Spontaneous seizures and seizure-induced death in mice with selective expression of mutant Nav1.6 in PV interneurons. We first sought to determine if expression of a GOF SCN8A variant selectively in PV interneurons would be sufficient for the development of spontaneous seizures. We used the conditional knockin Scn8aW/+ mouse model and crossed homozygous PV-Cre mice with Scn8aW/+.tdT mice to generate Scn8aW/+-PV mice, where the R1872W SCN8A variant is expressed exclusively in PV interneurons (Figure 1A). Scn8aW/+-PV mice were implanted with EEG recording electrodes and monitored for 10 weeks. To better conceptualize the phenotype of Scn8aW/+-PV mice with reference to another SCN8A DEE model, we also implanted EEG recording electrodes in Scn8aD/+ mice, which express the N1768D SCN8A variant globally, and monitored for 6–8 weeks. Spontaneous seizures were observed in all recorded Scn8aW/+-PV mice (n = 8; Figure 1, B and D) and Scn8aD/+ mice (n = 14, Figure 1, C and E). Median seizure onset in Scn8aW/+-PV mice was approximately 10 weeks of age. In Scn8aW/+-PV mice, seizures typically consisted of a wild running phase, which was immediately followed by a tonic-clonic phase in approximately 26% of seizures (23/89). Analysis of EEG signals from both Scn8aD/+ and Scn8aW/+-PV mice revealed spike wave discharges, a distinct aspect of electrographic seizures (Figure 1, B and C), highlighting similarities between a global mutation model and a model harboring an SCN8A variant exclusively in PV interneurons. Scn8aW/+-PV mice also died prematurely compared with WT littermates, with a median survival of 16.6 weeks (Figure 1F). Electrographic and video recordings verified Scn8aW/+-PV mice that died during monitoring succumbed to seizure-induced death (n = 3; Supplemental Videos 1 and 2). Interestingly, all fatal seizures exhibited a tonic phase before death, consistent with our previous findings in SCN8A DEE mice (31). In agreement with previous studies (29), Scn8aD/+ mice also died prematurely as a result of seizure-induced death (Figure 1F and Supplemental Video 3), which was significantly accelerated compared with Scn8aW/+-PV mice (P = 0.024). Overall, these findings show that a GOF variant exclusively expressed in PV interneurons can lead to seizures and seizure-induced death and support a previously unappreciated role for PV interneurons in seizure induction and seizure-induced death in a mouse model of SCN8A DEE.

Mice expressing the patient-derived SCN8A variant R1872W exclusively in PV interneurons exhibit spontaneous seizures and seizure-induced death. (A) Breeding strategy used to produce Scn8aW/+.tdT.PV-Cre mice (Scn8aW/+-PV mice, used for both in vivo and whole-cell patch clamp experiments) and age-matched littermate controls on a C57 background. These mice express the R1872W SCN8A mutation exclusively in PV interneurons, which are fluorescently labeled with tdTomato. (B) Example EEG recording of a spontaneous seizure (shown in blue) from an adult Scn8aW/+-PV mouse. Spontaneous seizure shown here resulted in seizure-induced death (Supplemental Videos 1 and 2). Purple box highlights spike wave discharges, expanded below. (C) Example EEG recording of a spontaneous seizure (red) from an adult Scn8aD/+ mouse, which expresses the N1768D SCN8A variant globally. Purple box highlights spike wave discharges, expanded below. (D) Seizure heatmap of (n = 8) Scn8aW/+-PV mice over a period of 10 weeks. (E) Seizure heatmap of (n = 14) Scn8aD/+ mice over a period of about 8 weeks. Monitoring began at slightly varying ages, indicated by white in heatmap. (F) Survival of Scn8aW/+-PV mice (n = 25) and Scn8aD/+ mice (n = 44) is significantly reduced when compared with WT (n = 27; ****, P < 0.0001; log-rank Mantel-Cox test). Survival of Scn8aD/+ mice is decreased compared with Scn8aW/+-PV mice (*, P < 0.05, log-rank Mantel-Cox test).

