Impaired fast-acting brain cells lead to communication problems and seizures in SCN8A-related developmental epilepsy

We first sought to determine if expression of a GOFvariant selectively in PV interneurons would be sufficient for the development of spontaneous seizures. We used the conditional knockinmouse model and crossed homozygous PV-Cre mice with.tdT mice to generate-PV mice, where the R1872Wvariant is expressed exclusively in PV interneurons ().-PV mice were implanted with EEG recording electrodes and monitored for 10 weeks. To better conceptualize the phenotype of-PV mice with reference to anotherDEE model, we also implanted EEG recording electrodes inmice, which express the N1768Dvariant globally, and monitored for 6–8 weeks. Spontaneous seizures were observed in all recorded-PV mice (= 8;) andmice (= 14,). Median seizure onset in-PV mice was approximately 10 weeks of age. In-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 bothand-PV mice revealed spike wave discharges, a distinct aspect of electrographic seizures (), highlighting similarities between a global mutation model and a model harboring anvariant exclusively in PV interneurons.-PV mice also died prematurely compared with WT littermates, with a median survival of 16.6 weeks (). Electrographic and video recordings verified-PV mice that died during monitoring succumbed to seizure-induced death (= 3;). Interestingly, all fatal seizures exhibited a tonic phase before death, consistent with our previous findings inDEE mice (). In agreement with previous studies (),mice also died prematurely as a result of seizure-induced death (and), which was significantly accelerated compared with-PV mice (= 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 ofDEE. SCN8A Scn8a Scn8a Scn8a SCN8A Scn8a Scn8a SCN8A Scn8a SCN8A Scn8a n Scn8a n Scn8a Scn8a Scn8a Scn8a SCN8A Scn8a Scn8a n SCN8A Scn8a Scn8a P SCN8A W/+ W/+ W/+ W/+ W/+ D/+ W/+ D/+ W/+ W/+ D/+ W/+ W/+ W/+ D/+ W/+ Figure 1A Figure 1, B and D Figure 1, C and E Figure 1, B and C Figure 1F Supplemental Videos 1 and 2 ^{31 29} Figure 1F Supplemental Video 3

To assess the intrinsic physiological function ofmutant 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),-PV, and age-matched WT littermates (). To verify that fluorescently labeled cells were indeed PV positive, we used immunohistochemistry to stain for PV in WT,, and-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 (). WT littermates from bothand-PV genotypes did not exhibit any differences in firing frequencies (= 0.656) and were pooled. Analysis of membrane and AP properties revealed thatPV interneurons had decreased downstroke velocity as well as increased AP width when compared with WT (). Using a series of depolarizing current injection steps to assess intrinsic excitability, we observed a difference in excitability (= 0.028) between WT,, andPV interneurons. Initially, PV interneurons expressing eithervariant were hyperexcitable compared with WT littermates at lower current injection steps (<100 pA inmice,= 0.045, and <360 pA in-PV mice,= 0.030). However, at higher current injection steps, bothandPV interneurons exhibited progressive AP failure as a result of depolarization block (>640 pA inmice,= 0.042; >840 pA in-PV mice,= 0.041;). BothandPV interneurons were more prone to depolarization block than their WT counterparts over the range of current injection magnitudes (< 0.0001 and= 0.016, respectively;). 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 (–). Here, the early onset of depolarization block inmutant PV interneurons indicates a PV hypoexcitability phenotype, similar to the phenotypes observed in PV interneurons in GOFDEE and in SST interneurons inDEE (,). Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a P Scn8a P Scn8a Scn8a Scn8a Scn8a P Scn8a P Scn8a Scn8a Scn8a P Scn8a P Scn8a Scn8a P P Scn8a SCN1A SCN8A D/+ W/+ D/+ W/+ D/+ W/+ D/+ D/+ W/+ D/+ W/+ D/+ W/+ D/+ W/+ D/+ W/+ Figure 2A Supplemental Figure 1 Table 1 Figure 2, B–F Figure 2F ^{32 37 11 32}

Previous studies have shown that excitatory pyramidal neurons in global knockinmice 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 (). To determine if firing is affected in excitatory neurons from-PV mice, which selectively express anvariant in PV interneurons, we recorded the intrinsic excitability of pyramidal neurons from cortical layers IV/V in adult mice (). Interestingly, we did not observe any differences in the intrinsic excitability of pyramidal neurons between the WT and-PV genotypes (). This suggests that alterations in the physiology of PV interneurons may be sufficient in facilitating seizures inDEE. Analysis of AP parameters revealed an increase in input resistance and a decrease in rheobase (, E and F, and), suggestive of some compensatory changes in excitatory PCs. Scn8a Scn8a Scn8a Scn8a SCN8A D/+ W/+ W/+ ¹⁰ Supplemental Figure 2 Supplemental Figure 2 Supplemental Figure 2 Supplemental Table 1

