion was 0.1 ml and was given immediately after the last of the three baseline distensions. We chose not to collect full stimulus response curves to minimize the number of times the colon would have to be stimulated before and after the infusion of test agents, as well as to minimize the possibility of changes in the response to the bolus infusion of a test agent over time.

Abdominal muscle contraction, referred to as the VMR, evoked in response to CRD was measured with EMG electrodes implanted in the abdominal muscle wall. The VMR activity was amplified (10,000X), filtered (0.3 to 10 kHz band pass), sampled at 20 kHz with 1401 interference (Cambridge Electronic Desing, Cambridge, UK) and stored in a PC for analysis off line. Using the Spike 2 software, the EMG waveform was rectified, and the integral of the resting activity subtracted waveform was used to quantify the VMR, where resting activity was the activity in the 10 sec prior to distention. VMR data collected after the infusion of the test compounds were analyzed as a percent change from the average of the three responses just prior to the infusion of test compounds. Following completion of the VMR experiments, mice were killed by decapitation.