Depolarization block in Scn8a mutant PV interneurons. To assess the intrinsic physiological function of Scn8a mutant PV interneurons, we performed electrophysiological recordings of fluorescently labeled PV interneurons in layer IV/V of the somatosensory cortex of adult (5 to 8 weeks) Scn8aD/+, Scn8aW/+-PV, and age-matched WT littermates (Figure 2A). To verify that fluorescently labeled cells were indeed PV positive, we used immunohistochemistry to stain for PV in WT, Scn8aD/+, and Scn8aW/+-PV mice with tdTomato as a Cre-dependent fluorescent marker driven by PV-Cre, where we found that more than 95% of cells were both PV and tdTomato positive (Supplemental Figure 1). WT littermates from both Scn8aD/+ and Scn8aW/+-PV genotypes did not exhibit any differences in firing frequencies (P = 0.656) and were pooled. Analysis of membrane and AP properties revealed that Scn8aD/+ PV interneurons had decreased downstroke velocity as well as increased AP width when compared with WT (Table 1). Using a series of depolarizing current injection steps to assess intrinsic excitability, we observed a difference in excitability (P = 0.028) between WT, Scn8aD/+, and Scn8aW/+ PV interneurons. Initially, PV interneurons expressing either Scn8a variant were hyperexcitable compared with WT littermates at lower current injection steps (<100 pA in Scn8aD/+ mice, P = 0.045, and <360 pA in Scn8aW/+-PV mice, P = 0.030). However, at higher current injection steps, both Scn8aD/+ and Scn8aW/+ PV interneurons exhibited progressive AP failure as a result of depolarization block (>640 pA in Scn8aD/+ mice, P = 0.042; >840 pA in Scn8aW/+-PV mice, P = 0.041; Figure 2, B–F). Both Scn8aD/+ and Scn8aW/+ PV interneurons were more prone to depolarization block than their WT counterparts over the range of current injection magnitudes (P < 0.0001 and P = 0.016, respectively; Figure 2F). Depolarization block of inhibitory interneurons has been previously implicated in seizure-like activity both in vitro and in vivo and has been proposed as a biophysical mechanism underlying approach of seizure threshold (32–37). Here, the early onset of depolarization block in Scn8a mutant PV interneurons indicates a PV hypoexcitability phenotype, similar to the phenotypes observed in PV interneurons in GOF SCN1A DEE and in SST interneurons in SCN8A DEE (11, 32).

Altered excitability and depolarization block in Scn8aD/+ and Scn8aW/+-PV interneurons. (A) Whole-cell recordings were collected from WT, Scn8aD/+, and Scn8aW/+-PV interneurons in layer IV/V of the somatosensory cortex in adult 5- to 8-week old mice. (B–D) Example traces of WT (B, black), Scn8aD/+ (C, red), and Scn8aW/+ (D, blue) PV interneuron firing at 200, 400, 600, and 800 pA current injections. Depolarization block is noted with arrows (DB). (E) Scn8aD/+ (n = 17 cells, 6 mice) and Scn8aW/+ PV (n = 17 cells, 5 mice) interneurons experience a decrease in firing via depolarization block (*, P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test) when compared with WT PV interneurons (n = 18 cells, 8 mice). Red or blue stars indicate individual points of significance for either Scn8aD/+ or Scn8aW/+-PV, respectively, by multiple comparisons test. (F) Cumulative distribution of PV interneuron entry into depolarization block relative to current injection magnitude for WT, Scn8aD/+, and Scn8aW/+-PV mice (****, P < 0.0001; *, P < 0.05, log-rank Mantel-Cox test).