Additionally, the role of development is an important consideration in understanding the pathophysiology ofDEE. In Dravet syndrome, differences in PV interneuron intrinsic excitability are observed only during a critical developmental time window (P18–P21) (). To determine if the same was true for PV interneurons inDEE, we measured intrinsic excitability at the critical P18–P21 time window (). Although no differences in intrinsic excitability were observed (), there were significant differences in AP waveform between WT,, and-PV interneurons at P18–P21. APs in P18–P21mutant mice were significantly wider, with slower upstroke and downstroke velocities, than in their WT counterparts (, C–F, and). 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. SCN8A SCN8A Scn8a Scn8a Scn8a ²¹ Supplemental Figure 3 Supplemental Figure 3B Supplemental Figure 3 Supplemental Table 2 D/+ W/+

Depolarization block inandPV 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 Ihave been identified as a major factor in many epileptic encephalopathy–causing variants, including both the N1768D and R1872W variants inDEE (,,,). Further, Iis a known determinant of depolarization block threshold (). In view of this, we recorded Iin PV interneurons in the whole-cell patch clamp configuration (). Iwas increased in both(–293.1 ± 38.0 pA;= 0.032) and(–347.1 ± 49.0 pA;= 0.004) PV interneurons when compared with WT (–166.6 ± 29.7 pA;). Half-maximal voltage of activation (V) did not differ from WT (–62.0 ± 1.0 mV) in either(–59.9 ± 1.1 mV;= 0.329) or-PV (–63.9 ± 1.2 mV;= 0.592) mice (). Another component of the sodium current that may affect excitability particularly in fast spiking cells is the I(,). Iis a slow inactivating depolarizing current that can contribute to increased AP frequency by providing additional depolarization during the falling phase of an AP (–). Ihas been previously implicated in TLE as well as in sodium channelopathies (,). Iwas significantly increased in-PV interneurons (–1,136.0 ± 178.5 pA;= 0.037), and while we observed an increasing trend, Iwas not significantly increased inPV interneurons (–952.8 ± 172.9 pA;= 0.219), when compared to WT (–595.9 ± 84.8 pA) PV interneurons (). Current-voltage relationship of Iwas not different between WT,, and-PV mice (= 0.631;). 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 Iand Iprobably provide a sustained level of depolarization, resulting in the accumulation of inactivated sodium channels and increased susceptibility to depolarization block (,,). Scn8a Scn8a SCN8A Scn8a P Scn8a P Scn8a P Scn8a P Scn8a P Scn8a P Scn8a Scn8a P D/+ W/+ D/+ W/+ D/+ W/+ W/+ D/+ D/+ W/+ NaP NaP NaP NaP 1/2 NaR NaR NaR NaR NaR NaR NaP NaR ^{5 10 30 38 11} Figure 3A Figure 3, B–E Figure 3F ^{39 40 39 41 42 43} Figure 3, G–J Figure 3K ^{11 32 44}

Alterations of both activation and steady-state inactivation parameters of the transient sodium channel current have been previously reported in cells expressing GOFvariants (,–). To examine PV interneuron sodium channel currents, we performed excised somatic patches in the outside-out configuration from PV interneurons (). Sodium current density, voltage-dependent activation, or steady-state inactivation were not different between WT,, and-PV mice (, and). SCN8A Scn8a Scn8a ^{5 45 47} Figure 4A Figure 4, B–H Table 2 D/+ W/+

Impaired excitability inmutant PV interneurons may lead to decreased inhibition onto excitatory PCs, as PV interneurons are known to directly inhibit PCs at the soma or AIS (,). To examine how alterations in PV interneuron excitability affect the cortical network, we recorded sIPSCs and mIPSCs from PCs () as a functional indicator of PV interneuron activity and connectivity. We found that PCs generated significantly fewer sIPSCs in both(4.22 ± 0.64 Hz;= 0.035) and-PV (4.07 ± 1.14 Hz;= 0.003) mice than their WT counterparts (7.97 ± 0.88 Hz;), suggesting a decrease in inhibitory input onto PCs. sIPSC frequencies betweenand-PV PCs were not different (> 0.99), which may imply that PV interneurons are largely responsible for the decrease in somatic inhibitory input in the globalmodel. sIPSC amplitude was not different between WT (–62.7 ± 4.3 pA),(–54.7 ± 5.8 pA), and-PV mice (–53.3 ± 8.4 pA;= 0.09,). Additionally, we calculated the total charge transfer from sIPSCs in WT,, and-PV PCs and found that the total spontaneous charge transfer onto PCs was significantly decreased in-PV mice (–15,346 ± 3,706 pA × s;= 0.008) compared with WT (–41,468 ± 7,641 pA × s,). Although it was not statistically significant, we also observed a decreasing trend in spontaneous inhibitory charge transfer inmice (–20,424 ± 4,895 pA × s;= 0.08;). 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 (,). 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 inmice (2.78 ± 0.57 Hz;= 0.821), but we did observe a significant reduction of mIPSC frequency in-PV mice (1.43 ± 0.22 Hz;= 0.027;), which could underlie impaired synaptic transmission in-PV mice. mIPSC amplitude did not differ between WT (–37.7 ± 3.7 pA),(–41.6 ± 3.0 pA;= 0.667), and-PV mice (–25.1 ± 3.5 pA;= 0.055,), though we did observe a decreasing trend in the mIPSC amplitude for-PV mice. Interestingly, we did not observe any significant differences in mIPSC total charge transfer between WT (–6,874 ± 1,194 pA × s),(–6,907 ± 1,426 pA × s;= 0.984), and-PV mice (–3,734 ± 872.6 pA × s;= 0.133,). Scn8a Scn8a P Scn8a P Scn8a Scn8a P Scn8a Scn8a Scn8a P Scn8a Scn8a Scn8a P Scn8a P Scn8a P Scn8a P Scn8a Scn8a P Scn8a P Scn8a Scn8a P Scn8a P ^{14 15} Figure 5A Figure 5, B and C Figure 5D Figure 5E Figure 5E ^{48 49} Figure 5F Figure 5G Figure 5H D/+ W/+ D/+ W/+ D/+ D/+ W/+ D/+ W/+ W/+ D/+ D/+ W/+ W/+ D/+ W/+ W/+ D/+ W/+

