General electrophysiology

Recordings were performed from small (~25 μm), capsaicin-sensitive (not shown) dissociated rat DRG neurons, up to 24 h after culturing. These neurons have been described in the literature to be nociceptive (Cardenas et al., 1995). Cell diameter was measured using Nikon Elements AI software (Nikon), from images acquired by a CCD camera (Q-Imaging). In experiments studying the role of I_M in nociceptor-like DRG neurons (Figures ​(Figures1,1, ​,2,2, 3A,B,E,F, Supplementary Figure 1), whole-cell membrane currents and voltages were recorded using a nystatin-based perforated patch technique (Horn and Marty, 1988) in voltage clamp and fast current-clamp modes, respectively, using a Multiclamp 700B amplifier (Molecular Devices), at room temperature (24 ± 2°C). All other experiments (Figures 3C,D, Supplementary Figure 2) were performed using voltage and current clamp in whole cell configuration. In all experiments, only the cells that showed less than 10% change in access resistance during the entire recording period were analyzed. Data were sampled at 50 kHz and were low-pass filtered at 20 kHz (-3 dB, 8 pole Bessel filter). Patch pipettes (3–5 MΩ) were pulled from borosilicate glass capillaries (1.5 mm/1.1 mm OD/ID, Sutter Instrument Co., Novato, CA, USA) on a P-1000 puller (Sutter Instrument Co.) and fire-polished (LWScientific). Access resistance was in the range of 4–8 MΩ. For voltage-clamp recordings, capacitive currents were minimized and series resistance was compensated by about 80%. Command voltage and current protocols were generated with a Digidata 1,440 A A/D interface (Molecular Devices). Data were digitized using pCLAMP 10.3 (Molecular Devices). Data averaging and peak detection were performed using Clampfit 10.3 software. Data were fitted using Origin 8 (OriginLab).

XE991 leads to spontaneous firing of nociceptor-like DRG neurons. (A) Left, Typical responses of nociceptor-like small (25 μm) DRG neurons to focal puff application of 10 μM XE991 (representative of 5 out of 6 experiments). Current clamp perforated-patch recordings performed from acutely dissociated DRG neurons perfused with extracellular solution. Note, membrane depolarization and onset of spike discharges shortly after application of XE991. Dashed line indicate resting potentials before drug application (−58 mV). Right, mean ± SEM (bar graphs) and individual neurons' changes in membrane potential before (gray) and after application of XE991 (red, measured before generation of first action potential). ^***p < 0.001, paired Student t-test, n = 6. Note that XE991-induced depolarization in all recorded neurons. (B) Same as in (A), but 3 μM XE991 was puff-applied on the neurons. Dashed line indicate resting potential before drug application (−59 mV). Left, representative of 10/11 neurons. ^***p < 0.001, paired Student t-test, n = 11. Note that 3 μM XE991 induced depolarization in all recorded neurons. (C) Control experiment. 10 min long puff application of vehicle onto nociceptor-like DRG neuron. Note, no spontaneous depolarization developed during this period, yet the cell fired normally upon injection of depolarizing current pulses (inset; representative of 9 experiments). Dotted boxes show time breaks in free-run recordings when current protocols where applied. Right, same as in (A,B), but measured 5 min and 10 min after puff application of vehicle; ns—not significant, one-way ANOVA, n = 9.

In nociceptor-like DRG neurons I_M is outward at resting-to-threshold potential range (A). Upper, Typical voltage-clamp perforated patch recordings of I_M from a nociceptor-like DRG neuron (see Methods, representative of 18/18 neurons). A family of currents was evoked by a series of 1 s, 5 mV hyperpolarizing voltage steps from a holding potential of −20 mV (voltage protocol is shown in inset). The peak current response obtained by stepping to −50 mV is shown at the bottom. The I_M relaxation was fitted with a bi-exponential line (red), which was extrapolated to the beginning of the voltage step. I_M amplitudes were measured as the differences between the instantaneous peak currents at command onset and the steady-state currents just before command offset (dotted line). (B) Averaged leak-subtracted peak I-V characteristics of I_M recorded from nociceptor-like DRG neurons (n = 18), calculated as described in Methods. ns—not significant; one-way ANOVA comparison between the current values obtained at −90 mV (zero current level) to currents at other command voltages. Note, significant outward current at −60 mV. Insets show box charts and individual values of resting membrane potentials (V_Rest, n = 10) and action potential thresholds (V_Th, n = 9), obtained from current clamp perforated patch recordings (see Methods). The middle line of the box charts represents the mean; the box represents 25 ~ 75% percentiles and caps delineate range within 1.5 interquartile range. The mean (in mV) is aligned to its values on the x-axis. The shadowed area indicates the range of membrane potentials between resting potential and threshold. (C) Mean ± SEM of I_M activation curves (g/g_max) fitted using the Boltzmann equation (see Methods) shows the onset of activation at −60 mV (ns—not significant; one-way ANOVA comparison between the conductance values obtained at −90 mV (zero conductance level) to conductances at other command voltages) and V_1/2 at −42 mV. The shadowed area indicates the range of membrane potentials between resting potential (mean, V_Rest) and threshold (mean, V_Th) taken from (B), insets.