Membrane and AP properties of adult layer IV/V PV interneurons

Previous studies have shown that excitatory pyramidal neurons in global knockin Scn8aD/+ mice are hyperexcitable compared with WT, suggesting that a global change in neuronal activity of both inhibitory and excitatory neurons likely contributes to the seizure phenotype (10). To determine if firing is affected in excitatory neurons from Scn8aW/+-PV mice, which selectively express an Scn8a variant in PV interneurons, we recorded the intrinsic excitability of pyramidal neurons from cortical layers IV/V in adult mice (Supplemental Figure 2). Interestingly, we did not observe any differences in the intrinsic excitability of pyramidal neurons between the WT and Scn8aW/+-PV genotypes (Supplemental Figure 2). This suggests that alterations in the physiology of PV interneurons may be sufficient in facilitating seizures in SCN8A DEE. Analysis of AP parameters revealed an increase in input resistance and a decrease in rheobase (Supplemental Figure 2, E and F, and Supplemental Table 1), suggestive of some compensatory changes in excitatory PCs.

Additionally, the role of development is an important consideration in understanding the pathophysiology of SCN8A DEE. In Dravet syndrome, differences in PV interneuron intrinsic excitability are observed only during a critical developmental time window (P18–P21) (21). To determine if the same was true for PV interneurons in SCN8A DEE, we measured intrinsic excitability at the critical P18–P21 time window (Supplemental Figure 3). Although no differences in intrinsic excitability were observed (Supplemental Figure 3B), there were significant differences in AP waveform between WT, Scn8aD/+, and Scn8aW/+-PV interneurons at P18–P21. APs in P18–P21 Scn8a mutant mice were significantly wider, with slower upstroke and downstroke velocities, than in their WT counterparts (Supplemental Figure 3, C–F, and Supplemental Table 2). These findings indicate early alterations in PV interneuron AP parameters before the onset of spontaneous seizures and may suggest a progression of PV interneuron physiology into adulthood.

GOF Nav1.6 mutations affect sodium channel currents in PV interneurons. Depolarization block in Scn8aD/+ and Scn8aW/+ PV interneurons likely arises from abnormal sodium channel activity as a result of the GOF variant, contributing to changes in membrane depolarization levels and subsequent sodium channel availability for AP initiation. Increases in the INaP have been identified as a major factor in many epileptic encephalopathy–causing variants, including both the N1768D and R1872W variants in SCN8A DEE (5, 10, 30, 38). Further, INaP is a known determinant of depolarization block threshold (11). In view of this, we recorded INaP in PV interneurons in the whole-cell patch clamp configuration (Figure 3A). INaP was increased in both Scn8aD/+ (–293.1 ± 38.0 pA; P = 0.032) and Scn8aW/+ (–347.1 ± 49.0 pA; P = 0.004) PV interneurons when compared with WT (–166.6 ± 29.7 pA; Figure 3, B–E). Half-maximal voltage of activation (V1/2) did not differ from WT (–62.0 ± 1.0 mV) in either Scn8aD/+ (–59.9 ± 1.1 mV; P = 0.329) or Scn8aW/+-PV (–63.9 ± 1.2 mV; P = 0.592) mice (Figure 3F). Another component of the sodium current that may affect excitability particularly in fast spiking cells is the INaR (39, 40). INaR is a slow inactivating depolarizing current that can contribute to increased AP frequency by providing additional depolarization during the falling phase of an AP (39–41). INaR has been previously implicated in TLE as well as in sodium channelopathies (42, 43). INaR was significantly increased in Scn8aW/+-PV interneurons (–1,136.0 ± 178.5 pA; P = 0.037), and while we observed an increasing trend, INaR was not significantly increased in Scn8aD/+ PV interneurons (–952.8 ± 172.9 pA; P = 0.219), when compared to WT (–595.9 ± 84.8 pA) PV interneurons (Figure 3, G–J). Current-voltage relationship of INaR was not different between WT, Scn8aD/+, and Scn8aW/+-PV mice (P = 0.631; Figure 3K). These results demonstrate an increase in 2 components of the overall sodium current in PV interneurons, which possibly contributes to their initial hyperexcitability and increased susceptibility to depolarization block. Increases in both INaP and INaR probably provide a sustained level of depolarization, resulting in the accumulation of inactivated sodium channels and increased susceptibility to depolarization block (11, 32, 44).