Impairment of synaptic transmission has been suggested as a disease mechanism in multiple epilepsy syndromes, notably Dravet syndrome (,,), and proper synaptic signaling is tightly linked to sodium channel function (). To assess how Na1.6 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 (). 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 (). The number of synaptically connected PV:PC pairs relative to the total number of pairs was not significantly different between WT,, and-PV mice (= 0.634,). 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. ^{23 50 51 52} Figure 6A Figure 6B Figure 6C v Scn8a Scn8a P D/+ W/+

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 (). PV interneurons are also known to fire reliably at high frequencies (). We found that stimulation of PV interneurons at a 1 Hz frequency reliably initiated single APs in WT mice. Although we detected some failures inand-PV mice, there was no significant difference in synaptic failure at a frequency of 1 Hz (= 0.160;) between the groups, suggesting no deficit in synaptic transmission at low stimulation frequencies. The amplitudes of the uIPSCs also did not differ between genotypes (,= 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 (,). We observed synaptic depression in WT,, and-PV connected pairs, with no significant difference in PPR between WT andmutant pairs (= 0.340 and= 0.189 respectively;). ^{53 15} Table 3 Table 3 ^{54 55} Table 3 Scn8a Scn8a P P Scn8a Scn8a Scn8a P P D/+ W/+ D/+ W/+

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;, and). 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,, and-PV mice. At lower frequencies (≤40 Hz), there were no differences in overall failure rate or last uIPSC failure rate between WT andmice; however, failure rates were significantly increased in-PV mice at 5, 10, and 20 Hz (), with an increasing trend at a 40 Hz stimulation frequency (). At 80 Hz, the overall failure rate in a 20-pulse train was increased in both(0.316 ± 0.062;= 0.039) and-PV (0.390 ± 0.048;= 0.009) mice compared with WT (0.101 ± 0.040;), with failures occurring approximately 3 times as frequently inmice when compared with WT. Similarly, at 120 Hz stimulation frequency with a 30-pulse train, failure rates observed inand-PV pairs were greater (0.382 ± 0.048 and 0.412 ± 0.068, respectively;= 0.016 and= 0.009) than those observed in their WT counterparts (0.123 ± 0.087;). The progression of total activity-dependent synaptic failure through increasing presynaptic stimulation frequencies is shown in–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(= 0.023) and-PV (= 0.025;), as well as in a 120 Hz train (= 0.043 and= 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(= 0.009) and-PV (= 0.012) mice when compared with WT mice (, and). Prolonged synaptic latency would suggest an impairment in conduction velocity or GABA release probability, potentially with a longer time lag to vesicle release (–). Efficient synaptic transmission and vesicle release are critical for overall network inhibition (). Figure 6, D–F Table 3 Figure 6, G–I Figure 6J Figure 6K Figure 6L Figure 6, G Table 3 Figure 6, M and N Table 3 ^{56 59 60} Scn8a Scn8a Scn8a Scn8a Scn8a P Scn8a P Scn8a Scn8a Scn8a P P Scn8a P Scn8a P P P Scn8a P Scn8a P D/+ W/+ D/+ W/+ D/+ W/+ W/+ D/+ W/+ D/+ W/+ D/+ W/+

