In vivo Extracellular Recordings of Dorsal Horn Neurons

The methods used for the in vivo extracellular recordings are described in detail elsewhere [31 (link)]. Briefly, extracellular single-unit recordings of the superficial dorsal horn (lamina II) neurons were performed as follows: recordings were conducted using superficial dorsal horn neurons at the depths of 20 to 150 µm from the surface. Unit signals were acquired using an amplifier (EX1; Dagan Corporation, Minneapolis, MN, USA). The data were digitized with an analog-to-digital converter (Digidata 1400A; Molecular Devices, Union City, CA, USA) and analyzed with Clampfit (version 10.2; Molecular Devices, Union City, CA, USA). To determine the site of stimulation, we searched for sites where tactile stimuli on the skin (debrided cotton) or unpleasant plucking stimuli (forceps) caused a neural response. For mechanical stimulation, the skin was bent with thin vFFs, and bending forces of 5.88, 9.8, 13.72, 39.2, 58.8, 78.4, 147, 255 mN (0.6, 1.0, 1.4, 4.0, 6.0, 8.0, 15.0, 26.0 g) were applied, respectively. Stimulation was applied for 10 s at the maximum response point of each receptive field in the ipsilateral hindlimb. For the lidocaine evaluation, 0.5% lidocaine (AstraZeneca Japan, Osaka, Japan) was administered at the vFF stimulation site.