INaP and INaR in WT, Scn8aD/+, and Scn8aW/+ PV interneurons. (A) INaP and INaR were recorded via whole-cell patch clamp onto PV interneurons in layer IV/V of the somatosensory cortex in adult, 5- to 8-week-old WT (n = 14 cells, 5 mice), Scn8aD/+ (n = 12 cells, 4 mice), and Scn8aW/+-PV mice (n = 15 cells, 5 mice). (B–D) Example traces of steady-state INaP evoked by slow voltage ramps from WT (B, black), Scn8aD/+ (C, red), and Scn8aW/+ (D, blue) PV interneurons. Traces in gray show slow voltage ramp in the presence of 500 nM tetrodotoxin (TTX). (E) Elevated maximum INaP in Scn8aD/+ (*, P < 0.05) and Scn8aW/+-PV (**, P < 0.01) interneurons compared with WT PV interneurons (Kruskal-Wallis test with Dunn’s multiple comparison test). (F) V1/2 values were not different between WT, Scn8aD/+, and Scn8aW/+-PV mice (P > 0.05, 1-way ANOVA with Dunnett’s multiple comparison test). (G–I) Example traces of TTX-subtracted INaR for WT (G, black), Scn8aD/+ (H, red), and Scn8aW/+ (I, blue). (J) Maximum INaR magnitude was increased between WT and Scn8aW/+-PV interneurons (*, P < 0.05), whereas INaR magnitude between WT and Scn8aD/+ PV interneurons was not significantly different (P > 0.05, Brown-Forsythe ANOVA with Dunnett’s multiple comparison test). (K) Current-voltage relationship for INaR is not significantly different between WT, Scn8aD/+, and Scn8aW/+-PV mice (P > 0.05, 2-way ANOVA).

Alterations of both activation and steady-state inactivation parameters of the transient sodium channel current have been previously reported in cells expressing GOF SCN8A variants (5, 45–47). To examine PV interneuron sodium channel currents, we performed excised somatic patches in the outside-out configuration from PV interneurons (Figure 4A). Sodium current density, voltage-dependent activation, or steady-state inactivation were not different between WT, Scn8aD/+, and Scn8aW/+-PV mice (Figure 4, B–H, and Table 2).

Transient sodium currents in WT, Scn8aD/+, and Scn8aW/+ PV interneurons. (A) Transient sodium current was assessed in PV interneurons using patch-clamp recordings in the outside-out configuration. (B–D) Example traces of sodium currents recorded from WT (B, black), Scn8aD/+ (C, red), and Scn8aW/+ (D, blue) PV interneurons. (E) Maximum transient sodium current was not significantly different between WT (n = 20 cells, 8 mice), Scn8aD/+ (n = 12 cells, 4 mice), and Scn8aW/+ (n = 12 cells, 4 mice) PV interneurons (P > 0.05, Kruskal-Wallis test with Dunn’s multiple comparison test). (F) Current-voltage relationship does not differ between WT, Scn8aD/+, and Scn8aW/+-PV interneurons (P > 0.05, 2-way ANOVA). (G) Voltage-dependent conductance curve does not differ significantly between WT, Scn8aD/+, and Scn8aW/+ PV interneurons (P > 0.05, 2-way ANOVA). (H) Steady-state inactivation does not differ significantly between WT (n = 12 cells, 4 mice), Scn8aD/+ (n = 11 cells, 4 mice), and Scn8aW/+ (n = 10 cells, 4 mice) PV interneurons (P > 0.05, 2-way ANOVA). Boltzmann curves shown are the average of individual curves generated from fits to data points.