PV interneurons are known to be the main drivers for seizure activity in Dravet syndrome, a disorder characterized by deficits in inhibitory neurons, primarily due to haploinsufficiency of Na1.1 (–). Selective deletion of Na1.1 in PV interneurons leads to reduced PV interneuron excitability, decreased spontaneous inhibition of excitatory neurons, and increased susceptibility to seizures (). Similar to impairments observed in mouse models of Dravet syndrome and inmice, which express the N1768Dvariant globally, we show here that selective expression of the GOF R1872W variant in PV interneurons is sufficient to induce spontaneous seizures and leads to seizure-induced death (SUDEP) in mice. Additionally,-PV mice exhibited a reduced seizure frequency and increased survival compared withmice, and global expression of the R1872W variant or exclusive expression in excitatory neurons leads to a more severe SUDEP phenotype than-PV mice (). This may indicate that although PV interneurons are an important contributor to theDEE phenotype, dysfunction of excitatory neurons remains a critical aspect of the disease physiology, as previously published (,,). Overall, these findings not only support an important role for PV interneurons in the seizure phenotype ofDEE but also provide support for a major role for Na1.6 channels in controlling PV interneuron excitability in addition to Na1.1 channels. v v v v ^{20 23 61 30 10 30 62} Scn8a SCN8A Scn8a Scn8a Scn8a SCN8A SCN8A D/+ W/+ D/+ W/+

Proper function ofis critical in repetitive firing (), and as such, we reasoned that mutations affecting the function ofwould affect the high-frequency, repetitive firing characteristic of PV interneurons. However, although at lower current injection magnitudes PV interneurons from both mutant mouse models were hyperexcitable, at higher magnitudes we observed PV interneuron AP failure through depolarization block, resulting in overall PV interneuron hypoexcitability. Increased susceptibility to depolarization block due to a GOF sodium channel mutation has been shown previously in bothDEE andDEE (,). Additionally, depolarization block in PV interneurons leads to hyperactivity and subsequent epileptic discharges in excitatory cells, and rescue of depolarization block via optogenetic stimulation leads to a reduction in epileptiform activity (–). Further, in vivo recording of PV interneurons shows evidence for PV depolarization block during seizure activity (). Scn8a Scn8a SCN8A SCN1A ^{41 11 32 34 36 37}

Interestingly, the susceptibility to depolarization block and subsequent hypoexcitability in inhibitory interneurons reported here indicates that a mechanism for seizures inDEE, a disorder characterized primarily by GOF sodium channel mutations, shares many similarities to that of Dravet syndrome, a disorder primarily characterized by sodium channel haploinsufficiency in inhibitory neurons. A study using a model of Dravet syndrome showed a similar pattern of initial hyperexcitability in PV interneurons followed by depolarization block (). However, impairment of PV interneuron excitability in Dravet syndrome is specific to the P18–P21 developmental time window (), whereas inDEE, PV interneuron activity is more markedly impaired in adulthood. The initial hyperexcitability of inhibitory interneurons seen in bothDEE and Dravet syndrome may play some role in the shared comorbidities between these severe developmental disorders. SCN8A SCN8A SCN8A ^{63 21}

We believe our study is the first to examine alterations in synaptic transmission between PV interneurons and excitatory neurons inDEE, and we show a distinct impairment of inhibitory synaptic transmission onto excitatory PCs in 2 patient-derived mutation models. Synaptic transmission was impaired in bothmutant mouse models:-PV connected pairs failed substantially more than WT at most frequencies, whereaspairs failed in an activity-dependent manner. Considering the fast-spiking nature of PV interneurons and the degree of inhibitory input they provide on neuronal excitatory networks, activity-dependent failure alone could have a substantial impact on overall seizure susceptibility. A likely mechanism for this failure could be impaired AP propagation, as proper signaling from PV interneurons requires a specific density and function of sodium channels (). This is further supported by the observed increase in synaptic latency, indicating that propagation may be slowed inmutant mice. Similarly, synaptic transmission between PV interneurons and PCs is also impaired in Dravet syndrome, though unlike our findings inDEE, intrinsic excitability deficits are restored in adult PV interneurons (,). A limitation of the study is the number of synaptically connected pairs recorded. It is possible that synaptic transmission is impaired in a non-activity-dependent manner in bothand-PV mice, as a slight increasing trend in the failure rates ofuIPSCs at low frequencies was observed. SCN8A Scn8a Scn8a Scn8a Scn8a SCN8A Scn8a Scn8a Scn8a W/+ D/+ D/+ W/+ D/+ ^{52 21 23}

