A dissociated DRG neuron preparation was used that we have repeatedly found in nociceptors from rats, mice, and humans to retain injury-induced, pain-related hyperexcitability for more than 24 hours in culture after in vivo induction of persistent neuropathic pain 8 (link)–11 (link), 42 (link), 52 (link), 53 (link), 81 (link), 84 (link). Rats were euthanized using intraperitoneal injection of pentobarbital/phenytoin solution (Euthasol, 0.9 ml, Virbac AH, Inc.) followed by transcardial perfusion of ice-cold phosphate buffered saline (PBS, Sigma-Aldrich). DRGs were harvested from below the vertebral T10 level to L6. Ganglia were surgically desheathed before transfer to high-glucose DMEM culture medium (Sigma-Aldrich) containing trypsin TRL (0.3 mg/ml, Worthington Biochemical Corporation) and collagenase D (1.4 mg/ml, Roche Life Science). After 40-min incubation under constant shaking at 34°C, digested DRGs were washed by two successive centrifugations and triturated with two fire-polished glass Pasteur pipettes of decreasing diameters. Cells were plated on 8-mm glass coverslips (Warner Instruments) coated with poly-L-ornithine (Sigma-Aldrich) 0.01% in DMEM without serum or growth factors, and incubated overnight at 37°C, 5% CO2 and 95% humidity.