Channel properties of layer IV/V PV interneurons

Decreased inhibitory input onto excitatory neurons in Scn8a mutant mice. Impaired excitability in Scn8a mutant PV interneurons may lead to decreased inhibition onto excitatory PCs, as PV interneurons are known to directly inhibit PCs at the soma or AIS (14, 15). To examine how alterations in PV interneuron excitability affect the cortical network, we recorded sIPSCs and mIPSCs from PCs (Figure 5A) as a functional indicator of PV interneuron activity and connectivity. We found that PCs generated significantly fewer sIPSCs in both Scn8aD/+ (4.22 ± 0.64 Hz; P = 0.035) and Scn8aW/+-PV (4.07 ± 1.14 Hz; P = 0.003) mice than their WT counterparts (7.97 ± 0.88 Hz; Figure 5, B and C), suggesting a decrease in inhibitory input onto PCs. sIPSC frequencies between Scn8aD/+ and Scn8aW/+-PV PCs were not different (P > 0.99), which may imply that PV interneurons are largely responsible for the decrease in somatic inhibitory input in the global Scn8aD/+ model. sIPSC amplitude was not different between WT (–62.7 ± 4.3 pA), Scn8aD/+ (–54.7 ± 5.8 pA), and Scn8aW/+-PV mice (–53.3 ± 8.4 pA; P = 0.09, Figure 5D). Additionally, we calculated the total charge transfer from sIPSCs in WT, Scn8aD/+, and Scn8aW/+-PV PCs and found that the total spontaneous charge transfer onto PCs was significantly decreased in Scn8aW/+-PV mice (–15,346 ± 3,706 pA × s; P = 0.008) compared with WT (–41,468 ± 7,641 pA × s, Figure 5E). Although it was not statistically significant, we also observed a decreasing trend in spontaneous inhibitory charge transfer in Scn8aD/+ mice (–20,424 ± 4,895 pA × s; P = 0.08; Figure 5E). sIPSC recordings include both AP-induced synaptic transients as well as mIPSCs, which occur because of spontaneous vesicle fusion in the absence of an AP (48, 49). To isolate AP-independent events, we performed recordings in the presence of TTX (500 nM). Relative to WT controls (3.52 ± 0.65 Hz), we found no significant difference in PC mIPSC frequency in Scn8aD/+ mice (2.78 ± 0.57 Hz; P = 0.821), but we did observe a significant reduction of mIPSC frequency in Scn8aW/+-PV mice (1.43 ± 0.22 Hz; P = 0.027; Figure 5F), which could underlie impaired synaptic transmission in Scn8aW/+-PV mice. mIPSC amplitude did not differ between WT (–37.7 ± 3.7 pA), Scn8aD/+ (–41.6 ± 3.0 pA; P = 0.667), and Scn8aW/+-PV mice (–25.1 ± 3.5 pA; P = 0.055, Figure 5G), though we did observe a decreasing trend in the mIPSC amplitude for Scn8aW/+-PV mice. Interestingly, we did not observe any significant differences in mIPSC total charge transfer between WT (–6,874 ± 1,194 pA × s), Scn8aD/+ (–6,907 ± 1,426 pA × s; P = 0.984), and Scn8aW/+-PV mice (–3,734 ± 872.6 pA × s; P = 0.133, Figure 5H).

IPSCs generated in PCs from WT, Scn8aD/+, and Scn8aW/+-PV mice. (A) Whole-cell recordings of IPSCs were collected from cortical layer V PCs in adult, 5- to 8-week-old WT, Scn8aD/+, and Scn8aW/+-PV mice. (B) Example traces of IPSCs generated in PCs from WT (black), Scn8aD/+ (red), and Scn8aW/+-PV (blue) mice. (C) Frequency of sIPSCs generated in PCs is decreased in Scn8aD/+ (n = 15 cells, 4 mice, *, P < 0.05) and Scn8aW/+-PV (n = 16 cells, 5 mice, **, P < 0.01) mice when compared with WT (n = 20 cells, 6 mice, Kruskal-Wallis test with Dunn’s multiple-comparison test). (D) Amplitude of sIPSCs generated in PCs is not significantly different between groups (P > 0.05, Kruskal-Wallis test with Dunn’s multiple-comparison test). (E) Total sIPSC charge transfer onto PCs was significantly decreased in Scn8aW/+-PV mice (**, P < 0.01), whereas total sIPSC charge transfer in Scn8aD/+ mice was not significantly different (P > 0.05, Kruskal-Wallis test with Dunn’s multiple-comparison test). (F) Frequency of mIPSCs recorded from PCs is decreased in Scn8aW/+-PV (n = 8 cells, 3 mice) mice when compared with WT (n = 7 cells, 3 mice, *, P < 0.05), whereas frequency of mIPSCs recorded from PCs in Scn8aD/+ (n = 7 cells, 3 mice) mice did not significantly differ from WT (P > 0.05, 1-way ANOVA with Dunnett’s multiple-comparison test). (G) Amplitude of mIPSCs recorded from PCs is not significantly different between WT, Scn8aD/+, and Scn8aW/+-PV mice (P > 0.05, Brown-Forsythe ANOVA with Dunnett’s multiple comparison test). (H) Total mIPSC charge transfer onto PCs did not significantly differ between WT, Scn8aD/+, and Scn8aW/+-PV mice (P > 0.05, Kruskal-Wallis test with Dunn’s multiple-comparison test).