Both depolarization block and synaptic transmission failure occurred at high PV interneuron firing frequencies, and as such, it is important to consider in vivo firing frequencies of PV interneurons. PV interneurons are a heterogeneous group made up of primarily basket cells and chandelier cells, which are named for their unique morphologies. These subtypes have slightly different firing patterns and synaptic targets (,). Since our recordings are focused within cortical layers IV/V, it is likely that we recorded primarily from PV-positive basket cells rather than chandelier cells. In vivo, PV interneurons, particularly basket cells, are phase-locked to gamma oscillations, which typically occur between 40 and 100 Hz (). Events such as sharp wave ripples (SWRs) can lead to PV firing frequencies of more than 120 Hz in vivo (). This demonstrates the relevance of both increased susceptibility of PV interneurons to depolarization block and of PV:PC synaptic transmission failure at high frequencies with expression of mutant. These gamma oscillations and SWRs are most often associated with the hippocampus; however, there is evidence for oscillations in the cortex (,). Recently, SWRs have been associated with epileptic discharges in Dravet syndrome: an increase in SWR amplitude may lead to inhibitory depolarization block and a shift into seizure-like activity (). Considering the frequencies at which we observe PV interneuron failure in bothmutant mouse models, increased susceptibility to depolarization block and failure of inhibitory synaptic transmission could underlie an additional mechanism of seizure generation inDEE. ^{14 15 64 65 66 67 68} Scn8a Scn8a SCN8A

We observed an increase in Iin bothand-PV interneurons with an increase in Iin-PV interneurons. However, we observed no difference in the transient sodium current inand-PV interneurons, though it is possible that excised somatic patches may not have recapitulated the high levels of Na1.6 in the axon. Previous studies suggest thatmay have a much larger role in Ithan transient current (). Increases in Ihave been implicated in various epilepsies (,,,), and prior computational modeling suggests that heightened Iunderlies the phenotype of increased susceptibility to depolarization block in inhibitory interneurons (). Ialso functions as an amplifier of synaptic currents (,), although we did not observe differences in amplitude of uIPSCs in recordings of synaptically connected pairs. Because Iis a consistent, non-inactivating component of the sodium current (), we hypothesize that elevations in Icontribute to premature failure of PV interneurons and subsequent entry into depolarization block. Additionally, Icurrents are crucial in facilitating repetitive, high-frequency firing, as they affect fast inactivation through an open channel block (,), and Na1.6 is a crucial contributor to I(). We only observed a significant increase in Iin-PV interneurons and not inPV interneurons, possibly due to mutation-specific effects: this has been observed previously in patient-derived neurons (). Increases in Ilikely provide excessive depolarizing current resulting in an increase in firing frequencies, which may be responsible for differences observed betweenand-PV interneuron firing, asPV interneurons enter depolarization block at lower current injections. NaP NaR v NaR NaP NaP NaP NaP NaP NaR v NaR NaR NaR Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a D/+ W/+ W/+ D/+ W/+ W/+ D/+ D/+ W/+ D/+ ^{41 38 42 44 69 11 70 71 44 40 72 41 73}

It is also important to consider the potential consequences of a GOF Na1.6 mutation on the structural composition of the AIS. Sodium channels are expressed together with potassium channels at the AIS, and both play crucial roles in controlling neuronal excitability (). Further, previous studies suggest interaction between sodium and potassium channels as a result of genetic mutations (–). Potassium channels such as K7.2, which is encoded by, interacts with Na1.6, and is an important mediator of M-type potassium current (,), or K3.1, which is important for repetitive, high-frequency firing (), could be affected by these changes in sodium channel function and may underlie some physiological differences observed inmutant PV interneurons. Interaction betweenandhas been shown previously in a DEE model: in DEE resulting from loss-of-function mutations in, an antisense oligonucleotide (ASO) to reduce the expression ofleads to a marked increase in survival (). v v v v ^{8 74 76 77 78 79 75} KNCQ2 Scn8a Scn8a Kcnq2 Kcnq2 Scn8a

Patients withvariants are typically treated with sodium channel blockers, and many are refractory to treatment, highlighting the need to further understand the basic mechanisms surrounding theDEE phenotype. Hyperexcitability of excitatory neurons has often been suggested as the underlying cause behind seizures inDEE, and, contradictory to our results here, a previous study suggests limited involvement of inhibitory interneurons due to the lack of seizures when the R1872Wvariant is expressed in all inhibitory interneurons (). However, in the previous study, the SUDEP phenotype of mice expressing the R1872W variant globally (; EIIa-Cre) is markedly more severe than that of mice expressing the R1872W variant exclusively in forebrain excitatory neurons (; EMX1-Cre), with median survival of 15 days and 46 days, respectively (), suggesting the involvement of additional cell types. While we acknowledge the critical contributions of excitatory neuron dysfunction to theDEE phenotype (,,), here we provide compelling support for a major involvement of PV inhibitory interneurons in the onset of spontaneous seizures and seizure-induced death inDEE. SCN8A SCN8A SCN8A SCN8A Scn8a Scn8a SCN8A SCN8A ^{30 30 10 30 62} W/+ W/+

