Effects of Cooling Temperatures via Thermal K2P Channels on Regeneration of High-Frequency Action Potentials at Nodes of Ranvier of Rat Aβ-Afferent Nerves

Cooling temperatures alter active membrane properties at the NR. A, Sample traces show APs (arrowhead indicated) recorded at a NR in response to step currents (inset) injected via recording electrode into the NR. The recording was performed under whole-cell current-clamp configuration at 24°C. B, Three overlay sample traces at expanded scale show APs recorded at an NR at 35°C (red), 24°C (black), and 15°C (blue). APs were evoked by rheobase step currents injected through recording electrode into the NR. C–G, Summary data at 35°C (n = 11), 24°C (n = 17), and 15°C (n = 14) of AP width (C), amplitude or upstroke (D), rheobase (E), threshold (F), and latency to AP threshold (G). Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey’s post hoc test. ns, not significant.

Thermal K2P activators partially counteract cooling temperature-induced changes of passive membrane properties of the NR. A, Two sets of sample traces show outward leak currents and membrane responses to testing pulses of ± 10-mV voltage steps at cooling temperature of 15°C in the absence (left) and presence the thermal K2P activator BL (right). Nodal membranes were held at −72 mV. Dashed line indicates the level of 0-pA current. B, Summary data of outward leak currents determined at the NR at 15°C in the absence (n = 6), presence of thermal K2P activators BL (10 μm, n = 9), AA (20 μm, n = 8), and protons ([pH]_i = 5, n = 8). Nodal membranes were held at −72 mV. C, Summary date of nodal membrane input resistance at 15°C in the absence (n = 6), presence of 10 μm BL (n = 9), 20 μm AA (n = 8), and protons ([pH]_i = 5, n = 8). D, Sample traces show RMPs at the NR at 15°C in the absence (Blue) and presence of 10 μm BL (orange). E, Summary data of RMP at 15°C in the absence (n = 6), presence of 10 μm BL (n = 7), 20 μm AA (n = 9), and protons ([pH]_i = 5, n = 8). In all experiments, BL was bath applied, AA and protons ([pH]_i = 5) were applied intracellularly through recording electrode internal solution. Data represent mean ± SEM, *p < 0.05, **p < 0.01, one-way ANOVA with the Tukey’s post hoc test.

Effects of thermal K2P activators on active membrane properties at the NR at cooling temperatures. A, Two overlay sample traces of APs recorded at a NR at cooling temperature of 15°C in the absence (blue) and presence of 10 μm BL (orange). APs were evoked by rheobase step currents injected through recording electrode into the NR. B–F, Summary data of active membrane properties at 15°C in the absence (n = 6), present of 10 μm BL (n = 8), 20 μm AA (n = 7), and protons ([pH]_i = 5, n = 8). Active membrane properties measured at the NR include AP width (B), amplitude (C), threshold (D), rheobase (E), and latency to AP threshold (F). Data represent mean ± SEM; ns, not significantly different, *p < 0.05, **p < 0.01, one-way ANOVA with the Tukey’s post hoc test.

Cooling temperatures reduce AP success rate at the NR in response to 200-Hz train stimulation. A, Schematic diagram illustrates experimental setting (left) and sample traces of APs evoked by electrical stimulation and recorded at a NR at 35°C (right). Electrical stimulation was applied at 1 Hz at a distal site of the nerve bundle. B, Three sets of sample traces show APs recorded at 35°C (top), 24°C (middle), and 15°C (bottom) in response to 200-Hz train stimulation for 20 s. Traces on right are at expanded scale. C, Time course of AP success rates in response to 200-Hz train stimulation for 20 s with experiments conducted at 35°C (red circles, n = 6), 24°C (black circles, n = 6), and 15°C (blue circles, n = 6). Time bin: 200 ms. D, Bar graph shows averaged AP success rate at 35°C (n = 6), 24°C (n = 6), and 15°C (n = 6) in response to the 200-Hz train stimulation for 20 s. Data represent mean ± SEM, *p < 0.05, ***p < 0.001, one-way ANOVA with the Tukey’s post hoc test.

