Spinal cord slice preparation for electrophysiological studies

8-wk-old male Wistar/ST rats (Japan SLC) weighing 230–280 g were used for electrophysiological studies. The rats were housed in a vivarium with a 12-h alternating light-dark cycle and were given food and water ad libitum. Electrophysiological experiments were conducted in accordance with the AAALAC guidelines for the care and use of laboratory animals; the protocol was approved by the Shionogi Research Laboratories IACUC. The rats were anesthetized with 3% isoflurane, and 40 ng/10 µl Netrin-4 protein solution (R&D Systems) was directly injected between lumbar vertebrae L4 and L5 using a 25-Ga needle attached to a Hamilton microsyringe. Intrathecal injection of Netrin-4 protein or saline solution was performed twice daily for 2 d. After injection, the rats were individually placed in a heated cage until recovery from anesthesia. The withdrawal threshold was measured by the up-down method of a von Frey monofilament stimulation to the plantar surface of each hind paw. Mechanical sensitivity was shown in the mean of the withdrawal threshold of each hind paw. 24–48 h after intrathecal administration, rats were decapitated under 60 mg/kg i.p. sodium pentobarbital anesthesia. The spinal cords were isolated and placed in ice-cold, low-sodium artificial cerebrospinal fluid (CSF) containing the following: 100 mM choline chloride, 13 mM NaCl, 3 mM KCl, 1 mM NaH₂PO₄, 25 mM NaHCO₃, 11 mM d-glucose, 1 mM CaCl₂, and 5 mM MgCl₂, pH 7.4, after bubbling with 95% O₂ and 5% CO₂. 300-µm transverse slices were prepared using a vibratome (VT1200S; Leica Biosystems) and maintained for at least 60 min in standard artificial CSF containing the following: 113 mM NaCl, 3 mM KCl, 1 mM NaH₂PO₄, 25 mM NaHCO₃, 11 mM d-glucose, 2 mM CaCl₂, and 1 mM MgCl₂, pH 7.4, after bubbling with 95% O₂ and 5% CO₂. Slices were transferred to a recording chamber mounted on the stage of a microscope (BX51-WI; Olympus) and superfused with standard artificial CSF (flow rate of 2.5 ml/min at 30–32°C).