Dorsal root ganglion (DRG) neuron preparation and electrophysiology

All experiments were carried out on adult (200–340 g) male Sprague-Dawley rats (Harlan, Indianapolis, IN and Charles River, Montreal, Quebec). A subset of animals received spinal nerve ligation (SNL) 2–5 days before terminal experiments [74]. Under isoflurane anesthesia, the paraspinal muscles were separated to access the L6 process, which was carefully removed. The L5 spinal nerve was tightly ligated with 6–0 silk suture. All nerve-injured animals maintained motor function but developed neuropathic pain as inferred by guarding of the affected paw.

To collect DRG neurons, rats were deeply anesthetized by subcutaneous injection of anesthetic cocktail (1 ml/kg of 55 mg/ml ketamine, 5.5 mg/ml xylazine, and 1.1 mg/ml acepromazine) or by isoflurane (4% for induction; 2.5% for maintenance). DRG (L4 and L5 in naïve animals; L5 in nerve-injured animals) were surgically removed to chilled MEM-FBS culture media and desheathed. DRG were then enzymatically treated for 45 minutes in culture media composed of 89% MEM, 370 units/ml penicillin and 370 μg/ml streptomycin, 1% MEM vitamin solution (all from Life Technologies), and 1.2 mg/ml collagenase Type 4 (Worthington Biochemical Corp). DRG were mechanically dissociated by trituration with a fire-polished Pasteur pipette, and further enzymatically treated for 5 minutes in Ca²⁺- and Mg²⁺-free Hanks’ balanced salt solution (HBSS; Life Technologies Inc), containing 2.5 mg/ml trypsin (Worthington Biochemical Corp) and 0.02% sterile ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich Canada Ltd). Trypsin activity was subsequently inhibited by the addition of MEM-FBS supplemented with 0.625 mg/ml MgSO₄ (Caledon Labs). Dissociated cells in MEM-FBS were plated on glass coverslips previously coated by a solution of 0.1 mg/ml poly-D-lysine, and incubated in MEM-FBS at 37°C, 5% CO₂, and 90% humidity for 2 h. Coverslips were then transferred to a HEPES-buffered Leibovitz’s L-15 media containing glutamine (Life Technologies Ltd), 10% FBS, 100 units/ml of penicillin and 100 μg/ml streptomycin, and 5 mM D-glucose (Caledon Labs) and stored at room temperature until used for experiments for 2–28 hours later. Spiking properties do not change appreciably over this period and nor do neurites develop based on storage at room temperature, omission of laminin from coverslips, and the growth factor-free culture medium used.

Coverslips with cultured cells were transferred to a recording chamber constantly perfused with room temperature, oxygenated (95% O₂ and 5% CO₂) artificial cerebral spinal fluid containing (in mM) 126 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 10 D-glucose, 26 NaHCO₃, 1.25 NaH₂PO₄. Cells were recorded in the whole-cell configuration with >70% series resistance compensation using an Axopatch 200B amplifier (Molecular Devices; Palo Alto, CA). Electrodes (2–5 MΩ) were filled with a recording solution containing (in mM) 125 KMeSO₄, 5 KCl, 10 HEPES, 2 MgCl₂, 4 ATP, 0.4 GTP as well as 0.1% Lucifer Yellow; pH was adjusted to 7.2 with KOH and osmolality was between 270 and 290 mosmol/L. For experiments on the contribution of ANO-1 channels, KMeSO₄ was reduced to 67 mM and KCl was increased to 63 mM to give E_Cl = -20 mV. Data were low-pass filtered at 2 KHz, digitized at 20 KHz using a CED 1401 computer interface (Cambridge Electronic Design, Cambridge, UK), and analyzed offline. Virtual GABA conductance was applied via dynamic clamp using Signal 5 software (CED). The virtual conductance was modeled as a step or as a synaptic waveform described by Eqn. 6. To express the virtual conductance as a density and thus exclude direct effects of cell size, we normalized absolute conductance values by membrane capacitance C because C is proportional to the surface area of the cell. Capacitance was measured for each cell based on responses to small (50 pA) hyperpolarizing current steps, where C = τ_membrane / R_in. To increase cellular excitability in neurons from naïve animals, potassium channels were blocked with bath applied 4-aminopyridine (4-AP). In a subset of experiments with 4-AP, a virtual voltage-dependent sodium conductance was also inserted via dynamic clamp using the equations and parameters reported by Ratté et al. [45]. Neurons from nerve-injured animals are already hyperexcitable and were not, therefore, subject to manipulations (i.e. 4-AP or virtual sodium conductance) intended to increase excitability.

All data and computer code are available from the corresponding author upon request.