Gene therapies are in development for bothDEE and Dravet syndrome, and downregulation ofhas been shown to reduce seizures in both disorders (–). Specifically, an ASO forwas able to significantly delay seizure onset and increase survival in mice that express the R1872Wvariant globally (). This ASO treatment targeted both excitatory and inhibitory neurons. Our previous studies have shown that ASO-mediated rescue of PV interneuron firing reduces seizures and prevents SUDEP in a model of Dravet syndrome (); a similar phenotype may be observed inDEE, where rescue of depolarization block prevents seizures and SUDEP. In a similar manner to Dravet syndrome, specific targeting of inhibitory interneurons inDEE may be a novel therapeutic strategy. SCN8A Scn8a Scn8a SCN8A SCN8A SCN8A ^{80 82 80 82}

In conclusion, we show here that PV interneurons play an important role inDEE. Elevations in Ilikely render PV interneurons more susceptible to AP failure, and subsequent depolarization block leads to a decrease in network inhibition. PV interneurons also exhibit impaired synaptic transmission, and together, we observe that a GOFvariant exclusively expressed in PV interneurons conveys susceptibility to spontaneous seizures and SUDEP. In the field ofDEE, prior research has focused primarily on the impact of GOFmutations on excitatory neurons (,,). These results, along with our previous work proposing that SST interneurons contribute to seizures (), shift the paradigm of theDEE field from primarily considering excitatory neuron hyperexcitability as the sole driver of the seizure phenotype and call for future studies to further explore the importance of inhibitory neuron activity inDEE. SCN8A SCN8A SCN8A SCN8A SCN8A SCN8A NaP ^{10 30 62 11}

Both male and female mice were used in this study. While roughly equal numbers of each sex were used in each experimental group, sex was not considered as a biological variable.

andmice were generated as previously described and maintained through crosses with C57BL/6J mice (The Jackson Laboratory [Jax], 000664) to keep all experimental mice on a C57BL/6J genetic background (,). Bothandtransgenic mice were previously gifted from Miriam Meisler at the University of Michigan, Ann Arbor, Michigan, USA. Cell type–specific expression of R1872W was achieved using males heterozygous for the R1872W allele and C57BL/6J females homozygous for PV-Cre (Jax, 017320) to generate mutant mice (-PV) (). Homozygous PV-IRES-Cre females were used for breeding to ensure minimal germline recombination due to Cre, as shown previously (,). Because certain transgenic mice entail the insertion of Cre directly into the coding sequence and because of the need for a fluorescent reporter to reliably identify PV interneurons in-slice, for all experiments we used WT controls that contained the same Cre allele but lacked the allele encoding thevariant. Fluorescent labeling of PV interneurons was achieved by first crossingormice with C57BL/6J mice homozygous for a Cre-dependent tdTomato reporter (Jax, 007909) to generatetdTomato ortdTomato mice. Then, maletdTomato ortdTomato mice were crossed with female mice homozygous for PV-Cre. Experimental groups used at least 3 randomly selected mice to achieve statistical power and roughly equal numbers of male and female mice. All genotyping was conducted through Transnetyx automated genotyping PCR services. Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a Scn8a D/+ W/+ D/+ W/+ W/+ D/+ W/+ D/+ W/+ D/+ W/+ ^{29 30 30 83 84}

Custom EEG headsets (PlasticsOne) were implanted in 5-week-old-PV mice and 6- to 8-week-oldmice using standard surgical techniques as previously described (). Anesthesia was induced with 5% and maintained with 0.5%–3% isoflurane. Adequacy of anesthesia was assessed by lack of toe-pinch reflex. A midline skin incision was made over the skull and connective tissue was removed. Burr holes were made at the lateral/anterior end of the left and right parietal bones to place EEG leads and at the interparietal bone for ground electrodes. EEG leads were placed bilaterally in the cortex or unilaterally placed in the cortex and superior colliculus using a twist. A headset was attached to the skull with dental acrylic (Jet Acrylic; Lang Dental). Mice received postoperative analgesia with ketoprofen (5 mg/kg, i.p.) and 0.9% saline (0.5 mL i.p.) and were allowed to recover a minimum of 2–5 days before seizure-monitoring experiments. Scn8a Scn8a W/+ D/+ ⁸⁵

Mice were then individually housed in custom-fabricated chambers and monitored for the duration of the experiment. The headsets were attached to a custom low-torque swivel cable, allowing mice to move freely in the chamber. EEG signals were amplified at 2,000× original magnification, and bandpass-filtered between 0.3 and 100 Hz, with an analog amplifier (Neurodata model 12, Grass Instruments). Biosignals were digitized with a Powerlab 16/35 and recorded using LabChart 7 software at 1 kS/s. Video acquisition was performed by multiplexing 4 miniature night vision–enabled cameras and then digitizing the video feed with a Dazzle Video Capture Device and recording at 30 fps with LabChart 7 software in tandem with biosignals.

