Sylgard base

The isolated mid-colon (1.0 cm) was rapidly transferred to a dish for dissection. This colon segment was opened along the mesenteric border and pinned (mucosa side up) to a Sylgard base (Dow Corning, Midland, MI, USA). The mucosa layer was removed, and the preparations were sliced into muscle strips in the circular or longitudinal axis. These test strips were placed in 10 ml of organ bath containing modified Krebs solution bubbled with 95% O₂ and 5% CO₂ at 37.0 ± 0.5°C, which was replaced every 20–30 min. An isometric force transducer (Harvard VF-1 Harvard Apparatus, Holliston, MA, USA) connected to a computer via an amplifier was used to record the motility of the muscle strips. These data were digitalized (25 Hz) using the Data 2001 software (Panlab, Barcelona, Spain) coupled to an A/D converter and were stored in the computer. The tension was set to 1 g to minimize local reflex stimulation of the bowel, and the strips were allowed to equilibrate for 1 h. Electrical field stimulation (0.5 ms, 10 Hz, and 0.5 mA) was applied using platinum electrodes connected via stimulus isolation units (Grass SIN5) to a square wave stimulator (Grass 588, Grass, Quincy, MA, USA). The concentration of all reagents is presented as the final concentration in 10 ml of organ bath.

Tissues were transferred to a bespoke rectangular recording chamber (100 mm (length)×60 mm (width)×20 mm (depth), with Sylgard base (Dow Corning, UK) and pinned flat with serosal facing up (figure 1A). Tissues were superfused with carbogenated Krebs buffer at a rate of 6 mL/min maintained at 32–34°C supplemented with atropine (10 µM) and nifedipine (10 µM) to prevent smooth muscle contractility. Mesenteric nerve bundles were dissected and recorded using suction electrodes as previously described.22 (link)

Lumbar splanchnic nerve (LSN) afferent preparations were conducted as previously described (47 (link)). Briefly, the distal colorectum (splenic flexure to rectum) and associated LSN (rostral to inferior mesenteric ganglia) were isolated from mice euthanized as described earlier. The colon was cannulated with fine thread (polyester, Gutermann) in a rectangular recording chamber with Sylgard base (Dow Corning, UK) and serosally superfused (7 mL/min; 32°C–34°C) and luminally perfused (200 μL/min) by a syringe pump (Harvard apparatus, MA) with carbogenated Krebs buffer solution (95% O₂–5% CO₂). Krebs buffer was supplemented with 10 μM atropine and 10 μM nifedipine to prevent smooth muscle activity (49 (link)).
Borosilicate suction electrodes were used to record the multiunit activity of LSN bundles. Signals were amplified (gain 5 kHz), band pass filtered (100–1,300 Hz; Neurolog, Digitimer Ltd, UK), and digitally filtered for 50 Hz noise (Humbug, Quest Scientific, Canada). Analog signals were digitized at 20 kHz (Micro1401; Cambridge Electronic Design, UK), and signals were visualized with Spike2 software (Cambridge Electronic Design, UK).