Aberrant Sodium Channel Currents and Hyperexcitability of Medial Entorhinal Cortex Neurons in a Mouse Model of SCN8A Encephalopathy

Abstract

SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Na_v1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8a^D/+), homozygous (Scn8a^D/D)), and WT (Scn8a^+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Na_v1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Na_v1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.

Keywords: SCN8A; action potential; entorhinal cortex; epilepsy; epileptic encephalopathy; sodium channels.

Figure 1.

Neuronal hyperexcitability of mEC layer II stellate neurons from Scn8a^N1768D mice. A–C, Representative traces of spikes elicited by 300 ms current injection steps of increasing current from a holding potential of −65 mV. D, E, Higher firing rates of D/+ (n = 20) and D/D (n = 21) neurons compared with WT (n = 20) at stimulation <300 pA. F, Firing rate of D/+ neurons is greater than that of D/D neurons at stimulation >460 pA. Data are shown as means ± SEM. *p < 0.05, two-way ANOVA with post hoc Tukey's test for multiple comparisons.

Figure 2.

Aberrant AP morphology in mutant mEC neurons. Ai–Ci, AP morphology elicited by a 300 ms depolarizing current injection of 290 pA. D, Superimposition of the first AP spike elicited by a depolarizing step to 290 pA from WT, D/+, and D/D neurons demonstrating the increase in AP duration in mutant neurons compared with WT. Note the delay in rising and falling phase of the AP in D/+ and D/D neurons. Aii–Cii, Superimposed traces of the first three AP spikes elicited by injection of 290 pA. The duration of spikes 2 and 3 is extended in the mutant neurons. Solid trace is the first AP, darker traces are the second AP, and broken lines are the third AP. Aiii–Ciii, Phase plots of dV/dt vs voltage for the first three AP spikes. *AP thresholds. Arrow indicates inflection of the rising phase of the phase plot indicative of AIS spike initiation; +, maximal conduction velocity as the spike invades the soma. The abrupt rise of dV/dt seen in WT neurons is less pronounced for the first spike in mutant neurons and further slowed for the second and third spikes. Aiv–Civ, First derivative (dV/dt) derived for the first AP spike. The peak of the first derivative is reduced in mutant neurons, indicating a slower upstroke velocity of the AP. Latency between spike initiation at the AIS and invasion into the soma was lengthened in mutant neurons, suggesting slower spike conduction. Second derivatives of the APs (superimposed, green) are also shown. The peaks are clearly discernable for the mutant neurons and are separated by a more pronounced latency. E, Plot of maximal hyperpolarizing voltages between AP spikes evoked by a 290 pA current injection (WT, n = 20; D/+, n = 20; D/D, n = 21). Fi–Fiii, Left, Na_v 1.6 expression along the AIS of WT, D/+, and D/D neurons (AnkG, green; Na_v1.6, red; Merge; yellow). Right, Graphs showing the localization and ROD of AnkG and Na_v1.6 (WT, n = 124 from 4 animals; D/+, n = 83 from 3 animals; D/D, n = 100 from 4 animals) staining along the length of the AIS for WT, D/+, and D/D neurons. Scale bars, 2 μm. Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons compared with WT; ^# p < 0.05 one-way ANOVA with post hoc Tukey test for multiple comparisons when D/+ were compared with D/D neurons.

Figure 3.

Synaptic stimulation of mutant mEC neurons elicits burst firing and prolonged depolarization. A–C, mEC deep layers were briefly stimulated at the AP threshold (1T), at twice the threshold value (2T), and at three times the threshold (3T). AP firing was measured in WT (n = 20), D/+ (n = 21), and D/D neurons (n = 22). Bursts of APs were elicited in the mutant neurons in response to 2T and 3T stimulation. D, AP frequency in response to increasing stimulation intensity. E, AUC for APs evoked at 1T stimulation is greater for D/D neurons than for D/+ or WT neurons. Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons.

Figure 4.

I_NaP and I_NaR Na channel currents are increased in N1768D mutant mEC layer II stellate neurons. Using recording solutions designed to reduce other types of inward and outward currents, voltage ramps were applied at a rate of 65 mV/s (inset) to elicit I_NaP currents. All I_NaP currents were abolished in the presence of 1 μm TTX (gray traces). Amplitudes of I_NaP currents were obtained by taking the original trace and subtracting from it the trace recorded in the presence of TTX (1 μm). A–C, I_NaP recordings from WT, D/+, and D/D mEC neurons. D, Superimposition of TTX subtracted I_NaP amplitudes for the three genotypes. E–G, I_NaR recordings from WT, D/+, and D/D mEC neurons. I_NaR currents were recorded using the indicated voltage protocol. Traces shown were obtained after subtracting traces recorded in the presence of TTX (1 μm). H, Superimposition of I_NaR of the three genotypes. I, Peak I_NaP currents in WT (n = 10), D/+ (n = 10), and D/D (n = 10) mEC neurons. J, Peak I_NaR currents in WT (n = 8), D/+ (n = 9), and D/D (n = 9) mEC neurons. K, Voltage dependence of I_NaR activation revealed no significant shifts in V_1/2 or k. Smooth lines correspond to the least-squares fit when average data were fit to a single Boltzmann equation. Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons.