Brain tissue for immunohistochemistry was processed as previously described (,). Mice were anesthetized and transcardially perfused with 10 mL PBS followed by 10 mL 4% paraformaldehyde (PFA). Brains were immersed in 4% PFA overnight at 4°C and stored in PBS. Coronal brain sections, 30 μm, were obtained using a cryostat. Sections were incubated with mouse anti-PV (MilliporeSigma, MAB1572) diluted in 2% goat serum (Jackson ImmunoResearch Laboratories) with 0.1% Triton X-100 (MilliporeSigma) at a concentration of 1:500 in Dulbecco's PBS. The secondary antibody, goat anti-mouse Alexa Fluor 488 (Invitrogen, A-11029), was diluted 1:1,000 in goat serum (2%) and Triton X-100 (0.1%) in Dulbecco's PBS. Sections were stained free-floating in primary antibody on a shaker at 4°C overnight and with secondary antibody for 1 hour at room temperature the following day. Tissues were counterstained with NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (Thermo Fisher Scientific, catalog R37606) included in the secondary antibody solution. Tissues were mounted on slides using AquaMount (Polysciences). ^{11 86}

Preparation of acute brain slices for patch-clamp electrophysiology experiments was modified from standard protocols previously described (,,). Mice were anesthetized with isoflurane and decapitated. The brains were rapidly removed and kept in chilled artificial cerebrospinal fluid (ACSF) (0°C) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaHPO, 2 CaCl, 1 MgCl, 0.5-ascorbic acid, 10 glucose, 25 NaHCO, and 2 Na-pyruvate. For dual-cell patch-clamp experiments, the slicing solution was modified to contain (in mM): 93-Methyl--glucamine, 2.5 KCl, 1.25 NaHPO, 20 HEPES, 5-ascorbic acid (sodium salt), 2 thiourea, 3 sodium pyruvate, 0.5 CaCl, 10 MgSO, 25-glucose, and 12-acetyl--cysteine, 30 NaHCO, with pH adjusted to 7.2–7.4 using HCl (osmolarity 310 mOsm). Slices were continuously oxygenated with 95% Oand 5% COthroughout the preparation. Coronal or horizontal brain sections, 300 μm, were prepared using a Leica Microsystems VT1200 vibratome. Slices were collected and placed in ACSF warmed to 37°C for about 30 minutes and then kept at room temperature for up to 6 hours. ^{10 11 30} 2 4 2 2 3 2 4 2 4 3 2 2 l d l d l N N

Brain slices were placed in a chamber superfused (~2 mL/min) with continuously oxygenated recording solution warmed to 32 ± 1°C. IntdTomato PV-Cre,tdTomato PV-Cre, or WT tdTomato PV-Cre mice, cortical layer IV/V PV interneurons were identified as red fluorescent cells, and pyramidal neurons were identified based on morphology and absence of fluorescence via video microscopy using a Carl Zeiss Axioscope microscope. Whole-cell recordings were performed using a Multiclamp 700B amplifier with signals digitized by a Digidata 1322A digitizer. Currents were amplified, lowpass-filtered at 2 kHz, and sampled at 100 kHz. Borosilicate electrodes were fabricated using a Brown-Flaming puller (model P1000, Sutter Instruments) to have pipette resistances between 1.5 and 3.5 MΩ. All patch-clamp electrophysiology data were analyzed using custom MATLAB scripts or ClampFit 10.7. Scn8a Scn8a D/+ W/+

Current-clamp recordings of neuronal excitability were collected in ACSF solution identical to that used for preparation of brain slices. The internal solution contained the following (in mM): 120 K-gluconate, 10 NaCl, 2 MgCl, 0.5 KEGTA, 10 HEPES, 4 NaATP, and 0.3 NaGTP, pH 7.2 (osmolarity 290 mOsm). Intrinsic excitability was assessed using methods adapted from those previously described (,). Briefly, resting membrane potential was manually recorded from the neuron at rest. Current ramps from 0 to 400 pA over 4 seconds were used to calculate passive membrane and AP properties, including threshold, upstroke and downstroke velocity, which are the maximum and minimum slopes on the AP, respectively; amplitude, which was defined as the voltage range between AP peak and threshold; APD, which is the duration of the AP at the midpoint between threshold and peak; input resistance, which was calculated using a –20 pA pulse in current-clamp recordings; and rheobase, which was defined as the maximum amount of depolarizing current that could be injected into neurons before eliciting an AP. AP frequency–current relationships were determined using 1-second current injections from –140 to 1,200 pA. Spikes were only counted if AP overshoot was >0 mV and amplitude was >20 mV. The threshold for depolarization block was operationally defined as the current injection step that elicited the maximum number of APs (i.e., subsequent current injection steps of greater magnitude resulted in fewer APs because of entry into depolarization block). 2 2 2 50 ^{10 11}

