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. 2025 Jul 15;14:RP105696. doi: 10.7554/eLife.105696

Figure 3. Recovery of action potential (AP) firing properties is greatly diminished following dual NaV1.6 and NaV1.2 inhibition compared to selective block of individual channels.

(A) Protocol used to characterize recovery of AP firing properties. Baseline spiking is determined by injecting current for 300 ms to elicit five to six APs. To promote NaV inactivation and maximal GNE-4076 onboarding, neurons are held at –12 mV in voltage-clamp for 30 s. Recovery of AP firing is evaluated by injecting the same current stimulus defined during baseline spiking with an inter-stimulus interval starting at 2 s, followed by 5, 15, 30, and 60 s. (B) Overlaid waveform of first AP at baseline or 18 s post GNE-4076 onboarding for all conditions (wildtype no drug, n = 12; wildtype + GNE-4076, n = 11; Scn8a/2aSR/SR + GNE-4076, n = 10; Scn2aSR/SR + GNE-4076, n = 11; Scn8aSR/SR. + GNE-4076, n = 11). (C) Overlaid phase plane of AP traces at baseline (100% transparency) or 18 s post GNE-4076 onboarding (20% transparency) for each condition in (B). Plots represent the AP velocity by taking the first derivative (dV/dt, y-axis) versus the membrane potential (mV, x-axis). Colors are matched to conditions represented in (B). (D) Recovery of AP threshold (Vm) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For Δ Vm, the baseline value is subtracted from individual timepoints throughout the recovery phase (Δ mV = recovery timepoint – baseline). Colors are matched to conditions represented in (B). Gray shaded bar represents recovery between 12 and 20 s. (E) Summary data for Δ Vm at 12–20 s post GNE-4076 onboarding (time period represented as gray bar in (D)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. ****p < 0.0001. (F) Recovery of AP peak velocity (dV/dt) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For Δ dV/dt, the baseline value is subtracted from individual time points throughout the recovery phase (Δ V/s = recovery timepoint – baseline). Colors are matched to conditions represented in (B). Gray shaded bar represents recovery between 12 and 20 s. (G) Summary data for Δ dV/dt at 12–20 s post GNE-4076 onboarding (time period represented as gray bar in (F)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. ****p < 0.0001.

Figure 3—source data 1. Data values for each experiment described in Figure 3.

Figure 3.

Figure 3—figure supplement 1. Depolarization across the axon initial segment (AIS) compartment is mediated by distinct NaV isoforms.

Figure 3—figure supplement 1.

(A) Schematic depicting different phases of an action potential (AP), including AP velocity (first derivative; dV/dt or V/s) and acceleration (second derivative; d2V/dt2 or V/s2). APs can be divided based on depolarization or repolarization phases, which largely depends on the opening of various voltage-gated ion channels. During cellular depolarization, two components exist that largely account for voltage change detected at either the AIS or soma, which are easily discerned by two humps present on a phase plane plot. The AIS component can further be separated into the AIS max or inflection point defined as the trough or peak of the second derivative and likely represents voltage change at distinct AIS regions like the proximal or distal AIS, respectively. (B) Example traces of first and second derivatives for all genotypes at baseline or 18 s post recovery. AIS inflection point (light green) is defined as corresponding dV/dt when acceleration (d2V/dt2) peaks during the AIS component. AIS max point (dark green) is defined as corresponding dV/dt when acceleration plateaus (visualized as a trough of d2V/dt2) during AIS component. (C) Summary data for Δ AIS inflection point (dV/dt) at 12–20 s post GNE-4076 onboarding (represented as light green bar in (B)). Box plots show median and 90% tails. Circles represent individual cells (wildtype no drug, n = 12; wildtype + GNE-4076, n = 11; Scn2aSR/SR + GNE-4076, n = 11; Scn8aSR/SR + GNE-4076, n = 11; Scn8a/2aSR/SR + GNE-4076, n = 11). One-way ANOVA, Holm–Šídák multiple comparisons test. ****p < 0.0001. (D) Recovery of AIS max velocity (dV/dt) represented as a delta value for individual cells plotted against time post GNE-4076 onboarding (log-scale). For Δ dV/dt, the baseline value is subtracted from individual time points throughout the recovery phase (Δ V/s = recovery timepoint − baseline). Gray shaded bar represents recovery between 12 and 20 s. (E) Summary data for Δ AIS max velocity (dV/dt) at 12–20 s post GNE-4076 onboarding (represented as gray bar in (D) or dark green bar in (B)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. ****p < 0.0001.
Figure 3—figure supplement 1—source data 1. Data values for each experiment described in Figure 3- figure supplement 1.
Figure 3—figure supplement 2. Recovery of action potential (AP) firing properties represented as absolute values.

Figure 3—figure supplement 2.

(A) Recovery of AP threshold (Vm) represented as absolute values for individual cells (wildtype no drug, n = 12; wildtype + GNE-4076, n = 11; Scn2aSR/SR + GNE-4076, n = 11; Scn8aSR/SR + GNE-4076, n = 11; Scn8a/2aSR/SR + GNE-4076, n = 11) plotted against time post GNE-4076 onboarding (log-scale). Colors are matched to conditions represented in Figure 3B. Gray shaded bar represents recovery between 12 and 20 s. (B) Summary data for Vm at 12–20 s post GNE-4076 onboarding (time period represented as gray bar in (A)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Recovery of AP peak velocity (dV/dt) represented as absolute values for individual cells plotted against time post GNE-4076 onboarding (log-scale). Colors are matched to conditions represented in Figure 3B. Gray shaded bar represents recovery between 12 and 20 s. (D) Summary data for peak dV/dt at 12–20 s post GNE-4076 onboarding (time period represented as gray bar in (C)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Recovery of axon initial segment (AIS) max velocity (dV/dt) represented as absolute values for individual cells plotted against time post GNE-4076 onboarding (log-scale). Colors are matched to conditions represented in Figure 3B. Gray shaded bar represents recovery between 12 and 20 s. (F) Summary data for AIS max velocity (dV/dt) at 12–20 s post GNE-4076 onboarding (time period represented as gray bar in (E)). Box plots show median and 90% tails. Circles represent individual cells. One-way ANOVA, Holm–Šídák multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 3—figure supplement 2—source data 1. Data values for each experiment described in Figure 3-figure supplement 2.