I_M is sufficient to prevent spontaneous firing. (A) Typical voltage responses to increasing current steps (showed in the middle panel) recorded from nociceptor-like cultured DRG neuron using perforated-patch, in current clamp mode (left) or from a single-compartment model (right, see Methods). Recordings were performed on the same DRG neuron before and after application of XE991 (shown in (E), left). Traces from the model were obtained using the model with intact g_Kv7/M. Note the similarity between the simulated to experimental responses. (B) Same as in (A), but showing responses to 1.5 s depolarizing current ramps (250 pA/s, showed below) recorded from nociceptor-like cultured DRG neuron using perforated patch in current clamp mode (left) or from the single-compartment model (right). Recordings were performed on the same DRG neuron before and after application of XE991 (shown in (F), left). Note the similarity between the simulated and experimental responses. (C,D) Validation of the capsaicin-like stimulation on a single compartmental model. (C) Current responses evoked by capsaicin-like stimulation (5 s, 1 μM, see Methods) when applied on a single-compartment model. (D). Capsaicin-like stimulation evokes a barrage of action potentials in the simulated neuron. Dotted line indicates the membrane potentials before the stimulation (−57.85 mV). (E,F) Voltage responses to increasing current steps (E, showed in the middle panel) or current ramps (F, showed below) recorded from the same neuron shown in (A,B), 10 min after application of 3 μM XE991 (left) or from the simulated neuron with g_Kv7/M = 0 (right). All measurements described in panels (E,F) were performed at the native resting potential, adjusted after XE991 application or when g_Kv7/M = 0, by injecting appropriate repolarizing currents. Note that inhibition of I_M leads to increase in neuronal excitability which was well reflected in the simulated neuron. (G) Free run recording of membrane potential in a simulated single-compartment modeled neuron during a gradual decrease in g_Kv7/M (the rate of decrease in g_Kv7/M shown below, see Methods). Dashed lines indicate resting potential before changes in g_Kv7/M (−57.85 mV). The vertical dotted line indicates time of first action potential. Horizontal dashed line indicates the level of g_Kv7/M at which the first action potential occurred. Note that in the simulated neuron, decrease in g_Kv7/M lead to slow depolarization followed by spontaneous firing, similar to the experimental results obtained after application of 3 and 10 μM XE991 on nociceptor-like DRG neurons (Figure ​(Figure11).

Nystatin-based pipette solution (290 mOsm) for perforated patch recordings was freshly prepared in the dark every 2–3 h and contained (in mM): 140 KCl, 1.6 MgCl₂, 2 EGTA, 10 HEPES, 2.5 Mg-ATP, 0.5 Na-GTP (pH = 7.4).

Nystatin (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) to obtain a 50 mg ml⁻¹ stock solution, which, after 1 min ultra-sonication, was diluted in pipette solution to obtain a working concentration of 125 μg ml⁻¹.

The intracellular solution for measuring capsaicin-induced current contained (in mM): 145 KCl, 5 NaCl, 2.5 MgCl₂, 2 MgATP, and 0.1 EGTA (pH = 7.4).

The intracellular solution for measuring capsaicin-evoked firing contained (in mM): 140 K-Aspartate, 10 NaCl, 2 MgCl₂, 4 MgATP, 10 HEPES (pH = 7.4).

The extracellular solution contained (in mM): 145 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 D-Glucose (pH = 7.4).

Pipette potential was zeroed before seal formation and membrane potential was corrected for liquid junction potential of −4.5 mV.

Only cells that generated at least one action potential in response to current injections were then used for capsaicin application and analysis.