Patch-clamp recordings from spinal cord neurons

The Animal Care and Use Committee of Hamamatsu University School of Medicine approved all animal experiments. All efforts were made to minimize the number of animals used and minimize suffering. Under deep isoflurane anesthesia, lumber spinal cords were removed from P0–1 mice and embedded in 3% agarose. Coronal lumber spinal cord slices (350 μm) were made using the vibrating microtome 7000 (Campden) in a cold, oxygenated sucrose solution containing (in mM): 220 sucrose, 120 NaCl, 2.5 KCl, 0.5 CaCl₂, 1.25 NaH₂PO₄, 1 MgCl₂, 26 NaHCO₃, 30 glucose, 10 MgSO₄ (pH 7.4). The slices were maintained in standard artificial cerebrospinal fluid (ACSF) consisting of (in mM) 120 NaCl, 2 KCl, 1 KH₂PO₄, 1 MgCl₂, 2 CaCl₂, 26 NaHCO₃, and 10 glucose (pH 7.4), and equilibrated with 95% O₂ and 5% CO₂ at room temperature prior to the recording. For recording, slices were transferred to a recording chamber, perfused with oxygenated ACSF in the presence of tetrodotoxin (500 nM). Electrophysiological recordings were performed using a MultiClamp 700B amplifier (Molecular Devives) and pClamp9 software (Molecular Devives). Currents were filtered at 2 kHz, digitized at 10 kHz using DigiData1322A. The data were analyzed off-line using Clampfit9 (Molecular Devices).

To estimate the reversal potential of GABA-stimulated currents (E_GABA) in ventral spinal cord neurons in acute lumber slices, we performed gramicidin-perforated patch-clamp recording to acquire GABA-evoked responses with native intracellular Cl⁻ concentrations (9). Patch electrode pipettes (2–4 MΩ) were pulled from borosilicate glass capillaries on a P-97 puller (Sutter Instruments) and filled with pipette solution composed of 150 mM KCl and 10 mM HEPES (pH 7.2), supplemented with gramicidin. Gramicidin (Sigma-Aldrich) was dissolved in methanol to prepare a stock solution of 10 mg/ml, then diluted in pipette solution to a final concentration of 30 μg/ml.

Reversal potential of the 100 μM GABA-induced current was measured at −50 mV holding potential (V_h), and 0.5 s voltage ramps from −100 to 0 mV were applied before and during GABA application. E_GABA was estimated by measuring the voltage at which the I-V relationships before and during GABA application intersected (40). 30 sec duration GABA puffs were applied through a patch pipette approximated to the soma, using an IM-300 programmable microinjector (Narishige).

To measure the efficacy of KCC2 mediated Cl⁻ extrusion, we performed transient Cl⁻loading by whole-cell voltage-clamp recording. Patch electrodes were filled with pipette solution containing (in mM) 123 K gluconate, 2 MgCl₂, 8 NaCl, 1 EGTA, 4 ATP, 0.3 GTP, and 10 HEPES (pH 7.2). Basal responses to 100 μM GABA puffs (3 psi and 3 sec duration) were recorded every 20 sec in current-clamp mode. Then, Cl⁻ was loaded by exposing the neurons to GABA for 20 sec in voltage-clamp mode (V_h=0 mV). After Cl⁻ loading, GABA responses were once again recorded every 20 sec in current-clamp mode to measure the rate of Cl⁻ extrusion (41).