Thermal K2P activators partially counteract cooling temperature-induced reduction of AP success rate in response to 200-Hz train stimulation. A, Two sets of sample traces show nodal APs evoked by 200-Hz train stimulation for 20 s in the absence (top), and presence (bottom) of 10 μm BL. Experiments were performed at 15°C. Traces on the right are at an expanded scale. B–E, Time course of success rate of nodal APs over 20 s in the absence (B, n = 6), presence of 10 μm BL (C, n = 6), 20 μm AA (D, n = 6), or protons (E, [pH]_i = 5, n = 6). Time bin: 200 ms. Dashed line in each panel indicates 50% AP success rate. F, Summary data of the time at which nodal AP success rate at 15°C reduced to 50% (T₅₀) in the absence (control, n = 6), present of 10 μm BL (n = 6), 20 μm AA (n = 6), and protons ([pH]_i = 5, n = 6). G, Summary data of averaged success rate of nodal APs in 20 s at 15°C in the absence (control, n = 6), presence of 10 μm BL (n = 6), 20 μm AA (n = 6), and protons ([pH]_i = 5, n = 6). Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey’s post hoc test.

Temperature-dependent and frequency-dependent changes of AP success rate at the NR and effects of thermal K2P activators. A, Plots of AP success rate against frequencies of train stimulation at 35°C (n = 6), 24°C (n = 6), and 15°C (n = 6). Train stimulation was applied at frequencies of 1, 10, 50, 100, 200, 500, and 1000 Hz each for a duration of 20 s. Dashed line indicates 50% AP success rate. B, Bar graph shows summary data (n = 6) of FS₅₀ values obtained from recordings performed at 35°C (n = 6), 24°C (n = 6), and 15°C (n = 6). FS₅₀, frequencies at which the AP success rate is 50%. C, Plots of AP success rate against different frequencies of train stimulation at 15°C in the absence (n = 6), presence of 10 μm BL (n = 6), 20 μm AA (n = 6), and protons (n = 5). Dashed line indicates 50% AP success rate. D, Summary data of FS₅₀ obtained from recordings at 15°C in the absence (n = 6), presence of BL (n = 6), AA (n = 6), and protons ([pH]_i= 5, n = 5). Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey’s post hoc test.

Effects of cooling temperatures and thermal K2P activators on voltage-gated Na⁺ current components at the NR. A, Three sets of sample traces show inward and outward currents following series of voltage steps; the recordings were performed at 35°C (right), 24°C (middle), and 15°C (left). Nodal membranes were held at −72 mV. Voltage steps were applied from –102 to 58 mV with a 10-mV increment each step. B, Three sets of sample traces show inward current components at an expanded scale with the recordings performed at 35°C (left), 24°C (middle), and 15°C (right). C, Three overlay sample traces of inward current components evoked by a voltage step from −72 to 18 mV; the recordings were performed at 35°C (red), 24°C (black), and 15°C (blue). D, Summary data of the amplitudes of inward current components recorded at 35°C (n = 8), 24°C (n = 8), and 15°C (n = 8). Voltage-step was from −72 to 18 mV. E, Summary data of the amplitudes of inward current components recorded at 15°C in the absence (n = 6), presence of 10 μm BL (n = 7), 20 μm AA (n = 7), and protons ([pH 5]_i, n = 8). Voltage-step was from −72 to 18 mV. Data represent mean ± SEM, * p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey’s post hoc test. ns, not significant.

Membrane potential-dependent and frequency-dependent alterations of AP-activated Na⁺ currents at the NR at 15°C. A, Three sets of sample traces of nodal membrane currents evoked by AP train stimulation at 10 Hz (first row), 50 Hz (second row), and 100 Hz (third row). Nodal membrane potentials were held at −75 mV and experiments performed at cooling temperature of 15°C. In each row, sample traces on left, middle, and right are the currents evoked at time points of 0 s (initial), 1 s, and 20 s, respectively. Traces in the fourth row are AP waveforms. In sample traces, AP-evoked voltage-gated Na⁺ currents are inward currents. Time scale: 5 ms. B, Time course of amplitudes of inward currents evoked by AP train stimulation at 10 Hz (solid circles, n = 9), 50 Hz (solid squares, n = 9), and 100 Hz (solid triangles, n = 9). C, D, Similar to A, B except membrane potentials were held at −84 mV. Inward currents were evoked by AP train stimulation at 10 Hz (n = 9, solid circles), 50 Hz (n = 9, solid squares), and 100 Hz (n = 9, solid triangles). E–G, Comparison of amplitudes of inward currents with membrane potentials held at −75 mV (open bars) and −84 mV (solid bars). Data were from inward currents at time points of 0, 1, and 20 s. Inward currents were evoked by AP train stimulation at 10 Hz (n = 9, E), 50 Hz (n = 9, F), and 100 Hz (n = 9, G). All recordings were performed at NRs at 15°C. Data represent mean ± SEM; ns, no significant difference, * p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test.