Iand Iwere recorded in the whole-cell patch clamp configuration in-slice, whereas transient sodium current was recorded in the outside-out configuration. The internal solution for all voltage-gated sodium channel recordings contained the following (in mM): 140 CsF, 2 MgCl, 1 EGTA, 10 HEPES, 4 NaATP, and 0.3 NaGTP with the pH adjusted to 7.3 and osmolality to 300 mOsm. The external solution for recording persistent and resurgent sodium currents has been previously described (,) and contained (in mM): 100 NaCl, 40 TEACl, 10 HEPES, 3.5 KCl, 2 CaCl, 2 MgCl, 0.2 CdCl, 4 of 4-aminopyridine, and 25-glucose. Outside-out recordings of transient sodium current were collected in ACSF as the external solution. Steady-state Is were elicited using a voltage ramp (20 mV/s) from –80 to –20 mV. To record resurgent sodium currents (I), PV interneurons were held at –100 mV, depolarized to 30 mV for 20 ms, and then stepped to voltages between −100 mV and 0 mV for 40 ms. After collecting recordings at baseline, protocols were repeated in the presence of 500 nM TTX (Alomone Labs) to completely block Iand I. Traces obtained in the presence of TTX were subtracted from those obtained in its absence. The Vof Iwas calculated as previously described (). Patch clamp recordings in the outside-out configuration were collected using a protocol modified from an approach previously described (,). Voltage-dependent activation and steady-state inactivation parameters were recorded using voltage protocols previously described (). For all sodium current recordings, we waited 2 minutes after achieving whole-cell configuration to account for initial shifts in the voltage dependence of activation. NaP NaR 2 2 2 2 2 NaP NaR NaP NaR 1/2 NaP ^{87 88 88 10 11 11} d

Patch-clamp recordings of IPSCs generated in PCs were performed using the same ACSF external solution and an internal solution containing (in mM): 70 K-Gluconate, 70 KCl, 10 HEPES, 1 EGTA, 2 MgCl, 4 MgATP, and 0.3 NaGTP, with the pH adjusted to 7.2–7.4 and osmolarity to 290 mOsm. Pyramidal cells were held at –70 mV and a 1-minute gap-free recording was performed in the voltage-clamp configuration to assess spontaneous IPSC frequencies before bath application of 500 nM TTX to record miniature IPSCs. After recording spontaneous and miniature IPSCs, 1 μM gabazine was bath applied to block currents and ensure that only inhibitory events were recorded. 2 3

uIPSCs were obtained via 2 simultaneous patch-clamp recordings from synaptically connected neurons located within 50 μm of one another in the somatosensory cortex of a horizontal slice. A 2 ms pulse at 1,000 pA elicited APs in the presynaptic neuron at 1, 5, 10, 20, 40, 80, and 120 Hz. The internal solution was modified to contain (in mM): 65 K-gluconate, 65 KCl, 2 MgCl, 10 HEPES, 0.5 EGTA, 10 phosphocreatine-Tris, 4 MgATP, and 0.3 NaGTP, with pH adjusted to 7.2–7.4 using KOH (osmolarity 290 mOsm) (). PPR was calculated as the amplitude of the second IPSC divided by the amplitude of the first IPSC. PPR was not calculated for trials in which the first and/or second IPSC event was a failure. uIPSC failures were identified by the absence of a transient current greater than 5 pA occurring within 5 ms of the presynaptic AP. Synaptically connected pairs were not used for analysis if resting membrane potential shifted more than 10 mV during recording. 2 2 ²³

Analysis of electrophysiological data was performed in a blinded manner. All statistical comparisons were made using the appropriate test in GraphPad Prism 9. Categorical data were analyzed using Fisher's exact test. For membrane and AP properties, spontaneous firing frequency, depolarization block threshold, peak sodium currents, half-maximal voltages, IPSC frequency and amplitude, and synaptic uIPSC properties, mouse genotypes were compared by 1-way ANOVA followed by Dunnett's multiple comparisons test when the data were normally distributed with equal variances, by Brown-Forsythe ANOVA with Dunnett's multiple comparisons test when the data were normally distributed with unequal variances, and by the nonparametric Kruskal–Wallis test followed by Dunn's multiple comparisons test when the data were not normally distributed. Data were assessed for normality using the Shapiro-Wilk test. Bartlett's test with= 0.05 was used to assess equal variance. Data were tested for outliers using the ROUT or Grubbs's method to identify outliers, and statistical outliers were not included in data analysis. A 2-way ANOVA followed by Tukey's test for multiple comparisons was used to compare groups in experiments in which repetitive measures were made from a single cell over various voltage commands or current injections. Cumulative distribution (survival) plots were analyzed by the log-rank Mantel-Cox test. Data are presented as individual data points and/or mean ± SEM. Exactandvalues are reported in figure legends. P n P

Animal experiments were performed in compliance with animal care guidelines issued by the NIH and Animal Care and Use Committee at the University of Virginia (protocol approval no. 3308).

Individual data values are available in thedocument. Supplemental material is available in the online version. Supporting Data Values