Extracellular amplifier

Extracellular suction electrode recordings were obtained by drawing the right abdominal-pleural connective and the siphon nerve each into a glass electrode filled with high-divalent cation saline (Fig. 2). An extracellular amplifier (model 1800, A-M Systems, Sequim, WA) was used to acquire and amplify extracellular recordings from nerves. These signals were digitized with a PowerLab 4/35 (AD Instruments, Dunedin, New Zealand) at a sampling rate of 1 kHz. Data were recorded using LabChart (v7.3.8, AD Instruments, Dunedin, New Zealand). When comparing FUS to electrical stimuli, nerve stimulation was produced with LabChart and applied via the A-M Systems extracellular amplifier. The siphon nerve was used for nerve stimulation, which consisted of 5–10 V, 1 ms pulses at 20 Hz for 0.25–1 s. Electrophysiological data were exported to Matlab (R2018b, MathWorks, Natick, MA) for analysis.

Whole-cell current-clamp recordings were made from spinal motoneurons localized in lumbosacral segments 1–3 as described previously (Gonzalez-Islas et al. 2010 (link)). Briefly, whole-cell recordings (electrodes, 5–10 MΩ) were obtained from motoneurons identified by their lateral position in the cord. Once whole-cell configuration was achieved, motoneurons were maintained under voltage clamp for a period of 5 minutes to allow stabilization before switching to current clamp configuration. Recordings were terminated whenever significant increases in resistance (≥20%) occurred. Cords were perfused with Tyrode’s solution as described above. The intracellular patch solution for these experiments contained the following (in mM): 5 NaCl, 100 K-gluconate, 36 KCl, 10 HEPES, 1 MgCl2, 0.1 CaCl2, 1 Na2ATP, 0.1 MgGTP. Pipette solution osmolarity was between 280 and 300 mOsm, and pH was adjusted to 7.3 with KOH. Tight-fitting glass suction electrodes were used to record from the ventrolateral funiculus (VLF) as described previously (O’Donovan & Landmesser 1987 (link), O’Donovan 1989 (link), Xu et al. 2005 (link)), in order to monitor episodes of SNA. VLF signals were amplified (1000X), filtered (0.1 Hz to 1 kHz) by an extracellular amplifier (A-M Systems Inc.) and assessed using Axograph Software.