PV interneuron synaptic transmission is impaired in Scn8a mutant mice. Impairment of synaptic transmission has been suggested as a disease mechanism in multiple epilepsy syndromes, notably Dravet syndrome (23, 50, 51), and proper synaptic signaling is tightly linked to sodium channel function (52). To assess how Nav1.6 function influences PV interneuron-mediated inhibitory synaptic transmission, we performed dual whole-cell patch clamp recordings of PV interneurons and nearby PCs to find synaptically connected pairs of cells (Figure 6A). Synaptically connected pairs were identified using a current ramp in the presynaptic PV interneuron to elicit inhibitory postsynaptic potentials (IPSPs) in the postsynaptic PC corresponding to each AP in the PV interneuron (Figure 6B). The number of synaptically connected PV:PC pairs relative to the total number of pairs was not significantly different between WT, Scn8aD/+, and Scn8aW/+-PV mice (P = 0.634, Figure 6C). In PV:PC connected pairs, we measured the properties of unitary inhibitory postsynaptic currents (uIPSCs) in PCs evoked by stimulation of PV interneurons. To accurately detect uIPSCs, a high-chloride internal solution was used to allow recording of uIPSCs as large inward currents and IPSPs as large membrane depolarizations, overall minimizing the possibility of inaccurately reporting a synaptic failure.

Increased synaptic transmission failure and synaptic latency in Scn8a mutant mice. (A) Image of dual whole-cell recording of a synaptically connected PV interneuron and pyramidal cell pair. (B) Example traces from a PV interneuron (gray) and synaptically coupled pyramidal cell (PC; black). (C) Proportion of successfully patched PV:PC pairs that were synaptically connected did not differ between WT (49 pairs from 13 mice), Scn8aD/+ (40 pairs from 8 mice), and Scn8aW/+-PV (54 pairs from 9 mice) in adult mice. (D–F) Example of presynaptic firing and evoked uIPSCs in WT (D; black), Scn8aD/+ (E; red), and Scn8aW/+-PV (F; blue) connected pairs at 5 Hz, 10 Hz, 20 Hz, 40 Hz, 80 Hz, and 120 Hz. Purple arrows denote uIPSC failures in the postsynaptic neuron. (G–L) Summary data for failure rates of evoked uIPSCs at various frequencies. In Scn8aD/+ connected pairs (n = 7, 5 mice), uIPSC failure rate is not significantly different from WT (n = 4, 3 mice) at 5, 10, 20, or 40 Hz (P > 0.05, G–J) but is significantly higher at PV interneuron firing frequencies of 80 and 120 Hz (*, P < 0.05, K and L). uIPSC failure rate in Scn8aW/+-PV pairs (n = 6, 5 mice) is significantly higher than WT at 5, 10, 20, 80, and 120 Hz (*, P < 0.05, G–I, K, and L) but did not significantly differ at 40 Hz (P < 0.05, J, 1-way ANOVA with Dunnett’s multiple-comparison test). (M and N) Example traces illustrating synaptic latency in WT, Scn8aD/+, and Scn8aW/+-PV, measured from the peak of the presynaptic AP to the onset of the evoked uIPSC (M). Gray dotted lines indicate this latency in WT. Latency is increased in Scn8aD/+ and Scn8aW/+-PV mice (N, 1-way ANOVA with Dunnett’s multiple-comparison test, *, P < 0.05, **, P < 0.01).

