Skin-Nerve Preparation for Single-Fiber Recording

Nav1.8^ChR2 mice of both males and females aged 8–11 weeks were used. Animals were anesthetized with 5% isoflurane and then sacrificed by decapitation. The hindpaw glabrous skin including plantar and finger regions together with medial planter nerve and tibial nerve before the branch from sciatic nerves were dissected out. The skin-nerve preparation was then placed in a Sylgard Silicone-coated bottom of a 60-mm recording chamber. The fat, muscle and connective tissues on the nerves and the skin were carefully removed with a pair of forceps. The skin was affixed to the bottom of the chamber by tissue pins with epidermis side facing up, and the nerve bundle was affixed by a tissue anchor in the same recording chamber. The cutting end of the nerve bundle was briefly exposed to a mixture of 0.05% dispase II plus 0.05% collagenase for 30–60 s, and the enzymes were then washed off by the normal Krebs solution (see below). This gentle enzyme treatment was to help separating individual afferent fibers at the cutting end of the nerve bundle so that a single fiber could be aspirated into the recording electrode and pressure-clamped for single-fiber recordings (see below). The recording chamber was then mounted on the stage of the Olympus BX51WI upright microscope. The skin-nerve preparation was superfused with a normal Krebs bath solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose (pH 7.3 and osmolarity 325 mOsm) and was saturated with 95% O₂ and 5% CO₂. The Krebs bath solution in the recording chamber was maintained at 28–32 °C during experiments.