Whole-cell Electrophysiology of Spinal Motoneurons

Spinal cord slices were prepared from P7–P9 rat cervical cords^{13 (link)}. Briefly, transverse slices (450–600 μm thick) of C6–C8 cervical segments were cut in oxygenated ice-cold cutting solution using a Linearslicer (PRO7, Dosaka EM, Kyoto, Japan) and transferred to a slice chamber perfused for more than 1 h at 15 ml/min with oxygenated artificial cerebrospinal fluid (ACSF).
Whole-cell currents were recorded from fluorescent MNs using patch-pipettes^{13 (link)} (Fig. 5A). The internal solution in the pipettes contained 50 μM N-(2-aminoethyl) biotinamide hydrochloride (Neurobiotin, Vector Laboratories, Burlingame, CA, USA), which was later visualized within MNs by reacting it with Texas Red-avidin (Fig. 5B). The whole-cell configuration was maintained for at least 20–40 min, which allowed the Neurobiotin to diffuse through the entire dendritic tree^{16 (link)}. Some of the morphological data presented in this study was obtained from common materials in our previous electrophysiological study^{13 (link)}.