Figure 5.

TTX (30 nm) decreases I_NaP and I_NaR currents. A–C, I_NaP currents were elicited by applying a voltage ramp at a rate of 65 mV/s before (WT, black; D/+, blue; D/D, red) and after application of TTX (30 nm; purple traces for all genotypes). Traces shown are those obtained after subtracting traces recorded in the presence of TTX (1 μm). D, Scatter plot of I_NaP peak amplitudes before and after the application of TTX (30 nm) (WT, n = 5; D/+, n = 5; D/D, n = 5). E–G, I_NaR measured before (WT, black; D/+, blue; D/D, red) and after the application of TTX (30 nm; purple traces for all the genotypes). H, Scatter plot of I_NaR current peak amplitude before and after the presence of TTX (30 nm) (WT, n = 6; D/+, n = 6; D/D, n = 5). Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons.

Figure 6.

TTX (30 nm) increases upstroke velocity and decreases AP duration in D/+ and D/D neurons. Ai–Ci, APs elicited by a 300 ms current injection pulse of 290 pA before (WT, black; D/+, blue; D/D, red) and after application of TTX (30 nm; purple traces for all genotypes). Aii–Cii, Plot of maximal hyperpolarizing voltages between AP spikes evoked by a 290 pA current injection before and after the application of TTX (30 nm) (WT, n = 10; D/+, n = 11; D/D, n = 13). Aiii–Ciii, Superimposed traces of the first two AP spikes elicited by injection of 290 pA (WT, black; D/+, blue; D/D, red) and after application of TTX (30 nm; purple traces for all genotypes). Aiv–Civ, Superimposed phase plots of the first two elicited APs before (WT, black; D/+, blue; D/D, red) and after application of TTX (30 nm; purple traces for all genotypes). Di–Fi, AP spikes elicited by a brief (4 ms) injection of suprathreshold current injection before (WT, black; D/+, blue; D/D, red) and after application of TTX (30; purple traces for all genotypes). Dii–Fii, Superimposed traces of AP spikes shown in Di–Fi. G, Scatter plot showing the AUC for AP spikes elicited by a brief (4 ms) current injection pulse before and after the application of TTX (30 nm) (WT, n = 5; D/+, n = 5; D/D, n = 5). Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons comparing with the pre-TTX condition. ♦p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons comparing D/D + TTX with D/+ + TTX. #p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons comparing WT + TTX.

Figure 7.

Altered Na channel currents in mutant mEC neurons. A, Representative current traces recorded using the outside-out patch-clamp configuration for WT, D/+, and D/D mEC layer II stellate neurons. B, Scatter plot of peak I_Na current from WT (n = 9), D/+ (n = 8), and D/D (n = 8) mEC neurons. C, Voltage dependence of channel activation for WT (n = 9), D/+ (n = 7), and D/D (n = 6) mEC neurons. Lines correspond to the least-squares fit when average data were fit to a single Boltzmann equation. D, Representative normalized current traces recorded during a 100 ms depolarizing pulse from a holding potential of −120 mV to −10 mV demonstrates elevated persistent Na current in mutant neurons. E, Ratio of I_Persistent/I_peak current 100 ms after stimulation in WT (n = 9), D/+ (n = 7), and D/D (n = 6) mEC neurons. F, Voltage dependence of channel inactivation for WT (n = 7), D/+ (n = 7), and D/D (n = 9) neurons. Lines correspond to the least-squares fit when average data were fit to a single Boltzmann equation. G, Representative steady-state inactivation traces elicited after prepulses to −110 mV, −80 mV, and −70 mV. Note the delay in channel inactivation for D/+ and D/D neurons compared with WT, indicating severe impairment of channel inactivation. H, Window current obtained by overlapping normalized activation and inactivation curves. Window current is elevated in D/+ (blue shaded area) and D/D (red checkered area) compared with WT (black shaded area) neurons. The voltage range for the window current is also shifted in a depolarizing direction. Data are shown as mean ± SEM. *p < 0.05, one-way ANOVA with post hoc Tukey's test for multiple comparisons.