Previous studies indicate that the PV:PC synapse is extremely reliable since PV interneurons have multiple synaptic boutons and a high release probability, indicative of a highly stable synapse (53). PV interneurons are also known to fire reliably at high frequencies (15). We found that stimulation of PV interneurons at a 1 Hz frequency reliably initiated single APs in WT mice. Although we detected some failures in Scn8aD/+ and Scn8aW/+-PV mice, there was no significant difference in synaptic failure at a frequency of 1 Hz (P = 0.160; Table 3) between the groups, suggesting no deficit in synaptic transmission at low stimulation frequencies. The amplitudes of the uIPSCs also did not differ between genotypes (Table 3, P = 0.427). Additionally, to identify any deficits in short-term synaptic plasticity, we used the first 2 IPSCs (IPSC1 and IPSC2) elicited by a presynaptic AP to quantify the paired-pulse ratio (PPR). The PV:PC synapse is known to experience short-term plasticity through synaptic depression (54, 55). We observed synaptic depression in WT, Scn8aD/+, and Scn8aW/+-PV connected pairs, with no significant difference in PPR between WT and Scn8a mutant pairs (P = 0.340 and P = 0.189 respectively; Table 3).

Properties of postsynaptic uIPSCs in synaptically connected PV:PC pairs

To analyze activity-dependent synaptic failure, we then used stimulation trains to elicit multiple APs at increasing frequencies (5, 10, 20, 40, 80, and 120 Hz; Figure 6, D–F, and Table 3). At each frequency, we measured the failure rate of the first and last uIPSC as well as the overall failure rate. Failure rate of the first uIPSC remained low and consistent between WT, Scn8aD/+, and Scn8aW/+-PV mice. At lower frequencies (≤40 Hz), there were no differences in overall failure rate or last uIPSC failure rate between WT and Scn8aD/+mice; however, failure rates were significantly increased in Scn8aW/+-PV mice at 5, 10, and 20 Hz (Figure 6, G–I), with an increasing trend at a 40 Hz stimulation frequency (Figure 6J). At 80 Hz, the overall failure rate in a 20-pulse train was increased in both Scn8aD/+ (0.316 ± 0.062; P = 0.039) and Scn8aW/+-PV (0.390 ± 0.048; P = 0.009) mice compared with WT (0.101 ± 0.040; Figure 6K), with failures occurring approximately 3 times as frequently in Scn8aW/+ mice when compared with WT. Similarly, at 120 Hz stimulation frequency with a 30-pulse train, failure rates observed in Scn8aD/+ and Scn8aW/+-PV pairs were greater (0.382 ± 0.048 and 0.412 ± 0.068, respectively; P = 0.016 and P = 0.009) than those observed in their WT counterparts (0.123 ± 0.087; Figure 6L). The progression of total activity-dependent synaptic failure through increasing presynaptic stimulation frequencies is shown in Figure 6, G–L. Additionally, synaptic failure of the last uIPSC in a stimulation train occurred in more than 40% of trials on average with a stimulation frequency of 80 or 120 Hz. We observed that this increase in synaptic failure was significant for the last uIPSC in an 80 Hz train in Scn8aD/+ (P = 0.023) and Scn8aW/+-PV (P = 0.025; Table 3), as well as in a 120 Hz train (P = 0.043 and P = 0.030, respectively), supporting a greater degree of activity-dependent failure. Analysis of synaptic latency times, measured from the peak of the presynaptic AP to the onset of the postsynaptic uIPSC, revealed an increase in synaptic latency in Scn8aD/+ (P = 0.009) and Scn8aW/+-PV (P = 0.012) mice when compared with WT mice (Figure 6, M and N, and Table 3). Prolonged synaptic latency would suggest an impairment in conduction velocity or GABA release probability, potentially with a longer time lag to vesicle release (56–59). Efficient synaptic transmission and vesicle release are critical for overall network inhibition (60).