Dissociation of DRG neurons and patch clamping experiments

To isolate DRG neurons, rats (two weeks after injection of tumor cells or NS) were sacrificed by cervical dislocation, followed by decapitation, as described previously.³³ In brief, L2-L5 DRGs ipsilateral to the injection tibia were dissected out and transferred to an ice-cold, oxygenated fresh dissecting solution (in mM): 130 NaCl, 5 KCl, 1.5 CaCl₂, 2 KH₂PO4, 6 MgSO₄, 10 HEPES, and 10 glucose. The pH of solution was adjusted to 7.2 and osmolarity was 305 mOsm. The DRGs were incubated in dissecting solution, which contained collagenase D (1.8 ∼ 2.0 mg/ml; Roche, Indianapolis, IN) and trypsin (1.5 mg/ml; Sigma, St. Louis, MO), for 1.5 h at 34.5℃. DRGs were taken out, washed, and transferred to 2 mL of dissecting solution containing DNase (0.5 mg/ml; Sigma). Single cell suspension was subsequently obtained by repeated trituration through a series of flame-polished glass pipettes. Cells were then plated onto acid-cleaned glass coverslips. Coverslips containing adherent DRG cells were then put in a small recording chamber (0.5 mL) and attached to the stage of an inverting microscope (Olympus IX71, Tokyo, Japan), which is fitted with both fluorescent and phase objectives. DiI-labeled neurons were identified by their fluorescence. For patch clamp recordings, cells were perfused at room temperature with normal external solution. The external solution contains (in mM) 130 NaCl, 5 KCl, 2 KH₂PO4, 2.5 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH = 7.2, adjusted with NaOH, osmolarity = 295–300 mOsm). Recording pipettes were pulled from borosilicate glass tubing using a horizontal puller (P-97, Sutter Instruments, California, USA) and typically had a resistance of 3.0 ∼ 5.0 MΩ when filled with normal internal solution. Resting membrane potential (RP) and rheobase were recorded under current clamp configuration. Whole cell voltages, filtered at 2 ∼ 5 kHz and sampled at 100 μs/point, were acquired with an EPC10 patch-clamp amplifier and stored on a computer using FitMaster (HEKA, Germany). All experiments were performed at room temperature (22 ∼ 24℃).